Febrile neutropenia (FN) is a major concern in patients undergoing chemotherapy for diffuse large B-cell lymphoma (DLBCL); however, the overall risk of FN is difficult to assess. This study aimed to develop a model for predicting the occurrence of FN in patients with DLBCL. In this multicenter, retrospective, observational analysis, a multivariate logistic regression model was used to analyze the association between FN incidence and pretreatment clinical factors. We included adult inpatients and outpatients (aged ≥ 18 years) diagnosed with DLBCL who were treated with chemotherapy. The study examined 246 patients. Considering FN occurring during the first cycle of chemotherapy as the primary outcome, a predictive model with a total score of 5 points was constructed as follows: 1 point each for viral hepatitis, extranodal involvement, and a high level of soluble interleukin-2 receptor and 2 points for lymphopenia. The area under the receiver operating characteristic curve of this model was 0.844 (95% confidence interval: 0.777–0.911). Our predictive model can assess the risk of FN before patients with DLBCL start chemotherapy, leading to better outcomes.

Hepatitis B virus (HBV) infection is a major factor in development of various liver diseases such as hepatocellular carcinoma (HCC). Among HBV encoded proteins, HBV X protein (HBx) is known to play key role in development of HCC. Hepatocyte nuclear factor 4α (HNF4α) is a nuclear transcription factor which is critical for hepatocyte differentiation. However, the expression level as well as its regulatory mechanism in HBV infection have yet to be clarified. Here, we observed the suppression of HNF4α in cells which stably express HBV whole genome or HBx protein alone, while transient transfection of HBV replicon or HBx plasmid had no effect on the HNF4α level. Importantly, in the stable HBV- or HBx-expressing hepatocytes, the downregulated level of HNF4α was restored by inhibiting ERK signaling pathway. Our data showed that HNF4α was suppressed during long-term HBV infection in cultured HepG2-NTCP cells as well as in mouse model following hydrodynamic injection of pAAV-HBV or in mice intravenously infected with rAAV-HBV. Importantly, HNF4α downregulation increased cell proliferation which contributed to the formation and development of tumor in xenograft nude mice. The data presented here provided several proofs for the effect of HBV infection in manipulating HNF4α regulatory pathway in HCC development.

Sporotrichosis is a subcutaneous mycosis affecting humans and other animals that can be transmitted a zoonosis with cats as the main vector. The conventional anti-fungal therapy is especially inefficient in immunocompromised patients, who tend to develop the most severe forms of the disease, thus prompting the search for alternative therapies. Given their antigen-presenting properties, dendritic cells (DCs) have been used in both prophylactic and therapeutic vaccination strategies. Hence, this study aims to assess the use of DCs as a prophylactic tool in sporotrichosis by evaluating the immune profile induced by Sporothrix schenckii cell wall proteins (SsCWP)-stimulated bone-marrow-derived DCs (BMDCs). Mouse BMDCs were stimulated with SsCWP for 24 hours and analyzed for the surface expression of co-stimulatory molecules and TLR-4, as well as the secretion of proinflammatory cytokines and IL-10. Following that, activated BMDCs were cocultured with splenocytes for 72 hours and had the same cytokines measured in the supernatant. SsCWP-stimulated BMDCs showed higher expression of CD80, CD86, and CD40, but not TLR-4, and higher secretion of IL-6, IL-17A, and TNF. On the other hand, higher levels of IFN-γ, IL-10, and IL-2 were found in the supernatants of the coculture as compared with the BMDCs alone; TNF secretion was almost completely abrogated, whereas IL-6 was only partially inhibited and IL-17A was unaffected. Our results thus suggest SsCWP-stimulated BMDCs are able to induce a Th1-prone cytokine profile, known to be protective against other fungal diseases. This result could lead to evaluate the development of prophylactic and/or therapeutic DC-based tools against sporotrichosis.

Current clinical treatment options for symptomatic, partial-thickness rotator cuff tear (sPTRCT) offer only limited potential for true tissue healing and improvement of clinical results. In animal models, injections of adult stem cells isolated from adipose tissue into tendon injuries evidenced histological regeneration of tendon tissue. However, it is unclear whether such beneficial effects could also be observed in a human tendon treated with fresh, uncultured, autologous, adipose derived regenerative cells (UA-ADRCs). A specific challenge in this regard is that UA-ADRCs cannot be labeled and, thus, not unequivocally identified in the host tissue. Therefore, histological regeneration of injured human tendons after injection of UA-ADRCs must be assessed using comprehensive, immunohistochemical and microscopic analysis of biopsies taken from the treated tendon a few weeks after injection of UA-ADRCs.

Coronavirus disease (COVID-19) is a new discovered strain where WHO officially declares the disease as COVID-19 while the virus responsible for it called Severe Acute Respiratory Syndrome Coronavirus 2 or SARS-CoV-2. The incubation period of this disease is between 14 days. Ordinary clinical symptoms that reported around the world include fever, cough, fatigue, diarrhoea and vomiting as well as asymptomatic for certain people. Infection is spread mainly through broad droplets. In early March 2020, WHO again has announced that COVID-19 is a pandemic with currently no specific treatment. The potential use of SARS-COV-2 proteome as a vaccine candidate by analysing through B-cell and T-cell antigenicity by using a immunoinformatics approach as a vaccine development early stage. In this study, we used consensus sequence for SARS-COV-2 proteome that was retrieved from NCBI database. VaxiJen 2.0 was mainly used to identify the antigenic property of SARS-COV-2 proteins. IEDB then used to analyse the B-cell epitope, the presence of T cell immunogenic epitope in SARS-COV-2 proteins was obtained by using compromise method of MHC class I and II tools that accessible respectively using ProPred-1 server and MHC II Binding Prediction in IEDB database. The best epitopes of B and T-cell epitopes were predicted with high antigencity and the information is disseminated through web-based database resource (https://covid-19.omicstutorials.com/epitopes/). This study will be useful to find a new epitope-based candidate for SARS-COV-2. However, further study needs to be done for the next stages of vaccine development.

Curcumin, a natural polyphenolic compound isolated from the plant Curcuma longa, is known to induce autophagy in various cancer cells, including lung cancer. In the present study, we also confirmed by LC3 immunofluorescence and immunoblotting analyses that curcumin triggers autophagy in human lung adenocarcinoma A549 cell line. In parallel with autophagy induction, gene expression of human GD3 synthase (hST8Sia I) responsible for ganglioside GD3 synthesis was markedly elevated in response to curcumin in A549 cells. To investigate transcriptional activation of hST8Sia I associated with autophagy formation in curcumin-treated A549 cells, functional characterization of the 5’-flanking region of the hST8Sia I gene was carried out using luciferase reporter assay system. Deletion analysis demonstrated that the -1146 to -646 region, which includes putative c-Ets-1, CREB, AP-1 and NF-κB binding sites, functions as the curcumin-responsive promoter of hST8Sia I in A549 cells. Site-directed mutagenesis and chromatin immunoprecipitation assay demonstrated that the NF-κB binding site at -731 to -722 was indispensable for the curcumin-induced hST8Sia I gene expression in A549 cells. Moreover, the transcriptional activation of hST8Sia I by curcumin A549 cells was strongly inhibited by compound C, an inhibitor of AMP-activated protein kinase (AMPK). These results suggest that curcumin controls hST8Sia I gene expression via AMPK signal pathway in A549 cells.

Premature ovarian insufficiency (POI) is a highly prevalent disorder, characterized by the development of menopause before age of 40. Most cases are idiopathic; however, in some women the cause of this condition (e.g. anticancer treatment, genetic disorders, and enzymatic defects) may be identified. Although hormone replacement therapy, the principal therapeutic approach for POI, helps to alleviate the related symptoms, this does not effectively solve the issue of fertility. Assisted reproductive techniques also lack efficacy in these women. Thus, the effective approach to manage the patients with POI is highly warranted. Several mechanisms, associated with POI, have been identified, including lack of FSH receptor functioning, alterations in the apoptosis control, mutations in Sal-like 4 genes, thymulin or basonuclin-1 deficiency etc. The above-mentioned may be good targets for gene therapy in order to correct defects, leading to POI. The goal of this review is to summarize the current experience on the POI studies, that employed gene therapy, and to discuss the possible future directions in this field.

Among the many Lysosomal Storage Disorders (LSDs) that would benefit from the establish-ment of novel cell models, either patient-derived or genetically engineered, is Mucopolysaccha-ridosis type II (MPS II). In fact, even though a specific therapeutic approach does exist for this disorder (Enzyme Replacement Therapy, ERT, with recombinant human IDS) and haematopoiet-ic stem cell transplantation (HSCT) may also hold promise whenever the disorder is diagnosed soon enough, both approaches have their own drawbacks. Here we present our results on the establishment and characterization of two MPS II pa-tient-derived stem cell line(s) from deciduous baby teeth. To the best of our knowledge, this is the first time a stem cell population is isolated from LSD patient samples obtained from the oral cavity. Taking into account our results on the molecular and biochemical characterization of those cells and our first insights on their subcellular MPS phenotype, we consider these cells will be a valuable tool for both pathophysiological assessments and drugs screenings. Ultimately, we believe patient-derived dental pulp stem cells (DPSC) may represent a feasible alternative to induced pluripotent stem cells (iPSC) in many labs with standard cell culture con-ditions and limited (human and economic) resources.

Soluble CD163 (sCD163) functions as a scavenger receptor for haptoglobin-hemoglobin complexes, and is mostly expressed by monocytes and macrophages. The level of soluble CD163 has been used to determine macrophage activation. Our goal was to determine whether the loading of M2 CD163 positive tumor-associated macrophages (TAMs) through the secretion of sCD163 affects overall survival in patients with DLBCL and eventually discriminates subgroups of patients with different prognosis. sCD163 levels were measured in 40 frozen serum samples from patients diagnosed with DLBCL and 30 healthy individuals (HI) using an enzyme-linked immunosorbent assay (ELISA). Statistical analyses were performed using SPSS version 28. The results showed that patients who achieved complete response after standard-of-care immuno-chemotherapy and were alive and disease-free after 12 months of follow-up, but had elevated sCD163 levels (above median) at diagnosis, presented a significantly worse overall survival compared to those with initial serum sCD163 levels below median (p=0.03). Consequently, serum sCD163 level in DLBCL patients may constitute a marker of long-term response to chemo-immunotherapy.

One of the hallmarks of microgravity-induced effects in several cellular models is represented by the alteration of oxidative balance with the consequent accumulation of Reactive Oxygen Species (ROS). It is well known that male germ cells are sensitive to oxidative stress and to changes of gravitational force even if published data on germ cell models are scarce. To gain more insights into the mechanisms of male germ cell response to altered gravity, a 3D cell culture model has been established from TCam-2 cells, a seminoma-derived cell line considered the only human cell line available to study in vitro mitotically active human male germ cells. TCam-2 cell spheroids were cultured for 24 hours under unitary gravity (Ctr) or simulated microgravity (s-microgravity) conditions, these last ones were obtained using the Random Positioning Machine (RPM). A significant increase in intracellular ROS and mitochondria superoxide anion levels has been observed after RPM exposure. In line with these results a trend of protein and lipid oxidation increase, and increased pCAMKII expression levels were observed after RPM exposure. The ultrastructural analysis by Transmission Electron Microscopy revealed that RPM-exposed mitochondria appeared enlarged and, even if seldom, disrupted. Notably, even the expression of the main enzymes involved in the redox homeostasis appears modulated by RPM exposure in a compensatory way, being GPX1, NCF1, and CYBB downregulated, whereas NOX4 and HMOX1 upregulated. Interestingly, HMOX1 is involved in the heme catabolism of mitochondria cytochromes, and therefore the positive modulation of this marker can be associated to the observed mitochondria alteration. All together, these data demonstrate TCam-2 spheroid sensitivity to acute SM exposure and indicate the capability of these cells to trigger compensatory mechanisms that allow to overcome the exposure to altered gravitational force.

Glioblastomas (GBM) are most common, primary brain tumors in adults. Despite advances in neurosurgery, radio- and chemotherapy, the median survival of GBM patients is 15 months. Recent large-scale genomic, transcriptomic and epigenetic analyses have shown the cellular and molecular heterogeneity of GBMs, which hampers the outcomes of standard therapies. We have established 13 GBM-derived cell cultures from fresh tumor specimens and characterized them molecularly using RNAseq, immunoblotting and immunocytochemistry. Evaluation of proneural (OLIG2, IDH1R132H, TP53 and PDGFRα), classical (EGFR) and mesenchymal markers (CHI3L1/YKL40, CD44 and phospho-STAT3), as well as expression of pluripotency (SOX2, OLIG2, NESTIN) and differentiation (GFAP, MAP2, β-Tubulin III) markers revealed the striking inter-tumor heterogeneity of primary GBM cell cultures. Upregulated expression of VIMENTIN, N-CADHERIN and CD44 at mRNA/protein levels suggested increased epithelial to mesenchymal transition (EMT) in most studied cell cultures. The effects of temozolomide (TMZ) or doxorubicin (DOX) were tested in three GBM-derived cell cultures with different methylation status of the MGMT promoter. Amongst TMZ- or DOX-treated cultures the strongest accumulation of apoptotic markers: caspase 7 and PARP were found in WG4 cells with methylated MGMT suggesting that its methylation status predicts vulnerability to both drugs. As many GBM-derived cells showed high EGFR levels, we tested the effects of AG1478, an EGFR inhibitor, on downstream signaling pathways. AG1478 caused decreased levels of phospho-STAT3, thus inhibition of active STAT3 augmented antitumor effects of DOX and TMZ in cells with methylated and intermediate status of MGMT. Altogether, our findings show that GBM-derived cell cultures mimic the considerable tumor heterogeneity and identifying patient-specific signaling vulnerabilities can assist in overcoming therapy resistance, by providing personalized combinatorial treatment recommendations.

Aims TRAIL is a promising anticancer agent that has the potential to sensitize a wide variety of cancer or transformed cells by inducing apoptosis. However, resistance to TRAIL is a growing concern. Current manuscript aimed to employing combination treatment to investigate resveratrol induced TRAIL sensitization in NSCLC. Method A549 and HCC-15 cells were used in experimental design. Cell viability was determined by morphological image, crystal violet staining and MTT assay. Apoptosis was evaluated by LDH assay, Annexin V and DAPI staining. Autophagy and apoptosis indicator protein were examined by western blotting. TEM and puncta assay were carried out to evaluate the autophagy. MTP and ROS activity were evaluated by JC-1 and H₂DCFDA staining. Findings Resveratrol is a polyphenolic compound capable of activation of tumor suppressor p53 and its pro-apoptotic modulator PUMA. Herein, we showed the p53-independent apoptosis by decrease the expression of phosphorylated Akt-mediated suppression of NF-κB that is also substantiated with the downregulation of anti-apoptotic factors Bcl-2 and Bcl-xl in NSCLC, resulting in an attenuation of TRAIL resistance in combined treatment. Furthermore, apoptosis was induced in TRAIL-resistant lung cancer cells with a co-treatment of resveratrol and TRAIL assessed by the loss of MMP, ROS generations which resulting the translocation of cytochrome c from the mitochondria into the cytosol due to mitochondrial dysfunction. Moreover, autophagy flux was not affected by resveratrol-induced TRAIL-mediated apoptosis in NSCLC. Significance Overall, targeting the NF-κB (p65) pathway via resveratrol attenuates TRAIL resistance and induces TRAIL-mediated apoptosis which could be the effective TRAIL-based cancer therapy regimen.

B and T lymphocytes demonstrate important alterations in patients with Systemic Lupus Erythematous (SLE), with significant up-regulation of Double Negative (DN) B cells. Aim of this study was to evaluate correlation of T cell immunity changes with the distinct B cell-pattern SLE. In the present study, Flow cytometry was performed in 30 patients with SLE, on remission, and 31 healthy controls, to detect DN B cells (CD19+IgD-CD27-) and a wide range of T lymphocyte subpopulations, based on the presence of CD45RA, CCR7, CD31, CD28, CD57, defined as naive, memory and advanced differentiated/senescent T cells. Both B and T lymphocytes were significantly reduced in SLE patients. However, the percentage of DN B cells were increased, compared to HC, 12.9(2.3-74.2) vs. 8(1.7-35), p=0.04. Distribution of CD4 and CD8 lymphocytes demonstrated a shift to advanced differentiated subsets. The population of DN B cells had significant positive correlation with most of early differentiated T lymphocytes, CD4CD31+, CD4CD45RA+CD28+, CD4CD45RA+CD57-, CD4CD45RA-CD57-, CD4CD28+CD57-, CD4CD28+CD57+, CD4 CM, CD8 CD31+, CD8 NAÏVE, CD8CD45RA-CD57-, CD8CD28+CD57-, CD8CD28+CD57+. Multiple Regression analysis revealed CD4CD31+, CD8CD45RA-CD57- and CD8CD28+CD57- cells as independent parameters contributing to DN B cells, adjusted R2=0.534, p<0.0001. The predominance of DN B cells in patients with SLE is closely associated with early differentiated T lymphocyte subsets, indicating a potential causality role of DN B cells in T lymphocyte activation.

The presence of premature termination codons (PTCs) in transcripts is dangerous for the cell as they encode potentially deleterious truncated proteins that can act with dominant-negative or gain-of-function effects. To avoid synthesis of these shortened polypeptides, several RNA surveillance systems can be activated to decrease the level of PTC-containing mRNAs. Nonsense-mediated mRNA decay (NMD) ensures an accelerated degradation of mRNAs harboring PTCs by using several key NMD factors such as up-frameshift (UPF) proteins. Another pathway called nonsense-associated altered splicing (NAS) upregulates transcripts that have skipped disturbing PTCs by alternative splicing. Therefore, these RNA quality control processes eliminate abnormal PTC-containing mRNAs from the cells by using positive and negative responses. In this review, we will describe the general mechanisms of NMD and NAS and their respective involvement in the decay of aberrant immunoglobulin and TCR transcripts in lymphoid cells.

Kidney fibrosis is a complex polyetiological progressive disease manifesting as an intraorgan proliferation of the connective tissue with the appearance of cicatricial changes. However, pathogenetic mechanisms of renal fibrosis remain poorly understood. The paper highlights features of the protease phenotype and histotopography of mast cells (MCs) found in the kidney, as the key players in extracellular matrix remodeling in nephrofibrosis, detected in a patient diagnosed with "nephrosclerosis with the non-functioning upper pole of the duplex right kidney in the junction of refluxing megaureter of the upper pole of the duplex right kidney". The highest content of MCs was found in the kidney areas with fibrotic changes. MCs were actively involved in the development of inflammatory and fibrotic changes in limited loci of the specific tissue microenvironment of the kidney through targeted secretion of tryptase, chymase, and carboxypeptidase A3 to the vascular endothelium, nephron epithelium, interstitium cells, and components of the intercellular substance. The formation of a profibrotic environment in the kidney interstitium was facilitated by epigenetic effects of tryptase promoting epithelial-mesenchymal transition (EMT) of the nephron epithelium. A selectively increased number and targeted secretory activity of MCs in limited loci of the kidney parenchyma without visible pathological changes evidence a trigger pathogenetic role of specific proteases in the formation of a local microenvironment with a profibrogenic metabolic profile. Thus, MCs are actively involved in the mechanisms of formation and evolution of fibrogenic niches in the kidney; this fact can be used to develop new strategies for therapeutic options of tubulointerstitial fibrosis resulted from chronic kidney injury. Further study of the molecular phenotype of mast cells reveals new fundamental mechanisms of fibrosis pathogenesis being a significant tool for personalized therapy in nephrology.

Lambertianic acid (LA) is a biologically active compound from the leaves of Pinus koraiensis. In the present study, apoptotic mechanisms of LA plus TNF-related apoptosis-inducing ligand (TRAIL) were elucidated in non-small cell lung cancer cells (NSCLCs). Cytotoxicity assay, flow cytometry, immunoprecipitation and Western blotting were performed. Here, combined treatment of LA and TRAIL increased cytotoxicity, sub-G1 population and cleaved poly (ADP-ribose) polymerase (PARP) and caspase3/8/9 in A549 and H1299 cells compared to LA or TRAIL alone. Furthermore, combined treatment of LA and TRAIL significantly decreased anti-apoptotic proteins such as B-cell lymphoma 2 (Bcl-2), Fas-like inhibitor protein (FLIP) and X-linked inhibitor of apoptosis protein (XIAP) and enhanced the activation of pro-apoptotic proteins Bid compared to LA or TRAIL alone. In addition, combined treatment of LA and TRAIL upregulated the expression of Death receptor 4 (DR4) and downregulated phosphorylation of nuclear factor kappa-light-chain-enhancer of activated B cells (p-NF-B), inhibitory protein of kB family (p-IB) and FLIP in A549 and H1299 cells along with disrupted binding of XIAP with caspase3 or NF-B. Overall, these findings suggest that lambertianic acid enhances TRAIL-induced apoptosis via inhibition of XIAP/NF-B in TRAIL resistant NSCLCs.

The influences of doped hole transporting layer (HTL) on the performances of perovskite solar cells were studied. The influences of electrostatic and surface roughness effect by solvent additive doping were compared. The electrostatic effect of solvent additive doping was compared by addition of 1,8-diiodooctane and 1,8-dichlorooctane to the HTL layer. The surface roughness effect of solvent additive doping was examined using atomic force microscopy. The perovskite crystallinity by doping HTL was studied by X-ray diffraction. The conductivity of HTL was measured using four probe methods and was higher for doped devices. Enhanced conductivity of PEDOT:PSS film was also proved using fluorescence emission quenching. Long-term stability of the device was enhanced by doping HTL. The device with solvent additive exhibited much enhanced stability, retained over 80% of the initial PCE for 400 hours, while PCE of the device without solvent additive decreased to less than 80% after 200 hours. A very weak power microwave treatment was used for post-annealing of the fully fabricated devices. Post-annealing enhanced the lifetime of the device. Comparing different size of cells, the device lifetime was decreased as the cell area increased and as the size of the cell increased, the extent of lifetime enhancement by doping was increased.

Severe COVID-19 alters the biochemical and morphological characteristics of blood cells in a wide variety of ways. To date, however, the vast majority of research has been devoted to the study of leukocytes, while erythrocyte morphological changes have received significantly less attention. The purpose of this research was to identify erythrocyte types that were unique to COVID-19, compare the number of different poikilocyte types, and measure erythrocyte sizes to provide data on size dispersion. Red blood cells obtained from 6 control donors (800-2200 cells for each donor) and 5 COVID-19 patients (800-1900 cells for each patient) were examined using low voltage scanning electron microscopy. We did not discover any forms of poikilocytes that would be unique to COVID-19. Among COVID-19 patients, we observed an increase in the number of acanthocytes (p=0.01) and a decrease in the number of spherocytes (p=0.03). In addition, our research demonstrates that COVID-19 causes an increase in the median (p=0.004) and interquartile range (p=0.009) when assessing erythrocyte size.

Familial Mediterranean fever (FMF) is a systemic autoinflammatory disorder caused by inherited mutations in the MEFV (Mediterranean FeVer) gene located on chromosome 16 (16p13.3) and coding pyrin protein. Despite the existing data on MEFV mutations, the exact mechanism of their effect on the development of pathological processes leading to autoinflammation observed in FMF, remains unclear. Induced pluripotent stem cells (iPSCs) are considered an important tool for studying the molecular genetic mechanisms of various diseases due to their ability to differentiate into any cell type, including macrophages, which contribute to the development of FMF. In this study, we developed iPSCs of an Armenian patient with FMF having M694V, p.(Met694Val) (c.2080A&gt;G, rs61752717) pathogenic mutation in exon 10 of the MEFV gene. As a result of direct differentiation, macrophages expressing CD14 and CD45 surface markers were obtained. In addition, we found that the morphology of patient macrophages derived from iPSCs with MEFV mutation was significantly different from that of differentiated from iPSCs of a healthy donor carrying wild-type MEFV gene.

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma and a heterogeneous disease characterized by mature B-cell neoplasms. The standard immunochemotherapy for DLBCL is R-CHOP, which includes rituximab, cyclophosphamide, doxoru-bicin, vincristine, and prednisone. Up to 30-40% of patients with DLBCL either exhibit primary refractory disease to standard treatment or experience relapse after the initial response. A 72-year-old man diagnosed with DLBCL involving chromosomal translocations t(1;22)(q21;q11.2) and t(6;18)(p25;q21) showed primary refractory disease after the fourth cycle of R-CHOP. The patient ultimately experienced cardiac involvement by the lymphoma and received salvage chemotherapy. He passed away about 15 months away after the diagnosis of DLBCL. We conducted fluorescence in situ hybridization (FISH) for further analysis of the chromosomal translocations. The breakpoint of chromosome 1q21 was located at a distance of around 151 Mb from the telomeric end of chromosome 1p. The breakpoint in chromosome 22q11 contains the immunoglobulin lambda locus. Furthermore, the breakpoint of chromosome 6p was in the telomeric region of chromosome 6p21. The breakpoint of chromosome 18q21 contains BCL2. This case report presents the first documented co-occurrence of chromosomal translocations t(1;22)(q21;q11.2) and t(6;18)(p25;q21) in a patient with DLBCL. These chromosomal translocations may indicate a worse clinical outcome.

Organoid engineering promises to revolutionize medicine with wide ranging applications of scientific, engineering, and clinical interest, including precision and personalized medicine, gene editing, drug development, disease modeling, cellular therapy, and a basic understanding of human development. Organoids are a three-dimensional (3D), miniature, caricature of a target organ, are initiated with stem/progenitor cells, and are extremely promising tools to model organ function. The biological basis for organoids is that they foster stem cell-self renewal, differentiation, and self-organization, recapitulating tissue structure or function better than 2D systems. In this review, we first discuss the importance of epithelial organs and the general properties of epithelial cells to provide context for the liver, pancreas, and gall bladder and rationale for organoid cultures. Next, we develop a general framework to understand self-organization, tissue hierarchy, and organoid cultivation. For each of these areas, we provide historical context, and review both a wide range of biological and/or biophysical/mathematic perspectives that enhances understanding of organoids. Next, we review existing techniques and progress in hepatobiliary and pancreatic organoid engineering. To do this, we review organoids from both primary tissues, cell lines, and stem cells, and introduce engineering studies when applicable. Noninvasive assessment of 1 organoids can reveal underlying biology and enable improved assays for growth, metabolism, and function. Applications of organoid for cell therapy are also discussed. Taken together, we establish a broad strong scientific foundation for organoids and provide an in-depth review of hepatic, biliary and pancreatic organoids.

One of the ways to regulate the sensitivity of human cells to the influenza virus is to knock out genes of the innate immune response. Promising targets for the knockout are genes of the interferon-inducible transmembrane protein (IFITM) family, in particular the IFITM3 gene, whose product limits the entry of a virus into the cell by blocking the fusion of the viral and endosomal membranes. In this study, by means of genome-editing system CRISPR/Cas9, monoclonal cell lines with an IFITM3 knockout were obtained based on WI-38 VA13 cells (human origin). It was found that such cell lines are more sensitive to infection by influenza A viruses of various subtypes. Nonetheless, this feature is not accompanied by an increased titer of newly formed viral particles in a culture medium.

The T cell composition of the tumor microenvironment (TME) of esophageal adenocarcinoma (EAC) in comparison to the T cell composition of the TME of Barrett esophagus (BE) and in the context of clinicopathologic features and patient survival is unknown. To help fill these knowledge gaps, we used a multiplex immunofluorescence platform to study adaptive T cells and T cell subtypes based on costimulatory/inhibitory markers in EAC (n=54) and matched BE (n=21) samples from 54 patients with EAC and correlated the T cell phenotype distribution with clinicopathologic features and disease-free survival. We observed significant enrichment of cytotoxic T cells (CTCs), memory T cells, effector memory T cells, memory T regulatory cells (MTregs), and T cells expressing costimulatory/inhibitory markers LAG3, TIM3, and ICOS in EAC compared to BE, in the stroma. In EAC, increased TIM3 expression on T cells significantly correlated with increased CTCs but not T regulatory cells (Tregs). In EAC, higher densities of Tregs and MTregs correlated with pT1 disease, low pathologic stage, and absence of lymphovascular invasion. In EAC, patients with higher density of TIM3-expressing T cells had shorter disease-free survival by univariate and multivariate analyses. Our results identify T cell subtypes that are relevant to EAC biology and patient outcome. Furthermore, our results identify TIM3 as a potential target for optimization of immunomodulatory therapy in EAC.

Proton exchange membrane (PEM) fuel cells have emerged as a viable alternative energy production source for stationary and transportation applications. Reliable and sustainable fuel cell operation requires effective water management. Membrane water content can vary along the stack during transients which can lead to losses in fuel cell performance. To control these variations a model that predicts the internal humidity dynamics of the stack is needed. In this study, a control-oriented model for predicting membrane water content variation was developed and implemented in MATLAB/Simulink. A lumped parameter model was initially developed and then further discretized into smaller control volumes to track humidity distribution along the stack. To validate the model’s predictions, the predicted results were compared to computer simulation results from GT-Suite. The root mean square error (RMSE) between the model’s prediction and GT-Suite’s simulation results was found to be within 1.5 membrane water content for all cases, demonstrating the model’s capability to capture the variation of membrane water content along the stack. The developed model will be useful for real-time control of membrane water content variation in PEM fuel cells.

Abstract : Aims: Regeneration of myocardial tissue and improvement of myocardial activity after ischemia is one of the major fields of interest in cardiovascular research. We have developed a novel experimental rat model, and used it to examine the effect of mesenchymal stem cell transplantation (MSCT) on myocardial ischemia evaluated by single photon emission computed tomography (SPECT/CT) and immunohistochemistry. Methods and results: Forty adult female Wistar rats underwent open thoracotomy with (n=30) or without (n=10) surgical ligation of the left anterior descending coronary artery (LAD), to create myocardial ischemia, which was confirmed with ECG. Myocardial viability was evaluated via SPECT/CT at 7 days before as well as at 7 and 14 days post-surgery. At day 0, 15 animals received homologous stem cells injected at the ischemic myocardium area. SPECT/CT evaluation confirmed the reduction of functional myocardial cells of the left ventricle one week post-infarction. Regeneration of the ischemic myocardium fifteen days post infarction was recorded, only in animals submitted to stem cell transplantation. Findings were also confirmed by histology and immunohistochemical analysis, with significant higher expression of GATA4 and Nkx2,5. Conclusion: The positive effect of mesenchymal stem cells transplantation in the ischemic myocardium was recorded. The application of SPECT- CT qualitative and quantitative evaluation of myocardial function may lead to a new approach in experimentation with an anticipated significant impact in the ongoing cardiovascular laboratory research. In clinical perspective, MSCT may be beneficial when accompanied with myocardial revascularization procedures.

Aim: Sca-1+CD31− cells were shown to be endothelial stem/progenitor cells, found in many mammalian tissues, including heart, were able to differentiate into cardiomyocytes in vitro and in vivo. Our previous work indicated that heart derived Sca-1+CD31− cells increased Nr1d1 mRNA level and decreased cells plasticity with aging. However, little is known about how NR1D1 affects Sca-1+CD31− cells plasticity. Methods: Lentiviral vector was used to stably overexpress Nr1d1 in young Sca-1+CD31− cells and to knockdown Nr1d1 in aged Sca-1+CD31− cells. Cell differentiation, proliferation, apoptosis, and cell cycle were evaluated. Results and Conclusions: The overexpression of Nr1d1 in young Sca-1+CD31− cells inhibited cell proliferation and promoted apoptosis. Knockdown of Nr1d1 in aged Sca-1+CD31− cells promoted cell proliferation and inhib-ited apoptosis. Using mouse cardiac myocytes cell line, confirmed the effect of Nr1d1 and indi-cated that Nr1d1 induce Serpina3 expression via Nr1d1 interaction with Nr4a3. Nr1d1 may there-fore be identified as a potent anti-aging receptor and be a therapeutic target for aging relative diseases.

Uniform water soluble MoS2 quantum dots (WS-MSQDs) are synthesized via a sequential combination of sintering/etching/exfoliation method and solvothermal route. The obtained WS-MSQDs with average size of approximately 3.4 nm exhibit sufficient water solubility and remarkable fluorescence properties. The WS-MSQDs have been utilized as a probe for detection of Fe3+ ions with high selectivity and specificity. Furthermore, the WS-MSQDs exhibit high fluorescence stability under different conditions. Finally, the WS-MSQDs are successfully applied for the fluorescence imaging of Fe3+ in living cells, which exhibited practical potential for biomedical applications.

Understanding the biology underlying the mechanisms and pathways regulating pancreatic β-cell development is necessary to understand the pathology of diabetes mellitus (DM), which is characterized by the progressive reduction in insulin producing β-cell mass. Pluripotent stem cells (PSCs) can potentially offer an unlimited supply of functional β-cells for cellular therapy and disease modeling of DM. Homeobox protein NKX6.1 is a transcription factor (TF) that plays a critical role in pancreatic β-cell function and proliferation. In human pancreatic islet, NKX6.1 expression is exclusive toβ-cells and is undetectable in other islet cells. Several reports showed that activation of NKX6.1 in PSC-derived pancreatic progenitors (MPCs), expressing PDX1 (PDX1⁺/NKX6.1⁺), warrants their future commitment to monohormonal β-cells. However, further differentiation of MPCs lacking NKX6.1 expression (PDX1⁺/NKX6.1^-) results in an undesirable generation of non-functional polyhormonal β-cells. The importance of NKX6.1 as a crucial regulator in MPC specification into functional β-cells directs attentions to further investigating its mechanism and enhancing NKX6.1 expression as a mean to increase β-cell function and mass. Here, we shed light on the role of NKX6.1 during pancreatic β-cell development and in directing the MPCs to functional monohormonal lineage. Furthermore, we address the transcriptional mechanisms and targets of NKX6.1 as well as its association with diabetes.

DNA methylation and other epigenetic factors are important in the pathogenesis of late-onset Alzheimer’s disease (LOAD). Methylenetetrahydrofolate reductase (MTHFR) gene mutations occur in most elderly patients with memory loss. MTHFR is critical for production of S-adenosyl-L-methionine (SAM), the principal methyl donor. A common mutation (1364T/T) of the cystathionine-γ-lyase (CTH) gene affects the enzyme that converts cystathionine to cysteine in the trans-sulfuration pathway causing plasma elevation of total homocysteine (tHcy) or hyperhomocysteinemia – a strong and independent risk factor for cognitive loss and AD. Other causes of hyperhomocysteinemia include aging, nutritional factors, and deficiencies of B vitamins. We emphasize the importance of supplementing vitamin B₁₂ (methylcobalamin), vitamin B₉ (folic acid), vitamin B₆ (pyridoxine), and SAM to patients in early stages of LOAD.

Noise induces oxidative stress in the cochlea followed by sensory cell death and hearing loss. The proof of principle that injections of antioxidant vitamins and Mg²⁺ prevent noise-induced hearing loss (NIHL) has been established. However, effectiveness of oral administration remains controversial and otoprotection mechanisms unclear. Using auditory evoked potentials, quantitative PCR and immunocytochemistry, we explored effects of oral administration of vitamins A, C, E and Mg²⁺ (ACEMg) on auditory function and sensory cell survival following NIHL in rats. Oral ACEMg reduced auditory thresholds shifts after NIHL. Improved auditory function correlated with increased survival of sensory outer hair cells. In parallel, oral ACEMg modulated the expression timeline of antioxidant enzymes in the cochlea after NIHL. There was increased expression of Glutathione peroxidase-1 and Catalase at 1 and 10 days, respectively. Also, pro-apoptotic Caspase-3 and Bax levels were diminished in ACEMg-treated rats, at 10 and 30 days, respectively, following noise overstimulation, whereas, at day 10 after noise exposure, the levels of anti-apoptotic Bcl-2, were significantly increased. Therefore, oral ACEMg improves auditory function by limiting sensory hair cell death in the auditory receptor following NIHL. Regulation of the expression of antioxidant enzymes and apoptosis-related proteins in cochlear structures is involved in such otoprotective mechanism.

Multiple lines of evidence indicate that CD8$^+$ T cells are important in the control of HIV-1 (HIV) replication. However, CD8$^+$ T cells induced by natural infection cannot eliminate the virus or reduce viral loads to acceptably low levels in most infected individuals. Understanding the basic quantitative features of CD8$^+$ T-cell responses induced during the course of HIV infection may therefore inform us about the limits that HIV vaccines, which aim to induce protective CD8$^+$ T-cell responses, must exceed. Using previously published experimental data from a cohort of HIV-infected individuals with sampling times from acute to chronic infection we defined the quantitative properties of CD8$^+$ T-cell responses to the whole HIV proteome. In contrast with a commonly held view, we found that the relative number of HIV-specific CD8$^+$ T-cell responses (response breadth) changed little over the course of infection (first 400 days post-infection), with moderate but statistically significant changes occurring only during the first 35 symptomatic days. This challenges the idea that a change in the T-cell response breadth over time is responsible for the slow speed of viral escape from CD8$^+$ T cells in the chronic infection. The breadth of HIV-specific CD8$^+$ T-cell responses was not correlated with the average viral load for our small cohort of patients. Metrics of relative immunodominance of HIV-specific CD8$^+$ T-cell responses such as Shannon entropy or the Evenness index were also not significantly correlated with the average viral load. Our mathematical-model-driven analysis suggested extremely slow expansion kinetics for the majority of HIV-specific CD8$^+$ T-cell responses and the presence of intra- and interclonal competition between multiple CD8$^+$ T-cell responses; such competition may limit the magnitude of CD8$^+$ T-cell responses, specific to different epitopes, and the overall number of T-cell responses induced by vaccination. Further understanding of mechanisms underlying interactions between the virus and virus-specific CD8$^+$ T-cell response will be instrumental in determining which T-cell-based vaccines will induce T-cell responses providing durable protection against HIV infection.

Cell secretome has been explored as a cell-free technique with high scientific and medical interest for Regenerative Medicine. In this work, the secretome produced and collected from Olfactory Mucosa Mesenchymal Stem Cells and Olfactory Ensheating Cells was analyzed and therapeutically applied to promote peripheral nerve regeneration. The analysis of the conditioned medium revealed the production and secretion of several factors with immunomodulatory functions, capable of intervening beneficially in the phases of nerve regeneration. Subsequently, the conditioned medium was applied to sciatic nerves of rats after neurotmesis, using Reaxon^® as tube-guides. Over 20 weeks, the animals were subjected to periodic functional assessments, and after this period, the sciatic nerves and cranial tibial muscles were evaluated stereologically and histomorphometrically, respectively. The results obtained allowed to confirm the beneficial effects resulting from the application of this therapeutic combination. The administration of conditioned medium from Olfactory Mucosal Mesenchymal Stem Cells led to the best results in motor performance, sensory recovery, and gait patterns. Stereological and histomorphometric evaluation also revealed the ability of this therapeutic combination to promote nervous and muscular histologic reorganization during the regenerative process. The therapeutic combination discussed in this work shows promising results and should be further explored to clarify irregularities found in the outcomes and to allow establishing the use of cell secretome as a new therapeutic field applied in the treatment of peripheral nerves after injury.

The main sources of intrinsic noise in excitable cells at the microcircuit and network levels are the stochastic characteristics of ion channel gating and activation of the synaptic conductance. Studies using in vivo, in vitro, and in silico methods to examine the effects of synaptic background activity were not adequately investigated in non-neuronal excitatory cells, where neurotransmitter-based innervation also occurs. We created a mathematical model to replicate the background synaptic noise dynamics in a non-neuronal cell. We utilized the stochastic Ornstein-Uhlenbeck process to represent excitatory synaptic conductance, which was incorporated into a whole-cell model to produce spontaneous and evoked cellular electrical activities. The single-cell model includes many biophysically detailed ion channels represented by a set of ordinary differential equations in Hodgkin-Huxley and Markov formalisms. This paradigm effectively induced irregular spontaneous depolarizations (SDs) and spontaneous action potential (sAP) resembling in vitro-like electrical activity in the cells. The input resistance decreased by multiple factors, and the spontaneous action potential firing rate elevated. The potential to reach the action potential threshold is altered. Background synaptic activity can alter the input/output characteristics of non-neuronal excitatory cells. Suppressing these baseline activities would facilitate the discovery of new pharmaceutical targets for different clinical diseases.

Human keratinocytes and melanoma can stick together to form clusters after eight days in co-culture. As in dynamic system concepts, one can consider cluster formation as the system attractor, cell seeding as the initial condition, and density change over time as a path within the basin of attraction. Herein, Cellular Automata, which is a class of Agent-Based Models, and Boolean Networks are discrete modeling methods used in population dynamics such that running the dynamics of cellular automata backward reveals an underlying network in terms of basin of attraction, also known as global dynamics. Thus, we hypothesize that one can estimate the local dynamic of an agent-based model from the basin of attraction of a boolean network. Here, we propose an approach to estimating these agent-based model rules, which consists of comparing the density states within each state transition and reaching a consensus among state transitions belonging to a basin of attraction of a boolean network. The objectives of this study are: (1) to infer a boolean network from the co-culture growth curve; (2) to estimate the rules of agent-based models; and (3) to implement spatial dynamics simulations. The binarization of the growth curve shows high population density after four days; the estimated agent-based model rules were compatible with cell proliferation and migration in agreement with literature, so we had to propose individual rules for survival and death. Spatial dynamics shows that (1) keratinocytes exhibit a higher density in neighborhoods where melanoma is present; (2) the chance of keratinocyte migration increases until the fourth day, then decreases, and the probability of survival increases substantially after four days; and (3) cells with low proliferation capacity die and free space for those with high ones. Our approach suggests that the attractor state of cell co-culture is induced mainly by an increase in keratinocyte migration and survival. Our approach has the potential to offer valuable clues about microenvironmental interactions or configurations that drive population dynamics.

Extracellular vesicles are considered a novel therapeutic tool, due to their ability to transfer their cargoes to target cells. Different strategies to directly load extracellular vesicles with RNA species have been proposed. Electroporation has been used for the loading of non-active vesicles, however the engineering of vesicles already carrying a therapeutically active cargo is still under investigation. We here set up a coincubation method to increase the anti-tumor effect of extracellular vesicles isolated from human liver stem cells (HLSC-EVs). Using the coincubation protocol, vesicles were loaded with the anti-tumor miRNA-145, and their effect was evaluated on renal cancer stem cell invasion. Loaded HLSC-EVs maintained their integrity and miR transfer ability, and miR-145 was protected by RNAse digestion possibly due to its binding to RNA-binding proteins on HLSC-EV surface, such as Annexin A2. Moreover, miR-145 coincubated HLSC-EVs were more effective in inhibiting the invasive properties of cancer stem cells, in comparison to naïve vesicles. The protocol reported here exploits a well-described property of extracellular vesicles to bind nucleic acids on their surface and protect them from degradation, in order to obtain an effective miRNA loading that results in the increase of the effect of naïve active extracellular vesicles.

The present study aimed to analyze novel mechanisms underlying Nrf2-mediated anti-apoptosis in periodontal ligament stem cells (PDLSCs) in the periodontitis oxidative microenvironment. We created an oxidative stress model with H2O2-treated PDLSCs. Herein, we used real-time PCR, western blotting, TUNEL staining, fluorogenic assay and transfer genetics to confirm the degree of oxidative stress and apoptosis as well as the Nrf2 function. Surprisingly, we demonstrated that with up-regulated ROS and MDA levels, the effect of oxidative stress was obvious under H2O2 treatment. Anti-oxidative molecules were changed after the H2O2 exposure, whereby the anti-oxidative signaling of Nrf2 was activated with the increase of its downstream effectors, HO-1, NQO1 and γ-GCS. Additionally, the apoptosis levels gradually increased with oxidative stress and changes in the caspase-9, caspase-3, Bax and c-Fos levels, but not with caspase-8 and down-regulated Bcl-2. The enhanced antioxidant effect could not resist the occurrence of apoptosis. Furthermore, Nrf2 overexpression effectively improved the anti-oxidative levels and increased cell proliferation. At the same time, overexpression effectively restrained TUNEL staining and decreased the molecular levels of caspase-9, caspase-3, et al, but not that of caspase-8. By contrast, silencing the expression Nrf2 levels had the opposite effect. Collectively, Nrf2 alleviates PDLSCs via its effects on anti-oxidative and anti-intrinsic apoptosis by the activation of anti-oxidative enzymes.

BACKGROUND&AIMS: Hepatitis B virus (HBV) infection remains a major public health problem. The interaction between HBV and the host inflammatory response is an important factor contributing to liver damage and disease development. We compared the correlation between the subclinical index and PBMCs concentration in two groups of pregnant women (HBsAg positive), which are different in HBV DNA concentration in Vietnam. METHODS: The Hierarchical cluster analysis (HCA) was run with 20 different clustering methods on data collected from 80 Vietnamese pregnant women and their babies (60/80 cord blood). RESULTS: In the high viral load group (HBV DNA ≥ 5x10^7 copies/ml), a strong correlation between CBMCs with serum maternal Haemoglobin concentration and maternal platelet and maternal ALT. Their R values are: -0.88, 0.82, and 0.84 with p=8.97E-03, 2.41E-02 and 1.75E-01, respectively. CONCLUSIONS: We found a significant correlation shift of subclinical index between the two groups, which may be important in diagnosing pregnant women with chronic hepatitis B virus infection.

Degradation is an inevitable companion in the operation of solid oxide fuel cell (SOFC) systems since it directly deteriorates the reliability of the system’s operation and the system’s durability. Both are seen as barriers that limit the extensive commercial use of SOFC systems. Therefore diagnosis and prognosis are valuable tools that can contribute to raising the reliability of the system operation, efficient health management, increased durability and implementation of predictive maintenance techniques. Remaining useful life (RUL) prediction has been extensively studied in many areas like batteries and proton-exchange membrane fuel cell (PEM) systems, and a range of different approaches has been proposed. On the other hand, results available in the domain of SOFC systems are still relatively limited. Moreover, methods relying on detailed process models and models of degradation turned out to have limited applicability for in-field applications. Therefore, in this paper, we propose an effective, data-driven approach to predicting RUL where the trend of the health index is modelled by an adaptive linear model, which is updated at all times during the system operation. This allows for a closed-form solution of the probability distribution of the RUL, which is the main novelty of this paper. Such a solution requires no computational load and is as such very convenient for the application in ordinary low-cost control systems. The performance of the approach is demonstrated first on the simulated case studies and then on the data obtained from a long-term experiment on a laboratory SOFC system. From the tests conducted so far it turns out that, the quality of the RUL prediction is usually rather low at the beginning of the system operation, but then gradually improves while the system is approaching the end-of-life (EOL), making it a viable tool for prognosis.

Background: The treatment of peripheral arterial disease (PAD) is focused on improving perfusion and oxygenation in the affected limb. Standard revascularization methods include bypass surgery, endovascular interventional procedures, or hybrid revascularization. Cell-based therapy can be an alternative strategy for patients with no-option critical limb ischemia who are not eligible for endovascular or surgical procedures. Aims: This review offers an up-to-date critical overview of the knowledge and data of evidence-based medicine on the position of cell therapy in the treatment of PAD. It provides an overview of current evidence and appraises the future perspectives of cell-based therapy, emphasizing the potential of exosomal cell-free approaches in patients with critical limb ischemia. Results: Meta-analyses focused on cell therapy problems in PAD treatment confirm a significantly greater chance of limb salvage in the first year after the cell therapy administration. The opportunity of defect healing is at least two times higher when compared with the standard conservative therapy. Secondary endpoints of the available meta-analyses are also included in this review. Improvement of perfusion and oxygenation parameters in the affected limb, pain regression, and claudication interval prolongation are discussed. Conclusions: The available evidence-based medicine data show that this technique is safe, associated with minimum complications or adverse events, and effective.

Ulcerative colitis is a chronic immune-mediated disease of unclear etiology, affecting people of different ages and significantly reducing the quality of life. Modern methods of therapy are mainly represented by anti-inflammatory drugs and are not aimed at a specific pathogenetic factor. In this study, we investigated the effect of transplantation of sterile stool filtrate from healthy donors on the induction of anti-inflammatory immune mechanisms. It was shown that performing such a procedure in patients with ulcerative colitis caused the appearance of T helper cells in the blood, which reacted to the content of sterile stool filtrates in an antigen-specific manner and produced IL10. At the same time, cells of the same patients before therapy in response to the addition of sterile stool filtrates were less reactive and predominantly produced IL4, indicating its pro-inflammatory skewing. The obtained data demonstrated the effect of an anti-inflammatory shift in the T-helper response after transplantation of sterile stool filtrate, which increased and persisted for at least three months after the procedure.

Alzheimer’s disease (AD), the most common cause of dementia, is characterized by disruptions in memory, cognition, and personality, significantly impacting morbidity and mortality rates among older adults. However, the exact pathophysiological mechanism of AD remains unknown, and effective treatment options for AD are still lacking. Human induced pluripotent stem cells (iPSC) are emerging as promising platforms for disease research, offering the ability to model genetic mutations associated with various conditions. Patient-derived iPSCs are useful for modeling neurodegenerative and neurodevelopmental disorders. In this study, we generated AD iPSCs from peripheral blood mononuclear cells obtained from a 65-year-old patient with AD carrying the E682K mutation in the amyloid precursor protein (APP) gene. Cerebral organoids derived from AD iPSCs recapitulated the AD phenotype, exhibiting significantly increased levels of tau protein. Our analysis revealed that an iPSC disease model of AD is a valuable assessment tool for pathophysiological research and drug screening.

The objective of this study was to determine the effects of supplementing L-isoleucine (L-Ile) on milk protein synthesis, using an immortalized bovine mammary epithelial (MAC-T) cell line. In this case, the cells were treated with 0, 0.3, 0.6, 0.9, 1.2 and 1.5 mM of supplemental Isoleucine (Ile), and the most efficient time for protein synthesis for each amino acid was determined by measuring the cell, medium and total protein at 0, 24, 48, 72 and 96 h. Confirmatory tests showed that 48h incubation time and 0.6 mM dosage of L-Ile are considered as the optimal time and dosage. The mechanism of milk protein synthesis was elucidated through proteomics analysis to clarify the metabolic pathway. When the L-Ile was supplemented, extracellular protein (medium protein) reached a peak at 48h, whereas in the case of the intracellular cell protein, it was shown to have reached to its peak at 24h in all L-Ile dosage treatments. In total, it is noted that there were 63 upregulated and 52 downregulated proteins. The results of the protein pathway analysis showed that the L-Ile group stimulated insulin/IGF pathway-mitogen activated protein kinase kinase/MAP kinase cascade, insulin/IGF pathway-protein kinase B signaling cascade, p53 pathway, de novo purine biosynthesis, Wnt signaling pathway, glycolysis, pentose phosphate pathway, and ATP synthesis which are pathways involved and related to protein and energy metabolism. Together, these results demonstrate that L-Ile supplementation was effective in stimulating β-casein synthesis by stimulating genes and pathways which are significantly related to protein and energy metabolism.

After peripheral nerve injury, axon and myelin regeneration are key events for optimal clinical improvements. We have previously shown that early bone marrow mononuclear cell (BMMC) transplantation exerts beneficial effects on myelin regeneration. In the present study we analyze whether there is a temporal window in which BMMC migrate more efficiently to damaged nerves while still retaining their positive effects. Adult Wistar rats of both sexes, with sciatic nerve crush were systemically transplanted with BMMC at different days post injury. Vehicle-treated, naïve, and sham rats were also included. Morphological, functional, and behavioral analyses were performed in nerves from each experimental group at different survival times. BMMC transplantation between 0 and 7 days after injury resulted in the largest number of nested cells within the injured sciatic nerve, which supports the therapeutic value of BMMC administration within the first week after injury. Most importantly, later BMMC administration 7 days after sciatic nerve crush was associated with neuropathic pain reversion, improved morphological appearance of the damaged nerves, and a tendency toward faster recovery in the sciatic functional index and electrophysiological parameters. Our results thus support the notion that even delayed BMMC treatment may represent a promising therapeutic strategy for peripheral nerve injuries.

Flow cytometric detection of intracellular (IC) signaling proteins and transcription factors (TFs) will help elucidate the regulation of B cell survival, proliferation and differentiation. However, simultaneous detection of signaling proteins or TFs, with membrane markers (MM) can be challenging as required fixation and permeabilization procedures can affect functionality of conjugated antibodies. Here, a phosphoflow method is presented for detection of activated NF-κB p65 and phosphorylated STAT1, STAT3, STAT5 and STAT6 together with B cell differentiation MM CD19, CD27 and CD38. Additionally, a TF-flow method is presented that allows detection of B cell TFs; PAX5, c-MYC, BCL6, AID and antibody-secreting cell (ASC) TFs BLIMP1 and XBP-1s together with MM. Applying these methods on in vitro induced human B cell differentiation cultures showed significantly different steady-state levels, and responses to stimulation, of phosphorylated signaling proteins in CD27-expressing B cell and ASC populations. The TF-flow protocol and UMAP analysis revealed heterogeneity in TF-expression within stimulated CD27 or CD38-expressing B cell subsets. The methods presented here allow for sensitive analysis of STAT and NF-κB p65 signaling and TFs together with B cell differentiation MM at single-cell resolution. This will aid further investigation of B cell responses in both health and disease.

Radiation therapy (RT) is one of the main approaches of cancer therapy, including non-small cell lung cancer (NSCLC). Radiation-induced DNA double-stranded breaks (DSBs), single-stranded DNA breaks (SSBs) and oxidized nucleobases cause replication fork stalling, chromosomal instability or point mutagenesis and can lead to cell death. Translesion DNA synthesis (TLS) is one of the DNA damage tolerance (DDT) pathways promoting replication of damaged DNA. Here, we explored the effects of the knockout of primase-polymerase PRIMPOL (PRIMPOL −/−) and TLS polymerase POLI (POLI −/−) genes in two different monoclonal lines derived from human NSCLC A549 cells. Our study aimed to investigate the impact of their absence on the susceptibility of these cells to ionizing radiation (IR)-induced stress. Without IR, cells that do not have PRIMPOL or POLI experience a considerable rise in apoptosis by day 7 of continuous cultivation and a noteworthy decline in cell migration under spatial confinement when compared to the parental wild-type cells. Despite this, we have found that PRIMPOL −/− exhibited higher clonogenic survival compared to wild-type cell lines in response to IR stress. Both of these cell lines demonstrated remarkable resilience to radiation stress, surpassing the survival rate of the original A549 cells. POLI deficiency reduces cellular γH2AX foci accumulation, while the presence of highest number of γH2AX foci in PRIMPOL−/− cells was ATM-independent. We report for the first time that increased post-irradiation clonogenic survival of PRIMPOL −/− A549 cells might be associated with the increase of CD133+ but not CD44+ populations of cancer stem-like cells.

CRCMSS/pMMR contain a significantly increased fraction of TREM2+ macrophages (TAMs) and CD66+ neutrophils (TANs) together with decrease of CD4-CD8-CD3+ double negative T lymphocytes (DNTs); no differences were revealed by the analysis of myeloid and plasmacytoid dendritic cell populations. A fraction of tumor-infiltrating T-cells display an exhausted phenotype, co-expressing PD-1 and TIM-3. Remarkably, expression of PD-L1 on fresh tumor cells and TAMs was undetectable even after in vitro stimulation with interferon-γ. These findings confirm the immune suppressive microenvironment of CRCMSS/pMMR characterized by dense infiltration of TAMs, occurrence of TANs, lack of DNTs, T-cell exhaustion and interferon-γ unresponsiveness by host and tumor cells. Appropriate bypass strategies should consider these combinations of immune escape mechanisms in CRCMSS/pMMR.

Background: The monocyte-phagocyte system (MPS), including monocytes/macrophages and dendritic cells (DCs), plays a key role in anti-viral immunity. We aimed to analyze the prognostic value of the MPS components on in-hospital mortality in a cohort of 58 patients (M/F ; mean ageSD years) with COVID-19 pneumonia and 22 age- and sex-matched healthy controls. Methods: We measured frequencies and absolute numbers of peripheral blood CD169+ monocytes, conventional CD1c+ and CD141+ (namely cDC2 and cDC1), and plasmacytoid CD303+ DCs by means of multi-parametric flow cytometry. A gene profile analysis of 770 immune-inflammatory related human genes and 20 SARS-CoV-2 genes was also performed. Results: Median frequencies and absolute counts of CD169-expressing monocytes were significantly higher in COVID-19 patients than in controls (p 0.04 and p 0.01, respectively). Conversely, percentages and absolute numbers of all DC subsets were markedly depleted in patients (p

Pertussis is a vaccine-preventable disease caused by the bacterium Bordetella pertussis. Over the past years, the incidence and mortality of pertussis increased significantly. A possible cause is the switch from whole cell to acellular pertussis vaccines, although other factors may also contribute. To develop future vaccines and improve current vaccination strategies, it is critical to understand factors influencing the generation of immunological memory. We applied high-dimensional flow cytometry to investigate changes in B cells in individuals of different ages and distinct priming backgrounds upon administration of an acellular pertussis booster vaccine. These findings were correlated to vaccine-specific plasma cells and serum Ig levels. Expansion and maturation of plasma cells 7 days post-vaccination was the most prominent cellular change in all age groups, and was most pronounced for more mature IgG1+ plasma cells. Cellular responses were stronger in individuals primed with whole cell vaccine than in individuals primed with acellular vaccine. Moreover, IgG1+ plasma cell expansion weakly correlated with Prn- and PT- specific serum IgG levels. Our study points at plasma cells as a potential early cellular marker of an immune response and contributes to understanding differences in immune responses between age groups and priming backgrounds.

Beta cells couple stimulation by glucose with insulin secretion and impairments in this coupling play a central role in diabetes mellitus. To clarify the effect of cAMP and the role of Epac2A in intracellular calcium signals and intercellular coupling, we performed functional multicellular calcium imaging in beta cells in mouse pancreas tissue slices after stimulation with glucose and forskolin in wild-type and Epac2A knock-out mice. Increased cAMP evoked calcium signals in otherwise sub-stimulatory glucose and beta cells from Epac2A knock-out mice displayed a faster activation. During the plateau phase, beta cells from Epac2A knock-out mice displayed a slightly higher active time in response to glucose compared with wild-type littermates, and increased cAMP increased the active time via a large increase in oscillation frequency and small decrease in oscillation duration in both Epac2A knock-out and wild-type mice. Functional network properties during stimulation with glucose did not differ in Epac2A knock-out mice, but the presence of Epac2A was crucial for the protective effect of increased cAMP in preventing a decline in beta cell functional connectivity with time. Finally, increased cAMP prolonged beta cell activity during deactivation in an Epac2A-independent manner.

Background: Prostate cancer (PCa) is characterized by significant heterogeneity in its molecular, genomic, and immunologic characteristics. Methods: Whole transcriptome RNAseq data from The Cancer Genome Atlas of prostate adenocarcinomas (n=496) was utilized. The immune microenvironment was characterized using the CIBERSORTX tool to identify immune cell type composition. Unsupervised hierarchical clustering was performed based on immune cell type content. Analyses of progression-free survival (PFS), distant metastases, and overall survival (OS) were performed using Kaplan-Meier estimates and Cox-regression multivariable analyses. Results: Four immune clusters were identified, largely defined by plasma cell, CD4+ Memory Resting T Cells (CD4 MR), M0 and M2 macrophage content (CD4 MRHighPlasma CellHighM0LowM2Low, CD4 MRLowPlasma CellHighM0LowM2Low, CD4 MRHighPlasma CellLowM0HighM2Low, and CD4 MRHighPlasma CellLowM0LowM2High). The two macrophage-enriched/plasma cell non-enriched clusters (3&4) demonstrated worse PFS (HR 2.24, 95% CI 1.46–3.45, p=0.0002) than the clusters 1&2. No metastatic events occurred in the non-macrophage-enriched clusters. Comparing clusters 3 vs 4, in patients treated by surgery alone, cluster 3 had zero progression events (p<0.0001). However, cluster 3 patients had worse outcomes after post-operative radiotherapy (p=0.018). Conclusion: Distinct tumor immune clusters with a macrophage-enriched phenotype and reduced plasma cell enrichment independently characterize an aggressive phenotype in localized prostate cancer that may differentially respond to treatment.

Over the last decades, cellular immunotherapy has revealed its curative potential. However, the inherent physiological characteristics of immune cells can limit the potency of this approach. Best defined in T cells, dysfunction associated with terminal differentiation, exhaustion, senescence, and activation-induced cell death undermine adoptive cell therapies. In this review, we concentrate on how the multiple mechanisms that articulate the various forms of immune dysfunction impact cellular therapies primarily involving conventional T cells, but also other lymphoid subtypes, in addition to the various strategies put in place to circumvent these effects. The repercussions of immune cell dysfunction across the full life cycle of cell therapy, from the source material, during manufacturing, and after adoptive transfer are discussed. Applicable to cellular products prepared from native and unmodified immune cells, as well as genetically engineered therapeutics, the understanding and potential modulation of dysfunctional features is key to the development of improved cellular immunotherapies.

The inability to efficiently metabolize homocysteine (Hcy), which occurs in nutritional and genetic deficiencies, leads to hyperhomocysteinemia (HHcy) thereby causing endothelial dysfunction, a hallmark of atherosclerosis which underpins cardiovascular disease (CVD). The dysregulation of mammalian target of rapamycin (mTOR) signaling, and impaired autophagy play important roles in CVD. Biochemically, HHcy is characterized by elevated levels of Hcy and its metabolites, Hcy-thiolactone (HTL) and N-Hcy-protein (N-Hcy). However, whether these metabolites can dysregulate mTOR signaling and autophagy in endothelial cells is not known. Here, we examined the influence of HTL, N-Hcy, and Hcy on the PHF8/H4K20me1/mTOR/autophagy pathway in human umbilical vein endothelial cells (HUVEC). We found that treatments with HTL, N-Hcy, or Hcy significantly reduced PHF8 protein and mRNA expression, increased H4K20me1, and upregulated mTOR signaling. Autophagy was also impaired (significantly downregulated BECN1, ATG5, ATG7, and LC3 protein and mRNA levels). We also found that these changes were mediated by PHF8-targeting microRNA (miR): miR-22-3p and miR-1229-3p. The effects of HTL, N-Hcy, or Hcy on the miR expression and on the PHF8/H4K20me1/mTOR/autophagy pathway were abrogated by treatments with an inhibitor of miR-22 or miR-1229. Taken together, these findings show that Hcy metabolites can upregulate miR-22-3p and miR-1229-3p expression, which then dysregulate the PHF8/H4K20me1/mTOR/autophagy pathway, important for vascular homeostasis.

Locally advanced rectal cancer (LARC) has traditionally been treated with trimodality therapy consisting of neoadjuvant radiation +/- chemotherapy, surgery and adjuvant chemotherapy. There is currently a clinical need for biomarkers to predict treatment response and outcomes, especially during neoadjuvant therapy. Liquid biopsy in the form of circulating tumour cells (CTCs) and circulating nucleic acids in particular microRNAs (miRNA) are novel, highly stable and clinically relevant regulators of disease. We studied a prospective cohort of 52 patients with LARC, and obtained samples at baseline, during treatment, and post-treatment. We enumerated CTCs during chemoradiation at these three time-points, using the IsofluxTM CTC Isolation and detection platform. We then subjected the isolated CTCs to miRNA expression analyses, using a panel of 106 miRNA candidates. We identified CTCs in 73% of patients at baseline, and numbers fell during treatment and miRNA expression profiles also changed during treatment. Between baseline and during treatment (week 3) time-points three microRNAs (hsa-miR-95, hsa-miR-10a and hsa-miR-16-1*) were highly differentially expressed. Importantly, hsa-miR-19b-3p and hsa-miR-483-5p were found to correlate with good response to treatment. The latter (hsa-miR-483-5p) was also found to be differentially expressed between good responders and poor responders. They represent potential predictive biomarkers and a potential miRNA-based treatment strategy. In this study, we demonstrate that CTCs are present and can be isolated in the non-metastatic early stage cancer setting, and their associated miRNA profiles can potentially be utilized to predict treatment response.

We describe a method for protein quantification and for mRNA quantification in small sample quantities of human induced pluripotent stem-cell-derived-cardiomyocytes (hiPSC-CMs). Demonstrated here is how the capillary-based protein detection system Wes^TM by ProteinSimple and the Power SYBR^TM Green Cells-to-CT^TM Kit by Invitrogen can be applied to individual samples in a 96-well microplate format and thus made compatible with high-throughput (HT) cardiomyocyte assays. As an example of the usage, we illustrate that Cx43 protein and GJA1 mRNA levels in hiPSC-CMs are enhanced when the optogenetic actuator, channelrodopsin-2 (ChR2), is genetically expressed in them. Instructions are presented for cell culture and lysate preparations from hiPSC-CMs, along with optimized parameter settings and experimental protocol steps. Strategies to optimize primary antibody concentrations as well as ways for signal normalization are discussed, i.e. antibody multiplexing and total protein assay. The sensitivity of both the Wes and Cells-to-CT kit enables protein and mRNA quantification in a HT format, which is important when dealing with precious small samples. In addition to being able to handle small cardiomyocyte samples, these streamlined and semi-automated processes enable quick mechanistic analysis.

Recent evidence indicates that the risk of being diagnosed with cancer in a tissue is strongly correlated (0.80) with the number of stem cell divisions accumulated by the tissue. Since cell division can generate random mutations during DNA replication, this correlation has been used to propose that cancer is largely caused by the accumulation of unavoidable mutations in driver genes. However, no correlation between the number of gene mutations and cancer risk across tissues has been reported. Because many somatic mutations in cancers originate prior to tumor initiation and the number of cell divisions occurring during tumor growth is similar among tissues, here I use whole genome sequencing information from 22,086 cancer samples and incidence data from the largest cancer registry in each continent to study the relationship between the number of gene mutations and the risk of cancer across 33 tissue types. Results show a weak positive correlation (mean = 0.14) between these two parameters in each of the five cancer registries. The correlation became stronger (mean = 0.50) when gender-related cancers were excluded. Results also show that 1,003 samples from 29 cancer types have zero mutations in genes. These data suggest that cancer etiology can be better explained by the accumulation of stem cell divisions than by the accumulation of gene mutations. Possible mechanisms by which the accumulation of cell divisions in stem cells increases the risk of cancer are discussed.

To evaluate whether autologous mesenchymal cells, adipose derived stem cells and platelet-rich plasma, grafted into the supracoroideal space by surgical treatment according to Limoli retinal restoration technique (LRRT), can produce growth factors in order to exert a beneficial effect in retinitis pigmentosa (RP) patients. Twenty-one eyes underwent surgery and divided based on retinal foveal thickness ≤ 190 or >190 µm into group A and group B, respectively. The specific LRRT triad was grafted in a deep scleral pocket above the choroid of each eye. At 6-month follow-up, group B showed an improvement in residual close-up visus and sensitivity at microperimetry compared to group A. After an in-depth review of molecular biology studies concerning degenerative phenomena underlying the etiopathogenesis of RP, it can be confirmed that further research is needed on tapeto-retinal degenerations both from a clinical and molecular point of view to obtain better functional results. In particular, it is necessary to increase the number of patients, extend observation times, and treat subjects in the presence of still trophic retinal tissue to allow adequate biochemical and functional catering.

The ongoing SARS-CoV-2 pandemic has raised concerns surrounding immunological protection against the virus, particularly for people with inborn errors of immunity (IEI). While COVID-19 vaccination induces robust antibody, memory B-cell, and T-cell responses in healthy individuals, how well vaccination protects people with IEI against infection and severe disease remains unclear – especially in the context of new viral variants and vaccine formulations – leading to anxiety, ongoing self-isolation, and repeated vaccination with limited evidence of increased efficacy. Whilst most people with IEI generate some level of cellular and/or humoral immunity to COVID-19 vaccination, the level of protection and durability of the response are unclear. Alongside vaccination, antibody-based therapeutics are aimed at limiting infection and severe disease in this cohort. Immunoglobulin replacement therapy (IgRT) provides passive immunity in antibody-deficient individuals. However, to successfully prevent SARS-CoV-2 infection, a sufficient number of neutralizing antibodies must be present in product. While antibodies against circulating variants are eventually present in IgRT products (both from natural infection and vaccination of the donors), evidence of their capacity to recognize new variants is limited. Furthermore, while antibody-based pre- and post-exposure prophylaxis can be effective, they are susceptible to decreased efficacy in the context of variant evolution. This review provides an in-depth overview of current knowledge about COVID-19 vaccine efficacy in IEIs, the efficacy of SARS-CoV-2-specific antibody products, and knowledge and technological advances required for continued protection of people with IEI.

In patients with presumed pancreatic ductal adenocarcinoma (PDAC), biomarkers that may open for personalised, risk-adapted treatment are lacking. The study analysed the impact of circulating tumour cells (CTCs) on the patterns of recurrence and survival in 98 patients resected for PDAC with 5-10 years of follow-up. Preoperative samples were analysed by the CellSearch® system for EpCAM+/DAPI+/CK+/CD45- CTCs. CTCs were detected in 7 of the 98 patients. CTCs predicted a significantly shorter median DFS of 3.3 vs. 9.2 months and a median CSS of 6.3 vs. 18.5 months. Relapse status was confirmed by imaging for 87 patients. Of these, 58 developed distant metastases (DM) and 29 cases isolated local recurrence (ILR) as first event. All patients with CTCs experienced DM. pN-status and histological grade &gt;2 were other independent risk factors for DM, but only CTCs predicted significantly shorter cancer-specific, disease-free and post-recurrence survival. We conclude that CTC presence in resected PDAC patients predicted early distant metastasis and impaired survival. The impact of CTCs was comparable to that of histopathological risk factors and exceeded the effect size of other preoperative parameters. Thus, preoperative CTCs alone or in combination with histopathological factors may guide initial treatment decisions in patients with resectable PDAC in the future.

For the replacement of lost contractile elements in the treatment of coronary heart disease, the most promising approaches to obtain cardiomyocytes by cardiac differentiation of pluripotent cells. The aim of this work is to develop a technology for the formation of a functional layer of cardiomyocytes differentiated from iPSCs, capable of generating rhythmic activity and synchronous contractions. To accelerate cardiomyocyte maturation, the renal subcapsular transplantation model was used in SCID mice. After explantation, the formation of cardiomyocyte contractile apparatus was assessed by fluorescence and electron microscopy, and calcium ion oscillation in the cytoplasm was assessed by visualization of the fluorescent calcium binding dye Fluo-8. It was shown that human iPSC-derived cardiomyocyte cell layers transplanted (for up to 6 weeks) under SCID mouse kidney fibrous capsules begin to form an ordered contractile apparatus and retain functional activity and the ability to oscillate calcium ion fluxes after explantation from the body.

Abstract: Sickle cell disease (SCD) is an autosomal recessive monogenic disorder caused by a homozygous mutation in the β-globin gene, which leads to erythrocyte sickling, hemolysis, vaso-occlusion and sterile inflammation. Oral l-glutamine administration has been shown to reduce the frequency of pain crisis in SCD patients, however, the long-term effect of l-glutamine in SCD remains to be determined. To understand the long-term effect of l-glutamine administration in SCD liver we used quantitative liver intravital microscopy and biochemical analysis in humanized SCD mice. We here show that chronic l-glutamine administration reduces hepatic hemoglobin-heme-iron level, but fails to ameliorate ischemic liver injury. Remarkably, we find that this failure in resolution of hepatobiliary injury and persistent liver fibrosis is associated with reduced expression of hepatic Kupffer cells post l-glutamine treatment. These findings establish the importance to investigate the long-term effects of l-glutamine therapy on liver pathophysiology in SCD patients.

Cachexia is a syndrome characterized by an ongoing loss of skeletal muscle mass associated with poor patient prognosis in non-small cell lung cancer (NSCLC). However, prognostic cachexia biomarkers in NSCLC are unknown. Here, we analyzed computed tomography (CT) images and tumor transcriptome data to identify potentially secreted cachexia biomarkers (PSCB) in NSCLC patients with low-muscularity. We integrated radiomics features (pectoralis muscle, sternum, and T10 vertebra) from CT of 89 NSCLC patients, which allowed us to identify an index for screening muscularity. Next, a tumor transcriptomic-based secretome analysis from these patients (discovery set) was evaluated to identify potential cachexia biomarkers in patients with low-muscularity. The prognostic value of these biomarkers for predicting recurrence and survival outcome was confirmed using expression data from eight lung cancer datasets (validation set). Finally, C2C12 myoblasts differentiated into myotubes were used to evaluate the ability of the selected biomarker, IL-8, in inducing muscle cell atrophy. We identified 75 over-expressed transcripts in patients with low-muscularity, which included IL6, CSF3, and IL8. Also, we identified NCAM1, CNTN1, SCG2, CADM1, IL8, NPTX1, and APOD as PSCB in the tumor secretome. These PSCB were capable of distinguishing worse and better prognosis (recurrence and survival) in NSCLC patients. IL8 was confirmed as a predictor of worse prognosis in all validation sets. In vitro assays revealed that IL-8 promoted C2C12 myotube atrophy. Tumors from low-muscularity patients presented a set of upregulated genes encoding for secreted proteins, including pro-inflammatory cytokines that predict worse overall survival in NSCLC. Among these up-regulated genes, IL8 expression in NSCLC tissues was associated with worse prognosis and the recombinant IL-8 was capable of triggering atrophy in C2C12 myotubes.

Geranylgeranyl acetone (GGA) protects against various types of cell damages by upregulating heat shock proteins. We investigated whether GGA protect neuronal cells from cell death induced by oxidative stress. Glutamate exposure was lethal to HT-22 cells which comprise a neuronal line derived from mouse hippocampus. This configuration is often used as a model for hippocampus neurodegeneration in vitro. In the present study, GGA protected HT-22 cells from glutamate-induced oxidative stress. GGA pretreatment did not induce Hsps. Moreover, reactive oxygen species increased to the same extent in both GGA-pretreated and untreated cells exposed to glutamate. In contrast, glutamate exposure and GGA pretreatment increased mitochondrial membrane potential. However, increases in intracellular Ca2+ concentration were inhibited by GGA pretreatment. In addition, the increase of phosphorylated ERKs by the glutamate exposure was inhibited by GGA pretreatment. These findings suggest that GGA protects HT-22 cells from glutamate-provoked cell death without Hsp induction and that the mitochondrial calcium buffering capacity plays an important role in this protective effect.

The past few years have brought substantial progress toward understanding how human cytomegalovirus (HCMV) enters the remarkably wide spectrum of cell types and tissues that the virus is observed to infect. Neuropilin-2 and platelet-derived growth factor receptor alpha (PDGFRα) were identified as receptors, respectively, for the trimeric and pentameric glycoprotein H / glycoprotein L (gH/gL) complexes that in large part govern HCMV cell tropism, while CD90 and CD147 were also found to play roles during entry. X-ray crystal structures for the proximal viral fusogen, glycoprotein B (gB), and for the pentameric gH/gL complex (pentamer) were solved. A novel virion gH complex consisting of gH bound to UL116 instead of gL was described, and findings supporting the existence of a stable complex between gH/gL and gB were reported. Additional work indicates that the pentamer promotes a mode of cell-associated spread that resists antibody neutralization, as opposed to the trimeric gH/gL complex (trimer), which appears to be broadly required for the infectivity of cell-free virions. Finally, viral factors such as UL148 and US16 were identified that can influence the incorporation of the alternative gH/gL complexes into virions. We will review these advances and their implications for understanding HCMV entry and cell tropism.

Abstract: Non-alcoholic fatty liver disease (NAFLD) begins with lipid accumulation within hepatocytes, but the relative contributions of different macronutrients is still unclear. We inves-tigated the impact of fatty acids, glucose and fructose on lipid accumulation in primary human hepatocytes (PHH) and three different hepatoma cell lines: HepG2, Huh7 and McA-RH7777 (McA). Cells were treated for 48 hours with fatty acids (0 or 200μM), glucose (5mM or 11mM) and fructose (0mM, 2mM or 8mM). Lipid accumulation was measured via Nile Red staining. All cell types accumulated lipid in response to fatty acids (P<0.001). PHH and McA, but not HepG2 or Huh7 cells, accumulated more lipid with 11mM glucose plus fatty acids (P=0.004, fatty acid x glucose interaction, for both), but only PHH increased lipid accumulation in response to fructose (P<0.001). Considerable variation was observed between PHH cells from different individuals. Lipid accumulation in PHH was increased by insulin (P=0.003) with inter-individual variability. Similarly, insulin increased lipid accumulation in both HepG2 and McA cells, with a bigger response in McA in the presence of fatty acids (P<0.001 for fatty acid x insulin). McA were more insulin sensitive than either HepG2 or Huh7 cells in terms of AKT phosphorylation (P<0.001 insulin x cell type interaction). Hence glucose and fructose can contribute to the accumulation of lipid in PHH with considerable inter-individual variation, but hepatoma cell lines are not good models of PHH.

Chimeric Antigen Receptor (CAR)-T cell immunotherapy revolutionized cancer treatment and consists of the genetic modification of T lymphocytes with a CAR gene, aiming to increase their ability to recognize and kill antigen-specific tumor cells. The dynamics of CAR-T cell responses in patients presents a multiphasic kinetics with distribution, expansion, contraction, and persistence phases. The characteristics and duration of each phase depend on the tumor type, the infused product, and on patient-specific characteristics. We present a mathematical model which describes the multiphasic CAR-T cell dynamics resulting from the interplay between CAR-T and tumor cells, considering patient and product heterogeneities. The CAR-T cell population is divided into functional (distributed and effector), memory, and exhausted CAR-T cell phenotypes. The model is able to describe the diversity of CAR-T cell dynamic behaviors in different patients and hematological cancers as well as their therapy outcomes. Our results indicate that the joint assessment of the area under the concentration-time curve in the first 28 days and the corresponding fraction of non-exhausted CAR-T cells may be considered as potential markers to classify therapy responses. Overall, the analysis of different CAR-T cell phenotypes can be a key aspect for a better understanding of the whole CAR-T cell dynamics.

Axonal degeneration resulting from optic nerve damage can lead to the progressive death of retinal ganglion cells (RGCs), culminating in irreversible vision loss. We contrasted two methods for inducing optic nerve damage: optic nerve compression (ONCo) and optic nerve crush (ONCr). These were assessed for their respective merits in simulating traumatic optic neuropathies and neurodegeneration. We also administered neural progenitor cells (NPCs) into the subtenon space to validate their potential in mitigating optic nerve damage. Our findings indicate that both ONCo and ONCr successfully induced optic nerve damage, as shown by increased ischemia and expression of genes linked to neuronal regeneration. Post NPCs injection, recovery in the expression of neuronal regeneration-related genes was more pronounced in the ONCo model than in the ONCr model, while inflammation-related gene expression saw a better recovery in ONCr. In addition, proteomic analysis of R28 cells in hypoxic conditions identified Vps35 and Syntaxin12 genes. Vps35 preserved mitochondrial function in ONCo, while Syntaxin12 appeared to restrain inflammation via the Wnt/β-catenin signaling pathway in ONCr. NPCs managed to restore damaged RGCs by elevating neuroprotection factors and controlling inflammation through mitochondrial homeostasis and Wnt/β-catenin signaling in hypoxia-injured R28 cells and in both animal models. Our results suggest that ischemic injury and crush injury cause optic nerve damage via different mechanisms, which can be effectively simulated using ONCo and ONCr, respectively. Moreover, cell-based therapies such as NPCs may offer promising avenues for treating various optic neuropathies, including ischemic and crush injuries.

Although metastasis is the primary cause of death on patients with malignant solid tumors, efficient antimetastatic therapies are not clinically available thus far. Sulfated glycosaminoglycans from marine sources have shown promising pharmacological effects, acting in different steps of the metastatic process. Oversulfated dermatan sulfate from ascidians is effective in preventing metastasis by inhibition of P-selectin, a platelet surface protein involved in the platelet-tumor cell emboli formation. We report in this work that the heparan sulfate isolated from the viscera of the ascidian Phallusia nigra drastically attenuates metastasis of colon carcinoma cells in mice. Our in vitro and in vivo assessments demonstrate that the P. nigra glycan has very low anticoagulant and antithrombotic activities and a reduced hypotension potential, although efficiently preventing metastasis. Therefore, it may be a promising candidate for the development of a novel anti-metastatic drug.

The amino acid transporters SLC38A5 and SLC7A11 are upregulated in triple-negative breast cancer (TNBC). SLC38A5 transports glutamine, methionine, glycine and serine, and therefore activates mTOR signaling and induces epigenetic modifications. SLC7A11 transports cystine and increases cellular levels of glutathione, which protects against oxidative stress and lipid peroxidation via glutathione peroxidase, a seleno (Se)-enzyme. The primary source of Se is dietary Se-methionine (Se-Met). Since SLC38A5 transports methionine, we examined its role in Se-Met uptake in TNBC cells. We found that SLC38A5 interacts with methionine and Se-Met with comparable affinity. We also examined the influence of Se-Met on Nrf2 in TNBC cells. Se-Met activated Nrf2 and induced expression of Nrf2-target genes, including SLC7A11. Our previous work discovered niclosamide, an antiparasitic drug, as a potent inhibitor of SLC38A5. Here we found SLC7A11 to be inhibited by niclosamide with an IC50 value in the range of 0.1-0.2 M. In addition to the direct inhibition of SLC38A5 and SLC7A11, pretreatment of TNBC cells with niclosamide reduced the expression of both transporters. Niclosamide decreased glutathione levels, inhibited proliferation, suppressed GPX4 expression, increased lipid peroxidation, and induced ferroptosis in TNBC cells. It also significantly reduced the growth of the TNBC cell line MB231 in mouse xenografts.

Studies of mast cell biology is dependent on relevant and validated in vitro models. We here present detailed information concerning the phenotype of both freshly isolated human skin mast cells (MCs), and of in vitro cultures of these cells that was obtained by analyzing their total transcriptome. Transcript levels of MC-related granule proteins and transcription factors were found to be remarkably stable over a 3-week culture period. Relatively modest changes were also seen for important cell surface receptors including the high affinity receptor for IgE, FCER1A, the low affinity receptor for IgG, FCGR2A, and the receptor for stem cell factor, KIT. FCGR2A was the only Fc receptor for IgG expressed by these cells. Comparisons of the present transcriptome against previously reported transcriptomes of mouse peritoneal MCs and mouse bone marrow derived MCs (BMMCs) revealed both similarities and major differences. Strikingly, cathepsin G was the most highly expressed granule protease in human skin MCs, in contrast to the almost total absent of this protease in both mouse MCs. Transcript levels for the majority of cell surface receptors were also very low compared to the granule proteases in both mouse and human MCs, with a difference of almost two orders of magnitude. An almost total absence of T cell granzymes was observed in human skin MCs, indicating that granzymes have no or only a minor role in human MC biology. Ex vivo skin MCs expressed high levels of selective immediate early genes and transcripts of heat-shock proteins. In validation experiments, we determined that this expression was an inherent property of the cells and not the result of the isolation process. Three to four weeks in culture results in an induction of cell growth related genes accompanying their expansion by 6-10-fold, which increases the number of cells for in vitro experiments. Collectively, we show that cultured human skin MCs resemble their ex vivo equivalents in many respects and are a more relevant in vitro model compared to mouse BMMCs for studies of MC biology, in particular human MC biology.

We conducted a retrospective analysis of the immunohistochemical (IHC) staining GRP94, an ER stress protein on large B-cell lymphoma (LBCL) cells, intracellular p53, and 15 factors involved in the metabolism of CHOP regimen :AKR1C3 (HO-metabolism), CYP3A4 (CHOP-metabolism) and HO efflux pumps (MDR1, MRP1). The study subjects were 42 patients with LBCL at our hospital. IHC staining using antibodies against the 17 factors. The odds ratios by logistic regression analysis using a dichotomous variable of CR, non-CR/relapse were statistically significant: for MDR1, MRP1, and AKR1C3. The overall survival (OS) after R-CHOP was compared by the log-rank test. The four groups showed that Very good (5-year OS, 100%) consisted of 4 patients showed negative IHC staining for both GRP94 and CYP3A4. Very poor (1-year OS, 0%), consisted of 3 patients who showed positive results of IHC for both GRP94 and CYP3A4. The remaining 35 patients comprised two subgroups: Good (5-year OS 60-80%.): 15 patients who showed negative staining for both MDR1 and AKR1C3, Poor: 5-year OS, 10-20%) :20 patients who showed positive staining for either MDR, AKR1C3,,MRP1 or p53. The Histological Prognostic Index (HPI) (The four groups: Very poor, Poor, Good, and Very pood) is a breakthrough method for stratifying patients based on factors involved in the development of treatment resistance.

Integrated photovoltaic-fuel cell (IPVFC) systems, amongst other integrated energy generation methodologies are renewable and clean energy technologies that have received diverse research and development attentions over the last few decades due to their potential applications in a hydrogen economy. This article systematically updates the state-of-the-art of IPVFC systems and provides critical insights into the research and development gaps needed to be filled/addressed to advance these systems towards full commercialisation. The design methodologies, renewable energy-based microgrid and off-grid applications, energy management strategies, optimisations and the prospects as self-sustaining power source were covered. IPVFC systems could play an important role in the upcoming hydrogen economy since they depend on solar hydrogen which has almost zero emissions during operation. Highlighted herein are the progresses as well as the technical challenges requiring research efforts to solve to realise numerous potential applications of IPVFC systems such as in unmanned aerial vehicles, hybrid electric vehicles, agricultural applications, telecommunications, desalination, synthesis of ammonia, boats, buildings, and distributed microgrid applications.

Ubiquitin-Specific-peptidase 22 (Usp22) cleaves ubiquitin moieties from numerous proteins, in particular transcription factors. Recently, it was reported that Usp22 acts as negative regulator of interferon-dependent responses. In the current study, we investigated the role of Usp22 deficiency upon acute viral infection with lymphocytic choriomeningitis (LCMV) virus. We found that lack of Usp22 on bone marrow derived cells (Usp22 fl/fl Vav1-Cre mice) reduced induction of type I and II interferons. Limited type I interferon response did not influence virus replication. However, restricted expression of PD-L1 led to increased frequencies of functional virus-specific CD8+ T cells and rapid death of Usp22 deficient mice. CD8+ T cell depletion experiments revealed that accelerated CD8+ T cells were responsible for enhanced lethality in Usp22 deficient mice. In conclusion, we found that the lack of Usp22 generated a pathological CD8+ T cell response, which gave rise to severe disease in mice.

Antibody-dependent cell-mediated cytotoxicity (ADCC) by natural killer (NK) lymphocytes eliminates cells infected with viruses. Antiviral ADCC requires three components: 1) antibody; 2) effector lymphocytes with the Fc-IgG receptor CD16A; and 3) viral proteins in infected cell membranes. Fc-afucosylated antibodies bind with greater affinity to CD16A than fucosylated antibodies; individuals’ variation in afucosylation contributes to differences in ADCC. Current assays for afucosylated antibodies involve expensive methods. We report an improved bioassay for antibodies that support ADCC which encompasses afucosylation. This assay utilizes externalization of CD107a by NK-92-CD16A cells after antibody recognition. We used anti-CD20 monoclonal antibodies, GA101 WT or glycoengineered (GE), 10% or ~50% afucosylated, and CD20-positive Raji target cells. CD107a increased detection 7-fold compared to flow cytometry to detect Raji-bound antibodies. WT and GE antibody effective concentrations (EC50s) for CD107a externalization differed by 20-fold, with afucosylated GA101-GE more detectable. The EC50s for CD107a-externalization vs. 51Cr cell death were similar for NK-92 CD16A and blood NK cells. Notably, the %CD107a-positive cells negatively correlated with dead Raji cells and was nearly undetectable at high NK:Raji ratios required for cytotoxicity. This bioassay is very sensitive and adaptable to assess anti-viral antibodies but unsuitable as a surrogate assay to monitor cell death after ADCC.

Leukemia, contributing to 3% of all cancer cases in the United States, presents a significant challenge in treatment due to its diverse subtypes, each dis-playing unique genetic and molecular abnormalities. The aggressive growth of ab-normal blood cells compromises immune function and poses challenges in developing effective therapeutic approaches. Unlike solid tumors, surgical resection is not an option, limiting treatment strategies to chemotherapy, immunotherapy, and radiation therapy, which may be nonspecific and lead to organ damage and toxicity. Anthracyclines, widely used in hematological tumors, face limitations due to severe cardiotoxicity, causing progressive cardiomyopathy and, in severe cases, congestive heart failure. Heart failure incidence is significantly higher in cancer patients treated with anthracyclines over 5, 10, and 15 years. Early detection of left ventricular dysfunction and cardiotoxicity is crucial to mitigate progression to congestive heart failure (CHF) in this population. Recent research by Bayrón-Marrero et al. reveals that lower levels of plasma soluble TREM-Like Transcript-1 (s-TLT-1), a platelet-specific receptor, correlate with left ven-tricular dysfunction in cardiovascular disease patients. Those with s-TLT-1 levels ex-ceeding 544 pg/mL exhibit a significant association with congestive heart failure. These findings suggest the potential use of s-TLT-1 as a diagnostic screening tool for early detection and treatment of anthracycline-induced CHF in leukemia patients.

Dietary fibers regulate intestinal barrier function; however, the precise mechanisms remain unclear. This study investigated the effects of psyllium fibers on antimicrobial protein expression, focusing on the type II immunity and tuft cell-group 2 innate lymphoid cell (ILC2) circuit in the small intestine of the mouse. Supplemental psyllium fiber upregulated antimicrobial proteins, such as small proline-rich protein 2A (SPRR2A) and resistin-like beta (RELMβ), in mouse small intestine, evidently affecting cecal microbiota composition. Psyllium fibers also increased the RNA and protein expression of molecules related to ILC2 and tuft cells, such as IL-13, IL-25, DCLK1, Gfi-1b, SH2 domain-containing protein 3C, and Spi-B. In addition, ILC2 inhibitor (disulfiram) and bitter taste receptor blocker administration reduced psyllium-induced SPRR2A and RELMβ expression. Collectively, psyllium supplementation upregulates antimicrobial proteins such as SPRR2A and RELMß via the type II immune response and tuft cell-ILC2 circuit in the mouse small intestine.

Adult neurogenesis involves generation of functional neurons from neural progenitor cells, which have the potential to complement and restore damaged neurons and neural circuits. Therefore, the development of drugs that stimulate neurogenesis represents a promising strategy in stem cell therapy and neural regeneration, greatly facilitating the reconstruction of neural circuits in cases of neurodegeneration and brain injury. Our study reveals that compound A5, previously designed and synthesized by our team, exhibits remarkable neuritogenic activities, effectively inducing neurogenesis in neural stem/progenitor cells (NSPCs). Subsequently, transcriptome analysis using high-throughput Illumina RNA-seq technology was performed to further elucidate the underlying molecular mechanisms by which compound A5 promotes neurogenesis. Notably, comparative transcriptome analysis showed that the up-regulated genes were mainly associated with neurogenesis and the down-regulated genes were mainly concerned with cell cycle progression. Furthermore, we confirmed that compound A5 significantly affected the expression of transcription factors related to neurogenesis and cell cycle regulatory proteins. Collectively, these findings identify a new compound with neurogenic activity and may provide insights into drug discovery for neural repair and regeneration.

A key feature of pulmonary arterial hypertension (PAH) is the hyperplastic proliferation exhibited by the vascular smooth muscle cells from patients (HPASMC). The growth inducers FOXM1 and PLK1 are highly upregulated in these cells. The mechanism by which these two proteins direct aberrant growth in these cells is not clear. Herein we identify cyclin dependent kinase 1 (CDK1) also termed cell division cycle protein 2 (CDC2), as having a primary role in promoting progress of the cell cycle leading to proliferation in HPASMC. HPASMC obtained from PAH patients and pulmonary arteries from Sugen/hypoxia rats were investigated for their expression of CDC2. Protein levels of CDC2 were much higher in PAH than in cells from normal donors. Knocking down FOXM1 or PLK1 protein expression with siRNA or pharmacological inhibitors lowered the cellular expression of CDC2 considerably. However, knockdown of CDC2 with siRNA or inhibiting its activity with RO-3306 did not reduce the protein expression of FOXM1 or PLK1. Expression of CDC2 and FOXM1 reached its maximum at G1/S, while PLK1 reached its maximum at G2/M phase of the cell cycle. The expression of other CDKs such as CDK2, CDK4, CDK6, CDK7 and CDK9 did not change in PAH HPASMC. Moreover, inhibition via Wee1 inhibitor adavosertib or siRNAs targeting Wee1, Myt1, CDC25A, CDC25B, or CDC25C led to dramatic decreases in CDC2 protein expression. Lastly, we found CDC2 expression at the RNA and protein level to be upregulated in pulmonary arteries during disease progression Sugen/hypoxia rats. In sum, our present results illustrate that the increased expression of FOXM1 and PLK1 in PAH leads directly to increased expression of CDC2 resulting in a potentiated growth hyperactivity of PASMC from patients with pulmonary hypertension. Our results further suggest that the regulation of CDC2, or associated regulatory proteins, will prove beneficial in the treatment of this disease.

Cancer researchers are fascinated by the chemistry of diverse natural products which show exciting potential as anticancer agents. In this study, we aimed to investigate the anticancer properties of watermelon rind extract (WRE) by examining its effects on cell proliferation, apoptosis, senescence, and global gene expression in human renal cell adenocarcinoma cells (HRAC-769-P) in vitro. Our metabolome data analysis of WRE exhibited untargeted phyto-constituents and targeted citrulline (22.29 µg/mg). HRAC-769-P cells were cultured in RPMI-1640 media and treated with 22.4, 44.8, 67.2, 88.6, 112, 134.4, and 156.8 mg mL-1 for 24, 48, and 72 hrs. At 24 hrs after treatment, (88.6 mg ml-1 of WRE) cell proliferation significantly reduced, more than 34% compared to the control. Cell viability decreased 48 and 72 hours after treatment to 45% and 37%, respectively. We also examined poly caspase, SA-beta-galactosidase (SA-beta-gal), and wound healing activities using WRE. All treatments induced an early poly caspase response and a significant reduction in cell migration. Further, we analyzed the transcript profile of the cells grown at 44.8 mg ml-1 of WRE after 6 hours using RNA sequencing (RNAseq) analysis. We identified 186 differentially expressed genes (DEGs), including 149 upregulated genes and 37 downregulated genes, in cells treated with WRE compared to the control. The differentially expressed genes were associated with NF-Kappa B signaling and TNF pathways. Crucial apoptosis-related genes such as BMF, NPTX1, NFKBIA, NFKBIE, and NFKBID might induce intrinsic and extrinsic apoptosis. Another possible mechanism is a high quantity of citrulline may lead to induction of apoptosis by the production of increased nitric oxide. Hence, our study suggests the potential anticancer properties of WRE and provides insights into its effects on cellular processes and gene expression in HRAC-769-P cells.

Recessive dystrophic epidermolysis (RDEB) is a rare, inherited and currently incurable skin blistering disorder characterized by cyclically recurring wounds coexisting with chronic non-healing wounds. In a recent clinical trial three intravenous infusions of skin-derived ABCB5+ mesenchymal stromal cells (MSCs) to 14 patients with RDEB improved healing of wounds that were present at baseline. Since in RDEB even minor mechanical forces perpetually provoke the development of new or recurrent wounds, a post-hoc analysis of patient photographs was performed to specifically assess the effects of ABCB5+ MSCs on new or recurrent wounds by evaluating 174 wounds that occurred after baseline. During 12 weeks of systemic treatment with ABCB5+ MSCs the number of newly occurring wounds declined. When compared to the previously reported healing responses of the wounds present at baseline, the newly occurring wounds healed faster, and a greater portion of healed wounds remained stably closed. These data suggest a previously undescribed skin-stabilizing effect of treatment with ABCB5+ MSCs and support repeated dosing of ABCB5+ MSCs in RDEB to continuously slow the wound development and accelerate the healing of new or recurrent wounds before they become infected or progress to a chronic, difficult-to-heal stage.

Mesenchymal stem cells (MSCs) showed in vitro mesoderm-lineage differentiation and self-renew capacity. However, no comparative study was reported on the biological characteristics of stem cells derived from skeletal muscle (SM-MSCs), dermal skin (DS-MSCs), and adipose tissues (A-MSCs) from a single donor in camels. The present study aimed to evaluate the influence of MSCs source on stem cell characteristics. We evaluated proliferation capacity and mesoderm-lineage differentiation potential from SM-MSCs, DS-MSCs, and A-MSCs. They showed spindle-like morphology after homogenization. The proliferation ability was no significant difference in all groups. Furthermore, the portion of the cell cycle and expression of pluripotent markers (Oct4, Sox2, and Nanog) were similar in all cell lines at passage 3. The differentiation capacity of A-MSCs into adipocytes was significantly higher than that of SM-MSCs and DS-MSCs. However, the osteoblast differentiation capacity of A-MSCs was significantly lower than that of SM-MSCs and DS-MSCs. Additionally, after osteoblast differentiation, the ALP activity and calcium content was significantly decreased in A-MSCs as compared to SM-MSCs and DS-MSCs. To the best of our knowledge, we primally established MSCs from the single camel and demonstrated their comparative characteristics including expression of pluripotent factors and proliferation, and in vitro differentiation capacity into adipocytes and osteoblasts.

Pancreatic ductal adenocarcinoma (PDAC) is mostly diagnosed at advanced or even metastasized stages limiting patient´s prognosis. Metastasis requires high tumor cell plasticity implying phenotypic switching in response to changing environments. Here, Epithelial-Mesenchymal-Transition (EMT), being associated with the gain of cancer stem cell (CSC) properties, and its reversion are important. Since it is poorly understood whether different CSC-phenotypes exist along the EMT-axis and how these impact malignancy-associated properties, we aimed to characterize CSC-populations of epithelial and mesenchymal PDAC cells. Single-cell cloning revealed CSC (Holoclone) and non-CSC (Paraclone) clones from the PDAC cell lines Panc1 and Panc89. Panc1 Holoclone cells showed a mesenchymal phenotype dominated by high expression of the stemness marker Nestin, while Panc89 Holoclone cells exhibited a SOX2-dominated epithelial phenotype. Panc89 Holoclone cells showed enhanced cell growth and self-renewal capacity but slow cluster-like invasion. Contrarily, Panc1 Holoclone cells showed slower cell growth and self-renewal ability but were highly invasive. Moreover, cell variants differentially responded to chemotherapy. In vivo, Panc1 and Panc89 cell variants significantly differed regarding number and size of metastases as well as organ manifestation leading to different survival outcomes. Overall, these data support the existence of different CSC-phenotypes along the EMT-axis in PDAC manifesting in different metastatic propensities.

There is a strong anticipated future for human pluripotent stem cell-derived hepatocytes (hiPS-HEP), but so far their use has been limited due to insufficient functionality. We investigated the potential of hiPS-HEP as an in vitro model for metabolic diseases by combining transcriptomics with multiple functional assays. The transcriptomics analysis revealed that 86% of the genes were expressed at similar levels in hiPS-HEP as in human primary hepatocytes (hphep). Adult characteristics of the hiPS-HEP were confirmed by the presence of important hepatocyte features, e.g. Albumin secretion and expression of major drug metabolizing genes. Normal energy metabolism is crucial for modeling metabolic diseases, and both transcriptomics data and functional assays showed that hiPS-HEP were similar to hphep regarding uptake of glucose, LDL and fatty acids. Importantly, the inflammatory state of the hiPS-HEP was low under standard conditions, but in response to lipid accumulation and ER stress the inflammation marker TNFα was upregulated. Furthermore, hiPS-HEP could be co-cultured with primary hepatic stellate cells both in 2D and in 3D spheroids, paving the way for using these co-cultures for modeling NASH. Taken together, hiPS-HEP have the potential to serve as an in vitro model for metabolic diseases. Furthermore, differently expressed genes identified in this study can serve as targets for future improvements of the hiPS-HEP.

A technique for fabrication of bacterial cellulose-based films with CeO₂ nanofiller has been developed. The structural and morphological characteristics of the materials have been studied, their thermal and mechanical properties in dry and swollen states having been determined. The preparation methodology makes it possible to obtain composites with a uniform distribution of nanoparticles. The catalytic effect of ceria regarding the thermal oxidative destruction of cellulose has been confirmed by TGA and DTA methods. An increase in CeO₂ content led to an increase in the elastic modulus (a 1.27-fold increase caused by the introduction of 5 wt.% of the nanofiller into the polymer) and strength of the films. This effect is explained by the formation of additional links between polymer macro-chains via the nanoparticles’ surface. The materials fabricated were characterised by a limited ability to swell in water. Swelling caused a 20- to 30-fold reduction in the stiffness of the material, the mechanical properties of the films in a swollen state remaining germane to their practical use. The application of the composite films in cell engineering as substrates for the stem cells’ proliferation has been studied. The increase in CeO₂ content in the films enhanced the proliferative activity of embryonic mouse stem cells. The cells cultured on the scaffold containing 5 wt.% of ceria demonstrated increased cell survival and migration activity. An analysis of gene expression confirmed improved cultivation conditions on CeO₂-containing scaffolds.

GeneVac-B® was recently licensed in Burkina Faso and is widely used for population vaccination. This study aimed to evaluate the immune response in people fully vaccinated against hepatitis B with the GeneVac-B® vaccine in Burkina Faso under actual conditions of use. This cross-sectional study included individuals fully vaccinated with GeneVac-B®. For each consenting participant, sociodemographic and clinical data were collected using a structured questionnaire. Approximately 4 mL of whole blood was collected for immunological (anti-HBs, HBsAg, anti-HCV and anti-HIV) and biochemical (blood glucose, total cholesterol, and triglycerides) testing. The anti-HBs titer was used to determine the immune response. A total of 392 participants were included in the study. The mean age was 35.5±15 years. Approximately 56.1% had been previously exposed to HBV. Three hundred eighty (380) participants had an anti-HBs titer ≥ 12 mIU/mL, a seroprotection rate of 96.9%, and 12 had a titer &lt; 12 mIU/mL, a nonresponse rate of 3.1%. Among the nonresponders, 11 (11/12; 91.7%) had high total cholesterol levels (&gt; 240 mg/dL), and 8 (8/12; 66.7%) were older than 40 years of age. The results showed an excellent immune response to GeneVac-B® in our study population. However, age &gt; 40 and high total cholesterol appear to be factors associated with nonresponse.

We present atomic structures and nonequilbrium synthesis of new class of materials, where the basic structural unit is a diamond tetrahedron. When units of one, two, and three tetrahedra are randomly packed, we create distinct phases of amorphous Q-carbon. Four tetrahedra in two adjacent layers lead to crystalline diamond lattice, which has four missing tetrahedra alternately. When these four missing tetrahedra are filled, we create subunit cell of crystalline Q-diamond. Theoretical calculations show that superconducting transition temperature (Tc) in 50 atomic % B-doped Q-diamond can reach near room temperature at ambient pressures. This is consistent with our earlier results using low-loss EELS measurements in 50 atomic % B-doped Q-carbon, which had mostly amorphous QB3 phase mixed with some crystalline Q-diamond phase. These EELS results showed that the Tc for these samples was in between 90K and 300K. Theoretical calculations of density of states, Eliashberg function, electron-phonon interaction parameter, and root-mean-square and logarithmic average of frequency in crystalline Q-diamond show Tc in the range of 268K to 300K, which is in a complete agreement with our EELS results in QB3.

FoxP3 is a transcription factor essential for the differentiation and function of T regulatory cells (Tregs). There are two major subsets of Tregs: natural Tregs (nTregs) generated in thymus and inducible Tregs (iTregs) produced in peripheral immune system. It has been documented that iTreg development is dependent on soluble mediators including interleukin 2 (IL2), transforming growth factor β (TGFβ) and all-trans-retinoic acid (ATRA). In our experiments we performed a gene expression array, followed by Real-time PCR experiments, to study the expression of genes regulated by 1,25-dihydroxyvitamin D (1,25D) or ATRA in cells of myeloid origin. Our experiments revealed that ATRA alone, but also a cocktail of mediators consisting of IL2, TGFβ and ATRA, upregulate the expression of FOXP3 gene in lymphoid cells, but also in normal and leukemic myeloid cells. The FoxP3 expression is followed by a phenotypic changes in cells of myeloid origin. Our results indicate that signaling pathways which are used in the late stages of T cell differentiation, are also active in the cells of myeloid lineage

Cancer stem cells play a crucial role in tumor initiation, metastasis and therapy resistance. Cellular heterogeneity and plasticity challenge the isolation of cancer stem cells. The impact of intratumoral cellular heterogeneity in the context of treatment resistance using a label-free approach remains understudied. Here, we use the sedimentation field-flow fractionation technique to separate, without labeling, cell subpopulations of colorectal cancer cell lines and primary cultures according to their biophysical properties. One of the three cell subpopulations sorted by SdFFF exhibits cancer stem cell traits, including high tumorigenicity in vivo, and a higher frequency of tumor-initiating cells compared to the other subpopulations. In vitro two- and three-dimensional chemosensitivity assays emphasize the therapeutic relevance of this cancer stem cell-like subpopulation due to its chemoresistance. Therefore, our findings highlight a label-free cell sorting approach to reveal intratumoral cellular heterogeneity and its implication in therapy resistance. This approach enables the study of the individualized response of each sorted cell subpopulation by breaking down the tumor, thus offering new perspectives for personalized therapy.

Cell-based therapy in regenerative medicine is a powerful tool that can be used both to restore various cell lost in a wide range of human disorders, and in renewal processes. Stem cells show promise for universal use in clinical medicine, potentially enabling the regeneration of numerous organs and tissues in the human body. This is possible due to their self-renewal capacity, and their ability to differentiate into various cell types. To date, pluripotent stem cells seem to be the most promising regeneration tool. Recently, a novel stem cell niche, called multilineage-differentiating stress-enduring (Muse) cells, is emerging. These cells are of particular interest because they are pluripotent and are found in adult human mesenchymal tissues. One of their most significant features is that they can produce cells representative of all the three germ layers. Furthermore, they can be easily harvested from fat, and isolated from the mesenchymal stem cells. This makes them very promising, allowing autologous treatments and avoiding the problems of rejection, typical of transplants. Muse cells have recently been employed, with encouraging results, in numerous preclinical studies, performed to test their efficacy in the treatment of various pathologies, exploiting their regenerative potential in different tissues. This systematic review aimed to 1) highlight the specific potential of Muse cells, and provide a better understanding of this niche, and 2) originate the first organized review of already tested applications of Muse cells in regenerative medicine. The obtained results could be useful to extend the possible therapeutic applications of Muse cells.

In the text, the synthesis and characteristics of the novel ONS-type vanadium(V) complexes with thioanilide derivatives of amino acids are described. They have shown inhibition of human protein tyrosine phosphatases (PTP1B, LAR, SHP1 and SHP2) in the submicromolar range, as well as inhibition of non-tyrosine phosphatases (CDC25A and PPA2) similar to bis(maltolato)oxovanadium(IV)(BMOV). The ONS complexes increased [14C]-deoxy-D-glucose transport into C2C12 myocytes, and one of them, VC070, also enhanced this transport in 3T3-L1 adipocytes. These complexes inhibited gluconeogenesis in hepatocytes HepG2, but none of them decreased lipid accumulation in the non-alcoholic fatty liver disease model using the same cells. Compared to the tested ONO-type vanadium complexes with 5-bromosalicylaldehyde and substituted benzhydrazides as Schiff base ligand components, the ONS complexes revealed stronger inhibition of protein tyrosine phosphatases, but the ONO complexes showed greater activity in the cell models in general. Moreover, the majority of the active complexes from both groups showed better effects than VOSO4 and BMOV. Complexes from both groups activated AKT and ERK signaling pathways in hepatocytes to a comparable extent. One of the ONO complexes, VC068, showed activity in all of the above models, also including glucose utilization in the myocytes and glucose transport in insulin-resistant hepatocytes. The discussion section explicates the results within the wider scope of the knowledge about vanadium complexes.

Healthy human subjects develop spontaneous CD8+ T cell responses to melanocyte antigens (MA) expressed by normal melanocytes, such as Tyrosinase, MAGE-A3, Melan/Mart-1, gp100, and NY-ESO-1. This natural autoimmunity directed against melanocytes might confer protection against the development of malignant melanoma (MM), where MA are present as overexpressed tumor-associated antigens. Consistent with this notion we report here that functional T cell reactivity to MA was found to be diminished in untreated MM patients: while 57.5% of healthy controls (n=40) exhibited high-frequency MA-specific T cell reactivity ex vivo, such was detected in only 12% of the untreated MM patients (n=24). Three lines of evidence suggest that the MA-reactive T cells present in healthy subjects undergo exhaustion once MM establishes itself. First, only the MA-specific T cell reactivity was affected in the MM patients; that to third party recall antigens was not. Second, in these patients, the residual MA-specific T cells, unlike third party antigen reactive T cells, were functionally impaired, showing a diminished per cell IFN-γ productivity. Third, successful immunotherapy with AGI-101H melanoma vaccine restored natural CD8+ T cell autoimmunity to MA in 85% of the vaccinated patients (n= 27). The role of natural T cell autoimmunity to tumor-associated MA is discussed based on discrete levels of T cell activation thresholds.

Bone marrow mononuclear cells (BMMNCs) have emerged as a potential therapeutic strategy for various incurable conditions including autism spectrum disorder (ASD). To study its safety and efficacy, we analyzed 1011 patients who underwent intrathecal administration of autologous BMMNCs along with neurorehabilitation. At mean follow-up of 19.3 months after intervention, 90.6% patients showed overall improvement. Improvements were observed in attention concentration, command following, eye contact, sitting tolerance, social interaction, hyperactivity, communication, speech, aggressiveness, stereotypical and self-injurious behavior. Statistically significant changes were observed in scores of Indian Scale of Autism Assessment and Childhood Autism Rating Scale (p<0.05). On subgroup analysis, improvements were recorded irrespective of age and disease severity and better outcome was observed in patients who took multiple doses of cell therapy compared to single dose. Comparative Brain PET-CT scan of 401 patients showed improved brain metabolism after the intervention. No major adverse events were reported. Only 9 out of 1011 patients experienced seizures which were managed using medications. 4 out of 9 had history of seizures while 5 had normal EEG with no history. However, improvements were still observed in these 9 patients. Thus, intrathecal transplantation of autologous BMMNCs along with neurorehabilitation is safe and efficacious in patients with ASD.

The life cycle of cancer follows the life cycle of the common ancestor of amoebozoans, metazoans, and fungi (AMF) and its systemic germline, which serves as a blueprint for all germlines capable of asymmetric cell division (ACD) and stem cell differentiation. Consequently, the oxygen sensitivity of the ancient non-gametogenic germline (Urgermline) was inherited by all germ and stem cell lines including the cancer germline. They all respond to Ugermline’s hyperoxia with loss of stemness and ACD ability and a dysregulated phenotype with irreparable DNA defects and defective symmetric cell divisions (DSCD). In protists, defective DSCD cells undergo an ancient MGRS repair program involving cell and nuclear fusion and hyperploid giant nuclei that restores the damaged genome to its former pre-DSCD state, with ACD potential and stemness. Human and metazoan DSCD use the same MGRS repair program inherited from the AMF ancestor. Ectopic DSCDs and DSCD-like phenotypes can survive in humans for many years in suitable niches. Under favorable environmental conditions, they also have access to the ancient MGRS repair mechanism including the ancient gene regulatory network (aGRN) and all other AMF genes. The aGRN takes control of cancer’s hybrid genome and represses human genes. It installs a G+S cancer life cycle of AMF imprinting that shapes the differentiation of naïve cancer stem cells (CSCs). CSCs are deeply homologous to the aGSCs of the AMF ancestor. Reprogramming of the DSCD genome by MGRS paves the way for oncogenesis. In this light, cancer is not a mutational or genetic disease, but a non-mutational genome-altering disease.

To map Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV 2) spreading and evaluate immune responses variations against this virus, it was essential to locally set up efficient serological tests. The SARS-CoV2 immunogenic proteins were very expensive and not affordable mainly for Low and Middle Income Countries (LMICs). For this purpose, the commonly used antigen, Re-ceptor-Binding Domain (RBD) of Spike S1 protein (S1RBD), was produced using the the Baculovirus Expression Vector System (BEVS) . During the current study, the expression of S1RBD was monitored using western blot under different culture conditions. Different parameters were studied: Multiplicity Of Infection (MOI), cell density at infection and harvest time. Hence, optimal conditions for efficient S1RBD production were identified: MOI 3; cell density at infection 2-3x 106 cells/mL and time post-infection (tpi or harvest time) of 72h and 72-96 h successively for expression in shake-flasks and 7L-bioreactor. A high production yield of S1RBD varying between 4mg and 70 mg per liter of crude cell culture supernatant was achieved, respectively, in shake-flasks and in 7L-bioreactor. Moreover, the produced S1RBD showed an excellent antigenicity potential against COVID-19 patient sera evaluated by western blot. Thus, additional serological assays, such as ELISA, were developed using the purified S1RDB (published by other research groups).

Background: Extracellular Vesicles are pivotal mediators in intercellular communication, facilitating the exchange of biological information among healthy, pathological and tumor cells. Between the diverse subtypes of extracellular vesicles, exosomes have unique properties, clinical and therapeutical applications, even towards cancer. Breast cancer ranks as one of the most prevalent malignancies across the globe. Both the tumor core and its surrounding microenvironment engage in a complex, orchestrated interaction that facilitates the cancer’s growth and spread. Methods: The most valuable and significant PubMed Literature about extracellular vesicles and adipose–derived stem cells exosomes was selected and analyzed in order to report their biological properties and potential applications, in particular on the Triple–Negative Breast Cancer. Results: Adipose–derived stem cells exosomes represent a potential tool to target the tumor core of the Triple–Negative Breast Cancer, its microenvironment and surrounding tissues including metastasis. The authors are developing a supportive Triple–Adipose–derived stem cells Exosomes [T.A.E.] Technology against breast cancer in order to perform a selective preconditioning of the cancer before or while the traditional oncological treatments are running. Conclusion: The possibility to exploit the impact of Adipose–derived stem cells exosomes within the T.A.E. Technology against the Triple–Negative breast cancer is real, highlighting a new era for supportive measures against breast cancer.Keywords: Extracellular vesicles; Exosomes; Adipose–derived stem cells, Tumor microenvironment, Triple–Negative Breast Cancer, T.A.E. Technology.

Solid tumor cells have an altered metabolism that can protect them from cytotoxic lymphocytes. The antidiabetic drug metformin modifies tumor cell metabolism and several clinical trials are testing its effectiveness for the treatment of solid cancers. The use of metformin in hematologic cancers has received much less attention, although allogeneic cytotoxic lymphocytes are very effective against these tumors. We show here that metformin induces expression of Natural Killer G2-D (NKG2D) ligands (NKG2DL) and intercellular adhesion molecule-1 (ICAM-1), a ligand of the lymphocyte function-associated antigen 1 (LFA-1). This leads to enhance sensitivity to cytotoxic lymphocytes. Overexpression of antiapoptotic Bcl-2 family members decrease both metformin effects. The sensitization to activated cytotoxic lymphocytes is mainly mediated by the increase on ICAM-1 levels, which favors cytotoxic lymphocytes binding to tumor cells. Finally, metformin decreases the growth of human hematological tumor cells in xenograft models, mainly in presence of monoclonal antibodies that recognize tumor antigens. Our results suggest that metformin could improve cytotoxic lymphocyte-mediated therapy.

In this work, we pursued the biological characterization of the venom of Montivipera born-muelleri, a viper from the Lebanese mountains. In relativity to its antibacterial potential, the in-hibitory effect of this venom on the F1F0-ATPase enzymes of Gram-positive Staphylocoocus epider-midis and Gram-negative Escherichia coli bacteria was examined. In order to determine the de-gree of cytotoxicity of the venom on the HCT116 human colon cancer cell lines, the biological MTT proliferation and cell viability test were implemented. After validation of the enzymatic F1F0-ATPase model by the spectrophotometric method, using quercetin as the reference ligand, re-sults revealed that M. bornmuelleri venom is able to inhibit the activity of the enzyme of these two bacteria with a concentration of the order of 100-150 µg/mL. In addition, a venom concentration of 10 µg/mL was sufficient to kill the totality of HCT116 cell lines cultivated in vitro. These data show that M. bornmuelleri venom is a mixture of diverse molecules presenting activities of interest and is a potential source to explore in order to discover new drug candidates.

Separation and interpretation of rebellious Circulating Tumor Cells (CTCs) originating from the primary tumor or cancer tissue plays a significant role in diagnostics, cancer progression analyses, suitable medicine exploration, and treatment proficiency examination. Cancer metastasis occurs when CTCs spread throughout the body and invade healthy tissues, leading to new tumors in that area. Although a dramatic rate of deaths begins from spreading CTCs around the body, valuable measures have been made to control their development. However, the first step is separating these harmful cells from the bloodstream and investigating their features. Having examined the characteristics of CTCs as cancer’s main strength, researchers can introduce complementary treatments that can affect cancerous cells without damaging the healthy cells. Therefore, according to their unique characteristics, numerous techniques have been established for continuous and fast separation and sorting of CTCs. Nevertheless, few separators enjoy the efficient performance and appropriate accuracy and can be produced in mass numbers due to the available fabrication equipment. Microfabrication advancements enable separators to combine the advantages of active and passive methods in a small-scale platform for probing individual cells and separation purposes. Reduction in reagents, sample volume, analysis time, and less harmfulness to patients are some of the motivations that encourage researchers to employ microfluidic instruments for CTCs separation from other blood cells over the last two decades. However, microfabrication limitations mean effective separators, and the diagnostic option they provide, are not readily available. Addressing these limitations requires optimizing the design and fabrication of separators such that they are reduced in their size and fabrication cost, while also maintaining high-throughput separating capability. The emergence of the Lab-On-a-Chip (LOC) and then Lab-On-a-CD (LOCD) technologies, having more inherent benefits than conventional microfluidic devices, has created new opportunities and become increasingly widespread in recent years. Evidence suggests that employing single methodologies or integrating approaches without sufficient understanding of potential outcomes is unlikely to result in successful diagnostic results. This paper contributes an extensive review of several separation systems, including fundamental theories and experimental details, and describes detailed operating principles and device performance.

Foot-and-mouth disease virus (FMDV) causes the highly contagious foot-and-mouth disease, which is characterized by the appearance of vesicles in and around the mouth and feet of cloven-hoofed animals. BHK21 cells are the cell line of choice for the propagation of FMDV for vaccine production world-wide but vary in their susceptibility for different FMDV strains. Previous studies showed that the FMDV resistance of a certain BHK cell line can be overcome by using a closely related but permissive cell line for the pre-adaptation of the virus, but the adapted strains were found to harbor several capsid mutations. In this study, these adaptive mutations were introduced into the original Asia-1 Shamir isolate individually or in combination to create a panel of 17 Asia-1 mutants by reverse genetics and examine the effects of the mutations on receptor usage, viral growth, immunogenicity and stability. A single amino acid exchange from glutamic acid to lysine at position 202 in VP1 turned out to be of major importance for productive infection of the suspension cell line BHK-2P. In consequence, two traditionally passage-derived strains and two recombinant viruses with a minimum set of mutations were tested in vivo. While the passaged-derived viruses showed a reduced particle stability, the genetically modified viruses were more stable but did not confer a protective immune response against the original virus isolate.

The anode of a microbial fuel cell (MFC) was formed on a graphite electrode and immobilized Gluconobacter oxydans VKM-1280 bacterial cells. Immobilization was performed in chitosan, poly(vinyl alcohol) or N-vinylpyrrolidone-modified poly(vinyl alcohol). Ethanol was used as substrate. The anode was modified using multiwalled carbon nanotubes. The aim of the modification was to create a conductive network between cell lipid membranes, containing exposed PQQ-dependent alcoholdehydrogenases, and the electrode to facilitate electron transfer in the system. The bioelectrochemical characteristics of modified anodes at various cell/polymer ratios were assessed via current density, power density, polarization curves and impedance spectres. MFCs based on chitosan at a matrix/cell volume ratio of 5:1 produced maximal power characteristics of the system (8.3 μW/cm2) at a minimal resistance (1111 Ohm cm2). Modification of the anode by multiwalled carbon nanotubes led to a slight decrease of internal resistance (down to 1078 Ohm cm2) and to an increase of generated power density up to 10.6 μW/cm2. We explored the possibility of accumulating electric energy from an MFC on a 6,800-μF capacitor via a boost converter. Generated voltage was increased from 0.3 V up to 3.2 V. Accumulated energy was used to power a Clark-type biosensor and a bluetooth transmitter with three sensors, a miniature electric motor and a light-emitting diode.

Background and Aim. Since virus-related hepatocellular carcinoma (HCC) pathogenesis involves liver inflammation, therefore, post-hepatitis C virus (HCV) infection would be a cause for liver cirrhosis that would progress to HCC. Cytotoxic T cells (Tc) are known to be involved in post-HCV complications and HCC pathogenesis. The inhibitory checkpoint Leukocyte-Associated Immunoglobulin-like Receptor-1 (LAIR-1) is expressed on Tc. Therefore, we aimed to determine whether the Tc expression level of LAIR-1 is associated with HCC progression post-HCV and moreover, to evaluate LAIR-1 expression as a non-invasive biomarker for HCC progression in the context of liver cirrhosis post-HCV genotype 4 (G4) in Egyptian patients’ peripheral venous blood liquid biopsy. We studied LAIR-1 expression on Tc related to the progression of liver cirrhosis in a case-controlled study enrolled 64 patients with post-HCV G4-HCC and 37 patients with post-HCV G4-liver cirrhosis. Methods: LAIR-1 expression was analyzed by flow cytometry. Results: LAIR-1 expression on Tc and the percentage of Tc positive for LAIR-1 (LAIR-1+Tc %) were significantly higher in the post-HCV G4-HCC group compared to the post-HCV G4-liver cirrhosis