B-1 cells, a subtype of B lymphocytes, are found in lymphoid tissues and peritoneal and pleural cavities. They play diverse roles, releasing cytokines, generating antibodies, regulating the immune response, engaging in phagocytosis, and moving to inflammatory sites. However, their physiology remains partially understood, especially in stressful conditions like sleep restriction. This study aimed to investigate murine B-1 cell activation and differentiation under sleep restriction. Male C57BL/6J mice underwent an 18-hour sleep restriction for 21 days, followed by Candida albicans or Paracoccidioides brasiliensis infection. Purified peritoneal B-1 cells were evaluated for activation markers, microbicidal molecules, cytokine gene expression, Toll-like receptors, and commitment factors. Results showed that sleep restriction minimally influenced activation marker expression in B-1 cells across infection models. However, under sleep restriction, microbicidal molecule production increased in P. brasiliensis (NO) and C. albicans (ROS) infections. Gene expression revealed elevated TLR-2 and cytokines (IL-12, IL-6, TNF-α) in sleep-restricted animals. Lineage commitment analysis indicated myeloid polarization in sleep-restricted B-1 cells for all infection models. These findings underscore the impact of sleep restriction on B-1 cell activation and differentiation, with outcomes varying based on infection type. This study highlights the importance of investigating immunological changes resulting from sleep restriction.

Aims: To study the effects of pyridoxal phosphate (PLP) on oxidative stress in isolated pancreatic acinar cells. We have previously shown that PLP has cytoprotective and insulinotropic effects on mice islet cells in vivo and in vitro studies. Main methods: Acinar cells were isolated from three months old WNIN male rats and were cultured in vitro for a period of 24 h in CO₂ incubator. Later the cells were divided into four groups as untreated (group 1), H₂O₂ treatment (group 2)_, PLP treatment (group 3) and PLP followed by H₂O₂ treatment (group 4). Cell viability was confirmed using MTT assays, oxidative stress levels were measured with ROS assay, change in different protein levels were recorded by flow cytometry. The acinar cells insulin secretion assay was performed with ELISA. The amylase protein expression was assessed using confocal microscopy. Key findings: The cell viability of acinar cell in group 1 was considered as 100%, while in group 2 it was reduced to 82% due to H₂O₂ effect, and in group 3 (99.8%) and group 4 (99.5%) were near to group 1 due to the cytoprotective effect of PLP. The ROS levels were increased by 1.47 folds in group 2, while PLP decreased to 1.02 fold in group 4, which was comparable with the changes in group 1. Beneficial effects of PLP were also observed from the increased expression levels of acinar cells are amylase -2.01, neurogenin-3-9.51, PDX-1- 23.6 and insulin-13.5 in group 3 compared to group 1. The specificity of PLP’s response was confirmed by amino oxy acetic acid (AOAA), a specific PLP inhibitor. The increased amylase protein localization with PLP was confirmed by confocal microscopy. Insulin secretion efficiency of acinar cells was observed to be 6.13 folds higher at basal and 24.63 fold higher at stimulated levels in group3 compared to group1. Significance: Our results advocate the antioxidant and cytoprotective effects of PLP on the pancreatic acinar cell along with increased pancreatic marker expressions of amylase,PDX-1, neurogenin-3 and insulin proteins.

Immune checkpoints are a set of molecules dysregulated in several human and canine cancers and aberrations of the PD-1/PD-L1 axis are often correlated with a worse prognosis. To gain insight into the role of immune checkpoints in canine diffuse large B-cell lymphoma (cDLBCL), we investigated PD-L1, PD-1 and CD8A expression by RNAscope. Results were correlated with several clinico-pathological features, including treatment, Ki67 index and outcome. A total of 33 dogs treated with CHOP (n=12) or CHOP plus APAVAC (n= 21) were included. PD-L1 signal was diffusely distributed among neoplastic cells, whereas PD-1 and CD8A were localized in tumor infiltrating lymphocytes. However, PD-1 mRNA was also retrieved in tumor cells. An association between PD-L1 and PD-1 scores was identified and a higher risk of relapse and lymphoma-related death was found in dogs treated with chemotherapy alone and dogs with higher PD-L1 and PD-1 scores. The correlation between PD-L1 and PD-1 is in line with the mechanism of immune checkpoints in cancers, where neoplastic cells overexpress PD-L1 that, in turn, binds PD-1 receptors in activated TIL. We also found that Ki67 index was significantly increased in dogs with the highest PD-L1 and PD-1 scores, indirectly suggesting a role in promoting tumor proliferation. Finally, even if the biological consequence of PD-1+ tumor cells is unknown, our findings suggest that PD-1 intrinsic expression in cDLBCL might contribute to tumor growth escaping adaptive immunity.

Background/methods: For mechanistic studies, in vitro human B cell differentiation and generation of plasma cells are invaluable techniques. However, the heterogeneity of both T cell-dependent (TD) and T cell-independent (TI) stimuli and the disparity of culture conditions used in existing protocols makes interpretation of results challenging. The aim of the present study was to achieve the most optimal B cell differentiation conditions using isolated CD19⁺ B cells and PBMC cultures. We addressed multiple seeding densities, different durations of culturing and various combinations of TD stimuli and TI stimuli including B cell receptor (BCR) triggering. B cell expansion, proliferation and differentiation was analyzed after 6 and 9 days by measuring B cell proliferation and expansion, plasmablast and plasma cell formation and immunoglobulin (Ig) secretion. In addition, these conditions were extrapolated using cryopreserved cells and differentiation potential was compared. Results: This study demonstrates improved differentiation efficiency after 9 days of culturing for both B cell and PBMC cultures using CD40L and IL-21 as TD stimuli and 6 days for CpG and IL-2 as TI stimuli. We arrived at optimized protocols requiring 2500 and 25.000 B cells per culture well for TD and TI assays, respectively. The results of the PBMC cultures were highly comparable to the B cell cultures, which allows dismissal of additional B cell isolation steps prior to culturing. In these optimized TD conditions, the addition of anti-BCR showed little effect on phenotypic B cell differentiation, however it interferes with Ig secretion measurements. Addition of IL-4 to the TD stimuli showed significantly lower Ig secretion. The addition of BAFF to optimized TI conditions showed enhanced B cell differentiation and Ig secretion in B cell but not in PBMC cultures. With this approach, efficient B cell differentiation and Ig secretion was accomplished when starting from fresh or cryopreserved samples. Conclusion: Our methodology demonstrates optimized TD and TI stimulation protocols for more indepth analysis of B cell differentiation in primary human B cell and PBMC cultures while requiring low amounts of B cells, making them ideally suited for future clinical and research studies on B cell differentiation of patient samples from different cohorts of B cell-mediated diseases.

Dengue is a viral mosquito-borne disease that rapidly spreads in tropical and subtropical countries, including the Philippines. One of its most distinguishing characteristics is the ability of the Dengue Virus (DENV) to easily surpass the innate responses of the body, thus activating B cells of the adaptive immunity to produce virus-specific antibodies. Moreover, Dengvaxia® is the only licensed vaccine for DENV, but recent studies showed that seronegative individuals become prone to increased disease severity and hospitalization. Owing to this limitation of the dengue vaccine, this study determined and compared consensus and unique B cell epitopes among each DENV (1-4) Philippine isolate to identify potential areas of interest for future vaccine studies and therapeutic developments. An in silico-based epitope prediction of forty (40) DENV 1-4 strains, each serotype represented by ten (10) sequences from The National Center for Biotechnology Information (NCBI), was conducted using Kolaskar and Tongaonkar antigenicity, Emini surface accessibility, and Parker hydrophilicity prediction in Immune Epitope Database (IEDB). Results showed that five (5) epitopes were consensus for DENV-1 with no detected unique epitope, one (1) consensus epitope for DENV-2 with two (2) unique epitopes, one (1) consensus epitope for DENV-3 plus two (2) unique epitopes, and two (2) consensus epitopes and one (1) unique epitope for DENV-4. The findings of this study would contribute to determining potential vaccine and diagnostic marker candidates for further research studies and immunological applications against DENV (1-4) Philippine isolates.

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.

Splenic micronodular T-cell/histiocyte-rich large B-cell lymphoma is possibly derived from nodal T-cell/histiocyte-rich large B-cell lymphoma; however, a transition between the nodal and splenic micronodular forms has not been described to date. Of note, the only lymph nodes to be involved in association with the splenic micronodular pattern of the disease are the splenic hilar lymph nodes, and that, with partial involvement only. Kan et al, in their series of articles, have raised the possibility that corticosteroids, when prescribed prior to splenectomy, cause histopathological and functional modulations (apoptosis, necrosis, tissue shrinkage), which modify or even obscure the diagnostic morphological features. The indications for glucocorticoid therapy are either related to the suspected lymphoma, or else to other comorbidities, like asthma and autoimmune disorders. We propose that patients with the splenic, rather than nodal subset of the disease are likely to have been prescribed corticosteroids prior to histopathologic examination of the involved tissue, causing disparate morphologies in the spleen. Apoptosis, as induced by corticosteroids, is hypothesized as the major mechanism initiating the histopa-thological and functional changes in the splenic micronodular variant of our patients.

The proliferation and survival signals emanating from the B-cell receptor (BCR) constitute a crucial aspect of mature lymphocyte’s life. Dysregulated BCR signaling is considered a potent contributor to tumor survival in different subtypes of B cell non-Hodgkin lymphomas (B-NHLs). In the last decade, emergence of BCR-associated kinases as rational therapeutic targets has led to the development and approval of several small molecule inhibitors targeting either Bruton's tyrosine kinase (BTK), spleen tyrosine kinase (SYK), or phosphatidylinositol 3 kinase (PI3K), offering alternative treatment options to standard chemoimmunotherapy, and making some of these drugs valuable assets in the anti-lymphoma armamentarium. Despite their initial effectiveness, these precision medicine strategies are limited by primary resistance in aggressive B-cell lymphoma like diffuse large B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL), especially in the case of first generation BTK inhibitors. In these patients, BCR-targeting drugs often fail to produce durable responses, and nearly all cases eventually progress with a dismal outcome, due to secondary resistance. This review will discuss our current understanding of the role of antigen-dependent and antigen-independent BCR signaling in DLBCL and MCL and will cover both approved inhibitors and investigational molecules being evaluated in early preclinical studies. We will discuss how the mechanisms of action of these molecules, and their off/on-target effects can influence their effectiveness and lead to toxicity, and how our actual knowledge supports the development of more specific inhibitors and new, rationally based, combination therapies, for the management of MCL and DLBCL patients.

Chronic lymphocytic leukemia (CLL) is characterized by dysfunctional B cells. Immune checkpoint molecules such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and programmed death-1 (PD-1) are upregulated in patients with CLL and may correlate with prognostic markers such as beta-2 microglobulin (B2M). The aim of this study was to evaluate the levels of immune checkpoints on B-cell subsets and to further correlate them with B2M levels in patients with CLL. We recruited 21 patients with CLL and 12 controls. B cell subsets and the levels of immune checkpoint expression were determined using conventional multi-color flowcytometry. Basal levels of B2M in patients with CLL were measured using an Enzyme-linked immunosorbent assay. Patients with CLL had increased levels of activated B cells when compared to the control group, p< 0.001. The expression of PD-1 and CTLA-4 were increased on activated B cells and memory B cells, p< 0.05. There were no associations between B2M levels and the measured immune checkpoints on B cell subsets, after adjusting for sex and age. African patients with CLL express elevated levels of PD-1 and CTLA-4 immune checkpoints on activated and memory B cell subsets. However, there is no correlation between these immune checkpoint expressions and B2M levels.

Background: Diffuse large B-cell lymphoma is the most common type of non-Hodgkin's lymphoma. Due to the variety of clinical manifestations, accurate diagnosis can be challenging. Here, we present a case of DLBCL that was initially misdiagnosed as a hematoma, highlighting the importance of considering malignancy in the setting of non-responsive soft tissue swelling. Methods: A 76-year-old man presented to the emergency department with right periorbital swelling and ecchymosis. The symptoms first appeared after an injury and did not resolve spontaneously. The patient was taking warfarin for atrial fibrillation. A facial bone CT revealed a large irregular homogeneous mass. An extraconal hematoma was suspected based on clinical history and radiologic findings. Incision and drainage were performed, resulting in evacuation of old blood. Despite continued drainage, the patient's symptoms worsened. A comparison with the previous CT revealed an increase in the size of the lesion. As a result, a surgical excisional biopsy was performed. Results: Biopsy of the mass showed a diffuse infiltrate of lymphocytes around the tissue. The diagnosis of diffuse large B-cell lymphoma was confirmed pathologically. The patient was referred to hematology for further management. Conclusions: DLBCL is a rare disease associated with a poor prognosis. Due to the previous injury and the use of anticoagulants, the patient was initially misdiagnosed as having a hematoma. This delayed the prompt diagnosis and affected the timing of treatment initiation. We must be aware that a malignant mass is a potential cause of unresponsive soft tissue swelling.

Food allergies are a growing public health concern worldwide, especially in children and young adults. Allergen-specific IgE plays a central role in the pathogenesis of food allergies, but their titers poorly correlate with allergy development. Host immune systems yield allergen-specific immunoglobulin (Ig)A, IgE and IgG subclasses with low or high affinities and differential Fc N-glycosylation patterns that can affect the allergic reaction to food in multiple ways. High affinity IgE is required to induce strong mast cell activation eventually leading to allergic anaphylaxis while low affinity IgE can even inhibit the development of clinically relevant allergic symptoms. IgA and IgG antibodies can inhibit IgE-mediated mast cell activation through various mechanisms, thereby protecting IgE-positive individuals from allergy development. The production of IgE and IgG with differential allergenic potential seems to be affected by the signaling strength of individual B cell receptors, and by cytokines from T cells. This review provides an overview of the diversity of the B cell response and the diverse roles of antibodies in food allergy.

Since the first description of SARS-CoV-2 in China in 2019, thousands of variants have emerged worldwide. For some of them, the constellation of mutations caused changes in virus biology, pathogenicity, infectivityity and transmissibility resulting in dissemination throughout the world. Gamma variant (P.1) differs from SARS-CoV-2 Wuhan strain (B.1) by 12 amino acids in the Spike (S) protein, and presented mutations related to greater affinity for the receptor angioten-sin-converting enzyme 2 (ACE-2) and/or immune escape. The Gamma variant and subvariants were responsible for the second wave of COVID-19 in the Brazilian city of Manaus, characterized by high mortality and rapid transmission. The ability of variants to induce cytokine production may be closely related to their pathogenicity. Herein we observed that there was no significant difference in the quantity of cytokines among macrophages or neutrophils infected with P.1 and B.1 strains. Also, no significant difference was observed in the absolute number of macrophages and neutrophils infected with these variants. Furthermore, no evidence of SARS-CoV-2 replication was observed in macrophages when infected by the two analyzed variants. Our findings suggest that the difference in the epidemiological outcome observed during the P.1 variant spread when compared to B.1, it is not explained by differences in the quantity of cytokines and absolute number of macrophages or neutrophils. Through bioinformatics analysis of the S protein, we observed that the physicochemical differences between the variants and subvariants of P.1, probably refer to the degree of infectivity, due to the impact caused in the recognition of antibodies and receptor af-finity

Pituitary adenylate cyclase-activating polypeptide receptor 1 (PAC1-R) has a higher affinity for pituitary adenylate cyclase- activating polypeptide (PACAP) than for vasoactive intestinal peptide (VIP). PACAP exerts its neuroprotective effects through PAC1-R. PAC1-R has become a target for drug development, and we targeted PAC1-R to screen small positive allosteric modulator 1 (SPAM1). SPAM1 has a neuroprotective effects, and in the D-galactose (D-gal) mouse model, SPAM1 improved the structure of the hippocampal dentate gyrus and restored the number of neurons. Compared with D-gal model mice, SPAM1 treatment upregulated the expression of Sirtuin 6 (SIRT6) and Lamin B1, and downregulated the expression of YinYang 1 (YY1) and p16. Consistent with this, the same results were obtained in senescent RGC-5 cells. Meanwhile, SPAM1 was also found to up-regulate SIRT6 and down-regulate YY1 expression in normal RGC-5 cells. Then, we further explored the mechanism of SPAM1 neuroprotection by whole transcriptome sequencing and proteomic analysis, and found that SPAM is involved in the longevity regulating pathway, and confirmed the up-regulation of neurofilament light polypeptide and neurofilament medium polypeptide by western blot, thus laying a pharmacological foundation for SPAM1 as a therapeutic agent for neurodegenerative diseases. polypeptide and neurofilament medium polypeptide. Thus, it lays a pharmacological foundation for SPAM1 as a therapeutic drug for neurodegenerative diseases.

Lamin A, B and C, the nuclear intermediate-filament proteins, play a role in epigenetic regulation. While Lamin B is expressed in all nucleated cells studied, Lamin A/C are transcribed in most somatic cell types except mature B lymphocytes. Since Epstein-Barr virus (EBV), a human gammaherpesvirus, is associated with tumorigenic processes and is known to alter the epigenotype of its host cells, we studied the expression of the LMNA gene and its epigenetic marks in EBV-carrying human lymphoid cell lines. We observed a high lamin A/C mRNA and protein expression in EBV-immortalized lymphoblastoid cell lines (LCLs) and in group III Burkitt lymphoma (BL) lines where hypomethylated first exons were observed with activating histone marks. In most cell lines with low promoter activity a highly methylated first exon could be detected. Our data showed that methylation of the first exon of LMNA was associated with the downregulation of LMNA expression whereas euchromatic histone marks were enriched at active LMNA promoters in EBV-immortalized LCLs. These data suggest a role for viral latency products to activate LMNAp in EBV-infected latency type III B cells in vitro. Expression of lamin A/C may contribute to the establishment of activated B cell phenotype that needs further explorations.

Chimeric Antigen Receptor (CAR) T-cell therapy has demonstrated high rates of response in recurrent B-cell Acute Lymphoblastic Leukemia in children and young adults. Despite this success, a fraction of patients experience relapse after treatment. Relapse is often preceded by recovery of healthy B cells, which suggests loss or dysfunction of CAR T cells in bone marrow. This site is harder to access, and thus is not monitored as frequently as peripheral blood. Understanding the interplay between B cells, leukemic cells and CAR T cells in bone marrow is paramount in ascertaining the causes of lack of response. In this paper, we put forward a mathematical model representing the interaction between constantly renewing B cells, CAR T cells and leukemic cells in the bone marrow. Our model accounts for the maturation dynamics of B cells and incorporates effector and memory CAR T cells. The model provides a plausible description of the dynamics of the various cellular compartments in bone marrow after CAR T infusion. After exploration of the parameter space, we found that the dynamics of CAR T product and disease were independent of the dose injected, initial B-cell load and tumor burden. We also show theoretically the importance of CAR T product attributes in determining therapy outcome, and have studied a variety of possible response scenarios, including second dosage schemes. We conclude by setting out ideas for the refinement of the model.

Non-coding RNAs (ncRNAs) comprise a diverse class of non-protein coding transcripts that regulate critical cellular processes associated with cancer. Advances in RNA-sequencing (RNA-Seq) have led to the characterization of non-coding RNA expression across different types of human cancers. Through comprehensive RNA-Seq profiling, a growing number of studies demonstrate that ncRNAs, including long non-coding RNA (lncRNAs) and microRNAs (miRNA), play central roles in progenitor B-cell Acute Lymphoblastic Leukemia (B-ALL) pathogenesis. Furthermore, due to their central roles in cellular homeostasis and their potential as biomarkers, the study of ncRNAs continues to provide new insight into the molecular mechanisms of B-ALL. This article reviews the ncRNA signatures reported for all B-ALL subtypes, focusing on technological developments in transcriptome profiling and recently discovered examples of ncRNAs with biologic and therapeutic relevance in B-ALL.

B lymphocyte development has two DNA recombination processes: V(D)J recombination of the immunoglobulin (Igh) gene variable region and class switching of the Igh constant regions from IgM to IgG, IgA, or IgE. V(D)J recombination is required for successful maturation of B cells from pro-B to pre-B to immature-B and then to mature B cells in the bone marrow. CSR occurs outside of the bone marrow when mature B cells migrate to peripheral lymphoid organs, such as spleen and lymph nodes. Both V(D)J recombination and CSR depend on an “open chromatin” state that makes DNA accessible to specific enzymes, recombination activating gene (RAG), and activation-induced cytidine deaminase (AID). Acetyltransferases GCN5 and PCAF possess redundant functions acetylating histone H3 lysine 9 (H3K9). Here, we generated a mouse model that lacks both GCN5 and PCAF in B cells. We found that double-deficient mice possess low levels of mature B cells in the bone marrow and peripheral organs, an accumulation of pro-B cells in bone marrow, and reduced CSR levels. We conclude that both GCN5 and PCAF are required for B cell development in vivo.

G‐quadruplexes (G4) are structures formed at the ends of the telomere, these are rich in guanines and were stabilized by molecules that bind to specific sites. TMPyP4 and Thymoquinone (TQ) are small molecules that bind to the G4, they have drawn attention because of their role as telomerase inhibitors. The aim of this study was to evaluate the effects of telomerase inhibitors on cellular proliferation, senescence, and death. Two cell lines LC‐HK2 (NSCLC) and RPE‐1 were treated with TMPyP4 (5μM) and TQ (10μM). Both inhibitors were effective in decreasing telomerase activity. TMPyP4 increased the percentage of cells with membrane damage associated with cell death and decreased the frequency of cells in the S‐phase. TMPyP4 changed the cell adhesion ability and modified the pattern of focal adhesion. TQ acted in a dose‐dependent manner, increasing the frequency of senescent cells, and inducing cell cycle arrest in the G1. In conclusion, the effects of both drugs on LC-HK2 and RPE-1 cell lines were different although both are telomerase inhibitors, because TMPyP4 decreased proteins of cell adhesion and TQ induces a decrease in cell viability.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive and biologically heterogeneous disease. Approximately 40% of patients with DLBCL will experience disease relapse or will be refractory to first line chemoimmunotherapy. In recent years there have been several new therapeutic agents approved for the treatment of relapsed/refractory (R/R) DLBCL. These agents include anti-CD19 chimeric antigen receptor T-cell therapies (CAR T-cells) and monoclonal antibodies as polatuzumab and tafasitamab. Nevertheless, despite the high efficacy of all these new therapies, there are still patients who do not respond or relapse, representing an unmet clinical need. This review describes new promising therapies that are under investigation to treat R/R DLBCL.

Canine lymphoma (CL) is one of the most common malignant tumors in dogs. The cause of CL remains unclear. Genetic mutations that have been suggested as possible causes of CL are not fully understood. Whole-exome sequencing (WES) is a time- and cost-effective method for detecting genetic variants targeting only protein-coding regions (exons) that are part of the entire genome region. A total of eight patients with B-cell lymphomas were recruited, and WES analysis was performed on whole blood and lymph node aspirate samples from each patient. A total of 17 somatic variants (GOLIM4, ITM2B, STN1, UNC79, PLEKHG4, BRF1, ENSCAFG00845007156, SEMA6B, DSC1, TNFAIP1, MYLK3, WAPL, ADORA2B, LOXHD1, GP6, AZIN1, and NCSTN) with moderate to high impact were identified by WES analysis. Through a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of 17 genes with somatic mutations, a total of 16 pathways were identified. Overall, the somatic mutations identified in this study suggest novel candidate mutations for CL, and further studies are needed to confirm the role of these mutations.

Immune checkpoint inhibitors (ICIs), including anti-programmed cell death 1 ligand 1 (PD-L1) antibodies, are significantly changing treatment strategies for human malignant diseases including oral cancer. Cancer cells usually escape from immune system and acquire proliferative capacity and invasive/metastatic potential. We have focused on the two immune checkpoints, PD-1/PD-L1 and CD47/SIRPα in the tumor microenvironment of oral squamous cell carcinoma (OSCC), and performed a retrospective analysis of the expression of seven immune-related factors (PD-L1, PD-1, CD4, CD8, CD47, CD56 and CD11c), and examined their correlation with clinicopathological status. As a result, there were no significant findings relating seven immune-related factors and several clinicopathological statuses. However, the immune-checkpoint related factors (PD-1, PD-L1, CD47) were highly expressed in non-keratinized epithelium-originated tumors when compared to those in keratinized epithelium-originated tumors. It is of interest that immunoediting via immune checkpoint-related factors was facilitated in non-keratinized sites. Several researchers reported that keratinization of oral mucosal epithelia affected the immune response, but our present finding is the first study to show a difference of the tumor immunity in the originating-epithelium of OSCC, keratinized or non-keratinized. Tumor immunity, an immune escape status of OSCC might be different in the originating-epithelium, keratinized or non-keratinized.

Clonal evolution and cellular plasticity are the genetic and non-genetic driving forces of tumor heterogeneity that in turn determines the tumor cell response towards therapeutic drugs. Sever-al lines of evidence suggest that therapeutic interventions foster the selection of drug resistant neural crest stem-like cells (NCSCs) that establish minimal residual disease (MRD) in melano-ma. Here we established a dual reporter system enabling the tracking of NGFR expression and mRNA stability, providing insights into the maintenance of NCSC-states. We observed that the transcriptional reporter that contained a 1kb fragment of the human NGFR promoter was acti-vated only in a minor subset (0.72±0.49%, range 0.3-1.5) and ~2-4% of A375 melanoma cells re-vealed stable NGFR mRNA. The combination of both reporters provided insights into pheno-type switching and revealed that both cellular subsets gave rise to cellular heterogeneity. Moreover, whole transcriptome profiling and gene set enrichment analysis (GSEA) of the minor cellular subset revealed hypoxia-associated genes serving as potential drivers of a NGFR-associated phenotype switching in vitro and in relapsed, post-BRAF inhibitor treated tu-mors. Concordantly, we observed that the minor cellular subset increased in response to dabrafenib over time. In summary, our reporter-based approach provided insights into plastici-ty and identified a cellular subset that might be responsible for the establishment of MRD in melanoma.

Growing evidence has revealed the implication of germline variation in cancer predisposition and prognostication. Here, we describe an analysis of putatively disruptive rare variants across the genomes of 726 patients with B-cell lymphoid neoplasms. We discovered a significant enrichment of 26 genes in germline protein truncating variants (PTVs), affecting cell signaling (MET, JAK2, ANGPT2), energy metabolism (ACO1) and nucleic acid metabolism and repair pathways (NT5E, DCK). Interestingly, some of these variants were restricted to either chronic lymphocytic leukemia (CLL) (i.e., ANGPT2 and AKR1C3) or B-cell lymphoma cases (PNMT, TPT1 and IGHMBP2). Additionally, we detected 1,675 likely disrupting variants in genes associated with cancer, of which 44.75% were novel events and 7.88% were PTVs. Among these, the most frequently affected genes were ATM, BIRC6, CLTCL1A and TSC2. Homozygous or compound heterozygous variants were detected in 28 cases; and coexisting somatic events were observed in 17 patients, some of which affected key lymphoma drivers such as ATM, KMT2D and MYC. Finally, we observed that variants in the helicase gene WRN were independently associated with shorter survival in CLL. Our study results support an important role for rare germline variation in the pathogenesis, clinical presentation and disease outcome of B-cell lymphoid neoplasms.

Acidic ostreolysin A/pleurotolysin B (OlyA/PlyB, formerly known as ostreolysin (Oly), and basic 20 kDa equinatoxins (EqTs) are cytolytic proteins isolated from the edible mushroom Pleurotus ostreatus and the sea anemone Actinia equina, respectively. Both toxins, although from different sources, share many similar biological activities: (i) colloid-osmotic shock by forming pores in cellular and artificial membranes enriched in cholesterol and sphingomyelin; (ii) increased vascular endothelial wall permeability in vivo and perivascular oedema; (iii) dose-dependent contraction of coronary vessels; (iv) haemolysis with pronounced hyperkalaemia in vivo; (v) bradycardia, myocardial ischemia and ventricular extrasystoles accompanied by progressive fall of arterial blood pressure and respiratory arrest in rodents. Both types of toxins are haemolytic within nanomolar range concentrations, and it seems that hyperkalaemia plays an important role in toxin cardiotoxicity. However, it was observed that the haemolytically more active EqT III is less toxic than EqT I, the most toxic and least haemolytic EqT. In mice, EqT II is more than 30 times more toxic than OlyA/PlyB when applied intravenously. These observations imply that haemolysis with hyperkalaemia is not the sole cause of the lethal activity of both toxins. Additional mechanisms responsible for lethal action of the two toxins are direct effects on heart, coronary vasoconstriction and related myocardial hypoxia. In this review, we appraise the pathophysiological mechanisms related to the chemical structure of OlyA/PlyB and EqTs, as well as their toxicity.

Background: The intrauterine and early extrauterine development represents a “window of opportunity” in the immunological development. The underlying mechanisms are still poorly understood. The aim of this study was to provide reference values B cell subpopulations in cord blood of term newborns, juveniles and in adults to find the spectrum of their physiological age-related variation. Methods: In this study, we used flow cytometry to evaluate human B lymphocytes and subpopulations in cord blood (n = 10), in peripheral blood from healthy juveniles aged 1 to 17 years (n=20) and from donors aged 24 to 62 years (n = 10). Results: Our findings showed increasing frequencies of IgM memory B cells, class-switched memory B cells, marginal zone B cells and plasmablasts, from cord blood to peripheral blood of juveniles and adults. In con-trast, the percentage of naïve B cells was higher in newborns than in juveniles and adults. The frequencies of were similar in cord blood and peripheral blood of adults. Interestingly, transitional B cells frequencies were similar in cord blood and adults but significantly lower in juveniles. Conclusions: The frequencies of circulating B cell subpopulation are subject to considerable changes during on-togeny, reflecting overlying effects of maturation and of the acquisition of an adaptive immune memory.

Cathepsins (Caths) are lysosomal proteases that participate in various physiological and pathological processes. Accumulating evidence suggests that cathepsins play a multifaceted role in radiotherapy response. Firstly, cathepsins contribute to the remodelling of the tumor microenvironment by degrading extracellular matrix components, promoting angiogenesis, and facilitating tumor invasion and metastasis. Their proteolytic activity influences the tumor's response to radiation by affecting oxygenation, nutrient availability, and immune cell infiltration within the tumor microenvironment. Furthermore, cathepsins have been implicated in DNA damage repair processes, including non-homologous end joining and homologous recombination. Cathepsin-mediated DNA repair mechanisms can promote radioresistance in cancer cells, limiting the efficacy of radiotherapy. Additionally, cathepsins have been associated with the activation of prosurvival signalling pathways, such as PI3K/Akt and NF-κB, which can confer resistance to radiation-induced cell death. Radiotherapy plays a crucial role in the treatment of various malignancies. However, the effectiveness of radiotherapy can be limited by intrinsic or acquired resistance mechanisms in cancer cells. In recent years, there has been growing interest in understanding the role of cathepsins, a family of proteases, in modulating the response of tumor cells to radiotherapy. In this study, we shed light on the regulation and expression of cathepsin B in colon carcinoma cell line (Caco-2) before and after exposure to radiation were investigated. Radiation-induced upregulation of cathepsin B in dose-independent manner. Proteolytic inhibition of cathepsin B by cathepsin B specific inhibitor CA074 has increased the cytotoxic effect of ionizing irradiation in caco-2 cells. Similar results were also obtained after cathepsin B knockout by CRISPR CAS9. These results showed that cathepsin B could contribute to ionizing radiation resistance and abolishing of cathepsin B either by inhibition its proteolytic activity or expression has increased in caco-2 cells susceptibility to ionizing irradiation.

Diffuse large B cell lymphoma (DLBCL), the most frequent type of non-Hodgkin lymphoma in adulthood, remains challenging clinical issues. Despite enhanced remission rates can be achieved, there are one-third of patients who will not respond to current treatment or will relapse with resistant disease, necessitating ongoing efforts on effective treatment strategies and agents. The vacuole H+-ATPase inhibitor bafilomycin A1 is broadly used to block late stage of autophagy flux at high concentration. In this study, we show that, to our surprise, bafilomycin A1 effectively inhibited and killed DLBCL cells at nanomolar concentrations (5nM). Bafilomycin A1 targeted cell cycle regulators cyclin D1 and cyclin E2 to induce cell cycle arrest in G0/G1 phase. Meanwhile, it induced caspase-dependent apoptosis with concomitant cleaved caspase-3 and Parp. Furthermore, we found that bafilomycin A1 inhibited autophagy flux at both early and late stages of the autophagy flux through activating ERK and mammalian target of rapamycin signaling, as well as by inhibiting the degradation of autolysosomes. We speculated that bafilomycin A1 as autophagy inhibitor might enhance the effect of DLBCL chemotherapeutic drug rituximab. Accordingly, our results provided evidence that the combination of bafilomycin A1 with rituximab enhanced the inhibition of DLBCL cells notably. Taken together, our data suggest that bafilomycin A1 may be a promising candidate drug in the therapy of diffuse large B cell lymphoma.

Cutaneous T cell lymphomas (CTCLs) are caused by malignant clonal proliferation of skin-tropic T cells. Most patients have an indolent disease course managed with skin-directed therapies, while some entities, or in advanced stages of disease, have aggressive progression and poor survival. To efficiently treat CTCL consistent entity markers are needed to prevent the delay in diagnosis and to provide disease specific treatment to patients. Recently, the introduction of high throughput parallel sequencing methods resulted in identification of numerous genetic and ep-igenetic alterations affecting the transcriptome of CTCL cells. In this review, most relevant genes, including TOX, CADM1, PLS3, DNM3, CXCL13, PD-1, BCL11B and TMEM244 are reported that are specifically expressed in CTCL. Upon verification their specificity and sensitivity in larger studies, their altered expression will be able to be recognized as novel biomarkers, that can im-prove the early diagnosis of CTCL.

Cell junction that is usually associated with dynamic cytoskeletons is essential for a wide range of cellular activities such as cell migration, cell communication, barrier function and signal transduction. Real-time observation of cell junction facilitates understanding of the mechanisms by which cell junction regulate relevant cellular activities. In this study, we examined the binding capacity of a modified tridecapeptide from Cx43 to cell junction protein ZO-1 and sought to create a fluorescent peptide that could be used to label cell junction. To introduce the fluorescent peptide into the cells, a cell-penetrating peptide was linked to the modified tridecapeptide and synthesized. The binding of the modified tridecapeptide was tested using pull down and immunoprecipitation assay. The ability of the peptide to label cell junction was assessed by adding it to fixed or cultured Caco-2 cells. Testing assay revealed that the Cx43-derived peptide can bind to ZO-1. Besides, the peptide was able to label cell junction of fixed cell, although no obvious cell junction labeling was observed clearly in living cells, probably due to the inadequate affinity. These results indicate that a peptide-based strategy for labeling cell junction may be feasible, and efforts to improve its affinity are warranted in the future.

Vascular cell adhesion molecule-1 (VCAM-1) is closely associated with tumor progression and metastasis. However, the relevance and role of VCAM-1 in lung cancer have not been clearly elucidated. In this study, we found that VCAM-1 was highly overexpressed in lung cancer tissue compared with that of normal lung, and high VCAM-1 expression correlated with poor survival of lung cancer patients. VCAM-1 knockdown reduced invasion in A549 human lung cancer cells, and competitive blocking experiments targeting the Ig-like domain 6 of VCAM-1 (VCAM-1-D6) demonstrated that the VCAM-1-D6 domain was critical for VCAM-1-mediated A549 cell invasion. Next, we developed a human monoclonal antibody specific to human and mouse VCAM-1-D6 (VCAM-1-D6 huMab), which was isolated from a human synthetic antibody library using phage display technology. Finally, we showed that VCAM-1-D6 huMab had a nanomolar affinity for VCAM-1-D6 and that it potently suppressed invasion in A549 and NCI-H1299 lung cancer cell lines. Taken together, these results suggest that VCAM-1-D6 is a novel therapeutic target in VCAM-1-mediated lung cancer invasion and that our newly developed VCAM-1-D6 huMab will be a useful tool for inhibiting VCAM-1-expressing lung cancer cell invasion.

The most common type of non-Hodgkin lymphoma in adults is diffuse large B-cell (DLBCL). There is a historical unmet need for more effective therapies in the 2nd and 3rd line setting. Emerging immunochemotherapies have shown activity in small studies of heavily pre-treated patients with prolonged remissions achieved in some patients. Anti-CD19 CAR (chimeric antigen receptor) T cells are potentially curative in the 3rd line and beyond setting and are under investigation in earlier lines of therapy.1 Antibody-drug conjugates (ADC’s) such as polatuzumab vedotin targeting the pan-B-cell marker CD79b has proven effectiveness in multiply-relapsed DLBCL patients. Tafasitamab (MOR208) is an anti-CD19 monoclonal antibody producing prolonged remissions when combined with Lenalidomide in patients who were not candidates for salvage chemotherapy or autologous stem cell transplant. Selinexor, an oral, small-molecule selective inhibitor of XPO1-mediated nuclear export (SINE), demonstrated prolonged activity against heavily-pretreated DLBCL without cumulative toxicity and is being investigated as part of an oral, chemotherapy-free regimen for relapsed aggressive lymphoma. This article reviews current strategies and novel therapies for relapsed/refractory DLBCL.

IQGAP1 is a multi-functional scaffold protein. However, its role in B cell development and function is not known. Here, we demonstrate Iqgap1mice contained significantly increased numbers of B220+ B, B220+IgM pro/pre-B, and B220+IgM+ immature-B cells in the bone marrow. Here, we show IQGAP1 as the essential scaffold that regulates early B cell development and function. Iqgap1mice contained significantly increased numbers of B220+ B, B220+IgM pro/pre-B, and B220+IgM+ immature-B cells in the bone marrow. New-forming and follicular B cell numbers were increased, while the marginal zone B cell numbers were significantly reduced in the spleen of the Iqgap1mice. Lack of IQGAP1 reduced T-dependent and T-independent humoral responses. Mechanistically, the lack of IQGAP1 considerably decreased the phosphorylation of Mek1/2, Erk1/2, and Jnk1/2. B cells from Iqgap1mice failed to suppress IL-7R-mediated activation of Stat5a/b, an essential step for cell-cycle exit and initiate light-chain recombination, leading to a reduction in RS rearrangement frequency. Lack of IQGAP1 resulted in a significant reduction of IRF4 and IRF8 in BM B cells. Our study provides the first evidence that IQGAP1-based signalosome is essential for the development and functions of B cells.

Adult T-cell leukemia (ATL) is an aggressive hematologic disease caused by human T-cell leukemia virus type 1 (HTLV-1) infection. Various animal models of HTLV-1 infection/ATL have been established to elucidate the pathogenesis of ATL and develop appropriate treatments. For analyses employing murine models, transgenic and immunodeficient mice are used because of the low infectivity of HTLV-1 in mice. HTLV-1 Tax and HBZ transgenic mice spontaneously develop tumors, and roles for both Tax and HBZ in cell transformation and tumor growth have been established. Severely immunodeficient mice were able to engraft ATL cell lines and have been used in preclinical studies of candidate molecules for the treatment of ATL. HTLV-1-infected humanized mice with an established human immune system are a suitable model to characterize cells in the early stages of HTLV-1 infection. This review outlines the characteristics of mouse models of HTLV-1 infection/ATL and describes progress made in elucidating the pathogenesis of ATL and developing related therapies using these mice.

Cardiac hypertrophy is an important and independent risk factor for the development of heart failure. To better understand the mechanisms and regulatory pathways involved in cardiac hypertrophy, there is a need for improved in vitro models. In this study, we investigated how hypertrophic stimulation affected human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs). The cells were stimulated with endothelin-1 (ET-1) for 8, 24, 48, 72, or 96h. Parameters including cell size, ANP-, proBNP-, and lactate concentration were analyzed. Moreover, transcriptional profiling using RNA-sequencing was performed to identify differentially expressed genes following ET-1 stimulation. The results show that the CMs increase in size by approximately 13% when exposed to ET-1 in parallel to increases in ANP and proBNP protein and mRNA levels. Furthermore, the lactate concentration in the media was significantly increased indicating that the CMs consume more glucose, a hallmark of cardiac hypertrophy. Using RNA-seq, a hypertrophic gene expression pattern was also observed in the stimulated CMs. Taken together, these results show that hiPSC-derived CMs stimulated with ET-1 display a hypertrophic response. The results from this study also provide new molecular insights about the underlying mechanisms of cardiac hypertrophy and may help accelerate the development of new drugs against this condition.

Treating relapsed and refractory diffuse large B-cell lymphoma is still challenging for clinicians, but the available CAR-T and bispecific antibodies revolutionized therapy. Autologous stem cell transplantation was the most effective treatment modality previously. The authors report data from a single center over ten years. The retrospective study included 116 patients. There were 53 relapsed, 39 primary refractory cases, 19 had CNS involvement, and 5 received primary consolidation transplants. The median duration of follow-up was 46 months. The median event-free survival was 75 months, and the median overall survival was 105 months for all cases. Five-year overall survival was 59%, and event-free survival was 54%. Pretreatment prognostic factors at diagnosis had no effect on the outcome of transplantation. The authors found no difference between survival in relapsed or refractory cases, and the number of salvage lines or the germinal center / activated B-cell type also did not influence the results. Complete metabolic response before transplant confirmed by 18FDG PET/CT strongly affected survival. The pretransplant creatinine and CRP levels significantly influenced the long-term outcome. The number of stem cells infused did not affect survival, but engraftment within nine days did result in better survival. These data support the finding that the response to salvage therapy did select a better prognostic group who may still benefit from autologous transplantation.

Primary cutaneous B-cell lymphomas (PCBCLs) are B-cell lymphomas that can occur in the skin without evidence of extracutaneous involvement. The 2005 WHO/EORTC classification of cutaneous lymphomas and its 2018 update have distinguished three main categories based on clinicopathological, immunohistochemical and genetic characteristics: primary cutaneous marginal zone lymphoma (PCMZL), primary cutaneous follicle centre lymphoma (PCFCL), and primary cutaneous diffuse large B-cell lymphoma, leg type (PCDLBCL-LT). PCMZL and PCFCL are clinically indolent, while PCDLBCL-LT is an aggressive lymphoma. Due to its low incidence and lack of prospective studies, it is difficult to establish a standard treatment for each subgroup. The objective of our study was to describe the clinical and pathological characteristics of 103 patients with cutaneous B-cell lymphoma from 12 centres belonging to the Spanish Lymphoma Oncology Group. The median age was 53 years (40-65). According to skin extension, 62% had single site lymphoma, 17% had regional lymphoma and 20% had multifocal lymphoma. Histology: 66% had PCMZL, 26% had PCFCL, and 8% had PCDLBCL-LT. Twenty-three percent of the patients were treated exclusively with surgery, 26% with radiotherapy only, 21% with surgery plus radiotherapy, 10% with polychemotherapy, and 5% with rituximab monotherapy. Overall, 96% of patients achieved a complete response, and 44% subsequently relapsed, most of them relapsing either locally or regionally. The 10-year OS was 94.5% for the entire cohort, 98% for the PCMZL cohort, 95% for the PCFCL cohort and 85.7% for the PCDLBCL-LT cohort. Our data are comparable to those of other published series, except for the high frequency of PCMZL. The expected heterogeneity in therapeutic management has been observed.

The KRAS mutant population has been undruggable for 40 years. G12C inhibitors and immunotherapy are the beginning of success. It is necessary to summarize the successful experience of the existing treatment model and explore the direction of the next treatment. In this review, we discuss the latest developments in targeted therapy and immunotherapy for KRAS-mutation NSCLC, aiming to provide direction or enlightenment for future treatment strategies.

The cyclin-dependent kinase (Cdk) 1–cyclin B (CycB) complex plays critical roles in cell cycle regulation. Before Drosophila male meiosis, CycB is exported from the nucleus to the cytoplasm via the nuclear porin 62kD (Nup62) subcomplex in the nuclear pore complex. When this is inhibited, Cdk1 is not activated, and meiosis does not initiate. We investigated the mechanism that controls cellular localization and activation of Cdk1. Cdk1–CycB continuously shuttled into and out of the nucleus in the prolonged G2 phase before meiosis. Overexpression of CycB, but not that with nuclear localization signal sequences, rescued reduced cytoplasmic CycB and inhibition of meiosis in Nup62-silenced cells. Full-scale Cdk1 activation occurred in the nucleus shortly after its rapid nuclear entry. Cdk1-dependent centrosome separation did not occur in Nup62-silenced cells, while Cdk1 interacted with Cdk-activating kinase and Twine/Cdc25C in the nucleus of Nup62-silenced cells, suggesting the involvement of another suppression mechanism. Silencing of roughex rescued Cdk1 inhibition and initiated meiosis. Nuclear exports of Cdk1 ensured its escape from inhibition by cyclin-dependent kinase inhibitor. The complex reentered the nucleus via importin β at the onset of meiosis. We propose a model regarding the dynamics and activation mechanism of Cdk1–CycB to initiate male meiosis.

Fluorescent bioprobes are invaluable tools for visualizing live cells and deciphering complex biological processes by targeting intracellular biomarkers without disrupting cellular functions. In addition to protein-binding concepts, fluorescent probes utilize various mechanisms, including membrane, metabolism, and gating-oriented strategies. This study introduces a novel fluorescent mechanism distinct from existing ways. Here, we developed a B-cell selective probe, CDrB, with unique transport mechanisms. Through SLC-CRISPRa screening, we identified two transporters, SLCO1B3 and SLC25A41, by sorting out populations exhibiting higher and lower fluorescence intensities, respectively, demonstrating contrasting activities. We confirmed that SLCO1B3, with comparable expression levels in T and B cells, facilitates the transport of CDrB into cells, while SLC25A41, overexpressed in T lymphocytes, actively exports CDrB. The observation suggests that SLC25A41 plays a crucial role in discriminating between T and B lymphocytes. Furthermore, it reveals the potential for reversible localization of SLC25A41 to demonstrate its distinct activity. This study is the first report to unveil a novel strategy of SLC by exporting the probe. We anticipate that this research will open up new avenues for developing fluorescent probes.

Here we first report, how cholera toxin (CT) A subunit (CTA), the bacterial enzyme moiety responsible of cell signaling alteration, can take over the exosomal pathway, spread extracellularly and be transmitted in a cell population. A first evidence for long-term transmission of CT toxic effect via extracellular vesicles was obtained in CHO cells. To follow CT intracellular route towards exosome secretion we adopted a strategy apt to convert multivesicular body (MVB) derived exosomes in traceable fluorescent vectors. CT treated Me665 cells, a human melanoma cell line highly expressing caveolin-1 (Cav-1) and GM1, were used to purify and characterize fluorescent exosomes. Our results clearly show association of CT with exosomes together with typical exosomal markers and the HSP90 and PDI molecules, the required membrane translocation elements of CTA to the cytoplasm. Confocal microscopy proved direct CT containing fluorescent exo transfer into CHO cells coupled with the morphological cell change characteristic of CT action. Moreover, direct assessment of cAMP levels in Me665 cells treated with CT containing exo showed an efficient induction of cAMP increase comparable with CT alone. From our results, we can infer that CT can exploit exosome-mediated cell communication to target and extend its pathophysiological action throughout cell tissues.

In green chemistry, filamentous fungi are regarded as a kind of robust microorganism for the biotransformation of natural products. Nonetheless, the screening of microorganisms is crucial for the effective biotransformation of natural products such as phenazine compounds. The precursor metabolite of most phenazine derivatives in Pseudomonas spp. is phenazine-1-carboxylic acid (PCA), the key constituent of shenqinmycin, widely used to control rice sheath blight in southern China. In this study, a new fungus strain Aspergillus sclerotiorum was isolated, which can efficiently convert PCA into 3-hydroxy-phenazine 1-carboxylic acid (3-OH-PCA). Moreover, an effective whole cells biotransformation system was constructed by screening optimal reaction conditions and carbon sources. Hence, Aspergillus sclerotiorum exhibited desirable adaptation by consumption of different carbon sources and maximum whole cell biomass (10.6 g/L DCW) was obtained as a biocatalyst from glucose. Optimal conditions for whole-cell biocatalysis of PCA were evaluated, including PCA concentration of 1120 mg/L, pH 7.0, temperature of 25°C, rotation rate 200 rpm and dry cell weight 15 g/L for 60 h, thus 1060 mg/L of 3-OH-PCA was obtained and the conversion efficiency of PCA was 94%. Hence, the results of repeated batch mood revealed that the biotransformation efficiency of fungus pellets reduced with each subsequent cycle but remained stable in all five cycles with the provision of glucose supplement. These findings open the prospective of using filamentous fungi for the whole cell biocatalysis of phenazine in enormous amount and efficient production of 3-OH-PCA. Moreover, these results laid the foundation for further research to disclose the genetic based mechanism of the strain responsible for PCA biotransformation.

Diffuse large B cell lymphoma (DLBCL) is curable with chemoimmunotherapy in ~65% of patients. One of the hallmarks of the pathogenesis and resistance to therapy in DLBCL is inhibition of apoptosis, which allows malignant cells to survive and acquire further alterations. Inhibition of apoptosis can be the result of genetic events inhibiting the intrinsic or extrinsic apoptotic pathways, as well as their modulators, such as the inhibitor of apoptosis proteins, P53, and components of the NF-kB pathway. Mechanisms of dysregulation include upregulation of anti-apoptotic proteins and downregulation of pro-apoptotic proteins via point mutations, amplifications, deletions, translocations, and influences of other proteins. Understanding the factors contributing to resistance to apoptosis in DLBCL is crucial in order to be able to develop targeted therapies that could improve outcomes by restoring apoptosis in malignant cells. This review describes the genetic events inhibiting apoptosis in DLBCL, provides a perspective of their interactions in lymphomagenesis, and discuss their implication for the future of DLBCL therapy.

We aimed to investigate whether the sequence length of HIV-1 increases over time. A longitudinal analysis of full-length coding region sequences (FLs) during an HIV-1 outbreak among pa-tients with hemophilia and local controls infected with the Korean subclade B of HIV-1 (KSB) was performed. Genes were amplified by overlapping RT-PCR or nested PCR and subjected to direct sequencing. Overall, 141 FLs were sequentially determined over 30 years in 62 KSB-infected patients. Phylogenetic analysis indicated that within KSB, two FLs from plasma donors O and P comprised two clusters together with 8 and 12 patients with hemophilia, respectively. Signature pattern analysis for the KSB of HIV-1 revealed 91 signature nucleotide residues (1.05%). In total, 48 and 43 signature nucleotides originated from clusters O and P, respectively. Only six positions contained 100% specific nucleotide(s) in clusters O and P. Additionally, in-depth FL analysis over 30 years indicates that the KSB FL significantly increased over time before combined antiretroviral therapy (cART) and decreased with cART. The increase occurred due to a significant increase in env and nef genes, originating in the variable regions of both genes. The increase in the sequence length of HIV-1 over time suggests that it has an evolutionary direction.

The objective of this study is to investigate whether the sequence length of HIV-1 increases over time. A longitudinal analysis of full-length coding region sequences (FLs) in an outbreak of HIV-1 infection among patients with hemophilia and local controls identified as infected with the Korean subclade B of HIV-1 (KSB). Genes amplified by overlapping RT-PCR or nested PCR were subjected to direct sequencing. In total, 141 FLs were sequentially determined over 30 years in 62 KSB-infected patients. Non-KSB sequences were retrieved from the Los Alamos National Laboratory HIV Database. Phylogenetic analysis indicated that within KSB, 2 FLs from plasma donors O and P comprised two clusters together with 8 and 12 patients with hemophilia, respectively. Signature pattern analysis for the KSB of HIV-1 revealed signature nucleotide residues at 1.05%, compared with local controls. Additionally, in-depth FLs sequence analysis over 30 years in KSB indicates that the KSB FL significantly increases over time before combined antiretroviral therapy (cART) and decreases on cART. Furthermore, the increase in FLs over time significantly occurred in the subtypes B, C and G, but, there was no increase in the subtypes D, A, and F1. Consequently, subtypes F1 and D had the shortest sequence length. Our analysis was extended to compare HIV-1 with HIV-2 and SIVs. Essentially, the longer the sequence length (SIVsm > HIV-2 > SIVcpz > HIV-1), the longer the survival period. The increase in the length of the HIV-1 sequence over time suggests that it might be an evolutionary direction toward attenuated pathogenicity.

Immunotherapy has been considered for years as a viable and attractive treatment option for patients with cancer. Among immunotherapy arsenal, the targeting of intratumoral immune cells by immune-checkpoint inhibitory agents has recently revolutionized the treatment of several subtypes of tumours. These approaches aimed at restoring an effective anti-tumour immunity, rapidly reached the market thanks to the simultaneous identification of inhibitory signals that dampen an effective antitumor response in a large variety of neoplastic cells, and the clinical development of monoclonal antibodies targeting checkpoint receptors. Leading therapies in solid tumours are mainly focused on the cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4) and programmed-death 1 (PD-1) pathways. These approaches have found a promising testing ground in both Hodgkin lymphoma and non-Hodgkin lymphoma, mainly because in these diseases the malignant cells interact with the immune system and commonly provide signals that regulate immune function. Although several trials have already demonstrated evidence of therapeutic activity with some checkpoint inhibitors in lymphoma, many of the immunologic lessons learned from solid tumours may not directly translate to lymphoid malignancies. In this sense, the mechanisms of effective antitumor responses are different between the different lymphoma subtypes, while the reasons for this substantial difference remain partially unknown. This review will discuss the current advances of immune-checkpoint blockade therapies in B-cell lymphoma and will build a projection of how the field may evolve in the near future. In particular, we will analyze the current strategies being evaluated both preclinically and clinically with the aim to foster the use of immune-checkpoint inhibitors in non-Hodgkin lymphoma, including combination approaches with chemotherapeutics, biological agents and/or different immunologic therapies.

Diffuse large B-cell lymphoma (DLBCL) requires a complete staging at diagnosis that may have prognostic and therapeutic implications. The role of bone marrow (BM) biopsy (BMB) is contro-versial in the era of nuclear-imaging techniques. We performed a comparative review of 25 studies focused on BM evaluation at DLBCL diagnosis, including at least two of the following techniques: BMB, flow cytometry, and positron emission tomography (PET-FDG). The report about BM involvement (BMi), diagnostic accuracy and prognostic significance was collected and compared among techniques. A concordance analysis between BMB, FCM and PET was also per-formed, and we deeply evaluated the implications of the different types of BMi: concordant by LBCL or discordant by low-grade B-cell lymphoma for both BMB and FCM, and focal or diffuse uptake pattern for PET. As main conclusion, BMB, FCM and PET are complementary tools which provide different and clinically relevant information in the assessment of BMi in newly DLBCL.

Soluble cell adhesion molecules (sCAMs) are secreted ectodomain fragments of surface adhesion molecules, ICAM1 and VCAM1. sCAMs have diverse immune functions beyond their primary function, impacting immune cell recruitment and activation. Elevated sVCAM1 levels have been associated with poor cardiovascular disease (CVD) outcomes, supporting its role as a potential diagnostic marker and therapeutic target. Inhibiting sVCAM1 release or interaction with immune cells could offer cardioprotection in conditions such as diabetes. Membrane bound surface adhesion molecules are widely expressed in a wide variety of cell types with higher expression in endothelial cells (ECs), but the source of sCAMs in the circulation is not clear. Hypothesizing that endothelial cells (ECs) could be a potential source of sCAMs, this study investigated whether dysfunctional EC signaling mechanisms during diabetes cause VCAM1 ectodomain shedding. Our results from samples from a inducible diabetic mouse model revealed increased sVCAM1 plasma levels in diabetes. Protein analysis indicated upregulated VCAM1 expression and metalloproteases ADAM10 and ADAM17 in diabetic ECs. ADAMs are known for proteolytic cleavage of adhesion molecules, contributing to inflammation. GSK3β, implicated in EC VCAM1 expression, was found to be activated in diabetic ECs. GSK3β overexpression in control ECs increased ADAM10/17 and VCAM1. A GSK3β inhibitor reduced active GSK3β and VCAM1 ectodomain shedding. These findings suggest that diabetic ECs with elevated GSK3β activity led to VCAM1 upregulation and ADAM10/17-mediated sVCAM1 shedding. This mechanism underscores the potential therapeutic role of GSK3β inhibition in reducing the levels of circulating sVCAM1. The complex roles of sCAMs extend well beyond CVD. Thus, unraveling the intricate involvement of sCAMs in the initiation and progression of vascular disease particularly in diabetes, holds significant therapeutic potential.

Programmed death ligand 1 (PD-L1) is a co-inhibitory molecule expressed on the surface of various cell types, known for its suppressive effect on T cells through interaction with PD-1. Neutrophils also express PD-L1, and its expression is elevated in specific situations; however, the immunobiological role of PD-L1+ neutrophil has not been fully characterized. Here, we report that PD-L1-expressing neutrophils increased in methicillin-resistant Staphylococcus aureus (MRSA) infection are highly functional in bacterial elimination and supporting inflammatory resolution. The frequency of PD-L1+ neutrophils was dramatically increased in MRSA-infected mice, and this population exhibited enhanced activity in bacterial elimination compared to PD-L1- neutrophils. An administration of PD-L1 monoclonal antibody did not impair PD-L1+ neutrophil function suggesting that PD-L1 expression itself does not influence neutrophil activity. However, PD-1/PD-L1 blockade significantly delayed liver inflammation resolution in MRSA-infected mice, as indicated by increased plasma alanine transaminase (ALT) level and frequencies of inflammatory leukocytes in the liver implying that neutrophil PD-L1 suppresses the inflammatory response of these cells during the acute phase of MRSA infection. Our results reveal that elevated PD-L1 expression can be a marker for enhanced anti-bacterial function of neutrophils. Moreover, PD-L1+ neutrophils are an indispensable population attenuating inflammatory leukocyte activities assisting in a smooth transition into the resolution phase in MRSA infection.

Separase is a key cysteine protease to separate sister chromatids through digestion of cohesion ring that inhibits chromosome segregation as trigger of metaphase–anaphase transition in eukaryotes and highly regulated its activity by binding with securin and cyclinB-CDK1 complex. Those bindings prevent proteolytic activity of separase until the onset of anaphase. Chromosome missegregation and aneuploidy are frequently observed in malignancies. However, there are some difficulties for biochemical examinations due to instability of separase in vitro and a few spatiotemporal resolution approaches monitoring live separase activity throughout mitotic processes. Here we have developed FRET-based molecular sensors including GFP variants with separase cleavable sequences as donor and covalently attached fluorescent dye as acceptor molecules applicable to conventional live cell imaging and flow cytometric analysis because of efficient live cell uptake. We investigated performance of equivalent molecular sensors localized or not localized inside nucleus under cell cycle control using flow cytometry. Synchronized cell cycle progression rendered significant separase activity detections in both molecular sensors. We obtained consistent outcomes with localized molecular sensor introduction and cell cycle control by fluorescent microscopic observations. We thus established live cell separase activity monitoring systems specifically or statistically that could lead to elucidate separase properties in detail.

The development of new diabetes drugs continues to be explored. Loureirin B, a flavonoid, extracted from Dracaena cochinchinensis, has been confirmed to increase insulin secretion and decrease blood glucose levels. For understanding the mechanism, a series of experiments had been employed based on computational methods and cell experiments. The insulin secretion significantly increased with the incubation of 0.01μM loureirin B for 4 hours. The viability of Ins-1 cells showed no significant difference with the treatment of loureirin B. Through computational methods, we hypothesized that loureirin B could interacts with K_ATP channels to promote insulin secretion. In cell experiments, it could be found that the current of K_ATP channel of Ins-1 cells was inhibited by the effect of loureirin B. After then, the voltage-dependent calcium channels were activated, the increase of Cx43 protein expression might mediate the Ca²⁺ to the intracellular. In summary, it could be concluded that loureirin B promoted insulin secretion mainly through inhibiting K_ATP current, the influx of Ca²⁺ to the Intracellular and the expression of Cx43.

Exportin-1, the ubiquitous nuclear protein transporter, is widely confirmed as an active chemotherapeutic target in non-small cell lung cancer condition while Juglans mandshurica is a well-studied anticancer plant in some lung cancer cell lines. We intend to find novel exportin-1 inhibitor from Juglans mandshurica with better potential tolerability and pharmaco-dynamo-kinetic properties than the current selective inhibitors of nuclear export in non-small cell lung cancer treatment. Osiris property explorer DataWarrior, Glide standard precision docking, quantum mechanics polarized ligand docking, MMGBSA binding free energy calculations, Jaguar density functional theory analysis, and the online web-based SwissADME were employed respectively in this study to filter the retrieved compounds based on tolerability, toxicity, and Lipinsky’s rule of five violation potential, determine their druggability, establish relative stability of the lead compound in water solvation model, and evaluates druglikeness, lead-likeness, as well as synthetic accessibility of the lead compound. This study reveals eriodictyol as having higher binding free energy (-40 kcal/mol) than that of standard (-39.56 kcal/mol) in exportin-1 active site, better synthetic accessibility score (3.15 versus 3.29), high GI absorption, non-blood brain barrier permeant, lacks Brenk and PAINS alert, obeying Lipinski’s, Ghose’s, Veber’s, Egan’s, and Muegge’s rule of druglikeness and lead-likeness as well as non-cytotoxic to HepG2 cells. We therefore found eriodictyol as a lead-like, non-toxic exportin-1 inhibitor with good predictive stability and pharmacokinetic potential and thus provided data for further validation of eriodictyol as a candidate exportin-1 inhibitor in both preclinical and clinical studies involving lung cancer therapy.

Resistance of tumor cells to anticancer drugs is a major obstacle in tumor therapy. In this study, we investigated the mechanism of cordycepin-mediated resensitization to cisplatin inT24R2, a derived T24 cell line. Treatment with cordycepin or cisplatin (2 g/ml) alone could not induce cell death of T24R2, but combination treatment of these drugs significantly induced apoptosis of the cells through mitochondrial pathway including depolarization of mitochondrial membrane, decrease of anti-apoptotic proteins, Bcl-2, Bcl-xL, and Mcl-1, and increase of pro-apoptotic proteins, Bak and Bax. . High expression of MDR1 was the cause of cisplatin resistance in T24R2, and cordycepin significantly reduced MDR1 expression through inhibition of MDR1 promoter activity. MDR1 promoter activity was dependent on a transcription factor, Ets-1, in T24R2 cells. Although there is a correlation between MDR1 and Ets-1 expression in bladder cancer patients, active Ets-1, Thr-38 phosphorylated form (pThr-38), was critical to induce MDR1 expression. Cordycepin decreased pThr-38 Ets-1 level through inhibition of AKT, which reduced MDR1 transcription and induced the resensitization of T24R2 to cisplatin. The results suggest that cordycepin effectively resensitizes cisplatin-resistant bladder cancer cells to cisplatin, thus serving as a potential strategy for treatment of anti-cancer drug resistant patients.

Ongoing beta cell death in type 1 diabetes (T1D) can be detected using biomarkers selectively discharged by dying beta cells into plasma. MicroRNA-375 (miR-375) ranks among top biomarkers based on studies in animal models and human islet transplantation. Our objective was to identify additional microRNAs that are co-released with miR-375 proportionate to the amount of beta cell destruction. RT-PCR profiling of 733 microRNAs in a discovery cohort of T1D patients 1 hour before/after islet transplantation indicated increased plasma levels of 22 microRNAs. Sub-selection for beta cell selectivity resulted in 15 microRNAs that were subjected to double-blinded multicenter analysis. This led to identification of 8 microRNAs that were consistently increased during early graft destruction: besides miR-375, these included miR-132/204/410/200a/429/125b, microRNAs with known function and enrichment in beta cells. Their potential clinical translation was investigated in a third independent cohort of 46 transplant patients, by correlating post-transplant microRNA levels to C-peptide levels 2 months later. Only miR-375 and miR-132 had prognostic potential for graft outcome and none of the newly identified microRNAs outperformed miR-375 in multiple regression. In conclusion, this study reveals multiple beta cell-enriched microRNAs that are co-released with miR-375 and can be used as complementary biomarkers of beta cell death.

In the present study, linear novel polymer poly(1-(2-((3-amino-2-hydroxypropyl)amino)ethyl)-1'-ethyl-[4,4'-bipyridine]-1,1'-di-ium) (poly(AHAEBD)) and its complex with iron (ΙΙΙ) ([Fe(poly(AHAEBD)2].Na3) were synthesized and then their anticancer effects on A375 human malignant melanoma cells line were evaluated. The structure of the synthesized compounds was confirmed using Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance spectroscopy (1HNMR), field emission scanning electron microscopy (FE-SEM), X-ray energy diffraction analysis (EDS) and gel permeation chromatography (GPC). Also, the cytotoxicity of cisplatin as a reference, on A375 melanoma cell line was tested. The IC50 of polymer-complex [Fe(poly(AHAEBD)2].Na3 (0.71 µg/mL), cisplatin (4.58 µg/mL) and poly(AHAEBD) (1.73 µg/mL) were obtained. Our results revealed that the polymer-complex [Fe(poly(AHAEBD)2].Na3 exhibited better performance compared to cisplatin. Furthermore, the coordination with iron (III) enhanced the cytotoxicity levels of poly(AHAEBD). According to these findings, the synthesized polymer-complex demonstrates remarkable potential as an anti-cancer agent. This study could provide the basis for future research focused on employing this new polymer-complex for in vivo testing, highlighting its potential for therapeutic applications.

Ovarian cancer poses a significant therapeutic challenge owing to the lack of specific targets for treatment. New treatment strategies are needed to improve patient outcomes and reduce the toxicity of existing therapies. This study aimed to investigate the synergistic effects of recombi-nant human apurinic/apyrimidinic endonuclease 1/redox factor 1 (rhAPE1/Ref-1) and aspirin on PEO14 and CAOV-3 ovarian cancer cells and human umbilical vein endothelial cells (HUVECs). Specifically, this study examined the mechanisms underlying the effect of rhAPE1/Ref-1 and as-pirin on cell death and the associated pathways. Cell viability was assessed using the MTT assay, and flow cytometry analysis and caspase-3/7 activity assays were conducted to determine apop-tosis. Western blotting was performed to evaluate the cleavage of poly(ADP-ribose) polymerase (PARP). Exposure to aspirin (3 mM) for 24 h did not affect cell viability in PEO14. Co-incubation of aspirin with rhAPE1/Ref-1 in PEO14 cells led to rhAPE1/Ref-1 acetylation and intracellular hyperacetylation. Co-treatment with aspirin (3 mM) and rhAPE1/Ref-1 induced a rhAPE1/Ref-1 concentration-dependent increase in cell death, particularly in PEO14 cells, as well as apoptosis, caspase-3/7 activation, and PARP cleavage. Furthermore, this combination treatment significantly increased paclitaxel-induced PEO14 cell death. Therefore, co-administration with rhAPE1/Ref-1 and aspirin is a potential strategy for ovarian cancer treatment.

Microglial dysfunction is one of the hallmarks and leading causes of common neurodegenerative diseases (NDD), including Alzheimer’s disease (AD) and Parkinson’s disease (PD). All these pathologies are characterized by aberrant aggregation of disease-causing proteins in the brain, which can directly activate microglia, trigger microglia-mediated neuroinflammation, and increase oxidative stress. Inhibition of glial activation may represent a therapeutic target to alleviate neurodegeneration. Recently, 3-iodothyronamine (T1AM), an endogenous derivative of thyroid hormone (TH) able to interact directly with a specific GPCR known as trace amine-associated receptor 1 (TAAR1), gained interest for its ability to promote neuroprotection in several models. Nevertheless, T1AM’s effects on microglial disfunction remain still elusive. In the present work we investigated whether T1AM could inhibit the inflammatory response of human HMC3 microglial cells to LPS/TNFα or β-amyloid peptide 25-35 (Aβ25-35) stimuli. The results of ELISA and qPCR assays revealed that T1AM was able to reduce microglia-mediate inflammatory response by inhibiting the release of proinflammatory factors, including IL-6, TNFα, NF-kB, MCP1 and MIP1, while promoting the release of anti-inflammatory mediators, such as IL-10. Notably, T1AM anti-inflammatory action in HMC3 cells resulted to be a TAAR1-mediated response, further increasing the relevance of the T1AM/TAAR1 system in the management of NDD.

T cells follow a triphasic distinct pathway of activation, proliferation and differentiation before becoming functionally and phenotypically ‘exhausted’ in settings of chronic infection, autoimmunity and in cancer. Exhausted T cells progressively lose canonical effector functions, exhibit altered transcriptional networks and epigenetic signatures and gain constitutive expression of a broad coinhibitory receptor suite. This review outlines recent advances in our understanding of exhausted T cell biology and examines cellular and molecular mechanisms by which a state of dysfunction or exhaustion is established, and mechanisms by which exhausted T cells may still contribute to pathogen or tumour control. Further, this review describes our understanding of exhausted T cell heterogeneity and outlines the mechanisms by which checkpoint blockade differentially engages exhausted T cell subsets to overcome exhaustion and recover T cell function.

Diffuse large B-cell lymphoma (DLBCL) is the most common haematological malignancy in humans and dogs. Several studies disclosed some similarities between the two species, including the constitutive activation of NF- κB pathway as a fundamental underlying pathogenetic mecha-nism. In humans, downregulation of IL-1R8 is implicated in DLBCL development, but its role in dogs has not been explored so far. To gain insight into the pathogenesis of this tumor in dogs, we evaluated the mRNA and protein expression of IL-1R8 in 12 hyperplastic lymph nodes ob-tained from dogs not bearing tumors and from 50 dogs with DLBCL. Moreover, we analysed through qRT-PCR the expression of TLR7, TLR9, MYC, and p52 genes that are known to be in-volved in the IL-1R8 regulatory network. IL-1R8 and p52 were downregulated in DLBCLs com-pared to control lymph nodes (p<0.001), while a higher expression of TLR7, TLR9 and MYC was observed in tumors (p<0.01). Immunohistochemistry confirmed gene expression results, reveal-ing a significantly lower IL-1R8 staining score in DLBCLs compared to control lymph nodes (p<0.0001). Taken together, these results suggest that IL-1R8 downregulation may represent one of the mechanisms driving DLBCL pathogenesis in dogs, mainly through dysregulation of the Toll-like/Interleukin receptors signalling cascade and the aberrant activation of classical NF-κB pathway.

Our recent studies showed that the treatment of human ovarian tumor cells with NCX4040 resulted in significant depletions of cellular glutathione, formation of reactive oxygen/nitrogen species and cell death. NCX4040 is also cytotoxic to several human colorectal cancer cells (CRC) in vitro and in vivo. Here, we have examined ferroptosis-dependent mechanism(s) of cytotoxicity of NCX4040 in HT-29 and K-RAS mutant HCT116 colon cell lines. Ferroptosis is characterized by accumulation of reactive oxygen species (ROS) within the cell, leading to an iron-dependent oxidative stress-mediated cell death. However, its relevance in the mechanism of NCX4040 cytotoxicity in CRC is not known. We found that NCX4040 generated ROS in CRC cells without any depletion of cellular GSH. Combinations of NCX4040 with erastin (ER) or RSL3 (RAS-selective lethal 3), known inducers of ferroptosis, enhanced CRC death. In contrast, ferrostatin-1, an inhibitor of ferroptosis, significantly inhibited NCX404-induced cell death. Treatment of CRC cells with NCX4040 resulted in induction of lipid peroxidation in a dose- and time-dependent manner. NCX4040 treatment induced several genes related to ferroptosis (e.g., CHAC1, GPX4 and NOX4) in both cell lines. Metabolomic studies also indicated significant increases in both lipid and energy metabolism following the drug treatment in HT-29 and HCT116 cells. These observations strongly suggest that NCX4040 causes ferroptosis-mediated cell death of CRC cells. Furthermore, combinations of NCX4040 and ER or RSL3 may contribute significantly for the treatment of CRC, including those that are difficult to treat due to the presence of Ras mutation in the clinic. NCX4040 induced ferroptosis may also be a dynamic form of cell death for the treatment of other cancers.

Accumulating reports have suggested that an inability to clear dead cells is a cause of inflammation and cancer and that the fascia is associated with cancer metastasis. Novel corpuscle-connected filiform structures (CCFSs) entangled on the fascia of the internal organs of rats were selectively visualized with high repeatability (93%) under vital staining with Janus Green B (JGB). Serial sections of CCFSs stained with haematoxylin and eosin and with Mattson Trichrome were microscopically examined. In parallel, the immunohistochemistry with CD31, Lyve 1, scanning electron microscopy, and transmission electron microscopy (TEM) were also used to investigate the novel properties of the CCFSs. All of the CCFSs enclosed JGB-stained granules, which was verified by the impromptu coupling of stereo and light microscopes and fluorescence-activated cell sorting. The functions of the CCFSs were discovered under fluorescence microscopy and confocal laser scanning microscopy to be implicated in cell death: fragmented DNAs and f-actins with extracellular DNAs. The dead cells in the CCFSs were verified by using terminal deoxynucleotidyl transferase dUTP nick end labelling and TEM. Moreover, the CCFS had a relatively-high concentration of calcium, a main element for cell death in tissue, which was measured in 2-dimensionally images using time-of-flight secondary ion mass spectrometry. Taken these results, we report for the first time that novel reticular CCFSs are widely entangled on the fascia of the internal organs of rats with the implication for cell death.

Akt1 is a proto-oncogene that is over active in most cancers. Akt1 activation requires phosphorylation at Thr308; phosphorylation at Ser473 further enhances catalytic activity. Akt1 activity is also regulated via interactions between the kinase domain and the N-terminal auto-inhibitory pleckstrin homology (PH) domain. As it was previously difficult to produce Akt1 in site-specifically phosphorylated forms, the contribution of each activating phosphorylation site to auto-inhibition was unknown. Using a combination of genetic code expansion and in vivo enzymatic phosphorylation, we produced Akt1 variants containing programmed phosphorylation to probe the interplay between Akt1 phosphorylation status and the auto-inhibitory function of the PH domain. Deletion of the PH domain increased the enzyme activity for all three phosphorylated Akt1 variants. For the doubly phosphorylated enzyme, deletion of the PH domain relieved auto-inhibition by 295-fold. We next found that phosphorylation at Ser473 provided resistance to chemical inhibition by Akti-1/2 inhibitor VIII. The Akti-1/2 inhibitor was most effective against pAkt1T308 and showed 4-fold decreased potency with Akt1 variants phosphorylated at Ser473. The data highlight the need to design more potent Akt1 inhibitors that are effective against the doubly phosphorylated and most pathogenic form of Akt1.

HAX1 is a multifunctional protein involved in the antagonism of apoptosis in cellular response to oxidative stress. In the present study we identified HAX1 as novel binding partner for Che-1/AATF, a pro-survival factor which plays a crucial role in fundamental processes, including response to multiple stresses and apoptosis. HAX1 and Che-1 proteins show extensive colocalization in mitochondria and we demonstrated that their association is strengthened after oxidative stress stimuli. Interestingly, in MCF-7 cells, resembling luminal estrogen receptor (ER) positive breast cancer, we found that Che-1 depletion correlates with decreased HAX1 mRNA and protein levels, and this event is not significantly affected by oxidative stress induction. Furthermore, we observed an enhancement of the previously reported interaction between HAX1 and estrogen receptor alpha (ERα) upon H2O2 treatment. These results indicate the two anti-apoptotic proteins HAX1 and Che-1 as coordinated players in cellular response to oxidative stress with a potential role in estrogen sensitive breast cancer cells.

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.

Background: Cord blood represents a link between intrauterine and early extrauterine development. Cord blood cells map an important time frame in human immune imprinting processes. It is unknown whether sex of the newborn affects the lymphocyte subpopulations in cord blood. Methods: 9 B and 21 T cell subpopulations were characterized by flow cytometry in human cord blood from 16 male and 21 female newborns, respectively. Results: Except for marginal zone B cells and transitional B cells, B cell count in all subsets was higher in cord blood of male newborns than in female newborns. Frequency of naive thymus negative Th cells was significantly higher in male cord blood whereas the remaining T cell subpopulations showed a higher count in cord blood of female newborns. Conclusion: Our study is the first revealing sex differences in B and T cell subpopulations of human cord blood. These results indicate that sex might have a higher impact for the developing immune system urging the need to expand research in this area.

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.

Erectile dysfunction (ED) is a common and feared complication of radical prostatectomy (RP) for prostate cancer. Recently, tissue engineering is being attempted for post-prostatectomy ED, with controlled interactions between cells, growth factors, and extracellular matrix (ECM) being important for nerve regeneration's structural integrity. In this study, we evaluated the effects of biomechanical ECM patch on human bone marrow-derived mesenchymal stem cells (hBMSCs) morphology and behavior in bilateral cavernous nerve injury (BCNI) rat model. The ECM patch, made of decellularized human fibroblast-derived ECM (hFDM) and a biocompatible polyvinyl alcohol (PVA) hydrogel, was tested with human bone marrow-derived mesenchymal stem cells (hBMSCs) on a bilateral cavernous nerve injury (BCNI) rat model. In vitro analysis showed that the hFDM/PVA + hBMSCs patches significantly increased neural development markers. In vivo experiments demonstrated that the rats treated with the hFDM/PVA patch had higher ICP/MAP ratio, a higher ratio of smooth muscle to collagen, increased content of nNOS, higher levels of eNOS protein expression, and a higher cGMP level compared to the BCNI group. These results indicate that the hFDM/PVA patch is effective in promoting angiogenesis, smooth muscle regeneration, and nitrergic nerve regeneration, which could contribute to improved erectile function in post-prostatectomy ED.

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.

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.

With an estimated 10 million people infected, the deltaretrovirus human T-cell lymphotropic virus type 1 (HTLV-1) is the second most prevalent pathogenic retrovirus in humans after HIV-1. Like HIV-1, HTLV-1 overwhelmingly persists in a host via a reservoir of latently infected CD4+ T cells. Although most patients are asymptomatic, HTLV-1-associated pathologies are often debilitating and include childhood infective dermatitis and adult T-cell leukaemia/lymphoma (ATLL), which presents in mature adulthood and is associated with poor prognosis with short overall survival despite treatment. Curiously, the strongest indicator for the development of ATLL is the acquisition of HTLV-1 through breastfeeding. There are no therapeutic or preventative regimens for HTLV-1. However, antiretrovirals (ARVs), which target the essential retrovirus enzymes, have been developed for and transformed HIV therapy. Since the architectures of retroviral enzyme active sites are highly conserved, some HIV-specific compounds may be active against HTLV-1. Here, we expand on our work showing that integrase strand transfer inhibitors (INSTIs) and some nucleoside reverse transcriptase inhibitors (NRTIs) block HTLV-1 transmission in cell culture. Specifically, we find that dolutegravir, the INSTI currently recommended as the basis of all new combination antiretroviral therapy prescriptions, and the latest prodrug formulation of the NRTI tenofovir, tenofovir alafenamide, also potently inhibit HTLV-1 infection in cell culture. Our results, if replicated in a clinical setting, could see transmission rates of HTLV-1 and future caseloads of HTLV-1-associated pathologies like ATLL dramatically cut via the simple redistribution of already widely available HIV pills to HTLV-1 endemic areas. Considering our findings with the old medical saying ‘it’s better to prevent than cure’, we highly recommend the inclusion of INSTIs and tenofovir prodrugs in upcoming HTLV-1 clinical trials as potential prophylactics.

Background: Oenothera biennis (evening primrose) produces bioactive substances with a diverse range of pharmacological functions. However, it is currently unknown whether extract prepared from the aerial parts of O. biennis (APOB) can protect the skin against oxidative stress. To investigate the protective effects of APOB against oxidative stress-induced damage in human skin keratinocytes (HaCaT) and elucidate the underlying mechanisms. Methods: We pretreated HaCaT cells with various concentrations of APOB or the antioxidant N-acetyl-L-cysteine before applying H2O2. We then compared the cell viability, intracellular reactive oxygen species (ROS) production, and DNA and mitochondrial damage between pretreated and untreated control cells using a range of assays, flow cytometry, and Western blot analysis and also examined the reducing power and DPPH free radical-scavenging activity of APOB. Results: APOB pretreatment significantly increased cell viability, effectively attenuated H2O2-induced comet tail formation, and inhibited H2O2-induced phosphorylation of the histone γH2AX, as well as the number of apoptotic bodies and Annexin V-positive cells. APOB was found to have a high reducing power and DPPH radical-scavenging activity and also exhibited scavenging activity against intracellular ROS accumulation and restored the loss of mitochondrial membrane potential caused by H2O2. APOB pretreatment almost totally reversed the enhanced cleavage of caspase-3, the degradation of poly (ADP-ribose)-polymerase (PARP), DNA fragmentation that usually occurs in the presence of H2O2 and increased the levels of heme oxygenase-1 (HO-1), a potent antioxidant enzyme that is associated with the induction of nuclear factor-erythroid 2-related factor 2 (Nrf2). Conclusions: APOB can protect HaCaT cells from H2O2-induced DNA damage and cell death by blocking cellular damage related to oxidative stress via a mechanism that affects ROS elimination and by activating the Nrf2/HO-1 signaling pathway.

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.

Small RNAs (sRNA) are epigenetic regulators of essential biological processes that have been linked to the onset and progression of leukemia, including acute adult T-cell leukemia-lymphoma (ATLL) caused by the retrovirus Human T-lymphotropic virus type 1. Here, we describe the sRNA profile of a 30-year-old female with ATLL at diagnosis and after maintenance therapy, with the goal of correlating their expression levels with response to therapy.

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.

: Immune checkpoint inhibitors (ICI) cemiplimab and pembrolizumab have revolutionized the treatment of advanced cutaneous squamous cell carcinoma (cSCC). We aimed to evaluate the effectiveness and safety of ICI in a real-world cSCC population, including patients with conditions which would exclude clinical trial participation. In this single-center, retrospective cohort study, we included all non-trial patients with advanced cSCC treated with ICI between 2017 and 2022. We evaluated investigator-assessed best overall response (BOR) and immune-related adverse events (irAEs). We correlated survival outcomes with age, performance status, immune status and irAEs. For the 36 patients identified, best overall response (BOR) to ICI was partial response (PR) in 41.7%, complete response (CR) in 27.8%, and stable disease in (SD) 13.9% of patients. The PFS rate at 1 year was 63%; median PFS was not reached (NR). The median OS was 38.6 months (95% CI 25.4-NR). Immune compromised patients, ECOG performance 2-3, and age ≥75 years were not significantly associated with PFS or OS. IrAE grade 3-4 were seen in 13.9% of patients. In our Canadian experience with real-world patients, ICI was an effective and safe treatment for advanced cSCC patients. Patients achieved great benefit with ICI regardless of age, immune status or ECOG performance status.

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.

Looking for effective synthetic methods for 1H-pyrazolo[3,4-b]quinolines preparation, we came across a procedure where in a three component reaction catalyzed by L-proline, 4-aryl-4,9-dihydro-1H-pyrazolo[3,4-b]quinolines are formed. These compounds can be easily oxidized to a fully aromatic system, which gave hope for a synthetic method that could replace e.g. Friedländer condensation, often used for this purpose, although severely limited by the availability of suitable substrates. However, after careful repetition of the procedures described in the publication, it turned out that the compounds described therein do not form at all. The actual compounds turned out to be 4,4-(phenylmethylene)-bis-(3-methyl-1-phenylpyrazol-5-oles). 4-Aryl-4,9-dihydro-1H-pyrazolo[3,4-b]quinolines were prepared by another method and used as standards to compare the products formed in the original procedure.

Ovarian cancer is among the most common causes of female mortality. Despite improvements in diagnostic methods, non-specific symptoms and delayed gynecological exams can lead to late-stage ovarian tumor discovery. In this study, the effect of an anti-cancer compound, 3-amino-N-(3-chloro-2-methylphenyl)-5-oxo-5,6,7,8-tetrahydrothieno[2,3-b]quinoline-2-carboxamide (Compound 1) was examined. Cytotoxicity, apoptosis, and metabolomic changes in ovarian cancer cell lines SK-OV-3 and OVCAR-3, as well as glycosphingolipid (GSL) expression, both on cancer stem cells (CSCs), marked as CD49f+ and non-CSCs (CD49f-) were explored. Treatment with Compound 1 reduced the percentage of CSCs compared to non-treated cells (P &lt; 0.001). The expression of eight GSLs on CSCs and non-CSCs was examined using flow cytometry. Both cell lines showed a change in the glycophenotype of the cells, either up or down of its expression, after the treatment. These findings raise the possibility of specifically targeting CSCs in ovarian cancer therapy. Additionally, treatment of Compound 1 resulted in a statistically significant increase in both early and late apoptosis (P&lt;0.001) suggesting a pivotal role in initiating programmed cell death by the apoptotic pathway. The metabolic analysis revealed that the treated cancer cells showed reduced metabolic activity compared to the control group (P &lt; 0.001)

Platinum-resistant high-grade serous ovarian cancer (HGSOC) is invariably a fatal disease, so a central goal in ovarian cancer research is to develop novel strategies to overcome platinum resistance. Treatment is thus moving towards personalized therapy. However, validated molecular biomarkers to predict patients’ risk of developing platinum resistance are still lacking. Extracellular vesicles (EVs) are promising candidates for biomarkers. EpCAM-specific EVs are largely unexplored biomarkers in predicting chemoresistance. Using transmission electron microscopy, nanoparticle tracking analysis and flow cytometry, we compared the characteristics of EVs released from a cell line derived from a clinically confirmed cisplatin-resistant patient (OAW28) and EVs released from two cell lines from tumors sensitive to platinum-based chemotherapy (PEO1, OAW42). We showed that characteristics of EVs released from the HGSOC cell line of chemoresistant patients had higher heterogeneity in size, a larger proportion of medium/large (&gt;200nm) EVs and a higher number of released EpCAM-positive EVs of different sizes, although expression of EpCAM predominated, especially on those larger than 400 nm. We also determined a strong positive correlation between the concentration of EpCAM-positive EVs and expression of cellular EpCAM. These results may contribute to prediction of platinum resistance in the future, although they should first be validated in clinical samples.

The uneven immunogenicity of the attenuated tetravalent dengue vaccine has created the diffi-culty to achieve a balanced protection against all four serotypes of dengue virus (DENV). To overcome this problem, non-replicative-based vaccines have come into focus as their immuno-genicity is adjustable. This approach is excellent for multivalent vaccines but commonly faces the issue of low immunogenicity. In this present study, we developed a non-replicating dengue vaccine composing of UV-inactivated dengue-2 virus (UV-DENV2) and DENV-2 NS1-279 protein encapsidated within nanoparticles. This vaccine candidate was administered in the presence of BCG cell wall cytoskeleton (BCG-CWS) as an adjuvant. We revealed, here, that encapsidated immunogens with BCG-CWS exerted potent activities on both B and T cells and elicited Th-1/Th-2 responses in mice. This was evidenced by strong neutralizing antibody activity with an unde-tectable antibody enhancing activity in a group of mice receiving encapsidated immunogens with BCG-CWS. Importantly, BCG-CWS significantly augmented antibody-mediated comple-ment-fixing activity, strongly stimulated the antigen-specific polyfunctional T cell responses as well as activated mixed Th-1/Th-2 responses specific to DENV2- and NS1-279 antigens. In conclu-sion, BCG-CWS potently adjuvanted the inactivated DENV-2 and subunit DENV immunogens. The mechanism of adjuvanticity remains unclear. This study revealed the potential use of BCG-CWS in vaccine development.

A large body of evidence indicates that environmental agents can induce alterations in DNA methylation (DNAm) profiles. Radiofrequency electromagnetic fields (RF-EMFs) are radiations emitted by everyday devices which have been classified as “possibly carcinogenic”, however their biological effects are unclear. As aberrant DNAm of genomic repetitive elements (RE) may promote genomic instability, here we sought to determine whether exposure to RF-EMFs could affect DNAm of different classes of RE, such as long interspersed nuclear element1s (LINE-1), Alu short interspersed nuclear element and ribosomal repeats. To this purpose, we analysed DNAm profiles of cervical cancer and neuroblastoma cell lines (HeLa, BE(2)C and SH-SY5Y) exposed to 900 MHz GSM-modulated RF-EMF through an Illumina-based targeted deep bisulfite sequencing approach. Our findings showed that radiofrequency exposure did not affect DNAm of Alu elements in any of the cell lines analysed. Conversely, it influenced DNAm of LINE1 and ribosomal repeats in terms of both average profiles and organization of methylated and un-methylated CpG sites, in different ways in each of the three cell lines studied.

Proprotein convertases activate various envelope glycoproteins and participate in cellular entry of many viruses. We recently showed that the convertase furin is critical for the infectivity of SARS-CoV-2. This study investigated the implication of the two cholesterol-regulating convertases SKI-1 and PCSK9 in SARS-CoV-2 entry. We used cell-to-cell fusion assays in HeLa cells and pseudoparticle entry into Calu-3 cells. SKI-1 increases cell-to-cell fusion by enhancing the activation of SREBP-2, whereas PCSK9 reduces cell-to-cell fusion by promoting the cellular degradation of ACE2. Metalloprotease activation is sensitive to enhanced cholesterol levels resulting from SKI-1-activated SREBP-2 that leads to enhanced S2’ formation. However, high metalloprotease activity results in S2’ shedding into a new C-terminal fragment (S2”), leading to reduced cell-to-cell fusion. Indeed, S-mutants that increase S2’’ formation, abolish S2’ and cell-to-cell fusion, as well as pseudoparticles entry, indicating that the formation of S2’’ prevents SARS-CoV-2 cell-to-cell fusion and entry. We next demonstrated that PCSK9 enhanced the cellular degradation of ACE2, thereby reducing cell-to-cell fusion. However, different from the LDLR, a canonical target of PCSK9, the C-terminal CHRD domain of PCSK9 is dispensable for the PCSK9-induced degradation of ACE2. Molecular modeling suggested binding of ACE2 to the Pro/Catalytic domains of mature PCSK9. Thus, both cholesterol-regulating convertases SKI-1 and PCSK9 can modulate SARS-CoV-2 entry via two independent mechanisms.

Cutaneous T cell lymphoma (CTCL) is a devastating, potentially fatal T lymphocyte malignancy affecting the skin. Despite all efforts, the etiology of the disease remains unknown. Infectious agents have long been suspected as factors or co-factors in CTCL pathogenesis. This review deals with the panel of bacterial and viral pathogens that have been investigated so far in an attempt to establish a potential link between infection/carriage and CTCL development. A special focus is given to a recently discovered human protoparvovirus, namely the cutavirus (CutaV), which has emerged as a plausible CTCL etiological agent. Available evidence in support of this hypothesis as well as alternative interpretations and uncertainties raised by some conflicting data are discussed. The complexity and the multifacetedness of the Parvoviridae family of viruses are illustrated by presenting another protoparvovirus, the rat H-1 parvovirus (H-1PV). H-1PV belongs to the same genus as the CutaV but carries a considerable potential for therapeutic applications in cutaneous lymphoma.

The human T-cell leukemia virus type 1 (HTLV-1) is the only known human oncogenic retrovirus. HTLV-1 can cause a cancer called adult T-cell leukemia/lymphoma (ATL). The virus is transmitted through body fluids of infected individuals, primarily breast milk, blood, and semen. At least 5–10 million people in the world are infected with HTLV-1. In addition to ATL, HTLV-1 infection can also cause HTLV-I-associated myelopathy (HAM /TSP). ATL is characterized by low viral ex-pression and poor prognosis. The oncogenesis triggered by HTLV-1 is extremely complex and the molecular mechanisms are not fully understood. However, viral regulatory proteins Tax and HTLV-1 bZIP factor (HBZ) have been shown to play key roles in the transformation of HTLV-1-infected T cells. Moreover, several studies have shown that the final fate of HTLV-1-infected transformed T cell clones is the result of a complex interplay of HTLV-1 oncogenic protein expression with cellular transcription factors that subvert the cell cycle and disrupt reg-ulated cell death, thereby exerting their transforming effects. This review provides updated in-formation on the mechanisms underlying the transforming action of HTLV-1 and the potential therapeutic targets to combat ATL.

Human T-lymphotropic virus type 1 (HTLV-1)–associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a slowly progressive spinal cord disorder with no effective treatment. There is much of interest in developing potential biomarkers for predicting the pathogenesis of HAM/TSP disorder. This study used Illumina massive parallel sequencing (MPS) technology to assess the cellular global noncoding RNAome expression profile in HAM/TSP patients (n = 10), asymptomatic HTLV-1 infected carriers (ASP, n = 8), and a second group of healthy controls (n = 5). Using various bioinformatics tools, the sRNA MPS reads were aligned, annotated, and profiled. There were 251 known and 50 potential novel sRNAs among the 402 detected sRNAs in the HAM and ASP groups versus the HC group. Sixty-eight known sRNAs were found to be significantly different between the ASP and HAM groups. In HAM vs ASP subjects, 88 mature miRNAs were downregulated. Three of these miRs (hsa-miR-185-5p, 32-5p, and 192-5p) have the potential to be used as biomarkers for predicting the pathogenesis of HAM/TSP. The top seven deregulated miRS target genes were linked to a variety of biological processes and molecular functions. Relevant reactome pathways to our findings provide a rich source of data and an opportunity to further understand sRNA regulation and function in HTLV-1 pathophysiology.

The advent of immunotherapy has revolutionized the treatment of advanced non-small cell lung cancer (NSCLC), improving the prognosis of this disease and becoming a key part of the treatment, especially in the population without an oncogenic driver mutation. Two different groups of immune checkpoint inhibitors (ICIs) are used in lung cancer: Anti- Cytotoxic T-lymphocyte antigen-4 (Anti-CTLA 4) and Anti- T-cell receptor programmed cell death-1 or its ligand (Anti-PD-1 and Anti PD-L1). Here in, we review an uptodate on the use of immunotherapy for advanced NSCLC.

Background: PD1 and its ligand PD-L1 are related to the prognosis in many solid tumors, whether its role in oral squamous cell carcinoma (OSCC) remains unclear. Methods: Retrospective monocentric study including all patients with OSCC diagnosed and treated between 01/2020 and 05/2022. PD-L1 expression was assessed by combined positive score (CPS), considering CPS > or equal to 1 as positive (1-20 “low expression”, and ≥20 “high”). Descriptive analysis of patient cohort and tumors was performed, including tumor size, stage, lymph node involvement, recur-rence and survival. Results: 65 patients (65 tumors) were analyzed. 66.15% of tumors were ad-vanced stages (III-IV), of them, 97.67% PD-L1+ versus 71.42% within early stages (I-II). T4 tumors expressed PD-L1 in 100% of cases, compared with 54’54% in T1 tumors. 50.79 % of tumors showed lymph node involvement (pN+), 100% of them showing PD-L1+. Prevalence of pN+ was 59.38% for PD-L1 high expression versus 40.63% for low expression. Patients' follow-up ranged from 2 to 34.5 months. No significant difference was seen between overall survival (OS) and PD-L1 +/- (CPS ≥1 vs CPS <1) neither PD-L1 high (CPS ≥20) vs low CPS <20) (p 0.97 and 0.64, re-spectively). Conclusions: The method used to measure (laboratory test with Dako 22C3 an-ti-PD-L1 primary antibody) PD-L1 was reliable and accurate, with a correlation coefficient be-tween the PD-L1 expression in the biopsy and surgical piece of 0.83 (p 0.0001). CPS≥1 was ob-served in large tumors (p<0.001), and was correlated with of lymph node metastases (p<0.004). Further analysis of PD-L1 expression and its relevance in tumor biology and prognosis is needed.

Breast cancer (BC) is the most common type of cancer in both men and women alike, but it is more prevalent in women. Natural compounds that can modulate the oncogenic process can be considered a significant anti-cancer agent for treating BC. These natural compounds are more effective than synthetic drugs, which have profound side effects on the normal cell and resistance to cancer cells. Genistein is a type of dietary phytoestrogen included in the flavonoid group with a similar structure of estrogen that might provide a strong alternative and complementary medicine to existing chemotherapeutics drugs. Several research studies demonstrated that it can target the estrogen receptor (ER), Human epidermal growth factor receptor-2 (HER2), and Breast cancer gene-1 (BRCA-1) in multiple BC cell lines, as well as sensitize cancer cell lines to this compound when used at an optimal inhibitory concentration. Genistein effectively showed anti-cancer activities through apoptosis induction, arresting cell cycle, inhibiting angiogenesis with metastasis, reducing inflammation, mammosphere formation, tumor growth, up-regulating tumor suppressor gene, and downregulating oncogene in suppressing cancer progression in vitro and animal model study. In addition, research studies have also suggested that these phytochemicals synergistically reverse the resistance mechanism of chemotherapeutic drugs, increasing the efficacy of some chemoinformatics drugs. Our review article aims to unbox and validate the molecular pharmacology in breast tissue, cell-specific anti-cancer mechanism with synergistic activity, and possible pharmacokinetic parameters of Genistein as a potential alternative therapeutic option for the treatment of BC.

HIV-1 transcribes only one kind of transcript – the full length genomic RNA. To make the mRNA transcripts for the accessory proteins Tat and Rev, the genomic RNA must completely splice. The mRNA transcripts for Vif, Vpr, and Env must splice but not completely. Genomic RNA (which also functions as mRNA for the Gag and Gag/Pro/Pol precursor polyproteins) must not splice at all. HIV-1 can tolerate a surprising range in the relative abundance of individual transcript types, and a surprising amount of aberrant and even odd splicing; however, it must not over-splice, which results in the loss of full length genomic RNA and has a dramatic fitness cost. Cells typically do not tolerate unspliced/incompletely spliced transcripts, so HIV-1 has to circumvent this cell policing mechanism to allow some splicing while suppressing most. Splicing is controlled by RNA secondary structure, cis-acting regulatory sequences which bind splicing factors, and the viral protein Rev. There is still much work to be done to clarify the combinatorial effects of these splicing regulators. These control mechanisms represent attractive targets to induce over-splicing as an antiviral strategy. Finally, splicing has been implicated in latency, but to date there is little supporting evidence for such a mechanism. In this review we apply what is known of cellular splicing to understand splicing in HIV-1, and also present data from our newer and more sensitive deep sequencing assays quantifying the different HIV-1 transcript types.

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.

Background: Recent researches have investigated the biological importance of RNA N1-methyladenosine (m1A) modifications in oncogenesis and progression of head and neck squamous cell carcinoma (HNSCC). However, whether m1A modifications also have latent effect in tumor microenvironment (TME) generation and immune regulation in HNSCC is unknown. Methods: We evaluated the m1A modification patterns and related to these modification patterns with TME cell infiltration features in 1041 HNSCC samples by bioinformatics approach. Results: The m1A score is an independent prognostic indicator. HNSCC patients with low m1A score group with poor overall survival (OS) was mainly characterized by stroma activation, lack of sufficient immune infiltration, and exhibited an immune- desert TME phenotype. Low m1A scores were also correlated with increased tumor mutation burden (TMB), and HNSCC patients with high TMB and low m1A scores had the worst OS. In addition, anti-CTLA-4 combined with anti-PD1 treatment was more effective in the high m1A score subgroup than in the low m1A score subgroup. Conclusions: This study revealed that m1A modifications play a non-negligible role in developing the TME versatility and complexity of HNSCC. Assessing m1A modification patterns in HNSCC helps improve our comprehension of its TME infiltration profile and guides more effective immunotherapeutic approaches.

Novel mechanism of ccRCC progression is essential, including PBRM1, BAP1, and SETD2 in histone-modifying and chromatin remodeling genes. The study aimed to investigate VHL, PD-1, PD-L1, PD-L2. BAP1, PBRM1, SETD2 expression in ccRCC primary cancers and metastatic tissues associated with the cancer dissemination. A total of 62 patients with RCC were enrolled in the study. Investigation of mRNA level of VHL, PD-1, PD-L1, PD-L2. PCR in real-time performed BAP1, PBRM1, SETD2 with the previous RNA isolation. Western Blotting analysis was used for detecting the p-VHL protein content in tissues. The VHL expression and p-VHL content determined the aggressive cancer behavior and elevated in disseminated tumors. The cancer dissemination was accompanied by an increase in both mRNA and VHL content. The PD-L2 prevalence in metastatic cancers is crucial in tumor progression. ccRCC progression in VHL overexpression is associated with the decrease in BAP1 gene expression. It is revealed the heterogeneity in molecular markers in primary tumors and metastases. The low mRNA level of BAP1, PBRM1, SETD2, PD-1, PD-L1, PD-L2 in metastases compared with primary tumors were found. We show a novel mechanism for VHL tumor progression and present a new instrument and factor targeting tumor-related pathologies with p-VHL/HIF altered function.

We conducted a pilot study to analyze the differential methylation status of 20 primary acinar adenocarcinomas of the lungs. These adenocarcinomas had to be wild type in mutation analysis and had either high (TPS&gt;50%; n=10) or low (TPS&lt;1%; n=10) PD-L1 status to be integrated into our study. To examine the methylation of 866,895 specific sites, we utilized the Illumina Infinium EPIC bead chip array. Both hypermethylation and hypomethylation play significant roles in tumor development, progression, and metastasis. They also impact the formation of the tumor microenvironment, which plays a decisive role in tumor differentiation, epigenetics, dissemination, and immune evasion. The gained methylation patterns were correlated with PD-L1 expression. Our analysis has identified distinct methylation patterns in lung adenocarcinomas with high and low PD-L1 expression. After analyzing the correlation between methylation results of genes and promoters with their pathobiology, we found that tumors with high expression of PD-L1 tend to exhibit oncogenic effects through hypermethylation. On the other hand, tumors with low PD-L1 expression show loss of their suppressor functions through hypomethylation. The suppressor functions of hypermethylated genes and promoters are ineffective compared to simultaneously activated dominant oncogenic mechanisms. The tumor microenvironment supports tumor growth in both groups.

Immune checkpoint inhibitors (ICIs) – anti-programmed death-1 (PD-1) and their ligands (PD-L1 and PD-L2) have become widely used in the treatment of several malignancies. Many immune-related adverse events have been linked to these agents. However, tuberculosis (TB) reactivation during their use is increasingly reported. Herein, we present a 58-year-old lady with advanced non-small cell lung cancer (NSCLC) ALK-negative, EGFR wild, and PD-L1 Immune histochemistry (IHC) strongly positive in 95% of tumor cells. The patient presented with high-grade fever and a history of productive cough for a 1-week duration. A few days later, she was diagnosed with pulmonary tuberculosis following the 6th cycle of Pembrolizumab, an anti-PD-1 monoclonal antibody. AFB smear and TB PCR from BAL were positive (rifampin resistance not detected), and she was accordingly started on Anti-TB medications. Immunotherapy was held. Of note, the patient had a history of sick contact with a patient with active TB infection ten years ago, but there was no documentation of latent TB or previous TB infection. Her HIV status is negative. Her sputum AFB smear continued to be positive after four weeks of anti-TB medications. Later, the patient was discharged after her sputum was cleared from AFB (negative x 2 sets). We assumed that our patient developed reactivation of pulmonary tuberculosis secondary to an immune checkpoint inhibitor (Pembrolizumab). She was not re-challenged with Pembrolizumab following TB diagnosis. To our knowledge, this is the first reported case from the Arab and the Middle East; it reinforces the previous observations of the association between ICIs administration and the development of MTB. Nevertheless, further studies in the clinical setting are necessary to establish the exact mechanism involved in this association. Oncologists' awareness & prompt recognition of this potential hazardous consequence are essential. Since there is no clear evidence whether LTBT prior PD-1/PD targeted immunotherapy is required, targeted LTBT before starting ICIs immunotherapy with TB chemoprophylaxis; yet to be explored, particularly in the regions where the MTB prevalence is high.

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.

BACKGROUND: Chronic hepatitis B infected with Hepatitis B virus remains a major health concern worldwide. Despite standard interferon-α and nucleotide analogues have been shown to reduce the deterioration of liver disease among chronic hepatitis B patients, covalently closed circular DNA was still difficult to eradicate. METHODS: A literature search of Pubmed and Web of science was performed with the following key words: ‘CRISPR’, ‘CRISPR/Cas9’, ‘hepatitis B’, ‘HBV’, ‘chronic hepatitis B’ and ‘HBV cccDNA’. The information about CRISPR/Cas9 for the treatment of HBV cccDNA or hepatitis B was reviewed. RESULTS: CRISPR/Cas9 could treat hepatitis B through suppressing or clearing HBV cccDNA with different gRNAs. CONCLUSION: With the emergence of CRISPR/Cas9 (the RNA-guided clustered regulatory interspaced short palindromic repeats, CRISPR) editing technology, clearance of hepatitis B virus and better prevention of liver carcinoma seemed to be possible.

Autoantibodies against specific lung cancer-associated antigens have been suggested for lung cancer diagnosis. This study aimed to evaluate the diagnostic performance of anti-gen-autoantibody immune complex (AIC) against its free antigens for CYFRA21-1, ProGRP, NGAL, and NSE in non-small cell lung cancer (NSCLC). In total, 85 patients with NSCLC and 120 healthy controls (HCs) were examined using a 9G DNA chip method. The ratios of AICs to their antigens and combinations of ratios consisting of two to four markers were calculated. The levels of AICs for CYFRA21-1, ProGRP, NGAL, and NSE were higher than their free antigens in all participants. The levels of each free antigen distinguished patients with NSCLC from HCs. The ratios of AIC to its antigen and seven combinations consisting of two to four ratios were significantly higher in patients with NSCLC than those in HCs. Excellent diagnostic performance was observed for all combination ratios (C4-1), with 85.9% sensitivity and 86.7% specificity at cutoff 3.51. Higher sensitivity was observed in the very early stages (0–I) and adenocarcinoma than in stages II–IV and other pathological types. The combination of all ratios of AICs and their antigens for all four markers is useful in the diagnosis of NSCLC.

Since the spread of the deadly virus SARS-CoV2 in late 2019, researchers have restlessly been seeking for unraveling how the virus factually enters the host cells. Some proteins on each side of the interaction between the virus and the host cells are involved as the major contributors to this process: 1- the nano-machine Spike protein on behalf of the virus, 2- angiotensin converting enzyme II, the mono-carboxypeptidase and the key component of renin angiotensin system on behalf of the host cell, 3- some host proteases and proteins exploited by SARS-CoV2, In this review, the complex process of SARS-CoV2 entrance into the host cells with the contribution of the involved host proteins as well as the sequential conformational changes in the Spike protein tending to increase the probability of complexification of the latter with angiotensin converting enzyme II, the receptor of the virus on the host cells, are discussed. Besides, the release of the catalytic ectodomain of angiotensin converting enzyme II as its soluble form in the extracellular space and its positive or negative impact on the infectivity of the virus are considered.

Pleural mesothelioma (PM) comprises three main subtypes: epithelioid, biphasic and sarcomatoid, which have a different impact on prognosis and treatment definition. However, PM subtyping can be complex given the inter- and intra-tumour morphological heterogeneity. We aim to use immunohistochemistry (IHC) to evaluate five markers (Mesothelin, Claudin-15, Complement Factor B, Plasminogen Activator Inhibitor 1, and p21-activated Kinase 4), whose encoding genes have been previously reported as deregulated among PM subtypes. Immunohistochemical expressions were determined in a case series of 73 PMs, and cut-offs for the epithelioid and non-epithelioid subtypes were selected. Further validation was performed on an independent cohort (30 PMs). For biphasic PM, the percentage of the epithelioid component was assessed, and IHC evaluation was also performed on the individual components separately. Mesothelin and Claudin-15 showed a good sensitivity (79% and 84%) and specificity (84% and 73%) for the epithelioid subtype. CFB and PAK4 had an inferior performance, with higher sensitivity (89% and 84%), but lower specificity (64% and 36%). In the biphasic group, all markers showed a different expression when comparing epithelioid with sarcomatoid areas. Mesothelin, Claudin-15 and CFB can be useful in subtype discrimination. PAI1 and PAK4 can improve component distinction in biphasic PM.

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.

Oral squamous cell carcinoma (OSCC) is one of the most prevalent human malignancies and a global health concern with a poor prognosis despite therapeutic advances, highlighting the need for a better understanding of its molecular background. The genomic landscape of OSCC is well-established and recent research has focused on miRNAs, which regulate gene expression and may be useful as non-invasive biomarkers. A plethora of findings regarding miRNA expression have been generated, posing challenges for their interpretation and identification of disease-specific molecules. Hence, we opted to identify the most important miRNA molecules by bridging genetics and epigenetics, focusing on the key genes implicated in OSCC development. Based on published reports, we have developed a custom panel of 15 major oncogenes and a second panel of 5 major tumor suppressor genes. Following a miRNA/target interaction analysis and a comprehensive study of the literature, we selected the miRNA molecules, which target the majority of each gene panel and are reported to be downregulated or upregulated in OSCC, respectively. As a result, miR-34a-5p, miR-155-5p, miR-124-3p, miR-1-3p and miR-16-5p appeared to be the most OSCC-specific. Their expressional patterns, their verified targets and the signaling pathways affected by their dysregulation in OSCC, are thoroughly discussed in this review.