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

Cardiovascular disease (CVD) comprises a group of heart and circulatory disorders, which are regarded as a global medical issue with high prevalence and mortality rates. Currently, vascular regenerative surgery represents the most employed therapeutic option to treat ischemic disorders, even though not all the patients are amenable to surgical revascularization. Therefore, more efficient therapeutic approaches are urgently required to promote neovascularization. Therapeutic angiogenesis represents an emerging strategy that aims at reconstructing the damaged vascular network by stimulating local angiogenesis and/or promoting de novo blood vessel formation according to a process known as vasculogenesis. Circulating endothelial colony forming cells (ECFCs), in turn, represent truly endothelial precursors able to aggregate into bidimensional tube networks and to originate patent vessels. Accordingly, ECFCs provide the most rationale and promising cellular candidate for therapeutic purposes. The current review provides a brief outline on the origin and characterization of ECFCs and a summary of the progress in preclinical studies aiming at assessing their efficacy in a variety of ischemic disorders, including AMI, PAD, ischemic brain disease and retinopathy. We also describe how to enhance the vasoreparative potential of ECFCs by boosting specific pro-angiogenic signalling pathways either pharmacologically or through gene manipulation. Taken together, these observations suggest that ECFCs represent a useful strategy to treat ischemic diseases.

Unanticipated errors in scientific research data can be attributed to the unwarranted assumption of uniformity in the polystyrene surface that is ubiquitously used in tissue culture flasks and dishes. We have shown that when adherent cells are subjected to fluid shear force, equivalent to rinsing the culture with a balanced salt solution, cells on some areas of the polystyrene surface will immediately rupture while still adherent on the surface. This heterogeneity on the polystyrene surface can cause unexpected variability in experimental results and in replicating experiments among labs. In this paper a novel quantitative method is described to measure the degree of heterogeneity on the polystyrene surface of tissue culture flasks. The results show significant variation among several brands of tissue culture flasks as well as large variability within the production lot of a manufacturer. The assay method involves loading the cells with a fluorescent marker that is released upon membrane rupture. Cell membrane rupture also causes the loss of marker proteins used in Westernblots. This novel assay method can be used to monitor the batch consistency and the manufacturing process of flasks and dishes. It may also be used to test new biomaterials.

Connexin 43 (Cx43) forms gap junctions that mediate the direct intercellular diffusion of ions and small molecules between adjacent cells. Cx43 displays both pro- and anti-tumorigenic properties, but the mechanisms underlying these characteristics are not fully understood. Tunneling nanotubes (TNTs) are long and thin membrane projections that connect cells, facilitating the exchange of not only small molecules, but also larger proteins, organelles, bacteria, and viruses. Typically, TNTs exhibit increased formation under conditions of cellular stress and are more prominent in cancer cells, where they are generally thought to be pro-metastatic and to provide growth and survival advantages. Cx43 has been described in TNTs, where it is thought to regulate small molecule diffusion through gap junctions. Here, we developed a high-fidelity CRISPR/Cas9 system to knockout (KO) Cx43. We found that loss of Cx43 expression was associated with significantly reduced TNT length and number in breast cancer cell lines. Notably, secreted factors present in conditioned medium stimulated TNTs more potently when derived from Cx43-expressing cells than from KO cells. Moreover, TNT formation was significantly induced by inhibition of several key cancer signaling pathways that both regulate Cx43 and are regulated by Cx43, including RhoA kinase (ROCK), protein kinase A (PKA), focal adhesion kinase (FAK), and p38. Intriguingly, drug-induced stimulation of TNTs was more potent in Cx43 KO cells than in wild-type cells. In conclusion, this work describes a novel non-canonical role for Cx43 in regulating TNTs, identifies key cancer signaling pathways that regulate TNTs in this setting, and provides mechanistic insight into a pro-tumorigenic role of Cx43 in cancer.

Methylenetetrahydrofolate reductase (MTHFR), an enzyme expressed in mammalian testes exerts direct effect on spermatogenesis; however, its protein characteristics in bovine testes remain unknown. Here, we analysed bovine testicular structure, MTHFR bioinformatics profile, mRNA, and protein expression characteristics in yellow-cattle (y-c) and yak testis using histological procedures, bioinformatics analysis, qRT-PCR, and western blot. Testes from 13 bovines, ≤ 2 years juvenile (y-c, n = 3; yak, n=3) and ≥ 3 years adult (y-c, n = 3; yak, n = 4) were collected and analysed. Anatomical characteristics of testis in y-c and yak were similar except the weight or size for which that of y-c was significantly higher or greater than yak. In y-c, an open reading frame (ORF) for 2600 nucleotides sequence, encoding 655 amino acids showed high homology with zebu cattle (99.51%) and wild yak (98.68%). Secondary and 3D protein structures were similar to that of humans with differences in number of nucleotides, amino acids, and some physico-chemical characteristics. MTHFR mRNA expression in y-c and yak were significantly higher in adult testes compared with juvenile ones. However, its protein expression was higher but not statistically significant in adult y-c and yak compared to the juvenile ones. The highlights and inferences of these and other findings are discussed.

Metazoans have an elaborate and functionally segmented body. It evolves from a single cell by systematic divisions. Metazoans attain structural complexity with exquisite precision, which is a molecular mystery. The indispensable role of centrioles in cell division and ciliogenesis can shed insight into this riddle. Cell division helps in growth of the body and is a highly regulated and integrated process. Its errors cause malignancies. The cell mass is organized during organogenesis. Prior to it, the centrioles are retrieved from the cell cycle to initiate ciliogenesis. The cilia-modulated developmental signaling pathways elaborate the body plan. The secluded compartment of the cilium reduces noise during signaling and is essential for a precise body plan development. The dysfunctional centrioles and cilia can distort body plan. Thus, centriole has a dual role in growth and cellular organization. This concept review analyses the comprehensive interactome and the key domain features (like C2 domain) of molecules which connect and disarm the centriole from the cell cycle and ciliogenesis by switching on or off the essential regulators of the pathways. The concentration of these signaling pathways at the centriole reinforces the hypothesis that centriole is the molecular workstation to carve out structural design and complexity in metazoans.

The consequences of Sickle Cell Disease (SCD), including ongoing hematopoietic stress and hemolysis, vascular damage and chronic therapies , such as blood transfusions and Hydroxyurea on hematopoietic stem and progenitor cell (HSPC) have not been characterized. We have quantified the frequencies of nine HSPC populations by flow cytometry in the peripheral blood of pediatric and adult patients stratified by treatment and controls. We observed broad differences between SCD patients and healthy controls. SCD is associated with 10 to 20-fold increase in CD34dim cells, and two to five-fold more CD34bright cells, a depletion in Megakaryocyte-Erythroid Progenitors and an increase in hematopoietic stem cells, when compared to controls. SCD is also associated with abnormal expression of CD235a and by very high levels of expression of the CD49f antigen. These findings were present to varying degrees in all patients, whether or not they were naïve or on chronic therapy. HU treatment tended to normalizes many of these parameters. Chronic stress erythropoiesis, inflammation caused by SCD and hydroxyurea therapy have long been suspected of causing premature aging of the hematopoietic system, and potentially increasing the risk of hematological malignancies. An important finding of this study was that the observed concentration of CD34bright cells and of all the HSPCs decreased logarithmically with time of treatment with HU. This correlation was independent of age and specific to HU treatment. Although the number of circulating HSPCs is influenced by many parameters, our findings suggest that HU treatment may decrease premature aging and hematologic malignancy risk compared to the other therapeutic modalities in SCD.

Multi-step evolution characterizes the carcinogenesis of GI malignancies, where the disease progresses through the accrual of genetic mutations, co-option of multiple host cellular factors and the acquisition of increasingly aggressive pathological features. Ensuing cancer complexity and plasticity are challenges for therapeutic approaches. On the other hand, disease inception is often triggered by a defined oncogenic event, which leads to escalating effects on gene expression, cellular metabolism, chromatin organization and ultimately cell identity. Decoding this molecular cascade of events is crucial to design strategies for early diagnosis as well as to tackle tumor onset. To this aim, the in vitro culture of non-transformed epithelial cells represents a major technical challenge. Genetic studies, combined with lineage-tracing experiments demonstrated that pancreatic cancerous lesions originate from acinar cells, a specialized cell type in the pancreatic epithelium, following mutations of the KRAS oncogene. Ex vivo culture of pancreatic acinar cells has allowed the identification of early mutant KRAS-driven alterations. Primary acinar cells survive in vitro in organoid-like 3D spheroids, which can recapitulate histological and molecular features of disease initiation. Here, we will discuss the isolation and culture of primary mouse pancreatic acinar cells, providing and historical and technical perspective. Impact for pancreatic cancer research will also be debated; in particular we will dissect the role of KRAS signaling in driving oncogenic transformation and highlight differences with canonical 2D culture of established cancer cell lines.

The study of neurodegenerative diseases using pluripotent stem cells requires new methods to assess neurodevelopment and neurodegeneration of specific neuronal subtypes. The cholinergic system, characterized by its use of the neurotransmitter acetylcholine, is one of the first to degenerate in Alzheimer’s disease and is also affected in frontotemporal dementia. We developed a differentiation protocol to generate basal forebrain cholinergic neurons (BFCNs) from induced pluripotent stem cells (iPSCs) aided by the use of small molecule inhibitors and growth factors. Ten iPSC lines were successfully differentiated into BFCNs using this protocol. The neuronal cultures were characterised through RNA and protein expression, and functional analysis of neurons was confirmed by whole-cell patch clamp. We have developed a reliable protocol using only small molecule inhibitors and growth factors, while avoiding transfection or cell sorting methods, to achieve a BFCN culture that expresses the characteristic markers of cholinergic neurons.

Nigella sativa, Carica papaya and Boswellia sacra are medicinal plants in the commonly used in folkloric medicine due to the presence of its immense therapeutic properties. Fifty (50) female albino mice weighing between 15-22g were divided into five groups of 10 mice each. Animal in group 1 served as control group and were administered distilled water while animal in group 2 were given 2ml of cisplatin (orally). Animal in group 3-5 were given orally; 100 mg/kg (low dose), 200 mg/kg (medium dose) and 400 mg/kg (high dose) of triherbal preparation. The feeding regimens lasted for 28 days. After 28 days, mammary gland and blood samples were collected for haematological and antioxidant analysis. The triherbal formula decreased the GSH and MDA levels of mice treated with 100 mg/kg and 400 mg/kg doses compare to control. The measurement of total protein content, SOD and CAT increased in treated animals compared to control. However, RBC (Red Blood Cell) counts significantly decreased in the low, medium and high dose groups (0.95±0.08, 6.57±0.08 and 3.55±0.55 x 106 cells/mm3 respectively) compared to control (7.34±0.40) at P<0.05. Also, significant decreases (P<0.05) in the level of the total WBC (White Blood Cell) count, platelet count, PCV (Packed Cell Volume) and Hb (haemoglobin) concentration were observed. The decreases were dose dependent. The MCH (Mean Corpuscular Haemoglobin) and MCHC (Mean Corpuscular Haemoglobin Concentration) except MCV (Mean Corpuscular Volume) significantly decreased in treated group only. The triherbal formulation exhibited significant antioxidant activities showing increased levels of SOD, CAT and Protein content due to activation of the enzyme involve in detoxification of free radicals and decreased in the level of GSH and MDA due to accumulation of peroxides and H2O2. Also, decreased in haematological parameters due to the presence of phytochemicals such as phenol, resins, saponins, sterols, tannis and terpenes in the triherbal formula. Therefore, it has potential to induce haematotoxicity hence consumption of high concentrations should be discouraged.

The identification and molecular characterization of cellular hierarchies in complex tissues is key to understanding both normal cellular homeostasis and tumorigenesis. The mammary epithelium is a heterogeneous tissue consisting of two main cellular compartments, an outer basal layer containing myoepithelial cells and an inner luminal layer consisting of estrogen receptor-negative (ER⁻) ductal cells and secretory alveolar cells (in the fully functional differentiated tissue) and hormone-responsive estrogen receptor-positive (ER⁺) cells. Recent publications have used single-cell RNA-sequencing (scRNA-seq) analysis to decipher epithelial cell differentiation hierarchies in human and murine mammary glands, and reported the identification of new cell types and states based on the expression of the luminal progenitor cell marker KIT (c-Kit). These studies allow for comprehensive and unbiased analysis of the different cell types that constitute a heterogeneous tissue. Here we discuss scRNA-seq studies in the context of previous research in which mammary epithelial cell populations were molecularly and functionally characterized, and identified c-Kit⁺ progenitors and cell states analogous to those reported in the recent scRNA-seq studies.

Alzheimer’s disease is a prominent neurological disorder, which leads to progressive dementia. The microtubule-associated protein Tau is been considered as one of the major causes of Alzheimer’s disease. Physiologically Tau assists in the stabilization of microtubules, contrary to this the pathological state of Tau results in the formation of neurotoxic tangles of Tau. The posttranslational modifications, such as GSK-3β-mediated Tau phosphorylation results in the generation of Tau pathology. Neuroinflammation generated in Alzheimer’s disease, contributes to elevated body temperature. The aim of present work is to study the effect of high temperature on Tau phosphorylation. The neuroblastoma cells were exposed to heat stress for 40 minutes. The immunofluorescence and western blot studies suggested that high temperature increases the levels of GSK-3β in cells. Heat stressed cells was also observed to have elevated levels of phosphorylated Tau. Additionally, heat stressed cells found to have modulated nuclear transport as the level of Ran was reduced. The results of present work suggested that increased temperature could be considered as a risk factor in Alzheimer’s disease as it elevated the GSK-3β levels in cells thus, resulting in increased Tau phosphorylation.

This article challenges the notion of the randomness of mutations in eukaryotic cells by unveiling stress-induced human non-random genome editing mechanisms. To account for the existence of such mechanisms, I have developed molecular concepts of the cell environment and cell environmental stressors and, making use of a large quantity of published data, hypothesized the origin of some crucial biological leaps along the evolutionary path of life on Earth under the pressure of natural selection, in particular, 1) virus-cell mating as a primordial form of sexual recombination and symbiosis; 2) Lamarckian CRISPR-Cas systems; 3) eukaryotic gene development; 4) antiviral activity of retrotransposon-guided mutagenic enzymes and finally; 5) the exaptation of antiviral mutagenic mechanisms to stress-induced genome editing mechanisms directed at “hypertranscribed” endogenous genes. Genes transcribed at their maximum rate (hypertranscribed), yet still unable to meet new chronic environmental demands generated by “pollution”, are inadequate and generate more and more intronic retrotransposon transcripts. In this scenario, RNA-guided mutagenic enzymes (e.g. AID/APOBECs), which have been shown to bind to retrotransposon RNA-repetitive sequences, would be surgically targeted by intronic retrotransposons on opened chromatin regions of the same “hypertranscribed” genes. RNA-guided mutagenic enzymes may therefore “Lamarkianly” generate single nucleotide polymorphisms (SNP) and copy number variations (CNV), as well as transposon transposition and chromosomal translocations in the restricted areas of hyperfunctional and inadequate genes, leaving intact the rest of the genome. CNV and SNP of hypertranscribed genes may allow cells to surgically explore a new fitness scenario, which increases their adaptability to stressful environmental conditions. Like the mechanisms of immunoglobulin somatic hypermutation, non-random genome editing mechanisms may generate several cell mutants, and those codifying for the most environmentally-adequate proteins would have a survival advantage and would therefore be Darwinianly selected. Non-random genome editing mechanisms represent a link between environmental changes and biological novelty and plasticity, and provide a molecular basis to reconcile gene-centered and “ecological” views of evolution.

To maintain proteostasis, cells must integrate information and activities that supervise protein synthesis, protein folding, conformational stability, and also protein degradation. Extrinsic and intrinsic conditions can both impact on normal proteostasis, causing the appearance of proteotoxic stress. Initially, proteotoxic stress elicits adaptive responses aimed to restore proteostasis, allowing cells to survival the stress condition. However, if the proteostasis restoration fails, a permanent and sustained proteotoxic stress can be deleterious and cell death ensues. Many cancer cells convive with high levels of proteotoxic stress and this condition could be exploited in a therapeutic perspective. Understanding the cell death pathways engaged by proteotoxic stress is instrumental to better hijack the proliferative fate of cancer cells.

The growth and dynamics of multicellular tissues involve tightly regulated and coordinated morphogenetic cell behaviours, such as shape changes, movement, and division, which are governed by subcellular machinery and involve coupling through short- and long-range signals. A key challenge in the fields of developmental biology, tissue engineering and regeneration is to understand how relationships between scales produce emergent tissue-scale behaviours. Recent advances in molecular biology, live-imaging and ex vivo techniques have revolutionised our ability to study these processes experimentally. To fully leverage these techniques and obtain a more comprehensive understanding of the causal relationships underlying tissue dynamics, computational modelling approaches are increasingly spanning multiple spatial and temporal scales, and are coupling cell shape, growth, mechanics and signalling. Yet such models remain technically challenging: modelling at each scale requires different areas of technical skills, while integration across scales necessitates the solution to novel mathematical and computational problems. This review aims to summarise recent progress in multiscale modelling of multicellular tissues and to highlight ongoing challenges associated with the construction, implementation, interrogation and validation of such models.

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

Life-threatening inflammatory conditions such as acute respiratory distress syndrome or sepsis often go hand in hand with severe vascular leakage. During inflammation, endothelial cell integrity and intact barrier function are crucial to limit leukocyte and plasma extravasation. Prostaglandin D₂ (PGD₂) is a potent inflammatory lipid mediator with vasoactive properties. It has been suggested that PGD^­₂ is involved in the regulation of endothelial barrier function; however, it is unclear whether this is also true for primary human pulmonary microvascular endothelial cells. Furthermore, as PGD₂ is a highly promiscuous ligand, we set out to determine which receptors are important in human pulmonary endothelial cells. In the current study, we found that PGD₂ and the DP1 agonist BW245c potently strengthened pulmonary and dermal microvascular endothelial cell barrier function and protected against thrombin-induced barrier disruption. Yet surprisingly, these effects were mediated only to a negligible extent via DP1 receptor activation. In contrast, we observed that the EP4 receptor was most important and mediated the barrier enhancement by PGD₂ and BW245c. These data demonstrate a novel mechanism by which PGD₂ may modulate inflammation and emphasizes the role of EP4 receptors in human endothelial cell function.

Astronauts at are risk of losing 1.0 – 1.5% of their bone mass for every month they spend in space despite their adherence to high impact exercise training programs designed to preserve the musculoskeletal system. This article reviews the basics of bone formation and resorption and details how exposure to microgravity or simulated microgravity affects the structure and function of osteoblasts, osteocytes, osteoclasts, and their mesenchymal and hematologic stem cell precursors. It details the critical roles that insulin-like growth facor-1 and its receptor IGFR1 play in maintaining bone homeostasis and how exposure of bone cells to microgravity affects the function of these growth factors. Lastly, it discusses the potential of tumor necrosis factor-related apoptosis-inducing ligand, syncytin-A, and sclerostin inhibitors and recombinant IGF-1 as a bone-saving treatment for astronauts in space and during their colonization of the Moon.

Myelin protein zero (P0), a type I transmembrane protein, is the most abundant protein in peripheral nervous system (PNS) myelin – the lipid-rich, periodic structure that concentrically encloses long axonal segments. Schwann cells, the myelinating glia of the PNS, express P0 throughout their development until the formation of mature myelin. In the intramyelinic compartment, the immunoglobulin-like domain of P0 bridges apposing membranes together via homophilic adhesion, forming a dense, macroscopic ultrastructure known as the intraperiod line. The C-terminal tail of P0 adheres apposing membranes together in the narrow cytoplasmic compartment of compact myelin, much like myelin basic protein (MBP). In mouse models, the absence of P0, unlike that of MBP or P2, severely disturbs the formation of myelin. Therefore, P0 is the executive molecule of PNS myelin maturation. How and when is P0 trafficked and modified to enable myelin compaction, and how disease mutations that give rise to incurable peripheral neuropathies alter the function of P0, are currently open questions. The potential mechanisms of P0 function in myelination are discussed, providing a foundation for the understanding of mature myelin development and how it derails in peripheral neuropathies.

Infection by the Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) results in the novel coronavirus disease COVID-19, which has posed a serious threat globally. Infection of SARS-CoV-2 during pregnancy is associated with complications like preterm labor and premature rupture of membranes; a proportion of neonates born to the infected mothers are also positive for the virus. During pregnancy, the placental barrier protects the fetus from pathogens and ensures healthy development. However, whether or not SARS-CoV-2 can infect the placenta is unknown. Herein, utilizing single-cell RNA-seq data, we report that the SARS-CoV-2 binding receptor ACE2 and the S protein priming protease TMPRSS2 are co-expressed by a subset of syncytiotrophoblasts (STB) in the first trimester and extra villous trophoblasts (EVT) in the second trimester human placenta. The ACE2- and TMPRSS2-positive (ACE2+TMPRSS2+) placental subsets express mRNA for proteins involved in viral budding and replication. These cells also express mRNA for proteins that interact with SARS-CoV-2 structural and non-structural proteins in the host cells. We also discovered unique signatures of genes in ACE2+TMPRSS2+ STBs and EVTs. The ACE2+TMPRSS2+ STBs are highly differentiated cells and express genes involved mitochondrial metabolism and glucose transport. The second trimester ACE2+TMPRSS2+ EVTs are enriched for markers of endovascular trophoblasts. Further, both these subtypes abundantly expressed genes in Toll like receptor pathway, the second trimester EVTs (but not first trimester STBs) are also enriched for component of the JAK-STAT pathway that drive inflammation. To conclude, herein we uncovered the cellular targets for SARS-CoV-2 entry and show that these cells can potentially drive viremia in the developing human placenta. Our results provide a basic framework towards understanding the paraphernalia involved in SARS-CoV-2 infections in pregnancy.

The generation of isogenic induced pluripotent stem cell (iPSC) lines using CRISPR-Cas9 technology is a technically challenging, time-consuming process with variable efficiency. Here we use fluorescence-activated cell sorting (FACS) to sort biallelic CRISPR-Cas9 edited single-cell iPS clones into high-throughput 96-well microtiter plates. We used high-content screening (HCS) technology and generated an in-house developed algorithm to select the correctly edited isogenic clones for continued expansion and validation. In our model we have gene-corrected the iPSCs of a Parkinson’s disease (PD) patient carrying the autosomal dominantly inherited heterozygous c.88G>C mutation in the SNCA gene, which leads to the pathogenic p.A30P form of the alpha-synuclein protein. Undertaking a PCR restriction-digest mediated clonal selection strategy prior to sequencing, we were able to post-sort validate each isogenic clone using a quadruple screening strategy. Subsequent transfection with mRNA encoding excision-only transposase allows for the generation of footprint-free isogenic iPSC lines. These monoclonal isogenic iPSC lines retain a normal molecular genotype, express pluripotency markers and have the ability to differentiate into the three germ layers. This combinatory approach of FACS, HCS and post-sorted restriction digestion facilitates the generation of isogenic cell lines for disease modelling to be scaled-up on an automated platform.

How to align leg segments between the four groups of arthropods (insects, crustaceans, myriapods, and chelicerates) has tantalized researchers for over a century. By comparing the loss-of-function phenotypes of leg patterning genes in diverged arthropod taxa, including a crustacean, insects, and spiders, we show that all arthropod legs can be aligned in a one-to-one fashion. We propose a model wherein insects incorporated two proximal leg segments into the body wall, which moved the ancestral leg lobe (exite) up onto the back to later form wings. For myriapods and chelicerates with seven leg segments, it appears that one proximal leg segment was incorporated into the body wall. According to this model, the chelicerate exopod and the crustacean exopod emerge from different leg segments, and are therefore proposed to have arisen independently. A framework for how to align arthropod appendages now opens up a powerful system for studying the origins of novel structures, the plasticity of developmental fields, and convergent evolution.

Bacteria participate in a wide diversity of symbiotic associations with eukaryotic hosts that require precise interactions for bacterial recognition and persistence. Most commonly, host-associated bacteria interfere with host gene expression to modulate the immune response to the infection. However, many of these bacteria also interfere with host cellular differentiation pathways to create a hospitable niche, resulting in the formation of novel cell types, tissues, and organs. In both of these situations, bacterial symbionts must interact with eukaryotic regulatory pathways. Here, we detail what is known about how bacterial symbionts, from pathogens to mutualists, control host cellular differentiation across the central dogma, from epigenetic chromatin modifications, to transcription and mRNA processing, to translation and protein modifications. We identify four main trends from this survey. First, mechanisms for controlling host gene expression appear to evolve from symbionts co-opting cross-talk between host signalling pathways. Second, symbiont regulatory capacity is constrained by the processes that drive reductive genome evolution in host-associated bacteria. Third, the regulatory mechanisms symbionts exhibit correlate with the cost/benefit nature of the association. And, fourth symbiont mechanisms for interacting with host genetic regulatory elements are not bound by native bacterial capabilities. Using this knowledge, we explore how the ubiquitous intracellular Wolbachia symbiont of arthropods and nematodes may modulate host cellular differentiation to manipulate host reproduction. Our survey of the literature on how infection alters gene expression in Wolbachia and its hosts revealed that, despite their intermediate-sized genomes, different strains appear capable of a wide diversity of regulatory manipulations. Given this and Wolbachia’s diversity of phenotypes and eukaryotic-like proteins, we expect that many symbiont-induced host differentiation mechanisms will be discovered in this system.

Stem cells are currently being used in many clinical trials for regenerative purposes. These are promising results for stem cells in the treatment of several diseases, including cancer. Nevertheless, there are still many variables which should be addressed before the application of stem cells for cancer treatment. One approach should be to establish well-characterized therapeutic stem cell banks to minimize the variation in results from different clinical trials and facilitate their effective use in basic and translational research.

Bacteria participate in a wide diversity of symbiotic associations with eukaryotic hosts that require precise interactions for bacterial recognition and persistence. Most commonly, host-associated bacteria interfere with host gene expression to modulate the immune response to the infection. However, many of these bacteria also interfere with host cellular differentiation pathways to create a hospitable niche, resulting in the formation of novel cell types, tissues, and organs. In both of these situations, bacterial symbionts must interact with eukaryotic regulatory pathways. Here, we detail what is known about how bacterial symbionts, from pathogens to mutualists, control host cellular differentiation across the central dogma, from epigenetic chromatin modifications, to transcription and mRNA processing, to translation and protein modifications. We identify four main trends from this survey. First, mechanisms for controlling host gene expression appear to evolve from symbionts co-opting cross-talk between host signalling pathways. Second, symbiont regulatory capacity is constrained by the processes that drive reductive genome evolution in host-associated bacteria. Third, the regulatory mechanisms symbionts exhibit correlate with the cost/benefit nature of the association. And, fourth symbiont mechanisms for interacting with host genetic regulatory elements are not bound by native bacterial capabilities. Using this knowledge, we explore how the ubiquitous intracellular Wolbachia symbiont of arthropods and nematodes may modulate host cellular differentiation to manipulate host reproduction. Our survey of the literature on how infection alters gene expression in Wolbachia and its hosts revealed that, despite their intermediate-sized genomes, different strains appear capable of a wide diversity of regulatory manipulations. Given this and Wolbachia’s diversity of phenotypes and eukaryotic-like proteins, we expect that many symbiont-induced host differentiation mechanisms will be discovered in this system.

The tumor organoid (tumoroid) model in three-dimensional (3D) culture systems has been developed to reflect more closely the in vivo tumors than 2D-cultured tumor cells. Notably, extracellular vesicles (EVs) are efficiently collectible from the culture supernatant of gel-free tumoroids. Matrix metalloproteinase (MMP) 3 is a multi-functional factor playing crucial roles in tumor progression. However, roles of MMP3 within tumor growth and EVs have not unveiled. Here, we investigated the protumorigenic roles of MMP3 on integrities of tumoroids and EVs. We generated MMP3-knockout (KO) cells using the CRISPR/Cas9 system from rapidly metastatic LuM1 tumor cells. Moreover, we established fluorescent cell lines with palmitoylation signal-fused fluorescent proteins (tdTomato and enhanced GFP). Then we confirmed the exchange of EVs between cellular populations and tumoroids. LuM1-tumoroids released large EVs (300-1000 nm) and small EVs (50-200 nm) while the knockout of MMP3 resulted in the additional release of broken EVs from tumoroids. The loss of MMP3 leads to a significant reduction in tumoroid size and to the development of the necrotic area within tumoroids. MMP3 and CD9 (a category-1 EV marker tetraspanin protein) were significantly down-regulated in MMP3-KO cells and their EV fraction. These weakened phenotypes by MMP3 KO were markedly rescued by the addition of MMP3-rich EVs or conditioned medium (CM) collected from LuM1-tumoroids, which caused a dramatic rise in the expression of MMP3, CD9, and Ki-67 (a marker of proliferating cells) in the MMP3-null/CD9-low tumoroids. Notably, MMP3 enriched in tumoroids-derived EVs and CM deeply penetrated into recipient MMP3-KO tumoroids, resulting in a remarkable enlargement of solid tumoroids, while MMP3-null EVs did not. These data demonstrate that EVs can mediate molecular transfer of MMP3 resulting in increasing the proliferation and CD9+ tumorigenesis, indicating crucial roles of MMP3 in tumor progression.

SARS-CoV2 (corona virus) has spread globally at an unprecedented rate; so far, increasing SARS-CoV2-infected individuals have been identified. Although the situation in China is improving and is currently under control, the outbreak in other countries and its pandemic management is only beginning to develop. Based on 154 SARS-CoV2 genome sequence analyses, we used receptor–ligand docking to identify one potential point mutation (V354F) on the spike structure which enhances spike binding to ACE2 receptors underlying potential super infection. Importantly, the V354F site on spike S1 had been identified in 5/10 infected French patients living in Paris, who sharing 100% identical SARS-CoV2 genomes. With Covid-19 cases increasing rapidly in France that could lead to a new explosion, we suggest that the French government should identify all potential super spreaders and treat them accordingly. In summary, our study provides on of the measures to avoid the potential second worldwide explosion of SARS-CoV2.

Coronavirus disease 2019 (COVID-19) is caused by infection with the 2019 novel coronavirus 2 (2019-nCoV, now referred to as SARS-CoV-2). COVID-19 has become a global pandemic since its outbreak at the end of Dec 2019. COVID-19 could lead to severe acute respiratory disease, especially to those who have reduced immunity. Binding of the viral Spike protein (S) to its receptor ACE2 (Angiotensin Converting Enzyme 2) on the surface of target cells has been proven to be key for virus entry and infection. Although ACE2 expression in the respiratory system is necessary for pneumonia infection by SARS-CoV-2, the regulation of ACE2 gene expression remains poorly investigated, especially for patients that are in pre-pathological conditions. Here, by analyzing The Gene Expression Omnibus (GEO) database, we investigated the expression regulation of ACE2 in various kinds of primary epithelial cells from the respiratory system after influenza A or respiratory Syncytial Virus (RSV) infection. Our analyses reveal that infection of influenza A, RSV or influenza vaccines greatly increased ACE2 expression, suggesting that influenza viral infection could represent a high risk factor for developing COVID-19. We also found that the regulatory effect of influenza A virus on ACE2 expression is associated with activation of the interferon beta-induced pathway and viral RNA-activated host response. Together, our data provide a theoretical framework for clinical classification for SARS-CoV-2 infection susceptibility and could be used for future prevention and therapy treatment for COVID-19.

Dietary vitamin A/all-trans retinol/ROL plays a critical role in human vision. ROL circulates bound to the plasma retinol-binding protein (RBP4) as RBP4-ROL. In the eye, the STRA6 membrane receptor binds to circulatory RBP4 and internalizes ROL. STRA6 is however not expressed in systemic tissues, where there is high-affinity RBP4 binding and ROL uptake. We tested the hypothesis, that the second retinol-binding protein 4 receptor 2 (Rbpr2) which is highly expressed in systemic tissues of zebrafish and mouse, contains a functional RBP4 binding domain, critical for ROL transport. As for STRA6, modeling and docking studies confirmed three conserved RBP4 binding residues in zebrafish Rbpr2. In cell culture studies, disruption of the RBP4 binding residues on Rbpr2 almost completely abolished uptake of exogenous vitamin A. CRISPR generated rbpr2-RBP4 domain zebrafish mutants showed microphthalmia, shorter photoreceptor outer segments, and decreased opsins, that were attributed to impaired ocular retinoid content. Injection of WT-Rbpr2 mRNA into rbpr2 mutant or all-trans retinoic acid treatments rescued the mutant eye phenotypes. In conclusion, zebrafish Rbpr2 contains a putative extracellular RBP4-ROL ligand-binding domain, critical for yolk vitamin A transport to the eye for ocular retinoid production and homeostasis, for photoreceptor cell survival.

Pre-implantation horse conceptuses require nutrients and signals from histotroph, the composition of which is regulated by luteal progesterone and conceptus-secreted factors. To distinguish progesterone and conceptus effects we shortened the period of endometrial progesterone-priming by asynchronous embryo transfer. Day 8 embryos were transferred to synchronous (day 8) or asynchronous (day 3) recipients, and RNA sequencing was performed on endometrium and conceptuses recovered 6 and 11 days later (embryo days 14 and 19). Asynchrony resulted in many more differentially expressed genes (DEGs) in conceptus membranes (3473) than endometrium (715). Gene ontology analysis identified upregulation in biological processes related to organogenesis and preventing apoptosis in synchronous conceptuses on day 14, and in cell adhesion and migration on day 19. Asynchrony also resulted in large numbers of DEGs related to ‘extracellular exosome’. In endometrium, genes involved in immunity, the inflammatory response, and apoptosis regulation were upregulated during synchronous pregnancy and, again, many genes related to extracellular exosome were differentially expressed. Interestingly, only 14 genes were differentially expressed in endometrium recovered 6 days after synchronous versus 11 days after asynchronous transfer (day 14 recipient in both). Among these, KNG1 and IGFBP3 were consistently up-regulated in synchronous endometrium. Furthermore bradykinin, an active peptide cleaved from KNG1, stimulated prostaglandin release by cultured trophectoderm cells. The horse conceptus thus responds to a negatively asynchronous uterus by extensively adjusting its transcriptome, whereas the endometrial transcriptome is modified only subtly by a more advanced conceptus.

In December 2019, a novel coronavirus (SARS-CoV-2) was identified in patients with pneumonia (called COVID-19) in Wuhan, Hubei Province, China. SARS-CoV-2 shares high sequence similarity and uses the same cell entry receptor, angiotensin-converting enzyme 2 (ACE2), as does severe acute respiratory syndrome coronavirus (SARS-CoV). Several studies have provided bioinformatic evidence of potential routes for SARS-CoV-2 infection in respiratory, cardiovascular, digestive and urinary systems. However, whether the reproductive system is a potential target of SARS-CoV-2 infection has not been determined. Here, we investigate the expression pattern of ACE2 in adult human testis at the level of single-cell transcriptomes. The results indicate that ACE2 is predominantly enriched in spermatogonia, Leydig and Sertoli cells. Gene ontology analyses indicate that GO categories associated with viral reproduction and transmission are highly enriched in ACE2-positive spermatogonia while male gamete generation related terms are down-regulated. Cell-cell junction and immunity related GO terms are increased in ACE2-positive Leydig and Sertoli cells, but mitochondria and reproduction related GO terms are decreased. These findings provide evidence that human testes are a potential target of SARS-CoV-2 infection which may have significant impact on our understanding of the pathophysiology of this rapidly spreading disease.

In this review, we discuss the insulin resistance (IR) and its development in the insulin target tissues that leads to diabetes. Also, we highlight the use of induced pluripotent stem cells (iPSCs) to understand the mechanisms underlying the development of IR. IR is associated with several metabolic disorders, including type 2 diabetes (T2D). The development of IR in insulin target tissues involves genetic and acquired factors. Persons at genetic risk for T2D tend to develop IR several years before glucose intolerance. Although there are currently several mouse models for both IR and T2D that had provided a lot of information about the disease, these models cannot recapitulate all the aspects of this complex disease as seen in each individual. Patient-specific iPSCs can overcome the hurdles faced with the classical mouse models for studying IR. iPSC technology can generate cells genetically identical to IR individuals, which can help in distinguishing between genetic and acquired defects in insulin sensitivity. Combining the technologies of the genome editing and iPSCs may provide important information about the inherited factors underlying the development of different forms of IR. Further studies are required to fill the gaps in understanding the pathogenesis of IR and diabetes.

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.

The signal transducer and activator of transcription 3 (Stat3) is activated in response to the phosphorylation of Y705 (pStat3) and has the dual function of signal transduction and activation of transcription. Our previous study suggested that pStat3 is functional during oocyte maturation when transcription is silenced. Therefore, we speculated that pStat3 may have another function. Immunocytochemical analysis revealed that pStat3 emerges at the microtubule asters and spindle and then localizes at the spindle poles concomitant with a Pericentrin during mouse oocyte maturation. When we examined conditionally knocked out Stat3−/− oocytes, we detected Stat3 and pStat3 proteins. The localization of the pStat3 was the same as that of Stat3+/+ oocytes, and the oocyte maturation proceeded normally, suggesting that pStat3 was still functioning. The oocytes were treated either with the Stat3 specific inhibitors, Stattic and BP-1-102, or anti-pStat3 antibody injection. This caused significant abnormal spindle assembly and chromosome mis-location in a dose-dependent manner, in which the pStat3 was either negative or localized improperly. Moreover, development of pre-implantation stage embryos derived from inhibitor-treated oocytes was also hampered significantly after in vitro fertilization. These findings indicate a novel function of pStat3 involved in spindle assembly.

Diabetes mellitus (DM) is one of the most prevalent metabolic disorders. In order to replace the function of the destroyed pancreatic beta cells in diabetes, islet transplantation is the widely practiced treatment; however, it has several limitations. As an alternative approach, human pluripotent stem cells (hPSCs) can provide an unlimited source of pancreatic cells that have the ability to secrete insulin in response to high blood glucose level. However, determination of the appropriate pancreatic lineage candidate for the purpose of cell therapy for treatment of diabetes is still debated upon. While hPSC-derived beta cells are perceived as the ultimate candidate, the efficiency needs further improvement in order to obtain a sufficient number of glucose responsive β-cells for transplantation therapy. On the other hand, hPSC-derived pancreatic progenitors can be efficiently generated in vitro and can further mature into glucose responsive beta cells in vivo after transplantation. Herein, we discuss the advantages and predicted challenges associated with the use of each of the two pancreatic lineage products for diabetes cell therapy. Furthermore, we address co-generation of functionally relevant islet cell subpopulations and structural properties contributing to glucose responsiveness of beta cells, as well as the available encapsulation technology for these cells.

Parkinson’s disease (PD) is a slowly progressing neurodegenerative disorder that is coupled to both widespread protein aggregation and to loss of substantia nigra dopamine (DA) neurons, resulting in a wide variety of motor and non-motor signs and symptoms. Recent findings suggest that the PD process is triggered several years before there is sufficient degeneration of DA neurons to cause onset of overt motor symptoms. According to this concept, the number of DA neurons present in the substantia nigra at birth could influence the time from the molecular triggering event until the clinical diagnosis with lower number of neurons at birth increasing the risk to develop the disease. Conversely, the risk for diagnosis would be reduced if the number of DA neurons is high at birth. In this commentary, we discuss the genetic and epigenetic factors that might influence the number of nigral DA neurons that each individual is born with and how these may be linked to PD risk.

Intermediate filaments constitute the third component of the cellular skeleton. Unlike actin and microtubule cytoskeletons, the intermediate filaments are composed of wide variety of structurally related proteins showing distinct expression patterns in tissues and cell types. Changes in expression patterns of intermediate filaments are often associated with cancer progression, in particular with phenotypes leading to increased cellular migration and invasion. In this review we will describe the role of vimentin intermediate filaments in cancer cell migration, cell adhesion structures, and metastasis formation. The potential for targeting vimentin in cancer treatment and the development of drugs targeting vimentin will be reviewed.

Corneal ulcer (CU) is an ophthalmopathy characterized by depression of the corneal surface with at least one stromal loss. CU is common in canine and feline species and is usually caused, among others, by trauma, infections, toxic contamination and endocrine disorders. They usually result from an increased inflammatory response and are associated with some clinical signs such as blepharospasm, photophobia, epiphora, pain and loss of corneal transparency. Despite advances in conventional and pharmacological therapy, in many cases indolent and recurrent ulcer treatments still lead to loss of visual acuity of the animal. This paper aims to report the effect of topical application of canine adipose tissue-derived mesenchymal stem cell (cATMSCs) as treatment of recurrent CU in a Poodle dog breed that showed clear difficulty in the healing process associated with diabetes. The animal was submitted to two applications of cATMSCs and showed improvement in the blepharospasm, conjunctival hyperemia, mucopurulent ocular secretion, photophobia, corneal opacity, chemosis, pigmentation, neovascularization, and pain parameters. Besides, Fluorescein test, Schirmer test and ocular fundus exam also showed improvement in their values concomitantly with lesion resolution. Due this, we showed that cATMSC therapy contribute to the regeneration of corneal tissue in CU and may contribute to the treatment to others ophthalmopathies.

Fucoidan is a brown algae-derived polysaccharide having several biomedical applications. This study simultaneously compares the anticancer activities of crude fucoidans from Fucus vesiculosus and Sargassum filipendula, and effects of low (LMW, 10-50kDa), medium (MMW, 50-100kDa) and high (HMW, >100kDa) molecular weight fractions of S. filipendula fucoidan against osteosarcoma cells. Glucose, fucose and acid levels were lower and sulphation was higher in F. vesiculosus crude fucoidan compared to S. filipendula crude fucoidan. MMW had highest the levels of sugars, acids and sulphation among molecular weight fractions. There was a dose dependent drop in focal adhesion formation and proliferation of cells for all fucoidan-types, but F. vesiculosus fucoidan and HMW had the strongest effects. G1-phase arrest was induced by F. vesiculosus fucoidan, MMW and HMW, however F. vesiculosus fucoidan treatment also caused accumulation in sub G1-phase. Mitochondrial damage occurred for all fucoidan-types, however F. vesiculosus fucoidan led to mitochondrial fragmentation. Annexin V/PI, TUNEL and cytochrome c staining confirmed stress induced apoptosis-like cell death for F. vesiculosus fucoidan but features of stress-induced necrosis-like cell death for S. filipendula fucoidans. There was also variation in penetrability of different fucoidans inside the cell. These differences in anti-cancer activity of fucoidans are applicable for osteosarcoma treatment.

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.

Despite the important role that the GH/IGF-I axis plays in vascular homeostasis, these kind of growth factors barely appear in articles addressing the neovascularization process. Currently, the vascular endothelium has turned to be considered as an authentic gland of internal secretion due to the wide variety of released factors and functions with local effect, including the paracrine/autocrine production of GH or IGF-I, for which the endothelium has specific receptors. In this comprehensive review, it will be described the evidence involving these proangiogenic hormones in arteriogenesis dealing with the arterial occlusion and making of them a potential therapy. It will be analyzed all those elements triggering the local and systemic production of GH/IGF-I and their possible role both in physiological and pathological conditions. The whole evidence will be combined with important data from the GHAS trial, in which GH or placebo were administrated to patients suffering from critical limb ischemia with no option for revascularization. We postulate that GH, alone or in combination, should be considered as a promising therapeutic agent for helping in the approach of the ischemic disease.

This article is a tribute to Lewis Wolpert on the occasion of the recent 50th anniversary of the publication of his article ‘Positional Information and the Spatial Pattern of Differentiation’. This tribute relates to another of his ideas: his early ‘Progress Zone’ timing model for limb development. Recent evidence is reviewed that a mechanism sharing features with this model patterns the main body axis in early vertebrate development. This tribute celebrates the golden era of Developmental Biology.

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

The Reeler mutation was described in mouse more than fifty year ago. Later, its causative gene (reln) was discovered in mouse, and its human orthologue (RELN) was demonstrated to be causative of lissencephaly 2 (LIS2) and about 20% of the cases of autosomal-dominant lateral temporal epilepsy (ADLTE). In both human and mice the gene encodes for a glycoprotein referred to as Reelin (Reln) that plays a primary role in neuronal migration during development and synaptic stabilization in adulthood. Besides LIS2 and ADLTE, RELN and/or other genes coding for the proteins of the Reln intracellular cascade have been associated more or less substantially to other conditions such as spinocerebellar ataxia type 7 and 37, VLDLR-associated cerebellar hypoplasia, PAFAH1B1-associated lissencephaly, autism and schizophrenia. According to their modalities of inheritances and with substantial differences among each other, these neuropsychiatric disorders can be modeled in the homozygous (reln-/-) or heterozygous (reln+/-) mouse. The usefulness of these mice as translational models is discussed, with focus on their construct and face validity. The latter is mainly treated directing the attention to the histological, neurochemical and functional observations in the cerebral cortex, hippocampus and cerebellum of Reeler mice and their human counterparts.

Except for a few species, the outer shape of vertebrates normally is characterized by bilateral symmetry. The inner organs, on the other hand, normally are arranged in bilaterally asymmetric patterns, which are of special importance for the normal function of the cardiovascular system of lung-breathing vertebrates. Deviations from the normal organ asymmetry can occur in the form of mirror imagery of the normal arrangement (situs inversus), or in the form of arrangements that have the tendency for development of bilateral symmetry, either in a pattern of bilateral left-sideness (left isomerism) or bilateral right-sidedness (right isomerism). The latter two forms of visceral situs anomalies are called “heterotaxy syndromes”. During the past 30 years, remarkable progress has been made in uncovering of the genetic etiology of heterotaxy syndromes. However, the pathogenetic mechanisms causing the spectrum of cardiovascular defects found in these syndromes remain poorly understood. In the present report, a spontaneous case of left cardiac isomerism found in a HH-stage 23 chick embryo is described. The observations made in this case suggest that hearts with left cardiac isomerism may have the tendency for development of a non-compaction cardiomyopathy caused by defective development of the proepicardium.

The vertebrate skeletal neuromuscular junction (NMJ) has long served as a model system for studying synapse structure, function and development. Over the last several decades a neuron-specific isoform of agrin, a heparan sulfate proteoglycan, has been identified as playing a central role in synapse formation at all vertebrate skeletal neuromuscular synapses. While agrin was initially postulated to be the inductive molecule that initiates synaptogenesis, this model has been modified in response to work showing that postsynaptic differentiation can develop in the absence of innervation, and that synapses can form in transgenic mice in which the agrin gene is ablated. In place of a unitary mechanism for neuromuscular synapse formation, studies in both mice and zebrafish have led to the proposal that two mechanisms mediate synaptogenesis, with some synapses being induced by nerve contact while others involve the incorporation of prepatterned postsynaptic structures. Moreover, the current model also proposes that agrin can serve two functions, to induce synaptogenesis and to stabilize new synapses, once these are formed. This review examines the evidence for these propositions, and concludes that it remains possible that a single molecular mechanism mediates synaptogenesis at all NMJs, and that agrin acts as a stabilizer, while its role as inducer is open to question. Moreover, if agrin does not act to initiate synaptogenesis, it follows that as yet uncharacterized molecular interactions are required to play this essential inductive role. Several alternatives to agrin for this function are suggested, including focal pericellular proteolysis and integrin signaling, but all require experimental validation.

The vertebrate skeletal neuromuscular junction (NMJ) has long served as a model system for studying synapse structure, function and development. Over the last several decades a neuron-specific isoform of agrin, a heparan sulfate proteoglycan, has been identified as playing a central role in synapse formation at all vertebrate skeletal neuromuscular synapses. While agrin was initially postulated to be the inductive molecule that initiates synaptogenesis, this model has been modified in response to work showing that postsynaptic differentiation can develop in the absence of innervation, and that synapses can form in transgenic mice in which the agrin gene is ablated. In place of a unitary mechanism for neuromuscular synapse formation, studies in both mice and zebrafish have led to the proposal that two mechanisms mediate synaptogenesis, with some synapses being induced by nerve contact while others involve the incorporation of prepatterned postsynaptic structures. Moreover, the current model also proposes that agrin can serve two functions, to induce synaptogenesis and to stabilize new synapses, once these are formed. This review examines the evidence for these propositions, and concludes that it remains possible that a single molecular mechanism mediates synaptogenesis at all NMJs, and that agrin acts as a stabilizer, while its role as inducer is open to question. Moreover, if agrin does not act to initiate synaptogenesis, it follows that as yet uncharacterized molecular interactions are required to play this essential inductive role. Several alternatives to agrin for this function are suggested, including focal pericellular proteolysis and integrin signaling, but all require experimental validation.

The cell’s capacity to integrate and respond to spatial information is a crucial feature of morphogenesis and development. The Planar Cell Polarity (PCP) pathway is a signaling mechanism, widely conserved across metazoans, providing spatial orientation along the plane of an epithelium in morphogenic processes ranging from insect wing patterning to mammalian cochleae. Although the core genes involved in the PCP pathway have been molecularly identified in the 1990s, the PCP signaling mechanism remains controversial. In this article I discuss the main players and previous models of PCP signaling reported in the literature, and propose a new model. According to it PCP is established through an homophobic signal by transmembrane protein Frizzled (Fz): 1) a Fz signal in one cell repeals Fz itself in the adjacent cell, thereby generating symmetry breaking; 2) the instructive PCP signal is conveyed through Fz interaction with atypical cadherin Flamingo (Fmi). More broadly, homophobic signaling may represent a novel mechanism for cell-cell signaling of spatial information through modulation of cell adhesion rather than canonical ligand-receptor binding.

Charcot-Marie-Tooth disease is a hereditary polyneuropathy caused by mutations in Mitofusin-2 (MFN2), a GTPase in the outer mitochondrial membrane involved in the regulation of mitochondrial fusion and bioenergetics. Autosomal-dominant inheritance of a R94Q mutation in MFN2 causes the axonal subtype 2A2A which is characterized by early onset and progressive atrophy of distal muscles caused by motoneuronal degeneration. Here, we studied mitochondrial shape, respiration, cytosolic and mitochondrial ATP content as well as mitochondrial quality control in MFN2-deficient fibroblasts stably expressing wildtype or R94Q MFN2. Under normal culture conditions, R94Q cells had slightly more fragmented mitochondria but a similar mitochondrial oxygen consumption, membrane potential and ATP production as wildtype cells. However, when inducing mild oxidative stress 24 h before analysis using 100 µM hydrogen peroxide, R94Q cells exhibited significantly increased respiration but decreased mitochondrial ATP production. This was accompanied by increased glucose uptake and an upregulation of hexokinase 1 and pyruvate kinase M2 suggesting increased pyruvate shuttling into mitochondria. As these changes coincided with decreased levels of PINK1/Parkin-mediated mitophagy in R94Q cells, we conclude that the disease-causing R94Q mutation in MFN2 causes uncoupling of mitochondrial respiration from ATP production by a less efficient mitochondrial quality control triggered by oxidative stress.

Charcot-Marie-Tooth disease is a hereditary polyneuropathy caused by mutations in Mitofusin-2 (MFN2), a GTPase in the outer mitochondrial membrane involved in the regulation of mitochondrial fusion and bioenergetics. Autosomal-dominant inheritance of a R94Q mutation in MFN2 causes the axonal subtype 2A2A which is characterized by early onset and progressive atrophy of distal muscles caused by motoneuronal degeneration. Here, we studied mitochondrial shape, respiration, cytosolic and mitochondrial ATP content as well as mitochondrial quality control in MFN2-deficient fibroblasts stably expressing wildtype or R94Q MFN2. Under normal culture conditions, R94Q cells had slightly more fragmented mitochondria but similar mitochondrial oxygen consumption, membrane potential and ATP production. Mild oxidative stress procured by 100 µM hydrogen peroxide applied 24 h before analysis, however, significantly increased respiration but decreased mitochondrial ATP production only in R94Q cells. This was accompanied by increased glucose uptake and an upregulation of hexokinase 1 and pyruvate kinase M2 suggesting increased pyruvate shuttling into mitochondria. As these changes coincided with decreased levels of PINK1/Parkin-mediated mitophagy in R94Q cells, we conclude that the disease-causing R94Q mutation in MFN2 causes uncoupling of mitochondrial respiration from ATP production by a less efficient mitochondrial quality control.

The rodent collecting duct (CD) expresses a 24p3/NGAL/lipocalin-2 (Lcn2) receptor (Slc22a17) apically to possibly mediate high-affinity reabsorption of filtered proteins by endocytosis, yet its functions remain uncertain. Recently, we showed that hyperosmolarity/-tonicity upregulates Slc22a17 in cultured mouse inner medullary CD cells, whereas activation of toll-like receptor 4 (TLR4) via bacterial lipopolysaccharides (LPS) downregulates Slc22a17. This is similar to the upregulation of Aqp2 by hyperosmolarity/-tonicity and arginine vasopressin (AVP) and downregulation by TLR4 signaling that occur via the transcription factors Nfat5 (TonEBP or OREBP), cAMP-responsive element binding protein (CREB), and nuclear factor-kappa B, respectively. The aim of the study was to determine the effects of osmolarity/tonicity via Nfat5, AVP via CREB and TLR4 signaling on the expression of Slc22a17 and its ligand Lcn2 in the mouse (m) cortical collecting duct cell line mCCD(cl.1). Normosmolarity/-tonicity was 300 mosmol/l whereas addition of 50-100 mmol/l NaCl for up to 72 h induced hyperosmolarity/-tonicity (400-500 mosmol/l). RT-PCR, qPCR, immunoblotting and immunofluorescence microscopy detected Slc22a17 and Lcn2 expression. RNAi silenced Nfat5, and the pharmacological agent 666-15 blocked CREB. Activation of TLR4 occurred with LPS. Similar to Aqp2, hyperosmotic/-tonic media and AVP upregulated Slc22a17 via activation of Nfat5 and CREB, respectively, and LPS/TLR4 signaling downregulated Slc22a17. Conversely, though Nfat5 mediated hyperosmolarity/-tonicity induced downregulation of Lcn2 expression, AVP reduced Lcn2 expression and predominantly apical Lcn2 secretion evoked by LPS, but through a posttranslational mode of action that was independent of cAMP signaling. In conclusion, the hyperosmotic/-tonic upregulation of Slc22a17 in mCCD(cl.1) cells via Nfat5 and by AVP via CREB suggests a contribution of Slc22a17 to adaptive osmotolerance, whereas Lcn2 downregulation could counteract increased proliferation and permanent damage of osmotically stressed cells.

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is an enigmatic condition characterized by fatigue that is unaided by rest and by exacerbation of symptoms after exertion (post-exertional malaise or “PEM”). There is no definitive molecular marker or known underlying pathological mechanism for the condition. Increasing evidence for aberrant energy metabolism suggests a role for mitochondrial dysfunction in ME/CFS. Our objective was therefore to measure mitochondrial function and cellular stress sensing in actively metabolising patient blood cells. We immortalized lymphoblasts isolated from 51 ME/CFS patients diagnosed according to the Canadian Consensus Criteria and an age- and gender-matched control group. Parameters of mitochondrial function and energy stress sensing were assessed by Seahorse extracellular flux analysis, proteomics, and an array of additional biochemical assays. As a proportion of the basal oxygen consumption rate (OCR), the rate of ATP synthesis by Complex V was significantly reduced in ME/CFS lymphoblasts, while significant elevations were observed in Complex I OCR, maximum OCR, spare respiratory capacity, nonmitochondrial OCR and “proton leak” as a proportion of the basal OCR. This was accompanied by an elevation of mitochondrial membrane potential, chronically hyperactivated TOR Complex I stress signalling and upregulated expression of mitochondrial respiratory complexes, fatty acid transporters and enzymes of the β-oxidation and TCA cycles. By contrast, mitochondrial mass and genome copy number, as well as glycolytic rates and steady state ATP levels were unchanged. Our results suggest a model in which ME/CFS lymphoblasts have a Complex V defect accompanied by compensatory upregulation of their respiratory capacity that includes the mitochondrial respiratory complexes, membrane transporters and enzymes involved in fatty acid β-oxidation. This homeostatically returns ATP synthesis and steady state levels to “normal” in resting cells, but may leave them unable to adequately respond to acute increases in energy demand as the relevant homeostatic pathways are already activated.

The epithelial cell tight junction structure is the site of the transepithelial movement of solutes and water between epithelial cells (paracellular permeability). Paracellular permeability can be divided into two distinct pathways, the Pore Pathway mediating the movement of small ions and solutes and the Leak Pathway mediating the movement of large solutes. Claudin proteins form the basic paracellular permeability barrier and mediate the movement of small ions and solutes via the Pore Pathway. The Leak Pathway remains less understood. Several proteins have been implicated in mediating the Leak Pathway, including occludin, ZO proteins, tricellulin, and actin filaments, but the proteins comprising the Leak Pathway remain unresolved. The properties of the Leak Pathway, such as its molecular mechanism, its regulation, and whether or not it has a size limit, remain controversial. This review will trace the evolution of the Leak Pathway concept from its origins, will discuss the current information about the properties of the Leak Pathway, and will discuss recent research suggesting a possible molecular basis for the Leak Pathway. Based on these findings, we propose a model for the molecular mechanism underlying the Leak Pathway and its regulation.

It is not understood how the numbers and identities of vertebrae are controlled during mammalian development. The remarkable robustness and conservation of segmental numbers may suggest a digital nature of the underlying process. Here I propose a mechanism that allows cells to obtain and store the segmental information in digital form, and to produce a pattern of chromatin accessibility that in turn regulates Hox gene expression specific to the metameric segment. The model requires that a regulatory element be present such that the number of occurrences of the motif between two consecutive Hox genes equals the number of segments under the control of the anterior gene. This is true for the recently discovered HRC3 motif, associated with histone modifications and developmental genes. The finding not only allows correctly predicting the numbers of segments using only sequence information, but also resolves the 40-year-old enigma of the function of temporal and spatial collinearity of Hox genes. The logic of the mechanism is illustrated in an animated video: https://youtu.be/4a3XOQ7Lz28. I also discuss how different aspects of the proposed mechanism can be tested experimentally.

Dietary contaminants are often an over-looked factor in the health of zebrafish. Typically, water is considered to be the source for most contaminants, especially within an aquatic environment. For this reason, source water for zebrafish recirculating systems is highly regulated and monitored daily. Most facilities use reverse osmosis or de-ionized water filtration systems to purify incoming water to ensure that contaminants, as well as pathogens, do not enter their zebrafish housing units. However, diets are rarely tested for contaminants and, in the case of manufactured zebrafish feeds, since the product is marketed for aquaculture or aquarium use it is assumed that the feed is acceptable for animals used for research. The following provides examples as to how contaminants could lead to negative effects on development and behavior of developing zebrafish.

Genetic activation of the class I PI3K pathway is very common in cancer. This mostly results from oncogenic mutations in PIK3CA, the gene encoding the ubiquitously expressed PI3Kα catalytic subunit, or from inactivation of the PTEN tumour suppressor, a lipid phosphatase which opposes class I PI3K signalling. The clinical impact of PI3K inhibitors in solid tumours, aimed at dampening cancer-cell-intrinsic PI3K activity, has thus far been limited. Challenges include poor drug tolerance, incomplete pathway inhibition and pre-existing or inhibitor-induced resistance. The principle of pharmacologically targeting cancer-cell-intrinsic PI3K activity also assumes that all cancer-promoting effects of PI3K activation are reversible, which might not be the case. Emerging evidence suggests that genetic PI3K pathway activation can induce and/or allow cells to tolerate chromosomal instability, which – even if occurring in a low fraction of the cell population – might help to facilitate and/or drive tumour evolution. While it is clear that such genomic events cannot be reverted pharmacologically, a role for PI3K in the regulation of chromosomal instability could be exploited by using PI3K pathway inhibitors to prevent those genomic events from happening and/or reduce the pace at which they are occurring, thereby dampening cancer development or progression. Such an impact might be most effective in tumours with clonal PI3K activation and achievable at lower drug doses than the maximum-tolerated doses of PI3K inhibitors currently used in the clinic.

Triploidy in cancer is associated with poor prognosis but its origins remain unclear. Here, we attempted to differentiate between random chromosomal and whole-genome origins of cancer triploidy. In silico meta-analysis was performed on 15 male malignant and 5 benign tumour cohorts (2928 karyotypes) extracted from the Mitelman Database, comparing their ploidy and combinations of sex chromosomes. A distinct near-triploid fraction was observed in all malignant tumour types, being especially high in seminoma. For all tumour types, X-chromosome doubling, predominantly observed as XXY, correlated strongly with the near-triploid state (r≈0.9, p<0.001), negatively correlated with near-diploidy, and did not correlate with near-tetraploidy. A smaller near-triploid component with a doubled X-chromosome was also present in 3 of 5 benign tumour types, especially notable in colon adenoma. Principal Component Analysis revealed a non-random correlation structure shaping the X-chromosome disomy distribution across all tumour types. We suggest that doubling of the maternal genome followed by pedogamic fusion with a paternal genome (a possible mimic of the fertilization aberration, 69, XXY digyny) associated with meiotic reprogramming may be responsible for the observed rearrangements of genome complements leading to cancer triploidy. The relatively frequent loss of the Y-chromosome results secondary from chromosome instability.

Androgens are key for pubertal development of the mammalian testis, a phenomenon tightly linked to Sertoli cell maturation. In this review, we discuss how androgen signalling affects Sertoli cell function and morphology by concomitantly inhibiting some processes and promoting others that contribute jointly to the completion of spermatogenesis. We focus on the molecular mechanisms that underlie AMH inhibition by androgens at puberty, as well as on the role androgens have on Sertoli cell tight junction formation and maintenance and, consequently, on its effect on proper germ cell differentiation and meiotic onset during spermatogenesis.

The identification and molecular characterization of cellular hierarchies in complex tissues is key to understanding both normal cellular homoeostasis and tumorigenesis. The mammary epithelium is a heterogeneous tissue consisting of two main cellular compartments, an outer basal layer containing myoepithelial cells and an inner luminal layer consisting of estrogen receptor negative (ER^-) ductal cells and secretory alveolar cells (in the fully functional differentiated tissue) and hormone responsive estrogen receptor positive (ER⁺) cells. Recent publications in Nature Communications used single cell RNA-sequencing (scRNA-seq) analysis to decipher epithelial cell differentiation hierarchies in human (Nguyen et al., 2018) and murine (Pal et al., 2017) mammary glands and report the identification of new cell types based on the expression of the luminal progenitor cell marker KIT (c-Kit). However, there are several inaccuracies and unfortunate omissions in the citation of previous research in each of these studies. As a result, the overall conclusions on the significance of these reports, in particular the claimed identification of new cell types is not accurate. Here we discuss these studies in the context of our previous research (Regan et al., 2012), in which we identified c-Kit as a luminal progenitor cell marker and functionally characterized cellular subpopulations analogous to those reported in the recent scRNA-seq studies.

Plant trichomes are specialized unicellular structures that originate and project from above ground epidermal tissues on the surfaces of leaves, petals, stems, petioles, peduncles, and seed coats depending on species. Trichomes (also called ‘hairs’) play well-recognized roles in defense against insect herbivores, both as a physical barrier that obstructs herbivore movement and by mediating chemical defenses. By virtue of their physical properties (size, density), trichome hairs can directly operate to protect buds of plants from insect damage, reduce leaf temperature, increase light reflectance, prevent water loss, and decrease leaf abrasion. Great variety of trichomes and their accessibility makes them a useful model for studying the molecular processes of cell fate determination, cell cycle control and cellular morphogenesis. In leaves, the developmental control of the trichomatous complement has highlighted a regulatory network based on four fundamental elements: (i) genes that activate and/or modify the normal cell cycle of epidermal cells (i.e. endoreduplication cycles); (ii) transcription factors that create activator/repressor complexes with a central role in determining cell fate, initiation and differentiation of an epidermal cell in trichome; (iii) evidences that point out the interplay of the aforesaid complexes with various phytohormones; (iv) epigenetic mechanisms involved in trichome development. Here, we describe trichome development in leaves, commonly subjected to environmental injury, and where most genetic regulators have been characterized.

Emerin is a nuclear envelope protein that contributes to genome organization and cell mechanics. Through its N-terminal LEM-domain, emerin interacts with the DNA-binding protein Barrier-to-Autointegration (BAF). Emerin also binds to members of the Linker of the Nucleoskeleton and Cytoskeleton (LINC) complex. Mutations in the gene encoding emerin are responsible for the majority of cases of X-linked Emery-Dreifuss Muscular Dystrophy (X-EDMD). Most of these mutations lead to an absence of emerin. A few missense and short deletion mutations in the disordered region of emerin are also associated with X-EDMD. More recently, missense and short deletion mutations P22L, ∆K37 and T43I were discovered in emerin LEM-domain, associated with isolated atrial cardiac defects (ACD). Here we reveal which defects, at both molecular and cellular levels, are elicited by these LEM-domain mutations. Whereas DK37 mutation impairs correct folding of the LEM-domain, P22L and T43I have no impact on the 3D structure of emerin. Surprisingly, all three mutants bind to BAF, albeit with a weaker affinity in the case of DK37. In human myofibroblasts derived from a patient’s fibroblasts, emerin ∆K37 is correctly localized at the inner nuclear membrane, but is present at a significantly lower level, indicating that this mutant is abnormally degraded. Moreover, SUN2 is reduced, and cells are defective in producing actin stress fibers when grown on a stiff substrate and after cyclic stretches. Altogether, our data suggest that the main effect of mutation DK37 is to perturb emerin function within the LINC complex in response to a mechanical stress.

Triploidy in cancer is associated with poor prognosis but its origins remain unclear. Here, based on frequent X-chromosome doubling in male tumours we attempted to differentiate between a random chromosomal origin and whole-genome origin of cancer triploidy. In silico meta-analysis was performed on 15 male malignant and 5 benign tumour cohorts (2928 karyotypes) extracted from the Mitelman Database, comparing their modal chromosome numbers (ploidy) and combinations of sex chromosomes. Karyotype heterogeneity with a distinct near-triploid fraction was observed in all malignant tumour types, especially high in seminoma. For all tumour types, X-chromosome doubling, dominantly presented by XXY, strongly correlated with the near-triploid state (r≈0.9, p<0.001), negatively correlated with near-diploidy, and did not correlate with near-tetraploidy. The proportion of XX,-Y near-triploid karyotypes was variably increased in somatic tumours. A smaller near-triploid component with a doubled X-chromosome was also present in 3 of 5 benign tumour types, especially notable in colon adenoma. We conclude that doubling of the maternal genome followed by fusion with a paternal genome (similar to digyny) is likely responsible for the observed whole genome triploidy and may be causative for cancer initiation. The Y-chromosome may subsequently be lost due to secondary chromosome instability processes.

Preventing muscle wasting in certain chronic diseases including cancer is an ongoing challenge. Studies have shown that polyphenols derived from fruits and vegetables show promise in reducing muscle loss in cellular and animal models of muscle wasting. We hypothesized that polyphenols derived from plum (Prunus domestica) could have anabolic and anti-catabolic benefits on skeletal muscle. The effects of a polyphenol-enriched plum extract (PE60) were evaluated in vitro on C2C12 and Colon-26 cancer cells. Treatment of myocytes with plum extract increased the cell size by ~3-fold (p<0.05) and stimulated myoblast differentiation by ~2-fold (p<0.05). Plum extract induced total protein synthesis by ~50% (p<0.05), reduced serum deprivation- induced total protein degradation by ~30% (P<0.05), and increased expression of IGF-1 by ~2-fold (p<0.05). Plum extract also reduced TNFα-induced NFkB activation by 80% (p<0.05) in A549/NFkB-luc cells. In addition, plum extract inhibited growth of Colon-26 cancer cells, and attenuated cytotoxicity in C2C12 myoblasts induced by soluble factors released from Colon-26 cells. In conclusion, our data suggest that plum extract may have pluripotent health benefits on muscle based on its demonstrated ability to promote myogenesis, stimulate muscle protein synthesis, and inhibit protein degradation. It also appears to protect muscle cell from tumor-induced cytotoxicity.

The nuclear lamina consists of a dense fibrous meshwork made of nuclear lamins, Type V intermediate filaments, and is ~14 nm thick according to recent cryo-electron tomography studies. Recent advances in light microscopy have extended the resolution to a scale allowing for the fine structure of the lamina to be imaged in the context of the whole nucleus. We review quantitative approaches to analyze the imaging data of the nuclear lamina as acquired by structured illumination microscopy (SIM) and single molecule localization microscopy (SMLM), as well as the requisite cell preparation techniques. In particular, we discuss the application of steerable filters and graph based methods to segment the structure of the four mammalian lamin isoforms (A, C, B1, and B2) and extract quantitative information.

Congenital anomalies of the kidney and urinary tract (CAKUT) are common birth defects deriving from abnormalities in renal differentiation during embryogenesis. CAKUT is the major cause of end-stage renal disease and chronic kidney diseases in children, but its genetic causes remain largely unresolved. Here we discuss advances in the understanding of how MAPK/ERK activity contributes to the regulation of ureteric bud branching morphogenesis, which dictates the final size, shape, and nephron number of the kidney. Recent studies also demonstrate that MAPK/ERK pathway is directly involved in nephrogenesis, regulating both the maintenance and differentiation of the nephrogenic mesenchyme. Interestingly, aberrant MAPK/ERK signaling is linked to many cancers, and recent studies suggest it also plays a role in the most common pediatric renal cancer, Wilms’ tumor.

This year marks the 20th anniversary of the discovery that proteins with various cellular functions can be temporarily immobilized in the nucleolus, a process known as nucleolar sequestration. This review reflects on the progress made to understand the physiological roles of nucleolar sequestration and the mechanisms involved in protein immobilization. We discuss how nucleolar sequestration consists of a highly choreographed amyloidogenic liquid-to-solid phase transition that converts the nucleolus into Amyloid bodies (A-bodies). The study of solid condensates A-bodies will offer unique perspectives on cellular assembly of membrane-less compartments and provide alternative insights on pathological amyloidogenesis involved in neurological disorders.

Artificial Intelligence based on Deep Learning is opening new horizons in Biomedical research and promises to revolutionize the Microscopy field. Slowly, it now transitions from the hands of experts in Computer Sciences to researchers in Cell Biology. Here, we introduce recent developments in Deep Learning applied to Microscopy, in a manner accessible to non-experts. We overview its concepts, capabilities and limitations, presenting applications in image segmentation, classification and restoration. We discuss how Deep Learning shows an outstanding potential to push the limits of Microscopy, enhancing resolution, signal and information content in acquired data. Its pitfalls are carefully discussed, as well as the future directions expected in this field.

Artificial Intelligence based on Deep Learning is opening new horizons in Biomedical research and promises to revolutionize the Microscopy field. Slowly, it now transitions from the hands of experts in Computer Sciences to researchers in Cell Biology. Here, we introduce recent developments in Deep Learning applied to Microscopy, in a manner accessible to non-experts. We overview its concepts, capabilities and limitations, presenting applications in image segmentation, classification and restoration. We discuss how Deep Learning shows an outstanding potential to push the limits of Microscopy, enhancing resolution, signal and information content in acquired data. Its pitfalls are carefully discussed, as well as the future directions expected in this field.

The early embryonic heart is a multi-layered tube consisting of (1) an outer myocardial tube; (2) an inner endocardial tube; and (3) an extracellular matrix layer interposed between myocardium and endocardium, called “cardiac jelly” (CJ). During the past decades, research on CJ has mainly focused on its molecular and cell biological aspects. This review focuses on the morphological and biomechanical aspects of CJ. Special attention is given to (1) the spatial distribution and fiber architecture of CJ; (2) the morphological dynamics of CJ during the cardiac cycle; and (3) the removal/remodeling of CJ during advanced heart looping stages, which leads to the formation of ventricular trabeculations and endocardial cushions. CJ acts as a hydraulic skeleton displaying striking structural and functional similarities with the mesoglea of jellyfish. CJ not only represents a filler substance, facilitating end-systolic occlusion of the embryonic heart lumen. Its elastic components antagonize the systolic deformations of the heart wall and thereby power the refilling phase of the ventricular tube. Non-uniform spatial distribution of CJ generates non-circular cross sections of the opened endocardial tube (initially elliptic, later deltoid), which seem to be advantageous for valveless pumping. Endocardial cushions arise from non-removed remnants of the original CJ.

The CCAAT/enhancer-binding protein β (C/EBPβ) is a transcription factor that regulates cellular proliferation, differentiation, apoptosis and tumorigenesis. Although the pro-oncogenic roles of C/EBPβ have been implicated in various human cancers, how it contributes to tumorigenesis or tumor progression has not been determined. Immunohistochemistry with human non-small cell lung cancer (NSCLC) tissues revealed that higher levels of C/EBPβ protein are expressed compared to normal lung tissues. Knockdown of C/EBPβ by siRNA reduced the proliferative capacity of NSCLC cells by delaying G₂/M transition of the cell cycle. In C/EBPβ-knockdown cells, a prolonged increase in phosphorylation of cyclin dependent kinase 1 at tyrosine 15 (Y15-pCDK1) was displayed with increased Wee1 and decreased Cdc25B expression, simultaneously. ChIP analysis showed that C/EBPβ bound to distal promoter regions of WEE1 and repressed WEE1 transcription through the interaction with histone deacetylase 2. Treatment of C/EBPβ-knockdown cells with a Wee1 inhibitor induced a decrease in Y15-pCDK1 and recovered cells from G₂/M arrest. In the xenograft tumors, the depletion of C/EBPβ significantly reduced tumor growth. Taken together, these results indicate that Wee1 is a novel transcription target of C/EBPβ that is required for the G₂/M phase of cell cycle progression, ultimately regulating proliferation of NSCLC cells.

Microbial symbioses exhibit astounding adaptations, yet all symbionts face the problem of how to reliably associate with host offspring every generation. A common strategy is vertical transmission, in which symbionts are directly transmitted from the female to her offspring. The diversity of symbionts and vertical transmission mechanisms is as expansive as the diversity of eukaryotic host taxa that house them. However, there are several common themes among these mechanisms based on the degree to which symbionts associate with the host germline during transmission. In this review, we detail three distinct vertical transmission strategies, starting with associations that are transmitted from host somatic cells to offspring somatic cells, either due to lacking a germline or avoiding it. A second strategy involves somatically-localized symbionts that migrate into the germline during host development. The third strategy we discuss is one in which the symbiont maintains continuous association with the germline throughout development. Unexpectedly, the vast majority of documented vertically inherited symbionts rely on the second strategy: soma-to-germline migration. Given that not all eukaryotes contain a sequestered germline and instead produce offspring from somatic stem cell lineages, this soma-to-germline migration is discussed in the context of multicellular evolution. Lastly, as recent genomics data have revealed an abundance of horizontal gene transfer events from symbiotic and non-symbiotic bacteria to host genomes, we discuss their impact on eukaryotic host evolution.

Aneuploidy should compromise cellular proliferation but paradoxically favours tumour progression and poor prognosis. Here, we consider this paradox in terms of our most recent observations of chemo/radio-resistant cells undergoing reversible polyploidy. The latter perform segregation of two parental groups of end-to-end linked dyads by pseudo-mitosis creating tetraploid cells through a dysfunctional spindle. This is followed by autokaryogamy and homologous pairing preceding a bi-looped endo-prophase. The associated RAD51 and DMC1/γ-H2AX double-strand break repair foci are tandemly situated on the AURKB/REC8/kinetochore doublets along replicated chromosome loops, indicative of recombination events. MOS-associated REC8-positive peri-nucleolar centromere cluster organises a monopolar spindle. The process is completed by reduction divisions (bi-polar or with radial cytotomy including pedogamic exchanges) and release of secondary cells and/or formation of an embryoid. Together this process preserves genomic integrity and chromosome pairing, while tolerating aneuploidy by by-passing the mitotic spindle and meiotic SC checkpoints. Concurrently, it reduces the chromosome number and facilitates recombination that decreases the mutation load of aneuploidy and lethality in the chemo-resistant tumour cells. This cancer life-cycle has parallels both within the cycling polyploidy of the asexual life cycles of ancient unicellular protists and cleavage embryos of early multicellulars, supporting the atavistic theory of cancer.

T-cell mediated immune responses should be regulated to avoid the development of autoimmune or chronic inflammation diseases. Several mechanisms have been described to regulate this process, namely death of overactivated T cells by cytokine deprivation, suppression by T regulatory cells (Treg), induction of expression of immune checkpoint molecules such as CTLA-4 and PD-1, or activation-induced cell death (AICD). In addition, activated T cells release membrane microvesicles called exosomes during these regulatory processes. In this review, we revise the role of exosome secretion in the different pathways of immune regulation described to date and its importance in the prevention of autoinmune disease. Expression of membrane-bound death ligands on the surface of exosomes during AICD, or the more recently described transfer of miRNA or even DNA inside T-cell exosomes are molecular mechanisms that will be analyzed.

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

Analyzed the literature devoted to the changes in the expression of the RAS proteins of cancer cells. A brief review of protein expression dynamics PAC in malignant tumors and the possible role of epigenetic mechanisms in these processes. Through research epigenetic mechanisms state for cancer have been developed principally new techniques for their correction, based on the use of selective regulators systems covalent modification-histone proteins (for example, deacetylase inhibitor) and microRNA synthesis technologies. Literature data show promising pharmacological correction epigenetic modification of chromatin in the treatment of cancer.

Caspase-3, onto which there is a convergence of the intrinsic and extrinsic apoptotic pathways, is the main executioner of apoptosis. We here review the current literature on the intervention of the protease in the execution of naturally occurring neuronal death (NOND) during cerebellar development. We will consider data on the most common altricial species (rat, mouse and rabbit), as well as humans. Among the different types of neurons and glia in cerebellum, there is ample evidence for an intervention of caspase-3 in the regulation of NOND of the post-mitotic cerebellar granule cells (CGCs) and Purkinje neurons as a consequence of failure to establish proper synaptic contacts with target (secondary cell death). It seems possible that also the GABAergic interneurons undergo a similar type of secondary cell death, but the intervention of caspase-3 in this case still remains to be clarified in full. Remarkably, CGCs also undergo primary cell death at the precursor/pre-migratory stage of differentiation, in this case without the intervention of caspase-3. Glial cells as well undergo a process of regulated cell death, but it seems possible that expression of caspase-3, at least in the Bergmann glia, is related to differentiation rather than death.

The cytostatic agent hydroxyurea (HU) has proven to be beneficial for a variety of conditions in the disciplines of oncology, hematology, infectious disease and dermatology. It disrupts the S-phase of the cell cycle by inhibiting the ribonucleotide reductase enzyme, thus blocking the transformation of ribonucleotides into deoxyribonucleotides, a rate limiting step in DNA synthesis. HU is listed as an essential medicine by the World Health Organization. Several studies have indicated that HU is well tolerated and safe in pregnant women and very young pediatric patients. To our knowledge, only a few controlled studies about the adverse effects of HU therapy have been done in humans. Despite this, the prevalence of central nervous system abnormalities, including ischemic lesions and stenosis have been reported. This review will summarize and present the effects of HU-exposure on the prenatal and perinatal development of the rat cerebellar cortex and deep cerebellar nuclei neurons. Our results call for the necessity to better understand HU effects and define the administration of this drug to gestating women and young pediatric patients.

Circulating tumor cells (CTCs) in the peripheral blood are the precursors to distant metastasis but the underlying mechanisms are poorly understood. This study aims at understanding the molecular features within CTCs in relation to their metastatic potential. Using in vitro CTC models, in which breast cancer cell lines are cultured in non-adherent conditions simulating the microenvironment in the blood stream, we found that suspension culture resulted in resistance to TNF-related apoptosis inducing ligand (TRAIL)-mediated cell death. Such a resistance was directly correlated with a reduction in surface and total levels of DR5 protein. In the non-adherent state, cells underwent rapid autophagic flux characterized by an accumulation of autophagosome organelles. Notably, DR5 was translocated to autophagosomes and underwent lysosomal degradation. Our data suggest that CTCs may evade TNF cytokine mediated immune surveillance through downregulation of DR expression. The data warrants further studies in cancer patients to find the status of DRs and other molecular features within primary CTCs in relation to disease progression or chemoresistance.

Senescence is a stable cell cycle arrest that is either tumor suppressive or tumor promoting depending on context. Epigenetic changes such as histone methylation are known to affect both induction and suppression of senescence by altering expression of genes that regulate the cell cycle and the senescence-associated secretory phenotype. A conserved group of proteins containing a Jumonji C (JmjC) domain alter chromatin state, and therefore gene expression, by demethylating histones. Here, we will discuss what is currently known about JmjC demethylases in induction of senescence and how these enzymes suppress senescence to contribute to tumorigenesis.

Copper, the highly toxicity micronutrient, plays two essential roles: it is a catalytic and structural cofactor for Cu-dependent enzymes, and it acts as a secondary messenger. In the cells, copper is imported by CTR1, a transmembrane high-affinity copper importer, and DMT1 (divalent metal transporter). In cytosol, enzyme-specific chaperones receive copper from CTR1 C-terminus and deliver it to their apoenzymes. DMT1 cannot be a donor of catalytic copper because it does not have cytosol domain which is required for copper transfer to the Cu-chaperons and following to cuproenzymes. Here we assume that DMT1 can mediate copper way required for regulatory copper pool. To verify this thought, we used CRISPR/Cas9 to generate H1299 cell line with CTR1 or DMT1 single knockout (KO) and CTR1/DMT1 double knockout (DKO). To confirm KOs of the genes qRT-PCR were used. Two independent clones for each gene were selected for further studies. In CTR1-KO cells, expression of the DMT1 gene was significantly increased. In subcellular compartments, copper concentration decreased dramatically in DKO cells. CTR1-KO cells, but not DMT1-KO, demonstrated reduced sensitivity to cisplatin and silver ions, agents that enter the cell through CTR1. The expression of genes, whose protein products require copper: HIF1α, XIAP, COMMD1, CCS, Cp, but not SOD1 and NF-kB, changed their level. Perhaps these data will help to understand how the disturbances of copper homeodynamics lead to the development of neurodegenerative and oncological disorders. Possibility of using CTR1 KO and DMT1 KO cells to study homeodynamics of catalytic and signaling copper selectively is discussed.

Mitochondrial functions are essential for life, critical for development, maintenance of stem cells, adaptation to physiological changes, responses to stress and aging. The complexity of mitochondrial biogenesis requires coordinated nuclear and mitochondrial gene expression, owing to the need of stoichiometrically assemble the OXPHOS system for ATP production. It requires, in addition, the import of thousands of proteins from the cytosol to keep optimal mitochondrial function and metabolism. Moreover, mitochondria require lipid supply for membrane biogenesis, while it is itself essential for the synthesis of membrane lipids. To achieve mitochondrial homeostasis, multiple mechanisms of quality control have evolved to ensure that mitochondrial function meets cell, tissue and organismal demands. Herein, we give an overview of mitochondrial mechanisms that are activated in response to stress, including mitochondrial dynamics, mitophagy and the mitochondrial unfolded protein response (UPR^mt). We then discuss the role of these stress responses in aging, with particular focus on Caenorhabditis elegans. Finally, we review observations that point to the mitochondrial PHB (prohibitin) complex as a key player in mitochondrial homeostasis, being essential for mitochondrial biogenesis and degradation, and responding to mitochondrial stress. Understanding how mitochondria responds to stress and how such responses are regulated is pivotal to combat aging and disease.

HD-Zip proteins are unique to plants, and contain a homeodomain closely linked to a leucine zipper motif, which are involved in dimerization and DNA binding. Based on homology in the HD-Zip domain, gene structure and the presence of additional motifs, HD-Zips are divided into four families, HD-Zip I–IV. Phylogenetic and bioinformatics analysis of HD-Zip genes using transcriptomic and genomic datasets from a wide range of plant species indicate that the HD-Zip protein class was already present in green algae. Later, HD-Zips experienced multiple duplication events that promoted neo- and sub-functionalizations. HD-Zip proteins are known to control key developmental and environmental responses, and a growing body of evidence indicates a strict link between members of the HD-Zip II and III families and the auxin machineries. Interactions of HD-Zip proteins with other hormones such as brassinolide and cytokinin have also been described. However, it is striking that among the genes regulated by REV, a HD-Zip III protein playing a key role in apical development, are genes that mediate ABA signaling. Furthermore, HAT1 and HAT3, two HD-Zip II proteins involved in key developmental processes, repress ABA biosynthesis and signaling, indicating an essential role of these factors in adjusting development to changing environment.

Single-particle tracking with quantum dots (QDs) constitutes a powerful tool to track the nanoscopic dynamics of individual cell membrane components unveiling their membrane diffusion characteristics. Here we tested the possibility of extracting from the nano-resolved (16 ms and 30 nm) population dynamics of several quantum dots, time-binned at the second time-scale, the rapid structural changes of the cell membrane surface. We used for this proof-of-concept study bright, small and stable biofunctional QD nanoconstructs recognizing the neuronal cannabinoid type 1 (CB1) receptor and a commercial point-localization microscope to reconstruct in 3D the dynamics of the plasma membrane surface of cultured cells with a spatial resolution of tens of nanometers. CB1 receptor was chosen because it’s a highly expressed and fast diffusing membrane protein. Therefore, rapid QD diffusion on the axonal plasma membrane of cultured hippocampal neurons allowed highly precise reconstruction of the membrane surface in less than one minute. QD nanoconstructs diffused into the membrane of synaptic clefts allowing the entire topological reconstruction of the presynaptic component. In addition, we demonstrated successful reconstruction of the remarkably high dynamics of membrane surface topology at the second time-scale both in HEK-293 cell filopodia and axons. Our results show that this novel technique, which we named nanoPaint, is a powerful precision tool for the study of the structural plasticity of cell membrane surfaces.

Plants have evolved two opposing strategies in response to competition for light: shade tolerance and shade avoidance. To detect the presence of neighboring vegetation, shade-avoiding plants have evolved the ability to perceive and integrate multiple signals. Among them, changes in light quality and quantity are central to elicit and regulate the shade avoidance response. Here, we describe recent advances in the understanding of photoperception and downstream signaling mechanisms underlying the shade avoidance response, focusing on Arabidopsis because most of our knowledge derives from studies conducted in this model plant. Shade avoidance is an adaptive response, resulting in phenotypes with high relative fitness in natural dense communities. However, it contributes to reduction in crop plant productivity, and the design of new strategies aimed at attenuating shade avoidance in a stage- and/or organ- specific manner in high-density crop plantings is a major challenge for the future. For this reason, in this review, we also report on recent advances in the molecular description of the shade avoidance response in crops, such as maize and tomato, and discuss similarity and differences with Arabidopsis.

Plants have evolved two opposing strategies in response to competition for light: shade tolerance and shade avoidance. To detect the presence of neighboring vegetation, shade-avoiding plants have evolved the ability to perceive and integrate multiple signals. Among them, changes in light quality and quantity are central to elicit and regulate the shade avoidance response. Here, we describe recent advances in the understanding of photoperception and downstream signaling mechanisms underlying the shade avoidance response, focusing on Arabidopsis because most of our knowledge derives from studies conducted in this model plant. Shade avoidance is an adaptive response, resulting in phenotypes with high relative fitness in natural dense communities. However, it contributes to reduction in crop plant productivity, and the design of new strategies aimed at attenuating shade avoidance in a stage- and/or organ- specific manner in high-density crop plantings is a major challenge for the future. For this reason, in this review, we also report on recent advances in the molecular description of the shade avoidance response in crops, such as maize and tomato, and discuss similarity and differences with Arabidopsis.

Osteoarthritis (OA) is a joint disease involving cartilage degeneration. This study aimed to compare properties of chondrocytes from less-affected (LA-Cartilage) and severely-affected (SA-Cartilage) of human OA articular cartilage. Based on Dougados classification, OA cartilage was classified into two groups; less-affected (Grade 0–1) and severely-affected (Grade 2–3). Chondrocytes from each group were cultured until passage (P) 4. Growth, migration, stem cell properties and chondrogenic properties under normal and inflammatory conditions, and the formation of in vitro 3D cartilage tissues were compared between groups. The growth and migratory properties of LA-chondrocytes and SA-chondrocytes were similar, except that the migration rate of SA-chondrocytes was significantly higher at P0 compared to LA-chondrocytes. Both LA-chondrocytes and SA-chondrocytes expressed mesenchymal stem cell markers and tri-lineage differentiation, but the expression of stem cell markers decreased significantly with increasing passage number. Exposure to inflammatory conditions induced distinct morphological changes and significant increases in expression of SOX9 at P4 and MMP3 at P1 for LA-chondrocytes. LA-chondrocytes and SA-chondrocytes able to develop into in vitro 3D constructs, but SA-chondrocytes exhibited superior cartilage-like properties. Chondrocytes from both less- and severely-affected regions are suitable to be used in clinical applications, however, chondrocytes from severely-affected regions could be a more favorable cell source.

In the present study, we have systematically examined the clinical significance of Nectin-4 (encoded by the PVRL-4 gene), a marker for breast cancer stem cells (CSCs), in cancer metastasis and angiogenesis using a variety of human specimens, including invasive duct carcinoma (IDC) with multiple grades, several types of primary tumors to local and distant relapses, lymph node metastases and circulating tumor cells (CTCs). Nectin-4 was overexpressed in more than 92% of samples with 65.2% Nectin-4 positive cells. The level of expression was increased with increasing tumor grade (GI-III) and size (T1-4) of IDC specimens. More induction of Nectin-4 was noted in relapsed samples from a variety of tumors (colon, tongue, liver, kidney, ovary, buccal mucosa) in comparison to primary tumors, while paired adjacent normal tissues do not express any Nectin-4. A high expression of Nectin-4 along with other representative markers in CTCs and lymph node metastasis was also observed in cancer specimens. An increased level of Nectin-4 along with representative metastatic (CD-44, Sca1, ALDH1, Nanog) and angiogenic (Ang-I, Ang-II, VEGF) markers was noted in metastatic tumors (local and distant) in comparison to primary tumors that were correlated with different grades of tumor progression. In addition, greater expression of Nectin-4 was observed in secondary tumors (distant metastasis, e.g., breast to liver or stomach to gallbladder) in comparison to primary tumors. Nectin-4 was overexpressed at all stages of metastasis and angiogenesis, thus appearing to play a major role in tumor relapse through the PI3K-Akt-NFκβ pathway.

Reactive oxygen species (ROS), which were originally classified as exclusively deleterious compounds, have gained increasing interest in the recent years given their action as bona fide signalling molecules. The main target of ROS action is the reversible oxidation of cysteines, leading to the formation of disulfide bonds, which modulate protein conformation and activity. ROS endowed with signalling properties are mainly produced by NADPH oxidases at the plasma membrane, but their action also involves a complex machinery of multiple redox-sensitive protein families that differs in their subcellular localization and their activity. Given that the levels and distribution of ROS are highly dynamic in part due to their limited stability, the development of various fluorescent ROS sensors, some of which are quantitative (ratiometric), represents a clear breakthrough in the field and have been adapted to both ex vivo and in vivo applications. The physiological implication of ROS signalling will be presented mainly in the frame of morphogenetics processes, embryogenesis, regeneration, and stem cell differentiation. Gain and loss of function as well as pharmacological strategies have demonstrated the wide but specific requirement of ROS signalling at multiple stages of these processes and its intricate relationship with other well-known signalling pathways.

Navigating growth cones are exposed to multiple signals simultaneously and have to integrate competing cues into a coherent navigational response. Integration of guidance cues is traditionally thought to occur at the level of cytoskeletal dynamics. Drosophila studies indicate that cells exhibit a low level of continuous caspase protease activation, and that axon guidance cues can activate or suppress caspase activity. We base a model for axon guidance on these observations. By analogy with other systems in which caspase signaling has non-apoptotic functions, we propose that caspase signaling can either reinforce repulsion or negate attraction in response to external guidance cues by cleaving cytoskeletal proteins. Over the course of an entire trajectory, incorrectly navigating axons may pass the threshold for apoptosis and be eliminated, whereas axons making correct decisions will survive. These observations would also explain why neurotrophic factors can act as axon guidance cues and why axon guidance systems such as Slit/Robo signaling may act as tumor suppressors in cancer.

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

Types of DNA recombination include homologous recombination and nonhomologous recombination. Homologous DNA recombination is a general term that includes exchange of information between chromatids: (reciprocal) crossing-over, gene conversion, and post-meiotic segregation. Gene conversion is now thought to be a type of non-Mendelian segregation of heterozygous markers near the recombination initiation site. Thus, it includes both gene conversion and post-meiotic segregation previously described. DNA non-HR including transpositional recombination and site-specific recombination. Our understanding of the molecular mechanism by which DNA recombination occurs has significantly increased in the past decades. Currently The synthesis-dependent strand annealing model is now thought to give rise to most or all noncrossovers, with the double-strand-break repair model forming mainly crossovers. The Shapiro model proposed by Dr. J. Shapiro explains the molecular mechanism of transpositional recombination. Site-specific recombination results from another distinct model. We previously proposed a novel theory which can provide a more reasonable and simpler explanation accounting for DNA HR including the 3 classes of recombinogenic events described above. In the new supplementedly molecular model, DNA meiotic recombination can be initiated by a copy choice mechanism, that is, copying part of 1 single-stranded DNA template, followed by DNA polymerase switching to another single-stranded DNA template, and then resuming the following DNA synthesis along the new template. The current review suggests that transpositional recombination and site-specific recombination should be initiated by copy choice during DNA synthesis rather than break/join mechanism. The work indicates that review of DNA nonhomologous recombination are very necessary. The novel theory would challenge earlier models accounting for transpositional recombination and site-specific recombination and would be critical to the understanding of the mechanisms. We hope copy choice initiating DNA nonhomologous recombination will be one of the concepts that are explored. Proper and specific experiments are required to reconstruct the detailed mechanism described here.

Neurodegenerative diseases like Alzheimer’s disease (AD) are poised to become a global health crisis, and therefore understanding the mechanisms underlying the pathogenesis is critical for the development of therapeutic strategies. Mutations in genes encoding presenilin occur in most familial Alzheimer’s disease but the role of PSEN in AD is not fully understood. In this review, the potential modes of pathogenesis of AD are discussed, focusing on calcium homeostasis and mitochondrial function. Moreover, research using Caenorhabditis elegans to explore the effects of calcium dysregulation due to presenilin mutations on mitochondrial function, oxidative stress and neurodegeneration is explored.

The devastating growth in the worldwide frequency of neurocognitive disorders and its allied difficulties such as decline in memory, spatial competency, and ability to focus poses a significant psychological public health problem. Inhibitor of Differentiation (ID) proteins are members of a family of helix-loop-helix (HLH) transcription factors. ID proteins have been demonstrated to be involved in neurodevelopmental & depressive diseases and thus may influence neurocognitive deficiencies due to environmental exposure. Previously, it has been demonstrated that environmental factors such as estrogenic endocrine disruptors (EEDs) have played an essential role in the influence of various neurocognitive disorders such as Alzheimer’s, Dementia, and Parkinson’s disease. Based on this increasing number of reports, we consider the impact of these environmental pollutants on ID proteins. Better understanding of how these ID proteins by which EED exposure can affect neurocognitive disorders in populations will prospectively deliver valuable information in the impediment and regulation of these diseases linked with environmental factor exposure.

Heparanase (HPSE) has been defined as a multitasking protein that exhibits a peculiar enzymatic activity towards HS chains but which simultaneously performs other non-enzymatic functions. Through its enzymatic activity, HPSE catalyzes the cutting of the side chains of heparan sulfate (HS) proteoglycans, thus contributing to the remodeling of the extracellular matrix and of the basal membranes. Furthermore, thanks to this activity, HPSE also promotes the release and diffusion of various HS-linked molecules as growth factors, cytokines and enzymes. In addition to being an enzyme HPSE has been shown to possess the ability to trigger different signaling pathways by interacting with transmembrane proteins. In normal tissue and in physiological conditions, HPSE exhibits only low levels of expression restricted only to keratinocytes, trophoblast, platelets and mast cells and leukocytes. On the contrary, in pathological conditions, such as in tumor progression and metastasis, inflammation and fibrosis, it is overexpressed. With this brief review, we intend to provide an update on current knowledge about the different role of HPSE protein exerted by its enzymatic and not-enzymatic activity.

In most Eukaryotes, ubiquitin either exists as free monoubiquitin or as a molecule that is covalently linked to other proteins. These two forms cycle between each other and due to the concerted antagonistic activity of ubiquitylating and deubiquitylating enzymes, an intracellular ubiquitin equilibrium is maintained that is essential for normal biological function. However, measuring the level and ratio of these forms of ubiquitin has been difficult and time consuming. In this paper, we have adapted a simple immunoblotting technique to monitor ubiquitin content and equilibrium dynamics in different developmental stages and tissues of Drosophila. Our data show that the level of total ubiquitin is distinct in different developmental stages, lowest at the larval-pupal transition and in three days old adult males, and highest in first instar larvae. Interestingly, the ratio of free mono-ubiquitin remains within 30-50% range of the total throughout larval development, but peaks to 70-80% at the larval-pupal and the pupal-adult transitions. It stays within the 70-80% range in adults. In developmentally and physiologically active tissues, the ratio of free ubiquitin is similarly high, most likely reflecting a high demand for ubiquitin availability. We also used this method to demonstrate the disruption of the finely tuned ubiquitin equilibrium by the abolition of proteasome function or the housekeeping deubiquitylase, Usp5. Our data support the notion that the ubiquitin equilibrium is regulated by tissue- and developmental stage-specific mechanisms.

Cells take decisions on their responses depending on the stimuli received by the surrounding extracellular environment, that provides the essential cues at the micro and the nano-lengthscales required for adhesion, orientation, proliferation and differentiation. In this study, discontinuous microcones on silicon (Si) and continuous microgrooves on polyethylene terephthalate (PET) substrates were fabricated via ultrashort-pulsed laser irradiation at various fluences, resulting in microstructures with different roughness and geometrical characteristics. The topographical models attained were specifically developed to imitate the guidance and alignment of Schwann cells for oriented axonal regrowth, towards nerve regeneration. At the same time, positive replicas of the silicon microstructures formed were successfully reproduced, via soft lithography, on the biodegradable polymer poly(lactide-co-glycolide) (PLGA). The anisotropic continuous (PET) and discontinuous (PLGA replicas) microstructured polymeric substrates were assessed in terms of their influence on the Schwann cells responses. It is shown that the developed micropatterned substrates enable control over the cellular adhesion, proliferation and orientation and are thus useful to engineer cell alignment in vitro. This property could be potentially useful in the fields of neural tissue engineering and for dynamic microenvironment systems that simulate in vivo conditions.

Stroke remains a major cause of death and disability in the United States and around the world. Solid safety and efficacy profiles of novel stroke therapeutics have been generated in the laboratory, but most failed in clinical trials. Investigations into the pathology and treatment of the disease remain a key research endeavor in advancing scientific understanding and clinical applications. In particular, cell-based regenerative medicine, specifically stem cells transplantation, may hold promise as stroke therapy because grafted cells and their components may recapitulate the growth and function of the neurovascular unit, which arguably represents the alpha and omega of stroke brain pathology and recovery. Recent evidence has implicated mitochondria, organelles with a central role in energy metabolism and stress response, in stroke progression. Recognizing that stem cells offer a source of healthy mitochondria, potentially transferrable into ischemic cells, may provide a new therapeutic tool. To this end, deciphering cellular and molecular processes underlying dysfunctional mitochondria may reveal innovative strategies for stroke therapy. Here, we review recent studies capturing the intimate participation of mitochondrial impairment in stroke pathology, and showcase promising methods of healthy mitochondria transfer into ischemic cells, to critically evaluate the potential of mitochondria-based stem cell therapy for stroke.

Glioblastoma (GBM) is often resistant to conventional and targeted therapeutics. ERBB4 is expressed throughout normal brain and is an oncogene in several pediatric brain cancers; therefore, we investigated ERBB4 as a prognostic marker and therapeutic target in GBM. Using RT-qPCR, we quantified mRNA encoding total ERBB4 and known ERBB4 variants in GBM and non-neoplastic normal brain (NNB) samples. Using immunohistochemistry, we characterized the localization of total and phosphorylated ERBB4 (p-ERBB4) and EGFR protein in archived GBM samples and assessed their association with patient survival. Furthermore, we evaluated the effect of ERBB4 phosphorylation on angiogenesis and tumorigenicity in GBM xenograft models. Total ERBB4 mRNA was significantly lower in GBM than NNB samples, with the JM-a and CYT-2 variants predominating. ERBB4 protein was ubiquitously expressed in GBM but was not associated with patient survival. However, high p-ERBB4 in 11% of archived GBM samples, independent of p-EGFR, was associated with shorter patient survival (12.0±3.2 months) than was no p-ERBB4 (22.5±9.5 months). Increased ERBB4 activation was also associated with increased proliferation, angiogenesis, tumorigenicity and reduced sensitivity to anti-EGFR treatment in xenograft models. Despite low ERBB4 mRNA in GBM, the functional effects of increased ERBB4 activation identify ERBB4 as a potential prognostic and therapeutic target.

Rhotekin is an effector protein for small GTPase Rho. This protein consists of a Rho binding domain (RBD), a pleckstrin homology (PH) domain, proline-rich regions and a C-terminal PDZ-binding motif. We and other groups have identified various binding partners for Rhotekin and proposed their possible physiological roles. However, functions of Rhotekin per se are largely unknown and information about its physiological roles are fragmentary. In this review, we summarize known features of Rhotekin in neuronal tissues and cancer cells. We also describe characteristics of binding partners for Rhotekin and predicted roles of their interaction.

TGF-β signaling transduces immunosuppressive biochemical and mechanical signals in the tumor microenvironment. In addition to canonical SMAD signaling, TGF-β can promote tumor growth and survival by inhibiting proinflammatory signaling and extracellular matrix remodeling. In this article, we will review how TAK1 activation lies at the intersection of proinflammatory signaling by immune receptors and anti-inflammatory signaling by TGF-β receptors. Additionally, we will discuss the role of TGF-β in the mechanobiology of cancer. Understanding how TGF-β dampens proinflammatory responses and induces pro-survival mechanical signals throughout cancer development will be critical in designing therapeutics which inhibit tumor progression while bolstering the immune response.