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Single cell transcriptomics were declared twice 'Method of the year' by Nature Methods journal, in 2013 and 2019. It marks the beginning of new avenue in fundamental and clinical research, as the classic biochemical approach is already seen as limited, as pooling up the contents of a large number of cells together depicts an averaged image that is missing the decision maker cells. Short-read sequencing has already revolutionized biology and medicine, providing an unsurpassed accuracy of the reads, and now third generation long-read nanopore sequencing completes and extends transcriptomics with the ability to discover all full-length isoforms at a single-cell level, providing a deeper insight in our understating of physiology and pathology of tissue functions. This review provides insight on how nanopore technological specifics and limitations have been explored so far in implementing the only direct nucleic acid sequencing platform in single cells sequencing. We review how single-cell sequencing was introduced, most widely used conventional and microfluidic approaches for single cell isolation, ways for further sequencing library generation tailored for Illumina, and how these were adopted for the nanopore technology. [ABSTRACT FROM AUTHOR]

Secreted by viable embryos, PIF is expressed by the placenta and found in maternal circulation. It promotes implantation and trophoblast invasion, achieving systemic immune homeostasis. Synthetic PIF successfully transposes endogenous PIF features to non-pregnant immune and transplant models. PIF affects innate and activated PBMC cytokines and genes expression. We report that PIF targets similar proteins in CD14+, CD4+ and CD8+ cells instigating integrated immune regulation. PIF-affinity chromatography followed by mass-spectrometry, pathway and heatmap analysis reveals that SET-apoptosis inhibitor, vimentin, myosin-9 and calmodulin are pivotal for immune regulation. PIF acts on macrophages down-stream of LPS (lipopolysaccharide-bacterial antigen) CD14/TLR4/MD2 complex, targeting myosin-9, thymosin-α1 and 14-3-3eta. PIF mainly targets platelet aggregation in CD4+, and skeletal proteins in CD8+ cells. Pathway analysis demonstrates that PIF targets and regulates SET, tubulin, actin-b, and S100 genes expression. PIF targets systemic immunity and has a short circulating half-life. Collectively, PIF targets identified; protective, immune regulatory and cytoskeleton proteins reveal mechanisms involved in the observed efficacy against immune disorders. [ABSTRACT FROM AUTHOR]

It is currently accepted that the Sertoli cells are proliferatively active only during the embryogenesis and early fetal development, seizing to divide after puberty, when the spermatogenic niche is prepared, and they become terminally differentiated. So far, only seasonal breeders from mammals have been reported as having season-dependent variations in adult Sertoli cells number and proliferation activity. In this review, we will try to shed light on testis somatic cell plasticity and discuss new evidence for some unique proliferative features Sertoli cells harbor. [ABSTRACT FROM AUTHOR]

Prostate cancer (PCa) stands as one of the most prevalent malignancies in men, ranking as the fifth leading cancer-associated mortality. Castration-resistance metastatic phenotype acquisition is substantiated by reprogramming of cellular responses to oxidative stress damage, involving evading apoptosis by losing p53 and modulating canonical mitophagy. Understanding the molecular underpinnings of mitochondria-related PCa, dysregulated miR-141 and autophagy-modulating MAPK1/ERK2 in canonical mitophagy altered context of PCa cell line model (LNCaP (p53+/+) and PC3 (p53−/−)) can provide insights into potential therapeutic strategies. Utilising nanopore transcriptome sequencing, differential gene expression was analysed post miR-141 mimic/inhibitor modulation. MiR-141 upregulation enriched pathways associated with TAK1-JNK-c-Jun and MAPK activation. However, its inhibition prioritized non-immune pathways, emphasising lysosome biogenesis. Using DAPGreen (a microtubule-associated protein 1A/1B-light chain 3 (LC3) marker analogue), flow cytometry and live imaging demonstrated that miR-141 rescue amplified macroautophagy in p53+/+ cells, which diminished under starvation; conversely, in p53−/− cells, the rescue initially suppressed macroautophagy but enhanced it when combined with starvation. MAPK1 silencing revealed that MAPK1 had suppressive effect in p53+/+ cells, which was lost in p53−/− cells. Combined with starvation it resulted in macroautophagy upregulation in both cell lines. Mitophagy flux detected by MtPhagy and Lyso dyes demonstrated that miR-141 rescue amplified mitophagy under normal and starved conditions in p53+/+ cells, while in p53−/− cells, amplification only occurred during starvation. In both cell lines, starvation alone supressed mitophagy. MAPK1 had suppressive effect on mitophagy only during starvation, regardless of p53 status. Modifying miR-141 and MAPK1/ERK2 dynamic duality axis could present a potential therapeutic strategy. [ABSTRACT FROM AUTHOR]

Abstract: Endometriosis is referred often as an angiogenic disease. The pivotal role of angiogenesis in the pathophysiology of this disease has been confirmed by many studies. This process has several steps, and VEGF is probably the most important in its initiation. There are others involved in its continuation and maintenance of the tight balance between a quiescent and activated blood vessel state. In the process of formation of new blood capillaries and arterioles, many different factors are involved in sometimes distinct pathways. Such factors are TGF-beta and endoglin—the latter being one of the main modulators of the TGF-beta signaling pathway. Endoglin is now not only established as a marker of active neo-angiogenesis and activated endothelium, but also turns to be an active player in the very process of endometriotic angiogenesis. Its signaling pathway of hypoxic activation is tightly interconnected with that of VEGF, and also some of the FGFs. FGF-1 and S100A13 are members of two distinct families of proteins – the FGFs, growth and angiogenic factors, and that of the S100 proteins, – Ca2+-binding proteins involved in cell function regulation, motility and signaling. These two particular members are quite unique in having no signal peptide sequence and being involved in common export pathway. Our hypothesis is that these two factors are involved in vascular remodeling in endometriotic angiogenesis, playing a role in vascular wall formation and migration of endothelial cells (ECs) and vascular smooth muscle cells (VSMCs). We believe also that endoglin is tightly involved in the new arteriolar formation in endometriosis, being expressed in VSMCs but not on the ECs of the middle-sized vessels. [Copyright &y& Elsevier]

An autoimmune response against myelin protein is considered one of the key pathogenic processes that initiates multiple sclerosis (MS). The currently available MS disease modifying therapies have demonstrated to reduce the frequency of inflammatory attacks. However, they appear limited in preventing disease progression and neurodegeneration. Hence, novel therapeutic approaches targeting both inflammation and neuroregeneration are urgently needed. A new pregnancy derived synthetic peptide, synthetic PreImplantation Factor (sPIF), crosses the blood-brain barrier and prevents neuro-inflammation. We report that sPIF reduces paralysis and de-myelination of the brain in a clinically-relevant experimental autoimmune encephalomyelitis mice model. These effects, at least in part, are due to post-translational modifications, which involve cyclic AMP dependent protein kinase (PKA), calcium-dependent protein kinase (PKC), and immune regulation. In terms of potential MS treatment, sPIF was successfully tested in neurodegenerative animal models of perinatal brain injury and experimental autoimmune encephalitis. Importantly, sPIF received a FDA Fast Track Approval for first in human trial in autommuninty (completed). [ABSTRACT FROM AUTHOR]

Simple Summary: We applied machine learning techniques to analyse the kinematics and enzyme activities of Muscovy duck sperm and the DNA methylation levels of the sperm cells. We aimed to find a reliable way to evaluate the quality of duck semen for artificial insemination. We defined good quality semen as having high motility and high methylation. We identified some key features that can predict semen quality, such as the amplitude of lateral head displacement, the wobble and the curvilinear velocity of sperm movement, and the levels of lactate dehydrogenase, alkaline phosphatase, and creatine kinase in the semen. This study aimed to develop a comprehensive approach for assessing fresh ejaculate from Muscovy duck (Cairina moschata) drakes to fulfil the requirements of artificial insemination in farm practices. The approach combines sperm kinetics (CASA) with non-kinetic parameters, such as vitality, enzyme activities (alkaline phosphatase (AP), creatine kinase (CK), lactate dehydrogenase (LDH), and γ-glutamyl-transferase (GGT)), and total DNA methylation as training features for a set of machine learning (ML) models designed to enhance the predictive capacity of sperm parameters. Samples were classified based on their progressive motility and DNA methylation features, exhibiting significant differences in total and progressive motility, curvilinear velocity (VCL), velocity of the average path (VAP), linear velocity (VSL), amplitude of lateral head displacement (ALH), beat-cross frequency (BCF), and live normal sperm cells in favour of fast motility ones. Additionally, there were significant differences in enzyme activities for AP and CK, with correlations to LDH and GGT levels. Although motility showed no correlation with total DNA methylation, ALH, wobble of the curvilinear trajectory (WOB), and VCL were significantly different in the newly introduced classification for "suggested good quality", where both motility and methylation were high. The performance differences observed while training various ML classifiers using different feature subsets highlight the importance of DNA methylation for achieving more accurate sample quality classification, even though there is no correlation between motility and DNA methylation. The parameters ALH, VCL, triton extracted LDH, and VAP were top-ranking for "suggested good quality" predictions by the neural network and gradient boosting models. In conclusion, integrating non-kinetic parameters into machine-learning-based sample classification offers a promising approach for selecting kinetically and morphologically superior duck sperm samples that might otherwise be hindered by a predominance of lowly methylated cells. [ABSTRACT FROM AUTHOR]

At present it is well-defined that autophagy is a fundamental process essential for cell life but its pro-viral and anti-viral role has been stated out with the COVID pandemic. However, viruses in turn have evolved diverse adaptive strategies to cope with autophagy driven host defense, either by blocking or hijacking the autophagy machinery for their own benefit. The mechanisms underlying autophagy modulation are presented in the current review which summarizes the accumulated knowledge on the crosstalk between autophagy and viral infections, with a particular emphasizes on SARS-CoV-2. The different types of autophagy related to infections and their molecular mechanisms are focused in the context of inflammation. In particular, SARS-CoV-2 entry, replication and disease pathogenesis are discussed. Models to study autophagy and to formulate novel treatment approaches and pharmacological modulation to fight COVID-19 are debated. The SARS-CoV-2—autophagy interplay is presented, revealing the complex dynamics and the molecular machinery of autophagy. The new molecular targets and strategies to treat COVID-19 effectively are envisaged. In conclusion, our finding underline the importance of development new treatment strategies and pharmacological modulation of autophagy to fight COVID-19. [ABSTRACT FROM AUTHOR]

Background/Aims: Pregnancy success requires mandatory maternal tolerance of the semi/allogeneic embryo involving embryo-derived signals. Expression levels of PreImplantation Factor (PIF), a novel peptide secreted by viable embryos, correlate with embryo development, and its early detection in circulation correlates with a favourable pregnancy outcome. PIF enhances endometrial receptivity to promote embryo implantation. Via the p53 pathway, it increases trophoblast invasion, improving cell survival / immune privilege. PIF also reduces spontaneous and LPS-induced foetal death in immune naïve murine model. We examined PIF effect on gene expression of human leukocyte antigen (HLA-G, -E -F and -C) and the influence of PIF on local progesterone activity in JEG-3 choriocarcinoma cells. Methods: PIF and progesterone (P4) effects on JEG-3 cells surface and intracellular HLA molecules was tested using monoclonal antibodies, flow cytometry, and Western blotting. PIF and IL17 effects on P4 and cytokines secretion was determined by ELISA. PIF and P4 effects on JEG-3 cells proteome was examined using 2D gel staining followed by spot analysis, mass spectrometry and bioinformatic analysis. Results: In cytotrophoblastic JEG-3 cells PIF increased intracellular expression of HLA-G, HLA-F, HLA-E and HLA-C and surface expression of HLA-G, HLA-E and HLA-C in dose and time dependent manner. In case of HLA-E, -F results were confirmed also by Western blot. Proteome analysis confirmed an increase in HLA-G, pro-tolerance FOXP3+ regulatory T cells (Tregs), coagulation factors and complement regulator. In contrast, PIF reduced PRDX2 and HSP70s to negate oxidative stress and protein misfolding. PIF enhanced local progesterone activity, increasing steroid secretion and the receptor protein. It also promoted the secretion of the Th1/Th2 cytokines (IL-10, IL-1β, IL-8, GM-CSF and TGF-β1), resulting in improved maternal signalling. Conclusion: PIF can generate a pro-tolerance milieu by enhancing the expression of HLA molecules and by amplifying endogenous progesterone activity. A Fast-Track clinical trial for autoimmune disease has been satisfactorily completed. The acquired data warrants PIF use for the treatment of early pregnancy disorders. [ABSTRACT FROM AUTHOR]

Sertoli cells constitutively maintain the spermatogonial stem cell niche and blood-testis barrier via impenetrable tight junctions under hormonal, growth factor and cytokine control. Immature Sertoli cell proliferation and survival benefit from FSH, NF-κB, and IL-1α-dependent signalling effective via the MAPK family of pathways and related to p38 MAPK dependent Ca2+ and K+ fluxes. Little is known about how the NF-κB and MAPK axis interact with pro-inflammatory NOD1/TLR4 and different voltage-gated channels. Silencing MAPK1, we found that MAPK1/ERK2 has a synergistic role with TLR4 in restricting the pro-inflammatory NF-κB/Caspase-1/IL-1β axis while having an antagonistic effect on TLR4 in promoting adult 15 P-1 Sertoli cell line proliferation and migration. In MAPK1/ERK2 absence, there was an unexpected TLR4-NF-κB behaviour, showing a strong NF-κB transactivation, assessed using a pNIFTY-SEAP reporter plasmid, accompanied by significantly activated cell migration and proliferation, as demonstrated by the wound healing assay. Voltage-gated potassium flux, most likely via Kv1.3, was significantly increased after TLR4 and NOD1 challenge in ATP DAMP signal presence, associated with inflammasome activation. TLR4 challenge also activated macroautophagy while inhibiting mitophagy. Thus, TLR4 activation restricted the NF-κB and Caspase-1/IL-1β pro-inflammatory axis, cell migration and proliferation, voltage-gated potassium efflux, and shifted mitophagy to macroautophagy (towards an inflammasome pathway-related cell death), promoting cell survival. MAPK1/ERK2 had a differential effect, as in its absence, TLR4 activation had the opposite effect on NF-κB transactivation and cell migration and proliferation. Thus, TLR4/MAPK1/NF-κB/Kv1.3 interaction provides optimal Sertoli cell maintenance conditions and allows cell death only in extreme circumstances of cumulative DAMP/PAMP signalling stimulation. [ABSTRACT FROM AUTHOR]

Male reproductive failure has been linked to successive development of various urologic diseases including prostate cancer. There is strong epidemiologic data in support of this association, it is important therefore to identify the fundamental grounds that lay beneath such a connection. Male reproductive biology, as sex determined, is significantly dependent upon the hormonal regulation of androgens. With the advancement of knowledge on androgen receptivity and epigenetic regulation, the role of new regulatory factors such as micro RNAs becomes essential. This review focuses on unraveling the role of micro RNA tight incorporation in androgen-dependent male reproductive biology in the context of recent prostate cancer data. [ABSTRACT FROM AUTHOR]

This review article summarizes current knowledge on regulation, functions, and capacities of stem cells in the female and male reproductive tract. Major locations in which pluripotent cells reside and from where they can be isolated are the ovaries, the endometrium, the decidua, and the testis. They include oocytes, embryonic stem cells, trophoblast stem cells, and spermatogonial stem cells, but also several side populations, which can be obtained after certain isolation and culture procedures. The potential of pluripotent cells in the reproductive tract to differentiate is manifold, but heterogenous, depending upon their respective origin. As stem cells have a potential for future application in transplantation and regenerative medicine, this article also reviews the literature on major histocompatibility complex expression on stem cells of the reproductive tract, because of its immunogenic effects, but also because of its potential expression of HLA- G, a potent immunomodulator mainly associated with trophoblast cells. [ABSTRACT FROM AUTHOR]

Problem Monocyte-derived dendritic cells (mDCs) are present in human decidua during the first month of pregnancy where they experience the effect of the increased concentration of progesterone (Pg) and estradiol (Estr). The aim of our study was to assess the effect of these reproductive hormones on the immunomodulatory role of mDCs. Methods of study The mDCs cultured in vitro in the presence of sex steroid hormones and characterized for their apoptosis and secretion of cytokines by flow cytometry, enzyme linked immunosorbent assay and proteome profiler kit. Results Female sex steroid hormones enhance the life cycle of mDCs, thus increasing the maturation and apoptosis, they also increase the production of interleukin (IL)-10 and IL-27 but only Pg increases the production of IL-13 and down regulates the secretion of IL-23. Conclusion The Pg and Estr participate in the regulation of the immune response during pregnancy by influencing apoptosis and the cytokine profile of the mDCs. [ABSTRACT FROM AUTHOR]

PreImplantation Factor (PIF9&15) secreted by viable embryos exerts an essential transplant acceptance and immune regulatory role in pregnancy. Synthetic PIF replicates endogenous PIF's effect in pregnant and non-pregnant immune disorder models. PIF binds macrophages to regulate CD3/CD28-induced T-cell response. We present evidence that PIF regulates the co-stimulatory T-cell receptor, CD2, which binds to and is activated by phytohemagglutinin (PHA), a potent mitogen, confirming PIF's ability to systemically respond to diverse immune stimulants. PIF's effect on PHA-activated PBMC (male and non-pregnant females) proliferation and cytokine secretion was tested, showing that both PIF9&15 block PHA-induced PBMC proliferation and promote anti-inflammatory IL10 secretion, while reducing pro-inflammatory IFNγ secretion. Thus favoring a TH2 cytokine bias. Surface plasmon resonance spectroscopy, immunocytochemistry and Flex station experiments reveal that PIF effect is direct. PIF targets intracellular targets but does not affect early Ca2+ mobilization. By promoting the CD2 receptor in activated T-cells and through inhibition of co-ligand CD58 expression, PIF regulates antigen-presenting cell (APC)-T-cell interactions required for PHA action. Structure-based design demonstrated that PIF15 offers improved target specificity as compared to PIF9. Collectively, PIF directly regulates mitogen-induced PBMC activation. Results support PIF translation for therapy of immune disorders. [ABSTRACT FROM AUTHOR]

Background: Endogenous PIF, upon which embryo development is dependent, is secreted only by viable mammalian embryos, and absent in non-viable ones. Synthetic PIF (sPIF) administration promotes singly cultured embryos development and protects against their demise caused by embryo-toxic serum. To identify and characterize critical sPIF-embryo protein interactions novel biochemical and bio-analytical methods were specifically devised. Methods: FITC-PIF uptake/binding by cultured murine and equine embryos was examined and compared with scrambled FITC-PIF (control). Murine embryo (d10) lysates were fractionated by reversed-phase HPLC, fractions printed onto microarray slides and probed with Biotin-PIF, IDE and Kv1.3 antibodies, using fluorescence detection. sPIF-based affinity column was developed to extract and identify PIF-protein interactions from lysates using peptide mass spectrometry (LC/MS/MS). In silico evaluation examined binding of PIF to critical targets, using mutation analysis. Results: PIF directly targets viable cultured embryos as compared with control peptide, which failed to bind. Multistep Biotin-PIF targets were confirmed by single-step PIF-affinity column based isolation. PIF binds protein disulfide isomerases a prolyl-4-hydroxylase β-subunit, (PDI, PDIA4, PDIA6-like) containing the antioxidant thioredoxin domain. PIF also binds protective heat shock proteins (70&90), co-chaperone, BAG-3. Remarkably, PIF targets a common RIPK site in PDI and HSP proteins. Further, single PIF amino acid mutation significantly reduced peptide-protein target bonding. PIF binds promiscuous tubulins, neuron backbones and ACTA-1,2 visceral proteins. Significant anti-IDE, while limited anti-Kv1.3b antibody-binding to Biotin-PIF positive lysates HPLC fractions were documented. Conclusion: Collectively, data identifies PIF shared targets on PDI and HSP in the embryo. Such are known to play a critical role in protecting against oxidative stress and protein misfolding. PIF-affinity-column is a novel utilitarian method for small molecule targets direct identification. Data reveals and completes the understanding of mechanisms involved in PIF-induced autotrophic and protective effects on the embryo. [ABSTRACT FROM AUTHOR]

Implantation is a major landmark in life. It involves the correct apposition of the embryo in the maternal endometrium. The cellular environment influences placenta development, and direct contact of the fetus with maternal tissues is achieved through decidual cells. At the decidua, and at systemic level, the correct balance of cells potentially acting as antigen-presenting cells and histocompatibility products play a pivotal role in achieving feto-maternal tolerance. Here, we review some of the current issues associated with the interplay between cells and molecules needed for pregnancy development. [ABSTRACT FROM AUTHOR]

Almost all transcribed human genes undergo alternative RNA splicing, which increases the diversity of the coding and non-coding cellular landscape. The resultant gene products might have distinctly different and, in some cases, even opposite functions. Therefore, the abnormal regulation of alternative splicing plays a crucial role in malignant transformation, development, and progression, a fact supported by the distinct splicing profiles identified in both healthy and tumor cells. Drug resistance, resulting in treatment failure, still remains a major challenge for current cancer therapy. Furthermore, tumor cells often take advantage of aberrant RNA splicing to overcome the toxicity of the administered chemotherapeutic agents. Thus, deciphering the alternative RNA splicing variants in tumor cells would provide opportunities for designing novel therapeutics combating cancer more efficiently. In the present review, we provide a comprehensive outline of the recent findings in alternative splicing in the most common neoplasms, including lung, breast, prostate, head and neck, glioma, colon, and blood malignancies. Molecular mechanisms developed by cancer cells to promote oncogenesis as well as to evade anticancer drug treatment and the subsequent chemotherapy failure are also discussed. Taken together, these findings offer novel opportunities for future studies and the development of targeted therapy for cancer-specific splicing variants. [ABSTRACT FROM AUTHOR]

Given the high risk, hemorrhagic disease virus (RHDV) leading to 100% mortality of the infected rabbits, special attention has to be paid to form an adequate and timely immunity after vaccination. The present investigation aimed to analyze the ability of the Spirulina (S.) platensis, enriched of phycocyanin, carotenoids, iron and selenium through modified technology of growth, to boost the immunity of the vaccinated rabbits against RHDV. The experimental white rabbits were fed 1g dry biomass S. platensis per head daily one month before and three months after vaccination with PESTORIN (Bioveta, Czech). The antibody titers against RHDV were determined at days 15, 30, 60 and 90 post-vaccination. Faster and higher production of the specific antibodies against RHDV was observed in animals treated with whole biomass of algae. The S. platensis, enriched with bioactive components, could be beneficial for supporting the immunization against RHDV. [ABSTRACT FROM AUTHOR]