Single-cell RNA sequencing data facilitates investigation of cell heterogeneity and subpopulations as well as differentially abundant states however modern single-cell RNA sequencing datasets are growing in size and complexity requiring advances in the bioinformatic methods that analyze them. Many methods exist for each step of analysis including read alignment, normalization, quality control, batch effect correction, imputation and dimensionality reduction. With so many options to choose from at each step of the analysis, benchmarking and a synthesis of the literature on the methods available is necessary to inform biological researchers on the most optimal workflow for their data. Here, recent key methods of analysis are highlighted with a focus on methods that facilitate identification of cell subpopulations and differentially abundant cell states. With a constantly expanding toolset for each step in single-cell RNA sequencing dataset analysis, biological researchers should stay informed to utilize the most applicable methods for their own analyses.

To guide analysts to select the right tool and parameters in differential gene expression analysis of single-cell RNA sequencing (scRNA-seq) data, we developed a novel simulator that recapitulates the data characteristics of real scRNA-seq datasets while accounting for all the relevant sources of variation in a multi-subject, multi-condition scRNA-seq experiment: the cell-to-cell variation within a subject, the variation across subjects, the variability across cell types, the mean/variance relationship of gene expression across genes, library size effects, group effects, and covariate effects. By applying it to benchmark 12 differential gene expression analysis methods (including cell-level and pseudo-bulk methods) on simulated multi-condition, multi-subject data of the 10x Genomics platform, we demonstrated that methods originating from the negative binomial mixed model such as glmmTMB and NEBULA-HL outperformed other methods. Utilizing NEBULA-HL in a statistical analysis pipeline (https://github.com/interactivereport/scRNAseq_DE) for single cell analysis will enable scientists to better understand cell-type specific transcriptomic response to disease or treatment effects and to discover new drug targets. Further, application to two real datasets showed the outperformance of our differential expression (DE) pipeline, with unified findings of differentially expressed genes (DEG) and a pseudo-time trajectory transcriptomic result. In the end, we made recommendations of filtering strategies of cells and genes based on simulation results to achieve optimal experimental goals.

Abstract: (1) Background: Cancer immune escape is associated with the metabolic reprogramming of TME, and combining metabolic targets with immunotherapy has great potential to improve clinical outcomes. Among all metabolic processes, lipid metabolism, especially fatty acid (FA) metabolism, has a huge role in cancer cell survival, migration, and proliferation, but its mechanism and role in the tumor immune microenvironment remain to be investigated. (2) Methods: We comprehensively analyzed 309 fatty acid-related genes, screened 121 different genes, and used one-way COX regression to select 15 genes with prognostic impact. Systematically evaluated the correlation between FMGs modification patterns and Tumor Microenvironment Infiltration, prognosis, and Immunotherapy. The FMGs-Score was constructed to quantify the FMGs modification patterns using principal component analysis. (3) Results: Three clusters based on FMGs-related genes were demonstrated in breast cancer, with three patterns of distinct immune cell infiltration and biological behavior. An FMGsScore signature was constructed to reveal that patients with a low FMGsScore had higher immune checkpoint expression, higher immune checkpoint inhibitor (ICI) scores, increased immune microenvironment infiltration, better survival advantage, and were more sensitive to immunotherapy than those with a high FMGsScore. Finally, the expression and function of the signature key gene NDUFAB1 were examined by in vitro experiments. (4) Conclusions: This study significantly demonstrates the non-negligible role of FMGs in the immune microenvironment of breast cancer, and that FMGsScores can be used to guide the prediction of immunotherapy in breast cancer patients. In in vitro experiments, knockdown of the NDUFAB1 gene resulted in reduced proliferation and migration of MCF-7 cell lines.

This project introduces a comprehensive pipeline that integrates various bioinformatics tools and methodologies derived from extensive bibliographic research to leverage Perturb-seq, a high-throughput sequencing method, for finding complex altered protein–protein interaction (PPi) subnetworks in cancer variants. The pipeline demonstrates excellent performance, successfully distinguishes impactful, identifies variants with similar expression profiles, and shows promising results for retrieving key affected modules already observed in KRAS and TP53 variants. These promising results indicate the potential of the Perturb-seq approach to become a standard strategy for uncovering not only common significantly expressed genes, but also interactors that could be targeted in future therapeutic strategies. All code to recapitulate the analysis is available along with documentation of pipeline usage at https://github.com/jesusch10/perturbseq_analysis.

During the last few years, advancement in the area of biochemistry, science of the material world, engineering and computer-aided testing has directed towards advancement of high-throughput tools for the sake of profiling information encoded in a gene. Single-cell RNA-sequencing (scRNA-seq) tools capable to examine the sequence data from each individual cells and it shows within population variety and permit exploring of cell conditions and transformation by extreme resolution, possibly give out cell sub-types or gene expression fluctuations that are shaded in mass sequencing processes, which shows population-averaged evaluations. Yet, the major disadvantage for this tool is the lack of success to pick out location related details of the RNA transcriptome, as this needs tissue detachment and cell isolation. Location based transcript determination is among the advancements in the area of medical biotechnology as this can find out the molecules like RNA dataset in their intact physical placement in tissue segment with spatial context at the scale of a single-cell, which is very advantageous as compare to single-cell sequencing techniques. These approaches give key observation in the areas of biomedical field sub-disciplines like neurology, embryology, carcinoma study, immune cell investigation and histological activities. Generally, this review mainly focused towards single-cell sequencing methods, technology development, its challenges observed, different expression data analysis mechanisms and their applications in different areas, such as in cancer research, microbes, central nervous system, sex organs and immune-biology, intensifying the essentiality of sequencing tools at single-cell level for the characterization of extremely dynamic individual cells.

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.

Objective To investigate the functional and biological significance of telomere-related active genes in cholangiocarcinoma (CCA) using single-cell sequencing and cell communication analysis. Methods First, we identified and annotated different cell types in CCA samples using single-cell sequencing data. Pseudotime analysis revealed the transcriptional status and differentiation process of CCA cells. We then explored interactions between CCA cells and their regulatory pathways through cell communication analysis. 47 key genes related to telomere activity were identified, and GO and KEGG pathway enrichment analysis was performed. Additionally, we constructed a prognostic model and verified the model's predictive efficacy. Finally, we identified genes associated with drug sensitivity in CCA patients and identified pathways associated with CCA prognosis through tumor TMB analysis. Results We identified 452 active telomere-associated cells. Then, we assessed telomere associated gene activity, obtaining 47 differentially expressed telomere genes by intersecting with CCA differentially expressed genes. Major up-regulated signaling pathways, including MIF-(CD74+CXCR4) and HLA-A-CD8A, were identified through pseudotime analysis. Three telomere-associated prognostic genes were identified using LASSO analysis, and prognostic-related models were built. In addition, PBRM1 mutation load was found to be highest in the high-risk group, making it a potential diagnostic target. Finally, drug sensitivity analysis revealed higher sensitivity to certain therapeutic drugs in the high-risk group, providing guidance for tailored CCA treatment. Conclusion The MIF-(CD74+CXCR4) and HLA-A-CD8A signaling pathways appear to be major pathways for CCA cell proliferation. Prognostic models based on telomere-associated genes show promise for application, indicating the biological and clinical significance of telomere activity genes in CCA.

Glioma is the most common primary malignant brain tumors. Despite advances in surgical techniques and treatment regimens, the therapeutic effects remain unsatisfactory. Immunotherapy has brought new hope to glioma patients, but the immunosuppressive nature of the tumor microenvironment (TME) has limited the therapeutic outcomes. This study aims to reveal the subpopulations and functional characteristics of tumor-associated macrophages (TAMs), to explore the regulatory effects of chlorogenic acid on the immune microenvironment, and investigate its potential for clinical application. In this study, Chlorogenic acid slowed tumor growth in the mouse model and extended the survival time of the mice. ScRNA-seq analysis elucidated the differentiation trajectories and functional characteristics of BMDMs and microglia, revealing the regulatory effects of chlorogenic acid on these two populations. It enhanced the antigen-presenting function of macrophages and the gene expression related to T-cell immune activation, activated microglia through the JAK-STAT pathway, and improved antitumor functions. Using the target prediction database, a PPI and molecular docking model were constructed, confirming the good affinity of chlorogenic acid with STAT1. A case of a patient treated with chlorogenic acid was reviewed, who survived for 5 years and 6 months, achieved partial remission (PR) after 9 months of treatment, and remains alive with no new symptoms or toxic side effects. Our study revealed the subtypes and differentiation trajectories of tumor-associated macrophages. Chlorogenic acid significantly improved the immune microenvironment of glioma by modulating the function of bone marrow-derived mono-macrophages (BMDMs) and microglia, demonstrating good antitumor effects. This study provides new insights for targeting the regulation of the TME and offers theoretical and practical support for the clinical application of chlorogenic acid.

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.

In the last decade, we have witnessed an upsurge in nuclei-based studies, particularly coupled with next-generation sequencing. Such studies aim at understanding the molecular states that exist in heterogeneous cell populations by applying increasingly more affordable sequencing approaches, in addition to optimized methodologies developed to isolate and select nuclei. Although these powerful new methods promise unprecedented insights, it is important to understand and critically consider the associated challenges. Here, we provide a comprehensive overview of the rise of nuclei-based studies and elaborate on their advantages and disadvantages. Improved designs and appropriate use of the various experimental strategies will result in acquiring biologically accurate and meaningful information.

Numerous dynamic and complicated processes characterize development from the oocyte to the embryo. However, given the importance of functional transcriptome profiles, long non-coding RNAs, single-nucleotide polymorphisms, and alternative splicing during embryonic development, the effect that these features have on the blastomeres of 2-, 4-, 8-, 16-cell, and morula stages of development have not been studied. Here, we conducted a scRNA-seq survey of cells from sheep from the oocyte to the blastocyst developmental stages. We then carried out experiments to identify and functionally analyze the transcriptome profiles, long non-coding RNAs, single-nucleotide polymorphisms (SNPs), and alternative splicing (AS). We founded that between the oocyte and zygote groups significantly down-regulated genes and the second-largest change in gene expression occurred between the 8- and 16-cell stages. We used various methods to construct a profile to characterize cellular and molecular features and systematically analyze the related GO and KEGG profile of cells of all stages from the oocyte to the blastocyst. This large-scale, single-cell atlas provides key cellular information and will likely assist clinical studies in improving preimplantation genetic diagnosis.

Atypical hemolytic uremic syndrome (aHUS) is a rare and life-threatening disease characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury, necessitating differentiation from other thrombotic microangiopathy disorders. Definitive biomarkers for disease diagnosis and activity are currently lacking, and identifying molecular markers is essential. We conducted single-cell sequencing on peripheral blood mononuclear cells from 13 aHUS patients, 3 aHUS family members, and 4 healthy controls. Analysis included clustering, cell type annotation, pseudotime estimation, and cell-cell communication. Immune cell populations differ among aHUS, aHUS families, and healthy controls. Disease activity and treatment influence T, NK, B, and monocyte subpopulations, with increased intermediate monocyte levels distinguishing aHUS from controls and aHUS groups with varying disease activity. Subclustering revealed differential gene expression in patients compared to controls; higher expression of mitochondria-related genes suggests cell metabolism may influence clinical course. Pseudotime trajectory analysis demonstrated unique immune cell differentiation, and cell-cell interaction analysis identified distinct signaling pathways among patients, family members, and controls. This single-cell sequencing study is the first to confirm immune cell dysregulation in aHUS pathogenesis, offering valuable insights into molecular mechanisms and potential new diagnostic and disease activity markers.

Chimeric antigen receptor (CAR) T-cell therapy has greatly improved outcomes for patients with relapsed or refractory hematological malignancies. However, challenges such as treatment resistance, relapse, and severe toxicity still hinder its widespread clinical application. Traditional transcriptome analysis has provided limited insights into the complex transcriptional landscape of both leukemia cells and engineered CAR-T cells, as well as their interactions within the tumor microenvironment. However, with the advent of single-cell sequencing techniques, a paradigm shift has occurred, providing robust tools to unravel the complexities of these factors. These techniques enable unbiased analysis of cellular heterogeneity and molecular patterns. These insights are invaluable for precise receptor design, guiding gene-based T cell modification, and optimizing manufacturing conditions. Consequently, this review utilizes modern single-cell sequencing techniques to clarify the transcriptional intricacies of leukemia cells and CAR-Ts. The aim of this manuscript is to discuss the potential mechanisms that contribute to the clinical failures of CAR-T immunotherapy. We will examine the biological characteristics of CAR-Ts, the mechanisms that govern clinical responses, and the intricacies of adverse events. By exploring these aspects, we hope to gain a deeper understanding of CAR-T therapy, which will ultimately lead to improved clinical outcomes and broader therapeutic applications.

[Objective] To investigate the expression of ALDH2 in hepatocellular carcinoma and its correlation with macrophage infiltration and with the activation of specific subtypes of macrophages. [Methods] Transcriptome and clinical data from patients with hepatocellular carcinoma were downloaded from The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) databases and analyzed and collated using R and Perl software. In the TCGA hepatocellular carcinoma dataset, the Wilcoxon test was used to identify macrophage activation-related gene sets that show differential expression between hepatocellular carcinoma and normal tissues. Single-cell sequencing combined with bulk data was used to analyze macrophage infiltration of hepatocellular carcinoma immune cells and the activation and recruitment of different subtypes of macrophages. Single-factor Cox regression and minimum absolute convergence and selection operator (Lasso) were used to identify survival-related genes and construct prognostic models of HCC. The patients were divided into two groups based on their model scores. The Wilcoxon test was used to identify differentially expressed genes (DEGs) between the two groups, and gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analyses of the DEGs were performed. Single-sample gene set enrichment analysis (ssGSEA) was performed to explore the possible mechanism underlying the differences in prognosis. The reliability of the model was verified using the ICGC hepatocellular carcinoma dataset. A Kaplan‒Meier curve was constructed and used to analyze the influence of macrophage activation-related gene expression on survival and prognosis in patients with hepatocellular carcinoma. The log-rank test and the independent t test were used to analyze survival time data and to compare immune cell infiltration in the two groups. [Results] In a comparison of hepatocellular carcinoma and paracancerous tissues, the expression of six of 137 macrophage activation-related genes (ALDH2, CCNB2, CYP2C9, CYP3A4, F9 and KLKB1) was found to be significantly correlated with overall survival time (OS) in patients with hepatocellular carcinoma. Based on this finding, a prognosis assessment model for hepatocellular carcinoma was constructed. The OS of the patients in the high-scoring group was significantly shorter than the OS of the patients in the low-scoring group (P<0.05), and the model predicted the prognosis of patients with hepatocellular carcinoma independently of sex, age, tumor grade and stage. The area under the receiver operating characteristic (ROC) curve of the model used to predict 1-, 2-, and 3-year survival was greater than 0.7 in both the test set and the validation set. GO enrichment of DEGs between the high- and low-scoring groups of the model suggested that the DEGs are mainly related to immune function. GSEA suggested that the tumor-infiltrating immune cells in the high-scoring group were macrophages, Th2 cells and Treg cells and showed that the number of tumor-infiltrating immune cells, including NK cells, was lower in the high-scoring group than in the low-scoring group. With respect to immune function, the immune checkpoint of the high-scoring group was higher than that of the low-scoring group. Single-cell sequencing data indicated the recruitment of B cells, M0 cells, M1 cells and M2 cells to the hepatocellular carcinoma microenvironment and suggested that ALDH2 regulates related signaling pathways in a way that promotes the differentiation of macrophages into M2-type macrophages. [Conclusion] M2-type macrophages are the dominant type of macrophages associated with immune infiltration in hepatocellular carcinoma. The macrophage-associated activation gene ALDH2 activates the macrophage polarization signaling pathway and negatively regulates the differentiation of macrophages into M2-type macrophages.

This article reviews the transformative impact of single-cell whole genome sequencing (scWGS) on cancer biology research. ScWGS has revolutionized our understanding of tumor heterogeneity, clonal evolution, and the tumor microenvironment by enabling high-resolution analysis of individual cells' genomic, transcriptomic, and epigenomic profiles. The technology's unique capabilities, including detection of rare genomic events, simultaneous capture of multiple genomic features, and association with phenotypic data, have opened new avenues for cancer research and precision medicine. Integration of scWGS with other single-cell omics technologies has provided a multidimensional view of cellular states and regulatory mechanisms in cancer. Advanced data analysis tools, including machine learning and AI algorithms, have been crucial in interpreting the vast amounts of data generated, leading to the identification of new biomarkers and development of predictive models for patient stratification. The article also discusses emerging technologies like spatial transcriptomics and in situ sequencing, which promise to further enhance our understanding of tumor spatial organization and cellular interactions. As scWGS and related technologies continue to evolve, they are expected to drive significant advances in personalized cancer diagnostics, prognosis, and therapy, ultimately improving patient outcomes in the era of precision oncology.

High resolution information on nucleotide arrangement in the genome of any or-ganism is critical to understand its genetic makeup, function, and evolutionary his-tory. For years, DNA sequencing has provided crucial breakthroughs in the field of genomics and molecular biology. With the advent of technology, Next Generation Sequencing has now transformed the whole genome sequencing prospects, thus unveiling the novel and interesting features of an organism genome that can behold. Next generation sequencing platforms offer realistic approach to identify and anal-yses whole genome with high throughput, accuracy, scalability in a very short time span. Information obtained can be further used in improving our existing healthcare systems, designing novel and personalized therapeutic modules, making the envi-ronment sustainable and proactively preparing ourselves for future pandemics and outbreaks. This article discusses the various NGS technologies and approaches, their advantages, drawbacks, and futuristic usage in different fields. Though still in nascent stages and marred with flaws, with ongoing advancement, NGS will indeed have promising impacts in the future.

Biological parameters extracted from electrical signals from various body parts have been used for many years to analyze the human body and its behavior. In addition, electrical signals from cancer cell lines, normal cells, and viruses, among others, have been widely used for the detection of various diseases. Single-cell parameters such as cell and cytoplasmic conductivity, relaxation frequency, and membrane capacitance are important. There are many techniques available to characterize biomaterials, such as scanning tunneling microscopy (STM), nanotechnology, microstrip cavity resonance measurement, dielectric impedance measurement (DEPIM), etc. This article reviews the single cell isolation and sorting techniques, summarizes the common single cell operations such as impedance flow cytometry, cell electrical impedance, electrical impedance sensing, and summarizes the current single cell analysis techniques and their applications.

Purpose: Pancreatic cancer (PAAD) is one of the most lethal malignancies. Immunotherapy is largely ineffective in PAAD. T-cell exhaustion contributes to the resistance for immunotherapies. We investigated the prognostic potential of T cell exhaustion-related genes (TEXGs). Methods: Single-cell RNA (scRNA) sequencing dataset from TISCH, and bulk sequencing datasets from TCGA and GTEx were applied to screen differently expressed TEXGs. Kaplan-Meier survival, LASSO regression, and Univariate/Multivariate Cox regression analysis were performed to construct TEXGs risk model. This model was used to predict the prognosis, tumor immune microenvironment, and immunotherapy response. PAAD cohorts from ICGC and GSE71729 datasets were used to validate this risk model. Pan-cancer expression of SPOCK2 was performed in TISCH database. Results: A six-gene (SPOCK2, MT1X, LIPH, RARRES3, EMP1, and MEG3) risk model was constructed. Low-risk PAAD patients had longer survival time in both training dataset (TCGA-PAAD, n=178) and validation datasets (ICGC-PACA-CA, ICGC-PAAD-US, and GSE71729, n=412). Multivariate Cox regression analysis demonstrated that the risk score was an independent prognostic variable for PAAD. High-risk patients correlated with cancer-related KEGG pathway enrichment, higher mutation frequency of KRAS and TP53 genes, and immunosuppressive status. Immunohistochemistry staining confirmed the changes of TEXGs in clinical samples. Moreover, pan-cancer scRNA sequencing datasets from TISCH analysis indicated that SPOCK2 might be a novel marker of exhausted CD8+ T cells. Conclusion: In conclusion, we established and validated a T cell exhaustion-related prognostic signature for PAAD patients. Moreover, our study suggests that SPOCK2 may be a novel marker of exhausted CD8+ T cells.

It is generally assumed that spirochetemia does not take place at the early localized stage of Lyme disease. This misassumption is due to the lack of sensitive and specific methods for detection. This paper reports that separation of the platelet-rich plasma immediately after venous blood collection to prevent the spirochetes from invading the lymphocytes in the test tube is crucial in molecular detection of a single Borrelia burgdorferi cell spun down in platelet pellets. In 2023, we tested the venous blood specimens of 145 people residing in Lyme disease-endemic areas in the United States. In 98 of them, who were suspected of having early localized Lyme disease irrespective of the absence or presence of a skin lesion, we found 33 specimens (33.7%) positive for B. burgdorferi, including 17 positive for FlaB gene only, 15 positive for both FlaB gene and 16S rRNA gene, and 1 positive for 16S rRNA gene only. Eight (17.0%) of the 47 asymptomatic resident controls were positive for FlaB PCR only. FlaB is a more sensitive chromosomal target than the 16S rRNA gene for the detection of 1-3 B. burgdorferi cells in 1 mL of platelet-rich plasma due to spirochetes gaining or retaining FlaB paralogs at the early localized stage of Lyme disease.

Background: Biological processes are based on complex networks of cells and molecules. Single cell multi-OMICs is a new tool aiming to provide new incites in the complex network of events controlling the functionality of the cell.; Methods: Since single cell technologies provide many sample measurements, they are the ideal environment for the application of deep learning and machine learning approaches. An autoencoder (AE) is composed of an encoder and a decoder sub-model. AE are very powerful in data compression and noise removal. However, the decoder model remains a black box from which is impossible to depict the contribution of the single input elements. We have recently developed a new class of autoencoders, called Sparsely Connected Autoencoders (SCA), which have the advantage of providing a controlled association among the input layer and the decoder module. This new architecture has the benefit that the decoder model is no anymore a black box and it can be used to depict new biologically interesting features from single cell data; Results: In this paper, we show that SCA hidden layer can grab new information usually hidden in single cell data, like as providing clustering on meta-features difficult, i.e. transcription factors expression, or impossible, miRNA expression, to depict in single cell RNAseq data. Furthermore, a SCA representation of cell clusters has the advantage of simulating a conventional bulk RNAseq, which is a data transformation allowing the identification of similarity among independent experiments; Conclusions: In our opinion, SCA represent the bioinformatics version of a “Swiss Army knife” for the extraction of hidden knowledgeable features from single cell OMICs data.

Scientific knowledge evolves in small steps, with occasional backsteps to correct inaccuracies, all occurring within a competitive environment. This perspective for the first time looks at the history of latency-related RNA (LR-RNA) that was later renamed latency-associated transcript (LAT). At the 1986 International Herpesvirus Workshop (IHW) meeting in Leeds, England, Daniel L Rock and Anthony B Nesburn first reported the discovery of human herpes virus 1 (HSV-1) latency-related (LR)-RNA that is antisense to ICP0. Less than a month after the IHW meeting a paper was submitted to Science magazine and 8 months later appeared in print thanking “D. Rock for suggesting RNA complementary to the ICP0 message may be present in latently infected cells”. This perspective is not a review of LAT literature but intends to clarify the timeline of LAT discovery and subsequent breakthroughs such as reactivation, apoptosis, CD8+ T cell exhaustion, and LAT expression in different cell types detected during latency. While many review articles have been written about LAT since 1987, the most comprehensive and balanced review about LAT was written by Dr. David Bloom’s group (1). In this overview, I will discuss our original collaboration with Dr. Dan Rock and subsequent work that our group performed, which is still ongoing. Finally, I will discuss the controversies associated with LAT from its inception to current times.

There have been several developments in the field of nanopore biosensor development and sequencing applications that address previous limitations that restricted widespread nanopore use. These innovations, paired with the large-scale commercialization of biological nanopore sequencing by Oxford Nanopore Technologies, are making these platforms a mainstay in contemporary research labs. Equipped with the ability to provide long and short-read sequencing information with quick turn-around times and simple sample preparation, nanopore sequencers are quickly improving our understanding of unsolved genetic, transcriptomic, and epigenetic problems. However, there remain some key obstacles that have yet to be improved. In this review, we provide a general introduction to nanopore sequencing principles, discussing biological and solid-state nanopore developments, obstacles to single-base detection, and library preparation considerations. We conclude with examples of important clinical applications to give perspective on the potential future of nanopore sequencing in the field of molecular diagnostics.

Single molecule de novo protein sequencing based on the 'superspecificity' of amino-acyl tRNA synthetases (aaRS) is proposed. An unfolded protein molecule is threaded through a nanopore in an electrolytic cell (e-cell) to expose the terminal residue in the e-cell's trans chamber. After the residue is cleaved with an exopeptidase, a set of tRNAs, their aaRSs, and ATP are added to trans. An aaRS charges a cognate tRNA molecule with the residue. The charged tRNA (along with the other reactants) is transferred to an extended e-cell with N (20 ≤ N ≤ 61) pores in N individual cis chambers and a single trans chamber. Each pore holds an RNA molecule ending in a unique codon that is exposed in trans. The charged tRNA's anticodon base-pairs with the terminal codon of an RNA. If tRNAs and residues are fluorescently tagged with two different colors, the residue can be identified from the observed position of the resulting color pair. As charging is 'superspecific' identification is unambiguous. The protein molecule in the first e-cell is advanced by one residue and the process repeated. In this approach there is no need for precise pore current or optical intensity measurements. Potential implementation issues are discussed. Other possibilities, including one in which the terminal residue is cleaved after charging, are also examined.

ACE2, the putative receptor for the novel coronavirus (2019-nCoV), played an important role in cell entry of 2019-nCoV. However, it is not yet clear what cell types within the human body express ACE2. Here, a systematic analysis was undertaken using published single cell datasets. In total, our study analyzed 229652 cells, from five different organs, derived from 88 donors. The top ACE2 expressing cells include proximal tubule cells in the kidney and enterocytes in the intestine. Other major ACE2 expressing cells in the kidney include podocytes, intercalated cells and endothelial cells. Our results offer a comprehensive atlas of ACE2 expression at the single cell level and unravel the enormous potential targets of 2019-nCoVinfection beyond the lung.

Spontaneous tumours in higher plants can develop depending on plant genotype without any pathogen invasion. Spontaneous tumour formation on the taproots is consistently observed in certain inbred lines of radish (Raphanus sativus var. radicula Pers.). In this work, we have sequenced the genomes of two closely related radish inbred lines that differ in their ability to spontaneously form tumours. We have identified numerous single nucleotide variants (amino acid substitutions, insertions or deletions) that are likely to be associated with the spontaneous tumour formation. Furthermore, we performed the identification of CLE and WOX genes the genomic sequences of the radish inbred lines and, as a result, identified two unique radish CLE genes which probably encode proteins with multiple CLE domains. The results obtained provide a basis for investigating the mechanisms of plant tumour formation and also for future genetic and genomic studies of radish.

Sperm gain fertilization ability and forward movement characteristics during epididymal transport. Sperm maturation is inseparable from the processing of the epididymis. Compared with other reproductive organs, relatively little is known about the epididymis. However, with the development of molecular biology and the improvement of single-cell sequence technology, we gradually realized that we should re-examine the importance of the epididymis for sperm mat-uration and for the entire male reproductive system. Recently, epididymis related reports have tended to be more regionalized and more refined. We can understand cell distribution and gene expression from the perspective of a single cell. It has been reported that different regions of the epididymis have different functions and different cell compositions, which may be determined by gene expression. Here, we comprehensively reviewed the epididymal studies. From the ana-tomical structure of the epididymis to the division of its regions, we focused on the cell compo-sition and region-specific genes differentially expressed in the epididymis. The gene expression of the epididymis extends to a series of biological processes of sperm in the epididymal cavity, and the serious effects of epididymal diseases on reproduction are discussed. Therefore, the importance of the epididymis in male reproduction is emphasized. With the maturity of single-cell spatial transcriptomics, it is hoped that the technology can be applied to epididymal research and bring about the spatiotemporal landscape of the epididymis at the single-cell level.

Hospital-based programs democratize mRNA therapeutics by facilitating the processes to translate a novel RNA idea from the bench to the clinic. Because mRNA is essentially biological software, therapeutic RNA constructs can be rapidly developed. The generation of small batches of clinical grade mRNA to support IND applications and first-in-man clinical trials, as well as personalized mRNA therapeutics delivered at the point-of-care, is feasible at a modest scale of cGMP manufacturing. Advances in mRNA manufacturing science and innovations in mRNA biology, are increasing the scope of mRNA clinical applications.

Head and neck squamous cell carcinoma (HNSCC) is a highly heterogeneous and the most common form of head and neck cancer, posing significant challenges for disease management. Single-cell RNA sequencing (sc-RNAseq) has emerged as a valuable tool for addressing these challenges by enabling detailed characterization of the tumor microenvironment (TME) at the cellular level. This review assesses the utility of sc-RNAseq in HNSCC research, compiling current strategies employing single-cell technologies. For HNSCC etiology, sc-RNAseq allows for the construction of cellular atlases, characterization of different cell types, and investigation of key genes and processes involved in cancer initiation, development, and progression within the TME. In terms of HNSCC diagnosis and prognosis, the high resolution offered by sc-RNAseq enables the identification of cell type-specific signatures, enhancing prognostic models and disease stratifiers for more accurate patient outcome assessments. Regarding HNSCC treatment, sc-RNAseq provides insights into cellular responses to various treatments, including radiotherapy, chemotherapy, and immunotherapy, contributing to improved patient stratification and personalized treatment strategies. This review highlights the unique contributions of sc-RNAseq to HNSCC research, addressing its cellular and biological complexity, and emphasizes its potential for advancing research and clinical practice in other cancer types.

Single cell proteins are the dead dried cells of microorganisms or purified protein isolated from microorganism’s cell culture, used as a food supplement to humans’ food and animals feed. World suffer from malnutrition particularly developing countries, due to rapid increase in population, increased the demand for protein and nutrients requirement. Bacteria is potential microorganism for SCP production due to high protein content, fast generation time, bio-active secondary metabolites production and can grow on various substrates. Actinobacteria species and strain have capability to produce biological active compounds, produced about two-thirds of antibiotics available in the market, actively used as antibiotics, antiprotozoal, antifungal, antiviral, anticancer, anticholesterol, antihelminth and immunosuppressant. Actinomycetes can be used as probiotic as well as single cell protein that will deal with antibiotic as well as protein source.

A sufficient amount of tissue sample is required to perform next-generation sequencing (NGS) with a high success rate, but the majority of patients with advanced non-small-cell lung cancer (NSCLC) are diagnosed with small biopsy specimens. Biopsy samples were collected from 184 patients with bronchoscopically diagnosed NSCLC. The tissue surface area, tumor cell count, and tumor content rate of each biopsy sample were evaluated. The impact of the cut-off criteria for the tissue surface area (≥ 1 mm2) and tumor content rate (≥ 30%) on the success rate of Oncomine Dx Target Test (ODxTT) was evaluated. The mean tissue surface area of the transbronchial biopsies was 1.23 ± 0.85 mm2 when small endobronchial ultrasonography with a guide sheath (EBUS-GS) was used, 2.16 ± 1.49 mm2 with large EBUS-GS, and 1.81 ± 0.75 mm2 with endobronchial biopsy (EBB). The proportion of samples with a tissue surface area of ≥ 1 mm2 was 48.8% for small EBUS-GS, 79.2% for large EBUS-GS, and 78.6% for EBB. Sixty-nine patients underwent ODxTT. The success rate of DNA sequencing was 84.1% and that of RNA sequencing was 92.7% over all patients. The success rate of DNA (RNA) sequencing was 57.1% (71.4%) for small EBUS-GS (n = 14), 93.4% (96.9%) for large EBUS-GS (n = 32), 62.5% (100%) for EBB (n = 8), and 100% (100%) for endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) (n = 15). Regardless of the device used, a tissue surface area of ≥ 1 mm2 is adequate for samples to be tested with NGS.

Cancer is the second leading cause of death worldwide. It is theorized that underlying genetic and epigenetic changes enable cells to proliferate out of control by escaping regulatory mechanisms. Although traditional molecular profiling techniques, i.e., bulk sequencing, can identify common mutations and gene expression patterns in cancer cells, they cannot detect tumour heterogeneity. However, single-cell technology has provided an ample opportunity to overcome this difficulty. Since this technology allows us to detect the heterogeneous properties of all cancer cells, this can further our knowledge of the signaling pathways in cancer cells. Indeed, single-cell transcriptomics technology has paved the road for identifying novel biomarkers and signaling pathways, which can serve as targets. This study aims to review the current knowledge about pathways involved in developing cancer cells and shed light on single-cell studies as promising therapeutic approaches.

Positive-sense single-stranded RNA (+ssRNA) viruses comprise many (re-)emerging human pathogens that pose a public health problem. Our innate immune system and in particular the interferon response form the important first line of defense against these viruses. Given their genetic flexibility, these viruses have therefore developed multiple strategies to evade the innate immune response in order to optimize their replication capacity. Already many molecular mechanisms of innate immune evasion by +ssRNA viruses have been identified. However, research addressing the effect of host innate immune evasion on the pathology caused by the viral infection is less prevalent in literature, though very relevant and interesting. Since interferons have been implicated in inflammatory diseases and immunopathology in addition to their protective role in infection, the influence of antagonizing the immune response may have an ambiguous effect on the clinical outcome of the viral disease. Therefore, this review discusses what is currently known about the role of interferons and host immune evasion in the pathogenesis of emerging viruses belonging to the coronaviruses, alphaviruses and flaviviruses.

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

Studies performed at single-cell resolution have demonstrated the physiological significance of cell-to-cell variability. Various types of mathematical models and systems analyses of biological networks have further been used to gain a better understanding of the sources and regulatory mechanisms of such variability. In this work, we present a novel sensitivity analysis method, called molecular density function perturbation (MDFP), for the dynamical analysis of cellular heterogeneity. The proposed analysis is based on introducing perturbations to the density or distribution function of the cellular state variables at specific time points, and quantifying how such perturbations affect the state distribution at later time points. We applied the MDFP analysis to a model of signal transduction pathway involved in TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)-induced apoptosis in HeLa cells. The MDFP analysis showed that caspase-8 activation regulates the timing of the switch-like increase of cPARP (cleaved poly(ADP-ribose) polymerase), an indicator of apoptosis. Meanwhile, the cell-to-cell variability in the commitment to apoptosis depended on mitochondrial outer membrane permeabilization (MOMP) and events following MOMP, including the release of Smac (second mitochondria-derived activator of caspases) and cytochrome-C from mitochondria, the inhibition of XIAP (X-linked inhibitor of apoptosis) by Smac and the formation of apoptosome.

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

The COVID-19 pandemic stimulated attention to the medicinal applications of messenger RNA (mRNA). mRNA is expected to be applied not only to vaccines but also to regenerative medicines. The purity of mRNA is important for its medicinal application. However, the current mRNA synthesis techniques have problems, e.g., contamination of undesired 5’-uncapped mRNA and double-stranded RNAs. Recently, our group developed a completely capped mRNA synthesis technology that contributed to the progress of mRNA research. The introduction of chemically modified nucleosides, e.g., N1-methylpseudouridine and 5-methylcytidine, have been demonstrated by Dr. Karikó and Dr. Weissman which opened the practical application of mRNA for vaccines and regenerative medicines. Dr. Yamanaka reported the production of Induced Pluripotent Stem Cells (iPSCs) by the introduction of 4 types of genes using a retrovirus vector. iPSCs are widely used for research about regenerative medicines and the preparation of disease models to screen new drug candidates. Of the Yamanaka factors, Klf4 and c-Myc are oncogenes, and there is a tumor development risk if these are integrated into genomic DNA. Therefore, regenerative medicines using mRNA, which has no risk of genome insertion, have attracted attention. In this review, we summarized the synthesis of mRNA and its application for regenerative medicine.

Esophageal cancer is an extremely deadly tumor that accounts for 5% of all cancer deaths. The two main subtypes of the disease are esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). To date, few studies analyse EAC for transcriptional signatures associated with diagnosis and/or prognosis, while most focus on the analysis of transcriptional profiles of ESCC. In this study, we used single-cell RNA sequencing (scRNA-seq) analysis of CD45+ cells enriched from Tumors and matched Non Tumor tissues from therapy naїve patients to identify all types of immune cells present in tumor immune infiltrate and its transcriptional profile. In addi-tion, we analysed the entire transcriptome in a cohort of 23 patients whose tissue biopsies were taken from Tumors and matched with Non Tumor tissue. The transcriptional signatures we obtained were then used to stratify a large cohort of TCGA EAC patients, demonstrating a strong association with their prognosis, as well as the ability to predict a patient's clinical outcome and better define the prognosis of EAC after surgery. In addition, these features may lead patients to effective therapies including immunotherapy approaches.

We investigated the potential genetic variants using whole exome sequencing (WES) and evalu-ated the disease course using T cell receptor (TCR) repertoire analysis in rheumatoid arthritis (RA). Fourteen patients with RA and five healthy controls (HCs) were enrolled. For patients with RA, only naïve patients were recruited before treatment, and data were collected at baseline and 6 and 12 months after the initiation of disease-modifying antirheumatic drug (DMARD) treatment. Laboratory data and disease parameters were also collected. Genetic variants were detected using WES, and the diversity of the TCR repertoire was assessed using Shannon-Weiner diversity. Some variants were detected by WES, but their clinical significance should be confirmed by further studies. The diversity of the TCR repertoire in patients with RA was lower than that in HCs; however, after DMARD treatment, it increased significantly. The diversity was negatively cor-related with laboratory findings and disease measures with statistical significance. Variants with a potential for RA pathogenesis were identified and the clinical significance of TCR repertoire was evaluated in Korean patients with RA. Further studies are required to confirm the findings of the present study.

Plant cells are omnipotent and breeding of new varieties can be achieved by protoplast fusion. Such fusions can be achieved by treatment with poly(ethylene glycol) or by applying an electric field. Microfluidic devices allow for controlled conditions and targeted manipulation of small batches of cells down to single cell analysis. To provide controlled conditions for protoplast fusions and achieve high reproducibility, we developed and characterized a microfluidic device to reliably trap few of Arabidopsis thaliana protoplasts and induced a cell fusion by controlled addition of poly(ethylene glycol) (PEG, with a molecular weight of 6000). Experiments were conducted to determine the survival rate of isolated protoplasts in our microfluidic system. Afterwards, PEG induced fusion was studied. Our results indicate that the following fusion parameters had significant impact on the fusion efficiency and duration: PEG concentration, osmolarity of solution, and flow velocity. PEG concentration below 10% led to only partial fusion. Osmolarity of the PEG fusion solution was found to strongly impact the fusion process; complete fusion of two source cells sufficiently took part in slightly hyper osmotic solutions, whereas iso-osmotic solutions led to only partial fusion at 20% PEG concentration. We observed accelerated fusion for higher fluid velocities. Up to this study, it was common sense that fusion is one directional, i.e. once two cells were fused into one cell they stay fused. Here, we present for the first time reversible fusion of protoplasts. Our microfluidic device paves the way to a deeper understanding on the kinetics and processes of cell fusion.

Related studies have shown that mitophagy plays a role in the progression of Atherosclerosis (AS). Utilizing GSE43292, GSE100927, and GSE159677 datasets, WGCNA and differential expression analysis identified mitophagy-related module genes and DEGs1, while single-cell analysis screened DEGs2. MR analysis revealed causal AS-linked genes in DEGs1. LASSO and expression validation identified biomarkers, evaluated via ROC curves. Further analyses included GSEA, immune infiltration, ANN, regulatory networks, and drug prediction. Four candidate genes were identified through WGCNA, differential expression analysis and MR analysis. The LASSO analysis identified CD36, IL1RN, and MME as biomarkers, while the MR analysis showed that IL1RN and MME are protective factors and CD36 is a risk factor in AS. The ROC curves disclosed that the ANN model had greater diagnostic power for AS than any other biomarker. The GSEA revealed that the biomarkers were enriched in lysosomes and hematopoietic cell lineage and were significantly positively correlated with monocytes. The regulatory network construct showed that small nucleolar RNA host gene 16 regulates MME via the micro RNAs. This study identified CD36, IL1RN, and MME as biomarkers of mitophagy in AS and indicated that they could be applied in the diagnosis and treatment of this condition.

The rise of data science in biology stimulates interdisciplinary collaborations to address fundamental questions. Here, we report the outcome of the first SINFONIA symposium focused on revealing the mechanisms governing plant reproductive development across biological scales. The intricate and dynamic target networks of known regulators of flower development remain poorly understood. To analyze development from the genome to the final floral organ morphology, high-resolution data that capture spatiotemporal regulatory activities are necessary and require advanced computational methods for analysis and modeling. Moreover, frameworks to share data, practices and approaches that facilitate the combination of varied expertise to advance the field are called for. Training young researchers in interdisciplinary approaches and science communication offers the opportunity to establish a collaborative mindset to shape future research.

Micro-blade design is an important factor in the cutting of single cells and other biological structures. This paper describes the fabrication process of three dimensional (3D) micro-blades for the cutting of single cells in a microfluidic “guillotine” intended for fundamental wound repair and regeneration studies. Our microfluidic guillotine consists of a fixed 3D micro-blade centered in a microchannel to bisect cells flowing through. We show that the Nanoscribe two-photon polymerization direct laser writing system is capable of fabricating complex 3D micro-blade geometries. However, structures made of the Nanoscribe IP-S resin have low adhesion to silicon, and they tend to peel off from the substrate after at most two times of replica molding in poly(dimethylsiloxane) (PDMS). Our work demonstrates that the use of a secondary mold replicates Nanoscribe printed features faithfully for at least 10 iterations. Finally, we show that complex micro-blade features can generate different degrees of cell wounding and cell survival rates compared with simple blades possessing a vertical cutting edge fabricated with conventional 2.5D photolithography. Our work lays the foundation for future applications in single cell analyses, wound repair and regeneration studies, as well as investigations of the physics of cutting and the interaction between the micro-blade and biological structures.

Proteomic technology has improved at a staggering pace in recent years, with even practitioners challenged to keep up with new methods and hardware. The most common metric used for method performance is the number of peptides and proteins identified. While this metric may be helpful for proteomics researchers shopping for new hardware, this is often not the most biologically relevant metric. Biologists often utilize proteomics in the search for protein regulators that are of lower relative copy number in the cell. In this review, I re-evaluate untargeted proteomics data using a simple graphical representation of the absolute copy number of proteins present in a single cancer cell as a metric. By comparing single shot proteomics data to the coverage of the most in-depth proteomic analysis of that cell line acquired to-date we can obtain a rapid metric of method performance. Using a simple copy number metric allows visualization of how proteomics has developed in both sensitivity and overall dynamic range when using both relatively long and short acquisition times. To enable reanalysis beyond what is presented here, two available web applications have been developed for single and multi-experiment comparisons with reference protein copy number data for multiple cell lines and organisms.

Single cell RNA-seq (scRNA-seq) profiles conceal temporal and spatial tissue developmental information. De novo reconstruction of single cell temporal trajectory has been fairly addressed, but reverse engineering single cell 3D spatial tissue localization is hitherto landmark based, and de novo spatial reconstruction is a compelling computational open problem. Here we show that a new algorithm - named D-CE - for coalescent embedding of single cell transcriptomic networks can address this open problem. We rely merely on the spatial information encoded in the expression patterns of developmental signal transcription factor (DST) genes, and we find that D-CE of cell-cell association DST-transcriptomic networks reliably reconstructs the Geo-seq or single cell samples’ 3D spatial tissue distribution. Comparison to the novoSpaRC and CSOmap (recent and only available de novo 3D spatial reconstruction methods) on 16 datasets and 681 reconstructions, reveals a significantly distinctive superior performance of D-CE.

Background: Recent developments in our understanding of the interactions between long non-coding RNA (lncRNA) and cellular components have improved treatment approaches for various human diseases including cancer, vascular diseases, and neurological diseases. Although investigation of specific lncRNAs revealed their role in the metabolism of cellular RNA, our understanding of their contribution to post-transcriptional regulation is relatively limited. In this study, we explore the role of lncRNAs in modulating alternative splicing and their impact on downstream protein-RNA interaction networks. Results: Analysis of alternative splicing events across 39 lncRNA wildtype and knockout RNA-sequencing datasets from three human cell lines: HeLa (Cervical Cancer), K562 (Myeloid Leukemia), and U87 (Glioblastoma), resulted in high confidence (fdr < 0.01) identification of 4432 skipped exon events and 2474 retained intron events, implicating 759 genes to be impacted at post-transcriptional level due to the loss of lncRNAs. We observed that a majority of the alternatively spliced genes in a lncRNA knockout were specific to the cell type, in agreement with the finding that genes affected by alternative splicing also displayed enriched functions in a cell type specific manner. To understand the mechanism behind this cell-type specific alternative splicing patterns, we analyzed RNA binding protein (RBP)-RNA interaction profiles across the spliced regions. Conclusions: Despite limited RBP binding data across cell lines, alternatively spliced events detected in lncRNA perturbation experiments were associated with RBPs binding in proximal intron-exon junctions, in a cell type specific manner. The cellular functions affected by alternative splicing were also affected in a cell type specific manner. Based on the RBP binding profiles in HeLa and K562 cells, we hypothesize that several lncRNAs are likely to exhibit a sponge effect in disease contexts, resulting in the functional disruption of RBPs due to altered titration of the RBPs from their target loci. We propose that such lncRNA sponges can extensively rewire the post-transcriptional gene regulatory networks by altering the protein-RNA interaction landscape in a cell-type specific manner.

This study proposes a redesign of asymmetric single-chamber microbial fuel cells (a-SCMFC) with the goal of optimizing energy production. The new approach is based on the introduction of an Intermediate Microfluidic Septum (IMS) as a relatively simple and inexpensive method to opti-mize both electrolyte flow and species transfer inside the devices. SCMFCs with the novel IMS, operated with sodium acetate as the carbon source, demonstrate to enhance the energy recovery (Erec) factor, defined as the ratio between the energy yield and the inner volume of electrolyte. In standard operative conditions, cells with IMS exhibit Erec value of (37±1) J/m3, with respect to (3.0±0.3) J/m3 of control cells. Furthermore, changing sodium acetate concentration the Erec values change accordingly. By monitoring the activity of a-SCMFCs for over one-year, beneficial impact of the IMS on both the initial inoculation phase and the long-term stability of electrical perfor-mance were observed. These improvements suggest the effectiveness of IMS to allow the de-velopment of efficient biofilms, likely due to the reduction in oxygen diffusion towards the anode. Electrochemical characterizations confirm that the presence of the IMS impacts the diffusion processes inside the electrolytic chamber, supporting the hypothesis of a beneficial effect on oxygen diffusion.

This paper is regarding design and program an Micro-controller Arduino Uno board by using Arduino software to work as a photo-sensor(Active) single axial solar tracker system(SASTS). A solar panel, two photo-resistors (LDR) in two sides (north/south) of the photo-voltaic(PV) and a servo motor are connected to the Uno board, which is running a code that prepared by Arduino software IDE in advanced then it works as a tracking system. Here, the LDRs send the signal of presence or absence of the light to the board and based on that sent signal the Uno reflects a new signal to the servo motor to rotate and finds the light source. Lastly, the photo-sensor single axis tracker is made while Continuously, the system tries to face the panel to the sun and whilst changing the irradiance intensity it starts searching to find the angle of highest irradiance. Based on results that are extracted from the data, the tracker system significantly boosts the output efficiency of the solar panel. By using the Micro-controller Uno board, LDRs, servo motor and special designed mechanical base, the tracking system is constructed, based on acquired data the influence of the STS on the increasing the solar panel efficiency is more obvious. Significantly, the tracker system rises the efficiency of the PV .

Receptor–ligand interactions at cell interfaces initiate signaling cascades essential for cellular communication and effector functions. Specifically, T cell receptor (TCR) interactions with pathogen-derived peptides presented by the major histocompatibility complex (pMHC) molecules on antigen-presenting cells are crucial for T cell activation. The binding duration, or dwell time, of TCR–pMHC interactions correlates with downstream signaling efficacy, with strong agonists exhibiting longer lifetimes compared to weak agonists. Traditional surface plasmon resonance (SPR) methods quantify 3D affinity but lack cellular context and fail to account for factors like membrane fluctuations. In the recent years, single-molecule Förster resonance energy transfer (smFRET) has been applied to measure 2D binding kinetics of TCR–pMHC interactions in a cellular context. Here, we introduce a rigorous mathematical model based on survival analysis to determine exponentially distributed receptor–ligand interaction lifetimes, verified through simulated data. Additionally, we developed a comprehensive analysis pipeline to extract interaction lifetimes from raw microscopy images, demonstrating the model’s accuracy and robustness across multiple TCR–pMHC pairs. Our new software suite automates data processing to enhance throughput and reduce bias. This methodology provides a refined tool for investigating T cell activation mechanisms, offering insights into immune response modulation.

Head and neck squamous cell carcinoma (HNSCC) arise from the mucosal epithelium in the oral cavity, pharynx, sino-nasal region, and larynx. Laryngeal squamous cell carcinoma (LSCC) represents one-third of all head and neck cancers. Dysregulated RNA-related pathways define an important molecular signature in this aggressive carcinoma. The Survival Motor Neuron (SMN) protein regulates fundamental aspects of the RNA metabolism but, curiously, its role in cancer is virtually unknown. For the first time, here we focus on SMN in cancer context. We conducted a pilot study in a total of 20 patients with LSCC where SMN was found overexpressed at both the protein and transcript levels. By a cellular model of human laryngeal carcinoma, we demonstrated that SMN impacts cancer-relevant behaviors and perturbs key players of cell migration, invasion, and adhesion. Furthermore, in LSCC we showed a physical interaction between SMN and the epidermal growth factor receptor (EGFR), whose overexpression is an important feature in these tumours. This study candidates SMN as novel therapeutic target in LSSC, and likely in the whole spectrum of HNSCC. Overall, we provide the first analysis of SMN in human cancer.

Ionizing radiation exposure can cause damage to diverse tissues and organs, with the hematopoietic system being the most sensitive. However, limited information is available regarding the radiosensitivity of various hematopoietic cell populations in the bone marrow due to the high heterogeneity of the hematopoietic system. In this study, we observed that granulocyte-macrophage progenitors, hematopoietic stem/progenitor cells and B cells within the bone marrow showed the highest sensitivity, exhibiting a rapid decrease in cell numbers following irradiation. Nonetheless, neutrophils, natural killer (NK) cells, T cells, and dendritic cells demonstrated a certain degree of radioresistance, with neutrophils exhibiting the most pronounced resistance. By employing single-cell transcriptome sequencing, we investigated the early responsive genes in various cell types following irradiation, revealing that distinct gene expression profiles emerged between radiosensitive and radioresistant cells. In B cells, radiation exposure led to a specific upregulation of genes associated with mitochondrial respiratory chain complexes, suggesting a connection between these complexes and cell radiosensitivity. In neutrophils, radiation exposure resulted in fewer gene alterations, indicating their potential for distinct mechanisms in radiation resistance. Collectively, this study provides insights into the molecular mechanism for the heterogeneity of radiosensitivity among the various bone marrow hematopoietic cell populations.

With the rising cost of animal feed protein, finding affordable and effective substitutes is crucial. Walnut kernel cake, a polyphenols, fiber, protein and fat-rich byproduct of walnut oil extraction, has been underexplored as a potential protein replacement in pig feed. In this study, we found that feeding large Diqing Tibetan pigs walnut kernel cake promoted adipose deposition and improved pork quality during pig growth. Transcriptome analysis revealed the upregulation of genes ANGPTL8, CCNP, ETV4, and TRIB3, associated with adipose deposition. Pathway analysis highlighted enrichment in adipose-deposition-related pathways, including PPAR, Insulin, PI3K-Akt, Wnt, and MAPK signaling. Further analysis identified DEGs (differentially expressed genes) positively correlated with adipose-related traits, such as PER2 and PTGES. Single-cell transcriptome data pointed to the specific expression of CD248 and PTGES in adipocyte progenitor/stem cells (APSCs), pivotal for adipocyte differentiation and adipose deposition regulation. This study demonstrates walnut kernel cake's potential to substitute soybean cake in pig feed, providing high-quality protein and promoting adipose deposition. It offers insights into feed protein replacement, human functional food, fat metabolism, and related diseases, with marker genes and pathways supporting pig breeding and pork quality improvement.

Intratumoral heterogeneity associates with more aggressive disease progression and worse patient outcomes. Understanding the reasons enabling the emergence of such heterogeneity remains incomplete, which restricts our ability to manage it from a therapeutic perspective. Technological advancements such as high-throughput molecular imaging, single-cell omics and spatial transcriptomics now allow recording the patterns of spatiotemporal heterogeneity in a longitudinal manner, thus offering insights into the multi-scale dynamics of its evolution. Here, we review latest technological trends and biological insights from molecular diagnostics as well as spatial transcriptomics, both of which have witnessed a burgeoning growth in recent past in terms of mapping heterogeneity within tumor cell types as well as stromal constitution. We also discuss ongoing challenges, indicating possible ways to integrate insights across these methods to have a systems-level spatiotemporal map of heterogeneity in each tumor, and a more systematic investigation of implications of heterogeneity for the patient outcomes.

Microscale surgery on single cells and small organisms have enabled major advances in fundamental biology and in engineering biological systems. Examples of applications range from wound healing and regeneration studies to the generation of hybridoma to produce monoclonal antibodies. Even today, these surgical operations are often performed manually, but they are labor-intensive and lack reproducibility. Microfluidics has emerged as a powerful technology to control and manipulate cells and multicellular systems at the micro- and nanoscale with high precision. Here, we review the physical and chemical mechanisms of microscale surgery, and the corresponding design principles, applications, and implementations in microfluidic systems. We consider four types of surgical operations: 1) Sectioning, which splits a biological entity into multiple parts, 2) ablation, which destroys part of an entity, 3) biopsy, which extracts materials from within a living cell, and 4) fusion, which joins multiple entities into one. For each type of surgery, we summarize the motivating applications and the microfluidic devices developed. Throughout the review, we highlight existing challenges and opportunities. We hope that this review will inspire scientists and engineers to continue to explore and improve microfluidic surgical methods.

To reveal rare phenotypes in bacterial populations conventional microbiology tools should be advanced to generate rapid, quantitative, accurate and high-throughput data. The main drawbacks of widely used traditional methods for antibiotic studies include low sampling rate and averaging data for population measurements. To overcome these limitations microfluidic-microscopy systems have great promise to produce quantitative single-cell data with high sampling rates. Using Mycobacterium smegmatis cells we applied both conventional assays and a microfluidic-microscopy method to reveal antibiotic-tolerance mechanisms of wild type and the msm2570::Tnmutant cells. Our results revealed that the enhanced antibiotic tolerance mechanism of the msm2570::Tn mutant was due to the low number of lysed cells during the antibiotic exposure compared with wild-type cells. This is the first study that characterized the antibiotic-tolerance phenotype of the msm2570::Tn mutant that has a transposon insertion in the msm2570 gene encoding a putative xanthine/uracil permease, which enrolls in uptake of nitrogen compound during nitrogen limitation. The experimental results indicate that the msm2570::Tn mutant can be further interrogated to reveal antibiotic killing mechanisms, in particularly, antibiotics those targets cell wall integrity.

An outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) occurred in China towards the end of 2019, and has spread rapidly ever since. Previous studies showed that some virus could affect the reproductive system and cause long-term complications. Recent studies exploring the source of SARS-CoV-2 using genomic sequencing have revealed that SARS-CoV-2 enters the host cells via the angiotensin-converting enzyme II (ACE2), the receptor that recognizes SARS-CoV. To investigate the expression of ACE2 and to explore the potential risk of infection in the reproductive system, we performed a thorough bioinformatic analysis on data from public databases involving RNA expression, protein expression, and single-cell RNA expression studies. The analyzed data showed high levels of ACE2 mRNA and protein expression in the testis and spermatids and equal levels of ACE2 expression in the uterus and lung. Comprehensive single-cell analysis identified ACE2 expression in the lung, testis, spermatids, and uterus. In conclusion, this study revealed the potential risk associated with the SARS-CoV-2 infection in the reproductive system and predicted that long-term complications might have a significant impact on the prevention and management of COVID-19, the disease caused upon infection with SARS-CoV-2.

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

The structural transitions RNAs undergo during trafficking are not well-understood. Here, we used the well-developed yeast Ty1 retrotransposon to provide the first structural model of genome (g) RNA in the nucleus from a retrovirus-like transposon. Through a detailed comparison of nuclear Ty1 gRNA structure with those established in the cytoplasm, virus-like particles (VLPs), and synthesized in vitro, we detected Ty1 gRNA structural alternations that occur during retrotransposition. Full-length Ty1 gRNA serves as the mRNA for Gag and Gag-Pol proteins and as the genome that is reverse transcribed within VLPs. We show that about 60% of base pairs predicted for the nuclear Ty1 gRNA appear in the cytoplasm, and active translation does not account for such structural differences. Most of the shared base pairs are represented by short-range interactions, while the long-distance pairings seem unique for each compartment. Highly structured motifs tend to be preserved after nuclear export of Ty1 gRNA. In addition, our study highlights the important role of Ty1 Gag in mediating critical RNA:RNA interactions required for retrotransposition.

Clear cell renal cell carcinoma (ccRCC) is the most prevalent form of renal cancer and its treatment is hindered by resistance to targeted and immunotherapies and combinations ob both. We have reported that knockdown of the antisense noncoding mitochondrial RNAs (ASncmtRNAs) with chemically-modified antisense oligonucleotides induces proliferative arrest and apoptotic death in tumor cells from many human and mouse cancer types. These studies have been mostly performed in vitro and in vivo on commercially-available cancer cell lines and have shown that, in mouse models, tumor growth is stunted by the treatment. The present work was performed on cells derived from primary and metastatic ccRCC tumors. We established primary cultures fom primary and metastatic ccRCC tumors, which were subjected to knockdown of ASncmtRNAs in vitro and in vivo in an orthotopic xenograft model in NOD/SCID mice. We found that these primary ccRCC cells are affected in the same way as tumor cell lines and, in the orthotopic model, tumor growth was significantly reduced by the treatment. This study on patient-derived ccRCC tumor cells represents a model closer to actual patient ccRCC tumors and shows that knockdown of ASncmtRNAs poses a potential treatment option for these patients.

Adult stem cells (ASCs) can be cultured with difficulty from most tissues, often requiring chemical or transgenic modification to achieve adequate quantities. We show here that mouse primary fibroblasts grown in suspension change from the elongated and flattened morphology observed under standard adherent culture conditions generating rounded cells with large nuclei and scant cytoplasm expressing the mesenchymal stem cell (MSC) marker (Sca1; Ly6A) within 24hrs. Based on this initial observation, we describe here a suspension culture method that, irrespective of the lineage used, mouse fibroblast, primary human somatic cells (fibroblasts, hepatocytes and keratinocytes), is capable of generating a high yield of cells in spheroid form which display expression of ASCs surface markers, circumventing the anoikis which often occurs at this stage. Moreover, mouse fibroblasts-derived spheroids can be differentiated into adipogenic and osteogenic lineages. Analysis of single cell RNA sequence data identified 8 distinct cell clusters with one in particular comprising approximately 10% of the cells showing high levels of proliferative capacity expressing high levels of genes related to MSCs and self-renewal as well as extracellular matrix (ECM). We believe the rapid, high-yield generation of proliferative, multi-potent ASC-like cells by the process we term suspension-induced stem cell transition (SIST) could have significant implications for regenerative medicine.

Contamination of the environment with nano- and microplastic particles exerts a threatened impact on the aquatic ecosystems and sustainable catfish aquaculture. The presence of nanoplastics has been found to have a detrimental impact on both aquatic and terrestrial ecosystems. The present study examines the effect of polystyrene nanoplastics on DNA, erythrocytes, oxidative status and renal of catfish in addition to the potential protective effects of Chlorella vulgaris bioremeditation and selenium to hinder this effect. Six equal groups of fish were used as follows: Group 1: served as a control group and received water free from PS NPs; Group 2: was exposed to PS NPs at a concentration of 5mg/L; Group 3: was exposed to PS NPs (5mg/L) + selenium (1mg/kg diet); Group 4: was exposed to PS NPs (5mg/L) + C. vulgaris (25g/kg diet); Group 5: was supplemented with C. vulgaris (25g/kg diet); and Group 6: was supplemented with selenium (1mg/kg diet). The results indicated that PS NPs induced oxidative stress by significantly elevating MDA activities and slightly reducing antioxidant biomarkers, resulting in DNA damage, increased frequency of micronuclei, erythrocyte alterations, and numerous histopathological alterations in kidney tissue. Selenium and C. vulgaris significantly ameliorated the oxidative/antioxidant status, reduced DNA damage, micronucleus frequency, erythrocyte alterations, and morphology of kidney tissue. Nevertheless, further research is needed to evaluate in detail the mechanism behind the toxicity with nano-microplatics in aquatic system.

The development of the ovarian antral follicle is a complex, highly regulated process. Oocytes orchestrate and coordinate the development of mammalian ovarian follicles, and the rate of follicular development is governed by a developmental program intrinsic to the oocyte. Characterizing oocyte signatures during this dynamic process is critical for understanding oocyte maturation and follicular development. Although the transcriptional signature of sheep oocytes matured in vitro and preovulatory oocytes have been previously described, the transcriptional changes of oocytes in antral follicles have not. Here, we used single-cell transcriptomics (SmartSeq2) to characterize sheep oocytes from small, medium, and large antral follicles. We characterized the transcriptomic landscape of sheep oocytes during antral follicle development, identifying unique features in the transcriptional atlas, stage-specific molecular signatures, oocyte-secreted factors, and transcription factor networks. Notably, we identified specific gene and signaling pathways in each antral follicle stage that may reflect oocyte quality and in vitro maturation competency. Additionally, we discovered key biological processes that drive the transition from small to large antral follicles, revealing hub genes involved in follicle recruitment and selection. Thus, our work provides a comprehensive characterization of the single-oocyte transcriptome, filling a gap in the mapping of the molecular landscape of sheep oogenesis. We also provide key insights into the transcriptional regulation of the critical stages of antral follicular development, which is essential for understanding how the oocyte orchestrates follicular development.

The snATAC + snRNA platform allows epigenomic profiling of open chromatin and gene expression with single-cell resolution. The most critical assay step is to isolate high-quality nuclei to proceed with droplet-base single nuclei isolation and barcoding. With the increasing popularity of multiomic profiling in various fields, there is a need for optimized and reliable nuclei isolation methods, mainly for human tissue samples. Herein we compared different nuclei isolation methods for cell suspensions, such as peripheral blood mononuclear cells (PBMC, n=18) and a solid tumor type, ovarian cancer (OC) (n=18), derived from debulking surgery. Nuclei morphology and sequencing output parameters were used to evaluate the quality of preparation. Our results show that NP-40 detergent-based nuclei isolation yields better sequencing results than collagenase tissue dissociation for OC, significantly impacting cell identification and analysis. Given the utility of applying such techniques to frozen samples, we also tested frozen preparation and digestion (n=6). A paired comparison between frozen and fresh samples validated the quality of both specimens. Finally, we demonstrate the reproducibility of scRNA and snATAC + snRNA platform, by comparing the gene expression profiling of PBMC. Our results highlight how the choice of nuclei isolation methods is critical for obtaining quality data in multiomic assays. It also shows that the measurement of expression between scRNA and snRNA is comparable and effective for cell type identification.

The following study reports on the first thraustochytrid isolates identified from Iceland. They were collected from three different locations off the northern coast of the country (Location A, Skagaströnd; Location B, Hveravík; and Location C, Eyjafjörður). Using 18S rDNA sequence analysis, isolates from Locations A and B were identified within the Thraustochytrium kinnei species while other isolates within the Sicyoidochytrium minutum species when compared to other known strains. Cells isolated from Locations A (2.10 ± 0.70 g/L) and B (1.54 ± 0.17 g/L) produced more biomass than the ones isolated from Location C (0.43 ± 0.02 g/L). This study offers the first-time examination of the utility of byproducts from fisheries as a nitrogen source in media formulation for thraustochytrids. Experiments showed that isolates produced more biomass (per unit of substrate) when cultured on nitrogen of marine (2.55 ± 0.74 g/L) as compared to of commercial origin (1.06 ± 0.57 g/L). Glycerol (2.43 ± 0.56 g/L) was a better carbon source than glucose (1.84 ± 0.57 g/L) in growth studies. Fatty acid (FA) profiles showed that the isolates from Location C (S. minutum) had low ratios of monounsaturated (4.21 ± 2.96%) and omega-6 (0.68 ± 0.59%) FAs. However, the isolates also had high ratios of docosahexaenoic acid (DHA; 35.65 ± 1.73%) and total omega-3 FAs (40.39 ± 2.39%), indicating that they could serve as a source of marine oils for human consumption and in aquaculture feeds. The T. kinnei isolates from Location A could be used in biodiesel production due to their high ratios of monounsaturated (18.38 ± 6.27%) long chain (57.43 ± 8.27%) FAs.

Ribosome assembly is one of the most fundamental processes in gene expression and has served as a playground to investigate the molecular mechanisms of how protein-RNA complexes (RNPs) assemble. The bacterial ribosome is composed of around 50 ribosomal proteins several of which are co-transcriptionally assembled on a ~4,500 nucleotides long pre-rRNA transcript that is further processed and modified during transcription, the entire process taking around 2 minutes in vivo and assisted by dozens of assembly factors. How this complex molecular process works so efficiently to produce an active ribosome has been investigated over decades and has resulted in the development of a plethora of novel approaches that can also be used to study the assembly of other RNPs. Here we review biochemical, structural and biophysical methods that have been developed and integrated to provide a detailed and quantitative understanding of this complex and intricate molecular process of assembly. We also discuss emerging cutting-edge approaches that could be used in the future to study how transcription, rRNA processing, cellular factors and the native cellular environment shape ribosome assembly and RNP assembly at large.

Safe sample transport is of great importance for infectious diseases diagnostics. Various treatments and buffers are used to inactivate pathogens in diagnostic samples. At the same time, adequate sample preservation, particularly of nucleic acids, is essential to allow an accurate laboratory diagnosis. For viruses with single-stranded RNA genomes of positive polarity, such as foot-and-mouth disease virus (FMDV), however, naked full-length viral RNA can itself be infectious. In order to assess the risk of infection from inactivated FMDV samples, two animal experiments were performed. In the first trial, six cattle were injected with FMDV RNA (isolate A22/IRQ/24/64) into the tongue epithelium. All animals developed clinical disease within two days and FMDV was reisolated from serum and saliva samples. In the second trial, another group of six cattle was exposed to FMDV RNA by instilling it on the tongue and spraying it into the nose. The animals were observed for 10 days after exposure. All animals remained clinically unremarkable and virus isolation as well as FMDV genome detection in serum and saliva were negative. No transfection reagent was used for any of the animal inoculations. In conclusion, cattle can be infected by injection with naked FMDV RNA, but not by non-invasive exposure to the RNA. Inactivated FMDV samples that contain full-length viral RNA carry only a negligible risk of infecting animals.

We remind about the dogma initially established with the nucleic acid double helix, i.e. the DNA structure as the primary source of life. However, we bring into the discussion those additional processes that were crucial to enable life and cell evolution. Studying chemosensory proteins (CSPs) and odor binding proteins (OBPs) of insects, we have found a high level of pinpoint mutations on the RNA and peptide sequences. Many of these mutations are found to be tissue-specific and induce subtle changes in the protein structure, leading to a new theory of cell multifunction and life evolution. Here, attention is given to RNA and peptide mutations in small soluble protein families known for carrying lipids and fatty acids as fuel for moth cells. A new phylogenetic analysis of mutations is presented and provides even more support to the pioneer work, i.e. the finding that mutations in binding proteins have spread through moths and various groups of insects. Then, focus is given to specific mechanisms of mutations that are not random, change α-helical profilings and bring new functions at the protein level. In conclusion, RNA and peptide mutations are not seen as representative of a multitude of diseases, but rather as an alternative way by which protocells developed to acquire multifunction and totipotency. This provides a basis for the theory of RNA/peptide mutations for birth and evolution of life on earth’s crust proposed here.

Iron oxide nanozymes are a form of nanomaterial with both superparamagnetic and enzyme-mimicking properties. Given the multifunctional nature of iron oxide nanozymes, it is attractive for creating iron oxide hybrid nanozymes through biomolecular modifications to imbue with auxillary properties. Such iron oxide hybrid nanozymes can be useful for rapid and cost-effective analysis of circulating tumor nucleic acids (ctNAs) in patient liquid biopsies during minimal residual disease (MRD) monitoring of cancer recurrence. Herein, the use of streptavidin-modified iron oxide hybrid nanozymes is reported for magnetic enrichment and bioelectrocatalytic sensing of three prostate cancer (PCa) ctRNA biomarkers with high detection specificity and sensitivity (10 copies) over an ultrabroad dynamic range (five orders of magnitude). Furthermore, the feasibility of ctRNA analysis for pre- and post-cancer treatment MRD monitoring is demonstrated using PCa urinary liquid biopsy samples.

Monoclonal antibodies (mAbs) have become an increasingly important therapeutic modality in recent years, with applications in oncology, hematology, and immunology. This review discusses the production of therapeutic mAbs, including the various types of mAbs (murine, chimeric, humanized, and fully human), their key applications, and the techniques used for their production, including hybridoma technology, single B cell antibody technology, and phage display technology. The review then proposes advanced techniques to overcome them. These include microfluidic sorting, alternative expression systems (including plants, bacteria, and yeast, and cell-free production systems), and the development of complex antibody formats like bi-specific monoclonal antibodies, antibody fragments, antibody-drug conjugates, and nanobody development. These advanced techniques promise to enhance the efficiency, cost-effectiveness, and scalability of mAb production, paving the way for more effective therapeutic applications. This review highlights the theoretical significance of integrating innovative technologies to overcome existing challenges in mAb production, ultimately advancing the field of precision medicine.

Once discovered, cancer stem cells have become a hot topic in the research of cancer therapy. These cells possess stem cell-like self-renewal and differentiation capacities and are important factors that dominate cancer metastasis, therapy- resistance and recurrence. What's worse, their own characteristics make them difficult to be eliminated. Colorectal cancer is the third most common cancer and the second leading cause of cancer death worldwide. Targeting colorectal cancer stem cells (CRCSCs) can inhibit colorectal cancer metastasis, enhance therapeutic efficacy, and reduce recurrence. Here, we introduced the origin, marker proteins, identification, cultivation and research techniques of CRCSCs, summarized the signaling pathways that regulate the stemness of CRCSCs, such as Wnt, JAK/STAT3, Notch, and Hh signaling pathway. In addition to these, we also reviewed anti-CRCSC drugs targeting signaling pathways，markers，and other regulators in recent years. These will help researchers gain insight into the current agents targeting to CRCSCs, explore new cancer drugs, and propose potential therapy.

Methanotrophic bacteria that possess a unique ability of using methane as a sole source of carbon and energy have attracted considerable attention as potential producers of a single-cell protein. So far, this biotechnology implied using freshwater methanotrophs, although many regions of the world have limited freshwater resources. This study aimed at searching for novel methanotrophs capable of fast growth in saltwater comparable in composition with seawater. A methane-oxidizing microbial consortium containing Methylomarinum- and Methylotuvimicrobium-like methanotrophs has been enriched from a sediment of the river Chernavka (water pH 7.5, total salt content 30 g L-1), a tributary river of the hypersaline Lake Elton, southern Russia. This microbial consortium, designated Ch1, demonstrated stable growth on natural gas in a bioreactor in media with a total salt content of 23 to 35.9 g L-1 at a dilution rate of 0.19-0.21 h-1. The highest biomass yield of 5.8 g CDW/L with a protein content of 63% was obtained during continuous cultivation of the consortium Ch1 in a medium with a total salt content of 29 g L-1. Isolation attempts resulted in obtaining a pure culture of methanotrophic bacteria, strain Ch1-1. The 16S rRNA gene sequence of strain Ch1-1 displayed 97.09-97.24% similarity to the corresponding gene fragments of characterized representatives of Methylomarinum vadi, methanotrophs isolated from marine habitats. The genome of strain Ch1-1 was 4.8 Mb in size and encoded 3 rRNA operons, and about 4400 proteins. The genome contained the gene cluster coding for ectoine biosynthesis, which explains the ability of strain Ch1-1 to tolerate high salt concentration.

Polyphosphate (polyP) accumulating organisms (PAOs) are the key agent to perform enhanced biological phosphorus removal (EBPR) activity, and intracellular polyP plays a key role in this process. Potential associations between EBPR performance and the polyP structure have been suggested, but are yet to be extensively investigated, mainly due to the lack of established methods for polyP characterization in the EBPR system. In this study, we explored and demonstrated that single-cell Raman spectroscopy (SCRS) can be employed for characterizing intracellular polyPs of PAOs in complex environmental samples such as EBPR systems. The results, for the first time, revealed distinct distribution patterns of polyP length (as Raman peak position) in PAOs in lab-scale EBPR reactors that were dominated with different PAO types, as well as among different full-scale EBPR systems with varying configurations. Furthermore, SCRS revealed distinctive polyP composition/features among PAO phenotypic sub-groups, which are likely associated with phylogenetic and/or phenotypic diversity in EBPR communities, highlighting the possible resolving power of SCRS at the microdiversity level. To validate the observed polyP length variations via SCRS, we also performed and compared bulk polyP length characteristics in EBPR biomass using conventional polyacrylamide gel electrophoresis (PAGE) and solution ³¹P nuclear magnetic resonance (³¹P-NMR) methods. The results are consistent with the SCRS findings and confirmed the variations in the polyP lengths among different EBPR systems. Compared to conventional methods, SCRS exhibited advantages as compared to conventional methods, including the ability to characterize in situ the intracellular polyPs at subcellular resolution in a label-free and non-destructive way, and the capability to capture subtle and detailed biochemical fingerprints of cells for phenotypic classification. SCRS also has recognized limitations in comparison with ³¹P-NMR and PAGE, such as the inability to quantitatively detect the average polyP chain length and its distribution. The results provided initial evidence for the potential of SCRS-enabled polyP characterization as an alternative and complementary microbial community phenotyping method to facilitate the phenotype-function (performance) relationship deduction in EBPR systems.

Kidneys of mice, rats and humans possess progenitors that maintain daily homeostasis and take part in endogenous regenerative processes following injury, owing to their capacity to proliferate and differentiate. In the glomerular and tubular compartments of the nephron, consistent studies demonstrated that well-characterized, distinct populations of progenitor cells, localized in the parietal epithelium of Bowman capsule and scattered in the proximal and distal tubules, could generate segment-specific cells in physiological conditions and following tissue injury. However, defective or abnormal regenerative responses of these progenitors can contribute to pathologic conditions. The molecular characteristics of renal progenitors have been extensively studied, revealing that numerous classical and evolutionarily conserved pathways, such as Notch or Wnt/β-catenin, play a major role in cell regulation. Others, such as retinoic acid, renin-angiotensin-aldosterone system, TLR2 (Toll-Like Receptor 2) and leptin, are also important in this process. In this review, we summarize the plethora of molecular mechanisms directing renal progenitor responses during homeostasis and following kidney injury. Finally, we will explore how single cell RNA sequencing could bring the characterization of renal progenitors to the next level, while knowing their molecular signature is gaining relevance in the clinic.

In current severe global emergency situation of 2019-nCov outbreak, it is imperative to identify vulnerable and susceptible groups for effective protection and care. Recently, studies found that 2019-nCov and SARS-nCov share the same receptor, ACE2. In this study, we analyzed five large-scale bulk transcriptomic datasets of normal lung tissue and two single-cell transcriptomic datasets to investigate the disparities related to race, age, gender and smoking status in ACE2 gene expression and its distribution among cell types. We didn’t find significant disparities in ACE2 gene expression between racial groups (Asian vs Caucasian), age groups (>60 vs <60) or gender groups (male vs female). However, we observed significantly higher ACE2 gene expression in former smoker’s lung compared to non-smoker’s lung. Also, we found higher ACE2 gene expression in Asian current smokers compared to non-smokers but not in Caucasian current smokers, which may indicate an existence of gene-smoking interaction. In addition, we found that ACE2 gene is expressed in specific cell types related to smoking history and location. In bronchial epithelium, ACE2 is actively expressed in goblet cells of current smokers and club cells of non-smokers. In alveoli, ACE2 is actively expressed in remodelled AT2 cells of former smokers. Together, this study indicates that smokers especially former smokers may be more susceptible to 2019-nCov and have infection paths different with non-smokers. Thus, smoking history may provide valuable information in identifying susceptible population and standardizing treatment regimen.

Classical models of gene expression were built using genetics and biochemistry. Although these approaches are powerful, they have very limited consideration of the spatial and temporal organization of gene expression. Although the spatial organization and dynamics of RNA polymerase II (RNAPII) transcription machinery has fundamental functional consequences for gene expression, its detailed studies have been for long time abrogated by the limits of classical light microscopy. The advent of super-resolution microscopy (SRM) techniques allowed for the visualization of the RNAPII transcription machinery with nanometer resolution and millisecond precision. In this review, we summarize the recent methodological advances in SRM, focus on its application for studies of the nanoscale organization in space and time of RNAPII transcription, and discuss its consequences for the mechanistic understanding of gene expression.

Genetic disorders are quite a major topic of discussion and debate in the recent world of biological sciences. Turner’s syndrome is one such disorder caused by a chromosome aneuploidy and it has characteristic symptoms in the patient or the affected individual. The amniotic fluid is a complex biological material found in the amniotic sac of pregnant women and they can provide valuable knowledge and understanding of the pathogenesis of this particular chromosomal abnormality. In this study, global gene expression analysis of cell-free RNA in amniotic fluid supernatant was used to detect genes/organ systems which may be significant in the pathophysiology of Turner’s syndrome. The cell-free RNA from the amniotic fluid of five mid-trimester Turner’s syndrome fetuses and five euploid female fetuses matched for age of gestation were extracted, amplified and hybridized onto Affymetrix U33 Plus 2.0. array. The paired t-test was used to identify the significantly differentially regulated genes. Biological interpretation was conducted using ingenuity pathway analysis and BioGPS gene expression atlas. Of the genes, XIST was especially downregulated and SHOX was not expressed differentially. One of the most highly represented organ systems was the hematologic/immune system, differentiating the transcriptome of Turner’s syndrome from other chromosomal aneuploidies that are discussed in this area of science. The differences in the transcriptome of the Turner’s syndrome are due to genome-wide dysregulation. The hematologic/immune system differences are significant in early-onset autoimmune dysfunction. There are other genes which have been identified that are associated with the cardiovascular and the skeletal system, as these are often seen to be affected in the female patients with turner’s syndrome. Hopefully, such knowledge gained from this study will help us to understand the deeper mechanisms of this disorder and the possible treatments of this disease.

This review explores recent advances in the design of fermentation processes for producing alternative proteins, with a focus on the utilization of agro-industrial waste and renewable substrates. Key bioprocess strategies, such as optimized bioreactor designs and strict fermentation parameter control, are discussed to enhance substrate conversion efficiency into protein biomass. The role of enzyme technologies in improving digestibility and protein quality is also highlighted. The article underscores the importance of developing cost-effective fermentation solutions that minimize environmental impact while addressing technical challenges such as scalability and contamination control. Furthermore, it emphasizes the growing need for innovations in fermentation process design to ensure the sustainability of industrial protein production. The review concludes that improvements in process design are fundamental to overcoming technological and regulatory barriers, particularly in increasing the efficiency and competitiveness of non-meat proteins in the global market.

Advanced genomics, transcriptomics and epigenomics techniques are providing unprecedented insights into the understanding of the molecular underpinnings of the central nervous system, including the neuro-sensory cochlea of the inner ear. Here, we report for the first time a comprehensive and updated overview of the most advanced omics techniques for the study of nucleic acids, and their applications in cochlear research. We describe the available in vitro and in vivo models for hearing research, the principles of genomics, transcriptomics and epigenomics, alongside their most advanced technologies (like single cell omics and spatial omics), which allows to investigate the molecular events that occur at single cell resolution retaining the spatial information.

The mutualistic interactions between the oleaginous yeast Lipomyces starkeyi and the green mi-croalga Chloroidium saccharophilum in mixed cultures were investigated to exploit possible syner-gistic effects. As a matter of facts, microalga could act as an oxygen generator for the yeast, while the yeast could provide carbon dioxide to microalga. A lignocellulosic hydrolysate from steam exploded Arundo donax (Giant reed) was used as low cost feedstock. The overall lipid content and lipid productivity obtained in the mixed culture treating the hydrolysate of Arundo donax were equal to 0.081 glipid.gbiomass-1 and 37.2 mglipid.L-1.d-1, respectively. They represented promising re-sults if compared to the model systems where synthetic media were used. This study provided new input for the integration of Single Cell Oil (SCO) production with agro-industrial feedstock and the fatty acid distribution mainly consisting of stearic (C18:0) and oleic acid (C18:1) allows promising applications in biofuels, cosmetics, food additives and other products of industrial interest.

Renal cell carcinoma (RCC) accounts for approximately 90-95% of all kidney cancers in adults, with the clear cell RCC (ccRCC) the most frequently identified subtype. RCC is known for its responsiveness to immunotherapy, making it an area of significant research interest. Immune checkpoint (IC) molecules, which regulate immune surveillance, are established therapeutic targets in RCC. The aim of this study was to analyze the influence of polymorphisms in genes HVEM and CD160 on susceptibility to ccRCC and patient overall survival (OS) with over a ten-year period of observation. We genotyped three HVEM single nucleotide polymorphisms (SNPs): rs1886730, rs2234167, and rs8725, and two CD160 SNPs: rs744877 and rs2231375, in 238 ccRCC patients and 521 controls. Our findings indicated that heterozygosity within rs2231375 and/or rs2234167 increases ccRCC risk. Furthermore, in women, heterozygosity within additional HVEM SNPs, rs8725 and rs1886730, is associated with an increased ccRCC risk. The presence of a minor allele for rs1886730, rs2234167, rs8725, and rs2231375 was also correlated with certain clinical features of the disease. Moreover, the rs1886730 SNP was found to be associated with patients' OS. In conclusion, our study highlights an association between HVEM and CD160 polymorphisms and the risk of developing ccRCC as well as patient’s OS.

The observation that certain therapeutic strategies for targeting inflammation benefit patients with distinct immune-mediated inflammatory diseases (IMIDs) is exemplified by the success of TNF blockade in conditions including rheumatoid arthritis, ulcerative colitis and skin psoriasis, albeit only for subsets of individuals with each condition. This suggests intersecting "nodes" in inflammatory networks at a molecular and cellular level may drive and/or maintain IMIDs, being “shared” between traditionally distinct diagnoses without mapping neatly to a single clinical phenotype. In line with this proposition, integrative tumour tissue analyses in oncology have highlighted novel cell states acting across diverse cancers, with important implications for precision medicine. Drawing upon advances in the oncology field, this narrative review will first summarise learnings from the Human Cell Atlas in health as a platform for interrogating IMID tissues. It will then review cross-disease studies to date that inform this endeavour, before considering future directions in the field.

Single-cell RNA sequencing (scRNA-seq) technique has enabled detailed analysis of gene expression at the single cell level, enhancing the understanding of subtle mechanisms that underly pathologies and drug resistance. To derive such biological meaning from sequencing data in oncology, some critical processing must be performed, including identification of the tumor cells by markers and algorithms that infer copy number variations (CNVs). We compared the performance of sciCNV, InferCNV, CopyKAT and SCEVAN tools that identify tumour cells by inferring CNVs from scRNA-seq data. Sequencing data from Pancreatic Ductal Adenocarcinoma (PDAC) patients, adjacent and healthy tissues were analysed, and the predicted tumor cells were compared to those identified by well-assessed PDAC markers. Results from InferCNV, CopyKAT and SCEVAN overlapped by less than 30% with InferCNV showing the highest sensitivity (0.72) and SCEVAN the highest specificity (0.75). We show that the predictions are highly dependent on the sample and the software used, and that they return so many false positives hence are of little use in verifying or filtering predictions made via tumor biomarkers. We highlight how critical this processing can be, warn against the blind use of these software and point out the great need for more reliable algorithms.

Circulating tumor cells (CTCs) are cells released from the primary tumor and intravasate into the blood or lymphatic vessels, where they are transported to distant sites and act as seeds that initiate cancer metastases. Recent advances in CTC research have shown their relevance as prognostic markers for early and metastatic disease detection, predictive biomarkers for relapse, and response to medical intervention or therapy. The rapidly evolving landscape of CTC biology has opened new avenues for understanding cancer progression, metastasis, and treatment response. Additionally, translating these findings into clinical applications holds promise for improving cancer diagnostics, prognosis, and personalized therapeutic strategies. This review discusses the significance of CTCs in cancer research and their associated challenges. We explore recent developments in the detection and characterization of CTCs and their implications in cancer research and clinical practice.

Tri-dimensional (3D) cell aggregates or spheroids are considered to be closer to physiological conditions than traditional 2D cell culture. Mesenchymal stem cells (MSCs) assembling in spheroids have increased the survival of transplanted cells. The regulation of the biological processes that maintain crucial physiological reactions of MSCs is closely related to the functioning of ion channels. The pattern of expression, role and regulatory mechanisms of ion channels could be significantly different in 3D compared to 2D culture, and, thus, needed to be properly analyzed on the level of ionic currents. We developed a specific approach that allowed us to register, for the first time, endogenous ion channels in endometrial MSCs (eMSCs) assembled in spheroids. Particularly, using the single-channel patch-clamp technique, we have recorded the activity of ion channels and observed their functional interplay in mechanosensitive clusters. Our experimental protocol could be applied for identification and studying of ion channels in 3D cell cultures.

Cancer immunotherapy reaches a remarkable achievement in various cancer types and brings new possibilities to improve cancer patients’ long-term survival. However, outcomes vary from case to case, and the present protocol benefits a small fraction of patients. One notable factor is the tumor microenvironment, especially the immune cell components, that may reflect the immune response's status quo on site. Thus, understanding the content of infiltrating immune cells in tumors is not only for research interesting but also a crucial subject toward precision medicine. We implement an algorithm for resolving relative proportions of twenty-one immune cell subclasses from a human tissue profiled transcriptome by microarray technology to reach the goal above. By selecting gene features and then adopting ?-Support Vector Regression, we can construct a deconvolution model and resolve the immune cell context. The excellent consistency between the estimated values and the correct immune-cell composition further demonstrates this approach provides a more natural alternative to revealing samples' immune cell content and reliable results like recent single-cell technologies. Based on this algorithm, the web-based deconvolution tool implemented named mySORT provides a user-friendly interface for estimating the immune cell content by uploading gene expression profiling. We also present comprehensive visualization 2D/3D plots in mySORT so that users can easily make a comparison between different samples. Finally, we synthesized pseudo-bulk expression data from single-cell transcriptomic datasets of 17 melanoma and 16 head and neck cancer patients. The deconvolution results of microarray-based data in the previous study and synthetic pseudo-bulk data all proved the excellent performance of mySORT. We believe that mySORT can help researchers in all fields easily understand complex immune microenvironment. The website of mySORT is freely accessible on https://symbiosis.iis.sinica.edu.tw/mySORT/.

Evidence indicates that the long noncoding RNAs are involved in the metformin-mediated anti-cancer processes. However, the potential effects of the long noncoding RNAs in metformin-mediated anti-tumor processes in esophageal squamous cell carcinomas (ESCC) are still elusive. This study uncovered that metformin decreases the level of long noncoding RNAs CCAT1 and SPRY4-IT1 thereby contributing to the down-regulation of c-Myc and vimentin. Also, the RNA level test of human ESCC tissue confirmed the positive correlation between CCAT1 and c-Myc. These findings demonstrated that metformin facilitated anti-cancer effects by targeting the 2 long noncoding RNAs (CCAT1 and SPRY4-IT1) and their consequential targets c-Myc and vimentin. Therefore, the CCAT1 and SPRY4-IT1 might act as novel molecular targets that mediate the anti-tumor effects in esophageal squamous cell carcinoma. This helps in predicting the treatment response of metformin in patients diagnosed with esophageal squamous cell carcinoma.

Single particle tracking (SPT) is a powerful class of methods for studying the dynamics of biomolecules inside living cells. The techniques reveal the trajectories of individual particles, with a resolution well below the diffraction limit of light, and from them the parameters defining the motion model, such as diffusion coefficients and confinement lengths. Most existing algorithms assume these parameters are constant throughout an experiment. However, it has been demonstrated that they often vary with time as the tracked particles move through different regions in the cell or as conditions inside the cell change in response to stimuli. In this work, we propose an estimation algorithm to determine time-varying parameters of systems that discretely switch between different linear models of motion with Gaussian noise statistics, covering dynamics such as diffusion, directed motion, and Ornstein-Uhlenbeck dynamics. Our algorithm consists of three stages. In the first stage, we use a sliding window approach, combined with Expectation Maximization (EM) to determine maximum likelihood estimates of the parameters as a function of time. These results are only used to roughly estimate the number of model switches that occur in the data to guide the selection of algorithm parameters in the second stage. In the second stage, we use change detection (CD) techniques to identify where the models switch, taking advantage of the off-line nature of the analysis of SPT data to create non-causal algorithms with better precision than a purely causal approach. Finally, we apply EM to each set of data between the change points to determine final parameter estimates. We demonstrate our approach using experimental data generated in the lab under controlled conditions.

Electrospinning is a simple versatile method to generate nanofibres. Remarkable progress has been made in the development of electrospinning process. The production of nanofibers is affected by many parameters, which influence the final material properties. Electrospun fibers have a wide range of applications such as energy storage and biomedical scaffolds. Polyacrylonitrile (PAN) has been widely used in the production of carbon nanofibers. PAN has received increasing interest in recent years, due to its excellent characteristics, such as spinnability, biocompatibility, and commercial viability, opening new applications in the biotechnological field. This paper provides an overview of the electrospinning process, its main parameters, and operation modes that affect nanofibers fabrication. A review of their biological applications is proposed. A focus is made on PAN fibers formation, functionalization, and application as scaffolds to allow cell growth. Overall, nanofibre scaffolds, made of stabilized PAN, appear as a powerful tool in medical applications needing controlled cell culture.

Intratumor heterogeneity (ITH) is associated with therapeutic resistance and poor prognosis in cancer patients, and attributed to genetic, epigenetic, and microenvironmental factors. We developed a new computational platform, GATHER, for geostatistical modeling of single cell RNA-seq data to synthesize high-resolution and continuous gene expression landscapes of a given tumor sample. Such landscapes allow GATHER to map the enriched regions of pathways of interest in the tumor space and identify genes that have spatial differential expressions at locations representing specific phenotypic contexts using measures based on optimal transport. GATHER provides new applications of spatial entropy measures for quantification and objective characterization of ITH. It includes new tools for insightful visualization of spatial transcriptomic phenomena. We illustrate the capabilities of GATHER using real data from breast cancer tumor to study hallmarks of cancer in the phenotypic contexts defined by cancer associated fibroblasts.

Tuberculosis (TB) remains a global healthcare crisis with an estimated 10 million new cases and 1.4 million deaths per year TB is caused by infection with the major human pathogen Mycobacte-rium tuberculosis, which is difficult to rapidly diagnose and treat. There is an urgent need for new methods of diagnosis, sufficient in vitro models which capably mimic all physiological condi-tions of the infection, and high-throughput drug screening platforms. Microfluidic-based tech-niques provide single-cell analysis which reduces experimental time, the cost of reagents, and have been extremely useful for gaining insight into monitoring microorganisms. This review out-lines the field of microfluidics and discusses the use of this novel technique so far in M. tuberculo-sis diagnostics, research methods, and drug discovery platforms. The practices of microfluidics have promising future applications for diagnosing and treating TB.

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.

The effects of hypothermia on neonatal encephalopathy may vary regionally, spatially, and cytopathologically in the gyrencephalic neocortex with manifestations potentially influenced by seizures that alter the severity and distribution of neuropathology. We developed a neonatal piglet survival model of hypoxic-ischemic (HI) encephalopathy and hypothermia with continuous encephalographic (cEEG) monitoring for seizures to study injury in neocortex. Neonatal piglets were randomized to naïve, HI-normothermia (NT), overnight hypothermia (HT) initiated 2 hours after HI, sham-NT, or sham-HT treatments. Some piglets within sham and HI groups received cEEG monitoring during recovery. Survival was 2-7 days (piglets were unmedicated and those with poor recovery and unresolving seizures were euthanized early); there was no differences in survival among groups (p&gt;0.078). Neuropathology was assessed by hematoxylin and eosin staining and immunohistochemistry for RNA Binding FOX-1 Homolog 3 (Rbfox3/NeuN). Normal and ischemic-necrotic neurons were counted (layers II-VI collectively) in somatosensory, motor, and prefrontal cortices, identified by cytoarchitecture and connectomics, and in inferior parietal cortex by layer. Seizure burden was determined. HI-NT piglets had reduced normal/total neuron ratio and increased ischemic-necrotic/total neuron ratio relative to naïve, sham-NT, and sham-HT piglets in anterior and posterior motor and somatosensory cortices. Frontal cortex was vulnerable in the prefrontal lateral bank after HI-NT with ischemic-necrosis. HI-HT piglets had higher normal/total neuron ratios and lower ischemic-necrotic/total neuron ratios than HI-NT piglets in anterior/posterior motor and somatosensory cortices. Total normal neuron density in layer III of inferior parietal cortex was reduced in HI-NT piglets compared to sham piglets and was protected by HT. Laminar analysis of Rbfox3 in somatosensory cortex revealed three types of neurons: Rbfox3-positive/normal, Rbfox3-positive/ischemic-necrotic, and Rbfox3-depleted. HI piglets had increased Rbfox3-depleted/total neuron ratio in layers II and III compared to sham-NT. Cortical neuron Rbfox3-depletion was partly rescued by HT. Seizure burden was more severe in HI piglets compared to sham piglets, but HI-NT and HI-HT piglets were similar. We conclude that 1) the neonatal piglet neocortex has a suprasylvian spatial vulnerability to HI and seizures; 2) HT protects against neuropathology in functionally different regions of the neonatal gyrencephalic neocortex; 3) neurons in neonatal neocortex have a limited cytopathology repertoire seen by H&amp;E staining; 4) higher seizure burden correlates with more ischemic-necrotic neurons in somatosensory cortex; 5) seizure presence associates with damage spread to inferior parietal cortex; 6) seizure presence is insensitive to HT, and 7) Rbfox3 immunophenotyping identifies a novel neuronal RNA splicing protein nuclear-depletion pathology that appears reversible by HT. This work demonstrates that HT protection of neocortex in neonatal HI is topographic and laminar, seizure unmitigating, and restores depleted neuronal RNA splicing factor.

Predictive biomarkers for immune checkpoint inhibitors (ICI), such as PD-L1 expression (TPS), remain limited in clinical applications. Improved novel ICI predictive biomarkers are urgently needed. We evaluated a live single-cell functional liquid biopsy cytokine profiling platform to track immunotherapy treatment response and outcomes. Peripheral blood mononuclear cell samples of healthy donors and NSCLC patients undergoing ICI-based therapies were collected longitudinally pre-/post-treatment under IRB-approved protocols. Samples were enriched for CD4+ and CD8+ T-lymphocytes and analyzed on the IsoLight platform. The single T-cells were captured in microchambers on IsoCode chips for cytokines profiling. Functional polyfunctionality data from 55,775 single cells were analyzed using the IsoSpeak software, kappa coefficient, and Kaplan-Meier survival plots. We found a significant difference between responders and non-responders in CD8+ T-cells’ changes in overall polyfunctionality and polyfunctional strength index (ΔPSI). ΔPSI score in CD8+ T-cells performed better than PD-L1 TPS and, when combined with PD-L1 TPS, strongly correlated with early ICI treatment response with a kappa coefficient of 1.0 (p=0.003). High CD4+ T-cells ΔPSI predicted a strong trend of improved PFS (3.9-fold) and OS (3-fold). In conclusion, single peripheral T-cell polyfunctionality dynamics analysis is a promising liquid biopsy profiling platform as NSCLC ICI predictive biomarker regardless of oncogene-addiction status.

Glioblastoma (GBM) is one of the most aggressive solid tumors, particularly due to the presence of cancer stem cells (CSCs). Today the characterization of this type of cells with an efficient, fast and low-cost method remains an issue. Hence, we have developed a microfluidic lab-on-a-chip based on dielectrophoresis (DEP) single cell electro-manipulation to measure the two crossover frequencies: f_x01 in low frequency range (below 500 kHz) and f_x02 in Ultra High Frequency range (UHF, above 50 MHz). First, in vitro conditions were investigated. U87-MG cell lines were cultured in different conditions in order to induce an undifferentiated phenotype. Then, ex vivo GBM cells from patients’ primary cell culture, were passed through the developed microfluidic system and characterized in order to reflect clinical conditions. This article demonstrates that the usual exploitation of low frequency range DEP does not allow the discrimination of the undifferentiated from the differentiated phenotypes of GBM cells. However, the presented study highlights the use of UHF-DEP as a relevant discriminant parameter. The proposed microfluidic lab-on-a-chip is able to follow the kinetic of U87-MG phenotype transformation in a CSC enrichment medium and their cancer stem cells phenotype acquirement.

C-C Chemokine receptor type 5 (CCR5) is expressed on the CD4 T cell surface where CXCR4 and CCR5 expressions are controlled differently during the activation of T cells and with the binding of interleukin type-2 (IL-2). IL-2 can upregulate CCR1 and CCR2 in CD45R01 T cells and increase the T lymphocyte chemotaxis toward CC-chemokines. CD4+ T cells are either apart of the T helper 1 or Th1 lymphocytes that release interferon gamma (IFNγ) and lymphotoxin that provide cellular immunity to internal pathogens and T helper cells type 2 (Th2), which secrete interleukins 4 and 5 (IL-4 and IL-5). IL-4 and IL-5 cause an allergic and humoral immune response to parasites. Th2 lymphocytes use CCR3 chemokine receptors. CCR5 and CXCR3 chemokine receptors are specific for CD4+ Th1 and Th2 lymphocytes. CCR5Δ32 is a 32-base-pair deletion of the CCR5 gene. CCR5 is a co-receptor for the entrance of the human immunodeficiency virus-1 (HIV-1). CCR5Δ32 creates a malfunction of the CCR5 protein that can prevent HIV-1 infection. However, the CCR5Δ32 32-base-pair deletion is not prevalent and predominant in many populations worldwide and there also exist more genetic variations of CCR5 known as CCR5-SNPs. An alternative polymorphism was identified based on the CCR5 gene that was identified as a A to G (A/G) point mutation. This point mutation is located at the 59029 locus on the promoter that lowers the expression rate of CCR5. Gurdol et al. found activity at the promoter of CCR5/590029G was 45% lesser than the CCR5/59029A. The genetic variations of CCR5 SNPs also include: 2459G>SNP of CCR5, C101X, CCR5 gene position at -2273, and the A to G SNP mutation found in two South African blacks. Therefore, more studies are needed to find more and varied chemokine polymorphisms that are present in many diverse populations in the world. The aim of this literature review is to describe the immense impact of CCR5 SNP mutations on viral infection susceptibility, on the pathogenesis of chronic conditions, to endorse the increased discovery of more novel CCR5 SNPs, and to show the significant potential of anti-CCR5 therapies to treat multiple diseased conditions.

The current spreading novel coronavirus SARS-CoV-2 is highly infectious and pathogenic and has attracted global attention. Recent studies have found that SARS-CoV-2 and SARS-CoV share around 80% of homology and use the same cell entry receptor, ACE2. These inspired us to study other receptors of SARS-CoV, which may be used for SARS-CoV-2 binding as well. In this study, we screened the gene expression of three receptors (ACE2, DC-SIGN and L-SIGN) in four datasets of normal lung tissue from lung adenocarcinoma patients and two single-cell RNA sequencing datasets from normal lung and bronchial epithelial cells separately. No significant difference in gene expression of these three receptors were found between gender groups (male vs female). We found higher gene expression of DC-SIGN in elder with age>60 and higher gene expression of L-SIGN in Caucasian than Asian. Similar to ACE2, we observed significantly higher DC-SIGN gene expression in the lungs of smokers, especially former smokers. However, smokers upregulate ACE2 and DC-SIGN gene expression in different cell types. In the whole lung, ACE2 is actively expressed in remodeled Alveolar Type II cells of former smokers, while DC-SIGN is largely expressed in monocytes of former smokers and dendritic cells of current smokers. In bronchial epithelium, no obvious gene expression of DC-SIGN and L-SIGN was observed while ACE2 was found to be actively expressed in goblet cells of current smokers and club cells of non-smokers. In conclusion, our findings may indicate that smokers, especially former smokers, and people over 60 have higher risk and are more susceptible to SARS-CoV-2 infection. Also, this study provides hints on possible SARS-CoV-2 pathogenicity mechanisms in lung infection.

The main property of tripodal ligands is the predictable type of coordination. Homo- and heteroleptic lanthanide complexes with tripodal ligands are a representative class of compounds. However, despite the fact that many of them are paramagnetic their magnetic behavior is poorly understood. This is because their photophysical and catalytic properties have been more attractive. In the present review, we are trying to summarize the available structural information and extremely few data on magnetic properties in order to draw some conclusions about the prospective of tripods using in the design of quantum molecular magnets based on Ln ions. We would like to draw the reader's attention to the fact that despite the consideration of a large part of the currently known lanthanide compounds with tripodal ligands, this review is not exhaustive. However, our goal was to draw the attention of researchers to the fact that a whole niche of air-stable Ln complexes remained outside the attention of magnetochemists and theoreticians.

We consider the problem of scheduling tasks on a single machine subject to an uncertain breakdown to minimize the flowtime. Assume the machine is unavailable during the breakdown; the starting time of the breakdown is a random variable s with distribution function D(s) and the terminating time of the breakdown has no any other information; jobs are non-resumable. Under the assumptions, starting from the perspective of statistical optimization, we first define the expected competitive ratio of an algorithm to find the optimized solution with the considered problem. Further, we propose and prove a certain result on the expected competitive ratio of the SPT rule. In particular, we show that under a quite loose condition the expected competitive ratio of the SPT rule is no more than 5/4. Meanwhile, we also make some discussions about our studies. What we have done will rich and improve the studying results on the area of scheduling to minimize flowtime and advance the development of online optimization and statistical optimization.

The investigation of the electromagnetic properties of biological particles in microfluidic platforms may enable wireless monitoring and interaction with functional activity of microorganisms. Of high relevance is the membrane potential as it is one of the most important parameters of living cells. In particular, the complex mechanisms of the cell’s membrane potential are comparable to the dynamics of bacteria membranes, providing a simplified platform for advancing the current techniques and knowledge of general bio-particle dynamics. In this work, we provide a theoretical analysis and experimental results on the microwave detection of bacteria on a microfluidic-based framework for sensing the membrane potential of bacteria. The results enable to further advance the state-of-the-art of electromagnetic bacteria sensing and microfluidic control, and their implication for measuring and interacting with the cell and its membrane potentials, which is of great importance for developing new biotechnological engineered systems and solutions.

Does cell clustering influence intrinsic and acquired multi-drug resistance (MDR) differently? To address this question, we studied cultured monolayers (representing individual cells) and cultured spheroids (representing clusters) formed by drug-naïve (intrinsic MDR) and drug-exposed (acquired MDR) lines of ovarian cancer A2780 cells by cytometry of reaction rate constant (CRRC). MDR efflux was characterized by accurate and robust “cell number vs. MDR efflux rate constant (kMDR)” histograms. Both drug-naïve and drug-exposed monolayer cells presented unimodal histograms; the histogram of drug-exposed cells was shifted towards higher kMDR value suggesting greater MDR activity. Spheroids of drug-naïve cells presented a bimodal histogram indicating the presence of two subpopulations with different MDR activity. In contrast, spheroids of drug-exposed cells presented a unimodal histogram qualitatively similar to that of the monolayers of drug-exposed cells but with a moderate shift towards greater MDR activity. The observed greater effect of cell clustering on intrinsic than on acquired MDR can help guide the development of new therapeutic strategies targeting clusters of circulating tumor cells.

Although early work on parental burnout in the 1980s did not link parental burnout to specific parenting situations, the idea that parents affected by specific vulnerability factors were particularly at risk of burnout quickly emerged. Based on the etiological model of parental burnout (i.e., the balance between risks and resources), the objective of this study was to determine if there were specific vulnerability factors that significantly increase the risk of parental burnout. 2,563 parents participated in the study. We compared parents with a child with special needs (n = 25), parents who had adopted a child (n = 130), and single parents (n = 109), to strictly matched control groups of parents. Parents with a child with special needs displayed higher burnout and lower balance between risk and resources than the control group; parents with an adopted child had similar levels of both parental burnout and balance; and single parents had higher parental burnout but similar balance. Parents who combined specific several vulnerability factors were at greater risk of burnout. Single parenthood and adoption do not in themselves trigger a process leading de facto to other risk factors, but having a child with special needs does.