Analysis Of Chromatin Structure Research Articles

An identification method of chromatin topological associated domains based on spatial density clustering

The rapid development of high-throughput chromatin conformation capture (Hi-C) technology provides rich genomic interaction data between chromosomal loci for chromatin structure analysis. However, existing methods for identifying topologically associated domains (TADs) based on Hi-C data suffer from low accuracy and sensitivity to parameters. In this context, a TAD identification method based on spatial density clustering was designed and implemented in this paper. The method preprocessed the raw Hi-C data to obtain normalized Hi-C contact matrix data. Then, it computed the distance matrix between loci, generated a reachability graph based on the core distance and reachability distance of loci, and extracted clustering clusters. Finally, it extracted TAD boundaries based on clustering results. This method could identify TAD structures with higher coherence, and TAD boundaries were enriched with more ChIP-seq factors. Experimental results demonstrate that our method has advantages such as higher accuracy and practical significance in TAD identification.

Deep learning with a small dataset predicts chromatin remodelling contribution to winter dormancy of apple axillary buds.

Epigenetic changes serve as a cellular memory for cumulative cold recognition in both herbaceous and tree species, including bud dormancy. However, most studies have discussed predicted chromatin structure with respect to histone marks. In the present study, we investigated the structural dynamics of bona fide chromatin to determine how plants recognise prolonged chilling during the initial stage of bud dormancy. The vegetative axillary buds of the 'Fuji' apple, which shows typical low temperature-dependent, but not photoperiod, dormancy induction, were used for the chromatin structure and transcriptional change analyses. The results were integrated using a deep-learning model and interpreted using statistical models, including Bayesian estimation. Although our model was constructed using a small dataset of two time points, chromatin remodelling due to random changes was excluded. The involvement of most nucleosome structural changes in transcriptional changes and the pivotal contribution of cold-driven circadian rhythm-dependent pathways regulated by the mobility of cis-regulatory elements were predicted. These findings may help to develop potential genetic targets for breeding species with less bud dormancy to overcome the effects of short winters during global warming. Our artificial intelligence concept can improve epigenetic analysis using a small dataset, especially in non-model plants with immature genome databases.

Individual transcription factors modulate both the micromovement of chromatin and its long-range structure

The control of eukaryotic gene expression is intimately connected to highly dynamic chromatin structures. Gene regulation relies on activator and repressor transcription factors (TFs) that induce local chromatin opening and closing. However, it is unclear how nucleus-wide chromatin organization responds dynamically to the activity of specific TFs. Here, we examined how two TFs with opposite effects on local chromatin accessibility modulate chromatin dynamics nucleus-wide. We combine high-resolution diffusion mapping and dense flow reconstruction and correlation in living cells to obtain an imaging-based, nanometer-scale analysis of local diffusion processes and long-range coordinated movements of both chromatin and TFs. We show that the expression of either an individual transcriptional activator (CDX2) or repressor (SIX6) with large numbers of binding sites increases chromatin mobility nucleus-wide, yet they induce opposite coherent chromatin motions at the micron scale. Hi-C analysis of higher-order chromatin structures shows that induction of the pioneer factor CDX2 leads both to changes in local chromatin interactions and the distribution of A and B compartments, thus relating the micromovement of chromatin with changes in compartmental structures. Given that inhibition of transcription initiation and elongation by RNA Pol II has a partial impact on the global chromatin dynamics induced by CDX2, we suggest that CDX2 overexpression alters chromatin structure dynamics both dependently and independently of transcription. Our biophysical analysis shows that sequence-specific TFs can influence chromatin structure on multiple architectural levels, arguing that local chromatin changes brought by TFs alter long-range chromatin mobility and its organization.

A systematic analyses of different bioinformatics pipelines for genomic data and its impact on deep learning models for chromatin loop prediction.

Genomic data analysis has witnessed a surge in complexity and volume, primarily driven by the advent of high-throughput technologies. In particular, studying chromatin loops and structures has become pivotal in understanding gene regulation and genome organization. This systematic investigation explores the realm of specialized bioinformatics pipelines designed specifically for the analysis of chromatin loops and structures. Our investigation incorporates two protein (CTCF and Cohesin) factor-specific loop interaction datasets from six distinct pipelines, amassing a comprehensive collection of 36 diverse datasets. Through a meticulous review of existing literature, we offer a holistic perspective on the methodologies, tools and algorithms underpinning the analysis of this multifaceted genomic feature. We illuminate the vast array of approaches deployed, encompassing pivotal aspects such as data preparation pipeline, preprocessing, statistical features and modelling techniques. Beyond this, we rigorously assess the strengths and limitations inherent in these bioinformatics pipelines, shedding light on the interplay between data quality and the performance of deep learning models, ultimately advancing our comprehension of genomic intricacies.

Current Advances in Single-Cell RNA Sequencing in Diabetic Retinopathy

With the development of high-throughput sequencing technology, humans have been able to conduct large-scale analysis of DNA sequence, chromatin structure, RNA transcripts, proteins, metabolites and other genomes and their products. Traditional high-throughput transcriptome sequencing techniques based on tissue samples (RNA Seq) are used to centrally sequence thousands of cells, each of which varies in size, protein levels, and mRNA expression transcription. Measuring the average of multiple cells grouped together can mask significant differences in gene expression between cells. Single-cell RNA sequencing is a technique for high-throughput sequencing of the genome, transcriptome, and epigenome at the single-cell level. Based on the single cell RNA transcription map, the intraocular cells can be distinguished from other subtypes, and the different subtypes are found to have significant differences in morphology, physiology and specific expression genes. In recent years, the application of single-cell RNA sequencing technology in the field of ophthalmology has increased, mainly including cell type and cell subtype identification, retinal development process, and eye disease research. This paper systematically summarized the latest application of single-cell sequencing technology in the field of diabetic retinopathy, and summarized marker genes and potential therapeutic targets. It has guiding significance for the clinical treatment of diabetic retinopathy.

Multiple strategies, including 6mA methylation, affecting plant alternative splicing in allopolyploid peanut.

Alternative splicing (AS), an important post-transcriptional regulation mechanism in eukaryotes, can significantly increase transcript diversity and contribute to gene expression regulation and many other complicated developmental processes. While plant gene AS events are well described, few studies have investigated the comprehensive regulation machinery of plant AS. Here, we use multi-omics to analyse peanut AS events. Using long-read isoform sequencing, 146 464 full-length non-chimeric transcripts were obtained, resulting in annotation corrections for 1782 genes and the identification of 4653 new loci. Using Iso-Seq RNA sequences, 271 776 unique splice junctions were identified, 82.49% of which were supported by transcriptome data. We characterized 50 977 polyadenylation sites for 23 262 genes, 12 369 of which had alternative polyadenylation sites. AS allows differential regulation of the same gene by miRNAs at the isoform level coupled with polyadenylation. In addition, we identified many long non-coding RNAs and fusion transcripts. There is a suppressed effect of 6mA on AS and gene expression. By analysis of chromatin structures, the genes located in the boundaries of topologically associated domains, proximal chromosomal telomere regions, inter- or intra-chromosomal loops were found to have more unique splice isoforms, higher expression, lower 6mA and more transposable elements (TEs) in their gene bodies than the other genes, indicating that chromatin interaction, 6mA and TEs play important roles in AS and gene expression. These results greatly refine the peanut genome annotation and contribute to the study of gene expression and regulation in peanuts. This work also showed AS is associated with multiple strategies for gene regulation.

Comparative interactome analysis of α-arrestin families in human and Drosophila

The α-arrestins form a large family of evolutionally conserved modulators that control diverse signaling pathways, including both G-protein-coupled receptor (GPCR)-mediated and non-GPCR-mediated pathways, across eukaryotes. However, unlike β-arrestins, only a few α-arrestin targets and functions have been characterized. Here, using affinity purification and mass spectrometry, we constructed interactomes for 6 human and 12 Drosophila α-arrestins. The resulting high-confidence interactomes comprised 307 and 467 prey proteins in human and Drosophila, respectively. A comparative analysis of these interactomes predicted not only conserved binding partners, such as motor proteins, proteases, ubiquitin ligases, RNA splicing factors, and GTPase-activating proteins, but also those specific to mammals, such as histone modifiers and the subunits of V-type ATPase. Given the manifestation of the interaction between the human α-arrestin, TXNIP, and the histone-modifying enzymes, including HDAC2, we undertook a global analysis of transcription signals and chromatin structures that were affected by TXNIP knockdown. We found that TXNIP activated targets by blocking HDAC2 recruitment to targets, a result that was validated by chromatin immunoprecipitation assays. Additionally, the interactome for an uncharacterized human α-arrestin ARRDC5 uncovered multiple components in the V-type ATPase, which plays a key role in bone resorption by osteoclasts. Our study presents conserved and species-specific protein–protein interaction maps for α-arrestins, which provide a valuable resource for interrogating their cellular functions for both basic and clinical research.

Comparative interactome analysis of α-arrestin families in human and Drosophila.

The α-arrestins form a large family of evolutionally conserved modulators that control diverse signaling pathways, including both G-protein-coupled receptor (GPCR)-mediated and non-GPCR-mediated pathways, across eukaryotes. However, unlike β-arrestins, only a few α-arrestin targets and functions have been characterized. Here, using affinity purification and mass spectrometry, we constructed interactomes for 6 human and 12 Drosophila α-arrestins. The resulting high-confidence interactomes comprised 307 and 467 prey proteins in human and Drosophila, respectively. A comparative analysis of these interactomes predicted not only conserved binding partners, such as motor proteins, proteases, ubiquitin ligases, RNA splicing factors, and GTPase-activating proteins, but also those specific to mammals, such as histone modifiers and the subunits of V-type ATPase. Given the manifestation of the interaction between the human α-arrestin, TXNIP, and the histone-modifying enzymes, including HDAC2, we undertook a global analysis of transcription signals and chromatin structures that were affected by TXNIP knockdown. We found that TXNIP activated targets by blocking HDAC2 recruitment to targets, a result that was validated by chromatin immunoprecipitation assays. Additionally, the interactome for an uncharacterized human α-arrestin ARRDC5 uncovered multiple components in the V-type ATPase, which plays a key role in bone resorption by osteoclasts. Our study presents conserved and species-specific protein-protein interaction maps for α-arrestins, which provide a valuable resource for interrogating their cellular functions for both basic and clinical research.

Fluorescent enzyme-based biosensor for sensitive analysis of DNA damage in cryopreserved sperm

The freeze-thaw process can induce irreversible structural and functional changes in human sperm, particularly sperm DNA damage. Selecting a more accurate and sensitive detection method for evaluating sperm DNA integrity is crucial. To accurately assess sperm DNA integrity following the freeze-thaw process and significantly improve the clinical and scientific utilization of cryopreserved sperm. In this study, we utilized a novel fluorescent biosensor, assisted by terminal deoxynucleotidyl transferase (TdT) and Endonuclease IV, to detect DNA breakpoints during sperm cryopreservation. We evaluated the biosensor's performance by comparing it with the conventional DNA fragmentation index (DFI) measured using sperm chromatin structure analysis (SCSA). The cryopreserved group exhibited a significantly higher sperm DFI compared to the fresh group. No significant difference was observed between the antioxidant group and the cryopreserved group. However, the new method revealed a significant reduction in the number of DNA breakpoints in the antioxidant group compared to the cryopreserved group. The novel biosensor demonstrated superior accuracy and effectiveness in assessing sperm DNA integrity during cryopreservation compared to the conventional SCSA method. We believe that the biosensor holds significant potential for widespread use in the field of reproductive medicine.

Evaluation of semen DNA integrity and related parameters with COVID-19 infection: a prospective cohort study

BackgroundIn the context of Corona Virus Disease 2019 (COVID-19) global pandemic, Its impact on male reproductive function should be concerned.MethodsOur study is a prospective cohort study that recruited participants infected or uninfected with COVID-19 between December 2022 and March 2023. All laboratory tests and questionnaire data were completed at the First Affiliated Hospital of Nanchang University. A total of 132 participants were enrolled, with 78 COVID-19 positive patients as the positive group and 54 COVID-19 negative participants as the negative group. Semen quality was assessed by the fifth World Health Organization criteria. The general characteristics of semen samples were assessed using CASA (computer-assisted sperm analysis). DNA damage and the high density stainability was assessed by sperm chromatin structure analysis (SCSA) based on flowcytometry.ResultsThe sperm concentration, progressive motility and motility in COVID-19 negative group were significantly higher than positive group. In the following DNA damage analysis, a remarkably lower sperm DNA fragmentation index (DFI) in the COVID-19 negative group. In the positive group, unhealthy lifestyles had no significant effect on semen parameters, DNA fragmentation and nuclear compaction.ConclusionsAfter excluding the interference of unhealthy lifestyle, the COVID-19 infection can have a significant impact on the quality of semen, especially the DFI,. Therefore, it shows that COVID-19 can adversely affects male fertility, and this result provides advisory guidance for clinicians.

Targeted insertion and reporter transgene activity at a gene safe harbor of the human blood fluke, Schistosoma mansoni

The identification and characterization of genomic safe harbor sites (GSHs) can facilitate consistent transgene activity with minimal disruption to the host cell genome. We combined computational genome annotation and chromatin structure analysis to predict the location of four GSHs in the human blood fluke, Schistosoma mansoni, a major infectious pathogen of the tropics. A transgene was introduced via CRISPR-Cas-assisted homology-directed repair into one of the GSHs in the egg of the parasite. Gene editing efficiencies of 24% and transgene-encoded fluorescence of 75% of gene-edited schistosome eggs were observed. The approach advances functional genomics for schistosomes by providing a tractable path for generating transgenics using homology-directed, repair-catalyzed transgene insertion. We also suggest that this work will serve as a roadmap for the development of similar approaches in helminths more broadly.

Association of embryo aneuploidy and sperm DNA damage in unexplained recurrent implantation failure patients under NGS-based PGT-A cycles.

Recurrent implantation failure (RIF) is one of the most common conditions affecting In Vitro Fertilization (IVF)/Intracytoplasmic sperm injection (ICSI) outcomes. Aneuploidy embryos, one of the main types of embryos-related factors, was reported to be a major contributor to RIF. The present study aimed to examine the association between sperm DNA fragmentation index (DFI) and outcomes of next-generation sequencing (NGS)-based preimplantation genetic testing for aneuploidy (PGT-A) in unexplained RIF patients. This study analyzed 119 couples with unexplained RIF who underwent 119 PGT-A cycles between January, 2017 and March, 2022. The 119 males were divided into 3 groups according to their sperm DFI levels: Group1 (low, DFI ≤ 15%, n = 50), Group2 (medium, 15% < DFI < 30%, n = 41) and Group3 (high, DFI ≥ 30%, n = 28). Sperm DFI was measured by sperm chromatin structure analysis (SCSA) technique. Trophectoderm biopsy on day 5 or 6 were performed with NGS technique. The following outcomes of PGT-A were analyzed and compared: fertilization, good-quality embryos, aneuploidy rate, miscarriage, live birth and newborn defects. The component of aneuploidy embryos was significantly higher in high DFI group (42.71%) than that of medium group (28.39%) and low group (27.80%). The miscarriage rate of high DFI group (27.27%) and medium group (14.29%) is significantly higher than that of low group (0.00%). No significant differences were found regarding fertility, good-quality embryo rate, pregnancy rate, live birth rate or newborn defects among three groups. The sperm DNA damage is associated with blastocyst aneuploidy and miscarriage rate in unexplained RIF cases. Embryo selection by PGT-A and efforts to decrease sperm DFI before IVF/ICSI treatments should be considered for those male patients with high DFI.

The 3D chromatin landscape of rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a pediatric soft tissue cancer with a lack of precision therapy options for patients. We hypothesized that with a general paucity of known mutations in RMS, chromatin structural driving mechanisms are essential for tumor proliferation. Thus, we carried out high-depth in situ Hi-C in representative cell lines and patient-derived xenografts (PDXs) to define chromatin architecture in each major RMS subtype. We report a comprehensive 3D chromatin structural analysis and characterization of fusion-positive (FP-RMS) and fusion-negative RMS (FN-RMS). We have generated spike-in in situ Hi-C chromatin interaction maps for the most common FP-RMS and FN-RMS cell lines and compared our data with PDXmodels. In our studies, we uncover common and distinct structural elements in large Mb-scale chromatin compartments, tumor-essential genes within variable topologically associating domains and unique patterns of structural variation. Our high-depth chromatin interactivity maps and comprehensive analyses provide context for gene regulatory events and reveal functional chromatin domains in RMS.

Effects of alcohol use on sperm chromatin structure, a retrospective analysis

BackgroundThe evaluation of the infertile couple is often complex as multiple factors in both the male and female can contribute, including social history. Previous studies have displayed that male ethanol consumption can disturb sperm motility, nuclear maturity, and deoxyribonucleic acid (DNA) integrity. The main purpose of this study is to evaluate the effects of male alcohol use on sperm chromatin structure analysis (SCSA®).This study was a retrospective chart review of 209 couples that presented to a midsize infertility clinic in the Midwest and had a semen analysis and SCSA® performed. Data extracted from the electronic medical record included demographics, tobacco use, alcohol use, occupational exposures, semen analysis results, and SCSA® results (DNA Fragmentation index (DFI) and High DNA stainability (HDS)). Statistical analysis was performed on this data set to determine significance with a p-level of 0.05, with the primary input being level of alcohol use and primary outcome being the SCSA® parameters.ResultsOverall, 11% of the cohort had heavy alcohol use (> 10 drinks/week), 27% moderate (3–10/week), 34% rare (0.5- < 3/week), and 28% none. 36% of the cohort had HDS > 10% (a marker of immature sperm chromatin). Level of alcohol use was not significantly associated with HDS > 10% or DFI. Heavier alcohol use was significantly associated with lower sperm count (p = 0.042). Increasing age was significantly associated with increasing DNA Fragmentation Index (p = 0.006), increased sperm count (p = 0.002), and lower semen volume (p = 0.022). Exposure to heat at work was significantly associated with lower semen volume (p = 0.042). Tobacco use was associated with lower sperm motility (p < 0.0001) and lower sperm count (p = 0.002).ConclusionsThere was not a significant association between the level of alcohol use and the High DNA Stainability or DNA Fragmentation Index of sperm. Increasing age was associated with semen parameters as expected, heat exposure was associated with lower semen volume, and tobacco use was associated with lower sperm motility and density. Further studies could investigate alcohol use and reactive oxidative species in sperm.

Regulation of matrix metalloproteinase-9 during monopoiesis and zinc deficiency by chromatin remodeling

IntroductionMatrix metalloproteinase-9 (MMP-9) cleaves various extracellular matrix proteins, hence significantly contributes to numerous physiological but also pathological processes. Monocytic differentiation is associated with increased MMP-9 gene expression. Interestingly, MMP-9 upregulation during monocytic differentiation is paralleled by a decline in intracellular zinc levels. Hence, an influence of zinc on the regulation of MMP-9 expression may exist. Although, previous studies suggest a vital role of zinc regarding MMP-9 activity, the possible relevance of zinc homeostasis during transcriptional regulation of MMP-9 for example via epigenetic mechanisms is rather unclear. AimThis study aims to find a correlation between zinc deficiency and MMP-9 transcriptional regulation, focusing on epigenetics as the possible mechanism behind zinc deficiency-induced changes. MethodsThe effect of differentiation and zinc deficiency on MMP-9 expression and MMP9 promoter accessibility was investigated using the acute promyelocytic cell line NB4. Intracellular free zinc levels were detected by flow cytometry. MMP-9 gene expression was measured by real-time PCR and ELISA. Analysis of chromatin structures was done using chromatin accessibility by real-time PCR (CHART) assay. ResultsDuring monocytic differentiation of NB4 cells, the decrease in intracellular zinc levels was paralleled by an increased production of MMP-9. Assessment of chromatin structure revealed increased accessibility of certain regions within the MMP-9 promoter in differentiated cells. Interestingly, upregulated activation-induced MMP-9 gene expression as well as a more accessible MMP-9 promoter were in zinc-deficient NB4 cells whereas zinc resupplementation reversed the effects. ConclusionThese data demonstrate an important role of epigenetic mechanisms in regulating MMP-9 expression under zinc deficiency. This could provide an encouraging step to expand the research on using zinc for the treatment of various pathological conditions such as inflammatory, vascular and autoimmune diseases resulting from MMP-9 deregulation.

Single layer centrifugation (SLC) for bacterial removal with Porcicoll positively modifies chromatin structure in boar spermatozoa

The storage of boar semen samples at 17 °C for artificial insemination (AI) doses enables the proliferation of the bacteria, making antibiotics necessary. This can contribute to the development of antimicrobial resistance (AMR). This study tested bacterial presence and sperm chromatin structure after using a low-density colloid (Porcicoll) as an antibiotic alternative to eliminate bacteria. Ejaculates (8 boars, 3 ejaculates each) were split as control and low-density colloid centrifugation (single layer centrifugation, SLC, 20%, and 30% Porcicoll) into 500 ml tubes. Analyses were carried out at days 0, 3, and 7 (17 °C) for microbial presence and sperm chromatin structure analysis: %DFI (DNA fragmentation) and %HDS (chromatin immaturity), monobromobimane (mBBr; free thiols and disulfide bridges), and chromomycin A3 (CMA3; chromatin compaction). Besides comparing bacterial presence (7 species identified) and chromatin variables between treatments, the associations between these sets of variables were described by canonical correlation analysis (CCA). Results showed a significant decrease of some bacteria or a complete removal after SLC (especially for P30). SLC also caused a decrease of %HDS and an increase of disulfide bridges and low and medium mBBr populations, suggesting the removal of immature sperm (poor chromatin compaction). CCA showed an association pattern compatible with the degradation of sperm chromatin parameters with bacterial contamination, especially Enterobacteria, P. aeuriginosa, and K. variicola. In conclusion, bacterial contamination affects sperm chromatin beyond DNA fragmentation; SLC with low-density colloid not only removes bacteria from boar semen, but also chromatin structure is enhanced after selection.

TGFβ1-Induced EMT in the MCF10A Mammary Epithelial Cell Line Model Is Executed Independently of SNAIL1 and ZEB1 but Relies on JUNB-Coordinated Transcriptional Regulation.

Epithelial-mesenchymal transition (EMT) fosters cancer cell invasion and metastasis, the main cause of cancer-related mortality. Growing evidence that SNAIL and ZEB transcription factors, typically portrayed as master regulators of EMT, may be dispensable for this process, led us to re-investigate its mechanistic underpinnings. For this, we used an unbiased computational approach that integrated time-resolved analyses of chromatin structure and differential gene expression, to predict transcriptional regulators of TGFβ1-inducible EMT in the MCF10A mammary epithelial cell line model. Bioinformatic analyses indicated comparatively minor contributions of SNAIL proteins and ZEB1 to TGFβ1-induced EMT, whereas the AP-1 subunit JUNB was anticipated to have a much larger impact. CRISPR/Cas9-mediated loss-of-function studies confirmed that TGFβ1-induced EMT proceeded independently of SNAIL proteins and ZEB1. In contrast, JUNB was necessary and sufficient for EMT in MCF10A cells, but not in A549 lung cancer cells, indicating cell-type-specificity of JUNB EMT-regulatory capacity. Nonetheless, the JUNB-dependence of EMT-associated transcriptional reprogramming in MCF10A cells allowed to define a gene expression signature which was regulated by TGFβ1 in diverse cellular backgrounds, showed positively correlated expression with TGFβ signaling in multiple cancer transcriptomes, and was predictive of patient survival in several cancer types. Altogether, our findings provide novel mechanistic insights into the context-dependent control of TGFβ1-driven EMT and thereby may lead to improved diagnostic and therapeutic options.

Overcoming CAR-T Cell Exhaustion and Improving Antitumoral Efficacy in Hematologic Neoplasms Utilizing CRISPR/Cas9 Technology

Introduction Chimeric antigen receptor (CAR)-T cell therapy is considered a therapeutic breakthrough in cancer immunotherapy especially targeting subsets of B cell leukemia and lymphomas. The barriers still limiting the clinical response to CAR-T cell therapy range from antigen escape, impaired tumor infiltration, severe toxicities, insufficient anti-tumoral effects, to CAR-T cell exhaustion. Varying approaches are underway to translate CAR-T cell therapy into the treatment of solid tumors and further hematological malignancies. Methods Based on literature review and single-cell analysis of chromatin structure, we identified potential genetic drivers of CAR-T cell exhaustion and insufficient anti-tumoral effects. To investigate the underlying cellular and genomic mechanisms, we established polyclonal gene knockouts utilizing CRISPR/Cas9 ribonucleoprotein (RNP) based genome editing. Knockout efficacy was validated on a genomic level using interference of CRISPR editing (ICE)-sequencing. To determine the killing capacity of modified anti-CD19-CD28ζ CAR-T cells of luciferase transgenic CD19+ Nalm6 ALL blasts, in vitro bioluminescence-based cytotoxicity assays were performed. Repetitive killing capacity was evaluated in vitro by co-culturing modified CAR-T cells with Nalm6 cells at a 1:1 E:T ratio over 48 hours followed by another co-culture with tumor cells at a 1:1 E:T ratio over 24 hours. Continuous stimulation of the T cell receptor (TCR) was assessed by stimulating 0.2x105 modified CAR-T cells with 2 µL anti-CD3/anti-CD28 antibodies over 48 hours. Expression of extracellular and transcriptional (exhaustion) markers were assessed by flow cytometry. To elucidate the underlying functional mechanisms, we performed single-cell RNA sequencing. Results Mean knockout efficacy across our candidate genes was >90%. Cell viability after expansion over 7 and 14 days was comparable between knockout CAR-T cells and unmodified control CAR-T cells (n≥4). The proliferative capacity of CAR-T knockout cells was comparable to control CAR-T cells (n≥4). Knockout of candidate 5 gene showed significantly enhanced killing capacity in cytotoxicity assays (n≥4; p= 0.0003) (Figure 1), as well as significant downregulation of the exhaustion markers programmed cell death protein-1 (PD-1) (n≥4; p= 0.037) and lymphocyte-activation gene-3 (LAG-3) (n≥4; p= 0.028) in flow cytometry after cytotoxicity assays. Levels of cytotoxic T cell mediators Granzyme B (n≥4; p= 0.489) and Interferon-gamma (IFNy) (n≥4; p= 0.361) were not reduced compared to control group. Except for PD-1 expression (p= 0.147), these findings were reproduceable when testing the repetitive killing capacity of candidate 5 knockout CAR-T cells (n= 3). Following continuous TCR stimulation PD-1 (n≥4; p= 0.013) and LAG-3 (n≥4; p= 0.031) were significantly downregulated compared to control CAR-T cells. Levels Granzyme B (n≥4; p= 0.637) and IFNy (n≥4; p= 0.501) were found to remain stable. Conclusions In summary, we present a novel promising target gene to enhance CAR-T cell efficacy with high potential for clinical translation, which is currently analyzed in vivo. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Applications of Single-Cell Sequencing Technology to the Enteric Nervous System.

With recent technical advances and diminishing sequencing costs, single-cell sequencing modalities have become commonplace. These tools permit analysis of RNA expression, DNA sequence, chromatin structure, and cell surface antigens at single-cell resolution. Simultaneous measurement of numerous parameters can resolve populations including rare cells, thus revealing cellular diversity within organs and permitting lineage reconstruction in developing tissues. Application of these methods to the enteric nervous system has yielded a wealth of data and biological insights. We review recent papers applying single-cell sequencing tools to the nascent neural crest and to the developing and mature enteric nervous system. These studies have shown significant diversity of enteric neurons and glia, suggested paradigms for neuronal specification, and revealed signaling pathways active during development. As technology evolves and multiome techniques combining two or more of transcriptomic, genomic, epigenetic, and proteomic data become prominent, we anticipate these modalities will become commonplace in ENS research and may find a role in diagnostic testing and personalized therapeutics.

Technical Evaluation of Commercial Sperm DFI Quality Control Products in SCSA Testing.

With the increase in population and the advancement of medicine, people even more hope that their offspring will be healthier. DNA fragmentation rate is currently one of the more common indicators for evaluating sperm fertility and predicting the outcome of pregnancy. In order to evaluate the technical performance and application value of commercial sperm DFI (DNA fragmentation index) quality control products in the flow cytometry sperm chromatin structure analysis (SCSA), this paper uses flow cytometry to test commercial sperm DFI quality control products (Celula) and laboratory routine self-made DFI quality control products. In this paper, the quality control of new commercial sperm DFI and laboratory-made quality control were compared and tested for 30 consecutive days. And this paper monitors the response of commercial quality control products to the interference of key reagent parameters. This paper compares the stability of the test results of two quality control products and their sensitivity to the interference of key performance parameters of the detection reagent. Experimental results show that commercial sperm DFI quality control products can simulate sperm DNA damage to achieve accurate detection of DNA integrity. The stability of commercial sperm DFI quality control (CV = 2.47%) is better than that of laboratory-made quality control (CV = 11.22%). The new commercial sperm DFI quality control product can sensitively detect changes in the concentration of acidified solution and staining solution at the same time. It can effectively control the quality of detection reagents and experimental procedures. The new commercial sperm DFI quality control product can effectively control sperm DNA integrity testing. It can be used as an external quality control product for quality control of test results to ensure that more accurate test results are provided to the clinic.