Single Cell Gel Electrophoresis Assay of Porcine Leydig Cell DNA Damage Induced by Zearalenone

[Objective] This study aimed to investigate the effect of zearalenone (ZEN) on DNA damage of porcine leydig cells. [Method] Porcine leydig cells cultured in vitro were collected to determine the median lethal dose (LD50) of ZEN with tetrazolium-based colorimetric assay (MTT assay). Comet assay was carried out to detect the DNA damage of porcine leydig cells exposed to at 0 (negative group), 1, 5, 10, 20, 40 μmol/L of ZEN. [Result] The percentage of cell tail was 16.67%, 34.00%, 40.67%, 52.00% and 64.67% under 0, 1, 5, 10 and 20 μmol/L of ZEN, respectively; the differences between the percentages of cell tail in various experimental groups had extremely significant statistical significance compared with the negative group (P<0.01), showing a significant dose-effect relationship; Tail length in various groups was 57.60±4.78, 57.75±6.25, 78.97±5.83, 100.50±6.94 and 146.83±12.31 μm, respectively; Tail DNA % in various groups was 21.29±2.25%, 22.24±2.43%, 31.21±6.27%, 37.45±4.33% and 60.68±9.83%, respectively; Tail length and Tail DNA % in experimental groups with ZEN concentration above 5 μmol/L showed significant differences (P<0.05) compared with the negative group, which showed an upward trend with the increase of ZEN concentration. [Conclusion] ZEN has genotoxic effect on porcine leydig cells, which can cause DNA damage, with a significant dose-effect relationship.

The Superiority of Like-rocket Immunoelectrophoresis Using in Single Cell Gel Electrophoresis Assay

[Objective] The like-rocket immunoelectrophoresis was used to explore a new feasible electrophoresis method for single cell gel electrophoresis assay （comet assay）.[Method] The like-rocket immunoelectrophoresis was used for single cell gel electrophoresis assay to detect DNA damage at single cell level,then it was compared with traditional electrophoresis method to analyze its advantage and disadvantages.[Result] Under cell DNA undamaged state,the results of two electrophoresis methods were consistent.When cell DNA was damaged,the comet tail divergence of some cells under traditional electrophoresis method were drifted,however,the comet tail image of like-rocket immunoelectrophoresis was concentrated and not shifted.[Conclusion] The like-rocket immunoelectrophoresis had some advantages.

Successful Nitrogen Doping of 1.3 GHz Single Cell Superconducting Radio-Frequency Cavities

A high intrinsic quality factor （Q0） of a superconducting radio-frequency cavity is beneficial to reducing the oper- ation costs of superconducting accelerators. Nitrogen doping （N-doping） has been demonstrated as a aseful way to improve Q0 of the superconducting cavity in recent years. N-doping researches with 1.3 GHz single cell cavities are carried out at Peking University and the preliminary results are promising. Our recipe is slightly different from other laboratories. After 250μm polishing, high pressure rinsing and 3 h high temperature annealing, the cavities are nitrogen doped at 2.7-4.0Pa for 20rain and then followed by 15μm electropolishing. Vertical test results show that Q0 of a 1.3 GHz single cell cavity made of large grain niobium has increased to 4 ×10 10 at 2.0K and medium gradient.

Detection of Sperm DNA Damage in Workers Exposed to Benzene by Modified Single Cell Gel Electrophoresis

Objective To assess the effect of benzene on sperm DNA damage ;Methods Twenty-seven benzene-exposed workers were selected as exposed group and 35 normal sperm donors as control group. Air concentration of benzene series in workshop was determined by gas chromatography. As an internal exposure dose of benzene, the concentration of trans, trans-muconic acid （ttMA） was determined by high performance liquid chromatography. DNA was detected by modified single cell gel electrophoresis （SCGE）. Results The air concentrations of benzene, toluene and xylene at the workplace were 86.49±2.83 mg/m^3, 97.20±3.52 mg/m^3 and 97.45± 2.10 mg/m^3, respectively. Urinary ttMA in exposed group （1.040 ± 0.617 mg/L） was significantly higher than that of control group （0.819 ± 0.157 mg/L）. The percentage of head DNA, determined by modified SCGE method, significantly decreased in the exposed group （n=13, 70.18% ± 7.36%） compared with the control （n=16, 90.62% ± 2.94%）（P〈0.001）. Conclusion The modified SCGE method can be used to investigate the damage of sperm DNA. As genotoxin and reprotoxins, benzene had direct effect on the germ cells during the spermatogenesiss.

Microfluidic-based single cell trapping using a combination of stagnation point flow and physical barrier

Single cell trapping in vitro by microfluidic device is an emerging approach for the study of the relationship between single cells and their dynamic biochemical microenvironments. In this paper, a hydrodynamic-based microfluidic device for single cell trapping is designed using a combination of stagnation point flow and physical barrier.The microfluidic device overcomes the weakness of the traditional ones, which have been only based upon either stagnation point flows or physical barriers, and can conveniently load dynamic biochemical signals to the trapped cell. In addition, it can connect with a programmable syringe pump and a microscope to constitute an integrated experimental system.It is experimentally verified that the microfluidic system can trap single cells in vitro even under flow disturbance and conveniently load biochemical signals to the trapped cell. The designed micro-device would provide a simple yet effective experimental platform for further study of the interactions between single cells and their microenvironments.

Single cell RNA sequencing reveals mesenchymal heterogeneity and critical functions of Cd271 in tooth development

BACKGROUND Accumulating evidence suggests that the maxillary process,to which cranial crest cells migrate,is essential to tooth development.Emerging studies indicate that Cd271 plays an essential role in odontogenesis.However,the underlying mechanisms have yet to be elucidated.AIM To establish the functionally heterogeneous population in the maxillary process,elucidate the effects of Cd271 deficiency on gene expression differences.METHODS p75NTR knockout(Cd271-/-)mice(from American Jackson laboratory)were used to collect the maxillofacial process tissue of p75NTR knockout mice,and the wildtype maxillofacial process of the same pregnant mouse wild was used as control.After single cell suspension,the cDNA was prepared by loading the single cell suspension into the 10x Genomics Chromium system to be sequenced by NovaSeq6000 sequencing system.Finally,the sequencing data in Fastq format were obtained.The FastQC software is used to evaluate the quality of data and CellRanger analyzed the data.The gene expression matrix is read by R software,and Seurat is used to control and standardize the data,reduce the dimension and cluster.We search for marker genes for subgroup annotation by consulting literature and database;explore the effect of p75NTR knockout on mesenchymal stem cells(MSCs)gene expression and cell proportion by cell subgrouping,differential gene analysis,enrichment analysis and protein-protein interaction network analysis;understand the interaction between MSCs cells and the differentiation trajectory and gene change characteristics of p75NTR knockout MSCs by cell communication analysis and pseudo-time analysis.Last we verified the findings single cell sequencing in vitro.RESULTS We identified 21 cell clusters,and we re-clustered these into three subclusters.Importantly,we revealed the cell–cell communication networks between clusters.We clarified that Cd271 was significantly associated with the regulation of mineralization.CONCLUSION This study provides comprehensive mechanistic insights into the maxillary-process-derived MSCs and demonstrates that Cd271 is significantly associated with the odontogenesis in mesenchymal populations.

Observing single cells in whole organs with optical imaging

Cells are the basic unit of human organs that are not fully understood.The revolutionary advancements of optical imaging alowed us to observe single cells in whole organs,revealing the complicated composition of cells with spatial information.Therefore,in this review,we revisit the principles of optical contrast related to those biomolecules and the optical techniques that transform optical contrast into detectable optical signals.Then,we describe optical imaging to achieve threedimensional spatial discrimination for biological tisutes.Due to the milky appearance of tissues,the spatial information burred deep in the whole organ.Fortunately,strategies developed in the last decade could circumvent this issue and lead us into a new era of investigation of the cells with their original spatial information.

COVID-19 Related Research by Data Mining in Single Cell Transcriptome Profiles

The outbreak of coronavirus disease 2019(COVID-2019)has drawn public attention all over the world.As a newly emerging area,single cell sequencing also exerts its power in the battle over the epidemic.In this review,the up-to-date knowledge of COVID-19 and its receptor is summarized,followed by a collection of the mining of single cell transcriptome profiling data for the information in aspects of the vulnerable cell types in humans and the potential mechanisms of the disease.

Fermentation Conditions on the Production of Single Cell Protein Feed by Water Chestnut Peel

In order to improve the comprehensive utilization value of water chestnut peel and the income of farmers. Contents of crude protein （ CP）, total sugar and reducing sugar were taken as indicators. Effects of initial pH, ratio of yeast to fungi species, incubation time and liquid volume on production of single cell pro- tein （SCP） feed was studied, and technological conditions on production of SCP feed by water chestnut peel were optimized by orthogonal test. Results showed that the production of SCP feed by water chestnut peel was optimal when pH was 5.0, ratio of yeast to fungi species was 2： 1, fermentation time was 2 d and the liquid volume was 70 mL / 250 mL. Under the optimum conditions, content of fermentation CP was 64.25%, content of total sugar was 19.8%, content of reducing sugar was 5.0%, content of coarse fibre was 0.0% and content of ash was 8.04%.

Conversion of Potato Peel Waste to Single Cell Protein by an Acidophilic Fungus

The aim of this research was to convert potato peel waste (PPW) to single cell protein (SCP), and to extract valuable phenolic compounds from the spent medium. PPW is an abundant by-product of potato processing industry, consisting mostly of starch, fibre and protein in a form of watery sludge. The PPW from a chip manufacturing plant was pre-treated with sulphuric acid, and used as a substrate for an acidophilic Scytalidium acidophilum fungus under non-aseptic conditions. The produced SCP had a promising amino acid composition to be used in animal feed. Phenolic compounds were not recovered from the spent medium, most likely due to the low pH in the medium. The present findings suggest that PPW is a suitable raw material for acidophilic SCP production, whilst the extraction of phenolic acids would require milder cultivation conditions or separation before pre-treatments of SCP production. The BOD5 of the PPW was reduced by in 98% due to fungal cultivation. Thus the feed production also served as an efficient means for reduction of organic load in the PPW.

A SARS-CoV-2 neutralizing antibody discovery by single cell sequencing and molecular modeling

Although vaccines have been developed,mutations of SARS-CoV-2,especially the dominant B.1.617.2(delta)and B.1.529(omicron)strains with more than 30 mutations on their spike protein,have caused a significant decline in prophylaxis,calling for the need for drug improvement.Antibodies are drugs preferentially used in infectious diseases and are easy to get from immunized organisms.The current study combined molecular modeling and single memory B cell sequencing to assess candidate sequences before experiments,providing a strategy for the fabrication of SARS-CoV-2 neutralizing antibodies.A total of 128 sequences were obtained after sequencing 196 memory B cells,and 42 sequences were left after merging extremely similar ones and discarding incomplete ones,followed by homology modeling of the antibody variable region.Thirteen candidate sequences were expressed,of which three were tested positive for receptor binding domain recognition but only one was confirmed as having broad neutralization against several SARS-CoV-2 variants.The current study successfully obtained a SARS-CoV-2 antibody with broad neutralizing abilities and provided a strategy for antibody development in emerging infectious diseases using single memory B cell BCR sequencing and computer assistance in antibody fabrication.

Single cell sequencing: technique, application,and future development

The progression of next generation sequencing is continuously changing the landscape of genomic, tran- scriptomic, and epigenomic studies. Particularly, advances in single cell manipulation and amplification techniques bring sequencing technology to the single-cell level. Single cell genome sequencing allows us to study tumor evolu- tion, gamete genesis, somatic mosaicism at genome-wide level; single cell transcriptome sequencing unveils the dynamic gene expression during early embryonic devel- opment, differentiation and reprogramming; single cell methylome sequencing is just taking off and shows great potential in cancer and stem cell studies. Lots of attempts are still being made in other dimensions of sequencing. The increasing need for single cell sequencing requires the future techniques with the following features： （1） high accuracy and fidelity; （2） able to perform multiple omics analyses in one cell; （3） high degree of automation and standardized pipeline. These progresses and improvements will lower the barrier for single cell sequencing to enter ordinary laboratories. The wide application of single cell sequencing techniques will substantially change biomedi- cal research in future.

Comprehensive integration of single-cell transcriptomic data illuminates the regulatory network architecture of plant cell fate specification

Plant morphogenesis relies on precise gene expression programs at the proper time and position which is orchestrated by transcription factors(TFs)in intricate regulatory networks in a cell-type specific manner.Here we introduced a comprehensive single-cell transcriptomic atlas of Arabidopsis seedlings.This atlas is the result of meticulous integration of 63 previously published scRNA-seq datasets,addressing batch effects and conserving biological variance.This integration spans a broad spectrum of tissues,including both below-and above-ground parts.Utilizing a rigorous approach for cell type annotation,we identified 47 distinct cell types or states,largely expanding our current view of plant cell compositions.We systematically constructed cell-type specific gene regulatory networks and uncovered key regulators that act in a coordinated manner to control cell-type specific gene expression.Taken together,our study not only offers extensive plant cell atlas exploration that serves as a valuable resource,but also provides molecular insights into gene-regulatory programs that varies from different cell types.

Micro/nanoelectrode-based electrochemical methodology for single cell and organelle analysis

Cells are the basic unit of life.Electrochemical analysis of single cells/organelles is essential for uncovering the molecular mechanisms of physiological and pathological processes that are difficult to elucidate on a larger scale.This paper provides an overview of the commonly used fabrication methods for micro/nanoelectrodes applied in the investigations of single cells/organelles as well as the corresponding electrochemical measurements over the last four years including extracellular measurement,combination of extra and intracellular measurement,intracellular reactive oxygen species and reactive nitrogen species(ROS/RNS)measurement,and isolated organelles measurement.

Valorisation of industrial hemp (Cannabis sativa L.) residues and cheese whey into volatile fatty acids for single cell protein production

The production of single cell protein(SCP)using lignocellulosic materials stands out as a promising route in the circular bioeconomy transition.However,multiple steps are necessary for lignocellulosics-to-SCP processes,involving chemical pretreatments and specific aerobic cultures.Whereas there are no studies that investigated the SCP production from lignocellulosics by using only biological processes and microbial biomass able to work both anaerobically and aerobically.In this view,the valorisation of industrial hemp(Cannabis sativa L.)biomass residues(HBRs),specifically hurds and a mix of leaves and inflorescences,combined with cheese whey(CW)was investigated through a semi-continuous acidogenic co-fermentation process(co-AF).The aim of this study was to maximise HBRs conversion into VFAs to be further used as carbon-rich substrates for SCP production.Different process conditions were tested by either removing CW or increasing the amount of HBRs in terms of VS(i.e.,two and four times)to evaluate the performance of the co-AF process.Increasing HBRs resulted in a proportional increase in VFA production up to 3115 mg HAc L^(-1),with experimental production nearly 40%higher than theoretical predictions.The synergy between HBRs and CW was demonstrated,proving the latter as essential to improve the biodegradability of the former.The produced VFAs were subsequently tested as substrates for SCP synthesis in batch aerobic tests.A biomass concentration of 2.43 g TSS L^(-1) was achieved with a C/N ratio of 5.0 and a pH of 9.0 after two days of aerobic fermentation,reaching a protein content of 42%(g protein per g TSS).These results demonstrate the overall feasibility of the VFA-mediated HBR-to-SCP valorisation process.

Valorization of durian peel as a carbon feedstock for a sustainable production of heterogeneous base catalyst,single cell oil and yeast-based biodiesel

The present study reports a successful attempt to produce single cell oil(SCO),heterogeneous base catalyst and yeast-based biodiesel from durian peel as a promising carbon feedstock by means of the waste-to-energy concept.For this purpose,first,durian peel(DP)was hydrolyzed by dilute sulfuric acid to obtain xylose-rich DP hydrolysate(XDPH)and post-hydrolysis DP solid residue(DPS).Candida viswanathii PSY8,a newly isolated oleaginous yeast,showed high SCO accumulation(5.1±0.1 g/L)and SCO content(35.3±0.13%)on undetoxified XDPH medium.A novel heterogeneous base catalyst(DPS-K)prepared from DPS by wet impregnation technique with KOH,exhibited considerable catalytic activity to convert SCO-rich wet yeast of C.viswanathii PSY8 into yeast-based biodiesel(FAME)via direct transesterification with a maximum FAME yield of 94.3%under optimal conditions(6 wt%catalyst,10:1 methanol to wet yeast ratio,75℃,and 2 h).Moreover,most of the yeast-based biodiesel properties obtained from the FAME profiles were correlated well with the biodiesel standards limit of Thai,ASTM D6751 and EN 14214.Additionally,the energy output of FAME produced about 37.5 MJ/kg was estimated.Thus,this present finding demonstrated the favorable strategy for sustainable and eco-friendly production of new generation biodiesel.

Single nucleus/cell RNA-seq of the chicken hypothalamicpituitaryovarian axis offers new insights into the molecular regulatory mechanisms of ovarian development

The hypothalamic-pituitary-ovarian(HPO)axis represents a central neuroendocrine network essential for reproductive function.Despite its critical role,the intrinsic heterogeneity within the HPO axis across vertebrates and the complex intercellular interactions remain poorly defined.This study provides the first comprehensive,unbiased,cell type-specific molecular profiling of all three components of the HPO axis in adult Lohmann layers and Liangshan Yanying chickens.Within the hypothalamus,pituitary,and ovary,seven,12,and 13 distinct cell types were identified,respectively.Results indicated that the pituitary adenylate cyclase activating polypeptide(PACAP),follicle-stimulating hormone(FSH),and prolactin(PRL)signaling pathways may modulate the synthesis and secretion of gonadotropin-releasing hormone(GnRH),FSH,and luteinizing hormone(LH)within the hypothalamus and pituitary.In the ovary,interactions between granulosa cells and oocytes involved the KIT,CD99,LIFR,FN1,and ANGPTL signaling pathways,which collectively regulate follicular maturation.The SEMA4 signaling pathway emerged as a critical mediator across all three tissues of the HPO axis.Additionally,gene expression analysis revealed that relaxin 3(RLN3),gastrin-releasing peptide(GRP),and cocaine-and amphetamine regulated transcripts(CART,also known as CARTPT)may function as novel endocrine hormones,influencing the HPO axis through autocrine,paracrine,and endocrine pathways.Comparative analyses between Lohmann layers and Liangshan Yanying chickens demonstrated higher expression levels of GRP,RLN3,CARTPT,LHCGR,FSHR,and GRPR in the ovaries of Lohmann layers,potentially contributing to their superior reproductive performance.In conclusion,this study provides a detailed molecular characterization of the HPO axis,offering novel insights into the regulatory mechanisms underlying reproductive biology.

Nanofluidics for sub-single cellular studies:Nascent progress,critical technologies,and future perspectives

In the field of cell studies,there is a burgeoning trend to further downscale the investigation from a single-cell level to a sub-single-cell level.Subcellular matter is the basic content in cells and correlates with cell heterogeneity.Sub-single cellular studies focus on the subcellular matter in single cells and aim to understand the details and heterogeneity of individual cells in terms of the subcellular matter or even at the single component/vesicle/molecule level.Hence,sub-single cellular studies can provide deeper insights into fundamental cell biology and the development of new diagnostic and therapeutic technologies and applications.Nonetheless,the contents of a single cell are not only ultra-small in volume but also extremely complex in composition,far exceeding the capabilities of most tools used in current cell studies.We believe that nanofluidics holds great potential in providing ideal tools for sub-single cellular studies,not only because of their capability to handle femtoliter/attoliter-scale samples,but also because of their possibility to manipulate and analyze subcellular matters at the single component/vesicle/molecule level in a high-throughput manner.In this review,we summarize the efforts in the field of nanofluidics for sub-single cellular studies,focusing on nascent progress and critical technologies that have the potential to overcome the technical bottlenecks.Some challenges and future opportunities to integrate with information sciences are also discussed.

Immune contexture defined by single cell technology for prognosis prediction and immunotherapy guidance in cancer

Tumor immune microenvironment is closely related to tumor initiation,prognosis,and response to immunotherapy.The immune landscapes,number of infiltrating immune cells,and the localization of lymphocytes in the tumor vary in across different types of tumors.The immune contexture in cancer,which is determined by the density,composition,functional state and organization of the leukocyte infiltrate of the tumor,can yield information relevant to the prediction of treatment response and patients’prognosis.Better understanding of the immune atlas in human tumors have been achieved with the development and application of single-cell analysis technology,which has provided a reference for prognosis,and insights on new targets for immunotherapy.In this review,we summarized the different characteristics of immune contexture in cancer defined by a variety of single-cell techniques,which have enhanced our understanding on the pathophysiology of the tumor microenvironment.We believe that there are much more to be uncovered in this rapidly developing field of medicine,and they will predict the prognosis of cancer patients and guide the rational design of immunotherapies for success in cancer eradication.

Complete disassociation of adult pancreas into viable single cells through cold trypsin-EDTA digestion

The in vitro isolation and analysis of pancreatic stem/progenitor cells are necessary for understanding their properties and function; however, the preparation of high-quality single-cell suspensions from adult pancreas is pre- requisite. In this study, we applied a cold trypsin-ethylenediaminetetraacetic acid （EDTA） digestion method to disas- sociate adult mouse pancreata into single cells. The yield of single cells and the viability of the harvested cells were much higher than those obtained via the two commonly used warm digestion methods. Flow cytometric analysis showed that the ratio of ductal or BCRPl-positive cells in cell suspensions prepared through cold digestion was con- sistent with that found in vivo. Cell culture tests showed that pancreatic epithelial cells prepared by cold digestion maintained proliferative capacity comparable to those derived from warm collagenase digestion. These results indicate that cold trypsin-EDTA digestion can effectively disassociate an adult mouse pancreas into viable single cells with minimal cell loss, and can be used for the isolation and analysis of pancreatic stem/progenitor cells.