Rapid advances in Ribonucleic Acid sequencing(or RNA-seq)technology for analyzing entire transcriptomes of desired tissue samples,or even of single cells at scale,have revolutionized biology in the past decade.Increas...Rapid advances in Ribonucleic Acid sequencing(or RNA-seq)technology for analyzing entire transcriptomes of desired tissue samples,or even of single cells at scale,have revolutionized biology in the past decade.Increasing accessibility and falling costs are making it possible to address many problems in biology that were once considered intractable,including the study of various social behaviors.RNA-seq is opening new avenues to understand long-standing questions on the molecular basis of behavioral plasticity and individual variation in the expression of a behavior.As whole transcriptomes are examined,it has become possible to make unbiased discoveries of underlying mechanisms with little or no necessity to predict genes involved in advance.However,researchers need to be aware of technical limitations and have to make specific decisions when applying RNA-seq to study social behavior.Here,we provide a perspective on the applications of RNA-seq and experimental design considerations for behavioral scientists who are unfamiliar with the technology but are considering using it in their research.展开更多
Organoids of the central nervous system,primarily derived from pluripotent stem cells or neural stem cells,are three-dimensional tissue cultures with self-organizing properties.When exposed to the right combinations o...Organoids of the central nervous system,primarily derived from pluripotent stem cells or neural stem cells,are three-dimensional tissue cultures with self-organizing properties.When exposed to the right combinations of signals,they differentiate into a 3D tissue consisting of complex cytoarchitecture and native cell types,including various neuron subtypes and glial cells.These features closely mimic native tissues,making them invaluable for developmental studies and disease modeling.In recent years,spinal cord organoids(SCOs)have been developed to investigate spinal cord development,injuries,and various neurological disorders.As an integral part of the central nervous system,SCOs play a vital role and serve as a site for studying both neurodevelopment and neurodegenerative diseases.展开更多
One of the main goals of the Human Genome Project is to identify all protein-coding genes.There are~20,5o0 protein-coding genes annotated in the human reference databases.However,in the last few years,proteogenomics s...One of the main goals of the Human Genome Project is to identify all protein-coding genes.There are~20,5o0 protein-coding genes annotated in the human reference databases.However,in the last few years,proteogenomics studies have predicted thousands of novel protein-coding regions,including low-molecular-weight proteins encoded by small open reading frames(sORFs)in untranslated regions of messenger RNAs and non-coding RNAs.Most of these predictions are based on bioinformatics analyses and ribosome footprint data.The validity of some of these sORF-encoded proteins(SEPs)has been established through functional characterization.With the growing number of predicted novel proteins,a strategy to identify reliable candidates that warrant further studies is needed.In this study,we developed an integrated proteoge-nomics workflow to identify a reliable set of novel protein-coding regions in the human genome based on their recurrent observations across multiple samples.Publicly available ribosome profiling and global proteomic datasets were used to establish protein-coding evidence.We pre-dicted protein translation from 4008 sORFs based on recurrent ribosome occupancy signals across samples.In addition,we identified 825 SEPs based on proteomic data.Some of the novel protein-coding regions identified were located in genome-wide association study(GWAS)loci associated with various traits and disease phenotypes.Peptides from SEPs are also presented by major histocompatibility complex class I(MHC-I),similar to canonical proteins.Novel protein-coding regions reported in this study expand the current catalog of protein-coding genes and warrant experimental studies to elucidate their cellular functions and potential roles in human diseases.展开更多
Pancreatic ductal adenocarcinoma(PDAC)remains one of medicine’s most urgent areas of unmet need.With 5-year survival rates of∼11%,PDAC is set to become the second leading cause of cancer related deaths by 2040[1].Th...Pancreatic ductal adenocarcinoma(PDAC)remains one of medicine’s most urgent areas of unmet need.With 5-year survival rates of∼11%,PDAC is set to become the second leading cause of cancer related deaths by 2040[1].The complex tumour microenvironment(TME)in PDAC,responsible for poor prognosis,is comprised of extracellular matrix(ECM)proteins and multiple cell types;with pancreatic stellate cells(PSCs),which become activated cancer associated fibroblasts(CAFs),being regarded as key orchestrators of the TME.We have demonstrated that treatment with all-trans retinoic acid(ATRA)can render activated PSCs(aPSC)to a quiescent(qPSC)phenotype(shift to G1 phase of cell cycle and other features[2]),resulting in stromal remodelling and thus,influencing cancer cell co-targeting with chemotherapy in patients[3].This has resulted in the use of ATRA along with standard-of-care chemotherapy in the Stromal TARgeting for PAncreatic Cancer(STARPAC)clinical trial,with promising results[4].These clinically relevant[5],exciting potential therapeutic benefits of stromal co-targeting through rendering PSCs quiescent[6],along with predictive inflammation-related biomarkers[7],and increased focus on cellular therapeutics such as NK cells,led us to postulate potential targetable PSC-immune cell interactions which may uncover a comprehensive therapeutic strategy for treating hitherto,incurable PDAC.展开更多
Conventional immunohistochemistry(IHC)is a widely used diagnostic technique in tissue pathology.However,this technique is associated with a number of limitations,including high inter-observer variability and the capac...Conventional immunohistochemistry(IHC)is a widely used diagnostic technique in tissue pathology.However,this technique is associated with a number of limitations,including high inter-observer variability and the capacity to label only one marker per tissue section.This review details various highly multiplexed techniques that have emerged to circumvent these constraints,allowing simultaneous detection of multiple markers on a single tissue section and the comprehensive study of cell composition,cellular functional and cell-cell interactions.Among these techniques,multiplex Immunohistochemistry/Immunofluorescence(mIHC/IF)has emerged to be particularly promising.mIHC/IF provides high-throughput multiplex staining and standardized quantitative analysis for highly reproducible,efficient and cost-effective tissue studies.This technique has immediate potential for translational research and clinical practice,particularly in the era of cancer immunotherapy.展开更多
Since December 2019,the world is increasingly facing an unprecedented challenge by coronavirus disease 2019(COVID-19)caused by a virus called severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),which is struct...Since December 2019,the world is increasingly facing an unprecedented challenge by coronavirus disease 2019(COVID-19)caused by a virus called severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),which is structurally related to the human severe acute respiratory syndrome CoV(SARS-CoV)and Middle East respiratory syn-drome CoV(MERS-CoV)(Zhu et al.,2020).The World Health Organization(WHO)has declared COVID-19 a pandemic and is calling for forceful action from all countries,as the number of infected patients and the number of deaths are increasing daily,causing catastrophic consequence to the daily life,economy,and society.展开更多
Although the nicotinamide nucleotides NAD（H） and NADP（H） are essential for various metabolic reactions that play major roles in maintenance of cellular homeostasis, the significance of NAD biosynthesis is not well...Although the nicotinamide nucleotides NAD（H） and NADP（H） are essential for various metabolic reactions that play major roles in maintenance of cellular homeostasis, the significance of NAD biosynthesis is not well under- stood. Here, we investigated the dynamics of pollen nicotinamide nucleotides in response to imbibition, a representative germination cue. Metabolic analysis with capillary electrophoresis electrospray ionization mass spectrometry revealed that excess amount of NAD＊ is accumulated in freshly harvested dry pollen, whereas it dramatically decreased immedi- ately after contact with water, importantly, excess of NAD~ impaired pollen tube growth. Moreover, NAD~ accumulation was retained after pollen was imbibed in the presence of NAD^-consuming reaction inhibitors and pollen germination was greatly retarded. Pollen deficient in the nicotinate/nicotinamide mononucleotide adenyltransferase （NMNAT） gene, encoding a key enzyme in NAD biosynthesis, and a lack of NAD＊ accumulation in the gametophyte, showed precocious pollen tube germination inside the anther Iocule and vigorous tube growth under high-humidity conditions. Hence, the accumulation of excess NAD＊ is not essential for pollen germination, but instead participates in regulating the timing of germination onset. These results indicate that NAD＋ accumulation acts to negatively regulate germination and a decrease in NAD＋ plays an important role in metabolic state transition.展开更多
With the advent of rapid automated in silico identification of biosynthetic gene clusters(BGCs),genomics pre-sents vast opportunities to accelerate natural product(NP)discovery.However,prolific NP producers,Strepto-my...With the advent of rapid automated in silico identification of biosynthetic gene clusters(BGCs),genomics pre-sents vast opportunities to accelerate natural product(NP)discovery.However,prolific NP producers,Strepto-myces,are exceptionally GC-rich(>80%)and highly repetitive within BGCs.These pose challenges in sequencing and high-quality genome assembly which are currently circumvented via intensive sequencing.Here,we outline a more cost-effective workflow using multiplex Illumina and Oxford Nanopore sequencing with hybrid long-short read assembly algorithms to generate high quality genomes.Our protocol involves subjecting long read-derived assemblies to up to 4 rounds of polishing with short reads to yield accurate BGC predictions.We successfully sequenced and assembled 8 GC-rich Streptomyces genomes whose lengths range from 7.1 to 12.1 Mb with a median N50 of 8.2 Mb.Taxonomic analysis revealed previous misrepresentation among these strains and allowed us to propose a potentially new species,Streptomyces sydneybrenneri.Further comprehensive characterization of their biosynthetic,pan-genomic and antibiotic resistance features especially for molecules derived from type I polyketide synthase(PKS)BGCs reflected their potential as alternative NP hosts.Thus,the genome assemblies and insights presented here are envisioned to serve as gateway for the scientific community to expand their avenues in NP discovery.展开更多
基金This work was supported by Yale-NUS College through grants R-607-265-225-121 and IG16-LR003.
文摘Rapid advances in Ribonucleic Acid sequencing(or RNA-seq)technology for analyzing entire transcriptomes of desired tissue samples,or even of single cells at scale,have revolutionized biology in the past decade.Increasing accessibility and falling costs are making it possible to address many problems in biology that were once considered intractable,including the study of various social behaviors.RNA-seq is opening new avenues to understand long-standing questions on the molecular basis of behavioral plasticity and individual variation in the expression of a behavior.As whole transcriptomes are examined,it has become possible to make unbiased discoveries of underlying mechanisms with little or no necessity to predict genes involved in advance.However,researchers need to be aware of technical limitations and have to make specific decisions when applying RNA-seq to study social behavior.Here,we provide a perspective on the applications of RNA-seq and experimental design considerations for behavioral scientists who are unfamiliar with the technology but are considering using it in their research.
基金supported by A*STAR Career Development Fund (C210112011)National Medical Research Council (MOH-001248-00)(to WHC)+2 种基金Singapore International Graduate Award (to YW)National Research Foundation (NRFNRFF-2018-003)Biomedical Research Council,A*STAR Research Entities (to SYN)
文摘Organoids of the central nervous system,primarily derived from pluripotent stem cells or neural stem cells,are three-dimensional tissue cultures with self-organizing properties.When exposed to the right combinations of signals,they differentiate into a 3D tissue consisting of complex cytoarchitecture and native cell types,including various neuron subtypes and glial cells.These features closely mimic native tissues,making them invaluable for developmental studies and disease modeling.In recent years,spinal cord organoids(SCOs)have been developed to investigate spinal cord development,injuries,and various neurological disorders.As an integral part of the central nervous system,SCOs play a vital role and serve as a site for studying both neurodevelopment and neurodegenerative diseases.
文摘One of the main goals of the Human Genome Project is to identify all protein-coding genes.There are~20,5o0 protein-coding genes annotated in the human reference databases.However,in the last few years,proteogenomics studies have predicted thousands of novel protein-coding regions,including low-molecular-weight proteins encoded by small open reading frames(sORFs)in untranslated regions of messenger RNAs and non-coding RNAs.Most of these predictions are based on bioinformatics analyses and ribosome footprint data.The validity of some of these sORF-encoded proteins(SEPs)has been established through functional characterization.With the growing number of predicted novel proteins,a strategy to identify reliable candidates that warrant further studies is needed.In this study,we developed an integrated proteoge-nomics workflow to identify a reliable set of novel protein-coding regions in the human genome based on their recurrent observations across multiple samples.Publicly available ribosome profiling and global proteomic datasets were used to establish protein-coding evidence.We pre-dicted protein translation from 4008 sORFs based on recurrent ribosome occupancy signals across samples.In addition,we identified 825 SEPs based on proteomic data.Some of the novel protein-coding regions identified were located in genome-wide association study(GWAS)loci associated with various traits and disease phenotypes.Peptides from SEPs are also presented by major histocompatibility complex class I(MHC-I),similar to canonical proteins.Novel protein-coding regions reported in this study expand the current catalog of protein-coding genes and warrant experimental studies to elucidate their cellular functions and potential roles in human diseases.
文摘Pancreatic ductal adenocarcinoma(PDAC)remains one of medicine’s most urgent areas of unmet need.With 5-year survival rates of∼11%,PDAC is set to become the second leading cause of cancer related deaths by 2040[1].The complex tumour microenvironment(TME)in PDAC,responsible for poor prognosis,is comprised of extracellular matrix(ECM)proteins and multiple cell types;with pancreatic stellate cells(PSCs),which become activated cancer associated fibroblasts(CAFs),being regarded as key orchestrators of the TME.We have demonstrated that treatment with all-trans retinoic acid(ATRA)can render activated PSCs(aPSC)to a quiescent(qPSC)phenotype(shift to G1 phase of cell cycle and other features[2]),resulting in stromal remodelling and thus,influencing cancer cell co-targeting with chemotherapy in patients[3].This has resulted in the use of ATRA along with standard-of-care chemotherapy in the Stromal TARgeting for PAncreatic Cancer(STARPAC)clinical trial,with promising results[4].These clinically relevant[5],exciting potential therapeutic benefits of stromal co-targeting through rendering PSCs quiescent[6],along with predictive inflammation-related biomarkers[7],and increased focus on cellular therapeutics such as NK cells,led us to postulate potential targetable PSC-immune cell interactions which may uncover a comprehensive therapeutic strategy for treating hitherto,incurable PDAC.
文摘Conventional immunohistochemistry(IHC)is a widely used diagnostic technique in tissue pathology.However,this technique is associated with a number of limitations,including high inter-observer variability and the capacity to label only one marker per tissue section.This review details various highly multiplexed techniques that have emerged to circumvent these constraints,allowing simultaneous detection of multiple markers on a single tissue section and the comprehensive study of cell composition,cellular functional and cell-cell interactions.Among these techniques,multiplex Immunohistochemistry/Immunofluorescence(mIHC/IF)has emerged to be particularly promising.mIHC/IF provides high-throughput multiplex staining and standardized quantitative analysis for highly reproducible,efficient and cost-effective tissue studies.This technique has immediate potential for translational research and clinical practice,particularly in the era of cancer immunotherapy.
文摘Since December 2019,the world is increasingly facing an unprecedented challenge by coronavirus disease 2019(COVID-19)caused by a virus called severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),which is structurally related to the human severe acute respiratory syndrome CoV(SARS-CoV)and Middle East respiratory syn-drome CoV(MERS-CoV)(Zhu et al.,2020).The World Health Organization(WHO)has declared COVID-19 a pandemic and is calling for forceful action from all countries,as the number of infected patients and the number of deaths are increasing daily,causing catastrophic consequence to the daily life,economy,and society.
文摘Although the nicotinamide nucleotides NAD（H） and NADP（H） are essential for various metabolic reactions that play major roles in maintenance of cellular homeostasis, the significance of NAD biosynthesis is not well under- stood. Here, we investigated the dynamics of pollen nicotinamide nucleotides in response to imbibition, a representative germination cue. Metabolic analysis with capillary electrophoresis electrospray ionization mass spectrometry revealed that excess amount of NAD＊ is accumulated in freshly harvested dry pollen, whereas it dramatically decreased immedi- ately after contact with water, importantly, excess of NAD~ impaired pollen tube growth. Moreover, NAD~ accumulation was retained after pollen was imbibed in the presence of NAD^-consuming reaction inhibitors and pollen germination was greatly retarded. Pollen deficient in the nicotinate/nicotinamide mononucleotide adenyltransferase （NMNAT） gene, encoding a key enzyme in NAD biosynthesis, and a lack of NAD＊ accumulation in the gametophyte, showed precocious pollen tube germination inside the anther Iocule and vigorous tube growth under high-humidity conditions. Hence, the accumulation of excess NAD＊ is not essential for pollen germination, but instead participates in regulating the timing of germination onset. These results indicate that NAD＋ accumulation acts to negatively regulate germination and a decrease in NAD＋ plays an important role in metabolic state transition.
基金supported by National Research Foundation,Singapore(NRF-CRP19-2017-05-00)Agency for Science,Technology and Research(A*STAR),Singapore(#21719).
文摘With the advent of rapid automated in silico identification of biosynthetic gene clusters(BGCs),genomics pre-sents vast opportunities to accelerate natural product(NP)discovery.However,prolific NP producers,Strepto-myces,are exceptionally GC-rich(>80%)and highly repetitive within BGCs.These pose challenges in sequencing and high-quality genome assembly which are currently circumvented via intensive sequencing.Here,we outline a more cost-effective workflow using multiplex Illumina and Oxford Nanopore sequencing with hybrid long-short read assembly algorithms to generate high quality genomes.Our protocol involves subjecting long read-derived assemblies to up to 4 rounds of polishing with short reads to yield accurate BGC predictions.We successfully sequenced and assembled 8 GC-rich Streptomyces genomes whose lengths range from 7.1 to 12.1 Mb with a median N50 of 8.2 Mb.Taxonomic analysis revealed previous misrepresentation among these strains and allowed us to propose a potentially new species,Streptomyces sydneybrenneri.Further comprehensive characterization of their biosynthetic,pan-genomic and antibiotic resistance features especially for molecules derived from type I polyketide synthase(PKS)BGCs reflected their potential as alternative NP hosts.Thus,the genome assemblies and insights presented here are envisioned to serve as gateway for the scientific community to expand their avenues in NP discovery.
