Background:Pistacia integerrima,a cornerstone of traditional medicine,is renowned for its therapeutic applications against various health conditions,including cancer and hepatitis.This study investigates the pharmacol...Background:Pistacia integerrima,a cornerstone of traditional medicine,is renowned for its therapeutic applications against various health conditions,including cancer and hepatitis.This study investigates the pharmacological potential of bioactive compounds derived from Pistacia integerrima in inhibiting 5-lipoxygenase(5-LOX),a key enzyme implicated in inflammation and cancer progression.The current study aimed to evaluate the lipoxygenase inhibitory activity of bioactive compounds from Pistacia integerrima and assess their potential for therapeutic development in the context of inflammation and cancer treatment.Methods:Three major compounds-spinacetin(1),patuletin(2),and pistagremic acid(3)-were isolated from Pistacia integerrima and analyzed for their lipoxygenase inhibitory activity.Biochemical assays and molecular docking studies were performed to assess their effectiveness in inhibiting 5-LOX.Results:All three compounds demonstrated significant inhibition of lipoxygenase activity.Spinacetin(1)and patuletin(2)exhibited the most potent inhibitory effects,with IC_(50)values of 40.34μM and 45.04μM,respectively.Molecular docking studies revealed that patuletin(2)had the highest binding affinity(−7.717 kcal/mol)against 5-LOX,followed by spinacetin(1)with a binding affinity of−6.074 kcal/mol.In-depth in silico analysis highlighted the drug-likeness of spinacetin(1)and its favorable toxicological profile,suggesting its suitability for therapeutic development.Conclusion:The study demonstrates that compounds from Pistacia integerrima,particularly spinacetin and patuletin,have significant lipoxygenase inhibitory activity,with spinacetin showing promise as a lead candidate for lipoxygenase-targeted therapies.The findings reinforce the therapeutic relevance of Pistacia integerrima and suggest that its bioactive compounds may serve as safer,plant-based alternatives to conventional anti-inflammatory and anticancer treatments.展开更多
Recent advances in our ability to design DNA binding factors with specificity for desired sequences have resulted in a revolution in genetic engineering, enabling directed changes to the genome to be made relatively e...Recent advances in our ability to design DNA binding factors with specificity for desired sequences have resulted in a revolution in genetic engineering, enabling directed changes to the genome to be made relatively easily. Traditional techniques for generating genetic mutations in most organisms have relied on selection from large pools of randomly induced mutations for those of particular interest, or time-consuming gene targeting by homologous recombination. Drosophila melanogaster has always been at the forefront of genetic analysis, and application of these new genome editing techniques to this organism will revolutionise our approach to performing analysis of gene function in the future. We discuss the recent techniques that apply the CRISPR/Cas9 system to Drosophila, highlight potential uses for this technology and speculate upon the future of genome engineering in this model organism.展开更多
CTP synthase is compartmentalized within a subcellular structure,termed the cytoophidium,in a range of organisms including bacteria, yeast,fruit fly and rat.Here we show that CTP synthase is also compartmentalized int...CTP synthase is compartmentalized within a subcellular structure,termed the cytoophidium,in a range of organisms including bacteria, yeast,fruit fly and rat.Here we show that CTP synthase is also compartmentalized into cytoophidia in human cells.Surprisingly,the occurrence of cytoophidia in human cells increases upon treatment with a glutamine analog 6-diazo-5-oxo-L-norleucine（DON）,an inhibitor of glutamine-dependent enzymes including CTP synthase.Experiments in flies confirmed that DON globally promotes cytoophidium assembly.Clonal analysis via CTP synthase RNA interference in somatic cells indicates that CTP synthase expression level is critical for the formation of cytoophidia.Moreover,DON facilitates cytoophidium assembly even when CTP synthase level is low.A second glutamine analog azaserine also promotes cytoophidum formation.Our data demonstrate that glutamine analogs serve as useful tools in the study of cytoophidia.展开更多
CTP synthase （CTPsyn） is a metabolic enzyme responsible for the de novo synthesis of the nucleotide CTE Several recent studies have shown that CTPsyn forms filamentous subcellular structures known as cytoophidia in ...CTP synthase （CTPsyn） is a metabolic enzyme responsible for the de novo synthesis of the nucleotide CTE Several recent studies have shown that CTPsyn forms filamentous subcellular structures known as cytoophidia in bacteria, yeast, fruit flies and humans. However, it remains elusive whether and how CTPsyn and cytoophidia play a role during development. Here, we show that cytoophidia are abundant in the neuroepithelial stem cells in Drosophila optic lobes. Optic lobes are underdeveloped in CTPsyn mutants as well as in CTPsyn RNAi. Moreover, overexpressing CTPsyn impairs the development of optic lobes, specifically by blocking the transition from neuro- epithelium to neuroblast. Taken together, our results indicate that CTPsyn is critical for optic lobe homeostasis in Drosophila.展开更多
The metabolic enzyme CTP synthase(CTPS) is able to compartmentalize into filaments,termed cytoophidia,in a variety of organisms including bacteria,budding yeast,fission yeast,fruit flies and mammals.A previous study i...The metabolic enzyme CTP synthase(CTPS) is able to compartmentalize into filaments,termed cytoophidia,in a variety of organisms including bacteria,budding yeast,fission yeast,fruit flies and mammals.A previous study in budding yeast shows that the filament-forming process of CTPS is not sensitive to temperature shift.Here we study CTPS filamentation in the fission yeast Schizosaccharomyces pombe.To our surprise,we find that both the length and the occurrence of cytoophidia in S.pombe decrease upon cold shock or heat shock.The temperature-dependent changes of cytoophidia are fast and reversible.Taking advantage of yeast genetics,we demonstrate that heat-shock proteins are required for cytoophidium assembly in S.pombe.Temperature sensitivity of cytoophidia makes S.pombe an attractive model system for future investigations of this novel membraneless organelle.展开更多
CTP synthase(CTPS), the rate-limiting enzyme in de novo CTP biosynthesis, has been demonstrated to assemble into evolutionarily conserved filamentous structures, termed cytoophidia, in Drosophila, bacteria, yeast and ...CTP synthase(CTPS), the rate-limiting enzyme in de novo CTP biosynthesis, has been demonstrated to assemble into evolutionarily conserved filamentous structures, termed cytoophidia, in Drosophila, bacteria, yeast and mammalian cells. However, the regulation and function of the cytoophidium remain elusive. Here, we provide evidence that the mechanistic target of rapamycin(mTOR) pathway controls cytoophidium assembly in mammalian and Drosophila cells. In mammalian cells, we find that inhibition of mTOR pathway attenuates cytoophidium formation. Moreover, CTPS cytoophidium assembly appears to be dependent on the mTOR complex 1(mTORC1) mainly. In addition, knockdown of the mTORC1 downstream target S6 K1 can inhibit cytoophidium formation, while overexpression of the constitutively active S6 K1 reverses mTOR knockdown-induced cytoophidium disassembly. Finally, reducing m TOR protein expression results in a decrease of the length of cytoophidium in Drosophila follicle cells.Therefore, our study connects CTPS cytoophidium formation with the mTOR signaling pathway.展开更多
The simplicity of the CRISPR/Cas9 system of genome engineering has opened up the possibility of performing genome-wide targeted mutagenesis in cell lines,enabling screening for cellular phenotypes resulting from genet...The simplicity of the CRISPR/Cas9 system of genome engineering has opened up the possibility of performing genome-wide targeted mutagenesis in cell lines,enabling screening for cellular phenotypes resulting from genetic aberrations.Drosophila cells have proven to be highly effective in identifying genes involved in cellular processes through similar screens using partial knockdown by RNAi.This is in part due to the lower degree of redundancy between genes in this organism,whilst still maintaining highly conserved gene networks and orthologs of many human disease-causing genes.The ability of CRISPR to generate genetic loss of function mutations not only increases the magnitude of any effect over currently employed RNAi techniques,but allows analysis over longer periods of time which can be critical for certain phenotypes.In this study,we have designed and built a genome-wide CRISPR library covering 13,501 genes,among which 8989 genes are targeted by three or more independent single guide RNAs（sg RNAs）.Moreover,we describe strategies to monitor the population of guide RNAs by high throughput sequencing（HTS）.We hope that this library will provide an invaluable resource for the community to screen loss of function mutations for cellular phenotypes,and as a source of guide RNA designs for future studies.展开更多
In order to investigate the influence of functional polymorphisms of macrophage migration inhibitory factor （M/F）, Fcg receptors CD16A （FCGR3A） and CD32A （FCGR2A） genes on susceptibility to pulmonary tuberculos...In order to investigate the influence of functional polymorphisms of macrophage migration inhibitory factor （M/F）, Fcg receptors CD16A （FCGR3A） and CD32A （FCGR2A） genes on susceptibility to pulmonary tuberculosis （PTB） in the Moroccan population, we analyzed 123 patients with PTB and 154 healthy controls. The genotyping for M/F-173 （G/C） （rs755622）, FCGR2A- 131H/R （rs 1801274） and FCGR3A-158V/F （rs396991） was carried out using TaqMan SNP Genotyping Assay method. We found a statistically significant in- crease of the MIF-173CC homozygote genotype and M/F-173＂C allele frequencies in PTB patients compared with healthy controls （17.07% versus 5.84%, P = 0.003; and 35.37% versus 26.30%, P = 0.02; respectively）. In contrast, no association was observed between FCGR2A-131H/R and FCGR3A-158V/F polymorphisms and tuberculosis disease. Our finding suggests that M/F-173℃ variant may play an important role in the development of active tuberculosis.展开更多
Human Myoblast Genome Therapy (HMGT) is a platform technology of cell transplantation, nuclear transfer, and tissue engineering. Unlike stem cells, myoblasts are differentiated, immature cells destined to become muscl...Human Myoblast Genome Therapy (HMGT) is a platform technology of cell transplantation, nuclear transfer, and tissue engineering. Unlike stem cells, myoblasts are differentiated, immature cells destined to become muscles. Myoblasts cultured from satellite cells of adult muscle biopsies survive, develop, and function to revitalize degenerative muscles upon transplantation. Injection injury activates regeneration of host myofibers that fuse with the engrafted myoblasts, sharing their nuclei in a common gene pool of the syncytium. Thus, through nuclear transfer and complementation, the normal human genome can be transferred into muscles of patients with genetic disorders to achieve phenotype repair or disease prevention. Myoblasts are safe and efficient gene transfer vehicles endogenous to muscles that constitute 50% of body weight. Results of over 280 HMGT procedures on Duchenne Muscular Dystrophy (DMD) subjects in the past 15 years demonstrated absolute safety. Myoblast-injected DMD muscles showed improved histology. Strength increase at 18 months post-operatively averaged 123%. In another application of HMGT on ischemic cardiomyopathy, the first human myoblast transfer into porcine myocardium revealed that it was safe and effective. Clinical trials on approximately 220 severe cardiomyopathy patients in 15 countries showed a <10% mortality. Most subjects received autologous cells implanted on the epicardial surface during coronory artery bypass graft, or injected on the endomyocardial surface percutaneously through guiding catheters. Significant increases in left ventricular ejection fraction, wall thickness, and wall motion have been reported, with reduction in perfusion defective areas, angina, and shortness of breath. As a new modality of treatment for disease in the skeletal muscle or myocardium, HMGT emerged as safe and effective. Large randomized multi-center trials are under way to confirm these preliminary results. The future of HMGT is bright and exciting.展开更多
The International Conference on Arginine and Pyrimidines （ICAP） had its origin as an arginine workshop organised by a group of microbiologists led by Werner Maas in 1972 （Rubio, 2003）. The shared requirement of ca...The International Conference on Arginine and Pyrimidines （ICAP） had its origin as an arginine workshop organised by a group of microbiologists led by Werner Maas in 1972 （Rubio, 2003）. The shared requirement of carbamoyl phosphate in both arginine and pyrimidine metabolism led to a natural association of researchers working in both fields.展开更多
＂Why do arginine and pyrimidines have to be considered together？＂ This was the question I asked when I was invited by Barbara Zimmermann to attend the 23rd Intemational Conference on Arginine and Pyrimidines （ICAP...＂Why do arginine and pyrimidines have to be considered together？＂ This was the question I asked when I was invited by Barbara Zimmermann to attend the 23rd Intemational Conference on Arginine and Pyrimidines （ICAP） three years ago. The meeting was held in the summer of 2012 at Bogota, Columbia, known as the ＂the Athens of South America＂. I am not alone in wanting to know more about the relationship between arginine and pyrimidines. Last summer, when ! was organising the next ICAP meeting at Oxford （Aughey et al., 2014）, one of my colleagues asked me, ＂Why such a weird name for a conference？＂ I am not sure if I had given her a satisfying answer and I was enthused to find out what the reasons may be.展开更多
Compartmentation of enzymes via filamentation has arisen as a mechanism for the regulation of metabolism.In 2010,three groups independently reported that CTP synthase(CTPS)can assemble into a filamentous structure ter...Compartmentation of enzymes via filamentation has arisen as a mechanism for the regulation of metabolism.In 2010,three groups independently reported that CTP synthase(CTPS)can assemble into a filamentous structure termed the cytoophidium.In searching for CTPS-interacting proteins,here we perform a yeast two-hybrid screening of Drosophila proteins and identify a putative CTPS-interacting protein,△~1-pyrroline-5-carboxylate synthase(P5CS).Using the Drosophila follicle cell as the in vivo model,we confirm that P5CS forms cytoophidia,which are associated with CTPS cytoophidia.Overexpression of P5CS increases the length of CTPS cytoophidia.Conversely,filamentation of CTPS affects the morphology of P5CS cytoophid ia.Finally,in vitro analyses confirm the filament-fo rming property of P5CS.Our work links CTPS with P5CS,two enzymes involved in the rate-limiting steps in pyrimidine and proline biosynthesis,respectively.展开更多
Developmental motifs in neurodegeneration:Neurodegeneration,the prominent feature of neurodegenerative disease,is characterized by the progressive and selective loss of neuronal function.As some of the pathologies cau...Developmental motifs in neurodegeneration:Neurodegeneration,the prominent feature of neurodegenerative disease,is characterized by the progressive and selective loss of neuronal function.As some of the pathologies caused by neurodegeneration may be irreversible,early intervention will be required for the treatments that aim to slow or halt the manifestation of these diseases.Traditionally,neurodegeneration evokes the idea of a progressive decline of brain function.展开更多
Compartmentation via filamentation has recently emerged as a novel mechanism for metabolic regulation. In order to identify filamentforming metabolic enzymes systematically, we performed a genome-wide screening of all...Compartmentation via filamentation has recently emerged as a novel mechanism for metabolic regulation. In order to identify filamentforming metabolic enzymes systematically, we performed a genome-wide screening of all strains available from an open reading frameGFP collection in Saccharomyces cerevisiae. We discovered nine novel filament-forming proteins and also confirmed those identified previously. From the 4159 strains, we found 23 proteins, mostly metabolic enzymes, which are capable of forming filaments in vivo. In silico protein-protein interaction analysis suggests that these filament-forming proteins can be clustered into several groups, including translational initiation machinery and glucose and nitrogen metabolic pathways. Using glutamine-utilising enzymes as examples, we found that the culture conditions affect the occurrence and length of the metabolic filaments. Furthermore, we found that two CTP synthases（Ura7p and Ura8p） and two asparagine synthetases（Asn1p and Asn2p） form filaments both in the cytoplasm and in the nucleus.Live imaging analyses suggest that metabolic filaments undergo sub-diffusion. Taken together, our genome-wide screening identifies additional filament-forming proteins in S. cerevisiae and suggests that filamentation of metabolic enzymes is more general than currently appreciated.展开更多
The ability of human deciduous tooth dental pulp cells（HDDPCs） to differentiate into odontoblasts that generate mineralized tissue holds immense potential for therapeutic use in the field of tooth regenerative medic...The ability of human deciduous tooth dental pulp cells（HDDPCs） to differentiate into odontoblasts that generate mineralized tissue holds immense potential for therapeutic use in the field of tooth regenerative medicine. Realization of this potential depends on efficient and optimized protocols for the genetic manipulation of HDDPCs. In this study, we demonstrate the use of a Piggy Bac（PB）-based gene transfer system as a method for introducing nonviral transposon DNA into HDDPCs and HDDPC-derived inducible pluripotent stem cells. The transfection efficiency of the PB-based system was significantly greater than previously reported for electroporation-based transfection of plasmid DNA. Using the neomycin resistance gene as a selection marker, HDDPCs were stably transfected at a rate nearly 40-fold higher than that achieved using conventional methods. Using this system, it was also possible to introduce two constructs simultaneously into a single cell. The resulting stable transfectants, expressing td Tomato and enhanced green fluorescent protein, exhibited both red and green fluorescence. The established cell line did not lose the acquired phenotype over three months of culture. Based on our results, we concluded that PB is superior to currently available methods for introducing plasmid DNA into HDDPCs. There may be significant challenges in the direct clinical application of this method for human dental tissue engineering due to safety risks and ethical concerns. However, the high level of transfection achieved with PB may have significant advantages in basic scientific research for dental tissue engineering applications, such as functional studies of genes and proteins. Furthermore, it is a useful tool for the isolation of genetically engineered HDDPC-derived stem cells for studies in tooth regenerative medicine.展开更多
AIM:To elucidate the influence of quasispecies on virological response and disease severity in patients with chronic hepatitis C. METHODS:Forty seven patients with hepatitis C [32 with chronic active hepatitis (CAH), ...AIM:To elucidate the influence of quasispecies on virological response and disease severity in patients with chronic hepatitis C. METHODS:Forty seven patients with hepatitis C [32 with chronic active hepatitis (CAH), 9 with cirrhosis, and 6 with hepatocellular carcinoma (HCC)] were screened for the presence of quasispecies by single stranded conformational polymorphism (SSCP) analysis in the hypervariable region (HVR) and non-structural 5B (NS5B) viral genes of hepatitis C virus. The 41 patients excluding those with HCC were on therapy and followed up for a year with the determination of virological response and disease severity. Virus isolated from twenty three randomly selected patients (11 non-responders and 12 showing a sustained virological response) was sequenced for the assessment of mutations. RESULTS:The occurrence of quasispecies was proportionately higher in patients with HCC and cirrhosis than in those with CAH, revealing a significant correlation between the molecular evolution of quasispecies and the severity of disease in patients with hepatitis C. The occurrence of complex quasispecies has a significant association (P < 0.05) with the non-responders, and leads to persistence of infection. Significant differences (P < 0.05) in viral load (log10 IU/mL) were observed among patients infected with complex quasispecies (CQS), those infected with simple quasispecies (SQS) and those with no quasispecies (NQS), after 12 wk (CQS-5.2 ± 2.3, SQS-3.2 ± 1.9, NQS-2.8 ± 2.4) and 24 wk (CQS-3.9 ± 2.2, SQS-3.0 ± 2.2, NQS-2.1 ± 2.3) in the HVR region. However, a statistically significant difference (P < 0.05) was observed between the viral loads of patients infected with CQS and those infected with NQS in NS5B viral gene after 24 wk (CQS-3.9 ± 2.2, SQS-3.0 ± 2.2, and NQS-2.1 ± 2.3) and 48 wk (CQS-3.1 ± 2.7, SQS-2.3 ± 2.4, NQS-2.0 ± 2.3) of therapy. Disease severity was significantly associated with viral load during therapy. The strains isolated from non-responders showed close pairing on phylogeny based on the NS5B gene, but dissimilar HVR regions. This revealed the possibility of the selection of resistant strains during the evolution of quasispecies in NS5B. CONCLUSION:Viral quasispecies may be an important predictor of virological responses to combination therapy in patients with chronic hepatitis C. Complex quasispecies and resistant strains may lead to high viral loads during therapy, with a concerted effect on disease severity.展开更多
Dear editor:Intraductalcarcinomaoftheprostate(IDC-P)ischaracterized by expansive growth of cancer cells in normal prostatic ducts with basal cell layer and associated with high grade invasive prostate cancer(PCa).Howe...Dear editor:Intraductalcarcinomaoftheprostate(IDC-P)ischaracterized by expansive growth of cancer cells in normal prostatic ducts with basal cell layer and associated with high grade invasive prostate cancer(PCa).However,the molecular profile or clinical character of IDC-P in progressive castration-resistant PCa(CRPC)was not fully characterized yet.展开更多
Dear Editor,Spinal and bulbar muscular atrophy(SBMA)or Kennedy’s disease is a rare X-linked,recessive,lower motor neuron disease caused by a CAG repeat expansion within the first exon of the androgen receptor(AR)gene...Dear Editor,Spinal and bulbar muscular atrophy(SBMA)or Kennedy’s disease is a rare X-linked,recessive,lower motor neuron disease caused by a CAG repeat expansion within the first exon of the androgen receptor(AR)gene.Instability of the CAG-triplet repeat impacts AR function;therefore,men with SBMA are thought to be at a very low risk of prostate cancer.We previously reported a case of high-risk prostate cancer with SBMA(repeat length 46).展开更多
Cleistogamy involves the shedding of pollen within an enclosed flower. In barley, this trait is determined by the presence of a recessive allele at the gene Clyl, a member of the AP2 gene family. Here we show that the...Cleistogamy involves the shedding of pollen within an enclosed flower. In barley, this trait is determined by the presence of a recessive allele at the gene Clyl, a member of the AP2 gene family. Here we show that the Clyl ortholog in einkorn （diploid） wheat （Triticum monococcum） TmAP2 shares a similar structure and identical pattern of transcription as Clyl. The transcript abundance of TmAP2 was high in the spike around the time of anthesis, but low in the leaf, plumule and radicle. The TmAP2 transcript was cleaved at its miR172 target site. Flower gaping at anthesis in einkorn wheat is induced by the expansion of the lodicules.展开更多
The classical linear filter is able to extract components from multi-component stochastic processes where the frequencies of components do not overlap over time, but fail for those processes where the frequencies over...The classical linear filter is able to extract components from multi-component stochastic processes where the frequencies of components do not overlap over time, but fail for those processes where the frequencies overlap over time. In this paper, we discuss two filtering methods for non-stationary processes: the G-filtering method and the Fractional Fourier transform (FrFT) method. The FrFT method is mainly designed for linear chirp signals where the frequency is linearly changing with time. The G-filter can be used to filter signals with wide range of frequency behaviors such as linear chirps, quadratic chirps and other type of chirp signals with strong time-varying frequency behavior. If frequencies of the components can be approximated or separated by a straight line or a polynomial curve, the G-filter can successfully extract components from the original series. We show that the G-filter is applicable to a wider variety of filtering applications than methods such as the FrFT which require data of a specified frequency behavior.展开更多
文摘Background:Pistacia integerrima,a cornerstone of traditional medicine,is renowned for its therapeutic applications against various health conditions,including cancer and hepatitis.This study investigates the pharmacological potential of bioactive compounds derived from Pistacia integerrima in inhibiting 5-lipoxygenase(5-LOX),a key enzyme implicated in inflammation and cancer progression.The current study aimed to evaluate the lipoxygenase inhibitory activity of bioactive compounds from Pistacia integerrima and assess their potential for therapeutic development in the context of inflammation and cancer treatment.Methods:Three major compounds-spinacetin(1),patuletin(2),and pistagremic acid(3)-were isolated from Pistacia integerrima and analyzed for their lipoxygenase inhibitory activity.Biochemical assays and molecular docking studies were performed to assess their effectiveness in inhibiting 5-LOX.Results:All three compounds demonstrated significant inhibition of lipoxygenase activity.Spinacetin(1)and patuletin(2)exhibited the most potent inhibitory effects,with IC_(50)values of 40.34μM and 45.04μM,respectively.Molecular docking studies revealed that patuletin(2)had the highest binding affinity(−7.717 kcal/mol)against 5-LOX,followed by spinacetin(1)with a binding affinity of−6.074 kcal/mol.In-depth in silico analysis highlighted the drug-likeness of spinacetin(1)and its favorable toxicological profile,suggesting its suitability for therapeutic development.Conclusion:The study demonstrates that compounds from Pistacia integerrima,particularly spinacetin and patuletin,have significant lipoxygenase inhibitory activity,with spinacetin showing promise as a lead candidate for lipoxygenase-targeted therapies.The findings reinforce the therapeutic relevance of Pistacia integerrima and suggest that its bioactive compounds may serve as safer,plant-based alternatives to conventional anti-inflammatory and anticancer treatments.
基金supported by the UK Medical Research Council and the European Research Council (DARCGENs, No. 249869)
文摘Recent advances in our ability to design DNA binding factors with specificity for desired sequences have resulted in a revolution in genetic engineering, enabling directed changes to the genome to be made relatively easily. Traditional techniques for generating genetic mutations in most organisms have relied on selection from large pools of randomly induced mutations for those of particular interest, or time-consuming gene targeting by homologous recombination. Drosophila melanogaster has always been at the forefront of genetic analysis, and application of these new genome editing techniques to this organism will revolutionise our approach to performing analysis of gene function in the future. We discuss the recent techniques that apply the CRISPR/Cas9 system to Drosophila, highlight potential uses for this technology and speculate upon the future of genome engineering in this model organism.
基金the TRiP at Harvard Medical School(NIH/ NIGMS R01-GM084947)
文摘CTP synthase is compartmentalized within a subcellular structure,termed the cytoophidium,in a range of organisms including bacteria, yeast,fruit fly and rat.Here we show that CTP synthase is also compartmentalized into cytoophidia in human cells.Surprisingly,the occurrence of cytoophidia in human cells increases upon treatment with a glutamine analog 6-diazo-5-oxo-L-norleucine（DON）,an inhibitor of glutamine-dependent enzymes including CTP synthase.Experiments in flies confirmed that DON globally promotes cytoophidium assembly.Clonal analysis via CTP synthase RNA interference in somatic cells indicates that CTP synthase expression level is critical for the formation of cytoophidia.Moreover,DON facilitates cytoophidium assembly even when CTP synthase level is low.A second glutamine analog azaserine also promotes cytoophidum formation.Our data demonstrate that glutamine analogs serve as useful tools in the study of cytoophidia.
文摘CTP synthase （CTPsyn） is a metabolic enzyme responsible for the de novo synthesis of the nucleotide CTE Several recent studies have shown that CTPsyn forms filamentous subcellular structures known as cytoophidia in bacteria, yeast, fruit flies and humans. However, it remains elusive whether and how CTPsyn and cytoophidia play a role during development. Here, we show that cytoophidia are abundant in the neuroepithelial stem cells in Drosophila optic lobes. Optic lobes are underdeveloped in CTPsyn mutants as well as in CTPsyn RNAi. Moreover, overexpressing CTPsyn impairs the development of optic lobes, specifically by blocking the transition from neuro- epithelium to neuroblast. Taken together, our results indicate that CTPsyn is critical for optic lobe homeostasis in Drosophila.
基金supported by the UK Medical Research Council(Grant No.MC_UU_12021/3 and MC_U137788471)University of OxfordShanghaiTech University
文摘The metabolic enzyme CTP synthase(CTPS) is able to compartmentalize into filaments,termed cytoophidia,in a variety of organisms including bacteria,budding yeast,fission yeast,fruit flies and mammals.A previous study in budding yeast shows that the filament-forming process of CTPS is not sensitive to temperature shift.Here we study CTPS filamentation in the fission yeast Schizosaccharomyces pombe.To our surprise,we find that both the length and the occurrence of cytoophidia in S.pombe decrease upon cold shock or heat shock.The temperature-dependent changes of cytoophidia are fast and reversible.Taking advantage of yeast genetics,we demonstrate that heat-shock proteins are required for cytoophidium assembly in S.pombe.Temperature sensitivity of cytoophidia makes S.pombe an attractive model system for future investigations of this novel membraneless organelle.
基金supported by Shanghai Tech University, the National Natural Science Foundation of China (81500266)the UK Medical Research Council (MC_UU_12021/3 and MC_U137788471)
文摘CTP synthase(CTPS), the rate-limiting enzyme in de novo CTP biosynthesis, has been demonstrated to assemble into evolutionarily conserved filamentous structures, termed cytoophidia, in Drosophila, bacteria, yeast and mammalian cells. However, the regulation and function of the cytoophidium remain elusive. Here, we provide evidence that the mechanistic target of rapamycin(mTOR) pathway controls cytoophidium assembly in mammalian and Drosophila cells. In mammalian cells, we find that inhibition of mTOR pathway attenuates cytoophidium formation. Moreover, CTPS cytoophidium assembly appears to be dependent on the mTOR complex 1(mTORC1) mainly. In addition, knockdown of the mTORC1 downstream target S6 K1 can inhibit cytoophidium formation, while overexpression of the constitutively active S6 K1 reverses mTOR knockdown-induced cytoophidium disassembly. Finally, reducing m TOR protein expression results in a decrease of the length of cytoophidium in Drosophila follicle cells.Therefore, our study connects CTPS cytoophidium formation with the mTOR signaling pathway.
基金the European Research Council(DARCGENs,project number 249869)the Medical Research Council for financial support
文摘The simplicity of the CRISPR/Cas9 system of genome engineering has opened up the possibility of performing genome-wide targeted mutagenesis in cell lines,enabling screening for cellular phenotypes resulting from genetic aberrations.Drosophila cells have proven to be highly effective in identifying genes involved in cellular processes through similar screens using partial knockdown by RNAi.This is in part due to the lower degree of redundancy between genes in this organism,whilst still maintaining highly conserved gene networks and orthologs of many human disease-causing genes.The ability of CRISPR to generate genetic loss of function mutations not only increases the magnitude of any effect over currently employed RNAi techniques,but allows analysis over longer periods of time which can be critical for certain phenotypes.In this study,we have designed and built a genome-wide CRISPR library covering 13,501 genes,among which 8989 genes are targeted by three or more independent single guide RNAs（sg RNAs）.Moreover,we describe strategies to monitor the population of guide RNAs by high throughput sequencing（HTS）.We hope that this library will provide an invaluable resource for the community to screen loss of function mutations for cellular phenotypes,and as a source of guide RNA designs for future studies.
基金supported in part by the grants SAF06-00398/CTS1880,Spainin another part by the National Institute of Hygiene,Rabat,Morocco
文摘In order to investigate the influence of functional polymorphisms of macrophage migration inhibitory factor （M/F）, Fcg receptors CD16A （FCGR3A） and CD32A （FCGR2A） genes on susceptibility to pulmonary tuberculosis （PTB） in the Moroccan population, we analyzed 123 patients with PTB and 154 healthy controls. The genotyping for M/F-173 （G/C） （rs755622）, FCGR2A- 131H/R （rs 1801274） and FCGR3A-158V/F （rs396991） was carried out using TaqMan SNP Genotyping Assay method. We found a statistically significant in- crease of the MIF-173CC homozygote genotype and M/F-173＂C allele frequencies in PTB patients compared with healthy controls （17.07% versus 5.84%, P = 0.003; and 35.37% versus 26.30%, P = 0.02; respectively）. In contrast, no association was observed between FCGR2A-131H/R and FCGR3A-158V/F polymorphisms and tuberculosis disease. Our finding suggests that M/F-173℃ variant may play an important role in the development of active tuberculosis.
文摘Human Myoblast Genome Therapy (HMGT) is a platform technology of cell transplantation, nuclear transfer, and tissue engineering. Unlike stem cells, myoblasts are differentiated, immature cells destined to become muscles. Myoblasts cultured from satellite cells of adult muscle biopsies survive, develop, and function to revitalize degenerative muscles upon transplantation. Injection injury activates regeneration of host myofibers that fuse with the engrafted myoblasts, sharing their nuclei in a common gene pool of the syncytium. Thus, through nuclear transfer and complementation, the normal human genome can be transferred into muscles of patients with genetic disorders to achieve phenotype repair or disease prevention. Myoblasts are safe and efficient gene transfer vehicles endogenous to muscles that constitute 50% of body weight. Results of over 280 HMGT procedures on Duchenne Muscular Dystrophy (DMD) subjects in the past 15 years demonstrated absolute safety. Myoblast-injected DMD muscles showed improved histology. Strength increase at 18 months post-operatively averaged 123%. In another application of HMGT on ischemic cardiomyopathy, the first human myoblast transfer into porcine myocardium revealed that it was safe and effective. Clinical trials on approximately 220 severe cardiomyopathy patients in 15 countries showed a <10% mortality. Most subjects received autologous cells implanted on the epicardial surface during coronory artery bypass graft, or injected on the endomyocardial surface percutaneously through guiding catheters. Significant increases in left ventricular ejection fraction, wall thickness, and wall motion have been reported, with reduction in perfusion defective areas, angina, and shortness of breath. As a new modality of treatment for disease in the skeletal muscle or myocardium, HMGT emerged as safe and effective. Large randomized multi-center trials are under way to confirm these preliminary results. The future of HMGT is bright and exciting.
基金sponsored by MRC Functional Genomics Unit at the University of Oxford, the Journal of Genetics and Genomics, and Dutscher Scientific
文摘The International Conference on Arginine and Pyrimidines （ICAP） had its origin as an arginine workshop organised by a group of microbiologists led by Werner Maas in 1972 （Rubio, 2003）. The shared requirement of carbamoyl phosphate in both arginine and pyrimidine metabolism led to a natural association of researchers working in both fields.
文摘＂Why do arginine and pyrimidines have to be considered together？＂ This was the question I asked when I was invited by Barbara Zimmermann to attend the 23rd Intemational Conference on Arginine and Pyrimidines （ICAP） three years ago. The meeting was held in the summer of 2012 at Bogota, Columbia, known as the ＂the Athens of South America＂. I am not alone in wanting to know more about the relationship between arginine and pyrimidines. Last summer, when ! was organising the next ICAP meeting at Oxford （Aughey et al., 2014）, one of my colleagues asked me, ＂Why such a weird name for a conference？＂ I am not sure if I had given her a satisfying answer and I was enthused to find out what the reasons may be.
基金supported by ShanghaiTech University,the UK Medical Research Council(Grant No.MC_UU_12021/3 and MC_U137788471)National Natural Science Foundation of China(Grant No.31771490)。
文摘Compartmentation of enzymes via filamentation has arisen as a mechanism for the regulation of metabolism.In 2010,three groups independently reported that CTP synthase(CTPS)can assemble into a filamentous structure termed the cytoophidium.In searching for CTPS-interacting proteins,here we perform a yeast two-hybrid screening of Drosophila proteins and identify a putative CTPS-interacting protein,△~1-pyrroline-5-carboxylate synthase(P5CS).Using the Drosophila follicle cell as the in vivo model,we confirm that P5CS forms cytoophidia,which are associated with CTPS cytoophidia.Overexpression of P5CS increases the length of CTPS cytoophidia.Conversely,filamentation of CTPS affects the morphology of P5CS cytoophid ia.Finally,in vitro analyses confirm the filament-fo rming property of P5CS.Our work links CTPS with P5CS,two enzymes involved in the rate-limiting steps in pyrimidine and proline biosynthesis,respectively.
文摘Developmental motifs in neurodegeneration:Neurodegeneration,the prominent feature of neurodegenerative disease,is characterized by the progressive and selective loss of neuronal function.As some of the pathologies caused by neurodegeneration may be irreversible,early intervention will be required for the treatments that aim to slow or halt the manifestation of these diseases.Traditionally,neurodegeneration evokes the idea of a progressive decline of brain function.
基金supported by the UK Medical Research Council (to J.L.L.), China Scholarship Council-University of Oxford Scholarship (to Q.J.S), Chinese Scholarship Council Studentship (to Y.H.), Malaysia Government Scholarship (to H.K.), the National Natural Science Foundation of China (No. 11304372) (to H.L., F. Y and P.Y.W.) and anonymous donation (to J.L.L.)
文摘Compartmentation via filamentation has recently emerged as a novel mechanism for metabolic regulation. In order to identify filamentforming metabolic enzymes systematically, we performed a genome-wide screening of all strains available from an open reading frameGFP collection in Saccharomyces cerevisiae. We discovered nine novel filament-forming proteins and also confirmed those identified previously. From the 4159 strains, we found 23 proteins, mostly metabolic enzymes, which are capable of forming filaments in vivo. In silico protein-protein interaction analysis suggests that these filament-forming proteins can be clustered into several groups, including translational initiation machinery and glucose and nitrogen metabolic pathways. Using glutamine-utilising enzymes as examples, we found that the culture conditions affect the occurrence and length of the metabolic filaments. Furthermore, we found that two CTP synthases（Ura7p and Ura8p） and two asparagine synthetases（Asn1p and Asn2p） form filaments both in the cytoplasm and in the nucleus.Live imaging analyses suggest that metabolic filaments undergo sub-diffusion. Taken together, our genome-wide screening identifies additional filament-forming proteins in S. cerevisiae and suggests that filamentation of metabolic enzymes is more general than currently appreciated.
基金supported in part by a Grant-in-Aid for Scientific Research (C) (grant no. 25463192) from the Ministry of Education, Science, Sports, Culture, and Technology of Japan
文摘The ability of human deciduous tooth dental pulp cells（HDDPCs） to differentiate into odontoblasts that generate mineralized tissue holds immense potential for therapeutic use in the field of tooth regenerative medicine. Realization of this potential depends on efficient and optimized protocols for the genetic manipulation of HDDPCs. In this study, we demonstrate the use of a Piggy Bac（PB）-based gene transfer system as a method for introducing nonviral transposon DNA into HDDPCs and HDDPC-derived inducible pluripotent stem cells. The transfection efficiency of the PB-based system was significantly greater than previously reported for electroporation-based transfection of plasmid DNA. Using the neomycin resistance gene as a selection marker, HDDPCs were stably transfected at a rate nearly 40-fold higher than that achieved using conventional methods. Using this system, it was also possible to introduce two constructs simultaneously into a single cell. The resulting stable transfectants, expressing td Tomato and enhanced green fluorescent protein, exhibited both red and green fluorescence. The established cell line did not lose the acquired phenotype over three months of culture. Based on our results, we concluded that PB is superior to currently available methods for introducing plasmid DNA into HDDPCs. There may be significant challenges in the direct clinical application of this method for human dental tissue engineering due to safety risks and ethical concerns. However, the high level of transfection achieved with PB may have significant advantages in basic scientific research for dental tissue engineering applications, such as functional studies of genes and proteins. Furthermore, it is a useful tool for the isolation of genetically engineered HDDPC-derived stem cells for studies in tooth regenerative medicine.
基金Indian Council of Medical Research No. 485/2003/ECD-I, New Delhi, India
文摘AIM:To elucidate the influence of quasispecies on virological response and disease severity in patients with chronic hepatitis C. METHODS:Forty seven patients with hepatitis C [32 with chronic active hepatitis (CAH), 9 with cirrhosis, and 6 with hepatocellular carcinoma (HCC)] were screened for the presence of quasispecies by single stranded conformational polymorphism (SSCP) analysis in the hypervariable region (HVR) and non-structural 5B (NS5B) viral genes of hepatitis C virus. The 41 patients excluding those with HCC were on therapy and followed up for a year with the determination of virological response and disease severity. Virus isolated from twenty three randomly selected patients (11 non-responders and 12 showing a sustained virological response) was sequenced for the assessment of mutations. RESULTS:The occurrence of quasispecies was proportionately higher in patients with HCC and cirrhosis than in those with CAH, revealing a significant correlation between the molecular evolution of quasispecies and the severity of disease in patients with hepatitis C. The occurrence of complex quasispecies has a significant association (P < 0.05) with the non-responders, and leads to persistence of infection. Significant differences (P < 0.05) in viral load (log10 IU/mL) were observed among patients infected with complex quasispecies (CQS), those infected with simple quasispecies (SQS) and those with no quasispecies (NQS), after 12 wk (CQS-5.2 ± 2.3, SQS-3.2 ± 1.9, NQS-2.8 ± 2.4) and 24 wk (CQS-3.9 ± 2.2, SQS-3.0 ± 2.2, NQS-2.1 ± 2.3) in the HVR region. However, a statistically significant difference (P < 0.05) was observed between the viral loads of patients infected with CQS and those infected with NQS in NS5B viral gene after 24 wk (CQS-3.9 ± 2.2, SQS-3.0 ± 2.2, and NQS-2.1 ± 2.3) and 48 wk (CQS-3.1 ± 2.7, SQS-2.3 ± 2.4, NQS-2.0 ± 2.3) of therapy. Disease severity was significantly associated with viral load during therapy. The strains isolated from non-responders showed close pairing on phylogeny based on the NS5B gene, but dissimilar HVR regions. This revealed the possibility of the selection of resistant strains during the evolution of quasispecies in NS5B. CONCLUSION:Viral quasispecies may be an important predictor of virological responses to combination therapy in patients with chronic hepatitis C. Complex quasispecies and resistant strains may lead to high viral loads during therapy, with a concerted effect on disease severity.
基金This study was supported in part by Grants-in-Aid for Sci-entific Research(#17K16813 to Hongo H and#17K11158 E.20H03817 to Kosaka T)from the Ministry of Education,Culture,Sports,Science,and Technology of JapanThe study was supported in part by research grant to Kosaka T from the Takeda Science Foundation,Japan.
文摘Dear editor:Intraductalcarcinomaoftheprostate(IDC-P)ischaracterized by expansive growth of cancer cells in normal prostatic ducts with basal cell layer and associated with high grade invasive prostate cancer(PCa).However,the molecular profile or clinical character of IDC-P in progressive castration-resistant PCa(CRPC)was not fully characterized yet.
基金Yoko Suzuki provided technical assistance.This study was supported in part by Grants-in-Aid for Scientific Research(#17K16813 to H Hongo and#17K11158 and#20H03817 to TK)from the Ministry of Education,Culture,Sports,Science,and Technology of JapanThe study was supported in part by research Grant to TK from the Takeda Science Foundation,Japan。
文摘Dear Editor,Spinal and bulbar muscular atrophy(SBMA)or Kennedy’s disease is a rare X-linked,recessive,lower motor neuron disease caused by a CAG repeat expansion within the first exon of the androgen receptor(AR)gene.Instability of the CAG-triplet repeat impacts AR function;therefore,men with SBMA are thought to be at a very low risk of prostate cancer.We previously reported a case of high-risk prostate cancer with SBMA(repeat length 46).
基金funded by the Japanese Ministry of Agriculture,Forestry and Fisheries (Genomics for Agricultural Innovation Grants No.TRG1004) to T.K.S.N.appreciates the award of a Japanese Government (Monbukagakusho:MEXT) scholarship
文摘Cleistogamy involves the shedding of pollen within an enclosed flower. In barley, this trait is determined by the presence of a recessive allele at the gene Clyl, a member of the AP2 gene family. Here we show that the Clyl ortholog in einkorn （diploid） wheat （Triticum monococcum） TmAP2 shares a similar structure and identical pattern of transcription as Clyl. The transcript abundance of TmAP2 was high in the spike around the time of anthesis, but low in the leaf, plumule and radicle. The TmAP2 transcript was cleaved at its miR172 target site. Flower gaping at anthesis in einkorn wheat is induced by the expansion of the lodicules.
文摘The classical linear filter is able to extract components from multi-component stochastic processes where the frequencies of components do not overlap over time, but fail for those processes where the frequencies overlap over time. In this paper, we discuss two filtering methods for non-stationary processes: the G-filtering method and the Fractional Fourier transform (FrFT) method. The FrFT method is mainly designed for linear chirp signals where the frequency is linearly changing with time. The G-filter can be used to filter signals with wide range of frequency behaviors such as linear chirps, quadratic chirps and other type of chirp signals with strong time-varying frequency behavior. If frequencies of the components can be approximated or separated by a straight line or a polynomial curve, the G-filter can successfully extract components from the original series. We show that the G-filter is applicable to a wider variety of filtering applications than methods such as the FrFT which require data of a specified frequency behavior.
