Human myoblast genome therapy

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.

Neurodevelopmental defects as a primer of neurodegeneration:lessons from spinal muscular atrophy and Huntington's disease

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.

PiggyBac transposon-mediated gene delivery efficiently generates stable transfectants derived from cultured primary human deciduous tooth dental pulp cells(HDDPCs) and HDDPC-derived iPS cells

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.

Influence of quasispecies on virological responses and disease severity in patients with chronic hepatitis C

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.

The first Japanese case of intraductal cancer of the prostate with checkpoint kinase 2 mutation

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.

Identification of an AP2 gene related to open flowering in diploid wheat(Triticum monococcum)

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.

Comparing the Time-Deformation Method with the Fractional Fourier Transform in Filtering Non-Stationary Processes

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.

A simplified protocol for the semi-large scale recovery of plasmids from <i>Escherichia coli</i>grown on agar plates

Semi-large scale liquid cultivation of transformed Escherichia coli (E. coli) in medium (100-200 ml) has been widely used for the acquisition of relatively large amounts of plasmid DNA (50-300 μg). However, this method requires an expensive high-speed centrifugation apparatus to precipitate E. coli before lysis, which is both laborious and time-consuming. Here, we demonstrate a method for agar plate-based cultivation of bacteria that does not employ a high-speed centrifugation apparatus. This procedure proves to be simple and reproducible, yielding an average of 82 μg of plasmid DNA per experiment. It may therefore be valuable for cloning/transfection experiments under limited financial backgrounds.

CRISPR/Cas9 and Genome Editing in Drosophila

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.

Glutamine analogs promote cytoophidium assembly in human and Drosophila cells

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.

CTP Synthase Is Required for Optic Lobe Homeostasis in Drosophila

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.

Temperature-sensitive cytoophidium assembly in Schizosaccharomyces pombe

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.

mTOR-S6K1 pathway mediates cytoophidium assembly

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.

A Genome-Wide CRISPR Library for High-Throughput Genetic Screening in Drosophila Cells

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.

Analysis of MIF,FCGR2A and FCGR3A gene polymorphisms with susceptibility to pulmonary tuberculosis in Moroccan population

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.

Evolution of Invertebrate Deuterostomes and Hox/ParaHox Genes

Transcription factors encoded by Antennapedia-class homeobox genes play crucial roles in controlling development of animals, and are often found clustered in animal genomes. The Hox and ParaHox gene clusters have been regarded as evolutionary sisters and evolved from a putative common ancestral gene complex, the ProtoHox cluster, prior to the divergence of the Cnidaria and Bilateria （bilaterally symmetrical animals）. The Deuterostomia is a monophyletic group of animals that belongs to the Bilateria, and a sister group to the Protostomia. The deuterostomes include the vertebrates （to which we belong）, invertebrate chordates, hemichordates, echinoderms and possibly xenoturbellids, as well as acoelomorphs. The studies of Hox and ParaHox genes provide insights into the origin and subsequent evolution of the bilaterian animals. Recently, it becomes apparent that among the Hox and ParaHox genes, there are significant variations in organization on the chromosome, expression pattern, and function. In this review, focusing on invertebrate deuterostomes, I first summarize recent findings about Hox and ParaHox genes. Next, citing unsolved issues, I try to provide clues that might allow us to reconstruct the common ancestor of deuterostomes, as well as understand the roles of Hox and ParaHox genes in the development and evolution of deuterostomes.

A Single JAZ Repressor Controls the Jasmonate Pathway in Marchantia polymorpha

JAZ proteins are negative regulators of jasm onate responses,acting both as repressors of transcription factors and as co-receptors of JA-lle. The high redundancy of JAZ genes in angiosperms has hindered the characterization of a complete depletion of JAZ function. Moreover, the recent discovery that dn- OPDA is the jasmonate ligand in Marchantia polymorpha demonstrates that JA-lle is not the sole COI1/ JAZ ligand in land plants and highlights the importance of studying JAZ co-receptors in bryophytes? Here, we have exploited the low gene redundancy of the liverwort M. polymorpha to characterize the single MpJ4Z in this early diverging plant lineage. We clarify the phylogenetic history of the TIFY family, demonstrate that MpJAZ is the ortholog of AtJAZ with a conserved function, and characterize its repressor activity of dn-OPDA resp on ses. Our results show that, con sistent with previous findings in Arabidopsis, MpJAZ represses jasmonates biosynthesis, senescence, and plant defenses, and promotes cell growth and reproductive fitness, highlighting the power of studies in Marchantia.

Gene expression analysis in the larval silk gland of the eri silkworm Samia ricini

Insects produce silk for a range of purposes. In the Lepidoptera, silk is utilized as a material for cocoon production and serves to protect larvae from adverse environmental conditions or predators. Species in the Saturniidae family produce an especially wide variety of cocoons, for example, large, golden colored cocoons and those with many small holes. Although gene expression in the silk gland of the domestic silkworm （Bombyx mori L.） has been extensively studied, considerably fewer investigations have focused on members of the saturniid family. Here, we established expression sequence tags from the silk gland of the eri silkworm （Samia ricini）, a saturniid species, and used these to analyze gene expression. Although we identified thefibroin heavy chain gene in the established library, genes for other major silk proteins, such asfibroin light chain andfibrohexamerin, were absent. This finding is consistent with previous reports that these latter proteins are lacking in saturniid silk. Recently, a series offibrohexamerin-like genes were identified in the Bombyx genome. We used this information to conduct a detailed analysis of the library established here. This analysis identified putative homologues of these genes. We also found several genes encoding small silk protein molecules that are also present in the silk of other Lepidoptera. Gene expression patterns were compared between eri and domestic silkworm, and both conserved and nonconserved expression patterns were identified for the tested genes. Such differential gene expression might be one of the major causes of the differences in silk properties between these species. We believe that our study can be of value as a basic catalogue for silk gland gene expression, which will yield to the further understanding of silk evolution.

Arabidopsis SWC4 Binds DNA and Recruits the SWR1 Complex to Modulate Histone H2A.Z Deposition at Key Regulatory Genes

Deposition of the H2A.Z histone variant by the SWR1 complex （SWRI-C） in regulatory regions of specific loci modulates transcription. Characterization of mutations in Arabidopsis thaliana homologs of yeast SWRI-C has revealed a role for H2A.Z exchange in a variety of developmental processes. Nevertheless, the exact composition of plant SWRI-C and how it is recruited to target genes remains to be established. Here we show that SWC4, the Arabidopsis homolog of yeast SANT domain protein Swc4/Eaf2, is a DNA-binding protein that interacts with SWR1-C subunits. We demonstrate that the swc4-1 knockout mutant is embryo- lethal, while SWC4 RNAi knockdown lines display pleiotropic phenotypic alterations in vegetative and repro- ductive traits, including acceleration of flowering time, indicating that SWC4 controls post-embryonic processes. Transcriptomic analyses and genome-wide profiling of H2A.Z indicate that SWC4 represses tran- scription of a number of genes, including the floral integrator FT and key transcription factors, mainly by modulating H2A.Z deposition. Interestingly, SWC4 silencing does not affect H2A.Z deposition at the FLC locus nor expression of this gene, a master regulator of flowering previously shown to be controlled by SWR1-C. Importantly, we find that SWC4 recognizes specific AT-rich DNA elements in the chromatin regions of target genes and that SWC4 silencing impairs SWRI-C binding at FT. Collectively, our data suggest that SWC4 regulates plant growth and development by aiding SWR1-C recruitment and modulating H2A.Z deposition.

Cellular Serpents and Dreaming Spires:New Frontiers in Arginine and Pyrimidine Biology

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.