参考文献

Electrospun poly-l-lactic acid (PLLA) nanofiber mats carrying surface amine groups,previously introduced by nitrogen atmospheric pressure nonequilibrium plasma,are embedded into aqueous solutions of oligomeric acrylamide-end capped AGMA1,a biocompatible polyamidoamine with arg-gly-asp (RGD)-reminiscent repeating units. The resultant mixture is finally cured giving PLLA-AGMA1 hydrogel composites that absorb large amounts of water and,in the swollen state,are translucent,soft,and pliable,yet as strong as the parent PLLA mat. They do not split apart from each other when swollen in water and remain highly flexible and resistant,since the hydrogel portion is covalently grafted onto the PLLA nanofibers via the addition reaction of the surface amine groups to a part of the terminal acrylic double bonds of AGMA1 oligomers. Preliminary tested as scaffolds,the composites prove capable of maintaining short-term undifferentiated cultures of human pluripotent stem cells in feeder-free conditions. View Publication

Mitochondrial DNA (mtDNA) mutations are maternally inherited and are associated with a broad range of debilitating and fatal diseases. Reproductive technologies designed to uncouple the inheritance of mtDNA from nuclear DNA may enable affected women to have a genetically related child with a greatly reduced risk of mtDNA disease. Here we report the first preclinical studies on pronuclear transplantation (PNT). Surprisingly,techniques used in proof-of-concept studies involving abnormally fertilized human zygotes were not well tolerated by normally fertilized zygotes. We have therefore developed an alternative approach based on transplanting pronuclei shortly after completion of meiosis rather than shortly before the first mitotic division. This promotes efficient development to the blastocyst stage with no detectable effect on aneuploidy or gene expression. After optimization,mtDNA carryover was reduced to textless2% in the majority (79%) of PNT blastocysts. The importance of reducing carryover to the lowest possible levels is highlighted by a progressive increase in heteroplasmy in a stem cell line derived from a PNT blastocyst with 4% mtDNA carryover. We conclude that PNT has the potential to reduce the risk of mtDNA disease,but it may not guarantee prevention. View Publication

Th2-eosinophil immune responses are well known for mediating host defence against helminths. Herein we describe a function of Th2-eosinophil responses in counteracting the development of arthritis. In two independent models of arthritis,Nippostrongylus brasiliensis infection leads to Th2 and eosinophil accumulation in the joints associated with robust inhibition of arthritis and protection from bone loss. Mechanistically,this protective effect is dependent on IL-4/IL-13-induced STAT6 pathway. Furthermore,we show that eosinophils play a central role in the modulation of arthritis probably through the increase of anti-inflammatory macrophages into arthritic joints. The presence of these pathways in human disease is confirmed by detection of GATA3-positive cells and eosinophils in the joints of rheumatoid arthritis patients. Taken together,these results demonstrate that eosinophils and helminth-induced activation of the Th2 pathway axis effectively mitigate the course of inflammatory arthritis. View Publication

CD8(+) T cells develop increased sensitivity following Ag experience,and differences in sensitivity exist between T cell memory subsets. How differential TCR signaling between memory subsets contributes to sensitivity differences is unclear. We show in mouse effector memory T cells (TEM) that textgreater50% of lymphocyte-specific protein tyrosine kinase (Lck) exists in a constitutively active conformation,compared with textless20% in central memory T cells (TCM). Immediately proximal to Lck signaling,we observed enhanced Zap-70 phosphorylation in TEM following TCR ligation compared with TCM Furthermore,we observed superior cytotoxic effector function in TEM compared with TCM,and we provide evidence that this results from a lower probability of TCM reaching threshold signaling owing to the decreased magnitude of TCR-proximal signaling. We provide evidence that the differences in Lck constitutive activity between CD8(+) TCM and TEM are due to differential regulation by SH2 domain-containing phosphatase-1 (Shp-1) and C-terminal Src kinase,and we use modeling of early TCR signaling to reveal the significance of these differences. We show that inhibition of Shp-1 results in increased constitutive Lck activity in TCM to levels similar to TEM,as well as increased cytotoxic effector function in TCM Collectively,this work demonstrates a role for constitutive Lck activity in controlling Ag sensitivity,and it suggests that differential activities of TCR-proximal signaling components may contribute to establishing the divergent effector properties of TCM and TEM. This work also identifies Shp-1 as a potential target to improve the cytotoxic effector functions of TCM for adoptive cell therapy applications. View Publication

Long-lived plasma cells are critical to humoral immunity as a lifelong source of protective antibodies. Antigen-activated B cells-with T-cell help-undergo affinity maturation within germinal centres and persist as long-lived IgG plasma cells in the bone marrow. Here we show that antigen-specific,induced IgM plasma cells also persist for a lifetime. Unlike long-lived IgG plasma cells,which develop in germinal centres and then home to the bone marrow,IgM plasma cells are primarily retained within the spleen and can develop even in the absence of germinal centres. Interestingly,their expressed IgV loci exhibit somatic mutations introduced by the activation-induced cytidine deaminase (AID). However,these IgM plasma cells are probably not antigen-selected,as replacement mutations are spread through the variable segment and not enriched within the CDRs. Finally,antibodies from long-lived IgM plasma cells provide protective host immunity against a lethal virus challenge. View Publication

The fact that Parkinson's disease (PD) can arise from numerous genetic mutations suggests a unifying molecular pathology underlying the various genetic backgrounds. To address this hypothesis,we took an integrated approach utilizing in vitro disease modeling and comprehensive transcriptome profiling to advance our understanding of PD progression and the concordant downstream signaling pathways across divergent genetic predispositions. To model PD in vitro,we generated neurons harboring disease-causing mutations from patient-specific,induced pluripotent stem cells (iPSCs). We observed signs of degeneration in midbrain dopaminergic neurons,reflecting the cardinal feature of PD. Gene expression signatures of PD neurons provided molecular insights into disease phenotypes observed in vitro,including oxidative stress vulnerability and altered neuronal activity. Notably,PD neurons show that elevated RBFOX1,a gene previously linked to neurodevelopmental diseases,underlies a pattern of alternative RNA-processing associated with PD-specific phenotypes. View Publication

Fibrodysplasia ossificans progressiva (FOP) syndrome is caused by mutation of the gene ACVR1,encoding a constitutive active bone morphogenetic protein type I receptor (also called ALK2) to induce heterotopic ossification in the patient. To genetically correct it,we attempted to generate the mutant ALK2-iPSCs (mALK2-iPSCs) from FOP-human dermal fibroblasts. However,the mALK2 leads to inhibitory pluripotency maintenance,or impaired clonogenic potential after single-cell dissociation as an inevitable step,which applies gene-correction tools to induced pluripotent stem cells (iPSCs). Thus,current iPSC-based gene therapy approach reveals a limitation that is not readily applicable to iPSCs with ALK2 mutation. Here we developed a simplified one-step procedure by simultaneously introducing reprogramming and gene-editing components into human fibroblasts derived from patient with FOP syndrome,and genetically treated it. The mixtures of reprogramming and gene-editing components are composed of reprogramming episomal vectors,CRISPR/Cas9-expressing vectors and single-stranded oligodeoxynucleotide harboring normal base to correct ALK2 c.617GtextgreaterA. The one-step-mediated ALK2 gene-corrected iPSCs restored global gene expression pattern,as well as mineralization to the extent of normal iPSCs. This procedure not only helps save time,labor and costs but also opens up a new paradigm that is beyond the current application of gene-editing methodologies,which is hampered by inhibitory pluripotency-maintenance requirements,or vulnerability of single-cell-dissociated iPSCs. View Publication

Thirdhand cigarette smoke (THS) was recently recognized as an environmental health hazard; however,little is known about it effects on cells. Mitochondria are sensitive monitors of cell health and report on environmentally-induced stress. We tested the effects of low levels of THS extracted from terry cloth on mitochondrial morphology and function using stem cells with well-defined mitochondria. Concentrations of THS that did not kill cells caused stress-induced mitochondrial hyperfusion (SIMH),which was characterized by changes in mitochondrial morphology indicative of fusion,increased mitochondrial membrane potential (MMP),increased ATP levels,increased superoxide production,and increased oxidation of mitochondrial proteins. SIMH was accompanied by a decrease in Fis1 expression,a gene responsible for mitochondrial fission,and a decrease in apoptosis-related genes,including Aifm2,Bbc3 and Bid There was also down regulation of Ucp2,Ucp4 and Ucp5,genes that decrease MMP thereby reducing oxidative phosphorylation,while promoting glycolysis. These effects,which collectively accompany SIMH,are a pro-survival mechanism to rescue damaged mitochondria and protect cells from apoptosis. Prolonged exposure to THS caused a reduction in MMP and decreased cell proliferation,which likely leads to apoptosis. View Publication

CD38,as a cell surface antigen is highly expressed in several hematologic malignancies including multiple myeloma (MM) and has been proven to be a good target for immunotherapy of the disease. CD38 is also a signaling enzyme responsible for the metabolism of two novel calcium messenger molecules. To be able to target this multifunctional protein,we generated a series of nanobodies against CD38 with high affinities. Crystal structures of the complexes of CD38 with the nanobodies were solved,identifying three separate epitopes on the carboxyl domain. Chromobodies,engineered by tagging the nanobody with fluorescence proteins,provide fast,simple and versatile tools for quantifying CD38 expression. Results confirmed that CD38 was highly expressed in malignant MM cells compared with normal white blood cells. The immunotoxin constructed by splicing the nanobody with a bacterial toxin,PE38 shows highly selective cytotoxicity against patient-derived MM cells as well as the cell lines,with half maximal effective concentration reaching as low as 10(-11) molar. The effectiveness of the immunotoxin can be further increased by stimulating CD38 expression using retinoid acid. These results set the stage for the development of clinical therapeutics as well as diagnostic screening for myeloma. View Publication

Disturbances in neuronal differentiation and function are an underlying factor of many brain disorders. Zinc homeostasis and signaling are important mediators for a normal brain development and function,given that zinc deficiency was shown to result in cognitive and emotional deficits in animal models that might be associated with neurodevelopmental disorders. One underlying mechanism of the observed detrimental effects of zinc deficiency on the brain might be impaired proliferation and differentiation of stem cells participating in neurogenesis. Thus,to examine the molecular mechanisms regulating zinc metabolism and signaling in differentiating neurons,using a protocol for motor neuron differentiation,we characterized the expression of zinc homeostasis genes during neurogenesis using human induced pluripotent stem cells (hiPSCs) and evaluated the influence of altered zinc levels on the expression of zinc homeostasis genes,cell survival,cell fate,and neuronal function. Our results show that zinc transporters are highly regulated genes during neuronal differentiation and that low zinc levels are associated with decreased cell survival,altered neuronal differentiation,and,in particular,synaptic function. We conclude that zinc deficiency in a critical time window during brain development might influence brain function by modulating neuronal differentiation. View Publication