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Mutations within the FUS gene (Fused in Sarcoma) are known to cause Amyotrophic Lateral Sclerosis (ALS),a neurodegenerative disease affecting upper and lower motoneurons. The FUS gene codes for a multifunctional RNA/DNA-binding protein that is primarily localized in the nucleus and is involved in cellular processes such as splicing,translation,mRNA transport and DNA damage response. In this study,we analyzed pathophysiological alterations associated with ALS related FUS mutations (mFUS) in human induced pluripotent stem cells (hiPSCs) and hiPSC derived motoneurons. To that end,we compared cells carrying a mild or severe mFUS in physiological- and/or stress conditions as well as after induced DNA damage. Following hyperosmolar stress or irradiation,mFUS hiPS cells recruited significantly more cytoplasmatic FUS into stress granules accompanied by impaired DNA-damage repair. In motoneurons wild-type FUS was localized in the nucleus but also deposited as small punctae within neurites. In motoneurons expressing mFUS the protein was additionally detected in the cytoplasm and a significantly increased number of large,densely packed FUS positive stress granules were seen along neurites. The amount of FUS mislocalization correlated positively with both the onset of the human disease (the earlier the onset the higher the FUS mislocalization) and the maturation status of the motoneurons. Moreover,even in non-stressed post-mitotic mFUS motoneurons clear signs of DNA-damage could be detected. In summary,we found that the susceptibility to cell stress was higher in mFUS hiPSCs and hiPSC derived motoneurons than in controls and the degree of FUS mislocalization correlated well with the clinical severity of the underlying ALS related mFUS. The accumulation of DNA damage and the cellular response to DNA damage stressors was more pronounced in post-mitotic mFUS motoneurons than in dividing hiPSCs suggesting that mFUS motoneurons accumulate foci of DNA damage,which in turn might be directly linked to neurodegeneration. View Publication

p53-related protein kinase (TP53RK,also known as PRPK) is an upstream kinase which phosphorylates (Ser15) and mediates p53 activity. Here we show that TP53RK confers poor prognosis in MM patients; and conversely,that TP53RK knockdown inhibits p53 phosphorylation and triggers multiple myeloma (MM) cell apoptosis,associated with downregulation of c-Myc and E2F-1-mediated upregulation of pro-apoptotic Bim. We further demonstrate that TP53RK downregulation also triggers growth inhibition in p53-deficient (KMS-11) and p53-mutant (U266) MM cell lines,and identify novel downstream targets of TP53RK including ribonucleotide reductase-1,telomerase reverse transcriptase,and cyclin dependent kinase inhibitor 2C (CDKN2C). Our previous studies showed that immunomodulatory drugs (IMiDs) downregulate p21 and trigger apoptosis in wt-p53 MM.1S cells,Importantly we here demonstrate by pull-down,nuclear magnetic resonance spectroscopy,differential scanning fluorimetry,and isothermal titration calorimetry,that IMiDs bind and inhibit TP53RK,with biologic sequelae similar to TP53RK knockdown. Our studies therefore demonstrate that either genetic or pharmacological inhibition of TP53RK triggers MM cell apoptosis via both p53-Myc axis-dependent and -independent pathways,validating TP53RK as a novel therapeutic target in patients with poor prognosis MM. View Publication

One of the key research areas surrounding HIV-1 concerns the regulation of the fusion event that occurs between the virus particle and the host cell during entry. Even if it is universally accepted that the large GTPase dynamin-2 is important during HIV-1 entry,its exact role during the first steps of HIV-1 infection is not well characterized. Here,we have utilized a multidisciplinary approach to study the DNM2 role during fusion of HIV-1 in primary resting CD4 T and TZM-bl cells. We have combined advanced light microscopy and functional cell-based assays to experimentally assess the role of dynamin-2 during these processes. Overall,our data suggest that dynamin-2,as a tetramer,might help to establish hemi-fusion and stabilizes the pore during HIV-1 fusion. View Publication

Numerous studies have demonstrated that administration of antigen (Ag)-pulsed dendritic cells (DCs) is an effective strategy for enhancing immunity to tumors and infectious disease organisms. However,the generation and/or isolation of DCs can require substantial time and expense. Therefore,using inactivated F. tularensis (iFt) Ag as a model immunogen,we first sought to determine if DCs could be replaced with peripheral blood mononuclear cells (PBMCs) during the ex-vivo pulse phase and still provide protection against Ft infection. Follow up studies were then conducted using the S. pneumoniae (Sp) vaccine Prevnar {\textregistered}13 as the Ag in the pulse phase followed by immunization and Sp challenge. In both cases,we demonstrate that PBMCs can be used in place of DCs when pulsing with iFt and/or Prevnar {\textregistered}13 ex vivo and re-administering the Ag-pulsed PBMCs as a vaccine. In addition,utilization of the i.n. route for Ag-pulsed PBMC administration is superior to use of the i.v. route in the case of Sp immunization,as well as when compared to direct injection of Prevnar {\textregistered}13 vaccine i.m. or i.n. Furthermore,this PBMC-based vaccine strategy provides a more marked and enduring protective immune response and is also capable of serving as a multi-organism vaccine platform. The potential for this ex-vivo vaccine strategy to provide a simpler,less time consuming,and less expensive approach to DC-based vaccines and vaccination in general is also discussed. View Publication

BACKGROUND Massive liquid crystal droplets have been found during embryonic development in more than twenty different tissues and organs,including the liver,brain and kidney. Liquid crystal deposits have also been identified in multiple human pathologies,including vascular disease,liver dysfunction,age-related macular degeneration,and other chronic illnesses. Despite the involvement of liquid crystals in such a large number of human processes,this phenomenon is poorly understood and there are no in vitro systems to further examine the function of liquid crystals in biology. RESULTS We report the presence of tubular birefringent structures in embryoid bodies (EBs) differentiated in culture. These birefringent tubular structures initiate at the EB surface and penetrated the cortex at a variety of depths. Under crossed polarized light,these tubules are seen as a collection of birefringent Maltese crosses and tubules with birefringent walls and a non-birefringent lumen. The fluidity of these birefringent structures under pressure application led to elongation and widening,which was partially recoverable with pressure release. These birefringent structures also displayed heat triggered phase transition from liquid crystal to isotropic status that is partially recoverable with return to ambient temperature. These pressure and temperature triggered changes confirm the birefringent structures as liquid crystals. The first report of liquid crystal so early in development. CONCLUSION The structure of the liquid crystal tubule network we observed distributed throughout the differentiated embryoid bodies may function as a transportation network for nutrients and metabolic waste during EB growth,and act as a precursor to the vascular system. This observation not only reveals the involvement of liquid crystals earlier than previously known,but also provides a method for studying liquid crystals in vitro. View Publication

Mouse embryonic fibroblasts (MEFs) and human foreskin fibroblasts (HFFs) are used for the culture of human embryonic stem cells (hESCs). MEFs and HFFs differed in their capacity to support the proliferation and pluripotency of hESCs and could affect cardiac differentiation potential of hESCs. The aim of this study was to evaluate the effect of MEFs and HFFs feeders on dopaminergic differentiation of hESCs lines. To minimize the impact of culture condition variation,two hESCs lines were cultured on mixed feeder cells (MFCs,MEFs: HFFs = 1:1) and HFFs feeder,respectively,and then were differentiated into dopaminergic (DA) neurons under the identical protocol. Dopaminergic differentiation was evaluated by immunocytochemistry,quantitative fluorescent real-time PCR,transmission and scanning electron microscopy,and patch clamp. Our results demonstrated that these hESCs-derived neurons were genuine and functional DA neurons. However,compared to hESCs line on MFCs feeder,hESCs line on HFFs feeder had a higher proportion of tyrosine hydroxylase (TH) positive cells and expressed higher levels of FOXA2,PITX3,NURR1,and TH genes. In addition,the values of threshold intensity and threshold membrane potential of DA neurons from hESCs line on HFFs feeder were lower than those of DA neurons from hESCs line on the MFCs feeder. In conclusion,HFFs feeder not only facilitated the differentiation of hESCs cells into dopaminergic neurons,but also induced hESCs-derived DA neurons to express higher electrophysiological excitability. Therefore,feeder cells could affect not only dopaminergic differentiation potential of different hESCs lines,but also electrophysiological properties of hESCs-derived DA neurons. View Publication

Postcardiac arrest acidosis can decrease survival. Effective medications without adverse side effects are still not well characterized. We aimed to analyze whether early administration of glutamine could improve survival and protect cardiomyocytes from postcardiac arrest acidosis using animal and cell models. Forty Wistar rats with postcardiac arrest acidosis (blood pH textless 7.2) were included. They were divided into study (500 mg/kg L-alanyl-L-glutamine,n = 20) and control (normal saline,n = 20) groups. Each of the rats received resuscitation. The outcomes were compared between the two groups. In addition,cardiomyocytes derived from human induced pluripotent stem cells were exposed to HBSS with different pH levels (7.3 or 6.5) or to culture medium (control). Apoptosis-related markers and beating function were analyzed. We found that the duration of survival was significantly longer in the study group (p textless 0.05). In addition,in pH 6.5 or pH 7.3 HBSS buffer,the expression levels of cell stress (p53) and apoptosis (caspase-3,Bcl-xL) markers were significantly lower in cardiomyocytes treated with 50 mM L-glutamine than those without L-glutamine (RT-PCR). L-glutamine also increased the beating function of cardiomyocytes,especially at the lower pH level (6.5). More importantly,glutamine decreased cardiomyocyte apoptosis and increased these cells' beating function at a low pH level. View Publication

Variability in induced pluripotent stem cell (iPSC) lines remains a concern for disease modeling and regenerative medicine. We have used RNA-sequencing analysis and linear mixed models to examine the sources of gene expression variability in 317 human iPSC lines from 101 individuals. We found that ∼50% of genome-wide expression variability is explained by variation across individuals and identified a set of expression quantitative trait loci that contribute to this variation. These analyses coupled with allele-specific expression show that iPSCs retain a donor-specific gene expression pattern. Network,pathway,and key driver analyses showed that Polycomb targets contribute significantly to the non-genetic variability seen within and across individuals,highlighting this chromatin regulator as a likely source of reprogramming-based variability. Our findings therefore shed light on variation between iPSC lines and illustrate the potential for our dataset and other similar large-scale analyses to identify underlying drivers relevant to iPSC applications. View Publication

BACKGROUND Oesophageal squamous cell carcinoma (ESCC) and colorectal cancer (CRC) are common types of human cancer. Spheroid colony formation is used to characterize cancer stem cell (CSCs). In the present study,we analyzed the significance of kinesin family 11 (KIF11 in human ESCC and CRC. MATERIALS AND METHODS Expression of KIF11 in 105 ESCC and 100 CRC cases was determined using immunohistochemistry. RNA interference was used to inhibit KIF11 expression in ESCC and CRC cell lines. RESULTS In total,61 out of 105 (58%) ESCC and 62 out of 100 (62%) CRC cases were positive for KIF11. Expression of KIF11 was not associated with any clinicopathological characteristics. Both the number and size of spheres produced by from TE-5 ESCC cells and DLD-1 CRC cells were significantly reduced upon KIF11 siRNA transfection compared to negative control siRNA transfection. CONCLUSION These results indicate that KIF11 plays an important role in CSCs of ESCC and CRC. View Publication

BACKGROUND Human rhinoviruses (HRVs) are a major trigger of asthma exacerbations,with the bronchial epithelium being the major site of HRV infection and replication. Mast cells (MCs) play a key role in asthma where their numbers are increased in the bronchial epithelium with increasing disease severity. OBJECTIVE In view of the emerging role of MCs in innate immunity and increased localization to the asthmatic bronchial epithelium,we investigated whether HRV infection of MCs generated innate immune responses which were protective against infection. METHODS The LAD2 MC line or primary human cord blood-derived MCs (CBMCs) were infected with HRV or UV-irradiated HRV at increasing multiplicities of infection (MOI) without or with IFN-β or IFN-λ. After 24 h,innate immune responses were assessed by RT-qPCR and IFN protein release by ELISA. Viral replication was determined by RT-qPCR and virion release by TCID50 assay. RESULTS HRV infection of LAD2 MCs induced expression of IFN-β,IFN-λ and IFN-stimulated genes. However,LAD2 MCs were permissive for HRV replication and release of infectious HRV particles. Similar findings were observed with CBMCs. Neutralization of the type I IFN receptor had minimal effects on viral shedding,suggesting that endogenous type I IFN signalling offered limited protection against HRV. However,augmentation of these responses by exogenous IFN-β,but not IFN-λ,protected MCs against HRV infection. CONCLUSION AND CLINICAL RELEVANCE MCs are permissive for the replication and release of HRV,which is prevented by exogenous IFN-β treatment. Taken together,these findings suggest a novel mechanism whereby MCs may contribute to HRV-induced asthma exacerbations. View Publication

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder in which the MECP2 (methyl CpG-binding protein 2) gene is mutated. Recent studies showed that RTT-derived neurons have many cellular deficits when compared to control,such as: less synapses,lower dendritic arborization and reduced spine density. Interestingly,treatment of RTT-derived neurons with Insulin-like Growth Factor 1 (IGF1) could rescue some of these cellular phenotypes. Given the critical role of IGF1 during neurodevelopment,the present study used human induced pluripotent stem cells (iPSCs) from RTT and control individuals to investigate the gene expression profile of IGF1 and IGF1R on different developmental stages of differentiation. We found that the thyroid hormone receptor (TRalpha 3) has a differential expression profile. Thyroid hormone is critical for normal brain development. Our results showed that there is a possible link between IGF1/IGF1R and the TRalpha 3 and that over expression of IGF1R in RTT cells may be the cause of neurites improvement in neural RTT-derived neurons. View Publication

Cellular therapy using stem cells in bone regeneration has gained increasing interest. Various studies suggest the clinical utility of osteoprogenitors-like mesenchymal stem cells in bone regeneration. However,limited availability of mesenchymal stem cells and conflicting evidence on their therapeutic efficacy limit their clinical application. Human embryonic stem cells (hESCs) are potentially an unlimited source of healthy and functional osteoprogenitors (OPs) that could be utilized for bone regenerative applications. However,limited ability to track hESC-derived progenies in vivo greatly hinders translational studies. Hence,in this study,we aimed to establish hESC-derived OPs (hESC-OPs) expressing green fluorescent protein (GFP) and to investigate their osteogenic differentiation potential in vitro. We fluorescently labelled H9-hESCs using a plasmid vector encoding GFP. The GFP-expressing hESCs were differentiated into hESC-OPs. The hESC-OPs(GFP+) stably expressed high levels of GFP,CD73,CD90,and CD105. They possessed osteogenic differentiation potential in vitro as demonstrated by increased expression of COL1A1,RUNX2,OSTERIX,and OPG transcripts and mineralized nodules positive for Alizarin Red and immunocytochemical expression of osteocalcin,alkaline phosphatase,and collagen-I. In conclusion,we have demonstrated that fluorescently labelled hESC-OPs can maintain their GFP expression for the long term and their potential for osteogenic differentiation in vitro. In future,these fluorescently labelled hESC-OPs could be used for noninvasive assessment of bone regeneration,safety,and therapeutic efficacy. View Publication