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Fragile X syndrome (FXS) is caused by the loss of fragile X mental retardation protein (FMRP), an RNA-binding protein that can regulate the translation of specific mRNAs. In this study, we developed an FXS human forebrain organoid model and observed that the loss of FMRP led to dysregulated neurogenesis, neuronal maturation and neuronal excitability. Bulk and single-cell gene expression analyses of FXS forebrain organoids revealed that the loss of FMRP altered gene expression in a cell-type-specific manner. The developmental deficits in FXS forebrain organoids could be rescued by inhibiting the phosphoinositide 3-kinase pathway but not the metabotropic glutamate pathway disrupted in the FXS mouse model. We identified a large number of human-specific mRNAs bound by FMRP. One of these human-specific FMRP targets, CHD2, contributed to the altered gene expression in FXS organoids. Collectively, our study revealed molecular, cellular and electrophysiological abnormalities associated with the loss of FMRP during human brain development.

High altitude hypobaric hypoxia environment impairs male’s reproductive function. Leptin is an adipose tissue-derived hormone which regulates body weight homeostasis. Its receptor (LepR) has been found in all levels of male reproductive axis, indicating that it can affect male reproductive system in a direct or (and) indirect way. However, the role of leptin signaling in hypobaric hypoxia induced male reproductive dysfunction remains to be elucidated. In this study, we investigated the changes of leptin levels in male SD rats in stimulated altitude of 5500 m hypobaric hypoxia environment and their effects on the hypothalamus-pituitary-gonad axis (HPG axis). A hypoxia animal model was established using a hypobaric hypoxia chamber. Rats were divided randomly into 1, 7, 14, 28-day hypoxia group, recovery group (14 days hypoxia+14 days normoxia) and their control groups. Hypoxia groups displayed obvious changes of testicular and epididymis index compared to control groups. The total number of sperm and sperm motility rate decreased dramatically, while sperm deformity rate increased in hypoxia groups. The flow cytometry analysis showed that the percentage of haploid in 1-day, 7-day and 28-day hypoxia groups increased while the proportion of diploid decreased in 14-day and 28-day hypoxia group. TUNEL staining showed that the testis cells apoptosis index (AI) of hypoxia groups increased significantly, and the apoptosis of cells mainly focus on spermatogonia and spermatocytes. The expression of GnRH in hypothalamus decreased dramatically under hypoxia condition, accompanied with the reduction of serum testosterone (T) level in 1-day and 28-day hypoxia groups and free-testosterone level (FT) in 1-day and 14-day hypoxia groups. Importantly, ELISA analysis showed that serum leptin level decreased in 7-day hypoxia groups and acylated-ghrelin, gastrin also changed, accompanying with reduction of LepR in hypothalamus in hypoxia groups. Immunohistochemical staining exhibited increased leptin and LepR in testis under hypobaric hypoxia conditions. Our results suggested that simulated high altitude hypobaric hypoxia environment decreased male reproductive function, depressed HPG axis activity and altered the serum concentration of hormones related to food intake in adult male rats. Additionally, hypobaric hypoxia induced the leptin-LepR expression in adult male rats’ testis, suggesting leptin-LepR signaling may mediate hypoxia-induced impairment in male rats’ reproductive system.

Background: Neonatal mortality rates remain high in Sub-Saharan African countries. Improving the newborn resuscitation skills of healthcare professionals is important in addressing this challenge. The aim of this study was to evaluate a neonatal resuscitation training programme delivered over a two-year period for healthcare professionals in Zanzibar, Tanzania.Methods: A pre- and post-intervention study was designed. We delivered neonatal resuscitation training over a 2-day period in 2017 and 2 days of refresher training in 2018. Knowledge was evaluated by a self-designed survey (11 items with a total score of 22) before and after the two training periods, and skills were evaluated by a skills checklist (six domains with 25 items with a total score of 50) completed by the trainers based on their observations. Statistical analysis included differences in the knowledge and skills scores before and after the training sessions and between the two periods.Results: A total of 23 healthcare professionals participated and completed both neonatal resuscitation training sessions. The knowledge mean scores before and after the training in 2017 increased from 9.60 to 13.60 (95% CI: −5.900; −2.099, p < 0.001), and in 2018, the scores increased from 10.80 to 15.44 (95% CI: −6.062; −3.217, p < 0.001). The mean knowledge scores post-training over time were 13.60 in 2017 and 15.44 in 2018 (95% CI: −3.489; 0.190, p = 0.030). The resuscitation skills performance between the two time periods increased from a mean of 32.26 (SD = 2.35) to a mean of 42.43 (SD = 1.73) (95% CI: −11.402; −8.945, p < 0.001).Conclusion: The neonatal resuscitation training programme increased the theoretical knowledge and resuscitation skills before and after the two training sessions and over time after a 9-month period. Continuous neonatal resuscitation training based on the local needs in resource-limited countries is essential to provide confidence in healthcare professionals to initiate resuscitation and to improve newborn outcomes.

Background Previous studies have suggested that Toxoplasma gondii (T. gondii) infection might be associated with fatty liver disease. However, the relationship between non-alcoholic fatty liver disease (NAFLD) and T. gondii infection has not been investigated in a large population. We aimed to study the relationship between those two diseases using a population-based dataset from the United States. Methods The data were collected from the third National Health and Nutrition Examination Survey (NHANES III) between 1988 and 1994. Statistical analysis was applied to compare the prevalence of NAFLD in anti-T. gondii antibody-positive participants with antibody-negative ones. Results A total of 9465 persons with a mean age of 44.33 ± 16.21 years, 46.9% of which were males, were included in the final analysis. Their mean BMI was 27.60 ± 5.96 kg/m2. A total of 2520 participants (26.62%) were positive for the T. gondii antibody. There was an increasing trend of seroprevalence of T. gondii with age (P for trend

Alisitipes phage SZW_AS01, a novel lytic phage that specifically infects Alistipes shahii, was isolated from wastewater samples in Shenzhen, China. The phage's genome consists of 45,392 bp, with a GC content of 47%. The genome encodes 56 putative open reading frames (ORFs) and 1 tRNA gene. Direct terminal repeats with a length of 55 bp are present at both ends of the genome. Phylogenetic analysis of the amino acid sequences of terminase large subunit shows that phage SZW_AS01 forms a distinct branch from the Siphoviridae family phages, but is far from the Podoviridae and Myoviridae family phages. Transmission electron microscopy confirmed that SZW_AS01 belongs to the Siphoviridae family. To the best of our knowledge, this is the first report of a lytic phage infecting bacteria in the Alistipes genus.

This study assessed neuropeptide Y (NPY) expression in the hippocampus after long-term survival following traumatic brain injury (TBI) induced by controlled cortical impact (CCI) with or without the development of posttraumatic epilepsy (PTE). We hypothesized that following long-term survival after CCI, the severity of tissue injury and the development of PTE would correlate with the degree of hippocampal neurodegeneration as reflected by NPY + and neuronal nuclear antigen (NeuN)+ cell loss. Adult Sprague–Dawley rats of 2–3 months of age were lesioned in the right parietal cortex and monitored for seizure activity by video and/or video-EEG. Neuropeptide Y and NeuN immunoreactivities (IRs) were quantified by light microscopy and semiautomatic image analysis approaches for unbiased quantification. Severely injured animals, marked by extensive tissue loss in the ipsilateral neocortex and adjacent hippocampus, resulted in significantly lower NeuN + hilar cell density and NPY + cell loss in the contralateral Cornu Ammonis (CA)-3 and dentate hilus (DH). The degree of NPY + cell loss was more severe in CCI-injured animals with PTE than those animals that did not develop PTE. Mildly injured animals demonstrated no significant change of NPY expression compared with control animals. Our findings of long-term alterations of NPY expression in the hippocampus of severely brain-injured animals can provide important insights into the cellular and molecular consequences of severe TBI and posttraumatic epileptogenesis.

Background & aims Leucine-rich repeat G-protein-coupled receptor 4 (LGR4) and its ligands R-spondin1-4 (Rspos) have been vastly investigated in embryonic development. The biological functions of Rspos-LGR4 system in liver remains largely unknown. Here, we explored whether it protects hepatocytes against hypoxia/reoxygenation (H/R) induced damage. Methods H/R injury was induced by dimethyloxalylglycine (DMOG) in AML12 cells and the effects of Rspo3 on cell proliferation and apoptosis were assessed. Specific shRNAs were used to interfere LGR4 or β-catenin. Results DMOG caused hepatocytes damage evidenced by increase in HIF-1α, cell death and apoptosis genes p27 and Bax, with concurrent decrease of cell proliferation genes PCNA and CyclinD1. Of all the Rspos, Rspo3 is predominantly expressed in AML12 hepatocytes. Importantly, Rspo3 demonstrated an alteration in a manner similar to proliferation-related genes during H/R injury. Rspo3 pretreatment rendered hepatocytes less vulnerable to DMOG induced H/R injury. Ablation of LGR4 using shRNA attenuated the protective effects of Rspo3. Wnt3a also protected AML12 cells from damages caused by H/R, showing enhanced proliferation activity. Notably, knockdown of β-catenin in hepatocytes completely abolished the effect of Rspo3 pretreatment on the expression levels of PCNA and CyclinD1. Conclusion Rspo3-LGR4 axis protects hepatocytes from H/R injury via activating β-catenin.

This study focused on identifying bladder cancer (BC)-associated genes, transcription factors (TFs), and microRNAs (miRNAs). Two microarray data sets GSE37815 and GSE40355 were utilized to screen common differentially expressed genes (DEGs) associated with BC. Then, functional enrichment analysis was performed for elucidating the involved functions of DEGs. Subsequently, the protein-protein interaction (PPI) network and submodule of PPI network were analyzed. Finally, the regulation relationships of TF-DEGs and miRNA-DEGs were obtained to construct miRNA-target-TF regulatory network. DEGs were identified across BC and normal bladder tissues samples. Functional enrichment analysis results showed that most upregulated DEGs were closely associated with the Gene Ontology function of "mitotic spindle assembly checkpoint" and pathway of "Cell cycle," whereas most downregulated DEGs were significantly associated with "Complement and coagulation cascades" pathway (e.g., A2M and F13A1) and "Ras signaling pathway" (e.g., GNG11). DEGs such as F13A1 and A2M were highlighted in the PPI network and Submodule 1. In addition, three centromere-associated CENPK, CENPF, and CENPO were enriched in Submodule 2. Moreover, miR-519d had high degree in the regulatory network and CENPO was predicted to be one target of miR-519d. The upregulated CENPK, CENPF, and CENPO, and downregulated A2M, F13A1, and GNG11 might contribute to the progression of BC. In addition, the downregulated miR-519d might lead to the development of BC by upregulating the expression of CENPO. However, future investigation of those findings should be needed.

BackgroundThe dilated cardiomyopathy with ataxia syndrome (DCMA) is an understudied autosomal recessive disease caused by loss-of-function mutations in the poorly characterized gene DNAJC19. Clinically, DCMA is commonly associated with heart failure and early death in affected children through an unknown mechanism. DCMA has been linked to Barth syndrome, a rare but well-studied disorder caused by deficient maturation of cardiolipin (CL), a key mitochondrial membrane phospholipid.MethodsPeripheral blood mononuclear cells from two children with DCMA and severe cardiac dysfunction were reprogrammed into induced pluripotent stem cells (iPSCs). Patient and control iPSCs were differentiated into beating cardiomyocytes (iPSC-CMs) using a metabolic selection strategy and mitochondrial structure and CL content before and after incubation with the mitochondrially-targeted peptide SS-31 were quantified.ResultsPatient iPSCs carry the causative DNAJC19 mutation (rs137854888) found in the Hutterite population and the iPSC-CMs demonstrated highly fragmented and abnormally-shaped mitochondria associated with an imbalanced isoform ratio of the mitochondrial protein OPA1, an important regulator of mitochondrial fusion. These abnormalities were reversible by incubation with SS-31 for 24 hours. Differentiation of iPSCs into iPSC-CMs increased the number of CL species observed but consistent, significant differences in CL content were not seen between patients and control.ConclusionsWe describe a unique and novel cellular model that provides insight into the mitochondrial abnormalities present in DCMA and identifies SS-31 as a potential therapeutic for this devastating disease.

Irisin, a myokine released during exercise, promotes browning of subcutaneous adipose tissue and regulates energy homeostasis. Although exercise constantly reduces blood cholesterol, whether irisin is involved in the regulation of cholesterol remains largely unknown. In the present study, subcutaneous infusion of irisin for 2 weeks induced a reduction in plasma and hepatic cholesterol in high fat diet-induced obese (DIO) mice. These alterations were associated with an activation of 5′ AMP-activated protein kinase (AMPK) and inhibition of sterol regulatory element-binding transcription factor 2 (SREBP2) transcription and nuclear translocation. In primary hepatocytes from either lean or DIO mice, irisin significantly decreased cholesterol content via sequential activation of AMPK and inhibition of SREBP2. Suppression of AMPK by compound C or AMPKα1 siRNA blocked irisin-induced alterations in cholesterol contents and SREBP2. In conclusion, irisin could suppress hepatic cholesterol production via a mechanism dependent of AMPK and SREBP2 signaling. These findings suggest that irisin is a promising therapeutic target for treatment of hypercholesterolemia., Graphical abstract Image 1, Highlights • Irisin inhibits hepatic cholesterol synthesis. • Irisin activates AMPK. • Irisin inhibits SREBP2 transcription and nuclear translocation. Irisin is a myokine released during exercise. Whether irisin contributes to the beneficial effects of exercise in reduction of blood cholesterol remains unknown. This study revealed that long-term infusion of irisin into obese mice ameliorated hypercholesterolemia and cholesterol content. The reduction of hepatic cholesterol induced by irisin may be mediated by activation of AMPK and subsequent suppression of SREBP2 transcription and nuclear translocation.

A myriad of drug delivery systems such as liposomes, micelles, polymers and inorganic nanoparticles (NPs) have been developed for cancer therapy. Very few of them, however, have the ability to integrate multiple functionalities such as specific delivery, high circulation stability, controllable release and good biocompatibility and biodegradability in a single system to improve the therapeutic efficacy. Herein, we report two types of stimuli-responsive nonporous silica prodrug NPs towards this goal for controlled release of anticancer drugs and efficient combinatorial cancer therapy. As a proof of concept, anticancer drugs camptothecin (CPT) and doxorubicin (DOX) were covalently encapsulated into silica matrices through glutathione (GSH)-responsive disulfide and pH-responsive hydrazone bonds, respectively, resulting in NPs with sizes tunable in the range of 50-200 nm. Both silica prodrug NPs showed stimuli-responsive controlled release upon exposure to a GSH-rich or acidic environment, resulting in improved anticancer efficacy. Notably, two prodrug NPs simultaneously taken up by HeLa cells showed a remarkable combinatorial efficacy compared to free drug pairs. These results suggest that the stimuli-responsive silica prodrug NPs are promising anticancer drug carriers for efficient cancer therapy.

Human induced pluripotent stem cells (hiPSCs) hold great hopes for application in regenerative medicine due to their inherent capacity to self-renew and differentiate into cells from the three embryonic germ layers. For clinical applications, a large quantity of hiPSCs produced in standardized and scalable culture processes is required. Several groups, including ours, have developed methodologies for scaled-up hiPSC production in stirred bioreactors in chemically defined medium. In this study, we optimized the critical steps and factors that affect hiPSC expansion and yield in stirred-suspension cultures, including inoculation conditions, seeding density, aggregate size, agitation rate, and cell passaging method. After multiple passages in stirred-suspension bioreactors, hiPSCs remained pluripotent, karyotypically normal, and capable of differentiating into all three germ layers.

High levels of S100A6 have been associated with poor outcome in some types of human cancers, but the role of S100A6 in the molecular pathogenesis of these cancers is largely unknown. This study was performed to explore the expression and functional roles of S100A6 in hepatocellular carcinoma (HCC). The expression level of S100A6 in HCC tumor and corresponding peritumoral tissues were determined by immunohistochemistry analysis. The potential functions of S100A6 in tumorigenesis and metastasis were analyzed by cell proliferation, migration, and invasion assays in human liver cancer cells. Moreover, through expression and purification of S100A6 recombinant protein tagged with cell-penetrating peptide, we analyzed its complex extracellular/intracellular effects in a S100A6-silenced cellular model. As a result, the expression of S100A6 was up-regulated in human HCC compared with adjacent peritumoral tissues. S100A6 silencing inhibited the growth and motility of HCC cells, while intracellular re-expression of S100A6 could rescue the proliferation and migration defects. Intracellular over-expression of S100A6 resulted in down-regulation of E-cadherin expression and promoted nuclear accumulation of β-catenin. Moreover, we found that the enhanced cell proliferation and motility after S100A6 stimulation were dependent on the activation of PI3K/AKT pathway. These results suggest that S100A6 may be involved in promotion and progression of human liver cancer.S100A6 is overexpressed in human hepatocellular carcinoma clinical specimens. S100A6 promotes proliferation and migration of human hepatoma cells. Overexpression of S100A6 results in alteration of E-cadherin and β-catenin. The multi-effects of S100A6 may be mediated in part by PI3K/AKT pathway activation.

We develop a new type of pH-responsive amphiphilic small molecular prodrug by conjugating folic acid with anti-tumour doxorubicin via a hydrazone bond. This prodrug is featured by high and precise drug loading (55.4wt%), which can self-assemble into micellar nanoparticles in neutral environment while disassemble in the presence of tumour cells expressing folic acid receptors or the acidic tumoral endosomal environment. The prodrug nanoparticles can effectively improve anticancer efficacy due to the features of pH-triggered drug release and targeted delivery. Moreover, in vitro cell study further indicated that the resulting prodrug nanoparticles had enhanced cytotoxicity for folic-acid-positive cells (HeLa) compared to folic-acid-negative cells (MCF-7). More importantly, the induced disassembly of prodrug nanoparticles can "switch on" the inherent fluorescence of the internalized doxorubicin drug in the tumour microenvironment, which can be used for the detection of tumour cells. We believe that this strategy can pave a new way for designing small molecular drug delivery systems and facilitate tumour diagnosis and treatment simultaneously.

An improved understanding of the pluripotency maintenance of embryonic stem (ES) cells is important for investigations of early embryo development and for cell replacement therapy, but the mechanism behind pluripotency is still incompletely understood. Recent findings show that zinc, an essential trace element in humans, is critically involved in regulating various signaling pathways and genes expression. However, its role in ES cell fate determination remains to be further explored. Here we showed that 2μM zinc chloride (ZnCl2) transiently maintained mouse ES cell pluripotency in vitro. The cultured mouse ES cells remained undifferentiated under 2μM ZnCl2 treatment in leukemia inhibitory factor (LIF) withdrawal, retinoic acid (RA) or embryoid bodies (EBs) differentiation assays. In addition, ZnCl2 increased pluripotency genes expression and inhibited differentiation genes expression. Further mechanistic studies revealed that ZnCl2 transiently activated signal transducers and activators of transcription 3 (Stat3) signaling through promoting Stat3 phosphorylation. Inhibition of Stat3 signaling abrogated the effects of ZnCl2 on mouse ES cell pluripotency. Taken together, this study demonstrated a critical role of zinc in the pluripotency maintenance of mouse ES cells, as well as an important regulator of Stat3 signaling.

Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), share the properties of unlimited self-renewal and the capacity to become any cell type in the body, making them well suited for regenerative medicine and cell therapy. So far, almost all hPSC lines have been directly or indirectly exposed to animal-derived products, which would hinder their use for clinical purposes. One of the biggest challenges in this area is to remove animal components from the derivation, propagation, and cryopreservation of hPSCs. Moreover, the presence of undefined components of animal or human origin in culture system may interfere with the interpretation of the effect of exogenous agents on the growth and differentiation of hPSCs and are prone to significant variability. To explore hPSC expansion in defined, xeno-free conditions, 2 different groups of culture systems were used to culture different hESC and hiPSC lines. Our results suggested that (1) medium, matrix, and exogenous factors have synergistic effects on the adhesion and growth of hPSCs; (2) cooperation of exogenous factors including basic fibroblast growth factor, Rho-associated kinase inhibitor (ROCK), and other growth factors is critical for hPSC adhesion and proliferation; (3) basal media have different effects on hPSC attachment to the culture surface; and (4) a medium or matrix component can work synergistically in one culture system, and not at all in another. In this study, we found that Vitronectin/TeSR2 and PDL/HEScGRO (Y-27632) systems were optimal for maintaining the long-term culture of 3 hESC lines and 2 hiPSC lines under defined, xeno-free conditions.

Pokemon is an important proto-oncogene that plays a critical role in cellular oncogenic transformation and tumorigenesis. Anoikis, which is regulated by Bim-mediated apoptosis, is critical to cancer cell invasion and metastasis. We investigated the role of Pokemon in anoikis, and our results show that Pokemon renders liver cells resistant to anoikis via suppression of Bim transcription. We knocked-down Pokemon in human hepatoma cells QGY7703 with small interfering RNAs (siRNA). Knockdown of Pokemon alone did not significantly affect the growth and survival of QGY7703 cells but notably enhanced their sensitivity to apoptotic stress due to the presence of chemical agents or cell detachment, thereby inducing anoikis, as evidenced by flow cytometry and caspase-3 activity assays. In contrast, ectopic expression of Pokemon in HL7702 cells led to resistance to anoikis. Dual-luciferase reporter and ChIP assays illustrated that Pokemon suppressed Bim transcription via direct binding to its promoter. Our results suggest that Pokemon prevents anoikis through the suppression of Bim expression, which facilitates tumor cell invasion and metastasis. This Pokemon-Bim pathway may be an effective target for therapeutic intervention for cancer.

Induced pluripotent stem cells (iPSCs) can provide an important source of cells for the next-generation of cell therapies in regenerative medicine, in part due to their similarity to embryonic stem cells (ESCs). Patient-specific iPSCs represent an opportunity for autologous cell therapies that are not restricted by immunological, ethical and technical obstacles. One of the technical hurdles that must be overcome before iPSCs can be clinically implemented is the scalable, reproducible production of iPSCs and their differentiated progeny. All of the iPSC lines established thus far have been generated and expanded with static tissue culture protocols, which are time-consuming and suffer from batch-to-batch variability. Alternatively, stirred suspension bioreactors propose several benefits and their homogeneous culture environment facilitates the large-scale expansion required for clinical studies at less cost. We have previously developed protocols for expanding murine and human ESCs as undifferentiated aggregates in stirred suspension bioreactors. The resulting cells were karyotypically normal, expressed pluripotency markers and could be differentiated into all three germ lineages, both in vitro and in vivo. In this study, we demonstrate that stirred suspension bioreactors yield 58-fold expansion of undifferentiated pluripotent iPSCs over 4 days. In vitro differentiation into cartilage, bone and cardiomyocytes lineages, in addition to in vivo teratoma formation, further confirmed the existence of fully functional and undifferentiated pluripotent iPSC aggregates following long-term passaging. Stirred suspension bioreactor culture represents an efficient process for the large-scale expansion and maintenance of iPSCs, which is an important first step in their clinical application.

Human embryonic stem (hES) cells hold great promise for application of human cell and tissue replacement therapy. However, the overwhelming majority of currently available hES cell lines have been directly or indirectly exposed to materials containing animal-derived components during their derivation, propagation, and cryopreservation. Unlike feeder-based cultures, which require the simultaneous growth of feeder and stem cells, resulting in mixed cell populations, stem cells grown on feeder-free systems are easily separated from the surface, presenting a pure population of cells for downstream applications. In this study, we have developed a novel method to expand hES cells in xeno-free, feeder-free conditions using 2 different matrices derived from xeno-free human foreskin fibroblasts (XF-HFFs). Using XF-HFF-derived extracellular matrix, together with 100 ng/mL recombinant bFGF-supplemented HEScGRO Basal Medium, long-term xeno-free expansion of hES cells is possible. Resulting hES cells were subjected to stringent tests and were found to maintain ES cell features, including morphology, pluripotency, stable karyotype, and expression of cell surface markers, for at least 20 passages. Xeno-free culturing practices are essential for the translation of basic hES cell research into the clinic. Therefore, the method presented in this study demonstrates that hES cells can be cultured in complete xeno-free conditions without the loss of pluripotency and furthermore, without the possibility of contamination from exogenous sources.

After slow freezing, the survival rate of human embryonic stem (hES) cells is poor and inconsistent. The aim of this study was to increase the freeze-thaw survival rate of hES cells by utilizing the ROCK inhibitor Y-27632. hES cell colonies were first treated with Y-27632, followed by collagenase IV and TrypLE Select dissociation whereupon small clumps were slow frozen using 90% Knockout serum replacement and 10% dimethyl sulfoxide. After thawing at 37 C, the clumps were cultured in medium supplemented with 10 microM Y-27632 for 1 day. Our results show that the use of Y-27632 significantly increases the survival of hES cells after thawing compared with that of the control group. Y-27632-treated freeze-thawed hES cells retain morphology, stable karyotype, expression of cell surface markers, and the potential to differentiate into derivatives of all three germ layers after long-term culture. We have concluded that conventional slow freezing with Y-27632 treatment is efficient and convenient for the cryopreservation of hES cells.

Long-term cultures of human embryonic stem (hES) cells require a feeder layer for maintaining cells in an undifferentiated state and increasing karyotype stability. In routine hES cell culture, mouse embryonic fibroblast (MEF) feeders and animal component-containing media (FBS or serum replacement) are commonly used. However, the use of animal materials increases the risk of transmitting pathogens to hES cells and therefore is not optimal for use in cultures intended for human transplantation. There are other limitations with conventional feeder cells, such as MEFs, which have a short lifespan and can only be propagated five to six passages before senescing. Several groups have investigated maintaining existing hES cell lines and deriving new hES cell lines on human feeder layers. However, almost all of these human source feeder cells employed in previous studies were derived and cultured in animal component conditions. Even though one group previously reported the derivation and culture of human foreskin fibroblasts (HFFs) in human serum-containing medium, this medium is not optimal because HFFs routinely undergo senescence after 10 passages when cultured in human serum. In this study we have developed a completely animal-free method to derive HFFs from primary tissues. We demonstrate that animal-free (AF) HFFs do not enter senescence within 55 passages when cultured in animal-free conditions. This methodology offers alternative and completely animal-free conditions for hES cell culture, thus maintaining hES cell morphology, pluripotency, karyotype stability, and expression of pluripotency markers. Moreover, no difference in hES cell maintenance was observed when they were cultured on AF-HFFs of different passage number or independent derivations.

According to the method used in our laboratory, our group synthesized (DIPP-Trp)2-Lys-OCH3. It inhibited the proliferation of K562 and HeLa cells in a dose-and time-dependent manner with an IC50 of 15.12 and 42.23 µM, respectively. (DIPP-Trp)2-Lys-OCH3 induced a dose-dependent increase of the G2/M cell population in K562 cells, and S cell population in HeLa cells; the sub-G0 population increased dramatically in both cell lines as seen by PI staining experiments using a FACS Calibur Flow cytometer (BeckmanCoulter, USA). Phosphatidylserine could significantly translocate to the surface of the membrane in (DIPP-Trp)2-Lys-OCH3-treated K562 and HeLa cells. The increase of an early apoptotic population was observed in a dose-dependent manner by both annexin-FITC and PI staining. It was concluded that (DIPP-Trp)2-Lys-OCH3 not only induced cells to enter into apoptosis, but also affected the progress of the cell cycle. It may have arrested the K562 and HeLa cells in the G2/M, S phases, respectively. The apoptotic pathway was pulsed at this point, resulting in the treated cells entering into programmed cell death. (DIPP-Trp)2-Lys-OCH3 is a potential anticancer drug that intervenes in the signalling pathway.

Thymidylate synthase (TS) is a critical enzyme for DNA biosynthesis and many nonclassical lipophilic antifolates targeting this enzyme are quite efficient and encouraging as antitumor drugs. In this paper, the binding model of 14 antifolates of substituted benzylamino- and heterocyclylmethylamino-carbodithioate derivatives of 4-(3H)-quinazolinone with TS is examined using molecular simulation methods—FlexiDock and SCORE2.0. The resulting conformation and orientation of these antifolates are directly applied to CoMFA study. The robust QSAR model, its three-dimensional contour map, and binding score of these antifolates derived from SCORE2.0 provide guidelines for structural optimization of current antifolates. The experiment indicates that deletion of cancer chemopreventive structure of dithiocarbamate is unfavorable for interaction between TS and antifolates.

To study associations between type 2 diabetes (T2DM) candidate genes and microvascular complications of diabetes (MVCDs), we performed case-control association studies for both T2DM and MVCDs in Han Chinese subjects. We recruited 1,939 unrelated Han Chinese T2DM patients and 918 individuals with normal blood glucose levels as nondiabetic controls. Among T2DM patients, 1116 have MVCDs, 266 have a history of T2DM of >10 years but never developed MVCDs. Eighty-two single-nucleotide polymorphisms (SNPs) in 54 candidate genes were genotyped. Discrete association studies were performed by the PLINK program for T2DM and MVCDs. Significant associations were found among candidate gene SNPs and T2DM, including rs1526167 of the TOX gene (allele A, P = 2.85 × 10−9, OR = 1.44). The SNP rs10811661 of the CDKN2A/B gene was also associated with T2DM (allele T, P = 4.09 × 10−7, OR = 1.36). When we used control patients with >10 years of T2DM history without MVCD, we found that the G allele of SNP rs1526167 of the TOX gene was associated with MVCD (nominal P = 4.33 × 10−4). In our study, significant associations were found between TOX and CDKN2A/B gene SNPs and T2DM. The TOX polymorphism might account for the higher risk of T2DM and the lower risk of MVCDs in the Han Chinese population.

Excessive secretion of glucagon is a major contributor to the development of diabetic hyperglycemia. Secretion of glucagon is regulated by various nutrients, with glucose being a primary determinant of the rate of alpha cell glucagon secretion. The intra-islet action of insulin is essential to exert the effect of glucose on the alpha cells since, in the absence of insulin, glucose is not able to suppress glucagon release in vivo. However, the precise mechanism by which insulin suppresses glucagon secretion from alpha cells is unknown. In this study, we show that insulin induces activation of GABAA receptors in the alpha cells by receptor translocation via an Akt kinase-dependent pathway. This leads to membrane hyperpolarization in the alpha cells and, ultimately, suppression of glucagon secretion. We propose that defects in this pathway(s) contribute to diabetic hyperglycemia.

The S1 serine protease family is one of the largest gene families known. Within this family there are several subfamilies that have been grouped together as a result of sequence comparisons and substrate identification. The grouping of related genes allows for the speculation of function for newly found members by comparison and for novel subfamilies by contrast. Analysis of the evolutionary patterns of genes indicates whether or not orthologs are likely to be identified in other species as well as potentially indicating that hypothesized orthologs are in fact not. Looking at subtle differences between subfamily members can reveal intricacies about function and expression. Previously, we have described genes encoding two novel serine proteinases, ISP1 and ISP2, which are most closely related to tryptases. The ISP1 gene encodes the embryo-derived enzyme strypsin, which is necessary for blastocyst hatching and invasion in vitro. Additionally both ISP1 and ISP2 are co-expressed in the endometrial gland during the time of hatching, suggesting that they may also both participate in zona lysis from within the uterine lumen. Here, we demonstrate that the ISPs are tandemly linked within the tryptase cluster on 17A3.3. We suggest that remarkable similarities within the 5'-untranslated and first intron regions of ISP1 and ISP2 may explain their intimate co-regulation in uterus. We also suggest that ISP genes have evolved through gene duplication and that the ISP1 gene has also begun to adopt an additional new function in the murine preimplantation embryo.

The Dlx gene family encodes homeodomain proteins that are required for forebrain and craniofacial development. Towards elucidating the roles for each of these genes, we have isolated cDNA clones encoding the full-coding sequence for murine Dlx-5 and partial coding sequence for murine Dlx-6. Three different classes of sense Dlx-5 cDNA clones were characterized, two of which lack the homeobox. We also identified an antisense Dlx-6 transcript. Genomic analysis shows that the Dlx-5 and -6 genes are linked. Biochemical analysis using gel shift assays demonstrate that DLX-1, -2 and -5 have very similar DNA-binding properties. The expression of Dlx-1, -2, -5, -6 and antisense Dlx-1 and -6 was studied in the midgestation mouse brain. We found that the Dlx genes are expressed in overlapping patterns at different stages of differentiation within the primordia of the basal ganglia. Dlx-1 and -2 are expressed in the least mature cells (in the ventricular and subventricular zones). Dlx-5 appears to be co-expressed with Dlx-1 and -2 in the SVZ, but is also expressed in the postmitotic cells of the mantle. Dlx-6 expression is strongest in the mantle. Antisense Dlx-1 and -6 have their highest expression in the SVZ. These results suggest that each of these Dlx genes may have a distinct role in different steps of differentiation in the basal ganglia.

Safety is the foremost issue in all human cell therapies, but human induced pluripotent stem cells (iPSCs) currently lack a useful safety indicator. Studies in chimeric mice have demonstrated that certain lines of iPSCs are tumorigenic; however a similar screen has not been developed for human iPSCs. Here, we show that in vitro cartilage tissue engineering is an excellent tool for screening human iPSC lines for tumorigenic potential. Although all human embryonic stem cells (ESCs) and most iPSC lines tested formed cartilage safely, certain human iPSCs displayed a pro-oncogenic state, as indicated by the presence of secretory tumors during cartilage differentiation in vitro. We observed five abnormal iPSC clones amoungst 21 lines derived from five different reprogramming methods using three cellular origins. We conclude that in vitro cartilage tissue engineering is a useful approach to identify abnormal human iPSC lines.

Conventionally, researchers remove spontaneously differentiated areas in human pluripotent stem cell (hPSC) colonies by using a finely drawn glass pipette or a commercially available syringe needle. However, when extreme differentiation occurs, it is inefficient to purify the remaining undifferentiated cells, as these undifferentiated areas are too small to be isolated completely with the mechanical method. Antibodies can be utilized to purify the rare undifferentiated cells; however, this type of purification cannot be used in xeno-free culture systems. To avoid the loss of valuable hPSCs, we developed a novel method to isolate undifferentiated hPSCs from extremely differentiated colonies that could be easily adapted to xeno-free culture conditions. This protocol involves dissecting away differentiated areas, dissociating the remaining colony into clumps, seeding small clumps into new dishes, and picking undifferentiated colonies for expansion. Using this method, we routinely achieve completely undifferentiated colonies in one passage without the use of antibody-based purification.

Breast carcinoma is the most common malignancy in women. The progression of tumor is associated with overexpression of inhibitors of apoptosis proteins (IAPs) such as survivin and multidrug resistant P-glycoprotein (P-gp). PI3K/Akt pathway is involved in cell cycle progression, apoptosis and neoplastic transformation. PI3K/Akt has been shown to regulate survivin in breast cancer. The aim of this study was to investigate the expression of survivin and P-gp, the modulation of survivin by P-gp in PI3K/Akt during the progression of drug resistance (MDR) in MCF-7 breast cancer cells and adriamycin (ADR)-resistant MCF-7/ADR cells. The expression of survivin and P-gp in MCF-7/ADR cells were higher than that of the MCF-7 cells using RT-PCR and Western blot analysis. Survivin transcription was associated with P-glycoprotein/MDR1 overexpression using promoter activity analysis. LY294002, specific inhibitor of PI3K could suppress survivin and P-gp expression, decreased survivin promoter activity and enhanced cell sensitivity to drugs. This study shows survivin transcription was associated with P-glycoprotein/MDR1 overexpression, PI3k/Akt pathway was involved in P-glycoprotein/MDR1 associated survivin transcription activity in the multidrug resistant MCF-7 breast cancer cells.

In embryonic stem (ES) cells, leukemia inhibitory factor (LIF)/STAT3, wnt and nodal/activin signaling are mainly active to control pluripotency during expansion. To maintain pluripotency, ES cells are typically cultured on feeder cells of varying origins. Murine ES cells are commonly cultured on murine embryonic fibroblasts (MEFs), which senesce early and must be frequently prepared. This process is laborious and leads to batch variation presenting a challenge for high-throughput ES cell expansion. Although some cell lines can be sustained by exogenous LIF, this method is costly. We present here a novel and inexpensive culture method for expanding murine ES cells on human foreskin fibroblast (HFF) feeders. After 20 passages on HFFs without LIF, ES cell lines showed normal expression levels of pluripotency markers, maintained a normal karyotype and retained the ability to contribute to the germline. As HFFs do not senesce for at least 62 passages, they present a vast supply of feeders.

Background We have recently reported the expression of murine Implantation Serine Proteinase genes in pre-implantation embryos (ISP1) and uterus (ISP1 and ISP2). These proteinases belong to the S1 proteinase family and are similar to mast cell tryptases, which function as multimers. Results Here, we report the purification and initial characterization of ISP1 and 2 with respect to their physico-chemical properties and physiological function. In addition to being co-expressed in uterus, we show that ISP1 and ISP2 are also co-expressed in the pre-implantation embryo. Together, they form a heterodimer with an approximate molecular weight of 63 kD. This complex is the active form of the enzyme, which we have further characterized as being trypsin-like, based on substrate and inhibitor specificities. In addition to having a role in embryo hatching and outgrowth, we demonstrate that ISP enzyme is localized to the site of embryo invasion during implantation and that its activity is important for successful implantation in vivo. Conclusion On the basis of similarities in structural, chemical, and functional properties, we suggest that this ISP enzyme complex represents the classical hatching enzyme, strypsin. Our results demonstrate a critical role for ISP in embryo hatching and implantation.

We have described two novel implantation serine proteinase (ISP) genes that are expressed during the implantation period. The ISP1 gene may encode the embryo-derived enzyme strypsin, which is necessary for blastocyst hatching in vitro and the initiation of invasion. The ISP2 gene, which encodes a related tryptase, is expressed in endometrial glands and is regulated by progesterone during the peri-implantation period. Based on similarities between ISP2 gene expression and that of a progesterone-regulated lumenal serine proteinase activity associated with lysis of the zona pellucida, we have suggested that the strypsin related protein, ISP2, may encode a zona lysin proteinase. Recently strypsin has also been found within uterine fluid, suggesting a second potential role in hatching. Consistently, we have discovered that ISP1 is also expressed in the uterine secretory gland at the time of hatching. In this study we demonstrate that both ISP1 and ISP2 are secreted together into the uterine lumen at peri-implantation, and that the appearance of ISP protein is regulated positively at the transcriptional level by progesterone and negatively at the posttranscriptional level by estrogen. This negative regulation by estrogen may be overridden in pregnancy as ISP protein expression is restored during oil-induced decidualization. ISP1 and ISP2 proteins are also expressed in proestrous suggesting additional roles in the endometrial cycle.

Induced pluripotent stem cells (iPSCs) are typically derived in adherent culture. Here we report fast and efficient derivation of mouse iPSCs in stirred suspension bioreactors, with and without the use of c-Myc. Suspension-reprogrammed cells expressed pluripotency markers, showed multilineage differentiation in vitro and in vivo, and contributed to the germline in chimeric mice. Suspension reprogramming has the potential to accelerate and standardize iPSC research.

Protein kinase catalyzes the transfer of the γ-phosphoryl group from ATP to the hydroxyl groups of protein side chains, which plays critical roles in signal transduction pathways by transmitting extracellular signals across the plasma membrane and nuclear membrane to the destination sites in the cytoplasm and the nucleus. Protein kinase C (PKC) is a superfamily of phospholipid-dependent Ser/Thr kinase. There are at least 12 isozymes in PKC family. They are distributed in different tissues and play different roles in physiological processes. On account of their concern with a variety of pathophysiologic states, such as cancer, inflammatory conditions, autoimmune disorder, and cardiac diseases, the inhibitors, which can inhibit the activity of PKC and the interaction of cytokine with receptor, and interfere signal transduction pathway, may be candidates of therapeutic drugs. Therefore, intense efforts have been made to develop specific protein kinase inhibitors as biological tools and therapeutic agents. This article reviews the recent development of some of PKC inhibitors based on their interaction with different conserved domains and different inhibition mechanisms.