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Chlamydia trachomatis (CT) infection is the most prevalent sexually transmitted bacterial disease worldwide. However, unlike that in female infertility, the role of CT infection in male infertility remains controversial. The objective of this retrospective study was to explore the impacts of CT infection in the genital tract on sperm quality, sperm acrosin activity, antisperm antibody levels, and inflammation in a large cohort of infertile males in China. A total of 7154 semen samples were collected from infertile male subjects, 416 of whom were CT positive (CT+ group) and 6738 of whom were CT negative (CT- group), in our hospital between January 2016 and December 2018. Routine semen parameters (semen volume, pH, sperm concentration, viability, motility, morphology, etc.), granulocyte elastase levels, antisperm antibody levels, and sperm acrosin activity were compared between the CT+ and CT- groups. Our results showed that CT infection was significantly correlated with an abnormally low semen volume, as well as an increased white blood cell count and granulocyte elastase level (all P < 0.05) in the semen of infertile males; other routine semen parameters were not negatively impacted. The antisperm antibody level and sperm acrosin activity were not affected by CT infection. These findings suggested that CT infection might contribute to inflammation and hypospermia but does not impair sperm viability, motility morphology, and acrosin activity or generate antisperm antibodies in the infertile males of China.

In the present study, we performed an exome-wide investigation of the burden of rare disease-causing variants for major depressive disorder (MDD) using 16,702 samples from UK biobank. Gene-based association analysis and candidate gene prioritization analysis indicated that FOXH1 have significant association with MDD. In addition, sphingolipid metabolism pathway was found to be less enriched with rare disease-causing variants in the MDD group, suggesting that this gene set may be involved in the pathophysiology of MDD.

To elucidate the symptoms and pathogens diversity of corn Fusarium sheath rot (CFSR), diseased samples were collected from 21 county-level regions in 12 prefecture-level districts of Sichuan Province from 2015 to 2018 in the present study. In the field, two symptom types appeared including small black spots with a linear distribution and wet blotches with a tawny or brown color. One hundred thirty-seven Fusarium isolates were identified based on morphological characteristics and phylogenetic analysis (EF1-α), and Koch’s postulates were also assessed. The results identified the isolates as 8 species in the Fusarium genus, including F. verticillioides, F. proliferatum, F. fujikuroi, F. asiaticum, F. equiseti, F. meridionale, F. graminearum and F. oxysporum, with isolation frequencies of 30.00, 22.67, 15.33, 7.33, 6.00, 5.33, 3.33 and 1.33%, respectively. Fusariumverticillioides and F. proliferatum were the dominant and subdominant species, respectively. Two or more Fusarium species such as F. verticillioides and F. proliferatum were simultaneously identified at a mixed infection rate of 14.67% in the present study. The pathogenicity test results showed that F. proliferatum and F. fujikuroi exhibited the highest virulence, with average disease indices of 30.28 ± 2.87 and 28.06 ± 1.96, followed by F. equiseti and F. verticillioides, with disease indices of 21.48 ± 2.14 and 16.21 ± 1.84, respectively. Fusarium asiaticum, F. graminearum and F. meridonale showed lower virulence, with disease indices of 13.80 ± 2.07, 11.57 ± 2.40 and 13.89 ± 2.49, respectively. Finally, F. orysporum presented the lowest virulence in CFSR, with a disease index of 10.14 ± 1.20. To the best of our knowledge, this is the first report of F. fujikuroi, F. meridionale and F. asiaticum as CFSR pathogens in China.

Myeloid-derived suppressor cells (MDSCs) suppress antitumor immune activities and facilitate cancer progression. Although the concept of immunosuppressive MDSCs is well established, the mechanism that MDSCs regulate non-small cell lung cancer (NSCLC) progression through the paracrine signals is still lacking. Here, we reported that the infiltration of MDSCs within NSCLC tissues was associated with the progression of cancer status, and was positively correlated with the Patient-derived xenograft model establishment, and poor patient prognosis. Intratumoral MDSCs directly promoted NSCLC metastasis and highly expressed chemokines that promote NSCLC cells invasion, including CCL11. CCL11 was capable of activating the AKT and ERK signaling pathways to promote NSCLC metastasis through the epithelial-mesenchymal transition (EMT) process. Moreover, high expression of CCL11 was associated with a poor prognosis in lung cancer as well as other types of cancer. Our findings underscore that MDSCs produce CCL11 to promote NSCLC metastasis via activation of ERK and AKT signaling and induction of EMT, suggesting that the MDSCs-CCL11-ERK/AKT-EMT axis contains potential targets for NSCLC metastasis treatment.

MSCs are kind of cultured cells that reside in different tissues as inducers or regulators of physiological and pathological processes. Here, we derived MSCs from amniotic fluid and compared their differentiation ability and immunosuppression effect on PHA‐activated PBMC with those of MSCs isolated from umbilical cords. Amniotic fluid MSCs were isolated and cultured on commercial AFC medium and classic MSC medium, and the number and size of colonies were used to evaluate differences in primary and passaged culture. Rate of proliferation, population doubling time, cell morphology, cell surface markers and mRNA expression were measured in subcultured cells. Furthermore, a comparative study was performed with umbilical cord MSCs to assess the ability of differentiation and immunosuppressive effect of PHA‐stimulated PBMCs. Amniotic fluid MSCs were isolated and expanded by three methods, and exhibited nearly all the characteristics of umbilical cord MSCs. Compared with umbilical cord MSCs, amniotic fluid MSCs had an enhanced osteogenic and chrondrogenic differentiation capability, and stronger immunosuppression effect of inhibition of PHA‐activated PBMC division. Culture with commercial AFCs medium yielded the highest percentage of CD105 expression and showed some advantages in primary cell isolation, cell source‐specific marker retention and cell proliferation. We demonstrated that amniotic fluid MSCs exhibited some advantages over umbilical cord MSCs, and different culture media caused cell proliferation, cell surface marker and cell morphology change, but were not associated with varying differentiation capability and immune effects.

Background: The quality of the early embryo is vital to embryonic development and implantation. As a highly conserved serine/threonine kinase, p21-activated kinase 2 (Pak2) participates in diverse biologic processes, especially in cytoskeleton remodeling and cell apoptosis. However, the role of Pak2 in preimplantation embryos remains unelucidated. Methods: In the present work, Pak2 was depleted using a specific small interfering RNA in early mouse embryos, validating the unique roles of Pak2 in spindle assembly and DNA repair during mice early embryonic development. We also employed immunoblotting, immunostaining, in vitro fertilization (IVF) and image quantification analyses to test the Pak2 knockdown on the embryonic development progression, spindle assembly, chromosome alignment, oxidative stress, DNA lesions and blastocyst cell apoptosis.Results: We found that Pak2 deletion significantly reduced blastocyst formation of early embryos. In addition, Pak2 depletion led to dramatically increased abnormal spindle assembly and chromosomal aberrations in the embryos. We noted the overproduction of reactive oxygen species (ROS) with Pak2 knockdown in embryos. Pak2 knockdown also resulted in the accumulation of phosphorylated γH2AX, indicative of increased embryonic DNA damage. Commensurate with this, a significantly augmented rate of blastocyst cell apoptosis was detected in Pak2-KD embryos compared to their controls. Conclusions: Collectively, our data suggest that Pak2 may serve as an important regulator of spindle assembly and DNA repair, and thus participate in the development of early mouse embryos.

β‐thalassaemia is a prevalent hereditary haematological disease caused by mutations in the human haemoglobin β (HBB) gene. Among them, the HBB IVS2‐654 (C > T) mutation, which is in the intron, creates an aberrant splicing site. Bone marrow transplantation for curing β‐thalassaemia is limited due to the lack of matched donors. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9), as a widely used tool for gene editing, is able to target specific sequence and create double‐strand break (DSB), which can be combined with the single‐stranded oligodeoxynucleotide (ssODN) to correct mutations. In this study, according to two different strategies, the HBB IVS2‐654 mutation was seamlessly corrected in iPSCs by CRISPR/Cas9 system and ssODN. To reduce the occurrence of secondary cleavage, a more efficient strategy was adopted. The corrected iPSCs kept pluripotency and genome stability. Moreover, they could differentiate normally. Through CRISPR/Cas9 system and ssODN, our study provides improved strategies for gene correction of β‐Thalassaemia, and the expression of the HBB gene can be restored, which can be used for gene therapy in the future.

Albeit microRNAs (miRNAs) have become increasingly appreciated for their essential roles in innate immune responses to viral infections; however, it is unknown how host miRNAs regulate influenza A virus (IAV)-induced inflammation. The aim of our study was to investigate the role of miR-146a in IAV replication in vitro and in vivo. In vitro, we found miR-146a was significantly upregulated in A549 cells with IAV infection. Overexpression of miR-146a promoted IAV replication, while downregulation of miR-146a repressed replication. We found that miR-146a diminished type I interferon (IFN) responses by decreasing IFN-β production and IFN-stimulated gene (ISG) expression. Furthermore, we found the IFNs level and IAV replication regulated by miR-146a inhibitor was partially reversed by depletion of interferon receptor (IFNAR) 1 or 2. In addition, we found that miR-146a directly targets tumor necrosis factor receptor association factor 6 (TRAF6), which is involved in the production of type I IFN, and TRAF6 overexpression reversed the replication-promoting effect of miR-146a on IAV. In vivo, inhibition of miR-146a alleviated IAV-induced mice lung injury and promoted survival rates by promoting type I antiviral activities. It is, therefore, concluded that downregulation of miR-146a inhibits IAV replication by enhancing type I IFN response through its target gene TRAF6 in vitro and in vivo, suggesting miR-146a antagomir might be a potential therapeutic target during IAV infection.

Background Neoadjuvant chemotherapy (NAC) induces a pathological complete response (pCR) in ~ 30% of patients with breast cancer. However, aberrant DNA methylation alterations are frequent events during breast cancer progression and acquisition of chemoresistance. We aimed to characterize the inter- and intra-tumor methylation heterogeneity (MH) in breast cancer following NAC. Methods DNA methylation profiles of spatially separated regions of breast tumors before and after NAC treatment were investigated using high-density methylation microarray. Methylation levels of genes of interest were further examined using multiplexed MethyLight droplet digital PCR (ddPCR). Results We have discovered different levels of intra-tumor MH in breast cancer patients. Moreover, NAC dramatically altered the methylation profiles and such changes were highly heterogeneous between the patients. Despite the high inter-patient heterogeneity, we identified that stem cell quiescence-associated genes ALDH1L1, HOPX, WNT5A and SOX9 were convergently hypomethylated across all the samples after NAC treatment. Furthermore, by using MethyLight ddPCR, we verified that the methylation levels of these 4 genes were significantly lower in breast tumor samples after NAC than those before NAC. Conclusions Our study has revealed that NAC dramatically alters epigenetic heterogeneity in breast cancer and induces convergent hypomethylation of stem cell quiescence-associated genes, ALDH1L1, HOPX, WNT5A and SOX9, which can potentially be developed as therapeutic targets or biomarkers for chemoresistance. Electronic supplementary material The online version of this article (10.1186/s13578-019-0278-y) contains supplementary material, which is available to authorized users.

Noninvasive prenatal testing (NIPT) for single gene disorders remains challenging. One approach that allows for accurate detection of the slight increase of the maternally inherited allele is the relative haplotype dosage (RHDO) analysis, which requires the construction of parental haplotypes. Recently, the nanopore sequencing technologies have become available and may be an ideal tool for direct construction of haplotypes. Here, we explored the feasibility of combining nanopore sequencing with the RHDO analysis in NIPT of β-thalassemia. Thirteen families at risk for β-thalassemia were recruited. Targeted region of parental genomic DNA was amplified by long-range PCR of 10 kb and 20 kb amplicons. Parental haplotypes were constructed using nanopore sequencing and next generation sequencing data. Fetal inheritance of parental haplotypes was classified by the RHDO analysis using data from maternal plasma DNA sequencing. Haplotype phasing was achieved in 12 families using data from 10 kb library. While data from the 20 kb library gave a better performance that haplotype phasing was achieved in all 13 families. Fetal status was correctly classified in 12 out of 13 families. Thus, targeted nanopore sequencing combined with the RHDO analysis is feasible to NIPT for β-thalassemia.

β-thalassemia, an autosomal recessive blood disorder that reduces the production of hemoglobin, is majorly caused by the point mutation of the HBB gene resulting in reduced or absent β-globin chains of the hemoglobin tetramer. Animal models recapitulating both the phenotype and genotype of human disease are valuable in the exploration of pathophysiology and for in vivo evaluation of novel therapeutic treatments. The docile temperament, short vital cycles, and low cost of rabbits make them an attractive animal model. However, β-thalassemia rabbit models are currently unavailable. Here, using CRISPR/Cas9-mediated genome editing, we point mutated the rabbit β-globin gene HBB2 with high efficiency and generated a β-thalassemia rabbit model. Hematological and histological analyses demonstrated that the genotypic mosaic F0 displayed a mild phenotype of anemia, and the heterozygous F1 exhibited typical characteristics of β-thalassemia. Whole-blood transcriptome analysis revealed that the gene expression was altered in HBB2-targeted when compared with WT rabbits. And the highly expressed genes in HBB2-targeted rabbits were enriched in lipid and iron metabolism, innate immunity, and hematopoietic processes. In conclusion, using CRISPR-mediated HBB2 knockout, we have created a β-thalassemia rabbit model that accurately recapitulates the human disease phenotype. We believe this tool will be valuable in advancing the investigation of pathogenesis and novel therapeutic targets of β-thalassemia and associated complications.

Mutations in the LPL gene lead to familial hypertriglyceridemia (FHTG) . We have previously generated an iPSC line (AHQUi001-A) from a FHTG patient with a heterozygous p.C310R (c.928 T > C) mutation in the LPL gene. Here we genetically corrected the C310R mutation in the LPL gene using CRISPR/Cas9 technology to generate AHQUi001-A-1, which demonstrates normal karyotype, morphology, pluripotency, and potential to differentiate towards three germ layers.

Dicarboximide fungicides mainly including procymidone, iprodione, vinclozolin, and dimethachlon are often applied as protective fungicides, 3,5-dichloroaniline (3,5-DCA) is their common metabolite in plant and environment. In this study, the acute toxicity of procymidone, iprodione and their metabolite of 3,5-DCA toward zebrafish was evaluated by semi-static method. The enrichment and metabolism of procymidone and iprodione in zebrafish were also clarified. The results indicated that procymidone and iprodione exhibited moderately toxic to adult zebrafish with the LC50 of 2.00 mg/L, 5.70 mg/L at 96 h. Both procymidone and iprodione could be metabolized to 3,5-DCA in zebrafish, which showed higher toxic to adult zebrafish with the LC50 of 1.64 mg/L at 96 h. From the perspective of histomorphology, for all treatment groups, the brain of the zebrafish was significantly damaged, while the damage to gut and gills was lighter. For procymidone, the biological concentration factor (BCF8d) were 236 and 246 at the exposure concentration of 0.2 mg/L and 0.04 mg/L, and the BCF8d were 3.2 and 2.4 for iprodione at the exposure concentration of 0.5 mg/L and 0.1 mg/L. Therefore, the procymidone and iprodione were moderate-enriched and low-enriched in zebrafish, respectively.

Background: X-linked adrenoleukodysrophy (ALD) is an inherited peroxisomal metabolism disorder, results from the loss-of-function mutation of ATP-binding cassette protein subfamily D1 (ABCD1) gene. The dysfunction of ALD protein, a peroxisomal ATP-binding cassette transporter, results in the excessive saturated very long chain fatty acids (VLCFAs) accumulation in organs including brain, spine and adrenal cortex. X-ALD is characterized as the childhood, adolescent, adult cerebral ALD, adrenomyeloneuropathy (AMN), adrenal insufficiency, and asymptomatic phenotypes, exhibiting a high variety of clinical neurological manifestations with or without adrenocortical insufficiency. Results: In this study, we reported two cases of X-ALD, which were firstly diagnosed as adrenal insufficiency (Addison’s disease) and treated with adrenocortical supplement. However, both of the cases progressed as neurological symptoms and signs after decades. Elevated VLCFAs level, brain MRI scan and genetic analysis confirmed final diagnosis. In addition, we identified two novel mutations of ABCD1 gene, c.874_876delGAG (p.Glu292del) and c.96_97delCT (p.Tyr33Profs*161) in exon 1 of ABCD1 gene. Sanger sequencing confirmed that the proband’s mother of the first case was heterozygous carrying the same variant. Adrenal insufficiency-only type is very rare, however, it may be the starting performance of X-ALD. Conclusions: The early warning manifestations should be noticed, and the probability of X-ALD should be considered. This report could be beneficial for the early diagnosis and genetic counseling for patients with X-ALD.

Anthracnose caused by Colletotrichum spp. is an important disease of blueberries and results in large economic losses for blueberry growers. Samples of anthracnose were collected from six main blueberry cultivation areas in Sichuan Province. In total, 74 Colletotrichum isolates were obtained through a single-spore purification method and identified to the species through morphological characteristics and phylogenetic analyses based on partial DNA sequences of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), internal transcribed spacer (ITS) regions, and the &beta, tubulin (TUB2), actin (ACT) and calmodulin (CAL) genes. Among all species, Colletotrichum fructicola was the most dominant species, with an isolation percentage of up to 66.2% in Sichuan, followed by Colletotrichum siamense (17.6%), C. kahawae (5.4%), C. karstii (5.4%), C. nymphaeae (2.7%) and C. sichuaninese (2.7%). Pathogenicity tests showed all species were able to cause typical anthracnose symptoms on blueberry leaves and stems. Colletotrichum fructicola was the predominant species with strong aggressiveness. Moreover, C. fructicola, C. kahawae, C. sichuaninese and C. nymphaeae are first reported here to cause blueberry anthracnose. This study provides a comprehensive reference for the association of different Colletotrichum spp., which may support the sustainable management of blueberry anthracnose.

Many pathogenic fungi depend on the development of specialized infection structures called appressoria to invade their hosts and cause disease. Impairing the function of fungal infection structures therefore provides a potential means by which diseases could be prevented. In spite of this extraordinary potential, however, relatively few anti-penetrant drugs have been developed to control fungal diseases, of either plants or animals. In the present study, we report the identification of compounds that act specifically to prevent fungal infection. We found that the organization of septin GTPases, which are essential for appressorium-mediated infection in the rice blast fungus Magnaporthe oryzae, requires very-long-chain fatty acids (VLCFAs), which act as mediators of septin organization at membrane interfaces. VLCFAs promote septin recruitment to curved plasma membranes and depletion of VLCFAs prevents septin assembly and host penetration by M. oryzae. We observed that VLCFA biosynthesis inhibitors not only prevent rice blast disease, but also show effective, broad-spectrum fungicidal activity against a wide range of fungal pathogens of maize, wheat and locusts, without affecting their respective hosts. Our findings reveal a mechanism underlying septin-mediated infection structure formation in fungi and provide a class of fungicides to control diverse diseases of plants and animals.

Primary Hypertriglyceridemia refers to a loss-of-function genetic defect which prevents the triglyceride (TG) in chylomicrons (CM) from lipolysis, leading to the accumulation of TG. The mutation of lipoprotein lipase (LPL) gene has been recognized as the main cause of primary hypertriglyceridemia. Recently, a new LPL gene mutation p.C310R(c. T928C) was identified in a family with hypertriglyceridemia. The proband was manifested by severe hypertriglyceridemia and diabetes. Skeletal muscle is the major LPL-synthesizing tissue and insulin response target tissue. However, little is known about the effects of LPL gene mutation on skeletal muscle. This study is intended to observe the effects of LPL-C310R mutation on glycolipid metabolism and skeletal muscle. We found that a significantly decreased LPL plasma concentration, activity and the expression levels in skeletal muscle were observed in LplC310R/+ mice comparing to wild type mice. Those mutant mice also exhibited increased fasting plasma TG, free fat acids (FFA) and insulin, as well as FFA in muscle, and decreased glucose tolerance. Enhanced expression of BIP and elevated phosphorylation of IRE1α were observed in skeletal muscle, suggesting increased endoplasmic reticulum stress (ERS). Consistent with this, increased phosphorylation of JNK was also observed. Meanwhile, remarkably enhanced phosphorylation of IRS-1 (Ser307) and decreased phosphorylation of AKT were observed in skeletal muscle of mutant mice, suggesting impaired insulin signaling. Significant lipid deposition and morphological changes in endoplasmic reticulum and mitochondria were observed in the skeletal muscle of mutant mice but not in wild type control. Results demonstrate Lpl C310R mutation caused impaired glucose tolerance, ER stress and impaired insulin signaling in skeletal muscle.

Many species of the genus Bipolaris are important plant pathogens and often cause leaf spot, root rot, and seedling blight in an extremely wide range of hosts around the world. In recent years, maize leaf spot caused by Bipolaris species has frequently occurred with complex symptoms and is becoming increasingly serious in Sichuan Province of China. To investigate the population diversity of Bipolaris spp. and their corresponding symptoms in maize, 747 samples of maize leaf spot were collected from 132 sampling sites in 19 administrative districts of Sichuan Province from 2011 to 2018. Based on morphological characteristics, pathogenicity testing, and phylogenetic analysis of the rDNA internal transcribed spacer (ITS) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes, a total of 1186 Bipolaris isolates were identified as B. maydis, B. zeicola, B. cynodontis, B. oryzae, B. setariae, and B. saccharicola, among which B. maydis and B. zeicola were the dominant pathogenic species, accounting for 57.34% and 42.07% of the isolates, respectively. We found that B. zeicola isolates were mainly distributed in high altitude and cool mountainous areas, while B. maydis was more widely distributed in Sichuan Province. The typical symptoms caused by the Bipolaris species were clearly distinct in maize. The typical symptoms caused by B. maydis were elongated strip lesions, or fusiform, elliptical lesions, and those caused by B. zeicola were narrow linear lesions. Herein, B. saccharicola was first reported on maize and caused subrotund lesions. This study provides useful information for disease diagnosis and management for Bipolaris leaf spot in maize.

Familial hypertriglyceridemia (FHTG) is an autosomal dominant disorder of lipoprotein metabolism, partly caused by mutations in the LPL gene, which encodes for the lipoprotein lipase. LPL deficiency can impair triglyceride hydrolysis which causes elevated plasma triglyceride levels. An induced pluripotent stem cell (iPSC) line was generated from peripheral blood mononuclear cells (PBMCs) of a 53 years-old male patient with FHTG who had a heterozygous p.C310R (c.928 T > C) mutation in the LPL gene based on the sendai virus delivery system. The cellular model will offer a powerful tool to investigate pathogenic mechanisms in FHTG and to develop a treatment for FHTG.

Animal models used to evaluate efficacies of immune checkpoint inhibitors are insufficient or inaccurate. We thus examined two xenograft models used for this purpose, with the aim of optimizing them. One method involves the use of peripheral blood mononuclear cells and cell line-derived xenografts (PBMCs-CDX model). For this model, we implanted human lung cancer cells into NOD-scid-IL2Rg−/− (NSI) mice, followed by injection of human PBMCs. The second method involves the use of hematopoietic stem and progenitor cells and CDX (HSPCs-CDX model). For this model, we first reconstituted the human immune system by transferring human CD34+ hematopoietic stem and progenitor cells (HSPCs-derived humanized model) and then transplanted human lung cancer cells. We found that the PBMCs-CDX model was more accurate in evaluating PD-L1/PD-1 targeted immunotherapies. In addition, it took only four weeks with the PBMCs-CDX model for efficacy evaluation, compared to 10–14 weeks with the HSPCs-CDX model. We then further established PBMCs-derived patient-derived xenografts (PDX) models, including an auto-PBMCs-PDX model using cancer and T cells from the same tumor, and applied them to assess the antitumor efficacies of anti-PD-L1 antibodies. We demonstrated that this PBMCs-derived PDX model was an invaluable tool to study the efficacies of PD-L1/PD-1 targeted cancer immunotherapies. Overall, we found our PBMCs-derived models to be excellent preclinical models for studying immune checkpoint inhibitors.

Spinocerebellar ataxia type 3 (SCA3) is caused by an abnormal expansion of the cytosine-adenine-guanine (CAG) triplet in ATXN3, which translates into a polyglutamine (polyQ) tract within ataxin-3 (ATXN3) protein. Although the pathogenic mechanisms remain unclear, it is well established that expression of mutant forms of ATXN3 carrying an expanded polyQ domain are involved in SCA3 pathogenesis, and several strategies to suppress mutant ATXN3 have shown promising potential for SCA3 treatment. In this study, we described successful clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated deletion of the expanded polyQ-encoding region of ATXN3 in induced pluripotent stem cells (iPSCs) derived from a SCA3 patient, and these patient-specific iPSCs retained pluripotency and neural differentiation following expanded polyQ deletion. Furthermore, the ubiquitin-binding capacity of ATXN3 was retained in the neural cells differentiated from the corrected iPSCs. For the first time, this work provides preliminary data for gene editing by CRISPR/Cas9 in SCA3, and demonstrates the feasibility of using a single-guide RNA pair to delete the expanded polyQ-encoding region of ATXN3, suggesting the potential efficacy of this method for future therapeutic application.

Objective To systematically investigate the prevalence and genetic characteristics of 4CMenB and rLP2086 vaccine candidates among Neisseria meningitidis serogroup B (NmB) in China. Methods A total of 485 NmB strains isolated in 29 provinces of China between 1968 and 2016 were selected from the culture collection of the national reference laboratory according to the isolation year, location, and source. Multi-locus sequence typing (MLST) and porA gene sequencing were performed on all 485 study strains; PCR was used to detect the fHbp, nadA, and nhba gene of 432 strains; positive amplification products from the fHbp and nadA genes from all strains, as well as those of the nhba gene from 172 representative strains, were sequenced. Results MLST results showed that the predominant (putative) clonal complexes (CCs) of NmB isolates have changed over time in China. While strains that could not be assigned to existing (p)CCs were the biggest proportion, CC4821 was the most prevalent lineage (36.0%) since 2005. PCR and sequence analysis revealed that the 4CMenB and rLP2086 vaccine candidates were highly diverse. Respectively, 152 PorA genotypes and 83 VR2 sequences were identified with significant diversity within a single CC; the complete nadA gene was found in ten of 432 study strains; fHbp was present in most strains (422/432) with variant 2 predominating (82.9%) in both patient- and carrier- derived isolates; almost all strains harbored the nhba gene while sequences were diverse. Conclusions With regards to clonal lineages and vaccine candidate proteins, NmB isolates from China were generally diverse. Further studies should be performed to evaluate the cross-protection of present vaccines against Chinese NmB strains.

Mitochondrial diseases are maternally inherited heterogeneous disorders that are primarily caused by mitochondrial DNA (mtDNA) mutations. Depending on the ratio of mutant to wild-type mtDNA, known as heteroplasmy, mitochondrial defects can result in a wide spectrum of clinical manifestations. Mitochondria-targeted endonucleases provide an alternative avenue for treating mitochondrial disorders via targeted destruction of the mutant mtDNA and induction of heteroplasmic shifting. Here, we generated mitochondrial disease patient-specific induced pluripotent stem cells (MiPSCs) that harbored a high proportion of m.3243A>G mtDNA mutations and caused mitochondrial encephalomyopathy and stroke-like episodes (MELAS). We engineered mitochondrial-targeted transcription activator-like effector nucleases (mitoTALENs) and successfully eliminated the m.3243A>G mutation in MiPSCs. Off-target mutagenesis was not detected in the targeted MiPSC clones. Utilizing a dual fluorescence iPSC reporter cell line expressing a 3243G mutant mtDNA sequence in the nuclear genome, mitoTALENs displayed a significantly limited ability to target the nuclear genome compared with nuclear-localized TALENs. Moreover, genetically rescued MiPSCs displayed normal mitochondrial respiration and energy production. Moreover, neuronal progenitor cells differentiated from the rescued MiPSCs also demonstrated normal metabolic profiles. Furthermore, we successfully achieved reduction in the human m.3243A>G mtDNA mutation in porcine oocytes via injection of mitoTALEN mRNA. Our study shows the great potential for using mitoTALENs for specific targeting of mutant mtDNA both in iPSCs and mammalian oocytes, which not only provides a new avenue for studying mitochondrial biology and disease but also suggests a potential therapeutic approach for the treatment of mitochondrial disease, as well as the prevention of germline transmission of mutant mtDNA. Electronic supplementary material The online version of this article (10.1007/s13238-017-0499-y) contains supplementary material, which is available to authorized users.

ZBTB7A plays important roles in several biological processes, including silencing of the fetal γ-globin genes, hematopoiesis, primed-to-naive transition, etc. Meanwhile, it is also associated with Oncogenic transformation and tumor progression. However, the mechanism of ZBTB7A function is not fully understood yet. Here, we generated a homozygous ZBTB7A knockout human induced pluripotent stem cell (iPSC) line, GZHMCi007-A by the CRISPR/Cas9-mediated homology-dependent DNA repair method. The iPSCs of ZBTB7A-/- established by us is a powerful tool for related research.

Increasing evidence suggests that undernutrition during the fetal period may lead to glucose intolerance, impair the insulin response and induce insulin resistance (IR). Considering the importance of chromium (Cr) in maintaining carbohydrate metabolism, the present study aimed to determine the effects of maternal low Cr (LC) on glucose metabolism in C57BL mice offspring, and the involved mechanisms. Weaned C57BL mice were born from mothers fed a control diet or LC diet, and were then fed a control or LC diet for 13 weeks. Subsequently, the liver microRNA (miRNA/miR) expression profile was analyzed by miRNA array analysis. A maternal LC diet increased fasting serum glucose (P

Human MSX1 gene is mapped to chromosome 4 and encodes a 303aa homeobox protein MSX1. MSX1 expression appears during early tooth development of vertebrate embryogenesis. Mutations in this protein are related to human tooth anomalie, cleft lip and palate and congenital ectodermal dysplasia syndrome. Most of the confirmed pathogenic mutations are located in exon2 encoded homeobox domain. Here, we report the establishment of MSX1 gene knockout human embryonic stem (hES) cell lines by CRISPR-Cas9 technology. These cell lines provide good materials for further studies of the roles MSX1 plays in human tooth development and congenital tooth agenesis.

47, XXX syndrome is one of several sex-chromosomal aneuploidies, and it has an incidence of approximately 1/1000 in newborn females. Because of heterogeneity in X-inactivation, these patients may exhibit a variety of clinical symptoms. Here, we report the generation of an integration-free human induced pluripotent stem cell line (GZHMUi001-A) by using Sendai virus to reprogram peripheral blood mononuclear cells from a 47, XXX syndrome patient with premature ovarian failure. This 47, XXX iPS cell line has characteristics of pluripotent stem cells and is a useful tool for the investigation of this X chromosome aneuploid disease.

Pre-maturation aging of immature oocytes may adversely affect the fate of an oocyte. Oxidative stress is one of the most detrimental factors affecting oocyte developmental competence and maturation during aging. In this study, experiments were designed to examine whether supplementation of antioxidants in a culture medium could protect immature mouse oocytes from damages caused by oxidative stress. Mouse oocytes at germinal vesicle stage were prevented from meiosis resumption and cultured in a medium with or without antioxidants for 12–36 h to allow oocytes to undergo aging. After aging, oocytes were cultured for maturation. Nuclear maturation, mitochondria activity, spindle morphology and DNA integrity were examined after maturation. It was found that antioxidants had protective effects on the oocytes in terms of nuclear maturation, functional mitochondria, spindle morphology and DNA integrity. As aging time was prolonged from 12 to 36 h, the protective effect of antioxidants became more obvious. However, as compared with oocytes without aging, it was found that aging significantly inhibited nuclear maturation, impaired mitochondria function, and damaged the spindle and DNA. These results indicate that pre-maturation aging is detrimental to oocytes’ competence to undergo maturation and other cellular activities, and antioxidants can protect oocytes from damages caused by aging.

// Yu Lun 1, * , Xiaofang Sun 1, * , Ping Wang 1 , Jingwei Chi 1 , Xu Hou 1 and Yangang Wang 1 1 Department of Endocrinology and Metabolic Diseases, The Affiliated Hospital of Qingdao University, Qingdao, China * Yu Lun and Xiaofang Sun contributed equally to this work Correspondence to: Yangang Wang, email: wangyangang9111@126.com Xu Hou, email: lunyu9111@gmail.com Keywords: lipoprotein lipase, hypertriglyceridemia, mutation, acute pancreatitis, compound heterozygosity Received: November 16, 2016 Accepted: April 11, 2017 Published: May 10, 2017 ABSTRACT Lipoprotein lipase (LPL) is widely expressed in skeletal muscles, cardiac muscles as well as adipose tissue and involved in the catabolism of triglyceride. Herein we have systematically characterized two novel loss-of-function mutations in LPL from a Chinese family in which afflicted members were manifested by severe hypertriglyceridemia and recurrent pancreatitis. DNA sequencing revealed that the proband was a heterozygote carrying a novel c.T928C (p.C310R) mutation in exon 6 of the LPL gene. Another member of the family was detected to be a compound heterozygote who along with the c.T928C mutation also carried a novel missense mutation c.A1187T (p.E396V) in exon 8 of the LPL gene. Furthermore, COS-1 cells were transfected with lentiviruses containing the mutant LPL genes. While C310R markedly reduced the overall LPL protein level, COS-1 cells carrying E396V or double mutations contained similar overall LPL protein levels to the wild-type. The specific activity of the LPL mutants remained at comparable magnitude to the wild-type. However, few LPL were detected in the culture medium for the mutants, suggesting that both mutations caused aberrant triglyceride catabolism. More specifically, E396V and double mutations dampened the transport of LPL to the cell surface, while for the C310R mutation, reducing LPL protein level might be involved. By characterizing these two novel LPL mutations, this study has expanded our understanding on the pathogenesis of familial hypertriglyceridemia (FHTG).

Ferroptosis is a form of regulated cell death driven by oxidative injury promoting lipid peroxidation, although detailed molecular regulators are largely unknown. Here, we show that heatshock 70-kDa protein 5 (HSPA5) negatively regulates ferroptosis in human pancreatic ductal adenocarcinoma (PDAC) cells. Mechanistically, activating transcription factor 4 (ATF4) resulted in the induction of HSPA5, which in turn bound glutathione peroxidase 4 (GPX4) and protected against GPX4 protein degradation and subsequent lipid peroxidation. Importantly, the HSPA5–GPX4 pathway mediated ferroptosis resistance, limiting the anticancer activity of gemcitabine. Genetic or pharmacologic inhibition of the HSPA5–GPX4 pathway enhanced gemcitabine sensitivity by disinhibiting ferroptosis in vitro and in both subcutaneous and orthotopic animal models of PDAC. Collectively, these findings identify a novel role of HSPA5 in ferroptosis and suggest a potential therapeutic strategy for overcoming gemcitabine resistance. Cancer Res; 77(8); 2064–77. ©2017 AACR.

Cadmium (Cd) is an environmental and industrial pollutant that induces a broad spectrum of toxicological effects, influences a variety of human organs, and is associated with poor semen quality and male infertility. Increasing evidence demonstrates that Cd induces testicular germ cell apoptosis in rodent animals. However, the specific effect of Cd exposure on autophagy in germ cells is poorly understood. We investigate the role of high-mobility group box 1 protein (HMGB1), a ubiquitous nuclear protein, on Cd-evoked autophagy in a mouse spermatocyte cell line (GC-2spd). Our data have shown that autophagy was significantly elevated in GC-2spd cells exposed to Cd. Furthermore, there was a reduction in rapamycin (RAP)-mediated apoptosis. In addition, Cd exposure reduced cell viability, which is an effect that could be significantly inhibited by RAP treatment. These results indicate that autophagy appears to serve a positive function in reducing Cd-induced cytotoxicity. In addition, HMGB1 increased coincident with the processing of LC3-I to LC3-II. Thus, the upregulation of HMGB1 increases LC3-II levels. Our data suggest that HMGB1-induced autophagy appears to act as a defense/survival mechanism against Cd cytotoxicity in GC-2spd cells.

Pomacea canaliculata is an invasive snail that can adversely affect biodiversity, agricultural crop yields and human health. Schima superba, which belongs to the Theaceae family, exhibits insecticidal and antibacterial activity, but few studies have focused on its potential molluscicidal activity. In this study, we systematically evaluated the molluscicidal activity of S. superba extract against P. canaliculata in a variety of conditions, including multiple exposure intervals (24, 48 and 72 h), temperatures (15, 20 and 25 °C) and shell diameters (1.5 ± 0.2, 2.5 ± 0.2 and 3.5 ± 0.2 cm). The molluscicidal activities of a 10% microemulsion of S. superba extract were tested on rice in plastic pots filled with mud at different exposure intervals (1, 3, 5 and 7 days). In addition, we isolated and purified the active components from S. superba extract and described them. S. superba was highly toxic to P. canaliculata; the LC50 values for the crude extract at 72 h were 12.25 mg L−1 (15 °C), 11.31 mg L−1 (20 °C), 8.33 mg L−1 (25 °C) with 2.5 ± 0.2 cm shell diameter and 9.43 mg L−1 (1.5 ± 0.2 cm shell diameter), 9.48 mg L−1 (2.5 ± 0.2 cm shell diameter), 10.06 mg L−1 (3.5 ± 0.2 cm shell diameter) at 25 °C. There was a positive correlation between the molluscicidal activity of the extract and treatment temperature after 24 h of treatment, but there was no clear correlation beyond 24 h. In addition, there was no statistically significant correlation between the molluscicidal activity of the extract and shell diameters. Pot trials showed that the crude extract effectively controlled P. canaliculata at 8 and 10 mg L−1. We isolated and characterized two pentacyclic triterpenoid saponins from S. superba, maetenoside B and sasanquasaponin III, which had high molluscicidal activity. This study provides an important foundation for future research on the potential value of S. superba as a molluscicide.

Glycyrrhizic acid ammonium salt (GAAS) is derived from glycyrrhizic acid, which is an active compound extracted from the Chinese traditional medicine licorice. GAAS is clinically applied to treat immune-mediated liver injury, but its mechanism remains elusive. Therefore, this study aimed to investigate the mechanism in which GAAS alleviates immune-mediated liver injury induced by Concanavalin A (ConA). After ten days of intragastric administration of GAAS, 20 mg/kg ConA was injected via tail vein to establish the immune-mediated liver injury model of BALB/C mice. Then, the concentrations of ALT, AST, and TBIL in the serum of mice were determined. H&E staining was performed to observe the pathological changes in the liver, and the expression of liver cytokines was detected by qPCR. Immunohistochemistry and Western blot analysis was employed to detect the expression of liver-related proteins. The apoptosis in liver tissue was detected by TUNEL. Our results suggest that GAAS demonstrated excellent protective effects in the liver. We found that GAAS down-regulated the mRNA expression of IL-1β, IL-6, TNF-α, IFN-γ, and IL-17A, and it up-regulated the mRNA expression of IL-4 and TGF-β. Additionally, GAAS may modulate the balance of four immune cells (Th1, Th2, Th17, and Treg) by regulating the expression of T-bet, GATA3, RORγt, and Foxp3 to alleviate liver injury in mice. Furthermore, GAAS decreased hepatocyte apoptosis by blocking the JAK1/STAT1/IRF1 pathway, suppressing oxidative stress, decreasing p-JNK expression, and regulating the expression of apoptosis-related proteins. In summary, the mechanism of GAAS in liver injury alleviation acts to regulate the balance of Th cells in the liver to inhibit hepatocyte apoptosis. This study may provide a new strategy for the treatment of immune-mediated liver injury.

Background Colchicine is a clinical medicine used for relief from gout and familial Mediterranean fever. Because of its toxic effects, intravenous injection of colchicine has been banned, but it is still widely administered orally. We assayed the toxic effects of colchicine in cultured primary chorionic villus cells and amniotic fluid cells to interpret its influence on the placenta and foetus. Methods Bright field record and cell count kit 8 were used to value cell viability. Flow cytometer was used to identify cells markers, cell cycle and cell apoptosis. G-banding was used for karyotype analysis for sample genetic and drug effect evaluation. Results Chorionic villus cells and amniotic fluid cells were characterized as mesenchymal cells that share most cell surface markers and have a similar response to colchicine. Colchicine did not induce a decline in cell viability at low concentrations but suppressed cell proliferation by arresting the cell cycle in the G2/M phase and increased the risk of tetraploid generation in a small subset of cases. Conclusions Our study revealed the results of a colchicine-induced toxicity test in prenatal cells and determined the anti-mitotic biologically functional dose and manner of administration that might reduce the risk of tetraploid generation.

Species of Botryosphaeriaceae fungi are important plant pathogens causing cankers, blight, and fruit rot in an extremely wide range of host. In recent years, kiwifruit rot has been a serious problem in Sichuan Province, one of the important kiwifruit production areas of China. Botryosphaeria dothidea has previously been associated with kiwifruit rot but little is known regarding whether other Botryosphaeriaceae genera also constitute kiwifruit rot pathogens in China. Accordingly, diseased fruit were collected from six different areas of Sichuan Province. Based on morphological characteristics, pathogenicity testing, and comparisons of DNA sequences of the internal transcribed spacer, transcription elongation factor 1-α, and β-tubulin genes, 135 isolates of Botryosphaeriaceae were identified as B. dothidea, Lasiodiplodia theobromae, and Neofusicoccum parvum. All of these species were found to cause kiwifruit rot. To understand the infection cycle of kiwifruit rot pathogens, these three species were used to inoculate leaves and shoots of kiwifruit. The results showed that these species could cause spots on leaves and lesions on shoots, producing abundant pycnidia on leaves and shoots surfaces. Moreover, B. dothidea conidia and ascospores from overwintered pycnidia and pseudothecia in kiwifruit orchards in April and August could cause fruit rot and spots on leaves of kiwifruit. Therefore, we concluded that overwintered pycnidia and pseudothecia of B. dothidea in kiwifruit orchards are the primary inoculum for kiwifruit rot, with new pycnidia that develop during the growing season serving as a secondary inoculum. This is the first report of N. parvum and L. theobromae causing kiwifruit rot in China and is also the first report that B. dothidea is able to overwinter as pycnidia and pseudothecia in kiwifruit orchards and serve as the primary inoculum for kiwifruit rot.

The deletions of the long arm of chromosome 14 involving the 14q24-q32 region have been identified as deletion 14q (del 14q) syndrome, but were rarely reported. The patients with del 14q syndrome are observed a peculiar facial appearance and neurological defects, but the molecular mechanisms were not clear. Here we generated a human iPSC line from the patient’s amniotic fluid cells with 24 Mb deletion in 14q24.3q32.31 which will serve as useful tools for studying the mechanism of del 14q syndrome and the genes involved, which will provide useful basic theory of prenatal diagnosis.

Monogenic disorders (MGDs), which are caused by single gene mutations, have a serious effect on human health. Among these, β-thalassemia (β-thal) represents one of the most common hereditary hematological diseases caused by mutations in the human hemoglobin β (HBB) gene. The technologies of induced pluripotent stem cells (iPSCs) and genetic correction provide insights into the treatments for MGDs, including β-thal. However, traditional approaches for correcting mutations have a low efficiency and leave a residual footprint, which leads to some safety concerns in clinical applications. As a proof of concept, we utilized single-strand oligodeoxynucleotides (ssODNs), high-fidelity CRISPR/Cas9 nuclease, and small molecules to achieve a seamless correction of the β-41/42 (TCTT) deletion mutation in β thalassemia patient-specific iPSCs with remarkable efficiency. Additionally, off-target analysis and whole-exome sequencing results revealed that corrected cells exhibited a minimal mutational load and no off-target mutagenesis. When differentiated into hematopoietic progenitor cells (HPCs) and then further to erythroblasts, the genetically corrected cells expressed normal β-globin transcripts. Our studies provide the most efficient and safe approach for the genetic correction of the β-41/42 (TCTT) deletion in iPSCs for further potential cell therapy of β-thal, which represents a potential therapeutic avenue for the gene correction of MGD-associated mutants in patient-specific iPSCs. Keywords: β thalassemia, induced pluripotent stem cells, CRISPR/Cas9, ssODNs

// Lin Huang 1, 2 , Zhen-Bo Wang 2 , Shu-Tao Qi 1 , Xue-Shan Ma 2 , Qiu-Xia Liang 2, 3 , Guo Lei 2 , Tie-Gang Meng 2 , Li-Feng Liang 1 , Ye-Xin Xian 1 , Yi Hou 2 , Xiao-Fang Sun 1 , Yong Zhao 4 , Wei-Hua Wang 1, 5 , Qing-Yuan Sun 2, 3 1 Key Laboratory of Major Obstetrics Diseases of Guangdong Province, Key Laboratory of Reproduction and Genetics of Guangdong Higher Education Institutes, The Third Hospital Affiliated to Guangzhou Medical University, Guangdong, China 2 State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China 3 University of the Chinese Academy of Sciences, Beijing, China 4 State Key Laboratory of Biomembrane, Institute of Zoology, Chinese Academy of Sciences, Beijing, China 5 Houston Fertility Institute/Houston Fertility Laboratory, Houston, Texas, USA Correspondence to: Qing-Yuan Sun, email: sunqy@ioz.ac.cn Wei-Hua Wang, email: wangweihua11@yahoo.com Keywords: RAD9A, Vasa, spermatogenesis, Sertoli cells, differentiation Received: June 16, 2016 Accepted: November 09, 2016 Published: November 16, 2016 ABSTRACT Spermatogenesis in testes requires precise spermatogonia differentiation. Spermatocytes lacking the Rad9a gene are arrested in pachytene prophase, implying a possible role for RAD9A in spermatogonia differentiation. However, numerous RAD9A -positive pachytene spermatocytes are still observed in mouse testes following Rad9a excision using the Stra8-Cre system, and it is unclear whether Rad9a deletion in spermatogonia interrupts differentiation. Here, we generated a mouse model in which Rad9a was specifically deleted in spermatogonial stem cells (SSCs) using Cre recombinase expression driven by the germ cell-specific Vasa promoter. Adult Rad9a -null male mice were infertile as a result of completely blocked spermatogonia differentiation. No early spermatocytes were detected in mutant testicular cords of 9-day-old mice. Mutant spermatogonia were prone to apoptosis, although proliferation rates were unaffected. Rad9a deletion also resulted in malformation of seminiferous tubules, in which cells assembled irregularly into clusters, and malformation led to testicular cord disruption. Our findings suggest that Rad9a is indispensable for spermatogonia differentiation and testicular development in mice.

Bone marrow injury remains a serious concern in traditional cancer treatment. Ferroptosis is an iron- and oxidative-dependent form of regulated cell death that has become part of an emerging strategy for chemotherapy. However, the key regulator of ferroptosis in bone marrow injury remains unknown. Here, we show that Fanconi anemia complementation group D2 (FANCD2), a nuclear protein involved in DNA damage repair, protects against ferroptosis-mediated injury in bone marrow stromal cells (BMSCs). The classical ferroptosis inducer erastin remarkably increased the levels of monoubiquitinated FANCD2, which in turn limited DNA damage in BMSCs. FANCD2-deficient BMSCs were more sensitive to erastin-induced ferroptosis (but not autophagy) than FANCD2 wild-type cells. Knockout of FANCD2 increased ferroptosis-associated biochemical events (e.g., ferrous iron accumulation, glutathione depletion, and malondialdehyde production). Mechanically, FANCD2 regulated genes and/or expression of proteins involved in iron metabolism (e.g., FTH1, TF, TFRC, HAMP, HSPB1, SLC40A1, and STEAP3) and lipid peroxidation (e.g., GPX4). Collectively, these findings indicate that FANCD2 plays a novel role in the negative regulation of ferroptosis. FANCD2 could represent an amenable target for the development of novel anticancer therapies aiming to reduce the side effects of ferroptosis inducers.

Dopamine is a neurotransmitter that has many functions in the nervous and immune systems. Ferroptosis is a non-apoptotic form of regulated cell death that is involved in cancer and neurodegenerative diseases. However, the role of dopamine in ferroptosis remains unidentified. Here, we show that the non-oxidative form of dopamine is a strong inhibitor of ferroptotic cell death. Dopamine dose-dependently blocked ferroptosis in cancer (PANC1 and HEY) and non-cancer (MEF and HEK293) cells following treatment with erastin, a small molecule ferroptosis inducer. Notably, dopamine reduced erastin-induced ferrous iron accumulation, glutathione depletion, and malondialdehyde production. Mechanically, dopamine increased the protein stability of glutathione peroxidase 4, a phospholipid hydroperoxidase that protects cells against membrane lipid peroxidation. Moreover, dopamine suppressed dopamine receptor D4 protein degradation and promoted dopamine receptor D5 gene expression. Thus, our findings uncover a novel function of dopamine in cell death and provide new insight into the regulation of iron metabolism and lipid peroxidation by neurotransmitters.

BACKGROUND Pueraria peduncularis (Grah. ex Benth.) Benth., which belongs to the Leguminosae family, exhibits resistance to many crop pests in agricultural production. Pomacea canaliculata is an important invasive snail in rice fields and causes severe yield losses. To evaluate the toxicity of P. peduncularis to P. canaliculata, in this study the molluscicidal activity of root extracts of P. peduncularis was tested against P. canaliculata; the active compounds were isolated, and the structures of these compounds were analysed using nuclear magnetic resonance (NMR) analysis and mass spectral analysis. RESULTS Our results showed that the molluscicidal activity of the root crude extract differed between P. canaliculata with different shell diameters after treatment for 72 h. The median lethal concentration (LC50) was 5.511 mg L−1 against snails of 1.5 ± 0.2 cm diameter and 12.383 mg L−1 against snails of 2.5 ± 0.2 cm diameter. Furthermore, two active ingredients isolated from root methanol extracts were identified as pedunsaponin A and pedunsaponin C. Both pedunsaponin A and pedunsaponin C showed strong molluscicidal activities, with LC50 values of 3.893 and 4.252 mg L−1, respectively, against snails with shell diameters of 1.5 ± 0.2 cm after treatment for 72 h. CONCLUSION Pueraria peduncularis extracts exhibit high molluscicidal activity and have great potential value for exploring a molluscicide to control Pomacea canaliculata. © 2016 Society of Chemical Industry

Aims: To demonstrate the value of a whole-genome high-resolution single-nucleotide polymorphism (SNP) array for the elucidation of genetic causes underlying pregnancy loss. Methods: The SNP array combined with SNPs and oligonucleotide probes was used to examine 60 samples of products of conception, including chorionic villi, fetal parts, and fetal blood. Results: The SNP array yielded a 38.3% (23/60) abnormality rate. In addition to the most common aneuploidy, it detected 16.7% additional aberrations involving copy number variation, triploidy, loss of heterozygosity or low-level mosaicism. Conclusion: This whole-genome high-resolution SNP array not only provides copy number information but also identifies the heterozygosity status, which facilitates the discovery of the novel genetic alterations associated with pregnancy failure and improves the management of subsequent pregnancies.

Macroautophagy/autophagy is an evolutionarily conserved degradation pathway that maintains homeostasis. Ferroptosis, a novel form of regulated cell death, is characterized by a production of reactive oxygen species from accumulated iron and lipid peroxidation. However, the relationship between autophagy and ferroptosis at the genetic level remains unclear. Here, we demonstrated that autophagy contributes to ferroptosis by degradation of ferritin in fibroblasts and cancer cells. Knockout or knockdown of Atg5 (autophagy-related 5) and Atg7 limited erastin-induced ferroptosis with decreased intracellular ferrous iron levels, and lipid peroxidation. Remarkably, NCOA4 (nuclear receptor coactivator 4) was a selective cargo receptor for the selective autophagic turnover of ferritin (namely ferritinophagy) in ferroptosis. Consistently, genetic inhibition of NCOA4 inhibited ferritin degradation and suppressed ferroptosis. In contrast, overexpression of NCOA4 increased ferritin degradation and promoted ferroptosis. These findings provide novel insight into the interplay between autophagy and regulated cell death.

In clinical diagnostics, single nucleotide polymorphism (SNP)-based microarray analysis enables the detection of copy number variations (CNVs), as well as copy number neutral regions, that are absent of heterozygosity throughout the genome. The aim of the present study was to evaluate the effectiveness and sensitivity of SNP-based microarray analysis in the diagnosis of hydatidiform mole (HM). By using whole-genome SNP microarray analysis, villous genotypes were detected, and the ploidy of villous tissue was determined to identify HMs. A total of 66 villous tissues and two twin tissues were assessed in the present study. Among these samples, 11 were triploid, one was tetraploid, 23 were abnormal aneuploidy, three were complete genome homozygosity, and the remaining ones were normal ploidy. The most noteworthy finding of the present study was the identification of six partial HMs and three complete HMs from those samples that were not identified as being HMs on the basis of the initial diagnosis of experienced obstetricians. This study has demonstrated that the application of an SNP-based microarray analysis was able to increase the sensitivity of diagnosis for HMs with partial and complete HMs, which makes the identification of these diseases at an early gestational age possible.

Ferroptosis is a recently recognized form of regulated cell death. It is characterized morphologically by the presence of smaller than normal mitochondria with condensed mitochondrial membrane densities, reduction or vanishing of mitochondria crista, and outer mitochondrial membrane rupture. It can be induced by experimental compounds (e.g., erastin, Ras-selective lethal small molecule 3, and buthionine sulfoximine) or clinical drugs (e.g., sulfasalazine, sorafenib, and artesunate) in cancer cells and certain normal cells (e.g., kidney tubule cells, neurons, fibroblasts, and T cells). Activation of mitochondrial voltage-dependent anion channels and mitogen-activated protein kinases, upregulation of endoplasmic reticulum stress, and inhibition of cystine/glutamate antiporter is involved in the induction of ferroptosis. This process is characterized by the accumulation of lipid peroxidation products and lethal reactive oxygen species (ROS) derived from iron metabolism and can be pharmacologically inhibited by iron chelators (e.g., deferoxamine and desferrioxamine mesylate) and lipid peroxidation inhibitors (e.g., ferrostatin, liproxstatin, and zileuton). Glutathione peroxidase 4, heat shock protein beta-1, and nuclear factor erythroid 2-related factor 2 function as negative regulators of ferroptosis by limiting ROS production and reducing cellular iron uptake, respectively. In contrast, NADPH oxidase and p53 (especially acetylation-defective mutant p53) act as positive regulators of ferroptosis by promotion of ROS production and inhibition of expression of SLC7A11 (a specific light-chain subunit of the cystine/glutamate antiporter), respectively. Misregulated ferroptosis has been implicated in multiple physiological and pathological processes, including cancer cell death, neurotoxicity, neurodegenerative diseases, acute renal failure, drug-induced hepatotoxicity, hepatic and heart ischemia/reperfusion injury, and T-cell immunity. In this review, we summarize the regulation mechanisms and signaling pathways of ferroptosis and discuss the role of ferroptosis in disease.

Hemophilia A (HA), is a X-linked recessive congenital bleeding disorder, caused by deficiency of the coagulation factor VIII (FVIII) which is encoded by coagulation factor 8 (F8). HA affects 1 of every 5,000 males worldwide. The intron 22 inversion (Inv22) mutation of F8 causes about 45% of severe HA cases. Here, we generated induced pluripotent stem cells (iPSCs) from a HA patient with Inv22 mutation by electroporation of urine-derived cells (UCs) with episomal plasmids under feeder-free, virus-free, serum-free condition and without oncogene c-MYC. This iPSCs line could facilitate future applications of human iPSCs by provide a valuable cell model.

EDA is a gene located at Xq13.1. It encodes different isoforms of tumor necrosis factor (TNF) superfamily member ectodysplasin A. Ectodysplasin A is a transmembrane protein which can be cleaved to form a secreted form and interact with EDA receptor to mediate the development of ectoderm. Mutations of the EDA gene are related to ectodermal dysplasia and tooth agenesis. Here, we report the establishment of the EDA gene knockout human embryonic stem (hES) cell line by CRISPR-Cas9 technology. This cell line provides good materials for further studies of the roles ectodysplasin A plays in ectoderm differentiation and tooth development.

RNA splicing is a critical mechanism by which to modify transcriptome, and its dysregulation is the underlying cause of many human diseases. It remains challenging, however, to genetically modulate a splicing event in its native context. Here, we demonstrate that a CRISPR-guided cytidine deaminase (i.e., targeted-AID mediated mutagenesis [TAM]) can efficiently modulate various forms of mRNA splicing. By converting invariant guanines to adenines at either 5' or 3' splice sites (SS), TAM induces exon skipping, activation of alternative SS, switching between mutually exclusive exons, or targeted intron retention. Conversely, TAM promotes downstream exon inclusion by mutating cytidines into thymines at the polypyrimidine tract. Applying this approach, we genetically restored the open reading frame and dystrophin function of a mutant DMD gene in patient-derived induced pluripotent stem cells (iPSCs). Thus, the CRISPR-guided cytidine deaminase provides a versatile genetic platform to modulate RNA splicing and to correct mutations associated with aberrant splicing in human diseases.