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For programmed cell death protein 1/programmed death-ligand 1 (PD-L1)-positive triple-negative breast cancer (TNBC), the addition of immune checkpoint inhibitors (ICIs) to chemotherapy has been shown to increase the objective response rate (ORR) by only 13.4-16.3%. Thus, examining converting tumor immunogenicity and targeting novel pathways may be needed to improve response to immunotherapy. B7-H4 is an emerging target for breast cancer immunotherapy. However, the antitumor effect of B7-H4 is unstable during cell generation and further research is necessary to strengthen the antitumor efficacy of B7-H4.To explore the potential of B7-H4-targeted immunotherapy of breast cancer, current study generated four monoclonal antibodies (mAbs) against B7-H4 through immunization of mice followed by phage display technology. T cell-engaged bispecific antibodies and immunotoxins were created based on these mAbs. To evaluate the anti-tumor activity of B7-H4-targeting bispecific antibody and immunotoxin, the anti-tumor efficacy testRigorousCollectively, our findings uncover a novel strategy of combining oncolytic virus HSV-2 with bispecific antibody reinforce antitumor immune response, which warranted further translational investigation.

Nonalcoholic steatohepatitis (NASH) is an aggressive liver disease threatening human health, yet no medicine is developed to treat this disease. In this study, we first discovered that Leptin mutant rats ( LepΔI14/ΔI14) exhibit characteristic NASH phenotypes including steatosis, lymphocyte infiltration, and ballooning after postnatal week 16. We then examined NASH progression by performing an integrated analysis of hepatic transcriptome in Leptin-deficient rats from postnatal 4 to 48 weeks. Initially, simple steatosis in LepΔI14/ΔI14 rats were observed with increased expression of the genes encoding for rate-limiting enzymes in lipid metabolism such as acetyl-CoA carboxylase and fatty acid synthase. When NASH phenotypes became well developed at postnatal week 16, we found gene expression changes in insulin resistance, inflammation, reactive oxygen species and endoplasmic reticulum stress. As NASH phenotypes further progressed with age, we observed elevated expression of cytokines and chemokines including C-C motif chemokine ligand 2, tumor necrosis factor ɑ, interleukin-6, and interleukin-1β together with activation of the c-Jun N-terminal kinase and nuclear factor-κB pathways. Histologically, livers in LepΔI14/ΔI14 rats exhibited increased cell infiltration of MPO+ neutrophils, CD8+ T cells, CD68+ hepatic macrophages, and CCR2+ inflammatory monocyte-derived macrophages associated with macrophage polarization from M2 to M1. Subsequent cross-species comparison of transcriptomes in human, rat, and mouse NASH models indicated that Leptin-deficient rats bear more similarities to human NASH patients than previously established mouse NASH models. Taken together, our study suggests that LepΔI14/ΔI14 rats are a valuable pre-clinical rodent model to evaluate NASH drug safety and efficacy.

EGFR and BRAF V600E mutations are both early driven and usually mutually exclusive. We report the case of a 59-year-old woman diagnosed with advanced lung adenocarcinoma harboring coexisting EGFR exon 18 G719A and BRAF V600E mutations. She experienced a long-term response to oral afatinib, with a progression-free survival rate of 33 months and an overall survival rate of 11 years. Lung adenocarcinoma with synchronous EGFR G719A and BRAF V600E mutations is rare and has not been previously reported. This case highlights the importance of an adequate response to afatinib and provides an optimal therapeutic option for such patients.

Background Genetic programs underlying preimplantation development and early lineage segregation are highly conserved across mammals. It has been suggested that nonhuman primates would be better model organisms for human embryogenesis, but a limited number of studies have investigated the monkey preimplantation development. In this study, we collect single cells from cynomolgus monkey preimplantation embryos for transcriptome profiling and compare with single-cell RNA-seq data derived from human and mouse embryos. Results By weighted gene-coexpression network analysis, we found that cynomolgus gene networks have greater conservation with human embryos including a greater number of conserved hub genes than that of mouse embryos. Consistently, we found that early ICM/TE lineage-segregating genes in monkeys exhibit greater similarity with human when compared to mouse, so are the genes in signaling pathways such as LRP1 and TCF7 involving in WNT pathway. Last, we tested the role of one conserved pre-EGA hub gene, SIN3A, using a morpholino knockdown of maternal RNA transcripts in monkey embryos followed by single-cell RNA-seq. We found that SIN3A knockdown disrupts the gene-silencing program during the embryonic genome activation transition and results in developmental delay of cynomolgus embryos. Conclusion Taken together, our study provided new insight into evolutionarily conserved and divergent transcriptome dynamics during mammalian preimplantation development.

Lung adenocarcinoma (LUAD) harboring EGFR mutations prevails in Asian population. However, the inter-patient and intra-tumor heterogeneity has not been addressed at single-cell resolution. Here we performed single-cell RNA sequencing (scRNA-seq) of total 125,674 cells from seven stage-I/II LUAD samples harboring EGFR mutations and five tumor-adjacent lung tissues. We identified diverse cell types within the tumor microenvironment (TME) in which myeloid cells and T cells were the most abundant stromal cell types in tumors and adjacent lung tissues. Within tumors, accompanied by an increase in CD1C+ dendritic cells, the tumor-associated macrophages (TAMs) showed pro-tumoral functions without signature gene expression of defined M1 or M2 polarization. Tumor-infiltrating T cells mainly displayed exhausted and regulatory T-cell features. The adenocarcinoma cells can be categorized into different subtypes based on their gene expression signatures in distinct pathways such as hypoxia, glycolysis, cell metabolism, translation initiation, cell cycle, and antigen presentation. By performing pseudotime trajectory, we found that ELF3 was among the most upregulated genes in more advanced tumor cells. In response to secretion of inflammatory cytokines (e.g., IL1B) from immune infiltrates, ELF3 in tumor cells was upregulated to trigger the activation of PI3K/Akt/NF-κB pathway and elevated expression of proliferation and anti-apoptosis genes such as BCL2L1 and CCND1. Taken together, our study revealed substantial heterogeneity within early-stage LUAD harboring EGFR mutations, implicating complex interactions among tumor cells, stromal cells and immune infiltrates in the TME.

BACKGROUND: The outcomes of advanced non-small cell lung cancer (NSCLC) patients after first- or second-line therapy are still discouraging due to a lack of effective treatment strategies. As a novel oral anti-angiogenesis drug, apatinib, approved by the National Medical Products Administration of China only for advanced gastric cancer, has been increasingly used in off-label treatment across various cancer types in recent years, especially advanced NSCLC. It has shown strong anti-tumor efficacy and acceptable safety. METHODS: This prospective study (NCT02974933) was conducted in patients with advanced NSCLC, who had suffered disease progression from the first- or second-line treatment, in Hubei Cancer Hospital. Eligible patients were enrolled and administrated with apatinib mesylate (500 mg qd) in combination with pemetrexed (500 mg/m(2), every 4 weeks). The primary endpoint was progression-free survival (PFS). RESULTS: From September 2016 to September 2019, a total of 21 advanced NSCLC patients were enrolled in Hubei Cancer Hospital. As of January 2021, treatment was discontinued in all patients, with 1 still in follow-up. There were 7/21 (33.3%) patients who achieved objective response. The median PFS and median overall survival (OS) were 7.0 months (95% CI: 6.15–7.85 months) and 13.0 months (95% CI: 7.39–18.6 months), respectively. Toxicities were tolerable or could be clinically managed. The most common grade 3–4 adverse events (AEs) were hypertension (14.3%, 3/21), hand-foot syndrome (4.7%, 1/21), and proteinuria (4.7%, 1/21). Hematological toxicities were moderate, with rare occurrences of grade 3/4 toxicities. During the period of treatment, there was no occurrence of treatment-related death. CONCLUSIONS: Apatinib plus pemetrexed demonstrated promising efficacy and a high level of safety profile in previously heavily-treated NSCLC patients. More definitive studies on the combination of apatinib and pemetrexed are warranted

Zebrafish (Danio rerio) are an attractive vertebrate model for the molecular dissection of disease mechanisms. Janus kinase (JAK)/signal transducer and activator of transcription (stat) has been defined through studies of cytokine signaling pathways in mammals. Here, we examined the expression level of Jak2a, which is a homolog of mammalian jak2 in zebrafish, by quantitative reverse transcriptase (RT)-PCR, and the peak of mRNA expression occurred at 3.75 hours post fertilization (hpf). The overexpression of Jak2a was proven by real-time Q-PCR and Western blot in 1-4-cell stage embryos injected with 400 ng/µl full-length jak2a mRNA as well as gfi1.1, gata1, mpo and β-embryonic hemoglobin as detected by real-time Q-PCR. Moreover, jak2a mRNA significantly increased the GFP+ population in the transgenic zebrafish lines Tg (gata1: gfp) (uninjected embryos: 17.22±1.70%; embryos injected with jak2a mRNA: 21.31±2.11%, p

Background: Circular RNAs (circRNAs) have been reported as the competing endogenous RNAs (ceRNAs) to sponge microRNAs (miRNAs) implicating in the initiation and progression of breast cancer. However, the functions of circRNAs in breast cancer have not been completely clarified. In the present study, we aimed to identify differentially expressed circRNAs in breast cancer tumor tissues, and their roles and downstream targets were investigated in the progression of breast cancer. Methods: High-throughput circRNA sequencing was performed to detect the differentially expressed circRNAs. The CCK-8 and flow cytometry were performed to measure the cell viability and apoptosis in breast cancer cells. Gene and protein expression were assayed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blotting, respectively. Results: hsa_circ_0004771 and Zinc finger E-box binding homeobox 2 (ZEB2) expression levels were up-regulated and positively correlated in breast cancer tumor tissues. In addition, the expression levels of miR-653 were reduced in breast cancer tumor tissues. We also found that hsa_circ_0004771 functioned as a sponge of miR-653 to inhibit its expression. miR-653 as a post-transcriptional regulator down-regulated the expression of ZEB2 by binding to its 3′-UTR. Interestingly, a significant inverse correlation was observed between miR-653 and hsa_circ_0004771 or ZEB2 expression in breast cancer tumor tissues. Knockdown of hsa_circ_0004771 and ZEB2 served as equally authentic of miR-653 mimics to induce growth inhibition and apoptosis in breast cancer cells. Conclusion: Hsa_circ_0004771/miR-653/ZEB2 regulatory feedback revealed a new molecular mechanism in the pathogenesis of breast cancer, which might provide novel therapeutic targets for the treatment of breast cancer.

Nonalcoholic steatohepatitis (NASH) is an aggressive liver disease threatening public health, however its natural history is poorly understood. Unlikeob/obmice,Lep∆I14/∆I14rats develop unique NASH phenotype with steatosis, lymphocyte infiltration and ballooning after postnatal week 16. UsingLep∆I14/∆I14rats as NASH model, we studied the natural history of NASH progression by performing an integrated analysis of hepatic transcriptome from postnatal week 4 to 48.Leptindeficiency results in a robust increase in expression of genes encoding 9 rate-limiting enzymes in lipid metabolism such as ACC and FASN. However, genes in positive regulation of inflammatory response are highly expressed at week 16 and then remain the steady elevated expression till week 48. The high expression of cytokines and chemokines including CCL2, TNFα, IL6 and IL1β is correlated with the phosphorylation of several key molecules in pathways such as JNK and NF-κB. Meanwhile, we observed cell infiltration of MPO+neutrophils, CD8+T cells, CD68+hepatic macrophages and CCR2+inflammatory monocyte-derived macrophages, together with macrophage polarization from M2 to M1. Importantly,Lep∆I14/∆I14rats share more homologous genes with NASH patients than previously established mouse models and crab eating monkeys with spontaneous hepatic steatosis. Transcriptomic analysis showed that many drug targets in clinical trials can be evaluated inLep∆I14/∆I14rats.ConclusionWe characterizeLep∆I14/∆I14rats as a unique NASH model by performing a long-term (i.e., 4 to 48 postnatal weeks) integrated transcriptomic analysis. This work reveal the temporal dynamics of hepatic gene expression in lipid metabolism and inflammation, and shed light on understanding the natural history of NASH in human beings.

Preimplantation genetic diagnosis/screening (PGD/PGS) aims to help couples lower the risks of transmitting genetic defects to their offspring, implantation failure, and/or miscarriage during in vitro fertilization (IVF) cycles. However, it is still being debated with regard to the practicality and diagnostic accuracy of PGD/PGS due to the concern of invasive biopsy and the potential mosaicism of embryos. Recently, several non-invasive and high-throughput assays have been developed to help overcome the challenges encountered in the conventional invasive biopsy and low-throughput analysis in PGD/PGS. In this mini-review, we will summarize the recent progresses of these new methods for PGD/PGS and discuss their potential applications in IVF clinics.

Rice leaf folder (Cnaphalocrocis medinalis Guenee) is one of the most important pests that endanger rice development and yield, which has characteristics of large outbreak areas, high occurrence frequencies and heavy damages. At present, the monitoring methods of rice leaf folder damage is based on artificial investigation, which has the advantages of objective truth and high reliability, while there is a drawback of time-consuming, and it cannot used for a wide range of rice damage monitoring. An ASD (Analytical Spectral Devices, Inc.) Hand-held Spectroradiometer was used at jointing stage of rice. The results showed that, reflectance from rice canopy significantly decreased in the green (530–570 nm) and near infrared (700–1000 nm) regions, and significantly increased in the blue (450–520 nm) and red (580–700 nm) regions as the rice leaf folder population increased. Reflectance from rice canopy significantly decreased in the spectral regions from 737 to 1000 nm as the infestation scale of pest population increased, and the most correlation appeared at 941 nm. The more the numbers of rice leaf folder, the higher the changes of such characteristic parameters. The positive correlations were found between the damage of rice leaf folder and the discrepancy of characteristic parameters in these experimental fields. With China Remote Sensing career advancement, a large number of independent researches and development satellites have launched. Among a new generation of high-resolution satellites, GaoFen-1 (GF-1) stands out. It sets high spatial resolution (2 m-16 m), multi-spectral and high temporal resolution (4-day) with 60 km-800 km swath in a fusion technology with strategic significance. In order to explore the adaptability of Chinese GF-1 images in monitoring rice damage from rice leaf folder, nine rice fields were selected by damage severity in Xinghua City, Jiangsu Province at full heading stage in 2015, and the Ratio Vegetation Index (RVI), Normalized Difference Vegetation Index (NDVI), Enhanced Vegetation Index (EVI), 2-band Enhanced Vegetation Index (EVI2), Soil Adjusted Vegetation Index (SAVI), Optimized Soil Adjusted Vegetation Index (OSAVI) were used to characterize the occurrence of rice leaf folder damages, which were calculated from the satellite GF-1 retrieval data. A series of analyses were performed to disclose the relationship among these six indices and the severity of rice leaf folder. Quantitative correlation analyses showed that NDVI, EVI, EVI2, SAVI, OSAVI and leaf folding population had a highly significant correlation (P

Patient-derived xenograft (PDX) animal models allow the exogenous growth of human tumors, offering an irreplaceable preclinical tool for oncology research. Mice are the most commonly used host for human PDX models, however their small body size limits the xenograft growth, sample collection, and drug evaluation. Therefore, we sought to develop a novel rat model that could overcome many of these limitations. We knocked out Rag1, Rag2, and Il2rg in Sprague Dawley (SD) rats by clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 technology. The development of lymphoid organs is significantly impaired in Rag1

Thyroid stimulating hormone receptor (TSHR), a G-protein-coupled receptor, is important for thyroid development and growth. In several cases, frameshift and/or nonsense mutations in TSHR were found in the patients with congenital hypothyroidism (CH), however they have not been functionally studied in an animal model. In the present work, we generated a unique Tshr Df/Df rat model that recapitulates the phenotypes in TSHR Y444X patient by CRISPR/Cas genome editing technology. In this rat model, TSHR is truncated at the second transmembrane domain, leading to CH phenotypes as what was observed in the patients, including dwarf, thyroid aplasia, infertility, TSH resistant as well as low serum thyroid hormone levels. The phenotypes can be reversed, at least partially, by levothyroxine (L-T4) treatment after weaning. The thyroid development is severely impaired in the Tshr Df/Df rats due to the suppression of the thyroid specific genes, i.e., thyroperoxidase (Tpo), thyroglobulin (Tg) and sodium iodide symporter (Nis), at both mRNA and protein levels. In conclusion, the Tshr Df/Df rat serves as a brand new genetic model to study CH in human, and will greatly help to shed light into the development of terminal organs that are sensitive to thyroid hormones.

To explore the material basis of conduction along meridian.Sixty SD rats(30 males,30 females) were randomly assigned into a normal group,an acupuncture group,a verapamil blocking group and a 0.9%NaCl blocking group(control group),15 rats in each one. Fluo 3-AM(calcium fluorescence probe) was injected at the observation part in femoral stomach meridian of foot-In the normal group, CaThe voltage-gated calcium channel at the meridian part is highly correlated with its tissue cells exciting conduction.

Most lung cancers are diagnosed at fairly advanced stages due to limited clinical symptoms. Platinum-based chemotherapy, either as single regimen or in combination with radiation, is one of the major recommendations for the patients. Earlier evaluation of the effectiveness of the chemotherapies is critical for developing better treatment plan given the toxicity of the chemotherapeutic reagents. Drug efficacy could be reflected in the systemic metabolism characteristics though knowledge about which remains scarce. In this study, serum metabolism influence of three types of commonly used platinum-based combination chemotherapy regimens, namely cisplatin with gemcitabine, vinorelbine or docetaxel, were studied using pattern recognition coupled with nuclear magnetic resonance techniques. The treated patients were divided into sensitive or insensitive subgroups according to their response to the treatments. We found that insensitive subjects can be identified from the sensitive ones with up-regulation of glucose and taurine but reduced alanine and lactate concentrations in serum. The combination chemotherapy of lung cancer is accompanied by disturbances of multiple metabolic pathways such as energy metabolism, phosphatidylcholine biosynthesis, so that the treated patients were marginally discriminated from the untreated. Serum metabolic profile of patients shows potential as an indicator of their response to platinum-based combination chemotherapy.

Severe aplastic anemia (SAA) is a life-threatening bone marrow failure syndrome characterized by pancytopenia and hypocellular bone marrow. Antithymocyte globulin (ATG) is the drug of choice for immunosuppressive therapy (IST) in patients with SAA ineligible for allogeneic stem cell transplantation. This study aimed to evaluate the efficacy and safety profile of porcine antilymphocyte globulin (p-ALG) plus CsA in the treatment of acquired SAA. Clinical information of 69 SAA patients treated with p-ALG plus CsA was collected and retrospectively analyzed for early mortality, response rate, survival rate, side effects, and other complications. The median age at diagnosis was 27 years (range 14 to 52). The overall response rate was 76.8 % with a 90-day median response time (range 30 ~ 360 days). Overall response rates at 3, 6, 12, 18, and 24 months were 63.8, 73.9, 76.8, 75.4, and 75.4 %, respectively. The median follow-up time for surviving patients was 24 months (range 4 ~ 44 months) and the 2-year overall survival (OS) rate was 88.4 %. The disease-free survival (DFS) rate at 2 years was 85.5 %. Older age (≥45 years), very (v)SAA subgroup, and lower baseline absolute lymphocyte count (1 × 10(9)/L) were independent unfavorable predictors of overall survival (p 0.05). Less than one third of patients had serum sickness or allergic reaction during ALG therapy, but symptoms could easily be relieved by steroid treatment; 27.54 % had mild hepatic impairment. Taken together, p-ALG showed similar efficacy and safety profiles to rabbit or horse ATG in IST of acquired SAA. It can be a suitable alternative preparation for rabbit ATG with the great advantage of lower medical expenses.

Severe aplastic anemia (SAA), which is considered to be an immune-mediated destruction of bone marrow stem cells with pancytopenia and hypoplasia, can be successfully treated with immunosuppressive therapy or hematopoietic stem cell transplantation (HSCT). Between January 2009 and December 2012, thirteen patients diagnosed with SAA were treated with tacrolimus plus rabbit antithymocyte globulin (ATG)-based immunosuppressive therapy (IST). The outcomes were then compared with our previous data for twenty-four patients administered with cyclosporine (CsA) plus rabbit ATG-based IST. All 37 cases accepted methylpredenisolone and recombinant human granulocyte colony-stimulating factor (rhG-CSF) from the first day that rabbit ATG was initiated. A total of 7 (54%) of the 13 patients in the tacrolimus group and 10 (42%) of the 24 cases in the ATG+CsA group achieved the criteria for complete response (CR); the partial response (PR) rate was 31% in the tacrolimus group and 33% in the ATG+CsA group. The median follow-up duration of the tacrolimus group and ATG+CsA group patients was 28 months and 27 months, respectively. Two patients in the tacrolimus group who were red blood cell- and platelet transfusion-dependent died, one of sepsis and the other of cerebral hemorrhage, whereas one patient died from serious infection on the 5th day after ATG was initiated in the ATG+CsA group. No clonal transformation to paroxysmal nocturnal hemoglobinuria (PNH) was observed in either group. Our data provide a possibility of using tacrolimus as part of an IST regimen for SAA in adults who have no opportunity of HSCT from human leukocyte antigen (HLA)-matched sibling donors.

The newly developed next-generation sequencing platforms, in combination with genome-scale amplification methods, provide a powerful tool to study genomics from a single cell. This mini-review summarizes the technologies of single cell genomics and their applications in several areas of biomedical research including stem cells, cancer biology and reproductive medicine. Particularly, it highlights recent advances in single cell exome sequencing, RNA-seq, and genome sequencing. The application of these powerful techniques will shed new light on the fundamental principles of gene transcription and genome organization at single-cell level and improve our understanding of cellular heterogeneity and diversity in multicellular organisms.

Peripheral nerve injury leads to various injury-induced responses in sensory neurons including physiological pain, neuronal cell death and nerve regeneration. In this study, we performed single-cell RNA-sequencing (scRNA-seq) analysis of mouse nonpeptidergic nociceptors (NP), peptidergic nociceptors (PEP) and large myelinated sensory neurons (LM) under both control and injury conditions at 3 days after sciatic nerve transection (SNT). After performing principle component and weighted gene co-expression network analysis, we categorized dorsal root ganglion (DRG) neurons into different subtypes and discovered co-regulated injury-response genes including novel regeneration associated genes (RAGs) in association with neuronal development, protein translation and cytoplasm transportation. In addition, we found significant up-regulation of the genes associated with cell death such as Pdcd2 in a subset of NP neurons after axotomy, implicating their actions in neuronal cell death upon nerve injury. Our study revealed the distinctive and sustained heterogeneity of transcriptomic responses to injury at single neuron level, implicating the involvement of different gene regulatory networks in nerve regeneration, neuronal cell death and neuropathy in different population of DRG neurons.

LEPTIN (LEP) is a circulating hormone released primarily from white adipocytes and is crucial for regulating satiety and energy homeostasis in humans and animals. Using the CRISPR technology, we created a set of Lep mutant rats that carry either null mutations or a deletion of the 14th Ile (LEP∆I14) in the mature LEP protein. We examined the potential off-target sites (OTS) by whole-genome high-throughput sequencing and/or Sanger-sequencing analysis and found no OTS in mutant rats. Mature LEP∆I14 is incessantly produced and released to blood at a much elevated level due to the feedback loop. Structure modeling of binding conformation between mutant LEP∆I14 and LEPTIN receptor (LEPR) suggests that the conformation of LEP∆I14 impairs its binding with LEPR, consistent with its inability to activate STAT3-binding element in the luciferase reporter assay. Phenotypic study demonstrated that Lep∆I14 rats recapitulate phenotypes of Lep-null mutant rats including obesity, hyperinsulinemia, hepatic steatosis, nephropathy and infertility. Compared to the existing ob/ob mouse models, this Lep∆I14/∆I14 rat strain provides a robust tool for further dissecting the roles of LEP in the diabetes related kidney disease and reproduction problem, beyond its well established function in regulating energy homeostasis.

A common theme in developmental biology is the repeated use of the same gene in diverse spatial and temporal domains, a process that generally involves transcriptional regulation mediated by multiple separate enhancers, each with its own arrangement of transcription factor (TF)-binding sites and associated activities. Here, by contrast, we show that the expression of the Drosophila Nidogen (Ndg) gene at different embryonic stages and in four mesodermal cell types is governed by the binding of multiple cell-specific Forkhead (Fkh) TFs – including Biniou (Bin), Checkpoint suppressor homologue (CHES-1-like) and Jumeau (Jumu) – to three functionally distinguishable Fkh-binding sites in the same enhancer. Whereas Bin activates the Ndg enhancer in the late visceral musculature, CHES-1-like cooperates with Jumu to repress this enhancer in the heart. CHES-1-like also represses the Ndg enhancer in a subset of somatic myoblasts prior to their fusion to form multinucleated myotubes. Moreover, different combinations of Fkh sites, corresponding to two different sequence specificities, mediate the particular functions of each TF. A genome-wide scan for the occurrence of both classes of Fkh domain recognition sites in association with binding sites for known cardiac TFs showed an enrichment of combinations containing the two Fkh motifs in putative enhancers found within the noncoding regions of genes having heart expression. Collectively, our results establish that different cell-specific members of a TF family regulate the activity of a single enhancer in distinct spatiotemporal domains, and demonstrate how individual binding motifs for a TF class can differentially influence gene expression.

Wogonoside (WO), a flavonoid extracted from Huangqin, plays multiple physiological roles. However, it has remained elusive how WO regulates hepatic fibrogenesis until now. The purpose of the study was to investigate the potential protective effects of WO against liver fibrosis induced by carbon tetrachloride (CCl4). In this study, male rats were randomly allocated into four groups: a control group, the CCl4 group, the CCl4 and WO (4 mg/kg) group, and CCl4 and WO (8 mg/kg) group. Hepatic fibrosis was induced by subcutaneous injection of CCl4 twice a week for a continuous 6-week period. Then the rats were intragastrically administrated with WO daily for 4 weeks before being killed. As expected, histopathological assessment, Masson trichrome staining, and Sirius red staining demonstrated that WO drastically ameliorated the hepatic fibrosis caused by CCl4. WO significantly attenuated the CCl4-induced upregulations of liver indices including alanine aminotransferase, aspartate aminotransferase, tumor necrosis factor-α, interleukin-1β, IL-6, hexadecenoic acid and laminin in serum, as well as hydroxyproline, malondialdehyde and phosphatidylinositol 3-kinase (PI3K)/protein Kinase B(Akt)/mechanistic target of rapamycin (mTOR)/nuclear factor-kappa B signalings in liver. Meanwhile, WO also effectively recovered the depletions of superoxide dismutase, glutathione and IL-10. Furthermore, we evaluated the effects of WO on the alpha smooth muscle actin, type I collagen expressions, and PI3K/Akt/ mTOR/ribosomal protein S6 kinase 70 kDa (p70S6K) signaling in transforming growth factor (TGF-β) stimulated hepatic stellate cell-T6 cells. These results suggested that WO had significant protective effects against liver fibrosis induced by CCl4.

A non-viral gene therapy vector, pcDNA3-EPO, was constructed by subcloning erythropoietin (EPO) cDNA into plasmid pcDNA3. After liposome-mediated transfection of the NIH 3T3 cells in vitro, EPO expression in the culture medium was detected by ELISA and amounted to 1.25 ± 0.3 IU ml−1. The biological activity of this EPO in the medium was detected after intramuscular injection of BALB/c mice. PCR of genomic DNA and RT-PCR of total RNA also confirmed that the plasmid pcDNA3-EPO had been transfected into the cells. A pool of pcDNA3-EPO transfectants, which stably expressed EPO, was obtained by G418 selection. When pcDNA3-EPO was combined into liposomes and intramuscularly injected into BALB/c mice, the reticulocyte ratio in the positive mice was three times higher than that in the control mice. In vivo expression was maintained in mice for at least one month.

Introduction L-type calcium channel (LTCC) is a unique and important factor in several cell lineages, whereas its role in the differentiation of dental pulp stem cells (DPSCs) is not well-known. In this study, we examined the function of LTCC α1C subunit (Ca v 1.2) and its distal C-terminus (DCT) during the in vitro differentiation of rat DPSCs (rDPSCs). Methods After fluorescence-activated cell sorting, rDPSCs were differentiated toward dentin sialophosphoprotein–positive odontoblasts and neural cells expressing specific neuronal markers. The inhibition of rDPSC differentiation via LTCC blocker nimodipine and Ca v 1.2 knockdown through short hairpin RNA was evaluated by using quantitative real-time polymerase chain reaction, Western blot, and immunofluorescence staining. Results Nimodipine treatment and Ca v 1.2 knockdown generated similar results. The number of positive calcium nodules and the protein and mRNA levels of dentin sialophosphoprotein were significantly reduced during odontogenic differentiation. The levels of microtubule-associated protein-2 and β-III-tubulin were reduced in neural differentiation. The expression of DCT decreased after odontogenic differentiation but significantly increased after neural differentiation ( P n = 9). Conclusions Our data showed that LTCC blocker nimodipine inhibits the odontogenic and neural differentiation of rDPSCs, and Ca v 1.2 is responsible for the activity of LTCC. The expression of DCT of Ca v 1.2 significantly changes during both odontogenic and neural differentiation. Thus, Ca v 1.2 of LTCC plays an essential role in differentiation of DPSCs, which might be mediated through the regulation of DCT levels in DPSCs.

The Drosophila heart is composed of two distinct cell types, the contractile cardial cells (CCs) and the surrounding non-muscle pericardial cells (PCs), development of which is regulated by a network of conserved signaling molecules and transcription factors (TFs). Here, we used machine learning with array-based chromatin immunoprecipitation (ChIP) data and TF sequence motifs to computationally classify cell type-specific cardiac enhancers. Extensive testing of predicted enhancers at single-cell resolution revealed the added value of ChIP data for modeling cell type-specific activities. Furthermore, clustering the top-scoring classifier sequence features identified novel cardiac and cell type-specific regulatory motifs. For example, we found that the Myb motif learned by the classifier is crucial for CC activity, and the Myb TF acts in concert with two forkhead domain TFs and Polo kinase to regulate cardiac progenitor cell divisions. In addition, differential motif enrichment and cis-trans genetic studies revealed that the Notch signaling pathway TF Suppressor of Hairless [Su(H)] discriminates PC from CC enhancer activities. Collectively, these studies elucidate molecular pathways used in the regulatory decisions for proliferation and differentiation of cardiac progenitor cells, implicate Su(H) in regulating cell fate decisions of these progenitors, and document the utility of enhancer modeling in uncovering developmental regulatory subnetworks.

Transcriptional enhancers are a primary mechanism by which tissue-specific gene expression is achieved. Despite the importance of these regulatory elements in development, responses to environmental stresses and disease, testing enhancer activity in animals remains tedious, with a minority of enhancers having been characterized. Here we describe 'enhancer-FACS-seq' (eFS) for highly parallel identification of active, tissue-specific enhancers in Drosophila melanogaster embryos. Analysis of enhancers identified by eFS as being active in mesodermal tissues revealed enriched DNA binding site motifs of known and putative, previously uncharacterized mesodermal transcription factors. Naive Bayes classifiers using transcription factor binding site motifs accurately predicted mesodermal enhancer activity. Application of eFS to other cell types and organisms should accelerate the cataloging of enhancers and understanding how transcriptional regulation is encoded in them.

Local catabolism of tryptophan (Trp) by indoleamine 2,3-dioxygenase (IDO) is considered an important mechanism of regulating T cell immunity. N-(3,4-dimethoxycinnamonyl) anthranilic acid (3,4-DAA) is an active synthetic anthranilic acid derivative which was proved to be effective to treat type1helper T lymphocyte (Th1) mediated autoimmune diseases such as multiple sclerosis. In this report, we investigated the effects of 3,4-DAA on the acute graft versus host disease (aGVHD) following allogeneic bone marrow transplantation (allo-BMT) and its potential mechanism of action. We established a murine aGVHD model, 3,4-DAA was injected intraperitoneally at 200mg/kg/day per mouse immediately after allo-BMT or at the onset of aGVHD for 14 consecutive days; the signs of aGVHD and the survival were recorded periodically. We revealed that administration of 3,4-DAA after allo-BMT significantly reduced the severity and the histological score of aGVHD; the survival for mice receiving 3,4-DAA prophylaxis and treatment was prolonged in comparison to the vehicle control mice. The plasma levels of IFN-γ, TNF-α, IL-12 and IL-2 in 3,4-DAA treatment group were found to be decreased, while the IDO activity, CD4(+)/CD25(+) Treg cells, and the IL-10, IL-5 and IL-4 levels elevated in these mice. In consistent with the in vivo results, 3,4-DAA also inhibited IFN-γ and IL-2 production of spleen T lymphocytes in vitro. Our findings suggest that 3,4-DAA can diminish the murine experimental aGVHD through regulating T cell proliferation and polarization; this property makes it a potential alternative agent for prevention and treatment of GVHD in the clinic.

Indoleamine 2,3-dioxygenase (IDO) is a rate-limiting enzyme for the tryptophan catabolism that plays an important role in the induction of immune tolerance. To evaluate the expression levels of IDO and interferon (IFN)-γ in patients receiving allogeneic hematopoietic stem cell transplantation (allo-HSCT) and to identify the correlation between IDO activity, IFN-γ, and acute graft-versus-host disease (aGVHD), we measured IDO mRNA expression in peripheral blood mononuclear cells in 89 allo-HSCT patients by reverse transcription-polymerase chain reaction. The IDO activity in plasma was also performed by reverse-phase high-performance liquid chromatography; plasma IFN-γ was detected by a standard enzyme-linked immunosorbent assay. IDO mRNA was detected in 55 of 74 patients (74.32%) with aGVHD. Of patients without aGVHD, only 2 of 26 expressed IDO mRNA (7.69%); none of 8 healthy volunteers was positive for IDO expression. Plasma IDO activity was much higher in aGVHD patients than in those without aGVHD (4.74 ± 3.35 vs 1.79 ± 1.02, respectively; P

Background Tip60 is a key histone acetyltransferase (HAT) enzyme that plays a central role in diverse biological processes critical for general cell function; however, the chromatin-mediated cell-type specific developmental pathways that are dependent exclusively upon the HAT activity of Tip60 remain to be explored. Methods and Findings Here, we investigate the role of Tip60 HAT activity in transcriptional control during multicellular development in vivo by examining genome-wide changes in gene expression in a Drosophila model system specifically depleted for endogenous dTip60 HAT function. Conclusions We show that amino acid residue E431 in the catalytic HAT domain of dTip60 is critical for the acetylation of endogenous histone H4 in our fly model in vivo, and demonstrate that dTip60 HAT activity is essential for multicellular development. Moreover, our results uncover a novel role for Tip60 HAT activity in controlling neuronal specific gene expression profiles essential for nervous system function as well as a central regulatory role for Tip60 HAT function in general metabolism.

Chromatin packaging directly influences gene programming as it permits only certain portions of the genome to be activated in any given developmental stage, cell, and tissue type. Histone acetyltransferases (HATs) are a key class of chromatin regulatory proteins that mediate such developmental chromatin control; however, their specific roles during multicellular development remain unclear. Here, we report the first isolation and developmental characterization of a Drosophila HAT gene (Dmel\TIP60) that is the homolog of the human HAT gene TIP60. We show that Dmel\TIP60 is differentially expressed during Drosophila development, with transcript levels significantly peaking during embryogenesis. We further demonstrate that reducing endogenous Dmel\TIP60 expression in Drosophila embryonic cells by RNAi results in cellular defects and lethality. Finally, using a GAL4-targeted RNAi system in Drosophila, we show that ubiquitous or mesoderm/muscle-specific reduction of Dmel\TIP60 expression results in lethality during fly development. Our results suggest a mechanism for HAT regulation involving developmental control of HAT expression profiles and show that Dmel\TIP60 is essential for multicellular development. Significantly, our inducible and targeted HAT knockdown system in Drosophila now provides a powerful tool for effectively studying the roles of TIP60 in specific tissues and cell types during development.

Using the paradigm of in vitro differentiation of hESCs/iPSCs into retinal pigment epithelial (RPE) cells, we have recently profiled mRNA and miRNA transcriptomes to define a set of RPE mRNA and miRNA signature genes implicated in directed RPE differentiation. In this study, in order to understand the role of DNA methylation in RPE differentiation, we profiled genome-scale DNA methylation patterns using the method of reduced representation bisulfite sequencing (RRBS). We found dynamic waves of de novo methylation and demethylation in four stages of RPE differentiation. Integrated analysis of DNA methylation and RPE transcriptomes revealed a reverse-correlation between levels of DNA methylation and expression of a subset of miRNA and mRNA genes that are important for RPE differentiation and function. Gene Ontology (GO) analysis suggested that genes undergoing dynamic methylation changes were related to RPE differentiation and maturation. We further compared methylation patterns among human ESC- and iPSC-derived RPE as well as primary fetal RPE (fRPE) cells, and discovered that specific DNA methylation pattern is useful to classify each of the three types of RPE cells. Our results demonstrate that DNA methylation may serve as biomarkers to characterize the cell differentiation process during the conversion of human pluripotent stem cells into functional RPE cells.