Three Alternaria Species, Including a New Species, Causing Leaf Spot Disease of Loquat (Eriobotrya japonica) in China.

Loquat (Eriobotrya japonica) is an economically important subtropical fruit crop in China. Field surveys conducted in different loquat orchards located in Chongqing, Sichuan and Fujian province between 2017-2020 resulted in a collection of 56 Alternaria-like isolates from trees exhibiting symptoms of loquat leaf spot. Multigene phylogenetic analyses using seven gene regions, namely ITS, gapdh, RPB2, tef1, Alt a 1, endoPG and OPA10-2, showed that all the isolates belonged to the genus Alternaria, and supporting morphological analysis identified them as members of species A. alternata, A. gaisen and A. chongqingensis sp. nov. In vitro- and in vivo- pathogenicity tests showed all the identified species to be pathogenic and able to cause leaf spot disease on loquat. Moreover, comprehensive phylogenetic analyses employing all combinations of the above seven gene sequences revealed the capability of Alt a 1-tef1-endoPG to provide a well-resolved gene tree for Alternaria spp. at the species level. This study adds to the current knowledge on an unknown species (A. chongqingensis sp. nov.) and the first report of A. gaisen in loquat worldwide.

Neoadjuvant checkpoint blockade in combination with Chemotherapy in patients with tripe-negative breast cancer: exploratory analysis of real-world, multicenter data.

PURPOSE: Despite the poor prognosis of triple-negative breast cancer (TNBC), it has been demonstrated that neoadjuvant immunotherapy in combination with chemotherapy can improve the pathologic complete response (pCR) rate and/or long-term outcome of TNBC. However, there have been no real-world studies reporting on the effectiveness of neoadjuvant checkpoint inhibitors in early TNBC. METHODS: Between November 2019 and December 2021, 63 early TNBC patients treated with anti-PD-1 antibodies (pembrolizumab or camrelizumab) or anti-PD-L1 antibody (atezolizumab) in combination with chemotherapy at seven institutions were included. PCR1 defined as ypT0/Tis and ypN0 was the primary endpoint. Secondary endpoints included pCR2 defined as ypT0/Tis, overall response rate (ORR), disease-free survival (DFS), drug-related adverse events (AEs) and biomarkers. RESULTS: Among the patients in the current study, 34.9% of patients were able to achieve pCR1, and 47.6% of patients had achieved pCR2. The ORR was 82.5%. 33 patients with non-pCR2 tumors were found to have a median DFS of 20.7 months (95% CI 16.3 months-not reached). The DFS of patients with pCR2 and non-pCR2 after neoadjuvant therapy was significantly different (HR = 0.28, 95% CI 0.10-0.79; P = 0.038). The most common AEs were nausea (63.4%), fatigue (42.7%), leucopenia (30.0%) and elevated transaminase (11.7%). CONCLUSION: It is possible to achieve a meaningful pCR rate and DFS by combining neoadjuvant checkpoint blockade with chemotherapy in patients with high-risk TNBC. Compared to clinical trials, however, there was a slightly lower pCR rate in this multicentered real-world study.

Multi-omics analyses of serum metabolome, gut microbiome and brain function reveal dysregulated microbiota-gut-brain axis in bipolar depression.

The intricate processes of microbiota-gut-brain communication in modulating human cognition and emotion, especially in the context of mood disorders, have remained elusive. Here we performed faecal metagenomic, serum metabolomics and neuroimaging studies on a cohort of 109 unmedicated patients with depressed bipolar disorder (BD) patients and 40 healthy controls (HCs) to characterise the microbial-gut-brain axis in BD. Across over 12,000 measured metabolic features, we observed a large discrepancy (73.54%) in the serum metabolome between BD patients and HCs, spotting differentially abundant microbial-derived neuroactive metabolites including multiple B-vitamins, kynurenic acid, gamma-aminobutyric acid and short-chain fatty acids. These metabolites could be linked to the abundance of gut microbiota presented with corresponding biosynthetic potentials, including Akkermansia muciniphila, Citrobacter spp. (Citrobacter freundii and Citrobacter werkmanii), Phascolarctobacterium spp., Yersinia spp. (Yersinia frederiksenii and Yersinia aleksiciae), Enterobacter spp. (Enterobacter cloacae and Enterobacter kobei) and Flavobacterium spp. Based on functional neuroimaging, BD-related neuroactive microbes and metabolites were discovered as potential markers associated with BD-typical features of functional connectivity of brain networks, hinting at aberrant cognitive function, emotion regulation, and interoception. Our study combines gut microbiota and neuroactive metabolites with brain functional connectivity, thereby revealing potential signalling pathways from the microbiota to the gut and the brain, which may have a role in the pathophysiology of BD.

Inversion of Forest Biomass Based on Multi-Source Remote Sensing Images.

Ecological forests are an important part of terrestrial ecosystems, are an important carbon sink and play a pivotal role in the global carbon cycle. At present, the comprehensive utilization of optical and radar data has broad application prospects in forest parameter extraction and biomass estimation. In this study, tree and topographic data of 354 plots in key nature reserves of Liaoning Province were used for biomass analysis. Remote sensing parameters were extracted from Landsat 8 OLI and Sentinel-1A radar data. Based on the strong correlation factors obtained via Pearson correlation analysis, a linear model, BP neural network model and PSO neural network model were used to simulate the biomass of the study area. The advantages of the three models were compared and analyzed, and the optimal model was selected to invert the biomass of Liaoning province. The results showed that 44 factors were correlated with forest biomass (p < 0.05), and 21 factors were significantly correlated with forest biomass (p < 0.01). The comparison between the prediction results of the three models and the real results shows that the PSO-improved neural network simulation results are the best, and the coefficient of determination is 0.7657. Through analysis, it is found that there is a nonlinear relationship between actual biomass and remote sensing data. Particle swarm optimization (PSO) can effectively solve the problem of low accuracy in traditional BP neural network models while maintaining a good training speed. The improved particle swarm model has good accuracy and speed and has broad application prospects in forest biomass inversion.

An evolutionarily conserved long noncoding RNA TUNA controls pluripotency and neural lineage commitment.

Here, we generated a genome-scale shRNA library targeting long intergenic noncoding RNAs (lincRNAs) in the mouse. We performed an unbiased loss-of-function study in mouse embryonic stem cells (mESCs) and identified 20 lincRNAs involved in the maintenance of pluripotency. Among these, TUNA (Tcl1 Upstream Neuron-Associated lincRNA, or megamind) was required for pluripotency and formed a complex with three RNA-binding proteins (RBPs). The TUNA-RBP complex was detected at the promoters of Nanog, Sox2, and Fgf4, and knockdown of TUNA or the individual RBPs inhibited neural differentiation of mESCs. TUNA showed striking evolutionary conservation of both sequence- and CNS-restricted expression in vertebrates. Accordingly, knockdown of tuna in zebrafish caused impaired locomotor function, and TUNA expression in the brains of Huntington's disease patients was significantly associated with disease grade. Our results suggest that the lincRNA TUNA plays a vital role in pluripotency and neural differentiation of ESCs and is associated with neurological function of adult vertebrates.

Potentials of CCL21 and CBS as Therapeutic Approaches for Breast Cancer.

OBJECTIVE: The present research set out to ascertain the roles of CC chemokine ligand 21 (CCL21) and cystathionine beta-synthase (CBS) in breast cancer (BC) cell biological behaviors and the relationship of CCL21 and CBS expression with the clinicopathological features of patients with BC. METHODS: Immunohistochemistry of CCL21 or CBS was performed in 18 intraductal cancer tissues, 124 invasive BC tissues, 50 paraneoplastic tissues, 50 lobular hyperplasia tissues, and 30 normal breast tissues. For cell experiments, two human BC cell lines (MDA-MB-231 and MCF-7) and a human breast epithelial cell line (MCF-10A) were utilized to detect the expression of CCL21 and CBS. After loss- and gain-of-function assays for CCL21 or CBS, the expression of CBS and CCL21 was measured by quantitative real-time polymerase chain reaction and western blot. Additionally, BC cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and 5-ethynyl-2'-deoxyuridine staining, and BC cell migration was determined by scratch test and Transwell assay. RESULTS: In the clinical data, the positive rate of CCL21 or CBS was significantly higher in invasive BC tissues than in intraductal BC tissues, lobular hyperplasia tissues, paraneoplastic tissues, and normal breast tissues (p < 0.05 or p < 0.01). CBS or CCL21 expression shared close association with the clinicopathological characteristics and the poor prognosis of BC patients. In cell experiments, overexpression of CCL21 or CBS enhanced the proliferative and migratory abilities of BC cells. CONCLUSION: CCL21 and CBS promoted BC cell migration and proliferation. CCL21 or CBS expression was strongly related to the poor prognosis of BC patients.

Genome-wide association study-based identification genes influencing agronomic traits in rice (Oryza sativa L.).

Rice is one of the most important cereal crops, providing the daily dietary intake for approximately 50% of the global human population. Here, we re-sequenced 259 rice accessions, generating 1371.65 Gb of raw data. Furthermore, we performed genome-wide association studies (GWAS) on 13 agronomic traits using 2.8 million single nucleotide polymorphisms (SNPs) characterized in 259 rice accessions. Phenotypic data and best linear unbiased prediction (BLUP) values of each of the 13 traits over two years of each trait were used for the GWAS. The results showed that 816 SNP signals were significantly associated with the 13 agronomic traits. Then we detected candidate genes related to target traits within 200 kb upstream and downstream of the associated SNP loci, based on linkage disequilibrium (LD) blocks in the whole rice genome. These candidate genes were further identified through haplotype block constructions. This comprehensive study provides a timely and important genomic resource for breeding high yielding rice cultivars.

[Cloning of transcription factor PcFBA-1 in Pogostemon cabin and its interaction with FPPS promoter].

Farnesyl diphosphate synthase(FPPS) is a key enzyme at the branch point of the sesquiterpene biosynthetic pathway, but there are no reports on the transcriptional regulation of FPPS promoter in Pogostemon cabin. In the early stage of this study, we obtained the binding protein PcFBA-1 of FPPS gene promoter in P. cabin. In order to explore the possible mechanism of PcFBA-1 involved in the regulation of patchouli alcohol biosynthesis, this study performed PCR-based cloning and sequencing analysis of PcFBA-1, analyzed the expression patterns of PcFBA-1 in different tissues by fluorescence quantitative PCR and its subcellular localization using the protoplast transformation system, detected the binding of PcFBA-1 protein to the FPPS promoter in vitro with the yeast one-hybrid system, and verified its transcriptional regulatory function by dual-luciferase reporter gene assay. The findings demonstrated that the cloned PcFBA-1 had an open reading frame(ORF) of 1 131 bp, encoding a protein of 376 amino acids, containing two conserved domains named F-box-like superfamily and FBA-1 superfamily, and belonging to the F-box family. Moreover, neither signal peptide nor transmembrane domain was contained, implying that it was an unstable hydrophilic protein. In addition, as revealed by fluorescence quantitative PCR results, PcFBA-1 had the highest expression in leaves, and there was no significant difference in expression in roots or stems. PcFBA-1 protein was proved mainly located in the cytoplasm. Furthermore, yeast one-hybrid screening and dual-luciferase reporter gene assay showed that PcFBA-1 was able to bind to FPPS promoter both in vitro and in vivo to enhance the activity of FPPS promoter. In summary, this study identifies a new transcription factor PcFBA-1 in P. cabin, which directly binds to the FPPS gene promoter to enhance the promoter activity. This had laid a foundation for the biosynthesis of patchouli alcohol and other active ingre-dients and provided a basis for metabolic engineering and genetic improvement of P. cabin.

Impaired blood-brain barrier in the microbiota-gut-brain axis: Potential role of bipolar susceptibility gene TRANK1.

Bipolar disorder (BD) is a common psychiatric illness with high prevalence and disease burden. Accumulating susceptibility genes for BD have been identified in recent years. However, the exact functions of these genes remain largely unknown. Despite its high heritability, gene and environment interaction is commonly accepted as the major contributing factor to BD pathogenesis. Intestine microbiota is increasingly recognized as a critical environmental factor for human health and diseases via the microbiota-gut-brain axis. BD individuals showed altered diversity and compositions in the commensal microbiota. In addition to pro-inflammatory factors, such as interleukin-6 and tumour necrosis factor-α, type 1 interferon signalling pathway is also modulated by specific intestinal bacterial strains. Disruption of the microbiota-gut-brain axis contributes to peripheral and central nervous system inflammation, which accounts for the BD aetiology. Administration of type 1 interferon can induce the expression of TRANK1, which is associated with elevated circulating biomarkers of the impaired blood-brain barrier in BD patients. In this review, we focus on the influence of intestine microbiota on the expression of bipolar gene TRANK1 and propose that intestine microbiota-dependent type 1 interferon signalling is sufficient to induce the over-expression of TRANK1, consequently causing the compromise of BBB integrity and facilitating the entrance of inflammatory mediators into the brain. Activated neuroinflammation eventually contributes to the occurrence and development of BD. This review provides a new perspective on how gut microbiota participate in the pathogenesis of BD. Future studies are needed to validate these assumptions and develop new treatment targets for BD.

Classification of PR-positive and PR-negative subtypes in ER-positive and HER2-negative breast cancers based on pathway scores.

PURPOSE: PR loss in ER+/HER2- breast cancer indicates worse prognosis and insensitivity to anti-estrogen therapy, while the mechanisms of PR loss in ER+/HER2- breast cancer remain unrevealed. METHODS: In this study, ER+/PR+/HER2- and ER+/PR-/HER2- breast cancer cases from TCGA were used. 1387 pathways were analyzed and used as variables for classifying the two groups with LASSO regression. RESULTS: ER+/PR+/HER2- and ER+/PR-/HER2- breast cancer groups can be classified by a combination of 13 pathways using their activity score. Among the 13 pathways, those involving growth factors and ion-channel transporters were most significant in the distinction, followed by pathways involving immune modulation and cell metabolism. Two growth factor pathways, EGF and IGF-1, were deferentially regulated in ER+/PR+/HER2- and ER+/PR-/HER2- groups. CONCLUSIONS: In conclusion, this study indicated in ER+/HER2- breast cancers the various status of PR expression can be an indication of molecular variation, particularly for the growth factor pathway activation.

First report of Fusarium solani Species Complex Causing Root Rot of Loquat (Eriobotrya japonica) in China.

Loquat (Eriobotrya japonica), a native fruit tree to China, is a popular edible fruit with medicinal properties (Badenes et al. 2013). A 2016-2019 field survey of ~13,000 loquat trees in two orchards in Chongqing and Fujian provinces showed about 5 to 10% root rot disease incidence. The disease symptoms included leaf yellowing, wilting, rotting of main root, and cracking of lateral roots, eventually leading to defoliation and death. To determine the causative agent, diseased roots from six trees were collected, washed in tap water, cut into 2-3 mm pieces, and disinfected for 3 min in 75% (v/v) EtOH. After rinsing in sterilized water, the root pieces were soaked in 10% NaClO (w/v) for 5-10 min, rinsed thrice in sterile water, and plated on potato dextrose agar (PDA). After 7 days of incubation at 25°C, individual spores were collected from the fungal colonies and replated. Single spore cultures growing on PDA gave rise to woolly-cottony, cream-white colored aerial mycelium and a yellowish pigmented mycelium. The average colony growth rate was 8.6 mm day-1 (n=3). Microscopic observation of the mycelium revealed septate and hyaline hyphae and long cylindrical monophialides. Macroconidia were moderately curved, stout, 3-4 septate, measuring 20.79-48.70 µm × 4.16-10.14 µm (n=50). Microconidia produced from long phialides were kidney-shaped, 0-2 septate, and 5.72-17.28 µm × 2.29-6.51 µm (n=50) in size. The mycelial characteristics and reproductive structures of the isolates fit the morphological description of Fusarium sp. (Summerell et al. 2003). To confirm this identification, translation elongation factor (EF-1α) and RNA polymerase I beta subunit (RPB1) and RNA polymerase II beta subunit (RPB2) regions of the genome were PCR amplified from 3 separate isolates (R2, R4 and R5) using EF1/ EF2, RPB1-Fa/G2R, RPB2-5f2/7cR & RPB2-7cF/11aR primer pairs (O'Donnell et al. 2010) and sequenced. BLASTn comparison of the EF-1α (MT976167), RPB1 (MT967271) and RPB2 (MW233052) regions from isolate R4 showed 99% identity with the EF-1α (GU170620, 675/676 bp), RPB1 (KC808270, 1543/1545 bp) and RPB2 (MK4419902, 1637/1638 bp) sequences of Fusarium solani species complex (FSSC) in GenBank database. The same species level identification was also found using FUSARIUM-ID and FUSARIUM-MLDT databases. Two-year-old seedlings (n=3) of two different cultivars, 'Hunanzaoshu' and 'Huabai No. 1', growing in pots indoors at 25-27 °C were inoculated by drenching the soil with a conidial suspension of isolate R4 (40 mL, 106 conidia mL-1 obtained from 6-10 day old cultures). Control plants (n=3) were inoculated with sterilized water. At 20 days after inoculation (DAI) the leaves of inoculated plants became chlorotic and wilted, defoliated over time, and by 53 DAI 91.67% of plants died. The taproot and lateral roots of inoculated plants appeared brown to black in color and most lateral roots died and decomposed at 53 DAI, whereas the control plant roots remained healthy. All control plants remained symptomless. Based on morphological and molecular characters (TEF-1, RPB1 and RPB2), the re-isolated pathogen from diseased plants was identical to the R4 isolate used for inoculation and the disease assays were repeated thrice. FSSC was recently reported to cause fruit rot disease on loquat in Pakistan (Abbas et al. 2017). Identifying Fusarium solani species complex as a disease agent in Chinese loquat will assist in future development of improved germplasm for this important worldwide tree crop.

Carbon disulfide induced decidualization disorder in the mice uterus at the window of implantation.

Carbon disulfide (CS2) is regarded as a common occupational poison that is widely used in the textile industry in China. Our previous research suggests that CS2 can induce significant implantation disorders in pregnant mice; however, the specific mechanism remains unclear. Uterine conception in mice must undergo decidualization, which is the prerequisite for propitious blastocyst implantation into the endometrium. Therefore, in this study, we established models of pregnant mice to explore the toxic effects of CS2 on decidualization to elucidate the basic mechanism of implantation disorder after CS2 exposure. The uterine tissues were immediately collected according to the predetermined endpoints to measure the expression levels of IGFBP1 and PRL (markers of decidualization differentiation), IL-11 (representing the secretory function of decidual cells), AKT and pAKT by western blotting, RT-PCR, immunohistochemical staining, H&E staining and ELISA. N-carbamoyl glutamic acid (NCG) acted as an agonist of AKT to verify the upstream regulatory mechanism of decidualization disorder by CS2. The results showed that the normal reaction of decidual transformation was obviously disrupted by CS2 upon 3.5 dpc and 4.5 dpc exposure. The blastocyst did not adhere to the epithelium after 3.5 dpc-exposure and did not invade the endometrium after 4.5 dpc-exposure, resulting in its suspension in the uterine cavity, stagnation and eventual loss. The proteins expression levels were decreased by 95.2% for IGFBP1 and 76.2% for PRL at the 4.5 dpc endpoint after 3.5 dpc CS2 exposure compared with the control. Simultaneously, the mRNA and protein expression levels of IL-11 in uterine tissues were significantly reduced by CS2, and consistent decreasing trends over time were observed for IGFBP1 and PRL, compared with the control. Additionally, the ratio of pAKT/AKT protein expression was decreased by 72.2% and 94.8% at 12 h and 18 h after 3.5 dpc exposure and by 53.3% and 74.3% at 6 h and 12 h after 4.5 dpc exposure, respectively, compared with the corresponding controls. Furthermore, NCG could recover the IGFBP1 and PRL protein expression, which was increased by 27.5% and 52.3% at 4.5 dpc and 6.5 dpc, respectively, after 3.5 dpc exposure for IGFBP1 and by 30.3% at 6.5 dpc after 4.5 dpc exposure for PRL, compared with CS2 exposure alone. Collectively, this study suggested that the decidualization disorder caused by CS2 at the window of implantation in pregnant mice, which is triggered by pAKT, contributed to the implantation disorder and eventually led to embryo loss. It is worth noting that our study may provide a new perspective and reference for exploring the toxic mechanism of implantation disorder and even infertility in harmful circumstances.

MiR-23a-3p-regulated abnormal acetylation of FOXP3 induces regulatory T cell function defect in Graves' disease.

This study aims to investigate the mechanism of miR-23a-3p in regulating Treg dysfunction in Graves' disease (GD). The percentage of Treg cells and interleukin (IL)-17+ T cells were determined by flow cytometry. The expression of forkhead box P3 (FOXP3), sirtuin 1 (SIRT1), RAR-related orphan receptor gamma t (RORγt) and miR-23a-3p was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) or Western blot. CD4+ T cells were treated with SIRT1 specific inhibitor EX-527 or left untreated. MiR-23a-3p mimic or inhibitor were transfected into CD4+ T cells. Acetylation expression of FOXP3 was analyzed by immunoprecipitation. The suppressive function of Treg was analyzed by the carboxyfluorescein succinimidyl ester (CFSE) assay. The results showed that GD patients have significantly less Treg cells and more IL-17+ T cells. FOXP3 and miR-23a-3p were significantly down-regulated meanwhile SIRT1 and RORγt were up-regulated in GD patients. FOXP3 acetylation level of the GD group was lower than that of control groups. After EX-527 treatment, the percentage of Treg cells, expression and acetylation level of FOXP3 were significantly increased in the GD group. GD Tregs exhibited weaker suppressive activity, miR-23a-3p mimic suppressed SIRT1 expression and suppressive-activity of Tregs whereas it promoted the expression and acetylation level of FOXP3 in the GD group. Our findings suggest that the Treg function defect in GD patients is mediated by the abnormal acetylation of FOXP3, which is regulated by miR-23a-3p via targeting SIRT1.

Forward and reverse mutations in stages of cancer development.

BACKGROUND: Massive occurrences of interstitial loss of heterozygosity (LOH) likely resulting from gene conversions were found by us in different cancers as a type of single-nucleotide variations (SNVs), comparable in abundance to the commonly investigated gain of heterozygosity (GOH) type of SNVs, raising the question of the relationships between these two opposing types of cancer mutations. METHODS: In the present study, SNVs in 12 tetra sample and 17 trio sample sets from four cancer types along with copy number variations (CNVs) were analyzed by AluScan sequencing, comparing tumor with white blood cells as well as tissues vicinal to the tumor. Four published "nontumor"-tumor metastasis trios and 246 pan-cancer pairs analyzed by whole-genome sequencing (WGS) and 67 trios by whole-exome sequencing (WES) were also examined. RESULTS: Widespread GOHs enriched with CG-to-TG changes and associated with nearby CNVs and LOHs enriched with TG-to-CG changes were observed. Occurrences of GOH were 1.9-fold higher than LOH in "nontumor" tissues more than 2 cm away from the tumors, and a majority of these GOHs and LOHs were reversed in "paratumor" tissues within 2 cm of the tumors, forming forward-reverse mutation cycles where the revertant LOHs displayed strong lineage effects that pointed to a sequential instead of parallel development from "nontumor" to "paratumor" and onto tumor cells, which was also supported by the relative frequencies of 26 distinct classes of CNVs between these three types of cell populations. CONCLUSIONS: These findings suggest that developing cancer cells undergo sequential changes that enable the "nontumor" cells to acquire a wide range of forward mutations including ones that are essential for oncogenicity, followed by revertant mutations in the "paratumor" cells to avoid growth retardation by excessive mutation load. Such utilization of forward-reverse mutation cycles as an adaptive mechanism was also observed in cultured HeLa cells upon successive replatings. An understanding of forward-reverse mutation cycles in cancer development could provide a genomic basis for improved early diagnosis, staging, and treatment of cancers.

Carbon Disulfide Induces Embryo Implantation Disorder by Disturbing the Polarization of Macrophages in Mice Uteri.

Carbon disulfide (CS2) induces embryo implantation disorders. Macrophages participate in the process of pregnancy. Therefore, we want to explore the effects of CS2 exposure on polarization and immune function of macrophages in pregnant mice uteri. The exposure times were gestation days 3 (GD3), 4 (GD4), and 5 (GD5), and the observation end points were arranged in a time series after CS2 exposure. The uterine tissues were collected to detect the expression levels of macrophages cytokines (IL-6, IL-12, TGF-ß1, and Vegf-a) and downstream regulatory cytokines of Th1-type (IL-2 and IFN-γ) and Th2-type (IL-10 and IL-4) by flow cytometry, ELISA, and q-PCR. The results showed that, compared with the controls, the ratios of M1/M2 macrophages in the endometrium significantly increased by 96%, 110%, and 177% at the GD4, GD6, and GD7 observation end points after GD3 exposure and increased about 3.88-fold and 2.37-fold at the GD6 and GD7 observation end points after GD4 exposure, respectively. In contrast, the ratio of M1 and M2 macrophages significantly reduced by 53% at the GD5 observation end point after GD3 exposure. Meanwhile, the expression levels of IL-6 were significantly increased about 2.00-fold for mRNA and 1.60-fold for protein at GD4 observation end points after GD3 exposure, and the mRNA levels of IL-12 increased about 3.61-fold at the GD6 observation end points after GD4 exposure. The mRNA levels of TGF-ß1 were significantly decreased by 41%, 25%, and 20% at the GD7 observation end points after exposure at GD3, GD4, and GD5, and the expression levels of Vegf-a mRNA and protein were decreased. Furthermore, the ratio of IL-2/IL4, IL-2/IL-10, IFN-γ/IL-4, and IFN-γ/IL-10 in the uterine tissue was significantly increased at the exposure groups. These findings suggest that the imbalanced polarization of macrophages is the key regulator in the progress of CS2-induced embryo loss.

A TP73-AS1/miR-200a/ZEB1 regulating loop promotes breast cancer cell invasion and migration.

Breast cancer (BC) is one of the leading causes of cancer deaths worldwide and the most common cancer among women. In our previous study, we revealed that lncRNA TP73-AS1 promotes breast cancer cell proliferation through directly binding to miR-200a. Herein, we evaluated the effect of TP73-AS1 in breast cancer cell invasion and migration, and further demonstrated the direct binding between TP73-AS1 and miR-200a, between miR-200a and 3'UTR of ZEB1, an essential metastasis-related transcription factor. TP73-AS1 promoted ZEB1 expression via competing with ZEB1 3'UTR for miR-200a binding. Moreover, ZEB1 could bind to the promoter region of TP73-AS1 to activate its expression. TP73-AS1 and ZEB1 expression was up-regulated, whereas miR-200a expression was down-regulated in breast cancer tissues. Taken together, we demonstrated a TP73-AS1/miR-200a/ZEB1 regulating loop in breast cancer cells, which promote cancer cell invasion and migration through regulating E-cadherin and Twist expression. Suppressing TP73-AS1 expression to rescue miR-200a expression, thus to inhibit ZEB1 and Twist expression and up-regulate E-cadherin might improve breast cancer cell invasion and migration.

TP73-AS1 promotes breast cancer cell proliferation through miR-200a-mediated TFAM inhibition.

P73 antisense RNA 1T (TP73-AS1 or PDAM) is a long non-coding RNA, which can regulate apoptosis through regulation of p53 signaling-related anti-apoptotic genes. An abnormal change of TP73-AS1 expression was noticed in cancers. The effects of TP73-AS1 in breast cancer (BC) growth and the underlying mechanism remain unclear so far. In the present study, the effect of TP73-AS1 in BC cell lines and clinical tumor samples was detected so as to reveal its role and function. In the present study, TP73-AS1 was specifically upregulated in BC tissues and BC cell lines and was correlated to a poorer prognosis in patients with BC. TP73-AS1 knocking down suppressed human BC cell proliferation in vitro through regulation of TFAM. In our previous study, we demonstrated that miR-200a inhibits BC cell proliferation through targeting TFAM; here we revealed that TP73-AS1 could regulate miR-200a through direct targeting. Moreover, TP73-AS1 might compete with TFAM for miR-200a binding thus to promote TFAM expression. Data from the present study revealed that TP73-AS1 promoted BC cell proliferation through acting as a competing endogenous RNA (ceRNA) by sponging miR-200a. In conclusion, we regarded TP73-AS1 as an oncogenic lncRNA promoting BC cell proliferation and a potential target for human BC treatment.

Identification of novel genes and networks governing hematopoietic stem cell development.

Hematopoietic stem cells (HSCs) are capable of giving rise to all blood cell lineages throughout adulthood, and the generation of engraftable HSCs from human pluripotent stem cells is a major goal for regenerative medicine. Here, we describe a functional genome-wide RNAi screen to identify genes required for the differentiation of embryonic stem cell (ESC) into hematopoietic stem/progenitor cells (HSPCs) in vitro We report the discovery of novel genes important for the endothelial-to-hematopoietic transition and subsequently for HSPC specification. High-throughput sequencing and bioinformatic analyses identified twelve groups of genes, including a set of 351 novel genes required for HSPC specification. As in vivo proof of concept, four of these genes, Ap2a1, Mettl22, Lrsam1, and Hal, are selected for validation, confirmed to be essential for HSPC development in zebrafish and for maintenance of human HSCs. Taken together, our results not only identify a number of novel regulatory genes and pathways essential for HSPC development but also serve as valuable resource for directed differentiation of therapy grade HSPCs using human pluripotent stem cells.

Specification of hepatopancreas progenitors in zebrafish by hnf1ba and wnt2bb.

Although the liver and ventral pancreas are thought to arise from a common multipotent progenitor pool, it is unclear whether these progenitors of the hepatopancreas system are specified by a common genetic mechanism. Efforts to determine the role of Hnf1b and Wnt signaling in this crucial process have been confounded by a combination of factors, including a narrow time frame for hepatopancreas specification, functional redundancy among Wnt ligands, and pleiotropic defects caused by either severe loss of Wnt signaling or Hnf1b function. Using a novel hypomorphic hnf1ba zebrafish mutant that exhibits pancreas hypoplasia, as observed in HNF1B monogenic diabetes, we show that hnf1ba plays essential roles in regulating ß-cell number and pancreas specification, distinct from its function in regulating pancreas size and liver specification, respectively. By combining Hnf1ba partial loss of function with conditional loss of Wnt signaling, we uncover a crucial developmental window when these pathways synergize to specify the entire ventrally derived hepatopancreas progenitor population. Furthermore, our in vivo genetic studies demonstrate that hnf1ba generates a permissive domain for Wnt signaling activity in the foregut endoderm. Collectively, our findings provide a new model for HNF1B function, yield insight into pancreas and ß-cell development, and suggest a new mechanism for hepatopancreatic specification.

Identification of Annexin A4 as a hepatopancreas factor involved in liver cell survival.

To gain insight into liver and pancreas development, we investigated the target of 2F11, a monoclonal antibody of unknown antigen, widely used in zebrafish studies for labeling hepatopancreatic ducts. Utilizing mass spectrometry and in vivo assays, we determined the molecular target of 2F11 to be Annexin A4 (Anxa4), a calcium binding protein. We further found that in both zebrafish and mouse endoderm, Anxa4 is broadly expressed in the developing liver and pancreas, and later becomes more restricted to the hepatopancreatic ducts and pancreatic islets, including the insulin producing ß-cells. Although Anxa4 is a known target of several monogenic diabetes genes and its elevated expression is associated with chemoresistance in malignancy, its in vivo role is largely unexplored. Knockdown of Anxa4 in zebrafish leads to elevated expression of caspase 8 and Δ113p53, and liver bud specific activation of Caspase 3 and apoptosis. Mosaic knockdown reveal that Anxa4 is required cell-autonomously in the liver bud for cell survival. This finding is further corroborated with mosaic anxa4 knockout studies using the CRISPR/Cas9 system. Collectively, we identify Anxa4 as a new, evolutionarily conserved hepatopancreatic factor that is required in zebrafish for liver progenitor viability, through inhibition of the extrinsic apoptotic pathway. A role for Anxa4 in cell survival may have implications for the mechanism of diabetic ß-cell apoptosis and cancer cell chemoresistance.