Oomycete pathogens produce a large number of effectors to promote infection. Their mode of action are largely unknown. Here we show that a Phytophthora sojae effector, PsCRN63, suppresses flg22-induced expression of FRK1 gene, a molecular marker in pathogen-associated molecular patterns (PAMP)-triggered immunity (PTI). However, PsCRN63 does not suppress upstream signaling events including flg22-induced MAPK activation and BIK1 phosphorylation, indicating that it acts downstream of MAPK cascades. The PsCRN63-transgenic Arabidopsis plants showed increased susceptibility to bacterial pathogen Pseudomonas syringae pathovar tomato (Pst) DC3000 and oomycete pathogen Phytophthora capsici. The callose deposition were suppressed in PsCRN63-transgenic plants compared with the wild-type control plants. Genes involved in PTI were also down-regulated in PsCRN63-transgenic plants. Interestingly, we found that PsCRN63 forms an dimer that is mediated by inter-molecular interactions between N-terminal and C-terminal domains in an inverted association manner. Furthermore, the N-terminal and C-terminal domains required for the dimerization are widely conserved among CRN effectors, suggesting that homo-/hetero-dimerization of Phytophthora CRN effectors is required to exert biological functions. Indeed, the dimerization was required for PTI suppression and cell death-induction activities of PsCRN63.

The calcifying shell is an excellent model for studying biomineralization and evolution. However, the molecular mechanisms of shell formation are only beginning to be elucidated in Mollusca. It is known that shell matrix proteins (SMPs) play important roles in shell formation. With increasing data of shell matrix proteomes from various species, we carried out a BLASTp bioinformatics analysis using the shell matrix proteome from Crassostrea gigas against 443 SMPs from nine other species. The highly conserved tyrosinase and chitin related proteins were identified in bivalve. In addition, the relatively conserved proteins containing domains of carbonic anhydrase, Sushi, Von Willebrand factor type A, and chitin binding, were identified from all the ten species. Moreover, 25 genes encoding SMPs were annotated and characterized that are involved in CaCO3 crystallization and represent chitin related or ECM related proteins. Together, data from these analyses provide new knowledge underlying the molecular mechanism of shell formation in C.gigas, supporting a refined shell formation model including chitin and ECM-related proteins.

A study was conducted to investigate the circulation of HRSV subgroup B (HRSVB) in China in recent years. HRSVB sequences from 365 samples collected in 1991, 2004 and 2008-2014 in China, together with 332 Chinese HRSVB sequences obtained from GenBank were analyzed to determine the geographic and yearly distribution of HRSVB. Phylogenetic analysis revealed these HRSVB sequences clustered into 4 genotypes with different frequencies: BA (83%), CB1 (11%), SAB (3.0%) and GB3 (0.7%). Between 2005 and 2013, there was a co-circulation of BA and non-BA genotypes in China. Genotypes BA9 and BA10 were two of the main BA genotypes detected in this study. Genotype BA9 was first detected in China in 2006 and became the predominant HRSVB genotype circulating in China from 2008 to 2014. Three different lineages were detected for both genotypes BA9 and BA10. Time to the most recent common ancestor for genotypes BA9 and BA10 was estimated for years 1997 and 1996, respectively. Results of this study not only contribute to the understanding of the circulation pattern, but also the phylogenetic pattern and evolution of HRSVB in China from 1991 to 2014.

Myeloid differentiation 1 (MD-1), a secreted protein interacting with radioprotective 105 (RP105), plays an important role in Toll-like receptor 4 (TLR4) signalling pathway. Previous studies showed that MD-1 may be restricted in the immune system. In this study, we demonstrated for the first time that MD-1 was highly expressed in both human and animal hearts. We also discovered that cardiac-specific overexpression of MD-1 significantly attenuated pressure overload-induced cardiac hypertrophy, fibrosis, and dysfunction, whereas loss of MD-1 had the opposite effects. Similar results were observed for in vitro angiotensin II-induced neonatal rat cardiomyocyte hypertrophy. The antihypertrophic effects of MD-1 under hypertrophic stimuli were associated with the blockage of MEK-ERK 1/2 and NF-κB signalling. Blocking MEK-ERK 1/2 signalling with a pharmacological inhibitor (U0126) greatly attenuated the detrimental effects observed in MD-1 knockout cardiomyocytes exposed to angiotensin II stimuli. Similar results were observed by blocking NF-κB signalling with a pharmacological inhibitor (BAY11-7082). Our data indicate that MD-1 inhibits cardiac hypertrophy and suppresses cardiac dysfunction during the remodelling process, which is dependent on its modulation of the MEK-ERK 1/2 and NF-κB signalling pathways. Thus, MD-1 might be a novel target for the treatment of pathological cardiac hypertrophy.

Glioma is the most prevalent malignant brain tumor. A comprehensive analysis of the glioma metabolome is still lacking. This study aims to explore new special metabolites in glioma tissues. A non-targeted human glioma metabolomics was performed by UPLC-Q-TOF/MS. The gene expressions of 18 enzymes associated with 3'-phosphoadenylate (pAp) metabolism was examined by qRT-PCR. Those enzymes cover the primary metabolic pathway of pAp. We identified 15 new metabolites (13 lipids and 2 nucleotides) that were significantly different between the glioma and control tissues. Glycerophosphatidylcholine [PC(36:1)] content was high and pAp content was significantly low in the control brain (p  < 0.01). In glioma tissues, PC(36:1) was not detected and pAp content was significantly increased. The gene expressions of 3'-nucleotidases (Inositol monophosphatase (IMPAD-1) and 3'(2'),5'-bisphosphate nucleotidase 1(BPNT-1)) were dramatically down-regulated. Meanwhile, the gene expression of 8 sulfotransferases (SULT), 2 phosphoadenosine phosphosulfate synthases (PAPSS-1 and PAPSS-2) and L-aminoadipate-semialdehyde dehydrogenase-phosphopante-theinyl transferase (AASDHPPT) were up-regulated. PC(36:1) absence and pAp accumulation are the most noticeable metabolic aberration in glioma. The dramatic down-regulation of IMPAD-1 and BPNT-1 are the primary cause for pAp dramatic accumulation. Our findings suggest that differential metabolites discovered in glioma could be used as potentially novel therapeutic targets or diagnostic biomarkers and that abnormal metabolism of lipids and nucleotides play roles in the pathogenesis of glioma.

Compound Opening Arrow Mixture (COAM) has demonstrated therapeutic effects in patients with breast cancer. We explored the underlying molecular mechanisms of COAM using a mouse model of breast cancer. Luciferase-labeled 4T1-Luc2 cells were inoculated into the breast pad of BALB/c-nu mice, which were divided into model group (saline), COAM (6 g/ml high-dose, 3 g/ml medium-dose, and 1.5 g/ml low-dose) groups, and low-molecular-weight heparin (LMWH, 1500 U/Kg) group. The number and distribution of 4T1-luc2 tumors were measured by an in vivo imaging system. Tumor cell apoptosis was measured through TUNEL and quantitating the expression of Caspase-3 mRNA and protein. Compared with the model group, in vivo tumor growth was lower in the LMWH- and COAM-treated groups. Tumor apoptosis was time-dependent and dose-dependent, as shown by a higher TUNEL apoptotic index and higher Caspase-3 mRNA and Caspase-3/cleaved-Caspase-3 proteins levels on the 14th day than the 7th day. The COAM high-dose group had the highest apoptotic index and the most activation of Caspase-3. Collectively, COAM significantly inhibits the growth of 4T1-luc2 breast cancer in mice and induces tumor apoptosis by activating Caspase-3, which provides a preliminary explanation of therapeutic effects of COAM.

Fireflies are among the most charismatic insects for their spectacular bioluminescence, but the origin and evolution of bioluminescence remain elusive. Especially, the genic basis of luciferin (D-luciferin) biosynthesis and light patterns is largely unknown. Here, we present the high-quality reference genomes of two fireflies Lamprigera yunnana (1053 Mb) and Abscondita terminalis (501 Mb) with great differences in both morphology and luminous behavior. We sequenced the transcriptomes and proteomes of luminous organs of two species. We created the CRISPR/Cas9-induced mutants of Abdominal B gene without luminous organs in the larvae of A. terminalis and sequenced the transcriptomes of mutants and wild-types. Combining gene expression analyses with comparative genomics, we propose a more complete luciferin synthesis pathway, and confirm the convergent evolution of bioluminescence in insects. Using experiments, the function of the firefly acyl-CoA thioesterase (ACOT1) to convert L-luciferin to D-luciferin was validated for the first time. Comparisons of three-dimension reconstruction of luminous organs and their differentially expressed genes among two species suggest that two positive genes in the calcium signaling pathway and structural difference of luminous organs may play an important role in the evolution of flash pattern. Altogether, our results provide important resources for further exploring bioluminescence in insects.

The hollow cylindrical nanohydroxyapatite/polyamide 66 strut (n-HA/PA66) has been used clinically for anterior cervical reconstruction. However, rates of occurrence of a "radiolucent gap" between the dense strut and adjacent endplates were reported. The aim of this in vivo study was to evaluate the viability and advantages of the novel porous n-HA/PA66 strut. The goat C3/4 partial discectomy and fusion model was built, and two groups of n-HA/PA66 struts were implanted into C3/4: group 1, porous n-HA/PA66 strut; and group 2, hollow cylindrical n-HA/PA66 strut filled with autogenous cancellous bone. CT evaluation was performed to assess the fusion status after 12 and 24 weeks. The cervical spines were harvested. Histomorphological analysis was performed to determine new bone formation. Biomechanical testing was performed to determine range of motion (ROM). CT confirmed the disappearance of the boundary of the porous strut and host bone, while the radiolucent gap remained clearly discernible in the dense strut group. The mean CT fusion scores of the porous group were significantly higher. Histologic evaluation showed that the porous struts promoted better osteointegration. Calcein fluorochrome labelling indicated faster bone ingrowth in the porous struts. Biomechanical tests revealed that the porous struts had significantly reduced micromotion. The porous n-HA/PA66 strut could offer interesting potential for cervical reconstruction after corpectomy.

The pyrite oxidation is crucial to the overall black shale oxidation process. A. ferrooxidans was documented an effective oxidation ability on pure pyrite, but its role in black shale oxidation is unclear. In this study, a comparative study of acid solution and A. ferrooxidans on the micro-surface characteristics at the initial stage (7 days) was conducted on black shale slices, a comprehensive approach combining the micro-morphologies, micro-structures, micro-environmental pH and micro-surface elemental content were investigated by using polarizing microscopies, SEM, fluorescent staining and EDX line scan analysis. The pyrite oxidation rate was employed to the index for black shale oxidation degree, and analyzed by XRD, aqueous pH, oxidation-reduction potential (ORP), ferrous and ferric ions concentrations measurement. The results show that the micro-surface characteristics are different in acid solution and A. ferrooxidans groups, which significantly impact the pyrite oxidation rate. A. ferrooxidans promote the jarosite formation and elemental C accumulation on the rocks micro-surface, which is assumed to inhibit further reactions. Two reaction phases named "pyrite oxidized phase" and "jarosite formation phase" are proposed to occur in the initial stage of A. ferrooxidans oxidizing black shale. These findings provide experimental data to evaluate the micro-surface reactions during black shale oxidation process.

The diverse modes of sexual reproduction in Bivalvia make it an excellent clade to understand the evolution of sex and sex determination. The cosmopolitan Pacific oyster Crassostrea gigas is an ideal model for bivalve sex determination studies because of its complicated sexuality, including dioecy, sex change and rare hermaphroditism. A major barrier to C. gigas sex determination study has been the lack of information on the type of sex determination. To identify its sex-determining system, sex observation by following the same individual in two consecutive years was conducted on 760 oysters from distinct populations. Stable sexuality and sex reversal in both directions were observed, which provides a case against the protandry of C. gigas. Restriction site-associated DNA sequencing (RAD-seq) based on 26 samples with unchanged and converted sexualities was carried out for identifying sex-linked marker. One SNP Cgsl-40 was proved to be sex-related, but sex-biased heterozygosity varied between populations for RAD-seq and validation, showing no evidence for sex chromosomes or single-locus models for C. gigas primary sex determination. Information obtained in our study provides novel insight into sex determination mechanism in C. gigas.

Trichomonas vaginitis (TV) is the most common non-viral sexually transmitted infection (STI) worldwide. The high prevalence of TV combined with mild or asymptomatic early symptoms leads to clinical vulnerability from delayed diagnosis. Latent infection can increase the incidence of pelvic infections, infertility, and adverse pregnancy complications. Data from 898 women who underwent vaginal flora testing from June 2014 to December 2014 were used to create a nomogram to assess the risk of TV in women in order to guide TV prevention and clinical intervention. The prediction model was evaluated in terms of identification, calibration, and clinical utility using the C-index, calibration plots, decision curve analysis, and internal validation. Predictors in the TV nomogram included age, occupation, yearly income, tea drinking, bathing frequency, menopause, spontaneous abortion, use of contraceptives, history of gynecological surgery, and HPV infection. The C-index of the TV risk prediction model was 0.732 (95% confidence interval: 0.695-0.768). It showed good discriminatory and predictive power. Decision curve analysis indicated that the nomogram had a good net benefit when the threshold probability of TV in women was 2-80%. The established TV prediction model easily, accurately, and quickly predicts the risk of TV onset.

MicroRNAs (miRNAs) regulate post-transcription gene expression by targeting genes and play crucial roles in diverse biological processes involving body color formation. However, miRNAs and miRNA-targets underlying shell color polymorphism remain largely unknown in mollusca. Using four shell colors full-sib families of the Pacific oyster Crassostrea gigas, we systematically identified miRNAs and miRNA-targets in the mantles, which organ could produce white, golden, black or partially pigmented shell. RNA sequencing and analysis identified a total of 53 known miRNA and 91 novel miRNAs, 47 of which were detected to differentially express among six pairwise groups. By integrating miRNA and mRNA expression profiles, a total of 870 genes were predicted as targets of differentially expressed miRNAs, mainly involving in biomineralization and pigmentation through functional enrichment. Furthermore, a total of four miRNAs and their target mRNAs were predicted to involve in synthesis of melanin, carotenoid or tetrapyrrole. Of them, lgi-miR-317 and its targets peroxidase and lncRNA TCONS_00951105 are implicated in acting as the competing endogenous RNA to regulate melanogenesis. Our studies revealed the systematic characterization of miRNAs profiles expressed in oyster mantle, which might facilitate understanding the intricate molecular regulation of shell color polymorphism and provide new insights into breeding research in oyster.

DNA methylation patterns in plants are dynamically regulated by DNA methylation and active DNA demethylation in response to both environmental changes and development of plant. Beginning with the removal of methylated cytosine by ROS1/DME family of 5-methylcytosine DNA glycosylases, active DNA demethylation in plants occurs through base excision repair. So far, many components involved in active DNA demethylation remain undiscovered. Through a forward genetic screening of Arabidopsis mutants showing DNA hypermethylation at the EPF2 promoter region, we identified the conserved iron-sulfur cluster assembly protein MET18. MET18 dysfunction caused DNA hypermethylation at more than 1000 loci as well as the silencing of reporter genes and some endogenous genes. MET18 can directly interact with ROS1 in vitro and in vivo. ROS1 activity was reduced in the met18 mutant plants and point mutation in the conserved Fe-S cluster binding motif of ROS1 disrupted its biological function. Interestingly, a large number of DNA hypomethylated loci, especially in the CHH context, were identified from the met18 mutants and most of the hypo-DMRs were from TE regions. Our results suggest that MET18 can regulate both active DNA demethylation and DNA methylation pathways in Arabidopsis.

This study represents the first comprehensive molecular assessment of northwestern Pacific molluscs. In total, 2801 DNA barcodes belonging to 569 species from China, Japan and Korea were analyzed. An overlap between intra- and interspecific genetic distances was present in 71 species. We tested the efficacy of this library by simulating a sequence-based specimen identification scenario using Best Match (BM), Best Close Match (BCM) and All Species Barcode (ASB) criteria with three threshold values. BM approach returned 89.15% true identifications (95.27% when excluding singletons). The highest success rate of congruent identifications was obtained with BCM at 0.053 threshold. The analysis of our barcode library together with public data resulted in 582 Barcode Index Numbers (BINs), 72.2% of which was found to be concordantly with morphology-based identifications. The discrepancies were divided in two groups: sequences from different species clustered in a single BIN and conspecific sequences divided in one more BINs. In Neighbour-Joining phenogram, 2,320 (83.0%) queries fromed 355 (62.4%) species-specific barcode clusters allowing their successful identification. 33 species showed paraphyletic and haplotype sharing. 62 cases are represented by deeply diverged lineages. This study suggest an increased species diversity in this region, highlighting taxonomic revision and conservation strategy for the cryptic complexes.

Human pluripotent stem cells (hPSCs) play important role in studying the function of human glutamatergic neurons and related disease pathogenesis. However, the current hPSC-derived cortical system produced a significant number of inhibitory GABAergic neurons that reduced the purity of excitatory neurons. In this study, we established a robust hPSC-derived cortical neurogenesis system by applying the SHH inhibitor cyclopamine. Cyclopamine specified the dorsal cortical fate in a dose-dependent manner and enhanced the generation of cortical glutamatergic neurons, expressing PAX6, TBR1, TBR2, CTIP2, SATB2, and vesicular glutamate transporters (vGLUT). In contrast, the ventral patterning was inhibited and the GABAergic neurons were significantly reduced to 12% with the treatment of cyclopamine. In addition, we applied our current method to generate trisomy 21 iPSC-derived glutamatergic neurons that showed a robust reduction of vesicular glutamate transporters in the glutamatergic neurons with trisomy 21, revealing the developmental deficits in cortical glutamatergic neurons. Our method enriched the generation of cortical glutamatergic neurons which may facilitate the study of human neurological diseases and cell therapy.

CARMIL2 is required for CD28-mediated co-stimulation of NF-κB signaling in T cells and its deficiency has been associated with primary immunodeficiency and, recently, very early onset inflammatory bowel disease (IBD). Here we describe the identification of novel biallelic CARMIL2 variants in three patients presenting with pediatric-onset IBD and in one with autoimmune polyendocrine syndrome (APS). None manifested overt clinical signs of immunodeficiency before their diagnosis. The first patient presented with very early onset IBD. His brother was found homozygous for the same CARMIL2 null variant and diagnosed with APS. Two other IBD patients were found homozygous for a nonsense and a missense CARMIL2 variant, respectively, and they both experienced a complicated postoperative course marked by severe infections. Immunostaining of bowel biopsies showed reduced CARMIL2 expression in all the three patients with IBD. Western blot and immunofluorescence of transfected cells revealed an altered expression pattern of the missense variant. Our work expands the genotypic and phenotypic spectrum of CARMIL2 deficiency, which can present with either IBD or APS, aside from classic immunodeficiency manifestations. CARMIL2 should be included in the diagnostic work-up of patients with suspected monogenic IBD.

Mollusca are the second largest phylum in the animal kingdom with different types of locomotion. Some molluscs are poor-migrating, while others are free-moving or fast-swimming. Most of the energy required for locomotion is provided by mitochondria via oxidative phosphorylation. Here, we conduct a comparative genomic analysis of 256 molluscs complete mitochondrial genomes and evaluate the role of energetic functional constraints on the protein-coding genes, providing a new insight into mitochondrial DNA (mtDNA) evolution. The weakly locomotive molluscs, compared to strongly locomotive molluscs, show significantly higher Ka/Ks ratio, which suggest they accumulated more nonsynonymous mutations in mtDNA and have experienced more relaxed evolutionary constraints. Eleven protein-coding genes (CoxI, CoxII, ATP6, Cytb, ND1-6, ND4L) show significant difference for Ka/Ks ratios between the strongly and weakly locomotive groups. The relaxation of selective constraints on Atp8 arise in the common ancestor of bivalves, and the further relaxation occurred in marine bivalves lineage. Our study thus demonstrates that selective constraints relevant to locomotive ability play an essential role in evolution of molluscs mtDNA.

Aspartate family amino acids (AFAAs) have important commercial values due to their wide spectrum of applications. Most if not all AFAAs are produced under aerobic conditions which is energy-intensive. To establish a cost-effective anaerobic process for production of AFAAs, it holds great promise to develop a new pathway enabling the conversion of oxoloacetate into aspartate through direct amination which is catalyzed by aspartate dehydrogenase (AspDH). Compared with the well studied aspartate aminotransferase and aspartate ammonia-lyase, only a few AspDHs are characterized till date, and failure to reproduce the high activity of AspDH from Rastonia eutropha documented in the literature encouraged us to screen and characterize novel AspDHs from different origins. Interestingly, the AspDHs from Klebsiella pneumoniae 34618 (KpnAspDH) and Delftia sp. Cs1-4 (DelAspDH) showed successful soluble expression. KpnAspDH and DelAspDH containing C-terminal hexa-histidine tags were purified and characterized for their catalytic properties. Notably, in addition to its high reductive amination activity, DelAspDH exhibited considerable stability as compared to the other source of AspDHs. This work thus provides novel enzyme resource for engineering strains capable of producing AFAAs under anaerobic conditions.

Paraquat (PQ) is a widely used fast-acting pyridine herbicide. Accidental ingestion or self-administration via various routes can cause severe organ damage. Currently, no effective antidote is available commercially, and the mortality rate of poisoned patients is exceptionally high. Here, the efficacy of anthrahydroquinone-2-6-disulfonate (AH2QDS) was observed in treating PQ poisoning by constructing in vivo and ex vivo models. We then explored the detoxification mechanism of AH2QDS. We demonstrated that, in a rat model, the PQ concentration in the PQ + AH2QDS group significantly decreased compared to the PQ only group. Additionally, AH2QDS protected the mitochondria of rats and A549 cells and decreased oxidative stress damage, thus improving animal survival and cell viability. Finally, the differentially expressed genes were analysed in the PQ + AH2QDS group and the PQ group by NextGen sequencing, and we verified that Nrf2's expression in the PQ + AH2QDS group was significantly higher than that in the PQ group. Our work identified that AH2QDS can detoxify PQ by reducing PQ uptake and protecting mitochondria while enhancing the body's antioxidant activity.

Long non-coding RNAs (lncRNAs) play crucial roles in diverse biological processes and have drawn extensive attention in the past few years. However, lncRNAs remain poorly understood about expression and roles in Crassostrea gigas, a potential model organism for marine molluscan studies. Here, we systematically identified lncRNAs in the mantles of C. gigas from four full-sib families characterized by white, black, golden, and partially pigmented shell. Using poly(A)-independent and strand-specific RNA-seq, a total of 441,205,852 clean reads and 12,243 lncRNA transcripts were obtained. LncRNA transcripts were relatively short with few exons and low levels of expression in comparison to protein coding mRNA transcripts. A total of 427 lncRNAs and 349 mRNAs were identified to differentially express among six pairwise groups, mainly involving in biomineralization and pigmentation through functional enrichment. Furthermore, a total of 6 mRNAs and their cis-acting lncRNAs were predicted to involve in synthesis of melanin, carotenoid, tetrapyrrole, or ommochrome. Of them, chorion peroxidase and its cis-acting lincRNA TCONS_00951105 are implicated in playing an essential role in the melanin synthetic pathway. Our studies provided the first systematic characterization of lncRNAs catalog expressed in oyster mantle, which may facilitate understanding the molecular regulation of shell colour diversity and provide new insights into future selective breeding of C. gigas for aquaculture.