In this study, NMR-based metabolomics in combination with multivariate pattern recognition technologies were employed to evaluate the physiological variations in the Wistar rats' plasma that are induced by pregnancy on the gestational days (GDs) 11, 14, 17 and 20. Untargeted metabolomics analysis revealed some possible mechanism of physiological effects for healthy pregnancies and showed a metabolic trajectory during pregnancy process. The levels of 24 metabolites were found to change significantly throughout pregnancy in maternal plasma. These metabolite changes involved in varied kinds of metabolic pathways including synthesis of biological substances, microbial metabolism in diverse environments, protein digestion and absorption, carbohydrate metabolism, digestion and absorption, mineral absorption, and ATP (Adenosine Triphosphate)-binding cassette transporters (ABC transporters). The substantial cores of all the metabolic pathways are promoting fetal growth and development and regulating maternal physiological state. This work showed relevant metabolic pathways perturbation in the maternal plasma due to normal pregnancy and provided the physical basis of time-dependent metabolic trajectory against which disease-related maternal physiological responses may be better understood in future studies.

From 2014 to 2015, three cases of highly pathogenic avian influenza infection occurred in zoo-housed north-east China tigers (Panthera tigris ssp.altaica) and four tigers died of respiratory distress in succession in Yunnan Province, China. We isolated and characterized three highly pathogenic avian influenza A(H5N1) viruses from these tigers. Phylogenetic analysis indicated that A/tiger /Yunnan /tig1404 /2014(H5N1) belongs to the provisional subclade 2.3.4.4e which were novel reassortant influenza A (H5N1) viruses with six internal genes from avian influenza A (H5N2) viruses. The HA gene of the isolated A/tiger /Yunnan /tig1412 /2014(H5N1) virus belongs to the subclade 2.3.2.1b. The isolated A/tiger /Yunnan /tig1508/2015 (H5N1) virus was a novel reassortant influenza A (H5N1) virus with three internal genes (PB2, PB1 and M) from H9N2 virus and belongs to the subclade 2.3.2.1c.

Dandruff is an unpleasant scalp disorder common to human populations. In this study, we systematically investigated the intra- and inter-associations among dandruff, physiological conditions such as sebum of the scalp, host demographics such as gender, age and the region of the scalp, and the microorganisms on the scalp. We found that the physiological conditions were highly relevant to the host age and varied in different regions of the same scalp. The sebum quantity and water content were negatively correlated with the formation of dandruff and had significant relationships with the two dominant but reciprocally inhibited bacteria on the scalp (Propionibacterium and Staphylococcus). The dominant fungus (Malassezia species) displayed contrary roles in its contribution to the healthy scalp micro-environment. Bacteria and fungi didn't show a close association with each other, but the intramembers were tightly linked. Bacteria had a stronger relationship with the severity of dandruff than fungi. Our results indicated that the severity of dandruff was closely associated with the interactions between the host and microorganisms. This study suggests that adjusting the balance of the bacteria on the scalp, particularly by enhancing Propionibacterium and suppressing Staphylococcus, might be a potential solution to lessen dandruff.

Electroencephalogram reactivity (EEG-R) is a positive predictive factor for assessing outcomes in comatose patients. Most studies assess the prognostic value of EEG-R utilizing visual analysis; however, this method is prone to subjectivity. We sought to categorize EEG-R with a quantitative approach. We retrospectively studied consecutive comatose patients who had an EEG-R recording performed 1-3 days after cardiopulmonary resuscitation (CPR) or during normothermia after therapeutic hypothermia. EEG-R was assessed via visual analysis and quantitative analysis separately. Clinical outcomes were followed-up at 3-month and dichotomized as recovery of awareness or no recovery of awareness. A total of 96 patients met the inclusion criteria, and 38 (40%) patients recovered awareness at 3-month followed-up. Of 27 patients with EEG-R measured with visual analysis, 22 patients recovered awareness; and of the 69 patients who did not demonstrated EEG-R, 16 patients recovered awareness. The sensitivity and specificity of visually measured EEG-R were 58% and 91%, respectively. The area under the receiver operating characteristic curve for the quantitative analysis was 0.92 (95% confidence interval, 0.87-0.97), with the best cut-off value of 0.10. EEG-R through quantitative analysis might be a good method in predicting the recovery of awareness in patients with post-anoxic coma after CPR.

Kashin-Beck disease (KBD) is an endemic degenerative osteoarthropathy. The mycotoxin of T-2 toxin is extensively accepted as a major etiological contributor to KBD. However, its function and mechanism in KBD remains unclearly elucidated. Here, T-2 toxin treatment induced chondrocyte injury in a time- and dose-dependent manner by repressing cell viability and promoting cell necrosis and apoptosis. Importantly, T-2 suppressed the transcription of type II collagen and aggrecan, as well as the release of sulphated glycosaminoglycan (sGAG). Furthermore, exposure to T-2 enhanced the transcription of matrix metalloproteinases (MMPs), including MMP-1, -2, -3 and -9. In contrast to control groups, higher expression of insulin-like growth factor binding protein 2 (IGFBP2) was observed in chondrocytes from KBD patients. Interestingly, T-2 toxin caused a dramatical elevation of IGFBP2 expression in chondrocytes. Mechanism analysis corroborated that cessation of IGFBP2 expression alleviated T-2-induced damage to chondrocytes. Simultaneously, transfection with IGFBP2 siRNA also attenuated matrix synthesis and catabolism-related gene expressions of MMPs. Together, this study validated that T-2 toxin exposure might promote the progression of KBD by inducing chondrocyte injury, suppressing matrix synthesis and accelerating cellular catabolism through IGFBP2. Therefore, this research will elucidate a new insight about how T-2 toxin participate in the pathogenesis of KBD.

T-lymphoma invasion and metastasis-inducing protein 1 (Tiam1) has been implicated in tumor occurrence and progression. Recent studies have shown that high expression levels of Tiam1 protein appear to be associated with the progression of numerous human tumors. This study attempted to explore the role of Tiam1 protein in tumor progression and the prognostic evaluation of breast cancer.

Species-specific antimicrobial therapy has the potential to combat the increasing threat of antibiotic resistance and alteration of the human microbiome. We therefore set out to demonstrate the beginning of a pathogen-selective drug discovery method using the periodontal pathogen Porphyromonas gingivalis as a model. Through our knowledge of metabolic networks and essential genes we identified a "druggable" essential target, meso-diaminopimelate dehydrogenase, which is found in a limited number of species. We adopted a high-throughput virtual screen method on the ZINC chemical library to select a group of potential small-molecule inhibitors. Meso-diaminopimelate dehydrogenase from P. gingivalis was first expressed and purified in Escherichia coli then characterized for enzymatic inhibitor screening studies. Several inhibitors with similar structural scaffolds containing a sulfonamide core and aromatic substituents showed dose-dependent inhibition. These compounds were further assayed showing reasonable whole-cell activity and the inhibition mechanism was determined. We conclude that the establishment of this target and screening strategy provides a model for the future development of new antimicrobials.

The silkmoth chorion was studied extensively by F.C. Kafatos' group for almost 40 years. However, the complete structure of the chorion locus was not obtained in the genome sequence of Bombyx mori published in 2008 due to repetitive sequences, resulting in gaps and an incomplete view of the locus. To obtain the complete sequence of the chorion locus, expressed sequence tags (ESTs) derived from follicular epithelium cells were used as probes to screen a bacterial artificial chromosome (BAC) library. Seven BACs were selected to construct a contig which covered the whole chorion locus. By Sanger sequencing, we successfully obtained complete sequences of the chorion locus spanning 871,711 base pairs on chromosome 2, where we annotated 127 chorion genes. The dataset reported here will recruit more researchers to revisit one of the oldest model systems which has been used to study developmentally regulated gene expression. It also provides insights into egg development and fertilization mechanisms and is relevant to applications related to improvements in breeding procedures and transgenesis.

Necroptosis as a molecular program, rather than simply incidental cell death, was established by elucidating the roles of receptor interacting protein (RIP) kinases 1 and 3, along with their downstream partner, mixed lineage kinase-like domain protein (MLKL). Previous studies suggested that phosphoglycerate mutase family member 5 (PGAM5), a mitochondrial protein that associates with RIP1/RIP3/MLKL complex, promotes necroptosis. We have generated mice deficient in the pgam5 gene and surprisingly found PGAM5-deficiency exacerbated rather than reduced necroptosis in response to multiple in vitro and in vivo necroptotic stimuli, including ischemic reperfusion injury (I/R) in the heart and brain. Electron microscopy, biochemical, and confocal analysis revealed that PGAM5 is indispensable for the process of PINK1 dependent mitophagy which antagonizes necroptosis. The loss of PGAM5/PINK1 mediated mitophagy causes the accumulation of abnormal mitochondria, leading to the overproduction of reactive oxygen species (ROS) that worsen necroptosis. Our results revise the former proposal that PGAM5 acts downstream of RIP1/RIP3 to mediate necroptosis. Instead, PGAM5 protects cells from necroptosis by independently promoting mitophagy. PGAM5 promotion of mitophagy may represent a therapeutic target for stroke, myocardial infarction and other diseases caused by oxidative damage and necroptosis.

Histone deacetylase (HDAC) 9, a member of class II HDACs, regulates a wide variety of normal and abnormal physiological functions, which is usually expressed at high levels in the brain and skeletal muscle. Although studies have highlighted the importance of HDAC-mediated epigenetic processes in the development of ischaemic stroke and very recent genome-wide association studies have identified a variant in HDAC9 associated with large-vessel ischemic stroke, the molecular events by which HDAC9 induces cerebral injury keep unclear. In this study, we found that HDAC9 was up-regulated in the ischaemic cerebral hemisphere after cerebral ischaemia/reperfusion (I/R) injury in rats and in vivo gene silencing of HDAC9 by recombinated lentivirus infection in the brain reduced cerebral injury in experimental stroke. We further demonstrated that HDAC9 contributed to oxygen-glucose deprivation-induced brain microvessel endothelial cell dysfunction as demonstrated by the increased inflammatory responses, cellular apoptosis and endothelial cell permeability dysfunction accompanied by reduced expression of tight-junction proteins. We further found that HDAC9 suppressed autophagy, which was associated with endothelial dysfunction. This study for the first time provides direct evidence that HDAC9 contributes to endothelial cell injury and demonstrates that HDAC9 is one of critical components of a signal transduction pathway that links cerebral injury to epigenetic modification in the brain.

Macrophages enhance glioma development and progression by shaping the tumor microenvironment. Class A1 scavenger receptor (SR-A1), a pattern recognition receptor primarily expressed in macrophages, is up-regulated in many human solid tumors. We found that SR-A1 expression in 136 human gliomas was positively correlated with tumor grade (P<0.01), but not prognosis or tumor recurrence. SR-A1-expressing macrophages originated primarily from circulating monocytes attracted to tumor tissue, and were almost twice as numerous as resident microglia in glioma tissues (P<0.001). The effects of SR-A1 on glioma proliferation and invasion were assessed in vivo using an SR-A1-deficient murine orthotopic glioma model. SR-A1 deletion promoted M2-like tumor-associated macrophage (TAM) polarization in mice by activating STAT3 and STAT6, which resulted in robust orthotopic glioma proliferation and angiogenesis. Finally, we found that HSP70 might be an endogenous ligand that activates SR-A1-dependent anti-tumorigenic pathways in gliomas, although its expression does not appear informative for diagnostic purposes. Our findings demonstrate a relationship between TAMs, SR-A1 expression and glioma growth and provide new insights into the pathogenic role of TAMs in glioma.

Both dichloroacetate (DCA) and metformin (Met) have shown promising antitumor efficacy by regulating cancer cell metabolism. However, the DCA-mediated protective autophagy and Met-induced lactate accumulation limit their tumor-killing potential respectively. So overcoming the corresponding shortages will improve their therapeutic effects. In the present study, we found that DCA and Met synergistically inhibited the growth and enhanced the apoptosis of ovarian cancer cells. Interestingly, we for the first time revealed that Met sensitized DCA via dramatically attenuating DCA-induced Mcl-1 protein and protective autophagy, while DCA sensitized Met through markedly alleviating Met-induced excessive lactate accumulation and glucose consumption. The in vivo experiments in nude mice also showed that DCA and Met synergistically suppressed the growth of xenograft ovarian tumors. These results may pave a way for developing novel strategies for the treatment of ovarian cancer based on the combined use of DCA and Met.

Alternative splicing (AS) contributes to the complexity of the mammalian proteome and plays an important role in diseases, including infectious diseases. The differential AS patterns of these transcript sequences between the healthy (HS3A) and mastitic (HS8A) cows naturally infected by Staphylococcus aureus were compared to understand the molecular mechanisms underlying mastitis resistance and susceptibility. In this study, using the Illumina paired-end RNA sequencing method, 1352 differentially expressed genes (DEGs) with higher than twofold changes were found in the HS3A and HS8A mammary gland tissues. Gene ontology and KEGG pathway analyses revealed that the cytokine-cytokine receptor interaction pathway is the most significantly enriched pathway. Approximately 16k annotated unigenes were respectively identified in two libraries, based on the bovine Bos taurus UMD3.1 sequence assembly and search. A total of 52.62% and 51.24% annotated unigenes were alternatively spliced in term of exon skipping, intron retention, alternative 5' splicing and alternative 3' splicing. Additionally, 1,317 AS unigenes were HS3A-specific, whereas 1,093 AS unigenes were HS8A-specific. Some immune-related genes, such as ITGB6, MYD88, ADA, ACKR1, and TNFRSF1B, and their potential relationships with mastitis were highlighted. From Chromosome 2, 4, 6, 7, 10, 13, 14, 17, and 20, 3.66% (HS3A) and 5.4% (HS8A) novel transcripts, which harbor known quantitative trait locus associated with clinical mastitis, were identified. Many DEGs in the healthy and mastitic mammary glands are involved in immune, defense, and inflammation responses. These DEGs, which exhibit diverse and specific splicing patterns and events, can endow dairy cattle with the potential complex genetic resistance against mastitis.

Adipocyte fatty acid binding protein (AFABP, FABP4) has been proven to be a potential therapeutic target for diabetes, atherosclerosis and inflammation-related diseases. In this study, a series of new scaffolds of small molecule inhibitors of FABP4 were identified by virtual screening and were validated by a bioassay. Fifty selected compounds were tested, which led to the discovery of seven hits. Structural similarity-based searches were then performed based on the hits and led to the identification of one high affinity compound 33b (Ki=0.29±0.07μM, ΔTm=8.5°C). This compound's effective blockade of inflammatory response was further validated by its ability to suppress pro-inflammatory cytokines induced by lipopolysaccharide (LPS) stimulation. Molecular dynamics simulation (MD) and mutagenesis studies validated key residues for its inhibitory potency and thus provide an important clue for the further development of drugs.

Salvia miltiorrhiza Bunge, which is also known as a traditional Chinese herbal medicine, is widely studied for its ability to accumulate the diterpene quinone Tanshinones. In addition to producing a variety of diterpene quinone, S. miltiorrhiza Bunge also accumulates sterol, brassinosteroid and triterpenoids. During their biosynthesis, squalene synthase (SQS, EC 2.5.1.21) converts two molecules of the hydrophilic substrate farnesyl diphosphate (FPP) into a hydrophobic product, squalene. In the present study, cloning and characterization of S. miltiorrhiza Bunge squalene synthase 2 (SmSQS2, Genbank Accession Number: KM408605) cDNA was investigated subsequently followed by its recombinant expression and preliminary enzyme activity. The full-length cDNA of SmSQS2 was 1 597 bp in length, with an open reading frame of 1 245 bp encoding 414 amino acids. The deduced amino acid sequence of SmSQS2 shared high similarity with those of SQSs from other plants. To obtain soluble recombinant enzymes, the truncated SmSQS2 in which 28 amino acids were deleted from the carboxy terminus was expressed as GST-Tag fusion protein in Escherichia coli BL21 (DE3) and confirmed by SDS-PAGE and Western Blot analysis, and the resultant bacterial crude extract was incubated with FPP and NADPH. Gas chromatograph-mass spectrometer analysis showed that squalene was detected in the in vitro reaction mixture. The gene expression level was analyzed through Quantitative real-time PCR, and was found to be higher in roots as compared to the leaves, and was up-regulated upon YE+ Ag(+) treatment. These results could serve as an important to understand the function of the SQS family. In addition, the identification of SmSQS2 is important for further studies of terpenoid and sterol biosynthesis in S. miltiorrhiza Bunge.

Osteosarcoma (OS) is the most common primary bone malignancy with a poor prognosis for all races and both sexes. In this study, we found that miR-381 is a positive prognosis factor for OS patients that OS patients with a low expression of miR-381 had a longer survival time after surgical intervention, and miR-381 expression promotes MG-63 cell proliferation and cell invasion ability. Our results also showed a strong negative correlation between the expression of miR-381 and LRRC4 (brain relative specific expression gene) in OS tissues. This demonstrated that LRRC4 is a direct target gene of miR-381, and suppressing the expression of miR-381 increases the sensitivity of OS cells to chemotherapeutic drugs through the LRRC4-mediated mTOR pathway. In summary, miR-381 is an important biomarker in directing therapeutic intervention and predicting prognosis in OS patients.

The SS18-SSX1 fusion gene has been shown to play important roles in the development of synovial sarcoma (SS), but the underlying molecular mechanisms and its downstream target genes are still not clear. Here SHC SH2-domain binding protein 1 (SHCBP1) was identified and validated to be a novel downstream target gene of SS18-SSX1 by using microarray assay, quantitative real-time (qPCR) and western blot. Expression of SHCBP1 was firstly confirmed in SS cell line and SS tissues. The effects of SHCBP1 overexpression or knockdown on SS cell proliferation and tumorigenicity were then studied by cell proliferation, DNA replication, colony formation, flow cytometric assays, and its in vivo tumorigenesis was determined in the nude mice. Meanwhile, the related signaling pathways of SHCBP1 were also examined in SS cells. The results indicated that SHCBP1 was significantly increased in SS cells and SS tissues compared with adjacent noncancerous tissues. The expression of SHCBP1 was demonstrated to be positively correlated with the SS18-SSX1 level. Overexpression and ablation of SHCBP1 promoted and inhibited, respectively, the proliferation and tumorigenicity of SS cells in vitro. SHCBP1 knockdown also significantly inhibited SS cell growth in nude mice, and lowered the MAPK/ERK and PI3K/AKT/mTOR signaling pathways and cyclin D1 expression. Our findings disclose that SHCBP1 is a novel downstream target gene of SS18-SSX1, and demonstrate that the oncogene SS18-SSX1 promotes tumorigenesis by increasing the expression of SHCBP1, which normally acts as a tumor promoting factor.

Ryanodine receptors (RyRs) are a class of giant ion channels with molecular mass over 2.2 mega-Daltons. These channels mediate calcium signaling in a variety of cells. Since more than 80% of the RyR protein is folded into the cytoplasmic assembly and the remaining residues form the transmembrane domain, it has been hypothesized that the activation and regulation of RyR channels occur through an as yet uncharacterized long-range allosteric mechanism. Here we report the characterization of a Ca(2+)-activated open-state RyR1 structure by cryo-electron microscopy. The structure has an overall resolution of 4.9 Å and a resolution of 4.2 Å for the core region. In comparison with the previously determined apo/closed-state structure, we observed long-range allosteric gating of the channel upon Ca(2+) activation. In-depth structural analyses elucidated a novel channel-gating mechanism and a novel ion selectivity mechanism of RyR1. Our work not only provides structural insights into the molecular mechanisms of channel gating and regulation of RyRs, but also sheds light on structural basis for channel-gating and ion selectivity mechanisms for the six-transmembrane-helix cation channel family.

Chlamydia trachomatis is an important human pathogen that undergoes a characteristic development cycle correlating with stage-specific gene expression profiles. Taking advantage of recent developments in the genetic transformation in C. trachomatis, we constructed a versatile green fluorescent protein (GFP) reporter system to study the development-dependent function of C. trachomatis promoters in an attempt to elucidate the mechanism that controls C. trachomatis adaptability. We validated the use of the GFP reporter system by visualizing the activity of an early euo gene promoter. Additionally, we uncovered a new ompA promoter, which we named P3, utilizing the GFP reporter system combined with 5' rapid amplification of cDNA ends (RACE), in vitro transcription assays, real-time quantitative RT-PCR (RT-qPCR), and flow cytometry. Mutagenesis of the P3 region verifies that P3 is a new class of C. trachomatis σ(66)-dependent promoter, which requires an extended -10 TGn motif for transcription. These results corroborate complex developmentally controlled ompA expression in C. trachomatis. The exploitation of genetically labeled C. trachomatis organisms with P3-driven GFP allows for the observation of changes in ompA expression in response to developmental signals. The results of this study could be used to complement previous findings and to advance understanding of C. trachomatis genetic expression.

We aimed to develop and translate a CD20-antigen-targeted radiopharmaceutical, Technetium-99 m-labeled (99mTc) rituximab, for sentinel lymph node (SLN) detection.