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BackgroundAfter fertilization, the fusion of gametes results in the formation of totipotent zygote. During sperm-egg fusion, maternal factors participate in parental chromatin remodeling. H3.3 is a histone H3 variant that plays essential roles in mouse embryogenesis.MethodsHere, we used transgenic early embryos expressing H3.3-eGFP or H2B-mCherry to elucidate changes of histone mobility.ResultsWe used FRAP analysis to identify that maternally stored H3.3 has a more significant change than H2B during maternal-to-embryonic transition. We also found that H3.3 mobile fraction, which may be regulated byde novoH3.3 incorporation, reflects chromatin compaction of parental genomes in GV oocytes and early embryos.ConclusionsOur results show that H3.3 kinetics in GV oocytes and early embryos is highly correlated with chromatin compaction status of parental genomes, indicating critical roles of H3.3 in higher-order chromatin organization.

An outbreak of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is leading to an unprecedented worldwide health crisis. SARS‐CoV‐2 cell entry depends on ACE2 and TMPRSS2. Our objectives are to analysis the expression profile of ACE2 and TMPRSS2 in human spermatogenic cells, follicle cells, and preimplantation embryos, thereby providing mechanistic insights into viral entry and viral impact on reproduction. We found that ACE2 is mainly expressed during gametogenesis in spermatogonia and oocytes of antral follicles, granulosa cells of antral follicles and pre‐ovulatory follicles, while TMPRSS2 almost has no expression in spermatogenic cells, oocytes or granulosa cells. In preimplantation embryos, ACE2 is expressed in early embryos before eight‐cell stage, and trophectoderm of late blastocysts, while TMPRSS2 initiates its robust expression in late blastocyst stage. ACE2 and TMPRSS2 only show significant co‐expression in trophectoderm of late blastocysts in all above cell types. We speculate that trophectoderm of late blastocysts is susceptible to SARS‐CoV‐2, and that the chance of SARS‐CoV‐2 being passed on to offspring through gametes is very low. Therefore, we propose that fertility preservation for COVID‐19 patients is relatively safe and rational. We also recommend embryo cryopreservation and embryo transfer into healthy recipient mother at cleavage stage instead of blastocyst stage. Moreover, we unexpectedly found that co‐expression pattern of ACE2 and TMPRSS2 in oocytes and preimplantation embryos in human, rhesus monkey and mouse are totally different, so animal models have significant limitations for evaluating transmission risk of SARS‐CoV‐2 in reproduction.

Abstract Embedding thread lift rhytidectomy, also known as “thread lifting” in China, with the natures of simple operation, less trauma and quick recovery, is progressively used in clinical practice as a new technology of face lifting. Herewith, a brief introduction of the previous advances of thread lifting techniques and materials in the facial beauty industry, combined with the discussion on various types of sutures, common complications, and the site of actions were provided. The main limitations of present thread lifting material include: (1) the use of non-absorbable sutures is liable to cause allergies and a series of complications; (2) the absorbable sutures are easily degradation, and people need to reshape in a relatively short period. Therefore, the high biocompatible spider silk was proposed as a novel material of thread lifting suture and related devices, the advantages and preliminary achievements on spider silk were also addressed. Graphic Abstract

Circular RNAs (circRNAs) produced from back-spliced exons are widely expressed, but individual circRNA functions remain poorly understood owing to the lack of adequate methods for distinguishing circRNAs from cognate messenger RNAs with overlapping exons. Here, we report that CRISPR–RfxCas13d can effectively discriminate circRNAs from mRNAs by using guide RNAs targeting sequences spanning back-splicing junction (BSJ) sites featured in RNA circles. Using a lentiviral library that targets sequences across BSJ sites of highly expressed human circRNAs, we show that a group of circRNAs are important for cell growth mostly in a cell-type-specific manner and that a common oncogenic circRNA, circFAM120A, promotes cell proliferation by preventing the mRNA for family with sequence similarity 120A (FAM120A) from binding the translation inhibitor IGF2BP2. Further application of RfxCas13d–BSJ-gRNA screening has uncovered circMan1a2, which has regulatory potential in mouse embryo preimplantation development. Together, these results establish CRISPR–RfxCas13d as a useful tool for the discovery and functional study of circRNAs at both individual and large-scale levels. This paper describes a CRISPR–Cas13 system to effectively target circRNAs and screen their functions in vitro and in vivo, which enables the study of relevant circRNA phenotypes in human cell proliferation and in mouse embryogenesis.

Hong Zhu, Jin-Hai Tang, Shi-Meng Zhang, Jia-Ping Qian, Xin Ling, Xiao-Ying Wu, Ling-Xia Yang Department of Gastroenterology, Suzhou Ninth People’s Hospital, Suzhou, Jiangsu Province, People’s Republic of ChinaCorrespondence: Ling-Xia Yang Email ylxnjq@163.comBackground: Gastric cancer (GC) is a common cancer with high incidence and mortality worldwide. In recent years, accumulating evidence has shown that long noncoding RNAs (lncRNAs) exert critical roles in the development and progression of cancer by acting as a tumor initiator or suppressor. LINC00963 is a newly reported lncRNA related to cancer, and its role in GC remains unclear.Materials and Methods: The expression levels of LINC00963, miR-612, and cell division cycle 5-like protein (CDC5L) were measured using quantitative real-time PCR or Western blot. The biological functions of LINC00963, miR-612, and CDC5L in GC cells were analyzed by transwell and proliferation experiments. The expression of CDC5L in patients with GC was evaluated using the Oncomine database. Bone marrow-derived dendritic cells (DCs) were derived from C57BL/6 mice.Results: LINC00963 expression was higher in GC tissues than in adjacent normal tissues. Similar results were found in GC cell lines and normal human gastric epithelial cells. Upregulation of LINC00963 was related to the poor prognosis of patients with GC. Knockdown of LINC00963 inhibited the proliferation, invasion, and metastasis but promoted the apoptosis of GC cells. Furthermore, silencing of LINC00963 in GC cells significantly suppressed the tumor growth of GC. Bioinformatics analysis indicated that LINC00963 could target miR-612 by functioning as a competing endogenous RNA. The expression of miR-612 decreased in GC tissues and cell lines. Meanwhile, LINC00963 expression was negatively associated with miR-612. CDC5L was a direct target of miR-612. miR-612 suppressed the expression of CDC5L in GC tissues and cells. Moreover, LINC00963 inhibited the differentiation and maturation of DCs by regulating miR-612 expression in DCs.Conclusion: LINC00963 promoted the progression of GC by competitively binding to miR-612 to regulate the expression of CDC5L and mediated DC-related anti-tumor immune response. Thus, targeting LINC00963 may be a promising therapeutic strategy for GC.Keywords: lncRNA LINC00963, gastric cancer, miR-612, CDC5L, dendritic cells

Purpose Chlorogenic acid (CGA), a phenolic acid isolated from fruits and vegetables, has been established to have neuroprotective properties in relation to Alzheimer's disease (AD). However, the precise mechanism by which CGA prevents cognitive deficits in AD has not been well studied. This study aimed to explore the potential molecular mechanism of CGA action using an Aβ25-35-induced SH-SY5Y neuron injury and cogxnitive deficits model in APP/PS1 mice. Methods Three-month-old male APP/PS1 double transgenic mice and a human neuroblastoma cell line (SH-SY5Y) were used to assess the effects of CGA on AD in vivo and in vitro, respectively. Cognitive function in mice was measured using a Morris water maze (MWM) test. Hematoxylin and eosin, monodansylcadaverine fluorescence, LysoTracker Red (LTR), and immunofluorescence staining were used to evaluate the morphological changes in vivo and in vitro. The protein expressions of autophagy markers (LC3B-II/LC3B-I, p62/SQSTM, beclin1 and Atg5) and lysosomal-function-related markers (cathepsin D, mTOR, p-mTOR P70S6K, p-p70s6k and TFEB) were analyzed with Western blot analyses. Results CGA treatment significantly improved spatial memory, relieved neuron damage, and inhibited autophagy in APP/PS1 mice (P

Colored common beans are associated with health promoting and chronic disease prevention effects. Male C57BL/6J mice were fed high-fat (HF) diets supplemented with cooked black turtle beans (HFB) to prevent obesity related insulin resistance. Mice on both HF and HFB were obese compared to mice fed a low-fat (LF) diet. Plasma low density lipoprotein (LDL) and triglyceride concentrations of mice fed HFB diet were 28% and 36.6% lower than those on HF diet. Homeostatic model assessment of insulin resistance (HOMA-IR) index of mice fed HFB diet was 87% lower than that of mice fed HF diet. Diabetes related biomarkers, gastric inhibitory polypeptide (GIP), leptin, glucagon, and inflammatory cytokines interleukin 4 (IL-4) and IL-5, 10 and 12, IFN-g and TNF-α were significantly affected by HFB diet. Pparα, Cyp7a1 and Fasn were down-regulated by HFB diet while LDL-R, Srebp-2, Adipoq and Slc2a4 were up-regulated by HFB diet. The ratio of Firmicutes/Bacteroidetes (F/B) was also decreased 64.1% by HFB diet compared to HF diet. The results indicated that cooked black turtle bean consumption could ameliorate insulin resistance and lower plasma LDL in mice fed HF diet through glucose signaling pathway and JNK/c-Jun pathway. Meanwhile, cooked black turtle bean consumption restored the gut microbiome.

Coronavirus disease 2019 (COVID-19) has infected over 124 million people worldwide. In addition to the development of therapeutics and vaccines, the evaluation of the sequelae in recovered patients is also important. Recent studies have indicated that COVID-19 has the ability to infect intestinal tissues and to trigger alterations of the gut microbiota. However, whether these changes in gut microbiota persist into the recovery stage remains largely unknown. Here, we recruited seven healthy Chinese men and seven recovered COVID-19 male patients with an average of 3-months after discharge and analyzed their fecal samples by 16S rRNA sequencing analysis to identify the differences in gut microbiota. Our results suggested that the gut microbiota differed in male recovered patients compared with healthy controls, in which a significant difference in Chao index, Simpson index, and β-diversity was observed. And the relative abundance of several bacterial species differed clearly between two groups, characterized by enrichment of opportunistic pathogens and insufficiency of some anti-inflammatory bacteria in producing short chain fatty acids. The above findings provide preliminary clues supporting that the imbalanced gut microbiota may not be fully restored in recovered patients, highlighting the importance of continuous monitoring of gut health in people who have recovered from COVID-19.

Trichinella spiralis is an important foodborne parasitic nematode distributed worldwide that infects humans and animals. Glutaminase (GLS) is an important gene in the glutamine-dependent acid resistance (AR) system; however, its role in T. spiralis muscle larvae (ML) remains unclear. The present study aimed to characterize T. spiralis GLS (TsGLS) and assess its function in T. spiralis ML AR both in vitro and in vivo using RNA interference. The results indicated that native TsGLS (72 kDa) was recognized by anti-rTsGLS serum at the muscle larvae stage; moreover, an immunofluorescence assay confirmed that TsGLS was located in the epidermis of ML. After silencing the TsGLS gene, the relative expression of TsGLS mRNA and the survival rate of T. spiralis ML were reduced by 60.11% and 16.55%, respectively, compared to those in the PBS and control groups. In vivo AR assays revealed that the worm numbers at 7 and 35 days post-infection (dpi) decreased by 61.64% and 66.71%, respectively, compared to those in the PBS group. The relative expression of TsGLS mRNA in F1 generation T. spiralis ML was reduced by 42.52%, compared to that in the PBS group. To the best of our knowledge, this is the first study to report the presence of the glutamine-dependent AR system in T. spiralis. Our results indicate that TsGLS plays a crucial role in the T. spiralis AR system; thus, it could be used as a potential candidate target molecule for producing vaccines against T. spiralis infection.

Autophagy serves an important role in amyloid-β (Aβ) metabolism and τ processing and clearance in Alzheimer's disease. The progression of Aβ plaque accumulation and hyperphosphorylation of τ proteins are enhanced by oxidative stress. A hydrogen peroxide (H2O2) injury cell model was established using SH-SY5Y cells. Cells were randomly divided into normal, H2O2 and chlorogenic acid (5-caffeoylquinic acid; CGA) groups. The influence of CGA on cell viability was evaluated using a Cell Counting Kit-8 assay and cell death was assessed using Hoechst 33342 nuclear staining. Autophagy induction and fusion of autophagic vacuoles assays were performed using monodansylcadaverine staining. Additionally, SH-SY5Y cells expressing Ad-mCherry-green fluorescent protein-LC3B were established to detect autophagic flow. LysoTracker Red staining was used to evaluate lysosome function and LysoSensor™ Green staining assays were used to assess lysosomal acidification. The results demonstrated that CGA decreased the apoptosis rate, increased cell viability and improved cell morphology in H2O2-treated SH-SY5Y cells. Furthermore, CGA alleviated the accumulation of autophagic vacuoles, reduced the LC3BII/I ratio and decreased P62 levels, resulting in increased autophagic flux. Additionally, CGA upregulated lysosome acidity and increased the expression levels of cathepsin D. Importantly, these effects of CGA on H2O2-treated SH-SY5Y cells were mediated via the mTOR-transcription factor EB signaling pathway. These results indicated that CGA protected cells against H2O2-induced oxidative damage via the upregulation of autophagosomes, which promoted autophagocytic degradation and increased autophagic flux.

After fertilization, highly differentiated sperm and oocyte are reprogrammed to totipotent embryo, which subsequently cleavages and develops into an individual through spatial-temporal differentiation. Histone modifications play critical roles to coordinate with other reprogramming events in early stages of embryogenesis. However, most of studies focus on modifications at N-terminus of histones, those at nucleosome core were not well understood. Here, we characterize the three key acetylation events in the histone H3 core, H3K56/K64/K122ac, in early human embryos. The three residues localize at DNA entry-exit position of the nucleosome. Globally, H3K56ac, H3K64ac and H3K122ac were detectable throughout preimplantation stages, with H3K64ac levels being relatively stronger and H3K122ac levels being much weaker. Besides, H3K56ac level had a peak at two-cell stage. Moreover, we found that LINEs also peak at two-cell stage, and H3K56ac was enriched at young LINE-1 in human ESCs, supporting that H3K56ac is an important driving force for young LINE-1 activation in human preimplantation embryos. Our results suggest that acetylation in the nucleosome core of histone H3 is dynamic and various during preimplantation development, and may drive diverse chromatin remodeling events in this developmental window.

Schistosomiasis is a parasitic helminth disease that can cause organ lesions leading to health damage. During a schistosome infection, schistosome eggs can flow into the liver along the portal vein. Numerous inflammatory cells gather around the eggs, causing granulomas and fibrosis in the liver. In this process, many molecules are involved in the initiation and regulation of the fibrous scar formation. However, the precise molecular mechanisms responsible for the progression of granuloma formation and fibrosis initiation caused by schistosome infection have not been extensively studied. In this study, C57BL/6 wild-type mice and Stat3flox/flox Alb-Cre mice were infected with cercariae of Schistosoma japonicum. Liver injury, effector molecule levels, and RNA transcriptome resequencing of liver tissue were detected at 4, 5, and 6 weeks postinfection. We investigated the role of STAT3 (signal transducer and activator of transcription 3) in Schistosoma-induced liver injury in mice. After 6 weeks postinfection, there was obvious liver fibrosis. A sustained pathological process (inflammation, oxidative stress, proliferation, and apoptosis) occurred in S. japonicum-induced liver fibrosis initiation. Meanwhile, we observed activation of the STAT3 pathway in hepatic injury during S. japonicum infection by RNA transcriptome resequencing. Liver deficiency of phospho-STAT3 alleviated infection-induced liver dysfunction, hepatic granuloma formation, and fibrosis initiation. It also promoted STAT3-dependent apoptosis and reduced liver inflammation, oxidative stress, and proliferation. Our results suggest that STAT3 signal pathway and its mediating inflammation, oxidative stress, proliferation, and apoptosis are involved in S. japonicum-induced liver injury and may be a new potential guideline for the treatment of schistosomiasis.

Helminths and their products can suppress the host immune response and other immunopathologies which may benefit parasite survival. Parasitic nematode Trichinella spiralis (T. spiralis) exert immunomodulatory effect on the host immune response through excretory-secretory (ES) products. However, the immunomodulatory mechanism is not yet completely understood or defined. Macrophages play a key role in modulating the host immune response to helminth parasite infection. Here, we focus on the effect of T. spiralis ES antigens on the immune response by studying the effect of ES antigens on RAW264.7 macrophages in vitro. RAW264.7 macrophages were incubated with ES antigens either alone or in combination with LPS. The cytokine production (TNF-α, IL-10 and IL-12 p70) and the expression of TLR were measured. Moreover, the nuclear translocation of MyD88 and NF-кB was assessed by Western blot analysis. Results indicate that ES products had impacts on reducing the expression of TLRs in LPS-induced macrophages. In addition, ES products inhibited the cytokine production of IL-12 p70 and TNF-α and alone boosted the expression of cytokine IL-10 in RAW264.7 macrophages. In conclusion, our results implied that T. spiralis ES antigens may regulate host immune response at the macrophages level in vitro.

Background CpGs, the major methylation sites in vertebrate genomes, exhibit a high mutation rate from the methylated form of CpG to TpG/CpA and, therefore, influence the evolution of genome composition. However, the quantitative effects of CpG to TpG/CpA mutations on the evolution of genome composition in terms of the dinucleotide frequencies/proportions remain poorly understood. Results Based on the neutral theory of molecular evolution, we propose a methylation-driven model (MDM) that allows predicting the changes in frequencies/proportions of the 16 dinucleotides and in the GC content of a genome given the known number of CpG to TpG/CpA mutations. The application of MDM to the 10 published vertebrate genomes shows that, for most of the 16 dinucleotides and the GC content, a good consistency is achieved between the predicted and observed trends of changes in the frequencies and content relative to the assumed initial values, and that the model performs better on the mammalian genomes than it does on the lower-vertebrate genomes. The model’s performance depends on the genome composition characteristics, the assumed initial state of the genome, and the estimated parameters, one or more of which are responsible for the different application effects on the mammalian and lower-vertebrate genomes and for the large deviations of the predicted frequencies of a few dinucleotides from their observed frequencies. Conclusions Despite certain limitations of the current model, the successful application to the higher-vertebrate (mammalian) genomes witnesses its potential for facilitating studies aimed at understanding the role of methylation in driving the evolution of genome dinucleotide composition.

To investigate the role of electrostatics in different temperature adaptations, we performed a comparative study on subtilisin-like serine proteases from psychrophilic Vibrio sp. PA-44 (VPR), mesophilic Engyodontium album (Tritirachium album) (PRK), and thermophilic Thermus aquaticus (AQN) using multiple-replica molecular dynamics (MD) simulations combined with continuum electrostatics calculations. The results reveal that although salt bridges are not a crucial factor in determining the overall thermostability of these three proteases, they on average provide the greatest, moderate, and least electrostatic stabilization to AQN, PRK, and VPR, respectively, at the respective organism growth temperatures. Most salt bridges in AQN are effectively stabilizing and thus contribute to maintaining the overall structural stability at 343 K, while nearly half of the salt bridges in VPR interconvert between being stabilizing and being destabilizing, likely aiding in enhancing the local conformational flexibility at 283 K. The individual salt bridges, salt-bridge networks, and calcium ions contribute differentially to local stability and flexibility of these three enzyme structures, depending on their spatial distributions and electrostatic strengths. The shared negatively charged surface potential at the active center of the three enzymes may provide the active-center flexibility necessary for nucleophilic attack and proton transfer. The differences in distributions of the electro-negative, electro-positive, and electro-neutral potentials, particularly over the back surfaces of the three proteases, may modulate/affect not only protein solubility and thermostability but also structural stability and flexibility/rigidity. These results demonstrate that electrostatics contributes to both heat and cold adaptation of subtilisin-like serine proteases through fine-tuning, either globally or locally, the structural stability and conformational flexibility/rigidity, thus providing a foundation for further engineering and mutagenesis studies.

Background This study aimed to evaluate the correlation between neutrophil to lymphocyte ratio (NLR) with overall survival (OS) of esophageal squamous cell carcinoma (ESCC) patients. Method Records of patients with diagnosed ESCC were reviewed. Leukocyte counts and patients' characteristics were extracted from their clinical records to calculate NLR. Correlation between NLR and baseline characteristics with overall survival (OS) was then analyzed using Cox regression. The patients were then separated into higher and lower NLR groups according to median NLR. OS was further compared between the 2 groups. Results A total of 1281 patients were included in the study. Cox regression analysis showed a significant correlation of NLR with OS of ESCC patients. The median pretreatment NLR was identified as 2.86. Higher NLR was associated with worse prognosis in terms of OS. Conclusions Pretreatment NLR is independently associated with OS of ESCC patients. Therefore, NLR may be used as a predictive indicator for pretreatment evaluation and adjustment of treatment regimen.

Whitmania pigra is an important medicinal resource that is widely farmed in aquaculture in Asia, and a decrease in body weight occurring during hibernation and hibernation lasting long time has serious impacts on production efficiency in aquaculture system. We examined energy metabolic and antioxidant enzymes of intestine from a hibernator (W. pigra) over cycles of hibernation-arousal. Results of the study demonstrated that hibernation in W. pigra was characterized by a profound decrease in energy metabolic during deep hibernation that was interrupted by rewarming arousal. And energy metabolic increased significantly during the rewarming arousal. Regulated suppression of energy metabolism probably contributes to energy savings. Oxidative stress decreased during deep hibernation along with a reduction in oxidative metabolism, but increased during entrance into hibernation and arousal from hibernation. This up-regulation of antioxidant defense (AD) during arousal was interpreted as protection of the intestine against oxidative damage to come with the enormous increase in metabolic activity during arousal from hibernation, and the up-regulation of AD during entrance into hibernation was interpreted that leech are exposed to significant stresses (cold acclimation) that must be dealt with appropriately to avoid irreversible tissue damage. It can be concluded that W. pigra has a strong AD system that protects it from the injurious effects of free radicals either during periods of entrance into hibernation and arousal. These results indicate the adaptive mechanism of hibernation that may be applied to increase production efficiency of leech by interrupting or shortening hibernation.

Malignant brain glioma is the most common and aggressive type of primary intracranial neoplasm. Regular chemotherapy cannot eradicate brain glioma cells and the residual glioma cells could form vasculogenic mimicry (VM) channels under hypoxic conditions to provide nutrients for tumor cell invasion. In addition, the existence of the blood-brain barrier (BBB) restricts most antitumor drugs into brain glioma. In this study, we developed a kind of lactoferrin (Lf) modified daunorubicin plus honokiol liposomes to transport antitumor drugs across BBB, eliminate the VM channels and block tumor cell invasion. The evaluations were performed on BBB model, brain glioma cells and glioma-bearing mice. In vitro results showed that the targeting liposomes with suitable physicochemical property could enhance the drug transportation acrossing the BBB, inhibit C6 cells invasion and destroy VM channels. Action mechanism studies indicated that Lf modified daunorubicin plus honokiol liposomes could activate apoptotic enzymes caspase 3 as well as down-regulate VM protein indicators (PI3K, MMP-2, MMP-9, VE-Cadherin and FAK). In vivo results displayed the targeting liposomes improved accumulation in brain tumor tissue and exhibited obvious antitumor efficacy. Therefore, Lf modified daunorubicin plus honokiol liposomes could be used as a potential therapy for treatment of brain glioma.

TIP60 K430 SUMOylation inactivates DNA-PKcs to facilitate DNA HR repair, and emerging target for radiation and PARPi cancer therapy., Nonhomologous end joining (NHEJ) and homologous recombination (HR) are major repair pathways of DNA double-strand breaks (DSBs). The pathway choice of HR and NHEJ is tightly regulated in cellular response to DNA damage. Here, we demonstrate that the interaction of TIP60 with DNA-PKcs is attenuated specifically in S phase, which facilitates HR pathway activation. SUMO2 modification of TIP60 K430 mediated by PISA4 E3 ligase blocks its interaction with DNA-PKcs, whereas TIP60 K430R mutation recovers its interaction with DNA-PKcs, which results in abnormally increased phosphorylation of DNA-PKcs S2056 in S phase and marked inhibition of HR efficiency, but barely affects NHEJ activity. TIP60 K430R mutant cancer cells are more sensitive to radiation and PARP inhibitors in cancer cell killing and tumor growth inhibition. Collectively, coordinated regulation of TIP60 and DNA-PKcs facilitates HR pathway choice in S-phase cells. TIP60 K430R mutant is a potential target of radiation and PARPi cancer therapy.

Ring1 and Yin Yang 1-Binding Protein (RYBP) is a member of non-canonical polycomb repressive complex 1 to mediate monoubiquitination of histone H2A at lysine 119. It plays an important role in development, but its role in reproduction remains illusive. In this study, we used Rybp conditional knockout mouse model to genetically ablate Rybp in male germ cells. We found that Rybp deficiency during spermatogenesis led to smaller testes, loss of germline cells, disturbed meiosis, increased apoptosis of spermatocytes, decreased sperm motility, and reduced global H3K9me3, without impacting retrotransposon expression. Meanwhile, we depleted Rybp during oogenesis, but oocyte maturation and preimplantation development were normal. Our findings demonstrate that RYBP plays important roles in spermatogenesis through regulating meiosis and sperm motility.

Hibernation is an energy-saving and adaptive strategy adopted by leech, an important medicinal resource in Asia, to survive low temperature. Reversible protein phosphorylation (RPP) plays a key role in the regulation of mammalian hibernation processes but has never been documented in freshwater invertebrate such as leech. In this study, we detected the effects of hibernation on the proteome and phosphoproteome of the leech Whitmania pigra. A total of 2184 proteins and 2598 sites were quantified. Deep-hibernation resulted in 85 up-regulated and 107 down-regulated proteins and 318 up-regulated and 204 down-regulated phosphosites using a 1.5-fold threshold (P0.05). Proteins involved in protein digestion and absorption, amino acid metabolism and N-glycan biosynthesis were significantly down-regulated during deep-hibernation. However, proteins involved in maintaining cell structure stability in hibernating animals were up-regulated. Differentially phosphorylated proteins provided the first global picture of a shift in energy metabolism, protein synthesis, cytoprotection and signaling during deep hibernation. Furthermore, AMP-activated protein kinase and protein kinase C play major roles in the regulation of these functional processes. These data significantly improve our understanding of the regulatory mechanisms of leech hibernation processes and provides substantial candidate phosphorylated proteins that could be important for functionally adapt in freshwater animals. SIGNIFICANCE: The leech Whitmania pigra as an important medicinal resource in Asia is an excellent model freshwater invertebrate for studies of environmentally-induced hibernation. The present study provides the first quantitative proteomics and phosphoproteomic analysis of leech hibernation using isobaric tag based TMT labeling and high-resolution mass spectrometry. These data significantly improve our understanding of the regulatory mechanisms when ectotherm animals face environmental stress and provides substantial candidate phosphorylated proteins that could be important for functionally adapt in freshwater animals.

As a folk medicine of the Jingpo minority in Yunnan province, the venom of Vespa magnifica has been commonly used for the treatment of rheumatoid arthritis. Quality standardization of the wasp venom is a necessary step for its pharmaceutical research and development. To control the quality of the wasp venom, a method based on high-performance liquid chromatography (HPLC) was developed for chemical fingerprint analysis. In the chromatographic fingerprinting, chemometrics procedures, including similarity analysis (SA), hierarchical clustering analysis (HCA), and principal component analysis (PCA), were applied to classify 134 batches (S1&ndash, S134) of wasp venom from different origins. The HPLC fingerprint method displayed good precision (Relative standard deviation, RSD &lt, 0.27%), stability (in 16 h, RSD &lt, 0.34%), and repeatability (RSD &lt, 1.00%). Simultaneously, four compounds (VMS1, VMS2, VMS3, and VMS4) in the wasp venom were purified and identified. VMS1 was 5-hydroxytryptamine, and the other compounds were three peptides that were sequenced as follows: Gly&ndash, Arg&ndash, Pro&ndash, Hyp&ndash, Gly&ndash, Phe&ndash, Ser&ndash, Ile&ndash, Asp&ndash, NH2 (VMS2), Ile&ndash, Asn&ndash, Leu&ndash, Lys&ndash, Ala&ndash, NH2 (VMS3), and Phe&ndash, NH2 (VMS4). The quantifications for these components were 110.2 mg/g, 26.9 mg/g, 216.3 mg/g, and 58.0 mg/g, respectively. The results of this work indicated that the combination of the chemical fingerprint and quantitative analysis offers a reasonable way to evaluate the quality of wasp venom.

B cells are involved in chronic graft-versus-host disease (cGVHD) pathogenesis, and Rituximab may have a therapeutic effect on cGVHD in allogeneic hematopoietic stem cell transplantation (allo-HSCT) patients. Herein, we retrospectively evaluated the prophylactic effect of Rituximab on cGVHD in a group of Chinese allo-HSCT patients. A total of 102 patients, who suffered Epstein Barr virus (EBV) viremia within 100 days after allo-HSCT, were included in this study. Fifty patients received Rituximab (375 mg/m2 weekly) for EBV viremia, while fifty-two patients did not receive Rituximab. A competing risk model was adopted to compare cumulative incidence of cGVHD, cumulative incidence of relapse (CIR) and transplantation-related mortality (TRM) between two groups. Cumulative incidence of cGVHD in the Rituximab group was lower than in controls (P = 0.0579). Multivariate analyses confirmed that Rituximab was an independent factor for the reduction of cumulative cGVHD incidence (P = 0.0069). No significant difference was observed in CIR (P = 0.39) or TRM (P = 0.48) between two groups and 2-year OS and DFS were comparable (OS, P = 0.667; DFS, P = 0.571). Administration of Rituximab in the early post-transplantation phase may protect against cGVHD in allo-HSCT patients without increasing CIR or TRM.

A series of new benzo[d]thiazole-hydrazones were synthesized and characterized by analytical and spectroscopic techniques. All the compounds were screened for their in vitro inhibition of H+/K+ ATPase and anti-inflammatory effects. The results revealed that compounds 6–8, 13–15, 18–20, 22, 23 and 27–30 displayed excellent inhibitory activity against H+/K+ ATPase, and their IC50 values were lower than those of the standard compound omeprazole. Compounds 2–5, 9–12, 28 and 30 exhibited better anti-inflammatory activity in comparison to the standard compound indomethacin. Studies of the structure–activity relationship (SAR) showed that electron-donating groups (OH and OCH3) favored inhibitory activity against H+/K+ ATPase, whereas electron-withdrawing groups (F, Cl, Br and NO2) favored anti-inflammatory activity, and derivatives with both electron-donating (OH and OCH3) and electron-withdrawing (Br) groups (16–18) displayed reasonable activity, whereas aliphatic analogues (24–26) exhibited less activity and heterocyclic analogues (27–30) displayed moderate activity in both biological studies. Molecular docking studies were performed for all the synthesized compounds, among which compounds 19 and 20 exhibited the highest docking scores for inhibitory activity against H+/K+ ATPase, whereas compounds 10 and 12 displayed the highest docking scores for anti-inflammatory activity.

Phoxinus lagowskii is a freshwater fish that is widely distributed in China. In this study, a comparative analysis of the mtDNA control region (D-loop) was performed to analyze the natural population structure and genetic diversity of 54 individuals from eleven locations (T1, T2, T3, T4, T5, T6, T7, T8, T9, T10 and T11) which was divided with reservoirs. The estimated haplotype and nucleotide diversity were 0.734 and 0.03514, respectively. An AMOVA indicated that 79.78% of the total variation originated from individual populations and 20.22% came from variation within the 11 geographic populations, which showed high genetic differentiation among the 11 geographic groups. A test of neutral evolution and mismatch distribution indicated that historical expansion occurred in these populations. However, the findings of low genetic diversity and high genetic differentiation demonstrated that the reproduction isolated by reservoir has showed a certain effect for the development of the populations, and th...

Food-grade soybeans with large seed size, uniformity, clear hilum and a high 11S/7S ratio are favoured by the food industry for making tofu. In order to search for soybean lines with desirable characteristics for making foods, 22 soybean lines were selected from the USDA-Soybean Germplasm Collection, were grown in Stoneville, MS for biochemical analysis and tofu texture and sensory quality tests. Eight lines were identified, from 22 lines harvested in 2014, to be suitable for tofu making, as judged by chemical composition and sensory quality of pressed tofu. In the filled tofu making and texture analysis study, the correlation between A3 subunit content and filled tofu firmness was significant (N=22, r=0.77, P0.001). The results indicated that the A3 subunit could be an indicator for predicting the firmness of tofu. The results provided important food quality information for the selection of soybean genotypes for improving food quality.

Molecular dynamics (MD) simulations of a subtilisin-like serine protease VPR from the psychrophilic marine bacterium Vibrio sp. PA-44 and its mesophilic homologue, proteinase K (PRK), have been performed for 20 ns at four different temperatures (300, 373, 473, and 573 K). The comparative analyses of MD trajectories reveal that at almost all temperatures, VPR exhibits greater structural fluctuations/deviations, more unstable regular secondary structural elements, and higher global flexibility than PRK. Although these two proteases follow similar unfolding pathways at high temperatures, VPR initiates unfolding at a lower temperature and unfolds faster at the same high temperatures than PRK. These observations collectively indicate that VPR is less stable and more heat-labile than PRK. Analyses of the structural/geometrical properties reveal that, when compared to PRK, VPR has larger radius of gyration (Rg), less intramolecular contacts and hydrogen bonds (HBs), more protein-solvent HBs, and smaller burial of nonpolar area and larger exposure of polar area. These suggest that the increased flexibility of VPR would be most likely caused by its reduced intramolecular interactions and more favourable protein-solvent interactions arising from the larger exposure of the polar area, whereas the enhanced stability of PRK could be ascribed to its increased intramolecular interactions arising from the better optimized hydrophobicity. The factors responsible for the significant differences in local flexibility between these two proteases were also analyzed and ascertained. This study provides insights into molecular basis of thermostability of homologous serine proteases adapted to different temperatures.

Although the GC content of the human genome is known, it remains undecided whether the base composition is already in equilibrium. This study aimed to examine the equilibrium GC content of the human genome based on 2,504 genomes from the 1000 Genomes Project. By recreating the results of some previous research on genome analysis, we revealed the problems in the results of those studies and the causes of the problems. The present results indicate that the GC content of the human genome has not yet reached equilibrium, and it is evolving toward a higher GC content. Furthermore, by analyzing the quantitative changes in CpG dinucleotides before and after mutations, we found that the effect of methylation on the GC content of a genome is very limited, even leading to an increase in the GC content of the genome rather than only a decrease. These findings, therefore, contradict the traditional perception.

Beclin1 (BECN1), which directly interacts with B‑cell lymphoma 2, serves an important role in autophagy and is involved in the tumorigenesis of various types of cancer. However, the definite role of BECN1 in breast cancer remains controversial. Bi-allelic knockout of Becn1 in a mouse model leads to an embryonic lethal phenotype, which hampers further investigation. To generate cell lines with knockout of BECN1, the CRISPR/Cas9 technique was used to disrupt BECN1 in human triple-negative breast cancer (TNBC) MDA‑MB‑231 cells. To the best of our knowledge, the present study was the first to successfully disrupt BECN1 in MDA‑MB‑231 cells and to screen three stable monoclonal BECN1‑knockout cell lines, suggesting that BECN1‑knockout is not lethal in TNBC cells. Functional analysis revealed that complete loss of BECN1 suppressed MDA‑MB‑231 proliferation and colony formation via inducing G0/G1 cell cycle arrest, not apoptosis, in vitro. On the other hand, BECN1‑knockout inhibited the migratory and invasive ability of MDA‑MB‑231 cells by partially reversing signals of epithelial-mesenchymal transition. Finally, analysis of publicly available gene expression datasets revealed increased expression of BECN1 in TNBC samples. Taken together, the results of the present study identified BECN1 as an oncogene, providing a novel potential target for the treatment of TNBC.

We aimed to assess the effect of Radix Isatidis dietary supplementation on resistance to Proteus mirabilis infection in Poecilobdella manillensis . Six groups of leeches housed in tanks were randomly divided into five treatment groups (fed a basal diet supplemented with 2.5, 5.0, 7.5, 10.0, or 12.5 g/kg of Radix Isatidis ) and one control group (fed a basal diet). Compared with the control group, the specific growth rate (SGR) and weight gain (WG) were significantly decreased in the treatment groups supplemented with Radix Isatidis . However, supplementation with 2.5 to 10.0 g/kg of Radix Isatidis did not produce an effect on mortality. Mortality was recorded after the leeches were challenged with P. mirabilis , and changes were observed in the serum cortisol, glucose, alanine aminotransferase (ALT), 3,5,30-triiodothyronine (T3), thyroxin (T4), alkaline phosphatase (ALP), total protein (TP), globin, and lysozyme contents. In the ingluvies, changes were observed in the total anti-oxidative capacity, superoxide dismutase (SOD) and catalase (CAT) activity, malondialdehyde (MDA) content and heat shock protein 70 (HSP70) gene expression over a period of 7 d. Treatment with 5.0 to 10.0 g/kg of Radix Isatidis increased the following parameters: total anti-oxidative capacity of the ingluvies prior to infection and at 1, 2 and 7 d after infection; serum ALP activity and serum globin concentrations prior to infection and at 1 and 2 d after infection; T4 concentrations prior to infection; serum lysozyme activity at 1 d after infection; SOD and CAT activity in the ingluvies prior to infection and 7 d after infection; T3 concentrations at 1 and 2 d after infection; and the relative level of HSP70 mRNA expression in the ingluvies at 2 d after infection. However, treatment with 5.0 to 10.0 g/kg of Radix Isatidis increased the following parameters: ALT contents prior to infection and 1, 2 and 7 d after infection; serum cortisol concentrations and serum glucose at 1, 2 and 7 d after infection; MDA contents of the ingluvies at 1 and 2 d after infection; and T4 concentrations at 2 and 7 d after infection. Compared with the control group, significantly lower mortality was observed in the groups supplemented with 5.0 to 10.0 g/kg of Radix Isatidis . These results suggest that supplementation with 5.0 to 10.0 g/kg of Radix Isatidis in the basal diet could enhance resistance to P. mirabilis infection in P. manillensis. Statement of relevance We believe that this manuscript is appropriate for publication in Aquaculture because it provides a natural method of controlling disease in P. manillensis , thereby avoiding the use of antibiotics and the development of antibiotic resistance. Furthermore, the results of our study provide new information on the physiological and molecular mechanisms that underlie the protective effect of Radix Isatidis .

Mineralocorticoid receptor (MR) antagonists continue to be a prevalent area of research in the pharmaceutical industry. Herein we report the discovery of various spirooxindole and dibenzoxazepine constructs as potent MR antagonists. SAR analysis of our spirooxindole hit led to highly potent compounds containing polar solubilizing groups, which interact with the helix-11 region of the MR ligand binding domain (LBD). Various dibenzoxazepine moieties were also prepared in an effort to replace a known dibenzoxepane system which interacts with the hydrophobic region of the MR LBD. In addition, an X-ray crystal structure was obtained from a highly potent compound which was shown to exhibit both partial agonist and antagonist modes of action against MR.

The envelope (Env) of HIV-1 plays critical roles in viral infection and immune evasion. Although structures of prefusion Env have been determined and phenotypes relevant to the CD4 dependency and the neutralization sensitivity for various HIV-1 isolates have been identified, the detailed structural dynamics and energetics underlying these two phenotypes have remained elusive. In this study, two unliganded structural models of gp120, one from the CD4-dependent, neutralization-resistant isolate H061.14 and the other from the CD4-independent, neutralization-sensitive R2 strain, were constructed, and subsequently were subjected to multiple-replica molecular dynamics (MD) simulations followed by free energy landscape (FEL) construction. Comparative analyses of MD trajectories reveal that during simulations R2-gp120 demonstrated larger structural fluctuations/deviations and higher global conformational flexibility than H061.14-gp120. Close comparison of local conformational flexibility shows that some of the structural regions involving direct interactions with gp41 and adjacent gp120 subunits in the context of the closed trimeric Env exhibit significantly higher flexibility in R2-gp120 than in H061.14-gp120, thus likely increasing the probability for R2-Env to open the trimer crown and prime gp41 fusogenic properties without induction by CD4. Collective motions derived from principal component analysis (PCA) reveal that R2-gp120 is prone to spontaneous transition to the neutralization-sensitive CD4-bound state while H061.14-gp120 tends to maintain the neutralization-resistant unliganded state. Finally, comparison between FELs reveals that R2-gp120 has larger conformational entropy, richer conformational diversity, and lower thermostability than H061.14-gp120, thus explaining why R2-gp120 is more structurally unstable and conformationally flexible, and has a higher propensity to transition to the CD4-bound state than H061.14-gp120. The present results reveal that the differences in dynamics and energetics between R2-gp120 and H061.14-gp120 impart Env trimers with distinct capacities to sample different states (i.e., R2-Env samples more readily the open state while H061.14-Env is more inclined to maintain the closed state), thus shedding light on the molecular mechanism underlying the HIV-1 phenotype associated with CD4 dependency/neutralization sensitivity.

Recombinant Helicobacter pylori neutrophil-activating protein fused with maltose-binding protein (rMBP-NAP), a potential TLR2 ligand, was reported to possess immunomodulatory effects on in situ tumors in our previous study. In the present work, we attempt to elucidate the effect of rMBP-NAP at the local immune modulation in B16-F10-induced metastatic lung cancer. Our results demonstrated that growth of B16-F10 melanoma metastases in the lung was significantly arrested after rMBP-NAP treatment, along with marked reduction in metastatic lung nodules and significant increase in survival. The treatment induced both local and systemic immune responses, which were associated with higher influx of CD4+/CD8+ T cells and drove toward Th1-like and cytotoxic immune environment. Moreover, rMBP-NAP also showed significant anti-angiogenic activity by reducing vascularization in lung tumor sections. rMBP-NAP could induce antitumor immunity through activating Th1 cells and producing pro-inflammatory cytokines, which are responsible for the effective cytotoxic immune response against cancer progression. Our findings indicate that rMBP-NAP might be a novel antitumor therapeutic strategy.

Lung fibrosis is an ultimate consequence of bronchopulmonary dysplasia (BPD) which shows the excessive proliferation of lung fibroblasts (LFs). To find a better model for studying the role of LFs in hyperoxia-induced lung fibrosis at the cellular level, we isolated LFs from the lung tissue of hyperoxia- and normoxia-exposed rat lungs on postnatal days 7, 14 and 21 for primary culture to study their proliferative behavior. In the present study, the LF predominance was > 95% in our culture method. The LFs isolated from rats exposed to hyperoxia in vivo showed significantly greater proliferation than that from normoxia-exposed rats. Flow cytometry revealed that percentage of LFs in S and G2/M stage increased, and proportion in the G0/G1 stage declined at the same time. A greater presence of myofibroblasts in LFs isolated from rats exposed to hyperoxia compared with those exposed to normoxia. In addition, elevated collagen level, transforming growth factor-β and connective tissue growth factor protein expression in conditioned medium were also found in hyperoxia LFs. These data demonstrate that hyperoxia promotes LFs proliferation, myofibroblast transdifferentiation and collagen synthesis in a time-dependent manner. The primary culture of LFs from hyperoxia-exposed rats is a feasible method for studying the pathogenesis and treatment of lung fibrosis caused by BPD at the cellular level.

BackgroundP16 methylation plays an important role in the pathogenesis of hyperoxia-induced lung fibrosis. 5-aza-2'-deoxycytidine (5-aza-CdR) is a major methyltransferase-specific inhibitor. In this study, the effects of 5-aza-CdR on a hyperoxia-induced lung fibrosis in neonatal rats were investigated.MethodsRat pups were exposed to 85% O2 for 21 days of and received intraperitoneal injections of 5-aza-CdR or normal saline (NS) once every other day. Survival rates and lung coefficients were calculated. Hematoxylin-eosin staining was performed to analyze the degree of lung fibrosis. Collagen content and TGF-β1 levels were determined. A methylation-specific polymerase chain reaction and western blotting were performed to determine P16 methylation status and P16, cyclin D1, and E2F1 protein expression.Results5-aza-CdR treatment during hyperoxia significantly improved the survival rate and weight gain, while it decreases the degree of lung fibrosis and levels of hydroxyproline and TGF-β1. Hyperoxia induced abnormal P16 methylation and 5-aza-CdR effectively reversed the hypermethylation of P16. Expression of the P16 protein in lung tissues was enhanced, while cyclin D1 and E2F1 protein were reduced by 5-aza-CdR treatment during hyperoxia.ConclusionThese data show that 5-aza-CdR attenuated lung fibrosis in neonatal rats exposed to hyperoxia by lowering hydroxyproline and TGF-β1 expression and via re-expression of P16 in neonatal rats.

A potent, in vivo efficacious 11β hydroxysteroid dehydrogenase type 1 (11β HSD1) inhibitor (11j) has been identified. Compound 11j inhibited 11β HSD1 activity in human adipocytes with an IC50 of 4.3nM and in primary human adipose tissue with an IC80 of 53nM. Oral administration of 11j to cynomolgus monkey inhibited 11β HSD1 activity in adipose tissue. Compound 11j exhibited >1000× selectivity over other hydroxysteroid dehydrogenases, displays desirable pharmacodynamic properties and entered human clinical trials in 2011.

The G protein-coupled receptor 55 (GPR55) is a novel cannabinoid receptor, whose exact role in anxiety remains unknown. The present study was conducted to explore the possible mechanisms by which GPR55 regulates anxiety and to evaluate the effectiveness of O-1602 in the treatment of anxiety-like symptoms. Mice were exposed to two types of acute stressors: restraint and forced swimming. Anxiety behavior was evaluated using the elevated plus maze and the open field test. We found that O-1602 alleviated anxiety-like behavior in acutely stressed mice. We used lentiviral shRNA to selective ly knockdown GPR55 in the medial orbital cortex and found that knockdown of GPR55 abolished the anxiolytic effect of O-1602. We also used Y-27632, a specific inhibitor of ROCK, and U73122, an inhibitor of PLC, and found that both inhibitors attenuated the effectiveness of O-1602. Western blot analysis revealed that O-1602 downregulated the expression of GluA1 and GluN2A in mice. Taken together, these results suggest that GPR55 plays an important role in anxiety and O-1602 may have therapeutic potential in treating anxiety-like symptoms.

Background B. napus (oilseed) is an important source of edible vegetable oil, and its nutritional and economic value is determined by its fatty acid composition and content. Results Using the Brassica 60 K SNP array, we performed a genome-wide association study of fatty acid composition in a population of 520 genetically diverse oilseed accessions. Using the PCA + K model in TASSEL 5.2.1, we identified 62 genomic regions that were significantly associated with the composition of seven fatty acids, and five consensus regions that mapped to the A2, A8, A9, C1, and C3 chromosomes, respectively, of the Brassica napus Darmor-bzh genome. We then identified 24 orthologs of the functional candidate genes involved in fatty acid biosynthesis, excluding BnaA.FAE1 and BnaC.FAE1 on the A8 and C3 homologous genome blocks, which are known to have critical roles in the fatty acid biosynthesis pathway, and potential orthologs of these genes (e.g., LACS9, KCR1, FAB1, LPAT4, KCS17, CER4, TT16, and ACBP5). Conclusions Our results demonstrate the power of association mapping in identifying genes of interest in B. napus and provide insight into the genetic basis of fatty acid biosynthesis in B. napus. Furthermore, our findings may facilitate marker-based breeding efforts aimed at improving fatty acid composition and quality in B. napus. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3607-8) contains supplementary material, which is available to authorized users.

Cucurbitacin IIa (CuIIa) is the major active component of the Helmseya amabilis root and is known to have antiviral and anti-inflammatory effects. In this study, we examined the antidepressant-like effects of CuIIa in a mouse model of chronic unpredictable mild stress (CUMS) and investigated the possible underlying mechanisms. To evaluate the antidepressant-like effects of CuIIa on depression-like behaviors, mice were subjected to the open-field test, the elevated plus-maze test, the forced-swimming test, and the tail-suspension test. We found that CuIIa treatment reversed the CUMS-induced behavioral abnormalities. Western blot analyses showed that CUMS significantly decreased brain-derived neurotrophic factor (BDNF) levels, cAMP-response element binding protein (CREB), and calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylation, and N-methyl-D-aspartate receptor subtype GluN2B and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor GluA1 expression in the amygdala; in addition, the expression of gamma-aminobutyric acid receptor A subunit α2 was upregulated in CUMS mice. These CUMS-induced changes were all normalized by CuIIa treatment and administration of the BDNF antagonist ANA-12 can block the antidepressant effect of CuIIa. Our findings suggest that the antidepressant-like effects of CuIIa may be exerted by regulation of the CaMKIIα-CREB-BDNF pathway and the balance between excitatory and inhibitory synaptic transmission in the amygdala.

Fragile X syndrome (FXS) is a form of inherited mental retardation that results from the absence of the fragile X mental retardation protein (FMRP), the product of the Fmr1 gene. Numerous studies have shown that FMRP expression in astrocytes is important in the development of FXS. Although astrocytes affect neuronal dendrite development in Fmr1 knockout (KO) mice, the factors released by astrocytes are still unclear. We cultured wild type (WT) cortical neurons in astrocyte-conditioned medium (ACM) from WT or Fmr1 KO mice. Immunocytochemistry and Western blotting were performed to detect the dendritic growth of both WT and KO neurons. We determined glutamate and γ-aminobutyric acid (GABA) levels using high-performance liquid chromatography (HPLC). The total neuronal dendritic length was reduced when cultured in the Fmr1 KO ACM. This neurotoxicity was triggered by an imbalanced release of glutamate and GABA from Fmr1 KO astrocytes. We found increased glutaminase and GABA transaminase (GABA-T) expression and decreased monoamine oxidase B expression in Fmr1 KO astrocytes. The elevated levels of glutamate contributed to oxidative stress in the cultured neurons. Vigabatrin (VGB), a GABA-T inhibitor, reversed the changes caused by glutamate and GABA release in Fmr1 KO astrocytes and the abnormal behaviors in Fmr1 KO mice. Our results indicate that the imbalance in the astrocytic glutamate and GABA release may be involved in the neuropathology and the underlying symptoms of FXS, and provides a therapeutic target for treatment.

Liver X receptor (LXR) agonists have been reported to lower brain amyloid beta (Aβ) and thus to have potential for the treatment of Alzheimer's disease. Structure and property based design led to the discovery of a series of orally bioavailable, brain penetrant LXR agonists. Oral administration of compound 18 to rats resulted in significant upregulation of the expression of the LXR target gene ABCA1 in brain tissue, but no significant effect on Aβ levels was detected.

Huntington's disease (HD) is an autosomal dominant inherited disease characterized by movement, psychiatric, and cognitive disorders. Previous research suggests that Praeruptorin C (Pra-C), an effective component in the root of Peucedanum praeruptorum dunn, a traditional Chinese medicine, may function in neuroprotection. The present study was conducted to evaluate the effectiveness of Pra-C in the treatment of HD-like symptoms in a 3-nitropropionic acid (3-NP) mouse model, and to explore the possible mechanism of the drug's activity. We treated 3-NP-injected mice with two different doses of Pra-C (1.5 and 3.0mg/kg) for 3 days. Motor behavior was tested using the open field test (OFT) and rotarod test, while psychiatric symptoms were tested using the forced swimming test (FST) and tail suspension test (TST). We found that Pra-C alleviated the motor deficits and depression-like behavior in the 3-NP-treated mice, and protected neurons from excitotoxicity. Western blot analysis revealed that Pra-C upregulated BDNF, DARPP32, and huntingtin protein in the striatum of 3-NP mice. These results taken together suggest that Pra-C may have therapeutic potential with respect to the movement, psychiatric, and cognitive symptoms of HD.

This article describes the application of Contour to the design and discovery of a novel, potent, orally efficacious liver X receptor β (LXRβ) agonist (17). Contour technology is a structure-based drug design platform that generates molecules using a context perceptive growth algorithm guided by a contact sensitive scoring function. The growth engine uses binding site perception and programmable growth capability to create drug-like molecules by assembling fragments that naturally complement hydrophilic and hydrophobic features of the protein binding site. Starting with a crystal structure of LXRβ and a docked 2-(methylsulfonyl)benzyl alcohol fragment (6), Contour was used to design agonists containing a piperazine core. Compound 17 binds to LXRβ with high affinity and to LXRα to a lesser extent, and induces the expression of LXR target genes in vitro and in vivo. This molecule served as a starting point for further optimization and generation of a candidate which is currently in human clinical trials for treating atopic dermatitis.

The effects of harvest and different processing methods on the anti-thrombin activity of Poecilobdella manillensis were respectively studied. The indicators included processing methods (vacuum freeze drying, fresh homogenate, drying under sunlight, freezing, scalding, baking under different temperatures), different parts (entire body, cephalon, pygidium, exudate) and body weights (≤10, 10-20, 20-30, 30-40, ≥40 g). The anti-thrombin activities of P. manillensis with different processing methods were evaluated by direct anti-thrombin titration. The results indicated that the processing methods significantly affected the anti-thrombin activities of P. manillensis. Among the 11 groups, the anti-thrombin activity of P. manillensis processed with vacuum freeze drying (1 303.56 U•g⁻¹) was significantly highest than the other groups (P

Although supplemental high-level oxygen treatment can promote the survival of premature infants, hyperoxia may adversely induce acute lung injury (ALI) in newborns. Our prior work illustrated that hyperoxic exposure could enhance the release of placental growth factor (PLGF) in the lungs of neonatal rats. We therefore postulated that PLGF contributed to hyperoxic ALI in newborns and evaluated the anti-PLGF treatment mediated by systematic delivery of lentivirus in hyperoxic ALI in this study. Lentivirus particles containing PLGF specific shRNA were injected into neonatal rats prior to hyperoxic exposure (90% oxygen for 72h) to inhibit PLGF expression. Hyperoxia induced oxidative damages in lung tissues as evidenced by the increased malondialdehyde and myeloperoxidase, and the decreased antioxidant superoxide dismutase. Also, hyperoxia caused excessive infiltration of inflammatory cells and overproduction of proinflammatory cytokines (tumor necrosis factor-α, interleukin-1β and interleukin-6) in rat lung tissue. These pathological alterations were partly reversed by PLGF shRNA delivery. The expression levels and activities of metalloproteinase (MMP)-2 and MMP9 were up-regulated in response to hyperoxia, whereas down-regulated when PLGF was inhibited. Moreover, PLGF shRNA inhibited nuclear factor kappa B (NFκB) signaling delivery in hyperoxic rat lungs. Additionally, exogenous PLGF-induced activation of MMPs in rat RLE-6TN alveolar epithelial cells was suppressed by NFκB inhibitor pyrrolidine dithiocarbamate. These results suggest that therapy targeting PLGF may be beneficial for infants with hyperoxic ALI.

Leeches are not only important medicinal animals worldwide but also are endangered. We aimed to (i) explore the level of genetic diversity within/among populations of three leeches, (ii) assess genetic differentiation among these three leeches, and (iii) discuss an appropriate strategy for conserving leech germplasm. A total of 315 individuals of Whitmania pigra, Hirudo nipponica and Poecilobdella manillensis from 21 populations were collected in China and Vietnam. The genetic structure and genetic diversity among and within the 21 populations were evaluated using target region amplified polymorphism (TRAP) and simple sequence repeat (SSR) markers. Sixteen pairs of TRAP primers generated a total of 398 fragments, of which 396 (99.50%) were polymorphic; fourteen pairs of SSR primers generated a total of 60 fragments, of which 59 (98.33%) were polymorphic. Shannon's index (I) and Nei's gene diversity index (H) for the three leeches were high at the species level (I=0.4980 and H=0.3323 for TRAPs, I=0.4487 and H=0.2969 for SSRs in W. pigra; I=0.4147/0.3769, H=0.2788/0.2566 for H. nipponica; and I=0.4616/0.4717, H=0.3099/0.3203 for P. manillensis). However, low genetic diversity was determined at the population level; the average genetic diversity measures within populations were H=0.1767/0.1376, I=0.2589/0.2043 for W. pigra, H=0.2149/0.2021, I=0.3184/0.3000 for H. nipponica and H=0.2850/0.2724, I=0.4152/0.3967 for P. manillensis. We conclude that there was limited gene exchange within/among populations and species, as the gene flow number (Nm) was 0.5493/0.5807. However, for all three species, the genetic diversity was different at the population level. Gene differentiation (Gst) and Nm were 0.4682 /0.5364 and 0.5678/0.4321 for W. pigra, 0.2294/0.2127 and 1.6797/1.8512 for H. nipponica and 0.1214/0.1496 and 3.6202/2.8412 for P. manillensis. STRUCTURE analysis, Unweighted Pair-Group Method with Arithmetic means (UPGMA) cluster analysis and Principal Coordinates Analysis (PCOA) all yielded similar results. The isolation-by-distance pattern was not significant for any of the three species by the Mantel test. These data emphasize the need for management, conservation, and rehabilitation of this animal species. Finally, an appropriate strategy for conserving leech is proposed.

The present study aimed to explore the mechanisms by which c-Myc is involved in mitotic catastrophe. HeLa-630 is a cell line stably silenced for c-Myc expression that was established in the laboratory of the School of Radiation Medicine and Protection. Multinucleated cells were observed in this line, and gene expression analysis was utilized to examine differences in gene expression in these cells compared with in the control cells transfected with the control plasmid. Gene ontology analysis was performed for differentially expressed genes. Expression profile analyses revealed that cells with silenced c-Myc exhibited abnormal expression patterns of genes involved in various functions, including the regulation of microtubule nucleation, centrosome duplication, the formation of pericentriolar material, DNA synthesis and metabolism, protein metabolism and the regulation of ion concentrations. Pathway analyses of differentially expressed genes demonstrated that these genes were primarily involved in diverse signal transduction pathways, including not only the adherens junction pathway, the transforming growth factor-β signaling pathway and the Wnt signaling pathway, among others, but also signaling pathways with roles in cytokine and immune regulation. The proportion of multinucleated cells with multipolar spindles was significantly higher in silenced c-Myc cells as compared with the control cells, and this discrepancy became more pronounced following cell irradiation. The inhibition of c-Myc in tumors may account for the radiosensitization of certain tumor cell types.

The development and/or progression of perihematomal edema (PHE) in patients with acute spontaneous intracerebral hemorrhage (ICH) vary substantially with different individuals. Although hematoma volume is a useful indicator for predicting PHE, its predictive power was not good at the early stage of ICH. Better predictors are urgently needed. In this study, we found that miR-130a was elevated in the serum of ICH patients and was an independent indicator positively associated with PHE volume within the first 3 days after onset. The R (2) was further evaluated when it is used in combination with hematoma mass. Serum miR-130a levels were associated with clinical outcome (National Institute of Health Stroke Scale (NIHSS) scores at day 14 and modified Rankin Scale (mRS) scores at day 90) only in patients with deep hematoma. Moreover, miR-130a was significantly increased in rat serum and perihematomal tissues and was in line with the change in brain edema. MiR-130a inhibitors reduced brain edema, blood-brain barrier (BBB) permeability, and increased neurological deficit scores, and miR-130a mimics increased monolayer permeability. Thrombin-stimulated brain microvascular endothelial cells (BMECs) were a main source of miR-130a under ICH. In the experimental model, the elevated miR-130a level was accompanied by the decreased caveolin-1 and increased matrix metalloproleinase (MMP)-2/9. Meanwhile, caveolin-1 (cav-1) was reduced by miR-130a mimics, accompanied by an increase in MMP-2/9 expression. The upregulated MMP-2/9 was then downregulated by cavtratin, a cav-1 scaffolding domain peptide. This regulation mechanism was authenticated in a thrombin-induced cellular ICH model. Our results suggest that serum miR-130a may serve as a useful early biomarker for monitoring post-ICH PHE and predicting prognosis and may be helpful in the decision-making of individualized therapy.

Molecular recognition, which is the process of biological macromolecules interacting with each other or various small molecules with a high specificity and affinity to form a specific complex, constitutes the basis of all processes in living organisms. Proteins, an important class of biological macromolecules, realize their functions through binding to themselves or other molecules. A detailed understanding of the protein-ligand interactions is therefore central to understanding biology at the molecular level. Moreover, knowledge of the mechanisms responsible for the protein-ligand recognition and binding will also facilitate the discovery, design, and development of drugs. In the present review, first, the physicochemical mechanisms underlying protein-ligand binding, including the binding kinetics, thermodynamic concepts and relationships, and binding driving forces, are introduced and rationalized. Next, three currently existing protein-ligand binding models--the "lock-and-key", "induced fit", and "conformational selection"--are described and their underlying thermodynamic mechanisms are discussed. Finally, the methods available for investigating protein-ligand binding affinity, including experimental and theoretical/computational approaches, are introduced, and their advantages, disadvantages, and challenges are discussed.

Background: Salvianolic acid B (SalB) represents the most abundant and bio-active phenolic constituent among the water-soluble compounds of Salvia miltiorrhiza. But the therapeutic potential of SalB has been significantly restricted by its poor absorption. Methods: In this study, chitosans (CS) and CS nanoparticles (NPs) with different molecular weights (MWs), which have influence on the absorption of SalB, was also investigated. Results: As a preliminary study, water-soluble CS with various MWs (3, 30, 50, and 100 kDa) was chosen. We investigated the MW-dependent Caco-2 cell layer transport phenomena in vitro of CS and NPs at concentrations (4 μg/ml, w/v). SalB, in presence CS or NPs has no significant toxic effect on Caco-2 cell. As the MW increases, the absorption enhancing effect of CS increases. However, as the MW decreases, the absorption enhancing effect of NPs increases. The AUC0–∞ of the SalB-100 kDa CS was 4.25 times greater than that of free SalB. And the AUC0–∞ of the SalB-3 kDa NPs was 16.03 times greater than that of free SalB. Conclusion: CS and NPs with different MWs as the absorption enhancers can promote the absorption of SalB. And the effect on NPs is better than CS. SUMMARY Formation mechanism for NPs