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STUDY QUESTION Are mutations in MOS (MOS proto-oncogene, serine/threonine kinase) involved in early embryonic arrest in infertile women? SUMMARY ANSWER We identified mutations in MOS that may cause human female infertility characterized by preimplantation embryonic arrest (PREMBA), and the effects of the mutations in human embryonic kidney 293T (HEK293T cells) and mouse oocytes provided evidence for a causal relation between MOS and female infertility. WHAT IS KNOWN ALREADY MOS, an activator of mitogen-activated protein kinase, mediates germinal vesicle breakdown and metaphase II arrest. Female MOS knockout mice are viable but sterile. Thus, MOS seems to be an important part of the mammalian cell cycle mechanism that regulates female meiosis. STUDY DESIGN, SIZE, DURATION Whole-exome sequencing, bioinformatics filtering analysis and genetic analysis were performed to identify two different biallelic mutations in MOS in two independent families. The infertile patients presenting with early embryonic arrest were recruited from October 2018 to June 2020. PARTICIPANTS/MATERIALS, SETTING, METHODS The female patients diagnosed with primary infertility were recruited from the reproduction centres of local hospitals. Genomic DNA from the affected individuals, their family members and healthy controls was extracted from peripheral blood. We performed whole-exome sequencing in patients diagnosed with PREMBA. Functional effects of the mutations were investigated in HEK293T cells by western blotting and in mouse oocytes by microinjection and immunofluorescence. MAIN RESULTS AND THE ROLE OF CHANCE We identified the homozygous missense mutation c.285C>A (p.(Asn95Lys)) and the compound heterozygous mutations c.467delG (p.(Gly156Alafs*18)) and c.956G>A (p.(Arg319His)) in MOS in two independent patients. The mutations c.285C>A (p.(Asn95Lys)) and c.467delG (p.(Gly156Alafs*18)) reduced the protein level of MOS, and all mutations reduced the ability of MOS to phosphorylate its downstream target, extracellular signal-regulated kinase1/2. In addition, the identified mutations reduced the capacity of exogenous human MOS to rescue the metaphase II exit phenotype, and the F-actin cytoskeleton of mouse oocytes was affected by the patient-derived mutations. LIMITATIONS, REASONS FOR CAUTION Owing to the lack of in vivo data from patient oocytes, the exact molecular mechanism affected by MOS mutations and leading to PREMBA is still unknown and should be further investigated using knock-out or knock-in mice. WIDER IMPLICATIONS OF THE FINDINGS We identified recessive mutations in MOS in two independent patients with the PREMBA phenotype. Our findings reveal the important role of MOS during human oocyte meiosis and embryonic development and suggest that mutations in MOS may be precise diagnostic markers for clinical genetic counselling. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the National Natural Science Foundation of China (81725006, 81822019, 81771581, 81971450, 81971382,82001538 and 82071642), the project supported by the Shanghai Municipal Science and Technology Major Project (2017SHZDZX01), the Project of the Shanghai Municipal Science and Technology Commission (19JC1411001), the Natural Science Foundation of Shanghai (19ZR1444500 and 21ZR1404800), the Shuguang Program of the Shanghai Education Development Foundation and the Shanghai Municipal Education Commission (18SG03), the Foundation of the Shanghai Health and Family Planning Commission (20154Y0162), the Capacity Building Planning Program for Shanghai Women and Children’s Health Service and the collaborative innovation centre project construction for Shanghai Women and Children’s Health. The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER N/A.

Peptidyl arginine deiminase, type VI (PADI6) is a member of the subcortical maternal complex (SCMC), which plays vital roles in mammalian embryogenesis. Most mutations in SCMC members have been reported to cause human embryonic arrest, and a total of 15 mutations in PADI6 have been shown to be responsible for early embryonic arrest according to previous studies. However, the genetic factors behind this phenotype remain to be understood in further detail. Here, we identified 13 novel mutations and 4 previously reported mutations of PADI6 in 14 patients who were diagnosed with abnormal embryonic development caused by early arrest, embryonic fragmentation, and recurrent implantation failure. Most of the mutations were predicted by in silico analysis to be deleterious or damaging to the function of PADI6. In addition, the total and East Asian population frequencies of the mutations were low or absent in the gnomAD database. Our study expands the mutational spectrum in PADI6 and will provide precise targets for genetic counseling in the future.

Mitochondrial damage in airway epithelial cells plays an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD). Sirtuin 3 (Sirt3) is a mitochondrial deacetylase regulating mitochondrial function, but its role in the pathogenesis of COPD is still unknown. The aim of the present study was to investigate the effect of Sirt3 on airway epithelial mitochondria in cigarette smoke-induced COPD. Our present study has shown serious airway inflammation, alveolar space enlargement, and mitochondrial damage of the airway epithelium in COPD rats. Compared to the control rats, Sirt3 protein expression was significantly decreased in the airway epithelium and lung tissue homogenate from COPD rats. In airway epithelial cells (BEAS-2B), cigarette smoke extract (CSE) treatment significantly decreased mRNA and protein expression of Sirt3 and manganese superoxide dismutase (MnSOD), as well as MnSOD activity in a concentration and time-dependent manner. Sirt3 siRNA further significantly intensified the decreases in MnSOD expression and activity and aggravated mitochondrial oxidative stress and cell injury when airway epithelial cells were treated with 7.5% CSE. In contrast, Sirt3 overexpression significantly prevented the decrease of MnSOD expression and activity and improved mitochondrial oxidative stress and cell injury in CSE-treated airway epithelial cells. These data suggest that Sirt3 inhibits airway epithelial mitochondrial oxidative stress possibly through the regulation of MnSOD, thereby contributing to the pathogenesis of COPD.

BACKGROUND The main endemic areas of alveolar echinococcosis (AE) are in Central Europe and Western China. Both the infiltration of intrahepatic vascular and bile duct structures as well as extrahepatic disease can lead to further complications and may increase morbidity in patients with AE. AIM To evaluate vascular/biliary involvement in hepatic AE and its distant extrahepatic disease manifestations in an international collective was the aim. METHODS Consecutively, five experienced examiners evaluated contrast-enhanced abdominal computed tomography (CT) scans for 200 patients with hepatic AE of each of four locations (n = 50) in Germany, France and China. Therefore, we retrospectively included the 50 most recent abdominal contrast-enhanced CT examinations at each center, performed because of hepatic AE from September 21, 2007 to March 21, 2018. AE liver lesions were classified according to the echinococcosis multilocularis Ulm classification for CT (EMUC-CT). Distant extrahepatic manifestations were documented either by whole body positron emission tomography–CT or with the addition of thoracic CT and cranial magnetic resonance imaging. Vascular/biliary involvement of the hepatic disease as well as the presence of distant extrahepatic manifestations were correlated with the EMUC-CT types of liver lesion. Statistical analysis was performed using SAS Version 9.4 (SAS Institute Inc., Cary, NC, United States). RESULTS Distant extrahepatic AE manifestations were significantly more frequent in China than in Europe (P = 0.0091). A significant relationship was found between the presence of distant extrahepatic disease and AE liver lesion size (P = 0.0075). Vascular/biliary structures were involved by the liver lesions significantly more frequently in China than in Europe (P < 0.0001), and vascular/biliary involvement depended on lesion size. Different morphological types of AE liver lesions led to varying frequencies of vascular/biliary involvement and were associated with different frequencies of distant extrahepatic manifestations: Vascular/biliary involvement as a function of lesions primary morphology ranged from 5.88% of type IV liver lesions to 100% among type III lesions. Type IV differed significantly in these associations from types I, II, and III (P < 0.0001). With respect to extrahepatic disease, the primary morphology types IV and V of liver lesions were not associated with any case of distant extrahepatic disease. In contrast, distant extrahepatic manifestations in types I–III were found to varying degrees, with a maximum of 22% for type III. CONCLUSION Different CT morphological patterns of hepatic AE lesions influence vascular/biliary involvement and the occurrence of distant extrahepatic manifestations. There are intercontinental differences regarding the characteristics of AE manifestation.

Due to the limited resource of bear bile powder, the major raw material of Tanreqing Capsules(TRQ), cultured bear bile powder is used as a replacement to develop the Tanreqing Capsules Substitute(TRQS). An LC-MS/MS method was established in this study for simultaneous quantitation of 8 compounds from TRQS in rat plasma: tauroursodeoxycholic acid(TUDCA), taurocheno-deoxycholic acid(TCDCA), ursodeoxycholic acid(UDCA), chenodeoxycholic acid(CDCA), ferulic acid, wogonoside, baicalin, and forsythoside A. Thereby, the pharmacokinetic behaviors of TRQ and TRQS were evaluated. Concentration of endogenous compounds TUDCA, TCDCA, UDCA, and CDCA was determined with the stable isotope surrogate analytes: D4-TUDCA, D4-TCDCA, D4-UDCA, and D4-CDCA. Plasma samples were extracted by acetonitrile-induced protein precipitation. The LC conditions are as follows: Waters BEH C_(18) column(2.1 mm×100 mm, 1.7 μm), mobile phase of 10 mmol·L~(-1) ammonium formate aqueous solution(containing 0.01% formic acid) and acetonitrile-methanol mixture(1∶5). MS conditions are as below: multiple reaction monitoring(MRM), ESI~(+/-). Concentration of UDCA, CDCA, TUDCA, and TCDCA was corrected with a response factor, which is the ratio between the responses recorded for the surrogate and the authentic analyte at the equal concentration. Each of the plasma components showed good linearity(rgt; 0.995 1). Accuracy and precision met the criteria(inter-day RSDlt;7.0%, RE 89.98%-112.0%; intra-day RSDlt;12%, RE 90.41%-111.2%). The recovery was 64.83%-119.9% and matrix effect was 87.15%-113.8%. The validated method was applied for pharmacokinetic study of TRQS and TRQ(po, 0.94 g·kg~(-1)). There was no significant difference in C_(max) and AUC_(0-24 h) of baicalin, UDCA, TUDCA, and TCDCA between the two groups, indicating similar pharmacokinetic behaviors between TRQS and TRQ in rats.

Ethnopharmacological relevance Natural bear bile powder (NBBP) is a traditional Chinese medicine used for treating liver dysfunction. Cultured bear bile powder (CBBP), which is produced using biotransformation of chicken bile, acts as an appropriate substitute for NBBP when treating cholestatic liver injury. Aim of the study To investigate the molecular mechanisms underlying the hepatoprotective effects of CBBP in an α-naphthylisothiocyanate (ANIT)-induced cholestatic mouse model. Materials and methods Cholestatic mice were pretreated with CBBP or NBBP via oral gavage once a day for two weeks. Their blood biochemistry and liver histopathology were then evaluated using standard protocols. Western blot analyses, real-time polymerase chain reaction, and immunohistochemistry were used to evaluate changes in the protein levels and gene expression profiles of factors associated with hepatic inflammation and apoptosis in cholestatic mice. Results CBBP significantly decreased the serum indices of liver injury, and ameliorated neutrophil infiltration and hepatocyte necrosis within liver tissue of cholestatic mice. Expression of the inflammatory factors, such as tumor necrosis factor-α, interleukin-1β (IL-1 β), IL-6, monocyte chemoattractant protein-1, and intercellular adhesion molecule 1, was significantly reduced in CBBP-treated cholestatic mice. Moreover, proteins involved in the toll-like receptor 4/myeloid differentiation factor 88/nuclear factor-kappa B (TLR4/Myd88/NF-κB) signaling pathway, such as CD14, TLR4, Myd88, and NF-κB, that were increased in cholestatic mice, were downregulated by CBBP. Meanwhile, increased expression of the apoptosis-related proteins, caspase-3 and Bax, in cholestatic mice was reversed by CBBP treatment. Conclusion CBBP treatment alleviates ANIT-induced cholestasis and liver injury by reducing hepatocyte inflammation and apoptosis.

Gardenia jasminoides Ellis is a well-known herbal medicine. In this study, the effect of G. jasminoides Ellis polysaccharide (GPS) on liver injury in an alpha-naphthylisothiocyanate (ANIT)-induced cholestatic mouse model and the associated molecular mechanisms were investigated. GPS administration dose-dependently ameliorated impaired hepatic function, including a 2-7-fold decrease in aminotransferase levels, ameliorating tissue damage, upregulating the expression of farnesoid X receptor (FXR) and pregnane X receptor (PXR) and their downstream efflux transporters, and decreasing the levels of 12 bile acids (BAs), in cholestatic mice. Furthermore, GPS ameliorated gut microbiota dysbiosis, improved intestinal barrier function, and reduced serum and hepatic lipopolysaccharide levels 1.5-fold. GPS also inhibited the Toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB) signaling, decreased the expression of inflammatory factor genes, and ameliorated hepatic inflammation. Notably, fecal microbiota transplantation from GPS-fed mice also increased the hepatic expression of FXR, PXR, and efflux transporters; decreased the levels of 12 BAs; restored intestinal barrier function; and decreased hepatic inflammation mediated by the TLR4/NF-κB pathway. In conclusion, GPS has a protective effect against cholestatic liver injury through modulation of gut microbiota and inhibition of the TLR4/NF-κB pathway. Regulating gut microbiota using herbal medicine polysaccharides may hold unique therapeutic promise for cholestatic liver diseases.

Perioperative neurocognitive dysfunction (PND) is a prevalent neurological complication after anesthesia and surgery. Ginkgolide B (GB) has been suggested to improve lipopolysaccharide‑induced learning and memory impairment. The present study aimed to investigate whether GB serves a protective role against PND by inhibiting inducible nitric oxide synthase (iNOS) and nitric oxide (NO). Abdominal surgery was performed on 10‑ to 12‑week‑old male C57BL/6 mice under isoflurane anesthesia. Prior to surgery, 1400W (a specific iNOS inhibitor) and GB were administered via intraperitoneal injection. Open field and fear conditioning tests were conducted to assess cognitive function on postoperative days 1 and 3. Biochemical assays were performed to evaluate alterations in NO, malondialdehyde (MDA) and superoxide dismutase (SOD) levels. Western blotting was performed to measure iNOS expression in the hippocampus on postoperative day 1. In addition, hematoxylin and eosin staining was performed to detect the neuronal morphology in the hippocampus. Following treatment with 1400W or GB, surgery‑induced cognitive dysfunction was improved. Compared with the control group, the surgery group exhibited significant overproduction of iNOS and MDA in the hippocampus on postoperative day 1. Higher levels of NO were also detected in the hippocampus and prefrontal cortex of the surgery group on postoperative day 1. Furthermore, pretreatment with 1400W or GB significantly inhibited the surgery‑induced elevation of NO and MDA in brain tissues. Moreover, GB pretreatment significantly inhibited surgery‑induced downregulation of SOD and upregulation of iNOS. Surgery‑induced increases in neuronal loss and the Bax/Bcl‑2 ratio in the hippocampus were significantly inhibited by pretreatment with GB. Collectively, the results of the present study demonstrated that the therapeutic effects of GB on PND were associated with inhibition of iNOS‑induced NO production, increased SOD, and the alleviation of neuronal loss and apoptosis.

Da-Huang-Xiao-Shi decoction (DHXSD), a traditional Chinese medicinal formula, has been used mainly to treat jaundice for more than 1700 years in China. In this study, we developed a rapid, sensitive, and accurate LC–MS/MS method to simultaneously determine multiple, potentially bioactive compounds of DHXSD, including five alkaloids (berberine, phellodendrine, palmatine, jatrorrhizine, and magnoflorine), five anthraquinones (rhein, aloe-emodin, emodin, chrysophanol, and physcion), two iridoid glycosides (geniposide and genipin 1-gentiobioside), and one iridoid aglycone (genipin) in rat plasma. Plasma samples collected from rats were treated immediately with 5% acetic acid to avoid the degradation of genipin. After protein precipitation with acetonitrile containing 5% acetic acid, the compounds were reconstituted in acetonitrile–water (50:50, v/v) solution containing 6.5% formic acid and separated on the ACQUITY™ UPLC BEH C18 column (2.1 × 100 mm; 1.7 μm) using a mobile phase composed of 2 mM ammonium formate in water (solvent A) and acetonitrile (solvent B) at a flow rate of 0.3 mL/min. Quantitation was performed on a Triple Quand 5500 tandem mass spectrometer coupled with an electrospray ionization (ESI) source. Multiple reaction monitoring (MRM) was used to quantify compounds in positive and negative ion modes. The method validation results showed that the specificity, linearity, precision and accuracy, recovery, matrix effect, and stability of the 13 compounds met the requirements for their quantitation in biological samples. This newly established method was successfully used in a pharmacokinetic study on rats orally treated with DHXSD. Besides, glucuronide and sulfate metabolites were also determined in rat plasma after hydrolysis. This is the first method developed for the simultaneous quantification of multiple compounds of DHXSD in vivo. Our study provides relevant information on the pharmacokinetics of DHXSD and the relationship between the compounds of DHXSD and their therapeutic effects.

Nucleus accumbens–associated protein-1 (NAC1) is a transcriptional repressor encoded by the NACC1 gene, which is amplified and overexpressed in various human cancers and plays critical roles in tumor development, progression, and drug resistance. NAC1 has therefore been explored as a potential therapeutic target for managing malignant tumors. However, effective approaches for effective targeting of this nuclear protein remain elusive. In this study, we identified a core unit consisting of Met(7) and Leu(90) in NAC1's N-terminal domain (amino acids 1–130), which is critical for its homodimerization and stability. Furthermore, using a combination of computational analysis of the NAC1 dimerization interface and high-throughput screening (HTS) for small molecules that inhibit NAC1 homodimerization, we identified a compound (NIC3) that selectively binds to the conserved Leu-90 of NAC1 and prevents its homodimerization, leading to proteasomal NAC1 degradation. Moreover, we demonstrate that NIC3-mediated down-regulation of NAC1 protein sensitizes drug-resistant tumor cells to conventional chemotherapy and enhances the antimetastatic effect of the antiangiogenic agent bevacizumab both in vitro and in vivo. These results suggest that small-molecule inhibitors of NAC1 homodimerization may effectively sensitize cancer cells to some anticancer agents and that NAC1 homodimerization could be further explored as a potential therapeutic target in the development of antineoplastic agents.

Purpose Human alveolar echinococcosis (AE) is a potentially lethal zoonosis caused by the cestode Echinococcus multilocularis. The aim of this systematic review is to establish a comprehensive global AE literature overview taking into account the epidemiologically relevant AE research of the twenty-first century. Methods We systematically searched the global literature published from 2001 through 2018 via MEDLINE, EMBASE, the Russian databases eLIBRARY.RU, CyberLeninka, the Chinese databases CNKI, VIP, Journals.research.ac.ir (Farsi language-based), Jordan E-Library (Arab language-based) and supplementary Google Scholar, in accordance with the PRISMA guidelines. QGIS software was used for the mapping of the affected countries. Results We have listed 154 relevant publications in the final literature synopsis in consideration of our quality assessment. Including non-autochthonous cases, human AE was reported in 36 countries within the northern hemisphere from 2001 to 2018. The first publication of AE in Tajikistan, Pakistan, South Korea, Belgium, the Netherlands, Slovakia, Hungary, Lithuania, Latvia, Slovenia and Morocco occurred in this century; further first cases in Taiwan, Thailand, and Denmark were considered to be non-autochthonous by the authors. The highest total case numbers (n ≥ 100 in a single article) were reported in France, Germany, Switzerland, Poland, and Lithuania, including China and Kyrgyzstan with by far the highest prevalence figures. Conclusions Our paper emphasises the increasing spread of reported cases and the rise in its numbers in the literature of the twenty-first century, especially in western, northern and eastern Europe, as well as in central Asia. Epidemiological studies on human infections are lacking in many parts of the world.

Metformin and berberine are often combined for treating diabetes. In the present study, we evaluated the drug-drug pharmacokinetic interaction between metformin and berberine after oral co-administration in vivo and the underlying mechanism. As revealed by comparison with the metformin-only group, berberine significantly decreased the maximum plasma concentration (Cmax), area under the curve from 0 to 4 h (AUC0–4h), and urinary and bile excretion, and increased the kidney tissue concentration of metformin in rats. The non-everted intestinal sac study showed that berberine inhibited the absorption of metformin, and in transfected Madin-Darby canine kidney (MDCK)-rat organic cation transporter 1 (MDCK-rOCT1), MDCK-rat organic cation transporter 2 (MDCK-rOCT2), and MDCK-rat multidrug and toxin extrusion 1 (MDCK-rMATE1) cells, berberine significantly inhibited metformin transport mediated by OCT1, OCT2, and MATE1 in a concentration-dependent manner with half-maximal inhibitory concentration (IC50) values of 18.8, 1.02, and 10.7 μM, respectively. In contrast, co-administration of metformin increased the Cmax and AUC0-4h of berberine with no significant difference in pharmacokinetics parameters between co-administration and berberine-only groups. Furthermore, metformin increased kidney and liver concentrations and reduced the urinary and biliary excretion of berberine. Metformin (≥1 or ≥0.3 mM) decreased berberine transport in MDCK-rOCT1, MDCK-rOCT2, and MDCK-rMATE1 cells. However, metformin did not affect berberine concentration in MDCK-multidrug resistance protein 1 cells. These results suggest that the combination of metformin and berberine induced a pharmacokinetic interaction by cooperatively inhibiting OCT and MATE1-mediated transport.

Cholestasis is characterized by obstruction of bile flow and can lead to serious liver injury. With sustained damage, cholestasis can progress to cholestatic liver fibrosis (CLF), and cirrhosis. Non-invasive, predictive, and reliable metabolites based on the early and progressive stages of CLF are urgently needed. Based on the 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-induced CLF mouse model, serum metabolic profiling via a time-series strategy with ultra-performance liquid chromatography-LTQ-Orbitrap-based metabolomics, combined with histological progression, was used to find CLF-specific metabolites, and explore their dynamic changes in progressive stages of CLF. Compared to those in the control group, DDC-induced groups showed a substantial elevation in cholestatic liver injury and fibrosis indices. Next, 21 differential serum metabolites were selected and identified between the normal (control) and DDC groups, and 12 of them were greatly altered over time. Among these, taurocholic acid, tauromuricholic acid, LysoPE (20:2), sulfoglycolithocholic acid, and taurohyodeoxycholic acid were associated with the progression of the hepatocyte injury index, alanine aminotransferase. More importantly, docosahexaenoic acid, arachidonic acid, proline, leucine, and linoleic acid were associated with the progression of liver fibrosis index, liver hydroxyproline. Moreover, the differential metabolites that were related to hepatocyte injury and liver fibrosis were further validated in DDC-induced mice at weeks 4 and 8. Overall, this work provides data on differential metabolites for the progressive pathology of CLF.

The mechanisms underlying postoperative pain differ from the inflammatory or neuropathic pain. Previous studies have demonstrated that intrathecal α-amino-3-hydroxy-5-methy-4-isoxazole propionate (AMPA) -kainate (KA) receptor antagonist inhibits the guarding pain behavior and mechanical hyperalgesia, indicating a critical role of spinal KA receptors in postoperative pain hypersensitivity. However, how the functional regulations of spinal KA receptor subunits are involved in the postoperative pain hypersensitivity remains elusive. Therefore, in the current study, we investigated the synaptic delivery of spinal KA receptor subunits and the interaction between KA receptor subunits and glutamate receptor-interacting protein (GRIP) during the postoperative pain. Our data indicated that plantar incision induced the synaptic delivery of GluK2, but not GluK1 or GluK3 in ipsilateral spinal cord dorsal horns. The co-immunoprecipitation showed an increased GluK2 –GRIP interaction in ipsilateral dorsal horn neurons at 6 h post-incision. Interestingly, Intrathecal pretreatment of GRIP siRNA increased the paw withdrawal thresholds to mechanical stimuli and decreased the cumulative pain scores in the paws ipsilateral to the incision at 6 h post-incision. Additionally, Intrathecal pretreatment of GRIP siRNA reduced the synaptic abundance of GluK2 in ipsilateral spinal dorsal horn at 6 h after plantar incision. In general, our data have demonstrated that the GluK2- GRIP interaction-mediated synaptic abundance of GluK2 in dorsal horn neurons plays an important role in the postoperative pain hypersensitivity. Disrupting the GluK2- GRIP interaction may provide a new approach for relieving postoperative pain.

A sensitive, specific, and accurate high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and validated for the quantification of prostaglandins D2 (PGD2) and E2 (PGE2) in a mouse ear edema model. We used activated charcoal to obtain PG-free ear samples. The chromatographic separation was performed using a Hypersil Gold C18 column. The limit of detection of each PG was 0.4 ng mL-1, and the intra- and inter-assay estimates of precision and accuracy were14.5 and 94.2-102.9%, respectively. Stability studies showed that all analytes were stable under various storage conditions and analytical processes. The developed and validated method was successfully used to investigate the anti-inflammatory effects of cultured bear bile powder (CBBP) by quantitatively determining PGE2 and PGD2 levels in mouse ear edema samples. These results showed that CBBP significantly inhibited the xylene-induced ear edema in mice and reversed the xylene-induced elevation of PGE2 and PGD2 levels. These results provide useful data about the anti-inflammatory bioactivities in tissues, mediated by the reduction of PGE2 and PGD2 levels, and may further encourage research and development studies of CBBP for its use as an anti-inflammatory agent.

Ethnopharmacological relevance Chronic cholestasis is a usual clinical pathological process in hepatopathy and has few treatment options; it is classified under the category of jaundice in Chinese medicine. Da-Huang-Xiao-Shi decoction (DHXSD) is a classic Chinese prescription which is used to treat jaundice. Aim of the study: We aimed to examine the protective effect of DHXSD on liver and its potential mechanism of action against chronic cholestasis. Materials and methods Chronic cholestasis was induced using 3, 5-diethoxycarbonyl-1,4-dihydroxychollidine (DDC) in mice. Mice were then administered DHXSD intragastrically at doses of 3.68, 7.35, and 14.70 g/kg for four weeks followed by further analyses. Serum biochemical indices and liver pathology were explored. Eighteen individual bile acids (BAs) in mice serum and liver were quantified using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The expression of BA related metabolic enzymes, transporters, along with nuclear receptor farnesoid X receptor (FXR) was detected by real-time qPCR and Western blot. Results DHXSD treatment reduced the serum biochemical indices, ameliorated pathological injury, and improved the disordered BA homeostasis. Mice treated with DHXSD showed significantly upregulated expression of the metabolic enzymes, cytochrome P450 2b10 (Cyp2b10), Cyp3a11, and UDP-glucuronosyltransferase 1a1 (Ugt1a1); and the bile acid transporters, multidrug resistance protein 2 (Mdr2), bile salt export pump (Bsep), and multidrug resistance-associated protein 3 (Mrp3). DHXSD treatment also significantly upregulated FXR expression in mice with DDC-induced chronic cholestasis. Conclusions DHXSD exerted protective effects on chronic cholestasis in DDC-treated mice by alleviating the disordered homeostasis of BAs through increased expression of BA related metabolic enzymes and efflux transporters.

Bear bile is a valuable medicinal material used in traditional Chinese medicine for over 2000 years. However, developing a substitute has become necessary because of protection measures for this endangered species. The ingredients of in vitro cultured bear bile powder (CBBP) include tauroursodeoxycholic acid (TUDCA), taurochenodeoxycholic acid (TCDCA), ursodeoxycholic acid (UDCA) and chenodeoxycholic acid (CDCA, and it has pharmacological properties that are similar to those of natural bear bile powder (NBBP). In this study, the pharmacokinetic parameters of both CBBP and NBBP were measured in rats with a new surrogate analyte LC-MS method using stable isotopes as surrogate analytes (D4-TUDCA, D4-TCDCA, D4-UDCA and D4-CDCA) with response factors validated in authentic matrix (plasma) for simultaneously monitoring the authentic analytes (TUDCA, TCDCA, UDCA and CDCA). The method validation was satisfactory for the linear regression (r, 0.9975-0.9994), precision (RSD intra-day, 0.72-9.35%; inter-day, 3.82-9.02%), accuracy (RE, -12.42-5.67%) and matrix effect (95.53-99.80%), along with analyte recovery (95.90-98.82%) and stability (89.48-101.81%) of surrogate analytes, and precision (RSD intra-day, 1.06- 11.51%; inter-day, 2.23- 11.38%), accuracy (RE, -7.40-10.76%) and stability (87.37-111.70%) of authentic analytes. We successfully applied this method to evaluate the pharmacokinetics of CBBP and NBBP in rats, which revealed the critical in vivo properties of both bear bile preparations.

Zygotic cleavage failure (ZCF) is a unique early embryonic phenotype resulting in female infertility and recurrent failure of in vitro fertilization (IVF) and/or intracytoplasmic sperm injection (ICSI). With this phenotype, morphologically normal oocytes can be retrieved and successfully fertilized, but they fail to undergo cleavage. Until now, whether this phenotype has a Mendelian inheritance pattern and which underlying genetic factors play a role in its development remained to be elucidated. B cell translocation gene 4 (BTG4) is a key adaptor of the CCR4-NOT deadenylase complex, which is involved in maternal mRNA decay in mice, but no human diseases caused by mutations in BTG4 have previously been reported. Here, we identified four homozygous mutations in BTG4 (GenBank: NM_017589.4 ) that are responsible for the phenotype of ZCF, and we found they followed a recessive inheritance pattern. Three of them—c.73C>T (p.Gln25Ter), c.1A>G (p.?), and c.475_478del (p.Ile159LeufsTer15)—resulted in complete loss of full-length BTG4 protein. For c.166G>A (p.Ala56Thr), although the protein level and distribution of mutant BTG4 was not altered in zygotes from affected individuals or in HeLa cells, the interaction between BTG4 and CNOT7 was abolished. In vivo studies further demonstrated that the process of maternal mRNA decay was disrupted in the zygotes of the affected individuals, which provides a mechanistic explanation for the phenotype of ZCF. Thus, we provide evidence that ZCF is a Mendelian phenotype resulting from mutations in BTG4. These findings contribute to our understanding of the role of BTG4 in human early embryonic development and provide a genetic marker for female infertility.

Ethnopharmacological relevance Natural bear bile powder (NBBP) has been used to treat seizures for thousands of years, but its application is greatly restricted due to ethical reasons. Cultured bear bile powder (CBBP), which is produced by biotransformation, may be an appropriate substitute for NBBP. However, the anti-convulsant effects of CBBP and its mechanisms remain unclear. Aim of the study This study aimed to investigate the anti-convulsant effects and possible mechanisms of CBBP in a febrile seizure (FS) rat model. Materials and methods FS was induced by placing the rats in a warm water bath (45.5 °C). The incidence rate and latency of FS, and hematoxylin-eosin staining (HE) were conducted for neurological damage. The levels of 4 bile acids and 8 main neurotransmitters in vivo were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The expression of bile acid related transports, neurotransmitter receptors, inflammatory factors, neurotrophic factors and glial fibrillary acidic protein (GFAP) in hippocampal tissues were detected by real-time PCR, western blotting, and immunohistochemistry. Results Pre-treatments with CBBP and similarly, NBBP, significantly reduced the incidence rate and prolonged the latency of FS. Additionally, CBBP alleviated the histological injury induced by FS in the rat hippocampus tissue. LC-MS/MS analyses revealed that CBBP markedly increased the levels of tauroursodeoxycholic acid (TUDCA), taurochenodeoxycholic acid (TCDCA), ursodeoxycholic acid (UDCA), and chenodeoxycholic acid (CDCA) in FS rats. Furthermore, the content of gamma-aminobutyric acid (GABA) was up-regulated in rats pre-treated with CBBP whereas GFAP was down-regulated. CBBP also significantly suppressed the expression of interleukin -1β (IL-1β), tumor necrosis factor α (TNF-α), nuclear factor kappa B (NF-κB), and brain-derived neurotrophic factor (BDNF) and its TrkB receptors, and improved the expression of GABA type A receptors (GABAAR) and farnesoid X receptors (FXR). Conclusions The present study demonstrated that CBBP had anti-convulsant effects in a FS rat model. CBBP may protect rats against FS, probably by up-regulating FXR, which was activated by increasing brain bile acids, up-regulating GABAergic transmission by inhibiting BDNF-TrkB signaling, and suppressing neuroinflammation by inhibiting the NF-κB pathway.

Ethnopharmacological relevance Intrahepatic cholestasis is a common condition of many liver diseases with few therapies. Yinchenzhufu decoction (YCZFD) is a representative traditional Chinese herbal formula used for treating jaundice and liver disease. Aim of the study To investigate the hepatoprotective effect of YCZFD against cholestatic liver injury and reveal its potential mechanism. Materials and methods Mice with alpha-naphthyl isothiocyanate (ANIT)-induced intrahepatic cholestasis were orally administered YCZFD at doses of 3, 6, and 12g crude drug/kg for 2 weeks followed by subsequent analyses. A serum metabolomics study was then performed to explore the different metabolites influenced by YCZFD using ultra-high-performance liquid chromatography coupled with linear ion trap-Orbitrap hybrid mass spectrometry (UPLC-LTQ-Orbitrap-MS/MS).The levels of individual bile acids in the serum, liver, and bile were determined by UPLC-MS/MS. The expression of metabolic enzymes, transporters, inflammatory factors, and cytokeratin-19 (CK-19) was determined by real-time PCR, western blotting, and immunohistochemistry. Results YCZFD administration decreased the serum biochemical indexes and ameliorated pathological damage, such as hepatic necrosis and inflammatory cell infiltration. Serum metabolomics revealed that the metabolites influenced by YCZFD were mainly associated with bile acid metabolism and inflammation. YCZFD administration effectively ameliorated the disordered bile acid homeostasis. The bile acid transporter, multidrug-resistance associated protein 2 (Mrp2), and the metabolic enzyme, cytochrome P450 2b10 (Cyp2b10), were upregulated in the YCZFD intervention group compared to those in the ANIT-induced group. YCZFD administration also significantly inhibited nuclear factor-κB (NF-κB) and its phosphorylation and decreased the expression of proinflammatory cytokines including tumor necrosis factor-α, interleukin-1β, and intercellular adhesion molecule-1 in ANIT-induced cholestatic mice. Additionally, the level of CK-19 was lower in the YCZFD intervention group than in the ANIT-induced cholestatic mice. Conclusion YCZFD administration ameliorated disordered bile acid homeostasis, inhibited NF-κB pathway-mediated inflammation, and protected the liver from bile duct injury. Therefore, YCZFD exerted a protective effect against cholestatic liver injury.

This review article summarized recent advances in the heat-induced antigen retrieval technique with numerous scientific fields in addition to immunohistochemistry. Particularly, proteomics including imaging mass spectrometry, extraction of proteins from formalin-fixed, paraffin-embedded (FFPE) tissues. Some novel approaches such as FFPE tissue-based renal immunopathology based on modified double heating protocols are also introduced in this review for further development. In general, the FFPE tissue housed in pathology worldwide is an invaluable treasure, and the simple method of heat-induced antigen retrieval is the gold key to open the door of this treasure.

The inflammatory response plays an important role in carcinogenesis. However, the functional role and mechanism of the UCHL3-associated inflammatory response in ovarian cancer remain to be characterized. Here, we report that increased expression of UCHL3 facilitates tumourigenesis by targeting TRAF2 protein, thereby enhancing the inflammatory response. The expression of UCHL3 is elevated in ovarian cancer patients and is associated with an unfavourable prognosis. Genetic ablation of UCHL3 was found to markedly block ovarian cancer cell proliferation, viability and migration both in vitro and in vivo. Mechanistically, luciferase pathway screening results show that NF-κB signalling is clearly activated compared with other pathways. UCHL3 was found to activate NF-κB signalling by deubiquitinating and stabilizing TRAF2, leading to tumourigenesis. Our results indicate that highly expressed UCHL3 enhances inflammation by stabilizing TRAF2, which in turn facilitates tumourigenesis in ovarian cancer, and that UCHL3 is a potential target for ovarian cancer patients with increased inflammation.

Objective To explore the effects of diallyl disulfide(DADS)-induced G2/M phase arrest on proliferation and apoptosis of ovarian cancer cells and its possible molecular mechanism.Methods DADS was used to incubate SK-OV-3 and OVCAR-3 cells,respectively,in different concentrations. Cell proliferation was measured by MTT assay and cell apoptosis rate was detected by flow cytometry assay. Xenograft model assay were performed to analyze the antitumor effect in vivo. Cell cycle phase distribution was detected by flow cytometry. Expressions of cell cycle G2/M phase as well as proliferation- and apoptosis-related proteins were measured by Western blotting.Results MTT assay showed that,after treatment of SK-OV-3(F=247.86,P=0.000)and OVCAR-3 cells(F=302.54,P=0.000)with different concentrations of DADS,the cell proliferation inhibition rate was significantly elevated with the increase of DADS concentrations in a concentration-dependent manner. The inhibition rate of SK-OV-3(F=335.12,P=0.000)and OVCAR-3 cells(F=347.43,P=0.000)at 24 h was significantly higher than that at 12 h and 48 h,showing a significant time-dependence manner. Flow cytometry showed that,after SK-OV-3 and OVCAR-3 cells were treated with different concentrations of DADS,the apoptosis rates increased significantly with the increase of DADS concentration in a concentration-dependent manner(P0.05). The apoptotic rates of SK-OV-3 and OVCAR-3 cells treated with DADS at 24 h was significantly higher than that at 12 h and 48 h in a significant time-dependence manner(P0.05). Compared with the blank treatment group,intraperitoneal injection of DADS solution significantly inhibited the xenograft volume of ovarian cancer cells in nude mice(F=548.23,P=0.000;F=311.84,P=0.000). After 30 mg/L of DADS was applied to SK-OV-3 and OVCAR-3 cells for 24 h,the percentage of cells in G2 phase of SK-OV-3 and OVCAR-3 cells increased significantly(F=375.11,P=0.000;F=256.48,P=0.000),compared with the blank cells. After 30 mg/L DADS was applied to SK-OV-3 and OVCAR-3 cells for 24 h,the expressions of p-Chk1(ser345)(F=108.89,P=0.013;F=97.58,P=0.018),p-CDC25C(ser216)(F=87.25,P=0.025;F=114.25,P=0.009),p-P53(ser15)(F=112.41,P=0.011;F=255.87,P=0.000),P21WAF1(F=246.38,P=0.001;F=141.36,P=0.005)and p-CDK1(Thr14/Tyr15)protein(F=298.12,P=0.000;F=233.15,P=0.000)were significantly increased,whereas the expressions of CDK1(F=308.24,P=0.000;F=257.55,P=0.000)and CyclinB1 protein(F=223.15,P=0.001;F=241.28,P=0.000)were significantly reduced.The expressions of proliferation and apoptosis-related proteins PCNA(F=77.36,P=0.031;F=157.28,P=0.001),Ki-67(F=205.64,P=0.007;F=315.22,P=0.000)and Survivin(F=122.13,P=0.013;F=188.24,P=0.000)were significantly decreased and Cleaved-caspase3 protein was significantly increased(F=86.46,P=0.023;F=99.11,P=0.009).Conclusion DADS can inhibit the proliferation of ovarian cancer cells and induce their apoptosis,which may be related to the activation of Chk1-CDC25C and P53-P21WAF1 signaling pathways in G2/M checkpoint,decreased kinase activity of CDK1,down-regulated expressions of CDK1 and CyclinB1 proteins,and ultimately cell cycle arrest at G2/M phase.

To investigate the effect of moderate-intensity aerobic exercise on the differential expression of rat atrial muscle Proteomics and genes, which provide research basis for the rehabilitation of chronic cardiovascular diseases and exercise -induced cardiac remodeling research.Twenty male SD rats were randomly divided into control group and experimental group (By software analysis, the experimental group compared with the control group, there were 8 protein points which their expression reduced more than 4/5 and 5 protein points up-regulated more than 5 times, 13 proteins were identified by mass spectrometry protein spots, the final identification results acquired 8 proteins and a unknown protein of molecular mass 54 KDa, such as:pyruvate dehydrogenase E1α1, mitochondrial aconitate hydratase, protein disulfide isomerase A3, methylmalonic acid semialdehyde dehydrogenase, mitochondrial dihydrolipoic acid dehydrogenase, isovaleryl coenzyme A dehydrogenase, glutathione synthetase, mitogen-activated protein kinase 3 and so on. Compared with the control group, the mRNA expression of methylmalonic acid semialdehyde dehydrogenase in the atrial muscle of rats was decreased after 4 weeks of moderate aerobic exercise (The 4 weeks moderate-intensity aerobic exercise induced ignificant changes of rats atrial muscle protemics. The majority of the 13 identified target proteins in this experiment are energy metabolism enzymes. The majority of the expression of the target protein and the mRNA expression in the atrial muscle is inconsistent and different. Exercise may affect the regulation of gene transcription or downstream translation and modification of these target proteins, resulting in the change of differential expression.

This study aimed at understanding the impact of different substitutions at codon 715 localized in the region II of the palm domain of herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) DNA polymerases (pol). Here, we report a new theoretical mutation V715S that confers resistance of HSV-1 to foscarnet/acyclovir (5.6- and 9.2-fold increases EC50 values compared to wild type, respectively) and of HCMV to foscarnet/ganciclovir (2.8- and 2.9-fold increases in EC50 values compared to wild type, respectively). To further analyze the importance of this amino acid, we investigated the impact of the already known mutations V715M and V715G on the replicative capacities and drug susceptibilities of both viruses as well as on the activity and drug inhibition of the DNA pol. The V715G recombinant HSV-1 mutant was resistant to foscarnet and acyclovir (3.4- and 4.6-fold EC50 increase, respectively) whereas the V715M mutant was susceptible to foscarnet and resistant to acyclovir (3.4-fold EC50 increase). The V715G recombinant HCMV mutant did not grow and the V715M mutant was resistant to foscarnet (3.7-fold EC50 increase) and susceptible to ganciclovir. Finally, we showed by three-dimensional modeling that the differential impact of these mutations on the viral replicative capacity and drug resistance profile was related to different hydrophobic local environments for V715 in the DNA pol of the two viruses. Furthermore, we hypothesize that the DNA pol of HSV-1 is more tolerant to changes at this residue compared to that of HCMV because of a more hydrophobic environment stabilizing the region.

Background Huangqi decoction (HQD) has been used to treat chronic liver diseases since the 11th century, but the effective components in HQD against liver fibrosis have not been definitively clarified. Purpose To investigate and identify multiple effective components in HQD against liver fibrosis using a pharmacokinetics-based comprehensive strategy. Methods The absorbed representative components in HQD and their metabolites were detected in human plasma and urine using high-resolution mass spectrometry combined with a database-directed method, and then pharmacokinetics in multiple HQD components in human plasma was analyzed by ultra-performance liquid chromatography coupled with triple-quadruple mass spectrometry. Furthermore, the anti-fibrotic effect of potential effective HQD components was studied in LX-2 cells and that of a multi-component combination of HQD (MCHD) was verified in a mouse CCl4-induced hepatic fibrosis model. Results Twenty-four prototype components in HQD and 17 metabolites were identified in humans, and the pharmacokinetic characteristics of 14 components were elucidated. Among these components, astragaloside IV, cycloastragenol, glycyrrhizic acid, glycyrrhetinic acid, liquiritigenin, and isoliquiritigenin downregulated the mRNA expression of α-SMA; cycloastragenol, calycosin-7-O-β-D-glucoside, formononetin, glycyrrhetinic acid, liquiritin, and isoliquiritin downregulated the mRNA expression of Col I; and calycosin, liquiritigenin, isoliquiritigenin, cycloastragenol, and glycyrrhetinic accelerated the apoptosis of LX-2 cells. MCHD reduced serum aminotransferase activity and hepatic collagen fibril deposition in mice with CCl4-induced hepatic fibrosis. Conclusion Using the pharmacokinetics-based comprehensive strategy, we revealed that multiple effective HQD components act together against liver fibrosis.

Repeated administration of morphine may result in opioid-induced hypersensitivity (OIH), which involves altered expression of numerous genes, including brain-derived neurotrophic factor (BDNF) in dorsal root ganglion (DRG) neurons. Yet, it remains unclear how BDNF expression is increased in DRG neurons after repeated morphine treatment. DNA methylation is an important mechanism of epigenetic control of gene expression. In the current study, we hypothesized that the demethylation regulation of certain BDNF gene promoters in DRG neurons may contribute to the development of OIH. Real-time RT-PCR was used to assess changes in the mRNA transcription levels of major BDNF exons including exon I, II, IV, VI, as well as total BDNF mRNA in DRGs from rats after repeated morphine administration. The levels of exon IV and total BDNF mRNA were significantly upregulated by repeated morphine administration, as compared to that in saline control group. Further, ELISA array and immunocytochemistry study revealed a robust upregulation of BDNF protein expression in DRG neurons after repeated morphine exposure. Correspondingly, the methylation levels of BDNF exon IV promoter showed a significant downregulation by morphine treatment. Importantly, intrathecal administration of a BDNF antibody, but not control IgG, significantly inhibited mechanical hypersensitivity that developed in rats after repeated morphine treatment. Conversely, intrathecal administration of an inhibitor of DNA methylation, 5-aza-2'-deoxycytidine (5-aza-dC) markedly upregulated the BDNF protein expression in DRG neurons and enhanced the mechanical allodynia after repeated morphine exposure. Together, our findings suggest that demethylation regulation of BDNF gene promoter may be implicated in the development of OIH through epigenetic control of BDNF expression in DRG neurons.

Erlong Zuoci decoction (ELZCD), a typical traditional Chinese medicine (TCM) prescription, has long been clinically used in treatment of deafness and tinnitus with the syndrome of "kidney yin deficiency". However, there are few studies to investigate its pharmacological mechanisms. Until now, there is not report about its effects on the age-related hearing loss (ARHL).The present study was conducted to observe the effects of ELZCD on the ARHL in C57BL/6J mice and explore the mechanisms.ELZCD was fed to C57BL/6J mice from 3 months to 6 months in ELZCD group as a dose of 6g/kg/d. And the same volume of saline was fed to mice in ARHL group. 3-months-old C57BL/6J mice were used as control group. High performance liquid chromatography (HPLC) was used for the quality control of ELZCD. Auditory brainstem response (ABR) was used to assess the hearing function of mice. The morphologic changes were observed by hematoxylin eosin (HE) staining. Apoptosis was tested by terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) method. Mitochondrial damage was detected by transmission electron microscopy (TEM). Quantitative RT-PCR (qRT-PCR) was used to observe the mRNA expression of p53 and Bak. Fluorescence immunohistochemical technique was used to test the protein expression of p53 and Bak.The hearing threshold of ARHL group was higher than that of control group (P0.001) and ELZCD decreased the rise of hearing threshold levels of ARHL mice (P0.001), which suggested ELZCD inhibited the hearing loss of ARHL mice. HE staining showed that ELZCD decreased the spiral ganglion (SG) cell damage and loss in ARHL. TUNEL test showed that the apoptotic SG cells increased in ARHL group compared to control group and decreased in ELZCD group compared to ARHL group. TEM observation showed that mitochondrial damage was obvious in SG cells of ARHL group and ELZCD inhibited the mitochondrial damage. The qRT-PCR results showed that the mRNA expression of p53 and Bak in ARHL group increased compared to that of control group (P0.05), and ELZCD reduced the elevated mRNA expression levels of p53 and Bak (P0.01, P0.05). In addition, ELZCD inhibited the increased proteins expression (green fluorescence) of p53 and Bak.The results demonstrated that ELZCD prevented ARHL in C57BL/6J mice and p53/Bak-mediated mitochondrial apoptosis of SG cells might be involved in the mechanisms.

Although MeHg is a confirmed neurotoxin at sufficiently high dosages, the effect of prenatal low-level Hg exposure on infant neurodevelopment is still unknown. Four hundred ten mother-infant pairs were recruited in rural northern China from September 2010 to September 2012. We investigated maternal diet during pregnancy, collected maternal and umbilical blood samples, and tested infant neurodevelopment using Gesell developmental schedules (GDS) at the age of 1 year old. The geometric means (GM) of Hg exposure in maternal and umbilical blood were 0.72 and 1.2 μg/L, respectively. Positive associations between Hg levels and the frequency of total fish consumption were found (Spearman's correlation, r = 0.09 for maternal Hg, r = 0.14 for umbilical Hg, p

A sensitive, specific, accurate HPLC-MS/MS method was developed and validated for the simultaneous quantification of catechin, epicatechin, liquiritin, isoliquiritin, liquiritigenin, isoliquiritigenin, piperine and glycyrrhetinic acid from Longhu Rendan pills in rat plasma. Chromatographic separation was performed with a Hypersil Gold C18 column using a gradient of methanol and 0.01% acetic acid containing 0.2 mm ammonium acetate as mobile phase. The analytes were quantified on a triple quadrupole mass spectrometer, operating in selected reaction monitoring mode and switching the electrospray ion source polarity between positive and negative modes in a single run. The calibration curves of catechin, epicatechin, liquiritin, isoliquiritin, liquiritigenin, isoliquiritigenin, piperine and glycyrrhetinic acid were linear over the concentration ranges of 5-2000, 5-2000, 0.5-200, 0.5-200, 0.25-100, 0.25-100, 0.025-10 and 0.50-200 ng mL(-1) , respectively. The intra- and inter-assay precisions and accuracies were

Multiple lines of evidence suggest that general anesthesia helps the recovery from sleep deprivation. However, little is known about the underlying neurochemical mechanisms. In the current study, we investigated the effect of anesthetic propofol on the release of glutamate (Glu) and γ-amino butyric acid (GABA) in the hippocampal CA1 region of rat with 24 h-paradoxical sleep deprivation (PSD).A guide cannula for microdialysis was inserted into the CA1 region of hippocampus in rats. At six days after cannula implantation, rats received 24 h-PSD by using the platform-water tank method. The rats were then subjected to natural sleep or propofol anesthesia (100 mg/kg, i.p.), respectively, after 24-h PSD. Microdialysis samples from hippocampus were collected before and at the end of PSD, and also at 1, 3, 6, and 8 h post-PSD. The concentrations of Glu and GABA in collected samples were determined by using high performance liquid chromatography.The current study showed that 24 h-PSD significantly increased the release of Glu and GABA in the hippocampus in rats. In both natural sleep and propofol anesthesia groups, the upregulated Glu and GABA levels after PSD gradually decreased and returned to the baseline level by 8 h post-PSD.Our data indicate that propofol anesthesia promotes the restoration of disturbed excitatory and inhibitory neurotransmitter release in the hippocampus after PSD, similar to the beneficial effects of natural sleep. This finding suggests that propofol anesthesia may be a viable pharmacotherapeutic strategy for the treatment of certain sleep disorders that share similar mechanisms with PSD.

SummaryIntracellular bacterial pathogens of a diverse nature share the ability to evade host immunity by impairing trafficking of endocytic cargo to lysosomes for degradation, a process that is poorly understood. Here, we show that the Salmonella enterica type 3 secreted effector SopD2 mediates this process by binding the host regulatory GTPase Rab7 and inhibiting its nucleotide exchange. Consequently, this limits Rab7 interaction with its dynein- and kinesin-binding effectors RILP and FYCO1 and thereby disrupts host-driven regulation of microtubule motors. Our study identifies a bacterial effector capable of directly binding and thereby modulating Rab7 activity and a mechanism of endocytic trafficking disruption that may provide insight into the pathogenesis of other bacteria. Additionally, we provide a powerful tool for the study of Rab7 function, and a potential therapeutic target.

An experiment was conducted to evaluate the effect of litter thickness on growth performance, immune status, environmental condition, and welfare quality in yellow broilers. In total, 1,800 one-day-old Suqin yellow broilers were raised for 21 d. On d 22, 1,600 birds of similar BW (404±12 g) were randomly selected and placed into 20 indoor pens (8 birds/m2, 10 m2/pen). These birds were assigned to a litter treatment of 4, 8, 12, and 16 cm. Each treatment was repeated in five pens. The results showed that a thicker litter was related to increased BW, daily weight gain, and daily feed intake (P

Analysis of Permian-Triassic brachiopod diversity and body size changes from different water depths spanning the continental shelf to basinal facies in South China provides insights into the process of environmental deterioration. Comparison of the temporal changes of brachiopod diversity between deepwater and shallow-water facies demonstrates that deepwater brachiopods disappeared earlier than shallow-water brachiopods. This indicates that high environmental stress commenced first in deepwater settings and later extended to shallow waters. This environmental stress is attributed to major volcanic eruptions, which first led to formation of a stratified ocean and a chemocline in the outer shelf and deeper water environments, causing the disappearance of deep marine benthos including brachiopods. The chemocline then rapidly migrated upward and extended to shallow waters, causing widespread mass extinction of shallow marine benthos. We predict that the spatial and temporal patterns of earlier onset of disappearance/extinction and ecological crisis in deeper water ecosystems will be recorded during other episodes of rapid global warming.

Ethnopharmacological relevance Xiexin decoction (XXD) has been used as a treatment for diabetes mellitus for more than 1300 years. XXD constituents with protective effects against diabetic nephropathy (DN) include Rhizoma Coptidis alkaloids (RA), Radix et Rhizoma Rhei polysaccharides (RP), and Radix Scutellaria flavones (RF). The aim of the study is to investigate the effects of combinations of RA, RP, and RF on DN and their mechanisms of action. Materials and Methods In vitro , high glucose-induced rat mesangial cells were treated with RA, RP, RF, and combinations thereof. Cell proliferation and levels of inflammatory factors were measured. In vivo , high-fat diet and streptozotocin-induced diabetic rats were treated with different combinations of RA, RP, and RF once per day for 12 weeks. Blood and urine biochemical parameters, renal tissue morphology, and inflammation were investigated. Results In vitro , the combination of the three groups of components inhibited mesangial cell proliferation and reduced the levels of monocyte chemotactic protein-1 (MCP-1) and collagen IV. The effects of the three constituent groups in combination were stronger than those of each group alone or combinations of two groups. In diabetic rats, combinations of the three groups of herb components ameliorated blood glucose, urinary albumin excretion and decreased renal mesangial matrix expansion and basement membrane thickening. In addition, the combinations reduced renal tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) protein levels, down-regulated the expression of nuclear factor κB (NF-κB) and transforming growth factor beta 1 (TGF-β1), and up-regulated the expression of inhibitor of nuclear factor κB (IκB) protein. Among the three groups of herb components, RA produced the strongest effects, followed by RP, and then by RF. Conclusions The combination of the three groups of herb components produced anti-DN effects through inhibition of inflammation mediated by NF-κB. Among the three groups of herb components, RA produced the strongest effect while RP and RF produced weaker effects.

Farnesyl diphosphate synthase (FPPS) is an enzyme from the class of short chain ( E)-prenyltransferases that catalyzes the condensation of two molecules of isopentenyl diphosphate (IPP, C5) with dimethylallyl diphosphate (DMAPP, C5) to generate the C15 product FPP. In insects, FPPS plays a key role in the biosynthesis of the morphogenetic and gonadotropic “juvenile hormone” (JH). Lepidopteran genomes encode two very distinct FPPS paralogs , one of which (“type-II”) is expressed almost exclusively in the JH-producing glands, the corpora allata . This paralog has been hypothesized to display structural features that enable the binding of the bulkier precursors required for the biosynthesis of lepidopteran ethyl-branched JHs. Here, we report on the first crystal structures of an insect FPPS solved to date. Apo, ligand-bound, and inhibitor-bound structures of type-II FPPS (FPPS2) from the spruce budworm , Choristoneura fumiferana (Order: Lepidoptera), were obtained. Comparison of apo and inhibitor-bound enzymes revealed differences in both inhibitor binding and structural plasticity of CfFPPS2 compared to other FPPSs. Our data showed that IPP is not essential to the closure of the C-terminal tail . Ortho-substituted pyridinium bisphosphonates, previously shown to inhibit CfFPPS2, bound to the allylic site, as predicted; however, their alkyl groups were oriented towards the homoallylic binding site, with the bulkier propyl-substituted inhibitor penetrating deeply into the IPP binding pocket. The current study sheds light on the structural basis of substrate specificity of type-II FPPS of the spruce budworm. Through a comparison with other inhibitor-bound FPPSs, we propose several approaches to improve inhibitor selectivity and potency.

International audience; Human metapneumovirus (HMPV) is an important cause of respiratory tract infections. The mechanism by which its fusion (F) protein is responsible for variable cytopathic effects in vitro remains unknown. We aligned the F sequences of the poorly fusogenic B2/CAN98-75 strain and the hyperfusogenic A1/C-85473 strain and identified divergent residues located in the two functional heptad repeats domains (HRA and HRB). We generated recombinant viruses by inserting the mutations N135T-G139N-T143K-K166E-E167D in HRA and/or K479R-N482S in HRB, corresponding to swapped sequences from C-85473, into CAN98-75 background and investigated their impact on in vitro phenotype and fusogenicity. We demonstrated that the five HRA mutations enhanced the fusogenicity of the recombinant rCAN98-75 virus, almost restoring the phenotype of the wild-type rC-85473 strain, whereas HRB substitutions alone had no significant effect on cell-cell fusion. Altogether, our results support the importance of the HRA domain for an HMPV-triggered fusion mechanism and identify key residues that modulate syncytium formation.

Berberine, a well-known plant alkaloid derived from Rhizoma coptidis, has potential applications as a therapeutic drug for diabetic nephropathy. However, the transporter-mediated renal transport of berberine remains largely unclear. This study aimed to investigate the renal transport mechanism of berberine using transfected cells, kidney slices and animal experiments. In Madin-Darby canine kidney (MDCK) cells stably expressing rat OCT2 (MDCK-rOCT2) and kidney slices, saturable and non-saturable uptake of berberine was observed, and corticosterone could inhibit the uptake of berberine, with IC50 values of 0.1 μm and 147.9 μm, respectively. In double-transfected cells, the cellular accumulation of berberine into MDCK-rOCT2 and MDCK-rOCT2-rMATE1 (MDCK cells stably expressing rOCT2 and rMATE1) cells was significantly higher than the uptake into MDCK cells. Meanwhile, berberine transcellular transport was considerably higher in double-transfected MDCK-rOCT2-rMATE1 cells than in MDCK and MDCK-rOCT2 cells. Corticosterone for MDCK-rMATE1 and MDCK-MDR1 and pyrimethamine for MDCK-rMATE1 at high concentrations could inhibit the efflux of berberine. In animal experiments, compared with the berberine alone group, the cumulative urinary excretion of berberine significantly decreased in the corticosterone or pyrimethamine pretreatment groups. In the rat kidney, pyrimethamine increased, and a low dose of corticosterone (5 mg/kg) decreased, the berberine concentration. However, there was no apparent change in the renal concentration of berberine in rats pretreated with corticosterone (10 or 20 mg/kg). Thus, berberine is not only a substrate of OCT2 and P-glycoprotein, but is also a substrate of MATE1. Both OCT2 and MATE1 mediate the renal vectorial transport of berberine.

To investigate the role of TLR3/PI3K signals in the occurrence and development of cervical cancer disease, TLR3-siRNA was used to block key signaling pathways involved in cervical cancer metastasis that are pivotal to metastatic tumor cells but not to normal cells under ordinary physiologic conditions. Results show that tumor U14 cell growth, migration and invasion in TLR3-siRNA treatment group were significantly decreased. Through LY294002 suppressing targeted gene, the LY294002 treatment specifically and significantly knocked down the expressions of tumor TLR3 and PI3K proteins in cervical cancer mice. Furthermore, expressions of tumor Survivin and FasL proteins were markedly suppressed, whereas expressions of Fas protein were upregulated in LY294002 treatment group mice. LY294002 treatment suppressed tumor growth and increased the thymus and spleen indeces and survival days of cervical cancer mice. This study demonstrates that TLR3-siRNA and LY294002 treatments can markedly suppress cervical cancer cell invasion and tumor growth and increase survival life by silencing targeted genes.

Ethnopharmacological relevance Yinchenzhufu decoction (YCZFD) is a classical Chinese herbal formula and has been used to treat severe jaundice in chronic liver injuries since the Qing Dynasty (18th century CE). To identify the components absorbed into the blood in YCZFD and explore their pharmacokinetic profile for understanding the effective ingredients of YCZFD. Materials and methods After rats were given YCZFD by intragastric administration, the plasma was processed by precipitation of protein. The compounds in YCZFD extract and the plasma were identified by using high-resolution mass spectrometry with a database-directed strategy. The pharmacokinetics of multiple compounds from YCZFD in rat plasma was studied by using the established UPLC-MS/MS method. Results Forty compounds in YCZFD extract and 21 prototype compounds with 11 metabolites in rat plasma were detected after oral administration. The pharmacokinetic parameters of glycyrrhizic acid, glycyrrhetic acid, cinnamic acid, ononin, atractylenolide III, and liquiritin from YCZFD were obtained in rats. Conclusions The identified constituents and the pharmacokinetic features of YCZFD are helpful for understanding the material bases of its therapeutic effects.

Xiexin decoction (XXD) is composed of Rhei Rhizoma (DH), Radix Scutellaria (HQ), and Coptis Chinensis (HL). Free anthraquinones in DH are the basic effective constituents in XXD. Reportedly, HL decreases the bioavailability of the anthraquinones, while HQ antagonizes the effect of HL. In this study, we aimed to determine the underlying mechanisms. The metabolisms of anthraquinones by intestinal flora were studied using rat fecal suspension (RFS); the metabolisms of rhein (a typical anthraquinone in DH) by rat intestine and liver were studied using rat intestine microsomes (RIMs) and rat liver microsomes (RLMs), respectively; the intestinal transport of rhein was studied using everted gut sacs. The results showed that HL decreased the amount of the free anthraquinones after incubation in RFS and inhibited the intestinal transport of rhein, but HQ antagonized the effect of HL. On the other hand, HQ strongly inhibited the glucuronidation of rhein in both RIMs and RLMs. The results suggested that HL decreased the oral bioavailability of the anthraquinones due to inhibiting the conversion of conjugated anthraquinones to free anthraquinones by intestinal flora and decreasing the intestinal transport of the anthraquinones; HQ confronted the effect of HL by inhibiting the glucuronidation of the anthraquinones in intestine and weakening the inhibitory effects of HL.

Accumulating evidence has revealed the link between age‑related hearing loss (presbycusis) and cognitive decline; however, their exact association remains unclear. The present study aimed to investigate the association between age‑related hearing loss and cognitive decline, and to explore the underlying mechanisms. Briefly, three groups of C57BL/6J mice were evaluated, based on their age, as follows: Young group, 3 months; adult group, 6 months; and middle‑aged group, 15 months. The results of an auditory brainstem response (ABR) test demonstrated that the hearing threshold levels of the mice were increased in those aged 6 and 15 months compared with those aged 3 months, thus suggesting that significant hearing loss occurred at 6 months, and worsened at 15 months. The results of a Morris water maze test demonstrated that spatial learning and memory function was significantly decreased in 15‑month‑old mice, but not in 6‑month‑old mice. Pearson analysis indicated that the escape latency was positively correlated with hearing threshold at 16 kHz and percentage of time in the target quadrant was negatively correlated with hearing threshold at 16 kHz, thus suggesting a correlation between age‑related hearing loss and cognitive decline. The auditory cortex and hippocampal CA1 region in 15‑month‑old mice exhibited significantly decreased cell numbers, abnormal arrangement and morphological alterations. Transmission electron microscopy revealed reduced synapse numbers and synaptic vesicle density in mice aged 15 months. Furthermore, the protein expression levels of matrix metalloproteinase (MMP)‑9 in the auditory cortex and hippocampus in the 15‑month‑old mice were significantly higher than in the 3‑month‑old mice. In conclusion, these findings support the correlation between age‑related hearing loss and cognitive decline in C57BL/6J mice, and indicated that MMP‑9 expression in the auditory cortex and hippocampus may be associated with the underlying mechanisms.

Deficits of protein phosphatase-2A (PP2A) play a crucial role in tau hyperphosphorylation, amyloid overproduction, and synaptic suppression of Alzheimer's disease (AD), in which PP2A is inactivated by the endogenously increased inhibitory protein, namely inhibitor-2 of PP2A (I2(PP2A)). Therefore, in vivo silencing I2(PP2A) may rescue PP2A and mitigate AD neurodegeneration. By infusion of lentivirus-shRNA targeting I2(PP2A) (LV-siI2(PP2A)) into hippocampus and frontal cortex of 11-month-old tg2576 mice, we demonstrated that expression of LV-siI2(PP2A) decreased remarkably the elevated I2(PP2A) in both mRNA and protein levels. Simultaneously, the PP2A activity was restored with the mechanisms involving reduction of the inhibitory binding of I2(PP2A) to PP2A catalytic subunit (PP2AC), repression of the inhibitory Leu309-demethylation and elevation of PP2AC. Silencing I2(PP2A) induced a long-lasting attenuation of amyloidogenesis in tg2576 mice with inhibition of amyloid precursor protein hyperphosphorylation and β-secretase activity, whereas simultaneous inhibition of PP2A abolished the antiamyloidogenic effects of I2(PP2A) silencing. Finally, silencing I2(PP2A) could improve learning and memory of tg2576 mice with preservation of several memory-associated components. Our data reveal that targeting I2(PP2A) can efficiently rescue Aβ toxicities and improve the memory deficits in tg2576 mice, suggesting that I2(PP2A) could be a promising target for potential AD therapies.

Protein phosphatase-2A (PP2A), an important phosphatase in dephosphorylating tau and preserving synapse, is significantly suppressed in Alzheimer's disease (AD), but the mechanism is not well understood. Here, we studied whether phosphotyrosyl phosphatase activator (PTPA) could activate PP2A by reducing its inhibitory phosphorylation at tyrosine 307 (P-PP2AC). We found that overexpression of PTPA activated PP2A by decreasing the level of P-PP2AC with reduced phosphorylation of tau, while knockdown of PTPA inhibited PP2A by increasing the level of P-PP2AC with enhanced tau phosphorylation. We also observed that expression of PTPA could upregulate the protein and mRNA levels of protein tyrosine phosphatase 1B (PTP1B) and simultaneous downregulation of PTP1B eliminated PTPA-induced PP2A activation. Importantly, we also found that the protein level of PTPA is downregulated in the brains of AD patients, and the AD transgenic mouse models with expression of mutant human amyloid precursor protein (hAPP) or the longest human tau (htau), respectively. Our data indicate that PTPA may activate PP2A through activating PTP1B and thus reducing the level of P-PP2AC, therefore upregulation of PTPA may represent a potential strategy in rescuing PP2A and arresting tau pathology in AD.

Jatrorrhizine is a protoberberine alkaloid derived from Coptis chinensis concentrated extremely in rat kidney. In the present study, the involvement of rat organic cation transporter 2 (rOCT2) in the renal uptake of jatrorrhizine in rat was investigated through in vitro and in vivo experiments. Saturable and nonsaturable uptakes of jatrorrhizine were observed in rat kidney slices and rOCT2-Madin-Darby canine kidney (MDCK) cells. Michaelis-Menten constants of 677.8 and 21.0 µM, maximum uptake rate of 123 (pmol/min)/mg kidney and 13.7 (pmol/min)/mg protein, and nonsaturable uptake clearance of 0.054 (µL/min)/mg kidney and 0.032 (µL/min)/mg protein were observed in rat kidney slices and rOCT2-MDCK cells, respectively. As inhibitors of rOCT2, corticosterone, verapamil, and cimetidine can inhibit jatrorrhizine uptake in rat kidney slices and rOCT2-MDCK cells. Their median inhibitory concentration in rat kidney slices was 7, 78, and 538 µM, whereas that in rOCT2-MDCK cells was 1.07, 86.5, and 151.8 µM. Coadministration with 20 mg/kg corticosterone, a selective inhibitor of rOCT2, reduced the jatrorrhizine concentration in the cortex and medulla in the in vivo experiment. Thus, rOCT2 is mainly responsible for the renal uptake of jatrorrhizine in rat and in the regulation of jatrorrhizine concentration in the kidney.

The db/db mouse is one of the most popular animal models for type 2 diabetes mellitus, but changes in the activities of important P450s and UGTs are still not completely clear. This study was designed to investigate the alterations of major hepatic cytochrome P450s and UDP-glucuronyltransferase enzymes in db/db mice. Mouse liver microsomes (MLMs) were obtained from male db/db mice and their wild type littermates. After incubation of the substrates separately with MLMs, the samples were pooled and analysed by high-throughput liquid chromatography-tandem mass spectrometry system for the simultaneous study of nine phase I metabolic reactions and three glucuronidation conjugation reactions to determine the activity of the metabolic enzymes. Compared with normal controls, the Cl

Pharmacological activities of some natural products diminish and even disappear after purification. In this study, we explored the mechanisms underlying the decrease of acute oral toxicity of Coptidis Rhizoma extract after purification. The water solubility, in vitro absorption and plasma exposure of berberine (the major active compound) in the Coptidis Rhizoma extract were much better than those of pure berberine. Scanning electron microscopy, laser scanning confocal microscopy (LSCM) and dynamic light scattering experiments confirmed that nanoparticles attached to very fine precipitates existed in the aqueous extract solution. The LSCM experiment showed that the precipitates were absorbed with the particles by the mouse intestine. High-speed centrifugation of the extract could not remove the nanoparticles and did not influence plasma exposure or acute oral toxicity. However, after extract dilution, the attached precipitates vanished, although the nanoparticles were preserved and there were no differences in the acute oral toxicity and plasma exposure between the extract and pure berberine. The nanoparticles were then purified and identified as proteinaceous. Furthermore, they could absorb co-dissolved berberine. Our results indicate that naturally occurring proteinaceous nanoparticles in Coptidis Rhizoma extract act as concentration-dependent carriers that facilitate berberine absorption. These findings should inspire related studies in other natural products.

Trichlorfon (TCF) is a widely used broad-spectrum agricultural organophosphate (OP) pesticide. Few studies have evaluated the effects of TCF on reproductive toxicity after low-level exposure, especially after long-term exposure. This study assessed the direct effects of TCF on estrous cycle, oocyte maturation in female mice, and developmental outcome in near-term fetuses after 30 consecutive days of maternal exposure to 2, 10, or 50 mg/kg body weight/d TCF via drinking water. Both male and female fetuses in the 50 mg/kg/d TCF-treated group had significantly reduced body weights; but this did not occur in the 2 mg/kg/d and 10 mg/kg/d TCF-treated groups. No difference in oocyte maturation, including the percentages of germinal vesicle breakdown (GVBD) and first polar body (PB1) extrusion, or in estrous cycle was found between the control and TCF-treated groups. No increased incidence of fetal external malformations was observed in the TCF-treated groups. Significant decreases in maternal liver weights occurred in the 10 and 50 mg/kg/d TCF-treated groups in a dose-dependent manner. No significant changes were found in the weight of organs such as the ovaries, thymus, kidneys, spleen, lungs, heart or brain. The lack of effects of 2 mg/kg/d and 10 mg/kg/d TCF on any in vivo reproductive and developmental endpoints examined suggest that no TCF reproductive toxicity occurs at exposures less than 10 mg/kg/d.

Monoammonium glycyrrhizinate (MAG) was the aglycone of glycyrrhizin derived from licorice. In this study, the anti-inflammatory effects of MAG on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice and the possible mechanisms involved in this protection were investigated. Pretreatment with MAG prior to the administration of intratracheal LPS significantly induced a decrease in lung wet weight/dry weight ratio, in total leukocyte number and neutrophil percent in the BALF, and in myeloperoxidase (MPO) activity of lung in dose-dependent manners. At the same time, pretreatment with MAG also significantly improved the super oxide dismutase (SOD) activity and induced the malondialdehyde (MDA) content in the bronchoalveolar lavage fluid (BALF). Importantly, pretreatment with MAG prevented an increase in cyclic adenosine monophosphate-phosphodiesterase (cAMP-PDE) activity of lung in a dose-dependent manner. In addition, it can up-regulate the interleukin-10 (IL-10) level and down-regulate the tumor neurosis factor-α (TNF-α) level in the lung tissue of ALI mice. These results showed that anti-inflammatory effects of MAG against the LPS-induced ALI may be due to its ability of primary inhibition of cAMP-PDE activity, oxidative stress and its regulation of cytokine effects. Thus the results support that use of MAG is beneficial in the treatment of ALI and ARDS.