Melittin analog p5RHH enhances recombinant adeno-associated virus transduction efficiency.

OBJECTIVE: Melittin and its derivative have been developed to support effective gene delivery systems. Their ability to facilitate endosomal release enhances the delivery of nanoparticle-based gene therapy. Nevertheless, its potential application in the context of viral vectors has not received much attention. Therefore, we would like to optimize the rAAV vector by Melittin analog to improve the transduction efficiency of rAAV in liver cancer cells and explore the mechanism of Melittin analog on rAAV. METHODS: Various melittin-derived peptides were inserted into loop VIII of the capsid protein in recombinant adeno-associated virus vectors. These vectors carrying either gfp or fluc genes were subjected to quantitative polymerase chain reaction assays and transduction assays in human embryonic kidney 293 (HEK293T) cells to investigate the efficiency of vector production and gene delivery. In addition, the ability of a specific p5RHH-rAAV vector to deliver genes was examined through in vitro transduction of different cultured cells and in vivo tail vein administration to C57BL/6 mice. Finally, the intricate details of the vector-mediated transduction mechanisms were explored by using pharmacological inhibitors of every stage of the rAAV2 intracellular life cycle. RESULTS: A total of 76 melittin-related peptides were identified from existing literature. Among them, CMA-3, p5RHH and aAR3 were found to significantly inhibit transduction of rAAV2 vector crude lysate. The p5RHH-rAAV2 vectors efficiently transduced not only rAAV-potent cell lines but also cell lines previously considered resistant to rAAV. Mechanistically, bafilomycin A1, a vacuolar endosome acidification inhibitor, completely inhibited the transgene expression mediated by the p5RHH-rAAV2 vectors. Most importantly, p5RHH-rAAV8 vectors also increased hepatic transduction in vivo in C57BL/6 mice. CONCLUSION: The incorporation of melittin analogs into the rAAV capsids results in a significant improvement in rAAV-mediated transgene expression. While further modifications remain an area of interest, our studies have substantially broadened the pharmacological prospects of melittin in the context of viral vector-mediated gene delivery. Please cite this article as: Meng J, He Y, Yang H, Zhou L, Wang S, Feng X, Al-shargi OY, Yu X, Zhu L, Ling, C. Melittin analog p5RHH enhances recombinant adeno-associated virus transduction efficiency. J Integr Med. 2024; 22(1): 72-82.

Alkaline arginine promotes the gentamicin-mediated killing of drug-resistant Salmonella by increasing NADH concentration and proton motive force.

Introduction: Antimicrobial resistance, especially the development of multidrug-resistant strains, is an urgent public health threat. Antibiotic adjuvants have been shown to improve the treatment of resistant bacterial infections. Methods: We verified that exogenous L-arginine promoted the killing effect of gentamicin against Salmonella in vitro and in vivo, and measured intracellular ATP, NADH, and PMF of bacteria. Gene expression was determined using real-time quantitative PCR. Results: This study found that alkaline arginine significantly increased gentamicin, tobramycin, kanamycin, and apramycin-mediated killing of drug-resistant Salmonella, including multidrug-resistant strains. Mechanistic studies showed that exogenous arginine was shown to increase the proton motive force, increasing the uptake of gentamicin and ultimately inducing bacterial cell death. Furthermore, in mouse infection model, arginine effectively improved gentamicin activity against Salmonella typhimurium. Discussion: These findings confirm that arginine is a highly effective and harmless aminoglycoside adjuvant and provide important evidence for its use in combination with antimicrobial agents to treat drug-resistant bacterial infections.

Prediction of phosphorus concentrations in shallow groundwater in intensive agricultural regions based on machine learning.

Excessive accumulation of phosphorus in soil profiles has become the main source of phosphorus in groundwater due to the application of phosphorus fertilizers in intensive agricultural regions (IARs). Elevated phosphorus concentrations in groundwater have become a global phenomenon, which places enormous pressure on the safe use of water resources and the safety of the aquatic environment. Currently, the prediction of pollutant concentrations in groundwater mainly focuses on nitrate nitrogen, while research on phosphorus prediction is limited. Taking the IARs approximately 8 plateau lakes in the Yunnan-Guizhou Plateau as an example, 570 shallow groundwater samples and 28 predictor variables were collected and measured, and a machine learning approach was used to predict phosphorus concentrations in groundwater. The performance of three machine learning algorithms and different sets of variables for predicting phosphorus concentrations in shallow groundwater was evaluated. The results showed that after all variables were introduced into the model, the R2, RMSE and MAE of support vector machine (SVM), random forest (RF) and neural network (NN) were 0.52-0.60, 0.101-0.108 and 0.074-0.081, respectively. Among them, the SVM model had the best prediction effect. The clay content and water-soluble phosphorus in soil and soluble organic carbon in groundwater had a high contribution to the prediction accuracy of the model. The prediction accuracy of the model with reduced number of variables showed that when the number of variables was equal to 6, the RF model had R2, RMSE and MAE values of 0.53, 0.108 and 0.074, respectively, and the number of variables increased again; there were small changes in R2, RMSE and MAE. Compared with the SVM and NN models, the RF model can achieve higher accuracy by inputting fewer variables.

Exogenous D-ribose promotes gentamicin treatment of several drug-resistant Salmonella.

The metabolic microenvironment of bacteria impacts drug efficacy. However, the metabolic mechanisms of drug-resistant Salmonella spp. remain largely unknown. This study characterized the metabolic mechanism of gentamicin-resistant Salmonella Choleraesuis and found that D-ribose increased the gentamicin-mediated killing of this bacteria. Non-targeted metabolomics of homologous gentamicin-susceptible Salmonella Choleraesuis (SCH-S) and gentamicin-resistant S. Choleraesuis (SCH-R) was performed using UHPLC-Q-TOF MS. The metabolic signature of SCH-R included disrupted central carbon metabolism and energy metabolism, along with dysregulated amino acid and nucleotide metabolism, vitamin and cofactor metabolism, and fatty acid synthesis. D-ribose, the most suppressed metabolite in SCH-R, was shown to strengthen gentamicin efficacy against SCH-R and a clinically isolated multidrug-resistant strain. This metabolite had a similar impact on Salmonella. Derby and Salmonella. Typhimurium. D-ribose activates central carbon metabolism including glycolysis, the pentose phosphate pathway (PPP), and the tricarboxylic acid cycle (TCA cycle), increases the abundance of NADH, polarizes the electron transport chain (ETC), and elevates the proton motive force (PMF) of cells, and induces drug uptake and cell death. These findings suggest that central carbon metabolism plays a critical role in the acquisition of gentamicin resistance by Salmonella, and that D-ribose may serve as an antibiotic adjuvant for gentamicin treatment of resistant bacterial infections.

Involvement of the gut-brain axis in vascular depression via tryptophan metabolism: A benefit of short chain fatty acids.

Cerebral hemodynamic dysfunction and hypoperfusion have been found to underlie vascular depression, but whether the gut-brain axis is involved remains unknown. In this study, a rat model of bilateral common carotid artery occlusion (BCCAO) was adopted to mimic chronic cerebral hypoperfusion. A reduced sucrose preference ratio, increased immobility time in the tail suspension test and forced swim test, and compromised gut homeostasis were found. A promoted conversion of tryptophan (Trp) into kynurenine (Kyn) instead of 5-hydroxytryptamine (5-HT) was observed in the hippocampus and gut of BCCAO rats. Meanwhile, 16S ribosomal RNA gene sequencing suggested a compromised profile of the gut SCFA-producing microbiome, with a decreased serum level of SCFAs revealed by targeted metabolomics analysis. With SCFA supplementation, BCCAO rats exhibited ameliorated depressive-like behaviors and improved gut dysbiosis, compared with the salt-matched BCCAO group. Enzyme-linked immunosorbent assays and quantitative RT-PCR suggested that SCFA supplementation suppressed the conversion of Trp to Kyn and rescued the reduction in 5-HT levels in the hippocampus and gut. In addition to inhibiting the upregulation of inflammatory cytokines, SCFA supplementation ameliorated the activated oxidative stress and reduced the number of microglia and the expression of its proinflammatory markers in the hippocampus post BCCAO. In conclusion, our data suggested the participation of the gut-brain axis in vascular depression, shedding light on the neuroprotective potential of treatment with gut-derived SCFAs.

Anti-epileptic effect of 2-deoxy-D-glucose by activation of miR-194/KATP signaling pathway. / 2-脱氧-D-葡萄糖激活miR-194/KATP信号通路发挥抗癫痫作用.

OBJECTIVES: Epilepsy is a syndrome of central nervous system dysfunction caused by many reasons, which is mainly characterized by abnormal discharge of neurons in the brain. Therefore, finding new targets for epilepsy therapy has always been the focus and hotspot in neurological research field. Studies have found that 2-deoxy-D-glucose (2-DG) exerts anti-epileptic effect by up-regulation of KATP channel subunit Kir6.1, Kir6.2 mRNA and protein. By using the database of TargetScan and miRBase to perform complementary pairing analysis on the sequences of miRNA and related target genes, it predicted that miR-194 might be the upstream signaling molecule of KATP channel. This study aims to explore the mechanism by which 2-DG exerts its anti-epileptic effect by regulating KATP channel subunits Kir6.1 and Kir6.2 via miR-194. METHODS: A magnesium-free epilepsy model was established and randomly divided into a control group, an epilepsy group (EP group), an EP+2-DG group, and miR-194 groups (including EP+miR-194 mimic, EP+miR-194 mimic+2-DG, EP+miR-194 mimic control, EP+miR-194 inhibitor, EP+miR-194 inhibitor+2-DG, and EP+miR-194 inhibitor control groups). The 2-DG was used to intervene miR-194 mimics, patch-clamp method was used to detect the spontaneous recurrent epileptiform discharges, real-time PCR was used to detect neuronal miR-194, Kir6.1, and Kir6.2 expressions, and the protein levels of Kir6.1 and Kir6.2were detected by Western blotting. RESULTS: Compared with the control group, there was no significant difference in the amplitude of spontaneous discharge potential in the EP group (P>0.05), but the frequency of spontaneous discharge was increased (P<0.05). Compared with the EP group, the frequency of spontaneous discharge was decreased (P<0.05). Compared with the EP+miR-194 mimic control group, the mRNA and protein expressions of Kir6.1 and Kir6.2 in the EP+miR-194 mimic group were down-regulated (all P<0.05). Compared with the EP+miR-194 inhibitor control group, the mRNA and protein expressions of Kir6.1 and Kir6.2 in the EP+miR-194 inhibitor group were up-regulated (all P<0.05). After pretreatment with miR-194 mimics, the mRNA and protein expression levels of KATP channel subunits Kir6.1 and Kir6.2 were decreased (all P<0.05). Compared with the EP+2-DG group, the mRNA and protein expression levels of Kir6.1 and Kir6.2 in the EP+miR-194 mimic+2-DG group were down-regulated (all P<0.05) and the mRNA and protein expression levels of Kir6.1 and Kir6.2 in the EP+miR-194 inhibitor+2-DG group were up-regulated (all P<0.05). CONCLUSIONS: The 2-DG might play an anti-epilepsy effect by up-regulating KATP channel subunits Kir6.1 and Kir6.2via miR-194.

Evidence construction of Chinese herbal formulae for the treatment of H. pylori positive peptic ulcer: A Bayesian network Meta-analysis.

PURPOSE: Helicobacter pylori (H. pylori) positive peptic ulcer disease (PUD) is a highly prevalent digestive disease with serious consequences of poor prognosis without rational medical intervention. Apparent advantages of Chinese herbal formulae application in PUDs have been appreciated by clinical practitioners recently in China. However, there is no enough viewpoint of evidence-based medicine for them to reach a broad consensus at the more favorable formulae. Therefore, we adopted the method of Bayesian network Meta-analysis (BNMA) in order to get a solution. METHOD: Literature retrieval of clinical randomized controlled trials (RCTs) was conducted with eight databases of PubMed, Embase, Cochrane Library, Web of Science, Chinese National Knowledge Infrastructure, the VIP medicine information system, the Wanfang Data Knowledge Service Platform and Chinese Biomedical Literature Service System. Review Manager 5.4 software, R project 4.1.1 software and STATA16.0 software were used to carry out this BNMA. The primary outcome indicator is H. pylori eradicated rate. The secondary outcome indicator is clinical effectiveness rate, recurrence rate and adverse reaction report. RESULT: A total of fifty-one RCTs involving 5172 patients and ten therapeutic strategies were included in this BNMA. The results showed that supplementation with Chinese herbal formulae significantly improved the eradication rate, clinical efficiency and reduced recurrence rate of H. pylori compared with the single conventional triple therapy (CON) group in a relatively safe manner. Comprehensive therapeutic efficacy analysis of H. pylori eradicated rate and clinical effectiveness rate showed that CON combined with Xiangsha Liujunzi decoction (XSD) manifests at the first grade among these ten therapeutic strategies. CONCLUSION: The efficacy and safety of Chinese herbal formulae and conventional triple therapy were validated by this BNMA. CON+XSD appears to be the most satisfactory therapeutic strategy for H. pylori-positive PUD, reminding clinicians of the potential value of the combination of Chinese herbal formulae and conventional chemotherapy. SYSTEMATIC REVIEW REGISTRATION: https://www.crd.york.ac.uk/PROSPERO/, identifier CRD42021268574.

[Shallow Groundwater Around Plateau Lakes: Spatiotemporal Distribution of Phosphorus and Its Driving Factors].

The extensive application of phosphorus fertilizers to croplands and the aggregation of towns and villages around plateau lakes has resulted in the continuous accumulation of phosphorus in the soil profile and the discharge of phosphorus pollutants, which causes phosphorus pollution in shallow groundwater around the lakes. The phosphorus entering the lake with shallow underground runoff in the region around the lake also affects the water quality safety of plateau lakes. The spatiotemporal differences in phosphorus concentrations in 452 shallow groundwater samples and the driving factors were analyzed by monitoring wells in croplands and residential areas around the eight lakes in Yunnan province during the rainy and dry seasons from 2019 to 2021. The results showed that seasonal changes and land use influenced phosphorus concentrations and their composition in shallow groundwater. The concentration of phosphorus in shallow groundwater in the rainy season was higher than that in the dry season, and it was also greater in cropland than that in residential areas. DTP was the dominant form of TP, accounting for 75%-81%, and DIP was the dominant form of DTP, accounting for 74%-80%. Nearly 30% of the samples around the eight lakes had TP concentrations exceeding the surface water Class Ⅲ standard (GB 3838); the exceeded rates of phosphorus in groundwater around the Erhai Lake (52%), Qiluhu Lake (45%), Xingyun Lake (42%), and Dianchi Lake (29%) were far higher than those of Yangzonghai Lake (16%), Fuxianhu Lake (13%), Chenghai Lake (6%), and Yilonghu Lake (5%). The key driving factors of phosphorus concentrations in shallow groundwater were water-soluble phosphorus (WEP), water content (MWC), soil organic matter (SOM), total nitrogen (TN), pH in the soil profile, and pH and groundwater level in the shallow groundwater (P<0.05). The increases in WEP, SOM, TN, and MWC in the soil and pH in groundwater significantly increased the concentrations of DIP and DTP in shallow groundwater, whereas the decrease in groundwater level significantly reduced the concentrations of DTP and DIP in the groundwater.

Evidence construction of Huangkui capsule against chronic glomerulonephritis: A systematic review and network pharmacology.

BACKGROUND: Chronic glomerulonephritis (CGN) is a relatively common primary glomerular disease. Huangkui capsule (HKC) combined with angiotensin receptor blocker (ARB) for CGN is frequently used in clinical practice, however, there is still lack of high-quality evidence-based evidence and network pharmacology to clarify the therapeutic efficacy and pharmacological mechanisms. PURPOSE: Integrating evidence-based medicine and network pharmacology to explain the therapeutic efficacy and pharmacological mechanisms of ARB combined with HKC for CGN. METHODS: Studies matching the topic were searched from PubMed, Web of Science, Embase database, the Cochrane Library, Chinese National Knowledge Infrastructure, CBM databases, the VIP medicine information system and the Wanfang database and screened according to inclusion and exclusion criteria. The data of the included studies were meta-analyzed by blood urea nitrogen (BUN), serum creatinine (SCR), 24-h urine protein (24hUP) and effective rate (ER). A meta-analysis of the data from the included studies was performed. Then, based on the network pharmacology, the chemical ingredients in HKC and their targets of action, disease targets, common targets and other relevant information were screened, and the key pathways were relevantly annotated based on bioinformatics technology to explore the potential mechanisms of HKC and ARB for CGN. RESULTS: The results showed that SCR index (p < 0.05), 24hUP index (p < 0.001) in the group treated with HKC and ARB were significantly lower than those in the control group. BUN index in the group treated with HKC and VAL were significantly lower than those in the control group (p < 0.001). Effective rate index in the group treated with HKC and ARB was significantly higher than those in the control group (p < 0.001). There was no significant difference in BUN treated with IRB, LOS, and TEL (p = 0.181; p = 0.811; p = 0.067). Based on network pharmacology, the results were as follows: The PPI network indicated that STAT3, AKT1, MAPK1, TP53 and JUN were key target proteins. The results of KEGG analysis suggested that the pharmacological mechanisms were mainly associated with AGE-RAGE signaling pathway in diabetic complications. CONCLUSION: The combination of ARB and HKC can achieve better therapeutic effects in the treatment of CGN, meanwhile, ARB and HKC have a significant improved effectiveness in the treatment of CGN compared with ARB or HKC alone. In addition, HKC and ARB synergistically treated CGN through a multi-pathway network.

The microbiota-gut-brain axis participates in chronic cerebral hypoperfusion by disrupting the metabolism of short-chain fatty acids.

BACKGROUND: Chronic cerebral hypoperfusion (CCH) underlies secondary brain injury following certain metabolic disorders and central nervous system (CNS) diseases. Dysregulation of the microbiota-gut-brain axis can exacerbate various CNS disorders through aberrantly expressed metabolites such as short-chain fatty acids (SCFAs). Yet, its relationship with CCH remains to be demonstrated. And if so, it is of interest to explore whether restoring gut microbiota to maintain SCFA metabolism could protect against CCH. RESULTS: Rats subjected to bilateral common carotid artery occlusion (BCCAO) as a model of CCH exhibited cognitive impairment, depressive-like behaviors, decreased gut motility, and compromised gut barrier functions. The 16S ribosomal RNA gene sequencing revealed an abnormal gut microbiota profile and decreased relative abundance of some representative SCFA producers, with the decreased hippocampal SCFAs as the further evidence. Using fecal microbiota transplantation (FMT), rats recolonized with a balanced gut microbiome acquired a higher level of hippocampal SCFAs, as well as decreased neuroinflammation when exposed to lipopolysaccharide. Healthy FMT promoted gut motility and gut barrier functions, and improved cognitive decline and depressive-like behaviors by inhibiting hippocampal neuronal apoptosis in BCCAO rats. Long-term SCFA supplementation further confirmed its neuroprotective effect in terms of relieving inflammatory response and hippocampal neuronal apoptosis following BCCAO. CONCLUSION: Our results demonstrate that modulating the gut microbiome via FMT can ameliorate BCCAO-induced gut dysbiosis, cognitive decline, and depressive-like behaviors, possibly by enhancing the relative abundance of SCFA-producing floras and subsequently increasing SCFA levels. Video abstract.

Comparative Analysis and Identification of Differentially Expressed microRNAs in the Hypothalamus of Kazakh Sheep Exposed to Different Photoperiod Conditions.

MicroRNAs (miRNA) plays an important role in several mammalian biological regulatory processes by post-transcriptionally regulating gene expression. However, there is little information on the miRNAs involved in the photoperiodism pathway that controls seasonal activity. To enhance our knowledge on the effect of different photoperiod conditions on miRNA, we divided Kazakh sheep into two groups: one exposed to a long photoperiod (LP, 16L:8D) and another with exposed to a short photoperiod (SP, 8L:16D) under supplemental feeding conditions. Further we compared the related miRNAs and target genes between the two groups. Fifteen differentially expressed miRNAs were identified, which were associated with 310 regulatory pathways covering photoperiodism, reproductive hormones, and nutrition. The miR-136-GNAQ pair was selected and validated as a differentially expressed, and a dual-luciferase reporter assay showed that the negative feedback loop existed between them. Examination of the expression profile revealed that the GNAQ expression was low in the estrous females both under LP and SP conditions, but high expression of GNAQ was observed in the anestrous females under LP conditions. Moreover, functional analysis revealed that KISS1 and GnRH expression was upregulated when GNAQ expression was downregulated in the hypothalamic cells, whereas DIO2 and TSHB expression was downregulated. Thus, miR-136-GNAQ might act as a switch in the regulation of seasonal estrus under different photoperiod conditions. These findings further enrich our understanding of the relationship between miRNAs and seasonal regulation of reproductive activity. Furthermore, our study provides novel insights into the miRNA-mediated regulatory mechanisms for overcoming photoinhibition in the seasonally breeding mammals, such as Kazakh sheep.

Three-Dimensional Arterial Pulse Signal Acquisition in Time Domain Using Flexible Pressure-Sensor Dense Arrays.

In this study, we developed a radial artery pulse acquisition system based on finger-worn dense pressure sensor arrays to enable three-dimensional pulse signals acquisition. The finger-worn dense pressure-sensor arrays were fabricated by packaging 18 ultra-small MEMS pressure sensors (0.4 mm × 0.4 mm × 0.2 mm each) with a pitch of 0.65 mm on flexible printed circuit boards. Pulse signals are measured and recorded simultaneously when traditional Chinese medicine practitioners wear the arrays on the fingers while palpating the radial pulse. Given that the pitches are much smaller than the diameter of the human radial artery, three-dimensional pulse envelope images can be measured with the system, as can the width and the dynamic width of the pulse signals. Furthermore, the array has an effective span of 11.6 mm-3-5 times the diameter of the radial artery-which enables easy and accurate positioning of the sensor array on the radial artery. This study also outlines proposed methods for measuring the pulse width and dynamic pulse width. The dynamic pulse widths of three volunteers were measured, and the dynamic pulse width measurements were consistent with those obtained by color Doppler ultrasound. The pulse wave velocity can also be measured with the system by measuring the pulse transit time between the pulse signals at the brachial and radial arteries using the finger-worn sensor arrays.

Transcriptome-based identification and expression characterization of RgABCC transporters in Rehmannia glutinosa.

ABCC multidrug resistance-associated proteins (ABCCs/MRPs), a subfamily of ABC transporters, are involved in multiple physiological processes. Although these proteins have been characterized in some plants, limited efforts have been made to address their possible roles in Rehmannia glutinosa, a medicinal plant. Here, we scanned R. glutinosa transcriptome sequences and identified 18 RgABCC genes by in silico analysis. Sequence alignment revealed that the RgABCCs were closely phylogenetically related and highly conserved with other plant ABCCs/MRPs. Subcellular localization revealed that most of the RgABCCs were deposited in vacuoles and a few in plasma membranes. Tissue-specific expression of the RgABCCs indicated significant specific accumulation patterns, implicating their roles in the respective tissues. Differential temporal expression patterns of the RgABCCs exhibited their potential roles during root development. Various abiotic stress and hormone treatment experiments indicated that some RgABCCs could be transcriptionally regulated in roots. Furthermore, the transcription of several RgABCCs in roots was strongly activated by cadmium (Cd), suggesting possible roles under heavy metal stresses. Functional analysis of RgABCC1 heterologous expression revealed that it may increase the tolerance to Cd in yeast, implying its Cd transport activity. Our study provides a detailed inventory and molecular characterization of the RgABCCs and valuable information for exploring their functions in R. glutinosa.

Integrated miRNA-mRNA analysis reveals the roles of miRNAs in the replanting benefit of Achyranthes bidentata roots.

The yield and quality of the medicinal plant Achyranthes bidentata can be increased when it is replanted into a field cultivated previously with the same crop, however, fundamental aspects of its biology (so-called "replanting benefit") still remain to be elucidated. miRNAs are sRNA molecules involved in the post-transcriptional regulation of gene expression in plant biological processes. Here, 267 conserved and 36 novel miRNAs were identified in A. bidentata roots. We compared the miRNA content of the roots (R1) from first-year planting with that of the roots (R2) of second-year replanting, and screened 21 differentially expressed (DE) miRNAs. Based on in silico functional analysis, integrated miRNA-mRNA datasets allowed the identification of 10 miRNA-target family modules, which might participate in the benefit. The expression profiles of the miRNA-target modules were potentially correlated with the presence of the replanting benefit. The indication was that the miRNA-responsive continuous monoculture could reprogram miRNA-mRNA expression patterns, which possibly promote the root growth and development, enhance its transport activity and strengthen its tolerance to various stresses, thereby improving A. bidentata productivity as observed in the replanting benefit. Our study provides basic data for further research on the molecular mechanisms of the benefit in A. bidentata.

Inhibition by microbial metabolites of Chinese dark tea of age-related neurodegenerative disorders in senescence-accelerated mouse prone 8 (SAMP8) mice.

Dark tea has a significant effect on the prevention and treatment of age-related degenerative diseases. At present, further exploration of its functional mechanisms is delayed because of the complexity of post-fermentation microbial metabolites during the production phase. In this study, new isolated microbial metabolites extracted from dark tea were used to explore the neuroprotective effects, and they also helped allow further exploration of the mechanism of dark tea. Taking senescence-accelerated mouse prone 8 (SAMP8) mice as a biological model, we examined the protective effect on brain neurons of post-fermentation microbial metabolites which were extracted from dark tea. The 4-month-old mice were given treatments of the same concentration (10 mg kg-1 d-1) which were l-theanine, 3,3'-azanediylbis(4-hydroxybenzoic acid) (CDT-1) and one of the 8-C N-ethyl-2-pyrrolidinone substituted flavan-3-ols (CDT-2) by gavage for 14 weeks. Relative measurements such as RT-PCR, ELISA, western blotting, and section staining (HE, Nissl and myelin) were carried out. The results showed that l-theanine, CDT-1 and CDT-2 could inhibit the decrease in body weight, and down-regulate the formation of 4-HNE and ubiquitinated protein aggregates and the Aß metabolic pathway. They could also increase endogenous antioxidant capacity, relieve cell hypoxia, and reduce the rate of neuronal apoptosis. This means that their protective activity regarding SAMP8 neurons was excellent and the activity of CDT-2 was the most significant.

Identification and analysis of microRNAs-mRNAs pairs associated with nutritional status in seasonal sheep.

Given the important role of nutritional status for reproductive performance, we aimed to explore the potential microRNA (miRNA)-mRNA pairs and their regulatory roles associated with nutritional status in seasonal reproducing sheep. Individual ewes were treated with and without 0.3 kg/day concentrates, and the body condition score, estrus rate, and related miRNAs and target genes were compared. A total of 261 differentially expressed miRNAs were identified, including 148 hypothalamus-expressed miRNAs and 113 ovary-expressed miRNAs, and 349 target genes were predicted to be associated with nutritional status and seasonal reproduction in sheep. Ultimately, the miR-200b-GNAQ pair was screened and validated as differentially expressed, and a dual luciferase reporter assay showed that miR-200b could bind to the 3'-untranslated region of GNAQ to mediate the hypothalamic-pituitary-ovarian axis. Thus, miR-200b and its target gene GNAQ likely represent an important negative feedback loop, providing a link between nutritional status and seasonal reproduction in sheep toward enhancing reproductive performance and productivity.

Nutritional status affects the microRNA profile of the hypothalamus of female sheep.

Recent studies on the seasonal regulation of the oestrous cycle in sheep have focussed mainly on the responses to photoperiod. However, the brain systems that control reproductive activity also respond to nutritional inputs, although the molecular mechanisms involved are not completely understood. One possibility is that small, non-coding RNAs, such as micro-RNAs (miRNAs), have significant influence. In the present study, the amounts and characteristics of miRNAs in hypothalamus from oestrous and anestrous ewes, fed low- or high-nutrient diets, were compared using Illumina HiSeq sequencing technology. In total, 398 miRNAs, including 261 novel miRNAs, were identified in ewes with an enhanced nutritional status (HEN), whereas 384 miRNAs, including 247 novel miRNAs, were identified in the ewes with a lesser nutritional status (HAN). There were eight conserved and 140 novel miRNAs expressed differentially between the two libraries. Based on quantitative real-time polymerase chain reaction, six miRNAs were assessed to verify the accuracy of the library database. Moreover, the correlation between the miRNA target and several upstream and downstream genes in the oestrus-related pathways were also verified in hypothalamus nerve cells. According to the results, nutritional status plays an important role in oestrous regulation in sheep, and the hypothalamic processes and pathways induced by nutritional signals (folic acid and tyrosine) are different from those induced by photoperiodic regulation of oestrus. We have expanded the repertoire of sheep miRNAs that could contribute to the molecular mechanisms that regulate the initiation of oestrous cycles in anestrous ewes in response to the influence of nutritional status.

Phenylisotertronic acids from the TCM endophytic fungus Phyllosticta sp.

Four new phenylisotertronic acids (1a/1b, 2a, and 3a) were isolated from a TCM endophytic fungal strain Phyllosticta sp. J13-2-12Y obtained from the leaves of Acorus tatarinowii, along with two known ones (2b and 3b). Compounds 1-3 all existed as mixtures of enantiomers, and their corresponding optically pure enantiomers were successfully isolated by chiral HPLC. The structures of isolated compounds were determined by comprehensive spectroscopic analyses and X-ray diffraction. Their absolute configurations were determined by ECD experiments and quantum chemical calculations. In addition, the antimicrobial activities and the cytotoxicities of these three pairs of optically pure enantiomers (1a/1b, 2a/2b, and 3a/3b) had been evaluated.

Phyllomeroterpenoids A-C, Multi-biosynthetic Pathway Derived Meroterpenoids from the TCM Endophytic Fungus Phyllosticta sp. and their Antimicrobial Activities.

Phyllomeroterpenoids A-C (1-3), multi-biosynthetic pathway derived meroterpenoids from amino acid/pentose phosphate/terpenoid pathways, were isolated from the TCM endophytic fungus Phyllosticta sp. J13-2-12Y, together with six biosynthetically related compounds (4-9). All structures were determined by extensive spectroscopic analysis, chemical derivatization, and ECD experiments. A plausible biosynthetic pathway of 1-3 was proposed. In addition, the antimicrobial activities of all isolated compounds were evaluated against Staphylococcus aureus 209P (bacterium) and Candida albicans FIM709 (fungus).

Effect of Water-Cooled Nd:YAG Laser on Dentinal Tubule Occlusion In Vitro.

OBJECTIVE: The objective of this study was to investigate the effect of a new water-cooled Nd:YAG laser on dentinal tubule occlusion. BACKGROUND DATA: The effect of water-cooled Nd:YAG laser on dentinal tubule occlusion has not been reported. METHODS: Acid-etched dentin samples were randomly divided into three groups: (1) dentin control, (2) dentin treated by Nd:YAG laser, (3) dentin treated by water-cooled Nd:YAG laser. After laser irradiation, half of the samples were immersed in a 6 wt% citric acid (pH 1.5) solution for 1 min to evaluate the acid resistance. The morphologies of dentin surfaces were characterized by scanning electron microscopy. The number and diameters of the open dentinal tubules were analyzed by one-way and two-way analyses of variance. RESULTS: Both the Nd:YAG laser and water-cooled Nd:YAG laser melted the superficial layer of dentin, which caused dentinal tubule occlusion in most areas and diameter reduction of the rest open tubules. Microcracks on the dentin surface were only observed in the Nd:YAG laser group. The tubule occlusion induced by the two lasers showed a good acid resistance. CONCLUSIONS: The effect of water-cooled Nd:YAG laser on dentinal tubule occlusion is similar to that of the Nd:YAG laser. The dentinal tubule occlusion induced by the two lasers could resist acid challenge to some extent.