Single cell landscape of parietal epithelial cells in healthy and diseased states.

The biology and diversity of glomerular parietal epithelial cells (PECs) are important for understanding podocyte regeneration and crescent formation. Although protein markers have revealed the morphological heterogeneity of PECs, the molecular characteristics of PEC subpopulations remain largely unknown. Here, we performed a comprehensive analysis of PECs using single-cell RNA sequencing (scRNA-seq) data. Our analysis identified five distinct PEC subpopulations: PEC-A1, PEC-A2, PEC-A3, PEC-A4 and PEC-B. Among these subpopulations, PEC- A1 and PEC-A2 were characterized as podocyte progenitors while PEC-A4 represented tubular progenitors. Further dynamic signaling network analysis indicated that activation of PEC-A4 and the proliferation of PEC-A3 played pivotal roles in crescent formation. Analyses suggested that upstream signals released by podocytes, immune cells, endothelial cells and mesangial cells serve as pathogenic signals and may be promising intervention targets in crescentic glomerulonephritis. Pharmacological blockade of two such pathogenic signaling targets, proteins Mif and Csf1r, reduced hyperplasia of the PECs and crescent formation in anti-glomerular basement membrane glomerulonephritis murine models. Thus, our study demonstrates that scRNA-seq-based analysis provided valuable insights into the pathology and therapeutic strategies for crescentic glomerulonephritis.

Novel role for epalrestat: protecting against NLRP3 inflammasome-driven NASH by targeting aldose reductase.

BACKGROUND: Nonalcoholic steatohepatitis (NASH) is a progressive and inflammatory subtype of nonalcoholic fatty liver disease (NAFLD) characterized by hepatocellular injury, inflammation, and fibrosis in various stages. More than 20% of patients with NASH will progress to cirrhosis. Currently, there is a lack of clinically effective drugs for treating NASH, as improving liver histology in NASH is difficult to achieve and maintain through weight loss alone. Hence, the present study aimed to investigate potential therapeutic drugs for NASH. METHODS: BMDMs and THP1 cells were used to construct an inflammasome activation model, and then we evaluated the effect of epalrestat on the NLRP3 inflammasome activation. Western blot, real-time qPCR, flow cytometry, and ELISA were used to evaluate the mechanism of epalrestat on NLRP3 inflammasome activation. Next, MCD-induced NASH models were used to evaluate the therapeutic effects of epalrestat in vivo. In addition, to evaluate the safety of epalrestat in vivo, mice were gavaged with epalrestat daily for 14 days. RESULTS: Epalrestat, a clinically effective and safe drug, inhibits NLRP3 inflammasome activation by acting upstream of caspase-1 and inducing ASC oligomerization. Importantly, epalrestat exerts its inhibitory effect on NLRP3 inflammasome activation by inhibiting the activation of aldose reductase. Further investigation revealed that the administration of epalrestat inhibited NLRP3 inflammasome activation in vivo, alleviating liver inflammation and improving NASH pathology. CONCLUSIONS: Our study indicated that epalrestat, an aldose reductase inhibitor, effectively suppressed NLRP3 inflammasome activation in vivo and in vitro and might be a new therapeutic approach for NASH.

Bergapten inhibits NLRP3 inflammasome activation and pyroptosis via promoting mitophagy.

Inhibition of NLRP3 inflammasome activation produces potent therapeutic effects in a wide array of inflammatory diseases. Bergapten (BeG), a furocoumarin phytohormone present in many herbal medicines and fruits, exibits anti-inflammatory activity. In this study we characterized the therapeutic potential of BeG against bacterial infection and inflammation-related disorders, and elucidated the underlying mechanisms. We showed that pre-treatment with BeG (20 µM) effectively inhibited NLRP3 inflammasome activation in both lipopolysaccharides (LPS)-primed J774A.1 cells and bone marrow-derived macrophages (BMDMs), evidenced by attenuated cleaved caspase-1 and mature IL-1ß release, as well as reduced ASC speck formation and subsequent gasdermin D (GSDMD)-mediated pyroptosis. Transcriptome analysis revealed that BeG regulated the expression of genes involved in mitochondrial and reactive oxygen species (ROS) metabolism in BMDMs. Moreover, BeG treatment reversed the diminished mitochondrial activity and ROS production after NLRP3 activation, and elevated the expression of LC3-II and enhanced the co-localization of LC3 with mitochondria. Treatment with 3-methyladenine (3-MA, 5 mM) reversed the inhibitory effects of BeG on IL-1ß, cleaved caspase-1 and LDH release, GSDMD-N formation as well as ROS production. In mouse model of Escherichia coli-induced sepsis and mouse model of Citrobacter rodentium-induced intestinal inflammation, pre-treatment with BeG (50 mg/kg) significantly ameliorated tissue inflammation and injury. In conclusion, BeG inhibits NLRP3 inflammasome activation and pyroptosis by promoting mitophagy and maintaining mitochondrial homeostasis. These results suggest BeG as a promising drug candidate for the treatment of bacterial infection and inflammation-related disorders.

Scoparone suppresses mitophagy-mediated NLRP3 inflammasome activation in inflammatory diseases.

Recent evidence shows that targeting NLRP3 inflammasome activation is an important means to treat inflammasome-driven diseases. Scoparone, a natural compound isolated from the Chinese herb Artemisia capillaris Thunb, has anti-inflammatory activity. In this study we investigated the effect of scoparone on NLRP3 inflammasome activation in inflammatory diseases. In LPS-primed, ATP or nigericin-stimulated mouse macrophage J774A.1 cells and bone marrow-derived macrophages (BMDMs), pretreatment with scoparone (50 µM) markedly restrained canonical and noncanonical NLRP3 inflammasome activation, evidenced by suppressed caspase-1 cleavage, GSDMD-mediated pyroptosis, mature IL-1ß secretion and the formation of ASC specks. We then conducted a transcriptome analysis in scoparone-pretreated BMDMs, and found that the differentially expressed genes were significantly enriched in mitochondrial reactive oxygen species (ROS) metabolic process, mitochondrial translation and assembly process, as well as in inflammatory response. We demonstrated in J774A.1 cells and BMDMs that scoparone promoted mitophagy, a well-characterized mechanism to control mitochondrial quality and reduce ROS production and subsequent NLRP3 inflammasome activation. Mitophagy blockade by 3-methyladenine (3-MA, 5 mM) reversed the protective effects of scoparone on mitochondrial damage and inflammation in the murine macrophages. Moreover, administration of scoparone (50 mg/kg) exerted significant preventive effects via inhibition of NLRP3 activation in mouse models of bacterial enteritis and septic shock. Collectively, scoparone displays potent anti-inflammatory effects via blocking NLRP3 inflammasome activation through enhancing mitophagy, highlighting a potential action mechanism in treating inflammasome-related diseases for further clinical investigation.

Capsaicin functions as a selective degrader of STAT3 to enhance host resistance to viral infection.

Although STAT3 has been reported as a negative regulator of type I interferon (IFN) signaling, the effects of pharmacologically inhibiting STAT3 on innate antiviral immunity are not well known. Capsaicin, approved for the treatment of postherpetic neuralgia and diabetic peripheral nerve pain, is an agonist of transient receptor potential vanilloid subtype 1 (TRPV1), with additional recognized potencies in anticancer, anti-inflammatory, and metabolic diseases. We investigated the effects of capsaicin on viral replication and innate antiviral immune response and discovered that capsaicin dose-dependently inhibited the replication of VSV, EMCV, and H1N1. In VSV-infected mice, pretreatment with capsaicin improved the survival rate and suppressed inflammatory responses accompanied by attenuated VSV replication in the liver, lung, and spleen. The inhibition of viral replication by capsaicin was independent of TRPV1 and occurred mainly at postviral entry steps. We further revealed that capsaicin directly bound to STAT3 protein and selectively promoted its lysosomal degradation. As a result, the negative regulation of STAT3 on the type I IFN response was attenuated, and host resistance to viral infection was enhanced. Our results suggest that capsaicin is a promising small-molecule drug candidate, and offer a feasible pharmacological strategy for strengthening host resistance to viral infection.

Single-cell RNA sequencing data locate ALDH1A2-mediated retinoic acid synthetic pathway to glomerular parietal epithelial cells.

Aldehyde dehydrogenase 1, family member A2, is a retinoic acid-synthesizing enzyme encoded by Aldh1a2 in mice and ALDH1A2 in humans. This enzyme is indispensable for kidney development, but its role in kidney physiology and pathophysiology remains to be fully defined. In this review, we mined single-cell and single-nucleus RNA sequencing databases of mouse and human kidneys and found that glomerular parietal epithelial cells (PECs) express a full set of genes encoding proteins needed for cellular vitamin A uptake, intracellular transport, and metabolism into retinoic acid. In particular, Aldh1a2/ALDH1A2 mRNAs are selectively enriched in mouse and human PECs. Aldh1a2 expression in PECs is greatly increased in a mouse model of anti-glomerular basement membrane glomerulonephritis and moderately induced in a mouse model of ischemia-reperfusion acute kidney injury. Aldh1a2 expression in PECs is substantially repressed in a chronic kidney disease mouse model combining diabetes, hypertension, and partial nephrectomy and is moderately repressed in mouse models of focal segmental glomerulosclerosis and diabetic nephropathy. Single-nucleus RNA sequencing data show that ALDH1A2 mRNA expression in PECs is diminished in patients with chronic kidney disease associated with diabetes, hypertension and polycystic kidney disease. In addition to data mining, we also performed Spearman's rank correlation coefficient analyses and identified gene transcripts correlated with Aldh1a2/ALDH1A2 transcripts in mouse PECs and PEC subtypes, and in human PECs of healthy subjects and patients with AKI or CKD. Furthermore, we conducted Gene Ontology pathway analyses and identified the biological pathways enriched among these Aldh1a2/ALDH1A2-correlated genes. Our data mining and analyses led us to hypothesize that ALDH1A2-mediated retinoic acid synthesis in PECs plays a yet-undefined role in the kidney and that its dysregulation mediates injury. Conditional, PEC-selective Aldh1a2 knockout, RNA silencing and transgenic mouse models will be useful tools to test this hypothesis. Clinical studies on genetics, epigenetics, expression and functions of ALDH1A2 and other genes needed for retinoic acid biosynthesis and signaling are also warranted.

Optimal transplantation strategy using human induced pluripotent stem cell-derived cardiomyocytes for acute myocardial infarction in nonhuman primates.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) have the potential to be a therapeutic option for myocardium restoration. However, hiPSC-CMs of varying maturation and transplantation routes exhibit different reactivity and therapeutic effects. We previously demonstrated that the saponin+ compound induces more mature hiPSC-CMs. The safety and efficacy of multi-route transplantation of saponin+ compound-induced hiPSC-CMs in a nonhuman primate with myocardial infarction will be investigated for the first time in this study. Our findings indicate that optimized hiPSC-CMs transplanted via intramyocardial and intravenous routes may affect myocardial functions by homing or mitochondrial transfer to the damaged myocardium to play a direct therapeutic role as well as indirect beneficial roles via anti-apoptotic and pro-angiogenesis mechanisms mediated by different paracrine growth factors. Due to significant mural thrombosis, higher mortality, and unilateral renal shrinkage, intracoronary transplantation of hiPSC-CMs requires closer attention to anticoagulation and caution in clinical use. Collectively, our data strongly indicated that intramyocardial transplantation of hiPSC-CMs is the ideal technique for clinical application; multiple cell transfers are recommended to achieve steady and protracted efficacy because intravenous transplantation's potency fluctuates. Thus, our study offers a rationale for choosing a therapeutic cell therapy and the best transplantation strategy for optimally induced hiPSC-CMs.

Can eastern wisdom resolve western epidemics? Traditional Chinese medicine therapies and the opioid crisis.

The widespread use of opioids to treat chronic pain led to a nation-wide crisis in the United States. Tens of thousands of deaths annually occur mainly due to respiratory depression, the most dangerous side effect of opioids. Non-opioid drugs and non-pharmacological treatments without addictive potential are urgently required. Traditional Chinese medicine (TCM) is based on a completely different medical theory than academic Western medicine. The scientific basis of acupuncture and herbal treatments as main TCM practices has been considerably improved during the past two decades, and large meta-analyses with thousands of patients provide evidence for their efficacy. Furthermore, opinion leaders in the United States favor non-pharmacological techniques including TCM for pain management to fight the opioid crisis. We advocate TCM as therapeutic option without addictive potential and without life-threatening side effects (e.g., respiratory depression) to treat chronic pain patients suffering from opioid misuse. The evidence suggests that: (1) opioid misuse cannot be satisfactorily managed with standard medication; (2) opinion leaders in the United States favor to consider non-opioid and non-pharmacological treatment strategies including those from TCM to treat acute and chronic pain conditions; (3) large meta-analyses provide scientific evidence for the clinical activity of acupuncture and herbal TCM remedies in the treatment of chronic pain. Future clinical trials should demonstrate the safety of TCM treatments if combined with Western medical practices to exclude negative interactions between both modalities.

Tracing the origins of Hakka and Chaoshanese by mitochondrial DNA analysis.

Hakka and Chaoshanese are two unique Han populations residing in southern China but with northern Han (NH) cultural traditions and linguistic influences. Although most of historical records indicate that both populations migrated from northern China in the last two thousand years, no consensus on their origins has been reached so far. To shed more light on the origins of Hakka and Chaoshanese, mitochondrial DNAs (mtDNAs) of 170 Hakka from Meizhou and 102 Chaoshanese from Chaoshan area, Guangdong Province, were analyzed. Our results show that some southern Chinese predominant haplogroups, e.g. B, F, and M7, have relatively high frequencies in both populations. Although median network analyses show that Hakka/Chaoshanese share some haplotypes with NH, interpopulation comparison reveals that both populations show closer affinity with southern Han (SH) populations than with NH. In consideration of previous results from nuclear gene (including Y chromosome) research, it is likely that matrilineal landscapes of both Hakka and Chaoshanese have largely been shaped by the local people during their migration southward and/or later colonization in southern China, and factors such as cultural assimilation, patrilocality, and even sex-bias in the immigrants might have played important roles during the process.

[Construction of genome BAC library for single Branchiostoma belcheri individual].

As one of the closest living invertebrate relatives of vertebrates, amphioxus (subphylum Cephalochordata) occupies a key position in animal evolution and is becoming the best available proxy and model animal for studying the last common ancestor of all chordates, especially vertebrates. As long-term continuous culturing of amphioxus in laboratory became reliable, for pushing this animal to be a more successful model system, whole-genome sequencing of one or more species derived from this branch will be another urgent issue needed to address. In the present study, we described the construction and characterization of a bacterial artificial chromosome (BAC) library, using a single individual of Chinese amphioxus (Branchiostoma belcheri). High quality genomic DNA extracted from the spermary was partially digested with EcoRand EcoRmethylase. Desirable DNA fragments were isolated by pulsed field gel electrophoresis (PFGE), ligated to linearized and phosphorylased carrier pCC1BAC, and then transformed to Escherichia coli EPI300. The constructed library consists of 44 706 clones with the average insert fragment size around 80 kb as estimated by PFGE. The representation of the library is about 9 equivalents to the amhioxus genome. These results indicate that the BAC library will be useful for functional genomic studies and facilitate the whole-genome sequencing of Chinese amphioxus.

In vitro screening and toxic mechanism exploring of leading components with potential hepatotoxicity of Herba Epimedii extracts.

Herba Epimedii is a famous Chinese edible herb, and due to its potential hepatotoxic effects, the safety associated with this herb has attracted a great deal of attention. In this study, the components of four types of the Herba Epimedii extracts were identified by HPLC-MS/MS. Among these components, 11 components that were present in all four extracts and could be obtained as reference substances were evaluated for their ability of cytotoxicity in HL-7702 and HepG2 cells, resulting in the identification of icarisid I and sagittatoside A as the most relevant with respect to the toxicity of the extracts. The targeted toxicological effects were further investigated using a series of correlated biological indicators to elucidate potentially hepatotoxic mechanisms. The results showed that the extracts and the selected compounds had varying degrees of influence on the leakage of ALT, AST and LDH; the activity of SOD, GSH and MDA; the increase in intercellular ROS; and the decrease in MMP. Among the tested substances, the ethanol extracts exhibited stronger hepatotoxicity, with icarisid I and sagittatoside A correlating with this toxic effect, and the hepatoxic mechanisms of which may be associated with damaged cell structure, increased oxidative stress and induction of apoptosis.

[Flavonoids from Diospyros kaki inhibit the adhesion between lymphocyte and dorsal root ganglion].

OBJECTIVE: To investigate the effects of flavonoids from the leaves of Diospyros kaki L (FLDK) on the adhesion between the lymphocyte and the neurone. METHODS: Centrifugal assay for fluorescence-bsaed cell adhesion was used to assay the adhesion between the lymphocyte and the dorsal root ganglion (DRG). RESULTS: The adhesion was significantly suppressed in the presence of FLDK dose-dependently at 5, 25 microg/mL concentration. FLDK was also effective to inhibit the adhesion under the challenge of ICAM-1 by 28.5% and 50.1%, respectively. Furthermore, FLDK enforced the inhibition of anti-NCAM antibody on the lymphocyte adhesion to DRG cells. CONCLUSION: FLDK might contribute to the prevention and treatment of the inflammation injury under neuron insult such as ischemia/reperfusion, neurotrauma and other neurodegenerative disease by inhibiting the adhesion between lymphocytes and neurons.

Combining the wisdoms of traditional medicine with cutting-edge science and technology at the forefront of medical sciences.

BACKGROUND: A central topic is to bring traditional medicine to a new horizon by integrating the latest advances in genomic, metabolomic, and system biological approaches, in order to re-examine the wisdom and knowledge of traditional Chinese medicine (TCM) and other traditional medicines. PURPOSE: A new consortium has been formed at a conference of the Harvard Medical School, Boston, on October 29-30, 2018. The main goal was to build a collaborative platform for the scientific investigation of traditional medicine with cutting edge sciences and technologies at the forefront of biomedicine. RESULTS: Traditional medicines are largely experience-based, but the scientific basis is largely non-satisfactory. Therefore, the transformation from experience-based to evidence-based medicine would be an important step forward. The consortium covers three main fields: TCM diagnostics, acupuncture and TCM pharmacology. Diseases occur because of regulatory imbalances of holistic physiological display and genetic information/expression related to systems biology and energy consumption/release (e.g. cold and hot) within body. As organs are interconnected by meridians, affecting the meridians by acupuncture and medicinal herbs restores healthy organ function and body balance. There are two concepts in herbal medicine: The traditional way is based on complex herbal mixtures. The second concept is related to Western pharmacological drug development including the isolation of bioactive phytochemicals, which are subjected to preclinical and clinical investigations. CONCLUSION: Development of collaborative scientific project to integrate the best of both worlds - Western and Eastern medicine into a "One World Integrative Medicine" for the sake of patients worldwide.

Recombinant interferon-alpha2b poly(lactic-co-glycolic acid) microspheres: pharmacokinetics-pharmacodynamics study in rhesus monkeys following intramuscular administration.

AIM: Investigation into pharmacokinetic-pharmacodynamic properties of interferon- alpha (IFN-alpha)2b-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres (MS) in rhesus monkey primates. METHOD: IFN-alpha2b was loaded with biodegradable PLGA with 3 inherent viscosities using a double emulsion and solvent evaporation method. The particle size, surface morphology, and in vitro release profiles were investigated. Two groups of rhesus monkeys (n=3) were injected intramuscularly with either 3 MIU/kg commercial IFN-alpha2b lyophilized powder or IFN-alpha2b-loaded PLGA microspheres (inherent viscosity of 0.89 dL/g). In vitro release was determined by Lowry protein assay. The serum IFN and neopterin levels were determined by the enzyme-linked immunosorbent assay (ELISA) method to evaluate biological activity of the microspheres in rhesus monkeys. RESULTS: The IFN-alpha2b microspheres with 3 inherent viscosities (0.39, 0.89, and 1.13 dL/g) were entirely spherical and had a smooth surface. The average diameter of each type was 45.55, 81.23, and 110.25 microm, respectively. The in vitro release was 30 d. The pharmacokinetic-pharmacodynamic properties between the IFN-alpha2b microspheres and IFN-alpha2b lyophilized powder were significantly different (P<0.05). CONCLUSION: The drug residence time for the IFN-alpha2b of the PLGA microsphere with an inherent viscosity of 0.89 dL/g in plasma significantly increased and had a longer time of biological effects in rhesus monkeys following intramuscular administration.

Reversal of resistance to oxazaphosphorines.

The oxazaphosphorines including cyclophosphamide (CPA), ifosfamide (IFO) and trofosfamide are one important group of alkylating agents. However, resistance is the major hindrance for success of oxazaphosphorine chemotherapy. The mechanism of resistance to oxazaphosphorines is not fully identified, but recently some novel insights into these aspects have been generated by using sensitive analytical techniques and powerful pharmacogenetic techniques. Potential mechanisms for oxazaphosphorine resistance include decreased activation by cytochrome P450s (e.g. CYP3A4, CYP2C9 and CYP2B6), increased deactivation of the agents by deactivating enzymes such as aldehyde dehydrogenases (ALDHs), increased cellular thiol level, increased DNA repair capacity, and altered cellular apoptotic response to DNA repair, e.g. deficient apoptosis due to lack of cellular mechanisms to result in cell death following DNA damage. In addition, decreased cellular accumulation of cytotoxic species of oxazaphosphorines may also play a role in the resistance. This review highlights the pharmacology of oxazaphosphorine anticancer drugs and possible agents that reverse the resistance to these agents. Possible agents that can overcome oxazaphosphorine resistance include inducers of CYPs, modulators of GSTs and ALDHs, modulators of DNA repair process, antisense oligonucleotides against genes encoding various enzymes and signalling proteins, and novel gene delivery systems. Most of these agents have been investigated in preclinical studies and promising results have been observed. To date, several types of these agents are being evaluated in Phase III trials in cancer patients. Further studies are needed to identify the molecular targets associated with resistance to oxazaphosphorines.

Analysis of expressed sequence tags from the venom ducts of Conus striatus: focusing on the expression profile of conotoxins.

Cone snails (genus Conus) are predatory marine gastropods that use venom peptides for interacting with prey, predators and competitors. A majority of these peptides, generally known as conotoxins demonstrate striking selectivity in targeting specific subtypes of ion channels and neurotransmitter receptors. So they are not only useful tools in neuroscience to characterize receptors and receptor subtypes, but offer great potential in new drug research and development as well. Here, a cDNA library from the venom ducts of a fish-hunting cone snail species, Conus striatus is described for the generation of expressed sequence tags (ESTs). A total of 429 ESTs were grouped into 137 clusters or singletons. Among these sequences, 221 were toxin sequences, accounting for 52.1% (corresponding to 19 clusters) of all transcripts. A-superfamily (132 ESTs) and O-superfamily conotoxins (80 ESTs) constitute the predominant toxin components. Some non-disulfide-rich Conus peptides were also found. The expression profile of conotoxins also explained to some extent the pharmacological and physiological reactions elicited by this typical piscivorous species. For the first time, a nonstop transcript of conotoxin was identified, which is suggestive that alternative polyadenylation may be a means of post-transcriptional regulation of conotoxin production. A comparison analysis of these conotoxins reveals the different variation and divergence patterns in these two superfamilies. Our investigations indicate that focal hyper-mutation, block substitution and exon shuffling are three main mechanisms leading to the conotoxin diversity in a species. The comprehensive set of Conus gene sequences allowed the identification of the representative classes of conotoxins and related components, which may lay the foundation for further research and development of conotoxins.

[Effects of a novel recombinant polypeptide from the sea anemone Anthopleura sp. on left ventricular function in dogs with acute cardiac insufficiency].

OBJECTIVE: To observe the effects of recombinant hk2a, a novel neurotoxin from the sea anemone Anthopleura sp., on left ventricular function of dogs with acute cardiac insufficiency. METHODS: Canine models of acute cardiac insufficiency were established by rapid ventricular pacing, in which the left ventricular ejection fraction (LVEF) was measured by Acuson ultrasound systems (Sequoia 512) at 0, 5, 15, 30 and 60 min, respectively, after intravenous injection of 30 microg/kg recombinant hk2a. The response of the canine models to hk2a treatments was observed in comparison with that of the dogs treated with Cedilanid (as positive control) and saline (as negative control). RESULTS: Intravenous injection of recombinant hk2a caused an immediate and significant increase in LVEF in the canine models of acute cardiac insufficiency (P<0.05), and the effect maintained for more than 30 min without significant effect on heart rate. Recombinant hk2a possessed such merits as quicker onset and greater potency in comparison with Cedilanid. CONCLUSION: Recombinant hk2a may significantly increase LVEF of the dogs with acute cardiac insufficiency.

[Effect of lat disruption on clavulanic acid production].

A 1.8kb fragment of lat was obtained from Streptomyces clavuligerus 27064, and replacement plasmid of pXAL1 and pXAL2 were constructed. PXAL1 and pXAL2 were used to disrupt the lat gene by bi-parental conjugation from E. coli to Streptomyces clavuligerus. A Am(r)Thio(S) transformant, named as XAL863, was obtained. The genome of Streptomyces clavuligerus 27064 and XAL863 was analyzed by southern blot technique, and the activity of lysine epsilon-aminotransferase in the two strains was also tested. Both results proved that the lat was disrupted in the XAL863. Streptomyces clavuligerus and XAL863 were cultured in the shaken flask respectively, and the production of clavulanic acid was analyzed by HPLC with the different incubation time interval, and the yield was approximately 1.8 times higher in the XAL863 at their highest production point.

Molecular analyses of HLA-DRB1, -DPB1, and -DQB1 in Jing ethnic minority of Southwest China.

In the present study, DNA typing for HLA-DRB1, DQB1 and DPB1 was performed using polymerase chain reaction-sequencing based typing (PCR-SBT) method in 144 random selected Jing ethnic individuals inhabiting in South China. Allele frequencies and two-locus haplotypes (DRB1-DQB1) were statistically analyzed and 20 DPB1 alleles, 27 DRB1 and 20 DQB1 were detected. The most frequent DPB1 allele was DPB1*0501 with the percentage of 36.9% followed by DPB1*1301 (15.7%), DPB1*0401 (11.0%) and DPB1*020102 (9.8%). Among the 27 detected DRB1 alleles, DRB1*120201 (13.8%) was most commonly observed followed by DRB1*150201, *030101 and *090102 alleles with the frequencies of 9.4%, 9.1% and 8.3%, respectively. Among the 20 detected DQB1 alleles the most predominant one was DQB1*030101/0309 (19.9%). DQB1*050201 (19.1%), DQB1*0201/0202 (16.1%) and DQB1*050101 (12.3%) were also frequently observed in Jing population. Statistical analysis of two-locus haplotypes showed that DRB1*120201-DQB1*030101/DRB1*120201-DQB1*0309 (HF = 9.4%, D = 6.65x10(-2)) was most predominant followed by DRB1*030101-DQB1*0201/DRB1*030101-DQB1*0202 (HF = 8.1%, D = 6.66 x 10(-2)). The comparison of HLA class II allele and haplotype frequencies in Jing with those in other populations all over the world and a dendrogram based on the DRB1, DQB1 and DPB1 genes suggested that Jing ethnic population has an origin of Southeast Asia and is belonged to the southern group of Chinese populations.

Molecular cloning, expression and characterization of three short chain alpha-neurotoxins from the venom of sea snake--Hydrophiinae Hydrophis cyanocinctus Daudin.

Three different genes named sn311, sn316 and sn285 were discovered by large-scale randomly sequencing the high quality cDNA library of the venom glands from Hydrophiinae Hydrophis cyanocinctus Daudin. Sequence analysis showed that these three genes encoded three different short chain alpha-neurotoxins of 81 amino acids, which contained a signal peptide of 21 amino acids and followed by a mature peptide of 60 amino acids. Amino acid comparison reveals that mature peptides of sn311 and sn316 are highly homologous, with the only variance at position 46, which is Lys46 and Ser46, respectively. Whereas the mature peptide of sn285 lacks the most conserved amino acids in short chain alpha-neurotoxins, Asp31 and Arg33. The coding sequences of three neurotoxins were cloned into a thioredoxin (TRX) fusion expression vector (pTRX) and expressed as soluble recombinant fusion proteins in E. coli. After purification, approximately 10 mg/l recombinant proteins with the purity up to 95% were obtained. These three recombinant proteins are designated as rSN311, rSN316 and rSN285, they have a molecular weight of 6.963, 6.920 and 6.756 kDa, respectively, which are similar to those predicted from amino acid sequences. LD50 values of rSN311, rSN316 and rSN285 are 0.0827, 0.095, and 0.0647 mg/kg to mice, respectively. Studies on effects of these recombinant proteins on neuromuscular transmission were carried out, and results indicate that they all can produce prompt blockade of neuromuscular transmission, but display distinct biological activity characteristic individually. The results from UV-circular dichroism (CD) spectra indicate that they share similar secondary structure compared to other identified alpha-neurotoxins, and no significant structural differences in these recombinant proteins are observed.