Diversification of freshwater crabs on the sky islands in the Hengduan Mountains Region, China.

The numerous naturally-fragmented sky islands (SIs) in the Hengduan Mountains Region (HMR) of southwestern China constitute discontinuous landscapes where montane habitats are isolated by dry-hot valleys which have fostered exceptional species diversification and endemicity. However, studies documenting the crucial role of SI on the speciation dynamics of native freshwater organisms are scarce. Here we used a novel set of comprehensive genetic markers (24 nuclear DNA sequences and complete mitogenomes), morphological characters, and biogeographical information to reveal the evolutionary history and speciation mechanisms of a group of small-bodied montane potamids in the genus Tenuipotamon. Our results provide a robustly supported phylogeny, and suggest that the vicariance events of these montane crabs correlate well with the emergence of SIs due to the uplift of the HMR during the Late Oligocene. Furthermore, ancestrally, mountain ridges provided corridors for the dispersal of these montane crabs that led to the colonization of moist montane-specific habitats, aided by past climatic conditions that were the crucial determinants of their evolutionary history. The present results illustrated that the mechanisms isolating SIs are reinforced by the harsh-dry isolating climatic features of dry-hot valleys separating SIs and continue to affect local diversification. This offers insights into the causes of the high biodiversity and endemism shown by the freshwater crabs of the HMR-SIs in southwestern China.

Role of KOH-activated biochar on promoting anaerobic digestion of biomass from Pennisetumgianteum.

Pennisetum giganteum is a promising non-food crop feedstock for biogas production due to its high productivity and bio-methane potential. However, the accumulation of volatile fatty acids (VFA) usually restricts the conversion efficiency of P. giganteum biomass (PGB) during anaerobic digestion (AD). Here, the role of KOH-activated biochar (KB) in improving the AD efficiency of PGB and the related mechanisms were investigated in detail. The results revealed that KB exhibited excellent electrical conductivity, electron transfer capacity and specific capacitance, which might be related to the decrease in the electron transfer resistance after adding KB to the AD process. In addition, the KB addition not only reinforced metabolisms of energy and VFAs but also promoted the conversion of VFAs to methane, leading to a 52% increase in the methane production rate. Bioinformatics analysis showed that Smithella and Methanosaeta were key players in the KB-mediated AD process of PGB. The stimulatory effect of methanogenesis probably resulted from the establishment of direct interspecies electron transfer (DIET) between VFA-oxidizing acetogens (e.g., Smithella) and Methanosaeta. These findings provided a key step to improve the PGB-based AD process.

Description of a New Species of Hua (Gastropoda: Semisulcospiridae) from Guizhou, China, Based on Morphology and Molecular Evidence.

A new species of Hua, Hua qiannanensis sp. nov., is described from Guizhou Province, China, based on morphological and molecular evidence. The new species can be distinguished from its congeners by the following combination of characters: the smooth shell, only three smaller cusps of lateral teeth on the inner side, outer marginal teeth with eight flattened and rounded denticles, an ovipositor pore in females, and BW/H ≥ 80%, B/H = 76.8-82.3%. Molecular analysis based on partial mitochondrial COI and 16S rDNA also supports the systematic position of the new taxon.

Low expression of glucocorticoid receptor a in oral lichen planus correlates with activation of nuclear factor κB: a preliminary study.

BACKGROUND: Emerging evidence indicates that the interaction between glucocorticoid receptor α (GRα) and nuclear factor κB (NF-κB) is a key pathogenetic cross talk in the autoimmune and inflammatory disorders. The objective of this study was to determine the GRα expression in patients with oral lichen planus (OLP) and investigate its correlation with NF-κB in OLP. METHODS: We compared the expression of GRα and NF-κB in oral biopsy specimens from patients with OLP(n = 32) against normal controls (n = 12) and investigated the correlation between the expression of GRα and NF-κB in OLP. RESULTS: Immunohistochemistry showed that GRα mainly expressed in the cytoplasm of keratinocytes of basal and spinosum layer of OLP. Both real-time quantitative PCR and Western blots revealed that the mRNA and protein expression levels of GRα were decreased compared with normal controls (both P < 0.001). Conversely, those levels of nuclear factor-kappa B (NF-κB) were increased compared with normal controls (both P < 0.001). Importantly, a significant inverse correlation between the GRα and NF-κB was found (P < 0.05). CONCLUSIONS: Our findings demonstrated that low expression of GRα in OLP correlates with activation of NF-κB, which indicates that the cross talk between GRα and NF-κB in OLP may become a new therapeutic target and represent a new approach to explore the pathogenesis of OLP.

In vitro culture system for keratinocytes obtained from oral lichen planus lesions.

OBJECTIVES: The aim of this study was to establish a stable in vitro culture system for keratinocytes obtained from oral lichen planus (OLP) lesions and evaluate cultured keratinocyte characteristics including cell morphology, ultrastructure, and expression of biomarkers. MATERIALS AND METHODS: OLP mucosa (histopathologically confirmed) was collected and cells isolated using the cold enzyme digestion method. Primary culture and serial passage were performed on serum-free keratinocyte medium. Morphological changes of cells were evaluated via inverted phase contrast microscopy, and cellular ultrastructure was observed by electron microscopy. Indirect immunofluorescence was used to detect expression of keratin and nuclear factor-kappaB (NF-κB). RESULTS: OLP type I keratinocytes was successfully cultured in vitro in serum-free medium. Cellular morphology was typically polygonal during the growth phase. Cells could be passaged continuously for five to six generations without losing viability. Transmission electron microscopy showed large nuclei and multiple vacuoles in the cultured cells consistent with histopathological features of OLP keratinocytes. Indirect immunofluorescence staining was positive for keratin and NF-κB. CONCLUSIONS: This study established that human OLP kera-tinocytes can be successfully cultured cells with histopathologic features and biomarker expression consistent with OLP type I keratinocytes. CLINICAL RELEVANCE: This culture system lays a foundation for the establishment of human OLP cell model in vitro.

[Codon optimization of recombinant staphylococcal enterotoxin O enhances the expression level in Escherichia coli].

OBJECTIVE: To enhance the expression level of staphylococcal enterotoxin O (SEO) by optimization of rare codons. METHODS: The gene of mature SEO (His-tag included) was cloned to pET28a, and 15 rare codons on the gene were optimized by PCR technology. These recombinant plasmids then were transformed into E.coli BL21(DE3), respectively. After IPTG induced, the expression levels of those mutants were analyzed by SDS-PAGE. The proteins were purified and their bioactivities were determined. RESULT: After the optimization of rare codons, the expression levels were increased from 7.49% to 19.8% in total cell proteins. The optimized SEO had bioactivity to stimulate the proliferation of murine lymphocytes, which was equivalent to that of non-optimized SEO in vitro. CONCLUSION: Optimization of rare codons can enhance the expression of SEO effectively.

A new genus and two new species of the subfamily Pauropodinae (Myriapoda: Pauropoda: Pauropodidae) from China.

A new genus Songius is established and two new species--Songius rugosus from Qixia Mountain and Laoshan Forest Park, Jiangsu, and Tiantangzhai, Dabie Mountain, Anhui, and Songius bicruris from Tiantangzhai--are described. A novel surface structure of the pygidial tergum was observed by scanning electron microscopy. The genus is established on the basis of the distinctive appearance of the modification of the surface structure of the pygidial tergum.

[Effect of different fluence rates applied in curcumin-photodynamic therapy on Candida albicans biofilms].

PURPOSE: In this study, the inactivation effect of different fluence rates on Candida albicans biofilms during curcumin-photodynamic therapy was investigated in vitro. METHODS: The standard Candida albicans and clinical isolated Candida albicans were selected as model fungus and different fluence rates (12, 22, 42, 62, 82, 102 mW/cm2) during curcumin-photodynamic therapy were applied to inactivate Candida albicans biofilm. To evaluate the inactivation effect, XTT assay and Live/Dead kit were employed to quantify and visualize the activities of Candida albicans biofilms. The data were analyzed with SPSS 19.0 software package. RESULTS: When 40 µmol/L of curcumin was applied followed by 4 min illumination, both standard Candida albicans and clinical isolated Candida albicans biofilms were greatly inactivated along with the increase of fluence rates. When fluence rate increased to 102 mW/cm2, there was no significant difference between the experimental group and the previous experimental group(P>0.05). CONCLUSIONS: Fluence rate plays an important role in inactivation of Candida albicans biofilms during curcumin-photodynamic therapy, with optimized value of fluence rate of 82 mW/cm2 in this study.

[Safety and immunological effect of domestic split influenza virus vaccine].

OBJECTIVE: To evaluate the safety and immunological effect of domestic split influenza virus vaccine. METHODS: All 606 subjects were divided into three groups by under 6, 16-60 and above 60 years old. Each age group was divided as study group (n = 213), control group 1 (n = 195) and control group 2 (n= 198) by Table of Random Number, one domestic vaccine and two imported vaccines were respectively inoculated in three group people. The differences of clinical side effect rate, antibody positive rate, protective rate and geometric mean titer (GMT) of these three vaccines were compared by using the statistical software with statistical significance of P < 0.05. RESULTS: The side effect rate of study group, control group 1 and control group 2 was 3.76% (8/213), 4.10% (8/195), and 3.54% (7/198), respectively without statistical significance(chi2 = 0.87, P =0.93). The positive seroconversion rates of H1N1, H3N2 and B in these three groups were respectively 89.2% (190/213), 63.4% (135/213), 86.4% (184/213), 88.7% (173/195), 61.5% (120/195), 87.2% (170/195), 87.9% (174/198), 61.6% (122/198) and 84.8% (168/198). There were no statistical significance in the total positive seroconversion rate of each antibody type (chi2(H1N1) = 0.94, P(H1N1) = 0.63; chi2(H3N2) = 0.94, P(H3N2) = 0.63; chi2(B) = 0.75, P(B) = 0.69). The average growth multiple of H1N1, H3N2 and B in these three groups were 10.7, 7.3, 8.4, 10.5, 6.3, 8.3, 10.2, 7.1, 8.8 times. There were no statistical significances in the GMT growth multiple of each antibody type (F(H1N1) = 0.35, P(H1N1) = 0.70; F(H3N2) = 2.22, P(H3N2) = 0.11; F(B) = 1.51, P(B) = 0.35). The antibody protective rates of H1N1, H3N2 and B were 100% (213/213), 70.0% (149/213), 95.3% (203/213), 100% (195/195), 66.7% (130/195), 97.9% (191/195), 99.5% (197/198), 66.2% (131/198), 96.5% (191/198) respectively. There was no statistical difference among the three vaccines (chi2(H1N1) = 2.04, P(H1N1) = 0.36; chi2(H3N2) = 0.74, P(H3N2) = 0.69; chi2(B) = 0.42, P(B) = 0.82). CONCLUSION: The domestic influenza split vaccine might be suitable for colony vaccination for its having clinical safety and immunological effect.

[Preparation and identification of monoclonal antibodies to staphylococcal enterotoxin I].

OBJECTIVE: To prepare and identify monoclonal antibodies against staphylococcal enterotoxin I (SEI). METHODS: Spleen cells obtained from mice immunized with the SEI protein were fused with the myeloma cells (SP2/0). Hybridomas were screened by enzyme-linked immunosorbent assay (ELISA) and the stable monoclonal hybridomas were isolated by limiting dilution at least three times. The characters of purified monoclonal antibodies were identified by indirect ELISA and Western blotting. RESULT: The monoclonal antibodies secreted by two hybridomas 8F7 and D8 belonged to IgG(2b) and IgG(1) subtypes. Both had high titer and specificity with no cross reaction to SEG, SEE and SEC. CONCLUSION: The monoclonal antibodies against SEI has been successfully prepared and identified in this study.

[Peptide bond scission of staphylococcal enterotoxin C2 and related factors].

OBJECTIVE: To investigate the limited digestion of recombinant staphylococcal enterotoxin C2 (SEC2-His)in different conditions. METHODS: The purified recombinant SEC2-His was treated with different reagents and the cleavage of rSEC2 molecule was observed by SDS-PAGE. RESULT: The cleavage occurred in positions Cys93-Cys110 of the disulfide loop. Complete auto-cleavage of recombinant SEC2 was observed in solution at 37degrees within 24 hrs, and that was accelerated under alkaline conditions. The auto-cleavage of the recombinant protein was inhibited in the presence of beta-ME (2%), PMSF (5-10 mmol/L), imidazole (1 mol/L) or crude E.coli lysate. Non-specific degradation of recombinant SEC2 was promoted with the increasing of the concentration of H(2)O(2). CONCLUSION: The recombinant SEC2-His is broken down in special site of protein, which may be associated with the protein structure.

[Preparation and application of antibody against staphylococcal enterotoxin C2].

The filtrate of Staphylococcus aureus culture has been used in an ampule form named as staphylococcal enterotoxin C injection for cancer therapy in clinic for ten years in China and proved to be effective. The active constituent of three kinds of injections is claimed to be staphylococcal enterotoxin C2 (SEC2), and the content of SEC2 is used as quality control. However, the correct content of SEC2 was not known and the relative amount of SEC2 was very low because of the complicated components of the filtrate. In this research, we established a proper ELISA system for the detection of SEC2 in staphylococcal enterotoxin C injection, which will improve the quality control of the injection. We produced and identified polyclonal and monoclonal antibodies of SEC2 and established BA-ELISA method based on the method of sandwich ELISA. It was found that the BA-ELISA method had good specificity, sensitivity and reproducibility, and being able to detect SEC2 at concentration from 2 to 20 ng x mL(-1), with an average CV value of 5.08%. The SEC2 content in staphylococcal enterotoxin C injection was calculated. There is some difference between the actual and labeled contents in the injections.

[Coexpression of PXRLBD with SRC88 and construction of equilibrium dialysis model of screening PXR ligands].

The aim of this study was to obtain the soluble protein of human pregnane X receptor ligand binding domain (PXRLBD) through the coexpression of PXRLBD and 88 amino acids of steroid receptor coactivator-1 (SRC88) and apply the protein to constructing a new model of screening PXR ligands. Expression plasmid of pETDuet-1-SRC88-PXRLBD was constructed and transformed into Escherichia coli Rosetta (DE3) to coexpress PXRLBD and SRC88 via induction by IPTG at low temperature. Then an equilibrium dialysis model was constructed to study the interaction between PXRLBD and drugs including clotrimazole and dexamethasone, using HPLC as the analysis method. The results showed that the soluble protein of PXRLBD was obtained and the HPLC data indicated that clotrimazole bound to PXRLBD, while dexamethasone did not bind to PXRLBD, which indicated the successful establishment of a new method for studying the interaction between PXR and drugs. The new method may be useful in the screening of PXR ligands in vitro.

[Alternatively spliced variants of constitutive androstane receptor in liver of mouse].

OBJECTIVE: To investigate the existence of alternatively spliced variants of constitutive androstane receptor (CAR) in liver of mouse. METHODS: The nucleotide from liver of mouse was purified and the CAR cDNA was amplified by PCR. The fragments of CAR cDNA were cloned to T vector and sequence analysis was performed. RESULT: Various spliced variants of CAR in liver mouse were confirmed by DNA sequencing. CONCLUSION: There are alternatively spliced variants in CAR, which are located in the ligand binding sequence of CAR.

[Construction, expression and characterization of recombinant fusion protein HSA-PTH (1-34) in Pichia pastoris].

OBJECTIVE: To obtain recombinant fusion protein HSA (human serum albumin)-PTH(1-34) in Pichia pastoris. METHODS: HSA and PTH(1-34) cDNA were obtained with PCR and the DNA segments were cloned into vector pPIC9 with linker. The linearized plasmids were transformed GS115 competent cells treated with LiCl, and mut+ transformants were screened on MD plate. With AOX promoter and alpha-MF signal sequences leading, fusion protein was expressed in GS115. PCR and SDS-PAGE were employed to confirm the integration and expression of HSA-PTH(1-34). The fusion protein was identified by Western blotting and classical adenylate cyclase assay. RESULT: The PCR results showed that the gene of HSA-PTH(1-34) was integrated into GS115 genome. Western bolt approved the existence of two domains of HSA and PTH(1-34). The bioactivity assay in rabbit cortical membranes indicated that HSA-PTH (1-34) activated adenylate cyclase, but the activity was lower than that of the synthetic PTH(1-34). CONCLUSION: Active fusion protein HSA-PTH (1-34) is successfully expressed in Pichia pastoris.

[Expression and bioactivity of the cloned staphylococcal enterotoxin O].

This study is to clone the gene of staphylococcal enterotoxins O, obtain recombinant protein (rSEO) and investigate its activity on mice lymphocyte. Staphylococcus aureus O gene is cloned into GST gene fusion vector pGEX-4T-1. The resultant plasmid pGEX-4T-SEO was used to transform E. coLi BL21, where the GST-SEO fusion protein was expressed efficiently. Then SEO was purified by Glutathione Sepharose 4B affinity column and digested with thrombin. The bioactivity of SEO was analyzed by MTT assay on mice lymphocyte and tumor cells. The nucleotide sequence was confirmed to code for the protein correctly, and soluble SEO was expressed efficiently in E. coli BL21 with pGEX-4T-SEO. The protein purified by affinity chromatography resulted to be one single band by SDS-PAGE detection. The MTT assay of the purified rSEO demonstrated that its abilities of stimulating T cells and inhibiting the proliferation of K562, K562-ADM and B16 cells were equivalent to that of SEC in vitro. The expression plasmid pGEX-4T-SEO was constructed and the recombinant superantigen was expressed successfully, which may provide a foundation for the further research of the anticancer activity of SEO.

Hypoxia-inducible factor-1α and glucose transporter 1 in the malignant transformation of oral lichen planus.

Hypoxia-inducible factor-1α (HIF-1α) and glucose transporter 1 (GLUT1) are key factors in numerous physiological and pathological processes. However, studies on their involvement in the pathogenesis of oral lichen planus (OLP) and its progression toward oral squamous cell carcinomas (OSCC) are scarce. In this study, we examined the protein and gene expressions of both HIF-1α and GLUT1 in normal mucosa, nonatrophic OLP (OLPI), atrophic OLP (OLPII), and OSCC resulting from OLP. Tissues were obtained from 60 cases of OLP patients (n=36 for OLPI, n=24 for OLPII), 20 cases of OSCC patients and 30 healthy control individuals. In addition, in order to investigate if the pathological changes are due to hypoxia, we cultured keratinocytes under hypoxia conditions and measured the expression of HIF-1α and GLUT1. The results indicated that the expressions of HIF-1α and GLUT1 were gradually amplified from normal mucosa to OLPI, OLPII, and OSCC. The expression of both HIF-1α and GLUT1 in OLPII was significantly greater than OLPII. Likewise, the HIF-1α and GLUT1 expressions in OSCC were markedly higher compared to both OLPI and OLPII. Similar trends were obtained in real time PCR and Western blot analyses. A progressive increased micro-vessel density (MVD) was also recorded from normal mucosa to OLPI, OLPII, and OSCC. Moreover, the correlation analysis revealed significant positive correlations between HIF-1α and GLUT1 which were both correlated with MVD in the OLP and OSCC groups. Culture of keratinocytes isolated from OLP tissues under hypoxic and normoxic conditions showed a time-dependent inhibition of keratinocyte proliferation and increased expression of HIF-1α and GLUT1 under hypoxia conditions. In summary, we provided new evidence that hypoxia markers HIF-1α and GLUT1 are upregulated in OLP and are potentially involved in pathological changes leading to malignant transformation of OLP. Further characterization of these factors will provide new ideas for the diagnosis and treatment of OLP.

Myriapod monophyly and relationships among myriapod classes based on nearly complete 28S and 18S rDNA sequences.

Myriapods play a pivotal position in the arthropod phylogenetic tree. The monophyly of Myriapoda and its internal relationships have been difficult to resolve. This study combined nearly complete 28S and 18S ribosomal RNA gene sequences (3,826 nt in total) to estimate the phylogenetic position of Myriapoda and phylogenetic relationships among four myriapod classes. Our data set consists of six new myriapod sequences and homologous sequences for 18 additional species available in GenBank. Among the six new myriapod sequences, those of the one pauropod and two symphylans are very important additions because they were such difficult taxa to classify in past molecular-phylogenetic studies. Phylogenetic trees were constructed with maximum parsimony, maximum likelihood, and Bayesian analyses. All methods yielded moderate to strong support for the monophyly of Myriapoda. Symphyla grouped strongly with Pauropoda under all analytical conditions. The KH test rejected the traditional view of Dignatha and Progoneata, and the topology obtained here, though not significantly supported, was Diplopoda versus ((Symphyla + Pauropoda) + Chilopoda).

[Expression and bioactivity analysis of staphylococcal enterotoxin C2].

AIM: To clone the gene of staphylococcal enterotoxin C2 and express it in the form of a soluble fusion protein in E. coli. Then the activation of SEC2 on mice lymphocyte and its lethal effects on tumor cells were studied. METHODS: Staphylococcus aureus SEC2 gene was cloned into GST gene fusion vector pGEX-4T-1. The resultant plasmid pGEX-4T-SEC2 was used to transform E. coli BL21, where the GST-SEC2 fusion protein was expressed efficiently. The rSEC2 protein was purified with Glutathione Sepharose 4B affinity column and digested with thrombin. The in vitro culture system was utilized to observe the activation of the SEC2 on mice lymphocyte and the lethal effects on tumor cells of the activated mice lymphocyte. RESULTS: The proper gene of SEC2 was cloned and purified rSEC2 was obtained. The MTT results indicated that rSEC2 have strong ability to stimulate mice lymphocyte to proliferate with a dose-dependent manner. With the proliferation of mice splenic lymphocyte, rSEC2 has a strong lethal effect on tumor cells B16, K562 and K562-AD. CONCLUSION: In this study, the gene of SEC2 was cloned and the rSEC2 protein was obtained, which had strong lethal effect on tumor cells B16, K562 and K562-AD.

Dimerization of human uridine diphosphate glucuronosyltransferase allozymes 1A1 and 1A9 alters their quercetin glucuronidation activities.

Uridine diphosphate glucuronosyltransferase 1A (UGT1A) is a major phase II drug-metabolism enzyme superfamily involved in the glucuronidation of endobiotics and xenobiotics in humans. Many polymorphisms in UGT1A genes are reported to inhibit or decrease UGT1A activity. In this study, two UGT1A1 allozymes, UGT1A1 wild-type and a splice mutant, as well as UGT1A9 wild-type and its three UGT1A9 allozymes, UGT1A9*2(C3Y), UGT1A9*3(M33T), and UGT1A9*5(D256N) were single- or double-expressed in a Bac-to-Bac expression system. Dimerization of UGT1A1 or UGT1A9 allozymes was observed via fluorescence resonance energy transfer (FRET) and co-immunoprecipitation analysis. SNPs of UGT1A altered the ability of protein-protein interaction, resulting in differential FRET efficiencies and donor-acceptor r distances. Dimerization changed the chemical regioselectivity, substrate-binding affinity, and enzymatic activity of UGT1A1 and UGT1A9 in glucuronidation of quercetin. These findings provide molecular insights into the consequences of homozygous and heterozygous UGT1A1 and UGT1A9 allozymes expression on quercetin glucuronidation.