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.

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.

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.

[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.

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.

Inter-isoform Hetero-dimerization of Human UDP-Glucuronosyltransferases (UGTs) 1A1, 1A9, and 2B7 and Impacts on Glucuronidation Activity.

Human UDP-glucuronosyltransferases (UGTs) play a pivotal role in phase II metabolism by catalyzing the glucuronidation of endobiotics and xenobiotics. The catalytic activities of UGTs are highly impacted by both genetic polymorphisms and oligomerization. The present study aimed to assess the inter-isoform hetero-dimerization of UGT1A1, 1A9, and 2B7, including the wild type (1A1*1, 1A9*1, and 2B7*1) and the naturally occurring (1A1*1b, 1A9*2/*3/*5, and 2B7*71S/*2/*5) variants. The related enzymes were double expressed in Bac-to-Bac systems. The fluorescence resonance energy transfer (FRET) technique and co-immunoprecipitation (Co-IP) revealed stable hetero-dimerization of UGT1A1, 1A9, and 2B7 allozymes. Variable FRET efficiencies and donor-acceptor distances suggested that genetic polymorphisms resulted in altered affinities to the target protein. In addition, the metabolic activities of UGTs were differentially altered upon hetero-dimerization via double expression systems. Moreover, protein interactions also changed the regioselectivity of UGT1A9 for querectin glucuronidation. These findings provide in-depth understanding of human UGT dimerization as well as clues for complicated UGT dependent metabolism in humans.

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.

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.

[Comparative study of sensitivity of different dental metal materials].

PURPOSE: To compare the sensitivity of different dental metal materials, in order to provide references for choosing of dental metal materials. METHODS: Patch test was performed on 92 patients wearing dental metal prosthesis. Pearson Chi-square test, corrected Chi-square test and Fisher exact test were used for statistical analysis with SPSS17.0 software package. RESULTS: (1)The sensitivity rates of different metal materials were different. The allergy rate of nickel (Ni) was the highest (22.8%), while the allergy rate of aluminum (Al) was 0. (2)More women were allergic to both palladium (Pd) and nickel (Ni) than men with significant difference (P>0.05). (3)Women with ear piercing were more allergic to nickel (Ni), but there was no significant correlation between ear piercing and nickel allergy (P>0.05).(4)There was cross reaction between nickel(Ni)and palladium (Pd), 83.3% of palladium (Pd) allergy patients were allergic to nickel (Ni), while 47.6% of nickel(Ni) allergy patients were allergic to palladium (Pd). (5)Patch test had a delayed reaction. CONCLUSIONS: Dental metal materials have certain allergies, women are more allergic to both palladium (Pd) and nickel (Ni) than men, with significant difference. Patch test may have a delayed reaction. If necessary, observation for 96 h, 7 days or even longer time, are needed to exclude false positivity. Supported by Research Fund of Science and Technology Committee of Shanghai Municipality (10411950900).

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.

The complete mitochondrial genome of the giant pill millipede, Sphaerotheriidae sp. (Myriapoda: Diplopoda: Sphaerotheriida).

The complete mitochondrial genome (mitogenome) of the millipede Sphaerotheriidae sp. has been studied. The genome is 14,970 bp long and contains the typical complement of 13 protein-coding genes, 22 transfer RNA genes, and 2 ribosomal RNA genes. Gene order in Sphaerotheriidae sp. mitogenome is assumed to represent the myriapod ground pattern, which is shared by myriapod-chelicerate clade.

[Antifungal susceptibility test of genotypes of Candida albicans from patients with atrophic or erosive oral lichen planus].

PURPOSE: To study the antifungal susceptibility of genotypes of Candida albicans from patients with atrophic or erosive oral lichen planus and provide evidence for the treatment of candidiasis. METHODS: Polymerase chain reaction(PCR) was adopted to analyze 101 Candida albicans which were isolated from atrophic or erosive oral lichen planus.Microdilution broth method was carried out for antifungal susceptibility test. SPSS16.0 software package was used for Chi-square test. RESULTS: A total of 101 strains of Candida albicans were divided into three types, 39 were genotype A strains,17 genotype B stains and 45 genotype C stains.Strains of genotype A were significantly more resistant to 5-fluorocytosine than strains of genotypes B and C(P<0.05). Strains of genotype B were significantly more resistant to fluconazole than strains of genotype A(P<0.05). Strains of genotype C were significantly more resistant to itraconazole than strains of genotype A(P<0.05).None of the strains of genotypes B and C presented drug resistance to nystatin. There was no significant difference among genotypes A,B and C(P>0.05). CONCLUSIONS: There is a correlation between Candida albicans genotypes and antifungal susceptibility. The use of antifungal agent should be based on the genotypes and antifungal susceptibility test of Candida albicans. For the treatment of candidiasis in patients with atrophic or erosive oral lichen planus, the value of nystatin should be addressed.

[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.

[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.

[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.

PAMAM-triamcinolone acetonide conjugate as a nucleus-targeting gene carrier for enhanced transfer activity.

The excellent transfection efficiency and viability are essential for successful gene therapy. It suggested that when bound to its glucocorticoid receptor, glucocorticoid steroid can dilate the nuclear pore complexes and facilitated the transport of pDNA into the nucleus. In this research, the two different degrees of substitution of PAMAM-triamcinolone acetonide (PAMAM-TA) conjugates were synthesised for efficient translocation of pDNA into the nucleus. The physicochemical properties of the polyplexes were investigated by agarose gel electrophoresis, Zeta-sizer and TEM. They both could form nano-size polyplexes with pDNA. The polyplexes were very stable and showed excellent buffering capacities, facilitating endosomal escape, and no obvious difference was found between them. The TA-conjugated PAMAM-mediated transfection of luciferase and EGFP genes showed better transfer activity than native PAMAM and was comparable to the PEI 25K (polyethylenimine), and lower cytotoxicity in HEK 293 and HepG 2 cells. Even with 10% serum, their transfer activity was still high relatively. In addition, confocal microscopy examination confirmed that the enhancing mechanism for enhanced gene transfer activity of PAMAM-TA conjugate may involve the nuclear translocation of the polyplex. The low substituted degree of TA to 0.22 did not interrupt its nuclear localization potency. These findings demonstrated that the TA-grafted PAMAM dendrimer is a potential candidate as a safe and efficient gene delivery carrier for gene therapy.

[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.

[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.