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This study focused on the microbiota and chemical compounds of the fermented Pinelliae Rhizoma produced in Longchang (LC), Zizhong (ZZ) and Xindu (XD), in Sichuan Province (China). High-throughput sequencing was used to analyze the microbiota. GC-MS and LC-MS were used to detect the compounds produced during the three different Pinelliae Rhizoma fermentation processes. The bacteria and fungi of the three fermented Pinelliae Rhizoma differed substantially, with the bacterial content mainly composed of the Bacillus genus, while the common fungi were only included in four OTUs, which belong to three species of Eurotiomycetes and Aspergillus cibarius. 51 volatile compounds were detected; they varied between LC, XD, and ZZ fermented Pinelliae Rhizoma. C10 and C15 terpenes were most frequently detected, and only curcumene and β-bisabolene were detected in the three fermented Pinelliae Rhizoma. 65 non-volatile compounds were detected by LC-MS, most were of C16, C18, C20, C21 and C22 structures. Cluster analysis showed more similarity between LC and XD fermented Pinelliae Rhizoma with regards to volatile compound content, but more similarity between the XD and ZZ fermented Pinelliae Rhizoma for non-volatiles. Moreover, no correlation between geographical distance and microflora or compounds of fermented Pinelliae Rhizoma was observed. These results showed that hundreds of compounds are produced by the natural mixed fermentation of Pinelliae Rhizoma, and may mostly relate to the microorganisms of five species. This study focused on the microbiota and chemical compounds of the fermented Pinelliae Rhizoma produced in Longchang (LC), Zizhong (ZZ) and Xindu (XD), in Sichuan Province (China). High-throughput sequencing was used to analyze the microbiota. GC-MS and LC-MS were used to detect the compounds produced during the three different Pinelliae Rhizoma fermentation processes. The bacteria and fungi of the three fermented Pinelliae Rhizoma differed substantially, with the bacterial content mainly composed of the Bacillus genus, while the common fungi were only included in four OTUs, which belong to three species of Eurotiomycetes and Aspergillus cibarius. 51 volatile compounds were detected; they varied between LC, XD, and ZZ fermented Pinelliae Rhizoma. C10 and C15 terpenes were most frequently detected, and only curcumene and β-bisabolene were detected in the three fermented Pinelliae Rhizoma. 65 non-volatile compounds were detected by LC-MS, most were of C16, C18, C20, C21 and C22 structures. Cluster analysis showed more similarity between LC and XD fermented Pinelliae Rhizoma with regards to volatile compound content, but more similarity between the XD and ZZ fermented Pinelliae Rhizoma for non-volatiles. Moreover, no correlation between geographical distance and microflora or compounds of fermented Pinelliae Rhizoma was observed. These results showed that hundreds of compounds are produced by the natural mixed fermentation of Pinelliae Rhizoma, and may mostly relate to the microorganisms of five species.

CUL4A regulate the termination of autophagy in a physical process. However, the relationship between CUL4A and autophagy in cancer is unclear. We retrospectively investigated 99 intrahepatic cholangiocarcinoma (iCCA) cases. Whole sections were used for immunohistochemical analysis for p62, and LC3B expression. Q-score was defined as the sum of the labeling intensity and proportion. The cut-off point for immunoreactivity was set. CUL4A was overexpressed in cell lines and autophagy reflux was compared after manipulation. The iCCA cases with CUL4A overexpression had significantly higher prevalence of intact activated autophagy (42.4 vs. 15.2%; p = 0.003), which was significantly associated with advance tumor stage (34.1% vs. 15.4%; p = 0.032), less extensive necrosis (8.3 vs. 49.3%; p < 0.001), and shortened disease-free survival (mean, 19.6 vs. 65.5 months, p = 0.015). In vitro, iCCA cells with CUL4A overexpression significantly increased LC3II level as compared to the cells under basal condition. Although both cell types showed intact autophagy with increased LC3II expression after bafilomycin A1 treatment, the accumulation of LC3II was higher in CUL4A-overexpressing cells. CUL4A overexpression increased the proliferation of cells as compared with control cells. After treatment with bafilomycin A1, proliferation was inhibited in both cell types, but the effects were more prominent in the cells overexpressing CUL4A. CUL4A promotes autophagy, and exhibits significantly higher autophagic flux which affects the proliferation of iCCA cells; these effects correlated with advance tumor stage and poor prognosis. Thus, targeting autophagy may be potentially therapeutic in iCCA.

Since early diagnosis of sepsis may assist clinicians in initiating timely, effective, and prognosis-improving antibiotic therapy, we developed an integrated microfluidic chip (IMC) for rapid isolation of both Gram-positive and Gram-negative bacteria from blood. The device comprised a membrane-based filtration module (90 min operating time), a bacteria-capturing module using a micro-mixer containing magnetic beads coated with "flexible neck" regions of mannose-binding lectin proteins for bacteria capture (20 min), and a miniature polymerase chain reaction (PCR) module for bacteria identification (90 min via TaqMan® probe technology). The filter separated all white blood cells and 99.5% of red blood cells from bacteria, which were captured at rates approaching 85%. The PCR assay's limit of detection was 5 colony-forming units (CFU) per reaction, and the entire process was completed in only 4 h. Since this is far less than that for culture-based approaches, this IMC may serve as a promising device for detection of sepsis.

This study reports an integrated microfluidic device that was capable of executing rapid antimicrobial susceptibility tests with one, two, or even three antibiotics against two clinically isolated multi-drug-resistant bacteria strains (including carbapenem-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus). Bacteria were automatically mixed for 10 min with serially diluted antibiotics with a novel, membrane-type micromixer consisting of two circular micropumps, and the minimum inhibitory concentrations (MIC) were then determined via simple colorimetric reactions in only 4.5–6 h using only 3 μL of bacteria sample of each reaction (as opposed to 24 h and 50 μL, respectively, with the conventional broth micro-dilution method). In addition to determining MICs of antibiotics (ceftazidime, gentamicin, meropenem, vancomycin and linezolid), interaction effects across antibiotics combinations (gentamicin/meropenem or ceftazidime/gentamicin/meropenem) at different dosages were explored. The efficacy of polypharmacy showed additivity when gentamicin or ceftazidime/gentamicin were combined with meropenem to treat carbapenem-resistant Escherichia coli. This represents the first time that the perplexing clinical decision to choose multiple antibiotics for combination therapy against drug resistant bacteria can be realized on an integrated microfluidic device within 6 h.

Objectives Severe hyperkalemia can cause life-threatening arrhythmia, cardiac arrest, or death. This study aimed to investigate the incidence and the associated factors relevant to critical hyperkalemia (≥6 mmol/L) among inpatients, outpatients, and emergency department. Their clinical outcomes were also analyzed. Methods All patients whose high serum potassium values had been reported as critical laboratory values in 2016 were enrolled. Their demographic data, comorbidities, clinical symptoms, biochemical data, and outcomes were reviewed and collected. The Charlson comorbidity score (CCS) and glomerular filtration rate (GFR) were computed to assess the comorbidity burden and renal function. Patients were divided into groups according to different settings, potassium and GFR levels, and their survival. Results Of the 293,830 total serum potassium tests, 1,382 (0.47%) reports were listed as critical laboratory values. The average reply time was 6.3 min. Their mean age was 67.2 years, while the average GFR was 12.2 mL/min/1.73 m2. The overall mortality rate was 34%. Patients in the emergency department had the highest incidence (0.92%), while inpatients had the worst outcome (51% mortality). The leading cause of mortality was septic shock. The fatal group had higher rates of clinical symptoms, higher potassium values, CCS, and eGFR (all p Conclusions Most of the responses for the reports were obtained within a short period of time. Patients with reported high critical serum potassium values were characterized by high rates of comorbidity, reduced eGFR, and mortality. The incidence, clinical manifestations, and outcomes varied in the different clinical settings.

Background The influenza virus is a highly infectious disease, with a notably rapid transmission rate. Autophagy is triggered by viral infection and is a survival mechanism exerted to maintain cellular homeostasis. Catechin is a representative phenolic acid which exerts anti-inflammatory responses against influenza A virus infection. The aim of this study is to explore the anti-H1N1 influenza virus effects by catechin associated with the restoration of autophagy. Methods XTT assay was used to detect cellular viability. The inhibitory effects on the H1N1 influenza virus were assessed by hemagglutination assay, neuraminidase activity, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The protein levels of H1N1 influenza virulence and autophagic markers were detected by Western blot. Results We herein demonstrated that catechin had no cytotoxic effect on both infected and noninfected A549 cells and exerted protective effects on infected A549 cells. The results of the hemagglutination assay, neuraminidase activity, and qRT-PCR to examine viral load demonstrated that catechin effectively inhibited the replication of the H1N1 influenza virus. The virulent M2 protein and viral nucleoprotein were also inhibited after treatment with catechin. As for the autophagic markers, the LC3B protein was notably decreased by catechin in a dose-dependent manner, while the amount of autophagic vacuoles in H1N1 influenza virus-infected cells also decreased after catechin treatment in a dose-dependent manner. Conclusion Collectively, the autophagy activated by the H1N1 influenza virus could be reversed after catechin treatment. This study indicates that catechin effectively inhibits H1N1 viral proliferation and thus may be applied as an adjuvant in future clinical application.

DNA modification is a naturally occurring DNA modification in prokaryotic and eukaryotic organisms and is involved in several biological processes. Although genome-wide methylation has been studied in many insects, the understanding of global and genomic DNA methylation during insect early embryonic development, is lacking especially for insect diapause. In this study, we analyzed the relationship between DNA methylomes and transcriptomes in diapause-destined eggs compared to diapause-terminated eggs in the silkworm, Bombyx mori (B. mori). The results revealed that methylation was sparse in this species, as previously reported. Moreover, methylation levels in diapause-terminated eggs (HCl-treated) were 0.05% higher than in non-treated eggs, mainly due to the contribution of CG methylation sites. Methylation tends to occur in the coding sequences and promoter regions, especially at transcription initiation sites and short interspersed elements. Additionally, 364 methylome- and transcriptome-associated genes were identified, which showed significant differences in methylation and expression levels in diapause-destined eggs when compared with diapause-terminated eggs, and 74% of methylome and transcriptome associated genes showed both hypermethylation and elevated expression. Most importantly, Kyoto Encyclopaedia of Genes and Genomes (KEGG) analyses showed that methylation may be positively associated with Bombyx mori embryonic development, by regulating cell differentiation, metabolism, apoptosis pathways and phosphorylation. Through analyzing the G2/M phase-specific E3 ubiquitin-protein ligase (G2E3), we speculate that methylation may affect embryo diapause by regulating the cell cycle in Bombyx mori. These findings will help unravel potential linkages between DNA methylation and gene expression during early insect embryonic development and insect diapause.

Trauma or infections associated critical bone defects lead to a huge economic burden in the healthcare system worldwide. Recent advances in tissue engineering have led to potential new strategies for the repair, replacement, and regeneration of bone defects, especially in biomaterials and decellularization protocols from xenogenic tissues. However, the complexity in bone structure and mechanical environment limits the synthesis of artificial bone with biomaterials. Thus, the purpose of our study is to develop a natural bone scaffold with great immunocompatibility. We combined decellularization techniques base on SC-CO2 to decellularize bovine bone. In order to study the immune response of mice to materials, the histology, spleen index, immune cells contents and in vitro proliferative performance, cytokine and immunoglobulin light chain expression of mice were characterized. Compared with the fresh bone group, the immune responses of decellularized group were significantly reduced. In conclusion, decellularization via this method can achieve a decellularized scaffold with great immunocompatibility. Our findings suggest the potential of using decellularized BB as a scaffold for bone bioengineering.

TiO2/g-C3N4 (TCN) heterojunction photocatalysts with dimensionality tuning was prepared, g-C3N4 successfully shifted from 3D (3DBG) to 2D (2DGN). Compared with 2DGN and TiO2, all the as-prepared heterostructure samples exhibited significantly enhanced photoactivities under light irradiation. Through the photocatalytic mercury removal experiment on samples with different doping ratios, the effect of oxygen species such as hydrocarbons or surface-adsorbing molecular H2O and doping amount on the performance of photocatalytic oxidation were investigated. Finally, the photocatalytic reaction mechanism of charge transfer in the dimensional adjustable Z-scheme heterojunction was proposed. This work provides a new strategy for developing efficient photocatalysts to control mercury.

Transition metal oxides (TMOs) have high theoretical specific capacity and excellent safety performance, and have been widely used in lithium ion batteries (LIBS). Today, research on transition metal oxides has not been interrupted. However, in practical applications, transition metal oxides still have the disadvantages of severe volume expansion and poor cycle stability. After continuous exploration, researchers have found that combining transition metal oxides with carbon matrix can solve this problem well. Therefore, this article reviews the latest developments in transition metal oxide carbon-based (Porous carbon-based, Biological carbon-based, Graphene carbon-based and MXene carbon-based) composite materials for lithium-ion battery anode materials. In addition, we analyzed the current challenges of composite materials research and put forward our own views. Finally, the future development direction of composite materials in the field of energy storage is prospected.

Bismuth-based nanomaterials exhibiting unique structures, which endow them with fascinating physicochemical properties, have received more and more interests as promising photocatalysts. Fabrication of BiOIO 3 photocatalysts by calcination method was investigated for the first time. XRD patterns showed that the crystallinity of BiOIO 3 photocatalysts could be controlled by calcination temperature. TGA demonstrated that calcining the precursor at a specific temperature range was appropriate for preparing BiOIO 3 photocatalysts. XPS and FT-IR characterization revealed that the BiOIO 3 photocatalysts prepared by calcination method possessed oxygen vacancies, which acted as the positive charge centers to trap the electron easily, inhibiting the recombination of photo electron-hole pairs. Furthermore, PL spectra confirmed the oxygen vacancies can favor for the separation of the electron-hole pairs and in turn enhance the photocatalytic performance. From the above analysis, the mechanism of preparing BiOIO 3 photocatalysts by calcination method was proposed. Meanwhile, the effect of oxygen vacancies on the photocatalytic activity of BiOIO 3 photocatalysts was investigated. The BiOIO 3 photocatalysts with oxygen vacancies were found to be efficiently photocatalytically remove gaseous Hg 0 and the relative photocatalysis mechanism was investigated.

At the upstream of electrostatic precipitator (ESP) or bag house (BH), using sorbents with excellent adsorption capacity is a common method for capturing mercury (Hg) from coal-fired power plant flue gas. ZnS, an inexpensively and conveniently prepared metal sulfide, can even be collected from nature, and is a promising candidate for its sulfur resistance. In this work, selenium ligands (Se2− and Se0) were introduced into ZnS for enhancing Hg0 capture capability. The experimental results showed that the Hg0 removal efficiency can reach 100% in a wide temperature range from 40 ℃ to 300 ℃ when the Se ratio was 30 wt% (30SZ). Compared with pure ZnS and Se powder, Se/ZnS were all exhibited an excellent absorbent capability. A long term experiment was imposed on 30SZ, and the sample adsorption capacity remained unchanged. Moreover, O2 and SO2 had insignificant effects on Hg0 adsorption, NO showed a slight competition for Hg0 adsorption. It was found that Se ligands (Se2− and Se0) and surface active S (S2- and S) sites immobilized Hg0 over the Se/ZnS surface, and in turn the adsorbed Hg0 was basically converted to HgSe and HgS, and the adsorption mechanism was also proposed. With these advantageous performances, 30SZ is expected to be an economically feasible and scientifically sound sorbent. This work also provides a promising way for Hg0 capture from coal combustion flue gas.

Exposure to phthalates has been demonstrated to cause reproductive toxicity in animals, but evidence of the association between phthalates and markers of male reproductive function have been inconsistent in human studies. Here we examined whether environmental exposure to phthalates contributes to altered reproductive hormone levels, sperm DNA damage and spermatozoa apoptosis in a Chinese population. From March to June 2013, repeated urine samples collected from male partners of couples attending an infertility clinic in Wuhan, China were analyzed for 8 phthalate metabolites. Associations of the urinary phthalate metabolites with serum hormone levels (n=483), sperm DNA damage parameters (n=509) and spermatozoa apoptosis measures (n=467) were assessed using multivariable linear regression models. After adjusting for potential confounders, mono-(2-ethylhexyl) phthalate (MEHP), a metabolite of di-(2-ethylhexyl)-phthalate (DEHP), was inversely associated with serum levels of estradiol, total testosterone (T) and free T (all P for trend

Polypharmacy is routinely administered to fight severe infections, though it has led to rampant multi-drug resistance in many bacterial strains. Preferably, antimicrobial susceptibility testing (AST) would be carried out prior to antibiotic prescription, though it is generally thought to be too complex and labor-intensive. In order to assist clinicians with better antibiotic administration for the effective treatment of bacterial infections, an integrated microfluidic system (IMS) capable of automating AST for 1-2 antibiotics against clinical bacterial pathogens was developed herein. Accurate determination of the minimum and fractional inhibitory concentrations of vancomycin, gentamicin, and linezolid were determined by assaying growth of two clinical methicillin-resistant Staphylococcus aureus isolates via a colorimetric assay on-chip. By applying various antibiotic combinations against a single pathogen in multiple chambers, the IMS could identify the optimal drug combination and the minimum effective dosage by evaluating the fractional inhibitory concentration index. This IMS possessed several advantages over conventional methods, including (1) a 50% reduction in bacterial sample and reagent volume (

Background/objectives Docosahexaenoic acid (DHA), an n-3 long chain polyunsaturated fatty acid (LCPUFA), is acquired by dietary intake or the in vivo conversion of α-linolenic acid. Many enzymes participating in LCPUFA synthesis are regulated by peroxisome proliferator-activated receptor alpha (PPARα). Therefore, it was hypothesized that the tissue accretion of endogenously synthesized DHA could be modified by PPARα. Materials/methods The tissue DHA concentrations and mRNA levels of genes participating in DHA biosynthesis were compared among PPARα homozygous (KO), heterozygous (HZ), and wild type (WT) mice (Exp I), and between WT mice treated with clofibrate (PPARα agonist) or those not treated (Exp II). In ExpII, the expression levels of the proteins associated with DHA function in the brain cortex and retina were also measured. An n3-PUFA depleted/replenished regimen was applied to mitigate the confounding effects of maternal DHA. Results PPARα ablation reduced the hepatic Acox, Fads1, and Fads2 mRNA levels, as well as the DHA concentration in the liver, but not in the brain cortex. In contrast, PPARα activation increased hepatic Acox, Fads1, Fads2 and Elovl5 mRNA levels, but reduced the DHA concentrations in the liver, retina, and phospholipid of brain cortex, and decreased mRNA and protein levels of the brain-derived neurotrophic factor in brain cortex. Conclusions LCPUFA enzyme expression was altered by PPARα. Either PPARα deficiency or activation-decreased tissue DHA concentration is a stimulus for further studies to determine the functional significance.

Periprosthetic joint infections (PJIs) are among the most severe complications emerging from prosthetic joint replacement surgeries. In order to possess a rapid means of diagnosing PJIs, an integrated microfluidic system was developed herein for detecting and identifying bacteria in human synovial fluid (HSF). The entire molecular diagnostic process, including (1) sample treatment, (2) bacterial isolation, (3) bacterial lysis, (4) nucleic acid amplification (via polymerase chain reaction (PCR)), and (5) optical detection, could be automated on a single chip. First, N-acetyl-L-cysteine was used to decrease the viscosity of HSF samples and consequently enhance bacterial isolation with vancomycin-coated nano-magnetic beads. Then, a universal 16S ribosomal ribonucleic acid PCR primer set and four species-specific primer sets were used for PCR-based detection and identification of four common bacteria previously associated with PJIs, including Staphylococcus aureus, methicillin-resistant S. aureus, Escherichia coli, and Acinetobacter baumannii. With this approach, the limit of detection was as low as 100 colony forming units (CFUs) per milliliter (or 20 CFUs per reaction), which is suitable for clinical diagnostics and for making informed decisions regarding post-operative antibiotic administration. More importantly, bacterial detection and identification data could be acquired within 90 minutes, representing a significant improvement over traditional culture-based methods (3–7 days). The developed microfluidic system may therefore serve as a promising tool for rapid diagnosis of PJIs.

Background Neurofibromatosis type 1 (NF1) is one of the most common hereditary neurocutaneous disorders. The malignant peripheral nerve sheath tumor (MPNST), transformed from NF1 related plexiform neurofibroma, is a rapidly growing and highly invasive tumor. No effective chemotherapeutic agent is currently available. Calebin-A is a derivative from turmeric Curcuma longa. Given the anti-inflammatory and anticancer potentials of curcumin, whether Calebin-A also had the tumoricidal effect upon MPNST cells is still elusive. Purpose To determine whether Calebin-A has the potential for anti-MPNST effect. Methods The MTT and FACS analysis of normal Schwann (HSC) and MPNST cells have been employed to determine the tumoricidal effect of Calebin-A. The expression of the signal pathway molecules was assessed by Western blotting. The CHIP with quantitative PCR assay was performed to quantify the promoter DNA binding to acetylated histone 3 (acetyl H3). The enzyme activities of histone acetyltransferase (HAT) and deacetylase (HDAC) have been evaluated by commercial kits. The measurements of tumor size of the xenograft mouse model were also performed. Results Calebin-A inhibited the proliferation of MPNST and primary neurofibroma cells in a dose-dependent manner. The flow cytometry analysis of the MPNST cells after treatment of 25 μm of Calebin-A demonstrated an increase of population in the G0/G1 phase but decrease in G2/M phase. Before treatment, the expression of Axl, Tyro3, and acetyl H3 was significantly higher in MPNST cells when compared to HSC. The expression of phosphorylated-AKT, -ERK1/2, survivin, hTERT, and acetyl H3 proteins were reduced after treatment. The CHIP assay shows the promoter DNA copies of survivin (BRIC5) and hTERT genes are significantly reduced post-treatment. The enzyme activity of HAT was significantly reduced, but not that of HDAC. Two HAT inhibitors, epigallocatechin-3-gallate (EGCG) and anacardic acid (AA) have also demonstrated a significant inhibitory effect on MPNST cells. Finally, the measurements of tumor size showed a significant reduction of the xenograft tumors after treatment of Calebin-A. Conclusion Both in vitro and in vivo studies showed Calebin-A could inhibit the proliferation of MPNST with suppression of survivin and hTERT. The reduced expression of these two factors might be through the epigenetic histone modification resulting from the decreased activity of HAT.

A series of NiMo/γ-Al 2 O 3 catalysts with different NiO loadings were prepared and characterized by XRD, BET, 27 Al-MAS NMR, Py-IR and HRTEM. The activity of these NiMo/γ-Al 2 O 3 catalysts in the hydrodesulphurization (HDS) of dibenzothiophene (DBT) was evaluated in a high pressure micro reactor; the interaction between Ni promoter and γ-Al 2 O 3 support as well as its effect on the nanostructure of active MoS 2 phase and HDS performance was then investigated. The results indicate that Ni promoter prefers to interact with the tetra-coordinated unsaturated aluminum sites on the support surface. With the increase of NiO loading, the average number of stacking layers for the MoS 2 clusters in the sulfided NiMo/γ-Al 2 O 3 catalysts is increased at the expense of the average length. As the slim MoS 2 clusters are more active for the HDS of DBT, the addition of Ni promoter is then effective to enhance the catalytic activity of NiMo/γ-Al 2 O 3 in HDS, but may lead to a slight decrease in the hydrogenation selectivity.

Background: Metals in single spot urine samples are often used to estimate individual exposure in human studies. However, measurements in urine could vary greatly over time due to variable exposure, potentially leading to exposure misclassification. Objective: We examined the variability of metal levels in the urine of 11 men who provided 529 samples on 8 days during a 3-month period, which corresponds to the duration of spermatogenesis. Method: The urinary levels of arsenic (As), cadmium (Cd), cobalt (Co), copper (Cu), lead (Pb), molybdenum (Mo), and nickel (Ni) were measured using inductively coupled plasma–mass spectrometry. We calculated the intraclass correlation coefficients (ICCs) to assess the reproducibility of metal measures and computed the sensitivity and specificity to evaluate how well spot urine samples determined the individuals’ 3-month average exposure. Results: Fair to good reproducibility was observed for the serial measurements of Cd (ICC = 0.53) in spot samples collected during the 3-month period, whereas the serial measurements of As, Co, Cu, Pb, Mo, and Ni showed poor reproducibility (ICCs = 0.01–0.29). Use of single spot urine samples to classify the high (top 33%) 3-month average metal levels had uniformly high specificities (0.70–0.84) but relatively low sensitivities (0.40–0.57), except for Cd (0.77). The minimum number of specimens (k) required to estimate the participant-specific mean for the seven metals within 20% of the “true” values ranged from 3 for Cd to 27 for Ni. Conclusions: The high variability observed in the urinary levels of As, Co, Cu, Pb, Mo, and Ni suggests that a single measurement provides only a brief snapshot in time of the exposure levels of an individual, which can result in a moderate degree of exposure misclassification. Citation: Wang YX, Feng W, Zeng Q, Sun Y, Wang P, You L, Yang P, Huang Z, Yu SL, Lu WQ. 2016. Variability of metal levels in spot, first morning, and 24-hour urine samples over a 3-month period in healthy adult Chinese men. Environ Health Perspect 124:468–476; http://dx.doi.org/10.1289/ehp.1409551

Exposure to phthalates has been found to have adverse effects on male reproductive function in animals. However, the findings from human studies are inconsistent. Here we examined the associations of phthalate exposure with semen quality and reproductive hormones in a Chinese population using phthalate metabolite concentrations measured in semen as biomarkers. Semen (n = 687) and blood samples (n = 342) were collected from the male partners of sub-fertile couples who presented to the Reproductive Center of Tongji Hospital in Wuhan, China. Semen quality parameters and serum reproductive hormone levels were determined. Semen concentrations of 8 phthalate metabolites were assessed using high-performance liquid chromatography and tandem mass spectrometry. Associations of the semen phthalate metabolites with semen quality parameters and serum reproductive hormones were assessed using confounder-adjusted linear and logistic regression models. Semen phthalate metabolites were significantly associated with decreases in semen volume [mono-n-butyl phthalate (MBP), mono-(2-ethylhexyl) phthalate (MEHP), mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono(2-ethyl-5-oxohexyl) phthalate (MEOHP)], sperm curvilinear velocity [monobenzyl phthalate (MBzP), MEHP, the percentage of di-(2-ethylhexyl)-phthalate metabolites excreted as MEHP (%MEHP)], and straight-line velocity (MBzP, MEHP, %MEHP), and also associated with an increased percentage of abnormal heads and tails (MBzP) (all p for trend

Exposure to phthalates has been demonstrated to have adverse effects on male reproduction in animal studies, but findings in human studies have been inconsistent. We recruited 1040 men from the Reproductive Center of Tongji Hospital in Wuhan, China from March to June 2013. Each man provided one semen sample and two urine samples. Semen quality parameters and the urinary concentrations of eight phthalate metabolites were determined. After multivariable adjustments, the urinary concentrations of monobutyl phthalate (MBP) were found to be positively associated with the below-reference sperm concentration and total sperm count, and the odds ratios (ORs) comparing extreme MBP quartiles were 2.01 (95% CI: 1.07, 3.79; p for trend=0.06) and 1.80 (95% CI: 1.05, 3.08; p for trend=0.02), respectively. The associations were confirmed by multivariable linear regression analysis, which showed that the MBP concentration was significantly associated with decreasing trends in the sperm concentration and total sperm count (both p for trend

Previous studies have revealed that some proteins in Bombyx mori larvae digestive juice show antiviral activity. Here, based on the label-free proteomics data, BmLipase member H-A (BmLHA) was identified as being involved in the response to BmNPV infection in B. mori larvae digestive juice. In the present study, a gene encoding the BmLHA protein in B. mori was characterized. The protein has an open reading fragment of 999 bp, encoding a predicted 332 amino acid residue-protein with a molecular weight of approximately 35.9 kDa. The phylogenetic analysis revealed that BmLHA shares a close genetic distance with Papilio xuthus Lipase member H-A. BmLHA was highly expressed in the middle part of the B. mori gut, and the expression level increased with instar rising in larvae. There was higher expression of BmLHA in A35 than in P50 strains, and it was upregulated in both A35 and P50 strains, following BmNPV infection. The expression level of VP39 decreased significantly in appropriate recombinant-BmLHA-treated groups compared with the PBS-treated group in B. mori larvae and BmN cells. Meanwhile, overexpression of BmLHA significantly reduced the infectivity of BmNPV in BmN cells. These results indicated that BmLHA did not have digestive function but had anti-BmNPV activity. Taken together, our work provides valuable data for the clarification of the molecular characterization BmLHA and supplements research on proteins of anti-BmNPV activity in B. mori.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a major pathogen causing severe economic loss. Previous studies have revealed that some proteins in silkworm digestive juice show antiviral activity. In this study, antiviral activity examination of different resistant strains showed that the digestive juice of the resistant strain (A35) had higher inhibition to virus than the susceptible strain (P50). Subsequently, the label-free quantitative proteomics was used to study the midgut digestive juice response to BmNPV infection in P50 and A35 strains. A total of 98 proteins were identified, of which 80 were differentially expressed proteins (DEPs) with 54 enzymes and 26 nonenzymatic proteins by comparing the proteomes of infected and non-infected P50 and A35 silkworms. These DEPs are mainly involved in metabolism, proteolysis, neuroactive ligand receptor interaction, starch and sucrose metabolism and glutathione metabolism. After removing the genetic background and individual immune stress response proteins, 9 DEPs were identified potentially involved in resistance to BmNPV. Further studies showed that a serine protease, an alkaline phosphatase and serine protease inhibitor 2 isoform X1 were differentially expressed in A35 compared to P50 or post BmNPV infection. Taken together, these results provide insights into the potential mechanisms for silkworm digestive juice to provide resistance to BmNPV infection. Signifcance: Bombyx mori nucleopolyhedrovirus (BmNPV) is highly pathogenic, which has a great impact on the sericulture. BmNPV entered the midgut lumen and exposed to digestive juices after oral infection. Previous studies have revealed that some proteins in silkworm digestive juice show antiviral activity, however, current information on the digestive juice proteome of high resistant silkworm strain after BmNPV challenge compared to susceptible strain is incomprehensive. Here, we combined label-free quantification method, bioinformatics, RT-qPCR and western blot analysis and found that BmNPV infection causes some protein changes in the silkworm midgut digestive juice. The DEPs were identified in the digestive juices of different resistant strains following BmNPV infection, and screened out some proteins potentially related to resistance to BmNPV. Three important differentially expression proteins were validated by independent approaches. These findings uncover the potential role of silkworm digestive juice in providing resistance to BmNPV and supplemented the profile of the proteome of the digestive juices in B. mori., Graphical abstract Unlabelled Image, Highlights • The digestive juice antiviral activity of resistant strain was higher than susceptible strain. • Silkworm digestive juice was analyzed by label-free quantitative proteomics. • Eighty DEPs were identified in different resistant strains following BmNPV infection. • Some DEPs were validated by RT-qPCR and western blot. • These results provide insights into the potential mechanisms for B. mori resistant to BmNPV infection.

Background and Purpose: Drynaria fortunei J. Sm (D. fortunei), known as Gu-Sui-Bu, is used in traditional Chinese medicine to treat common injuries, including bone fractures and bruising. The specific functional mechanisms of the angiogenic and endothelial cell migration properties of D. fortunei are currently unclear. Thus, the purpose of this study is to validate the potential angiogenic and cellular migration properties and related mechanisms by D. fortunei both in vivo and in vitro. Experimental Approach: The present study investigates, both in vivo and in vitro, the wound healing effects of D. fortunei as associated with angiogenesis, specifically by the modulation of matrix metalloproteinases (MMPs) and upregulation of vascular endothelial growth factor (VEGF) ligand/receptors. In order to determine the potential angiogenic effects of D. fortunei, in vivo neovascularization of chick chorioallantoic membranes (CAMs) assay, and directed in vivo angiogenesis assay (DIVVA) were performed, while in vitro scratch wound healing, migration, and matrix-induced tube formation assays were performed by using human umbilical vascular endothelial cells (HUVECs). Furthermore, we used qPCR to analyze the gene expressions and Western blot to observe protein expressions of MMP-2, MMP-14, TIMP-2, RECK, and VEGF/VEGFRs. Results: This study identified five major compounds from the water extract of D. fortunei: protocatechuic acid, caffeic acid 4-O-β-D-glucopyranoside, 5,7-dihydroxychromone-7-O-rutinoside, neoeriocitrin, and naringin. D. fortunei was confirmed to activate in vivo angiogenesis by CAM and DIVVA assays. D. fortunei further exhibited in vitro angiogenic effects associated with cell migration, as demonstrated by the tube formation assay, transwell migration assay, and scratch wound healing assay. The extracellular MMP-2 activity was found to be dose-dependently augmented both in vitro and in vivo by D. fortunei. The mRNA and protein expressions of MMP-2, and MMP-14 were increased; while the tissue inhibitor metalloproteinase-2 (TIMP-2), and reversion-inducing cysteine-rich protein with kazal motifs (RECK) were both decreased. Furthermore, D. fortunei activated the gene and protein expressions of VEGF-A, -B, and VEGFR-2, -3. Conclusion: D. fortunei increased MMP-2 activity, thereby stimulating angiogenesis and cell migration, both in vivo and in vitro, as a result of MMP-2 and TIMP-2 balance modulation and the activation of VEGF/VEGFRs expression.

Carnosic acid (CA), a major polyphenolic diterpene present in Rosmarinus officinalis, has been reported to have multiple functions, including antitumor activity. The MTT assay, BrdU incorporation, wound healing, and colony formation were used to detect melanoma B16F10 cell growth and proliferation. Flow cytometry was used for cell cycle detection. p21 and p27 expression was detected by Western blotting. B16F10 cell xenograft model was established, and treated with CA, carmustine (BCNU), or lomustine (CCNU). The present study found that CA exhibits significant growth inhibition and cell cycle arrest in melanoma B16F10 cells. We also found that CA triggers cell cycle arrest at G0/G1 phase, and enhances p21 expression. Additionally, CA can enhance BCNU- and CCNU-mediated cytotoxicity and cell cycle arrest in B16F10 cells. Finally, we found that CA inhibits tumor growth, and reduces the values of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in vivo. The present study study concluded that CA may be safe and useful as a novel chemotherapeutic agent.

Acute leukemia is one of the commonly diagnosed neoplasms and causes human death. However, the treatment for acute leukemia is not yet satisfactory. Studies have shown that mushroom-derived polysaccharides display low toxicity and have been used clinically for cancer therapy. Therefore, we set out to evaluate the anti-cancerous efficacy of a water-soluble polysaccharide extract from Inonotus taiwanensis (WSPIS) on human acute monocytic leukemia THP-1 and U937 cell lines in vitro. Under our experimental conditions, WSPIS elicited dose-dependent growth retardation and induced apoptotic cell death. Further analysis showed that WSPIS-induced apoptosis was associated with a mitochondrial apoptotic pathway, such as the disruption of mitochondrial membrane potential (MMP), followed by the activation of caspase-9, caspase-3, and PARP (poly(ADP-ribose) polymerase) cleavage. However, a broad caspase inhibitor, Z-VAD.fmk, could not prevent WSPIS-induced apoptosis. These data imply that mechanism(s) other than caspase might be involved. Thus, the involvement of endonuclease G (endoG), a mediator arbitrating caspase-independent oligonucleosomal DNA fragmentation, was examined. Western blotting demonstrated that WSPIS could elicit nuclear translocation of endoG. MMP disruption after WSPIS treatment was accompanied by intracellular reactive oxygen species (ROS) generation. However, pretreatment with N-acetyl-l-cysteine (NAC) could not attenuate WSPIS-induced apoptosis. In addition, our data also show that WSPIS could inhibit autophagy. Activation of autophagy by rapamycin decreased WSPIS-induced apoptosis and cell death. Taken together, our findings suggest that cell cycle arrest, endonuclease G-mediated apoptosis, and autophagy inhibition contribute to the anti-cancerous effect of WSPIS on human acute monocytic leukemia cells.

To explore the in situ metabolic characteristics of yeasts involved in the spontaneous fermentation process of Chinese strong-flavoured liquor, a comparison was conducted between solid-state fermentation (SSF) and submerged fermentation (SmF) when supplemented with 24 indigenous yeast strains, with a focus on the production of ethanol and a broad range of volatile compounds responsible for the characteristics of Chinese strong-flavoured liquor. Under the various experimental conditions, the 24 indigenous yeast strains showed different influences on the mixed fermentation system. The fluctuations caused by different yeast strains in the mixed system were less than those caused by the different fermentation modes relative to the formation of flavour compounds. SSF was found to be more suitable for the production of ethanol, methanol and ethyl lactate, whereas SmF was more suitable for the production of 10 higher alcohols, four esters and four acids. This study revealed the relationships amongst the indigenous yeasts, SSF, and the distinctive flavour profiles of Chinese strong-flavoured liquor. This work provides evidence of the existence of internal stability in spontaneous SSF, thereby facilitating a better understanding of the fermentative mechanism in the SSF process for Chinese strong-flavoured liquor production Copyright © 2015 The Institute of Brewing & Distilling

Cyclic GMP-dependent protein kinase II (cGKII; PRKG2) phosphorylates a variety of biological targets and has been identified as a gout-susceptible gene. However, the regulatory role of cGKII in triggering gout disease has yet to be clarified. Thus, we plan to explore the specific function of cGKII in macrophages related to gout disease. By using cGKII gene knockdown method, we detected macrophage M1/M2 polarization, phagocytosis, and their responses to stimulation by monosodium urate (MSU). cGKII was highly expressed in M1 phenotype, but not in M2, and cGKII knockdown significantly inhibited macrophage M1 polarization by decreasing M1 chemokine markers (CXCL10 and CCL2) and downregulating phagocytosis function. We further identified that cGKII-associated phagocytosis was mediated by upregulating toll-like receptor 2 (TLR2) expression, but not by TLR4. Mimicking gout condition by MSU treatments, we found that MSU alone induced cGKII and TLR2 expression with increased M1 polarization markers and phagocytosis activity. It means that cGKII knockdown significantly inhibited this MSU-induced cGKII-TLR2-phagocytosis axis. Our study showed that cGKII plays a key role in M1 polarization, especially in TLR2-mediated phagocytosis under MSU exposure. The findings provide evidence for the possible role of cGKII as an inflammation exciter in gout disease.Gout-susceptible gene cGKII is necessary for macrophage M1 polarization. cGKII regulates M1 phagocytosis function via TLR2. Monosodium urate treatments increase cGKII expression and related function. This study reveals the role of cGKII in enhancing gouty inflammatory responses.

Periprosthetic joint infection (PJI) is one of the severe complications of prosthetic joint replacement. Delayed PJI diagnosis may anchor bacteria in periprosthetic tissues, and removal of the prosthesis might be inevitable. The diagnosis of PJI depends on the identification of microorganisms by standard microbiological cultures or more advanced molecular diagnostic methods for detection of bacterial genes. However, these methods are relatively time-consuming, labor-intensive and not human error-free. Moreover, it is challenging to distinguish live from dead bacteria by using DNA-based molecular diagnostics since bacterial DNA will be remained in the tissue even after the death of the bacteria. In this work, an integrated microfluidic system has been developed to perform the entire molecular diagnostic process for the PJI diagnosis in a single chip. We combined the loop-mediated isothermal amplification (LAMP) with ethidium monoazide (EMA) in an integrated microfluidic system to identify live bacteria with reasonable sensitivity and high specificity. All the diagnostic processes including bacteria isolation, cell lysis, DNA amplification and optical detection can be automatically performed on the integrated microfluidic system by using a compact custom-made control system. The integrated system can accommodate four primers complementary to six regions of the target genes and improve the detection limit by using LAMP. The limit of detection in this multiple EMA-LAMP assay could be as low as 5 fg/reaction (~1 CFU/reaction) when choosing an optimized primer set as we demonstrated in mecA gene detection. Thus, the developed system for PJI diagnosis has great potential to become a point-of-care device.

This paper reports an improved fermentation process that includes simultaneous saccharification, detoxification, and cofermentation as steps for producing bioethanol. Rice straw was first steam exploded (SE) or butanone solution exploded (BSE) and then cofermented with Saccharomyces cerevisiae and Candida shehatae. To overcome the inhibitors, the exploded rice straw was continuously and slowly introduced into a 10 L ventilated fermenter. When the fermentation conditions were set to 1.0% initial dry matter, 10% total dry matter, addition rate of 120 mg/min, total fermentation time of 234 h, and dose of 0.1% (NH4)2SO4, yields of 25.8 g/100 g of dry matter ethanol and 88% total sugar use were obtained for BSE rice straw. The ethanol yields were not significantly different between detoxified materials and non-detoxified materials. Most of the furfural, hydroxymethylfurfural (5-HMF), acetic acid, and butanone were removed during the fermentation of non-detoxified materials, and the sugar concentrations were very low. The in situ detoxification and fermentation was effective and inexpensive when the pre-detoxification of exploded materials and the pre-adaptation of strains steps were omitted.

Aiming at the removal of chlorinated disinfection by-products in water and the difficulty of separation of powder bentonite from water, the polyacrylamide bentonite composite was prepared and used as absorbent. The mechanism of adsorption and regeneration was discussed. The adsorption of chloroacetic acid on polyacrylamide bentonite composite followed Langmuir isothermal and Pseudo second order kinetic equation, and the adsorption capacity was 63.01mg/g at optimal temperature 30°C. The sorption heat were at the range of 9~16kJ/mol and decreased with the increase of temperature. It was indicated the adsorption mainly belonged to chemical adsorption, and the proportion of physical adsorption increased with the temperature increase. The thermodynamic parameters were ΔH0=-5.226kJ/mol, ΔG0=-27.753kJ/mol. These showed the adsorption was spontaneous and exothermal process. The composites were characterized by XRD and FT-IR. It was found the acrylamide intercalated in interlayer of bentonite and polymerized, then interacted with bentonite by hydrogen bonds. The d001 increased and decreased with adsorption and regeneration, the chloroacetic acid was considered to intercalate in the interlayer of polyacrylamide bentonite composite. The hydrogen bonds may exist between chloroacetic acid and CH2 groups. The polyacrylamide bentonite composite adsorbed chloroacetic acid could be regenerated by NaOH solution.

Digital loop-mediated amplification (LAMP) is an isothermal, quantitative DNA amplification approach which has high sensitivity and specificity and could be used to analyze extremely low amount of DNA within a short period of time. In this study, an integrated self-driven microfluidic chip was proposed herein for performing a digital LAMP process for vancomycin-resistant Enterococcus (VRE) diagnosis. The entire bacterial diagnosis could be automatically performed by capillary forces through the new polydimethylsiloxane (PDMS) surface treatment without using external pumps. Moreover, digitized droplets could be separated from each other by normally-close valves such that it could be quantitated. The contact angle of the treated PDMS surface was measured to be only 24°, which was the lowest value in literatures. This is the first time that a rapid (within 1 hour), simple, and hydrophilic PDMS surface modification method has been reported, which could be used in self-driven microfluidic devices for digital LAMP. This device may become a promising tool for clinical diagnosis and point-of-care applications.

An efficient system for inducing somatic embryogenesis in Panax notoginseng was established using shaker flasks and bioreactor cultures; furthermore, regenerated plantlets were successfully transferred to ex vitro soil conditions. Embryogenic callus was induced from segments of adventitious roots incubated on Murashige and Skoog (MS) medium containing 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) after 5 weeks of culturing. The highest frequency (100%) of somatic embryogenesis, with a mean of 32.7 somatic embryos per callus, was obtained on embryogenic callus incubated on a medium containing 0.5 mg/L 2,4-D. To scale-up somatic embryo formation, 10 g (~1.65 × 104) of early globular-stage somatic embryos were incubated in a 3 L airlift bioreactor containing 1.5 L 1/2 MS medium without plant growth regulators (PGRs) for a period of 4 weeks; these globular-stage somatic embryos then developed into cotyledonary embryos. When maintained on PGR-free medium, the cotyledonary embryos developed roots but did not develop shoots. However, when they were treated with gibberellic acid (GA3), they continued to germinate and transformed into plantlets after 2 weeks of culture. Plantlets with well-developed shoots and roots were transferred to an autoclaved vermiculite and perlite mixture, acclimatized for a period of 3 months and successfully transferred to forest mountain soil. Following overwintering, these plants produced new growth.

The experiments on ultraprecision turning of SiC reinforced aluminium composites have been done by using polycrystalloid diamond (PCD) tool with the three-dimensional cutting forces measured with a Kistler dynamometer, the quality of the machined surfaces was examined under Atom Force Microscope (AFM) and Scanning Electron Microscope (SEM) and the roughness of the machined surfaces was measured with roughometer of Talysurf-6. The experimental results show that the quality of the machined surfaces of SiC reinforced aluminium composites is predominantly controlled by the deformation mechanisms of SiC reinforcements, and the quality of the machined surfaces is better when reinforcements deform in the form of direct cutoff than in the form of pullout. The higher the volume fraction of SiC reinforcements, the worse the quality of the machined surfaces and the greater the amplitude of vibration and the average value of the cutting forces. On the same condition of volume fraction and cutting, the quality of the machined surfaces of SiC whiskers reinforced aluminium matrix composites is better than that of SiC particles reinforced aluminium matrix composites.

In previous study we reported that pretreatment with plasmolysis enhanced somatic embryo formation in hypocotyls of Eleutherococcus senticosus. In the present study, the expression level of callose synthase gene in embryos of E. senticosus in response to 2,4-D, sucrose and mannitol treatments was analyzed by RT-PCR. The results show that plasmolysis pretreatment using sucrose and mannitol significantly promoted the expression of callose synthase gene. Also, the thicker cell walls of explant plasmolyzed compared with controls were observed during the somatic embryogenesis. We suggest that the callose may make the cells in epidermis separate from neighboring cells and then develop into embryogenic potential cells.

Exposure to metals, including essential and nonessential elements, is widespread and may be associated with altered semen quality. This study aimed to examine the association between urinary metal concentrations and semen quality in a Chinese population. We measured semen quality parameters (sperm concentration, count, motility, normal morphology, and abnormal head) and 13 metals [arsenic (As), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), iron (Fe), lead (Pb), manganese (Mn), molybdenum (Mo), mercury (Hg), nickel (Ni), selenium (Se), and zinc (Zn)] in the urine of 394 men from an infertility clinic. Multivariable logistic and linear regressions were used to assess the relationship between the creatinine-adjusted urinary metal concentrations and semen quality parameters. We found a significant trend for decreased odds ratios (ORs) for below-reference sperm count with increasing Se quartiles (p for trend = 0.04) and a significant trend for increased sperm percent abnormal head with increasing Ni quartiles (p for trend = 0.03). These associations persisted, even when considering multiple metals. Our results suggest that Ni exposure may be associated with deteriorated sperm morphology and that Se exposure may be associated with better semen quality. However, our findings warrant further studies in a larger and general population.

Toxicological studies have shown that phthalates, a class of widely used chemicals, can impair male reproductive function, but epidemiological evidence is inconsistent. This study aimed to investigate the associations of semen phthalate metabolites with sperm apoptosis and DNA damage in a Chinese population. We assessed sperm apoptosis markers with Annexin V/PI analysis and sperm DNA integrity with comet assay before measuring eight phthalate metabolites in semen by high-performance liquid chromatography and tandem mass spectrometry (HPLC-MS/MS) among 463 men from Wuhan, China. We found a suggestive dose-response relationship between semen mono-(2-ethylhexyl) phthalate (MEHP) and an increased percentage of Annexin V+/PI- sperm (p for trend of0.10). We also observed that semen monomethyl phthalate (MMP) and monoethyl phthalate (MEP) were associated with significant dose-related increases in tail length of the comet (both p for trend of0.01). In conclusion, our data indicate that semen MEHP is associated with increased sperm apoptosis and that semen MMP and MEP are associated with increased sperm DNA damage in a Chinese population.

Eleutherococcus senticosus zygotic embryos were pretreated with 1.0 M mannitol or sucrose for 3-24 h. This pretreatment resulted in a high frequency of somatic-embryo formation on hormone-free medium. All the somatic embryos developed directly and independently from single epidermal cells on the surface of zygotic embryos after plasmolyzing pretreatment. Scanning electron microscopic observation revealed that the epidermal cells of hypocotyls rapidly became irregular and showed a random orientation before somatic-embryo development commenced. At the same time, the epidermal cells in the untreated control remained regular. Callose concentration determined by fluorometric analysis increased sharply in E. senticosus zygotic embryos after plasmolyzing pretreatment but remained low in the untreated control. Aniline blue fluorescent staining of callose showed that the plasmolyzing pretreatment of zygotic embryos resulted in heavy accumulation of callose between the plasma membrane and cell walls. On the basis of these results, we suggest that plasmolyzing pretreatment of zygotic embryos induces the accumulation of callose, and the interruption of cell-to-cell communication imposed by this might stimulate the reprogramming of epidermal cells into embryogenically competent cells and finally induce somatic-embryo development from single cells.

Siberian ginseng (Eleutherococcus senticosus) is endangered mainly because of overexploitation. A multiplication system was established via in vitro nodal cutting of the stem. When cotyledonary seedling nodes were cultured on Murashige and Skoog (MS) medium with different concentrations of GA3 (1.0–30.0 mg/L) for 3 weeks, shoots with nodes were formed from the cotyledonary nodes. Thereafter, nodal segments (including an axillary bud) excised from these shoots were incubated on MS, Lloyd and McCown (WPM) and Schenk and Hildebrandt (SH) media with various concentrations of auxin, cytokinin and GA3. The most effective medium and hormonal combination for the shoot formation from axillary buds of nodal segments was MS medium containing BA (2.0 mg/L), IBA (0.5 mg/L) and GA3 (0.5 mg/L). In this environment, 99.93 % of axillary buds produced shoots, with an average shoot length of 16.95 mm and 2.75 nodes were formed per shoot. Rooting of axillary shoots was most effectively achieved on NH4NO3‐free, 1/2 MS medium ...

We report the first case of prosthetic joint infection caused by Mycobacterium alvei , which was identified by PCR-restriction fragment length polymorphism and verified by analysis of nucleotide sequences of its amplified 16S ribosomal DNA. The pathogen was susceptible to linezolid, amikacin, ciprofloxacin, tigecycline, and trimethoprim-sulfamethoxazole. The clinical implications are discussed.

Arsenic is a class I human carcinogen (such as inducing skin cancer) by its prominent chemical interaction with protein thio (-SH) group. Therefore, arsenic may compromise protein S-nitrosylation by competing the -SH binding activity. In the present study, we aimed to understand the influence of arsenic on protein S-nitrosylation and the following proteomic changes. By using primary human skin keratinocyte, we found that arsenic treatment decreased the level of protein S-nitrosylation. This was coincident to the decent expressions of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS). By using LC-MS/MS, around twenty S-nitrosoproteins were detected in the biotin-switched eluent. With the interest that arsenic not only regulates posttranslational S-nitrosylation but also separately affects protein’s translation expression, we performed two-dimensional gel electrophoresis and found that 8 proteins were significantly decreased during arsenic treatment. Whether these decreased proteins are the consequence of protein S-nitrosylation will be further investigated. Taken together, these results provide a finding that arsenic can deplete the binding activity of NO and therefore reduce protein S-nitrosylation.

Urinary trichloroacetic acid (TCAA) and baseline blood trihalomethanes (THMs) have been measured as biomarkers of exposure to drinking water disinfection by-products (DBPs) that have been associated with increased risk of cancers and adverse reproductive outcomes. This study aimed to identify predictors of urinary TCAA and baseline blood THMs among men in China. Urine samples, blood samples, and information on socio-demographic factors and water-use activities were collected from 2216 men who participated in a cross-sectional study of exposure to drinking water DBPs and reproductive health during 2011 to 2012. Urinary TCAA and baseline blood THMs including chloroform (TCM), bromodichloromethane (BDCM), dibromochloromethane (DBCM), and bromoform (TBM) were analyzed. Multivariable linear regression was used to evaluate predictors of urinary TCAA and baseline blood THM concentrations. Tap water consumption was significantly associated with creatinine-adjusted urinary TCAA concentration (β = 0.23 μg/g creatinine per log10 unit; 95% CI: 0.12, 0.35). Men with surface water source had 0.13 (95% CI: 0.00, 0.27) higher mean creatinine-adjusted urinary TCAA concentrations than those with ground water source. Smoking was associated with lower concentration of creatinine-adjusted urinary TCAA. Age was significantly associated with baseline blood Br-THM (sum of BDCM, DBCM, and TBM) concentration (β = 0.01 ng/L per unit; 95% CI: 0.00, 0.02). Increased household income was associated with decreased concentrations of baseline blood BDCM and Br-THMs. Our results suggest that tap water consumption, water source, smoking, age, and household income as the primary determinants of exposure to drinking water DBPs should be considered in exposure assessment.

The C-terminal end of the heavy chain of human plasma prekallikrein or kallikrein contains a binding site for high-molecular-weight kininogen, the nonenzymatic procofactor of contact activation. To further map this binding site, a series of overlapping peptides were synthesized. The amount of kallikrein that bound to kininogen-coated microtiter plate wells in the presence of increasing concentrations of each peptide was determined by kallikrein amidolytic activity. A peptide encompassing Lys266-Gly295 of kallikrein, conformationally constrained by a disulfide bond, displayed the lowest Kd value (approximately 67 microM). The linear peptide, Leu262-Gly295, displayed lower affinity (129 microM). N-terminal or C-terminal truncation/extension peptides of this sequence diminished binding activity. Since the closely related protein, factor XI, has been shown to bind kininogen, a kallikrein-based peptide (Phe56-Gly86) homologous to the binding domain of FXI, was examined and found to possess less, but significant, binding affinity for kininogen (Kd 530 microM). Isothermal titration calorimetry was used to assess binding between the kallikrein-based peptides and a peptide encompassing the kallikrein binding domain in kininogen (Ser565-Lys595). Leu262-Gly295 possesses potent binding activity (Kd 52 microM), while Phe56-Gly86 displays poorer binding activity (Kd 400 microM). These interactions are endothermic and entropically favored, suggesting that a conformational rearrangement takes place upon binding. We conclude that the binding site for kininogen within prekallikrein is composed of discontinuous linear segments that form a contiguous surface in the folded protein.

In order to investigate hypoglycemic effects of Momordica charantia(MC) leaves saponins, saponins with purity of 86.0% were extracted from leaves of MC. The extracts were used to treat hyperglycemic rats and the damaged insulinoma cell RIN-M5F in vitro. The effects of MC leaves extracts on the metabolic parameters (serum glucose, insulin, glycogen, Superoxide Dismutase(SOD) activity and Malonedialdehyde (MDA) levels of hyperglycemic rats and the effects on RIN-M5F cells were observed. The results showed that there was a significant increase in serum insulin and glycogen, accompanied by a decrease in serum glucose and MDA in treated rats with MC leaves saponins. And the extracts also exhibited obvious protection and repairation effects on RIN-M5F cells.

Homogeneous pre-pro-GnRH/GAP protein was recently synthesized in 100 mg quantities by solid-phase methods and surprisingly, the synthetic pre-pro-protein, which normally does not escape the endoplasmic recticulum, was found to inhibit the release of prolactin from cultured pituitary cells. This is the first demonstration of significant biological activity associated with a precursor protein and provides the rationale for its further study. We now report the results of our initial examination of the conformational properties of pre-pro-GnRH/GAP protein as a prelude to solving its solution phase conformation by homonuclear1H-NMR protocols. Thermal andpH titration fluorescence and circular dichroism spectroscopies reveal that the protein is resistant to thermal-induced conformational changes but is particularly sensitive topH-induced conformational changes; while Asp/Glu and Arg residues may contribute to structural stability, His and Lys residues predominate. Pre-pro-GnRH/GAP is about 30% helix in the range of 2–40°C; however, even at 90°C, the peptide retains nearly 50% of its helix character. There is no evidence for a cooperative transition; for this reason, differential scanning calorimetry failed to yield a defined transition thermogram. Pre-pro-GnRH/GAP apparently does not pass through a transition state as a function of temperature but appears to flex and retain a high percentage of helix structure, resulting in subtle changes in secondary structure. There is no discernible isodichroic point. On either side of the neutralpH range, however, there are dramatic changes in structure that result in nonreversible denaturation of the protein. Relative to N(Ac)Trp-amide, the emission position of intrinsic Trp fluorescence of pre-pro-GnRH/GAP is blue shifted to 338 nm, indicating that the microenvironment(s) encompassing the 2 Trp residues are buried within the protein structure. Synthetic pre-pro-GNRH/GAP is a substrate for GAP-releasing enzyme (the proposed physiologically relevant processing enzyme of the precursor protein) and yields GAP peptide (D14–I69). Of the other serine proteinases tested (trypsin, plasmin, kallikrein), only GAP-releasing enzyme shows this specificity of cleavage. Hierarchical cleavage observed in the time course of proteolysis with trypsin, however, suggests that other peptide products might be formed from GAP once it is processed from the precursor protein by cleavage at sites other than the primary processing site catalyzed by enzymes other than GAP-releasing enzyme. The primary processing site for GAP-releasing enzyme (GLRPGGKR) is thus accessible in the precursor protein, consistent with our hypothesis that the recognition sequence is located at the surface of the protein and acts as a recognition element for the processing endoproteinase. The conformation of the precursor protein is dynamic, supporting the idea that intracellular (and/or intragranular) conditions may play a role in regulation of endoproteolysis. Conformational flexing of the pro-hormone in response to intracellular conditions may serve to differentially expose various processing sites which may help explain tissue specificity of processing.

Unique sequence-binding sites are exposed on the surface of high molecular weight kininogen which complex prekallikrein or factor XI with high affinity and specificity. A sequence comprising 31 residues of the mature kininogen molecule (Asp565-Lys595) retains full binding activity for prekallikrein (KD = 20 nM) and assumes a complex folded structure in solution which is stabilized by long-range interactions between N- and C-terminal residues. The sequence Trp569-Lys595 (27 residues) shows only 28% of this binding affinity and lacks the key structural features required for protein recognition (Scarsdale, J. N., and Harris, R. B., J. Prot. Chem. 9, 647-659, 1990). We were thus able to predict that N- or C-terminal truncations of the binding-site sequence would disrupt the conformational integrity required for binding. Two new peptides of 20- and 22- residues have now been synthesized and their solution phase structures examined. These peptides are N- and C-terminal truncations, respectively, of the 27-residue sequence and correspond to the sequences Asp576-Lys595 and Trp569-Asp590 of high molecular weight kininogen. The results of fluorescence emission and circular dichroism (CD) spectroscopies in the range 25-90 degrees C and from differential scanning calorimetry (DSC) all substantiate the idea that the C-terminal truncation peptide binds prekallikrein 35-fold poorer than the 31-residue peptide because it is relatively unordered and possesses a less stable structure. Surprisingly, the N-terminal truncation peptide (20-mer) shows structural stability even at elevated temperatures and, like the 31-residue peptide, undergoes cold-induced denaturation observable in the DSC. 2D-NMR analysis of the 20-residue peptide revealed two distinct structures; one conformer possesses a more compact, folded structure than the other. However, the predicted structures assumed by either conformer are very different from those of either the 31- or 27-residue peptides. Hence, the binding affinity of the 20-residue peptide is 60-fold poorer than that for the 31-residue peptide because it assumes a nonproductive binding conformation(s).

The TSG101 protein has been implicated in multiple biological functions including regulation of gene transcription, vesicular trafficking, cellular growth and differentiation. However, the cellular signals that control TSG101 functions are unclear. Here, we demonstrate that TSG101 is upregulated during keratinocyte differentiation in both human foreskin tissue and reconstructed organotypic skin cultures. In addition, we found that TSG101 siRNA inhibits calcium-induced early differentiation of human foreskin keratinocytes, indicating an essential and downstream role for TSG101 in this process. Furthermore, the PKC agonist TPA promotes expression of TSG101 and keratin 10 in keratinocytes under low calcium conditions, while co-treatment with the PKC inhibitor GF 109203X blocks TPA-induced TSG101 and keratin 10 upregulation. Previous work has established that the TSG101 gene is controlled by a TATA-less promoter that harbors a Sp1-binding site. Here we show that both calcium and TPA activate PKC, stimulate phosphorylation of Sp1, and augment the activity of the TSG101 promoter in a manner dependent on its Sp1-binding site. Release of calcium from intracellular stores with thapsigargin, an endoplasmic reticulum Ca2+-ATPase inhibitor that elevates intracellular free Ca2+ without activating PKC, does not affect Sp1 phosphorylation and TSG101 promoter activity. Taken together, these data suggest that an intracellular calcium store independent PKC-Sp1 signaling pathway induces early keratinocyte differentiation through upregulation of TSG101.

Objective:To explore the hemostatic and analgesic effect of Gonghuan Zhixue Tablet (GHZXT) on mice and to produce experimental evidence for exploiting new drug for endome trorrhagia caused by Cu-intrauterine contraceptive device (Cu-IUD). Meth ods: Compared with 6-aminocaproic acid and notoginseng, the effects of GH ZXT on clotting and bleeding time of mice with capillary method and severed tail were investigated; and compared with aspirin, the analgesic effects of GHZXT on mice were investigated with hot plate and torsive body method. Results: The clotting time of mice was remarkably shortened with a rising of the dos age of GHZXT and the difference between each therapeutic group and distilled wat er group was remarkable. As compared with distilled water group, the bleeding ti me of each dosage group of GHZXT was obviously shortened; and each dosage of GHZ XT could prolong the time of pain reaction to hot plate and decrease the degree of torsive body of the mice. Conclusion: Pharmacological experiment has proved that GHZXT has evident hemostatic and analgesic function.

Silver nanoparticles (AgNPs) are one of the most effective disinfectants for eliminating pathogenic microbial contamination from water. The difficult separation of Ag-based disinfectants from water impedes their large-scale application. In this study, Ag-coated Fe3O4@SiO2 magnetic composite material (MCM-Ag) is prepared by a facile route and its antibacterial activity against Escherichia coli (E. coli) is also investigated by analyzing the growth and morphology of cells after treatment. Results show that the fixed AgNPs (10–20 nm) are well dispersed on the surface of MCM-Ag. Some AgNPs are also embedded in the structure of the silica shell to avoid oxidation and release, which enhances the stability of this disinfectant. The antibacterial effect of released Ag+ is distinguished from that of AgNPs by monitoring E. coli growth in presence or absence of O2. MCM-Ag with its ferromagnetic property can be repetitively used 5 times and the possible contamination of disinfectant to environment is avoided. MCM-Ag can damage the functional groups of vital enzymes and proteins of E. coli by released Ag+ and destroy the structure of cell membrane by the generated reactive oxygen species. It is concluded that the easily-separated MCM-Ag is an efficient and environment-friendly antibacterial agent, deserving further application.

High molecular weight kininogen, a plasma glycoprotein, circulates as a noncovalent complex with either prekallikrein or factor XI, two other plasma glycoproteins. The binding domain for factor XI within kininogen, Pro556-Met613 (58 residues), wholly contains the binding domain for prekallikrein, Ser565-Lys595 (31 residues), but Trp569-Lys595 (27 residues) retains some ability to bind prekallikrein. Complex formation between these proteins is mediated by recognition between complementary domains. The 58-residue factor XI peptide domain has now been prepared following a strategy of condensation of long-chain peptide fragments prepared using orthogonal chemistry protocols. The 58-, 31-, and 27-residue peptides assume very different structures in aqueous solution as revealed by differential scanning calorimetry, intrinsic fluorescence emission, and circular dichroism spectroscopies. Thus, the 31-residue peptide shows a broad endothermic transition in differential scanning calorimetry (DSC), but the 58-mer undergoes a well-defined, two-state transition (Tm 43 degrees C; transition enthalpy approximately 30 kcal/mol). The 58- and 27-residue peptides continuously lose structure with increasing temperature, but the 31-mer retains significant structure even at temperatures approaching 90 degrees C. Lys595 plays a critical role in maintaining structure through electrostatic contacts, probably with Asp572 in the N-terminal segment of the 31-residue sequence. Isothermal ligand titration calorimetry was used to directly assess the ability of the 31-, 27-, and 58-residue peptides to bind prekallikrein. The 31-residue peptide binds prekallikrein with 25-fold higher affinity (Kd = 1.0 x 10(-6) M) than the 58-residue peptide and with 5.4-fold higher affinity than the 27-residue peptide. Hence, the essential features of the 31-residue peptide domain required for binding prekallikrein are absent in the 58-residue peptide, which is optimized for binding factor XI. The results suggest that a conformational change may occur within kininogen that causes expression of one domain structure in preference to the other.