Exploring the impact of gut microbiota on liver health in mice and patients with Wilson disease.

BACKGROUND AND AIMS: Distinctive gut microbial profiles have been observed between patients with Wilson disease (WD) and healthy individuals. Despite this, the exact relationship and influence of gut microbiota on the advancement of WD-related liver damage remain ambiguous. This research seeks to clarify the gut microbiota characteristics in both human patients and mouse models of WD, as well as their impact on liver injury. METHODS: Gut microbial features in healthy individuals, patients with WD, healthy mice and mice with early- and late-stage WD were analysed using 16S rRNA gene sequencing. Additionally, WD-afflicted mice underwent treatment with either an antibiotic cocktail (with normal saline as a control) or healthy microbiota (using disease microbiota as a control). The study assessed gut microbiota composition, hepatic transcriptome profiles, liver copper concentrations and hepatic pathological injuries. RESULTS: Patients with hepatic WD and mice with WD-related liver injury displayed altered gut microbiota composition, notably with a significant reduction in Lactobacillus abundance. Additionally, the abundances of several gut genera, including Lactobacillus, Veillonella and Eubacterium coprostanoligenes, showed significant correlations with the severity of liver injury in patients with WD. In WD mice, antibiotic treatment or transplantation of healthy microbiota altered the gut microbial structure, increased Lactobacillus abundance and modified the hepatic transcriptional profile. These interventions resulted in reduced hepatic copper concentration and alleviation of WD-related liver injury. CONCLUSIONS: Individuals and mice with pronounced WD-related liver injury exhibited shifts in gut microbial composition. Regulating gut microbiota through healthy microbiota transplantation emerges as a promising therapeutic approach for treating WD-related liver injury.

Initiating antiretroviral therapy within 2 weeks of anti-Pneumocystis treatment does not increase mortality or AIDS-defining events in patients with HIV-associated moderate to severe Pneumocystis pneumonia: results of a prospective observational multicenter study.

BACKGROUND: The mortality rate remains high among patients with coinfection with Pneumocystis pneumonia (PCP) and HIV. The timing for initiation of antiretroviral therapy (ART) after a diagnosis of moderate to severe PCP remains controversial, however. We therefore designed the present study to determine the optimal timing for ART initiation in AIDS-associated PCP (AIDS/PCP) patients. METHODS: This was a multicenter, observational, prospective clinical trial. Eligible participants were recruited from 14 hospitals in mainland China, and assigned to an Early ART arm (initiation of ART ≤ 14 days after PCP diagnosis) and a Deferred ART arm (initiation of ART > 14 days after PCP diagnosis). The primary outcomes were death and the incidence of AIDS-defining events at week 48. The secondary outcomes were the changes in CD4+ T-cell counts from baseline values at weeks 12, 24, and 48, the virological suppression rate at week 24 and week 48, the rate of development of PCP-associated immune reconstitution inflammatory syndrome (PCP/IRIS), and the rate of adverse events over 48 weeks. RESULTS: The present study was performed using the data of 363 participants, with 169 participants in the Early ART arm, and 194 participants in the Deferred ART arm. Immunological and virological outcomes were found to be similar in both treatment arms. At week 48, there were no significant differences for the incidence of mortality (20 vs. 26, p = 0.860), and AIDS-defining events (17 vs. 26, p = 0.412). Over 48 weeks, the rates of PCP/IRIS (2 vs. 3, p = 1.000), adverse events (70 vs. 72, p = 0.465), and grade 3 or 4 adverse events (28 vs. 34, p = 0.919) did not reach statistical significance. A significant difference observed between two study arms was that 11 participants (55.0%) in the Early ART arm compared to 23 participants (88.5%) in the Deferred ART arm (p = 0.026) succumbed before ART had ever been started. CONCLUSIONS: Early ART initiation results in no increase in mortality, AIDS-defining events, IRIS, adverse events, and immunological or virological outcomes. These results support the early initiation of ART in patients with moderate to severe AIDS/PCP. Clinical trial registration The present trial was registered at Chinese Clinical Trial Registry (ChiCTR1900021195). Registered 1 February 2019, http://www.chictr.org.cn/showproj.aspx?proj=35362 .

Thrombotic antiphospholipid syndrome in a child with human immunodeficiency virus: a rare case report.

BACKGROUND: Antiphospholipid syndrome (APS) is a non-inflammatory autoimmune disorder induced by antiphospholipid antibodies, which occurs exceedingly rarely in pediatric population and even more rarely reported in HIV positive children. A case of 11 years old boy had a sudden onset of swelling in his left lower leg along with pain which were worsening gradually. Initially, topical ointment was applied for 1 month which were ineffective in reducing pain and swelling. Instead, the symptoms were aggravated and suddenly spread to the proximal thigh, accompanied by dyskinesia of left lower leg. Both color doppler ultrasonography and vascular CT scan of left lower leg revealed deep venous thrombosis. His serum anti-phospholipid antibodies (aPLs) were tested positive. He was a known case of HIV virological failure with substantial HIV viral load (VL) despite receiving regular antiretroviral therapy (ART). His symptoms improved after giving aggressive antithrombotic and high dose corticosteroid treatments. CONCLUSION: When pediatric patients develop thrombotic disease, APS also needs to be ruled out. The autoantibodies levels should be routinely tested to look for recurrent thrombosis in children with HIV/AIDS.

AIDS-associated Talaromyces marneffei central nervous system infection in patients of southwestern China.

BACKGROUND: The clinical and laboratory characteristics of AIDS-associated Talaromyces marneffei infection, a rare but a fatal mycosis disease of the central nervous system, remain unclear. CASE PRESENTATION: Herein, we conducted a retrospective study of ten AIDS patients with cerebrospinal fluid culture-confirmed central nervous system infection caused by Talaromyces marneffei. All 10 patients were promptly treated with antifungal treatment for a prolonged duration and early antiviral therapy (ART). Among them, seven patients were farmers. Nine patients were discharged after full recovery, while one patient died during hospitalization, resulting in a mortality rate of 10%. All patients initially presented symptoms and signs of an increase in intracranial pressure, mainly manifesting as headache, dizziness, vomiting, fever, decreased muscle strength, diplopia or even altered consciousness with seizures in severe patients. Nine patients (90%) showed lateral ventricle dilatation or intracranial infectious lesions on brain CT. Cerebrospinal fluid findings included elevated intracranial pressure, increased leukocyte count, low glucose, low chloride and high cerebrospinal fluid protein. The median CD4+ T count of patients was 104 cells/µL (IQR, 36-224 cells/µL) at the onset of the disease. The CD4+ T cell counts of three patients who eventually died were significantly lower (W = 6.00, p = 0.020) than those of the patients who survived. CONCLUSIONS: The common clinical symptoms of T. marneffei central nervous system infection are associated with high intracranial pressure and intracranial infectious lesions. Earlier recognition and diagnosis and a prolonged course of amphotericin B treatment followed by itraconazole combined with early ART might reduce the mortality rate.

[De novo domestication to create new crops].

Crop improvement by domestication and traditional breeding often results in fitness penalties and loss of genetic diversity, which greatly threatens crop production and food security under the challenging global climate. De novo domestication has been proposed as a novel strategy for crop breeding. By combining multi-omics, genome editing and synthetic biology approaches, domestication of wild or semi-wild plant species can be accelerated by rapidly introducing desirable traits without causing an associated drag on their inherent traits. In this review, we summarize the history of crop domestication, emphasize the urgency for breeding strategy innovation, and discuss recent progress of de novo crop domestication.

Synthesis and Biological Evaluation of Novel Dehydroabietic Acid-Oxazolidinone Hybrids for Antitumor Properties.

Novel representatives of the important group of biologically-active, dehydroabietic acid-bearing oxazolidinone moiety were synthesized to explore more efficacious and less toxic antitumor agents. Structures of all the newly target molecules were confirmed by IR, ¹H-NMR, 13C-NMR, and HR-MS. The inhibitory activities of these compounds against different human cancer cell lines (MGC-803, CNE-2, SK-OV-3, NCI-H460) and human normal liver cell line LO2 were evaluated and compared with the commercial anticancer drug cisplatin, using standard MTT (methyl thiazolytetrazolium) assay in vitro. The pharmacological screening results revealed that most of the hybrids showed significantly improved antiproliferative activities over dehydroabietic acid and that some displayed better inhibitory activities compared to cisplatin. In particular, compound 4j exhibited promising cytotoxicity with IC50 values ranging from 3.82 to 17.76 µM against all the test cell lines and displayed very weak cytotoxicity (IC50 > 100 µM) on normal cells, showing good selectivity between normal and malignant cells. Furthermore, the action mechanism of the representative compound 4j was preliminarily investigated by Annexin-V/PI dual staining, Hoechst 33258 staining, which indicated that the compound can induce cell apoptosis in MGC-803 cells in a dose-dependent manner and arrest the cell cycle in G1 phase. Therefore, 4j may be further exploited as a novel pharmacophore model for the development of anticancer agents.

Asymptomatic oral yeast carriage and antifungal susceptibility profile of HIV-infected patients in Kunming, Yunnan Province of China.

BACKGROUND: Oral Candida colonization and its relation with predisposing factors in HIV-infected patients have received wide concerns during recent decades. In this study, we investigated asymptomatic oral Candida carriage rate, species distribution and antifungal susceptibility of 604 HIV-infected patients and 851 healthy individuals in Kunming, Yunnan Province of China. METHODS: Mucosal swab sampling was taken from each subject and CHROMagar Candida agar medium and API 20C AUX system were used to identify yeast isolates. In vitro antifungal susceptibility was tested by the broth microdilution method according to the M27-A2 document of the Clinical and Laboratory Standard Institute (CLSI). RESULTS: The oral yeast colonization rate in HIV-infected patients (49.5%) was higher than that of healthy subjects (20.7%). Candida albicans constituted the most frequent species, accounting for 82.2% of yeast isolates. The remaining species were composed of C. glabrata, C. parapsilosis, C. krusei, C. tropicalis, C. rugosa, C. norvegensis, Pichia ohmeri and Saccharomyces cerevisiae. In HIV-infected patients, asymptomatic oral yeast colonization was associated with low CD4 cell count (<200 cells/mm3) and lack of highly active antiretroviral therapy (HAART). Different Candida species isolated from our samples presented different susceptibility to voriconazole, fluconazole and itraconazole. Amphotericin B had the best inhibiting effect for all isolates. CONCLUSION: Oral yeast colonization in Han Chinese patients with HIV from Kunming had common and unique features and was associated with CD4 cell number and HARRT. Amphotericin B should be used with first priority in controlling Candida infection in Han Chinese patients from Kunming. Our results provide first hand information on monitoring oral yeasts colonization in HIV-infected patients from Kunming, China.

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Livestock wastewater is an important reservoir of antibiotic resistance genes (ARGs), with high environmental risks. We investigated the seasonal variations of distribution and removal of swine wastewater originated high-risk tetracycline resistance genes (TRGs) in horizontal subsurface flow constructed wetlands. The effects of exogenous addition of tetracycline (TC) and copper ion (Cu2+) on the abundance of TRGs in effluent with single and combined pollution of antibiotic and heavy metal were studied. The results showed that all the three high-risk TRGs (tetM, tetO and tetW) were detected in swine wastewater. Wetlands could effectively reduce the ARGs, with the absolute abundance of TRGs in effluent being decreased by 1.1-2.4 and 1.7-2.9 orders of magnitude in summer and winter compared with the influent, respectively. The abundance of TRGs in wetland soils showed the characte-ristics that the outflow side was lower than the inflow side, the non-rhizosphere area was lower than the rhizosphere area, and lower in winter than in summer. In summer and winter, single and combined pollution of TC and Cu2+ in swine wastewater would increase the abundance of TRGs in effluent compared with that in the control. The constructed wetland is suitable for controlling the environmental diffusion of ARGs in livestock wastewater.

Prevalent and highly mobile antibiotic resistance genes in commercial organic fertilizers.

Compost-based organic fertilizers made from animal manures may contain high levels of antibiotic resistance genes (ARGs). However, the factors affecting the abundance and profile of ARGs in organic fertilizers remain unclear. We conducted a national-wide survey in China to investigate the effect of material type and composting process on ARG abundance in commercial organic fertilizers and quantified the contributions of bacterial composition and mobile genetic elements (MGEs) to the structuring of ARGs, using quantitative PCR and Illumina sequencing of 16S rRNA gene amplicons. The tetracycline, sulfonamide, aminoglycoside and macrolide resistance genes were present at high levels in all organic fertilizers. Seven ARGs that confer resistance to clinically important antibiotics, including three ß-lactam resistance genes, three quinolone resistance genes and the colistin resistance gene mcr-1, were detected in 8 - 50% the compost samples, whereas the vancomycin resistance gene vanC was not detected. Raw material type had a significant (p < 0.001) effect on the ARG abundance, with composts made from animal feces except some cattle feces generally having higher loads of ARGs than those from non-animal raw materials. Composting process type showed no significant (p > 0.05) effect on ARG abundance in the organic fertilizers. MGEs exerted a greater influence on ARG composition than bacterial community, suggesting a strong mobility of ARGs in the organic fertilizers. Our study highlights the need to manage the risk of ARG dissemination from agricultural wastes.

Spatiotemporal visualization for the global COVID-19 surveillance by balloon chart.

BACKGROUND: Considering the widespread of coronavirus disease 2019 (COVID-19) pandemic in the world, it is important to understand the spatiotemporal development of the pandemic. In this study, we aimed to visualize time-associated alterations of COVID-19 in the context of continents and countries. METHODS: Using COVID-19 case and death data from February to December 2020 offered by Johns Hopkins University, we generated time-associated balloon charts with multiple epidemiological indicators including crude case fatality rate (CFR), morbidity, mortality and the total number of cases, to compare the progression of the pandemic within a specific period across regions and countries, integrating seven related dimensions together. The area chart is used to supplement the display of the balloon chart in daily new COVID-19 case changes in UN geographic regions over time. Javascript and Vega-Lite were chosen for programming and mapping COVID-19 data in browsers for visualization. RESULTS: From February 1st to December 20th 2020, the COVID-19 pandemic spread across UN subregions in the chronological order. It was first reported in East Asia, and then became noticeable in Europe (South, West and North), North America, East Europe and West Asia, Central and South America, Southern Africa, Caribbean, South Asia, North Africa, Southeast Asia and Oceania, causing several waves of epidemics in different regions. Since October, the balloons of Europe, North America and West Asia have been rising rapidly, reaching a dramatically high morbidity level ranging from 200 to 500/10 000 by December, suggesting an emerging winter wave of COVID-19 which was much bigger than the previous ones. By late December 2020, some European and American countries displayed a leading mortality as high as or over 100/100 000, represented by Belgium, Czechia, Spain, France, Italy, UK, Hungary, Bulgaria, Peru, USA, Argentina, Brazil, Chile and Mexico. The mortality of Iran was the highest in Asia (over 60/100 000), and that of South Africa topped in Africa (40/100 000). In the last 15 days, the CFRs of most countries were at low levels of less than 5%, while Mexico had exceptional high CFR close to 10%. CONCLUSIONS: We creatively used visualization integrating 7-dimensional epidemiologic and spatiotemporal indicators to assess the progression of COVID-19 pandemic in terms of transmissibility and severity. Such methodology allows public health workers and policy makers to understand the epidemics comparatively and flexibly.

[Expression of B7-H1 on peripheral myeloid dendritic cells in patients with HIV infection and its correlation with diseases progression].

OBJECTIVE: To investigate the features of B7-H1 expression on peripheral myeloid dendritic cells (mDCs) in patients with HIV infection and evaluate the correlations between B7-H1 expression and disease progression. METHODS: Peripheral blood samples from 82 treatment-naïve patients with HIV infection, 28 viral complete responders (CRs) under antiretroviral therapy (ART) and 14 healthy controls (HCs) were collected. Flow cytometry was applied to investigate the expression of B7-H1 on mDCs and CD4 cell counts. Plasma HIV-1 viral load was detected by bDNA. RESULTS: The frequency of B7-H1 expression on mDCs were 14.15% +/- 2.63%, 3.31% +/- 0.51% and 0.52% +/- 0.10% in AIDS patients, asymptomatic HIV infected individuals and HCs respectively. As compared with HCs, B7-H1 was significantly up-regulated on mDCs in HIV/AIDS patients. The order was as follows: AIDS patients > asymptomatic HIV infected individuals > HCs (all P < 0.05). Interestingly, the expression of B7-H1 on mDCs in long-term nonprogressors (LTNPs) was 3.12% +/- 1.14%. And it was lower than that in typical progressors (TPs) [8.12% +/- 1.37% (P = 0.001)]. Moreover, the expression of B7-H1 was negatively correlated with CD4 cell counts and positively correlated with plasma viral load in these patients (r = -0.631, P < 0.01 and r = 0.482, P < 0.01 respectively). The expression of B7-H1 on mDCs was significantly lower in ART complete responders than that in AIDS patients (6.59% +/- 1.43% vs 14.15% +/- 2.63%) (P < 0.01). Expression of B7-H1 on mDCs decreased markedly in patients whose CD4 cell counts greatly elevated after a successful antiretroviral treatment. CONCLUSION: The expression of B7-H1 on mDCs is significantly up-regulated in HIV/AIDS patients. With a close correlation with disease status, it acts as a marker of disease progression.

[Reproduction of a murine model of hyperthermic treatment].

OBJECTIVE: To find out the most suitable conditions for a whole body hyperthermia (WBH) model and the influence of these conditions on the blood brain barrier (BBB) disruption and brain edema in rats. METHODS: Forty male Sprague-Dawley (SD) rats were randomly assigned to four groups (n=10 in each group): control group, group A, group B and group C. After anesthesia with pentobarbital, rats were subjected to femoral artery and vein cannulation. Rats of control group were housed at a controlled room temperature (25-26 degrees C) for 4 hours. Rats of group A, group B and group C were exposed to WBH in a biological oxygen supply heated container (relative humidity 65%, wind velocity 25 cm/s) maintained at 34, 36 and 38 degrees C for 3 hours, respectively. Then the rats were removed from the heated container and their body temperature was cooled down for 1 hour. During heating, rectal temperature, heart rate (HR), mean arterial pressure (MAP), pH, partial pressure of oxygen in artery (PaO(2)), partial pressure of carbon dioxide in artery (PaCO(2)), the dosage of anesthetic, and the mortality rate in each group were recorded. Evans blue (EB) was administered into the femoral vein and allowed to circulate for 5 minutes. At the end of the experiment, the animals were perfused with 0.9% saline and heparin through the heart, and the brain was harvested for the examination of BBB permeability, water content and morphological alterations in brain tissues and neurons. RESULTS: The total dosage of pentobarbital was not significantly different among all groups. After WBH for 3 hours, the average rectal temperature was higher than rats without WBH, and the mortality rate was 0, 10%, 10% and 40% in groups control, A, B, C, respectively. HR of groups A, B and C were significantly higher than those of control group; MAP, pH of group A, B and C were significantly lower than those of control group (all P<0.05). Compared to that of control group, water content of the brain and permeability of EB in groups A, B and C were significantly increased (P<0.05 or P<0.01), but there was no marked difference on PaO(2), PaCO(2) and haematocrit (HCT) among groups A, B and C. Morphological investigation showed that there were different degrees of structural changes in brain tissue in groups A, B and C under light microscopy. Under transmission election microscopy, the structure of nerve cells and BBB in group B and group C showed moderate to profound alterations, but there were no changes in group A. CONCLUSION: Rats housed in a biological oxygen supply heat container with the temperature maintained at 36 degrees C for 3 hours could establish an ideal WBH model with notable BBB breakdown, moderate brain edema, and histological changes in brain.

[Studies on extraction process of the main saponin constituents from the stem bark of Kalopanax septemlobus in Guangxi].

Using orthogonal experiment design, the total saponin constituents were obtained by refluxing extraction with alcohol and separated by macroporous adsorption resin and n-Butyl alcohol from the stem bark of Kalopanax septemlobus. According to the purity analysis and the yield, the extraction process was optimized. The results showed that the main saponin constituents were gained with a yield of 1.32% by using macroporous adsorption resin but 1.05% by using n-Butyl alcohol. The former was more efficient than the latter on both yield and color. The optimal process with isolation by macroporous adsorption resin is cheap, simple and practical.

Deficient TP53 expression, function, and cisplatin sensitivity are restored by quinacrine in head and neck cancer.

PURPOSE: To determine the nature and potential pharmacologic reversibility of deficient TP53 expression and function in head and neck squamous cell carcinomas (HNSCC) with wild-type TP53, previously associated with decreased sensitivity to cisplatin therapy. EXPERIMENTAL DESIGN: TP53 genotype, mRNA and protein expression, TP53-induced p21 expression, and TP53 DNA-binding and reporter gene function were determined in a panel of nine previously characterized HNSCC cell lines from the University of Michigan squamous cell carcinoma (UM-SCC) series. The genotoxic drug doxorubicin and the anti-inflammatory and antimalarial drug quinacrine, previously identified as inducers of TP53, were used to examine the nature and potential reversibility of deficient TP53 expression and function. The specific role of inducible TP53 on function and cellular proliferation was confirmed using selective TP53 inhibitor pifithrin-alpha or short hairpin RNA knockdown. The capability of quinacrine to sensitize HNSCC to the cytotoxic effects of cisplatin was assessed. RESULTS: UM-SCC cell lines with wild-type TP53 genotype underexpressed TP53 mRNA and protein when compared with normal human keratinocytes or UM-SCC with mutant TP53. Although doxorubicin failed to induce TP53 expression or functional activity, quinacrine induced TP53 mRNA and protein expression, increased TP53 reporter activity and p21 protein expression, and induced growth inhibition in these wild-type TP53 cell lines. Quinacrine-induced TP53 reporter activity and growth suppression were attenuated by pifithrin-alpha and TP53 short hairpin RNA knockdown. Furthermore, quinacrine sensitized UM-SCC to cisplatin in vitro. CONCLUSIONS: Deficient TP53 mRNA and protein expression underlies decreased function in a subset of HNSCC with wild-type TP53 and can be restored together with cisplatin sensitization by quinacrine.

A novel nuclear factor-kappaB gene signature is differentially expressed in head and neck squamous cell carcinomas in association with TP53 status.

PURPOSE: To determine if gene signatures differentially expressed in head and neck squamous cell carcinomas (HNSCC) are related to alterations in transcription factors nuclear factor-kappaB (NF-kappaB) and TP53 previously associated with decreased cell death, response to therapy, and worse prognosis. EXPERIMENTAL DESIGN: Unique gene signatures expressed by HNSCC lines were identified by cDNA microarray, principal components, and cluster analyses and validated by quantitative reverse transcription-PCR (RT-PCR) and in situ hybridization. Bioinformatic analysis of the promoters and ontogeny of these clustered genes was done. Expression of proteins encoded by genes of a putative NF-kappaB signature, NF-kappaB p65, and TP53 were examined in HNSCC tissue specimens by immunostaining. Predicted promoter binding and modulation of expression of candidate NF-kappaB genes and cell survival were evaluated by p65 chromatin immunoprecipitation (ChIP) and small interfering RNA (siRNA) knockdown. RESULTS: Two groups of HNSCC exhibiting distinct gene signatures were identified: cluster A enriched for histone genes, with a higher prevalence of TP53 promoter binding motifs; and cluster B enriched for injury response genes with NF-kappaB regulatory motifs. Coexpression of cluster B proteins was observed with strong NF-kappaB phospho-p65 and weak TP53 staining, and NF-kappaB phospho-p65 was inversely associated with TP53 (P = 0.02). Promoter binding of the NF-kappaB signature genes was confirmed by p65 ChIP, and down-modulation of their expression and cell death were induced by p65 siRNA. CONCLUSION: NF-kappaB promotes expression of a novel NF-kappaB-related gene signature and cell survival in HNSCC that weakly express TP53, a subset previously associated with inactivated wild-type TP53, greater resistance to chemoradiotherapy, and worse prognosis.

Chronic deep brain stimulation in the rat nucleus accumbens and its effect on morphine reinforcement.

In order to explore a novel method for the treatment of drug abuse, we evaluated the effect of chronic deep brain stimulation (DBS) of the rat nucleus accumbens (NAc) on morphine reinforcement, using a DBS apparatus and an implant method we developed. Thirty-two adult rats weighing 240-260 g were divided into three groups, which included a DBS group (n = 10, administration of surgery, morphine and DBS), a sham DBS group (n = 12, administration of surgery and morphine) and a control group (n = 10, administration of physiological saline). The DBS electrode was stereotaxically implanted into the core of unilateral NAc and connected to an implantable pulse generator. Then, they were fixed to the rat skull. One week later, the rats in each group were intraperitoneally injected with morphine at an increasing dose (10-60 mg/kg) once daily. The rats in the DBS group were administered a 130-Hz high-frequency stimulation (HFS) once daily. A 900-second conditioned place preference (CPP) paradigm was used for determining the effect of electrical stimulation on morphine reinforcement in rats. The data showed that 7-10 days later, the preference score of the DBS group was significantly lower than that of the sham DBS group. The results suggest that chronic HFS of the rat NAc can block CPP induced by morphine and attenuate morphine reinforcement.

[Comparison of Extraction Methods of Extracellular Polymeric Substances from Activated Sludge].

Extracellular polymeric substances (EPSs) are biosynthetic polymers of microbial origin in the sludge activation process and crucially affect the properties of sludge in biological wastewater treatment reactors, such as the formation of sludge flocs, stabilization of sludge structure, and protection of microbes against noxious environmental conditions. However, the EPS extraction efficiency differs significantly according to the extraction method used. In this study, soluble EPSs and loosely bound EPSs can be extracted by centrifugation first and tightly bound EPSs in activated sludge require additional eight treatments for extraction, respectively. Three physical methods (centrifugation, sonication, and heating) and five chemical methods (cation exchange resin, NaOH, formaldehyde+NaOH, EDTA, and formaldehyde+EDTA) were tested, and the content and composition of TB-EPS were analyzed. Meanwhile, the functional groups and elements in TB-EPS were investigated. Results showed that the heating method did not introduce exogenous substances during the EPS extraction process and that the destruction of cells from this method was relatively slight. Heating was shown to be a gentle and efficient method in this study. The three-dimensional excitation and emission matrix (EEM) fluorescence spectroscopy showed that the cation exchange resin method had good extraction effect on humic-like and protein-like substances. As to fulvic-acid-like substances, NaOH was better than the other seven methods. Infrared spectroscopy showed that no notable difference appeared in the functional groups of the TB-EPS extracted by physical methods, whereas chemical methods induced big differences and showed particular bands that did not appear in the TB-EPS extracted by physical methods. Overall, the amounts of TB-EPS elements extracted using chemical methods were greater than those extracted using physical methods. In conclusion, a method must be selected and established for each case, taking into consideration the experimental purpose, and the most appropriate method should be chosen carefully.

cDNA microarray and bioinformatic analysis of nuclear factor-kappaB related genes in squamous cell carcinoma.

Squamous cell carcinomas and several other cancers have been found to exhibit microarray expression profiles that include genes related to nuclear factor (NF)-kappaB, a signal activated transcription factor that is evolutionarily important in regulating early response gene programs to injury and infection. Inhibition of NF-kappaB by expression of a dominant negative signal phosphorylation site mutant of inhibitor-kappaB, IkappaBalphaM, under a tetracycline inducible promoter, established the role of NF-kappaB as an essential molecular switch modulating multiple genes important in the malignant phenotype. Bioinfomatic analysis of the promoter and coding region of IkappaBalphaM-modulated genes has enabled identification of new candidates with and without known NF-kappaB related motifs for validation and functional studies of their relationship to NF-kappaB. These studies illustrate how microarray data can be used to generate a hypothesis regarding regulation of genes by a specific signal transcription factor, and how genetic mutants and bioinformatic analysis can be used to analyze the relative importance of the regulatory molecule to expression of genes involved in the malignant phenotype.

Hepatocyte growth factor/scatter factor differentially regulates expression of proangiogenic factors through Egr-1 in head and neck squamous cell carcinoma.

Hepatocyte growth factor/scatter factor (HGF) and the angiogenesis factors platelet-derived growth factors (PDGF), vascular endothelial growth factor (VEGF), and interleukin-8 (IL-8) are found in elevated concentrations in serum or tumor tissue of patients with head and neck squamous cell carcinomas (HNSCC), suggesting these factors may be coregulated. A cDNA microarray analysis for HGF-inducible genes revealed that HGF also modulates PDGFA expression, a gene recently shown to be inducible by the transcription factor, early growth response-1 (Egr-1). In the present study, we investigated the potential role of HGF-induced Egr-1 in expression of PDGF, VEGF, and IL-8. HGF induced expression of all three factors and Egr-1 expression and DNA-binding activity. The analysis of promoter sequences showed putative Egr-1 binding sites in the PDGFA or VEGF but not in the IL-8 promoter, and HGF-induced Egr-1-binding activity was confirmed by chromatin immunoprecipitation (ChIP) assay. The maximal basal and HGF-induced promoter activity for the PDGFA gene existed within -630 bp of the promoter region, and overexpression of Egr-1 significantly increased such activity. Consistent with this, expression of PDGFA and VEGF but not IL-8 showed corresponding differences with Egr-1 expression in HNSCC tumor specimens and were strongly suppressed by transfection of Egr-1-antisense or small interference RNA (siRNA) oligonucleotides. HGF-induced expression of Egr-1, PDGFA, and VEGF was suppressed by pharmacologic and siRNA inhibitors of mitogen-activated protein kinase kinase 1/2 (MEK1/2) and protein kinase C (PKC) pathways. We conclude that the HGF-induced activation of transcription factor Egr-1 by MEK1/2- and PKC-dependent mechanisms differentially contributes to expression of PDGF and VEGF, which are important angiogenesis factors and targets for HNSCC therapy.

[Evolution of Extracellular Polymeric Substances of the Activated Sludge with Calcium Ion Addition During Set-up Period of Sequencing Batch Reactors].

Great attention has been paid to accelerate the start-up period and enhance floc properties and structural stability in activated sludge reactors with the aid of inorganic chemical agents such as calcium ion. The laboratory-scale sequencing batch reactors (SBRs) were operated continuously for 35 days to investigate the effect of calcium ion (Ca2+) on the physicochemical properties and evolution of extracellular polymeric substances (EPS) of activated sludge during set-up period. When compared to the control (non-calcium ion addition), the addition of 150 mg·L-1Ca2+ to the influent significantly increased the mixed liquid suspended solids (MLSS) and the mixed liquid volatile suspended solids (MLVSS) by 89.6% and 75.6% on 28 d, respectively, and decreased the sludge volume index (SVI) by 47.9% following SBRs set-up. Compared with the control system, the contents of EPS, polysaccharides (PS) and proteins (PN) were increased by 76.4%, 28.8% and 31.6% under the condition of Ca2+ dosage of 150 mg·L-1. The PS/PN ratio was 68.8 for Ca2+ addition, compared to only 36.6 for the control reactor. Analysis using three-dimensional excitation emission matrix fluorescence spectroscopy and Fourier transform infrared spectrum revealed that Ca2+ addition changed the compositional characteristics of EPS. Results from this study provided a fundamental knowledge basis for the improvement of the settling properties of activated sludge with calcium ion addition.