Association of non-alcoholic fatty liver disease with total testosterone in non-overweight/obese men with type 2 diabetes mellitus.

PURPOSE: Non-alcoholic fatty liver disease (NAFLD) is considered as both a vital risk factor and a consequence of type 2 diabetes mellitus (T2DM). Low total testosterone (TT) is common in men with T2DM, contributing to increased risks of metabolic diseases. This study aimed to investigate the association between TT levels and the prevalence of NAFLD in men with T2DM. METHODS: In this cross-sectional study, 1005 men with T2DM were enrolled in National Metabolic Management Center (MMC) of First Affiliated Hospital of Wenzhou Medical University between January 2017 and August 2021. NAFLD was diagnosed using ultrasound as described by the Chinese Liver Disease Association. Overweight/obesity was defined as body mass index (BMI) ≥ 25 kg/m2 according to WHO BMI classifications. RESULTS: Individuals without NAFLD had higher serum TT levels than those with NAFLD. After adjustments for potential confounding factors, the top tertile was significantly associated with lower prevalence of NAFLD compared with the bottom tertile of TT level [odds ratio (OR) 0.303, 95% confidence interval (CI) 0.281-0.713; P < 0.001]. The association between TT with NAFLD in individuals with normal weight (OR 0.175, 95% CI 0.098-0.315; P < 0.001) was stronger than in individuals with overweight/obesity (OR 0.509, 95% CI 0.267-0.971; P = 0.040). There was a significant interaction of TT with overweight/obesity (P for interaction = 0.018 for NAFLD). CONCLUSION: Higher serum TT was significantly associated with a lower prevalence of NAFLD in men with T2DM. We found that the relationship of TT and NAFLD was stronger in individuals with non-overweight/obesity.

[Expression and regulatory role of ultraconserved long non-coding RNA uc.77 in lung cancer].

Objective: To investigate the effect and molecular mechanism of ultra-conservative long non-coding RNA uc.77 in lung cancer. Methods: Lung cancer tissues and adjacent normal tissues were obtained from 61 patients with lung cancer who were diagnosed with lung cancer and underwent surgery from 2014 to 2016 in the General Hospital of the Southern Theater Command of the People's Liberation Army. Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the uc.77 relative expressions in normal human bronchial epithelial cells 16HBE, lung cancer cell lines, and 61 pair lung cancer tissues. Uc.77 siRNA was transfected into lung cancer cells to interfere with the expression of uc.77, qRT-PCR was used to verify the interference effect, CCK8 method and clone formation experiment were used to detect cell proliferation ability, flow cytometry was used to detect apoptosis and cell cycle changes. H1299 cells transfected with uc.77 siRNA were injected into the subcutaneous right side of BALB/c nude mice to construct a tumor-bearing model for exploring the role of uc.77 on tumor growth. Western blot and qRT-PCR methods were used to detect the protein and mRNA expressions of p21. Results: The relative expression levels of uc.77 in lung cancer cell lines 95D, H1299, A549, H460, H446 and 16HBE-T were significantly higher than that of 16HBE cells (P<0.05). The uc.77 RNA expression levels of lung cancer tissues was significantly higher than that of the adjacent normal tissues (P<0.001). In addition, increased lncRNA uc.77 expression was significantly associated with big tumor size, lymph node metastasis and advanced TNM stage (P<0.05). After transfection with uc.77 siRNA, the expressions of uc.77 in H1299, 95-D and 16HBE-T cells were reduced (P<0.05), and the cell proliferation capacities were reduced at 48 hours and 72 hours (P<0.05). After transfection with uc.77 siRNA-1, the G(0)/G(1) phase cell ratio of H1299 siRNA-1 group [(71.86±3.46)%] was higher than those of H1299-control group [(47.62±5.48)%] and H1299 siRNA-NC group [(61.38±5.62)%, P<0.05], S phase cell ratio of H1299 siRNA-1 group [(14.99±3.61)%] was lower than those of H1299-control group [(34.95±7.05)%] and H1299 siRNA-NC group [(23.75±5.87)%, P<0.05], the apoptosis rate of H1299 siRNA-1 group [(4.90±1.80)%] was higher than those of H1299-control group [(3.30±0.80)%] and H1299 siRNA-NC group [(2.80±1.20)%, P<0.05], the colony formation rate of H1299 siRNA-1 group [(19.20±2.00)%] was lower than those of H1299 control group [(32.60±2.00)%] and H1299 siRNA-NC group [(34.40±1.00)%, P<0.05]. The results of the nude mice tumor formation experiment showed that the tumor volume of the H1299 siRNA-1 group was significantly lower than those of the H1299-control group and the H1299-negative control group (P<0.05), the average tumor weight of H1299 siRNA-1 group was significantly lower than those of H1299-control group and H1299-negative control group (P<0.05), tumor cell growth marker Ki-67 in the H1299 siRNA-1 group showed weak positive, and Ki-67 in the H1299-control group and H1299-negative control group showed positive. The result of qRT-PCR analysis showed that the mRNA expression level of p21 in H1299 siRNA-1 group (2.57±0.45) was higher than those in H1299 control group (1.00±0.00, P=0.001) and H1299 siRNA-NC group (1.52±0.37, P=0.009). The result of western blotting analysis also showed that the expression of p21 protein level in H1299 siRNA-1 group increased. Conclusions: The expression of ultraconserved long non-coding RNA uc.77 is elevated in lung cancer cell lines and lung cancer tissues. Silencing the expression of ultraconservative long noncoding RNA uc.77 can inhibit tumor growth, and blocking uc.77 expression may be a potential therapeutic target for lung cancer.

Taxonomic and functional shifts in the beech rhizosphere microbiome across a natural soil toposequence.

It has been rarely questioned as to whether the enrichment of specific bacterial taxa found in the rhizosphere of a given plant species changes with different soil types under field conditions and under similar climatic conditions. Understanding tree microbiome interactions is essential because, in contrast to annual plants, tree species require decades to grow and strongly depend on the nutritive resources of the soil. In this context, we tested using a natural toposequence the hypothesis that beech trees select specific taxa and functions in their rhizosphere based on the soil conditions and their nutritive requirements. Our 16S rRNA gene pyrosequencing analyses revealed that the soil type determines the taxa colonizing the beech rhizosphere. A rhizosphere effect was observed in each soil type, but a stronger effect was observed in the nutrient-poor soils. Although the communities varied significantly across the toposequence, we identified a core beech rhizosphere microbiome. Functionally, GeoChip analyses showed a functional redundancy across the toposequence, with genes related to nutrient cycling and to the bacterial immune system being significantly enriched in the rhizosphere. Altogether, the data suggest that, regardless of the soil conditions, trees enrich variable bacterial communities to maintain the functions necessary for their nutrition.

Role of carboxylic acid groups in the reduction of nitric oxide by carbon at low temperature, as exemplified by graphene oxide.

Graphene oxide (GO) was utilized to investigate the role of carboxylic acid groups in the reduction of nitric oxide (NO) for the first time. As a result, GO with sufficient carboxylic acid groups reduced 45% of NO at 100 °C. However, GO without these oxygen-containing groups barely reduced NO (lower than 5%) under the same conditions. After reduction of NO, the carboxylic acid group content on GO decreased from 8.32 to 5.22 mmol g-1. Simultaneously, the anhydride group content increased from 0.14 to 0.28 mmol g-1. FTIR spectroscopy also indicated that the carboxylic acid groups transformed into anhydride and lactone groups. Moreover, both transient kinetics and TG-MS studies demonstrated that reactive intermediates formed during the reaction between NO and GO at 100 °C. Based on these results, it was proposed that the carboxylic acid groups participated in NO reduction by consumption and regeneration. This mechanism explains why carbon is usually an effective reductant and catalyst support for NO removal at low temperature.

[Epidemiological investigation of two leptospirosis death cases in Guizhou Province].

Objective: To conduct an epidemiological investigation of two leptospirosis death cases reported in Guizhou Province in 2014. Methods: The information of the patients were investigated and analyzed. The serological detection, samples of the two patients was detected using ELISA and microscopic agglutination test (MAT). Leptospira carrier status of murine host animal in the living environment of the two patients was investigated in October and November of 2014. Leptospires in the kidney were cultured and isolated, the isolates were identified using Leptospira specific PCR and further identified with serogroup specific PCR and the conventional MAT. The relativity between the carrier status of murine and the death cases of human leptospirosis was analyzed. Results: The two death cases of human leptospirosis came from Liping County and the clinical symptoms were consistent with the diagnosis criteria for Leptospirosis. The results of ELISA detection showed that the anti-Leptospira antibody was positive for one of the death cases, MAT identified the serum reacted with sera-group icterohaemorrhagiae Leptospira, while the serum sample of the other case was failed to perform antibody detection due to hemolysis. 1 600 traps were placed in the living environment of the two death cases and 183 murine rodents were trapped. The murine density was 11.44% (183/1 600); 40 leptospirea suspected strains were isolated and all of them were isolated from Apodemus agrarius. The positive rate was 21.86% (40/183); 95 Apodemus agrarius were trapped and the murine density was 5.93% (95/1 600). Species specific PCR identified all the 40 strains as Leptospire. Serogroup specific PCR further identification showed that they were iterohaemorrahgiae serogroup Leptospria. interrogans. Conclusion: Anti-iterohaemorrahgiae Leptospira antibody was detected from one of the two patients. 40 strains of iterohaemorrahgiae serogroup Leptospira interrogans were isolated and all of them were isolated from Apodemus agrarius in the living environment and the serogroup of the Leptospira matched with the serological detection results from patients, which indicated that the two death cases were caused by the infection of iterohaemorrahgiae serogroup Leptospira interrogans, and Apodemus agrarius were the potential source of infection.

[The roles of serum free light chain ratio in the diagnosis and prognosis of newly diagnosed multiple myeloma].

OBJECTIVE: The roles of serum free light chain ratio (sFLCR) in the diagnosis and prognosis of newly diagnosed multiple myeloma (NDMM) patients were analyzed. METHODS: The clinical data was retrospectively analyzed for 82 newly diagnosed multiple myeloma (NDMM) patients in the first affiliated hospital of Soochow University from September 28, 2012 to July 18, 2105. The serum free light chain levels were measured and κ/λ ratios were calculated, so we could analyze the roles of sFLCR in the diagnosis and prognosis of newly diagnosed multiple myeloma (NDMM) patients. RESULTS: It was 85.5% (70/82) positive of M protein by serum protein electrophoresis (SFE) and 93.9%(77/82) by serum immunofixation electrophoresis (IFE). Both sFLC and sFLCR abnormalities were 96.3% (79/82). The estimated 40-months overall survival was 87% for the high free light chain ratio group (sFLCR ≥100 or≤0.01) and 61% for the low free light chain ratio group (0.01

Osteocytic connexin hemichannels suppress breast cancer growth and bone metastasis.

Although the skeleton is one of the predominant sites for breast cancer metastasis, why breast cancer cells often become dormant after homing to bone is not well understood. Here, we reported an intrinsic self-defense mechanism of bone cells against breast cancer cells: a critical role of connexin (Cx) 43 hemichannels in osteocytes in the suppression of breast cancer bone metastasis. Cx43 hemichannels allow passage of small molecules between the intracellular and extracellular environments. The treatment of bisphosphonate drugs, either alendronate (ALN) or zoledronic acid (ZOL), opened Cx43 hemichannels in osteocytes. Conditioned media (CM) collected from MLO-Y4 osteocyte cells treated with bisphosphonates inhibited the anchorage-independent growth, migration and invasion of MDA-MB-231 human breast cancer cells and Py8119 mouse mammary carcinoma cells, and this inhibitory effect was attenuated with Cx43(E2), a specific hemichannel-blocking antibody. The opening of osteocytic Cx43 hemichannels by mechanical stimulation had similar inhibitory effects on breast cancer cells and this inhibition was attenuated by Cx43(E2) antibody as well. These inhibitory effects on cancer cells were mediated by ATP released from osteocyte Cx43 hemichannels. Furthermore, both Cx43 osteocyte-specific knockout mice and osteocyte-specific Δ130-136 transgenic mice with impaired Cx43 gap junctions and hemichannels showed significantly increased tumor growth and attenuated the inhibitory effect of ZOL. However, R76W transgenic mice with functional hemichannels but not gap junctions in osteocytes did not display a significant difference. Together, our studies establish the specific inhibitory role of osteocytic Cx43 hemichannels, and exploiting the activity of this channel could serve as a de novo therapeutic strategy.

[Effect of hydrogen peroxide on the lateral line hair cell regeneration of zebrafish].

Objective:To investigate effect of hydrogen peroxide (H2O2) in the lateral line hair cell growth and regeneration after damage on zebrafish. Method:Select 5 dpf zebrafish, each group of 10, randomly divided into A control group: the system of water culture. B H2O2 group: 10 µmol/L, 20 µmol/L H2O2 solution to replace three times a day. C neomycin group: treatment with system water after 1 h culture by 200 µmol/L neomycin. D neomycin + H2O2 group: 20 µmol/L H2O2 solution to replace three times a day after 200 µmol/L neomycin treatment for 1 h. E cisplatin group: treatment with system water after 3 h culture by 1 000 µmol/L cisplatin. F cisplatin + H2O2 group: 20 µmol/L H2O2 solution to replace three times a day after 1 000 µmol/L cisplatin treatment for 3 h. Each group in H2O2 treatment for 0 h, 24 h, 48 h was marked their hair cells by immunofluorescence method and count the P1, P7, P8 neuromasts under the fluorescence microscope. Repeat 3 times. Result:The number of hair cells on P1, P7, P8 three neuramasts among 5 to 7 dpf zebrafish were 9.364±0.901(n=11),9.645±0.598(n=15),9.922±0.862(n=13), no obvious difference (P>0.05); 10µmol/L, 20µmol/L H2O2 treated zebrafish for 48 h, the numbers were 11.540±0.741,11.905±0.607,compaired with the control group(10.841±0.389), P<0.05; neomycin+ H2O2 48 h and neomycin 48 h respectively were 10.600±0.689,8.767±0.603, P<0.01; cisplatin+ H2O2 48 h and cisplatin 48 h were 5.967±1.086,5.633±1.548, P>0.05. Conclusion:20 µmol/L H2O2 promotes the development of lateral line hair cells of zebrafish; H2O2 promotes the regeneration of the lateral line hair cells after injury of neomycin, but not cisplatin.

Management with willow short rotation coppice increase the functional gene diversity and functional activity of a heavy metal polluted soil.

We studied the microbial functional diversity, biochemical activity, heavy metals (HM) availability and soil toxicity of Cd, Pb and Zn contaminated soils, kept under grassland or short rotation coppice (SRC) to attenuate the risks associated with HM contamination and restore the soil ecological functions. Soil microbial functional diversity was analyzed by the GeoChip, a functional gene microarray containing probes for genes involved in nutrient cycling, metal resistance and stress response. Soil under SRC showed a higher abundance of microbial genes involved in C, N, P and S cycles and resistance to various HM, higher microbial biomass, respiration and enzyme activity rates, and lower HM availability than the grassland soil. The linkages between functional genes of soil microbial communities and soil chemical properties, HM availability and biochemical activity were also investigated. Soil toxicity and N, P and Pb availability were important factors in shaping the microbial functional diversity, as determined by CCA. We concluded that in HM contaminated soils the microbial functional diversity was positively influenced by SRC management through the reduction of HM availability and soil toxicity increase of nutrient cycling. The presented results can be important in predicting the long term environmental sustainability of plant-based soil remediation.

Differential impact of adenosine nucleotides released by osteocytes on breast cancer growth and bone metastasis.

Extracellular ATP has been shown to either inhibit or promote cancer growth and migration; however, the mechanism underlying this discrepancy remained elusive. Here we demonstrate the divergent roles of ATP and adenosine released by bone osteocytes on breast cancers. We showed that conditioned media (CM) collected from osteocytes treated with alendronate (AD), a bisphosphonate drug, inhibited the migration of human breast cancer MDA-MB-231 cells. Removal of the extracellular ATP by apyrase in CM abolished this effect, suggesting the involvement of ATP. ATP exerted its inhibitory effect through the activation of purinergic P2X receptor signaling in breast cancer cells evidenced by the attenuation of the inhibition by an antagonist, oxidized ATP, as well as knocking down P2X7 with small interfering RNA (siRNA), and the inhibition of migration by an agonist, BzATP. Intriguingly, ATP had a biphasic effect on breast cancer cells-lower dosage inhibited but higher dosage promoted its migration. The stimulatory effect on migration was blocked by an adenosine receptor antagonist, MRS1754, ARL67156, an ecto-ATPase inhibitor, and A2A receptor siRNA, suggesting that in contrast to ATP, adenosine, a metabolic product of ATP, promoted migration of breast cancer cells. Consistently, non-hydrolyzable ATP, ATPγS, only inhibited but did not promote cancer cell migration. ATP also had a similar inhibitory effect on the Py8119 mouse mammary carcinoma cells; however, adenosine had no effect owing to the absence of the A2A receptor. Consistently, ATPγS inhibited, whereas adenosine promoted anchorage-independent growth of MDA-MB-231 cells. Our in vivo xenograft study showed a significant delay of tumor growth with the treatment of ATPγS. Moreover, the extent of bone metastasis in a mouse intratibial model was significantly reduced with the treatment of ATPγS. Together, our results suggest the distinct roles of ATP and adenosine released by osteocytes and the activation of corresponding receptors P2X7 and A2A signaling on breast cancer cell growth, migration and bone metastasis.

Identification and antibacterial characteristics of an endophytic fungus Fusarium oxysporum from Lilium lancifolium.

AIMS: The aim of this study is to isolate and identify an endophytic fungus with antibacterial activity from the Asian medicinal and culinary plant Lilium lancifolium and to study the characteristics of its major antibacterial fractions. METHODS AND RESULTS: After strict sample sterilization, an endophytic fungus BH-3 with great antibacterial activity against Leuconostoc mesenteroides was isolated from the bulbs of L. lancifolium and was identified as Fusarium oxysporum on the basis of internal transcribed spacer (ITS) rDNA sequence and morphological traits. After partial purification including superfiltration and gel filtration, the major antibacterial fractions were found to be the substances with the molecular mass ranging from 35 to 60kDa, mainly 55kDa. The partially purified antibacterial fractions were stable at thermal processes, with more than 80% of activity left at 60°C for 1h, and even 70·75% left at 121°C for 15min. 90·33-98·97% of activity was observed in the pH range of 4·0-7·0. But the fractions were sensitive to different proteases. CONCLUSIONS: Endophytic strain F. oxysporum BH-3 isolated from the bulbs of L. lancifolium produced protein-like antibacterial metabolites. The antibacterial assay against Leuc. mesenteroides indicated that the fractions were stable at thermal processes and wide pH conditions, but sensitive to proteolyses. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides an increasing understanding of endophytic F. oxysporum in L. lancifolium and its metabolites, which have a great potential in food industry as antibacterial agents.

Effectiveness and safety of patient initiated single-dose versus continuous low-dose antibiotic prophylaxis for recurrent urinary tract infections in postmenopausal women: a randomized controlled study.

This study evaluated patient-initiated single-dose antibiotic prophylaxis and continuous long-term low-dose daily antibiotic use for the prevention of recurrent urinary tract infections (UTI) in 68 postmenopausal women. The women were randomized to take a low-dose antibiotic each night (continuous group, n = 37) or a single-dose antibiotic each time they experienced conditions predisposing to UTI (intermittent group, n = 31). During the 12-month study, 1.4 and 1.9 UTIs/patient developed in the continuous and the intermittent groups, respectively, which was significantly lower than the incidence of UTIs in the previous 12 months in these patients (4.7 and 5.1 UTIs/patient, respectively). The incidence of gastro intestinal adverse events was significantly lower in the intermittent group compared with the continuous group (9.1% versus 30.0%). In conclusion, patient-initiated single-dose intermittent antibiotic prophylaxis was as effective as low-dose daily antibiotic prophylaxis in the treatment of recurrent UTIs in post menopausal women and was associated with fewer gastrointestinal adverse events.

[Epidemic situation analysis of Japanese encephalitis in 2005.].

BACKGROUND: To investigate the epidemic situation of Japanese encephalitis (JE) in three provinces, Guizhou, Sichuan, and Hubei in 2005. METHODS: Information about epidemic situation of JE, mosquitoes specimens were collected and titers of JE virus in hosts in the above three surveillance sites were determined. RESULTS: The reported cases of JE in Guizhou, Sichuan, and Hubei province accounted for 40.7% of total cases in 2005 in China. The numbers of cases in Guizhou and Sichuan ranked at the first two in China, morbidity exceeded 1/100,000, which was higher than average level in China. Zero to 10 years old children accounted for 90% in reported cases. Most of the cases were children lived at home. Almost all JE cases were presented from June to September, but most cases were reported between July and August. Investigaton of the density of vector showed that the dominant mosquitoes were Culex, especially the Culex tritaeniorhynchus. CONCLUSION: The epidemic status of JE was similar among the three provinces and the whole country. The number of JEV cases in Guizhou and Sichuan were the highest in China.

Temporal transcriptomic analysis as Desulfovibrio vulgaris Hildenborough transitions into stationary phase during electron donor depletion.

Desulfovibrio vulgaris was cultivated in a defined medium, and biomass was sampled for approximately 70 h to characterize the shifts in gene expression as cells transitioned from the exponential to the stationary phase during electron donor depletion. In addition to temporal transcriptomics, total protein, carbohydrate, lactate, acetate, and sulfate levels were measured. The microarray data were examined for statistically significant expression changes, hierarchical cluster analysis, and promoter element prediction and were validated by quantitative PCR. As the cells transitioned from the exponential phase to the stationary phase, a majority of the down-expressed genes were involved in translation and transcription, and this trend continued at the remaining times. There were general increases in relative expression for intracellular trafficking and secretion, ion transport, and coenzyme metabolism as the cells entered the stationary phase. As expected, the DNA replication machinery was down-expressed, and the expression of genes involved in DNA repair increased during the stationary phase. Genes involved in amino acid acquisition, carbohydrate metabolism, energy production, and cell envelope biogenesis did not exhibit uniform transcriptional responses. Interestingly, most phage-related genes were up-expressed at the onset of the stationary phase. This result suggested that nutrient depletion may affect community dynamics and DNA transfer mechanisms of sulfate-reducing bacteria via the phage cycle. The putative feoAB system (in addition to other presumptive iron metabolism genes) was significantly up-expressed, and this suggested the possible importance of Fe2+ acquisition under metal-reducing conditions. The expression of a large subset of carbohydrate-related genes was altered, and the total cellular carbohydrate levels declined during the growth phase transition. Interestingly, the D. vulgaris genome does not contain a putative rpoS gene, a common attribute of the delta-Proteobacteria genomes sequenced to date, and the transcription profiles of other putative rpo genes were not significantly altered. Our results indicated that in addition to expected changes (e.g., energy conversion, protein turnover, translation, transcription, and DNA replication and repair), genes related to phage, stress response, carbohydrate flux, the outer envelope, and iron homeostasis played important roles as D. vulgaris cells experienced electron donor depletion.

Confidence intervals of similarity values determined for cloned SSU rRNA genes from environmental samples.

The goal of this research was to investigate the influence of the error rate of sequence determination on the differentiation of cloned SSU rRNA gene sequences for assessment of community structure. SSU rRNA cloned sequences from groundwater samples that represent different bacterial divisions were sequenced multiple times with the same sequencing primer. From comparison of sequence alignments with unedited data, confidence intervals were obtained from both a 'double binomial' model of sequence comparison and by non-parametric methods. The results indicated that similarity values below 0.9946 are likely derived from dissimilar sequences at a confidence level of 0.95, and not sequencing errors. The results confirmed that screening by direct sequence determination could be reliably used to differentiate at the species level. However, given sequencing errors comparable to those seen in this study, sequences with similarities above 0.9946 should be treated as the same sequence if a 95% confidence is desired.

Coupling of functional gene diversity and geochemical data from environmental samples.

Genomic techniques commonly used for assessing distributions of microorganisms in the environment often produce small sample sizes. We investigated artificial neural networks for analyzing the distributions of nitrite reductase genes (nirS and nirK) and two sets of dissimilatory sulfite reductase genes (dsrAB1 and dsrAB2) in small sample sets. Data reduction (to reduce the number of input parameters), cross-validation (to measure the generalization error), weight decay (to adjust model parameters to reduce generalization error), and importance analysis (to determine which variables had the most influence) were useful in developing and interpreting neural network models that could be used to infer relationships between geochemistry and gene distributions. A robust relationship was observed between geochemistry and the frequencies of genes that were not closely related to known dissimilatory sulfite reductase genes (dsrAB2). Uranium and sulfate appeared to be the most related to distribution of two groups of these unusual dsrAB-related genes. For the other three groups, the distributions appeared to be related to pH, nickel, nonpurgeable organic carbon, and total organic carbon. The models relating the geochemical parameters to the distributions of the nirS, nirK, and dsrAB1 genes did not generalize as well as the models for dsrAB2. The data also illustrate the danger (generating a model that has a high generalization error) of not using a validation approach in evaluating the meaningfulness of the fit of linear or nonlinear models to such small sample sizes.

Detection and quantification of copper-denitrifying bacteria by quantitative competitive PCR.

We developed a quantitative competitive PCR (QC-PCR) system to detect and quantify copper-denitrifying bacteria in environmental samples. The primers were specific to copper-dependent nitrite reductase gene (nirK). We were able to detect about 200 copeis of nirK in the presence of abundant non-specific target DNA and about 1.2 x 10(3)Pseudomonas sp. G-179 cells from one gram of sterilized soil by PCR amplification. A 312-bp nirK internal standard (IS) was constructed, which showed very similar amplification efficiency with the target nirKfragment (349 bp) over 4 orders of magnitude (10(3)-10(6)). The accuracy of this system was evaluated by quantifying various known amount of nirK DNA. The linear regressions were obtained with a R(2) of 0.9867 for 10(3)copies of nirK, 0.9917 for 10(4) copies of nirK, 0.9899 for 10(5) copies of nirK and 0.9846 for 10(6) copies of nirK. A high correlation between measured nirK and calculated nirK (slope of 1.0398, R(2)=0.9992) demonstrated that an accurate measurement could be achieved with this system. Using this method, we quantified nirK in several A-horizon and stream sediment samples from eastern Tennessee. In general, the abundance of nirK was in the range of 10(8)-10(9) copies g soil(-1) dry weight. The nirK content in the soil samples appeared correlated with NH(4)(N) content in the soil. The activities of copper-denitrifying bacteria were evaluated by quantifying cDNA of nirK. In most of sample examined, the content of nirK cDNA was less than 10(5) copies g soil(-1) dry weight. Higher nirK cDNA content (>10(6) copies g soil(-1) dry weight) was detected from both sediment samples at Rattlebox Creek and the Walker Branch West Ridge. Although the stream sediment samples at the Walker Branch West Ridge contained less half of the nirK gene content as compared to A-horizon sample, the activities of copper-denitrifying bacteria were almost 600 times higher than in the A-horizon sample.

Laser desorption mass spectrometry for microbial DNA analysis.

Recently, we demonstrated that a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS) can be used to determine the molecular weight of polymerase chain reaction (PCR) products of intact 16S rRNA regions and to profile their restriction digests. This is the first time that MALDI-TOF MS with ultraviolet (UV) photoionization has been used to analyze a PCR product of approximately 1600 nucleotides in length.

Interaction of 3,4-dienoyl-CoA thioesters with medium chain acyl-CoA dehydrogenase: stereochemistry of inactivation of a flavoenzyme.

The medium chain acyl-CoA dehydrogenase is rapidly inhibited by racemic 3,4-dienoyl-CoA derivatives with a stoichiometry of two molecules of racemate per enzyme flavin. Synthesis of R- and S-3,4-decadienoyl-CoA shows that the R-enantiomer is a potent, stoichiometric, inhibitor of the enzyme. alpha-Proton abstraction yields an enolate to oxidized flavin charge-transfer intermediate prior to adduct formation. The crystal structure of the reduced, inactive enzyme shows a single covalent bond linking the C-4 carbon of the 2,4-dienoyl-CoA moiety and the N5 locus of reduced flavin. The kinetics of reversal of adduct formation by release of the conjugated 2,4-diene were evaluated as a function of both acyl chain length and truncation of the CoA moiety. The adduct is most stable with medium chain length allenic inhibitors. However, the adducts with R-3,4-decadienoyl-pantetheine and -N-acetylcysteamine are some 9- and >100-fold more kinetically stable than the full-length CoA thioester. Crystal structures of these reduced enzyme species, determined to 2.4 A, suggest that the placement of H-bonds to the inhibitor carbonyl oxygen and the positioning of the catalytic base are important determinants of adduct stability. The S-3,4-decadienoyl-CoA is not a significant inhibitor of the medium chain dehydrogenase and does not form a detectable flavin adduct. However, the S-isomer is rapidly isomerized to the trans-trans-2,4-conjugated diene. Protein modeling studies suggest that the S-enantiomer cannot approach close enough to the isoalloxazine ring to form a flavin adduct, but can be facilely reprotonated by the catalytic base. These studies show that truncation of CoA thioesters may allow the design of unexpectedly potent lipophilic inhibitors of fatty acid oxidation.