Reproducibility and quantitation of amplicon sequencing-based detection

To determine the reproducibility and quantitation of the amplicon sequencing-based detection approach for analyzing microbial community structure, a total of 24 microbial communities from a long-term global change experimental site were examined. Genomic DNA obtained from each community was used to amplify 16S rRNA genes with two or three barcode tags as technical replicates in the presence of a small quantity (0.1% wt/wt) of genomic DNA from Shewanella oneidensis MR-1 as the control. The technical reproducibility of the amplicon sequencing-based detection approach is quite low, with an average operational taxonomic unit (OTU) overlap of 17.2%±2.3% between two technical replicates, and 8.2%±2.3% among three technical replicates, which is most likely due to problems associated with random sampling processes. Such variations in technical replicates could have substantial effects on estimating β-diversity but less on α-diversity. A high variation was also observed in the control across different samples (for example, 66.7-fold for the forward primer), suggesting that the amplicon sequencing-based detection approach could not be quantitative. In addition, various strategies were examined to improve the comparability of amplicon sequencing data, such as increasing biological replicates, and removing singleton sequences and less-representative OTUs across biological replicates. Finally, as expected, various statistical analyses with preprocessed experimental data revealed clear differences in the composition and structure of microbial communities between warming and non-warming, or between clipping and non-clipping. Taken together, these results suggest that amplicon sequencing-based detection is useful in analyzing microbial community structure even though it is not reproducible and quantitative. However, great caution should be taken in experimental design and data interpretation when the amplicon sequencing-based detection approach is used for quantitative analysis of the β-diversity of microbial communities.     

Combined Genomics and Experimental Analyses of Respiratory Characteristics of Shewanella putrefaciens W3-18-1

It has previously been shown that the Shewanella putrefaciens W3-18-1 strain produces remarkably high current in microbial fuel cells (MFCs) and can form magnetite at 0°C. To explore the underlying mechanisms, we developed a genetic manipulation method by deleting the restriction-modification system genes of the SGI1 (Salmonella genome island 1)-like prophage and analyzed the key genes involved in bacterial respiration. W3-18-1 has less respiratory flexibility than the well-characterized S. oneidensis MR-1 strain, as it possesses fewer cytochrome c genes and lacks the ability to oxidize sulfite or reduce dimethyl sulfoxide (DMSO) and timethylamine oxide (TMAO). W3-18-1 lacks the hydrogen-producing Fe-only hydrogenase, and the hydrogen-oxidizing Ni-Fe hydrogenase genes were split into two separate clusters. Two periplasmic nitrate reductases (NapDAGHB and NapDABC) were functionally redundant in anaerobic growth of W3-18-1 with nitrate as the electron acceptor, though napDABC was not regulated by Crp. Moreover, nitrate respiration started earlier in W3-18-1 than in MR-1 (with NapDAGHB only) under microoxic conditions. These results indicate that Shewanella putrefaciens W3-18-1 is well adapted to habitats with higher oxygen levels. Taken together, the results of this study provide valuable insights into bacterial genome evolution.     

Association of p53 Arg72Pro polymorphism with bladder cancer: A meta-analysis

Background

p53 tumor suppressor gene Arg72Pro polymorphism has been associated with bladder cancer. However, results were inconsistent. We performed this meta-analysis to estimate the association between p53 Arg72Pro polymorphism and bladder cancer.

Methods

Electronic search of PubMed was conducted to select studies. Studies containing available genotype frequencies of Arg72Pro were chosen, and pooled odds ratio (OR) with 95% confidence interval (CI) was used to assess the association.

Results

The final meta-analysis included 14 published studies with 2176 bladder cancer cases and 2798 controls. The results suggested that the variant genotype was associated with the bladder cancer risk (additive model: OR = 1.72, 95% CI: 1.036–1.325, P = 0.011; dominant model: OR = 1.268, 95% CI: 1.003–1.602, P = 0.047) in Asian subgroup. However, the association was not significant between this polymorphism and bladder cancer risk in Caucasian (additive model: OR = 0.773, 95% CI: 0.564–1.059, P = 0.109; dominant model: OR = 0.685, 95% CI: 0.418–1.124, P = 0.134).

Conclusion

This meta-analysis suggests that p53 Arg72Pro polymorphism is associated with increased risk of bladder cancer in Asians. To validate the association between this polymorphism and bladder cancer, further studies with larger participants worldwide are needed.     

Structure-based virtual screening for glycosyltransferase51

Glycosyltransferase is an essential and easily accessible drug target for antibiotic-resistance. The crystal structures of glycosyltransferase (GT₅₁) provide us with the chance to develop new antibiotics that interrupt a yet unexplored molecular target. Based on the crystal structure of GT₅₁, we have carried out computational screening of GT₅₁ in order to look for novel GT₅₁ inhibitors. The present study was accomplished by using advance docking and scoring methodology. It is the first example of virtual screening of GT₅₁ inhibitors. Two docking procedures (Surflex-Dock and FlexX-Pharm dockings) were applied and nine novel potential leads are proposed after thorough examination by a combination of methods.     

The Long Noncoding RNA HOTAIR Contributes to Cisplatin Resistance of Human Lung Adenocarcinoma Cells via downregualtion of p21WAF1/CIP1 Expression

HOTAIR, a long intervening non-coding RNA (lincRNA), associates with the Polycomb Repressive Complex 2 (PRC2) and is reported to reprogram chromatin organization and promote tumor progression. However, little is known about the roles of this gene in the development of chemoresistance phenotype of lung adenocarcinoma (LAD). Thus, we investigated the involvement of HOTAIR in the resistance of LAD cells to cisplatin. In this study, we show that HOTAIR expression was significantly upregulated in cisplatin-resistant A549/DDP cells compared with in parental A549 cells. Knockdown of HOTAIR by RNA interference could resensitize the responses of A549/DDP cells to cisplatin both in vitro and in vivo. In contrast, overexpression of HOTAIR could decrease the sensitivity of A549 and SPC-A1 cells to cisplatin. We also found that the siRNA/HOTAIR1-mediated chemosensivity enhancement was associated with inhibition of cell proliferation, induction of G₀/G₁ cell-cycle arrest and apoptosis enhancement through regulation of p21^WAF1/CIP1 (p21) expression. Also, pcDNA/p21or siRNA/p21 could mimic the effects of siRNA/HOTAIR1 or pcDNA/HOTAIR on the sensitivity of LAD cells to cisplatin. Importantly, siRNA/p21 or pcDNA/p21 could partially rescue the effects of siRNA/HOTAIR1 or pcDNA/HOTAIR on both p21 expression and cisplatin sensitivity in LAD cells. Further, HOTAIR was observed to be significantly downregulated in cisplatin-responding LAD tissues, and its expression was inversely correlated with p21 mRNA expression. Taken together, our findings suggest that upregulation of HOTAIR contributes to the cisplatin resistance of LAD cells, at least in part, through the regulation of p21 expression.     

Biochemical characterization of a calcium-sensitive protein kinase LeCPK2 from tomato

LeCPK2 (GenBank GQ205414), a versatile calcium-dependent protein kinase (CDPK or CPK) gene was isolated from tomato in our previous study. In this study, the biochemical properties of LeCPK2 were further investigated. To examine the role of the C-terminal calmodulin-like domain (CLD) of LeCPK2 with respect to Ca2+ activation, the kinase activities of recombinant full-length and truncated LeCPK2 were measured by Kinase-Glo Luminescent kinase assay (Promega). The results showed that LeCPK2 activity was Ca(2+)-dependent and the C-terminal CLD of 161 residues was essential for the activation of LeCPK2. The activity of LeCPK2 was sharply stimulated by Ca2+ with K0.5 (concentration of Ca2+ for half-maximal activity) of 48.8 and 45.5 nM with substrate histone IIIs and syntide 2, respectively. The optimal concentration of Mg2+ for LeCPK2 activity was 20 and 10 mM for substrate histone IIIs and syntide 2, respectively. The K(m) value of LeCPK2 towards histone IIIs and syntide 2 was 44.9 microg/ml and 89.52 microM, respectively. The determination of biochemical properties of LeCPK2 would provide some clues on how its activity was regulated in vivo.     

Microarray-based evaluation of whole-community genome DNA amplification methods

Three whole-community genome amplification methods, Bst, REPLI-g, and Templiphi, were evaluated using a microarray-based approach. The amplification biases of all methods were <3-fold. For pure-culture DNA, REPLI-g and Templiphi showed less bias than Bst. For community DNA, REPLI-g showed the least bias and highest number of genes, while Bst had the highest success rate and was suitable for low-quality DNA.     

Microtransponders, the miniature RFID electronic chips, as platforms for cell growth in cytotoxicity assays.

BACKGROUND: An electronic radio frequency (RF) microchip, the microtransponder (MTP), has been developed as a platform for assays in the fields of genomics and proteomics. Upon activation by light, each MTP provides a unique RF identification (ID) signal that matches a chip to the specific biological material attached to it. The MTP is powered by a photocell and has an antenna that transmits the signal. The aim of the present study was to explore utility of MTPs as a platform for cell growth in cytotoxicity assays. METHODS: The MCF-7, MCF-116, A549, or T-24 cells growing on MTPs placed in petri dishes or slide chambers were cultured untreated or exposed to antitumor drugs topotecan, mitoxantrone, or onconase for up to 4 days. Their attachment to- and growth on- MTPs was assessed by fluorescence microscopy and laser scanning cytometry (LSC) and compared with growth on the dish surface in the MTP neighborhood. The MTPs were fixed in ethanol, stained with propidium iodide (PI), and interrogated in flow in the instrument capable to rapidly (up to 103 MTPs/s) identify their ID signal and measure fluorescence. RESULTS: The cells plated on MTPs exhibited similar attachment properties to those plated in culture dishes. When measured by LSC, they had similar mitotic activity, growth rate, and cell cycle distributions as the cells adhering to the culture dish in the neighborhood of MTPs. The fluorescence intensity of MTPs provided information about the cell number per MTP, which made it possible to assess cell growth rate and monitor the cytostatic/cytotoxic effects of the tested drugs. CONCLUSIONS: The MTP-based system holds promise for the multiplexed cell assays in which numerous different cell lines can be screened for their growth rate or sensitivity while exposed to particular agents in the same vessel. Other advantages of the system are the rapidity of the screening and a very large number of ID codes. Because many cell lines/types can be assayed in a single dish, the system also offers cost savings on tissue culture reagents.     

Chronic daily administration of ethyl docosahexaenoate protects against gerbil brain ischemic damage through reduction of arachidonic acid liberation and accumulation

Recently, we reported that dietary ethyl docosahexaenoate (Et-DHA) intake decreases the level of membrane arachidonic acid (AA), which reduces the generation of AA metabolites in ischemic gerbil brain. As an extended study, we further investigated the influence of the chronic administration of Et-DHA on free AA levels after ischemia. In addition, Na,K-ATPase activity, cation content, cerebral edema and brain damage were also evaluated. Weanling male gerbils were orally pretreated with either Et-DHA (200 mg/kg) or vehicle, once a day for 10 weeks, and subjected to transient forebrain ischemia by bilateral common carotid occlusion for 30 min. Time-course analyses revealed that pretreatment with Et-DHA, compared with pretreatment with the vehicle, significantly decreased the brain's free AA levels during ischemia (5, 15 and 30 min) and after reperfusion (5, 10, 15 and 30 min), and attenuated the decline of Na,K-ATPase activity at examined time points. Pretreatment with Et-DHA significantly prevented an increase in Na(+) concentration and a decrease in K(+) concentration after 24 h of reperfusion, which resulted in lower cerebral water content. Reduced brain infarct volume and low animal mortality were also observed in Et-DHA-treated animals. These data suggest that the reduction of ischemia-induced AA liberation and accumulation by Et-DHA pretreatment may be attributable to (a) protection against the decline of Na,K-ATPase activity, (b) postischemic cerebral edema and brain damage and (c) animal mortality.