A Pit-1 Binding Site Adjacent to E-box133 in the Rat PRL Promoter is Necessary for Pulsatile Gene Expression Activity

Recent evidence reveals that prolactin gene expression (PRL-GE) in mammotropes occurs in pulses, but the molecular process(es) underlying this phenomenon remains unclear. Earlier, we have identified an E-box (E-box133) in the rat PRL promoter that binds several circadian elements and is critical for this dynamic process. Preliminary analysis revealed a Pit-1 binding site (P2) located immediately adjacent to this E-box133 raising the possibility that some type of functional relationship may exist between these two promoter regions. In this study, using serum shocked GH₃ cell culture system to synchronize PRL-GE activity, we determined that Pit-1 gene expression occurred in pulses with time phases similar to that for PRL. Interestingly, EMSA analysis not only confirmed Pit-1 binding to the P2 site, but also revealed an interaction with factor(s) binding to the adjacent E-box133 promoter element. Additionally, down-regulation of Pit-1 by siRNA reduced PRL levels during pulse periods. Thus, using multiple evidences, our results demonstrate clearly that the Pit-1 P2 site is necessary for PRL-GE elaboration. Furthermore, the proximity of this critical Pit-1 binding site (P2) and the E-box133 element coupled with the evidences of a site-to-site protein interactions suggest that the process of PRL-GE pulse activity might involve more dynamic and intricate cross-talks between promoter elements that may span some, or all, of the proximal region of the PRL promoter in driving its pulsatile expression.     

The Role of Hemosiderin Excision in Seizure Outcome in Cerebral Cavernous Malformation Surgery: A Systematic Review and Meta-Analysis

Background and Purpose

Whether the excision of hemosiderin surrounding cerebral cavernous malformations (CCMs) is necessary to achieve a seizure-free result has been the subject of debate. Here, we report a systematic review of related literature up to Jan 1, 2015 including 594 patients to assess the effect of hemosiderin excision on seizure outcome in patients with CCMs by meta-analysis.

Methods

Ten studies comparing extended hemosiderin excision with only lesion resection were identified by searching the English-language literature. Meta-analyses, subgroup analyses and sensitivity analysis were conducted to determine the association between hemosiderin excision and seizure outcome after surgery.

Results

Seizure outcome was significantly improved in the patients who underwent an extended excision of the surrounding hemosiderin (OR, 0.62; 95% CI: 0.42–0.91; P = 0.01). In subgroup analysis, studies from Asia (OR, 0.42; 95% CI: 0.25–0.71; P = 0.001), male-majority (female ratio < 50%) studies (OR, 0.56; 95% CI: 0.33–0.96; P = 0.04), low occurrence rate of multiple CCMs (OR, 0.37; 95% CI: 0.20–0.71; P = 0.003), cohort studies (OR, 0.44; 95% CI: 0.28–0.68; P = 0.78), longer duration of seizure symptoms (> 1 year) before surgery (OR, 0.43; 95% CI: 0.22–0.84; P = 0.01), lesion diameter > 2 cm (OR, 0.41; 95% CI: 0.19–0.87; P = 0.02) and short-term (< 3 years) follow-up (OR, 0.48; 95% CI: 0.29–0.80; P = 0.005) tended to correlate with a significantly favorable outcome.

Conclusion

Patients who underwent extended surrounding hemosiderin excision could exhibit significantly improved seizure outcomes compared to patients without hemosiderin excision. However, further well-designed prospective multiple-center RCT studies are still needed.     

Successful operation of continuous reactors at short retention times results in high-density,fast-rate Dehalococcoides dechlorinating cultures

The discovery of Dehalococcoides mccartyi reducing perchloroethene and trichloroethene (TCE) to ethene was a key landmark for bioremediation applications at contaminated sites. D. mccartyi-containing cultures are typically grown in batch-fed reactors. On the other hand, continuous cultivation of these microorganisms has been described only at long hydraulic retention times (HRTs). We report the cultivation of a representative D. mccartyi-containing culture in continuous stirred-tank reactors (CSTRs) at a short, 3-d HRT, using TCE as the electron acceptor. We successfully operated 3-d HRT CSTRs for up to 120 days and observed sustained dechlorination of TCE at influent concentrations of 1 and 2 mM TCE to ≥97 % ethene, coupled to the production of 10¹² D. mccartyi cells L_culture ^?1. These outcomes were possible in part by using a medium with low bicarbonate concentrations (5 mM) to minimize the excessive proliferation of microorganisms that use bicarbonate as an electron acceptor and compete with D. mccartyi for H₂. The maximum conversion rates for the CSTR-produced culture were 0.13?±?0.016, 0.06?±?0.018, and 0.02?±?0.007 mmol Cl^? L_culture ^?1 h^?1, respectively, for TCE, cis-dichloroethene, and vinyl chloride. The CSTR operation described here provides the fastest laboratory cultivation rate of high-cell density Dehalococcoides cultures reported in the literature to date. This cultivation method provides a fundamental scientific platform for potential future operations of such a system at larger scales.     

Establishment and characterization of a new <Emphasis Type="Italic">Aedes aegypti</Emphasis> (L.) (Diptera: Culicidae) cell line with special emphasis on virus susceptibility

A new cell line from the neonate larvae of Aedes aegypti (L) mosquito was established and characterized. The cell line at the 50th passage (P) level consisted of three prominent cell types, i.e., epithelial-like cells (92%), fibroblast-like cells (7%), and giant cells (∼1%). Karyological analysis showed diploid (2n = 6) number of chromosomes in >75% cells at P-50. The growth kinetics studied at 52nd passage level showed approximately tenfold increase in cell number over a 10-d study period. The species specificity studies using DNA amplification fingerprinting profile analysis using RAPD primers demonstrated 100% homology with the host profile showing the integrity of the cell line. Electron microscopy revealed the absence of mycoplasma or other adventitious agents. The cell line supported the multiplication of seven arboviruses, i.e., Chikungunya (CHIK), Japanese encephalitis, West Nile, dengue 2 (DEN-2), Chandipura, vesicular stomatitis, and Chittoor viruses. The cell line did not replicate Ganjam and Kaisodi viruses. CHIK virus yield in the new cell line was approximately 3log and 0.5log 50% tissue culture infective dose (TCID₅₀)/mL higher than Vero E6 and C6/36 cell lines, respectively. In the case of DEN-2 virus, it yielded 1log TCID₅₀/mL higher than Vero E6, but lesser than C6/36 cell line. Due to its high susceptibility to a broad spectrum of viruses, the new cell line may find application in virus isolation during epidemics and in antigen production.     

Role of coupled dynamics in the catalytic activity of prokaryotic-like prolyl-tRNA synthetases

Prolyl-tRNA synthetases (ProRSs) have been shown to activate both cognate and some noncognate amino acids and attach them to specific tRNA(Pro) substrates. For example, alanine, which is smaller than cognate proline, is misactivated by Escherichia coli ProRS. Mischarged Ala-tRNA(Pro) is hydrolyzed by an editing domain (INS) that is distinct from the activation domain. It was previously shown that deletion of the INS greatly reduced cognate proline activation efficiency. In this study, experimental and computational approaches were used to test the hypothesis that deletion of the INS alters the internal protein dynamics leading to reduced catalytic function. Kinetic studies with two ProRS variants, G217A and E218A, revealed decreased amino acid activation efficiency. Molecular dynamics studies showed motional coupling between the INS and protein segments containing the catalytically important proline-binding loop (PBL, residues 199-206). In particular, the complete deletion of INS, as well as mutation of G217 or E218 to alanine, exhibited significant effects on the motion of the PBL. The presence of coupled dynamics between neighboring protein segments was also observed through in silico mutations and essential dynamics analysis. Altogether, this study demonstrates that structural elements at the editing domain-activation domain interface participate in coupled motions that facilitate amino acid binding and catalysis by bacterial ProRSs, which may explain why truncated or defunct editing domains have been maintained in some systems, despite the lack of catalytic activity.     

Managing methanogens and homoacetogens to promote reductive dechlorination of trichloroethene with direct delivery of H2 in a membrane biofilm reactor

A study with H(2)-based membrane biofilm reactors (MBfRs) was undertaken to examine the effectiveness of direct H(2) delivery in ex-situ reductive dechlorination of chlorinated ethenes. Trichloroethene (TCE) could be reductively dechlorinated to ethene with up to 95% efficiency as long as the pH-increase effects of methanogens and homoacetogens were managed and dechlorinators were selected for during start-up by creating H(2) limitation. Based on quantitative PCR, the dominant bacterial groups in the biofilm at the end of reactor operation were Dehalococcoides, Geobacter, and homoacetogens. Pyrosequencing confirmed the dominance of the dechlorinators and identified Acetobacterium as the key homoacetogen. Homoacetogens outcompeted methanogens for bicarbonate, based on the effluent concentration of acetate, by suppressing methanogens during batch start-up. This was corroborated by the methanogenesis functional gene mcrA, which was 1-2 orders of magnitude lower than the FTHFS functional gene for homoacetogens. Imaging of the MBfR fibers using scanning electron microscopy showed a distinct Dehalococcoides-like morphology in the fiber biofilm. These results support that direct addition of H(2) can allow for efficient and complete reductive dechlorination, and they shed light into how H(2)-fed biofilms, when operated to manage methanogenic and homoacetogenic activity, can be used for ex-situ bioremediation of chlorinated ethenes.     

Caerulomycin A Enhances Transforming Growth Factor-β (TGF-β)-Smad3 Protein Signaling by Suppressing Interferon-γ (IFN-γ)-Signal Transducer and Activator of Transcription 1 (STAT1) Protein Signaling to Expand Regulatory T Cells (Tregs)

Cytokines play a very important role in the regulation of immune homeostasis. Regulatory T cells (Tregs) responsible for the generation of peripheral tolerance are under the tight regulation of the cytokine milieu. In this study, we report a novel role of a bipyridyl compound, Caerulomycin A (CaeA), in inducing the generation of Tregs. It was observed that CaeA substantially up-regulated the pool of Tregs, as evidenced by an increased frequency of CD4⁺ Foxp3⁺ cells. In addition, CaeA significantly suppressed the number of Th1 and Th17 cells, as supported by a decreased percentage of CD4⁺/IFN-γ⁺ and CD4⁺/IL-17⁺ cells, respectively. Furthermore, we established the mechanism and observed that CaeA interfered with IFN-γ-induced STAT1 signaling by augmenting SOCS1 expression. An increase in the TGF-β-mediated Smad3 activity was also noted. Furthermore, CaeA rescued Tregs from IFN-γ-induced inhibition. These results were corroborated by blocking Smad3 activity, which abolished the CaeA-facilitated generation of Tregs. In essence, our results indicate a novel role of CaeA in inducing the generation of Tregs. This finding suggests that CaeA has enough potential to be considered as a potent future drug for the treatment of autoimmunity.     

Gene expression profiling of coronary artery disease and its relation with different severities

Global gene expression profiling is a powerful tool enabling the understanding of pathophysiology and subsequent management of diseases. This study aims to explore functionally annotated differentially expressed genes (DEGs); their biological processes for coronary artery disease (CAD) and its different severities of atherosclerotic lesions. This study also aims to identify the change in expression patterns of DEGs in atherosclerotic lesions of single-vessel disease (SVD) and triple-vessel disease (TVD). The weight of different severities of lesion was estimated using a modified Gensini score. The gene expression profiling was performed using the Affymetrix microarray platform. The functional annotation for CAD was performed using DAVID v6.8. The biological network gene ontology tool (BiNGO) and ClueGO were used to explore the biological processes of functionally annotated genes of CAD. The changes in gene expression from SVD to TVD were determined by evaluating the fold change. Functionally annotated genes were found in an unique set and could be distinguishing two distinct severities of CAD. The biological processes such as cellular migration, locomotion, cell adhesion, cytokine production, positive regulation of cell death etc. enriched the functionally annotated genes in SVD, whereas, wound healing, negative regulation of cell death, blood coagulation, angiogenesis and fibrinolysis were enriched significantly in TVD patients. The genes THBS1 and CAPN10 were functionally annotated for CAD in both SVD and TVD. The 61 DEGs were identified, those have changes their expression with different severities of atherosclerotic lesions, in which 13 genes had more than two-fold change in expression between SVD and TVD. The consistent findings were obtained on validation of microarray gene expression of selected 10 genes in a separate cohort using real-time PCR. This study identified putative candidate genes and their biological processes predisposing toward and affecting the severity of CAD.     

Patterns in Benthic Biodiversity Link Lake Trophic Status to Structure and Potential Function of Three Large,Deep Lakes

Relative to their scarcity, large, deep lakes support a large proportion of the world’s freshwater species. This biodiversity is threatened by human development and is in need of conservation. Direct comparison of biodiversity is the basis of biological monitoring for conservation but is difficult to conduct between large, insular ecosystems. The objective of our study was to conduct such a comparison of benthic biodiversity between three of the world’s largest lakes: Lake Tahoe, USA; Lake Hövsgöl, Mongolia; and Crater Lake, USA. We examined biodiversity of common benthic organism, the non-biting midges (Chironomidae) and determined lake trophic status using chironomid-based lake typology, tested whether community structure was similar between the three lakes despite geographic distance; and tested whether chironomid diversity would show significant variation within and between lakes. Typology analysis indicated that Lake Hövsgöl was ultra-oligotrophic, Crater Lake was oligotrophic, and Lake Tahoe was borderline oligotrophic/mesotrophic. These results were similar to traditional pelagic measures of lake trophic status for Lake Hövsgöl and Crater Lake but differed for Lake Tahoe, which has been designated as ultra-oligotrophic by traditional pelagic measures such as transparency found in the literature. Analysis of similarity showed that Lake Tahoe and Lake Hövsgöl chironomid communities were more similar to each other than either was to Crater Lake communities. Diversity varied between the three lakes and spatially within each lake. This research shows that chironomid communities from these large lakes were sensitive to trophic conditions. Chironomid communities were similar between the deep environments of Lake Hövsgöl and Lake Tahoe, indicating that chironomid communities from these lakes may be useful in comparing trophic state changes in large lakes. Spatial variation in Lake Tahoe’s diversity is indicative of differential response of chironomid communities to nutrient enrichment which may be an indication of changes in trophic state within and across habitats.