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.     

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.     

Cytochrome P450 reductase: a harbinger of diffusible reduced oxygen species

The bi-enzymatic system of cytochrome P450 (CYP, a hemoprotein) and cytochrome P450 reductase (CPR, a diflavoenzyme) mediate the redox metabolism of diverse indigenous and xenobiotic molecules in various cellular and organ systems, using oxygen and NADPH. Curiously, when a 1:1 ratio is seen to be optimal for metabolism, the ubiquitous CYP:CPR distribution ratio is 10 to 100:1 or higher. Further, the NADPH equivalents consumed in these in vitro or in situ assemblies usually far exceeded the amount of substrate metabolized. We aimed to find the rationale to explain for these two oddities. We report here that CPR is capable of activating molecular oxygen on its own merit, generating diffusible reduced oxygen species (DROS). Also, in the first instance for a flavoprotein, CPR is shown to deplete peroxide via diffusible radical mediated process, thereby leading to the formation of water (but without significant evolution of oxygen). We also quantitatively demonstrate that the rate of oxygen activation and peroxide depletion by CPR accounts for the major reactivity in the CYP+CPR mixture. We show unambiguously that CPR is able to regulate the concentration of diffusible reduced oxygen species in the reaction milieu. These findings point out that CPR mediated processes are bound to be energetically 'wasteful' and potentially 'hazardous' owing to the unavoidable nature of the CPR to generate and deplete DROS. Hence, we can understand that CPR is distributed at low densities in cells. Some of the activities that were primarily attributed to the heme-center of CYP are now established to be a facet of the flavins of CPR. The current approach of modeling drugs to minimize "uncoupling" on the basis of erstwhile hypothesis stands questionable, considering the ideas brought forth in this work.     

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.     

Synthesis and evaluation of β-carboline derivatives as inhibitors of human immunodeficiency virus

A series of β-carboline derivatives were synthesized by utilizing aromatization and chemoselective alkylation method recently reported from our laboratory. Synthesized derivatives were evaluated for anti-HIV activity in human CD4+ T cell line (CEM-GFP) infected with HIV-1 NL_4.3 virus. 1-Formyl-β-carboline-3-carbxylic acid methyl ester (15) showed inhibition of human immunodeficiency virus at IC₅₀ = 2.9 μM.     

Valley‐scale hydrogeomorphology drives river fish assemblage variation in Mongolia

River hydrogeomorphology is a major driver shaping biodiversity and community composition. Here, we examine how hydrogeomorphic heterogeneity expressed by Functional Process Zones (FPZs) in river networks is associated with fish assemblage variation. We examined this association in two distinct ecoregions in Mongolia expected to display different gradients of river network hydrogeomorphic heterogeneity. We delineated FPZs by extracting valley‐scale hydrogeomorphic variables at 10 km sample intervals in forest steppe (FS) and in grassland (G) river networks. We sampled fish assemblages and examined variation associated with changes in gradients of hydrogeomorphology as expressed by the FPZs. Thus, we examined assemblage variation as patterns of occurrence‐ and abundance‐based beta diversities for the taxonomic composition of assemblages and as functional beta diversity. Overall, we delineated 5 and 6 FPZs in river networks of the FS and G, respectively. Eight fish species were found in the FS river network and seventeen in the G, four of them common to both ecoregions. Functional richness was correspondingly higher in the G river network. Variation in the taxonomic composition of assemblages was driven by species turnover and was only significant in the G river network. Abundance‐based taxonomic variation was significant in river networks of both ecoregions, while the functional beta diversity results were inconclusive. We show that valley‐scale hydrogeomorphology is a significant driver of variation in fish assemblages at a macrosystem scale. Both changes in the composition of fish assemblages and the carrying capacity of the river network were driven by valley‐scale hydrogeomorphic variables. River network hydrogeomorphology as accounted for in the study has, therefore, the potential to inform macrosystem scale community ecology research and conservation efforts.