Functional analysis of early secreted antigenic target-6, the dominant T-cell antigen of Mycobacterium tuberculosis, reveals key residues involved in secretion, complex formation, virulence, and immunogenicity

Proteins of the 6-kDa early secreted antigenic target (ESAT-6) secretion system-1 of Mycobacterium tuberculosis are not only strongly involved in the anti-mycobacterial Th1-host immune response but are also key players for virulence. In this study, protein engineering together with bioinformatic, immunological, and virulence analyses allowed us to pinpoint regions of the ESAT-6 molecule that are critical for its biological activity in M. tuberculosis. Mutation of the Trp-Xaa-Gly motif, conserved in a wide variety of ESAT-6-like proteins, abolished complex formation with the partner protein CFP-10, induction of specific T-cell responses, and virulence. Replacement of conserved Leu residues interfered with secretion, coiled-coil formation, and virulence, whereas certain mutations at the extreme C terminus did not affect secretion but caused attenuation, possibly because of altered ESAT-6 targeting or trafficking. In contrast, the mutation of several residues on the outer surface of the four-helical bundle structure of the ESAT-6.CFP-10 complex showed much less effect. Construction of recombinant BCG expressing ESAT-6 with a C-terminal hexahistidine tag allowed us to co-purify ESAT-6 and CFP-10, experimentally confirming their strong interaction both in and outside of the mycobacterial cell. The strain induced potent, antigen-specific T-cell responses and intermediate in vivo growth in mice, suggesting that it remained immunogenic and biologically active despite the tag. Together with previous NMR data, the results of this study have allowed a biologically relevant model of the ESAT-6.CFP-10 complex to be constructed that is critical for understanding the structure-function relationship in tuberculosis pathogenesis.     

The Possible Role of Staphylococcus epidermidis LPxTG Surface Protein SesC in Biofilm Formation

Staphylococcus epidermidis is the most common cause of device-associated infections. It has been shown that active and passive immunization in an animal model against protein SesC significantly reduces S. epidermidis biofilm-associated infections. In order to elucidate its role, knock-out of sesC or isolation of S. epidermidis sesC-negative mutants were attempted, however, without success. As an alternative strategy, sesC was introduced into Staphylococcus aureus 8325–4 and its isogenic icaADBC and srtA mutants, into the clinical methicillin-sensitive S. aureus isolate MSSA4 and the MRSA S. aureus isolate BH1CC, which all lack sesC. Transformation of these strains with sesC i) changed the biofilm phenotype of strains 8325–4 and MSSA4 from PIA-dependent to proteinaceous even though PIA synthesis was not affected, ii) converted the non-biofilm-forming strain 8325–4 ica::tet to a proteinaceous biofilm-forming strain, iii) impaired PIA-dependent biofilm formation by 8325–4 srtA::tet, iv) had no impact on protein-mediated biofilm formation of BH1CC and v) increased in vivo catheter and organ colonization by strain 8325–4. Furthermore, treatment with anti-SesC antibodies significantly reduced in vitro biofilm formation and in vivo colonization by these transformants expressing sesC. These findings strongly suggest that SesC is involved in S. epidermidis attachment to and subsequent biofilm formation on a substrate.     

Major histocompatibility complex heterozygosity reduces fitness in experimentally infected mice

It is often suggested that heterozygosity at major histocompatibility complex (MHC) loci confers enhanced resistance to infectious diseases (heterozygote advantage, HA, hypothesis), and overdominant selection should contribute to the evolution of these highly polymorphic genes. The evidence for the HA hypothesis is mixed and mainly from laboratory studies on inbred congenic mice, leaving the importance of MHC heterozygosity for natural populations unclear. We tested the HA hypothesis by infecting mice, produced by crossbreeding congenic C57BL/10 with wild ones, with different strains of Salmonella, both in laboratory and in large population enclosures. In the laboratory, we found that MHC influenced resistance, despite interacting wild-derived background loci. Surprisingly, resistance was mostly recessive rather than dominant, unlike in most inbred mouse strains, and it was never overdominant. In the enclosures, heterozygotes did not show better resistance, survival, or reproductive success compared to homozygotes. On the contrary, infected heterozygous females produced significantly fewer pups than homozygotes. Our results show that MHC effects are not masked on an outbred genetic background, and that MHC heterozygosity provides no immunological benefits when resistance is recessive, and can actually reduce fitness. These findings challenge the HA hypothesis and emphasize the need for studies on wild, genetically diverse species.     

Establishment of a biomimetic device based on tri-layer polymer actuators--propulsion fins

We propose to use bending type tri-layer polymer actuators as propulsion fins for a biomimetic device consisting of a rigid body, like a box fish having a carapace, and paired fins running through the rigid body, like a fish having pectoral fins. The fins or polymer bending actuators can be considered as individually controlled flexible membranes. Each fin is activated with sinusoidal inputs such that there is a phase lag between the movements of successive fins to create enough thrust force for propulsion. Eight fins with 0.125 aspect ratio have been used along both sides of the rigid body to move the device in the direction perpendicular to the longitudinal axis of the body. The designed device with the paired fins was successfully tested, moving in an organic solution consisting of solvent, propylene carbonate (PC), and electrolyte. The design procedure outlined in this study is offered as a guide to making functional devices based on polymer actuators and sensors.     

Oleoylethanolamide: A novel pharmaceutical agent in the management of obesity-an updated review

Obesity as a multifactorial disorder has been shown a dramatically growing trend recently. Besides genetic and environmental factors, dysregulation of the endocannabinoid system tone is involved in the pathogenesis of obesity. This study reviewed the potential efficacy of Oleoylethanolamide (OEA) as an endocannabinoid-like compound in the energy homeostasis and appetite control in people with obesity. OEA as a lipid mediator and bioactive endogenous ethanolamide fatty acid is structurally similar to the endocannabinoid system compounds; nevertheless, it is unable to induce to the cannabinoid receptors. Unlike endocannabinoids, OEA negatively acts on the food intake and suppress appetite via various mechanisms. Indeed, OEA as a ligand of PPAR-α, GPR-119, and TRPV1 receptors participates in the regulation of energy intake and energy expenditure, feeding behavior, and weight gain control. OEA delays meal initiation, reduces meal size, and increases intervals between meals. Considering side effects of some approaches used for the management of obesity such as antiobesity drugs and surgery as well as based on sufficient evidence about the protective effects of OEA in the improvement of common abnormalities in people with obese, its supplementation as a novel efficient and FDA approved pharmaceutical agent can be recommended.     

Promoter methylation and functional variants in arachidonate 5-lipoxygenase and forkhead box protein O1 genes associated with coronary artery disease

Coronary artery disease (CAD) is a multifactorial chronic inflammatory disease, which is the most common form of heart disease. This is one of the main causes of death in the United States. Inflammation is one of the key drivers of atherosclerotic plaque development. Forkhead box protein O1 (FOXO1s) family and 5-lipoxygenase make an important contribution to atherosclerosis. The aim of this study was to investigate the methylation pattern and polymorphism analysis of FOXO1 and arachidonate 5-lipoxygenase (ALOX5) promoter genes. We studied 50 patients with CAD and 50 age- and sex-matched healthy controls by high resolution melt technique. Overall, we found significant differences between patients and controls in terms of the promoter methylation of ALOX5 (P > 0.05). But there was no significant difference in FOXO1 promoter methylation between patient and controls. Single nucleotide polymorphisms genotyping of rs12762303 and rs2297627, in ALOX5 and FOXO1 genes were demonstrated a significant correlation between mutant allele and the risk of CAD, respectively. Furthermore, there were significant associations between CT + CC genotype and ALOX5 expression. Our findings demonstrated functional effects of single nucleotide polymorphisms (SNPs) and DNA methylation in ALOX5 on mentioned genes expression and they resulted in CAD progression.     

Development of α4 integrin DNA aptamer as a potential therapeutic tool for multiple sclerosis

One of the most important molecules for multiple sclerosis pathogenesis is α4 integrin, which is responsible for autoreactive leukocytes migration into the brain. The monoclonal antibody, natalizumab, was introduced to market for blocking the extravasation of autoreactive leukocytes via inhibition of α4 integrin. However, the disadvantages of antibodies provided a suitable background for other agents to be replaced with antibodies. Considering the profound advantages of aptamers over antibodies, aptamer isolation against α4 integrin was intended in the current study. The α4 integrin-specific aptamers were selected using cell-systematic evolution of ligands by exponential enrichment (SELEX) method with human embryonic kidney (HEK)-293T overexpressing α4 integrin and HEK-293T as target and control cells, respectively. Evaluation of selected aptamer was performed through flow cytometric analysis. The selected clones were then sequenced and analyzed for any possible secondary structure and affinity. The results of this study led to isolation of 13 different single-stranded DNA clones in 11 rounds of selection which were categorized to three clusters based on common structural motifs and the equilibrium dissociation constant (K _d) of the most stable structure was calculated. The evaluation of SELEX progress showed growth in aptamer affinity with increasing of the number of cycles. Taken together, the findings of this study demonstrated the isolation of α4-specific single-stranded DNA aptamers with suitable affinity for ligand, which can further be replaced with natalizumab.     

Sparse networks of directly coupled,polymorphic, and functional side chains in allosteric proteins

Recent studies have highlighted the role of coupled side‐chain fluctuations alone in the allosteric behavior of proteins. Moreover, examination of X‐ray crystallography data has recently revealed new information about the prevalence of alternate side‐chain conformations (conformational polymorphism), and attempts have been made to uncover the hidden alternate conformations from X‐ray data. Hence, new computational approaches are required that consider the polymorphic nature of the side chains, and incorporate the effects of this phenomenon in the study of information transmission and functional interactions of residues in a molecule. These studies can provide a more accurate understanding of the allosteric behavior. In this article, we first present a novel approach to generate an ensemble of conformations and an efficient computational method to extract direct couplings of side chains in allosteric proteins, and provide sparse network representations of the couplings. We take the side‐chain conformational polymorphism into account, and show that by studying the intrinsic dynamics of an inactive structure, we are able to construct a network of functionally crucial residues. Second, we show that the proposed method is capable of providing a magnified view of the coupled and conformationally polymorphic residues. This model reveals couplings between the alternate conformations of a coupled residue pair. To the best of our knowledge, this is the first computational method for extracting networks of side chains' alternate conformations. Such networks help in providing a detailed image of side‐chain dynamics in functionally important and conformationally polymorphic sites, such as binding and/or allosteric sites. Proteins 2015; 83:497–516. © 2014 Wiley Periodicals, Inc.     

Biodegradation of gasoline and BTEX in a microaerophilic biobarrier

Continuous bioremediation of gasoline-contaminatedwater in a packed-bed biobarrier system underoxygen-limited conditions is discussed. This studywas part of an extensive effort to develop analternative technology for the in situbioremediation of hydrocarbons where there is alimited supply of oxygen. Protruded stainless steelpieces and granulated peat moss were used as packingmaterial to support microbial growth in twobiobarriers. The inoculum was an enrichment culture ofan indigenous microbial population from a soil sample.The biobarriers' inlet gasoline concentrations and thelinear liquid velocities were similar to thosecommonly found at in situ conditions. Gasolineremoval efficiencies ranged from 94% to 99.9% in thestainless steel-packed biobarrier, and from 86.6% to99.6% in the peat moss-packed biobarrier. Effluentgasoline concentrations below 0.03 mg/l were obtainedat gasoline loading rates less than 27.5 mg/l.d in thestainless steel-packed biobarrier. The remainingfraction of gasoline in the effluent consisted mainlyof three aliphatic compounds and not the aromaticcompounds. Both biobarrier packings supported nearcomplete removal of the most soluble aromatichydrocarbons of gasoline (BTEX) under all theconditions examined. The consumption of sulfate andthe presence of sulfate-reducing microorganismssuggested the presence of anaerobic metabolism duringthe degradation of gasoline. Up to 92% gasoline wasremoved during the first 3 cm of the biobarriers'length.