Serum antibody responses to Wolbachia surface protein in patients with human lymphatic filariasis

Wolbachia surface protein (WSP), which is the most abundantly expressed protein of Wolbachia from the human filarial parasite Brugia malayi, was chosen for the present study. B‐cell epitope prediction of the WSP protein sequence indicates a high antigenicity, surface probability and hydrophilicity by DNA STAR software analysis. ProPred analysis suggests the presence of HLA class II binding regions in the WSP protein that contribute to T‐cell responses and isotype reactivity. In order to validate these findings, the gene coding for endosymbiont WSP was PCR‐amplified from the genomic DNA of the human filarial parasite Brugia malayi and cloned in T‐7 expression vector pRSET‐A. Western blot and ELISA at the total IgG level with recombiant WSP indicated a significantly elevated reactivity in CP compared to MF, EN and NEN individuals. Isotype ELISA also suggested an elevated reactivity in CP patients at the IgG1 level. In contrast, WSP‐specific IgG4 levels were found to be elevated in MF patients compared to CP and EN. Besides this, WSP‐specific IgE levels indicated an elevated reactivity in CP and MF patients compared to normals. Observations from ELISA supported the in silico predictions that indicate the presence of B‐ and T‐cell epitopes. Hence, a combinatorial approach of in silico predictions and wet‐lab studies provides interesting insights into the role of Wolbachia proteins in filarial pathogenesis.     

Immunolocalization and serum antibody responses to Brugia malayi pepsin inhibitor homolog (Bm-33)

cDNA coding for Brugia malayi pepsin inhibitor homolog (Bm-33) from the human filarial parasite was cloned in pRSET for large-scale expression and functional characterization. The pRSET-B cloned gene did not yield recombinant protein expression and the reason was attributed to the presence of an N-terminal signal peptide. The gene was subcloned in pRSET-A without a signal peptide and the 33 kDa histidine-tagged recombinant protein was purified by IMAC. All individuals from an endemic area generated IgG responses against Bm-33 in the order MF>CP>EN. Isotype analysis indicated an elevated IgG4 reactivity in the order MF>EN>CP. Bm-33-specific IgE levels were elevated in MF, CP and EN compared to non-endemic normals with no significant differences among the groups. Paraffin-embedded sections of Setaria digitata (cattle filarial parasite) stained with mouse anti-Bm-33 antibodies exhibited the hypodermal nature of Bm-33. These findings suggest that Bm-33 is an immunodominant antigen and contributes to filarial pathogenesis.     

Epigallocatechin-3-gallate exhibits anti-fibrotic effect by attenuating bleomycin-induced glycoconjugates, lysosomal hydrolases and ultrastructural changes in rat model pulmonary fibrosis

Pulmonary fibrosis is characterized by excessive deposition of extracellular matrix components in the alveolar space, which hampers normal respiration process. Pathophysiological enzymes, glycoprotein moieties and matrix degrading lysosomal hydrolases are key markers and play a crucial role in the progression of fibrosis. Bleomycin is an anti-neoplastic drug, used for the treatment of various types of cancers and induces pulmonary fibrosis due its deleterious side effect. Tea catechin epigallocatechin-3-gallate (EGCG) is known for its wide array of beneficial effects. The present study was intended to evaluate the beneficial efficacy of EGCG against bleomycin-induced glycoconjugates, lysosomal hydrolases and ultrastructural changes in the lungs of Wistar rats. Intratracheal instillation of bleomycin (6.5 U/kg body weight) to rats increased the activities of pathophysiological enzymes such as aspartate transaminase, alanine transaminase, lactate dehydrogenase and alkaline phosphatase, which were attenuated upon EGCG treatment. The increased level of hydroxyproline and histopathological parameters in bleomycin-induced rats were decreased upon EGCG treatment. Bleomycin-induced increase in the level of glycoconjugates was restored closer to normal levels on EGCG treatment. Furthermore, the increased activities of matrix degrading lysosomal enzymes in bleomycin-induced rats were reduced upon EGCG supplementation. Treatment with EGCG also attenuated bleomycin-induced ultrastructural changes as observed from transmission electron microscopy studies. The results of the present study put-forward EGCG as a potential anti-fibrotic agent due to its attenuating effect on potential fibrotic markers.     

Solvent Friction Changes the Folding Pathway of the Tryptophan Zipper TZ2

Because the rate of a diffusional process such as protein folding is controlled by friction encountered along the reaction pathway, the speed of folding is readily tunable through adjustment of solvent viscosity. The precise relationship between solvent viscosity and the rate of diffusion is complex and even conformation-dependent, however, because both solvent friction and protein internal friction contribute to the total reaction friction. The heterogeneity of the reaction friction along the folding pathway may have subtle consequences. For proteins that fold on a multidimensional free-energy surface, an increase in solvent friction may drive a qualitative change in folding trajectory. Our time-resolved experiments on the rapidly and heterogeneously folding β-hairpin TZ2 show a shift in the folding pathway as viscosity increases, even though the energetics of folding is unaltered. We also observe a nonlinear or saturating behavior of the folding relaxation time with rising solvent viscosity, potentially an experimental signature of the shifting pathway for unfolding. Our results show that manipulations of solvent viscosity in folding experiments and simulations may have subtle and unexpected consequences on the folding dynamics being studied.     

Alleviation of Proteolytic Sensitivity To Enhance Recombinant Lipase Production in Escherichia coli

Two amino acids, Leu149 and Val223, were identified as proteolytically sensitive when Pseudozyma antarctica lipase (PalB) was heterologously expressed in Escherichia coli. The functional expression was enhanced using the double mutant for cultivation. However, the recombinant protein production was still limited by PalB misfolding, which was resolved by DsbA coexpression.Though Escherichia coli retains popularity as a host for recombinant protein production, it has technical limitations for expressing eukaryotic proteins. Host/protein incompatibility is critical, since eukaryotic proteins heterologously expressed in E. coli are recognized as a foreign object that can induce heat shock responses and drive protease-mediated degradation. E. coli contains several proteases situated in various intracellular compartments, including the cytoplasm, periplasm, inner membrane, and outer membrane (7, 9). While these proteases play a pivotal role in maintaining cell physiology by clearing up abnormal proteins (4), they can potentially degrade recombinant proteins (6). Nevertheless, the mechanisms associated with protein degradation and protein substrate specificity are incompletely understood.Since the proteolysis of recombinant proteins is a common issue, approaches to overcome this limitation have been extensively explored. These include using protease-deficient mutants as an expression host (10, 18), lowering the cultivation temperature (3), coexpressing folding factors (19, 20), applying protein fusion technology (2, 8), eliminating protease cleavage sites (5, 15), and modifying target protein hydrophobicity (11). While genetic elimination of proteolytically sensitive sequences without impacting the target protein''s bioactivity appears to be technically attractive, there is no effective prediction or identification of potential protease cleavage sites. Proteolytically sensitive sequences might not be specific to certain proteases. Various factors affecting the folding, solubility, steric hindrance, and binding of the substrate protein, e.g., neighboring domains or amino acids, can contribute to the sensitivity of proteolytic sites, making sequence manipulation for proteolysis alleviation more difficult.Herein, the recombinant protein''s sensitivity toward intracellular proteolysis was genetically alleviated to enhance the functional expression of PalB, an industrial enzyme with justified applications (1), in E. coli and to understand the relationship between proteolytic specificity and substrate protein sequence. Since there are three intramolecular disulfide bonds whose formation can potentially affect PalB structure and bioactivity, PalB expression in the oxidative periplasm of E. coli was explored (20, 22). However, the expressed PalB protein was extremely sensitive to intracellular proteolysis, resulting in complete degradation. To overcome this problem, PalB mutant variants that were stable against proteolysis were derived (Table ​(Table11).

TABLE 1.

Strains, plasmids, and oligonucleotides

Fusobacterium necrophorum: a ruminal bacterium that invades liver to cause abscesses in cattle

Fusobacterium necrophorum, a Gram-negative, rod-shaped, and an aerotolerant anaerobe, is a normal inhabitant of the rumen of cattle. The organism is in ruminal contents and adherent to the ruminal wall. Its role in ruminal fermentation is to metabolize lactic acid and degrade feed and epithelial proteins. The ruminal concentration is higher in grain-fed than forage-fed cattle. From the rumen, the organism gains entry into the portal circulation and is trapped in the liver to cause abscesses. The organism is an opportunistic pathogen and a primary causative agent of liver abscesses, an economically important disease of grain-fed cattle. Liver abscesses are often secondary to ruminal acidosis and rumenitis in grain-fed cattle. Two subspecies of F. necrophorum, subsp. necrophorum (biotype A) and subsp. funduliforme (biotype B), are recognized that can be differentiated based on morphological, biochemical, biological and molecular characteristics. The subsp. necrophorum is more virulent and is isolated more frequently from infections than the subsp. funduliforme. Several toxins or secreted products have been implicated as virulence factors. The major factors contributing to ruminal colonization and invasion into the liver are hemagglutinin, endotoxin and leukotoxin, of which leukotoxin is the protective antigen. In some conditions, the organism synergistically interacts with Arcanobacterium pyogenes, a facultative anaerobic organism and a secondary etiologic agent, to cause liver abscesses.     

Calcium store depletion induces persistent perisomatic increases in the functional density of h channels in hippocampal pyramidal neurons

The regulation of intracellular calcium by the endoplasmic reticulum (ER) plays a critical role in neuronal function. While the consequences associated with depleting calcium from the ER have been studied in multiple systems, it is not known whether the intrinsic properties of a neuron change in response to such perturbations. In this study, we demonstrate that the depletion of calcium from the ER of hippocampal CA1 pyramidal neurons induces a persistent, perisomatic increase in the density of functional h channels resulting in a reduction in intrinsic excitability and an increase in the optimal response frequency. This form of intrinsic plasticity is dependent on the elevation of cytoplasmic calcium, inositol triphosphate receptors, store-operated calcium channels, and the protein kinase A pathway. We postulate that this form of depletion-induced intrinsic plasticity is a neuroprotective mechanism that reduces excitability after depletion of calcium stores triggered through altered network activity during pathological conditions.     

Real-time fluorescence loop mediated isothermal amplification for the diagnosis of malaria

BACKGROUND: Molecular diagnostic methods can complement existing tools to improve the diagnosis of malaria. However, they require good laboratory infrastructure thereby restricting their use to reference laboratories and research studies. Therefore, adopting molecular tools for routine use in malaria endemic countries will require simpler molecular platforms. The recently developed loop-mediated isothermal amplification (LAMP) method is relatively simple and can be improved for better use in endemic countries. In this study, we attempted to improve this method for malaria diagnosis by using a simple and portable device capable of performing both the amplification and detection (by fluorescence) of LAMP in one platform. We refer to this as the RealAmp method. METHODOLOGY AND SIGNIFICANT FINDINGS: Published genus-specific primers were used to test the utility of this method. DNA derived from different species of malaria parasites was used for the initial characterization. Clinical samples of P. falciparum were used to determine the sensitivity and specificity of this system compared to microscopy and a nested PCR method. Additionally, directly boiled parasite preparations were compared with a conventional DNA isolation method. The RealAmp method was found to be simple and allowed real-time detection of DNA amplification. The time to amplification varied but was generally less than 60 minutes. All human-infecting Plasmodium species were detected. The sensitivity and specificity of RealAmp in detecting P. falciparum was 96.7% and 91.7% respectively, compared to microscopy and 98.9% and 100% respectively, compared to a standard nested PCR method. In addition, this method consistently detected P. falciparum from directly boiled blood samples. CONCLUSION: This RealAmp method has great potential as a field usable molecular tool for diagnosis of malaria. This tool can provide an alternative to conventional PCR based diagnostic methods for field use in clinical and operational programs.