Helminth Infections Coincident with Active Pulmonary Tuberculosis Inhibit Mono- and Multifunctional CD4+ and CD8+ T Cell Responses in a Process Dependent on IL-10

Tissue invasive helminth infections and tuberculosis (TB) are co-endemic in many parts of the world and can trigger immune responses that might antagonize each other. We have previously shown that helminth infections modulate the Th1 and Th17 responses to mycobacterial-antigens in latent TB. To determine whether helminth infections modulate antigen-specific and non-specific immune responses in active pulmonary TB, we examined CD4⁺ and CD8⁺ T cell responses as well as the systemic (plasma) cytokine levels in individuals with pulmonary TB with or without two distinct helminth infections—Wuchereria bancrofti and Strongyloides stercoralis infection. By analyzing the frequencies of Th1 and Th17 CD4⁺ and CD8⁺ T cells and their component subsets (including multifunctional cells), we report a significant diminution in the mycobacterial–specific frequencies of mono- and multi–functional CD4⁺ Th1 and (to a lesser extent) Th17 cells when concomitant filarial or Strongyloides infection occurs. The impairment in CD4⁺ and CD8⁺ T cell cytokine responses was antigen-specific as polyclonal activated T cell frequencies were equivalent irrespective of helminth infection status. This diminution in T cell responses was also reflected in diminished circulating levels of Th1 (IFN-γ, TNF-α and IL-2)- and Th17 (IL-17A and IL-17F)-associated cytokines. Finally, we demonstrate that for the filarial co-infections at least, this diminished frequency of multifunctional CD4⁺ T cell responses was partially dependent on IL-10 as IL-10 blockade significantly increased the frequencies of CD4⁺ Th1 cells. Thus, co-existent helminth infection is associated with an IL-10 mediated (for filarial infection) profound inhibition of antigen-specific CD4⁺ T cell responses as well as protective systemic cytokine responses in active pulmonary TB.     

Directed evolution of poly[(R)-3-hydroxybutyrate] depolymerase using cell surface display system: functional importance of asparagine at position 285

Poly[(R)-3-hydroxybutyrate] (PHB) depolymerase from Ralstonia pickettii T1 (PhaZ_RpiT1) consists of three functional domains to effectively degrade solid PHB materials, and its catalytic domain catalyzes the ester bond cleavage of the substrate. We performed the directed evolution of PhaZ_RpiT1 targeted at the catalytic domain in combination with the cell surface display method to effectively screen for mutants with improved p-nitrophenyl butyrate (pNPC4) activity. Mutated PhaZ_RpiT1 genes generated by error-prone PCR were fused to the oprI gene to display them as fusion proteins on Escherichia coli cell surface. Some cells displaying the mutant enzymes showed a two- to fourfold increase in pNPC4 hydrolysis activity relative to cells displaying wild-type enzyme. These mutant genes were recombined by a staggered extension process and the recombined enzymes were displayed to result in a five- to eightfold higher pNPC4 hydrolysis activity than the wild type. To further evaluate the mutation effects, unfused and undisplayed enzymes were prepared and applied to the hydrolysis of p-nitrophenyl esters having different chain lengths (pNPCn; n?=?2–6) and PHB degradation. One specific second-generation mutant showed an approximately tenfold increase in maximum rate for pNPC3 hydrolysis, although its PHB degradation efficiency at 1 μg/mL of enzyme concentration was approximately 3.5-fold lower than that of the wild type. Gene analysis showed that N285D or N285Y mutations were found in six of the seven improved second-generation mutants, indicating that Asn285 probably participates in the regulation of substrate recognition and may be more favorable for PHB degradation process than other amino acid residues.     

Therapeutic management of Mycobacterium avium subspecies paratuberculosis infection with complete resolution of symptoms and disease in a patient with advanced inflammatory bowel syndrome

Molecular Biology Reports - A 26-year-old male had a history of frequent bowel movements, mushy stool with mucus and loss of 25 kg body weight in 6 months was diagnosed as a case...     

Length–weight relationships of six indigenous fish species from the River Cauvery and its estuary,India

The present study estimated length–weight relationships (LWRs) for six indigenous fish species (Barilius gatensis, Salmostoma acinaces, S. boopis, Puntius amphibius, Hemibagrus punctatus and Ambassis miops) based on specimens collected from River Cauvery (including estuary) during July 2017–January 2020. The sampling surveys were carried out in three distinct sampling seasons, viz., the pre-monsoon (March–May), the monsoon (July–October) and the post-monsoon (November–February). Majority of the fish specimens dealt in the study were collected from multi-meshed monofilament gill nets (mesh sizes 18, 30, 45, 60, 90, 110, 120 and 150 mm) operated by local fishers. For those sites situated in the protected areas, sampling was carried out by cast nets with prior permission from the local administration and the collected fishes were released back into river after length–weight measurements. The length measurements were noted as total length (TL) measured to the nearest 0.1 cm by using a digital Vernier caliper. A digital balance was used for weight measurements with an accuracy of 0.01 g. The study recorded a new maximum length of 48 cm for H. punctatus. The LWR data generated from the present study are significant for proper assessment of the stock status and their management, if collected together with other essential biological and physical parameters.     

Purification and characterization of Mn-peroxidase from Musa paradisiaca (banana) stem juice

Mn-peroxidase (MnP), a biotechnologically important enzyme was purified for the first time from a plant source Musa paradisiaca (banana) stem, which is an agro-waste easily available after harvest of banana fruits. MnP was earlier purified only from the fungal sources. The enzyme was purified from stem juice by ultrafiltration and anion-exchange column chromatography on diethylamino ethylcellulose with 8-fold purification and purification yield of 65%. The enzyme gave a single protein band in SDS-PAGE corresponding to molecular mass 43 kDa. The Native-PAGE of the enzyme also gave a single protein band, confirming the purity of the enzyme. The UV/VIS spectrum of the purified enzyme differed from the other heme peroxidases, as the Soret band was shifted towards lower wavelength and the enzyme had an intense absorption band around 250 nm. The K(m) values using MnSO4 and H2O2 as the substrates of the purified enzyme were 21.0 and 9.5 microM, respectively. The calculated k(cat) value of the purified enzyme using Mn(II) as the substrate in 50 mM lactate buffer (pH 4.5) at 25 degrees C was 6.7s(-1), giving a k(cat)/K(m) value of 0.32 microM(-1)s(-1). The k(cat) value for the MnP-catalyzed reaction was found to be dependent of the Mn(III) chelator molecules malonate, lactate and oxalate, indicating that the enzyme oxidized chelated Mn(II) to Mn(III). The pH and temperature optima of the enzyme were 4.5 and 25 degrees C, respectively. The enzyme in combination with H2O2 liberated bromine and iodine in presence of KBr and KI respectively. All these enzymatic characteristics were similar to those of fungal MnP. The enzyme has the potential as a green brominating and iodinating agent in combination with KBr/KI and H2O2.     

Src-mediated Post-translational Regulation of Endoglin Stability and Function Is Critical for Angiogenesis

Endoglin is a transforming growth factor β (TGF-β) co-receptor essential for angiogenesis and tumor vascularization. Endoglin modulates the crucial balance between pro- and anti-angiogenic signaling by activin receptor-like kinase (ALK) 1, 5, and TGF-β type II (TβRII) receptors. Despite its established role in physiology and disease, the mechanism of endoglin down-regulation remains unknown. Here we report that the conserved juxtamembrane cytoplasmic tyrosine motif (⁶¹²YIY⁶¹⁴) is a critical determinant of angiogenesis. Src directly phosphorylates this motif to induce endoglin internalization and degradation via the lysosome. We identified epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) as Src-activators that induce endoglin turnover following ⁶¹²YIY⁶¹⁴ phosphorylation. Interestingly, Src phosphorylation of endoglin-⁶¹²YIY⁶¹⁴ was also an important process for receptor down-regulation by TRACON105 (TRC105), an endoglin-targeting antibody currently in clinical trials. The regulation of ⁶¹²YIY⁶¹⁴ phosphorylation was critical for angiogenesis, as both the phosphomimetic and unphosphorylatable mutants impaired endothelial functions including proliferation, migration, and capillary tube formation. Collectively, these findings establish Src and pro-angiogenic mitogens as critical mediators of endoglin stability and function.     

Development and characterization of a human articular cartilage‐derived chondrocyte cell line that retains chondrocyte phenotype

Chondrocytes, the only cell type present in articular cartilage, regulate tissue homeostasis by a fine balance of metabolism that includes both anabolic and catabolic activities. Therefore, the biology of chondrocytes is critical for understanding cartilage metabolism. One major limitation when studying primary chondrocytes in culture is their loss of phenotype. To overcome this hurdle, limited attempts have been made to develop human chondrocyte cell lines that retain the phenotype for use as a good surrogate model. In this study, we report a novel approach to the establishment and characterization of human articular cartilage‐derived chondrocyte cell lines. Adenoviral infection followed by culture of chondrocytes in 3‐dimensional matrix within 48 h post‐infection maintained the phenotype prior to clonal selection. Cells were then placed in culture either as monolayer, or in 3‐dimensional matrix of alginate or agarose. The clones were characterized by their basal gene expression profile of chondrocyte markers. Based on type II collagen expression, 21 clones were analyzed for gene expression following treatment with IL‐1 or BMP‐7 and compared to similarly stimulated primary chondrocytes. This resulted in selection of two clones that retained the chondrocyte phenotype as evidenced by expression of type II collagen and other extra‐cellular matrix molecules. In addition, one clone (AL‐4‐17) showed similar responses as primary chondrocytes when treated with IL‐1 or BMP‐7. In summary, this report provides a novel procedure to develop human articular cartilage‐derived chondrocyte cell lines, which preserve important characteristics of articular chondrocytes and represent a useful model to study chondrocyte biology. J. Cell. Physiol. 222: 695–702, 2010. © 2009 Wiley‐Liss, Inc.     

Characterization of the Vaginal Microbiota among Sexual Risk Behavior Groups of Women with Bacterial Vaginosis

Background

The pathogenesis of bacterial vaginosis (BV) remains elusive. BV may be more common among women who have sex with women (WSW). The objective of this study was to use 454 pyrosequencing to investigate the vaginal microbiome of WSW, women who have sex with women and men (WSWM), and women who have sex with men (WSM) with BV to determine if there are differences in organism composition between groups that may inform new hypotheses regarding the pathogenesis of BV.

Methods

Vaginal swab specimens from eligible women with BV at the Mississippi State Department of Health STD Clinic were used. After DNA extraction, 454 pyrosequencing of PCR-amplified 16S rRNA gene sequences was performed. Sequence data was classified using the Ribosomal Database Program classifer. Complete linkage clustering analysis was performed to compare bacterial community composition among samples. Differences in operational taxonomic units with an abundance of ≥2% between risk behavior groups were determined. Alpha and beta diversity were measured using Shannon’s Index implemented in QIIME and Unifrac analysis, respectively.

Results

33 WSW, 35 WSWM, and 44 WSM were included. The vaginal bacterial communities of all women clustered into four taxonomic groups with the dominant taxonomic group in each being Lactobacillus, Lachnospiraceae, Prevotella, and Sneathia. Regarding differences in organism composition between risk behavior groups, the abundance of Atopobium (relative ratio (RR)=0.24; 95%CI 0.11-0.54) and Parvimonas (RR=0.33; 95%CI 0.11-0.93) were significantly lower in WSW than WSM, the abundance of Prevotella was significantly higher in WSW than WSWM (RR=1.77; 95%CI 1.10-2.86), and the abundance of Atopobium (RR=0.41; 95%CI 0.18-0.88) was significantly lower in WSWM than WSM. Overall, WSM had the highest diversity of bacterial taxa.

Conclusion

The microbiology of BV among women in different risk behavior groups is heterogeneous. WSM in this study had the highest diversity of bacterial taxa. Additional studies are needed to better understand these differences.     

Development of doubled haploids from an elite <Emphasis Type="Italic">indica</Emphasis> rice hybrid (BS6444G) using anther culture

A total of 200 doubled haploids (DHs) were generated from an elite rice hybrid, ‘BS6444G’ for which an androgenic method was developed by manipulating the physical and chemical factors. The spike pretreated at 10?°C for 7–8 days was effective for callusing and green plant regeneration. The maximum callus frequency was achieved when the anthers cultured in N6 medium supplemented with 2.0 mg L^?1 2,4-diclorophenoxyacetic acid, 0.5 mg L^?1 6-benzylaminopurine and 3% maltose. Calli induced in N6 media also showed significant green shoot regeneration in MS medium supplemented with 0.5 mg L^?1 1-napthalene acetic acid, 0.5 mg L^?1 kinetin, 1.5 mg L^?1 benzylaminopurine and 3% sucrose producing 210 green plants. Assessment of the ploidy status showed 95.71% fertile diploids and 4.2% polyploids; no haploids were observed. A total of 38 sequence-tagged microsatellite (STMS) markers proved able to discriminate a heterozygote from all the 200 DHs. The DHs grown in the field showed significant variation for their agronomic traits. Comparison of traits with control indicates homogeneity within each DH line and significant variance of traits between DH lines. Nine DH lines produce higher grain yield than the hybrid parent which suggests the possibility of exploiting hybrid vigor in indica rice through the development of DH lines of high yielding hybrids.