Pyrazinamidase activity of Mycobacterium tuberculosis--a test of sensitivity to pyrazinamide

Pyrazinamidase activity has been found to correlate with pyrazinamide sensitivity in strains of Mycobacterium tuberculosis. In vitro sensitivity to pyrazinamide in acidified L?wenstein-Jensen medium, and pyrazinamidase activity by the Wayne method, were determined in 378 clinical isolates of M. tuberculosis. A close correlation was observed between the results of both tests. This method of detecting pyrazinamidase activity was found to be a rapid, simple and reliable substitute for pyrazinamide sensitivity testing, and it overcomes the difficulty of growing M. tuberculosis at pH 5.5, as required in the standard method.     

Novel therapy for insulin-dependent diabetes mellitus: infusion of in vitro-generated insulin-secreting cells

Insulin-dependent diabetes mellitus (IDDM) is a metabolic disease usually resulting from autoimmune-mediated β-cell destruction requiring lifetime exogenous insulin replacement. Mesenchymal stem cells (MSC) hold promising therapy. We present our experience of treating IDDM with co-infusion of in vitro autologous adipose tissue-derived MSC-differentiated insulin-secreting cells (ISC) with hematopoietic stem cells (HSC). This was an Institutional Review Board approved prospective non-randomized open-labeled clinical trial after informed consent from ten patients. ISC were differentiated from autologous adipose tissue-derived MSC and were infused with bone marrow-derived HSC in portal, thymic circulation by mini-laparotomy and in subcutaneous circulation. Patients were monitored for blood sugar levels, serum C-peptide levels, glycosylated hemoglobin (Hb1Ac) and glutamic acid decarboxylase (GAD) antibodies. Insulin administration was made on sliding scale with an objective of maintaining FBS < 150 mg/dL and PPBS around 200 mg/dL. Mean 3.34 mL cell inoculums with 5.25 × 10⁴ cells/μL were infused. No untoward effects were observed. Over a mean follow-up of 31.71 months, mean serum C-peptide of 0.22 ng/mL before infusion had sustained rise of 0.92 ng/mL with decreased exogenous insulin requirement from 63.9 international units (IU)/day to 38.6 IU/day. Improvement in mean Hb1Ac was observed from 10.99 to 6.72 %. Mean GAD antibodies were positive in all patients with mean of 331.10 IU/mL, which decreased to mean of 123 IU/mL. Co-infusion of autologous ISC with HSC represents a viable novel therapeutic option for IDDM.     

Kapetansky-Juri Technique to Correct the Whistlers Lip in the Multiple Operated Cleft Patient

Purpose

One of the common sequels of a cleft lip repair may be “whistling lip deformity” but other deformities are also seen particularly in failed or multiple resurgery cases. This retrospective study was carried out to evaluate the usefulness of “Kapetansky-Juri” advancement flap technique to correct such deformities.

Methods

Ten patients of bilateral cleft lip with history of minimum five failed cleft lip surgeries and having residual lip deformity were operated using “Kapetansky-Juri” advancement flap technique and were followed up to minimum 36 months.

Results

All patients showed good tissue fullness and complete correction of the deformity. There was no contracture of surrounding skin or vermilion during follow-up period. In eight patients minimal scar formation was seen while two showed midline scar formation.

Conclusion

No tissue loss due to vascular insufficiency was observed. The technique gives good tissue distribution and minimal surface scar formation.     

Toll‐like receptor 6 mediated inflammatory and functional responses of zinc oxide nanoparticles primed macrophages

Macrophages are among the most sensitive immune cells because of their phagocytic activity and are prone to become dysfunctional or not able to perform properly if nanoparticle load increases. We have previously reported that zinc oxide nanoparticles (ZNPs) induce inflammatory responses in macrophages that contribute to their death. Recognition of ZNPs by pattern recognition receptors such as toll‐like receptors (TLRs) might be a factor in the initiation of these responses in macrophages. Therefore, in this study we explored the role played by TLR6 and mitogen‐activated protein kinase (MAPKs) pathways in the inflammatory responses of macrophages during ZNPs exposure. ZNPs‐activated macrophages showed enhanced expression of activation and maturation markers (CD1d, MHC‐II, CD86 and CD71). Among various TLRs screened, TLR6 emerged as the most potent activator for ZNPs‐induced inflammatory responses. Downstream signalling proteins myeloid differentiation 88, interleukin‐1 receptor associated kinase and tumour necrosis factor receptor‐associated factor were also enhanced. On inhibiting MAPKs pathways individually, the inflammatory responses such as interleukin‐1β, interleukin‐6, tumour necrosis factor‐α, cyclooxygenase‐2 and inducible nitric oxide synthase were suppressed. TLR6 silencing significantly inhibited the pro‐inflammatory cytokine levels, reactive nitrogen species generation and inducible nitric oxide synthase expression. Also, inhibition of MAPKs in the absence of TLR6 signalling validated the link between TLR6 and MAPKs in ZNPs‐induced inflammatory responses. TLR6 was found to be co‐localized with autophagosomes. Macrophages lacking TLR6 inhibited the autophagosome marker protein‐microtubule‐associated protein1 light chain 3‐isoform II formation and phagocytosis. These results demonstrate that inflammatory responses caused by ZNPs‐activated macrophages strongly depend on TLR6‐mediated MAPK signalling.     

Fractional exhaled nitric oxide in children with acute exacerbation of asthma

Objective

To determine whether fractional exhaled nitric oxide (FENO) has a utility as a diagnostic or predictive maker in acute exacerbations of asthma in children.

Design

Analysis of data collected in a pediatric asthma cohort.

Setting

Pediatric Chest Clinic of a tertiary care hospital

Methods

A cohort of children with asthma was followed up every 3 months in addition to any acute exacerbation visits. Pulmonary function tests (PFT) and FENO were obtained at all visits. We compared the FENO values during acute exacerbations with those at baseline and those during the follow up.

Results

243 asthmatic children were enrolled from August 2009 to December 2011 [mean (SD) follow up — 434 (227) days]. FENO during acute exacerbations was not different from FENO during follow up; however, FENO was significantly higher than personal best FENO during follow up (P < 0.0001). FENO during acute exacerbation did not correlate with the severity of acute exacerbation (P=0.29). The receiver operating characteristics curve for FENO as a marker for acute exacerbation had an area under the curve of 0.59. Cut-off of 20 ppb had a poor sensitivity (44%) and specificity (68.7%) for acute exacerbation.

Conclusions

FENO levels during acute exacerbation increase from their personal best levels. However, no particular cut off could be identified that could help in either diagnosing acute exacerbation or predicting its severity.     

From the Cover: Defense mutualisms enhance plant diversification

The ability of plants to form mutualistic relationships with animal defenders has long been suspected to influence their evolutionary success, both by decreasing extinction risk and by increasing opportunity for speciation through an expanded realized niche. Nonetheless, the hypothesis that defense mutualisms consistently enhance plant diversification across lineages has not been well tested due to a lack of phenotypic and phylogenetic information. Using a global analysis, we show that the >100 vascular plant families in which species have evolved extrafloral nectaries (EFNs), sugar-secreting organs that recruit arthropod mutualists, have twofold higher diversification rates than families that lack species with EFNs. Zooming in on six distantly related plant clades, trait-dependent diversification models confirmed the tendency for lineages with EFNs to display increased rates of diversification. These results were consistent across methodological approaches. Inference using reversible-jump Markov chain Monte Carlo (MCMC) to model the placement and number of rate shifts revealed that high net diversification rates in EFN clades were driven by an increased number of positive rate shifts following EFN evolution compared with sister clades, suggesting that EFNs may be indirect facilitators of diversification. Our replicated analysis indicates that defense mutualisms put lineages on a path toward increased diversification rates within and between clades, and is concordant with the hypothesis that mutualistic interactions with animals can have an impact on deep macroevolutionary patterns and enhance plant diversity.Ever since the key innovation hypothesis was first proposed in the 1940s (1, 2), the origination of novel traits has been a popular yet controversial explanation for the exceptional disparity in species richness observed across clades in the tree of life. Despite decades of research linking traits to diversification, we have remarkably few examples of traits that have been convincingly demonstrated to spur diversification repeatedly across independent, distantly related groups. Notable exceptions include a number of ecologically important traits mediating interactions between plants and animals (3–6), suggesting that these interactions may be particularly important drivers of macroevolutionary patterns. Here, we test the hypothesis that plant defense mutualisms, a widespread and classically studied ecological interaction whereby plants provide food rewards to arthropod bodyguards in return for protection against natural enemies (7), increase the evolutionary diversification rate of the plant lineages that participate in them. The morphological traits that mediate defense mutualisms represent well-studied examples of characters hypothesized to expand a plant’s niche via interactions with mutualists and influence species success in various environmental contexts (8). Although the costs and benefits of participating in defense mutualisms are well studied (9), the hypothesis that the ecological impact of defense mutualisms leaves a predictable macroevolutionary signature, increasing lineage diversification within and among clades of plants, has only been examined in a single genus (10).Defense mutualisms may have an impact on plant speciation and extinction rates via several mechanisms. Unlike the evolution of traits related to reproduction, which, more intuitively, could have an impact on lineage diversification (e.g., refs. 5, 11), the direct mechanism by which defense mutualisms are hypothesized to influence diversification is less obvious. One direct mechanism is a decreased incidence of damage and disease due to an enhanced defensive repertoire, which may allow for increased population sizes and, in turn, lower extinction rates (6). Additionally, by expanding the realized niche of a plant (12), defense mutualisms may broaden the range of habitats a plant can occupy (10), thereby increasing instances of allopatric speciation.However, in addition to these direct mechanisms, the evolution of mutualistic traits may facilitate diversification indirectly. First, if niche expansion results in the successful occupation of more environments, mutualistic traits may increase the probability a lineage will encounter conditions ripe with ecological opportunity (e.g., new adaptive zones), which, in turn, will drive increases in diversification. In other words, the evolution of a trait may enable subsequent diversification via increasing exposure to new environments, some of which will harbor external drivers of radiation, such as the uplift of a mountain range or unoccupied niches. Second, the evolution of defense mutualisms may free up resources for the plant, and thereby facilitate the evolution of other innovative traits that subsequently enhance diversification. These indirect effects need not be contingent on the existence of the direct effects mentioned above, and represent a largely overlooked hypothesis concerning how traits can affect diversification (13–15).We suggest that indirect impacts of trait evolution on diversification should be reflected in a phylogenetic pattern in which the origination of a trait is followed by an increased probability of subsequent, downstream rate shifts relative to clades that lack the trait (Fig. 1). Because the indirect effect of the trait is contingent upon additional conditions (e.g., ecological opportunity, the evolution of another trait), there may be a substantial lag between the origin of the trait and rate shifts. Alternatively, a direct effect of the trait on the diversification rate is consistent with a pattern whereby a sustained rate shift occurs concomitantly with, or on the same branch as, the origin of the trait on the phylogeny (Fig. 1). Direct and indirect patterns are not mutually exclusive, and both patterns may be detectable on a single phylogeny (Fig. 1).Open in a separate windowFig. 1.A conceptualization of phylogenetic patterns consistent with direct or indirect effects of EFNs (or any trait) on lineage diversification. A net change in diversification may be due to direct or indirect mechanisms. In the Upper Right, a rate shift occurs concomitantly with the origin of EFNs, consistent with a direct effect. If one or more shifts occur with some delay (Lower), this is consistent with a hypothesis that a trait has an indirect or context-dependent effect on diversification rates.We focus on the macroevolutionary consequences of the repeated origination of extrafloral nectaries (EFNs), nectar-secreting glands found on nonfloral plant tissues that provide food for a wide array of beneficial arthropod bodyguards (16). EFNs are well studied ecologically, and their only known function is defense against herbivores and microbial pathogens by attracting natural enemies (17). Such features have evolved hundreds of times and occur in about a quarter of all vascular plant families (18). Here, we first ask whether, across all vascular plants, families containing species with EFNs are associated with higher diversification rates than families without EFNs. We then focus in on the phylogenetic history and evolution of EFNs in six distantly related plant clades to evaluate whether EFNs are linked, directly or indirectly, to increased lineage diversification rates. As such, this study represents a replicated, multiscale test of the macroevolutionary consequences of a convergently evolved and ecologically important mutualistic trait.     

Community-wide convergent evolution in insect adaptation to toxic cardenolides by substitutions in the Na,K-ATPase

The extent of convergent molecular evolution is largely unknown, yet is critical to understanding the genetics of adaptation. Target site insensitivity to cardenolides is a prime candidate for studying molecular convergence because herbivores in six orders of insects have specialized on these plant poisons, which gain their toxicity by blocking an essential transmembrane carrier, the sodium pump (Na,K-ATPase). We investigated gene sequences of the Na,K-ATPase α-subunit in 18 insects feeding on cardenolide-containing plants (spanning 15 genera and four orders) to screen for amino acid substitutions that might lower sensitivity to cardenolides. The replacement N122H that was previously shown to confer resistance in the monarch butterfly (Danaus plexippus) and Chrysochus leaf beetles was found in four additional species, Oncopeltus fasciatus and Lygaeus kalmii (Heteroptera, Lygaeidae), Labidomera clivicollis (Coleoptera, Chrysomelidae), and Liriomyza asclepiadis (Diptera, Agromyzidae). Thus, across 300 Myr of insect divergence, specialization on cardenolide-containing plants resulted in molecular convergence for an adaptation likely involved in coevolution. Our screen revealed a number of other substitutions connected to cardenolide binding in mammals. We confirmed that some of the particular substitutions provide resistance to cardenolides by introducing five distinct constructs of the Drosophila melanogaster gene into susceptible eucaryotic cells under an ouabain selection regime. These functional assays demonstrate that combined substitutions of Q(111) and N(122) are synergistic, with greater than twofold higher resistance than either substitution alone and >12-fold resistance over the wild type. Thus, even across deep phylogenetic branches, evolutionary degrees of freedom seem to be limited by physiological constraints, such that the same molecular substitutions confer adaptation.