How Do Urban Indian Private Practitioners Diagnose and Treat Tuberculosis? A Cross-Sectional Study in Chennai

Setting

Private practitioners are frequently the first point of healthcare contact for patients with tuberculosis (TB) in India. Inappropriate TB management practices among private practitioners may contribute to delayed TB diagnosis and generate drug resistance. However, these practices are not well understood. We evaluated diagnostic and treatment practices for active TB and benchmarked practices against International Standards for TB Care (ISTC) among private medical practitioners in Chennai.

Design

A cross-sectional survey of 228 practitioners practicing in the private sector from January 2014 to February 2015 in Chennai city who saw at least one TB patient in the previous year. Practitioners were randomly selected from both the general community and a list of practitioners who referred patients to a public-private mix program for TB treatment in Chennai. Practitioners were interviewed using standardized questionnaires.

Results

Among 228 private practitioners, a median of 12 (IQR 4–28) patients with TB were seen per year. Of 10 ISTC standards evaluated, the median of standards adhered to was 4.0 (IQR 3.0–6.0). Chest physicians reported greater median ISTC adherence than other MD and MS practitioners (score 7.0 vs. 4.0, P<0.001), or MBBS practitioners (score 7.0 vs. 4.0, P<0.001). Only 52% of all practitioners sent >5% of patients with cough for TB testing, 83% used smear microscopy for diagnosis, 33% monitored treatment response, and 22% notified TB cases to authorities. Of 228 practitioners, 68 reported referring all patients with new pulmonary TB for treatment, while 160 listed 27 different regimens; 78% (125/160) prescribed a regimen classified as consistent with ISTC. Appropriate treatment practices differed significantly between chest physicians and other MD and MS practitioners (54% vs. 87%, P<0.001).

Conclusion

TB management practices in India’s urban private sector are heterogeneous and often suboptimal. Private providers must be better engaged to improve diagnostic capacity and decrease TB transmission in the community.     

Sphingolipids are required for the stable membrane association of glycosylphosphatidylinositol-anchored proteins in yeast

Ongoing sphingolipid synthesis is specifically required in vivo for the endoplasmic reticulum (ER) to Golgi transport of glycosylphosphatidylinositol (GPI)-anchored proteins. However, the sphingolipid intermediates that are required for transport nor their role(s) have been identified. Using stereoisomers of dihydrosphingosine, together with specific inhibitors and a mutant defective for sphingolipid synthesis, we now show that ceramides and/or inositol sphingolipids are indispensable for GPI-anchored protein transport. Furthermore, in the absence of sphingolipid synthesis, a significant fraction of GPI-anchored proteins is no longer associated tightly with the ER membrane. The loose membrane association is neither because of the lack of a GPI-anchor nor because of prolonged ER retention of GPI-anchored proteins. These results indicate that ceramides and/or inositol sphingolipids are required to stabilize the association of GPI-anchored proteins with membranes. They could act either by direct involvement as membrane components or as substrates for the remodeling of GPI lipid moieties.     

Functions of fights in territory establishment

Fights are often observed when prospective territory owners settle in patches of vacant habitat, but the function of these fights in space acquisition is obscure. This study tests two hypotheses about the effect of fights on subsequent space use patterns: first, that settlers win space by winning fights and, second, that fights encourage the establishment of mutually exclusive home ranges between opponents (i.e., "fights make neighbors"). The behavior of juvenile Anolts aeneus lizards was recorded as they established territories in patches of habitat in the field. In support of the fights-make-neighbors hypothesis, opponents whose last aggressive interaction was a fight were six times more likely to have mutually exclusive home ranges at the end of the settlement period than were otherwise equivalent dyads whose last encounter was a chase. Contra the hypothesis that settlers win space by winning fights, most last fights ended in a draw, and there was no discernable relationship between the outcome of last fights and the subsequent space use of the contestants. These and previous analyses of settlement behavior in this species suggest that fights during the settlement period encourage the formation of symmetrical social and spatial relationships between neighboring settlers.     

Effect of oilseed cakes on alpha-amylase production by Bacillus licheniformis CUMC305.

The effects of oilseed cakes on extracellular thermostable alpha-amylase production by Bacillus licheniformis CUMC305 was investigated. Each oilseed cake was made of groundnut, mustard, sesame, linseed, coconut copra, madhuca, or cotton. alpha-Amylase production was considerably improved in all instances and varied with the oilseed cake concentration in basal medium containing peptone and beef extract. Maximum increases were effected by a low concentration (0.5 to 1.0%) of groundnut or coconut, a high concentration (3%) of linseed or mustard, and an Rintermediate concentration (2%) of cotton, madhuca, or sesame. The oilseed cakes made of groundnut or mustard could completely replace the conventional peptone-beef extract medium as the fermentation base for the production of alpha-amylase by B. licheniformis. The addition of corn steep liquor to cotton, linseed, sesame, or madhuca cake in the medium improved alpha-amylase production.     

Novel Mutations that Enhance or Repress the Aggregation Potential of SOD1

Mutations in SOD1 cause FALS by a gain of function likely related to protein misfolding and aggregation. SOD1 mutations encompass virtually every domain of the molecule, making it difficult to identify motifs important in SOD1 aggregation. Zinc binding to SOD1 is important for structural integrity, and is hypothesized to play a role in mutant SOD1 aggregation. To address this question, we mutated the unique zinc binding sites of SOD1 and examined whether these changes would influence SOD1 aggregation. We generated single and multiple mutations in SOD1 zinc binding residues (H71, H80 and D83) either alone or in combination with an aggregate forming mutation (A4V) known to cause disease. These SOD1 mutants were assayed for their ability to form aggregates.Using an in vitro cellular SOD1 aggregation assay, we show that combining A4V with mutations in non-zinc binding domains (G37R or G85R) increases SOD1 aggregation potential. Mutations at two zinc binding residues (H71G and D83G) also increase SOD1 aggregation potential. However, an H80G mutation at the third zinc binding residue decreases SOD1 aggregation potential even in the context of other aggregate forming SOD1 mutations. These results demonstrate that various mutations have different effects on SOD1 aggregation potential and that the H80G mutation appears to uniquely act as a dominant inhibitor of SOD1 aggregation.     

Activation of insect anti-oxidative mechanisms by mammalian glucagon

Resembling the main function of insect adipokinetic hormones (AKHs), the vertebrate hormone glucagon mobilizes energy reserves and participates in the control of glucose level in the blood. Considering the similarities, the effect of porcine glucagon was evaluated in an insect model species, the firebug Pyrrhocoris apterus. Using the mouse anti-glucagon antibody, presence of immunoreactive material was demonstrated for the first time in the firebug CNS and gut by ELISA. Mammalian (porcine) glucagon injected into the adult bugs showed no effect on hemolymph lipid level or on the level of AKH in CNS and hemolymph, however, it activated an antioxidant response when oxidative stress was elicited by paraquat, a diquaternary derivative of 4, 4′-bipyridyl. Glucagon elicited the antioxidant response by increasing glutathione and decreasing protein carbonyl levels in hemolymph, decreasing both protein carbonyl and protein nitrotyrosine levels in CNS. Additionally, when co-injected with paraquat, glucagon partially eliminated oxidative stress markers elicited by this redox cycling agent and oxidative stressor. This indicates that glucagon might induce an antioxidant defense in insects, as recently described for AKH. Failure of glucagon to alter AKH level in the bug's body indicates employment of an independent pathway without involving the native AKH.     

Oxidation of methionine residues in recombinant human interleukin-1 receptor antagonist: implications of conformational stability on protein oxidation kinetics

Oxidation of methionine residues is involved in several biochemical processes and in degradation of therapeutic proteins. The relationship between conformational stability and methionine oxidation in recombinant human interleukin-1 receptor antagonist (rhIL-1ra) was investigated to document how thermodynamics of unfolding affect methionine oxidation in proteins. Conformational stability of rhIL-1ra was monitored by equilibrium urea denaturation, and thermodynamic parameters of unfolding (DeltaGH2O, m, and Cm) were estimated at different temperatures. Methionine oxidation induced by hydrogen peroxide at varying temperatures was monitored during "coincubation" of rhIL-1ra with peptides mimicking specific regions of the reactive methionine residues in the protein. The coincubation study allowed estimation of oxidation rates in protein and peptide at each temperature from which normalized oxidation rate constants and activation energies were calculated. The rate constants for buried Met-11 in the protein were lower than for methionine in the peptide with an associated increase in activation energy. The rate constants and activation energy of solvent exposed methionines in protein and peptide were similar. The results showed that conformational stability, monitored using the Cm value, has an effect on oxidation rates of buried methionines. The rate constant of buried Met-11 correlated well with the Cm value but not DeltaGH2O. No correlation was observed for the oxidation rates of solvent-exposed methionines with any thermodynamic parameters of unfolding. The findings presented have implications in protein engineering, in design of accelerated stability studies for protein formulation development, and in understanding disease conditions involving protein oxidation.     

Mathematical model based estimation of volumetric oxygen transfer coefficient(s) in the production of proteolytic enzymes in Bacillus amyloliquefaciens

Alkaline protease is a class of important hydrolytic enzymes having wide applications in bioprocess industries. Their optimum pH in the alkaline range and stability at higher temperatures make them ideal in detergent and leather processing industries. These enzymes have excellent depilating capacity. The present study aims at process optimization for the production of alkaline protease from Bacillus amyloliquefaciens ATCC 23844. Information on the optimal operating temperature and pH were elicited from specific growth rates and alkaline protease yields. It was also observed that besides pH and temperature, the oxygen transfer rate is another important limiting variable for the production of protease. Volumetric oxygen transfer coefficient (k _L a) was estimated at various impeller speeds and aeration rates. The optimal impeller speed and aeration rates were determined from k _L a and the relative protease yield data. It was understood that the oxygen transfer rate is one of the crucial parameters for the production of proteolytic enzymes by B. amyloliquefaciens.     

Characterization of liver disease and lipid metabolism in the Niemann-Pick C1 mouse

Niemann-Pick type C1 (NPC1) disease is an autosomal-recessive cholesterol-storage disorder characterized by liver dysfunction, hepatosplenomegaly, and progressive neurodegeneration. The NPC1 gene is expressed in every tissue of the body, with liver expressing the highest amounts of NPC1 mRNA and protein. A number of studies have now indicated that the NPC1 protein regulates the transport of cholesterol from late endosomes/lysosomes to other cellular compartments involved in maintaining intracellular cholesterol homeostasis. The present study characterizes liver disease and lipid metabolism in NPC1 mice at 35 days of age before the development of weight loss and neurological symptoms. At this age, homozygous affected (NPC1(-/-)) mice were characterized with mild hepatomegaly, an elevation of liver enzymes, and an accumulation of liver cholesterol approximately four times that measured in normal (NPC1(+/+)) mice. In contrast, heterozygous (NPC1(+/-)) mice were without hepatomegaly and an elevation of liver enzymes, but the livers had a significant accumulation of triacylglycerol. With respect to apolipoprotein and lipoprotein metabolism, the results indicated only minor alterations in NPC1(-/-) mouse serum. Finally, compared to NPC1(+/+) mouse livers, the amount and processing of SREBP-1 and -2 proteins were significantly increased in NPC1(-/-) mouse livers, suggesting a relative deficiency of cholesterol at the metabolically active pool of cholesterol located at the endoplasmic reticulum. The results from this study further support the hypothesis that an accumulation of lipoprotein-derived cholesterol within late endosomes/lysosomes, in addition to altered intracellular cholesterol homeostasis, has a key role in the biochemical and cellular pathophysiology associated with NPC1 liver disease.     

Localization and characterization of prolipoprotein diacylglyceryl transferase (Lgt) critical in bacterial lipoprotein biosynthesis

Lipid modification of proteins is an essential post-translational event that can be targeted for protein engineering and pharmaceutical applications. In this regard, the unique and ubiquitous bacterial N-terminal lipid modification (N-acyl S-diacylglyceryl modification of N-terminal cysteine) is particularly attractive. It is initiated by phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (Lgt) and therefore its properties, which remain uninvestigated, largely define the specifics of the modification. A synthetic peptide-substrate (MKATKSAVGSTLAGCSSHHHHHH) with a short hydrophilic h-region, unlike that of the prototypical substrate used so far, demonstrated lack of enzyme's substrate preference based on hydrophobicity, perhaps accounting for a significant number of lipoproteins possessing hydrophilic signal peptides. Solubilization experiments revealed a peripheral and possibly reversible hydrophobic association of Lgt with the inner-membrane on the cytosolic side contradicting its deduced transmembrane topology. Except for heat stability, the soluble enzyme was indistinguishable from the membrane-bound enzyme in kinetic behaviour, indicating that the committed first step of bacterial lipid modification may be aqueous compatible. The direct, more accurate, precise and easier paper electrophoretic assay, designed anew, and Lgt's ready extraction with water or low ionic strength solutions from inverted vesicles could aid better understanding and exploitation of the enzyme and bacterial lipid modification.