Use of in-house PCR for identification of Mycobacterium tuberculosis in BACTEC broth cultures of respiratory specimens

We evaluated the ability of a PCR assay to identify Mycobacterium tuberculosis complex (MTBC) from positive BACTEC(R) 12B broth cultures. A total of 107 sputum samples were processed and inoculated into Ogawa slants and BACTEC(R) 12B vials. At a growth index (GI) > 30, 1.0 ml of the 12B broth was removed, stored, and assayed with PCR. Molecular results were compared to those obtained by phenotypic identification methods, including the BACTEC(R) NAP method. The average times required to perform PCR and NAP were compared. Of the 107 broth cultures evaluated, 90 were NAP positive, while 91 were PCR positive for MTBC. Of particular interest were three contaminated BACTEC(R) 12B broth cultures yielding microorganisms other than acid-fast bacilli growth with a MTBC that were successfully identified by PCR, resulting in a mean time of 14 days to identify MTBC before NAP identification. These results suggest that PCR could be used as an alternative to the NAP test for the rapid identification of MTBC in BACTEC(R) 12B cultures, particularly in those that contained both MTBC and nontuberculous mycobacteria.     

Strong cross-bridges potentiate the Ca(2+) affinity changes produced by hypertrophic cardiomyopathy cardiac troponin C mutants in myofilaments: a fast kinetic approach

This spectroscopic study examined the steady-state and kinetic parameters governing the cross-bridge effect on the increased Ca(2+) affinity of hypertrophic cardiomyopathy-cardiac troponin C (HCM-cTnC) mutants. Previously, we found that incorporation of the A8V and D145E HCM-cTnC mutants, but not E134D into thin filaments (TFs), increased the apparent Ca(2+) affinity relative to TFs containing the WT protein. Here, we show that the addition of myosin subfragment 1 (S1) to TFs reconstituted with these mutants in the absence of MgATP(2-), the condition conducive to rigor cross-bridge formation, further increased the apparent Ca(2+) affinity. Stopped-flow fluorescence techniques were used to determine the kinetics of Ca(2+) dissociation (k(off)) from the cTnC mutants in the presence of TFs and S1. At a high level of complexity (i.e. TF + S1), an increase in the Ca(2+) affinity and decrease in k(off) was achieved for the A8V and D145E mutants when compared with WT. Therefore, it appears that the cTnC Ca(2+) off-rate is most likely to be affected rather than the Ca(2+) on rate. At all levels of TF complexity, the results obtained with the E134D mutant reproduced those seen with the WT protein. We conclude that strong cross-bridges potentiate the Ca(2+)-sensitizing effect of HCM-cTnC mutants on the myofilament. Finally, the slower k(off) from the A8V and D145E mutants can be directly correlated with the diastolic dysfunction seen in these patients.     

Cytoplasmic glutathione redox status determines survival upon exposure to the thiol-oxidant 4,4'-dipyridyl disulfide

Dipyridyl disulfide (DPS) is a highly reactive thiol oxidant that functions as electron acceptor in thiol-disulfide exchange reactions. DPS is very toxic to yeasts, impairing growth at low micromolar concentrations. The genes TRX2 (thioredoxin), SOD1 (superoxide dismutase), GSH1 (gamma-glutamyl-cysteine synthetase) and, particularly, GLR1 (glutathione reductase) are required for survival on DPS. DPS is uniquely thiol-specific, and we found that the cellular mechanisms for DPS detoxification differ substantially from that of the commonly used thiol oxidant diamide. In contrast to this oxidant, the full antioxidant pools of glutathione (GSH) and thioredoxin are required for resistance to DPS. We found that DPS-sensitive mutants display increases in the disulfide form of GSH (GSSG) during DPS exposure that roughly correlate with their more oxidizing GSH redox potential in the cytosol and their degree of DPS sensitivity. DPS seems to induce a specific disulfide stress, where an increase in the cytoplasmic/nuclear GSSG/GSH ratio results in putative DPS target(s) becoming sensitive to DPS.     

A potent modifier of liver cancer risk on distal mouse chromosome 1: linkage analysis and characterization of congenic lines

The C3H/HeJ (C3H) and CBA/J (CBA) mouse strains are classical mouse models of cancer susceptibility, exhibiting high risks for both spontaneous and chemically induced liver cancer. By analysis of backcrosses and intercrosses between C3H or CBA and resistant B6 mice, we have mapped a potent modifier of hepatocellular carcinoma development to distal chromosome 1, linked to the marker D1Mit33 with combined LOD(W) scores of approximately 5.9 (C3H) and 6.5 (CBA). We previously identified this region as one of two that modify susceptibility in the more distantly related C57BR/cdJ (BR) strain. Congenic B6.C3H(D1Mit5-D1Mit17) and B6.BR(D1Mit5-D1Mit17) mice developed significantly more liver tumors than B6 mice did (6- to 13-fold, P < 10(-11), in males; 3- to 4-fold, P < 10(-3), in females). Thus, distal chromosome 1 carries one or more genes that are sufficient to confer susceptibility to liver cancer.     

Improved pharmacological properties for superoxide dismutase modified with β-cyclodextrin–carboxymethylcellulose polymer

Superoxide dismutase was glycosidated with cyclodextrin-branched carboxymethylcellulose. The modified enzyme contained 1.4 mol polymer per mol protein and retained 87% of the initial activity. The anti-inflammatory activity of superoxide dismutase was 2.2-times increased after conjugation and its plasma half-life time was prolonged from 4.8 min to 7.2 h.     

Supramolecular-mediated immobilization of l-phenylalanine dehydrogenase on cyclodextrin-coated Au electrodes for biosensor applications

A supramolecular approach was used for adsorbing a monolayer of adamantane-modified phenylalanine dehydrogenase on β-cyclodextrin-coated Au electrodes. The enzyme electrode (poised at +200 mV vs. Ag/AgCl) showed a linear amperometric response up to 3 mM l-phenylalanine (l-Phe) with a lower detection limit of 15 μM. The reversible nature of this immobilization approach was confirmed.     

Optimization of Ellagitannase Production by Aspergillus niger GH1 by Solid-State Fermentation

Ellagic acid is one of the most bioactive antioxidants with important applications in pharmaceutical, cosmetic, and food industries. However, there are few biotechnological processes developed for its production, because it requires precursors (ellagitannins) and the corresponding biocatalyst (ellagitannase). The aim of this study was to optimize the culture conditions for ellagitannase production by Aspergillus niger in solid-state fermentation (SSF). The bioprocess was carried out into a column bioreactor packed with polyurethane foam impregnated with an ellagitannins solution as carbon source. Four strains of Aspergillus niger (PSH, GH1, HT4, and HC2) were evaluated for ellagitannase production. The study was performed in two experimental steps. A Plackett–Burman design was used to determine the influencing parameters on ellagitannase production. Ellagitannins concentration, KCl, and MgSO₄ were determined to be the most significant parameters. Box–Behnken design was used to define the interaction of the selected parameters. The highest enzyme value was obtained by A. niger PSH at concentrations of 7.5 g/L ellagitannins, 3.04 g/L KCl, and 0.76 g/L MgSO₄. The methodology followed here allowed increasing the ellagitannase activity 10 times over other researcher results (938.8 U/g ellagitannins). These results are significantly higher than those reported previously and represent an important contribution for the establishment of a new bioprocess for ellagic acid and ellagitannase production.     

Infant Feeding and School Attainment in Five Cohorts from Low- and Middle-Income Countries

Background

Performance in intelligence tests tends to be higher among individuals breastfed as infants, but little is known about the association between breastfeeding and achieved schooling. We assessed the association of infant feeding with school achievement in five cohorts from low- and middle-income countries. Unlike high-income country settings where most previous studies come from, breastfeeding is not positively associated with socioeconomic position in our cohorts, thus reducing the likelihood of a spurious positive association.

Methodology and Principal Findings

Participants included 10,082 young adults from five birth cohorts (Brazil, India, Guatemala, the Philippines, and South Africa). The exposures variables were whether the subject was ever breastfed, total duration of breastfeeding, and age at introduction of complementary foods. We adjusted the estimates for age at follow up, sex, maternal age, smoking during pregnancy, birthweight and socioeconomic position at birth. The key outcome was the highest grade achieved at school. In unadjusted analyses, the association between ever breastfeeding and schooling was positive in Brazil, inverse in the Philippines, and null in South Africa; in adjusted analyses, these associations were attenuated. In Brazil, schooling was highest among individuals breastfed for 3–12 months whereas in the Philippines duration of breastfeeding was inversely associated with schooling; and null associations were observed in South Africa and Guatemala. These associations were attenuated in adjusted models. Late introduction of solid foods was associated with lower schooling achievement in Brazil and South Africa.

Conclusion

Measures of breastfeeding are not consistently related to schooling achievement in contemporary cohorts of young adults in lower and middle-income countries.     

Thermal responses and body fluid balance of competitive male swimmers during a training session

Thermoregulatory and body fluid balance (BFB) responses of competitive swimmers were studied during a typical interval training session under natural field conditions. Subjects were 9 males (18.0 +/- 1.7 years; VO(2)max = 3.8 +/- 0.9 L x min(-1)) who covered 9,000 m in 180 minutes in an outdoor pool (mean water temperature = 26.8 +/- 0.3 degrees C; mean wet bulb globe temperature = 29.8 +/- 2.8 degrees C). Mean body weight (BWt) decreased by 1.8 +/- 0.5 kg (P < 0.05), and rectal temperature increased by 1.0 +/- 1.0 degrees C (P < 0.05). Volitional water intake (WI) (0.1 +/- 0.2 kg) did not maintain BFB (-0.5 kg per hour) and plasma volume decreased 10.7 +/- 5.4%. During a typical training session, swimmers experienced significant body fluid losses, and WI was not enough to prevent involuntary dehydration. The magnitude of the fluid losses (2.5% of BWt) was sufficient to compromise convective thermoregulation because of the decreased plasma volume. Hence, to prevent involuntary dehydration, swimmers should be encouraged to consume an amount of fluids that equals losses throughout the training sessions.     

Primate models to study eccrine sweating

The histochemistry and histology of the eccrine sweat gland in the rhesus monkey (Macaca mulatta) are described. The histochemical distribution and localization of enzymes and substrates are very similar to those found in the human; innervation is cholinergic. Active eccrine glands on the general body surface average 136 glands/cm². Above the thermal neutral zone (TNZ), sweating is the major avenue for heat loss and the role of panting in dissipating heat is relatively insignificant. The intrahypothalamic administration of prostaglandin E₁ (PGE₁) suppresses sweating and leads to an increase in core temperature. A linear relation is found between local sweat rates on the general body surface and clamped hypothalamic temperature. Studies also provide direct support for the concept that brain temperature and skin temperature interact additively in the control of sweating in higher primates. The functional characteristics of eccrine sweating in the patas monkey (Erythocebus) are qualitatively similar to those in the rhesus monkey. The patas monkey maintains a relatively constant rectal temperature (37.6–38.4°C) when equilibrated to a wide range of ambient temperaures of 15–40°C. Eccrine sweating is the main effector system for heat dissipation above the TNZ. We emphasize here that evaporative heat loss that is due to sweating is related to both mean skin and mean body temperature and at 40°C is 40% higher than that recorded from the rhesus monkey. These results indicate that the patas monkey, because of its high sweating capacity and other similarities with the human eccrine system, is a most appropriate animal model for comparative studies of eccrine sweat gland function in primates in general.