EspR-dependent ESAT-6 Protein Secretion of Mycobacterium tuberculosis Requires the Presence of Virulence Regulator PhoP.

Attenuation of Mycobacterium bovis BCG strain is related to the loss of the RD1-encoded ESX-1 secretion system. The ESX-1 system secretes virulence factor ESAT-6 that plays a critical role in modulation of the host immune system, which is essential for establishment of a productive infection. Previous studies suggest that among the reasons for attenuation of Mycobacterium tuberculosis H37Ra is a mutation in the phoP gene that interferes with the ESX-1 secretion system and inhibits secretion of ESAT-6. Here, we identify a totally different and distinct regulatory mechanism involving PhoP and transcription regulator EspR on transcriptional control of the espACD operon, which is required for ESX-1-dependent ESAT-6 secretion. Although both of these regulators are capable of influencing espACD expression, we show that activation of espACD requires direct recruitment of both PhoP and EspR at the espACD promoter. The most fundamental insights are derived from the inhibition of EspR binding at the espACD regulatory region of the phoP mutant strain because of PhoP-EspR protein-protein interactions. Based on these results, a model is proposed suggesting how PhoP and EspR protein-protein interactions contribute to activation of espACD expression and, in turn, control ESAT-6 secretion, an essential pathogenic determinant of M. tuberculosis Together, these results have significant implications on the mechanism of virulence regulation of M. tuberculosis.

Transcriptome analysis highlights the conserved difference between embryonic and postnatal-derived alveolar macrophages.

Alveolar macrophages (AMs) reside on the luminal surfaces of the airways and alveoli where they maintain host defense and promote alveolar homeostasis by ingesting inhaled particulates and regulating inflammatory responses. Recent studies have demonstrated that AMs populate the lungs during embryogenesis and self-renew throughout life with minimal replacement by circulating monocytes, except under extreme conditions of depletion or radiation injury. Here we demonstrate that on a global scale, environment appears to dictate AM development and function. Indeed, transcriptome analysis of embryonic host-derived and postnatal donor-derived AMs coexisting within the same mouse demonstrated >98% correlation and overall functional analyses were similar. However, we also identified several genes whose expression was dictated by origin rather than environment. The most differentially expressed gene not altered by environment was Marco, a gene recently demonstrated to have enhancer activity in embryonic-derived but not postnatal-derived tissue macrophages. Overall, we show that under homeostatic conditions, the environment largely dictates the programming and function of AMs, whereas the expression of a small number of genes remains linked to the origin of the cell.

Flow Cytometric Analysis of Mononuclear Phagocytes in Nondiseased Human Lung and Lung-Draining Lymph Nodes.

RATIONALE: The pulmonary mononuclear phagocyte system is a critical host defense mechanism composed of macrophages, monocytes, monocyte-derived cells, and dendritic cells. However, our current characterization of these cells is limited because it is derived largely from animal studies and analysis of human mononuclear phagocytes from blood and small tissue resections around tumors. OBJECTIVES: Phenotypic and morphologic characterization of mononuclear phagocytes that potentially access inhaled antigens in human lungs. METHODS: We acquired and analyzed pulmonary mononuclear phagocytes from fully intact nondiseased human lungs (including the major blood vessels and draining lymph nodes) obtained en bloc from 72 individual donors. Differential labeling of hematopoietic cells via intrabronchial and intravenous administration of antibodies within the same lobe was used to identify extravascular tissue-resident mononuclear phagocytes and exclude cells within the vascular lumen. Multiparameter flow cytometry was used to identify mononuclear phagocyte populations among cells labeled by each route of antibody delivery. MEASUREMENTS AND MAIN RESULTS: We performed a phenotypic analysis of pulmonary mononuclear phagocytes isolated from whole nondiseased human lungs and lung-draining lymph nodes. Five pulmonary mononuclear phagocytes were observed, including macrophages, monocyte-derived cells, and dendritic cells that were phenotypically distinct from cell populations found in blood. CONCLUSIONS: Different mononuclear phagocytes, particularly dendritic cells, were labeled by intravascular and intrabronchial antibody delivery, countering the notion that tissue and blood mononuclear phagocytes are equivalent systems. Phenotypic descriptions of the mononuclear phagocytes in nondiseased lungs provide a precedent for comparative studies in diseased lungs and potential targets for therapeutics.

Phosphorylation of PhoP protein plays direct regulatory role in lipid biosynthesis of Mycobacterium tuberculosis.

Mycobacterium tuberculosis PhoP is essential for virulence and intracellular growth of the tubercle bacilli. Genetic evidence suggests that PhoP regulates complex lipid biosynthesis, and absence of some of these lipid molecules in a phoP mutant partly accounts for its attenuated growth in macrophages and/or mice. To investigate the mechanism of regulation, here we demonstrate the essentiality of phosphorylation of PhoP in the regulation of complex lipid biosynthesis. We show that phosphorylated PhoP activates transcription of pks2 and msl3, gene(s) encoding polyketide ß-ketoacyl synthases through direct DNA binding at the upstream regulatory region(s) of the target genes. Our results identify the genetic determinants recognized by PhoP and show that activation of target genes requires interaction(s) of the phosphorylated regulator at the cognate binding sites. The fact that these sites within the regulatory region of respective genes do not bind in vitro with either unphosphorylated or phosphorylation-deficient PhoP protein is consistent with phosphorylation-dependent assembly of the transcription initiation complex leading to in vivo transcriptional activation. Together, these results reveal so far unknown molecular mechanisms of how PhoP contributes to M. tuberculosis cell wall composition by regulating complex lipid biosynthesis.

Efficacy of barley based probiotic food mixture in treatment of pathogenic E.coli induced diarrhoea in mice.

An indigenous food mixture was developed by mixing barley flour (raw and germinated), whey powder and tomato pulp in the ratio of 2:1:1:1 (w/w). The developed food (100 g) was mixed with water (500 ml) and autoclaved at 1.5 kg/cm(2) for 15 min. It was then cooled and inoculated with Lactobacillus acidophilus curd (5%) and incubated at 37°C for 12 h containing 10(6) cfu/ml broth. Fermented food mixture formulated from germinated barley flour maintained adequate cell viability (8.88 cfu/ml) as compared to non-germinated food mixture. To study the therapeutic effect of the food mixture, diarrhoea was induced in mice using 0.5 ml of aqueous suspension orally with the help of sterilized syringe to each of the overnight fasted mice and the mice were examined till onset of diarrhoea. The aqueous suspension was prepared by using 10 ml of six h old culture of E. coli cells (5 × 10(11) cfu/ml) and 6 ml alkaline solution (powdered chalk (40%), colloidal kaolin (43%) and magnesium trisilicate (17%) and both were mixed in 10:6 (v/v) proportions. After induction of diarrhoea, the mice were divided in two groups, control and experimental. The control group was fed on unfermented food mixture whereas experimental group was fed on fermented food mixture. Faecal ash, nitrogen, moisture and coliform count increased while faecal lactobacilli count decreased in mice having diarrhoea. In the experimental group, which was fed on fermented food mixture, normal values were reached within 7 days of feeding but no such changes were observed in control group which was fed on unfermented food mixture. Liver and kidney showed lesions due to E. coli infection were significantly alleviated on feeding of probiotic food mixture.

Domain structure of virulence-associated response regulator PhoP of Mycobacterium tuberculosis: role of the linker region in regulator-promoter interaction(s).

The PhoP and PhoR proteins from Mycobacterium tuberculosis form a highly specific two-component system that controls expression of genes involved in complex lipid biosynthesis and regulation of unknown virulence determinants. The several functions of PhoP are apportioned between a C-terminal effector domain (PhoPC) and an N-terminal receiver domain (PhoPN), phosphorylation of which regulates activation of the effector domain. Here we show that PhoPN, on its own, demonstrates PhoR-dependent phosphorylation. PhoPC, the truncated variant bearing the DNA binding domain, binds in vitro to the target site with affinity similar to that of the full-length protein. To complement the finding that residues spanning Met(1) to Arg(138) of PhoP constitute the minimal functional PhoPN, we identified Arg(150) as the first residue of the distal PhoPC domain capable of DNA binding on its own, thereby identifying an interdomain linker. However, coupling of two functional domains together in a single polypeptide chain is essential for phosphorylation-coupled DNA binding by PhoP. We discuss consequences of tethering of two domains on DNA binding and demonstrate that linker length and not individual residues of the newly identified linker plays a critical role in regulating interdomain interactions. Together, these results have implications for the molecular mechanism of transmission of conformation change associated with phosphorylation of PhoP that results in the altered DNA recognition by the C-terminal domain.

Need for Changes in the Practical Physiology Curriculum of Medical Undergraduates.

INTRODUCTION: Physiology is one of the foundation sciences for the medical curriculum. It forms the basis of all life sciences. Physiology practical classes in India are divided into three sections: exercises in haematology, exercises on humans and experiments on amphibians. Faculty and students of medical schools in India are of the opinion that animal experiments should be discontinued, that the curriculum in experimental physiology and pharmacology needs to be updated, that the same understanding of topics could be obtained by using alternative methods and that there is an urgent need to introduce alternatives of animal experiments. AIM: To assess the need for changes in the practical physiology curriculum of medical undergraduates. MATERIALS AND METHODS: The study was conducted among 300 MBBS, second and third professional students using a semi-structured questionnaire. The questionnaire consisted of two sections: the first section was based on the current experimental physiology curriculum regarding the relevance of topics taught in haematology, amphibian and human practical classes. In the second section, the opinion was also sought regarding introduction of new topics related to recent advances in clinical physiology and basic clinical skills. RESULTS: More than 60% of the students marked most of the haematology experiments useful. All the amphibian practicals were considered irrelevant/not useful by more than 75% of the students. Regarding the human experiments, more than 70% of the students marked useful. More than 65% students were of the opinion that basic clinical skills should be introduced at undergraduate level. More than 50% students favoured introduction of topics from recent advances in physiology. CONCLUSION: The present study concludes that there is an imperative need to implement radical changes in the experimental physiology curriculum which should be in consonance with patient care for the doctors of tomorrow to render better health service.

Effect of Menstrual Cycle Phases on Plasma Lipid and Lipoprotein Levels in Regularly Menstruating Women.

INTRODUCTION: Physiological cyclic variability in levels of sex hormones during a menstrual cycle may possibly have an impact on lipids, lipoprotein levels and therefore on cardiovascular health status of females. AIM: To ascertain the effect of menstrual cycle phases on the levels of cardiovascular risk predictors i.e., plasma lipids and lipoproteins in healthy menstruating women. MATERIALS AND METHODS: In the present study, 111 healthy regularly menstruating females aged 15-45 years were examined at two points of a menstrual cycle i.e., in the follicular phase (10th day) and in the luteal phase (22nd day) to find out the plasma levels of lipids and lipoproteins. The data were statistically analysed using paired t-test. RESULTS: A significant decrease in the mean levels of Total Cholesterol (TC) (p=0.006), Low Density Lipoprotein Cholesterol (LDL-C) (p=0.004), TC/High Density Lipoprotein Cholesterol (HDL-C) (p=0.006), Low Density Lipoprotein Cholesterol (LDL)/HDL (p=0.01), and Triglyceride (TG)/HDL (p=0.02) was observed in the luteal phase compared to the follicular phase while TG (p=0.18), HDL-C (p=0.16). VLDL-C (p=0.17) did not show a significant difference although lower levels were observed in the luteal phase of TG and VLDL-C and higher levels for HDL-C. CONCLUSION: The present findings reveal high levels of oestrogen in the luteal phase compared to follicular phase of menstrual cycle. Clinicians should take menstrual cycle phase in account when inferring a women biomarker measurement in the analysis of cardiovascular diseases, particularly females possessing marginal levels. Moreover research studies should take the menstrual cycle phase in consideration which may be a confounding factor in framing and concluding studies on reproductive age group women.

Nutritional attributes of agaricus Bisporus and Pleurotus sajor caju mushrooms.

Agaricus bisporus and Pleurotus sajor caju mushrooms were procured from the Department of Plant Pathology, CCS Haryana Agricultural University, Hisar (INDIA) and analysed for various nutritional attributes. The fat and ash content were significantly higher in Agaricus bisporus, whereas, crude fibre and crude protein contents were significantly higher in Pleurotus sajor caju. Total and protein nitrogen was significantly higher in Pleurotus sajor caju than Agaricus bisporus mushroom as a result its true protein content was also significantly higher. No significant differences were found in the energy, carbohydrates and non-protein nitrogen contents of both the varieties of mushroom. Both varieties contained low phytic acid and oxalate however, it was significantly higher in Pleurotus sajor caju mushroom. The in vitro protein digestibility of both was not differing significantly.

Nutritional status of obese hypercholesterolemic adults.

The study was conducted to collect information regarding food and nutrient intake of hypercholesterolemic adults (male n = 30, female n = 20). The Body Mass Index (BMI) of the respondents indicated that 70% of them had a BMI more than 26. The food intake of the respondents was recorded for three consecutive days using 24 hour dietary recall and questionnaire method. The intake of cereals, sugar and oil was significantly higher than the Recommended Dietary Intake (RDI) prescribed for Indians, whereas intake of pulses was at par with RDI. The consumption of green leafy and other vegetable and roots and tubers was significantly lower among both male and female respondents. The intake of protein was at par with recommended dietary allowance (RDA), but fat and energy intake were significantly higher in both male and female respondents. The subjects were given diet counselling and this received a good response from the respondents.

Nutritional and sensory evaluation of nutritious porridge prepared using combinations of soy and sorghum grits.

A study was conducted to determine the acceptability of porridge developed from various proportions of soy and sorghum. Partially defatted soydhal was soaked in four salt solutions namely, sodium carbonate, sodium bicarbonate, sodium tripolyphosphate and sodium chloride. Soaked dhal was dried and made into grits. Soy and sorghum grits were mixed in three proportions i.e. 10:90, 20:80 and 30:70 to develop nutritious porridge. Porridge prepared from wheat grits served as a control. The sensory evaluation of the developed porridge was done using a 9-point hedonic scale. Porridge prepared using sodium carbonate soaked soydhal was found to fall in the 'like slightly' category whereas other three types of porridges as well as control porridge were 'liked moderately' by the panellists. Nutritional evaluation of the developed porridge demonstrated that protein, fat, crude fibre and ash contents increased significantly (P < 0.05) as the level of soy was increased from 10 to 20 and 30 per cent.

The influence of teent (Capparis decidua) on human plasma triglycerides, total lipids and phospholipids.

Teent is an unripe fruit of Ker (Capparis decidua) a xerophytic bush. It contains 15.1% protein and 42.88% fibre. Being a rich source of fibre, the process for preparation of teent powder and other recipes were standardized for feeding hyperlipidemic subjects. The diet of 15 hyperlipidemic adults (40-60 yrs.) was supplemented with teent for three months and plasma triglycerides, total lipids and phospholipids were analysed before and at the end of the experiment. Significant reductions in plasma triglycerides, total lipids and phospholipids concentration were noticed.

Effect of pranayama on rate pressure product in mild hypertensives.

CONTEXT: The modern living life-style is known to produce various physical and psychological stresses resulting in increased blood pressure (BP) and heart rate (HR). This can lead to increased myocardial oxygen demand (MVO2). MVO2 correlated best with rate pressure product (RPP). RPP is a product of HR and systolic BP. OBJECTIVE: The present study was conducted to evaluate the effect of relaxation in the form of pranayama on RPP in mild hypertensives. MATERIALS AND METHODS: Mild hypertensive patients were divided into two groups. Group A received antihypertensive drugs for 6 weeks and Group B received antihypertensive drugs along with pranayama training for 6 weeks. RESULTS: BP decreased significantly in Group B (148 ± 8.09-127 ± 12.10 mm of Hg) where pranayama was added. The decrease was significant as compared to Group A. HR decreased significantly in both the groups as compared to baseline, however the decrease was similar in both groups. RPP decreased significantly in both groups as compared to baseline, however the decrease was significantly more (P < 0.01) when pranayama was added to antihypertensive drugs (96.73 ± 20.53) when compared to antihypertensive drugs alone (114.66 ± 26.30). CONCLUSION: The pranayama produces relaxed state and in this state parasympathetic activity overrides sympathetic activity. Hence, addition of pranayama can be a useful addition to antihypertensive drugs for better control of hypertension in mild hypertensives.