Tetracycline-inducible gene expression in mycobacteria within an animal host using modified Streptomyces tcp830 regulatory elements

Inducible expression systems are powerful tools for studying gene function. Though several inducible expression systems are now available for mycobacteria, none have been used to modulate bacterial gene expression during an animal infection. A tetracycline-inducible expression system from Streptomyces coelicolor was successfully adapted for use in mycobacteria. To prevent baseline expression without induction, S. coelicolor tetR gene was overexpressed using the acetamidase promoter and regulatory gene block. Target gene expression was controlled by the S. coelicolor tcp830 promoter and operator allele. The −10 promoter consensus sequence of the tcp830 promoter was modified to better resemble known strong mycobacterial promoters. Using this system, induction of tetR fully repressed tcp830-dependent expression of green fluorescent protein (GFP) to baseline levels. Addition of anhydrotetracycline led to a 62-fold induction of GFP expression in vitro and 15-fold induction in a mouse mycobacterial peritonitis model in the presence of maximal tetR expression. Chemically regulatable gene expression during animal infection may be a useful tool in studying mycobacterial pathogenesis.     

The water characteristics of the Nile in the Sudan with a note on the effect of Eichhornia crassipes on the hydrobiology of the Nile

Summary The study of both the physical and chemical water characteristics of the Nile and its tributaries in the Sudan (south of Khartoum) was carried out. The main changes along the White Nile are due to tributary contribution especially that of the riverain swamps and River Sobat. The Gazal has no effect due to its negligible contribution to the White Nile. The influence of the Zeraf, although showing marked characteristics, is restricted to 50–80 km north its mouth. This is due to its low discharge and it is being diluted with the Sobat water.The study of Bahr el Gazal showed a significant longitudinal succession of this tributary where the water characteristics of its upper reaches differ considerably from those at Lake No. At the Gazal mouth a decrease in the dissolved phosphates, silicon, iron and chlorides takes place.The water of Bahr el Jebel, as it passes through the Zeraf, becomes enriched with nutrient salts which account for the dense plankton population of this tributary. As the water passes through the swamps at the sudd region depletion of its oxygen and sulphate content takes place. The characteristics of the water at the swamps affect the animal life and may be important factors in the distribution of animals living in these waters.The influence of Jebel Aulia dam on the water characteristics was studied and results show that significant modifications take place during the seasonal storage of water.The effect of the water hyacinth (Eichhornia crassipes Sol ms.) on the hydrobiology of the Nile was pointed out. The marked decrease of the nitrate nitrogen at Jebel Aulia basin may be partly attributed to its uptake by this plant; especially the reservoir provides ideal conditions for the spread of Eichhornia. The stunted growth of the water hyacinth was recorded at the upper reaches of Bahr el Gazal. This could not be explained due to the low salt content or low pH of the Gazal as Eichhornia is widely spread and shows maximum growth at the Sobat which has more or less the same salt content, pH, oxygen content and alkalinity.

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Résumé Une étude des caractéristiques physiques et chimiques de l'eau du Nil et ses tributaires au Sudan (Sud de Khartoum) a été faite. Les changements principaux au long du Nil Blanc sont dues à la contribution des tributaires spécialement celle des marais et du fleuve Sobat. Le Gazal n'a pas d'effet étant donné sa contribution négligeable au Nil Blanc. Le Zaraf, malgre qu'il montre des caractéristiques marqués, a un effet limité à 50–80 kms au nord de son embouchure. Ceci est du à son faible écoulement et à son étendue avec l'eau du Sobat.L'étude de l'eau du Bahr el Gazal a montré une succession longidutinale de ses tributaires où les caractéristiques de l'eau de ses hautes ressources diffèrent de celle du lac No. A l'embouchure du Gazal se produit une réduction des phosphates diluées, silice, fer et chlorides. L'eau du Bahr el Jebel étant donné qu'elle passe dans le Zeraf enrich de sels nutritif qui alimentent le plankton dans ce tributaire. Quand l'eau passe dans les marais du Sud une dépletion de son contenu en oxygen et sulphates se présente. Les caractéristiques de l'eau des marais affectent la vie de l'animal et peuvent être d'importants facteurs dans la distribution des animaux qui vivent dans ces eaux.L'influence du Barrage de Jebel Aulia sur les caractéristiques a été étudiée et les résultats montrent des modifications signifiantes qui ont lieu durant l'emmagasinage de la saison.L'effet de l'Eichhornia crassipes Solms sur l'hydrobiologie du Nil a été remarqué. La diminution marquée du nitrogen au bassin de Jebel Aulia peut être attribuée à son absorbtion par cette plante surtout que le resérvoir fournit des conditions idéales pour l'étendue d'Eichhornia.La lente croissance de cette dernière plante a été remarquée aux hautes embouchures de Bahr el Gazal. Ceci ne peut pas être expliqué étant donnée la faible quantité de sel et au bas pH de l'eau de Gazal. Au Sobat où l'eau contient plus ou moins le même sel, pH, oxygen et alcalinité, l'Eichhorina est largement développé et s'accroit rapidement.

     

Prostaglandin I2 is less relaxant than prostaglandin E2 on the lamb ductus arteriosus

Prostaglandin(PG) I₂ and its stable metabolite, 6-keto-PGF_1α, were tested on the isolated ductus arteriosus from mature fetal lambs. PGI₂ relaxed the ductus in high doses (threshold 10⁻⁶M) and its activity disappeared on standing at room temperature for 30 minutes. 6-keto-PGF_1α was inactive at all doses. By contrast, PGE₂ produced a dose-dependent relaxation over a range between 10⁻¹⁰ and 10⁻⁶ M. These findings confirm that PGE₂ is the most potent ductal relaxant among the known derivatives of arachidonic acid. PGE₂ probably maintains ductus patency in the fetus and, together with PGE₁, remains the compound of choice in the management of newborns requiring a viable ductus for survival.     

Method to integrate multiple plasmids into the mycobacterial chromosome

In order to create a system in which two independent plasmids can be integrated into a mycobacterial chromosome, a mycobacterial plasmid was constructed containing the phage attachment site attP from the mycobacteriophage L5 genome and additionally containing the bacterial attachment site, attB. This plasmid will integrate into the mycobacterial chromosome via recombination of the plasmid-borne attP site with the chromosomal attB site in the presence of a mycobacterial vector carrying the L5 integrase (int) gene. The integrated plasmid has a plasmid-borne attB site that is preserved and will accept the integration of additional mycobacterial plasmids containing the L5 attP site. This system should be useful in the construction of novel mycobacterial strains. In particular, this system provides a method by which several recombinant antigens or reporter constructs can be sequentially inserted into a mycobacterial strain and subsequently tested.     

Targeting the cell wall of Mycobacterium tuberculosis: a molecular modeling investigation of the interaction of imipenem and meropenem with L,D-transpeptidase 2

The single crystal X-ray structure of the extracellular portion of the L,D-transpeptidase (ex-Ldt_Mt2 – residues 120–408) enzyme was recently reported. It was observed that imipenem and meropenem inhibit activity of this enzyme, responsible for generating L,D-transpeptide linkages in the peptidoglycan layer of Mycobacterium tuberculosis. Imipenem is more active and isothermal titration calorimetry experiments revealed that meropenem is subjected to an entropy penalty upon binding to the enzyme. Herein, we report a molecular modeling approach to obtain a molecular view of the inhibitor/enzyme interactions. The average binding free energies for nine commercially available inhibitors were calculated using MM/GBSA and Solvation Interaction Energy (SIE) approaches and the calculated energies corresponded well with the available experimentally observed results. The method reproduces the same order of binding energies as experimentally observed for imipenem and meropenem. We have also demonstrated that SIE is a reasonably accurate and cost-effective free energy method, which can be used to predict carbapenem affinities for this enzyme. A theoretical explanation was offered for the experimental entropy penalty observed for meropenem, creating optimism that this computational model can serve as a potential computational model for other researchers in the field.     

High-level expression of a proteolytically sensitive diphtheria toxin fragment in Escherichia coli.

ABM508 is a recombinant fusion protein consisting of the N-terminal 485 amino acids of diphtheria toxin joined to alpha-melanocyte-stimulating hormone. When expressed in Escherichia coli under the control of the tox promoter and signal sequence, ABM508 is severely degraded. When overexpressed from a thermoinducible lambda pR promoter fusion, ABM508 is largely insoluble. We compared the expression of ABM508 (501 amino acids) to a full-length mutant form of the toxin (CRM197; 535 amino acids) and found that CRM197 showed minimal proteolysis. Thus, the removal of the C-terminal 50 amino acids of the toxin destabilizes the protein, making it a target for proteases. Proteolysis of ABM508 could be reduced by removal of the tox signal sequence (thereby directing the protein to the cytoplasm) and growth in lon and htpR mutant strains of E. coli. We also showed that the solubility of tox gene products expressed in E. coli was directly related to the growth temperature of the culture. Thus, a fragment A fusion protein (223 amino acids), ABM508, and CRM197 were found in soluble extracts when expressed at 30 degrees C but could not be released by the same procedures after growth at 42 degrees C. On the basis of these observations, we fused the coding sequences for mature ABM508 to the trc promoter (inducible at 30 degrees C by isopropyl-beta-D-thiogalactoside) and expressed this construct in a lon htpR strain of E. coli. This plasmid made 10 mg of soluble tox protein per liter of culture (7.7% of the total cell protein) or 14 times more than our previous maximal level. Extracts from lon htpR cells harboring this plasmid had high levels of ADP-ribosyltransferase activity, and although proteolysis still occurred, the major tox product corresponded to full-length ABM508.     

Disease-modifying antirheumatic drugs are associated with a reduced risk for cardiovascular disease in patients with rheumatoid arthritis: a case control study

Rheumatoid arthritis (RA) is characterized by inflammation and an increased risk for cardiovascular disease (CVD). This study investigates possible associations between CVD and the use of conventional disease-modifying antirheumatic drugs (DMARDs) in RA. Using a case control design, 613 RA patients (5,649 patient-years) were studied, 72 with CVD and 541 without CVD. Data on RA, CVD and drug treatment were evaluated from time of RA diagnosis up to the first cardiovascular event or the end of the follow-up period. The dataset was categorized according to DMARD use: sulfasalazine (SSZ), hydroxychloroquine (HCQ) or methotrexate (MTX). Odds ratios (ORs) for CVD, corrected for age, gender, smoking and RA duration, were calculated per DMARD group. Patients who never used SSZ, HCQ or MTX were used as a reference group. MTX treatment was associated with a significant CVD risk reduction, with ORs (95% CI): 'MTX only', 0.16 (0.04 to 0.66); 'MTX and SSZ ever', 0.20 (0.08 to 0.51); and 'MTX, SSZ and HCQ ever', 0.20 (0.08 to 0.54). The risk reductions remained significant after additional correction for the presence of rheumatoid factor and erosions. After correction for hypertension, diabetes and hypercholesterolemia, 'MTX or SSZ ever' and 'MTX, SSZ and HCQ ever' showed significant CVD risk reduction. Rheumatoid factor positivity and erosions both increased CVD risk, with ORs of 2.04 (1.02 to 4.07) and 2.36 (0.92 to 6.08), respectively. MTX and, to a lesser extent, SSZ were associated with significantly lower CVD risk compared to RA patients who never used SSZ, HCQ or MTX. We hypothesize that DMARD use, in particular MTX use, results in powerful suppression of inflammation, thereby reducing the development of atherosclerosis and subsequently clinically overt CVD.     

Mycobacterium tuberculosis Directs T Helper 2 Cell Differentiation by Inducing Interleukin-1�� Production in Dendritic Cells

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), resides and replicates within phagocytes and persists in susceptible hosts by modulating protective innate immune responses. Furthermore, M. tuberculosis promotes T helper 2 (Th2) immune responses by altering the balance of T cell polarizing cytokines in infected cells. However, cytokines that regulate Th2 cell differentiation during TB infection remain unknown. Here we show that IL-1β, produced by phagocytes infected by virulent M. tuberculosis strain H37Rv, directs Th2 cell differentiation. In sharp contrast, the vaccine strain bacille Calmette-Guérin as well as RD-1 and ESAT-6 mutants of H37Rv failed to induce IL-1β and promote Th2 cell differentiation. Furthermore, ESAT-6 induced IL-1β production in dendritic cells (DCs), and CD4⁺ T cells co-cultured with infected DCs differentiated into Th2 cells. Taken together, our findings indicate that IL-1β induced by RD-1/ESAT-6 plays an important role in the differentiation of Th2 cells, which in turn facilitates progression of TB by inhibiting host protective Th1 responses.     

Use of the Non-Pneumatic Anti-Shock Garment (NASG) for Life-Threatening Obstetric Hemorrhage: A Cost-Effectiveness Analysis in Egypt and Nigeria

Objective

To assess the cost-effectiveness of a non-pneumatic anti-shock garment (NASG) for obstetric hemorrhage in tertiary hospitals in Egypt and Nigeria.

Methods

We combined published data from pre-intervention/NASG-intervention clinical trials with costs from study sites. For each country, we used observed proportions of initial shock level (mild: mean arterial pressure [MAP] >60 mmHg; severe: MAP ≤60 mmHg) to define a standard population of 1,000 women presenting in shock. We examined three intervention scenarios: no women in shock receive the NASG, only women in severe shock receive the NASG, and all women in shock receive the NASG. Clinical data included frequencies of adverse health outcomes (mortality, severe morbidity, severe anemia), and interventions to manage bleeding (uterotonics, blood transfusions, hysterectomies). Costs (in 2010 international dollars) included the NASG, training, and clinical interventions. We compared costs and disability-adjusted life years (DALYs) across the intervention scenarios.

Results

For 1000 women presenting in shock, providing the NASG to those in severe shock results in decreased mortality and morbidity, which averts 357 DALYs in Egypt and 2,063 DALYs in Nigeria. Differences in use of interventions result in net savings of $9,489 in Egypt (primarily due to reduced transfusions) and net costs of $6,460 in Nigeria, with a cost per DALY averted of $3.13. Results of providing the NASG for women in mild shock has smaller and uncertain effects due to few clinical events in this data set.

Conclusion

Using the NASG for women in severe shock resulted in markedly improved health outcomes (2–2.9 DALYs averted per woman, primarily due to reduced mortality), with net savings or extremely low cost per DALY averted. This suggests that in resource-limited settings, the NASG is a very cost-effective intervention for women in severe hypovolemic shock. The effects of the NASG for mild shock are less certain.