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There is a compelling need to find drugs active against Mycobacterium tuberculosis ( Mtb ). 4'-Phosphopantetheinyl transferase (PptT) is an essential enzyme in Mtb that has attracted interest as a potential drug target. We optimized a PptT assay, used it to screen 422,740 compounds, and identified raltitrexed, an antineoplastic antimetabolite, as the most potent PptT inhibitor yet reported. While trying unsuccessfully to improve raltitrexed's ability to kill Mtb and remove its ability to kill human cells, we learned three lessons that may help others developing antibiotics. First, binding of raltitrexed substantially changed the configuration of the PptT active site, complicating molecular modeling of analogs based on the unliganded crystal structure or the structure of cocrystals with inhibitors of another class. Second, minor changes in the raltitrexed molecule changed its target in Mtb from PptT to dihydrofolate reductase (DHFR). Third, the structure-activity relationship for over 800 raltitrexed analogs only became interpretable when we quantified and characterized the compounds' intrabacterial accumulation and transformation.

New drugs and new targets are urgently needed to treat tuberculosis. We discovered that d-phenylalanine-benzoxazole Q112 displays potent antibacterial activity against Mycobacterium tuberculosis ( Mtb ) in multiple media and in macrophage infections. A metabolomic profiling indicates that Q112 has a unique mechanism of action. Q112 perturbs the essential pantothenate/coenzyme A biosynthetic pathway, depleting pantoate while increasing ketopantoate, as would be expected if ketopantoate reductase (KPR) were inhibited. We searched for alternative KPRs, since the enzyme annotated as PanE KPR is not essential in Mtb . The ketol-acid reductoisomerase IlvC catalyzes the KPR reaction in the close Mtb relative Corynebacterium glutamicum , but Mtb IlvC does not display KPR activity. We identified the essential protein Rv3603c as an orthologue of PanG KPR and demonstrated that a purified recombinant Rv3603c has KPR activity. Q112 inhibits Rv3603c, explaining the metabolomic changes. Surprisingly, pantothenate does not rescue Q112 -treated bacteria, indicating that Q112 has an additional target(s). Q112 -resistant strains contain loss-of-function mutations in the twin arginine translocase TatABC, further underscoring Q112 's unique mechanism of action. Loss of TatABC causes a severe fitness deficit attributed to changes in nutrient uptake, suggesting that Q112 resistance may derive from a decrease in uptake.

Endotoxin removal using detergent washes and extractions are well-established, efficient, and cost-effective methods; however, removing residual detergent post treatment has been shown to be a challenge. In this communication, we show a simple and fast method for determining the detergent concentration in a protein solution post treatment and highlight strategies for detergent removal to achieve levels below the critical micelle concentration (CMC), the minimum concentration at which detergent micelles form., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Objective: Almost one-third of pregnant women develop symptoms of restless legs syndrome (RLS), which could have a negative impact on quality of life and the course of pregnancy and/or labor. The aim of our study was to determine possible risk factors for developing RLS in pregnant women., Methods: A total of 300 random women in the third trimester of gravidity filled out a simple questionnaire based on the official diagnostic criteria for RLS. Respondents positive for RLS were interviewed to further characterize their symptoms. The parameters of iron metabolism were examined based on blood samples. All data were statistically analyzed., Results: We detected significantly lower hemoglobin levels with signs of hypochromic anemia typical of iron deficiency in RLS-positive respondents, although oral iron supplementation was significantly higher in this group. The overall severity of symptoms correlated inversely with hemoglobin level., Conclusion: According to our results, it appears that serum iron level disturbances play a crucial role in the pathophysiology of secondary RLS in pregnant women., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Restless legs syndrome (RLS) is a frequent neurological disorder which predominantly affects women. Pregnancy is one of the most common conditions leading to secondary RLS. Severe symptoms of RLS may lead to complications of pregnancy and/or labor. The aim of this study was to determine the prevalence and characteristics of RLS in pregnant women., Methods: Women in the third trimester of gravidity filled out a simple questionnaire based on the official diagnostic criteria for RLS. Positive responders were interviewed in order to further characterize their symptoms. Afterwards information on changes in frequency and/or intensity of the symptoms after delivery was obtained by a telephone follow-up. All data were statistically analysed., Results: A total of 300 questionnaires were completed. All 94 RLS-positives met the four diagnostic criteria (31.33%). There was no difference in age, body mass index, or the number of previous pregnancies between RLS-positives and RLS-negatives, but weight gain during pregnancy was significantly higher in RLS-positives. More than 30% of positives had clinically significant symptoms, and 50% reported sleep disturbances. Almost 75% of the cases of RLS were secondary, i.e., symptoms occurred only during pregnancy (with a peak in the third trimester). More complications of pregnancy or labor occurred in women with RLS, but this was only marginally significant., Conclusions: Our study confirmed the relatively high prevalence of RLS in pregnant women compared with the general population. Although almost three-fourths of the symptoms were only transient throughout pregnancy, the impact of the severe symptoms and sleep deprivation on the course of pregnancy and delivery was not negligible. Early detection and adequate treatment of severe RLS are necessary to prevent maternal discomfort and possible health risks. The questionnaire method is a simple, reliable diagnostic tool.