Acetazolamide-based fungal chitinase inhibitors

Chitin is an essential structural component of the fungal cell wall. Chitinases are thought to be important for fungal cell wall remodelling, and inhibition of these enzymes has been proposed as a potential strategy for development of novel anti-fungals. The fungal pathogen Aspergillus fumigatus possesses two distinct multi-gene chitinase families. Here we explore acetazolamide as a chemical scaffold for the inhibition of an A. fumigatus ‘plant-type’ chitinase. A co-crystal structure of AfChiA1 with acetazolamide was used to guide synthesis and screening of acetazolamide analogues that yielded SAR in agreement with these structural data. Although acetazolamide and its analogues are weak inhibitors of the enzyme, they have a high ligand efficiency and as such are interesting leads for future inhibitor development

Genomic and proteomic analysis of the Alkali-Tolerance Response (AlTR) in Listeria monocytogenes 10403S

<p>Abstract</p> <p>Background</p> <p>Information regarding the Alkali-Tolerance Response (AlTR) in <it>Listeria monocytogenes </it>is very limited. Treatment of alkali-adapted cells with the protein synthesis inhibitor chloramphenicol has revealed that the AlTR is at least partially protein-dependent. In order to gain a more comprehensive perspective on the physiology and regulation of the AlTR, we compared differential gene expression and protein content of cells adapted at pH 9.5 and un-adapted cells (pH 7.0) using complementary DNA (cDNA) microarray and two-dimensional (2D) gel electrophoresis, (combined with mass spectrometry) respectively.</p> <p>Results</p> <p>In this study, <it>L. monocytogenes </it>was shown to exhibit a significant AlTR following a 1-h exposure to mild alkali (pH 9.5), which is capable of protecting cells from subsequent lethal alkali stress (pH 12.0). Adaptive intracellular gene expression involved genes that are associated with virulence, the general stress response, cell division, and changes in cell wall structure and included many genes with unknown functions. The observed variability between results of cDNA arrays and 2D gel electrophoresis may be accounted for by posttranslational modifications. Interestingly, several alkali induced genes/proteins can provide a cross protective overlap to other types of stresses.</p> <p>Conclusion</p> <p>Alkali pH provides therefore <it>L. monocytogenes </it>with nonspecific multiple-stress resistance that may be vital for survival in the human gastrointestinal tract as well as within food processing systems where alkali conditions prevail. This study showed strong evidence that the AlTR in <it>L. monocytogenes </it>functions as to minimize excess alkalisation and energy expenditures while mobilizing available carbon sources.</p

20-HETE Mediates Ozone-Induced, Neutrophil-Independent Airway Hyper-Responsiveness in Mice

Background Ozone, a pollutant known to induce airway hyper-responsiveness (AHR), increases morbidity and mortality in patients with obstructive airway diseases and asthma. We postulate oxidized lipids mediate in vivo ozone-induced AHR in murine airways.Methodology/Principal Findings Male BALB/c mice were exposed to ozone (3 or 6 ppm) or filtered air (controls) for 2 h. Precision cut lung slices (PCLS; 250 µm thickness) containing an intrapulmonary airway (∼0.01 mm$^2$ lumen area) were prepared immediately after exposure or 16 h later. After 24 h, airways were contracted to carbachol (CCh). Log EC$_{50}$ and E$_{\text{max}}$ values were then calculated by measuring the airway lumen area with respect to baseline. In parallel studies, dexamethasone (2.5 mg/kg), or 1-aminobenzotriazol (ABT) (50 mg/kg) were given intraperitoneal injection to naïve mice 18 h prior to ozone exposure. Indomethacin (10 mg/kg) was administered 2 h prior. Cell counts, cytokine levels and liquid chromatography-mass spectrometry (LC-MS) for lipid analysis were assessed in bronchoalveolar lavage (BAL) fluid from ozone exposed and control mice. Ozone acutely induced AHR to CCh. Dexamethasone or indomethacin had little effect on the ozone-induced AHR; while, ABT, a cytochrome P450 inhibitor, markedly attenuated airway sensitivity. BAL fluid from ozone exposed animals, which did not contain an increase in neutrophils or interleukin (IL)-6 levels, increased airway sensitivity following in vitro incubation with a naïve PCLS. In parallel, significant increases in oxidized lipids were also identified using LC-MS with increases of 20-HETE that were decreased following ABT treatment.Conclusions/Significance These data show that ozone acutely induces AHR to CCh independent of inflammation and is insensitive to steroid treatment or cyclooxygenase (COX) inhibition. BAL fluid from ozone exposed mice mimicked the effects of in vivo ozone exposure that were associated with marked increases in oxidized lipids. 20-HETE plays a pivotal role in mediating acute ozone-induced AHR

Risks of adverse perinatal and maternal outcomes among women with hypertensive disorders of pregnancy in southwestern Uganda.

INTRODUCTION: Hypertensive disorders of pregnancy (HDP) are a leading cause of global perinatal (fetal and neonatal) and maternal morbidity and mortality. We sought to describe HDP and determine the magnitude and risk factors for adverse perinatal and maternal outcomes among women with HDP in southwestern Uganda. METHODS: We prospectively enrolled pregnant women admitted for delivery and diagnosed with HDP at a tertiary referral hospital in southwestern Uganda from January 2019 to November 2019, excluding women with pre-existing hypertension. The participants were observed and adverse perinatal and maternal outcomes were documented. We used multivariable logistic regression models to determine independent risk factors associated with adverse perinatal and maternal outcomes. RESULTS: A total of 103 pregnant women with a new-onset HDP were enrolled. Almost all women, 93.2% (n = 96) had either pre-eclampsia with severe features or eclampsia. The majority, 58% (n = 60) of the participants had an adverse perinatal outcome (36.9% admitted to the neonatal intensive care unit (ICU), 20.3% stillbirths, and 1.1% neonatal deaths). Fewer participants, 19.4% (n = 20) had an adverse maternal outcome HELLP syndrome (7.8%), ICU admission (3%), and postpartum hemorrhage (3%). In adjusted analyses, gestational age of < 34 weeks at delivery and birth weight <2.5kg were independent risk factors for adverse perinatal outcomes while referral from another health facility and eclampsia were independent risk factors for adverse maternal outcomes. CONCLUSION: Among women with HDP at our institution, majority had preeclampsia with severe symptoms or eclampsia and an unacceptably high rate of adverse perinatal and maternal outcomes; over a fifth of the mothers experiencing stillbirth. This calls for improved antenatal surveillance of women with HDP and in particular improved neonatal and maternal critical care expertise at delivering facilities. Earlier detection and referral, as well as improvement in initial management at lower level health units and on arrival at the referral site is imperative

(13)C MRS and LC-MS Flux Analysis of Tumor Intermediary Metabolism.

We present the first validated metabolic network model for analysis of flux through key pathways of tumor intermediary metabolism, including glycolysis, the oxidative and non-oxidative arms of the pentose pyrophosphate shunt, the TCA cycle as well as its anaplerotic pathways, pyruvate-malate shuttling, glutaminolysis, and fatty acid biosynthesis and oxidation. The model that is called Bonded Cumomer Analysis for application to (13)C magnetic resonance spectroscopy ((13)C MRS) data and Fragmented Cumomer Analysis for mass spectrometric data is a refined and efficient form of isotopomer analysis that can readily be expanded to incorporate glycogen, phospholipid, and other pathways thereby encompassing all the key pathways of tumor intermediary metabolism. Validation was achieved by demonstrating agreement of experimental measurements of the metabolic rates of oxygen consumption, glucose consumption, lactate production, and glutamate pool size with independent measurements of these parameters in cultured human DB-1 melanoma cells. These cumomer models have been applied to studies of DB-1 melanoma and DLCL2 human diffuse large B-cell lymphoma cells in culture and as xenografts in nude mice at 9.4 T. The latter studies demonstrate the potential translation of these methods to in situ studies of human tumor metabolism by MRS with stable (13)C isotopically labeled substrates on instruments operating at high magnetic fields (≥7 T). The melanoma studies indicate that this tumor line obtains 51% of its ATP by mitochondrial metabolism and 49% by glycolytic metabolism under both euglycemic (5 mM glucose) and hyperglycemic conditions (26 mM glucose). While a high level of glutamine uptake is detected corresponding to ~50% of TCA cycle flux under hyperglycemic conditions, and ~100% of TCA cycle flux under euglycemic conditions, glutaminolysis flux and its contributions to ATP synthesis were very small. Studies of human lymphoma cells demonstrated that inhibition of mammalian target of rapamycin (mTOR) signaling produced changes in flux through the glycolytic, pentose shunt, and TCA cycle pathways that were evident within 8 h of treatment and increased at 24 and 48 h. Lactate was demonstrated to be a suitable biomarker of mTOR inhibition that could readily be monitored by (1)H MRS and perhaps also by FDG-PET and hyperpolarized (13)C MRS methods