In Silico-Based High-Throughput Screen for Discovery of Novel Combinations for Tuberculosis Treatment

There are currently 18 drug classes for the treatment of tuberculosis, including those in the development pipeline. An in silico simulation enabled combing the innumerably large search space to derive multidrug combinations. Through the use of ordinary differential equations (ODE), we constructed an in silico kinetic platform in which the major metabolic pathways in Mycobacterium tuberculosis and the mechanisms of the antituberculosis drugs were integrated into a virtual proteome. The optimized model was used to evaluate 816 triplets from the set of 18 drugs. The experimentally derived cumulative fractional inhibitory concentration (∑FIC) value was within twofold of the model prediction. Bacterial enumeration revealed that a significant number of combinations that were synergistic for growth inhibition were also synergistic for bactericidal effect. The in silico-based screen provided new starting points for testing in a mouse model of tuberculosis, in which two novel triplets and five novel quartets were significantly superior to the reference drug triplet of isoniazid, rifampin, and ethambutol (HRE) or the quartet of HRE plus pyrazinamide (HREZ).     

Diagnostic probes for Bacillus anthracis spores selected from a landscape phage library

BACKGROUND: Recent use of Bacillus anthracis spores as a bioweapon has highlighted the need for a continuous monitoring system. Current monitoring systems rely on antibody-derived probes, which are not hardy enough to withstand long-term use under extreme conditions. We describe new, phage-derived probes that can be used as robust substitutes for antibodies. METHODS: From a landscape phage library with random octapeptides displayed on all copies of the major phage coat protein of the phage fd-tet, we selected clones that bound to spores of B. anthracis (Sterne strain). ELISA, micropanning, and coprecipitation assays were used to evaluate the specificity and selectivity with which these phage bound to B. anthracis spores. RESULTS: Peptides on the selected clones directed binding of the phage to B. anthracis spores. Most clones exhibited little or no binding to spores of distantly related Bacillus species, but some binding was observed with spores of closely related species. Our most specific spore-binding phage displayed a peptide EPRLSPHS (several thousand peptides per phage) and bound 3.5- to 70-fold better to spores of B. anthracis Sterne than to spores of other Bacillus species. CONCLUSIONS: The selected phage probes bound preferentially to B. anthracis Sterne spores compared with other Bacillus species. These phage could possibly be further developed into highly specific and robust probes suitable for long-term use in continuous monitoring devices and biosorbents.     

Inherent variations in CO-H2S-mediated carotid body O2 sensing mediate hypertension and pulmonary edema

Oxygen (O₂) sensing by the carotid body and its chemosensory reflex is critical for homeostatic regulation of breathing and blood pressure. Humans and animals exhibit substantial interindividual variation in this chemosensory reflex response, with profound effects on cardiorespiratory functions. However, the underlying mechanisms are not known. Here, we report that inherent variations in carotid body O₂ sensing by carbon monoxide (CO)-sensitive hydrogen sulfide (H₂S) signaling contribute to reflex variation in three genetically distinct rat strains. Compared with Sprague-Dawley (SD) rats, Brown-Norway (BN) rats exhibit impaired carotid body O₂ sensing and develop pulmonary edema as a consequence of poor ventilatory adaptation to hypobaric hypoxia. Spontaneous Hypertensive (SH) rat carotid bodies display inherent hypersensitivity to hypoxia and develop hypertension. BN rat carotid bodies have naturally higher CO and lower H₂S levels than SD rat, whereas SH carotid bodies have reduced CO and greater H₂S generation. Higher CO levels in BN rats were associated with higher substrate affinity of the enzyme heme oxygenase 2, whereas SH rats present lower substrate affinity and, thus, reduced CO generation. Reducing CO levels in BN rat carotid bodies increased H₂S generation, restoring O₂ sensing and preventing hypoxia-induced pulmonary edema. Increasing CO levels in SH carotid bodies reduced H₂S generation, preventing hypersensitivity to hypoxia and controlling hypertension in SH rats.Oxygen, an essential substrate for generating ATP, is vital for sustaining much of life on earth. A low availability of oxygen directs vertebrates’ complex respiratory and cardiovascular systems to maintain optimal oxygenation of tissues by increasing ventilation and blood pressure. Interestingly, ventilatory responses to hypoxia are not uniform but, instead, exhibit substantial variation among humans (1). These varied ventilatory responses to hypoxia result in dire physiological consequences: a diminished hypoxic ventilatory response can result in poor adaptation to low O₂ environments (2) and high-altitude pulmonary edema (3–5), whereas a heightened response is associated with essential hypertension (6). Similar variations in hypoxic response have also been documented in different strains of rodents (7–10). In comparison with Sprague-Dawley (SD) rats, Brown-Norway (BN) rats display a markedly reduced ventilatory response to hypoxia (8, 9), whereas Spontaneous Hypertensive (SH) rats exhibit an augmented response (11). SH rats also present enhanced sympathetic nerve activity and hypertension (7). Despite the physiological significance, the mechanisms underlying interindividual variation in systemic responses to hypoxia are not known.The carotid body is the key sensor of arterial blood oxygen, and its chemosensory reflex is a critical regulator of breathing, sympathetic tone, and blood pressure (12, 13). Differing responses in ventilation and sympathetic nerve activity to ambient oxygen levels may reflect inherent variations in the O₂ sensing ability of the carotid body. Although a number of hypotheses have been proposed to explain carotid body-mediated O₂ sensing (12, 13), emerging evidence suggests that the gasotransmitter hydrogen sulfide (H₂S) is required for O₂ sensing by the carotid body (14–17). Hypoxia results in increased H₂S generation in the carotid body. Glomus cells, the primary O₂ sensing cells in the carotid body, express cystathionine-γ-lyase (CSE), an H₂S catalyzing enzyme (15, 16). Homozygous CSE-null mice display diminished H₂S generation and a severely impaired response to hypoxia (15, 16). We hypothesized that variations in the chemosensory reflex are a result of differences in H₂S signaling in the carotid body. We tested this possibility by examining the carotid body response to hypoxia in SD, BN, and SH rats and further assessed the physiological consequences of variations in carotid body O₂ sensing on ventilatory adaptations to hypoxia and blood pressure.     

Predicting the onset of Addison's disease: ACTH, renin, cortisol and 21-hydroxylase autoantibodies

Context Autoantibodies to 21‐hydroxylase (21OH‐AA) precede onset of autoimmune Addison’s disease (AD). Progression to AD can take months to years, and early detection of metabolic decompensation may prevent morbidity and mortality. Objective To define optimal methods of predicting progression to overt AD (defined by subnormal peak cortisol response to Cosyntropin) in 21OH‐AA+ individuals. Design, Setting and Participants Individuals were screened for 21OH‐AA at the Barbara Davis Center from 1993 to 2011. Subjects positive for 21OH‐AA (n = 87) were tested, and the majority prospectively followed for the development of Addison’s disease, including seven diagnosed with AD upon 21OH‐AA discovery (discovered), seven who progressed to AD (progressors) and 73 nonprogressors. Main Outcome Measured Plasma renin activity (PRA), ACTH, baseline cortisol, peak cortisol and 21OH‐AA were measured at various time points relative to diagnosis of AD or last AD‐free follow‐up. Results Compared with nonprogressors, in the time period 2 months–2 years prior to the onset of AD, progressors were significantly more likely to have elevated ACTH (11–22 pm , P < 1E‐4), with no significant differences in mean PRA (P = 0·07) or baseline cortisol (P = 0·08), and significant but less distinct differences seen with 21OH‐AA levels (P < 1E‐4) and poststimulation cortisol levels (P = 6E‐3). Conclusion Moderately elevated ACTH is a more useful early indicator of impending AD than 21OH‐AA, PRA or peak cortisol, in the 2 months–2 years preceding the onset of AD.