Fault Detection for Vaccine Refrigeration via Convolutional Neural Networks Trained on Simulated Datasets.

In low-and middle-income countries, the cold chain that supports vaccine storage and distribution is vulnerable due to insufficient infrastructure and interoperable data. To bolster these networks, we developed a convolutional neural network-based fault detection method for vaccine refrigerators using datasets synthetically generated by thermodynamic modelling. We demonstrate that these thermodynamic models can be calibrated to real cooling systems in order to identify system-specific faults under a diverse range of operating conditions. If implemented on a large scale, this portable, flexible approach has the potential to increase the fidelity and lower the cost of vaccine distribution in remote communities.

Small Molecules Targeting Mycobacterium tuberculosis Type II NADH Dehydrogenase Exhibit Antimycobacterial Activity.

The generation of ATP through oxidative phosphorylation is an essential metabolic function for Mycobaterium tuberculosis (Mtb), regardless of the growth environment. The type II NADH dehydrogenase (Ndh-2) is the conduit for electrons into the pathway, and is absent in the mammalian genome, thus making it a potential drug target. Herein, we report the identification of two types of small molecules as selective inhibitors for Ndh-2 through a multicomponent high-throughput screen. Both compounds block ATP synthesis, lead to effects consistent with loss of NADH turnover, and importantly, exert bactericidal activity against Mtb. Extensive medicinal chemistry optimization afforded the best analogue with an MIC of 90 nm against Mtb. Moreover, the two scaffolds have differential inhibitory activities against the two homologous Ndh-2 enzymes in Mtb, which will allow precise control over Ndh-2 function in Mtb to facilitate the assessment of this anti-TB drug target.

Contemporary Data and Trends in the Economic Costs of Mental Disabilities.

This article addresses the economic effects of mental disabilities by analyzing contemporary data in the context of micro- and macroeconomic thought and relevant statistical literature. Within the parameters of these conceptual and statistical reference points, the authors seek to discern current trends in the direct, indirect, and opportunity costs posed by mental disabilities, not only to the individuals who suffer from them, but also to their families, to employers, and to society as a whole. The authors also discuss uncertainties that inhere in available data concerning both the prevalence of these conditions and the related costs of treatment, as well as the complexity of drawing correlations among variables with respect to these costs and the difficulty of identifying a meaningful measure of the economic consequences that attend mental disabilities. Copyright © 2017 John Wiley & Sons, Ltd.

Stringent Response Factors PPX1 and PPK2 Play an Important Role in Mycobacterium tuberculosis Metabolism, Biofilm Formation, and Sensitivity to Isoniazid In Vivo.

Mycobacterium tuberculosis remains a global health threat largely due to the lengthy duration of curative antibiotic treatment, contributing to medical nonadherence and the emergence of drug resistance. This prolonged therapy is likely due to the presence of M. tuberculosis persisters, which exhibit antibiotic tolerance. Inorganic polyphosphate [poly(P)] is a key regulatory molecule in the M. tuberculosis stringent response mediating antibiotic tolerance. The polyphosphate kinase PPK1 is responsible for poly(P) synthesis in M. tuberculosis, while the exopolyphosphatases PPX1 and PPX2 and the GTP synthase PPK2 are responsible for poly(P) hydrolysis. In the present study, we show by liquid chromatography-tandem mass spectrometry that poly(P)-accumulating M. tuberculosis mutant strains deficient in ppx1 or ppk2 had significantly lower intracellular levels of glycerol-3-phosphate (G3P) and 1-deoxy-xylulose-5-phosphate. Real-time PCR revealed decreased expression of genes in the G3P synthesis pathway in each mutant. The ppx1-deficient mutant also showed a significant accumulation of metabolites in the tricarboxylic acid cycle, as well as altered arginine and NADH metabolism. Each poly(P)-accumulating strain showed defective biofilm formation, while deficiency of ppk2 was associated with increased sensitivity to plumbagin and meropenem and deficiency of ppx1 led to enhanced susceptibility to clofazimine. A DNA vaccine expressing ppx1 and ppk2, together with two other members of the M. tuberculosis stringent response, M. tuberculosis rel and sigE, did not show protective activity against aerosol challenge with M. tuberculosis, but vaccine-induced immunity enhanced the killing activity of isoniazid in a murine model of chronic tuberculosis. In summary, poly(P)-regulating factors of the M. tuberculosis stringent response play an important role in M. tuberculosis metabolism, biofilm formation, and antibiotic sensitivity in vivo.

Characterization of the type 2 NADH:menaquinone oxidoreductases from Staphylococcus aureus and the bactericidal action of phenothiazines.

Methicillin-resistant Staphylococcus aureus (MRSA) is currently one of the principal multiple drug resistant bacterial pathogens causing serious infections, many of which are life-threatening. Consequently, new therapeutic targets are required to combat such infections. In the current work, we explore the type 2 Nicotinamide adenine dinucleotide reduced form (NADH) dehydrogenases (NDH-2s) as possible drug targets and look at the effects of phenothiazines, known to inhibit NDH-2 from Mycobacterium tuberculosis. NDH-2s are monotopic membrane proteins that catalyze the transfer of electrons from NADH via flavin adenine dinucleotide (FAD) to the quinone pool. They are required for maintaining the NADH/Nicotinamide adenine dinucleotide (NAD(+)) redox balance and contribute indirectly to the generation of proton motive force. NDH-2s are not present in mammals, but are the only form of respiratory NADH dehydrogenase in several pathogens, including S. aureus. In this work, the two putative ndh genes present in the S. aureus genome were identified, cloned and expressed, and the proteins were purified and characterized. Phenothiazines were shown to inhibit both of the S. aureus NDH-2s with half maximal inhibitory concentration (IC50) values as low as 8µM. However, evaluating the effects of phenothiazines on whole cells of S. aureus was complicated by the fact that they are also acting as uncouplers of oxidative phosphorylation. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.

Acinetobacter baumannii OxPhos inhibitors as selective anti-infective agents.

The Gram-negative bacterium Acinetobacter baumannii is an opportunistic pathogen in humans and infections are poorly treated by current therapy. Recent emergence of multi-drug resistant strains and the lack of new antibiotics demand an immediate action for development of new anti-Acinetobacter agents. To this end, oxidative phosphorylation (OxPhos) was identified as a novel target for drug discovery research. Consequently, a library of ∼10,000 compounds was screened using a membrane-based ATP synthesis assay. One hit identified was the 2-iminobenzimidazole 1 that inhibited the OxPhos of A. baumannii with a modestly high selectivity against mitochondrial OxPhos, and displayed an MIC of 25µM (17µg/mL) against the pathogen. The 2-iminobenzimidazole 1 was found to inhibit the type 1 NADH-quinone oxidoreductase (NDH-1) of A. baumannii OxPhos by a biochemical approach. Among various derivatives that were synthesized to date, des-hydroxy analog 5 is among the most active with a relatively tight SAR requirement for the N'-aminoalkyl side chain. Analog 5 also showed less cytotoxicity against NIH3T3 and HepG2 mammalian cell lines, demonstrating the potential for this series of compounds as anti-Acinetobacter agents. Additional SAR development and target validation is underway.

Mycobacterium tuberculosis type II NADH-menaquinone oxidoreductase catalyzes electron transfer through a two-site ping-pong mechanism and has two quinone-binding sites.

Type II NADH-quinone oxidoreductase (NDH-2) catalyzes the transfer electrons from NADH to the quinone pool and plays an essential role in the oxidative phosphorylation system of Mycobacterium tuberculosis (Mtb). The absence of NDH-2 in the mammalian mitochondrial electron transport chain makes this enzyme an attractive target for antibiotic development. To fully establish the kinetic properties of this enzyme, we studied the interaction of Mtb NDH-2 with substrates, NADH, and various quinone analogues and their products in both membrane and soluble environments. These studies, and comparative analyses of the kinetics with thio-NAD(+) and quinone electron acceptors, provided evidence that Mtb NDH-2 catalyzes the transfer electrons from NADH to quinone substrates by a nonclassical, two-site ping-pong kinetic mechanism whereby substrate quinones bind to a site that is distinct from the NADH-binding site. Furthermore, the effects of quinols on Mtb NDH-2 catalytic activity demonstrate the presence of two binding sites for quinone ligands, one favoring the reduced form and the other favoring the oxidized form.

Reduction of clofazimine by mycobacterial type 2 NADH:quinone oxidoreductase: a pathway for the generation of bactericidal levels of reactive oxygen species.

The mechanism of action of clofazimine (CFZ), an antimycobacterial drug with a long history, is not well understood. The present study describes a redox cycling pathway that involves the enzymatic reduction of CFZ by NDH-2, the primary respiratory chain NADH:quinone oxidoreductase of mycobacteria and nonenzymatic oxidation of reduced CFZ by O(2) yielding CFZ and reactive oxygen species (ROS). This pathway was demonstrated using isolated membranes and purified recombinant NDH-2. The reduction and oxidation of CFZ was measured spectrally, and the production of ROS was measured using a coupled assay system with Amplex Red. Supporting the ROS-based killing mechanism, bacteria grown in the presence of antioxidants are more resistant to CFZ. CFZ-mediated increase in NADH oxidation and ROS production were not observed in membranes from three different Gram-negative bacteria but was observed in Staphylococcus aureus and Saccharomyces cerevisiae, which is consistent with the known antimicrobial specificity of CFZ. A more soluble analog of CFZ, KS6, was synthesized and was shown to have the same activities as CFZ. These studies describe a pathway for a continuous and high rate of reactive oxygen species production in Mycobacterium smegmatis treated with CFZ and a CFZ analog as well as evidence that cell death produced by these agents are related to the production of these radical species.

Tracing and testing multiple generations of contacts to COVID-19 cases: cost-benefit trade-offs.

Traditional contact tracing tests the direct contacts of those who test positive. But, by the time an infected individual is tested, the infection starting from the person may have infected a chain of individuals. Hence, why should the testing stop at direct contacts, and not test secondary, tertiary contacts or even contacts further down? One deterrent in testing long chains of individuals right away may be that it substantially increases the testing load, or does it? We investigate the costs and benefits of such multi-hop contact tracing for different number of hops. Considering diverse contact networks, we show that the cost-benefit trade-off can be characterized in terms of a single measurable attribute, the initial epidemic growth rate. Once this growth rate crosses a threshold, multi-hop contact tracing substantially reduces the outbreak size compared with traditional tracing. Multi-hop even incurs a lower cost compared with the traditional tracing for a large range of values of the growth rate. The cost-benefit trade-offs can be classified into three phases depending on the value of the growth rate. The need for choosing a larger number of hops becomes greater as the growth rate increases or the environment becomes less conducive toward containing the disease.

Countering the potential re-emergence of a deadly infectious disease-Information warfare, identifying strategic threats, launching countermeasures.

OBJECTIVES: Eradicated infectious diseases like smallpox can re-emerge through accident or the designs of bioterrorists, and cause heavy casualties. Presently, the populace is largely susceptible as only a small percentage is vaccinated, and their immunity is likely to have waned. And when the disease re-emerges, the susceptible individuals may be manipulated by disinformation on Social Media to refuse vaccines. Thus, a combination of countermeasures consisting of antiviral drugs and vaccines and a range of policies for their application need to be investigated. Opinions regarding whether to receive vaccines evolve over time through social exchanges via networks that overlap with but are not identical to the disease propagation networks. These couple the spread of the biological and information contagion and necessitate a joint investigation of the two. METHODS: We develop a computationally tractable metapopulation epidemiological model that captures the joint spatio-temporal evolution of an infectious disease (e.g., smallpox, COVID-19) and opinion dynamics. RESULTS: Considering smallpox, the computations based on the model show that opinion dynamics have a substantial impact on the fatality count. Towards understanding how perpetrators are likely to seed the infection, we identify a) the initial distribution of infected individuals that maximize the overall fatality count; and b) which habitation structures are more vulnerable to outbreaks. We assess the relative efficacy of different countermeasures and conclude that a combination of vaccines and drugs minimize the fatalities, and by itself, drugs reduce fatalities more than the vaccine. Accordingly, we assess the impact of increase in the supply of drugs and identify the most effective among a collection of policies for administering of drugs for various parameter combinations. Many of the observed patterns are stable to variations of a diverse set of parameters. CONCLUSIONS: Our findings provide a quantitative foundation for various important elements of public health discourse that have largely been conducted qualitatively.

Inhibiting the stringent response blocks Mycobacterium tuberculosis entry into quiescence and reduces persistence.

The stringent response enables Mycobacterium tuberculosis (Mtb) to shut down its replication and metabolism under various stresses. Here we show that Mtb lacking the stringent response enzyme RelMtb was unable to slow its replication rate during nutrient starvation. Metabolomics analysis revealed that the nutrient-starved relMtb -deficient strain had increased metabolism similar to that of exponentially growing wild-type bacteria in nutrient-rich broth, consistent with an inability to enter quiescence. Deficiency of relMtb increased the susceptibility of mutant bacteria to killing by isoniazid during nutrient starvation and in the lungs of chronically infected mice. We screened a pharmaceutical library of over 2 million compounds for inhibitors of RelMtb and showed that the lead compound X9 was able to directly kill nutrient-starved M. tuberculosis and enhanced the killing activity of isoniazid. Inhibition of RelMtb is a promising approach to target M. tuberculosis persisters, with the potential to shorten the duration of TB treatment.

Characterization of nucleotide pools as a function of physiological state in Escherichia coli.

Using a modified method that involves minimal manipulation of cells, we report new information about nucleotide pool sizes and changes throughout the Escherichia coli growth curve. Nucleotide pool sizes are critically dependent on sample manipulation and extraction methods. Centrifugation and even short (2 min) lapses in sample preparation can dramatically affect results. The measured ATP concentration at three different growth rates is at least 3 mM, well above the 0.8 mM needed to saturate the rRNA promoter P1 in vitro. Many of the pools, including ATP, GTP, and UTP, begin to decrease while the cells are still in mid-log growth. After an almost universal drop in nucleotide concentration as the cells transition from logarithmic to stationary phase, there is a "rebound" of certain nucleotides, most notably ATP, after the cells enter stationary phase, followed by a progressive decrease. UTP, in contrast, increases as the cells transition into stationary phase. The higher UTP values might be related to elevated UDP-glucose/galactose, which was found to be at higher concentrations than expected in stationary phase. dTTP is the most abundant deoxynucleoside triphosphate (dNTP) in the cell despite the fact that its precursors, UDP and UTP, are not. All dNTPs decrease through the growth curve but do not have the abrupt drop, as seen with other nucleotides when the cells transition into stationary phase.

Delayed bet-hedging resilience strategies under environmental fluctuations.

Many biological populations, such as bacterial colonies, have developed through evolution a protection mechanism, called bet hedging, to increase their probability of survival under stressful environmental fluctuation. In this context, the concept of preadaptation refers to a common type of bet-hedging protection strategy in which a relatively small number of individuals in a population stochastically switch their phenotypes to a dormant metabolic state in which they increase their probability of survival against potential environmental shocks. Hence, if an environmental shock took place at some point in time, preadapted organisms would be better adapted to survive and proliferate once the shock is over. In many biological populations, the mechanisms of preadaptation and proliferation present delays whose influence in the fitness of the population are not well understood. In this paper, we propose a rigorous mathematical framework to analyze the role of delays in both preadaptation and proliferation mechanisms in the survival of biological populations, with an emphasis on bacterial colonies. Our theoretical framework allows us to analytically quantify the average growth rate of a bet-hedging bacterial colony with stochastically delayed reactions with arbitrary precision. We verify the accuracy of the proposed method by numerical simulations and conclude that the growth rate of a bet-hedging population shows a nontrivial dependency on their preadaptation and proliferation delays. Contrary to the current belief, our results show that faster reactions do not, in general, increase the overall fitness of a biological population.

Discovery of Imidazo[1,2-a]pyridine Ethers and Squaramides as Selective and Potent Inhibitors of Mycobacterial Adenosine Triphosphate (ATP) Synthesis.

The approval of bedaquiline to treat tuberculosis has validated adenosine triphosphate (ATP) synthase as an attractive target to kill Mycobacterium tuberculosis (Mtb). Herein, we report the discovery of two diverse lead series imidazo[1,2-a]pyridine ethers (IPE) and squaramides (SQA) as inhibitors of mycobacterial ATP synthesis. Through medicinal chemistry exploration, we established a robust structure-activity relationship of these two scaffolds, resulting in nanomolar potencies in an ATP synthesis inhibition assay. A biochemical deconvolution cascade suggested cytochrome c oxidase as the potential target of IPE class of molecules, whereas characterization of spontaneous resistant mutants of SQAs unambiguously identified ATP synthase as its molecular target. Absence of cross resistance against bedaquiline resistant mutants suggested a different binding site for SQAs on ATP synthase. Furthermore, SQAs were found to be noncytotoxic and demonstrated efficacy in a mouse model of tuberculosis infection.

Deficiency of the novel exopolyphosphatase Rv1026/PPX2 leads to metabolic downshift and altered cell wall permeability in Mycobacterium tuberculosis.

UNLABELLED: Mycobacterium tuberculosis can persist for decades in the human host. Stringent response pathways involving inorganic polyphosphate [poly(P)], which is synthesized and hydrolyzed by polyphosphate kinase (PPK) and exopolyphosphatase (PPX), respectively, are believed to play a key regulatory role in bacterial persistence. We show here that M. tuberculosis poly(P) accumulation is temporally linked to bacillary growth restriction. We also identify M. tuberculosis Rv1026 as a novel exopolyphosphatase with hydrolytic activity against long-chain poly(P). Using a tetracycline-inducible expression system to knock down expression of Rv1026 (ppx2), we found that M. tuberculosis poly(P) accumulation leads to slowed growth and reduced susceptibility to isoniazid, increased resistance to heat and acid pH, and enhanced intracellular survival during macrophage infection. By transmission electron microscopy, the ppx2 knockdown strain exhibited increased cell wall thickness, which was associated with reduced cell wall permeability to hydrophilic drugs rather than induction of drug efflux pumps or altered biofilm formation relative to the empty vector control. Transcriptomic and metabolomic analysis revealed a metabolic downshift of the ppx2 knockdown characterized by reduced transcription and translation and a downshift of glycerol-3-phosphate levels. In summary, poly(P) plays an important role in M. tuberculosis growth restriction and metabolic downshift and contributes to antibiotic tolerance through altered cell wall permeability. IMPORTANCE: The stringent response, involving the regulatory molecules inorganic polyphosphate [poly(P)] and (p)ppGpp, is believed to mediate Mycobacterium tuberculosis persistence. In this study, we identified a novel enzyme (Rv1026, PPX2) responsible for hydrolyzing long-chain poly(P). A genetically engineered M. tuberculosis strain deficient in the ppx2 gene showed increased poly(P) levels, which were associated with early bacterial growth arrest and reduced susceptibility to the first-line drug isoniazid, as well as increased bacterial survival during exposure to stress conditions and within macrophages. Relative to the control strain, the mutant showed increased thickness of the cell wall and reduced drug permeability. Global gene expression and metabolite analysis revealed reduced expression of the transcriptional and translational machinery and a shift in carbon source utilization. In summary, regulation of the poly(P) balance is critical for persister formation in M. tuberculosis.

Crystal structure of the biologically active form of class Ib ribonucleotide reductase small subunit from Mycobacterium tuberculosis.

Two nrdF genes of Mycobacterium tuberculosis code for different R2 subunits of the class Ib ribonucleotide reductase (RNR). The proteins are denoted R2F-1 and R2F-2 having 71% sequence identity. The R2F-2 subunit forms the biologically active RNR complex with the catalytic R1E-subunit. We present the structure of the reduced R2F-2 subunit to 2.2 A resolution. Comparison of the R2F-2 structure with a model of R2F-1 suggests that the important differences are located at the C-terminus. We found that within class Ib, the E-helix close to the iron diiron centre has two preferred conformations, which cannot be explained by the redox-state of the diiron centre. In the R2F-2 structure, we also could see a mobility of alphaE in between the two conformations.

Cysteine proteinases from distinct cellular compartments are recruited to phagocytic vesicles by Entamoeba histolytica.

Cysteine proteinases, which are encoded by at least seven genes, play a critical role in the pathogenesis of invasive amebiasis caused by Entamoeba histolytica. The study of these enzymes has been hampered by the inability to obtain significant quantities of the individual native proteinases. We have now expressed functionally active recombinant ACP1 (EhCP3) and ACP2 (EhCP2) proteinases in baculoviral expression vectors. The purified recombinant ACP1 and ACP2 proteinases exhibited similar activities for fluorogenic peptide substrates, especially in their preference for an arginine residue at the P2 position. Although ACP1 and ACP2 are structurally cathepsin L, homology modeling revealed that the aspartic acid in the S2 pocket would result in a substrate specificity for positively charged amino acids, like cathepsin B. The hydrolysis of peptide substrates was strongly inhibited by small peptidyl inhibitors specifically designed for parasitic cysteine proteinases. Confocal and immunoelectron microscopy localization of the proteinases with monoclonal and monospecific antibodies raised to the recombinant enzymes and peptides demonstrated that ACP2 was membrane-associated while ACP1 was cytoplasmic. Following phagocytosis of erythrocytes, ACP1, as well as the membrane-associated cysteine proteinase, ACP2, were incorporated into phagocytic vesicles. These studies suggest that E. histolytica has a redundancy of cysteine proteinases for intracellular digestion and that they may be recruited from different cellular compartments to the site of digestion of phagocytosed cells. The production of active proteinases in baculovirus and large scale recombinant enzymes in bacteria should further our understanding of the role of different cysteine proteinase gene products in virulence.

Recurrence of a solitary fibrous tumor of the pleura: a case report.

We report a case of an 80-year-old female with two prior thoracotomies for benign solitary fibrous tumor of the pleura (SFTP) presenting with a two-month history of shortness of breath. Computed tomography revealed a pleural-based recurrence and operative excision revealed multiple adherent tumors throughout the thoracic cavity. Pathologic examination demonstrated malignant degeneration of this previously benign tumor. We consider the importance of recurrence of benign SFTP and the significance of surgical care and follow up of patients with this rare tumor.