Rational Design of Selective and Bioactive Inhibitors of the Mycobacterium Tuberculosis Proteasome

The 20S core particle of the proteasome in Mycobacterium tuberculosis (Mtb) is a promising, yet unconventional, drug target. This multimeric peptidase is not essential, yet degrades proteins that have become damaged and toxic via reactions with nitric oxide (and/or the associated reactive nitrogen intermediates) produced during the host immune response. Proteasome inhibitors could render Mtb susceptible to the immune system, but they would only be therapeutically viable if they do not inhibit the essential 20S counterpart in humans. Selective inhibitors of the Mtb 20S were designed and synthesized on the bases of both its unique substrate preferences and the structures of substrate-mimicking covalent inhibitors of eukaryotic proteasomes called syringolins. Unlike the parent syringolins, the designed analogues weakly inhibit the human 20S (Hs 20S) proteasome and preferentially inhibit Mtb 20S over the human counterpart by as much as 74-fold. Moreover, they can penetrate the mycobacterial cell envelope and render Mtb susceptible to nitric oxide-mediated stress. Importantly, they do not inhibit the growth of human cell lines in vitro and thus may be starting points for tuberculosis drug development.National Institutes of Health (U.S.) (Grant AI-16892

A network of Rab GTPases controls phagosome maturation and is modulated by Salmonella enterica serovar Typhimurium

Members of the Rab guanosine triphosphatase (GTPase) family are key regulators of membrane traffic. Here we examined the association of 48 Rabs with model phagosomes containing a non-invasive mutant of Salmonella enterica serovar Typhimurium (S. Typhimurium). This mutant traffics to lysosomes and allowed us to determine which Rabs localize to a maturing phagosome. In total, 18 Rabs associated with maturing phagosomes, each with its own kinetics of association. Dominant-negative mutants of Rab23 and 35 inhibited phagosome–lysosome fusion. A large number of Rab GTPases localized to wild-type Salmonella-containing vacuoles (SCVs), which do not fuse with lysosomes. However, some Rabs (8B, 13, 23, 32, and 35) were excluded from wild-type SCVs whereas others (5A, 5B, 5C, 7A, 11A, and 11B) were enriched on this compartment. Our studies demonstrate that a complex network of Rab GTPases controls endocytic progression to lysosomes and that this is modulated by S. Typhimurium to allow its intracellular growth

Whole cell screen for inhibitors of pH homeostasis in Mycobacterium tuberculosis

Bacterial pathogens like Mycobacterium tuberculosis (Mtb) encounter acidic microenvironments in the host and must maintain their acid-base homeostasis to survive. A genetic screen identified two Mtb strains that cannot control intrabacterial pH (pHIB) in an acidic environment; infection with either strain led to severe attenuation in mice. To search for additional proteins that Mtb requires to survive at low pH, we introduced a whole-cell screen for compounds that disrupt pHIB, along with counter-screens that identify ionophores and membrane perturbors. Application of these methods to a natural product library identified four compounds of interest, one of which may inhibit novel pathway(s). This approach yields compounds that may lead to the identification of pathways that allow Mtb to survive in acidic environments, a setting in which Mtb is resistant to most of the drugs currently used to treat tuberculosis

Structure of human immunoproteasome with a reversible and noncompetitive inhibitor that selectively inhibits activated lymphocytes

Proteasome inhibitors benefit patients with multiple myeloma and B cell-dependent autoimmune disorders but exert toxicity from inhibition of proteasomes in other cells. Toxicity should be minimized by reversible inhibition of the immunoproteasome β5i subunit while sparing the constitutive β5c subunit. Here we report β5i-selective inhibition by asparagine-ethylenediamine (AsnEDA)-based compounds and present the high-resolution cryo-EM structural analysis of the human immunoproteasome. Despite inhibiting noncompetitively, an AsnEDA inhibitor binds the active site. Hydrophobic interactions are accompanied by hydrogen bonding with β5i and β6 subunits. The inhibitors are far more cytotoxic for myeloma and lymphoma cell lines than for hepatocarcinoma or non-activated lymphocytes. They block human B-cell proliferation and promote apoptotic cell death selectively in antibody-secreting B cells, and to a lesser extent in activated human T cells. Reversible, β5i-selective inhibitors may be useful for treatment of diseases involving activated or neoplastic B cells or activated T cells

Improved Control of Tuberculosis and Activation of Macrophages in Mice Lacking Protein Kinase R

Host factors that microbial pathogens exploit for their propagation are potential targets for therapeuic countermeasures. No host enzyme has been identified whose genetic absence benefits the intact mammalian host in vivo during infection with Mycobacterium tuberculosis (Mtb), the leading cause of death from bacterial infection. Here, we report that the dsRNA-dependent protein kinase (PKR) is such an enzyme. PKR-deficient mice contained fewer viable Mtb and showed less pulmonary pathology than wild type mice. We identified two potential mechanisms for the protective effect of PKR deficiency: increased apoptosis of macrophages in response to Mtb and enhanced activation of macrophages in response to IFN-gamma. The restraining effect of PKR on macrophage activation was explained by its mediation of a previously unrecognized ability of IFN-gamma to induce low levels of the macrophage deactivating factor interleukin 10 (IL10). These observations suggest that PKR inhibitors may prove useful as an adjunctive treatment for tuberculosis

Analysis, design and implementation of a non-isolated switched inductor/capacitor converter with low voltage spikes

In order to take the advantage of boost capability and filtering characteristics of the switched inductor/capacitor, a non-isolated switched inductor/capacitor (NSI/C) converter is proposed. The topological structure of the non-isolated switched inductor/capacitor converter is analyzed in detail, and the DC steady-state model is established in continuous conduction mode (CCM). The voltage gain of the converter and the stress of the switch in the converter are theoretically deduced and compared with the existing converters; the parameter design of the inductance and capacitance is calculated. The simulation model was built in MATLAB/Simulink, and the correctness of the theoretical calculation for the proposed converter was verified by simulation results. Finally, the experimental prototypes of the proposed converter and the non-isolated high step-up (NHS) converter were built. Through the comparison of experimental results, the feasibility and validity of the design scheme for the proposed converter are verified.Published versionThis work was financially supported by Key Research and Development Project of Hebei Province (18214302D) and (19212101D), Open Project of Provincial Collaborative Innovation Center of Industrial Energy-saving and Power Quality Control, Anhui Province (KFKT201504), Natural Science Foundation of Hebei Province (F2018205178)

Correction: Improved Control of Tuberculosis and Activation of Macrophages in Mice Lacking Protein Kinase R.

[This corrects the article DOI: 10.1371/journal.pone.0030512.]

Salmonella-Induced Filament Formation Is a Dynamic Phenotype Induced by Rapidly Replicating Salmonella enterica Serovar Typhimurium in Epithelial Cells

Salmonella enterica serovar Typhimurium has the fascinating ability to form tubular structures known as Salmonella-induced filaments (Sifs) in host cells. Here, we show that the prevalence of the Sif phenotype in HeLa cells is affected by host cell density, growth, and the multiplicity of infection. Sif formation was observed in cells that displayed rapid intracellular bacterial replication and was found to be dynamic, being maximal 8 to 10 h postinfection and declining thereafter. The virulence factors SpvB and SseJ were found to negatively modulate Sif formation. Our findings demonstrate the complex and dynamic nature of the Sif phenotype

Spatial-temporal potential exposure risk analytics and urban sustainability impacts related to COVID-19 mitigation: A perspective from car mobility behaviour

Coronavirus disease-2019 (COVID-19) poses a significant threat to the population and urban sustainability worldwide. The surge mitigation is complicated and associates many factors, including the pandemic status, policy, socioeconomics and resident behaviours. Modelling and analytics with spatial-temporal big urban data are required to assist the mitigation of the pandemic. This study proposes a novel perspective to analyse the spatial-temporal potential exposure risk of residents by capturing human behaviours based on spatial-temporal car park availability data. Near real-time data from 1,904 residential car parks in Singapore, a classical megacity, are collected to analyse car mobility and its spatial-temporal heat map. The implementation of the circuit breaker, a COVID-19 measure, in Singapore has reduced the mobility and heat (daily frequency of mobility) significantly at about 30.0%. It contributes to a 44.3%–55.4% reduction in the transportation-related air emissions under two scenarios of travelling distance reductions. Urban sustainability impacts in both environment and economy are discussed. The spatial-temporal potential exposure risk mapping with space-time interactions is further investigated via an extended Bayesian spatial-temporal regression model. The maximal reduction rate of the defined potential exposure risk lowers to 37.6% by comparison with its peak value. The big data analytics of changes in car mobility behaviour and the resultant potential exposure risks can provide insights to assist in (a) designing a flexible circuit breaker exit strategy, (b) precise management via identifying and tracing hotspots on the mobility heat map, and (c) making timely decisions by fitting curves dynamically in different phases of COVID-19 mitigation. The proposed method has the potential to be used by decision-makers worldwide with available data to make flexible regulations and planning.</p

Energy, environmental, economic and social equity (4E) pressures of COVID-19 vaccination mismanagement: a global perspective

Vaccination now offers a way to resolve the COVID-19 pandemic. However, it is critical to recognise the full energy, environmental, economic and social equity (4E) impacts of the vaccination life cycle. The full 4E impacts include the design and trials, order management, material preparation, manufacturing, cold chain logistics, low-temperature storage, crowd management and end-of-life waste management. A life cycle perspective is necessary for sustainable vaccination management because a prolonged immunisation campaign for COVID-19 is likely. The impacts are geographically dispersed across sectors and regions, creating real and virtual 4E footprints that occur at different timescales. Decision-makers in industry and governments have to act, unify, resolve, and work together to implement more sustainable COVID-19 vaccination management globally and locally to minimise the 4E footprints. Potential practices include using renewable energy in production, storage, transportation and waste treatment, using better product design for packaging, using the Internet of Things (IoT) and big data analytics for better logistics, using real-time database management for better tracking of deliveries and public vaccination programmes, and using coordination platforms for more equitable vaccine access. These practices raise global challenges but suggest solutions with a 4E perspective, which could mitigate the impacts of global vaccination campaigns and prepare sustainably for future pandemics and global warming