Exosomes and microvesicles (MV) are cell membranous sacs originating from multivesicular bodies and plasma membranes that facilitate long-distance intercellular communications. Their functional biology, however, remains incompletely understood. Macrophage exosomes and MV isolated by immunoaffinity and sucrose cushion centrifugation were characterized by morphologic, biochemical, and molecular assays. Lipidomic, proteomic, and cell biologic approaches uncovered novel processes by which exosomes and MV facilitate HIV-1 infection and dissemination. HIV-1 was “entrapped” in exosome aggregates. Robust HIV-1 replication followed infection with exosome-enhanced fractions isolated from infected cell supernatants. MV- and exosome-facilitated viral infections are affected by a range of cell surface receptors and adhesion proteins. HIV-1 containing exosomes readily completed its life cycle in human monocyte-derived macrophages but not in CD4− cells. The data support a significant role for exosomes as facilitators of viral infection.

Conventional homogeneous catalysis relies on one transition metal/ligand combination to promote all steps within a catalytic cycle. This approach is suboptimal when different steps within the cycle place different demands on the catalyst. Herein we report for the first time a serial ligand catalysis mechanism in which two different ligands interact sequentially with the metal to promote different product-forming steps of the same catalytic cycle. We recently reported sulfoxide-promoted, catalytic Pd(OAc) 2 / benzoquinone (BQ)/AcOH R-olefin allylic oxidation systems 1 that have the interesting feature of furnishing either predominantly linear or branched allylic acetates depending on whether DMSO or bissulfoxide ligands are used, respectively. 1a While investigating the bis-sulfoxide-promoted system, we discovered that 1 partially decomposes 2 under the reaction conditions to generate vinyl sulfoxide 2 (Table 1). We tested commercially available 2 and found that 10 mol % 2/Pd(OAc) 2 effectively promotes the oxidation reaction to furnish branched products with no decomposition. 1b,3 Reducing the equivalents of AcOH significantly improves regioselectivities (Table 1, entries 3a,b) by suppressing a background Pd(II)-mediated isomerization. 4 We now report a vinyl sulfoxidepromoted catalytic system for the mild, chemo-(R-versus internal olefins), and highly regioselective C-H oxidation of R-olefins to furnish allylic alkyl and aryl esters that proceeds via a mechanism in which two different ligands are responsible for promoting different steps in the catalytic cycle (Table 3). Mechanistic studies were carried out to establish the fundamental steps of this catalytic cycle and the role of vinyl sulfoxide 2 and BQ therein. When stoichiometric mixtures of 1-undecene, Pd(OAc) 2 , and 2 were heated and monitored by 1 H NMR, dimeric π-allylpalladium acetate complex A was observed in ca. 59% yield (eq 1). 5a-c When BQ was then added to this reaction mixture, formation of allylic acetate product was observed with yields and regioselectivities similar to those observed for the stoichiometric reaction run in the presence of BQ (eq 1, Table 1, entry 3h). In the absence of 2, with and without BQ, formation of complex A was not observed. These data are consistent with 2, and not BQ, acting as a ligand to effect Pd-mediated allylic C-H cleavage to likely form a monomeric π-allylpalladium intermediate that is detected in the form of dimeric complex A.

Summary Understanding the basis of bacterial persistence in latent infections is critical for eradication of tuberculosis. Analysis of Mycobacterium tuberculosis mRNA expression in an in vitro model of non-replicating persistence indicated that the bacilli require electron transport chain components and ATP synthesis for survival. Additionally, low μM concentrations of aminoalkoxydiphenylmethane derivatives inhibited both the aerobic growth and survival of non-replicating, persistent M. tuberculosis. Metabolic labeling studies and quantitation of cellular menaquinone levels suggested that menaquinone synthesis, and consequently electron transport, is the target of the aminoalkoxydiphenylmethane derivatives. This hypothesis is strongly supported by the observations that treatment with these compounds inhibits oxygen consumption and that supplementation of growth medium with exogenous menaquinone rescued both growth and oxygen consumption of treated bacilli. In vitro assays indicate that the aminoalkoxydiphenylmethane derivatives specifically inhibit MenA, an enzyme involved in the synthesis of menaquinone. Thus, the results provide insight into the physiology of mycobacterial persistence and a basis for the development of novel drugs that enhance eradication of persistent bacilli and latent tuberculosis.

The DosR regulon in Mycobacterium tuberculosis is involved in respiration-limiting conditions, its induction is controlled by two histidine kinases, DosS and DosT, and recent experimental evidence indicates DosS senses either molecular oxygen or a redox change. Under aerobic conditions, induction of the DosR regulon by DosS, but not DosT, was observed after the addition of ascorbate, a powerful cytochrome c reductant, demonstrating that DosS responds to a redox signal even in the presence of high oxygen tension. During hypoxic conditions, regulon induction was attenuated by treatment with compounds that occluded electron flow into the menaquinone pool or decreased the size of the menaquinone pool itself. Increased regulon expression during hypoxia was observed when exogenous menaquinone was added, demonstrating that the menaquinone pool is a limiting factor in regulon induction. Taken together, these data demonstrate that a reduced menaquinone pool directly or indirectly triggers induction of the DosR regulon via DosS. Biochemical analysis of menaquinones upon entry into hypoxic/anaerobic conditions demonstrated the disappearance of the unsaturated species and low-level maintenance of the mono-saturated menaquinone. Relative to the unsaturated form, an analog of the saturated form is better able to induce signaling via DosS and rescue inhibition of menaquinone synthesis and is less toxic. The menaquinone pool is central to the electron transport system (ETS) and therefore provides a mechanistic link between the respiratory state of the bacilli and DosS signaling. Although this report demonstrates that DosS responds to a reduced ETS, it does not rule out a role for oxygen in silencing signaling.Mycobacteria are strict aerobes, but Mycobacterium tuberculosis encounters microaerobic to anaerobic environments during the course of infection. Oxygen-limited microenvironments occur in mature granulomas, which are known to be avascular, inflammatory, and necrotic (1,5,20,40,56). Recent reports have detected mycobacterial DNA in visibly normal lung tissue (17), as well as adipose tissue (32). Interestingly, adipose tissue has been associated with hypoxia as well as the presence of nitric oxide (NO), both of which inhibit respiration (38,51,57,60). Further, M. tuberculosis is able to survive for long periods of time in a nonreplicating and nonrespiring state (15, 55).The DosR regulon is expressed in response to hypoxia, NO, and carbon monoxide (CO) and is thought to be important for early adaptation to these stimuli as well as long-term survival in the host (2,25,26,46,47,53,58). Despite induction by other gases, the presence of oxygen itself inhibits induction of the regulon (26,41,43,48). The DosR regulon is regulated by the response regulator DosR (DevR; Rv3133c) and was recently shown to be positively regulated by PhoP (Rv0757) to a basal level during aerobic growth (16). DosR is activated by two sensor histidine kinases, DosT (Rv2027c) and DosS (DevS; Rv3132c). The activation of DosR occurs through autophosphorylatio...

New treatments and novel drugs are required to counter the growing problem of drug-resistant strains of Mycobacterium tuberculosis (M.tb). Our approach against drug resistant M.tb, as well as other intracellular pathogens, is by targeted drug delivery using nanoformulations of drugs already in use, as well as drugs in development. Among the latter are gallium (III) (Ga)-based compounds. In the current work, six different types of Ga and rifampin nanoparticles were prepared in such a way as to enhance targeting of M.tb infected-macrophages. They were then tested for their ability to inhibit growth of a fully pathogenic strain (H37Rv) or a non-pathogenic strain (H37Ra) of M.tb. Encapsulating Ga in folate- or mannose-conjugated block copolymers provided sustained Ga release for 15 days and significantly inhibited M.tb growth in human monocyte-derived macrophages. Nanoformulations with dendrimers encapsulating Ga or rifampin also showed promising anti-tuberculous activity. The nanoparticles co-localized with M.tb containing phagosomes, as measured by detection of mature cathepsin D (34 kDa, lysosomal hydrogenase). They also promoted maturation of the phagosome, which would be expected to increase macrophage-mediated killing of the organism. Delivery of Ga or rifampin in the form of nanoparticles to macrophages offers a promising approach for the development of new therapeutic anti-tuberculous drugs.