Novel Assay Platform to Evaluate Intracellular Killing of Mycobacterium tuberculosis: In Vitro and In Vivo Validation

One of the main hallmarks of tuberculosis (TB) is the ability of the causative agent to transform into a stage of dormancy and the capability of long persistence in the host phagocytes. It is believed that approximately one-third of the population of the world is latently infected with Mycobacterium tuberculosis (Mtb), and 5%–10% of these individuals can develop clinical manifestations of active TB even decades after the initial infection. In this latent, intracellular form, the bacillus is shielded by an extremely robust cell wall and becomes phenotypically resistant to most antituberculars. Therefore, there is a clear rationale to develop novel compounds or carrier-conjugated constructs of existing drugs that are effective against the intracellular form of the bacilli. In this paper, we describe an experimental road map to define optimal candidates against intracellular Mtb and potential compounds effective in the therapy of latent TB. To validate our approach, isoniazid, a first-line antitubercular drug was employed, which is active against extracellular Mtb in the submicromolar range, but ineffective against the intracellular form of the bacteria. Cationic peptide conjugates of isoniazid were synthesized and employed to study the host-directed drug delivery. To measure the intracellular killing activity of the compounds, Mtb-infected MonoMac-6 human monocytic cells were utilized. We have assessed the antitubercular activity, cytotoxicity, membrane interactions in combination with internalization efficacy, localization, and penetration ability on interface and tissue-mimicking 3D models. Based on these in vitro data, most active compounds were further evaluated in vivo in a murine model of TB. Intraperitoneal infectious route was employed to induce a course of slowly progressive and systemic disease. The well-being of the animals, monitored by the body weight, allows a prolonged experimental setup and provides a great opportunity to test the long-term activity of the drug candidates. Having shown the great potency of this simple and suitable experimental design for antimicrobial research, the proposed novel assay platform could be used in the future to develop further innovative and highly effective antituberculars.

Lysosome Sensing Is a Key Mechanism in Leishmania Intracellular Development

Phagolysosomes of macrophages are the niche where the parasitic protozoan Leishmania resides and causes human leishmaniasis. During infection, this organism encounters dramatic environmental changes. These include heat shock (from 26°C in the vector to 33°C or 37°C in the host, for cutaneous and visceral species, respectively) and acidic pH typical to the lysosome and nutrient availability. Leishmania cells developed ways to sense the lysosome-specific environment (acidic pH and body temperature) as means of recognition and, subsequently, initiation of differentiation into the intracellular form. Recent studies have indicated that protein kinase A plays a role as the gatekeeper that enables differentiation initiation. This review provides an update on the lysosome signaling pathway-mediated Leishmania intracellular development.

DBSCAN-SWA: an integrated tool for rapid prophage detection and annotation

SummaryAs an intracellular form of a bacteriophage in the bacterial host genome, a prophage is usually integrated into bacterial DNA with high specificity and contributes to horizontal gene transfer (HGT). Phage therapy has been widely applied, for example, using phages to kill bacteria to treat pathogenic and resistant bacterial infections. Therefore, it is necessary to develop effective tools for the fast and accurate identification of prophages. Here, we introduce DBSCAN-SWA, a command line software tool developed to predict prophage regions of bacterial genomes. DBSCAN-SWA runs faster than any previous tool. Importantly, it has great detection power based on analysis using 184 manually curated prophages, with a recall of 85% compared with Phage_Finder (63%), VirSorter (74%) and PHASTER (82%) for raw DNA sequences. DBSCAN-SWA also provides user-friendly visualizations including a circular prophage viewer and interactive DataTables.Availability and implementationDBSCAN-SWA is implemented in Python3 and is freely available under an open source GPLv2 license from https://github.com/HIT-ImmunologyLab/DBSCAN-SWA/.

Antileishmanial activity of sp2-iminosugar derivatives

sp2-iminosugar S-linked pseudoglycosides selectively inhibit growth of the intracellular form of Leishmania donovani.

Divergent mechanism regulating fluid intake and metabolism by the brain renin-angiotensin system

The purpose of this review is two-fold. First, I will highlight recent advances in our understanding of the mechanisms regulating angiotensin II (ANG II) synthesis in the brain, focusing on evidence that renin is expressed in the brain and is expressed in two forms: a secreted form, which may catalyze extracellular ANG I generation from glial or neuronal angiotensinogen (AGT), and an intracellular form, which may generate intracellular ANG in neurons that may act as a neurotransmitter. Second, I will discuss recent studies that advance the concept that the renin-angiotensin system (RAS) in the brain not only is a potent regulator of blood pressure and fluid intake but may also regulate metabolism. The efferent pathways regulating the blood pressure/dipsogenic effects and the metabolic effects of elevated central RAS activity appear different, with the former being dependent upon the hypothalamic-pituitary-adrenal axis, and the latter being dependent upon an interaction between the brain and the systemic (or adipose) RAS.

The Immunogenicity of the Tumor-Associated Antigenα-Fetoprotein Is Enhanced by a Fusion with a Transmembrane Domain

Aim. To investigate the ability of recombinant modified vaccinia virus Ankara (rMVA) vector to induce an immune response against a well-tolerated self-antigen.Methods. rMVA vectors expressing different form ofα-fetoprotein (AFP) were produced and characterized. Naïve mice were vaccinated with MVA vectors expressing the AFP antigen in either a secreted, or a membrane-bound, or an intracellular form. The immune response was monitored by an IFNΓ ELISpot assay and antibody detection.Results. Vaccination with the membrane-associated form of AFP induced a stronger CD8+T-cell response compared to the ones obtained with the MVA encoding the secreted or the intracellular forms of AFP. Moreover, the vaccination with the membrane-bound AFP elicited the production of AFP-specific antibodies.Conclusions. The AFP transmembrane form is more immunogenic. Expressing a membrane-bound form in the context of an MVA vaccination could enhance the immunogenicity of a self-antigen.

Autocrine Effects of Visfatin on Hepatocyte Sensitivity to Insulin Action

Visfatin was originally described as an adipokine with insulin mimetic effects. Recently, it was found that visfatin is identical with the Nampt (nicotinamide phosphoribosyltransferase) gene that codes for an intra- and extracellular NAD biosynthetic enzyme and is predominantly expressed outside the adipose tissue. In the current study, we found strong protein and mRNA expression of visfatin in rat heart, liver, kidney, and muscle, while the expression of visfatin in visceral fat was significantly lower and undetectable in subcutaneous fat. The insulin-mimetic effects of visfatin (extracellular form of Nampt or eNampt) are controversial and even less is known about autocrine effects of visfatin (intracellular form of Nampt or iNampt). Since liver plays a major role in glucose metabolism, we studied visfatin effects on insulin-stimulated cellular glucose uptake in Fao rat hepatocytes using RNA interference (RNAi). RNAi-mediated downregulation of visfatin expression in Fao cells was associated with significantly reduced NAD biosynthesis (0.3±0.01 vs. 0.5±0.01 mmol/h/g, P<0.05) and with significantly decreased incremental glucose uptake after stimulation with insulin when compared to controls with normal expression of visfatin (0.6±0.2 vs. 2.2±0.5 nnmol/g/2 h, P=0.02). These results provide evidence that visfatin exhibits important autocrine effects on sensitivity of liver cells to insulin action possibly through its effects on NAD biosynthesis.

Osteopontin localizes to the nucleus of 293 cells and associates with polo-like kinase-1

Osteopontin (OPN) is a secreted phosphoprotein involved in cellular proliferation and associated with tumor progression. Although an intracellular form of OPN has been described, its function remains unknown. In this study, a novel nuclear location for intracellular OPN and a correlation with cell division were demonstrated. OPN distinctly localized to the nucleus in a subset of transiently transfected human embryonic kidney 293 cells. Immunoblotting confirmed the nuclear location of native OPN, and results from immunofluorescence studies suggested an association between nuclear OPN and cell cycle progression. Flow cytometry revealed that nuclear and cellular OPN content rose significantly during the S and G2/M phases, respectively. Treatment of cells with the DNA polymerase inhibitor aphidicolin prevented cell cycling and greatly reduced cellular OPN content. The intracellular location of OPN coincided with polo-like kinase-1 (Plk-1), a member of the polo-like kinase family, which, in part through their regulation of centrosome-related events, are integral to successful cellular mitosis. OPN and Plk-1 were coimmunoprecipitated from nuclear, but not cystoslic, extracts, demonstrating an interaction that is limited to the nucleus, presumably during mitosis. Deletion of the COOH terminus of OPN militated against nuclear localization and Plk-1 interaction. Elevated expression of OPN was also associated with an increase in the number of multinucleate 293 cells, whereas transfection of the COOH-terminal-deleted OPN decreased the percentage of multinucleate cells below basal levels. These findings implicate intranuclear OPN as a participant in the process of cell duplication.