Leishmania exploits host cAMP/EPAC/calcineurin signaling to induce an IL-33-mediated anti-inflammatory environment for the establishment of infection.

Host anti-inflammatory responses are critical for the progression of visceral leishmaniasis, and the pleiotropic cytokine interleukin (IL)-33 was found to be upregulated in infection. Here, we documented that IL-33 induction is a consequence of elevated cAMP-mediated exchange protein activated by cAMP (EPAC)/calcineurin-dependent signaling and essential for the sustenance of infection. Leishmania donovani-infected macrophages showed upregulation of IL-33 and its neutralization resulted in decreased parasite survival and increased inflammatory responses. Infection-induced cAMP was involved in IL-33 production and of its downstream effectors PKA and EPAC, only the latter was responsible for elevated IL-33 level. EPAC initiated Rap-dependent phospholipase C activation, which triggered the release of intracellular calcium followed by calcium/calmodulin complex formation. Screening of calmodulin-dependent enzymes affirmed involvement of the phosphatase calcineurin in cAMP/EPAC/calcium/calmodulin signaling-induced IL-33 production and parasite survival. Activated calcineurin ensured nuclear localization of the transcription factors, nuclear factor of activated T cell 1 and hypoxia-inducible factor 1 alpha required for IL-33 transcription, and we further confirmed this by chromatin immunoprecipitation assay. Administering specific inhibitors of nuclear factor of activated T cell 1 and hypoxia-inducible factor 1 alpha in BALB/c mouse model of visceral leishmaniasis decreased liver and spleen parasite burden along with reduction in IL-33 level. Splenocyte supernatants of inhibitor-treated infected mice further documented an increase in tumor necrosis factor alpha and IL-12 level with simultaneous decrease of IL-10, thereby indicating an overall disease-escalating effect of IL-33. Thus, this study demonstrates that cAMP/EPAC/calcineurin signaling is crucial for the activation of IL-33 and in effect creates anti-inflammatory responses, essential for infection.

G-quadruplex structural dynamics at MAPK12 promoter dictates transcriptional switch to determine stemness in breast cancer.

P38γ (MAPK12) is predominantly expressed in triple negative breast cancer cells (TNBC) and induces stem cell (CSC) expansion resulting in decreased survival of the patients due to metastasis. Abundance of G-rich sequences at MAPK12 promoter implied the functional probability to reverse tumorigenesis, though the formation of G-Quadruplex (G4) structures at MAPK12 promoter is elusive. Here, we identified two evolutionary consensus adjacent G4 motifs upstream of the MAPK12 promoter, forming parallel G4 structures. They exist in an equilibria between G4 and duplex, regulated by the binding turnover of Sp1 and Nucleolin that bind to these G4 motifs and regulate MAPK12 transcriptional homeostasis. To underscore the gene-regulatory functions of G4 motifs, we employed CRISPR-Cas9 system to eliminate G4s from TNBC cells and synthesized a naphthalene diimide (NDI) derivative (TGS24) which shows high-affinity binding to MAPK12-G4 and inhibits MAPK12 transcription. Deletion of G4 motifs and NDI compound interfere with the recruitment of the transcription factors, inhibiting MAPK12 expression in cancer cells. The molecular basis of NDI-induced G4 transcriptional regulation was analysed by RNA-seq analyses, which revealed that MAPK12-G4 inhibits oncogenic RAS transformation and trans-activation of NANOG. MAPK12-G4 also reduces CD44High/CD24Low population in TNBC cells and downregulates internal stem cell markers, arresting the stemness properties of cancer cells.

A ratiometric small-molecule fluorescent probe for the selective detection of hypochlorite by an oxidative cyclization reaction: application to commercial disinfectants and live cells.

A highly selective thiophene-thioimidazole hydrazine-based ratiometric chemodosimeter (TPBN) was designed and synthesized to detect hypochlorite (ClO-). The probe showed yellow fluorescence and exhibited ultra sensitivity towards hypochlorite (detection limit 8.74 nM) through the oxidative intramolecular cyclization process to give a blue fluorescent triazole product (TPBN-P). Additionally, the as-designed sensor displayed a fast response (80 s) to hypochlorite with excellent selectivity over other competing analytes. DFT calculations, ESI-MS, and 1H NMR titration experiments supported the detection mechanism. The probe was a valuable and practical ratiometric sensor for test strips, commercial disinfectants, and water samples. The probe was successfully used in the bio-imaging of hypochlorite in human breast cancer cells due to its noteworthy photophysical characteristics and good cell permeability.

Translationally Controlled Tumor Protein-Mediated Stabilization of Host Antiapoptotic Protein MCL-1 Is Critical for Establishment of Infection by Intramacrophage Parasite Leishmania donovani.

In the early phase of infection, the intramacrophage pathogen Leishmania donovani protects its niche with the help of the antiapoptotic protein myeloid cell leukemia-1 (MCL-1). Whether Leishmania could exploit MCL-1, an extremely labile protein, at the late phase is still unclear. A steady translational level of MCL-1 observed up to 48 h postinfection and increased caspase-3 activity in MCL-1-silenced infected macrophages documented its importance in the late hours of infection. The transcript level of MCL-1 showed a sharp decline at 6 h postinfection, and persistent MCL-1 expression in cyclohexamide-treated cells negates the possibility of de novo protein synthesis, thereby suggesting infection-induced stability. Increased ubiquitination, a prerequisite for proteasomal degradation of MCL-1, was also found to be absent in the late hours of infection. Lack of interaction with its specific E3 ubiquitin ligase MULE (MCL-1 ubiquitin ligase E3) and specific deubiquitinase USP9X prompted us to search for blockade of the ubiquitin-binding site in MCL-1. To this end, TCTP (translationally controlled tumor protein), a well-known binding partner of MCL-1 and antiapoptotic regulator, was found to be strongly associated with MCL-1 during infection. Phosphorylation of TCTP, a requirement for MCL-1 binding, was also increased in infected macrophages. Knockdown of TCTP decreased MCL-1 expression and short hairpin RNA-mediated silencing of TCTP in an infected mouse model of visceral leishmaniasis showed decreased parasite burden and induction of liver cell apoptosis. Collectively, our investigation revealed a key mechanism of how L. donovani exploits TCTP to establish infection within the host.

Pyridyl-Isonicotinoyl Hydrazone-Bridged Zn(II) Coordination Framework with Thiophenedicarboxylato Link: Structure, Biological Activity, and Electrical Conductivity.

Multidimensional applicability of functional materials is one of the focal attractions in today's scientific research. Highly stable and crystalline coordination polymers served as one of the active members in the club of multifunctional materials. Toward this concept, a 3-dimensional (3D) coordination framework, {[Zn2(tdc)2(pcih)2]n} (1) (tdc2-, 2,5-thiophene dicarboxylate; pcih, pyridine-4-carboxaldehyde isonicotinoyl hydrazine), is designed and has been structurally well characterized by single crystal X-ray crystallography. One of the carboxylate groups of tdc2- chelates to Zn(II), while the other carboxylato group (-COO) acts as bridging-O to neighboring Zn(II); the pcih serves as pyridyl-N bridging motif to two Zn(II) centers. The optical band gap, 3.83 eV (Tauc's plot), implies probable semiconducting ability of the material. Interestingly, the device fabricated using compound 1 measures the electrical conductivity, 2.21 × 10-5 S cm-1, and series resistance (Rs), 807 Ω, at the dark phase, which are improved significantly to 6.36 × 10-5 S cm-1 and 460 Ω, respectively, under illumination conditions. Isoniazid, used to synthesize pcih and hence the Zn(II) compound 1, is a medicine; so, the medicinal efficiency of 1 is checked by measuring the anticancer activity against MDA-MB-231, HeLa, HCT-116, and HepG2 cells. It is observed that drug efficacy is highest on MDA-MB-231 cells (IC50: 19.43 ± 1.36 µM) than other cancer cells [IC50: 24.43 ± 2.02 µM (HeLa), 26.06 ± 3.48 µM (HCT-116), and 44.28 ± 3.04 µM (HepG2)]. Therefore, the material has significant contribution in the area of energy and health toward the sustainable development goals.

Near-infrared fluorescent turn-on probe for hydrazine detection: environmental samples and live cell imaging.

An acetoxy naphthaldehyde conjugated benzophenoxazinium chloride chromophore-based-donor-π-acceptor (D-π-A) fluorescent probe BPN (benzophenoxazinium naphthoxy imine) displaying near-infrared (NIR) emission was reported for hydrazine detection. The chosen water-soluble benzophenoxazinium chloride chromophore has excellent photostability, a high molar extinction coefficient and fluorescence quantum yield (Φ = from 0.0075 to 0.6193), higher selectivity towards hydrazine and a longer fluorescence lifetime. In the presence of hydrazine, BPN exhibits near infrared fluorescence emission at 725 nm along with color change from light blue to red, as detected by the naked eye. Moreover, the BPN probe can selectively detect hydrazine (DL = 4.5 × 10-10 M) in a 90% aqueous DMSO solution without interfering with other analytes. As proof of real samples, the probe is successfully applied to sense hydrazine in thin layer chromatography (TLC) paper strips (both solution and vapor phases) and water and soil samples, suggesting its significant potential application. Also, due to its NIR emission and aqueous solubility, the BPN probe can be successfully used in live cell imaging with low cytotoxicity.

A turn-on fluorescent probe for selective detection of H2S in environmental samples and bio-imaging in human breast cancer cells.

A triphenylamine-benzothaizole-based turn-on fluorescent probe TPB-NO2 was designed and synthesized for tracking H2S in both environmental and biological samples depending upon the sensing strategy of thiolysis of 2,4-dinitrophenyl (DNP) ether. Due to PET (photoinduced electron transfer), occurring from donor triphenylamine moiety to acceptor DNP moiety, the probe TPB-NO2 itself is very weakly fluorescent and colorless in DMSO/H2O solution (1 : 1, v/v; 10 mM HEPES buffer, pH 7.4). But the addition of H2S leads to thiolysis of 2,4-dinitrophenyl ether to block the initial PET process and hence it exhibits naked eye detectable turn-on response with bright cyan fluorescence and intense brown color. Not only that, the probe exhibits excellent selectivity over other bio-thiols like Cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), fast response time (<2 min), and high sensitivity with a detection limit of 9.81 nM. Moreover, to explore the practical applicability of our probe we employed it to monitor H2S successfully in environmental water samples, solid-state TLC strip study, Quantitative determination of H2S in eggs, and in the bioimaging of human breast cancer cells (MDA-MB 231).

A 'double locked' ratiometric fluorescent probe for detection of cysteine in a viscous system and its application in cancer cells.

Intracellular viscosity is a physicochemical property that regulates the consequences of several biological progressions. Cysteine (Cys) is an important signaling molecule that commands many cellular activities, such as antioxidant generation. Predicting that both may be interconnected with a diversity of pathological processes, their contemporaneous measurement would be valuable for studying the pathological ailment of cells. Herein, we have synthesized a 'double locked' probe, acrylic acid 6-[4-(2-benzothiazol-2-yl-2-cyano-vinyl)-phenyl]-naphthalen-2-yl ester (ABN) for the detection of Cys in a viscous medium and explored its application to living cells that were exposed to dexamethasone to regulate the intracellular viscosity level. ABN displayed a satisfactory ratiometric (blue to orange) fluorescence response in solution and in living cells when Cys and viscosity coexisted. A turn-on fluorescence signal was visualized when the probe was individually treated with Cys and glycerol (a standard viscosity source). Therefore, we propose that ABN is a fluorescent probe that permits the monitoring of variations in intracellular viscosity and Cys levels in a biological environment, and it can be utilized in innumerable cellular damage models.

Increased host ATP efflux and its conversion to extracellular adenosine is crucial for establishing Leishmania infection.

Intracellular survival of Leishmania donovani demands rapid production of host ATP for its sustenance. However, a gradual decrease in intracellular ATP in spite of increased glycolysis suggests ATP efflux during infection. Accordingly, upon infection, we show here that ATP is exported and the major exporter was pannexin-1, leading to raised extracellular ATP levels. Extracellular ATP shows a gradual decrease after the initial increase, and analysis of cell surface ATP-degrading enzymes revealed induction of the ectonucleotidases CD39 and CD73. Ectonucleotidase-mediated ATP degradation leads to increased extracellular adenosine (eADO), and inhibition of CD39 and CD73 in infected cells decreased adenosine concentration and parasite survival, documenting the importance of adenosine in infection. Inhibiting adenosine uptake by cells did not affect parasite survival, suggesting that eADO exerts its effect through receptor-mediated signalling. We also show that Leishmania induces the expression of adenosine receptors A2AR and A2BR, both of which are important for anti-inflammatory responses. Treating infected BALB/c mice with CD39 and CD73 inhibitors resulted in decreased parasite burden and increased host-favourable cytokine production. Collectively, these observations indicate that infection-induced ATP is exported, and after conversion into adenosine, propagates infection via receptor-mediated signalling.

Spectrophotometric Determination of Trace Amount of Total FeII/FeIII and Live Cell Imaging of a Carboxylato Zn(II) Coordination Polymer.

The coordination polymer, (Zn(II)-CP, 1), {[Zn(2,6-NDC)(4-Cltpy)](H2O)4} (1) (2,6-H2NDC = 2,6-naphthalene dicarboxylic acid and 4-Cltpy = 4'-chloro-[2,2';6',2″]terpyridine) is structurally characterized by single crystal X-ray diffraction measurement and other physicochemical studies (PXRD, FTIR, thermal analysis, microanalytical data). 4-Cltpy acts as end-capping ligand, and NDC2- is a carboxylato bridging motif to constitute ZnN3O2 distorted trigonal bipyramid core that propagates to construct 1D chain. The coordination polymer, 1, detects total iron (Fe3+ and Fe2+) in aqueous solution by visual color change, colorless to pink. Absorption spectrophotometric technique in aqueous medium measures the limit of detection (LOD) 0.11 µM (Fe2+) and 0.15 µM (Fe3+), and binding constants (Kd) are 6.7 × 104 M-1 (Fe3+) and 3.33 × 104 M-1 (Fe2+). Biocompatibility of 1 is examined in live cells, and intracellular Fe2+ and Fe3+ are detected in MDA-MB 231 cells. Zn(II) substitution is assumed upon addition of FeIII/FeII solution to the suspension of the coordination polymer, 1, in water-acetonitrile (41:1) (LZnII + FeIII/II → LFeIII + ZnII, where L is defined as coordinated ligands), which is accompanied by changing from colorless to pink at room temperature. The color of the mixture may be assumed to the charge transfer transition from carboxylate-O to Cltpy via Fe(II/III) bridging center (carboxylate-O-Fe-CltPy). The product isolated from the reaction is finally characterized as Fe(III)@1-CP. It is presumed that product Fe(II)@1-CP may undergo fast aerial oxidation to transform Fe(III)@1-CP. The FeIII exchanged framework (Fe(III)@1-CP) has been characterized by PXRD, IR, TGA and energy dispersive X-ray analysis (EDX)-SEM. The MTT assay calculates the cell viability (%), and the tolerance limit is 100 µM to total Fe2+ and Fe3+.

A benzothiazole-based dual reaction site fluorescent probe for the selective detection of hydrazine in water and live cells.

As hydrazine is an environmental pollutant and highly toxic to living organisms, selective and rapid detection is highly needed for the benefit of living organisms as well as the environment. Here, we first introduced a novel benzothiazole conjugated methyldicyanovinyl coumarin probe BTC, with dual recognition sites for hydrazine detection. The incorporation of the methyldicyanovinyl group into the benzocoumarin fluorophore increased the electrophilicity of the lactone ring of the probe BTC facilitating the nucleophilic attack of hydrazine and rapid (within 1 min, low detection limit = 1.7 nM) turn-on sky blue fluorescence with 700-fold fluorescence intensity enhancement was observed via hydrazine-induced lactone ring-opening followed by selective cleavage of the dicyanovinyl group. According to the literature, dicyanovinyl group assisted lactone ring opening has revealed the possibility of hydrazine recognition with a large Stokes shift (140 nm) and a high fluorescence quantum yield (0.67). Here, the DFT study and practical applications of the probe BTC in different water samples have been presented. The probe BTC was also successfully applied for the detection of hydrazine in the vapor phase using paper strips and in live MDA-MB 231 cells.

A dual-channel chemodosimetric sensor for discrimination between hypochlorite and nerve-agent mimic DCP: application on human breast cancer cells.

A styryl bridge containing a triphenylamine-thioimidazole hydrazine-based dual-analyte-responsive fluorescent sensor was designed and synthesized for the detection of the nerve gas simulant diethyl chlorophosphate (DCP) and hypochlorite (OCl-) for the first time. Hypochlorite induces oxidative intramolecular cyclization to give a triazole structure, which exhibited blue fluorescence with excellent selectivity and a low detection limit (8.05 × 10-7 M) in solution. Conversely, the probe forms a phosphorylated intermediate with diethyl chlorophosphate, which undergoes further hydrolyzation and presents green fluorescence in a ratiometric mode with a low detection limit (3.56 × 10-8 M). Additionally, the as-designed sensor was utilized to construct a portable kit for real-time monitoring of DCP in a discriminatory, simple and safe manner. Lastly, the probe was also productively employed for in situ imaging of OCl- and DCP in the living cell.

Crude protein fraction with high thioredoxin reductase (TrxR) enzyme activity from filarial parasite Setaria cervi counters lipopolysaccharide (LPS)-induced inflammation in macrophages.

Host-parasite interaction has always been an area of interest to the parasite biologists. The complex immune interactions between the parasite and/or the parasite-derived products with the host immune cells determine the fate of the disease biology. Parasitic organisms are widely equipped with a vast array of protective machineries including antioxidant enzymes to withstand the hostile condition inside the host body. The reactive oxygen species (ROS) generated inside the host as a result of parasitic intervention can be endured by the parasite by their own tools to ensure their survival. One such antioxidant enzyme in the filarial parasite that plays a significant role in redox homeostasis, survivability and disease progression is the thioredoxin reductase (TrxR). Herein, we have projected a crude lysate of the bovine filarial parasite Setaria cervi enriched with high TrxR enzyme activity has the capacity to downregulate lipopolysaccharide (LPS)-induced inflammatory macrophages. TrxR-mediated inhibition of the TLR4-NF-κB axis resulting into downregulation of the pro-inflammatory cytokines with concomitant upregulation of the anti-inflammatory cytokines supports the filarial parasite to produce an anti-inflammatory milieu which ultimately promotes worm survivability inside the host and pathogenesis.

Modulatory Effects of Biosynthesized Gold Nanoparticles Conjugated with Curcumin and Paclitaxel on Tumorigenesis and Metastatic Pathways-In Vitro and In Vivo Studies.

BACKGROUND: Breast cancer is the most common cancer in women globally, and diagnosing it early and finding potential drug candidates against multi-drug resistant metastatic breast cancers provide the possibilities of better treatment and extending life. METHODS: The current study aimed to evaluate the synergistic anti-metastatic activity of Curcumin (Cur) and Paclitaxel (Pacli) individually, the combination of Curcumin-Paclitaxel (CP), and also in conjugation with gold nanoparticles (AuNP-Curcumin (Au-C), AuNP-Paclitaxel (Au-P), and AuNP-Curcumin-Paclitaxel (Au-CP)) in various in vitro and in vivo models. RESULTS: The results from combination treatments of CP and Au-CP demonstrated excellent synergistic cytotoxic effects in triple-negative breast cancer cell lines (MDA MB 231 and 4T1) in in vitro and in vivo mouse models. Detailed mechanistic studies were performed that reveal that the anti-cancer effects were associated with the downregulation of the expression of VEGF, CYCLIN-D1, and STAT-3 genes and upregulation of the apoptotic Caspase-9 gene. The group of mice that received CP combination therapy (with and without gold nanoparticles) showed a significant reduction in the size of tumor when compared to the Pacli alone treatment and control groups. CONCLUSIONS: Together, the results suggest that the delivery of gold conjugated Au-CP formulations may help in modulating the outcomes of chemotherapy. The present study is well supported with observations from cell-based assays, molecular and histopathological analyses.

Identification and biochemical characterization of Asp t 36, a new fungal allergen from Aspergillus terreus.

Aspergillus terreus is an allergenic fungus, in addition to causing infections in both humans and plants. However, the allergens in this fungus are still unknown, limiting the development of diagnostic and therapeutic strategies. We used a proteomic approach to search for allergens, identifying 16 allergens based on two-dimensional immunoblotting with A. terreus susceptible patient sera. We further characterized triose-phosphate isomerase (Asp t 36), one of the dominant IgE (IgE)-reactive proteins. The gene was cloned and expressed in Escherichia coli. Phylogenetic analysis showed Asp t 36 to be highly conserved with close similarity to the triose-phosphate isomerase protein sequence from Dermatophagoides farinae, an allergenic dust mite. We identified four immunodominant epitopes using synthetic peptides, and mapped them on a homology-based model of the tertiary structure of Asp t 36. Among these, two were found to create a continuous surface patch on the 3D structure, rendering it an IgE-binding hotspot. Biophysical analysis indicated that Asp t 36 shows similar secondary structure content and temperature sensitivity with other reported triose-phosphate isomerase allergens. In vivo studies using a murine model displayed that the recombinant Asp t 36 was able to stimulate airway inflammation, as demonstrated by an influx of eosinophils, goblet cell hyperplasia, elevated serum Igs, and induction of Th2 cytokines. Collectively, our results reveal the immunogenic property of Asp t 36, a major allergen from A. terreus, and define a new fungal allergen more broadly. This allergen could serve as a potent candidate for investigating component resolved diagnosis and immunotherapy.

Leishmania donovani Targets Host Transcription Factor NRF2 To Activate Antioxidant Enzyme HO-1 and Transcriptional Repressor ATF3 for Establishing Infection.

We showed previously that antioxidant enzyme heme oxygenase 1 (HO-1) is critical for Leishmania survival in visceral leishmaniasis. HO-1 inhibits host oxidative burst and inflammatory cytokine production, leading to parasite persistence. In the present study, screening of reported HO-1 transcription factors revealed that infection upregulated (4.1-fold compared to control [P < 0.001]) nuclear factor erythroid 2 (NFE2)-related factor 2 (NRF2). Silencing of NRF2 reduced both HO-1 expression and parasite survival. Investigation revealed that infection-induced transient reactive oxygen species (ROS) production dissociated NRF2 from its inhibitor KEAP1 and enabled phosphorylation-dependent nuclear translocation. Both NRF2 and HO-1 silencing in infection increased production of proinflammatory cytokines. But the level was greater in NRF2-silenced cells than in HO-1-silenced ones, suggesting the presence of other targets of NRF2. Another stress responsive transcription factor ATF3 is also induced (4.6-fold compared to control [P < 0.001]) by NRF2 during infection. Silencing of ATF3 reduced parasite survival (59.3% decrease compared to control [P < 0.001]) and increased proinflammatory cytokines. Infection-induced ATF3 recruited HDAC1 into the promoter sites of tumor necrosis factor alpha (TNF-α) and interleukin 12b (IL-12b) genes. Resulting deacetylated histones prevented NF-κB promoter binding, thereby reducing transcription of inflammatory cytokines. Administering the NRF2 inhibitor trigonelline hydrochloride to infected BALB/c mice resulted in reduced HO-1 and ATF3 expression, decreased spleen and liver parasite burdens, and increased proinflammatory cytokine levels. These results suggest that Leishmania upregulates NRF2 to activate both HO-1 and ATF3 for disease progression.

Activating transcription factor 3 modulates the macrophage immune response to Mycobacterium tuberculosis infection via reciprocal regulation of inflammatory genes and lipid body formation.

Infection of macrophages by Mycobacterium tuberculosis elicits an immune response that clears the bacterium. However, the bacterium is able to subvert the innate immune response. Differential expression of transcription factors (TFs) is central to the dynamic balance of this interaction. Among other functions, TFs regulate the production of antibacterial agents such as nitric oxide, pro-inflammatory cytokines and neutral lipids which are stored in lipid bodies (LBs) and favour bacterial survival. Here, we demonstrate that the TF activating transcription factor 3 (ATF3) is upregulated early during infection of macrophages or mice. Depletion of ATF3 enhances mycobacterial survival in macrophages suggesting its host-protective role. ATF3 interacts with chromatin remodelling protein brahma-related gene 1 and both associate with the promoters of interleukin-12p40, interleukin-6 and nitric oxide synthase 2, to activate expression of these genes. Strikingly, ATF3 downregulates LB formation by associating at the promoters of positive regulators of LB formation such as cholesterol 25 hydroxylase and the microRNA-33 locus. ATF3 represses the association of the activating mark, acetyl histone H4 lysine 8 at the promoter of cholesterol 25 hydroxylase. Our study suggests opposing roles of ATF3 in regulation of distinct sets of macrophage genes during infection, converging on a host-protective immune response.

Leishmania donovani Exploits Macrophage Heme Oxygenase-1 To Neutralize Oxidative Burst and TLR Signaling-Dependent Host Defense.

Suppression of host oxidative burst is essential for survival of the intracellular parasite Leishmania donovani Screening of macrophage antioxidant enzymes during infection revealed marked upregulation of the heme-degrading enzyme, heme oxygenase-1 (HO-1). Moreover, HO-1-silenced RAW macrophages depicted increased superoxide production and decreased parasite survival. HO-1 induction decreased cellular heme content, thereby inhibiting the heme-dependent maturation of gp91phox, a catalytic component of major reactive oxygen species-producing enzyme NAD(P)H oxidase. Decreased gp91phox expression resulted in reduced stability of p22phox, another component of the catalytic center of NAD(P)H oxidase. Replenishing infected cells with exogenous heme reversed these effects and restored NAD(P)H oxidase activity. Persistent HO-1 expression at late hour of infection prompted us to investigate its effect on other host defense parameters, and inhibition study revealed a reciprocal relationship of HO-1 with host proinflammatory responses. Among all the HO-1-mediated heme degradation products (CO, Fe, and biliverdin), only CO documented potent anti-inflammatory effects. Quenching of CO during infection increased the production of disease-resolving cytokines IL-12 and TNF-α. Coimmunoprecipitation experiments revealed that CO inhibited the interaction of TLR4 with MyD88 and TIR domain-containing adapter-inducing IFN-ß, thereby dampening the activation of NF-κB and IFN regulatory factor 3-mediated production of proinflammatory cytokines. Administration of HO-1 inhibitor tin protoporphyrin IX dichloride in infected BALB/c mice led to a decrease in liver and spleen parasite burden along with increased production of IL-12 and TNF-α. These results suggest that HO-1 on one hand inhibits reactive oxygen species generation and on the other hand downregulates host favorable cytokine responses, thereby facilitating intramacrophage parasite survival.

The sensor kinase MtrB of Mycobacterium tuberculosis regulates hypoxic survival and establishment of infection.

Paired two-component systems (TCSs), having a sensor kinase (SK) and a cognate response regulator (RR), enable the human pathogen Mycobacterium tuberculosis to respond to the external environment and to persist within its host. Here, we inactivated the SK gene of the TCS MtrAB, mtrB, generating the strain ΔmtrB We show that mtrB loss reduces the bacterium's ability to survive in macrophages and increases its association with autophagosomes and autolysosomes. Notably, the ΔmtrB strain was markedly defective in establishing lung infection in mice, with no detectable lung pathology following aerosol challenge. ΔmtrB was less able to withstand hypoxic and acid stresses and to form biofilms and had decreased viability under hypoxia. Transcriptional profiling of ΔmtrB by gene microarray analysis, validated by quantitative RT-PCR, indicated down-regulation of the hypoxia-associated dosR regulon, as well as genes associated with other pathways linked to adaptation of M. tuberculosis to the host environment. Using in vitro biochemical assays, we demonstrate that MtrB interacts with DosR (a noncognate RR) in a phosphorylation-independent manner. Electrophoretic mobility shift assays revealed that MtrB enhances the binding of DosR to the hspX promoter, suggesting an unexpected role of MtrB in DosR-regulated gene expression in M. tuberculosis Taken together, these findings indicate that MtrB functions as a regulator of DosR-dependent gene expression and in the adaptation of M. tuberculosis to hypoxia and the host environment. We propose that MtrB may be exploited as a chemotherapeutic target against tuberculosis.

Anti-cancer potential of (1,2-dihydronaphtho[2,1-b]furan-2-yl)methanone derivatives.

A series of 1,2-dihydronaphtho[2,1-b]furan derivatives were synthesized by cyclizing 1-(aryl/alkyl(arylthio)methyl)-naphthalen-2-ol and pyridinium bromides in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in very good yield. The synthesized compounds were evaluated for their anti-proliferative potential against human triple negative MDA-MB-468 and MCF-7 breast cancer cells and non-cancerous WI-38 cells (lung fibroblast cell) using MTT experiments. Among 21 synthesized compounds, three compounds (3a, 3b and 3 s) showed promising anti-cancer potential and compound 3b was found to have best anti-proliferative activities based on the results of several biochemical and microscopic experiments.