Clonal hematopoiesis driven by mutated DNMT3A promotes inflammatory bone loss.

Clonal hematopoiesis of indeterminate potential (CHIP) arises from aging-associated acquired mutations in hematopoietic progenitors, which display clonal expansion and produce phenotypically altered leukocytes. We associated CHIP-DNMT3A mutations with a higher prevalence of periodontitis and gingival inflammation among 4,946 community-dwelling adults. To model DNMT3A-driven CHIP, we used mice with the heterozygous loss-of-function mutation R878H, equivalent to the human hotspot mutation R882H. Partial transplantation with Dnmt3aR878H/+ bone marrow (BM) cells resulted in clonal expansion of mutant cells into both myeloid and lymphoid lineages and an elevated abundance of osteoclast precursors in the BM and osteoclastogenic macrophages in the periphery. DNMT3A-driven clonal hematopoiesis in recipient mice promoted naturally occurring periodontitis and aggravated experimentally induced periodontitis and arthritis, associated with enhanced osteoclastogenesis, IL-17-dependent inflammation and neutrophil responses, and impaired regulatory T cell immunosuppressive activity. DNMT3A-driven clonal hematopoiesis and, subsequently, periodontitis were suppressed by rapamycin treatment. DNMT3A-driven CHIP represents a treatable state of maladaptive hematopoiesis promoting inflammatory bone loss.

Maladaptive innate immune training of myelopoiesis links inflammatory comorbidities.

Bone marrow (BM)-mediated trained innate immunity (TII) is a state of heightened immune responsiveness of hematopoietic stem and progenitor cells (HSPC) and their myeloid progeny. We show here that maladaptive BM-mediated TII underlies inflammatory comorbidities, as exemplified by the periodontitis-arthritis axis. Experimental-periodontitis-related systemic inflammation in mice induced epigenetic rewiring of HSPC and led to sustained enhancement of production of myeloid cells with increased inflammatory preparedness. The periodontitis-induced trained phenotype was transmissible by BM transplantation to naive recipients, which exhibited increased inflammatory responsiveness and disease severity when subjected to inflammatory arthritis. IL-1 signaling in HSPC was essential for their maladaptive training by periodontitis. Therefore, maladaptive innate immune training of myelopoiesis underlies inflammatory comorbidities and may be pharmacologically targeted to treat them via a holistic approach.

Novel Agents against Miltefosine-Unresponsive Leishmania donovani.

Visceral leishmaniasis is a deadly endemic disease. Unresponsiveness to the only available oral drug miltefosine poses a big challenge for the chemotherapy of the disease. We report a novel molecule, PS-203 {4-(4,4,8-trimethyl-7-oxo-3-oxabicyclo[3.3.1]non-2-yl)-benzoic acid methyl ester}, as effective against a miltefosine-unresponsive strain of the parasite. Further, combinations of PS-203 with miltefosine were also evaluated and showed promising results against a miltefosine-unresponsive strain.

In Vitro Assay for the Assessment of Oxygen Depletion Triggers in Human Cell Lines, Associated with Improving Responses to Cancer Therapy.

Tumors are usually associated with oxygen-deficient regions (hypoxia) which results from reduced and disorganized intratumoral vasculature, increased diffusion distances, and growing tumor masses. The proteomic and metabolomic landscape of the hypoxic cells is reprogrammed through hypoxia-induced transcription factor 1 which is activated in hypoxic conditions and is inactive when oxygen is abundant. This transcription factor has also been shown to inhibit or even reverse cell differentiation. Hypoxia impedes chemotherapy as it hampers the formation of cytotoxic free radicals due to the lesser availability of molecular oxygen. The metastatic and invasive attributes of cancer cells in hypoxic conditions are exacerbated, which results in poor therapeutic outcomes. Various cell-based assays for measuring hypoxia have been developed which give an estimate of the hypoxic state of cancer cells. Prior knowledge of these assays will improve the efficacy of the treatment regimens for cancers. This article provides exhaustive information on the hypoxia-based assays which are sensitive, robust, reliable, and give easy readout with choice of cell type for these assays may be dictated by the procedural or endpoint selection.

Gene Alterations Induced by Glutamine (Q) Encoding CAG Repeats Associated with Neurodegeneration.

Several studies have been reported linking the role of polyglutamine (polyQ) disease-associated proteins with altered gene regulation induced by an unstable trinucleotide (CAG) repeat. Owing to their dynamic nature of expansion, these DNA repeats form secondary structures interfering with the normal cellular mechanisms like replication and transcription and, thereby, have become the underlying cause of numerous neurodegenerative disorders involving mental retardation and/or muscular or neuronal degeneration. Despite the widespread expression of the disease-causing protein, specific subsets of neurons are susceptible to specific patterns of inheritance and clinical symptoms. Although this cell-type selectivity is still elusive and less understood, it has been found that aberrant transcriptional regulation is one of the primary causes of polyQ diseases where the functions of histone-modifying complexes are disrupted. Besides, epigenetic modifications play a critical role in the pathogenesis of these diseases. In this chapter, we will be delving into how these polyQ repeats induce the self-assembly and aggregation of altered carrier proteins based on gene alterations, causing neuronal toxicity and cellular deaths. Besides, genomic instability in CAG repeats due to altered chromatin-related enzymes will be highlighted, along with epigenetic changes present in many polyQ disorders. Understanding the underlying molecular mechanisms in the root cause of these disorders will culminate in identifying therapeutic approaches for the treatment of these neurodegenerative disorders.

Mutational studies on Leishmania donovani dihydrolipoamide dehydrogenase (LdBPK291950.1) indicates that the enzyme may not be classical class-I pyridine nucleotide-disulfide oxidoreductase.

We report biochemical studies on two Cys residues mutation (Cys15Thr, Cys38Gly) nearest to the active site and three other amino acid substitution mutations expected to be the part of active site of LdDLDH_Variant1. Our biochemical studies show that the replacement of Cys15 increases the Km for dihydrolipoamide (DLD) substrate by five folds and NAD+ by three fold indicating that this mutation affects the binding of DLD and NAD+ significantly. Cys38 was also mutated to 'Gly' which resulted in nine fold greater Km for NAD+ without affecting Km for DLD. However, even after these mutations (Cys15Thr and Cys38Gly), reduced enzyme activity suggests that both the 'Cys' residues are not involved in disulfide bond formation but affect the binding of substrates. The data hints towards the possibility of a different catalytic mechanism from the classical class I - pyridine nucleotide-disulfide oxidoreductase. Remaining other mutated residues Ala48Ile, Asp49Gly, and Ala54Ile showed an increase in two to three-folds Km value for NAD+, which means these residues are important for the binding of NAD+ to the enzyme. However, Ala48Ile and Asp49Gly mutations showed a decrease of Km for DLD. Apart from the mutational studies, localization of LdDLDH_Variant2 of LdDLDH was also analyzed.

BLIMP-1 Mediated Downregulation of TAK1 and p53 Molecules Is Crucial in the Pathogenesis of Kala-Azar.

Precise regulation of inflammasome is critical during any pathogenic encounter. The whole innate immune system comprising of pattern recognition receptors (PRRs) relies on its ability to sense microbes. The fate of cellular death in infected cells depends mostly on the activation of these inflammasome, the dysregulation of which, due to functional manipulation by various pathogens, leads to be the cause of many human diseases. Here, an interesting finding has been observed which is related to how Leishmania donovani parasites exploit various host mediator molecules to cause immunosuppression. Here we report for the first time that the parasites check pyroptosis in the infected cells in-vitro by BLIMP-1 mediated suppression of TAK1 and p53 proteins. This might be one of the reasons how parasites evade the pro-inflammatory response of the host cells. Further understandings and validations are required to come up with better therapeutic approaches against kala-azar.

Biochemical characterization of a stable azoreductase enzyme from Chromobacterium violaceum: Application in industrial effluent dye degradation.

The presence of dye, including azo functional group (NN) containing dyes, in industrial waste water is one of the major causes of water pollution. This report showcases the functional role of azoreductase from Chromobacterium violaceum (MTCC No: 2656) as a valuable enzyme for degradation of azo dyes. The enzyme was cloned, expressed, purified and biochemically characterized and further tested for degradation efficiency of azo group containing dyes like methyl red, amaranth and methyl orange. The degraded azo dye products (metabolites) resulted by the action of azoreductase enzyme had reduced toxicity on fibroblast cell lines (L929) as compared to raw and intact dye. Further, good stability of the enzyme makes it more suitable for various applications related to the degradation and decolourisation of effluent dyes.

Leishmania donovani evades Caspase 1 dependent host defense mechanism during infection.

Significant advances have been made in understanding the regulation of inflammasomes and its involvement in innate immunity during pathogenic infections. Inflammasome activation is a tightly regulated process that provides defense against pathogenic infection and important for inflammatory response. Very few studies on the involvement of NLRP3 inflammasome protein complex have been reported in leishmanial infections with contradictory results and without much mechanistic insights. However, the role of NLRP3 inflammasome and its components has not been well deciphered in Leishmania donovani infection. Here we report for the first time a detailed mechanism and plausible impairment of caspase 1 activation during L. donovani infection leading to the survival of these parasites inside the host cells. Low mRNA expression of pro-caspase 1 and lack of caspase 1 maturation were observed after infection, hindering the processing of pro-IL-1ß and pro-IL-18 into their mature counter parts. Further, siRNA mediated knock-down of caspase 1 in macrophage cells (THP-1) resulted in significantly higher parasitic burden validating the importance of caspase 1 in the host defense mechanism. Taken together, our data suggests that the parasite inhibits caspase 1 activation to evade the inflammatory nature of pyroptosis.

BLIMP-1 Plays Important Role in the Regulation of Macrophage Pyroptosis for the Growth and Multiplication of Leishmania donovani.

Visceral leishmaniasis, one of the fatal forms of the disease, is caused by Leishmania donovani and presents morbid clinical manifestations. The parasite evades pro-inflammatory immune responses by several reported mechanisms and modulates the host immune system to cause fatal symptoms. A plethora of reports related to the role of BLIMP-1 and its involvement in suppressing the immune response in various infectious diseases have been documented. Higher parasitic burden due to increased BLIMP-1 production has been reported earlier for malaria and leishmaniasis with no detailed information. We report for the first time the role of BLIMP-1 in suppressing macrophage pyroptosis during L. donovani infection and thereby tweaking the tight regulation of the NFκß-NLRP3 signaling pathway. Expression analyses of BLIMP-1 and NFκß have been measured using real-time PCR and Western blotting. The importance of BLIMP-1 has been validated using a siRNA-mediated experiment along with caspase 1 activity, LDH release assay, and infectivity index analyses. An inverse relationship between BLIMP-1 and NFκß expression has been highlighted during L. donovani infection, which is reversed in blimp-1 deficient cells infected with promastigotes. The above fact has been further validated with caspase 1 activity assay, and LDH release along with IFNγ and TNF-α release assay. Finally, resumption of pyroptosis has been concluded in infected blimp-1 deficient cells in contrast to wild type infected cells. We conjecture that parasites modulate the NFκß-NLRP3 signaling pathway by taking advantage of BLIMP-1 dependent IL-10 production and finally disrupting an inflammation-mediated pyroptosis cell death pathway in infected cells.

Biochemical characterization and chemical validation of Leishmania MAP Kinase-3 as a potential drug target.

Protozoan parasites of the Leishmania genus have evolved unique signaling pathways that can sense various environmental changes and trigger stage differentiation for survival and host infectivity. MAP kinase (MAPK) plays a critical role in various cellular activities like cell differentiation, proliferation, stress regulation, and apoptosis. The Leishmania donovani MAPK3 (LdMAPK3) is involved in the regulation of flagella length and hence plays an important role in disease transmission. Here, we reported the gene cloning, protein expression, biochemical characterizations, inhibition studies and cell proliferation assay of LdMAPK3. The recombinant purified LdMAPK3 enzyme obeys the Michaelis-Menten equation with Km and Vmax of LdMAPK3 was found to be 20.23 nM and 38.77 ± 0.71 nmoles ATP consumed/mg LdMAPK3/min respectively. The maximum kinase activity of LdMAPK3 was recorded at 35 °C and pH 7. The in-vitro inhibition studies with two natural inhibitors genistein (GEN) and chrysin (CHY) was evaluated against LdMAPK3. The Ki value for GEN and CHY were found to be 3.76 ± 0.28 µM and Ki = 8.75 ± 0.11 µM respectively. The IC50 value for the compounds, GEN and CHY against L. donovani promastigotes were calculated as 9.9 µg/mL and 13 µg/mL respectively. Our study, therefore, reports LdMAPK3 as a new target for therapeutic approach against leishmaniasis.

Episomal expression of human glutathione reductase (HuGR) in Leishmania sheds light on evolutionary pressure for unique redox metabolism pathway: Impaired stress tolerance ability of Leishmania donovani.

Trypanothione based redox metabolism is unique to the Trypanosomatida family. Despite extensive studies on redox metabolism of Leishmania parasites, a prominent question of why Leishmania adopt this unique redox pathway remains elusive. We have episomally expressed human glutathione reductase (HuGR) in Leishmania donovani (LdGR+) and investigated its effect. LdGR+ strain has slower growth compared to the wild type (Ld) indicating decreased survival ability of the strain. Further, LdGR+ strain showed enhanced accumulation of intracellular reactive oxygen species (ROS) and more sensitivity to the anti-leishmanial drug, Miltefosine, inferring increased stress level. In contrast, the expression analyses of genes specific to redox metabolism were increased significantly in LdGR+ strain compared to wild type. Lower infectivity index of the LdGR+ strain substantiated the above findings and indicated that the expression of HuGR reduces the stress tolerance ability of the parasite. From molecular docking studies with HuGR, it was observed that oxidized trypanothione (TS2) binds much better than oxidized glutathione (GS2). These results also give us hints that the parasite is losing infectivity potential due to an overall increase in intracellular stress caused with the expression of HuGR, showcasing a possible role of evolutionary pressure on the Leishmania parasites posed by HuGR.

Apoptosis: Mediator Molecules, Interplay with Other Cell Death Processes and Therapeutic Potentials.

Apoptosis, a form of programmed cell death, plays a very crucial role in various physiological processes for maintaining cell homeostasis. This process has several characteristic features like membrane blebbing, nuclear condensation, DNA fragmentation and cell shrinkage. Any defect in this highly regulated process eventually leads to extended cell survival and could result in neoplastic cell expansion followed by genetic instability. The apoptotic machinery is mainly processed and regulated by various caspases, a family of cysteine proteases. Significant advancement has been made towards understanding the molecular mechanisms of apoptosis which provides new insights in modulating the life or death of a cell. The main goal of this review is to highlight recent updates on apoptosis, the cross-talk with other cellular death processes and its therapeutic potentials.