Expression of Plasmodium major facilitator superfamily protein in transporters - Δ Candida identifies a drug transporter.

Aim: To assess the functional relevance of a putative Major Facilitator Superfamily protein (PF3D7_0210300; 'PfMFSDT') as a drug transporter, using Candida glabrata for orthologous protein expression.Methods: Complementary Determining Sequence encoding PfMFSDT was integrated into the genome of genetically engineered C. glabrata strain MSY8 via homologous recombination, followed by assessing its functional relevance as a drug transporter.Results & conclusion: The modified C. glabrata strain exhibited plasma membrane localization of PfMFSDT and characteristics of an Major Facilitator Superfamily transporter, conferring resistance to antifungals, ketoconazole and itraconazole. The nanomolar inhibitory effects of the drugs on the intra-erythrocytic growth of Plasmodium falciparum highlight their antimalarial properties. This study proposes PfMFSDT as a drug transporter, expanding the repertoire of the currently known antimalarial 'resistome'.

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Long-lived lung megakaryocytes contribute to platelet recovery in thrombocytopenia models.

Lung megakaryocytes (Mks) are largely extravascular with an immune phenotype (1). Because bone marrow (BM) Mks are short-lived it has been assumed that extravascular lung Mks are constantly 'seeded' from the BM. To investigate lung Mk origins and how that impacts their functions, we developed methods to specifically label lung Mks using CFSE dye and biotin delivered oropharyngeal. Labeled lung Mks were present for up to four months, while BM Mks had a <1 week lifespan. In a parabiosis model, lung Mks were partially replaced over 1-month from a circulating source. Unlike tissue-resident macrophages, using MDS1-Cre-ERT2 TdTomato mice, we found that lung Mks arise from hematopoietic stem cells. However, studies with FlkSwitch mTmG mice showed that lung Mks are derived from a Flt3-independent lineage that does not go through a multipotent progenitor. CFSE labeling to track lung Mk-derived platelets showed that about 10% of circulating platelets are lung resident Mk-derived at steady state, but in sterile thrombocytopenia this was doubled (about 20%). Lung-derived platelets were similarly increased in a malaria infection model (Plasmodium yoelii) typified by thrombocytopenia. These studies indicate that lung Mks arise from a Flt3-negative BM source, are long-lived, and contribute more platelets during thrombocytopenia.

Platelet Ido1 expression is induced during Plasmodium yoelii infection, altering plasma tryptophan metabolites.

Platelets are immune responsive in many diseases as noted by changes in platelet mRNA in conditions such as sepsis1, atherosclerosis2, COVID-193,4, and many other inflammatory and infectious etiologies5. The malaria causing Plasmodium parasite is a persistent public health threat and significant evidence shows that platelets participate in host responses to infection. Using a mouse model of non-lethal/uncomplicated malaria, P. yoelii XNL (PyNL), infected, but not control mouse platelets expressed Ido1, a rate limiting enzyme in tryptophan metabolism that increases kynurenine at the expense of serotonin. Interferon-gamma (IFN) is a potent inducer of Ido1 and mice treated with recombinant IFN had increased platelet Ido1 and IDO1 activity. PyNL infected mice treated with anti-IFN antibody had similar platelet Ido1 and metabolic profiles to that of uninfected controls. PyNL infected mice become thrombocytopenic by day 7 post-infection and transfusion of platelets from IFN treated wild type mice, but not Ido1-/- mice, increased the plasma kynurenine to tryptophan ratio, indicating platelets are a source of post-infection IDO1 activity. We generated platelet specific Ido1 knockout mice to assess the contribution of platelet Ido1 during PyNL infection. Platelet specific Ido1-/- mice had increased death and evidence of lung thrombi which were not present in infected WT mice. Platelet Ido1 may be a significant contributor to plasma KYN in IFN driven immune processes and the loss of platelets may limit total Ido1, leading to immune and vascular dysfunction.

Prefoldins are novel regulators of the unfolded protein response in artemisinin resistant Plasmodium falciparum malaria.

Emerging Artemisinin (ART) resistance in Plasmodium falciparum (Pf) poses challenges for the discovery of novel drugs to tackle ART-resistant parasites. Concentrated efforts toward the ART resistance mechanism indicated a strong molecular link of ART resistance with upregulated expression of unfolded protein response pathways involving Prefoldins (PFDs). However, a complete characterization of PFDs as molecular players taking part in ART resistance mechanism, and discovery of small molecule inhibitors to block this process have not been identified to date. Here, we functionally characterized all Pf Prefoldin subunits (PFD1-6) and established a causative role played by PFDs in ART resistance by demonstrating their expression in intra-erythrocytic parasites along with their interactions with Kelch13 protein through immunoprecipitation coupled MS/MS analysis. Systematic biophysical interaction analysis between all subunits of PFDs revealed their potential to form a complex. The role of PFDs in ART resistance was confirmed in orthologous yeast PFD6 mutants, where PfPFD6 expression in yeast mutants reverted phenotype to ART resistance. We identified an FDA-approved drug "Biperiden" that restricts the formation of Prefoldin complex and inhibits its interaction with its key parasite protein substrates, MSP-1 and α-tubulin-I. Moreover, Biperiden treatment inhibits the parasite growth in ART-sensitive Pf3D7 and resistant Pf3D7k13R539T strains. Ring survival assays that are clinically relevant to analyze ART resistance in Pf3D7k13R539T parasites demonstrate the potency of BPD to inhibit the growth of survivor parasites. Overall, our study provides the first evidence of the role of PfPFDs in ART resistance mechanisms and opens new avenues for the management of resistant parasites.

Targeting PfProhibitin 2-Hu-Hsp70A1A complex as a unique approach towards malaria vaccine development.

Malaria parasite invasion to host erythrocytes is mediated by multiple interactions between merozoite ligands and erythrocyte receptors that contribute toward the development of disease pathology. Here, we report a novel antigen Plasmodium prohibitin "PfPHB2" and identify its cognate partner "Hsp70A1A" in host erythrocyte that plays a crucial role in mediating host-parasite interaction during merozoite invasion. Using small interfering RNA (siRNA)- and glucosamine-6-phosphate riboswitch (glmS) ribozyme-mediated approach, we show that loss of Hsp70A1A in red blood cells (RBCs) or PfPHB2 in infected red blood cells (iRBCs), respectively, inhibit PfPHB2-Hsp70A1A interaction leading to invasion inhibition. Antibodies targeting PfPHB2 and monoclonal antibody therapeutics against Hsp70A1A efficiently block parasite invasion. Recombinant PfPHB2 binds to RBCs which is inhibited by anti-PfPHB2 antibody and monoclonal antibody against Hsp70A1A. The validation of PfPHB2 to serve as antigen is further supported by detection of anti-PfPHB2 antibody in patient sera. Overall, this study proposes PfPHB2 as vaccine candidate and highlights the use of monoclonal antibody therapeutics for future malaria treatment.

Thrombocytopenia Independently Leads to Changes in Monocyte Immune Function.

BACKGROUND: While platelets have well-studied hemostatic functions, platelets are immune cells that circulate at the interface between the vascular wall and white blood cells. The physiological implications of these constant transient interactions are poorly understood. Activated platelets induce and amplify immune responses, but platelets may also maintain immune homeostasis in healthy conditions, including maintaining vascular integrity and T helper cell differentiation, meaning that platelets are central to both immune responses and immune quiescence. Clinical data have shown an association between low platelet counts (thrombocytopenia) and immune dysfunction in patients with sepsis and extracorporeal membrane oxygenation, further implicating platelets as more holistic immune regulators, but studies of platelet immune functions in nondisease contexts have had limited study. METHODS: We used in vivo models of thrombocytopenia and in vitro models of platelet and monocyte interactions, as well as RNA-seq and ATAC-seq (assay for transposase-accessible chromatin with sequencing), to mechanistically determine how resting platelet and monocyte interactions immune program monocytes. RESULTS: Circulating platelets and monocytes interact in a CD47-dependent manner to regulate monocyte metabolism, histone methylation, and gene expression. Resting platelet-monocyte interactions limit TLR (toll-like receptor) signaling responses in healthy conditions in an innate immune training-like manner. In both human patients with sepsis and mouse sepsis models, thrombocytopenia exacerbated monocyte immune dysfunction, including increased cytokine production. CONCLUSIONS: Thrombocytopenia immune programs monocytes in a manner that may lead to immune dysfunction in the context of sepsis. This is the first demonstration that sterile, endogenous cell interactions between resting platelets and monocytes regulate monocyte metabolism and pathogen responses, demonstrating platelets to be immune rheostats in both health and disease.

Tackling the emerging Artemisinin-resistant malaria parasite by modulation of defensive oxido-reductive mechanism via nitrofurantoin repurposing.

Oxidative stress-mediated cell death has remained the prime parasiticidal mechanism of front line antimalarial, artemisinin (ART). The emergence of resistant Plasmodium parasites characterized by oxidative stress management due to impaired activation of ART and enhanced reactive oxygen species (ROS) detoxification has decreased its clinical efficacy. This gap can be filled by development of alternative chemotherapeutic agents to combat resistance defense mechanism. Interestingly, repositioning of clinically approved drugs presents an emerging approach for expediting antimalarial drug development and circumventing resistance. Herein, we evaluated the antimalarial potential of nitrofurantoin (NTF), a clinically used antibacterial drug, against intra-erythrocytic stages of ART-sensitive (Pf3D7) and resistant (PfKelch13R539T) strains of P. falciparum, alone and in combination with ART. NTF exhibited growth inhibitory effect at submicro-molar concentration by arresting parasite growth at trophozoite stage. It also inhibited the survival of resistant parasites as revealed by ring survival assay. Concomitantly, in vitro combination assay revealed synergistic association of NTF with ART. NTF was found to enhance the reactive oxygen and nitrogen species, and induced mitochondrial membrane depolarization in parasite. Furthermore, we found that exposure of parasites to NTF disrupted redox balance by impeding Glutathione Reductase activity, which manifests in enhanced oxidative stress, inducing parasite death. In vivo administration of NTF, alone and in combination with ART, in P. berghei ANKA-infected mice blocked parasite multiplication and enhanced mean survival time. Overall, our results indicate NTF as a promising repurposable drug with therapeutic potential against ART-sensitive as well as resistant parasites.

Targeting an essential Plasmodium cold shock protein to block growth and transmission of malaria parasite.

Cold shock proteins are characterized by the presence of one or more cold shock domains that bestow them with nucleic acid binding ability. Although cold shock proteins are well characterized in bacteria, plants and humans, there is no information on their existence and role in malaria parasite. Here, we have identified and delineated the function of a cold shock protein of Plasmodium falciparum (Pf) 'PfCoSP'. We demonstrate that PfCoSP exhibits nucleic acid binding properties and regulates gene expression. PfCoSP promotes microtubule assembly by interacting with Pf α/ß tubulin. We identified a human cold shock protein LIN28A inhibitor 'LI71' as a binding partner of PfCoSP which inhibited PfCoSP-DNA and α/ß tubulin interactions and, also inhibited the development of asexual blood stages and gametocyte stage of malaria parasite. Because PfCoSP is essential for parasite survival, characterization of its interacting partners may form the basis for development of future anti-malarials.

Thrombocytopenia Independently Leads to Monocyte Immune Dysfunction.

In addition to their well-studied hemostatic functions, platelets are immune cells. Platelets circulate at the interface between the vascular wall and leukocytes, and transient platelet-leukocyte complexes are found in both healthy and disease states, positioning platelets to provide physiologic cues of vascular health and injury. Roles for activated platelets in inducing and amplifying immune responses have received an increasing amount of research attention, but our past studies also showed that normal platelet counts are needed in healthy conditions to maintain immune homeostasis. We have now found that thrombocytopenia (a low platelet count) leads to monocyte dysfunction, independent of the cause of thrombocytopenia, in a manner that is dependent on direct platelet-monocyte CD47 interactions that regulate monocyte immunometabolism and gene expression. Compared to monocytes from mice with normal platelet counts, monocytes from thrombocytopenic mice had increased toll-like receptor (TLR) responses, including increased IL-6 production. Furthermore, ex vivo co-incubation of resting platelets with platelet naïve bone marrow monocytes, induced monocyte metabolic programming and durable changes in TLR agonist responses. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq) on monocytes from thrombocytopenic mice showed persistently open chromatin at LPS response genes and resting platelet interactions with monocytes induced histone methylation in a CD47 dependent manner. Using mouse models of thrombocytopenia and sepsis, normal platelet numbers were needed to limit monocyte immune dysregulation and IL6 expression in monocytes from human patients with sepsis also inversely correlated with patient platelet counts. Our studies demonstrate that in healthy conditions, resting platelets maintain monocyte immune tolerance by regulating monocyte immunometabolic processes that lead to epigenetic changes in TLR-related genes. This is also the first demonstration of sterile cell interactions that regulate of innate immune-metabolism and monocyte pathogen responses.

Transfusion of Adult, but Not Neonatal, Platelets Promotes Monocyte Trafficking in Neonatal Mice.

BACKGROUND: Thrombocytopenia is common in preterm neonates. Platelet transfusions are sometimes given to thrombocytopenic neonates with the hope of reducing the bleeding risk, however, there are little clinical data to support this practice, and platelet transfusions may increase the bleeding risk or lead to adverse complications. Our group previously reported that fetal platelets expressed lower levels of immune-related mRNA compared with adult platelets. In this study, we focused on the effects of adult versus neonatal platelets on monocyte immune functions that may have an impact on neonatal immune function and transfusion complications. METHODS: Using RNA sequencing of postnatal day 7 and adult platelets, we determined age-dependent platelet gene expression. Platelets and naive bone marrow-isolated monocytes were cocultured and monocyte phenotypes determined by RNA sequencing and flow cytometry. An in vivo model of platelet transfusion in neonatal thrombocytopenic mice was used in which platelet-deficient TPOR (thrombopoietin receptor) mutant mice were transfused with adult or postnatal day 7 platelets and monocyte phenotypes and trafficking were determined. RESULTS: Adult and neonatal platelets had differential immune molecule expression, including Selp. Monocytes incubated with adult or neonatal mouse platelets had similar inflammatory (Ly6Chi) but different trafficking phenotypes, as defined by CCR2 and CCR5 mRNA and surface expression. Blocking P-sel (P-selectin) interactions with its PSGL-1 (P-sel glycoprotein ligand-1) receptor on monocytes limited the adult platelet-induced monocyte trafficking phenotype, as well as adult platelet-induced monocyte migration in vitro. Similar results were seen in vivo, when thrombocytopenic neonatal mice were transfused with adult or postnatal day 7 platelets; adult platelets increased monocyte CCR2 and CCR5, as well as monocyte chemokine migration, whereas postnatal day 7 platelets did not. CONCLUSIONS: These data provide comparative insights into adult and neonatal platelet transfusion-regulated monocyte functions. The transfusion of adult platelets to neonatal mice was associated with an acute inflammatory and trafficking monocyte phenotype that was platelet P-sel dependent and may have an impact on complications associated with neonatal platelet transfusions.

Post-Translation Modifications and Mutations of Human Linker Histone Subtypes: Their Manifestation in Disease.

Linker histones (LH) are a critical component of chromatin in addition to the canonical histones (H2A, H2B, H3, and H4). In humans, 11 subtypes (7 somatic and 4 germinal) of linker histones have been identified, and their diverse cellular functions in chromatin structure, DNA replication, DNA repair, transcription, and apoptosis have been explored, especially for the somatic subtypes. Delineating the unique role of human linker histone (hLH) and their subtypes is highly tedious given their high homology and overlapping expression patterns. However, recent advancements in mass spectrometry combined with HPLC have helped in identifying the post-translational modifications (PTMs) found on the different LH subtypes. However, while a number of PTMs have been identified and their potential nuclear and non-nuclear functions explored in cellular processes, there are very few studies delineating the direct relevance of these PTMs in diseases. In addition, recent whole-genome sequencing of clinical samples from cancer patients and individuals afflicted with Rahman syndrome have identified high-frequency mutations and therefore broadened the perspective of the linker histone mutations in diseases. In this review, we compile the identified PTMs of hLH subtypes, current knowledge of the relevance of hLH PTMs in human diseases, and the correlation of PTMs coinciding with mutations mapped in diseases.

Complementary crosstalk between palmitoylation and phosphorylation events in MTIP regulates its role during Plasmodium falciparum invasion.

Post-translational modifications (PTMs) including phosphorylation and palmitoylation have emerged as crucial biomolecular events that govern many cellular processes including functioning of motility- and invasion-associated proteins during Plasmodium falciparum invasion. However, no study has ever focused on understanding the possibility of a crosstalk between these two molecular events and its direct impact on preinvasion- and invasion-associated protein-protein interaction (PPI) network-based molecular machinery. Here, we used an integrated in silico analysis to enrich two different catalogues of proteins: (i) the first group defines the cumulative pool of phosphorylated and palmitoylated proteins, and (ii) the second group represents a common set of proteins predicted to have both phosphorylation and palmitoylation. Subsequent PPI analysis identified an important protein cluster comprising myosin A tail interacting protein (MTIP) as one of the hub proteins of the glideosome motor complex in P. falciparum, predicted to have dual modification with the possibility of a crosstalk between the same. Our findings suggested that blocking palmitoylation led to reduced phosphorylation and blocking phosphorylation led to abrogated palmitoylation of MTIP. As a result of the crosstalk between these biomolecular events, MTIP's interaction with myosin A was found to be abrogated. Next, the crosstalk between phosphorylation and palmitoylation was confirmed at a global proteome level by click chemistry and the phenotypic effect of this crosstalk was observed via synergistic inhibition in P. falciparum invasion using checkerboard assay and isobologram method. Overall, our findings revealed, for the first time, an interdependence between two PTM types, their possible crosstalk, and its direct impact on MTIP-mediated invasion via glideosome assembly protein myosin A in P. falciparum. These insights can be exploited for futuristic drug discovery platforms targeting parasite molecular machinery for developing novel antimalarial therapeutics.

Dynamic Palmitoylation of Red Cell Membrane Proteins Governs Susceptibility to Invasion by the Malaria Parasite, Plasmodium falciparum.

Phosphorylation and other post-translational modifications of red blood cell (RBC) proteins govern membrane function and have a role in the invasion of RBCs by the malaria parasite, Plasmodium falciparum. Furthermore, a percentage of RBC proteins are palmitoylated, although the functional consequences are unknown. We establish dynamic palmitoylation of 118 RBC membrane proteins using click chemistry and acyl biotin exchange (ABE)-coupled LC-MS/MS and characterize their involvement in controlling membrane organization and parasite invasion. RBCs were treated with a generic palmitoylation inhibitor, 2-bromopalmitate (2-BMP), and then analyzed using ABE-coupled LC-MS/MS. Only 42 of the 118 palmitoylated proteins detected were palmitoylated in the 2-BMP-treated sample, indicating that palmitoylation is dynamically regulated. Interestingly, membrane receptors such as semaphorin 7A, CR1, and ABCB6, which are known to be involved in merozoite interaction with RBCs and parasite invasion, were found to be dynamically palmitoylated, including the blood group antigen, Kell, whose antigenic abundance was significantly reduced following 2-BMP treatment. To investigate the involvement of Kell in merozoite invasion of RBCs, a specific antibody to its extracellular domain was used. The antibody targeting Kell inhibited merozoite invasion of RBCs by 50%, implying a role of Kell, a dynamically palmitoylated potent host-derived receptor, in parasite invasion. Furthermore, a significant reduction in merozoite contact with the RBC membrane and a consequent decrease in parasite invasion following 2-BMP treatment demonstrated that palmitoylation does indeed regulate RBC susceptibility to parasite invasion. Taken together, our findings revealed the dynamic palmitoylome of RBC membrane proteins and its role in P. falciparum invasion.

ß2M Signals Monocytes Through Non-Canonical TGFß Receptor Signal Transduction.

RATIONALE: Circulating monocytes can have proinflammatory or proreparative phenotypes. The endogenous signaling molecules and pathways that regulate monocyte polarization in vivo are poorly understood. We have shown that platelet-derived ß2M (ß-2 microglobulin) and TGF-ß (transforming growth factor ß) have opposing effects on monocytes by inducing inflammatory and reparative phenotypes, respectively, but each bind and signal through the same receptor. We now define the signaling pathways involved. OBJECTIVE: To determine the molecular mechanisms and signal transduction pathways by which ß2M and TGF-ß regulate monocyte responses both in vitro and in vivo. METHODS AND RESULTS: Wild-type- (WT) and platelet-specific ß2M knockout mice were treated intravenously with either ß2M or TGF-ß to increase plasma concentrations to those in cardiovascular diseases. Elevated plasma ß2M increased proinflammatory monocytes, while increased plasma TGFß increased proreparative monocytes. TGF-ßR (TGF-ß receptor) inhibition blunted monocyte responses to both ß2M and TGF-ß in vivo. Using imaging flow cytometry, we found that ß2M decreased monocyte SMAD2/3 nuclear localization, while TGF-ß promoted SMAD nuclear translocation but decreased noncanonical/inflammatory (JNK [jun kinase] and NF-κB [nuclear factor-κB] nuclear localization). This was confirmed in vitro using both imaging flow cytometry and immunoblots. ß2M, but not TGF-ß, promoted ubiquitination of SMAD3 and SMAD4, that inhibited their nuclear trafficking. Inhibition of ubiquitin ligase activity blocked noncanonical SMAD-independent monocyte signaling and skewed monocytes towards a proreparative monocyte response. CONCLUSIONS: Our findings indicate that elevated plasma ß2M and TGF-ß dichotomously polarize monocytes. Furthermore, these immune molecules share a common receptor but induce SMAD-dependent canonical signaling (TGF-ß) versus noncanonical SMAD-independent signaling (ß2M) in a ubiquitin ligase dependent manner. This work has broad implications as ß2M is increased in several inflammatory conditions, while TGF-ß is increased in fibrotic diseases. Graphic Abstract: A graphic abstract is available for this article.

Lin28B Regulates Angiotensin II-Mediated Let-7c/miR-99a MicroRNA Formation Consequently Affecting Macrophage Polarization and Allergic Inflammation.

Angiotensin-II (Ang-II) receptor plays a role in allergic airway inflammation; however, the underlying mechanism and role of macrophages need better understanding. In the present study, angiotensin-II infusion (1 µg/kg/min) in ovalbumin-induced airway inflammation mice model significantly decreased immune cell infiltration, goblet cell hyperplasia, and eosinophil numbers in lungs. Ang-II infusion increased M1 and decreased M2 macrophage population in bronchoalveolar lavage fluid and respective macrophage markers in lung macrophages. Similarly, in vitro Ang-II treatment in murine bone marrow-derived macrophages (BMDMs) induced M1 and reduced M2 macrophage phenotype with enhanced bactericidal activity. Mechanistically, Ang-II inhibits Let-7c and miR-99a expression in BMDMs and in vivo as well. Lentiviral overexpression of Let-7c and miR-99a miRNAs in BMDMs abrogated Ang-II-induced M1 phenotype activation and promoted M2 phenotype, which is governed by targeting TNFα by miR-99a. In lung macrophages, ovalbumin-induced TNFα inhibition was rescued after Ang-II treatment. In BMDMs, knockdown of TNFα abrogated Ang-II-induced M2 to M1 macrophage phenotype switch and associated bactericidal activity. Ang-II affects mature miRNA formation by enhancing Lin28B levels in macrophages in vivo and in vitro. Furthermore, Lin28B knockdown prevented Ang-II-mediated inhibition of mature Let-7c/miR-99a miRNA formation, M2 to M1 macrophage phenotype switch, and increased bactericidal activity. Therefore, present study suggests a role of Lin28B in Ang-II-induced Let-7c/miR-99a miRNA formation that consequently affects TNFα production, M1 phenotype activation, and allergic airway inflammation. Graphical Abstract Ovalbumin inhibits LIN28B expression thereby fails to inhibit premature to mature Let-7c/miR-99a miRNA formation. Mature miR-99a miRNA that inhibits TNFα consequently promotes M2 polarization and allergic airway inflammation. While Ang-II induces Lin28B, which inhibits Let-7c/miR-99a miRNA processing and mature miRNA formation, this results in increased TNFα levels that lead to M1 polarization and allergic airway inflammation inhibition.

Chronic toxicity of methamphetamine: Oxidative remodeling of pulmonary arteries.

Methamphetamine (MA) has a high uptake in lung, but the precise mechanism of MA-induced lung toxicity remains unclear. The aim of this study is to investigate the role of MA abuse in remodeling of pulmonary arteries and to explore the possible correlation of the association of the remodeling with the redox imbalance in pulmonary arterial smooth muscle cells (PASMCs). Wistar rats were randomly divided into control group and MA group for the experimental study. We employed H&E staining, western blot, immunofluorescence, knockdown, flow in our experimental approach. Our studies shows that chronic exposure to MA led to weight loss, increased pulmonary arterial pressure, hypertrophy of right ventricle and remodeling of pulmonary arterial wall of rats. Our cell culture study with PASMCs indicates that MA significantly induced the imbalance between proliferation and apoptosis by upregulating the level of PCNA, Bcl-2 and reduction in the expression of BAX and Caspase 3. MA markedly prevented the nuclear translocation of Nrf2 to inhibit antioxidation. The knockdown of Nrf2 expression using siRNA significantly elevated the expression of SOD2/GCS and the production of ROS in PASMCs and even scaled up the amount of PASMCs induced by MA. Linear regression analysis showed that knockdown of Nrf2 promoted the positive correlation of relative ROS level with proliferation of PASMCs. Therefore, chronic exposure to MA induces pulmonary arterial remodeling by Nrf2-mediated imbalance of redox system to aggravate oxidative stress, and Nrf2 is a possible target for the treatment of MA-lung toxicity.

MicroRNA-99a mimics inhibit M1 macrophage phenotype and adipose tissue inflammation by targeting TNFα.

In human adipose tissue and obesity, miR-99a expression is negatively correlated with inflammation. Therefore, the present study investigated the role of miR-99a in macrophage phenotype activation and adipose tissue inflammation. M2 BMDMs showed a significant increase in miR-99a expression when compared to the M0 and M1 phenotypes. Phenotype-switching experiments established an association between upregulated miR-99a expression and the M2 phenotype. Overexpression of miR-99a prevented M1 phenotype activation and attenuated bactericidal activity. Likewise, knockdown of miR-99a abolished M2 phenotype activation. By means of in silico target prediction tools and a luciferase reporter assay, TNFα was identified as a direct target of miR-99a. Knockdown of TNFα recapitulated the effect of miR-99a overexpression in M1 BMDMs. In a db/db mice model, miR-99a expression was reduced in eWAT and F4/80+ ATMs. Systemic overexpression of miR-99a in db/db mice attenuated adipocyte hypertrophy with increased CD301 and reduced CD86 immunostaining. Flow cytometry analysis also showed an increased M2 and a reduced M1 macrophage population. Mimics of miR-99a also improved the diabetic dyslipidemia and insulin signaling in eWAT and liver, with an attenuated expression of gluconeogenesis and cholesterol metabolism genes in the liver. Furthermore, adoptive transfer of miR-99a-overexpressing macrophages in the db/db mice recapitulated in vivo miR-99a mimic effects with increased M2 and reduced M1 macrophage populations and improved systemic glucose, insulin sensitivity, and insulin signaling in the eWAT and liver. The present study demonstrates that miR-99a mimics can regulate macrophage M1 phenotype activation by targeting TNFα. miR-99a therapeutics in diabetic mice reduces the adipose tissue inflammation and improves insulin sensitivity.

Detrimental effects of atherogenic and high fat diet on bone and aortic calcification rescued by an isoflavonoid Caviunin ß-d-glucopyranoside.

OBJECTIVE: Atherogenic diet (AD) and high fat diet (HFD) cause deleterious effect on bone micro-architecture and this phenomenon prompts aortic calcification. This study aims to show the effects of Caviunin ß-d-glucopyranoside (CAFG), against bone loss and its associated aortic calcification in presence of AD and HFD challenged diets. METHODS: Five groups of C57BL/6 male mice with 8 animals in each group, comprising of chow, AD, HFD, AD+CAFG and HFD+CAFG were fed with respective diets for 16 weeks. At the end of the treatment period, preventive effects of CAFG on bone tissue were analyzed by assessing the osteogenic potential of bone marrow cells, bone micro-architecture, ability of new bone formation and histomorphometry studies. Aortic calcification was assessed by transcription and translation analysis of osteogenic key markers in aortic tissue and assessment of aortic endothelial function. Plasma lipid profiling was done to assess the effects of diets as its role in both bone loss and aortic calcification. RESULTS: Bone marrow stromal cell (BMSC's) dynamics showed that AD and HFD decreased osteoblast number that led to bone loss, deterioration in bone micro-architecture with up-regulated bone resorptive genes that lead to increase in aortic calcification. CAFG treatment rescued the bone health by modulating BMSC's towards osteogenic lineage. It increased the osteogenic gene expression with simultaneous decrease in osteoclastic genes thus stabilized the receptor activator of nuclear factor-kappa-B ligand/osteoprotegerin ratio that eventually reduced the amount of calcification in aorta. Biochemical studies showed that CAFG reduced the TC, TG and LDL-C content with no marked changes in HDL-C. Moreover, CAFG decreased the osteogenic key markers in the aortic tissue and enhanced endothelial function. CONCLUSION: Overall, this study indicates that CAFG protected against physiologically challenged diet induced bone loss with associated vascular calcification in mice. Moreover, data revealed that atherogenic diet is more detrimental as compared to the excess fatty acid diet to the bone and aorta.

Coagulin-L ameliorates TLR4 induced oxidative damage and immune response by regulating mitochondria and NOX-derived ROS.

Withanolides possess diverse biological and pharmacological activity but their immunomodulatory function is less realized. Hence, coagulin-L, a withanolide isolated from Withania coagulans Dunal has been studied for such an effect in human and murine cells, and mice model. Coagulin-L (1, 3, 10µM) exhibited immunomodulatory effect by suppressing TLR4 induced immune mediators such as cytokines (GMCSF, IFNα, IFNγ, IL-1α, IL-1Rα, IL-1ß, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12 (p40/p70), IL-13, IL-15, IL-17), chemokines (IL-8/CXCL8, MIG/CXCL9, IP-10/CXCL10, KC, MCP-1/CCL2, MIP-1α/CCL3, MIP-1ß/CCL4, RANTES/CCL5, eotaxin/CCL11), growth factors (FGF-basic, VEGF), nitric oxide and intracellular superoxide. Mechanistically, coagulin-L abrogated LPS induced total and mitochondrial ROS generation, NOX2, NOX4 mRNA expression, IRAK and MAPK (p38, JNK, ERK) activation. Coagulin-L also attenuated IκBα degradation, which prevented NFκB downstream iNOS expression and pro-inflammatory cytokine release. Furthermore, coagulin-L (10, 25, 50mg/kg, p.o.), undermined the LPS (10mg/kg, i.p.) induced endotoxemia response in mice as evinced from diminished cytokine release, nitric oxide, aortic p38 MAPK activation and endothelial tissue impairment besides suppressing NOX2 and NOX4 expression in liver and aorta. Moreover, coagulin-L also alleviated the ROS mediated oxidative damage which was assessed through protein carbonyl, lipid hydroperoxide, 8-isoprostane and 8-hydroxy-2-deoxyguanosine quantification. To extend, coagulin-L also suppressed carrageenan-induced paw edema and thioglycollate-induced peritonitis in mice. Therefore, coagulin-L can be of therapeutic importance in pathological conditions induced by oxidative damage.