Chronic chikungunya disease (CCD): clinical insights, immunopathogenesis and therapeutic perspectives.

Chikungunya virus, an arthropod-borne pathogen is recognized by the World Health Organization as a top priority Emerging Infectious Disease and is ranked fourth in public health needs according to the Coalition for Epidemic Preparedness Innovations. Despite its substantial impact, as evidenced by an annual estimate of 120 274 disability-adjusted life years, our understanding of the chronic aspects of chikungunya disease remains limited. This review focuses on chronic chikungunya disease, emphasizing its clinical manifestations, immunopathogenesis, therapeutic options and disease burden.

TLR-mediated aggresome-like induced structures comprise antimicrobial peptides and attenuate intracellular bacterial survival.

Immune cells employ diverse mechanisms for host defense. Macrophages, in response to TLR activation, assemble aggresome-like induced structures (ALIS). Our group has shown TLR4-signaling transcriptionally upregulates p62/sequestome1, which assembles ALIS. We have demonstrated that TLR4-mediated autophagy is, in fact, selective-autophagy of ALIS. We hypothesize that TLR-mediated autophagy and ALIS contribute to host-defense. Here we show that ALIS are assembled in macrophages upon exposure to different bacteria. These structures are associated with pathogen-containing phagosomes. Importantly, we present evidence of increased bacterial burden, where ALIS assembly is prevented with p62-specific siRNA. We have employed 3D-super-resolution structured illumination microscopy (3D-SR-SIM) and mass-spectrometric (MS) analyses to gain insight into the assembly of ALIS. Ultra-structural analyses of known constituents of ALIS (p62, ubiquitin, LC3) reveal that ALIS are organized structures with distinct patterns of alignment. Furthermore, MS-analyses of ALIS identified, among others, several proteins of known antimicrobial properties. We have validated MS data by testing the association of some of these molecules (Bst2, IFITM2, IFITM3) with ALIS and the phagocytosed-bacteria. We surmise that AMPs enrichment in ALIS leads to their delivery to bacteria-containing phagosomes and restricts the bacteria. Our findings in this paper support hitherto unknown functions of ALIS in host-defense.

Optomicrofluidic detection of cancer cells in peripheral blood via metabolic glycoengineering.

The currently existing label-based techniques for the detection of circulating tumor cells (CTCs) target natural surface proteins of cells and are therefore applicable to only limited cancer cell types. We report optomicrofluidic detection of cancer cells in the pool of peripheral blood mononuclear cells (PBMCs) by exploiting the difference in their cell metabolism. We employ metabolic glycoengineering as a click chemistry tool for tagging cells that yields several fold-higher fluorescence signals from cancer cells compared to that from PBMCs. The effects of concentrations of the tagging compounds and cell incubation time on the fluorescence signal intensity are studied. The tagged cells were encapsulated in droplets ensuring that cells enter the detection region two-dimensionally focused in single-file and optically detected with a high detection efficiency and low coefficient of variation of the signals. The metabolic tagging approach showed a significantly higher tagging efficiency and average fluorescence signal compared to the well-established and widely adopted anti-EpCAM-FITC-based tagging. We demonstrated the detection of three different cancer cell lines - EpCAM-negative cervical cancer cell, HeLa, weakly EpCAM positive, and triple-negative breast cancer cell, MDA-MB-231, and strongly EpCAM positive breast cancer cell, MCF7, highlighting that the proposed technique is independent of naturally occurring cell surface proteins and widely applicable. The metabolically tagged and optically detected cells were successfully recultured, proving the compatibility of the proposed technique with downstream assays. The proposed technique is then utilised for the detection of CTCs in metastatic cancer patients' blood. The current work provides a new strategy for detecting cancer cells in the blood that can find potential applications in both fundamental research and clinical studies involving CTCs as well as in single-cell sequencing.

Polyamine metabolism impacts T cell dysfunction in the oral mucosa of people living with HIV.

Metabolic changes in immune cells contribute to both physiological and pathophysiological outcomes of immune reactions. Here, by comparing protein expression, transcriptome, and salivary metabolome profiles of uninfected and HIV+ individuals, we found perturbations of polyamine metabolism in the oral mucosa of HIV+ patients. Mechanistic studies using an in vitro human tonsil organoid infection model revealed that HIV infection of T cells also resulted in increased polyamine synthesis, which was dependent on the activities of caspase-1, IL-1ß, and ornithine decarboxylase-1. HIV-1 also led to a heightened expression of polyamine synthesis intermediates including ornithine decarboxylase-1 as well as an elevated dysfunctional regulatory T cell (TregDys)/T helper 17 (Th17) cell ratios. Blockade of caspase-1 and polyamine synthesis intermediates reversed the TregDys phenotype showing the direct role of polyamine pathway in altering T cell functions during HIV-1 infection. Lastly, oral mucosal TregDys/Th17 ratios and CD4 hyperactivation positively correlated with salivary putrescine levels, which were found to be elevated in the saliva of HIV+ patients. Thus, by revealing the role of aberrantly increased polyamine synthesis during HIV infection, our study unveils a mechanism by which chronic viral infections could drive distinct T cell effector programs and Treg dysfunction.

Bisdemethoxycurcumin promotes apoptosis in human platelets via activation of ERK signaling pathway.

Curcumin, a major bioactive component of turmeric (Curcuma longa), is known for its multiple health benefits. Curcumin as such is a mixture of its analogs: bisdemethoxycurcumin (BDMC)-3%, and demethoxycurcumin (DMC)-17%. Although the effect of curcumin on platelets is documented, the effect of BDMC and DMC on platelets is less studied. Considering the indispensable role played by platelets in hemostasis, thrombosis, inflammation, and immunity, the present study evaluates the effect of curcumin, DMC and BDMC on platelet apoptosis. The components of curcumin were purified by silica-gel column chromatography. The purity and mass analysis of the purified curcuminoids was determined by RP-HPLC and LC-MS respectively. When analyzed for platelet apoptotic markers, only BDMC demonstrated increased incidence of platelet apoptotic markers including increase in intracellular Ca2+, decrease in ∆ψm, alteration in BCl-2 family proteins, the release of cytochrome c, caspase activation, and PS externalization via activation of ERK activation. ERK inhibitor PD98059 significantly alleviated BDMC induced decrease in ∆ψm, alteration in BCl-2, caspase-8 activation and PS externalization. Our results demonstrate that curcumin, DMC and BDMC differentially act on platelet in inducing apoptosis and the study highlights that the toxicity associated with curcumin therapy might be attributed to BDMC in the mammalian system.

Guggulipid ameliorates adjuvant-induced arthritis and liver oxidative damage by suppressing inflammatory and oxidative stress mediators.

BACKGROUND: Arthritis is a common degenerative joint disease characterized by deterioration of articular cartilage, subchondral bone, and associated with immobility, pain and inflammation. The incessant action of reactive oxygen species (ROS) during progressive arthritis causes severe oxidative damage to vital organs and circulatory system. PURPOSE: In this study we investigated the ability of guggulipid (GL), a lipid rich extract from the gum resin of the plant Commiphora whighitii to suppress the progressive arthritis and associated liver oxidative stress both in vivo and in vitro. STUDY DESIGN/METHODS: The anti-arthritic ability of GL was demonstrated in vitro using IL-1ß stimulated bovine nasal cartilage model and in vivo Freund's complete adjuvant-induced arthritic rat model. Collagen/proteoglycan degradation and pro-inflammatory mediators were monitored in the harvested culture medium of nasal cartilage by estimating the levels of matrix metalloproteinases (MMPs), hydroxy proline, glycosaminoglycans and inflammatory mediators. Further, anti-arthritic ability of GL was evaluated in vivo by measuring enzymatic and non-enzymatic mediators of cartilage degradation, inflammation and oxidative stress markers. RESULTS: GL significantly inhibited the IL-1ß stimulated cartilage degradation in vitro by mitigating the MMPs activity, collagen degradation and secretion of pro-inflammatory mediators. Further, GL significantly reduced the adjuvant-induced paw swelling and body weight loss in vivo. GL remarkably reduced the MMPs and hyaluronidases activities in serum and bone homogenate along with altered hematological parameters. GL also mitigated the elevated bone resorbing enzymes cathepsins, exoglycosidases and phosphatases. Additionally, GL effectively mitigated ROS and oxidative stress-mediators recuperating the altered serum/liver oxidative stress and liver damage incurred during arthritic progression. CONCLUSION: In summary, the study clearly demonstrates the protective efficacy of GL against arthritis and its associated oxidative stress, particularly, liver oxidative damage. Hence, GL could be a potential alternative and complementary medicine to treat inflammatory joint diseases.

Lentiviral Vpx induces alteration of mammalian cell nuclear envelope integrity.

Vpx, a virion-associated protein of Human Immunodeficiency Virus 2 (HIV-2) and Simian Immunodeficiency Virus (SIV) counteracts host restriction factor SAMDH1 for efficient viral DNA synthesis in the cytoplasm and mediates subsequent nuclear translocation of the viral genome. Vpx was found to be indispensable in the viral infection of terminally differentiated target cells and macaques infected with virions carrying truncated Vpx showed delayed pathogenesis, suggesting multiple roles of Vpx at different steps in the virus life cycle. The current study demonstrates a novel function of SIVsmPBj1.9 Vpx on the integrity of the nuclear envelope in HeLa cells. Results from the Super-Resolution Structured Illumination Microscopy (SR-SIM) analysis showed that Vpx puncta alter HeLa cell nuclear envelope assembly. Furthermore, three-dimensional (3D) SIM analysis of such regions suggests that Vpx is primed in a specific way to disrupt the nuclear envelope integrity. The nuclear incursion of cytoplasmic proteins through Vpx mediated ruptured nuclear envelope regions suggest that these events might play a critical role in the nuclear entry of otherwise cytoplasmically sequestered molecules and theirby may be assisting Vpx functions including the transport of viral genome into the nucleus, which is critical for the establishment of virus infection and pathogenesis.

Benzimidazole tethered pyrrolo[3,4-b]quinoline with broad-spectrum activity against fungal pathogens.

Fungal infections caused by Candida and Cryptococcus are particularly dangerous for immunocompromised individuals. In this study, we identified that benzimidazole fused pyrrolo[3,4-b]quinoline compounds have potent antifungal activity against several clinical isolates of pathogenic fungal strains. Specifically, the compound 6a did not show cytotoxicity against mammalian cells at a concentration that inhibits the growth of fungal strains. In addition, the compound 6a also significantly reduced the metabolic activity of fungal cells in the Candida albicans biofilms. Collectively, our results indicate that benzimidazole fused quinoline compounds have a potential to develop as an antifungal agents.

Regio and stereoselective synthesis of anticancer spirooxindolopyrrolidine embedded piperidone heterocyclic hybrids derived from one-pot cascade protocol.

BACKGROUND: Spiropyrrolidine tethered piperidone heterocyclic hybrids were synthesized with complete regio- and stereoselectively in excellent yield via a tandem three-component 1,3-dipolar cycloaddition and subsequent enamine reaction in [bmim]Br. The synthesized compounds were evaluated for their anticancer activity against FaDu hypopharyngeal tumor cells. FINDINGS: Interestingly, most compounds displayed cytotoxicities similar to the standard anticancer agent bleomycin, with two of them (5a and 5g) being slightly more active than the reference drug. CONCLUSION: Synthesized compounds have also been evaluated for their apoptosis-inducing properties in a cancer cell model, finding that treatment with compounds 5a-e led to apoptotic cell death.

Multicomponent Domino Synthesis, Anticancer Activity and Molecular Modeling Simulation of Complex Dispirooxindolopyrrolidines.

A series of spirooxindolopyrrolidine fused N-styrylpiperidone heterocyclic hybrids has been synthesized in excellent yield via a domino multicomponent protocol that involves one-pot three component 1,3-dipolar cycloaddition and concomitant enamine reactions performed in an inexpensive ionic liquid, namely 1-butyl-3-methylimidazolium bromide ([bmim]Br). Compounds thus synthesized were evaluated for their cytotoxicity against U-937 tumor cells. Interestingly; compounds 5i and 5m exhibited a better cytotoxicity than the anticancer drug bleomycin. In addition; the effect of the synthesized compounds on the nuclear morphology of U937 FaDu cells revealed that treatment with compounds 5a⁻m led to their apoptotic cell death.

Bone Degeneration, Inflammation and Secondary Complications of Arthritis: Potential Targets and their Natural Inhibitors.

Arthritis is marked by joint deterioration that affects articular cartilage and subchondral bone. Though cartilage degradation does the major damage during arthritis, subsequent bone degeneration cannot be neglected. Recent progress in arthritis research has identified the clinical importance of bone erosion in destructive arthritis. Studies have showed the key role played by osteoclasts and receptor activator of nuclear factor kappaB ligand (RANKL) signaling in bone erosion. Cathepsins and tartrate resistant acid phosphatase (TRAP) are considered key enzymatic factors contributing to bone erosion. Further, reactive oxygen species (ROS) formed at the ruffled border of osteoclasts also causes bone resorption and matrix degradation. Besides, severe inflammation during arthritis induces bone erosion by aiding in Ca2+ removal and activating osteoclastogenesis. The inflammatory cytokines and ROS influence osteoclast differentiation by regulating osteoclast-lineage cells or by acting on other cells to regulate the expression of RANKL and osteoprotegerin (OPG). The enhanced production of pro-inflammatory cytokines and ROS in arthritis stimulates tissue injury by means of oxidative damage leading to vital organ damage and synovial and circulatory cell apoptosis. Thus, blocking enzymatic and non-enzymatic factors responsible for bone erosion and inflammation is considered a prime strategy in the management of arthritis. In this review we provide an overview of the mechanisms of bone erosion, inflammation and associated oxidative stress/damage during arthritis perpetuation along with shedding light on potential targets. The article also describes the possible natural therapeutic agents that could prevent bone loss and inflammation, and related secondary complications of arthritis.

Novel oxolane derivative DMTD mitigates high glucose-induced erythrocyte apoptosis by regulating oxidative stress.

Chronic hyperglycemia is one of the characteristic conditions associated with Diabetes Mellitus (DM), which often exerts deleterious effects on erythrocyte morphology and hemodynamic properties leading to anemia and diabetes-associated vascular complications. High glucose-induced over production of reactive oxygen species (ROS) can alter the blood cell metabolism and biochemical functions subsequently causing eryptosis (red blood cell death), yet another complication of concern in DM. Therefore, blocking high glucose-induced oxidative damage and subsequent eryptosis is of high importance in the better management of DM and associated vascular complications. In this study, we synthesized an oxolane derivative 1-(2,2-dimethyltetrahydrofuro[2,3][1,3]dioxol-5-yl)ethane-1,2-diol (DMTD), and demonstrated its efficacy to mitigate hyperglycemia-induced ROS generation and subsequent eryptosis. We showed that DMTD effectively inhibits high glucose-induced ROS generation, intracellular calcium levels, phosphaditylserine (PS) scrambling, calpain and band 3 activation, LDH leakage, protein glycation and lipid peroxidation, meanwhile enhances the antioxidant indices, osmotic fragility and Na+/K+-ATPase activity in erythrocytes. DMTD dose dependently decreased the glycated hemoglobin level and enhances the glucose utilization by erythrocytes in vitro. Further, DMTD alleviated the increase in ROS production, intracellular Ca2+ level and PS externalization in the erythrocytes of human diabetic subjects and enhanced the Na+/K+-ATPase activity. Taken together, the synthesized oxolane derivative DMTD could be a novel synthetic inhibitor of high glucose-induced oxidative stress and eryptosis. Considering the present results DMTD could be a potential therapeutic to treat DM and associated complications and open new avenues in developing synthetic therapeutic targeting of DM-associated complications.

Design, synthesis and antiproliferative activity of decarbonyl luotonin analogues.

A small library of benzimidazole-fused pyrrolo[3,4-b]quinoline has been synthesized from readily available benzimidazole 2-carbaldehyde and various substituted arylamines in good to excellent yields utilizing an intramolecular Povarov reaction catalyzed by boron trifluoride diethyl etharate as the key final step. The compounds thus synthesized can be considered as decarbonyl analogues of the anticancer alkaloid luotonin A and were evaluated in a DNA relaxation assay for their ability to inhibit human topoisomerase I. Interestingly, two of the compounds showed a remarkable activity that is comparable to that of the standard drug camptothecin. The compounds were also evaluated for their cytotoxic effect in four highly aggressive human cancer cell lines, namely KB, MDA-MB231 (breast), LNCap (prostate), and HT1080 (fibrosarcoma). Some of the compounds obtained showed promising cytotoxicities for these four cell lines.

Cell-free methemoglobin drives platelets to apoptosis via mitochondrial ROS-mediated activation of JNK and p38 MAP kinase.

Cell-free hemoglobin (Hb), a well-known marker of intravascular hemolysis, is eventually oxidized to methemoglobin (MtHb). Elevated levels of MtHb have been noted, alongside depleted levels of platelets, in several hemolytic diseases. The current study aims to probe the possible role of MtHb in platelet death, based on the facts that it is a pro-inflammatory and pro-apoptotic agent, as well as the sensitive nature of platelets and their tendency to undergo apoptosis under oxidative stress. An attempt is made to establish the link between hemolysis and thrombocytopenia, by deciphering the underlying molecular signaling pathways. The results of this study demonstrate that MtHb, not Hb exerts oxidative stress on platelets, which triggers their death via ROS-mediated mitochondrial apoptotic pathway. It was further established that the MtHb-induced platelet apoptotic events mediate through JNK and p38 MAPK activation. Thus, the study presents a mechanistic insight into the previous studies that reported the incidence of thrombocytopenia in hemolytic diseases. This study highlights the fate of platelets in intravascular hemolytic conditions, which demands the need for a specific treatment strategy considering the risks associated with thrombocytopenia during severe hemolytic diseases.

Correction: Novel Synthetic Oxazines Target NF-κB in Colon Cancer In Vitro and Inflammatory Bowel Disease In Vivo.

[This corrects the article DOI: 10.1371/journal.pone.0163209.].

Gyrospun antimicrobial nanoparticle loaded fibrous polymeric filters.

A one step approach to prepare hybrid nanoparticle embedded polymer fibres using pressurised gyration is presented. Two types of novel antimicrobial nanoparticles and poly(methylmethacrylate) polymer were used in this work. X-ray diffraction analysis of the nanoparticles revealed Ag, Cu and W are the main elements present in them. The concentration of the polymer solution and the nanoparticle concentration had a significant influence on the fibre diameter, pore size and morphology. Fibres with a diameter in the range of 6-20µm were spun using 20wt% polymer solutions containing 0.1, 0.25 and 0.5 wt% nanoparticles under 0.3MPa working pressure and a rotational speed of 36,000rpm. Continuous, bead-free fibre morphologies were obtained for each case. The pore size in the fibres varied between 36 and 300nm. Successful incorporation of the nanoparticles in polymer fibres was confirmed by energy dispersive x-ray analysis. The fibres were also gyrospun on to metallic discs to prepare filters which were tested for their antibacterial activity on a suspension of Pseudomonas aeruginosa. Nanoparticle loaded fibres showed higher antibacterial efficacy than pure poly(methylmethacrylate) fibres.

Melatonin inhibits snake venom and antivenom induced oxidative stress and augments treatment efficacy.

Snakebite is a neglected health hazard. Its patho-physiology has largely been focused on systemic and local toxicities; whereas, venom and antivenom induced oxidative stress has long been ignored. Antivenom therapy although neutralizes venom lethality and saves many lives, remains ineffective against oxidative stress. This prompted us to complement antivenom with an antioxidant molecule melatonin that would protect against oxidative stress and increase the efficacy of the existing snakebite therapy. Here we show that D. russelli and E. carinatus venoms induce strong oxidative stress that persists even after antivenom administration in mice model. Additionally, antivenoms also induce oxidative stress. Polyvalent antivenom induce more oxidative stress than monovalent antivenom. Strikingly, antivenom and melatonin together not only inhibit venom and antivenom induced oxidative stress but also significantly reduce the neutralizing antivenom dose. This study provides a therapeutic potential for enhancing the existing snakebite therapy. The combined treatment of antivenom+melatonin would prevent the upsurge of oxidative stress as well as minimize the antivenom load. Thus the investigation offers immense scope for physicians and toxinologists to reinvestigate, design new strategies and think beyond the conventional mode of antivenom therapy.

Medium-term outcomes of the Universal-2 total wrist arthroplasty in patients with rheumatoid arthritis.

AIMS: The aims of this study were to evaluate the clinical and radiological outcomes of the Universal-2 total wrist arthroplasty (TWA) in patients with rheumatoid arthritis. PATIENTS AND METHODS: This was a retrospective review of all 95 Universal-2 TWAs which were performed in our institution between 2003 to 2012 in patients with rheumatoid arthritis. A total of six patients were lost to follow-up and two died of unrelated causes. A total of ten patients had bilateral procedures. Accordingly, 75 patients (85 TWAs) were included in the study. There were 59 women and 16 men with a mean age of 59 years (26 to 86). The mean follow-up was 53 months (24 to 120). Clinical assessment involved recording pain on a visual analogue score, range of movement, grip strength, the Quick Disabilities of the Arm, Shoulder and Hand (DASH) and Wrightington wrist scores. Any adverse effects were documented with particular emphasis on residual pain, limitation of movement, infection, dislocation and the need for revision surgery. Radiographic assessment was performed pre-operatively and at three, six and 12 months post-operatively, and annually thereafter. Arthroplasties were assessed for distal row intercarpal fusion and loosening. Radiolucent zones around the components were documented according to a system developed at our institution. RESULTS: The mean worst pain was 8.1 (3 to 10) pre-operatively and 5.4 (0 to 10) at latest follow-up (p < 0.001). Movements were preserved with mean dorsiflexion of 29o (0 o to 70 o) and palmar flexion of 21o (0o to 50o). The mean grip strength was 4.8 kg (1.7 to 11.5) pre-operatively and 10 kg (0 to 28) at final follow-up (p < 0.001). The mean QuickDASH and Wrightington wrist scores improved from 61 (16 to 91) to 46 (0 to 89) and 7.9 (1.8 to 10) to 5.7 (0 to 7.8) (p < 0.001). A total of six patients (7%) had major complications; three required revision arthroplasty and three an arthrodesis. The Kaplan-Meier probability of survival using removal of the components as the endpoint was 91% at 7.8 years (95% confidence interval 84 to 91). CONCLUSION: The Universal-2 TWA is recommended for use in patients with rheumatoid arthritis. Cite this article: Bone Joint J 2016;98-B:1642-7.

Novel Synthetic Oxazines Target NF-κB in Colon Cancer In Vitro and Inflammatory Bowel Disease In Vivo.

Aberrant activation of nuclear factor kappa B (NF-κB) has been linked with the pathogenesis of several proinflammatory diseases including number of cancers and inflammatory bowel diseases. In the present work, we evaluated the anticancer activity of 1,2-oxazines derivatives against colorectal cancer cell lines and identified 2-((2-acetyl-6,6-dimethyl-4-phenyl-5,6-dihydro-2H-1,2-oxazin-3-yl)methyl)isoindoline-1,3-dione (API) as the lead anticancer agent among the tested compounds. The apoptosis inducing effect of API was demonstrated using flow cytometry analysis and measuring the caspase 3/7 activity in API treated cells. Based on the literature on inhibition of NF-κB by oxazines, we evaluated the effect of 1,2-oxazines against the ability of NF-κB binding to DNA, NF-κB-dependent luciferase expression and IκBα phosphorylation. We found that, API abrogate constitutive activation of NF-κB and inhibits IκBα phosphorylation in HCT116 cells. Our in silico analysis revealed the binding of oxazines to the hydrophobic cavity that present between the interface of p65 and IκBα. Given the relevance with aberrant activation of NF-κB in inflammation bowel disease (IBD), we evaluated the effect of API on dextran sulphate sodium-induced IBD mice model. The treatment of IBD induced mice with API decreased the myeloperoxidase activity in colonic extract, modulated the colon length and serum levels of pro- and anti-inflammatory cytokines such as TNF-α, IFN-γ, IL-6, IL-1ß and IL-10. Furthermore, the histological analysis revealed the restoration of the distorted cryptic epithelial structure of colon in the API treated animals. In conclusion, we comprehensively validated the NF-κB inhibitory efficacy of API that targets NF-κB in in vitro colon cancer and an in vivo inflammatory bowel disease model.