Transcriptomic Analysis of Inflammatory Cardiomyopathy Identifies Molecular Signatures of Disease and Informs in silico Prediction of a Network-Based Rationale for Therapy.

Inflammatory cardiomyopathy covers a group of diseases characterized by inflammation and dysfunction of the heart muscle. The immunosuppressive agents such as prednisolone, azathioprine and cyclosporine are modestly effective treatments, but a molecular rationale underpinning such therapy or the development of new therapeutic strategies is lacking. We aimed to develop a network-based approach to identify therapeutic targets for inflammatory cardiomyopathy from the evolving myocardial transcriptome in a mouse model of the disease. We performed bulk RNA sequencing of hearts at early, mid and late time points from mice with experimental autoimmune myocarditis. We identified a cascade of pathway-level events involving early activation of cytokine and chemokine-signaling pathways that precede leucocyte infiltration and are followed by innate immune, antigen-presentation, complement and cell-adhesion pathway activation. We integrated these pathway events into a network-like representation from which we further identified a 50-gene subnetwork that is predominantly induced during the course of autoimmune myocardial inflammation. We developed a combinatorial attack strategy where we quantify network tolerance to combinatorial node removal to determine target-specific therapeutic potential. We find that combinatorial attack of Traf2, Nfkb1, Rac1, and Vav1 disconnects 80% of nodes from the largest network component. Two of these nodes, Nfkb1 and Rac1, are directly targeted by prednisolone and azathioprine respectively, supporting the idea that the methodology developed here can identify valid therapeutic targets. Whereas Nfkb1 and Rac1 removal disconnects 56% of nodes, we show that additional removal of Btk and Pik3cd causes 72% node disconnection. In conclusion, transcriptome profiling, pathway integration, and network identification of autoimmune myocardial inflammation provide a molecular signature applicable to the diagnosis of inflammatory cardiomyopathy. Combinatorial attack provides a rationale for immunosuppressive therapy of inflammatory cardiomyopathy and provides an in silico prediction that the approved therapeutics, ibrutinib and idelalisib targeting Btk and Pik3cd respectively, could potentially be re-purposed as adjuncts to immunosuppression.

A Comprehensive Review and Perspective on Anticancer Mechanisms of Withaferin A in Breast Cancer.

Withaferin A (hereafter abbreviated as WA) is a promising anticancer steroidal lactone abundant in a medicinal plant (Withania somnifera) native to Asia. The root/leaf extract of Withania somnifera, which belongs to the Solanaceae family, continues to be included in the Ayurvedic medicine formulations of alternative medicine practice. Numerous chemicals are detectable in the root/leaf extract of Withania somnifera [e.g., withanolides (WA, withanone, withanolide A, etc.), alkaloids, sitoindosides, etc.], but the anticancer effect of this medicinal plant is largely attributed to WA. Anticancer effect of WA was initially reported in the early 70s in the Ehrlich ascites tumor cell model in vitro Since then, numerous preclinical studies have been performed using cellular and animal models of different cancers including breast cancer to determine cancer therapeutic and chemopreventive effects of WA. Chemoprevention, a word first introduced by Dr. Michael B. Sporn, was intended to impede, arrest, or reverse carcinogenesis at its earliest stages with pharmacologic agents. This review succinctly summarizes the published findings on anticancer pharmacology of WA in breast cancer focusing on pharmacokinetic behavior, in vivo efficacy data in preclinical models in a therapeutic and chemoprevention settings, and its known effects on cancer-relevant cellular processes (e.g., growth arrest, apoptosis induction, autophagy, metabolic adaptation, immune function, etc.) and molecular targets (e.g., suppression of oncogenes such as estrogen receptor-α, STAT3, etc.). Potential gaps in knowledge as well as future research directions essential for clinical development of WA for chemoprevention and/or treatment of breast cancer are also discussed.

A knottin scaffold directs the CXC-chemokine-binding specificity of tick evasins.

Tick evasins (EVAs) bind either CC- or CXC-chemokines by a poorly understood promiscuous or "one-to-many" mechanism to neutralize inflammation. Because EVAs potently inhibit inflammation in many preclinical models, highlighting their potential as biological therapeutics for inflammatory diseases, we sought to further unravel the CXC-chemokine-EVA interactions. Using yeast surface display, we identified and characterized 27 novel CXC-chemokine-binding evasins homologous to EVA3 and defined two functional classes. The first, which included EVA3, exclusively bound ELR+ CXC-chemokines, whereas the second class bound both ELR+ and ELR- CXC-chemokines, in several cases including CXC-motif chemokine ligand 10 (CXCL10) but, surprisingly, not CXCL8. The X-ray crystal structure of EVA3 at a resolution of 1.79 Å revealed a single antiparallel ß-sheet with six conserved cysteine residues forming a disulfide-bonded knottin scaffold that creates a contiguous solvent-accessible surface. Swapping analyses identified distinct knottin scaffold segments necessary for different CXC-chemokine-binding activities, implying that differential ligand positioning, at least in part, plays a role in promiscuous binding. Swapping segments also transferred chemokine-binding activity, resulting in a hybrid EVA with dual CXCL10- and CXCL8-binding activities. The solvent-accessible surfaces of the knottin scaffold segments have distinctive shape and charge, which we suggest drives chemokine-binding specificity. These studies provide structural and mechanistic insight into how CXC-chemokine-binding tick EVAs achieve class specificity but also engage in promiscuous binding.

Genetically engineered two-warhead evasins provide a method to achieve precision targeting of disease-relevant chemokine subsets.

Both CC and CXC-class chemokines drive inflammatory disease. Tick salivary chemokine-binding proteins (CKBPs), or evasins, specifically bind subsets of CC- or CXC-chemokines, and could precisely target disease-relevant chemokines. Here we have used yeast surface display to identify two tick evasins: a CC-CKBP, P1243 from Amblyomma americanum and a CXC-CKBP, P1156 from Ixodes ricinus. P1243 binds 11 CC-chemokines with Kd < 10 nM, and 10 CC-chemokines with Kd between 10 and 100 nM. P1156 binds two ELR + CXC-chemokines with Kd < 10 nM, and four ELR + CXC-chemokines with Kd between 10 and 100 nM. Both CKBPs neutralize chemokine activity with IC50 < 10 nM in cell migration assays. As both CC- and CXC-CKBP activities are desirable in a single agent, we have engineered "two-warhead" CKBPs to create single agents that bind and neutralize subsets of CC and CXC chemokines. These results show that tick evasins can be linked to create non-natural proteins that target subsets of CC and CXC chemokines. We suggest that "two-warhead" evasins, designed by matching the activities of parental evasins to CC and CXC chemokines expressed in disease, would achieve precision targeting of inflammatory disease-relevant chemokines by a single agent.

The N-terminal domain of a tick evasin is critical for chemokine binding and neutralization and confers specific binding activity to other evasins.

Tick chemokine-binding proteins (evasins) are an emerging class of biologicals that target multiple chemokines and show anti-inflammatory activities in preclinical disease models. Using yeast surface display, we identified a CCL8-binding evasin, P672, from the tick Rhipicephalus pulchellus We found that P672 binds CCL8 and eight other CC-class chemokines with a Kd < 10 nm and four other CC chemokines with a Kd between 10 and 100 nm and neutralizes CCL3, CCL3L1, and CCL8 with an IC50 < 10 nm The CC chemokine-binding profile was distinct from that of evasin 1 (EVA1), which does not bind CCL8. We also show that P672's binding activity can be markedly modulated by the location of a StrepII-His purification tag. Combining native MS and bottom-up proteomics, we further demonstrated that P672 is glycosylated and forms a 1:1 complex with CCL8, disrupting CCL8 homodimerization. Homology modeling of P672 using the crystal structure of the EVA1 and CCL3 complex as template suggested that 44 N-terminal residues of P672 form most of the contacts with CCL8. Replacing the 29 N-terminal residues of EVA1 with the 44 N-terminal residues of P672 enabled this hybrid evasin to bind and neutralize CCL8, indicating that the CCL8-binding properties of P672 reside, in part, in its N-terminal residues. This study shows that the function of certain tick evasins can be manipulated simply by adding a tag. We conclude that homology modeling helps identify regions with transportable chemokine-binding functions within evasins, which can be used to construct hybrid evasins with altered properties.

Yeast surface display identifies a family of evasins from ticks with novel polyvalent CC chemokine-binding activities.

Chemokines function via G-protein coupled receptors in a robust network to recruit immune cells to sites of inflammation. Due to the complexity of this network, targeting single chemokines or receptors has not been successful in inflammatory disease. Dog tick saliva contains polyvalent CC-chemokine binding peptides termed evasins 1 and 4, that efficiently disrupt the chemokine network in models of inflammatory disease. Here we develop yeast surface display as a tool for functionally identifying evasins, and use it to identify 10 novel polyvalent CC-chemokine binding evasin-like peptides from salivary transcriptomes of eight tick species in Rhipicephalus and Amblyomma genera. These evasins have unique binding profiles compared to evasins 1 and 4, targeting CCL2 and CCL13 in addition to other CC-chemokines. Evasin binding leads to neutralisation of chemokine function including that of complex chemokine mixtures, suggesting therapeutic efficacy in inflammatory disease. We propose that yeast surface display is a powerful approach to mine potential therapeutics from inter-species protein interactions that have arisen during evolution of parasitism in ticks.

Mitochondrial dysfunction in cancer chemoprevention by phytochemicals from dietary and medicinal plants.

Cancer chemoprevention, a scientific term coined by Dr. Sporn in the late seventies, implies use of natural or synthetic chemicals to block, delay or reverse carcinogenesis. Phytochemicals derived from edible and medicinal plants have been studied rather extensively for cancer chemoprevention using preclinical models in the past few decades. Nevertheless, some of these agents (e.g., isothiocyanates from cruciferous vegetables like broccoli and watercress) have already entered into clinical investigations. Examples of widely studied and highly promising phytochemicals from edible and medicinal plants include cruciferous vegetable constituents (phenethyl isothiocyanate, benzyl isothiocyanate, and sulforaphane), withaferin A (WA) derived from a medicinal plant (Withania somnifera) used heavily in Asia, and an oriental medicine plant component honokiol (HNK). An interesting feature of these structurally-diverse phytochemicals is that they target mitochondria to provoke cancer cell-selective death program. Mechanisms underlying cell death induction by commonly studied phytochemicals have been discussed rather extensively and thus are not covered in this review article. Instead, the primary focus of this perspective is to discuss experimental evidence pointing to mitochondrial dysfunction in cancer chemoprevention by promising phytochemicals.

Genetic affinities of the Jewish populations of India.

Due to the lack of written records or inscription, the origin and affiliation of Indian Jewish populations with other world populations remain contentious. Previous genetic studies have found evidence for a minor shared ancestry of Indian Jewish with Middle Eastern (Jewish) populations. However, these studies (relied on limited individuals), haven't explored the detailed temporal and spatial admixture process of Indian Jewish populations with the local Indian populations. Here, using large sample size with combination of high resolution biparental (autosomal) and uniparental markers (Y chromosome and mitochondrial DNA), we reconstructed genetic history of Indian Jewish by investigating the patterns of genetic diversity. Consistent with the previous observations, we detected minor Middle Eastern specific ancestry component among Indian Jewish communities, but virtually negligible in their local neighbouring Indian populations. The temporal test of admixture suggested that the first admixture of migrant Jewish populations from Middle East to South India (Cochin) occurred during fifth century. Overall, we concluded that the Jewish migration and admixture in India left a record in their genomes, which can link them to the 'Jewish Diaspora'.

Broccoli-Derived Sulforaphane and Chemoprevention of Prostate Cancer: From Bench to Bedside.

Sulforaphane (SFN) is a metabolic by product of cruciferous vegetables and is the biologically active phytochemical found in high concentrations in broccoli. It has been studied extensively for its anticancer efficacy and the underlying mechanisms using cell culture and preclinical models. The immediate precursor of SFN is glucoraphanin, a glucosinolate which requires metabolic conversion to SFN. SFN and other notable isothiocyanates, including phenethyl isothiocyanate and benzyl isothiocyanate found in various cruciferous vegetables, have also been implicated to have a chemopreventive role for breast, colon and prostate cancer. In-vitro and in-vivo anti-cancer activity of this class of compounds summarizing the past two decades of basic science research has previously been reviewed by us and others. The present review aims to focus specifically on SFN and its chemopreventive and antineoplastic activity against prostate cancer. Particular emphasis in this communication is placed on the current status of clinical research and prospects for future clinical trials with the overall objective to better understand the clinical utility of this promising chemopreventive nutraceutical in the context of mechanisms of prostate carcinogenesis.

Osteopontin deficiency affects imiquimod-induced psoriasis-like murine skin inflammation and lymphocyte distribution in skin, draining lymph nodes and spleen.

Osteopontin (OPN) that enhances autoimmunity is expressed in psoriasis lesions; however, its functions in psoriatic inflammation are unknown. We investigated the role of OPN in OPN deficient mice (OPN-/-) by inducing psoriasis-like inflammation through skin application of imiquimod (IMQ). OPN-/- mice treated with IMQ showed delayed onset ear swelling and attracted less inflammatory cells to the skin. IMQ-induced lymph node swelling was reduced in the absence of OPN, and IMQ-mediated expansion of B cells was inhibited. Further, reduction of CD4(+) T-cell numbers by IMQ in lymph nodes was suppressed in OPN-/- mice, with an increase in the CD4/CD8 ratio. A comparable pattern was found in spleen. Importantly, IMQ-induced IL-17 and IL-4 expression by CD4(+) lymph node T cells was reduced in OPN-/- mice. In conclusion, OPN may modulate psoriasis-like inflammation through altering lymphocyte distribution in skin and draining lymph nodes and by inducing IL-17 expression of inflammatory T cells.

Unravelling the distinct strains of Tharu ancestry.

The northern region of the Indian subcontinent is a vast landscape interlaced by diverse ecologies, for example, the Gangetic Plain and the Himalayas. A great number of ethnic groups are found there, displaying a multitude of languages and cultures. The Tharu is one of the largest and most linguistically diverse of such groups, scattered across the Tarai region of Nepal and bordering Indian states. Their origins are uncertain. Hypotheses have been advanced postulating shared ancestry with Austroasiatic, or Tibeto-Burman-speaking populations as well as aboriginal roots in the Tarai. Several Tharu groups speak a variety of Indo-Aryan languages, but have traditionally been described by ethnographers as representing East Asian phenotype. Their ancestry and intra-population diversity has previously been tested only for haploid (mitochondrial DNA and Y-chromosome) markers in a small portion of the population. This study presents the first systematic genetic survey of the Tharu from both Nepal and two Indian states of Uttarakhand and Uttar Pradesh, using genome-wide SNPs and haploid markers. We show that the Tharu have dual genetic ancestry as up to one-half of their gene pool is of East Asian origin. Within the South Asian proportion of the Tharu genetic ancestry, we see vestiges of their common origin in the north of the South Asian Subcontinent manifested by mitochondrial DNA haplogroup M43.

Reduction of CD18 promotes expansion of inflammatory γδ T cells collaborating with CD4+ T cells in chronic murine psoriasiform dermatitis.

IL-17 is a critical factor in the pathogenesis of psoriasis and other inflammatory diseases. The impact of γδ T cells, accounting for an important source of IL-17 in acute murine IL-23- and imiquimod-induced skin inflammation, in human psoriasis is still unclear. Using the polygenic CD18(hypo) PL/J psoriasis mouse model spontaneously developing chronic psoriasiform dermatitis due to reduced CD18/ß2 integrin expression to 2-16% of wild-type levels, we investigated in this study the influence of adhesion molecule expression on generation of inflammatory γδ T cells and analyzed the occurrence of IL-17-producing γδ and CD4(+) T cells at different disease stages. Severity of CD18(hypo) PL/J psoriasiform dermatitis correlated with a loss of skin-resident Vγ5(+) T cells and concurrent skin infiltration with IL-17(+), IL-22(+), and TNF-α(+) γδTCR(low) cells preceded by increases in Vγ4(+) T cells in local lymph nodes. In vitro, reduced CD18 levels promoted expansion of inflammatory memory-type γδ T cells in response to IL-7. Similar to IL-17 or IL-23/p19 depletion, injection of diseased CD18(hypo) PL/J mice with anti-γδTCR Abs significantly reduced skin inflammation and largely eliminated pathological γδ and CD4(+) T cells. Moreover, CD18(hypo) γδ T cells induced allogeneic CD4(+) T cell responses more potently than CD18(wt) counterparts and, upon adoptive transfer, triggered psoriasiform dermatitis in susceptible hosts. These results demonstrate a novel function of reduced CD18 levels in generation of pathological γδ T cells that was confirmed by detection of increases in CD18(low) γδ T cells in psoriasis patients and may also have implications for other inflammatory diseases.

Reduced CD18 levels drive regulatory T cell conversion into Th17 cells in the CD18hypo PL/J mouse model of psoriasis.

Defective development and function of CD4(+)CD25(high+)Foxp3(+) regulatory T cells (Tregs) contribute to the pathogenesis of psoriasis and other autoimmune diseases. Little is known about the influence of adhesions molecules on the differentiation of Foxp3(+) Tregs into proinflammatory Th17 cells occurring in lesional skin and blood of psoriasis patients. In the CD18(hypo) PL/J mouse model of psoriasis, reduced expression of CD18/ß2 integrin to 2-16% of wild-type levels is associated with progressive loss of Tregs, impaired cell-cell contact between Tregs and dendritic cells (DCs), as well as Treg dysfunction as reported earlier. In the present investigation, Tregs derived from CD18(hypo) PL/J mice were analyzed for their propensity to differentiate into IL-17-producing Th17 cells in vivo and in in vitro Treg-DC cocultures. Adoptively transferred CD18(hypo) PL/J Tregs were more inclined toward conversion into IL-17-producing Th17 cells in vivo in an inflammatory as well as noninflammatory environment compared with CD18(wt) PL/J Tregs. Addition of neutralizing Ab against CD18 to Treg-DC cocultures in vitro promoted conversion of CD18(wt) PL/J Tregs to Th17 cells in a dose-dependent manner similar to conversion rates of CD18(hypo) PL/J Tregs. Reduced thymic output of naturally occurring Tregs and peripheral conversion of Tregs into Th17 cells therefore both contribute to the loss of Tregs and the psoriasiform dermatitis observed in CD18(hypo) PL/J mice. Our data overall indicate that CD18 expression levels impact Treg development as well as Treg plasticity and that differentiation of Tregs into IL-17-producing Th17 cells is distinctly facilitated by a subtotal deficiency of CD18.

Cancer chemoprevention with dietary isothiocyanates mature for clinical translational research.

Inverse association between dietary intake of cruciferous vegetables and cancer risk observed in population-based case-control studies is partly attributable to structurally simple but mechanistically complex phytochemicals with an isothiocyanate (-N=C=S) functional group. Cancer protective role for dietary isothiocyanates (ITCs) is substantiated by preclinical studies in rodent models. A common feature of many naturally occurring ITCs relates to their ability to cause growth arrest and cell death selectively in cancer cells. At the same time, evidence continues to accumulate to suggest that even subtle change in chemical structure of the ITCs can have a profound effect on their activity and mechanism of action. Existing mechanistic paradigm stipulates that ITCs may not only prevent cancer initiation by altering carcinogen metabolism but also inhibit post-initiation cancer development by suppressing many processes relevant to tumor progression, including cellular proliferation, neoangiogenesis, epithelial-mesenchymal transition, and self-renewal of cancer stem cells. Moreover, the ITCs are known to suppress diverse oncogenic signaling pathways often hyperactive in human cancers (e.g. nuclear factor-κB, hormone receptors, signal transducer and activator of transcription 3) to elicit cancer chemopreventive response. However, more recent studies highlight potential adverse effect of Notch activation by ITCs on their ability to inhibit migration of cancer cells. Mechanisms underlying ITC-mediated modulation of carcinogen metabolism, growth arrest, and cell death have been reviewed extensively. This article provides a perspective on bench-cage-bedside evidence supporting cancer chemopreventive role for some of the most promising ITCs. Structure-activity relationship and mechanistic complexity in the context of cancer chemoprevention with ITCs is also highlighted.