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Non-conventional fracture test geometries such as single cantilever bending, clamped beam bending and clamped wire bending have been developed over the years to suit the needs of micro- and nano-scale testing. The same geometries can also be used at the macro-scale if testing standards are well established. Development of length scale compatible geometries helps the materials engineer to seamlessly extract material properties across multiple length scales. This article proposes the crack driving force (or energy release rate G) criteria for the above three geometries. These geometries have found widespread use at the micro-scale and can be adapted to the macro-scale. Use of a global energy-based criterion instead of the local stress-based criteria (stress intensity factor K) has its own advantages, especially in multi-phase materials and interface-dominated structures which display an R-curve. Aspects of crack stability even under load control arising because of the geometric factors, especially the beam or wire aspect ratio, are discussed in this context. Validation of the compliance approach is carried out by comparing it to the J-integral extracted directly for a linear elastic material using extended finite element modeling and also using the analytical formulation for a double-cantilever beam specimen. Experimental evidence of the validity of the solutions in determining the fracture energy of a linear elastic brittle material, PMMA, as a homogeneous model system is shown.

It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient quality for further biochemical and structural characterization, as well as for use in consumer products, chemical processes and therapeutics. Native mass spectrometry (nMS) has become an important tool in protein analysis due to its ability to retain non-covalent interactions during measurements, making it possible to obtain protein structural information with high sensitivity and at high speed. Interferences from the presence of non-volatiles are typically alleviated by offline buffer exchange, which is time-consuming and difficult to automate. We provide a protocol for rapid online buffer exchange (OBE) nMS to directly screen structural features of pre-purified proteins, protein complexes or clarified cell lysates. In the liquid chromatography coupled to mass spectrometry (LC-MS) approach described in this protocol, samples in MS-incompatible conditions are injected onto a short size-exclusion chromatography column. Proteins and protein complexes are separated from small molecule non-volatile buffer components using an aqueous, non-denaturing mobile phase. Eluted proteins and protein complexes are detected by the mass spectrometer after electrospray ionization. Mass spectra can inform regarding protein sample purity and oligomerization, and additional tandem mass spectra can help to further obtain information on protein complex subunits. Information obtained by OBE nMS can be used for fast (

Heterodimeric interaction specificity between two DNA strands, and between protein and DNA, is often achieved by varying side chains or bases coming off the protein or DNA backbone -- for example, the bases participating in Watson-Crick base pairing in the double helix, or the side chains of protein contacting DNA in TALEN-DNA complexes. This modularity enables the generation of an essentially unlimited number of orthogonal DNA-DNA and protein-DNA heterodimers. In contrast, protein-protein interaction specificity is often achieved through backbone shape complementarity (1), which is less modular and hence harder to generalize. Coiled coil heterodimers are an exception, but the restricted geometry of interactions across the heterodimer interface (primarily at the heptad a and d positions (2)) limits the number of orthogonal pairs that can be created simply by varying sidechain interactions (3,4). Here we demonstrate that heterodimeric interaction specificity can be achieved using extensive and modular buried hydrogen bond networks. We used the Crick generating equations (5) to produce millions of four helix backbones with varying degrees of supercoiling around a central axis, identified those accommodating extensive hydrogen bond networks, and used Rosetta to connect pairs of helices with short loops and optimize the remainder of the sequence. 65 of 97 such designs expressed in E. coli formed constitutive heterodimers, and crystal structures of four designs were in close agreement with the computational models and confirmed the designed hydrogen bond networks. In cells, a set of six heterodimers were found to be fully orthogonal, and in vitro, following mixing of 32 chains from sixteen heterodimer designs, denaturation in 5M GdnHCl and reannealing, the vast majority of the interactions observed by native mass spectrometry were between the designed cognate pairs. The ability to design orthogonal protein heterodimers should enable sophisticated protein based control logic for synthetic biology, and illustrates that nature has not fully explored the possibilities for programmable biomolecular interaction modalities.

Nucleoside diphosphate kinases (NDKs) are ubiquitous enzymes that catalyze the transfer of the γ-phosphate moiety from an NTP donor to an NDP acceptor, crucial for maintaining the cellular level of nucleoside triphosphates (NTPs). The inability of trypanosomatids to synthesize purines de novo and their dependence on the salvage pathway makes NDK an attractive target to develop drugs for the diseases they cause. Here we report the discovery of novel inhibitors for Leishmania NDK based on the structural and functional characterization of purified recombinant NDK from Leishmania amazonensis. Recombinant LaNDK possesses auto-phosphorylation, phosphotransferase and kinase activities with Histidine 117 playing an essential role. LaNDK crystals were grown by hanging drop vapour diffusion method in a solution containing 18% PEG-MME 500, 100 mM Bis-Tris propane pH 6.0 and 50 mM MgCl2. It belongs to the hexagonal space group P6322 with unit cell parameters a = b = 115.18, c = 62.18 A and α = β = 90°, γ = 120°. The structure solved by molecular replacement methods was refined to crystallographic R-factor and Rfree values of 22.54 and 26.52%, respectively. Molecular docking and dynamics simulation-based virtual screening identified putative binding compounds. Protein inhibition studies of selected hits identified five inhibitors effective at micromolar concentrations. One of the compounds showed ~45% inhibition of Leishmania promastigotes proliferation. Analysis of inhibitor-NDK complexes reveals the mode of their binding, facilitating design of new compounds for optimization of activities as drugs against leishmaniasis.

The aryl hydrocarbon receptor (AHR) is a multi-domain cytosolic protein that belongs to the basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) family of transcription factors. Ligand binding induces a conformational change in AHR and promotes nuclear translocation of the receptor (Kewley et al. 2004). AHR can either bind to exogenous (polycyclic aromatic hydrocarbons, dioxins, cigarette smoke) or endogenous ligands (arachidonic acid and leukotrienes, heme metabolites, UV photoproducts of tryptophan) within the cytoplasm. Exogenous ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3-methylcholanthrene are known to activate AHR and mediate cellular toxic response. AHR is also activated by dietary compounds including indole-3-carbinol and flavonoids that mediate various physiological activities in the body (Nguyen and Bradfield 2008; Marconett et al. 2010). AHR ligands are classified as agonists and antagonists depending on the ability of ligands to activate or inhibit AHR induced activity. Previous reports indicate that ligands such as kaempferol, resveratrol, galangin, chrysin and quercetin act either as agonists or antagonists based on the ligand concentration and type of cells induced (Zhang et al. 2003). Thus, diversity of ligands makes AHR signaling a very dynamic and complex. AHR in its inactive state is located in the cytoplasm and forms a complex with molecular chaperones, such as heat shock protein 90 (HSP90) and co-chaperons such as p23 and AHR-interacting protein (AIP) (Trivellin and Korbonits 2011). In presence of ligand, AHR undergoes nuclear translocation where it interacts with AHR nuclear transporter (ARNT) or AHR repressor (AHRR) through the PAS domain. It has been reported that nucleocytoplasmic transport mechanism of AHR varies between humans and mice. In humans, AHR both in stimulated or unstimulated state can undergo nuclear translocation complexed with AIP. In contrast, association of AIP prevents nucleocytoplasmic shuttling of AHR in mice in both stimulated and unstimulated states (Ramadoss et al. 2004). Once AHR is translocated to the nucleus it forms a heterodimer complex with ARNT and binds to xenobiotic response elements located in the promoter region of the target genes. This complex induces coordinated transcription of detoxifying enzymes for efficient absorption, distribution and elimination of xenobiotics from the body (Abel and Haarmann-Stemmann 2010). Apart from this, AHR is known to exhibit endogenous functions such as cell proliferation, cell differentiation and apoptosis. It also acts as an endogenous regulator in several developmental and physiological processes including neurogenesis, hematopoietic stem cell regulation, cellular stress response, immunoregulation and reproductive health (Lindsey and Papoutsakis 2012; Kadow et al. 2011; Hansen et al. 2014). AHR is associated with various pathological and physiological disorders in the body including autoimmune diseases (Veldhoen et al. 2008), inflammation (Podechard et al. 2008; Ovrevik et al. 2014), cardiovascular diseases (Kerley-Hamilton et al. 2012; Savouret et al. 2003) and cancer. Activation of AHR in presence of cigarette smoke has been well documented in lung cancer (Martey et al. 2005; Tsay et al. 2013). Cigarette smoke induced AHR is also known to mediate immune signaling mechanism in chronic obstructive pulmonary disease (COPD) (Chen et al. 2011). Apart from playing an essential role in COPD and lung cancer, AHR expression has been reported in other cancers and adenomas. AHR is reported to be downregulated in growth hormone secreting pituitary adenomas (Jaffrain-Rea et al. 2009), while, increased expression levels of AHR are associated with tumorigenesis in medulloblastoma (Dever and Opanashuk 2012). The enhancement of AHR levels under ligand stimulation induced cell cycle arrest has been reported in pancreatic and gastric cancer (Koliopanos et al. 2002; Peng et al. 2009). Depending upon the type of ligand stimulation, AHR is either known to promote or inhibit tumor progression. Stimulation of AHR with TCDD, 2,3,7,8-tetrachlorodibenzofuran and 3,3′-diindolylmethane inhibits invasiveness and cell growth in breast cancer (Hall et al. 2010). In contrast, stimulation of AHR with n-butyl benzyl phthalate and dibutyl phthalate ligands enhances tumorigenic properties in breast cancer cells (Hsieh et al. 2012). Therefore, AHR could serve as a potential therapeutic target in several cancers (Murray et al. 2014) and hence it is important to develop AHR signaling pathway to understand the mechanism of AHR mediated tumor progression and regression. Although, the diverse role of AHR is documented in literature however a detailed network of AHR signaling is lacking. In this study, we have curated literature information pertaining to AHR induced signaling and developed a pathway map to facilitate better understanding of this receptor.

Obesity is implicated as an important factor in the rising incidence of liver cancer in the United States. Bariatric surgery is increasingly used for treating morbid obesity and comorbidities. Using administrative data from UHC, a consortium of academic medical centers in the US, we compared the prevalence of liver cancer among admissions with and without a history of bariatric surgery within a 3-year period. Admissions with a history of bariatric surgery had a 61% lower prevalence of liver cancer compared to those without a history of bariatric surgery (prevalence ratio 0.39, 95% confidence interval 0.35 – 0.44), and these inverse associations persisted within strata of sex, race, and ethnicity. This hospital administrative records-based analysis suggests that bariatric surgery could play a role in liver cancer prevention.

Interleukin-17 (IL-17) belongs to a relatively new family of cytokines that has garnered attention as the signature cytokine of Th17 cells. This cytokine family consists of 6 ligands, which bind to 5 receptor subtypes and induce downstream signaling. Although the receptors are ubiquitously expressed, cellular responses to ligands vary across tissues. The cytokine family is associated with various autoimmune disorders including rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, asthma and psoriasis in addition to being implicated in the pathogenesis of cancer. In addition, this family plays a role in host defense against bacterial and fungal infections. The signaling mechanisms of the IL-17 family of proinflammatory cytokines are not well explored. In this study, we present a resource of literature-annotated reactions induced by IL-17. The reactions are catalogued under 5 categories, namely; molecular association, catalysis, transport, activation/inhibition and gene regulation. A total of 93 molecules and 122 reactions have been annotated. The IL-17 pathway is freely available through NetPath, a resource of signal transduction pathways previously developed by our group.

Background: Hepatocellular carcinoma (HCC) occurs less commonly among women than men in almost all regions of the world. The disparity in risk is particularly notable prior to menopause suggesting that hormonal exposures during reproductive life may be protective. Exogenous oestrogenic exposures such as oral contraceptives (OCs), however, have been reported to increase risk, suggesting that estrogens may be hepatocarcinogenic. To examine the effects of reproductive factors and exogenous hormones on risk, we conducted a prospective analysis among a large group of US women. Methods: In the Liver Cancer Pooling Project, a consortium of US-based cohort studies, data from 799 500 women in 11 cohorts were pooled and harmonised. Cox proportional hazards regression models were used to generate hazard ratios (HRs) and 95% confidence intervals (CIs) for the associations of reproductive factors and exogenous hormones with HCC (n=248). Results: Bilateral oophorectomy was associated with a significantly increased risk of HCC (HR=2.67, 95% CI=1.22–5.85), which did not appear to be related to a shorter duration of exposure to endogenous hormones or to menopausal hormone therapy use. There was no association between OC use and HCC (HR=1.12, 95% CI=0.82–1.55). Nor were there associations with parity, age at first birth, age at natural menopause, or duration of fertility. Conclusions: The current study suggests that bilateral oophorectomy increases the risk of HCC but the explanation for the association is unclear. There was no association between OC use and HCC risk. Examination of endogenous hormone levels in relation to HCC may help to clarify the findings of the current study.

EZH2 (enhancer of zeste homolog 2) is a critical enzymatic subunit of the polycomb repressive complex 2 (PRC2), which trimethylates histone H3 (H3K27) to mediate gene repression. Somatic mutations, overexpression and hyperactivation of EZH2 have been implicated in the pathogenesis of several forms of cancer. In particular, recurrent gain-of-function mutations targeting EZH2 Y641 occur most frequently in follicular lymphoma and aggressive diffuse large B-cell lymphoma and are associated with H3K27me3 hyperactivation, which contributes to lymphoma pathogenesis. However, the post-translational mechanisms of EZH2 regulation are not completely understood. Here we show that EZH2 is a novel interactor and substrate of the SCF E3 ubiquitin ligase β-TrCP (FBXW1). β-TrCP ubiquitinates EZH2 and Jak2-mediated phosphorylation on Y641 directs β-TrCP-mediated EZH2 degradation. RNA interference-mediated silencing of β-TrCP or inhibition of Jak2 results in EZH2 stabilization with attendant increase in H3K27 trimethylation activity. Importantly, the EZH2(Y641) mutants recurrently implicated in lymphoma pathogenesis are unable to bind β-TrCP. Further, endogenous EZH2(Y641) mutants in lymphoma cells exhibit increased EZH2 stability and H3K27me3 hyperactivity. Our studies demonstrate that β-TrCP has an important role in controlling H3K27 trimethylation activity and lymphoma pathogenesis by targeting EZH2 for degradation.

Background Poor prognosis in gallbladder cancer is due to late presentation of the disease, lack of reliable biomarkers for early diagnosis and limited targeted therapies. Early diagnostic markers and novel therapeutic targets can significantly improve clinical management of gallbladder cancer. Methods Proteomic analysis of four gallbladder cancer cell lines based on the invasive property (non-invasive to highly invasive) was carried out using the isobaric tags for relative and absolute quantitation labeling-based quantitative proteomic approach. The expression of macrophage migration inhibitory factor was analysed in gallbladder adenocarcinoma tissues using immunohistochemistry. In vitro cellular assays were carried out in a panel of gallbladder cancer cell lines using MIF inhibitors, ISO-1 and 4-IPP or its specific siRNA. Results The quantitative proteomic experiment led to the identification of 3,653 proteins, among which 654 were found to be overexpressed and 387 were downregulated in the invasive cell lines (OCUG-1, NOZ and GB-d1) compared to the non-invasive cell line, TGBC24TKB. Among these, macrophage migration inhibitory factor (MIF) was observed to be highly overexpressed in two of the invasive cell lines. MIF is a pleiotropic proinflammatory cytokine that plays a causative role in multiple diseases, including cancer. MIF has been reported to play a central role in tumor cell proliferation and invasion in several cancers. Immunohistochemical labeling of tumor tissue microarrays for MIF expression revealed that it was overexpressed in 21 of 29 gallbladder adenocarcinoma cases. Silencing/inhibition of MIF using siRNA and/or MIF antagonists resulted in a significant decrease in cell viability, colony forming ability and invasive property of the gallbladder cancer cells. Conclusions Our findings support the role of MIF in tumor aggressiveness and suggest its potential application as a therapeutic target for gallbladder cancer. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1855-z) contains supplementary material, which is available to authorized users.

Background Rheumatoid arthritis and osteoarthritis are two common musculoskeletal disorders that affect the joints. Despite high prevalence rates, etiological factors involved in these disorders remain largely unknown. Dissecting the molecular aspects of these disorders will significantly contribute to improving their diagnosis and clinical management. In order to identify proteins that are differentially expressed between these two conditions, a quantitative proteomic profiling of synovial fluid obtained from rheumatoid arthritis and osteoarthritis patients was carried out by using iTRAQ labeling followed by high resolution mass spectrometry analysis. Results We have identified 575 proteins out of which 135 proteins were found to be differentially expressed by ≥3-fold in the synovial fluid of rheumatoid arthritis and osteoarthritis patients. Proteins not previously reported to be associated with rheumatoid arthritis including, coronin-1A (CORO1A), fibrinogen like-2 (FGL2), and macrophage capping protein (CAPG) were found to be upregulated in rheumatoid arthritis. Proteins such as CD5 molecule-like protein (CD5L), soluble scavenger receptor cysteine-rich domain-containing protein (SSC5D), and TTK protein kinase (TTK) were found to be upregulated in the synovial fluid of osteoarthritis patients. We confirmed the upregulation of CAPG in rheumatoid arthritis synovial fluid by multiple reaction monitoring assay as well as by Western blot. Pathway analysis of differentially expressed proteins revealed a significant enrichment of genes involved in glycolytic pathway in rheumatoid arthritis. Conclusions We report here the largest identification of proteins from the synovial fluid of rheumatoid arthritis and osteoarthritis patients using a quantitative proteomics approach. The novel proteins identified from our study needs to be explored further for their role in the disease pathogenesis of rheumatoid arthritis and osteoarthritis. Sartaj Ahmad and Raja Sekhar Nirujogi contributed equally to this article.

Methods We modeled our infrastructure after a successful PEPFARfunded HIV care and treatment program and selected HIVinfected women as our initial target population. Zambian nurses underwent classroom and clinically-mentored training to become primary service providers for screening women for cervical lesions using visual inspection with acetic acid (VIA) and treatment with cryotherapy, when indicated. Women with cryotherapy-ineligible lesions were referred to the university hospital where physicians were trained to perform punch biopsy and loop electrosurgical excision procedure (LEEP) for histologic evaluation. We utilized telecervicography for distance consultation as well as reviewing digital images weekly for quality assurance and continuing education. Patients with invasive cancer were referred for hysterectomy, radiation or palliation, depending on the stage of their disease