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Background Lameness is a debilitating condition in equine athletes that leads to more performance limitation and loss of use than any other medical condition. There are a limited number of non-terminal experimental models that can be used to study early inflammatory and synovial fluid biophysical changes that occur in the equine joint. Here, we compare the well-established carpal IL-1β-induced synovitis model to a tarsal intra-articular lavage model, focusing on serial changes in synovial fluid inflammatory cytokines/chemokines and the synovial fluid lubricating molecules lubricin/proteoglycan 4 and hyaluronic acid. The objectives of this study were to evaluate clinical signs; synovial membrane and synovial fluid inflammation; and synovial fluid lubricants and biophysical properties in response to carpal IL-1β synovitis and tarsal intra-articular lavage. Results Hyaluronic acid (HA) concentrations, especially high molecular weight HA, and synovial fluid viscosity decreased after both synovitis and lavage interventions. Synovial fluid lubricin concentrations increased 17–20-fold for both synovitis and lavage models, with similar changes in both affected and contralateral joints, suggesting that repeated arthrocentesis alone resulted in elevated synovial fluid lubricin concentrations. Synovitis resulted in a more severe inflammatory response based on clinical signs (temperature, heart rate, respiratory rate, lameness and joint effusion) and clinicopathological and biochemical parameters (white blood cell count, total protein, prostaglandin E2, sulfated glycosaminoglycans, tumor necrosis factor-α and CC chemokine ligands − 2, − 3, − 5 and − 11) as compared to lavage. Conclusions Synovial fluid lubricin increased in response to IL-1β synovitis and joint lavage but also as a result of repeated arthrocentesis. Frequent repeated arthrocentesis is associated with inflammatory changes, including increased sulfated glycosaminoglycan concentrations and decreased hyaluronic acid concentrations. Synovitis results in more significant inflammatory changes than joint lavage. Our data suggests that synovial fluid lubricin, TNF-α, CCL2, CCL3, CCL5, CCL11 and sGAG may be useful biomarkers for synovitis and post-lavage joint inflammation. Caution should be exercised when performing repeated arthrocentesis clinically or in experimental studies due to the inflammatory response and loss of HA and synovial fluid viscosity.

For many years it has been postulated the role of dietary factors, in particular the consumption of fatty acids, in the development of cancer. It has been confirmed in numerous epidemiological observations, as well as the experimental studies on cell lines. Despite numerous studies the role of various fatty acids and their metabolites, has not been clearly established. Among others there are still under investigation on the importance of linoleic acid, belonging to the group of polyunsaturated omega-6. Because of the diversity of scientific reports authors of this publication are trying to make a critical synthesis analysis of current knowledge of the postulated role of linoleic acid in development of colon cancer. And they also attempt to draw practical conclusions from them. The ability to modify their lifestyle, including dietary habits, and their real impact on the development of malignancies is a very important area of research, and can have an impact on the cancer epidemiology. Based on currently available data, dietary recommendations can be made for individual polyunsaturated fatty acid. It is recommended that in adults the LA share was 4% of the energy value of the diet, while the α-linolenic acid share was 0.5% of the energy value of the diet. In addition, in order to provide the right amount of docosahexaenoic acid and eicosapentaenoic acid, it is recommended to include at least 2 portions of fish per week, including 1 portion of oily fish.

Heat shock can induce either cytoprotective mechanisms or cell death. We found that in certain human and mouse cells, including spermatocytes, activated heat shock factor 1 (HSF1) binds to sequences located in the intron(s) of the PMAIP1 (NOXA) gene and upregulates its expression which induces apoptosis. Such a mode of PMAIP1 activation is not dependent on p53. Therefore, HSF1 not only can activate the expression of genes encoding cytoprotective heat shock proteins, which prevents apoptosis, but it can also positively regulate the proapoptotic PMAIP1 gene, which facilitates cell death. This could be the primary cause of hyperthermia-induced elimination of heat-sensitive cells, yet other pro-death mechanisms might also be involved.

Background Ability to adapt to temperature changes trough the Heat Shock Response (HSR) pathways is one of the most fundamental and clinically relevant cellular response systems. Heat Shock (HS) affects the signalling and gene expression responses of the Nuclear Factor κB (NF-κB) transcription factor, a critical regulator of proliferation and inflammation, however, our quantitative understanding of how cells sense and adapt to temperature changes is limited. Methods We used live-cell time-lapse microscopy and mathematical modelling to understand the signalling of the NF-κB system in the human MCF7 breast adenocarcinoma cells in response to pro-inflammatory Interleukin 1β (IL1β) and Tumour Necrosis Factor α (TNFα) cytokines, following exposure to a 37–43 °C range of physiological and clinical temperatures. Results We show that exposure to 43 °C 1 h HS inhibits the immediate NF-κB signalling response to TNFα and IL1β stimulation although uptake of cytokines is not impaired. Within 4 h after HS treatment IL1β-induced NF-κB responses return to normal levels, but the recovery of the TNFα-induced responses is still affected. Using siRNA knock-down of Heat Shock Factor 1 (HSF1) we show that this stimulus-specificity is conferred via the Inhibitory κB kinase (IKK) signalosome where HSF1-dependent feedback regulates TNFα, but not IL1β-mediated IKK recovery post HS. Furthermore, we demonstrate that through the temperature-dependent denaturation and recovery of IKK, TNFα and IL1β-mediated signalling exhibit different temperature sensitivity and adaptation to repeated HS when exposed to a 37–43 °C temperature range. Specifically, IL1β-mediated NF-κB responses are more robust to temperature changes in comparison to those induced by TNFα treatment. Conclusions We demonstrate that the kinetics of the NF-κB system following temperature stress is cytokine specific and exhibit differential adaptation to temperature changes. We propose that this differential temperature sensitivity is mediated via the IKK signalosome, which acts as a bona fide temperature sensor trough the HSR cross-talk. This novel quantitative understanding of NF-κB and HSR interactions is fundamentally important for the potential optimization of therapeutic hyperthermia protocols.

Environmental toxicants such as heavy metals from contaminated water or soil and isothiocyanates (ITC) from dietary sources act as pro-oxidants by directly generating reactive oxygen species (ROS) or through depleting cellular antioxidants such as glutathione. Toxicants can alter drug metabolism, and it was reported that CYP2B10 and UGT1A1 are induced by phenethyl isothiocyanate (PEITC) through the constitutive androstane receptor (CAR). The possibility that nuclear factor erythroid 2-related factor 2 (NRF2), the master regulator of the antioxidant response, could coactivate CAR was investigated in neonatal hUGT1/Nrf2(−/−) mice. Neonatal mice were treated with PEITC or cadmium (Cd(2+)) by oral gavage for 2 days. Both PEITC and Cd(2+) induced UGT1A1 RNA and protein in intestinal tissues in both hUGT1/Nrf2(+/−) and hUGT1/Nrf2(−/−) neonates, indicating NRF2-independent regulation of UGT1A1. Increases in CYP2B10 RNA in intestinal tissues were observed following PEITC or Cd(2+) exposure. Activation of intestinal CAR by Cd(2+) exposure was directly assessed by nuclear fractionation and Western blot analyses at 0.5, 1, 2, and 4 hours after treatment in hUGT1 neonates and after 48 hours in hUGT1/Nrf2(+/−) and hUGT1/Nrf2(−/−) neonates. CAR localized to the nucleus independently of NRF2 48 hours after exposure. Substantial CAR localization to the nucleus occurred at the 2- and 4-hour time points, coinciding with a decrease in phosphorylation of cytoplasmic extracellular signal-regulated kinases 1 and 2 and a nuclear increase in P38/p-P38 content. This suggests that a novel oxidative stress-MAPK-CAR axis exists with phenotypic consequences. SIGNIFICANCE STATEMENT: Pro-oxidant toxicants can alter drug metabolism through activation of CAR, independent of the NRF2-KEAP1 signaling pathway. Changes in proteins associated with drug metabolism and linked to increases in intestinal maturation are mediated through an oxidative stress-MAPK-CAR axis.

Recent advances in fluorescence microscopy have enabled the visualization of subcellular structures at unprecedented resolution. However, the complexity of state-of-the-art microscopes has increased considerably, often requiring the precise control and synchronization of multiple peripheral devices at high speeds. Drawing inspiration from open-source prototyping systems, like the Arduino, we describe the development of a new control platform that adopts the best features of these systems – affordability, facile programmability, and flexible connectivity – but with the scientific-grade inputs and outputs (I/O) and built-in routines that are necessary to control peripherals in advanced microscopy applications. Notably, our platform includes waveform generators and I/O for point-and azimuthal-scanning of excitation in laser-based applications. As a proof of concept, we show how the integration of waveform generation, multiplexed analog outputs, and native hardware triggers into a single central hub provides a versatile platform for performing fast circle-scanning acquisitions, including ring scanning-angle interference microscopy (SAIM), total internal reflection fluorescence (TIRF and ring TIRF) microscopy, and multiangle TIRF (MA-TIRF). We also demonstrate how the low communication latency of our hardware platform can reduce image intensity and reconstruction artifacts arising from synchronization errors produced by software control. Our complete platform, including hardware design files, firmware, API, software, and all associated source code, will be hosted for community-based development and collaboration.

Early detection of the most common pediatric neoplasm, B-cell precursor lymphoblastic leukemia (BCP-ALL), is challenging and requires invasive bone marrow biopsies. The purpose of this study was to establish new biomarkers for early screening to detect pediatric leukemia. In this small cohort study, Fourier transform infrared (FTIR) spectra were obtained from blood sera of 10 patients with BCP-ALL and were compared with the control samples from 10 children with some conditions other than neoplasm. Using various analytical approaches, including a new physical model, some significant differences were observable. The most important include: the different peak area ratio 2965/1645 cm−1 (p = 0.002), the lower average percentage of both β-sheet and β-turn protein structures in the sera of BCP-ALL patients (p = 0.03), an AdaBoost-based predictive model for classifying healthy vs. BCP-ALL patients with 85% accuracy, and the phase shift of the first derivative in the spectral range 1050–1042 cm−1 correlating with white blood cell (WBC) and blast cell count in BCP-ALL patients contrary to the samples obtained from healthy controls. Although verification in larger groups of patients will be necessary, these promising results suggest that FTIR spectroscopy may have future potential for the early screening of BCP-ALL.

Atherosclerosis remains a large health and economic burden. Even though it has been studied for more than a century, its complex pathophysiology has not been elucidated. The relatively well-established contributors include: chronic inflammation in response to oxidized cholesterol, reactive oxygen species-induced damage and apoptosis. Recently, profilin 1, a regulator of actin dynamics emerged as a potential new player in the field. Profilin is abundant in stable atherosclerotic plaques and in thrombi extracted from infarct-related arteries in patients with acute myocardial infarction. The exact role of profilin in atherosclerosis and its complications, as well as its mechanisms of action, remain unknown. Here, we summarize several pathways in which profilin may act through mitochondria in a number of processes implicated in atherosclerosis.

Background and Objectives:Currently, diabetes is becoming a very serious challenge in medicine, type 2 diabetes mellitus (T2DM) is referred to as a noncontagious epidemic of the 21st century. The aim of the study is to assess the lifestyle of patients with type 2 diabetes, in particular eating habits, physical activity, and tobacco smoking, and to compare the obtained values with the latest recommendations of the Polish Society of Diabetology. Materials and Methods: The study group was comprised of 460 patients with type 2 diabetes, treated in the Diabetes Outpatient Clinic (n = 299) and the Clinical Department of Internal Medicine (n = 166). The study was conducted using a questionnaire, which included the Food Intake Frequency Questionnaire, 24h dietary recall, and the International Physical Activity Questionnaire. Results: Abnormal excessive consumption of saturated fatty acids and salt and insufficient intake of dietary fiber was observed in the study group. Physical activity was insufficient in over 50% of the study group. In the study group, 17% of patients were smokers and the mean number of pack-years was 23. Conclusions:In the study group, excessive fat consumption (i.e.,saturated fatty acids) and dietary fiber deficiency were found in the daily diet.

Summary Background & aims Abnormalities of parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23) secretion may cause calcium-phosphate (Ca–P) metabolism disorders in nephrolithiasis. Post-phosphate-load alterations in serum Ca, P and PTH, phosphaturia and calciuria enable monitoring hormonal regulation of Ca–P homeostasis. Our study aimed to determine differences in: 1.selected Ca–P metabolism parameters between healthy and kidney-stone-forming individuals, 2.PTH and FGF23 secretion induced by sodium-phosphate-load(NaP-load) in patients with/without hypercalciuria, 3.secretion of Ca–P related hormones in patients with low and normal/high serum concentrations of 25-hydroxyvitamin D3 (25OHD3). Methods Sodium phosphates NaH2PO4/Na2HPO4-100mmol were administered orally for five days in 19 hypercalciuric [urinary Ca(U–Ca) 6.5 ± 1.7 mmol/d]–HSF, 35 normocalciuric (2.5±1 mmol/d)–NSF stone-forming patients and 19 controls (U–Ca 2.5 ± 1.4 mmol/d)–CG. On days 1 and 5 PTH-,FGF23-,Ca-,P were determined before and after NaP-load. The areas under PTH, FGF23 curves (AUC) were calculated. U–Ca, urinary phosphate (U–P) and sodium (U–Na) were also determined. Results Following NaP-load, patients and controls exhibited expected alterations in Ca–P homeostasis. Despite changes in phosphate and PTH, no differences in FGF23 concentrations were observed. Patients differed from controls in having higher AUCPTH, calciuria and natriuresis, taking longer for PTH and P to normalize and lack of correlation between AUCPTH and phosphaturia. Post-NaP-load hypocalciuric effect of PTH secretion in NSF was less pronounced than in CG. In the HSFs, the hypocalciuric effect was more pronounced than in NSFs, but insufficient to correct hypercalciuria. In all stone-formers with low 25OHD3 concentrations, the AUCFGF23 was significantly increased on first (1215 ± 605vs766 ± 315 p = 0.0457) and fifth days (1211 ± 641vs777 ± 299 p = 0.041) of NaP-load, compared to normal/high 25OHD3-patients. Hypercalciuric patients with low 25OHD3 concentrations had greater AUCPTH5 than those with normal/high 25OHD3 (1005 ± 401vs835 ± 220 p = 0.0341). Conclusions Compared to controls, kidney-stone-forming patients exhibited enhanced PTH secretion after NaP-load. The HSFs showed a more pronounced hypocalciuric effect than NSFs, but insufficient to correct hypercalciuria. In hypercalciuric stone-formers with low 25OHD3, FGF23 engagement in hyperphosphatemia reduction increased.

The glycocalyx coating the outside of most cells is a polymer meshwork comprising proteins and complex sugar chains called glycans. From a physical perspective, the glycocalyx has long been considered a simple 'slime' that protects cells from mechanical disruption or against pathogen interactions, but the great complexity of the structure argues for the evolution of more advanced functionality: the glycocalyx serves as the complex physical environment within which cell-surface receptors reside and operate. Recent studies have demonstrated that the glycocalyx can exert thermodynamic and kinetic control over cell signalling by serving as the local medium within which receptors diffuse, assemble and function. The composition and structure of the glycocalyx change markedly with changes in cell state, including transformation. Notably, cancer-specific changes fuel the synthesis of monomeric building blocks and machinery for production of long-chain polymers that alter the physical and chemical structure of the glycocalyx. In this Review, we discuss these changes and their physical consequences on receptor function and emergent cell behaviours.

The mouse 3110001I22Rik gene located in the first intron of Bfar is considered as a Bfar variant coding for the BFARv3 protein. However, it differs from other BFAR isoforms and resembles periphilin 1 (PPHLN1) due to its two (Lge1 and serine-rich) conserved domains. We identified the BFARv3/EGFP-interacting proteins by co-immunoprecipitation coupled to mass spectrometry, which revealed 40S ribosomal proteins (RPS3, RPS14, RPS19, RPS25, RPS27), histones (H1.2, H1.4, H3.3C), proteins involved in RNA processing and splicing (SFPQ, SNRPA1, HNRNPA3, NONO, KHDRBS3), calcium signaling (HPCAL1, PTK2B), as well as HSD17B4, GRB14, POSTN, and MYO10. Co-immunoprecipitation revealed that both Lge1 and Ser-rich domains of BFARv3 were necessary for binding to RNA-interacting factors NONO and SFPQ, known to be components of paraspeckles. Reciprocal co-immunoprecipitation and the proximity ligation assay confirmed that both BFARv3 and PPHLN1 could interact with NONO and SFPQ, suggesting a new function for PPHLN1 as well. BFARv3 and its Lge1 or Ser-rich-deficient mutants preferentially localize in the nucleus. We found an accumulation of BFARv3/EGFP (but not its mutated forms) in the nuclear granules, which was enhanced in response to arsenite treatment and ionizing radiation. Although Bfar v3 is expressed ubiquitously in mouse tissues, its expression is the highest in metaphase II oocytes. The BFARv3 interactome suggests its role in RNA metabolism, which is critical for the transcriptionally silent MII oocyte. Mouse BFARv3 has no ortholog in the human genome, thus it may contribute to the differences between these two species observed in oocyte maturation and early embryonic development.

Elevated temperature induces the heat shock (HS) response, which modulates cell proliferation, apoptosis, the immune and inflammatory responses. However, specific mechanisms linking the HS response pathways to major cellular signaling systems are not fully understood. Here we used integrated computational and experimental approaches to quantitatively analyze the crosstalk mechanisms between the HS-response and a master regulator of inflammation, cell proliferation, and apoptosis the Nuclear Factor κB (NF-κB) system. We found that populations of human osteosarcoma cells, exposed to a clinically relevant 43°C HS had an attenuated NF-κB p65 response to Tumor Necrosis Factor α (TNFα) treatment. The degree of inhibition of the NF-κB response depended on the HS exposure time. Mathematical modeling of single cells indicated that individual crosstalk mechanisms differentially encode HS-mediated NF-κB responses while being consistent with the observed population-level responses. In particular “all-or-nothing” encoding mechanisms were involved in the HS-dependent regulation of the IKK activity and IκBα phosphorylation, while others involving transport were “analogue”. In order to discriminate between these mechanisms, we used live-cell imaging of nuclear translocations of the NF-κB p65 subunit. The single cell responses exhibited “all-or-nothing” encoding. While most cells did not respond to TNFα stimulation after a 60 min HS, 27% showed responses similar to those not receiving HS. We further demonstrated experimentally and theoretically that the predicted inhibition of IKK activity was consistent with the observed HS-dependent depletion of the IKKα and IKKβ subunits in whole cell lysates. However, a combination of “all-or-nothing” crosstalk mechanisms was required to completely recapitulate the single cell data. We postulate therefore that the heterogeneity of the single cell responses might be explained by the cell-intrinsic variability of HS-modulated IKK signaling. In summary, we show that high temperature modulates NF-κB responses in single cells in a complex and unintuitive manner, which needs to be considered in hyperthermia-based treatment strategies., Author summary Hyperthermia has been considered a promising strategy to sensitize cancer cells to treatment. As such, it might potentially increase treatment efficacy while reducing negative side effects. So far, this potential has not been fully realized. One of the major obstacles is a lack of quantitative understanding of crosstalk mechanisms involved in the heat shock and other cellular stress-factor systems. In this work, we used experimental population-level and live-cell imaging approaches to characterize the NF-κB signaling system response to physiologically high temperature. We then iteratively applied mathematical modeling of single cells to infer the underlying regulatory mechanisms. We showed experimentally that elevated temperature attenuated cytokine-induced NF-κB system responses, resulting in complex and unintuitive single cell behavior. Our analyses suggested that these responses are consistent with stochastic cell-intrinsic heat-shock-mediated regulation of the NF-κB system via the IKK signaling module. Considering the oncogenic associations of the NF-κB pathway, the crosstalk mechanisms described in this work are important for the understanding of chemo- and radio-resistance to treatment. However, more but similar, quantitative studies are required to ultimately change currently used therapy protocols.

Objectives: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited treatment options and poor prognosis. TNBC is usually diagnosed at a relatively young age and is characterized by high risk of developing metastases. Some epigenetic regulation of gene expression is associated with TNBC. Expression of microRNAs (miRNAs) can serve as a potential tool for identifying critical biomarkers in TNBC. The aim of our study is to examine expression of selected miRNAs in TNBC and to assess the relationship between miRNA expression and clinicopathological factors. Material and methods: Expression levels of 19 selected miRNAs were compared between cancerous and normal breast tissues by use of qPCR method. We have evaluated the relationship between the expression level of miRNAs and clinicopathological factors such as: age, tumor size and lymph node status. Results: We found that in TNBC tissues, when compared with normal breast tissues, the expression of miR-190a, miR- 136-5p and miR-126-5p was significantly reduced (p = 0.0041, p = 0.0007, p = 0.0007, respectively) whereas expression of miR-135b-5p and miR-182-5p was significantly increased (p = 0.0194, p = 0.0041, respectively). We found a linear trend for tumor size and expression of miR-126-5p (p = 0.0296) and miR-135b-5p (p = 0.0241). Conclusions: Our study confirms that miRNA expression profile is dysregulated in TNBC patients compared to healthy controls. MiR-190a, miR-136-5p, miR-126-5p, miR-135b-5p and miR-182-5p may be associated with development and progression of TNBC

The gastrointestinal tract is enriched with xenobiotic processing proteins that play important roles in xenobiotic bioactivation, metabolism, and detoxification. The application of genetically modified mouse models has been instrumental in characterizing the function of xenobiotic processing genes (XPG) and their proteins in drug metabolism. Here, we report the utilization of three-dimensional crypt organoid cultures from these animal models to study intestinal drug metabolism and toxicity. With the successful culturing of crypt organoids, we profiled the abundance of Phase I and Phase II XPG expression, drug transporter gene expression, and xenobiotic nuclear receptor (XNR) gene expression. Functions of XNRs were examined by treating crypt cells with XNR prototypical agonists. Real-time quantitative polymerase chain reaction demonstrated that the representative downstream target genes were induced. These findings were validated from cultures developed from XNR-null mice. In crypt cultures isolated from Pxr-/- mice, pregnenolone 16α-carbonitrile failed to induce Cyp3a11 gene expression; similarly, WY14643 failed to induce Cyp4a10 in the Pparα-/- crypts. Crypt cultures from control (Ugt1F/F ) and intestinal epithelial cell (IEC) specific Ugt1 null mice (Ugt1ΔIEC ) were treated with camptothecin-11, an anticancer prodrug with severe intestinal toxicity that originates from insufficient UGT1A1-dependent glucuronidation of its active metabolite SN-38. In the absence of Ugt1 gene expression, Ugt1ΔIEC crypt cultures exhibit very limited production of SN-38 glucuronide, concordant with increased apoptosis in comparison with Ugt1F/F crypt cultures. This study suggests crypt organoid cultures as an effective in vitro model for studying intestinal drug metabolism and toxicity.

Turner syndrome (TS) is characterized by the partial or complete loss of one sex chromosome and results in growth failure, gonadal insufficiency and cardiac anomalies. Treatment with growth hormone (GH) during childhood has indisputable benefits when taking into account the low stature of TS women. Medical records and biochemical findings of 33 TS women treated with GH in childhood (GH+) were compared to those of 124 TS women who did not receive GH (GH−). It seems that the GH-treated group might have had a more severe initial phenotype than the untreated group, as evidenced by higher FSH, more feeding issues in infancy, more lymphedema cases and urinary system malformations. GH+ women were significantly taller and had a better lipid profile and lower prevalence of arterial hypertension than GH− . However, they also had lower thrombocyte counts, a greater prevalence of retrognathism and nail anomalies, especially when the GH treatment was delayed. Long-term GH use was not as effective for growth as GH treatment during the initial period and seemed to have resulted in elevated creatinine levels. GH treatment in childhood has benefits in adulthood; however, adverse effects may occur, especially in individuals with treatment that is delayed or is too long.

Few approaches exist for the stable and controllable synthesis of customized mucin glycoproteins for glycocalyx editing in eukaryotic cells. Taking advantage of custom gene synthesis and a biology-by-parts approach to cDNA construction, we build a library of swappable DNA bricks for mucin leader tags, membrane anchors, cytoplasmic motifs, and optical reporters, as well as codon-optimized native mucin repeats and newly designed domains for synthetic mucins. We construct a library of over 50 mucins, each with unique chemical, structural, and optical properties and describe how additional permutations could readily be constructed. We apply the library to explore sequence-specific effects on glycosylation for engineering of mucins. We find that the extension of the immature α-GalNAc Tn-antigen to Core 1 and Core 2 glycan structures depends on the underlying peptide backbone sequence. Glycosylation could also be influenced through recycling motifs on the mucin cytoplasmic tail. We expect that the mucin parts inventory presented here can be broadly applied for glycocalyx research and mucin-based biotechnologies.

Background & Aims Colonic stem cells are essential for producing the mucosal lining, which in turn protects stem cells from insult by luminal factors. Discovery of genetic and biochemical events that control stem cell proliferation and differentiation can be leveraged to decipher the causal factors of ulcerative colitis and aid the development of more effective therapy. Methods We performed in vivo and in vitro studies from control (nuclear receptor corepressor 1 [NCoR1F/F]) and intestinal epithelial cell–specific NCoR1-deficient mice (NCoR1ΔIEC). Mice were challenged with dextran sodium sulfate to induce experimental ulcerative colitis, followed by colitis examination, barrier permeability analysis, cell proliferation immunostaining assays, and RNA sequencing analysis. By using crypt cultures, the organoid-forming efficiency, cell proliferation, apoptosis, and histone acetylation were analyzed after butyrate and/or tumor necrosis factor α treatments. Results NCoR1ΔIEC mice showed a dramatic increase in disease severity in this colitis model, with suppression of proliferative cells at the crypt base as an early event and a concomitant increase in barrier permeability. Genome expression patterns showed an important role for NCoR1 in colonic stem cell proliferation and secretory cell differentiation. Colonic organoids cultured from NCoR1ΔIEC mice were more sensitive to butyrate-induced cell growth inhibition and apoptosis, which were exaggerated further by tumor necrosis factor α co-treatment, which was accompanied by increased histone acetylation. Conclusions NCoR1 regulates colonic stem cell proliferation and secretory cell differentiation. When NCoR1 is disrupted, barrier protection is weakened, allowing luminal products such as butyrate to penetrate and synergistically damage the colonic crypt cells. Transcript profiling: RNA sequencing data have been deposited in the GEO database, accession number: GSE136153., Graphical abstract

The heterogeneous nature of inflammatory bowel disease (IBD) presents challenges, particularly when choosing therapy. Activation of the NF-κB transcription factor is a highly-regulated, dynamic event in IBD pathogenesis. We expressed the human NF-κB/p65 subunit in blood-derived macrophages, using lentivirus. Confocal imaging of p65 activation revealed that a higher proportion of macrophages from Crohn’s patients responded to lipid-A compared to controls. In contrast, cells from ulcerative colitis (UC) patients exhibited a shorter duration of p65 nuclear localisation compared to healthy controls and Crohn’s donors. Using a similar lentivirus approach, NF-κB-regulated luciferase was expressed in patient macrophages, isolated from frozen peripheral blood mononuclear cell samples. Following activation, samples could be segregated into three clusters based on the NF-κB-regulated luciferase response. The majority of UC samples appeared in hypo-responsive cluster 1, with Crohn’s patients representing the majority of hyper-responsive cluster 3. A positive correlation was seen between NF-κB-induced luciferase activity and cytokine levels released to medium from stimulated macrophages, but not in serum or biopsy. Analysis of macrophage cytokine responses and patient metadata revealed a strong correlation between Crohn’s patients who smoked and hyper-activation of p65. Thesein vitrodynamic assays of NF-κB activation in blood-derived macrophages segregate IBD patients into groups with different phenotypes and therefore may help determine response to therapy.Significance statementThis manuscript describes two dynamic assays of NF-κB activation in blood-derived macrophages that can segregate IBD patients into groups with different phenotypes. For the first time we introduce the use of dynamic measurements of a transcription factor activation as a method to stratify patients and we are confident that our approach will lead in future to early patient stratification and prediction of treatment outcome.

The specific targeting of differentially expressed glycans in malignant cells has emerged as an attractive anticancer strategy. One such target is the oncodevelopmental antigen polysialic acid (polySia), a polymer of α2,8-linked sialic acid residues that is largely absent during postnatal development but is re-expressed during progression of several malignant human tumors, including small-cell and non–small cell lung carcinomas, glioma, neuroblastoma, and pancreatic carcinoma. In these cancers, expression of polySia correlates with tumor progression and poor prognosis and appears to modulate cancer cell adhesion, invasiveness, and metastasis. To evaluate the potential of PolySia as a target for anticancer therapy, we developed a chimeric human polySia-specific mAb that retained low nanomolar (nmol/L) target affinity and exhibited exquisite selectivity for polySia structures. The engineered chimeric mAb recognized several polySia-positive tumor cell lines in vitro and induced rapid endocytosis of polySia antigens. To determine whether this internalization could be exploited for delivery of conjugated cytotoxic drugs, we generated an antibody–drug conjugate (ADC) by covalently linking the chimeric human mAb to the tubulin-binding maytansinoid DM1 using a bioorthogonal chemical reaction scheme. The resulting polySia-directed ADC demonstrated potent target-dependent cytotoxicity against polySia-positive tumor cells in vitro. Collectively, these results establish polySia as a valid cell-surface, cancer-specific target for glycan-directed ADC and contribute to a growing body of evidence that the tumor glycocalyx is a promising target for synthetic immunotherapies. Significance: These findings describe a glycan-specific antibody-drug conjugate that establishes polySia as a viable cell surface target within the tumor glycocalyx.

IntroductionDiabetes mellitus (DM) is the most common metabolic disease. A WHO report from 2016 indicates that 422 million people worldwide suffer from DM or hyperglycaemia because of impaired glucose metabolism. Chronic hyperglycaemia leads to micro- and macrovessel damage, which may result in life-threatening complications. The Wnt pathway regulates cell proliferation and survival by modulating the expression of genes that control cell differentiation. Three linked Wnt pathways have been discovered thus far: aβ-catenin-dependent pathway and two pathways independent ofβ-catenin – the planar cell polarity pathway and calcium-dependent pathway. The Wnt pathway regulates genes associated with inflammation, cell cycle, angiogenesis, fibrinolysis and other molecular processes.Areas coveredThis review presents the current state of knowledge regarding the contribution of the Wnt pathway to endothelial ageing under hyperglycaemic conditions and provides new insights into the molecular basis of diabetic endothelial dysfunction.ConclusionTheβ-catenin-dependent pathway is a potential target in the prophylaxis and treatment of early-stage diabetes-related vascular complications. However, the underlying molecular mechanisms remain largely undetermined and require further investigation.

Introduction Long and diffuse coronary lesions (LDCLs) are routinely subjected to percutaneous management, but long‑term clinical outcomes and complication predictors with the use of contemporary stents and techniques remain undetermined. Objectives The aim of the study was to address long‑term effects of percutaneous management of LDCLs, using contemporary devices and optimization techniques. Patients and methods Long and diffuse coronary lesion was defined as a lesion requiring an implantation of 30 mm or longer total stent(s) length (TSL) into one coronary artery (bailouts excluded). There were 290 LDCL interventions with the use of newer generation drug‑eluting stents (DESs; cobalt chromium everolimus- or zotarolimus-eluting stents) performed between January 2013 and January 2016. Results The mean (SD) TSL was 55.5 (16.8) mm. The use of intravascular ultrasound / optical coherence tomography was 17.1%, rotablation, 6.9%, and noncompliant balloon, 88.9%. The median (range) follow‑up duration was 831 (390-1373) days. All‑cause mortality and cardiac death rates were 11.7% and 6.9%, respectively. The myocardial infarction (MI) rate was 6.6%, including target‑vessel MI in 4.1%. The rate of clinically‑driven repeat revascularization was 13.8%, and of definite or probable LDCL stent thrombosis, 7.2%. Overall patient‑oriented adverse event rate (any death, MI, or repeat revascularization) was 25.5%, and device‑oriented rate (cardiac death, target vessel‑MI, or target lesion restenosis), 13.4%. Adverse outcome predictors were chronic kidney disease, acute coronary syndrome as an indication for the procedure, chronic heart failure with reduced left ventricular ejection fraction, multivessel disease, and coexisting peripheral artery disease, but not lesion‑related factors, such as bifurcation, calcification, chronic total occlusion, or TSL. Conclusions Adverse outcomes following contemporary LDCL management using newer generation DESs in routine clinical practice are associated with clinical patient characteristics rather than lesion characteristics or TSL. We identified high‑risk patient cohorts that may benefit from enhanced surveillance.

Fencing is a combat sport in which fencers compete in three weapons: foil, saber and epee. They differ from each other in blade type, valid target and scoring technique. Although the fundamental role of speed, coordination and sensory-motor skills in fencing is indisputable, merely rapid and correct accomplishment of a movement does not ensure success. Outright bout against an opponent requires high level of perceiving, information processing and accurate decision-making. Thus, in fencing, as a ‘game of anticipation and deception’, technical abilities are just a tool used to execute chosen strategy and tactics. The repeatability of joints’ movements relevant to the lunge performance was measured. Shoulder, elbow and wrist joint of the upper arm, hip joint and knee leg movements were checked in order to match and determine the number of necessary recordings. The study covered 10 athletes, 6 saber and 4 epee fencers, members of Polish National Team. There is a repeatability in measured movements. The weak and negative correlation coefficient is observed just between several subjects. The strong correlation coefficient was noted across trials within a single subject. This result implies, that frequently repeated movements create stable movement patterns which is individual for each of the subjects. According to individual character of performed actions, animation database should contain possible highest number of recorded athletes. Just widening of database can provide expected level of diversity to fencing training and prevent the effect of ‘learning’ just one opponent.

SummaryCancer metastasis, i.e., the spreading of tumor cells from the primary tumor to distant organs, is responsible for the vast majority of cancer deaths. In the process, cancer cells migrate through narrow interstitial spaces substantially smaller in cross-section than the cell. During such confined migration, cancer cells experience extensive nuclear deformation, nuclear envelope rupture, and DNA damage. The molecular mechanisms responsible for the confined migration-induced DNA damage remain incompletely understood. While in some cell lines, DNA damage is closely associated with nuclear envelope rupture, we show that in others, mechanical deformation of the nucleus is sufficient to cause DNA damage, even in the absence of nuclear envelope rupture. This deformation-induced DNA damage, unlike nuclear envelope rupture-induced DNA damage, occurs primarily in S/G2 phase of the cell cycle and is associated with replication forks. Nuclear deformation, resulting from either confined migration or external cell compression, increases replication stress, possibly by increasing replication fork stalling, providing a molecular mechanism for the deformation-induced DNA damage. Thus, we have uncovered a new mechanism for mechanically induced DNA damage, linking mechanical deformation of the nucleus to DNA replication stress. This mechanically induced DNA damage could not only increase genomic instability in metastasizing cancer cells, but could also cause DNA damage in non-migrating cells and tissues that experience mechanical compression during development, thereby contributing to tumorigenesis and DNA damage response activation.

Background The genomic duplication study is fundamental to understand the process of evolution. In evolutionary molecular biology, many approaches focus on discovering the occurrences of gene duplications and multiple gene duplication episodes and their locations in the Tree of Life. To reconstruct such episodes, one can cluster single gene duplications inferred by reconciling a set of gene trees with a species tree. Results We propose an efficient quadratic time algorithm to solve the problem of genomic duplication clustering, in which input gene trees are rooted, episode locations are restricted to preserve the minimal number of single gene duplications, clustering rules are described by minimum episodes method, and the goal is based on the recently introduced new approach to minimize the maximal number of duplication episodes on a single path, called here the M P score. Based on our theoretical results, we show new algorithmic relationships between the M P score and the minimum episodes ( M E ) score, defined as the minimal number of duplication episodes. Conclusions Our evaluation analysis on three empirical datasets demonstrates, that under the model in which the minimal number of duplications is preserved, the duplication clusterings with minimal M P score support the clusterings with the minimal total number of duplication episodes. Availability: The software is available at https://bitbucket.org/pgor17/rmp .

Interleukin-1β (IL-1β) is a critical regulator of the inflammatory response. IL-1β is not secreted through the conventional ER-Golgi route of protein secretion, and to date its mechanism of release has been unknown. Crucially, its secretion depends upon the processing of a precursor form following the activation of the multimolecular inflammasome complex. Using a novel and reversible pharmacological inhibitor of the IL-1β release process, in combination with biochemical, biophysical, and real-time single-cell confocal microscopy with macrophage cells expressing Venus-labelled IL-1β, we have discovered that the secretion of IL-1β after inflammasome activation requires membrane permeabilisation, and occurs in parallel with the death of the secreting cell. Thus, in macrophages the release of IL-1β in response to inflammasome activation appears to be a secretory process independent of nonspecific leakage of proteins during cell death. The mechanism of membrane permeabilisation leading to IL-1β release is distinct from the unconventional secretory mechanism employed by its structural homologues fibroblast growth factor 2 (FGF2) or IL-1α, a process that involves the formation of membrane pores but does not result in cell death. These discoveries reveal key processes at the initiation of an inflammatory response and deliver new insights into the mechanisms of protein release.Cell Death and Differentiation advance online publication, 12 February 2016; doi:10.1038/cdd.2015.176.

Summary Single-cell gene expression is inherently variable, but how this variability is controlled in response to stimulation remains unclear. Here, we use single-cell RNA-seq and single-molecule mRNA counting (smFISH) to study inducible gene expression in the immune toll-like receptor system. We show that mRNA counts of tumor necrosis factor α conform to a standard stochastic switch model, while transcription of interleukin-1β involves an additional regulatory step resulting in increased heterogeneity. Despite different modes of regulation, systematic analysis of single-cell data for a range of genes demonstrates that the variability in transcript count is linearly constrained by the mean response over a range of conditions. Mathematical modeling of smFISH counts and experimental perturbation of chromatin state demonstrates that linear constraints emerge through modulation of transcriptional bursting along with gene-specific relationships. Overall, our analyses demonstrate that the variability of the inducible single-cell mRNA response is constrained by transcriptional bursting.

As interest in the role of extracellular vesicles in cell-to-cell communication has increased, so has the use of microscopy and analytical techniques to assess their formation, release, and morphology. In this study, we evaluate scanning electron microscopy (SEM) and cryo-SEM for characterizing the formation and shedding of vesicles from human breast cell lines, parental and hyaluronan synthase 3-(HAS3)-overexpressing MCF10A cells, grown directly on transmission electron microscopy (TEM) grids. While cells imaged with conventional and cryo-SEM exhibit distinct morphologies due to the sample preparation process for each technique, tubular structures protruding from the cell surfaces were observed with both approaches. For HAS3-MCF10A cells, vesicles were present along the length of membrane protrusions. Once completely shed from the cells, extracellular vesicles were characterized using nanoparticle tracking analysis (NTA) and cryo-TEM. The size distributions obtained by each technique were different not only in the range of vesicles analyzed, but also in the relative proportion of smaller-to-larger vesicles. These differences are attributed to the presence of biological debris in the media, which is difficult to differentiate from vesicles in NTA. Furthermore, we demonstrate that cryo-TEM can be used to distinguish between vesicles based on their respective surface structures, thereby providing a path to differentiating vesicle subpopulations and identifying their size distributions. Our study emphasizes the necessity of pairing several techniques to characterize extracellular vesicles.

Optimization of host-cell production systems with improved yield and production reliability is desired to meet the increasing demand for biologics with complex posttranslational modifications. Aggregation of suspension-adapted mammalian cells remains a significant problem that can limit the cellular density and per volume yield of bioreactors. Here, we propose a genetically encoded technology that directs the synthesis of antiadhesive and protective coatings on the cellular surface. Inspired by the natural ability of mucin glycoproteins to resist cellular adhesion and hydrate and protect cell and tissue surfaces, we genetically encode new cell-surface coatings through the fusion of engineered mucin domains to synthetic transmembrane anchors. Combined with appropriate expression systems, the mucin-coating technology directs the assembly of thick, highly hydrated barriers to strongly mitigate cell aggregation and protect cells in suspension against fluid shear stresses. The coating technology is demonstrated on suspension-adapted human 293-F cells, which resist clumping even in media formulations that otherwise would induce extreme cell aggregation and show improved performance over a commercially available anticlumping agent. The stable biopolymer coatings do not show deleterious effects on cell proliferation rate, efficiency of transient transfection with complementary DNAs, or recombinant protein expression. Overall, our mucin-coating technology and engineered cell lines have the potential to improve the single-cell growth and viability of suspended cells in bioreactors.

Cells bend their plasma membranes into highly curved forms to interact with the local environment, but how shape generation is regulated is not fully resolved. Here, we report a synergy between shape-generating processes in the cell interior and the external organization and composition of the cell-surface glycocalyx. Mucin biopolymers and long-chain polysaccharides within the glycocalyx can generate entropic forces that favor or disfavor the projection of spherical and finger-like extensions from the cell surface. A polymer brush model of the glycocalyx successfully predicts the effects of polymer size and cell-surface density on membrane morphologies. Specific glycocalyx compositions can also induce plasma membrane instabilities to generate more exotic undulating and pearled membrane structures and drive secretion of extracellular vesicles. Together, our results suggest a fundamental role for the glycocalyx in regulating curved membrane features that serve in communication between cells and with the extracellular matrix.

Toll-like receptor (TLR) signaling regulates macrophage activation and effector cytokine propagation in the constrained environment of a tissue. In macrophage populations, TLR4 stimulates the dose-dependent transcription of nuclear factor B (NF-B) target genes. However, using single-RNA counting, we found that individual cells exhibited a wide range (three orders of magnitude) of expression of the gene encoding the proinflammatory cytokine tumor necrosis factor  (TNF-). The TLR4-induced TNFA transcriptional response correlated with the extent of NF-B signaling in the cells and their size. We compared the rates of TNF- production and uptake in macrophages and mouse embryonic fibroblasts and generated a mathematical model to explore the heterogeneity in the response of macrophages to TLR4 stimulation and the propagation of the TNF- signal in the tissue. The model predicts that the local propagation of the TLR4-dependent TNF- response and cellular NF-B signaling are limited to small distances of a few cell diameters between neighboring tissue-resident macrophages. In our predictive model, TNF- propagation was constrained by competitive uptake of TNF- from the environment, rather than by heterogeneous production of the cytokine. We propose that the highly constrained architecture of tissues enables effective localized propagation of inflammatory cues, while avoiding out-of-context responses at longer distances.

Yersiniosis is a foodborne infection caused by Yersinia enterocolitica or Yersinia pseudotuberculosis. Although yersiniosis is most often self-limiting, some patients develop chronic infections, such as reactive arthritis, glomerulonephritis, or myocarditis, which require an antibiotic treatment. Whereas early infections can be diagnosed by direct detection of bacteria, chronic infections can only be identified by serological tests. At this point, a serological method for differentiation between infections with the two Yersinia species is important since antibiotic susceptibility of these bacteria is different. Traditional immunoassays do not distinguish between infections with Y. enterocolitica and Y. pseudotuberculosis. The only test that allows for this differentiation is Mikrogen's strip test where discrimination between the two types of infection is based on two recombinant bacterial proteins, MyfA and PsaA (specific for Y. enterocolitica and Y. pseudotuberculosis, respectively). Here, we show that Y. enterocolitica and Y. pseudotuberculosis, cultured under the conditions that mimic the natural rout of infection, express surface antigens different from MyfA and PsaA that can also be used in a discrimination test. Further, we describe a new ELISA that is based on the whole bacteria and recombinant MyfA and PsaA as antigens, and that allows the differentiation between infections with Y. enterocolitica and Y. pseudotuberculosis and simultaneous detection of yersiniosis.

Breast cancer accounts for 22%-25% of all female cancers diagnosed worldwide. The aim of study was to compare the 5-year relative survival rates for breast cancer patients treated in the years 2008-2010, 2000-2002, and 2005-2007, and to determine their relationships with the methods and costs of treatment. Data were collected from the National Cancer Registry and the Narodowy Fundusz Zdrowia (National Health Fund) data bases. An increase in the 5-year survival rate was observed. The results show the impact of some factors on the survival and treatment costs. It is necessary to create data bases being a platform for further comprehensive analyses.

Background One of evolutionary molecular biology fundamental issues is to discover genomic duplication events and their correspondence to the species tree. Such events can be reconstructed by clustering single gene duplications inferred by reconciling a set of gene trees with a species tree. Results Here we propose the first solutions to the genomic duplication problem in which every reconciliation with the minimal number of single gene duplications is allowed and the method of clustering called minimum episodes under the assumption that input gene trees are unrooted. Conclusions We showed new theoretical properties of unrooted reconciliation for the duplication cost and apply them to design several exact and heuristic algorithms for solving the problem. Our evaluation study on empirical dataset confirmed several genomic duplication events from the literature and demonstrate that algorithms can be successfully applied.

Background Antiphospholipid syndrome (APS) is an autoimmune disease characterised by the presence of antiphospholipid (aPL) antibodies that have prothrombotic activity. Antiphospholipid antibodies are associated with an increased risk of pregnancy complications (recurrent miscarriage, premature birth, intrauterine growth retardation) and thrombotic events (both arterial and venous). The most common thrombotic events include brain ischaemia (stroke or transient ischaemic attack) and deep vein thrombosis. To diagnose APS, the presence of aPL antibodies in two measurements and at least one thrombotic event or pregnancy complication are required. It is unclear if people with positive aPL antibodies but without any previous thrombotic events should receive primary antithrombotic prophylaxis. Objectives To assess the effects of antiplatelet or anticoagulant agents versus placebo or no intervention or other intervention on the development of thrombosis in people with aPL antibodies who have not had a thrombotic event. We did not address obstetric outcomes in this review as these have been thoroughly addressed by other Cochrane Reviews. Search methods We searched the Cochrane Vascular Specialised Register (4 December 2017), the Cochrane Central Register of Controlled Trials (CENTRAL) (last search 29 November 2017), MEDLINE Ovid, Embase Ovid, CINAHL, and AMED (searched 4 December 2017), and trials registries (searched 29 November 2017). We also checked reference lists of included studies, systematic reviews, and practice guidelines, and contacted experts in the field. Selection criteria We included randomised controlled trials (RCTs) that compared any antiplatelet or anticoagulant agents, or their combinations, at any dose and mode of delivery with placebo, no intervention, or other intervention. We also included RCTs that compared antiplatelet or anticoagulant agents with each other or that compared two different doses of the same drug. We included studies performed in people of any age and with no history of thrombosis (as defined by APS Sapporo classification criteria or updated Sydney classification criteria), but with aPL antibodies confirmed on at last two separate measurements. The studies included both pregnant women who tested positive for aPL antibodies and had a history of recurrent obstetric complications, as well as non-pregnancy related cases with positive screening for antibodies, in accordance with the criteria mentioned above. Data collection and analysis Pairs of authors independently selected studies for inclusion, extracted data, and assessed the risk of bias for the included studies and quality of evidence using GRADE. Any discrepancies were resolved through discussion or by consulting a third review author when necessary. In addition, one review author checked all the extracted numerical data. Main results We included nine studies involving 1044 randomised participants. The studies took place in several countries and had different funding sources. No study was at low risk of bias in all domains. We classified all included studies as at unclear or high risk of bias in two or more domains. Seven included studies focused mainly on obstetric outcomes. One study included non-pregnancy-related cases, and one study included both pregnancy-related cases and other patients with positive results for aPL antibodies. The remaining studies concerned women with aPL antibodies and a history of pregnancy failure. Four studies compared anticoagulant with or without acetylsalicylic acid (ASA) versus ASA only and observed no clear difference in thrombosis risk (risk ratio (RR) 0.98, 95% confidence interval (CI) 0.25 to 3.77; 4 studies; 493 participants; low-quality evidence). No major bleeding was reported, but minor bleeding risk (nasal bleeding, menorrhagia) was higher in the anticoagulant with ASA group as compared with ASA alone in one study (RR 22.45, 95% CI 1.34 to 374.81; 1 study; 164 participants; low-quality evidence). In one study ASA was compared with placebo, and there were no clear differences in thrombosis (RR 5.21, 95% CI 0.63 to 42.97; 1 study; 98 participants; low-quality evidence) or minor bleeding risk between the groups (RR 3.13, 95% CI 0.34 to 29.01; 1 study; 98 participants; low-quality evidence), and no major bleeding was observed. Two studies compared ASA with low molecular weight heparin (LMWH) versus placebo or intravenous immunoglobulin (IVIG), and no thrombotic events were observed in any of the groups. Moreover, there were no clear differences in the risk of bleeding requiring transfusion (RR 9.0, 95% CI 0.49 to 164.76; 1 study; 180 participants; moderate-quality evidence) or postpartum bleeding (RR 1.30, 95% CI 0.60 to 2.81; 1 study; 180 participants; moderate-quality evidence) between the groups. Two studies compared ASA with high-dose LMWH versus ASA with low-dose LMWF or unfractionated heparin (UFH); no thrombotic events or major bleeding was reported. Mortality and quality of life data were not reported for any of the comparisons. Authors' conclusions There is insufficient evidence to demonstrate benefit or harm of using anticoagulants with or without ASA versus ASA alone in people with aPL antibodies and a history of recurrent pregnancy loss and with no such history; ASA versus placebo in people with aPL antibodies; and ASA with LMWH versus placebo or IVIG, and ASA with high-dose LMWH versus ASA with low-dose LMWH or UFH, in women with aPL antibodies and a history of recurrent pregnancy loss, for the primary prevention of thrombotic events. In a mixed population of people with a history of previous pregnancy loss and without such a history treated with anticoagulant combined with ASA, the incidence of minor bleeding (nasal bleeding, menorrhagia) was increased when compared with ASA alone. Studies that are adequately powered and that focus mainly on thrombotic events are needed to draw any firm conclusions on the primary prevention of thrombotic events in people with antiphospholipid antibodies.

Introduction Anxiety and depression correlate with poor asthma control. There is a cyclical relationship: poor asthma control increasing likelihood of emotional distress, which in turn reduces patient adherence and healthcare utilisation. In this study we assessed quality of life and the prevalence of depression and anxiety in patients with severe asthma. Methods 55 patients on step 4–5 therapy attending the severe asthma clinic at North Manchester General Hospital, completed two questionnaires anonymously: Hospital Anxiety and Depression Scale (HADS) and Health Survey Short-Form (SF-36). The HADS yields scores for anxiety and depression with scores greater than or equal to 8/21 in either domain considered clinically significant. SF-36 scores were transformed into composite mental and physical component scores, with scores Results 49% and 31% of the sample had clinically significant anxiety and depression respectively. 33% had scores falling in the moderate-severe range for anxiety and/or depression with 5 patients having co-existing moderate-severe anxiety and depression. Patients reported poor quality of life relating to their physical (50%) and mental health (33%). Conclusion The prevalence of anxiety in patients with severe asthma is significantly higher than in patients with chronic respiratory disorders (49% cf. 29%) 1 and is linked to poorer health and quality of life outcomes. Extrapolating from our findings, we expect a third of patients in the asthma service (≥500 patients seen per year including 125 new referrals), to benefit from individual psychological support. NICE guidance recommends each should receive initial assessment and 12 therapy sessions (cognitive behavioural therapy/psychodynamic psychotherapies). The position paper by the Kings Fund 2 argued that mental health services should be delivered as part of an integrated approach to health. This study supports a clear role for an integrated physical health clinical psychology service as part of routine care for patients with severe asthma. References Naylor, Haines, Vyas, Fowler. M3 anxiety and depression in patients with breathing pattern disorders or chronic respiratory disease. Thorax2015;70:A227–A228. Naylor, Das, Ross, Honeyman, Thompson, Gilburt. (2016). Bringing together physical and mental health . A New Frontier for Integrated Care . London: The Kings Fund.

Background Mesenchymal stromal cells (MSCs) can be used intra-articularly to quell inflammation and promote cartilage healing; however, mechanisms by which MSCs mitigate joint disease remain poorly understood. Galectins, a family of β-galactoside binding proteins, regulate inflammation, adhesion and cell migration in diverse cell types. Galectin-1 and galectin-3 are proposed to be important intra-articular modulators of inflammation in both osteoarthritis and rheumatoid arthritis. Here, we asked whether equine bone marrow-derived MSCs (BMSCs) express higher levels of galectin-1 and -3 relative to synovial fibroblasts and chondrocytes and if an inflammatory environment affects BMSC galectin expression and motility. Methods Equine galectin-1 and -3 gene expression was quantified using qRT-PCR in cultured BMSCs, synoviocytes and articular chondrocytes, in addition to synovial membrane and articular cartilage tissues. Galectin gene expression, protein expression, and protein secretion were measured in equine BMSCs following exposure to inflammatory cytokines (IL-1β 5 and 10 ng/mL, TNF-α 25 and 50 ng/mL, or LPS 0.1, 1, 10 and 50 μg/mL). BMSC focal adhesion formation was assessed using confocal microscopy, and BMSC motility was quantified in the presence of inflammatory cytokines (IL-1β or TNF-α) and the pan-galectin inhibitor β-lactose (100 and 200 mM). Results Equine BMSCs expressed 3-fold higher galectin-1 mRNA levels as compared to cultured synovial fibroblasts (p = 0.0005) and 30-fold higher galectin-1 (p

Objective To assess possible changes in leptin and ghrelin secretion due to etanercept in juvenile idiopathic arthritis (JIA). Methods 50 patients with JIA and 16 age-matched controls were enrolled into this prospective, cross-sectional study. Serum leptin, total and acyl ghrelin were measured in addition to white blood cell (WBC) and lymphocyte counts. Results 25 patients received etanercept and 25 conventional therapies (including methotrexate) for JIA. There was no difference between treatment and control groups in leptin or ghrelin levels and no evidence of a relationship between leptin and ghrelin in patients with JIA. In all children with JIA there was a correlation between leptin and body mass index (BMI). However, compared with children in the conventional treatment group, children in the etanercept group showed a positive correlation between total ghrelin and BMI and those with a low BMI showed a negative correlation between acyl ghrelin and BMI. Conclusion No differences in leptin and ghrelin concentrations were found when patients with JIA and controls were compared or when patients who received etanercept were compared with those who received conventional treatment for JIA.

Isothiocyanates, such as phenethyl isothiocyanate (PEITC), are formed following the consumption of cruciferous vegetables and generate reactive oxygen species (ROS) that lead to the induction of cytoprotective genes such as the UDP-glucuronosyltransferases (UGTs). The induction of ROS activates the Nrf2-Keap 1 pathway leading to the induction of genes through antioxidant response elements (AREs). UGT1A1, the sole enzyme responsible for the metabolism of bilirubin, can be induced following activation of Nrf2. When neonatal humanized UGT1 (hUGT1) mice, which exhibit severe levels of total serum bilirubin (TSB) because of a developmental delay in expression of the UGT1A1 gene, were treated with PEITC, TSB levels were reduced. Liver and intestinal UGT1A1 were induced, along with murine CYP2B10, a consensus CAR target gene. In both neonatal and adult hUGT1/Car−/− mice, PEITC was unable to induce CYP2B10. A similar result was observed following analysis of UGT1A1 expression in liver. However, TSB levels were still reduced in hUGT1/Car−/− neonatal mice because of ROS induction of intestinal UGT1A1. When oxidative stress was blocked by exposing mice to N-acetylcysteine, induction of liver UGT1A1 and CYP2B10 by PEITC was prevented. Thus, new findings in this report link an important role in CAR activation that is dependent upon oxidative stress.

The glycocalyx is a coating of protein and sugar on the surface of all living cells. Dramatic perturbations to the composition and structure of the glycocalyx are frequently observed in aggressive cancers. However, tools to experimentally mimic and model the cancer-specific glycocalyx remain limited. Here, we develop a genetically encoded toolkit to engineer the chemical and physical structure of the cellular glycocalyx. By manipulating the glycocalyx structure, we are able to switch the adhesive state of cells from strongly adherent to fully detached. Surprisingly, we find that a thick and dense glycocalyx with high O-glycan content promotes cell survival even in a suspended state, characteristic of circulating tumor cells during metastatic dissemination. Our data suggest that glycocalyx-mediated survival is largely independent of receptor tyrosine kinase and mitogen activated kinase signaling. While anchorage is still required for proliferation, we find that cells with a thick glycocalyx can dynamically attach to a matrix scaffold, undergo cellular division, and quickly disassociate again into a suspended state. Together, our technology provides a needed toolkit for engineering the glycocalyx in glycobiology and cancer research.

Severe neonatal hyperbilirubinemia (SNH) and the onset of bilirubin encephalopathy and kernicterus result in part from delayed expression of UDP-glucuronosyltransferase 1A1 (UGT1A1) and the inability to metabolize bilirubin. Although there is a good understanding of the early events after birth that lead to the rapid increase in serum bilirubin, the events that control delayed expression of UGT1A1 during development remain a mystery. Humanized UGT1 (hUGT1) mice develop SNH spontaneously, which is linked to repression of both liver and intestinal UGT1A1. In this study, we report that deletion of intestinal nuclear receptor corepressor 1 (NCoR1) completely diminishes hyperbilirubinemia in hUGT1 neonates because of intestinal UGT1A1 gene derepression. Transcriptomic studies and immunohistochemistry analysis demonstrate that NCoR1 plays a major role in repressing developmental maturation of the intestines. Derepression is marked by accelerated metabolic and oxidative phosphorylation, drug metabolism, fatty acid metabolism, and intestinal maturation, events that are controlled predominantly by H3K27 acetylation. The control of NCoR1 function and derepression is linked to IKKβ function, as validated in hUGT1 mice with targeted deletion of intestinal IKKβ. Physiological events during neonatal development that target activation of an IKKβ/NCoR1 loop in intestinal epithelial cells lead to derepression of genes involved in intestinal maturation and bilirubin detoxification. These findings provide a mechanism of NCoR1 in intestinal homeostasis during development and provide a key link to those events that control developmental repression of UGT1A1 and hyperbilirubinemia.

This is a protocol for a Cochrane Review (Intervention). The objectives are as follows: To assess the effects of antiplatelet and anticoagulant agents versus placebo or no intervention or other intervention for the development of thrombosis in people with antiphospholipid (aPL) antibodies but who have not had a thrombotic event.

One of evolutionary molecular biology fundamental issues is to discover genomic duplication events and their correspondence to the species tree. Such events can be reconstructed by clustering single gene duplications that are inferred by reconciling a set of gene trees with a species tree. Here we propose the first solution to the genomic duplication problem in which every reconciliation with the minimal number of single gene duplications is allowed and the method of clustering called minimum episodes under the assumption that input gene trees are unrooted. We also present an evaluation study of proposed algorithms on empirical datasets.

Recent studies suggest that cells make stochastic choices with respect to differentiation or division. However, the molecular mechanism underlying such stochasticity is unknown. We previously proposed that the timing of vertebrate neuronal differentiation is regulated by molecular oscillations of a transcriptional repressor, HES1, tuned by a post-transcriptional repressor, miR-9. Here, we computationally model the effects of intrinsic noise on the Hes1/miR-9 oscillator as a consequence of low molecular numbers of interacting species, determined experimentally. We report that increased stochasticity spreads the timing of differentiation in a population, such that initially equivalent cells differentiate over a period of time. Surprisingly, inherent stochasticity also increases the robustness of the progenitor state and lessens the impact of unequal, random distribution of molecules at cell division on the temporal spread of differentiation at the population level. This advantageous use of biological noise contrasts with the view that noise needs to be counteracted. DOI: http://dx.doi.org/10.7554/eLife.16118.001

Cells respond dynamically to pulsatile cytokine stimulation. Here we report that single, or well-spaced pulses of TNFα (>100 min apart) give a high probability of NF-κB activation. However, fewer cells respond to shorter pulse intervals (, In biological systems, timing is often critical to the interpretation of signals that determine cell fate. Here the authors demonstrate how single cells and cellular populations respond dynamically to pulsatile stimulation by TNFα and IL-1β, and suggest a mechanism by which the two cytokines can synergistically modulate inflammation.

The minimizing-deep-coalescence (MDC) approach infers a median (species) tree for a given set of gene trees under the deep coalescence cost. This cost accounts for the minimum number of deep coalescences needed to reconcile a gene tree with a species tree where the leaf-genes are mapped to the leaf-species through a function called leaf labeling. In order to better understand the MDC approach we investigate here the diameter of a gene tree, which is an important property of the deep coalescence cost. This diameter is the maximal deep coalescence costs for a given gene tree under all leaf labelings for each possible species tree topology. While we prove that this diameter is generally infinite, this result relies on the diameter’s unrealistic assumption that species trees can be of infinite size. Providing a more practical definition, we introduce a natural extension of the gene tree diameter that constrains the species tree size by a given constant. For this new diameter, we describe an exact formula, present a complete classification of the trees yielding this diameter, derive formulas for its mean and variance, and demonstrate its ability using comparative studies.

Reconciliation based cost functions play crucial role in comparing gene family trees with their species tree. To provide a better understanding of tree reconciliation we derive mean formulas for gene duplication, gene loss and gene duplication-loss cost functions, for a fixed species tree under the uniform model of gene trees. We then analyse the time complexity and study mathematical properties of these formulas. Finally, we provide several computational experiments on empirical datasets for the duplication, duplication-loss and deep coalescence means under the uniform model.

The pro-inflammatory cytokine interleukin (IL)-1β is an important mediator of the inflammatory response. In order to perform its role in the inflammatory cascade, IL-1β must be secreted from the cell, yet it lacks a signal peptide that is required for conventional secretion, and the exact mechanism of release remains undefined. Conventional biochemical methods have limited the investigation into the processes involved in IL-1β secretion to population dynamics, yet heterogeneity between cells has been observed at a single-cell level. Here, greater sensitivity is achieved with the use of a newly developed vector that codes for a fluorescently labelled version of IL-1β. Combining this with real-time single-cell confocal microscopy using the methods described here, we have developed an effective protocol for investigating the mechanisms of IL-1β secretion and the testing of the hypothesis that IL-1β secretion requires membrane permeabilisation.

Dynamic cellular systems reprogram gene expression to ensure appropriate cellular fate responses to specific extracellular cues. Here we demonstrate that the dynamics of Nuclear Factor kappa B (NF-κB) signalling and the cell cycle are prioritised differently depending on the timing of an inflammatory signal. Using iterative experimental and computational analyses, we show physical and functional interactions between NF-κB and the E2 Factor 1 (E2F-1) and E2 Factor 4 (E2F-4) cell cycle regulators. These interactions modulate the NF-κB response. In S-phase, the NF-κB response was delayed or repressed, while cell cycle progression was unimpeded. By contrast, activation of NF-κB at the G1/S boundary resulted in a longer cell cycle and more synchronous initial NF-κB responses between cells. These data identify new mechanisms by which the cellular response to stress is differentially controlled at different stages of the cell cycle. DOI: http://dx.doi.org/10.7554/eLife.10473.001, eLife digest Investigating how cells adapt to the constantly changing environment inside the body is vitally important for understanding how the body responds to an injury or infection. One of the ways in which human cells adapt is by dividing to produce new cells. This takes place in a repeating pattern of events, known as the cell cycle, through which a cell copies its DNA (in a stage known as S-phase) and then divides to make two daughter cells. Each stage of the cell cycle is tightly controlled; for example, a family of proteins called E2 factors control the entry of the cell into S phase. “Inflammatory” signals produced by a wound or during an infection can activate a protein called Nuclear Factor-kappaB (NF-κB), which controls the activity of genes that allow cells to adapt to the situation. Research shows that the activity of NF-κB is also regulated by the cell cycle, but it has not been clear how this works. Here, Ankers et al. investigated whether the stage of the cell cycle might affect how NF-κB responds to inflammatory signals. The experiments show that the NF-κB response was stronger in cells that were just about to enter S-phase than in cells that were already copying their DNA. An E2 factor called E2F-1 –which accumulates in the run up to S-phase – interacts with NF-κB and can alter the activity of certain genes. However, during S-phase, another E2 factor family member called E2F-4 binds to NF-κB and represses its activation. Next, Ankers et al. used a mathematical model to understand how these protein interactions can affect the response of cells to inflammatory signals. These findings suggest that direct interactions between E2 factor proteins and NF-κB enable cells to decide whether to divide or react in different ways to inflammatory signals. The research tools developed in this study, combined with other new experimental techniques, will allow researchers to accurately predict how cells will respond to inflammatory signals at different points in the cell cycle. DOI: http://dx.doi.org/10.7554/eLife.10473.002