Targeting danger molecules in tendinopathy: the HMGB1/TLR4 axis

Objectives: To seek evidence of the danger molecule, high-mobility group protein B1 (HMGB1) expression in human tendinopathy and thereafter, to explore mechanisms where HMGB1 may regulate inflammatory mediators and matrix regulation in human tendinopathy. Methods: Torn supraspinatus tendon (established pathology) and matched intact subscapularis tendon (representing ‘early pathology’) biopsies were collected from patients undergoing arthroscopic shoulder surgery. Control samples of subscapularis tendon were collected from patients undergoing arthroscopic stabilisation surgery. Markers of inflammation and HMGB1 were quantified by reverse transcriptase PCR (RT-PCR) and immunohistochemistry. Human tendon-derived primary cells were derived from hamstring tendon tissue obtained during hamstring tendon anterior cruciate ligament reconstruction and used through passage 3. In vitro effects of recombinant HMGB1 on tenocyte matrix and inflammatory potential were measured using quantitative RT-PCR, ELISA and immunohistochemistry staining. Results: Tendinopathic tissues demonstrated significantly increased levels of the danger molecule HMGB1 compared with control tissues with early tendinopathy tissue showing the greatest expression. The addition of recombinant human HMGB1 to tenocytes led to significant increase in expression of a number of inflammatory mediators, including interleukin 1 beta (IL-1β), IL-6, IL-33, CCL2 and CXCL12, in vitro. Further analysis demonstrated rhHMGB1 treatment resulted in increased expression of genes involved in matrix remodelling. Significant increases were observed in Col3, Tenascin-C and Decorin. Moreover, blocking HMGB1 signalling via toll-like receptor 4 (TLR4) silencing reversed these key inflammatory and matrix changes. Conclusion: HMGB1 is present in human tendinopathy and can regulate inflammatory cytokines and matrix changes. We propose HMGB1 as a mediator driving the inflammatory/matrix crosstalk and manipulation of the HMGB1/TLR4 axis may offer novel therapeutic approaches targeting inflammatory mechanisms in the management of human tendon disorders

Genome-wide association studies for methane production in dairy cattle

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. Genomic selection has been proposed for the mitigation of methane (CH4) emissions by cattle because there is considerable variability in CH4 emissions between individuals fed on the same diet. The genome-wide association study (GWAS) represents an important tool for the detection of candidate genes, haplotypes or single nucleotide polymorphisms (SNP) markers related to characteristics of economic interest. The present study included information for 280 cows in three dairy production systems in Mexico: 1) Dual Purpose (n = 100), 2) Specialized Tropical Dairy (n = 76), 3) Familiar Production System (n = 104). Concentrations of CH4 in a breath of individual cows at the time of milking (MEIm) were estimated through a system of infrared sensors. After quality control analyses, 21,958 SNPs were included. Associations of markers were made using a linear regression model, corrected with principal component analyses. In total, 46 SNPs were identified as significant for CH4 production. Several SNPs associated with CH4 production were found at regions previously described for quantitative trait loci of composition characteristics of meat, milk fatty acids and characteristics related to feed intake. It was concluded that the SNPs identified could be used in genomic selection programs in developing countries and combined with other datasets for global selection

S100A8 & S100A9: alarmin mediated inflammation in tendinopathy

Alarmins S100A8 and S100A9 are endogenous molecules released in response to environmental triggers and cellular damage. They are constitutively expressed in immune cells such as monocytes and neutrophils and their expression is upregulated under inflammatory conditions. The molecular mechanisms that regulate inflammatory pathways in tendinopathy are largely unknown therefore identifying early immune effectors is essential to understanding the pathology. Based on our previous investigations highlighting tendinopathy as an alarmin mediated pathology we sought evidence of S100A8 & A9 expression in a human model of tendinopathy and thereafter, to explore mechanisms whereby S100 proteins may regulate release of inflammatory mediators and matrix synthesis in human tenocytes. Immunohistochemistry and quantitative RT-PCR showed S100A8 & A9 expression was significantly upregulated in tendinopathic tissue compared with control. Furthermore, treating primary human tenocytes with exogenous S100A8 & A9 significantly increased protein release of IL-6, IL-8, CCL2, CCL20 and CXCL10; however, no alterations in genes associated with matrix remodelling were observed at a transcript level. We propose S100A8 & A9 participate in early pathology by modulating the stromal microenvironment and influencing the inflammatory profile observed in tendinopathy. S100A8 and S100A9 may participate in a positive feedback mechanism involving enhanced leukocyte recruitment and release of pro-inflammatory cytokines from tenocytes that perpetuates the inflammatory response within the tendon in the early stages of disease

Vancomycin wrap for anterior cruciate ligament surgery

Background: The use of the vancomycin wrap to pretreat the hamstring graft in anterior cruciate ligament reconstruction (ACLR) has grown in popularity since it was first described in 2012 and has significantly reduced rates of postoperative infection. However, it remains unknown if this antibiotic treatment affects the molecular composition of the graft. Purpose: To establish whether treatment with vancomycin at 5 mg/mL, the most commonly used concentration, alters the molecular function of the hamstring graft in ACLR. Study Design: Controlled laboratory study. Methods: Surplus hamstring tendon collected after routine ACLR surgery was used for in vitro cell culture and ex vivo tissue experiments. Vancomycin was used at 5 mg/mL in RPMI or saline diluent to treat cells and tendon tissue, respectively, with diluent control conditions. Cell viability at 30, 60, and 120 minutes was assessed via colorimetric viability assay. Tendon cells treated with control and experimental conditions for 1 hour was evaluated using semiquantitative reverse transcription analysis, immunohistochemistry staining, and protein quantitation via enzyme-linked immunosorbent assay for changes in apoptotic, matrix, and inflammatory gene and protein expression. Results: Vancomycin treatment at 5 mg/mL significantly reduced tenocyte viability in vitro after 60 minutes of treatment (P < .05); however, this was not sustained at 120 minutes. Vancomycin-treated tendon tissue showed no significant increase in apoptotic gene expression, or apoptotic protein levels in tissue or supernatant, ex vivo. Vancomycin was associated with a reduction in inflammatory proteins from treated tendon supernatants (IL-6; P < .05). Conclusion: Vancomycin did not significantly alter the molecular structure of the hamstring graft. Reductions in matrix protein and inflammatory cytokine release point to a potential beneficial effect of vancomycin in generating a homeostatic environment. Clinical Relevance: Vancomycin ACL wrap does not alter the molecular structure of the ACL hamstring graft and may improve graft integrity

Surgical treatment for colorectal cancer: Analysis of the influence of an enhanced recovery programme on long-term oncological outcomes-a study protocol for a prospective, multicentre, observational cohort study

Introduction The evidence currently available from enhanced recovery after surgery (ERAS) programmes concerns their benefits in the immediate postoperative period, but there is still very little evidence as to whether their correct implementation benefits patients in the long term. The working hypothesis here is that, due to the lower response to surgical aggression and lower rates of postoperative complications, ERAS protocols can reduce colorectal cancer-related mortality. The main objective of this study is to analyse the impact of an ERAS programme for colorectal cancer on 5-year survival. As secondary objectives, we propose to analyse the weight of each of the predefined items in the oncological results as well as the quality of life. Methods and analysis A multicentre prospective cohort study was conducted in patients older than 18 years of age who are scheduled to undergo surgery for colorectal cancer. The study involved 12 hospitals with an implemented enhanced recovery protocol according to the guidelines published by the Spanish National Health Service. The intervention group includes patients with a minimum implementation level of 70%, and the control group includes those who fail to reach this level. Compliance will be studied using 18 key performance indicators, and the results will be analysed using cancer survival indicators, including overall survival, cancer-specific survival and relapse-free survival. The time to recurrence, perioperative morbidity and mortality, hospital stay and quality of life will also be studied, the latter using the validated EuroQol Five questionnaire. The propensity index method will be used to create comparable treatment and control groups, and a multivariate regression will be used to study each variable. The Kaplan-Meier estimator will be used to estimate survival and the log-rank test to make comparisons. A p value of less than 0.05 (two-tailed) will be considered to be significant. Ethics and dissemination Ethical approval for this study was obtained from the Aragon Ethical Committee (C.P.-C.I. PI20/086) on 4 March 2020. The findings of this study will be submitted to peer-reviewed journals (BMJ Open, JAMA Surgery, Annals of Surgery, British Journal of Surgery). Abstracts will be submitted to relevant national and international meetings. Trial registration number NCT04305314

Gene expression profiling integrated into network modelling reveals heterogeneity in the mechanisms of BRCA1 tumorigenesis

Background: gene expression profiling has distinguished sporadic breast tumour classes with genetic and clinical differences. Less is known about the molecular classification of familial breast tumours, which are generally considered to be less heterogeneous. Here, we describe molecular signatures that define BRCA1 subclasses depending on the expression of the gene encoding for oestrogen receptor, ESR1. Methods: for this purpose, we have used the Oncochip v2, a cancer-related cDNA microarray to analyze 14 BRCA1-associated breast tumours. Results: signatures were found to be molecularly associated with different biological processes and transcriptional regulatory programs. The signature of ESR1-positive tumours was mainly linked to cell proliferation and regulated by ER, whereas the signature of ESR1-negative tumours was mainly linked to the immune response and possibly regulated by transcription factors of the REL/NFκB family. These signatures were then verified in an independent series of familial and sporadic breast tumours, which revealed a possible prognostic value for each subclass. Over-expression of immune response genes seems to be a common feature of ER-negative sporadic and familial breast cancer and may be associated with good prognosis. Interestingly, the ESR1-negative tumours were substratified into two groups presenting slight differences in the magnitude of the expression of immune response transcripts and REL/NFκB transcription factors, which could be dependent on the type of BRCA1 germline mutation. Conclusion: this study reveals the molecular complexity of BRCA1 breast tumours, which are found to display similarities to sporadic tumours, and suggests possible prognostic implications

Surgical treatment for colorectal cancer: Analysis of the influence of an enhanced recovery programme on long-term oncological outcomes-a study protocol for a prospective, multicentre, observational cohort study

Introduction The evidence currently available from enhanced recovery after surgery (ERAS) programmes concerns their benefits in the immediate postoperative period, but there is still very little evidence as to whether their correct implementation benefits patients in the long term. The working hypothesis here is that, due to the lower response to surgical aggression and lower rates of postoperative complications, ERAS protocols can reduce colorectal cancer-related mortality. The main objective of this study is to analyse the impact of an ERAS programme for colorectal cancer on 5-year survival. As secondary objectives, we propose to analyse the weight of each of the predefined items in the oncological results as well as the quality of life. Methods and analysis A multicentre prospective cohort study was conducted in patients older than 18 years of age who are scheduled to undergo surgery for colorectal cancer. The study involved 12 hospitals with an implemented enhanced recovery protocol according to the guidelines published by the Spanish National Health Service. The intervention group includes patients with a minimum implementation level of 70%, and the control group includes those who fail to reach this level. Compliance will be studied using 18 key performance indicators, and the results will be analysed using cancer survival indicators, including overall survival, cancer-specific survival and relapse-free survival. The time to recurrence, perioperative morbidity and mortality, hospital stay and quality of life will also be studied, the latter using the validated EuroQol Five questionnaire. The propensity index method will be used to create comparable treatment and control groups, and a multivariate regression will be used to study each variable. The Kaplan-Meier estimator will be used to estimate survival and the log-rank test to make comparisons. A p value of less than 0.05 (two-tailed) will be considered to be significant. Ethics and dissemination Ethical approval for this study was obtained from the Aragon Ethical Committee (C.P.-C.I. PI20/086) on 4 March 2020. The findings of this study will be submitted to peer-reviewed journals (BMJ Open, JAMA Surgery, Annals of Surgery, British Journal of Surgery). Abstracts will be submitted to relevant national and international meetings.The present research study was awarded a Ministerio de Ciencia e Innovación health research project grant (PI19/00291) from the Carlos III Institute of the Spanish National Health Service as part of the 2019 call for Strategic Action in Health

Strategies to reengage patients lost to follow up in HIV care in high income countries, a scoping review

Background: Despite remarkable achievements in antiretroviral therapy (ART), losses to follow-up (LTFU) might prevent the long-term success of HIV treatment and might delay the achievement of the 90-90-90 objectives. This scoping review is aimed at the description and analysis of the strategies used in high-income countries to reengage LTFU in HIV care, their implementation and impact. Methods: A scoping review was done following Arksey & O'Malley's methodological framework and recommendations from Joanna Briggs Institute. Peer reviewed articles were searched for in Pubmed, Scopus and Web of Science; and grey literature was searched for in Google and other sources of information. Documents were charted according to the information presented on LTFU, the reengagement procedures used in HIV units in high-income countries, published during the last 15 years. In addition, bibliographies of chosen articles were reviewed for additional articles. Results: Twenty-eight documents were finally included, over 80% of them published in the United States later than 2015. Database searches, phone calls and/or mail contacts were the most common strategies used to locate and track LTFU, while motivational interviews and strengths-based techniques were used most often during reengagement visits. Outcomes like tracing activities efficacy, rates of reengagement and viral load reduction were reported as outcome measures. Conclusions: This review shows a recent and growing trend in developing and implementing patient reengagement strategies in HIV care. However, most of these strategies have been implemented in the United States and little information is available for other high-income countries. The procedures used to trace and contact LTFU are similar across reviewed studies, but their impact and sustainability are widely different depending on the country studied

Frequency, prognostic impact, and subtype association of 8p12, 8q24, 11q13, 12p13, 17q12, and 20q13 amplifications in breast cancers

BACKGROUND: Oncogene amplification and overexpression occur in tumor cells. Amplification status may provide diagnostic and prognostic information and may lead to new treatment strategies. Chromosomal regions 8p12, 8q24, 11q13, 17q12 and 20q13 are recurrently amplified in breast cancers. METHODS: To assess the frequencies and clinical impact of amplifications, we analyzed 547 invasive breast tumors organized in a tissue microarray (TMA) by fluorescence in situ hybridization (FISH) and calculated correlations with histoclinical features and prognosis. BAC probes were designed for: (i) two 8p12 subregions centered on RAB11FIP1 and FGFR1 loci, respectively; (ii) 11q13 region centered on CCND1; (iii) 12p13 region spanning NOL1; and (iv) three 20q13 subregions centered on MYBL2, ZNF217 and AURKA, respectively. Regions 8q24 and 17q12 were analyzed with MYC and ERBB2 commercial probes, respectively. RESULTS: We observed amplification of 8p12 (amplified at RAB11FIP1 and/or FGFR1) in 22.8%, 8q24 in 6.1%, 11q13 in 19.6%, 12p13 in 4.1%, 17q12 in 9.9%, 20q13(Z )(amplified at ZNF217 only) in 9.9%, and 20q13(Co )(co-amplification of two or three 20q13 loci) in 8.5% of cases. The 8q24, 12p13, and 17q12 amplifications were correlated with high grade. The most frequent single amplifications were 8p12 (9.8%), 8q24 (3.3%) and 12p13 (3.3%), 20q13(Z )and 20q13(Co )(1.6%) regions. The 17q12 and 11q13 regions were never found amplified alone. The most frequent co-amplification was 8p12/11q13. Amplifications of 8p12 and 17q12 were associated with poor outcome. Amplification of 12p13 was associated with basal molecular subtype. CONCLUSION: Our results establish the frequencies, prognostic impacts and subtype associations of various amplifications and co-amplifications in breast cancers

Genome-wide inference of regulatory networks in Streptomyces coelicolor

Background: The onset of antibiotics production in Streptomyces species is co-ordinated with differentiation events. An understanding of the genetic circuits that regulate these coupled biological phenomena is essential to discover and engineer the pharmacologically important natural products made by these species. The availability of genomic tools and access to a large warehouse of transcriptome data for the model organism, Streptomyces coelicolor, provides incentive to decipher the intricacies of the regulatory cascades and develop biologically meaningful hypotheses. Results: In this study, more than 500 samples of genome-wide temporal transcriptome data, comprising wild-type and more than 25 regulatory gene mutants of Streptomyces coelicolor probed across multiple stress and medium conditions, were investigated. Information based on transcript and functional similarity was used to update a previously-predicted whole-genome operon map and further applied to predict transcriptional networks constituting modules enriched in diverse functions such as secondary metabolism, and sigma factor. The predicted network displays a scale-free architecture with a small-world property observed in many biological networks. The networks were further investigated to identify functionally-relevant modules that exhibit functional coherence and a consensus motif in the promoter elements indicative of DNA-binding elements. Conclusions: Despite the enormous experimental as well as computational challenges, a systems approach for integrating diverse genome-scale datasets to elucidate complex regulatory networks is beginning to emerge. We present an integrated analysis of transcriptome data and genomic features to refine a whole-genome operon map and to construct regulatory networks at the cistron level in Streptomyces coelicolor. The functionally-relevant modules identified in this study pose as potential targets for further studies and verification.