Proteomic Profiling and Label-Free Quantification of Bovine Milk Proteins during Experimentally Induced Escherichia coli Mastitis

Coliform mastitis has been a primary focus of dairy cattle disease research due to staggering affiliated losses, severe systemic complications arising from host inflammatory response to lipopolysaccharide, and the poor response of coliform pathogens to antimicrobials. Reliable biomarkers are needed to evaluate the efficacy of adjunctive therapies for the treatment of inflammation associated with coliform mastitis, and to aid in the approval of new veterinary drugs. The aims of the current analyses were to utilize proteomic methodologies to evaluate protein expression in whey from cows with experimentally induced coliform mastitis, and to employ label-free quantification strategies to estimate changes in relative abundance of proteins identified in milk over the course of clinical infection. Two-dimensional gel electrophoresis followed by matrix-assisted laser desorption/ionization time-of-flight (MALDI- TOF) mass spectrometry (MS) resulted in the identification of complement factors, antimicrobial proteins, and acute phase proteins in mastitic milk. Analysis using liquid chromatography (LC) inline with electrospray ionization - quadrupole TOF tandem mass spectrometry (MS/MS) resulted primarily in the identification of abundant whey and casein proteins, and the transient detection of proteins related to host response. Nano-LC- nanospray-MS/MS using a linear ion trap, however, led to the robust discovery of over fifty inflammatory proteins in whey from mastitic milk, including the novel markers kininogen-2 and inter-alpha trypsin inhibitor heavy chain-4. Normalized spectral counts were compared to enzyme-linked immunosorbant assay (ELISA) for select proteins to assess the accuracy of the spectral count data. Similar expression patterns were detected using spectral counts and ELISA. Results indicate that proteomic methodologies can detect biomarkers of coliform mastitis in bovine milk during clinical infections, and that spectral counts are a viable means of evaluating relative changes in protein biomarkers of mastitis, including those for which no antibody currently exists

Proteomic Analysis of Goat Milk

The advancement of electrophoresis and chromatography, along with technological developments in mass spectrometry, has widened the potential application of proteomics to study milk from smaller ruminants. The aim of this chapter is to provide an in-depth overview of the development and progress of proteomics applications in goat milk. After examining various proteomic approaches that are currently applied to this field, we narrow our focus on proteomic investigations of mastitis in goat milk. A summary of protein modulation in goat milk during experimentally-induced endotoxin mastitis is discussed. Because the molecular function of proteins is disrupted during disease due to changes in post-translational modifications, we also review the phosphorylation of caseins, which are the predominant phosphoproteins in milk, and discuss the implications of casein modifications during mastitis. These results offer new insights into the changes of protein expression in goat milk during infection

Proteomic Analyses of Host and Pathogen Responses during Bovine Mastitis

The pursuit of biomarkers for use as clinical screening tools, measures for early detection, disease monitoring, and as a means for assessing therapeutic responses has steadily evolved in human and veterinary medicine over the past two decades. Concurrently, advances in mass spectrometry have markedly expanded proteomic capabilities for biomarker discovery. While initial mass spectrometric biomarker discovery endeavors focused primarily on the detection of modulated proteins in human tissues and fluids, recent efforts have shifted to include proteomic analyses of biological samples from food animal species. Mastitis continues to garner attention in veterinary research due mainly to affiliated financial losses and food safety concerns over antimicrobial use, but also because there are only a limited number of efficacious mastitis treatment options. Accordingly, comparative proteomic analyses of bovine milk have emerged in recent years. Efforts to prevent agricultural-related food-borne illness have likewise fueled an interest in the proteomic evaluation of several prominent strains of bacteria, including common mastitis pathogens. The interest in establishing biomarkers of the host and pathogen responses during bovine mastitis stems largely from the need to better characterize mechanisms of the disease, to identify reliable biomarkers for use as measures of early detection and drug efficacy, and to uncover potentially novel targets for the development of alternative therapeutics. The following review focuses primarily on comparative proteomic analyses conducted on healthy versus mastitic bovine milk. However, a comparison of the host defense proteome of human and bovine milk and the proteomic analysis of common veterinary pathogens are likewise introduced

alphabeta T cell receptors as predictors of health and disease

The diversity of antigen receptors and the specificity it underlies are the hallmarks of the cellular arm of the adaptive immune system. T and B lymphocytes are indeed truly unique in their ability to generate receptors capable of recognizing virtually any pathogen. It has been known for several decades that T lymphocytes recognize short peptides derived from degraded proteins presented by major histocompatibility complex (MHC) molecules at the cell surface. Interaction between peptide-MHC (pMHC) and the T cell receptor (TCR) is central to both thymic selection and peripheral antigen recognition. It is widely assumed that TCR diversity is required, or at least highly desirable, to provide sufficient immune coverage. However, a number of immune responses are associated with the selection of predictable, narrow, or skewed repertoires and public TCR chains. Here, we summarize the current knowledge on the formation of the TCR repertoire and its maintenance in health and disease. We also outline the various molecular mechanisms that govern the composition of the pre-selection, naive and antigen-specific TCR repertoires. Finally, we suggest that with the development of high-throughput sequencing, common TCR \u27signatures\u27 raised against specific antigens could provide important diagnostic biomarkers and surrogate predictors of disease onset, progression and outcome

Convalescent plasma in patients admitted to hospital with COVID-19 (RECOVERY): a randomised controlled, open-label, platform trial

SummaryBackground Azithromycin has been proposed as a treatment for COVID-19 on the basis of its immunomodulatoryactions. We aimed to evaluate the safety and efficacy of azithromycin in patients admitted to hospital with COVID-19.Methods In this randomised, controlled, open-label, adaptive platform trial (Randomised Evaluation of COVID-19Therapy [RECOVERY]), several possible treatments were compared with usual care in patients admitted to hospitalwith COVID-19 in the UK. The trial is underway at 176 hospitals in the UK. Eligible and consenting patients wererandomly allocated to either usual standard of care alone or usual standard of care plus azithromycin 500 mg once perday by mouth or intravenously for 10 days or until discharge (or allocation to one of the other RECOVERY treatmentgroups). Patients were assigned via web-based simple (unstratified) randomisation with allocation concealment andwere twice as likely to be randomly assigned to usual care than to any of the active treatment groups. Participants andlocal study staff were not masked to the allocated treatment, but all others involved in the trial were masked to theoutcome data during the trial. The primary outcome was 28-day all-cause mortality, assessed in the intention-to-treatpopulation. The trial is registered with ISRCTN, 50189673, and ClinicalTrials.gov, NCT04381936.Findings Between April 7 and Nov 27, 2020, of 16 442 patients enrolled in the RECOVERY trial, 9433 (57%) wereeligible and 7763 were included in the assessment of azithromycin. The mean age of these study participants was65·3 years (SD 15·7) and approximately a third were women (2944 [38%] of 7763). 2582 patients were randomlyallocated to receive azithromycin and 5181 patients were randomly allocated to usual care alone. Overall,561 (22%) patients allocated to azithromycin and 1162 (22%) patients allocated to usual care died within 28 days(rate ratio 0·97, 95% CI 0·87–1·07; p=0·50). No significant difference was seen in duration of hospital stay (median10 days [IQR 5 to >28] vs 11 days [5 to >28]) or the proportion of patients discharged from hospital alive within 28 days(rate ratio 1·04, 95% CI 0·98–1·10; p=0·19). Among those not on invasive mechanical ventilation at baseline, nosignificant difference was seen in the proportion meeting the composite endpoint of invasive mechanical ventilationor death (risk ratio 0·95, 95% CI 0·87–1·03; p=0·24).Interpretation In patients admitted to hospital with COVID-19, azithromycin did not improve survival or otherprespecified clinical outcomes. Azithromycin use in patients admitted to hospital with COVID-19 should be restrictedto patients in whom there is a clear antimicrobial indication

Association between state physical education (PE) requirements and PE participation, physical activity, and body mass index change

OBJECTIVE: To determine if state physical education (PE) laws are associated with student physical education attendance and physical activity (PA), and whether physical education and competitive food laws, in conjunction, are associated with lower BMI change. METHOD: State laws regarding physical education time requirements and competitive foods in 2003 and 2006 were classified as strong, weak, or none, based on codified law ratings obtained from the Classification of Laws Associated with School Students. Laws were linked to student data on PE attendance and physical activity (8th grade, Spring 2007) and BMI change (5th-8th grade, 2004-2007), obtained from the Early Childhood Longitudinal Study (n=5510 students in 40 states). RESULTS: Girls reported 0.31 more days of activity (95% CI: 0.02, 0.61) and were more likely to attend physical education ≥3days/week (74.1% versus 52.1%, difference=22.0, 95% CI: 2.1, 42.0) if they resided in states with strong physical education laws compared to no physical education laws. Weak physical education laws had modest associations with PE and activity, and there was no evidence that weak laws reduce BMI gain regardless of competitive food laws. CONCLUSION: Strong physical education laws with specific time requirements may increase physical education attendance and activity in girls. There is insufficient evidence that physical education laws reduce student weight gain