MVMRmode: Introducing an R package for plurality valid estimators for multivariable Mendelian randomisation.

BACKGROUND: Mendelian randomisation (MR) is the use of genetic variants as instrumental variables. Mode-based estimators (MBE) are one of the most popular types of estimators used in univariable-MR studies and is often used as a sensitivity analysis for pleiotropy. However, because there are no plurality valid regression estimators, modal estimators for multivariable-MR have been under-explored. METHODS: We use the residual framework for multivariable-MR to introduce two multivariable modal estimators: multivariable-MBE, which uses IVW to create residuals fed into a traditional plurality valid estimator, and an estimator which instead has the residuals fed into the contamination mixture method (CM), multivariable-CM. We then use Monte-Carlo simulations to explore the performance of these estimators when compared to existing ones and re-analyse the data used by Grant and Burgess (2021) looking at the causal effect of intelligence, education, and household income on Alzheimer's disease as an applied example. RESULTS: In our simulation, we found that multivariable-MBE was generally too variable to be much use. Multivariable-CM produced more precise estimates on the other hand. Multivariable-CM performed better than MR-Egger in almost all settings, and Weighted Median under balanced pleiotropy. However, it underperformed Weighted Median when there was a moderate amount of directional pleiotropy. Our re-analysis supported the conclusion of Grant and Burgess (2021), that intelligence had a protective effect on Alzheimer's disease, while education, and household income do not have a causal effect. CONCLUSIONS: Here we introduced two, non-regression-based, plurality valid estimators for multivariable MR. Of these, "multivariable-CM" which uses IVW to create residuals fed into a contamination-mixture model, performed the best. This estimator uses a plurality of variants valid assumption, and appears to provide precise and unbiased estimates in the presence of balanced pleiotropy and small amounts of directional pleiotropy.

The learning curve for robotic-assisted total hip arthroplasty in low, medium, and high-volume surgeons.

Background: Robotic systems have been designed to increase the accuracy of implant alignment in total knee and hip arthroplasty. This technology is associated with a learning curve for the operative time to reach peak efficiency in its use. Prior studies done on high-volume orthopedic surgeons have suggested a learning curve of 14-35 cases for robotic-assisted total hip arthroplasty (THA). It is unclear if this learning curve is different for surgeons with lower volumes. Methods: Data was collected retrospectively from 299 THA procedures done by three different surgeons, with low (1-15 cases/year), medium (16-50), and high (51+) volume caseload. The learning curve was assessed primarily by average operative time from cases 1-20, 21-50, and 51+. Results: The high-volume surgeon had a learning curve of 20 cases, while the low and medium volume surgeons had no significant decrease in their operative time through the cases included in the study (20 and 63, respectively). Conclusions: High volume surgeons have a learning curve of about 20 cases, while low and medium volume surgeons have a longer curve, which was not able to be measured in this study.

A novel family of defensin-like peptides from Hermetia illucens with antibacterial properties.

BACKGROUND: The world faces a major infectious disease challenge. Interest in the discovery, design, or development of antimicrobial peptides (AMPs) as an alternative approach for the treatment of bacterial infections has increased. Insects are a good source of AMPs which are the main effector molecules of their innate immune system. Black Soldier Fly Larvae (BSFL) are being developed for large-scale rearing for food sustainability, waste reduction and as sustainable animal and fish feed. Bioinformatic studies have suggested that BSFL have the largest number of AMPs identified in insects. However, most AMPs identified in BSF have not yet undergone antimicrobial evaluation but are promising leads to treat critical infections. RESULTS: Jg7197.t1, Jg7902.t1 and Jg7904.t1 were expressed into the haemolymph of larvae following infection with Salmonella enterica serovar Typhimurium and were predicted to be AMPs using the computational tool ampir. The genes encoding these proteins were within 2 distinct clusters in chromosome 1 of the BSF genome. Following removal of signal peptides, predicted structures of the mature proteins were superimposed, highlighting a high degree of structural conservation. The 3 AMPs share primary sequences with proteins that contain a Kunitz-binding domain; characterised for inhibitory action against proteases, and antimicrobial activities. An in vitro antimicrobial screen indicated that heterologously expressed SUMO-Jg7197.t1 and SUMO-Jg7902.t1 did not show activity against 12 bacterial strains. While recombinant SUMO-Jg7904.t1 had antimicrobial activity against a range of Gram-negative and Gram-positive bacteria, including the serious pathogen Pseudomonas aeruginosa. CONCLUSIONS: We have cloned and purified putative AMPs from BSFL and performed initial in vitro experiments to evaluate their antimicrobial activity. In doing so, we have identified a putative novel defensin-like AMP, Jg7904.t1, encoded in a paralogous gene cluster, with antimicrobial activity against P. aeruginosa.

Do social media use and patient satisfaction scores correlate with online award recognition among hip and knee arthroplasty specialists?

INTRODUCTION: The physician-patient interaction now begins before patients arrive in the office. Online ratings, social media profiles, and online award status are all components of physician online reputation which contributes to the patient's initial impressions. Therefore, it is important to understand the interplay of these factors and determine if there is a consistent trend indicating the value of this information. METHODS: We Identified all (N â€‹= â€‹160) registered American Association of Hip and Knee Surgeons (AAHKS) in New England using the https://findadoctor.aahks.net/tool for Massachusetts (MA), Connecticut (CT), Rhode Island (RI), Vermont (VT), New Hampshire (NH), and Maine (ME) on 6/26/2023. We collected surgeon age, fellowship graduation year, and practice type (i.e. Academic or Private). The average 5-star rating and number of ratings were collected from four websites. Any professional-use Facebook, Instagram, Twitter, LinkedIn, YouTube Channel, Personal Websites, or Institutional Websites were identified and a modified SMI Score was calculated. Finally, Castle Connolly Top Doctor, Local Magazine (e.g. Boston Magazine) Top Doctor, or the presence of having any award was noted for each surgeon. RESULTS: We identified several significant trends indicating that online awards were associated with higher online ratings. Social media presence, as determined by SMI Score, was also correlated with higher ratings overall and a higher likelihood of having an online award. CONCLUSION: Given the observed trends and reported importance patients place on ratings and awards, surgeons may consider increasing online engagement via social media and encouraging patients to share their experience via online ratings.

Identification of Campylobacter jejuni and Campylobacter coli genes contributing to oxidative stress response using TraDIS analysis.

BACKGROUND: Campylobacter jejuni and Campylobacter coli are the major causative agents of bacterial gastroenteritis worldwide and are known obligate microaerophiles. Despite being sensitive to oxygen and its reduction products, both species are readily isolated from animal food products kept under atmospheric conditions where they face high oxygen tension levels. RESULTS: In this study, Transposon Directed Insertion-site Sequencing (TraDIS) was used to investigate the ability of one C. jejuni strain and two C. coli strains to overcome oxidative stress, using H2O2 to mimic oxidative stress. Genes were identified that were required for oxidative stress resistance for each individual strain but also allowed a comparison across the three strains. Mutations in the perR and ahpC genes were found to increase Campylobacter tolerance to H2O2. The roles of these proteins in oxidative stress were previously known in C. jejuni, but this data indicates that they most likely play a similar role in C. coli. Mutation of czcD decreased Campylobacter tolerance to H2O2. The role of CzcD, which functions as a zinc exporter, has not previously been linked to oxidative stress. The TraDIS data was confirmed using defined deletions of perR and czcD in C. coli 15-537360. CONCLUSIONS: This is the first study to investigate gene fitness in both C. jejuni and C. coli under oxidative stress conditions and highlights both similar roles for certain genes for both species and highlights other genes that have a role under oxidative stress.

Plasma protein signatures of adult asthma.

BACKGROUND: Adult asthma is complex and incompletely understood. Plasma proteomics is an evolving technique that can both generate biomarkers and provide insights into disease mechanisms. We aimed to identify plasma proteomic signatures of adult asthma. METHODS: Protein abundance in plasma was measured in individuals from the Agricultural Lung Health Study (ALHS) (761 asthma, 1095 non-case) and the Atherosclerosis Risk in Communities study (470 asthma, 10,669 non-case) using the SOMAScan 5K array. Associations with asthma were estimated using covariate adjusted logistic regression and meta-analyzed using inverse-variance weighting. Additionally, in ALHS, we examined phenotypes based on both asthma and seroatopy (asthma with atopy (n = 207), asthma without atopy (n = 554), atopy without asthma (n = 147), compared to neither (n = 948)). RESULTS: Meta-analysis of 4860 proteins identified 115 significantly (FDR<0.05) associated with asthma. Multiple signaling pathways related to airway inflammation and pulmonary injury were enriched (FDR<0.05) among these proteins. A proteomic score generated using machine learning provided predictive value for asthma (AUC = 0.77, 95% CI = 0.75-0.79 in training set; AUC = 0.72, 95% CI = 0.69-0.75 in validation set). Twenty proteins are targeted by approved or investigational drugs for asthma or other conditions, suggesting potential drug repurposing. The combined asthma-atopy phenotype showed significant associations with 20 proteins, including five not identified in the overall asthma analysis. CONCLUSION: This first large-scale proteomics study identified over 100 plasma proteins associated with current asthma in adults. In addition to validating previous associations, we identified many novel proteins that could inform development of diagnostic biomarkers and therapeutic targets in asthma management.

A Bayesian approach to Mendelian randomization using summary statistics in the univariable and multivariable settings with correlated pleiotropy.

Mendelian randomization uses genetic variants as instrumental variables to make causal inferences on the effect of an exposure on an outcome. Due to the recent abundance of high-powered genome-wide association studies, many putative causal exposures of interest have large numbers of independent genetic variants with which they associate, each representing a potential instrument for use in a Mendelian randomization analysis. Such polygenic analyses increase the power of the study design to detect causal effects; however, they also increase the potential for bias due to instrument invalidity. Recent attention has been given to dealing with bias caused by correlated pleiotropy, which results from violation of the "instrument strength independent of direct effect" assumption. Although methods have been proposed that can account for this bias, a number of restrictive conditions remain in many commonly used techniques. In this paper, we propose a Bayesian framework for Mendelian randomization that provides valid causal inference under very general settings. We propose the methods MR-Horse and MVMR-Horse, which can be performed without access to individual-level data, using only summary statistics of the type commonly published by genome-wide association studies, and can account for both correlated and uncorrelated pleiotropy. In simulation studies, we show that the approach retains type I error rates below nominal levels even in high-pleiotropy scenarios. We demonstrate the proposed approaches in applied examples in both univariable and multivariable settings, some with very weak instruments.

Socioeconomic Disadvantage Predicts Decreased Likelihood of Maintaining a Functional Knee Arthroplasty Following Treatment for Prosthetic Joint Infection.

BACKGROUND: Prosthetic joint infection (PJI) carries major morbidity and mortality as well as a complicated and lengthy treatment course. In patients who have high degrees of socioeconomic disadvantage, this may be a particularly devastating complication. Our study sought to evaluate the impact of socioeconomic deprivation on outcomes following treatment for PJI of the knee. METHODS: We conducted a retrospective review of revision total knee arthroplasty (TKA) procedures performed for the treatment of initial PJI between 2008 and 2020 at a single tertiary care center in the United States. The Area Deprivation Index (ADI) was used to quantify socioeconomic deprivation. The primary outcome measure was presence of a functional knee joint at the time of most recent follow-up defined as TKA components or an articulating spacer. A total of 96 patients were included for analysis. The median follow-up duration was 26.5 months. RESULTS: There was no significant difference in the rate of treatment failure (P = .63). However, the proportion of patients who had a functional knee arthroplasty (in contrast to having undergone arthrodesis, amputation, or retention of a static spacer) declined significantly with increasing ADI index (81.8% for the least disadvantaged group, 58.7% for the middle group, 42.9% for the most disadvantaged group, P = .021). CONCLUSIONS: Patients who have a higher socioeconomic disadvantage as measured by ADI are less likely to maintain a functional knee arthroplasty following treatment for TKA PJI. These findings support continued efforts to improve access to care and optimize treatment plans for patients who have socioeconomic disadvantage.

The Relative Importance of Factors That Applicants Weigh When Ranking Adult Reconstruction Fellowships as Well as Their Perspectives on Robotic-Assisted Arthroplasty.

BACKGROUND: Orthopedic Surgery Fellowship programs offer highly specialized training that varies based on the training environment and surgical experience. Additionally, for Adult Reconstruction programs, robotic-assisted surgery exposure has been a widely discussed topic. The purpose of this study was to determine the relative value of various factors to Adult Hip and Knee Fellowship applicants, and their perceptions of robotic-assisted arthroplasty. METHODS: We surveyed 780 applicants who applied to our fellowship to matriculate in 2020 to 2024. We received 158 responses (20.3% response rate). We assessed factors concerning people and perceptions, logistics, salary and benefits, program reputation and curriculum, and surgical experience. Additionally, we surveyed fellows' attitudes toward using robotic surgery and its impact on patient outcomes. RESULTS: The highest-rated factors were Level of Hands-On Operative Experience (4.83), Revision Hip Volume (4.72), Revision Knee Volume (4.71), Multiple Surgical Exposures to the Hip (4.59), and Clinical Case Variety (4.59). Respondents who were postfellowship matriculation placed significantly more value on Exposure to Multiple Attendings with Surgical Diversity (P = .01), and Anterior Hip Volume (P = .04), and less value on Geographic Location (P = .04) and Patient-Specific Instrumentation (P = .02) than prematriculates. Overall, 65% of applicants plan to or currently use robotics, 7.6% do not, and 27.2% said "Maybe". Those who plan to or currently use robotics most cited procedure fidelity, patient-preference, and marketability as reasons to use robotics. CONCLUSIONS: Hands-on surgical experience and revision volume were the most important factors for fellowship applicants. Applicants placed lower importance on robotics exposure and their perspectives on robotics in their future practice were highly variable. Our results will inform fellowship programs and future applicants what previous applicants have valued in their training to help guide fellowship program structure, resource management, as well as recruitment.

Product warranties by orthopaedic surgery companies. Quality control or a new fad?

Introduction: Product guarantees are known to the manufacturing industry, however warranties have been rare in Orthopaedic surgery. Over the last 10 years, select manufacturers of implants have instituted warranties of varying scope, length, and reimbursement. This phenomenon prompted us to investigate the landscape of warranties in Orthopaedics and compare that to other medical industries to better inform their impact on patient care. Methods: We conducted a systematic review of patient access material of over 120 Orthopaedic manufacturers including that of the Top 25 grossing companies of 2022 to identify the prevalence and scope of these warranties. Results: We identified eight companies that offer a warranty on implants. The expiration time for the implant warranties ranged from one year to lifetime. The scope of the warranties ranged from coverage of a one-time component replacement to outcome-based guarantees that cover any complications and revisions that may result from the surgery. Discussion: While the use of warranties remains uncommon in orthopaedics, their utility is expanding and evolving. Contemporary warranties appear to have a focus on enhancing product-marketability and improving quality-control.

An attacin antimicrobial peptide, Hill_BB_C10074, from Hermetia illucens with anti-Pseudomonas aeruginosa activity.

BACKGROUND: There is a global need to develop new therapies to treat infectious diseases and tackle the rise in antimicrobial resistance. To date, the larvae of the Black Solider Fly, Hermetia illucens, have the largest repertoire of antimicrobial peptides derived from insects. Antimicrobial peptides are of particular interest in the exploration of alternative antimicrobials due to their potent action and reduced propensity to induce resistance compared with more traditional antibiotics. RESULTS: The predicted attacin from H. illucens, Hill_BB_C10074, was first identified in the transcriptome of H. illucens populations that had been fed a plant-oil based diet. In this study, recombinant Hill_BB_C10074 (500 µg/mL), was found to possess potent antimicrobial activity against the serious Gram-negative pathogen, Pseudomonas aeruginosa. Sequence and structural homology modelling predicted that Hill_BB_C10074 formed a homotrimeric complex that may form pores in the Gram-negative bacterial outer membrane. In vitro experiments defined the antimicrobial action of Hill_BB_C10074 against P. aeruginosa and transmission electron microscopy and electrochemical impedance spectroscopy confirmed the outer membrane disruptive power of Hill_BB_C10074 which was greater than the clinically relevant antibiotic, polymyxin B. CONCLUSIONS: Combining predictive tools with in vitro approaches, we have characterised Hill_BB_C10074 as an important insect antimicrobial peptide and promising candidate for the future development of clinical antimicrobials.

Models of care and the advanced practice nurse role in caring for children and adolescents with a cancer predisposition syndrome: a scoping review protocol.

OBJECTIVE: This scoping review will examine the literature describing models of care, barriers and facilitators of care, and gaps in care delivery for children and adolescents with a cancer predisposition syndrome (CPS). It will also explore how advanced practice nurses contribute to the delivery of care for children and adolescents with a CPS. INTRODUCTION: Cancer remains a leading cause of death in children and adolescents. Pediatric CPS clinics proactively aim for early diagnosis or prevention of cancer in children and adolescents with a CPS. Additionally, the holistic well-being of individuals requires a multidisciplinary team, including advanced practice nurses, to manage their complex health care needs. INCLUSION CRITERIA: This review will consider both published and unpublished literature exploring aspects of models of care and the role of the nurse in pediatric CPS clinics. Literature published in English from 1991 will be considered. METHODS: This scoping review will follow the JBI methodology for scoping reviews. The review will include searches in MEDLINE, Embase, and CINAHL Complete. Gray literature searches will be conducted in OAIster and Social Science Research Network (SSRN), as well as websites of hospitals in the USA and the UK with large pediatric cancer centers. Two reviewers will screen titles, abstracts, and full-text articles. An extraction table will be used to extract relevant data from all included articles and facilitate data analysis. Results will be presented in narrative and tabular format. REVIEW REGISTRATION: Open Science Framework osf.io/axkp7/.

Mendelian randomization for nephrologists.

Confounding is a major limitation of observational studies. Mendelian randomization (MR) is a powerful study design that uses genetic variants as instrumental variables to enable examination of the causal effect of an exposure on an outcome in observational data. With the emergence of large-scale genome-wide association studies in nephrology over the past decade, MR has become a popular method to establish causal inferences. However, MR is a complex and challenging methodology that requires careful consideration to ensure robust results. This review article aims to summarize the basic concepts of MR, its application and relevance in nephrology, and the methodological challenges and limitations as well as discuss the current guidelines for design and reporting. With reference to a clinically relevant example of examining the causal relationship between the estimated glomerular filtration rate and cancer, this review outlines the key steps to conducting an MR study, including the key considerations and potential pitfalls at each step. These include defining the clinical question, selecting the data sources, identifying and refining appropriate genetic variants by considering linkage disequilibrium and associations with potential confounders, harmonization of variants across data sets, validation of the genetic instrument by assessing its strength, estimation of the causal effects, confirming the validity of the findings, and interpreting and reporting results.

Genetically Predicted Vegetable Intake and Cardiovascular Diseases and Risk Factors: An Investigation with Mendelian Randomization.

BACKGROUND: The associations between vegetable intake and cardiovascular diseases have been demonstrated in observational studies, but less sufficiently in randomized trials. Mendelian randomization has been considered a promising alternative in causal inference. The separate effects of cooked and raw vegetable intake remain unclear. This study aimed to investigate the associations between cooked and raw vegetable intake with cardiovascular outcomes using MR. METHODS: We identified 15 and 28 genetic variants statistically and biologically associated with cooked and raw vegetable intake, respectively, from previous genome-wide association studies, which were used as instrumental variables to estimate associations with coronary heart disease (CHD), stroke, heart failure (HF), and atrial fibrillation (AF). The independent effects of genetically predicted cooked and raw vegetable intake were examined using multivariable MR analysis. We performed one-sample and two-sample MR analyses and combined their results using meta-analysis. Bonferroni correction was applied for multiple comparisons. We performed two-sample MR analysis for cardiometabolic risk factors (serum lipids, blood pressure, body mass index, and glycemic traits) to explore the potential mechanisms. RESULTS: In the MR meta-analysis of 1.2 million participants, we found null evidence for associations between genetically predicted cooked and raw vegetable intake with CHD, HF, or AF. Raw vegetable intake was nominally associated with stroke (odds ratio [95% confidence interval] 0.82 [0.69-0.98] per 1 daily serving increase, p = 0.03), but this association did not pass the corrected significance level. We found consistently null evidence for associations with serum lipids, blood pressure, body mass index, or glycemic traits. CONCLUSIONS: We found null evidence for associations between genetically predicted vegetable intake with CHD, AF, HF, or cardiometabolic risk factors in this MR study. Raw vegetable intake may reduce risk of stroke, but this warrants more research. True associations between vegetable intake and CVDs cannot be completely ruled out, and future investigations are required for causal inference in nutritional research.

CVS Health's Acquisition of Oak Street Health Reconfirms Market Viability of Private Equity Investment in Value-Based Payment Models for Primary Care.

For over a decade and for the foreseeable future, federal agencies have made efforts to promote value-based care through various incentive schemes, such as the recent "Regulatory Sprint to Coordinated Care." Federal incentive schemes and other "macro tailwinds" have brought in private equity investors, especially in the context of primary care for Medicare beneficiaries. Oak Street Health and its private equity backers were pioneers in this space, applying buy-and-build strategies to create "next-generation" primary care networks "that focus largely or entirely on Medicare Advantage enrollees." Although Oak Street Health persuasively established a workable "playbook" for private equity investment in value-based care, and forecasts have been favorable, the ultimate market viability of this value-based playbook hinges on whether or not private equity investors can locate corporate buyers. The market viability of such a strategy has now been reconfirmed by the acquisition of Oak Street Health by CVS Health ("CVS"), announced February 8, 2023, and closed May 2, 2023, especially given that the incentives and the efficiencies associated with this deal are likely to be applicable to large-scale vertically integrated "payvider" corporations more generally. This Recent Transaction Comment examines CVS's acquisition of Oak Street Health to consider what factors might lead vertically integrated health care corporations to acquire value-based primary care networks in the future, and what knock on effects such acquisitions might have on future private equity buyouts in health care.

Application of TraDIS to define the core essential genome of Campylobacter jejuni and Campylobacter coli.

Campylobacter species are the major cause of bacterial gastroenteritis. As there is no effective vaccine, combined with the rapid increase in antimicrobial resistant strains, there is a need to identify new targets for intervention. Essential genes are those that are necessary for growth and/or survival, making these attractive targets. In this study, comprehensive transposon mutant libraries were created in six C. jejuni strains, four C. coli strains and one C. lari and C. hyointestinalis strain, allowing for those genes that cannot tolerate a transposon insertion being called as essential. Comparison of essential gene lists using core genome analysis can highlight those genes which are common across multiple strains and/or species. Comparison of C. jejuni and C. coli, the two species that cause the most disease, identified 316 essential genes. Genes of interest highlighted members of the purine pathway being essential for C. jejuni whilst also finding that a functional potassium uptake system is essential. Protein-protein interaction networks using these essential gene lists also highlighted proteins in the purine pathway being major 'hub' proteins which have a large number of interactors across the network. When adding in two more species (C. lari and C. hyointestinalis) the essential gene list reduces to 261. Within these 261 essential genes, there are many genes that have been found to be essential in other bacteria. These include htrB and PEB4, which have previously been found as core virulence genes across Campylobacter species in other studies. There were 21 genes which have no known function with eight of these being associated with the membrane. These surface-associated essential genes may provide attractive targets. The essential gene lists presented will help to prioritise targets for the development of novel therapeutic and preventative interventions.

Enterohemorrhagic Escherichia coli responds to gut microbiota metabolites by altering metabolism and activating stress responses.

Enterohemorrhagic Escherichia coli (EHEC) is a major cause of severe bloody diarrhea, with potentially lethal complications, such as hemolytic uremic syndrome. In humans, EHEC colonizes the colon, which is also home to a diverse community of trillions of microbes known as the gut microbiota. Although these microbes and the metabolites that they produce represent an important component of EHEC's ecological niche, little is known about how EHEC senses and responds to the presence of gut microbiota metabolites. In this study, we used a combined RNA-Seq and Tn-Seq approach to characterize EHEC's response to metabolites from an in vitro culture of 33 human gut microbiota isolates (MET-1), previously demonstrated to effectively resolve recurrent Clostridioides difficile infection in human patients. Collectively, the results revealed that EHEC adjusts to growth in the presence of microbiota metabolites in two major ways: by altering its metabolism and by activating stress responses. Metabolic adaptations to the presence of microbiota metabolites included increased expression of systems for maintaining redox balance and decreased expression of biotin biosynthesis genes, reflecting the high levels of biotin released by the microbiota into the culture medium. In addition, numerous genes related to envelope and oxidative stress responses (including cpxP, spy, soxS, yhcN, and bhsA) were upregulated during EHEC growth in a medium containing microbiota metabolites. Together, these results provide insight into the molecular mechanisms by which pathogens adapt to the presence of competing microbes in the host environment, which ultimately may enable the development of therapies to enhance colonization resistance and prevent infection.

Using genetic association data to guide drug discovery and development: Review of methods and applications.

Evidence on the validity of drug targets from randomized trials is reliable but typically expensive and slow to obtain. In contrast, evidence from conventional observational epidemiological studies is less reliable because of the potential for bias from confounding and reverse causation. Mendelian randomization is a quasi-experimental approach analogous to a randomized trial that exploits naturally occurring randomization in the transmission of genetic variants. In Mendelian randomization, genetic variants that can be regarded as proxies for an intervention on the proposed drug target are leveraged as instrumental variables to investigate potential effects on biomarkers and disease outcomes in large-scale observational datasets. This approach can be implemented rapidly for a range of drug targets to provide evidence on their effects and thus inform on their priority for further investigation. In this review, we present statistical methods and their applications to showcase the diverse opportunities for applying Mendelian randomization in guiding clinical development efforts, thus enabling interventions to target the right mechanism in the right population group at the right time. These methods can inform investigators on the mechanisms underlying drug effects, their related biomarkers, implications for the timing of interventions, and the population subgroups that stand to gain the most benefit. Most methods can be implemented with publicly available data on summarized genetic associations with traits and diseases, meaning that the only major limitations to their usage are the availability of appropriately powered studies for the exposure and outcome and the existence of a suitable genetic proxy for the proposed intervention.

High-Throughput Mutagenesis Reveals a Role for Antimicrobial Resistance- and Virulence-Associated Mobile Genetic Elements in Staphylococcus aureus Host Adaptation.

Methicillin-resistant Staphylococcus aureus (MRSA) clonal-complex 398 (CC398) is the dominant livestock-associated (LA) MRSA lineage in European livestock and an increasing cause of difficult-to-treat human disease. LA-CC398 MRSA evolved from a diverse human-associated methicillin-sensitive population, and this transition from humans to livestock was associated with three mobile genetic elements (MGEs). In this study, we apply transposon-directed insertion site sequencing (TraDIS), a high-throughput transposon mutagenesis approach, to investigate genetic signatures that contribute to LA-CC398 causing disease in humans. We identified 26 genes associated with LA-CC398 survival in human blood and 47 genes in porcine blood. We carried out phylogenetic reconstruction on 1,180 CC398 isolates to investigate the genetic context of all identified genes. We found that all genes associated with survival in human blood were part of the CC398 core genome, while 2/47 genes essential for survival in porcine blood were located on MGEs. Gene SAPIG0966 was located on the previously identified Tn916 transposon carrying a tetracycline resistance gene, which has been shown to be stably inherited within LA-CC398. Gene SAPIG1525 was carried on a phage element, which in part, matched phiSa2wa_st1, a previously identified bacteriophage carrying the Panton-Valentine leucocidin (PVL) virulence factor. Gene deletion mutants constructed in two LA-CC398 strains confirmed that the SAPIG0966 carrying Tn916 and SAPIG1525 were important for CC398 survival in porcine blood. Our study shows that MGEs that carry antimicrobial resistance and virulence genes could have a secondary function in bacterial survival in blood and may be important for host adaptation. IMPORTANCE CC398 is the dominant type of methicillin-resistant Staphylococcus aureus (MRSA) in European livestock and a growing cause of human infections. Previous studies have suggested MRSA CC398 evolved from human-associated methicillin-sensitive Staphylococcus aureus and is capable of rapidly readapting to human hosts while maintaining antibiotic resistance. Using high-throughput transposon mutagenesis, our study identified 26 and 47 genes important for MRSA CC398 survival in human and porcine blood, respectively. Two of the genes important for MRSA CC398 survival in porcine blood were located on mobile genetic elements (MGEs) carrying resistance or virulence genes. Our study shows that these MGEs carrying antimicrobial resistance and virulence genes could have a secondary function in bacterial survival in blood and may be important for blood infection and host adaptation.

Development and validation of a CRISPR interference system for gene regulation in Campylobacter jejuni.

BACKGROUND: Campylobacter spp. are the leading cause of bacterial food-borne illness in humans worldwide, with Campylobacter jejuni responsible for 80% of these infections. There is an urgent need to understand fundamental C. jejuni biology for the development of new strategies to prevent and treat infections. The range of molecular tools available to regulate gene expression in C. jejuni is limited, which in turn constrains our ability to interrogate the function of essential and conditionally essential genes. We have addressed this by developing and utilising a CRISPR-based interference system known as CRISPRi in C. jejuni to control gene expression. To achieve this, a catalytically inactive ("dead") cas9 and sgRNA backbone from the Streptococcus pyogenes CRISPRi system was combined with C. jejuni-derived promoters of predetermined expression activities to develop a CRISPRi-based repression tool in C. jejuni strains M1Cam and 81-176. RESULTS: The CRISPRi tool was validated through successful repression of the arylsulphatase-encoding gene astA using a range of sgRNA target sequences spanning the astA gene. The tool was also applied to target astA in an M1Cam CRISPR-Cas9 deletion strain, which showed that the presence of an endogenous CRISPR-Cas9 system did not affect the activity of the CRISPRi-based repression tool. The tool was further validated against the hippicurase-encoding gene hipO. Following this, the flagella genes flgR, flaA, flaB and both flaA and flaB were targeted for CRISPRi-based repression, which resulted in varying levels of motility reduction and flagella phenotypes as determined by phenotypical assays and transmission electron microscopy (TEM). CONCLUSIONS: This is the first report of a CRISPRi-based tool in C. jejuni, which will provide a valuable resource to the Campylobacter community.