Quantifying the fitness effects of resistance alleles with and without anthelmintic selection pressure using Caenorhabditis elegans.

Albendazole (a benzimidazole) and ivermectin (a macrocyclic lactone) are the two most commonly co-administered anthelmintic drugs in mass-drug administration programs worldwide. Despite emerging resistance, we do not fully understand the mechanisms of resistance to these drugs nor the consequences of delivering them in combination. Albendazole resistance has primarily been attributed to variation in the drug target, a beta-tubulin gene. Ivermectin targets glutamate-gated chloride channels (GluCls), but it is unknown whether GluCl genes are involved in ivermectin resistance in nature. Using Caenorhabditis elegans, we defined the fitness costs associated with loss of the drug target genes singly or in combinations of the genes that encode GluCl subunits. We quantified the loss-of-function effects on three traits: (i) multi-generational competitive fitness, (ii) fecundity, and (iii) development. In competitive fitness and development assays, we found that a deletion of the beta-tubulin gene ben-1 conferred albendazole resistance, but ivermectin resistance required the loss of two GluCl genes (avr-14 and avr-15). The fecundity assays revealed that loss of ben-1 did not provide any fitness benefit in albendazole conditions and that no GluCl deletion mutants were resistant to ivermectin. Next, we searched for evidence of multi-drug resistance across the three traits. Loss of ben-1 did not confer resistance to ivermectin, nor did loss of any single GluCl subunit or combination confer resistance to albendazole. Finally, we assessed the development of 124 C. elegans wild strains across six benzimidazoles and seven macrocyclic lactones to identify evidence of multi-drug resistance between the two drug classes and found a strong phenotypic correlation within a drug class but not across drug classes. Because each gene affects various aspects of nematode physiology, these results suggest that it is necessary to assess multiple fitness traits to evaluate how each gene contributes to anthelmintic resistance.

Quantitative tests of albendazole resistance in beta-tubulin mutants.

Benzimidazole (BZ) anthelmintics are among the most important treatments for parasitic nematode infections in the developing world. Widespread BZ resistance in veterinary parasites and emerging resistance in human parasites raise major concerns for the continued use of BZs. Knowledge of the mechanisms of resistance is necessary to make informed treatment decisions and circumvent resistance. Benzimidazole resistance has traditionally been associated with mutations and natural variants in the C. elegans beta-tubulin gene ben-1 and orthologs in parasitic species. However, variants in ben-1 alone do not explain the differences in BZ responses across parasite populations. Here, we examine the roles of five C. elegans beta-tubulin genes (tbb-1, mec-7, tbb-4, ben-1, and tbb-6) to identify the role each gene plays in BZ response. We generated C. elegans strains with a loss of each beta-tubulin gene, as well as strains with a loss of tbb-1, mec-7, tbb-4, or tbb-6 in a genetic background that also lacks ben-1 to test beta-tubulin redundancy in BZ response. We found that only the individual loss of ben-1 conferred a substantial level of BZ resistance, although the loss of tbb-1 was found to confer a small benefit in the presence of albendazole (ABZ). The loss of ben-1 was found to confer an almost complete rescue of animal development in the presence of 30 µM ABZ, likely explaining why no additive effects caused by the loss of a second beta-tubulin were observed. We demonstrate that ben-1 is the only beta-tubulin gene in C. elegans where loss confers substantial BZ resistance.

Quantifying the fitness effects of resistance alleles with and without anthelmintic selection pressure using Caenorhabditis elegans.

Albendazole and ivermectin are the two most commonly co-administered anthelmintic drugs in mass-drug administration programs worldwide. Despite emerging resistance, we do not fully understand the mechanisms of resistance to these drugs nor the consequences of delivering them in combination. Albendazole resistance has primarily been attributed to variation in the drug target, a beta-tubulin gene. Ivermectin targets glutamate-gated chloride channel (GluCl) genes, but it is unknown whether these genes are involved in ivermectin resistance in nature. Using Caenorhabditis elegans, we defined the fitness costs associated with loss of the drug target genes singly or in combinations of the genes that encode GluCl subunits. We quantified the loss-of function effects on three traits: (i) multi-generational competitive fitness, (ii) fecundity, and (iii) development. In competitive fitness and development assays, we found that a deletion of the beta-tubulin gene ben-1 conferred albendazole resistance, but ivermectin resistance required loss of two GluCl genes (avr-14 and avr-15) or loss of three GluCl genes (avr-14, avr-15, and glc-1). The fecundity assays revealed that loss of ben-1 did not provide any fitness benefit in albendazole and that no GluCl deletion mutants were resistant to ivermectin. Next, we searched for evidence of multi-drug resistance across the three traits. Loss of ben-1 did not confer resistance to ivermectin, nor did loss of any single GluCl subunit or combination confer resistance to albendazole. Finally, we assessed the development of 124 C. elegans wild strains across six benzimidazoles and seven macrocyclic lactones to identify evidence of multi-drug resistance between the two drug classes and found a strong phenotypic correlation within a drug class but not across drug classes. Because each gene affects various aspects of nematode physiology, these results suggest that it is necessary to assess multiple fitness traits to evaluate how each gene contributes to anthelmintic resistance.

Behavioral, metabolic, and lipidomic characterization of the 5xFADxTg30 mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is associated with both extracellular amyloid-ß (Aß) plaques and intracellular tau-containing neurofibrillary tangles (NFT). We characterized the behavioral, metabolic and lipidomic phenotype of the 5xFADxTg30 mouse model which contains overexpression of both Aß and tau. Our results independently reproduce several phenotypic traits described previously for this model, while providing additional characterization. This model develops many aspects associated with AD including frailty, decreased survival, initiation of aspects of cognitive decline and alterations to specific lipid classes and molecular lipid species in the plasma and brain. Notably, some sex-specific differences exist in this model and motor impairment with aging in this model does compromise the utility of the model for some movement-based behavioral assessments of cognitive function. These findings provide a reference for individuals interested in using this model to understand the pathology associated with elevated Aß and tau or for testing potential therapeutics for the treatment of AD.

KatG catalase deficiency confers bedaquiline hyper-susceptibility to isoniazid resistant Mycobacterium tuberculosis.

Multidrug-resistant tuberculosis (MDR-TB) is a growing source of global mortality and threatens global control of tuberculosis (TB) disease. The diarylquinoline bedaquiline (BDQ) recently emerged as a highly efficacious drug against MDR-TB, defined as resistance to the first-line drugs isoniazid (INH) and rifampin. INH resistance is primarily caused by loss-of-function mutations in the catalase KatG, but mechanisms underlying BDQ's efficacy against MDR-TB remain unknown. Here we employ a systems biology approach to investigate BDQ hyper-susceptibility in INH-resistant Mycobacterium tuberculosis . We found hyper-susceptibility to BDQ in INH-resistant cells is due to several physiological changes induced by KatG deficiency, including increased susceptibility to reactive oxygen species and DNA damage, remodeling of transcriptional programs, and metabolic repression of folate biosynthesis. We demonstrate BDQ hyper-susceptibility is common in INH-resistant clinical isolates. Collectively, these results highlight how altered bacterial physiology can impact drug efficacy in drug-resistant bacteria.

Internally versus externally trained residents and fellows hired as attendings at a large integrated healthcare system: a 20-year retrospective study.

BACKGROUND: There remains a question of whether graduates trained internally are different than those trained elsewhere. We examine the difference between physicians trained within our Graduate Medical Education (GME) programs versus physicians trained elsewhere. Our large integrated healthcare system is unique in addressing this objective due to its large physician labor hiring needs across different specialties of GME graduates. METHODS: A retrospective review was performed from Jan 2000 to August 2020 of Kaiser Permanente Southern California (KPSC) physicians hired: KPSC GME trained versus non-KPSC GME trained. We examined five variables: retention, leadership (current or historical), physician relations cases, member appraisal of physician and provider services survey (MAPPS) scores, and rate of board certification. Chi-square test of proportions was used for comparison, p < 0.05 was significant. RESULTS: From Jan 2000 to August 2020, 2940 residents and fellows graduated from KPSC GME programs, of which 1127 (38%) were hired on at KPSC as full time attendings. Across all five metrics (Retention 82% vs 76% (p = < 0.01), Leadership [current 13% vs 10% (p = < 0.01)or historical 17% vs 14% (p = 0.01)], Physician Relations 23% vs 26% (p = 0.04), MAPPS 75% vs 69% (p = < 0.01), and Board Certification 81% vs 74% (p = < 0.01)), KPSC outperformed non-KPSC GME-trained physicians to a statistically significant degree. CONCLUSIONS: We have shown that an internally sponsored GME program can represent an opportunity for recruitment of physicians that may have higher retention rates, higher probability of being physician leaders, decreased likelihood of physician relations issues, improved patient satisfaction, and increased rates of board certification.

A tetranuclear polypyridylruthenium(II) complex as a selective stain for extracellular vesicle penetration through brain microvascular endothelium.

A new photoluminescent polypyridylruthenium(II) stain for extracellular vesicles (EVs) released from lipopolysaccharide-stimulated THP-1 monocytes enabled important new insights into how the bacteria-induced immune system affects the blood-brain barrier (BBB). These included previously unknown aspects of EV interactions with BBB microvascular endothelial cells and the extracellular matrix relevant to human brain diseases.

Quantifying sustained pain worsening in knee osteoarthritis.

OBJECTIVE: Recent work suggests that many persons with knee osteoarthritis (OA) experience stable symptoms over time. Whether patients experience periods of symptom exacerbation or flare which interrupt this stable course, and how long such periods last, has received little study. Our objective is to describe the frequency and duration of episodes of pain worsening in persons with knee OA. METHODS: We selected participants from the Osteoarthritis Initiative with radiographic, symptomatic knee OA. We defined a clinically relevant increase in knee pain as an increase in Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) pain of ≥9 points. We defined sustained worsening as maintaining at least 80% of the initial increase. We used Poisson regression to estimate the incidence rate (IR) of episodes of pain worsening. RESULTS: 1093 participants were included in the analysis. Eighty-eight percent had ≥1 increase in WOMAC pain ≥9 points (IR: 26.3 per 100 person years (95% CI: 25.2, 27.4)). Forty-eight percent had ≥1 episode of sustained worsening (IR: 9.7 per 100 person-years (95% CI: 8.9, 10.5)). Elevated pain was maintained an average of 2.4 years after the initial increase. CONCLUSION: Most participants with knee OA reported at least one clinically relevant increase in WOMAC pain, but fewer than half experienced an episode of sustained pain worsening. These individual-level data portray a more nuanced and fluctuating course of OA pain than suggested by trajectory studies. These data could be useful in shared decision-making regarding prognosis and treatment choices in persons affected by symptomatic knee OA.

Controlled Continuous Evolution of Enzymatic Activity Screened at Ultrahigh Throughput Using Drop-Based Microfluidics.

Enzymes are highly specific catalysts delivering improved drugs and greener industrial processes. Naturally occurring enzymes must typically be optimized which is often accomplished through directed evolution; however, this is still a labor- and capital-intensive process, due in part to multiple molecular biology steps including DNA extraction, in vitro library generation, transformation, and limited screening throughput. We present an effective and broadly applicable continuous evolution platform that enables controlled exploration of fitness landscape to evolve enzymes at ultrahigh throughput based on direct measurement of enzymatic activity. This drop-based microfluidics platform cycles cells between growth and mutagenesis followed by screening with minimal human intervention, relying on the nCas9 chimera with mutagenesis polymerase to produce in vivo gene diversification using sgRNAs tiled along the gene. We evolve alditol oxidase to change its substrate specificity towards glycerol, turning a waste product into a valuable feedstock. We identify a variant with a 10.5-fold catalytic efficiency.

Research participants may not recall their participation but have a better understanding of alternative management options than patients in routine care.

BACKGROUND: Patient understanding of care interventions, of the clinical uncertainty, and of their participation in clinical research is often poor. We hypothesized that compared to routine care, patients would better understand the prevailing uncertainty when they participated in research. METHODS: A questionnaire was administered to patients at the time they attended a follow-up neurovascular clinic 4 to 52 weeks after a care episode where they did or did not participate in a clinical trial. Patients were not reminded whether they had previously participated in a clinical trial. Questions concerned their understanding of the risks/benefits of interventions, the availability of alternative options, whether their personal opinion was taken into consideration, the reason for the final decision, their confidence at having received the best management, and whether they had been research participants. RESULTS: Between June 2019 and June 2020, 167 patients were recruited; 71 had truly been research participants, while 96 had not. A greater proportion of research patients were aware of the existence of management alternatives (65% versus 44%; P=0.008). Patients of both groups believed their personal opinion counted in the final decision (76% versus 70%), and patients were equally confident that they had received the best management (94%). Research patients believed they had participated in research 46% of the time, compared to 12% of routine care patients (P=0.003). CONCLUSION: Many patients do not recall that they participated in a clinical trial, but they have a better understanding of the clinical uncertainty and of the availability of alternative management options.

Does gabapentin provide benefit for patients with knee OA? A benefit-harm and cost-effectiveness analysis.

OBJECTIVE: Gabapentin can treat neuropathic pain syndromes and has increasingly been prescribed to treat nociplastic pain. Some patients with knee osteoarthritis (OA) suffer from both nociceptive and nociplastic pain. We examined the cost-effectiveness of adding gabapentin to knee OA care. METHOD: We used the Osteoarthritis Policy Model, a validated Monte Carlo simulation of knee OA, to examine the value of gabapentin in treating knee OA by comparing three strategies: 1) usual care, gabapentin sparing (UC-GS); 2) targeted gabapentin (TG), which provides gabapentin plus usual care for those who screen positive for nociplastic pain on the modified PainDETECT questionnaire (mPD-Q) and usual care only for those who screen negative; and 3) universal gabapentin plus usual care (UG). Outcomes included cumulative quality-adjusted life years (QALYs), lifetime direct medical costs, and incremental cost-effectiveness ratios (ICERs), discounted at 3% annually. We derived model inputs from published literature and national databases and varied key input parameters in sensitivity analyses. RESULTS: UC-GS dominated both gabapentin-containing strategies, as it led to lower costs and more QALYs. TG resulted in a cost increase of $689 and a cumulative QALY reduction of 0.012 QALYs. UG resulted in a further $1,868 cost increase and 0.036 QALY decrease. The results were robust to plausible changes in input parameters. The lowest TG strategy ICER of $53,000/QALY was reported when mPD-Q specificity was increased to 100% and AE rate was reduced to 0%. CONCLUSION: Incorporating gabapentin into care for patients with knee OA does not appear to offer good value.

The role of RBC antigen transgene integration sites on RBC biology in mice.

BACKGROUND: Transgenic mice expressing RBC specific antigens are widely used in mechanistic studies of RBC alloimmunization. Existing RBC donor strains have random transgene integration, potentially disrupting host elements that can confound biological interpretation. STUDY DESIGN AND METHODS: Integration site and genomic alterations were characterized by both targeted locus amplification and congenic backcrossing in the five most commonly used RBC alloantigen donor strains (KEL-K2hi , KEL-K2med , and KEL-K2lo , and KEL-K1). A targeted transgenic approach was developed to allow RBC specific transgene expression from a safe harbor locus (ROSA26). Alloimmune responses were assessed by transfusing alloantigen expressing RBCs into wild-type recipients and measuring alloantibodies by flow cytometry. RESULTS/FINDINGS: Four of the five analyzed strains had at least one gene disrupted by the transgene integration but none of the disrupted genes are known to be involved in RBC biology. The integration of KEL-K2med potentially altered the immunological properties of RBCs, although the biological significance of the observed changes is unclear. The ROSA26 targeted approach resulted in a single copy of the transgene that maintains RBC specific expression without random disruption of genomic elements. CONCLUSION: These findings provide a detailed characterization of genomic disruption by transgene integration found in commonly used RBC donor strains that is relevant to numerous previous publications as well as future studies. With the possible exception of KEL-K2med , transgene integration is not predicted to affect RBC biology in existing models, and new models can avoid this concern using the described targeted transgenic approach.

The turkey ascarid, Ascaridia dissimilis, as a model genetic system.

Parasitic nematodes cause significant effects on humans each year, with the most prevalent being Ascaris lumbricoides. Benzimidazoles (BZ) are the most widely used anthelmintic drug in humans, and although the biology of resistance to this drug class is understood in some species, resistance is poorly characterized in ascarids. Models such as Caenorhabditis elegans were essential in developing our current understanding of BZ resistance, but more closely related model nematodes are needed to understand resistance in ascarids. Here, we propose a new ascarid model species that infects turkeys, Ascaridia dissimilis, to develop a better understanding of BZ resistance.

Understanding the research-care demarcation and why it must be revised.

BACKGROUND: A clean-cut separation between research and care was artificially created at the time of the Belmont report more than 40 years ago. The demarcation was initially controversial but eventually was implemented for political reasons. We examine why it must be revised. METHODS: We review historical research scandals as well as the theoretical basis for the Belmont demarcation. We then discuss consequences on medical practice and propose an alternative. DISCUSSION: Most research scandals involved abusing human beings supposedly for the sake of science. Belmont commissioners were aware the research/care problem was double-headed. While research subjects should be protected from abuse in the research context, patients need to be protected from unvalidated medical and surgical interventions in the care context. For political reasons the Commission recommended the regulation of research but to leave medical practice untouched. Thus the Commission had to distinguish research from care. The notion of 'generalizable knowledge' was introduced to define and regulate research, but the inadvertent result was that by trying to protect research subjects, the regulation has not only failed to protect all other patients, but also encouraged the widespread practice of unvalidated interventions within the care context. The notion of validated care should be re-introduced into a proper analysis of the care-research demarcation, for care research is an integral ingredient of a good medical practice. CONCLUSION: The research-care demarcation should be revised to leave room for the validated/unvalidated care distinction. Care research, essential to guide medical practice, should be facilitated at all levels.

A Systematic Review of Autistic People and the Criminal Justice System: An Update of King and Murphy (2014).

The purpose of this paper was to determine whether recommendations made by King & Murphy (Journal of Autism and Developmental Disorders 44:2717-2733, 2014) in their review of the evidence on autistic people in contact with the criminal justice system (CJS) have been addressed. Research published since 2013 was systematically examined and synthesised. The quality of 47 papers was assessed using the Mixed Methods Appraisal Tool. Findings suggest a limited amount of good quality research has been conducted that has focused on improving our understanding of autistic people in contact with the CJS since 2013. Methodological limitations make direct comparisons between autistic and non-autistic offenders difficult. Autistic people commit a range of crimes and appear to have unique characteristics that warrant further exploration (i.e., vulnerabilities, motivations for offending).

"If I don't have credit, I don't have anything": Perspectives on the credit scoring system among mothers with low incomes.

This mixed-methods study examines consumer perspectives on the credit scoring system drawn from in-depth interviews with 72 mothers with low incomes and national survey data from the National Financial Capability Study. Interviewees express strong awareness of credit scoring and a desire to have good credit. National survey data corroborate these findings, showing that most mothers with low incomes are knowledgeable about their credit scores. They know what behaviors improve credit standing and recognize the tradeoffs between present consumption and longer-run goals. They do not reject the credit scoring system's legitimacy and seek to work within this system to pursue their financial goals, despite obstacles to success. This evidence enriches our understanding of the perspectives and values that motivate consumer financial behaviors and highlights the systemic challenges to people's financial well-being that are embedded in a seemingly widely accepted credit scoring system.

"I Don't Like All Those Fees" Pragmatism About Financial Services Among Low-Income Parents.

Basic financial services facilitate people's ability to manage their finances, save, and receive payments from employers or the government. Drawing on survey data as well as qualitative interviews with 80 mothers with limited incomes, we find that parents take a pragmatic view and use a wide range of financial services to meet their needs including fintech, prepaid cards, and mobile phone-based solutions, as well as traditional banks. Mistrust in institutions is an important factor in shaping the services mothers avoid. Structural factors, like employers' payment methods, also play a role in financial service use. These low-income parents of young children are actively using a range of financial services, much broader than those provided by traditional banks. Many mothers engaged in complex financial management practices to receive income and pay their bills. This opens room for potentially costly errors and is, at least, taxing their cognitive bandwidth. Researchers must attend to the diverse set of financial services with which parents engage and investigate how this affects families' financial wellbeing and inclusion.