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.

Dietary trehalose enhances virulence of epidemic Clostridium difficile.

Clostridium difficile disease has recently increased to become a dominant nosocomial pathogen in North America and Europe, although little is known about what has driven this emergence. Here we show that two epidemic ribotypes (RT027 and RT078) have acquired unique mechanisms to metabolize low concentrations of the disaccharide trehalose. RT027 strains contain a single point mutation in the trehalose repressor that increases the sensitivity of this ribotype to trehalose by more than 500-fold. Furthermore, dietary trehalose increases the virulence of a RT027 strain in a mouse model of infection. RT078 strains acquired a cluster of four genes involved in trehalose metabolism, including a PTS permease that is both necessary and sufficient for growth on low concentrations of trehalose. We propose that the implementation of trehalose as a food additive into the human diet, shortly before the emergence of these two epidemic lineages, helped select for their emergence and contributed to hypervirulence.

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.

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.

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.

"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.

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.

The Adapted Firesetting Assessment Scale: reliability and validity.

BACKGROUND: The Adapted Firesetting Assessment Scale was developed for use with adults with developmental disabilities targeting fire-related factors thought to be associated with deliberate firesetting behaviour (i.e. attitudes towards fire, fire interest, fire normalisation, identification with fire and fire safety awareness). However, the psychometric properties of the scale are yet to be evaluated. METHOD: The reliability, validity, comprehensibility, relevance and comprehensiveness of the Adapted Firesetting Assessment Scale were evaluated. Fifty-nine adults with developmental disabilities, some of whom had a history of firesetting, completed the Adapted Firesetting Assessment Scale on two occasions. Feedback about the questionnaire was sought from both participants and professionals. RESULTS: The AFAS has acceptable internal consistency and excellent test-retest reliability. The attitudes towards fire, fire normalisation, poor fire safety subscales and total scores discriminated firesetters from non-firesetters. Content analysis of feedback indicated items of the AFAS were understood, relevant, accessible and comprehensible. CONCLUSION: A larger study is needed to examine the factor structure of the AFAS.

Needs assessment of community health workers to enhance efficient delivery of their services for community case management of malaria in Kenya.

BACKGROUND: Malaria continues to be the leading cause of morbidity and mortality in Africa. Community Case Management of malaria (CCMm) which is undertaken by engaging Community Health Workers (CHWs) to effectively address management of malaria cases in some endemic communities was explored in this study. The aim was to assess the needs of CHWs that would help sustain and retain their services to enhance the efficient delivery of CCMm. METHODS: Using semi-structured questionnaires, data on the needs of CHWs was gathered through a qualitative study consisting of in-depth interviews and focus group discussions (FGDs) conducted among study participants in five districts in western Kenya. The study participants comprised of 100 CHWs, 100 mothers of children under five years and 25 key informants made up of public health officers and clinicians involved in the CCMm. The interviews were conducted in English and Swahili or Dholuo, the local language. The recorded audio interviews were transcribed later. The analysis was done using NVivo version 7 software and transcripts were coded after which themes related to the objectives of the study were identified. RESULTS: All the study participants recognized the need to train and update CHWs on their work as well as remunerating them for their services to enhance efficient delivery of services. The CHWs on their part perceived the provision of gloves, rapid diagnostic test kits (RDTs), lancets, cotton wool and ethanol, bins (to dispose of RDTs and lancets), together with drugs for treating clients as the essential needs to undertake CCMm in the communities. Other logistical needs and incentives mentioned by CHWs and key informants for the successful delivery of CCMm included: gumboots, raincoats, torch lights, mobile phones, means of transportation (bicycles and motorbikes), uniforms and ID cards for identification. CONCLUSIONS: CHWs would perform tasks better and their services retained for a sustainable CCMm if: properly incentivized; offered refresher trainings (and updates) on malaria; and equipped with the requisite tools identified in this study.

Do images of dietitians on the Internet reflect the profession?

BACKGROUND: The image of dietitians presented on the Internet shapes public perception of the profession, which in turn may influence engagement with professionals. The aim was to describe the portrayal of the dietetic profession on the Internet and how this aligns with international professional demographics. METHODS: In this cross-sectional observational study, images appearing in two Google image searches using the word 'dietitian' were analysed for content by two independent researchers. A coding framework was used to identify personal characteristics of professionals and others presented in the image, as well as the work setting. These were compared to demographic data of members of professional associations in Australia, the United Kingdom (UK) and the United States of America (USA). RESULTS: The dietitian portrayed in the images (n = 339) was most often female (88%), Caucasian (72%), aged between 26-39 years (63%), pictured alone (78%), pictured with food (78%) and in a setting that could not be determined (76%). The age and gender profile presented matches the characteristics of the international workforce; however, there was an absence of images illustrating dietitians in authentic work roles. CONCLUSIONS: The images resulting from an online search for 'dietitian' do not fully illustrate the profession. There are opportunities to create and share authentic images online that show the breadth of work roles and diversity of professionals' age, gender, cultural background and size.

A Study of GP Workload and Satisfaction.

Aims In this novel study in the Irish setting, we quantified the number items managed per General Practitioner (GP) consult, how each item is managed, and impact on a GP's job satisfaction. Methods Participating GPs at two surgeries completed a questionnaire - integrated into the practice management software - after each consultation that satisfied the inclusion criteria during a four-week period. Results Due to feasibility constraints, 500 of 857 (58.3%) completed questionnaires were randomly selected for our sample. GPs manage an average of 1.76 items per consultation. Older patients presented with more items. Greater number of presenting items led to less being managed on the day 71% (n=5) for 5 items vs. 95.2% (n= 246) for 1 item, longer consultation duration (mean = 14.63 minutes (4-45) and decreased GP satisfaction, mean 8/10 (2-10). Conclusion Increasing the number of items in a GP consultation has a statistically significant effect on duration of consultation, how each item is managed, and even GP satisfaction.

Increased care at discharge from COVID-19: The association between pre-admission frailty and increased care needs after hospital discharge; a multicentre European observational cohort study.

BACKGROUND: The COVID-19 pandemic has placed significant pressure on health and social care. Survivors of COVID-19 may be left with substantial functional deficits requiring ongoing care. We aimed to determine whether pre-admission frailty was associated with increased care needs at discharge for patients admitted to hospital with COVID-19. METHODS: Patients were included if aged over 18 years old and admitted to hospital with COVID-19 between 27 February and 10 June 2020. The Clinical Frailty Scale (CFS) was used to assess pre-admission frailty status. Admission and discharge care levels were recorded. Data were analysed using a mixed-effects logistic regression adjusted for age, sex, smoking status, comorbidities, and admission CRP as a marker of severity of disease. RESULTS: Thirteen hospitals included patients: 1671 patients were screened, and 840 were excluded including, 521 patients who died before discharge (31.1%). Of the 831 patients who were discharged, the median age was 71 years (IQR, 58-81 years) and 369 (44.4%) were women. The median length of hospital stay was 12 days (IQR 6-24). Using the CFS, 438 (47.0%) were living with frailty (≥ CFS 5), and 193 (23.2%) required an increase in the level of care provided. Multivariable analysis showed that frailty was associated with an increase in care needs compared to patients without frailty (CFS 1-3). The adjusted odds ratios (aOR) were as follows: CFS 4, 1.99 (0.97-4.11); CFS 5, 3.77 (1.94-7.32); CFS 6, 4.04 (2.09-7.82); CFS 7, 2.16 (1.12-4.20); and CFS 8, 3.19 (1.06-9.56). CONCLUSIONS: Around a quarter of patients admitted with COVID-19 had increased care needs at discharge. Pre-admission frailty was strongly associated with the need for an increased level of care at discharge. Our results have implications for service planning and public health policy as well as a person's functional outcome, suggesting that frailty screening should be utilised for predictive modelling and early individualised discharge planning.

Association of knee OA structural phenotypes to risk for progression: a secondary analysis from the Foundation for National Institutes of Health Osteoarthritis Biomarkers study (FNIH).

PURPOSE: Aim was to stratify the knee MRIs of the Foundation for National Institutes of Health Osteoarthritis Biomarkers Consortium (FNIH) cohort into distinct structural phenotypes based on semiquantitative assessment and to determine risk for pain and structural progression over 48 months. METHODS: The study sample from the FNIH project was selected as a nested case-control study with knees showing either 1) radiographic and pain progression (i.e., "composite" cases), 2) radiographic progression only ("JSL"), 3) pain progression only, and 4) neither radiographic nor pain progression. MRI was performed on 3T systems. MRIs were read according to the MOAKS scoring system. Knees were stratified into subchondral bone, cartilage/meniscus and inflammatory phenotypes using the baseline visits. The relation of each phenotype to risk of being in the combined JSL plus composite outcome or composite case only group compared to those not having that phenotype was determined using logistic regression. Only KL2 and 3 and those without root tears were included. RESULTS: 485 knees were included. 362 (75%) did not have any phenotype, while 95 (20%) had the bone phenotype, 22 (5%) the cartilage/meniscus phenotype and 19 (4%) the inflammatory phenotype. The bone phenotype was associated with a higher odds of the combined JSL plus composite outcome and composite outcome only (OR 1.81; [95%CI 1.14,2.85] and 1.65; 95%CI [1.04,2.61]) while the inflammatory (OR 0.96 [95%CI 0.38,2.42] and 1.25; 95%CI [0.48,3.25]) and the cartilage/meniscus phenotypes were not significantly associated with outcome (OR 1.30 95%CI [0.55,3.07] and 0.99; 95%CI [0.40,2,49]). CONCLUSIONS: The bone phenotype was associated with increased risk of having both radiographic and pain progression. Phenotypic stratification may be useful to consider when selecting patients for inclusion in clinical trials.

Estimating contextual effect in nonpharmacological therapies for pain in knee osteoarthritis: a systematic analytic review.

OBJECTIVE: Conduct a systematic review and use meta-analytic techniques to estimate the proportion of total treatment effect that can be attributable to contextual effects (PCE) in adults receiving nonpharmacological, nonsurgical (NPNS) treatments for knee osteoarthritis (OA). DESIGN: We reviewed the published literature to identify five frequently studied NPNS treatments for knee OA: exercise, acupuncture, ultrasound, laser, and transcutaneous electrical nerve stimulation (TENS). We searched for randomized controlled trials (RCTs) of these treatments and abstracted pre- and post-intervention pain scores for groups receiving placebo and active treatments. For each study we calculated the PCE by dividing the change in pain in the placebo group by the change in pain in the active treatment group. We log transformed the PCE measure and pooled across studies using a random effects model. RESULTS: We identified 25 studies for analysis and clustered the RCTs into two groups: acupuncture and topical energy modalities (TEM). 13 acupuncture studies included 1,653 subjects and 12 TEM studies included 572 subjects. The combined PCE was 0.61 (95% CI 0.46-0.80) for acupuncture and 0.69 (95% CI 0.54-0.88) for TEM. CONCLUSION: Our findings suggest that about 61% and 69% of the total treatment effect experienced by subjects receiving acupuncture and TEM treatments, respectively, for knee OA pain may be explained by contextual effects. Contextual effects may include the placebo effect, changes attributable to natural history, and effects of co-therapies. These data highlight the important role of contextual effects in the response to NPNS OA treatments.

Association of baseline and change in tibial and femoral cartilage thickness and development of widespread full-thickness cartilage loss in knee osteoarthritis - data from the Osteoarthritis Initiative.

OBJECTIVE: To investigate whether baseline cartilage thickness and its longitudinal change are associated with incident widespread full-thickness cartilage loss (wsFTCL) in knee osteoarthritis, and whether there are optimal cut-off values for predicting wsFTCL. METHODS: Central medial tibial (cMT) and femoral (cMF) cartilage were assessed using quantitative magnetic resonance imaging data from the Osteoarthritis Initiative cohort (N = 600 knees). Cartilage thickness was measured at baseline and 12 months. wsFTCL was defined semi-quantitatively (scores 2 and 3 from the MRI Osteoarthritis Knee Score) and its incidence at 24 months recorded. Logistic regression was used to determine the odds of developing wsFTCL for baseline and for each 0.1 mm decrease in cartilage thickness. Cut-off values were investigated using the minimal-p method and area under the Receiver Operating Characteristic curves (AUC). RESULTS: Incident wsFTCL was observed in 66 (12%) and 73 (14%) knees in cMT and cMF, respectively. Lower baseline cMT and cMF cartilage thickness values were associated with wsFTCL (OR = 1.20; 95% CI: 1.11, 1.28 and OR = 1.15; 95% CI: 1.06 to 1.24, respectively). Optimal cut-off AUCs for the tibia and femur were 0.64 (0.57-0.70) and 0.63 (0.57-0.69), respectively. Longitudinal decrease in femoral, but not tibial, cartilage thickness was associated with incident wsFTCL (OR = 1.77; 95% CI: 1.30 to 2.40); optimal cut-off AUC 0.65 (95% CI: 0.58-0.72). CONCLUSION: Lower baseline cMT and baseline/change (decrease) over 12 months in cMF cartilage thickness were associated with incident, location-specific, wsFTCL at 24 months. Optimal cut-off values were relatively low and of uncertain utility for predicting incident wsFTCL.

MRI-based screening for structural definition of eligibility in clinical DMOAD trials: Rapid OsteoArthritis MRI Eligibility Score (ROAMES).

PURPOSE: Our aim was to introduce a simplified MRI instrument, Rapid OsteoArthritis MRI Eligibility Score (ROAMES), for defining structural eligibility of patients for inclusion in disease-modifying osteoarthritis drug trials using a tri-compartmental anatomic approach that enables stratification of knees into different structural phenotypes and includes diagnoses of exclusion. We also aimed to define overlap between phenotypes and determine reliability. METHODS: 50 knees from the Foundation for National Institutes of Health Osteoarthritis Biomarkers study, a nested case-control study within the Osteoarthritis Initiative, were selected within pre-defined definitions of phenotypes as either inflammatory, subchondral bone, meniscus/cartilage, atrophic or hypertrophic. A focused scoring instrument was developed covering cartilage, meniscal damage, inflammation and osteophytes. Diagnoses of exclusion were meniscal root tears, osteonecrosis, subchondral insufficiency fracture, tumors, malignant marrow infiltration and acute traumatic changes. Reliability was determined using weighted kappa statistics. Descriptive statistics were used for determining concordance between the a priori phenotypic definition and ROAMES and overlap between phenotypes. RESULTS: ROAMES identified 43 of 50 (86%) pre-defined phenotypes correctly. Of the 50 participants, 27 (54%) had no additional phenotypes other than the pre-defined phenotype. 18 (36%) had one and 5 (10%) had two additional phenotypes. None had three or four additional phenotypes. All features of ROAMES showed almost perfect agreement. One case with osteonecrosis and one with a tumor were detected. CONCLUSIONS: ROAMES is able to screen and stratify potentially eligible knees into different structural phenotypes and record relevant diagnoses of exclusion. Reliability of the instrument showed almost perfect agreement.

Formaldehyde-activated WEHI-150 induces DNA interstrand crosslinks with unique structural features.

Mitoxantrone is an anticancer anthracenedione that can be activated by formaldehyde to generate covalent drug-DNA adducts. Despite their covalent nature, these DNA lesions are relatively labile. It was recently established that analogues of mitoxantrone featuring extended side-chains terminating in primary amino groups typically yielded high levels of stable DNA adducts following their activation by formaldehyde. In this study we describe the DNA sequence-specific binding properties of the mitoxantrone analogue WEHI-150 which is the first anthracenedione to form apparent DNA crosslinks mediated by formaldehyde. The utility of this compound lies in the versatility of the covalent binding modes displayed. Unlike other anthracenediones described to date, WEHI-150 can mediate covalent adducts that are independent of interactions with the N-2 of guanine and is capable of adduct formation at novel DNA sequences. Moreover, these covalent adducts incorporate more than one formaldehyde-mediated bond with DNA, thus facilitating the formation of highly lethal DNA crosslinks. The versatility of binding observed is anticipated to allow the next generation of anthracenediones to interact with a broader spectrum of nucleic acid species than previously demonstrated by the parent compounds, thus allowing for more diverse biological activities.

Articular chondrocytes isolated from the knee and ankle joints of human tissue donors demonstrate similar redox-regulated MAP kinase and Akt signaling.

OBJECTIVE: To compare key intracellular redox-regulated signaling pathways in chondrocytes derived from knee joint femoral cartilage and ankle joint talar cartilage in order to determine if differences exist that might contribute to the lower prevalence of ankle osteoarthritis (OA). METHODS: Femoral and talar chondrocytes were treated with H2O2 generators (menadione or 2-3-dimethoxy-1,4-napthoquinone (DMNQ), fragments of fibronectin (FN-f)) to stimulate MAP kinase signaling (MAPK), or with IGF-1 to stimulate the Akt signaling pathway. Hyperoxidation of the peroxiredoxins, used as a measure of redox status, and phosphorylation of proteins pertinent to MAPK (p38, ERK, JNK, c-Jun) and Akt (Akt, PRAS40) signaling cascades were detected by immunoblotting. RESULTS: Treatment of femoral and talar chondrocytes with menadione, DMNQ or FN-f led to a time dependent increase in extracellular-regulated kinase (ERK) and p38 phosphorylation. DMNQ and FN-f stimulation enhanced phosphorylation of JNK and its downstream substrate, c-Jun. Menadione treatment did not stimulate JNK activity but hyperoxidized the peroxiredoxins and inhibited IGF-1-induced Akt activation. In all experiments, chondrocytes derived from the femur and talar joints displayed comparable MAP kinase responses after treatment with various catabolic stimuli, as well as similar Akt signaling responses after IGF-1 treatment. CONCLUSIONS: MAP kinase and Akt signaling in response to factors that modulate the intracellular redox status were similar in chondrocytes from knee and ankle joints suggesting that redox signaling differences do not explain differences in OA prevalence. Talar chondrocytes, when isolated from their native matrix, can be used to examine redox-regulated cell signaling events relevant to OA in either joint.

Evaluation of potential health effects associated with occupational and environmental exposure to styrene - an update.

The potential chronic health risks of occupational and environmental exposure to styrene were evaluated to update health hazard and exposure information developed since the Harvard Center for Risk Analysis risk assessment for styrene was performed in 2002. The updated hazard assessment of styrene's health effects indicates human cancers and ototoxicity remain potential concerns. However, mechanistic research on mouse lung tumors demonstrates these tumors are mouse-specific and of low relevance to human cancer risk. The updated toxicity database supports toxicity reference levels of 20 ppm (equates to 400 mg urinary metabolites mandelic acid + phenylglyoxylic acid/g creatinine) for worker inhalation exposure and 3.7 ppm and 2.5 mg/kg bw/day, respectively, for general population inhalation and oral exposure. No cancer risk value estimates are proposed given the established lack of relevance of mouse lung tumors and inconsistent epidemiology evidence. The updated exposure assessment supports inhalation and ingestion routes as important. The updated risk assessment found estimated risks within acceptable ranges for all age groups of the general population and workers with occupational exposures in non-fiber-reinforced polymer composites industries and fiber-reinforced polymer composites (FRP) workers using closed-mold operations or open-mold operations with respiratory protection. Only FRP workers using open-mold operations not using respiratory protection have risk exceedances for styrene and should be considered for risk management measures. In addition, given the reported interaction of styrene exposure with noise, noise reduction to sustain levels below 85 dB(A) needs be in place.