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Simulations of single photon avalanche diodes (SPADs) based on the Ge-on-Si material platform are presented, highlighting the potential performance enhancement achievable with nano-hole array photonic crystal structures. Such structures can be used to enhance photon absorption and therefore increase single photon detection efficiencies (SPDE). However, there is yet to be a study of these structures in application to Ge-on-Si SPADs to determine if the optical enhancements can be realized as SPDE or to evaluate the change in dark count rate due to the nano-holes that form the photonic crystal. This work establishes an optimization and analysis platform for investigating photonic crystal structures on SPAD devices. Both a direct Ge etch method, and an etched amorphous Si design are compared to a reference device with an optimized anti-reflection coating. Finite difference time domain simulations were used to optimize the photonic crystal parameters for these structures, finding a potential absorption of up to 37.09 % at wavelengths of 1550 nm for a 1 µm absorption layer, compared to 11.33 % for the reference device. Subsequently, TCAD simulations and custom code were used to calculate the effective enhancement to SPAD performance metrics, as a function of material and passivation quality, showing up to 2.41x higher SPDE and 2.57x better noise-equivalent power is achievable provided etched surfaces are sufficiently well passivated.

Background and Objectives: Physicians have long been considered valued members of a solid US health care system. Significant changes in medical education, health care, and society at-large suggest that current medical students may face a different future. To help guide educators and policy makers, we set out to understand medical students' perceptions of the future of health care and their place in it., Methods: In year one of a longitudinal study, we conducted in-depth interviews of Case Western Reserve University medical students. A multidisciplinary team performed iterative thematic analyses and sampling until reaching saturation on major themes., Results: Eleven medical student participants described social and health care issues as major influences on their professional futures. Concerns included health care system failings, unsustainable costs, climate change, demographic shifts, disinformation, and public distrust in health care. Students looked forward to team practice and using technology, data, and artificial intelligence in care delivery. They hoped for greater access and equity in health care, with a focus on prevention and social, behavioral, and environmental drivers of health. Most students expected to be employed rather than in private practice and sought time/flexibility for professional and personal interests. Paying off medical school debt and advocating for patients and change were priorities. Many saw primary care as important, but fewer envisioned it as their career path of choice., Conclusions: Medical students envision a future shaped by health care systems and social issues. These findings can inform those helping students prepare for uncertainty and rapid change in their careers, their lives, and the lives of their patients.

Objectives: The main objective of this study was to compare the postoperative analgesic effects of grapiprant with those of robenacoxib in cats undergoing ovariohysterectomy (OVH)., Methods: In total, 37 female cats (age range 4 months-10 years, weighing ⩾2.5 kg) were enrolled in a prospective, randomized, masked, non-inferiority (NI) clinical trial. Cats received oral robenacoxib (1 mg/kg) or grapiprant (2 mg/kg) 2 h before OVH. Analgesia was assessed via the Feline Grimace Scale (FGS), the Glasgow Composite Measure Pain Scale-Feline (CMPS-F), von Frey monofilaments (vFFs) and pressure algometry (ALG) 2 h before treatment administration, at extubation, and 2, 4, 6, 8, 18 and 24 hours after extubation. Hydromorphone (<8 h postoperatively) or buprenorphine (>18 h postoperatively) were administered to cats with scores of ⩾5/20 on CMPS-F and/or ⩾4/10 on FGS. NI margins for CMPS-F and vFFs were set at 3 and -0.2, respectively. A mixed-effect ANOVA was used for FGS scores ( P  <0.05). Data are reported as mean ± SEM., Results: The data from 33 cats were analyzed. The upper limit of the 95% confidence interval (CI) (0.35) was less than the NI margin of 3 for CMPS-F, and the lower limit of the 95% CI (0.055) was greater than the NI margin of -0.2 for vFFs, indicating NI of grapiprant. The FGS scores were greater than baseline at extubation for both treatments (1.65 ± 0.63; P  = 0.001); however, there was no difference between treatments. There was no difference between treatments, nor treatment by time interaction, for vFFs ( P  <0.001). The CMPS-F scores for both treatments were higher at extubation but returned to baseline after 4 h ( P  <0.001). For ALG, there was no difference in treatment or treatment by time interaction. The robenacoxib group had lower pressure readings at extubation and 6 h compared with baseline., Conclusions and Relevance: These results indicate that grapiprant was non-inferior to robenacoxib for mitigating postsurgical pain in cats after OVH performed via ventral celiotomy. The impact of grapiprant for analgesia in OVH via the flank is unknown., Competing Interests: Conflict of interestThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

The aim of this retrospective cohort study was to provide a single-center clinical audit of complications for single chamber permanent pacemaker implantation (PPI) techniques and determine if the clinical parameters, PPI technique or complications were associated with outcome. The electronic medical records were searched for dogs treated for bradyarrhythmia with PPI. Data related to presenting complaint, signalment of the dog, ECG diagnosis, echocardiographic findings, PPI technique, and programing of the pacemaker were recorded. Survival length (days) was recorded as the last veterinary visit; if the dog was dead the reason was documented. Cumulative survival of each pacemaker was examined by a Kaplan-Meier survival curve and the two techniques compared with a logrank test. Chi-square was used to determine the association between major complications and death. A total of 66 dogs with 52 transvenous and 30 epicardial PPIs were included. All epicardial pacemakers were implanted via transdiaphragmatic approach. A total of 31 life-threatening complications were reported. There were nine deaths related to major complications (13.6% of the study sample). The median follow-up period was 366 days, with a median survival of 255 days, and a significant difference in cumulative survival of each pacemaker (P = 0.01) between epicardial (93 days, range 0-1882 days) and transvenous (334 days, range 0-2745) PPIs but no significant difference in cumulative survival between the two techniques when only the first pacemaker was considered (p = 0.07). The presence of a major complications had a significant association with death due to pacemaker complications (P<0.001). The decision to perform epicardial PPI in failed transvenous PPI patients may have skewed the cumulative survival as was evident in the lack of significant difference in survival when only first PPI were examined. Major complication rates between the two techniques were similar and the authors consider both techniques equally reliable to manage symptomatic bradycardia in dogs., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Köster et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Limited data are available on the effects of pregnancy on the maternal metabolome. Therefore, the objective of this study was to use metabolomics analysis to determine pathways impacted by pregnancy followed by targeted confirmatory analysis to provide more powerful conclusions about metabolic alterations during pregnancy. Forty-seven pregnant women, 18-50 years of age were included in this study, with each subject serving as their own control. Plasma samples were collected between 25 and 28 weeks gestation and again ≥3 months postpartum for metabolomics analysis utilizing an HILIC/UHPLC/MS/MS assay with confirmatory targeted specific concentration analysis for 10 of the significantly altered amino acids utilizing an LC/MS assay. Principle component analysis (PCA) on metabolomics data clearly separated pregnant and postpartum groups and identified outliers in a preliminary assessment. Of the 980 metabolites recorded, 706 were determined to be significantly different between pregnancy and postpartum. Pathway analysis revealed three significantly impacted pathways, arginine biosynthesis ( p = 2 × 10 -5 and FDR = 1 × 10 -3 ), valine, leucine, and isoleucine metabolism ( p = 2 × 10 -5 and FDR = 2 × 10 -3 ), and xanthine metabolism ( p = 4 × 10 -5 and FDR = 4 × 10 -3 ). Of these we focused analysis on arginine biosynthesis and branched-chain amino acid (BCAA) metabolism due to their clinical importance and interconnected roles in amino acid metabolism. In the confirmational analysis, 7 of 10 metabolites were confirmed as significant and all 10 confirmed the direction of change of concentrations observed in the metabolomics analysis. The data support an alteration in urea nitrogen disposition and amino acid metabolism during pregnancy. These changes could also impact endogenous nitric oxide production and contribute to diseases of pregnancy. This study provides evidence for changes in both the ammonia-urea nitrogen and the BCAA metabolism taking place during pregnancy.

Background: Workers of color experience a disproportionate share of work-related injuries and illnesses (WRII), however, most workers' compensation systems do not collect race and ethnicity information, making it difficult to monitor trends over time, or to investigate specific policies and procedures that maintain or could eliminate the unequal burden of WRII for workers of color. The purpose of this study is to apply a Bayesian method to Washington workers' compensation claims data to identify racial and ethnic disparities of WRII by industry and occupation, improving upon existing surveillance limitations. Measuring differences in risk for WRII will better inform prevention efforts and target prevention to those at increased risk., Methods: To estimate WRII by race/ethnicity, we applied the Bayesian Improved Surname Geocode (BISG) method to surname and residential address data among all Washington workers' compensation claims filed for injuries in 2013-2017. We then compare worker and injury characteristics by imputed race/ethnicity, and estimate rates of WRII by imputed race/ethnicity within industry and occupation., Results: Black/African Americans had the highest rates of WRII claims across all industry and occupational sectors. Hispanic/Latino WRII claimants also had higher rates than Whites and Asian/Pacific Islanders in almost all industry and occupational sectors. For accepted claims with both medical and non-medical compensation, Bodily reaction/overexertion injuries accounted for almost half of the claims during this reporting period., Discussion: The high rates of injury we report by racial/ethnic categories is a cause for major concern. Nearly all industry and occupation-specific rates of workers' compensation claims are higher for Black/African American and Hispanic/Latino workers compared to Whites. More work is needed to identify work-related, systemic, and individual characteristics., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Smith et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Objective: To evaluate and compare postoperative analgesic effects of grapiprant and carprofen in dogs undergoing ovariohysterectomy., Animals: 42 sexually intact female healthy dogs (< 35 kg and 0.5 to 7 years old) were enrolled., Procedures: In a masked, randomized, noninferiority clinical trial, dogs received either 2 mg/kg of grapiprant or 4.4 mg/kg of carprofen orally 2 hours prior to ovariohysterectomy. Postoperative pain was assessed using the Glasgow Composite Pain Scale-Short Form (GCPS-SF) at extubation and 2, 4, 6, 8, 18, and 24 hours postextubation and compared to baseline. After each pain scoring, mechanical nociceptive testing with von Frey monofilaments (vF) was performed to assess hyperalgesia. Hydromorphone (0.05 mg/kg, IM) was administered to any dog with a GCPS-SF of ≥ 5/24. The noninferiority limit (NI) for the GCPS-SF was Δ = 3. The NI for vF was Δ = -0.2. Following noninferiority, a mixed-effect ANOVA and post hoc comparisons were made with the Tukey correction method (P < .05)., Results: 3 dogs required rescue analgesia and were excluded from statistical analysis. Of the remaining 39 dogs, the upper CI for GCPS-SF was below the NI of 3 and the lower CI for vF was greater than the NI of -0.2, indicating noninferiority of grapiprant as compared to carprofen. There was no difference between treatment (P = .89) nor treatment by time (P = .62) for GCPS-SF. There was no difference between groups at any time point or over time when vF were used., Clinical Relevance: Our study results support the use of grapiprant as an analgesic alternative to carprofen in dogs undergoing ovariohysterectomy.

Workers performing cleaning duties experience higher injury rates, especially in the form of musculoskeletal disorders (MSDs), than other industries. It is essential to understand the inherent risks associated with the nature of this occupation. Based on the Balance Theory (Smith & Carayon-Sainfort, 1989), this review surveys the current literature, especially those published since the previous review paper (Kumar & Kumar, 2008), and identifies which elements contributing to MSD risks were examined: task, technology, organization, environment, individual, and their interactions. Thirty-nine research papers published between 2005 and 2021 are identified and summarized. Among these papers, task and individual elements received the most attention, at 42 and 34 occurrences, respectively. The interaction elements of technology-organization, technology-environment, and organization-environment received less than three mentions. The goal of this literature review is to update the knowledge base and identify current trends for the cleaning occupation. Possible interventions for risk reduction and future research directions are suggested., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Activation of T cell responses is essential for effective tumor clearance; however, inducing targeted, potent antigen presentation to stimulate T cell responses remains challenging. We generated Activating Antigen Carriers (AACs) by engineering red blood cells (RBCs) to encapsulate relevant tumor antigens and the adjuvant polyinosinic-polycytidylic acid (poly I:C), for use as a tumor-specific cancer vaccine. The processing method and conditions used to create the AACs promote phosphatidylserine exposure on RBCs and thus harness the natural process of aged RBC clearance to enable targeting of the AACs to endogenous professional antigen presenting cells (APCs) without the use of chemicals or viral vectors. AAC uptake, antigen processing, and presentation by APCs drive antigen-specific activation of T cells, both in mouse in vivo and human in vitro systems, promoting polyfunctionality of CD8+ T cells and, in a tumor model, driving high levels of antigen-specific CD8+ T cell infiltration and tumor killing. The efficacy of AAC therapy was further enhanced by combination with the chemotherapeutic agent Cisplatin. In summary, these findings support AACs as a potential vector-free immunotherapy strategy to enable potent antigen presentation and T cell stimulation by endogenous APCs with broad therapeutic potential., Competing Interests: All listed authors are current or former SQZ Biotechnologies Company employees., (Copyright © 2022 Blagovic, Smith, Ramakrishnan, Moore, Soto, Thompson, Stockmann, Kruszelnicki, Thakkar, Murray, Torres, Wondimagegnhu, Yi, Dadgar, Paracha, Page, Clear, Chaudhry, Myint, Bridgen, Gilbert, Seidl, Sharei, Loughhead, Bernstein and Yarar.)

Vitamin A is vital to maternal-fetal health and pregnancy outcomes. However, little is known about pregnancy associated changes in maternal vitamin A homeostasis and concentrations of circulating retinol metabolites. The goal of this study was to characterize retinoid concentrations in healthy women ( n = 23) during two stages of pregnancy (25-28 weeks gestation and 28-32 weeks gestation) as compared to ≥3 months postpartum. It was hypothesized that plasma retinol, retinol binding protein 4 (RBP4), transthyretin and albumin concentrations would decline during pregnancy and return to baseline by 3 months postpartum. At 25-28 weeks gestation, plasma retinol (-27%), 4-oxo-13- cis -retinoic acid (-34%), and albumin (-22%) concentrations were significantly lower, and all-trans -retinoic acid (+48%) concentrations were significantly higher compared to ≥3 months postpartum in healthy women. In addition, at 28-32 weeks gestation, plasma retinol (-41%), retinol binding protein 4 (RBP4; -17%), transthyretin (TTR; -21%), albumin (-26%), 13- cis -retinoic acid (-23%) and 4-oxo-13- cis -retinoic acid (-48%) concentrations were significantly lower, whereas plasma all-trans -retinoic acid concentrations (+30%) were significantly higher than ≥3 months postpartum. Collectively, the data demonstrates that in healthy pregnancies, retinol plasma concentrations are lower, but all-trans -retinoic acid concentrations are higher than postpartum.

Melanoma causes most skin cancer-related deaths, yet melanoma mortality rates can be decreased by life-long reduction of ultraviolet radiation exposure and early detection. The disease is readily detectable through skin examinations by trained medical providers; however, the U.S. Preventive Services Task Force cites insufficient evidence to recommend "visual skin examination by a clinician to screen for skin cancer" in asymptomatic adults in the United States. As a coastal state with much outdoor occupational and recreational exposure to ultraviolet light, Rhode Island has developed a coordinated statewide partnership of stakeholders who provide valuable resources and expertise that maximize the reach and efficacy of targeted skin cancer prevention and screening programs. These programs include public skin cancer screening events, shade planning efforts, distribution of sunscreen at state parks and beaches, and educational programming.

In canine and feline patients presenting in a state of hemodynamic collapse, obtaining vascular access can be challenging. Delays in achieving vascular access interfere with delivery of patient care. In human medicine, definitions of difficult vascular access are variable and include the need for multiple placement attempts or involvement of specialized teams and equipment. Incidence and risk factors for difficult vascular access have not been well studied in veterinary patients, which limits understanding of how best to address this issue. Alternatives to percutaneous peripheral or central intravenous catheterization in dogs and cats include venous cutdowns, umbilical access in newborns, corpus cavernosum access in males, ultrasound-guided catheterization, and intraosseous catheterization. In recent years, advances in ultrasonography and intraosseous access techniques have made these more accessible to veterinary practitioners. These vascular access techniques are reviewed here, along with advantages, limitations, and areas for future study of each technique., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lee, Guieu, Bussières and Smith.)

Objective: To evaluate the level of agreement (LOA) between direct and oscillometric blood pressure (BP) measurements and the ability of oscillometric measurements to accurately detect hypotension in anesthetized chimpanzees (Pan troglodytes)., Animals: 8 captive, adult chimpanzees., Procedures: During prescheduled annual examinations, each chimpanzee underwent general anesthesia and patient monitoring for their examination, echocardiography for a concurrent study, and measurement of direct BP with the use of tibial artery catheterization and oscillometry with the use of a cuff placed around a brachium and a cuff placed around the second digit of the contralateral forelimb for the present study. Bland-Altman plots were generated to compare results for direct and oscillometric BP measurements. Mean bias and 95% LOAs were calculated for oscillometric measurements of systolic arterial pressure (SAP), diastolic arterial pressure (DAP), and mean arterial pressure (MAP) for each cuff site. Sensitivity and specificity in detecting hypotension were also determined for each cuff site., Results: There were 74 paired direct and brachial oscillometric measurements of each, SAP, MAP, and DAP and 66 paired direct and digit oscillometric measurements of each, SAP, MAP, and DAP. Only brachial oscillometric measurements of MAP had adequate sensitivity (78%) and specificity (95%) to accurately detect hypotension, and this technique also had the least mean bias (0.8 mm Hg; 95% LOA, -29 to 31 mm Hg)., Conclusions and Clinical Relevance: Results indicated that brachial oscillometric measurement of MAP provided reasonable agreement with tibial arterial direct MAP measurement and performed well in diagnosing hypotension in anesthetized chimpanzees.

Background: In Rhode Island, malignant melanoma of skin causes about 30 deaths a year. Early detection has been shown to reduce mortality risk., Methods: Dermatology volunteers and public health professionals convened 27 free skin cancer screenings at public beaches in 2015-2019 to raise skin cancer awareness and screen patients for malignancy. Participants with suspicious lesions were referred for follow-up and later telephoned to ascertain outcomes., Results: Of 2354 people screened, 597 (25%) were referred. 319 of 597 (53%) were later reached by telephone. 196 of 319 (61%) who had kept appointments by the time of the telephone call reported the following diagnoses: 7 malignant melanomas, 32 keratinocyte carcinomas, and 34 actinic keratoses, yielding 3.0 as number needed to biopsy (NNB), and 18.3 as number needed to screen (NNS)., Conclusions: Our results demonstrate the value of convenient skin cancer screening events, suggesting the desirability of additional interventions of this type.

The role of the microbiota in many diseases including cancer has gained increasing attention. Paired with this is our expanding appreciation for the heterogeneity of the neutrophil compartment regarding surface marker expression and functionality. In this review, we will discuss the influence of the microbiota on granulopoiesis and consequent activity of neutrophils in cancer. As evidence for this microbiota-neutrophil-cancer axis builds, it exposes new therapeutic targets to improve a cancer patient's outcome., (©2018 Society for Leukocyte Biology.)

OBJECTIVE To investigate use of the plethysmographic variability index (PVI) and perfusion index (PI) for evaluating changes in arterial blood pressure in anesthetized tigers (Panthera tigris). ANIMALS 8 adult tigers. PROCEDURES Each tiger was anesthetized once with a combination of ketamine, midazolam, medetomidine, and isoflurane. Anesthetic monitoring included assessment of PI, PVI, direct blood pressure measurements, anesthetic gas concentrations, esophageal temperature, and results of capnography and ECG. Mean arterial blood pressure (MAP) was maintained for at least 20 minutes at each of the following blood pressure conditions: hypotensive (MAP = 50 ± 5 mm Hg), normotensive (MAP = 70 ± 5 mm Hg), and hypertensive (MAP = 90 ± 5 mm Hg). Arterial blood gas analysis was performed at the beginning of anesthesia and at each blood pressure condition. RESULTS Mean ± SD PI values were 1.82 ± 2.38%, 1.17 ± 0.77%, and 1.71 ± 1.51% and mean PVI values were 16.00 ± 5.07%, 10.44 ± 3.55%, and 8.17 ± 3.49% for hypotensive, normotensive, and hypertensive conditions, respectively. The PI values did not differ significantly among blood pressure conditions. The PVI value for the hypotensive condition differed significantly from values for the normotensive and hypertensive conditions. The PVI values were significantly correlated with MAP (r = -0.657). The OR of hypotension to nonhypotension for PVI values ≥ 18% was 43.6. CONCLUSIONS AND CLINICAL RELEVANCE PVI was a clinically applicable variable determined by use of noninvasive methods in anesthetized tigers. Values of PVI ≥ 18% may indicate hypotension.

In 2015, more than 27 million people in the United States reported that they currently used illicit drugs or misused prescription drugs, and more than 66 million reported binge drinking during the previous month. Data from public health surveillance systems on drug and alcohol abuse are crucial for developing and evaluating interventions to prevent and control such behavior. However, public health surveillance for behavioral health in the United States has been hindered by organizational issues and other factors. For example, existing guidelines for surveillance evaluation do not distinguish between data systems that characterize behavioral health problems and those that assess other public health problems (eg, infectious diseases). To address this gap in behavioral health surveillance, we present a revised framework for evaluating behavioral health surveillance systems. This system framework builds on published frameworks and incorporates additional attributes (informatics capabilities and population coverage) that we deemed necessary for evaluating behavioral health-related surveillance. This revised surveillance evaluation framework can support ongoing improvements to behavioral health surveillance systems and ensure their continued usefulness for detecting, preventing, and managing behavioral health problems.

Introduction: We analyzed workers' compensation (WC) data to identify characteristics related to workers' compensation claim outcomes among janitorial service workers in Washington State., Method: We analyzed WC data from the Washington State Department of Labor & Industries (L&I) State Fund (SF) from January 1, 2003 through December 31, 2013, for janitorial service workers employed in the National Occupational Research Agenda (NORA) Services Sector. We constructed multivariable models to identify factors associated with higher medical costs and increased time lost from work., Results: There were 2,390 janitorial service compensable claims available for analysis. There were significant differences in injury type and other factors by gender, age, and language preference. Linguistic minority status was associated with longer time loss and higher median medical costs. Women were estimated to account for 35% of janitorial service workers but made up 55% of the compensable claims in this study., Conclusions: Janitorial service workers comprise a large vulnerable occupational group in the U.S. workforce. Identifying differences by injury type and potential inequitable outcomes by gender and language is important to ensuring equal treatment in the workers' compensation process., Practical Applications: There were significant differences in injury and individual characteristics between men and women in this study. Women had twice the estimated rate of injury to men, and were more likely to require Spanish language materials. Improving communication for training and knowledge about the workers' compensation system appear to be high priorities in this population of injured janitorial service workers., (Copyright © 2017 Elsevier Ltd and National Safety Council. All rights reserved.)

Objective: To identify a genetic cause of early-onset systemic lupus erythematosus (SLE) in a large consanguineous family from Turkey and to study the mechanisms of the disease., Methods: We performed whole-exome sequencing and single-nucleotide polymorphism array genotyping in family members with and without SLE. Protein and gene expression, cytokine profile, neutrophil extracellular trap (NET) formation, and presence of low-density granulocytes were evaluated in patient primary cells and serum samples., Results: We identified a novel, homozygous, loss-of-function mutation (p.Pro445Leufs*11) in the C1R gene. Using the Sanger method of DNA sequencing in 14 family members, we confirmed the presence of the mutation in 4 patients with SLE and in an asymptomatic 9-year-old girl. Complement levels were low in sera from patients with truncated C1r protein. Two siblings with SLE who were available for detailed evaluation exhibited strong type I interferon (IFN) inflammatory signatures despite their disease being clinically inactive at the time of sampling. The type I IFN transcriptional signature in the patients' blood correlated with disease expressivity, whereas the neutrophil signature in peripheral blood mononuclear cells was likely associated with disease severity. The female patient with SLE with the most severe phenotype presented with a stronger neutrophil signature, defined by enhanced NET formation and the presence of low-density granulocytes. Analysis of exome data for modifying alleles suggested enrichment of common SLE-associated variants in the more severely affected patients. Lupus-associated HLA alleles or HLA haplotypes were not shared among the 4 affected subjects., Conclusion: Our findings revealed a novel high-penetrance mutation in C1R as the cause of monogenic SLE. Disease expressivity in this family appears to be influenced by additional common and rare genetic variants., (© 2017, American College of Rheumatology.)

Objective: Dysregulation of innate and adaptive immune responses contributes to the pathogenesis of systemic lupus erythematosus (SLE) and its associated premature vascular damage. No drug to date targets both systemic inflammatory disease and the cardiovascular complications of SLE. Tofacitinib is a JAK inhibitor that blocks signaling downstream of multiple cytokines implicated in lupus pathogenesis. While clinical trials have shown that tofacitinib exhibits significant clinical efficacy in various autoimmune diseases, its role in SLE and the associated vascular pathology remains to be characterized., Methods: MRL/lpr lupus-prone mice were administered tofacitinib or vehicle by gavage for 6 weeks (therapeutic arm) or 8 weeks (preventive arm). Nephritis, skin inflammation, serum levels of autoantibodies and cytokines, mononuclear cell phenotype and gene expression, neutrophil extracellular traps (NETs) release, endothelium-dependent vasorelaxation, and endothelial differentiation were compared in treated and untreated mice., Results: Treatment with tofacitinib led to significant improvement in measures of disease activity, including nephritis, skin inflammation, and autoantibody production. In addition, tofacitinib treatment reduced serum levels of proinflammatory cytokines and interferon responses in splenocytes and kidney tissue. Tofacitinib also modulated the formation of NETs and significantly increased endothelium-dependent vasorelaxation and endothelial differentiation. The drug was effective in both preventive and therapeutic strategies., Conclusion: Tofacitinib modulates the innate and adaptive immune responses, ameliorates murine lupus, and improves vascular function. These results indicate that JAK inhibitors have the potential to be beneficial in SLE and its associated vascular damage., (© 2016, American College of Rheumatology.)

Objective: Patients with systemic lupus erythematosus (SLE) have an increased risk of cardiovascular disease (CVD) and impaired endothelial repair. Although vitamin D deficiency is associated with increased CVD risk in the general population, a causal relationship has not been demonstrated. We aimed to determine whether vitamin D deficiency directly modulates endothelial dysfunction and immune responses in a murine model of SLE., Methods: Vitamin D deficiency was induced in lupus-prone MRL/lpr mice by dietary restriction for 6 weeks. Endothelium-dependent vasorelaxation was quantified using aortic ring myography, and endothelial repair mechanisms were assessed by evaluating the phenotype and function of bone marrow endothelial progenitor cells (EPCs) and with the use of an in vivo Matrigel plug model. Lupus disease activity was determined by evaluating expression of interferon-stimulated genes (ISGs) in splenic tissue, positivity for serum autoantibodies, and renal histology. To validate the findings, expression of ISGs was also measured in whole blood from vitamin D-deficient and vitamin D-sufficient patients with SLE., Results: Vitamin D deficiency resulted in impaired endothelium-dependent vasorelaxation and decreases in neoangiogenesis without a change in the total number of EPCs. There were no differences in anti-double-stranded DNA titers, proteinuria, or glomerulonephritis (activity or chronicity) between vitamin D-deficient or sufficient mice. Vitamin D deficiency was associated with a trend toward increased ISG expression both in mice and in patients with SLE., Conclusion: These findings indicate that vitamin D deficiency is associated with hampered vascular repair and reduced endothelial function, and may modulate type I interferon responses., (© 2016, American College of Rheumatology.)

Neutrophil extracellular traps (NETs) are implicated in autoimmunity, but how they are generated and their roles in sterile inflammation remain unclear. Ribonucleoprotein immune complexes (RNP ICs), inducers of NETosis, require mitochondrial reactive oxygen species (ROS) for maximal NET stimulation. After RNP IC stimulation of neutrophils, mitochondria become hypopolarized and translocate to the cell surface. Extracellular release of oxidized mitochondrial DNA is proinflammatory in vitro, and when this DNA is injected into mice, it stimulates type I interferon (IFN) signaling through a pathway dependent on the DNA sensor STING. Mitochondrial ROS are also necessary for spontaneous NETosis of low-density granulocytes from individuals with systemic lupus erythematosus. This was also observed in individuals with chronic granulomatous disease, who lack NADPH oxidase activity but still develop autoimmunity and type I IFN signatures. Mitochondrial ROS inhibition in vivo reduces disease severity and type I IFN responses in a mouse model of lupus. Together, these findings highlight a role for mitochondria in the generation not only of NETs but also of pro-inflammatory oxidized mitochondrial DNA in autoimmune diseases.

Rationale: Autologous and allogeneic hematopoietic stem cell transplant (HSCT) patients are susceptible to pulmonary infections, including bacterial pathogens, even after hematopoietic reconstitution. We previously reported that murine bone marrow transplant (BMT) neutrophils overexpress cyclooxygenase-2, overproduce prostaglandin E2 (PGE2), and exhibit defective intracellular bacterial killing. Neutrophil extracellular traps (NETs) are DNA structures that capture and kill extracellular bacteria and other pathogens., Objectives: To determine whether NETosis was defective after transplant and if so, whether this was regulated by PGE2 signaling., Methods: Neutrophils isolated from mice and humans (both control and HSCT subjects) were analyzed for NETosis in response to various stimuli in the presence or absence of PGE2 signaling modifiers., Measurements and Main Results: NETs were visualized by immunofluorescence or quantified by Sytox Green fluorescence. Treatment of BMT or HSCT neutrophils with phorbol 12-myristate 13-acetate or rapamycin resulted in reduced NET formation relative to control cells. NET formation after BMT was rescued both in vitro and in vivo with cyclooxygenase inhibitors. Additionally, the EP2 receptor antagonist (PF-04418948) or the EP4 antagonist (AE3-208) restored NET formation in neutrophils isolated from BMT mice or HSCT patients. Exogenous PGE2 treatment limited NETosis of neutrophils collected from normal human volunteers and naive mice in an exchange protein activated by cAMP- and protein kinase A-dependent manner., Conclusions: Our results suggest blockade of the PGE2-EP2 or EP4 signaling pathway restores NETosis after transplantation. Furthermore, these data provide the first description of a physiologic inhibitor of NETosis.

Objective: SLE is an autoimmune disease characterized by autoantibody generation, organ damage and an increased risk of cardiovascular disease. Generally considered an anti-inflammatory cytokine, IL-10 is increased in SLE and correlates with poor cardiovascular outcomes in the general population. The aim of this study was to explore the putative role of IL-10 in modulating endothelial function in SLE by examining the effects of this cytokine on endothelial progenitor cell/circulating angiogenic cell (EPC/CAC) differentiation., Methods: Human and murine control and lupus EPCs/CACs were differentiated into mature endothelial cells (ECs) in the presence or absence of graded concentrations of recombinant IL-10 with or without recombinant IFN-α or a neutralizing antibody to IL-10. IL-10-deficient mice were examined to assess the role of this cytokine in type I IFN-mediated inhibition of EC differentiation and neo-angiogenesis using an in vivo Matrigel plug assay. Serum IL-10 concentrations were measured via ELISA., Results: IL-10 hampers EC differentiation in a dose-dependent manner. In murine EPC cultures, IL-10 is required to observe the inhibitory effects of type I IFNs on EPC function and neo-angiogenesis. In human SLE EPC/CAC cultures, neutralization of IL-10 significantly improved the differentiation of EPCs, and IL-10 enhanced type I IFN-mediated EPC/CAC dysfunction. The presence of IL-10 in serum inversely correlated with EPC/CAC function in SLE but not in control cells., Conclusion: IL-10 interferes with endothelial differentiation and may enhance the effects of type I IFN on vascular repair in SLE. IL-10 may be a relevant target for improving cardiovascular risk in SLE., (© The Author 2014. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Objective: Oxidative stress and oxidized high-density lipoprotein (HDL) are implicated as risk factors for cardiovascular disease (CVD) in systemic lupus erythematosus (SLE). Yet, how HDL is oxidized and rendered dysfunctional in SLE remains unclear. Neutrophil extracellular traps (NETs), the levels of which are elevated in lupus, possess oxidant-generating enzymes, including myeloperoxidase (MPO), NADPH oxidase (NOX), and nitric oxide synthase (NOS). We hypothesized that NETs mediate HDL oxidation, impairing cholesterol efflux capacity (CEC)., Methods: Plasma MPO levels and CEC activity were examined in controls and lupus patients, and 3-chlorotyrosine (MPO specific) and 3-nitrotyrosine (derived from reactive nitrogen species) were quantified in human HDL. Multivariable linear models were used to estimate and test differences between groups. HDL was exposed to NETs from control and lupus neutrophils in the presence or absence of MPO, NOX, NOS inhibitors, and chloroquine (CQ). Murine HDL oxidation was quantified after NET inhibition in vivo., Results: SLE patients displayed higher MPO levels and diminished CEC compared to controls. SLE HDL had higher 3-nitrotyrosine and 3-chlorotyrosine content than control HDL, with site-specific oxidation signatures on apolipoprotein A-I. Experiments with human and murine NETs confirmed that chlorination was mediated by MPO and NOX, and nitration by NOS and NOX. Mice with lupus treated with the NET inhibitor Cl-amidine displayed significantly decreased HDL oxidation. CQ inhibited NET formation in vitro., Conclusion: Active NOS, NOX, and MPO within NETs significantly modify HDL, rendering the lipoprotein proatherogenic. Since NET formation is enhanced in SLE, these findings support a novel role for NET-derived lipoprotein oxidation in SLE-associated CVD and identify additional proatherogenic roles of neutrophils and putative protective roles of antimalarials in autoimmunity., (Published 2014. This article is a U.S. Government work and is in the public domain in the USA.)

Objective: The objective of this prospective study is to investigate the exposure-response relationships between various workplace physical exposures of force, repetition, and their combination assessed at an individual level with lateral epicondylitis (LE)., Background: Workplace upper extremity musculoskeletal disorders (UEMSDs) are prevalent, disabling, and expensive. LE is one of the major UEMSDs in active workers., Method: We used detailed health history, symptoms, and physical examination for identifying incidence and detailed exposure assessment to capture work tasks of each worker and to measure quantitative mechanical workload. We used counting process style input of proportional hazards regression for modeling cumulative incidence that accounts for changed exposure estimates during the follow-up period when respondents change jobs., Results: The incidence rate of LE on the dominant side was 4.91 per 100 person-years. Adjusted for age and gender, the combined effect of forearm pronation > or = 45 degrees for > or = 40% of time and time spent with forceful exertion, including any power grip (hazard ratio [HR] = 2.8, 95% confidence interval [CI] = [1.35-5.77]), lifting for > or = 3% of time (HR = 2.50, 95% CI = [1.19-5.24]), and duty cycle for forceful exertion for > or = 10% (HR = 2.25, 95% CI = [1.09-4.66]), were significant predictors of dominant side LE, whereas neither the awkward posture nor the forceful exertion alone was significant. Older workers with jobs requiring a high percentage of time working with force in combination with awkward postures of forearm were more likely to predict LE., Conclusion: This study shows the evidence of the etiologic role of strenuous manual tasks in the occurrence of LE.

Objective: Patients with systemic lupus erythematosus (SLE) have a notable increase in atherothrombotic cardiovascular disease (CVD) which is not explained by the Framingham risk equation. In vitro studies indicate that type I interferons (IFNs) may play prominent roles in increased CV risk in SLE. However, the in vivo relevance of these findings, with regard to the development of CVD, has not been characterized. This study was undertaken to examine the role of type I IFNs in endothelial dysfunction, aberrant vascular repair, and atherothrombosis in murine models of lupus and atherosclerosis., Methods: Lupus-prone New Zealand mixed 2328 (NZM) mice and atherosclerosis-prone apolipoprotein E- knockout (apoE(-/-) ) mice were compared to mice lacking type I IFN receptor (INZM and apoE(-/-) IFNAR(-/-) mice, respectively) with regard to endothelial vasodilatory function, endothelial progenitor cell (EPC) function, in vivo neoangiogenesis, plaque development, and occlusive thrombosis. Similar experiments were performed using NZM and apoE(-/-) mice exposed to an IFNα-containing or empty adenovirus., Results: Loss of type I IFN receptor signaling improved endothelium-dependent vasorelaxation, lipoprotein parameters, EPC numbers and function, and neoangiogenesis in lupus-prone mice, independent of disease activity or sex. Further, acute exposure to IFNα impaired endothelial vasorelaxation and EPC function in lupus-prone and non-lupus-prone mice. Decreased atherosclerosis severity and arterial inflammatory infiltrates and increased neoangiogenesis were observed in apoE(-/-) IFNAR(-/-) mice, compared to apoE(-/-) mice, while NZM and apoE(-/-) mice exposed to IFNα developed accelerated thrombosis and platelet activation., Conclusion: These results support the hypothesis that type I IFNs play key roles in the development of premature CVD in SLE and, potentially, in the general population, through pleiotropic deleterious effects on the vasculature., (Copyright © 2012 by the American College of Rheumatology.)

Objective: The aim of this study was to assess prevalence, incidence, and persistence of carpal tunnel syndrome (CTS) and associated symptoms over a one-year period in a working population., Methods: We conducted a one-year prospective study of 418 active workers in 12 worksites. Detailed health interviews, psychosocial questionnaires, and electrophysiological studies [ie, nerve conduction velocity (NCV) tests] were conducted at baseline and one-year follow-up. Individual-observed exposure assessments of wrist posture, hand activity, and hand forces were conducted., Results: Prevalence of CTS cases at baseline was 10.8% [95% confidence interval (95% CI) 7.8-13.7%] on the dominant side and 6.0% (95% CI 3.7-87.3%) on the non-dominant side; CTS symptoms were 14.1% (95% CI 10.8-17.5%) and 11.0% (95% CI 8.0-14.0%), respectively. Incidence of CTS cases were 7.5% (95% CI 4.8-10.2%) and 5.6% (95% CI 3.3-7.9%), respectively. Higher proportions of subjects with current symptoms or positive electrophysiological test findings at baseline became CTS cases at one year compared to those with neither (28.6% and 13.9%, respectively, versus 0.5%). One-year persistence of CTS case status was 44.4% (95% CI 29.9-59.0%) for the dominant hand and 52.0% (95% CI 32.4-71.6) for non-dominant hand. There were significant differences at baseline between asymptomatic subjects and CTS cases with respect to higher job demands (P=0.027), lower job satisfaction (P=0.036), lower general health (P=0.0009), higher exposure to vibrating hand tool use (P=0.039), and greater time using a forceful power grip (P=0.035) among cases., Conclusions: CTS symptoms more than positive NCV test results alone appear to predict CTS at one year. Persistence of CTS at one year is high.

Objectives: The aim was to explore whether "adjustment" for gender masks important exposure differences between men and women in a study of rotator cuff syndrome (RCS) and carpal tunnel syndrome (CTS) and work exposures., Methods: This cross-sectional study of 733 subjects in 12 health care and manufacturing workplaces used detailed individual health and work exposure assessment methods. Multiple logistic regression analysis was used to compare gender stratified and adjusted models., Results: Prevalence of RCS and CTS among women was 7.1% and 11.3% respectively, and among men 7.8% and 6.4%. In adjusted (gender, age, body mass index) multivariate analyses of RCS and CTS, gender was not statistically significantly different. For RCS, upper arm flexion >/=45 degrees and forceful pinch increased the odds in the gender-adjusted model (OR 2.66, 95% CI 1.26-5.59) but primarily among women in the stratified analysis (OR 6.68, 95% CI 1.81-24.66 versus OR 1.45, 95% CI 0.53-4.00). For CTS, wrist radial/ulnar deviation >/=4% time and lifting >/=4.5kg >3% time, the adjusted OR was higher for women (OR 4.85, 95% CI 2.12-11.11) and in the gender stratified analyses, the odds were increased for both genders (women OR 5.18, 95% CI 1.70-15.81 and men OR 3.63, 95% CI 1.08-12.18)., Conclusions: Gender differences in response to physical work exposures may reflect gender segregation in work and potential differences in pinch and lifting capacity. Reduction in these exposures may reduce prevalence of upper extremity disorders for all workers.

Background: Hand symptom diagrams (HSDs) for rating the distribution of paraesthesias are proposed for use in epidemiological studies of carpal tunnel syndrome (CTS)., Aim: To assess the validity of HSDs in a working population of manufacturing and service workers participating in a prospective study of musculoskeletal disorders., Methods: Assessment of each subject involved completing a HSD, a heath assessment and electrodiagnostic studies (EDSs). HSDs were rated for CTS blinded to the health assessment and EDS results. The validity assessments of HSD used EDS as the sole confirmatory standard for CTS., Results: A total of 733 subjects (65% of those eligible) participated in the study and 720 underwent EDSs. Dominant hand prevalence of a positive HSD and delayed nerve conduction studies in this working population was 9.2 per 100 workers. The sensitivity of a positive HSD for all workers was 0.28. By restricting the population to those workers with any current hand symptoms or to any worker with neuropathic hand symptoms, the sensitivities of HSD improved to 0.61 and 0.79, respectively. The positive predictive value of a HSD, with our study prevalence, was 0.48., Conclusions: The HSD classification schema has poor validity when applied to a general working population but improves when applied to workers with current neuropathic symptoms. The high number of false-negative HSDs in the general study population is most likely to be due to the inadequacies of using EDS as the confirmatory test. With a low prevalence of CTS, the positive predictive value for HSDs is poor.

Background: Tissue morphogenesis is a complex process whereby tissue structures self-assemble by the aggregate behaviors of independently acting cells responding to both intracellular and extracellular cues in their environment. During embryonic development, morphogenesis is particularly important for organizing cells into tissues, and although key regulatory events of this process are well studied in isolation, a number of important systems-level questions remain unanswered. This is due, in part, to a lack of integrative tools that enable the coupling of biological phenomena across spatial and temporal scales. Here, we present a new computational framework that integrates intracellular signaling information with multi-cell behaviors in the context of a spatially heterogeneous tissue environment., Results: We have developed a computational simulation of mesendoderm migration in the Xenopus laevis explant model, which is a well studied biological model of tissue morphogenesis that recapitulates many features of this process during development in humans. The simulation couples, via a JAVA interface, an ordinary differential equation-based mass action kinetics model to compute intracellular Wnt/beta-catenin signaling with an agent-based model of mesendoderm migration across a fibronectin extracellular matrix substrate. The emergent cell behaviors in the simulation suggest the following properties of the system: maintaining the integrity of cell-to-cell contact signals is necessary for preventing fractionation of cells as they move, contact with the Fn substrate and the existence of a Fn gradient provides an extracellular feedback loop that governs migration speed, the incorporation of polarity signals is required for cells to migrate in the same direction, and a delicate balance of integrin and cadherin interactions is needed to reproduce experimentally observed migratory behaviors., Conclusion: Our computational framework couples two different spatial scales in biology: intracellular with multicellular. In our simulation, events at one scale have quantitative and dynamic impact on events at the other scale. This integration enables the testing and identification of key systems-level hypotheses regarding how signaling proteins affect overall tissue-level behavior during morphogenesis in an experimentally verifiable system. Applications of this approach extend to the study of tissue patterning processes that occur during adulthood and disease, such as tumorgenesis and atherogenesis.

Campylobacter jejuni is a major cause of human inflammatory enteritis. During the course of human disease numerous proinflammatory cytokines are produced. Little is known, however, about the cytokine responses produced during the interaction of this bacterium with the avian host. Campylobacter has been considered a commensal of the avian host. Any differences in innate responses to this pathogen between the human and avian hosts should lead to a greater understanding of the disease process in humans. We have demonstrated expression of proinflammatory cytokines and chemokines in response to Campylobacter infection in avian primary chick kidney cells and the avian macrophage cell line HD11. The data indicate that Campylobacter can stimulate the avian host in a proinflammatory manner. The data strongly suggest that the lack of pathology in vivo is not due to an inability of Campylobacter to stimulate a proinflammatory response from avian cells.

In an effort to identify gene products involved in transcriptional regulation in apicomplexan parasites, the Toxoplasma gondii expressed sequence tag (EST) database was examined for sequences containing similarity to known transcriptional components. One EST (dbEST ID #466792) exhibited strong similarity to yeast GCN5 and other histone acetyltransferases (HATs). Primers were designed based on the EST sequence and used to amplify an 850 bp fragment (containing an intron) from T. gondii genomic DNA which was used to identify four cDNA clones from a tachyzoite cDNA library. The complete open reading frame (ORF) of 3.5 kb was elucidated using 5' RACE and genomic sequence. The deduced amino acid sequence of the coding region shows that the C-terminal domain possesses unequivocal similarity to GCN5 family members. However, unlike other lower eukaryotes, T. gondii GCN5 has an extended N-terminal domain similar in length, but not in composition, to metazoan HAT proteins. These features distinguish T. gondii GCN5 as a novel member of the GCN5 family. A portion of the cDNA sequence was used as a probe to isolate three overlapping clones from a T. gondii genomic library, generating a approximately 7.5 kb map of the GCN5 locus which contains seven exons separated by six introns. Southern analysis verifies the predicted map and suggests that a similar locus may be present elsewhere in the genome.

A non-reducing trisaccharide, alpha-D-mannopyranosyl-(1 --> 4)-alpha-D-glucuronopyranosyl-(1 --> 2)-myo-inositol (MGI) accumulated in the spent medium of cell-suspension cultures of 'Paul's Scarlet' rose (Rosa sp.) predominantly during the period of rapid cell growth. This trisaccharide was also produced by cultures of sycamore (Acer pseudoplatanus L.) but not by those of the graminaceous monocots maize (Zea mays L.) and tall fescue grass (Festuca arundinacea Schreb.). When added to cultured Rosa cells, [(14)C]MGI was neither taken up by the cells nor bound to the cell surface and was not metabolised extracellularly. When D-[6-(14)C]glucuronic acid was fed to cultured Rosa cells, extracellular [(14)C]MGI started to appear only after a 5-h lag period, compared with a 0.5-h lag period for labelling of extracelluar polysaccharides. Furthermore, [(14)C]MGI continued to accumulate in the medium for at least 20 h after the accumulation of (14)C-polymers had ceased. These observations indicate that extracellular MGI was produced from a slowly turning-over pool of a pre-formed intermediate. Structural considerations indicate that the intermediate could be a glucuronomannan or a phytoglycolipid (glycophosphosphingolipid). No Rosa polysaccharides could be found that generated MGI in the presence of living Rosa cells. We therefore favour phytoglycolipids as the probable biosynthetic origin of MGI.

Lon protease and members of the Clp family of molecular chaperones and protease regulatory subunits contain homologous regions with properties expected for substrate-binding domains. Fragments corresponding to these sequences are stably and independently folded for Lon, ClpA, and ClpY. The corresponding regions from ClpB and ClpX are unstable. All five fragments exhibit distinct patterns of binding to three proteins that are protease substrates in vivo: the heat shock transcription factor sigma32, the SOS mutagenesis protein UmuD, and Arc repressor bearing the SsrA degradation tag. Recognition of UmuD is mediated through peptide sequences within a 24-residue N-terminal region whereas recognition of both sigma32 and SsrA-tagged Arc requires sequences at the C terminus. These results indicate that the Lon and Clp proteases use the same mechanism of substrate discrimination and suggest that these related ATP-dependent bacterial proteases scrutinize accessible or disordered regions of potential substrates for the presence of specific targeting sequences.

Pig seminal proteins PSP-I and PSP-II are major protein components of boars' ejaculate and are present as heterodimers (PSP-dimer) in seminal plasma. These proteins were examined for their ability to modulate pig lymphocyte activity in vitro in mitogen-induced lymphocyte proliferation assays and in one-way mixed lymphocyte reactions. Pig lymphocytes were cultured with phytohaemagglutinin, concanavalin A, or pokeweed mitogen (PWM) in the presence or absence of pig seminal proteins and the amount of cellular [3H]thymidine was used as an indication of proliferation. In the absence of mitogens, none of the three pig seminal proteins affected lymphocyte proliferation suggesting that these proteins are not antigenic or mitogenic. PSP-dimer enhanced lymphocyte proliferation induced by PWM (156-227%, P < 0.05) in a concentration-dependent manner, but had no effect on phytohaemagglutinin- or concanavalin A-induced proliferation. PSP-I enhanced (127-185%, P < 0.05) phytohaemagglutinin-induced proliferation. PSP-II augmented (130-240%, P < 0.05) lymphocyte proliferation induced by concanavalin A and PWM. Lymphocytes from gilts were significantly more responsive to concanavalin A- and PWM-induced lymphocyte proliferation in the presence of PSP-I compared with boars (concanavalin A: gilts 131%, boars 91%; PWM: gilts 188%, boars 134%; P < 0.05). In the mixed lymphocyte reaction, pretreating stimulating cells with increasing concentrations of PSP-I or PSP-II elicited a 400% concentration-dependent increase (P < 0.01) in lymphocyte proliferation. The abundance of pig seminal proteins in boar seminal plasma, their ability to enhance lymphocyte proliferation, and their previously reported ability to bind to lymphocytes suggest that these proteins are immunostimulatory and supports the hypothesis that they modulate uterine immune activity to ensure reproductive success.

ClpX, a molecular chaperone and the regulatory subunit of the ClpXP protease, is shown to contain tandem modular domains that bind to the C-terminal sequences of target proteins in a manner that parallels functional specificity. Nuclear magnetic resonance studies show that these C-terminal sequences are displayed as disordered peptides on the surface of otherwise folded proteins. The ClpX substrate-binding domains are homologous to sequences in other Clp/Hsp100 proteins and are related more distantly to PDZ domains, which also mediate C-terminal specific protein-protein interactions. Conservation of these binding domains indicates that the mode of substrate recognition characterized here for ClpX will be a conserved feature among Clp/Hsp100 family members and a distinguishing characteristic between this chaperone family and the Hsp70 chaperones.

Through the use of two internal controls, we have developed an improved method of quantitating ribonuclease protection assay (RPA) results. A truncated sense RNA fragment and an antisense RNA fragment for the gene of interest were transcribed from PCR fragments containing T7 bacterial promoters. An 18S ribosomal RNA fragment was also used. When radiolabeled antisense and 18S probes, along with sense fragment and sample RNA, were hybridized, digested with RNase A/T1 and gel-electrophoresed, three distinct bands resulted. The antisense RNA fragment bound to the sense RNA fragment confirmed the integrity of the reaction. The antisense RNA fragment bound to endogenous mRNA measured the amount of specific gene expression in the sample. The 18S RNA fragment bound to endogenous mRNA determined the actual amount of sample added to the gel. Using the specific activities of the antisense and 18S transcripts, and scintillation counts of the protected fragments, we calculated the amounts of message and total RNA on the gel, determining picogram of message per microgram of total RNA. Final results were not based on assumed original amounts of RNA placed in the assay nor were they biased by lane-to-lane variations. Through the described adaptations, we have developed a well-controlled RPA that accurately and reproducibly quantifies gene expression.

Significantly different m values (1.9-2.7 kcal mol-1 M-1) were observed for point mutations at a single, solvent-exposed site (T53) in a variant of the B1 domain of streptococcal Protein G using guanidine hydrochloride (GuHCl) as a denaturant. This report focuses on elucidating the energetic and structural implications of these m-value differences in two Protein G mutants, containing Ala and Thr at position 53. These two proteins are representative of the high (m+) and low (m-) m-value mutants studied. Differential scanning calorimetry revealed no evidence of equilibrium intermediates. A comparison of GuHCl denaturation monitored by fluorescence and circular dichroism showed that secondary and tertiary structure denatured concomitantly. The rates of folding (286 S-1 for the m+ mutant and 952 S-1 for the m- mutant) and the rates of unfolding (11 S-1 for m+ mutant and 3 S-1 for the m- mutant) were significantly different, as determined by stopped-flow fluorescence. The relative solvation free energies of the transition states were identical for the two proteins (alpha ++ = 0.3). Small-angle X-ray scattering showed that the radius of gyration of the denatured state (Rgd) of the m+ mutant did not change with increasing denaturant concentrations (Rgd approximately 23 A); whereas, the Rgd of the m- mutant increased from approximately 17 A to 23 A with increasing denaturant concentration. The results indicate that the mutations exert significant effects in both the native and GuHCl-induced denatured state of these two proteins.

To determine the interaction energy between cross-strand pairs of side chains on an antiparallel beta sheet, pairwise amino acid substitutions were made on the solvent-exposed face of the B1 domain of streptococcal protein G. The measured interaction energies were substantial (1.8 kilocalories per mole) and comparable to the magnitude of the beta sheet propensities. The experimental results paralleled the statistical frequency with which the residue pairs are found in beta sheets of known structure.

Background: The 1989 recommendations of the US Preventive Services Task Force (USPSTF) represent an emerging consensus about which clinical preventive services should be delivered. However, practicing physicians disagree with a number of the recommendations in the Task Force prevention guidelines, and the reasons for disagreement have not been widely explored., Methods: A survey questionnaire assessing physician agreement or disagreement with the USPSTF recommendations was sent to all 1784 active members of the Ohio Academy of Family Physicians in October 1990. A factor analysis was performed on the items with which at least 5% of physicians disagreed. Associations of physician demographics and attitudes with the factor scores were then examined., Results: At least 5% of the 898 responding physicians disagreed with 67 of 150 USPSTF recommendations. Physicians disagreed with the USPSTF recommendations in three ways: (1) they believed that screening for some cancers is appropriate, even though not recommended by the USPSTF; (2) they believed that screening for other diseases in some populations is appropriate, even though not recommended by the USPSTF; and (3) they disagreed with some USPSTF recommendations for screening that is considered time-consuming or intrusive. Further analyses showed that practice setting and experience with the USPSTF guidelines were predictive of all three disagreement factors. Physician age, race, residency training, and reasons for disagreement were associated with two of the three factors., Conclusions: Physician disagreement with the USPSTF recommendations was not random but clustered into three distinct factors. An opportunity exists to design educational interventions for targeted subgroups of physicians. The views of practicing physicians should be incorporated into future guidelines.

myf5 is one of a family of four myogenic determination genes that control skeletal muscle differentiation. To study the role of myf5 in vivo, we generated transgenic mice harboring the bovine homolog, bmyf, under control of the murine sarcoma virus promoter. Ectopic expression of the full-length bmyf transgene was detected in brain and heart tissue samples of F1 progeny from transgenic founder mice. Ectopic bmyf expression activated endogenous skeletal myogenic determination genes in the hearts and brains of transgenic animals. Incomplete skeletal myogenesis in most hearts gave rise to cardiomegaly and focal areas of cardiomyopathy. In brains in which ectopic expression led to a more complete myogenesis, focal areas of multinucleated, striated myotubes containing actin, desmin, and myosin were observed. These unexpected results show that myf5 can initiate myogenic differentiation in vivo, supporting the hypothesis that myf5 is responsible for determination of cells to the myogenic lineage in normal embryogenesis.