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The loss of function of AAA (ATPases associated with diverse cellular activities) mechanoenzymes has been linked to diseases, and small molecules that activate these proteins can be powerful tools to probe mechanisms and test therapeutic hypotheses. Unlike chemical inhibitors that can bind a single conformational state to block enzyme function, activator binding must be permissive to different conformational states needed for mechanochemistry. However, we do not know how AAA proteins can be activated by small molecules. Here, we focus on valosin-containing protein (VCP)/p97, an AAA unfoldase whose loss of function has been linked to protein aggregation-based disorders, to identify druggable sites for chemical activators. We identified VCP ATPase Activator 1 (VAA1), a compound that dose-dependently stimulates VCP ATPase activity up to ~threefold. Our cryo-EM studies resulted in structures (ranging from ~2.9 to 3.7 Å-resolution) of VCP in apo and ADP-bound states and revealed that VAA1 binds an allosteric pocket near the C-terminus in both states. Engineered mutations in the VAA1-binding site confer resistance to VAA1, and furthermore, modulate VCP activity. Mutation of a phenylalanine residue in the VCP C-terminal tail that can occupy the VAA1 binding site also stimulates ATPase activity, suggesting that VAA1 acts by mimicking this interaction. Together, our findings uncover a druggable allosteric site and a mechanism of enzyme regulation that can be tuned through small molecule mimicry., Competing Interests: Competing interests statement:T.M.K. is a co-founder of and has an ownership interest in RADD Pharmaceuticals, Inc.

Type 1 diabetes management is intricately influenced by social determinants of health. Economic status impacts access to vital resources like insulin and diabetes technology. Racism, social injustice, and implicit biases affect equitable delivery of care. Education levels affect understanding of self-care, leading to disparities in glycemic outcomes. Geographic location can limit access to health care facilities. Stressors from discrimination or financial strain can disrupt disease management. Addressing these social factors is crucial for equitable diabetes care, emphasizing the need for comprehensive strategies that go beyond medical interventions to ensure optimal health outcomes for all individuals with type 1 diabetes., Competing Interests: Conflict of interest None., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Chemical probes can be valuable tools for studying protein targets, but addressing concerns about a probe's cellular target or its specificity can be challenging. A reliable strategy is to use a mutation that does not alter a target's function but confers resistance (or sensitizes) to the inhibitor in both cellular and biochemical assays. However, challenges remain in finding such mutations. Here, we discuss structure- and cell-based approaches to identify resistance- and sensitivity-conferring mutations. Further, we describe how resistance-conferring mutations can help with compound design, and the use of saturation mutagenesis to characterize a compound binding site. We highlight how genetic approaches can ensure the proper use of chemical inhibitors to pursue mechanistic studies and test therapeutic hypotheses., Competing Interests: Declaration of competing interest T.M.K. is a co-founder of and has an ownership interest in RADD Pharmaceuticals, Inc., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Abstract: Untreated congenital hypothyroidism (CH) leads to intellectual disabilities. Prompt diagnosis by newborn screening (NBS) leading to early and adequate treatment results in grossly normal neurocognitive outcomes in adulthood. However, NBS for hypothyroidism is not yet established in all countries globally. Seventy percent of neonates worldwide do not undergo NBS.The initial treatment of CH is levothyroxine, 10 to 15 mcg/kg daily. The goals of treatment are to maintain consistent euthyroidism with normal thyroid-stimulating hormone and free thyroxine in the upper half of the age-specific reference range during the first 3 years of life. Controversy remains regarding detection of thyroid dysfunction and optimal management of special populations, including preterm or low-birth weight infants and infants with transient or mild CH, trisomy 21, or central hypothyroidism.Newborn screening alone is not sufficient to prevent adverse outcomes from CH in a pediatric population. In addition to NBS, the management of CH requires timely confirmation of the diagnosis, accurate interpretation of thyroid function testing, effective treatment, and consistent follow-up. Physicians need to consider hypothyroidism in the face of clinical symptoms, even if NBS thyroid test results are normal. When clinical symptoms and signs of hypothyroidism are present (such as large posterior fontanelle, large tongue, umbilical hernia, prolonged jaundice, constipation, lethargy, and/or hypothermia), measurement of serum thyroid-stimulating hormone and free thyroxine is indicated, regardless of NBS results., (Copyright © 2023 by the American Academy of Pediatrics.)

Achieving glycemic targets in youth and young adults with type 1 diabetes (T1D) is challenging. Diabetes devices, including continuous glucose monitors (CGM) may impact glycemic control. We analyzed the proportion of CGM use in youth and young adults with T1D at nine U.S. T1D Exchange Quality Improvement (T1DX-QI) Collaborative centers and 402 European diabetes prospective follow-up registry (Diabetes-Patienten-Verlaufsdokumentation [DPV]) sites from 2017 to 2020 and examined the association of CGM use to glycemic control as measured by hemoglobin A1c (HbA1c). CGM use increased each year from 2017 to 2020 across all age ranges (<6, 6-<12, 12-<18, 18-<25 years) in both registries and lower mean HbA1c was observed in CGM users compared with nonusers regardless of insulin delivery method for all years analyzed. CGM use appeared to increase more so in the European DPV than the U.S. T1DX-QI, which may be due to transatlantic differences in health care systems, insurance coverage, and prescriber habits.

Southeast Asia is considered to have some of the highest levels of marine plastic pollution in the world. It is therefore vitally important to increase our understanding of the impacts and risks of plastic pollution to marine ecosystems and the essential services they provide to support the development of mitigation measures in the region. An interdisciplinary, international network of experts (Australia, Indonesia, Ireland, Malaysia, the Philippines, Singapore, Thailand, the United Kingdom, and Vietnam) set a research agenda for marine plastic pollution in the region, synthesizing current knowledge and highlighting areas for further research in Southeast Asia. Using an inductive method, 21 research questions emerged under five non-predefined key themes, grouping them according to which: (1) characterise marine plastic pollution in Southeast Asia; (2) explore its movement and fate across the region; (3) describe the biological and chemical modifications marine plastic pollution undergoes; (4) detail its environmental, social, and economic impacts; and, finally, (5) target regional policies and possible solutions. Questions relating to these research priority areas highlight the importance of better understanding the fate of marine plastic pollution, its degradation, and the impacts and risks it can generate across communities and different ecosystem services. Knowledge of these aspects will help support actions which currently suffer from transboundary problems, lack of responsibility, and inaction to tackle the issue from its point source in the region. Being profoundly affected by marine plastic pollution, Southeast Asian countries provide an opportunity to test the effectiveness of innovative and socially inclusive changes in marine plastic governance, as well as both high and low-tech solutions, which can offer insights and actionable models to the rest of the world., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Tyrosine kinase inhibitors that target vascular endothelial growth factor receptor [VEGFR-TKI] are a class of targeted therapies approved for treatment of several malignancies and are increasingly used in the pediatric population. Development of hypothyroidism during VEGFR-TKI therapy is well described in adults; however, there are no available data in children. Importantly, hypothyroidism during childhood can negatively impact growth and neurodevelopment. This retrospective study is the first to document frequency and severity of VEGFR-TKI induced hypothyroidism in pediatric and young adult patients. Patients included were ≤25 years of age and treated with at least one VEGFR-TKI between 2010 and 2018 at Cincinnati Children's Hospital Medical Center. After review of clinical and demographic data, 69 patients were identified. Of these, 19 (27.5%) developed thyroid dysfunction defined as Thyroid-stimulating hormone≥5 mIU/mL during therapy. Twelve of those patients had overt hypothyroidism with documentation of low free thyroxine and/or levothyroxine initiation. Mean exposure time to VEGFR-TKI before thyroid dysfunction was 2.8 (0.5-10.4) months. These results suggest moderate risk of developing thyroid dysfunction during VEGFR-TKI therapy in pediatric and young adult patients. Baseline thyroid hormone screening should be performed and repeated frequently during the first year of therapy in the pediatric population., Competing Interests: The authors declare no conflict of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Context: The impact of the COVID-19 pandemic on individuals with type 1 diabetes remains poorly defined., Objective: We examined United States trends in diabetic ketoacidosis (DKA) among individuals with type 1 diabetes (T1D) during the COVID-19 pandemic at 7 large US medical centers and factors associated with these trends., Methods: We compared DKA events among children and adults with T1D during COVID-19 surge 1 (March-May 2020) and COVID-19 surge 2 (August-October 2020) to the same periods in 2019. Analysis was performed using descriptive statistics and chi-square tests., Results: We found no difference in the absolute number of T1D patients experiencing DKA in 2019 vs 2020. However, a higher proportion of non-Hispanic Black (NHB) individuals experienced DKA in 2019 than non-Hispanic White (NHW) individuals (44.6% vs 16.0%; P < .001), and this disparity persisted during the COVID-19 pandemic (48.6% vs 18.6%; P < .001). DKA was less common among patients on continuous glucose monitor (CGM) or insulin pump in 2020 compared to 2019 (CGM: 13.2% vs 15.0%, P < .001; insulin pump: 8.0% vs 10.6%, P < .001). In contrast to annual DKA totals, a higher proportion of patients had DKA during COVID-19 surges 1 and 2 compared to the same months in 2019 (surge 1: 7.1% vs 5.4%, P < .001; surge 2: 6.6% vs 5.7%, P = .001)., Conclusion: DKA frequency increased among T1D patients during COVID-19 surges with highest frequency among NHB patients. DKA was less common among patients using CGM or insulin pumps. These findings highlight the urgent need for improved strategies to prevent DKA among patients with T1D-not only under pandemic conditions, but under all conditions-especially among populations most affected by health inequities., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society.)

The Irish Sea is an important area for Norway Lobster Nephrops norvegicus fisheries, which are the most valuable fishing resource in the UK. Norway lobster are known to ingest microplastic pollution present in the sediment and have displayed reduced body mass when exposed to microplastic pollution. Here, we identified microplastic pollution in the Irish Sea fishing grounds through analysis of 24 sediment samples from four sites of differing proximity to the Western Irish Sea Gyre in both 2016 and 2019. We used µFTIR spectroscopy to identify seven polymer types, and a total of 77 microplastics consisting of fibres and fragments. The mean microplastics per gram of sediment ranged from 0.13 to 0.49 and 0 to 1.17 MP/g in 2016 and 2019, respectively. There were no differences in the microplastic counts across years, and there was no correlation of microplastic counts with proximity to the Western Irish Sea Gyre. Considering the consistently high microplastic abundance found in the Irish Sea, and the propensity of N. norvegicus to ingest and be negatively impacted by them, we suggest microplastic pollution levels in the Irish Sea may have adverse impacts on N. norvegicus and negative implications for fishery sustainability in the future., (© 2022. The Author(s).)

Plastic pollution and climate change have commonly been treated as two separate issues and sometimes are even seen as competing. Here we present an alternative view that these two issues are fundamentally linked. Primarily, we explore how plastic contributes to greenhouse gas (GHG) emissions from the beginning to the end of its life cycle. Secondly, we show that more extreme weather and floods associated with climate change, will exacerbate the spread of plastic in the natural environment. Finally, both issues occur throughout the marine environment, and we show that ecosystems and species can be particularly vulnerable to both, such as coral reefs that face disease spread through plastic pollution and climate-driven increased global bleaching events. A Web of Science search showed climate change and plastic pollution studies in the ocean are often siloed, with only 0.4% of the articles examining both stressors simultaneously. We also identified a lack of regional and industry-specific life cycle analysis data for comparisons in relative GHG contributions by materials and products. Overall, we suggest that rather than debate over the relative importance of climate change or marine plastic pollution, a more productive course would be to determine the linking factors between the two and identify solutions to combat both crises., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Objectives: Physical examinations to characterize pubertal maturation may be unacceptable for children enrolled in research studies. Studies confirm the utility of pubertal self staging for research, but there has been limited comparison of self examination with hormone biomarkers. Our objective was to assess concordance of pubertal self staging with hormone biomarkers of puberty., Methods: Participants were enrolled in the Health Outcomes and Measures of the Environment Study, a longitudinal pregnancy and birth cohort study. At age 12 years, 139 females and 112 males completed pubertal self staging including breast and pubic hair development in females and pubic hair development in males. No clinical physical examination was performed. Hormone concentrations were measured in 102 females and 96 males including serum dehydroepiandrosterone sulfate, luteinizing hormone, and follicle-stimulating hormone in all; estradiol in females; and testosterone in males., Results: Estradiol was significantly associated with female breast stage, even when adjusted for BMI, with geometric least squares means (95%CI) of 13.2 (8.7, 20.2), 38.3 (29.9, 49.1), 59.4 (39.8, 88.6), and 81.2 (45.6, 144) pg/mL for breast stage 1-2, 3, 4, and 5, respectively. Testosterone was significantly associated with male pubic hair stage, with adjusted geometric least squares means (95%CI) of 37.6 (19.9, 71.1), 43.4 (27.7, 68.3), 126 (78.4, 203), 275 (146, 521), and 559 (237, 1319) ng/dL for pubic hair stage 1, 2, 3, 4, and 5, respectively., Conclusions: Self assessed pubertal development was positively associated with hormonal biomarkers of puberty., (© 2021 Walter de Gruyter GmbH, Berlin/Boston.)

The structural conservation across the AAA (ATPases associated with diverse cellular activities) protein family makes designing selective chemical inhibitors challenging. Here, we identify a triazolopyridine-based fragment that binds the AAA domain of human katanin, a microtubule-severing protein. We have developed a model for compound binding and designed ASPIR-1 (allele-specific, proximity-induced reactivity-based inhibitor-1), a cell-permeable compound that selectively inhibits katanin with an engineered cysteine mutation. Only in cells expressing mutant katanin does ASPIR-1 treatment increase the accumulation of CAMSAP2 at microtubule minus ends, confirming specific on-target cellular activity. Importantly, ASPIR-1 also selectively inhibits engineered cysteine mutants of human VPS4B and FIGL1-AAA proteins, involved in organelle dynamics and genome stability, respectively. Structural studies confirm our model for compound binding at the AAA ATPase site and the proximity-induced reactivity-based inhibition. Together, our findings suggest a chemical genetics approach to decipher AAA protein functions across essential cellular processes and to test hypotheses for developing therapeutics.

Spleen tyrosine kinase (SYK) is a non-receptor cytosolic kinase. Due to its pivotal role in B cell receptor and Fc-receptor signaling, inhibition of SYK has been targeted in a variety of disease areas. Herein, we report the optimization of a series of potent and selective SYK inhibitors, focusing on improving metabolic stability, pharmacokinetics and hERG inhibition. As a result, we identified 30, which exhibited no hERG activity but unfortunately was poorly absorbed in rats and mice. We also identified a SYK chemical probe, 17, which exhibits excellent potency at SYK, and an adequate rodent PK profile to support in vivo efficacy/PD studies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Drug-like inhibitors are often designed by mimicking cofactor or substrate interactions with enzymes. However, as active sites are comprised of conserved residues, it is difficult to identify the critical interactions needed to design selective inhibitors. We are developing an approach, named RADD (resistance analysis during design), which involves engineering point mutations in the target to generate active alleles and testing compounds against them. Mutations that alter compound potency identify residues that make key interactions with the inhibitor and predict target-binding poses. Here, we apply this approach to analyze how diaminotriazole-based inhibitors bind spastin, a microtubule-severing AAA (ATPase associated with diverse cellular activities) protein. The distinct binding poses predicted for two similar inhibitors were confirmed by a series of X-ray structures. Importantly, our approach not only reveals how selective inhibition of the target can be achieved but also identifies resistance-conferring mutations at the early stages of the design process., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background Children with congenital hypothyroidism (CH) are at risk for preventable intellectual disability without adequate medical management. The purpose of this manuscript is to discuss quality improvement (QI)-based processes for improving provider adherence to practice guidelines and ultimately identifying at-risk patients with chronic illness prior to the occurrence of adverse events. Methods Our study population included patients ages ≤3 years diagnosed with CH; lost to follow-up was defined as >180 days since last evaluation by an endocrinology provider. Iterative testing of interventions focused on establishing standardized care through (1) registry-based identification, (2) scheduling future appointments during current visits, (3) outreach to patients lost to follow-up and (4) provider and family education of current practice guidelines. Results A population-validated, electronic medical registry identified approximately 100 patients ages ≤3 years diagnosed with CH; initially, 12% of patients met criteria for lost to follow-up. Through serial testing of interventions, the rate of loss to follow-up declined to the goal of <5% within 8 months. Additional measures showed improvement in provider adherence to standard of care. All patients identified as lost to follow-up initially were seen within the first 3 months of intervention. Conclusions Applying QI methodology, a multidisciplinary team implemented a process to identify and contact high-risk CH patients with inadequate follow-up. Focused interventions targeting population management, scheduling and patient/provider education yield sustained improvement in the percentage of patients with a chronic condition who are lost to follow-up.

Germinal heterozygous activating STAT3 mutations represent a novel monogenic defect associated with multi-organ autoimmune disease and, in some cases, severe growth retardation. By using whole-exome sequencing, we identified two novel STAT3 mutations, p.E616del and p.C426R, in two unrelated pediatric patients with IGF-I deficiency and immune dysregulation. The functional analyses showed that both variants were gain-of-function (GOF), although they were not constitutively phosphorylated. They presented differences in their dephosphorylation kinetics and transcriptional activities under interleukin-6 stimulation. Both variants increased their transcriptional activities in response to growth hormone (GH) treatment. Nonetheless, STAT5b transcriptional activity was diminished in the presence of STAT3 GOF variants, suggesting a disruptive role of STAT3 GOF variants in the GH signaling pathway. This study highlights the broad clinical spectrum of patients presenting activating STAT3 mutations and explores the underlying molecular pathway responsible for this condition, suggesting that different mutations may drive increased activity by slightly different mechanisms., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Cyclin-dependent kinase (CDK) 12 knockdown via siRNA decreases the transcription of DNA-damage-response genes and sensitizes BRCA wild-type cells to poly(ADP-ribose) polymerase (PARP) inhibition. To recapitulate this effect with a small molecule, we sought a potent, selective CDK12 inhibitor. Crystal structures and modeling informed hybridization between dinaciclib and SR-3029, resulting in lead compound 5 [(S)-2-(1-(6-(((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)amino)-9-ethyl-9H-purin-2-yl)piperidin-2-yl)ethan-1-ol]. Further structure-guided optimization delivered a series of selective CDK12 inhibitors, including compound 7 [(S)-2-(1-(6-(((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)amino)-9-isopropyl-9H-purin-2-yl)piperidin-2-yl)ethan-1-ol]. Profiling of this compound across CDK9, 7, 2, and 1 at high ATP concentration, single-point kinase panel screening against 352 targets at 0.1 μm, and proteomics via kinase affinity matrix technology demonstrated the selectivity. This series of compounds inhibits phosphorylation of Ser2 on the C-terminal repeat domain of RNA polymerase II, consistent with CDK12 inhibition. These selective compounds were also acutely toxic to OV90 as well as THP1 cells., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Purpose: This study delineates clinical and psychosocial characteristics of adolescents with type 1 diabetes and suicidal ideation (SI) and reports clinical and psychosocial outcomes after mental health intervention (safety assessment and brief in-clinic intervention)., Methods: Adolescents aged 13-17 years with type 1 diabetes completed the Children's Depression Inventory (CDI) from January 2011 to 2012. Youth with significant depressive symptoms and/or SI endorsement underwent mental health intervention. Two control subjects were matched to each adolescent endorsing SI and compared using t-tests to assess clinical and psychosocial variables. Trajectory of depressive symptoms and outcomes for case subjects were observed through January 2013., Results: Twenty-seven percent (127/473) exhibited moderate to high risk for depression based on CDI scores and 38 (8%) endorsed SI. Adolescents who endorsed SI were more likely to have higher CDI scores and public insurance when compared with youth who denied SI. There was no difference in glycemic control, measured by hemoglobin A1c, between case and control groups. During the year after intervention, 28 participants who initially endorsed SI underwent repeat assessment; mean CDI scores declined by 10.57 (standard deviation: 6.92) points and 78% no longer endorsed SI., Conclusions: Given the potential lethality of insulin when taken in intentional overdose, the need for consistent identification of suicidality is an important feature of depression screening. Study findings indicate statistically significant differences in depressive symptoms and insurance status, when comparing adolescents who endorsed SI to those who denied. Improvement in depressive symptoms and SI endorsement occurred after integrating brief mental health intervention into diabetes visits., (Copyright © 2017 Society for Adolescent Health and Medicine. Published by Elsevier Inc. All rights reserved.)

A visible-light-promoted iridium photoredox and nickel dual-catalyzed cross-coupling procedure for the formation C-N bonds has been developed. With this method, various aryl amines were chemoselectively cross-coupled with electronically and sterically diverse aryl iodides and bromides to forge the corresponding C-N bonds, which are of high interest to the pharmaceutical industries. Aryl iodides were found to be a more efficient electrophilic coupling partner. The coupling reactions were carried out at room temperature without the rigorous exclusion of molecular oxygen, thus making this newly developed Ir-photoredox/Ni dual-catalyzed procedure very mild and operationally simple., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Multicellular biofilms are an ancient bacterial adaptation that offers a protective environment for survival in hostile habitats. In microaerophilic organisms such as Campylobacter, biofilms play a key role in transmission to humans as the bacteria are exposed to atmospheric oxygen concentrations when leaving the reservoir host gut. Genetic determinants of biofilm formation differ between species, but little is known about how strains of the same species achieve the biofilm phenotype with different genetic backgrounds. Our approach combines genome-wide association studies with traditional microbiology techniques to investigate the genetic basis of biofilm formation in 102 Campylobacter jejuni isolates. We quantified biofilm formation among the isolates and identified hotspots of genetic variation in homologous sequences that correspond to variation in biofilm phenotypes. Thirteen genes demonstrated a statistically robust association including those involved in adhesion, motility, glycosylation, capsule production and oxidative stress. The genes associated with biofilm formation were different in the host generalist ST-21 and ST-45 clonal complexes, which are frequently isolated from multiple host species and clinical samples. This suggests the evolution of enhanced biofilm from different genetic backgrounds and a possible role in colonization of multiple hosts and transmission to humans., (© 2015 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Objective: Depression is common among adolescents, but rates increase significantly in the presence of chronic health conditions. Outpatient screening for depression is recommended but rarely formally conducted due to barriers of implementation., Methods: To provide a model for depression screening of youth with chronic health conditions, a standard process using a self-administered electronic version of the Children's Depression Inventory (CDI) was developed. Quality improvement methodology and traditional analytic approaches were used to test the feasibility and outcomes of routine screening in patients 13 to 17 years of age with type 1 diabetes., Results: Of the 528 eligible adolescents, 509 (96%) received at least 1 depression screen during the first year. The process was tested and refined in over 1200 patient encounters, which resulted in an increase in depression screening rates from <5% to a median of 85% over the initial 12 months. Both patients and staff reported acceptance of screening on qualitative surveys. Elevated CDI scores (≥ 16) were found in 8% of the sample; moderate scores (10-15) in 12% of the sample. Low risk scores were found in 80% of the sample. Higher CDI scores correlated with lower blood glucose monitoring frequency and higher hemoglobin A1c, confirming the link between more depression symptoms and poorer diabetes management and control. Suicidal ideation was endorsed in 7% of the population., Conclusions: Systematic depression screening in adolescents with type 1 diabetes can be reliably implemented with clinically significant results. A systematic approach, such as described in this study, can serve as a model for other chronic health conditions.

Background: The late onset of pelvic visceral prolapse and incontinence after childbirth injury could be explained by menopause-associated connective tissue weakening. Uterosacral ligament resilience (UsR) was assessed to determine whether it influenced uterine or pelvic floor mobility, or varied with age, vaginal delivery, menopause or histological variations in the ligament., Methods: UsR was measured by tensiometry in ligaments from 85 hysterectomy specimens, and was correlated with the presence of symptomatic uterocervical prolapse, prehysterectomy uterine and anorectal mobility, patient age, history of vaginal delivery and menopause. Forty-five of these ligaments were examined for ligament thickness, muscle to collagen ratio, and oestrogen and progesterone receptor density. The results were correlated with UsR., Results: UsR was significantly reduced (P = 0.02) in symptomatic uterovaginal prolapse, but there was no correlation with either uterocervical or anorectal descent in women without symptomatic prolapse. There was a significant decrease in UsR with vaginal delivery (P = 0.003), menopause (P = 0.009) and older age (P = 0.005). The uterosacral ligament was significantly thinner and contained fewer oestrogen and progesterone receptors after menopause, but this did not affect UsR., Conclusion: Where pelvic floor muscles are weakened, decreases in pelvic connective tissue resilience related to the menopause may facilitate progression to symptomatic pelvic visceral prolapse., (Copyright 2003 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd.)

We observed coherent proton tunneling in the cyclic network of four hydrogen bonds in calix[4]arene. The tunneling frequency of 35 megahertz was revealed by a peak in the magnetic field dependence of the proton spin-lattice relaxation rate measured with field-cycling nuclear magnetic resonance in the solid state at temperatures below 80 kelvin. The amplitude of the coherent tunneling peak grows with temperature according to a Boltzmann law with energy D/kB = (125 +/- 10) kelvin (where kB is Boltzmann's constant). The tunneling peak can be interpreted in the context of level crossings in the region where the tunneling frequency matches the proton Larmor frequency. The tunneling spectrum reveals fine structure that we attribute to coupling between the hydrogen bonds in the network. The characteristics of the tunneling peak are interpreted in the context of the potential energy surface experienced by the hydrogen atoms in the network.

We induced in allergic humans the counterpart of murine experimental T-cell tolerance. T-cell lines from cat-allergic humans were used to map T-cell epitopes for the principal allergen of cat dander, Fel d 1. Two peptides of 27 amino acids each were synthesized to contain the dominant epitopes (ALLERVAX CAT). After a safety trial, we carried out a blinded study of the dose required for efficacy. We randomly divided 95 cat-sensitive patients into placebo, 7.5 micrograms, 75 micrograms, and 750 micrograms groups. Patients received a subcutaneous injection weekly for 4 wk. Before and after treatment, patients were exposed in a room inhabited by live cats and scored by nose and lung symptoms. Baseline nasal and lung scores (+/-SEM) were 6.2 +/- 0.56 and 5.4 +/- 0.73 in the 750 micrograms group; 7.8 +/- 0.53 and 4.7 +/- 0.68 in the placebo group. Six weeks after treatment, scores adjusted for baseline differences were reduced in the 750 micrograms group: -2.3 +/- 4.9 and -2.3 +/- 0.59 compared with -0.84 +/- 0.50 and -0.85 +/- 0.62 in the placebo group. The 75 micrograms group showed intermediate effects and the 7.5 micrograms group no effect. Linear trend analysis indicated a significant dose response effect: p = 0.05 for nose and 0.03 for lung symptoms. Allergic side effects occurred an hour or more after the first 750 micrograms dose in 16 of 24 patients but required little or no treatment with one exception. T-cell reactive treatment peptides safely improved allergic responses to cats.

Complementary DNA (cDNA) clones encoding the heavy chain of the heterodimeric human membrane glycoprotein 4F2 have been isolated by immunoscreening of a lambda gt11 expression library. The identity of these clones has been confirmed by hybridization to RNA and DNA prepared from mouse L-cell transfectants, which were produced by whole cell gene transfer and selected for cell-surface expression of the human 4F2 heavy chain. DNA sequence analysis suggests that the 4F2 heavy-chain cDNAs encode an approximately 526-amino acid type II membrane glycoprotein, which is composed of a large C-terminal extracellular domain, a single potential transmembrane region, and a 50-81 amino acid N-terminal intracytoplasmic domain. Southern blotting experiments have shown that the 4F2 heavy-chain cDNAs are derived from a single-copy gene that has been highly conserved during mammalian evolution.

A monoclonal antibody (DU-ALL-1) was generated to common acute lymphoblastic leukemia (cALL) cells by microcytotoxicity and indirect immunofluorescence, DU-ALL-1 reacted only with cALL cell lines and not with the other hematopoietic cell lines tested. Peripheral blood lymphocytes, monocytes, granulocytes and mitogen-activated lymphocytes did not react significantly with this antibody. However, platelets (100%) and normal bone marrow cells (8.5%) reacted with DU-ALL-1. Microcytotoxicity testing of human leukemia cells showed that DU-ALL-1 reacted with cells from a majority of null and pre-B ALL patients (63/77) and with cells from some patients with acute myeloblastic leukemia (4/7) and T-ALL (4/20). DU-ALL-1 was generally non-reactive with cells from patients with B-cell leukemias (2/16) and chronic myelogenous leukemia in blast crisis (0/4). By an indirect immunoperoxidase technique, DU-ALL-1 reacted with a variety of non-hematopoietic tissues, including smooth and cardiac muscle and epithelia from several organs. The DU-ALL-1 antigen had an apparent mol. wt of 24,000 and did not bind to lectins or label with [3H]glucosamine. Thus, DU-ALL-1 defines a 24,000-mol. wt protein which is absent from most peripheral blood mononuclear cells, is expressed on normal platelets and several non-hematopoietic tissues, and may be a useful for subclassifying leukemias.

The common acute lymphoblastic leukemia antigen (CALLA), as defined by J-5 murine monoclonal antibodies, was detected on renal tubular and glomerular cells from fetal and adult donors by an indirect immunoperoxidase technique. CALLA could also be detected on epithelial cells of the fetal small intestine and on myoepithelial cells of adult breast but not on myoepithelial cells of the salivary gland. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of immunoprecipitated 125I-labeled membrane antigens from dissociated renal cells demonstrated that the antigen migrated as a 90,000 mol wt antigen rather than the 98,000-100,000 mol wt antigen noted on CALLA-positive tissue culture cell lines. The data suggest that the determinant defined by the J-5 monoclonal antibody is neither a lymphoid cell-specific differentiation antigen nor a leukemia-specific antigen.

The T1/Leu-1/CD5 molecule, a human T-cell surface glycoprotein of relative molecular mass (Mr) 67,000, has been implicated in the proliferative response of activated T cells and in T-cell helper function. A similar involvement in T-cell proliferation has been reported for Ly-1, the murine homologue of T1. Here we report the complete amino-acid sequence of the T1 precursor molecule deduced from complementary DNA clones. The protein contains a classical signal peptide; a 347-amino-acid extracellular segment; a transmembrane region; and a 93-amino-acid intracellular segment. The extracellular segment contains many cysteine residues and is composed of two related structural domains separated by a proline/threonine-rich region. The T1 molecule has structural features characteristic of other receptor molecules.

Rabbit and monkey antisera after appropriate absorption were rendered specific for normal or leukemic lymphoid- and myeloid-associated antigens. Antisera defining a common peripheral blood T-cell antigen, a thymus leukemia antigen, HLA-DR or Ia-like antigen, common acute lymphoblastic leukemia antigen (CALLA), and a myeloid-monocyte (M) antigen were used in a microcytotoxicity assay to classify leukemic cells from 30 patients in a double blind study. The antisera to the M antigen reacted with adherent peripheral blood cells and polymorphonuclear leukocytes and failed to react with nonadherent mononuclear cells and enriched T-cells and chronic lymphocytic leukemia cells. The M antisera also reacted with U937, a monocytic-type cell line, and with HL60, a promyelocytic-type cell line, but failed to react with T and B lymphoblastoid cell lines. The specificities of the other antisera have been described in previous reports. Cells from three of the patients could not be phenotyped by microcytotoxicity testing. Cells from 25 patients had a consensus morphological or histochemical diagnosis of either acute lymphoblastic leukemia or acute nonlymphocytic leukemia. The serological classification of these patients using the five types of antisera listed above were consistent with the consensus diagnosis. In addition, the lymphoid cancers were further subclassified as to T-, B-, or thymus antigen types. There was no consensus lymphoid versus myeloid diagnosis on cells from two patient. The serological classification in both cases favored a diagnosis of myeloid rather than lymphoid leukemia.

The human 4F2 cell surface antigen is a 120-kilodalton (kDa) disulfide-linked heterodimer which is composed of an 80- to 90-kDa glycosylated heavy chain (4F2HC) and a 35- to 40-kDa nonglycosylated light chain (4F2LC). 4F2 belongs to a family of inducible cell surface molecules which are involved in T-lymphocyte activation and growth. To better understand the molecular mechanism(s) that controls 4F2HC gene expression in both resting and activated T cells, a 4F2HC human genomic clone was isolated and structurally characterized. The 4F2HC gene spans 8 kilobases of chromosome 11 and is composed of nine exons. The 5' upstream region of the gene displays several properties which are characteristic of housekeeping genes. It is G+C rich and hypomethylated in peripheral blood lymphocyte DNA and contains multiple binding sites for the Sp1 transcription factor while lacking TATA or CCAAT sequences. This region of the gene also displays sequence homologies with several other inducible T-cell genes, including the interleukin-2, interleukin-2 receptor alpha chain, dihydrofolate reductase, thymidine kinase, and transferrin receptor genes. A 255-base-pair fragment of the 4F2HC gene which contains 154 base pairs of the 5' flanking sequence was able to efficiently promote expression of the bacterial chloramphenicol acetyltransferase gene in human Jurkat T cells, indicating that it contains promoter or enhancer (or both) sequences. Analyses of chromatin structure in resting and lectin-activated T cells revealed the presence of stable DNase I-hypersensitive sites within both the 5' flanking and intron 1 regions of the 4F2HC gene. Although the 4F2HC gene displayed many of the structural features characteristic of a constitutively expressed gene, lectin-mediated activation of resting peripheral blood T lymphocytes resulted in a dramatic increase in steady-state levels of 4F2HC mRNA.

We report the isolation of cDNA clones of the mouse lymphocyte differentiation antigen Ly-1. One of these cDNA clones was confirmed to be full-length by DNA sequencing and by expression of Ly-1 by L cells transfected with this clone. Analysis of the predicted amino acid sequence indicated that the Ly-1 polypeptide is synthesized with a 23 amino acid leader and that the mature protein consists of an amino-terminal region of 347 amino acids, a transmembrane sequence of 30 residues, and a carboxyl-terminal region of 94 amino acids. The amino-terminal region appears to be divided into two subregions by a threonine- and proline-rich sequence of 23 amino acids that is highly conserved between Ly-1 and its human homologue Leu-1 (CD5) in position and amino acid composition. The first amino-terminal subregion of 111 amino acids is predicted to be arranged in a beta-pleated sheet structure of six strands. The entire amino-terminal region is rich in cysteine, with all of its 22 cysteine residues conserved between Ly-1 and Leu-1. The carboxyl-terminal region has no cysteines. Ly-1 and Leu-1 are 63% identical, with a gradient of identical residues from 43% for the first amino-terminal subregion to 58% for the second amino-terminal subregion and 90% for the carboxyl-terminal region. The predicted secondary structure of the first amino-terminal subregion and identities of certain conserved residues among most members of the immunoglobulin gene superfamily suggest that Ly-1 and Leu-1 are distant members of this family.

In order to define the function of the CD 5 (T1, Leu 1, Tp 67 in the human; Ly-1 in the mouse) molecule, a cDNA clone of human CD 5 was expressed in a CD5-deficient Jurkat cell line and in a murine T cell hybridoma. A Jurkat subclone (Jurkat 9.9) produced interleukin 2 (IL 2) in response to anti-CD 3 monoclonal antibody (mAb) cross-linked to solid support. IL 2 production was enhanced by co-culture with the anti-CD 5 mAb OKT 1. A CD 5-deficient mutant clone Jurkat 1.15 was generated by treatment with ethyl methanesulfonate followed by selection with anti-CD 5 mAb plus complement. Jurkat 1.15 did not demonstrate enhancement of IL 2 production by OKT 1 in the presence of cross-linked anti-CD 3 mAb. A cDNA encoding human CD 5 was introduced into a defective retrovirus which was used to infect Jurkat 1.15. A Jurkat clone stably expressing CD 5 was established. In response to OKT 1, a rise in intracellular calcium was observed in both the parent Jurkat 9.9 and the CD 5+ infectant but not in the CD 5- mutant or a G 418- resistant control. Furthermore, expression of CD 5 restored the augmentation of IL 2 production by OKT 1 in response to cross-linked anti-CD 3 mAb. A murine T cell hybridoma By 155.16 which produces IL 2 in response to HLA-DR antigens was also infected with the CD 5-recombinant retrovirus and three stable CD 5+ infectants were generated. These hybridomas showed enhancement of IL 2 production by stimulation with OKT 1 in the presence of suboptimal concentrations of soluble anti-murine CD 3 mAb. These results provide further evidence that CD 5 provides a co-stimulatory signal for T cell activation.