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Background: Blood products form the cornerstone of contemporary hemorrhage control but are limited resources. Freeze‐dried plasma (FDP), which contains coagulation factors, is a promising adjunct in hemostatic resuscitation. We explore the association between FDP alone or in combination with other blood products on 24‐h mortality. Study Design and Methods: This is a secondary data analysis from a cross‐sectional prospective observational multicenter study of adult trauma patients in the Western Cape of South Africa. We compare mortality among trauma patients at risk of hemorrhage in three treatment groups: Blood Products only, FDP + Blood Products, and FDP only. We apply inverse probability of treatment weighting and fit a multivariable Cox proportional hazards model to assess the hazard of 24‐h mortality. Results: Four hundred and forty‐eight patients were included, and 55 (12.2%) died within 24 h of hospital arrival. Compared to the Blood Products only group, we found no difference in 24‐h mortality for the FDP + Blood Product group (p =.40) and a lower hazard of death for the FDP only group (hazard = 0.38; 95% CI, 0.15–1.00; p =.05). However, sensitivity analyses showed no difference in 24‐h mortality across treatments in subgroups with moderate and severe shock, early blood product administration, and accounting for immortal time bias. Conclusion: We found insufficient evidence to conclude there is a difference in relative 24‐h mortality among trauma patients at risk for hemorrhage who received FDP alone, blood products alone, or blood products with FDP. There may be an adjunctive role for FDP in hemorrhagic shock resuscitation in settings with significantly restricted access to blood products. [ABSTRACT FROM AUTHOR]

Context Antidrug antibodies (ADA) can potentially affect drug pharmacokinetics, safety, and efficacy. Objective This work aimed to evaluate treatment-emergent (TE) ADA in tirzepatide (TZP)-treated participants across 7 phase 3 trials and their potential effect on pharmacokinetics, efficacy, and safety. Methods ADA were assessed at baseline and throughout the study until end point, defined as week 40 (SURPASS-1, -2, and -5) or week 52 (SURPASS-3, -4, Japan-Mono, and Japan-Combo). Samples for ADA characterization were collected at SURPASS trial sites. Participants included ADA-evaluable TZP-treated patients with type 2 diabetes (N = 5025). Interventions included TZP 5, 10, or 15 mg. ADA were detected and characterized for their ability to cross-react with native glucose-dependent insulinotropic polypeptide (nGIP) and glucagon-like peptide-1 (nGLP-1), neutralize tirzepatide activity on GIP and GLP-1 receptors, and neutralize nGIP and nGLP-1. Results TE ADA developed in 51.1% of tirzepatide-treated patients. Proportions were similar across dose groups. Maximum ADA titers ranged from 1:20 to 1: 81 920 among TE ADA+ patients. Neutralizing antibodies (NAb) against TZP activity on GIP and GLP-1 receptors were observed in 1.9% and 2.1% of patients, respectively. Less than 1.0% of patients had cross-reactive NAb against nGIP or nGLP-1. TE ADA status, ADA titer, and NAb status had no effect on the pharmacokinetics or efficacy of TZP. More TE ADA+ patients experienced hypersensitivity reactions or injection site reactions than TE ADA– patients. The majority of hypersensitivity and injection site reactions were nonserious and nonsevere, and most events occurred and/or resolved irrespective of TE ADA status or titer. Conclusion Immunogenicity did not affect TZP pharmacokinetics or efficacy. The majority of hypersensitivity or injection site reactions experienced by TE ADA+ patients were mild to moderate in severity. [ABSTRACT FROM AUTHOR]

Introduction: Melorheostosis is a rare bone disorder with limited literature that describes the effect of this disease on functional and motor abilities. As part of a natural history study, four outcome measures were administered to better understand the burden this disease has on a person's ability to engage in basic and instrumental activities of daily living. Objective: To investigate the relationship between functional engagement, fatigue, and motor ability in patients with melorheostosis. Design: Cross‐sectional data gathered from a longitudinal natural history observational study. Setting Rehabilitation department within a single institution. Participants: Forty‐seven adult volunteers with melorheostosis were enrolled. Two participants were removed for failure to meet diagnosis eligibility. Thirty patients had lower extremity (LE) osteosclerotic bone lesions, 14 had upper extremity (UE) lesions, and one had lesions in both UEs and LEs. Interventions: Not applicable. Main Outcome Measures: Activity Card Sort, Second Edition (ACS); Multi‐Dimensional Fatigue Inventory; Lower Extremity Functional Scale; Upper Extremity Functional Index. Results: On the ACS, high‐demand leisure (HDL) activities were the least retained (p <.001). Of the activities rated most important, HDL activities were the most likely to have been given up (27%). General fatigue (μ = 11.8) and physical fatigue (μ = 11.0) were the two most limiting fatigue constructs. There were moderate negative correlations with HDL activities compared to physical fatigue (r = −0.524, p <.001) and reduced activity fatigue (r = −0.58, p =.001). LE lesions had a large effect on completing LE tasks (d = 0.95) and UE lesions had a medium effect on completing tasks involving the UE (d = 0.69). Conclusions: Patients with melorheostosis experience fatigue and low engagement in HDL activities. The results of this study underscore the importance of acknowledging activity domain, fatigue constructs, and lesion location to support and provide targeted evidence‐based rehabilitative therapy. Clinical Trial Registration Number: NCT02504879. [ABSTRACT FROM AUTHOR]

A clear scientific and operational need exists for harmonized bioanalytical immunogenicity study reporting to facilitate communication of immunogenicity findings and expedient review by industry and health authorities. To address these key bioanalytical reporting gaps and provide a report structure for documenting immunogenicity results, this cross-industry group was formed to establish harmonized recommendations and a develop a submission template to facilitate agency filings. Provided here are recommendations for reporting clinical anti-drug antibody (ADA) assay results using ligand-binding assay technologies. This publication describes the essential bioanalytical report (BAR) elements such as the method, critical reagents and equipment, study samples, results, and data analysis, and provides a template for a suggested structure for the ADA BAR. This publication focuses on the content and presentation of the bioanalytical ADA sample analysis report. The interpretation of immunogenicity data, including the evaluation of the impact of ADA on safety, exposure, and efficacy, is out of scope of this publication. [ABSTRACT FROM AUTHOR]

Recent reflection on the role of museums and galleries has focused on their socially situated nature; and that as a social construct, co-produced with its audiences, heritage is in part discursively constituted. This has included acknowledgement that the inherited discourse is hegemonic and exclusive of divergent narratives, leading to moves to create alternatives to contest it, which include temporary exhibitions. These provide a potentially democratic space for discursive incursions freed from the constraints of the permanent museum. But they are also spatially and temporally peripheral, occupying a discursive space outside the standard visit. This raises questions as to whether, once the exhibition is over, the alternative will be subsumed once more. This article explores this issue using a dataset of TripAdvisor reviews to analyse the discursive responses of visitors to temporary and permanent collections, using the Musée d'Orsay's 2019 exhibition Le Modèle Noir as a case study. Analysis shows that Le Modèle Noir reviews exhibit greater discursive fragmentation, reveal a relative lack of appeals to collective identity, and do not connect the exhibition with the permanent collection. Potential implications of this for initiatives that seek to counter the hegemonic narrative are discussed. [ABSTRACT FROM AUTHOR]

Despite the positive impacts of an increasing number of organisational initiatives and campaign groups, unequal access to the countryside remains an intransigent issue. Contesting the countryside's normative associations is thus not just a conceptual challenge but a practical one for organisations managing rural sites. Taking the National Trust-run site of Wembury in Devon, UK, as a case study, I use critical discourse analysis to uncover institutions' (including the National Trust and other charities, news media, and factual programmes) and individuals' (using TripAdvisor data) discursive constructions of the landscape. Emerging themes include discourses of place, activities, and people, that—despite some dissonance and seeming contestation—cohere and (re)produce ideologies based on normative narratives of rural landscapes. I suggest the potential value of discourse analysis in surfacing rural storyscapes, and leveraging them to disrupt discourses which further exclusionary ideologies, as a tool to enable locally contextualised, practical means of advancing inclusion. [ABSTRACT FROM AUTHOR]

Background: National Ugly Mugs (NUM) is a UK-wide violence prevention and victim support charity that provides a mechanism for sex workers to share safety information and obtain support for harms that they may experience during the course of their work. Over the past several years, NUM has witnessed a decline in sex workers willing to access police as part of their recovery journeys after experiencing victimisation. In 2012, 28% of those reporting to NUM chose to engage with the legal system; in 2020, this was down to 7.7% amongst off-street independent workers. Statistics for 2021 indicate a continuation of this downward trend. Furthermore, anonymous consent to share information with police also declined from 95% in 2012 to 69% in 2020. Methods: NUM conducted a survey of 88 sex working members in 2020. This information combined with our data on victimisation provides insights of the factors that deter sex workers from involving police as part of their justice-seeking efforts. Results: Survey results reveal that sex workers feel alienated and untrusting of police and courts. Conclusions: The implications of sex workers not sharing information about dangerous individuals with police and choosing not to participate in court processes signal significant flaws in our legal system regarding safe and inequitable access and pose dangers for all of us. [ABSTRACT FROM AUTHOR]

Background: Individuals with obesity frequently present for anaesthesia and surgery. Good communication during the preoperative consultation can optimise the provision of relevant health information and guide improvement of health status preoperatively. Methods: We planned a systematic literature review to assess existing guidelines and evidence of effectiveness for how anaesthetists should communicate with patients who have obesity in the preoperative period about perioperative risks and weight management. Database searches used keywords related to perioperative weight loss conversations. We found no papers that directly addressed our aim. The literature identified as most relevant was analysed in the form of a narrative review. Results: The majority of suggestions for weight loss conversations came from primary care. Four primary themes potentially relevant to anaesthetists were identified: barriers to such conversations, communication tools, language and communication and specific recommendations. Identified barriers included lack of skills, training, poor remuneration, pessimism and time constraints for clinicians. Established discussion tools including the '5A's' approach (Assess, Advise, Agree, Assist, Arrange) and motivational interviewing may hold promise to improve preoperative conversations. The papers highlighted a need for empathetic language, including use of patient-specific language where possible. Conclusions: There are currently no published guidelines for how anaesthetists could most effectively discuss weight in the perioperative period with patients who have obesity. Much of the literature for obesity communication is based on the primary care setting. The perioperative period may represent an increased time of receptiveness for patients. Guidelines for discussions about weight management and associated perioperative risk are suggested. [ABSTRACT FROM AUTHOR]

Previous research (Orange & Laviolette [2010] A disgruntled tourist in King Arthur's court: Archaeology and identity at Tintagel, Cornwall. Public Archaeology, 9(2), 85–107; Robb [1998] Tourism and legends: Archaeology of heritage. Annals of Tourism Research, 25(3), 579–596) has described the interpretative tension at Tintagel Castle between history and Arthurian legend; since these articles were written, a sculpture that English Heritage says is 'inspired by the legend of King Arthur, by the historic kings and royal figures associated with Tintagel,' and a carving of Merlin's face have been added to the site. Using discourse analysis of TripAdvisor reviews, this article explores how legend and history are experienced by visitors. Despite an 'inextricable' link with Arthur, his actual absence here in both physical and narrative realms equals an absence of imaginative stimulus, for which the statue, while enabling superficial physical interaction, cannot compensate. Likewise, many reviewers see the mediaeval remains not as a presence of ruins but an absence of castle, and are similarly uninspired to transport themselves into a historical narrative. It is only reviewers inspired by history who engage the ruins as a 'thing' whose imaginations immerse them in their visit. [ABSTRACT FROM AUTHOR]

Phenomenon: Factors related to individual circumstance and organizational climate are contributing to a worsening burnout problem among providers in healthcare settings. In the academic health center, junior faculty may be at particular risk for burnout given intersecting responsibilities of clinical expertise, research rigor, teaching commitments, and service expectations. To date, much of the focus on preventing and mitigating burnout has been located at the individual level, addressing lifestyle modification and self-regulation skills. We sought to examine relationships between institutional context and burnout qualities as a means to identify opportunities for organizational leadership to address faculty burnout. Approach: Data are from a baseline survey of assistant professors (faculty with diverse ratios for clinical, research, and teaching responsibilities) located within a pediatrics department in an academic medical center. Pearson correlation coefficients and logistic regression models were used to examine relationships between institutional factors (mentorship, collaboration opportunities, feelings of empowerment, value, and support of well-being) and experiences of burnout as measured by the original 22-item Maslach Burnout Inventory (subscales: Emotional Exhaustion, Depersonalization, and Low Personal Accomplishment). Findings: Three perceived institutional characteristics were significantly associated with all three dimensions of burnout, particularly emotional exhaustion, which decreased with increasing perception of (a) empowerment to communicate professional needs, (b) feeling valued for contributions to the department, and (c) department commitment to support faculty well-being. In multivariate logistic regression models, adjusted for gender identity and years since training, increased positive perceptions of these three institutional characteristics were associated with significantly lower odds of burnout. For example, for each unit increase along a 5-point rating scale in feeling empowered to communicate needs and feeling valued for contributions to the department, the odds of meeting cutoff scores for burnout were reduced by 78% (p =.002) to 84% (p =.002), respectively. Insights: Although much of the focus on addressing burnout in healthcare settings has been on promoting coping skills and building resilience at the individual level, our findings add to a growing literature documenting a significant role for institutional leadership in identifying and facilitating strategies to promote faculty well-being. Findings also support leadership's role for improving institutional climate via creating opportunities to increase faculty perceptions of empowerment and value in the department. [ABSTRACT FROM AUTHOR]

People who have cancer diagnoses often need care throughout their lives through all stages of their illness. These stages include diagnosis, primary treatment, survivorship, and end of life. The management of people with cancer, now a common and chronic illness with long‐term survival improving, is complex, challenging, and rapidly changing. Rehabilitation for people with cancer diagnoses is a new specialty and is charged with providing care throughout the trajectory of illness and wellness to maximize potential for function and mitigate disability. Rehabilitation interventions include the application of physical and occupational therapeutics, speech and language interventions, and physical medicine in order to help patients reach their individual goals and to promote life satisfaction. The Department of Rehabilitation in the Clinical Center of the National Institutes of Health has pioneered this field through research and clinical care models over the past 40 years. Staff of this department has supported clinical research investigators at the National Institutes of Health in their exploration of new treatments using chemotherapies, surgery, radiation, and psychosocial interventions. They have also engaged in research specific to rehabilitation to devise and improve functional outcome measures, design exercise interventions, devise orthotics, and prosthetic devices for adaptation to functional loss. Collectively, the staff has published widely in oncology textbooks and professional journals in order to share findings and improve the quality of cancer rehabilitation treatment across the continuum of care. [ABSTRACT FROM AUTHOR]

Telematic performance offers significant potential for musicians in remote communities to perform together, increasing access to the type of ensemble music-making that is commonplace in urban areas. This article presents a range of perspectives taken from interviews with participants in the Online Orchestra pilot performance. Participants highlight the significant potential of telematic performance to overcome the challenge of music-making in geographically remote communities. The feasibility of making music in latency-rich environments is corroborated, as is the importance of the conductor in telematic performance. Suggestions are given for the fine tuning of peripheral equipment, and a preference emerges for the more traditional and simple music commissioned by the project. [ABSTRACT FROM AUTHOR]

Naturopathic oncology is a relatively new and emerging field capable of providing professional integrative or alternative services to cancer patients. Foundational research is critical to identify topics in the clinical and research development of naturopathic oncology for future growth of the field. Study design. This study implements a modified Delphi protocol to develop expert consensus regarding ethics, philosophy, and research development in naturopathic oncology. Methods. The modified protocol implements a nomination process to select a panel of 8 physicians and to assist in question formulation. The protocol includes an in-person discussion of 6 questions with multiple iterations to maintain the concept of the Delphi methodology as well as a postdiscussion consensus survey. Results. The protocol identified, ranked, and established consensus for numerous themes per question. Underlying key topics include integration with conventional medicine, evidence-based medicine, patient education, patient safety, and additional training requirements for naturopathic oncologists. Conclusions. The systematic nomination and questioning of a panel of experts provides a foundational and educational resource to assist in clarification of clinical ethics, philosophy, and research development in the emerging field of naturopathic oncology. [ABSTRACT FROM AUTHOR]

PC2 (polycystin-2) forms a Ca2+-permeable channel in the cell membrane and its function is regulated by cytosolic Ca2+ levels. Mutations in the C-terminal tail of human PC2 (HPC2 Cterm) lead to autosomal dominant polycystic kidney disease. The HPC2 Cterm protein contains a Ca2+-binding site responsible for channel gating and function. To provide the foundation for understanding how Ca2+ regulates the channel through the HPC2 Cterm, we characterized Ca2+ binding and its conformational and dynamic responses within the HPC2 Cterm. By examining hydrogen-deuterium (H-D) exchange profiles, we show that part of the coiled-coil domain in the HPC2 Cterm forms a stable helix bundle regardless of the presence of Ca2+. The HPC2 L1EF construct contains the Ca2+-binding EF-hand and the N-terminal linker 1 region without the downstream coiled coil. We show that the linker stabilizes the Ca2+-bound conformation of the EF-hand, thus enhancing its Ca2+-binding affinity to the same level as the HPC2 Cterm. In comparison, the coiled coil is not required for the high-affinity binding. By comparing the conformational dynamics of the HPC2 Cterm and HPC2 L1EF with saturating Ca2+, we show that the HPC2 Cterm and HPC2 L1EF share a similar increase in structural stability upon Ca2+ binding. Nevertheless, they have different profiles of H–D exchange under non-saturating Ca2+ conditions, implying their different conformational exchange between the Ca2+-bound and -unbound states. The present study, for the first time, provides a complete map of dynamic responses to Ca2+-binding within the full-length HPC2 Cterm. Our results suggest mechanisms for functional regulation of the PC2 channel and PC2's roles in the pathophysiology of polycystic kidney disease. [ABSTRACT FROM AUTHOR]

Analyzing data from a fifteen-year follow-up survey of high school students originally surveyed in 1957-58 and resurveyed in 1973-74, this paper examines the effects of the timing of marriage and first birth on subsequent childspacing, holding constant the effects of other variables that may be sources of spuriousness. The results suggest that age at first marriage has a causal effect on the occurrence of a short first birth interval and that age at first marriage and premarital pregnancy interact in their effect on the occurrence of a short second birth interval. Age at first marriage has no causal effect on the spacing of the second birth for those whose first child was maritally conceived. The spacing of the first birth, however, appears to have a causal effect on the spacing of the second. [ABSTRACT FROM AUTHOR]

Context. Cardiac glycosides of plant origin are implicated in toxic ingestions that may result in hospitalization and are potentially lethal. The utility of commonly available digoxin serum assays for detecting foxglove and oleander ingestion has been demonstrated, but no studies have evaluated the structurally similar convallatoxin found in Convallaria majalis (lily of the valley) for rapid laboratory screening, nor has digoxin immune Fab been tested as an antidote for this ingestion. Objective. We aimed to (1) evaluate multiple digoxin assays for cross-reactivity to convallatoxin, (2) identify whether convallatoxin could be detected in vivo at clinically significant doses, and (3) determine whether digoxin immune Fab could be an effective antidote to convallatoxin. Materials and methods. Cross-reactivities of purified convallatoxin and oleandrin with five common digoxin immunoassays were determined. Serum from mice challenged with convallatoxin was tested for apparent digoxin levels. Binding of convallatoxin to digoxin immune Fab was determined in vitro. Results. Both convallatoxin and oleandrin were detectable by a panel of commonly used digoxin immunoassays, but cross-reactivity was variable between individual assays. We observed measurable apparent digoxin levels in serum of convallatoxin intoxicated mice at sublethal doses. Convallatoxin demonstrated no binding by digoxin immune Fab. Conclusion. Multiple digoxin immunoassays detect botanical cardiac glycosides including convallatoxin and thus may be useful for rapid determination of severe exposures, but neutralization of convallatoxin by digoxin immune Fab is unlikely to provide therapeutic benefit. [ABSTRACT FROM AUTHOR]

Polycystin 2 (PC2) is a calcium-dependent calcium channel, and mutations to human PC2 (hPC2) are associated with polycystic kidney disease. The C-terminal tail of hPC2 contains 2 EF hand motifs, but only the second binds calcium. Here, we investigate whether these EF hand motifs serve as a calcium sensor responsible for the calcium dependence of PC2 function. Using NMR and bioinformatics, we show that the overall fold is highly conserved, but in evolutionarily earlier species, both EF hands bind calcium. To test whether the EF hand motif is truly a calcium sensor controlling PC2 channel function, we altered the number of calcium binding sites in hPC2. NMR studies confirmed that modified hPC2 binds an additional calcium ion. Single-channel recordings demonstrated a leftward shift in the calcium dependence, and imaging studies in cells showed that calcium transients were enhanced compared with wild-type hPC2. However, biophysics and functional studies showed that the first EF hand can only bind calcium and be functionally active if the second (native) calcium-binding EF hand is intact. These results suggest that the number and location of calcium-binding sites in the EF hand senses the concentration of calcium required for PC2 channel activity and cellular function. [ABSTRACT FROM AUTHOR]

A letter to the editor regarding acquired haemophilia A after stem cell transplant for sickle cell disease is presented.

The La protein binds the 3' ends of many newly synthesized noncoding RNAs, protecting these RNAs from nucleases and influencing folding, maturation, and ribonucleoprotein assembly. Although 3' end binding by La involves the N-terminal La domain and adjacent RNA recognition motif (RRM), the mechanisms by which La stabilizes diverse RNAs from nucleases and assists subsequent events in their biogenesis are unknown. Here we report that a conserved feature of La proteins, an intrinsically disordered C terminus, is required for the accumulation of certain noncoding RNA precursors and for the role of the Saccharomyces cerevisiae La protein Lhp1p in assisting formation of correctly folded pre-tRNA anticodon stems in vivo. Footprinting experiments using purified Lhp1p reveal that the C terminus is required to protect a pre-tRNA anticodon stem from chemical modification. Although the C terminus of Lhp1p is hypersensitive to proteases in vitro, it becomes protease-resistant upon binding pre-tRNAs, U6 RNA, or pre-5S rRNA. Thus, while high affinity binding to 3' ends requires the La domain and RRM, a conformationally flexible C terminus allows La to interact productively with a diversity of noncoding RNA precursors. We propose that intrinsically disordered domains adjacent to well characterized RNA-binding motifs in other promiscuous RNA-binding proteins may similarly contribute to the ability of these proteins to influence the cellular fates of multiple distinct RNA targets. [ABSTRACT FROM AUTHOR]

AV411 (ibudilast; 3-isobutyryl-2-isopropylpyrazolo-[1,5-a]pyridine) is an antiinflammatory drug that was initially developed for the treatment of bronchial asthma but which also has been used for cerebrovascular and ocular indications. It is a nonselective inhibitor of various phosphodiesterases (PDEs) and has varied antiinflammatory activity. More recently, AV411 has been studied as a possible therapeutic for the treatment of neuropathic pain and opioid withdrawal through its actions on glial cells. As described herein, the PDE inhibitor AV411 and its PDE-inhibition-compromised analog AV1013 inhibit the catalytic and chemotactic functions of the proinflammatory protein, macrophage migration inhibitory factor (MIF). Enzymatic analysis indicates that these compounds are noncompetitive inhibitors of the p-hydroxyphenylpyruvate (HPP) tautomerase activity of MIF and an allosteric binding site of AV411 and AV1013 is detected by NMR. The allosteric inhibition mechanism is further elucidated by X-ray crystallography based on the MIF/AV1013 binary and MIF/AV1013/HPP ternary complexes. In addition, our antibody experiments directed against MIF receptors indicate that CXCR2 is the major receptor for MIF-mediated chemotaxis of peripheral blood mononuclear cells. [ABSTRACT FROM AUTHOR]

Women with ovarian cancer have a low survival rate and develop resistance to chemotherapy, so new approaches to treatment are needed. We unexpectedly found administration of a replication-deficient adenovirus containing human growth hormone sequences (AdXGH) was beneficial in a mouse model of human ovarian cancer. Intraperitoneal injections of AdXGH prolonged median survival from a mean of 31 ± 1.2 to 40 ± 1.4 days in immunodeficient SCID mice given SKOV3.ip1 human ovarian cancer cells in the peritoneal cavity. Adenovirus containing human prolactin or del32-71growth hormone sequences had no effect. Repeated injection of growth hormone or implantation of tablets with sustained growth hormone release did not increase survival. Control mice had overlapping tumors throughout the peritoneal cavity and liver and frequent lung metastases 24 days after tumor cell injection. Mice that received two injections of AdXGH had no lung metastases. Mice that received four injections had no lung or liver metastases and peritoneal fibrosis. They did not survive longer than mice that received two injections, but they had enlarged livers with hepatocellular changes, indicating that a limitation of increasing the dose is liver toxicity. [ABSTRACT FROM AUTHOR]

The C-terminal cytoplasmic tail of polycystin-2 (PC2/TRPP2), a Ca2+-permeable channel, is frequently mutated or truncated in autosomal dominant polycystic kidney disease. We have previously shown that this tail consists of three functional regions: an EF-hand domain (PC2-EF, 720-797), a flexible linker (798-827), and an oligomeric coiled coil domain (828-895). We found that PC2-EF binds Ca2+ at a single site and undergoes Ca2+-dependent conformational changes, suggesting it is an essential element of Ca2+-sensitive regulation of PC2 activity. Here we describe the NMR structure and dynamics of Ca2+-bound PC2-EF. Human PC2-EF contains a divergent non-Ca2+-binding helix-loop-helix (HLH) motif packed against a canonical Ca2+-binding EF-hand motif. This HLH motif may have evolved from a canonical EF-hand found in invertebrate PC2 homologs. Temperature-dependent steady-state NOE experiments and NMR R1 and R2 relaxation rates correlate with increased molecular motion in the EF-hand, possibly due to exchange between apo and Ca2+-bound states, consistent with a role for PC2-EF as a Ca2+-sensitive regulator. Structure-based sequence conservation analysis reveals a conserved hydrophobic surface in the same region, which may mediate Ca2+-dependent protein interactions. We propose that Ca2+-sensing by PC2-EF is responsible for the cooperative nature of PC2 channel activation and inhibition. Based on our results, we present a mechanism of regulation of the Ca2+ dependence of PC2 channel activity by PC2-EF. [ABSTRACT FROM AUTHOR]

OCRL, whose mutations are responsible for Lowe syndrome and Dent disease, and INPP5B are two similar proteins comprising a central inositol 5-phosphatase domain followed by an ASH and a RhoGAP-like domain. Their divergent NH2-terminal portions remain uncharacterized. We show that the NH2-terminal region of OCRL, but not of INPP5B, binds clathrin heavy chain. OCRL, which in contrast to INPP5B visits late stage endocytic clathrin-coated pits, was earlier shown to contain another binding site for clathrin in its COOH-terminal region. NMR structure determination further reveals that despite their primary sequence dissimilarity, the NH2-terminal portions of both OCRL and INPP5B contain a PH domain. The novel clathrin-binding site in OCRL maps to an unusual clathrin-box motif located in a loop of the PH domain, whose mutations reduce recruitment efficiency of OCRL to coated pits. These findings suggest an evolutionary pressure for a specialized function of OCRL in bridging phosphoinositide metabolism to clathrin-dependent membrane trafficking. [ABSTRACT FROM AUTHOR]

A member of the family of hematopoietic cytokines human prolactin (hPRL) is a 23k kDa polypeptide hormone, which displays pH dependence in its structural and functional properties. The binding affinity of hPRL for the extracellular domain of its receptor decreases 500-fold over the relatively narrow, physiologic pH range from 8 to 6; whereas, the affinity of human growth hormone (hGH), its closest evolutionary cousin, does not. Similarly, the structural stability of hPRL decreases from 7.6 to 5.6 kcal/mol from pH 8 to 6, respectively, whereas the stability of hGH is slightly increased over this same pH range. hPRL contains nine histidines, compared with hGH's three, and they are likely responsible for hPRL's pH-dependent behavior. We have systematically mutated each of hPRL's histidines to alanine and measured the effect on pH-dependent global stability. Surprisingly, a vast majority of these mutations stabilize the native protein, by as much as 2-3 kcal/mol. Changes in the overall pH dependence to hPRL global stability can be rationalized according to the predominant structural interactions of individual histidines in the hPRL tertiary structure. Using double mutant cycles, we detect large interaction free energies within a cluster of nearby histidines, which are both stabilizing and destabilizing to the native state. Finally, by comparing the structural locations of hPRL's nine histidines with their homologous residues in hGH, we speculate on the evolutionary role of replacing structurally stabilizing residues with histidine to introduce pH dependence to cytokine function. [ABSTRACT FROM AUTHOR]

The self-association of human growth hormone (hGH) was investigated using 15N NMR relaxation. The investigation relies on the 15N R1 and R2 relaxation rates and the heteronuclear {1H}-15N NOEs of the backbone amide groups at multiple protein concentrations. It is shown that the rotational correlation time of hGH in solution depends strongly on its concentration, indicating a significant degree of self-association. The self-association is reversible and the monomers in the aggregates are noncovalently linked. Extrapolation of the relaxation data to zero concentration predicts a correlation time of 13.4 ns and a rotational diffusion anisotropy of 1.26 for monomeric hGH, in agreement with the rotational diffusion properties estimated by hydrodynamic calculations. Moreover, the extrapolation allows characterization of the backbone dynamics of monomeric hGH without interference from self-association phenomena, and it is found that hGH is considerably more flexible than originally thought. A concerted least-squares analysis of the 15N relaxations and their concentration dependence reveals that the self-association goes beyond a simple monomer-dimer equilibrium, and that tetramers or other multimeric states co-exist in fast exchange with the monomeric and dimeric hGH at sub-millimolar concentrations. Small changes in the 1H and 15N amide chemical shifts suggest that a region around the C-terminus is involved in the oligomer formation. Proteins 2008. © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

The GLUT4-regulating protein, TUG, functions to retain GLUT4-containing membrane vesicles intracellularly and, in response to insulin stimulation, releases these vesicles to the cellular exocytic machinery for translocation to the plasma membrane. As part of our on going effort to describe the molecular basis for TUG function, we have determined the tertiary structure and characterized the backbone dynamics for an N-terminal ubiquitin-like domain (TUG-UBL1) using NMR spectroscopy. A well-ordered conformation is observed for residues 10-83 of full-length TUG, and confirms a β-grasp or ubiquitin-like topology. Although not required for in vitro association with GLUT4, the functional role of the TUG-UBL1 domain has not yet been described. We undertook a limited literature review of similar N-terminal UBL domains and noted that a majority participate in protein-protein interactions, generally functioning as adaptor modules to physically associate the over all activity of the protein with a specific cellular process, such as the ubiquitin-proteasome pathway. In consistent fashion, TUG-UBL1 is not expected to participate in a covalent protein modification reaction as it lacks the characteristic C-terminal diglycine ('GG') motif required for conjugation to an acceptor lysine, and also lacks the three most common acceptor lysine residues involved in polyubiquitination. Additionally, analysis of the TUG-UBL1 molecular surface reveals a lack of conservation of the 'Ile-44 hydrophobic face' typically involved in ubiquitin recognition. Instead, we speculate on the possible significance of a concentrated area of negative electrostatic potential with increased backbone mobility, both of which are features suggestive of a potential protein-protein interaction site. [ABSTRACT FROM AUTHOR]

Spinocerebellar ataxia type 3 is a human neurodegenerative disease resulting from polyglutamine tract expansion. The affected protein, ataxin-3, which contains an N-terminal Josephin domain followed by tandem ubiquitin (Ub)-interacting motifs (UIMs) and a polyglutamine stretch, has been implicated in the function of the Ub proteasome system. NMR-based structural analysis has now revealed that the Josephin domain binds Ub and has a papain-like fold that is reminiscent of that of other deubiquitinases, despite primary sequence divergence but consistent with its deubiqutinating activity. Mutation of the catalytic Cys enhances the stability of a complex between ataxin-3 and polyubiquitinated proteins. This effect depends on the integrity of the UIM region, suggesting that the UIMs are bound to the substrate polyubiquitin during catalysis. We propose that ataxin-3 functions as a polyubiquitin chain-editing enzyme. [ABSTRACT FROM AUTHOR]

Dinitrophenol, a chemical currently used as an insecticide, is known to uncouple mitochondrial oxidative phosphorylation. A component of explosives, it has also been used in the past as a food coloring and clothing dye. In the 1930s, physicians prescribed it for weight loss, but this practice was discontinued when reports of cataracts, deaths, and other adverse outcomes came to light. We describe in our report the overdose and fatality of a teenager who purchased the product as a weight loss dietary supplement by mail order. We also describe a laboratory method that allowed postmortem determination of the dinitrophenol concentration in the victim's serum. Her death, despite prompt medical treatment, underscores the danger of dinitrophenol. The easy accessibility and apparent resurgent interest in dinitrophenol as a weight loss agent is extremely timely and troubling. [ABSTRACT FROM AUTHOR]

Information about drug residues and pharmacokinetic parameters in aquatic species is relatively sparse. In addition, it is difficult to rapidly compare data between studies due to differences in experimental conditions, such as water temperatures and salinity. To facilitate the study of aquatic species drug metabolism, we constructed a Fish Drug/Chemical Analysis Phish-Pharm (FDA-PP) database. This database consists of more than 400 articles that include data from 90 species (64 genera) of fish. Data fields include genus, species, water temperatures, the average animal weight, sample types analyzed, drug (or chemical) name, dosage, route of administration, metabolites identified, method of analysis, protein binding, clearance, volume of distribution in a central compartment (Vc) or volume of distribution at steady-state (Vd), and drug half-lives (t1/2). Additional fields list the citation, authors, title, and Internet links. The document will be periodically updated, and users are invited to submit additional data. Updates will be announced in future issues of The AAPS Journal. This database will be a valuable resource to investigators of drug metabolism in aquatic species as well as government and private organizations involved in the drug approval process for aquatic species. [ABSTRACT FROM AUTHOR]

Identifying correlates of immunity or susceptibility to disease promotes understanding of pathogenesis and development of diagnostic tools, treatments, and vaccines. There is evidence that type 1 cytokine responses are associated with protection against tuberculosis, and suppression of type 1, or switching to type 2 responses, with susceptibility, but this has not been studied prospectively. We studied a cohort of 631 HIV-1-infected Ugandan adults. At enrolment we performed whole blood cultures for type 1 (interferon [IFN]-γ, interleukin [IL]-2) and type 2/immunosuppressive (IL-5, IL-10) responses to mycobacterial antigens (purified protein derivative [PPD] and culture filtrate proteins [CFP]). The incidence of tuberculosis was not associated with IFN-γ responses, but was higher among participants with IL-2 responses (adjusted rate ratios [RR]: PPD 3.48; CFP 3.99; P < 0.001). For tuberculin skin test-positive participants, high incidence was also associated with an IL-10 response to PPD (adjusted RR 6.24, P = 0.03); for those with a BCG scar, high incidence was associated with positive IL-5 responses (adjusted RRs: PPD 3.64, P = 0.006; CFP 3.44, P = 0.04). The association with IL-2 production may reflect a response to tuberculous infection or to activating disease; the associations with IL-10 and IL-5 are in keeping with the expected role of immunosuppressive or type 2 cytokines. [Copyright &y& Elsevier]

There are several steps that must occur for secretory granules to form: (1) Secretory proteins that make up the dense cores of the granules must be concentrated; (2) membrane proteins necessary for granule function must accumulate in the correct location; and (3) inappropriate membrane proteins and excess membrane must be removed. Reversible aggregation of secretory granule proteins provides a mechanism for concentrating and sorting these proteins. There is specificity in the way secretory granule proteins are treated in cells that make granules. The specificity has been shown in some cases to occur after the aggregation process, so that granules containing different aggregates function differently. An explanation could be that a property of the aggregate, such as a surface motif, might influence the accumulation of membrane proteins necessary for granule function. Such a conclusion implies that the aggregates are not amorphous but have structure. Use of NMR spectroscopy to investigate changes in the environment of amino acid residues in secretory granule proteins as they form oligomers by using 15N relaxation times might provide a means to determine which residues are specifically involved in aggregation. [ABSTRACT FROM AUTHOR]

The article discusses studies on the impact of gut microbiota examination in the development of effective conventional cancer treatments and mentions the considerations in adjunctive cancer care.