Phosphorylation of USP20 on Ser334 by IRAK1 promotes IL-1ß-evoked signaling in vascular smooth muscle cells and vascular inflammation.

Reversible lysine-63 (K63) polyubiquitination regulates proinflammatory signaling in vascular smooth muscle cells (SMCs) and plays an integral role in atherosclerosis. Ubiquitin-specific peptidase 20 (USP20) reduces NFκB activation triggered by proinflammatory stimuli, and USP20 activity attenuates atherosclerosis in mice. The association of USP20 with its substrates triggers deubiquitinase activity; this association is regulated by phosphorylation of USP20 on Ser334 (mouse) or Ser333 (human). USP20 Ser333 phosphorylation was greater in SMCs of atherosclerotic segments of human arteries as compared with nonatherosclerotic segments. To determine whether USP20 Ser334 phosphorylation regulates proinflammatory signaling, we created USP20-S334A mice using CRISPR/Cas9-mediated gene editing. USP20-S334A mice developed ∼50% less neointimal hyperplasia than congenic WT mice after carotid endothelial denudation. WT carotid SMCs showed substantial phosphorylation of USP20 Ser334, and WT carotids demonstrated greater NFκB activation, VCAM-1 expression, and SMC proliferation than USP20-S334A carotids. Concordantly, USP20-S334A primary SMCs in vitro proliferated and migrated less than WT SMCs in response to IL-1ß. An active site ubiquitin probe bound to USP20-S334A and USP20-WT equivalently, but USP20-S334A associated more avidly with TRAF6 than USP20-WT. IL-1ß induced less K63-linked polyubiquitination of TRAF6 and less downstream NFκB activity in USP20-S334A than in WT SMCs. Using in vitro phosphorylation with purified IRAK1 and siRNA-mediated gene silencing of IRAK1 in SMCs, we identified IRAK1 as a novel kinase for IL-1ß-induced USP20 Ser334 phosphorylation. Our findings reveal novel mechanisms regulating IL-1ß-induced proinflammatory signaling: by phosphorylating USP20 Ser334, IRAK1 diminishes the association of USP20 with TRAF6 and thus augments NFκB activation, SMC inflammation, and neointimal hyperplasia.

The ubiquitination status of the glucagon receptor determines signal bias.

The pancreatic hormone glucagon activates the glucagon receptor (GCGR), a class B seven-transmembrane G protein-coupled receptor that couples to the stimulatory heterotrimeric G protein and provokes PKA-dependent signaling cascades vital to hepatic glucose metabolism and islet insulin secretion. Glucagon-stimulation also initiates recruitment of the endocytic adaptors, ßarrestin1 and ßarrestin2, which regulate desensitization and internalization of the GCGR. Unlike many other G protein-coupled receptors, the GCGR expressed at the plasma membrane is constitutively ubiquitinated and upon agonist-activation, internalized GCGRs are deubiquitinated at early endosomes and recycled via Rab4-containing vesicles. Herein we report a novel link between the ubiquitination status and signal transduction mechanism of the GCGR. In the deubiquitinated state, coupling of the GCGR to Gs is diminished, while binding to ßarrestin is enhanced with signaling biased to a ßarrestin1-dependent p38 mitogen activated protein kinase (MAPK) pathway. This ubiquitin-dependent signaling bias arises through the modification of lysine333 (K333) on the cytoplasmic face of transmembrane helix V. Compared with the GCGR-WT, the mutant GCGR-K333R has impaired ubiquitination, diminished G protein coupling, and PKA signaling but unimpaired potentiation of glucose-stimulated-insulin secretion in response to agonist-stimulation, which involves p38 MAPK signaling. Both WT and GCGR-K333R promote the formation of glucagon-induced ßarrestin1-dependent p38 signaling scaffold that requires canonical upstream MAPK-Kinase3, but is independent of Gs, Gi, and ßarrestin2. Thus, ubiquitination/deubiquitination at K333 in the GCGR defines the activation of distinct transducers with the potential to influence various facets of glucagon signaling in health and disease.

WHO-Point Prevalence Survey of Antibiotic Use Among Inpatients at a Core National Antimicrobial Consumption Network Site in North India: Findings and Implications.

Data on Point Prevalence Surveys (PPSs) in India are limited yet. We report findings of a PPS conducted in a core "National Antimicrobial Consumption Network site" under National Centre for Disease Control - WHO project "Point prevalence survey of antimicrobial consumption at healthcare facilities." A cross-sectional survey was conducted as per the "WHO methodology for PPS on antibiotic use in hospitals" in a tertiary care hospital in India in December 2021. Data were collected using predesigned and pretested questionnaire in separate hospital, ward, and patient forms. Eight hundred two inpatients (excluding ICUs) were covered out of whom 299 (37.3%) were on antibiotics with 11.7% receiving 3 or more antibiotics. Surgical prophylaxis (SP) (42.5%) and community acquired infections (32.8%) were the most common indications for antibiotic use. Of the patients, 92.5% received SP for more than 24 hrs. Most commonly prescribed antibiotics were penicillins with beta-lactamase inhibitors (22.3%). Of the total antibiotic prescriptions, 81.5% were from WHO essential medicines list and 12% from "not recommended" WHO AWaRe classification. Of the antibiotic prescriptions, 84.6% were parenteral. Few prescriptions complied with standard treatment guidelines (1.9%), documented indication for antibiotic use (11.6%), and stop/review date (4.4%) in notes. Double anaerobic cover accounted for 6.8% of the total prescriptions. Some identified areas for improvement were: formulation of hospital antibiotic guidelines, promoting culture of sending cultures, improvement in surgical antibiotic prophylaxis, decreasing use of antibiotic combinations and double anaerobic cover, fostering IV to oral switch of antibiotics, and ensuring effective communication among health care workers by documenting adequate information in medical notes.

Foodborne illness outbreak linked to a rural community kitchen in a rural area of Patiala District, Punjab, India, 2018.

BACKGROUND: In December 2018, an acute gastroenteritis outbreak was reported from Faridpur-Gujjran village, Patiala district, Punjab, India. OBJECTIVE: The objective of this study was to describe the epidemiology and risk factors of the outbreak and recommend prevention measures. METHODS: We conducted a descriptive study and a retrospective cohort study in the village. We defined a case as vomiting or ≥3 loose feces in 24 h plus abdominal pain and/or fever in a resident of the village during December 23-28, 2018. To find cases, we conducted a house-to-house survey; to identify risk factors, we conducted a retrospective cohort study. Fecal specimens were tested for enteric pathogens; water samples were tested for fecal contamination. We also interviewed food handlers. We compared attack rates by level of exposure. From the cohort study, we calculated risk ratios with 95% confidence intervals. RESULTS: From the 261 residents of the village, we identified 116 cases (attack rate 44%) and no deaths. The median age of affected persons was 27.5 years (range 0.5-80 years). The illness was associated with eating in a community kitchen of a temple during December 23-24, 2018. Eating mixed vegetables was associated with illness. We found no pathogens in fecal specimens. All three water samples showed coliform contamination. Cooked food had been left at room temperature before serving. CONCLUSION: Improper storage practices might have led to microbial proliferation of the food served. Our findings will help guide the enforcement of food safety policies for community kitchens.

Addressing Reemergence of Diphtheria among Adolescents through Program Integration in India.

We report a diphtheria outbreak mostly among children (median 12 years; range 4-26 years) of a religious minority in urban India. Case-fatality rate (15%, 19/124) was higher among unimmunized patients (relative risk 4.1, 95% CI 1.5-11.7). We recommend mandating and integrating immunization into school health programs to prevent reemergence.

Disease surveillance during a large religious mass gathering in India: The Prayagraj Kumbh 2019 experience.

BACKGROUND: Mass gathering (MG) events are associated with public health risks. During the period January 14 to March 4, 2019, Kumbh Mela in Prayagraj, India was attended by an estimated 120 million visitors. An onsite disease surveillance was established to identify and respond to disease outbreaks. METHODS: A health coordination committee was established for planning. Disease surveillance was prioritized and risk assessment was done to identify diseases/conditions based on epidemic potential, severity of illness, and reporting requirement under the International Health Regulations (IHR) of 2005. A daily indicator and event-based disease surveillance was planned. The indicator-based surveillance (IBS) manually and electronically recorded data from patient hospital visits and collected MG area water testing data to assess trends. The event-based surveillance (EBS) helped identify outbreak signals based on pre-identified event triggers from the media, private health facilities, and the food safety department. Epidemic intelligence was used to analyse the data and events to detect signals, verify alerts, and initiate the response. RESULTS: At Kumbh Mela, disease surveillance was established for 22 acute diseases/syndromes. Sixty-five health facilities reported 156 154 illnesses (21% of a total 738 526 hospital encounters). Among the reported illnesses, 95% (n = 148 834) were communicable diseases such as acute respiratory illness (n = 52 504, 5%), acute fever (n = 41 957, 28%), and skin infections (n = 27 094, 18%). The remaining 5% (n = 7300) were non-communicable diseases (injuries n = 6601, 90%; hypothermia n = 224, 3%; burns n = 210, 3%). Water samples tested inadequate for residual chlorine in 20% of samples (102/521). The incident command centre generated 12 early warning signals from IBS and EBS: acute diarrheal disease (n = 8, 66%), vector-borne disease (n = 2, 16%), vaccine-preventable disease (n = 1, 8%), and thermal event (n = 1, 8%). There were two outbreaks (acute gastroenteritis and chickenpox) that were investigated and controlled. CONCLUSIONS: This onsite disease surveillance imparted a public health legacy by successfully implementing an epidemic intelligence enabled system for early disease detection and response to monitor public health risks. Acute respiratory illnesses emerged as a leading cause of morbidity among visitors. Future MG events should include disease surveillance as part of planning and augment capacity for acute respiratory illness diagnosis and management.

Agonist-activated glucagon receptors are deubiquitinated at early endosomes by two distinct deubiquitinases to facilitate Rab4a-dependent recycling.

The glucagon receptor (GCGR) activated by the peptide hormone glucagon is a seven-transmembrane G protein-coupled receptor (GPCR) that regulates blood glucose levels. Ubiquitination influences trafficking and signaling of many GPCRs, but its characterization for the GCGR is lacking. Using endocytic colocalization and ubiquitination assays, we have identified a correlation between the ubiquitination profile and recycling of the GCGR. Our experiments revealed that GCGRs are constitutively ubiquitinated at the cell surface. Glucagon stimulation not only promoted GCGR endocytic trafficking through Rab5a early endosomes and Rab4a recycling endosomes, but also induced rapid deubiquitination of GCGRs. Inhibiting GCGR internalization or disrupting endocytic trafficking prevented agonist-induced deubiquitination of the GCGR. Furthermore, a Rab4a dominant negative (DN) that blocks trafficking at recycling endosomes enabled GCGR deubiquitination, whereas a Rab5a DN that blocks trafficking at early endosomes eliminated agonist-induced GCGR deubiquitination. By down-regulating candidate deubiquitinases that are either linked with GPCR trafficking or localized on endosomes, we identified signal-transducing adaptor molecule-binding protein (STAMBP) and ubiquitin-specific protease 33 (USP33) as cognate deubiquitinases for the GCGR. Our data suggest that USP33 constitutively deubiquitinates the GCGR, whereas both STAMBP and USP33 deubiquitinate agonist-activated GCGRs at early endosomes. A mutant GCGR with all five intracellular lysines altered to arginines remains deubiquitinated and shows augmented trafficking to Rab4a recycling endosomes compared with the WT, thus affirming the role of deubiquitination in GCGR recycling. We conclude that the GCGRs are rapidly deubiquitinated after agonist-activation to facilitate Rab4a-dependent recycling and that USP33 and STAMBP activities are critical for the endocytic recycling of the GCGR.

In-frame fusion of SUMO1 enhances ßarrestin2's association with activated GPCRs as well as with nuclear pore complexes.

Small ubiquitin like modifier (SUMO) conjugation or SUMOylation of ßarrestin2 promotes its association with the clathrin adaptor protein AP2 and facilitates rapid ß2 adrenergic receptor (ß2AR) internalization. However, disruption of the consensus SUMOylation site in ßarrestin2, did not prevent ßarrestin2's association with activated ß2ARs, dopamine D2 receptors (D2Rs), angiotensin type 1a receptors (AT1aRs) and V2 vasopressin receptors (V2Rs). To address the role of SUMOylation in the trafficking of ßarrestin and GPCR complexes, we generated and characterized a yellow fluorescent protein (YFP) tagged ßarrestin2-SUMO1 chimeric protein, which is resistant to de-SUMOylation. In HEK-293 cells, YFP-SUMO1 predominantly localized in the nucleus, whereas YFP-ßarrestin2 is cytoplasmic. YFP-ßarrestin2-SUMO1 in addition to being cytoplasmic, is localized at the nuclear membrane. Nonetheless, ßarrestin2-SUMO1 associated robustly with agonist-activated ß2ARs as evaluated by co-immunoprecipitation, confocal microscopy and bioluminescence resonance energy transfer (BRET). ßarrestin2-SUMO1 associated strongly with the D2R, which forms transient complexes with ßarrestin2. But, ßarrestin2-SUMO1 and ßarrestin2 showed equivalent binding with the V2R, which forms stable complexes with ßarrestin2. ßarrestin2 expression level directly correlated with the steady state levels of the unmodified form of RanGAP1, which upon SUMOylation associates with nuclear membrane. On the other hand, ßarrestin2-SUMO1 not only localized at the nuclear membrane, but also formed a macromolecular complex with RanGAP1. Taken together, our data suggest that SUMOylation of ßarrestin2 promotes its protein interactions at both cell and nuclear membranes. Furthermore, ßarrestin2-SUMO1 presents as a useful tool to characterize ßarrestin2 recruitment to GPCRs, which form transient and unstable complex with ßarrestin2.

The deubiquitinase ubiquitin-specific protease 20 is a positive modulator of myocardial ß1-adrenergic receptor expression and signaling.

Reversible ubiquitination of G protein-coupled receptors regulates their trafficking and signaling; whether deubiquitinases regulate myocardial ß1-adrenergic receptors (ß1ARs) is unknown. We report that ubiquitin-specific protease 20 (USP20) deubiquitinates and attenuates lysosomal trafficking of the ß1AR. ß1AR-induced phosphorylation of USP20 Ser-333 by protein kinase A-α (PKAα) was required for optimal USP20-mediated regulation of ß1AR lysosomal trafficking. Both phosphomimetic (S333D) and phosphorylation-impaired (S333A) USP20 possess intrinsic deubiquitinase activity equivalent to WT activity. However, unlike USP20 WT and S333D, the S333A mutant associated poorly with the ß1AR and failed to deubiquitinate the ß1AR. USP20-KO mice showed normal baseline systolic function but impaired ß1AR-induced contractility and relaxation. Dobutamine stimulation did not increase cAMP in USP20-KO left ventricles (LVs), whereas NKH477-induced adenylyl cyclase activity was equivalent to WT. The USP20 homolog USP33, which shares redundant roles with USP20, had no effect on ß1AR ubiquitination, but USP33 was up-regulated in USP20-KO hearts suggesting compensatory regulation. Myocardial ß1AR expression in USP20-KO was drastically reduced, whereas ß2AR expression was maintained as determined by radioligand binding in LV sarcolemmal membranes. Phospho-USP20 was significantly increased in LVs of wildtype (WT) mice after a 1-week catecholamine infusion and a 2-week chronic pressure overload induced by transverse aortic constriction (TAC). Phospho-USP20 was undetectable in ß1AR KO mice subjected to TAC, suggesting a role for USP20 phosphorylation in cardiac response to pressure overload. We conclude that USP20 regulates ß1AR signaling in vitro and in vivo Additionally, ß1AR-induced USP20 phosphorylation may serve as a feed-forward mechanism to stabilize ß1AR expression and signaling during pathological insults to the myocardium.

Encoding the ß-Arrestin Trafficking Fate of Ghrelin Receptor GHSR1a: C-Tail-Independent Molecular Determinants in GPCRs.

G-protein-coupled receptors (GPCRs) can bias signaling through distinct biochemical pathways that originate from G-protein/receptor and ß-arrestin/receptor complexes. Receptor conformations supporting ß-arrestin engagement depend on multiple receptor determinants. Using ghrelin receptor GHR1a, we demonstrate by bioluminescence resonance energy transfer and fluorescence microscopy a critical role for its second intracellular loop 2 (ICL2) domain in stabilizing ß-arrestin/GHSR1a core interactions and determining receptor trafficking fate. We validate our findings in ICL2 gain- and loss-of-function experiments assessing ß-arrestin and ubiquitin-dependent internalization of the CC chemokine receptor, CCR1. Like all CC and CXC subfamily chemokine receptors, CCR1 lacks a critical proline residue found in the ICL2 consensus domain of rhodopsin-family GPCRs. Our study indicates that ICL2, C-tail determinants, and the orthosteric binding pocket that regulates ß-arrestin/receptor complex stability are sufficient to encode a broad repertoire of the trafficking fates observed for rhodopsin-family GPCRs, suggesting they provide the essential elements for regulating a large fraction of ß-arrestin signaling bias.

USP20 (Ubiquitin-Specific Protease 20) Inhibits TNF (Tumor Necrosis Factor)-Triggered Smooth Muscle Cell Inflammation and Attenuates Atherosclerosis.

Objective- Signaling that activates NFκB (nuclear factor κB) in smooth muscle cells (SMCs) is integral to atherosclerosis and involves reversible ubiquitination that activates proteins downstream of proatherogenic receptors. Deubiquitination of these proteins is mediated by USP20 (ubiquitin-specific protease 20), among other deubiquitinases. We sought to determine whether USP20 activity in SMCs decreases atherosclerosis. Approach and Results- To address this question, we used male Ldlr-/- mice without (control) or with SMC-specific expression of murine USP20 (SMC-USP20-transgenic) or its dominant-negative (DN; C154S/H643Q) mutant (SMC-DN-USP20-transgenic). Before the appearance of intimal macrophages, NFκB activation in aortic medial SMCs was greater in SMC-DN-USP20-transgenic than in control mice. After 16 weeks on a Western diet, SMC-DN-USP20-transgenic mice had 46% greater brachiocephalic artery atheroma area than control mice. Congruently, aortic atherosclerosis assessed en face was 21% greater than control in SMC-DN-USP20-transgenic mice and 13% less than control in SMC-USP20-transgenic mice. In response to TNF (tumor necrosis factor), SMCs from SMC-DN-USP20-transgenic mice showed ≈3-fold greater NFκB activation than control SMCs. Silencing USP20 in SMCs with siRNA (small interfering RNA) augmented NFκB activation by ≈50% in response to either TNF or IL-1ß (interleukin-1ß). Coimmunoprecipitation experiments revealed that USP20 associates with several components of the TNFR1 (TNF receptor-1) signaling pathway, including RIPK1 (receptor-interacting protein kinase 1), a critical checkpoint in TNF-induced NFκB activation and inflammation. TNF evoked ≈2-fold more RIPK1 ubiquitination in SMC-DN-USP20-transgenic than in control SMCs, and RIPK1 was deubiquitinated by purified USP20 in vitro. Conclusions- USP20 attenuates TNF- and IL-1ß-evoked atherogenic signaling in SMCs, by deubiquitinating RIPK1, among other signaling intermediates.

Manifold roles of ß-arrestins in GPCR signaling elucidated with siRNA and CRISPR/Cas9.

G protein-coupled receptors (GPCRs) use diverse mechanisms to regulate the mitogen-activated protein kinases ERK1/2. ß-Arrestins (ßArr1/2) are ubiquitous inhibitors of G protein signaling, promoting GPCR desensitization and internalization and serving as scaffolds for ERK1/2 activation. Studies using CRISPR/Cas9 to delete ßArr1/2 and G proteins have cast doubt on the role of ß-arrestins in activating specific pools of ERK1/2. We compared the effects of siRNA-mediated knockdown of ßArr1/2 and reconstitution with ßArr1/2 in three different parental and CRISPR-derived ßArr1/2 knockout HEK293 cell pairs to assess the effect of ßArr1/2 deletion on ERK1/2 activation by four Gs-coupled GPCRs. In all parental lines with all receptors, ERK1/2 stimulation was reduced by siRNAs specific for ßArr2 or ßArr1/2. In contrast, variable effects were observed with CRISPR-derived cell lines both between different lines and with activation of different receptors. For ß2 adrenergic receptors (ß2ARs) and ß1ARs, ßArr1/2 deletion increased, decreased, or had no effect on isoproterenol-stimulated ERK1/2 activation in different CRISPR clones. ERK1/2 activation by the vasopressin V2 and follicle-stimulating hormone receptors was reduced in these cells but was enhanced by reconstitution with ßArr1/2. Loss of desensitization and receptor internalization in CRISPR ßArr1/2 knockout cells caused ß2AR-mediated stimulation of ERK1/2 to become more dependent on G proteins, which was reversed by reintroducing ßArr1/2. These data suggest that ßArr1/2 function as a regulatory hub, determining the balance between mechanistically different pathways that result in activation of ERK1/2, and caution against extrapolating results obtained from ßArr1/2- or G protein-deleted cells to GPCR behavior in native systems.

G Protein-Coupled Receptor Signaling Through ß-Arrestin-Dependent Mechanisms.

ß-arrestin1 (or arrestin2) and ß-arrestin2 (or arrestin3) are ubiquitously expressed cytosolic adaptor proteins that were originally discovered for their inhibitory role in G protein-coupled receptor (GPCR) signaling through heterotrimeric G proteins. However, further biochemical characterization revealed that ß-arrestins do not just "block" the activated GPCRs, but trigger endocytosis and kinase activation leading to specific signaling pathways that can be localized on endosomes. The signaling pathways initiated by ß-arrestins were also found to be independent of G protein activation by GPCRs. The discovery of ligands that blocked G protein activation but promoted ß-arrestin binding, or vice-versa, suggested the exciting possibility of selectively activating intracellular signaling pathways. In addition, it is becoming increasingly evident that ß-arrestin-dependent signaling is extremely diverse and provokes distinct cellular responses through different GPCRs even when the same effector kinase is involved. In this review, we summarize various signaling pathways mediated by ß-arrestins and highlight the physiologic effects of ß-arrestin-dependent signaling.

Characterization of an Indole-3-Acetamide Hydrolase from Alcaligenes faecalis subsp. parafaecalis and Its Application in Efficient Preparation of Both Enantiomers of Chiral Building Block 2,3-Dihydro-1,4-Benzodioxin-2-Carboxylic Acid.

Both the enantiomers of 2,3-dihydro-1,4-benzodioxin-2-carboxylic acid are valuable chiral synthons for enantiospecific synthesis of therapeutic agents such as (S)-doxazosin mesylate, WB 4101, MKC 242, 2,3-dihydro-2-hydroxymethyl-1,4-benzodioxin, and N-[2,4-oxo-1,3-thiazolidin-3-yl]-2,3-dihydro-1,4-benzodioxin-2-carboxamide. Pharmaceutical applications require these enantiomers in optically pure form. However, currently available methods suffer from one drawback or other, such as low efficiency, uncommon and not so easily accessible chiral resolving agent and less than optimal enantiomeric purity. Our interest in finding a biocatalyst for efficient production of enantiomerically pure 2,3-dihydro-1,4-benzodioxin-2-carboxylic acid lead us to discover an amidase activity from Alcaligenes faecalis subsp. parafaecalis, which was able to kinetically resolve 2,3-dihydro-1,4-benzodioxin-2-carboxyamide with E value of >200. Thus, at about 50% conversion, (R)-2,3-dihydro-1,4-benzodioxin-2-carboxylic acid was produced in >99% e.e. The remaining amide had (S)-configuration and 99% e.e. The amide and acid were easily separated by aqueous (alkaline)-organic two phase extraction method. The same amidase was able to catalyse, albeit at much lower rate the hydrolysis of (S)-amide to (S)-acid without loss of e.e. The amidase activity was identified as indole-3-acetamide hydrolase (IaaH). IaaH is known to catalyse conversion of indole-3-acetamide (IAM) to indole-3-acetic acid (IAA), which is phytohormone of auxin class and is widespread among plants and bacteria that inhabit plant rhizosphere. IaaH exhibited high activity for 2,3-dihydro-1,4-benzodioxin-2-carboxamide, which was about 65% compared to its natural substrate, indole-3-acetamide. The natural substrate for IaaH indole-3-acetamide shared, at least in part a similar bicyclic structure with 2,3-dihydro-1,4-benzodioxin-2-carboxamide, which may account for high activity of enzyme towards this un-natural substrate. To the best of our knowledge this is the first application of IaaH in production of industrially important molecules.

Novel immunosuppressive agent caerulomycin A exerts its effect by depleting cellular iron content.

BACKGROUND AND PURPOSE: Recently, we have described the use of caerulomycin A (CaeA) as a potent novel immunosuppressive agent. Immunosuppressive drugs are crucial for long-term graft survival following organ transplantation and treatment of autoimmune diseases, inflammatory disorders, hypersensitivity to allergens, etc. The objective of this study was to identify cellular targets of CaeA and decipher its mechanism of action. EXPERIMENTAL APPROACH: Jurkat cells were treated with CaeA and cellular iron content, iron uptake/release, DNA content and deoxyribonucleoside triphosphate pool determined. Activation of MAPKs; expression level of transferrin receptor 1, ferritin and cell cycle control molecules; reactive oxygen species (ROS) and cell viability were measured using Western blotting, qRT-PCR or flow cytometry. KEY RESULTS: CaeA caused intracellular iron depletion by reducing its uptake and increasing its release by cells. CaeA caused cell cycle arrest by (i) inhibiting ribonucleotide reductase (RNR) enzyme, which catalyses the rate-limiting step in the synthesis of DNA; (ii) stimulating MAPKs signalling transduction pathways that play an important role in cell growth, proliferation and differentiation; and (iii) by targeting cell cycle control molecules such as cyclin D1, cyclin-dependent kinase 4 and p21(CIP1/WAF1) . The effect of CaeA on cell proliferation was reversible. CONCLUSIONS AND IMPLICATIONS: CaeA exerts its immunosuppressive effect by targeting iron. The effect is reversible, which makes CaeA an attractive candidate for development as a potent immunosuppressive drug, but also indicates that iron chelation can be used as a rationale approach to selectively suppress the immune system, because compared with normal cells, rapidly proliferating cells require a higher utilization of iron.

Gender differences in health care utilization among veterans with chronic pain.

BACKGROUND: Previous research reports that 48% of veterans regularly experience and express concern over pain. Outpatient service use is higher for veterans with pain than for veterans without pain. Our study objective was to identify differences in outpatient utilization between men and women veterans with chronic pain. METHODS: We identified all men and women veterans at the Durham Veterans Affairs Medical Center in fiscal year (FY) 2002 between the ages of 21 and 60 that had two visits for the same pain location at least 6 weeks apart as determined by ICD-9 coding. Men and women were age-matched at a 2:1 ratio. We then compared the number of outpatient visits between genders in FY 2003. RESULTS: We identified 406 female and 812 male veterans. The mean number of clinic visits for women was 25.2 (SD 30.2) and for men 17.6 (SD 24.1). After adjusting for multiple pain sites, psychiatric diagnoses, age, and comorbidities, women veterans had a 27% higher rate of outpatient visits than men (incidence rate ratio [RR] 1.27, 95% confidence [CI] 1.15 to 1.41). Specifically, women had higher rates of visits to primary care (RR 1.36, 95% CI 1.24 to 1.50), physical therapy (RR 1.67, 95% CI 1.20 to 2.33), and other clinics (RR 1.28, 95% CI 1.14 to 1.44), and had a higher rate of visits to address pain (RR 1.15, 95% CI 1.02 to 1.30) than men. CONCLUSIONS: This is the first study to examine gender differences in chronic pain and utilization in the veteran population. Women veterans with chronic pain may need more resources to adequately manage chronic pain conditions as well as associated comorbidities and psychiatric disease.

The impact of environmental tobacco smoke on women's risk of dying from heart disease: a meta-analysis.

OBJECTIVE: To review systematically and analyze the association between environmental tobacco smoke (ETS) exposure and the risk of dying from heart disease in women. METHODS: We searched the English-language literature using MEDLINE (1966-April 2004), CINAHL, PsychInfo, and bibliographies of selected studies. We included studies that specifically addressed the association of ETS and heart disease mortality in women and had adequate controls and retrievable risk estimates. We looked for either cohort studies or randomized controlled trials. Studies were evaluated independently by two of the authors. Nine cohort studies were finally selected for analysis. We estimated the summary relative risk (RR) and associated 95% confidence intervals (95% CI) using a random-effects model. RESULTS: Mean follow-up periods for these cohorts ranged from 6 to 39 years. Among non-smoking women, exposure to ETS was associated with a 15% increase in the risk of dying from heart disease compared with nonsmoking women not exposed to ETS (RR = 1.15, 95% CI 1.03-1.28, p < 0.05). CONCLUSIONS: Among nonsmoking women, exposure to passive smoke increases the risk of dying from heart disease. In accordance with the newly developed guidelines by the American Heart Association for prevention of cardiovascular disease (CVD) in women, we recommend counseling women on reducing or avoiding ETS exposure.