Targeted degradation of zDHHC-PATs decreases substrate S-palmitoylation.

Reversible S-palmitoylation of protein cysteines, catalysed by a family of integral membrane zDHHC-motif containing palmitoyl acyl transferases (zDHHC-PATs), controls the localisation, activity, and interactions of numerous integral and peripheral membrane proteins. There are compelling reasons to want to inhibit the activity of individual zDHHC-PATs in both the laboratory and the clinic, but the specificity of existing tools is poor. Given the extensive conservation of the zDHHC-PAT active site, development of isoform-specific competitive inhibitors is highly challenging. We therefore hypothesised that proteolysis-targeting chimaeras (PROTACs) may offer greater specificity to target this class of enzymes. In proof-of-principle experiments we engineered cell lines expressing tetracycline-inducible Halo-tagged zDHHC5 or zDHHC20, and evaluated the impact of Halo-PROTACs on zDHHC-PAT expression and substrate palmitoylation. In HEK-derived FT-293 cells, Halo-zDHHC5 degradation significantly decreased palmitoylation of its substrate phospholemman, and Halo-zDHHC20 degradation significantly diminished palmitoylation of its substrate IFITM3, but not of the SARS-CoV-2 spike protein. In contrast, in a second kidney derived cell line, Vero E6, Halo-zDHHC20 degradation did not alter palmitoylation of either IFITM3 or SARS-CoV-2 spike. We conclude from these experiments that PROTAC-mediated targeting of zDHHC-PATs to decrease substrate palmitoylation is feasible. However, given the well-established degeneracy in the zDHHC-PAT family, in some settings the activity of non-targeted zDHHC-PATs may substitute and preserve substrate palmitoylation.

Cysteine post-translational modifications regulate protein interactions of caveolin-3.

Caveolae are small flask-shaped invaginations of the surface membrane which are proposed to recruit and co-localize signaling molecules. The distinctive caveolar shape is achieved by the oligomeric structural protein caveolin, of which three isoforms exist. Aside from the finding that caveolin-3 is specifically expressed in muscle, functional differences between the caveolin isoforms have not been rigorously investigated. Caveolin-3 is relatively cysteine-rich compared to caveolins 1 and 2, so we investigated its cysteine post-translational modifications. We find that caveolin-3 is palmitoylated at 6 cysteines and becomes glutathiolated following redox stress. We map the caveolin-3 palmitoylation sites to a cluster of cysteines in its C terminal membrane domain, and the glutathiolation site to an N terminal cysteine close to the region of caveolin-3 proposed to engage in protein interactions. Glutathiolation abolishes caveolin-3 interaction with heterotrimeric G protein alpha subunits. Our results indicate that a caveolin-3 oligomer contains up to 66 palmitates, compared to up to 33 for caveolin-1. The additional palmitoylation sites in caveolin-3 therefore provide a mechanistic basis by which caveolae in smooth and striated muscle can possess unique phospholipid and protein cargoes. These unique adaptations of the muscle-specific caveolin isoform have important implications for caveolar assembly and signaling.

Palmitoylation of the pore-forming subunit of Ca(v)1.2 controls channel voltage sensitivity and calcium transients in cardiac myocytes.

Mammalian voltage-activated L-type Ca2+ channels, such as Ca(v)1.2, control transmembrane Ca2+ fluxes in numerous excitable tissues. Here, we report that the pore-forming α1C subunit of Ca(v)1.2 is reversibly palmitoylated in rat, rabbit, and human ventricular myocytes. We map the palmitoylation sites to two regions of the channel: The N terminus and the linker between domains I and II. Whole-cell voltage clamping revealed a rightward shift of the Ca(v)1.2 current-voltage relationship when α1C was not palmitoylated. To examine function, we expressed dihydropyridine-resistant α1C in human induced pluripotent stem cell-derived cardiomyocytes and measured Ca2+ transients in the presence of nifedipine to block the endogenous channels. The transients generated by unpalmitoylatable channels displayed a similar activation time course but significantly reduced amplitude compared to those generated by wild-type channels. We thus conclude that palmitoylation controls the voltage sensitivity of Ca(v)1.2. Given that the identified Ca(v)1.2 palmitoylation sites are also conserved in most Ca(v)1 isoforms, we propose that palmitoylation of the pore-forming α1C subunit provides a means to regulate the voltage sensitivity of voltage-activated Ca2+ channels in excitable cells.

Improving the Efficiency of Medication Reconciliation in Two Taiwanese Hospitals by Using the Taiwan National Health Insurance PharmaCloud Medication System.

Purpose: Medication reconciliation (MedRec) is a process to ensure complete and accurate communication of patient medication information throughout care transitions to prevent medication errors. Hospitals in Taiwan have stride to implement a universal protocol for MedRec. To establish a feasible protocol indigenously, the World Health Organization (WHO) protocol was incorporated with the Taiwan National Health Insurance (NHI) PharmaCloud patient medication profile. The efficiency and error detection capability of this modified protocol was evaluated in two hospitals. Methods: A prospective, non-randomized, unblinded, multicenter cohort study was conducted. Subjects were recruited among patients admitted for colorectal or orthopedic surgery with at least 4 or more chronic drugs. To obtain the best possible medication history (BPMH), the control group was conducted according to the WHO protocol, and the experimental group used the modified WHO protocol with the medication data from the PharmaCloud system. The time spent on the two protocols was recorded. Admission and discharge orders were reconciled against the BPMH to identify any discrepancies. Discrepancies were evaluated by appropriateness, prescribing intentions, and types of inappropriateness. The levels of potential harm were classified for inappropriate discrepancies. Results: The mean time to obtain BPMH in the control group was 34.3±10.8 minutes and in the experimental group 27.5±11.5 minutes (P = 0.01). The experimental group had more subjects with discrepancies (87.9%) than the control (58.3%) (p < 0.001). The discrepancies in both admission and discharge orders for the experimental group (84.5 and 67.2%) were higher than those of the control (47.9 and 37.5%). Many inappropriate discrepancies were classified as the potential harm of level 2 (77.8%). Conclusion: Through the establishment of BPMH with the medication data from the Taiwan NHI PharmaCloud, MedRec could be achieved with greater efficiency and error detection capability in both the admission and discharge order validation processes.

Dynamic but discordant alterations in zDHHC5 expression and palmitoylation of its substrates in cardiac pathologies.

S-palmitoylation is an essential lipid modification catalysed by zDHHC-palmitoyl acyltransferases that regulates the localisation and activity of substrates in every class of protein and tissue investigated to date. In the heart, S-palmitoylation regulates sodium-calcium exchanger (NCX1) inactivation, phospholemman (PLM) inhibition of the Na+/K+ ATPase, Nav1.5 influence on membrane excitability and membrane localisation of heterotrimeric G-proteins. The cell surface localised enzyme zDHHC5 palmitoylates NCX1 and PLM and is implicated in injury during anoxia/reperfusion. Little is known about how palmitoylation remodels in cardiac diseases. We investigated expression of zDHHC5 in animal models of left ventricular hypertrophy (LVH) and heart failure (HF), along with HF tissue from humans. zDHHC5 expression increased rapidly during onset of LVH, whilst HF was associated with decreased zDHHC5 expression. Paradoxically, palmitoylation of the zDHHC5 substrate NCX1 was significantly reduced in LVH but increased in human HF, while palmitoylation of the zDHHC5 substrate PLM was unchanged in all settings. Overexpression of zDHHC5 in rabbit ventricular cardiomyocytes did not alter palmitoylation of its substrates or overall cardiomyocyte contractility, suggesting changes in zDHHC5 expression in disease may not be a primary driver of pathology. zDHHC5 itself is regulated by post-translational modifications, including palmitoylation in its C-terminal tail. We found that in HF palmitoylation of zDHHC5 changed in the same manner as palmitoylation of NCX1, suggesting additional regulatory mechanisms may be involved. This study provides novel evidence that palmitoylation of cardiac substrates is altered in the setting of HF, and that expression of zDHHC5 is dysregulated in both hypertrophy and HF.

Palmitoylation regulates cellular distribution of and transmembrane Ca flux through TrpM7.

The bifunctional cation channel/kinase TrpM7 is ubiquitously expressed and regulates embryonic development and pathogenesis of several common diseases. The TrpM7 integral membrane ion channel domain regulates transmembrane movement of divalent cations, and its kinase domain controls gene expression via histone phosphorylation. Mechanisms regulating TrpM7 are elusive. It exists in two populations in the cell: at the cell surface where it controls divalent cation fluxes, and in intracellular vesicles where it controls zinc uptake and release. Here we report that TrpM7 is palmitoylated at a cluster of cysteines at the C terminal end of its Trp domain. Palmitoylation controls the exit of TrpM7 from the endoplasmic reticulum and the distribution of TrpM7 between cell surface and intracellular pools. Using the Retention Using Selective Hooks (RUSH) system, we demonstrate that palmitoylated TrpM7 traffics from the Golgi to the surface membrane whereas non-palmitoylated TrpM7 is sequestered in intracellular vesicles. We identify the Golgi-resident enzyme zDHHC17 and surface membrane-resident enzyme zDHHC5 as responsible for palmitoylating TrpM7 and find that TrpM7-mediated transmembrane calcium uptake is significantly reduced when TrpM7 is not palmitoylated. The closely related channel/kinase TrpM6 is also palmitoylated on the C terminal side of its Trp domain. Our findings demonstrate that palmitoylation controls ion channel activity of TrpM7 and that TrpM7 trafficking is dependant on its palmitoylation. We define a new mechanism for post translational modification and regulation of TrpM7 and other Trps.

Insights into the molecular basis of the palmitoylation and depalmitoylation of NCX1.

Catalyzed by zDHHC-PAT enzymes and reversed by thioesterases, protein palmitoylation is the only post-translational modification recognized to regulate the sodium/calcium exchanger NCX1. NCX1 palmitoylation occurs at a single site at position 739 in its large regulatory intracellular loop. An amphipathic ɑ-helix between residues 740-756 is a critical for NCX1 palmitoylation. Given the rich background of the structural elements involving in NCX1 palmitoylation, the molecular basis of NCX1 palmitoylation is still relatively poorly understood. Here we found that (1) the identity of palmitoylation machinery of NCX1 controls its spatial organization within the cell, (2) the NCX1 amphipathic ɑ-helix directly interacts with zDHHC-PATs, (3) NCX1 is still palmitoylated when it is arrested in either Golgi or ER, indicating that NCX1 is a substrate for multiple zDHHC-PATs, (4) the thioesterase APT1 but not APT2 as a part of NCX1-depalmitoylation machinery governs subcellular organization of NCX1, (5) APT1 catalyzes NCX1 depalmitoylation in the Golgi but not in the ER. We also report that NCX2 and NCX3 are dually palmitoylated, with important implications for substrate recognition and enzyme catalysis by zDHHC-PATs. Our results could support new molecular or pharmacological strategies targeting the NCX1 palmitoylation and depalmitoylation machinery.

An amphipathic α-helix directs palmitoylation of the large intracellular loop of the sodium/calcium exchanger.

The electrogenic sodium/calcium exchanger (NCX) mediates bidirectional calcium transport controlled by the transmembrane sodium gradient. NCX inactivation occurs in the absence of phosphatidylinositol 4,5-bisphosphate and is facilitated by palmitoylation of a single cysteine at position 739 within the large intracellular loop of NCX. The aim of this investigation was to identify the structural determinants of NCX1 palmitoylation. Full-length NCX1 (FL-NCX1) and a YFP fusion protein of the NCX1 large intracellular loop (YFP-NCX1) were expressed in HEK cells. Single amino acid changes around Cys-739 in FL-NCX1 and deletions on the N-terminal side of Cys-739 in YFP-NCX1 did not affect NCX1 palmitoylation, with the exception of the rare human polymorphism S738F, which enhanced FL-NCX1 palmitoylation, and D741A, which modestly reduced it. In contrast, deletion of a 21-amino acid segment enriched in aromatic amino acids on the C-terminal side of Cys-739 abolished YFP-NCX1 palmitoylation. We hypothesized that this segment forms an amphipathic α-helix whose properties facilitate Cys-739 palmitoylation. Introduction of negatively charged amino acids to the hydrophobic face or of helix-breaking prolines impaired palmitoylation of both YFP-NCX1 and FL-NCX1. Alanine mutations on the hydrophilic face of the helix significantly reduced FL-NCX1 palmitoylation. Of note, when the helix-containing segment was introduced adjacent to cysteines that are not normally palmitoylated, they became palmitoylation sites. In conclusion, we have identified an amphipathic α-helix in the NCX1 large intracellular loop that controls NCX1 palmitoylation. NCX1 palmitoylation is governed by a distal secondary structure element rather than by local primary sequence.

Cardiac abnormalities after aneurysmal subarachnoid hemorrhage: effects of ß-blockers and angiotensin-converting enzyme inhibitors.

BACKGROUND: Cardiac abnormalities attributed to adrenergic surge are common after aneurysmal subarachnoid hemorrhage. Prescribed medications that block adrenergic stimulation may suppress the onset of cardiopulmonary compromise in patients after aneurysmal subarachnoid hemorrhage. OBJECTIVES: To compare the incidence of early cardiac complications between patients who reported prescribed use of ß-blockers and/or angiotensin-converting enzyme inhibitors before aneurysmal subarachnoid hemorrhage and patients who did not. METHODS: A retrospective review of 254 adult patients after acute aneurysmal subarachnoid hemorrhage who were enrolled in an existing R01 study. Demographic data and history were obtained from patients'/proxies' reports and charts. Cardiac enzyme levels, 12-lead electrocardiograms, and chest radiographs were obtained on admission. Holter monitoring and echocardiograms were completed as a part of the R01 study. RESULTS: Patients reporting prescribed use of angiotensin-converting enzyme inhibitors or ß-blockers before aneurysmal subarachnoid hemorrhage had more ventricular and supraventricular ectopy on a Holter report than did patients who did not (P < .05). When age, race, sex, and injury (Fisher grade) were controlled for, patients reporting use of ß-blockers were 8 times more likely than others to have occasional to frequent ventricular ectopy (P = .02). CONCLUSION: No concrete evidence was found that exposure to adrenergic blockade before aneurysmal subarachnoid hemorrhage provides protection from neurocardiac injury.

Evaluation of chinese-herbal-medicine-induced herb-drug interactions: focusing on organic anion transporter 1.

The consumption of Chinese herbal medicines (CHMs) is increasing exponentially. Many patients utilize CHMs concomitantly with prescription drugs in great frequency. Herb-drug interaction has hence become an important focus of study. Transporter-mediated herb-drug interactions have the potential to seriously influence drug efficacy and toxicity. Since organic anion transporter 1 (OAT1) is crucial in renal active secretion and drug-drug interactions, the possibility of modulation of OAT1-mediated drug transport should be seriously concerned. Sixty-three clinically used CHMs were evaluated in the study. An hOAT1-overexpressing cell line was used for the in vitro CHMs screening, and the effective candidates were administered to Wistar rats to access renal hemodynamics. The regulation of OAT1 mRNA expression was also examined for further evidence of CHMs affecting OAT1-mediated transport. Among all the 63 CHMs, formulae Gui Zhi Fu Ling Wan (GZ) and Chia Wei Hsiao Yao San (CW) exhibited significant inhibitions on hOAT1-mediated [(3)H]-PAH uptake in vitro and PAH clearance and net secretion in vivo. Moreover, GZ showed concentration-dependent manners both in vitro and in vivo, and the decrease of rOAT1 mRNA expression indicated that GZ not only inhibited function of OAT1 but also suppressed expression of OAT1.

Correlation between ED symptoms and clinical outcomes in the patient with aneurysmal subarachnoid hemorrhage.

INTRODUCTION: Aneurysmal subarachnoid hemorrhage (aSAH) is a devastating neurologic insult often presenting to the emergency department as a headache. Recognition and prompt treatment are important to good outcomes. The purpose of this analysis was to examine the presentation of aSAH patients to the emergency department and determine whether presentation predicts length of stay or death. METHODS: This is a retrospective review of data gathered from 2 existing studies. Data from patients diagnosed with acute aSAH were reviewed for symptoms, clinical presentation, history, demographics, and laboratory results. Statistical analysis was completed by use of χ(2) and regression analysis. RESULTS: This sample of 193 adult aSAH patients confirmed headache as well as meningeal signs as the most frequent symptom on presentation to the emergency department, and this was cited as the most common reason for seeking medical treatment. Symptom presentation did not appear to affect length of stay; however, survival analysis showed that patients who presented with a Hunt and Hess grade greater than 3 along with bradycardia were 15.6 times more likely to die within the first month of aSAH. DISCUSSION: Although aSAH presentation remains the same, this analysis did find a correlation between poor clinical grade and bradycardia to be a significant predictor of death at 30 days. Additional study may help to determine whether any intervention could lessen this effect. Although patient diagnosis and referral from the community emergency department to a tertiary center were relatively quick, there was a wide window of time between patient recognition of symptoms and seeking medical treatment.

Cognitive behavioral symptom management intervention in patients with cancer: survival analysis.

PURPOSE: Associations between psychological factors and cancer survival have been under debate. We retrospectively explored the effect of the Cancer Care Intervention (CCI), an individually delivered cognitive behavioral symptom management intervention on survival in individuals with cancer. METHODS: Data were obtained from a randomized controlled trial (R01 CA79280; 1997-2003) that were originally designed to evaluate the CCI to reduce symptom severity in 237 individuals with solid tumors during their first course of chemotherapy. Participants were randomized into: (1) ten-contact, 20-week CCI plus usual care (n = 118) and (2) usual care only (n = 119). Survival data as of June 2009 were censored based upon Social Security Death Index. RESULTS: Participants were mostly female (73.4%), Caucasian (92.8%), and 59.6 ± 10.5 years old. Breast (38.8%) and lung (35%) cancer were the most common cancer types. At enrollment, 66.7% of the participants had cancers at stage III or greater. Overall mortality was 53.2% (126 of 237). The CCI did not significantly affect survival (median survival, CCI = 88 months; usual care = 53.3 months; log rank = 0.30, p = 0.58). Age, stage of cancer, and surgical removal of the tumor were the only factors significantly associated with survival. Post hoc analysis stratified by cancer site and gender (women with breast cancer, women with lung cancer, men with lung cancer, and others) showed no survival effect from the CCI. CONCLUSION: In this analysis, the CCI was not associated with better survival. For future research, studies exploring survival outcomes need to consider specific characteristics of each intervention and cancer type.

Antibodies to the envelope glycoprotein of human T cell leukemia virus type 1 robustly activate cell-mediated cytotoxic responses and directly neutralize viral infectivity at multiple steps of the entry process.

Infection of human cells by human T cell leukemia virus type 1 (HTLV-1) is mediated by the viral envelope glycoproteins. The gp46 surface glycoprotein binds to cell surface receptors, including heparan sulfate proteoglycans, neuropilin 1, and glucose transporter 1, allowing the transmembrane glycoprotein to initiate fusion of the viral and cellular membranes. The envelope glycoproteins are recognized by neutralizing Abs and CTL following a protective immune response, and therefore, represent attractive components for a HTLV-1 vaccine. To begin to explore the immunological properties of potential envelope-based subunit vaccine candidates, we have used a soluble recombinant surface glycoprotein (gp46, SU) fused to the Fc region of human IgG (sRgp46-Fc) as an immunogen to vaccinate mice. The recombinant SU protein is highly immunogenic and induces high titer Ab responses, facilitating selection of hybridomas that secrete mAbs targeting SU. Many of these mAbs recognize envelope displayed on the surface of HTLV-1-infected cells and virions and several of the mAbs robustly antagonize envelope-mediated membrane fusion and neutralize pseudovirus infectivity. The most potently neutralizing mAbs recognize the N-terminal receptor-binding domain of SU, though there is considerable variation in neutralizing proficiency of the receptor-binding domain-targeted mAbs. By contrast, Abs targeting the C-terminal domain of SU tend to lack robust neutralizing activity. Importantly, we find that both neutralizing and poorly neutralizing Abs strongly stimulate neutrophil-mediated cytotoxic responses to HTLV-1-infected cells. Our data demonstrate that recombinant forms of SU possess immunological features that are of significant utility to subunit vaccine design.

Role of endothelin-1 in human aneurysmal subarachnoid hemorrhage: associations with vasospasm and delayed cerebral ischemia.

BACKGROUND: Endothelin-1 (ET-1) is a potent vasoconstrictor implicated in the pathogenesis of vasospasm and delayed cerebral ischemia (DCI) in aneurysmal subarachnoid hemorrhage (aSAH) patients. The aim of this study was to investigate the relationship between cerebrospinal fluid (CSF) ET-1 levels and angiographic vasospasm and DCI. METHODS: Patients with aSAH were consented (n = 106). Cerebral vasospasm was determined by angiography. DCI was determined by transcranial Doppler (TCD) results and/or angiogram results with corresponding clinical deterioration. CSF ET-1 levels over 14 days after the initial insult was quantified by ELISA. ET-1 analysis included a group-based trajectory analysis and ET-1 exposure rate during 24, 48, and 72 h prior to, as well as 72 h post angiography, or clinical deterioration. RESULTS: Trajectory analysis revealed two distinct groups of subjects with 56% of patients in the low ET-1 trajectory group (mean at day 1 = 0.31 pg/ml; SE = 0.04; mean at day 14 = 0.41 pg/ml; SE = 0.15) and 44% of patients in the high ET-1 trajectory group (mean at day 1 = 0.65 pg/ml; SE = 0.08; mean at day 14 = 0.61 pg/ml; SE = 0.06). Furthermore, we observed that ET-1 exposure rate 72 h before angiography and clinical spasm was a significant predictor of both angiographic vasospasm and DCI, whereas, ET-1 exposure after angiography and clinical spasm was not associated with either angiographic vasospasm or DCI. CONCLUSION: Based on these results we conclude that ET-1 concentrations are elevated in a sub-group of patients and that the acute (72 h prior to angiography and clinical neurological deterioration), but not chronic, elevations in CSF ET-1 concentrations are indicative of the pathogenic alterations of vasospasm and DCI in aSAH patients.

Development and validation of a liquid chromatography-tandem mass spectrometry method for simultaneous quantification of p-aminohippuric acid and inulin in rat plasma for renal function study.

The first liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous quantification of p-aminohippuric acid and inulin, both typical biomarkers of kidney function. 5-(Hydroxymethyl)furfural, generated from inulin by acid and heat preparation, was used as an inulin substitute for the quantification. Acetaminophen was used as the internal standard. Solid-phase extraction was carried out with 5% methanol as the washing solution to optimize the retention of the analytes and to avoid obstruction of the orifice plate of the mass spectrometer caused by any unreacted inulin residue remaining from the sample preparation process. Chromatography separation was performed on a Symmetry C(18) column and a mobile phase composed of 2 mM ammonium formate and 0.1% formic acid in water (solvent A) and 2 mM ammonium formate and 0.1% formic acid in acetonitrile (solvent B) (30:70, v/v). Detection was performed with a triple-quadrupole tandem mass spectrometer using positive ion mode electrospray ionization in the multiple reaction monitoring mode. The selected transitions were m/z 195.2 → 120.2, 127.1 → 109.1, and 152.1 → 110.0 for p-aminohippuric acid, inulin [measured as 5-(hydroxymethyl)furfural], and acetaminophen, respectively. The linearity ranged from 10 to 140 µg/mL and from 100 to 1,400 µg/mL for p-aminohippurric acid and inulin (r > 0.99), respectively. The precisions and accuracies were all within 12 and 11% for the lower limit of quantification and quality control samples, respectively. This application was proven to be reliable and accurate and was successfully applied to a renal function study.

Predictors of delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage: a cardiac focus.

BACKGROUND: Myocardial injury after aneurysmal subarachnoid hemorrhage (aSAH) is associated with poor outcomes. Delayed cerebral ischemia (DCI) is also a complication of aSAH. We sought to determine whether (1) DCI could be predicted by demographics, aSAH severity/aneurysm location, or aSAH-associated myocardial injury (SAHMI), and (2) DCI is associated with increased mortality after aSAH. METHODS: Prospective longitudinal study of 149 aSAH subjects with definitive DCI evaluation, age 18-75 years, Hunt and Hess (HH) ≥ 3, and/or Fisher ≥ 2, and admitted to the Neurovascular ICU. DCI was defined by the presence of neurological deterioration accompanied by evidence of abnormal cerebral blood flow. RESULTS: Subjects were 48% DCI(+) and 52% DCI(-). DCI(+) subjects had more severe aSAH [HH (P = 0.002), Fisher (P = 0.004), admission Glasgow Coma Scale (P = 0.018)]. More DCI(+) subjects had pulmonary congestion than DCI(-) subjects (63 vs. 39%, P = 0.003). On echocardiogram, cardiac output (CO, liters per minute [LPM]) was significantly higher in DCI(+) than in DCI(-) subjects (6 ± 2 vs. 5 ± 1 LPM; P = 0.015). Multivariate analysis identified CO and Fisher grade as independent predictors of DCI (P = 0.02, 0.019). For each 1 LPM increase in CO, the odds of DCI increased by 1.5 (95% CI: 1.1-2.1). Fisher grade 4 increased the odds of DCI by 6.5 compared to Fisher grade 2 (95% CI: 1.6-25.8). After controlling for Fisher grade, CO remained an independent predictor of DCI (P = 0.02). Three-month mortality rate was not significantly different between DCI groups, P = 0.786. CONCLUSION: Elevated CO and Fisher grade are predictors of DCI after aSAH. However, prevention of DCI may not decrease mortality.

Women's awareness of ovarian cancer risks and symptoms.

BACKGROUND: The vast majority of cases of ovarian cancer are diagnosed at stage III or IV, and five-year survival rates after diagnosis at these stages are 71% and 31%, respectively. Although a consensus among researchers on the signs and symptoms of ovarian cancer has evolved over time, whether women themselves know them isn't clear. OBJECTIVE: To assess how well informed women ages 40 and older are of ovarian cancer symptoms and risk factors. METHODS: In 2006 the National Ovarian Cancer Coalition developed an online survey with a private research firm that asked respondents about their familiarity with ovarian cancer symptoms and risk factors. Women were also asked whether they thought the Papanicolaou test diagnosed ovarian cancer (a common misconception) and whether they had discussed ovarian cancer with a physician. If they had discussed the issue, they were asked who had initiated the conversation. Data from a convenience sample of 1,235 responses to the online survey were analyzed, using descriptive and comparative statistics. Respondents were categorized by age, education level, race or ethnicity, and whether or not they knew someone with ovarian cancer. Comparisons were made to determine whether demographic factors were associated with women's knowledge of specific symptoms and risk factors associated with ovarian cancer. RESULTS: Only 15% of respondents were familiar with ovarian cancer symptoms, and more than two-thirds incorrectly believed that the Papanicolaou test diagnoses the disease. Four out of five had never had a conversation with a physician about symptoms and risk factors; among these, more than half assumed that because their physician had not initiated such a discussion, ovarian cancer was "not an issue." Of the 19% of women who'd had such discussions, two-thirds had initiated them themselves. Respondents were more knowledgeable about risk factors; 59% correctly identified personal or family history of breast, ovarian, or colon cancer, and half of respondents correctly identified genetic predisposition, as risk factors. CONCLUSIONS: Awareness of ovarian cancer symptoms and risk factors among women in the general population is low. Ovarian cancer is often diagnosed at late stages, when cure is difficult; consequently, heightening women's awareness of risk factors and symptoms might help to reduce delays in diagnosis. Nurses should provide women with specific information on symptoms and risk factors in educating them on ovarian cancer.

Resistance to neutralization by antibodies targeting the coiled coil of fusion-active envelope is a common feature of retroviruses.

The human T-cell leukemia virus transmembrane glycoprotein (TM) is a typical class 1 membrane fusion protein and a subunit of the viral envelope glycoprotein complex. Following activation, the TM undergoes conformational transitions from a native nonfusogenic state to a fusion-active pre-hairpin intermediate that subsequently resolves to a compact trimer-of-hairpins or six-helix bundle. Disruption of these structural transitions inhibits membrane fusion and viral entry and validates TM as an anti-viral and vaccine target. To investigate the immunological properties of fusion-active TM, we have generated a panel of monoclonal antibodies that recognize the coiled-coil domain of the pre-hairpin intermediate. Antibody reactivity is highly sensitive to the conformation of the coiled coil as binding is dramatically reduced or lost on denatured antigen. Moreover, a unique group of antibodies are 100-1000-fold more reactive with the coiled coil than the trimer-of-hairpins form of TM. The antibodies recognize virally expressed envelope, and significantly, some selectively bind to envelope only under conditions that promote membrane fusion. Most importantly, many of the antibodies potently block six-helix bundle formation in vitro. Nevertheless, viral envelope was remarkably resistant to neutralization by antibodies directed to the coiled coil. The data imply that the coiled coil of viral envelope is poorly exposed to antibody during membrane fusion. We suggest that resistance to neutralization by antibodies directed to fusion-associated structures is a common property of retroviral TM and perhaps of other viral class I fusion proteins. These observations have significant implications for vaccine design.

An antibody that blocks human T-cell leukemia virus type 1 six-helix-bundle formation in vitro identified by a novel assay for inhibitors of envelope function.

Fusion of the viral and cellular membranes is a critical step in the infection of cells by the human T-cell leukemia virus type 1 (HTLV-1) and this process is catalysed by the viral envelope glycoproteins. During fusion, the transmembrane glycoprotein (TM) is thought to undergo a transition from a rod-like pre-hairpin conformation that is stabilized by a trimeric coiled coil to a more compact six-helix-bundle or trimer-of-hairpins structure. Importantly, synthetic peptides that interfere with the conformational changes of TM are potent inhibitors of membrane fusion and HTLV-1 entry, suggesting that the pre-hairpin motif is a valid target for antiviral therapy. Here, a stable, trimeric TM derivative that mimics the coiled-coil structure of fusion-active TM has been used to develop a plate-based assay to identify reagents that interfere with the formation of the six-helix bundle. The assay discriminates effectively between strong, weak and inactive peptide inhibitors of membrane fusion and has been used to identify a monoclonal antibody (mAb) that disrupts six-helix-bundle formation efficiently in vitro. The mAb is reactive with the C-helical region of TM, indicating that this region of TM is immunogenic. However, the mAb failed to neutralize HTLV-1 envelope-mediated membrane fusion, suggesting that, on native viral envelope, the epitope recognized by the mAb is obscured during fusion. This novel mAb will be of value in the immunological characterization of fusion-active structures of HTLV-1 TM. Moreover, the assay developed here will aid the search for therapeutic antibodies, peptides and small-molecule inhibitors targeting envelope and the HTLV-1 entry process.