Conformational activation and inhibition of von Willebrand factor by targeting its autoinhibitory module.

ABSTRACT: Activation of von Willebrand factor (VWF) is a tightly controlled process governed primarily by local elements around its A1 domain. Recent studies suggest that the O-glycosylated sequences flanking the A1 domain constitute a discontinuous and force-sensitive autoinhibitory module (AIM), although its extent and conformation remains controversial. Here, we used a targeted screening strategy to identify 2 groups of nanobodies. One group, represented by clone 6D12, is conformation insensitive and binds the N-terminal AIM (NAIM) sequence that is distal from A1; 6D12 activates human VWF and induces aggregation of platelet-rich plasma at submicromolar concentrations. The other group, represented by clones Nd4 and Nd6, is conformation sensitive and targets the C-terminal AIM (CAIM). Nd4 and Nd6 inhibit ristocetin-induced platelet aggregation and reduce VWF-mediated platelet adhesion under flow. A crystal structure of Nd6 in complex with AIM-A1 shows a novel conformation of both CAIM and NAIM that are primed to interact, providing a model of steric hindrance stabilized by the AIM as the mechanism for regulating GPIbα binding to VWF. Hydrogen-deuterium exchange mass spectrometry analysis shows that binding of 6D12 induces the exposure of the GPIbα-binding site in the A1 domain, but binding of inhibitory nanobodies reduces it. Overall, these results suggest that the distal portion of NAIM is involved in specific interactions with CAIM, and binding of nanobodies to the AIM could either disrupt its conformation to activate VWF or stabilize its conformation to upkeep VWF autoinhibition. These reported nanobodies could facilitate future studies of VWF functions and related pathologies.

Design, Synthesis, and Evaluation of New 1H-Benzo[d]imidazole Based PqsR Inhibitors as Adjuvant Therapy for Pseudomonas aeruginosa Infections.

Pseudomonas aeruginosa is one of the top priority pathogens that requires immediate attention according to the World Health Organisation (WHO). Due to the alarming shortage of novel antimicrobials, targeting quorum sensing (QS), a bacterial cell to cell signaling system controlling virulence, has emerged as a promising approach as an antibiotic adjuvant therapy. Interference with the pqs system, one of three QS systems in P. aeruginosa, results in reduction of bacterial virulence gene expression and biofilm maturation. Herein, we report a hit to lead process to fine-tune the potency of our previously reported inhibitor 1 (IC50 3.2 µM in P. aeruginosa PAO1-L), which led to the discovery of 2-(4-(3-((6-chloro-1-isopropyl-1H-benzo[d]imidazol-2-yl)amino)-2-hydroxypropoxy)phenyl)acetonitrile (6f) as a potent PqsR antagonist. Compound 6f inhibited the PqsR-controlled PpqsA-lux transcriptional reporter fusion in P. aeruginosa at low submicromolar concentrations. Moreover, 6f showed improved efficacy against P. aeruginosa CF isolates with significant inhibition of pyocyanin, 2-alkyl-4(1H)-quinolones production.

Assessing real-time positive subjective effects of alcohol using high-resolution ecological momentary assessment in risky versus light drinkers.

BACKGROUND: High-resolution ecological momentary assessment (HR-EMA) can assess acute alcohol responses during naturalistic heavy drinking episodes. The goal of this study was to use HR-EMA to examine drinking behavior and subjective responses to alcohol in risky drinkers (moderate-severe alcohol use disorder [MS-AUD], heavy social drinkers [HD]) and light drinkers (LD). We expected that risky drinkers would endorse greater alcohol stimulation and reward, with lower sedation, than LD, even when controlling for amount of alcohol consumed. METHODS: Participants (N = 112; 54% male, M ± SD age = 27.2 ± 4.2 years) completed smartphone-based HR-EMA during one typical alcohol drinking occasion and one non-alcohol-drinking occasion in their natural environment. Participants were prompted to complete next-day surveys that assessed drinking-related outcomes, study acceptability, and safety. RESULTS: HR-EMA prompt completion rates were excellent (92% and 89% for the alcohol and nonalcohol episodes, respectively). The MS-AUD group consumed the most alcohol with the highest estimated blood alcohol concentration (eBAC) by the end of the alcohol drinking episode (0.14 g/dL) versus LD (0.02 g/dL), with HD intermediate (0.10 g/dL). Relative to LD, MS-AUD and HD endorsed greater positive effects of alcohol (stimulation, liking, and wanting). CONCLUSIONS: This study is the first to use HR-EMA to measure and compare real-world acute alcohol responses across diverse drinker subgroups, including persons with MS-AUD. Results demonstrate that risky drinkers experience heightened pleasurable effects measured in real-time during natural-environment alcohol responses. Rather than drinking excessively to eventually achieve desirable subjective effects, risky drinkers show sensitivity to positive alcohol effects throughout a heavy drinking episode.

Courage to Quit® rolling group: Implementation in an urban medical center in primarily low-income Black smokers.

BACKGROUND: Continuous "rolling" tobacco group treatments may help reduce cessation disparities by increasing access among underserved people who smoke cigarettes. We evaluated the implementation of a rolling enrollment adaptation of an evidence-based tobacco treatment group intervention, Courage to Quit®-Rolling (CTQ®-R). METHODS: The 4-session CTQ®-R incorporating psychoeducation, motivational enhancement, and cognitive behavioral skills was evaluated by examining feasibility and preliminary program outcomes with a pre-post design using the SQUIRE method in a sample of 289 primarily low-income, Black people who smoke. Feasibility was measured by examining program retention. Paired t-tests evaluated changes in behavioral intentions and knowledge about smoking cessation and differences in average daily cigarettes smoked from first to last session attended. RESULTS: CTQ-R was feasible to implement in an urban medical center program enrolling primarily low-income Black people who smoke, with 52% attending at least 2 sessions and 24% completing the full program. Participants demonstrated improvements in knowledge of smoking cessation strategies and confidence in quitting (ps < .004). Preliminary effectiveness analyses showed a 30% reduction in average daily cigarette use, with group completers reporting greater reduction than non-completers. CONCLUSIONS: CTQ®-R is feasible and showed preliminary effectiveness for increasing knowledge about stop smoking skills and reducing cigarette smoking. IMPLICATIONS: A rolling enrollment smoking group treatment is feasible and may be effective among people who smoke who face historical and systemic barriers to tobacco treatment engagement. Evaluation in other settings and over longer periods of time is needed.

Inherited ADAMTS13 mutations associated with Thrombotic Thrombocytopenic Purpura: a short review and update.

ADAMTS13 is a plasma metalloprotease with the primary function of cleaving VWF to maintain hemostasis. Circulating ADAMTS13 is in the closed conformation until blood vessel injury triggers a VWF-dependant activation to the open active form of the protein. ADAMTS13 is a multi-domain protein with the domains broadly functioning to interact and cleave VWF or maintain global latency of ADAMTS13. Thrombotic Thrombocytopenic Purpura is a disease characterized by excessive thrombi formation in the microvasculature, diagnosis is made when ADAMTS13 activity is <10%. In the hereditary form, a variety of mutations are found throughout all domains of ADAMTS13, examples are given alongside details of each domain in this article. ADAMTS13 mutations can inhibit the binding and cleavage of VWF directly or indirectly through reduced secretion, leading to increased size of VWF multimers and platelet recruitment. Molecular characterization of ADAMTS13 may provide insight into the mechanisms of TTP to aid in both scientific and clinical research.

Cue Reactivity Effects of Heated Tobacco Product Use in Current, Former, and Never Smokers in the United States.

INTRODUCTION: Heated tobacco products (HTPs) share similar characteristics as combustible cigarettes and electronic nicotine delivery systems (ENDS) and thus may serve as cues for smoking and vaping. While HTP familiarity is low in the United States, HTPs may be perceived as a less harmful alternative to cigarettes. AIMS AND METHODS: The present study examined if visual exposure to HTP use influenced cigarette and e-cigarette craving in a large national sample of adults with varied smoking patterns. Current, former, and never cigarette smokers (N = 515; Mage = 40) were recruited from online crowd-sourcing panels throughout the United States from January to April 2020. Participants completed surveys before and after watching a video depicting the use of an HTP, cigarette, or bottled water. Main outcomes were changes in cigarette craving after exposure to the video cue. Secondary outcomes included changes in e-cigarette craving. RESULTS: Relative to the water cue, the HTP and cigarette cues increased cigarette craving in current smokers. The HTP cue also increased e-cigarette craving (desire for a mod or vape pen and JUUL) across all subgroups. CONCLUSIONS: Current smokers demonstrated cue reactivity to the use of an HTP as they reported increases in both cigarette and e-cigarette craving after exposure. All smoking groups reported e-cigarette reactivity to the HTP cue. As HTPs gain traction globally, it is crucial to consider how their use may influence active users and passive viewers to inform future health policies. IMPLICATIONS: Noncombustible nicotine delivery systems are known cues for cigarette smoking and e-cigarette vaping, and this study examined whether relatively novel heated tobacco products (HTPs) may also act as a smoking or vaping cue in adults across varied smoking backgrounds. Results showed that passive exposure to HTP use increased desire for both a cigarette and an e-cigarette in current smokers and also increased desire for an e-cigarette in former and nonsmokers. Thus given its similarity to smoking and vaping, HTP use may affect passive observers and could play a role in perpetuating the dual use of cigarettes and vape products.

Hearing loss increases with size but not site of tympanic membrane perforation in Aboriginal Australian children in remote locations.

Objective: To investigate the effect of size, site, and activity of tympanic membrane (TM) perforation on hearing loss (HL) in Aboriginal and Torres Strait Islander (ATSI) children. Design: Observational study. Methodology: Children aged 5-18 years who identified as ATSI at seven Anangu community schools within the Anangu Pitjantjatjara Yankunytjatjara Lands and Maralinga Lands of South Australia underwent 4-frequency pure-tone audiometry (0.5, 1, 2, and 4 kHz) and video-otoscopy (VO). VO data was reviewed by surgeons for a middle ear diagnosis and VO files with TM perforations were then classified by perforation site (AS, AI, PS, PI, A, P, I) and size (<25%, 25%-50%, 50%-75%, or 75%-100%). Results: Five hundred seventy-five VO files with matching audiological data were obtained. Active perforations (35 dBHL; 28-44 IQR) demonstrated greater HL than inactive perforations (31 dBHL; 29-39 IQR) p = .0029. For inactive perforations there was a significant difference between <25% and all larger perforations (p < .0001) whereas for active perforations the significance changed to between <25% (p < .0001) and 25%-50% (p < .05) when compared to larger perforations. When perforation site was compared within all size/activity groups, no statistically different findings were identified. In all analyses, findings did not change when individual frequencies were compared to 4-frequency pure-tone average dBHL. Conclusion: In ATSI children from remote communities, HL is greater in ears with larger perforations and active middle ear disease but there was no relationship between perforation site and HL.Level of evidence: Level 4.

The effect of acute alcohol intoxication on alcohol cue salience: An event-related brain potential study.

OBJECTIVE: Alcohol cue salience is considered core to the broader understanding of drinking behaviors. In the present research, we sought to build the knowledge of alcohol cue salience by exploring P3 responses to alcohol images among social drinkers within a large-scale alcohol-administration study. METHOD: Participants (N = 246) were randomly assigned to receive either a moderate dose of alcohol (target BAC = .08%) or a nonalcoholic control beverage. Following beverage administration, participants engaged in image-viewing tasks while EEG was recorded. We examined the impact of alcohol on the amplitude of P3 responses to pictures of alcoholic versus nonalcoholic beverages, exploring both beverage-manipulation and individual-difference moderators of these effects. RESULTS: Results revealed a significant effect of acute alcohol intoxication on P3 responses across stimulus types, with the overall amplitude of P3 being significantly smaller among participants consuming alcohol versus a nonalcoholic beverage. In addition, results revealed a significant main effect of image type, such that P3 amplitude was larger for alcohol images compared to nonalcohol images. No interactions emerged between stimulus type and beverage condition or stimulus type and AUD risk level. CONCLUSIONS: With the aim of better understanding the potential influence of the broader context on responses to individual cues, the present study examined the perceived salience of alcohol cues within a drinking setting. Findings provide evidence for alcohol cue salience that is both robust and also widespread across drinkers. More generally, the present study's findings may offer new directions for understanding neurocognitive processes of alcohol cue salience across contexts. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Parent's anxiety links household stress and young children's behavioral dysregulation.

Young children rely heavily on their caregivers to gain information about the environment, especially during times of duress. Therefore, considering parental assessments of behavior in the context of stressful environments may better facilitate our understanding of the longstanding association between early environmental stressors and changes in child behavior and physiology. Confirming many previous reports, a higher degree of household stress exposure was associated with elevated mental health symptoms in 2- to 6-year-old children (N = 115; anxiety and externalizing behaviors), which were verified in a subset of children with laboratory-based behaviors (N = 46). However, these associations were mediated by parental anxiety symptoms, which were also associated with increased cortisol levels in children. A closer look at the stressors indicated that it was the adult-targeted, and not the child-targeted, stressors that correlated most with children's behavior problems. These results highlight the importance of considering the mediating effect of parents, when examining associations between household stress and young children's behavioral development.

Optimizing the Interaction of Exercise Volume and Metformin to Induce a Clinically Significant Reduction in Metabolic Syndrome Severity: A Randomised Trial.

Insulin resistance is a central mediating factor of the metabolic syndrome (MetS), with exercise training and metformin proven antidotes to insulin resistance. However, when the two therapies are combined there is conflicting data regarding whether metformin blunts or improves exercise training-induced adaptations. The volume of exercise (duration, intensity, and frequency) on the interaction of exercise training and metformin has yet to be investigated. The aim of this study is therefore to explore the impact of a combination of different exercise volumes and metformin on MetS severity. This is a secondary analysis of data from one of the sites of the 'Exercise in Prevention of Metabolic Syndrome' (EX-MET) study. Ninety-nine adults with MetS were randomized into a 16-week exercise program completing either: (i) moderate-intensity continuous training (MICT) at 60-70% of peak heart rate (HRpeak) for 30 min/session (n = 34, 150 min/week); (ii) high-volume high-intensity interval training (HIIT) consisting of 4 × 4 min bouts at 85-95% HRpeak, interspersed with 3 min of active recovery at 50-70% HRpeak (n = 34, 38 min/session, 114 min/week); or (iii) low volume HIIT, 1 × 4 min bout of HIIT at 85-95% HRpeak (n = 31, 17 min/session, 51 min/week). Metformin intake was monitored and recorded throughout the trial. MetS severity was calculated as z-scores derived from MetS risk factors assessed at pre- and post-intervention. Sixty-five participants had complete pre- and post-intervention data for MetS z-score, of which 18 participants (28%) were taking metformin. Over the 16-week intervention, a similar proportion of participants clinically improved MetS severity (Δ ≥ -0.87) with metformin (8/18, 44%) or without metformin (23/47, 49%) (p = 0.75). While there were no between-group differences (p = 0.24), in those who did not take metformin low-volume HIIT had more likely responders (10/15, 67%) compared to MICT (6/16, 38%) and high-volume HIIT (7/16, 44%). In those taking metformin, there was a lower proportion of participants who clinically improved MetS severity following high-volume HIIT (1/6, 17%) compared to MICT (2/4, 50%) and low-volume HIIT (5/8, 63%), but with no between-group difference (p = 0.23). Moreover, in those who performed high-volume HIIT, there was a statistically significantly higher proportion (p = 0.03) of likely non-responders with improved MetS severity in participants taking metformin (4/6, 67%) compared to those not taking metformin (3/16, 19%). In individuals with MetS, the effect of high volume HIIT on MetS severity may be blunted in those taking metformin. These findings need to be confirmed in a larger study.

Free-Wilson Analysis of Comprehensive Data on Phosphoinositide-3-kinase (PI3K) Inhibitors Reveals Importance of N-Methylation for PI3Kδ Activity.

Phosphoinositide-3-kinase δ (PI3Kδ) is a critical regulator of cell growth and transformation and has been explored as a therapeutic target for a range of diseases. Through the exploration of the thienopyrimidine scaffold, we have identified a ligand-efficient methylation that leads to remarkable selectivity for PI3Kδ over the closely related isoforms. Interrogation through the Free-Wilson analysis highlights the innate selectivity the thienopyrimidine scaffold has for PI3Kδ and provides a predictive model for the activity against the PI3K isoforms.

Systemic hypersensitivity to fosaprepitant - A report of two cases.

Fosaprepitant is a widely administered antiemetic used mainly for moderately to highly emetogenic chemotherapy. Local injection site reactions are the most common type of infusion reaction reported from fosaprepitant. At our institution, two separate patients have experienced systemic hypersensitivity reactions to their infusions of fosaprepitant. We report a review of the literature and the details of these reactions.

Effectiveness of a lifestyle modification programme in weight maintenance in obese subjects after cessation of treatment with Orlistat.

OBJECTIVE: To examine the efficacy of a lifestyle modification programme in weight maintenance for obese subjects after cessation of treatment with Orlistat. METHODS: Fifty-five subjects with and without diabetes mellitus were randomized to a lifestyle modification programme or to usual care at the end of 6 months' treatment with Orlistat. The intervention programme was nutritionist led, consisting of components of dietary management, physical activity, peer group support and discussion using techniques of self-monitoring, stimulus control and cognitive restructuring. Anthropometric indices, body composition, basal metabolic rate, blood pressure, fasting glucose, glycosylated haemoglobin, lipid profile, 24-hour urinary albumin excretion, dietary intake, physical activity level, and quality of life were assessed before and after the intervention period. Results Subjects in the intervention group maintained their weight loss and favourable anthropometric, metabolic, dietary intake, physical activity and quality of life profiles, while most parameters deteriorated in the usual care group, being more marked in subjects with diabetes. The magnitude of weight gain was comparable to that lost during Orlistat treatment. CONCLUSION: A specially designed nutritionist-led lifestyle modification programme for obese subjects is effective in weight maintenance after treatment with Orlistat, in the absence of which the benefits of drug treatment were lost. The magnitude of the effect of lifestyle modification is comparable to that observed with Orlistat.

Molecular biology of Rh proteins and relevance to molecular medicine.

The Rhesus (Rh) blood group system is expressed by a pair of 12-transmembrane-domain-containing proteins, the RhCcEe and RhD proteins. RhCcEe and RhD associate as a Rh core complex that comprises one RhD/CcEe protein and most likely two Rh-associated glycoproteins (RhAG) as a trimer. All these Rh proteins are homologous and share this homology with two human non-erythroid proteins, RhBG and RhCG. All Rh protein superfamily members share homology and function in a similar manner to the Mep/Amt ammonium transporters, which are highly conserved in bacteria, plants and invertebrates. Significant advances have been made in our understanding of the structure and function of Rh proteins, as well as in the clinical management of Rh haemolytic disease. This review summarises our current knowledge concerning the molecular biology of Rh proteins and their role in transfusion and pregnancy incompatibility.

Ligation of CD47 mediates phosphatidylserine expression on erythrocytes and a concomitant loss of viability in vitro.

CD47 ligation has been shown to induce phosphatidylserine (PS) expression as part of a death pathway in nucleated blood cells. Using Annexin V binding assays we showed that ligation of CD47 with the specific CD47-binding peptide 41NK, anti-CD47 monoclonal antibody, and its natural ligand thrombospondin-1 also induced PS expression on enucleated erythrocytes. PS expression was associated with a concomitant loss of erythrocyte viability in vitro. Further characterisation of the CD47-PS signalling pathway on erythrocytes may help develop clinical strategies to further preserve the life of blood donations and improve our understanding of certain types of haemolytic anaemias.

Expression of phosphatidylserine (PS) on wild-type and Gerbich variant erythrocytes following glycophorin-C (GPC) ligation.

Glycophorin-C (GPC) is a 40 kDa glycoprotein expressed on erythrocytes and is a receptor for the malarial parasite Plasmodium falciparum to invade these cells. A link between GPC binding (ligation) and phosphatidylserine (PS) expression on erythrocytes has been suggested by its appearance on P. falciparum-infected erythrocytes. Phosphatidylserine expression has also been shown to be a marker of cellular death in a number of biological pathways including some in erythrocytes. Using Annexin V binding, we demonstrated that ligation of GPC with mouse mAb (BRIC-10) induced PS expression on normal erythrocytes. Phosphatidylserine exposure was prevented following tryptic digestion of intact erythrocytes. In addition, GPC variant phenotypes Yus (Delta exon 2) and Gerbich (Delta exon 3), which express a truncated extracellular domain, did not express PS following BRIC-10 binding, whereas PS was exposed on Ls(a) erythrocytes (duplication of exon 3). GPC ligation was also shown to result in a concomitant loss of erythrocyte viability in wild-type erythrocytes after 24 h in vitro. These results identify a potential pathway linking GPC to PS exposure on erythrocytes that may have a role in regulating red cell turnover. Further characterization of this pathway may also identify new targets for the treatment of P. falciparum malaria.

Association of the D8S282 marker near the lipoprotein lipase gene locus with systolic blood pressure in healthy Chinese subjects.

OBJECTIVE: To investigate the association between the marker D8S282 near the lipoprotein lipase (LPL) gene locus, and blood pressure, anthropometric and biochemical parameters in 229 healthy Chinese subjects. METHOD Genotyping was performed using an automated DNA sequencer and the Base ImageIR software. Eight different alleles were identified (272-286 bp) resulting in 15 genotypes in our population. We investigated the association between the common (28.8%) 278 bp allele and the anthropometric and biochemical parameters. RESULTS: In a tertile analysis, the frequency of the 278 bp allele increased linearly ( P = 0.003) with increasing systolic blood pressure (SBP). The relationship was most evident in the females ( n = 141); SBP was higher in homozygotes for the 278 bp allele (117 +/- 10 mmHg, = 12) than those without this allele (109 +/- 9 mmHg, = 77, 0.05) and was gene-dose dependent, and this difference was more significant after adjusting for age (P = 0.004). No relationship between the locus and the anthropometric or biochemical parameters investigated was observed. CONCLUSION: The D8S282 marker near the LPL gene locus contributes to the variance of SBP in healthy Hong Kong Chinese subjects, particularly in females.

The effect of orlistat-induced weight loss, without concomitant hypocaloric diet, on cardiovascular risk factors and insulin sensitivity in young obese Chinese subjects with or without type 2 diabetes.

BACKGROUND: We examined the weight-losing effect of orlistat treatment on insulin sensitivity and cardiovascular risk factors in a group of severely obese young Chinese patients with or without type 2 diabetes mellitus. METHODS: Obese patients with diabetes (n = 33) and obese nondiabetic patients (n = 27) were given orlistat, 120 mg 3 times daily, without a concomitant hypocaloric diet for 6 months (body mass index [calculated as weight in kilograms divided by the square of height in meter; kg/m2] range, 27.8-47.4). The efficacy measures were (1) insulin sensitivity indices derived from the homeostasis model assessment and a composite measure of whole-body insulin sensitivity index; (2) glycemic control; (3) cardiovascular risk factors, including anthropometry, blood pressure, lipid profiles, and albuminuria; and (4) body composition determined by dual-energy x-ray absorptiometry. RESULTS: At baseline, patients with diabetes had lower body mass index and percentage of body fat but higher waist-hip ratios and were more insulin resistant. Orlistat therapy reduced body weight, waist and hip circumferences, percentage of total body fat, blood pressure, fasting plasma glucose and lipid levels, albuminuria, and insulin sensitivity indices in both groups (all, P<.05). Despite less weight reduction, we found a greater percentage of reduction from baseline in glycosylated hemoglobin level (-11.6% vs -3.6%; P<.001), fasting plasma glucose level (-18.2% vs -5.0%; P<.001), and systolic blood pressure (-7.1% vs -3.1%; P =.02) in patients with diabetes. Obese subjects without diabetes had greater improvements in triglyceride levels, albuminuria, and the homeostasis model assessment (all, P<.01). CONCLUSION: Short-term orlistat treatment without the use of a hypocaloric diet significantly improved insulin sensitivity and cardiovascular risk profiles in severely obese Chinese patients with or without type 2 diabetes.