Active Recruitment Research Articles

Increased Circulating Extracellular Superoxide Dismutase Attenuates Platelet-Neutrophil Interactions.

Acute respiratory distress syndrome (ARDS) is a serious illness accounting for 10% of ICU admissions and high mortality of 31-45% with a paucity of pharmacologic treatment options. Dysregulated inflammation and oxidative stress are hallmark features of ARDS. We previously showed that transgenic mice expressing a naturally occurring polymorphism of the antioxidant enzyme extracellular superoxide dismutase (EC-SOD), are protected against Staphylococcus aureus (S. aureus) pneumonia, acute lung injury, and pulmonary neutrophilia. In this mouse strain, an R213G amino acid substitution leads to lower tissue binding affinity and elevated alveolar and plasma EC-SOD levels, though the redox-regulated mechanisms responsible for protection against S. aureus are not yet elucidated. Neutrophils are recruited to the areas of injury and inflammation, in part by activated platelets, which contain multiple redox-sensitive targets. Thus, we hypothesize that increased circulating EC-SOD due to the EC-SOD R213G variant protects against S. aureus pneumonia by reducing platelet activation and subsequent neutrophil recruitment to the lung. We demonstrate that, compared to WT mice with S. aureus pneumonia, platelet activation, formation of platelet-neutrophil aggregates (PNAs), and influx of neutrophils and PNAs into the lung are decreased in the infected R213G mice. Furthermore, pre-treatment with a MnTE-2-PyP SOD mimetic protects against S. aureus-induced platelet activation, pulmonary neutrophilia, and acute lung injury. Our data highlight the redox regulation of platelet activation as a driver of S. aureus-induced acute lung injury.

A CDSS based Integrated Pathway for Influenza Vaccination Across Primary and Secondary Care

Abstract Background In Italy, influenza vaccination coverage in people ≥ 65 years is unsatisfactory (56.7%), underlining the need to improve current vaccination strategies. The objectives of this study have been: to define a novel pathway to increase influenza vaccination adherence among ≥ 65 years population in the Lazio region; to provide a predictive estimate of the epidemiological and economic pathway’s potential impact. Methods A multidisciplinary working group (WG) featuring cross-sectoral expertise was created and WG periodic meetings were held to define the patient journey and its flow-chart representation. The pathway’s potential impact was assessed through epidemiological and economic indicators and scenario analyses. Results An integrated pathway across primary and secondary care was defined, based on the active patient in-Hospital recruitment and vaccination and enhanced by a Clinical Decision Support System relying on a digital algorithm to identify eligible patients. Assuming an increase of influenza vaccination coverage from the current rate of 60% (scenario 1) to 65% (scenario 2) in ≥ 65 years population in the Lazio region thanks to the pathway implementation, an increase of 8% in avoided influenza cases, influenza- or pneumonia-related hospitalizations and influenza-related outpatient visits was estimated with a relative increase in savings for hospitalizations and outpatients visits of up 2,367,310 euros. Setting the vaccination coverage at 70% (scenario 3), an increase of 16% in avoided influenza cases, hospitalizations and outpatient visits was estimated with a relative increase in savings for hospitalizations and outpatients’ visits of up to 4,833,259 euros. Conclusions Alongside offering a predictive estimate of the relevant pathway’s potential impact, both epidemiological and economic, this project, with its robust methodology, may serve as a scalable and transferable model for enhancing vaccination coverage at national and international level. Key messages • The proposed pathway, offering the option of receiving flu vaccination within the Hospital, supports the paradigm shift towards primary prevention pathways in secondary care settings. • Another relevant aspect of the integrated pathway is the adoption of an Artificial Intelligence tool to identify suitable patients and improve their recruitment and adherence to vaccination.

Strategies to improve recruitment to multimodal group programmes for obesity: a systematic review

Abstract Background The World Health Organization describes obesity as one of the greatest public health challenges in the 21st century. Possible therapies include multimodal (group) programmes with nutrition, exercise, and behavioural therapy elements. However, programme providers often have difficulties recruiting the target groups and motivating them to participate. This systematic review aimed to identify barriers and facilitators as well as strategies to improve recruitment. Methods We systematically searched five databases (Medline, CINAHL, Cochrane, PsycInfo, Web of Science). Based on predefined inclusion criteria, we selected primary studies of different study designs (e.g. [randomised] controlled trials, qualitative studies). We extracted barriers and facilitators as well as recruitment strategies into predefined tables, assessed the quality of the studies and summarised the results narratively. Results Of the 1,082 articles identified, 16 met the inclusion criteria. The main active recruitment strategies were writing directly to potential participants and referrals from health professionals. The most frequently reported passive strategies included media adverts, websites, flyers, social media, and word of mouth. The information on how many people approached actually participated was heterogeneous, which illustrates that recruitment depends on the setting/context and the people involved. Some studies came to contradictory results, e.g., regarding whether passive or active methods are more successful. In most cases, a combination of strategies was recommended. The studies reported numerous recruitment barriers, e.g., lack of staff time, lack of motivation, or fear of discrimination. Conclusions The review provides a broad overview of recruitment methods that can be applied depending on the framework conditions and target groups. The summary of possible barriers can serve as an overview of which aspects could be considered when designing or adapting a programme. Key messages • To improve recruitment to obesity programmes, a combination of several recruitment strategies, including active and passive methods, should be applied. • Results on the “effectiveness” of the strategies were inconclusive, which indicates that the success of methods depends on the context, setting and the people involved.

The impact of healthcare worker migration in source countries: Colombia, Indonesia, and Jordan

Abstract Background To address domestic shortages, high-income countries are increasingly recruiting healthcare workers from low- and middle-income countries. This practice is much debated, yet empirical evidence is rare. This study fills this knowledge gap by reporting high-level stakeholders’ perspectives on health system impacts of international migration and active recruitment of HCWs in Colombia, Indonesia, and Jordan. Methods We used a multiple case study methodology, based on qualitative methods integrated with information available in the published literature. Results All respondents decried a lack of robust and detailed data as a serious challenge in ascertaining their perspectives on impacts of healthcare workersmigration. Stakeholders described current emigration levels as not substantially aggravating existing healthcare workforce availability challenges. This is due to the fact that all three countries are faced with healthcare worker unemployment grounded in unwillingness to work in rural areas and/or overproduction of certain cadres. Respondents, however, pleaded against targeting very experienced and specialised individuals. While observing little harm of healthcare worker migration at present, stakeholders also noted few benefits such as brain gain, describing how various barriers to skill enhancement, return, and reintegration into the health system hamper in practice what may be possible in theory. Conclusions Improved availability of data on healthcare workers migration, including their potential return and reintegration into their country of origin’s health system, is urgently necessary to understand and monitor costs and benefits in dynamic national and international health labour markets. Our results imply that potential benefits of migration do not come into being automatically, but need in-country supportive policy, such as favourable reintegration policies or programs targeting engagement of the diaspora.

Ferroptosis-Mediated Inflammation Promotes Pulmonary Hypertension.

Mitochondrial dysfunction, characterized by impaired lipid metabolism and heightened reactive oxygen species generation, results in lipid peroxidation and ferroptosis. Ferroptosis is an inflammatory mode of cell death that promotes complement activation and macrophage recruitment. In pulmonary arterial hypertension (PAH), pulmonary arterial endothelial cells exhibit cellular phenotypes that promote ferroptosis. Moreover, there is ectopic complement deposition and inflammatory macrophage accumulation in the pulmonary vasculature. However, the effects of ferroptosis inhibition on these pathogenic mechanisms and the cellular landscape of the pulmonary vasculature are incompletely defined. Multiomics and physiological analyses evaluated how ferroptosis inhibition-modulated preclinical PAH. The impact of adeno-associated virus 1-mediated expression of the proferroptotic protein ACSL (acyl-CoA synthetase long-chain family member) 4 on PAH was determined, and a genetic association study in humans further probed the relationship between ferroptosis and pulmonary hypertension. Ferrostatin-1, a small-molecule ferroptosis inhibitor, mitigated PAH severity in monocrotaline rats. RNA-sequencing and proteomics analyses demonstrated ferroptosis was associated with PAH severity. RNA-sequencing, proteomics, and confocal microscopy revealed complement activation and proinflammatory cytokines/chemokines were suppressed by ferrostatin-1. In addition, ferrostatin-1 combatted changes in endothelial, smooth muscle, and interstitial macrophage abundance and gene activation patterns as revealed by deconvolution RNA-sequencing. Ferroptotic pulmonary arterial endothelial cell damage-associated molecular patterns restructured the transcriptomic signature and mitochondrial morphology, promoted the proliferation of pulmonary artery smooth muscle cells, and created a proinflammatory phenotype in monocytes in vitro. Adeno-associated virus 1-Acsl4 induced an inflammatory PAH phenotype in rats. Finally, single-nucleotide polymorphisms in 6 ferroptosis genes identified a potential link between ferroptosis and pulmonary hypertension severity in the Vanderbilt BioVU repository. Ferroptosis promotes PAH through metabolic and inflammatory mechanisms in the pulmonary vasculature.

Role of the Kölliker-Fuse/parabrachial complex in the generation of postinspiratory vagal and sympathetic nerve activities and their recruitment by hypoxemic stimuli in the rat.

In the rat, the activity of laryngeal adductor muscles, the crural diaphragm, and sympathetic vasomotor neurons is entrained to the postinspiratory (post-I) phase of the respiratory cycle, a mechanism thought to enhance cardiorespiratory efficiency. The identity of the central neurons responsible for transmitting respiratory activity to these outputs remains unresolved. Here we explore the contribution of the Kölliker-Fuse/parabrachial nuclei (KF-PBN) in the generation of post-I activity in vagal and sympathetic outputs under steady-state conditions and during acute hypoxemia, a condition that potently recruits post-I activity. In artificially ventilated, vagotomized, and urethane-anesthetized rats, bilateral KF-PBN inhibition by microinjection of the GABAA receptor agonist isoguvacine evoked stereotypical responses on respiratory pattern, characterized by a reduction in phrenic nerve burst amplitude, a modest lengthening of inspiratory time, and an increase in breath-to-breath variability, while post-I vagal nerve activity was abolished and post-I sympathetic nerve activity diminished. During acute hypoxemia, KF-PBN inhibition attenuated tachypneic responses and completely abolished post-I vagal activity while preserving respiratory-sympathetic coupling. Furthermore, KF-PBN inhibition disrupted the decline in respiratory frequency that normally follows resumption of oxygenation. These findings suggest that the KF-PBN is a critical hub for the distribution of post-I activities to vagal and sympathetic outputs and is an important contributor to the dynamic adjustments to respiratory patterns that occur in response to acute hypoxia. Although KF-PBN appears essential for post-I vagal activity, it only partially contributes to post-I sympathetic nerve activity, suggesting the contribution of multiple neural pathways to respiratory-sympathetic coupling.NEW & NOTEWORTHY Inhibition of neurons in the pontine Kölliker-Fuse/parabrachial complex (KF-PBN) differentially inhibited postinspiratory (post-I) activity in vagal and sympathetic outputs. The strong recruitment of post-I vagal activity that occurs in response to hypoxemia is selectively abolished by KF-PBN inhibition. This suggests that 1) post-I activity in vagal and sympathetic outputs may be generated by partially independent mechanisms and 2) neurons in the KF-PBN are a preeminent source of drive for the generation of eupneic post-I activity.

An acceptability and feasibility investigation of a community-based motor program for autistic children with moderate and high support needs

BackgroundGeneral motor impairments are ubiquitous in Autism and are positively correlated with autism symptom severity. This study assessed the feasibility and acceptability of a 13-week community-based motor program for autistic children with moderate and high support needs (MHS). MethodIn this exploratory single arm within-subject study, 10 autistic children with MHS, ages 4–10 years, completed a community-based motor program delivered in a one-on-one format by therapists. Feasibility was determined through recruitment, retention, attendance, enjoyment, engagement and useability of motor, physical activity (PA), and behavioural assessment tools. Thematic analysis evaluated post-intervention semi-structured parent interviews. ResultsHigh program attendance and retention were demonstrated, and therapists reported child engagement and enjoyment were high. Identified themes revealed parents perceived unexpected substantial positive impacts including improvements in social, motor, and behavioural outcomes. They highly valued both the program and the therapeutic relationships fostered within it. Although parents of the children with the higher support needs reported the most substantial improvements, these children were unable to score on motor assessments due to difficulties following instructions and off-task behaviours. Additionally, only four participants wore an accelerometer-based PA monitor. ConclusionsThis community-based motor program was considered feasible and acceptable by the children’s parents, and qualitative findings provided valuable insights into clinical practice for children with MHS. However, recruitment challenges and assessment tools being unfeasible for children with higher support needs led to low participant numbers. Future evaluations should explore alternative assessment measures and study designs, given the challenges associated with studying such a heterogenous and complex group.

Improving Recruitment Of Older Patients For Patient And Public Involvement And Engagement Work (PPIE): Ensuring A Representative Voice

Abstract Background Older people are under-represented in research, despite this demographic being the biggest users of healthcare resources. Service design needs to be patient-centred and therefore engaging this underserved group is crucial, albeit challenging. We describe a quality improvement project aimed at improving recruitment of patients into a local PPIE group in a perioperative geriatric service. Methods Clinicians opportunistically consent patients for the PPIE group during their perioperative geriatric clinic consultations. A baseline level of recruitment was established before implementing a series of PDSA cycles. The interventions were firstly, to adapt the paper clinic proforma making the consent section clearer, secondly to give weekly reminders to clinicians, and finally discussing PPIE with patients while they wait for their appointment. A focus group with clinicians was run using nominal group technique to explore barriers and potential solutions to recruitment. Results An average of three patients were being recruited per month from April 2022 to September 2023. Two weeks following the first intervention, no new patients were consented. Two weeks following the second intervention, this improved to two new patients. Following the third intervention, five new patients were recruited after two clinic sessions. From the focus group, the top three barriers to recruitment were forgetting, clinical responsibilities taking priority, and lack of awareness by new staff. The highest rated solution was utilising non-clinical staff to recruit patients in the waiting room. Conclusion During busy clinics, it is easy for clinicians to forget about issues not directly related to the patient in front of them. However, PPIE is vital in co-designing healthcare. There are both staff and patient-related barriers in recruiting and maintaining a PPIE group in older patients. From our project, we found a combination of passive (e.g. a tickbox proforma) and active recruitment by dedicated staff the best way to improve PPIE.

Ferroptosis Integrates Mitochondrial Derangements and Pathological Inflammation to Promote Pulmonary Hypertension.

Mitochondrial dysfunction, characterized by impaired lipid metabolism and heightened reactive oxygen species (ROS) generation, results in lipid peroxidation and ferroptosis. Ferroptosis is an inflammatory mode of cell death that promotes complement activation and macrophage recruitment. In pulmonary arterial hypertension (PAH), pulmonary arterial endothelial cells (PAEC) exhibit cellular phenotypes that promote ferroptosis. Moreover, there is ectopic complement deposition and inflammatory macrophage accumulation in the pulmonary vasculature. However, the effects of ferroptosis inhibition on these pathogenic mechanisms and the cellular landscape of the pulmonary vasculature are incompletely defined. Multi-omics and physiological analyses evaluated how ferroptosis inhibition modulated preclinical PAH. The impact of AAV1-mediated expression of the pro-ferroptotic protein ACSL4 on PAH was determined, and a genetic association study in humans further probed the relationship between ferroptosis and pulmonary hypertension (PH). Ferrostatin-1, a small-molecule ferroptosis inhibitor, mitigated PAH severity in monocrotaline rats. RNA-seq and proteomics analyses demonstrated ferroptosis was associated with PAH severity. RNA-seq, proteomics, and confocal microscopy revealed complement activation and pro-inflammatory cytokines/chemokines were suppressed by ferrostatin-1. Additionally, ferrostatin-1 combatted changes in endothelial, smooth muscle, and interstitial macrophage abundance and gene activation patterns as revealed by deconvolution RNA-seq. Ferroptotic PAEC damage associated molecular patterns restructured the transcriptomic signature, mitochondrial morphology, and promoted proliferation of pulmonary artery smooth muscle cells, and created a pro-inflammatory phenotype in monocytes in vitro. AAV1-Acsl4 induced an inflammatory PAH phenotype in rats. Finally, single-nucleotide polymorphisms in six ferroptosis genes identified a potential link between ferroptosis and PH severity in the Vanderbilt BioVU repository. Ferroptosis promotes PAH through metabolic and inflammatory mechanisms in the pulmonary vasculature.

Characterization and spatial distribution of infiltrating lymphocytes in medullary, and lymphocyte-predominant triple negative breast cancers

Medullary carcinoma of the breast (MedBC) is a rare histological type that accounts for less than 5% of all invasive breast cancers. Here, we performed an exploratory study aimed to determine whether imaging mass cytometry (IMC) can be used to characterize the immune infiltration and the spatial distribution heterogeneity in the rare subtype of MedBC compared to atypical MedBC and TNBC-TILS+ tumors. In both MedBC and TNBC-TILs+, there was a notable enrichment of immune cells in the peripheral regions of the tumors, whereas in atypical MedBC, the immune cells exhibited a central enrichment pattern. This distribution of infiltrated cells reflects an active immune recruitment correlated to more favorable prognosis. In MedBC, spatial analysis shows that immune cells are localized at a greater distance from the tumor cells. IMC highlights the heterogeneity of immune microenvironment across three main subtypes of breast tumors and could help to define distinct immune patterns.

BV2 Microglial Cell Activation/Polarization Is Influenced by Extracellular Vesicles Released from Mutated SOD1 NSC-34 Motoneuron-like Cells.

Microglia-mediated neuroinflammation is a key player in the pathogenesis of amyotrophic lateral sclerosis (ALS) as it can contribute to the progressive degeneration of motor neurons (MNs). Here, we investigated the role of mSOD1 NSC-34 MN-like cell-derived extracellular vesicles (EVs) in inducing the activation of BV2 microglial cells. NSC-34-released EVs were isolated by culture medium differential ultracentrifugation to obtain two fractions, one containing small EVs (diameter < 200 nm) and the other containing large EVs (diameter > 200 nm). BV2 cells were incubated with the two EV fractions for 12, 24, and 48 h to evaluate 1) the state of microglial inflammation through RT-PCR of IL-1β, IL-6, IL-4, and IL-10 and 2) the expression of proteins involved in inflammasome activation (IL-β and caspase 1), cell death (caspase 3), and glial cell recruitment (CXCR1), and presence of the TGFβ cytokine receptor (TGFβ-R2). The obtained results suggest a mSOD1 type-dependent polarization of BV2 cells towards an early neurotoxic phenotype and a late neuroprotective status, with an appearance of mixed M1 and M2 microglia subpopulations. A significant role in driving microglial cell activation is played by the TGFβ/CX3CR1 axis. Therefore, targeting the dysregulated microglial response and modulating neuroinflammation could hold promise as a therapeutic strategy for ALS.

Antibodies and complement are key drivers of thrombosis

Venous thromboembolism (VTE) is a common, deadly disease with an increasing incidence despite preventive efforts. Clinical observations have associated elevated antibody concentrations or antibody-based therapies with thrombotic events. However, how antibodies contribute to thrombosis is unknown. Here, we show that reduced blood flow enabled immunoglobulin M (IgM) to bind to FcμR and the polymeric immunoglobulin receptor (pIgR), initiating endothelial activation and platelet recruitment. Subsequently, the procoagulant surface of activated platelets accommodated antigen- and FcγR-independent IgG deposition. This leads to classical complement activation, setting in motion a prothrombotic vicious circle. Key elements of this mechanism were present in humans in the setting of venous stasis as well as in the dysregulated immunothrombosis of COVID-19. This antibody-driven thrombosis can be prevented by pharmacologically targeting complement. Hence, our results uncover antibodies as previously unrecognized central regulators of thrombosis. These findings carry relevance for therapeutic application of antibodies and open innovative avenues to target thrombosis without compromising hemostasis.

Atg16l2 augments Nlrc4 inflammasome activation by facilitating NAIPs-NLRC4 association.

As cytoplasmic protein complexes that are pivotal for innate immunity, inflammasomes act primarily through the detection of pathogen- or danger-associated molecular patterns. Nucleotide oligomerisation domain-like receptor family and caspase activation recruitment domain-containing protein 4 (NLRC4) inflammasomes identify and eliminate intracellular pathogens, a process contingent on the ligand-recognition capabilities of neuronal apoptosis inhibitory proteins (NAIPs). Upon detection of specific molecules indicative of intracellular infection, NAIPs discern distinct pathogenic components and subsequently transmit signals to NLRC4, thus initiating their activation and triggering an inflammatory response. However, the mechanisms underlying NLRC4 inflammasome remain unclear. In this study, we elucidated the critical role of ATG16L2 in activating the NLRC4 inflammasome. ATG16L2-deficient macrophages exhibited reduced NLRC4 inflammasome activation, characterised by decreased oligomerisation of apoptosis-associated speck-like protein containing a CARD and attenuated cleavage of Pro-caspase-1, Pro-IL-1β and gasdermin D. Co-immunoprecipitation assays revealed an interaction between ATG16L2 and NAIPs. Furthermore, ATG16L2 enhanced the association between NAIPs and NLRC4 by binding to NAIPs. For ATG16L2-knockout mice infected with Salmonella typhimurium, pathogen clearance and survival rates markedly decreased. Collectively, our findings suggest that ATG16L2 is a significant modulator of the innate immune system, influencing the activity of the NLRC4 inflammasome and the host's defensive response to intracellular pathogens.

Mechanism for controlled assembly of transcriptional condensates by Aire

Transcriptional condensates play a crucial role in gene expression and regulation, yet their assembly mechanisms remain poorly understood. Here, we report a multi-layered mechanism for condensate assembly by autoimmune regulator (Aire), an essential transcriptional regulator that orchestrates gene expression reprogramming for central T cell tolerance. Aire condensates assemble on enhancers, stimulating local transcriptional activities and connecting disparate inter-chromosomal loci. This functional condensate formation hinges upon the coordination between three Aire domains: polymerization domain caspase activation recruitment domain (CARD), histone-binding domain (first plant homeodomain (PHD1)), and C-terminal tail (CTT). Specifically, CTT binds coactivators CBP/p300, recruiting Aire to CBP/p300-rich enhancers and promoting CARD-mediated condensate assembly. Conversely, PHD1 binds to the ubiquitous histone mark H3K4me0, keeping Aire dispersed throughout the genome until Aire nucleates on enhancers. Our findings showed that the balance between PHD1-mediated suppression and CTT-mediated stimulation of Aire polymerization is crucial to form transcriptionally active condensates at target sites, providing new insights into controlled polymerization of transcriptional regulators.

Co-Designing a Smoking Cessation Chatbot: Focus Group Study of End Users and Smoking Cessation Professionals.

Our prototype smoking cessation chatbot, Quin, provides evidence-based, personalized support delivered via a smartphone app to help people quit smoking. We developed Quin using a multiphase program of co-design research, part of which included focus group evaluation of Quin among stakeholders prior to clinical testing. This study aimed to gather and compare feedback on the user experience of the Quin prototype from end users and smoking cessation professionals (SCPs) via a beta testing process to inform ongoing chatbot iterations and refinements. Following active and passive recruitment, we conducted web-based focus groups with SCPs and end users from Queensland, Australia. Participants tested the app for 1-2 weeks prior to focus group discussion and could also log conversation feedback within the app. Focus groups of SCPs were completed first to review the breadth and accuracy of information, and feedback was prioritized and implemented as major updates using Agile processes prior to end user focus groups. We categorized logged in-app feedback using content analysis and thematically analyzed focus group transcripts. In total, 6 focus groups were completed between August 2022 and June 2023; 3 for SCPs (n=9 participants) and 3 for end users (n=7 participants). Four SCPs had previously smoked, and most end users currently smoked cigarettes (n=5), and 2 had quit smoking. The mean duration of focus groups was 58 (SD 10.9; range 46-74) minutes. We identified four major themes from focus group feedback: (1) conversation design, (2) functionality, (3) relationality and anthropomorphism, and (4) role as a smoking cessation support tool. In response to SCPs' feedback, we made two major updates to Quin between cohorts: (1) improvements to conversation flow and (2) addition of the "Moments of Crisis" conversation tree. Participant feedback also informed 17 recommendations for future smoking cessation chatbot developments. Feedback from end users and SCPs highlighted the importance of chatbot functionality, as this underpinned Quin's conversation design and relationality. The ready accessibility of accurate cessation information and impartial support that Quin provided was recognized as a key benefit for end users, the latter of which contributed to a feeling of accountability to the chatbot. Findings will inform the ongoing development of a mature prototype for clinical testing.

Phosphorylation patterns in the AT1R C-terminal tail specify distinct downstream signaling pathways.

Different ligands stabilize specific conformations of the angiotensin II type 1 receptor (AT1R) that direct distinct signaling cascades mediated by heterotrimeric G proteins or β-arrestin. These different active conformations are thought to engage distinct intracellular transducers because of differential phosphorylation patterns in the receptor C-terminal tail (the "barcode" hypothesis). Here, we identified the AT1R barcodes for the endogenous agonist AngII, which stimulates both G protein activation and β-arrestin recruitment, and for a synthetic biased agonist that only stimulates β-arrestin recruitment. The endogenous and β-arrestin-biased agonists induced two different ensembles of phosphorylation sites along the C-terminal tail. The phosphorylation of eight serine and threonine residues in the proximal and middle portions of the tail was required for full β-arrestin functionality, whereas phosphorylation of the serine and threonine residues in the distal portion of the tail had little influence on β-arrestin function. Similarly, molecular dynamics simulations showed that the proximal and middle clusters of phosphorylated residues were critical for stable β-arrestin-receptor interactions. These findings demonstrate that ligands that stabilize different receptor conformations induce different phosphorylation clusters in the C-terminal tail as barcodes to evoke distinct receptor-transducer engagement, receptor trafficking, and signaling.

CD3-engaging bispecific antibodies trigger a paracrine regulated wave of T-cell recruitment for effective tumor killing

The mechanism of action of bispecific antibodies (bsAbs) directing T-cell immunity to solid tumors is incompletely understood. Here, we screened a series of CD3xHER2 bsAbs using extracellular matrix (ECM) embedded breast cancer tumoroid arrays exposed to healthy donor-derived T-cells. An initial phase of random T-cell movement throughout the ECM (day 1–2), was followed by a bsAb-dependent phase of active T-cell recruitment to tumoroids (day 2–4), and tumoroid killing (day 4–6). Low affinity HER2 or CD3 arms were compensated for by increasing bsAb concentrations. Instead, a bsAb binding a membrane proximal HER2 epitope supported tumor killing whereas a bsAb binding a membrane distal epitope did not, despite similar affinities and intra-tumoroid localization of the bsAbs, and efficacy in 2D co-cultures. Initial T-cell-tumor contact through effective bsAbs triggered a wave of subsequent T-cell recruitment. This critical surge of T-cell recruitment was explained by paracrine signaling and preceded a full-scale T-cell tumor attack.

Ohnut vs waitlist control for the self-management of endometriosis-associated deep dyspareunia: a pilot randomized controlled trial.

Deep dyspareunia affects 50% of people with endometriosis. The Ohnut is a set of interlocking rings that fit over the penis/insertive object. One or more rings can be used to limit insertion depth and reduce deep dyspareunia. We conducted a pilot, parallel, open-label randomized controlled trial (RCT) to investigate the feasibility of the study design and the acceptability and preliminary efficacy of the Ohnut. Participants were recruited from a tertiary center for endometriosis. Eligibility criteria were surgically confirmed endometriosis, age 19-49years, monogamous sexual relationship with a partner willing to participate in the study, and no comorbid superficial dyspareunia, anxiety, or depression. Couples were randomized into an intervention group or a waitlist control group using a 1:1 allocation ratio. All couples had sex as normal during weeks 1 to 4 (baseline period), and couples in the intervention group used the Ohnut with sex during weeks 5 to 10 (intervention period) while controls had sex as normal. Patient participants used daily diaries to record sexual activity and deep dyspareunia score (0-10) for the 10-week study. Intervention group participants completed an acceptability questionnaire at the end of the study. The primary outcomes were feasibility of the study and acceptability of the Ohnut. We also assessed differences in deep dyspareunia scores in the participants who used the Ohnut compared to the control participants who did not. We recruited approximately 5 couples per month of active recruitment. Of 864 potentially eligible participants, we successfully contacted 44.7% (n = 386), of whom 8.0% (n = 31) consented, 64.8% (n = 250) were ineligible, and 27.2% (n = 105) declined. Thirty-one couples were randomly assigned to the intervention or control group, and 17 couples completed the study. Intervention group couples used the Ohnut for an average of 72.4% (32.7%) of sexual encounters during the intervention period. The mean acceptability index score for the Ohnut was 0.83 (0.078) among patients and 0.83 (0.049) among partners (index between 0 and 1). After controlling for baseline deep dyspareunia, there was a significant difference in the intervention period mean deep dyspareunia scores between the control and intervention group (4.69 (2.44) vs 2.46 (1.82), P = .012). We identified preliminary evidence for the acceptability and efficacy of the Ohnut among both patients and partners, suggesting that the Ohnut may be a useful stand-alone or adjuvant management tool for endometriosis-associated deep dyspareunia. Strengths of this study were the "real-world" use of the Ohnut and data collection from both patients and partners. Limitations of the study design included the strict eligibility criteria that affected feasibility and generalizability. This pilot RCT indicated that the Ohnut may be an acceptable and effective intervention to reduce endometriosis-associated deep dyspareunia. We identified opportunities to improve design for a larger RCT. This clinical trial was registered with clinicaltrials.gov (#NCT04370444).

Distinct regulation of ATM signaling by DNA single-strand breaks and APE1

In response to DNA double-strand breaks or oxidative stress, ATM-dependent DNA damage response (DDR) is activated to maintain genome integrity. However, it remains elusive whether and how DNA single-strand breaks (SSBs) activate ATM. Here, we provide direct evidence in Xenopus egg extracts that ATM-mediated DDR is activated by a defined SSB structure. Our mechanistic studies reveal that APE1 promotes the SSB-induced ATM DDR through APE1 exonuclease activity and ATM recruitment to SSB sites. APE1 protein can form oligomers to activate the ATM DDR in Xenopus egg extracts in the absence of DNA and can directly stimulate ATM kinase activity in vitro. Our findings reveal distinct mechanisms of the ATM-dependent DDR activation by SSBs in eukaryotic systems and identify APE1 as a direct activator of ATM kinase.

A therapeutic small molecule enhances γ-oscillations and improves cognition/memory in Alzheimer’s disease model mice

Brain rhythms provide the timing for recruitment of brain activity required for linking together neuronal ensembles engaged in specific tasks. The γ-oscillations (30 to 120 Hz) orchestrate neuronal circuits underlying cognitive processes and working memory. These oscillations are reduced in numerous neurological and psychiatric disorders, including early cognitive decline in Alzheimer's disease (AD). Here, we report on a potent brain-permeable small molecule, DDL-920 that increases γ-oscillations and improves cognition/memory in a mouse model of AD, thus showing promise as a class of therapeutics for AD. We employed anatomical, in vitro and in vivo electrophysiological, and behavioral methods to examine the effects of our lead therapeutic candidate small molecule. As a novel in central nervous system pharmacotherapy, our lead molecule acts as a potent, efficacious, and selective negative allosteric modulator of the γ-aminobutyric acid type A receptors most likely assembled from α1β2δ subunits. These receptors, identified through anatomical and pharmacological means, underlie the tonic inhibition of parvalbumin (PV) expressing interneurons (PV+INs) critically involved in the generation of γ-oscillations. When orally administered twice daily for 2 wk, DDL-920 restored the cognitive/memory impairments of 3- to 4-mo-old AD model mice as measured by their performance in the Barnes maze. Our approach is unique as it is meant to enhance cognitive performance and working memory in a state-dependent manner by engaging and amplifying the brain's endogenous γ-oscillations through enhancing the function of PV+INs.