Acceptability of a rectal microbicide douche for HIV prevention: a mixed-methods analysis of a first-in-human formulation pilot study.

OBJECTIVES: DREAM-01 was an open label, dose-escalation and variable osmolarity study to identify a tenofovir HIV-prevention douche/enema that could achieve protective colon tissue cell concentrations and high acceptability. To assess impact on sexual enjoyment, iso-osmolar and hypo-osmolar placebo douches were provided for at-home use before receptive anal sex (RAS). METHODS: Eighteen HIV-uninfected men who have RAS were administered three tenofovir douches at the research clinic: Product A, an iso-osmolar dose; Product B, an iso-osmolar escalation dose; and Product C, a hypo-osmolar escalation dose. Following Products A and C, participants were given a saline douche of matching osmolarity to use at home before RAS. Participants reported acceptability via a computer-assisted self-interview and in-depth interview in this mixed-methods study. RESULTS: All three products were rated acceptable by 17 (95%) of the participants. A majority (94%) would be likely or very likely to use any of the three products before RAS. Of those who used the saline douches before RAS and then rated their sexual enjoyment, most reported that their sexual enjoyment was not affected. Interview data revealed that participants found the product easy to incorporate into their regular routine, but would prefer to use more liquid for cleansing. CONCLUSIONS: These findings indicate that the hypo-osmolar Product C, which also provides the most rapid delivery of tenofovir for HIV prevention, is acceptable for future safety trials and that our sample reports high likelihood of using a rectal microbicide douche for HIV prevention. Our findings support continued pursuit of a tenofovir rectal microbicide douche. TRIAL REGISTRATION NUMBER: NCT02750540.

Association of optic disc pallor and RNFL thickness with cerebral small vessel disease in the PREVENT-Dementia study.

INTRODUCTION: We tested associations between two retinal measures (optic disc pallor, peripapillary retinal nerve fiber layer [pRNFL] thickness) and four magnetic resonance imaging markers of cerebral small vessel disease (SVD; lacunes, microbleeds, white matter hyperintensities, and enlarged perivascular spaces [ePVSs]). METHODS: We used PallorMetrics to quantify optic disc pallor from fundus photographs, and pRNFL thickness from optical coherence tomography scans. Linear and logistic regression assessed relationships between retinal measures and SVD markers. Participants (N = 108, mean age 51.6) were from the PREVENT Dementia study. RESULTS: Global optic disc pallor was linked to ePVSs in the basal ganglia in both left (ß = 0.12, standard error [SE] = 0.05, P < 0.05) and right eyes (ß = 0.13, SE = 0.05, P < 0.05). Associations were also noted in different disc sectors. No pRNFL associations with SVD markers were found. DISCUSSION: Optic disc pallor correlated with ePVSs in the basal ganglia, suggesting retinal examination may be a useful method to study brain health changes related to SVD. Highlights: Optic disc pallor is linked to enlarged perivascular spaces in basal ganglia.There is no association between peripapillary retinal nerve fiber layer thickness and cerebral small vessel disease markers.Optic disc examination could provide insights into brain health.The sample included 108 midlife adults from the PREVENT Dementia study.

Alteration of Neuropilin-1 and Heparan Sulfate Interaction Impairs Murine B16 Tumor Growth.

Neuropilin-1 acts as a coreceptor with vascular endothelial growth factor receptors to facilitate binding of its ligand, vascular endothelial growth factor. Neuropilin-1 also binds to heparan sulfate, but the functional significance of this interaction has not been established. A combinatorial library screening using heparin oligosaccharides followed by molecular dynamics simulations of a heparin tetradecasaccharide suggested a highly conserved binding site composed of amino acid residues extending across the b1 and b2 domains of murine neuropilin-1. Mutagenesis studies established the importance of arginine513 and lysine514 for binding of heparin to a recombinant form of Nrp1 composed of the a1, a2, b1, and b2 domains. Recombinant Nrp1 protein bearing R513A,K514A mutations showed a significant loss of heparin-binding, heparin-induced dimerization, and heparin-dependent thermal stabilization. Isothermal calorimetry experiments suggested a 1:2 complex of heparin tetradecasaccharide:Nrp1. To study the impact of altered heparin binding in vivo, a mutant allele of Nrp1 bearing the R513A,K514A mutations was created in mice (Nrp1D) and crossbred to Nrp1+/- mice to examine the impact of altered heparan sulfate binding. Analysis of tumor formation showed variable effects on tumor growth in Nrp1D/D mice, resulting in a frank reduction in tumor growth in Nrp1D/- mice. Expression of mutant Nrp1D protein was normal in tissues, suggesting that the reduction in tumor growth was due to the altered binding of heparin/heparan sulfate to neuropilin-1. These findings suggest that the interaction of neuropilin-1 with heparan sulfate modulates its stability and its role in tumor formation and growth.

Neuroimaging and Clinical Findings in Healthy Middle-Aged Adults With Mild Traumatic Brain Injury in the PREVENT Dementia Study.

Importance: Traumatic brain injuries (TBI) represent an important, potentially modifiable risk factor for dementia. Despite frequently observed vascular imaging changes in individuals with TBI, the relationships between TBI-associated changes in brain imaging and clinical outcomes have largely been overlooked in community cases of TBI. Objective: To assess whether TBI are associated with and interact with midlife changes in neuroimaging and clinical features in otherwise healthy individuals. Design, Setting, and Participants: This cross-sectional analysis used baseline data from the PREVENT Dementia program collected across 5 sites in the UK and Ireland between 2014 and 2020. Eligible participants were cognitively healthy midlife adults aged between 40 and 59 years. Data were analyzed between January 2023 and April 2024. Exposure: Lifetime TBI history was assessed using the Brain Injury Screening Questionnaire. Main Outcomes and Measures: Cerebral microbleeds and other markers of cerebral small vessel disease (white matter hyperintensities [WMH], lacunes, perivascular spaces) were assessed on 3T magnetic resonance imaging. Clinical measures were cognition, sleep, depression, gait, and cardiovascular disease (CVD) risk, assessed using Computerized Assessment of Information Processing (COGNITO), Pittsburgh Sleep Quality Index, Center for Epidemiologic Studies Depression Scale, clinical interviews, and the Framingham Risk Score, respectively. Results: Of 617 participants (median [IQR] age, 52 [47-56] years; 380 female [61.6%]), 223 (36.1%) had a history of TBI. TBI was associated with higher microbleed count (ß = 0.10; 95% CI, 0.01-0.18; P = .03), with a dose-response association observed with increasing number of TBI events (ß = 0.05; 95% CI, 0.01-0.09; P = .03). Conversely, TBI was not associated with other measures of small vessel disease, including WMH. Furthermore, TBI moderated microbleed associations with vascular risk factors and clinical outcomes, such that associations were present only in the absence of TBI. Importantly, observations held when analyses were restricted to individuals reporting only mild TBI. Conclusions and Relevance: In this cross-sectional study of healthy middle-aged adults, detectable changes in brain imaging and clinical features were associated with remote, even mild, TBI in the general population. The potential contribution of vascular injury to TBI-related neurodegeneration presents promising avenues to identify potential targets, with findings highlighting the need to reduce TBI through early intervention and prevention in both clinical care and policymaking.

Lunar rhythms and their carry-over effects may shape environmental sex determination in a coral reef fish.

Lunar rhythms shape spawning phenology and subsequent risks and rewards for early life-history stages in the sea. Here, we consider a perplexing spawning phenology of the sixbar wrasse (Thalassoma hardwicke), in which parents spawn disproportionately around the new moon, despite the low survival of these larvae. Because primary sex determination in this system is highly plastic and sensitive to social environments experienced early in development, we ask whether this puzzling pattern of spawning is explained by fitness trade-offs associated with primary sexual maturation. We used otoliths from 871 fish to explore how spawning on different phases of the moon shapes the environments and phenotypes of settling larvae. Offspring that were born at the new moon were more likely to settle (i) before other larvae, (ii) at a larger body size, (iii) at an older age, (iv) to the best quality sites, and (v) as part of a social group-all increasing the likelihood of primary maturation to male. Selection of birthdates across life stage transitions suggests that the perplexing spawning phenology of adults may reflect an evolutionarily stable strategy that includes new moon spawning for compensatory benefits later in life, including preferential production of primary males at certain times.

CIB2 function is distinct from Whirlin in the development of cochlear stereocilia staircase pattern.

Variations in genes coding for calcium and integrin binding protein 2 (CIB2) and whirlin cause deafness both in humans and mice. We previously reported that CIB2 binds to whirlin, and is essential for normal staircase architecture of auditory hair cells stereocilia. Here, we refine the interacting domains between these proteins and provide evidence that both proteins have distinct role in the development and organization of stereocilia bundles required for auditory transduction. Using a series of CIB2 and whirlin deletion constructs and nanoscale pulldown (NanoSPD) assays, we localized the regions of CIB2 that are critical for interaction with whirlin. AlphaFold 2 multimer, independently identified the same interacting regions between CIB2 and whirlin proteins, providing a detailed structural model of the interaction between the CIB2 EF2 domain and whirlin HHD2 domain. Next, we investigated genetic interaction between murine Cib2 and Whrn using genetic approaches. Hearing in mice double heterozygous for functionally null alleles (Cib2 KO/+ ;Whrn wi/+ ) was similar to age-matched wild type mice, indicating that partial deficiency for both Cib2 and Whrn does not impair hearing. Double homozygous mutant mice (Cib2 KO/KO ;Whrn wi/wi ) had profound hearing loss and cochlear stereocilia exhibited a predominant phenotype seen in single Whrn wi/wi mutants. Furthermore, over-expression of Whrn in Cib2 KO/KO mice did not rescue the stereocilia morphology. These data suggest that, CIB2 is multifunctional, with key independent functions in development and/or maintenance of stereocilia staircase pattern in auditory hair cells.

Growth of complete ammonia oxidizers on guanidine.

Guanidine is a chemically stable nitrogen compound that is excreted in human urine and is widely used in manufacturing of plastics, as a flame retardant and as a component of propellants, and is well known as a protein denaturant in biochemistry1-3. Guanidine occurs widely in nature and is used by several microorganisms as a nitrogen source, but microorganisms growing on guanidine as the only substrate have not yet been identified. Here we show that the complete ammonia oxidizer (comammox) Nitrospira inopinata and probably most other comammox microorganisms can grow on guanidine as the sole source of energy, reductant and nitrogen. Proteomics, enzyme kinetics and the crystal structure of a N. inopinata guanidinase homologue demonstrated that it is a bona fide guanidinase. Incubation experiments with comammox-containing agricultural soil and wastewater treatment plant microbiomes suggested that guanidine serves as substrate for nitrification in the environment. The identification of guanidine as a growth substrate for comammox shows an unexpected niche of these globally important nitrifiers and offers opportunities for their isolation.

Modeling of HIV-1 prophylactic efficacy and toxicity with islatravir shows non-superiority for oral dosing, but promise as a subcutaneous implant.

HIV prevention with pre-exposure prophylaxis (PrEP) constitutes a major pillar in fighting the ongoing epidemic. While daily oral PrEP adherence may be challenging, long-acting (LA-)PrEP in oral or implant formulations could overcome frequent dosing with convenient administration. The novel drug islatravir (ISL) may be suitable for LA-PrEP, but dose-dependent reductions in CD 4 + $$ \mathrm{CD}{4}^{+} $$ T cell and lymphocyte counts were observed at high doses. We developed a mathematical model to predict ISL pro-drug levels in plasma and active intracellular ISL-triphosphate concentrations after oral vs. subcutaneous implant dosing. Using phase II trial data, we simulated antiviral effects and estimated HIV risk reduction for multiple dosages and dosing frequencies. We then established exposure thresholds where no adverse effects on immune cells were observed. Our findings suggest that implants with 56-62 mg ISL offer effective HIV risk reduction without reducing lymphocyte counts. Oral 0.1 mg daily, 3-5 mg weekly, and 10 mg biweekly ISL provide comparable efficacy, but weekly and biweekly doses may affect lymphocyte counts, while daily dosing regimen offered no advantage over existing oral PrEP. Oral 0.5-1 mg on demand provided > 90 % $$ >90\% $$ protection, while not being suitable for post-exposure prophylaxis. These findings suggest ISL could be considered for further development as a promising and safe agent for implantable PrEP.

Alterations of the AKT Pathway in Sporadic Human Tumors, Inherited Susceptibility to Cancer, and Overgrowth Syndromes.

The AKT kinases are critical signaling molecules that regulate cellular physiology upon the activation of tyrosine kinase receptors and phosphatidylinositol 3-kinases (PI3K). AKT kinases govern many cellular processes considered hallmarks of cancer, including cell proliferation and survival, cell size, tumor invasion, metastasis, and angiogenesis. AKT signaling is regulated by multiple tumor suppressors and oncogenic proteins whose loss or activation, respectively, leads to dysregulation of this pathway, thereby contributing to oncogenesis. Herein, we review the enormous body of literature documenting how the AKT pathway becomes hyperactivated in sporadic human tumors and various hereditary cancer syndromes. We also discuss the role of activating mutations of AKT pathway genes in various chimeric overgrowth disorders, including Proteus syndrome, hypoglycemia with hypertrophy, CLOVES and SOLAMEN syndromes, and hemimegalencephaly.

Navigating Real-World Randomized Clinical Trials: The 'Parents as Teachers' Experience.

The Parents as Teachers Randomized Controlled Trial (PAT RCT) Case Study investigates the multifaceted impact of implementing the PAT RCT in Arizona, U.S.A., shedding light on both the positive and negative effects. There has been a recent focus on improving the implementation of RCTs in community settings, as this issue has not been fully addressed. This research presents a case study examining the implementation of a community-based RCT in home visitation. This study also addresses the strategies that can be employed to mitigate some of the challenges in the implementation of an RCT, offering valuable insights for future RCTs in the domain of home visiting. The PAT program, aimed at providing parent education and family engagement for children from birth to kindergarten, encompasses a range of services, including personal visits, group connections, child screenings, and community resource linkages. The Parents as Teachers Randomized Controlled Trial (PAT RCT) directly promotes health by educating parents about health and wellness as well as providing early child screenings and heath referrals, all of which enhance health outcomes through timely interventions and improved parental practices. Lessons from the study also aim to improve the implementation of future health-related RCTs, ensuring effective delivery and impactful results.

Discovery of VU6007496: Challenges in the Development of an M1 Positive Allosteric Modulator Backup Candidate.

Herein we report progress toward a backup clinical candidate to the M1 positive allosteric modulator (PAM) VU319/ACP-319. Scaffold-hopping from the pyrrolo[2,3-b]pyridine-based M1 PAM VU6007477 to isomeric pyrrolo[3,2-b]pyridine and thieno[3,2-b]pyridine congeners identified several backup contenders. Ultimately, VU6007496, a pyrrolo[3,2-b]pyridine, advanced into late stage profiling, only to be plagued with unanticipated, species-specific metabolism and active/toxic metabolites which were identified in our phenotypic seizure liability in vivo screen, preventing further development. However, VU6007496 proved to be a highly selective and CNS penetrant M1 PAM, with minimal agonism, that displayed excellent multispecies IV/PO pharmacokinetics (PK), CNS penetration, no induction of long-term depression (or cholinergic toxicity) and robust efficacy in novel object recognition (minimum effective dose = 3 mg/kg p.o.). Thus, VU6007496 can serve as another valuable in vivo tool compound in rats and nonhuman primates, but not mouse, to study selective M1 activation.

Development of VU6036864: A Triazolopyridine-Based High-Quality Antagonist Tool Compound of the M5 Muscarinic Acetylcholine Receptor.

While the muscarinic acetylcholine receptor mAChR subtype 5 (M5) has been studied over decades, recent findings suggest that more in-depth research is required to elucidate a thorough understanding of its physiological function related to neurological and psychiatric disorders. Our efforts to identify potent, selective, and pharmaceutically favorable next-generation M5 antagonist tool compounds have led to the discovery of a novel triazolopyridine-based series. In particular, VU6036864 (45) showed exquisite potency (human M5 IC50 = 20 nM), good subtype selectivity (>500 fold selectivity against human M1-4), desirable brain exposure (Kp = 0.68, Kp,uu = 0.65), and high oral bioavailability (%F > 100%). VU6036864 (45) and its close analogues will support further studies of M5 as advanced antagonist tool compounds and play an important role in the emerging biology of M5.

Discovery of VU6008677: A Structurally Distinct Tricyclic M4 Positive Allosteric Modulator with Improved CYP450 Profile.

This Letter details our efforts to develop novel tricyclic muscarinic acetylcholine receptor subtype 4 (M4) positive allosteric modulator (PAM) scaffolds with improved pharmacological properties. This endeavor involved a "tie-back" strategy to replace the 3-amino-5-chloro-4,6-dimethylthieno[2,3-b]pyridine-2-carboxamide core, which led to the discovery of two novel tricyclic cores: an 8-chloro-9-methylpyrido[3',2':4,5]thieno[3,2-d]pyrimidin-4-amine core and 8-chloro-7,9-dimethylpyrido[3',2':4,5]furo[3,2-d]pyrimidin-4-amine core. Both tricyclic cores displayed low nanomolar potency against human M4 and greatly reduced cytochrome P450 inhibition when compared with parent compound ML253.

Patterns of spawning and settlement of reef fishes as strategic responses to post-settlement competition.

Settlement is a critical transition in the life history of reef fish, and the timing of this event can have a strong effect on fitness. Key factors that influence settlement timing are predictable lunar cyclic variation in tidal currents, moonlight, and nocturnal predation risk as larvae transition from pelagic to benthic environments. However, populations typically display wide variation in the arrival of settlers over the lunar cycle. This variation is often hypothesized to result from unpredictable conditions in the pelagic environment and bet-hedging by spawning adults. Here, we consider the hypothesis that the timing of spawning and settlement is a strategic response to post-settlement competition. We use a game theoretic model to predict spawning and settlement distributions when fish face a tradeoff between minimizing density-independent predation risk while crossing the reef crest vs. avoiding high competitor density on settlement habitat. In general, we expect competition to spread spawning over time such that settlement is distributed around the lunar phase with the lowest predation risk, similar to an ideal free distribution in which competition spreads competitors across space. We examine the effects of overcompensating density dependence, age-dependent competition, and competition among daily settler cohorts. Our model predicts that even in the absence of stochastic variation in the larval environment, competition can result in qualitative divergence between spawning and settlement distributions. Furthermore, we show that if competitive strength increases with settler age, competition results in covariation between settler age and settlement date, with older larvae settling when predation risk is minimal. We predict that competition between daily cohorts delays peak settlement, with priority effects potentially selecting for a multimodal settlement distribution.

The crosstalk between neuropilin-1 and tumor necrosis factor-α in endothelial cells.

Tumor necrosis factor-α (TNFα) is a master cytokine which induces expression of chemokines and adhesion molecules, such as intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), in endothelial cells to initiate the vascular inflammatory response. In this study, we identified neuropilin-1 (NRP1), a co-receptor of several structurally diverse ligands, as a modulator of TNFα-induced inflammatory response of endothelial cells. NRP1 shRNA expression suppressed TNFα-stimulated leukocyte adhesion and expression of ICAM-1 and VCAM-1 in human umbilical vein endothelial cells (HUVECs). Likewise, it reduced TNFα-induced phosphorylation of MAPK p38 but did not significantly affect other TNF-induced signaling pathways, such as the classical NFκB and the AKT pathway. Immunofluorescent staining demonstrated co-localization of NRP1 with the two receptors of TNF, TNFR1 and TNFR2. Co-immunoprecipitation further confirmed that NRP1 was in the same protein complex or membrane compartment as TNFR1 and TNFR2, respectively. Modulation of NRP1 expression, however, neither affected TNFR levels in the cell membrane nor the receptor binding affinities of TNFα. Although a direct interface between NRP1 and TNFα/TNFR1 appeared possible from a protein docking model, a direct interaction was not supported by binding assays in cell-free microplates and cultured cells. Furthermore, TNFα was shown to downregulate NRP1 in a time-dependent manner through TNFR1-NFκB pathway in HUVECs. Taken together, our study reveals a novel reciprocal crosstalk between NRP1 and TNFα in vascular endothelial cells.

How fishes and invertebrates impact coral resilience.

Increasingly intense and frequent ocean heatwaves are causing widespread coral mortality. These heatwaves are just one of the many stressors - among for instance ocean acidification, nutrient pollution and destructive fishing practices - that have caused widespread decline of coral reefs over the past century. This destruction of reefs threatens the remarkable biodiversity of organisms that depend upon coral reefs. However, recent research suggests that many of the fishes and invertebrates that inhabit coral reefs may play an underappreciated role in influencing the resistance and recovery of corals to stressors, especially those caused by global climate change such as ocean heatwaves. Unraveling the threads that link these coral inhabitants to the corals' response to stressors has the potential to weave a more comprehensive model of resilience that integrates the plight of coral reefs with the breathtaking diversity of life they host. Here, we aim to elucidate the critical roles that coral-associated fishes and invertebrates play in mediating coral resilience to environmental stressors. By integrating recent research findings, we aim to showcase how these often-overlooked organisms influence coral resilience in the face of climate change.

Plant roots affect free-living diazotroph communities in temperate grassland soils despite decades of fertilization.

Fixation of atmospheric N2 by free-living diazotrophs accounts for an important proportion of nitrogen naturally introduced to temperate grasslands. The effect of plants or fertilization on the general microbial community has been extensively studied, yet an understanding of the potential combinatorial effects on the community structure and activity of free-living diazotrophs is lacking. In this study we provide a multilevel assessment of the single and interactive effects of different long-term fertilization treatments, plant species and vicinity to roots on the free-living diazotroph community in relation to the general microbial community in grassland soils. We sequenced the dinitrogenase reductase (nifH) and the 16S rRNA genes of bulk soil and root-associated compartments (rhizosphere soil, rhizoplane and root) of two grass species (Arrhenatherum elatius and Anthoxanthum odoratum) and two herb species (Galium album and Plantago lanceolata) growing in Austrian grassland soils treated with different fertilizers (N, P, NPK) since 1960. Overall, fertilization has the strongest effect on the diazotroph and general microbial community structure, however with vicinity to the root, the plant effect increases. Despite the long-term fertilization, plants strongly influence the diazotroph communities emphasizing the complexity of soil microbial communities' responses to changing nutrient conditions in temperate grasslands.

SARS-CoV-2 population dynamics in immunocompetent individuals in a closed transmission chain shows genomic diversity over the course of infection.

BACKGROUND: SARS-CoV-2 remains rapidly evolving, and many biologically important genomic substitutions/indels have characterised novel SARS-CoV-2 lineages, which have emerged during successive global waves of the pandemic. Worldwide genomic sequencing has been able to monitor these waves, track transmission clusters, and examine viral evolution in real time to help inform healthcare policy. One school of thought is that an apparent greater than average divergence in an emerging lineage from contemporary variants may require persistent infection, for example in an immunocompromised host. Due to the nature of the COVID-19 pandemic and sampling, there were few studies that examined the evolutionary trajectory of SARS-CoV-2 in healthy individuals. METHODS: We investigated viral evolutionary trends and participant symptomatology within a cluster of 16 SARS-CoV-2 infected, immunocompetent individuals with no co-morbidities in a closed transmission chain. Longitudinal nasopharyngeal swab sampling allowed characterisation of SARS-CoV-2 intra-host variation over time at both the dominant and minor genomic variant levels through Nimagen-Illumina sequencing. RESULTS: A change in viral lineage assignment was observed in individual infections; however, there was only one indel and no evidence of recombination over the period of an acute infection. Minor and dominant genomic modifications varied between participants, with some minor genomic modifications increasing in abundance to become the dominant viral sequence during infection. CONCLUSIONS: Data from this cohort of SARS-CoV-2-infected participants demonstrated that long-term persistent infection in an immunocompromised host was not necessarily a prerequisite for generating a greater than average frequency of amino acid substitutions. Amino acid substitutions at both the dominant and minor genomic sequence level were observed in immunocompetent individuals during infection showing that viral lineage changes can occur generating viral diversity.