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Abstract Kinetoplastids are a clade of eukaryotic protozoans that include human parasitic pathogens like trypanosomes and Leishmania species. In these organisms, protein-coding genes are transcribed as polycistronic pre-mRNAs, which need to be processed by the coupled action of trans-splicing and polyadenylation to yield monogenic mature mRNAs. During trans-splicing, a universal RNA sequence, the spliced leader RNA (SL RNA) mini-exon, is added to the 5’-end of each mRNA. The 5’-end of this mini-exon carries a hypermethylated cap structure and is bound by a trypanosomatid-specific cap-binding complex (CBC). The function of three of the kinetoplastid CBC subunits is unknown, but an essential role in cap-binding and trans-splicing has been suggested. Here, we report cryo-EM structures that reveal the molecular architecture of the Trypanosoma brucei CBC (TbCBC) complex. We find that TbCBC interacts with two distinct features of the SL RNA. The TbCBP20 subunit interacts with the m7G cap while TbCBP66 recognizes double-stranded portions of the SL RNA. Our findings pave the way for future research on mRNA maturation in kinetoplastids. Moreover, the observed structural similarities and differences between TbCBC and the mammalian cap-binding complex will be crucial for considering the potential of TbCBC as a target for anti-trypanosomatid drug development.

Abstract Eukaryotic translation initiation factor eIF4B is required for efficient cap-dependent translation, it is overexpressed in cancer cells, and may influence stress granule formation. Due to the high degree of intrinsic disorder, eIF4B is rarely observed in cryo-EM structures of translation complexes and only ever by its single structured RNA recognition motif domain, leaving the molecular details of its large intrinsically disordered region (IDR) unknown. By integrating experiments and simulations we demonstrate that eIF4B IDR orchestrates and fine-tunes an intricate transition from monomers to a condensed phase, in which large-size dynamic oligomers form before mesoscopic phase separation. Single-molecule spectroscopy combined with molecular simulations enabled us to characterize the conformational ensembles and underlying intra- and intermolecular dynamics across the oligomerization transition. The observed sensitivity to ionic strength and molecular crowding in the self-association landscape suggests potential regulation of eIF4B nanoscopic and mesoscopic behaviors such as driven by protein modifications, binding partners or changes to the cellular environment.

Abstract Protein copy numbers constrain systems-level properties of regulatory networks, but proportional proteomic data remain scarce compared to RNA-seq. We related mRNA to protein statistically using best-available data from quantitative proteomics and transcriptomics for 4366 genes in 369 cell lines. The approach starts with a protein’s median copy number and hierarchically appends mRNA–protein and mRNA–mRNA dependencies to define an optimal gene-specific model linking mRNAs to protein. For dozens of cell lines and primary samples, these protein inferences from mRNA outmatch stringent null models, a count-based protein-abundance repository, empirical mRNA-to-protein ratios, and a proteogenomic DREAM challenge winner. The optimal mRNA-to-protein relationships capture biological processes along with hundreds of known protein-protein complexes, suggesting mechanistic relationships. We use the method to identify a viral-receptor abundance threshold for coxsackievirus B3 susceptibility from 1489 systems-biology infection models parameterized by protein inference. When applied to 796 RNA-seq profiles of breast cancer, inferred copy-number estimates collectively re-classify 26–29% of luminal tumors. By adopting a gene-centered perspective of mRNA–protein covariation across different biological contexts, we achieve accuracies comparable to the technical reproducibility of contemporary proteomics.

Biotic and abiotic disturbances modify tree structure and degrade stand health. Accurate geospatial data on stand structure is important for monitoring tree growth, forest health, progression and severity of diseases and pests, and estimating resilience to climate stress. The live crown ratio (LCR) of trees serves as a key health indicator but has been understudied at the landscape level using remote sensing data. This study generated the leaf area index (LAI) and a novel spatial layer of LCR at site and landscape scales using a combination of satellite data and ground observations. We conducted field surveys to collect plot-level (10 m × 10 m) data in four eastern white pine (EWP; Pinus strobus L.)-dominated sites in the state of Maine, USA. The plot-level data were used to develop regression models for LAI and LCR estimation using microwave (Sentinel-1) and optical (Sentinel-2) remote sensing data and applying the Random Forest (RF) and Support Vector Machine (SVM) machine learning algorithms. The RF model showed higher prediction accuracy than the SVM model at the site level. Moreover, the prediction accuracy at the site and landscape levels were comparable for LAI (R2 > 0.76) and LCR (R2 > 0.71) using the RF model. Furthermore, the predicted LAI and LCR were integrated with canopy height and stand density to develop a novel health index map for EWP. The resulting health index map successfully delineated patches representing various health categories. Forestry practitioners and decision-makers can use the derived health index map and intermediate spatial data layers (LAI and LCR) to guide stand management. The developed framework can potentially be applied to other coniferous and broadleaved species for remote sensing-based LCR estimation and forest health assessment upon further studies and verification.

ABSTRACTIntroduction: Pituitary adenylate cyclase-activating polypeptide (PACAP) regulates plasticity in brain systems underlying arousal and memory and is associated with posttraumatic stress disorder (PTSD). Research in animal models suggests that PACAP modulates entorhinal cortex (EC) input to the hippocampus, contributing to impaired contextual fear conditioning. In PTSD, PACAP is associated with higher activity of the amygdala to threat stimuli and lower functional connectivity of the amygdala and hippocampus. However, PACAP-affiliated structural alterations of these regions have not been investigated in PTSD. Here, we examined whether peripheral PACAP levels were associated with neuronal morphology of the amygdala and hippocampus (primary analyses), and EC (secondary) using Neurite Orientation Dispersion and Density Imaging.Methods: Sixty-four (44 female) adults (19 to 54 years old) with DSM-5 Criterion A trauma exposure completed the Clinician-Administered PTSD Scale (CAPS-5), a blood draw, and magnetic resonance imaging. PACAP38 radioimmunoassay was performed and T1-weighted and multi-shell diffusion-weighted images were acquired. Neurite Density Index (NDI) and Orientation Dispersion Index (ODI) were quantified in the amygdala, hippocampus, and EC. CAPS-5 total score and anxious arousal score were used to test for clinical associations with brain structure.Results: Higher PACAP levels were associated with greater EC NDI (β = 0.0099, q = 0.032) and lower EC ODI (β = −0.0073, q = 0.047), and not hippocampal or amygdala measures. Neither EC NDI nor ODI was associated with clinical measures.Conclusions: Circulating PACAP levels were associated with altered neuronal density of the EC but not the hippocampus or amygdala. These findings strengthen evidence that PACAP may impact arousal-associated memory circuits in PTSD.

Abstract Seaweed farming is widely promoted as an approach to mitigating climate change despite limited data on carbon removal pathways and uncertainty around benefits and risks at operational scales. We explored the feasibility of climate change mitigation from seaweed farming by constructing five scenarios spanning a range of industry development in coastal British Columbia, Canada, a temperate region identified as highly suitable for seaweed farming. Depending on growth rates and the fate of farmed seaweed, our scenarios sequestered or avoided between 0.20 and 8.2 Tg CO2e year−1, equivalent to 0.3% and 13% of annual greenhouse gas emissions in BC, respectively. Realisation of climate benefits required seaweed-based products to replace existing, more emissions-intensive products, as marine sequestration was relatively inefficient. Such products were also key to reducing the monetary cost of climate benefits, with product values exceeding production costs in only one of the scenarios we examined. However, model estimates have large uncertainties dominated by seaweed production and emissions avoided, making these key priorities for future research. Our results show that seaweed farming could make an economically feasible contribute to Canada’s climate goals if markets for value-added seaweed based products are developed. Moreover, our model demonstrates the possibility for farmers, regulators, and researchers to accurately quantify the climate benefits of seaweed farming in their regional contexts.

Abstract Loss-of-function mutations in the progranulin (GRN) gene are an autosomal dominant cause of Frontotemporal Dementia (FTD). These mutations typically result in haploinsufficiency of the progranulin protein. Grn +/– mice provide a model for progranulin haploinsufficiency and develop FTD-like behavioral abnormalities by 9–10 months of age. In previous work, we demonstrated that Grn +/– mice develop a low dominance phenotype in the tube test that is associated with reduced dendritic arborization of layer II/III pyramidal neurons in the prelimbic region of the medial prefrontal cortex (mPFC), a region key for social dominance behavior in the tube test assay. In this study, we investigated whether progranulin haploinsufficiency induced changes in dendritic spine density and morphology. Individual layer II/III pyramidal neurons in the prelimbic mPFC of 9–10 month old wild-type or Grn +/– mice were targeted for iontophoretic microinjection of fluorescent dye, followed by high-resolution confocal microscopy and 3D reconstruction for morphometry analysis. Dendritic spine density in Grn +/– mice was comparable to wild-type littermates, but the apical dendrites in Grn +/– mice had a shift in the proportion of spine types, with fewer stubby spines and more thin spines. Additionally, apical dendrites of Grn +/– mice had longer spines and smaller thin spine head diameter in comparison to wild-type littermates. These changes in spine morphology may contribute to altered circuit-level activity and social dominance deficits in Grn +/– mice.

Abstract Palaeontologists have long sought to explain the diversification of individual clades to whole biotas at global scales. Advances in our understanding of the spatial distribution of the fossil record through geological time, however, has demonstrated that global trends in biodiversity were a mosaic of regionally heterogeneous diversification processes. Drivers of diversification must presumably have also displayed regional variation to produce the spatial disparities observed in past taxonomic richness. Here, we analyse the fossil record of ammonoids, pelagic shelled cephalopods, through the Late Cretaceous, characterised by some palaeontologists as an interval of biotic decline prior to their total extinction at the Cretaceous-Paleogene boundary. We regionally subdivide this record to eliminate the impacts of spatial sampling biases and infer regional origination and extinction rates corrected for temporal sampling biases using Bayesian methods. We then model these rates using biotic and abiotic drivers commonly inferred to influence diversification. Ammonoid diversification dynamics and responses to this common set of diversity drivers were regionally heterogeneous, do not support ecological decline, and demonstrate that their global diversification signal is influenced by spatial disparities in sampling effort. These results call into question the feasibility of seeking drivers of diversity at global scales in the fossil record.

The elastic stiffness of bulk concrete materials results from the complex interaction of aggregates, voids, and hydrated cement (which can have multiple hardened phases at multiple length scales). Given the complexities associated with understanding the arrangement of these particles within bulk concrete volumes, estimations for elastic modulus often rely on empirical correlations with unit weight and compressive strength. Such estimations are inherently scale-dependent and fail to capture variability in mix designs, particularly the variability found in specialty concrete mixes. To develop a scale-independent method for estimating elastic modulus from mix-design volume fraction information, this study explores a novel bottom-up approach using cement paste phase stiffness values determined through micro-mechanical experimentation and randomized Monte-Carlo spring arrangement simulations. Statistical representations of cement paste phase stiffness distributions and bulk volume fraction data are combined to provide estimations for elastic stiffness in both the composite cement paste and bulk concrete containing fine aggregate and fibers. Resulting a priori estimations of UHPC cement paste stiffness from the micro-mechanical upscaling simulations were within 4% of measured values (based on mix-design and void volume fraction information alone) for a selected sample of mix proportions. When applied to the two UHPC mixes containing fibers and fine aggregate, upscaling simulations consistently overpredicted the measured elastic modulus, likely due to the aggregate-cement interfacial transition zone (ITZ) properties that were not captured in the micro-mechanical testing.

Mild cognitive impairment (MCI) affects nearly 20% of older adults worldwide, with no targetable interventions for prevention. COVID-19 adversely affects cognition, with >70% of older adults with Long COVID presenting with cognitive complaints. Neurovascular coupling (NVC), an essential mechanism of cognitive function, declines with aging and is further attenuated in neurocognitive disorders. The effect of COVID-19 on NVC responses has yet to be addressed in older adults who are vulnerable to dementia progression. Participants with MCI and a history of COVID-19 (COV+, N = 31) and MCI participants with no history of infection (COV− N = 11) participated in this cross-sectional study to determine if COVID-19 affects cerebrocortical NVC responses and vascular function. Functional near-infrared spectroscopy was used to measure cerebrocortical NVC responses, and endothelial function was assessed via insonation of the brachial artery during a flow-mediated dilation protocol. NVC responses were elicited by the working memory n-back paradigm. NVC in the left dorsolateral prefrontal cortex and endothelial function was decreased in the COV+ group compared to the COV− group. These data provide mechanistic insight into how COVID-19 may exacerbate long-term cognitive sequela seen in older adults, highlighting the urgent need for further research and clinical trials to explore novel therapeutic interventions aimed at preserving/restoring NVC.

Biomaterials that can be reversibly stiffened and shaped could be useful in broad biomedical applications where form-fitting scaffolds are needed. Here we investigate the combination of strong non-linear elasticity in biopolymer networks with the reconfigurability of packed hydrogel particles within a composite biomaterial. By packing microgels into collagen-1 networks and characterizing their linear and non-linear material properties, we empirically determine a scaling relationship that describes the synergistic dependence of the material's linear elastic shear modulus on the concentration of both components. We perform high-strain rheological tests and find that the materials strain stiffen and also exhibit a form of programmability, where no applied stress is required to maintain stiffened states of deformation after large strains are applied. We demonstrate that this non-linear rheological behavior can be used to shape samples that do not spontaneously relax large-scale bends, holding their deformed shapes for days. Detailed analysis of the frequency-dependent rheology reveals an unexpected connection to the rheology of living cells, where models of soft glasses capture their low-frequency behaviors and polymer elasticity models capture their high-frequency behaviors.

Abstract Dual antiplatelet therapy (DAPT) with aspirin and a P2Y12 receptor inhibitor (clopidogrel, prasugrel, or ticagrelor) is indicated after percutaneous coronary intervention (PCI) to reduce the risk of atherothrombotic events. Approximately 30% of the US population has a CYP2C19 no‐function allele that reduces the effectiveness of clopidogrel, but not prasugrel or ticagrelor, after PCI. We have shown improved outcomes with the integration of CYP2C19 genotyping into clinical care to guide the selection of prasugrel or ticagrelor in CYP2C19 no‐function allele carriers. However, the influence of patient‐specific demographic, clinical, and other genetic factors on outcomes with genotype‐guided DAPT has not been defined. In addition, the impact of genotype‐guided de‐escalation from prasugrel or ticagrelor to clopidogrel in patients without a CYP2C19 no‐function allele has not been investigated in a diverse, real‐world clinical setting. The Precision Antiplatelet Therapy after Percutaneous Coronary Intervention (Precision PCI) Registry is a multicenter US registry of patients who underwent PCI and clinical CYP2C19 testing. The registry is enrolling a diverse population, assessing atherothrombotic and bleeding events over 12 months, collecting DNA samples, and conducting platelet function testing in a subset of patients. The registry aims to define the influence of African ancestry and other patient‐specific factors on clinical outcomes with CYP2C19‐guided DAPT, evaluate the safety and effectiveness of CYP2C19‐guided DAPT de‐escalation following PCI in a real‐world setting, and identify additional genetic influences of clopidogrel response after PCI, with the ultimate goal of establishing optimal strategies for individualized antiplatelet therapy that improves outcomes in a diverse, real‐world population.

Background Cytochrome P450 2C19 (CYP2C19) intermediate and poor metabolizer patients exhibit diminished clopidogrel clinical effectiveness after percutaneous coronary intervention (PCI). However, outcome studies to date have lacked racial diversity. Thus, the impact of CYP2C19 genotype on cardiovascular outcomes in patients treated with clopidogrel who identify as Black or African American remains unclear. Methods and Results Adults among 5 institutions who self‐identified as Black or African American, underwent PCI and clinical CYP2C19 genotyping, and were treated with clopidogrel were included. Data were abstracted from health records. Major atherothrombotic (composite of death, myocardial infarction, ischemic stroke, stent thrombosis, or revascularization for unstable angina) and bleeding event rates within 1 year after PCI were compared across CYP2C19 metabolizer groups using multivariable Cox regression adjusted for potential confounders and baseline variables meeting a threshold of P

Abstract Background Mosquitoes are the deadliest organisms in the world, killing an estimated 750,000 people per year due to the pathogens they can transmit. Mosquitoes also pose a major threat to other vertebrate animals. Culex territans is a mosquito species found in temperate zones worldwide that feeds almost exclusively on amphibians and can transmit parasites; however, little is known about its ability to transmit other pathogens, including fungi. Batrachochytrium dendrobatidis (Bd) is a topical pathogenic fungus that spreads through contact. With amphibian populations around the world experiencing mass die-offs and extinctions due to this pathogen, it is critical to study all potential modes of transmission. Because Cx. territans mosquitoes are in contact with their hosts for long periods of time while blood-feeding, we hypothesize that they can transmit and pick up Bd. Methods In this study, we first assessed Cx. territans ability to transfer the fungus from an infected surface to a clean one under laboratory conditions. We also conducted a surveillance study of Bd infections in frogs and mosquitoes in the field (Mountain Lake Biological station, VA, USA). In parallel, we determined Cx. territans host preference via blood meal analysis of field caught mosquitoes. Results We found that this mosquito species can carry the fungus to an uninfected surface, implying that they may have the ability to transmit Bd to their amphibian hosts. We also found that Cx. territans feed primarily on green frogs (Rana clamitans) and bullfrogs (Rana catesbeiana) and that the prevalence of Bd within the frog population at our field site varied between years. Conclusions This study provides critical insights into understanding the role of amphibian-biting mosquitoes in transmitting pathogens, which can be applied to disease ecology of susceptible amphibian populations worldwide. Graphical Abstract

Abstract Impaired cerebrovascular function contributes to the genesis of age‐related cognitive decline. In this study, the hypothesis is tested that impairments in neurovascular coupling (NVC) responses and brain network function predict cognitive dysfunction in older adults. Cerebromicrovascular and working memory function of healthy young (n = 21, 33.2±7.0 years) and aged (n = 30, 75.9±6.9 years) participants are assessed. To determine NVC responses and functional connectivity (FC) during a working memory (n‐back) paradigm, oxy‐ and deoxyhemoglobin concentration changes from the frontal cortex using functional near‐infrared spectroscopy are recorded. NVC responses are significantly impaired during the 2‐back task in aged participants, while the frontal networks are characterized by higher local and global connection strength, and dynamic FC (p < 0.05). Both impaired NVC and increased FC correlate with age‐related decline in accuracy during the 2‐back task. These findings suggest that task‐related brain states in older adults require stronger functional connections to compensate for the attenuated NVC responses associated with working memory load.

Abstract Nonnative species are a key agent of global change. However, nonnative invertebrates remain understudied at the community scales where they are most likely to drive local extirpations. We use the North American NEON pitfall trapping network to document the number of nonnative species from 51 invertebrate communities, testing four classes of drivers. We sequenced samples using the eDNA from the sample's storage ethanol. We used AICc informed regression to evaluate how native species richness, productivity, habitat, temperature, and human population density and vehicular traffic account for continent‐wide variation in the number of nonnative species in a local community. The percentage of nonnatives varied 3‐fold among habitat types and over 10‐fold (0%–14%) overall. We found evidence for two types of constraints on nonnative diversity. Consistent with Capacity rules (i.e., how the number of niches and individuals reflect the number of species an ecosystem can support) nonnatives increased with existing native species richness and ecosystem productivity. Consistent with Establishment Rules (i.e., how the dispersal rate of nonnative propagules and the number of open sites limits nonnative species richness) nonnatives increased with automobile traffic—a measure of human‐generated propagule pressure—and were twice as common in pastures than native grasslands. After accounting for drivers associated with a community's ability to support native species (native species richness and productivity), nonnatives are more common in communities that are regularly seasonally disturbed (pastures and, potentially deciduous forests) and those experiencing more vehicular traffic. These baseline values across the US North America will allow NEON's monitoring mission to document how anthropogenic change—from disturbance to propagule transport, from temperature to trends in local extinction—further shape biotic homogenization.

Abstract Body size is a key functional trait governing how an animal community transforms resources and conditions into performance, abundance, and fitness. Here we use the National Ecological Observatory Network of pitfall traps to explore how an ecosystem's plant productivity, temperature, and growing season length accounts for the range of body size across 99 ground invertebrate communities. The 19‐fold continental variation in mean body size failed to covary with latitude, while common ordinal subtaxa grew smaller (e.g., myriapods) to larger (e.g., acari) from Puerto Rico to Alaska. Communities with a larger mean size arose when winters were longer and gross primary productivity was high. The diversity of body sizes in a community (measured as the CV) varied ninefold and decreased with latitude (r2 = 0.24) consistently across common orders. Size‐diverse communities were less likely in ecosystems with long winters (suggesting constraints on the time to build, r2 = 0.34) and those with high invertebrate activity (and hence trap catch, r2 = 0.12). Body size distributions thus appeared to arise from conflicting combinations of constraint (i.e., the ability to build large bodies) and performance (utility of large size in surviving long winters). As warming promotes growing season length, populations of larger, rarer individuals may benefit.

IntroductionGiven the role of microbiomes in promoting host health and homeostasis, understanding the factors shaping skin microbial communities in wild vertebrates has become increasingly important in conservation. This goal is even more pressing for amphibians, for which the skin has multiple critical functions, and pathogens currently decimating populations are linked to significant changes in skin microbiomes. However, because microbiomes are also shaped by environmental and ecological influences, as well as by host phylogeny, it is important to quantify these contributions to microbiome structure in the presence of infection.MethodsTo understand the joint influence of these diverse factors shaping microbiomes, we used 16S rRNA sequencing to characterize the skin microbial communities of six salamander species (families Plethodontidae and Salamandridae) found in Oklahoma and contrasted the effects of infection status, phylogeny, host ecology, and host environment (i.e., climate) on skin microbiomes.ResultsDifferences at the level of host family were the main factor influencing microbiome diversity; however, we did not detect a substantial phylogenetic signal. Instead, host ecology and environment were more important in driving microbiome differences among species and genera. Salamanders that tested positive for the skin fungal parasite Batrachochytrium dendrobatidis (Bd) also had slightly less diverse microbiomes than Bd-free animals, but no such differences were associated with the systemic pathogen ranavirus (RV).DiscussionTogether, these results indicate a nuanced relationship between the number and type of microbes present on salamander skin and the factors influencing them. By developing a baseline assessment of the microbiome diversity and richness present on the skin of these focal species, this work also provides a foundation for monitoring and evaluating changes in skin microbiomes as populations continue to experience stressors and diseases.

Previous research demonstrated the distribution of distinct microbial communities in the equine hindgut surrounding the pelvic flexure. The current study evaluated gene expression in epithelial tissues surrounding the pelvic flexure to characterize patterns that might correlate with microbial distribution. Gene expression was determined by analyzing RNA sequence data from the pelvic flexure, the left and right ventral colon, and the left and right dorsal colon. An average of 18,330 genes were expressed across the five tissues sampled. Most of the genes showed some level of expression in all five tissues. Tissue-restricted patterns of expression were also observed. Genes with restricted expression in the left ventral and left dorsal colons have communication, signaling, and regulatory functions that correlate with their known physiology. In contrast, genes expressed exclusively in the pelvic flexure have diverse functions. The ontology of genes differentially expressed between the pelvic flexure and the surrounding tissues was associated with immune functions and signaling processes. Despite being non-significant, these enrichment trends were reinforced by the functions of statistically significant expression differences between tissues of the hindgut. These results provide insight into the physiology of the equine hindgut epithelium that might influence the microbiota and its distribution.

In the United States (U.S.), newborn screening (NBS) for spinal muscular atrophy (SMA) is implemented by individual states. There is likely variation in the practice patterns of state NBS programs and among the providers caring for newborns with SMA. This is a prospective, descriptive, observational study that seeks to quantify and describe practice patterns and heterogeneities in state NBS programs and provider practices in the U.S. We surveyed U.S. state NBS programs and care providers of newborns with SMA. Thirty states and 41 practitioners responded. NBS program practices vary by state. Most (74%) state programs provide results to both primary care and specialist providers and also defer confirmatory SMA testing to those providers. Two states had relatively high rates of false-positive or inclusive results. The total birth prevalence of SMA was 1:13,862. Most providers were in tertiary care centers (90%) and were child neurologists (81%) and/or had fellowship training in Neuromuscular Medicine or Electromyography (76%). All providers see new referrals in less than a week, but many do not initiate treatment until >3 weeks of age (39%), with most commonly reported delays related to insurance processes. Most (81%) prefer onasemnogene abeparvovec-xioi (OA) as the treatment of choice, mainly due to perceived efficacy and the route/frequency of administration. NBS practice patterns in the U.S. vary by state but overall yielded the predicted birth prevalence of positive results. Providers evaluate these newborns urgently, but many do not initiate therapy until after 3 weeks of age. Treatment delays are mainly related to insurance processes.

Age-related cerebromicrovascular endothelial dysfunction underlies the initiation and progression of cognitive dysfunction and dementia, thus increasing the susceptibility of older adults to such conditions. Normal brain function requires dynamic adjustment of cerebral blood flow to meet the energetic demands of active neurons, which is achieved the homeostatic mechanism neurovascular coupling (NVC). In this context, therapeutical strategies aimed at rescuing or preserving NVC responses can delay the incidence or mitigate the severity of age-related cognitive dysfunction, and time-restricted eating (TRE) is a potential candidate for such a strategy. Studies have reported that TRE can improve cardiometabolic risk factors in older adults. However, the effect of TRE on cerebrovascular endothelial function remains unexplored. Thus, this protocol outlines the study procedures to test our hypothesis that a 6-month TRE regimen of 10-h eating window will improve NVC responses and endothelial function in community-dwelling older adults. This is a single-arm, open-label interventional trial. We aim to recruit 32 adults aged 55-80 years. Participants are instructed to maintain a TRE regimen of 10 h of free eating followed by 14 h of fasting for 6 months. Before and after fasting, participants are assessed for cognitive performance, peripheral micro- and macrovascular endothelial function, and NVC responses, as well as for several confounding factors, including body composition, dietary, and physical activity data. We expect that 6 months of TRE will improve NVC response and endothelial function in older adults compared with baseline, and that these improvements will be accompanied by improvements in cognitive performance. The study proposed herein will provide critical insight into a new potential therapeutical strategy for targeting age-related cognitive dysfunction. Ultimately, slowing down or alleviating cognitive decline will translate into improved quality of life and longer healthspan for aging adults. This study was prospectively registered at ClinicalTrials.gov (NCT06019195) on August 24, 2023.

Despite the constant advances in fluorescence imaging techniques, monitoring endogenous proteins still constitutes a major challenge in particular when considering dynamics studies or super-resolution imaging. We have recently evolved specific protein-based binders for PSD-95, the main postsynaptic scaffold proteins at excitatory synapses. Since the synthetic recombinant binders recognize epitopes not directly involved in the target protein activity, we consider them here as tools to develop endogenous PSD-95 imaging probes. After confirming their lack of impact on PSD-95 function, we validated their use as intrabody fluorescent probes. We further engineered the probes and demonstrated their usefulness in different super-resolution imaging modalities (STED, PALM, and DNA-PAINT) in both live and fixed neurons. Finally, we exploited the binders to enrich at the synapse genetically encoded calcium reporters. Overall, we demonstrate that these evolved binders constitute a robust and efficient platform to selectively target and monitor endogenous PSD-95 using various fluorescence imaging techniques.

Abstract Lung cancer is the leading cause of cancer‐related deaths worldwide. Surgery and chemoradiation are the standard of care in early stages of non‐small cell lung cancer (NSCLC), while immunotherapy is the standard of care in late‐stage NSCLC. The immune composition of the tumor microenvironment (TME) is recognized as an indicator for responsiveness to immunotherapy, although much remains unknown about its role in responsiveness to surgery or chemoradiation. In this pilot study, we characterized the NSCLC TME using mass cytometry (CyTOF) and bulk RNA sequencing (RNA‐Seq) with deconvolution of RNA‐Seq being performed by Kassandra, a recently published deconvolution tool. Stratification of patients based on the intratumoral abundance of B cells identified that the B‐cell rich patient group had increased expression of CXCL13 and greater abundance of PD1+ CD8 T cells. The presence of B cells and PD1+ CD8 T cells correlated positively with the presence of intratumoral tertiary lymphoid structures (TLS). We then assessed the predictive and prognostic utility of these cell types and TLS within publicly available stage 3 and 4 lung adenocarcinoma (LUAD) RNA‐Seq datasets. As previously described by others, pre‐treatment expression of intratumoral 12‐chemokine TLS gene signature is associated with progression free survival (PFS) in patients who receive treatment with immune checkpoint inhibitors (ICI). Notably and unexpectedly pre‐treatment percentages of intratumoral B cells are associated with PFS in patients who receive surgery, chemotherapy, or radiation. Further studies to confirm these findings would allow for more effective patient selection for both ICI and non‐ICI treatments.

Introduction: The American Cancer Society estimates 79,000 individuals will be diagnosed with kidney cancer in 2022, most of which are initially found as small renal masses (SRMs). Proper management of SRM patients includes careful evaluation of risk factors such as medical comorbidities and renal function. To investigate the importance of these risk factors, we examined their effect on crossover to delayed intervention (DI) and overall survival (OS) in patients undergoing active surveillance (AS) for SRMs. Methods: This is an Institutional Review Board-approved retrospective analysis of AS patients presented at kidney tumor conferences with SRMs between 2007 and 2017. Univariable and multivariable logistic regression analyses were performed to determine how factors including estimated glomerular filtration rate (eGFR), diabetes, and chronic kidney disease are associated with DI and OS. Results: A total of 111 cases were reviewed. In general, AS patients were elderly and had significant comorbidities. On univariate analysis, intervention was more likely to occur in patients with a younger age (P = 0.01), better kidney function (P = 0.01), and higher tumor growth rates (GRs) (P = 0.02). Higher eGFR was associated with better survival (P = 0.03), while higher tumor GRs (P = 0.014), greater Charlson Comorbidity Index (P = 0.01), and larger tumors (P = 0.01) were associated with worse OS. Of the comorbidities, diabetes was found to be an independent predictor of worse OS (P = 0.01). Conclusions: Patient-level factors – such as diabetes and eGFR – are associated with the rate of DI and OS among SRM patients. Consideration of these factors may facilitate better AS protocols and improve patient outcomes for those with SRMs.

In Eukarya, immature mRNA transcripts (pre-mRNA) often contain coding sequences, or exons, interleaved by non-coding sequences, or introns. Introns are removed upon splicing, and further regulation of the retained exons leads to alternatively spliced mRNA. The splicing reaction requires the stepwise assembly of the spliceosome, a macromolecular machine composed of small nuclear ribonucleoproteins (snRNPs). This review focuses on the early stage of spliceosome assembly, when U1 snRNP defines each intron 5’-splice site (5ʹss) in the pre-mRNA. We first introduce the splicing reaction and the impact of alternative splicing on gene expression regulation. Thereafter, we extensively discuss splicing descriptors that influence the 5ʹss selection by U1 snRNP, such as sequence determinants, and interactions mediated by U1-specific proteins or U1 small nuclear RNA (U1 snRNA). We also include examples of diseases that affect the 5ʹss selection by U1 snRNP, and discuss recent therapeutic advances that manipulate U1 snRNP 5ʹss selectivity with antisense oligonucleotides and small-molecule splicing switches.

Eukaryotic pre-mRNA is processed by a large multiprotein complex to accurately cleave the 3′ end, and to catalyse the addition of the poly(A) tail. Within this cleavage and polyadenylation specificity factor (CPSF) machinery, the CPSF73/CPSF3 endonuclease subunit directly contacts both CPSF100/CPSF2 and the scaffold protein Symplekin to form a subcomplex known as the core cleavage complex or mammalian cleavage factor. Here we have taken advantage of a stable CPSF73-CPSF100 minimal heterodimer from Encephalitozoon cuniculi to determine the solution structure formed by the first and second C-terminal domain (CTD1 and CTD2) of both proteins. We find a large number of contacts between both proteins in the complex, and notably in the region between CTD1 and CTD2. A similarity is also observed between CTD2 and the TATA-box binding protein (TBP) domains. Separately, we have determined the structure of the terminal CTD3 domain of CPSF73, which also belongs to the TBP domain family and is connected by a flexible linker to the rest of CPSF73. Biochemical assays demonstrate a key role for the CTD3 of CPSF73 in binding Symplekin, and structural models of the trimeric complex from other species allow for comparative analysis and support an overall conserved architecture.

Anurans (frogs and toads) are an ecologically diverse group of vertebrate organisms that display a myriad of reproductive modes and life history traits. To persist in such an expansive array of habitats, these organisms have evolved specialized skin that is used for respiration while also protecting against moisture loss, pathogens, and environmental contaminants. Anuran skin is also colonized by communities of symbiotic microorganisms, and these skin microbiota serve critical roles in numerous processes associated with anuran host health and persistence such as pathogen resistance and immunity. However, gaps remain in our understanding of the environmental and evolutionary processes that shape frog skin microbial communities. Here, we combined existing anuran disease data with 16S rRNA skin microbial inventories to elucidate the roles that geographic location, host evolutionary history, host ecology, and pathogen presence play in the microbial community assemblage of five co-distributed frog host species in Oklahoma. These focal species possess distinct ecological preferences: aquatic, semi-aquatic, and arboreal, and our results indicate that host ecology is the primary driver of frog skin microbial community structure. Additionally, compositional differences were observed among select host species based on geographic location, but this was not consistent among all five frog species. We did not find evidence of phylogenetic signal among our samples and results from the Classification and Regression Tree Analysis revealed that the presence of the amphibian pathogen Batrachochytrium dendrobatidis and the severity of infection were not drivers of skin microbiome differences among our focal host species. Results from this comparative study contribute to our growing understanding of the environmental and host-associated drivers of skin microbial community assemblage and represents one of the first studies on landscape-level variation in skin microbial communities among North American frogs.

Previous research has explored the importance of effective course advising to academic success. Course advising that is student-focused and takes a “whole-of-student” approach has been found to be especially important for students that have historically been minoritised in tertiary education. This includes students who are Indigenous, first-in-family, or from low socio-economic backgrounds. However, no research, to our knowledge, has investigated how Pacific students and staff envision course advising. Given Pacific students are among the fastest growing academic cohort in New Zealand and Australia, it is important for universities to understand how to foster Pacific student success. This research involved a series of talanoa (conversations) with both Pacific students and staff to better understand the current role of course advising in student decision-making, future hopes for the development of course advising, and suggestions to improve Pacific support in course advice. Understanding the perspective of Pacific students and staff helps to highlight the current gaps in course advising systems and encourages universities to acknowledge the importance of relationship building, the development of cultural competencies, and increasing Pacific representation in the course advising process. Results support reassessing course advising systems to help improve retention rates of Pacific students. ... How Can Course Advising Better Support Pacific Student Success? by Marea S. Colombo, Ruthie E. Holmes, Cameron D. Young & Stephen Scott is licensed under CC BY 4.0

Abstract Opioid prescribing for postoperative pain management is challenging because of inter‐patient variability in opioid response and concern about opioid addiction. Tramadol, hydrocodone, and codeine depend on the cytochrome P450 2D6 (CYP2D6) enzyme for formation of highly potent metabolites. Individuals with reduced or absent CYP2D6 activity (i.e., intermediate metabolizers [IMs] or poor metabolizers [PMs], respectively) have lower concentrations of potent opioid metabolites and potentially inadequate pain control. The primary objective of this prospective, multicenter, randomized pragmatic trial is to determine the effect of postoperative CYP2D6‐guided opioid prescribing on pain control and opioid usage. Up to 2020 participants, age ≥8 years, scheduled to undergo a surgical procedure will be enrolled and randomized to immediate pharmacogenetic testing with clinical decision support (CDS) for CYP2D6 phenotype‐guided postoperative pain management (intervention arm) or delayed testing without CDS (control arm). CDS is provided through medical record alerts and/or a pharmacist consult note. For IMs and PM in the intervention arm, CDS includes recommendations to avoid hydrocodone, tramadol, and codeine. Patient‐reported pain‐related outcomes are collected 10 days and 1, 3, and 6 months after surgery. The primary outcome, a composite of pain intensity and opioid usage at 10 days postsurgery, will be compared in the subgroup of IMs and PMs in the intervention (n = 152) versus the control (n = 152) arm. Secondary end points include prescription pain medication misuse scores and opioid persistence at 6 months. This trial will provide data on the clinical utility of CYP2D6 phenotype‐guided opioid selection for improving postoperative pain control and reducing opioid‐related risks.

Despite multiple recent field studies, herpetological species diversity of the Romblon Island Group in the central Philippines—particularly Sibuyan Island—has remained underestimated. Recently, we investigated the diversity of the herpetofauna of Mount Guiting-Guiting Natural Park, based on an elevational transect (10–1557 m a.s.l.). Our surveys resulted in a total of 47 species of amphibians and reptiles, including 14 new island records and one atypical occurrence of a snake species recorded for the first time from a high elevation (939 m a.s.l). These new records constitute a notable increase (21%) in Sibuyan’s herpetological species diversity as compared to surveys from a decade ago. We also provide updates of the taxonomy and identification of species endemic to this island (e.g., members of the genera Platymantis Günther, 1858, Brachymeles Duméril & Gibron, 1839, and Pseudogekko Taylor, 1922), and discuss the importance of continued surveys and field-derived data to inform conservation status assessments of Sibuyan’s unique assemblage of amphibians and reptiles.

Abstract Dormant propagules can provide a rapid colonization source for temporary aquatic habitats and set the trajectory for community dynamics, yet the egg banks of stormwater management systems have received little attention. We asked which species hatched from the sediment of drainage ditches in Champaign County, IL, and found bdelloid rotifers and ostracods (Heterocypris incongruens) to be the most common taxa. These sites also are colonized by mosquitoes, and we established laboratory experiments to examine interspecific interactions between common co‐occurring taxa. Culex restuans larvae were reared in the presence or absence of H. incongruens at two intra‐ and interspecific densities (20 or 40 total individuals) and their survivorship to adulthood, development time to adulthood, adult body size, and sex ratio were determined. Survival for Cx. restuans was significantly lower at high larval density than at low larval density in both treatments. Culex restuans larvae reared in the presence of H. incongruens had a shorter development time to adulthood and emerged as larger adults compared to those reared in the absence of H. incongruens. The sex ratios in the H. incongruens treatments were female‐biased whereas those in the Culex‐only treatments were male‐biased. These differences may have epidemiological implications, as only female mosquitoes serve as disease vectors. Our results emphasize the importance of understanding interspecific interactions in influencing larval mosquito development traits.

Unwanted RNA transcripts are targeted for degradation by nuclear complexes such as MTREC/PAXT. Here, the authors structurally and functionally characterized three interfaces of the scaffold protein Red1, providing mechanistic insights into conserved features of MTREC/PAXT architecture.

Abstract Many studies report atypical responses to sensory information in autistic individuals, yet it is not clear which stages of processing are affected, with little consideration given to decision-making processes. We combined diffusion modelling with high-density EEG to identify which processing stages differ between 50 autistic and 50 typically developing children aged 6–14 years during two visual motion tasks. Our pre-registered hypotheses were that autistic children would show task-dependent differences in sensory evidence accumulation, alongside a more cautious decision-making style and longer non-decision time across tasks. We tested these hypotheses using hierarchical Bayesian diffusion models with a rigorous blind modelling approach, finding no conclusive evidence for our hypotheses. Using a data-driven method, we identified a response-locked centro-parietal component previously linked to the decision-making process. The build-up in this component did not consistently relate to evidence accumulation in autistic children. This suggests that the relationship between the EEG measure and diffusion-modelling is not straightforward in autistic children. Compared to a related study of children with dyslexia, motion processing differences appear less pronounced in autistic children. Exploratory analyses also suggest weak evidence that ADHD symptoms moderate perceptual decision-making in autistic children.

Abstract We describe the development and validation of a qPCR assay to detect Poecilia reticulata, a highly invasive species of freshwater fish invasive to the Caribbean islands, through environmental DNA (eDNA) sampling. Originating from Trinidad, this species is invasive and detrimental to countless native tropical fish communities. A qPCR assay, consisting of a set of primers and a fluorescent probe, amplifying a 214 base pair target region of the mitochondrial Cytochrome B gene was designed for P. reticulata from existing DNA sequence data. The assay was assessed for target specificity, with no evidence of amplification in closely related or sympatrically distributed non‐target species. In vitro tests indicate that the assay consistently detects P. reticulata down to concentrations of 2.0 × 10−5 ng/μl. The developed assay provides a new, practical tool for monitoring freshwater habitats throughout the Caribbean, allowing for early and rapid detection of invasive fish species of conservation concern.

Macular edema is the pathological accumulation of fluid in the central retina. It is a complication of many retinal diseases, including diabetic retinopathy, retinal vascular occlusions and uveitis, among others. Macular edema causes decreased visual acuity and, when chronic or refractory, can cause severe and permanent visual impairment and blindness. In most instances, it develops due to dysregulation of the blood-retinal barrier which permits infiltration of the retinal tissue by proteins and other solutes that are normally retained in the blood. The increase in osmotic pressure in the tissue drives fluid accumulation. Current treatments include vascular endothelial growth factor blockers, corticosteroids, and non-steroidal anti-inflammatory drugs. These treatments target vasoactive and inflammatory mediators that cause disruption to the blood-retinal barrier. In this review, a clinical overview of macular edema is provided, mechanisms of disease are discussed, highlighting processes targeted by current treatments, and areas of opportunity for future research are identified.

Hypothalamic-pituitary adrenal (HPA)axis dysregulation has long been implicated in stress-related disorders such as major depression and post-traumatic stress disorder. Glucocorticoids (GCs) are released from the adrenal glands as a result of HPA-axis activation. The release of GCs is implicated with several neurobiological changes that are associated with negative consequences of chronic stress and the onset and course of psychiatric disorders. Investigating the underlying neurobiological effects of GCs may help to better understand the pathophysiology of stress-related psychiatric disorders. GCs impact a plethora of neuronal processes at the genetic, epigenetic, cellular, and molecular levels. Given the scarcity and difficulty in accessing human brain samples, 2D and 3D in vitro neuronal cultures are becoming increasingly useful in studying GC effects. In this review, we provide an overview of in vitro studies investigating the effects of GCs on key neuronal processes such as proliferation and survival of progenitor cells, neurogenesis, synaptic plasticity, neuronal activity, inflammation, genetic vulnerability, and epigenetic alterations. Finally, we discuss the challenges in the field and offer suggestions for improving the use of in vitro models to investigate GC effects.

Beech leaf disease (BLD) damage is associated with the parasitic nematode Litylenchus crenatae ssp. mccannii. Foliar symptoms manifest as darkened or chlorotic galls in the interveinal portions in the leaf, which become leathery and crinkled under high severity of infection. Though nearly a decade has passed since the discovery of this disease, little is known regarding the impact of BLD on leaf function and physiology. This study assesses the variation in leaf gas exchange and physiological leaf traits among asymptomatic and BLD-infected leaves across a gradient of symptom severity within a natural forested stand in central Connecticut, USA. Leaves with BLD symptoms are found to have significantly reduced carbon assimilation and instantaneous water use efficiency, with increased levels of stomatal conductance as symptom severity progresses. Leaf response to light manipulation is also affected, with an increase in dark respiration and the light compensation point among banded and crinkled leaves. Additionally, BLD symptoms are found to have a significant influence on leaf water content, specific leaf area, and leaf nitrogen content. Relationships between gas exchange and these leaf traits yield linear correlations that are used to infer functional relationships impacted by the disease.

Abstract Non‐forest ecosystems, dominated by shrubs, grasses and herbaceous plants, provide ecosystem services including carbon sequestration and forage for grazing, and are highly sensitive to climatic changes. Yet these ecosystems are poorly represented in remotely sensed biomass products and are undersampled by in situ monitoring. Current global change threats emphasize the need for new tools to capture biomass change in non‐forest ecosystems at appropriate scales. Here we developed and deployed a new protocol for photogrammetric height using unoccupied aerial vehicle (UAV) images to test its capability for delivering standardized measurements of biomass across a globally distributed field experiment. We assessed whether canopy height inferred from UAV photogrammetry allows the prediction of aboveground biomass (AGB) across low‐stature plant species by conducting 38 photogrammetric surveys over 741 harvested plots to sample 50 species. We found mean canopy height was strongly predictive of AGB across species, with a median adjusted R2 of 0.87 (ranging from 0.46 to 0.99) and median prediction error from leave‐one‐out cross‐validation of 3.9%. Biomass per‐unit‐of‐height was similar within but different among, plant functional types. We found that photogrammetric reconstructions of canopy height were sensitive to wind speed but not sun elevation during surveys. We demonstrated that our photogrammetric approach produced generalizable measurements across growth forms and environmental settings and yielded accuracies as good as those obtained from in situ approaches. We demonstrate that using a standardized approach for UAV photogrammetry can deliver accurate AGB estimates across a wide range of dynamic and heterogeneous ecosystems. Many academic and land management institutions have the technical capacity to deploy these approaches over extents of 1–10 ha−1. Photogrammetric approaches could provide much‐needed information required to calibrate and validate the vegetation models and satellite‐derived biomass products that are essential to understand vulnerable and understudied non‐forested ecosystems around the globe.

AbstractNon-mammalian model organisms have been essential for our understanding of the mechanisms that control development, disease, and physiology, but they are underutilized in pharmacological and toxicological phenotypic screening assays due to their low throughput in comparison with cell-based screens. To increase the utility of using Drosophila melanogaster

Abstract Characterised by intraocular inflammation, non‐infectious uveitis includes a large group of autoimmune and autoinflammatory diseases that either involve the eye alone or have both ocular and systemic manifestations. When non‐infectious uveitis involves the posterior segment of the eye, specifically the retina, there is substantial risk of vision loss, often linked to breakdown of the inner blood‐retinal barrier. This barrier is formed by non‐fenestrated retinal vascular endothelial cells, reinforced by supporting cells that include pericytes, Müller cells and astrocytes. Across the published literature, a group of inflammatory cytokines stand out as prominent mediators of intraocular inflammation, with effects on the retinal endothelium that may contribute to breakdown of the inner blood‐retinal barrier, namely tumour necrosis factor (TNF)‐α, interleukin (IL)‐1β, IL‐6, IL‐8, IL‐17 and chemokine C‐C motif ligand (CCL)2. This article reviews the function of each cytokine and discusses the evidence for their involvement in retinal endothelial barrier dysfunction in non‐infectious uveitis, including basic laboratory investigations, studies of ocular fluids collected from patients with non‐infectious uveitis, and results of clinical treatment trials. The review also outlines gaps in knowledge in this area. Understanding the disease processes at a molecular level can suggest treatment alternatives that are directed against appropriate biological targets to protect the posterior segment of eye and preserve vision in non‐infectious uveitis.

IntroductionMild cognitive impairment (MCI) is a prodromal stage to dementia, affecting up to 20% of the aging population worldwide. Patients with MCI have an annual conversion rate to dementia of 15-20%. Thus, conditions that increase the conversion from MCI to dementia are of the utmost public health concern. The COVID-19 pandemic poses a significant impact on our aging population with cognitive decline as one of the leading complications following recovery from acute infection. Recent findings suggest that COVID-19 increases the conversion rate from MCI to dementia in older adults. Hence, we aim to uncover a mechanism for COVID-19 induced cognitive impairment and progression to dementia to pave the way for future therapeutic targets that may mitigate COVID-19 induced cognitive decline.MethodologyA prospective longitudinal study is conducted at the University of Oklahoma Health Sciences Center. Patients are screened in the Department of Neurology and must have a formal diagnosis of MCI, and MRI imaging prior to study enrollment. Patients who meet the inclusion criteria are enrolled and followed-up at 18-months after their first visit. Visit one and 18-month follow-up will include an integrated and cohesive battery of vascular and cognitive measurements, including peripheral endothelial function (flow-mediated dilation, laser speckle contrast imaging), retinal and cerebrovascular hemodynamics (dynamic vessel retinal analysis, functional near-infrared spectroscopy), and fluid and crystalized intelligence (NIH-Toolbox, n-back). Multiple logistic regression will be used for primary longitudinal data analysis to determine whether COVID-19 related impairment in neurovascular coupling and increases in white matter hyperintensity burden contribute to progression to dementia.

Swarm intelligence has promising applications for firm search and decision-choice problems and is particularly well suited for examining how other firms influence the focal firm’s search. To evaluate search performance, researchers examining firm search through simulation models typically build a performance landscape. The NK model is the leading tool used for this purpose in the management science literature. We assess the usefulness of the NK landscape for simulated swarm search. We find that the strength of the swarm model for examining firm search and decision-choice problems—the ability to model the influence of other firms on the focal firm—is limited to the NK landscape. Researchers will need alternative ways to create a performance landscape in order to use our full swarm model in simulations. We also identify multiple opportunities—endogenous landscapes, agent-specific landscapes, incomplete information, and costly movements—that future researchers can include in landscape development to gain the maximum insights from swarm-based firm search simulations.

Ruthenium complexes of phosphinocarboxamide ligands, and their use to form metallacycles using halide abstraction/deprotonation reactions are reported. Thus, [Ru(p-cym){PPh2C(=O)NHR}Cl2; R = iPr (1), Ph (2), p-tol (3)] and [Ru(p-cym){PPh2C(=O)N(R)C(=O)N(H)R}Cl2; R = Ph (4), p-tol (5)] were synthesized from [(p-cym)RuCl2]2 (p-cym = para-cymene) and phosphinocarboxamides or phosphinodicarboxamides, respectively. Single-crystal X-ray diffraction measurements on 1–5 reveal coordination to ruthenium through the phosphorus donor, with an intramolecular hydrogen bond between the amine-bound proton and a metal-bound chloride. Six-membered metallacycles formed by halide abstraction/deprotonation of complexes 4 and 5 afforded [Ru(p-cym){κ2-P,N-PPh2C(=O)N(R)C(=O)NR}Cl] [R = Ph (6), p-tol (7)]. These species exist as a mixture of two rotational isomers in solution, as demonstrated by NMR spectroscopy.