Mice expressing nonpolymerizable fibrinogen have reduced arterial and venous thrombosis with preserved hemostasis.

ABSTRACT: Elevated circulating fibrinogen levels correlate with increased risk for both cardiovascular and venous thromboembolic diseases. In vitro studies show that formation of a highly dense fibrin matrix is a major determinant of clot structure and stability. Here, we analyzed the impact of nonpolymerizable fibrinogen on arterial and venous thrombosis as well as hemostasis in vivo using FgaEK mice that express normal levels of a fibrinogen that cannot be cleaved by thrombin. In a model of carotid artery thrombosis, FgaWT/EK and FgaEK/EK mice were protected from occlusion with 4% ferric chloride (FeCl3) challenges compared with wild-type (FgaWT/WT) mice, but this protection was lost, with injuries driven by higher concentrations of FeCl3. In contrast, fibrinogen-deficient (Fga-/-) mice showed no evidence of occlusion, even with high-concentration FeCl3 challenge. Fibrinogen-dependent platelet aggregation and intraplatelet fibrinogen content were similar in FgaWT/WT, FgaWT/EK, and FgaEK/EK mice, consistent with preserved fibrinogen-platelet interactions that support arterial thrombosis with severe challenge. In an inferior vena cava stasis model of venous thrombosis, FgaEK/EK mice had near complete protection from thrombus formation. FgaWT/EK mice also displayed reduced thrombus incidence and a significant reduction in thrombus mass relative to FgaWT/WT mice after inferior vena cava stasis, suggesting that partial expression of nonpolymerizable fibrinogen was sufficient for conferring protection. Notably, FgaWT/EK and FgaEK/EK mice had preserved hemostasis in multiple models as well as normal wound healing times after skin incision, unlike Fga-/- mice that displayed significant bleeding and delayed healing. These findings indicate that a nonpolymerizable fibrinogen variant can significantly suppress occlusive thrombosis while preserving hemostatic potential in vivo.

Vaccination against neoantigens induced in cross-priming cDC1 in vivo.

The conventional type 1 dendritic cells (cDC1) play a pivotal role in protective immunity against pathogens and cancer. However, their low frequency in the blood and tissues limits their use in immune therapy. We have recently described a method to vaccinate against neoantigens that are induced in tumor cells by targeted delivery of a TAP siRNA to dendritic cells using a TLR9 binding CpG oligonucleotide. Since TLR9 is also expressed in immune suppressive myeloid populations TLR9 targeting could reduce the effectiveness of this approach. Here, we describe a modular multivalent antibody platform to target the TAP siRNA to resident Clec9a expressing cDC1 and show that it leads to selective and sustained TAP downregulation in cDC1 and inhibits tumor growth in mice more effectively than CpG targeted siRNA. To induce DC maturation an agonistic CD40 antibody was administered to the siRNA treated mice. To obviate the need for a second drug formulation and reduce the risk of toxicity, we exploited the multivalent nature of this targeting platform to co-deliver the TAP siRNA and a DC maturation agent, a CpG containing oligonucleotide, to cDC1 in vivo and show that it was more effective than Clec9a targeting of TAP siRNA in combination with CD40 antibody. This study describes a way to manipulate the function of cDC1 cells in vivo using a broadly applicable antibody-based targeting platform to deliver multiple biological agents to specific cells in vivo to potentiate (immune) therapy and to probe the biology of specific cell types in their natural settings.

Hypofibrinogenemia with preserved hemostasis and protection from thrombosis in mice with an Fga truncation mutation.

Genetic variants within the fibrinogen Aα chain encoding the αC-region commonly result in hypodysfibrinogenemia in patients. However, the (patho)physiological consequences and underlying mechanisms of such mutations remain undefined. Here, we generated Fga270 mice carrying a premature termination codon within the Fga gene at residue 271. The Fga270 mutation was compatible with Mendelian inheritance for offspring of heterozygous crosses. Adult Fga270/270 mice were hypofibrinogenemic with ∼10% plasma fibrinogen levels relative to FgaWT/WT mice, linked to 90% reduction in hepatic Fga messenger RNA (mRNA) because of nonsense-mediated decay of the mutant mRNA. Fga270/270 mice had preserved hemostatic potential in vitro and in vivo in models of tail bleeding and laser-induced saphenous vein injury, whereas Fga-/- mice had continuous bleeding. Platelets from FgaWT/WT and Fga270/270 mice displayed comparable initial aggregation following adenosine 5'-diphosphate stimulation, but Fga270/270 platelets quickly disaggregated. Despite ∼10% plasma fibrinogen, the fibrinogen level in Fga270/270 platelets was ∼30% of FgaWT/WT platelets with a compensatory increase in fibronectin. Notably, Fga270/270 mice showed complete protection from thrombosis in the inferior vena cava stasis model. In a model of Staphylococcus aureus peritonitis, Fga270/270 mice supported local, fibrinogen-mediated bacterial clearance and host survival comparable to FgaWT/WT, unlike Fga-/- mice. Decreasing the normal fibrinogen levels to ∼10% with small interfering RNA in mice also provided significant protection from venous thrombosis without compromising hemostatic potential and antimicrobial function. These findings both reveal novel molecular mechanisms underpinning fibrinogen αC-region truncation mutations and highlight the concept that selective fibrinogen reduction may be efficacious for limiting thrombosis while preserving hemostatic and immune protective functions.

Features of databases that supported searching for rapid evidence synthesis during COVID-19: implications for future public health emergencies.

BACKGROUND: As evidence related to the COVID-19 pandemic surged, databases, platforms, and repositories evolved with features and functions to assist users in promptly finding the most relevant evidence. In response, research synthesis teams adopted novel searching strategies to sift through the vast amount of evidence to synthesize and disseminate the most up-to-date evidence. This paper explores the key database features that facilitated systematic searching for rapid evidence synthesis during the COVID-19 pandemic to inform knowledge management infrastructure during future global health emergencies. METHODS: This paper outlines the features and functions of previously existing and newly created evidence sources routinely searched as part of the NCCMT's Rapid Evidence Service methods, including databases, platforms, and repositories. Specific functions of each evidence source were assessed as they pertain to searching in the context of a public health emergency, including the topics of indexed citations, the level of evidence of indexed citations, and specific usability features of each evidence source. RESULTS: Thirteen evidence sources were assessed, of which four were newly created and nine were either pre-existing or adapted from previously existing resources. Evidence sources varied in topics indexed, level of evidence indexed, and specific searching functions. CONCLUSION: This paper offers insights into which features enabled systematic searching for the completion of rapid reviews to inform decision makers within 5-10 days. These findings provide guidance for knowledge management strategies and evidence infrastructures during future public health emergencies.

Mobilizing community-driven health promotion through community granting programs: a rapid systematic review.

BACKGROUND: Effective health promotion responds to the unique needs of communities. Community granting programs that fund community-driven health promotion initiatives are a potential mechanism to meet those unique needs. While numerous community health-focused programs are available, the various strategies used by granting programs to foster engagement, administer grants and support awardees have not been systematically evaluated. This rapid systematic review explores the administration of community granting programs and how various program components impact process and population health outcomes. METHODS: A systematic search was conducted across three databases: Medline, SocINDEX, and Political Science Database. Single reviewers completed screening, consistent with a rapid review protocol. Studies describing or evaluating community granting programs for health or public health initiatives were included. Data regarding program characteristics were extracted and studies were evaluated for quality. A convergent integrated approach was used to analyze quantitative and qualitative findings. RESULTS: Thirty-five community granting programs, described in 36 studies, were included. Most were descriptive reports or qualitative studies conducted in the USA. Program support for grant awardees included technical assistance, workshops and training, program websites, and networking facilitation. While most programs reported on process outcomes, few reported on community or health outcomes; such outcomes were positive when reported. Programs reported that many funded projects were likely sustainable beyond program funding, due to the development of awardee skills, new partnerships, and securing additional funding. From the perspectives of program staff and awardees, facilitators included the technical assistance and workshops provided by the programs, networking amongst awardees, and the involvement of community members. Barriers included short timelines to develop proposals and allocate funds. CONCLUSIONS: This review provides a comprehensive overview of health-related community granting programs. Grant awardees benefit from technical assistance, workshops, and networking with other awardees. Project sustainability is enhanced by the development of new community partnerships and grant-writing training for awardees. Community granting programs can be a valuable strategy to drive community health, with several key elements that enhance community mobilization. REGISTRATION: PROSPERO #CRD42023399364.

Strategies to implement evidence-informed decision making at the organizational level: a rapid systematic review.

BACKGROUND: Achievement of evidence-informed decision making (EIDM) requires the integration of evidence into all practice decisions by identifying and synthesizing evidence, then developing and executing plans to implement and evaluate changes to practice. This rapid systematic review synthesizes evidence for strategies for the implementation of EIDM across organizations, mapping facilitators and barriers to the COM-B (capability, opportunity, motivation, behaviour) model for behaviour change. The review was conducted to support leadership at organizations delivering public health services (health promotion, communicable disease prevention) to drive change toward evidence-informed public health. METHODS: A systematic search was conducted in multiple databases and by reviewing publications of key authors. Articles that describe interventions to drive EIDM within teams, departments, or organizations were eligible for inclusion. For each included article, quality was assessed, and details of the intervention, setting, outcomes, facilitators and barriers were extracted. A convergent integrated approach was undertaken to analyze both quantitative and qualitative findings. RESULTS: Thirty-seven articles are included. Studies were conducted in primary care, public health, social services, and occupational health settings. Strategies to implement EIDM included the establishment of Knowledge Broker-type roles, building the EIDM capacity of staff, and research or academic partnerships. Facilitators and barriers align with the COM-B model for behaviour change. Facilitators for capability include the development of staff knowledge and skill, establishing specialized roles, and knowledge sharing across the organization, though staff turnover and subsequent knowledge loss was a barrier to capability. For opportunity, facilitators include the development of processes or mechanisms to support new practices, forums for learning and skill development, and protected time, and barriers include competing priorities. Facilitators identified for motivation include supportive organizational culture, expectations for new practices to occur, recognition and positive reinforcement, and strong leadership support. Barriers include negative attitudes toward new practices, and lack of understanding and support from management. CONCLUSION: This review provides a comprehensive analysis of facilitators and barriers for the implementation of EIDM in organizations for public health, mapped to the COM-B model for behaviour change. The existing literature for strategies to support EIDM in public health illustrates several facilitators and barriers linked to realizing EIDM. Knowledge of these factors will help senior leadership develop and implement EIDM strategies tailored to their organization, leading to increased likelihood of implementation success. REVIEW REGISTRATION: PROSPERO CRD42022318994.

Rethinking Data Collection Methods During the Pandemic: Development and Implementation of CATI for the All of Us Research Program.

The All of Us Research Program is a longitudinal cohort study aiming to build a diverse database to advance precision medicine. The COVID-19 pandemic hindered the ability of participants to receive in-person assistance at enrollment sites to complete digital surveys. Therefore, the program implemented Computer-Assisted Telephone Interviewing (CATI) to facilitate survey completion remotely to combat the disrupted data collection procedures. In January 2021, All of Us implemented a 1-year CATI Pilot supporting 9399 participants and resulting in 16 337 submitted surveys. The pilot showed that CATI was successful in increasing survey completion and retention activities for the All of Us Research Program, given the additional remote support offered to participants. Given the success of the CATI Pilot, multimodal survey administration will continue.

Recent advances in the genomic resources for sheep.

Sheep (Ovis aries) provide a vital source of protein and fibre to human populations. In coming decades, as the pressures associated with rapidly changing climates increase, breeding sheep sustainably as well as producing enough protein to feed a growing human population will pose a considerable challenge for sheep production across the globe. High quality reference genomes and other genomic resources can help to meet these challenges by: (1) informing breeding programmes by adding a priori information about the genome, (2) providing tools such as pangenomes for characterising and conserving global genetic diversity, and (3) improving our understanding of fundamental biology using the power of genomic information to link cell, tissue and whole animal scale knowledge. In this review we describe recent advances in the genomic resources available for sheep, discuss how these might help to meet future challenges for sheep production, and provide some insight into what the future might hold.

Novel Paracrine Action of Endothelium Enhances Glucose Uptake in Muscle and Fat.

[Figure: see text].

An outbreak of Salmonella Typhimurium associated with the consumption of raw liver at an Eid al-Adha celebration in Wales (UK), July 2021.

In July 2021, Public Health Wales received two notifications of salmonella gastroenteritis. Both cases has attended the same barbecue to celebrate Eid al-Adha, two days earlier. Additional cases attending the same barbecue were found and an outbreak investigation was initiated. The barbecue was attended by a North African community's social network. On same day, smaller lunches were held in three homes in the social network. Many people attended both a lunch and the barbecue. Cases were defined as someone with an epidemiological link to the barbecue and/or lunches with diarrhoea and/or vomiting with date of onset following these events. We undertook a cohort study of 36 people attending the barbecue and/or lunch, and a nested case-control study using Firth logistic regression. A communication campaign, sensitive towards different cultural practices, was developed in collaboration with the affected community. Consumption of a traditional raw liver dish, 'marrara', at the barbecue was the likely vehicle for infection (Firth logistic regression, aOR: 49.99, 95%CI 1.71-1461.54, p = 0.02). Meat and offal came from two local butchers (same supplier) and samples yielded identical whole genome sequences as cases. Future outbreak investigations should be relevant to the community affected by considering dishes beyond those found in routine questionnaires.

Btk Supports Autoreactive B Cell Development and Protects against Apoptosis but Is Expendable for Antigen Presentation.

Bruton's tyrosine kinase (Btk) propagates B cell signaling, and BTK inhibitors are in clinical trials for autoimmune disease. Although autoreactive B cells fail to develop in the absence of Btk, its role in mature cells is unknown. To address this issue, a model of conditional removal (Btk flox/Cre-ERT2 ) was used to excise Btk from mature transgenic B cells that recognize the pathophysiologic autoantigen insulin. Anti-insulin B cells escape central tolerance and promote autoimmune diabetes, mimicking human autoreactive cells. Lifelong Btk deficiency was previously shown to eliminate 95% of anti-insulin B cells, but in this model, mature anti-insulin B cells survived for weeks after targeted Btk deletion, even when competing with a polyclonal repertoire. BCR-stimulated cells could still signal via Syk, PLCy2, and CD22, but failed to upregulate the antiapoptotic protein Bcl-xL, and proliferation was impaired. Surprisingly, Btk-depleted anti-insulin B cells could still present Ag and activate T cells, a critical function in promoting T cell-mediated islet cell destruction. Thus, pharmacologic targeting of Btk may be most effective by blocking expansion of established autoreactive cells, and preventing emergence of new ones.

Genetic characterisation of the Connemara pony and the Warmblood horse using a within-breed clustering approach.

BACKGROUND: The Connemara pony (CP) is an Irish breed that has experienced varied selection by breeders over the last fifty years, with objectives ranging from the traditional hardy pony to an agile athlete. We compared these ponies with well-studied Warmblood (WB) horses, which are also selectively bred for athletic performance but with a much larger census population. Using genome-wide single nucleotide polymorphism (SNP) and whole-genome sequencing data from 116 WB (94 UK WB and 22 European WB) and 36 CP (33 UK CP and 3 US CP), we studied the genomic diversity, inbreeding and population structure of these breeds. RESULTS: The k-means clustering approach divided both the CP and WB populations into four genetic groups, among which the CP genetic group 1 (C1) associated with non-registered CP, C4 with US CP, WB genetic group 1 (W1) with Holsteiners, and W3 with Anglo European and British WB. Maximum and mean linkage disequilibrium (LD) varied significantly between the two breeds (mean from 0.077 to 0.130 for CP and from 0.016 to 0.370 for WB), but the rate of LD decay was generally slower in CP than WB. The LD block size distribution peaked at 225 kb for all genetic groups, with most of the LD blocks not exceeding 1 Mb. The top 0.5% harmonic mean pairwise fixation index (FST) values identified ontology terms related to cancer risk when the four CP genetic groups were compared. The four CP genetic groups were less inbred than the WB genetic groups, but C2, C3 and C4 had a lower proportion of shorter runs of homozygosity (ROH) (74 to 76% < 4 Mb) than the four WB genetic groups (80 to 85% < 4 Mb), indicating more recent inbreeding. The CP and WB genetic groups had a similar ratio of effective number of breeders (Neb) to effective population size (Ne). CONCLUSIONS: Distinct genetic groups of individuals were revealed within each breed, and in WB these genetic groups reflected population substructure better than studbook or country of origin. Ontology terms associated with immune and inflammatory responses were identified from the signatures of selection between CP genetic groups, and while CP were less inbred than WB, the evidence pointed to a greater degree of recent inbreeding. The ratio of Neb to Ne was similar in CP and WB, indicating the influence of popular sires is similar in CP and WB.

Exploring the feasibility of establishing a core set of sexual, reproductive, maternal, newborn, child, and adolescent health indicators in humanitarian settings: results from a multi-methods assessment in Jordan.

BACKGROUND: Reliable and rigorously collected sexual, reproductive, maternal, newborn, child, and adolescent health (SRMNCAH) data in humanitarian settings is often sparse and variable in quality across different humanitarian settings. To address this gap in data quality, the World Health Organization (WHO) developed a core set of indicators for monitoring and evaluating SRMNCAH services and outcomes in humanitarian settings, and assessed their feasibility in the field in Jordan, in addition to three other countries; with the goal of aggregating information from global consultations and field-level assessments to reach consensus on a set of core SRMNCAH indicators for services and outcome evaluation in humanitarian settings among WHO global partners. METHODS: The feasibility assessment in Jordan focused on the following constructs: relevance/usefulness, feasibility of measurement, systems and resources, and ethical issues. The multi-methods assessment included five components; a desk review, key informant interviews, focus group discussions, facility assessments, and observational sessions. RESULTS: Findings suggest that there is widespread support among regional, national, as well as global stakeholders for developing a core list of SRMNCAH indicators for monitoring and evaluation of services and outcomes in humanitarian settings in Jordan. There are numerous resources and data collection systems that could be leveraged, built upon, and improved to ensure the feasibility of collecting this proposed set of indicators. However, the data collection load requested from donors, the national government, international and UN agencies, coordination/cluster systems must be better harmonized, standardized, and less burdensome. CONCLUSIONS: Despite stakeholder support in developing a core set of indicators, this would only be useful if it has the buy-in from the international community. Greater harmonization and coordination, alongside increased resource allocation, would improve data collection efforts and allow stakeholders to meet indicators' reporting requirements.

Natural adaptation and human selection of northeast African sheep genomes.

African sheep manifest diverse but distinct physio-anatomical traits, which are the outcomes of natural- and human-driven selection. Here, we generated 34.8 million variants from 150 indigenous northeast African sheep genomes sequenced at an average depth of ∼54× for 130 samples (Ethiopia, Libya) and ∼20× for 20 samples (Sudan). These represented sheep from diverse environments, tail morphology and post-Neolithic introductions to Africa. Phylogenetic and model-based admixture analysis provided evidence of four genetic groups corresponding to altitudinal geographic origins, tail morphotypes and possible historical introduction and dispersal of the species into and across the continent. Running admixture at higher levels of K (6 ≤ K ≤ 25), revealed cryptic levels of genome intermixing as well as distinct genetic backgrounds in some populations. Comparative genomic analysis identified targets of selection that spanned conserved haplotype structures overlapping clusters of genes and gene families. These were related to hypoxia responses, ear morphology, caudal vertebrae and tail skeleton length, and tail fat-depot structures. Our findings provide novel insights underpinning morphological variation and response to human-driven selection and environmental adaptation in African indigenous sheep.

KLB dysregulation mediates disrupted muscle development in intrauterine growth restriction.

Intrauterine growth restriction (IUGR) is a leading cause of neonatal morbidity and mortality in humans and domestic animals. Developmental adaptations of skeletal muscle in IUGR lead to increased risk of premature muscle loss and metabolic disease in later life. Here, we identified ß-Klotho (KLB), a fibroblast growth factor 21 (FGF21) co-receptor, as a novel regulator of muscle development in IUGR. Using the pig as a naturally-occurring disease model, we performed transcriptome-wide profiling of fetal muscle (day 90 of pregnancy) from IUGR and normal-weight (NW) littermates. We found that, alongside large-scale transcriptional changes comprising multiple developmental, tissue injury and metabolic gene pathways, KLB was increased in IUGR muscle. Moreover, FGF21 concentrations were increased in plasma in IUGR fetuses. Using cultures of fetal muscle progenitor cells (MPCs), we showed reduced myogenic capacity of IUGR compared to NW muscle in vitro, as evidenced by differences in fusion indices and myogenic transcript levels, as well as mechanistic target of rapamycin (mTOR) activity. Moreover, transfection of MPCs with KLB small interfering RNA promoted myogenesis and mTOR activation, whereas treatment with FGF21 had opposite and dose-dependent effects in porcine and also in human fetal MPCs. In conclusion, our results identify KLB as a novel and potentially critical mediator of impaired muscle development in IUGR, through conserved mechanisms in pigs and humans. Our data shed new light onto the pathogenesis of IUGR, a significant cause of lifelong ill-health in humans and animals. KEY POINTS: Intrauterine growth restriction (IUGR) is associated with large-scale transcriptional changes in developmental, tissue injury and metabolic gene pathways in fetal skeletal muscle. Levels of the fibroblast growth factor 21 (FGF21) co-receptor, ß-Klotho (KLB) are increased in IUGR fetal muscle, and FGF21 concentrations are increased in IUGR fetal plasma. KLB mediates a reduction in muscle development through inhibition of mechanistic target of rapamycin signalling. These effects of KLB on muscle cells are conserved in pig and human, suggesting a vital role of this protein in the regulation of muscle development and function in mammals.

Targeting mitophagy in Parkinson's disease.

The genetics and pathophysiology of Parkinson's disease (PD) strongly implicate mitochondria in disease aetiology. Elegant studies over the last two decades have elucidated complex molecular signaling governing the identification and removal of dysfunctional mitochondria from the cell, a process of mitochondrial quality control known as mitophagy. Mitochondrial deficits and specifically reduced mitophagy are evident in both sporadic and familial PD. Mendelian genetics attributes loss-of-function mutations in key mitophagy regulators PINK1 and Parkin to early-onset PD. Pharmacologically enhancing mitophagy and accelerating the removal of damaged mitochondria are of interest for developing a disease-modifying PD therapeutic. However, despite significant understanding of both PINK1-Parkin-dependent and -independent mitochondrial quality control pathways, the therapeutic potential of targeting mitophagy remains to be fully explored. Here, we provide a summary of the genetic evidence supporting the role for mitophagy failure as a pathogenic mechanism in PD. We assess the tractability of mitophagy pathways and prospects for drug discovery and consider intervention points for mitophagy enhancement. We explore the numerous hit molecules beginning to emerge from high-content/high-throughput screening as well as the biochemical and phenotypic assays that enabled these screens. The chemical and biological properties of these reference compounds suggest many could be used to interrogate and perturb mitochondrial biology to validate promising drug targets. Finally, we address key considerations and challenges in achieving preclinical proof-of-concept, including in vivo mitophagy reporter methodologies and disease models, as well as patient stratification and biomarker development for mitochondrial forms of the disease.

Geographical contrasts of Y-chromosomal haplogroups from wild and domestic goats reveal ancient migrations and recent introgressions.

By their paternal transmission, Y-chromosomal haplotypes are sensitive markers of population history and male-mediated introgression. Previous studies identified biallelic single-nucleotide variants in the SRY, ZFY and DDX3Y genes, which in domestic goats identified four major Y-chromosomal haplotypes, Y1A, Y1B, Y2A and Y2B, with a marked geographical partitioning. Here, we extracted goat Y-chromosomal variants from whole-genome sequences of 386 domestic goats (75 breeds) and seven wild goat species, which were generated by the VarGoats goat genome project. Phylogenetic analyses indicated domestic haplogroups corresponding to Y1B, Y2A and Y2B, respectively, whereas Y1A is split into Y1AA and Y1AB. All five haplogroups were detected in 26 ancient DNA samples from southeast Europe or Asia. Haplotypes from present-day bezoars are not shared with domestic goats and are attached to deep nodes of the trees and networks. Haplogroup distributions for 186 domestic breeds indicate ancient paternal population bottlenecks and expansions during migrations into northern Europe, eastern and southern Asia, and Africa south of the Sahara. In addition, sharing of haplogroups indicates male-mediated introgressions, most notably an early gene flow from Asian goats into Madagascar and the crossbreeding that in the 19th century resulted in the popular Boer and Anglo-Nubian breeds. More recent introgressions are those from European goats into the native Korean goat population and from Boer goat into Uganda, Kenya, Tanzania, Malawi and Zimbabwe. This study illustrates the power of the Y-chromosomal variants for reconstructing the history of domestic species with a wide geographical range.

Clinical Trial-Ready Patient Cohorts for Multiple System Atrophy: Coupling Biospecimen and iPSC Banking to Longitudinal Deep-Phenotyping.

Multiple system atrophy (MSA) is a fatal neurodegenerative disease of unknown etiology characterized by widespread aggregation of the protein alpha-synuclein in neurons and glia. Its orphan status, biological relationship to Parkinson's disease (PD), and rapid progression have sparked interest in drug development. One significant obstacle to therapeutics is disease heterogeneity. Here, we share our process of developing a clinical trial-ready cohort of MSA patients (69 patients in 2 years) within an outpatient clinical setting, and recruiting 20 of these patients into a longitudinal "n-of-few" clinical trial paradigm. First, we deeply phenotype our patients with clinical scales (UMSARS, BARS, MoCA, NMSS, and UPSIT) and tests designed to establish early differential diagnosis (including volumetric MRI, FDG-PET, MIBG scan, polysomnography, genetic testing, autonomic function tests, skin biopsy) or disease activity (PBR06-TSPO). Second, we longitudinally collect biospecimens (blood, CSF, stool) and clinical, biometric, and imaging data to generate antecedent disease-progression scores. Third, in our Mass General Brigham SCiN study (stem cells in neurodegeneration), we generate induced pluripotent stem cell (iPSC) models from our patients, matched to biospecimens, including postmortem brain. We present 38 iPSC lines derived from MSA patients and relevant disease controls (spinocerebellar ataxia and PD, including alpha-synuclein triplication cases), 22 matched to whole-genome sequenced postmortem brain. iPSC models may facilitate matching patients to appropriate therapies, particularly in heterogeneous diseases for which patient-specific biology may elude animal models. We anticipate that deeply phenotyped and genotyped patient cohorts matched to cellular models will increase the likelihood of success in clinical trials for MSA.

A Glucose Biosensor Based on Phosphorescence Lifetime Sensing and a Thermoresponsive Membrane.

The adoption of existing continuous glucose monitors (CGMs) is limited by user burden. Herein, a design for a glucose biosensor with the potential for subcutaneous implantation, without the need for a transcutaneous probe or affixed transmitter, is presented. The design is based on the combination of an enzyme-driven phosphorescence lifetime-based glucose-sensing assay and a thermoresponsive membrane anticipated to reduce biofouling. The metalloporphyrin, Pd meso-tetra(sulfophenyl)-tetrabenzoporphyrin ([PdPh4 (SO3 Na)4 TBP]3 , HULK) as well as glucose oxidase (GOx) are successfully incorporated into the UV-cured double network (DN) membranes by leveraging electrostatic interactions and covalent conjugation, respectively. The oxygen-sensitive metalloporphyrin is incorporated at different levels within the DN membranes. These HULK-containing membranes retain the desired thermosensitivity, as well as glucose diffusivity and primary optical properties of the metalloporphyrin. After subsequently modifying the membranes with GOx, glucose-sensing experiments reveal that membranes prepared with the lowest GOx level exhibit the expected increase in phosphorescent lifetime for glucose concentrations up to 200 mg dL-1 . For membranes prepared with relatively higher GOx, oxygen-limited behavior is considered the source of diminished sensitivity at higher glucose levels. This proof-of-concept study demonstrates the promising potential of a biosensor design integrating a specific optical biosensing chemistry into a thermoresponsive hydrogel membrane.

Investigation of USP30 inhibition to enhance Parkin-mediated mitophagy: tools and approaches.

Mitochondrial dysfunction is implicated in Parkinson disease (PD). Mutations in Parkin, an E3 ubiquitin ligase, can cause juvenile-onset Parkinsonism, probably through impairment of mitophagy. Inhibition of the de-ubiquitinating enzyme USP30 may counter this effect to enhance mitophagy. Using different tools and cellular approaches, we wanted to independently confirm this claimed role for USP30. Pharmacological characterisation of additional tool compounds that selectively inhibit USP30 are reported. The consequence of USP30 inhibition by these compounds, siRNA knockdown and overexpression of dominant-negative USP30 on the mitophagy pathway in different disease-relevant cellular models was explored. Knockdown and inhibition of USP30 showed increased p-Ser65-ubiquitin levels and mitophagy in neuronal cell models. Furthermore, patient-derived fibroblasts carrying pathogenic mutations in Parkin showed reduced p-Ser65-ubiquitin levels compared with wild-type cells, levels that could be restored using either USP30 inhibitor or dominant-negative USP30 expression. Our data provide additional support for USP30 inhibition as a regulator of the mitophagy pathway.