ß-Triketones as Reactive Handles for Polymer Diversification via Dynamic Catalyst-Free Diketoenamine Click Chemistry.

The spontaneous condensation of amines with ß-triketones (TK), forming ß,ß'-diketoenamines (DKE) and releasing water as the sole byproduct, exhibits many of the hallmarks of "click" reactions. Such characteristics render TKs as a highly advantageous platform for efficient polymer diversification, even in biological contexts. Leveraging reversible addition-fragmentation chain transfer (RAFT) and photoiniferter polymerization of novel TK-containing vinylic monomers, we synthesized polymers containing pendent TKs with excellent control of molecular weights, even in excess of 106 g mol-1. Under mild, catalyst-free conditions, poly(ß-triketone methacrylate) could be modified with a diverse scope of amines containing a plethora of functional groups. The high efficiency of this functionalization approach was further emphasized when grafting-to with poly(ethylene glycol)-amine resulting in bottlebrushes with molecular weights reaching 2.0 × 107 g mol-1. Critically, while the formed DKE linkages are stable under ambient conditions, they undergo catalyst-free, dynamic transamination at elevated temperatures, paving the way for associative covalent adaptable networks. Overall, we introduce pendent triketone moieties into methacrylate and acrylamide polymers, establishing a novel postpolymerization modification technique that facilitates catalyst-free ligation of amines under highly permissible conditions.

Hybrid Photoiniferter and Ring-Opening Polymerization Yields One-Pot Anisotropic Nanorods.

Polymerization-induced self-assembly (PISA) has emerged as a scalable one-pot technique to prepare block copolymer (BCP) nanoparticles. Recently, a PISA process, that results in poly(l-lactide)-b-poly(ethylene glycol) BCP nanoparticles coined ring-opening polymerization (ROP)-induced crystallization-driven self-assembly (ROPI-CDSA), was developed. The resulting nanorods demonstrate a strong propensity for aggregation, resulting in the formation of 2D sheets and 3D networks. This article reports the synthesis of poly(N,N-dimethyl acrylamide)-b-poly(l)-lactide BCP nanoparticles by ROPI-CDSA, utilizing a two-step, one-pot approach. A dual-functionalized photoiniferter is first used for controlled radical polymerization of the acrylamido-based monomer, and the resulting polymer serves as a macroinitiator for organocatalyzed ROP to form the solvophobic polyester block. The resulting nanorods are highly stable and display anisotropy at higher molecular weights (>12k Da) and concentrations (>20% solids) than the previous report. This development expands the chemical scope of ROPI-CDSA BCPs and provides readily accessible nanorods made with biocompatible materials.

Estimated capital costs of fish exclusion technologies for hydropower facilities.

Hydropower is a reliable source of renewable energy, and its future expansion is likely to be in the form of either smaller new stream development (NSD) projects or powering existing non-powered dams. Thresholds for entrainment risk to fish and the requirements for fish exclusion at hydropower facilities often differ depending on the species involved, the characteristics of the facility, and the goals of stakeholders, but little quantitative information is present within the literature regarding the specific costs of fish exclusion measures. Cost data associated with protection, mitigation, and enhancement (PM&E) measures related to positive barrier screening were identified using keyword searches of an existing environmental mitigation cost data set and manual extraction from regulatory licensing documents available in the Federal Energy Regulatory Commission (FERC) eLibrary. This approach yielded a total of 50 p.m.&E mitigation measures with estimated capital construction costs pertaining to positive barrier screens and represented <10% of the 171 total FERC project dockets available in the data set. These data were highly skewed toward conventional relicensing projects, as <7% were associated with NSD projects. Results indicate highly variable costs are associated with fish screening, with flow-normalized costs one to two orders of magnitude higher for screening with the highest exclusion capability (≤0.09 in. spacing) compared with coarser screening (1-2 in.). These data provide an initial baseline for estimating exclusion costs for hydropower development and may help developers consider options for more fish-friendly generation technologies, though gaps remain relating to a lack of data, particularly for NSD projects.

Sodium-glucose cotransporter 2 inhibition does not improve the acute pressure natriuresis response in rats with type 1 diabetes.

NEW FINDINGS: What is the central question of this study? Sodium-glucose cotransporter 2 (SGLT2) inhibitors reduce cardiovascular risk in patients with both diabetic and non-diabetic kidney disease: can SGLT2 inhibition improve renal pressure natriuresis (PN), an important mechanism for long-term blood pressure control, which is impaired in type 1 diabetes mellitus (T1DM)? What is the main finding and its importance? The SGLT2 inhibitor dapagliflozin did not enhance the acute in vivo PN response in either healthy or T1DM Sprague-Dawley rats. The data suggest that the mechanism underpinning the clinical benefits of SGLT2 inhibitors on health is unlikely to be due to an enhanced natriuretic response to increased blood pressure. ABSTRACT: Type 1 diabetes mellitus (T1DM) leads to serious complications including premature cardiovascular and kidney disease. Hypertension contributes importantly to these adverse outcomes. The renal pressure natriuresis (PN) response, a key regulator of blood pressure (BP), is impaired in rats with T1DM as tubular sodium reabsorption fails to down-regulate with increasing BP. We hypothesised that sodium-glucose cotransporter 2 (SGLT2) inhibitors, which reduce cardiovascular risk in kidney disease, would augment the PN response in T1DM rats. Non-diabetic or T1DM (35-50 mg/kg streptozotocin i.p.) adult male Sprague-Dawley rats were anaesthetised (thiopental 50 mg/kg i.p.) and randomised to receive either dapagliflozin (1 mg/kg i.v.) or vehicle. Baseline sodium excretion was measured and then BP was increased by sequential arterial ligations to induce the PN response. In non-diabetic animals, the natriuretic and diuretic responses to increasing BP were not augmented by dapagliflozin. Dapagliflozin induced glycosuria, but this was not influenced by BP. In T1DM rats the PN response was impaired. Dapagliflozin again increased urinary glucose excretion but did not enhance PN. Inhibition of SGLT2 does not enhance the PN response in rats, either with or without T1DM. SGLT2 makes only a minor contribution to tubular sodium reabsorption and does not contribute to the impaired PN response in T1DM.

MIR503HG Loss Promotes Endothelial-to-Mesenchymal Transition in Vascular Disease.

[Figure: see text].

Kidney Single-Cell Atlas Reveals Myeloid Heterogeneity in Progression and Regression of Kidney Disease.

BACKGROUND: Little is known about the roles of myeloid cell subsets in kidney injury and in the limited ability of the organ to repair itself. Characterizing these cells based only on surface markers using flow cytometry might not provide a full phenotypic picture. Defining these cells at the single-cell, transcriptomic level could reveal myeloid heterogeneity in the progression and regression of kidney disease. METHODS: Integrated droplet- and plate-based single-cell RNA sequencing were used in the murine, reversible, unilateral ureteric obstruction model to dissect the transcriptomic landscape at the single-cell level during renal injury and the resolution of fibrosis. Paired blood exchange tracked the fate of monocytes recruited to the injured kidney. RESULTS: A single-cell atlas of the kidney generated using transcriptomics revealed marked changes in the proportion and gene expression of renal cell types during injury and repair. Conventional flow cytometry markers would not have identified the 12 myeloid cell subsets. Monocytes recruited to the kidney early after injury rapidly adopt a proinflammatory, profibrotic phenotype that expresses Arg1, before transitioning to become Ccr2+ macrophages that accumulate in late injury. Conversely, a novel Mmp12+ macrophage subset acts during repair. CONCLUSIONS: Complementary technologies identified novel myeloid subtypes, based on transcriptomics in single cells, that represent therapeutic targets to inhibit progression or promote regression of kidney disease.

Impaired pressure natriuresis and non-dipping blood pressure in rats with early type 1 diabetes mellitus.

KEY POINTS: Type 1 diabetes mellitus increases cardiovascular risk; hypertension amplifies this risk, while pressure natriuresis regulates long-term blood pressure. We induced type 1 diabetes in rats by streptozotocin injection and demonstrated a substantial impairment of pressure natriuresis: acute increases in blood pressure did not increase renal medullary blood flow, tubular sodium reabsorption was not downregulated, and proximal tubule sodium reabsorption, measured by lithium clearance, was unaffected. Insulin reduced blood glucose in diabetic rats, and rescued the pressure natriuresis response without influencing lithium clearance, but did not restore medullary blood flow. Radiotelemetry showed that diastolic blood pressure was increased in diabetic rats, and its diurnal variation was reduced. Increases in medullary blood flow and decreases in distal tubule sodium reabsorption that offset acute rises in BP are impaired in early type 1 diabetes, and this impairment could be a target for preventing hypertension in type 1 diabetes. ABSTRACT: Type 1 diabetes mellitus (T1DM) substantially increases cardiovascular risk, and hypertension amplifies this risk. Blood pressure (BP) and body sodium homeostasis are linked. T1DM patients have increased total exchangeable sodium, correlating directly with BP. Pressure natriuresis is an important physiological regulator of BP. We hypothesised that pressure natriuresis would be impaired, and BP increased, in the early phase of T1DM. Male Sprague-Dawley rats were injected with streptozotocin (30-45 mg/kg) or citrate vehicle. After 3 weeks, pressure natriuresis was induced by serial arterial ligation. In non-diabetic controls, this increased fractional excretion of sodium from ∼1% to ∼25% of the filtered load (P < 0.01); in T1DM rats, the response was significantly blunted, peaking at only ∼3% (P < 0.01). Mechanistically, normal lithium clearance suggested that distal tubule sodium reabsorption was not downregulated with increased BP in T1DM rats. The pressure dependence of renal medullary perfusion, considered a key factor in the integrated response, was abolished. Insulin therapy rescued the natriuretic response in diabetic rats, restoring normal downregulation of tubular sodium reabsorption when BP was increased. However, the pressure dependence of medullary perfusion was not restored, suggesting persistent vascular dysfunction despite glycaemic control. Radiotelemetry showed that T1DM did not affect systolic BP, but mean diastolic BP was ∼5 mmHg higher than in non-diabetic controls (P < 0.01), and normal diurnal variation was reduced. In conclusion, functional impairment of renal sodium and BP homeostasis is an early manifestation of T1DM, preceding hypertension and nephropathy. Early intervention to restore pressure natriuresis in T1DM may complement reductions in cardiovascular risk achieved with glycaemic control.

Green Methodologies for Copper(I)-Catalyzed Azide-Alkyne Cycloadditions: A Comparative Study.

Successful copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reactions may be achieved by several methods. In this paper, four synthetic protocols were performed for direct comparison of time required for the synthesis, yield, and purity of the 1H-1,2,3-triazole products. The methods with Cu(I) catalysts were conventional, microwave heating, solvent-free, and a method using glycerol solvent. The compounds synthesized in this paper were known non-fluorinated triazoles and new fluorinated triazoles. The results lead to the conclusion that the microwave method should be strongly considered for CuAAC syntheses.

Weekly variation in health-care quality by day and time of admission: a nationwide, registry-based, prospective cohort study of acute stroke care.

BACKGROUND: Studies in many health systems have shown evidence of poorer quality health care for patients admitted on weekends or overnight than for those admitted during the week (the so-called weekend effect). We postulated that variation in quality was dependent on not only day, but also time, of admission, and aimed to describe the pattern and magnitude of variation in the quality of acute stroke care across the entire week. METHODS: We did this nationwide, registry-based, prospective cohort study using data from the Sentinel Stroke National Audit Programme. We included all adult patients (aged >16 years) admitted to hospital with acute stroke (ischaemic or primary intracerebral haemorrhage) in England and Wales between April 1, 2013, and March 31, 2014. Our outcome measure was 30 day post-admission survival. We estimated adjusted odds ratios for 13 indicators of acute stroke-care quality by fitting multilevel multivariable regression models across 42 4-h time periods per week. FINDINGS: The study cohort comprised 74,307 patients with acute stroke admitted to 199 hospitals. Care quality varied across the entire week, not only between weekends and weekdays, with different quality measures showing different patterns and magnitudes of temporal variation. We identified four patterns of variation: a diurnal pattern (thrombolysis, brain scan within 12 h, brain scan within 1 h, dysphagia screening), a day of the week pattern (stroke physician assessment, nurse assessment, physiotherapy, occupational therapy, and assessment of communication and swallowing by a speech and language therapist), an off-hours pattern (door-to-needle time for thrombolysis), and a flow pattern whereby quality changed sequentially across days (stroke-unit admission within 4 h). The largest magnitude of variation was for door-to-needle time within 60 min (range in quality 35-66% [16/46-232/350]; coefficient of variation 18·2). There was no difference in 30 day survival between weekends and weekdays (adjusted odds ratio 1·03, 95% CI 0·95-1·13), but patients admitted overnight on weekdays had lower odds of survival (0·90, 0·82-0·99). INTERPRETATION: The weekend effect is a simplification, and just one of several patterns of weekly variation occurring in the quality of stroke care. Weekly variation should be further investigated in other health-care settings, and quality improvement should focus on reducing temporal variation in quality and not only the weekend effect. FUNDING: None.

Optomechanical Quantum Control of a Nitrogen-Vacancy Center in Diamond.

We demonstrate optomechanical quantum control of the internal electronic states of a diamond nitrogen-vacancy (NV) center in the resolved-sideband regime by coupling the NV to both optical fields and surface acoustic waves via a phonon-assisted optical transition and by taking advantage of the strong excited-state electron-phonon coupling of a NV center. Optomechanically driven Rabi oscillations as well as quantum interferences between the optomechanical sideband and the direct dipole-optical transitions are realized. These studies open the door to using resolved-sideband optomechanical coupling for quantum control of both the atomlike internal states and the motional states of a coupled NV-nanomechanical system, leading to the development of a solid-state analog of trapped ions.

Learning from the design and development of the NHS Safety Thermometer.

QUALITY ISSUE: Research indicates that 10% of patients are harmed by healthcare but data that can be used in real time to improve safety are not routinely available. INITIAL ASSESSMENT: We identified the need for a prospective safety measurement system that healthcare professionals can use to improve safety locally, regionally and nationally. CHOICE OF SOLUTION: We designed, developed and implemented a national tool, named the NHS Safety Thermometer (NHS ST) with the goal of measuring the prevalence of harm from pressure ulcers, falls, urinary tract infection in patients with catheters and venous thromboembolism on one day each month for all NHS patients. IMPLEMENTATION: The NHS ST survey instrument was developed in a learning collaborative involving 161 organizations (e.g. hospitals and other delivery organizations) using a Plan, Do, Study, Act method. EVALUATION: Testing of operational definitions, technical capability and use were conducted and feedback systems were established by site coordinators in each participating organization. During the 17-month pilot, site coordinators reported a total of 73,651 patient entries. LESSONS LEARNED: It is feasible to obtain national data through standardized reporting by site coordinators at the point of care. Some caution is required in interpreting data and work is required locally to ensure data collection systems are robust and data collectors were trained. Sampling is an important strategy to optimize efficiency and reduce the burden of measurement.

Exploring the Fibrous Nature of Single-Stranded DNA-Collagen Complexes: Nanostructural Observations and Physicochemical Insights.

Nucleic acid-collagen complexes (NACCs) are a self-assembled biomimetic fibrillary platform arising from the spontaneous complexation of single-stranded DNA (ssDNA) oligonucleotides and collagen. NACCs merge the extracellular matrix functionality of collagen with the tunable bioactivity of ssDNA as aptamers for broad biomedical applications. We hypothesize that NACCs offer a hierarchical architecture across multiple length scales that significantly varies compared to native collagen. We investigate this using atomic force microscopy and electron microscopy (transmission electron microscopy and cryogenic electron microscopy). Results demonstrate key topographical differences induced by adding ssDNA oligonucleotides to collagen type I. NACCs form a dense network of intertwined collagen fiber bundles in the microscale and nanoscale while retaining their characteristic D-band periodicities (∼67 nm). Additionally, our exploration of thermodynamic parameters governing the interaction indicates an entropically favorable NACC formation driven by ssDNA. Thermal analysis demonstrates the preservation of collagen's triple helical domains and a more stabilized polypeptide structure at higher temperatures than native collagen. These findings offer important insights into our understanding of the ssDNA-induced complexation of collagen toward the further establishment of structure-property relationships in NACCs and their future development into practical biomaterials. They also provide pathways for manipulating and enhancing collagenous matrices' properties without requiring complex chemical modifications or fabrication procedures.

Polysquaramides.

Thermoplastics, while advantageous for their processability and recyclability, often compromise thermochemical stability and mechanical strength compared to thermosets. Addressing this limitation, we introduce an innovative approach employing reversibly cross-linked polymers, utilizing squaramide moieties to reconcile recyclability and robustness. Herein, we detail the synthesis of supramolecularly cross-linked polysquaramides through the condensation polymerization of diethyl squarate with primary and secondary diamines. This methodology embeds hydrogen-bonding squaramide motifs into the polymer chains, yielding materials with significantly enhanced storage moduli, reaching up to 1.2 GPa. Material characterization via dynamic mechanical analysis, creep-recovery, and stress relaxation experiments delineate a distinctive rubbery plateau across a broad temperature range, excellent creep resistance, and multimodal viscoelastic flow, respectively, attributable to the dynamic nature of the supramolecular cross-links. Additionally, the study showcases the modulation of glass transition temperature (Tg) by altering the monomer composition and stoichiometry, demonstrating the tunability of polymer viscoelastic properties through precise control over hydrogen bonding interactions. Overall, the incorporation of squaramide motifs not only provides the structural integrity and mechanical performance of these thermoplastics but also leads to engineering materials with tailored viscoelastic characteristics.

P2X7 receptor knockout does not alter renal function or prevent angiotensin II-induced kidney injury in F344 rats.

P2X7 receptors mediate immune and endothelial cell responses to extracellular ATP. Acute pharmacological blockade increases renal blood flow and filtration rate, suggesting that receptor activation promotes tonic vasoconstriction. P2X7 expression is increased in kidney disease and blockade/knockout is renoprotective. We generated a P2X7 knockout rat on F344 background, hypothesising enhanced renal blood flow and protection from angiotensin-II-induced renal injury. CRISPR/Cas9 introduced an early stop codon into exon 2 of P2rx7, abolishing P2X7 protein in kidney and reducing P2rx7 mRNA abundance by ~ 60% in bone-marrow derived macrophages. The M1 polarisation response to lipopolysaccharide was unaffected but P2X7 receptor knockout suppressed ATP-induced IL-1ß release. In male knockout rats, acetylcholine-induced dilation of the renal artery ex vivo was diminished but not the response to nitroprusside. Renal function in male and female knockout rats was not different from wild-type. Finally, in male rats infused with angiotensin-II for 6 weeks, P2X7 knockout did not reduce albuminuria, tubular injury, renal macrophage accrual, and renal perivascular fibrosis. Contrary to our hypothesis, global P2X7 knockout had no impact on in vivo renal hemodynamics. Our study does not indicate a major role for P2X7 receptor activation in renal vascular injury.

Dynamic Ablative Networks: Shapeable Heat-Shielding Materials.

Thermoset materials sacrifice recyclability and reshapeability for increased chemical and mechanical robustness because of an immobilized, cross-linked polymeric matrix. The robust material properties of thermosets make them well-suited for applications such as heat-shielding materials (HSMs) or ablatives where excellent thermal stability, good mechanical strength, and high charring ability are paramount. Many of these material properties are characteristic of covalent adaptable networks (CANs), where the static connectivity of thermosets has been replaced with dynamic cross-links. This dynamic connectivity allows network mobility while retaining cross-link connectivity to permit damage repair and reshaping that are traditionally inaccessible for thermoset materials. Herein, we report the synthesis of hybrid inorganic-organic enaminone vitrimers that contain an exceptionally high weight percent of polyhedral oligomeric silsesquioxane (POSS)-derivatives. Polycondensation of ß-ketoester-containing POSS with various diamine cross-linkers led to materials with facile tunability, shapeability, predictable glass transition temperatures, good thermal stability, and high residual char mass following thermal degradation. Furthermore, the char materials show notable retention of their preordained shape following decomposition, suggesting their future utility in the design of HSMs with complex detailing.

High-performance polyimine vitrimers from an aromatic bio-based scaffold.

Bio-based vitrimers represent a promising class of thermosetting polymer materials, pairing the recyclability of dynamic covalent networks with the renewability of non-fossil fuel feedstocks. Vanillin, a low-cost lignin derivative, enables facile construction of polyimine networks marked by rapid exchange and sensitivity to acid-catalyzed hydrolysis. Furthermore, the aromatic structure makes it a promising candidate for the design of highly aromatic networks capable of high-performance thermal and dimensional stability. Such properties are paramount in polymeric thermal protection systems. Here, we report on the fabrication of polyimine networks with particularly high aromatic content from a novel trifunctional vanillin monomer prepared from the nucleophilic aromatic substitution of perfluoropyridine (PFP) on a multi-gram scale (>20 g) in high yield (86%). The trifunctional aromatic scaffold was then crosslinked with various diamines to demonstrate tunable viscoelastic behavior and thermal properties, with glass transition temperatures (Tg) ranging from 9 to 147 °C, degradation temperatures (5% mass loss) up to approximately 370 °C, and excellent char yields up to 68% at 650 °C under nitrogen. Moreover, the vitrimers displayed mechanical reprocessability over five destruction/healing cycles and rapid chemical recyclability following acidic hydrolysis at mild temperatures. Our findings indicate that vitrimers possessing tunable properties and high-performance thermomechanical behavior can be easily constructed from vanillin and electrophilic aromatic scaffolds for applications in heat-shielding materials and ablative coatings.

Demonstration of continuous gradient elution functionality with automated liquid handling systems for high-throughput purification process development.

Various high-throughput systems and strategies are employed by the biopharmaceutical industry for early to late-stage process development for biologics manufacturing. The associated increases to experiment productivity and reduction in material consumption makes high throughput tools integral for bioprocess development. While these high-throughput systems have been successfully leveraged to generate high quality data representative of manufacturing scale processes, their data interpretation often requires complex data transformation and time-intensive system characterization. With respect to high throughput purification development, RoboColumns by Repligen operated on Tecan automated liquid handling systems offer superior performance scalability, but lack an optimized liquid delivery system that is representative of preparative chromatography. Particularly, stock Tecan liquid handling systems lack the capability to provide high-capacity continuous liquid flow and ideal linear gradient chromatography conditions. These limitations impact protein chromatography performance and hinder the application of high-throughput gradient elution experiments. In this work, we describe a Tecan Freedom EVO high-throughput purification tool that provides more continuous liquid delivery enabling continuous gradient elution capability for RoboColumn experiments as demonstrated by generation of highly linear conductivity gradients. Results demonstrate that the tool can provide RoboColumn performance and product quality data that is in agreement with larger, bench scale chromatography formats for two model purification methods. The described gradient purification method also provides more consistent performance between RoboColumns and larger column formats compared to step elution methods using the same optimized Tecan system. Lastly, new insights into the impact of discontinuous flow on RoboColumn elution performance are introduced, which may help further improve application of these data towards bioprocess development.

What is the NHS Safety Thermometer?

The English National Health Service (NHS) announced a new programme to incentivize use of the NHS Safety Thermometer (NHS ST) in the NHS Operating Framework for 2012/13. For the first time, the NHS is using the Commissioning for Quality and Innovation (CQUIN) scheme, a contract lever, to incentivize ALL providers of NHS care to measure four common complications (harms) using the NHS ST in a proactive way on one day per month. This national CQUIN scheme provides financial reward for the collection of baseline data with a view to incentivizing the achievement of improvement goals in later years. In this paper, we describe the rationale for this large-scale data collection, the purpose of the instrument and its potential contribution to our current understanding of patient safety. It is not a comprehensive description of the method or preliminary data. This will be published separately. The focus of the NHS ST on pressure ulcers, falls, catheters and urine infection and venous thromboembolism is broadly applicable to patients across all healthcare settings, but is specifically pertinent to older people who, experiencing more healthcare intervention, are at risk of not one but multiple harms. In this paper, we also describe an innovative patient-level composite measure of the absence of harm from the four identified, termed as "harmfreecare" which is unique to the NHS ST and is under development to raise standards for patient safety.

Single-cell RNA sequencing profiling of mouse endothelial cells in response to pulmonary arterial hypertension.

AIMS: Endothelial cell (EC) dysfunction drives the initiation and pathogenesis of pulmonary arterial hypertension (PAH). We aimed to characterize EC dynamics in PAH at single-cell resolution. METHODS AND RESULTS: We carried out single-cell RNA sequencing (scRNA-seq) of lung ECs isolated from an EC lineage-tracing mouse model in Control and SU5416/hypoxia-induced PAH conditions. EC populations corresponding to distinct lung vessel types, including two discrete capillary populations, were identified in both Control and PAH mice. Differential gene expression analysis revealed global PAH-induced EC changes that were confirmed by bulk RNA-seq. This included upregulation of the major histocompatibility complex class II pathway, supporting a role for ECs in the inflammatory response in PAH. We also identified a PAH response specific to the second capillary EC population including upregulation of genes involved in cell death, cell motility, and angiogenesis. Interestingly, four genes with genetic variants associated with PAH were dysregulated in mouse ECs in PAH. To compare relevance across PAH models and species, we performed a detailed analysis of EC heterogeneity and response to PAH in rats and humans through whole-lung PAH scRNA-seq datasets, revealing that 51% of up-regulated mouse genes were also up-regulated in rat or human PAH. We identified promising new candidates to target endothelial dysfunction including CD74, the knockdown of which regulates EC proliferation and barrier integrity in vitro. Finally, with an in silico cell ordering approach, we identified zonation-dependent changes across the arteriovenous axis in mouse PAH and showed upregulation of the Serine/threonine-protein kinase Sgk1 at the junction between the macro- and microvasculature. CONCLUSION: This study uncovers PAH-induced EC transcriptomic changes at a high resolution, revealing novel targets for potential therapeutic candidate development.

A simple and rapid method for identifying and semi-quantifying peptide hormones in isolated pancreatic islets by direct-tissue matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

We describe a new, simple, robust and efficient method based on direct-tissue matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry that enables consistent semi-quantitation of peptide hormones in isolated pancreatic islets from normal and diabetic rodents. Prominent signals were measured that corresponded to all the main peptide hormones present in islet-endocrine cells: (α-cells) glucagon, glicentin-related polypeptide/GRPP; (ß-cells) insulin I, insulin II, C-peptide I, C-peptide II, amylin; (δ-cells) somatostatin-14; and (PP-cells), and pancreatic polypeptide. The signal ratios coincided with known relative hormone abundances. The method demonstrated that severe insulin deficiency is accompanied by elevated levels of all non-ß-cell-hormones in diabetic rat islets, consistent with alleviation of paracrine suppression of hormone production by non-ß-cells. It was also effective in characterizing hormonal phenotype in hemizygous human-amylin transgenic mice that express human and mouse amylin in approx. equimolar quantities. Finally, the method demonstrated utility in basic peptide-hormone discovery by identifying a prominent new Gcg-gene-derived peptide (theoretical monoisotopic molecular weight 3263.5 Da), closely related to but distinct from GRPP, in diabetic islets. This peptide, whose sequence is HAPQDTEENARSFPASQTEPLEDPNQINE in Rattus norvegicus, could be a peptide hormone whose roles in physiology and metabolic disease warrant further investigation. This method provides a powerful new approach that could provide important new insights into the physiology and regulation of peptide hormones in islets and other endocrine tissues. It has potentially wide-ranging applications that encompass endocrinology, pharmacology, phenotypic analysis in genetic models of metabolic disease, and hormone discovery, and could also effectively limit the numbers of animals required for such studies.