Chromophobe renal cell carcinoma (chRCC) is generally considered an indolent malignancy; however, studies have produced conflicting results regarding the differences among its histologic patterns. The prognostic significance of eosinophilic morphology in chRCC remains controversial, in part due to inconsistent diagnostic criteria and inclusion of morphologic mimickers. Cases were classified by 3 expert genitourinary pathologists into eosinophilic, classic, and mixed chRCC patterns based on strict morphologic and immunohistochemical criteria (n=110). Clinicopathologic features, copy number variations (CNVs), and targeted sequencing of TP53 , PTEN , and RB1 were assessed, along with pathway analyses using RNAseq and multiplex gene expression profiling. Clinicopathological and survival analysis were assessed using GraphPad Prism. The final cohort included 24 eosinophilic, 47 classic, and 39 mixed chRCC. CNV frequencies were similar across patterns, while RB1 aberrations and enrichment of oncogenic pathways (TP53, PTEN, RB1, DNA repair, mTOR, and cell cycle) were increased in both mixed and eosinophilic chRCC, with the greatest prominence in eosinophilic chRCC. Clinically, eosinophilic chRCC and mixed chRCC were associated with larger tumour size, more advanced clinical stage, and significantly worse disease-free survival (DFS) and overall survival (OS) compared with classic chRCC. On multivariate analysis, histologic pattern and stage remained prognostic of DFS. Therefore, eosinophilic chRCC showed a distinct molecular phenotype with enriched oncogenic pathways and poorer outcomes, while mixed chRCC displayed an intermediate but similarly aggressive profile. These findings indicate that eosinophilic morphology has potentially more aggressive biology than classic chRCC, underscoring the need for the introduction of specific diagnostic criteria for eosinophilic chRCC and its differentiation clinically.

Peripheral nerve injury denervates muscle, resulting in muscle paralysis and atrophy. This is reversible if timely muscle reinnervation occurs. With delayed reinnervation, the muscle’s reparative ability declines, and muscle-resident fibro-adipogenic progenitor cells (FAPs) proliferate and differentiate, inducing fibro-fatty muscle degradation and thereby physical disability. The mechanisms by which the peripheral nerve regulates FAPs expansion and differentiation are incompletely understood. Using the rat tibial neve transection model, we demonstrated an increased FAPs content and a changing FAPs phenotype, with an increased capacity for adipocyte and fibroblast differentiation, in gastrocnemius muscle post-denervation. The FAPs response was inhibited by immediate tibial nerve repair with muscle reinnervation via neuromuscular junctions (NMJs) and sensory organs (e.g., muscle spindles) or the sensory protection of muscle (where a pure sensory nerve is sutured to the distal tibial nerve stump) with reinnervation by muscle spindles alone. We found that both procedures reduced denervation-mediated increases in glial-cell-line-derived neurotrophic factor (GDNF) in muscle and that GDNF promoted FAPs adipogenic and fibrogenic differentiation in vitro. These results suggest that the peripheral nerve controls FAPs recruitment and differentiation via the modulation of muscle GDNF expression through NMJs and muscle spindles. GDNF can serve as a therapeutic target in the management of denervation-induced muscle injury.

Sepsis is the bodys dysfunctional response to infection associated with organ failure. Delays in diagnosis have a substantial impact on survival. Herein, samples from 586 in-house patients were used in conjunction with machine learning and cross-validation to narrow a gene expression signature of immune cell reprogramming to predict clinical deterioration in patients with suspected sepsis within the first 24 hours (h) of clinical presentation using just six genes (Sepset). The accuracy of the test (~90% in early intensive care unit (ICU) and 70% in emergency room patients) was validated in 3,178 patients from existing independent cohorts. A real-time reverse transcriptase polymerase chain reaction (RT-PCR)-based test was shown to have a 98% sensitivity in >230 patients to predict worsening of the sequential organ failure scores or admission to the ICU within the first 24 h following Sepset detection. A stand-alone centrifugal microfluidic instrument that integrates the entire automated workflow for detection of the Sepset classifier in whole blood using digital droplet PCR was developed and tested. This PREcision meDIcine for CriTical care (PREDICT) system had a high sensitivity of 92%, specificity of 89%, and an overall accuracy of 88% in identifying the risk of imminent clinical deterioration in patients with suspected sepsis. ### Competing Interest Statement Conflicts of Interest REWH is an inventor of the Sepset signature that has been patented in 17 countries (e.g. US patent 11,851,717 issued Dec 26, 2023) and is CEO and a shareholder of Asep Medical and its subsidiary Sepset BioSciences Inc. that have licensed in these patents and are actively commercially developing sepsis diagnostics. REWH also has a contract from Sepset Biosciences for development of diagnostic assays for adult sepsis. PGYZ and EFH are employees of Sepset Biosciences Inc. and/or Asep Medical. ### Clinical Protocols ### Funding Statement Funding statement included in manuscript ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Unity Health Toronto REB approval I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Previously published data can be obtained from GEO. Data from internal validation cohort can be obtained upon request

The COVID-19 pandemic has created an urgency to study the host gene response that leads to variable clinical presentations of the disease, particularly the critical illness response. miRNAs have been implicated in the mechanism of host immune dysregulation and thus hold potential as biomarkers and/or therapeutic agents with clinical application. Hence, further analyses of their altered expression in COVID-19 is warranted. An important basis for this is identifying appropriate reference genes for high quality expression analysis studies. In the current report, NanoString technology was used to study the expression of 798 miRNAs in the peripheral blood of 24 critically ill patients, 12 had COVID-19 and 12 were COVID-19 negative. A list of potentially stable candidate reference genes was generated that included ten miRNAs. The top six were analyzed using reverse transcription quantitative polymerase chain reaction (RT-qPCR) in a total of 41 patients so as to apply standard computational algorithms for validating reference genes, namely geNorm, NormFinder, BestKeeper and RefFinder. There was general agreement among all four algorithms in the ranking of four stable miRNAs: miR-186-5p, miR-148b-3p, miR-194-5p and miR-448. A detailed analysis of their output rankings led to the conclusion that miR-186-5p and miR-148b-3p are appropriate reference genes for miRNA expression studies using PaxGene tubes in the peripheral blood of patients critically ill with COVID-19 disease.

The recent COVID-19 pandemic has highlighted the need for diagnostic platforms that are rapidly adaptable, affordable, and accessible globally, especially for low-resource settings. To address this need, we describe the development and functional validation of a novel immunoassay technique termed the cell-free dot blot (CFDB) method.

Virus-induced lung injury is associated with loss of pulmonary epithelial-endothelial tight junction integrity. While the alveolar-capillary membrane may be an indirect target of injury, viruses may interact directly and/or indirectly with miRs to augment their replication potential and evade the host antiviral defense system. Here, we expose how the influenza virus (H1N1) capitalizes on host-derived interferon-induced, microRNA (miR)-193b-5p to target occludin and compromise antiviral defenses. Lung biopsies from patients infected with H1N1 revealed increased miR-193b-5p levels, marked reduction in occludin protein, and disruption of the alveolar-capillary barrier. In C57BL/6 mice, the expression of miR-193b-5p increased, and occludin decreased, 5-6 days post-infection with influenza (PR8). Inhibition of miR-193b-5p in primary human bronchial, pulmonary microvascular, and nasal epithelial cells enhanced antiviral responses. miR-193b-deficient mice were resistant to PR8. Knockdown of occludin, both in vitro and in vivo, and overexpression of miR-193b-5p reconstituted susceptibility to viral infection. miR-193b-5p inhibitor mitigated loss of occludin, improved viral clearance, reduced lung edema, and augmented survival in infected mice. Our results elucidate how the innate immune system may be exploited by the influenza virus and how strategies that prevent loss of occludin and preserve tight junction function may limit susceptibility to virus-induced lung injury.

Abstract Background Intensive care unit (ICU)‐acquired weakness is characterized by muscle atrophy and impaired contractility that may persist after ICU discharge. Dysregulated muscle repair and regeneration gene co‐expression networks are present in critical illness survivors with persistent muscle wasting and weakness. We aimed to identify microRNAs (miRs) regulating the gene networks and determine their role in the self‐renewal of muscle in ICU survivors. Methods Muscle whole‐transcriptome expression was assessed with microarrays in banked quadriceps biopsies obtained at 7 days and 6 months post‐ICU discharge from critically ill patients (n = 15) in the RECOVER programme and healthy individuals (n = 8). We conducted an integrated miR–messenger RNA analysis to identify miR/gene pairs associated with muscle recovery post‐critical illness and evaluated their impact on myoblast proliferation and differentiation in human AB1167 and murine C2C12 cell lines in vitro. Select target genes were validated with quantitative PCR. Results Twenty‐two miRs were predicted to regulate the Day 7 post‐ICU muscle transcriptome vs. controls. Thirty per cent of all differentially expressed genes shared a 3'UTR regulatory sequence for miR‐424‐3p/5p, which was 10‐fold down‐regulated in patients (P < 0.001) and correlated with quadriceps size (R = 0.86, P < 0.001), strength (R = 0.75, P = 0.007), and physical function (Functional Independence Measures motor subscore, R = 0.92, P < 0.001) suggesting its potential role as a master regulator of early recovery of muscle mass and strength following ICU discharge. Network analysis demonstrated enrichment for cellular respiration and muscle fate commitment/development related genes. At 6 months post‐ICU discharge, a 14‐miR expression signature, including miRs‐490‐3p and ‐744‐5p, identified patients with muscle mass recovery vs. those with sustained atrophy. Constitutive overexpression of the novel miR‐490‐3p significantly inhibited AB1167 and C2C12 myoblast proliferation (cell count AB1167 miR‐490‐3p mimic or scrambled‐miR transfected myoblasts 7926 ± 4060 vs. 14 159 ± 3515 respectively, P = 0.006; proportion Ki67‐positive nuclei AB1167 miR‐490‐3p mimic or scrambled‐miR transfected myoblasts 0.38 ± 0.07 vs. 0.54 ± 0.06 respectively, P < 0.001; proliferating cell nuclear antigen expression AB1167 miR‐490‐3p mimic or scrambled‐miR transfected myoblasts 11.48 ± 1.97 vs. 16.75 ± 1.19 respectively, P = 0.040). Constitutive overexpression of miR‐744‐5p, a known regulator of myogenesis, significantly inhibited AB1167 and C2C12 myoblast differentiation (fusion index AB1167 miR‐744‐5p mimic or scrambled‐miR transfected myoblasts 8.31 ± 7.00% vs. 40.29 ± 9.37% respectively, P < 0.001; myosin heavy chain expression miR‐744‐5p mimic or scrambled‐miR transfected myoblasts 0.92 ± 0.39 vs. 13.53 ± 5.5 respectively, P = 0.01). Conclusions Combined functional transcriptomics identified 36 miRs including miRs‐424‐3p/5p, ‐490‐3p, and ‐744‐5p as potential regulators of gene networks associated with recovery of muscle mass and strength following critical illness. MiR‐490‐3p is identified as a novel regulator of myogenesis.

With sunitinib treatment of metastatic renal cell carcinoma, most patients end up developing resistance over time. Recent clinical trials have shown that individualizing treatment protocols could delay resistance and result in better outcomes. We developed an in vivo xenograft tumor model and compared tumor growth rate, morphological, and transcriptomic differences between alternative and traditional treatment schedules. Our results show that the alternative treatment regime could delay/postpone cancer progression. Additionally, we identified distinct morphological changes in the tumor with alternative and traditional treatments, likely due to the significantly dysregulated signaling pathways between the protocols. Further investigation of the signaling pathways underlying these morphological changes may lead potential therapeutic targets to be used in a combined treatment with sunitinib, which offers promise in postponing/reversing the resistance of sunitinib.

Angiogenesis is a critical process in tumor progression. Inhibition of angiogenesis by blocking VEGF signaling can impair existing tumor vessels and halt tumor progression. However, the benefits are transient, and most patients who initially respond to these therapies develop resistance. Accordingly, there is a need for new anti-angiogenesis therapeutics to delay the processes of resistance or eliminate the resistive effects entirely. This manuscript presents the results of a screen of the National Institutes of Health Clinical Collections Libraries I & II (NIHCCLI&II) for novel angiogenesis inhibitors. The 727 compounds of the NIHCCLI&II library were screened with a high-throughput drug discovery platform (HTP) developed previously with angiogenesis-specific protocols utilizing zebrafish. The screen resulted in 14 hit compounds that were subsequently narrowed down to one, with PD 81,723 chosen as the lead compound. PD 81,723 was validated as an inhibitor of angiogenesis in vivo in zebrafish and in vitro in human umbilical vein endothelial cells (HUVECs). Zebrafish exposed to PD 81,723 exhibited several signs of a diminished endothelial network due to the inhibition of angiogenesis. Immunochemical analysis did not reveal any significant apoptotic or mitotic activity in the zebrafish. Assays with cultured HUVECs elucidated the ability of PD 81,723 to inhibit capillary tube formation, migration, and proliferation of endothelial cells. In addition, PD 81,723 did not induce apoptosis while significantly down regulating p21, AKT, VEGFR-2, p-VEGFR-2, eNOS, and p-eNOS, with no notable change in endogenous VEGF-A in cultured HUVECs.

Introduction There remains a paucity of data comparing ambulatory initiation of home non-invasive ventilation (NIV) with a model requiring inpatient admission.1 In our institution, a Quality Improvement (QI) project was performed where an ambulatory model for NIV initiation was developed and evaluated. Methods Ambulatory pathways were formulated for NIV initiation in the outpatient setting, alongside outreach and initiation of NIV for inpatients referred within regional hospitals. The primary outcome measure was 'compliance with NIV' defined as NIV use ≥4 hours/night for ≥75% of nights.2 Results Between 6.1.20 and 1.7.20, 76 referrals for home NIV were assessed within the ambulatory model. Of these, NIV was not indicated in 3 cases and contraindicated in 1 case, while 2 trialled NIV and declined it, leaving 70 patients who commenced home NIV (n=36 following COVID-19 ward 'closure'). Neuromuscular disease was the principal diagnosis in 41% (29/70) with MND comprising 20/29 neuromuscular cases; see table 1. Ventilator interaction data was available for 68 patients where mean NIV use was 5.21 (SD 3.98) hours/night. Of those established by ambulatory pathway, 62% (42/68) were deemed 'compliant' with NIV in comparison to previous data reporting compliance in 62% (56/90) of subjects established through inpatient admission2. It was calculated that delivery of the ambulatory pathway resulted in a cost saving of £197,967 for this period, achieved principally by admission avoidance based on previous length of stay data and Level 2 bed costings. Conclusions An ambulatory model for initiation of home NIV appears to be as effective in achieving compliance as inpatient admission, while carrying health economic benefits. Ambulatory treatment pathways enabled us to deliver service continuity during the COVID-19 pandemic. References Mandal S, Arbane G, Murphy P. et al. Medium-term cost-effectiveness of an automated non-invasive ventilation outpatient set-up versus a standard fixed level non-invasive ventilation inpatient set-up in obese patients with chronic respiratory failure: a protocol description. BMJ Open. 2015; 5; e007082. Ward K, Chakrabarti B, Ashcroft H. et al. Compliance with non-invasive ventilation (NIV) at 90 days may be associated with use of NIV in the first 2 weeks: investigation of telemonitoring data. AJRCCM. 2018; 197; A1463.

Object: Quantification of urinary miRNAs can be challenging especially for low abundance miRNAs. We aimed to optimize the quantification of urinary exosomal miRNAs and compare the performance efficiency between droplet digital PCR (ddPCR) and real-time quantitative PCR (qPCR). Methods: We optimized a number of parameters for ddPCR such as annealing temperatures, annealing time and PCR cycle number. We also compared the performance of ddPCR and qPCR. Results: By comparing the fluorescence amplification separation, the optimal annealing temperature was 59 degrees C, optimal annealing time was 60s and optimal cycle number was 45 for measuring urinary exosomal miRNAs. ddPCR had much higher technical sensitivity compared to qPCR. The minimal detectable concentration of miR-29a was < 50 copies/mu L by ddPCR compared to 6473 copies/mu L for qPCR. Also, ddPCR generated more consistent results for serially diluted samples compared to qPCR. ddPCR generated smaller within-run variations than qPCR though this did not reach statistical significance. It also resulted in better reproducibility with smaller between-run variations. Conclusions: Optimization of urinary exosomal miRNA ddPCR assay is dependent on assessing key variables including experimental annealing temperature and time as well as the number of PCR cycles. ddPCR has a higher sensitivity, reproducibility, and accuracy in comparison to qPCR.

Papillary renal cell carcinoma (PRCC) is the most common type of RCC in end-stage kidney disease (ESKD). Papillary adenoma (PA) is a small benign lesion morphologically similar to PRCC and is suggested to be its precursor. PA is also prevalent in ESKD. The evolution of PAs to PRCCs and their relationship to ESKD are poorly understood. A total of 140 PAs, normal kidneys, ESKDs, and PRCCs were analyzed. Previously described markers of renal tubular progenitor cells were analyzed using immunohistochemistry and quantified with digital analysis. Progenitor cells were significantly increased in ESKD (P < 0.0001) and PAs (P = 0.02) in comparison with the normal kidney. Pathway analysis using global miRNA and chromosomal copy number variations revealed a common developmental theme between PA and the PRCCs. Whole exome sequencing showed a KMT2C-specific pathogenic mutation among all PAs and PRCCs. KMT2C is a chromosome 7 epigenetic regulator implicated in development and oncogenesis. Collectively, results show possible connection of PRCCs to PA and the progenitor-like cell population, which are increased in response to renal tubular injury. In addition, each PRCC histologic subtype had its own set of mutational changes, indicating divergence from a common precursor. The study reports previously unknown biological aspects of PRCC development and could influence current surveillance criteria and early detection strategies of PRCC tumors.

Objective: The pathophysiologic processes of abdominal aortic aneurysms (AAAs) and atherosclerosis often intersect. Given that anomalies in vascular smooth muscle cell (SMC) autophagy have been noted in models of atherosclerosis, we sought to evaluate the potential role that SMC autophagy may play in the initiation and progression of AAAs. Methods: Studies were conducted in ATG7(flx/flx) SM22 alpha-Cre(tg/+) (SMC ATG7 knockout [SMC-ATG7-KO]) and ATG7(WT/WT); SM22 alpha-Cre(tg/+) (SMC ATG7 wild-type [SMC-ATG7-WT]) littermates that were continuously infused with angiotensin II (Ang II; 1.5mg/kg/d) for up to 12weeks. Mortality, morbidity, hemodynamics, and aortic remodeling were documented. Results: During the 12-week observation window, all of the Ang II-treated SMC-ATG-WT mice (n = 6) survived, whereas 10 of the 19 Ang II-treated SMC-ATG-KO mice had died by week 7 (log-rank test, P <.001). Mean arterial pressure (128.07 +/- 3.4mm Hg for Ang II-treated SMC-ATG-KO vs 138.5 +/- 5.87mm Hg for Ang II-treated SMC-ATG-WT mice) and diastolic arterial pressure (109.7 +/- 2.55mm Hg for Ang II-treated SMC-ATG7-KO vs 119.4 +/- 2.12mm Hg for Ang II-treated SMC-ATG7-WT mice) were significantly different between the two groups (P <.01). Cardiac rupture, myocardial infarct, end-organ damage, pleural effusion, and venous distention were noted in Ang II-treated SMC-ATG7-KO but not in Ang II-treated SMC-ATG7-WT mice. Although the suprarenal aortic diameters of the Ang II-treated SMC-ATG7-KO group demonstrated a trending increase (at week 4, 1.26 +/- 0.06mm [n = 14] for Ang II-treated SMC-ATG-KO mice vs 1.09 +/- 0.02mm [n = 5] for Ang II-treated SMC-ATG-WT mice; P <.05), only 2 of the 19 developed abdominal aortic dissections. Conclusions: Mice with SMC ATG7 deficiency that are chronically infused with Ang II do not tend to develop dissecting AAA but do exhibit adverse aortic remodeling and appreciable cardiac failure-associated mortality. (J Vasc Surg 2018; 68: 859-71.) Clinical Relevance: Although loss of smooth muscle cell ATG7 had little impact on the development and progression of an experimental model of abdominal aortic aneurysm, it did promote both adverse aortic remodeling and cardiac failure-associated mortality.

Background OSA is an increasingly recognised disease and successfully treated with CPAP. There are ever increasing demands on sleep services in the UK, in terms of provision, compliance and safety. One possible solution is the use of remote monitoring soon after CPAP set-up to determine usage patterns, residual apnoea-hypopnoea index (AHI) and mask problems. We have analysed our remote monitoring database to assess new patients with OSA set-up on CPAP. Method Retrospective data was collected from patients with OSA commenced on CPAP from start of June 2017 for thirty days. All patients referred to the Aintree University Hospital Sleep service had a cardio-respiratory sleep study and subsequently, if appropriate, referral for CPAP. All patients with OSA were commenced on a Resmed Airsense S10 device with humidification, using a predictive algorithm, and had an assessment for an appropriate interface. All data was collected with patient consent using Resmed Airview. Results Data was available in 71 patients (male 70%) for at least two weeks within this time period. Mean CPAP usage was 5 hours 4 mins with 84.5% using full face masks. Mean Residual AHI was low at 8.5 with small numbers of residual central events recorded (Table 1). See Table for more details Conclusion Remote monitoring provides a large amount of useful data which can potentially help improve CPAP provision in the UK. There is large proportion of patient with nocturnal hypoventilation, and despite effective treatment with CPAP, a group with a residual increase in AHI. Non-compliance and mask leak are identified issues and twenty patients used CPAP for less than an hour a night, with seven of those not at all. Remote monitoring allows early detection of non-compliance and an opportunity for earlier intervention to improve management in this patient group