Generation and Study of Antibodies against Two Triangular Trimers Derived from Aß.

Monoclonal antibodies (mAbs) that target the P-amyloid peptide (Aß) are important Alzheimer's disease research tools and are now being used as Alzheimer's disease therapies. Conformation-specific mAbs that target oligomeric and fibrillar Aß assemblies are of particular interest, as these assemblies are associated with Alzheimer's disease pathogenesis and progression. This paper reports the generation of rabbit mAbs against two different triangular trimers derived from Aß. These antibodies are the first mAbs generated against Aß oligomer mimics in which the high-resolution structures of the oligomers are known. We describe the isolation of the mAbs using single B-cell sorting of peripheral blood mononuclear cells (PBMCs) from immunized rabbits, the selectivity of the mAbs for the triangular trimers, the immunoreactivity of the mAbs with aggregated Aß42, and the immunoreactivity of the mAbs in brain tissue from the 5xFAD Alzheimer's disease mouse model. The characterization of these mAbs against structurally defined trimers derived from Aß enhances understanding of antibody-amyloid recognition and may benefit the development of diagnostics and immunotherapies in Alzheimer's disease.

Effectiveness of an anti-inflammatory diet versus low-fat diet for knee osteoarthritis: the FEAST randomised controlled trial protocol.

INTRODUCTION: Chronic inflammation plays a key role in knee osteoarthritis pathophysiology and increases risk of comorbidities, yet most interventions do not typically target inflammation. Our study will investigate if an anti-inflammatory dietary programme is superior to a standard care low-fat dietary programme for improving knee pain, function and quality-of-life in people with knee osteoarthritis. METHODS AND ANALYSIS: The eFEct of an Anti-inflammatory diet for knee oSTeoarthritis study is a parallel-group, assessor-blinded, superiority randomised controlled trial. Following baseline assessment, 144 participants aged 45-85 years with symptomatic knee osteoarthritis will be randomly allocated to one of two treatment groups (1:1 ratio). Participants randomised to the anti-inflammatory dietary programme will receive six dietary consultations over 12 weeks (two in-person and four phone/videoconference) and additional educational and behaviour change resources. The consultations and resources emphasise nutrient-dense minimally processed anti-inflammatory foods and discourage proinflammatory processed foods. Participants randomised to the standard care low-fat dietary programme will receive three dietary consultations over 12 weeks (two in-person and one phone/videoconference) consisting of healthy eating advice and education based on the Australian Dietary Guidelines, reflecting usual care in Australia. Adherence will be assessed with 3-day food diaries. Outcomes are assessed at 12 weeks and 6 months. The primary outcome will be change from baseline to 12 weeks in the mean score on four Knee injury and Osteoarthritis Outcome Score (KOOS4) subscales: knee pain, symptoms, function in daily activities and knee-related quality of life. Secondary outcomes include change in individual KOOS subscale scores, patient-perceived improvement, health-related quality of life, body mass and composition using dual-energy X-ray absorptiometry, inflammatory (high-sensitivity C reactive protein, interleukins, tumour necrosis factor-α) and metabolic blood biomarkers (glucose, glycated haemoglobin (HbA1c), insulin, liver function, lipids), lower-limb function and physical activity. ETHICS AND DISSEMINATION: The study has received ethics approval from La Trobe University Human Ethics Committee. Results will be presented in peer-reviewed journals and at international conferences. TRIAL REGISTRATION NUMBER: ACTRN12622000440729.

Stem cell dynamics and cellular heterogeneity across lineage subtypes of castrate resistant prostate cancer.

To resist lineage-dependent therapies such as androgen receptor inhibition, prostate luminal epithelial adenocarcinoma cells often adopt a stem-like state resulting in lineage-plasticity and phenotypic heterogeneity. Castrate resistant prostate adenocarcinoma can transition to neuroendocrine and occasionally to amphicrine, co-expressed luminal and neuroendocrine, phenotypes. We developed CRPC patient-derived organoid models that preserve heterogeneity of the originating tumor, including an amphicrine model displaying a range of luminal and neuroendocrine phenotypes. To gain biological insight and to identify potential treatment targets within heterogeneous tumor cell populations, we assessed the lineage hierarchy and molecular characteristics of various CRPC tumor subpopulations. Transcriptionally similar stem/progenitor cells were identified for all lineage populations. Lineage tracing in amphicrine CRPC showed that heterogeneity originated from distinct subclones of infrequent stem/progenitor cells that produced mainly quiescent differentiated amphicrine progeny. By contrast, adenocarcinoma CRPC progeny originated from stem/progenitor cells and self-renewing differentiated luminal cells. NEPC was composed almost exclusively of self-renewing stem/progenitor cells. Amphicrine subpopulations were enriched for secretory luminal, mesenchymal, and enzalutamide treatment persistent signatures that characterize clinical progression. Finally, the amphicrine stem/progenitor subpopulation was specifically depleted with an AURKA inhibitor, which blocked tumor growth. These data illuminate distinct stem cell characteristics for subtype-specific CRPC in addition to demonstrating a context for targeting differentiation-competent prostate stem cells.

Adiposity and cartilage lesions following ACL reconstruction.

OBJECTIVE: To determine if global, central, or peripheral adiposity is associated with prevalent and worsening cartilage lesions following anterior cruciate ligament reconstruction (ACLR). METHODS: In 107 individuals one-year post-ACLR, adiposity was assessed globally (body mass index, BMI), centrally (waist circumference), and peripherally (knee subcutaneous adipose tissue thickness) from magnetic resonance imaging (MRI). Tibiofemoral and patellofemoral cartilage lesions were assessed from knee MRIs at 1- and 5-years post-ACLR. Poisson regression evaluated the relation of adiposity with prevalent and worsening tibiofemoral and patellofemoral cartilage lesions adjusting for age, sex, and activity level. RESULTS: The prevalence ratios of adiposity with tibiofemoral (presence in 49%) and patellofemoral (44%) cartilage lesions ranged from 0.99 to 1.03. Adiposity was more strongly associated with longitudinal changes in tibiofemoral (worsening in 21%) and patellofemoral (44%) cartilage lesions. One-unit increase in global (kg/m2), central (cm), and peripheral (mm) adiposity was associated with a higher risk of worsening tibiofemoral cartilage lesions by 17% (risk ratios [95% CI]: 1.17 [1.09 to 1.23]), 5% (1.05 [1.02 to 1.08]), and 9% (1.09 [1.03 to 1.16]), and patellofemoral cartilage lesions by 5% (1.05 [1.00 to 1.12]) 2% (1.02 [1.00 to 1.04]) and 2% (1.02 [1.00 to 1.04]), respectively. CONCLUSION: Greater adiposity was a risk factor for worsening cartilage lesions up to 5 years post-ACLR. Clinical interventions aimed at mitigating excess adiposity may be beneficial in preventive approaches for early post-traumatic OA.

Recruitment and baseline characteristics of young adults at risk of early-onset knee osteoarthritis after ACL reconstruction in the SUPER-Knee trial.

Objectives: The study aims to (1) report the process of recruiting young adults into a secondary knee osteoarthritis prevention randomised controlled trial (RCT) after anterior cruciate ligament reconstruction (ACLR); (2) determine the number of individuals needed to be screened to include one participant (NNS) and (3) report baseline characteristics of randomised participants. Methods: The SUpervised exercise-therapy and Patient Education Rehabilitation (SUPER)-Knee RCT compares SUPER and minimal intervention for young adults (aged 18-40 years) with ongoing symptoms (ie, mean score of <80/100 from four Knee injury and Osteoarthritis Outcome Score subscales (KOOS4)) 9-36 months post-ACLR. The NNS was calculated as the number of prospective participants screened to enrol one person. At baseline, participants provided medical history, completed questionnaires (demographic, injury/surgery, rehabilitation characteristics) and underwent physical examination. Results: 1044 individuals were screened to identify 567 eligible people, from which 184 participants (63% male) enrolled. The sample of enrolled participants was multicultural (29% born outside Australia; 2% Indigenous Australians). The NNS was 5.7. For randomised participants, mean±SD age was 30±6 years. The mean body mass index was 27.3±5.2 kg/m2, with overweight (43%) and obesity (21%) common. Participants were, on average, 2.3 years post-ACLR. Over half completed <8 months of postoperative rehabilitation, with 56% having concurrent injury/surgery to meniscus and/or cartilage. The most affected KOOS (0=worst, 100=best) subscale was quality of life (mean 43.7±19.1). Conclusion: Young adults post-ACLR were willing to participate in a secondary osteoarthritis prevention trial. Sample size calculations should be multiplied by at least 5.7 to provide an estimate of the NNS. The SUPER-Knee cohort is ideally positioned to monitor and intervene in the early development and trajectory of osteoarthritis. Trial registration number: ACTRN12620001164987.

Expert Opinion on Current Trends in the Use of Insulin in the Management of People with Type 2 Diabetes from the South-Eastern European Region and Israel.

Despite the availability of various antihyperglycaemic therapies and comprehensive guidelines, glycaemic control in diabetes management has not improved significantly during the last decade in the real-world clinical setting. Treatment inertia arising from a complex interplay among patient-, clinician- and healthcare-system-related factors is the prime reason for this suboptimal glycaemic control. Also, the key factor leading to inadequate glycaemic levels remains limited communication between healthcare professionals (HCPs) and people with type 2 diabetes (PwT2D). Early insulin administration has several advantages including reduced glucotoxicity, high efficacy and preserved ß-cell mass/function, leading to lowering the risk of diabetes complications. The current publication is based on consensus of experts from the South-Eastern European region and Israel who reviewed the existing evidence and guidelines for the treatment of PwT2D. Herein, the experts emphasised the timely use of insulin, preferably second-generation basal insulin (BI) analogues and intensification using basal-plus therapy, as the most-potent glucose-lowering treatment choice in the real-world clinical setting. Despite an increase in the use of glucagon-like peptide-1 receptor agonists (GLP-1 RAs), the experts urged timely insulin initiation for inadequate glycaemic control in PwT2D. Furthermore, the combination of BI and GLP-1 RA addressing both fasting plasma glucose and post-prandial excursions as a free- or fixed-ratio combination was identified to reduce treatment complexity and burden. To minimise discontinuation and improve adherence, the experts reiterated quality, regular interactions and discussions between HCPs and PwT2D/carers for their involvement in the diabetes management decision-making process. Clinicians and HCPs should consider the opinions of the experts in accordance with the most recent recommendations for diabetes management.

No sign of weakness: a systematic review and meta-analysis of hip and calf muscle strength after anterior cruciate ligament injury.

OBJECTIVE: We aimed to determine hip and lower-leg muscle strength in people after ACL injury compared with an uninjured control group (between people) and the uninjured contralateral limb (between limbs). DESIGN: Systematic review with meta-analysis. DATA SOURCES: MEDLINE, EMBASE, CINAHL, Scopus, Cochrane CENTRAL and SportDiscus to 28 February 2023. ELIGIBILITY CRITERIA: Primary ACL injury with mean age 18-40 years at time of injury. Studies had to measure hip and/or lower-leg muscle strength quantitatively (eg, dynamometer) and report muscle strength for the ACL-injured limb compared with: (i) an uninjured control group and/or (ii) the uninjured contralateral limb. Risk of bias was assessed according to Cochrane Collaboration domains. RESULTS: Twenty-eight studies were included (n=23 measured strength ≤12 months post-ACL reconstruction). Most examined hip abduction (16 studies), hip extension (12 studies) and hip external rotation (7 studies) strength. We found no meaningful difference in muscle strength between people or between limbs for hip abduction, extension, internal rotation, flexion or ankle plantarflexion, dorsiflexion (estimates ranged from -9% to +9% of comparator). The only non-zero differences identified were in hip adduction (24% stronger on ACL limb (95% CI 8% to 42%)) and hip external rotation strength (12% deficit on ACL limb (95% CI 6% to 18%)) compared with uninjured controls at follow-ups >12 months, however both results stemmed from only two studies. Certainty of evidence was very low for all outcomes and comparisons, and drawn primarily from the first year post-ACL reconstruction. CONCLUSION: Our results do not show widespread or substantial muscle weakness of the hip and lower-leg muscles after ACL injury, contrasting deficits of 10%-20% commonly reported for knee extensors and flexors. As it is unclear if deficits in hip and lower-leg muscle strength resolve with appropriate rehabilitation or no postinjury or postoperative weakness occurs, individualised assessment should guide training of hip and lower-leg strength following ACL injury. PROSPERO REGISTRATION NUMBER: CRD42020216793.

HaplotagLR: An efficient and configurable utility for haplotagging long reads.

Understanding the functional effects of sequence variation is crucial in genomics. Individual human genomes contain millions of variants that contribute to phenotypic variability and disease risks at the population level. Because variants rarely act in isolation, we must consider potential interactions of neighboring variants to accurately predict functional effects. We can accomplish this using haplotagging, which matches sequencing reads to their parental haplotypes using alleles observed at known heterozygous variants. However, few published tools for haplotagging exist and these share several technical and usability-related shortcomings that limit applicability, in particular a lack of insight or control over error rates, and lack of key metrics on the underlying sources of haplotagging error. Here we present HaplotagLR: a user-friendly tool that haplotags long sequencing reads based on a multinomial model and existing phased variant lists. HaplotagLR is user-configurable and includes a basic error model to control the empirical FDR in its output. We show that HaplotagLR outperforms the leading haplotagging method in simulated datasets, especially at high levels of specificity, and displays 7% greater sensitivity in haplotagging real data. HaplotagLR advances both the immediate utility of haplotagging and paves the way for further improvements to this important method.

Identifying distinct neural features between the initial and corrective phases of precise reaching using AutoLFADS.

Many initial movements require subsequent corrective movements, but how motor cortex transitions to make corrections and how similar the encoding is to initial movements is unclear. In our study, we explored how the brain's motor cortex signals both initial and corrective movements during a precision reaching task. We recorded a large population of neurons from two male rhesus macaques across multiple sessions to examine the neural firing rates during not only initial movements but also subsequent corrective movements. AutoLFADS, an auto-encoder-based deep-learning model, was applied to provide a clearer picture of neurons' activity on individual corrective movements across sessions. Decoding of reach velocity generalized poorly from initial to corrective submovements. Unlike initial movements, it was challenging to predict the velocity of corrective movements using traditional linear methods in a single, global neural space. We identified several locations in the neural space where corrective submovements originated after the initial reaches, signifying firing rates different than the baseline before initial movements. To improve corrective movement decoding, we demonstrate that a state-dependent decoder incorporating the population firing rates at the initiation of correction improved performance, highlighting the diverse neural features of corrective movements. In summary, we show neural differences between initial and corrective submovements and how the neural activity encodes specific combinations of velocity and position. These findings are inconsistent with assumptions that neural correlations with kinematic features are global and independent, emphasizing that traditional methods often fall short in describing these diverse neural processes for online corrective movements.Significance Statement We analyzed submovement neural population dynamics during precision reaching. Using an auto-encoder-based deep-learning model, AutoLFADS, we examined neural activity on a single-trial basis. Our study shows distinct neural dynamics between initial and corrective submovements. We demonstrate the existence of unique neural features within each submovement class that encode complex combinations of position and reach direction. Our study also highlights the benefit of state-specific decoding strategies, which consider the neural firing rates at the onset of any given submovement, when decoding complex motor tasks such as corrective submovements.

Continuous home terlipressin infusion increases handgrip strength and reduces ascites-A prospective randomized crossover study.

BACKGROUND AND AIMS: Observational studies suggest a beneficial effect of continuous terlipressin infusion (CTI) on ascites and sarcopenia in decompensated cirrhosis with portal hypertension. APPROACH AND RESULTS: This single-center, prospective, cross-over study randomized 30 patients with cirrhosis, ascites, and sarcopenia to commence on 12 weeks of home CTI or 12 weeks of observation prior to cross-over. The co-primary outcomes were change in handgrip strength and paracentesis volume. Secondary outcomes included quality of life, sarcopenia measures, renal function, safety, and hospitalization. The median age of participants was 62 years (IQR: 57-64), the median Model for End-Stage Liver Disease-Sodium was 16 (12.3-20.8), and 22 (73%) were male. Handgrip strength increased by a mean adjusted difference (MAD) of 3.09 kg (95% CI: 1.11-5.08 kg) between CTI and observation ( p =0.006); an 11.8% increase from baseline. The total volume of ascites drained decreased by a MAD of 11.39L (2.99-19.85, p =0.01), with 1.75 fewer episodes of paracentesis (0.925-2.59, p <0.001) on CTI. Serum creatinine decreased, urinary sodium excretion increased, and quality of life was significantly higher on CTI (all p <0.001), with an increase in Chronic Liver Disease Questionnaire score of 0.41 points (0.23-0.59). There were 7 minor line-related complications but no cardiac events or pulmonary edema. CONCLUSIONS: This novel study demonstrates a significant increase in handgrip strength, reduction in paracentesis volume, and improved quality of life in patients with decompensated cirrhosis treated with continuous terlipressin infusion. These findings provide a strong rationale for the use of ambulatory CTI in appropriately selected patients with cirrhosis.

Supramolecular Interactions of Teixobactin Analogues in the Crystal State.

This Note presents the X-ray crystallographic structure of the N-methylated teixobactin analogue N-Me-d-Gln4,Lys10-teixobactin (1). Eight peptide molecules comprise the asymmetric unit, with each peptide molecule binding a chloride anion through hydrogen bonding with the amide NH group of residues 7, 8, 10, and 11. The peptide molecules form hydrogen-bonded antiparallel ß-sheet dimers in the crystal lattice, with residues 1-3 comprising the dimerization interface. The dimers further assemble end-to-end in the crystal lattice.

Online health information behaviour and its association with statin adherence in patients with high cardiovascular risk: A prospective cohort study.

Objective: Statins are effective for preventing cardiovascular disease. However, many patients decide not to take statins because of negative influences, such as online misinformation. Online health information may affect decisions on medication adherence, but measuring it is challenging. This study aimed to examine the associations between online health information behaviour and statin adherence in patients with high cardiovascular risk. Methods: A prospective cohort study involving 233 patients with high cardiovascular risk was conducted at a primary care clinic in Malaysia. Participants used a digital information diary tool to record online health information they encountered for 2 months and completed a questionnaire about statin necessity, concerns and adherence at the end of the observation period. Data were analysed using structural equation modelling. Results: The results showed that 55.8% (130 of 233 patients) encountered online health information. Patients who actively sought online health information (91 of 233 patients) had higher concerns about statin use (ß = 0.323, p = 0.023). Participants with higher concern about statin use were also more likely to be non-adherent (ß = -0.337, p < 0.001). Patients who actively sought online health information were more likely to have lower statin adherence, mediated by higher concerns about statin use (indirect effect, ß = -0.109, p = 0.048). Conclusions: Our results suggest that patients with higher levels of concern about statins may be actively seeking online information about statins, and their concerns might influence how they search, what they find, and the potential to encounter misinformation. Our study highlights the importance of addressing patients' concerns about medications to improve adherence.

Toward designing human intervention studies to prevent osteoarthritis after knee injury: A report from an interdisciplinary OARSI 2023 workshop.

Objective: The global impact of osteoarthritis is growing. Currently no disease modifying osteoarthritis drugs/therapies exist, increasing the need for preventative strategies. Knee injuries have a high prevalence, distinct onset, and strong independent association with post-traumatic osteoarthritis (PTOA). Numerous groups are embarking upon research that will culminate in clinical trials to assess the effect of interventions to prevent knee PTOA despite challenges and lack of consensus about trial design in this population. Our objectives were to improve awareness of knee PTOA prevention trial design and discuss state-of-the art methods to address the unique opportunities and challenges of these studies. Design: An international interdisciplinary group developed a workshop, hosted at the 2023 Osteoarthritis Research Society International Congress. Here we summarize the workshop content and outputs, with the goal of moving the field of PTOA prevention trial design forward. Results: Workshop highlights included discussions about target population (considering risk, homogeneity, and possibility of modifying osteoarthritis outcome); target treatment (considering delivery, timing, feasibility and effectiveness); comparators (usual care, placebo), and primary symptomatic outcomes considering surrogates and the importance of knee function and symptoms other than pain to this population. Conclusions: Opportunities to test multimodal PTOA prevention interventions across preclinical models and clinical trials exist. As improving symptomatic outcomes aligns with patient and regulator priorities, co-primary symptomatic (single or aggregate/multidimensional outcome considering function and symptoms beyond pain) and structural/physiological outcomes may be appropriate for these trials. To ensure PTOA prevention trials are relevant and acceptable to all stakeholders, future research should address critical knowledge gaps and challenges.

Lineage-specific canonical and non-canonical activity of EZH2 in advanced prostate cancer subtypes.

Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase and emerging therapeutic target that is overexpressed in most castration-resistant prostate cancers and implicated as a driver of disease progression and resistance to hormonal therapies. Here we define the lineage-specific action and differential activity of EZH2 in both prostate adenocarcinoma (PRAD) and neuroendocrine prostate cancer (NEPC) subtypes of advanced prostate cancer to better understand the role of EZH2 in modulating differentiation, lineage plasticity, and to identify mediators of response and resistance to EZH2 inhibitor therapy. Mechanistically, EZH2 modulates bivalent genes that results in upregulation of NEPC-associated transcriptional drivers (e.g., ASCL1) and neuronal gene programs, and leads to forward differentiation after targeting EZH2 in NEPC. Subtype-specific downstream effects of EZH2 inhibition on cell cycle genes support the potential rationale for co-targeting cyclin/CDK to overcome resistance to EZH2 inhibition.

Identifying distinct neural features between the initial and corrective phases of precise reaching using AutoLFADS.

Many initial movements require subsequent corrective movements, but how motor cortex transitions to make corrections and how similar the encoding is to initial movements is unclear. In our study, we explored how the brain's motor cortex signals both initial and corrective movements during a precision reaching task. We recorded a large population of neurons from two male rhesus macaques across multiple sessions to examine the neural firing rates during not only initial movements but also subsequent corrective movements. AutoLFADS, an auto-encoder-based deep-learning model, was applied to provide a clearer picture of neurons' activity on individual corrective movements across sessions. Decoding of reach velocity generalized poorly from initial to corrective submovements. Unlike initial movements, it was challenging to predict the velocity of corrective movements using traditional linear methods in a single, global neural space. We identified several locations in the neural space where corrective submovements originated after the initial reaches, signifying firing rates different than the baseline before initial movements. To improve corrective movement decoding, we demonstrate that a state-dependent decoder incorporating the population firing rates at the initiation of correction improved performance, highlighting the diverse neural features of corrective movements. In summary, we show neural differences between initial and corrective submovements and how the neural activity encodes specific combinations of velocity and position. These findings are inconsistent with assumptions that neural correlations with kinematic features are global and independent, emphasizing that traditional methods often fall short in describing these diverse neural processes for online corrective movements. Significance Statement: We analyzed submovement neural population dynamics during precision reaching. Using an auto- encoder-based deep-learning model, AutoLFADS, we examined neural activity on a single-trial basis. Our study shows distinct neural dynamics between initial and corrective submovements. We demonstrate the existence of unique neural features within each submovement class that encode complex combinations of position and reach direction. Our study also highlights the benefit of state-specific decoding strategies, which consider the neural firing rates at the onset of any given submovement, when decoding complex motor tasks such as corrective submovements.

The Impact of Patterns in Linkage Disequilibrium and Sequencing Quality on the Imprint of Balancing Selection.

Regions under balancing selection are characterized by dense polymorphisms and multiple persistent haplotypes, along with other sequence complexities. Successful identification of these patterns depends on both the statistical approach and the quality of sequencing. To address this challenge, at first, a new statistical method called LD-ABF was developed, employing efficient Bayesian techniques to effectively test for balancing selection. LD-ABF demonstrated the most robust detection of selection in a variety of simulation scenarios, compared against a range of existing tests/tools (Tajima's D, HKA, Dng, BetaScan, and BalLerMix). Furthermore, the impact of the quality of sequencing on detection of balancing selection was explored, as well, using: (i) SNP genotyping and exome data, (ii) targeted high-resolution HLA genotyping (IHIW), and (iii) whole-genome long-read sequencing data (Pangenome). In the analysis of SNP genotyping and exome data, we identified known targets and 38 new selection signatures in genes not previously linked to balancing selection. To further investigate the impact of sequencing quality on detection of balancing selection, a detailed investigation of the MHC was performed with high-resolution HLA typing data. Higher quality sequencing revealed the HLA-DQ genes consistently demonstrated strong selection signatures otherwise not observed from the sparser SNP array and exome data. The HLA-DQ selection signature was also replicated in the Pangenome samples using considerably less samples but, with high-quality long-read sequence data. The improved statistical method, coupled with higher quality sequencing, leads to more consistent identification of selection and enhanced localization of variants under selection, particularly in complex regions.

Candidate circulating microRNA biomarkers in dogs with chronic pancreatitis.

BACKGROUND: Pancreatitis is an important cause of disease and death in dogs. Available circulating biomarkers are not sufficiently sensitive and specific for a definitive diagnosis. HYPOTHESIS: Circulating microRNAs would be differentially expressed in dogs with chronic pancreatitis and could have potential as diagnostic biomarkers. ANIMALS: Healthy controls (n = 19) and dogs with naturally occurring pancreatitis (n = 17). METHODS: A retrospective case-control study. Dogs with pancreatitis were included if they satisfied diagnostic criteria for pancreatitis as adjudicated by 3 experts. MicroRNA was extracted from stored serum samples and sequenced. Reads were mapped to mature microRNA sequences in the canine, mouse, and human genomes. Differentially expressed microRNAs were identified and the potential mechanistic relevance explored using Qiagen Ingenuity Pathway Analysis (IPA). RESULTS: Reads mapping to 196 mature microRNA sequences were detected. Eight circulating microRNAs were significantly differentially expressed in dogs with pancreatitis (≥2-fold change and false discovery rate <0.05). Four of these mapped to the canine genome (cfa-miR-221, cfa-miR-222, cfa-miR-23a, and cfa-miR-205). Three mapped to the murine genome (mmu-miR-484, mmu-miR-6240, mmu-miR-101a-3p) and 1 to the human genome (hsa-miR-1290). Expression in dogs with pancreatitis was higher for 7 microRNAs and lower for mmu-miR-101a-3p. Qiagen IPA demonstrated a number of the differently expressed microRNAs are involved in a common pancreatic inflammatory pathway. CONCLUSIONS: The significantly differentially expressed microRNAs represent promising candidates for further validation as diagnostic biomarkers for canine pancreatitis.

Overexpression of VEGFα as a biomarker of endothelial dysfunction in aortic tissue of α-GAL-Tg/KO mice and its upregulation in the serum of patients with Fabry's disease.

Introduction: Fabry's disease is an X-linked lysosomal storage disorder caused by reduced activity of α-galactosidase A (GAL), leading to premature death on account of renal, cardiac, and vascular organ failure. Accumulation of the GAL substrate globotriaosylceramide (Gb3) in endothelial and smooth muscle cells is associated with early vascular cell damage, suggesting endothelial dysfunction as a driver of cardiorenal organ failure. Here, we studied the vascular expression of the key angiogenic factors, VEGFα and its antagonist angiostatin, in Fabry α-GAL-Tg/KO mice and determined circulating VEGFα and angiostatin serum levels in patients with Fabry's disease and healthy controls. Methods: Cryopreserved aortic vessels from six α-GAL-Tg/KO and six wild-type (WT) mice were obtained and VEGFα and angiostatin levels were determined by performing Western blot analysis. VEGFα expression was visualized by an immunohistochemical staining of paraffin aortic rings. In addition, VEGFα and angiostatin serum levels were measured by using an enzyme-linked immunosorbent assay in 48 patients with genetically verified Fabry's disease (50% male) and 22 healthy controls and correlated with disease severity markers such as lyso-Gb3, albuminuria, NTproBNP, high-sensitive troponin T (hsTNT), and myocardial wall thickness. Results: It was found that there was a significant increase in VEGFα protein expression (1.66 ± 0.35 vs. 0.62 ± 0.16, p = 0.0009) and a decrease in angiostatin expression (0.024 ± 0.007 vs. 0.053 ± 0.02, p = 0.038) in aortic lysates from α-GAL-Tg/KO compared with that from WT mice. Immunohistochemical staining revealed an adventitial VEGFα signal in α-GAL-Tg/KO mice, whereas no VEGFα signal could be detected in WT mice aortas. No differences in aortic angiostatin expression between α-GAL-Tg/KO- and WT mice could be visualized. The serum levels of VEGFα were significantly upregulated in patients with Fabry's disease compared with that in healthy controls (708.5 ± 426.3 vs. 458.5 ± 181.5 pg/ml, p = 0.048) and positively associated with albuminuria (r = 0.82, p < 0.0001) and elevated NTproBNP (r = 0.87, p < 0.0001) and hsTNT values (r = 0.41, p = 0.048) in male patients with Fabry's disease. For angiostatin, no significant difference was found between patients with Fabry's disease and healthy controls (747.6 ± 390.3 vs. 858.8 ± 599.3 pg/ml). Discussion: In conclusion, an overexpression of VEGFα and downregulation of its counter player angiostatin in aortic tissue of α-GAL-Tg/KO mice support the hypothesis of an underlying vasculopathy in Fabry's disease. Elevated VEGFα serum levels were also observed in patients with Fabry's disease and were positively associated with elevated markers of organ manifestation in males. These findings suggest that angiogenetic markers, such as VEGFα, may be potentially useful biomarkers for the detection of endothelial dysfunction in classical Fabry's disease.