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Background Systemic sclerosis-associated interstitial lung disease (SSc-ILD) is difficult to manage due to the heterogeneous disease course. There is a high need for new biomarkers to identify patients at high risk for ILD progression. Fibroblast activation protein (FAP) has gained interest as a biomarker to reflect fibrotic activity. As circulating FAP (cFAP) can be measured in blood, we aimed to investigate the value of cFAP in SSc-ILD. Methods cFAP concentrations were determined in plasma samples of 210 patients with systemic sclerosis (SSc) using an enzyme-linked immunosorbent assay. We compared cFAP in baseline and repeated longitudinal samples between SSc patients with ( n  = 63) and without ILD ( n  = 147). Furthermore, we investigated the correlation between cFAP and ILD progression at follow-up. In an exploratory analysis, we also investigated if cFAP was associated with other disease-related clinical features and all-cause mortality. Results cFAP levels were not different between SSc patients with- and without ILD, both at baseline (median 91.5 and 97.7 ng/mL respectively; p  = 0.80) or during follow-up. Furthermore, we found no association between cFAP at baseline and ILD progression at 1, 2 and 5 years of follow-up. Notably, cFAP levels were elevated at baseline in patients with higher skin scores (mRSS ≥ 10 compared to mRSS < 10; p  = 0.01). Finally, we did not find an association between cFAP and all-cause mortality at 5 and 10 years of follow-up. Conclusion In conclusion, cFAP does not seem useful as a biomarker in SSc-ILD. However, the association between cFAP and skin score deserves further investigation.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to LDL receptors, leading to their degradation. Genetics studies have shown that loss-of-function mutations in PCSK9 result in reduced plasma LDL cholesterol and decreased risk of coronary heart disease. We aimed to investigate the safety and efficacy of ALN-PCS, a small interfering RNA that inhibits PCSK9 synthesis, in healthy volunteers with raised cholesterol who were not on lipid-lowering treatment. We did a randomised, single-blind, placebo-controlled, phase 1 dose-escalation study in healthy adult volunteers with serum LDL cholesterol of 3·00 mmol/L or higher. Participants were randomly assigned in a 3:1 ratio by computer algorithm to receive one dose of intravenous ALN-PCS (with doses ranging from 0·015 to 0·400 mg/kg) or placebo. The primary endpoint was safety and tolerability of ALN-PCS. Secondary endpoints were the pharmacokinetic characteristics of ALN-PCS and its pharmacodynamic effects on PCSK9 and LDL cholesterol. Study participants were masked to treatment assignment. Analysis was per protocol and we used ANCOVA to analyse pharmacodynamic endpoint data. This trial is registered with ClinicalTrials.gov, number NCT01437059. Of 32 participants, 24 were randomly allocated to receive a single dose of ALN-PCS (0·015 mg/kg [n=3], 0·045 mg/kg [n=3], 0·090 mg/kg [n=3], 0·150 mg/kg [n=3], 0·250 mg/kg [n=6], or 0·400 mg/kg [n=6]) and eight to placebo. The proportions of patients affected by treatment-emergent adverse events were similar in the ALN-PCS and placebo groups (19 [79%] vs seven [88%]). ALN-PCS was rapidly distributed, with peak concentration and area under the curve (0 to last measurement) increasing in a roughly dose-proportional way across the dose range tested. In the group given 0·400 mg/kg of ALN-PCS, treatment resulted in a mean 70% reduction in circulating PCSK9 plasma protein (p<0·0001) and a mean 40% reduction in LDL cholesterol from baseline relative to placebo (p<0·0001). Our results suggest that inhibition of PCSK9 synthesis by RNA interference (RNAi) provides a potentially safe mechanism to reduce LDL cholesterol concentration in healthy individuals with raised cholesterol. These results support the further assessment of ALN-PCS in patients with hypercholesterolaemia, including those being treated with statins. This study is the first to show an RNAi drug being used to affect a clinically validated endpoint (ie, LDL cholesterol) in human beings. Alnylam Pharmaceuticals.

Chronological age is an imprecise proxy for reproductive capacity, necessitating biomarkers that reflect the underlying pathophysiology of the ovary. Fibrotic remodeling of the ovarian stroma is a key hallmark of biological ovarian aging, yet it cannot be assessed by current clinical tools. This study aimed to identify and validate a novel serum biomarker for fibrotic ovarian aging by applying supervised machine learning (ML) to human ovarian transcriptomic data. Transcriptomic data from the Genotype-Tissue Expression (GTEx) database were analyzed using ML algorithms to identify candidate genes predictive of ovarian aging, and finally, fibroblast activation protein (FAP) and collectin-11 (COLEC11) were selected for clinical validation. In a cross-sectional study, serum levels of FAP and COLEC11, along with key hormonal indices, were measured in two nested patient cohorts, and their associations with ovarian reserve and clinical parameters were analyzed. Serum FAP levels did not correlate with age but showed a strong inverse correlation with anti-Müllerian hormone (AMH) (r = −0.61, p = 0.001), a finding accentuated in women with decreased ovarian reserve (DOR). While COLEC11 correlated with age, it failed to differentiate DOR status. FAP levels were independent of central hormonal regulation, consistent with preclinical fibrotic models. Circulating FAP reflects age-independent, fibrotic ovarian aging, offering stromal-specific information not captured by conventional hormonal markers. This study provides the first clinical validation of FAP as a biomarker for ovarian stromal aging, holding potential for improved reproductive risk assessment.

Two siblings presented with cardiomyopathy, hypertension, arrhythmia, and fibrosis of the left atrium. Each had a homozygous null variant in CORIN , the gene encoding atrial natriuretic peptide (ANP)–converting enzyme. A plasma sample obtained from one of the siblings had no detectable levels of corin or N-terminal pro-ANP but had elevated levels of B-type natriuretic peptide (BNP) and one of the two protein markers of fibrosis that we tested. These and other findings support the hypothesis that BNP cannot fully compensate for a lack of activation of the ANP pathway and that corin is critical to normal ANP activity, left atrial function, and cardiovascular homeostasis. Findings from a study involving two siblings homozygous for a null variant in CORIN have implications for understanding the extent to which the BNP and ANP pathways overlap.

Abstract Background Stress precipitates mood disorders, characterized by a range of symptoms present in different combinations, suggesting the existence of disease subtypes. Using an animal model, we previously described that repetitive stress via restraint or immobilization induced depressive-like behaviors in rats that were differentially reverted by a serotonin- or noradrenaline-based antidepressant drug, indicating that different neurobiological mechanisms may be involved. The forebrain astrocyte protein aldolase C, contained in small extracellular vesicles, was identified as a potential biomarker in the cerebrospinal fluid; however, its specific origin remains unknown. Here, we propose to investigate whether serum small extracellular vesicles contain a stress-specific protein cargo and whether serum aldolase C has a brain origin. Methods We isolated and characterized serum small extracellular vesicles from rats exposed to restraint, immobilization, or no stress, and their proteomes were identified by mass spectrometry. Data available via ProteomeXchange with identifier PXD009085 were validated, in part, by western blot. In utero electroporation was performed to study the direct transfer of recombinant aldolase C-GFP from brain cells to blood small extracellular vesicles. Results A differential proteome was identified among the experimental groups, including aldolase C, astrocytic glial fibrillary acidic protein, synaptophysin, and reelin. Additionally, we observed that, when expressed in the brain, aldolase C tagged with green fluorescent protein could be recovered in serum small extracellular vesicles. Conclusion The protein cargo of serum small extracellular vesicles constitutes a valuable source of biomarkers of stress-induced diseases, including those characterized by depressive-like behaviors. Brain-to-periphery signaling mediated by a differential molecular cargo of small extracellular vesicles is a novel and challenging mechanism by which the brain might communicate health and disease states to the rest of the body.

Preeclampsia results from placental insufficiency and causes maternal endothelial dysfunction and multi-organ damage. Our in-silico analysis identified Cystatin 6 (CST6), a cysteine protease inhibitor, as located on the placental surface where it might be released into maternal circulation. This study aimed to characterise CST6 and one of its high affinity targets, Legumain (LGMN), in preeclampsia and assess its biomarker potential by measuring levels in maternal circulation. Placental CST6 mRNA expression was significantly increased in 78 pregnancies complicated by early-onset preeclampsia (delivering at < 34 weeks’ gestation) relative to 30 gestation matched controls (P  < 0.0001). LGMN mRNA expression was significantly decreased ( P  = 0.0309). Circulating CST6 was increased in 35 pregnancies complicated by early-onset preeclampsia (< 34 weeks’ gestation) relative to 27 gestation matched controls ( P  = 0.0261), and LGMN levels remained unchanged. At 36 weeks’ gestation, circulating CST6 was significantly increased (P =  0.001), while LGMN was significantly decreased ( P  = 0.0135) in 21 pregnancies preceding diagnosis of preeclampsia at term, compared to 184 pregnancies that did not develop preeclampsia. Human trophoblast stem cells (hTSC) were differentiated into syncytiotrophoblast or extravillous trophoblast (EVT) to evaluate CST6 and LGMN expression in these trophoblast lineages. CST6 and LGMN mRNA expression were significantly increased across 96 h after syncytiotrophoblast ( P  = 0.0066 and P  = 0.0010 respectively) and EVT differentiation (P =  0.0618 and P  = 0.0016 respectively), with the highest expression in syncytiotrophoblast. Computational analysis of two publicly available single-cell and single-nuclei RNA sequencing datasets correlated with the expression pattern observed in vitro. When syncytiotrophoblast cells were exposed to hypoxia (1% O 2 vs. 8% O 2 ), CST6 expression significantly increased ( P  = 0.0079), whilst LGMN expression was unchanged. The vascular endothelium may serve as an additional source of circulating CST6 and LGMN in preeclampsia.  Induction of dysfunction in endothelial cells by TNFα, caused reduced CST6 expression ( P  = 0.0036), whilst LGMN expression remained unchanged. Administering recombinant CST6 to endothelial cells enhanced markers of endothelial dysfunction and LGMN expression in the presence of TNFα. These findings indicate an inverse relationship between CST6 and LGMN in the placenta and maternal circulation in preeclampsia. We suggest elevated circulating levels of CST6 may be induced by placental hypoxia. This study provides novel insight into the dysregulation of CST6 and LGMN in preeclampsia and introduces their potential roles in human pregnancy and associated pathology.

Upon vascular injury, locally controlled haemostasis prevents life-threatening blood loss and ensures wound healing. Intracellular material derived from damaged cells at these sites will become exposed to blood components and could contribute to blood coagulation and pathological thrombus formation. So far, the functional and mechanistic consequences of this concept are not understood. Here, we present in vivo and in vitro evidence that different forms of eukaryotic and prokaryotic RNA serve as promoters of blood coagulation. Extracellular RNA was found to augment (auto-)activation of proteases of the contact phase pathway of blood coagulation such as factors XII and XI, both exhibiting strong RNA binding. Moreover, administration of exogenous RNA provoked a significant procoagulant response in rabbits. In mice that underwent an arterial thrombosis model, extracellular RNA was found associated with fibrin-rich thrombi, and pretreatment with RNase (but not DNase) significantly delayed occlusive thrombus formation. Thus, extracellular RNA derived from damaged or necrotic cells particularly under pathological conditions or severe tissue damage represents the long sought natural \"foreign surface\" and provides a procoagulant cofactor template for the factors XII/XI-induced contact activation/amplification of blood coagulation. Extracellular RNA thereby reveals a yet unrecognized target for antithrombotic intervention, using RNase or related therapeutic strategies.

Sjogren's syndrome (SS) is considered as a chronic, autoimmune disorder, that can present with various manifestations both intra and extra-glandular. Cognitive dysfunction is pivotal in recognizing neurological complications in SS. A study involving 44 SS cases and 37 controls was conducted to evaluate cognitive dysfunction further. Participants underwent multiple cognitive tests and blood tests for evaluation. Also, the β-site amyloid precursor protein cleaving enzyme 1 (BACE1), Interleukin 6 (IL-6), total antioxidant capacity (TAC), nitric oxide (NO), and malondialdehyde (MDA) serum levels were measured. Multiple analyses were done by PRISM 10 and SPSS 22. The MoCA and SDLT scores were lower in Sjogren patients (P < 0.001). Serum BACE1, IL-6, NO, TAC, and MDA did not statistically vary in the SS patients. The only variables varied by medication therapy with methotrexate (MTX), hydroxychloroquine (HCQ), and prednisone were WBC count (P = 0.03) and triglyceride levels (in MTX and HCQ, P = 0.04), with no effect on neurocognitive factors. IL-6 was strongly correlated with the duration of symptoms (r = 0.99, P-value < 0.001). BACE1 had a positive correlation with IL-6 level (r = 0.4, P-value = 0.027). SS patients demonstrated significantly lower performance in neurocognitive tests, while BACE1 and inflammatory markers were not altered. This indicates that cognitive decline in SS is present but the mechanism still requires further evaluation. MTX, HCQ, and Prednisone use did not alter neurocognitive factors. Important correlations were found between hematological and cognitive tests in this study which provides new insights in the field of SS.

The RNA-guided endonuclease Cas9 has emerged as a versatile genome-editing platform. However, the size of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for basic research and therapeutic applications that use the highly versatile adeno-associated virus (AAV) delivery vehicle. Here, we characterize six smaller Cas9 orthologues and show that Cas9 from Staphylococcus aureus (SaCas9) can edit the genome with efficiencies similar to those of SpCas9, while being more than 1 kilobase shorter. We packaged SaCas9 and its single guide RNA expression cassette into a single AAV vector and targeted the cholesterol regulatory gene Pcsk9 in the mouse liver. Within one week of injection, we observed >40% gene modification, accompanied by significant reductions in serum Pcsk9 and total cholesterol levels. We further assess the genome-wide targeting specificity of SaCas9 and SpCas9 using BLESS, and demonstrate that SaCas9-mediated in vivo genome editing has the potential to be efficient and specific. The physical size of the commonly used Cas9 from Streptococcus pyogenes poses challenges for CRISPR-Cas genome editing systems that use the adeno-associated virus as a delivery vehicle; here, smaller Cas9 orthologues are characterized, and Cas9 from Staphylococcus aureus allowed targeting of the cholesterol regulatory gene Pcsk9 in the mouse liver. A small Cas9 gene editor enzyme The RNA-guided endonuclease CRISPR-Cas9 is being widely adopted as the genome-editing tool of choice. However, the physical size of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) poses problems for applications that use the adeno-associated virus (AAV) as a delivery vehicle. Feng Zhang and colleagues now characterize six smaller Cas9 orthologues. Focusing on Cas9 from Staphylococcus aureus , they packaged it and its single guide RNA expression cassette into a single AAV vector and targeted the cholesterol regulatory gene Pcsk9 in the mouse liver. Greater than 40% gene modification was observed within a week of injection, accompanied by significant reductions in serum Pcsk9 and total cholesterol levels.

Clearance of amyloid-beta (Aβ) from the brain is an important therapeutic strategy for Alzheimer’s disease (AD). Current studies mainly focus on the central approach of Aβ clearance by introducing therapeutic agents into the brain. In a previous study, we found that peripheral tissues and organs play important roles in clearing brain-derived Aβ, suggesting that the peripheral approach of removing Aβ from the blood may also be effective for AD therapy. Here, we investigated whether peritoneal dialysis, a clinically available therapeutic method for chronic kidney disease (CKD), reduces brain Aβ burden and attenuates AD-type pathologies and cognitive impairments. Thirty patients with newly diagnosed CKD were enrolled. The plasma Aβ concentrations of the patients were measured before and after peritoneal dialysis. APP/PS1 mice were subjected to peritoneal dialysis once a day for 1 month from 6 months of age (prevention study) or 9 months of age (treatment study). The Aβ in the interstitial fluid (ISF) was collected using microdialysis. Behavioural performance, long-term potentiation (LTP), Aβ burden and other AD-type pathologies were measured after 1 month of peritoneal dialysis. Peritoneal dialysis significantly reduced plasma Aβ levels in both CKD patients and APP/PS1 mice. Aβ levels in the brain ISF of APP/PS1 mice immediately decreased after reduction of Aβ in the blood during peritoneal dialysis. In both prevention and treatment studies, peritoneal dialysis substantially reduced Aβ deposition, attenuated other AD-type pathologies, including Tau hyperphosphorylation, glial activation, neuroinflammation, neuronal loss, and synaptic dysfunction, and rescued the behavioural deficits of APPswe/PS1 mice. Importantly, the Aβ phagocytosis function of microglia was enhanced in APP/PS1 mice after peritoneal dialysis. Our study suggests that peritoneal dialysis is a promising therapeutic method for AD, and Aβ clearance using a peripheral approach could be a desirable therapeutic strategy for AD.