Exosomal miRNAs in patients with chronic heart failure and hyperuricemia and the underlying mechanisms.

Chronic heart failure (CHF) combined with hyperuricemia (HUA) is a comorbidity that is hard to diagnose by a single biomarker. Exosomal miRNAs are differentially expressed in cardiovascular diseases and are closely associated with regulating most biological functions. This study aimed to provide evidence for miRNA as a new molecular marker for precise diagnosis of the comorbidity of CHF with HUA and further analyze the potential targets of differentially expressed miRNA. This controlled study included 30 CHF patients combined with HUA (Group T) and 30 healthy volunteers (Group C). 6 peripheral blood samples from Group T and Group C were analyzed for exosomal miRNAs by high-throughput sequencing and then validated in the remaining 24 peripheral blood samples from Group T and Group C by applying real-time PCR (RT-PCR). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed using R software to predict the differential miRNAs' action targets. 42 differentially expressed miRNAs were detected (18 upregulated and 24 downregulated), in which miR-27a-5p was significantly upregulated (P<0.01), and miR-139-3p was significantly downregulated (P<0.01) in Group T. The combination of miR-27a-5p and miR-139-3p predicted the development of CHF combined with HUA with a maximum area under the curve (AUC) of 0.899 (95 % CI: 0.812-0.987, SEN=79.2 %, SPE=91.7 %, J value = 0.709). GO and KEGG enrichment analysis revealed that the differentially expressed miRNAs had a role in activating the AMPK-mTOR signaling pathway to activate the autophagic response. Collectively, our findings suggest that upregulated exosomal miR-27a-5p combined with downregulated exosomal miR-139-3p can be used as a novel molecular marker for precise diagnosis of CHF combined with HUA and enhanced autophagy by AMPK-mTOR signaling pathway may be one pathogenesis of the differentially expressed miRNAs.

AMP kinase: A promising therapeutic drug target for post-COVID-19 complications.

The COVID-19 pandemic, caused by SARS-CoV-2, has resulted in severe respiratory issues and persistent complications, particularly affecting glucose metabolism. Patients with or without pre-existing diabetes often experience worsened symptoms, highlighting the need for innovative therapeutic approaches. AMPK, a crucial regulator of cellular energy balance, plays a pivotal role in glucose metabolism, insulin sensitivity, and inflammatory responses. AMPK activation, through allosteric or kinase-dependent mechanisms, impacts cellular processes like glucose uptake, fatty acid oxidation, and autophagy. The tissue-specific distribution of AMPK emphasizes its role in maintaining metabolic homeostasis throughout the body. Intriguingly, SARS-CoV-2 infection inhibits AMPK, contributing to metabolic dysregulation and post-COVID-19 complications. AMPK activators like capsaicinoids, curcumin, phytoestrogens, cilostazol, and momordicosides have demonstrated the potential to regulate AMPK activity. Compounds from various sources improve fatty acid oxidation and insulin sensitivity, with metformin showing opposing effects on AMPK activation compared to the virus, suggesting potential therapeutic options. The diverse effects of AMPK activation extend to its role in countering viral infections, further highlighting its significance in COVID-19. This review explores AMPK activation mechanisms, its role in metabolic disorders, and the potential use of natural compounds to target AMPK for post-COVID-19 complications. Also, it aims to review the possible methods of activating AMPK to prevent post-COVID-19 diabetes and cardiovascular complications. It also explores the use of natural compounds for their therapeutic effects in targeting the AMPK pathways. Targeting AMPK activation emerges as a promising avenue to mitigate the long-term effects of COVID-19, offering hope for improved patient outcomes and a better quality of life.

Evaluation of anti-diabetic effects of Glimepiride/metformin cocrystal.

Emerging data suggest that cocrystal of two compounds may have a different pharmacological effect from two compounds alone or their physical combination. Glimepiride (Gli) and metformin (Met) are two types of anti-diabetic drugs. Previously we generated the glimepiride/metformin cocrystal (GM). In this study, we evaluated the anti-diabetic effects of GM and explored the underlying mechanisms. Our result showed that GM reduced the blood glucose and HbA1c levels in db/db mice, and low doses of GM can achieve the hypoglycemic effect as Gli or Met alone, and high dose of GM was better than Gli and Met alone in improving the pathological changes of liver. In vivo studies showed that GM activated AMPK and STAT3 signaling, downregulated TXNIP expression and upregulated MaFA expression. Moreover, GM promoted the secretion of insulin in pancreas of db/db mice and in high glucose-treated INS-1 and MIN-6 cells. Together, GM possesses slightly better anti-diabetic effects than Met or Gli alone in db/db mice, and the mechanism of GM protecting ß-cell dysfunction induced by glucotoxicity may be associated with activation of the AMPK/TXNIP/MaFA pathway.

ALTERED MERISTEM PROGRAM1 impairs RNA silencing by repressing the biogenesis of a subset of inverted repeat-derived siRNAs.

RNA silencing negatively regulates gene expression at the transcriptional and post-transcriptional levels through DNA methylation, histone modification, mRNA cleavage, and translational inhibition. Small interfering RNAs (siRNAs) of 21 to 24 nucleotides are processed from double-stranded RNAs by Dicer-like enzymes and play essential roles in RNA silencing in plants. Here, we demonstrated that ALTERED MERISTEM PROGRAM1 (AMP1) and its putative paralog LIKE AMP1 (LAMP1) impair RNA silencing by repressing the biogenesis of a subset of inverted repeat (IR)-derived siRNAs in Arabidopsis (Arabidopsis thaliana). AMP1 and LAMP1 inhibit Pol II-dependent IR gene transcription by suppressing ARGONAUTE 1 (AGO1) protein levels. Genetic analysis indicates that AMP1 acts upstream of RNA polymerase IV subunit 1 (NRPD1), RNA-dependent RNA polymerase 2 (RDR2), and Dicer-Like 4 (DCL4), which are required for IR-induced RNA silencing. We also show that AMP1 and LAMP1 inhibit siRNA-mediated silencing in a different mechanism from AGO4 and DCL3. Together, these results reveal two previously unknown players in siRNA biogenesis from IRs - AGO1, which promotes IR transcription, and AMP1, which inhibits IR transcription indirectly through the repression of AGO1 expression.

Rab2A-mediated Golgi-lipid droplet interactions support very-low-density lipoprotein secretion in hepatocytes.

Lipid droplets (LDs) serve as crucial hubs for lipid trafficking and metabolic regulation through their numerous interactions with various organelles. While the interplay between LDs and the Golgi apparatus has been recognized, their roles and underlying mechanisms remain poorly understood. Here, we reveal the role of Ras-related protein Rab-2A (Rab2A) in mediating LD-Golgi interactions, thereby contributing to very-low-density lipoprotein (VLDL) lipidation and secretion in hepatocytes. Mechanistically, our findings identify a selective interaction between Golgi-localized Rab2A and 17-beta-hydroxysteroid dehydrogenase 13 (HSD17B13) protein residing on LDs. This complex facilitates dynamic organelle communication between the Golgi apparatus and LDs, thus contributing to lipid transfer from LDs to the Golgi apparatus for VLDL2 lipidation and secretion. Attenuation of Rab2A activity via AMP-activated protein kinase (AMPK) suppresses the Rab2A-HSD17B13 complex formation, impairing LD-Golgi interactions and subsequent VLDL secretion. Furthermore, genetic inhibition of Rab2A and HSD17B13 in the liver reduces the serum triglyceride and cholesterol levels. Collectively, this study provides a new perspective on the interactions between the Golgi apparatus and LDs.

A modified linear capacitive displacement transducer for any range.

-In the present paper, a modified linear capacitive displacement transducer is developed, where the measurement errors and non-linearity errors due to the various effects such as the effect of the change of atmospheric parameters, the effect of parasitic fringe capacitance, the effect of stray capacitance, the effect of parallel position shifting of the sensing plates, etc. are minimized, and the output is linearly related with displacement for any range. The transducer consists of a sensing capacitor and a similar reference capacitor, each of which has a noninductive double layer short-circuited winding wound on an insulating uniform hollow cylinder. The sensing cylinder has a thin circular conducting disk, which is easily displaceable by the moving object along the inside walls of the cylinder from the outside by means of a thin conducting disk rod attached to the central position of the disk. The reference cylinder has a similar disk with a disk rod fixed at the reference zero position. The transducer circuit measures the difference of the combined cylindrical-perpendicular plate capacitances between the short-circuited terminal of vertical noninductive winding and the horizontal conducting disk of the two capacitors and gives an output that is found to be linearly related with displacement for the entire length of the sensing cylinder. The measurement is free from the non-linearity error for any range because the factors producing nonlinearity are similar for the two capacitors and tend to cancel each other. The derived characteristic equations are supported by the experimental results with very good accuracy and repeatability.

Classification of Variants of Reduced Penetrance in High Penetrance Cancer Susceptibility Genes: Framework for Genetics Clinicians and Clinical Scientists by CanVIG-UK (Cancer Variant Interpretation Group-UK).

PURPOSE: Current practice is to report and manage likely pathogenic/pathogenic variants in a given cancer susceptibility gene (CSG) as though having equivalent penetrance, despite increasing evidence of inter-variant variability in risk associations. Using existing variant interpretation approaches, largely based on full-penetrance models, variants where reduced penetrance is suspected may be classified inconsistently and/or as variants of uncertain significance (VUS). We aimed to develop a national consensus approach for such variants within the Cancer Variant Interpretation Group UK (CanVIG-UK) multidisciplinary network. METHODS: A series of surveys and live polls were conducted during and between CanVIG-UK monthly meetings on various scenarios potentially indicating reduced penetrance. These informed the iterative development of a framework for the classification of variants of reduced penetrance by the CanVIG-UK Steering and Advisory Group (CStAG) working group. RESULTS: CanVIG-UK recommendations for amendment of the 2015 ACMG/AMP variant interpretation framework were developed for variants where (A) Active evidence suggests a reduced penetrance effect size (e.g. from case-control or segregation data) (B) Reduced penetrance effect is inferred from weaker/potentially-inconsistent observed data. CONCLUSIONS: CanVIG-UK propose a framework for the classification of variants of reduced penetrance in high-penetrance genes. These principles, whilst developed for CSGs, are potentially applicable to other clinical contexts.

Metformin Mitigates Trimethyltin-Induced Cognition Impairment and Hippocampal Neurodegeneration.

The neurotoxicant trimethyltin (TMT) triggers cognitive impairment and hippocampal neurodegeneration. TMT is a useful research tool for the study of Alzheimer's disease (AD) pathogenesis and treatment. Although the antidiabetic agent metformin has shown promising neuroprotective effects, however, its precise modes of action in neurodegenerative disorders need to be further elucidated. In this study, we investigated whether metformin can mitigate TMT cognition impairment and hippocampal neurodegeneration. To induce an AD-like phenotype, TMT was injected i.p. (8 mg/kg) and metformin was administered daily p.o. for 3 weeks at 200 mg/kg. Our results showed that metformin administration to the TMT group mitigated learning and memory impairment in Barnes maze, novel object recognition (NOR) task, and Y maze, attenuated hippocampal oxidative, inflammatory, and cell death/pyroptotic factors, and also reversed neurodegeneration-related proteins such as presenilin 1 and p-Tau. Hippocampal level of AMP-activated protein kinase (AMPK) as a key regulator of energy homeostasis was also improved following metformin treatment. Additionally, metformin reduced hippocampal acetylcholinesterase (AChE) activity, glial fibrillary acidic protein (GFAP)-positive reactivity, and prevented the loss of CA1 pyramidal neurons. This study showed that metformin mitigated TMT-induced neurodegeneration and this may pave the way to develop new therapeutics to combat against cognitive deficits under neurotoxic conditions.

AAV vector-derived elements integrate into Cas9-generated double-strand breaks and disrupt gene transcription.

We previously developed an adeno-associated virus (AAV) Cas9 gene therapy for Angelman syndrome that integrated into the genome and prematurely terminated Ube3a-ATS. Here, we assessed the performance of 3 additional AAV vectors containing S. aureus Cas9 in vitro and in vivo, and 25 vectors containing N. meningitidis Cas9 in vitro, all targeting single sites within Ube3a-ATS. We found that none of these single-target gRNA vectors were as effective as multi-target gRNA vectors at reducing Ube3a-ATS expression in neurons. We also developed an anchored multiplex PCR sequencing method and analysis pipeline to quantify the relative frequency of all possible editing events at target sites, including AAV integration and unresolved double-strand breaks. We found that integration of AAV was the most frequent editing event (67%-89% of all edits) at three different single target sites, surpassing insertions and deletions (indels). None of the most frequently observed indels were capable of blocking transcription when incorporated into a Ube3a-ATS minigene reporter, whereas two vector derived elements-the poly(A) and reverse promoter-reduced downstream transcription by up to 50%. Our findings suggest that the probability that a gene trapping AAV integration event occurs is influenced by which vector-derived element(s) are integrated and by the number of target sites.

Lack of AMP-activated protein kinase-α1 reduces nitric oxide synthesis in thoracic aorta perivascular adipose tissue.

OBJECTIVE: Perivascular adipose tissue (PVAT) releases anti-contractile bioactive molecules including NO. PVAT anti-contractile activity is attenuated in mice lacking AMPKα1 (AMP-activated protein kinase-α1). As AMPK regulates endothelial NO synthase (eNOS) activity in cultured cells, NO synthesis was examined in PVAT from AMPKα1 knockout (KO) mice. METHODS AND RESULTS: Endothelium-denuded thoracic or abdominal aortic rings were isolated from wild type (WT) and KO mice. NOS inhibition enhanced vasoconstriction in PVAT-intact thoracic aortic rings from mice of either genotype yet had no effect on abdominal rings as assessed by wire myography. Thoracic aorta PVAT exhibited increased NO production, NOS activity and levels of the brown adipose tissue marker uncoupling protein-1 (UCP1) compared to abdominal PVAT. In KO mice, NO production was significantly reduced in thoracic but not abdominal PVAT. Reduced NO production in KO thoracic PVAT was not due to altered levels or phosphorylation of eNOS but was associated with increased caveolin-1:eNOS association and caveolin-1 Tyr14 phosphorylation. A peptide that disrupts eNOS:caveolin-1 association increased NO synthesis and reduced vasoconstriction of PVAT-intact thoracic but not abdominal aortic rings. KO thoracic PVAT also exhibited reduced UCP1 levels. CONCLUSIONS: Murine thoracic aorta PVAT exhibits higher NO synthesis and UCP1 levels than abdominal aortic PVAT. Downregulation of AMPK suppresses NO synthesis which may contribute to the reduced anticontractile activity and reduced brown adipose tissue phenotype of KO thoracic PVAT. The mechanism underlying the effect of AMPK downregulation likely results from increased caveolin-1:eNOS association associated with caveolin-1 Tyr14 phosphorylation.

Induction of long-term hyperexcitability by memory-related cAMP signaling in isolated nociceptor cell bodies.

Persistent hyperactivity of nociceptors is known to contribute significantly to long-lasting sensitization and ongoing pain in many clinical conditions. It is often assumed that nociceptor hyperactivity is mainly driven by continuing stimulation from inflammatory mediators. We have tested an additional possibility: that persistent increases in excitability promoting hyperactivity can be induced by a prototypical cellular signaling pathway long known to induce late-phase long-term potentiation (LTP) of synapses in brain regions involved in memory formation. This cAMP-PKA-CREB-gene transcription-protein synthesis pathway was tested using whole-cell current clamp methods on small dissociated sensory neurons (primarily nociceptors) from dorsal root ganglia (DRGs) excised from previously uninjured ("naïve") male rats. Six-hour treatment with the specific Gαs-coupled 5-HT4 receptor agonist, prucalopride, or with the adenylyl cyclase activator forskolin induced long-term hyperexcitability (LTH) in DRG neurons that manifested 12-24 h later as action potential (AP) discharge (ongoing activity, OA) during artificial depolarization to -45 mV, a membrane potential that is normally subthreshold for AP generation. Prucalopride treatment also induced significant long-lasting depolarization of resting membrane potential (from -69 to -66 mV), enhanced depolarizing spontaneous fluctuations (DSFs) of membrane potential, and produced trends for reduced AP threshold and rheobase. LTH was prevented by co-treatment of prucalopride with inhibitors of PKA, CREB, gene transcription, or protein synthesis. As in the induction of synaptic memory, many other cellular signals are likely to be involved. However, the discovery that this prototypical memory induction pathway can induce nociceptor LTH, along with reports that cAMP signaling and CREB activity in DRGs can induce hyperalgesic priming, suggest that early, temporary, cAMP-induced transcriptional and translational mechanisms can induce nociceptor LTH that might last for long periods. The present results also raise the question of whether reactivation of primed signaling mechanisms by re-exposure to inflammatory mediators linked to cAMP synthesis during subsequent challenges to bodily integrity can "reconsolidate" nociceptor memory, extending the duration of persistent hyperexcitability.

A Piezoresistive-Sensor Nonlinearity Correction on-Chip Method with Highly Robust Class-AB Driving Capability.

This paper presents a thorough robust Class-AB power amplifier design and its application in pressure-mode sensor-on-chip nonlinearity correction. Considering its use in piezoresistive sensing applications, a gain-boosting-aided folded cascode structure is utilized to increase the amplifier's gain by a large amount as well as enhancing the power rejection ability, and a push-pull structure with miller compensation, a floating gate technique, and an adaptive output driving limiting structures are adopted to achieve high-efficiency current driving capability, high stability, and electronic environmental compatibility. This amplifier is applied in a real sensor nonlinearity correction on-chip system. With the help of a self-designed 7-bit + sign DAC and a self-designed two-stage operational amplifier, this system is compatible with nonlinear correction at different signal conditioning output values. It can also drive resistive sensors as small as 300 ohms and as high as tens of thousands of ohms. The designed two-stage operational amplifier utilizes the TSMC 0.18 um process, resulting in a final circuit power consumption of 0.183 mW. The amplifier exhibits a gain greater than 140 dB, a phase margin of 68°, and a unit gain bandwidth exceeding 199.76 kHz. The output voltage range spans from 0 to 4.6 V. The final simulation results indicate that the nonlinear correction system designed in this paper can correct piezoresistive sensors with a nonlinearity of up to ±2.5% under various PVT (Process-Voltage-Temperature) conditions. After calibration by this system, the maximum error in the output voltage is 4 mV, effectively reducing the nonlinearity to 4% of its original value in the worst-case scenario.

Fibroblast Growth Factor 21 Suppressed Neutrophil Extracellular Traps Induced by Myocardial Ischemia/Reperfusion Injury via Adenosine Monophosphate-Activated Protein Kinase.

Background: Previous investigations have established the anti-inflammatory properties of fibroblast growth factor 21 (FGF21). However, the specific mechanism through which FGF21 mitigates myocardial ischemia/reperfusion (I/R) injury by inhibiting neutrophil extracellular traps (NETs) remains unclear. Methods: A mice model of myocardial I/R injury was induced, and myocardial tissue was stained with immunofluorescence to assess NETs. Serum NETs levels were quantified using a PicoGreen kit. In addition, the expression levels of adenosine monophosphate (AMP)-activated protein kinase (AMPK) and FGF21 were evaluated by Wes fully automated protein blotting quantitative analysis system. Moreover, a hypoxia/reoxygenation (H/R) model was established using AMPK inhibitor and agonist pretreated H9c2 cells to further explore the relationship between FGF21 and AMPK. Results: Compared with the control group, serum NETs levels were significantly higher in I/R mice, and a large number of NETs were formed in myocardial tissues (97.63 ± 11.45 vs. 69.65 ± 3.33, P < 0.05). However, NETs levels were reversed in FGF21 pretreated mice (P < 0.05). Further studies showed that FGF21 enhanced AMPK expression, which was significantly increased after inhibition of AMPK and decreased after promotion of AMPK (P < 0.05). Conclusions: FGF21 may exert cardioprotective effects by inhibiting I/R injury-induced NETs via AMPK.

ULK1-regulated AMP sensing by AMPK and its application for the treatment of chronic kidney disease.

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a central kinase involved in energy homeostasis. Increased intracellular AMP levels result in AMPK activation through the binding of AMP to the γ-subunit of AMPK. Recently, we reported that AMP-induced AMPK activation is impaired in the kidneys in chronic kidney disease (CKD) despite an increase in the AMP/ATP ratio. However, the mechanisms by which AMP sensing is disrupted in CKD are unclear. Here, we identified mechanisms of energy homeostasis in which Unc-51-like kinase 1 (ULK1)-dependent phosphorylation of AMPKγ1 at Ser260/Thr262 promoting AMP sensitivity of AMPK. AMPK activation by AMP was impaired in Ulk1 knockout mice despite an increased AMP/ATP ratio. ULK1 expression is markedly downregulated in CKD kidneys, leading to AMP sensing failure. Additionally, MK8722, an allosteric AMPK activator, stimulated AMPK in the kidneys of a CKD mouse model (5/6th nephrectomy) via a pathway that is independent of AMP sensing. Thus, our study shows that MK8722 treatment significantly attenuates the deterioration of kidney function in CKD and may be a potential therapeutic option in CKD therapeutics.

Large-scale application of ClinGen-InSiGHT APC-specific ACMG/AMP variant classification criteria leads to substantial reduction in VUS.

Pathogenic constitutional APC variants underlie familial adenomatous polyposis, the most common hereditary gastrointestinal polyposis syndrome. To improve variant classification and resolve the interpretative challenges of variants of uncertain significance (VUSs), APC-specific variant classification criteria were developed by the ClinGen-InSiGHT Hereditary Colorectal Cancer/Polyposis Variant Curation Expert Panel (VCEP) based on the criteria of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP). A streamlined algorithm using the APC-specific criteria was developed and applied to assess all APC variants in ClinVar and the International Society for Gastrointestinal Hereditary Tumours (InSiGHT) international reference APC Leiden Open Variation Database (LOVD) variant database, which included a total of 10,228 unique APC variants. Among the ClinVar and LOVD variants with an initial classification of (likely) benign or (likely) pathogenic, 94% and 96% remained in their original categories, respectively. In contrast, 41% ClinVar and 61% LOVD VUSs were reclassified into clinically meaningful classes, the vast majority as (likely) benign. The total number of VUSs was reduced by 37%. In 24 out of 37 (65%) promising APC variants that remained VUS despite evidence for pathogenicity, a data-mining-driven work-up allowed their reclassification as (likely) pathogenic. These results demonstrated that the application of APC-specific criteria substantially reduced the number of VUSs in ClinVar and LOVD. The study also demonstrated the feasibility of a systematic approach to variant classification in large datasets, which might serve as a generalizable model for other gene- or disease-specific variant interpretation initiatives. It also allowed for the prioritization of VUSs that will benefit from in-depth evidence collection. This subset of APC variants was approved by the VCEP and made publicly available through ClinVar and LOVD for widespread clinical use.

Intranasal Trans-Sialidase Vaccine Mitigates Acute and Chronic Pathology in a Preclinical Oral Chagas Disease Model.

Chagas disease, caused by Trypanosoma cruzi, leads to severe complications in 30% of infected individuals, including acute myocarditis and chronic fibrosing cardiomyopathy. Despite the significant burden of this disease, there is currently no licensed vaccine available to prevent it. This study aimed to evaluate the mucosal and systemic immunogenicity as well as the prophylactic efficacy of a mucosal vaccine candidate and its impact on both acute and chronic cardiomyopathy. The results showed that the nasal administration of trans-sialidase (TS) plus c-di-AMP (TS+A) vaccine elicited a NALT expression of IFN-γ, IL-17a and IL-4 mRNA as well as a nasal-specific production of IgA. An in vivo challenge with TS also triggered increased proliferation of lymphocytes from the NALT, sentinel cervical lymph node, and spleen. TS+A immunization increased the plasma levels of Th1/Th2/Th17 cytokines and elicited an evident cellular response by which to judge enhanced delayed-type hypersensitivity responses following a TS footpad challenge. After oral infection, TS+A-vaccinated mice showed significantly reduced parasitemia and parasite load in the heart, muscles and intestines, while markers of hepatic and muscle damage as well as clinical manifestations of acute infection were strongly diminished. TS+A also attenuated acute myocarditis and the expression of inflammatory markers in the heart. The protection conferred by TS+A extended into the chronic phase, where it resulted in a clear reduction in chronic myocarditis, fibrosis and functional electrocardiographic abnormalities, associated with a decreased expression of the pro-fibrotic TGF-ß. These results revealed that it is possible to develop a mucosal vaccine against T. cruzi based on TS and c-di-AMP that is capable of reducing the development of Chagas cardiomyopathy, the hallmark of Chagas disease.

Immunohistochemistry Study of Antimicrobial Peptides as a Future Diagnostic and Prognostic Tool for Periprosthetic Joint Infections.

Periprosthetic joint infection (PJI) is a reputable complication of arthroplasty surgery. Septic loosening is an implant biofilm-related infection with different characteristics and treatment than aseptic loosening. Misdiagnosing PJI results in choosing an inappropriate treatment and, in most cases, failure to achieve asepsis. The worldwide increase of arthroplasty surgeries forces us to research more accurate ways to detect PJIs earlier, cheaper, and faster. In the current study, we investigated 52 arthroplasty revision surgeries (septic and aseptic) and, using immunohistochemistry staining of periprosthetic tissue, successfully demonstrated an important increase in antimicrobial peptides human ß defensin-3 (HBD-3) and cathelicidin (LL-37) in the PJI group. Furthermore, we observed that patients with a positive LL-37 stain were associated with a more reserved prognosis at one-year follow-up. These promising results suggest that antimicrobial peptides HBD-3 and LL-37 could be used as future biomarkers for PJI detection.

AMPK agonism optimizes the in vivo persistence and anti-leukemia efficacy of chimeric antigen receptor T cells.

BACKGROUND: Chimeric antigen receptor T cell (CART) therapy has seen great clinical success. However, up to 50% of leukemia patients relapse and long-term survivor data indicate that CART cell persistence is key to enforcing relapse-free survival. Unfortunately, ex vivo expansion protocols often drive metabolic and functional exhaustion, reducing in vivo efficacy. Preclinical models have demonstrated that redirecting metabolism ex vivo can improve in vivo T cell function and we hypothesized that exposure to an agonist targeting the metabolic regulator AMP-activated protein kinase (AMPK), would create CARTs capable of both efficient leukemia clearance and increased in vivo persistence. METHODS: CART cells were generated from healthy human via lentiviral transduction. Following activation, cells were exposed to either Compound 991 or DMSO for 96 hours, followed by a 48-hour washout. During and after agonist treatment, T cells were harvested for metabolic and functional assessments. To test in vivo efficacy, immunodeficient mice were injected with luciferase+ NALM6 leukemia cells, followed one week later by either 991- or DMSO-expanded CARTs. Leukemia burden and anti-leukemia efficacy was assessed via radiance imaging and overall survival. RESULTS: Human T cells expanded in Compound 991 activated AMPK without limiting cellular expansion and gained both mitochondrial density and improved handling of reactive oxygen species (ROS). Importantly, receipt of 991-exposed CARTs significantly improved in vivo leukemia clearance, prolonged recipient survival, and increased CD4+ T cell yields at early times post-injection. Ex vivo, 991 agonist treatment mimicked nutrient starvation, increased autophagic flux, and promoted generation of mitochondrially-protective metabolites. DISCUSSION: Ex vivo expansion processes are necessary to generate sufficient cell numbers, but often promote sustained activation and differentiation, negatively impacting in vivo persistence and function. Here, we demonstrate that promoting AMPK activity during CART expansion metabolically reprograms cells without limiting T cell yield, enhances in vivo anti-leukemia efficacy, and improves CD4+ in vivo persistence. Importantly, AMPK agonism achieves these results without further modifying the expansion media, changing the CART construct, or genetically altering the cells. Altogether, these data highlight AMPK agonism as a potent and readily translatable approach to improve the metabolic profile and overall efficacy of cancer-targeting T cells.

Hypoxanthine ameliorates diet-induced insulin resistance by improving hepatic lipid metabolism and gluconeogenesis via AMPK/mTOR/PPARα pathway.

AIM: Insulin resistance (IR) is a pivotal metabolic disorder associated with type 2 diabetes and metabolic syndrome. This study investigated the potential of hypoxanthine (Hx), a purine metabolite and uric acid precursor, in ameliorating IR and regulating hepatic glucose and lipid metabolism. METHODS: We utilized both in vitro IR-HepG2 cells and in vivo diet-induced IR mice to investigate the impact of Hx. The HepG2 cells were treated with Hx to evaluate its effects on glucose production and lipid deposition. Activity-based protein profiling (ABPP) was applied to identify Hx-target proteins and the underlying pathways. In vivo studies involved administration of Hx to IR mice, followed by assessments of IR-associated indices, with explores on the potential regulating mechanisms on hepatic glucose and lipid metabolism. KEY FINDINGS: Hx intervention significantly reduced glucose production and lipid deposition in a dose-dependent manner without affecting cell viability in IR-HepG2 cells. ABPP identified key Hx-target proteins engaged in fatty acid and pyruvate metabolism. In vivo, Hx treatment reduced IR severities, as evidenced by decreased HOMA-IR, fasting blood glucose, and serum lipid profiles. Histological assessments confirmed reduced liver lipid deposition. Mechanistic insights revealed that Hx suppresses hepatic gluconeogenesis and fatty acid synthesis, and promotes fatty acid oxidation via the AMPK/mTOR/PPARα pathway. SIGNIFICANCE: This study delineates a novel role of Hx in regulating hepatic metabolism, offering a potential therapeutic strategy for IR and associated metabolic disorders. The findings provide a foundation for further investigation into the role of purine metabolites in metabolic regulation and their clinical implications.