State Of Tissue Research Articles

Role of RAS/BRAF and PIK3CA mutations in tissue and plasma for prognostic assessment in metastatic colorectal cancer (mCRC).

281 Background: Mutations in RAS/BRAF ( RAS/BRAFm ) and PIK3CA ( PIK3CAm ) genes have been described as mechanisms of resistance to anti-EGFR agents in mCRC. However, the utility of liquid biopsy in their baseline detection remains unknown. We aim to analyse oncological outcomes according to RAS/BRAF and PIK3CA mutational status in tissue & plasma in mCRC. Methods: We conducted aretrospective study in patients (pts) with mCRC from 2017 to 2024. RAS/BRAF and PIK3CA mutational status assessment was performed at baseline using Idylla (Biocartis) and Cobas PIK3CA kits, respectively. Cox models were used to analyse overall survival (OS) in RAS/BRAFm and PIK3CAm vs wild-type (WT) population. Results: We analysed 404 paired samples (tissue & plasma) from 202p with mCRC. RAS/BRAFm was described in 45% (91p) in tissue and in 43.6% (88p) in plasma. PIK3CA was tested in 84p, with PIK3CAm detection in 19% (16p) in tissue and 11.9% (10p) in plasma. Liver involvement was more frequent in RAS/BRAFm vs WT (80.6% vs 63.9%; p=0.02), and in PIK3CAm vs WT population (94.4% vs 63.6%; p=0.01). Concordance study (tissue & plasma) for RAS/BRAF and PIK3CA are presented (Table). Subjects with concordant results had worse OS in RAS/BRAFm (18.1m vs 51.1m, HR=2.60; p<0.001) regardless of liver involvement, and showed the same trend in PIK3CAm (22.6m vs 28.1m, p=0.81). Patients with discordant results and plasma RAS/BRAFm or plasma PIK3CAm showed worse OS (23.7 m vs 51.1m; p = 0.006 and 16.4 m vs 42.5 m; p=0.18, respectively) and a lower disease control rate with anti-EGFR therapy (p = 0.04 and p=0.42, respectively). PIK3CAm were evenly distributed in RAS/BRAF WT vs mutated tumors (50% vs 51.5%; p=0.92) and had no independent prognostic impact in OS after stratifying by RAS/BRAF mutational status; RAS/BRAF WT (p=0,93) and RAS/BRAFm (p=0,91). Conclusions: Baseline determination of RAS/BRAFm and PIK3CAm by liquid biopsy in mCRC provide valuable prognostic and predictive information, extending its utility beyond tissue-based analysis. Correlation study of RAS/BRAF and PIK3CA mutational status in tissue and plasma in mCRC. RAS/BRAF tissue PIK3CA tissue RAS/BRAF Plasma Mutated n (%) WT n (%) Total n (%) PIK3CA Plasma Mutated n (%) WT n (%) Total n (%) Mutated n (%) 72 (80) 15 (13.4) 87 (43.1) Mutated n (%) 7 (43.8) 2 (2.9) 9 (10.7) WT n (%) 18 (20) 97 (86.6) 115 (56.9) WT n (%) 9 (56.2) 66 (97.1) 75 (89.3) Total n (%) 90 (100) 112 (100) 202 (100) Total n (%) 16 (100) 68 (100) 84 (100) Positive agreement: 72/90: 80% Positive agreement: 7/16: 43.8% Negative agreement: 97/112: 86.6% Negative agreement: 66/68: 97.1% Overall agreement: 169/202: 83.6% Overall agreement: 73/84: 86.9% KI*: 0.67 KI: 0.49 KI: Kappa Index.

Long-Term Clinical Outcomes for Bone-Anchored Hearing Implants: 3-Year Comparison Between Minimally Invasive Ponto Surgery and the Linear Incision Technique With Tissue Preservation.

To compare the 3-year outcomes of the modified minimally invasive Ponto surgery (m-MIPS) to both the original MIPS (o-MIPS) and linear incision technique with soft tissue preservation (LIT-TP) for inserting bone-anchored hearing implants (BAHIs). Prospective study with three patient groups: m-MIPS, o-MIPS, and LIT-TP. Tertiary referral center. In the m-MIPS group, 24 patients with 25 implants were enrolled. The o-MIPS and LIT-TP groups included 25 patients (25 implants) each. The patients underwent BAHI surgery using m-MIPS, o-MIPS, or LIT-TP. Implant survival and implant stability were compared between all groups. Soft tissue status, skin sensibility, subjective numbness, and hearing-related quality of life (HRQoL) were compared between m-MIPS and LIT-TP and o-MIPS and LIT-TP, respectively. Implant survival was comparable between m-MIPS and LIT-TP (96 versus 100%), with o-MIPS showing nonsignificant lower survival (88%). Both MIPS groups exhibited fewer (adverse) skin reactions, better skin sensibility, and less subjective numbness than LIT-TP throughout visits. At 3 years, soft tissue status, sensibility, and numbness were comparable between groups. Device use was consistent among groups (83-86% daily users). All groups demonstrated significant improvement in HRQoL post-surgery based on GBI, GHSI, and APHAB total scores. Compared with LIT-TP, m-MIPS showed comparable long-term implant survival, fewer (adverse) skin reactions, and earlier sensibility and numbness recovery. M-MIPS resulted in favorable clinical and QoL outcomes with low intra- and postoperative complication rates until 3 years after surgery. It is therefore considered a safe technique for BAHI insertion. Moreover, with a shorter surgery time and the ability to operate under local anesthesia in a controlled outpatient setting, m-MIPS appears to be a more efficient alternative to LIT-TP.

Modified titanium post with polished sidewalls and prefabricated shoulders can effectively preserve the teeth with subgingival defects

Objective of this study was to examine the clinical efficacy and mechanical characteristics of the modified titanium post in the restoration of subgingival defect teeth. Teeth with subgingival defects depth ≥ 2 mm were randomly restored using a fiber post after crown lengthening and a modified titanium post, respectively. Gingival index, sulcus bleeding index, probing depth, tooth mobility, and gingival papilla height were recorded before and after restoration. Tooth models with a 4 mm subgingival defect, which then underwent fiber post restoration after crown lengthening and modified titanium post restoration respectively, was established. The stresses in the tooth and tooth-supporting structures were measured under vertical and horizontal loads. The gingival index, sulcus bleeding index, and probing depth in the titanium post groups did not show significant differences compared to those of the crown lengthening group. However, the tooth mobility, gingival papilla height, and stress distribution in the tooth and periodontal tissue were significantly better in the titanium post than those in crown lengthening group. Teeth restored with modified titanium post had a healthy periodontal status, clinical efficacy and stress distribution in dental and periodontal tissues were superior to the teeth restored with crown lengthening. However, longer term observation was still needed.

PLK3 weakens antioxidant defense and inhibits proliferation of porcine Leydig cells under oxidative stress

Aging is characterized by cellular degeneration and impaired physiological functions, leading to a decline in male sexual desire and reproductive capacity. Oxidative stress (OS) lead to testicular aging by impairing the male reproductive system, but the potential mechanisms remain unclear. In the present study, the functional status of testicular tissues from young and aged boars was compared, and the transcriptional responses of Leydig cells (LCs) to hydrogen peroxide (H2O2)-induced senescence were explored, revealing the role of OS in promoting aging of the male reproductive system. 601 differentially expressed genes (DEGs) associated with OS, cell cycle regulation, and intracellular processes were identified. These DEGs were significantly enriched in critical aging pathways, including the p53 signaling pathway, autophagy, and cellular senescence. Protein-protein interaction (PPI) network analysis unveiled 15 key genes related to cell cycle and DNA replication, with polo-like kinase 3 (PLK3) exhibiting increased expression under OS. In vitro, PLK3 knockdown significantly enhanced the viability and antioxidant capacity of LCs under OS. This study deepens our understanding of how LCs respond to OS and provides new therapeutic targets for enhancing cellular resistance to oxidative damage and promoting tissue health.

Development Validation and Verification of Dhatu Sarata Assessment Questionnaire (DSAQ): Structural and functional status of Tissues

Development Validation and Verification of Dhatu Sarata Assessment Questionnaire (DSAQ): Structural and functional status of Tissues

INSIGHTS INTO THE MOLECULAR AND BIOCHEMICAL ROLE OF QUINIC ACID IN ALLEVIATING ETHANOL-INDUCED LIVER TOXICITY IN A RAT MODEL: EXPLORING OXIDATIVE STRESS, INFLAMMATION, AND APOPTOSIS SIGNALING PATHWAYS

Objective: The study aimed to evaluate the effects of quinic acid, a natural bioactive compound, on tissue and circulatory antioxidant status, lipid peroxidation, and its anti-apoptotic and anti-inflammatory mechanisms in ethanol-induced hepatotoxicity in rats. Methods: The rats were divided into four groups. Groups 1 and 4 were administered isocaloric glucose. Groups 2 and 3 received 30% ethanol at a dose of 5 g/kg body weight daily. In addition, Groups 3 and 4 were treated with quinic acid (50 mg/kg body weight) dissolved in 2% dimethyl sulfoxide. Results: The results demonstrated significantly elevated levels of tissue thiobarbituric acid reactive substances (TBARS), conjugated dienes (CD), and lipid hydroperoxides (LOOH), along with significantly reduced enzymatic and non-enzymatic antioxidant activities, including superoxide dismutase (SOD), catalase (CAT), and glutathione-related enzymes such as glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione-S-transferase (GST), as well as reduced levels of glutathione (GSH), Vitamin C, and Vitamin E in ethanol-treated rats compared to the control group. Administration of quinic acid to rats with ethanol-induced liver injury significantly reduced the levels of TBARS, LOOH, and CD while markedly increasing the activity of SOD, CAT, GPx, GR, GST, and levels of GSH, Vitamin C, and Vitamin E in liver tissues compared to untreated ethanol-exposed rats. In addition, ethanol-treated rats showed increased mast cell accumulation, which was reduced by quinic acid treatment, along with elevated expressions of inflammatory and apoptotic markers, including Bax, Caspase-9, tumor necrosis factor-alpha, Nuclear factor kappa B, and interleukin-6, and a decreased expression of Bcl2 in the liver. Quinic acid supplementation in ethanol-fed rats reversed these ethanol-induced changes. Immunohistochemical studies further supported these findings. Conclusion: Quinic acid, with its antioxidant, anti-inflammatory, and anti-apoptotic properties, may offer a therapeutic option for protecting against ethanol-induced hepatotoxicity

Roles of perivascular adipose tissue in the pathogenesis of atherosclerosis - an update on recent findings.

Lifestyle-related diseases, such as atherosclerosis and diabetes, are now considered to be a series of diseases caused by chronic inflammation. Adipose tissue is considered to be an endocrine organ that not only plays a role in lipid storage, heat production, and buffering, but also produces physiologically active substances and is involved in chronic inflammation. Perivascular adipose tissue (PVAT) surrounding blood vessels similarly produces inflammatory and anti-inflammatory physiologically active substances that act on blood vessels either directly or via the bloodstream. Epicardial adipose tissue (EAT), which is in direct contact with the coronary arteries inside the pericardium, is thought to have a direct effect on the coronary arteries as well. The presence and inflammatory status of these adipose tissues can be evaluated by imaging tests, and has been shown to be associated with the presence of current cardiovascular disease (CVD) and to be a prognostic factor. It is also expected to become a new diagnostic and therapeutic target for CVD.

MORPHOLOGICAL ASPECTS OF POST-TRAUMATIC LOWER JAW REMODELING IN THE SETTING OF OSTEOPLASTY

Bone tissue regeneration remains a complex and crucial challenge for modern medicine. The unique properties of chitosan, its effects on bone regeneration in the maxillofacial region, as well as the underlying mechanisms and dynamics, remain poorly understood and warrant further investigation. Aim. The aim of the study is to determine the dynamics of morphological, radiological, and lectin-histochemical characteristics of bone-ceramic regenerate after the transplantation of octacalcium phosphate with the addition of chitosan acetate with ampicillin into an experimental defect in the rabbit mandible. Materials and methods. Adult male rabbits aged 6-7 months and weighing 2.5-3 kg were used for the study. The control group consisted of animals with a bone defect that healed under a blood clot. The experimental group consisted of rabbits in which the bone defect was filled with an osteotropic material containing octacalcium phosphate, chitosan acetate, and ampicillin (OCР-N-Chitosan-Ampicillin). Post-traumatic bone tissue status within the defect area was monitored for 84 days. The following methods were employed: bone defect modeling, assessment of jaw macrostructure, radiographic examination, radiovisiography, examination of bone sections under a microscope, and lectin-histochemical analysis of decalcified bone sections. Results. Examination of the macrostructure of the experimental bone defect in the rabbit mandible after implantation of material with octacalcium phosphate, chitosan acetate, and ampicillin (OCР-N -Chitosan-Ampicillin) revealed numerous regenerative changes that occurred after the injury and correlated with the data of radiographic and radiovisiographic examinations. Microscopic examination of bone sections allowed us to establish the phased nature of the dynamics of the studied regenerative changes and the composition of the regenerate. Conclusions. It was established that in the experimental group of animals in which defect repair was performed using OCP-N-Chitosan-Ampicillin material, a shortening of the reactive phase of the post-traumatic inflammatory process was observed, which ensured the active onset of osteoregeneration. The use of this combination of drugs showed accelerated reparative regeneration of the mandible and adjacent mucosa.

Extensive methylation analysis of circulating tumor DNA in plasma of patients with gastric cancer

DNA methylation is known to be involved in tumor progression. This is the first study to perform an extensive methylation analysis of plasma circulating tumor DNA (ctDNA) using targeted bisulfite sequencing in gastric cancer (GC) patients to evaluate the usefulness of ctDNA methylation as a new biomarker. Sixteen patients who received chemotherapy for recurrent GC were included. After confirmation of the methylation status of 63 genes using the Cancer Genome Atlas (TCGA) dataset, the methylation status in paired tumor and non-tumor tissues and plasma were investigated using targeted bisulfite sequencing in these genes. Forty-four of the 63 genes were significantly hypermethylated in GC patients in the TCGA cohort. Of these 44 genes, hierarchical clustering showed that five (SPG20, FBN1, SDC2, TFPI2, SEPT9) were particularly hypermethylated in tumor compared to non-tumor tissues in our GC cohort. In plasma methylation analysis, patients with high methylation of these genes had significantly worse overall survival than those with low methylation (log-rank P = 0.009). In a patient who underwent blood sampling at multiple points, the methylation levels of these five genes varied closely with clinical tumor status. The plasma ctDNA methylation levels of these five genes could be useful as a noninvasive prognostic biomarker for GC.

Platelet-rich plasma effects on in vitro cells derived from pediatric patients with andrological diseases

Undescended testis and testicular torsion represent two frequent andrological diseases that affect the pediatric age. Despite these testicular disorders having different causes, they both negatively influence fertility in adulthood mainly due to the accumulation of reactive oxygen species (ROS), which represents the primary molecular damage underlying their long-term effects. The gold standard of treatment for both pathologies is surgery; however, it cannot guarantee an optimal fertility outcome in all clinical cases, underscoring the need to identify effective adjuvant therapies that may target the augmented ROS levels. For this aim, we investigated the pro-proliferative and anti-oxidant effects of PRP (platelet-rich plasma), a hemoderivative product used in regenerative medicine. We confirmed the increased oxidative status in testicular tissue by directly analyzing patients’ biopsies with mass spectrometry and highlighting that three antioxidant proteins are significantly overexpressed compared to healthy testicles. Afterward, we in vitro treated cells derived from patients with cryptorchidism or testicular torsion with PRP, showing that it consistently decreases ROS levels and slightly induces cell proliferation. This study supports the potential use of PRP in patients with testis torsion or cryptorchidism, encouraging its future clinical application as adjuvant therapy to preserve the functionality of this organ by decreasing its ROS levels.

The role of oxidative stress in bone tissue in the pathogenesis of osteopenia in patients with chronic lymphocytic leukemia

Introduction. A decrease in bone mineral density (BMD), the development of osteopenia and osteoporosis is observed in patients with chronic lymphocytic leukemia (CLL). Patients with CLL are at a higher risk of developing fractures due to osteoporosis compared to healthy age-matched individuals. The pathogenesis of the osteodestructive process in CLL has been poorly studied and may be associated with excessive generation of reactive oxygen species and/or inhibition of antioxidant defense.Aim: to investigate the relationship between indicators of oxidative stress in bone tissue and indicators of osteopenia in patients with CLL.Materials and methods. The study included 48 male patients with CLL aged 50–70 years, divided into group 1 (n = 34) without signs of osteopenia and group 2 (n = 14) with signs of osteopenia based on osteodensitometry (T-score from –1.0 SD to –2.5 SD). BMD, T- and Z-scores were assessed in the lumbar spine, proximal femoral neck (PFC), and proximal femur. In the bone tissue homogenate, the content of products of oxidative modification of proteins (OMP) was determined spectrophotometrically in spontaneous and metal-catalyzed modes, reserve-adaptation potential, and general antioxidant status.Results. Osteopenia was detected in 30 % of patients with CLL according to osteodensitometry in the neck of the proximal femur. In patients with CLL and osteopenia, signs of oxidative stress were observed in the bone tissue: early OMP products of a neutral and basic nature, late products of a neutral nature accumulated in the spontaneous detection mode; early and late OMP products of a neutral and basic nature accumulated in the induced mode; reserve-adaptive potential, the general antioxidant status decreased. Signs of osteopenia in the PFC in patients with CLL in the femoral neck increased as the content of early and late OMP products in the bone tissue increased in spontaneous and metal-induced detection modes, and the general antioxidant status in the bone tissue decreased.Conclusion. Based on the data obtained, it is possible to modernize diagnostic criteria and therapeutic approaches.

Morphological characteristics of regeneration of the lower jaw bone in an experiment using natural collagen with lincomycin

This study aimed to investigate the dynamic changes in the histo­morphological architecture of bone-ceramic regeneration following the transplantation of natural collagen combined with lincomycin into an experimental defect of the rabbit mandible. 45 adult male rabbits aged 6-7 months and weighing 2.5-3 kg were used for the study. 20 animals were in control group and 20 animals in experimental. An additional 5 intact animals were used to study the normal structure of bone tissue in the studied area of the lower jaw. The control group consisted of animals with a bone defect that healed under a blood clot. The experimental group consisted of rabbits with the bone defect filled with natural collagen, with simultaneous intramuscular injections of lincomycin at a dose of 12 mg/kg body weight once a day for 6 days (Col-C-lincomycin). Post-traumatic bone tissue status within the defect area was monitored for 84 days. Ultrastructural changes were studied using scanning electron microscopy. To determine changes in the composition in the bone regeneration, three parameters were calculated – the relative volume of: 1) bone tissue, 2) osteoplastic material and 3) connective tissue in the bone regeneration. The data were analyzed using the Student's t-test, and a difference at p<0.05 was considered statistically significant. The research of the surface topography of the experimental bone defect in the rabbit mandible, following the implantation of Col-C material in combination with lincomycin, revealed numerous regenerative changes that occurred after injury and correlated with the dynamic changes in the relative volume of bone tissue, osteoplastic material, and connective tissue within the regeneration. Morphometric analysis of the relative volume of the constituent components of the regeneration in the experimental defect revealed a phased nature of the dynamics of the observed changes. The osteocyte lacuno-canalicular system that formed after material implantation acquired characteristics of typical structure. Foci of incomplete osteogenesis were not visualized. Unlike the control group, after the application of Col-C material in combination with lincomycin, the majority of osteons in the regeneration near the outer bone plate did not differ from the typical structure of the maternal bone in terms of both its structure and geometry.

Increased ROS levels, antioxidant defense disturbances and bioenergetic disruption induced by thiosulfate administration in the brain of neonatal rats.

Sulfite oxidase deficiencies, either caused by deficiency of the apoenzyme or the molybdenum cofactor, and ethylmalonic encephalopathy are inherited disorders that impact sulfur metabolism. These patients present with severe neurodeterioration accompanied by cerebral cortex and cerebellum abnormalities, and high thiosulfate levels in plasma and tissues, including the brain. We aimed to clarify the mechanisms of such abnormalities, so we assessed the ex vivo effects of thiosulfate administration on energetic status and oxidative stress markers in cortical and cerebellar tissues of newborn rats. Thiosulfate (0.5 µmol/g) or PBS (vehicle) was injected into the fourth ventricle of rat pups. Thirty minutes after the injection, animals were euthanized and the brain structures were utilized for the experiments. Our data showed that thiosulfate decreased the reduced glutathione (GSH) concentrations, and superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST) activities in the cortical structure. Thiosulfate also increased DCFH oxidation, hydrogen peroxide generation and glutathione reductase activity. In the cerebellum, thiosulfate reduced SOD and glutathione peroxidase activities but increased GST and CAT activities as well as DCFH oxidation. Regarding energy metabolism, thiosulfate specifically decreased complex IV activity in the cortex, whereas it increased cerebellar complex I and creatine kinase activities, indicating bioenergetic disturbances. The results suggest that the accumulation of thiosulfate causing redox disruption and bioenergetic alterations has a prominent role in the pathogenesis of sulfur metabolism deficiencies.

Functional and phenotypic changes in natural killer cells expressing immune checkpoint receptors PD-1, CTLA-4, LAG-3, and TIGIT in non-small cell lung cancer: the comparative analysis of tumor microenvironment, peripheral venous blood, and tumor-draining veins.

Natural killer (NK) cells are a cytotoxic subset of innate lymphoid cells and have key roles in antitumoral immunity. This study evaluates the roles of immune checkpoint receptors on NK cell phenotype and functions both before and after circulation through tumor tissue. Twenty non-small cell lung cancer patients undergoing surgery and 21 healthy controls were included. Lymphocytes were isolated from peripheral venous blood, tumor-draining venous blood, and tumor tissue. Immune checkpoint receptor (ICR) expressions, intracellular cytokines, and cytotoxic capacity of NK cell subsets were analyzed by flow cytometry. Circulatory levels of sPD-1, sCTLA-4, sLAG-3, and sTIGIT were determined by ELISA. PD-1, CTLA-4, and LAG-3 expressions of both cytotoxic (CD56neg/dimCD16bright) and cytokine-producing (CD56bright/dimCD16neg) NK cells increased in tumor tissue compared to both peripheral and tumor-draining veins. NK cells expressing PD-1, CTLA-4, or LAG-3 had significantly lower IFN- and TNF- and increased IL-10 expressions in tumor tissue compared to peripheral venous blood. The cytotoxic activity (perforin and granzyme A expressions) of NK cells from tumor tissue was significantly reduced compared to peripheral blood. Soluble ICRs decreased in peripheral blood and tumor-draining vein of the patients compared to peripheral blood of healthy individuals. However, NK cell phenotype and functions were similar in peripheral blood and tumor-draining vein. NSCLC tumor microenvironment impacts ICR expressions in NK cells, and ICR-expressing NK cells have impaired inflammatory cytokine secretion and cytotoxic activities with a regulatory phenotype. However, tumor-draining venous blood did not reflect the immune status of the tumor tissue.

ABCG1 orchestrates adipose tissue macrophage plasticity and insulin resistance in obesity by rewiring saturated fatty acid pools.

The mechanisms governing adipose tissue macrophage (ATM) metabolic adaptation during diet-induced obesity (DIO) are poorly understood. In obese adipose tissue, ATMs are exposed to lipid fluxes, which can influence the activation of specific inflammatory and metabolic programs and contribute to the development of obesity-associated insulin resistance and other metabolic disorders. In the present study, we demonstrate that the membrane ATP-binding cassette g1 (Abcg1) transporter controls the ATM functional response to fatty acids (FAs) carried by triglyceride-rich lipoproteins, which are abundant in high-energy diets. Mice genetically lacking Abcg1 in the myeloid lineage presented an ameliorated inflammatory status in adipose tissue and reduced insulin resistance. Abcg1-deficient ATMs exhibited a less inflammatory phenotype accompanied by a low bioenergetic profile and modified FA metabolism. A closer look at the ATM lipidome revealed a shift in the handling of FA pools, including a redirection of saturated FAs from membrane phospholipids to lipid droplets, leading to a reduction in membrane rigidity and neutralization of proinflammatory FAs. ATMs from human individuals with obesity presented the same reciprocal relationship between ABCG1 expression and this inflammatory and metabolic status. Abolition of this protective, anti-inflammatory phenotype in Abcg1-deficient ATMs was achieved through restoration of lipoprotein lipase (Lpl) activity, thus delineating the importance of the Abcg1/Lpl axis in controlling ATM metabolic inflammation. Overall, our study identifies the rewiring of FA pools by Abcg1 as a major pathway orchestrating ATM plasticity and insulin resistance in DIO.

Role of Redox Homeostasis in the Communication Between Brain and Liver Through Extracellular Vesicles.

Extracellular vesicles (EVs) are small, membrane-bound particles secreted by cells into the extracellular environment, playing an increasingly recognized role in inter-organ communication and the regulation of various physiological processes. Regarding the redox homeostasis context, EVs play a pivotal role in propagating and mitigating oxidative stress signals across different organs. Cells under oxidative stress release EVs containing signaling molecules that can influence the redox status of distant cells and tissues. EVs are starting to be recognized as contributors to brain-liver communication. Therefore, in this review, we show how redox imbalance can affect the release of EVs in the brain and liver. We propose EVs as mediators of redox homeostasis in the brain-liver axis.

Huazhi Rougan Granule Alleviates Liver and Intestinal Damage in Non-Alcoholic Fatty Liver Disease by Regulating miR-122 Expression and TLR4/MyD88/NF-κB Pathway Activation.

miR-122 is upregulated in non-alcoholic fatty liver disease (NAFLD) liver tissue, and knockdown of miR-122 protects hepatocytes from lipid metabolism disorders. This study aimed to investigate whether Huazhi Rougan Granule (HRG) alleviates NAFLD liver and intestinal injury by regulating the miR-122-mediated TLR4/MyD88/NF-κB pathway. Rats with NAFLD were constructed by high-fat feeding. Serum levels of total cholesterol (TC), triglycerides (TG), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were measured using a fully automated biochemical instrument. Histopathological changes in the liver and small intestine were observed by HE staining. QRT-PCR detected the expression level of miR-122 in the liver tissues. The protein expression of TLR4, MyD88, NF- κB p65, and p-p65 in liver tissues was detected by western blotting. HRG slowed down the weight gain of NAFLD rats, decreased (P<0.05) the levels of TC, TG, ALT, AST, TNF-α, IL-1β, IL-6, LPS, and Hpt, improved the pathological status of liver and small intestine tissues, upregulated (P<0.05) the expression of ZO-1 and Occludin, downregulated (P<0.05) the protein expression of TLR4, MyD88, and p-p65, and inhibited (P<0.05) the expression of miR-122. HRG may alleviate hepatic and intestinal injuries in rats with NAFLD by regulating the miR-122-mediated TLR4/MyD88/NF-κB pathway.

Unraveling the DNA methylation landscape in dog blood across breeds

BackgroundDNA methylation is a covalent bond modification that is observed mainly at cytosine bases in the context of CG pairs. DNA methylation patterns reflect the status of individual tissues, such as cell composition, age, and the local environment, in mammals. Genetic factors also impact DNA methylation, and the genetic diversity among various dog breeds provides a valuable platform for exploring this topic. Compared to those in the human genome, studies on the profiling of methylation in the dog genome have been less comprehensive.ResultsOur study provides extensive profiling of DNA methylation in the whole blood of three dog breeds using whole-genome bisulfite sequencing. The difference in DNA methylation between breeds was moderate after removing CpGs overlapping with potential genetic variation. However, variance in methylation between individuals was common and often occurred in promoters and CpG islands (CGIs). Moreover, we adopted contextual awareness methodology to characterize DNA primary sequences using natural language processing (NLP). This method could be used to effectively separate unmethylated CGIs from highly methylated CGIs in the sequences that are identified by the conventional criteria.ConclusionsThis study presents a comprehensive DNA methylation landscape in the dog blood. Our observations reveal the similar methylation patterns across dog breeds, while CGI regions showed high variations in DNA methylation level between individuals. Our study also highlights the potential of NLP approach for analyzing low-complexity DNA sequences, such as CGIs.

Abstract A038: Investigating PD-L1 status in circulating tumor cells isolated from blood samples of lung cancer patients

Abstract Background: This research study aimed to assess the performance of a research use only (RUO) immunofluorescence (IF) assay targeting programmed death-ligand 1 (PD-L1) evolution in epithelial and/or mesenchymal circulating tumor cells (CTCs) isolated from blood using Parsortix® microfluidic technology. Upregulation of PD-L1 enables cancer cells to evade the host immune response. Assessment of PD-L1 status in the tumor, as determined by traditional tissue biopsy, can indicate if immunotherapy has the potential to be an effective treatment. However, PD-L1 expression in tumor biopsies may not reflect metastatic sites in their entirety and can become outdated during tumor evolution, as the process cannot be repeated due its invasive nature. Liquid biopsy offers a minimally invasive option for dynamic testing of PD-L1 in CTCs as patients undergo treatment. Methods: Analytical linearity, and specificity and sensitivity were assessed by spiking HCC1954 and Hs 578T cancer cell lines into the blood samples of 34 healthy volunteers. The assay was then evaluated in samples from 47 metastatic lung cancer patients, among which 32 had a known PD-L1 tissue status (19 positive, 13 negative). Up to six successive draws were taken for each patient, collected into Streck Cell-Free DNA blood collection tubes. ANGLE’s Parsortix® instruments were used to isolate spiked cancer cells or CTCs from blood samples based on their size and deformability. The CTC- enriched harvests were processed onto ANGLE’s CellKeepTM slides and IF stained with ANGLE’s Portrait® PD-L1 RUO assay for PD-L1 identification on CTCs. Slides were imaged on a BioView automated imaging system. Results: Analytical specificity of PD-L1 was 98% and analytical sensitivity was 81%. Analytical linearity (R2=0.91) was shown over a 0–500 cells range. In metastatic lung cancer patients, ≥1 CTC was identified in at least one draw for 91% of donors, with 55% of those donors exhibiting ≥1 PD-L1 positive CTC. A majority (81%) of donors exhibited a positive correlation between PD-L1 tissue status and CTC expression. However, the remaining 19% of donors exhibited discordance with a PD-L1 negative tissue status but PD-L1 positivity identified in ≥1 CTC. Of the PD-L1 positive CTCs identified, 98% expressed mesenchymal markers. Dynamic change was shown in all but one donor, with PD-L1 status of the CTCs changing over time. Conclusions: This study demonstrated the ability to determine PD-L1 status in CTCs from the blood of metastatic lung cancer patients and, subject to further study, the future potential to develop dynamic PD-L1 testing for advancement of more personalized cancer treatments. Patients with a PD-L1 negative tissue status exhibited PD-L1 positive CTCs, demonstrating the feasibility of a more repeatable and accurate assessment of PD-L1 status than can be achieved through a traditional tissue biopsy. Citation Format: Morgan Spode, Chloe Goodwin, Aarabhi Varatharajah, Aaron Cottingham, Elisha Duhig, Laura Kaja, David Greaves, Mariacristina Ciccioli, Anne-Sophie Pailhes-Jimenez. Investigating PD-L1 status in circulating tumor cells isolated from blood samples of lung cancer patients [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A038.

Abstract 4148070: Clonal Hematopoeisis of Indeterminate Potential and Risk of Autopsy-defined Sudden Cardiac Death

Introduction: Clonal hematopoiesis of indeterminate potential (CHIP) has been associated with an increased risk of coronary artery disease (CAD) and cardiovascular (CV) mortality. Whether CHIP may affect myocardium beyond promoting CAD, or its potential association with sudden cardiac death (SCD), the most feared manifestation of CV disease, is unknown. Research Questions/Hypothesis: We hypothesize that CHIP-mutant macrophages infiltrate the myocardium and are associated with autopsy-defined arrhythmic death. Goals/Aims: (1) determine rate of CHIP at time of death in an unselected, countywide study of all incident sudden deaths; (2) evaluate whether CHIP-mutant macrophages infiltrate into myocardium; and (3) evaluate potential association of CHIP with autopsy-confirmed arrhythmic causes among countywide sudden deaths Methods/Approach: We used autopsy to adjudicate arrhythmic from non-arrhythmic (PE, stroke, overdose, tamponade) causes in 1,148 sudden deaths and 141 trauma control deaths in San Francisco County from 2011-23. DNA was extracted from frozen blood samples collected at time of death from 399 consented cases. Sequencing of 22 CHIP-associated genes was performed to ~2000x mean target coverage. All pathogenic/likely pathogenic CHIP variants at &gt;2% variant allele frequency (VAF) were considered CHIP+. DNA was then isolated from left ventricular (LV) tissue sampled at autopsy in all CHIP+ blood cases and sequenced as above. Results: Of 399 consented cases, 70 blood samples (17.5%) were CHIP+ (range 2%-39.6%; mean age 70.6 years vs. 58.1 years for CHIP-). The most frequent mutant genes were DNMT3A, TET2, and ASXL1. Proportion of arrhythmic causes among sudden deaths was similar for CHIP+ vs. CHIP- cases (36 of 70 [52%] vs. 171 of 329 [51%]). Sequencing of available LV tissue from 61 CHIP+ blood cases revealed 67% (n=41) harbored the same CHIP mutation identified in blood at &gt;0.2% VAF (range 0.2%-4.3%). Proportion of arrhythmic causes among sudden deaths was significantly higher in cases where both blood and LV were CHIP+ (24 of 41 [58.5%] vs. 5 of 19 [26.3%] CHIP+ blood/CHIP- LV, p=0.02). Conclusions: Prevalence of CHIP positivity at sudden death is similar to published studies and correlated with age. CHIP+ status in myocardial tissue but not peripheral blood was associated with arrhythmic causes of sudden death, suggesting a direct tissue effect of CHIP-mutant macrophages.