DNA Fragmentation Factor Research Articles

DNA fragmentation factor 40-based therapeutic approaches for cancer: a review article.

DNA Fragmentation Factor (DFF) is a heterodimer protein involved in DNA fragmentation during apoptosis, which acts as a trigger downstream of caspase-3 activation. DFF40 catalytically active homo-oligomers break down chromosomal DNA. Previous scientific investigations have revealed a link between DFF40 expression changes and various cancers. DFF40 deletion or down-regulation has been observed in some cancers. Consequently, therapeutic strategies involving the DFF40 molecule compensating led to an increased rate of cancer cell apoptosis. In this review article, we aimed to introduce cancers with low expression of this protein first. The second part of this paper focuses on studies that utilized exogenous DFF40 protein produced by recombinant DNA technology and surveyed during in vitro and in vivo tests. Finally, compensation for diminished expression of the mentioned protein via gene therapy-based techniques to make up for this apoptotic molecule's low expression is the topic of thelast part of this review article.

DFF-ChIP: a method to detect and quantify complex interactions between RNA polymerase II, transcription factors, and chromatin.

Recently, we introduced a chromatin immunoprecipitation (ChIP) technique utilizing the human DNA Fragmentation Factor (DFF) to digest the DNA prior to immunoprecipitation (DFF-ChIP) that provides the precise location of transcription complexes and their interactions with neighboring nucleosomes. Here we expand the technique to new targets and provide useful information concerning purification of DFF, digestion conditions, and the impact of crosslinking. DFF-ChIP analysis was performed individually for subunits of Mediator, DSIF, and NELF that that do not interact with DNA directly, but rather interact with RNA polymerase II (Pol II). We found that Mediator was associated almost exclusively with preinitiation complexes (PICs). DSIF and NELF were associated with engaged Pol II and, in addition, potential intermediates between PICs and early initiation complexes. DFF-ChIP was then used to analyze the occupancy of a tight binding transcription factor, CTCF, and a much weaker binding factor, glucocorticoid receptor (GR), with and without crosslinking. These results were compared to those from standard ChIP-Seq that employs sonication and to CUT&RUN which utilizes MNase to fragment the genomic DNA. Our findings indicate that DFF-ChIP reveals details of occupancy that are not available using other methods including information revealing pertinent protein:protein interactions.

Relationship between the Cell Death-Inducing DNA Fragmentation Factor 45-Like Effector Protein Family and the Risk of Dyslipidemia.

According to the research, obesity is associated with hyperlipidemia, hypertension, and type 2 diabetes mellitus, which are grouped as metabolic syndrome. Notably, under the obese status, the adipocyte could accumulate excessive lipid as lipid droplets (LDs), leading the dysfunctional fat mass. Recently, emerging evidence has shown that the cell death-inducing DNA fragmentation factor 45-like effector protein (CIDE) family played an important role in regulating lipid metabolism. In addition, diverse CIDE proteins were also confirmed to influence the intracellular lipid metabolism, such as within adipocyte, hepatocyte, and macrophage. Nevertheless, the results which showed the regulatory influence of CIDE proteins are significantly contradictory from in vitro experiments and in vivo clinical studies. Similarly, recent studies have changed the perception of these proteins, redefining them as regulators of lipid droplet dynamics and fat metabolism, which contribute to a healthy metabolic phenotype in humans. However, the underlying mechanisms by which the diverse CIDE proteins alter lipid metabolism are not elucidated. In the current review, the understandings of CIDE proteins in lipid catabolism were well-summarized. On the other hand, the relatively mechanisms were also proposed for the further understandings of the CIDE protein family.

Spatial multi-omics characterizes GPR35-relevant lipid metabolism signatures across liver zonation in MASLD

Abstract Metabolic dysfunction-associated steatotic liver disease (MASLD) is a metabolic disease that can progress to metabolic dysfunction-associated steatohepatitis (MASH), cirrhosis, and cancer. The zonal distribution of biomolecules in the liver is implicated in mediating the disease progression. Recently, G-protein-coupled receptor 35 (GPR35) has been highlighted to play a role in MASLD, but the precise mechanism is not fully understood, particularly, in a liver-zonal manner. Here, we aimed to identify spatially distributed specific genes and metabolites in different liver zonation that are regulated by GPR35 in MASLD, by combining lipid metabolomics, spatial transcriptomics (ST), and spatial metabolomics (SM). We found that GPR35 influenced lipid accumulation, inflammatory and metabolism-related factors in specific regions, notably affecting the anti-inflammation factor ELF4 (E74 like E-twenty six (ETS) transcription factor 4), lipid homeostasis key factor CIDEA (cell death-inducing DNA fragmentation factor alpha (DFFA)-like effector A), and the injury response-related genes SAA1/2/3 (serum amyloid A1/2/3), thereby impacting MASLD progression. Furthermore, SM elucidated specific metabolite distributions across different liver regions, such as C10H11N4O7P (3ʹ,5ʹ-cyclic inosine monophosphate (3ʹ,5ʹ-IMP)) for the central vein, and this metabolite significantly decreased in the liver zones of GPR35-deficient mice during MASLD progression. Taken together, GPR35 regulates hepatocyte damage repair, controls inflammation, and prevents MASLD progression by influencing phospholipid homeostasis and gene expression in a zonal manner.

Cell-autonomous Action of the FGF19 Promotes UCP1 Expression and Function in Human Brown Adipocytes

Fibroblast growth factor 19 (FGF19) is an intestine-derived hormone that regulates glucose and energy metabolism. Central and systemic administration of human FGF19 in mice promotes increased energy expenditure and body weight loss through mechanisms that are not fully understood. In this study, we investigated whether FGF19 can activate thermogenesis in brown adipocytes (BA) in a cell-autonomous manner. We differentiated immortalized human brown preadipocytes into mature adipocytes with adipogenic induction media in the presence and absence of FGF19 for 18 days to evaluate the effects of FGF19 on adipogenesis and thermogenesis. Through qPCR and RNA-sequencing analyses, we observed that FGF19 receptors were expressed in both brown preadipocytes and mature BA. However, the obligatory FGF19 co-receptor, β-klotho, was not expressed in preadipocytes, suggesting that the effects of FGF19 must occur preferentially in mature BA. Lipid staining with Oil Red O as well as gene expression of the adipogenic markers fatty acid binding protein (FABP4) and peroxisome proliferator activated receptor-γ (PPAR-γ) showed that adipogenesis was not affected by FGF19. We then treated mature BA with FGF19 (100 ng/mL) acutely and chronically to evaluate FGF19-induced changes in signaling, gene transcription, and thermogenic function. FGF19 signaling was assessed by measuring ERK1/2 phosphorylation levels, which increased within 5 min and remained elevated for at least 30 min after treatment. Using Seahorse assays, we observed that 24-hour FGF19 treatment promoted increased basal respiration, ATP production, and proton leak in human BA, indictive of enhanced mitochondrial function. Since proton leak across the inner membrane through the uncoupling protein 1 (UCP1) generates heat, we assessed the expression of UCP1 and other thermogenic genes. Interestingly, FGF19 treatment specifically increased UCP1 gene expression without affecting other classical adrenergic-mediated thermogenic genes, such as deiodinase 2 (DIO2), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α), PR domain containing 16 (PRDM16), and cell death-inducing DNA fragmentation factor A (CIDEA). These findings suggest that FGF19 regulates UCP1 expression through non-canonical pathways. Future work will elucidate the mechanism by which FGF19 promotes UCP1 gene expression in mature BA. Taken together, FGF19 stimulates ERK1/2 phosphorylation and UCP1 expression in human BA, leading to enhanced mitochondrial respiration. These results provide new mechanistic insights into FGF19’s effects on metabolic regulation. Topic subcategories: Adipocyte biology. Funding sources: Sao Paulo Research Foundation (FAPESP): 2022/15148-2 and NIH R01 DK102898. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Differential expression of DNA fragmentation factor 45 in leukoplakia and oral squamous cell carcinoma and its correlation with TNF-α levels

BackgroundDNA fragmentation factor (DFF) - 45, a heterodimeric protein, involved in apoptotic processes is found to play important role in various human cancers. Its role in the development and progression of oral cavity squamous cell carcinoma (OSCC) is still unclear. Tumour necrosis factor-α (TNF-α) has been found to be higher in OSCC, yet, the mechanism of circumventing the antitumor effect of this cytokine is not understood. Thus, the aim of this study was to evaluate the expression of DFF-45 in samples of OSCC, leukoplakia, and healthy oral mucosa (control) and correlate it with salivary and serum TNF-α. Materials and methodsSurgically excised specimens from patients with primary OSCC (n = 20) leukoplakia (n = 30) and normal oral mucosa (n = 25) were immuno stained for DFF-45. The relationship of DFF-45expression with clinical parameters and histological grading was also evaluated. TNF-α levels were measured in serum and saliva collected from study subjects using cytokine ELISA technique. ResultsThe expression of DFF-45 in OSCC was either absent or mild, while a significantly up-regulated expression of this protein was observed in leukoplakia lesions. TNF-α levels in both saliva and serum were significantly higher in OSCC group compared to control and leukoplakia. We observed a negative correlation between TNF-α levels and DFF-45 expression in OSCC, similar observation was found with histological staging of OSCC. ConclusionThe absence of DFF-45 in oral cancer lesion suggests that this protein may suppress tumour progression. Down regulation of DFF-45 by tumor cells may be a mechanism to evade the anti-cancer effect of TNF-α and further investigations may unravel its role into malignant transition.

The Interplay of Varicoceles, Sperm Epigenetics, and Male Infertility: A Focused, Contemporary Review

Despite the wide recognition of varicocele as a contributor to infertility, its exact pathophysiologic mechanism is not fully understood. As the field of male infertility continues to see progress in new diagnostic and therapeutic technologies, we sought to obtain a robust understanding of the interplay of varicoceles, oxidative stress, DNA methylation, and male infertility. We performed a comprehensive review of the contemporary literature in PubMed using search terms and keywords associated with varicoceles, DNA methylation, and male infertility. The most important findings from the literature review were included in this report. Articles were selected based on quality and relevance to the topic. Eight studies were included in this review. These studies discuss DNA methylation and varicocele-associated male infertility through evaluation of sperm parameters, DNA methylation, DNA fragmentation, and other relevant factors. Our review of the literature on varicocele-associated sperm DNA methylation indicates that global hypomethylation occurs in association with varicoceles. Some data suggest a dose-dependent effect of varicocele grade on sperm DNA methylation, with the greatest magnitude changes seen in grades II and III varicoceles. While changes in DNA methylation patterns would be expected following varicocelectomy, such changes were not found to be statistically significant in one study reviewed here. However, current human studies are limited by small sample size and short follow-up time. Future clinical and preclinical studies with robust sample sizes and hypothesis-driven investigations are necessary to further understand the relationship between DNA methylation patterns and varicocele-associated male infertility.

Production of recombinant DNA fragmentation factor 40 in fusion to an antimicrobial peptide from spider venom and evaluation of its cytotoxic effects.

DNA fragmentation factor 40 (DFF40) as an apoptotic molecule can represent a novel approach to cancer treatment. Lycosin-I (LYC-I), a peptide derived from spider venom, was considered for the targeted delivery of DFF40 to cancer cells. This study attempted to produce soluble DFF40-LYC-I and evaluate its selective lethal effects on HeLa cells. pTWINl vector was used to produce LYC-I and DFF40-LYC-I in E. coli BL21 (DE3) fused to inteins 1 and 2. IPTG concentration and incubation temperature were optimized to achieve the highest level of soluble product. To remove inteins 1 and 2 from the recombinant peptide or protein, pH shift and dithiothreitol were used for a 24-h incubation period at room temperature, respectively. MTT assay was performed to assess the biological effects of these bio-molecules on HeLa and HUVEC cell lines. LYC-I and DFF40-LYC-I were detected in SDS-PAGE with bands of approximately 57 and 97 kDa, respectively. Furthermore, the 3 and 43 kDa bands showed the purified molecules. The IC50 value of DFF40-LYC-I and DFF40 was determined as 6.6 and 17.03 μg/mL for HeLa, respectively. LYC-I had no cytotoxic effects on both cell lines, even at high concentrations. A new fusion protein with targeted cancer treatment potential was produced for the first time by LYC-I with a safe profile on normal cells. This fusion protein exhibited higher cytotoxic effects in cancer cells compared to normal cells. However, additional investigations are required to determine the apoptosis induction and evaluate selective toxicity against other cancer and normal cell lines.

Cotadutide improves brown adipose tissue thermogenesis in obese mice

We studied the effect of cotadutide, a dual agonist glucagon-like peptide 1 (GLP1)/Glucagon, on interscapular brown adipose tissue (iBAT) remodeling and thermogenesis of obese mice. Twelve-week-old male C57BL/6 mice were fed a control diet (C group, n = 20) or a high-fat diet (HF group, n = 20) for ten weeks. Then, animals were redivided, adding cotadutide treatment: C, CC, HF, and HFC for four additional weeks. The multilocular brown adipocyte structure showed fat conversion (whitening), hypertrophy, and structural disarray in the HF group, which was reverted in cotadutide-treated animals. Cotadutide enhances the body temperature, thermogenesis, and sympathetic innervation (peroxisome proliferator-activated receptor-α, β3 adrenergic receptor, interleukin 6, and uncoupled protein 1), reduces pro-inflammatory markers (disintegrin and metallopeptidase domain, morphogenetic protein 8a, and neuregulin 4), and improves angiogenesis (vascular endothelial growth factor A, and perlecan). In addition, cotadutide enhances lipolysis (perilipin and cell death-inducing DNA fragmentation factor α), mitochondrial biogenesis (nuclear respiratory factor 1, transcription factor A mitochondrial, mitochondrial dynamin-like GTPase, and peroxisome proliferator-activated receptor gamma coactivator 1α), and mitochondrial fusion/fission (dynamin-related protein 1, mitochondrial fission protein 1, and parkin RBR E3 ubiquitin protein ligase). Cotadutide reduces endoplasmic reticulum stress (activating transcription factor 4, C/EBP homologous protein, and growth arrest and DNA-damage inducible), and extracellular matrix markers (lysyl oxidase, collagen type I α1, collagen type VI α3, matrix metallopeptidases 2 and 9, and hyaluronan synthases 1 and 2). In conclusion, the experimental evidence is compelling in demonstrating cotadutide's thermogenic effect on obese mice's iBAT, contributing to unraveling its action mechanisms and the possible translational benefits.

3-tert-Butyl-4-hydroxyanisole Perturbs Differentiation of C3H10T1/2 Mesenchymal Stem Cells into Brown Adipocytes through Regulating Smad Signaling.

3-tert-Butyl-4-hydroxyanisole (3-BHA), one of the most commonly used antioxidants in foodstuffs, has been identified as an environmental endocrine disruptor (EED) with obesogenic activity. Given the increasing concern on EED-caused dysfunction in lipid metabolism, whether 3-BHA could influence the development of brown adipocytes is worthy of being explored. In this study, the effect of 3-BHA on the differentiation of C3H10T1/2 mesenchymal stem cells (MSCs) into brown adipocytes was investigated. Exposure to 3-BHA promoted lipogenesis of the differentiated cells, as evidenced by the increased intracellular lipid accumulation and elevated expressions of adipogenic biomarkers, including peroxisome proliferator-activated receptor γ (PPARγ), Perilipin, Adiponectin, and fatty acid binding protein 4 (FABP4). Surprisingly, the thermogenic capacity of the differentiated cells was compromised as a result of 3-BHA exposure, because neither intracellular mitochondrial contents nor expressions of thermogenic biomarkers, including uncoupling protein 1 (UCP1), peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α), cell-death-inducing DNA fragmentation factor α subunit-like effector A (CIDEA), and PR domain containing 16 (PRDM16), were increased by this chemical. The underlying molecular mechanism exploration revealed that, in contrast to p38 MAPK, 3-BHA stimulation induced phosphorylation of Smad1/5/8 in an exposure time-dependent manner, suggesting that this chemical-triggered Smad signaling was responsible for the shift of C3H10T1/2 MSC differentiation from a brown to white-like phenotype. The finding herein, for the first time, revealed the perturbation of 3-BHA in the development of brown adipocytes, uncovering new knowledge about the obesogenic potential of this emerging chemical of concern.

Features of the response to subchronic low-dose exposure to copper oxide nanoparticles in rats

Copper is an essential trace element for human health and, at the same time, a major industrial metal widely used both in its elemental form and in compounds. We conducted a dose-dependent assessment of the response of outbred albino male rats to subchronic low-dose exposure to copper oxide nanoparticles administered intraperitoneally at cumulative doses of 18 and 36 mg/kg during 6 weeks to exposure groups 1 and 2, respectively. We observed disorders at different levels of organization of the body in the exposed animals, from molecular to organismal. The observed decrease in the activity of succinate dehydrogenase in nucleated blood cells gave evidence of impaired bioenergetics processes. In view of the results of the metabolomics analysis, we assume mitochondrial damage and contribution of apoptotic processes to the pathology induced by copper poisoning. We also assume neurodegenerative effects based on the assessed morphological parameters of the nervous system, results of behavioral tests, and a decreased level of expression of genes encoding NMDA receptor subunits in the hippocampus. The hepatotoxic effect noted by a number of metabolomics-based, biochemical, and cytological indicators was manifested by the impaired protein-synthesizing function of the liver and enhanced degenerative processes in its cells. We also observed a nephrotoxic effect of nanosized copper oxide with a predominant lesion of proximal kidney tubules. At the same time, both doses tested demonstrated such positive health effects as a statistically significant decrease in the activity of alkaline phosphatase and the nucleated blood cell DNA fragmentation factor. Judging by the changes observed, the cumulative dose of copper oxide nanoparticles of 18 mg/kg body weight administered intraperitoneally approximates the threshold one for rats. The established markers of health impairments may serve as a starting point in the development of techniques of early diagnosis of copper poisoning.

Increasing the Sensitivity of MCF-7 Breast Cancer Cells to Quercetin by Declining DFF45 Expression Level

Background and Objectives In recent years, flavonoids such as quercetin have been considered as new anticancer drugs. The mechanisms of action of quercetin include cell cycle arrest, inhabitation of cell proliferation, and induction of apoptosis. This study aims to reduce quercetin’s side effects by increasing MCF-7 breast cancer cells’ sensitivity to this drug and facilitating the cytotoxic effects of quercetin at lower concentrations. Subjects and Methods In this study, the MTT assay was used to determine the concentration that reduced the cell viability by 50% (i.e. lethal concentration 50 or LC50). Then, the expression of the DNA fragmentation factor-45 (DFF45) and some genes in the apoptosis pathway (caspase3, p53, BAX, BCL-2, AIF), the autophagy pathway (LC3, ATG5, Beclin, DRAM) and the AKT/mTOR pathway (AKT1, mTOR, and PTEN), in cells treated with siRNA, quercetin, and quercetin+siRNA using the real-time PCR. Results According to the results of MTT assay, the LC50 value for quercetin was determined 220 μM. The results indicated the initiation of cell death through autophagy pathways. The combined treatment (quercetin+siRNA) increased the mechanism of cancer cell death more than the quercetin treatment alone. Conclusion One of the regulating pathways of apoptosis is forcing the inhibitory effect of DFF45 on DFF40/CAD nuclease. Down regulation of DFF45, along with quercetin administration, can lead to induction of breast cancer cell death which can be a novel technique for the treatment of breast cancer.

Identification of the immune-related biomarkers in Behcet’s disease by plasma proteomic analysis

BackgroundThis study aimed to investigate the expression profile of immune response-related proteins of Behcet’s disease (BD) patients and identify potential biomarkers for this disease.MethodsPlasma was collected from BD patients and healthy controls (HC). Immune response-related proteins were measured using the Olink Immune Response Panel. Differentially expressed proteins (DEPs) were used to construct prediction models via five machine learning algorithms: naive Bayes, support vector machine, extreme gradient boosting, random forest, and neural network. The prediction performance of the five models was assessed using the area under the curve (AUC) value, recall (sensitivity), specificity, precision, accuracy, F1 score, and residual distribution. Subtype analysis of BD was performed using the consensus clustering method.ResultsProteomics results showed 43 DEPs between BD patients and HC (P < 0.05). These DEPs were mainly involved in the Toll-like receptor 9 and NF-κB signaling pathways. Five models were constructed using DEPs [interleukin 10 (IL10), Fc receptor like 3 (FCRL3), Mannan-binding lectin serine peptidase 1 (MASP1), NF2, moesin-ezrin-radixin like (MERLIN) tumor suppressor (NF2), FAM3 metabolism regulating signaling molecule B (FAM3B), and O-6-methylguanine-DNA methyltransferase (MGMT)]. Among these models, the neural network model showed the best performance (AUC = 0.856, recall: 0.692, specificity: 0.857, precision: 0.900, accuracy: 0.750, F1 score: 0.783). BD patients were divided into two subtypes according to the consensus clustering method: one with high disease activity in association with higher expression of tripartite motif-containing 5 (TRIM5), SH2 domain-containing 1A (SH2D1A), phosphoinositide-3-kinase adaptor protein 1 (PIK3AP1), hematopoietic cell-specific Lyn substrate 1 (HCLS1), and DNA fragmentation factor subunit alpha (DFFA) and the other with low disease activity in association with higher expression of C–C motif chemokine ligand 11 (CCL11).ConclusionsOur study not only revealed a distinctive immune response-related protein profile for BD but also showed that IL10, FCRL3, MASP1, NF2, FAM3B, and MGMT could serve as potential immune biomarkers for this disease. Additionally, a novel molecular disease classification model was constructed to identify subsets of BD.

Filbertone, (2E)-5-methyl-2-hepten-4-one, regulates thermogenesis and lipid metabolism in skeletal muscle of a high-fat diet fed mice

Filbertone, the principal flavor compound of hazelnuts, is known to have preventive effects against hypothalamic inflammation, obesity and adipocity in vitro and in vivo. However, the effect of filbertone in skeletal muscle remains unknown. In the present study, we determined the effect of filbertone in skeletal muscle of mice fed a high-fat diet (HFD). To identify the underlying molecular and cellular processes of filbertone, we performed whole transcriptome sequencing in skeletal muscle. The muscle transcriptome analysis revealed that the upregulated differentially expressed genes (DEGs) in filbertone-fed mice were substantially associated with several pathways including thermogenesis, fatty acid degradation, oxidative phosphorylation, and branched chain amino acids (BCAAs) degradation. Furthermore, the expression level of thermogenic genes such as uncoupling protein 1 (Ucp1; p < 0.05), cell death-inducing DNA fragmentation factor alpha-like effector A (Cidea; p < 0.05), peroxisome proliferator-activated receptor alpha (Ppara; p < 0.05) and lipid droplet-associated protein genes such as Plin3 (p < 0.05), Plin4 (p < 0.05), and Plin5 (p < 0.05) were significantly upregulated in muscle tissue of HFD with filbertone fed mice compared to HFD only fed mice. Filbertone also elevated the protein level of UCP1 (p < 0.05) and PPARα (p < 0.05). In addition, filbertone reduced the accumulation of intracellular lipids in C2C12 myotubes (p < 0.05). On the basis of these results, we suggest that filbertone has a crucial effect in the regulation of muscle lipid metabolism and energy balance.

MEG3 Regulates CSE-Induced Apoptosis by Regulating miR-421/DFFB Signal Axis.

Chronic obstructive pulmonary disease (COPD) is a common respiratory disease with irreversible and progressive obstruction of airflow. Currently, there are no clinically available treatments to prevent COPD progression. Apoptosis of human lung microvascular endothelial cells (HPMECs) and bronchial epithelial cells (HBECs) is often observed in COPD, but its pathogenesis has not been fully elucidated. LncRNA maternally expressed gene 3 (MEG3) is closely related to CSE-induced apoptosis, but the specific mechanism of MEG3 in COPD is still unknown. In the present study, cigarette smoke extract (CSE) is used to treat HPMECs and HBECs. Flow cytometry assay is used to detect the apoptosis of these cells. The expression of MEG3 in CSE-treated HPMECs and HBECs is detected by qRT-PCR. LncBase v.2 is used to predict miRNAs binding to MEG3, and miR-421 is found to bind to MEG3. Dual luciferase report analysis and RNA immunoprecipitation experiment jointly clarified the binding relationship between MEG3 and miR-421. MiR-421 was downregulated in CSE-treated HPMECs/HBECs, and miR-421 overexpression mitigated CSE-induced apoptosis in these cells. Subsequently, DFFB was found to be directly targeted by miR-421. The overexpression of miR-421 dramatically reduced the expression level of DNA fragmentation factor subunit beta (DFFB). DFFB was found downregulated in CSE-treated HPMECs and HBECs. MEG3 contributed to the apoptosis of HPMECs and HBECs induced by CSE by regulating the miR-421/DFFB axis. This study presents a new perspective on the diagnosis and treatment of COPD caused by CSE.

Fragmentation landscape of cell-free DNA revealed by deconvolutional analysis of end motifs

Cell-free DNA (cfDNA) fragmentation is nonrandom, at least partially mediated by various DNA nucleases, forming characteristic cfDNA end motifs. However, there is a paucity of tools for deciphering the relative contributions of cfDNA cleavage patterns related to underlying fragmentation factors. In this study, through non-negative matrix factorization algorithm, we used 256 5' 4-mer end motifs to identify distinct types of cfDNA cleavage patterns, referred to as "founder" end-motif profiles (F-profiles). F-profiles were associated with different DNA nucleases based on whether such patterns were disrupted in nuclease-knockout mouse models. Contributions of individual F-profiles in a cfDNA sample could be determined by deconvolutional analysis. We analyzed 93 murine cfDNA samples of different nuclease-deficient mice and identified six types of F-profiles. F-profiles I, II, and III were linked to deoxyribonuclease 1 like 3 (DNASE1L3), deoxyribonuclease 1 (DNASE1), and DNA fragmentation factor subunit beta (DFFB), respectively. We revealed that 42.9% of plasma cfDNA molecules were attributed to DNASE1L3-mediated fragmentation, whereas 43.4% of urinary cfDNA molecules involved DNASE1-mediated fragmentation. We further demonstrated that the relative contributions of F-profiles were useful to inform pathological states, such as autoimmune disorders and cancer. Among the six F-profiles, the use of F-profile I could inform the human patients with systemic lupus erythematosus. F-profile VI could be used to detect individuals with hepatocellular carcinoma, with an area under the receiver operating characteristic curve of 0.97. F-profile VI was more prominent in patients with nasopharyngeal carcinoma undergoing chemoradiotherapy. We proposed that this profile might be related to oxidative stress.

Creatine ameliorates high-fat diet-induced obesity by regulation of lipolysis and lipophagy in brown adipose tissue and liver

Diet-induced obesity in mice and humans is commonly associated with an imbalance between energy intake and expenditure. Reportedly, creatine can enhance energy expenditure in brown adipose tissue and reduce hepatic triglycerides accumulation; however, the molecular mechanism underlying the role of exogenous creatine supplementation in regulating lipid droplet mobilization remains elusive. Herein, we employed a high-fat diet (HFD)- induced mouse model to investigate the role of creatine in regulating lipolysis and lipophagy in brown adipose tissue and the liver. Exogenous creatine supplementation ameliorated HFD-induced obesity, increased insulin sensitivity and improved glucose homeostasis. Creatine supplementation enhanced the expression of uncoupling protein 1 (UCP1), cell death-inducing DNA fragmentation factor alpha-like effector A (CIDEA), and other brown adipose tissue-specific thermogenic genes Cpt1a, Gyk, and Pgc1β in brown adipose tissue. Furthermore, creatine inhibited the expression of CIDEA, which promotes hepatic lipid accumulation. Creatine stimulated the expression of triglyceride lipase adipose triglyceride lipase, and phospho-hormone-sensitive lipase (HSL) induced increased lipolysis in brown adipose tissue and the liver. Meanwhile, reduced LC3B expression was accompanied by an increased level of p62 in HFD-fed mice, indicating diminished basal autophagy in brown adipose tissue and the liver; however, creatine enhanced P62/LC3B induced lipophagy in brown adipose tissue and the liver. Collectively, our results suggest that creatine may function as a brown adipose tissue activator to increase whole-body energy metabolism via coordinated lipolysis and lipophagy in brown adipose tissue and the liver.

Meloxicam Inhibits Apoptosis in Neurons by Deactivating Tumor Necrosis Factor Receptor Superfamily Member 25, Leading to the Decreased Cleavage of DNA Fragmentation Factor Subunit α in Alzheimer's Disease.

Neuronal apoptosis is considered to be a critical cause of Alzheimer's disease (AD). Recently, meloxicam has shown neuroprotective effects; however, the inherent mechanisms are highly overlooked. Using APP/PS1 transgenic (Tg) mice as in vivo animal models, we found that meloxicam inhibits apoptosis in neurons by deactivating tumor necrosis factor receptor superfamily member 25 (TNFRSF25), leading to the suppression of the expression of fas-associated protein with death domain (FADD) and the cleavage of DNA fragmentation factor subunit α (DFFA) and cysteine aspartic acid protease-3 (caspase 3) via β-amyloid protein (Aβ)-depressing mechanisms. Moreover, the meloxicam treatment blocked the effects of β-amyloid protein oligomers (Aβo) on stimulating the synthesis of tumor necrosis factor α (TNF-α) and TNF-like ligand 1A (TL1A) in neuroblastoma (N) 2a cells. TNF-α and TL1A induce apoptosis in neurons via TNFR- and TNFRSF25-dependent caspase 3-activating mechanisms, respectively. Knocking down the expression of TNFRSF25 blocked the effects of TL1A on inducing apoptosis in neurons by deactivating the signaling cascades of FADD, caspase 3, and DFFA. Consistently, TNFRSF25 shRNA blocked the effects of Aβo on inducing neuronal apoptosis, which was corroborated by the efficacy of meloxicam in inhibiting Aβo-induced neuronal apoptosis. By ameliorating neuronal apoptosis, meloxicam improved memory loss in APP/PS1 Tg mice.

Application of an antibody microarray for serum protein profiling of coronary artery stenosis

Protein expression profiling in the serum is used to identify novel biomarkers and investigate the signaling pathways in various diseases. The aim of the present study was to evaluate serum biomarkers associated with coronary artery stenosis resulting from atherosclerosis.The study included 4 groups of subjects: group A and B with and without coronary lesions, respectively, were selected from a previously reported cohort study on coronary atherosclerosis, control group C comprised of asymptomatic subjects and group D was used for independent validation of the microarray data by ELISA. Labeled serum proteins were profiled by an Explorer antibody array, which included 656 specific antibodies in two replicates (FullMoon Biosystems, USA). Cadherin-P, interleukin-5, glutathione S-transferase Mu, and neuronal nitric oxide synthase were sex-independently increased in Group A compared with those in group B. The microarray data on cadherin-P were externally validated in an independent group D using ELISA. Fibroblast growth factor-1, FGF-2, collagen II, granulocyte-macrophage colony-stimulating factor, IL-1 alpha, angiopoietin-2, granulocyte colony-stimulating factor, lymphocyte cell-specific protein tyrosine kinase, and IkappaB kinase b were increase in men in group A compared with group B. Cyclin-dependent kinase 1, DNA fragmentation factor subunit alpha DFF45/ICAD, adenovirus type 2 E1A, calponin, ADP-ribosylation factor-6, muscle-specific actin, thyroid hormone receptor alpha, and alpha-methylacyl-CoA racemase were specifically increased in women in Group A compared with group B. Alterations in the levels of specific proteins may point to the signaling pathways contributing to coronary atherosclerosis, and these proteins will be useful biomarkers for the progression of cardiovascular diseases.

O-252 Next generation sequencing analysis of the seminal microbiome in male partners of women with idiopathic recurrent pregnancy loss: results of a prospective cohort study

Abstract Study question Is there an association between the semen microbiome, seminal reactive oxygen species (ROS) and DNA fragmentation in men with recurrent pregnancy loss (RPL)? Summary answer This pilot study outlines the subtle role that microbiota play in influencing ROS and sperm DNA damage for male partners of women with RPL What is known already RPL is defined as the loss of two or more consecutive pregnancies. This devastating condition impacts approximately 1% of couples. Paternal causes are not routinely screened for and an underlying cause is not found in up to 50% of cases. Recent studies have reported an association between elevated seminal ROS and sperm DNA fragmentation in the male partner, and RPL. We hypothesised that seminal microbiota contribute to increased ROS and sperm DNA damage. To test this, we investigated the relationship between seminal bacterial composition and ROS levels in men with proven fertility versus men with a history of RPL. Study design, size, duration We conducted a prospective, case-control study and recruited participants between November 2018 and March 2020 at Imperial College Healthcare NHS Trust. A total of 109 men participated in the study; 46 men with RPL and 63 men with proven fertility and no history of RPL. Each participant attended for a single study visit which consisted of a full medical history, assessment of testicular volume, height, weight, blood samples and production of a semen sample. Participants/materials, setting, methods Routine semen analysis (WHO) and endocrine and lipid profiles were performed for all patients. Semen ROS and DNA fragmentation were performed (luminol and TUNEL methodologies, respectively). ROS were classified as high (&amp;gt;3.77 RLU/s) or low (&amp;lt;3.77 RLU/s). Metataxonomic profiling of samples was performed using Illumina Miseq-based sequencing of the V1-V2 hypervariable regions of bacterial 16S rRNA gene amplicons. Multivariate and univariate modelling was performed to explore associations between metataxonomic profiles, ROS levels and clinical metadata. Main results and the role of chance Men with RPL had higher mean semen volume (p = 0.02) and increased prevalence of high ROS (p = 0.02, Fisher's exact) compared with controls; but other clinical characteristics were similar between groups. A total of 3,700,136 high quality sequence reads were generated for the dataset with an average of 33,946 reads/sample. Hierarchical clustering of bacterial genera relative abundances identified 4 distinct microbial signatures characterised by high relative abundance of 1. Streptococcus, 2. Lactobacillus and Gardnerella, 3. polymicrobial (including Prevotella), and 4. Corynebacterium and Finegoldia. Prevalences of these groups were similar in control and RPL groups (p = 0.11). Additionally, no association between the bacterial genera groups and elevated ROS, DNA fragmentation, or clinical factors such as age, ethnicity, or semen volume were observed (chi-square tests). At species taxonomy level relative abundance of L. crispatus was higher in controls compared to RPL, but did not withstand false discovery rate correction for multiple testing (p = 0.006, q = 0.67). Higher relative abundance of Microbacterium was detected in semen samples with high DNA fragmentation (p = 8.7E-4, q = 0.08). This relationship was even stronger within the RPL cohort (p = 2.8E-5, q = 0.002). No significant enrichment of specific taxa was observed between high or low ROS samples however, low ROS was associated with Corynebacterium relative abundance &amp;gt;20%. Limitations, reasons for caution More patients are required to enhance statistical power. Duplicate sample collection may establish the robustness of seminal compositions observed. Time since last sexual intercourse samples may affect the analysis. Concomitant analysis of the vaginal microbiome of female partners may improve our understanding of how partners may affect each other’s fertility. Wider implications of the findings Our data suggests interactions between microbiota composition, ROS and sperm DNA damage which may be implicated in the pathogenesis of recurrent miscarriage. Further studies are needed to determine if seminal microbiota play causal roles in RPL, and whether interventions modifying the seminal microbiome may modify pregnancy outcomes in affected couples Trial registration number not applicable