Specific Kits Research Articles

Two novel variants in GRN: the relevance of CNV analysis and genetic screening in FTLD patients with a negative family history

BackgroundFrontotemporal lobar degeneration (FTLD) is one of the leading causes of early onset dementia. Pathogenic variants in GRN have been reported to cause 5–25% of familial and 5% of sporadic FTLD. Here, we present two novel, likely pathogenic variants in GRN.MethodsFour patients from four different families underwent whole exome sequencing (WES) with additional copy-number variance (CNV) analysis in a clinical setting. TMEM106B rs1990622 and rs3173615 SNPs and 3’UTR insertion were tested in one presymptomatic carrier. In three probands and one presymptomatic carrier, plasma progranulin (PGRN) levels were measured using a specific ELISA kit. In two probands, neuropathological diagnosis was established using current neuropathological criteria.ResultsThrough CNV analysis on WES data, we identified a partial deletion, NM_002087.2 (GRN):c.1179 + 104_1536delinsCTGA, p.(?), in three patients with primary progressive aphasia and/or corticobasal syndrome. Haplotype analysis revealed a shared haplotype block, suggesting that the deletion represents a founder mutation. Additionally, we found a novel, missense variant, NM_002087.2 (GRN):c.23 T > A, p.(Val8Glu), in one proband with a negative family history. The proband’s unaffected parent—in their 80 s—carried the same variant, yet was homozygous for the TMEM106B risk haplotype. The pathogenicity of both GRN variants was supported by typical neuropathological features and reduced PGRN levels.ConclusionWe recommend a thorough genetic screening, including CNV analysis, for both familial and apparent sporadic FTLD patients. Furthermore, the presymptomatic carrier homozygous for the TMEM106B risk haplotype exemplifies the presence of other protective factors that modify disease onset and urges caution in genetic counselling based on the TMEM106B haplotype.

Comparison of the Effects of Brief Intervention (BI) on Relapse Prevention and Withdrawal Permanency in Opioid Addicts and Stimulant Users: A Quasi-Experimental Study

Background: Addiction to narcotics and stimulants is a major social and health challenge worldwide. Objectives: Considering the importance of non-medical treatments in addiction rehabilitation and the necessity of psychotherapy programs in substance abuse rehabilitation centers, the present study aimed to compare the effects of Brief Intervention (BI) on relapse prevention and sustained abstinence in opioid addicts and stimulant abusers seeking help from drop-in-centers (DICs) dedicated to harm reduction in Ahvaz, a city in southwestern Iran. Methods: This applied, quasi-experimental study employed a pre-test, post-test, and follow-up design. The statistical population included 120 male substance abusers and opioid addicts aged 18 to 59, systematically selected from 467 individuals with active files in Iran’s Drug and Alcohol Treatment Information System (IDATIS) under the Ministry of Health in 2023. Participants were selected based on inclusion and exclusion criteria and randomized into four groups: Two experimental groups and two control groups, each with 30 participants. Brief Intervention was provided only to the two experimental groups in four 60-minute sessions, while no intervention was offered to the control groups. Testing tools included specific kits for screening morphine and amphetamines in the urine of participants. Data were analyzed using analysis of variance (ANOVA) with repeated measures and descriptive statistical methods, utilizing SPSS version 24. Results: The study demonstrated that BI was significantly effective in relapse prevention and promoting sustained abstinence in opioid addicts attending harm reduction centers in Ahvaz (P < 0.001). However, BI was not effective in preventing relapse or promoting abstinence in stimulant abusers (P = 0.235). Conclusions: The findings suggest that BI is an effective intervention for preventing relapse and promoting abstinence in opioid abusers. It is recommended that executive managers of addiction harm reduction centers guide their experienced social workers to implement this method as a selective psychotherapy approach complementary to medical treatment for opioid addicts.

METTL14 Promotes Lipopolysaccharide-Induced Myocardial Damage via m6A-Dependent Stabilization of TRPM7 mRNA.

Sepsis-induced myocardial injury (SIMI) is a vital pathological component of severe sepsis and septic shock. As a prevalent internal mRNA modification in eukaryotic cells, N6-methyladenosine (m6A) modification is implicated in sepsis and immune disorders. Methyltransferase-like 14 (METTL14), a core subunit of the methyltransferase complex that catalyzes messenger RNA m6A modification, is involved in the regulation of human cardiomyocyte cell line (AC16) injury. This study aimed to explore the role and mechanism of METTL14 in lipopolysaccharide (LPS) -induced myocardial injury.Cell viability and apoptosis were analyzed via 3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT), flow cytometry, and TdT-mediated dUTP nick-end labeling (TUNEL) assay. The Tumor necrosis factor alpha (TNF-α) and Interleukin-1beta (IL-1β) levels were analyzed via Enzyme linked immunosorbent assay (ELISA). Caspase-3 activity, reactive oxygen species activity, malondialdehyde level, and glutathione level were assessed using special assay kits. The levels of transient receptor potential melastatin 7 (TRPM7) and METTL14 mRNA were determined via Real-time quantitative polymerase chain reaction (RT-qPCR). Meanwhile, the protein levels of TRPM7, METTL14, phospho-p65 (p-p65), total p65 (p65), p-IκBα, and total IκBα (IκBα) were examined via western blot assay.LPS treatment repressed AC16 cell viability and induced cell apoptosis, inflammatory response, oxidative stress, and ferroptosis in vitro. METTL14 and TRPM7 were upregulated in LPS-treated AC16 cells. At the molecular level, METTL14 could increase the stability of TRPM7 mRNA via m6A methylation. Moreover, METTL14 deficiency could abolish LPS-triggered AC16 cell injury and ferroptosis via TRPM7 regulation.METTL14 knockdown reversed LPS-caused myocardial cell damage mainly by regulating the stability of TRPM7 mRNA, providing a novel therapeutic target for septic cardiomyopathy treatment.

Assessment of IL-38 And IL-40 Levels in Gingival Crevicular Fluid in Gingivitis and Stage III Grade C Periodontitis: A Comparative Clinical Study

Periodontal diseases are infections of the supporting apparatus of teeth caused by microbial agents, and cytokines have an important role in the inflammatory response. The purpose of this study is to measure the concentrations of interleukin-38 (IL-38) and interleukin-40 (IL-40) in the gingival crevicular fluid (GCF) in patients with stage III grade C periodontitis (SIII-GC), gingivitis and those with healthy periodontium. The study included a total of 75 people, 26 PH patients, 26 G patients, and 23 SIII-GC chronic periodontitis patients. Clinical parameters of a patient such as plaque index (PI), gingival index (GI), bleeding upon probing (BOP), probing pocket depth (PPD) and clinical attachment loss (CAL) were analyzed. GCF was collected and measured using the ELISA technique with IL-38 and IL-40 specific antibodies and kits. Interleukin-38 and interleukin-40 of the SIII-GC periodontitis group was significantly higher (P < 0.05) than other groups as regards normal gingival crevicular fluid IL-38 and IL-40 levels. However, differences in IL-38 and IL-40 levels were not significant between the two other groups: PH and G ones (P > 0.05). Positive correlation was found between GCF derived IL-38 and IL-40 levels with clinical periodontal parameters from sampled areas (P < 0.001). The strongest findings of this research indicate that within GCF, both IL-38 and IL-40 levels were elevated as total GCF IL-38 and total GCF IL-40 levels in different patients were high.

A méh- vagy darázscsípés okozta anafilaxiás sokk vizsgálata a triptáz enzim és a méh-, illetve darázsméreg specifikus immunglobulin E szintek vérből történő meghatározása alapján, egyes rendkívüli haláleseti eljárásokban

Aim: Systemic allergic reactions triggered by bee and wasp venom affect approximately 3% of adults, and in severe cases, can lead to anaphylactic reactions and death. The exact cause of extraordinary deaths resulting from insect stings is often not determined in a significant number of cases. The aim of this study is to provide guidance through methodological recommendations to investigate the precise cause of deaths presumably resulting from insect stings, specifically focusing on uncovering the facts and causes of anaphylaxis. Methodology: The study reviews literature related to anaphylaxis caused by insect stings and animal toxins, as well as its immunological background. It seeks to translate the information found in the literature into the realm of official activities related to extraordinary deaths. The study also summarises knowledge about groups of animals in Hungary that are medically significant due to their venom, providing guidance for on-site recognition, search, documentation, and recording of the most common Hymenoptera species with venom capable of frequently inducing anaphylactic reactions. Findings: The key to diagnosing deaths caused by anaphylaxis lies in crime scene investigation. The attending medical doctor must recognise the symptoms of insect stings and ensure the collection of a blood sample for allergological purposes. A special blood collection kit has been assembled for this purpose. The blood sample, which must be stored at 2–8ºC, should be sent to the Semmelweis University Institute of Laboratory Medicine within 2 days. By measuring the level of tryptase enzyme in the blood sample and detecting the presence of specific immunoglobulin E for the venom of the respective bee or wasp species, the cause of the anaphylactic reaction can be confirmed. The crime scene investigation team should also make an effort to locate and secure the potential insect species responsible for the sting. If the forensic entomologist successfully identifies the species, it facilitates the specific IgE allergological examination for the respective venom. Value: In cases of death resulting from insect stings, the cause is often not determined due to lack of proper caution and appropriate methods. The procedural approach recommended in the study establishes the basis for determining the precise cause of death in cases of suspected insect stings.

Cytokine profile, differential somatic cell count, and oxidative status of Italian Mediterranean buffalo milk affected by the temperature-humidity index.

In the context of climate change, there has been an increased interest in improving management practices for animals genetically adapted to extreme environmental conditions, such as buffaloes. The temperature-humidity index (THI) is used to determine the severity of heat stress in livestock. This study aimed to evaluate the cytokine profile, oxidative staus, differential somatic cell count (DCC), and the surface expression and activity of myeloperoxidase (MPO) in the somatic cells (SCs) of buffalo. Milk samples (n = 216) were collected from the spring to summer season under three different THI classes (THI < 72; ≤72 THI < 76, and THI ≥ 76). The cytokine profile was determined using ELISA, and the expression of DSCC and MPO was determined by flow cytometry. MPO activity was performed on SC extracts using a specific ELISA kit. Oxidative status was determined by the antioxidant/oxidant balance combining the free radical scavenging activity levels, and reactive oxygen and nitrogenous species. The results on the cytokine profile showed that at the THI ≥ 76 the levels of both IL-10 and IFN-γ were highest. IL-1β secretion was lower at the THI < 72 than at the THI values ranging from ≤72 THI < 76. Higher levels of both TNF-α and IL-12 were registered in both THI < 72 and THI ≥ 76 classes. The level of IL-4 was higher in the THI ≥ 76 class than in the ≤72 to <76 range. Data on DCC showed a decrease in the percentage of macrophages and lymphocytes as the THI increased from the ≤72 to <76 range to THI ≥ 76. Furthermore, the highest percentage of polymorphonuclear leukocyte (PMNLs) was registered in both ≤72 to <76 and THI ≥ 76 classes. The MPO activity and surface expression on SC were lower at a THI above 76, which could be associated with an absence of inflammation. A condition of oxidative imbalance was registered as demonstrated by the lower levels of antioxidant/oxidant balance along with increasing THI. Present data demonstrated that buffaloes were able to modulate the alteration of immune response activated by heat stress throughout a series of cross-linked mechanisms involving cytokine networks, different somatic cell distribution, and oxidative status.

TRIM46 accelerates H1N1 influenza virus-induced ferroptosis and inflammatory response by regulating SLC7A11 ubiquitination.

Influenza A (H1N1) virus is an acute respiratory infection responsible for enormous morbidity and mortality worldwide. The tripartite motif-containing protein 46 (TRIM46) has an antiviral function that inhibits various viral infections. This study is designed to explore the role and mechanism of TRIM46 in the progress of H1N1 infection. Herein, we infected A549 or 16HBE cells with the H1N1 virus at different times to assess TRIM46 and solute carrier family 7 member 11 (SLC7A11) expression. TRIM46 and Influenza A nucleoprotein mRNA levels were detected by real-time quantitative polymerase chain reaction (RT-qPCR). TRIM46, solute carrier family 7 member 11 (SLC7A11), and Nucleoprotein protein levels were detected using protein level were detected by western blot assay. Cell virulence was determined using Virulence assay (TCID50) assay. Cell viability was determined using Cell Counting Kit-8 (CCK-8) assay. Reactive oxygen species (ROS), intracellular iron content, Malondialdehyde (MDA), and Glutathione (GSH) levels were determined using special assay kits. The stability of SLC7A11 was assessed by Cycloheximide (CHX) assay. Interaction between TRIM46 and SLC7A11 was verified using Co-immunoprecipitation (CoIP) assay. The biological role of TRIM46 was assessed in H1N1 virus-challenged lung injury mice in vivo. TRIM46 level was significantly increased during H1N1 virus infection, and SLC7A11 expression was decreased. TRIM46 downregulation could suppress H1N1 virus replication and relieve H1N1 infection-induced ferroptosis and inflammation in A549 or 16HBE cells. Mechanistically, TRIM46 could promote SLC7A11 ubiquitination and decrease its stability. TRIM46 knockdown repressed H1N1 virus-induced lung injury in vivo. TRIM46 could contribute to influenza A H1N1 virus infection by promoting SLC7A11 ubiquitination in A549 cells, which indicates that targeting TRIM46 may improve the prognosis of patients.

Neutrophil elastase activates macrophage calpain as a mechanism for phagocytic failure.

Neutrophil elastase (NE), elevated in the cystic fibrosis (CF) airway, causes macrophage phagocytic failure. We previously reported that NE increases the release of protease calcium ion-dependent papain-like cysteine protease-2 (Calpain-2) in macrophages. We hypothesized that NE mediates macrophage failure through activation of Calpains. We demonstrate that Calpain inhibition rescued NE-induced macrophage phagocytic failure in murine alveolar macrophages in both cftr-null and wild-type genotypes. We then sought to determine how NE regulates Calpain-2. Human monocyte-derived macrophages (hMDMs) from persons with CF (PwCF) and non-CF subjects were treated with NE or control vehicle, and cell lysates were prepared to evaluate Calpain-2 protein abundance by Western and Calpain activity by a specific activity kit. Calpain is activated by intracellular calcium and inactivated by an endogenous inhibitor, Calpastatin. hMDMs were thus treated with NE or control vehicle and cell lysates were analyzed for increased intracellular calcium by Fluo-4 assay and for Calpastatin protein abundance by Western. NE increased Calpain-2 protein and activity, degraded Calpastatin, and increased intracellular calcium in macrophages. At baseline, there are no differences in Calpain activity, Calpain-2 and Calpastatin expression, and intracellular calcium between CF and non-CF macrophages. NE increased macrophage Calpain-2 protein and Calpain activity by two potential mechanisms: degradation of Calpastatin and/or increased intracellular calcium. In summary, Calpain inhibition restored NE-induced macrophage phagocytic failure suggesting a potential CFTR-independent target for phagocytic failure in the CF airway.NEW & NOTEWORTHY Neutrophil elastase, a cystic fibrosis airway inflammation biomarker, increases macrophage Calpain activity, and Calpain inhibition partially restores the decreased phagocytosis in neutrophil elastase-challenged macrophages. Neutrophil elastase increases Calpain-2 protein, degrades the Calpain inhibitor, Calpastatin, and increases intracellular calcium as potential mechanisms of Calpain activation. This presents a novel mechanism for macrophage dysfunction relevant to cystic fibrosis.

The green formulation of silver nanoparticles for the anticancer and lupus nephritis treatment applications and reducing the inflammatory cytokines in the rat periodontal model

In this study, a novel bio-inspired synthesis for the preparation of Ag NPs by Calendula persica flower extract has been researched. Also, the effects of the recent composite on lupus nephritis by following the TGF-β1 signaling pathway in mice and gingival amounts of IL1-β and TNF-α in the animal periodontal model were determined. To assess the crystal nature, morphology and size of the prepared nanoparticles, X-ray diffraction (XRD), transmission electron microscopy (TEM), ultraviolet–visible (UV–Vis) and field emission-scanning electron microscopy (FE-SEM) analyses were applied in this study. The prepared Ag NPs was employed for its effectiveness against colon cancer cell line (C26). The bio-synthesized Ag NPs had also cytotoxic property versus colon cancer based on MTT protocol. Collectively, the Ag NPs fabricated by biological way has potential applications in inflammatory treatment. In lupus nephritis section, indexes in animal tissues and serum were identified using specific kits, while pathological alterations in the mice kidney were examined through H&E staining. Additionally, qPCR was employed to determine the expression related to TGF-β 1 in kidney, thereby providing further insight into the silver nanoparticles mechanism. The findings indicated that silver nanoparticles reduced the mRNA expression levels of TGF-β 1 and NF-κB in the kidneys, while simultaneously increasing the Mn-SOD and IκB-α expression. Histological examination of H&E-stained sections revealed that silver nanoparticles mitigated the morphological abnormalities of the glomeruli and the inflammatory infiltration associated with nephritis. Silver nanoparticles demonstrated an inhibitory effect on the dsDNA positive rate in animal suffering from nephritis. Silver nanoparticles were found to reduce the increase in urinary protein and the pro-inflammatory cytokines concentrations in both the kidney and serum. In the periodontal model study, 0-3 ligatures were applied to induce the inflammatory periodontitis in right mandibular first molar neck in rats. The levels of inflammatory cytokines (IL1-β, CINC-1, IL-10 and TNF-α) were determined by common immunological test, ELISA. The findings revealed that Ag NPs administration could decease the IL1-β and TNF-α production in the rat periodontal model gum tissue. In addition, the outcome of this research indicated that IL1-β and TNF-α levels raised in the rat periodontal model gum tissue compared to control group. Ag NPs increased the levels of superoxide dismutase, catalase, and plasma bone alkaline phosphatase and reduced the IL1-β, TNF-α, inducible nitric oxide synthase and RANK genes mRNA expression. The results indicate that pre/post-treatment with Ag NPs because of its direct activity on pro-inflammatory cytokines inhibition improved the inflammatory symptoms in the periodontitis and lupus nephritis animals.

Isocitrate dehydrogenase 2 mutation promotes cytarabine resistance in acute myeloid leukemia by Warburg effect.

Mutation of isocitrate dehydrogenase 2 (IDH2) is a key factor in promoting cytarabine (Ara-C) resistance in acute myeloid leukemia (AML), however the underly mechanism remains unclear. Acute myeloid leukemia cells, were cultured with either IDH2 knockdown (KD-IDH2) or overexpression (OE-IDH2) to elucidate the role of IDH2 in these leukemic cell lines. Additionally, mutant cell lines were engineered to replicate clinically relevant IDH2 mutations. To investigate cellular responses, the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) was administered to the cells. Cell proliferation was quantified using a Cell Counting Kit-8 (CCK-8), while apoptosis was evaluated through propidium iodide staining followed by flow cytometry. Glycolytic metabolism levels were measured using a specific reagent kit, and Western blotting was employed to determine the expression levels of glycolysis-related proteins. Transcriptome sequencing was conducted to elucidate the mechanisms by which IDH2 mutations influence glycolysis. Furthermore, both in vitro cell experiments and in vivo subcutaneous transplantation tumor models in nude mice were utilized to validate these mechanisms. OE-IDH2 in AML cells, enhances resistance to the Ara-C, promotes cell proliferation and glycolysis, and inhibits apoptosis. KD-IDH2 exhibits opposite effects. Both IDH2 mutations and OE-IDH2 produce similar effects on these cellular processes. The increase in glycolysis levels following IDH2 mutation may contribute to the reduced efficacy of Enasidenib in inhibiting the proliferation of IDH-mutant AML cells. Transcriptome sequencing results indicate an enrichment of the PI3K/Akt signaling pathway in IDH2-mutant AML cells. BEZ235 significantly inhibits the expression of phosphorylated PI3K (p-PI3K), phosphorylated Akt (p-Akt), mTOR, glycolytic metabolism, and Ara-C resistance both in vitro and in vivo. Overexpression and mutation of IDH2 coordinate with the Warburg effect through the PI3K/Akt/mTOR pathway to promote Ara-C resistance in AML.

Correlation of Interleukin-10 and Interleukin-8 with Cytomegalovirus and Epstein–Barr Virus Infection in Hemodialysis Patients

Abstract Background: Herpes viruses like cytomegalovirus (CMV) and Epstein–Barr virus (EBV) can cause serious illness in those who already have impaired immune systems. Objectives: The purpose of this research was to examine the impact of CMV and EBV infection on specific immunological markers in individuals undergoing hemodialysis. Materials and Methods: Blood sample was collected from 91 hemodialysis patients and 50 healthy individuals as a control group for comparison. Serum was separated and examined for the confirmation of CMV and EBV infection, and then the sera was tested by using specific ELISA kits (human interleukin-10 and human IL-8, Sunlong Biotech Co., HangZhou, China). Results: Highest mean of IL-8 and IL-10 was obtained in CMV IgM positive patients in comparison with IgG positive and control group with P value = 0.00002, 0.0008, respectively. The results in patients with EBV IgM positive were also highly significant in comparison with control, P value for IL-8 was 0.0002 and for IL-10 was 0.00004. Conclusion: The study concluded that, the level of IL-10 and IL-8 was higher among hemodialysis patients with acute EBV and CMV infection, and this indicated that the ability of CMV and EBV to enhance IL-8 and IL-10 production which may play an important role in immune inflammatory states associated with CMV and EBV infection.

Digital Workflow for Rehabilitation of Severely Discolored Teeth Due to Red Staining from Endodontic Material.

The aim of this short communication is to present a technique to rehabilitate severely discolored teeth with computer-aided designed and computer-aided manufactured (CAD-CAM) zirconia crowns. After confirming the absence of periapical lesions and sufficient crown structure, any caries or fractured restorations can be removed and replaced by an interim composite restoration. A shoulder subgingival preparation is performed and scanned with an intraoral scanner to design a CAD-CAM zirconia crown using a monolithic zirconia material. This crown is highly polished using a specific polishing kit, but not glazed. This technique is suggested to be useful in cases of dark discolored teeth due to staining endodontic materials such as resorcinol-formaldehyde resin.

Assessment of the humoral immunity against diphtheria, tetanus, and hepatitis B among children with acute lymphocytic leukemia

Background: Approximately all systemic therapies for childhood affect the immune system. The behavior of the immune system in leukemia patients following chemotherapy is not yet clearly defined. The probability of vaccination failure and the need for revaccination remain challenging for these patients. Aim: To evaluate the humoral immunity against diphtheria, tetanus, and hepatitis B in children with acute lymphocytic leukemia (ALL) immediately and 6 months after chemotherapy. Materials and Methods: In the present prospective cohort study, 21 patients with ALL referred to Mofid Children’s Hospital were studied immediately and 6 months after chemotherapy. Serum samples were collected from patients, and the levels of immunoglobulins (IgG, IgM, IgE, and IgA) antibodies against diphtheria, tetanus, and hepatitis B were determined using specific enzymelinked immunosorbent assay kits. The obtained data were analyzed using Statistical Package for Social Sciences 21 software. Results: A total of 13 males and 8 females with an average age of 8.6 ± 2.5 years were included in the present study. Six months after chemotherapy, the mean level of IgG, IgM, IgE, and IgA displayed an increase of 563.1 units in IgG, 11 units in IgM, 11.3 units in IgE, and 5 units in IgA levels. Moreover, data revealed that 6 months after chemotherapy, the mean level of IgG antibodies displayed an increase of 7.09, 3.43, and 1.03 units against hepatitis B, diphtheria, and tetanus, respectively. A significant relationship was found between the antibody level against diphtheria and the age group of the patients (p = 0.003). Conclusion: Humoral immune status was boosted after 6 months of chemotherapy, though all patients had some extent of lasting immune dysfunction. We indicate that survivors of childhood cancer have ongoing humoral immunological defects and may remain at risk for infectious complications after completion of therapy. Relevance for Patients: The present study indicated that systemic therapies for pediatrics with leukemia affect the immune system. Pediatrics with leukemia may remain at risk for infectious complications after completion of therapy.

Modulating ferroptosis and mycobactericidal activity in lung epithelial cells via YY1/iNOS pathway

BackgroundMycobacterium tuberculosis infection triggers various forms of host cell death, including ferroptosis in lung epithelial cells; YY1, a critical transcription factor, plays a pivotal role in regulating ferroptosis, however, the underlying mechanisms are not fully understood. MethodsTo investigate Mycobacterium marinum (M.marinum) infection in lung epithelial cells A549 and H1299, we utilized flow cytometry to evaluate cell death and measure reactive oxygen species (ROS). Colony-forming unit (CFU) assays determined the intracellular bacterial load. Ferroptosis was analyzed using a specific detection kit to measure malondialdehyde (MDA) and glutathione (GSH) levels. The interaction between the transcription factor YY1 and the iNOS promoter was assessed through a dual-luciferase reporter assay. ResultsM.marinum induced ferroptosis in lung epithelial cells through invasion. This effect is most pronounced at 8 h of infection and decreases over time but increased with a higher multiplicity of infection (MOI). YY1 knockdown decreases the expression of SLC7A11 and GPX4, attenuates cellular ferroptosis, while YY1 overexpression has the opposite phenomenon, enhancing the expression of bactericidal molecules such as iNOS and MPEG1, thereby markedly reducing the intracellular bacterial load. We identified substantial binding of YY1 to the iNOS promoter region (−655 to −1018 bp), enhancing mycobactericidal activity in YY1-overexpressing cells. ConclusionsOur study demonstrates that YY1 inhibits ferroptosis induced by Mycobacterium marinum infection and reduces intracellular bacterial proliferation in lung epithelial cells. These findings provide a crucial basis for developing anti-tuberculosis therapies that target YY1 modulation, potentially offering new clinical avenues for the treatment of tuberculosis.

PSAT1 is upregulated by METTL3 to attenuate high glucose-induced retinal pigment epithelial cell apoptosis and oxidative stress

BackgroundDiabetic retinopathy (DR) is a major ocular complication of diabetes mellitus, and a significant cause of visual impairment and blindness in adults. Phosphoserine aminotransferase 1 (PSAT1) is an enzyme participating in serine synthesis, which might improve insulin signaling and insulin sensitivity. Furthermore, it has been reported that the m6A methylation in mRNA controls gene expression under many physiological and pathological conditions. Nevertheless, the influences of m6A methylation on PSAT1 expression and DR progression at the molecular level have not been reported.MethodsHigh-glucose (HG) was used to treat human retinal pigment epithelial cells (ARPE-19) to construct a cell injury model. PSAT1 and Methyltransferase-like 3 (METTL3) levels were detected by real-time quantitative polymerase chain reaction (RT-qPCR). PSAT1, B-cell lymphoma-2 (Bcl-2), Bcl-2 related X protein (Bax), and METTL3 protein levels were examined by western blot assay. Cell viability and apoptosis were detected by Cell Counting Kit-8 (CCK-8) and TUNEL assays. Reactive oxygen species (ROS), malondialdehyde (MDA), and Glutathione peroxidase (GSH-Px) levels were examined using special assay kits. Interaction between METTL3 and PSAT1 was verified using methylated RNA immunoprecipitation (MeRIP) and dual-luciferase reporter assay.ResultsPSAT1 and METTL3 levels were decreased in DR patients and HG-treated ARPE-19 cells. Upregulation of PSAT1 might attenuate HG-induced cell viability inhibition and apoptosis and oxidative stress promotion in ARPE-19 cells. Moreover, PSAT1 was identified as a downstream target of METTL3-mediated m6A modification. METTL3 might improve the stability of PSAT1 mRNA via m6A methylation.ConclusionMETTL3 might mitigate HG-induced ARPE-19 cell damage partly by regulating the stability of PSAT1 mRNA, providing a promising therapeutic target for DR.

B-324 Pilot Study Result of EQA for Fecal Immunochemical Test for Hemoglobin Using a Novel Ready-to-Use Sample Material

Abstract Background The primary screening method for colorectal cancer is fecal immunochemical test (FIT) detecting human hemoglobin (Hb) in feces. There are several tests on the market both for automated analyzers and point-of-care testing (POCT). To ensure high quality results, the tests should be monitored with appropriate quality assessment procedures including participation in external quality assessment (EQA) programs. By participating in an EQA program using a stool-like material, clinical laboratories and POCT sites can fulfil the ISO 15189 requirements of monitoring the complete laboratory process from pre- to postanalytical phases in addition to the quantitative analytical phase of FIT. Here we present the first results of an international EQA pilot study for quantitative FIT using a novel ready-to-use stool-like material, performed by EQA provider Labquality. Methods 40 participants from 8 countries using 9 different FIT reagents were recruited for the pilot study performed in 2023. A set of artificial stool samples containing human hemoglobin uniformly was shipped to the participants at ambient temperature. The sample material is developed by HECTEF (Health Care Technology Foundation, Japan). The participants were instructed to collect the sample with their test specific sample collection kit and perform analysis according to the manufacturer’s instructions as a regular patient sample. Quantitative results (µg Hb/g feces) and qualitative interpretations (positive/negative) were reported in Labquality’s electronic platform LabScala. The quantitative target value was a method group consensus mean. A questionnaire regarding the usability of the sample material was sent to all participants. Results Sample S001 contained no Hb and all participants (40/40) reported a result corresponding to the lower detection limit of their test in use or a low numeric result most likely explained by unspecific background. All reported a negative interpretation. Sample S002 was a high positive sample for Hb. The measured value exceeded the upper detection limit of 4/9 tests (Exdia iFOB, Precision Biosensor; Fecal Occult Blood (iFOB) Neo, Boditech; FOB Test, VedaLab; NADAL FOB Quant test, Nal von Minden). The mean values and CV% were 116 µg/g and 8% for iFOBT, Aidian; 192 ug/g and 4% for Standard iFOB FIA, SD Biosensor; 144 µg/g and 45% for FOB assay kit FOB Gold, Sentinel Diagnostics (different clinical chemistry analyzers used); 91 µg/g and 26% for OC-Sensor FIT, Eiken Chemical. All participants responded a positive qualitative clinical interpretation. The majority of the responders on the feedback questionnaire (26/35) found the samples to be easy to use including comments of the sample being user-friendly and ready-to-use, resembling a patient sample and easy to apply with the test specific sample collection kit. Conclusions The fecal-like sample used in this Labquality’s EQA pilot study evaluates the complete analytical process including the preanalytical, analytical and postanalytical phases. An EQA scheme using this sample material is more compliant to ISO 15189 requirements compared to schemes where liquid or lyophilized samples are used. The new sample material used on this pilot study was positively received by most of the participants and it was compatible with all tests included in the study.

B-059 Development and Validation of Multiplex Quantitative Allergy Tests based on Automated Flow Fluorescence Immunoassay Analyzer

Abstract Background Serological allergy tests measuring allergen-specific IgE (sIgE) are simpler and safer than skin prick tests, and the results are not influenced by skin condition or human factors. Quantitative detection of sIgE can also aid in evaluating patient allergies and aiding subsequent treatment. Dymind has researched and developed novel quantitative allergy tests based on the automated flow fluorescence immunoassay analyzer system (MF1280), enabling quantitative detection of multiple sIgE in just one test with one sample. Here, we present the performance of tIgE and two specific IgE test kits using the Dymind MF1280 system. Methods This study evaluated the performance of total IgE (tIgE) and two allergen-specific IgE (sIgE) panels of inhalant and food allergens, inhalant allergen panel including d1 (Dermatophagoides pteronyssinus), d2 (Dermatophagoides farina), i6 (Cockroach), e1 (Cat epithelium), e5 (Dog epithelium), w1(Common ragweed), and food allergen panel including f1 (Egg white), f2 (Cow’s milk), f3 (cod), f23 (Crab), 24 (Shrimp), and f27 (beef). Evaluation criteria included precision, linearity, limit of detection (LOD) and method comparison. As clinical performance of 425 clinical samples collected from 2 hospital laboratory in China was evaluated by both of Dymind allergen IgE MF1280 system and Phadia ImmunoCAP. Results The validated linearity of total IgE and sIgE were 2.0-5000.0 IU/mL and 0.1-100.0 IU/mL respectively. The LOD of total IgE and sIgE determined to be 2 IU/mL and 0.1 IU/mL respectively. The precision was evaluated by assaying a low and a high samples of every sIgE assay and the result of d1, d2, i6, e1, e5, w1, f1, f2, f3, f23, f24, f27 were 3.73% / 1.34% (low sample / high sample), 3.26% / 1.95%, 2.18% / 0.87%, 1.31% / 1.40%, 3.63% / 1.13%, 2.84% / 4.76%, 4.50% / 1.53%, 5.97% / 1.64%, 3.46% / 1.11%, 1.58% / 1.73%, 3.77% / 1.28%, 1.78% / 0.96% respectively. The total mean coefficient of measurements was below 7% to ensure high precision in multiplex quantitative analysis. Method comparison of 425 clinical samples between Phadia ImmunoCAP and Dymind allergen IgE showed total concordance rate was over 85% except f2 (over 80%). Conclusions The Dymind Allergen-specific IgE test kits on the automated flow fluorescence immunoassay analyzer system provide fast and accurate in vitro diagnostic detection of tIgE and sIgE for aiding diagnostics specific allergens sensitization and treatment for allergy patients, with good precision and excellent agreement compared to ImmunoCAP. The MF1280 system has advantages including random access, fully automated, multiplex quantitative detection of allergen sIgE with reducing assay time, sample consumption and lower costs.

Insulin-Like Growth Factor Binding Protein 2 Drives Neurodegeneration in Parkinson's Disease: Insights From InVivo and InVitro Studies.

Insulin-like growth factor binding protein 2 (IGFBP2) is implicated in various neurodegenerative diseases. However, its role in Parkinson's disease (PD) is unclear. PD rat model was established by 6-OHDA injection. After 3 weeks, mRNA-seq was conducted. Rats received rIGFBP2 via intra-MFB injection 6 h prior to 6-OHDA infusion, and the effect of IGFBP2 in PD rats was investigated by western blotting, IHC, specific kits, JC-1 staining, and TUNEL analysis. Invitro, PC12 cells were treated with 6-OHDA, and CCK-8, specific kits, Hoechst 33258 staining, Western blotting, and JC-1 staining were performed to assess the IGFBP2's role. mRNA-seq revealed DEGs in PD, with attention to downregulated IGFBP2. rIGFBP2 treatment aggravated neurobehavioral deficits, decreased TH expression, Ψm, ATP level and SOD, GSH-Px activities but increased α-synuclein, ROS, MDA, mitochondrial cytochrome c contents, cell apoptosis in 6-OHDA-lesioned rats, which might be mediated through inactivating IGF-1R/AKT pathway. In 6-OHDA-treated PC12 cells, rIGFBP2 aggravated cell injury, demonstrated by decreased cell viability and increased apoptosis, oxidative stress, and mitochondrial dysfunction. Co-treatment with rIGFBP2 and rIGF-1 partially reversed the effect of rIGFBP2 on cell damage. IGFBP2 exacerbates neurodegeneration in PD through increasing oxidative stress, mitochondrial dysfunction, and apoptosis via inhibiting IGF-1R/AKT pathway.

Protective effect and mechanism of lycium barbarum polysaccharide against UVB-induced skin photoaging.

Cellular senescence can be categorized into two main types, including exogenous and endogenous aging. Photoaging, which is aging induced by ultraviolet (UV) radiation, significantly contributes to exogenous aging, accounting for approximately 80% of such cases. Superoxide Dismutase (SOD) is a class of antioxidant enzymes, with SOD2 being predominantly localized in the mitochondrial matrix. Ultraviolet radiation (UVR) inhibits SOD2 activity by acetylating the key lysine residues on SOD2. Sirtuin3 (SIRT3), the principal mitochondrial deacetylase, enhances the anti-oxidant capacity of SOD2 by deacetylating. Lycium barbarum polysaccharide (LBP) is the main bioactive component extracted from Lycium barbarum (LB). It has been reported to have numerous potential health benefits, such as anti-oxidation, anti-aging, anti-inflammatory and anti-apoptotic properties. Furthermore, LBP has been shown to regulate hepatic oxidative stress via the SIRT3-SOD2 pathway. The aim of this study was to construct a UVB-Stress-induced Premature Senescence (UVB-SIPS) model to investigate the protective effects and underlying mechanisms of LBP against UVB-induced skin photoaging. Irradiated with different UVB doses to select the suitable dose for constructing the UVB-SIPS model. Cell morphology was observed using a microscope. The proportion of senescent cells was assessed by senescence-associated β-galactosidase (SA-β-gal) staining. Cell viability was studied using the Cell Counting Kit-8 (CCK-8). Intracellular levels of reactive oxygen species (ROS) were observed using flow cytometry and an inverted fluorescence microscope. Expression of γ-H2AX was investigated using flow cytometry. Western blot (WB) was used to verify the expression of senescence-associated proteins (p21, p53, MMP-1, and MMP-3). Enzyme-Linked Immunosorbnent Assay (ELISA) was used to measure pro-inflammatory cytokines levels (IL-6, TNF-α). WB was also used to analyze the expression of SIRT3, SOD2, and Ac-SOD2, and a specific kit was employed to detect SOD2 activity. Our results suggested that the UVB-SIPS group pre-treated with LBP exhibited a reduced proportion of cells positive for SA-β-gal staining, mitigated production of intracellular ROS, an amelioration in γ-H2AX expression, and down-regulated expression of senescence-associated proteins and pro-inflammatory cytokines as compared to the UVB-SIPS group. Moreover, in contrast to the control group, the UVB-SIPS group showed regulated SIRT3 expression and SOD activity, elevated Ac-SOD2 expression and an increased ratio of Ac-SOD2/SOD2. However, the UVB-SIPS group pre-treated with LBP showed an upregulation of SIRT3 expression and enhanced SOD activity, a reduction in AC-SOD2 expression, and a decreased ratio of AC-SOD2/SOD2, compared to the untreated UVB-SIPS group. Additionally, the photo-protective effect of LBP was diminished following treatment with 3-TYP, a SIRT3-specific inhibitor. This study suggested that LBP, a natural component, exhibits anti-oxidant and anti-photoaging properties, potentially mediated through the SIRT3-SOD2 pathway.

Bu Shen Huo Xue Formula Provides Neuroprotection Against Spinal Cord Injury by Inhibiting Oxidative Stress by Activating the Nrf2 Signaling Pathway.

Spinal cord injury (SCI) is an irreversible neurological disease that can result in severe neurological dysfunction. The Bu Shen Huo Xue Formula (BSHXF) has been clinically shown to assist in the recovery of limb function in patients with SCI. However, the underlying mechanisms of BSHXF's therapeutic effects remain unclear. This study aimed to evaluate the effects of BSHXF in a mouse model of SCI and to identify potential therapeutic targets. The composition of BSHXF was analyzed using high-performance liquid chromatography (HPLC). In vivo, SCI was induced in mice following established protocols, followed by administration of BSHXF. Motor function was assessed using the Basso-Beattie-Bresnahan (BBB) and footprint tests. Levels of superoxide dismutase (SOD) and malondialdehyde (MDA) were quantified with specific assay kits. Protein expression analysis was performed using Western blot and immunofluorescence. Additionally, reactive oxygen species (ROS) levels and apoptosis rates were evaluated with dedicated staining kits. In vitro, neurons were exposed to lipopolysaccharide (LPS) to investigate the effects of BSHXF on neuronal oxidative stress. The protective effects of BSHXF against LPS-induced neuronal injury were examined through RT-PCR, Western blot, and immunofluorescence. The eight primary bioactive constituents of BSHXF were identified using HPLC. BSHXF significantly reduced tissue damage and enhanced functional recovery following SCI. Meanwhile, BSHXF treatment led to significant reductions in oxidative stress and apoptosis rates. It also reversed neuronal loss and reduced glial scarring after SCI. LPS exposure induced neuronal apoptosis and axonal degeneration; however, after intervention with BSHXF, neuronal damage was reduced, and the protective effects of BSHXF were mediated by the activation of the Nrf2 pathway. BSHXF decreased tissue damage and enhanced functional recovery after SCI by protecting neurons against oxidative stress and apoptosis. The effects of BSHXF on SCI may be related to the activation of the Nrf2 pathway.