Detection Kit Research Articles

Transcriptome analyses reveal molecular mechanisms of novel compound heterozygous ACO2 variants causing infantile cerebellar retinal degeneration

Background and purposeInfantile cerebellar retinal degeneration (ICRD) (OMIM #614559) is a rare autosomal recessive inherited disease associated with mutations in the aconitase 2 (ACO2) gene. We report a Chinese girl with novel compound heterozygous variants in ACO2, who presented at 7 months of age with psychomotor retardation, truncal hypotonia, and ophthalmologic abnormalities. This study aims to investigate the potential molecular mechanisms underlying ACO2 deficiency-induced neuropathy.MethodsWhole exome sequencing was performed on family members to screen for potential pathogenic mutations, followed by Sanger sequencing for validation. Mitochondrial aconitase activity and mitochondrial DNA (mtDNA) copy number were measured using an aconitase activity detection kit and quantitative PCR, respectively. Transcriptome expression profiles from patient cells, and cerebellar and retinal organoids retrieved from the GEO database were integrated. Functional enrichment analysis and protein-protein interaction networks were used to identify key molecules, and their expression levels were validated using Western blot analysis.ResultsGenetic testing revealed novel compound heterozygous variations in the proband's ACO2 gene (NM:001098), including c.854A>G (p.Asn285Ser) and c.1183C>T (p.Arg395Cys). Predictive analysis of the tertiary structure of the ACO2 protein suggests that both p.Asn285Ser and p.Arg395Cys affect the binding ability of ACO2 to ligands. The mitochondrial aconitase activity and mtDNA copy number in the proband's leukocytes were significantly reduced. Transcriptomic data analysis identified 80 key candidate genes involved in ACO2-related neuropathy. Among these, LRP8 and ANK3, whose gene expression levels were significantly positively correlated with ACO2, were further validated by Western blot analysis.ConclusionsThis study expands the spectrum of pathogenic ACO2 variants, elucidates the potential molecular mechanisms underlying ACO2-related neuropathy, provides in-depth support for the pathogenicity of ACO2 genetic variations, and offers new insights into the pathogenesis of ICRD.

EXTH-39. A BENCH-TOP MODEL FOR THE OPTIMIZATION OF ULTRASOUND PARAMETERS FOR SONODYNAMIC THERAPY OF GLIOBLASTOMA

Abstract BACKGROUND In-vitro studies have demonstrated that low frequency sound waves can interact with hemo-porphyrin molecule to induce the production of reactive oxygen species. This biophysical phenomenon can induce tumour cell death in glioblastoma cells in-vitro. The purpose of this study was to evaluate the production of reactive oxygen species and expression of apoptotic markers in glioblastoma cells in response to varying total treatment time and ultrasound power using a bench-top focused ultrasound (FUS) device. METHODS U251 cells were exposed to 5-Aminolevulenic Acid (5-ALA) at varying concentrations for up to 24 hours. The time point with peak proto-porphyrin IX (PPIX) fluorescence was established using fluorescence activated cell sorting (FACS). A transducer utilized at 0.91MHz was placed on an inverted stage with the focus targeted to the bottom of a standard 96-well cell culture plate. Pressure wave delivery was confirmed using a hydrophone. GBM cells were treated with 5-ALA alone, FUS alone, or 5-ALA + FUS and the cells were incubated for 24 hours prior to measuring ROS induction and cell death. ROS was measured using FACS with the CellROX Green Kit for Oxidative Stress Detection and Annexin-V was measured using the Dead Cell Stain Kit. RESULTS Peak fluorescence intensity for PPIX in the U251 cell line was found to be induced through incubation with 5-ALA for 24 hours with a concentration 200mg/mL. The fluorescence intensity of PPIX was found to be 313% greater in the treated group compared to the control group. Peak ROS was observed using a 0.91MHz transducer frequency for a total time of 120s with 6W average power. ROS was 61% and 66% greater in the SDT group compared to the FUS and 5-ALA group respectively. CONCLUSION Using this bench-top set up we successfully induced ROS and apoptosis in U251 cells. Establishing a reliable in-vitro model will allow us to further investigate the effects of other sonication parameters such as burst length.

TMET-22. OVERCOMING RESISTANCE: PANOBINOSTAT SENSITIZES 2DG-RESISTANT PEDIATRIC HIGH-GRADE GLIOMAS WITH H3.3 K27M/G34R MUTATIONS TO APOPTOSIS

Abstract BACKGROUND Pediatric high-grade gliomas (pHGG) harboring H3.3 K27M/G34R mutations present formidable challenges in treatment due to their aggressiveness and resistance to standard chemotherapy. Our preliminary investigations have shown promising results, indicating that combining 2-deoxyglucose (2DG), a glycolytic inhibitor, with DNA-damaging agents synergistically inhibits pHGG growth. However, prolonged exposure to 2DG induces resistance, necessitating the exploration of alternative therapies. This study aims to investigate the potential of panobinostat, a histone deacetylase inhibitor, in sensitizing 2DG-resistant pHGG with H3.3 K27M/G34R mutations to apoptosis. METHODS Transcriptomic analysis via Illumina NovaSeq 6000 and metabolomic profiling employing Liquid Chromatography-High-Resolution Mass Spectrometry was used to delineate the molecular landscape of naive and 2DG-resistant pHGG cell lines. Functional assays, including the Agilent Seahorse Mito Stress Test and flow cytometry, assessed bioenergetics and mitochondrial dynamics. Intracellular NAD+ and NADH levels were quantified utilizing NAD+/NADH Glo assays, while apoptotic markers and metabolic stress were measured using the Annexin V-FITC Conjugates for Apoptosis Detection kit and western blot. RESULTS Comparative analysis revealed increased transketolase activity in 2DG-resistant cells, augmenting the interaction between the pentose phosphate pathway and glycolysis, resulting in elevated levels of NADPH and ATP. Additionally, these resistant cells displayed enhanced reliance on mitochondrial energy metabolism, as evidenced by substantial increases in mitochondrial membrane potential and mass, coupled with elevated NAD+ levels. Subsequent treatment with panobinostat induced a significant decrease in NAD+ biosynthesis and ATP generation. Furthermore, this intervention triggered the upregulation of pro-apoptotic BCL-2 family proteins, particularly BAX and BAK, concomitant with a notable reduction in mitochondrial membrane potential and the accumulation of reactive oxygen species. Consequently, heightened caspase-3 activity ensued, leading to significantly elevated cell death rates in 2DG-resistant gliomas. CONCLUSION Our findings highlight panobinostat as a promising therapy to resensitize 2DG-resistant pHGG cells to apoptosis, offering hope for treating these aggressive tumors with H3.3 K27M/G34R mutations.

USP15 as a Potential Therapeutic Target in Cerebral Ischemia: Modulation of Ferroptosis and Cognitive Dysfunction via the Nrf2/GPX4 Axis in Mice.

This study aimed to investigate the role of ubiquitin-specific peptidase 15 (USP15) in ischemic cognitive dysfunction using a mouse model and a cerebral ischemia (CI) cell model, its impact on ferroptosis and the underlying mechanisms. Oxygen-glucose deprivation/reoxygenation (OGD/ R)-induced HT-22 cells were used to establish the CI cell model, and mice induced with CI were used as the animal model for ischemic cognitive dysfunction. Cell damage was evaluated using Cell Counting Kit-8 (CCK-8), flow cytometry (FCM), immunoblotting, and immunofluorescence assays. Cognitive dysfunction in the CI mice was assessed through water maze experiments. Ferroptosis was examined with an iron detection kit and immunoblotting, oxidative stress was evaluated using 2',7'-dichlorofluorescin diacetate (DCF) and enzyme-linked immunosorbent assay (ELISA), and mechanistic experiments were performed via immunoblotting. USP15 knockdown alleviated OGD/ R-induced damage in HT-22 cells. In vivo, USP15 depletion mitigated brain injury in middle cerebral artery occlusion (MCAO) mice and improved learning and memory function. The absence of USP15 reduced oxidative stress in MCAO mice and attenuated ferroptosis by activating nuclear factor erythroid 2-related factor 2 (Nrf2). Mechanistic investigations confirmed that USP15 depletion ameliorated cognitive impairment and ferroptosis through the activation of the Nrf2/ GPX4 axis. USP15 is associated with ferroptosis and cognitive dysfunction in mice and could serve as a potential therapeutic target in CI.

Epidemiology of Acute Infectious Diarrhea-Associated Viruses in Children in Hangzhou, China, 2023

Abstract Objective To investigate the epidemiological characteristics of children infected with diarrheal virus in Hangzhou, China, 2023. Methods From January 2023 to December 2023, 20,939 stool samples from children with acute infectious diarrhea were collected for the detection of rotavirus A and human adenovirus (HAdV), using latex agglutination detection kits; 7,584 stool samples were collected for the detection of norovirus, using real-time polymerase chain reaction reagent. Results A total of 639 (3.0%, 639/20,939) tested positive for rotavirus A, and 1,201 (5.7%, 1,201/20,939) positive samples were detected by colloidal gold method. The positive rates of norovirus were 42.2% (3,203/7,585). Among all age groups, the rotavirus A positive detection rate was the highest in participants aged 3 to 6 years (6.46%, 260/4,024). The monthly distribution of patients with rotavirus A showed that the number of cases was the lowest in October (0.45%, 8/1,779) and reached the peak in April (7.97%, 144/1,806). The highest and lowest positive rates among all age groups for HAdV were found in children aged 3 to 6 years(8.27%, 333/4,024) and in those aged 0 to 6 months (2.21%, 60/2,717). The monthly positivity rates of HAdV spanning from January to November 2023 were 1.38, 1.44, 2.34, 3.65, 6.64, 7.71, 7.54, 7.13, 6.82, 4.15, and 6.50%, and reached the peak in December (8.17%). For norovirus, children aged 1 to 3 years had the highest positive detection rate (57.95%, 1,349/2,328), while infants aged 0 to 6 months had the lowest positive detection rate (19.60%, 205/1,046). The results show that January had the lowest number of cases (14.63%, 6/41), while September had the highest (50.51%, 545/1,079). Conclusion The detection rate of rotavirus A and HAdV was highest among participants aged 3 to 6 years, whereas the detection rate of norovirus was highest among those aged 1 to 3 years. The monthly distribution peaks for the three enteric viruses varied significantly.

Tanshinone II A Facilitates Chemosensitivity of Osteosarcoma Cells to Cisplatin via Activation of p38 MAPK Pathway.

To examine the mechanism of action of tanshinone II A (Tan II A) in promoting chemosensitization of osteosarcoma cells to cisplatin (DDP). The effects of different concentrations of Tan II A (0-80 µ mol/L) and DDP (0-2 µ mol/L) on the proliferation of osteosarcoma cell lines (U2R, U2OS, 143B, and HOS) at different times were examined using the cell counting kit-8 and colony formation assays. Migration and invasion of U2R and U2OS cells were detected after 24 h treatment with 30 µ mol/L Tan II A, 0.5 µ mol/L DDP alone, and a combination of 10 µ mol/L Tan II A and 0.25 µ mol/L DDP using the transwell assay. After 48 h of treatment of U2R and U2OS cells with predetermined concentrations of each group of drugs, the cell cycle was analyzed using a cell cycle detection kit and flow cytometry. After 48 h treatment, apoptosis of U2R and U2OS cells was detected using annexin V-FITC apoptosis detection kit and flow cytometry. U2R cells were inoculated into the unilateral axilla of nude mice and then the mice were randomly divided into 4 groups of 6 nude mice each. The 4 groups were treated with equal volume of Tan II A (15 mg/kg), DDP (3 mg/kg), Tan II A (7.5 mg/kg) + DDP (1.5 mg/kg), and normal saline, respectively. The body weight of the nude mice was weighed, and the tumor volume and weight were measured. Cell-related gene and signaling pathway expression were detected by RNA sequencing and Kyoto Encyclopedia of Genes and Genomes pathway analysis. p38 MAPK signaling pathway proteins and apoptotic protein expressions were detected by Western blot. In vitro studies have shown that Tan II A, DDP and the combination of Tan II A and DDP inhibit the proliferation, migration and invasion of osteosarcoma cells. The inhibitory effect was more pronounced in the Tan II A and DDP combined treatment group (P<0.05 or P<0.01). Osteosarcoma cells underwent significantly cell-cycle arrest and cell apoptosis by Tan II A-DDP combination treatment (P<0.05 or P<0.01). In vivo studies demonstrated that the Tan II A-DD combination treatment group significantly inhibited tumor growth compared to the Tan II A and DDP single drug group (P<0.01). Additionally, we found that the combination of Tan II A and DDP treatment enhanced the p38 MAPK signaling pathway. Western blot assays showed higher p-p38, cleaved caspase-3, and Bax and lower caspase-3, and Bcl-2 expressions with the combination of Tan II A and DDP treatment compared to the single drug treatment (P<0.01). Tan II A synergizes with DDP by activating the p38/MAPK pathway to upregulate cleaved caspase-3 and Bax pro-apoptotic gene expressions, and downregulate caspase-3 and Bcl-2 inhibitory apoptotic gene expressions, thereby enhancing the chemosensitivity of osteosarcoma cells to DDP.

Prevalence of inhaled allergen sensitization among patients with suspected allergic diseases in Sichuan province: 3-year data from a single center

Allergic diseases are becoming increasingly common, and they are a threat to people’s health. Exploring the distribution characteristics of allergen-specific immunoglobulin E (sIgE) in people from Sichuan province, southwest China, can provide clinical epidemiological data. This study enrolled 12,204 consecutive patients with suspected allergies from May 2018 to May 2021. Among the patients, 4206 were diagnosed with allergic rhinitis or asthma. The Rayto Lumiray 1600 detection system and sIgE and total IgE (tIgE) detection kits were used to measure the levels of nine common sIgE and tIgE. sIgE ≥ 0.35 IU/mL was considered positive. The sensitization rate of D1 (Dermatophagoides pteronyssinus) is the highest (22.97%), followed by D2 (Dermatophagoides farinae) (23.33%); M3 (Aspergillus fumigatus) has the lowest positive rate (1.5%). E5 (Canis familiaris) and I6 (Blatella germanica) exhibited relatively high sensitization rates (8.64% and 12.35%, respectively). Allergen sensitization was significantly higher in men than in women. Moreover, 90.3% of sensitization samples from D1 were combined with at least one of the other eight allergens. Similarly, a positive correlation was demonstrated between D1, D2, and E1 (Felis domesticus). In addition, patients with allergic rhinitis (91.88% and 91.54%, respectively), allergic asthma (72.44% and 74.01%, respectively), and allergic rhinitis with asthma (92.12% and 93.22%, respectively) were mainly sensitized to D1 and D2. D1, D2, I6, and E5 are the main allergens. Moreover, D1 and D2 are the main allergens in patients with respiratory allergic diseases. Research focusing on the distribution of allergens and the characteristics of hypersensitivity in different people helps prevent, diagnose, and treat allergic diseases.

BRD7 regulates cellular senescence and apoptosis in ALS by modulating p21 expression and p53 mitochondrial translocation respectively

Cellular senescence is involved in the progression of neurodegenerative diseases. Motor neurons exhibit senescence-like alterations in ALS. BRD7, identified as a regulatory factor associated with cellular senescence, its function in ALS remains unclear. This study aims to investigate the potential role and mechanisms of BRD7 in ALS. We analyzed RNA levels using qRT-PCR, protein levels through immunofluorescence and western blot, and apoptosis via TUNEL staining. Cell transfection was conducted for in vitro experiments. The level of β-galactosidase was measured by β-galactosidase activity detection kit. ALS motor neurons exhibited senescence-like alterations, characterized by increased activity of p53, p21, and β-galactosidase, as well as reduced lamin B1 staining. Additionally, the expression of BRD7 was upregulated and induced cellular senescence and apoptosis. Downregulation of BRD7 alleviates the cellular senescence by inhibiting p21 rather than p53. Knockdown of BRD7 inhibited p53 mitochondrial translocation, leading to reduced apoptosis. Our results suggest that BRD7 plays an important role in the survival of ALS motor neurons. BRD7 knockdown can reduce cellular senescence and apoptosis by inhibiting p21 and p53 mitochondrial translocation.

Naringin promotes osteogenic potential in bone marrow-derived mesenchymal stem cells via mediation of miR-26a/Ski axis

BackgroundOsteonecrosis of the femoral head (ONFH) is a common orthopedic disease, which seriously affects the quality of life of patients. Naringin has protective effect on ONFH. In present study, we aimed to investigate the mechanism of Naringin regulating miR-26a in ONFH. MethodsTwo sequencing datasets (GSE89587 for micro-RNA, GSE123568 for mRNA) related to ONFH were obtained from the GEO database for bioinformatics analysis. Bone marrow-derived mesenchymal stem cells (BMSCs) were treated with adipogenic medium (AM) or osteogenic medium (OM), and then treated with 10 μM, 100 μM or 1000 μM Naringin. Gene and protein levels were detected by RT-qPCR and Western blotting. ALP activity and alizarin red staining (ARS) were applied to investigate the osteogenic differentiation of BMSCs. Oil red O staining was performed to test adipogenic differentiation. The content of triglycerides (TG) in BMSCs was detected by TG detection kit. Double luciferase reporter gene measured the interaction between miR-26a and Ski. ResultsBioinfomatic analyses indicated a significant downregulation of miR-26a and a significant upregulation of Ski in the peripheral blood of patients with ONFH. Naringin significantly promoted the osteogenic differentiation, and increased the ALP activity and ARS. Meanwhile, it decreased the adipogenic differentiation and inhibited TG levels. In addition, miR-26a was downregulated and Ski was increased in AM-treated BMSCs, while miR-26a was upregulated and Ski was decreased in OM-treated BMSCs. Furthermore, miR-26a promoted the osteogenic differentiation and suppressed the adipogenic differentiation in BMSCs. Moreover, Naringin enhanced osteogenic potential in BMSCs was reversed by knockdown of miR-26a or overexpression of Ski. ConclusionNaringin could promote osteogenic differentiation of BMSCs via mediation of miR-26a/Ski axis. Thereby, Naringin might be a new agent for ONFH treatment.

Effect of Bioplastic Material on Adhesion, Growth, and Proliferative Activity of Human Fibroblasts When Incubated in Solutions Mimic the Acidity of Wound an Acute and Chronic Inflammation.

: The aim of this study was to characterize the effect of bioplastic material based on collagen, hyaluronic acid, and elastin on the viability and proliferative activity of human fibroblasts in conditions simulating the acidity of acute and chronic wounds. : Bioplastic material was made according to the method described in RF patent no.2722744. Adhesive properties and proliferative activity of human fibroblasts were assessed visually using fluorescent microscopy. The number of apoptotic and necrotic cells was assessed by flow cytometry (BD FACSCanto II) using the commercial FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen). The strength, Young's modulus, and elasticity of the gels were determined on a TA.XT-plus texture analyzer (Stable Micro Systems, Great Britain). : Using the methods of luminescent microscopy and flow cytometry, we found that the cell viability (namely, adhesive properties and proliferative activity) decreases after incubation on condition mimic of physiological acidosis. We found that bioplastic material contributes to the preservation of adhesive properties, viability, and proliferative activity of fibroblasts in physiological acidosis conditions. The results obtained indicate that bioplastic material based on soluble dermis components can be used as a biologically active component of wound dressings to increase the effectiveness of reparative regeneration, especially in cases of excessive acute inflammation.

Performance of DNA methylation and blood-borne tumor indicators in detecting colorectal neoplasia and adenomas: a comparative study with the fecal occult blood test

ObjectivesTo evaluate the performance of stool methylated syndecan2 (mSDC2), methylated septin9 (mSEPT9), fecal occult blood test (FOBT), carcinoembryonic antigen (CEA), carbohydrate antigen 125 (CA125) and carbohydrate antigen 199 (CA199) in detecting colorectal neoplasia and adenomas.MethodsBlood-borne CEA, CA125, and CA199 levels were measured by electrochemiluminescence. The SDC2 methylation was detected by Methylation Detection Kit for Human SDC2 Gene (Real time PCR), and the SEPT9 methylation was detected by the Septin9 Gene Methylation Detection Kit based on PCR fluorescent probe assay. The colonoscopy combined with tissue biopsy pathology was used as a validation criterion for colorectal neoplasia.ResultsIn detecting colorectal neoplasia, the AUCs of mSDC2, FOBT and mSEPT9 were 0.935 (95% CI: 0.915-0.956, P&amp;lt;0.001), 0.824 (95% CI: 0.617-1.000, P&amp;lt;0.001) and 0.671 (95% CI: 0.511-0.831, P&amp;lt;0.001), respectively. The sensitivity of mSDC2, FOBT and mSEPT9 were 100.0%, 66.7% and 40.0%, respectively. But the AUC of CEA, CA125 and CA199 were not statistically significant for colorectal neoplasia (all P&amp;gt;0.05). The combined application of mSEPT9 and mSDC2 showed the best predictive performance (AUC: 0.956, 95% CI: 0.887~1.000). For adenomas, the AUC of FOBT was extremely low (AUC: 0.524, 95% CI: 0.502-0.545, P=0.004). The CEA, CA125, CA199, mSEPT9 and mSDC2 were not statistically significant in detecting adenomas (all P&amp;gt;0.05).ConclusionsFor individual tests, FOBT and mSDC2 are relatively better indicators for detecting colorectal neoplasia compared to mSEPT9, CEA, CA125 and CA199. The combined form of mSEPT9 and mSDC2 to detect colorectal neoplasia has good predictive performance. However, none of these indicators demonstrated significant predictive power for detecting adenomas in our study.

Epidemiological Dynamics of Canine Morbillivirus in Resident Dogs of Makurdi Metropolis, Benue State, Nigeria

Canine distemper is an endemic viral disease of dogs in Nigeria. Knowledge of the carrier status of dogs is key to successful control of the disease. In this study, the carrier status of apparently healthy dogs’ resident in parts of Makurdi metropolis was determined using the immunochromatographic rapid antigen assay kit for qualitative detection of canine morbillivirus antigens in ante-mortem samples. Ocular-, nasal-, and rectal- swabs, as well as serum were taken from each of 204 dogs bringing a total of 816 samples. The results showed that 26.96 % of the dogs sampled were positive for Canine morbillivirus antigens. Viral antigens were detected in 9.8%, 11.27%, and 4.41% of nasal, ocular, and rectal swaps respectively, and in 6.37% of the serum samples collected. An age-related susceptibility was observed as viral antigens detection rate was higher in younger dogs compared to older ones. Similarly, 40.43% of vaccinated and 22.93% of unvaccinated dogs were positive for canine morbillivirus antigens. Of significant importance is the prevalence rate in unvaccinated population of dogs. In terms of breed-related detection rate, 23.93% of Nigerian local dogs and 40.63% of exotic breeds tested positive for viral antigens, and it was observed that 25.54% of dogs which had history of contact with other dogs and 40% of dogs which had no such history carried canine morbillivirus antigens. The significance of this study is that it details the current epidemiological dynamics of canine morbillivirus in resident dogs of Makurdi metropolitan area, and the findings are discussed. Subject Areas: Animal infectious disease epidemiology.

The resurgence of pertussis in Central Serbia in the period from June to December 2023

Abstract Background There was a registered increase in the number of confirmed pertussis cases in Central Serbia between June and December 2023. Among the possible causes of pertussis resurgence are increased awareness of pertussis among healthcare workers, availability of the sensitive RT PCR method in diagnostic, the limited duration of vaccine-induced immunity as well as decline of immunization coverage influenced by the COVID-19 pandemic. Methods The nasopharyngeal samples from clinics and primary care departments were transferred to Institute of Public Health of Serbia. DNA was extracted from each sample using the MagMAX Viral/Pathogen kit. The presence of B. pertussis was determined using two PCR assays, VIASURE Bordetella Real Time PCR Detection Kit and Bosphore B. pertussis/parapertussis Genotyping Kit, Anatolia Geneworks. Results RT-PCR confirmed a total of 338 pertussis cases in Institute of Public Health of Serbia during the observed period. Most of confirmed cases were among children 9-14 years old (49,7%) followed by infants younger than 12 months (21,9%). The more severe cases were among the unvaccinated and uncomplete vaccinated infants, 78,4% of which required hospitalization with two deaths in this age group. Conclusions Based on the analysis of the obtained results, we can highlight the importance of using sensitive methods in pathogen detection. Rapid diagnosis, laboratory confirmation and notification ensure early public health intervention to minimize the spread of disease. Despite long tradition of mandatory immunization against pertussis in the Republic of Serbia, wanning of vaccine induced immunity poses potential risk for the resurgence of pertussis. For pertussis control, in the future, it will be essential to achieve high immunization coverage as well as to introduce a booster dose of pertussis containing vaccine for children aged 14 and to implement vaccination of pregnant women. Key messages • For the better pertussis control vaccination of adolescents and adults, as well as vaccination of pregnant women should be considered in the future vaccination policies. • Clinicians should consider pertussis in the differential diagnosis of persistent cough illness in people of all ages, even those previously immunized.

Detection of Chlamydia trachomatis and Neisseria gonorrhoeae (and Its Resistance to Ciprofloxacin): Validation of a Molecular Biology Tool for Rapid Diagnosis and Treatment

Background and Objectives: Neisseria gonorrhoeae and Chlamydia trachomatis can cause similar clinical syndromes and may coexist in infections. In emergency medicine, empirical treatment targeting both pathogens is often employed, potentially contributing to antibiotic resistance. Gonococcal resistance has emerged against first-line antimicrobials, necessitating prior testing for fluoroquinolone susceptibility. Certest Biotec developed two molecular diagnostic products for simultaneous detection: VIASURE C. trachomatis &amp; N. gonorrhoeae Real-Time PCR Detection Kit and VIASURE Neisseria gonorrhoeae Ciprofloxacin-Resistant Real-Time PCR Detection Kit. To evaluate these products, clinical performance assessments were conducted at the Clinical Microbiology Laboratory of Miguel Servet University Hospital in Zaragoza, Spain. Results and Conclusions: Both VIASURE assays under study demonstrated high clinical sensitivity and specificity compared to reference molecular assays and Sanger sequencing. These kits offer an accurate diagnosis, facilitating appropriate treatment choices while addressing concerns about emerging antibiotic resistance. Methods: A total of 540 clinical samples from 540 patients already characterized as positive or negative for CT and NG by a molecular assay and by antibiotic susceptibility testing for ciprofloxacin using a concentration gradient diffusion method were used for the clinical evaluation. In cases where sensitivity results were unavailable, conventional PCR and Sanger sequencing were employed.

In Vitro Assessment of the Neuroprotective Effects of Pomegranate (Punica granatum L.) Polyphenols Against Tau Phosphorylation, Neuroinflammation, and Oxidative Stress

Background: Oxidative stress and chronic inflammation, at both the systemic and the central level, are critical early events in atherosclerosis and Alzheimer’s disease (AD). Purpose: To investigate the oxidative stress-, inflammation-, and Tau-phosphorylation-lowering effects of pomegranate polyphenols (PPs) (punicalagin, ellagic acid, peel, and aril extracts). Methods: We used flow cytometry to quantify the protein expression of proinflammatory cytokines (IL-1β) and anti-inflammatory mediators (IL-10) in THP-1 macrophages, as well as M1/M2 cell-specific marker (CD86 and CD163) expression in human microglia HMC3 cells. The IL-10 protein expression was also quantified in U373-MG human astrocytes. The effect of PPs on human amyloid beta 1-42 (Aβ1-42)-induced oxidative stress was assessed in the microglia by measuring ROS generation and lipid peroxidation, using 2′,7′-dichlorofluorescein diacetate (DCFH-DA) and thiobarbituric acid reactive substance (TBARS) tests, respectively. Neuronal viability and cell apoptotic response to Aβ1-42 toxicity were assayed using the MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) assay and the annexin-V-FITC apoptosis detection kit, respectively. Finally, flow cytometry analysis was also performed to evaluate the ability of PPs to modulate Aβ1-42-induced Tau-181 phosphorylation (pTau-181). Results: Our data indicate that PPs are significantly (p &lt; 0.05) effective in countering Aβ1-42-induced inflammation through increasing the anti-inflammatory cytokines (IL-10) in U373-MG astrocytes and THP1 macrophages and decreasing proinflammatory marker (IL-1β) expression in THP1 macrophages. The PPs were also significantly (p &lt; 0.05) effective in inducing the phenotypic transition of THP-1 macrophages and microglial cells from M1 to M2 by decreasing CD86 and increasing CD163 surface receptor expression. Moreover, our treatments have a significant (p &lt; 0.05) beneficial impact on oxidative stress, illustrated in the reduction in TBARS and ROS generation. Our treatments have significant (p &lt; 0.05) cell viability improvement capacities and anti-apoptotic effects on human H4 neurons. Furthermore, our results suggest that Aβ1-42 significantly (p &lt; 0.05) increases pTau-181. This effect is significantly (p &lt; 0.05) attenuated by arils, peels, and punicalagin and drastically reduced by the ellagic acid treatment. Conclusion: Overall, our results attribute to PPs anti-inflammatory, antioxidant, anti-apoptotic, and anti-Tau-pathology potential. Future studies should aim to extend our knowledge of the potential role of PPs in Ab1-42-induced neurodegeneration, particularly concerning its association with the tauopathy involved in AD.

Emodin regulated lactate metabolism by inhibiting MCT1 to delay non-small cell lung cancer progression.

Lung cancer is one of the most common malignant tumors in the world, with high incidence rate and mortality. Monocarboxylate transporter (MCT) 1 has been found to be widely expressed in various tumors and plays a crucial role in regulating energy metabolism. Emodin, as an important traditional Chinese medicine in China, has been reported to inhibit the progression of lung cancer. However, its potential mechanism has not been fully elucidated. The effects of emodin and MCT1 inhibitor AZD3965 on the proliferation, migration, and invasion of lung cancer cells were detected using cell counting kit-8 (CCK-8) assay, wound-healing assay, and transwell small chamber assay. The content of glucose, lactate, and pyruvate in the cell culture medium was detected using a glucose, lactate, and pyruvate detection kit, and also detected protein expression using western blotting. In addition, to investigate the effects of emodin and AZD3965 on lung cancer in vivo, we constructed nude mice subcutaneous transplant tumor model by subcutaneous injection of lung cancer cells. The results showed that emodin and AZD3965 could inhibit the proliferation, migration, and invasion of lung cancer cells. At the same time, they could inhibit the expression of MCT1 in lung cancer cells and promote the release of lactate, but did not affect the content of glucose and pyruvate. In vivo experiments had shown that emodin and AZD3965 could effectively inhibit the growth of lung cancer and inhibit the expression of MCT1. All in all, our data suggested that emodin inhibited the proliferation, migration, and invasion of lung cancer cells, possibly by inhibiting MCT1, providing important theoretical basis for elucidating the mechanism of emodin in treating lung cancer.

Rapid and Efficient Dual Detection Of Zn2+ Ions and Oxytetracycline Hydrochloride Using a Responsive Fluorescent "On-Off" Sensor Based on Simple Salen-Type Schiff Base Ligand.

This research has progressed to an effective detection chemosensor of zinc, aluminum ions and oxytetracycline hydrochloride antibiotic based on the fluorescence technique. A straightforward method utilizing microwave irradiation was employed to synthesize the salen-type Schiff base ligand N,N'-bis(salicylaldehyde)4,5-dichloro-1,2-phenylenediamine (H2I), providing a good 70 % yield. In ethanol, the H2I sensor demonstrated remarkable rapidity, selectivity, and sensitivity in detecting zinc ions. The fluorescence spectrum exhibited a 44-fold substantial enhancement at 522 nm and achieved a low limit of detection (LOD) of 1.47 μM. Furthermore, the H2I probe's emission intensity increased by 124 times when compared to the ligand's ability to detect Al3+ ions at 494 nm with a LOD value of 7.4 μM. Additional research was done using the H2I probe's effective Zn2+ detection capability. The ability to recognize zinc ions in different real water samples demonstrated a recovery rate of 98.67 % to 103.31 %. Interestingly, a naked-eye visible fluorescence color of H2I solution impregnated filter papers turned colorless into yell ow under UV irradiation by adding Zn2+ ions, renders it suitable for developing a practical zinc ion detection kit test. In particular, the I-Zn2+ complex effectively quenched the fluorescence toward oxytetracycline hydrochloride (OTC) with a LOD value of 1.49×10-2 μM in DMSO:H2O (6 : 4, v/v). This is a novel and effective procedure for sensing OTC antibiotic by the I-Zn2+ complex. These findings hold immense potential for the development of dual fluorescent probes, thereby enhancing sensitivity and specificity in identifying metal ions and antibiotics in a wide range of applications.

Comparative evaluation of DNA synthesis for qPCR analysis from oral squamous cell carcinoma tissues - A rapid and robust isolation technique for gene expression studies

BackgroundDNA isolation from biological materials is the initial step in several bioanalytical processes, including the polymerase chain reaction (PCR). This procedure works best with pure DNA devoid of potential amplification reaction inhibitors. Since the quantity and quality of biological samples are limited, it is essential to extract as much DNA as possible from the original sample. The solid-phase extraction technique is frequently used to purify DNA. In this process, the DNA is adsorbed onto a solid support, any contaminants from the reaction are eliminated by washing, and the purified DNA is then eluted from the support. The quality and concentration of DNA have a direct effect on the gene analysis procedure. Therefore, before interpreting the results of PCR-based diagnostic assays, it is imperative to confirm the DNA preparation and integrity, particularly if no pathogenic DNA is identified. MethodsA comparative study was carried out using two standardized protocols for DNA synthesis in comparison with a test protocol incorporating carrier RNA and proteinase K from a viral detection kit. The quality and quantity of the synthesized DNA are assessed by qPCR analysis for gene amplification of metastasis suppressor genes NDRG1, RhoGDI, and KISS 1. Results and conclusionThe test protocol showed higher yields of DNA in comparison to the standard protocol. The concentration of DNA obtained was validated by the concentration of gene expressed from q PCR analysis. The gene expression was significantly higher when the test protocol method was followed for DNA synthesis. Thus the study validates the potential of nucleic acid carrier RNA molecules to improve DNA extraction processes used in tissue samples for gene analysis procedures.

Modified perylene diimide for femto molar level detection of glucose: smartphone-assisted colorimetric glucose detection kits.

In this report, a functionalized hydroxyphenyl benzothiazole (HBT) derivative has been synthesized and anchored onto the perylene diimide (PDI) core at the -bay position (PHI). PHI has been explored for the generation of radical anions (PH1˙-) and dianions (PH12-) in 20% HEPES buffer-DMSO solution using H2S as a sacrificial electron donor. The PH1˙- has a half-life (t1/2) of 1.5 h and 3 h in oxygenated and hypoxic conditions, respectively. The formation of radical anions has been confirmed by optical (absorbance and fluorescence) methods, cyclic voltammetry (CV), differential pulse voltammetry (DPV), and femtosecond transient absorbance spectroscopy along with current-voltage (I-V) and NOBF4 studies. The PH1˙- showed peroxidase-like activity for the reduction of H2O2 as low as 170 fmol L-1 (fM) giving a colour change from sea green to pink. The biochemical assay which consists of PH1˙-+ GOx has been further utilized as a glucose sensor. Upon addition of glucose (0-8 nM) in the biochemical assay, the in-situ produced H2O2 (after oxidation of glucose with GOx) oxidized PH1˙- to PH1 giving a sea green to pink colorimetric read out along with a decrease in the absorption intensities at 720, 815, 880 and 950 nm and the emergence of absorption intensity at 541 nm. The lowest limit of detection is 85 fM. We also explored this biochemical assay for the detection of 860 fM of glucose in a 10% blood serum. Similarly, fluorometric, CV and DPV studies were carried out for the detection of glucose using this biochemical assay. The smartphone-assisted RGB colour analyser showed large variations in the red colour and this RGB based colour differentiation can be used for the detection of 1 nM of glucose.

Assessment of Nine Real-Time PCR Kits for African Swine Fever Virus Approved in Republic of Korea.

The African swine fever virus (ASFV) causes severe disease in wild and domestic pigs, with high mortality rates, extensive spread, and significant economic losses globally. Despite ongoing efforts, an effective vaccine remains elusive. Therefore, effective diagnostic methods are needed to rapidly detect and prevent the further spread of ASF. This study assessed nine commercial kits based on real-time polymerase chain reaction (PCR) approved in the Republic of Korea using the synthesized ASFV plasmid, 20 food waste samples, and artificially spiked samples (ASSs). The kits were evaluated for their diagnostic sensitivity, specificity, cost per reaction, and reaction running time. In addition, the results were compared with those of the World Organization for Animal Health (WOAH) standard methods. Three commercial kits (VDx® ASFV qPCR Kit, Palm PCR™ ASFV Fast PCR Kit, and PowerChek™ ASFV Real-time PCR Detection Kit Ver.1.0) demonstrated the highest sensitivity (100 ag/μL), cost-effectiveness (less than KRW 10,000), and shortest running time (less than 70 min). These kits are suitable for the monitoring, early diagnosis, and prevention of the spread of ASF. This is the first report on the performance comparison of ASFV diagnostic kits approved in the Republic of Korea, providing valuable information for selecting kits for testing with food waste samples.