Enzyme-linked Immunoassay Research Articles

Gold-grafted melamine sponge as surface-enhanced Raman spectroscopy substrate for enzyme-linked immunoassay of mycotoxins in cereal samples.

Gold-grafted melamine sponge (AuSp) was fabricated and applied as surface-enhanced Raman spectroscopy (SERS) substrate for enzyme-linked immunoassay of 2 typical mycotoxins, zearalenone (ZEN) and T-2 toxin. The gold nanoparticles were in situ grown on the sponge, and the product showed good effect in enhancing Raman signal and good stability. COMSOL and Gaussian simulations were used to analyze the enhancing effect. Some of the experimental conditions that may affect the determination results were studied in order to meet the maximum response to mycotoxins. At optimized conditions, the limits of detection of the method were 1μg·kg-1 and 0.05μg·kg-1 for ZEN and T-2 toxin, with linear ranges of 5-100μg·kg-1 and 0.1-20μg·kg-1, respectively, and relative standard deviations lower than 7.0%. The established method was used to determine ZEN and T-2 toxin in six kinds of cereal samples, the recoveries were 85-120%, and relative errors were less than 13.0%. These results indicated the accuracy of the established method. After 1-year storage, signal strength of AuSp for mycotoxins determination decreased by less than 10%, showing stability over a long time. Our work provided a new method for SERS determination of mycotoxins, which reduces the complexity of substrate preparation and the sample consumption compared with traditional methods, and thus has potential for on-line SERS determination.

Epidemiological Study of Respiratory Virus Infections in Pediatric Patients with Systemic Inflammatory Response Syndrome

Background: Respiratory infections are a common cause of systemic inflammatory response syndrome (SIRS) in pediatric patients. However, literature specifically addressing respiratory viruses that lead to SIRS or sepsis in children is limited, primarily consisting of case reports and lacking data for more comprehensive conclusions. Objectives: This study aims to investigate the relationship between respiratory viral infections and SIRS in children, focusing on virus detection rates, patient profiles, and the incidence of septic shock associated with different respiratory viruses. Methods: A retrospective analysis was conducted from January 2022 to December 2023 on hospitalized children diagnosed with respiratory infections and SIRS. Respiratory viruses were detected through qualitative PCR testing, and procalcitonin levels were measured using an enzyme-linked fluorescent immunoassay. Results: A total of 931 pediatric patients with SIRS were tested for respiratory viruses and/or other pathogens. Among these, 298 (32.0%) were positive for respiratory viruses, with 263 (28.2%) cases attributed solely to respiratory viruses. Human rhinovirus (HRV) had the highest infection rate at 37.3% (98/263), followed by human adenovirus (HAdV) at 24.3% (64/263). Seasonal distribution among the 263 SIRS cases associated with respiratory viral infections showed the highest incidence in spring (39.5%), followed by summer (25.5%), winter (17.9%), and autumn (17.1%). Septic shock rates were highest among cases of influenza A) FluA(, human metapneumovirus (hMP), and co-infections, with incidences of 27.8%, 18.2%, and 22.7%, respectively. There was significant variation in septic shock rates across different viral infections in pediatric patients (P < 0.001). Conclusions: Many hospitalized children with respiratory viral infections develop SIRS, and a subset of these cases can progress to septic shock.

Assessment of Sex Hormones (FSH, LH, Prolactin, Estrogen, Progesterone and Testosterone) of Pulmonary Tuberculosis Patients

Background: Tuberculosis (TB) is a one of the major global health problems ranking as the eighth leading cause of death in low- and middle-income countries. This study was carried out to evaluate the Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH), Prolactin, Estrogen, Progesterone and Testosterone of pulmonary tuberculosis patients in Edo State. Objectives: The objectives of the study are to assess the FSH, LH, Prolactin, Estrogen, Progesterone and Testosterone levels of Pulmonary Tuberculosis (PTB) patients compared with control, hormonal levels of PTB patients of new case, 2 months and 6 months therapy compared with control, hormonal levels of PTB patients in relation to gender and hormonal levels of PTB patients in relation to age. Material and methods: A total of 120 samples were used in this study comprising PTB new cases (30), PTB 2 months therapy (30), PTB 6 months therapy (30) and Control (30) respectively. FSH, LH, Prolactin, Estrogen, Progesterone and Testosterone were measured by Enzyme-Linked Immunoassay Test (ELISA). The results were presented using tables as mean ± standard deviation. Statistical analysis was done using one-way Analysis of Variance (ANOVA) and the student’s t-test. Significant difference was accepted at p<0.05. Results: The results obtained showed that the FSH (mIU/ml) of new case subjects, 2 months on therapy, 6 months on therapy and control were 13.42±3.58, 11.38±3.04, 6.52±2.57 and 8.71±3.15; LH (mIU/ml) was 10.43±2.95, 8.32±2.44, 5.21±2.23 and 6.05±2.44; prolactin (µg/L) was 10.17±4.04, 9.47±2.56, 10.11±6.74 and 12.96±7.09; estrogen (pg/ml) was 35.97±9.27, 41.50±12.65, 57.60±17.46 and 64.97±29.24, progesterone (ng/ml) was 0.18±0.05, 0.25±0.06, 0.40±0.10 and 0.37±0.08; and testosterone (ng/ml) was 2.49±0.95, 2.48±1.46, 4.00±2.92 and 4.32±3.84 respectively. Conclusion: In conclusion, there was significant difference (p<0.05) in the FSH, LH, Prolactin, Estrogen, Progesterone and Testosterone of the subjects in the different groups compared with control. FSH, LH, Prolactin, Estrogen and Progesterone were significantly higher (p<0.05) in female subjects compared with male subjects, while Testosterone was non-significantly (p>0.05) higher in male subjects compared with female subjects.

20-Hydroxyeicosatetraenoic Acid Regulates the Src/EGFR/NF-κB Signaling Pathway Via GPR75 to Activate Microglia and Promote TBI in the Immature Brain.

20-Hydroxyeicosatetraenoic acid (20-HETE) is associated with secondary damage in traumatic brain injury (TBI) of the immature brain. Microglial activation is pivotal in this process. However, the underlying mechanism of action remains unknown. While 20-HETE interacts with G protein-coupled receptor 75 (GPR75) in some pathological processes, their interaction in brain tissue remains uncertain. This study aimed to investigate whether 20-HETE can activate microglia by binding to GPR75 in TBI of the immature brain. Drug affinity responsive molecular target stability (DARTS) assays, cycloheximide (CHX) chase assays, and auto-dock assays were employed to analyze the interaction between 20-HETE and GPR75. The expression levels of cytochrome P450 4A (CYP4A) and GPR75 in activated microglia in an immature brain TBI model were observed by western blot and multiple immunofluorescence staining. The effects of different levels of 20-HETE expression and lentivirus-mediated GPR75 gene silencing on 20-HETE-induced inflammatory factor release from BV-2 cells were observed by enzyme-linked immunoassay (ELISA). The phosphorylation levels of the downstream Src kinase, epidermal growth factor receptor (EGFR), and nuclear factor (NF)-κB were assessed using western blot. Cell viability and apoptosis were detected by CCK-8 and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays. 20-HETE bound to the GPR75 protein and inhibited its degradation. GPR75 gene silencing reversed the 20-HETE-induced inflammatory activation of BV-2 cells, effectively inhibiting the activation of the Src/EGFR/NF-κB pathway and the effects of 20-HETE on cell viability and the apoptosis rate. In contrast, overexpression of GPR75 had the opposite effect. In addition, after immature brain TBI, the 20-HETE and GPR75 expression levels were upregulated in microglia, with significant activation of the Src/EGFR/NF-κB pathway. Inhibition of 20-HETE synthesis with N-hydroxy-N'-(4-n-butyl-2-methylphenyl) formamidine (HET0016) produced the opposite effect. 20-HETE regulates the Src/EGFR/NF-κB signaling pathway via GPR75 to activate microglia, promoting immature brain TBI. These findings offer a novel target for promoting the brain injury effect of 20-HETE.

Abstract 4134426: Targeting the TRIM29-PERK signaling axis for treating viral myocarditis

Introduction: Viral myocarditis is an inflammatory disease of the myocardium that significantly contributes to sudden death in children and young adults. The COVID-19 pandemic underscores the critical need to understand the pathogenesis and develop effective treatment strategies for viral myocarditis. Methods: To investigate the role of the novel E3 ligase TRIM29 in viral myocarditis, we used wild-type and TRIM29 knockout mice infected with coxsackievirus B3 (CVB3) and encephalomyocarditis virus (EMCV), to induce the myocarditis. Additionally, wild-type mice infected with CVB3 were treated with the protein kinase RNA-like endoplasmic reticulum kinase (PERK) inhibitor GSK2656157 or a DMSO control to evaluate potential therapeutic interventions. Mice survival and heart function were monitored using transthoracic echocardiography, and hearts were harvested for histological and immunohistochemical analysis. Techniques including real-time PCR, western blotting, co-immunoprecipitation, enzyme-linked immunoassay, flow cytometry, over-expression, and knockdown were employed to elucidate the pathogenic mechanisms by which TRIM29 regulates the endoplasmic reticulum stress response after viral infection. Results: We found that TRIM29 was highly induced by cardiotropic viruses, including CVB3 and EMCV, and promoted PERK-mediated endoplasmic reticulum (ER) stress, apoptosis, and reactive oxygen species (ROS) responses, which restricted viral replication in cardiomyocytes in vitro . TRIM29 deficiency protected mice from viral myocarditis by enhancing cardiac antiviral functions and reducing PERK-mediated inflammation and immunosuppressive cells in vivo . Mechanistically, TRIM29 interacted with PERK to catalyze the SUMOylation of PERK, maintaining its stability and thereby promoting PERK-mediated signaling pathways. Furthermore, we demonstrated that the PERK inhibitor GSK2656157 mitigated viral myocarditis by disrupting the TRIM29-PERK interaction, which improved cardiac function, enhanced cardiac antiviral responses, and reduced inflammation and immunosuppressive cells in vivo . Conclusions: Our findings provide the first evidence that the TRIM29-PERK signaling axis can be targeted for the treatment of viral myocarditis. This suggests that targeting the TRIM29-PERK axis could effectively mitigate viral myocarditis and associated cardiovascular diseases.

Abstract 4114198: Humanin Level is Inversely Correlated with NT-proBNP Level and Mortality in Heart Failure with Reduced Ejection Fraction

Background: Mitochondrial dysfunction contributes to heart failure (HF) progression via diminished energy supply, release of reactive oxygen species, and increased cell death. However, there has yet to be clinically relevant biomarkers that address mitochondrial-mediated cardiac injury in patients. Humanin (HN) is a mitochondria-derived peptide that protects cells through the interference of apoptosis and reduction of oxidative stress. The objectives of our study were to measure circulating plasma HN levels in patients with HF with reduced ejection fraction and describe the relationship of HN to validated, clinically relevant measures of HF. Methods: We conducted a prospective cohort study. To be included in the study, patients must have: 1) left ventricular ejection fraction <40%, 2) high risk of future clinical instability, 3) medication and device treatment according to guidelines, and 4) stable medical therapy for at least 30 days prior to screening. Plasma HN levels were measured at time of study enrollment by an enzyme-linked immunoassay. Statistical analyses were performed to evaluate the relationships between HN levels and demographic variables, quantitative measures of HF, and clinical outcomes. Results: 54 HF patients were included in the study. Age and sex were not significantly associated with HN level. There was a significant difference in mean HN level by race with highest HN level seen in Caucasians and the lowest HN level seen in Hispanics (1310 pg/ml vs. 630 pg/ml, p=0.012). There was a significant inverse correlation between NT-proBNP level and HN level (r=-0.46, p <0.001). After adjusting for race, higher NT-proBNP level, as both a continuous and categorical variable, was significantly associated with lower HN level. Furthermore, HN level was significantly lower in patients who died within 6 months compared to those who did not (520.7 pg/ml vs. 1146.9 pg/ml, p=0.013). After adjusting for race, lower HN level was significantly associated with increased odds of 6-month mortality (OR=0.784, 95% CI 0.620-0.943, p=0.018). Conclusions: To the best of our knowledge, this is the first study to describe plasma HN levels in HF patients. Its significant correlation with mortality and NT-proBNP level supports a potential role for HN as a novel prognostic biomarker in HF with reduced ejection fraction.

Anti-Müllerian hormone as a predictor of oocyte yield following controlled ovarian stimulation in the rhesus macaque.

Anti-Müllerian hormone (AMH) is widely used in the clinic as a biomarker for ovarian reserve and to predict ovarian response to gonadotropin stimulation. Patients with higher AMH levels tend to yield more oocytes and have better outcomes from assisted reproductive technology (ART) procedures. The goal of this study is to determine if AMH can be used to predict the outcome of controlled ovarian stimulation in rhesus macaques, which are commonly used in biomedical research, to refine animal use while maximizing oocyte yield. We hypothesized that pre-stimulation AMH values can be used to predict oocyte yield and quality. Regularly cycling adult macaques underwent controlled ovarian stimulation and baseline (pre-stimulation) plasma AMH levels were determined using an AMH-specific enzyme-linked immunoassay. Oocytes were collected by laparoscopic or ultrasound-guided aspiration, then counted and evaluated for quality and stage of meiosis. Sperm from established fertile males were used to inseminate the oocytes in vitro with fertilization success checked 14 - 16 hours later. Females were grouped by oocyte yield: low ≤ 17; mid = 18 - 41; high ≥ 42. We found that high and mid yielders had significantly higher AMH than low yielders (p<0.0001) and the percent of mature oocytes was greater in the high and mid yielders. There were no significant differences in oocyte quality or ova fertilization rate. These data suggest that AMH is a useful measure for controlled ovarian stimulation success in rhesus macaques and can be used to identify suitable animals for oocyte donation before entering them into a stimulation protocol.

Correlation of Eight (8) Polymorphisms and Their Genotypes with the Risk Factors of Cardiovascular Disease in a Black Elderly Population.

Single nucleotide polymorphisms (SNPs) have been associated with the development of cardiovascular diseases (CVDs). This study correlated eight SNPs with the risk factors of CVD in a black elderly population. Genotyping was used to detect eight polymorphisms; rs675 (ApoA-IV), rs699 (Angiotensinogen (AGT)), rs247616 and rs1968905 (Cholesteryl ester transfer protein (CETP)), rs1801278 (Insulin receptor substrate 1 (IRS-1)), rs1805087 (Methylenetetrahydrofolate reductase (MTHFR)) and rs28362286 and rs67608943 (Proprotein convertase subtilisin/kexin type 9 (PCSK9)), as well as their genotypes in deoxyribonucleic acid (DNA) extracted from peripheral blood. The cardiovascular risk (CVR) measurements were conducted on a Konelab 20i Thermo Scientific autoanalyzer and an enzyme-linked immunoassay (ELISA) assay. International Business Machines Corporation (IBM)® Statistical Package for the Social Sciences ® (SPSS) version 28 was used for statistical analysis. The heterozygous and homozygous genotypes of the eight polymorphisms were detected with the corresponding CVD risk factors. Subgroup analysis indicated that certain genotype carriers exhibited variations in their concentrations of CVR factors compared to others; however, these differences did not reach statistical significance. For example, carriers of the G genotype of the rs699 polymorphism showed marginally different blood pressure readings compared to the AG genotype carriers. The multiple linear regression analysis indicated that the only significant association was between PCSK9 and the rs28362286 (p = 0.029) polymorphism. The findings of our study show that single nucleotide polymorphisms are disseminated across the human genome. The heterozygous and homozygous genotypes of the SNPs require further investigation as they may have independent and possible collective roles in increasing the risk of CVDs.

Compact Digital Immunoassay Platform Integrating ELISA with a Lateral Flow Strip.

On-site diagnosis of infection in their early stages requires assays with high sensitivities that are compact and easy to operate out of the laboratory and hospital environments. However, current assay technologies fall short of these requirements and require highly skilled technicians to set up, operate, and interpret the results. To address these challenges, we developed and evaluated a Point-of-Care-Testing (PoCT) immunoassay platform called the D-strip. The D-strip platform combines the capabilities of a digital enzyme-linked immunoassay (ELISA) with a lateral flow assay (LFA). The D-strip sample flow cell is composed of the same components found in conventional LFAs, and its high sensitivity is due to its efficient implementation of ELISA. The fully integrated platform is simple and requires minimal user intervention to operate. The D-strip exhibited a sample-to-result time of 15 min with a limit of detection (LOD) of 1.7 × 103 copies/mL for severe acute respiratory syndrome coronavirus 2 (SARS-2-CoV) antigen. The LOD of the D-strip is 488-fold higher than that for conventional LFAs and is comparable to a clinical laboratory test. The D-strip is a compact and highly sensitive immunoassay platform with a strong potential for application as a confirmatory assay outside the clinical laboratory.

Gain-of-Function Variant in Spleen Tyrosine Kinase Regulates Macrophage Migration and Functions to Promote Intestinal Inflammation.

Spleen tyrosine kinase (Syk) is a widely-expressed cytoplasmic non-receptor tyrosine kinase involved in regulating various signaling pathways and plays an important role in chronic inflammation and autoimmune diseases. Gain-of-function SYK variants have been implicated in pediatric inflammatory bowel diseases. This study aimed to investigate the effects of gain-of-function SYK variants on the susceptibility to experimental colitis and macrophage function. Colitis was induced using dextran sodium sulfate and dinitrobenzene sulfonic acid in mice harboring a gain-of-function variant in SYK (SykS544Y). Intestinal inflammation was assessed via disease activity index, histological analysis, and Western blotting. The frequencies of macrophages, phagocytosis, and reactive oxygen species (ROS) production in bone marrow-derived macrophages (BMDM) were measured via flow cytometry. Chemokines and BMDM chemotaxis were analyzed using real-time quantitative reverse transcription polymerase chain reaction and Transwell assays. The expression of nucleotide-binding domain leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome-related proteins were detected using immunohistochemistry, enzyme-linked immunoassay and Western blotting. SykS544Y mice exhibited increased susceptibility to experimental colitis, and macrophage infiltration in colon tissues significantly increased. We observed increased expression of macrophage chemokines in colon tissues and enhanced chemotaxis in SykS544Y BMDM. Additionally, we detected increased levels of fluorescent microspheres and 2.7-dichloride-hydro fluorescein diacetate-labeled ROS in SykS544Y BMDM. Moreover, enhanced levels of NLRP3 inflammasome-related proteins were observed in the colon tissues and BMDM from SykS544Y mice. Gain-of-function variant in SYK may contribute to the pathogenesis of pediatric inflammatory bowel diseases by promoting macrophage migration, phagocytosis, ROS production and activation of NLRP3 inflammasomes.

In situ construction of oriented Au/PPy nanorod arrays-based aptasensor for fast and ultrasensitive determination of thrombin

The dynamic changes in coagulation function indicators provide important references for early warning, disease progression, and prognosis evaluation of clinical cardio-cerebrovascular disease (CCVD) patients. However, current assay methods, such as enzyme-linked immunosorbent assay (ELISA), fluorescence immunoassay and colorimetry, are time-consuming, require bulky equipment, tedious operations, and long analytical period, which are difficult in achieving an on-site and rapid detection. To address above issues, we proposed an ultrasensitive and fast-responded aptasensor for the detection of a typical coagulation function indicator (Thrombin, Tob) through constructing a unique oriented Au/polypyrrole (PPy) nanorod arrays. This architecture has provided both high electrocatalysis and abundant active sites as the signal transfer and DNA carrier. Meanwhile, through the specific DNA-binding representative design, we also designed a target-induced chain release strategy based on the competitive interaction between Tob and cDNA, which remarkably accelerated the Tob recognition process. This aptasensor enables to present an ultralow detection limit of 3 fg/mL and fast detection time of 20 min in real human serum samples, together with the favorable repeatability, stability and anti-interference ability for early warning and prognosis prediction of CCVD. It is promising that this aptasensor can be extended to analyze more disease markers through the change of different bases sequence of the applied DNA stands, which will provide an on-site and convenient technique and device for clinical disease diagnosis.

Histomorphometric changes in pituitary gonadotropic endocrinocytes when exposed to dark deprivation

Aim. To assess the effect of 30-day dark deprivation on functional and histomorphometric changes in adenohypophysis gonadotropic endocrinocytes and their reversibility in mature male rats.Materials and methods. Mongrel white male rats (n = 36) weighing 365–375 g at 4 months of age were randomly divided into three groups (each n = 12). For 30 days the control group was in automatic light-dark mode 12/12, and the rats of experimental groups 1 and 2 were in round-the-clock artificial lighting (24/0, 300 Lux), then the rats of group 2 were returned to 12/12 mode for the next 14 days. In the animals of the control and group 1 during their lifetime on the 31st day, and in group 2 on the 45th day, blood was taken from the abdominal aorta and levels of follicle-stimulating (FSH) and luteinizing (LH) hormones, melatonin, and Klotho protein were determined an enzyme-linked immunosorbent assay and immunoassay and after which they were removed from the experiment by decapitation. Postmortem histological and immunohistochemical examination of the pituitary gland was done using rabbit polyclonal antibodies targeting caspase-3 and Klotho protein, as well as morphometry. Statistical data processing was performed using the Kruskal-Wallis test with post-hoc Dunn’s test.Results. Light desynchronization in the form of 30 days of dark deprivation increased FSH and LH levels and decreased melatonin and Klotho protein levels in the blood of male rats; increased gonadotropic endocrine cell area, volume, and perimeter by 23.1% (p &lt; 0.001), 48.7% (p &lt; 0.001), and 10.9% (p &lt; 0.001), respectively; and increased nucleus area, volume, and perimeter by 16%, 11.7%, and 2.5%, respectively. An immunohistochemical study showed an increase in the specific area of caspase-3-immunoreactive gonadotropic endocrinocytes by 25.2% without obvious morphological signs of apoptosis, and a decrease in the expression of Klotho protein by 25.7%. All indicators were reversible, the levels of FSH and Klotho protein in the blood of animals almost reached their initial values after 14 days of restoration of the light-dark cycle 12/12.Conclusion. Dark deprivation for 30 days in male rats induced reversible processes of accelerated aging and apoptosis in cells, as evidenced by changes in the expression of aging markers in gonadotropic endocrinocytes and levels of gonadotropic hormones in the blood. When the light-dark mode is restored, the levels of FSH and Klotho protein normalize as early as 14 days.

Measurement of α-synuclein as protein cargo in plasma extracellular vesicles

Extracellular vesicles (EVs) are released by all cells and hold great promise as a class of biomarkers. This promise has led to increased interest in measuring EV proteins from both total EVs as well as brain-derived EVs in plasma. However, measuring cargo proteins in EVs has been challenging because EVs are present at low levels, and EV isolation methods are imperfect at separating EVs from free proteins. Thus, knowing whether a protein measured after EV isolation is truly inside EVs is difficult. In this study, we developed methods to measure whether a protein is inside EVs and quantify the ratio of a protein in EVs relative to total plasma. To achieve this, we combined a high-yield size-exclusion chromatography protocol with an optimized protease protection assay and Single Molecule Array (Simoa) digital enzyme-linked immunoassays (ELISAs) for ultrasensitive measurement of proteins inside EVs. We applied these methods to analyze α-synuclein and confirmed that a small fraction of the total plasma α-synuclein is inside EVs. Additionally, we developed a highly sensitive Simoa assay for phosphorylated α-synuclein (phosphorylated at the Ser129 residue). We found enrichment in the phosphorylated α-synuclein to total α-synuclein ratio inside EVs relative to outside EVs. Finally, we applied the methods we developed to measure total and phosphorylated α-synuclein inside EVs from Parkinson's disease and Lewy body dementia patient samples. This work provides a framework for determining the levels of proteins in EVs and represents an important step in the development of EV diagnostics for diseases of the brain, as well as other organs.

Brain-Derived Neurotrophic Factor (BDNF) is Associated with Self-Reported Cognitive Function in Adults with HIV.

Background: People with HIV (PWH) are at risk of developing HIV-associated neurocognitive disorder (HAND) despite receiving combination antiretroviral therapy. Brain-derived neurotrophic factor (BDNF) has been implicated in cognitive function and neuroplasticity, but its role in HIV-related neuroinflammation remains understudied. Methods: This study analyzed data from the CHAMPS study, assessing BDNF serum levels and cognitive function in 140 adults with HIV at baseline. Cognitive function was evaluated using the PROMIS Applied Cognition-Abilities 8-item questionnaire. BDNF levels (pg/ml) were measured using high sensitivity Enzyme-Linked Immunoassay (ELISA) kits. Linear regression analyses were conducted to explore the associations between BDNF levels, cognitive function, and AIDS diagnosis, adjusting for demographic variables. Results: A significant positive association was found between BDNF levels and cognitive function scores in PWH (p = .03). Additionally, PWH with a history of AIDS diagnosis showed significantly lower BDNF levels (p = .02). Other demographic factors did not significantly impact cognitive function or BDNF levels in this cohort. Conclusions: Our results highlight the potential of BDNF as a biomarker for cognitive decline in PWH and suggest its relevance in understanding HAND pathophysiology. Further research is warranted to explore the multifaceted interactions influencing cognitive outcomes in this population and to develop targeted interventions for improving cognitive health in PWH.

Immunogenicity and Efficacy of Combined mRNA Vaccine Against Influenza and SARS-CoV-2 in Mice Animal Models.

Background. The combined or multivalent vaccines are actively used in pediatric practice and offer a series of advantages, including a reduced number of injections and visits to the doctor, simplicity of the vaccination schedule and minimization of side effects, easier vaccine monitoring and storage, and lower vaccination costs. The practice of widespread use of the combined vaccines has shown the potential to increase vaccination coverage against single infections. The mRNA platform has been shown to be effective against the COVID-19 pandemic and enables the development of combined vaccines. There are currently no mRNA-based combined vaccines approved for use in humans. Some studies have shown that different mRNA components in a vaccine can interact to increase or decrease the immunogenicity and efficacy of the combined vaccine. Objectives. In the present study, we investigated the possibility of combining the mRNA vaccines, encoding seasonal influenza and SARS-CoV-2 antigens. In our previous works, both vaccine candidates have shown excellent immunogenicity and efficacy profiles in mice. Methods. The mRNA-LNPs were prepared by microfluidic mixing, immunogenicity in mice was assessed by hemagglutination inhibition assay, enzyme-linked immunoassay and virus neutralization assay. Immunological efficacy was assessed in a mouse viral challenge model. Results. In this work, we demonstrated that the individual mRNA components of the combined vaccine did not affect the immunogenicity level of each other. The combined vaccine demonstrated excellent protective efficacy, providing a 100% survival rate when mice were infected with the H1N1 influenza virus and reducing the viral load in the lungs. Four days after the challenge with SARS-CoV-2 EG.5.1.1., no viable virus and low levels of detectable viral RNA were observed in the lungs of vaccinated mice. Conclusions. The combination does not lead to mutual interference between the individual vaccines. We believe that such a combined mRNA-based vaccine could be a good alternative to separated human vaccinations for the prevention of COVID-19 and influenza.

Identification of new targets for the diagnosis of cysts (four) and trophozoites (one) of the eye pathogen Acanthamoeba.

Free-living amoeba in the soil or water cause Acanthamoeba keratitis, which is diagnosed by identification of cysts by microscopy of the eye or of corneal scrapings, using calcofluor-white that unfortunately cross-reacts with fungi and plants. Alternatively, Acanthamoeba infections are diagnosed by identification of trophozoites in cultures of scrapings. Here we showed that rabbit antibodies to four abundant cyst wall proteins (Jonah, Luke, Leo, and laccase) each efficiently detect calcofluor-white-labeled cysts of 10 of 11 Acanthamoeba isolates obtained from the ATCC. Further, laccase released into the medium by encysting Acanthamoebae was detected by an enzyme-linked immunoassay. We also showed that rabbit antibodies to the mannose-binding domain of the Acanthamoeba mannose-binding protein, which mediates adherence of trophozoites to keratinocytes, efficiently identifies trophozoites of all 11 ATCC isolates. In summary, four wall proteins and the ManBD appear to be excellent targets for diagnosis of Acanthamoeba cysts and trophozoites, respectively.

Comparative assessment of neurotrophic proteins in vibration disease

Introduction. The problem of health impairment due to the impact of physical factors, including vibration disease (VD), retains medical and social relevance due to the significant specific gravity in the structure of occupational morbidity and high prevalence. Under vibration exposure, the acid-base equilibrium, immune status are disturbed, rheological properties and properties of the neurohumoral system, as well as functional indices of the central and peripheral nervous system change. Neurotrophins are known to play an important role in regulating the development and functioning of the central and peripheral nervous systems. In this regard, research is needed to determine in VD patients the levels of neurospecific proteins, which may serve as one of the markers for tracing changes in the nervous system in workers. The aim of the study was to identify the peculiarities of changes in neurospecific proteins (BDNF, S100β, and MBP) in the serum of patients with VD of different genesis. Materials and methods. Serum concentrations of neurotrophic proteins: brain neurotrophic factor – BDNF, S100β protein and myelin basic protein (MBP) were studied by solid-phase enzyme-linked immunoassay. Results. There was an increase in serum BDNF, S100β protein, and MBP concentrations in persons with VD due to combined exposure to local and general vibration, and an increase in MBP was observed in patients with VD due to exposure to local vibration. Limitations. The limitations of this work are small groups of employees. Conclusion. As a result of studies, it can be assumed that in VD due to exposure to combined vibration, changes are observed in the peripheral and central parts of the nervous system, and in VD due to local vibration, disorders seem to be mainly associated with the peripheral nervous system. The general pattern of the revealed disorders in the content of MBP lies in the neurodestructive processes occurring in the nerve fibers in patients with VD, regardless of genesis. Summarizing the above mentioned, the validity of studying the concentration of neurospecific proteins in vibration disease occurring with damaging processes in the nervous tissue should be recognized.

Therapeutic Effects of Proanthocyanidins on Diabetic Erectile Dysfunction in Rats.

The rising occurrence of erectile dysfunction related to diabetes mellitus (DMED) has led to the creation of new medications. Proanthocyanidins (PROs) is a potential agent for DMED. In this study, the DMED rat model was established using streptozotocin (STZ) and erectile function was assessed using apomorphine (APO) in rats. Following this, the rats were subjected to oral treatment with PRO. Then, we evaluated the influence of PROs on DMED rats. The findings suggest that PROs significantly enhance erectile function in DMED rats. PROs modulated glucose and lipid metabolism in DMED rats by decreasing blood glucose and lipid levels while increasing liver glycogen and serum insulin levels. Furthermore, PROs enhanced vascular endothelial function in DMED rats by augmenting nitric oxide (NO) levels and reducing the levels of endothelin-1 (ET-1) and lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1). Additionally, PROs have been shown to elevate testosterone (T) levels, mitigate pathological testicular damage, and enhance sperm concentration and survival rates. Finally, the core targets were screened using network pharmacology, followed by validation through molecular docking, enzyme-linked immunoassay (ELISA), and real-time PCR methodologies. Our findings imply that PROs may treat DMED by elevating AKT1 levels while concurrently diminishing CASP3 levels, thereby effectively regulating the PI3K-Akt signaling pathway. Overall, these results support using PROs as a potential candidate for the treatment of DMED.

Urinary cyclophilin A as an early marker of chronic kidney disease with underlying type 2 diabetes

Cyclophilin A (CypA) is a novel renal inflammation biomarker, with levels altered in various diseases, particularly in patients with diabetes mellitus (DM) and kidney damage. This study aimed to investigate the correlation between urinary cyclophilin A (uCypA) and chronic kidney disease (CKD) conditions with and without type 2 diabetes mellitus (T2DM) using an in-house enzyme-linked immunoassay (ELISA) method. A uCypA strip-test prototype was also developed. An indirect ELISA was performed to determine the uCypA levels. A 0.48 µg/mL uCypA cutoff differentiated healthy patients from those with early-stage CKD (stages I and II). The uCypA levels were significantly increased in patients with progression of renal deterioration, especially in the T2DM with late-stage CKD group, compared to the control group. Fasting blood sugar (FBS), estimated glomerular filtration rate (eGFR), albumin/creatinine ratio, and metformin use were associated with uCypA levels. Multinomial logistic regression analysis revealed an association between uCypA levels and T2DM diagnosed for over five years and early-stage CKD. This finding shows that uCypA could be used as a biomarker for distinguishing early-stage CKD as well as T2DM complications, which is beneficial for patients to be aware of their health status and change their behavior to slow kidney deterioration.

Smartphone-based dual-mode aptasensor with bifunctional metal-organic frameworks as signal probes for ochratoxin A detection

Ochratoxin A (OTA) poses significant risks to human health, being potentially nephrotoxic, carcinogenic, genotoxic, and immuno-toxic. In this work, we developed a dual-mode aptasensor for OTA analysis, integrating colorimetric, electrochemical, and smartphone-based detection. The bifunctional Fe-MIL-88 metal-organic framework, acting as both a nanozyme capable of catalyzing the 3,3′,5,5′-tetramethylbenzidine substrate and an electrochemical signal amplifier, enabled OTA quantification through current response or changes in color and absorbance intensity. Besides, the spatial confinement effect enhances the local concentration of Fe-MIL-88 signal probes through rolling circle amplification reaction, thereby contributing to a substantial enhancement in sensitivity. The proposed technique is simple, disposable, highly sensitive and selective, enabling OTA detection in the range of 1 fg/mL to 250 ng/mL, with a limit of detection of 0.22 fg/mL (3σ rule). Furthermore, we successfully detected OTA in corn, wheat, and red wine samples, with results good concordance with those obtained using commercial enzyme-linked immunoassay kits.