Enzyme-linked Immunosorbent Assay Method Research Articles

Extracorporeal Shock Wave Therapy as Regenerative Medicine in Knee Osteoarthritis: Pre-Experiment for Matrix Metalloproteinase-3 Evaluation.

Knee osteoarthritis (KOA) is currently treated by regenerative therapies that aim to inhibit arthritic degeneration. Extracorporeal shock wave therapy (ESWT) is one of the physical regenerative approaches used for KOA management. However, little is known regarding the impact of shock wave treatment on matrix metalloproteinase-3 (MMP-3), which is one of the enzymes mediating cartilage degradation. To evaluate the effect of ESWT on MMP-3 levels and pain intensity in patients with KOA. Fourteen patients diagnosed with Kellgren Lawrence, grades 2 and 3 KOA were recruited for the study. ESWT piezo shockwave was applied once a week for six weeks. MMP-3 levels in the blood were measured pre-test, mid-test (three weeks after therapy) and post-test (one week after the last session) by enzyme-linked immunosorbent assay (ELISA). The perceived pain was recorded at each session by the Wong Becker Face Scale. The median pre-test, mid-test and post-test MMP-3 levels were 19.92 ng/mL, 15.89 ng/mL and 18.82 ng/mL, respectively, and there were significant differences between the pre-test and mid-test, and the pre-test and post-test values (p < 0.05). The pain scores also decreased significantly over the period of intervention. MMP-3 levels decreased significantly in KOA patients after ESWT, and the decline was most obvious after 3 weeks of therapy. Therefore, EWST should be considered as a suitable treatment option for KOA.

Real and statistical processing of barley yield data naturally contaminated with fusariotoxin

Fumonisins are produced by fungi of the genus Fusarium spp., which are widely spread out in the nature. They are produced during grain growth, ripening and harvesting and are known as “field mycotoxins“. Fusarium species can produce compounds that are toxic to animals and humans. In this study, the development of Fusarium toxins was determined in healthy and mechanically damaged barley grains treated and untreated with fungicide, respectively, under natural contamination. It was found that healthy kernels treated with fungicide had the highest yield of 6700 kg.ha-1 followed by the yield of damaged kernels treated with fungicide. The lowest yield of 4850 kg.ha-1 was reported for the damaged and untreated barley grains. The two treatments whose plants were treated with fungicide had a higher yield than their counterparts, i.e. the plants not treated with fungicide. In confirmation of this by mycotoxicological analysis was established the presence of fumonisin 0.234 mg.kg-1 in healthy seeds untreated with fungicide and 0.274 mg.kg-1 in mechanically damaged untreated seeds. Fumonisin concentrations were measured by using Enzyme-Linked Immunosorbent Assay (ELISA) method. The used approach in this study shows that fungicide treatments effectively reduce mycotoxin levels in barley and increase grain yield. The use of fungicides should be combined with other infection mitigation strategies, such as avoiding mechanically damaged grains, which are a suitable substrate for mycotoxins production. These measures will help to improve food and feed safety.

Preconditioning Local Injection of Activated Platelet-Rich Plasma Increases Angiogenesis, VEGF Levels, and Viability of Modified McFarlane Flap in Diabetes-Induced Rats

Background The risk of flap necrosis in tissue reconstruction surgery is elevated in patients with vascular disorders, such as diabetes mellitus. Chronic hyperglycaemia causes endothelial cell dysfunction and increases inflammatory process, causing vascular insufficiency. Platelet-rich plasma (PRP) contains high levels of platelets, growth factors and fibrinogens. Its regenerative properties spark interest in supporting flap survival in relation to diabetic complications. Methods Thirty Wistar rats were divided into three groups. The first group included diabetic rats without PRP injection, which underwent flap procedure. The second group included diabetes-induced rats receiving PRP subcutaneous injection one day prior to flap procedure. The third group included non-diabetic rats receiving PRP injection one day prior to flap procedure. Flap tissue samples were taken on the seventh day to measure VEGF levels using enzyme-linked immunosorbent assay (ELISA) method; angiogenesis and collagen density were measured from histopathology examination, and flap viability was analysed using digital measurements. Results Analysis showed that flap viability, angiogenesis and VEGF levels were significantly higher in the PRP-injected diabetic rats compared to diabetic rats that did not receive PRP. The levels of VEGF, angiogenesis and viability of flaps in diabetic rats given PRP did not differ significantly compared to non-diabetic rats that received PRP. Conclusion Flap preconditioning through local injection of activated PRP enhances flap viability, VEGF levels and angiogenesis, in random skin flaps in diabetic rats, to the level where it does not differ significantly to non-diabetic rats that were given PRP. Keywords. Platelet-rich plasma, diabetes mellitus, VEGF, angiogenesis, random skin flap

Relationship Between Serum Brain-Derived Neurotrophic Factor and Neurotrophin-3 Levels and Hearing Thresholds in Age-Related Patients With Hearing Loss.

Age-related hearing loss (ARHL) is a general term used to describe the sensorineural type of hearing loss occurring in both ears in older adults. Neurotrophins are the most promising candidates for supporting the auditory nerve by increasing neuronal survival. This study aimed to help elucidate the pathophysiology of ARHL by determining whether any relationship exists between brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) levels in serum samples from patients diagnosed with ARHL. Seventy-seven individuals, a study group of 41 patients diagnosed with ARHL, and a control group of 36 participants without hearing loss were evaluated. Serum samples were collected and used to measure serum BDNF and NT-3 levels with the new Nepenthe enzyme-linked immunosorbent assay method. Median pure-tone average results in the 2000, 4000, and 6000 Hz ranges were 52.5 (44.3-67.3) dB HL in the ARHL group and 13.5 (11.1-17.1) dB HL in the control group. The difference was statistically significant (p = .001). Although NT-3 and BDNF levels were both lower in ARHL patients than in participants without hearing loss, only the BDNF levels were significantly (p = .002) lower. Mean left and right ear word recognition scores were also lower in ARHL patients than in control groups. The ARHL group was further divided into two subgroups based on word recognition scores to evaluate significant differences in BDNF and NT-3 levels. No statistically significant difference was observed in BDNF and NT-3 levels between these subgroups. However, there was a significant difference in word recognition scores. Low BDNF levels in the ARHL group suggest that BDNF may play a role in the pathogenesis of ARHL. Patients with low (ARHL1) and high (ARHL2) word recognition scores were compared for the first time in the literature in terms of BDNF and NT-3 levels. However, the results were not statistically significant. This article is a preliminary study and was written to provide guidance for our next comprehensive project.

Both High-Intensity Interval and Moderate-Intensity Continuous Training Decrease Fetuin-A Levels in High Fat Diet Fed Male Rats

Background: Fetuin-A is a hepatokine that increases in obesity, and a high-fat diet (HFD) contributes to this condition. Obesity is characterized by increased body mass index (BMI) and is correlated to insulin resistance. This study aims to analyze the difference between High-Intensity Interval Training (HIIT) and Moderate-Intensity Continuous Training (MICT) on fetuin-A, insulin, fasting blood glucose (FBG) levels, and BMI in HFD-fed rats. Methods: Twenty-four male Wistar rats were divided into four groups: CD (standard diet), HFD (HFD only), HFD-IT (HFD and HIIT), and HFD-CT (HFD and MICT). HFD consisted of a standard diet with an additional 2 mL/200-gram body weight of lard oil daily. In the HFD-IT group, swimming was performed with a 9% body weight load with short duration and intermittent rest periods, while the HFD-CT group was given a 6% body weight load and continuous swimming. Swimming was conducted five days a week for four weeks. Fetuin-A and insulin levels were measured using enzyme-linked immunosorbent assay (ELISA) method, and FBG levels were measured using a glucometer. Results: Fetuin-A levels were significantly lower in the HFD-IT and HFD-CT groups compared to the HFD group (p&lt;0.05). The HFD-CT group had a significant decrease in FBG levels (p&lt;0.05), but the HFD-IT group did not. There were no differences in BMI and insulin levels between groups after four weeks of treatment (p&gt;0.05). Conclusion: HIIT and MICT have similar effectiveness in reducing fetuin-A levels. In addition, MICT also managed to reduce FBG levels. Keywords: interval training, continuous training, high-fat diet, fetuin-A, insulin, healthy lifestyle.

Changes in High-Sensitivity C-Reactive Protein Levels After Two-Weeks of Moderate-Intensity Endurance Exercise in Obese Women

Study purpose. The study purpose was to analyze the effect of moderate-intensity endurance exercise on reducing inflammation in obese women. Materials and methods. Twenty obese women selected according to the established criteria were used as subjects. Then the subjects were divided into two groups: the control group (G1) and the endurance exercise group (G2). Moderate-intensity endurance exercise was performed by running on a treadmill for 40 minutes, with a frequency of 5 times/week for 2 weeks. Moderate-intensity was performed with 60%-70% HRmax (HRmax formula: 220-age). Blood sampling for inflammation measurement using the biomarker High-sensitivity C-reactive protein (hs-CRP) was performed before and after the intervention for 2 weeks. hs-CRP was measured using the enzyme-linked immunosorbent assay (ELISA) method. A paired sample t-test with a significant level (p ≤ 0.05) was used for data analysis. Results. The results showed the mean hs-CRP levels between before and after the intervention in G1 (6.76±4.40 vs. 6.43±4.89 ng/mL, (p=0.641)) and G2 (6.56±3.34 vs. 2.12±1.14 ng/mL, (p=0.004)). Conclusions. The study indicates that moderate-intensity endurance exercise has been shown to be effective in reducing inflammation levels in obese women, so individuals with obesity are advised to reduce inflammation levels through moderate-intensity endurance exercise.

Serum GAS6, sAXL, IL-10, NO, and BCL-2 levels are decreased in patients with Behçet's disease.

Behçet's disease (BD) is an autoimmune chronic systemic inflammatory disease characterized by a versatile clinical spectrum. Growth arrest specific protein 6 (GAS6)/soluble AXL (sAXL) signaling pathway draws attention in the resolution of inflammation, and its deficiency is associated with chronic inflammatory, autoimmune diseases, as well as clearance of apoptotic cells by phagocytes - efferocytosis. In this study, it was aimed to investigate whether GAS6/sAXL, interleukin (IL)-10, nitric oxide (NO), and BCL-2 levels were associated with inflammation and efferocytosis contributes to the pathogenesis of BD. A total of 37 Behçet patients with ocular involvement and 30 healthy control subjects were included in this study. GAS6, sAXL, IL-10, NO, and BCL-2 levels were quantified using enzyme-linked immunosorbent assay (ELISA) method. Serum GAS6, sAXL, IL-10, NO, and BCL-2 levels were significantly lower in patients with BD compared to the controls (P < 0.005, P < 0.001, P < 0.001, P < 0.001, and P < 0.001, respectively). In correlation analysis, research parameters decreased in patients with BD was significantly correlated with each other: GAS6-IL-10 (r = 0.585, P < 0.001), GAS6-BCL-2 (r = 0.541, P < 0.001), sAXL-BCL-2 (r = 0.696, P < 0.001), IL-10-NO (r = 0.717, P < 0.001), IL-10-BCL-2 (r = 0.759, P < 0.001), and NO-BCL-2 (r = 0.541, P < 0.001). In conclusion, decreased serum BCL-2 level may be an indicator of increased apoptosis in these patients and decreased levels of GAS6/sAXL, IL-10, and NO may indicate insufficient clearance of apoptotic bodies released as a result of increased apoptosis in BD patients.

Insight into the Interplay of Gd-IgA1, HMGB1, RAGE and PCDH1 in IgA Vasculitis (IgAV).

The pathogenesis of IgAV, the most common systemic vasculitis in childhood, appears to be complex and requires further elucidation. We aimed to investigate the potential role of galactose-deficient immunoglobulin A1 (Gd-IgA1), high-mobility group box 1 (HMGB1), receptor for advanced glycation end products (RAGE) and protocadherin 1 (PCDH1) in the pathogenesis of IgAV. Our prospective study enrolled 86 patients with IgAV and 70 controls. HMGB1, RAGE, Gd-IgA1 and PCDH1 in serum and urine were determined by the enzyme-linked immunosorbent assay (ELISA) method at the onset of the disease and after a six-month interval in patients and once in the control group. Serum concentrations of HMGB1, RAGE and PCDH1 and urinary concentrations of HMGB1, RAGE, Gd-IgA1 and PCDH1 were significantly higher in patients with IgAV than in the control group (p < 0.001). Concentrations of HMGB1 (5573 pg/mL vs. 3477 pg/mL vs. 1088 pg/mL, p < 0.001) and RAGE (309 pg/mL vs. 302.4 pg/mL vs. 201.3 pg/mL, p = 0.012) in the serum of patients remained significantly elevated when the disease onset was compared with the six-month follow-up interval, and thus could be a potential marker of disease activity. Urinary concentration of HMGB1 measured in the follow-up period was higher in patients with nephritis compared to IgAV without nephritis (270.9 (146.7-542.7) ng/mmol vs. 133.2 (85.9-318.6) ng/mmol, p = 0.049) and significantly positively correlated with the urine albumine to creatinine ratio (τ = 0.184, p < 0.05), the number of erythrocytes in urine samples (τ = 0.193, p < 0.05) and with the outcome of nephritis (τ = 0.287, p < 0.05); therefore, HMGB1 could be a potential tool for monitoring patients with IgAV who develop nephritis. Taken together, our results imply a possible interplay of Gd-IgA1, HMGB1, RAGE and PCDH1 in the development of IgAV. The identification of sensitive biomarkers in IgAV may provide disease prevention and future therapeutics.

Efficacy of high-intensity interval and continuous endurance trainings on cecal microbiota metabolites and inflammatory factors in diabetic rats induced by high-fat diet.

Physical exercise is known to modulate the intestinal microbiota composition and control the symptoms of metabolic syndrome. In this research, we intend to investigate and compare the effect of high-intensity interval and continuous endurance trainings (HIIT and CET) on cecal microbiota metabolites and inflammatory factors in diabetic rats. A number of Wistar rats were made diabetic by a high-fat diet and trained under two types of exercise protocols, HIIT and CET. After taking samples from the cecal tissue and serum of rats to reveal the effect of exercise, three microbial species from the Firmicute and Bacteroid phyla, which are the main types of intestinal microbes, and their metabolites include two short-chain fatty acids (SCFAs), butyrate and propionate and also, the inflammatory factors TLR4 and IL6 were analyzed through quantitative polymerase chain reaction (qPCR), high-performance liquid chromatography (HPLC), and Enzyme-linked immunosorbent assay (ELISA) methods. In general, exercise while increasing the representative of Firmicute has caused a relative reduction of Bacteroides and improved the concentration of SCFAs. In this regard, HIIT outperforms CET in up-regulating Akkermansia and Butyrivibrio expression, and butyrate and propionate metabolites concentration. Also, both exercises significantly reduced cecal expression of TLR4 and sera concentration of IL6 compared to the diabetic group, although the reduction rate was higher in the CET group than in HIIT. Our findings suggest that some symptoms of metabolic syndrome such as intestinal dysbiosis and the resulting metabolic disorders are better controlled by HIIT and inflammation by CET. Certainly, more extensive research on other contributing factors could help clarify the results.

EVALUATION OF SOME LIVER ENZYMES AND ALPHA-FETOPROTEIN IN WORKERS OCCUPATIONALLY EXPOSED TO E-WASTE IN NNEWI, ANAMBRA STATE NIGERIA

BACKGROUND: The advent of electricity has spurred significant technological advancements, leading to the production of numerous electrical and electronic devices. This progress, however, has resulted in a surge of largely irreparable electronic waste (e-waste) being constantly shipped to Nnewi, a metropolitan city with large-scale commercial and industrialactivities. E-waste contains thousands of chemicals, many of which are toxic to humans upon exposure.AIM OF THE STUDY: To evaluate the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and serum levels of alpha-fetoprotein (AFP) in workers occupationally exposed to e-waste.METHODOLOGY: The study comprised 40 e-waste workers (exposed group) and 40 healthy individuals (control group). Approximately 5 ml of venous blood was collected from each participant. The liver enzymes – ALT, AST, and ALP were evaluated colorimetrically, while AFP was assayed by Enzyme-linked Immunosorbent Assay (ELISA) method. Data obtained was statistically analyzed using SPSS windows version 25 with significance set as p&lt;0.05 at 95% confidence interval; Independent T-test, descriptive statistics and Pearson correlation were employed where relevant. Results were presented as means ± standard deviation in contingency tables.RESULT: There was no significant difference (p&gt;0.05) in the activities of ALT, AST, ALP and serum level of AFP between the exposed subjects and the control. However, significantly negative correlation was found between age and ALT (r = -0.294; p= 0.024), while DOE also had negative correlation with ALP (r= -0.294; P= 0.024) - all among e-waste exposed group.CONCLUSION: This study has shown that exposure to e-waste may not have a harmful effect on the liver function, since there was no significant difference in the liver function enzymes, and serum levels of AFP. However, age and longer duration of exposure to e-waste could be detrimental to health.

A declining trend of hepatitis A and hepatitis E at tertiary care hospital in South Gujarat

Objectives: Hepatitis A virus (HAV) and Hepatitis E virus (HEV) both are spread through the fecal-oral route and cause acute viral hepatitis (AVH) and pose a major public health problem in India. This study was done to find out the proportion of positivity of HAV and HEV in patients with AVH and its seasonal trend. Materials and Methods: A retrospective study was carried out at Surat Municipal Institute of Medical Education and Research Medical College, Department of Microbiology, Surat, Gujarat. Result of 3615 blood samples of suspected AVH patients of the past 5 years (January 2018–December 2022) were taken from hospital data records. The enzyme-linked immunosorbent assay method was used to test serum samples for immunoglobulin M (IgM) HAV and IgM HEV antibodies for HAV and HEV, respectively. All samples were evaluated for liver function as well. Results: The positivity of HAV and HEV was 15.13% and 10.26%, respectively. The coinfection rate was 2.07%. HAV and HEV both affected males more than females. Among pregnant females, HEV infection had more positivity (6.77%) than HAV, which had 1.08% positivity. HAV and HEV infections had a seasonal trend, with the highest infection rate in the monsoon. Conclusion: The declining trend of cases of HAV and HEV was found in Surat city of south Gujarat which indicates increased awareness about hepatitis among people and better public health management by the civic authorities.

The Effect of Anti-fatigue Decoction on the Behaviors and Serological Indicators in a Central Fatigue Rat Model.

This study aimed to assess the effects of Anti-fatigue Decoction (AFD) against central fatigue by observing the behaviors and serological indicators of rats modeled by the modified multiple platform method (MMPM) after drug intervention. Grip strength measurements were used to evaluate the muscle strength of rats. The open field test was utilized to assess anxiety-like behavior, while the Morris water maze test was conducted to evaluate the memory function of the rats. Following the behavioral assessments, rat serum samples were collected to measure the concentrations of corticosterone (CORT) and lactic acid (LAC). The concentration of LAC was determined using the colorimetric method, while the concentration of CORT was measured using the enzyme-linked immunosorbent assay (ELISA) method. Compared to the blank control group, following MMPM modeling, rats exhibited significant reductions in grip strength and impaired ability to memory. The serum analysis revealed increased levels of LAC and CORT in the model group rats. AFD can noticeably reverse these adverse changes to a certain extent. These findings highlight the positive effects of AFD and coenzymeQ10 on physical and cognitive abilities and alterations in serum biomarker levels of central fatigue rats.

Development and validation of ELISA method for quantification of Q-1802 in serum and its application to pharmacokinetic study in ICR Mouse

Q-1802 is a humanized bispecific antibody targeting programmed death-ligand 1 (PD-L1) and Claudin 18.2 (CLDN18.2). It can bind to CLDN18.2 and mediate antibody-dependent cell-mediated cytotoxicity against tumor cells. The Fc segment of the antibody recognizing PD-L1 blocks PD-1 signaling and activates innate immunity and adaptive immunity. In this study, we report the development, validation, and application of sensitive and high-throughput enzyme-linked immunosorbent assays (ELISA) to measure the concentrations of Q-1802 in ICR mouse serum. The assay is sensitive, with a lower limit of quantification of 50 ng/mL, has a broad dynamic range of 50–3200 ng/mL, and exhibits excellent precision and accuracy. These assays were successfully applied to in vitro serum stability and pharmacokinetic (PK) studies. In conclusion, we have developed and validated a highly sensitive and selective method for measuring Q-1802 in ICR mouse serum. The development and validation steps of assays met the required criteria for validation, which suggested that these can be applied to quantify Q-1802, as well as in PK studies.

Distribution of ABO/Rhesus Blood Groups Among Hepatitis B and C Virus (HBV Ag, HCV Ab) Positive Patients in Anbar Providence, Iraq

This study aimed to retrospectively investigate the prevalence of Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) infections among blood donors at Anbar Blood Bank in Iraq. The study also explored the correlation between ABO and Rh blood groups with Transfusion-Transmissible Infections (TTIs) and sociodemographic factors and cross-sectional retrospective study was conducted on 100 blood donors at Anbar Blood Bank from March 24, 2023, to June 21, 2023. Inclusion criteria encompassed HBV and HCV-positive patients who provided consent, while exclusion criteria included individuals without HBV and HCV infections. Blood samples (5 mL) were collected, and tests for TTI markers (HBV, HCV) and ABO and Rh blood grouping were performed using enzyme-linked immunosorbent assay (ELISA) and slide method, respectively. Sociodemographic data, TTI marker results, and ABO and Rh blood types were recorded for analysis. Descriptive analysis of sociodemographic data was performed, and a chi-square test was employed to assess the correlation between ABO and Rh blood groups with TTI markers and sociodemographic factors (Age, sex, and living area). Statistical significance was set at P&lt;0.05. The results revealed a male predominance (71%), with the majority aged between 20-30 years (39%) and residing in rural areas (52%). The most common blood group was O+ (39%), while AB- was the least prevalent (0%). In terms of viral infections, 80% of participants were found to be infected with HBs Ag, and 20% with HCV Ab. Notably, blood group O+ exhibited the highest infection rates for both HBs (28%) and HCV (11%), while blood group A- demonstrated the lowest HBs infection rate (3%) and no HCV infection. The study provides valuable insights into the prevalence of HBV and HCV infections among blood donors in Anbar, Iraq. Additionally, correlations between ABO and Rh blood groups, TTI markers, and sociodemographic factors were explored. The findings contribute to the understanding of transfusion safety and may inform blood screening and donor selection protocols.

ELISA assay for the quantification of ipilimumab in human serum, plasma, milk, and cerebrospinal fluid

Ipilimumab is an immune checkpoint inhibitor of the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Ipilimumab has become part of the standard of care for different types of cancer. The efficacy of these treatments is limited due to immune-related toxicity and high economic costs. Dose rationalization studies based on pharmacokinetic data may help to address these limitations. For this purpose, more sensitive analytical methods are needed. We report the development and validation of the first enzyme-linked immunosorbent assay (ELISA) for sensitive determination of ipilimumab concentrations in human serum, plasma, cerebrospinal fluid (CSF), and milk. Our assay is based on the specific capture of ipilimumab by immobilized CTLA-4. The lower limit of quantifications of ipilimumab in serum, plasma, and milk are 50 ng/mL and 10 ng/mL in CSF. The ELISA method showed long-term storage stability for at least one year at −80°C and was successfully cross-validated with ultraperformance liquid chromatography coupled with tandem mass spectrometry. The ELISA method is reliable, relatively inexpensive, and can be used in serum, plasma, CSF, and milk from patients treated with ipilimumab, as evidenced by the analysis of real clinical samples.

Assessment of the functional state of gut microbiota in patients with metabolic-associated fatty liver disease in combination with type 2 diabetes mellitus

Background. The purpose of the study was to assess the functional state of gut microbiota in patients with metabolic-associated fatty liver disease (MAFLD) combined with type 2 diabetes mellitus (T2DM). Materials and methods. The prospective interventional randomized study included 71 patients with MAFLD in combination with T2DM, who were examined and divided into the 2 groups. The first group included 39 people with MAFLD and T2DM without small intestinal bacterial overgrowth (SIBO). The second group consisted of 32 patients with MAFLD in combination with T2DM and SIBO. The control group included 25 practically healthy patients. The content of serum zonulin was determined by enzyme-linked immunosorbent assay (ELISA) using test systems IDK Zonulin ELISA, KR5601. Serum concentration of interleukin (IL) 6 and IL-10 was assessed by the ELISA method using the Human Interleukin 6 and 10 ELISA Kit test systems. The content of short-chain fatty acids in feces was determined by gas chromatography with mass spectrometry in the PerkinElmer Clarus 680 GC Gas Chromatograph. Results. Patients of the first and second groups had an increase in hepatic aminotransferases, total cholesterol, triglycerides, fasting blood glucose, insulin, HOMA-IR, glycosylated hemoglobin, serum zonulin, leptin, IL-6 and liver attenuation coefficient and a decrease in high-density lipoprotein. The results of steatometry revealed a severe degree of hepatic steatosis (S3) in patients with MAFLD combined with T2DM and SIBO and without SIBO. During the quantitative study of the level of butyric acid in feces, it was found to be significantly reduced, by 2.3 times in the first group and by 3.4 times in the second one, compared to the controls (p &lt; 0.001). The content of butyric acid was decreased by 1.4 times in the second group compared to the first one (p &lt; 0.001). When evaluating the level of acetic acid, it was found to be significantly increased compared to the controls, by 1.2 times in the first group and by 1.3 times in the second one (p &lt; 0.001). The concentration of propionic acid was increased by 1.3 times in the first group and by 1.5 times in the second one compared the control group (p &lt; 0.05). When comparing the level of acetic acid, a significant increase by 1.3 times was observed in patients of the first and second groups compared to patients of the control group (p &lt; 0.001). The content of propionic and acetic acids was increased by 1.1 times in the second group compared to patients of the first group (p &lt; 0.05). When analyzing the correlation coefficients in patients with MAFLD combined with T2DM and SIBO and without SIBO, a direct proportional strong correlation was revealed between the levels of butyric, acetic and propionic acids and serum zonulin, IL-6, IL-10. A direct moderate correlation was found between the content of short-chain fatty acids and leptin, indicators of lipid metabolism (total cholesterol, triglycerides), carbohydrate metabolism (fasting blood glucose, HOMA-IR, HbA1c) and inverse weak correlation with high-density lipoprotein. A direct moderate correlation was revealed between levels of short-chain fatty acids and indicators of functional activity of the liver (alanine aminotransferase, aspartate aminotransferase), liver attenuation coefficient in patients with MAFLD combined with T2DM and SIBO and without SIBO. Conclusions. Increased intestinal permeability and hyperleptinemia have been found in patients with МAFLD combined with T2DM and SIBO and without SIBO. When studying the functional state of the gut microbiota, an increase was found in propionic and acetic acids and a decrease in butyric acid, which indicates an increase in the number of Bacteroidetes in the intestine and a decrease in Firmicutes. Patients with MAFLD combined with Т2DM and SIBO and without SIBO had a direct proportional strong correlation between the levels of butyric, acetic and propionic acids and serum zonulin, IL-6, IL-10, which is associated with pro-inflammatory and anti-inflammatory processes in the intestinal mucosa and a violation of the permeability of the intestinal barrier. It was found that concentrations of short-chain fatty acids correlate with indicators of functional activity of the liver (alanine aminotransferase, aspartate aminotransferase), leptin, indicators of carbohydrate and lipid metabolism, degree of hepatic steatosis.

Synthetic protein protease sensor platform.

Introduction: Protease activity can serve as a highly specific biomarker for application in health, biotech, and beyond. The aim of this study was to develop a protease cleavable synthetic protein platform to detect protease activity in a rapid cell-free setting. Methods: The protease sensor is modular, with orthogonal peptide tags at the N and C terminal ends, which can be uncoupled via a protease responsive module located in between. The sensor design allows for several different readouts of cleavage signal. A protein 'backbone' [Green fluorescent protein (GFP)] was designed in silico to have both a C-terminal Flag-tag and N-Terminal 6x histidine tag (HIS) for antibody detection. A protease cleavage site, which can be adapted for any known protease cleavage sequence, enables the uncoupling of the peptide tags. Three different proteases-Tobacco, Etch Virus (TEV), the main protease from coronavirus SARS-COV-2 (Mpro) and Matrix Metallopeptidase 9 (MMP9)-a cancer-selective human protease-were examined. A sandwich Enzyme-Linked Immunosorbent Assay (ELISA) was developed based on antibodies against the HIS and Flag tags. As an alternative readout, a C-terminal quencher peptide separable by protease cleavage from the GFP was also included. Purified proteins were deployed in cell-free cleavage assays with their respective protease. Western blots, fluorescence assays and immunoassay were performed on samples. Results: Following the design, build and validation of protein constructs, specific protease cleavage was initially demonstrated by Western blot. The novel ELISA proved to afford highly sensitive detection of protease activity in all cases. By way of alternative readout, activation of fluorescence signal upon protease cleavage was also demonstrated but did not match the sensitivity provided by the ELISA method. Discussion: This platform, comprising a protease-responsive synthetic protein device and accompanying readout, is suitable for future deployment in a rapid, low-cost, lateral flow setting. The modular protein device can readily accommodate any desired protease-response module (target protease cleavage site). This study validates the concept with three disparate proteases and applications-human infectious disease, cancer and agricultural crop infection.

MATERNAL SERUM LEPTIN LEVELS IN SUBJECTS WITH GESTATIONAL DIABETES MELLITUS

Objectives: To determine serum leptin levels and its correlation with pre-pregnancy BMI, BMI, and insulin resistance at 24 - 28 weeks of gestation and before delivery in subjects with gestational diabetes mellitus (GDM). Methods: A cross-sectional descriptive, longitudinal follow-up study with a control group on 115 pregnant women with GDM and 115 pregnant women with normal glucose tolerance (NGT) enrolled at 24 - 28 weeks of gestation and completed the study. The subjects' serum leptin levels were measured using the enzyme-linked immunosorbent assay (ELISA) method at 24 - 28 weeks of pregnancy and before delivery. The clinical characteristics, serum insulin levels, and homeostatic model assessment of insulin resistance (HOMA-IR) were also performed at both study time points. Results: Serum leptin levels in subjects with GDM were significantly higher than those in normal pregnant women at 24 - 28 weeks of gestation and before delivery (p &lt; 0.001). At both study time points, there was a positive correlation between serum leptin levels and pre-pregnancy BMI, maternal weight, serum insulin levels, and HOMA-IR index in subjects with GDM (p &lt; 0.05). Multivariable logistic regression showed that pre-pregnancy BMI and serum insulin levels were associated with hyperleptinemia       (p &lt; 0.05). Conclusion: Serum leptin levels were higher in GDM and had a positive correlation with pre-pregnancy BMI, BMI, and insulin resistance at the time of the study. Pre-pregnancy BMI and serum insulin levels were independent factors related to increased serum leptin levels.

Development of an ELISA method to determine adulterated cow milk in camel milk

Camel milk might be adulterated with cheaper cow's milk because of its high price. The establishment of a rapid and reliable method for detecting cow milk in camel milk is a matter of great concern. In the study, the chromatogram of camel milk with different adulteration ratios was analyzed by High Performance Liquid Chromatography (HPLC). The peak area of liquid chromatography was analyzed by Principal Component Analysis (PCA). β-Lactoglobulin (β-Lg) was screened as a characteristic protein. Enzyme-Linked Immunosorbent Assay (ELISA) was established selected β-Lg as the antigenic indicator. This study was aimed at developing an ELISA method that enabled rapid and highly sensitive detection of the proportion of adulterated milk in camel milk. The minimum detection limit was 1% of cow milk in camel milk, and the results were highly reproducible with no significant difference between true and measured values (P > 0.05).

An automated fluorescent immunoassay for on-site screening of AFM1 in raw milk at the ppt level

In the dairy industry, the quality of raw milk as it is collected from dairy farmers must be strictly ensured. Therefore, when on-site screening of typical carcinogens in raw milk, this technology must simultaneously be convenient to operate and highly sensitive. Here, an automated and fluorescent immunoassay system for screening trace amounts of aflatoxin M1 (AFM1) in raw milk was developed. The whole immunoassay procedure can be processed in a true “sample-to-results” paradigm, avoiding the tedious manual operation of the traditional indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) method. In addition, we designed an integrated fluorescent spectrometer that can quantitatively measure fluorescent signals with high sensitivity. The automated and fluorescent immunoassay system can screen AFM1 in raw milk samples with an ultra low limit of detection (4.7 pg/mL in raw milk). The half maximal inhibitory concentration (IC50) was 8.3 pg/mL, which is approximately 4-fold lower than that of traditional ic-ELISA. When the system was applied to screen actual raw milk samples, the recovery rates ranged from 79.4% to 104.6%, with a coefficient of variation ranging from 8.9% to 15.2%. Overall, we believe that the automated and fluorescent immunoassay system can provide a one-stop solution that is user-friendly and highly sensitive for screening trace AFM1 contaminants in raw milk.