Enzyme-linked Immunosorbent Assay Research Articles

Serological and Molecular Investigation of Infectious Laryngotracheitis Virus in Chickens from Robe Town, Southeastern Ethiopia

Infectious laryngotracheitis virus (ILTV) is responsible for avian infectious laryngotracheitis (ILT), a highly contagious acute respiratory disease affecting chickens. However, there is limited information on ILTV and its distribution in Ethiopia, particularly in the southeastern region. The aim of this study was to establish the serological prevalence and molecular evidence in commercial and backyard chickens from Robe town, Southeastern Ethiopia. A cross-sectional study was conducted between December 2021 and June 2022, collecting 240 serum samples from randomly selected chickens belonging to eight kebeles (farms) using systematic random sampling. ILTV-specific antibodies were detected using a commercial indirect enzyme-linked immunosorbent assay (ELISA). From 240 serum samples, 26.7% were positive for ILTV antibodies. Logistic regression analysis identified the type of poultry farm (backyard) and the introduction of chickens from other farms as potential risk factors associated with ILTV exposure. Tracheal tissue and oropharyngeal and tracheal swabs were collected from suspected chickens for isolation and molecular detection. A total of six samples were successfully isolated in embryonated eggs (40%), with four of them verified with a specific PCR. These findings documented the presence of ILTV in the study area, which needs further insight to fully understand the actual spread of ILTV and quantify the damage caused to the poultry sector.

Does salivary cortisol serve as a potential biomarker for temporomandibular disorders in adults?

BackgroundThe etiology of temporomandibular disorders (TMD) is multifactorial, involving a complex interplay of psychological and physiological factors. While salivary biomarkers, particularly cortisol, play an important role in TMD pathophysiology, evidence in the literature is still scarce and inconsistent. Hence, this study aims to evaluate the applicability of salivary cortisol as a potential biomarker for TMD in adults.MethodsThe Diagnostic Criteria for Temporomandibular Disorders (DC/TMD) were used to accurately diagnose TMD patients. The study included adults, both male and female, aged 18–40 in the TMD group (n = 66) and non TMD participants (n = 66) matched for age and gender. Salivary samples were collected from participants at two time points: early and late morning. Cortisol levels in the samples were quantified using enzyme-linked immunosorbent assay (ELISA). Statistical analysis was performed using a one-way ANOVA to evaluate the correlations between cortisol levels and the study variables. Tukey’s post-hoc tests were applied to adjust for multiple comparisons.ResultsSalivary cortisol levels were significantly higher in the TMD group than in the corresponding controls (p = 0.034). In the TMD group, the mean cortisol levels early in the morning were 29.95 ± 75.05 (0-398.64), while the late morning levels were recorded as 4.87 ± 3.96 (0-17.13). In the control group, the mean cortisol levels early in the morning were 10.98 ± 16.83 (2.16–92.90), and late morning mean cortisol levels were 6.15 ± 6.13 (0-20.42). This indicates that the early morning levels of cortisol are higher in TMD patients (p = 0.046). In the subgroup analysis of the TMD, the mean salivary cortisol levels recorded were highest at 82.49 ± 124.34 (8.32-398.64) in patients having disc displacement without reduction with limited mouth opening. Furthermore, the mean salivary cortisol levels in the early morning were statistically higher (84.83 ± 132.80) in males compared to females (9.36 ± 9.01) (p = 0.008) with TMD.ConclusionThe result of this study suggests that salivary cortisol could be a potential biomarker for a specific TMD subtype (disc displacement without reduction with limited mouth opening). However, further studies are needed to better understand the role of cortisol biomarker in the underlying pathogenesis of TMD.

Analysis of associations of personality traits and stress with the level of serum cortisol in Sakha (Yakut) men

The purpose of this study is to test the hypothesis that personality traits and stressful situations experienced in childhood could be associated with the level of serum cortisol. The sample included 121 healthy adult men of Yakut (Sakha) ethnicity aged 18 to 27 years. To assess personality traits, the TCI temperament and character questionnaire by R. Cloninger was used. Serum cortisol levels were assessed by enzyme-linked immunosorbent assay (ELISA). It was found that such a temperament trait as “reward dependence” is associated with higher level of cortisol in the blood (p = 0.04). Experienced stressful situations are associated with the character trait “self-transcendence” (p = 0.049) but do not significantly affect cortisol levels. In individuals with high levels of stress, significant correlations were found between the “novelty seeking” (r = 0.33) and “self-directedness” (r = 0.36) with serum cortisol levels, which may reflect the prolonged effect of stress on increasing the sensitivity of the adrenal cortex. The results indicate a possible connection between one of the human temperament traits “reward dependence” and higher levels of cortisol in the blood. A high level of experienced stress situations reduces scores on the character trait “self-transcendence.”

Development and Validation of an Indirect and Blocking ELISA for the Serological Diagnosis of African Swine Fever

African swine fever (ASF) is an economically devastating viral disease of pigs caused by the ASF virus (ASFV). The rapid global spread of ASF has increased the demand for ASF diagnostics to be readily available and accessible. No commercial ASF enzyme-linked immunosorbent assay (ELISA) kits are manufactured and licensed in North America. Here, we report the development of two serological diagnostic assays, a blocking ELISA (bELISA) based on ASFV glycoprotein p54 and an indirect ELISA (iELISA) based on ASFV glycoproteins p54 and p72. The assays showed high sensitivity and specificity and detected anti-ASFV antibodies in serum samples from experimentally infected animals as early as 8 days post-infection. The two assays were produced commercially (AsurDx™ bELISA and iELISA) and subjected to extensive validation. Based on data from a set of characterized reference sera, the prototype commercial assays, while maintaining 100.00% specificity, showed 97.67% (AsurDx™ bELISA) and 83.72% (AsurDx™ iELISA) sensitivity. Both prototype assays detected anti-ASFV antibodies in serum samples collected from pigs experimentally infected with multiple ASFV strains and field samples collected from sick, recovering, and vaccinated animals. The two commercially available assays can be used in routine ASF diagnostics, serological surveys, and for evaluating serological responses to ASF vaccine candidates.

Seroprevalence of anti-SARS-CoV-2 IgM and IgG and COVID-19 vaccine uptake in healthy volunteers in Nairobi, Kenya: a cross-sectional study

IntroductionSeroprevalence of anti-severe acute respiratory syndrome coronavirus 2 (anti-SARS-CoV-2) antibodies in the postvaccination period in Kenya remains to be elucidated. This study aimed to determine the seroprevalence of anti-SARS-CoV-2 IgM and IgG and evaluate Covid-19 vaccination uptake in a university setting in Nairobi.MethodsThis cross-sectional study assayed serum anti-SARS-CoV-2 IgM and IgG levels using enzyme-linked immunosorbent assays. A structured questionnaire was used to determine vaccine uptake, vaccine hesitancy and reasons for hesitancy.ResultsA total of 189 participants were enrolled (median age, 21 years; female, 50.8%). The seroprevalence of anti-SARS-CoV-2 was 12.7% for IgM and 87.8% for IgG. Anti-SARS-CoV-2 IgG titers were higher among the vaccinated vs. non-vaccinated individuals (p < 0.001, U = 2817.5), females vs. males (p = 0.024, U = 3616), and those vaccinated ≤ 6 months before the study vs. those vaccinated >1 year earlier (p = 0.002, H = 12.359). The vaccination hesitancy rate was 43.4% and the underlying reasons included mistrust (22.4%), health concerns (19.7%), and lack of information (18.4%).DiscussionThe high seroprevalence of anti-SARS-CoV-2 IgG is an indication of high exposure to SARS-CoV-2 either through natural infection or through vaccination. The high vaccine hesitancy noted necessitates community engagement, and public education to dispel myths and misinformation prior to roll out of new vaccines and other health interventions.

Comparison of Different Keratinocyte Cell Line Models for Analysis of NLRP1 Inflammasome Activation

The NLRP1 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-1) inflammasome is the most important inflammasome in human keratinocytes. It plays a crucial role in regulating innate immunity in the skin. This study aimed to evaluate NLRP1 inflammasome activation and the corresponding levels of detection in different keratinocyte cell lines to identify a suitable in vitro model for analyzing inflammasome activation in keratinocytes. We compared NLRP1 inflammasome activation, expression, and cell death among primary keratinocytes and immortalized keratinocyte cell lines HaCaT, HaSKpw, and SVTERT upon stimulation with ultraviolet B (UVB) irradiation or talabostat. The effects of both NLRP1 inducers on cell death and the modification of NLRP1 molecules were examined using fluorescence-activated cell sorting analysis, Western blotting, and an enzyme-linked immunosorbent assay. The key inflammasome components had varied expression levels among the keratinocyte cell models, with the highest expression observed in primary keratinocytes. Moreover, our data showed that both UVB and talabostat triggered cell death, and NLRP1 inflammasome activation was readily detected in primary keratinocytes but not in the analyzed immortalized keratinocyte cell lines. Therefore, we do not recommend the use of the immortalized keratinocyte cell lines HaCaT, HaSKpw, and SVTERT for analyzing inflammasome activation in keratinocytes; we strongly recommend the use of primary keratinocytes for these studies.

Elevated plasma progranulin levels in the acute phase are correlated with recovery of left ventricular function in the chronic phase in patients with acute myocardial infarction

Background Progranulin is a secreted glycoprotein that regulates inflammation and wound healing. However, plasma progranulin levels in the acute phase and their clinical significance in patients with acute myocardial infarction (AMI) remain to be elucidated. Objective We aimed to investigate the relationship between the increase in plasma progranulin levels in the acute phase and the recovery of left ventricular function in the chronic phase in AMI patients. Method and result Eighteen AMI patients were followed up for 6 months. Blood samples were collected from the antecubital vein on day 0 (on admission) and day 7 in the acute phase. The control group consisted of patients without significant coronary artery stenosis, as assessed by cardiac catheterization (n = 16). Plasma progranulin levels were measured by enzyme-linked immunosorbent assay. Echocardiography was performed in the acute (within 7 days) and chronic (6 months) phases of AMI to evaluate left ventricular ejection fraction using the modified Simpson’s method. Plasma progranulin levels in the AMI group on day 0 (69.5 ± 24.6 ng/mL) were similar to those in the control group (84.2 ± 47.1 ng/mL). There was a significant increase in progranulin levels in the AMI group on day 7 (104.2 ± 52.0 ng/mL) compared with day 0. The increase in plasma progranulin levels in the acute phase was positively correlated with the increase in left ventricular ejection fraction between the acute and chronic phases. Among various factors, only plasma progranulin levels were favorably correlated with left ventricular functional recovery in the chronic phase. Conclusion The increase in plasma progranulin levels in the acute phase may serve as a predictive biomarker and a contributer for the recovery of left ventricular function in the chronic phase in patients with AMI.

SPP1 is a plasma biomarker associated with the dia gnosis and prediction of prognosis in sepsis

In this study, peripheral whole blood samples from 22 hospitalized patients and 10 healthy individuals were analyzed using a combination of Data Independent Acquisition (DIA) and Enzyme-Linked Immunosorbent Assay (ELISA) techniques to identify differentially expressed proteins (DEPs) in sepsis patients’ plasma. The aim was to provide accurate and detailed biomarkers, such as SPP1, for determining the pathological stages of sepsis. SPP1, known as osteopontin1 is a pleiotropic protein with a wide distribution and multifunctional effects. Its protein expression is associated with inflammatory changes, including variations in expression levels in infectious diseases, allergic diseases, and situations involving tissue damage. The registration number was ChiCTR1900021261.The full date of first registration year is 2018. In the affiliated hospital of southwest medical university, 22 sepsis patients were hospitalized from January 2019 to September 2020 and 10 normal healthy individuals were selected for DIA-based quantitative proteomics analysis. In addition to gene ontology analysis and Kyoto Encyclopedia of genes and genomes analysis, enrichment analysis of data was performed and target protein network was screened through joint protein–protein interaction and visualization techniques. The selected protein targets were then validated by Elisa kit. The software was used to analyze the differences comparing the control group to the sepsis group and the sepsis group, as well as between sepsis survivals and non-survivals, and a ROC curve was drawn to evaluate the diagnostic value and prognostic effect of the method of the corresponding target proteins. A total of 174 DEPs were screened by bioinformatics analysis. An analysis of go pathway enrichment revealed the following: These proteins were mainly involved in biological processes among them are the inflammation response, the metabolism of extracellular matrix, the secretion of cell secretions, the activation of cells, and the immune response. According to the Kegg pathway analysis, they were mainly involved in complement cascade polymerization, extracellular protease and glycosylase activation, protein synthesis process, biotin metabolism, leukocyte transmembrane migration, bacterial infection and phagosome formation. SPP1 was identified as a possible plasma biomarker and was therefore further validated using Elisa. As a result of experiments, it has been demonstrated that level in sepsis patients is significantly compared to the normal control group and the level is also higher in non-survivals of sepsis. The ROC curve can be used to see that it can diagnose sepsis more accurately and improve prognostic ability prediction. Cell experiments confirm that SPP1 is highly expressed in sepsis. There is a significant difference in the levels of SPP1 protein between the normal group and the sepsis group; it not only has good diagnostic significance for sepsis, but also provides corresponding reference value for patient prognosis; Therefore, it is more likely to become a biological marker of sepsis over time.

Circ_0068655 silencing ameliorates hypoxia-induced human cardiomyocyte injury by regulating apoptotic and inflammatory responses

Abstract Background There is growing evidence suggesting that the dysregulation of circular RNAs (circRNAs) plays a significant role in various myocardial disorders, including myocardial ischemia (MI). This study aimed to explore the function of hsa_circ_0068655 (circ_0068655) in hypoxia-induced cardiomyocyte injury. Methods Human AC16 cardiomyocyte cells were cultured under anaerobic condition to induce an in vitro model of MI. Cell apoptosis was assessed by Annexin V-fluorescein isothiocyanate staining and caspase-3 and caspase-9 activity assays. Cell proliferation was analyzed by 5-Ethynyl-2’-deoxyuridine (EdU) incorporation assay. Inflammation was evaluated by enzyme-linked immunosorbent assays. Circ_0068655, miR-370-3p and BCL-2-like 11 (BCL2L11) expression were detected by real-time quantitative polymerase chain reaction or western blotting. The target interactions among circ_0068655, miR-370-3p and BCL2L11 were predicted using bioinformatics tools and validated using dual-luciferase reporter assays and RNA immunoprecipitation assays. Results Hypoxia treatment led to upregulated expression of circ_0068655 and BCL2L11, and downregulated expression of miR-370-3p in AC16 cells. This treatment also resulted in reduced cell viability, increased apoptosis rate, elevated caspase-9/3 activities and cleavage, and enhanced production of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β. Notably, knockdown of circ_0068655 alleviated these detrimental effects. In addition, circ_0068655 silencing-mediated effects were restored by decreasing miR-370-3p expression in hypoxia-treated AC16 cells. Moreover, ectopic BCL2L11 expression remitted the effects of miR-370-3p overexpression on hypoxia-treated AC16 cells. Mechanistically, circ_0068655 was found to act as a sponge for miR-370-3p, thereby regulating BCL2L11 expression. Conclusion Circ_0068655 silencing ameliorated hypoxia-induced human cardiomyocyte injury through the miR-370-3p/BCL2L11 axis.

Determination of the Level of von Willebrand Factor, ADAMTS13, and Ratio of ADAMTS13:von Willebrand Factor in Sickle Cell Disease Patients

Abstract Background: Sickle cell anemia (SCA) is a hypercoagulable state characterized by a significant alteration in hemostatic parameters which may predispose an increased risk of vaso-occlusive crisis (VOC). Sickle cell disease (SCD) is the most common genetic disorder in sub-Saharan Africa. Nigeria bears a high disease burden with an estimated prevalence of 1%–3% of its population being affected by the disease. The study seeks to determine the role of von Willebrand factor (VWF), ADAMTS13, and the ratio of ADAMTS13:VWF antigen in the pathogenesis of VOC. Objective: The objective of this study is to evaluate the level of VWF, ADAMTS13, and their ratio in SCD subjects in Calabar and to determine their role in the pathogenesis of VOC. Methodology: This is a comparative study carried out at the University of Calabar Teaching Hospital (UCTH), Calabar. Sixty SCA patients were evaluated in VOC and steady states as well as five parented healthy controls. VWF: Ag and ADAMTS13:Ag were evaluated using Assaypro enzyme-linked immunosorbent assay kits with Lot nos. 01751728 and 04222167R, respectively. Data were analyzed by IBM SPSS Chicago software version 21. The study was approved by the UCTH Institution Ethical Review Board. Results: The mean ages of the SCA subjects and controls were 23.5 ± 7.2 years and 26.5 ± 5.6 years, respectively (P = 0.706). There were 23 (38.3%) males in the SCA group and 21 (42.0%) females in the controls. There was no significant difference in their sex distribution (P = 0.063). The mean (standard deviation [SD]) of VWF in VOC, steady state, and controls were 2.52 ± 0.34, 1.34 ± 0.23, and 1.41 ± 0.23 IU/mL, respectively. The differences in mean were significantly higher in VOC state (P = 0.003). The mean ± SD of ADAMTS13 in VOC, steady state, and controls were 0.61 ± 0.10, 0.44 ± 0.06, and 0.62 ± 0.10 μg/L, respectively. ADAMTS13 levels did not differ significantly across the groups (P = 0.270). Similarly, there was no significant difference between ADAMTS13:VWF ratios across the groups (P = 0.318). Conclusion: VWF level is elevated in VOC state and thus may be implicated in the pathogenesis of VOC. ADAMTS13 and the ratio of ADAMTS13:VWF are not significantly affected in VOC.

Protective efficacy of a recombinant adenovirus expressing novel dual F and HN proteins of bovine parainfluenza virus type 3

Bovine parainfluenza virus type 3 (BPIV3) is a viral respiratory pathogen that infects cattle and causes significant economic losses. We generated a recombinant adenovirus called rHAd5-F + HN by expressing the fusion (F) and hemagglutinin-neuraminidase (HN) glycoprotein of BPIV3 using the human adenovirus serotype 5 (rHAd5). We evaluated its effects on humoral and cellular immune responses in mice (n = 45) and calves (n = 9). Serum antibody responses were assessed by enzyme-linked immunosorbent assay (ELISA), hemagglutination inhibition (HI), and neutralising antibodies (NAb). After boosting immunity with rHAd5-F + HN, mice produced significantly higher levels of antibodies against the BPIV3 genotype A and genotype C strains. The production of antibodies exceeded those produced by adenoviruses rHAd5-F and rHAd5-HN, which express the F and HN glycoprotein, respectively. The percentages of splenic CD3+/CD8+T lymphocytes and IL-4+ cytokines in rHAd5-F + HN mice were considerably higher than those in the control group. Mice immunised with rHAd5-F + HN exhibited much lower viral loads in the lungs and tracheas compared to the control group. Additionally, the lungs of mice vaccinated with rHAd5-F + HN showed no notable histopathological changes. On the other hand, rHAd5-F + HN produced a humoral immune response in calves. Following the booster intramuscular injection with the rHAd5-F + HN, the serum antibody levels against BPIV3 genotype C strain were 1:20 452, 1:1024, and 1:426 in calves, as detected by ELISA, HI, and NAb, respectively. The HI and NAb levels against the BPIV3 genotype A strain were 1:213 and 1:85 in calves, respectively. These results indicate that rHAd5-F + HN effectively induced immunity against BPIV3 infection.

Amino acid deletions at positions 893 and 894 of cytotoxin-associated gene A protein affect Helicobacter pylori gastric epithelial cell interactions

BACKGROUND Helicobacter pylori (H. pylori ) persistently colonizes the human gastric mucosa in more than 50% of the global population, leading to various gastroduodenal diseases ranging from chronic gastritis to gastric carcinoma. Cytotoxin-associated gene A (CagA) protein, an important oncoprotein, has highly polymorphic Glu-Pro-Ile-Tyr-Ala segments at the carboxyl terminus, which play crucial roles in pathogenesis. Our previous study revealed a significant association between amino acid deletions at positions 893 and 894 and gastric cancer. AIM To investigate the impact of amino acid deletions at positions 893 and 894 on CagA function. METHODS We selected a representative HZT strain from a gastric cancer patient with amino acid deletions at positions 893 and 894. The cagA gene was amplified and mutated into cagA -NT and cagA -NE (sequence characteristics of strains from nongastric cancer patients), cloned and inserted into pAdtrack-CMV, and then transfected into AGS cells. The expression of cagA and its mutants was examined using real-time polymerase chain reaction and Western blotting, cell elongation via cell counting, F-actin cytoskeleton visualization using fluorescence staining, and interleukin-8 (IL-8) secretion via enzyme-linked immunosorbent assay. RESULTS The results revealed that pAdtrack/cagA induced a more pronounced hummingbird phenotype than pAdtrack/cagA -NT and pAdtrack/cagA -NE (40.88 ± 3.10 vs 32.50 ± 3.17, P < 0.001 and 40.88 ± 3.10 vs 32.17 ± 3.00, P < 0.001) at 12 hours after transfection. At 24 hours, pAdtrack/cagA- NE induced significantly fewer hummingbird phenotypes than pAdtrack/cagA and pAdtrack/cagA- NT (46.02 ± 2.12 vs 53.90 ± 2.10, P < 0.001 and 46.02 ± 2.12 vs 51.15 ± 3.74, P < 0.001). The total amount of F-actin caused by pAdtrack/cagA was significantly lower than that caused by pAdtrack/cagA- NT and pAdtrack/cagA- NE (27.54 ± 17.37 vs 41.51 ± 11.90, P < 0.001 and 27.54 ± 17.37 vs 41.39 ± 14.22, P < 0.001) at 12 hours after transfection. Additionally, pAdtrack/cagA induced higher IL-8 secretion than pAdtrack/cagA- NT and pAdtrack/cagA- NE at different times after transfection. CONCLUSION Amino acid deletions at positions 893 and 894 enhance CagA pathogenicity, which is crucial for revealing the pathogenic mechanism of CagA and identifying biomarkers of highly pathogenic H. pylori .

Immunological quality of colostrum and specific antibodies against enteropathogens in the colostrum and transition milk of crossbred Gir × Holstein cows.

Colostrum management is crucial for enhancing the immune response against enteropathogens and the survival of dairy calves during the first few weeks of life. However, few physiological studies have investigated the dynamics of general and specific immunoglobulin G (IgG) content in cow milk during early lactation stages, particularly in that of crossbred Gir × Holstein dairy cows, the most predominant dairy cattle population in tropical countries, such as Brazil. Therefore, this study evaluated the effects of parity and milking order on the volume and quality of colostrum, transition milk, and mature milk in crossbred Gir × Holstein cows using three traditional on-farm tests. The dynamics of IgG in the mammary secretions and the specific antibody levels against enteropathogens were also determined using sandwich enzyme-linked immunosorbent assay (ELISA) during the early stages of lactation. Fifty healthy Gir × Holstein cows were divided into two groups based on parity number, i.e., primiparous (n = 18) and multiparous (n = 33). They were monitored from the first to the 43rd milking. The colostrum volume and quality were evaluated using a colostrometer, Brix refractometer, and Colostro Balls after the first milking, in addition to the colostral IgG levels measured using sandwich ELISA as a reference standard. On-farm tests showed that the colostrum samples obtained from Gir × Holstein cows exhibited an optimal colostrum quality based on the literature criteria, regardless of parity number; however, the IgG mass was higher in the colostrum of multiparous (201 ± 67.03g) cows than in that of primiparous (144 ± 32.40g) cows. The volume and composition of transition and whole milk were also assessed at the second to ninth, 11th, 13th, 15th, 29th, and 43rd milkings. Multiparous cows produced higher volumes of transition milk than primiparous cows. In addition, multiparous cows exhibited a higher total solids percentage in their postpartum mammary secretions than primiparous cows. A higher percentage of inhibition of specific antibodies against Escherichia coli K99 was observed in the blood serum of multiparous cows than in that of primiparous cows. The volume and composition of mammary secretions changed over time; milk production increased, whereas total solids, total IgG, and specific antibody levels against most enteropathogens decreased, regardless of parity. Additionally, an association between parity and time was observed with respect to milk yield, the Brix score (%), and specific antibody levels against the Clostridium perfringens alpha toxin in mammary sections and against coronavirus and rotavirus in blood samples. This association indicated higher values in multiparous cattle than in primiparous cattle at specific time points. In conclusion, this study reveals postpartum time-dependent changes in the physiological and immunological components in the mammary secretions and blood of crossbred Gir × Holstein cows from the first to the 43rd milking. These results will contribute to the development of future research in Gir × Holstein-specific neonatology, which is genetically adapted to tropical and subtropical countries.

Expression of Five Important Biomarkers in Non-small Cell Lung Cancer: MicroRNAs-128, miR-335-5p, miR-1254, VEGF, and K-RAS

Background: Lung cancers, such as non-small cell lung cancer (NSCLC), are the most common malignancy and leading cause of cancer-related death worldwide. MicroRNAs (miRNAs) play a role in the occurrence of lung cancer by targeting both tumor suppressor genes and oncogenes. Among them, miR-128, miR-335-5p, and miR-1254 are mainly reported to function in regulating lung cancer cell behavior. Objectives: The aim of the study was to investigate the existence and expression levels of miRNA-128, miR-335-5p, and miR-1254 as well as mRNA and protein expression levels of VEGF and mRNA expression levels of K-RAS in both peripheral blood and cancerous tissue of NSCLC patients compared to the healthy subjects. Methods: Blood and tissue samples were collected from 50 healthy donors and 50 NSCLS patients. The serum level of VEGF was measured using the commercial enzyme-linked immunosorbent assay (ELISA). The mRNA expression levels of K-RAS and VEGF, as well as the expression of miRNA-128, miR-335-5p, and miR-1254, were determined, using real-time PCR. Results: Lower mRNA expression levels of miRNA-128, miR-335-5p, and miR-1254, as well as higher mRNA expression levels of VEGF and K-RAS, were detected in both peripheral blood and tissue samples from NSCLC patients compared to those of the healthy subjects. The VEGF protein levels in the peripheral blood of the patients were also found to be significantly lower than those in the healthy subjects. Conclusions: The findings from this study suggest that miR-335-5p, miR-1254, and miR-128, may contribute to the pathogenesis and tumorigenesis of NSCLC at least in part by modulation of proliferation, migration, and angiogenesis via targeting VEGF and K-RAS signaling pathways. K-RAS and VEGF may be target genes for miR-335-5p, miR-1254, and miR-128 in NSCLC patients.

Sex ratios of olive ridley sea turtles in the North Pacific high seas: implications for climate change research

Size-class distributions and sex ratio data provide critical information to assess the demography and reproductive potential of animal populations, such as sea turtles. Sea turtle sex is determined by incubation temperature, whereby warmer temperatures during a certain period of embryonic development produce more female hatchlings. Whereas hatchling sex ratios have been well-studied, sex ratios of sea turtle foraging aggregations are less known for most populations. Here we report on sex ratios of immature and mature olive ridley sea turtles Lepidochelys olivacea in the Eastern Tropical Pacific (ETP) and Central North Pacific (CNP) based on blood plasma hormone analysis, refined with Bayesian modeling, or gonad examination. Our findings established that (1) the commercial enzyme-linked immunosorbent assay used in the present study was appropriate to analyze testosterone concentration in olive ridley blood plasma to determine foraging ground sex ratios (via a Bayesian model); (2) size-at-maturity is generally larger in males than females in the ETP; (3) the overall sex ratio among all turtles was 1.2F:1.0M; and (4) the sex ratio of smaller-sized immature turtles from both study regions was female-biased (ETP, 1.6F:1.0M and CNP, 2.1F:1.0M). These are the first sex ratio estimates for olive ridleys foraging in the high seas of the North Pacific Ocean. The data can inform population models for species conservation, particularly those that contribute to the development of conservation plans that consider climate change projections.

A Long-Term Follow-Up Study of Serum NFATc3 Levels in Pediatric Patients with Bronchial Asthma: A Prospective Observational Case-Control Investigation.

The early and precise diagnosis of asthma significantly impacts the long-term health outcomes of pediatric patients. The sensitivity and specificity of current biomarkers, however, are frequently limited. Our study aimed to evaluate the clinical significance of nuclear factor of activated T cells, cytoplasmic 3 (NFATc3), in pediatric bronchial asthma, focusing on its diagnostic and prognostic value for disease severity and recurrence. This observational, prospective case-control study involved 200 pediatric patients with bronchial asthma and 200 age- and sex-matched healthy controls, from January 2020 to January 2023. Follow-up varied from 1 to 3 years. We measured levels of NFATc3 and inflammatory cytokines interleukin-1β (IL-1β), IL-6, and TNF-α via enzyme-linked immunosorbent assay. NFATc3 and IL-1β levels at enrollment were markedly higher in patients with acute exacerbations and those classified as severe, compared with their less severe counterparts. Throughout the study, NFATc3, IL-1β, and IL-6 levels significantly increased in severe or acutely exacerbating cases. The diagnostic value of NFATc3 was assessed through receiver operating characteristic curve analysis, which showed its potential in diagnosing bronchial asthma and identifying severe cases. Spearman's analysis confirmed positive associations between peak NFATc3 and cytokine levels. Importantly, disease type, NFATc3 values at enrollment, as well as peak IL-6 levels were identified as independent risk factors for severe bronchial asthma. Elevated NFATc3 is linked with the severity of pediatric bronchial asthma and serves as a potential biomarker for diagnosis and severity prediction, emphasizing its role in guiding treatment strategies.

Association Study of rs1632947, rs1233334, and rs371194629 Polymorphisms in Human Leukocyte Antigen GGene Expression and soluble Human Leukocyte Antigen Gwith Lupus.

Systemic lupus erythematosus is a chronic autoimmune disease that has been associated with human leukocyte antigen G (HLA-G) in previous studies on immunological diseases. This study aimed to investigate the association between three HLA-G gene polymorphisms (rs1632947, rs1233334, and rs371194629) and their impact on HLA-G mRNA expression and soluble HLA-G levels in serum. Genotyping was performed using TaqMan probe PCR. RNA extraction, reverse transcription PCR, and real-time PCR assays were conducted to assess the expression of the HLA-G gene in tissue samples. Soluble HLA-G was measured using enzyme-linked immunosorbent assay in serum. Results show a significant difference in the frequency of the G allele for two 5'-untranslated region (UTR) polymorphisms of the HLA-G gene (rs1632947 and rs1233334) located at positions -964 and -725, respectively, between lupus patients and controls, with p-values of 0.009 and 0.040, respectively. In addition, the study identified the 14 bp insertion allele of the rs371194629 polymorphism located in the 3' UTR of the gene as a risk factor for lupus, with a p-value of 0.001. Our results also indicate that lupus-related alleles may increase the risk of developing the disease by upregulating the expression of HLA-G and increasing soluble HLA-G levels in serum. The findings of the study suggest that the identified genetic variants may play a role in the development of lupus and could be useful in identifying individuals at risk for the disease. These results are important for advancing our understanding of the genetic basis of lupus and may have implications for the development of new treatments and diagnostic tools for the disease.

Canine Brucellosis: A Comprehensive Review

Canine brucellosis, caused by Brucella canis, is a global infectious and zoonotic disease that poses a significant public health risk due to the close interactions between dogs and humans. In dogs, the disease can lead to abortion outbreaks, reproductive failures, lymphadenopathy, and occasionally osteoarticular issues. Asymptomatic infections are also relatively common. In humans, brucellosis typically presents with a febrile illness and non-specific symptoms such as splenomegaly, fatigue, and weakness. The disease is notably problematic in breeding programs due to reproductive failures, including infertility and abortion, leading to substantial economic losses. Transmission occurs through direct contact with infected bodily fluids, particularly during mating, or via contaminated environments. Diagnosing canine brucellosis accurately is critical and can be achieved using various methods, including bacterial cultures, serological tests like the Rose Bengal Plate Test (RBPT) and Enzyme-Linked Immunosorbent Assay (ELISA), and molecular techniques such as polymerase chain reaction (PCR). Treatment is limited, with neutering and antibiotic therapy being the main approaches, though these do not always ensure complete recovery. Preventive measures are essential and include regular screening of breeding dogs, isolation of infected animals, and strict biosecurity protocols. Given its zoonotic potential, it is crucial for veterinary professionals and pet owners to be aware of the disease. Increased awareness and future research on new diagnostic methods and effective vaccines are vital for improving control strategies and reducing the impact of canine brucellosis on both canine and human health.

Topical Reconstituted High-Density Lipoproteins Elicit Anti-Inflammatory Effects in Diabetic Wounds.

Objective: Reconstituted high-density lipoproteins (rHDL) improve wound healing in diabetes. We aimed to determine if rHDL elicit anti-inflammatory effects in diabetic wounds, as a mechanism to explain their wound healing benefits. Approach: Diabetes was induced using streptozotocin in C57Bl6/J mice. Two full-thickness wounds were placed on the subflanks of diabetic and nondiabetic (ND) mice. Phosphate-buffered saline (PBS) or rHDL (50 µg/wound/day) were applied topically. Wound closure was assessed daily. Inflammatory gene transcripts were measured by qPCR and proteins by Western blotting and enzyme-linked immunosorbent assay in wounds collected at baseline, 24 h, and 3 days postwounding. Wound macrophages were assessed by flow cytometry 7 days postwounding. The fate of fluorescent 3,3-dioctadecyloxacarbocyanine, perchlorate (DiO)-labeled rHDL was tracked by flow cytometry, fluorescent imaging, and microscopy. Results: In diabetic mice, rHDL increased wound closure rates at days 6 (+288%, p < 0.01) and 7 (+639%, p < 0.0001) postwounding, compared with PBS controls. After 3 days, rHDL-treated diabetic wounds had lower Rela (-65%) and C-C motif chemokine ligand 2 (Ccl2) (-59%) mRNA levels and CCL2 protein (29%) than PBS controls, p < 0.05 for all. Wound macrophage content was higher in diabetic than ND wounds, but rHDL did not change macrophage content or polarity. DiO-rHDL were taken up by key wound cells including fibroblasts, macrophages, keratinocytes and endothelial cells, and retained in wounds for at least 48 h. Innovation: rHDL exerts anti-inflammatory effects in diabetic wounds early postwounding, which may contribute to its wound healing properties. Conclusion: The anti-inflammatory properties of rHDL in diabetic wounds present topical rHDL as a novel treatment option for improving healing in patients with diabetic foot ulcers.

Homocysteine aggravates intestinal inflammation through promotion of 5-LOX and COX-2 in IBD

BackgroundHomocysteine (Hcy) is a pro-inflammatory molecule that has the potential to induce oxidative damage to cells and stimulate the release of inflammatory mediators. Hcy has been observed to enhance the production of inflammatory agents in vascular endothelial cells. However, the impact of Hcy on intestinal mucosal inflammation remains largely unexplored. Therefore, the objective of this study was to examine the potential of Hcy to stimulate the synthesis of inflammatory mediators and elucidate the underlying mechanisms in the intestinal mucosa.MethodsA total of 99 patients diagnosed with inflammatory bowel disease (IBD) and 10 healthy individuals were included in this study to assess the impact of homocysteine (Hcy) on the levels of leukotriene E4 (LTE4) and prostaglandin E2 (PGE2). The underlying mechanism responsible for the generation of LTE4 and PGE2 induced by Hcy was investigated using colitis rats and Caco-2 cells. 32 Sprague–Dawley rats were categorized into four groups: normal control, TNBS model, normal with Hcy injection, and TNBS model with Hcy injection. The mRNA expressions of 5-LOX, COX-2, and NF-κB were assessed using reverse transcription quantitative polymerase chain reaction (RT-qPCR). Caco-2 cells were subjected to treatment with varying concentrations (10, 20, 50, 100 μmol/L) of Hcy and incubated for different durations (1, 3, 6 h). The alterations in NF-κB activity, as well as the levels of Hcy, LTE4, and PGE2, were measured using enzyme-linked immunosorbent assay (ELISA).ResultsThe excretion of Hcy, LTE4, and PGE2 in urine exhibited significant increases in patients with inflammatory bowel disease (IBD), Crohn's disease (CD), and ulcerative colitis (UC). In addition, Hcy demonstrated a significant increase in the expression of 5-LOX, COX-2, and NF-κB, as well as elevated levels of LTE4 and PGE2 in rats with colitis. Furthermore, Hcy was found to induce NF-κB activation and nuclear translocation, thereby contributing to the enhanced synthesis of LTE4 and PGE2 in Caco-2 cells.ConclusionsHcy was found to enhance the expression of 5-LOX and COX-2 by activating NF-κB, thereby augmenting the production of LTE4 and PGE2, which ultimately exacerbates colonic inflammation in individuals with inflammatory bowel disease (IBD).