ELISA Research Articles

Novel Tricyclo[4.2.1]Nonane and Bicyclo[2.2.1]Heptane Derivatives: Synthesis, in Vitro Biological Activities and in Silico Studies.

The target benzothiazole derivatives (8a-g) were synthesized starting from norbornene. The addition of dichloroketene to norbornene followed by the reduction of chlorine atoms were synthesized tricyclo[4.2.1.02,5]non-7-en-3-one (4). The condensation of benzaldehyde derivatives with compound 4 were obtained chalcone analogs (6a-g). Finally, benzothiazole derivatives (8a-g) were obtained by the reaction of the chalcone analogs with 2-aminobenzothiol in an acidic medium. The antiproliferative activities of compounds 6a-g and 8a-g were determined against C6 (rat brain tumor) and HeLa (human cervical carcinoma) cell lines using a BrdU cell proliferation ELISA assay with 5-fluorouracil (5-FU) as a standard. In both series, when compared with 5-FU (IC50=3.8 μM for C6 and 16.33 μM for HeLa), the most active compounds against C6 cells were 6a and 8g with IC50 values of 14.13 μM and 29.99 μM, respectively. With this, 6a, 6e, 6f, and 8b were the most active compounds against HeLa cells with IC50 values of 0.8, 1.21, 19.33 and 18.13 μM, respectively. Additionally, the SwissADME online web tool was used to predict the physicochemical and ADME properties of the tested compounds. The results showed that all compounds possess promising predicted physiochemical and pharmacokinetic properties and comply with Lipinski's rule of 5, indicating that they are predicted to be orally bioavailable that they possess a predicted bioavailability score of 0.55. Furthermore, in the SwissADME Boiled-Egg chart, all compounds showed high predicted GIT absorption, while compounds 6a-g showed blood-brain barrier (BBB) permeation, and compounds 8a-g did not. Moreover, none of the compounds was (P-gp) substrates. This study investigated the potential interactions between the antiapoptotic proteins Bcl-2, Bcl-xl, Bcl-w, Brag-1, Bfl-1, and Mcl-1 and compounds 6a, 8b, and 8g through molecular docking studies. The findings suggest that these compounds may effectively inhibit antiapoptotic proteins, as evidenced by significant hydrogen bonds and hydrophobic interactions, particularly with Bcl-xl.

Study on anti-adhesion effect and mechanism of dynamic and static stress stimulation during early healing process of rat Achilles tendon injury

To investigate the anti-adhesive effect and underlying mechanism of dynamic and static stress stimulation on the early healing process of rat Achilles tendon injury. Achilles tendon tissues of 15 male Sprague Dawley (SD) rats aged 4-6 weeks were isolated and cultured by enzyme digestion method. Rat Achilles tendon cells were treated with tumor necrosis factor α to construct the Achilles tendon injury cell model, and dynamic stress stimulation (dynamic group) and static stress stimulation (static group) were applied respectively, while the control group was not treated. Live/dead cell double staining was used to detect cell activity, ELISA assay was used to detect the expression of α smooth muscle actin (α-SMA), and real-time fluorescence quantitative PCR was used to detect the mRNA expression of collagen type Ⅰ (COL1A1), collagen type Ⅲ (COL3A1), and Scleraxis (SCX). Thirty male SD rats aged 4-6 weeks underwent Achilles tendon suture and were randomly divided into dynamic group (treated by dynamic stress stimulation), static group (treated by static stress stimulation), and control group (untreated), with 10 rats in each group. HE staining and scoring were performed to evaluate the healing of Achilles tendon at 8 days after operation. COL1A1 and COL3A1 protein expressions were detected by immunohistochemical staining, α-SMA and SCX protein expressions were detected by Western blot, and maximum tendon breaking force and tendon stiffness were detected by biomechanical stretching test. In vitro cell experiment, when compared to the static group, the number of living cells in the dynamic group was higher, the expression of α-SMA protein was decreased, the relative expression of COL3A1 mRNA was decreased, and the relative expression of SCX mRNA was increased, and the differences were all significant ( P<0.05). In the in vivo animal experiment, when compared to the static group, the tendon healing in the dynamic group was better, the HE staining score was lower, the expression of COL1A1 protein was increased, the expression of COL3A1 protein was decreased, the relative expression of SCX protein was increased, the relative expression of α-SMA protein was decreased, and the tendon stiffness was increased, the differences were all significant ( P<0.05). Compared with static stress stimulation, the dynamic stress stimulation improves the fibrosis of the scar tissue of the rat Achilles tendon, promote the recovery of the biomechanical property of the Achilles tendon, and has obvious anti-adhesion effect.

Role of R-spondin 2 on osteogenic differentiation of bone marrow mesenchymal stem cells and bone metabolism in ovariectomized mice

To investigate the effects of R-spondin 2 (Rspo2) on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and bone mineral content in ovariectomized mice. BMSCs were extracted from the bone marrow of the long bones of 7 4-week-old female C57BL/6 mice using whole bone marrow culture and passaged. After the cell phenotype was identified by flow cytometry, the 3rd generation cells were co-cultured with 10, 20, 40, 80, and 100 nmol/L Rspo2. Then, the cell activity and proliferative capacity were determined by cell counting kit 8 (CCK-8), and the intervention concentration of Rspo2 was screened for the subsequent experiments. The osteogenic differentiation ability of BMSCs was detected by alkaline phosphatase (ALP) staining, and the mRNA levels of osteogenesis-related genes [RUNX family transcription factor 2 (Runx2), collagen type Ⅰ alpha 1 (Col1), osteocalcin (OCN)] were detected by real-time fluorescence quantitative PCR (RT-qPCR). In addition, 18 10-week-old female C57BL/6 mice were randomly divided into sham operation group (sham group), ovariectomy group (OVX group), and OVX+Rspo2-intervention group (OVX+Rspo2 group), with 6 mice in each group. The sham group only underwent bilateral back incision and suturing, while the other two groups established osteoporosis mouse models by bilateral ovarian castration. Then, the mice were given a weekly intraperitoneal Rspo2 (1 mg/kg) treatment in OVX+Rspo2 group and saline at the same dosage in sham group and OVX group. After 12 weeks of treatment, the body mass and uterus mass of the mice were weighed in the 3 groups to assess whether the OVX model was successfully prepared; the tibia bones were stained with HE and immunohistochemistry staining to observe the changes in tibial bone mass and the expression level of Runx2 protein in the bone tissues. Blood was collected to detect the expressions of bone metabolism markers [ALP, OCN, type Ⅰ procollagen amino-terminal peptide (PINP)] and bone resorption marker [β-collagen degradation product (β-CTX)] by ELISA assay. Micro-CT was used to detect the bone microstructure changes in the tibia, and three-dimensional histomorphometric analyses were performed to analyze the trabeculae thickness (Tb.Th), trabeculae number (Tb.N), trabeculae separation (Tb.Sp), and bone volume fraction (BV/TV). CCK-8 assay showed that Rspo2 concentrations below 80 nmol/L were not cytotoxic ( P>0.05), and the cell viability of 20 nmol/L Rspo2 group was significantly higher than that of the control group ( P<0.05). Based on the above results, 10, 20, and 40 nmol/L Rspo2 were selected for subsequent experiments. ALP staining showed that the positive cell area of each concentration of Rspo2 group was significantly larger than that of the control group ( P<0.05), with the highest showed in the 20 nmol/L Rspo2 group. The expression levels of the osteogenesis-related genes (Runx2, Col1, OCN) significantly increased, and the differences were significant between Rspo2 groups and control group ( P<0.05) except for Runx2 in the 40 nmol/L Rspo2 group. In animal experiments, all groups of mice survived until the completion of the experiment, and the results of the body mass and uterus mass after 12 weeks of treatment showed that the OVX model was successfully prepared. Histological and immunohistochemical staining showed that the sparseness and connectivity of bone trabecula and the expression of Runx2 in the OVX group were lower than those in the sham group, whereas they were reversed in the OVX+Rspo2 group after treatment with Rspo2, and the differences were significant ( P<0.05). ELISA assay showed that compared with the sham group, the serum bone metabolism markers in OVX group had an increase in ALP and a decrease in PINP ( P<0.05). After Rspo2 intervention, PINP expression significantly reversed and increased, with significant differences compared to the sham group and OVX group ( P<0.05). The bone resorption marker (β-CTX) was significantly higher in the OVX group than in the sham group ( P<0.05), and it was significantly decreased in the OVX+Rspo2 group when compared with the OVX group ( P<0.05). Compared with the sham group, Tb.Th, Tb.N, and BV/TV significantly decreased in the OVX group, while Tb.Sp significantly increased ( P<0.05); after Rspo2 intervention, all of the above indexes significantly improved in the OVX+Rspo2 group ( P<0.05) except Tb.Th. Rspo2 promotes differentiation of BMSCs to osteoblasts, ameliorates osteoporosis due to estrogen deficiency, and promotes bone formation in mice.

Approaching onchocerciasis elimination in Equatorial Guinea: Near zero transmission and public health implication.

Onchocerciasis and lymphatic filariasis (LF) are endemic in Equatorial Guinea with notable variations in disease incidence between island and mainland regions. Historically, efforts to control and map these diseases were concentrated in Bioko Island, where loiasis is absent, allowing for targeted onchocerciasis interruption strategies. With the cessation of onchocerciasis transmission on Bioko and no reported cases on Annobon island, assessing the transmission status in the previously unaddressed mainland region has become imperative. Mapping efforts in mainland Equatorial Guinea have proven low to moderate level of transmission for LF and onchocerciasis, although the results so far have not been very conclusive. The current study aims to update the prevalence estimates for onchocerciasis and LF in mainland Equatorial Guinea using various diagnostic techniques. This is the first cross-sectional study carried out to estimate the prevalence of onchocerciasis and LF in the mainland area of Equatorial Guinea, from September to December 2019, based on the combination of skin snip biopsies, thick blood smears, laboratory serological tests (ELISA tests for the detection of IgG4 antibodies against Onchocerca volvulus recombinant antigen Ov16 and Wuchereria bancrofti recombinant antigen Wb123) and molecular laboratory tests. Frequencies and prevalence rates, along with 95% confidence intervals for interval estimation of a binomial proportion, were computed. The overall onchocerciasis seroprevalence calculated for the study was 0.3% (95% CI: 0.1 to 0.5%). Microscopic examination of skin biopsies from the eight individuals seropositive for Ov16, out of the 3951 individuals initially tested, revealed no O. volvulus microfilariae. However, DNA extracted from one skin snip was successfully amplified, with subsequent sequencing confirming the presence of O. volvulus. Among the 3951 individuals, 182 were found to have anti-Wb123 antibodies, suggesting exposure to W. bancrofti, with an estimated seroprevalence of 4.6% (95% CI: 4.0 to 5.3%). Microscopy and Filaria-real time-PCR (F-RT-PCR) analysis for W. bancrofti were negative across all samples. The findings indicate that onchocerciasis may no longer constitutes a public health problem in Equatorial Guinea, positioning the country on the verge of achieving elimination. Additionally, the mapped prevalence of LF will facilitate the formulation of national strategies aimed at eradicating filariases countrywide.

Saliva NIR spectroscopy and Aquaphotomics: a novel diagnostic approach to Paratuberculosis in dairy cattle

IntroductionParatuberculosis is a granulomatous intestinal infection that affects ruminant animals worldwide. The disease is often detected when most animals are already infected due to the long incubation period and the high transmissibility of the infectious agent. The lack of a comprehensive method to diagnose Paratuberculosis is a global challenge. Therefore, a non-destructive, fast, and cost-effective diagnostic method for early detection of Paratuberculosis is crucial.MethodsNear-infrared spectroscopy (NIRS) and Aquaphotomics have the potential to diagnose the disease by detecting changes in biological fluids. This study aimed to investigate the diagnostic ability of NIRS and Aquaphotomics for Paratuberculosis in dairy cattle by monitoring and data mining of saliva. The diagnostic models were developed according to saliva spectra of dairy cattle in the NIR range and 12 water absorbance bands from 100 to 200 days after calving in two groups: positive and negative, based on the same results of seven ELISA tests of blood plasma, as a reference test.ResultsBoth NIRS and Aquaphotomics methods had high diagnostic accuracy. Using QDA and SVM models, 99% total accuracy, 98% sensitivity, and 100% specificity were achieved in internal validation. The total accuracy in external validation was 90%. This study presents two novel approaches to diagnosing Paratuberculosis in dairy cattle using saliva.DiscussionThe study found that changes in water absorbance spectral patterns of saliva caused by complex physiological changes, such as the amount of antibody related to Paratuberculosis in dairy cattle as biomarkers, are crucial in detecting Paratuberculosis using NIRS and Aquaphotomics.

Abstract 4134539: Hybrid Cardiomyocyte Targeting Peptide Down-regulates NF-κB Pathway in Human Cardiomyocytes

Introduction: Role of NF-κB driven inflammation leading to heart failure with preserved ejection fraction has received little attention. Our previous in vivo work with cardiac targeting peptide (CTP) in guinea pigs identified suppression of NF- κB pathway by CTP. In the current work we sought to further study the effects of a hybrid version of CTP (hCTP: amino acids at positions 3 and 11 substituted with D-amino acids) on TNF-α mediated NF-κB activation, as well as expression of its downstream inflammatory markers IL-1β and IL-8. Methods: Human cardiomyocytes (CMCs) were transfected using Lipofectamine 3000 with a reporter luciferase plasmid carrying an NF-κB promoter site upstream of the luciferase gene. Cells were treated with various concentrations of hCTP for 24 hours, followed by a TNF-α challenge (20ng/mL), addition of luciferin as substrate, and measurement of luminescence. Cell viability in response to above manipulations was assessed by FACS using live-dead stain. CMCs were treated as above, and cell culture supernatants analyzed for IL-8 levels using human ELISA kit. In another set of experiments, CMCs were transfected with NF-κB reporter plasmid, and after treatment for 24 hours with various concentrations of hCTP, incubated with 10nM angiotensin 2 (Ang2) and 100nM phenylephrine (PE) for 24hrs to assess levels of IL-1β in the cell culture media using an ELISA assay. Results: hCTP inhibited TNF-α mediated NF-κB activation with a dose-response curve, with maximum inhibition with hCTP seen at 10µM concentrations (p&lt;0.001). Treatment with hCTP inhibited the TNF-α induced IL-8 secretion with significant decreases seen even at the 1µM dose. Similarly, secretion of IL-1β production by CMCs in response to Ang2/PE stimulation was suppressed by hCTP even at the lowest tested dose (1µM; p&lt;0.5). Conclusions: Our data suggests that hCTP inhibited NF-κB pathway transcriptional activity in response to TNF-α stimulation in a dose-dependent fashion in human CMCs. Furthermore, treatment with hCTP inhibited TNF-α induced IL-8 and IL-1β secretion even at doses as low as 1µM. The ability of hCTP to efficiently targeting this inflammatory pathway suggests that it has therapeutic potential in the treatment of heart failure. Further in vivo studies are ongoing to test this hypothesis.

Abstract 4139148: Mechanisms of Maternal Cardiovascular Disease Development in a Novel Mouse Model of Pregnancy-Induced Diabetes

Background: Women with gestational diabetes mellitus (GDM) are more likely to develop cardiovascular disease (CVD) within 3 to 10 years following pregnancy. Endothelial dysfunction is a mechanism implicated in both hyperglycemia and CVDs, and it may constitute the missing link in this interaction. One reason for the lack of progress into the cardiovascular complications of GDM is the need for appropriate preclinical models that mimic human GDM. Also, the exact mechanisms mediating increased future cardiovascular risk in maternal with pregnancy-induced diabetes is unclear. Aim: To determine the mechanisms underlying cardiovascular disease in hormonal model of GDM. Methods: Sixteen week-old C57BL/6J female mice were treated with insulin receptor antagonist (S961) or saline (n=4) from the second trimester of pregnancy until delivery to develop a model of GDM. Glucose tolerance test was determined by glucometer and fasting insulin by colorimetry. Cardiac structure and function were measured by echocardiography (VEVO 3100) and pulse wave velocity was measured by Pulse Wave Doppler. Exercise tolerance test was performed on treadmill. Plasma endothelin-1 and nitrite were measured by ELISA and colorimetric assay. Aortic SIRT-1 was determined by western blot and aortic vascular reactivity was determined by wire myograph. Results: S961-induced GDM mice exhibit hyperglycemia (169.2 ± 3.6 vs. 105.5 ± 10.8mg/dL, P&lt;0.004 ) and hyperinsulinemia (1.9 ± 0.2 vs. 0.8 ± 0.1ng/ml , p&lt;0.01 ) which mimics the condition observed in human GDM. Interestingly, S961-induced GDM mice went on to develop systolic and diastolic dysfunction, vascular stiffness (3.7 ± 0.1 vs. 3.1 ± 0.08m/s, p&lt;0.005 ), exercise intolerance and increased thickness of the cardiac wall at 10 weeks post-delivery. Plasma ET-1 was elevated in our model during pregnancy (28.1 ± 1.7 vs. 5.2 ± 0.2pg/ml, p&lt;0.002 ) and at 10 weeks post-delivery (19.9 ± 2.9 vs. 6.7 ± 0.7pg/ml, p&lt;0.05 ). Circulating NO and aortic expression of sirtuin-1 (SIRT 1), that is involved in endothelial longevity is reduced in GDM mice. Additionally, vascular relaxation to acetylcholine was attenuated in S961-induced GDM mice. Conclusion: We have developed a novel mouse model of GDM similar to that observed in pregnant women by treatment with S961. Moreover, S961 treated mice develop cardiac dysfunction and vascular stiffness at 10 weeks post-delivery. Alterations in ET-1, SIRT 1, and NO levels may be responsible for the development of CVD in this model.

MiR203a-3p as a potential biomarker for synovial pathology associated with osteoarthritis: a pilot study.

Osteoarthritis (OA) is a degenerative musculoskeletal disease that significantly impacts the quality of life. Currently, no validated biomarkers for early detection of OA are defined. The possibility of discovering OA biomarkers is the focus of this study. Human primary OA synovial cells (SVs), isolated from discarded joint tissue of patients with Kellgren & Lawrence score (KL) < 3, (mild/moderate) and KL ≥ 3 (severe), were characterized by FACS analysis. Through qRT-PCR and ELISA assays the inflammation, fibrosis status and the different miRNAs expression has been investigated. The role of miR-203a-3p and its precursors were evaluated through gain and loss of function study, IL-1β synoviocytes treatments and methylation analysis of miR203a promoter. The qRT-PCR analysis of miR203a-3p and pre-miR203a on plasma (isolated 24h before surgery, 3days and 1month after surgery) and synovial fluid (recovered during the surgery) were done to support our in vitro data. MiR203a-3p expression is inversely correlated with the aggressiveness of OA, modulating the expression of epithelial to mesenchymal transition (EMT) and pro-inflammatory factors, as well as regulating the expression of secreted protein acidic and rich in cysteine (SPARC) mRNA. Methylation analysis of the miR203a promoter and SVs IL-1β treatment's highlighted the impact of inflammation on miR203a-3p and pre-miR203a expression; as confirmed by both miRNAs detection in biological fluids derived from patients with severe OA. Our preliminary results suggest that miR-203a-3p might be a potential candidate for staging OA progression and a new protective/predictive biomarker for synovial OA degeneration. Further studies are needed to validate its potential impact on OA.

Koala ocular disease grades are defined by chlamydial load changes and increases in Th2 immune responses

IntroductionThis study employs bulk RNA sequencing, PCR, and ELISA assays to analyze the pathological factors affecting the outcomes of C. pecorum ocular infections in koalas. It investigates the immune responses and gene expression profiles associated with various stages of koala ocular chlamydiosis.MethodsA cohort of 114 koalas from Queensland, Australia were assessed, with 47% displaying clinical signs of ocular disease. Animals were classified into three cohorts: acute active disease (G1), chronic active disease (G2), and chronic inactive disease (G3), along with subclinical Chlamydia pecorum positive (H2) and healthy (H1) cohorts.ResultsAnalysis of clinical, microbiological, humoral immune and cellular immune biomarkers revealed varying chlamydial loads and anti-chlamydial IgG levels across disease grades, with a negative correlation observed between ocular chlamydial load and anti-chlamydial IgG. Koala ocular mucosa gene expression analysis from 27 koalas identified shared expression pathways across disease cohorts, with a significant upregulation of IFNγ expression and tryptophan metabolism in all disease stages.DiscussionThese findings help elucidate immune response dynamics and molecular pathways underlying koala ocular chlamydiosis, providing insights crucial for disease management strategies.

TMIC-12. LITT RESULTS IN ROBUST T CELL INFILTRATION WITHIN THE TUMOR MICROENVIRONMENT IN A BRAIN METASTASIS MURINE MODEL

Abstract INTRODUCTION Laser interstitial thermal therapy (LITT) is a novel minimally invasive neurosurgical technique used to ablate intra-axial brain tumors with real-time thermometry. The impact of LITT on the brain metastatic tumor microenvironment (TME) is unknown in patients due to limitations of tumor tissue collection post-LITT. The primary objective of this study was to describe the kinetics of immunologic infiltration within the TME in a melanoma brain metastasis model. METHOD: C57BL6 mice underwent intra-cranial implantation of B16F10-OVA tumor cells and treated with LITT using a custom-made murine LITT system developed in our laboratory. The tumors were treated to 42°C (near infra-red 1064 nm DPSS laser system) 7 days post-surgery. Tumors were collected 2 days and 10 days post-LITT for immune-phenotyping using flow cytometry. Blood was collected 6 h and 48 h post-LITT to perform the ELISA for IFN-g and TNF-a. RESULTS LITT treatment triggered a cascade of local infiltration of immune cells at different time points. 2-days post-LITT resulted in increased CD8 T cells (p&amp;lt;0.05), macrophage (p&amp;lt;0.05) and microglia infiltration (p&amp;lt;0.01) within the TME compared to control and sham surgery animals. By 10 days after LITT, significant infiltration by CD8 T cells (p&amp;lt;0.0001) and M1 macrophages (p&amp;lt;0.0001) was observed. The CD8 T cells were T central memory (TCM) and T effector memory (TEM). Moreover, comparison between 2 days and 10 days after LITT revealed significant increase of TEM (p&amp;lt;0.0001) and TCM (p&amp;lt;0.001) 10 days post-LITT. In an ELISA assay, non-significant differences were observed in IFN-g and TNF-a level at 48 h. CONCLUSIONS LITT enhances the recruitment of cytotoxic CD8 T cells within TME in a time-dependent manner. The ability of LITT to recruit TCM and TEM to the TME strongly support its potential therapeutic benefit, especially in the context of immunotherapy.

Exploring the mechanism of Suxin Hugan Fang in treating ulcerative colitis based on network pharmacology

As a traditional Chinese medicine formula used in clinical practice for an extended period, Suxin-Hugan-Fang (SXHGF) exhibits excellent anti-inflammatory properties. However, the efficacy of SXHGF in treating ulcerative colitis (UC) and its mechanism of action are still unclear. In this study, the therapeutic effects of SXHGF on UC were evaluated using network pharmacology and experimental validation, while also investigating its mechanism of action. By administering DSS to C57BL/6 mice to construct a mouse model of ulcerative colitis, the therapeutic effect of SXHGF on ulcerative colitis was evaluated based on weight loss percentage, disease activity index, colon length changes, and pathological conditions as indicators. The main chemical components of SXHGF were determined by LC-MS-QTOF method. The potential targets and mechanisms of action of SXHGF in the treatment of UC were inferred using bioinformatics methods, and further validated through ELISA, IHC, and Western blotting assays. The experimental results demonstrate that SXHGF can suppress oxidative stress and oxidative damage in the colon tissue of UC mice, and alleviate DSS-induced ulcerative colitis by inhibiting the JAK2/STAT3 and NFκB pathways.

Chemical analysis, antibacterial and anti-inflammatory effect of Achillea fragrantissima essential oil growing wild in Egypt

BackgroundAchillea fragrantissima (F. Asteraceae) is traditionally used to treat skin infections and inflammation. The present work intended to prepare essential oils (EOs) from A. fragrantissima aerial parts growing widely in Egypt and investigate its antibacterial activity against skin-related pathogens and in vitro cell-based anti-inflammatory activity.MethodsEOs of the fresh aerial parts were extracted by hydrodistillation (HD), microwave-assisted hydrodistillation (MAHD), and head-space (HS), while those of the dried ones were prepared by supercritical fluid (SF). The result EOs were analyzed using GC/MS. The antibacterial activity was evaluated alongside Pseudomonas aeruginosa ATCC 9027, Escherichia coli ATCC 8739, Staphylococcus aureus ATCC 25923, Streptococcus pyogenes ATCC 12344, Clostridium perfringens ATCC 13124 by agar diffusion, microwell dilution, and biofilm formation tests. The anti-inflammatory activity was evaluated by measuring tumor necrosis factor-alpha (TNF-α), interleukin 2 (IL-2), and 6 (IL-6) in lipopolysaccharides (LPS)- stimulated RAW 264.7 cells using ELISA assays in addition, expression of nitric oxide synthase (iNOS) was measured via western blot.ResultsThe SF method gave the highest EO yield (1.50 mL v/w). Oxygenated components constituted the highest percentage in the four methods, 84.14, 79.21, 73.29 and 33.57% in the HS, HD, MAHD, and SF, respectively. Moreover, variation in the amount of identified compounds was apparent; in HS EO α-thujone (29.37%), artemisia ketone (19.59%), and santolina alcohol (14.66%) are major components, while α-thujone (20.38%) and piperatone (12.09%) were significant in HD. Moreover, ( +)-spathulenol (12.22%) and piperatone (10.48%) were significant in MAHD, while piperatone (14.83%) and β-sitosterol (11.07%) were significant in SF EO. HD, MAHD, and SF EOs exhibited susceptibility against P. aeruginosa (IZ = 9–14 mm), E. coli (11–13 mm), and C. perfringens (IZ = 10–14 mm) in agar diffusion assay. MAHD EOs demonstrated potent growth inhibition (MICs = 0.25–2 mg/mL), followed by HD EOs (MICs = 13–52 mg/mL) to all tested microorganisms in well microdilution assay. Also, they exert MBC values equal to or higher than the MICs. Furthermore, SF EOs inhibited the biofilm formation of all tested microorganisms by 65.12—80.84%. Specifically, MAHD and HD EOs efficiently suppress the biofilm of S. pyogenes (77.87%) and P. aeruginosa (60. 29%), respectively. Ultimately, HD and SF EOs showed anti-inflammatory activity by suppressing the TNF-α, IL-2, and IL-6 release and iNOS expression in LPS-stimulated RAW 264.7 macrophages.ConclusionA. fragrantissima EO is rich in oxygenated volatile compounds with antibacterial and anti-inflammatory activities. It is encouraged as a bioactive agent for adjusting skin infections, though additional studies are essential for their safety in clinical settings.

Corynoxine suppresses lung adenocarcinoma proliferation and metastasis via inhibiting PI3K/AKT pathway and suppressing Cyclooxygenase-2 expression

BackgroundLung adenocarcinoma (LUAD) is the most common lung cancer subtype, and the prognosis of affected patients is generally poor. The traditional Chinese medicine Uncaria rhychophaylla has been reported to exhibit anti-lung cancer properties. Accordingly, the main bioactive ingredient in Uncaria rhychophaylla, Corynoxine, may hold great value as a treatment for lung cancer.MethodsThe impact of Corynoxine on the viability of LUAD cells was assessed using the Cell Counting Kit-8 (CCK-8) assay. Apoptosis in A549 cells was evaluated via flow cytometry. Migration and invasion capabilities were determined through wound healing and Transwell assays, respectively. The key pathways targeted by Corynoxine in LUAD were identified using a network pharmacology approach. Additionally, Western immunoblotting, quantitative real-time PCR (qRT-PCR), and ELISA assays were conducted to validate the underlying mechanisms. The in vivo anti-tumor efficacy of Corynoxine was assessed in xenograft nude mice.ResultsIn this study, Corynoxine treatment was found to markedly suppress in vitro LUAD cell proliferative, migratory, and invasive activity. It additionally downregulated Vimentin and promoted E-cadherin upregulation consistent with the disruption of epithelial-mesenchymal transition (EMT) induction while also accelerating apoptotic death. Furthermore, network pharmacology analysis revealed that the PI3K/AKT pathway is a potential target of Corynoxine in LUAD. In vitro assays demonstrated that treatment with Corynoxine resulted in the suppression of PI3K/AKT signaling and a consequent drop in cyclooxygenase-2 (COX-2) expression. These findings were further confirmed in vivo in mice harboring A549 tumor xenografts in which Corynoxine was able to interfere with the PI3K/AKT/COX-2 signaling axis.ConclusionThis study elucidated the potential effects of Corynoxine in suppressing proliferation and metastasis in LUAD, along with investigating the underlying mechanisms. These data highlight the promise of Corynoxine as a novel therapeutic tool for the treatment of individuals diagnosed with LUAD.

Pigment epithelium-derived factor exerts neuroprotection in oxygen-induced retinopathy by targeting endoplasmic reticulum stress and oxidative stress

Endoplasmic reticulum (ER) stress and oxidative stress have been involved in the occurrence of neuronal apoptosis in ischemic retinopathy. Pigment epitheliu-derived factor (PEDF) is well known for its multifunctional properties, including neuroprotection, anti-inflammation and antioxidant. However, the association between PEDF and ER stress or oxidative stress in ischemic retinopathy remain incompletely understood. In this study, the concentration of the key factor of ER stress C/EBP homologous protein (CHOP) in aqueous humor (AqH) and vitreous samples of proliferative diabetic retinopathy (PDR) patients were measured by ELISA assays. Oxygen-induced retinopathy (OIR) mice model was established and PEDF intravitreal injections were conducted. Primary bone marrow derived macrophages (BMDMs) were isolated and cultured under hypoxic conditions in vitro. Western blotting, real-time RT-PCR, immunofluorescence, transmission electron microscopy (TEM), TUNEL assays were performed to explore roles of PEDF on ER stress and oxidative stress, as well as subsequently neuronal apoptosis under hypoxic conditions in vivo and in vitro. The results revealed that ER stress and oxidative stress were notably activated under hypoxic conditions. We also observed that hypoxia evoked ultrastructural damage of ER and mitochondrion in the retina. However, PEDF significantly prevented ER stress and oxidative stress, as well as the damage of ultrastructure, resulting in diminution of photoreceptor apoptosis in OIR retinas. These results indicate that PEDF may play its neuroprotection role through inhibiting ER stress and oxidative stress in ischemic retinopathy, which is a novel molecular mechanism of PEDF protecting photoreceptors from ischemic damage, thereby suggesting that PEDF is an effective therapeutic agent for the treatment of neuron damage in ischemic retinal diseases.

Simultaneous Stimulation of Peripheral Blood Mononuclear Cells with CpG ODN2006 and α-IgM Antibodies Leads to Strong Immune Responses in Monocytes Independent of B Cell Activation

CpG ODN2006 is widely used both in vitro and in vivo to achieve B cell activation and has been previously applied in clinical trials as an adjuvant and anti-cancer agent. Recent studies have demonstrated the benefit of combining CpG ODN2006 with α-IgM antibodies to obtain optimal B cell activation in vitro. In this study, we expanded the knowledge of how both agents affect other types of peripheral blood mononuclear cells (PBMCs), thereby highlighting beneficial and potentially unfavorable properties of the combination of CpG ODN2006 and α-IgM when applied beyond isolated B cells. We elucidated the effects of both compounds on mixed PBMCs, as well as on B cell- and monocyte-depleted PBMCs, allowing us to distinguish between direct effects and indirect influences mediated by other interacting immune cells. Flow cytometry was used to measure the expression of surface markers and intracellular cytokines, while ELISA and multiplex assays were performed to determine cytokine secretion. Our results revealed that stimulation of mixed PBMCs with CpG ODN2006 and α-IgM strongly increased cytokine secretion, primarily originating from α-IgM-stimulated monocytes. Monocyte activation was confirmed by increased CD86 and HLA-DR expression and occurred independently of B cells. The high level of monocyte-derived cytokines after α-IgM exposure did not affect B cell activation. However, it represents a rather unfavorable property for clinical applications. In conclusion, α-IgM is a potent inducer of cytokine production in monocytes. Based on our findings we hypothesize that significant side effects on monocytes can occur when using α-IgM to enhance CpG ODN2006’s efficacy on B cells, particularly in clinical settings.

Study on the mechanism of OSM participating in myocardial fibrosis by inhibiting TGFβ-induced EndMT of cardiac microvascular endothelial cells through SPARC/SMAD signaling.

Cardiac fibrosis constitutes a crucial element in the progression of diverse chronic cardiac conditions. Notably, a significant correlation has been observed between the endothelial-to-mesenchymal transition (EndMT) and the emergence of cardiac fibrosis. To investigate mechanisms, we employed immunofluorescence for α-SMA and CD31 analysis, Western blotting for CD34, vimentin, and SPARC overexpression. CCK8, wound healing, and transwell assay-assessed cell viability, invasion, and migration. SPARC overexpression plasmid was constructed and validated by Western blotting. Fibrosis levels were quantified via Masson staining, and collagen 1 and 3 expressions were measured using ELISA assays. Notably, in TGF-β-induced H5V cells, the downregulation of CD31 and CD34 expression, along with the upregulation of α-SMA and vimentin, suggests the induction of EndMT in cardiac fibrosis. Interestingly, OSM treatment mitigated EndMT progression, cell invasion, migration, and the expression of p-SMAD2, p-SMAD3, and SPARC in TGF-β-treated H5V cells. Further analysis revealed that OSM alleviated TGFβ-induced EndMT, invasion, and migration of cardiac microvascular endothelial cells by suppressing SPARC/SMAD signaling. Moreover, OSM therapy notably mitigated myocardial tissue fibrosis, along with a reduction in the expression of collagen 1, collagen 3, α-SMA, and CD34, while augmenting CD31 and vimentin expression in ISO-induced myocardial tissue. Additionally, OSM exhibited the ability to suppress myocardial tissue fibrosis and the expression of EndMT markers as well as SPARC/SMAD signals in ISO-induced myocardial tissue. Our comprehensive analysis unveiled that OSM contributes significantly to myocardial fibrosis modulation by inhibiting TGFβ-mediated EndMT in myocardial microvascular endothelial cells via SPARC/SMAD signaling.

Astaxanthin mediated repair of tBHP-Induced cellular injury in chondrocytes

ABSTRACT Objective This study investigates how astaxanthin (AST) counters tert-butyl hydroperoxide (tBHP)-induced cellular damage in C28/I2 chondrocytes, focusing on the circ-HP1BP3/miR-139-5p/SOD1 signaling pathway and its use in sustained-release microspheres for osteoarthritis treatment. Methods We employed a variety of techniques including real-time quantitative PCR, Western blot, ELISA, and dual-luciferase reporter gene assays to explore AST's molecular effects. Additionally, the efficacy of AST-loaded sustained-release microspheres was evaluated in vitro and in a mouse model of osteoarthritis. Results AST significantly enhanced SOD1 expression, reducing apoptosis and inflammation in damaged cells. The AST-loaded microspheres showed promising in vitro drug release, improved cell viability, and reduced oxidative stress. In the osteoarthritis mouse model, they effectively decreased joint inflammation and increased the expression of chondrocyte markers. Conclusion Astaxanthin effectively mitigates oxidative stress and inflammation in chondrocytes via the circ-HP1BP3/miR-139-5p/SOD1 pathway. The development of AST-loaded microspheres offers a novel and promising approach for osteoarthritis therapy, potentially extending to osteoarthritis treatment.

Prokaryotic Expression of Coat Protein Gene of Grapevine Berry Inner Necrosis Virus and Preparation of Its Polyclonal Antibody

Grapevine berry inner necrosis virus (GINV) and grapevine Pinot gris virus (GPGV) are prevalent viral diseases affecting viticulture, posing significant threats in grape-producing regions of China. Previous studies have emphasized the harmful effects of grape viruses on the grape industry all over the world. However, few reports have focused specifically on GINV. In wild grapevines, GINV infection frequently leads to grapevine fanleaf degeneration disease (GFDD). GINV often co-occurs with other grape viruses, exacerbating its harmful effects on the grapevine industry in China. In this study, we collected grapevine samples from Taizhou city, Jiangsu Province, where GINV infection was confirmed. Based on the GINV coat protein (CP) gene, we developed a high-throughput and high-sensitivity direct antigen-coated ELISA and Dot blot assay for field diagnosis of GINV CP in grape samples. The CP gene was cloned from GINV-infected grape samples, and the GINV CP was expressed using the pET30(a) vector. Specific polyclonal antiserum CPGINV was generated by immunizing rabbits with the purified protein, and its sensitivity was determined to be satisfactory. Leveraging the high accuracy and sensitivity of the CPGINV antiserum, we developed a rapid, precise, and scalable diagnostic method for GINV in the grapevine industry. The established ELISA and Dot blot assays successfully detected GINV-infected grapevine samples. The occurrence of GINV is relatively common in China, which poses a risk of transmission and threatens the healthy development of the grape industry. Therefore, this study prepared CPGINV antiserum and established an efficient, rapid, sensitive, accurate, and high-throughput diagnostic method, providing a foundational approach for the prevention and control of vitis viral diseases.

A single dose recombinant AAV based CHIKV vaccine elicits robust and durable protective antibody responses in mice.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that is responsible for Chikungunya fever, which is characterized by fever, rash, and debilitating polyarthralgia. Since its re-emergence in 2004, CHIKV has continued to spread to new regions and become a severe health threat to global public. Development of safe and single dose vaccines that provide durable protection is desirable to control the spread of virus. The recombinant adeno-associated virus (rAAV) vectors represent promising vaccine platform to provide prolonged protection with a single-dose immunization. In this study, we developed a rAAV capsid serotype 1 vector based CHIKV vaccine and evaluated its protection effect against CHIKV challenge. The recombinant AAV1 encoding the full-length structural proteins of CHIKV (named as rAAV1-CHIKV-SP) was generated in vitro by transfecting the plasmids of AAV helper-free system into HEK-293T cells. The safety and immunogenicity of rAAV1-CHIKV-SP were tested in 4-week-old C57BL/6 mice. The antibody responses of the mice receiving prime-boost or single-dose immunization of the vaccine were determined by ELISA and plaque reduction neutralizing test. The immunized mice were challenged with CHIKV to evaluate the protection effect of the vaccine. The rAAV1-CHIKV-SP showed remarkable safety and immunogenicity in C57BL/6 mice. A single dose intramuscular injection of rAAV1-CHIKV-SP elicited high level and long-lasting antibody responses, and conferred complete protection against a heterologous CHIKV strain challenge. These results suggest rAAV1-CHIKV-SP represents a promising vaccine candidate against different CHIKV clades with a simplified immunization strategy.

Faecal biomarkers in children with coeliac disease: A way forward?

Novel markers to reflect the intestinal damage in coeliac disease are needed. We studied the potential of faecal and serum neopterin, and faecal myeloperoxidase, human β-defensin-2, and lipocalin-2 in a case-control study. Data were collected from medical records and a biobank including newly diagnosed coeliac disease patients, potential coeliac disease patients and non-coeliac controls. Commercially available ELISA assays were used for measuring the biomarkers. Altogether 19 patients with coeliac disease (median age 9.0 years), 8 with potential coeliac disease (4.0 years) and 18 controls (6.5 years) were included. The highest faecal neopterin levels were seen in potential coeliac disease, followed by controls and coeliac disease (median 513 vs. 372 vs. 255 nmol/L, respectively, p = 0.016). Also, serum neopterin was highest in the potential coeliac disease group (9.8 vs. 5.5 vs. 5.9 nmol/L, p = 0.022). After age adjustment and robust variance estimation, only differences in serum neopterin remained significant. Other markers did not differ between the groups. None of the markers were significantly associated with serum transglutaminase-2 antibody levels. Differences in neopterin levels amongst patients with potential coeliac disease, coeliac disease, and controls suggest that neopterin might serve as an early disease marker.