Enzyme-linked Immunosorbent Assay Analysis Research Articles

Different polyphenols on cold plasma-induced covalent ovalbumin-polyphenols conjugates and affecting the antigenicity of OVA and its functionalities

The study investigated cold plasma (CP) treatment to modulate the antigenicity and functionality of ovalbumin (OVA) through CP-induced covalent conjugation with five structurally distinct dietary polyphenols, gallic acid (GA), syringic acid (SA), (-)-epigallocatechin gallate (EGCG), (-)-epicatechin gallate (EGC), and (-)-epicatechin (EC). Results indicated that a maximum polyphenol content of 176.63 ± 1.83 μmol/g in OVA-EGCG conjugates was obtained following CP treatment (60 s). The generation of covalent OVA-polyphenol conjugates was confirmed through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The binding sites of the five polyphenols in OVA were identified using LC-MS/MS. Different regions and quantities of binding sites were observed for the various polyphenols. Seventeen binding sites were detected in OVA-EC conjugates, whereas only one site (His371) of EGC was found in OVA-EGC conjugates. Additionally, a maximum reduction in IgE binding capacity (63.13 ± 0.87%) of OVA was achieved after GA grafting, supported by western blotting (WB) and enzyme-linked immunosorbent assay (ELISA) analyses. The masking and steric-hindrance effect on linear epitopes by polyphenol grafting play key roles in reducing OVA’s antigenicity. Moreover, the emulsifying properties and antioxidant activity of OVA were substantially improved following polyphenol grafting. Correlation analysis revealed that the hydroxyl group (-OH) content in polyphenol molecules was the critical factor determining covalent binding efficiency between OVA and polyphenols, consequently resulting in antigenicity elimination and functionality improvement of OVA. This study demonstrates that CP-induced covalent grafting of polyphenols is an effective strategy for eliminating antigenicity and modulating the functionalities of proteins.

FAP+ gastric cancer mesenchymal stromal cells via paracrining INHBA and remodeling ECM promote tumor progression

Gastric cancer (GC) mesenchymal stromal cells (GCMSCs) are the predominant components of the tumor microenvironment (TME) and play a role in the occurrence, development, and metastasis of tumors. However, GCMSCs exhibit phenotypic and functional heterogeneity. The key population of GCMSCs which are vital to tumor progression remains elusive. The expression of fibroblast activation protein (FAP) in gastric cancer was analyzed and verified using clinical pathology data and single-cell RNA sequencing database of gastric cancer patients. FAP positive GCMSCs (FAP+ GCMSCs) were isolated via flow cytometry and characterized through transcriptomic sequencing. The impact of conditioned medium from FAP+ GCMSCs on gastric cancer cell lines was assessed using Enzyme-linked immunosorbent assay (ELISA) and Western blot analyses. Additionally, immunohistochemistry (IHC) and Masson’s trichrome staining were employed to explore the association between FAP+ GCMSCs and extracellular matrix (ECM) deposition in gastric cancer tissues. Our study demonstrates that FAP is predominantly expressed in the mesenchymal stromal cells within the gastric cancer milieu. FAP+ GCMSCs exhibited enhanced proliferation, migration, contraction, and tumor-promoting capabilities compared to their FAP− counterparts. These cells significantly increased proliferation and migration of gastric cancer cells through the paracrine secretion of Inhibin Subunit Beta A (INHBA) and activation of the SMAD2/3 signaling pathway. Moreover, FAP+ GCMSCs also induced collagen deposition in ECM and then up-regulated invasion and stemness of GC cells. Mechanistically, this process was mediated by the interaction of collagen with Integrin Subunit Beta 1 (ITGB1), triggering the phosphorylation of Focal Adhesion Kinase (FAK) and Yes Associated Transcriptional Regulator (YAP). Our findings reveal that FAP+ GCSMCs enhanced the GC progression via releasing cytokine INHBA and remodeling ECM providing a theoretical basis for further exploration of tumor stromal-targeting therapy of gastric cancer.

Therapeutic potential of green tea catechins on the development of Parkinson’s disease symptoms in a transgenic A53T mouse model

ABSTRACT Objectives: This study aimed to evaluate the effects of green tea catechins on the prevention of Parkinson's disease neurobehavioral symptoms and α-synuclein blood plasma concentration in a hemizygous transgenic A53T mouse model. Methods: Thirty 6-month-old male mice were randomly assigned to three groups (n = 10/group): control, low-dose, and high-dose, receiving green tea polyphenol (GTP) treatment in their drinking water at 0%, 0.5%, and 1.5%, respectively, over a 90-day period. The efficacy of ad libitum dosing was assessed by analyzing the bioaccumulation of tea catechins in urine samples collected from metabolic cages on days 0, 30, 60, and 90, using LC/Q-TOF analysis. PD-related behavioral impairments were measured with open field and rotarod performance tests on days 0, 45, and 90. On day 90, plasma α-synuclein levels were analyzed via enzyme-linked immunosorbent assay (ELISA) to assess treatment effects. Results: Circulating tea catechin metabolites were detected in treated groups by day 30, with levels progressively increasing through day 90. By day 90, control mice exhibited significant deficits in rotarod performance, while both low- and high-dose groups maintained or improved their maximum time on the rotarod. Open field testing indicated reduced anxiety-related behavior in control mice compared to treated groups. ELISA analysis revealed significantly lower circulating α-synuclein levels in high-dose mice compared to controls. Conclusion: Our findings indicate that sustained administration of tea catechins significantly reduces circulating α-synuclein levels in blood plasma, improves motor coordination in a dose-dependent manner, and modulates anxiety-related behaviors in a PD mouse model.

Materials and Techniques of the Mural Paintings in the Church-Ossuary of the Rila Monastery, Bulgaria

This presented research examined the wall paintings in the Church-Ossuary Presentation of the Blessed Virgin, part of the most important Rila Monastery complex in Bulgaria, painted by the painters from Mount Athos in 1795. The painting materials used to create the unique murals were studied for the first time by optical microscopy (OM), attenuated total reflectance Fourier transform infrared (ATR–FTIR), scanning electron microscopy energy dispersive X-ray spectroscopy (SEM–EDS), and enzyme-linked immunosorbent assay (ELISA). The vibrational and elemental analysis showed that the color palette of the paintings is composed of pigments traditional for Orthodox church wall paintings such as natural pigments, including yellow ochre, red ochre, green earth, and calcite, as well as other historical pigments of synthetic origin, including smalt, red lead, cinnabar, and verdigris. The analysis of the binders by the ATR–FTIR spectroscopy and enzyme-linked immunosorbent assay (ELISA) analysis implied the use of the post-Byzantine egg tempera technique. Only the blue backgrounds in the murals were painted using a smalt-based paint mixed with a carbohydrate binder. Based on the current analysis and comparison with the successive paintings in the other churches of the Rila Monastery, it could be concluded that the technology of the painting process followed by the Athonite artists during the work in the Church-Ossuary became a point of reference for many Bulgarian icon-painters later.

G-Protein-Coupled Receptor-Associated Sorting Protein 1 Overexpression Is Involved in the Progression of Benign Prostatic Hyperplasia, Early-Stage Prostatic Malignant Diseases, and Prostate Cancer.

Background/Objectives: Prostate cancer (PCa) is a prevalent malignancy, necessitating accurate diagnostic methods to distinguish it from benign conditions such as benign prostatic hyperplasia (BPH). Current diagnostic tools, relying primarily on serum prostate-specific antigen (PSA) levels, lack specificity, leading to an over-diagnosis and unnecessary treatment of patients with benign conditions. This study explores G-protein-coupled receptor-associated sorting protein 1 (GASP-1) as a more sensitive biomarker for PCa detection. Methods: Prostate tissue microarrays of healthy, BPH, and prostate cancer patients with different Gleason scores were studied. Polyclonal antibodies targeted against GASP-1 were used for routine immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA) analyses. Results: The results indicated a 5-fold difference in serum GASP-1 levels between BPH and PCa, which was validated through GASP-1 IHC. Furthermore, a novel scoring system, the H-score, assesses GASP-1 granules' intensity and size, revealing a clear distinction between BPH and PCa. An additional analysis of GASP-1 expression between PCa cases with different Gleason scores reveals that GASP-1 overexpression correlates with PCa severity, providing insights into disease progression. Conclusions: The study supports GASP-1's role as a promising diagnostic marker, supplementing PSA testing, and offering improved risk stratification for PCa. Additionally, an open-source software system is introduced for an efficient GASP-1 granule color analysis, enhancing diagnostic accuracy.

Preventive effects of matrine on LPS-induced inflammation in RAW 264.7 cells and intestinal damage in mice through the TLR4/NF-κB/MAPK pathway

BackgroundMatrine is a tetracyclic quinolizidine alkaloid with diverse bioactive properties, including anti-inflammatory and neuroprotective properties. However, the underlying anti-inflammatory mechanisms remain unclear. PurposeThis study aimed to explore how matrine reduces Lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 cells and to assess its protective effects against LPS-induced intestinal damage. MethodsThe effect of matrine on cell viability was assessed using the cell counting kit-8 (CCK-8) assay. Additionally, its impact on inflammatory cytokines and macrophage polarization was assessed using enzyme-linked immunosorbent assay (ELISA), flow cytometry, and quantitative real-time polymerase chain reaction (qRT-PCR) analyses. The effects on intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), nitric oxide (NO) production, and oxidative stress were evaluated using 2′,7′-dichlorodihydrofluorescein diacetate staining and JC-1 and Griess assays. Immunofluorescence staining was used to observe the translocation of the NF-κB p65 subunit. Western blotting (WB) and qRT-PCR were employed to analyze the expression levels of proteins related to the toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB)/mitogen-activated protein kinase (MAPK) pathway. An LPS-induced mouse model was established to study the intestinal inflammation and barrier injury. Mouse feces characteristics, colon length, and disease activity index (DAI) were recorded. Hematoxylin-eosin (H&E) and alcian blue/periodic acid schiff (AB/PAS) staining were used to observe morphological changes and barrier damage in the duodenum, jejunum, ileum, and colon and to measure villus length, crypt depth, goblet cell count, and positive areas in the duodenum, jejunum, and ileum. The content of short-chain fatty acids (SCFAs) in the colon was determined using gas chromatography (GC). ResultsMatrine inhibited LPS-induced inflammatory cytokine levels, suppressed macrophage M1 polarization, and promoted M2 macrophage polarization. Matrine reduced LPS-induced increases in ROS and NO levels and regulates oxidative stress. Additionally, matrine inhibited the nuclear translocation of the NF-κB p65 subunit and exerted anti-inflammatory effects by suppressing the activation of the TLR4/NF-κB/MAPK pathway. In vivo experiments indicated that matrine significantly alleviated LPS-induced diarrhea, increased DAI, and shortened the colon. Matrine reduced the production of the pro-inflammatory cytokine interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α and the pro-inflammatory mediator NO in mouse intestinal tissues while promoting the content of the anti-inflammatory cytokine IL-10. Furthermore, it improved intestinal tissue structure and alleviated LPS-induced intestinal barrier damage. Finally, matrine increased the SCFA levels in the intestine. ConclusionMatrine exerted its anti-inflammatory effects and protects against intestinal injury through the TLR4/NF-κB/MAPK signaling pathway.

Pyrazolone-nicotinic acid derivative (4Z)-4-(2-hydroxybenzylidine)-5-methyl-2-(pyridine-3-ylcarbonyl)-2, 4-dihydro-3H-pyrazole-3-one (IIc) as multitarget inhibitor of neurodegeneration and behavioural impairment in Dementia.

The study was aimed at the synthesis and pharmacological investigation of (4Z)-4-(2-hydroxybenzylidine)-5-methyl-2-(pyridine-3-ylcarbonyl)-2, 4-dihydro-3H-pyrazole-3-one (IIc) in mice model of scopolamine-induced neurodegeneration and cognition impairment. The behavioural studies included Y-Maze Test, Water Morris Test, and Novel Object Recognition Test in Albino mice (20-25g). Scopalamine was used as an inducing agent. The acetylcholinesterase (AChE) inhibitory assay was used to assess the role of the test compounds in vitro. The Crystal Violet Staining (Nissl staining) was used to assess the neuroprotective and antiapoptotic effect through quantifying the number of neurons and viability. The expression of the anti-inflammatory enzyme cyclooxygenase-2 (COX-2), cytokine tumour necrotic factor (TNF-α), key transcription factor producing pro-inflammatory signals nuclear factor kappa B (P-NFkB), and apoptosis marker p-JNK was validated through enzyme-linked immunosorbent assay (ELISA) and immunohistochemical (IHC) analysis. The tested compound reverted cognitive and behavioural impairment through inhibiting scopolamine-induced inflammation and oxidative stress. We found that the compound IIc improved the short-term memory and learning behaviour of the experimental animals. Further investigation into molecular mechanisms showed that this effect was the manifestation of immunomodulatory, antioxidant, and consequently, of downsizing of inflammatory cytokines. These results were further validated through docking analysis. Finally, we conclude that the pyrazolone-nicotinic acid derivative IIc reversed the scopolamine-induced cognitive and behavioural deficits, attributed to acetylcholinesterase inhibition, neuronal recovery, antioxidant potential, and through downregulating the neuroinflammatory mediators p-NF-kB, cytokine TNF-α, and anti-inflammatory enzyme COX-2.

Investigation of the effects of electrical stimulation on BDNF and NGF levels in the sciatic nerve injury rat model

The current study aimed to investigate the effects of electrical stimulation on brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) levels in rats with sciatic nerve injury. Twenty-eight rats were divided into four groups of sham (S, n=7), electrical stimulation (ES, n=7), sciatic nerve injury (SNI, n=7) and sciatic nerve injury+electrical stimulation (SNI+ES, n=7). An experimental nerve damage model was produced by applying a closing force to compress the sciatic nerve. Electrical stimulation was applied for twenty minutes at 200 μs, 2mA, and 20 Hz for fifteen days. Enzyme-linked immunosorbent assay analysis was used to evaluate the levels of NGF and BDNF. It was shown that the SNI group had higher brain BDNF levels than the other groups, while the S group had lower brain BDNF levels than the other groups (P

Weitiao No. 3 (3) enhances the efficacy of anti-programmed cell death protein-1 immunotherapy by modulating the intestinal microbiota in an orthotopic model of gastric cancer mice.

To explore the effects of Weitiao No. 3 (3, WD-3) on anti-programmed cell death protein-1 (PD-1) immunotherapy in gastric cancer (GC). The intestinal microbiota was analyzed by 16S rDNA sequencing of fecal samples from three groups: healthy people (Health), GC patients (GC), and WD-3-treated GC patients (WD-3). Next, we established an orthotopic model of GC mice, which were treated with anti-PD-1, WD-3, or an inoculation of intestinal bacteria. Immune markers CD3, CD4, CD8, and forkhead box protein P3 (FOXP3), and the cell proliferation marker Ki67, were evaluated by immunohistochemistry. Cell apoptosis in GC tumors was assessed by terminal-deoxynucleotidyl-transferase-mediated deoxyuridine triphosphate nick end labeling staining. Enzyme-linked immunosorbent assays (ELISAs) were performed to analyze the serum levels of the following cytokines in GC mice: tumor necrosis factor (TNF)-α, interleukin (IL)-2, IL-6, IL-10, interferon (IFN)-γ, and transforming growth factor (TGF)-β. Sequencing data showed that there were significant differences in the composition of the gut microbial community among the three human groups. The gut bacteria in the three groups mainly comprised the phyla Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria. At the genus level, the relative abundances of Bifidobacterium and Coprococcus showed significant decreases in the GC group, and an obvious increase in the WD-3 group, compared with the Health group. Interestingly, the relative abundance of Saccharopolyspora was only detected in the WD-3 group. The results of in vivo experiments in GC mice showed that WD-3 or anti-PD-1 treatment increased the levels of CD3+, CD4+, and CD8+ T cells, but decreased the levels of FOXP3+ regulatory T cells. Furthermore, WD-3 or PD-1 antibody treatment inhibited proliferation and promoted apoptosis of GC tumor cells. ELISA analysis showed that WD-3 or PD-1 antibody treatment facilitated TNF-α, IL-2, and IFN-γ expression, while suppressing IL-6, IL-10, and TGF-β expression. Combination therapy with WD-3 and anti-PD-1 intensified all of these effects. WD-3 enhanced the immunotherapeutic efficacy of anti-PD-1 by modulating the intestinal microbiota in an orthotopic model of GC mice.

Exogenous Reactive Oxygen Species Augments SMAD4 Expression And TGF-β Paradox in Human Breast Cancer

Background: The Transforming growth factor-β (TGF-β) functions to induce apoptosis, cell cycle arrest, and differentiation is central to sustaining tissue homeostasis and maintaining genomic stability. TGF-β normally, an effective tumor-suppressor that restricts the uncontrolled division of cells augments the development and progression of human malignancies when cytostatic activities of TGF-β are resisted by genetic and epigenetic events caused by tumorigenesis. This dichotomic nature of TGF-β during oncogenesis termed as “TGF-β Paradox,” persists to be the most crucial and puzzling query regarding its physio-pathological function and the role of cellular antioxidant status is highly interrelated which warrants more studies on the role of endogenous reactive oxygen species (ROS) in deciding epithelial-mesenchymal transition (EMT) process. The objective of the study was to check whether enhanced ROS augments the TGF-β pathway facilitating EMT. Methods: In vitro toxicity assay was performed to assess the appropriate concentration of hydrogen peroxide (H2O2) imparting oxidative stress. Comet assay and 8-OHdG (8-hydroxy-2’-deoxyguanosine) enzyme-linked immunosorbent assay (ELISA) were performed to check the extent of DNA damage and adduct production respectively. Mitogen-activated protein kinase (MAPK) p38 ELISA and mRNA gene expression analysis of TGF-β and SMAD were done to verify the effect of H2O2 on these signaling. Results: The objective of the study was to check whether enhanced ROS augments the TGF-β pathway facilitating EMT. Along with morphological alterations, a dose-dependent decrease in cell viability was seen at 300µM of H2O2 compared to 75µM. DCFDA labeling discovered the dose-dependent gradation of intracellular ROS generation and this was correlated to increased cellular DNA damage and DNA adduct production which was increased linearly with increasing H2O2 as evident with comet test and 8-OHdG ELISA. Significantly reduced MAPK p38 activity revealed by indirect ELISA analysis suggests lessened suppression of cell growth. Conclusions: The study establishes that higher intracellular ROS will facilitate the TGF-β paradox leading to epithelial mesenchymal transition which can adversely affect therapeutic strategies targeting EMT

Integration of UPLC-MS/MS-based metabolomics and desorption electrospray ionization-mass spectrometry imaging reveals that Shouhui Tongbian Capsule alleviates slow transit constipation by regulating bile acid metabolism

Slow transit constipation (STC) is a common intestinal disorder. Some studies reported that Shouhui Tongbian Capsule (SHTB) can effectively mitigate STC symptoms. A detailed understanding of the changes in the endogenous metabolite profile of rats is crucial for a more accurate comprehension of the molecular pathological characteristics of SHTB in treating STC. In the present study, a method integrating metabolomics based on Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and Desorption electrospray ionization (DESI)-mass spectrometry imaging (MSI) was proposed to investigate serum, feces and colon tissue metabolic alterations of STC rats induced by diphenoxylate and the effect of SHTB treatment on metabolism. Then, Enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) analysis for verifying the potential mechanism of SHTB in treating STC. As a result, we first indicated that SHTB significantly improved intestinal peristalsis and low fecal water content in STC rats. Furthermore, after treatment with SHTB, the thickness of muscle layers was increased, demonstrated SHTB’s effectiveness in reducing intestinal injury in STC rats. Besides, bile acid (BA) metabolomics based on UPLC-MS/MS revealed significant increase in serum levels of Cholic acid (CA), Deoxycholic acid (DCA), Chenodeoxycholic acid (CDCA), Ursodeoxycholic acid (UDCA), and Glycolithocholic acid (GLCA), whereas the contents of CA and DCA in feces were significantly decreased in STC rats. Nonetheless, they returned to the control levels after the SHTB administration. ELISA results showed that SHTB significantly hindered the excessive reabsorption of BAs by inhibiting apical sodium-dependent bile acid transporter (ASBT), organic solute transporter alpha (OSTα) and organic solute transporter beta (OSTβ) in the ileum tissue of STC rats. Furthermore, the DESI-MSI analysis revealed that SHTB remarkably enhanced DCA in the colon tissue of STC rats. The WB results indicated that SHTB reinstated Takeda G-protein–coupled receptor 5 (TGR5) expression, a receptor for BAs and a key regulator of colonic motility. Consequently, DCA exerted its effects on TGR5, leading to the promotion of colonic motility. This study provided more comprehensive and detailed information about the BA metabolomics in the serum, feces and colon of STC rats. These findings highlighted the promising potential of metabolomics based on UPLC-MS/MS and DESI-MSI method for application in the study of STC diseases.

Molecular Biomarkers for In Vitro Thrombogenicity Assessment of Medical Device Materials.

To develop standardized in vitro thrombogenicity test methods for evaluating medical device materials, three platelet activation biomarkers, beta-thromboglobulin (β-TG), platelet factor 4 (PF4), soluble p-selectin (CD62P), and a plasma coagulation marker, thrombin-antithrombin complex (TAT), were investigated. Whole blood, drawn from six healthy human volunteers into Anticoagulant Citrate Dextrose Solution A was recalcified and heparinized over a concentration range of 0.5-1.5 U/mL. The blood was incubated with test materials with different thrombogenic potentials for 60 min at 37°C, using a 6 cm2/mL material surface area to blood volume ratio. After incubation, the blood platelet count was measured before centrifuging the blood to prepare platelet-poor plasma (PPP) and platelet-free plasma (PFP) for enzyme-linked immunosorbent assay analysis of the biomarkers. The results show that all four markers effectively differentiated the materials with different thrombogenic potentials at heparin concentrations from 1.0 to 1.5 U/mL. When a donor-specific heparin concentration (determined by activated clotting time) was used, the markers were able to differentiate materials consistently for blood from all the donors. Additionally, using PFP instead of PPP further improved the test method's ability to differentiate the thrombogenic materials from the negative control for β-TG and TAT. Moreover, the platelet activation markers were able to detect reversible platelet activation induced by adenosine diphosphate (ADP). In summary, all three platelet activation markers (β-TG, PF4, and CD62P) can distinguish thrombogenic potentials of different materials and detect ADP-induced reversible platelet activation. Test consistency and sensitivity can be enhanced by using a donor-specific heparin concentration and PFP. The same test conditions are applicable to the measurement of coagulation marker TAT.

Decreased plasma gelsolin fosters a fibrotic tumor microenvironment and promotes chemoradiotherapy resistance in esophageal squamous cell carcinoma

BackgroundStromal fibrosis is highly associated with therapeutic resistance and poor survival in esophageal squamous cell carcinoma (ESCC) patients. Low expression of plasma gelsolin (pGSN), a serum abundant protein, has been found to correlate with inflammation and fibrosis. Here, we evaluated pGSN expression in patients with different stages of cancer and therapeutic responses, and delineated the molecular mechanisms involved to gain insight into therapeutic strategies for ESCC.MethodsCirculating pGSN level in ESCC patients was determined by enzyme-linked immunosorbent assay analysis, and the tissue microarray of tumors was analyzed by immunohistochemistry staining. Cell-based studies were performed to investigate cancer behaviors and molecular mechanisms, and mouse models were used to examine the pGSN-induced tumor suppressive effects in vivo.ResultsCirculating pGSN expression is distinctively decreased during ESCC progression, and low pGSN expression correlates with poor therapeutic responses and poor survival. Methylation-specific PCR analysis confirmed that decreased pGSN expression is partly attributed to the hypermethylation of the GSN promoter, the gene encoding pGSN. Importantly, cell-based immunoprecipitation and protein stability assays demonstrated that pGSN competes with oncogenic tenascin-C (TNC) for the binding and degradation of integrin αvβ3, revealing that decreased pGSN expression leads to the promotion of oncogenic signaling transduction in cancer cells and fibroblasts. Furthermore, overexpression of pGSN caused the attenuation of TNC expression and inactivation of cancer-associated fibroblast (CAF), thereby leading to tumor growth inhibition in mice.ConclusionsOur results demonstrated that GSN methylation causes decreased secretion of pGSN, leading to integrin dysregulation, oncogenic TNC activation, and CAF formation. These findings highlight the role of pGSN in therapeutic resistance and the fibrotic tumor microenvironment of ESCC.

Association of the vitamin D metabolism gene polymorphism with the severity of coronary lesions assessed by SYNTAX score

This study aimed to determine the association of vitamin D serum blood levels and vitamin D gene polymorphism with the severity of coronary lesions in patients with stable coronary artery disease (CAD). Material and methods. 260 patients with stable CAD (average age was 58 years) were examined in the presented research. All patients were divided into two groups according to the SYNTAX score: low-risk patients with SYNTAX score ≤ 31 (n = 224) and high-risk patients with SYNTAX score > 31 (n = 36). For enzyme-linked immunosorbent assay and genetic analysis, peripheral blood was collected from the cubital vein into vacuum tubes containing coagulation activator and K3-EDTA, respectively. Serum blood level of 25-hydroxyvitamin D (DiaSource Diagnostics, Belgium) and 1,25-dihydroxyvitamin D (Immunodiagnostic Systems, Great Britain) were determined by enzyme-linked immunosorbent assay according to the manufacturers’ protocols. Genomic DNA was isolated by phenol-chloroform extraction method from whole blood. The quality and quantity of isolated DNA were assessed using NanoDrop spectrophotometer (Thermo Fisher Scientific, USA). Five polymorphic variants in the VDR (rs2228570 and rs73123) and GC (rs7041, rs1155563 and rs2298849) genes were selected for analysis. Genotyping was performed by real-time PCR in a 96-well plate with fluorescently labeled TaqMan probes. The quality of PCR was controlled by repeated genotyping of 10 % of the analyzed samples. Results. We found no statistically significant differences in serum blood level of the studied markers in patients from low-risk and high-risk groups. One polymorphic variant in the GC gene associated with the multiple coronary lesions (rs2298849) (odds ratio 2.26, 95 % confidence interval 1.28–3.99, p = 0.006) according to an additive inheritance model was identified. In addition, we determined the association between low serum blood level of 1,25-dihydroxyvitamin D in patients with CAD with multiple lesions of the coronary vascular system with A/A – A/G genotypes of the rs2228570 polymorphism in the VDR gene, A/A genotype of the rs7041 polymorphism and A/A genotype of the rs2298849 polymorphism in the GC gene. Conclusions. Allelic variants in the vitamin D metabolism genes are associated with the degree of coronary artery lesions assessed by the SYNTAX score in patients with stable CAD. Also, serum blood level of the active form of vitamin D (1,25-dihydroxyvitamin D) is less in carriers of homozygous genotypes for the major alleles of the VDR and GC genes.

RNA interference-mediated silencing of ctl13 inhibits innate immunity and development in stored pest Tribolium castaneum

C-type lectins (CTLs) play a pivotal role in the regulation of insect immunity and growth, making them potential molecular targets for RNA interference (RNAi)-mediated pest control. Although multiple CTLs have been identified in the genomes of various insects, their specific functions and underlying molecular mechanisms remain unclear. In the present study, a novel CTL, Tcctl13 with a single CRD, was identified in Tribolium castaneum. Tcctl13 is expressed in diverse immune-related tissues and developmental stages, with a notable increase in its expression upon exposure to lipopolysaccharides (LPS) and peptidoglycan (PGN). Molecular docking and enzyme-linked immunosorbent assay (ELISA) analyses revealed that TcCTL13 possesses the ability interacted with LPS and PGN. The binding and agglutinating activities of recombinant TcCTL13 (rTcCTL13) were demonstrated against both gram-negative and positive bacteria. After using RNAi to silence Tcctl13, the expression of the eight antimicrobial peptide (AMP) genes was significantly reduced. In addition, knocking down Tcctl13 during the early larval or pupal stage hindered, the normal metamorphosis process in T. castaneum, ultimately leading to the demise of all beetles. Further research showed that Tcctl13 and nine AMPs were significantly downregulation after 20-Hydroxyecdysone (20E) injection. Instead, the up-regulation of Tcctl13 and six AMPs was observed following interference with the 20E receptor (ecdysone receptor, EcR), indicating that the function of Tcctl13 is regulated by 20E in T. castaneum. Collectively, these findings suggest that Tcctl13 plays a role in the regulation of innate immunity and development in T. castaneum, offering a promising molecular target for managing insect pests using RNAi-based approaches.

Effects of Mucuna pruriens (L.) DC. and Levodopa in Improving Parkinson's Disease in Rotenone Intoxicated Mice.

Parkinson's disease (PD) is the second leading neurodegenerative disease after Alzheimer's disease. Mucuna pruriens (L.) DC. (MP) is a plant that contains Levodopa (L-DOPA) and has been known to improve the symptoms of PD. In this preliminary study, we investigated the anti-parkinsonian potential of MP to compare the effects of L-DOPA. We first developed an in vivo model of the PD in C57BL/6 male mice using rotenone. A total of twelve mice were used for this experiment. Nine mice were injected with rotenone (28 mg/kg) daily for 28 days. The mice experiments were performed to validate the effectiveness of MP to treat PD. Synthetic L-DOPA in a ratio of 1:20 with MP was used as MP contains 5% L-DOPA by weight in it. MP and L-DOPA were injected for 19 days on a daily basis. Cognitive function was evaluated using beam balance and olfactory tests. Serum analysis was performed using serum enzyme-linked immunosorbent assay (ELISA) analysis test. IL-12, IL-6, and TGF-β 1 were evaluated to validate the PD inducement and treatment. The levels of IL-12, IL-6, and TGF-β1 (p < 0.0001) in the PD mice group were significantly higher than those in the control group. The PD mice also showed higher latencies in beam balance and olfactory tests (p < 0.0001) compared to the control group. Both MP and L-DOPA-treated groups showed alleviation in latencies in beam balance and olfactory tests and decreased neuroinflammation in ELISA analysis (p < 0.001). The results treated by MP and L-DOPA showed insignificant differences in their values (p > 0.05). This proved that the MP and L-DOPA had similar effects in improving the symptoms of PD when used in the ratio of 1:20. Furthermore, both MP and L-DOPA reduced the level of IL-6 and TGF-β1 in this study. It may be inferred that a reduction in the level of IL-6 and TGF-β1 eventually leads to a reduction in the Th17 cells. The pathogenic Th17 is thought to be present in virtually all chronic inflammatory disorders. This can be an interesting area of research in further understanding the immunological effect of MP in ameliorating PD symptoms.

Heterologous Prime-Boost Immunization Strategies Using Varicella-Zoster Virus gE mRNA Vaccine and Adjuvanted Protein Subunit Vaccine Triggered Superior Cell Immune Response in Middle-Aged Mice.

Heterologous immunization using different vaccine platforms has been demonstrated as an efficient strategy to enhance antigen-specific immune responses. In this study, we performed a head-to-head comparison of both humoral and cellular immune response induced by different prime-boost immunization regimens of mRNA vaccine and adjuvanted protein subunit vaccine against varicella-zoster virus (VZV) in middle-aged mice, aiming to get a better understanding of the influence of vaccination schedule on immune response. VZV glycoprotein (gE) mRNA was synthesized and encapsulated into SM-102-based lipid nanoparticles (LNPs). VZV-primed middle-aged C57BL/6 mice were then subjected to homologous and heterologous prime-boost immunization strategies using VZV gE mRNA vaccine (RNA-gE) and protein subunit vaccine (PS-gE). The antigen-specific antibodies were evaluated using enzyme-linked immunosorbent assay (ELISA) analysis. Additionally, cell-mediated immunity (CMI) was detected using ELISPOT assay and flow cytometry. Besides, in vivo safety profiles were also evaluated and compared. The mRNA-loaded lipid nanoparticles had a hydrodynamic diameter of approximately 130 nm and a polydispersity index of 0.156. Total IgG antibody levels exhibited no significant differences among different immunization strategies. However, mice received 2×RNA-gE or RNA-gE>PS-gE showed a lower IgG1/IgG2c ratio than those received 2×PS-gE and PS-gE> RNA-gE. The CMI response induced by 2×RNA-gE or RNA-gE>PS-gE was significantly stronger than that induced by 2×PS-gE and PS-gE> RNA-gE. The safety evaluation indicated that both mRNA vaccine and protein vaccine induced a transient body weight loss in mice. Furthermore, the protein vaccine produced a notable inflammatory response at the injection sites, while the mRNA vaccine showed no observable inflammation. The heterologous prime-boost strategy has demonstrated that an mRNA-primed immunization regimen can induce a better cell-mediated immune response than a protein subunit-primed regimen in middle-aged mice. These findings provide valuable insights into the design and optimization of VZV vaccines with the potentials to broaden varicella vaccination strategies in the future.

Evaluation of the cognitive, physiological, and biomarker effects of heavy metal exposure in Wistar rats.

Individuals exposed to heavy metals are known to experience physiological and biochemical changes, which raise questions regarding possible health effects. In our earlier research, significant concentrations of vanadium (V), mercury (Hg), cadmium (Cd), and arsenic (As) were found in food and medical packaging materials. This study aimed to evaluate the cognitive, physiological, and biomarker effects of select heavy metal exposure in Wistar rats. Over a 13-week period, five groups of rats (six rats per group, with both males and females) were assessed to study the effects of oral exposure to V, Hg, Cd, and As. The study focused on evaluating physiological, cognitive, and biochemical markers, with the results compared to those of a control group. Comparing all groups of rats treated with heavy metals, the study revealed significant deficits in learning and spatial orientation (water maze test); rats treated with V, Cd, and Hg showed signs of depression. Rats treated with As also showed signs of hyperactivity, which may indicate a connection to attention-deficit hyperactivity disorder (rat tail suspension test). The groups exposed to different heavy metals varied in their physiological (water and food intake, urine and feces output) and biochemical responses (enzyme-linked immunosorbent assay, prostate-specific antigen, T3, T4, thyroid-stimulating hormone, carcinoembryonic antigen, and blood glucose analysis), with Hg exhibiting the strongest impacts. Rats given Hg showed signs of hypothyroidism, such as increased food intake and weight gain. This study clarifies the complex relationships between exposure to heavy metals and various biological systems, shedding light on their potential health impacts. The findings provide insight into the effects of heavy metals on neural and thyroid tissues, as well as their propensity to cause cellular dedifferentiation. However, the study has certain limitations, such as the relatively short duration of exposure and the use of only a few selected biomarkers. Future research should focus on long-term exposure studies, incorporate a broader range of biomarkers, and explore the underlying mechanisms at a molecular level to better understand the full spectrum of health risks associated with heavy metal exposure.

Increase of HCN current in SOD1-associated amyotrophic lateral sclerosis.

The clinical manifestations of sporadic amyotrophic lateral sclerosis (ALS) vary widely. However, the current classification of ALS is mainly based on clinical presentations, while the roles of electrophysiological and biomedical biomarkers remain limited. Herein, we investigated a group of patients with sporadic ALS and an ALS mouse model with superoxide dismutase 1 (SOD1)/G93A transgenes using nerve excitability tests (NET) to investigate axonal membrane properties and chemical precipitation, followed by enzyme-linked immunosorbent assay analysis to measure plasma misfolded protein levels. Six of 19 patients (31.6%) with sporadic ALS had elevated plasma misfolded SOD1 protein levels. In sporadic ALS patients, only those with elevated misfolded SOD1 protein levels showed an increased inward rectification in the current-threshold (I/V) curve and an increased threshold reduction in the hyperpolarizing threshold electrotonus (TE) in the NET study. Two familial ALS patients with SOD1 mutations also exhibited similar electrophysiological patterns of NET. For patients with sporadic ALS showing significantly increased inward rectification in the I/V curve, we noted an elevation in plasma misfolded SOD1 level, but not in total SOD1, misfolded C9orf72, or misfolded phosphorylated TDP43 levels. Computer simulations demonstrated that the aforementioned axonal excitability changes are likely associated with an increase in hyperpolarization-activated cyclic nucleotide-gated (HCN) current. In SOD1/G93A mice, NET also showed an increased inward rectification in the I/V curve, which could be reversed by a single injection of the HCN channel blocker, ZD7288. Daily treatment of SOD1/G93A mice with ZD7288 partially prevented the early motor function decline and spinal motor neuron death. In summary, sporadic ALS patients with elevated plasma misfolded SOD1 exhibited similar patterns of motor axonal excitability changes as familial ALS patients and ALS mice with mutant SOD1 genes, suggesting the existence of SOD1-associated sporadic ALS. The observed NET pattern of increased inward rectification in the I/V curve was attributable to an elevation in the HCN current in SOD1-associated ALS.

A miniaturized platform based on reciprocating-flowing immunobinding for ultrafast, highly-sensitive and on-site immunoassay

Immunoassays are considered important methods for detecting disease biomarkers for biomedical diagnosis. Particularly, enzyme-linked immunosorbent assay (ELISA) is a commonly-used immunoassay method due to its high reliability and low cost. Nowadays, automatic analyzers integrating all steps of ELISA can achieve detection without manual operation to save labor, increase detection efficiency and avoid manual error. However, most ELISA analyzers require more than 30 min for one detection, which are not fast enough for on-site point-of-care testing (POCT). Herein, based on the reciprocating-flowing immunobinding strategy, we developed a miniaturized reciprocating-flowing POCT platform (RF-POCT platform) for rapid immunodetection. RF-POCT platform, which included an electronic control module, a fluid control module, a mechanical control module and a detection module, enabled fast immunobinding of antigen and antibody. Taking ELISA as an example of immunoassay methods for RF-POCT platform, we developed a miniaturized reciprocating-flowing ELISA analyzer (RF-ELISA analyzer) to automatically detect SARS-CoV-2 nucleocapsid protein (SARS-CoV-2 N protein) and dust mite-specific immunoglobulin E (DM-IgE), with the LOD of 5.2–6.6 pg/mL and the CV less than 3 %. The detection required only 3.5 min. Afterwards, we detected the SARS-CoV-2 N protein in nasal swab samples and the DM-IgE in serum samples; the specificities were 85.7–100 %, the sensitivities were 96.3–100 %. The quantitative results from RF-ELISA analyzer were generally-consistent with those from large ELISA analyzers in hospitals. Therefore, RF-ELISA analyzer demonstrated excellent performance on ultrafast, highly-sensitive, and on-site immunoassay, with potential to support on-site POCT for primary hospitals and community clinics.