Enzyme-linked Immunosorbent Assay Analysis Research Articles

Prestin in Human Perilymph, Cerebrospinal Fluid, and Blood as a Biomarker for Hearing Loss.

Determining the concentration of prestin in human blood, cerebrospinal fluid (CSF), and perilymph (PL), and evaluating its suitability as a clinical biomarker for sensori-neural hearing loss (SNHL). Human blood, CSF, and PL samples were intraoperatively collected from 42 patients with tumors of the internal auditory canal or with intracochlear tumors undergoing translabyrinthine or middle fossa tumor removal. Prestin concentration was measured using enzyme-linked immunosorbent assay and linear regression analyses were performed to investigate its associations with audiological as well as vestibular test results. Tertiary referral center. The median prestin concentration in blood samples of the 42 study participants (26 women, mean ± standard deviation age, 52.7 ± 12.5 years) was 1.32 (interquartile range, IQR, 0.71-1.99) ng/mL. CSF prestin levels were significantly higher with 4.73 (IQR, 2.45-14.03) ng/mL (P = .005). With 84.74 (IQR, 38.95-122.00) ng/mL, PL prestin concentration was significantly higher compared to blood (P = .01) and CSF (P = .03) levels. Linear regression analyses showed significant associations of CSF prestin concentration with preoperative hearing levels (pure-tone average and word recognition; P = .008, R2 = 0.1894; P = .03, R2 = 0.1857), but no correlations with blood or PL levels. This study's findings highlight the volatile nature of prestin levels and provide the first insights into this potential biomarker's concentrations in body fluids apart from blood. Future investigations should comprehensively assess human prestin levels with different etiologies of SNHL, prestin's natural homeostasis and systemic circulation, and its temporal dynamics after cochlear trauma. Finally, clinically approved detection kits for prestin are urgently required prior to considering a potential translational implementation of this diagnostic technique.

New Insights into Hepatic and Intestinal Microcirculation and Pulmonary Inflammation in a Model of Septic Shock and Veno-Arterial Extracorporeal Membrane Oxygenation in the Rat

Despite significant efforts toward improving therapy for septic shock, mortality remains high. Applying veno-arterial (V-A) extracorporeal membrane oxygenation (ECMO) in this context remains controversial. Since the cannulation of the femoral artery for V-A ECMO return leads to lower body hyperoxia, this study investigated the impact of V-A ECMO therapy on the intestinal and hepatic microcirculation during septic shock in a rodent model. Thirty male Lewis rats were randomly assigned to receive V-A ECMO therapy with low (60 mL/kg/min) or high (90 mL/kg/min) blood flow or a sham procedure. Hemodynamic data were collected through a pressure-volume catheter in the left ventricle and a catheter in the lateral tail artery. Septic shock was induced by intravenous administration of lipopolysaccharide (1 mg/kg). The rats received lung-protective ventilation during V-A ECMO therapy. The hepatic and intestinal microcirculation was measured by micro-lightguide spectrophotometry after median laparotomy for two hours. Systemic and pulmonary inflammation was detected via enzyme-linked immunosorbent assays (ELISA) of the plasma and bronchoalveolar lavage (BAL), respectively, measuring tumor necrosis factor-alpha (TNF-α), interleukins 6 (IL-6) and 10 (IL-10), and C-X-C motif ligands 2 (CXCL2) and 5 (CXCL5). Oxygen saturation and relative hemoglobin concentration were reduced in the hepatic and intestinal microcirculation during V-A ECMO therapy, independent of the blood flow rate. Further, rats treated with V-A ECMO therapy also presented elevated systolic, diastolic, and mean arterial blood pressure and increased stroke volume, cardiac output, and left ventricular end-diastolic volume. However, left ventricular end-diastolic pressure was only elevated during high-flow V-A ECMO therapy. Blood gas analysis revealed a dilutional anemia during V-A ECMO therapy. ELISA analysis showed an elevated plasma CXCL2 concentration only during high-flow V-A ECMO therapy and elevated BAL CXCL2 and CXCL5 concentrations only during low-flow V-A ECMO therapy. Rats undergoing V-A ECMO therapy exhibited impaired microcirculation of the intestine and liver during septic shock despite increased blood pressure and cardiac output. Increased pulmonary inflammation was detected only during low-flow V-A ECMO therapy in septic shock.

New Insights into Hepatic and Intestinal Microcirculation and Pulmonary Inflammation in a Model of Septic Shock and Veno-Arterial Extracorporeal Membrane Oxygenation in the Rat.

Despite significant efforts toward improving therapy for septic shock, mortality remains high. Applying veno-arterial (V-A) extracorporeal membrane oxygenation (ECMO) in this context remains controversial. Since the cannulation of the femoral artery for V-A ECMO return leads to lower body hyperoxia, this study investigated the impact of V-A ECMO therapy on the intestinal and hepatic microcirculation during septic shock in a rodent model. Thirty male Lewis rats were randomly assigned to receive V-A ECMO therapy with low (60 mL/kg/min) or high (90 mL/kg/min) blood flow or a sham procedure. Hemodynamic data were collected through a pressure-volume catheter in the left ventricle and a catheter in the lateral tail artery. Septic shock was induced by intravenous administration of lipopolysaccharide (1 mg/kg). The rats received lung-protective ventilation during V-A ECMO therapy. The hepatic and intestinal microcirculation was measured by micro-lightguide spectrophotometry after median laparotomy for two hours. Systemic and pulmonary inflammation was detected via enzyme-linked immunosorbent assays (ELISA) of the plasma and bronchoalveolar lavage (BAL), respectively, measuring tumor necrosis factor-alpha (TNF-α), interleukins 6 (IL-6) and 10 (IL-10), and C-X-C motif ligands 2 (CXCL2) and 5 (CXCL5). Oxygen saturation and relative hemoglobin concentration were reduced in the hepatic and intestinal microcirculation during V-A ECMO therapy, independent of the blood flow rate. Further, rats treated with V-A ECMO therapy also presented elevated systolic, diastolic, and mean arterial blood pressure and increased stroke volume, cardiac output, and left ventricular end-diastolic volume. However, left ventricular end-diastolic pressure was only elevated during high-flow V-A ECMO therapy. Blood gas analysis revealed a dilutional anemia during V-A ECMO therapy. ELISA analysis showed an elevated plasma CXCL2 concentration only during high-flow V-A ECMO therapy and elevated BAL CXCL2 and CXCL5 concentrations only during low-flow V-A ECMO therapy. Rats undergoing V-A ECMO therapy exhibited impaired microcirculation of the intestine and liver during septic shock despite increased blood pressure and cardiac output. Increased pulmonary inflammation was detected only during low-flow V-A ECMO therapy in septic shock.

Maternal Zinc Diet Impairs Learning and Memory in Offspring Rats through the CREB/BDNF Pathway

BACKGROUND: Zinc released into the synaptic cleft able to modulate various signaling pathways, including brain derived neurotrophic factor (BDNF) and its receptor tropomyosin receptor kinase B (TrkB). Zinc binding to its receptor, G-protein coupled receptor 39 (GPR39), may trigger biochemical pathways leading to cAMP response element binding protein (CREB)-dependent gene transcription that subsequently promotes BDNF upregulation. Therefore, zinc dyshomeostasis should be considered as a condition that induces disruption of CREB/BDNF signaling. This study was conducted to examine the effect of maternal zinc diet on hippocampal expression levels of CREB and BDNF in offspring.METHODS: One-day pregnant rats were randomly divided into five groups: zinc-deficient (D), zinc-restricted (R), zinc-adequate (A), zinc-supplemented (S), and excess zinc-supplemented (ES). The groups had different zinc diets during pregnancy and lactation. The behavioral function of the offspring was tested with Y-maze at the 43th postnatal. Hippocampus was isolated, BDNF was assessed by quantitative real-time polymerase chain reaction (qRT-PCR), and CREB was examined using sandwich enzyme-linked immunosorbent assay (ELISA).RESULTS: Spatial working memory measurement demonstrated that D and ES group exhibited a significantly lower spontaneous alternation than other groups. The qRT-PCR and ELISA analysis revealed the hippocampal expression level of BDNF and CREB decreased in groups D and ES, but tended to increase in groups R and S, until the highest expression peak was found in group A.CONCLUSION: High and low intake of zinc induces lower expression of BDNF and CREB in hippocampus, which further impairs learning and memory. Our findings suggest the signaling pathway of CREB/BDNF is involved in zinc dyshomeostasis-induced cognitive impairments.KEYWORDS: hippocampus, BDNF, CREB, TrkB, GPR39, zinc, diet, LTP

Reprogramming Filamentous fd Viruses to Capture Copper Ions.

C-terminal truncated variants (A, VA, NVA, ANVA, FANVA and GFANVA) of our recently identified Cu(II) specific peptide "HGFANVA" were displayed on filamentous fd phages. Wild type fd-tet and engineered virus variants were treated with 100 mM Cu(II) solution at a final phage concentration of 1011 vir/ml and 1012 vir/ml. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) imaging before Cu(II) exposure showed ≈6-8 nm thick filamentous virus layer formation. Cu(II) treatment resulted in aggregated bundle-like assemblies with mineral deposition. HGFANVA phage formed aggregates with an excessive mineral coverage. As the virus concentration was 10-fold decreased, nanowire-like assemblies were observed for shorter peptide variants A, NVA and ANVA. Wild type fd phages did not show any mineral formation. Energy dispersive X-ray spectroscopy (EDX) analyses revealed the presence of C and N peaks on phage organic material. Cu peak was only detected for engineered viruses. Metal ion binding of viruses was next investigated by enzyme-linked immunosorbent assay (ELISA) analyses. Engineered viruses were able to bind Cu(II) forming mineralized intertwined structures although no His (H) unit was displayed. Such genetically reprogrammed virus based biological materials can be further applied for bioremediation studies to achieve a circular economy.

Artemisia argyi essential oil alleviates asthma by regulating 5-LOX-CysLTs and IDO-1-KYN pathways: Insights from metabolomics

Ethnopharmacological relevanceArtemisia argyi essential oil (AAEO) is a traditional herbal remedy for asthma. However, the potential effect of AAEO on asthma has not been elucidated. Aim of the studyTo investigate the protective properties of AAEO upon asthma and elucidate its mechanism. Materials and methodsThe effects of AAEO in asthma were assessed by histology and biochemical analysis. Then, we integrated real-time reverse transcription-quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, immunohistochemistry and metabolomics analysis to reveal its mechanism. ResultsIn vivo, AAEO reduced the counts of white blood cells (WBCs) and cytokines in bronchoalveolar lavage fluid (BALF), ameliorated pathologic alterations in lung tissues, and inhibited secretion of OVA-sIgE and muc5ac. Metabolomics results showed that AAEO can exert therapeutic effects on asthmatic mice by regulating disordered arachidonic acid metabolism and tryptophan metabolism. Further studies shown that AAEO inhibited the expression of 5-LOX and reduced the accumulation of CysLTs in mice. Meanwhile, AAEO promoted the activity of IDO-1, facilitated the conversion of tryptophan to kynurenine, and regulated the imbalance of Treg/Th17 immunity. Immunohistochemical results showed that AAEO promoted the expression of IDO-1. RT-qPCR results showed that AAEO promoted the expression of IL-10 and Foxp3 mRNA, and inhibited the expression of IL-17A and RORγt mRNA, thus regulated the imbalance of Treg/Th17 immunity and exerted its therapeutic effects. ConclusionAAEO treatment not only attenuates the clinical symptoms of asthma but is also involved in regulating lung tissue metabolism. The anti-asthmatic activity of AAEO may be achieved by reprogramming 5-LOX-CysLTs and IDO-1-KYN pathways.

Molecular characteristics of non-human papillomavirus driven cervical adenocarcinoma and adenosquamous carcinoma.

e17532 Background: Cervical cancer is the fourth most common malignancy in women worldwide. Although infection from human papillomavirus (HPV) has been the major cause of cervical cancer, about 3-8% of cervical cancer cases are HPV-negative. Clinical data showed that patients with HPV-negative cervical cancer had significantly lower DFS than those with HPV-positive cervical cancer. The detection of HPV DNA tends to be lower in adenosquamous carcinoma (AC/ASC) than in squamous cell carcinoma. Because the mechanism of HPV-negative cervical cancer development is unclear, this study aims to find the pattern of differential gene expression in HPV-negative AC/ASC and verify the underlying potential mechanism. Methods: In this study, we aimed to exam the differential gene expression profiling by exome-capture RNA sequencing on formalin-fixed, paraffin-embedded tumor tissue of prospectively enrolled cervical AC/ASC patients (HPV-negative: HPV-positive: 1:1) and validate the selected biomarkers on the remaining (unmatched; n = 110) prospective cohort and the 274 retrospective cohort patients. Subsequently, dysregulated differentially expressed genes specifically existed in HPV-negative cervical AC/ASC tissues and HPV-negative cell lines were validated by Western blotting, enzyme-linked immunosorbent assay (ELISA), and immunohistochemical staining (IHC staining). Results: Exome-capture RNA sequencing on HPV-negative and age/grade matched HPV-positive AC/ASC (10:6) for discovery initially. We found differentially activated WNT pathway from HPV positive/negative AC/ASC in the prospective cohort. In the preliminary results of 28 prospective cohort patients, SOX17 and MMP7 highly expressed genes in HPV-negative (n = 13) then the HPV-positive (n = 11) cervical AC/ASC by IHC staining (p = 0.1029 and p = 0.0306, respectively). Moreover, plasma MMP7 levels were upregulated in HPV-negative AC/ASC (n = 10) compared with HPV-positive AC/ASC (n = 17) (p = 0.0086) by ELISA analysis in the prospective cohort. Conclusions: Further studies and more analyses will be continued to investigate the effects of changes in the expression of SOX17 and MMP7 related mechanisms, and targeting WNT/SOX17/MMP7 pathways specifically associated with HPV-negative AC/ASC to provide new insights into the early screening and effective treatment of HPV-negative AC/ASC.

Molecular characteristics of non-human papillomavirus driven cervical adenocarcinoma and adenosquamous carcinoma.

e17532 Background: Cervical cancer is the fourth most common malignancy in women worldwide. Although infection from human papillomavirus (HPV) has been the major cause of cervical cancer, about 3-8% of cervical cancer cases are HPV-negative. Clinical data showed that patients with HPV-negative cervical cancer had significantly lower DFS than those with HPV-positive cervical cancer. The detection of HPV DNA tends to be lower in adenosquamous carcinoma (AC/ASC) than in squamous cell carcinoma. Because the mechanism of HPV-negative cervical cancer development is unclear, this study aims to find the pattern of differential gene expression in HPV-negative AC/ASC and verify the underlying potential mechanism. Methods: In this study, we aimed to exam the differential gene expression profiling by exome-capture RNA sequencing on formalin-fixed, paraffin-embedded tumor tissue of prospectively enrolled cervical AC/ASC patients (HPV-negative: HPV-positive: 1:1) and validate the selected biomarkers on the remaining (unmatched; n = 110) prospective cohort and the 274 retrospective cohort patients. Subsequently, dysregulated differentially expressed genes specifically existed in HPV-negative cervical AC/ASC tissues and HPV-negative cell lines were validated by Western blotting, enzyme-linked immunosorbent assay (ELISA), and immunohistochemical staining (IHC staining). Results: Exome-capture RNA sequencing on HPV-negative and age/grade matched HPV-positive AC/ASC (10:6) for discovery initially. We found differentially activated WNT pathway from HPV positive/negative AC/ASC in the prospective cohort. In the preliminary results of 28 prospective cohort patients, SOX17 and MMP7 highly expressed genes in HPV-negative (n = 13) then the HPV-positive (n = 11) cervical AC/ASC by IHC staining (p = 0.1029 and p = 0.0306, respectively). Moreover, plasma MMP7 levels were upregulated in HPV-negative AC/ASC (n = 10) compared with HPV-positive AC/ASC (n = 17) (p = 0.0086) by ELISA analysis in the prospective cohort. Conclusions: Further studies and more analyses will be continued to investigate the effects of changes in the expression of SOX17 and MMP7 related mechanisms, and targeting WNT/SOX17/MMP7 pathways specifically associated with HPV-negative AC/ASC to provide new insights into the early screening and effective treatment of HPV-negative AC/ASC.

Exploring the multicomponent synergy mechanism of Zuogui Wan on postmenopausal osteoporosis by a systems pharmacology strategy.

To explore the multi-component synergistic mechanism of Zuogui Wan (, ZGW) in treating postmenopausal osteoporosis (PMOP). The main components and target genes of ZGW were screened via the Traditional Chinese Medicine Systems Pharmacology (TCMSP). In addition, the target gene sets of PMOP were derived from the GeneCards and Online Mendelian Inheritance in Man databases. The search tool for recurring instances of neighbouring genes (STRING) 11.0 software was used to analyze the interaction among intersecting genes. Cytoscape 3.6.1 software and the Matthews correlation coefficient (MCC) algorithm were used to screen the core genes. Fifty Sprague-Dawley female rats were randomly divided into the sham-operated (Sham) group and the four ovariectomized (OVX) subgroups. Rats subjected to Sham or OVX were administered with the vehicle (OVX, 1 mL water/100 g weight), 17β-estradiol (E2, 50 μg·kg-1·d-1), and lyophilized powder of ZGW at a low dose of 2.3 (ZGW-L) and high dose of 4.6 (ZGW-H) g·kg-1·d-1 for three months. The bone density and bone strength were assessed using dual-energy X-ray and three-point bending tests, respectively. Furthermore, enzyme-linked immun-osorbent assay, Hematoxylin-eosin staining, and western blot analysis were used to determine the potential pharmacological mechanisms of action of ZGW in PMOP. A total of 117 active compounds of ZGW were screened from the TCMSP. Furthermore, 108 intersecting genes of drugs and diseases were identified. Using STRING software and the MCC algorithm, ten core genes, including C-X-C chemokine living 8 (CXCL8), C-C chemokine receptor type 2 (CCR2), alpha-2a active receptor (ADRA2A), melatonin receptor type 1B (MTNR1B), and amyloid-beta A4 protein (APP), were identified. The anti-osteoporosis regulation network of ZGW was constructed using the Cytoscape software. The animal experiments demonstrated that ZGW groups significantly reduced the serum levels of β-C-terminal telopeptide of type I collagen (β-CTX) and increased serum levels of bone-specific alkaline phosphatase (BALP) (P < 0.05, P < 0.01). The OVX group exhibited a significant decrease in bone mineral density and bone strength compared with the Sham group (P < 0.01). Moreover, treatment with ZGW resulted in increased trabecular thickness, improved arrangement of trabecular structure, and reduced empty bone lacunae. Furthermore, treatment with ZGW significantly increased the protein expression of CXCL8, ADRA2A, and CCR2 (P < 0.05, P < 0.01), and significantly decreased the protein expression of Runx2 (P < 0.01). Furthermore, the ZGW and E2 groups demonstrated significantly increased BMD (P < 0.05, P < 0.01), improved bone strength (P < 0.05, P < 0.01), reduced expression of CXCL8, ADRA2A, and CCR2, and increased runt-related transcription factor 2 levels in bone tissue (P < 0.05, P < 0.01) compared with the OVX group. However, there were no significant differences in MTNR1B and APP expression among the groups. ZGW shows synergistic mechanisms in PMOP through multiple components, targets, and pathways.

Molecular mechanisms of poor osseointegration in irradiated bone: In vivo study in a rat tibia model.

Radiotherapy is associated with cell depletion and loss of blood supply, which are linked to compromised bone healing. However, the molecular events underlying these effects at the tissue-implant interface have not been fully elucidated. This study aimed to determine the major molecular mediators associated with compromised osseointegration due to previous exposure to radiation. Titanium implants were placed in rat tibiae with or without pre-exposure to 20 Gy irradiation. Histomorphometric, biomechanical, quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay analyses were performed at 1 and 4 weeks after implantation. The detrimental effects of irradiation were characterized by reduced bone-implant contact and removal torque. Furthermore, pre-exposure to radiation induced different molecular dysfunctions such as (i) increased expression of pro-inflammatory (Tnf) and osteoclastic (Ctsk) genes and decreased expression of the bone formation (Alpl) gene in implant-adherent cells; (ii) increased expression of bone formation (Alpl and Bglap) genes in peri-implant bone; and (iii) increased expression of pro-inflammatory (Tnf) and pro-fibrotic (Tgfb1) genes in peri-implant soft tissue. The serum levels of pro-inflammatory, bone formation and bone resorption proteins were greater in the irradiated rats. Irradiation causes the dysregulation of multiple biological activities, among which perturbed inflammation seems to play a common role in hindering osseointegration.

Linagliptin Mitigates TGF-β1 Mediated Epithelial-Mesenchymal Transition in Tacrolimus-Induced Renal Interstitial Fibrosis via Smad/ERK/P38 and HIF-1α/LOXL2 Signaling Pathways.

The dipeptidyl peptidase-4 (DPP-4) inhibitors, a novel anti-diabetic medication family, are renoprotective in diabetes, but a comparable benefit in chronic non-diabetic kidney diseases is still under investigation. This study aimed to elucidate the molecular mechanisms of linagliptin's (Lina) protective role in a rat model of chronic kidney injury caused by tacrolimus (TAC) independent of blood glucose levels. Thirty-two adult male Sprague Dawley rats were equally randomized into four groups and treated daily for 28 d as follows: The control group; received olive oil (1 mL/kg/d, subcutaneously), group 2; received Lina (5 mg/kg/d, orally), group 3; received TAC (1.5 mg/kg/d, subcutaneously), group 4; received TAC plus Lina concomitantly in doses as the same previous groups. Blood and urine samples were collected to investigate renal function indices and tubular injury markers. Additionally, signaling molecules, epithelial-mesenchymal transition (EMT), and fibrotic-related proteins in kidney tissue were assessed by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis, immunohistochemical and histological examinations. Tacrolimus markedly induced renal injury and fibrosis as indicated by renal dysfunction, histological damage, and deposition of extracellular matrix (ECM) proteins. It also increased transforming growth factor β1 (TGF-β1), Smad4, p-extracellular signal-regulated kinase (ERK)1/2/ERK1/2, and p-P38/P38 mitogen-activated protein kinase (MAPK) protein levels. These alterations were markedly attenuated by the Lina administration. Moreover, Lina significantly inhibited EMT, evidenced by inhibiting Vimentin and α-smooth muscle actin (α-SMA) and elevating E-cadherin. Furthermore, Lina diminished hypoxia-related protein levels with a subsequent reduction in Snail and Twist expressions. We concluded that Lina may protect against TAC-induced interstitial fibrosis by modulating TGF-β1 mediated EMT via Smad-dependent and independent signaling pathways.

#1933 Urinary proteomics identifying the novel biomarker associated with crescentic glomerulonephritis

Abstract Background and Aims As a severe progressive acute renal injury, crescentic glomerulonephritis requires timely pathology diagnosis and treatment. New non-invasive biomarkers will be crucial when the renal biopsy is not available or unacceptable. Urinary proteomics, specifically data-independent acquisition (DIA) proteomics, might provide some potential indicators. Method Urine proteomics examination was on patients with crescentic glomerulonephritis(n = 15), chronic glomerulonephritis (n = 15), acute kidney injury (AKI) without glomerular crescentic(n = 10), and 15 healthy controls. The crescentic glomerulonephritis group is the patients with extensive glomerular crescents over 50%. We performed LC-MS/MS analysis to identify differentially expressed proteins (DEPs), Ingenuity Pathway Analysis (IPA), and the proteome map for significant pathways and crucial proteins among patients and controls. The signature proteins were validated using enzyme-linked immunosorbent assay (ELISA) analysis. Results A total of 55 population, males occupied 45.45%, with the average age being 53 ± 13 years old. A volcano plot among crescentic glomerulonephritis and control groups is in Fig. 1. IPA analysis showed that neutrophil degranulation and complement cascade were the top two pathways in crescentic glomerulonephritis but not in the healthy and nephritis groups. The activated neddylation pathway in patients with crescentic glomerulonephritis compared with AKI patients. After integrating the DEPs among three groups via the Venn plot, we observed eight DEPs significantly associated with the crescentic glomerulonephritis, among which Coagulation factor V (F5), Phospholipid transfer protein (PLTP), and Alcohol dehydrogenase 1C (ADH1C) were significant proteins. Biomarker analysis showed the area under the curve values for predicting crescentic glomerulonephritis were 0.867, 0.851, and 0.815, respectively (P &amp;lt; 0.001). Conclusion Urine proteomics holds promise in identifying the crescentic glomerulonephritis patient, thus providing a potential value as a noninvasive biomarker.

Improving stroke prognosis by TLR4 KO to enhance N2 neutrophil infiltration and reduce M1 macrophage polarization.

Cerebral ischemic stroke remains a leading cause of mortality and morbidity worldwide. Toll-like receptor 4 (TLR4) has been implicated in neuroinflammatory responses poststroke, particularly in the infiltration of immune cells and polarization of macrophages. This study aimed to elucidate the impact of TLR4 deficiency on neutrophil infiltration and subsequent macrophage polarization after middle cerebral artery occlusion (MCAO), exploring its role in stroke prognosis. The objective was to investigate how TLR4 deficiency influences neutrophil behavior poststroke, its role in macrophage polarization, and its impact on stroke prognosis using murine models. Wild-type and TLR4-deficient adult male mice underwent MCAO induction, followed by various analyses, including flow cytometry to assess immune cell populations, bone marrow transplantation experiments to evaluate TLR4-deficient neutrophil behaviors, and enzyme-linked immunosorbent assay and Western blot analysis for cytokine and protein expression profiling. Neurobehavioral tests and infarct volume analysis were performed to assess the functional and anatomical prognosis poststroke. TLR4-deficient mice exhibited reduced infarct volumes, increased neutrophil infiltration, and reduced M1-type macrophage polarization post-MCAO compared to wild-type mice. Moreover, the depletion of neutrophils reversed the neuroprotective effects observed in TLR4-deficient mice, suggesting the involvement of neutrophils in mediating TLR4's protective role. Additionally, N1-type neutrophils were found to promote M1 macrophage polarization via neutrophil gelatinase-associated lipocalin (NGAL) secretion, a process blocked by TLR4 deficiency. The study underscores the protective role of TLR4 deficiency in ischemic stroke, delineating its association with increased N2-type neutrophil infiltration, diminished M1 macrophage polarization, and reduced neuroinflammatory responses. Understanding the interplay between TLR4, neutrophils, and macrophages sheds light on potential therapeutic targets for stroke management, highlighting TLR4 as a promising avenue for intervention in stroke-associated neuroinflammation and tissue damage.

Enzyme-linked immunosorbent assay and immunohistochemical analysis of mast cell related biochemicals in oral submucous fibrosis.

Oral submucous fibrosis (OSMF), a potentially malignant disorder, is developed by progressive fibrous tissue deposition in connective tissue along with atrophy of oral mucosa. Histological sections also show the mast cell infiltration in submucosa which may indicate their possible role in this entity. Abundant availability of biochemicals in mast cells like histamine and serine proteases like chymase may be released and play specific pathways in the disease pathophysiology. Possibly, if the histamine release has some part to play, diamine oxidase may also be found to have a relationship as it metabolizes histamine. The present study is proposed to identify the presence of chymase, histamine, and diamine oxidase in both, serum as well as tissue by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC) respectively. This study may provide probable insight into the mast cell-related chemicals and their association with OSMF.

Polarized M2 macrophages induced by glycosylated nano-hydroxyapatites activate bone regeneration in periodontitis therapy.

To investigate the association between periodontal macrophage polarization states and the alveolar bone levels, and to assess whether glycosylated nano-hydroxyapatites (GHANPs) could improve bone regeneration in periodontitis by inducing macrophage M2 polarization. The change of macrophage polarization state in inflammatory periodontal tissues (with bone loss) was examined using clinical gingival samples. The relationship between macrophage phenotype and bone level in periodontal bone loss and repair was evaluated using a mouse periodontitis model. The effect of GHANPs on macrophage polarization was assessed by the in vitro model of lipopolysaccharide (LPS)-stimulated inflammation. The polarization-related markers were detected by immunofluorescence staining, real-time polymerase chain reaction and enzyme-linked immunosorbent assay analysis. The therapeutic effect of GHANPs on alveolar bone loss was explored in experimental periodontitis by histological staining and micro-CT analysis. A lower macrophage M2/M1 ratio was observed in periodontitis-affected human gingival tissues. The results of animal experiments demonstrated a positive correlation between a lower Arg-1/iNOS ratio and accelerated alveolar bone loss; also, the proportion of Arg-1-positive macrophages increased during bone repair and regeneration. The administration of GHANPs partially restored M2 macrophage polarization after LPS stimulation. GHANPs increased alveolar bone repair and regeneration in experimental periodontitis induced by ligation, potentially related to their macrophage M2 transition regulation. The findings of this study indicate that the induction of macrophage M2 polarization can be considered a viable approach for enhancing inflammatory bone repair. Additionally, GHANPs show potential in the clinical treatment of periodontitis.

Troxerutin suppress inflammation response and oxidative stress in jellyfish dermatitis by activating Nrf2/HO-1 signaling pathway.

Stomolophus meleagris envenomation causes severe cutaneous symptoms known as jellyfish dermatitis. The potential molecule mechanisms and treatment efficiency of dermatitis remain elusive because of the complicated venom components. The biological activity and molecular regulation mechanism of Troxerutin (TRX) was firstly examined as a potential treatment for jellyfish dermatitis. We examined the inhibit effects of the TRX on tentacle extract (TE) obtained from S. meleagris in vivo and in vitro using the mice paw swelling models and corresponding assays for Enzyme-Linked Immunosorbent Assay (ELISA) Analysis, cell counting kit-8 assay, flow cytometry, respectively. The mechanism of TRX on HaCaT cells probed the altered activity of relevant signaling pathways by RNA sequencing and verified by RT-qPCR, Western blot to further confirm protective effects of TRX against the inflammation and oxidative damage caused by TE. TE significantly induced the mice paw skin toxicity and accumulation of inflammatory cytokines and reactive oxygen species in vivo and vitro. Moreover, a robust increase in the phosphorylation of mitogen-activated protein kinase (MAPKs) and nuclear factor-kappa B (NF-κB) signaling pathways was observed. While, the acute cutaneous inflammation and oxidative stress induced by TE were significantly ameliorated by TRX treatment. Notablly, TRX suppressed the phosphorylation of MAPK and NF-κB by initiating the nuclear factor erythroid 2-related factor 2 signaling pathway, which result in decreasing inflammatory cytokine release. TRX inhibits the major signaling pathway responsible for inducing inflammatory and oxidative damage of jellyfish dermatitis, offering a novel therapy in clinical applications.

Isolation and Characterization of the Vascular Endothelial Growth Factor Receptor Targeting ScFv Antibody Fragments Derived from Phage Display Technology.

Angiogenesis, as a tumor hallmark, plays an important role in the growth and development of the tumor vasculature system. There is a huge amount of evidence suggesting that the vascular endothelial growth factor receptor (VEGFR-2)/VEGF-A axis is one of the main contributors to tumor angiogenesis and metastasis. Thus, inhibition of the VEGFR-2 signaling pathway by anti-VEGFR-2 mAb can retard tumor growth. In this study, we employ phage display technology and solution-phase biopanning (SPB) to isolate specific single-chain variable fragments (scFvs) against VEGFR-2 and report on the receptor binding characteristics of the candidate scFvs A semisynthetic phage antibody library to isolate anti-VEGFR-2 scFvs through an SPB performed with decreasing concentrations of the VEGFR-2-His tag and VEGFR-2-biotin. After successful expression and purification, the specificity of the selected scFv clones was further analyzed by enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunoblotting. The competition assay was undertaken to identify the VEGFR-2 receptor-blocking properties of the scFvs. Furthermore, the molecular binding characteristics of candidate scFvs were extensively studied by peptide-protein docking. Polyclonal ELISA analysis subsequent to four rounds of biopanning showed a significant enrichment of VEGFR-2-specific phage clones by increasing positive signals from the first round toward the fourth round of selection. The individual VEGFR-2-reactive scFv phage clones were identified by monoclonal phage ELISA. The sequence analysis and complementarity-determining region alignment identified the four unique anti-VEGFR-2-scFv clones. The soluble and purified scFvs displayed binding activity against soluble and cell-associated forms of VEGFR-2 protein in the ELISA and flow cytometry assays. Based on the inference from the molecular docking results, scFvs D3, E1, H1, and E9 recognized domains 2 and 3 on the VEGFR-2 protein and displayed competition with VEGF-A for binding to VEGFR-2. The competition assay confirmed that scFvs H1 and D3 can block the VEGFR-2/VEGF-A interaction. In conclusion, we identified novel VEGFR-2-blocking scFvs that perhaps exhibit the potential for angiogenesis inhibition in VEGFR-2-overexpressed tumor cells.

Downregulation of lncRNA MIR17HG reduced tumorigenicity and Treg-mediated immune escape of non-small-cell lung cancer cells through targeting the miR-17-5p/RUNX3 axis.

Long noncoding RNA MIR17HG was involved with the progression of non-small-cell lung cancer (NSCLC), but specific mechanisms of MIR17HG-mediated immune escape of NSCLC cells were still unknown. The present study investigated the function of MIR17HG on regulatory T cell (Treg)-mediated immune escape and the underlying mechanisms in NSCLC. Expression of MIR17HG and miR-17-5p in NSCLC tissue samples were detected using quantitative real-time PCR (qRT-PCR). A549 and H1299 cells were transfected with sh-MIR17HG, miR-17-5p inhibitor, or sh-MIR17HG + miR-17-5p inhibitor, followed by cocultured with Tregs. Cell proliferation was measured using 5-ethynyl-20-deoxyuridine (Edu) staining assay and cell counting kit-8 (CCK-8) assay. Flow cytometry was used for determining positive numbers of FOXP3+CD4+/CD25+/CD8+ Tregs. Through subcutaneous injection with transfected A549 cells, a xenograft nude mouse model was established. Weights and volumes of xenograft tumors were evaluated. Additionally, the expressions of immune-related factors includingtransforming growth factor beta (TGF-β), vascular endothelial growth factor A (VEGF-A), interleukin-10 (IL-10), IL-4, and interferon-gamma (IFN-γ) in cultured cells, were evaluated by enzyme-linked immunosorbent assay and western blot analysis. Then, miR-17-5p was decreased and MIR17HG was enhanced in both NSCLC tissues and cell lines. MIR17HG knockdown significantly suppressed cell proliferation, tumorigenicity, and immune capacity of Tregs in A549 and H1299 cells, whereas sh-MIR17HG significantly reduced expression levels of VEGF-A, TGF-β, IL-4, and IL-10 but promoted the IFN-γ level in vitro and in vivo. Moreover, downregulation of miR-17-5p significantly reversed the effects of sh-MIR17HG. Additionally, we identified that runt- related transcription factor 3 (RUNX3) was a target of miR-17-5p, and sh-MIR17HG and miR-17-5p mimics downregulated RUNX3 expression. In conclusion, downregulation of MIR17HG suppresses tumorigenicity and Treg-mediated immune escape in NSCLC through downregulating the miR-17-5p/RUNX3 axis, indicating that this axis contains potential biomarkers for NSCLC.

Mitochondrial ROS participates in Porphyromonas gingivalis-induced pyroptosis in cementoblasts

This study aimed to investigate correlation between mitochondrial reactive oxygen species and Porphyromonas gingivalis in the process of cementoblast pyroptosis. Lactate dehydrogenase activity assay, enzyme-linked immunosorbent assay, western blotting and flow cytometry analysis were utilized to explore whether Porphyromonas gingivalis triggered pyroptosis in cementoblasts. Reactive oxygen species and mitochondrial reactive oxygen species were detected using flow cytometry and fluorescence staining. The effect of mitochondrial reactive oxygen species on the Porphyromonas gingivalis-induced pyroptosis of cementoblasts was assessed by Mito-Tempo, mitochondrion-targeted superoxide dismutase mimetic. Phosphorylation levels of p65 were measured by western blotting. SC75741, a nuclear factor-kappa B inhibitor, was added to block the nuclear factor-kappa B in the Porphyromonas gingivalis-infected cementoblasts. Porphyromonas gingivalis triggered pyroptosis of cementoblasts, and an elevation in reactive oxygen species generation in the mitochondria was observed. Inhibition of mitochondrial reactive oxygen species reduced pyroptosis and nuclear factor-kappa B signaling pathway mediated the pyroptotic cell death in Porphyromonas gingivalis-infected cementoblasts. Together, our findings demonstrate that mitochondrial reactive oxygen species increased by Porphyromonas gingivalis participated in the pyroptosis of cementoblasts. Targeting mitochondrial reactive oxygen species may offer therapeutic strategies for root surface remodeling or periodontal regeneration.

Enzyme-linked immunosorbent assay and immunohistochemical analysis of mast cell related biochemicals in oral submucous fibrosis

Oral submucous fibrosis (OSMF), a potentially malignant disorder, is developed by progressive fibrous tissue deposition in connective tissue along with atrophy of oral mucosa. Histological sections also show the mast cell infiltration in submucosa which may indicate their possible role in this entity. Abundant availability of biochemicals in mast cells like histamine and serine proteases like chymase may be released and play specific pathways in the disease pathophysiology. Possibly, if the histamine release has some part to play, diamine oxidase may also be found to have a relationship as it metabolizes histamine. The present study is proposed to identify the presence of chymase, histamine, and diamine oxidase in both, serum as well as tissue by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC) respectively. This study may provide probable insight into the mast cell-related chemicals and their association with OSMF.