Underlying mechanisms behind the neuroprotective effect of vanillic acid against diabetes-associated cognitive decline: An in vivo study in a rat model.

Hippocampal synaptic dysfunction, oxidative stress, neuroinflammation, and neuronal loss play critical roles in the pathophysiology of diabetes-associated cognitive decline (DACD). The study aimed to investigate the effects of vanillic acid (VA), a phenolic compound, against DACD and explore the potential underlying mechanisms. Following confirmation of diabetes, rats were treated with VA (50 mg/kg/day; P.O.) or insulin (6 IU/rat/day; S.C.) for 8 consecutive weeks. The cognitive performance of the rats was evaluated using passive-avoidance and water-maze tasks. Long-term potentiation (LTP) was induced at hippocampal dentate gyrus (DG) synapses in response to high-frequency stimulation (HFS) applied to the perforant pathway (PP) to evaluate synaptic plasticity. Oxidative stress factors, inflammatory markers, and histological changes were evaluated in the rat hippocampus. This study showed that streptozotocin (STZ)-induced diabetes caused cognitive decline that was associated with inhibition of LTP induction, suppression of enzymatic antioxidant activities, enhanced lipid peroxidation, elevated levels of inflammatory proteins, and neuronal loss. Interestingly, chronic treatment with VA alleviated blood glucose levels, improved cognitive decline, ameliorated LTP impairment, modulated oxidative-antioxidative status, inhibited inflammatory response, and prevented neuronal loss in diabetic rats at a level comparable to insulin therapy. The results suggest that the antihyperglycemic, antioxidative, anti-inflammatory, and neuroplastic properties of VA may be the mechanisms behind its neuroprotective effect against DACD.

Detection of different variants of SARS-CoV-2 RNA (genome) on inanimate surfaces in high-touch public environmental surfaces.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) disease started in late 2019 and still continues as a global pandemic, spreading among people around the world. There is limited knowledge about the role of contaminated environmental surfaces, especially high-touch public surfaces, in the transmission of the disease. The objective of the present investigation was detection of different variants (Delta, UK, and Omicron) of SARS-CoV-2 RNA (genome) on inanimate surfaces in high-touch public environmental surfaces in different seasons. Automated teller machines of banks (ATM), point-of-sale (POS) machine, gas station pump nozzles, and escalator handrails of malls were selected as high-touch environmental surfaces in public places. Overall, 75 samples were collected from these places and examined for the presence of SARS-CoV-2 RNA (genome), and 21 samples (28%) were positive. Although the role of fomite transmission of COVID-19 is understood, more studies should be conducted to determine the virus survival rate as well as the required efforts to prevent the spread of SARS-CoV-2 such as frequent cleaning and the use of efficient disinfectants on environmental surfaces, especially high-touch public places. In conclusion, the results address the importance of touching contaminated inanimate objects as well as transmission through environmental surfaces, and they could be used to establish an effective protocol to prevent indirect environmental transmission of SARS-CoV-2, slow down the spread of the virus, and reduce the risk of infection.

The Effect of Long-Term Iron Chelator Therapy on Serum Levels of Hepcidin and Ferritin in Patients with Thalassemia Major and Intermediate.

Serum hepcidin is a good predictor of iron overload compared with serum ferritin. However, serum hepcidin levels may change under different conditions. The current study aims to determine the role of long-term iron chelator therapy on serum levels of hepcidin and ferritin in patients with thalassemia major (TM) and intermediate (TI). In this cross-sectional study 91 patients with thalassemia TM and TI, who referred to the thalassemia center were chosen. The serum levels of hepcidin and ferritin were measured after two years of iron chelator therapy by ELISA and ECL methods, respectively. The patients' demographic information was extracted from their records. After treatment with iron chelator, ferritin levels decreased in 44 patients (48.4%), and increased in 47 patients (%51.6). Median serum levels of hepcidin decreased in all patients (%100). Also, there was a significant association between serum levels of hepcidin and ferritin (p value = 0.034). Furthermore, while a significant difference was observed between ferritin changes (p = 0.01), no difference was found between changes in hepcidin based on the type of iron chelator (p value = 0.94). Increased levels of hepcidin and ferritin in ß-thalassemia patients are significantly ameliorated by iron chelator.

Endothelin-1 Stimulates PAI-1 Protein Expression via Dual Transactivation Pathway Dependent ROCK and Phosphorylation of Smad2L.

Objective: In addition to the carboxy region, Smad2 transcription factor can be phosphorylated in the linker region as
well. Phosphorylation of Smad2 linker region (Smad2L) promotes the expression of plasminogen activator inhibitor type
1 (PAI-1) which leads to cardiovascular disorders such as atherosclerosis. The purpose of this study was to evaluate the role of dual transactivation of EGF and TGF-ß receptors in phosphorylation of Smad2L and protein expression of PAI-1 induced by endothelin-1 (ET-1) in bovine aortic endothelial cells (BAECs). In addition, as an intermediary of G protein-coupled receptor (GPCR) signaling, the functions of ROCK and PLC were investigated in dual transactivation pathways.
Materials and Methods: The experimental study is an in vitro study performed on BAECs. Proteins were investigated
by western blotting using protein-specific antibodies against phospho-Smad2 linker region residues (Ser245/250/255),
phospho-Smad2 carboxy residues (465/467), ERK1/(Thr202/Thr204), and PAI-1.
Results: TGF (2 ng/ml), EGF (100 ng/ml) and ET-1 (100 nM) induced the phosphorylation of Smad2L. This response was
blocked in the presence of AG1478 (EGFR antagonists), SB431542 (TGFR inhibitor), and Y27632 (Rho-associated protein kinase (ROCK antagonist). Moreover, ET-1-increased protein expression of PAI-1 was decreased in the presence of bosentan (ET receptor inhibitor), AG1478, SB431542, and Y27632.
Conclusion: The results indicated that ET-1 increases the phosphorylation of Smad2L and protein expression of PAI-1
via induced the transactivation pathways of EGFR and TGFR. This study is the first attempt to scrutinize the significant role of ROCK in the protein expression of PAI-1.

Endothelin-1 dependent expression of GAG genes involves NOX and p38 mediated Smad linker region phosphorylation.

Endothelin-1 (ET-1) is implicated in the development of atherosclerosis and mediates glycosaminoglycan (GAG) chain hyperelongation on proteoglycans. Our aim was to identify the ET-1-mediated signalling pathway involving NADPH oxidase (NOX), p38 MAP kinsae and Smad2 linker region phosphorylation (phospho-Smad2L) regulate GAG synthesising enzymes mRNA expression (C4ST-1 and ChSy1) involved in GAG chains hyperelongation in human vascular smooth muscle cells (VSMCs). Signalling intermediates were detected and quantified by Western blotting and the mRNA levels of GAG synthesising enzymes were assessed by quantitative real-time polymerase chain reaction (qRT-PCR). ET-1 treatment of human VSMCs resulted in an increase in phospho-Smad2L level. The TGF-ß receptor antagonist, SB431542 and the mixed ETA and ETB receptor antagonist bosentan, inhibited ET-1-mediated phospho-Smad2L level. In the presence of apocynin and diphenyleneiodonium chloride (DPI) (NOX inhibitors) and SB239063 (p38 inhibitor) ET-1-mediated phospho-Smad2L levels were inhibited. The gene expression levels of GAG synthesising enzymes post-ET-1 treatment were increased compared to untreated controls (p < 0.01). The ET-mediated the mRNA levels of these enzymes were blocked by the bosentan, SB431542, SB239063, DPI, apocynin and antioxidant N-acetyl-L-cysteine (NAC). ET-1-mediated signalling to GAG synthesising enzymes gene expression occurs via transactivation-dependent pathway involving NOX, p38 MAP kinsae and Smad2 linker region phosphorylation.

Air Surveillance for Viral Contamination with SARS-CoV-2 RNA at a Healthcare Facility.

The transmission pathway of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 also called COVID-19 disease) in indoor environments are the main area of contention between health systems and scientists. In this context, little has been investigated about the collection of airborne viral shedding. Here, we collected air samples from 24 locations inside the sole COVID-19 patient care center in Zabol, Iran, for screening SARS-CoV-2 RNA from March to May 2021. Locations included the ICU, COVID-19 wards (CWs) rooms, corridors, nearby nurses' stations, and toilets. We identified the SARS-CoV-2 RNA in breathing zone of CW, in room air, with the positivity rate of 2.5% at a concentration of 17 × 103 virus genome copies/m3 air. It also investigates the relationship between local climate conditions [i.e., temperature and relative humidity] and COVID-19 transmission with the evolution of daily official data on the number of new cases, hospitalizations, and deaths. Current data explained that the difference of temperature and humidity may affect the behavior of virus along with other factors, i.e., population density, individual viral shedding, and infectious dose of SARS-CoV-2 (both indoor and outdoor). Our data support the potential SARS-CoV-2 airborne transmission indoors suggesting the specific safety assessment of building to improve ventilation solutions besides proper using face masks and extensive public health interventions.

Endothelin-1 Induced Phosphorylation of Caveolin-1 and Smad2C in Human Vascular Smooth Muscle Cells: Role of NADPH Oxidases, c-Abl, and Caveolae Integrity in TGF-ß Receptor Transactivation.

Caveolin-1(Cav-1) is one of the most important components of caveolae in the cell membrane, which plays an important role in cell signaling transduction, such as EGFR and TGF-ß receptor transactivation. The purpose of this study was to evaluate the effect of c-Abl and NAD(P)H oxidases (NOX) on phosphorylation of Cav-1 and consequently their effect on phosphorylation of Smad2C induced by Endothelin-1 in human vascular smooth muscle cells (VSMCs). In this study, all experiments were performed using human VSMCs. The phosphorylation level of the Caveolin-1 and Smad2C proteins were assessed by western blotting using Phospho-Caveolin-1 (Tyr14) antibody and phospho-Smad2 (Ser465/467) antibody. The data were reported as mean ± SEM. The VSMCs treated with endothelin-1(ET-1) (100 nanomolar (nmol)) demonstrated a time-dependent increase in the pCav-1 level (p<0.05). The inhibitors of NOX (diphenyleneiodonium) (p<0.05), cholesterol depleting agent (beta-cyclodextrin) (p<0.05) and c-Abl inhibitor (PP1) (p<0.01) were able to reduce the level of the phospho-Cav-1 and phospho-Smad2C induced by Et-1 (p<0.05). Our results proposed that caveolae structure, NOX, c-Abl played an important role in the phosphorylation of Cav-1 induced by ET-1 in the human VSMCs. Furthermore, our findings showed that phosphoCav-1 involved in TGFR transactivation. Thus, Et-1 via a transactivation-dependent mechanism can cause phosphorylation of Smad2C.

EGF Receptor Transactivation by Endothelin-1 Increased CHSY-1 Mediated by NADPH Oxidase and Phosphorylation of ERK1/2.

OBJECTIVE: Growth factors [transforming growth factor-ß (TGF-ß), epidermal growth factor (EGF), endothelin-1 (ET1)] stimulate proteoglycan synthesis resulting in retention and accumulation of low density lipoprotein (LDL) in vessel intima and leading to atherosclerosis development. This study investigated the role of ET-1 on the expression of CHSY1, proteoglycan synthesizing enzyme, through both EGF and TGF-ß receptor transactivation in human vascular smooth muscle cells (VSMCs). Also, we explored the involvement of NADPH oxidase (NOX), an important intermediate of redox signaling, in ET-1 transactivated EGF receptor (EGFR) through endothelin receptors. MATERIALS AND METHODS: In this experimental study, phosphorylated ERK1/2 and CHSY1 protein levels in the human VSMCs were measured by Western blot analysis using anti phospho-ERK1/2 (Thr202/Tyr204) and anti CHSY1 antibodies. RESULTS: ET-1 (100 nM) and EGF (100 ng/ml) stimulated ERK1/2 phosphorylation and inhibited in the presence of bosentan (ET receptor inhibitor), AG1478 (EGFR inhibitor), and DPI (NOX antagonist). Also, ET-1 treatment increased CHSY1 enzyme level; this response was suppressed by bosentan, AG1478, DPI, and SB431542, TGF-ß receptor antagonist. This study revealed that ET-1 increases expression of CHSY1 through transactivation of EGF and TGF-ß receptors. CONCLUSION: Transactivation through the EGF receptor mediated by phospho-ERK1/2 leads to expression of CHSY1 protein. EGF receptor transactivation by ET-1 is shown for the first time, to be dependent on NOX enzymes.

Protective Effects of Co-administration of Zinc and Selenium Against Streptozotocin-Induced Alzheimer's Disease: Behavioral, Mitochondrial Oxidative Stress, and GPR39 Expression Alterations in Rats.

Changes in the concentrations of trace metals such as zinc (Zn) and selenium (Se) can pathologically lead to neurodegenerative conditions such as the Alzheimer's disease (AD). Previous studies have shown that mitochondrial dysfunction plays an important role in the pathogenesis of AD. Several male Wistar rats were randomly divided into five groups: sham group, AD group that received 3 mg/kg of streptozotocin (STZ) intracerebroventricularly, AD + Zn group that received 10 mg/kg of Zn intraperitoneally (i.p.) for 1 week, AD + Se group that received 0.1 mg/kg of Se i.p. for 1 week, and AD + Zn + Se group that received 10 mg/kg of Zn i.p. plus 0.1 mg/kg of Se i.p. for 1 week. At end of the study, behavioral tests and mitochondrial oxidative stress and GPR39 gene expression evaluations were carried out. Co-administration of Zn and Se significantly decreased the potential collapse of mitochondrial membrane, reactive oxygen species levels, and lipid peroxidation levels while significantly increased cognitive performance, superoxide dismutase (SOD), glutathione peroxidase, and catalase activity in the brain mitochondria compared with the STZ group. In addition, no significant changes were observed in GPR39 expression in the co-treated group. Findings of the current study showed that ZnR/GPR39 receptor, mitochondrial dysfunction, and oxidative stress play important roles in the pathogenesis of AD. Co-treatment of Zn and Se improved the cognitive performance, mitochondrial dysfunction, and oxidative stress caused by STZ-induced AD. Therefore, therapeutic approaches to improve mitochondrial function could be effective in preventing the initiation and progression of AD.

Endothelin-1 increases CHSY-1 expression in aortic endothelial cells via transactivation of transforming growth factor ß type I receptor induced by type B receptor endothelin-1.

OBJECTIVES: TGF-ß through hyperelongation of glycosaminoglycan (GAG) chains leads to binding of low-density lipoproteins to the proteoglycans. The vasoactive peptide, endothelin-1 (ET-1), plays a key role in the development of atherosclerosis. This study addressed the question whether ET-1 by activating the Rho kinase and cytoskeletal rearrangement can transactivate the TGF-ß receptor leading to phosphorylation of the transcription factor Smad2 and increased expression of the GAG chain synthesizing enzyme such as chondroitin synthase-1 (CHSY-1) in bovine aortic endothelial cells (BAECs). METHODS: In this study, intermediates in ET-1-induced Smad2C phosphorylation and the protein level of CHSY-1 were identified and quantified by Western blotting. KEY FINDINGS: Endothelin-1 caused time-dependent phosphorylation of Smad2C which was inhibited in the presence of the endothelin B receptor antagonist, BQ788. The response to ET-1 was inhibited by the Rho/ROCK kinase antagonist, Y27632 and by cytochalasin D, an inhibitor of actin polymerization but the ET-1-mediated pSmad2C was not inhibited by the matrix metalloproteinase (MMP) inhibitor, GM6001. ET-1 increased CHSY-1 protein level, which was inhibited in the presence of BQ788, cytochalasin D and Y27632. CONCLUSIONS: Endothelin-1 signalling via the ETB receptor utilizes cytoskeletal rearrangement and Rho kinase but not MMPs leading to TßRI transactivation signalling and phosphorylation of Smad2C and through this pathway increased the level of CHSY-1.

Transforming growth factor-ß1 mediated CHST11 and CHSY1 mRNA expression is ROS dependent in vascular smooth muscle cells.

Transforming growth factor (TGF)-ß1 mediates glycosaminoglycan (GAG) chain hyperelongation on secreted proteoglycans and these modifications are associated with increased lipid binding in the vessel wall and the development of atherosclerosis. In vascular smooth muscle cells (VSMCs), TGF-ß1 regulated GAG elongation via extracellular signal-regulated kinase (ERK) and p38 as well as Smad2 linker region phosphorylation. In this study, our aim was to identify the TGF-ß1 mediated signalling pathway involving reactive oxygen species (ROS) and Smad2 linker region phosphorylation that regulate the mRNA expression of GAG synthesizing enzymes, chondroitin 4-O-sulfotransferase 1 (CHST11) and chondroitin sulfate synthase 1 (CHSY1) which are the rate limiting enzymes involved in GAG chain elongation. Signalling molecules were assessed by western blotting, quantitative real-time PCR was used for analysis of gene expression and intracellular ROS level was measured by a fluorescence based assay. TGF-ß1 induced ROS production in VSMCs. Nicotinamide adenine dinucleotide phosphate oxidase (Nox) inhibitors, diphenyleneiodonium (DPI) and apocynin blocked TGF-ß1 mediated Smad2 linker region phosphorylation. TGF-ß1 treatment increased the mRNA levels of CHST11 and CHSY1. Pharmacological inhibition of Nox blocked TGF-ß1 mediated mitogen activated protein kinases (MAPKs) phosphorylation and TGF-ß1 stimulated CHST11 and CHSY1 mRNA expression. These findings demonstrated that TGF-ß1 mediated expression of CHST11 and CHSY1 can occur via Nox-dependent pathways and Smad2 linker region phosphorylation.

Reactive Oxygen Species and p38MAPK Have a Role in the Smad2 Linker Region Phosphorylation Induced by TGF-ß.

Background: Transforming growth factor-ß (TGF-ß) in addition to the C-terminal region can phosphorylate receptor-regulated Smads (R-Smads) in their linker region. The aim of the present study was to evaluate the role of signaling mediators such as NAD(P)H oxidases (reactive oxygen species [ROS] generators), ROS, and ROS-sensitive p38 mitogen-activated protein kinase (p38MAPK) in this signaling pathway in cultured human vascular smooth muscle cells (VSMCs). Methods: The present in vitro study was performed on human VSMCs. Proteins were detected by western blotting utilizing an anti-phospho-Smad2 (Ser245/250/255) rabbit polyclonal antibody and a horseradish peroxidase-labeled secondary antibody. Glyceraldehyde-3-phosphate dehydrogenase was used as a loading control. The phospho-Smad2 linker region (pSmad2L) was detected in all the experimental groups: a control group (untreated group), a group treated with TGF-ß (2 ng/mL), and a group treated with TGF-ß plus different inhibitors. The data were normalized and presented as mean±SEM. The statistical analyses were performed using SPSS, version 16.0, and the nonparametric Kruskal-Wallis test. A P value smaller than 0.05 was considered statistically significant. Results: The VSMCs treated with TGF-ß (2 ng/mL) showed a time-dependent increase in the pSmad2L level. The highest level was observed at 15 minutes (P=0.03). The inhibitors of NAD(P)H oxidases (diphenyleneiodonium and apocynin) (P=0.04), ROS scavenger (N-acetylcysteine) (P=0.04), and p38MAPK inhibitor (SB-202190) (P=0.04) were able to reduce the increased level of the pSmad2L by TGF-ß. Conclusion: Our results suggested that NAD(P)H oxidases played an important role in the Smad2L phosphorylation in the human VSMCs. Furthermore, our results confirmed that ROS and p38MAPK were involved in this signaling pathway. Thus, TGF-ß via a ROS-dependent mechanism can transmit its signals to the pSmad2L.