Detection of SARS-CoV-2 in Environment: Current Surveillance and Effective Data Management of COVID-19.

Since diagnostic laboratories handle large COVID-19 samples, researchers have established laboratory-based assays and developed biosensor prototypes. Both share the same purpose; to ascertain the occurrence of air and surface contaminations by the SARS-CoV-2 virus. However, the biosensors further utilize internet-of-things (IoT) technology to monitor COVID-19 virus contamination, specifically in the diagnostic laboratory setting. The IoT-capable biosensors have great potential to monitor for possible virus contamination. Numerous studies have been done on COVID-19 virus air and surface contamination in the hospital setting. Through reviews, there are abundant reports on the viral transmission of SARS-CoV-2 through droplet infections, person-to-person close contact and fecal-oral transmission. However, studies on environmental conditions need to be better reported. Therefore, this review covers the detection of SARS-CoV-2 in airborne and wastewater samples using biosensors with comprehensive studies in methods and techniques of sampling and sensing (2020 until 2023). Furthermore, the review exposes sensing cases in public health settings. Then, the integration of data management together with biosensors is well explained. Last, the review ended with challenges to having a practical COVID-19 biosensor applied for environmental surveillance samples.

Hybrid Inorganic Organic PSF/Hap Dual-Layer Hollow Fibre Membrane for the Treatment of Lead Contaminated Water.

Lead (Pb) exposure can be harmful to public health, especially through drinking water. One of the promising treatment methods for lead contaminated water is the adsorption-filtration method. To ensure the cost-effectiveness of the process, naturally derived adsorbent shall be utilised. In this study, hydroxyapatite particles, Ca10(PO4)6(OH)2 (HAP) derived from waste cockle shell, were incorporated into the outer layer of polysulfone/HAP (PSf/HAP) dual-layer hollow fibre (DLHF) membrane to enhance the removal of lead from the water source due to its hydrophilic nature and excellent adsorption capacity. The PSf/HAP DLHF membranes at different HAP loadings in the outer layer (0, 10, 20, 30 and 40 wt%) were fabricated via the co-extrusion phase inversion technique. The performance of the DLHF membranes was evaluated in terms of pure water flux, permeability and adsorption capacity towards lead. The results indicated that the HAP was successfully incorporated into the outer layer of the membrane, as visibly confirmed by microscopic analysis. The trend was towards an increase in pure water flux, permeability and lead adsorption capacity as the HAP loading increased to the optimum loading of 30 wt%. The optimized DLHF membrane displayed a reduced water contact angle by 95%, indicating its improved surface hydrophilicity, which positively affects the pure water flux and permeability of the membrane. Furthermore, the DLHF membrane possessed the highest lead adsorption capacity, 141.2 mg/g. The development of a hybrid inorganic-organic DLHF membrane via the incorporation of the naturally derived HAP in the outer layer is a cost-effective approach to treat lead contaminated water.

Marginal Fit of Porcelain Laminate Veneer Materials under Thermocycling Condition: An In-Vitro Study.

Objective: The purpose of this study was to evaluate the cervical marginal fit of porcelain laminate veneer (PLV) restorations made from two different types of CAD/CAM ceramic laminates: CEREC C PC and E.max (LD). Materials and Methods: This in-vitro experiment used a total of 32 human maxillary first premolars that were clean and free of any cracks or caries, extracted for orthodontic purposes. The samples were divided in a random way into two study groups: A and B (n = 16). Each sample was mounted on a dental surveyor and a silicon impression was made to create a silicone index for each tooth in both groups. Standardized preparation was carried out for all the samples by using preparation bur kit for the ceramic veneer system. Subsequently, digital impressions were made for all the samples by using Trios 3 shape intraoral camera (Sirona Dental Systems). The design of veneer restorations was made using Sirona inLab CAD SW 16.1 with CEREC inLab MC XL (Dentsply, Sirona Dental Systems, Bensheim, Germany). The veneer restorations were cemented using 3M RelyX veneer resin cement (3M ESPE, Seefeld, Germany) and the samples kept in distilled water for two weeks at 37 °C. All the specimens were subjected to thermocycling in a water bath with temperature varying between 5 °C and 55 °C for 500 cycles. The cervical marginal fit of veneers was evaluated by a digital microscope after sectioning the embedded teeth in acrylic resin. Results: The lowest mean of cervical marginal gap was recorded for Group A (91.59431 ± 1.626069) which was restored with CEREC CAD/CAM veneers, while the highest mean value of the gap was recorded for Group B (106.48863 ± 2.506684) which was restored with IPS E.max CAD. The t-test showed that the type of porcelain veneer restoration had a highly significant effect on the cervical marginal fit (p ≤ 0.01). Conclusions: CEREC CAD/CAM veneers showed smaller cervical marginal gaps, indicating a better fit compared to the IPS E.max CAD.

Evaluation of Shear Bond Strength of Resin-Based Composites to Biodentine with Three Types of Seventh-Generation Bonding Agents: An In Vitro Study.

Methods: Forty-eight acrylic blocks having central holes with a nominal diameter of 4 mm and a depth of 2 mm were prepared. The holes of the acrylic blocks were filled with Biodentine, which was prepared following the guidelines provided by the manufacturer. Then, the specimens were divided into six groups (n = 8). Groups 1, 2, and 3, Tetric N-Ceram composite bonded to Biodentine with Tetric N-bond, Xeno V+, Bond Force bond, respectively. Group 4, 5, and 6, Filtek Z350 bonded to Biodentine with the same three adhesives. The specimens were placed in distilled water for 24 hours and tested for the SBS in a universal testing machine at a crosshead speed of 1 mm/min. The test data were listed in a table and independent samples t-test and analysis of variance (ANOVA) were conducted as a part of the statistical analysis. Results: The Tetric N bonding agent achieved the highest SBS followed by Bond Force, and Xeno V and highly significant difference was found. On the other hand, an overall increase in the SBS values of the Tetric N-Ceram resin was noticed in comparison with the Filtek Z350 and the differences was statistically significant. Although the specimens failed in adhesive, cohesive and mixed fracture modes but the cohesive was found to be the dominant failure mode in all groups. Conclusion: Among the tested bonding agents and resin composites, the Tetric N-Ceram composite bonded by Tetric N-bond self-etch adhesive with the Biodentine showed the highest SBS compared to the other combinations.

Direct and Sensitive Detection of Dopamine Using Carbon Quantum Dots Based Refractive Index Surface Plasmon Resonance Sensor.

Abnormality of dopamine (DA), a vital neurotransmitter in the brain's neuronal pathways, causes several neurological diseases. Rapid and sensitive sensors for DA detection are required for early diagnosis of such disorders. Herein, a carbon quantum dot (CQD)-based refractive index surface plasmon resonance (SPR) sensor was designed. The sensor performance was evaluated for various concentrations of DA. Increasing DA levels yielded blue-shifted SPR dips. The experimental findings revealed an excellent sensitivity response of 0.138°/pM in a linear range from 0.001 to 100 pM and a high binding affinity of 6.234 TM-1. The effects of varied concentrations of DA on the optical characteristics of CQD thin film were further proved theoretically. Increased DA levels decreased the thickness and real part of the refractive index of CQD film, according to fitting results. Furthermore, the observed reduction in surface roughness using AFM demonstrated that DA was bound to the sensor layer. This, in turn, explained the blue shift in SPR reflectance curves. This optical sensor offers great potential as a trustworthy solution for direct measurement due to its simple construction, high sensitivity, and other sensing features.

Bisphenol A Removal Using Visible Light Driven Cu2O/PVDF Photocatalytic Dual Layer Hollow Fiber Membrane.

Bisphenol A (BPA) is amongst the endocrine disrupting compounds (EDCs) that cause illness to humans and in this work was removed using copper (I) oxide (Cu2O) visible light photocatalyst which has a narrow bandgap of 2.2 eV. This was done by embedding Cu2O into polyvinylidene fluoride (PVDF) membranes to generate a Cu2O/PVDF dual layer hollow fiber (DLHF) membrane using a co-extrusion technique. The initial ratio of 0.25 Cu2O/PVDF was used to study variation of the outer dope extrusion flowrate for 3 mL/min, 6 mL/min and 9 mL/min. Subsequently, the best flowrate was used to vary Cu2O/PVDF for 0.25, 0.50 and 0.75 with fixed outer dope extrusion flowrate. Under visible light irradiation, 10 mg/L of BPA was used to assess the membranes performance. The results show that the outer and inner layers of the membrane have finger-like structures, whereas the intermediate section of the membrane has a sponge-like structure. With high porosity up to 63.13%, the membrane is hydrophilic and exhibited high flux up to 13,891 L/m2h. The optimum photocatalytic membrane configuration is 0.50 Cu2O/PVDF DLHF membrane with 6 mL/min outer dope flowrate, which was able to remove 75% of 10 ppm BPA under visible light irradiation without copper leaching into the water sample.

Oilfield-produced water treatment using conventional and membrane-based technologies for beneficial reuse: A critical review.

Oilfield produced water (OPW) is one of the most important by-products, resulting from oil and gas exploration. The water contains a complex mixture of organic and inorganic compounds such as grease, dissolved salt, heavy metals as well as dissolved and dispersed oils, which can be toxic to the environment and public health. This article critically reviews the complex properties of OPW and various technologies for its treatment. They include the physico-chemical treatment process, biological treatment process, and physical treatment process. Their technological strengths and bottlenecks as well as strategies to mitigate their bottlenecks are elaborated. A particular focus is placed on membrane technologies. Finally, further research direction, challenges, and perspectives of treatment technologies for OPW are discussed. It is conclusively evident from 262 published studies (1965-2021) that no single treatment method is highly effective for OPW treatment as a stand-alone process however, conventional membrane-based technologies are frequently used for the treatment of OPW with the ultrafiltration (UF) process being the most used for oil rejection form OPW and oily waste water. After membrane treatment, treated effluents of the OPW could be reused for irrigation, habitant and wildlife watering, microalgae production, and livestock watering. Overall, this implies that target pollutants in the OPW samples could be removed efficiently for subsequent use, despite its complex properties. In general, it is however important to note that feed quality, desired quality of effluent, cost-effectiveness, simplicity of process are key determinants in choosing the most suitable treatment process for OPW treatment.

Expert guidance on prophylaxis and treatment of dermatologic adverse events with Tumor Treating Fields (TTFields) therapy in the thoracic region.

Tumor Treating Fields (TTFields) are electric fields, delivered via wearable arrays placed on or near the tumor site, that exert physical forces to disrupt cellular processes critical for cancer cell viability and tumor progression. As a first-in-class treatment, TTFields therapy is approved for use in newly diagnosed glioblastoma, recurrent glioblastoma, and pleural mesothelioma. Additionally, TTFields therapy is being investigated in non-small cell lung cancer (NSCLC), brain metastases from NSCLC, pancreatic cancer, ovarian cancer, hepatocellular carcinoma, and gastric adenocarcinoma. Because TTFields therapy is well tolerated and delivery is locoregional, there is low risk of additive systemic adverse events (AEs) when used with other cancer treatment modalities. The most common AE associated with TTFields therapy is mild-to-moderate skin events, which can be treated with topical agents and may be managed without significant treatment interruptions. Currently, there are no guidelines for oncologists regarding the management of TTFields therapy-related skin AEs in the thoracic region, applicable for patients with pleural mesothelioma or NSCLC. This publication aims to provide guidance on preventing, minimizing, and managing dermatologic AEs in the thoracic region to help improve patient quality of life and reduce treatment interruptions that may impact outcomes with TTFields therapy.

Fabrication of High Performance PVDF Hollow Fiber Membrane Using Less Toxic Solvent at Different Additive Loading and Air Gap.

Existing toxic solvents in the manufacturing of polymeric membranes have been raising concerns due to the risks of exposure to health and the environment. Furthermore, the lower tensile strength of the membrane renders these membranes unable to endure greater pressure during water treatment. To sustain a healthier ecosystem, fabrication of polyvinylidene fluoride (PVDF) hollow fiber membrane using a less toxic solvent, triethyl phosphate (TEP), with a lower molecular weight polyethylene glycol (PEG 400) (0-3 wt.%) additive were experimentally demonstrated via a phase inversion-based spinning technique at various air gap (10, 20 and 30 cm). Membrane with 2 wt.% of PEG 400 exhibited the desired ultrafiltration asymmetric morphology, while 3 wt.% PEG 400 resulting microfiltration. The surface roughness, porosity, and water flux performance increased as the loading of PEG 400 increased. The mechanical properties and contact angle of the fabricated membrane were influenced by the air gap where 20 cm indicate 2.91 MPa and 84.72°, respectively, leading to a stronger tensile and hydrophilicity surface. Lower toxicity TEP as a solvent helped in increasing the tensile properties of the membrane as well as producing an eco-friendly membrane towards creating a sustainable environment. The comprehensive investigation in this study may present a novel composition for the robust structure of polymeric hollow fiber membrane that is suitable in membrane technology.

Assessing Fracture Resistance of Restored Premolars with Novel Composite Materials: An In Vitro Study.

During restorative treatment, premolars restored with resin filling materials using the conventional incremental-fill technique take longer restoration time and undermine the integrity of the tooth. The aim of this study was to assess fracture resistance of premolars restored by various types of novel bulk-fill composite resin materials. Forty-eight (n = 48) freshly extracted sound maxillary first premolars were used in this in vitro study. The teeth were divided into six groups, each having 8 specimens. Group A (positive control) was allocated for the intact teeth. For specimens in Groups B to F, a large cavity (Class-II MOD) was prepared with a standardized dimension of cavity (3 mm depth on the pulpal floor, 4 mm at the gingival seat, and 3 mm cavity width). Group B represented prepared teeth without any restoration. Group C, Group D, Group E, and Group F were restored with Tetric EvoCeram® incremental-fill (conventional), Beautifil bulk-fill, Filtek posterior bulk-fill, and SonicFill 2 bulk-fill restorative materials, respectively. All samples were finished and polished with an enhanced finishing kit and stored in distilled water for a month before the fracture resistance testing. All the samples were exposed to the axial loading (the speed of crosshead was 1 mm/min) in a computer-controlled universal testing machine (LARYEE, China) via a steel bar (6 mm in diameter) and the maximum applied force in Newton was recorded as the fracture resistance. One-way analysis of variance (SPSS 21) was used to compare the fracture resistance within the groups, and Tukey's post hoc test was used to determine the difference between the groups. The lowest value of fracture resistance was recorded for Group B, and the highest value was recorded for Group A followed by the values of Group D, Group C, Group F, and Group E. One-way ANOVA revealed a statistically significant difference between the groups (P < 0.05). Nonsignificant difference was found between the premolars restored by bulk-fill and conventional composites. Among the bulk-fill restored specimens, Beautifil restorative demonstrated significantly higher fracture resistance in comparison with the other two bulk-fill restored specimen groups (SonicFill 2 and Filtek). Bulk-fill composite such as Beautifil could be an alternative option to conventional incremental-fill composite for premolar restoration.

The effects of pollen, propolis, and caffeic acid phenethyl ester on tyrosine hydroxylase activity and total RNA levels in hypertensive rats caused by nitric oxide synthase inhibition: experimental, docking and molecular dynamic studies.

The objective of the present study was to evaluate the effects of propolis, pollen, and caffeic acid phenethyl ester (CAPE) on tyrosine hydroxylase (TH) activity and total RNA levels of Nω-nitro-L-arginine methyl ester (L-NAME) inhibition of nitric oxide synthase in the heart, adrenal medulla, and hypothalamus of hypertensive male Sprague dawley rats. The TH activity in the adrenal medulla, heart, and hypothalamus of the rats was significantly increased in the L-NAME group vs. control (p < 0.05). Treatment with L-NAME led to a significant increase in blood pressure (BP) in the L-NAME group compared to control (p < 0.05). These data suggest that propolis, pollen, and CAPE may mediate diminished TH activity in the heart, adrenal medulla, and hypothalamus in hypertensive rats. The decreased TH activity may be due to the modulation and synthesis of catecholamines and BP effects. In addition, the binding mechanism of CAPE within the catalytic domain of TH was investigated by means of molecular modeling approaches. These data suggest that the amino acid residues, Glu429 and Ser354 of TH may play a pivotal role in the stabilization of CAPE within the active site as evaluated by molecular dynamics (MD) simulations. Gibbs binding free energy (ΔGbinding) of CAPE in complex with TH was also determined by post-processing MD analysis approaches (i.e. Poisson-Boltzmann Surface Area (MM-PBSA) method).

Effects of propolis, caffeic acid phenethyl ester, and pollen on renal injury in hypertensive rat: An experimental and theoretical approach.

The objective of this study was to evaluate the antioxidant effects of propolis, caffeic acid phenethyl ester (CAPE; active compound in propolis), and pollen on biochemical oxidative stress biomarkers in rat kidney tissue inhibited by Nω -nitro-L-arginine methyl ester (L-NAME). The biomarkers evaluated were paraoxonase (PON1), oxidative stress index (OSI), total antioxidant status (TAS), total oxidant status (TOS), asymmetric dimethylarginine (ADMA), and nuclear factor kappa B (NF-κB). TAS levels and PON1 activity were significantly decreased in kidney tissue samples in the L-NAME-treated group (P < 0.05). The levels of TAS and PONI were higher in the L-NAME plus propolis, CAPE, and pollen groups compared with the L-NAME-treated group. TOS, ADMA, and NF-κB levels were significantly increased in the kidney tissue samples of the L-NAME-treated group (P < 0.05). However, these parameters were significantly lower in the L-NAME plus propolis, CAPE, and pollen groups (P < 0.05) compared with rats administered L-NAME alone (P < 0.05). Furthermore, the binding energy of CAPE within catalytic domain of glutathione reductase (GR) enzyme as well as its inhibitory mechanism was determined using molecular modeling approaches. In conclusion, experimental and theoretical data suggested that oxidative alterations occurring in the kidney tissue of chronic hypertensive rats may be prevented via active compound of propolis, CAPE administration.

Synthesis of a nano-silver metal ink for use in thick conductive film fabrication applied on a semiconductor package.

The success of printing technology in the electronics industry primarily depends on the availability of metal printing ink. Various types of commercially available metal ink are widely used in different industries such as the solar cell, radio frequency identification (RFID) and light emitting diode (LED) industries, with limited usage in semiconductor packaging. The use of printed ink in semiconductor IC packaging is limited by several factors such as poor electrical performance and mechanical strength. Poor adhesion of the printed metal track to the epoxy molding compound is another critical factor that has caused a decline in interest in the application of printing technology to the semiconductor industry. In this study, two different groups of adhesion promoters, based on metal and polymer groups, were used to promote adhesion between the printed ink and the epoxy molding substrate. The experimental data show that silver ink with a metal oxide adhesion promoter adheres better than silver ink with a polymer adhesion promoter. This result can be explained by the hydroxyl bonding between the metal oxide promoter and the silane grouping agent on the epoxy substrate, which contributes a greater adhesion strength compared to the polymer adhesion promoter. Hypotheses of the physical and chemical functions of both adhesion promoters are described in detail.

Integrated technologies for solid waste bin monitoring system.

The integration of communication technologies such as radio frequency identification (RFID), global positioning system (GPS), general packet radio system (GPRS), and geographic information system (GIS) with a camera are constructed for solid waste monitoring system. The aim is to improve the way of responding to customer's inquiry and emergency cases and estimate the solid waste amount without any involvement of the truck driver. The proposed system consists of RFID tag mounted on the bin, RFID reader as in truck, GPRS/GSM as web server, and GIS as map server, database server, and control server. The tracking devices mounted in the trucks collect location information in real time via the GPS. This information is transferred continuously through GPRS to a central database. The users are able to view the current location of each truck in the collection stage via a web-based application and thereby manage the fleet. The trucks positions and trash bin information are displayed on a digital map, which is made available by a map server. Thus, the solid waste of the bin and the truck are being monitored using the developed system.

Calcium-sensing receptor signaling pathways in medullary thick ascending limb cells mediate COX-2-derived PGE2 production: functional significance.

We determined the functional implications of calcium-sensing receptor (CaR)-dependent, Gq- and Gi-coupled signaling cascades, which work in a coordinated manner to regulate activity of nuclear factor of activated T cells and tumor necrosis factor (TNF)-alpha gene transcription that cause expression of cyclooxygenase (COX)-2-derived prostaglandin E2 (PGE2) synthesis by rat medullary thick ascending limb cells (mTAL). Interruption of Gq, Gi, protein kinase C (PKC), or calcineurin (CaN) activities abolished CaR-mediated COX-2 expression and PGE2 synthesis. We tested the hypothesis that these pathways contribute to the effects of CaR activation on ion transport in mTAL cells. Ouabain-sensitive O2 consumption, an in vitro correlate of ion transport in the mTAL, was inhibited by approximately 70% in cells treated for 6 h with extracellular Ca2+ (1.2 mM), an effect prevented in mTAL cells transiently transfected with a dominant negative CaR overexpression construct (R796W), indicating that the effect was initiated by stimulation of the CaR. Pretreatment with the COX-2-selective inhibitor, NS-398 (1 microM), reversed CaR-activated decreases in ouabain-sensitive O2 consumption by approximately 60%, but did not alter basal levels of ouabain-sensitive O2 consumption. Similarly, inhibition of either Gq, Gi, PKC, or CaN, which are components of the mechanism associated with CaR-stimulated COX-2-derived PGE2 synthesis, reversed the inhibitory effects of CaR on O2 consumption without affecting basal O2 consumption. Our findings identified signaling elements required for CaR-mediated TNF production that are integral components regulating mTAL function via a mechanism involving COX-2 expression and PGE2 production.

CaR activation increases TNF production by mTAL cells via a Gi-dependent mechanism.

We evaluated the contribution of calcium-sensing receptor (CaR)-mediated G(i)-coupled signaling to TNF production in medullary thick ascending limb (mTAL) cells. A selective G(i) inhibitor, pertussis toxin (PTX), but not the inactive B-oligomer binding subunit, abolished CaR-mediated increases in TNF production. The inhibitory effect of PTX was partially reversed by using an adenylate cyclase inhibitor. CaR-mediated TNF production also was partially reversed by a cAMP analog, 8-Br-cAMP. IP(1) accumulation was CaR dependent and blocked by PI-PLC; partial inhibition also was observed with PTX. CaR increased calcineurin (CaN) activity by approximately threefold, and PTX prevented CaR-mediated increases in CaN activity, an nuclear factor of activated T cells (NFAT)-cis reporter construct, and a TNF promoter construct. The interaction between G(i) and PKC was determined, as we previously showed that CaR-mediated TNF production was CaN and NFAT- mediated and G(q) dependent. CaR activation increased PKC activity by twofold, an effect abolished by transient transfection with a dominant negative CaR construct, R796W, or pretreatment with PTX. Inhibition with the pan-specific PKC inhibitor GF 109203X (20 nM) abolished CaR-mediated increases in activity of CaN, an NFAT reporter, and a TNF promoter construct. Collectively, the data suggest that G(i)-coupled signaling contributes to NFAT-mediated TNF production in a CaN- and PKC-dependent manner and may be part of a CaR mechanism to regulate mTAL function. Moreover, concurrent G(q) and G(i) signaling is required for CaR-mediated TNF production in mTAL cells via a CaN/NFAT pathway that is PKC dependent. Understanding CaR-mediated signaling pathways that regulate TNF production in the mTAL is crucial to defining novel mechanisms that regulate extracellular fluid volume and salt balance.

Effects of the components of hormone therapy on matrix metalloproteinases in breast-cancer cells: an in vitro study.

A combination of E and progestogens significantly increased matrix metalloproteinase (MMP)-2 in both T47D cells (E(2)-medroxyprogesteroneacetate [MPA] and E(2)-P) and MCF-7 cells (E(2)-MPA, E(2)-P, and equilin-MPA). All combinations resulted in higher MMP-9 levels in MCF-7 cells, but higher MMP-9 levels resulted only with equilin-norethinderone in T47D cells.

NFAT regulates calcium-sensing receptor-mediated TNF production.

Because nuclear factor of activated T cells (NFAT) has been implicated in TNF production as well as osmoregulation and salt and water homeostasis, we addressed whether calcium-sensing receptor (CaR)-mediated TNF production in medullary thick ascending limb (mTAL) cells was NFAT dependent. TNF production in response to addition of extracellular Ca(2+) (1.2 mM) was abolished in mTAL cells transiently transfected with a dominant-negative CaR construct (R796W) or pretreated with the phosphatidylinositol phospholipase C (PI-PLC) inhibitor U-73122. Cyclosporine A (CsA), an inhibitor of the serine/threonine phosphatase calcineurin, and a peptide ligand, VIVIT, that selectively inhibits calcineurin-NFAT signaling, also prevented CaR-mediated TNF production. Increases in calcineurin activity in cells challenged with Ca(2+) were inhibited after pretreatment with U-73122 and CsA, suggesting that CaR activation increases calcineurin activity in a PI-PLC-dependent manner. Moreover, U-73122, CsA, and VIVIT inhibited CaR-dependent activity of an NFAT construct that drives expression of firefly luciferase in transiently transfected mTAL cells. Collectively, these data verify the role of calcineurin and NFAT in CaR-mediated TNF production by mTAL cells. Activation of the CaR also increased the binding of NFAT to a consensus oligonucleotide, an effect that was blocked by U-73122 and CsA, suggesting that a calcineurin- and NFAT-dependent pathway increases TNF production in mTAL cells. This mechanism likely regulates TNF gene transcription as U-73122, CsA, and VIVIT blocked CaR-dependent activity of a TNF promoter construct. Elucidating CaR-mediated signaling pathways that regulate TNF production in the mTAL will be crucial to understanding mechanisms that regulate extracellular fluid volume and salt balance.

Calcium-sensing receptor-mediated TNF production in medullary thick ascending limb cells.

Medullary thick ascending limb (mTAL) cells in primary culture express the Ca(2+)-sensing receptor (CaR), a G protein-coupled receptor that senses changes in extracellular Ca(2+) (Ca(o)(2+)) concentration, resulting in increases of intracellular Ca(2+) concentration and PKC activity. Exposure of mTAL cells to either Ca(o)(2+) or the CaR-selective agonist poly-L-arginine increased TNF-alpha synthesis. Moreover, the response to Ca(o)(2+) was enhanced in mTAL cells transfected with a CaR overexpression vector. Transfection of mTAL cells with a TNF promoter construct revealed an increase in reporter gene activity after exposure of the cells to Ca(o)(2+), suggesting that intracellular signaling pathways initiated by means of activation of a CaR contribute to TNF synthesis by a mechanism that involves transcription of the TNF gene. Neutralization of TNF activity with an anti-TNF antibody attenuated Ca(2+)-mediated increases in cyclooxygenase-2 (COX-2) protein expression and PGE(2) synthesis, suggesting that TNF exerts an autocrine effect in the mTAL, which contributes to COX-2-mediated PGE(2) production. Preincubation with the PKC inhibitor bisindolylmaleimide I inhibited Ca(2+)-mediated TNF production. Significant inhibition of COX-2 protein expression and PGE(2) synthesis also was observed when cells were challenged with Ca(o)(2+) in the presence of bisindolylmaleimide I. The data suggest that increases in TNF production subsequent to activation of the CaR may be the basis of an important renal mechanism that regulates salt and water excretion.