Highly sensitive two-dimensional ion chromatography mass spectrometry method for nitrite determination in hydroxypropyl methylcellulose.

Due to their potential adverse health effects, some N-nitrosamines in drug products are strictly regulated with very low maximum daily intake limits. Nitrosamines can be formed from the reaction of nitrite and secondary or tertiary amines when both species co-exist in the drug synthesis or formulation process. One key strategy to mitigate nitrosamine risk in drugs is to select low-nitrite containing pharma excipients for formulation. It is necessary to develop a sensitive method for trace nitrite determination in pharma excipients as it enables drug producers to study nitrosamine formation kinetics and select excipient suppliers. This study details the development and validation of a two-dimensional ion chromatography mass spectrometry (2D-IC/MS) method for trace nitrite determination in hydroxypropyl methylcellulose (HPMC), one of the most important pharmaceutical excipients used in many drug formulations. The 2D-IC system was operated in heart-cutting mode with a concentrator column coupling the two dimensions. A standard bore anion-exchange column was used in the first dimension (1D) to enable a large volume injection for increased sensitivity and provide improved resolution between nitrite and the interfering chloride peak. A high efficiency microbore anion-exchange column with different selectivity was used in the second dimension (2D) to resolve nitrite from other interfering species. The use of 2D-IC resulted in significantly improved resolution, solving the sensitivity loss issue due to ion suppression from an otherwise 1D separation. MS detection with selective ion monitoring and isotope labeled nitrite internal standard further improve the method specificity, accuracy, and ruggedness, as compared with conductivity detection. For trace determination, it is also extremely important to have a clean blank. For this purpose, a novel cleaning procedure using a strong anion wash was developed to remove nitrite contamination from labware. The optimized method was validated with linearity of nitrite in the concentration range of 18.5-5005.8 ng/g having a regression coefficient of >0.9999, precision with RSD at 3.5-10.1 % and recovery of 90.5-102.4 %. The limit of detection and limit of quantitation were 8.9 and 29.6 ng/g relative to the HPMC sample, or equivalent to 89 and 296 pg/g in the sample solution, respectively.

Momordin Ic ameliorates psoriasis skin damage in mice via the IL-23/IL-17 axis.

Psoriasis, a chronic and easily recurring inflammatory skin disease, causes a great economic burden to the patient's family because the etiology and mechanism are still unclear and the treatment cycle is long. In this study, the function and related mechanisms of Momordin Ic in psoriasis were investigated. The IMQ-induced mouse psoriasis model was constructed. The protective effects of different doses of Momordin Ic on psoriasis skin damage in mice were detected by PASI score, HE staining and Ki-67 staining. A psoriasis-like keratinocyte model was established at the cellular level using M5 (IL-17A, IL-22, oncostatin M, IL-1α, and TNF-α) triggered HaCaT. The effects of Momordin Ic upon HaCaT cell biological behavior were examined using MTT and CCK-8 assays. In terms of mechanism, the expression level of each inflammatory factor was assessed using IHC staining and/or ELISA, qRT-PCR, the expression of oxidative stress-related indicators was detected biochemically, and western blot was performed to detect the levels of key proteins of the Wnt signaling and VEGF. As the results shown,  at the in vivo level, Momordin Ic significantly alleviated skin damage, reduced PASI score and inhibited hyperproliferation of keratinized cells in psoriasis mice. At the cellular level, Momordin Ic also significantly reversed M5-induced hyperproliferation of HaCaT keratinocytes. In terms of mechanism, Momordin Ic significantly inhibited the IL-23/IL-17 axis, dramatically elevated the levels of intracellular antioxidants including SOD, GSH-Px, and CAT, and significantly down-regulated the levels of the indicator of oxidative damage, malondialdehyde (MDA). In addition, Momordin Ic also significantly inhibited the level of ß-catenin, a pivotal protein of the Wnt signaling, C-Myc, a target gene of the Wnt signaling, and VEGF, a critical protein of angiogenesis. In conclusion, Momordin Ic can be involved in the skin-protective effects of psoriasis by multiple mechanisms, including inhibition of the Wnt signaling pathway and the IL-23/IL-17 axis, and suppression of oxidative damageand VEGF expression. Momordin Ic has been proven to be an underlying therapeutic drug for the treatment of psoriasis.

DTR-SHIELD: Mutual Synchronization for Protecting against DoS Attacks on the SHIELD Protocol with AES-CTR Mode.

To enhance security in the semiconductor industry's globalized production, the Defense Advanced Research Projects Agency (DARPA) proposed an authentication protocol under the Supply Chain Hardware Integrity for Electronics Defense (SHIELD) program. This protocol integrates a secure hardware root-of-trust, known as a dielet, into integrated circuits (ICs). The SHIELD protocol, combined with the Advanced Encryption Standard (AES) in counter mode, named CTR-SHIELD, targets try-and-check attacks. However, CTR-SHIELD is vulnerable to desynchronization attacks on its counter blocks. To counteract this, we introduce the DTR-SHIELD protocol, where DTR stands for double counters. DTR-SHIELD addresses the desynchronization issue by altering the counter incrementation process, which previously solely relied on truncated serial IDs. Our protocol adds a new AES encryption step and requires the dielet to transmit an additional 100 bits, ensuring more robust security through active server involvement and message verification.

Adaptive Laboratory Evolution to Improve Recombinant Protein Production Using Insect Cells.

Adaptive laboratory evolution (ALE) is a powerful tool for enhancing the fitness of cell lines in specific applications, including recombinant protein production. Through adaptation to nonstandard culture conditions, cells can develop specific traits that make them high producers. Despite being widely used for microorganisms and, to lesser extent, for mammalian cells, ALE has been poorly leveraged for insect cells. Here, we describe a method for adapting insect High Five and Sf9 cells to nonstandard culture conditions via an ALE approach. Aiming to demonstrate the potential of ALE to improve productivity of insect cells, two case studies are demonstrated. In the first, we adapted insect High Five cells from their standard pH (6.2) to neutral pH (7.0); this adaptation allowed to improve production of influenza virus-like particles (VLPs) by threefold, using the transient baculovirus expression vector system. In the second, we adapted insect Sf9 cells from their standard culture temperature (27 °C) to hypothermic growth (22 °C); this adaptation allowed to improve production of influenza VLPs by sixfold, using stable cell lines. These examples demonstrate the potential of ALE for enhancing productivity within distinct insect cell hosts and expression systems by manipulating different culture conditions.

Recombinant AAV Production.

The insect cell-baculovirus expression vector (IC-BEV) platform has enabled small research-scale and large commercial-scale production of recombinant proteins and therapeutic biologics including recombinant adeno-associated virus (rAAV)-based gene delivery vectors. The wide use of this platform is comparable with other mammalian cell line-based platforms due to its simplicity, high-yield, comparable quality attributes, and robust bioprocessing features. In this chapter, we describe a rAAV production protocol employing one of the recent modifications of the One-Bac platform that consists of a stable transformed Sf9 cell line carrying AAV Rep2/Cap5 genes that are induced upon infection with a single recombinant baculovirus expression vector harboring the transgene of interest (rAAV genome). The overall protocol consists of essential steps including rBEV working stock preparation, rAAV production, and centrifugation-based clarification of cell culture lysate. The same protocol can also be applied for rAAV vector production using traditional Three-Bac, Two-Bac, and Mono-Bac platforms without requiring significant changes.

One-Step Purification Strategy for Cowpea Chlorotic Mottle Virus-Like Particles Produced by the IC-BEVS.

Virus-like particles (VLP) of the cowpea chlorotic mottle virus (CCMV), a plant virus, have been shown to be safe and noncytotoxic vehicles for delivering various cargos, including nucleic acids and peptides, and as scaffolds for presenting epitopes. Thus, CCMV-VLP have acquired increasing attention to be used in fields such as gene therapy, drug delivery, and vaccine development. Regardless of their production method, most reports purify CCMV-VLP through a series of ultracentrifugation steps using sucrose density gradient ultracentrifugation, which is a complex and time-consuming process. Here, the use of anion exchange chromatography is described as a one-step protocol for purification of CCMV-VLP produced by the insect cell-baculovirus expression vector system (IC-BEVS).

Network pharmacology- and cell-based assessments identify the FAK/Src pathway as a molecular target for the antimetastatic effect of momordin Ic against cholangiocarcinoma.

Previous studies have indicated the efficacy of momordin Ic (MIc), a plant-derived triterpenoid, against several types of cancers, implying its potential for further development. However, comprehensive insights into the molecular mechanisms and targets of MIc in cholangiocarcinoma (CCA) are lacking. This study aimed to investigate the actions of MIc against CCA at the molecular level. Network pharmacology analysis was first employed to predict the mechanisms and targets of MIc. The results unveiled the potential involvement of MIc in apoptosis and cell migration, pinpointing Src and FAK as key targets. Subsequently, cell-based assays, in accordance with FAK/Src-associated metastasis, were conducted, demonstrating the ability of MIc to attenuate the metastatic behaviours of KKU-452 cells. The in vitro results further indicated the capability of MIc to suppress the epithelial-mesenchymal transition (EMT) process, notably by downregulating EMT regulators, including N-cadherin, vimentin, ZEB2 and FOXC1/2 expression. Furthermore, MIc suppressed the activation of the FAK/Src signalling pathway, influencing critical downstream factors such as MMP-9, VEGF, ICAM-1, and c-Myc. Molecular docking simulations also suggested that MIc could interact with FAK and Src domains and restrain kinases from being activated by hindering ATP binding. In conclusion, this study employs a comprehensive approach encompassing network pharmacology analysis, in vitro assays, and molecular docking to unveil the mechanisms and targets of MIc in CCA. MIc mitigates metastatic behaviours and suppresses key pathways, offering a promising avenue for future therapeutic strategies against this aggressive cancer.

Synergy of Rapamycin and Methioninase on Colorectal Cancer Cells Requires Simultaneous and Not Sequential Administration: Implications for mTOR Inhibition.

Background/Aim: Rapamycin inhibits the mTOR protein kinase. Methioninase (rMETase), by degrading methionine, targets the methionine addiction of cancer cells and has been shown to improve the efficacy of chemotherapy drugs, reducing their effective doses. Our previous study demonstrated that rapamycin and rMETase work synergistically against colorectal-cancer cells, but not on normal cells, when administered simultaneously in vitro. In the present study, we aimed to further our previous findings by exploring whether  synergy exists between rapamycin and rMETase when used sequentially against HCT-116 colorectal-carcinoma cells, compared to simultaneous administration, in vitro. Materials and Methods: The half-maximal inhibitory concentrations (IC50) of rapamycin alone and rMETase alone against the HCT-116 human colorectal-cancer cell line were previously determined using the CCK-8 cell viability assay (11). We then examined the efficacy of rapamycin and rMETase, both at their IC50, administered simultaneously or sequentially on the HCT-116 cell line, with rapamycin administered before rMETase and vice versa. Results: The IC50 for rapamycin and rMETase, determined from previous experiments (11), was 1.38 nM and 0.39 U/ml, respectively, of HCT-116 cells. When rMETase was administered four days before rapamycin, both at the IC50, there was a 30.46% inhibition of HCT-116 cells. When rapamycin was administered four days before rMETase, both at the IC50, there was an inhibition of 41.13%. When both rapamycin and rMETase were simultaneously administered, both at the IC50, there was a 71.03% inhibition. Conclusion: Rapamycin and rMETase have synergistic efficacy against colorectal-cancer cells in vitro when administered simultaneously, but not sequentially.

Vortioxetine improved schizophrenia-like behavioral deficits in a Poly I:C-induced maternal immune activation model of schizophrenia in rats.

BACKGROUND: Several studies provide clear evidence that exposure to various infections during pregnancy are linked with an increased risk for schizophrenia. In preclinical studies, administration of polyinosinic-polycytidylic acid (Poly I:C) in pregnant rodents can induce maternal immune activation leading to impairments in brain function in the offspring. OBJECTIVES: The aim of this study was to investigate the effect of vortioxetine, a multimodal selective serotonin reuptake inhibitor (SSRI), in the pathophysiology of Poly I:C-induced schizophrenia-like model in rats. METHODS: For this purpose, Poly I:C (8 mg/kg, ip) was injected into pregnant animals 14 days after mating, and tail blood was taken for determination of IL-6 levels after 2 h. At postnatal days 83-86, behavioral tests were performed. RESULTS: Our results revealed that Poly I:C caused impairments in prepulse inhibition, novel object recognition, social interaction, and open-field tests. Chronic administration of vortioxetine (2.5, 5, and 10 mg/kg, ip, postnatal days 69-83) caused significant improvements in these deficits. CONCLUSION: Overall, our findings indicate that vortioxetine may provide new therapeutic approaches for the treatment of schizophrenia. We think that increased serotonergic activity in frontal brain regions may provide the ameliorative effect of vortioxetine, especially on negative and cognitive symptoms. Therefore, it will be useful to determine the efficacy of vortioxetine with combined drugs with further studies.

How Intravesical Platelet-Rich Plasma Can Help Patients with Interstitial Cystitis/Bladder Pain Syndrome: A Comprehensive Scoping Review.

INTRODUCTION AND HYPOTHESIS: Interstitial cystitis/bladder pain syndrome (IC/BPS) is a condition characterized by chronic inflammation that affects the bladder. The study was aimed at evaluating the effectiveness of intravesical platelet-rich plasma (PRP) injections in patients with IC/BPS. METHODS: We conducted a comprehensive search strategy to involve studies that investigate the efficacy of intravesical PRP injections or instillations over different time intervals. Various outcome measures were assessed, including pain scores, functional outcomes, urodynamic parameters, and surface expressions on the urothelium. RESULTS: Our search strategy revealed 1,125 studies. After screening, ten articles met the inclusion criteria. Intravesical PRP significantly reduced the visual analog scale (VAS) compared with baseline scores. Several clinical trials reported significant improvements in the global response rate (GRA), O'Leary-Sant Symptom (OSS) questionnaire, Interstitial Cystitis Symptom Index (ICSI), and Interstitial Cystitis Problem Index (ICPI). Urodynamic parameters such as maximum flow rate (Qmax) and post-voiding residual (PVR) showed significant improvements in some studies. CONCLUSION: The study concluded that intravesical PRP injections could be a promising effective treatment option for IC/BPS patients by their significant ability to reduce pain. However, improvement of urodynamic and functional outcomes is still not clear. Further large comparative trials are still warranted to assess the efficacy of PRP instillation.

Investigation of the use of aluminum oxide nanoparticle-enhanced waste cooking oil blends in compression ignition engines.

The sustainable utilization of waste cooking oil (WCO) as an alternative to fossil fuels has gained considerable attention due to its potential for delivering substantial environmental and economic benefits. This research attempts to explore the impact of incorporating aluminum oxide nanoparticles (AONP) into WCO on the emissions, combustion characteristics, and overall performance of a single-cylinder compression ignition (CI) engine. Comparative analyses were conducted against conventional commercial diesel fuel and pure WCO, as well as varying blends of WCO with AONP at 25 ppm, 50 ppm, and 75 ppm concentrations. The experimental results demonstrate a notable enhancement in brake thermal efficiency (BTE), with a 13.2% increase observed in the WCO + 75 AONP fuel blend compared to neat WCO. Engines fueled by WCO nanoparticle blends showed significant augmentation in-cylinder pressure and heat release rates. Furthermore, these blends exhibited a substantial reduction in carbon monoxide (CO), hydrocarbons (HC), and soot emissions by 44%, 31%, and 48%, respectively, while nitrogen oxide (NO) emissions increased by 7% compared to neat WCO. Among the assessed fuel mixtures, the WCO + 75 AONP blend demonstrated higher engine performance. This study underscores the potential of aluminum oxide nanoparticle-enhanced WCO blends as viable and environmentally responsible options for sustainable energy solutions. However, challenges such as production costs and long-term fuel stability must be addressed to establish nano-fuels as financially viable alternatives.

Endogenous Type I-C CRISPR-Cas system of Streptococcus equi subsp. zooepidemicus promotes biofilm formation and pathogenicity.

Streptococcus equi subsp. zooepidemicus (SEZ) is a significant zoonotic pathogen that causes septicemia, meningitis, and mastitis in domestic animals. Recent reports have highlighted high-mortality outbreaks among swine in the United States. Traditionally recognized for its adaptive immune functions, the CRISPR-Cas system has also been implicated in gene regulation, bacterial pathophysiology, virulence, and evolution. The Type I-C CRISPR-Cas system, which is prevalent in SEZ isolates, appears to play a pivotal role in regulating the pathogenicity of SEZ. By constructing a Cas3 mutant strain (ΔCas3) and a CRISPR-deficient strain (ΔCRISPR), we demonstrated that this system significantly promotes biofilm formation and cell adhesion. However, the deficiency in the CRISPR-Cas system did not affect bacterial morphology or capsule production. In vitro studies showed that the CRISPR-Cas system enhances pro-inflammatory responses in RAW264.7 cells. The ΔCas3 and ΔCRISPR mutant strains exhibited reduced mortality rates in mice, accompanied by a decreased bacterial load in specific organs. RNA-seq analysis revealed distinct expression patterns in both mutant strains, with ΔCas3 displaying a broader range of differentially expressed genes, which accounted for over 70% of the differential genes observed in ΔCRISPR. These genes were predominantly linked to lipid metabolism, the ABC transport system, signal transduction, and quorum sensing. These findings enhance our understanding of the complex role of the CRISPR-Cas system in SEZ pathogenesis and provide valuable insights for developing innovative therapeutic strategies to combat infections.

Implementation of an ionization chamber array for linear accelerator monthly dosimetry QA.

PURPOSE: The IC Profiler (ICP) manufactured by Sun Nuclear Corporation (SNC) is an ionization chamber (IC) array used for linear accelerator dosimetry measurements. Previous work characterized response of the ICP under various conditions, but there is limited work of its implementation into monthly QA measurement procedures. This work quantifies ICP accuracy and variables that affect accuracy for beam output measurements, and demonstrates feasibility of using the ICP for all recommended monthly dosimetry measurements. METHODS: A total of 1985 output measurements on six Varian TrueBeam and Edge linear accelerators were performed using three ICP with quad wedges (QWs) and were compared with conventional IC measurements. The accuracy of the ICP for beam output was characterized as the difference between the ICP and IC. Variables that affect ICP accuracy, including gain settings, calibrations, and template baselining as well as machine or energy-specific bias were investigated. Measurements of profile constancy, energy, dose rate constancy, wedge factors, and gating were performed. RESULTS: The initially observed mean output difference between the ICP and IC was 0.16% (0.61%). When gain settings were optimized, the output difference accuracy improved to -0.02% (0.38%). The output accuracy of the ICP was not dependent on array, dose, temperature and pressure calibrations, or template baselining. Statistically, ICP output accuracy was dependent on machine and beam energy, but clinically, all measurements fell within 0.5% of unity. ICP measurements of energy, dose rate constancy, and wedge factors matched passing results with conventional IC in water measurements. Gating and beam profile constancy measurements demonstrated good stability using the ICP. Finally, monthly dosimetry QA using ICP was completed in an average of 33 min compared to 66 min using the IC. CONCLUSION: This work demonstrated the feasibility and efficiency of using the ICP, with specific considerations, as a measurement device for dosimetric linear accelerator monthly QA.

Doping control analysis of myo-inositol trispyrophosphate and 10 bisphosphonates in equine plasma by ion chromatography-mass spectrometry and its application to clodronic acid horse administration.

Bisphosphonates and myo-inositol trispyrophosphate (ITPP) are two classes of difficult-to-detect polar drugs that are prohibited under the rules of racing. ITPP is a drug capable of increasing the amount of oxygen in hypoxic tissues, and studies have shown that administration of ITPP increases the maximal exercise capacity in mice. The properties of ITPP make it an ideal candidate as a doping agent to enhance performance in racehorses. In recent years, ITPP had indeed been detected in racehorses and confiscated items. As for bisphosphonates, it is especially critical to control their use as since February 2019, the International Agreement on Breeding, Racing and Wagering (IABRW) by the International Federation of Horseracing Authorities (IFHA) had identified specific conditions on which bisphosphonates should not be administered to a racehorse. A recent review of literature shows that there is yet a simultaneous screening method for detecting ITPP and bisphosphonates in equine samples. This paper describes an efficient ion chromatography high-resolution mass spectrometry (IC-HRMS) method for the simultaneous detection of ITPP and 10 bisphosphonates at sub-parts-per-billion (ppb) to low-ppb levels in equine plasma after solid-phase extraction (SPE) and its application to an administration study of clodronic acid in horses.

Role of the gel spacer in safely delivering whole pelvic radiation therapy without central shielding in computed tomography-based image-guided adaptive brachytherapy for uterine cervical cancer patients.

BACKGROUND: To protect the rectum and bladder from high dose exposure, the Japanese guidelines for managing uterine cervical carcinoma recommend pelvic irradiation with central shielding (CS). Conversely, the European Society for Radiotherapy and Oncology (ESTRO) and the American Brachytherapy Society (ABS) guidelines recommend delivering ≥85 Gy to high-risk clinical target volume D90 (CTVHR D90%). In this study, we investigated whether a gel spacer can enable the safe delivery of the ESTRO/ABS-recommended doses to the target while observing dose constraints for the OARs without using CS in external beam radiation therapy (EBRT). MATERIALS AND METHODS: Twenty patients who received definitive radiation therapy without CS and were treated by brachytherapy with a gel spacer between 2017 and 2022 were retrospectively reviewed. The cumulative doses of EBRT and brachytherapy treatment outcomes and incidence of adverse events were also examined. RESULTS: The median cumulative CTVHR D90%, rectum D2cm3, and bladder D2cm3 were 86.6 Gy, 62.9 Gy, and 72.0 Gy, respectively. The 2-year local control rate was 95%. There were no CTCAE ≥Grade 3 late gastrointestinal or genitourinary adverse events. CONCLUSIONS: The use of gel spacer can enable ESTRO/ABS-recommended dose constraints even without using CS in EBRT, with favorable outcomes and low adverse event rates.

Simple Determination of Affinity Constants of Antibodies by Competitive Immunoassays.

The affinity constant, also known as the equilibrium constant, binding constant, equilibrium association constant, or the reciprocal value, the equilibrium dissociation constant (Kd), can be considered as one of the most important characteristics for any antibody-antigen pair. Many methods based on different technologies have been proposed and used to determine this value. However, since a very large number of publications and commercial datasheets do not include this information, significant obstacles in performing such measurements seem to exist. In other cases where such data are reported, the results have often proved to be unreliable. This situation may indicate that most of the technologies available today require a high level of expertise and effort that does not seem to be available in many laboratories. In this paper, we present a simple approach based on standard immunoassay technology that is easy and quick to perform. It relies on the effect that the molar IC50 approaches the Kd value in the case of infinitely small concentrations of the reagent concentrations. A two-dimensional dilution of the reagents leads to an asymptotic convergence to Kd. The approach has some similarity to the well-known checkerboard titration used for the optimization of immunoassays. A well-known antibody against the FLAG peptide, clone M2, was used as a model system and the results were compared with other methods. This approach could be used in any case where a competitive assay is available or can be developed. The determination of an affinity constant should belong to the crucial parameters in any quality control of antibody-related products and assays and should be mandatory in papers using immunochemical protocols.

Optimization of DAC-ELISA and IC-RT-PCR using the developed polyclonal antibody and one-step RT-PCR assays for detection of Indian citrus ringspot virus in kinnow orange of Punjab, India.

Indian citrus ringspot virus (ICRSV), a member of the Mandarivirus genus, causes citrus ringspot disease, impacting kinnow orange quality and yield. Early and accurate detection methods are crucial before visible symptoms manifest in plants. In this study, a 507 bp partial coat protein gene (pCPG) segment was amplified from infected kinnow leaf tissues, cloned into a pET28a vector, and transformed into E. coli BL21(DE3) cells. Induced with IPTG, the cells overexpressed a recombinant partial coat protein (rpCP) of approximately 23 kDa, purified using Ni-NTA resin via affinity chromatography. Validated in western blot with an anti-His antibody, rpCP was used to generate an ICRSV-specific polyclonal antibody (PAb) in rabbits. PAb, optimized at 1:1000 dilution, successfully detected ICRSV in infected kinnow orange leaf extracts via DAC-ELISA and IC-RT-PCR assays. ICRSV was detectable in sample dilutions up to 1:640 and 1:10240 (w/v, g mL-1) by DAC-ELISA and IC-RT-PCR, respectively. One-step RT-PCR assays were also optimized, confirming the presence of ICRSV by amplifying a 507 bp pCPG fragment from total RNA extracted from kinnow orange leaves, with dilution up to 1:5120 (w/v, g mL-1). The result demonstrated that IC-RT-PCR has a 16-fold and 2-fold higher sensitivity than DAC-ELISA and one-step RT-PCR assays.

Distinct Effects of Respiratory Viral Infection Models on miR-149-5p, IL-6 and p63 Expression in BEAS-2B and A549 Epithelial Cells.

Respiratory viruses cause airway inflammation, resulting in epithelial injury and repair. miRNAs, including miR-149-5p, regulate different pathological conditions. We aimed to determine how miR-149-5p functions in regulating pro-inflammatory IL-6 and p63, key regulators of airway epithelial wound repair, in response to viral proteins in bronchial (BEAS-2B) and alveolar (A549) epithelial cells. BEAS-2B or A549 cells were incubated with poly (I:C, 0.5 µg/mL) for 48 h or SARS-CoV-2 spike protein-1 or 2 subunit (S1 or S2, 1 µg/mL) for 24 h. miR-149-5p was suppressed in BEAS-2B challenged with poly (I:C), correlating with IL-6 and p63 upregulation. miR-149-5p was down-regulated in A549 stimulated with poly (I:C); IL-6 expression increased, but p63 protein levels were undetectable. miR-149-5p remained unchanged in cells exposed to S1 or S2, while S1 transfection increased IL-6 expression in BEAS-2B cells. Ectopic over-expression of miR-149-5p in BEAS-2B cells suppressed IL-6 and p63 mRNA levels and inhibited poly (I:C)-induced IL-6 and p63 mRNA expressions. miR-149-5p directly suppressed IL-6 mRNA in BEAS-2B cells. Hence, BEAS-2B cells respond differently to poly (I:C), S1 or S2 compared to A549 cells. Thus, miR-149-5p dysregulation may be involved in poly (I:C)-stimulated but not S1- or S2-stimulated increased IL-6 production and p63 expression in BEAS-2B cells.

Exploring the binding properties of DNA/BSA and cytotoxicity studies with new terpyridine-ester-based metal complexes (M = Fe(III), Ni(II), Cu(II) and Ru(III)) - A comparative analysis.

Many terpyridines and their metal complexes are known to exhibit remarkable potential for the interaction of biological targets. Notably, a subtle change in the structure of the ligand can influence these interactions significantly. In this regard, it would be very interesting to assess the binding affinity of functionalized molecules with DNA/BSA. In this work, a novel ester-based terpyridine (L) and the corresponding four metal complexes with Ni(II) (MC1), Cu(II) (MC2), Fe(III) (MC3) and Ru(III) (MC4) were prepared and structurally characterized using various spectroscopic and analytical techniques including the validation of molecular structures of ligand (L) and Ni(II)-Tpy complex (MC1). The EPR data demonstrate that MC1 is diamagnetic and other complexes (MC2-MC4) exhibit paramagnetic behavior. Additionally, the structures of ligands and metal complexes were determined using DFT studies and the same were utilized for the docking studies. Interestingly, MC3 and MC4 exhibit a predominant lowest binding energy of -9.62 Kcal/mol (with DNA) and -10.05 Kcal/mol (with BSA) respectively. The binding affinity of the ligand and its complexes with protein and DNA was evaluated by spectroscopic techniques. Notably, the cytotoxicity studies of L and MC1-MC4 were performed against the MCF-7 (human breast cancer) cell lines. The complex MC4 displayed great activity with an IC50 of 3.5 ±â€¯1.75 µM among all synthesized compounds and comparable with cisplatin.