Ammonium chloride-induced hypothermia is attenuated by transient receptor potential channel vanilloid-1, but augmented by ankyrin-1 in rodents.

AIMS: Systemic administration of ammonium chloride (NH4Cl), an acidifying agent used in human patients and experimental conditions, causes hypothermia in mice, however, the mechanisms of the thermoregulatory response to NH4Cl and whether it develops in other species remained unknown. MAIN METHODS: We studied body temperature (Tb) changes in rats and mice induced by intraperitoneal administration of NH4Cl after blockade of transient receptor potential vanilloid-1 (TRPV1) or ankyrin-1 (TRPA1) channels. KEY FINDINGS: In rats, NH4Cl decreased Tb by 0.4-0.8°C (p < 0.05). The NH4Cl-induced hypothermia also developed in Trpv1 knockout (Trpv1-/-) and wild-type (Trpv1+/+) mice, however, the Tb drop was exaggerated in Trpv1-/- mice compared to Trpv1+/+ controls with maximal decreases of 4.0 vs. 2.1°C, respectively (p < 0.05). Pharmacological blockade of TRPV1 channels with AMG 517 augmented the hypothermic response to NH4Cl in genetically unmodified mice and rats (p < 0.05 for both). In contrast, when NH4Cl was infused to mice genetically lacking the TRPA1 channel, the hypothermic response was significantly attenuated compared to wild-type controls with maximal mean Tb difference of 1.0°C between the genotypes (p = 0.008). Pretreatment of rats with a TRPA1 antagonist (A967079) also attenuated the NH4Cl-induced Tb drop with a maximal difference of 0.7°C between the pretreatment groups (p = 0.003). SIGNIFICANCE: TRPV1 channels limit, whereas TRPA1 channels exaggerate the development of NH4Cl-induced hypothermia in rats and mice, but other mechanisms are also involved. Our results warrant for regular Tb control and careful consideration of NH4Cl treatment in patients with TRPA1 and TRPV1 channel dysfunctions.

Salmonella-induced microbiome profile in response to sanitation by quaternary ammonium chloride.

Salmonella enterica is a prominent cause of foodborne disease in the United States. However, the mechanism and route of pathogen transmission that leads to Salmonella infection in commercial processing plants are poorly understood. This study aimed to investigate the effect of mixed-species biofilms on S. enterica survival and persistence under sanitizer stress [Quaternary ammonium compounds (QACs)] by analyzing 78 floor drain samples from a meat processing facility and three S. enterica strains (serovars Cerro, Montevideo, and Typhimurium) isolated from that facility and an unrelated source. The four test groups were as follows: control, QAC treatment, Salmonella addition, and QAC treatment with Salmonella addition. DNAs were extracted, and 16S rRNA gene based on the variable region V4 amplicon sequencing was performed to analyze the relative abundance, core microbiome, and Alpha and Beta diversity using the qiime2 pipeline. At the genus level, the Brochothrix (45.56%), Pseudomonas (38.94%), Carnobacterium (6.18%), Lactococcus (4.68%), Serratia (3.14%), and Staphylococcus (0.82%) were shown to be the most prevalent in all drain samples. The results demonstrate that the relative abundance of different bacterial genera was affected by both QAC treatment and Salmonella addition, with some genera showing increases or decreases in abundance. Notably, the correlation network was constructed to understand the relationships between the different bacteria. Nitrospira had the greatest number of connections in the floor drain environment network, with two negative and eight positive correlations. The results suggest that Nitrospira in the mixed-species biofilm community may play a role in converting ammonium in the QAC sanitizer into nitrites. Thus, Nitrospira could be a potentially important genus in providing sanitizer resistance to pathogen-encompassed mixed-species biofilms.IMPORTANCESalmonella contamination in meat processing facilities can lead to foodborne illness outbreaks. Our study characterized the microbiome dynamics in beef facility drains and their response to Salmonella addition and common sanitizer (QAC). Nitrospira could be an important genus in providing sanitizer resistance to pathogen-encompassed mixed-species biofilms. The results provide insight into the impact of mixed-species biofilms on Salmonella survival and persistence under sanitizer stress in meat processing facilities. The results highlight the need to consider mixed-species biofilm effects when developing targeted interventions to enhance food safety.

The protective effect of Luteolin on chicken spleen lymphocytes from ammonia poisoning through mitochondria and balancing energy metabolism disorders.

Ammonia poses a significant challenge in the contemporary intensive breeding industry, resulting in substantial economic losses. Despite this, there is a dearth of research investigating efficacious strategies to prevent ammonia poisoning in poultry. Consequently, the objective of this study was to investigate the molecular mechanisms through which Luteolin (Lut) safeguards mitochondria and restores equilibrium to energy metabolism disorders, thereby shielding chicken spleen lymphocytes from the detrimental effects of ammonia poisoning. Chicken spleen lymphocytes were categorized into 3 distinct groups: the control group, the ammonia group (with the addition of 1 mmol/L of ammonium chloride), and the Lut group (with the treatment of 0.5 µg/mL of Lut for 12 h followed by the addition of 1 mmol/L of ammonium chloride). These groups were then cultured for a duration of 24 h. To investigate the potential protective effect of Lut on lymphocytes exposed to ammonia, various techniques were employed, including CCK-8 analysis, ultrastructural observation, reagent kit methodology, fluorescence microscopy, and quantitative real-time PCR (qRT-PCR). The findings indicate that Lut has the potential to mitigate the morphological damage of mitochondria caused by ammonia poisoning. Additionally, it can counteract the decline in mitochondrial membrane potential, ATP content, and ATPase activities (specifically Na+/K+-ATPase, Ca2+-ATPase, Mg2+-ATPase, and Ca/Mg2+-ATPase) following exposure to ammonia in lymphocytes. Lut also has the ability to regulate the expression of genes involved in mitochondrial fusion (Opa1, Mfn1, and Mfn2) and division (Drp1 and Mff) in spleen lymphocytes after ammonia exposure. This regulation leads to a balanced energy metabolism (HK1, HK2, LDHA, LDHB, PFK, PK, SDHB, and ACO2) and provides protection against ammonia poisoning.

The effect of decontamination using quaternary ammonium chloride on the bacterial burden of hospital privacy curtains.

This quasi-experimental study aimed to identify the effect of decontamination using quaternary ammonium chloride (QAC) on bacterial burden on hospital privacy curtains. The objects were the high-touch edges of 66 polyester curtains in inpatient wards. The decontamination was performed daily (n = 22), twice-weekly (n = 22), or not performed (n = 22) for 28 days. The bacterial burden on the curtains was measured based on the number of bacteria, the proportion of curtains with >2.5 colony-forming unit/cm2 , and the proportion of curtains with multidrug-resistant organisms (MDROs). As a result, the daily or twice-weekly decontamination groups showed a significantly lower increase in bacterial burden than the no-decontamination group overall and at all four posttest times. On day 28, daily decontamination showed a lower increase in the number of bacteria (p < 0.001) and proportions of curtains with >2.5 colony form units/cm2 (p < 0.001) than the no-decontamination condition, and in the number of curtains with MDROs than twice-weekly decontamination. In conclusion, decontamination of curtains using QAC helps reduce bacterial burden, and daily decontamination is recommended up to 28 days after installation.

Assessment of antibacterial properties and skin irritation potential of anodized aluminum impregnated with various quaternary ammonium.

The importance of the inert environment in the transmission of pathogens has been reassessed in recent years. To reduce cross-contamination, new biocidal materials used in high touch surfaces (e.g., stair railings, door handles) have been developed. However, their impact on skin remains poorly described. The present study aimed to evaluate the antibacterial properties and the risk of skin irritation of two materials based on hard-anodized aluminum (AA) impregnated with quaternary ammonium compound solutions (QAC#1 or QAC#2). The QAC#1 or QAC#2 solutions vary in composition, QAC#2 being free of dioctyl dimethyl ammonium chloride (Dio-DAC) and octyl decyl dimethyl ammonium chloride (ODDAC). Unlike AA used as a control, both AA-QAC#1 and AA-QAC#2 had excellent and rapid antibacterial efficacy, killing 99.9 % of Staphylococcus aureus and Escherichia coli bacteria, in 15 s and 1 min, respectively. The impregnation solutions (QAC#1 and QAC#2) did not show any skin sensitizing effect on transformed human keratinocytes. Nevertheless, these solutions as well as the materials (AA-QAC#1, AA-QAC#2), and the liquid extracts derived from them, induced a very rapid cytotoxicity on L929 murine fibroblasts (>70 % after 1 h of contact) as shown by LDH, MTS and neutral red assays. This cytotoxicity can be explained by the fast QACs release occurring when AA-QAC#1 and AA-QAC#2 were immersed in aqueous medium. To overcome the limitation of assays based on liquid condition, an in vitro skin irritation assay on reconstructed human epidermis (RHE) was developed. The effect of the materials upon their direct contact with the epidermis grown at the liquid-air interface was determined by evaluating tissue viability and quantifying interleukin-1 alpha (IL-1α) which is released in skin during injury or infection. AA-QAC#1 induced a significant decrease in RHE viability, close to OECD and ISO 10993-10 acceptability thresholds and enhanced the pro-inflammatory IL-1α secretion compared with AA-QAC#2. Finally, these results were corroborated by in vivo assays on mice using erythema and edema visual scores, histological observations, and epidermal thickness measurement. AA had no effect on the skin, while a stronger irritation was induced by AA-QAC#1 compared with AA-QAC#2. Hence, these materials were classified as moderate and slight irritants, respectively. In summary, this study revealed that AA-QAC#2 without Dio-DAC and ODDAC could be a great candidate for high touch surface applications, showing an extremely effective and rapid bactericidal activity, without inducing adverse effects for skin tissue.

Effect of Substituents on the Homopolymerization Activity of Methyl Alkyl Diallyl Ammonium Chloride.

Among nitrogen-containing cationic electrolytes, diallyl quaternary ammonium salt is a typical monomer with the highest positive charge density, which has attracted the most attention, especially in the research on homopolymers and copolymers of dimethyl diallyl ammonium chloride (DMDAAC), which occupy a very unique and important position. In order to improve the lipophilicity of substituted diallyl ammonium chloride monomers under the premise of high cationic charge density, the simplest, most direct, and most efficient structure design strategy was selected in this paper. Only one of the substituents on DMDAAC quaternary ammonium nitrogen was modified by alkyl; the substituents were propyl and amyl groups, and their corresponding monomers were methyl propyl diallyl ammonium chloride (MPDAAC) and methyl amyl diallyl ammonium chloride (MADAAC), respectively. The effect of substituent structure on the homopolymerization activity of methyl alkyl diallyl ammonium chloride was illustrated by quantum chemical calculation and homopolymerization rate determination experiments via ammonium persulfate (APS) as the initiator system. The results of quantum chemistry simulation showed that, with the finite increase in substituted alkyl chain length, the numerical values of the bond length and the charge distribution of methyl alkyl diallyl ammonium chloride monomer changed little, with the activation energy of the reactions in the following order: DMDAAC < MPDAAC < MADAAC. The polymerization activities measured by the dilatometer method were in the order DMDAAC > MPDAAC > MADAAC. The activation energies Ea of homopolymerization were 96.70 kJ/mol, 97.25 kJ/mol, and 100.23 kJ/mol, and the rate equation of homopolymerization of each monomer was obtained. After analyzing and comparing these results, it could be easily found that the electronic effect of substituent was not obvious, whereas the effect of the steric hindrance was dominant. The above studies have laid a good foundation for an understanding of the polymerization activity of methyl alkyl diallyl ammonium chloride monomers and the possibility of preparation and application of these polymers with high molecular weight.

The Impact of Ammonium Chloride-Based Erythrocyte Lysis Process on Banked Adipose-Derived Stem Cells.

The safety of banked human adipose-derived stem cells (hADSCs) purified by 155 mM ammonium chloride (NH4Cl)-based erythrocyte lysis has not been evaluated. This study was conducted to determine the impact of NH4Cl-based erythrocyte lysis on the biological characteristics of cryopreserved hADSCs. Stromal vascular fractions (SVFs) were obtained from lipoaspirates and purified with NH4Cl-based erythrocyte lysis (lysis group) or without (nonlysis group). The hADSCs were freshly isolated (fresh group) from SVFs and/or cryopreserved for 2 weeks (cryo group). The morphologies, immunophenotypes, viability, apoptosis, and growth kinetics of each group were compared. The cell cycle and differentiation capacity assays were performed in both cryopreserved groups. All groups showed similar cell morphology, immunological phenotypes, and viability. However, the main effect of lysis and its interaction with cryopreservation were observed when early apoptosis was regarded as a dependent variable in two-way repeated-measures analysis of variance. After cryopreservation, significant growth retardation and S-phase fraction reduction were observed in lytic hADSCs compared with those in nonlytic hADSCs. No significant differences in the adipogenic and osteogenic differentiation capacities were found between the two groups. Although NH4Cl-based erythrocyte lysis did not affect the cell morphology, immunological phenotypes, viability, and adipogenic and osteogenic differentiation capacities of cryopreserved hADSCs, exposure to NH4Cl-based erythrocyte lysis or its synergistic action with cryopreservation may induce apoptosis and inhibit the proliferation and mitosis of cryopreserved hADSCs. These results indicate that NH4Cl-based erythrocyte lysis is not suitable for high-quality banked collection of hADSCs for future clinical applications. Further development of safe, convenient, and cost-effective purification methods of hADSCs is warranted.

Structurally Conserved Domains between Flavivirus and Alphavirus Fusion Glycoproteins Contribute to Replication and Infectious-Virion Production.

Alphaviruses and flaviviruses have class II fusion glycoproteins that are essential for virion assembly and infectivity. Importantly, the tip of domain II is structurally conserved between the alphavirus and flavivirus fusion proteins, yet whether these structural similarities between virus families translate to functional similarities is unclear. Using in vivo evolution of Zika virus (ZIKV), we identified several novel emerging variants, including an envelope glycoprotein variant in ß-strand c (V114M) of domain II. We have previously shown that the analogous ß-strand c and the ij loop, located in the tip of domain II of the alphavirus E1 glycoprotein, are important for infectivity. This led us to hypothesize that flavivirus E ß-strand c also contributes to flavivirus infection. We generated this ZIKV glycoprotein variant and found that while it had little impact on infection in mosquitoes, it reduced replication in human cells and mice and increased virus sensitivity to ammonium chloride, as seen for alphaviruses. In light of these results and given our alphavirus ij loop studies, we mutated a conserved alanine at the tip of the flavivirus ij loop to valine to test its effect on ZIKV infectivity. Interestingly, this mutation inhibited infectious virion production of ZIKV and yellow fever virus, but not West Nile virus. Together, these studies show that shared domains of the alphavirus and flavivirus class II fusion glycoproteins harbor structurally analogous residues that are functionally important and contribute to virus infection in vivo.IMPORTANCE Arboviruses are a significant global public health threat, yet there are no antivirals targeting these viruses. This problem is in part due to our lack of knowledge of the molecular mechanisms involved in the arbovirus life cycle. In particular, virus entry and assembly are essential processes in the virus life cycle and steps that can be targeted for the development of antiviral therapies. Therefore, understanding common, fundamental mechanisms used by different arboviruses for entry and assembly is essential. In this study, we show that flavivirus and alphavirus residues located in structurally conserved and analogous regions of the class II fusion proteins contribute to common mechanisms of entry, dissemination, and infectious-virion production. These studies highlight how class II fusion proteins function and provide novel targets for development of antivirals.

Therapeutic Antitumor Efficacy of Cancer Stem Cell-Derived DRibble Vaccine on Colorectal Carcinoma.

Dendritic cell (DC)-based immunotherapy has been a promising strategy for colon cancer therapy, but the efficacy of dendritic cell vaccines is in part limited by immunogenicity of loaded antigens. In this study, we aimed to identify a putative tumor antigen that can generate or enhance anti-tumor immune responses against colon cancer. CD44+ colon cancer stem cells (CCSCs) were isolated from mouse colorectal carcinoma CT-26 cell cultures and induced to form defective ribosomal products-containing autophagosome-rich blebs (DRibbles) by treatment with rapamycin, bortezomib, and ammonium chloride. DRibbles were characterized by western blot and transmission electron microscopy. DCs generated from the mice bone marrow monocytes were cocultured with DRibbles, then surface markers of DCs were analyzed by flow cytometry. Meanwhile, the efficacy of DRibble-DCs was examined in vivo. Our results showed that CCSC-derived DRibbles upregulated CD80, CD86, major histocompatibility complex (MHC)-I, and MHC-II on DCs and induced proliferation of mouse splenic lymphocytes and CD8+ T cells. In a model of colorectal carcinoma using BALB/c mice with robust tumor growth and mortality, DC vaccine pulsed with CCSC-derived DRibbles suppressed tumor growth and extended survival. A lactate dehydrogenase test indicated a strong cytolytic activity of cytotoxic T-cells derived from mice vaccinated with CCSC-derived DRibbles against CT-26 cells. Furthermore, flow cytometry analyses showed that the percentages of IFN-γ-producing CD8+ T-cells were increased in SD-DC group compare with the other groups. These findings provide a rationale for novel immunotherapeutic anti-tumor approaches based on DRibbles derived from colon cancer stem cells.

Strategies to target SARS-CoV-2 entry and infection using dual mechanisms of inhibition by acidification inhibitors.

Many viruses utilize the host endo-lysosomal network for infection. Tracing the endocytic itinerary of SARS-CoV-2 can provide insights into viral trafficking and aid in designing new therapeutic strategies. Here, we demonstrate that the receptor binding domain (RBD) of SARS-CoV-2 spike protein is internalized via the pH-dependent CLIC/GEEC (CG) endocytic pathway in human gastric-adenocarcinoma (AGS) cells expressing undetectable levels of ACE2. Ectopic expression of ACE2 (AGS-ACE2) results in RBD traffic via both CG and clathrin-mediated endocytosis. Endosomal acidification inhibitors like BafilomycinA1 and NH4Cl, which inhibit the CG pathway, reduce the uptake of RBD and impede Spike-pseudoviral infection in both AGS and AGS-ACE2 cells. The inhibition by BafilomycinA1 was found to be distinct from Chloroquine which neither affects RBD uptake nor alters endosomal pH, yet attenuates Spike-pseudovirus entry. By screening a subset of FDA-approved inhibitors for functionality similar to BafilomycinA1, we identified Niclosamide as a SARS-CoV-2 entry inhibitor. Further validation using a clinical isolate of SARS-CoV-2 in AGS-ACE2 and Vero cells confirmed its antiviral effect. We propose that Niclosamide, and other drugs which neutralize endosomal pH as well as inhibit the endocytic uptake, could provide broader applicability in subverting infection of viruses entering host cells via a pH-dependent endocytic pathway.

Rice amino acid transporter-like 6 (OsATL6) is involved in amino acid homeostasis by modulating the vacuolar storage of glutamine in roots.

Glutamine is a product of ammonium (NH4+ ) assimilation catalyzed by glutamine synthetase (GS) and glutamate synthase (GOGAT). The growth of NH4+ -preferring paddy rice (Oryza sativa L.) depends on root NH4+ assimilation and the subsequent root-to-shoot allocation of glutamine; however, little is known about the mechanism of glutamine storage in roots. Here, using transcriptome and reverse genetics analyses, we show that the rice amino acid transporter-like 6 (OsATL6) protein exports glutamine to the root vacuoles under NH4+ -replete conditions. OsATL6 was expressed, along with OsGS1;2 and OsNADH-GOGAT1, in wild-type (WT) roots fed with sufficient NH4 Cl, and was induced by glutamine treatment. We generated two independent Tos17 retrotransposon insertion mutants showing reduced OsATL6 expression to determine the function of OsATL6. Compared with segregants lacking the Tos17 insertion, the OsATL6 knock-down mutant seedlings exhibited lower root glutamine content but higher glutamine concentration in the xylem sap and greater shoot growth under NH4+ -replete conditions. The transient expression of monomeric red fluorescent protein-fused OsATL6 in onion epidermal cells confirmed the tonoplast localization of OsATL6. When OsATL6 was expressed in Xenopus laevis oocytes, glutamine efflux from the cell into the acidic bath solution increased. Under sufficient NH4+ supply, OsATL6 transiently accumulated in sclerenchyma and pericycle cells, which are located adjacent to the Casparian strip, thus obstructing the apoplastic solute path, and in vascular parenchyma cells of WT roots before the peak accumulation of GS1;2 and NADH-GOGAT1 occurred. These findings suggest that OsATL6 temporarily stores excess glutamine, produced by NH4+ assimilation, in root vacuoles before it can be translocated to the shoot.

A real-time and high-throughput neutralization test based on SARS-CoV-2 pseudovirus containing monomeric infrared fluorescent protein as reporter.

Neutralizing antibodies to SARS-CoV-2 have been shown to correlate with protection in animals and humans, disease severity, survival, and vaccine efficacy. With the ongoing large-scale vaccination in different countries and continuous surge of new variants of global concerns, a convenient, cost-effective and high-throughput neutralization test is urgently needed. Conventional SARS-CoV-2 neutralization test is tedious, time-consuming and requires a biosafety level 3 laboratory. Despite recent reports of neutralizations using different pseudoviruses with a luciferase or green fluorescent protein reporter, the laborious steps, inter-assay variability or high background limit their high-throughput potential. In this study we generated lentivirus-based pseudoviruses containing a monomeric infrared fluorescent protein reporter to develop neutralization assays. Similar tropism, infection kinetics and mechanism of entry through receptor-mediated endocytosis were found in the three pseudoviruses generated. Compared with pseudovirus D614, pseudovirus with D614G mutation had decreased shedding and higher density of S1 protein present on particles. The 50% neutralization titers to pseudoviruses D614 or D614G correlated with the plaque reduction neutralization titers to live SARS-CoV-2. The turn-around time of 48-72 h, minimal autofluorescence, one-step image quantification, expandable to 384-well, sequential readouts and dual quantifications by flow cytometry support its high-throughput and versatile applications at a non-reference and biosafety level 2 laboratory, in particular for assessing the neutralization sensitivity of new variants by sera from natural infection or different vaccinations during our fight against the pandemic.

Inhibition of PI3K/Akt/mTOR pathway by ammonium chloride induced apoptosis and autophagy in MAC-T cell.

Ammonia is a harmful gas with a pungent odor, participates in the regulation of a variety of apoptosis and autophagy, which in turn affects the growth and differentiation of cells. To test the regulation of NH3 on the apoptosis and autophagy of mammary epithelial cells, we selected NH4Cl as NH3 donor in vitro model. MTT and CCK-8 assay kits were employed to detect cell activity. Real-time quantitative PCR and western blot methods were used to detect the abundance of inflammatory molecules, apoptosis markers, and autophagy genes. We selected TUNEL kit and the Annexin-FITC/PI method to detect apoptosis. TEM analysis was used to detect autophagic vesicles, and MDC stain evaluated the formation of autophagosome. The results indicated that NH4Cl reduced cell viability in a concentration-dependent manner and promoted cell inflammatory response, apoptosis, and autophagy. NH4Cl stimulation notable increased the autophagosomes number. Interestingly, we also detected that the addition of LY294002 and Rapamycin inhibited the PI3K/Akt pathway and the mTOR pathway, respectively, resulting in changes in both apoptosis and autophagy. Therefore, we draw a conclusion that NH3 may regulate the apoptosis and autophagic response of bovine mammary epithelial cells through the PI3K/Akt/mTOR signaling pathway. Further investigations on ammonia's function in other physiological respects, will be critical to provide theoretical help for the improvement of production performance. It will be also helpful for controlling the harmful gas ammonia concentration in the livestock house to protect the health of dairy cows.

Inhibitors of endosomal acidification suppress SARS-CoV-2 replication and relieve viral pneumonia in hACE2 transgenic mice.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2 and broke out as a global pandemic in late 2019. The acidic pH environment of endosomes is believed to be essential for SARS-CoV-2 to be able to enter cells and begin replication. However, the clinical use of endosomal acidification inhibitors, typically chloroquine, has been controversial with this respect. METHODS: In this study, RT-qPCR method was used to detect the SARS-CoV-2N gene to evaluate viral replication. The CCK-8 assay was also used to evaluate the cytotoxic effect of SARS-CoV-2. In situ hybridization was used to examine the distribution of the SARS-CoV-2 gene in lung tissues. Hematoxylin and eosin staining was also used to evaluate virus-associated pathological changes in lung tissues. RESULTS: In this study, analysis showed that endosomal acidification inhibitors, including chloroquine, bafilomycin A1 and NH4CL, significantly reduced the viral yields of SARS-CoV-2 in Vero E6, Huh-7 and 293T-ACE2 cells. Chloroquine and bafilomycin A1 also improved the viability and proliferation of Vero E6 cells after SARS-CoV-2 infection. Moreover, in the hACE2 transgenic mice model of SARS-CoV-2 infection, chloroquine and bafilomycin A1 reduced viral replication in lung tissues and alleviated viral pneumonia with reduced inflammatory exudation and infiltration in peribronchiolar and perivascular tissues, as well as improved structures of alveolar septum and pulmonary alveoli. CONCLUSIONS: Our research investigated the antiviral effects of endosomal acidification inhibitors against SARS-CoV-2 in several infection models and provides an experimental basis for further mechanistic studies and drug development.

Direct current stimulation enhances neuronal alpha-synuclein degradation in vitro.

Despite transcranial Direct Current Stimulation (DCS) is currently proposed as a symptomatic treatment in Parkinson's disease, the intracellular and molecular mechanisms elicited by this technique are still unknown, and its disease-modifying potential unexplored. Aim of this study was to elucidate the on-line and off-line effects of DCS on the expression, aggregation and degradation of alpha-synuclein (asyn) in a human neuroblastoma cell line under basal conditions and in presence of pharmachologically-induced increased asyn levels. Following DCS, gene and protein expression of asyn and its main autophagic catabolic pathways were assessed by real-time PCR and Western blot, extracellular asyn levels by Dot blot. We found that, under standard conditions, DCS increased monomeric and reduced oligomeric asyn forms, with a concomitant down-regulation of both macroautophagy and chaperone-mediated autophagy. Differently, in presence of rotenone-induced increased asyn, DCS efficiently counteracted asyn accumulation, not acting on its gene transcription, but potentiating its degradation. DCS also reduced intracellular and extracellular asyn levels, increased following lysosomal inhibition, independently from autophagic degradation, suggesting that other mechanisms are also involved. Collectively, these findings suggest that DCS exerts on-line and off-line effects on the expression, aggregation and autophagic degradation of asyn, indicating a till unknown neuroprotective role of tDCS.

Impact of Direction of Transport on the Evaluation of Inhibition Potencies of Multidrug and Toxin Extrusion Protein 1 Inhibitors.

Multidrug and toxin extrusion (MATE) transporters are expressed on the luminal membrane of renal proximal tubule cells and extrude their substrates into the luminal side of the tubules. Inhibition of MATE1 can reduce renal secretory clearance of its substrate drugs and lead to drug-drug interactions (DDIs). To address whether IC50 values of MATE1 inhibitors with regard to their extracellular concentrations are affected by the direction of MATE1-mediated transport, we established an efflux assay of 1-methyl-4-phenylpyridinium (MPP+) and metformin using the human embryonic kidney 293 model transiently expressing human MATE1. The efflux rate was defined by reduction of the cellular amount of MPP+ and metformin for 0.25 minutes shortly after the removal of extracellular MPP+ and metformin. Inhibition potencies of 12 inhibitors toward MATE1-mediated transport were determined in both uptake and efflux assays. When MPP+ was used as a substrate, 8 out of 12 inhibitors showed comparable IC50 values between assays (<4-fold). IC50 values from the efflux assays were higher for cimetidine (9.9-fold), trimethoprim (10-fold), famotidine (6.4-fold), and cephalexin (>3.8-fold). When metformin was used as a substrate, IC50 values of the tested inhibitors when evaluated using uptake and efflux assays were within 4-fold of each other, with the exception of cephalexin (>4.7-fold). IC50 values obtained from the uptake assay using metformin showed smaller IC50 values than those from the efflux assay. Therefore, the uptake assay is recommended to determine IC50 values for the DDI predictions. SIGNIFICANCE STATEMENT: In this study, a new method to evaluate IC50 values of extracellular added inhibitors utilizing an efflux assay was established. IC50 values were not largely different between uptake and efflux directions but were smaller for uptake. This study supports the rationale for a commonly accepted uptake assay with metformin as an in vitro probe substrate for multidrug and toxin extrusion 1-mediated drug-drug interaction risk assessment in drug development.

Effects of Ammonium Chloride on Ozone-induced Airway Inflammation: the Role of Slc26a4 in the Lungs of Mice.

BACKGROUND: Exposure to ozone (O3) induces neutrophilic inflammation and goblet cell hyperplasia in humans and experimental animals. Because the solute carrier family 26-member 4 (Slc26a4; pendrin) gene induces mucin production and intraluminal acidification in the airways, it was hypothesized to be a key molecule in O3-induced airway injury. Thus, we evaluated the role of Slc26a4 and the protective effects of ammonium chloride (NH4Cl) in O3-induced airway injury in mice. METHODS: Six-week-old female BALB/c mice were exposed to filtered air or O3 for 21 days (2 ppm for 3 hr/day). NH4Cl (0, 0.1, 1, and 10 mM) was administered intratracheally into the airways. Airway resistance was measured using a flexiVent system, and bronchoalveolar lavage fluid (BALF) cells were differentially counted. Slc26a4 and Muc5ac proteins and mRNA were measured via western blotting, real-time polymerase chain reaction, and immunostaining. Tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-17, IL-1ß, and caspase-1 were analyzed via western blotting. RESULTS: The levels Slc26a4 protein and mRNA significantly increased in lung tissues from Day 7 to Day 21 of O3 exposure, with concomitant increases in lung resistance, numbers of goblet cells in lung tissues, and inflammatory cells and thiocyanate (SCN-) levels in BALF in a time-dependent manner. Treatment with NH4Cl significantly reduced these changes to levels similar to those of sham-treated mice, with a concomitant reduction of Slc26a4 proteins in lung lysates and SCN- levels in BALF. Slc26a4 protein was co-expressed with muc5ac protein in the bronchial epithelium, as indicated by immunofluorescence staining. NH4Cl treatment also significantly attenuated the O3-induced increases in IFN-γ, TNF-α, IL-17, IL-1ß, and p20-activated caspase-1. CONCLUSION: Slc26a4 may be involved in O3-induced inflammatory and epithelial changes in the airways via activation of the inflammasome and the induction of IL-17 and IFN-γ. NH4Cl shows a potential as a therapeutic agent for controlling O3-induced airway inflammation and epithelial damage by modulating Slc26a4 expression.

Water Soluble Coumarin Quaternary Ammonium Chlorides: Synthesis and Biological Evaluation.

In the present study, coumarin-bearing three pyridinium and three tetra-alkyl ammonium salts were synthesized. The compounds were fully characterized by 1 H- and 13 C-NMR, LC/MS and IR spectroscopic methods and elemental analyses. The cytotoxic properties of all compounds were tested against human liver cancer (HepG2), human colorectal cancer (Caco-2) and non-cancer mouse fibroblast (L-929) cell lines. Some compounds performed comparable cytotoxicity with standard drug cisplatin. Antibacterial properties of the compounds were tested against Gram-negative Escherichia coli and Gram-positive Bacillus subtilis bacteria, but the compounds did not have any antibacterial effect against both bacteria. Enzyme inhibitory properties of all compounds were tested on the activities of human carbonic anhydrase I and II, and xanthine oxidase. All compounds inhibited both enzymes more effectively than standard drugs, acetazolamide and allopurinol, respectively. The biological evaluation results showed that ionic and water soluble coumarin derivatives are promising structures for further investigations especially on enzyme inhibition field.

Quantitative Proteomics Analysis by Sequential Window Acquisition of All Theoretical Mass Spectra-Mass Spectrometry Reveals a Cross-Protection Mechanism for Monascus To Tolerate High-Concentration Ammonium Chloride.

To achieve the accumulation of targeted secondary metabolites, microorganisms must adopt various protection mechanisms to avoid or reduce damage to cells caused by abiotic stresses, which formed from the changes of physical and chemical culture conditions. The protection mechanism of Monascus sp. to tolerate high-concentration ammonium chloride was analyzed by sequential window acquisition of all theoretical mass spectra-mass spectrometry proteomics in this work, and the results indicated that abiotic stresses caused by high-concentration ammonium chloride inhibited the synthesis of chitin and glycoprotein, leading to a decrease in cell wall integrity and, thus, affecting cell growth. At the same time, it also inhibited the complex enzyme III and IV activities of the mitochondrial cytochrome respiratory chain, leading to an increase in reactive oxygen species (ROS) levels. With the aim to respond to abiotic stresses, the cross-protection mechanism was implemented in Monascus, including self-protection of the Monascus cell by promoting synthesis of trehalose, a molecular chaperone that facilitates protein folding (such as heat-shock protein) and autophagy-related proteins, through not the enzyme protection system (superoxide dismutase, peroxidase, catalase, NADPH oxidase, and alternative oxidase) but the glutathione/glutaredoxin system, to maintain the intracellular redox state and then eliminate or reduce ROS damage to the cell. At the same time, an alternative respiratory pathway related to NADH dehydrogenase was activated to balance the material and energy metabolism.

A modified lysosomal organelle mediates nonlytic egress of reovirus.

Mammalian orthoreoviruses (reoviruses) are nonenveloped viruses that replicate in cytoplasmic membranous organelles called viral inclusions (VIs) where progeny virions are assembled. To better understand cellular routes of nonlytic reovirus exit, we imaged sites of virus egress in infected, nonpolarized human brain microvascular endothelial cells (HBMECs) and observed one or two distinct egress zones per cell at the basal surface. Transmission electron microscopy and 3D electron tomography (ET) of the egress zones revealed clusters of virions within membrane-bound structures, which we term membranous carriers (MCs), approaching and fusing with the plasma membrane. These virion-containing MCs emerged from larger, LAMP-1-positive membranous organelles that are morphologically compatible with lysosomes. We call these structures sorting organelles (SOs). Reovirus infection induces an increase in the number and size of lysosomes and modifies the pH of these organelles from ∼4.5-5 to ∼6.1 after recruitment to VIs and before incorporation of virions. ET of VI-SO-MC interfaces demonstrated that these compartments are connected by membrane-fusion points, through which mature virions are transported. Collectively, our results show that reovirus uses a previously undescribed, membrane-engaged, nonlytic egress mechanism and highlights a potential new target for therapeutic intervention.