CFTR-rich ionocytes mediate chloride absorption across airway epithelia.

The volume and composition of a thin layer of liquid covering the airway surface defend the lung from inhaled pathogens and debris. Airway epithelia secrete Cl- into the airway surface liquid through cystic fibrosis transmembrane conductance regulator (CFTR) channels, thereby increasing the volume of airway surface liquid. The discovery that pulmonary ionocytes contain high levels of CFTR led us to predict that ionocytes drive secretion. However, we found the opposite. Elevating ionocyte abundance increased liquid absorption, whereas reducing ionocyte abundance increased secretion. In contrast to other airway epithelial cells, ionocytes contained barttin/Cl- channels in their basolateral membrane. Disrupting barttin/Cl- channel function impaired liquid absorption, and overexpressing barttin/Cl- channels increased absorption. Together, apical CFTR and basolateral barttin/Cl- channels provide an electrically conductive pathway for Cl- flow through ionocytes, and the transepithelial voltage generated by apical Na+ channels drives absorption. These findings indicate that ionocytes mediate liquid absorption, and secretory cells mediate liquid secretion. Segregating these counteracting activities to distinct cell types enables epithelia to precisely control the airway surface. Moreover, the divergent role of CFTR in ionocytes and secretory cells suggests that cystic fibrosis disrupts both liquid secretion and absorption.

Inflammation as a Regulator of the Airway Surface Liquid pH in Cystic Fibrosis.

The airway surface liquid (ASL) is a thin sheet of fluid that covers the luminal aspect of the airway epithelium. The ASL is a site of several first-line host defenses, and its composition is a key factor that determines respiratory fitness. Specifically, the acid-base balance of ASL has a major influence on the vital respiratory defense processes of mucociliary clearance and antimicrobial peptide activity against inhaled pathogens. In the inherited disorder cystic fibrosis (CF), loss of cystic fibrosis transmembrane conductance regulator (CFTR) anion channel function reduces HCO3- secretion, lowers the pH of ASL (pHASL), and impairs host defenses. These abnormalities initiate a pathologic process whose hallmarks are chronic infection, inflammation, mucus obstruction, and bronchiectasis. Inflammation is particularly relevant as it develops early in CF and persists despite highly effective CFTR modulator therapy. Recent studies show that inflammation may alter HCO3- and H+ secretion across the airway epithelia and thus regulate pHASL. Moreover, inflammation may enhance the restoration of CFTR channel function in CF epithelia exposed to clinically approved modulators. This review focuses on the complex relationships between acid-base secretion, airway inflammation, pHASL regulation, and therapeutic responses to CFTR modulators. These factors have important implications for defining optimal ways of tackling CF airway inflammation in the post-modulator era.

Interplay of eco-friendly factors and islamic religiosity towards recycled package products: A cross-cultural study.

Climate change has increasingly been recognised and associated with consumer behaviour: Practitioners are developing their strategies to reduce environmental degradation while increasing the management of sustainable consumption; it needs to better understand consumer attitudes and eco-friendly factors about the issue. Therefore, the current study focused to understand the effects of pro-environmental factors on individuals' environmental attitudes (purchase behaviour towards products with recycled packaging) through the lens of theory of planned behaviour in a cross-cultural setting. Moreover, present research focuses on the moderating role that religiosity plays in causal pathways between certain determinants (attitude, subjective norms, and perceived behavioural control) and intentions in this context. A multi-wave time-lagged research design was employed in this study, and university students from two developing countries were surveyed (N = 324, 266). The findings revealed pronounced similarities between the two examined countries. Overwhelmingly, pro-environmental factors examined (environmental values, environmental knowledge, and environmental concern) were found to be positively related to attitude formation. Further results showed that attitude and subjective norms are significant predictors of the intention to purchase products with recycled packaging. Moreover, with the exception of perceived behavioural control, religiosity moderates the relationships between all the determinants of TPB and intention to purchase recycled packaged products. Present study offers insightful implications to management of these emerging and/or similar cultural markets regarding customer value for green products. Using TPB, present study broadened and deepen extant stream of literature on consumption of recycled packaged products in two highly emerging markets; Pakistan and Malaysia.

Detection of linezolid resistance cfr gene among MRSA isolates.

BACKGROUND: Linezolid (Oxazolidinones) is commonly used against a variety of Gram-positive infections, especially methicillin-resistant Staphylococcus aureus (MRSA). The emerging resistance to linezolid curtail the treatment of infections caused by MRSA and other Gram-positive bacteria. Presence of cfr gene plays a crucial role in Linezolid resistance. OBJECTIVE: Present study was aimed to detect cfr gene among clinical MRSA isolates. MATERIALS AND METHODS: The suspected Staphylococcus aureus isolates were processed through Kirby Bauer disc diffusion methods for the confirmation of MRSA strains. Phenotypic Linezolid resistance was determined through broth micro-dilution method. The plasmid and DNA of Linezolid resistant isolates were subjected to molecular characterization for the presence of cfr gene. RESULTS: Among 100 Staphylococcus aureus isolates, 85 of them were confirmed as MRSA isolates. Categorically, 65% MRSA isolates were sensitive to linezolid with MIC lower than 8 µg/ml, whereas, 35% of them were resistant to linezolid having MIC greater than 8 µg/ml. MIC level of 128 µg/ml was observed among 3.5% of the resistant isolates. Similarly, MIC level of 64 µg/ml, 32 µg/ml, 16 µg/ml and 8 µg/ml were noted for 3.5%, 4.7%, 8.2% and 15.3% isolates respectively. Linezolid resistance cfr gene was detected only in 9.4% of the resistant isolates. CONCLUSION: Multi drug resistance among MRSA isolates is keenly attributed to the presence of cfr gene as evident in the present study, and horizontal dissemination of cfr gene among MRSA strains is accredited to cfr-carrying transposons and plasmids.

WNK Inhibition Increases Surface Liquid pH and Host Defense in Cystic Fibrosis Airway Epithelia.

In cystic fibrosis (CF), reduced HCO3- secretion acidifies the airway surface liquid (ASL), and the acidic pH disrupts host defenses. Thus, understanding the control of ASL pH (pHASL) in CF may help identify novel targets and facilitate therapeutic development. In diverse epithelia, the WNK (with-no-lysine [K]) kinases coordinate HCO3- and Cl- transport, but their functions in airway epithelia are poorly understood. Here, we tested the hypothesis that WNK kinases regulate CF pHASL. In primary cultures of differentiated human airway epithelia, inhibiting WNK kinases acutely increased both CF and non-CF pHASL. This response was HCO3- dependent and involved downstream SPAK/OSR1 (Ste20/SPS1-related proline-alanine-rich protein kinase/oxidative stress responsive 1 kinase). Importantly, WNK inhibition enhanced key host defenses otherwise impaired in CF. Human airway epithelia expressed two WNK isoforms in secretory cells and ionocytes, and knockdown of either WNK1 or WNK2 increased CF pHASL. WNK inhibition decreased Cl- secretion and the response to bumetanide, an NKCC1 (sodium-potassium-chloride cotransporter 1) inhibitor. Surprisingly, bumetanide alone or basolateral Cl- substitution also alkalinized CF pHASL. These data suggest that WNK kinases influence the balance between transepithelial Cl- versus HCO3- secretion. Moreover, reducing basolateral Cl- entry may increase HCO3- secretion and raise pHASL, thereby improving CF host defenses.

Photoperiodic Modulation in Immune and Reproductive Systems in Japanese Quails (Coturnix japonica): A Morphometric Perspective.

The present study was designed to elucidate a relationship between lymphoid organs and reproductive activity in male Japanese quails (Coturnix japonica) bred in a temperate region of Pakistan (30.3753° N, 69.3451° E) in response to photoperiodic changes. The research focused primarily on the relative morphological changes in primary (thymus and bursa of Fabricius) and secondary (spleen) lymphoid organs with respect to seasonal variations in the histomorphometry of testicular tissue. For this purpose, a comparable number of clinically healthy Japanese quails were exsanguinated during active (April-May), regressive (September-October) and inactive (January-February) reproductive phases. Following an extensive gross measurement of lymphoid and reproductive organs, a histomorphometric analysis was performed on sampled tissues by employing ImageJ® software. Blood was collected for hormonal and leukocytic analysis. One-way ANOVA was used for statistical comparison. Testes had the highest parenchymal development in the active phase (80.66 ± 21.22 µm) and the lowest in the inactive phase (27.80 ± 7.22 µm). Conversely, a percentage change was evident in the sizes of primary (bursa: 61.5%, thymus: 46.9%) and secondary (spleen: 23.9%) lymphoid organs during inactive and active reproductive phases. This study demonstrated that a physiological trade-off is imperative between immune and reproductive systems for optimum survivability and reproductive performance.

Inflammatory cytokines TNF-α and IL-17 enhance the efficacy of cystic fibrosis transmembrane conductance regulator modulators.

Without cystic fibrosis transmembrane conductance regulator-mediated (CFTR-mediated) HCO3- secretion, airway epithelia of newborns with cystic fibrosis (CF) produce an abnormally acidic airway surface liquid (ASL), and the decreased pH impairs respiratory host defenses. However, within a few months of birth, ASL pH increases to match that in non-CF airways. Although the physiological basis for the increase is unknown, this time course matches the development of inflammation in CF airways. To learn whether inflammation alters CF ASL pH, we treated CF epithelia with TNF-α and IL-17 (TNF-α+IL-17), 2 inflammatory cytokines that are elevated in CF airways. TNF-α+IL-17 markedly increased ASL pH by upregulating pendrin, an apical Cl-/HCO3- exchanger. Moreover, when CF epithelia were exposed to TNF-α+IL-17, clinically approved CFTR modulators further alkalinized ASL pH. As predicted by these results, in vivo data revealed a positive correlation between airway inflammation and CFTR modulator-induced improvement in lung function. These findings suggest that inflammation is a key regulator of HCO3- secretion in CF airways. Thus, they explain earlier observations that ASL pH increases after birth and indicate that, for similar levels of inflammation, the pH of CF ASL is abnormally acidic. These results also suggest that a non-cell-autonomous mechanism, airway inflammation, is an important determinant of the response to CFTR modulators.

Detection of blaNDM-1 gene in ESBL producing Escherichia coli and Klebsiella pneumoniae isolated from urine samples.

INTRODUCTION: Enterobacteriaceae such as Escherichia coli and Klebsiella pneumoniae are the most prominent bacterial species resistant to almost all commonly used antibiotics. Carbapenem is one of the last resort drugs for treating such emerging multidrug-resistant bacteria. This study aimed to detect carbapenem-resistant blaNDM-1 gene in ESBL producing E. coli and K. pneumoniae isolates. METHODOLOGY: A total of 190 E. coli and 350 K. pneumoniae isolates were screened for extended spectrumß-lactamase (ESBL), carbapenemase and metallo ß-lactamase (MBL) production via double-disk synergy test (DDST), modified Hodge test and combined-disk diffusion method. The blaNDM-1 gene was detected by PCR and confirmed via Sanger sequencing method. RESULTS: Of the 540 isolates tested, 71.8% were found to be multidrug-resistant. Overall rate of ESBL-positive isolates were 57.89% E. coli and 31.42% K. pneumoniae. Among ESBL positive isolates, 49.09% E. coli and 40% K. pneumoniae were positive for carbapenemase production whereas MBL production was detected in 29% E. coli and 22% K. pneumoniae isolates. In MBL positive isolates, (37%) E. coli and (40%) K. pneumoniae isolates harboured blaNDM-1 gene. The pair-wise DNA was aligned with the NDM-1 sequence from GenBank. The alignment score was 243 and the blast nucleotide sequencing results showed 97% sequence similarity with the sequences in GenBank for the blaNDM-1 gene. CONCLUSIONS: The blaNDM-1 gene was found to be the most prevalent in urine samples. There is a dire need to conduct screening tests in hospitals and communities to find out the exact prevalence of the blaNDM-1 spread in our population.

Molecular Epidemiology of mcr-1, bla KPC-2, and bla NDM-1 Harboring Clinically Isolated Escherichia coli from Pakistan.

PURPOSE: The multiple-drug resistant Escherichia coli are among the deadliest pathogens causing life-threatening infections. This study was planned to determine the molecular epidemiology of mcr-1, bla KPC-2, and bla NDM-1 harboring clinically isolated E. coli from Pakistan. METHODS: In total, 545 strains of E. coli from clinical samples were collected from June 2018 to September 2019. All the isolates were screened for colistin-resistance, extended-spectrum-ß-lactamases (ESBL), and carbapenemases through the micro-dilution method, Double-Disk-Synergy-Test (DDST), and Modified-Hodge-Test (MHT). The detection, sequence-typing, conjugal transfer, S1-PFGE, plasmid-replicon-typing, and southern-blotting for mcr, ESBL, and carbapenemase-encoding genes were performed. FINDINGS: A total of four (0.73%) colistin-resistant strains carrying alongside mcr-1 and bla CTX-M-15 genes, three of these strains also had the bla TEM-1 gene. The presence of ESBL genes was detected in 139 (25.5%) isolates harboring bla CTXM-15 (74.82%), bla TEM (34.53%), bla SHV (28.06%) and bla OXA-1 (28.78%). In 129 carbapenemase-producers, 35.83% possessed bla NDM-1, 26.67% bla KPC-2, 8.3% bla OXA-48, 25% bla VIM-1, and 20.83% bla IMP-1 genes. The sequence typing revealed that mcr-1 harboring isolates belonged to ST405, ST117, and ST156. Fifty percent of bla KPC-2 and 48.83% of bla NDM-1 were found on ST131 and ST1196, respectively. Two rare types of STs, ST7584, and ST8671 were also identified in this study. The mcr-1 gene was located on Incl2 (60-kb) plasmid. The bla KPC-2 was present on (140-kb) IncH12, (100-kb) IncN, (90-kb) Incl1, while bla NDM-1 was located on (70-kb) IncFIIK, (140-kb) IncH12, (100-kb) IncN, (60-kb) IncA/C, and (45-kb) IncFII plasmids, which were successfully trans-conjugated. Among the plasmid types, the Incl1 carrying bla KPC-2, IncH12 harboring bla KPC-2 and bla NDM-1, and IncFIIK carrying bla NDM-1 were for the first time detected in Pakistan. CONCLUSION: The mcr-1, bla KPC-2, and bla NDM-1 genes finding in various clonal and plasmids types indicate that a substantial selection of the resistance genes had occurred in our clinical strains.

First Report of blaNDM-1 Bearing IncX3 Plasmid in Clinically Isolated ST11 Klebsiella pneumoniae from Pakistan.

The New Delhi Metallo-ß-lactamase (NDM) is among the most threatening forms of carbapenemases produced by K. pneumoniae, well-known to cause severe worldwide infections. The molecular epidemiology of blaNDM-1-harboring K. pneumoniae is not well elucidated in Pakistan. Herein, we aim to determine the antibiotics-resistance profile, genes type, molecular type, and plasmid analysis of 125 clinically isolated K. pneumoniae strains from urine samples during July 2018 to January 2019 in Pakistan. A total of 34 (27.2%) K. pneumoniae isolates were carbapenemases producers, and 23 (18.4%) harbored the blaNDM-1 gene. The other carbapenemases encoding genes, i.e., blaIMP-1 (7.2%), blaVIM-1 (3.2%), and blaOXA-48 (2.4%) were also detected. The Multi Locus Sequence Typing (MLST) results revealed that all blaNDM-1-harboring isolates were ST11. The other sequence types detected were ST1, ST37, and ST105. The cluster analysis of Xbal Pulsed Field Gel Electrophoresis (PFGE) revealed variation amongst the clusters of the identical sequence type isolates. The blaNDM-1 gene in all of the isolates was located on a 45-kb IncX3 plasmid, successfully transconjugated. For the first time, blaNDM-1-bearing IncX3 plasmids were identified from Pakistan, and this might be a new primary vehicle for disseminating blaNDM-1 in Enterobacteriaceae as it has a high rate of transferability.

Antibiotic resistance in Pakistan: a systematic review of past decade.

BACKGROUND: During the last six decades, extensive use of antibiotics has selected resistant strains, increasing the rate of fatal infectious diseases, and exerting an economic burden on society. This situation is widely accepted as a global problem, yet its degree is not well elucidated in many regions of the world. Up till now, no systemic analysis of Antimicrobial resistance (AMR) in Pakistan has been published. The current study aims to describe the antibiotic-resistance scenario of Pakistan from human samples of the last 10 y, to find the gaps in surveillances and methodology and recommendations for researchers and prescribers founded on these outcomes. METHODS: Original research articles analyzed the pattern of Antibiotic resistance of any World Health Organization (WHO) enlisted priority pathogens in Pakistan (published onward 2009 till March 2020), were collected from PubMed, Google scholar, and PakMedi Net search engines. These articles were selected based on predefined inclusion and exclusion criteria. Data about the study characteristics and antibiotic-resistance for a given bacterium were excluded from literature. Antibiotic resistance to a particular bacterium was calculated as a median resistance with 95% Confidence Interval (CI). RESULTS: Studies published in the last 10 y showed that Urinary Tract Infection (UTI) is the most reported clinical diagnosis (16.1%) in Pakistan. E. coli were reported in 28 (30.11%) studies showing high resistance to antibiotics' first line. Methicillin-resistant Staphylococcus aureus (MRSA) was found in 49% of S. aureus' total reported cases. Phenotypic resistance pattern has mostly been evaluated by Disk Diffusion Method (DDM) (82.8%), taken Clinical Laboratory Standards Institute (CLSI) as a breakpoint reference guideline (in 79.6% studies). Only 28 (30.11%) studies have made molecular identification of the resistance gene. blaTEM (78.94% in Shigella spp) and blaNDM-1 (32.75% in Klebsiella spp) are the prominent reported resistant genes followed by VanA (45.53% in Enterococcus spp), mcr-1 (1.61% in Acinetobacter spp), and blaKPC-2 (31.67% in E. coli). Most of the studies were from Sindh (40.86%), followed by Punjab (35.48%), while Baluchistan's AMR data was not available. CONCLUSION: Outcomes of our study emphasize that most of the pathogens show high resistance to commonly used antibiotics; also, we find gaps in surveillances and breaches in methodological data. Based on these findings, we recommend the regularization of surveillance practice and precise actions to combat the region's AMR.

Expression of antimicrobial peptide Cecropin P1 in Saccharomyces cerevisiae and its antibacterial and antiviral activity in vitro

BACKGROUND: Cecropin P1, acting as an antimicrobial, has a broad-spectrum antibacterial activity with some antiviral and antifungal properties. It is a promising natural alternative to antibiotics which is originally isolated from the pig intestinal parasitic nematode Ascaris suum. Many studies have shown that Cecropin P1 is helpful for the prevention or treatment of clinical diseases. Therefore, it is very necessary to establish a safe, nontoxic, and efficient expression method of Cecropin P1. RESULTS: The results indicated that the recombinant protein was about 5.5 kDa showed by Tricine­SDS­ PAGE and Western blot. And Cecropin P1 was efficiently secreted and expressed after 12 h of induction, with an increasing yield over the course of the induction. Its maximum concentration was 7.83 mg/L after concentration and purification. In addition, in vitro experiments demonstrated that Cecropin P1 not only exerted a strong inhibitory effect on Escherichia coli, Salmonella sp., Shigella sp., and Pasteurella sp., but also displayed an antiviral activity against PRRSV NADC30-Like strain. CONCLUSIONS: Collectively, the strategy of expressing Cecropin P1 in Saccharomyces cerevisiae is harmless, efficient, and safe for cells. In addition, the expressed Cecropin P1 has antiviral and antibacterial properties concurrently.

A structure-based virtual screening and molecular docking by using potent inhibitors against nucleoprotein of Crimean-Congo hemorrhagic fever virus.

BACKGROUND & OBJECTIVES: Crimean-Congo Hemorrhagic Fever Virus (CCHFV) is a vector borne pathogen, well-known for causing endemic hemorrhagic fever in Asia, Europe and Africa. There is no specific drug or vaccine available against CCHFV. The recent upsurge of Crimean-Congo Hemorrhagic Fever around the globe has made it a major health issue and this demands investigation for specific inhibitors to viral proteins. The objective of this study was to assess inhibitors that may have the potential to dock CCHFV nucleoprotein which plays an important role in viral assembly. METHODS: We performed structure-based virtual screening and molecular docking by using potent inhibitors against nucleoprotein of CCHFV. Screening was performed by a webserver, MtiOpenScreen which gave 1000 drug-like molecules from PubChem. PyRx Autodock vina was utilized to dock the protein. The docking poses were observed for interaction analysis by LigPlot+. This study provided ten potential candidates capable of binding to the active site of NP of CCHFV. The selected hits were then subjected to toxicity prediction by ProTox-II. RESULTS: Four hits were identified that specifically dock nucleoprotein at the presumed binding site. Furthermore, these compounds have less binding energy i.e., 9.7 kcal/mol, 9.8 kcal/mol and 10.4 kcal/mol and with equal toxicity measures when compared to an FDA approved drug. INTERPRETATION & CONCLUSION: This study illustrates that virtual screening is an efficient in silico approach to identify target-specific inhibitors. Researchers in this area who investigate drugs or synthesize agents against CCHFV with better efficacy could utilize reported inhibitors rather than trying random compounds ambivalently.

Characterisation of Bacteriocins Produced by Lactobacillus spp. Isolated from the Traditional Pakistani Yoghurt and Their Antimicrobial Activity against Common Foodborne Pathogens.

Lactic acid bacteria (LAB) are widely known for their probiotic activities for centuries. These bacteria synthesise some secretory proteinaceous toxins, bacteriocins, which help destroy similar or interrelated bacterial strains. This study was aimed at characterising bacteriocins extracted from Lactobacillus spp. found in yoghurt and assessing their bactericidal effect on foodborne bacteria. Twelve isolated Lactobacillus spp. were examined to produce bacteriocins by the organic solvent extraction method. Bacteriocins produced by two of these strains, Lactobacillus helveticus (BLh) and Lactobacillus plantarum (BLp), showed the most significant antimicrobial activity, especially against Staphylococcus aureus and Acinetobacter baumannii. Analysis of SDS-PAGE showed that L. plantarum and L. helveticus bacteriocins have a molecular weight of ~10 kDa and ~15 kDa, respectively. L. plantarum (BLp) bacteriocin was heat stable while L. helveticus (BLh) bacteriocin was heat labile. Both bacteriocins have shown activity at acidic pH. Exposure to a UV light enhances the activity of the BLh; however, it had negligible effects on the BLp. Different proteolytic enzymes confirmed the proteinaceous nature of both the bacteriocins. From this study, it was concluded that bacteriocin extracts from L. helveticus (BLh) can be considered a preferable candidate against foodborne pathogens as compared to L. plantarum (BLp). These partially purified bacteriocins should be further processed to attain purified product that could be useful for food spoilage and preservation purposes.

Phylogeny of hospital acquired MRSA, and its comparative phenotypic clinico-epidemiology with vancomycin resistant S. aureus (VRSA).

Staphylococcus aureus is emerging as complicated pathogen because of its wide-ranging origin, multiple variants, and compromised antibiotic susceptibilities. Current study was planned to find lineage of hospital acquired methicillin resistant Staphylococcus aureus (HA-MRSA), and its comparative phenotypic clinico-epidemiology with vancomycin resistant S. aureus (VRSA). A total of (n = 200) samples were aseptically collected from wound, nose, and cerebrospinal fluid of patients from metropolitan and rural background hospitals along with on spot filling in of questionnaire. Phylogenetic analysis of HA-MRSA was identified by targeting mecA gene in S. aureus. At optimal tree branch length of 1.91 and evolutionary distance 0.1, high level sequence similarity (97%-99%) was observed with different strains of S. aureus isolated from both human and animal. Non-descriptive statistics at 5% probability found 61% S. aureus, while 43.44% of them were HA-MRSA, 92.62% VRSA, and 42.62% were both MRSA and VRSA. Among assumed risk factors, use of antibiotics, venous catheterization, chronic disease, pre-hospital visits, and ICU admitted patients showed significant association (p<0.05) with pathogen. HA-MRSA was 37.50%, 80%, and 37.50% sensitive to chloramphenicol, gentamicin, and oxacillin, respectively. While <50% of VRSA were sensitive against oxacillin, enoxacin, and chloramphenicol. A significant difference (p<0.05) of percentage responses of MRSA and VRSA at resistant, intermediate, and sensitive cadre against all antibiotics except chloramphenicol was obvious in this study. The Current study concluded higher prevalence of MRSA & VRSA, significant association of risk factors, limiting antibiotic susceptibility profile, and genetic transfer at animal-human interface which suggests further studies cum preventive strategies to be planned.

Paracellular bicarbonate flux across human cystic fibrosis airway epithelia tempers changes in airway surface liquid pH.

KEY POINTS: Cl- and HCO3- had similar paracellular permeabilities in human airway epithelia. PCl /PNa of airway epithelia was unaltered by pH 7.4 vs. pH 6.0 solutions. Under basal conditions, calculated paracellular HCO3- flux was secretory. Cytokines that increased airway surface liquid pH decreased or reversed paracellular HCO3- flux. HCO3- flux through the paracellular pathway may counterbalance effects of cellular H+ and HCO3- secretion. ABSTRACT: Airway epithelia control the pH of airway surface liquid (ASL), thereby optimizing respiratory defences. Active H+ and HCO3- secretion by airway epithelial cells produce an ASL that is acidic compared with the interstitial space. The paracellular pathway could provide a route for passive HCO3- flux that also modifies ASL pH. However, there is limited information about paracellular HCO3- flux, and it remains uncertain whether an acidic pH produced by loss of cystic fibrosis transmembrane conductance regulator anion channels or proinflammatory cytokines might alter the paracellular pathway function. To investigate paracellular HCO3- transport, we studied differentiated primary cultures of human cystic fibrosis (CF) and non-CF airway epithelia. The paracellular pathway was pH-insensitive at pH 6.0 vs. pH 7.4 and was equally permeable to Cl- and HCO3- . Under basal conditions at pH ∼6.6, calculated paracellular HCO3- flux was weakly secretory. Treating epithelia with IL-17 plus TNFα alkalinized ASL pH to ∼7.0, increased paracellular HCO3- permeability, and paracellular HCO3- flux was negligible. Applying IL-13 increased ASL pH to ∼7.4 without altering paracellular HCO3- permeability, and calculated paracellular HCO3- flux was absorptive. These results suggest that HCO3- flux through the paracellular pathway counterbalances, in part, changes in the ASL pH produced via cellular mechanisms. As the pH of ASL increases towards that of basolateral liquid, paracellular HCO3- flux becomes absorptive, tempering the alkaline pH generated by transcellular HCO3- secretion.

TNFα and IL-17 alkalinize airway surface liquid through CFTR and pendrin.

The pH of airway surface liquid (ASL) is a key factor that determines respiratory host defense; ASL acidification impairs and alkalinization enhances key defense mechanisms. Under healthy conditions, airway epithelia secrete base ([Formula: see text]) and acid (H+) to control ASL pH (pHASL). Neutrophil-predominant inflammation is a hallmark of several airway diseases, and TNFα and IL-17 are key drivers. However, how these cytokines perturb pHASL regulation is uncertain. In primary cultures of differentiated human airway epithelia, TNFα decreased and IL-17 did not change pHASL. However, the combination (TNFα+IL-17) markedly increased pHASL by increasing [Formula: see text] secretion. TNFα+IL-17 increased expression and function of two apical [Formula: see text] transporters, CFTR anion channels and pendrin Cl-/[Formula: see text] exchangers. Both were required for maximal alkalinization. TNFα+IL-17 induced pendrin expression primarily in secretory cells where it was coexpressed with CFTR. Interestingly, significant pendrin expression was not detected in CFTR-rich ionocytes. These results indicate that TNFα+IL-17 stimulate [Formula: see text] secretion via CFTR and pendrin to alkalinize ASL, which may represent an important defense mechanism in inflamed airways.

Protective effects of metallothionein and vitamin E in the trunk kidney and blood of cadmium poisoned Ctenopharyngodon idellus.

Cadmium (Cd), a substance with one of the most critical health hazard indices, can cause damage to both the blood and kidneys and accumulates in the body at last. The present work studied the toxicological effects of Cd and the therapeutic effects of metallothionein (MT) and vitamin E (VE) on the trunk kidney and blood of freshwater grass carp (Ctenopharyngodon idellus). Grass carp were divided into three groups: Cd + phosphate-buffered saline (PBS) group, Cd + VE group, and the Cd + MT group. Fish were injected with CdCl2 on the first day and then VE, MT, or PBS was administered 4 days post-injection. Fish not injected with Cd were used as a negative control. The blood and trunk kidney amassed Cd and suffered severe damage in the forms of organ toxicity cytotoxicity, and immunotoxicity. However, the MT reduced the Cd content in the trunk kidney and blood and partially stabilized the damaged organs. Treatment with VE, however, only demonstrated weaker protection against on Cd-induced toxicity. The results indicate that exogenous MT may play an essential role in restoring homeostasis of the Cd-poisoned urinary and circulatory system and that it may help eliminate Cd in aquatic animals.

Thymol Inhibits Biofilm Formation, Eliminates Pre-Existing Biofilms, and Enhances Clearance of Methicillin-Resistant Staphylococcus aureus (MRSA) in a Mouse Peritoneal Implant Infection Model.

Methicillin-resistant Staphylococcus aureus (MRSA) is a common human pathogen that causes several difficult-to-treat infections, including biofilm-associated infections. The biofilm-forming ability of S. aureus plays a pivotal role in its resistance to most currently available antibiotics, including vancomycin, which is the first-choice drug for treating MRSA infections. In this study, the ability of thymol (a monoterpenoid phenol isolated from plants) to inhibit biofilm formation and to eliminate mature biofilms, was assessed. We found that thymol could inhibit biofilm formation and remove mature biofilms by inhibiting the production of polysaccharide intracellular adhesin (PIA) and the release of extracellular DNA (eDNA). However, cotreatment with thymol and vancomycin was more effective at eliminating MRSA biofilms, in a mouse infection model, than monotherapy with vancomycin. Comparative histopathological analyses revealed that thymol reduced the pathological changes and inflammatory responses in the wounds. Assessments of white blood cell counts and serum TNF-α and IL-6 levels showed reduced inflammation and an increased immune response following treatment with thymol and vancomycin. These results indicate that combinatorial treatment with thymol and vancomycin has the potential to serve as a more effective therapy for MRSA biofilm-associated infections than vancomycin monotherapy.

The antibacterial mechanism of oridonin against methicillin-resistant Staphylococcus aureus (MRSA).

Context: Methicillin-resistant Staphylococcus aureus (MRSA) is a very harmful bacterium. Oridonin, a component in Rabdosia rubescens (Hemsl.) Hara (Lamiaceae), is widely used against bacterial infections in China. Objective: We evaluated oridonin effects on MRSA cell membrane and wall, protein metabolism, lactate dehydrogenase (LDH), DNA and microscopic structure. Materials and methods: Broth microdilution and flat colony counting methods were used to measure oridonin minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against USA300 strain. Electrical conductivity and DNA exosmosis were analysed to study oridonin effects (128 µg/mL) on cell membrane and wall for 0, 1, 2, 4 and 6 h. Sodium dodecyl sulphate polyacrylamide gel electrophoresis was used to detect effects on soluble protein synthesis after 6, 10 and 16 h. LDH activity was examined with an enzyme-linked immunosorbent assay. Effects of oridonin on USA300 DNA were investigated with DAPI staining. Morphological changes in MRSA following oridonin treatment were determined with scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Results: Oridonin MIC and MBC values against USA300 were 64 and 512 µg/mL, respectively. The conductivity and DNA exosmosis level of oridonin-treated USA300 improved by 3.20±0.84% and increased by 58.63 ± 1.78 µg/mL, respectively. LDH and soluble protein levels decreased by 30.85±7.69% and 27.51 ± 1.39%, respectively. A decrease in fluorescence intensity was reported with time. Oridonin affected the morphology of USA300. Conclusions: Oridonin antibacterial mechanism was related to changes in cell membrane and cell wall permeability, disturbance in protein and DNA metabolism, and influence on bacterial morphology. Thus, oridonin may help in treating MRSA infection.