Airway hyperresponsiveness and inflammation are fundamental hallmarks of allergic asthma that are accompanied by increases in certain polycations, such as eosinophil cationic protein. Levels of these cations in body fluids correlate with asthma severity. We show that polycations and elevated extracellular calcium activate the human recombinant and native calcium-sensing receptor (CaSR), leading to intracellular calcium mobilization, cyclic adenosine monophosphate breakdown, and p38 mitogen-activated protein kinase phosphorylation in airway smooth muscle (ASM) cells. These effects can be prevented by CaSR antagonists, termed calcilytics. Moreover, asthmatic patients and allergen-sensitized mice expressed more CaSR in ASMs than did their healthy counterparts. Indeed, polycations induced hyper-reactivity in mouse bronchi, and this effect was prevented by calcilytics and absent in mice with CaSR ablation from ASM. Calcilytics also reduced airway hyperresponsiveness and inflammation in allergen-sensitized mice in vivo. These data show that a functional CaSR is up-regulated in asthmatic ASM and targeted by locally produced polycations to induce hyperresponsiveness and inflammation. Thus, calcilytics may represent effective asthma therapeutics.

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There has been an explosion of probiotic incorporated based product. However, many reports indicated that most of the probiotics have failed to survive in high quantity, which has limited their effectiveness in most functional foods. Thus, to overcome this problem, microencapsulation is considered to be a promising process. In this study, Lactococcus lactis Gh1 was encapsulated via spray-drying with gum Arabic together with Synsepalum dulcificum or commonly known as miracle fruit. It was observed that after spray-drying, high viability (~109 CFU/mL) powders containing L. lactis in combination with S. dulcificum were developed, which was then formulated into yogurt. The tolerance of encapsulated bacterial cells in simulated gastric juice at pH 1.5 was tested in an in-vitro model and the result showed that after 2 h, cell viability remained high at 1.11 × 106 CFU/mL. Incubation of encapsulated cells in the presence of 0.6% (w/v) bile salts showed it was able to survive (~104 CFU/mL) after 2 h. Microencapsulated L. lactis retained a higher viability, at ~107 CFU/mL, when incorporated into yogurt compared to non-microencapsulated cells ~105 CFU/mL. The fortification of microencapsulated and non-microencapsulated L. lactis in yogurts influenced the viable cell counts of yogurt starter cultures, Lactobacillus delbrueckii subs. bulgaricus and Streptococcus thermophilus.

Autosomal dominant hypocalcemia type 1 (ADH1) is a rare form of hypoparathyroidism caused by heterozygous, gain‐of‐function mutations of the calcium‐sensing receptor gene (CAR). Individuals are hypocalcemic with inappropriately low parathyroid hormone (PTH) secretion and relative hypercalciuria. Calcilytics are negative allosteric modulators of the extracellular calcium receptor (CaR) and therefore may have therapeutic benefits in ADH1. Five adults with ADH1 due to four distinct CAR mutations received escalating doses of the calcilytic compound NPSP795 (SHP635) on 3 consecutive days. Pharmacokinetics, pharmacodynamics, efficacy, and safety were assessed. Parallel in vitro testing with subject CaR mutations assessed the effects of NPSP795 on cytoplasmic calcium concentrations (Ca2+i), and ERK and p38MAPK phosphorylation. These effects were correlated with clinical responses to administration of NPSP795. NPSP795 increased plasma PTH levels in a concentration‐dependent manner up to 129% above baseline (p = 0.013) at the highest exposure levels. Fractional excretion of calcium (FECa) trended down but not significantly so. Blood ionized calcium levels remained stable during NPSP795 infusion despite fasting, no calcitriol supplementation, and little calcium supplementation. NPSP795 was generally safe and well‐tolerated. There was significant variability in response clinically across genotypes. In vitro, all mutant CaRs were half‐maximally activated (EC50) at lower concentrations of extracellular calcium (Ca2+o) compared to wild‐type (WT) CaR; NPSP795 exposure increased the EC50 for all CaR activity readouts. However, the in vitro responses to NPSP795 did not correlate with any clinical parameters. NPSP795 increased plasma PTH levels in subjects with ADH1 in a dose‐dependent manner, and thus, serves as proof‐of‐concept that calcilytics could be an effective treatment for ADH1. Albeit all mutations appear to be activating at the CaR, in vitro observations were not predictive of the in vivo phenotype or the response to calcilytics, suggesting that other parameters impact the response to the drug. © 2019 American Society for Bone and Mineral Research.

The calcium-sensing receptor (CaR) modulates renal calcium reabsorption and parathyroid hormone (PTH) secretion and is involved in the etiology of secondary hyperparathyroidism in CKD. Supraphysiologic changes in extracellular pH (pH o ) modulate CaR responsiveness in HEK-293 (CaR-HEK) cells. Therefore, because acidosis and alkalosis are associated with altered PTH secretion in vivo, we examined whether pathophysiologic changes in pH o can significantly alter CaR responsiveness in both heterologous and endogenous expression systems and whether this affects PTH secretion. In both CaR-HEK and isolated bovine parathyroid cells, decreasing pH o from 7.4 to 7.2 rapidly inhibited CaR-induced intracellular calcium (Ca 2+ i ) mobilization, whereas raising pH o to 7.6 potentiated responsiveness to extracellular calcium (Ca i mobilization. Intracellular pH was unaffected by acute 0.4-unit pH o changes, and the presence of physiologic albumin concentrations failed to attenuate the pH o -mediated effects. None of the individual point mutations created at histidine or cysteine residues in the extracellular domain of CaR attenuated pH o sensitivity. Finally, pathophysiologic pH o elevation reversibly suppressed PTH secretion from perifused human parathyroid cells, and acidosis transiently increased PTH secretion. Therefore, pathophysiologic pH o changes can modulate CaR responsiveness in HEK-293 and parathyroid cells independently of extracellular histidine residues. Specifically, pathophysiologic acidification inhibits CaR activity, thus permitting PTH secretion, whereas alkalinization potentiates CaR activity to suppress PTH secretion. These findings suggest that acid-base disturbances may affect the CaR-mediated control of parathyroid function and calcium metabolism in vivo.

Keratinase is a type of proteolytic enzyme with broad application in industry. The main objective of this work is the optimization of keratinase production from Bacillus sp. strain UPM-AAG1 using Plackett-Burman (PB) and central composite design (CCD) for parameters, such as pH, temperature, feather concentration, and inoculum size. The optimum points for temperature, pH, and inoculum and feather concentrations were 31.66 °C, 6.87, 5.01 (w/v), and 4.53 (w/v), respectively, with an optimum keratinase activity of 60.55 U/mL. The keratinase activity was further numerically optimized for commercial application. The best numerical solution recommended a pH of 5.84, temperature of 25 °C, inoculums’ size of 5.0 (v/v), feather concentration of 4.97 (w/v). Optimization resulted an activity of 56.218 U/mL with the desirability value of 0.968. Amino acid analysis profile revealed the presence of essential and non-essential amino acids. These properties make Bacillus sp. UPM-AAG1 a potential bacterium to be used locally for the production of keratinase from feather waste.

Molybdenum and heavy metals are toxicants that are needed to be removed from the environment as they are non-biodegradable and pose a serious threat to the ecology. A previously isolated keratin-degrading Bacillus sp. strain khayat was shown to be able to reduce molybdenum (sodium molybdate) to molybdenum blue (Mo-blue). Reduction occurred optimally at the pHs of between 5.8 and 6.8 and temperatures of between 25 and 34 °C. Glucose was the best electron donor for supporting molybdate reduction followed by sucrose, fructose, glycogen, lactose, meso-inositol and glycerol in descending order. Other requirements include a phosphate concentration between 5.0 and 7.5 mM and a molybdate concentration of between 10 and 20 mM. The absorption spectrum of the Mo-blue produced was similar to previous Mo-reducing bacterium, and closely resembles a reduced phosphomolybdate. Molybdenum reduction was inhibited by Hg (ii), Ag (i), Cu (ii), As (v) and Pb (ii) at 84.7, 78.9, 53.5, 36.8 and 27.7 %, respectively. Analysis using phylogenetic analysis resulted in a tentative identification of the bacterium as Bacillus sp. strain khayat. The bacterium was unable utilize any of the xenobiotics as sources of electron donor for reduction but the bacterium was able to grow on diesel and SDS. The ability of this bacterium to detoxify several toxicants makes this bacterium an important tool for bioremediation of multiple toxicants.

Microglial cells are the primary immune cell resident in the brain. Growing evidence indicates that microglial cells play a prominent role in alcohol-induced brain pathologies. However, alcohol-induced effects on microglial cells and the underlying mechanisms are not fully understood, and evidence exists to support generation of oxidative stress due to NADPH oxidases (NOX_-mediated production of reactive oxygen species (ROS). Here, we investigated the role of the oxidative stress-sensitive Ca2+-permeable transient receptor potential melastatin-related 2 (TRPM2) channel in ethanol (EtOH)-induced microglial cell death using BV2 microglial cells. Like H2O2, exposure to EtOH induced concentration-dependent cell death, assessed using a propidium iodide assay. H2O2/EtOH-induced cell death was inhibited by treatment with TRPM2 channel inhibitors and also treatment with poly(ADP-ribose) polymerase (PARP) inhibitors, demonstrating the critical role of PARP and the TRPM2 channel in EtOH-induced cell death. Exposure to EtOH, as expected, led to an increase in ROS production, shown using imaging of 2’,7’-dichlorofluorescein fluorescence. Consistently, EtOH-induced microglial cell death was suppressed by inhibition of NADPH oxidase (NOX) as well as inhibition of protein kinase C. Taken together, our results suggest that exposure to high doses of ethanol can induce microglial cell death via the NOX/ROS/PARP/TRPM2 signaling pathway, providing novel and potentially important insights into alcohol-induced brain pathologies.