Phosphatase Inhibitors Function as Novel,Broad Spectrum Botulinum Neurotoxin Antagonists in Mouse and Human Embryonic Stem Cell-Derived Motor Neuron-Based Assays

There is an urgent need to develop novel treatments to counter Botulinum neurotoxin (BoNT) poisoning. Currently, the majority of BoNT drug development efforts focus on directly inhibiting the proteolytic components of BoNT, i.e. light chains (LC). Although this is a rational approach, previous research has shown that LCs are extremely difficult drug targets and that inhibiting multi-serotype BoNTs with a single LC inhibitor may not be feasible. An alternative approach would target neuronal pathways involved in intoxication/recovery, rather than the LC itself. Phosphorylation-related mechanisms have been implicated in the intoxication pathway(s) of BoNTs. However, the effects of phosphatase inhibitors upon BoNT activity in the physiological target of BoNTs, i.e. motor neurons, have not been investigated. In this study, a small library of phosphatase inhibitors was screened for BoNT antagonism in the context of mouse embryonic stem cell-derived motor neurons (ES-MNs). Four inhibitors were found to function as BoNT/A antagonists. Subsequently, we confirmed that these inhibitors protect against BoNT/A in a dose-dependent manner in human ES-MNs. Additionally, these compounds provide protection when administered in post-intoxication scenario. Importantly, the inhibitors were also effective against BoNT serotypes B and E. To the best of our knowledge, this is the first study showing phosphatase inhibitors as broad-spectrum BoNT antagonists.     

Age-related regulation of excitation–contraction coupling in rat heart

Hearts from subjects with different ages have different Ca²⁺ signaling. Release of Ca²⁺ from intracellular stores in response to an action potential initiates cardiac contraction. Both depolarization-stimulated and spontaneous Ca²⁺ releases, Ca²⁺ transients and Ca²⁺ sparks, demonstrate the main events of excitation–contraction coupling (ECC). Global increase in free Ca²⁺ concentration ([Ca²⁺] _i ) consists of summation of Ca²⁺ release events in cardiomyocytes. Since the Ca²⁺ flux induced by Ca²⁺ sparks reports a summation of ryanodine-sensitive Ca²⁺ release channels (RyR2s)’s behavior in a spark cluster, evaluation of the properties of Ca²⁺ sparks and Ca²⁺ transients may provide insight into the role of RyR2s on altered heart function between 3-month-old (young adult) and 6-month-old (mature adult) rats. Basal [Ca²⁺] _i and Ca²⁺ sparks frequency were significantly higher in mature adult rats compared to those of young adults. Moreover, amplitudes of Ca²⁺ sparks and Ca²⁺ transients were significantly smaller in mature adults than those of young adults with longer time courses. A smaller L-type Ca²⁺ current density and decreased SR Ca²⁺ load was observed in mature adult rats. In addition, RyR2s were markedly hyperphosphorylated, and phosphorylation levels of PKA and CaMKII were higher in heart from mature adults compared to those of young adults, whereas their SERCA protein levels were similar. Our data demonstrate that hearts from rats with different ages have different Ca²⁺ signaling including hyperphosphorylation of RyR2s and higher basal [Ca²⁺] _i together with increased oxidized protein-thiols in mature adult rats compared to those of young adults, which play important roles in ECC. Finally, we report that ECC efficiency changes with age during maturation, partially related with an increased cellular oxidation level leading to reduced free protein-thiols in cardiomyocytes.     

Profound cardioprotection with timolol in a female rat model of aging-related altered left ventricular function

Increasing evidence shows a marked beneficial effect with β-blockers in heart dysfunction via scavenging reactive oxygen species. Previously we showed that chronic treatment with either timolol or propranolol possessed similar beneficial effects for heart function in male rats as age increased, whereas only timolol exerted similar benefits in female rats. Therefore, in this study, we aimed first to examine the cellular bases for age-related alterations in excitation-contraction coupling in ventricular myocytes from female rats and, second, to investigate the hypothesis that age-related changes in [Ca(2+)](i) homeostasis and receptor-mediated system can be prevented with chronic timolol treatment. Chronic timolol treatment of 3-month-old female rats abolished age-related decrease in left ventricular developed pressure and the attenuated responses to β-adrenoreceptor stimulation. It also normalized the altered parameters of [Ca(2+)](i) transients, decreased Ca(2+) loading of sarcoplasmic reticulum and increased basal [Ca(2+)](i), and decreased L-type Ca(2+) currents in 12-month-old female rats compared with the 3-month-old group. Adenylyl cyclase activity, β-adrenoreceptor affinity to its agonist, and β-adrenoreceptor density of the 12-month-old group are normalized to those of the 3-month-old group. Moreover, timolol treatment prevented dysfunction of the antioxidant system, including increased lipid peroxidation, decreased ratio of reduced glutathione to oxidized glutathione, and decreased activities of thioredoxin reductase and glucose-6-phosphate dehydrogenase, in the left ventricle of hearts from the 12-month-old group. Our data confirmed that aging-related early myocardial impairment is primarily related to a dysfunctional antioxidant system and impairment of Ca(2+) homeostasis, which can be prevented with chronic timolol treatment.     

Medium optimization and kinetic modeling for the production of Aspergillus niger inulinase

Bioprocess and Biosystems Engineering - The goals of this study were to optimize the medium formulation for enhanced production of Aspergillus niger inulinase using Plackett–Burman Design...     

Cardioprotective effect of propranolol on diabetes-induced altered intracellular Ca2+ signaling in rat

We have previously shown that chronic treatment with propranolol had beneficial effects on heart function in rats during increasing-age in a gender-dependent manner. Herein, we hypothesize that propranolol would improve cardiac function in diabetic cardiomyopathy and investigated the benefits of chronic oral administration of propranolol on the parameters of Ca²⁺ signaling in the heart of streptozotocin-diabetic rats. Male diabetic rats received propranolol (25 mg/kg, daily) for 12 weeks, 1 week after diabetes induction. Treatment of the diabetic rats with propranolol did not produce a hypoglycaemic effect whereas it attenuated the increased cell size. Basal and β-agonist response levels of left ventricular developed pressure were significantly higher in propranolol-treated diabetic rats relative to untreated diabetics while left ventricular end diastolic pressure of the treated diabetics was comparable to the controls. Propranolol treatment normalized also the prolongation of the action potential in papillary muscles from the diabetic rat hearts. This treatment attenuated the parameters of Ca²⁺ transients, depressed Ca²⁺ loading of the sarcoplasmic reticulum, and of the basal intracellular Ca²⁺ level of diabetic cardiomyocytes. Furthermore, Western blot data indicated that the diabetes-induced alterations in the cardiac ryanodine receptor Ca²⁺ release channel’s hyperphosphorylation decreased the FKBP12.6 protein level. Also, the high phosphorylated levels of PKA and CaMKII were prevented with propranolol treatment. Chronic treatment with propranolol seems to prevent diabetes-related changes in heart function by controlling intracellular Ca²⁺ signaling and preventing the development of left ventricular remodeling in diabetic cardiomyopathy.     

A discriminative method for remote homology detection based on n-peptide compositions with reduced amino acid alphabets

In this study, n-peptide compositions are utilized for protein vectorization over a discriminative remote homology detection framework based on support vector machines (SVMs). The size of amino acid alphabet is gradually reduced for increasing values of n to make the method to conform with the memory resources in conventional workstations. A hash structure is implemented for accelerated search of n-peptides. The method is tested to see its ability to classify proteins into families on a subset of SCOP family database and compared against many of the existing homology detection methods including the most popular generative methods; SAM-98 and PSI-BLAST and the recent SVM methods; SVM-Fisher, SVM-BLAST and SVM-Pairwise. The results have demonstrated that the new method significantly outperforms SVM-Fisher, SVM-BLAST, SAM-98 and PSI-BLAST, while achieving a comparable accuracy with SVM-Pairwise. In terms of efficiency, it performs much better than SVM-Pairwise. It is shown that the information of n-peptide compositions with reduced amino acid alphabets provides an accurate and efficient means of protein vectorization for SVM-based sequence classification.     

Sex-related effects on diabetes-induced alterations in calcium release in the rat heart

The present study was designed to determine whether the properties of local Ca(2+) release and its related regulatory mechanisms might provide insight into the role of sex differences in heart functions of control and streptozotocin-induced diabetic adult rats. Left ventricular developed pressure, the rates of pressure development and decay (+/-dP/dt), basal intracellular Ca(2+) level ([Ca(2+)](i)), and spatiotemporal parameters of [Ca(2+)](i) transients were found to be similar in male and female control rats. However, spatiotemporal parameters of Ca(2+) sparks in cardiomyocytes isolated from control females were significantly larger and slower than those in control males. Diabetes reduced left ventricular developed pressure to a lower extent in females than in males, and the diabetes-induced depressions in both +dP/dt and -dP/dt were less in females than in males. Diabetes elicited a smaller reduction in the amplitude of [Ca(2+)](i) transients in females than in males, a smaller reduction in sarcoplasmic reticulum-Ca(2+) load, and less increase in basal [Ca(2+)](i). Similarly, the elementary Ca(2+) events and their control proteins were clearly different in both sexes, and these differences were more marked in diabetes. Diabetes-induced depression of the Ca(2+) spark amplitude was significantly less in females than in matched males. Levels of cardiac ryanodine receptors (RyR2) and FK506-binding protein 12.6 in control females were significantly higher than those shown in control males. Diabetes induced less RyR2 phosphorylation and FK506-binding protein 12.6 unbinding in females. Moreover, total and free sulfhydryl groups were significantly less reduced, and PKC levels were less increased, in diabetic females than in diabetic males. The present data related to local Ca(2+) release and its related proteins describe some of the mechanisms that may underlie sex-related differences accounting for females to have less frequent development of cardiac diseases.     

Gender related differential effects of Omega-3E treatment on diabetes-induced left ventricular dysfunction

The present study was designed to determine whether there are beneficial effects of intake of Ω-3E (containing 70% pure omega-3 and 2% natural vitamin E) in cardiac dysfunction of diabetic rats. We also examined whether there are gender-related differences in the responses to the intake of Ω-3E on the heart dysfunction. Experiments were performed by using Langendorff-perfused hearts from normal, diabetic (with 50 mg/kg streptozotocin), and Ω-3E (50 mg/kg body weight/day) treated diabetic 3-month-old Wistar rats. Ω-3E treatment of the diabetics caused small, but significant decrease (13% and 14% female versus male) in the blood glucose level. Ω-3E treatment of the diabetic female rats did not prevent diabetes-induced decrease in left ventricular developed pressure (LVDP) and increase in left ventricular end-diastolic pressure (LVEDP) with respect to the control female rats. On the other hand, the treatment of diabetic male rats caused significant recovery in depressed LVDP. Furthermore, such treatment of diabetic female and male rats caused significant recovery in depressed rates of changes of developed pressure. This effect was more significant in males. Besides, Ω-3E caused significant further lengthening in the diabetes-induced increased time to the peak of the developed pressure in females, while it normalized the lengthening in the relaxation of the developed pressure in diabetic males. In addition, Ω-3E treatment caused significant restorations in the diabetes-induced altered activities of antioxidant enzymes without any significant gender discrepancy. Present data show that there are gender related differences in diabetic heart dysfunction and the response to antioxidant treatment.     

Neural network prediction of thermophilic (65 degrees C) sulfidogenic fluidized-bed reactor performance for the treatment of metal-containing wastewater

The performance of a fluidized-bed reactor (FBR) based sulfate reducing bioprocess was predicted using artificial neural network (ANN). The FBR was operated at high (65 degrees C) temperature and it was fed with iron (40-90 mg/L) and sulfate (1,000-1,500 mg/L) containing acidic (pH = 3.5-6) synthetic wastewater. Ethanol was supplemented as carbon and electron source for sulfate reducing bacteria (SRB). The wastewater pH of 4.3-4.4 was neutralized by the alkalinity produced in acetate oxidation and the average effluent pH was 7.8 +/- 0.8. The oxidation of acetate is the rate-limiting step in the sulfidogenic ethanol oxidation by thermophilic SRB, which resulted in acetate accumulation. Sulfate reduction and acetate oxidation rates showed variation depending on the operational conditions with the maximum rates of 1 g/L/d (0.2 g/g volatile solids (VS)/d) and 0.3 g/L/d (0.06 g/g VS/d), respectively. This study presents an ANN model predicting the performance of the reactor and determining the optimal architecture of this model; such as best back-propagation (BP) algorithm and neuron numbers. The Levenberg-Marquardt algorithm was selected as the best of 12 BP algorithms and optimal neuron number was determined as 20. The developed ANN model predicted acetate (R=0.91), sulfate (R=0.95), sulfide (R=0.97), and alkalinity (R=0.94) in the FBR effluent. Hence, the ANN based model can be used to predict the FBR performance, to control the operational conditions for improved process performance.     

UV-C irradiation reduces microbial populations and deterioration in Cucurbita pepo fruit tissue

Tissue slices of zucchini squash (Cucurbita pepo L., cv. Tigress) fruit were exposed to ultraviolet-C (UV-C) radiation from germicidal lamps for 1, 10 or 20 min; however, only 10 and 20 min UV-C exposure significantly reduced microbial activity and deterioration during subsequent storage at 5 or 10 degrees C. UV-C treated slices had higher respiration rates than controls; however, the ethylene production of the slices was not affected by UV-C treatments. Slight UV-C irradiation damage (reddish brown discoloration) was detected on the surface of 10 and 20 min-treated slices after 12 days of storage at 10 degrees C. Slices stored at 5 degrees C did not show UV-C damage. Chilling injury was not observed until after 20 days of storage at 5 degrees C. The symptoms of chilling injury appeared as dried sunken brown spots on the surface of cortex tissue. UV-C treatments did not affect the degree of chilling injury during storage at 5 degrees C. UV-C treatment also had no consistent effect on sugar or malic acid concentrations. The most pronounced effect of UV-C irradiation was to retard microbial growth thereby providing a basis for the frequently observed delay in senescence and subsequent deterioration in fruit tissues.