Role of Ca2+ in H+ transport by rabbit gastric glands studied with A23187 and BAPTA, an incorporated Ca2+ chelator

The role of Ca2+ in stimulation of H+ gastric secretion by cAMP-dependent and -independent secretagogues was studied in isolated rabbit glands using Ca2+ ionophore, A23187, and an intracellular Ca2+ chelator (BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) incorporated as its acetoxymethyl ester (BAPTA-AM). Acetylcholine (ACh), tetragastrin (TG), histamine and forskolin induced a transitory increase of intracellular Ca2+ concentration, [Ca2+]i, measured in gastric glands loaded with Ca2+-sensitive dye fura-2, and provoked an acid secretory response evaluated with aminopyrine accumulation ratio (AP ratio). The Ca2+-ionophore A23187 also induced an increase in [Ca2+]i and in AP ratio. cAMP-dependent secretagogues were more potent stimulants of acid secretion than cAMP-independent secretagogues. cAMP analogue, 8-bromo-adenosine 3',5'-cyclic monophosphate (8-BR-cAMP) induced an increase in AP ratio without modifying [Ca2+]i. BAPTA-AM (5-25 microM) induced a transient decrease of resting [Ca2+]i which returned to basal level due to extracellular Ca2+ entry. Increases in [Ca2+]i produced by ACh and TG were abolished by BAPTA and those produced by Ca2+ ionophore A23187 were partially buffered. BAPTA inhibited in a dose-dependent manner H+ secretion induced by cholinergic and gastrinergic stimulants in the presence of cimetidine. A23187 increased the AP ratio to values similar to those obtained with ACh or TG and was not inhibited by BAPTA. BAPTA partially inhibited (40%) the increase in AP ratio induced by forskolin and histamine inspite of the complete inhibition of the Ca2+ response. BAPTA did not inhibit the response to 8-BR-cAMP. BAPTA inhibition of forskolin stimulation was reversed by A23187 and the response was potentiated. These results indicate that ACh and TG response are completely dependent on an increase of [Ca2+]i. The response to cAMP-dependent agonists histamine and forskolin depend both on Ca2+ and cAMP. For forskolin stimulation the response may be the result of a potentiation between Ca2+ and cAMP.     

Preparation of rat liver plasma membrane with respect to glucagon-sensitive adenylate cyclase

Further modification of Neville's method of preparation of rat liver plasma membrane has been made in order to study glucagon-sensitive adenylate cyclase. This modified method introduces two additional steps to the Neville procedures. One involves saving an intermediate layer above the pellet following 1500g centrifugation. The other adds a centrifugation step at 20,000g. The improved method increases the yield of membrane protein by 5-fold and increases forskolin, glucagon, and 5'-guanyl imidodiphosphate stimulated activity of adenylate cyclase by 2.8-fold. A 13-fold increase in the yield of total adenylate cyclase activity above the current method was obtained. The membrane adenylate cyclase and its hormone sensitivity was stable in liquid nitrogen for at least 8 months. This modified method appears to be useful in preparing a better yield and quality of rat liver plasma membrane from given starting hepatic tissue for studies of glucagon-sensitive adenylate cyclase.     

Effects of stilbene optical brighteners on the insecticidal activity of Bacillus thuringiensis and a single nucleopolyhedrovirus on Helicoverpa armigera

The incorporation of certain stilbene optical brighteners into virus-based formulations has been demonstrated to increase viral pathogenicity (as indicated by reduced LD/LC₅₀ values) but their effect on Bacillus thuringiensis activity has been scarcely investigated. We determined the effect of nine optical brighteners on the insecticidal activity of B. thuringiensis ser. kurstaki HD-1 strain (Bt HD-1) on Helicoverpa armigera and also compared the effect of two optical brighteners on the insecticidal activity of Bt HD-1 and occlusion bodies (OBs) of a Spanish isolate of H. armigera single nucleocapsid nucleopolyhedrovirus (HearNPV-SP1). Blankophor CLE, Blankophor DRS, Blankophor ER, and Leucophor SAC significantly increased the pathogenicity of Bt HD-1. In contrast, Tinopal UNPA-GX, Tinopal CBS, Blankophor BA, Leucophor AP, and Leucophor UO had an adverse or no effect on its insecticidal activity. Mixtures of HearNPV-SP1 OBs with Tinopal UNPA-GX or Leucophor UO resulted in 31.4- and 11.4-fold increases in pathogenicity, respectively, at 1%, and 11.4- and 6.3-fold increases in pathogenicity, respectively, at 0.1%, compared to the OBs alone. However, none of these brighteners increased Bt HD-1 activity. These results appear consistent with the hypothesis that the enhancement of HearNPV-SP1 pathogenicity and the null or antagonistic effects observed in Bt HD-1 against H. armigera were due to optical brightener-mediated degradation of the peritrophic membrane, but additional systematic studies involving a broad range of brighteners and electron microscope observations are required to confirm this premise.     

Controlled gene expression in bifidobacteria by use of a bile-responsive element

The promoter activity of the upstream region of the bile-inducible gene betA from Bifidobacterium longum subsp. longum NCC2705 was characterized. DNA fragments were cloned into the reporter vector pMDYAbfB, and the arabinofuranosidase activity was determined under different in vitro conditions. A segment of 469 bp was found to be the smallest operational unit that retains bile inducibility. The reporter activity was strongly affected by the presence of ox gall, cholate, and conjugated cholate, but not by other bile salts and cell-surface-acting compounds. Remarkably, this bile-inducible system was also active in other bifidobacteria containing betA homologs.     

An artificial intelligence model to identify snakes from across the world: Opportunities and challenges for global health and herpetology

BackgroundSnakebite envenoming is a neglected tropical disease that kills an estimated 81,000 to 138,000 people and disables another 400,000 globally every year. The World Health Organization aims to halve this burden by 2030. To achieve this ambitious goal, we need to close the data gap in snake ecology and snakebite epidemiology and give healthcare providers up-to-date knowledge and access to better diagnostic tools. An essential first step is to improve the capacity to identify biting snakes taxonomically. The existence of AI-based identification tools for other animals offers an innovative opportunity to apply machine learning to snake identification and snakebite envenoming, a life-threatening situation.MethodologyWe developed an AI model based on Vision Transformer, a recent neural network architecture, and a comprehensive snake photo dataset of 386,006 training photos covering 198 venomous and 574 non-venomous snake species from 188 countries. We gathered photos from online biodiversity platforms (iNaturalist and HerpMapper) and a photo-sharing site (Flickr).Principal findingsThe model macro-averaged F1 score, which reflects the species-wise performance as averaging performance for each species, is 92.2%. The accuracy on a species and genus level is 96.0% and 99.0%, respectively. The average accuracy per country is 94.2%. The model accurately classifies selected venomous and non-venomous lookalike species from Southeast Asia and sub-Saharan Africa.ConclusionsTo our knowledge, this model’s taxonomic and geographic coverage and performance are unprecedented. This model could provide high-speed and low-cost snake identification to support snakebite victims and healthcare providers in low-resource settings, as well as zoologists, conservationists, and nature lovers from across the world.     

Quantification of Mesenchymal Stem Cell (MSC) Delivery to a Target Site Using In Vivo Confocal Microscopy

The ability to deliver cells to appropriate target tissues is a prerequisite for successful cell-based therapy. To optimize cell therapy it is therefore necessary to develop a robust method of in vivo cell delivery quantification. Here we examine Mesenchymal Stem Cells (MSCs) labeled with a series of 4 membrane dyes from which we select the optimal dye combination for pair-wise comparisons of delivery to inflamed tissue in the mouse ear using confocal fluorescence imaging. The use of an optimized dye pair for simultaneous tracking of two cell populations in the same animal enables quantification of a test population that is referenced to an internal control population, thereby eliminating intra-subject variations and variations in injected cell numbers. Consistent results were obtained even when the administered cell number varied by more than an order of magnitude, demonstrating an ability to neutralize one of the largest sources of in vivo experimental error and to greatly reduce the number of cells required to evaluate cell delivery. With this method, we are able to show a small but significant increase in the delivery of cytokine pre-treated MSCs (TNF-α & IFN-γ) compared to control MSCs. Our results suggest future directions for screening cell strategies using our in vivo cell delivery assay, which may be useful to develop methods to maximize cell therapeutic potential.