Isolation and Identification of Bacillus thuringiensis from Aedesaegypti Larvae as Potential Source of Endotoxin to Control Dengue Vectors

Dengue is an emergent disease transmitted by Aedes aegypti mosquitos prominent in tropical countries. Numerous methods have been used to prevent the spread of Dengue fever, such as fogging and treatment using anti-larvae chemicals, yet these methods are harmful. Bacillus thuringiensis found in Aedes aegypti larvae is capable of producing endotoxin that able to kill insects without any side effect on humans, thus it is able to control Dengue vectors without any adverse effects to the environment. Aedes aegypti larvae were crushed and mixed with saline solution to isolate the bacteria in the larvae. From all bacterial colonies extracted from the larvae, 13 colonies with appearance closest to Bacillus colonies were screened using gram staining, spore staining, and biochemical testing. From 13 colonies, 8 of them were further analysed using ARDRA and cry1A gene amplification. These analyses showed one of the colonies had cry1A gene, which indicated the colony was Bacillus thuringiensis. The isolated Bacillus thuringiensis was used for endotoxin production and efficacy assay

X-ray measurement of intracellular chloride and other ions in mammalian cells

Intracellular chloride ion (Cl−) is known to have various roles in the cellular growth, modulation of cell cycles and volume, and in pathological disorders such as cancers, neurological and cardiovascular disorders. Various methods employed to measure intracellular Cl− requires lengthy procedure and measurement duration, bulky and advanced instruments, or incapable to measure low to single-cell sample quantity. In this study, we report the measurement of intracellular chloride and other ions in mammalian cells using the FROZEN freeze-drying method for preparing adherent cell cultures for analysis. The sample preparation does not require chemical fixation and enables direct measurement by X-Ray analytical methods such as X-Ray Fluorescence (XRF) or Energy Dispersive X-Ray Fluorescence (EDS). Cell staining showed that the cells retained their content and location after preparation, which enables single-cell level analysis. Treatment of Furosemide to the cells disrupted the Cl−, K+ and Na+ transport, causing changes in the intracellular ions. Additionally, the intracellular Cl− of the dried cells was successfully measured, as low as 0.9 mM. The XRF and EDS measurements showed that treated cells displayed a significant reduction of intracellular Cl− on all tested cell lines. However, no significant changes were detected in intracellular K+. Furthermore, EDS analysis on single cells showed a significant decrease in intracellular Na+ in most cell lines. These results demonstrate the efficiency and simplicity of the proposed method in intracellular ion analysis, allowing quick and simple preparation to measure biological samples with high sensitivity for various intracellular ions, which may be applicable in the diagnosis of diseases

Intracellular and extracellular ion variation measurement in alpha(α)-particle irradiated cells using t-FROZEN! and laser-induced breakdown spectroscopy

Many radiobiological changes occur in unirradiated cells due to concealed signals transmitted from the irradiated cells through the Radiation-induced bystander effect (RIBE). Calcium ions (Ca2+) remain the only ion–molecule extensively studied so far amongst the ions that control cell function. This is partly due to using fluorescent indicators to investigate the signaling process that suffers limitations in labelling procedure and ion specificity. To understand the ion imbalance in irradiated cells and cell medium serving as donor and carrier of the bystander signals. Here, we investigate ion concentrations in the extracellular and intracellular space of α-particle (Am-241) irradiated Chinese hamster ovary (CHO) cells exposed to two different radiation doses (0.3 nGy, and 0.7 nGy). Laser-induced breakdown spectroscopy (LIBS) with our novel t-FROZEN! method separating the cells from its extracellular matrix was employed as a tool. Also, X-ray fluorescence spectroscopy (XRF), inductively coupled plasma-optical emission spectrometry (ICP-OES), and electrical impedance measurements were employed as complementary techniques. Our results show that influx of K+ and outflux of Na+, Mg2+, Cl- and Ca2+ occur in the irradiated cells, compared with controls, leading to an imbalance in the concentration of crucial signaling ions across the cell membrane. Our study has provided vital insights into comprehensive composition of ion-based radiation-induced bystander signals and guide the development of Na+, Mg2+, Cl- and K+ channel targeting drugs

Lithium in breast milk transiently affects the renal electrolytic balance of infants

Background: The use of lithium during breast-feeding has not been comprehensively investigated in humans due to concerns about lithium toxicity. Procedure: We analyzed lithium in the kidneys of nursed pups of lithium medicated mothers, using analytical spectroscopy in a novel rat model. The mothers were healthy rats administered lithium via gavage (1000 mg/day Li2CO3 per 50 kg body weight). Results: Lithium was detected in the breast milk, and in the blood of pups (0.08 mM), of lithium-exposed dams at post-natal day 18 (P18), during breast-feeding. No lithium was detected after breast-feeding, at P25 (4 days after cessation of nursing). The lithium pups blood had elevated urea nitrogen at P18 and reduced total T4 at P18 and P25, indicating a longer-term effect on the kidneys and the thyroid gland. Multivariate machine-learning analysis of spectroscopy data collected from the excised kidneys of pups showed elevated potassium in lithium-exposed animals both during- and after breast-feeding. The elevated renal potassium was associated with low nephrin expression in the kidneys measured immunohistochemically during breast-feeding. After lithium exposure is stopped, the filtration of lithium from the kidneys reverses these effects. Our study showed that breastfeeding during lithium use has an effect on the kidneys of the offspring in rats