Uptake of zinc and copper by halophilic bacteria isolated from the dead sea shore,Jordan

Ten Gram-positive and gram-negative bacterial cultures were recovered from nine water, mud, and soil samples collected from the Dead Sea shore at Suwaymah. All bacterial cultures were able to grow at 10% NaCl and at 45°C. They were able to grow in nutrient media supplemented with 1250 ppm of Zn. Most of them, except cultures 2 and 8, were able to grow in nutrient medium supplemented with 1000 ppm of Cu. After 2 wk of incubation of these 10 cultures at different concentrations (5, 25, 100, and 500 ppm), stock solutions of both Zn and Cu elements, the maximum absorption using atomic absorption spectrometry for Zn was achieved by culture 7 at 11.2%, 1.0%, 38.4%, and 84.54%, respectively, from the previous stock solutions, whereas the maximum absorption of the same concentration of Cu was achieved by culture 3 at 6.2%, 55.56%, 85.66%, and 90.82%, respectively, of the different concentrations. After 3 wk of incubation, the estimated absorption for Zn was achieved by cultures 2, 9, and 10 at 19.2%, 16.68%, 42.92%, and 76.5%, 18.2%, 21.56%, 32.22%, and 77.43%, and 20.8%, 23.52%, 32.22%, and 82.84% of the previous stocks. The maximum absorption of the same concentration of Cu was achieved by culture 3 at 32.6%, 49.88%, 90.44%, and 91.86%, respectively. The accumulation of the absorbed metals was found to be maximum in the protoplast of all cultures. The accumulation at the cell wall was maximum for cultures 2 and 6 for Zn and Cu, respectively, and between the cell wall and the plasma membrane, it was maximum for cultures 2 and 8 for Zn and Cu, respectively.     

Identification of Cys385 in the isolated kinase insertion domain of heme-regulated eIF2 alpha kinase (HRI) as the heme axial ligand by site-directed mutagenesis and spectral characterization

Heme-regulated eIF2alpha kinase (HRI) is an important enzyme that modulates protein synthesis during cellular emergency/stress conditions, such as heme deficiency in red cells. It is essential to identify the heme axial ligand(s) and/or binding sites to establish the heme regulation mechanism of HRI. Previous reports suggest that a His residue in the N-terminal region and a Cys residue in the C-terminal region trans to the His are axial ligands of the heme. Moreover, mutational analyses indicate that a residue located in the kinase insertion (KI) domain between Kinase I and Kinase II domains in the C-terminal region is an axial ligand. In the present study, we isolate the KI domain of mouse HRI and employ site-directed mutagenesis to identify the heme axial ligand. The optical absorption spectrum of the Fe(III) hemin-bound wild-type KI displays a broad Soret band at around 373nm, while that of the Fe(II) heme-bound protein contains a band at 422nm. Spectral titration studies conducted for both the Fe(III) hemin and Fe(II) heme complexes with KI support a 1:1 stoichiometry of heme iron to protein. Resonance Raman spectra of Fe(III) hemin-bound KI suggest that thiol is the axial ligand in a 5-coordinate high-spin heme complex as a major form. Electron spin resonance (ESR) spectra of Fe(III) hemin-bound KI indicate that the axial ligands are OH(-) and Cys. Since Cys385 is the only cysteine in KI, the residue was mutated to Ser, and its spectral characteristics were analyzed. The Soret band position, heme spectral titration behavior and ESR parameters of the Cys385Ser mutant were markedly different from those of wild-type KI. Based on these spectroscopic findings, we conclude that Cys385 is an axial ligand of isolated KI.     

Rewired Metabolism in Drug-resistant Leukemia Cells: A METABOLIC SWITCH HALLMARKED BY REDUCED DEPENDENCE ON EXOGENOUS GLUTAMINE*

Cancer cells that escape induction therapy are a major cause of relapse. Understanding metabolic alterations associated with drug resistance opens up unexplored opportunities for the development of new therapeutic strategies. Here, we applied a broad spectrum of technologies including RNA sequencing, global untargeted metabolomics, and stable isotope labeling mass spectrometry to identify metabolic changes in P-glycoprotein overexpressing T-cell acute lymphoblastic leukemia (ALL) cells, which escaped a therapeutically relevant daunorubicin treatment. We show that compared with sensitive ALL cells, resistant leukemia cells possess a fundamentally rewired central metabolism characterized by reduced dependence on glutamine despite a lack of expression of glutamate-ammonia ligase (GLUL), a higher demand for glucose and an altered rate of fatty acid β-oxidation, accompanied by a decreased pantothenic acid uptake capacity. We experimentally validate our findings by selectively targeting components of this metabolic switch, using approved drugs and starvation approaches followed by cell viability analyses in both the ALL cells and in an acute myeloid leukemia (AML) sensitive/resistant cell line pair. We demonstrate how comparative metabolomics and RNA expression profiling of drug-sensitive and -resistant cells expose targetable metabolic changes and potential resistance markers. Our results show that drug resistance is associated with significant metabolic costs in cancer cells, which could be exploited using new therapeutic strategies.     

In-vitro diagnosis of single and poly microbial species targeted for diabetic foot infection using e-nose technology

Background

Effective management of patients with diabetic foot infection is a crucial concern. A delay in prescribing appropriate antimicrobial agent can lead to amputation or life threatening complications. Thus, this electronic nose (e-nose) technique will provide a diagnostic tool that will allow for rapid and accurate identification of a pathogen.

Results

This study investigates the performance of e-nose technique performing direct measurement of static headspace with algorithm and data interpretations which was validated by Headspace SPME-GC-MS, to determine the causative bacteria responsible for diabetic foot infection. The study was proposed to complement the wound swabbing method for bacterial culture and to serve as a rapid screening tool for bacteria species identification. The investigation focused on both single and poly microbial subjected to different agar media cultures. A multi-class technique was applied including statistical approaches such as Support Vector Machine (SVM), K Nearest Neighbor (KNN), Linear Discriminant Analysis (LDA) as well as neural networks called Probability Neural Network (PNN). Most of classifiers successfully identified poly and single microbial species with up to 90% accuracy.

Conclusions

The results obtained from this study showed that the e-nose was able to identify and differentiate between poly and single microbial species comparable to the conventional clinical technique. It also indicates that even though poly and single bacterial species in different agar solution emit different headspace volatiles, they can still be discriminated and identified using multivariate techniques.     

Physico-mechanical properties of chemically treated palm and coir fiber reinforced polypropylene composites

In this work, palm and coir fiber reinforced polypropylene bio-composites were manufactured using a single extruder and injection molding machine. Raw palm and coir were chemically treated with benzene diazonium salt to increase their compatibility with the polypropylene matrix. Both raw and treated palm and coir fiber at five level of fiber loading (15, 20, 25, 30 and 35 wt.%) was utilized during composite manufacturing. Microstructural analysis and mechanical tests were conducted. Comparison has been made between the properties of the palm and coir fiber composites. Treated fiber reinforced specimens yielded better mechanical properties compared to the raw composites, while coir fiber composites had better mechanical properties than palm fiber ones. Based on fiber loading, 30% fiber reinforced composites had the optimum set of mechanical properties.     

KNApSAcK family databases: integrated metabolite-plant species databases for multifaceted plant research

A database (DB) describing the relationships between species and their metabolites would be useful for metabolomics research, because it targets systematic analysis of enormous numbers of organic compounds with known or unknown structures in metabolomics. We constructed an extensive species-metabolite DB for plants, the KNApSAcK Core DB, which contains 101,500 species-metabolite relationships encompassing 20,741 species and 50,048 metabolites. We also developed a search engine within the KNApSAcK Core DB for use in metabolomics research, making it possible to search for metabolites based on an accurate mass, molecular formula, metabolite name or mass spectra in several ionization modes. We also have developed databases for retrieving metabolites related to plants used for a range of purposes. In our multifaceted plant usage DB, medicinal/edible plants are related to the geographic zones (GZs) where the plants are used, their biological activities, and formulae of Japanese and Indonesian traditional medicines (Kampo and Jamu, respectively). These data are connected to the species-metabolites relationship DB within the KNApSAcK Core DB, keyed via the species names. All databases can be accessed via the website http://kanaya.naist.jp/KNApSAcK_Family/. KNApSAcK WorldMap DB comprises 41,548 GZ-plant pair entries, including 222 GZs and 15,240 medicinal/edible plants. The KAMPO DB consists of 336 formulae encompassing 278 medicinal plants; the JAMU DB consists of 5,310 formulae encompassing 550 medicinal plants. The Biological Activity DB consists of 2,418 biological activities and 33,706 pairwise relationships between medicinal plants and their biological activities. Current statistics of the binary relationships between individual databases were characterized by the degree distribution analysis, leading to a prediction of at least 1,060,000 metabolites within all plants. In the future, the study of metabolomics will need to take this huge number of metabolites into consideration.     

Adaptation of <Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> to Thermal Stress Yields a Thermotolerant Variant Which Also Exhibits Improved Survival at pH 2

Loss in probiotic viability upon exposure to stressful storage and transport conditions has plagued the probiotic market worldwide. Lactobacillus acidophilus is an important probiotic that is added to various functional foods. It is known to be fairly labile and susceptible to temperature variations that it encounters during processing and storage which increases production cost. It has been repeatedly demonstrated that pre-exposure to sub-lethal doses of stress, particularly, temperature and pH, leads to improved survival of various probiotics when they subsequently encounter the same stress of a much greater magnitude. Attempts to adapt L. acidophilus to temperatures as high as 65 °C to arrive at a thermotolerant variant have not been reported previously. To improve viability at elevated temperatures, we gradually adapted the L. acidophilus NCFM strain to survival at 65 °C for 40 min. Following adaptation, the variant showed a 2-log greater survival compared to wild-type at 65 °C. Interestingly, this thermotolerant variant also demonstrated a 2-log greater stability compared to wild-type at pH 2.0. The improved pH and temperature stress tolerance exhibited by this variant remained unaltered even when the strain was lyophilized. Moreover, the thermotolerant variant demonstrated improved stability compared to wild-type when stored for up to a week at 37 and 42 °C. Probiotic properties of the variant such as adherence to epithelial cells and antibacterial activity remained unaltered. This strain can potentially help address the issue of significant loss in viable cell counts of L. acidophilus which is typically encountered during probiotic manufacture and storage.     

Evaluation of Neurotransmitter Alterations in Four Distinct Brain Regions After Rapid Eye Movement Sleep Deprivation (REMSD) Induced Mania-Like Behaviour in Swiss Albino Mice

A number of neurotransmitter systems have been implicated in contributing to the pathology of mood disorders, including those of dopamine (DA), serotonin (5-HT), norepinephrine (NE) and γ-aminobutyric acid (GABA). Rapid eye movement sleep deprivation (REMSD) alters most of the neurotransmitters, which may have adverse behavioural changes and other health consequences like mania and other psychiatric disorders. The exact role of REMSD altered neurotransmitter levels and the manner in which emerging consequences lead to mania-like behaviour is poorly understood. Thus, we sought to verify the levels of neurotransmitter changes after 48, 72 and 96 h of REMSD induced mania-like behaviour in mice. We performed modified multiple platform (MMP) method of depriving the REM sleep and one group maintained as a control. To measure the hyperactivity through locomotion, exploration and behavioural despair, we performed the Open Field Test (OFT) and the Forced Swim Test (FST). Quantitative determinations of DA, 5-HT, NE and GABA concentrations in four distinct brain regions (cerebral cortex, hippocampus, midbrain, and pons) were determined by the spectrofluorimetric method. These experiments showed higher locomotion and increased swimming, struggling/climbing and decreased mobility among REMSD animals as well as disrupted concentrations of the majority of the studied neurotransmitters during REMSD. Our study indicated that REMSD results in mania-like behaviour in mice and associated disruption to neurotransmitter levels, although the exact mechanisms by which these take place remain to be determined.