Isolation and characterization of the mycobacterial phagosome: segregation from the endosomal/lysosomal pathway

Mycobacteria have the ability to persist within host phagocytes, and their success as intracellular pathogens is thought to be related to the ability to modify their intracellular environment. After entry into phagocytes, mycobacteria-containing phagosomes acquire markers for the endosomal pathway, but do not fuse with lysosomes. The molecular machinery that is involved in the entry and survival of mycobacteria in host cells is poorly characterized. Here we describe the use of organelle electrophoresis to study the uptake of Mycobacterium bovis bacille Calmette Guerin (BCG) into murine macrophages. We demonstrate that live, but not dead, mycobacteria occupy a phagosome that can be physically separated from endosomal/lysosomal compartments. Biochemical analysis of purified mycobacterial phagosomes revealed the absence of endosomal/lysosomal markers LAMP-1 and β-hexosaminidase. Combining subcellular fractionation with two-dimensional gel electrophoresis, we found that a set of host proteins was present in phagosomes that were absent from endosomal/lysosomal compartments. The residence of mycobacteria in compartments outside the endosomal/lysosomal system may explain their persistence inside host cells and their sequestration from immune recognition. Furthermore, the approach described here may contribute to an improved understanding of the molecular mechanisms that determine the intracellular fate of mycobacteria during infection.     

AMP-activated protein kinase is a key regulator of obesity-associated factors

An imbalance between caloric intake and energy expenditure leads to obesity. Obesity is an important risk factor for the development of several metabolic diseases including insulin resistance, metabolic syndrome, type 2 diabetes mellitus, and cardiovascular disease. So, controlling obesity could be effective in the improvement of obesity-related diseases. Various factors are involved in obesity, such as AMP-activated protein kinases (AMPK), silent information regulators, inflammatory mediators, oxidative stress parameters, gastrointestinal hormones, adipokines, angiopoietin-like proteins, and microRNAs. These factors play an important role in obesity by controlling fat metabolism, energy homeostasis, food intake, and insulin sensitivity. AMPK is a heterotrimeric serine/threonine protein kinase known as a fuel-sensing enzyme. The central role of AMPK in obesity makes it an attractive molecule to target obesity and related metabolic diseases. In this review, the critical role of AMPK in obesity and the interplay between AMPK and obesity-associated factors were elaborated.     

Time‐lapse microscopy of oxidative stress demonstrates metabolic sensitivity of retinal pericytes under high glucose condition

Hyperglycemia affects retinal vascular cell function, promotes the development and progression of diabetic retinopathy and ultimately causes vision loss. Oxidative stress, reactive oxygen species (ROS) in excess, is a key biomarker for diabetic retinopathy. Using time‐lapse fluorescence microscopy, ROS dynamics was monitored and the metabolic resistivity of retinal endothelial cells (REC) and pericytes (RPC) was compared under metabolic stress conditions including high glucose (HG). In the presence of a mitochondrial stressor, REC exhibited a significant increase in the rate of ROS production compared with RPC. Thus, under normal glucose (NG), REC may utilize oxidative metabolism as the bioenergetic source, while RPC metabolic activity is independent of mitochondrial respiration. In HG condition, the rate of ROS production in RPC was significantly higher, whereas this rate remained unchanged in REC. Thus, under HG condition RPC may preferentially utilize oxidative metabolism, which results in increased rate of ROS production. In contrast, REC use glycolysis as their major bioenergetic source for ATP production, and consequently HG minimally affects their ROS levels. These observations are consistent with our previous studies where we showed HG condition has minimal effect on apoptosis of REC, but results in increased rate of apoptosis in RPC. Collectively, our results suggest that REC and RPC exhibit different metabolic activity preferences under different glucose conditions. Thus, protection of RPC from oxidative stress may provide an early point of intervention in development and progression of diabetic retinopathy.      

Phytoremediation Effect and Growth Responses of Cynodon spp. and Agropyron desertorum in a Petroleum-Contaminated Soil

In order to compare the petroleum tolerance and phytoremediation ability of a native grass, Agropyron desertorum (desert Wheatgrass) with Cynodon spp. (Bermuda grass) in a petroleum hydrocarbon-contaminated soil, a 7-month greenhouse experiment was performed. There were 4 soil treatments with 0% (uncontaminated soil), 2%, 4%, and 12% (w_oil/w_soil) petroleum concentration. Parameters including shoot and root fresh weight and dry weight, root penetration depth and root density depth, soil respiration, and total petroleum hydrocarbons (TPH) degradation were measured during and after experiments. The results showed an increase in shoot fresh weight of A. desertorum in soil polluted with 2% petroleum sludge compared to the uncontaminated soil, whereas the growth of Bermuda grass significantly decreased in corresponding treatment. Root growth of A. desertorum was decreased in 2% and 4% petroleum sludge, whereas it was increased in Bermuda grass species. Overall, root fresh weight of Bermuda grass was higher than that of A. desertorum in all treatments. Significant increase in microorganisms' activity was observed in the presence of petroleum sludge and plants in soil compared with uncontaminated soil without plants, and the highest soil respiration (37.6 mg C-CO₂/kg soil day) has been observed in the rhizosphere of Bermuda grass in treatment with 12% petroleum sludge. Plants had a significant role in the degradation of soil contaminants as TPH degradation in planted soils was significantly higher than that in unplanted soil (TPH degradation (%) was 30.4 and 38.9 in A. desertorum and Bermuda grass, respectively, whereas it was just 13.3 in unplanted soil). The rhizosphere of Bermuda grass had significantly less residual TPHs compared to A. desertorum. The results indicated that both Cynodon spp. and A. desertorum had a peculiar tolerance to petroleum pollution. Therefore, as Bermuda grass has already been suggested to be a typical and efficient species for phytoremediating petroleum-contaminated sites, A. desertorum may also prove to be a suitable native alternative.     

Optimization of staged bioleaching of low-grade chalcopyrite ore in the presence and absence of chloride in the irrigating lixiviant: ANFIS simulation

In this investigation, copper was bioleached from a low-grade chalcopyrite ore using a chloride-containing lixiviant. In this regard, firstly, the composition of the bacterial culture media was designed to control the cost in commercial application. The bacterial culture used in this process was acclimated to the presence of chloride in the lixiviant. Practically speaking, the modified culture helped the bio-heap-leaching system operate in the chloridic media. Compared to the copper recovery from the low-grade chalcopyrite by bioleaching in the absence of chloride, bioleaching in the presence of chloride resulted in improved copper recovery. The composition of the lixiviant used in this study was a modification with respect to the basal salts in 9 K medium to optimize the leaching process. When leaching the ore in columns, 76.81 % Cu (based on solid residues of bioleaching operation) was recovered by staged leaching with lixiviant containing 34.22 mM NaCl. The quantitative findings were supported by SEM/EDS observations, X-ray elemental mapping, and mineralogical analysis of the ore before and after leaching. Finally, Adaptive neuro-fuzzy inference system (ANFIS) was used to simulate the operational parameters affecting the bioleaching operation in chloride–sulfate system.     

Reactivation of mutant p53: Constraints on mechanism highlighted by principal component analysis of the DNA binding domain

Most p53 mutations associated with cancer are located in its DNA binding domain (DBD). Many structures (X‐ray and NMR) of this domain are available in the protein data bank (PDB) and a vast conformational heterogeneity characterizes the various free and complexed states. The major difference between the apo and the holo‐complexed states appears to lie in the L1 loop. In particular, the conformations of this loop appear to depend intimately on the sequence of DNA to which it binds. This conclusion builds upon recent observations that implicate the tetramerization and the C‐terminal domains (respectively TD and Cter) in DNA binding specificity. Detailed PCA analysis of the most recent collection of DBD structures from the PDB have been carried out. In contrast to recommendations that small molecules/drugs stabilize the flexible L1 loop to rescue mutant p53, our study highlights a need to retain the flexibility of the p53 DNA binding surface (DBS). It is the adaptability of this region that enables p53 to engage in the diverse interactions responsible for its functionality. Proteins 2016; 84:1443–1461. © 2016 Wiley Periodicals, Inc.     

DFT study of the interaction of cytidine and 2′-deoxycytidine with Li, Na, and K: effects of metal cationization on sugar puckering and stability of the N-glycosidic bond

Density functional theory (DFT) calculations were performed at the B3LYP level with a 6-311++G(d,p) basis set to systematically explore the geometrical multiplicity and binding strength for complexes formed by Li⁺, Na⁺, and K⁺ with cytidine and 2′-deoxycytidine. All computational studies indicate that the metal ion affinity (MIA) decreases from Li⁺ to Na⁺ and K⁺ for cytosine nucleosides. For example, for cytidine the affinity for the above metal ions are 79.5, 55.2, and 41.8 and for 2′-deoxycytidine, 82.8, 57.4, and 42.2 kcal/mol, respectively. It is also interesting to mention that linear correlations between calculated MIA values and the atomic numbers (Z) of the above metal ions were found. The influence of metal cationization on the coordination modes and the strength of the N-glycosidic bond in cytosine nucleosides have been studied. In all cases, the N1-C1′ bond distance changes upon introducing a positive charge in the nucleosides. It has been found that metal binding significantly changes the values of the phase angle of pseudorotation P in the sugar unit of these nucleosides. With respect to the sugar ring, metal binding changes the values of the glycosyl torsion angle and sugar ring conformation. The present calculations in the gas phase provide the first clues on the intrinsic chemistry of these systems and may be of value for studies of the influence of metal cations on the conformational behavior and function of nucleic acids.     

Effects of Gibberellic Acid on Primary Terpenoids and Δ9-Tetrahydrocannabinol in Cannabis sativa at Flowering Stage

Plants synthesize an astonishing diversity of isoprenoids, some of which play essential roles in photosynthesis, respiration, and the regulation of growth and development. Two independent pathways for the biosynthesis of isoprenoid precursors coexist within the plant cell: the cytosolic mevalonic acid (MVA) pathway and the plastidial methylerythritol phosphate (MEP) pathway. However, little is known about the effects of plant hormones on the regulation of these pathways. In the present study we investigated the effect of gibberellic acid (GA₃) on changes in the amounts of many produced terpenoids and the activity of the key enzymes, 1-deoxy-D-xylulose 5-phosphate synthase (DXS) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), in these pathways. Our results showed GA₃ caused a decrease in DXS activity in both sexes that it was accompanied by a decrease in chlorophylls, carotenoids and Δ⁹-tetrahydrocannabinol (THC) contents and an increase in α-tocopherol content. The treated plants with GA₃ showed an increase in HMGR activity. This increase in HMGR activity was followed by accumulation of stigmasterol and β-sitosterol in male and female plants and campestrol in male plants. The pattern of the changes in the amounts of sterols was exactly similar to the changes in the HMGR activity. These data suggest that GA₃ can probably influence the MEP and MVA pathways oppositely, with stimulatory and inhibitory effects on the produced primary terpenoids in MVA and DXS pathways, respectively.     

Comparative ontogeny of perfect and pistillate florets in Senecio vernalis (Asteraceae)

We studied the inflorescence, and in particular ontogeny and development of the florets in Senecio vernalis as a representative member of Asteraceae, using epi-illumination microscopy. Initiation and subsequent development of florets on the highly convex inflorescence apex occur acropetally, except for pistillate ray florets, which show a lag in initiation. Receptacular bracts derive from the receptacular surface after development of all florets. The order of whorl initiation in both disc and ray florets include corolla, androecium and finally the pappus, together with the gynoecium. Development of corolla lobes from a ring meristem occurs in bidirectional order starting from the lateral side, whereas stamens incept unidirectionally from the abaxial side. Concurrently with the inception of two median carpel primordia, a ring meristem develops at the base of the corolla from which pappus bristles differentiate in later stages. Pistillate ray florets show significant differences from perfect disc florets as reflected by the zygomorphic shape of the floral apex and a shift of floral merosity from pentamery to tetramery. Loss of stamens in ray florets occurs due to abortion of primordia after initiation.