Hepatic overexpression of murine Abcb11 increases hepatobiliary lipid secretion and reduces hepatic steatosis

Abcb11 encodes for the liver bile salt export pump, which is rate-limiting for hepatobiliary bile salt secretion. We employed transthyretin-Abcb11 and BAC-Abcb11 transgenes to develop mice overexpressing the bile salt export pump in the liver. The mice manifest increases in bile flow and biliary secretion of bile salts, phosphatidylcholine, and cholesterol. Hepatic gene expression of cholesterol 7alpha-hydroxylase and ileal expression of the apical sodium bile salt transporter are markedly reduced, whereas gene expression of targets of the nuclear bile salt receptor FXR (ileal lipid-binding protein, short heterodimer partner (SHP) is increased. Because these changes in gene expression are associated with an increased overall hydrophobicity of the bile salt pool and a 4-fold increase of the FXR ligand taurodeoxycholate, they reflect bile salt-mediated regulation of FXR and SHP target genes. Despite the increased biliary secretion of bile salts, fecal bile salt excretion is unchanged, suggestive of an enhanced enterohepatic cycling of bile salts. Abcb11 transgenic mice fed a lithogenic (high cholesterol/fat/cholic acid) diet display markedly reduced hepatic steatosis compared with wild-type controls. We conclude that mice overexpressing Abcb11 display an increase in biliary bile salt secretion and taurodeoxycholate content, which is associated with FXR/SHP-mediated changes in hepatic and ileal gene expression. Because these mice are resistant to hepatic lipid accumulation, regulation of Abcb11 may be important for the pathogenesis and treatment of steatohepatitis.     

Fungal metabolites of (E)-guggulsterone and their antibacterial and radical-scavenging activities

Fungal transformation of (E)-guggulsterone (= (17E)-pregna-4,17-diene-3,16-dione; 1) by Rhizopus stolonifer, Fusarium lini, Cunninghamella elegans, or Gibberella fujikuroi afforded ten hydroxylated metabolites (2-11; Scheme), which were fully characterized. Compounds 4-11 have not been described yet. Some of these novel hydroxylated metabolites, as well as acetylated derivatives thereof, exhibited significant antibacterial and radical-scavenging activities (Table 3).     

Maternal cigarette smoking during pregnancy and offspring DNA methylation in midlife

Maternal smoking in pregnancy (MSP) has been associated with DNA methylation in specific CpG sites (CpGs) in infants and children. We investigated whether MSP, independent of own personal active smoking, was associated with midlife DNA methylation in CpGs that were previously identified in studies of MSP-DNA methylation in children. We used data on MSP collected from pregnant mothers of 89 adult women born in 1959–1964 and measured DNA methylation in blood (granulocytes) collected in 2001–2007 (mean age: 43 years). Seventeen CpGs were differentially methylated by MSP, with multiple CpGs mapping to CYP1A1, MYO1G, AHRR, and GFI1. These associations were consistent in direction with prior studies (e.g., MSP associated with more and less methylation in AHRR and CYP1A1, respectively) and, with the exception of AHRR CpGs, were not substantially altered by adjustment for active smoking. These preliminary results confirm prior prospective reports that MSP influences the offspring DNA methylation, and extends the timeframe to midlife, and suggest that these effects may persist into adulthood, independently of active smoking.     

Studies on Degradation of 7-ketocholesterol by Environmental Bacterial Isolates

Medical bioremediation is a unique strategy of targeting pathogenic compounds with an exogenous enzyme of microbial origin. The objective of this study was to isolate and screen the microorganisms from diverse environmental samples for their ability to catabolize 7-ketocholesterol. Isolation of bacterial strains was performed and molecular identification was carried out by amplification and sequencing of 16S rDNA for 4 the best degrader isolates. Degradation was confirmed on the basis of UV spectrophotometric and HPLC analysis. Four bacterial isolates, showing high catabolic activity towards 7-ketocholesterol were isolated: Alcanivorax jadensis IP4 (accession number KP309836; sea water sediment), Streptomyces auratus IP2 (accession number KP309837; soil), Serratia marcescens IP3 (accession number KP309838; soil) and Thermobifida fusca IP1 (accession number KM677184; manure piles). All the isolates were capable of utilizing 7-ketocholesterol as the sole organic substrate, resulting in its mineralisation. The most rapid degradation was observed with A. jadensis IP4 followed by T. fusca IP1. The degradation was followed and analyzed by HPLC. A. jadensis IP4 removed 7-ketocholesterol below detection levels within 8 days.     

Genotype Analysis of Bacillus anthracis Strains Circulating in Bangladesh

In Bangladesh, anthrax, caused by the bacterium Bacillus anthracis, is considered an endemic disease affecting ruminants with sporadic zoonotic occurrences in humans. Due to the lack of knowledge about risks from an incorrect removal of infected carcasses, the disease is not properly monitored, and because of the socio-economic conditions, the situation is under-reported and under-diagnosed. For sensitive species, anthrax represents a fatal outcome with sudden death and sometimes bleeding from natural orifices. The most common source of infection for ruminants is ingestion of spores during grazing in contaminated pastures or through grass and water contaminated with anthrax spores. Domestic cattle, sheep and goats can also become infected through contaminated bone meal (used as feed) originating from anthrax-infected carcasses. The present investigation was conducted to isolate B. anthracis organisms from 169 samples (73 soil, 1 tissue, 4 bone and 91 bone meal samples) collected from 12 different districts of Bangladesh. The sampling was carried out from 2012 to 2015. Twelve samples resulted positive for B. anthracis. Biomolecular analyses were conducted starting from the Canonical Single Nucleotide Polymorphism (CanSNP) to analyze the phylogenetic origin of strains. The analysis of genotype, obtained through the Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) with the analysis of 15 Variable Number Tandem Repeats (VNTR), demonstrated four different genotypes: two of them were previously identified in the district of Sirajganj. The sub-genotyping, conducted with Single Nucleotide Repeats analysis, revealed the presence of eight subgenotypes. The data of the present study concluded that there was no observed correlation between imported cattle feed and anthrax occurrence in Bangladesh and that the remarkable genetic variations of B. anthracis were found in the soil of numerous outbreaks in this country.     

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a fluorescently tagged-protein of interest is transiently expressed in plant tissue following biolistic delivery of its encoding DNA construct. The intra- and intercellular distribution of the tagged protein is then analyzed by confocal microscopy. We describe this technology in detail, providing step-by-step protocols to assay and evaluate the extent of symplastic protein transport in three plant species, Arabidopsis thaliana, Nicotiana benthamiana and N. tabacum (tobacco).Download video file.^{(44M, mov)}     

Marine bacterial isolates inhibit biofilm formation and disrupt mature biofilms of Pseudomonas aeruginosa PAO1

According to the Centers for Disease Control and Prevention, biofilms cause 65% of infections in developed countries. Pseudomonas aeruginosa biofilm cause life threatening infections in cystic fibrosis infection and they are 1,000 times more tolerant to antibiotic than the planktonic cells. As quorum sensing, hydrophobicity index and extracellular polysaccharide play a crucial role in biofilm formation, extracts from 46 marine bacterial isolates were screened against these factors in P. aeruginosa. Eleven extracts showed antibiofilm activity. Extracts of S6-01 (Bacillus indicus = MTCC 5559) and S6-15 (Bacillus pumilus = MTCC 5560) inhibited the formation of PAO1 biofilm up to 95% in their Biofilm Inhibitory Concentration(BIC) of 50 and 60 μg/ml and 85% and 64% in the subinhibitory concentrations (1/4 and 1/8 of the BIC, respectively). Furthermore, the mature biofilm was disrupted to 70–74% in their BIC. The antibiofilm compound from S6-15 was partially purified using solvent extraction followed by TLC and silica column and further characterized by IR analysis. Current study for the first time reveals the antibiofilm and antiquorum-sensing activity of B. pumilus, B. indicus, Bacillus arsenicus, Halobacillus trueperi, Ferrimonas balearica, and Marinobacter hydrocarbonoclasticus from marine habitat.     

Role of EGF receptor transactivation in phosphoinositide 3-kinase-dependent activation of MAP kinase by GPCRs

Many G protein coupled receptors (GPCRs) cause phosphorylation of MAP kinases through transactivation of the epidermal growth factor receptor (EGF-R), leading to increased cell survival and growth, motility, and migration. Phosphoinositide 3-kinase (PI3K) is one of the important cell survival signaling molecules activated by EGF-R stimulation. However, the extent to which EGF-R transactivation is essential for GPCR agonist-stimulated PI3K activation is not known. Here we examined the mechanism of PI3K activation that elicits GPCR-mediated ERK1/2 activation by pathways dependent and/or independent of EGF-R transactivation in specific cell types. Immortalized hypothalamic neurons (GT1-7 cells) express endogenous gonadotropin-releasing hormone receptors (GnRH-R) and their stimulation causes marked phosphorylation of ERK1/2 and Akt (Ser 473) through transactivation of the EGF-R and recruitment of PI3K. In C9 hepatocytes, agonist activation of AT1 angiotensin II (AT1-R), lysophosphatidic acid (LPA), and EGF receptors caused phosphorylation of Akt through activation of the EGF-R in a PI3K-dependent manner. However, ERK1/2 activation by these agonists in these cells was independent of PI3K activation. In contrast, agonist stimulation of HEK 293 cells stably expressing AT1-R caused ERK1/2 phosphorylation that was independent of EGF-R transactivation but required PI3K activation. LPA signaling in these cells showed partial and complete dependence on EGF-R and PI3K, respectively. These data indicate that GPCR-induced ERK1/2 phosphorylation is dependent or independent of PI3K in specific cell types, and that the involvement of PI3K during ERK1/2 activation is not dependent solely on agonist-induced transactivation of the EGF-R.     

Effects of residual chlorfluazuron on haemolymph-borne oviposition-stimulating factors in Spodoptera litura

Acylurea insecticides can have various effects on insect reproduction and may even interfere with haemolymph‐borne oviposition‐stimulating factors (OSFs). This study describes the effects of injecting haemolymph into females of the common cutworm, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae), the effects of chlorfluazuron on the activities of OSFs, when sublethal doses (LD₁₀: 1.00 ng larva^?1 or LD₃₀: 3.75 ng larva^?1) were applied topically to newly ecdysed fifth instars under laboratory conditions. The haemolymph of moths of varying age, sex, and mating status resulted in the following increases in oviposition rates within the first 24 h, listed in decreasing order: the haemolymph of mated females injected into mated females (56%), mated females into newly emerged virgin females (53%), virgin males into virgin females (49%), and virgin females into mated females (29%), compared with uninjected mated or virgin females. Two factors are involved in the activation of ovipositon: OSF‐I is found in virgin females, whereas OSF‐II may be formed or received by males during mating. By contrast, the haemolymph taken from chlorfluazuron‐treated adults (treated as fifth instars with sublethal doses) injected into females resulted in the following percentage decreases in oviposition rates within the first 24 h, listed in decreasing order: the haemolymph of LD₁₀‐ and LD₃₀‐treated virgin males injected into untreated virgin females (77 and 84%, respectively), LD₁₀ and LD₃₀‐treated virgin females into untreated mated females (70 and 80%, respectively), LD₁₀ and LD₃₀‐treated mated females into untreated mated females (61 and 69%, respectively), LD₁₀ and LD₃₀‐treated mated females into untreated virgin females (59 or 68%, respectively), compared with untreated ones. Hence, residual chlorfluazuron decreases the activities of OSFs by significantly decreasing the oviposition rates. Moreover, virgin females’ or males’ OSFs are more sensitive to chlorfluazuron than those of mated cutworms. Both OSFs are most likely proteins.