Hepatic protein kinase Cbeta deficiency mitigates late-onset obesity.

Although aging is associated with progressive adiposity and a decline in liver function, the underlying molecular mechanisms and metabolic interplay are incompletely understood. Here, we demonstrate that aging induces hepatic protein kinase Cbeta (PKCß) expression, while hepatocyte PKCß deficiency (PKCßHep-/-) in mice significantly attenuates obesity in aged mice fed a high-fat diet. Compared with control PKCßfl/fl mice, PKCßHep-/- mice showed elevated energy expenditure with augmentation of oxygen consumption and carbon dioxide production which was dependent on ß3-adrenergic receptor signaling, thereby favoring negative energy balance. This effect was accompanied by induction of thermogenic genes in brown adipose tissue (BAT) and increased BAT respiratory capacity, as well as a shift to oxidative muscle fiber type with an improved mitochondrial function, thereby enhancing oxidative capacity of thermogenic tissues. Furthermore, in PKCßHep-/- mice, we determined that PKCß overexpression in the liver mitigated elevated expression of thermogenic genes in BAT. In conclusion, our study thus establishes hepatocyte PKCß induction as a critical component of pathophysiological energy metabolism by promoting progressive hepatic and extrahepatic metabolic derangements in energy homeostasis, contributing to late-onset obesity. These findings have potential implications for augmenting thermogenesis as a means of combating aging-induced obesity.

Role of hepatic PKCß in nutritional regulation of hepatic glycogen synthesis.

The signaling mechanisms by which dietary fat and cholesterol signals regulate central pathways of glucose homeostasis are not completely understood. By using a hepatocyte-specific PKCß-deficient (PKCßHep-/-) mouse model, we demonstrated the role of hepatic PKCß in slowing disposal of glucose overload by suppressing glycogenesis and increasing hepatic glucose output. PKCßHep-/- mice exhibited lower plasma glucose under the fed condition, modestly improved systemic glucose tolerance and mildly suppressed gluconeogenesis, increased hepatic glycogen accumulation and synthesis due to elevated glucokinase expression and activated glycogen synthase (GS), and suppressed glucose-6-phosphatase expression compared with controls. These events were independent of hepatic AKT/GSK-3α/ß signaling and were accompanied by increased HNF-4α transactivation, reduced FoxO1 protein abundance, and elevated expression of GS targeting protein phosphatase 1 regulatory subunit 3C in the PKCßHep-/- liver compared with controls. The above data strongly imply that hepatic PKCß deficiency causes hypoglycemia postprandially by promoting glucose phosphorylation via upregulating glucokinase and subsequently redirecting more glucose-6-phosphate to glycogen via activating GS. In summary, hepatic PKCß has a unique and essential ability to induce a coordinated response that negatively affects glycogenesis at multiple levels under physiological postprandial conditions, thereby integrating nutritional fat intake with dysregulation of glucose homeostasis.

Hepatocyte-specific PKCß deficiency protects against high-fat diet-induced nonalcoholic hepatic steatosis.

OBJECTIVE: Nonalcoholic hepatic steatosis, also known as fatty liver, is a uniform response of the liver to hyperlipidic-hypercaloric diet intake. However, the post-ingestive signals and mechanistic processes driving hepatic steatosis are not well understood. Emerging data demonstrate that protein kinase C beta (PKCß), a lipid-sensitive kinase, plays a critical role in energy metabolism and adaptation to environmental and nutritional stimuli. Despite its powerful effect on glucose and lipid metabolism, knowledge of the physiological roles of hepatic PKCß in energy homeostasis is limited. METHODS: The floxed-PKCß and hepatocyte-specific PKCß-deficient mouse models were generated to study the in vivo role of hepatocyte PKCß on diet-induced hepatic steatosis, lipid metabolism, and mitochondrial function. RESULTS: We report that hepatocyte-specific PKCß deficiency protects mice from development of hepatic steatosis induced by high-fat diet, without affecting body weight gain. This protection is associated with attenuation of SREBP-1c transactivation and improved hepatic mitochondrial respiratory chain. Lipidomic analysis identified significant increases in the critical mitochondrial inner membrane lipid, cardiolipin, in PKCß-deficient livers compared to control. Moreover, hepatocyte PKCß deficiency had no significant effect on either hepatic or whole-body insulin sensitivity supporting dissociation between hepatic steatosis and insulin resistance. CONCLUSIONS: The above data indicate that hepatocyte PKCß is a key focus of dietary lipid perception and is essential for efficient storage of dietary lipids in liver largely through coordinating energy utilization and lipogenesis during post-prandial period. These results highlight the importance of hepatic PKCß as a drug target for obesity-associated nonalcoholic hepatic steatosis.

Evaluation of the Antibacterial Activity of 75 Mushrooms Collected in the Vicinity of Oxford, Ohio (USA).

Antibiotic-resistant bacteria are an increasing and serious health concern worldwide, and multidrug-resistant pathogens are increasingly emerging among patients across the United States. Researchers are exploring sources of traditional medicines, including mushrooms, to find new antibiotic compounds. In this study, the antibiotic activities of 75 mushrooms collected in the area surrounding Oxford, Ohio (USA), were assayed for antibiotic activity against 6 bacterial strains (Pseudomonas aeruginosa reference strains PAO1 and PA14, P. fluorescens, Bacillus subtilis, Staphylococcus epidermidis, and Micrococcus luteus). Mushroom samples were identified by using DNA ribotyping. We used methanol and water extracts of mushrooms in agar diffusion assays to screen for antibiotic activity toward each bacterial strain. A total of 25 mushroom species had antibacterial activity against at least 1 bacterium. Water extracts of Polyporus squamosus, Ganoderma applanatum, Lentinellus subaustralis, Laetiporus sulphureus, G. lucidum, and Trametes versicolor exhibited strong antibiotic activity against all bacterial strains tested. Water and methanol extracts from 25 mushroom species had significant activity against most of the bacteria tested. A minimum inhibitory concentration (MIC) against S. epidermidis was determined for all samples that exhibited antibiotic activity in the disk assay. The G. lucidum and L. sulphureus extracts displayed the strongest inhibition, with an MIC of 0.1 mg/mL.

Adenovirus-Mediated Delivery of Decoy Hyper Binding Sites Targeting Oncogenic HMGA1 Reduces Pancreatic and Liver Cancer Cell Viability.

High mobility group AT-hook 1 (HMGA1) protein is an oncogenic architectural transcription factor that plays an essential role in early development, but it is also implicated in many human cancers. Elevated levels of HMGA1 in cancer cells cause misregulation of gene expression and are associated with increased cancer cell proliferation and increased chemotherapy resistance. We have devised a strategy of using engineered viruses to deliver decoy hyper binding sites for HMGA1 to the nucleus of cancer cells with the goal of sequestering excess HMGA1 at the decoy hyper binding sites due to binding competition. Sequestration of excess HMGA1 at the decoy binding sites is intended to reduce HMGA1 binding at the naturally occurring genomic HMGA1 binding sites, which should result in normalized gene expression and restored sensitivity to chemotherapy. As proof of principle, we engineered the replication defective adenovirus serotype 5 genome to contain hyper binding sites for HMGA1 composed of six copies of an individual HMGA1 binding site, referred to as HMGA-6. A 70%-80% reduction in cell viability and increased sensitivity to gemcitabine was observed in five different pancreatic and liver cancer cell lines 72 hr after infection with replication defective engineered adenovirus serotype 5 virus containing the HMGA-6 decoy hyper binding sites. The decoy hyper binding site strategy should be general for targeting overexpression of any double-stranded DNA-binding oncogenic transcription factor responsible for cancer cell proliferation.

A mouse model study of toxicity and biodistribution of a replication defective adenovirus serotype 5 virus with its genome engineered to contain a decoy hyper binding site to sequester and suppress oncogenic HMGA1 as a new cancer treatment therapy.

The HGMA1 architectural transcription factor is highly overexpressed in many human cancers. Because HMGA1 is a hub for regulation of many oncogenes, its overexpression in cancer plays a central role in cancer progression and therefore HMGA1 is gaining increasing attention as a target for development of therapeutic approaches to suppress either its expression or action in cancer cells. We have developed the strategy of introducing decoy hyper binding sites for HMGA1 into the nucleus of cancer cells with the goal of competetively sequestering overexpressed HMGA1 and thus suppressing its oncogenic action. Towards achieving this goal, we have introduced an HMGA1 decoy hyper binding site composed of six copies of a high affinity HMGA1 binding site into the genome of the replication defective adenovirus serotype 5 genome and shown that the engineered virus effectively reduces the viability of human pancreatic and cancer cells. Here we report the first pre-clinical measures of toxicity and biodistribution of the engineered virus in C57BL/6J Black 6 mice. The immune response to exposure of the engineered virus was determined by assaying the serum levels of key cytokines, IL-6 and TNF-α. Toxicity due to exposure to the virus was determined by measuring the serum levels of the liver enzymes aspartate aminotransferase and alanine aminotransferase. Biodistribution was measured following direct injection into the pancreas or liver by quantifying viral loads in the pancreas, liver, spleen and brain.

A Computational Approach for Designing a Universal Epitope-Based Peptide Vaccine Against Nipah Virus.

Nipah virus (NiV) is highly pathogenic single-stranded negative sense RNA virus. It can cause severe encephalitis and respiratory disease in humans. In addition, NiV infects a large range of host including mammals. As a result of its higher zoonotic potential and pathogenicity for human, it has been rated as an alert in recent days. A therapeutic treatment or vaccines has become elusive to fight against this virus. In this study, the attachment (G) and fusion (F) glycoproteins of NiV, responsible for the viral attachment and entry to the host cell, were selected to develop epitope-based vaccine against Nipah virus. Epitopes were identified from the conserved region of G and F protein of NiV. Both B-cell and T-cell immunity were checked to affirm it that these epitopes will be able to induce humoral and cellular immunity. A total of 6 T-cell epitopes and 19 significant HLA-epitope interactions were identified. Eventually it has shown an acceptable percentage in population coverage (46.45 %) and efficient binding with HLA molecule by molecular docking study.

Arsenosugar induced blood and brain oxidative stress, DNA damage and neurobehavioral impairments.

The effect of Arsenosugar on motor function and contextual memory-related to place and event; the extent of DNA damage and oxidative stress in male swiss albino mice was investigated. Passive avoidance test was used for memory test; rota motor test was used for motor function. Several biochemical parameters were used for assessing oxidative stress due to arsenosugar ingestion. Decreased passive avoidance time and decreased retention time in rotating rod indicated disruption of normal neurobehavior. Significant dose-dependent DNA damage was found in mice blood and brain. Decreased super oxide dismutase, increased lipid peroxidation, decreased protein sulfohydryl content, increased protein carbonyl content in blood and hippocampal tissue; glutathione in blood and glutathione peroxidase in hippocampal tissue indicated the ability of arsenosugar to cause oxidative stress. This study concludes with evidence that arsenosugar ingestion causes higher oxidative stress, increases DNA damage in the blood and hippocampus in vivo. This might be responsible for the dysfunction of cognitive and motor functions. However, further investigation is suggested for deciphering the biomolecular mechanism.

Satellite phage TLCφ enables toxigenic conversion by CTX phage through dif site alteration.

Bacterial chromosomes often carry integrated genetic elements (for example plasmids, transposons, prophages and islands) whose precise function and contribution to the evolutionary fitness of the host bacterium are unknown. The CTXφ prophage, which encodes cholera toxin in Vibrio cholerae, is known to be adjacent to a chromosomally integrated element of unknown function termed the toxin-linked cryptic (TLC). Here we report the characterization of a TLC-related element that corresponds to the genome of a satellite filamentous phage (TLC-Knφ1), which uses the morphogenesis genes of another filamentous phage (fs2φ) to form infectious TLC-Knφ1 phage particles. The TLC-Knφ1 phage genome carries a sequence similar to the dif recombination sequence, which functions in chromosome dimer resolution using XerC and XerD recombinases. The dif sequence is also exploited by lysogenic filamentous phages (for example CTXφ) for chromosomal integration of their genomes. Bacterial cells defective in the dimer resolution often show an aberrant filamentous cell morphology. We found that acquisition and chromosomal integration of the TLC-Knφ1 genome restored a perfect dif site and normal morphology to V. cholerae wild-type and mutant strains with dif(-) filamentation phenotypes. Furthermore, lysogeny of a dif(-) non-toxigenic V. cholerae with TLC-Knφ1 promoted its subsequent toxigenic conversion through integration of CTXφ into the restored dif site. These results reveal a remarkable level of cooperative interactions between multiple filamentous phages in the emergence of the bacterial pathogen that causes cholera.

A computational approach for identification of epitopes in dengue virus envelope protein: a step towards designing a universal dengue vaccine targeting endemic regions.

A major problem in designing vaccine for the dengue virus has been the high antigenic variability in the envelope protein of different virus strains. In this study, a computational approach was adopted to identify a multi-epitope vaccine candidate against dengue virus that may be suitable for large populations in the dengue-endemic regions. Different bioinformatics tools were exploited that helped the identification of a conserved immunological hot-spot in the dengue envelope protein. The tools also rendered the prediction of immunogenicity and population coverage to the proposed 'in silico' vaccine candidate against dengue. A peptide region, spanning 19 amino acids, was identified in the envelope protein which found to be conserved in all four types of dengue viruses. Ten proteasomal cleavage sites were identified within the 19-mer conserved peptide sequence and a total of 8 overlapping putative cytotoxic T cell (CTL) epitopes were identified. The immunogenicity of these epitopes was evaluated in terms of their binding affinities to and dissociation half-time from respective human leukocyte antigen (HLA) molecules. The HLA allele frequencies were studied among populations in the dengue endemic regions and compared with respect to HLA restriction patterns of the overlapping epitopes. The cumulative population coverage for these epitopes as vaccine candidates was high ranging from approximately 80% to 92%. Structural analysis suggested that a 9-mer epitope fitted well into the peptide-binding groove of HLA-A*0201. In conclusion, the 19-mer epitope cluster was shown to have the potential for use as a vaccine candidate against dengue.

Assessment of the evolutionary origin and possibility of CRISPR-Cas (CASS) mediated RNA interference pathway in Vibrio cholerae O395.

Bacteria have developed several defense mechanisms against bacteriophages over evolutionary time, but the concept of prokaryotic RNA interference mediated defense mechanism against phages and other invading genetic elements has emerged only recently. Clustered regularly interspaced short palindromic repeats (CRISPR) together with closely associated genes (cas genes) constitute the CASS system that is believed to provide a RNAi-like defense mechanism against bacteriophages within the host bacterium. However, a CASS mediated RNAi-like pathway in enteric pathogens such as Vibrio cholerae O395 or Escherichia coli O157 have not been reported yet. This study specifically was designed to investigate the possibility and evolutionary origin of CASS mediated RNAi-like pathway in the genome of a set of enteric pathogens, especially V. cholerae. The results showed that V. cholerae O395 and also other related enteric pathogens have the essential CASS components (CRISPR and cas genes) to mediate a RNAi-like pathway. The functional domains of a V. cholerae Cas3 protein, which is believed to act as a prokaryotic Dicer, was revealed and compared with the domains of eukaryotic Dicer proteins. Extensive homology in several functional domains provides significant evidence that the Cas3 protein has the essential domains to play a vital role in RNAi like pathway in V. cholerae. The secondary structure of the pre-siRNA for V. cholerae O395 was determined and its thermodynamic stability also reinforced the previous findings and signifies the probability of a RNAi-like pathway in V. cholerae O395.