Impact of dengue virus (serotype DENV-2) infection on liver of BALB/c mice: A histopathological analysis

In this research, we characterized the histopathological impact of dengue virus (serotype DENV-2) infection in livers of BALB/c mice. The mice were infected with different doses of DENV-2 via intraperitoneal injection and liver tissues were processed for histological analyses and variation was documented. In the BALB/c mouse model, typical liver tissues showed regular hepatocyte architecture, with normal endothelial cells surrounding sinusoid capillary. Based on histopathological observations, the liver sections of BALB/c mice infected by DENV-2 exhibited a loss of cell integrity, with a widening of the sinusoidal spaces. There were marked increases in the infiltration of mononuclear cells. The areas of hemorrhage and micro- and macrovesicular steatosis were noted. Necrosis and apoptosis were abundantly present. The hallmark of viral infection, i.e., cytopathic effects, included intracellular edema and vacuole formation, cumulatively led to sinusoidal and lobular collapse in the liver. The histopathological studies on autopsy specimens of fatal human DENV cases are important to shed light on tissue damage for preventive and treatment modalities, in order to manage future DENV infections. In this framework, the method present here on BALB/c mouse model may be used to study not only the effects of infections by other DENV serotypes, but also to investigate the effects of novel drugs, such as recently developed nano-formulations, and the relative recovery ability with intact immune functions of host.     

The critical role played by endotoxin-induced liver autophagy in the maintenance of lipid metabolism during sepsis

Macroautophagy/autophagy is a central mechanism by which cells maintain integrity and homeostasis, and endotoxin-induced autophagy plays important roles in innate immunity. Although TLR4 stimulation mediated by lipopolysaccharide (LPS) also upregulates autophagy in hepatocytes and liver, its physiological role remains elusive. The objective of this study was to determine the role of LPS-induced autophagy in the regulation of liver lipid metabolism. LPS treatment (5 mg/kg) increased autophagy, as detected by LC3 conversion and transmission electron microscopy (TEM) analysis in C57BL6 mouse livers. AC2F hepatocytes also showed increased autophagic flux after LPS treatment (1 μg/ml). To investigate the role of LPS-induced autophagy further, liver lipid metabolism changes in LPS-treated mice and fasted controls were compared. Interestingly, LPS-treated mice showed less lipid accumulation in liver than fasted mice despite increased fatty acid uptake and lipid synthesis-associated genes. In vitro analysis using AC2F hepatocytes demonstrated LPS-induced autophagy influenced the degradation of lipid droplets. Inhibition of LPS-induced autophagy using bafilomycin A₁ or Atg7 knockdown significantly increased lipid accumulation in AC2F hepatocytes. In addition, pretreatment with chloroquine aggravated LPS-induced lipid accumulation and inflammation in C57BL6 mouse livers. The physiological importance of autophagy was verified in LPS-treated young and aged rats. Autophagic response was diminished in LPS-treated aged rats and lipid metabolism was impaired during sepsis, indicating autophagy response is important for regulating lipid metabolism after endotoxin challenge. Our findings demonstrate endotoxin-induced autophagy is important for the regulation of lipid metabolism, and suggest that autophagy helps maintain lipid metabolism homeostasis during sepsis.     

AMP-activated protein kinase β-subunit requires internal motion for optimal carbohydrate binding

AMP-activated protein kinase interacts with oligosaccharides and glycogen through the carbohydrate-binding module (CBM) containing the β-subunit, for which there are two isoforms (β(1) and β(2)). Muscle-specific β(2)-CBM, either as an isolated domain or in the intact enzyme, binds carbohydrates more tightly than the ubiquitous β(1)-CBM. Although residues that contact carbohydrate are strictly conserved, an additional threonine in a loop of β(2)-CBM is concurrent with an increase in flexibility in β(2)-CBM, which may account for the affinity differences between the two isoforms. In contrast to β(1)-CBM, unbound β(2)-CBM showed microsecond-to-millisecond motion at the base of a β-hairpin that contains residues that make critical contacts with carbohydrate. Upon binding to carbohydrate, similar microsecond-to-millisecond motion was observed in this β-hairpin and the loop that contains the threonine insertion. Deletion of the threonine from β(2)-CBM resulted in reduced carbohydrate affinity. Although motion was retained in the unbound state, a significant loss of motion was observed in the bound state of the β(2)-CBM mutant. Insertion of a threonine into the background of β(1)-CBM resulted in increased ligand affinity and flexibility in these loops when bound to carbohydrate. However, these mutations indicate that the additional threonine is not solely responsible for the differences in carbohydrate affinity and protein dynamics. Nevertheless, these results suggest that altered protein dynamics may contribute to differences in the ligand affinity of the two naturally occurring CBM isoforms.     

Hypoxia-inducible factor-1α mediates oral squamous cell carcinoma invasion via upregulation of α5 integrin and fibronectin

With progressive and rapid growth of malignant tumors, cancer cells in an ischemic condition are expected to develop an increased potential for local invasive growth. To address this hypothesis, we first examined the effect of hypoxia on the invasiveness of oral squamous cell carcinoma (OSCC) cells using the Matrigel invasion assay. We then investigated the effect of hypoxia on the protein and mRNA expression of α5 integrin and fibronectin, which are major factors involved in tumor cell invasion. We showed that (i) hypoxia increased the invasiveness of OSCC cells, (ii) α5 integrin and fibronectin protein and mRNA expression levels were increased in OSCC cells under hypoxic conditions, (iii) hypoxia stimulated autocrine secretion of fibronectin in OSCC cells, (iv) administration of siRNA_HIF-1α caused a significant decrease in α5 integrin and fibronectin protein, confirming that HIF-1α plays a role in their induction, and (v) siRNA_HIF-1α abrogated hypoxia-induced cell invasion. Collectively, these data suggest that hypoxia promotes OSCC cell invasion that is elicited by HIF-1α-dependent α5 integrin and fibronectin induction.     

Phylogeny of Glaucosomatidae inferred from molecular evidence

Most species of glaucosomatids (Teleostei: Glaucosomatidae) are endemic to Australia, except Glaucosoma buergeri that is widely distributed from Australia to Japan. This study elucidated phylogenetic relationships among glaucosomatids based on the morphological characters of the saccular‐otolith sagitta, in addition to molecular evidence of mitochondrial 16S rDNA, cytochrome oxidase I (COI) and cytochrome b (cyt b) sequences, and nuclear rhodopsin sequences. The topologies of individuals' phylogenetic trees, based on 16S rDNA, COI and cyt b sequences, were statistically indistinguishable from one another, and were only slightly different from a tree based on rhodopsin sequences. These molecular tree topologies, however, differed from species relationships in morphology‐based phylogenetic hypothesis proposed in previous studies. Specimens of G. buergeri from Australia and Taiwan showed differences in the sagitta and molecular differentiation at the four genes, suggesting a possible speciation event. Both molecular and morphological evidences indicate that Glaucosoma magnificum is the plesiomorphic sister species of other glaucosomatid species. Glaucosoma hebraicum is the sister species of a clade composed of G. buergeri and Glaucosoma scapulare. Molecular and morphological evidences also support the species status of G. hebraicum.     

A Survey of Cryptosporidium Oocysts in Water Supplies during a 10-Year Period (2000-2009) in Seoul

This study has been conducted to estimate the occurrence of Cryptosporidium oocysts in water supplies in the Metropolitan area of Seoul, South Korea, for 10 years from 2000 to 2009. Water samples were collected quarterly at 6 intakes in the Han River and its largest stream and 6 conventional Water Treatment Plants (WTPs) serving drinking water for 10 million people of Seoul. Cryptosporidium oocysts were found in 22.5% of intake water samples and arithmetic mean was 0.65 oocysts/10 L (range 0-22 oocysts/10 L). Although the annual mean of oocyst number was as low as 0.04-1.90 oocysts/10 L, 3 peaks in 2004 and 2007 were observed and the pollution level was a little higher in winter. The lowest density was observed at Paldang intake and the pollution level increased at Kuui and Jayang intakes. At the end of the largest stream, oocysts were found in 70% of collected samples (mean 5.71 oocysts/10 L) and it seemed that its joining the Han River resulted in the increase at Kuui intake and downstream. Oocyst removal by physical process exceeded 2.0-2.3 log and then all finished water samples collected at 6 WTPs were negative for Cryptosporidium in each 100 L sample for 10 years. These results suggested that domestic wastewater from the urban region could be a source of Cryptosporidium pollution and separating sewage systems adjacent to the intakes could be meaningful for some intakes having weakness related to parasitological water quality.     

Multifunctional siRNA delivery system: Polyelectrolyte complex micelles of six‐arm PEG conjugate of siRNA and cell penetrating peptide with crosslinked fusogenic peptide

For therapeutic applications of small interfering RNA (siRNA), serum stability, enhanced cellular uptake, and facile endosome escape are key issues for designing carriers. In this study, green fluorescent protein (GFP) siRNA was conjugated to a six‐arm polyethylene glycol (PEG) derivative via a reducible disulfide linkage (6PEG‐siRNA). The 6PEG‐siRNA conjugate was also functionalized with a cell penetrating peptide, Hph1 to enhance its cellular uptake property (6PEG‐siRNA‐Hph1). The 6PEG‐siRNA‐Hph1 conjugate was electrostatically complexed with cationic self‐crosslinked fusogenic KALA peptide (cl‐KALA) to form multifunctional polyelectrolyte complex micelles for gene silencing. The resultant siRNA complex formulation with multiple PEG chains showed superior physical stability and resistance to enzymatic degradation. The 6PEG‐siRNA‐Hph1/cl‐KALA complexes exhibited enhanced GFP gene silencing efficiency for MDA‐MB‐435 cells in the serum containing condition. The current reducible and multifunctional polyelectrolyte complex micelles are expected to have high potential for efficient delivery of therapeutic siRNA. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010