Inguinal Fat Compensates Whole Body Metabolic Functionality in Partially Lipodystrophic Mice with Reduced PPARγ Expression.

Peroxisome proliferator-activated receptor γ (PPARγ) gene mutations in humans and mice lead to whole-body insulin resistance and partial lipodystrophy. It is unclear whether preserved fat depots in partial lipodystrophy are beneficial for whole-body metabolic homeostasis. We analyzed the insulin response and expression of metabolic genes in the preserved fat depots of PpargC/- mice, a familial partial lipodystrophy type 3 (FPLD3) mouse model resulting from a 75% decrease in Pparg transcripts. Perigonadal fat of PpargC/- mice in the basal state showed dramatic decreases in adipose tissue mass and insulin sensitivity, whereas inguinal fat showed compensatory increases. Preservation of inguinal fat metabolic ability and flexibility was reflected by the normal expression of metabolic genes in the basal or fasting/refeeding states. The high nutrient load further increased insulin sensitivity in inguinal fat, but the expression of metabolic genes became dysregulated. Inguinal fat removal resulted in further impairment of whole-body insulin sensitivity in PpargC/- mice. Conversely, the compensatory increase in insulin sensitivity of the inguinal fat in PpargC/- mice diminished as activation of PPARγ by its agonists restored insulin sensitivity and metabolic ability of perigonadal fat. Together, we demonstrated that inguinal fat of PpargC/- mice plays a compensatory role in combating perigonadal fat abnormalities.

Analysis of prescription pattern and guideline adherence in the management of asthma among medical institutions and physician specialties in Taiwan between 2000 and 2010.

PURPOSE: The aim of this study was to evaluate prescription patterns of antiasthmatic medications in ambulatory care, guideline adherence by physician specialties and medical institutions, and the rate of hospitalization and emergency department visits due to asthma exacerbation. METHODS: The ambulatory visits between 2000 and 2010 from the Taiwan Longitudinal Health Insurance Database 2000 were analyzed for prescription trends. Seven classes of antiasthmatic medications were identified for prescription trend analysis. Prescription patterns of different medical institutions and physician specialties were further evaluated. FINDINGS: We studied 4495 patients with newly diagnosed asthma in 2000. Estimates indicated an increased use in fixed-dose combination of inhaled corticosteroids and long-acting ß2-agonists (3.6% in 2002 to 28.8% in 2010) with decreased use of inhaled corticosteroids (14.5% in 2001 to 7.3% in 2010). Xanthine was still the most frequently used medication for asthmatic patients (60.2% in 2001 and 45.2% in 2010). Another marked increase was the use of leukotriene receptor antagonists (2.6% in 2001 to 6.0% in 2010). In the studied population, the rate of hospital admission or emergency department visit moderately decreased from 1.42% to 0.59% during 10 years. Physicians in medical centers and regional hospitals, as well as asthma specialists, dominated the increased use of fixed-dose combinations of inhaled corticosteroids and long-acting ß2-agonists and leukotriene receptor antagonists. IMPLICATIONS: Physicians in academic medical centers and asthma specialists achieved better adherence to the core recommendations of the international guidelines for asthma management. The reasons for guideline nonadherence among physicians in district hospitals and primary care clinics deserve health care professionals' attention and require further investigation.

Iridovirus CARD Protein Inhibits Apoptosis through Intrinsic and Extrinsic Pathways.

Grouper iridovirus (GIV) belongs to the genus Ranavirus of the family Iridoviridae; the genomes of such viruses contain an anti-apoptotic caspase recruitment domain (CARD) gene. The GIV-CARD gene encodes a protein of 91 amino acids with a molecular mass of 10,505 Daltons, and shows high similarity to other viral CARD genes and human ICEBERG. In this study, we used Northern blot to demonstrate that GIV-CARD transcription begins at 4 h post-infection; furthermore, we report that its transcription is completely inhibited by cycloheximide but not by aphidicolin, indicating that GIV-CARD is an early gene. GIV-CARD-EGFP and GIV-CARD-FLAG recombinant proteins were observed to translocate from the cytoplasm into the nucleus, but no obvious nuclear localization sequence was observed within GIV-CARD. RNA interference-mediated knockdown of GIV-CARD in GK cells infected with GIV inhibited expression of GIV-CARD and five other viral genes during the early stages of infection, and also reduced GIV infection ability. Immunostaining was performed to show that apoptosis was effectively inhibited in cells expressing GIV-CARD. HeLa cells irradiated with UV or treated with anti-Fas antibody will undergo apoptosis through the intrinsic and extrinsic pathways, respectively. However, over-expression of recombinant GIV-CARD protein in HeLa cells inhibited apoptosis induced by mitochondrial and death receptor signaling. Finally, we report that expression of GIV-CARD in HeLa cells significantly reduced the activities of caspase-8 and -9 following apoptosis triggered by anti-Fas antibody. Taken together, these results demonstrate that GIV-CARD inhibits apoptosis through both intrinsic and extrinsic pathways.

Recognition of Linear B-Cell Epitope of Betanodavirus Coat Protein by RG-M18 Neutralizing mAB Inhibits Giant Grouper Nervous Necrosis Virus (GGNNV) Infection.

Betanodavirus is a causative agent of viral nervous necrosis syndrome in many important aquaculture marine fish larvae, resulting in high global mortality. The coat protein of Betanodavirus is the sole structural protein, and it can assemble the virion particle by itself. In this study, we used a high-titer neutralizing mAB, RG-M18, to identify the linear B-cell epitope on the viral coat protein. By mapping a series of recombinant proteins generated using the E. coli PET expression system, we demonstrated that the linear epitope recognized by RG-M18 is located at the C-terminus of the coat protein, between amino acid residues 195 and 338. To define the minimal epitope region, a set of overlapping peptides were synthesized and evaluated for RG-M18 binding. Such analysis identified the 195VNVSVLCR202 motif as the minimal epitope. Comparative analysis of Alanine scanning mutagenesis with dot-blotting and ELISA revealed that Valine197, Valine199, and Cysteine201 are critical for antibody binding. Substitution of Leucine200 in the RGNNV, BFNNV, and TPNNV genotypes with Methionine200 (thereby simulating the SJNNV genotype) did not affect binding affinity, implying that RG-M18 can recognize all genotypes of Betanodaviruses. In competition experiments, synthetic multiple antigen peptides of this epitope dramatically suppressed giant grouper nervous necrosis virus (GGNNV) propagation in grouper brain cells. The data provide new insights into the protective mechanism of this neutralizing mAB, with broader implications for Betanodavirus vaccinology and antiviral peptide drug development.

Molecular characterization and expression analysis of four isotypes of immunoglobulin light chain genes in orange-spotted grouper, Epinephelus coioides.

To date, many immunoglobulin (Ig) genes have been identified in diverse teleost species, but the contributions of different types of light chain (IgL) to the immune response remain unclear. Screening of a stimulated kidney cDNA library from orange-spotted grouper (Osg, Epinephelus coioides) resulted in the identification of 26 full Ig light chain (OsgIgL) coding sequences. These 26 OsgIgLs encoded peptides from 235 to 248 amino acid residues and could be grouped into five variable (V(L)) and four constant (C(L)) isotypes. The C(L) regions contained three conserved cysteine residues that may participate in intra- or inter-chain disulfide bond formation. The four C(L) isotypes could be sub-grouped into two serological types: κ (C(L)-I, C(L)-II and C(L)-III) and σ (C(L)-IV), by phylogenetic analysis. The OsgIgL genes were found to be expressed in various tissues, with greatest levels of expression observed in the head-kidney and spleen. The major expression type was C(L)-I, which comprised 92% and 91% of total OsgIgL gene expression in the head-kidney and spleen, respectively. Transcription of all four C(L) isotypes was differentially affected in response to various immunostimulators, including lipopolysaccharide (LPS), poly I:C and grouper iridovirus (GIV). Induction of OsgIgL genes in response to immunostimulators was particularly dramatic in the spleen, suggesting this organ holds particular importance for the regulation of OsgIgL expression. Furthermore, vaccination of grouper with formalin-inactivated GIV also induced differential patterns of expression in all four OsgIgL isotypes. In summary, the significant and diverse patterns of transcriptional induction observed for OsgIgL isotypes in the spleen and head-kidney imply that each isotype may have unique roles in the immune response.

Transcriptional analysis of orange-spotted grouper reacting to experimental grouper iridovirus infection.

Disease caused by grouper iridovirus (GIV) has resulted in economic losses due to high mortality in grouper culture. Thirty-eight up- and 48 down-regulated known entities have been identified using a GIV-infected grouper kidney cDNA microarray chip. Further quantitative validation was executed in the head-kidney and spleen for 24 candidate genes and 7 immune factors following GIV inoculation. Significant induction with various patterns could be seen in 30 tested genes in the spleen. However, only 23 genes had induction in the head-kidney and meanwhile 5 genes showed reduction. Transcriptional expression profiles of selected genes in response to lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (PIC) were also established to compare with the GIV-stimulated expression. The results indicated that the responses of most genes facing GIV invasion have more similarities to PIC treatment than LPS. Seven genes are thought to be interferon-related factors: RNA helicase DHX58, ISG15, viperin, HECT E3 ligase (HECT), CD9, urokinase plasminogen activator surface receptor (PLAUR) and Mx-1. Following immunization with inactivated GIV, significant induction could be seen in DHX58, viperin, IL-1ß, IL-8, COX-2, HECT, PLAUR, IgM, Mx-1, very large inducible GTPase-1 (VLIG1) and TNF-α in the head-kidney or spleen, and the latter 6 genes also had a gradual increasing pattern by a boosting immunization. These factors might play important roles in adaptive antiviral protection. Thus, we have characterized the temporal response patterns of virus responsive genes and have also identified several potential immune markers to further investigate host antiviral defense mechanisms.

Differential display of grouper iridovirus-infected grouper cells by immunostaining.

Grouper iridovirus (GIV) is one of the most devastating infectious pathogens of aquaculture fish. When infecting a susceptible cell line, such as GK-2, GIV causes antigenic changes in host cellular proteins. To understand the host gene expression characteristics after viral infection, we developed an immunostaining method to screen differentially expressed genes of fish cells in response to GIV infection using phage display complementary DNA libraries. In total, 66 genes were identified from grouper kidney and brain cell lines. These genes are related to replication, transcription, translation, immunity, apoptosis, structure proteins, metabolism, energy, protein modification, and homeostasis. Four dynamic antigenic patterns were observed among these immunocloned genes upon GIV infection. Microarray analysis further confirmed the transcriptional patterns of 80% of the identified genes. This immunostaining screening method provides insights into a host's cellular protein response to viral infection on a translational basis.

Characterization of serum immunoglobulin M of grouper and cDNA cloning of its heavy chain.

Immunoglobulin M (IgM) from the whole serum of grouper fish, Epinephelus coioides was purified by affinity chromatography using protein A-Sepharose column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions revealed that the relative molecular masses (Mr) of the equimolar heavy and light chains of IgM were 78,000 and 27,000, respectively. The cDNAs encoding IgM heavy chain comprising its variable (VH) and constant (CH) regions have been cloned and sequenced from a grouper kidney cDNA library by antibody screening method. Five VH (130-142 amino acids) and four CH (450-454 amino acids) families were identified. The variable and constant regions were conserved with their putative domains. All the four constant region domains (CH1-CH2-CH3-CH4) contained each three conserved cysteine residues, which are considered to form the inter- and intra-chain disulfide bridges. There were three carbohydrate acceptor sites in the constant region. In general, the pattern of IgM gene organization seems to resemble that of other teleosts. Moreover, the CH genes in grouper IgM occur as multifamily as reported in Atlantic salmon and common carp.