Strain-based and sex-biased differences in adrenal and pancreatic gene expression between KK/HlJ and C57BL/6 J mice.

BACKGROUND: The ever-increasing prevalence of diabetes and associated comorbidities serves to highlight the necessity of biologically relevant small-animal models to investigate its etiology, pathology and treatment. Although the C57BL/6 J model is amongst the most widely used mouse model due to its susceptibility to diet-induced obesity (DIO), there are a number of limitations namely [1] that unambiguous fasting hyperglycemia can only be achieved via dietary manipulation and/or chemical ablation of the pancreatic beta cells. [2] Heterogeneity in the obesogenic effects of hypercaloric feeding has been noted, together with sex-dependent differences, with males being more responsive. The KK mouse strain has been used to study aspects of the metabolic syndrome and prediabetes. We recently conducted a study which characterized the differences in male and female glucocentric parameters between the KK/HlJ and C57BL/6 J strains as well as diabetes-related behavioral differences (Inglis et al. 2019). In the present study, we further characterize these models by examining strain- and sex-dependent differences in pancreatic and adrenal gene expression using Affymetrix microarray together with endocrine-associated serum analysis. RESULTS: In addition to strain-associated differences in insulin tolerance, we found significant elevations in KK/HlJ mouse serum leptin, insulin and aldosterone. Additionally, glucagon and corticosterone were elevated in female mice of both strains. Using 2-factor ANOVA and a significance level set at 0.05, we identified 10,269 pancreatic and 10,338 adrenal genes with an intensity cut-off of ≥2.0 for all 4 experimental groups. In the pancreas, gene expression upregulated in the KK/HlJ strain related to increased insulin secretory granule biofunction and pancreatic hyperplasia, whereas ontology of upregulated adrenal differentially expressed genes (DEGs) related to cell signaling and neurotransmission. We established a network of functionally related DEGs commonly upregulated in both endocrine tissues of KK/HlJ mice which included the genes coding for endocrine secretory vesicle biogenesis and regulation: PCSK2, PCSK1N, SCG5, PTPRN, CHGB and APLP1. We also identified genes with sex-biased expression common to both strains and tissues including the paternally expressed imprint gene neuronatin. CONCLUSION: Our novel results have further characterized the commonalities and diversities of pancreatic and adrenal gene expression between the KK/HlJ and C57BL/6 J strains as well as differences in serum markers of endocrine physiology.

IL-12 immunotherapy of Braf(V600E)-induced papillary thyroid cancer in a mouse model.

Papillary thyroid carcinoma (PTC) accounts for >80% thyroid malignancies, and BRAF(V600E) mutation is frequently found in >40% PTC. Interleukin-12 (IL-12) is a proinflammatory heterodimeric cytokine with strong antitumor activity. It is not known whether IL-12 immunotherapy is effective against Braf(V600E)-induced PTC. In the present study, we investigated the effectiveness of IL-12 immunotherapy against Braf(V600E)-induced PTC in LSL-Braf(V600E)/TPO-Cre mice. LSL-Braf(V600E)/TPO-Cre mice were created for thyroid-specific expression of Braf(V600E) under the endogenous Braf promoter, and spontaneous PTC developed at about 5 weeks of age. The mice were subjected to two treatment regimens: (1) weekly intramuscular injection of 50 µg plasmid DNA expressing a single-chain IL-12 fusion protein (scIL-12/CMVpDNA), (2) daily intraperitoneal injection of mouse recombinant IL-12 protein (mrIL-12, 100 ng per day). The role of T cells, natural killer (NK) cells, and transforming growth factor-ß (TGF-ß) in IL-12-mediated antitumor effects was determined by a (51)Cr-release cytotoxicity assay. Tumor size and weight were significantly reduced by either weekly intramuscular injection of scIL-12/CMVpDNA or daily intraperitoneal injection of mrIL-12, and tumor became more localized. Survival was significantly increased when treatment started at 1 week of age as compared with that at the 6 weeks of age. Both NK and CD8(+) T cells were involved in the cytotoxicity against tumor cells and their antitumor activity was significantly reduced in tumor-bearing mice. TGF-ß also inhibited the antitumor activity of NK and CD8(+) T cells. The immune suppression was completely reversed by IL-12 treatment and partially recovered by anti-TGF-ß antibody. We conclude that both IL-12 gene therapy and recombinant protein therapy are effective against PTC. Given that the immune response is significantly suppressed in tumor-bearing mice and can be restored by IL-12, the current study raises a possibility of the application of IL-12 as an adjuvant therapy for thyroid cancer.

ISCA2 mutation causes infantile neurodegenerative mitochondrial disorder.

BACKGROUND: There are numerous nuclear genes that cause mitochondrial disorders and clinically and genetically heterogeneous disorders whose aetiology often remains unsolved. In this study, we aim to investigate an autosomal recessive syndrome causing leukodystrophy and neuroregression. We studied six patients from five unrelated consanguineous families. METHODS: Patients underwent full neurological, radiological, genetic, metabolic and dysmorphological examinations. Exome sequencing coupled with autozygosity mapping, Sanger sequencing, microsatellite haplotyping, standard and molecular karyotyping and whole mitochondrial DNA sequencing were used to identify the genetic cause of the syndrome. Immunohistochemistry, transmission electron microscopy, confocal microscopy, dipstick assays, quantitative PCR, reverse transcription PCR and quantitative reverse transcription PCR were performed on different tissue samples from the patients. RESULTS: We identified a homoallelic missense founder mutation in ISCA2 leading to mitochondrial depletion and reduced complex I activity as well as decreased ISCA2, ISCA1 and IBA57 expression in fibroblasts. MRI indicated similar white matter abnormalities in the patients. Histological examination of the skeletal muscle showed mild to moderate variation in myofibre size and the presence of many randomly distributed atrophic fibres. CONCLUSIONS: Our data demonstrate that ISCA2 deficiency leads to a hereditary mitochondrial neurodegenerative white matter disease in infancy.

KRAS(G12D)-mediated oncogenic transformation of thyroid follicular cells requires long-term TSH stimulation and is regulated by SPRY1.

KRAS(G12D) can cause lung cancer rapidly, but is not sufficient to induce thyroid cancer. It is not clear whether long-term serum thyroid stimulating hormone (TSH) stimulation can promote KRAS(G12D)-mediated thyroid follicular cell transformation. In the present study, we investigated the effect of long-term TSH stimulation in KRAS(G12D) knock-in mice and the role of Sprouty1 (SPRY1) in KRAS(G12D)-mediated signaling. We used TPO-KRAS(G12D) mice for thyroid-specific expression of KRAS(G12D) under the endogenous KRAS promoter. Twenty TPO-KRAS(G12D) mice were given anti-thyroid drug propylthiouracil (PTU, 0.1% w/v) in drinking water to induce serum TSH and 20 mice were without PTU treatment. Equal number of wild-type littermates (TPO-KRAS(WT)) was given the same treatment. The expression of SPRY1, a negative regulator of receptor tyrosine kinase (RTK) signaling, was analyzed in both KRAS(G12D)-and BRAF(V600E)-induced thyroid cancers. Without PTU treatment, only mild thyroid enlargement and hyperplasia were observed in TPO-KRAS(G12D) mice. With PTU treatment, significant thyroid enlargement and hyperplasia occurred in both TPO-KRAS(G12D) and TPO-KRAS(WT) littermates. Thyroids from TPO-KRAS(G12D) mice were six times larger than TPO-KRAS(WT) littermates. Distinct thyroid histology was found between TPO-KRAS(G12D) and TPO-KRAS(WT) mice: thyroid from TPO-KRAS(G12D) mice showed hyperplasia with well-maintained follicular architecture whereas in TPO-KRAS(WT) mice this structure was replaced by papillary hyperplasia. Among 10 TPO-KRAS(G12D) mice monitored for 14 months, two developed follicular thyroid cancer (FTC), one with pulmonary metastasis. Differential SPRY1 expression was demonstrated: increased in FTC and reduced in papillary thyroid cancer (PTC). The increased SPRY1 expression in FTC promoted TSH-RAS signaling through PI3K/AKT pathway whereas downregulation of SPRY1 by BRAF(V600E) in PTC resulted in both MAPK and PI3K/AKT activation. We conclude that chronic TSH stimulation can enhance KRAS(G12D)-mediated oncogenesis, leading to FTC. SPRY1 may function as a molecular switch to control MAPK signaling and its downregulation by BRAF(V600E) favors PTC development.

Identification of the tetraspanin CD82 as a new barrier to xenotransplantation.

Significant immunological obstacles are to be negotiated before xenotransplantation becomes a clinical reality. An initial rejection of transplanted vascularized xenograft is attributed to Galα1,3Galß1,4GlcNAc-R (Galα1,3-Gal)-dependent and -independent mechanisms. Hitherto, no receptor molecule has been identified that could account for Galα1,3-Gal-independent rejection. In this study, we identify the tetraspanin CD82 as a receptor molecule for the Galα1,3-Gal-independent mechanism. We demonstrate that, in contrast to human undifferentiated myeloid cell lines, differentiated cell lines are capable of recognizing xenogeneic porcine aortic endothelial cells in a calcium-dependent manner. Transcriptome-wide analysis to identify the differentially expressed transcripts in these cells revealed that the most likely candidate of the Galα1,3-Gal-independent recognition moiety is the tetraspanin CD82. Abs to CD82 inhibited the calcium response and the subsequent activation invoked by xenogeneic encounter. Our data identify CD82 on innate immune cells as a major "xenogenicity sensor" and open new avenues of intervention to making xenotransplantation a clinical reality.

Increased CYP24A1 expression is associated with BRAF(V600E) mutation and advanced stages in papillary thyroid carcinoma.

OBJECTIVE: 1α, 25(OH)2 D3 (calcitriol), the active form of vitamin D, has been shown to exert antiproliferative effects in many cancers. Overexpression of CYP24A1, the primary vitamin D-inactivating enzyme, is also observed in a variety of human cancers, thus potentially neutralizing the antitumour effect of 1α, 25(OH)2 D3. This study investigates the expression of CYP24A1 and the effect of BRAF(V600E) on its expression in thyroid cancer. METHODS: We investigated 60 papillary thyroid carcinoma (PTC) specimens for CYP24A1 expression and its association with BRAF mutation and disease progression. CYP24A1 expression was measured by real-time RT-PCR, and BRAF(V600E) mutation was detected by PCR-DNA sequencing analysis. The interaction between BRAF(V600E) and CYP24A1 expression was determined by Western blot analysis and real-time RT-PCR. RESULTS: CYP24A1 expression was increased in PTC as compared to benign multinodular goitre. The expression was further increased in stage III and IV tumours. There is a strong correlation between CYP24A1 overexpression and BRAF(V600E) mutation (P < 0·01). In thyroid cancer cell lines expressing BRAF(V600E) , CYP24A1 expression was significantly higher when compared to those without BRAF(V600E) expression. BRAF(V600E) transgene expression in CAL62 cell line can induce CYP24A1 expression. Furthermore, BRAF(V600E) inhibitor PLX4720 can significantly down-regulate CYP24A1 expression and enhance the antiproliferative effects of calcitriol in thyroid cancer cell lines. CONCLUSION: CYP24A1 overexpression is a poor prognostic indicator for PTC and may reflect BRAF(V600E) mutation and MARK activation. The crosstalk between vitamin D and MAPK signalling pathways results in resistance to calcitriol-mediated antitumour effects, and the resistance can be reversed by BRAF(V600E) inhibitor PLX4720.

The interplay of intracellular calcium and zinc ions in response to electric field stimulation in primary rat cortical neurons in vitro.

Recent pharmacological studies demonstrate a role for zinc (Zn2+) in shaping intracellular calcium (Ca2+) dynamics and vice versa in excitable cells including neurons and cardiomyocytes. Herein, we sought to examine the dynamic of intracellular release of Ca2+ and Zn2+ upon modifying excitability of primary rat cortical neurons using electric field stimulation (EFS) in vitro. We show that exposure to EFS with an intensity of 7.69 V/cm induces transient membrane hyperpolarization together with transient elevations in the cytosolic levels of Ca2+ and Zn2+ ions. The EFS-induced hyperpolarization was inhibited by prior treatment of cells with the K+ channel opener diazoxide. Chemical hyperpolarization had no apparent effect on either Ca2+ or Zn2+. The source of EFS-induced rise in Ca2+ and Zn2+ seemed to be intracellular, and that the dynamic inferred of an interplay between Ca2+ and Zn2+ ions, whereby the removal of extracellular Ca2+ augmented the release of intracellular Ca2+ and Zn2+ and caused a stronger and more sustained hyperpolarization. We demonstrate that Zn2+ is released from intracellular vesicles located in the soma, with major co-localizations in the lysosomes and endoplasmic reticulum. These studies further support the use of EFS as a tool to interrogate the kinetics of intracellular ions in response to changing membrane potential in vitro.

Molecular characterization of a novel p.R118C mutation in the insulin receptor gene from patients with severe insulin resistance.

CONTEXT: Mutations of the insulin receptor gene (INSR) can cause genetic syndromes associated with severe insulin resistance. OBJECTIVES: We aimed to analyse INSR mutations in Saudi patients with severe insulin resistance. DESIGN: Ten patients with Type A insulin resistance syndrome from five unrelated Saudi families were investigated. The entire coding region of INSR was sequenced. The founder effect was assessed by microsatellite haplotype analysis. The functional effect of the mutation was investigated by in vitro functional assays. RESULTS: A novel biallelic c.433 C>T (p.R118C) mutation was detected in all patients. The c.433 C>T (p.R118C) sequence variation was not found in 100 population controls. The arginine residue at position 118 is located in the ligand-binding domain of INSR and is highly conserved across species. Microsatellite haplotype analysis of these patients indicated that p.R118C was a founder mutation created approximately 2900 years ago. The wild-type and mutant (R118C) INSR were cloned and expressed in CHO cells for functional analysis. Specific insulin binding to the mutant receptor was reduced by 83% as compared to wild-type (WT), although the mutant receptor was processed and expressed on the cell surface. Insulin-mediated receptor autophosphorylation was also significantly reduced in CHO(R118C) cells. CONCLUSIONS: Biallelic c.433 C>T (p.R118C) mutation of INSR causes significant damage to insulin binding and insulin-mediated signal transduction. p.R118C is a founder mutation frequently present in the Saudi patients with severe insulin resistance.

Toward allogenizing a xenograft: Xenogeneic cardiac scaffolds recellularized with human-induced pluripotent stem cells do not activate human naïve neutrophils.

The limited availability of human donor organs suitable for transplantation has resulted in ever-increasing patient waiting lists globally. Xenotransplantation is considered a potential option, but is yet to reach clinical practice. Although remarkable progress has been made in overcoming immunological rejection, issues with functionality are still to be resolved. Bioengineering approaches have been used to create cardiac tissues with optimized functions. The use of decellularized xenogeneic cardiac tissues seeded with donor-derived cardiac cells may prove to be a viable strategy as supporting structures of the native tissue such as vasculature can be utilized. Here we used sequential perfusion to decellularize adult rat hearts. The acellular scaffolds were reseeded with human endothelial cells, human fibroblasts, human mesenchymal stem cells, and cardiac cells derived from human-induced pluripotent stem cells. The ability of the resultant recellularized rat scaffolds to activate human naïve neutrophils in vitro was investigated to measure xenogeneic recognition. Our results demonstrate that in contrast to cadaveric xenogeneic hearts, acellular and recellularized xenogeneic scaffolds did not activate human naïve neutrophils and suggest that decellularization removes the xenogeneic antigens that lead to human naïve neutrophil activation thus allowing human cells to populate the now "allogenized" xenogeneic scaffolds.

Sex-dimorphism in cardiac nutrigenomics: effect of trans fat and/or monosodium glutamate consumption.

BACKGROUND: A paucity of information on biological sex-specific differences in cardiac gene expression in response to diet has prompted this present nutrigenomics investigation. Sexual dimorphism exists in the physiological and transcriptional response to diet, particularly in response to high-fat feeding. Consumption of Trans-fatty acids (TFA) has been linked to substantially increased risk of heart disease, in which sexual dimorphism is apparent, with males suffering a higher disease rate. Impairment of the cardiovascular system has been noted in animals exposed to Monosodium Glutamate (MSG) during the neonatal period, and sexual dimorphism in the growth axis of MSG-treated animals has previously been noted. Processed foods may contain both TFA and MSG. METHODS: We examined physiological differences and changes in gene expression in response to TFA and/or MSG consumption compared to a control diet, in male and female C57BL/6J mice. RESULTS: Heart and % body weight increases were greater in TFA-fed mice, who also exhibited dyslipidemia (P < 0.05). Hearts from MSG-fed females weighed less than males (P < 0.05). 2-factor ANOVA indicated that the TFA diet induced over twice as many cardiac differentially expressed genes (DEGs) in males compared to females (P < 0.001); and 4 times as many male DEGs were downregulated including Gata4, Mef2d and Srebf2. Enrichment of functional Gene Ontology (GO) categories were related to transcription, phosphorylation and anatomic structure (P < 0.01). A number of genes were upregulated in males and downregulated in females, including pro-apoptotic histone deacetylase-2 (HDAC2). Sexual dimorphism was also observed in cardiac transcription from MSG-fed animals, with both sexes upregulating approximately 100 DEGs exhibiting sex-specific differences in GO categories. A comparison of cardiac gene expression between all diet combinations together identified a subset of 111 DEGs significant only in males, 64 DEGs significant in females only, and 74 transcripts identified as differentially expressed in response to dietary manipulation in both sexes. CONCLUSION: Our model identified major changes in the cardiac transcriptional profile of TFA and/or MSG-fed mice compared to controls, which was reflected by significant differences in the physiological profile within the 4 diet groups. Identification of sexual dimorphism in cardiac transcription may provide the basis for sex-specific medicine in the future.

Effect of trans-fat, fructose and monosodium glutamate feeding on feline weight gain, adiposity, insulin sensitivity, adipokine and lipid profile.

The incidence of obesity and type 2 diabetes mellitus (T2DM) is increasing, and new experimental models are required to investigate the diverse aspects of these polygenic diseases, which are intimately linked in terms of aetiology. Feline T2DM has been shown to closely resemble human T2DM in terms of its clinical, pathological and physiological features. Our aim was to develop a feline model of diet-induced weight gain, adiposity and metabolic deregulation, and to examine correlates of weight and body fat change, insulin homeostasis, lipid profile, adipokines and clinical chemistry, in order to study associations which may shed light on the mechanism of diet-induced metabolic dysregulation. We used a combination of partially hydrogenated vegetable shortening and high-fructose corn syrup to generate a high-fat-high-fructose diet. The effects of this diet were compared with an isoenergetic standard chow, either in the presence or absence of 1.125 % dietary monosodium glutamate (MSG). Dual-energy X-ray absorptiometry body imaging and a glucose tolerance test were performed. The present results indicate that dietary MSG increased weight gain and adiposity, and reduced insulin sensitivity (P < 0.05), whereas high-fat-high-fructose feeding resulted in elevated cortisol and markers of liver dysfunction (P < 0.01). The combination of all three dietary constituents resulted in lower insulin levels and elevated serum ß-hydroxybutyrate and cortisol (P < 0.05). This combination also resulted in a lower first-phase insulin release during glucose tolerance testing (P < 0.001). In conclusion, markers of insulin deregulation and metabolic dysfunction associated with adiposity and T2DM can be induced by dietary factors in a feline model.

ß-Catenin Attenuation Inhibits Tumor Growth and Promotes Differentiation in a BRAFV600E-Driven Thyroid Cancer Animal Model.

BRAFV600E mutation is the most frequent genetic alteration in papillary thyroid cancer (PTC). ß-Catenin (Ctnnb1) is a key downstream component of canonical Wnt signaling pathway and is frequently overexpressed in PTC. BRAF V600E-driven tumors have been speculated to rely on Wnt/ß-catenin signaling to sustain its growth, although many details remain to be elucidated. In this study, we investigated the role of ß-catenin in BrafV600E -driven thyroid cancer in a transgenic mouse model. In Braf V600E mice with wild-type (WT) Ctnnb1 (BVE-Ctnnb1WT or BVE), overexpression of ß-catenin was observed in thyroid tumors. In Braf V600E mice with Ctnnb1 knockout (BVE-Ctnnb1null), thyroid tumor growth was slowed with significant reduction in papillary architecture. This was associated with increased expression of genes involved in thyroid hormone synthesis, elevated 124iodine uptake, and serum T4. The survival of BVE-Ctnnb1null mice was increased by more than 50% during 14-month observation. Mechanistically, downregulation of MAPK, PI3K/Akt, and TGFß pathways and loss of epithelial-mesenchymal transition (EMT) were demonstrated in the BVE-Ctnnb1null tumors. Treatment with dual ß-catenin/KDM4A inhibitor PKF118-310 dramatically improved the sensitivity of BVE-Ctnnb1WT tumor cells to BRAFV600E inhibitor PLX4720, resulting in significant growth arrest and apoptosis in vitro, and tumor regression and differentiation in vivo These findings indicate that ß-catenin signaling plays an important role in thyroid cancer growth and resistance to BRAFV600E inhibitors. Simultaneously targeting both Wnt/ß-catenin and MAPK signaling pathways may achieve better therapeutic outcome in BRAFV600E inhibitor-resistant and/or radioiodine-refractory thyroid cancer.

Human Properdin Released By Infiltrating Neutrophils Can Modulate Influenza A Virus Infection.

The complement system is designed to recognise and eliminate invading pathogens via activation of classical, alternative and lectin pathways. Human properdin stabilises the alternative pathway C3 convertase, resulting in an amplification loop that leads to the formation of C5 convertase, thereby acting as a positive regulator of the alternative pathway. It has been noted that human properdin on its own can operate as a pattern recognition receptor and exert immune functions outside its involvement in complement activation. Properdin can bind directly to microbial targets via DNA, sulfatides and glycosaminoglycans, apoptotic cells, nanoparticles, and well-known viral virulence factors. This study was aimed at investigating the complement-independent role of properdin against Influenza A virus infection. As one of the first immune cells to arrive at the site of IAV infection, we show here that IAV challenged neutrophils released properdin in a time-dependent manner. Properdin was found to directly interact with haemagglutinin, neuraminidase and matrix 1 protein Influenza A virus proteins in ELISA and western blot. Furthermore, modelling studies revealed that properdin could bind HA and NA of the H1N1 subtype with higher affinity compared to that of H3N2 due to the presence of an HA cleavage site in H1N1. In an infection assay using A549 cells, properdin suppressed viral replication in pH1N1 subtype while promoting replication of H3N2 subtype, as revealed by qPCR analysis of M1 transcripts. Properdin treatment triggered an anti-inflammatory response in H1N1-challenged A549 cells and a pro-inflammatory response in H3N2-infected cells, as evident from differential mRNA expression of TNF-α, NF-κB, IFN-α, IFN-ß, IL-6, IL-12 and RANTES. Properdin treatment also reduced luciferase reporter activity in MDCK cells transduced with H1N1 pseudotyped lentiviral particles; however, it was increased in the case of pseudotyped H3N2 particles. Collectively, we conclude that infiltrating neutrophils at the site of IAV infection can release properdin, which then acts as an entry inhibitor for pandemic H1N1 subtype while suppressing viral replication and inducing an anti-inflammatory response. H3N2 subtype can escape this immune restriction due to altered haemagglutinin and neuraminindase, leading to enhanced viral entry, replication and pro-inflammatory response. Thus, depending on the subtype, properdin can either limit or aggravate IAV infection in the host.

Sugar-sweetened carbonated beverage consumption correlates with BMI, waist circumference, and poor dietary choices in school children.

BACKGROUND: The prevalence of obesity and overweight is increasing globally. Frequently coexisting with under-nutrition in developing countries, obesity is a major contributor to chronic disease, and will become a serious healthcare burden especially in countries with a larger percentage of youthful population. 35% of the population of Saudi Arabia are under the age of 16, and adult dietary preferences are often established during early childhood years. Our objective was to examine the dietary habits in relation to body-mass-index (BMI) and waist circumference (W_C), together with exercise and sleep patterns in a cohort of male and female Saudi school children, in order to ascertain whether dietary patterns are associated with obesity phenotypes in this population. METHODS: 5033 boys and 4400 girls aged 10 to 19 years old participated in a designed Food Frequency Questionnaire. BMI and W_C measurements were obtained and correlated with dietary intake. RESULTS: The overall prevalence of overweight and obesity was 12.2% and 27.0% respectively, with boys having higher obesity rates than girls (P

C4b Binding Protein Acts as an Innate Immune Effector Against Influenza A Virus.

C4b Binding Protein (C4BP) is a major fluid phase inhibitor of the classical and lectin pathways of the complement system. Complement inhibition is achieved by binding to and restricting the role of activated complement component C4b. C4BP functions as a co-factor for factor I in proteolytic inactivation of both soluble and cell surface-bound C4b, thus restricting the formation of the C3-convertase, C4b2a. C4BP also accelerates the natural decay/dissociation of the C3 convertase. This makes C4BP a prime target for exploitation by pathogens to escape complement attack, as seen in Streptococcus pyogenes or Flavivirus. Here, we examined whether C4BP can act on its own in a complement independent manner, against pathogens. C4BP bound H1N1 and H3N2 subtypes of Influenza A Virus (IAV) most likely via multiple sites in Complement Control Protein (CCP) 1-2, 4-5, and 7-8 domains of its α-chain. In addition, C4BP CCP1-2 bound H3N2 better than H1N1. C4BP bound three IAV envelope proteins: Haemagglutinin (~70 kDa), Neuraminidase (~55 kDa), and Matrix protein 1 (~25kDa). C4BP suppressed H1N1 subtype infection into the lung epithelial cell line, A549, while it promoted infection by H3N2 subtype. C4BP restricted viral entry for H1N1 but had the opposite effect on H3N2, as evident from experiments using pseudo-typed viral particles. C4BP downregulated mRNA levels of pro-inflammatory IFN-α, IL-12, and NFκB in the case of H1N1, while it promoted a pro-inflammatory immune response by upregulating IFN- α, TNF-α, RANTES, and IL-6 in the case of H3N2. We conclude that C4BP differentially modulates the efficacy of IAV entry, and hence, replication in a target cell in a strain-dependent manner, and acts as an entry inhibitor for H1N1. Thus, CCP containing complement proteins such as factor H and C4BP may have additional defense roles against IAV that do not rely on the regulation of complement activation.

Complement-Independent Modulation of Influenza A Virus Infection by Factor H.

The complement system is an ancient innate immune defense mechanism that can recognize molecular patterns on the invading pathogens. Factor H, as an inhibitor of the alternative pathway, down-regulates complement activation on the host cell surface. Locally synthesized factor H at the site of infection/injury, including lungs, can act as a pattern recognition molecule without involving complement activation. Here, we report that factor H, a sialic acid binder, interacts with influenza A virus (IAV) and modulates IAV entry, as evident from down-regulation of matrix protein 1 (M1) in H1N1 subtype-infected cells and up-regulation of M1 expression in H3N2-infected A549 cells. Far-western blot revealed that factor H binds hemagglutinin (HA, ~70 kDa), neuraminidase (NA, ~60 kDa), and M1 (~25 kDa). IAV-induced transcriptional levels of IFN-α, TNF-α, IL-12, IL-6, IFN-α, and RANTES were reduced following factor H treatment for the H1N1 subtype at 6 h post-infection. However, for the H3N2 subtype, mRNA levels of these pro-inflammatory cytokines were enhanced. A recombinant form of vaccinia virus complement control protein (VCP), which like factor H, contains CCP modules and has complement-regulatory activity, mirrored the results obtained with factor H. Both factor H (25%), and VCP (45%) were found to reduce luciferase reporter activity in MDCK cells transduced with H1N1 pseudotyped lentiviral particles. Factor H (50%) and VCP (30%) enhanced the luciferase reporter activity for H3N2, suggesting an entry inhibitory role of factor H and VCP against H1N1, but not H3N2. Thus, factor H can modulate IAV infection and inflammatory responses, independent of its complement-related functions.

Mutation of SGK3, a Novel Regulator of Renal Phosphate Transport, Causes Autosomal Dominant Hypophosphatemic Rickets.

CONTEXT: Hypophosphatemic rickets (HR) is a group of rare hereditary renal phosphate wasting disorders caused by mutations in PHEX, FGF23, DMP1, ENPP1, CLCN5, SLC9A3R1, SLC34A1, or SLC34A3. OBJECTIVE: A large kindred with 5 HR patients was recruited with dominant inheritance. The study was undertaken to investigate underlying genetic defects in HR patients. DESIGN: Patients and their family members were initially analyzed for PHEX and FGF23 mutations using polymerase chain reaction sequencing and copy number analysis. Exome sequencing was subsequently performed to identify novel candidate genes. RESULTS: PHEX and FGF23 mutations were not detected in the patients. No copy number variation was observed in the genome using CytoScan HD array analysis. Mutations in DMP1, ENPP1, CLCN5, SLC9A3R1, SLC34A1, or SLC34A3 were also not found by exome sequencing. A novel c.979-96 T>A mutation in the SGK3 gene was found to be strictly segregated in a heterozygous pattern in patients and was not present in normal family members. The mutation is located 1 bp downstream of a highly conserved adenosine branch point, resulted in exon 13 skipping and in-frame deletion of 29 amino acids, which is part of the protein kinase domain and contains a Thr-320 phosphorylation site that is required for its activation. Protein tertiary structure modelling showed significant structural change in the protein kinase domain following the deletion. CONCLUSIONS: The c.979-96 T>A splice mutation in the SGK3 gene causes exon 13 skipping and deletion of 29 amino acids in the protein kinase domain. The SGK3 mutation may cause autosomal dominant HR.

Effect of dietary monosodium glutamate on trans fat-induced nonalcoholic fatty liver disease.

The effects of dietary monosodium glutamate (MSG) on trans-fatty acid (TFA)-induced nonalcoholic fatty liver disease (NAFLD) are addressed in an animal model. We used Affymetrix microarray analysis to investigate hepatic gene expression and the contribution of visceral white adipose tissue (WAT) to diet-induced NAFLD. Trans-fat feeding increased serum leptin, FFA, HDL-cholesterol (HDL-C), and total cholesterol (T-CHOL) levels, while robustly elevating the expression of genes involved in hepatic lipogenesis, including the transcription factor sterol-regulatory element binding protein 1c. Histological examination revealed hepatic macrosteatosis in TFA-fed animals. Conversely, dietary MSG at doses similar to human average daily intake caused hepatic microsteatosis and the expression of beta-oxidative genes. Serum triglyceride, FFA, and insulin levels were elevated in MSG-treated animals. The abdominal cavities of TFA- or MSG-treated animals had increased WAT deposition compared with controls. Microarray analysis of WAT gene expression revealed increased lipid biosynthetic gene expression, together with a 50% decrease in the key transcription factor Ppargc1a. A combination of TFA+MSG resulted in the highest levels of serum HDL-C, T-CHOL, and leptin. Microarray analysis of TFA+MSG-treated livers showed elevated expression of markers of hepatic inflammation, lipid storage, cell damage, and cell cycle impairment. TFA+MSG mice also had a high degree of WAT deposition and lipogenic gene expression. Levels of Ppargc1a were further reduced to 25% by TFA+MSG treatment. MSG exacerbates TFA-induced NAFLD.

Strain and sex-based glucocentric & behavioral differences between KK/HlJ and C57BL/6J mice.

INTRODUCTION: Small-animal models are the most widely used preclinical model for studying the etiology, pathology and treatment of diabetes, prediabetes and diabetic comorbidities. Diabetic patients are burdened with higher rates of depression, anxiety and cognitive decline due to inadequate control of blood glucose levels, vascular damage and aberrant CNS insulin signaling. The C57BL/6J model is amongst the most widely used mouse model due to its susceptibility to diet-induced obesity (DIO). This strain has also been well-characterized in behavioral research studies. However the C57BL/6J model has a number of limitations: [1] overt fasting hyperglycemia can only be induced by dietary manipulation and/or chemical ablation of the pancreatic beta cells. [2] There is heterogeneity in the obesogenic response to hypercaloric feeding as well as sex-dependent differences, with males being more responsive. The KK inbred strain has been used to study aspects of the metabolic syndrome and prediabetes due to inherent glucose intolerance, hyperinsulinemia and insulin resistance. However KK/HlJ mice are less well-characterized and there have been fewer behavioral studies reported. The aim of this study was to examine differences in male and female glucocentric parameters between KK/HlJ and C57BL/6J mice, and to compare their performance in a variety of standard behavioral tests relating to general, anxiogenic and cognitive paradigms. METHODS: Strain differences in male and female KK/HlJ and C57BL/6J mouse adiposity, glucose and insulin parameters were studied together with group differences in standard Open Field, Object Recognition, Elevated Plus Maze, Light-Dark Transition, Porsolt test, Marble Burying, Social Recognition and Morris Water Maze tests. Correlations between behavioral variables were analyzed. RESULTS AND CONCLUSION: In addition to being uniformly larger, hyperinsulinemic and more insulin intolerant than C57BL/6J mice, we observed marked strain and sex-differences in KK/HlJ behavior. KK/HlJ mice exhibited less locomotor and vertical exploratory behavior in comparison to C57BL/6J, whereas object exploration and novel object discrimination were superior in KK/HlJ mice. Female KK/HlJ mice were faster swimmers, whereas the males exhibited greater spatial cognition and place-learning during the MWM test.

Modifying inter-cistronic sequence significantly enhances IRES dependent second gene expression in bicistronic vector: Construction of optimised cassette for gene therapy of familial hypercholesterolemia.

Internal ribosome entry site (IRES) sequences have become a valuable tool in the construction of gene transfer and therapeutic vectors for multi-cistronic gene expression from a single mRNA transcript. The optimal conditions for effective use of this sequence to construct a functional expression vector are not precisely defined but it is generally assumed that the internal ribosome entry site dependent expression of the second gene in such as cassette is less efficient than the cap-dependent expression of the first gene. Mainly tailoring inter-cistronic sequence significantly enhances IRES dependent second gene expression in bicistronic vector further in construction of optimised cassette for gene therapy of familial hypercholesterolemia. We tailored the size of the inter-cistronic spacer sequence at the 5' region of the internal ribosome entry site sequence using sequential deletions and demonstrated that the expression of the 3' gene can be significantly increased to similar levels as the cap-dependent expression of the 5' gene. Maximum expression efficiency of the downstream gene was obtained when the spacer is composed of 18-141 base pairs. In this case a single mRNA transcriptional unit containing both the first and the second Cistron was detected. Whilst constructs with spacer sequences of 216 bp or longer generate a single transcriptional unit containing only the first Cistron. This suggests that long spacers may affect transcription termination. When the spacer is 188 bp, both transcripts were produced simultaneously in most transfected cells, while a fraction of them expressed only the first but not the second gene. Expression analyses of vectors containing optimised cassettes clearly confirm that efficiency of gene transfer and biological activity of the expressed transgenic proteins in the transduced cells can be achieved. Furthermore, Computational analysis was carried out by molecular dynamics (MD) simulation to determine the most emerges as viable containing specific binding site and bridging of 5' and 3' ends involving direct RNA-RNA contacts and RNA-protein interactions. These results provide a mechanistic basis for translation stimulation and RNA resembling for the synergistic stimulation of cap-dependent translation.