Fahad Zadjali

Use of Germ-Free Animal Models in Microbiota-Related Research.

The large intestine is a home for trillions of microbiota, which confer many benefits on the host, including production of vitamins, absorption of nutrients, pathogen displacement, and development of the immune system. For several decades, germ-free animals have been used to study the interaction between the host and its microbiota. This minireview describes the technical aspects for establishing and maintaining germ-free animals and highlights the advantages and disadvantages for germ-free animals as experimental models.

Gut microbial communities modulating brain development and function

Mammalian brain development is initiated in utero and internal and external environmental signals can affect this process all the way until adulthood. Recent observations suggest that one such external cue is the indigenous microbiota which has been shown to affect developmental programming of the brain. This may have consequences for brain maturation and function that impact on cognitive functions later in life. This review discusses these recent findings from a developmental perspective.

Suppressor of Cytokine Signaling 6 (SOCS6) Negatively Regulates Flt3 Signal Transduction through Direct Binding to Phosphorylated Tyrosines 591 and 919 of Flt3

Background: Flt3 is an important regulator of hematopoiesis and is often found mutated and constitutively active in patients with acute myeloid leukemia. Results: SOCS6 is up-regulated by Flt3 activation and binds to phosphorylated Flt3. Conclusion: SOCS6 is a negative regulator of Flt3 signaling. Significance: Our results provide a role for SOCS6 in Flt3 signaling. The absence of SOCS6 promotes transformation of cells by Flt3 ITD. The receptor tyrosine kinase Flt3 is an important growth factor receptor in hematopoiesis, and gain-of-function mutations of the receptor contribute to the transformation of acute myeloid leukemia. SOCS6 (suppressor of cytokine signaling 6) is a member of the SOCS family of E3 ubiquitin ligases that can regulate receptor tyrosine kinase signal transduction. In this study, we analyzed the role of SOCS6 in Flt3 signal transduction. The results show that ligand stimulation of Flt3 can induce association of SOCS6 and Flt3 and tyrosine phosphorylation of SOCS6. Phosphopeptide fishing indicated that SOCS6 binds directly to phosphotyrosines 591 and 919 of Flt3. By using stably transfected Ba/F3 cells with Flt3 and/or SOCS6, we show that the presence of SOCS6 can enhance ubiquitination of Flt3, as well as internalization and degradation of the receptor. The presence of SOCS6 also induces weaker activation of Erk1/2, but not Akt, in transfected Ba/F3 and UT-7 cells and in OCI-AML-5 cells. The absence of SOCS6 promotes Ba/F3 and UT-7 cell proliferation induced by oncogenic internal tandem duplications of Flt3. Taken together, these results suggest that SOCS6 negatively regulates Flt3 activation, the downstream Erk signaling pathway, and cell proliferation.

Structural Basis for c-KIT Inhibition by the Suppressor of Cytokine Signaling 6 (SOCS6) Ubiquitin Ligase

The c-KIT receptor tyrosine kinase mediates the cellular response to stem cell factor (SCF). Whereas c-KIT activity is important for the proliferation of hematopoietic cells, melanocytes and germ cells, uncontrolled c-KIT activity contributes to the growth of diverse human tumors. Suppressor of cytokine signaling 6 (SOCS6) is a member of the SOCS family of E3 ubiquitin ligases that can interact with c-KIT and suppress c-KIT-dependent pathways. Here, we analyzed the molecular mechanisms that determine SOCS6 substrate recognition. Our results show that the SH2 domain of SOCS6 is essential for its interaction with c-KIT pY568. The 1.45-Å crystal structure of SOCS6 SH2 domain bound to the c-KIT substrate peptide (c-KIT residues 564–574) revealed a highly complementary and specific interface giving rise to a high affinity interaction (Kd = 0.3 μm). Interestingly, the SH2 binding pocket extends to substrate residue position pY+6 and envelopes the c-KIT phosphopeptide with a large BG loop insertion that contributes significantly to substrate interaction. We demonstrate that SOCS6 has ubiquitin ligase activity toward c-KIT and regulates c-KIT protein turnover in cells. Our data support a role of SOCS6 as a feedback inhibitor of SCF-dependent signaling and provides molecular data to account for target specificity within the SOCS family of ubiquitin ligases.

SOCS2 deletion protects against hepatic steatosis but worsens insulin resistance in high-fat-diet-fed mice

Hepatic steatosis is a prominent feature in patients with growth hormone (GH) deficiency. The ubiquitin ligase SOCS2 attenuates hepatic GH signaling by inhibiting the Janus kinase 2 (JAK2)‐signal transducer and activator of transcription 5b (STAT5b) axis. Here, we investigated the role of SOCS2 in the development of diet‐induced hepatic steatosis and insulin resistance. SOCS2‐knockout (SOCS2−/−) mice and wild‐type littermates were fed for 4 mo with control or high‐fat diet, followed by assessment of insulin sensitivity, hepatic lipid content, and expression of inflammatory cytokines. SOCS2−/− mice exhibited increased hepatic TG secretion by 77.6% (P< 0.001) as compared with wild‐type control mice and were protected from high‐fat‐diet (HFD)‐induced hepatic steatosis, showing 49.3% (P<0.01) reduction in liver TG levels compared to HFD‐fed wild‐type littermates. In contrast, we found that HFD‐triggered attenuation of systemic insulin sensitivity was more marked in SOCS2−/− mice. Livers from the HFD‐fed SOCS2−/− mice showed increased NF‐κB activity as well as elevated expression of genes for the inflammatory cytokines IFN‐γ and IL‐6. An inhibitory role of SOCS2 on Toll‐like receptor 4 signaling was demonstrated in macrophages obtained from the SOCS2−/− and wild‐type mice. This study identified SOCS2 as an important regulator of hepatic homeostasis under conditions of high‐fat dietary stress.—Zadjali, F., Santana‐Farre, R., Vesterlund, M., Carow, B., Mirecki‐Garrido, M., Hernandez‐Hernandez, I., Flodström‐Tullberg, M., Parini, P., Rottenberg, M., Norstedt, G., Fernandez‐Perez, L., Flores‐Morales, A. SOCS2 deletion protects against hepatic steatosis but worsens insulin resistance in high‐fat‐diet‐fed mice. FASEB J. 26, 3282–3291 (2012). www.fasebj.org

Association of adiponectin promoter variants with traits and clusters of metabolic syndrome in Arabs: Family-based study

Plasma levels of adiponectin are decreased in type 2 diabetes, obesity and hypertension. Our aim was to use a family-based analysis to identify the genetic variants of the adiponectin (ADIPOQ) gene that are associated with obesity, insulin resistance, dyslipidemia and hypertension, among Arabs. We screened 328 Arabs in one large extended family for single nucleotide polymorphisms (SNPs) in the promoter region of the ADIPOQ gene. Two common SNPs were detected: rs17300539 and rs266729. Evidences of association between traits related to the metabolic syndrome and the SNPs were studied by implementing quantitative genetic association analysis. Results showed that SNP rs266729 was significantly associated with body weight (p-value=0.001), waist circumference (p-value=0.037), BMI (p-value=0.015) and percentage of total body fat (p-value=0.003). Up to 4.1% of heritability of obesity traits was explained by the rs266729 locus. Further cross-sectional analysis showed that carriers of the G allele had significantly higher values of waist circumference, BMI and percentage of total body fat (p-values 0.014, 0.004 and 0.032, respectively). No association was detected between SNP rs266729 and other clusters of metabolic syndrome or their traits except for HOMA-IR and fasting plasma insulin levels, p-values 0.035 and 0.004, respectively. In contrast, both measured genotype and cross-sectional analysis failed to detect an association between the SNP rs17300539 with traits and clusters of metabolic syndrome. In conclusion, we showed family-based evidence of association of SNP rs266729 at ADIPOQ gene with traits defining obesity in Arab population. This is important for future prediction and prevention of obesity in population where obesity is in an increasing trend.

Busulphan-Cyclophosphamide Cause Endothelial Injury, Remodeling of Resistance Arteries and Enhanced Expression of Endothelial Nitric Oxide Synthase

Stem cell transplantation (SCT) is a curative treatment for malignant and non malignant diseases. However, transplantation-related complications including cardiovascular disease deteriorate the clinical outcome and quality of life. We have investigated the acute effects of conditioning regimen on the pharmacology, physiology and structure of large elastic arteries and small resistance-sized arteries in a SCT mouse model. Mesenteric resistance arteries and aorta were dissected from Balb/c mice conditioned with busulphan (Bu) and cyclophosphamide (Cy). In vitro isometric force development and pharmacology, in combination with RT-PCR, Western blotting and electron microscopy were used to study vascular properties. Compared with controls, mesenteric resistance arteries from the Bu-Cy group had larger internal circumference, showed enhanced endothelium mediated relaxation and increased expression of endothelial nitric oxide synthase (eNOS). Bu-Cy treated animals had lower mean blood pressure and signs of endothelial injury. Aortas of treated animals had a higher reactivity to noradrenaline. We conclude that short-term consequences of Bu-Cy treatment divergently affect large and small arteries of the cardiovascular system. The increased noradrenaline reactivity of large elastic arteries was not associated with increased blood pressure at rest. Instead, Bu-Cy treatment lowered blood pressure via augmented microvascular endothelial dependent relaxation, increased expression of vascular eNOS and remodeling toward a larger lumen. The changes in the properties of resistance arteries can be associated with direct effects of the compounds on vascular wall or possibly indirectly induced via altered translational activity associated with the reduced hematocrit and shear stress. This study contributes to understanding the mechanisms that underlie the early effects of conditioning regimen on resistance arteries and may help in designing further investigations to understand the late effects on vascular system.

Nutraceuticals as Lipid-Lowering Treatment in Pregnancy and Their Effects on the Metabolic Syndrome.

Maternal nutrition and lifestyle before and during pregnancy influence both mother and offsprings health and can be correlated with the metabolic syndrome in later life. Findings from animal and human studies indicate that nutrition during pregnancy has an important role in microbiological, metabolic, physiologic and immunologic development and homeostasis. A low nutritional intake in early pregnancy may represent a risk for adverse effects during pregnancy as well as on birth outcome. It seems that dietary supplementation with probiotics in perinatal period may represent safe and practical approach in dealing with the most common adverse pregnancy outcomes such as obesity and gestational diabetes. The SPRING (Study of Probiotics in the prevention of Gestational diabetes) will give important answers about potential benefits of probiotics in pregnant women who are obese and overweight and otherwise at the high risk for complications during pregnancy. Fish oil supplementation during the last trimester of pregnancy showed no effects on plasma lipids and lipoproteins in offspring, as well as on their adiposity. The effect of hypercholesterolemia during pregnancy on both mothers and child needs to be further investigated as it could have a biological role. The guidelines for the eventual clinical approach currently do not exist. Potential benefits of nutraceuticals on several metabolic parameters have been suggested. Limited evidence does not allow to draw final conclusions on preventive health strategies and dietary patterns that should be promoted during pregnancy. Further prospective and intervention studies are needed to establish it. Healthy lifestyle and dietary advice with appropriate supplements usage should be considered.

Frequencies of the Arg16Gly, Gln27Glu and Thr164Ile Adrenoceptor β2 Polymorphisms among Omanis.

OBJECTIVES This study aimed to assess the distribution of missense mutations in the adrenoceptor β2 (ADRB2) gene in an Omani cohort. METHODS This study was carried out between May 2014 and March 2015 at the Sultan Qaboos University, Muscat, Oman. Blood samples were taken from 316 unrelated Omani subjects. Genotyping for rs1042713 (c.46A>G, p.Arg16Gly), rs1042714 (c.79C>G, p.Gln27Glu) and rs1800888 (c.491C>T, p.Thr164Ile) polymorphisms was performed by real-time polymerase chain reaction using single nucleotide polymorphism (SNP) genotyping assays. The allelic frequencies of these polymorphisms were estimated on the basis of the observed numbers of specific alleles from the genotype data for male and female subjects. The genotype frequencies for each polymorphism were tested for deviation from the Hardy-Weinberg equilibrium. RESULTS Gly16 and Glu27 were the most frequent variants found among the cohort (63% and 75%, respectively). The Ile164 variant was not detected in the study population. There was a significant linkage disequilibrium between the rs1042713 and rs1042714 SNPs (r(2) = 0.209; P ≤0.001). The most observed haplotypes were Gly16-Gln27 and Arg16-Gln27 (0.37 and 0.38, respectively). The frequency of Gly16-Glu27 was 0.25, comprising all Glu27 carriers. CONCLUSION The allelic distribution of variants in this Omani cohort was similar to distributions reported among Caucasian populations.

The ubiquitin ligase Cullin5 SOCS2 regulates NDR1/STK38 stability and NF-κB transactivation

SOCS2 is a pleiotropic E3 ligase. Its deficiency is associated with gigantism and organismal lethality upon inflammatory challenge. However, mechanistic understanding of SOCS2 function is dismal due to our unawareness of its protein substrates. We performed a mass spectrometry based proteomic profiling upon SOCS2 depletion and yield quantitative data for ~4200 proteins. Through this screen we identify a novel target of SOCS2, the serine-threonine kinase NDR1. Over-expression of SOCS2 accelerates turnover, while its knockdown stabilizes, endogenous NDR1 protein. SOCS2 interacts with NDR1 and promotes its degradation through K48-linked ubiquitination. Functionally, over-expression of SOCS2 antagonizes NDR1-induced TNFα-stimulated NF-κB activity. Conversely, depletion of NDR1 rescues the effect of SOCS2-deficiency on TNFα-induced NF-κB transactivation. Using a SOCS2−/− mice model of colitis we show that SOCS2-deficiency is pro-inflammatory and negatively correlates with NDR1 and nuclear p65 levels. Lastly, we provide evidence to suggest that NDR1 acts as an oncogene in prostate cancer. To the best of our knowledge, this is the first report of an identified E3 ligase for NDR1. These results might explain how SOCS2-deficiency leads to hyper-activation of NF-κB and downstream pathological implications and posits that SOCS2 induced degradation of NDR1 may act as a switch in restricting TNFα-NF-κB pathway.