Qatar Foundation Annual Research Conference Proceedings Volume 2016 Issue 1

Klinefelter Syndrome (KS) was first described 73 years ago. Surprisingly KS yet remains underdiagnosed in the population. Lack of awareness about symptoms and the guidelines of KS diagnosis among paediatricians and General Practitioners (GP) could be the main reason. Failing to perform a thorough physical examination, not considering or unable to get cytogenetic confirmation on suspected patients are causative factors for delayed diagnosis. Hence long standing comordities such as learning disabilities, psychosocial problems and Attention Deficit Disorder in children remain under-recognized behind an undiagnosed clinical condition. The benefits of early diagnosis at prepubertal age are much higher with the available technologies such as sperm retrieval from testes biopsy and intracytoplasmic sperm injection. This could drastically improve fertility and quality of life (QoL). As prenatal diagnosis is not promising, population screening is suggested to be the way of ensuring timely diagnosis. We reviewed KS patients in Oman presented over a decade at our tertiary care centre and compared our finding with review of literature available. We realized that lack of awareness, cultural reasons and early marriage among men is some of causes for late diagnosis. Hence we propose that prepubertal diagnosis should surely be the focus to improve fertility and QoL in KS patients. This is the first report of KS from the Omani population.

Klinefelter syndrome (KS) is a well-known sex chromosomal disorder in men, affecting 0.1–0.2% of the male population. About 80–90% of KS patients show 47,XXY and the remaining lesser percentage of them present in either mosaic (47,XXY/46,XY) forms or with additional sex chromosomes (48,XXXY,48,XXYY,49,XXXXY) or structurally abnormal X chromosome acquired through non-disjunction during maternal or paternal gametogenesis. KS results from a nondisjunction event in the father, in nearly half of the cases. The prevalence of KS is much higher even among men with infertility (azoospermia) ranging from ∼3%–13%. It remains underdiagnosed with significant number of patients being unidentified, while only a 10% of prepubertal KS being diagnosed. Moreover in adulthood it was estimated that only one fourth (25%) of affected males are diagnosed, mostly due to their investigation for infertility.

Comorbidities of this syndrome are more prominent than symptoms of hypogonadism and hence the underlying disorder remains undiagnosed while the comorbidity gets treated. KS is characterized by a genetic, whole gonadal dysfunction affecting germ cells from early fetal life. Signs may vary depending upon gonadal dysfunction and sex hormone deficiency during early life (young adults) to infertility in an adult male without any other signs of hypogonadism. During pre-pubertal years, the condition is underdiagnosed because of low androgen levels and non-production of sperms. Any mild developmental disorders or cryptorchidism should be an alerting signs. There are no symptoms that reliably indicate KS in prepubertal patients. Hence, suspected KS can be diagnosed by thorough physical examination (testosterone deficiency and testicular size) or chance observation during treatment of concomitant disease. The goal should be to achieve timely diagnosis. Isidori et al, (2008) claims ultrasound can suggest KS as early as puberty stage, before any signs are noticed on clinical examination.

Prenatal diagnosis of KS is a chance finding with no specific indication of ultrasound marker feature or serum screening marker. Hence, no effective way is currently available to screen the population prenatally, apart from cytogenetic(karyotype) analysis. Thus, it is only possible when amniocenteses are performed due to advanced maternal age and are likely to have the opportunity of ‘chance-diagnosis’. Although maternal age is a well-known risk factor for meiotic non-disjunction (especially for Down Syndrome); Bojesen et al found a 4-fold increase in the prevalence of KS cases with maternal age being greater than 40 years when compared to those below 24 years.

KS is also the most frequent genetic cause of azoospermia. A significant proportion of men with KF remain undiagnosed, probably because of the wide phenotypic variability and lack of knowledge of the symptoms among health professionals. From the data based on patient registries in Denmark, it is suspected that, only 25% of all KS patients are diagnosed in their lifetime. Therfore, awareness regarding KS is important among health care professionals and General Practitioners (GP). Careful clinical examination to rule out KS, followed by cytogenetic confirmation helps in early detection. This might enable early treatment which in turn might improve fertility and their quality of life (QoL). The incidence of KS may vary across different populations, however studies suggest a higher prevalence in Australian and Asian population. To date, there are no reports on KS published from Omani population.

Therefore, we have made an observation/assessment of age at diagnosis of KS, and the reasons for delayed presentation/diagnosis, from patients encountered at our centre, over the past decade. Based on our experience and the review of data available from other population, we hereby present our data from Omani population studied at our tertiary care centre. As prenatal diagnosis is not promising in detecting KS foetus, population screening is likely to the best option of ensuing timely diagnosis for these patients. We also suggest early diagnosis could possibly improve fertility and QoL among KS patients.

Background

Vascular calcification and increased mortality is now well linked in hemodialysis (HD) patients. Among the inhibitors and promoters of calcium-phosphate and bone metabolism, we studied Sclerostin, a glycoprotein mainly expressed by osteocytes, involved in regulating bone formation by inhibiting Wnt–β-catenin signaling. Studies demonstrated, in experimental CDK animal model, a positive association between Sclerostin production and dietary phosphate intake.

Materials and Methods

We investigated the relation between Sclerostin levels (SL) and dietary habits in 49 Caucasian HD patients (age 36–90, M/F 31/18, dialysis vintage 1–144 months). Nutrient intake was analyzed with 24 h-recall method. Serum Sclerostin was measured by ELISA method. Statistically analysis was performed by SPSS software.

Results

Mean calcium and phosphate intake were 705 ± 584 and 1196 ± 498 mg/day, respectively. Serum SL (3356 ± 2118 pg/ml) were increased in HD patients. Positive correlation was found between serum SL and calcium (r = 0.33, p = 0.025) and phosphate intake (r = 0.34, p = 0.021). Patients with SL higher were older (p = 0.003), showed higher protein (p = 0.012), calcium (p = 0.017) and phosphate (p = 0.004) intake. Multiple linear regression testing Sclerostin as dependent variable (dialysis vintage, age, body-weight, serum calcium and phosphate, serum PTH, calcium and phosphate intake as independent variables) found that dietary phosphate intake was the only significant determinant of SL (r = 0.356, B = 1.5, p = 0.004). In Patients with calcium carbonate or acetate therapy (n = 31), serum SL was positively correlated with dietary calcium (r = 0.433, p = 0.017) and phosphate intake (r = 0.377, p = 0.04).

Conclusions

The dietary phosphate and calcium intake may influence serum Sclerostin levels and potentially affect bone remodeling or soft tissue calcification in HD patients.

Introduction

Cirrhosis is an abnormal liver condition, mainly caused by viral hepatitis B or C, fatty liver diseases and alcoholism. Ascites is common complication of cirrhosis, associated with poor quality of life, abnormal cognitive functions, increased work disability and increased risk of infection, consequently development of hepatic encephalopathy1. Studies have suggested that inflammation caused by secondary infection with hyper ammonia act as synergistic factor responsible for hepatic encephalopathy in cirrhotic patients2. Magnetic resonance imaging (MRI) is the most commonly used method to observe brain abnormalities in in cirrhotic patients. These patients showed hyperintensities in basal ganglion on T1-weighted MRI and abnormal brain metabolites such as increased Glx, decreased myo-inositol and choline level on MR spectroscopy (MRS), decreased magnetization transfer ratio, and increased mean diffusivity on diffusion tensor imaging MRI 3,4,5,6,7,8. Though different neuroimaging studies investigated structural, diffusion, functional and metabolic brain changes in adult cirrhotic, the regional changes in gray matter and structural brain connectivity are not yet studied in pediatric cirrhotic patients. In this study, we evaluated the gray matter changes and global and regional topological properties of structural brain networks in pediatric cirrhotic compared to pediatric controls.

Materials and methods

Institutional regulatory board and ethics committee approved the current study protocol. 22 pediatric cirrhotic (mean age 11.6 ± 3.4 years, no prior HE), and 17 age and sex matched heathy controls were included in this study. Written informed consent was obtained from each individual prior study. Cirrhosis was diagnosed by the presence of a combination of high serum-ascites albumin gradient ascites, splenomegaly, large varices without EHPVO, irregular liver surface, portal vein ≥  13 mm and collaterals. Magnetic resonance imaging (MRI) was performed at 3-T clinical MR Scanner (GE Healthcare Technologies, Milwaukee, WI, United States) using a standard quadrature head coil. Conventional T2-, T1-weighted imaging and high-resolution T1-weighted structural imaging using a fast spoiled gradient echo BRAVO pulse sequence (TR =  8.4 ms; TE =  3.32 ms; inversion time =  400 ms; FA =  13°; matrix size =  512′512; FOV =  240′240 mm²; slice-thickness =  1.0 mm), were performed on each subject. T2-, T1-weighted images were examined for any gross brain pathology, such as cysts, tumors, or any other mass lesions, and presence of such anomaly was used as an exclusion criteria. We used high-resolution T1-weighted structural images for measuring regional gray matter changes and construction of structural network. Brain imaging data were processed using the statistical parametric mapping package (SPM8, http://www.fil.ion.ucl.ac.uk/spm/), MRIcroN, and MATLAB-based (The Math Works Inc, Natick, MA) custom software. High-resolution T1-weighted images from all subjects were visually examined for the presence of tumors and cysts. High-resolution T1-weighted images corrected for any bias and inhomogeneity-corrected images were partitioned into gray, white, and cerebrospinal fluid tissue types using a unified segmentation approach9,10. Gray matter tissues maps were normalized to the Montreal Neurological Institute (MNI) space and were modulated and smoothed using a Gaussian filter (FWHM, 10 mm). For the strctural networks construction we used graph theory based analysis using GAT software by using gray matter maps as described in details elsewhere11. In brief we generated 90 cortical and subcortical regions of interest (ROIs), excluding the cerebellum, from the Automated Anatomical Labeling (AAL) atlas using the WFU PickAtlas Toolbox. The extracted residual volumes of all 90 anatomical ROIs were used for construction of structural correlation networks.

Statistical analysis

All statistical computations were performed using the Statistical Package for Social Sciences (SPSS) version 16.0 (SPSS Inc., Chicago, USA). The normalized and smoothed gray matter tissue probability maps were compared between groups using analysis of covariance (ANCOVA; uncorrected threshold, p =  0.01; extended threshold, 100 voxels), with age and gender included as covariates. A p value of less than 0.05 was considered to be statistically significant.

Results

Glass brain images in Fig. 1 are showing significantly lower gray matter volumes (GMV) in multiple brain sites with few brain areas showing significantly higher GMV in pediatric cirrhotic compared to those of controls (Fig. 1). The correlation matrix of the cirrhotic group showed overall lower correlation strength than the control group (Fig. 2). In pediatric cirrhotic reduced brain network characteristic across a range of network densities was observed compared to control (Fig. 3). Pediatric cirrhotic also showed altered structural connectivity networks and hubs (Fig. 4).

Discussion

Cirrhotic patients showed altered gray matter volumes suggesting the brain tissue injury and decreased regional connectivity (clustering coefficient), while reduced global network organization (small worldness) and integration (hubs) suggesting decreased robustness and efficiency of the brain network. These results contribute to novel insights regarding the neurobiological mechanisms underlying cognitive deficits in these patients. The pathophysiological mechanism of brain tissue injury may include hyper ammonia secondary to inflammation resulting neuronal tissue injury2. This structural analysis using voxel based and graph theory might provide a more appropriate paradigm for understanding complicated neurobiological mechanism of cirrhotic patients, and may help to improve the clinical managements of these patients 12.

References

1. Ferenci P, Lockwood A, Mullen K, et al. Hepatic encephalopathy: definition, nomenclature, diagnosis, and quantification-final report of the working party at the 11th World Congresses of Gastroenterology, Vienna, 1998.Hepatology 2002;35:716–21.

2. Butterworth RF. Pathogenesis of hepatic encephalopathy in cirrhosis: the concept of synergism revisited. Metab Brain Dis. 2015.

3. Lai PH, Chen C, Liang HL, et al. Hyperintense basal ganglia on T1-weighted MR imaging. AJR Am J Roentgenol 1999;172:1109–15.

4. Geissler A, Lock G, Fründ R, et al. Cerebral abnormalities in patients with cirrhosis detected by proton magnetic resonance spectroscopy and magnetic resonance imaging. Hepatology 1997;25:48–54.

5. Rovira A, Grivé E, Pedraza S, et al. Magnetization transfer ratio values and proton MR spectroscopy of normal-appearing cerebral white matter in patients with liver cirrhosis. AJNR Am J Neuroradiol 2001;22:1137–42.

6. Laubenberger J, Häussinger D, Bayer S, et al. Proton magnetic resonance spectroscopy of the brain in symptomatic and asymptomatic patients with liver cirrhosis. Gastroenterology 1997;112:1610–16.

7. Miese F, Kircheis G, Wittsack HJ, et al. 1H-MR spectroscopy, magnetization transfer, and diffusion-weighted imaging in alcoholic and nonalcoholic patients with cirrhosis with hepatic encephalopathy. AJNR Am J Neuroradiol 2006;27:1019–26.

8. Kale RA, Gupta RK, Saraswat VA, et al. Demonstration of interstitial cerebral edema with diffusion tensor MR imaging in type C hepatic encephalopathy. Hepatology 2006;43:698–706.

9. Ashburner J., Friston K. Multimodal image coregistration and partitioning—a unified framework. Neuroimage. 1997;6:209–217

10. Friston K.J., Holmes A., Worsley K.J., Poline J.B., Frith C.D., Frackowiak R.S. Statistical parametric maps in functional imaging: a general linear approach. Hum. Brain Mapp.1995;2:189–210.

11. Hosseini SM, Hoeft F, Kesler SR GAT: a graph-theoretical analysis toolbox for analyzing between-group differences in large-scale structural and functional brain networks. PLoS One. 2012;7:e40709.

12. Petrella JR: Use of graph theory to evaluate brain networks: a clinical tool for a small world? Radiology 2011, 259:317–320.

Critically ill patients in the intensive care units (ICUs) are often in acutely disturbed state of mind characterized by restlessness, illusions, and nervousness. Such patients, for instance, those who are mechanically ventilated may incur difficulties during treatment procedures such as endotracheal tube intubation/extubation. Apart from critical illness, treatment induced delirium may cause them to dislodge themselves from life-saving equipment and thus hinder cooperative and safe treatment in the ICU. Hence, it is often recommended to moderately sedate such patients for several days to reduce patient anxiety, facilitate sleep, aid treatment and thus endure patient safety. However, most anesthetics affect cardiac and respiratory functions. Hence, it is important to monitor and control the infusion of anesthetics to meet sedation requirements while keeping patient vital parameters within safe limits. The critical task of anesthesia administration also necessitates that drug dosing be optimal, patient specific, and robust.

Towards this end, we propose to use a reinforcement learning based approach to develop a closed-loop anesthesia controller that accounts for hemodynamic parameter variations. Main advantage of the proposed approach is that it does not require a model, it involves optimization, and is robust to interpatient variabilities. We formulate the problem of deriving control laws that track a desired trajectory as a sequential decision making problem represented by a finite Markov decision process (MDP) and then use reinforcement learning-based approach to solve the MDPs for goal oriented decision making. Specifically, we use reinforcement learning approaches, such as Q-learning, to develop a closed-loop anesthesia controller using the bispectral index (BIS) as a control variable while concurrently accounting for the mean arterial pressure (MAP). Moreover, the proposed method monitors and controls the infusion of anesthetics by minimizing a weighted combination of the error of the BIS and MAP signals. Account for two variables by considering the error reduces the computational complexity of the reinforcement learning algorithm and consequently the controller processing time.

We present simulation results and statistical results using the 30 simulated patients. For our simulations, the pharmacokinetic and the pharmacodynamic values of the simulated patients are chosen randomly from a predefined range. To quantify the performance of the trained agent in the closed-loop anesthesia control, we use the median performance error (MDPE), median absolute performance error (MDAPE), root mean square error (RMSE), and interquartile range (IQ). In order to further investigate the effect of simultaneous regulation of the BIS and MAP parameters on the sedation level (BIS) of a patient, we also conducted three different in silico case studies. In the first case study, a hemodynamic disturbance is considered in which the MAP is altered by d units. This case study considers the effect of other factors such as hemorrhage on MAP as an exogenous disturbance. In the second case study, the MAP is set to a constant value irrespective of propofol infusion, which corresponds to patients that remain intubated in the ICU with post-aortic aneurysm repair or septic patients with respiratory failure. In the third case study, a disturbance due to administration of a synergetic drug such as remifentanil is considered during the administration of propofol. This case study considers the effect of drug interaction on the closed-loop control of hypnotic agent administration.

In order to provide a protective host response to a vast array of invading pathological microorganisms the ability of naïve CD4⁺T cells to differentiate into discrete effector subsets, each able to mediate specific aspects of immunity is a central tenet of the adaptive immune response. T helper (Th) 1 cells mediate protection against intracellular pathogens, such as bacterial and viral infections, whereas Th2 cells mediate protective responses against extracellular pathogens such as helminthic parasites. Additionally, naive T cells may be induced to differentiate into T regulatory (Treg) cells, with Treg cells serving as an immunological checkpoint to dampen the immune response and protect against inappropriate activation of the immune system. As in the case of autoimmune diseases where aberrant Th1 cell differentiation occurs in response to self antigens or in the case of asthma and allergic responses where aberrant Th2 cell differentiation occurs in response to non-self environmental antigens. In order to activate naïve T cells and induce them to differentiate to combat a specific invading pathogen dendritic cells (DC), which are present throughout the body serving as cellular sentinels constantly surveying there surrounding for evidence of infection must be activated. Activation of DC occurs through ligand mediated activation of pathogen associated molecular pattern receptors or danger associated molecular pattern receptors. Allowing for the engagement of specific downstream patterns of effector molecule regulation that play an instructive role in the decision making process which occurs during the activation, division and differentiation of naïve T cells. Current dogma suggests that the generation of differentiated effector CD4⁺T cells takes place over a 3–4 day period following the initial engagement of the T cell receptor (TCR). Activation of CD4⁺T cells is thought to occur in three distinct functional phases, priming, proliferation and differentiation. Through the use of an in vitro culture system that allows for precise control of the factors which regulate the activation of naïve CD4⁺T cells we initially assessed the requirements for cytokine signaling vs. strength of TCR signaling during differentiation. Here, we determined that Th2 differentiation can be driven following activation with a weak TCR stimuli in the absence of additional cytokine inputs, indicating that Th2 differentiation occurs through a default endogenous pathway following activation. Whereas, Th1 differentiation required both a strong TCR signal and the presence of an instructive cytokine, in this case IFNg in order for differentiation to occur. We additionally investigated the kinetics of upregulation of the master transcription factors that regulate commitment to a specific T-helper phenotype. CD4⁺T cells acquire the disposition to commit to either Th1, Th2 or Treg lineages very soon after activation >24 hr and prior to the induction of division, as evidenced by increased expression of the master transcription factor proteins Tbet, GATA3 or Foxp3. Further, we show that entrance into cellular division is not required for the induction of a full program of differentiation to occur. By interrupting the activation of naïve CD4⁺T cells at different time points following stimulation through the use of anti-MHCII antibody. We were able to probe the effects that both alteration of TCR signal strength and alteration of TCR signal length have on the induction of differentiation as compared to division. This approach allowed us to demonstrate that TCR signaling is responsible for two distinct activation programs, whereby the strength of signal that a naïve T cell receives working in concert with or without cytokine at an early phase can induce Th1 or Th2 differentiation respectively. Whereas the length of the TCR signal can be construed as a secondary component of activation which controls the ability of CD4⁺T cells to enter into division. Here, we determined that at low concentrations of antigen TCR signaling must occur for 2 hr. Demonstrating that the signal delivered through the TCR represents not only an essential component for inducing an immune response through the initial activation of naïve CD4⁺T cells, but also acts as a rheostat that is able to determine both the strength and the length of TCR signal received. In turn controlling cellular decision making by dictating the outcome of differentiation and whether a cell will enter into cycle. As such these results have important implications for the rational design of vaccination strategies, in order to modulate components of TCR signaling cascade to direct an optimal response against the target vaccine antigens.

Background

Melanoma is an aggressive neoplasm characterized by a complex etiology. Several molecular alterations occur during melanoma progression. The most commonly mutated pathway is the mitogen-activated protein kinases (MAPK)/ERK cascade. The activation of the MAPK/ERK signaling occurs either through gain-of-function mutations in BRAF and NRAS gene or through autocrine growth factor stimulation. Documented mutations have been found in the kinase domain of BRAF gene encoded by exon 11 and 15 with a frequency of 50–70%. The majority of these mutations affect one critical amino acid, resulting in the V600E substitution which account for more than 90% of all BRAF mutations. Given the high incidence of BRAF V600E mutation in melanoma, patient management is based on the use of specific inhibitors when patients carry BRAF V600E mutation.

By comparing RNA-seq and DNA Sanger sequencing data, we found that among 15 melanoma cell lines 3 were discordant in the mutation detection (BRAF V600E at DNA level/Sanger sequencing and BRAF WT on RNA-seq). We initially postulated that those cell lines may express only the WT allele at the RNA level although mutated at the DNA level. A more careful analysis showed that these cell lines express very low level of BRAF RNA and the expression may be in favor of the WT allele.

Given the low BRAF V600E RNA expression, we tested in this study whether the three discordant cell lines may respond differently to BRAF-specific inhibitors compared to the concordant BRAF WT and BRAF V600E control cell lines.

Methods

The three discordant cell lines, one BRAF V600E and one BRAF WT control cell lines were treated with three BRAF inhibitors, including two BRAF V600E specific (vemurafenib and PLX4720) and one aspecific (sorafenib).

Measurement of cell proliferation was performed by MTT assay. Quantitative real time PCR and Western Blot were also performed to detect BRAF V600E RNA and protein expression and to assess MAPK pathway activation.

Results

The three discordant cell lines showed BRAF V600E expression both at the RNA and protein level and MAPK pathway activation although at a lower level as compared to the BRAF V600E control cell line. The proliferation rate of the discordant cell lines decreased after treatment with vemurafenib and PLX4720 but was not affected by treatment with sorafenib, suggesting a BRAF V600E biological behavior. Yet, responsiveness to the BRAF specific inhibitors was lower as compared to the BRAF V600E control.

All together these data suggest that the cell lines carrying BRAF V600E mutations at the DNA level may respond differently to BRAF targeted treatment and the differential responsiveness may be related to a lower BRAF V600E RNA and protein expression.

Background

Monoallelic expression (MAE) is a frequent genomic phenomenon in normal tissues, however its role in cancer is yet to be fully understood. MAE is defined as the expression of a gene that is restricted to one allele in the presence of a diploid heterozygous genome. Constitutive MAE occur for imprinted genes, odorant receptors and random X inactivation. Several studies in normal tissues have showed MAE in about 5–20% of the cases. However, little information exists on the MAE rate in cancer. In this study we assessed the presence and rate of MAE in melanoma. The genetic basis of melanoma has been studied in depth over the past decades, leading to the identification of mutations/genetic alterations responsible for melanoma development. To examine the role of MAE in melanoma we used 15 melanoma cell lines and compared their RNA-seq data with genotyping data obtained by the parental TIL (tumor infiltrating lymphocytes).

Methods

Genotyping was performed by Illumina HumanOmni1 beadchip. For the RNA-seq experiment, library preparation and sequencing was performed using the Illumina TruSeq Stranded Total RNA Human Kit and subsequently sequenced using a HiSeq 2500 according to manufacturer's guidelines. Genotype calling and subsequent quality filtering was performed in Illumina's Genome Studio software. IMPUTE2 (University of Oxford) was used for imputation of the genotyped data. RNA-Seq analysis was performed using the Broad Best Practice Workflows for RNA-Seq with the exception that a genotype was created for every available base pair in order to compare to the genotyped array data. In house custom perl scripts were then created to compare the genotyping data to the processed RNA-Seq data. The genotype and the B-allele frequency were calculated the latter creating a range of expression to evaluate.

By comparing genotyping data with RNA-seq data, we identified SNPs in which DNA genotypes were heterozygous and corresponding RNA genotypes were homozygous. All homozygous DNA genotypes were removed prior to the analysis. To confirm the validity to detect MAE, we examined heterozygous DNA genotypes from X chromosome of female samples as well as for imprinted and olfactory receptor genes and confirmed MAE.

Results

MAE was detected in all 15 cell lines although to a different rate (spanning from approximately 17% to 75% MAE). When looking at the B-allele frequencies we found a preferential pattern of complete monoallelic expression rather then differential monoallelic expression across the 15 melanoma cell lines. As some samples showed high differences in the homozygous and heterozygous call rate, we looked at the single chromosomes and showed that MAE may be explained by underlying large copy number imbalances and isodisomy in some instances. Nevertheless, some chromosome regions showed MAE without CN imbalances suggesting that additional mechanisms (including epigenetic silencing) may explain MAE in melanoma.

Conclusion

The biological implications of MAE are yet to be realized. Nevertheless our findings suggest that MAE is a common phenomenon in melanoma cell lines. Further analyses are currently being undertaken to evaluate whether MAE is gene/pathway specific and to understand whether MAE can be employed by cancers to achieve a more aggressive phenotype.

Background

Obesity related diseases including type 2 diabetes mellitus (T2DM) and non-alcoholic fatty liver disease have become major health problems. Inappropriate insulin production and dyslipidemia are commonly associated with obesity. It is multifactorial and heterogeneous in origin. While 60–80% of obese subjects are insulin resistant (IR) and rapidly develop metabolic diseases, called pathologically obese (PO), a proportion retain sensitivity to the hormone and remain relatively healthy (MHO), either because the progression to disease is slower in these individuals or they have developed pathways that renders them immune. Stratification of this disease, depending on the range of associated pathologies, would help identify mediators, design targeted therapies, in the understanding of mechanisms for this apparent protection. Also some ethnicities, such as South Asians and Arabs, appear susceptible to both obesity and its associated pathologies, perhaps largely determined by lifestyle factors, such as diet and exercise. However weight loss, mainly surgical, is proving to be the most successful means of reducing body weight and improving insulin sensitivity.

Therefore the aims of this study were to compare morbidly obese patients of Caucasian and Arab origin prior to and after weight loss to identify biomarkers of insulin sensitivity and inflammation in Arabs and Caucasians.

Methods

Subjects: Morbidly obese patients of Arab and Caucasian origins awaiting bariatric surgery (gastric bypass, gastric sleeve, or gastric band) were recruited from the pre-operative clinics: Al-Emadi Hospital, Hamad Medical Corporation, (Doha, Qatar) and Whittington Hospital (North London Obesity Surgery Service, Whittington Hospital, London, UK). Morbid obesity was defined as BMI ≥ 40 kg.m^− 2 or BMI ≥ 35 kg.m^− 2 with significant co-morbidities. All studies were approved by the relevant National Ethics Committees.

The studies included both males and females over the age of 18 years. Patients with coronary artery disease, uncontrolled hypertension, malignancy or terminal illness, connective tissue disease or other inflammatory conditions likely to affect cytokine levels, immuno-compromised subjects and those with substance abuse or other causes for poor compliance were excluded.

Anthropometric measurements were recorded: age (years), weight (kg), and height (m) systolic and diastolic blood pressure (mmHg). BMI was calculated (kg.m^− 2). Patient information including demographic data (date of birth, gender, ethnicity), surgery type (gastric bypass, gastric band, gastric sleeve), co-morbidities, current medication, weight loss history, smoking habits and alcohol consumption were recorded from hospital notes.

Samples: Blood samples (EDTA, NaF, no anti-coagulent), following an overnight fast, were drawn from an ante-cubital vein on the day of the operation, immediately after anesthesia. Samples were centrifuged (3000 rpm, 15 minutes, 25°C), and the plasma or serum collected and stored at − 80 °C prior to assay.

Assays: Blood samples were used to determine glucose (hexokinase), lipids (Total Cholesterol, LDL and HDL: Roche) and insulin (ELISA, Mercodia). Adipokines (leptin, adiponectin, interleukin-6, and MCP1) were assayed by ELISA (R&D Systems, Oxon, UK). Insulin resistance was calculated using the homeostatic model assessment where HOMA =  (glucose in mmol/L ×  insulin in miU/ml)/22.5.

The criteria for classification: Subjects were considered MHO if free of T2DM, dyslipidaemia, and cardiovascular disease, and exhibited systolic blood pressure less than 140 mmHg, diastolic blood pressure less than 85 mmHg, fasting plasma glucose less than 6.8 mmol/l and insulin less than 6.5 miU/ml.

Statistics: Data were entered in SPSS version 22.0 for statistical analysis. Parametric tests were used for normally distributed data and non-parametric analysis for skewed data.

Results

Effect of ethnicity on cardiometabolic risk factors.

Despite the Arab cohort being significantly younger, they were hyperglycemic hyperinsulemic and hyperleptinaemic, compared to the Caucasians. This population also had elevated β-cell function and insulin resistance, while insulin sensitivity was lower. However there was no significant difference in blood pressure. Total-, LDL- and HDL-cholesterol were higher, but triglycerides lower, in Arabs. Leptin, a marker of adipose tissue mass and adipocyte hypertrophy, was elevated in Arabs. Furthermore the proinflammatory adipokines (MCP-1, IL-6) were higher and the anti-inflammatory adipokines (adiponectin) lower in this population.

Weight loss

In Caucasians, there was an increase in both HDL- and LDL-cholesterol in the PO group, and in serum adiponectin in both MHO and PO patients, following surgery. However, the insulin levels and HOMA-IR index decreased significantly in the PO group. Fasting plasma glucose did not change after weight loss in either group, whereas the total cholesterol increased in both groups significantly. In Arabs, three months after surgery, both MHO and PO subjects showed significant reduction in BMI, which was accompanied by lower systemic insulin, HOMA-IR and leptin. There was no change in total-, LDL- and HDL-cholesterol, whereas triglycerides were reduced after the weight loss. The inflammatory biomarkers CRP and IL-6 were unchanged.

Conclusion

Despite both populations being equally obese the Arabs had greater prevalence of risk factors for cardio metabolic complications, with fewer having a metabolically healthy phenotype. In non-diabetic Arabs, compared to Caucasians, insulin resistance and inflammation appeared to be predominant lesions. Interestingly higher leptin levels in the BMI-matched Arabs points to adipocyte hypertrophy and adipose tissue dysfunction as causal factors in these lesions. The young age at which Arabs develop obesity perhaps explains the greater susceptibility to its pathological consequences.

Store-operated calcium entry (SOCE) is a ubiquitous Ca²⁺ influx pathway essential for many physiological functions and failure to maintain normal calcium homeostasis is one of the leading causes of cellular dysfunction in a wide variety of pathological conditions. Orai1, a key regulator of SOCE, constitutively recycles at steady state in the frog oocyte and internalizes into intracellular vesicular compartments during meiosis, leading to inactivation of SOCE. Such mechanism provides proper regulation of Ca²⁺ signaling in preparation for fertilization and embryonic development. Previous data showed a role for Orai1 C-terminus in its internalization during meiosis. However, the minimal region required for Orai1 internalization at steady state and during meiosis is not known.

We began the study of the molecular determinants of Orai1 trafficking in Xenopus oocytes by comparing the localization of multiple GFP-Orai1 C-terminal mutants 1-266, 1-275, and 1-285 intracellularly. Orai1 mutants 1-275 and 1-285 were both internalized during meiosis behaving similarly to WT, however, Orai1 1-266 was significantly enriched at the plasma membrane and did not internalize during oocyte maturation. To further map the region required for Orai1 internalization, we will generate specific mutants in the region 267-275 and evaluate the contribution of these signals in Orai1 internalization during meiosis. Mutants showing defect in internalization will be tested for phenotype rescue by co-injecting Orai1 with different potential candidates. These proteins include: 1) Rab5, a member of Rab family of GTPases has been shown to play a role as a regulator of intracellular trafficking during endocytosis. Previous data from our group has shown that Rab5 colocalizes with Orai1 in both oocytes and eggs. 2) Caveolin-1, a coat protein mediating caveolin-dependent endocytosis. 3) Flotilin-1, a membrane-associated protein involved in flotilin-dependent endocytosis.

As a second step in exploring the molecular and biochemical mechanisms underlying Orai1 internalization during meiosis, we will identify Orai1 associated proteins by co-immunoprecipitation of Orai1 complexes followed by quantitative proteomic analysis using dimethyl labeling coupled with tandem liquid chromatography-mass spectrometry (LC-MS). Candidates that are enriched in wild-type Orai1 immunoprecipitation but missing in internalization-deficient Orai1 mutants are suggested to play role in Orai1 endocytosis.

In conclusion, our results here suggest that residues 266–275 at C-terminus of Orai1 are essential for its internalization during meiosis. Using specific mutant(s) within this region, we will test the rescue of mutant(s) phenotype with potential candidates, and by quantitative proteomic analysis identify proteins associated with Orai1 and potentially important in its internalization.