Qatar Foundation Annual Research Conference Proceedings Volume 2016 Issue 1

Summary

Insulin resistance manifest in skeletal muscle, liver and adipocytes. Insulin stimulates muscle glucose uptake, suppresses the rate of hepatic glucose production (HGP) and restrains lipolysis. In insulin resistant individuals, insulin-stimulated glucose uptake is markedly reduced in skeletal muscle, impaired insulin-mediated suppression of hepatic glucose production (HGP) and inhibition of lipolysis. It is evident from human and animal studies that diabetes alters miRNAs expression in metabolically important organs (adipose, liver and skeletal muscle).micRNA are small non coding RNA molecules. However, they play critical role in the regulation of gene expression via post-transcriptional mechanisms. They are small in size (21?23 nucleotides) and recent studies have demonstrated that they play important role in many human complex diseases including diabetes. The ability of miRNA that simultaneously regulate many target genes makes them attractive candidates for regulating insulin production/secretion and action. mRNA are secreted to the circulation and are taken up from the circulation by several tissues, making them an attractive mechanism for inter-tissue signaling molecules. Thus, if micro RNA play role in the pathogenesis of insulin resistance or in communicating between insulin resistant tissues and the beta cell to regulate the level of beta cell function in concert with the prevailing level of insulin resistance, we anticipate a change in the circulatory profile of micro RNA in insulin resistant individuals compared to insulin sensitive individuals. Such a distinct circulatory profile of micro RNA in insulin sensitive versus insulin resistant individuals could provide a signature for the identification of insulin resistant individuals. Moreover, differentially expressed micro RNA molecules in insulin resistant individuals could serve signaling molecules which convey messages between insulin resistant tissues, i.e. liver, skeletal muscle and adipocytes and the beta cell. Insulin sensitivity is commonly assessed to predict the risk and evaluate the management of type 2 diabetes in clinical and research settings. Insulin sensitivity can be measured using a variety of techniques that are commonly employed in diabetes research and care. Among them, euglycemic hyperinsulemic clamp is the gold-standard method to quantify the sensitivity to insulin.

The aim of the present study is to determine the profile of miRNA in the plasma in insulin sensitive Arab individuals and compare the result to that in insulin resistant Arab individuals.

specific aims are;

1. To discover the circulatory profile of microRNA in insulin sensitive and insulin resistant Arab individuals by using next generation sequencing technology.

2. To identify novel microRNA/targets which are differentially expressed in insulin resistant individuals by real time Taq-Man PCR.

3. To explore the effects changes of these miRNAs in insulin resistance diabetes β-Cell Biology. The experiments range from relevant tissue and cell cultures systems to human physiology. Design and methods: The circulating miRNA profile was assessed in a pilot study of 20 healthy volunteers whom glucose tolerance status was determined by the OGTT, and their insulin sensitivity was quantitated by the gold standard method the euglycemic hyperinsulemic clamp. Based on Glucose Infusion rate (GIR: mg/kg/min), before and after a 6-h hyperinsulinemeic euglycemic clamp, we selected two groups: 10 with high levels of GIR (insulin-sensitive group) and 10 with Very low levels of GIR (insulin resistant group).The serum samples was collected from the two groups for miRNA extraction and subsequent sequencing using the True-seq Illumina protocol. A small RNA library was generated from samples using the Illumina Truseq™ Small. The purified cDNA library was used for cluster generation on Illumina's Cluster Station and then sequenced on Illumina GAIIx following vendor's instruction for running the instrument. Raw sequencing reads (40 nts) were obtained using Illumina's Sequencing Control Studio software version 2.8 (SCS v2.8) following real-time sequencing image analysis and base-calling by Illumina's Real-Time Analysis version 1.8.70 (RTA v1.8.70). Result: Sequencing of the small circulating RNA isolated from insulin-resistant and insulin-sensitive healthy samples produced 69,559,764 raw reads. A total of 41,697,300 mappable reads was obtained after sequence filtering. Using a 2-fold expression difference as a cutoff, 33 miRNAs showed significally differential expression between insulin sensitive and insulin-resistant group. Among which, 10 were up-regulated and 22 were down-regulated. This RNA-seq discovery study shows that the most changes in miRNAs expression is in accordance with previous studies performed to explore circulating miRNAs in human insulin resistance or T2DM. Indeed, among the 54 selected miRNAs, we have identified the same changes in mir-126 expression that has described by Zampetaki and colleagues. This group reported that mir-126 is a unique plasma miRNA signature in 800 patients with T2D and observed that the reduced levels of mir-126 showed the strongest association with T2D. From the 33 miRNAs, a relative number of them are much documented to be associated with insulin resistance and type 2 diabetes. Nevertheless, some identified miRNAs are potential novel candidates and need to be deeply exploring during the event of the pathogenesis of T2D. Conclusion: This study provides a comparative profiling of circulating miRNAs in insulin-resistant versus insulin–sensitive subjects and the identification of specific miRNAs in circulation that changes are associated with insulin action. To our knowledge, this is the first study to investigate insulin resistance in Arab population using euglycemic hyperinsulemic clamp the gold-standard method to assess insulin sensitivity and aim to identify changes in circulating miRNAs expression associated to insulin action and signaling. The study also provides evidence that Insulin Resistance in human is related to changes in multiples microRNAs. Functional Validation studies of the differentially expressed miRNAs and relevant target and signaling pathways are ongoing.

Neural stem cells (NSC) have self-renewal and multipotent properties and may serve as an ideal cell source for transplantation to treat neurodegenerative insults such as Parkinson's, Alzheimer's, and spinal cord injury (SCI). Obtaining NSCs from adult human olfactory bulb (OB) would avoid ethical issues associated with the use of embryonic tissue, and provide an easily accessible cell source that would preclude the need for invasive brain surgery.

We used Agilent and Illumina Whole Human Genome Oligonucleotide Microarray to compare the genomic profiles of human embryonic NSC at a single time point in culture; and a multicellular tissue from postmortem adult substantia nigra (SN), an area rich in dopaminergic (DA) neurons. We identified 13525 up-regulated genes in both cell types of which 3737 (27.6%) genes were up-regulated in the hENSC, 4116 (30.4%) genes were up-regulated in the human substantia nigra dopaminergic cells, and 5672 (41.93%) were significantly up-regulated in both cell populations. Careful analysis of the data using DAVID has permitted us to distinguish several genes and pathways involved in dopaminergic (DA) differentiation, and to identify the crucial signaling pathways that direct this process. The set of genes expressed more highly at hENSC is enriched in molecules known or predicted to be involved in the M phase of the mitotic cell cycle. On the other hand, the genes expressed in SN cells include a different set of functional categories, namely synaptic transmission, central nervous system development, structural constituents of the myelin sheath, the internode region of axons, myelination, cell projection, cell somata, ion transport, and the voltage-gated ion channel complex. These data were compared with data from various databases, and between different types of arrays, Agilent versus Illumina. This approach has allowed us to confirm the consistency of our results for a large number of genes that delineate the phenotypical differences of embryonic NSCs, and SN cells.

Next we studied global gene expression profiling using RNA from human embryonic neural stem cells (hENSC), and adult human olfactory bulb-derived neural stem cells (OBNSCs), to define the gene expression pattern and signaling pathways specific for each cell lineage. We demonstrated large differences in the gene expression profile of human embryonic NSC, and adult human OBNSCs, but less variability between parallel cultures. Transcripts of genes involved in neural tube development and patterning (ALDH1A2, FOXA2), progenitor marker genes (LMX1a, ALDH1A1, SOX10), proliferation of neural progenitors (WNT1 and WNT3a), neuroplastin (NPTN), POU3F1 (OCT6), neuroligin (NLGN4X), MEIS2, and NPAS1 were up-regulated in both cell populations. Using Gene Ontology, 325 out of 3875 investigated gene sets were different. 41 out of the 307 investigated Cellular Component (CC) categories, 45 out of the 620 investigated Molecular Function (MF) categories, and 239 out of the 2948 investigated Biological Process (BP) categories were significant. KEGG Pathway Class Comparison revealed that 75 out of 171 investigated gene sets passed the 0.005 significance threshold. Levels of gene expression were explored in three signaling pathways, Notch, Wnt, and mTOR, all known to be involved in NS cell fate determination. The transcriptional signature also clarifies the role of genes involved in epigenetic modifications. The SWI/SNF DNA chromatin remodeling complex family, including SMARCC1 and SMARCE1, was specifically up-regulated in OBNSC but not in hENSC. Differences in the gene expression profile of transcripts controlling epigenetic modifications and signaling pathways might indicate differences in the therapeutic potential of the two cell populations with respect to potential cell survival, proliferation, migration, and differentiation following engraftments in different CNS insults.

Next, the transcriptional factors (TF) and genomic markers associated with neurogenesis, proliferation, differentiation, and epigenetic control in human embryonic neural stem cells (hENSC), and adult human olfactory bulb neural stem cells (OBNSC) were studied using immunohistochemistry (IHC) and DNA microarrays. The biological impact of TF gene changes in the examined cell types was estimated using DAVID to specify a different GO class and signaling pathway based on KEGG database. Eleven, and twenty eight TF genes were up-regulated (fold change ≤ 2–39) in OBNSC, and hENSC respectively. KEGG pathway analysis for the up-regulated TF genes revealed significant enrichments for the basal transcription factor pathway, and Notch signaling pathway in OBNSCs, and hENSCs, respectively. Immunofluorescence analysis revealed a significantly greater expression of β-tubulin III (TUBB3),MAP, glial fibrillary acidic protein (GFAP), and O4 in hENSC compared to OBNSC. Furthermore, the expression of epigenetic-related TF-genes SMARCC1, TAF12, and UHRF1 increased significantly in OBNSC when compared with hENSC.

This suggests that exogenous application of NGF may be a promising therapeutic strategy for traumatic and neurodegenerative diseases. However, effective delivery of NGF into the CNS parenchyma is still challenging due to its limited ability to cross the blood–brain barrier, and the intolerable side effects if administered into the brain ventricular system. An effective method to ensure delivery of NGF into the parenchyma of CNS might be genetic modification of NSC to overexpress the NGF gene. Overexpression of NGF in adult human OBNSC is expected to alter their proliferation and differentiation, possibly enhancing their therapeutic potential. We genetically modified adult human OBNS/PC to overexpress human NGF (hNGF) and green fluorescent protein (GFP) genes, hoping to provide insight about the effects of hNGF and GFP gene overexpression in adult human OBNS/PC and on their in vitro multipotentiality using DNA microarray, immunophenotyping, and Western blot (WB) protocols. Our analysis revealed that OBNS/PC-GFP and OBNS/PCGFP- hNGF differentiation is a multifaceted process involving changes in major biological processes as reflected in alteration of the gene expression levels of crucial markers such as cell cycle and survival markers, stemness markers, and differentiation markers. The differentiation of both cell classes was also associated with modulations of key signaling pathways such MAPK signaling pathway, ErbB signaling pathway, and neuroactive ligand-receptor interaction pathway for OBNS/PC-GFP, and axon guidance, calcium channel, voltage-dependent, gamma subunit 7 for OBNS/PC-GFP-hNGF, as revealed by GO and KEGG. Differentiated OBNS/PC-GFP-hNGF displayed extensively branched cytoplasmic processes, a significantly faster growth rate and up modulated the expression of oligodendroglia precursor cells markers (PDGFRα, NG2 and CNPase) respect to OBNS/PC-GFP counterparts. These findings suggest an enhanced proliferation and oligodendrocytic differentiation potential for OBNS/PC-GFP-hNGF as compared to OBNS/PC-GFP.

To assess the therapeutic potential of OBNSCs, we studied the fate of allogenic adult human olfactory bulb neural stem/progenitor cells (OBNSC/NPCs) transplanted into the rat hippocampus treated with ibotenic acid (IBO), a neurotoxicant specific to hippocampal cholinergic neurons that are lost in Alzheimer's disease. We assessed their possible ability to survive, integrate, proliferate, and differentiate into different neuronal and glial elements: we also evaluated their possible therapeutic potential, and the mechanism(s) relevant to neuroprotection following their engraftment into the CNS milieu. The isolated OBNSC/NPCs-hNGF were stereotaxically transplanted into the hippocampus of IBO-treated animals and controls. Stereological analysis of engrafted OBNSCs eight weeks post transplantation revealed a 1.89 fold increase with respect to the initial cell population, indicating a marked ability for survival and proliferation. In addition, 54.71_11.38%, 30.18_6.00%, and 15.09_5.38% of engrafted OBNSCs were identified by morphological criteria suggestive of mature neurons, oligodendrocytes and astrocytes respectively. Taken together, this work demonstrated that human OBNSCs expressing NGF ameliorate the cognitive deficiencies associated with IBO-induced lesions in AD model rats, and the improvement can probably be attributed primarily to neuronal and glial cell replacement as well as the trophic influence exerted by the secreted NGF.

Next, the hNFG-GFP-OBNSCs were transplanted into the striatum of 6-OHDA Parkinsonian rats. The grafted cells survived in the lesion environment for more than eight weeks after implantation with no tumor formation. The grafted cells differentiated in vivo into oligodendrocyte-like (25 ± 2.88%), neuron-like (52.63 ± 4.16%), and astrocytic-like (22.36 ± 1.56%) lineages based on morphological criteria. Transplanted rats exhibited a significant partial correction in stepping and placing non-pharmacological behavioral tests, using a pole and rotarod. Taken together, our data encourage further investigations for the possible use of OBNSCs as a promising cell-based therapeutic strategy for Parkinson's disease.

Finally, hNGF-GFP-OBNSCs were engrafted in a rat model of SCI at day 7 post injury. All transplanted animals exhibited successful engraftment. The survival rate was about 30% relevant to initially transplanted cells. 27% of the engrafted cells differentiated along the oligodendrocyte, nearly as many (16%) differentiated into neurons, and about 56% of the cells displayed astrocyte morphology. The study revealed that hNGF-GFP-OBNSCs were able to survive in the lesion environment for more than eight weeks after implantation; this was supported by transgenic overexpression of hNGF on engrafted cells. We didn't observe locomotor recovery by BBB test, footprint analysis and grid walk tests three months post-treatment. No sign of immunorejections were recorded during the 8 week time window of the study. Engrafted cells were distributed throughout gray and white matter of the cord with no evidence of abnormal morphology or any mass formation indicative of tumorigenesis.

In Algeria, there is about seventy percent of the population that lives in remote areas and the desert. These areas are cut from health services, and the people do not have access to modern health services existing in Northern Algeria. The limited health care budget-delivered to these desert areas, and the shortage of doctors (health care specialists) jeopardize the improvement of health care system in the desert areas in Southern Algeria. Recently, although the Centre du Development des Technologies Avancées (CDTA) was granted the right to develop the feasibility of telemedicine in the country, the project centered in the city of Ouarlga, 600 km South of Algiers, giving only medical consultation and diagnostic to pediatrics. However, this initial project does not cover all the Southern remote and desert areas in the country. Also, it does not provide universal health care coverage to all the people living there. The paper discusses the challenges faced by the local authorities to develop health care system coverage in the South of Algeria. Also, it introduces how ICTs and internet-based technologies can integrated into health care system to help people with delivering most of the modern health services while living the desert areas. It proposes a telemedicine project which is based on WINDOWS NET, Adruino, and other ICT applications to develop an eHealth strategy in the country.

Keywords

ICT applications, internet-based technologies, Health care system, Arduino, desert areas.

Background

Acute myeloid leukemia (AML) is a heterogeneous disease with respect to clinical picture and therapeutic outcome, partly reflected by differences in molecular and cyto-genetics. Clonal cytogenetic abnormalities are one of the most important factors predicting clinical outcome in acute myeloid leukemia and are used to guide risk-adapted treatment strategies.

Deregulated expression of genes coding transcription factors involved in cell proliferation, survival or differentiation is known to be implicated in the process of leukomogenesis. Brain and acute leukemia, cytoplasmic gene (BAALC) and ETS-related gene (ERG) are examples of these transcription factors BAALC gene, located on chromosome band 8q22.3, is considered as a marker of early hematopoietic progenitor cells. High levels of BAALC expression were found in AML patients with trisomy 8, as well as in a subset of cytogenetically normal–AML(CN-AML) patients in which it was considered as poor prognostic factor The role of BAALC in leukemogenesis is not fully understood, but it was proposed that BAALC blocks myeloid differentiation ERG gene, located on chromosome band 21q22, belongs to the ETS family of transcription factors required for normal hematopoiesis. It is a transforming proto-oncogene that is expressed in stem cells and endothelial cells. It is frequently overexpressed in AML patients with complex karyotypes and amplification of chromosome 21. The ERG gene has been found to be involved in atypical chromosomal rearrangements with alterations of the transcription factor in several cancers. Aberrant expression of full-length ERG protein has been found in acute myeloid leukemia and acute T-lymphoblastic Leukemia. Chromosomal rearrangements driving the formation of EWS/ERG and FUS/ERG fusion proteins have been described in a subset of Ewing sarcoma and in acute myeloid leukemia.

Most of the previous researches were concerned about levels and prognostic effect of BAALC and ERG in CN-AML patients, while data about their incidence in AML and relation to cases with abnormal karyotype were lacking. The objective of this study was to detect the incidence of BAALC and ERG in a group of AML patients with abnormal karyotype in comparison to normal individuals, their levels and distribution among AML FAB subtypes as well as to study their impact on the disease outcome and reliability in detection of minimal residual disease in relation to the cytogenetic markers.

Methods

The current study was carried out on 44 patients with acute myeloid leukemia (AML), in the period between, July 2011 and July 2012 among cases referred to nuclear medicine and oncology unit of Kasr Alainy School of medicine, Cairo University with follow up period of 18 months, as well as 44 age and sex matched controls. They were patients suspected to have hyperspleenism or idiopathic thrombocytopenia coming for BM aspirate. Cases were diagnosed according to WHO criteria. The diagnosis of AML was based on morphological and phenotypic data. Subtypes according to the French–American–British classification were available for all the patients. Patients were 21 males and 23 females. Age ranged from 21 to 65 years. The control group included 17 male and 27 females with no medical history of any type of cancer. Their ages ranged between 22 and 50 years. All patients and controls were analyzed for clinical and laboratory findings, including full history taking, clinical examination, routine laboratory investigations, LDH, abdominal ultrasound for detection of organomegaly and lymphadenopathy. The patients were subjected as well to cytochemical, immunophenotypic and cytogenetic analysis to confirm diagnosis and to divide the patients into their subtypes. Local institutional research board approval as well as written informed consent was obtained from all the participants before including them in the study. All patients included in the study were treated according to the protocol of the nuclear medicine and oncology department, Cairo University, using ongoing induction and consolidation regimens for treatment of adult AML cases.

Induction of remission

Patients were subjected to 7–3 protocol for induction of remission: Novantrone: 12 mg/m², IV on day 1 and 3. ARA – C: 100 mg/m², continuous IV infusion, from day l–7. If remission is not achieved, this protocol was repeated again. If no or minimal response, patients were shifted to high dose chemotherapy. Induction therapy for acute promyelocytic leukemia (PML) included oral administration of all-trans-retinoic acid (ATRA) 45 mg/m²/day until induces remission.

Consolidation

High dose ARA-C for 4 cycles. ARA-C: 2 g/m², over 2 hours infusions, every 12 hours on days 1–4. Significant association to relapse free survival and overall survival were estimated for studied genes at a median follow-up of 18 months. Complete remission (CR) was defined as recovery of morphologically normal BM and peripheral blood cells with white cell counts ≥ 1,500/L, and platelets ≥ 100,000/L, less than 5% BM blasts and no evidence of extramedullary leukemia. Relapse was defined by ≥ 5% BM or peripheral blood blasts, or development of extramedullary leukemia in patients with previously documented CR. Relapse free survival (RFS) was measured from the date of CR until date of relapse or death. Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) assay was performed for estimation of BAALC and ERG expression.

Results

BAALC was expressed in 36(81.82%) of AML cases versus 10(22.72%) of the control group which was highly statistically significant (P <  0.001). While ERG was positive in 39(88.64%) of cases and 8(18.18 %) of controls and that was also highly statistically significant (P <  0.001). The distribution of the positive cases among FAB subtypes did not show any significant differences. The lower values of BAALC and ERG in the cases compared to controls means higher levels in the cases as the numerical value of the CT is inversely related to the amount of amplicon in the reaction. Follow up of the patients revealed 12 cases of CR and 32 with unfavorable outcome; 17 showed partial recovery (PR), 8 cases relapsed and 7 patients died. Highly significant correlation was detected between positive genes expression and presence of bulky tumor and organomegaly. The levels of each gene were expressed as 2-ΔΔ CT i.e. number of fold increased above the mean level of the control group. Patients were divided into two groups according to these levels (high and low) as described previously in statistic paragraph. BAALC levels median and range were 1479.8(220.8–7550.2) for high levels group and 10.1(0.13–54.9) for low levels group. While levels of ERG were 425.5(27–1985) and 2.44(0.021–8.17) for high and low levels groups respectively. There were statistically no significant differences between the 2 studied groups regarding the BAALC gene expression before and after treatment (P value = 0.86) or the ERG gene expression before and after treatment (P value = 0.75). Multivariate regression test had revealed that BAALC and ERG are independent risk factors of acute leukemia, because (table7). Although we abolished the effect of bad prognostic factors (age>60 years, high serum LDH and WBC levels, high bone marrow blast percentage, presence of bulky tumor), still high levels of these genes are associated with lower CR, RFS, and shorter OS.

Conclusion

As regard ERG, the levels in AML cases did not differ significantly from theirs in normal BM. So determination of cut off value to detect MRD will not be applicable. Further researches still needed to clarify the role of BAALC and ERG in the pathogenesis of leukemia and their importance as targets for treatment of AML that could be promising due to their high incidence of expression in AML.

Keywords

BAALC, ERG, gene expression, Quantitative real-time RT-PCR, AML.

Background

Pesticide poisoning is a global public health problem. Annually, 3 million people are hospitalized due to pesticide poisoning with over 250,000 deaths all over the world 1. With the growing population and rapid industrialization in Qatar, there has been a natural increase in pesticide usage and hence, exposure amongst the workers handling them. It is therefore vital to not only understand the existing knowledge, attitudes and practices about handling pesticides, but also to explore the effectiveness of measures like education and personal protective equipment on the general well being of the workers. We hypothesize that training the workers to handle pesticides is vital in maintaining high safety standards, thereby preventing associated toxicity while handling them that the use of personal protective equipment (PPE) improves the general health of pesticide handlers in the long run.

Methods

100 municipality employees in Qatar who work with pesticides, were interviewed in person by trained bilingual staff using a structured questionnaire model (Table 4). The questionnaire included basic demographic data, questions related to methods of pesticide storage, retrieval, application and disposition, usage of PPEs, adherence to safety practices and views of the worker on the company safety protocols and their implementation. The data was then entered into excel format and analyzed using descriptive statistics and associations inferred by odds ratios.

Results

The mean age of the workers was 37.4(SD-9.9)(Table1). Amongst workers nearly 2.9 % of them who did not use personal protective equipment visited hospital annually when compared to 1.4% of workers who used personal protective equipment (Table 2). Of the interviewed workers 81.1% did not know the contents or the name of the pesticides they were handling at work (Table 3). Unsafe behaviors such as preparation of pesticides at the site of its usage rather than in a specified preparation room (29.6%), noncompliance with wearing protective clothing (38.8%), handling of drinking water (22%) and food (10%) on site where pesticides are used and not washing clothes every day after work (45.9%) were observed. Around a quarter of the interviewees did not receive any training on preparation and handling of pesticides (Table 3). Workers who received training in pesticides usage were more likely to be aware of its effects on the environment (61.6%)(OR–3.9), less likely to eat or drink while handling pesticides(83.6%) (OR–4.3) and more likely to give household members appropriate instructions prior to application of pesticides (90.4%)(OR–5.0)(Table3).Workers who did not wear special protective clothes at work were found to be, more than twice as likely to visit hospitals per year, than those who wore(RR-2.2)(Table3). A third of the workers were not aware if their company had safety protocols in place, whereas a similar proportion felt no such protocols exist.

Conclusion

Unsafe practices were found to be significantly common amongst the personnel using pesticides in Qatar. Workers who received prior training, handled pesticides with better safety standards, highlighting the importance of training in pesticide handling. Wearing protective clothing most likely has a positive impact on the general well being of the workers.

Keywords

Improved safety standards, municipality workers, pesticide exposure, protective clothing.

Resources 1-World Health Organization

The Impacts of Pesticides on Health: Preventing Intentional and Unintentional Deaths from Pesticide Poisoning (fact sheet) 2- Gunnell, David and Eddleston, Michael. “Suicide by intentional ingestion of pesticides: a continuing tragedy in developing countries.” International Journal of Epidemiology 32 (2003): 902–909.

Table 1 (DEMOGRAPHIC DATA) Demographic variables (n = 100) Percentage* Age Less than 20 1 21 to 35 48.9 36 to 50 36.7 More than 50 13.3 Mean 37.4 years Education Primary 33.7 Secondary 44.9 Not educated 11.2 Other 8.2 Number of years working with pesticides < 1 11.2 1 to 5 30.6 6 to 10 24.5 >10 32.7 Living with Family 14.3 Friends 30.6 Colleagues 54.1 Children 0.00 Other 2.0 *Total may not be 100% if there were missing values

Table 2 (Comparison of variables for those wore special protective clothes to those who didn't) Variable Wore Special protective clothes (n,%) Did not wear(n,%) Number 59(59) 38(38) Age(mean) 37.4 37.5 Education Primary 27(45.8) 5(13.2) Secondary 20(33.9) 24(63.2) Not educated 8(13.6) 3(7.9) Years Working with pesticides < 1 5(8.5) 6(15.8) 1–5 20(33.9) 10(26.3) 6–10 14(23.7) 10(26.3) >10 20(33.9) 11(28.9) Co morbid conditions 16(27.1) 9(23.7) Knowledge on content of pesticides Yes 7(11.9) 11(28.9) No 52(88.1) 27(71.1) Any training received? Yes 46(77.9) 27(71.1) No 13(22.0) 11(28.9) Average hospital visit/year 1.4(0.8–1.9) 2.9(1.9–4.1)

Table 3 (ODDS RATIO) Variable Trained in pesticides (n = 73) Not trained in pesticides (n = 24) Odds Ratio with 95%CI Workers view on effect on environment YES 45 7 3.9(1.4 to 10.6) NO 28 17 Do not handle food/water at site 61 13 4.3(1.6–11.9) Wear special clothes 46 13 1.4(0.6–3.7) Knowledge of contents in pesticides present 16 2 3.1(0.7 – 14.5) Instruction to Household members 66 15 5.7(1.8–17.6) Washing clothes after work 40 13 1.02(0.41–2.59) Average visits to hospital/year 2.05(1.35–2.77) 2.04(0.68–3.41)

Table 4 (RESPONSE TO QUESTIONNAIRE IN PERCENTAGE) Affirmative response Do you know the contents of the pesticide you are using? 18.4 When spraying inside do you give any instructions to the household members? 82.7 If yes what are they? Stay away from home 56.3 Don't prepare food at home 7.5 Don't get into the room for a specified period of time 35 Pesticides are poisonous 1.3 Where do you prepare the pesticides? On site 29.6 Comes prepared 3.1 Preparation room 58.2 Other 0.00 Did you receive any education on how to prepare/apply the pesticides? 74.5 If yes, who educated you? Foreman 19.7 Supervisor 40.9 Senior co worker 27.3 Other staff 12.1 When were you educated? At the start of job 55.3 Periodically 28.7 Where do you store the pesticides? Home 0.00 At the site 15.3 Buy and use immediately 1.0 Specific store 83.7 How do you dispose the containers? Yourself 15.3 By the company 73.5 Other 6.1 Do you wear special protective clothes, while using the pesticides? 60.2 Do you wash your clothes daily after work? 64 Do you wash them separately? 59 Powder 75.5 Solid tablets 41.8 Spray 74.5 Liquid 26.5 Smoke 33.7 Other 3.1 Which form of pesticide do you use out door? Powder 52.6 Solid tablets 38.1 Spray 82.5 Liquid 32.9 Smoke 60.8 Other 1.0 What personal protective equipment are you using? Masks 95.9 Gloves 93.9 Clothes 67.4 Shoes 83.7 Goggles 51.0 Other 2.0 Do you know the effects of pesticides? On Humans 81 On the environment 53 Pesticides can be absorbed through? Skin 46.3 Mouth 75.8 Inhalation 93.7 Other 6.3 Pesticides have? Only acute effects 42.3 Only chronic effects 14.4 Both 29.9 Neither 13.4 In case of accident exposure, what do you do? Wash with water and soap 78.5 Drink milk 13.3 Call ambulance 8.1 Go to hospital 11.2 Inform supervisor 7.1 How do you dispose this equipment? General waste 28.9 Specific site for disposal 25.3 Company does it 30.1 Plastic bags 6 Reuse/keep it to oneself 3.6 During application, do you? Eat 10.3 Drink 22.7 Both 1.0 None 76.3 After application, do you wash hands? 98.9 Do you have any co morbid conditions? 25.5 Are you taking any medications? 20.4 How many visits to the Health care/E.D/Admissions per year? 1.9(1.4–2.5) Are there any safety protocols at your company/work place? Yes 29.6 No 39.8 Don't know 30.6 Is your Company/Employer strict about compliance of safety protocols? Yes 48.5 No 23.7 Don't Know 27.8 If any untoward incident occurs, are they documented/informed to respective officials? Yes 43.9 No 43.9 Don't Know 13.3 Did you ever witness any case of sever morbidity or death in your company workers due to pesticide poisoning? Yes 17.4 No 82.7 Does your company provide compensation for pesticide poisoned workers? Yes 1.0 No 40.8 Don't Know 58.2.

A variety of climatic factors are known to influence respiratory health, usually through their impact on air quality. High temperatures, for example, are often associated with an increase in ground levels of ozone, which in turn negatively impact respiratory function (Thurston and Ito 1999; WHO 2000). In New York City, high temperatures were correlated with increased hospital admissions for both chronic airway obstruction and asthma (as well as some cardiovascular problems) (Lin et al. 2009). Humidity levels are often positively correlated with allergenic spores (e.g., Hasnain et al. 2012) and other particulates (e.g., Kulshrestha et al. 2012) and thus can trigger asthmatic attacks or other forms of respiratory distress. More recently, investigators have begun to examine the relationship between dust-sand storms and respiratory health. For example, Waness et al. (2011) cited evidence suggesting that exposure to dust-sand storms in the Middle East may contribute to pulmonary alveolar proteinosis and silicosis. In this study, we conducted a detailed correlational analysis between various climatic variables, the density of airborne particulate matter, and the incidence of hospital admissions related to respiratory problems.Inhalation of air particulates may result in serious health problems, most notably with the respiratory system. Due to a variety of factors (traffic, construction, weather patterns), the air in Doha is of poor quality and is significantly over targets for PM2.5 and PM10 (WHO, 2014 rankings). We examined the levels of particulate matter to determine if there was a daily or weekly pattern. We then compared the number of air particulates in the summer and winter and attempted to determine if small (0.5–2.5 mg/l) or large (>2.5 mg) particulate matter was related to weather patterns and resulted in increased use of the nebulizer at a local clinic. We found that particle number (both small and large) was highest around 6 am and lowest around 12 pm, a pattern that didn't change between months. The number of large and small particles was generally lower on Friday when human activity was lowest. The temperature decreased in winter while humidity increased; wind speed remained relatively constant. Data suggest that nebulizer use was higher in the winter and lower in the summer although the reasons for this are speculative. There was no relationship found between the number of people using the nebulizer and the number of air particles.

Obesity and type 2 diabetes is characterized by insulin resistance which has been reported as the major risk factors associated with the development of the endothelial dysfunction and vascular complications such as atherosclerosis. Induction of the vascular dysfunction is obviously a proved metabolic consequence of insulin resistance. Diabetes leads to altered retinal microvascular function and ultimately diabetic retinopathy. Insulin signaling may play a role in this process, and animal studies indicated a role of the insulin in the pathogenesis of retinal neovascularization through its effect on endothelial cells. Endothelial dysfunction impairs ocular hemodynamics by reducing the bioavailability of NO and increasing the production of reactive oxygen species (ROS) and may be responsible for the pathogenesis of vascular dysfunction in retinopathy. Diabetic retinopathy (DR) a major consequence of diabetes is considered the leading cause of vision loss and blindness worldwide among working adults. Endothelial dysfunction expediting imbalance in vascular homeostasis, is one of the primary manifestation leading to the pathogenesis of DR. NO a major vasodilator involved in the regulation of vascular homeostasis is reported to be released by insulin dependent PI3K/Akt signaling pathway. Endothelial dysfunction impairs ocular hemodynamics by reducing the bioavailability of NO and increasing the production of reactive oxygen species (ROS) and may be responsible for the pathogenesis of vascular dysfunction in retinopathy. Insulin stimulated NO productions are reported to be well established in cardiovascular and macrovascular endothelium and its association with PI3K/Akt pathway. Contrary to this insulin signaling in retinal endothelium have minimal reports with some studies suggesting it to be an important physiology player in hyperglycemia or insulin resistance induced DR. This prompted us to investigate the association between hyperglycemia and insulin mediated PI3K/Akt pathway eNOS directed NO production and associated multivariate effect on HRMECs survival, proliferation, angiogenesis, adhesion, apoptotic and inflammatory markers. The aim of this study is to examine the examine the effect of insulin on NO production in human retinal microvascular endothelial cells cultured in hyperglcemic conditions. In the current study in order to examine the effect of insulin on NO production, HRECs cells were cultured and grown in high glucose (30 mM) and normal (5 mM) glucose for 24 hours. Subsequently, the cells were treated with 100 nM insulin for 10 minutes, 1, 2, and 4 hours. The various parameters of PI3K/Akt signaling pathway were analyzed. IRS-1, IRS-2, PI3K, Akt, eNOS, VEFGA, NFkB were analyzed for gene expression. Adhesion molecules such as P-selectin and ICAM-1 were assessed by flow cytometry. ROS and NO production were measured by immunofluorescence and fluorometry respectively. The cell viability, cell cycle, apoptosis and total oxidative stress were evaluated by imaging flowcytometery. This study demonstrated that hyperglycemia causes an increase in ROS/oxidative stress and apoptosis, while insulin promotes a significant decrease in ROS and apoptosis. eNOS mediated NO production increases with hyperglycemia but remarkably decreases with insulin treatment after 1 hour, 2 hours and 4 hours. This dissimilarity of the results to previously reported studies could be due to different endothelial cell types used or varied duration of experimental hyperglycemia. The most significant reason for the variation may be due to different method of NO measurement where, most previous studies measured NO production by isolated NO meters. However, in the current study fluorometry a more sensititve method to measure oxidized product of NO, nitrite an indicator direct NO production was utilized and confirmed by the immunoflouresence technique using the dye DAF-FM Diacetate. The assessment of PI3K/Akt pathway gene expression revealed that this study demonstrates a slight increase but insignificant elevation of IRS-1 and IRS-2 genes expression in hyperglycemic condition compared to the basal control condition, while gene expression of PI3K, Akt and eNOS were significantly upregulated in the presence of high glucose. Insulin treatment caused an up regulation of IRS-1 and IRS-2 genes after 1 hour however, PI3K, Akt and eNOS were significantly reduced. The analysis of angiogenic and proapoptotic markers VEGFA and NFkB by RT-PCR showed no significant change in the expression of NFkB in hyperglycemic alone, hyperglycemic and normoglycemic cells with insulin treatment at all-time points. However, hyperglycemia significantly increased the expression of VEGF. Though compared to basal control group insulin treatment significantly increased the expression of VEGFA in both hyperglycemic and normoglycemic cells yet the expression was low compared to HG state. Consistent with the VEGFA gene expression HG significantly increased the increase the cell migration and angiogenesis while insulin treatment significantly improves barrier function. Hyperglycemia significantly increased adhesion protein P-selectin with significant reduction at 4hrs insulin treatment in the current study. This study demonstrated a significant reduction in P-selectin after insulin suggesting that insulin could participate in preventing leukocyte adhesion thereby attenuating the progression of DR. This study could not demonstrate significance change in the ICAM-1 protein. This could be due to difference in the endothelial cells used, the duration and the type of insulin treatment used. This study reported that short acting insulin commonly used in the treatment of DR could control the metabolic fluxes thereby leading to improvement in oxidative stress and apoptosis. This could prevent early changes in vasodilator, adhesion and angiogenic markers such as NO, VEGFA, P-selectin involved in the angiogenesis, inflammation and neovascularization involved in retinal vascular functioning. The study shows the potent effect of short-acting insulin treatment to counteract these biomarkers and factors involved in the pathogenesis of DR and conserving microvascular function in HRMECs exposed to hyperglycemia (30 mM) and were reported to be improved. Thus, the diabetic interventions using insulin as a key therapy with others may have the potential to be utilized as a readily available, safe and inexpensive medicine to protect against microvascular complications of DR and delay its onset. In conclusion, this study demonstrated that Hyperglycemia causes an increase in ROS/oxidative stress and apoptosis, while insulin promotes a significant decrease in ROS and apoptosis, eNOS mediated NO production increases with hyperglycemia but remarkably decreases with insulin treatment after 1 hour, 2 hours and 4 hours, insulin could counteract the hyperglycemic effect on AKT/pI3 kinase which mediates NO production and VEGF-A, decreased adhesion molecules p-selectin involved in barrier disorder of retinal endothelial cells. Thus it could be proposed that insulin could be considered as regulators of angiogenesis.

People in developing countries of the world are facing a changing environment due to urbanization – changes that include lifestyle changes as well as changes in the dietary habits (and content). These lifestyle changes are now known to contribute to an epidemic of metabolic disease. The underlying mechanisms are unclear. The burden of type 2 diabetes mellitus (T2D) is increasing worldwide, particularly in developing countries, where it is predicted that over 70% of the global burden of T2D will exist by 2030 (Echouffo-Tcheugui and Dagogo-Jack, 2012). Although reasons for the increasing rates of T2D in developing countries are not fully elucidated, important factors include lifestyle changes involving rural-to-urban migration (“urbanization”), intra-uterine undernutrition and foetal programming (epigenetic changes progressively introduced over multiple generations and associated with maternal undernutrition). During the past two decades, increasing evidence arising from multiple clinical studies conducted by the research teams of Yajnik and Barker support an important role of early life undernutrition, and specifically disturbances of one-carbon metabolism, in the heightened susceptibility of Indians to diabetes at a younger age, and in the absence of generalized obesity (Kulkarni et al., 2007; Yajnik et al., 2014; Yajnik and Deshmukh, 2012; Yajnik et al., 2003; Yajnik et al., 1995). The Pune Maternal Nutrition Studies have highlighted the body composition and nutritional-metabolic peculiarities of multigenerationally undernourished Indians: a thin-fat (low lean mass, high fat mass) phenotype compared to Europeans, with predominant visceral deposition of fat. This body composition is strongly associated with insulin resistance and related metabolic-endocrine abnormalities. Importantly, this ‘thin-fat’ phenotype was present at birth and therefore, programmed during intrauterine life, possibly through epigenetic mechanisms over multiple generations. Maternal intergenerational undernutrition, evident in stunting, low BMI, and a disturbance of dietary methyl donors (low protein and vitamin B12 and high folate status related to vegetarian food habits) appear contributory to the increased risk of diabetes and CVD in Indians (Yajnik, 2004; Yajnik and Deshmukh, 2012; Yajnik et al., 2008; Yajnik et al., 2003). It is now well-appreciated that the intra-uterine environment can induce heritable alterations that may be retained over generations (Aiken and Ozanne, 2014; Good speed et al., 2014; Ng et al., 2010). A primate maternal high-fat diet supplemented with calorically dense treats leading to obesity has been shown to epigenetically alter chromatin structure in their progeny via SIRT1 mediated covalent modifications of histones (Aagaard-Tillery et al., 2008; Cox et al., 2009; Suter et al., 2012). Increased adiposity and insulin resistance have also been reported in high fat diet fed rodent models. Intra-uterine programming may involve epigenetic changes, which are passed over generations, and may promote the development of adiposity and T2D. Recently, we demonstrated (cover story; August 2015 issue of Cell Metabolism) that the metabolic effects introduced over multiple (50) generations of undernutrition cannot be reversed after two generations of unrestricted access to nutrients. Undernourished rats demonstrated low birth-weight, high visceral adiposity (DXA/MRI), and insulin resistance (hyperinsulinemic-euglycemic clamps), compared to age/gender-matched Control rats. Relative to Controls, Undernourished rats had higher circulating insulin, homocysteine, endotoxin and leptin levels, lower adiponectin, vitamin B12 and folate levels, and an eight-fold increased susceptibility to diabetes, after Streptozotocin (STZ) exposure. These metabolic abnormalities were not reversed after two generations of recuperation. Adverse epigenetic (histone modification) profiles in insulin gene promoter region of Undernourished rats are not reversed following two generations of macro-nutrient availability and might explain the persistent detrimental metabolic profiles in similar multigenerational undernourished human populations. One of the major strengths of this study is that the current animal model of diabetes was not generated by genetic manipulations but rather by feeding Control rats with an undernourished died for 50 generations (over 12 years) and then a normal diet for two generations. The Undernourished rats showed visceral adiposity and were less sensitivity to insulin. They also demonstrated several markers of metabolic disease. In order to correct this state, we allowed Undernourished rats to have unrestricted access to Control chow and water (now called as “Recuperation” rats). Recuperation rats, surprisingly, show increased visceral adiposity, continue to be less sensitive to insulin, and have the same level of diabetes susceptibility as Undernourished rats. Analysis of epigenetic signatures at insulin gene promoter region in the pancreatic islet cells confirms that although a slight improvement is seen in histone modifications as well as the recruitment of histone methyl transferases at the insulin gene promoter region, two generations of normal nutrient availability is in itself not sufficient to reverse the epigenetic changes to Control levels. These studies underscore the importance of epigenetic regulation of gene expression and indicate that micronutrient or other appropriate supplementation should be considered in dietary intervention studies on populations that have faced undernutrition over multiple generations. I will present our recent data from animal models as well as our data from two independent clinical cohorts; first, the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) trial of around 10,000 Type 2 diabetic individuals followed over 12 years and secondly, the Pune Maternal Nutrition Study comprising of around 700 women and their babies over a period of 18 years of follow-up. Our studies (Discovery Analysis of MicroRNAs in Gene-Environment Interactions: DAMAGE-I study) involve analysis of DNA methylation changes (using the Illumina 450 K methylation arrays), GWAS analysis (using the Affymetrix platform), Telomere length assay (using quantitative real-time PCR) and microRNA analysis (using high throughput high sensitivity real-time TaqMan-PCR panels/chips, in addition to several (over 100) clinical biomarkers. Studies are also in progress to understand the role of gut microbiota – our inner environment that significantly influences human physiology. These studies demonstrate that the change in diet and lifestyle (urbanization) leads to significant changes in the short-chain fatty acid-producing gut bacteria and influence our lifestyle through epigenetic programming of gut cells. Overall, the studies discussed above, demonstrate the potential for integrative research technologies that we have used in understanding newer molecular biomarkers of diabetes and cardiovascular health and will allow future application of these molecular biomarkers to understanding the progression of diabetes and its complications in other (non-Australian) cohorts of at-risk of diabetes individuals worldwide. Hopefully, these technologies will help in better understanding the progression of Diabetes in individuals at risk of diabetes, provide newer tools to clinicians for predicting treatment efficacies and patient stratification and enable the development of newer and effective strategies to manage and cure diabetes.

Declarations

The current research discussed here is funded via three different grants to Professor Hardikar from the Australian Research Council (ARC), the National Health and Medical Research Council (NHMRC) and the Juvenile Diabetes Research Foundation (JDRF). Professor Hardikar is also on the advisory committee for Abbott Pharmaceuticals in relation to a probiotic formulation that has the potential for improving metabolic health in diabetes.

Chicken meat from the shelves of supermarkets in Qatar was tested for the presence of Camplobacter spp., and the presence of five virulence genes (htrB, cdtB, clpB, cadF and ciaB) was assessed in isolates. Forty eight percent of the chickens provided for supermarkets by Saudi (53%) and Qatari (45.9%) producers were found to be contaminated and the most important factor affecting the overall prevalence of contaminated chickens was the store from which chicken samples originated. Variation in prevalence of Camplylobacter in chicken meat from different stores was evident even when the same producer supplied the three stores in our survey. Differences in the prevalence and in the combinations of virulence genes in isolates that can and cannot grow in a classic maintenance medium (karmali) were identified, providing a starting point for linking presence/absence of particular virulence genes with actual in vivo virulence and pathogenicity. Because of the relatively low infective doses of Canpylobacter that are required to initiate infection in humans, it will be important to explore further the relationships we identified between certain Campylobacter virulence genes and their capacity for survival in poultry meat, and hence their contribution to the incidence of campylobacteriosis.