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Abstract Centrin1 gene deleted Leishmania donovani parasite (LdCen1 −/− ) was developed and extensively tested experimentally as an intracellular stage-specific attenuated and immunoprotective live parasite vaccine candidate ex vivo using human PBMCs and in vivo in animals. Here we report manufacturing and pre-clinical evaluation of current Good-Laboratory Practice (cGLP) grade LdCen1 −/− parasites, as a prerequisite before proceeding with clinical trials. We screened three batches of LdCen1 −/− parasites manufactured in bioreactors under cGLP conditions, for their consistency in genetic stability, attenuation, and safety. One such batch was preclinically tested using human PBMCs and animals (hamsters and dogs) for its safety and protective immunogenicity. The immunogenicity of the CGLP grade LdCen1 −/− parasites was similar to one grown under laboratory conditions. The cGLP grade LdCen1 −/− parasites were found to be safe and non-toxic in hamsters and dogs even at 3 times the anticipated vaccine dose. When PBMCs from healed visceral leishmaniasis (VL) cases were infected with cGLP LdCen1 −/− , there was a significant increase in the stimulation of cytokines that contribute to protective responses against VL. This effect, measured by multiplex ELISA, was greater than that observed in PBMCs from healthy individuals. These results suggest that cGLP grade LdCen1 −/− manufactured under cGMP complaint conditions can be suitable for future clinical trials.

Abstract Type 1 diabetes (T1D) is characterized by the progressive destruction of pancreatic β-cells, leading to insulin deficiency and lifelong dependency on exogenous insulin. Higher estimates of heritability rates in monozygotic twins, followed by dizygotic twins and sib-pairs, indicate the role of genetics in the pathogenesis of T1D. The incidence and prevalence of T1D are alarmingly high in Kuwait. Consanguineous marriages account for 50–70% of all marriages in Kuwait, leading to an excessive burden of recessive allele enrichment and clustering of familial disorders. Thus, genetic studies from this Arab region are expected to lead to the identification of novel gene loci for T1D. In this study, we performed linkage analyses to identify the recurrent genetic variants segregating in high-risk Kuwaiti families with T1D. We studied 18 unrelated Kuwaiti native T1D families using whole exome sequencing data from 86 individuals, of whom 37 were diagnosed with T1D. The study identified three potential loci with a LOD score of ≥ 3, spanning across four candidate genes, namely SLC17A1 (rs1165196:pT269I), SLC17A3 (rs942379: p.S370S), TATDN2 (rs394558:p.V256I), and TMEM131L (rs6848033:p.R190R). Upon examination of missense variants from these genes in the familial T1D dataset, we observed a significantly increased enrichment of the genotype homozygous for the minor allele at SLC17A3 rs56027330_p.G279R accounting for 16.2% in affected children from 6 unrelated Kuwaiti T1D families compared to 1000 genomes Phase 3 data (0.9%). Data from the NephQTL database revealed that the rs1165196, rs942379, rs394558, and rs56027330 SNPs exhibited genotype-based differential expression in either glomerular or tubular tissues. Data from the GTEx database revealed rs942379 and rs394558 as QTL variants altering the expression of TRIM38 and IRAK2 respectively. Global genome-wide association studies indicated that SLC17A1 rs1165196 and other variants from SLC17A3 are associated with uric acid concentrations and gout. Further evidence from the T1D Knowledge portal supported the role of shortlisted variants in T1D pathogenesis and urate metabolism. Our study suggests the involvement of SLC17A1, SLC17A3, TATDN2, and TMEM131L genes in familial T1D in Kuwait. An enrichment selection of genotype homozygous for the minor allele is observed at SLC17A3 rs56027330_p.G279R variant in affected members of Kuwaiti T1D families. Future studies may focus on replicating the findings in a larger T1D cohort and delineate the mechanistic details of the impact of these novel candidate genes on the pathophysiology of T1D.

Abstract Studies have established the association between increased plasma levels of matrix metalloproteinase (MMP)-9 and adipose tissue inflammation. Tumor necrosis factor α (TNFα) was elevated in obesity and is involved in the induction of MMP-9 in monocytic cells. However, the underlying molecular mechanism was incompletely understood. As per our recent report, TNFα mediates inflammatory responses through long-chain acyl-CoA synthetase 1 (ACSL1). Therefore, we further investigated the role of ACSL1 in TNFα-mediated MMP-9 secretion in monocytic cells. THP-1 cells and primary monocytes were used to study MMP-9 expression. mRNA and protein levels of MMP-9 were determined by qRT-PCR and ELISA, respectively. Signaling pathways were studied using Western blotting, inhibitors, and NF-kB/AP1 reporter cells. We found that THP-1 cells and primary human monocytes displayed increased MMP-9 mRNA expression and protein secretion after incubation with TNFα. ACSL1 inhibition using triacsin C significantly reduced the expression of MMP-9 in the THP-1 cells. However, the inhibition of β-oxidation and ceramide biosynthesis did not affect the TNFα-induced MMP-9 production. Using small interfering RNA-mediated ACSL1 knockdown, we further confirmed that TNFα-induced MMP-9 expression/secretion was significantly reduced in ACSL1-deficient cells. TNFα-mediated MMP-9 expression was also significantly reduced by the inhibition of ERK1/ERK2, JNK, and NF-kB. We further observed that TNFα induced phosphorylation of SAPK/JNK (p54/46), ERK1/2 (p44/42 MAPK), and NF-kB p65. ACSL1 inhibition reduced the TNFα-mediated phosphorylation of SAPK/JNK, c-Jun, ERK1/2, and NF-kB. In addition, increased NF-κB/AP-1 activity was inhibited in triacsin C treated cells. Altogether, our findings suggest that ACSL1/JNK/ERK/NF-kB axis plays an important role in the regulation of MMP-9 induced by TNFα in monocytic THP-1 cells.

Abstract Toll-like receptors (TLRs) have been targeted for therapeutic drug development for several disorders, including cardiovascular diseases (CVD), and diabetes mellitus. Daily levels physical activity (PA) has been purported to influence the systemic circulation of cytokines, affecting the overall activation of TLRs and influencing the inflammatory milieu. Objective and self-reported daily PA was tracked in 69 normal-weight adults. Freedson's cut-offs categorized daily PA intensity into the 25th lowest, medium, and top percentiles. Monocytic TLR2 expression was quantified by flow cytometry in fresh whole blood. Cross-sectional associations between flow cytometry measured TLR2+ subsets and clinical biomarkers were evaluated. PA increased circulation of TLR2+ monocytes. TLR2 expression was adversely corelated with reduced diastolic blood pressure (DBP), triglyceride (TG), and matrix metallopeptidase 9 (MMP9) levels. However, regression analysis indicated that only TG levels were independently linked with TLR2+ subsets in circulation in active participants. Higher daily levels of physical activity are associated with improved cardiovascular blood markers and elevated circulatory monocytic TLR2+ subsets. These findings suggest that TLR2 may play a role in modulating CVD risk factors in individuals leading physically active lifestyles.

Abstract Lepidopteran insect pest Helicoverpa armigera is one of the most destructive pests of crop plants and several biotechnological approaches are being developed for its control. Plant defensins are small cationic and cysteine-rich peptides that play a role in plant defense. Ingestion of a defensin from Capsicum annuum (CanDef-20) induced a dose-dependent reduction in larval and pupal mass, delayed metamorphosis and also severely reduced fecundity and fertility in H. armigera. To understand the molecular mechanisms of CanDef-20 ingestion-mediated antibiosis in H. armigera larvae, a comparative transcriptomics analysis was carried out. Predominant downregulation of GOs represents serine-type endopeptidases, structural constituents of ribosomes and integral membrane components and differential upregulation of ATP binding, nucleus and translation, while up-regulation of nucleic acid binding represented by transposable elements, were detected. Different isoforms of lipase, serine endopeptidase, glutathione S-transferase, cadherin, alkaline phosphatase and aminopeptidases were found to be upregulated as a compensatory response to CanDef-20 ingestion. In vitro enzyme assays and qPCR analysis of some representative genes associated with vital cellular processes like metamorphosis, food digestion and gut membrane indicated adaptive differential regulations in CanDef-20 fed H. armigera larvae. We conclude that CanDef-20 ingestion affects insect metabolism in a number of ways through its interaction with cell membrane, enzymes, cytoplasmic proteins and triggering transposon mobilization which are linked to growth retardation and adaptive strategies in H. armigera.

Abstract Organic anion transporting polypeptides (OATP), which are encoded by SLCO genes, participate in the hepatic elimination of drugs and xenobiotics. SLCO1B1 is an important pharmacogenomic gene (encoding OATP1B1) associated with response to the uptake of endogenous compounds, such as statin and bilirubin. Ethnicity of the patient modulates the response to these drugs; the frequency and haplotype data for SLCO1B1 genetic variants in the Arab population is lacking. Therefore, we determined the frequencies of two well-characterized SLCO1B1 single nucleotide polymorphisms (SNP) and haplotypes that affect the OATP1B1 drugs transportation activity in Qatari population. Genotyping data for two SLCO1B1 SNPs (c.388A > G, c.521 T > C) were extracted from whole exome data of 1050 Qatari individuals, who were divided into three ancestry groups, namely Bedouins, Persians/South Asians, and Africans. By way of using Fisher's exact and Chi-square tests, we evaluated the differences in minor allele frequency (MAF) of the two functional SNPs and haplotype frequencies (HF) among the three ancestry groups. The OATP1B1 phenotypes were assigned according to their function by following the guidelines from the Clinical Pharmacogenetics Implementation Consortium for SLCO1B1 and Simvastatin-Induced Myopathy.The MAF of SLCO1B1:c.388A > G was higher compared to that of SLCO1B1:c.521 T > C in the study cohort. It was significantly high in the African ancestry group compared with the other two groups, whereas SLCO1B1:c.521 T > C was significantly low in the African ancestry group compared with the other two groups. The SLCO1B1 *15 haplotype had the highest HF, followed by *1b, *1a, and *5. Only the SLCO1B1 *5 haplotype showed no significant difference in frequency across the three ancestry groups. Furthermore, we observed that the OATP1B1 normal function phenotype accounted for 58% of the Qatari individuals, the intermediate function phenotype accounted for 35% with significant differences across the ancestry groups, and the low function phenotype accounted for 6% of the total Qatari individuals with a higher trend observed in the Bedouin group.The results indicate that the phenotype frequencies of the OATP1B1 intermediate and low function in the Qatari population appear at the higher end of the frequency range seen worldwide. Thus, a pharmacogenetic screening program for SLCO1B1 variants may be necessary for the Qatari population.

Abstract There has recently been a growing interest in examining the role of epigenetic modifications, such as DNA methylation, in the etiology of type 1 diabetes (T1D). This study aimed to delineate differences in methylation patterns between T1D-affected and healthy individuals by examining the genome-wide methylation of individuals from three Arab families from Kuwait with T1D-affected mono-/dizygotic twins and non-twinned siblings. Bisulfite sequencing of DNA from the peripheral blood of the affected and healthy individuals from each of the three families was performed. Methylation profiles of the affected individuals were compared to those of the healthy individuals Principal component analysis on the observed methylation profiling based on base-pair resolution clustered the T1D-affected twins together family-wide. The sites/regions that were differentially methylated between the T1D and healthy samples harbored 84 genes, of which 18 were known to be differentially methylated in T1D individuals compared to healthy individuals in publicly available gene expression data resources. We further validated two of the 18 genes—namely ICA1 and DRAM1 that were hypermethylated in T1D samples compared to healthy samples—for upregulation in T1D samples from an extended study cohort of familial T1D. The study confirmed that the ICA1 and DRAM1 genes are differentially expressed in T1D samples compared to healthy samples.

Abstract The neuropeptide urocortin 3 (UCN3) has a beneficial effect on metabolic disorders, such as obesity, diabetes, and cardiovascular disease. It has been reported that UCN3 regulates insulin secretion and is dysregulated with increasing severity of obesity and diabetes. However, its function in the adipose tissue is unclear. We investigated the overexpression of UCN3 in 3T3-L1 preadipocytes and differentiated adipocytes and its effects on heat shock response, ER stress, inflammatory markers, and glucose uptake in the presence of stress-inducing concentrations of palmitic acid (PA). UCN3 overexpression significantly downregulated heat shock proteins (HSP60, HSP72 and HSP90) and ER stress response markers (GRP78, PERK, ATF6, and IRE1α) and attenuated inflammation (TNFα) and apoptosis (CHOP). Moreover, enhanced glucose uptake was observed in both preadipocytes and mature adipocytes, which is associated with upregulated phosphorylation of AKT and ERK but reduced p-JNK. Moderate effects of UCN3 overexpression were also observed in the presence of 400 μM of PA, and macrophage conditioned medium dramatically decreased the UCN3 mRNA levels in differentiated 3T3-L1 cells. In conclusion, the beneficial effects of UCN3 in adipocytes are reflected, at least partially, by the improvement in cellular stress response and glucose uptake and attenuation of inflammation and apoptosis.

Abstract Genetic variants responsible for Maturity-Onset-Diabetes of the Young (MODY) in Kuwait were investigated. A newly established a National Referral Clinic, the Dasman Diabetes Institute (DDI-NRC), assessed forty-five members from 31 suspected MODY families by whole exome sequencing. Thirty-three of the 45 samples were independently sequenced at the DDI-NRI, Exeter University, UK ( https://www.diabetesgenes.org/ ) using targeted 21-gene panel approach. Pathogenic mutations in GCK, HNF1A, HNF1B, HNF4A, and PDX1 confirmed MODY in 7 families, giving an overall positivity rate of 22.6% in this cohort. Novel variants were identified in three families in PDX1, HNF1B, and HNF1B. In this cohort, Multiplex Ligation-dependent Probe Amplification assay did not add any value to MODY variant detection rate in sequencing negative cases. In highly selected familial autoantibody negative diabetes, known MODY genes represent a minority and 77.3% of the familial cases have yet to have a causal variant described.

Abstract The role of leukocyte inflammatory markers and toll like receptors (TLRs)2/4 in pathologies associated with elevated resting heart rate (RHR) levels in healthy obese (HO) individuals is not well elucidated. Herein, we investigated the relationship of RHR with expression of leukocyte-inflammatory markers and TLRs in HO individuals. 58-obese and 57-lean participants with no history of a major medical condition, were recruited in this study. In HO individuals, the elevated-RHR correlated positively with diastolic blood pressure, cholesterol, pro-inflammatory monocytes CD11b+CD11c+CD206− phenotype (r = 0.52, P = 0.0003) as well as with activated T cells CD8+HLA-DR+ phenotype (r = 0.27, P = 0.039). No association was found between RHR and the percentage of CD16+CD11b+ neutrophils. Interestingly, elevated RHR positively correlated with cells expressing TLR4 and TLR2 (CD14+TLR4+, r = 0.51, P ≤ 0.0001; and CD14+TLR2+, r = 0.42, P = 0.001). TLR4+ expressing cells also associated positively with the plasma concentrations of proinflammatory or vascular permeability/matrix modulatory markers including TNF-α (r = 0.36, P = 0.005), VEGF (r = 0.47, P = 0.0002), and MMP-9 (r = 0.53, P ≤ 0.0001). Multiple regression revealed that RHR is independently associated with CD14+TLR4+ monocytes and VEGF. We conclude that in HO individuals, increased CD14+TLR4+ monocytes and circulatory VEGF levels associated independently with RHR, implying that RHR monitoring could be used as a non-invasive clinical indicator to identify healthy obese individuals at an increased risk of developing inflammation and cardiovascular disease.

Abstract The imipramine ONC201 has antiproliferative effects in several cancer cell types and activates integrated stress response pathway associated with the induction of Damage Inducible Transcript 3 (DDIT3, also known as C/EBP homologous protein or CHOP). We investigated the signaling pathways through which ONC201/CHOP crosstalk is regulated in ONC201-treated nonmetastatic and metastatic cancer cell lines (Dukes' type B colorectal adenocarcinoma nonmetastatic SW480 and metastatic LS-174T cells, respectively). Cell proliferation and apoptosis were evaluated by MTT assays and flow cytometry, gene expression was assessed by Affymetrix microarray, signaling pathway perturbations were assessed in silico, and key regulatory proteins were validated by Western blotting. Unlike LS-174T cells, SW480 cells were resistant to ONC201 treatment; Gene Ontology analysis of differentially expressed genes showed that cellular responsiveness to ONC201 treatment also differed substantially. In both ONC201-treated cell lines, CHOP expression was upregulated; however, its upstream regulatory mechanisms were perturbed. Although, PERK, ATF6 and IRE1 ER-stress pathways upregulated CHOP in both cell types, the Bak/Bax pathway regulated CHOP only LS-174T cells. Additionally, CHOP RNA splicing profiles varied between cell lines; these were further modified by ONC201 treatment. In conclusion, we delineated the signaling mechanisms by which CHOP expression is regulated in ONC201-treated non-metastatic and metastatic colorectal cell lines. The observed differences could be related to cellular plasticity and metabolic reprogramming, nevertheless, detailed mechanistic studies are required for further validations.

Abstract Ceramide kinase (CERK) phosphorylates ceramide to produce ceramide-1-phosphate (C1P), which is involved in the development of metabolic inflammation. TNF-α modulates inflammatory responses in monocytes associated with various inflammatory disorders; however, the underlying mechanisms remain not fully understood. Here, we investigated the role of CERK in TNF-α-induced inflammatory responses in monocytes. Our results show that disruption of CERK activity in monocytes, either by chemical inhibitor NVP-231 or by small interfering RNA (siRNA), results in the defective expression of inflammatory markers including CD11c, CD11b and HLA-DR in response to TNF-α. Our data show that TNF-α upregulates ceramide phosphorylation. Inhibition of CERK in monocytes significantly reduced the secretion of IL-1β and MCP-1. Similar results were observed in CERK-downregulated cells. TNF-α-induced phosphorylation of JNK, p38 and NF-κB was reduced by inhibition of CERK. Additionally, NF-κB/AP-1 activity was suppressed by the inhibition of CERK. Clinically, obese individuals had higher levels of CERK expression in PBMCs compared to lean individuals, which correlated with their TNF-α levels. Taken together, these results suggest that CERK plays a key role in regulating inflammatory responses in human monocytes during TNF-α stimulation. CERK may be a relevant target for developing novel therapies for chronic inflammatory diseases.

Abstract Surface area and surface active sites are two important key parameters in enhancing the gas sensing as well as photocatalytic properties of the parent material. With this motivation, herein, we report a facile synthesis of Reduced Graphene Oxide/Tungsten Oxide RGO/WO3 hierarchical nanostructures via simple hydrothermal route, and their validation in accomplishment of improved H2S sensing and highly efficient solar driven photo-degradation of RhB Dye. The self-made RGO using modified Hummer’s method, is utilized to develop the RGO/WO3 nanocomposites with 0.15, 0.3 and 0.5 wt% of RGO in WO3 matrix. As-developed nanocomposites were analyzed using various physicochemical techniques such as XRD, FE-SEM, TEM/HRTEM, and EDAX. The creation of hierarchic marigold frameworks culminated in a well affiliated mesoporous system, offering efficient gas delivery networks, leading to a significant increase in sensing response to H2S. The optimized sensor (RGO/WO3 with 0.3 wt% loading) exhibited selective response towards H2S, which is ~ 13 times higher (Ra/Rg = 22.9) than pristine WO3 (Ra/Rg = 1.78) sensor. Looking at bi-directional application, graphene platform boosted the photocatalytic activity (94% degradation of Rhodamine B dye in 210 min) under natural sunlight. The RGO’s role in increasing the active surface and surface area is clarified by the H2S gas response analysis and solar-driven photo-degradation of RhB dye solution. The outcome of this study provides the new insights to RGO/WO3 based nanocomposites’ research spreadsheet, in view of multidisciplinary applications.

Abstract Obesity, a major risk factor for metabolic disorders, is highly prevalent in Qatari population. Maternal transmission of obesity traits can be significant; for example, X haplogroup is known to be associated with lower BMI and body fat mass in Northern Europeans and T haplogroup which is a sister haplogroup of J is known to be associated with obesity in Caucasian subjects from Austria and Southern Italy. We aimed to delineate the mitochondrial haplogroups and variants associated with obesity in Qatari population. Mitochondrial genomes of 864 Qatari individuals were extracted from whole exome sequencing data with an average coverage of 77X. We distributed the participants into 2 sub-cohorts: obese (BMI ≥ 30) and non-obese (BMI

Abstract Segmental bone defects caused by trauma and disease represent a major clinical problem worldwide. Current treatment options are limited and often associated with poor outcomes and severe complications. Bone engineering is a promising alternative solution, but a number of technical challenges must be addressed to allow for effective and reproducible construction of segmental grafts that meet the size and geometrical requirements needed for individual patients and routine clinical applications. It is important to devise engineering strategies and standard operating procedures that make it possible to scale up the size of bone-engineered grafts, minimize process and product variability, and facilitate technology transfer and implementation. To address these issues, we have combined traditional and modular tissue engineering approaches in a strategy referred to as Segmental Additive Tissue Engineering (SATE). To demonstrate this approach, a digital reconstruction of a rabbit femoral defect was partitioned transversally to the longitudinal axis into segments (modules) with discoidal geometry and defined thickness to enable protocol standardization and effective tissue formation in vitro. Bone grafts corresponding to each segment were then engineered using biomimetic scaffolds seeded with human induced pluripotent stem cell-derived mesodermal progenitors (iPSC-MPs) and a novel perfusion bioreactor with universal design. The SATE strategy enables the effective and reproducible engineering of segmental bone grafts for personalized skeletal reconstruction, and will facilitate technology transfer and implementation of a tissue engineering approach to segmental bone defect therapy.

Abstract The limited information about the sludge quality has made its management a top environmental challenge. In the present study, occurrence and the spatiotemporal variations of 52 inorganic elements were investigated in the sludge samples from three wastewater treatment plants (WWTPs) in Xiamen city, China. The results showed, the occurrence of 49 elements with the concentrations in the range of >125–53500 mg kg−1 dry sludge (DS) for commonly used industrial metals, 1.22–14.0 mg kg−1 DS for precious metals, and 1.12–439.0 mg kg−1 DS for rare earth elements. The geo-accumulation studies indicated a moderate to high levels of buildup of some elements in the sewage sludge. Principal components analysis (PCA) indicated strong spatial and weak temporal variations in the concentrations of the elements. Therefore, the sludge disposal operations, based on the element concentrations, geoaccumulation and economic potential are suggested for each WWTP. Sludge from W1 and W2 were found suitable for agricultural usage, while that from W3 showed a higher economic potential for the recovery of precious metals. This study concludes that a comprehensive analysis of the elements in the sewage sludge could provide critical information for the disposal and management of the sludge.

Abstract Biventricular pacing is an important modality to improve left ventricular (LV) synchronization and long-term function. However, the biological effects of this treatment are far from being elucidated and existing animal models are limited and demanding. Recently, we introduced an implanted device for double-site epicardial pacing in rats and echocardiographically demonstrated favorable effects of LV and biventricular (LV-based) pacing modes typically observed in humans. Here, this new animal model was further characterized. Electrodes were implanted either on the right atria (RA) and right ventricle (RV) or on the RV and LV. Following recovery, rats were either used for invasive hemodynamic measurements (pressure-volume analysis) or exposed to sustained RV vs. biventricular tachypacing for 3 days. RV pacing compromised, while LV-based pacing modes markedly enhanced cardiac performance. Changes in LV performance were associated with prominent compensatory changes in arterial resistance. Sustained RV tachypacing increased the electrocardiogram QTc interval by 7.9 ± 3.1 ms (n = 6, p