In vitro photoprotective potential of aryl-sandwiched (thio)semicarbazones against UVA mediated cellular and DNA damage.

The most prevalent solar ultraviolet radiation is ultraviolet-A (UVA) radiation. It is the inducer of reactive oxygen species (ROS), a potent mediator of inflammation and photocarcinogenesis. Regular application of sunscreens containing UVA filters is an effective preventive measure in mitigating the risk associated with the formation of dermal carcinoma. Therefore, the development of new photoprotective agents is of great need. The current work examined the in vitro photoprotection of the aryl-linked (thio)semicarbazone derivatives against UVA-mediated DNA damage, inflammation, reactive nitrogen species (RNS), and ROS. Except for the inflammatory cytokine assay, which was carried out on the human monocytic leukemia (THP-1) cell line, all tests were conducted on the human dermal fibroblast (BJ) cell line. In comparison to benzophenone (reference compound), the compound (2Z, 2'Z)-2,2'-(1,3-Phenylenebis (methanylylidene)) bis (hydrazine-1-carbothioamide) (DD-21) demonstrated considerable protection against UVA-induced damage. Compared to the UVA-irradiated control, DD-21 significantly decreased the levels of nitric oxide (NO) and ROS (p < 0.001). In the presence of DD-21, the release of UVA-induced pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß), was also significantly reduced (p < 0.05). Moreover, it was observed that DD-21 protected the cells from UVA-mediated DNA strand breaks and also inhibited the formation of cyclobutane pyrimidine dimers (CPDs) upon comparison to the UVA-exposed control cells (p < 0.001). In conclusion, the findings of this study revealed that DD-21 exhibits remarkable photoprotective properties, thus demonstrating its potential as a candidate UVA filter.

Part-II: an update of Schiff bases synthesis and applications in medicinal chemistry-a patent review (2016-2023).

INTRODUCTION: Schiff bases are compounds with characteristic features of azomethine linkage (-C=N-). Schiff bases are capable of coordinating with metal ions via azomethine nitrogen. Schiff base derivatives and their metal complexes are known for intriguing novel therapeutic properties. In organic synthesis, the Schiff base reaction is prime in creating the C-N bond. Synthetic accessibility and structural diversity are the salient features for facile synthesis of Schiff base hybrids via a condensation reaction between an aldehyde/ketone and primary amines. AREA COVERED: This review aims to provide a comprehensive overview of the commendable medicinal applications of Schiff base derivatives and their metal complexes patented from 2016 to 2023. EXPERT OPINION: Schiff base derivatives are exceptional molecules for their assorted applications in medicinal chemistry. Several Schiff base products are marketed as drugs, and plenty of room is available for the purposive synthesis of new compounds in a diverse pool of disciplines. Expansion in the derivatization of Schiff bases in innumerable directions with multitudinous applications makes them 'magical molecules.' These compounds have proved extraordinary, from medicinal chemistry to other fields outside medicine. This review covers the therapeutic importance of Schiff base derivatives and aims to cover the patents published in recent years (2016-2023).

Association of lipid metabolism-related metabolites with overweight/obesity based on the FTO rs1421085.

Globally, obesity is a severe health issue. A more precise and practical approach is required to enhance clinical care and drug development. The FTO (fat mass and obesity-associated) gene variant rs1421085 is strongly associated with an increased susceptibility to obesity in numerous populations; however, the precise mechanism behind this association concerning metabolomics is still not understood. This study aims to examine the association between metabolites and obesity-related anthropometric traits based on the variant FTO rs1421085. This study was based on a case-control design involving a total of 542 participants including overweight/obese cases and healthy controls. The blood samples were collected from all the participants. The isolated serum samples were subjected to untargeted metabolomics using GC-MS. The isolated DNA samples were genotyped for the FTO rs1421085 variant. Initially, a total of 42 metabolites were identified on GC-MS, which were subjected to further association analyses. The study observed a significant association of two metabolites, glycerol and 2,3-dihydroxypropyl stearate with FTO gene variant rs1421085 and obesity-related anthropometric traits including % BF, WHtR, WC, and HC. The CT genotype of FTO rs1421085 may greatly increase the risk of overweight/obesity by changing the lipid metabolism-related metabolites. Therefore, this study highlights the significance of biochemical networks in the progression of obesity in carriers of the FTO rs1421085 risk genotype.

Interactive effects of FTO rs9939609 and obesogenic behavioral factors on adiposity-related anthropometric and metabolic phenotypes.

Numerous genetic variants have been linked to obesity predisposition, however, the interplay of genetic and behavioral factors is very crucial in determining the final phenotype. Therefore, this study examined the interactive effects of the FTO rs9939609 and various obesogenic behavioral factors on adiposity-related anthropometric and metabolic phenotypes in a sample of Pakistani population. A total of 612 participants encompassing 306 overweight/obese (cases) and an equal number (306) of age- and sex-matched normal-weight (controls) individuals were included in the study. Adiposity-related anthropometric parameters were collected by taking corresponding body measurements by following standard procedures. The metabolic parameters were assessed by performing corresponding biochemical assays. A standard questionnaire was devised for the collection of adiposity-related behavioral information. The FTO rs9939609 was genotyped by employing TaqMan allelic discrimination assay. The data was analyzed by using SPSS software. Interactive effects of the FTO rs9939609 and behavioral factors on obesity-related anthropometric and metabolic phenotypes were examined via linear regression by adjusting potential confounders and making correction for multiple comparisons. The results implied that the interaction between the FTO rs9939609 and low physical activity may significantly increase various adiposity-related anthropometric variables (p < 0.05). However, no such interactive effects were found on any adiposity-related metabolic variable. In conclusion, the interaction between the FTO rs9939609 and low physical activity may have a significant impact on obesity-related anthropometric traits in the Pakistani population.

Probing photoprotection properties of lipophilic chain conjugated thiourea-aryl group molecules to attenuate ultraviolet-A induced cellular and DNA damages.

Ultraviolet-A (UVA) radiation is a major contributor to reactive oxygen species (ROS), reactive nitrite species (RNS), inflammation, and DNA damage, which causes photoaging and photocarcinogenesis. This study aimed to evaluate the UVA protective potential of lipophilic chain conjugated thiourea-substituted aryl group molecules against UVA-induced cellular damages in human dermal fibroblasts (BJ cell line). We tested a series of nineteen (19) molecules for UVA photoprotection, from which 2',5'-dichlorophenyl-substituted molecule DD-04 showed remarkable UVA protection properties compared to the reference (benzophenone). The results indicate that DD-04 significantly reduced intracellular ROS and nitric oxide (NO) as compared to the UVA-irradiated control (p < 0.001). Moreover, the compound DD-04 showed anti-inflammatory activity as it significantly reduced the levels of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) pro-inflammatory cytokines produced by THP-1 (human monocytic) cells (p < 0.05). DNA damage was also prevented by DD-04 treatment in the presence of UVA. It was observed that DD-04 significantly reduced the number of cyclobutane pyrimidine dimers (CPDs) when compared to the UVA-irradiated control (p < 0.001). Finally, the DNA strand breaks were checked and a single intact DNA band was seen upon treatment with DD-04 in the presence of UVA. In conclusion, DD-04 can be considered a potential candidate UVA filter due to its photoprotective potential.

Effect of interaction between obesity-promoting genetic variants and behavioral factors on the risk of obese phenotypes.

The studies investigating gene-gene and gene-environment (or gene-behavior) interactions provide valuable insight into the pathomechanisms underlying obese phenotypes. The Pakistani population due to its unique characteristics offers numerous advantages for conducting such studies. In this view, the current study was undertaken to examine the effects of gene-gene and gene-environment/behavior interactions on the risk of obesity in a sample of Pakistani population. A total of 578 adult participants including 290 overweight/obese cases and 288 normal-weight controls were involved. The five key obesity-associated genetic variants namely MC4R rs17782313, BDNF rs6265, FTO rs1421085, TMEM18 rs7561317, and NEGR1 rs2815752 were genotyped using the TaqMan allelic discrimination assays. The data related to behavioral factors, such as eating pattern, diet consciousness, the tendency toward fat-dense food (TFDF), sleep duration, sleep-wake cycle (SWC), shift work (SW), and physical activity levels were collected via a questionnaire. Gene-gene and gene-behavior interactions were analyzed by multifactor dimensionality reduction and linear regression, respectively. In our study, only TMEM18 rs7561317 was found to be significantly associated with anthropometric traits with no significant effect of gene-gene interactions were observed on obesity-related phenotypes. However, the genetic variants were found to interact with the behavioral factors to significantly influence various obesity-related anthropometric traits including BMI, waist circumference, hip circumference, waist-to-hip ratio, waist-to-height ratio, and percentage of body fat. In conclusion, the interaction between genetic architecture and behavior/environment determines the outcome of obesity-related anthropometric phenotypes. Thus, gene-environment/behavior interaction studies should be promoted to explore the risk of complex and multifactorial disorders, such as obesity.

Predicting anthropometric and metabolic traits with a genetic risk score for obesity in a sample of Pakistanis.

Obesity is an outcome of multiple factors including environmental and genetic influences. Common obesity is a polygenic trait indicating that multiple genetic variants act synergistically to influence its expression. We constructed a genetic risk score (GRS) based on five genetic variants (MC4R rs17782313, BDNF rs6265, FTO rs1421085, TMEM18 rs7561317, and NEGR1 rs2815752) and examined its association with obesity-related traits in a sample of Pakistanis. The study involved 306 overweight/obese (OW/OB) and 300 normal-weight (NW) individuals. The age range of the study participants was 12-63 years. All anthropometric and metabolic parameters were measured for each participant via standard procedures and biochemical assays, respectively. The genetic variants were genotyped by allelic discrimination assays. The age- and gender-adjusted associations between the GRS and obesity-related anthropometric and metabolic measures were determined using linear regression analyses. The results showed that OW/OB individuals had significantly higher mean ranks of GRS than NW individuals. Moreover, a significant association of the GRS with obesity-related anthropometric traits was seen. However, the GRS did not appear to affect any obesity-related metabolic parameter. In conclusion, our findings indicate the combined effect of multiple genetic variants on the obesity-related anthropometric phenotypes in Pakistanis.

Association of metabolites with obesity based on two gene variants, MC4R rs17782313 and BDNF rs6265.

Previous genome-wide association analyses for obesity related genes demonstrated the association of BDNF gene variant rs6265 and MC4R gene variant rs17782313 with body mass index (BMI). However, the associated metabolite pathways are still behind the curtain. The aim of the current study is to investigate the associations of metabolic changes in obesity with MC4R gene variant rs17782313 and BDNF variant rs6265. Gas chromatography-mass spectrometry based untargeted metabolomics approach was used and 42 identified serum metabolites were selected for statistical analyses. Significant association of seven metabolites with MC4R gene variant rs17782313 based on obesity and thirty metabolites with obesity dependent BDNF variant rs6265 using additive model (adjusted p < 0.05) was observed. This study highlights the importance of alteration of fatty acid biosynthesis, probably due to high consumption of fats may cause to develop obesity. But obesity is a complex disorder and the full clarification of this complex machinery is still distant. To understand the obesity in a better way, more studies are required to identify remaining metabolites and also mechanism of these metabolic entities.

Multicomponent reactions (MCR) in medicinal chemistry: a patent review (2010-2020).

Introduction: Multicomponent reactions (MCR) has been utilized to synthesize a vast range of analogs belonging to diverse classes of heterocyclic compounds offering multidimensional pharmaceutical applications. The unique feature of MCR includes the synthesis of highly functionalized molecules in a single pot to build quick libraries of compounds of biological interest to identify new leads as potential therapeutic agents.Area covered: The current review article covers the patents published in the last decade in order to highlight the importance of multicomponent reactions for synthesizing complex-functionalized molecules of high biological significance.Expert opinion: Easily automated one-pot multicomponent reactions (MCRs) has demonstrated successful impact at different stages of the lead discovery, lead optimization, and pre-clinical process development arenas. Application of MCRs is the recent advancement in the field of drug design and discovery which will expectedly lead to the development of medicinally important heterocyclic compounds with a vast range of biological activities.

Association of the NEGR1 rs2815752 with obesity and related traits in Pakistani females.

Introduction: The variant NEGR1 rs2815752 has recently been linked with obesity in Caucasians. However, a very limited number of studies have examined the association of the NEGR1 rs2815752 with overweight/obesity in non-Caucasians with no such study ever performed in Pakistani population. Therefore, the present study was undertaken to seek the association of the rs2815752 with overweight, obesity, and related traits in Pakistanis.Subjects and methods: The study involved 112 overweight/control pairs (total 224) and 194 obese/control pairs (total 388). Anthropometric parameters were measured by employing standard procedures. Metabolic parameters were determined by biochemical assays. Behavioral information was collected through a questionnaire. The rs2815752 was genotyped via TaqMan allelic discrimination assay. Regression analyses were employed to analyze the data in SPSS software.Results: The study revealed significant gender-specific association of the rs2815752 with obesity (OR 3.03; CI 1.19-7.72, p = 0.020) and some obesity-related anomalous anthropometric traits (weight, BMI, waist circumference, hip circumference, and abdominal and supra-iliac skinfold thicknesses) in females according to dominant model (h = 0.023). However, no association of the rs2815752 with obesity-related behavioral and metabolic parameters was observed.Conclusion: The NEGR1 rs2815752 may be associated with obese phenotype and some of the related anthropometric traits in Pakistani females.

Metabolic implications of circadian disruption.

Circadian rhythms are generated by the circadian clock, a self-sustained internal timing system that exhibits 24-h rhythms in the body. In mammals, circadian rhythms are driven by a central clock located in suprachiasmatic nucleus and various peripheral clocks located in different tissues and organs of the body. Many cellular, behavioral, and physiological processes are regulated by the circadian clock in coordination with environmental cues. The process of metabolism is also under circadian regulation. Loss of synchronization between the internal clock and environmental zeitgebers results in disruption of the circadian rhythms that seriously impacts metabolic homeostasis leading to changed eating behavior, altered glucose and lipid metabolism, and weight gain. This in turn augments the risk of having various cardio-metabolic disorders such as obesity, diabetes, metabolic syndrome, and cardiovascular disease. This review sheds light on circadian rhythms and their role in metabolism with the identification of gaps in the current knowledge that remain to be explored in these fields. In this review, the molecular mechanisms underlying circadian rhythms have been elaborated first. Then, the focus has been kept on explaining the physiological significance of circadian rhythms in regulating metabolism. Finally, the implications for metabolism when these rhythms are disrupted due to genetic mutations or social and occupational needs enforced by modern lifestyle have been discussed.

Association and interaction of the FTO rs1421085 with overweight/obesity in a sample of Pakistani individuals.

PURPOSE: Genetic variants determine the predisposition of an individual to obesity in a given environment. The present study was conducted to seek an association of the FTO variant rs1421085 with overweight/obesity and related traits in 612 Pakistani subjects in a case-control manner (overweight/obese = 306 and non-obese = 306). Moreover, interaction effects of the rs1421085 and overweight/obesity on multiple metabolic traits were also investigated, which were never explored before in Pakistani as well as in any other population. MATERIALS AND METHODS: Anthropometric traits were measured by standard procedures, while metabolic parameters were determined by biochemical assays. Genotyping of the rs1421085 was carried out by TaqMan allelic discrimination assay. The data were analysed using SPSS software version 19. RESULTS: The study revealed a significant association of the rs1421085 with overweight/obese phenotype with respect to over-dominant model indicated by h-index. The CT genotype of the rs1421085 was observed to increase the risk of being overweight/obese by 1.583 times (95% CI 1.147-2.185, p = 0.005). The CT genotype was also found to be associated with higher values of all anthropometric variables (except height and waist-to-hip ratio). Moreover, the interaction between the CT genotype of the rs1421085 and overweight/obesity was found to influence several metabolic parameters (raised blood pressure, product of triglyceride and glucose index, triglyceride levels, LDL-C, VLDL-C, coronary risk index, atherogenic index, and triglyceride-to-HDL-C ratio). CONCLUSION: In conclusion, the rs1421085 was found to be associated with overweight/obesity and related anthropometric traits independent of age and gender in Pakistani population. Moreover, this variant was found to influence various metabolic traits in the presence of overweight/obesity. LEVEL OF EVIDENCE: Level III, case-control analytic study.

Association of the Variant rs7561317 Downstream of the TMEM18 Gene with Overweight/Obesity and Related Anthropometric Traits in a Sample of Pakistani Population.

Obesity is a multifactorial disorder and requires favorable environment for its expression. However, some individuals are more prone to weight gain than others in an obesogenic environment. Thus, at individual level, who becomes overweight or obese is mostly determined by genetic factors. The current study was undertaken to explore for the first time the association of the TMEM18 rs7561317 variant with overweight/obesity and related anthropometric, metabolic, physical, and behavioral traits in a sample of Pakistani population with association between the rs7561317 and many traits was not investigated before in any population. The current study involved a total of 612 subjects including 306 overweight/obese and equal number of age- and sex-matched normal weight individuals. Obesity-related parameters were determined and the variant was genotyped by allelic discrimination assay. All the aforementioned associations were assessed by regression analyses adjusted for covariates and corrected for multiple comparisons. The results revealed a significant association of the TMEM18 rs7561317 with overweight/obese phenotype in more than one genetic model. Therefore, h-index (degree of dominance) was calculated, which indicated the recessive mode of inheritance for the above-said association. Similarly, a significant association of the rs7561317 with obesity-related anthropometric traits and clinical surrogate markers of visceral adiposity was observed. Thus, GG genotype of the rs7561317 was found to increase 1.74 times the risk of overweight/obesity in Pakistani population (OR = 1.74, 95% CI 1.210-2.496, p = 0.003) while low physical activity seemed to accentuate the TMEM18 rs7561317-associated risk of overweight/obesity (OR = 2.696, 95% CI 1.485-4.896, p = 0.004).

Association of BDNF rs6265 and MC4R rs17782313 with metabolic syndrome in Pakistanis.

The current case-control study sought the association of BDNF rs6265 and MC4R rs17782313 with metabolic syndrome (MetS), MetS components and other related metabolic parameters in a sample of Pakistani subjects. Fasting high-density lipoprotein cholesterol (HDL-C) and homeostatic model assessment of insulin sensitivity showed a significantly lower mean whereas body mass index (BMI), waist circumference, systolic blood pressure (SBP), diastolic blood pressure (DBP), fasting blood glucose, insulin, total cholesterol (TC), low-density lipoprotein cholesterol, very-low-density lipoprotein cholesterol, triglycerides (TG), cholesterol to HDL-C ratio, TG to HDL-C ratio, homeostatic model assessment of insulin resistance, visceral adiposity index, lipid accumulation product and the product of TG and glucose showed a significantly higher mean in the presence of MetS. Reduced HDL-C appeared as the most frequent and hypertriglyceridemia as the least frequent component of MetS whereas clustering of reduced HDL-C + abdominal obesity (AO) + hyperglycemia appeared as the most prevalent combination of MetS components. Moreover, BDNF rs6265 showed BMI and gender independent association with increased risk of MetS in Pakistani individuals whereas MC4R rs17782313 showed BMI and gender dependent association with increased risk of MetS in Pakistani females. In addition, BDNF rs6265 and MC4R rs17782313 showed gender-dependent associations with decreased risk of having low HDL-C in males and increased risk of having abdominal obesity in females, respectively. However, no association was observed for metabolic variables other than components of MetS across genotypes of both BDNF rs6265 and MC4R rs17782313.

The BDNF rs6265 variant may interact with overweight and obesity to influence obesity-related physical, metabolic and behavioural traits in Pakistani individuals.

BACKGROUND: The association of the variant rs6265 (G>A) in the brain-derived neurotrophic factor (BDNF) gene with obesity and other obesity-related parameters is not known for the Pakistani population. Moreover, the effects of interaction between BDNF rs6265 and overweight/obesity on obesity-related traits have never been investigated before. AIM: To find the association of the BDNF rs6265 with obesity and related traits and to explore the effect of rs6265 × obesity interaction on obesity-related traits in Pakistanis. SUBJECTS AND METHODS: The study involved a total of 606 subjects, including 306 overweight and obese (OW/OB) cases and 300 normal weight (NW) controls. The genotyping of the BDNF rs6265 was done and obesity-related anthropometric, physical, behavioural and metabolic parameters were determined. Statistical analyses using SPSS software were performed to find the associations. RESULTS: The study revealed a lack of association of the BDNF rs6265 with obesity and obesity-related traits. On the other hand, the interaction between the BDNF rs6265 and overweight/obesity was found to be significantly associated with some of the obesity-related anomalous traits. However, no association between rs6265 and these anomalous traits was seen in either group when the association test was performed in NW and OW/OB groups separately. CONCLUSION: The BDNF rs6265, in the presence of obesity, may be associated with elevated risk of anomalous metabolic, behavioural and physical traits and obesity-related co-morbidities, but it needs to be validated in a significantly larger Pakistani sample population.

MC4R variant rs17782313 and manifestation of obese phenotype in Pakistani females.

MC4R represents a key player involved in melanocortin-mediated control of energy balance. Recently identified near MC4R variant rs17782313 (T > C) can serve as a contributing factor for obese phenotype but its association with obesity has never been sought in a sample of the Pakistani population. The role of genetic variants as causal factors varies across populations. Association studies in a specific population can help us to distinguish global from local gene-gene and gene-environment interactions. This is the first study that investigated the association of rs17782313 with obesity and various obesity-linked anthropometric, metabolic, physical, and behavioural traits in Pakistani subjects including 306 OW/OB (overweight and obese) and 300 NW (normal weight) individuals. The comparison of various aforementioned obesity-linked continuous and categorical variables between OW/OB and NW subjects revealed that almost all variables were found significantly aberrant (p < 0.05) in OW/OB subjects as compared to their age- and gender-matched NW controls indicating greater risk of developing various cardio-metabolic disorders. The genotyping of rs17782313 showed significant association of this variant with obesity and obesity-linked anthropometric traits in females suggesting the gender-specific effect of this variant in our population. The minor allele C increased the risk of obesity by 1.55 times (95% CI = 1.1-2.18, p = 0.01) whereas homozygous CC genotype increased the risk by 2.43 times (95% CI = 1.19-4.96, p = 0.015) in females. However, no association of rs17782313 was observed with any of the obesity-linked metabolic, physical, and behavioural traits except random eating timings. In conclusion, the current study significantly contributes to the knowledge of the genetic proneness to obesity in Pakistani females. This could also be helpful for forthcoming meta-analysis studies elucidating which variants are truly associated with the susceptibility to develop an obese phenotype.

Role of leptin G-2548A polymorphism in age- and gender-specific development of obesity.

Leptin is involved in the regulation of food intake and energy expenditure, and therefore, is central to adipositysensing pathway. We examined the relationship of the leptin G-2548A polymorphism with obesity and obesityrelated anthropometric and metabolic parameters in a total of 394 (239 obese and 155 non-obese) subjects between 5 and 45 years of age. Body weight, height, waist circumference (WC), hip circumference (HC) and blood pressure (BP) were measured. Body mass index (BMI) and waist-to-hip ratio (WHR) were calculated. Levels of fasting blood glucose (FBG), insulin, leptin and leptin receptor were determined, and homeostasis model assessment of insulin resistance (HOMA-IR) was calculated. Genotyping was carried out by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The LEP G-2548A polymorphism showed association with obesity in children and adolescents (less than or equal to 18 years of age) but not in adults. However, analysis by gender stratification revealed association with obesity in girls only. In addition, G-2548A polymorphism showed association with BMI, WC, HC, fasting blood glucose and serum leptin levels. This suggests that G-2548A polymorphism may influence the susceptibility to metabolic disturbances and obesity at an early life. Further investigation with a larger sample size is required to validate the effect of LEP G-2548A polymorphism in obese Pakistani girls.

Lack of association of the NPAS2 gene Ala394Thr polymorphism (rs2305160:G>A) with risk of chronic lymphocytic leukemia.

BACKGROUND: NPAS2 is a product of the circadian clock gene. It acts as a putative tumor suppressor by playing an important role in DNA damage responses, cell cycle control and apoptosis. Chronic lymphocytic leukemia (CLL) appears to be an apoptosis related disorder and alteration in the NPAS2 gene might therefore be directly involved in the etiology of CLL. Here, the Ala394Thr polymorphism (rs2305160:G>A) in the NPAS2 gene was genotyped and melatonin concentrations were measured in a total of seventy-four individuals, including thirty-seven CLL cases and an equal number of age- and sex-matched healthy controls in order to examine the effect of NPAS2 polymorphism and melatonin concentrations on CLL risk in a Pakistani population. MATERIALS AND METHODS: Genotyping of rs2305160:G>A polymorphism at NPAS2 locus was carried out by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR). Melatonin concentrations were determined by enzyme linked immunosorbent assay (ELISA). Statistical analysis was performed using Statistical Package for Social Sciences software. RESULTS: Our results demonstrated no association of the variant Thr genotypes (Ala/ Thr and Thr/Thr) with risk of CLL. Similarly, no association of rs2305160 with CLL was observed in either females or males after stratification of study population on a gender basis. Moreover, when the subjects with CLL were further stratified into shift-workers and non-shift-workers, no association of rs2305160 with CLL was seen in either case. However, significantly low serum melatonin levels were observed in CLL patients as compared to healthy subjects (p<0.05). Also, lower melatonin levels were seen in shift-workers as compared to non-shift-workers (p<0.05). There was no significant difference (p>0.05) in the melatonin levels across NPAS2 genotypes in all subjects, subjects with CLL who were either shift workers or non-shift-workers. General Linear Model (GLM) univariate analysis revealed no significant association (p>0.05) of the rs2305160 polymorphism of the NPAS2 gene with melatonin levels in any of the groups. CONCLUSIONS: While low melatonin levels and shift-work can be considered as one of the risk factors for CLL, the NPAS2 rs2305160 polymorphism does not appear to have any association with risk of CLL in our Pakistani population.

Deregulated expression of circadian clock and clock-controlled cell cycle genes in chronic lymphocytic leukemia.

Circadian rhythms are endogenous and self-sustained oscillations of multiple biological processes with approximately 24-h rhythmicity. Circadian genes and their protein products constitute the molecular components of the circadian oscillator that form positive/negative feedback loops and generate circadian rhythms. The circadian regulation extends from core clock genes to various clock-controlled genes that include various cell cycle genes. Aberrant expression of circadian clock genes, therefore, may lead to genomic instability and accelerated cellular proliferation potentially promoting carcinogenesis. The current study encompasses the investigation of simultaneous expression of four circadian clock genes (Bmal1, Clock, Per1 and Per2) and three clock-controlled cell cycle genes (Myc, Cyclin D1 and Wee1) at mRNA level and determination of serum melatonin levels in peripheral blood samples of 37 CLL (chronic lymphocytic leukemia) patients and equal number of age- and sex-matched healthy controls in order to indicate association between deregulated circadian clock and manifestation of CLL. Results showed significantly down-regulated expression of Bmal1, Per1, Per2 and Wee1 and significantly up-regulated expression of Myc and Cyclin D1 (P < 0.0001) in CLL patients as compared to healthy controls. When expression of these genes was compared between shift-workers and non-shift-workers within the CLL group, the expression was found more aberrant in shift-workers as compared to non-shift-workers. However, this difference was found statistically significant for Myc and Cyclin D1 only (P < 0.05). Serum melatonin levels were found significantly low (P < 0.0001) in CLL subjects as compared to healthy controls whereas melatonin levels were found still lower in shift-workers as compared to non-shift-workers within CLL group (P < 0.01). Our results suggest that aberrant expression of circadian clock genes can lead to aberrant expression of their downstream targets that are involved in cell proliferation and apoptosis and hence may result in manifestation of CLL. Moreover, shift-work and low melatonin levels may also contribute in etiology of CLL by further perturbing of circadian clock.

Common variant of FTO gene, rs9939609, and obesity in Pakistani females.

Numerous studies confirmed the association of FTO (fat mass and obesity associated gene) common variant, rs9939609, with obesity in European populations. However, studies in Asian populations revealed conflicting results. We examined the association of rs9939609 variant of FTO gene with obesity and obesity-related anthropometric and metabolic parameters in Pakistani population. Body weight, height, waist circumference, hip circumference, and blood pressure (BP) were measured. BMI and waist-to-hip ratio (WHR) were calculated. Levels of fasting blood glucose (FBG), insulin, leptin, and leptin receptors were measured by enzyme linked immunosorbent assay (ELISA), and homeostasis model assessment of insulin resistance (HOMA-IR) was calculated. The results showed association of FTO gene, rs9939609, with obesity in females (>18 years of age). FTO minor allele increased the risk of obesity by 2.8 times (95% CI = 1.3-6.0) in females. This allele showed association with body weight, BMI, waist circumference, hip circumference, WHR, BP, plasma FBG levels, HOMA-IR, plasma insulin levels, and plasma leptin levels. In conclusion, FTO gene, rs9939609, is associated with BMI and risk of obesity in adult Pakistani females. Association of rs9939609 variant with higher FBG, plasma insulin, and leptin levels indicates that this polymorphism may disturb the metabolism in adult females and predispose them to obesity and type 2 diabetes. However, the above-mentioned findings were not seen in children or males.