Particular guanine rich DNA sequences can fold into stable four-stranded G-quadruplex structures, under physiological concentrations of Na + and K + , in vitro.1 Such sequence motifs are found in the telomeres where they can fold into quadruplexes under the control of specific telomere binding proteins.2 G-quadruplex motifs have been identified throughout the genome3 and concentrate immediately upstream of transcription initiation sites.3a A number of these so-called "promoter quadruplex" sequences have been studied for several proto-oncogenes, including c-MYC,4 BCL2,5 VEGF,6 KRAS7 and two G-quadruplexes in the c-kit promoter8 (c-kit1 and c-kit2). One working hypothesis couples quadruplex formation in promoters to transcription, suggesting an opportunity for chemical intervention of gene expression using small molecule G-quadruplex ligands. Some proof-of-concept has been provided for the case of c-MYC where small molecule ligands, TmPyP4,4 and quindoline9 derivatives have been shown to inhibit gene expression, while KRAS gene expression was inhibited by TmPyP4.7The c-kit proto-oncogene encodes a tyrosine kinase receptor for the growth-promoting cytokine SCF (stem cell factor) which plays an important biological role in the control of differentiation.10 A small molecule inhibitor of c-kit, Gleevec (imatinib mesylate), is being effectively used in the treatment of gastrointestinal stromal tumors (GIST).11 A small molecule that inhibits c-kit expression at the transcriptional level would provide further evidence to support the promoter-quadruplex hypothesis and might inspire the exploration of quadruplex-based therapeutic approaches to address GIST. Herein, we report the design, synthesis, biophysical evaluation with primary biological data on 3,8,10-trisubstituted isoalloxazines ( Figure 1).The design of isoalloxazines as potential G-quadruplex ligands was inspired by the observation, arising from SELEX studies, that oxidized riboflavin (7,8-dimethyl-10-ribitylisoalloxazine) binds to an intramolecular G-quartet with moderate binding affinity (K d ) of 1-5 μM.12 Our design principles maintain the planar isoalloxazine scaffold, to enable interactions with G-quartet.13 Amine side chains were introduced to provide potential for interactions with quadruplex loops and grooves and the negatively charged sugar-phosphate backbone. We developed and employed a short and robust synthetic route to 3,8,10-trisubstituted isoalloxazines to prepare ligands of general structure 1 (Figure 1). The key isoalloxazine building blocks were synthesized using modified literature procedures.14 An efficient method was developed for the introduction of amino alkyl side chains to afford 1a-f in good overall yields (see Supporting Information).To evaluate the interaction properties of isoalloxazines 1a-f with a number of DNA targets, we employed surface plasmon resonance (SPR) to evaluate equilibrium binding15 and a fluorescence resonance energy transfer (FRET) melting assay16 to evaluate the stabilizing influence of the ligand. We included thr...

The BCL-2 gene encodes a 25 kDa membrane protein that plays critical roles in the control of apoptosis. The regulation of BCL-2 gene expression is highly complex and occurs both transcriptionally and posttranscriptionally. In particular, the 5′ upstream region of BCL-2 contains a number of elements that control its expression. We have identified a highly conserved 25-nucleotide G-rich sequence (BCL2Q), with potential to fold into a RNA G-quadruplex structure, located 42 nucleotides upstream of the translation start site of human BCL-2. In this study, we used a series of biophysical experiments to show that the BCL2Q sequence folds into a stable RNA G-quadruplex in vitro, and we conducted functional luciferase reporter-based assays, in a cell-free lysate and in three types of human cell lines, to demonstrate that the BCL2Q sequence modulates protein expression in the context of the 493-nucleotide native 5′ untranslated region of BCL-2.

Type 2 diabetes mellitus (T2DM) accounts for 90% of diabetes cases worldwide. The majority of T2DM patients are obese. Dysbiosis in the gut microflora is strongly associated with the pathogenesis of obesity and T2DM; however, the microbiome of obese-T2DM individuals in the Pakistani population remains unexplored. The gut microbiota signature of 60 Pakistani adults was studied using 16S rRNA sequencing targeting V3–V4 hypervariable regions. The sequence analysis revealed that bacteria from Firmicutes were predominant along with those from Clostridia and Negativicutes, whereas bacteria from Verrucomicrobia, Bacteroidetes, Proteobacteria, and Elusimicrobia were less abundant among the obese T2DM patients. These data distinctively vary from those in reports on the Indian population. The difference in gut microbiota could presumably be related to the distinct lifestyle and eastern dietary habits (high carbohydrate and fat intake, low fiber intake) and unregulated antibiotic consumption. This is the first study carried out to understand the gut microbiome and its correlation with individual life style of obese T2DM patients in the Pakistani population.

No abstract

No abstract