Enhancement of telomerase extension via quadruple nucleic acid recycling to develop a novel colorimetric biosensing method for kanamycin assay.

BACKGROUND: Colorimetric biosensors have important value for antibiotic residue testing. However, many previous methods were constructed based on the optical density change of certain unstable single-colored products with poor discrimination for visual measurements. Moreover, their low extinction coefficients usually result in low sensitivity of biosensors. In addition, many conventional signal amplification strategies often involve sophisticated nanomaterial preparation, inconvenient multi-step assay manipulation and limited signal amplification ability. Therefore, the development of new colorimetric biosensing strategies with excellent visual discrimination, high sensitivity and convenient manipulation is highly desirable. RESULTS: We designed a target recycling accelerated cascade DNA walking amplification mechanism to trigger a telomerase extension-related enzymatic reaction, and developed a novel colorimetric biosensing strategy for kanamycin (Kana) assay. The target recycling was induced by an exonuclease III-assisted aptamer recognition reaction, which could also trigger the successive DNA walking at the streptavidin (SA)- and magnetic bead (MB)-based tracks. This not only caused the quantitative exposure of the telomeric substrate primers on MB surfaces but also released another strand to accelerate the SA-based DNA walking. By using the telomerase extension product to link numerous alkaline phosphatases and induce the plasmonic property change of gold nanobipyramids (Au NBPs), a colorimetric signal output strategy was constructed. This method could be applied for the high-resolution visual screening of Kana, and it also showed a very low detection limit of 17.6 fg mL-1 for assaying Kana over a wide, five-order-magnitude linear range. SIGNIFICANCE: The quadruple nucleic acid recycling-enhanced telomerase extension resulted in the ultrahigh sensitivity of the method and also excluded the sophisticated manipulations involved in conventional biosensing strategies. The multiple enzyme catalysis-induced plasmonic property change of Au NBPs realized the stable and multicolor visual signal transduction. Together with its low cost, simple operation, high selectivity, excellent repeatability, and reliable performances, this method exhibits great potential for use in practical applications.

Evolution of GCGR family ligand-receptor extensive cross-interaction systems suggests a therapeutic direction for hyperglycemia in mammals.

Glucose is essential to the physiological processes of vertebrates. Mammalian physiological stability requires a relatively stable blood glucose level (~5 mM), whereas other vertebrates have greater flexibility in regulating blood glucose (0.5-25 mM). GCGR family receptors play an important role in vertebrate glucose regulation. Here, we examine the evolution of the GCGR family ligand-receptor systems in different species. Comparatively, we discover that the conserved sequences among GCG family ligands lead to the non-specific activation of ligands across species. In particular, we observe that glucagon-like peptide 1 receptor (GLP1R), glucagon-like peptide 2 receptor (GLP2R), and glucagon-like receptor (GCGLR, also called GCRPR) are arbitrarily activated by other members of the ligand family in birds. Moreover, we reveal that Gallus gallus GLP2 (gGLP2) effectively activates mammalian GLP1R and improves glucose tolerance in diabetic mice. Our study has important implications for understanding blood glucose stabilization in vertebrates and demonstrates that gGLP2 may be a potential drug for treating type 2 diabetes.

Predicting late-onset preeclampsia by detecting ELABELA content using an immunochromatographic colloidal gold test strip: Blood ELABELA content predicts the risk of pre-eclampsia.

Preeclampsia is a pregnancy disorder that seriously affects the outcome of mothers and infants and lacks effective prediction and diagnosis methods. ELABELA is the second endogenous ligand of the apelin receptor (APJ) and is associated with the pathogenesis of preeclampsia. In a previous study, the authors found that the downregulation of ELABELA expression is closely related to late-onset preeclampsia, which may be a marker for the clinical diagnosis of late-onset preeclampsia. In this study, the authors again collected 120 maternal blood samples, including 60 pregnant women with a medical diagnosis of late-onset preeclampsia. ELISA results showed that the serum ELABELA concentration in late-onset preeclampsia pregnant women (12.57 ± 7.77 ng/mL) was significantly lower than that in normal pregnant women (36.99 ± 23.58 ng/mL), which was consistent with previously reported results. Therefore, the authors used an ELABELA monoclonal antibody to label four colloidal gold nanoparticles with different diameters (15, 30, 55, and 150 nm) and developed a transverse-flow immunochromatographic band for the rapid and accurate detection of serum ELABELA levels. The strip test shows that colloidal gold with a diameter of 30 nm can be used as a good ELABELA detection marker and had more than 90% positive detection effect. Therefore, the authors hope that the colloidal gold strip with ELABELA as the diagnostic index developed by us will be popularized and applied in clinical diagnosis.

Multifunctional AIE Nanosphere-Based "Nanobomb" for Trimodal Imaging-Guided Photothermal/Photodynamic/Pharmacological Therapy of Drug-Resistant Bacterial Infections.

Injudicious or inappropriate use of antibiotics has led to the prevalence of drug-resistant bacteria, posing a huge menace to global health. Here, a self-assembled aggregation-induced emission (AIE) nanosphere (AIE-PEG1000 NPs) that simultaneously possesses near-infrared region II (NIR-II) fluorescence emissive, photothermal, and photodynamic properties is prepared using a multifunctional AIE luminogen (AIE-4COOH). The AIE-PEG1000 NPs were encapsulated with teicoplanin (Tei) and ammonium bicarbonate (AB) into lipid nanovesicles to form a laser-activated "nanobomb" (AIE-Tei@AB NVs) for the multimodal theranostics of drug-resistant bacterial infections. In vivo experiments validate that the "nanobomb" enables high-performance NIR-II fluorescence, infrared thermal, and ultrasound (AB decomposition during the photothermal process to produce numerous CO2/NH3 bubbles, which is an efficient ultrasound contrast agent) imaging of multidrug-resistant bacteria-infected foci after intravenous administration of AIE-Tei@AB NVs followed by 660 nm laser stimulation. The highly efficient photothermal and photodynamic features of AIE-Tei@AB NVs, combined with the excellent pharmacological property of rapidly released Tei during bubble generation and NV disintegration, collectively promote broad-spectrum eradication of three clinically isolated multidrug-resistant bacteria strains and rapid healing of infected wounds. This multimodal imaging-guided synergistic therapeutic strategy can be extended for the theranostics of superbugs.

The vertebrate- and testis- specific transmembrane protein C11ORF94 plays a critical role in sperm-oocyte membrane binding.

Sperm-oocyte membrane fusion is necessary for mammalian fertilization. The factors that determine the fusion of sperm with oocytes are largely unknown. So far, spermatozoon factor IZUMO1 and the IZUMO1 counter-receptor JUNO on the oocyte membrane has been identified as a protein requiring fusion. Some sperm membrane proteins such as FIMP, SPACA6 and TEME95, have been proved not to directly regulate fusion, but their knockout will affect the fusion process of sperm and oocytes. Here, we identified a novel gene C11orf94 encoding a testicular-specific small transmembrane protein that emerges in vertebrates likely acquired via horizontal gene transfer from bacteria and plays an indispensable role in sperm-oocyte binding. We demonstrated that the deletion of C11orf94 dramatically decreased male fertility in mice. Sperm from C11orf94-deficient mice could pass through the zona pellucida, but failed to bind to the oocyte membrane, thus accumulating in the perivitelline space. In consistence, when the sperm of C11orf94-deficient mice were microinjected into the oocyte cytoplasm, fertilized oocytes were obtained and developed normally to blastocysts. Proteomics analysis revealed that C11orf94 influenced the expression of multiple gene products known to be indispensable for sperm-oocyte binding and fusion, including IZUMO1, EQTN and CRISP1. Thus, our study indicated that C11ORF94 is a vertebrate- and testis-specific small transmembrane protein that plays a critical role in sperm binding to the oolemma.

Treadmill exercise decreases cerebral edema in rats with local cerebral infarction by modulating AQP4 polar expression through the caveolin-1/TRPV4 signaling pathway.

Rehabilitation therapy is beneficial for patients with ischemic stroke. Our previous study showed that treadmill training is conducive to neurological function in rats that underwent middle cerebral artery occlusion (MCAO). However, whether exercise benefits cerebral edema and the underlying mechanism remain unclear. This study investigated the influence of treadmill exercise on brain edema and the mechanism of its formation and elimination. The MCAO model was established with Sprague-Dawley (SD) rats, and lentivirus-mediated caveolin-1 shRNA was used to investigate the role of caveolin-1 in brain edema. As expected, we found that treadmill exercise has a beneficial effect on brain edema after stroke. Training led to a significant increase in the expression of caveolin-1 and TRPV4; and reduced brain water content and blood-brain barrier (BBB) damage. This treatment also changed the localization of aquaporin-4 (AQP4). Moreover, the effect of treadmill training on the polar expression of AQP4 differed over time. The results showed that early treadmill training inhibited the polar expression of AQP4, and later promoted its expression. However, the rats that were injected with the caveolin-1 shRNA lentivirus exhibited enhanced edema. Caveolin-1 shRNA eliminated the protective effect induced by exercise, which is consistent with the downregulation of TRPV4 expression. The findings indicate that treadmill training improves brain edema through the caveolin-1/TRPV4/AQP4 pathway.

Magnetoelectric Polymer Membrane-Based Electrical Microenvironment with Magnetically Controlled Antibacterial Activity.

The "hard to clean" parts of food processing devices (e.g., the corners of pipes) are difficult to disinfect. This challenge might be overcome through the application of a positive electrical environment. However, the chemical modification of a material surface is complex and difficult. In this work, we developed a smart electroactive TbxDy1-xFe alloy/poly(vinylidene fluoride-trifluoroethylene) (TD/P(VDF-TrFE)) magnetoelectric coating to endow stainless steel with the function of a smart adjustable electrical environment, which was realized by the introduction of a magnetic field of various intensities (0-1800 Oe). An antibacterial assay showed that the polarized coating@stainless steel (P-CS) exhibited antibacterial effects, with the highest antibacterial effect observed at 1800 Oe. Furthermore, in this study, we have, for the first time, explored the antibacterial mechanism of TD/P(VDF-TrFE)-assisted electrical stimulation based on the bacterial intracellular reactive oxygen species (ROS) level, cell respiratory chain, and membrane potential. The results showed that a microelectric field was formed on the P-CS sample in an aqueous solution, which not only generated ROS on the cathode surface but also caused H+ consumption in the electrochemical gradient of the bacterial membrane, leading to OH- production and inhibition of adenosine triphosphate (ATP) synthesis. In addition, the electric field also induced hyperpolarization of the membrane potential in Escherichia coli cells via a K+ efflux, thus inducing rearrangement of the outer membrane. In conclusion, an adjustable surface potential was established through the introduction of magnetoelectric polymer coatings, which endowed stainless steel with magnetically controlled antibacterial effects.

pH-responsive curcumin-based nanoscale ZIF-8 combining chemophotodynamic therapy for excellent antibacterial activity.

Antimicrobial photodynamic therapy (aPDT) is a highly attractive therapy due to its advantages of being a non-antibiotic procedure for reducing drug-resistant microbes. Curcumin (CCM) has been considered as a natural photosensitizer for PDT with prominent antibacterial, antifungal, and anti-proliferative activity. However, its excellent biological and pharmacological activities are limited because of its low solubility, rapid metabolization and instability. Herein, we reported a promising agent based on CCM-incorporated into zeolitic imidazolate framework-8 (ZIF@CCM). The as-prepared nanoparticle exhibited high drug loading capability (11.57%) and drug loading encapsulation (82.76%). Additionally, ZIF@CCM displayed a pH-responsive drug release behavior and chemophotodynamic therapy for excellent antibacterial activity. The underlying mechanism elucidated that Zn2+ released from ZIF-8 increased the permeability of the bacterial cell membrane with leakages of K+. The overproduction of extracellular ROS further resulted in the disrupted bacterial cell membrane and distorted bacterial morphology. Thus, ZIF@CCM-mediated photodynamic activation might be a promising treatment strategy for microbial inactivation.

High-speed rail model reveals the gene tandem amplification mediated by short repeated sequence in eukaryote.

The occurrence of gene duplication/amplification (GDA) provide potential material for adaptive evolution with environmental stress. Several molecular models have been proposed to explain GDA, recombination via short stretches of sequence similarity plays a crucial role. By screening genomes for such events, we propose a "SRS (short repeated sequence) *N + unit + SRS*N" amplified unit under USCE (unequal sister-chromatid exchange) for tandem amplification mediated by SRS with different repeat numbers in eukaryotes. The amplified units identified from 2131 well-organized amplification events that generate multi gene/element copy amplified with subsequent adaptive evolution in the respective species. Genomic data we analyzed showed dynamic changes among related species or subspecies or plants from different ecotypes/strains. This study clarifies the characteristics of variable copy number SRS on both sides of amplified unit under USCE mechanism, to explain well-organized gene tandem amplification under environmental stress mediated by SRS in all eukaryotes.

LGR5 constitutively activates NF-κB signaling to regulate the growth of intestinal crypts.

Mammalian LGR5 and LGR4, markers of adult stem cells, are involved in many physiological functions by enhancing WNT signaling. However, whether LGR5 and LGR4 are coupled to other intracellular signaling pathways to regulate stem cell function remains unknown. Here, we show that LGR5 and LGR4 can constitutively activate NF-κB signaling in a ligand-independent manner, which is dependent on their C-termini, but independent of receptor endocytosis. Moreover, the C-termini of LGR5/4 interact with TROY, which is required for activating NF-κB signaling. In small intestinal crypt organoids, overexpression of a C-terminal deletion mutant of LGR5 inhibits the growth and bud formation of organoids, whereas overexpression of the R-spondin-binding mutant of LGR5 that is defective for WNT signaling can still promote organoid growth. Our study reveals that NF-κB signaling, regulated by LGR5 and LGR4, plays an important role in the survival of colon cancer cells and the growth of intestinal crypts. Our findings also suggest that LGR5/4-induced NF-κB signaling and WNT signaling may co-regulate the growth of LGR5+ adult stem cells and intestinal crypts.

Adaptive evolution of GPR39 in diverse directions in vertebrates.

G protein-coupled receptors (GPCRs) play an important role in physiology and disease and represent productive drug targets. Orphan GPCRs, which have unknown endogenous ligands, are considered drug targets and consequently have attracted great interest in identifying their endogenous cognate ligands for deorphanization. However, additional studies have shown that GPCRs, including many orphan GPCRs, can constitutively activate G protein signaling in a ligand-independent manner. GPR39 is such an orphan GPCR with constitutive activity. Here, we performed a phylogenetic and selection analysis of GPR39 in vertebrates, and we found that GPR39 underwent positive selection in different branches of vertebrates. Using luciferase reporter assays, we demonstrated that human, frog and chicken GPR39 can constitutively activate Gq and G12 signaling pathways in a ligand-independent manner. Zebrafish GPR39 can constitutively activate Gs, Gq and G12 signaling pathways in a ligand-independent manner. We further found that the zebrafish-H2967.35 site is crucial for the activity of the Gs signaling pathway. In addition, our mutagenesis studies indicated that the positive selection sites of GPR39 from different species had important effects on the constitutive activity of the receptor. Our results revealed the adaptive evolution of GPR39 in diverse directions, which led to differences in constitutive activity.

Proteomic-based identification of oocyte maturation-related proteins in mouse germinal vesicle oocytes.

Oocyte proteins play an important role in oocyte maturation, fertilization and embryonic development. However, the protein composition of mouse germinal vesicle (GV) oocytes is still unclear. Using one-dimensional Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (1D SDS-PAGE) and Reverse-phase liquid chromatography tandem mass spectrometry (RP-LC-MS/MS), we constructed a protein profile of mouse GV oocytes. First, our proteomics profile identified 1,405 different proteins from 11,000 mouse GV oocytes lacking zona pellucida. Second, with detailed bioinformatics analysis, a group of proteins that play an essential role in oocyte maturation was screened. In addition, the expression and localization of suppressor of G2 allele of skp1(SUGT1, also called SGT1), heterogeneous nuclear ribonucleoprotein K (Hnrpk), Seruin, Cullin1(Clu1) and nuclear distribution protein C (Nudc) in mouse ovaries and early embryos were also captured and investigated in this study. Moreover, the protein profile was submitted to the Proteomics Identifications Database (PRIDE) and is available via ProteomeXchange with the identifier PXD014314. Our research provides valuable resources for the study of oocyte proteins and oocyte maturation and helps to clarify the mechanisms of oocyte maturation.

Anti-diabetic effect of oligosaccharides from seaweed Sargassum confusum via JNK-IRS1/PI3K signalling pathways and regulation of gut microbiota.

Brown seaweed Sargassum confusum (C. Agardh) has been used in traditional Chinese medicine to treat a variety of diseases. The aim of the present study was to evaluate the anti-diabetic effect of oligosaccharides from brown seaweed S. confusum (SCO). The anti-diabetic effect of SCO was evaluated in vivo using high-fat/high-sucrose fed hamsters. Molecular mechanisms of modulating gene expression of specific members of insulin signaling pathways were determined. The components of the intestinal microflora in diabetic animals were also analyzed by high-throughput 16S rRNA gene sequencing. And it was found that SCO had a sequence of sulfated anhydrogalactose and methyl sulfated galactoside units. Fasting blood glucose levels were significantly decreased after SCO administration. Histology showed that SCO could protect the cellular architecture of the liver. SCO could also significantly increase the relative abundance of Lactobacillus and Clostridium XIVa and decrease that of Allobaculum, Bacteroides and Clostridium IV. The active role of SCO in anti-diabetic effect was revealed by its regulation of insulin receptor substrate 1/phosphatidylinositol 3-kinase and c-Jun N-terminal kinase pathways. These results suggested that SCO might be used as a functional material to regulate gut microbiota in obese and diabetic individuals.

HSC-specific knockdown of GGPPS alleviated CCl4-induced chronic liver fibrosis through mediating RhoA/Rock pathway.

Hepatic stellate cells (HSCs) play a critical role in the pathogenesis and reversal of liver fibrosis. Targeting HSCs is of great significance in the treatment of hepatic fibrosis, and has attracted wide attention of scholars. Here we demonstrated that expression of geranylgeranyldiphosphate synthase (GGPPS) predominantly increased in HSCs in murine fibrotic liver. HSC-specific knockdown of GGPPS using vitamin A-coupled liposome carrying siRNA-ggpps decreased activation of HSCs and alleviated fiber accumulation in vivo. Furthermore, our in vitro studies showed that GGPPS was up-regulated during HSCs activation in TGF-ß1-dependent manner. Inhibition of GGPPS suppressed TGF-ß1 induced F-actin reorganization and HSCs activation in LX-2 cells. Further, we found that GGPPS regulated HSCs activation and liver fibrosis possibly by enhancing RhoA/Rock kinase signaling. So its concluded that GGPPS promotes liver fibrosis by activating HSCs, which may represent a potential target for anti-fibrosis therapies.

ELABELA, as a potential diagnostic biomarker of preeclampsia, regulates abnormally shallow placentation via APJ.

Preeclampsia (PE) is a major cause of maternal mortality and morbidity worldwide. Although there has been great progress in the understanding of PE, the exact cause for the disease development is still unclear. Recently, studies showed that genetic deletion of ELABELA (ELA, also known as APELA) could induce PE-like symptoms in mice. However, the role of ELA in the disease development of PE remains elusive. Our objective was to measure the changes of ELA levels in maternal serum, urine, and placenta from preeclamptic pregnant women and healthy pregnant women and evaluate the correlation between ELA levels and the occurrence of PE. Additionally, we investigated the effect of ELA on the migration and proliferation of human trophoblast cells. ELA levels are significantly decreased in late-onset PE pregnancies compared with normal pregnancies. The mRNA and protein expressions of ELA and the apelin receptor (APLNR or APJ) in late-onset PE placental tissues are also decreased. Furthermore, our in vitro study showed that the addition of ELA significantly increased the invasion ability and proliferation of trophoblast cells, which were inhibited by the APJ-specific antagonist ML221. Our study identified ELA as significantly decreased in late-onset PE; therefore, it might play an important role in the pathogenesis of late-onset PE.

Hypoglycemic activity and gut microbiota regulation of a novel polysaccharide from Grifola frondosa in type 2 diabetic mice.

GFP-N, a novel heteropolysaccharide with a molecular weight of 1.26 × 107 Da, was isolated from maitake mushroom and purified by anion-exchange chromatography on a DEAE cellulose-52 column and gel-filtration chromatography on a Sephadex G-100 column. Its structure was characterized by Fourier transform infrared spectroscopy and one-dimensional (1H- and 13C-) NMR spectra, 1H1H correlation spectroscopy, and 1H13C heteronuclear single-quantum coherence spectroscopy. The structure of GFP-N consisted of L-arabinose, D-mannose and D-glucose and mainly contained three kinds of linkage type units as →2,6)-α-D-Manp-(1 → 4, α-L-Araf-C1→, and →3,6)-ß-D-Glcp-(1 → . GFP-N could activate insulin receptor substrate 1, phosphatidylinositol-3-kinase, and glucose transporter 4 and inhibit c-Jun N-terminal kinase 1/2 for hypoglycemic effects in diabetic mouse livers. This is also the first report of the regulatory efficacy of Grifola frondosa polysaccharide on intestinal microflora in vivo using single-molecule real-time sequencing. These results indicated that polysaccharide from maitake mushroom could be as an enhancer to improve type 2 diabetes and a healthy food option to help regulate gut microbiota in diabetic individuals.

Evidence for a role of geranylgeranylation in renal angiomyolipoma and renal epithelioid angiomyolipoma.

Research on mevalonate kinase deficiency has revealed that it may lead to the development of renal angiomyolipomas (RAMLs). Thus, it was suspected that geranylgeranyl pyrophosphate synthase (GGPPS), a key enzyme in the mevalonate pathway, may be involved in the development of RAMLs. In the present study, the expression of GGPPS in RAMLs and renal epithelioid angiomyolipomas (REAs) was assessed, and paraffin embedded specimens from 60 patients, including 9 cases with REA and 51 cases with RAML, were examined. Immunoreactivity was evaluated semi-quantitatively according to the intensity of staining and the percentage of positively stained cells. The results indicated that GGPPS was predominantly present in the cytoplasm, and REA tissues exhibited higher expression of GGPPS in the cytoplasm compared with RAML tissues. It was also identified that GGPPS was upregulated in TSC2-null cells, and inhibition of GGPPS could induce apoptosis of TSC2-null cells by autophagy. In conclusion, the increased expression of GGPPS in RAMLs and REAs indicated that mevalonate pathways may be involved in disease progression. GGPPS may serve as a potential therapeutic target and the current results may provide a novel therapeutic strategy for RAML and lymphangioleiomyomatosis.

Early growth response-1 negative feedback regulates skeletal muscle postprandial insulin sensitivity via activating Ptp1b transcription.

Postprandial insulin desensitization plays a critical role in maintaining whole-body glucose homeostasis by avoiding the excessive absorption of blood glucose; however, the detailed mechanisms that underlie how the major player, skeletal muscle, desensitizes insulin action remain to be elucidated. Herein, we report that early growth response gene-1 ( Egr-1) is activated by insulin in skeletal muscle and provides feedback inhibition that regulates insulin sensitivity after a meal. The inhibition of the transcriptional activity of Egr-1 enhanced the phosphorylation of the insulin receptor (InsR) and Akt, thus increasing glucose uptake in L6 myotubes after insulin stimulation, whereas overexpression of Egr-1 decreased insulin sensitivity. Furthermore, deletion of Egr-1 in the skeletal muscle improved systemic insulin sensitivity and glucose tolerance, which resulted in lower blood glucose levels after refeeding. Mechanistic analysis demonstrated that EGR-1 inhibited InsR phosphorylation and glucose uptake in skeletal muscle by binding to the proximal promoter region of protein tyrosine phosphatase-1B (PTP1B) and directly activating transcription. PTP1B knockdown largely restored insulin sensitivity and enhanced glucose uptake, even under conditions of EGR-1 overexpression. Our results indicate that EGR-1/PTP1B signaling negatively regulates postprandial insulin sensitivity and suggest a potential therapeutic target for the prevention and treatment of excessive glucose absorption.-Wu, J., Tao, W.-W., Chong, D.-Y., Lai, S.-S., Wang, C., Liu, Q., Zhang, T.-Y., Xue, B., Li, C.-J. Early growth response-1 negative feedback regulates skeletal muscle postprandial insulin sensitivity via activating Ptp1b transcription.

A novel type 2 diabetes risk allele increases the promoter activity of the muscle-specific small ankyrin 1 gene.

Genome-wide association studies have identified Ankyrin-1 (ANK1) as a common type 2 diabetes (T2D) susceptibility locus. However, the underlying causal variants and functional mechanisms remain unknown. We screened for 8 tag single nucleotide polymorphisms (SNPs) in ANK1 between 2 case-control studies. Genotype analysis revealed significant associations of 3 SNPs, rs508419 (first identified here), rs515071, and rs516946 with T2D (P < 0.001). These SNPs were in linkage disequilibrium (r(2) > 0.80); subsequent analysis indicated that the CCC haplotype associated with increased T2D susceptibility (OR 1.447, P < 0.001). Further mapping showed that rs508419 resides in the muscle-specific ANK1 gene promoter. Allele-specific mRNA and protein level measurements confirmed association of the C allele with increased small ANK1 (sAnk1) expression in human skeletal muscle (P = 0.018 and P < 0.001, respectively). Luciferase assays showed increased rs508419-C allele transcriptional activity in murine skeletal muscle C2C12 myoblasts, and electrophoretic mobility-shift assays demonstrated altered rs508419 DNA-protein complex formation. Glucose uptake was decreased with excess sAnk1 expression upon insulin stimulation. Thus, the ANK1 rs508419-C T2D-risk allele alters DNA-protein complex binding leading to increased promoter activity and sAnk1 expression; thus, increased sAnk1 expression in skeletal muscle might contribute to T2D susceptibility.