Receptor L1 Research Articles

Characterization of the Ovarian Development and Associated Factors during the Breeding Migration of Coilia nasus in the Yangtze River

Coilia nasus is a typical anadromous migratory fish found in the lower reaches of the Yangtze River. Every year, C. nasus clusters offshore and swims upstream along the Yangtze River into the tributaries and lakes in the middle and lower reaches of the Yangtze River to breed. In this study, female C. nasus were collected as study subjects from the Chongming section of Shanghai, the Taizhou section of Jiangsu, and the Anqing section of Anhui. Their ovaries were used to examine tissue sections and investigate gene expression, including the follicle-stimulating hormone receptor (fshr), the luteinizing hormone receptor (lhr), kisspeptin-1 (kiss1), and forkhead box l2 (foxl2), which are related to reproductive development, while the serum levels of estrogen (including estradiol, E2) and progestins (including 17α,20β-dihydroxy-4-pregenen-3-one, 17α,20β-DHP) were also analyzed. Our results showed that, first, the growth period of the oocytes was small in stage II of ovarian development, in which both E2 and 17α,20β-DHP levels and gene expression were low. Then, in stage III, the growth period of the oocytes became large, and the yolk granules and oil droplets began to appear. Simultaneously, E2 and the expression of kiss1 and foxl2 were significantly elevated. Finally, stage IV was the period of a large amount of accumulation of nutrients in the oocytes, and 17α,20β-DHP levels and the expression of fshr and lhr were significantly elevated. These results enrich the theoretical study of ovarian development in the natural population of C. nasus, supplementing the biological basis of C. nasus reproduction and scientifically supporting the study of C. nasus population ecology and resource conservation.

Omniligase-1-Mediated Phage-Peptide Library Modification and Insulin Engineering.

Chemical and enzymatic modifications of peptide-displayed libraries have been successfully employed to expand the phage display library. However, the requirement of specific epitopes and scaffolds has limited the scope of protein engineering using phage display. In this study, we present a novel approach utilizing omniligase-1-mediated selective and specific ligation on the phage pIII protein, offering a high conversion rate and compatibility with commercially available phage libraries. We applied this method to perform high-throughput engineering of insulin analogues with randomized B chain C-terminal regions. Insulin analogues with different B chain C-terminal segments were selected and exhibited biological activity equivalent to that of human insulin. Molecular dynamics studies of insulin analogues revealed a novel interaction between the insulin B27 residue and insulin receptor L1 domain. In summary, our findings highlight the potential of omniligase-1-mediated phage display in the development and screening of disulfide-rich peptides and proteins. This approach holds promise for the creation of novel insulin analogues with enhanced therapeutic properties and exhibits potential for the development of other therapeutic compounds.

OR12-06 Hypothalamic Adgrl1 Is A Potential Glucoreceptor Involved In Regulating Energy Balance, Glucose Homeostasis, And Leptin Sensitivity In Mice

Abstract Disclosure: D.J. Samuel: None. T.S. Faniyan: None. M.U. Raza: None. S. Bathina: None. K.H. Chhabra: None. The hypothalamus regulates energy and glucose homeostasis by influencing glucose-stimulated insulin secretion, insulin sensitivity, and activating the hypoglycemia counterregulatory response. Although hypothalamic glucose transporters coupled with glucose metabolism are involved in mediating some of these effects, it is unknown whether a plasma membrane receptor that directly binds to glucose can contribute to hypothalamic glucose signaling. We used affinity chromatography and biotin-labeled glucose to enrich glucose-binding plasma membrane proteins/receptors in the mouse hypothalamus and then performed proteomic analyses. Adhesion G-protein Coupled Receptor L1 (Adgrl1) was a top candidate as a potential glucoreceptor. Therefore, we generated Adgrl1loxp/loxp mice to knockout Adgrl1 in the ventromedial hypothalamus (VMH; a region known for glucose sensing) using the Cre-Lox system to determine the role of Adgrl1VMH in glucose homeostasis, energy balance, and leptin sensitivity. We measured body weight, food intake, 6h fasting blood glucose and plasma insulin levels. We also determined leptin sensitivity in the Adgrl1VMH deficient mice. We observed that 24 weeks old male Adgrl1VMH deficient mice had higher (p<0.05) body weight (49 ±3.6 vs 35 ±1.3 g), blood glucose (210 ±4 vs 180 ±5 mg/dl), plasma insulin levels (2.92 ±0.24 vs 1.63 ±0.09 ng/ml), and leptin levels (21.11 ±1.72 vs 8.38 ±0.80 ng/ml) compared to their control littermates. Hyperinsulinemia and hyperleptinemia persisted in the Adgrl1VMH knockout mice even when they were weight-matched to their littermate controls, indicating the direct role of Adgrl1VMH in regulating plasma insulin and leptin levels. Ovariectomy in female mice was necessary to unmask the effects of Adgrl1VMH deficiency on energy and glucose homeostasis. To assess acute leptin sensitivity, we injected Adgrl1VMH deficient mice and their littermate control mice with recombinant mouse leptin (5 mg/Kg, i.p., twice a day for two days), and monitored their body weight and food intake. Male Adgrl1VMH deficient mice were more susceptible (p<0.05) to leptin mediated decrease in food intake (-1 ±0.1 vs -0.5 ±0.005, g/day; these values are relative to each group’s corresponding baseline food intake) than their littermate controls. Decrease in body weight was similar in both groups with this acute leptin treatment. These findings suggest that Adgrl1VMH is essential in regulating energy and glucose homeostasis in a sex-specific manner. Moreover, Adgrl1VMH contributes to leptin’s acute effects on food intake. Therefore, hypothalamic Adgrl1 could be a target to enhance leptin sensitivity in future therapies for treating type 2 diabetes and obesity. Presentation: Friday, June 16, 2023

Adgrl3.1-deficient zebrafish show noradrenaline-mediated externalizing behaviors, and altered expression of externalizing disorder-candidate genes, suggesting functional targets for treatment

Externalizing disorders (ED) are a cause of concern for public health, and their high heritability makes genetic risk factors a priority for research. Adhesion G-Protein-Coupled Receptor L3 (ADGRL3) is strongly linked to several EDs, and loss-of-function models have shown the impacts of this gene on several core ED-related behaviors. For example, adgrl3.1−/− zebrafish show high levels of hyperactivity. However, our understanding of the mechanisms by which this gene influences behavior is incomplete. Here we characterized, for the first time, externalizing behavioral phenotypes of adgrl3.1−/− zebrafish and found them to be highly impulsive, show risk-taking in a novel environment, have attentional deficits, and show high levels of hyperactivity. All of these phenotypes were rescued by atomoxetine, demonstrating noradrenergic mediation of the externalizing effects of adgrl3.1. Transcriptomic analyses of the brains of adgrl3.1−/− vs. wild-type fish revealed several differentially expressed genes and enriched gene clusters that were independent of noradrenergic manipulation. This suggests new putative functional pathways underlying ED-related behaviors, and potential targets for the treatment of ED.

An Increase in Mucin2 Expression Is Required for Colon Cancer Progression Mediated by L1.

An induction in the expression of the cell adhesion receptor L1, a Wnt target gene, is a characteristic feature of Wnt/β-catenin activation in colon cancer cells at later stages of the disease. We investigated the proteins secreted following L1 expression in colon cancer cells and identified Mucin2 among the most abundant secreted proteins. We found that suppressing Mucin2 expression in L1-expressing colon cancer cells inhibits cell proliferation, motility, tumorigenesis, and liver metastasis. We detected several signaling pathways involved in Mucin2 induction in L1-expressing cells. In human colon cancer tissue, Mucin2 expression was significantly reduced or lost in the adenocarcinoma tissue, while in the mucinous subtype of colon cancer tissue, Mucin2 expression was increased. An increased signature of L1/Mucin2 expression reduced the survival rate of human colon cancer patients. Thus, induction of Mucin2 expression by L1 is required during mucinous colon cancer progression and can serve as a marker for diagnosis and a target for therapy.

Simultaneous visual and spectroscopic multi-analyte detection of Al3+ and AsO2− using simple salicylidene based D-π-A chromophore

The salicylidene-isoniazide Schiff base D-π-A chromophore ( L1 ) exhibits chromogenic and fluorogenic dual responsive behaviour with multiple ICT-PET-ESIPT interplaying sensing mechanism and provides diverse optical signals promising to selectively and simultaneously track multiple analytes in water rich media. • Salicylidene-isoniazide Schiff base ( L1) exhibited naked-eye detectable distinctive colour changes for Al 3+ and AsO 2 - • It displayed turn-on type distinct fluorogenic response for both Al 3+ and AsO 2 - • Multiple ICT-PET-ESIPT interplaying mechanism provided diverse optical signals to simultaneously track multiple analytes. • Applied for fluorescence imaging of Al 3+ and AsO 2 - in living cells. The present communication reports the efficacy of a salicylidene-isoniazide Schiff base based donor-π-acceptor (D-π-A) chemosensor ( L1 ) for chromogenic and fluorogenic dual mode sensing of Al 3+ and AsO 2 - in water rich semi-aqueous solution. L1 offers multiple benefits of simplicity, ease of synthesis, inexpensive, suitable binding sites for cations and anions, high sensitivity, noteworthy colourimetric response, naked-eye detection, selectivity, and good detection limits of 1.89 μM for Al 3+ and 2.2 μM for AsO 2 - . It shows selective turn-on fluorescence response for both Al 3+ and AsO 2 - , attributed to CHEF due to the combined effect of arrest of PET, inhibition of -CH=N- geometrical isomerization, prevention of ESIPT process and increase in conformational rigidity. Owing to specific interaction between Mo(VI) and arsenite, L1 -Mo(VI) ensemble system is successfully utilized for colourimetric detection of arsenite through displacement approach. The binding modes of the receptor L1 to these analytes are established through Job’s plot and 1 H-NMR spectroscopic analysis. The reversible off-on-off fluorescence switching behaviour of L1 upon binding to Al 3+ and AsO 2 - are applied to construct combinational OR, AND, and NOT molecular logic gates. Fluorescence turn-on sensing, cell permeability and low toxicity of the chemosensor allows monitoring of these analytes in living cells.

Fluorescent sensing of non-steroidal anti-inflammatory drugs naproxen and ketoprofen by dansylated squaramide-based receptors.

Bis-squaramide receptors L1-L4 bearing a dansyl moiety were synthesised and their potential applications as fluorescent probes towards non steroidal anti-inflammatory drugs naproxen and ketoprofen was investigated. A detailed photophysical characterization in CH3CN/DMSO solution (9 : 1 v/v) was conducted and demonstrated that the two macrocyclic receptors L1 and L2 show good sensitivity towards ketoprofen with an ON-OFF fluorescent response, while the two open chain receptors L3 and L4 behave similarly with the three guests considered. DFT theoretical calculations carried out on L2 and L4 as model receptors allowed to propose a possible coordination mode towards the guests. Finally, 1H-NMR spectroscopy in DMSO-d6/0.5% water solution demonstrated that the four receptors interact with the considered guests via H-bonds.

ADGRL3 genomic variation implicated in neurogenesis and ADHD links functional effects to the incretin polypeptide GIP

Attention deficit/hyperactivity disorder (ADHD) is the most common childhood neurodevelopmental disorder. Single nucleotide polymorphisms (SNPs) in the Adhesion G Protein-Coupled Receptor L3 (ADGRL3) gene are associated with increased susceptibility to developing ADHD worldwide. However, the effect of ADGRL3 non-synonymous SNPs (nsSNPs) on the ADGRL3 protein function is vastly unknown. Using several bioinformatics tools to evaluate the impact of mutations, we found that nsSNPs rs35106420, rs61747658, and rs734644, previously reported to be associated and in linkage with ADHD in disparate populations from the world over, are predicted as pathogenic variants. Docking analysis of rs35106420, harbored in the ADGLR3-hormone receptor domain (HRM, a common extracellular domain of the secretin-like GPCRs family), showed that HRM interacts with the Glucose-dependent insulinotropic polypeptide (GIP), part of the incretin hormones family. GIP has been linked to the pathogenesis of diabetes mellitus, and our analyses suggest a potential link to ADHD. Overall, the comprehensive application of bioinformatics tools showed that functional mutations in the ADGLR3 gene disrupt the standard and wild ADGRL3 structure, most likely affecting its metabolic regulation. Further in vitro experiments are granted to evaluate these in silico predictions of the ADGRL3-GIP interaction and dissect the complexity underlying the development of ADHD.

A molecular tuner: Substituent controlled π/π···hole induced selective colorimetric intramolecular push-pull fluoride sensor

Controlling the anion selectivity of synthetic anion receptors towards a particular anion is challenging task for researcher. In the case of bipodal receptors this become trickier since anion can approach the recognition elements of receptors from any direction. In order to overcome this problem, we adopted the new strategy in which the receptor molecules are functionalized with electron donating and/or withdrawing group. In the way of hunting such a receptors, herein Thio-semicarbazide based chemosensors 1-(2,4-dinitrophenyl)-4-(4-nitrophenyl) thiosemicarbazide L1, and 1-(2,4-dimethylphenyl)-4-(4-nitrophenyl) thiosemicarbazide L2 with electron withdrawing (EW) and donating (ED) groups have been synthesized for selective colorimetric anion sensing. Substituent effects on the strength of those receptors L1 and L2 have been studied by naked-eye colorimetric experiments, Ultraviolet-Visible (UV-Vis) spectrometric titration studies, Fourier-Transform Infrared experiments (FT-IR), 1H-NMR, 19F-NMR spectroscopic studies and Molecular Electrostatic Potential (MEP) surface analysis. When we have nitro substitution on both end of the thio-semicarbazide receptor L1, there were no selectivity was found with anions and receptor L1 shows visible color change with the all the tested anions. On the other hand, instead of nitro, when we have methyl substitution on one end and nitro substitution at an another end of the thio-semicarbazide receptor L2, then the receptor L2 show excellent selectivity towards fluoride ion with high association constant. Additionally, for azide considerable bathochromic shift of 70 nm also observed with reference to absorption values of cyanide and fluoride anion which will clearly distinguish the presence of azide ion from cyanide and fluoride. The observed bathochromic shift is mainly attributed due to the intramolecular charge transfer (ICT). In this work, we have successfully tuned the selectivity of synthesized receptors via generating a push-pull effect originated by electron withdrawing and donation substitutions on both end of the receptor L2. The enhanced selectivity of L2 is achieved by altering the acidity of -NHs of thio-semicarbazide derivative L2 by substituting EW and ED groups. In this present work, we have tuned the selectivity of receptor is via intramolecular push-pull effect by electron withdrawing and donating substituent.

Driver mutations in ADGRL3 are involved in the evolution of ependymoma

Although there have been recent advances in the molecular pathology of ependymomas, little is known about the underlying molecular evolution during its development. Here, we assessed the clinical, pathological and molecular evolutionary process of ependymoma recurrence in a 9-year-old patient who had seven recurrences of supratentorial ependymoma and died from intracranial multiregional recurrences at the age of 19 years old. Whole-genome sequencing (WGS) of 7 tumor samples (1 primary and 6 subsequent recurrent tumors) was performed to elucidate the mutation landscape and identify potential driver mutations for tumor evolution. The genetic profiles of the seven tumor specimens showed significant heterogeneity and suggested a highly branched evolutionary pattern. The mutational signatures and chromothripsis changed with treatments. Strikingly, adhesion G protein-coupled receptor L3 (ADGRL3, also known as Latrophilins 3, LPNH3) was found to be consistently mutated during the entire disease process. However, Sanger sequencing of other 78 ependymoma patients who underwent surgery at our institution showed no genetic alteration of ADGRL3, as found in the present case. The mRNA levels of ADGRL3 were significantly lower in ependymomas (n = 36), as compared with normal brain tissue (n = 3). Grade III ependymomas had the lowest ADGRL3 expression. Moreover, ependymomas with lower mRNA level of ADGRL3 had shorter overall survival. Our findings, therefore, demonstrate a rare evolutionary process of ependymoma involving ADGRL3.

Loss of Adhesion G‐Protein‐Coupled Receptor L2 Expression Impacts Colonic Epithelial Proliferation

BackgroundThe role of adhesion G‐protein‐coupled receptor L2 (ADGRL2, a.k.a. Latrophilin‐2) in human health has been understudied despite discoveries that the receptor is required for both heart development and neural synaptogenesis. This project tests the physiological function of ADGRL2 in the colon where our prior findings that ADGRL2 is lost in human colorectal cancer (CRC), downregulated during hyperproliferative recovery from murine DSS colitis, enriched in stem and progenitor cells, and is inversely correlated with growth arrest. This raises the intriguing possibility that ADGRL2 restricts stem cell expansion and therefore downregulation of ADGRL2 is required for stem‐cell driven regeneration and repair.MethodsThe effect of Adgrl2 on cell proliferation was assessed by histological staining for Ki‐67 antigen or for the thymidine analogue EdU (5‐ethynyl‐2’‐deoxyuridine, administered by intraperitoneal injection 2hr prior to harvest) in intestinal‐epithelial specific Adgrl2 knockout mice (Adgrl2 IE KO). Percent positive cells were quantified per crypt and per crypt region (crypt base, mid crypt, upper crypt) in Image J.Results/ConclusionsAdgrl2 IE KO mice have overall reduced numbers of Ki‐67+ cells (8.2% difference, p = 0.0016) in the colonic epithelium. This seems paradoxical given that we hypothesize a tumor suppressor role for ADGRL2. However, this effect is driven by a pronounced decrease in Ki‐67+ cells in the post‐mitotic upper crypt of Adgrl2 IE KO mice (15.2% difference, p = 0.0121) while percentages of Ki‐67+ cells in the more proliferative mid crypt and crypt base were determined to be roughly equal between groups, suggesting that the observed effect is likely not driven by a reduction in proliferating stem or progenitor cells but is perhaps due to increased cell cycle exit or terminal differentiation with Adgrl2 knockout. Given that Ki‐67 antigen is expressed in all active cell cycle phases, we used EdU labeling of S‐phase proliferating cells to further assess the impact of Adgrl2 loss on colonocyte proliferation. Proliferation is not impaired with loss of Adgrl2 and instead showed a trend towards increased EdU positive cells in the crypt base (6.7% increase, p= 0.0907). As expected, overall number of S‐phase cells in the upper crypt compartment was low for both Adgrl2 KO and WT animals, however a significant decrease in EdU positive cells was still detected in Adgrl2 KO mice (2.98% change, p=0.0500). Based on these data, the loss of Adgrl2 does not decrease proliferative capacity of the stem cell or progenitor niche, but does effect the distribution of active cycling or differentiating cells along the crypt axis, likely by promoting cell cycle exit or terminal differentiation. Further studies will investigate this possibility and the role of Adgrl2 loss on cell renewal in the context of disease. These data support our hypothesis that ADGRL2 modulates epithelial regeneration in the colon, potentially as a stem‐cell specific negative regulator. Mechanistic studies on dysregulated growth and repair in the colon may also illuminate targets to prevent colitis‐associated cancer in IBD patients.

Pyrene-Hydrazone-π···Hole Coupled Turn-on Fluorescent and Naked- Eye Colorimetric Sensor for Cyanide: Role of Homogeneous π-Hole Dispersion in Anion Selectivity.

A pyrene based probe associated with π···hole - hydrazone as one of the recognizing elements is synthesized and its turn in to a selective colorimetric and turn-on fluorescent sensor, (L3) for cyanide anion. This chemo sensor show high selectivity towards cyanide anion through photo electron transfer (PET) mechanism. The binding strength and sensitivity of the chemo sensor L3 towards cyanide are found to be 2.0 X 104, and 4.44 x 10-4 respectively. We have compared this high selectivity of the receptor towards cyanide, with our previously reported receptors L1 and L2. The detailed UV-Vis, Emission, 1H-NMR, IR spectroscopic and Molecular Electrostatic Potential (MEP) studies reveals that the homogeneous π···hole dispersion in the aromatic ring governing the selectivity of the receptor towards cyanide anion. Such a positive π···hole homogeneous dispersion is missing in the case of sensor L2, instead we have polarized π···hole dispersion towards 2ndand 4thposition of di-nitrophenyl chromophoric unit in L2.

A new 2-hydroxynaphthalene based Schiff base receptor for detection of Cu2+, Fe3+, HSO4−, CN− ions and D–amino acids in aqueous DMSO solution

A new Schiff base receptor L2 was prepared and characterized by elemental analyses, IR, 1H/13C NMR, UV–vis, HRMS and PL data. It exhibited one emission band (λem = 711 nm) with red-point luminescence in the solid state, and one emission band (λem = 603–613 nm) with yellow luminescence in DMSO solution, which point out the existence of excited state intramolecular proton transfer (ESIPT) reaction between tautomeric forms in the excited state. The recognizing ability of receptor against metal ions (Cr3+, Al3+, Fe3+, Pb2+, Co2+, Cu2+, Mn2+, Cd2+, Zn2+ and Ni2+), α–D–amino acids (glycine, alanine, phenylalanine, valine, serine and threonine) and inorganic anions (F−, Cl−, Br−, CH3COO−, H2PO4−, SO32–, S2O32−, NO3−, HSO4−, CN− and OH−) was determined in DMSO–H2O solution by colorimetric and UV–vis experiments. L2 selectively detected Cu2+ and Fe3+ ions through visual color change as well as UV–vis spectra. Addition of acidic and basic amino acid solution to L2 caused the change of light orange color of receptor to bright light orange and pale lemon color, respectively. L2 had a better selectivity for detecting of cationic form of glycine, while it showed good recognition towards anionic form of all the tested D–amino acids. Besides, L2 exhibited a highly selective naked-eye sensing of hydrogen sulfate, cyanide and hydroxide ion comparing to the other ions.

Modulatory effect of Artocarpus camansi on ILP-2, InR, and Imp-L2 genes of sucrose –induced diabetes mellitus in Drosophila melanogaster

Diabetes mellitus continues to be a menace, being one of the five major causes of death in the world. In this study, the common fruit fly, Drosophila melanogaster, a well-studied genetic model organism for understanding molecular mechanisms of human diseases, and Artocarpus camansi (breadnut), an underutilized fruit, was used. This study was aimed at investigating the antihyperglycemic potentials of Artocarpus camansi fruit in sucrose-induced diabetic Drosophila melanogaster. Phytochemical screening was carried out after the fruit has been pulverized and freeze-dried. Total phenol content and total flavonoid content were carried out in vitro on the aqueous extract of Artocarpus camansi, and the result obtained showed that its phenol content is low, and its flavonoid content increases at increasing concentrations. Alpha-amylase inhibitory activity was carried out in vivo on sucrose-induced diabetic Drosophila melanogaster tissue. Gene expression profiling of Insulin-like peptide-2 (ILP-2), Insulin-like receptor (InR) and Ecdysone inducible gene L2 (Imp-L2) was carried out on trizol homogenate of Drosophila melanogaster tissue. In this study, Drosophila melanogaster was divided into nine groups. Group1 served as the basal control as they were fed with normal basal diet, group II served as the negative control which were fed with basal diet and 0.5 mL sucrose/100 mL distilled water, group III served as the positive control which were fed with basal diet 0.5 mL sucrose/100 mL distilled water and metformin, groups IV and V which were fed with basal diet and 0.1 and 1% Artocarpus camansi respectively, groups VI and VII were fed with basal diet, sucrose and 0.1 and 1% Artocarpus camansi respectively, groups VIII and IX served the purpose of the synergistic effect which were fed with basal diet, sucrose, metformin and 0.1 and 1% Artocarpus camansi respectively. All the groups were left for seven days. The experiment was conducted for 3 months and the fruit fly meals were changed every 5 days. Gene expression profiling results showed that the dietary inclusion of fruit downregulated the expression of ILP-2 and InR and upregulated the expression of Imp-L2 when the diabetic group were compared with the normal control. The results suggest that Artocarpus camansi fruit could possess antihyperglycemic properties and its use as a nutraceutical in the alleviation of diabetes should be encouraged.

Adhesion G protein-coupled receptor L3 gene variants: Statistically significant association observed in the male Indo-caucasoid Attention deficit hyperactivity disorder probands.

Primary symptoms of Attention Deficit Hyperactivity Disorder (ADHD) are age inappropriate inattention, hyperactivity and impulsivity. Caucasoid individuals showed increased susceptibility to ADHD and disruptive behaviour in presence of Adhesion G-protein-coupled receptor L3 (ADGRL3) gene variants. We investigated ADGRL3 rs1868790, rs6551665, rs2345039 in Indo-Caucasoid families with ADHD probands (N = 249) and controls (N = 350). Behavioural traits, executive function, and IQ of probands were measured through Conner's Parent Rating Scale-Revised, Parental Account of Children's Symptoms, Barkley Deficit in Executive Functioning-Child & Adolescent Scale, and Wechsler Intelligence Scale for Children-III respectively. After obtaining informed written consent, peripheral blood was collected for genomic DNA isolation and target sites were analyzed by PCR based methods or TaqMan assay. Case-control analysis showed higher frequency of rs2345039 'C' allele, 'CC' genotype and A-A-C haplotype in the ADHD probands, principally due to higher occurrence of the 'C' allele and A-A-C haplotype in the male probands (P < 0.05). Mother of the probands also showed higher occurrence of the 'C' allele and "CC" genotype (P < 0.01). Executive function was better in presence of rs2345039 "GG" (P = 0.04) while IQ score was higher in presence of rs6551665 "AA" (P = 0.06). Linkage disequilibrium between rs6551665 and rs2345039 was stronger in the ADHD cases, chiefly in the male probands. Multifactor dimensionality reduction analysis showed strong interaction between rs6551665 and rs2345039 in the male probands while in the female probands rs1868790 and rs6551665 revealed non-linear interaction. Based on these observations, we infer that ADGRL3 may have a role in the aetiology of ADHD in this population warranting further in depth investigation.

Brain structural and functional substrates of ADGRL3 (latrophilin 3) haplotype in attention-deficit/hyperactivity disorder

Previous studies have shown that the gene encoding the adhesion G protein-coupled receptor L3 (ADGRL3; formerly latrophilin 3, LPHN3) is associated with Attention-Deficit/Hyperactivity Disorder (ADHD). Conversely, no studies have investigated the anatomical or functional brain substrates of ADGRL3 risk variants. We examined here whether individuals with different ADGRL3 haplotypes, including both patients with ADHD and healthy controls, showed differences in brain anatomy and function. We recruited and genotyped adult patients with combined type ADHD and healthy controls to achieve a sample balanced for age, sex, premorbid IQ, and three ADGRL3 haplotype groups (risk, protective, and others). The final sample (n = 128) underwent structural and functional brain imaging (voxel-based morphometry and n-back working memory fMRI). We analyzed the brain structural and functional effects of ADHD, haplotypes, and their interaction, covarying for age, sex, and medication. Individuals (patients or controls) with the protective haplotype showed strong, widespread hypo-activation in the frontal cortex extending to inferior temporal and fusiform gyri. Individuals (patients or controls) with the risk haplotype also showed hypo-activation, more focused in the right temporal cortex. Patients showed parietal hyper-activation. Disorder-haplotype interactions, as well as structural findings, were not statistically significant. To sum up, both protective and risk ADGRL3 haplotypes are associated with substantial brain hypo-activation during working memory tasks, stressing this gene’s relevance in cognitive brain function. Conversely, we did not find brain effects of the interactions between adult ADHD and ADGRL3 haplotypes.

Synthesis of calix[4]arenes bearing thiosemicarbazone moieties with naphthalene groups: Highly selective turn off/on fluorescent sensor for Cu(II) recognition

Calix[4]arenes-based fluorescent chemosensors containing thiosemicarbazone as a binding site and naphthalene as a fluorogenic unit in a 1,3-alternate (L1) and cone (L2) conformation, were synthesized and examined for their binding abilities towards anions and cations using different spectroscopic techniques. Both receptors L1 and L2, exhibited an off/on fluorescence behaviour due to complex formation with Cu(II). They showed selective recognition of Cu(II) in presence of other cations and anions detecting up to 4.25 µg/L for L1 and 1.58 µg/L for L2, both in acetonitrile. The complexation behaviour was studied by 1H NMR and FT-IR spectral analysis, indicating that copper interacts with sulphur and with imine nitrogen of the thiosemicarbazone. No major differences in the selectivity and sensitivity were found between calix[4]arene receptors in 1,3-alternate and cone conformation. The 1H NMR spectra indicates that receptor L2 changed its conformation from cone to pinched cone, stiffening the conformation due to interaction of the thiosemicarbazone moiety with copper.

The Difference in Sensing Properties Towards Anions between Two Pyridinium Amide-Based Receptors with a Subtle Change of H-Bond Position

Two pyridinium amide-based receptors L1 and L2 with a small difference of H-bond position of the amide have been synthesized and characterized. Interestingly, they exhibited a huge difference in sensing towards AcO- and H2PO4 -, respectively. Receptor L1 was found to be ‘naked-eye’ selective for AcO- anions, while receptor L2 showed clear fluorescence enhancement selective to H2PO4 - anion. The recognition ability has been established by fluorescence emission, UV-vis spectra, and 1HNMR titration.

Adhesion G‐Protein‐Coupled Receptor L2 Expression is Lost in Colorectal Cancer but is Also Associated with Colonic Stem Cells

Colorectal cancer (CRC) is driven by uncontrolled expansion and differentiation of crypt stem cells. We are testing the role of adhesion G‐protein‐coupled receptor L2 (ADGRL2), whose function has yet to be studied in the intestine, as a potential gatekeeper for proliferating colonic stem cells and renewal.MethodsTo assess receptor regulation during disease, mRNA levels were measured in matched normal and tumor specimens from human CRC. We corroborated findings by mining Gene Expression Omnibus (GEO) datasets and examined receptor modulation in response to growth arrest conditions (serum starvation and MAP kinase inhibition) in human CRC cell lines. We further interrogated ADGRL2 function using a stable inducible shRNA knockout line using SW480 cells and compared the gene expression of colonocyte markers in the induced ADGRL2 KO cells versus non‐induced cells. To assess possible inflammatory links, mice were given DSS colitis with 3% DSS in drinking water for 3 days and colons were collected on day 7 in the hyperproliferative recovery phase.ResultsADGRL2 was significantly and selectively reduced in tumors relative to adjacent matched normal tissue (5.14 fold, p &lt; 0.0001). This was confirmed using two GEO datasets, one of which also revealed a pronounced loss in adenomas (3.82 fold, p &lt; 0.0001). ADGRL2 was induced by growth arrest conditions in CRC cell lines, both by serum starvation (2.79 fold, p = 0.0115) and map kinase inhibition (5.59 fold, p = 0.0086). Knockdown in SW480 cells resulted in a loss of the colonic stem cell marker LGR5, while other markers for colonic cell types remained unchanged. Analysis of publicly available microarray data confirmed that ADGRL2 and LGR5 levels are associated in the colonic mucosa. Adgrl2 levels were suppressed in recovery from DSS colitis (1.74 fold, p &lt; 0.0001), suggesting a role in restricting repair mechanisms.ConclusionsDownregulation during recovery from colonic injury, induction by antiproliferative stimuli, and association with LGR5 together suggest that ADGRL2 may have a role in orchestrating epithelial regeneration in the colon, potentially as a stem‐cell specific negative regulator. The substantial loss of ADGRL2 in human CRC adenoma and adenocarcinoma raises the possibility that ADGRL2 functions as a tumor suppressor. Thus, while modulation of ADGRL2 levels may mediate epithelial repair following injury, chronic loss could give way to uncontrollable stem cell expansion and loss of crypt architecture, leading to dysplasia and tumorigenesis.Support or Funding InformationThis work was supported by: NIH R01DK095004 and a Senior Research Award from the Crohn’s and Colitis Foundation (MRF). Support for EB includes The Saban Research Institute Predoctoral Intramural Funding Grant and The Roy E. Thomas Foundation Graduate Scholarship. Support for CM includes an Undergraduate Summer Research Fellowship from the Crohn’s and Colitis Foundation.

Meta-analysis and systematic review of ADGRL3 (LPHN3) polymorphisms in ADHD susceptibility.

The gene encoding adhesion G protein-coupled receptor L3 (ADGRL3, also referred to as latrophilin 3 or LPHN3) has been associated with ADHD susceptibility in independent ADHD samples. We conducted a systematic review and a comprehensive meta-analysis to summarize the associations between the most studied ADGRL3 polymorphisms (rs6551665, rs1947274, rs1947275, and rs2345039) and both childhood and adulthood ADHD. Eight association studies (seven published and one unpublished) fulfilled criteria for inclusion in our meta-analysis. We also incorporated GWAS data for ADGRL3. In order to avoid overlapping samples, we started with summary statistics from GWAS samples and then added data from gene association studies. The results of our meta-analysis suggest an effect of ADGRL3 variants on ADHD susceptibility in children (n = 8724/14,644 cases/controls and 1893 families): rs6551665 A allele (Z score = -2.701; p = 0.0069); rs1947274 A allele (Z score = -2.033; p = 0.0421); rs1947275 T allele (Z score = 2.339; p = 0.0978); and rs2345039 C allele (Z score = 3.806; p = 0.0026). Heterogeneity was found in analyses for three SNPs (rs6551665, rs1947274, and rs2345039). In adults, results were not significant (n = 6532 cases/15,874 controls): rs6551665 A allele (Z score = 2.005; p = 0.0450); rs1947274 A allele (Z score = 2.179; p = 0.0293); rs1947275 T allele (Z score = -0.822; p = 0.4109); and rs2345039 C allele (Z score = -1.544; p = 0.1226). Heterogeneity was found just for rs6551665. In addition, funnel plots did not suggest publication biases. Consistent with ADGRL3's role in early neurodevelopment, our findings suggest that the gene is predominantly associated with childhood ADHD.