Human D2 Research Articles

Vitex agnus castus Extract Ze 440: Diterpene and Triterpene’s Interactions with Dopamine D2 Receptor

Pre-clinical studies suggest that extracts prepared from the fruits of Vitex agnus castus (VAC) interact with dopamine D2 receptors, leading to reduced prolactin secretion. In previous experiments, dopaminergic activity was mostly evaluated using radioligand binding assays or via the inhibition of prolactin release from rat pituitary cells. Diterpenes featuring a clerodadienol scaffold were identified as major active compounds, but no conclusive data regarding their potency and intrinsic activity are available. Utilising advances in chromatography, we re-examined this topic using HPLC-based tracking of bioactivity via microfractionation of the VAC extract Ze 440. Using a cAMP-based assay, we measured dopaminergic activity in CHO-K1 cells that overexpress the human D2 receptor. Six diterpenes were isolated from two active HPLC microfractions. Viteagnusin I emerged as the most potent diterpene (EC50: 6.6 µM), followed by rotundifuran (EC50: 12.8 µM), whereas vitexilactone was inactive (EC50: >50 µM). Interestingly, triterpenes were also identified as active, with 3-epi-maslinic acid being the most active compound (EC50: 5.1 µM). To better understand these interactions at the molecular level, selected diterpenes and triterpenes were analysed through molecular docking against D2 receptor structures. Our data show that the dopaminergic activity of VAC diterpenes seems to depend on the configuration and on ring substitution in the side chain. This study also highlights for the first time the dopaminergic contribution of triterpenes such as 3-epi-maslinic acid.

Discovery and structure − activity relationships of 2,4,5-trimethoxyphenyl pyrimidine derivatives as selective D5 receptor partial agonists

Dopamine receptors are therapeutic targets for the treatment of various neurological and psychiatric disorders, including Parkinson’s and Alzheimer’s. Previously, PF-06649751 (tavapadon), PF-2562 and PW0464 have been discovered as potent and selective G protein-biased D1/D5 receptor agonists with optimal pharmacokinetic properties. However, no selective D5R agonist has been reported yet. In this context, we designed and synthesized forty non-catecholamines-based pyrimidine derivatives and identified four pyrimidine derivatives as selective D5R partial agonists. Using cAMP-based GloSensor assay in transiently transfected HEK293T cells with human D1 or D5 receptors, we discovered that compound 5c (4-(4-bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-amine) exhibited modest D5R agonist activity. This leads us to explore various modifications of this scaffold to improve the D5 agonist potency and efficacy. Using molecular docking, and rational design followed by their evaluation at D1 and D5 receptors for agonist activity, we identified three new derivatives, 5j, 5h, and 5e. The most potent compound of this series 5j (4-(4-iodophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-amine), exhibited EC50 of 269.7 ± 6.6 nM. Mice microsomal stability studies revealed that 5j is quite stable (>70 % at 1 hr). Furthermore, pharmacokinetic analysis of 5j (20 mg/kg, p.o) in C57BL/6j mice showed that 5j is readily absorbed via oral route of dosing and also enters into the brain (plasma Tmax: 1 h, Cmax: 51.10 ± 13.51 ng/ml; Brain Tmax: 0.5 h, Cmax: 22.54 ± 4.08 ng/ml). We further determined the in-vivo effect of 5j on cognition in scopolamine-induced amnesia in C57BL/6j mice. We observed that 5j (10 mg/kg, p.o) alleviated scopolamine-induced impairment in short-term memory and social recognition, which were blocked by D1/D5 antagonist SCH23390 (0.1 mg/kg, i.p.). Furthermore, 5j did not exhibit any cytotoxicity (up to 10 µM) or in vivo acute toxicity up to 200 mg/kg (p.o). These results strongly suggest that 5j could be further developed for treating neurological disorders wherein the D5 receptors play pivotal roles.

D2 dopamine receptor expression, reactivity to rewards, and reinforcement learning in a complex value-based decision-making task.

Different dopamine (DA) subtypes have opposing dynamics at postsynaptic receptors, with the ratio of D1 to D2 receptors determining the relative sensitivity to gains and losses, respectively, during value-based learning. This effective sensitivity to different reward feedback interacts with phasic DA levels to determine the effectiveness of learning, particularly in dynamic feedback situations where the frequency and magnitude of rewards need to be integrated over time to make optimal decisions. We modeled this effect in simulations of the underlying basal ganglia pathways and then tested the predictions in individuals with a variant of the human dopamine receptor D2 (DRD2; -141C Ins/Del and Del/Del) gene that associates with lower levels of D2 receptor expression (N = 119) and compared their performance in the Iowa Gambling Task to noncarrier controls (N = 319). Ventral striatal (VS) reactivity to rewards was measured in the Cards task with fMRI. DRD2 variant carriers made less effective decisions than noncarriers, but this effect was not moderated by VS reward reactivity as is hypothesized by our model. These results suggest that the interaction between DA receptor subtypes and reactivity to rewards during learning may be more complex than originally thought.

Comparison of the function of two novel human dopamine D2 receptor variants identifies a likely mechanism for their pathogenicity

Two recently discovered DRD2 mutations, c.634A > T, p.Ile212Phe and c.1121T > G, p.Met374Arg, cause hyperkinetic movement disorders that have overlapping features but apparently differ in severity. The two known carriers of the Met374Arg variant had early childhood disease onset and more severe motor, cognitive, and neuropsychiatric deficits than any known carriers of the Ile212Phe variant, whose symptoms were first apparent in adolescence. Here, we evaluated if differences in the function of the two variants in cultured cells could explain differing pathogenicity. Both variants were expressed less abundantly than the wild type receptor and exhibited loss of agonist-induced arrestin binding, but differences in expression and arrestin binding between the variants were minor. Basal and agonist-induced activation of heterotrimeric Gi/o/z proteins, however, showed clear differences; agonists were generally more potent at Met374Arg than at the Ile212Phe or wild type variants. Furthermore, all Gα subtypes tested were constitutively activated more by Met374Arg than by Ile212Phe. Met374Arg produced greater constitutive inhibition of cyclic AMP accumulation than Ile212Phe or the wild type D2 receptor. Met374Arg and Ile212Phe were more sensitive to thermal inactivation than the wild type D2 receptor, as reported for other constitutively active receptors, but Ile212Phe was affected more than Met374Arg. Additional pharmacological characterization suggested that the mutations differentially affect the shape of the agonist binding pocket and the potency of dopamine, norepinephrine, and tyramine. Molecular dynamics simulations provided a structural rationale for enhanced constitutive activation and agonist potency. Enhanced constitutive and agonist-induced G protein-mediated signaling likely contributes to the pathogenicity of these novel variants.

Humanized dopamine D4.7 receptor male mice display risk-taking behavior and deficits of social recognition and working memory in light/dark-dependent manner.

The dopamine D4 receptor 7-repeat allele (D4.7 R) has been linked with psychiatric disorders such as attention-deficit-hyperactivity disorder, autism, and schizophrenia. However, the highly diverse study populations and often contradictory findings make it difficult to draw reliable conclusions. The D4.7 R has the potential to explain individual differences in behavior. However, there is still a great deal of ambiguity surrounding whether it is causally connected to the etiology of psychiatric disorders. Therefore, humanized D4.7 R mice, with the long third intracellular domain of the human D4.7 R, may provide a valuable tool to examine the relationship between the D4.7 R variant and specific behavioral phenotypes. We report that D4.7 R male mice carrying the humanized D4.7 R variant exhibit distinct behavioral features that are dependent on the light-dark cycle. The behavioral phenotype was characterized by a working memory deficit, delayed decision execution in the light phase, decreased stress and anxiety, and increased risk behavior in the dark phase. Further, D4.7 R mice displayed impaired social recognition memory in both the light and dark phases. These findings provide insight into the potential causal relationship between the human D4.7 R variant and specific behaviors and encourage further consideration of dopamine D4 receptor (DRD4) ligands as novel treatments for psychiatric disorders in which D4.7 R has been implicated.

Stapling Amantadine to Melanostatin Neuropeptide: Discovery of Potent Positive Allosteric Modulators of the D2 Receptors.

This work describes the synthesis and pharmacological and toxicological evaluation of melanostatin (MIF-1) bioconjugates with amantadine (Am) via a peptide linkage. The data from the functional assays at human dopamine D2 receptors (hD2R) showed that bioconjugates 1 (EC50 = 26.39 ± 3.37 nM) and 2 (EC50 = 17.82 ± 4.24 nM) promote a 3.3- and 4.9-fold increase of dopamine potency, respectively, at 0.01 nM, with no effect on the efficacy (Emax = 100%). In this assay, MIF-1 was only active at the highest concentration tested (EC50 = 23.64 ± 6.73 nM, at 1 nM). Cytotoxicity assays in differentiated SH-SY5Y cells showed that both MIF-1 (94.09 ± 5.75%, p < 0.05) and carbamate derivative 2 (89.73 ± 4.95%, p < 0.0001) exhibited mild but statistical significant toxicity (assessed through the MTT reduction assay) at 200 μM, while conjugate 1 was found nontoxic at this concentration.

OR14-04 Musashi-1 Is A Cell-type Specific Regulator Of Type 2 Deiodinase-mediated Thyroid Hormone Activation

Abstract Disclosure: P. Mohacsik: None. E. Halmos: None. Y. Ruska: None. C. Fekete: None. B. Gereben: None. Thyroid hormone (TH) exerts a profound effect on the regulation of energy homeostasis, metabolism, brain development and the determination of cell fate in various tissues. Type 2 deiodinase enzyme (D2) is the crucial regulator of TH activation by converting the prohormone thyroxine to T3 that can bind the TH receptor. This represents a fundamental step of cell-type specific TH action. D2 regulation is tightly controlled at various molecular levels to allow quick adaptation of TH action to specific challenges. Within the brain, D2 is expressed in hypothalamic tanycytes that play a crucial role in the regulation of the hypothalamo-pituitary-thyroid (HPT) axis and in astrocytes of extrahypothalamic brain regions. Under changing TH economy, D2 activity undergoes homeostatic alteration in astrocytes while remains largely unaffected in hypothalamic tanycytes. Our aim was to identify regulators that could underlie this phenomenon as a cell-type specific regulator of D2-dependent TH action. Musashi (Msi)-1 is a highly conserved RNA-binding protein that regulates cell cycle and play a crucial role during brain development. We found, that Msi-1 was co-expressed with D2 both in hypothalamic tanycytes and cortical astrocytes in mice and T3 treatment (24h T3 1µg/bwg, i.p.) increased Msi-1 protein level in both cell types indicating its TH-dependent regulation. Then we identified multiple potential Msi-1 binding sites in the extremely long 3’UTR both of the human, mouse, rat and chicken D2 mRNA and used RNA electromobility shift assay that revealed direct binding of Msi-1 to the 3’UTR of the D2 mRNA. Studies on the impact of Msi-1 level on D2 activity showed, that Msi-1 overexpression decreased D2 activity in HEK293 cells while this effect was abolished by the deletion of a 3 kb region of the 3’UTR of D2 containing the Msi-1 binding sites. Furthermore, Msi-1 knock down by shRNA in H4 glioma cells increased endogenous D2 activity. A mouse model, lacking Msi-1 protein exclusively in the brain and pituitary, showed increased D2 activity in the cortex accompanied with a slightly decreased serum fT4 and decreased liver dio1 expression. Increased cortical D2 activity was not a consequence of the mildly hypothyroid periphery since cortical expression of dio3, the T3-regulated T3-degrading enzyme remained unchanged along with enpp2, another T3-regulated gene. In contrast, D2 activity remained unchanged in hypothalamic tanycytes. Our results identify Msi-1 as a TH-dependent cell-type specific regulator of D2 activity in the mouse brain that acts via direct binding to the 3’UTR of the D2 mRNA. In summary, Msi-1 regulates D2 activity cell type specifically and may contribute to the homeostatic regulation of cortical D2 while at the same time allows TH-resistant D2 function in tanycytes that is required by proper HPT function. Presentation: Friday, June 16, 2023

Computer-Aided Designing Peptide Inhibitors of Human Hematopoietic Prostaglandin D2 Synthase Combined Molecular Docking and Molecular Dynamics Simulation.

Human hematopoietic prostaglandin D2 synthase (HPGDS) is involved in the production of prostaglandin D2, which participates in various physiological processes, including inflammation, allergic reactions, and sleep regulation. Inhibitors of HPGDS have been investigated as potential anti-inflammatory agents. For the investigation of potent HPGDS inhibitors, we carried out a computational modeling study combining molecular docking and molecular dynamics simulation for selecting and virtual confirming the designed binders. We selected the structure of HPGDS (PDB ID: 2CVD) carrying its native inhibitor compound HQL as our research target. The random 5-mer peptide library was created by building the 3-D structure of random peptides using Rosetta Buildpeptide and performing conformational optimization. Molecular docking was carried out by accommodating the peptides into the location of their native binder and then conducting docking using FlexPepDock. The two peptides RMYYY and VMYMI, which display the lowest binding energy against HPGDS, were selected to perform a comparative study. The interaction of RMYYY and VMYMI against HPGDS was further confirmed using molecular dynamics simulation and aligned with its native binder, HQL. We show the selected binders to have stronger binding energy and more frequent interactions against HPGDS than HQL. In addition, we analyzed the solubility, hydrophobicity, charge, and bioactivity of the generated peptides, and we show that the selected strong binder may be further used as therapeutic drugs.

Incorporation of a recirculating mobile lipid pool description into the cyclic volatile siloxane (cVMS) PBPK model captures terminal clearance of D4 after repeated inhalation exposure in F344 and SD Rats

The most recent version of the octamethylcyclotetrasiloxane (D4) physiologically based pharmacokinetic (model) was developed using the available kinetic studies in male and female F344 rats. Additional data, which had not been included in the D4 model development, allowed for a more detailed assessment of the loss of D4 following long-term exposure in both SD and F344 rats. This new data demonstrated a deficiency in the published PBPK model predictions of terminal concentrations of D4 in plasma and fat 14 days after the end of exposures for 28-days, 6 h/day, where the model predictions were an order of magnitude lower than the data. To capture this time-point without altering the end-of-exposure peak concentrations in blood and fat required conversion of the one-way (liver to fat) mobile lipoprotein pool (MLP) into a bi-directional pool between liver and fat. Simulation of the D4 pharmacokinetics in the SD rat, as opposed to the F344 rat, also required a reduction of both fold induction of liver metabolism (KMAX: 5- to 2-fold) and maximal rate of metabolism (VMAXC: 5.0–1.54 mg/kg0.75). The revised MLP description was extended to the human D4 model using a parallelogram approach between rat and human MLP parameters to establish the parameters for the current model in the absence of similar long-term clearance data in the human. The revised human D4 model provided good fits to the human inhalation and dermal exposure studies while not appreciably altering cross-species dose metrics based on the free concentration of D4 in blood.

Mutual Cooperativity of Three Allosteric Sites on the Dopamine D1 Receptor.

An amine-containing molecule called Compound A has been reported by a group from Bristol-Myers Squibb to act as a positive allosteric modulator (PAM) at the dopamine D1 receptor. We synthesized the more active enantiomer of Compound A (BMS-A1) and compared it with the D1 PAMs DETQ and MLS6585, which are known to bind to intracellular loop 2 and the extracellular portion of transmembrane helix 7, respectively. Results from D1/D5 chimeras indicated that PAM activity of BMS-A1 tracked with the presence of D1 sequence in the N-terminal/extracellular region of the D1 receptor, a unique location compared with either of the other PAMs. In pairwise combinations, BMS-A1 potentiated the small allo-agonist activity of each of the other PAMs, while the triple PAM combination (in the absence of dopamine) produced a cAMP response about 64% of the maximum produced by dopamine. Each of the pairwise PAM combinations produced a much larger leftward shift of the dopamine EC50 than either single PAM alone. All three PAMs in combination produced a 1000-fold leftward shift of the dopamine curve. These results demonstrate the presence of three non-overlapping allosteric sites that cooperatively stabilize the same activated state of the human D1 receptor. SIGNIFICANCE STATEMENT: Deficiencies in dopamine D1 receptor activation are seen in Parkinson disease and other neuropsychiatric disorders. In this study, three positive allosteric modulators of the dopamine D1 receptor were found to bind to distinct and separate sites, interacting synergistically with each other and dopamine, with the triple combination causing a 1000-fold leftward shift of the response to dopamine. These results showcase multiple opportunities to modulate D1 tone and highlight new pharmacological approaches for allosteric modulation of G-protein-coupled receptors.

An innovative strategy to identify new targets for delivering antibodies to the brain has led to the exploration of the integrin family.

Increasing brain exposure of biotherapeutics is key to success in central nervous system disease drug discovery. Accessing the brain parenchyma is especially difficult for large polar molecules such as biotherapeutics and antibodies because of the blood-brain barrier. We investigated a new immunization strategy to identify novel receptors mediating transcytosis across the blood-brain barrier. We immunized mice with primary non-human primate brain microvascular endothelial cells to obtain antibodies. These antibodies were screened for their capacity to bind and to be internalized by primary non-human primate brain microvascular endothelial cells and Human Cerebral Microvascular Endothelial Cell clone D3. They were further evaluated for their transcytosis capabilities in three in vitro blood-brain barrier models. In parallel, their targets were identified by two different methods and their pattern of binding to human tissue was investigated using immunohistochemistry. 12 antibodies with unique sequence and internalization capacities were selected amongst more than six hundred. Aside from one antibody targeting Activated Leukocyte Cell Adhesion Molecule and one targeting Striatin3, most of the other antibodies recognized β1 integrin and its heterodimers. The antibody with the best transcytosis capabilities in all blood-brain barrier in vitro models and with the best binding capacity was an anti-αnβ1 integrin. In comparison, commercial anti-integrin antibodies performed poorly in transcytosis assays, emphasizing the originality of the antibodies derived here. Immunohistochemistry studies showed specific vascular staining on human and non-human primate tissues. This transcytotic behavior has not previously been reported for anti-integrin antibodies. Further studies should be undertaken to validate this new mechanism in vivo and to evaluate its potential in brain delivery.

Development of GPC2-directed chimeric antigen receptors using mRNA for pediatric brain tumors

BackgroundPediatric brain tumors are the leading cause of cancer death in children with an urgent need for innovative therapies. Glypican 2 (GPC2) is a cell surface oncoprotein expressed in neuroblastoma...

2-(Fluoromethoxy)-4'-(S-methanesulfonimidoyl)-1,1'-biphenyl (UCM-1306), an Orally Bioavailable Positive Allosteric Modulator of the Human Dopamine D1 Receptor for Parkinson's Disease.

Tolerance development caused by dopamine replacement with l-DOPA and therapeutic drawbacks upon activation of dopaminergic receptors with orthosteric agonists reveal a significant unmet need for safe and effective treatment of Parkinson’s disease. In search for selective modulators of the D1 receptor, the screening of a chemical library and subsequent medicinal chemistry program around an identified hit resulted in new synthetic compound 26 [UCM-1306, 2-(fluoromethoxy)-4′-(S-methanesulfonimidoyl)-1,1′-biphenyl] that increases the dopamine maximal effect in a dose-dependent manner in human and mouse D1 receptors, is inactive in the absence of dopamine, modulates dopamine affinity for the receptor, exhibits subtype selectivity, and displays low binding competition with orthosteric ligands. The new allosteric modulator potentiates cocaine-induced locomotion and enhances l-DOPA recovery of decreased locomotor activity in reserpinized mice after oral administration. The behavior of compound 26 supports the interest of a positive allosteric modulator of the D1 receptor as a promising therapeutic approach for Parkinson’s disease.

Insights into the role of N‐linked glycosylation in directing plasma membrane localization of GPCRs expressed in Saccharomyces cerevisiae

Glycosylation is the biochemical process of the attachment of sugar moieties or carbohydrates to proteins during co‐translational or post‐translational modification (PTM). It is one of many types of protein modifications that naturally occurs during the process of protein expression in organisms like Saccharomyces cerevisiae (Baker’s yeast). For natively expressed proteins, glycosylation can be critical for proper protein processing and can also be important for the production of proteins in a stable and biologically active state. For proteins that are non‐natively, or heterologously, expressed in these organisms, glycosylation can lead to improper processing and ultimately produce proteins with little to no biological relevance. This proves to be an undesirable effect for the elucidation of protein structure. However, large quantities of protein are required for structural analysis, presenting a significant obstacle for structural biologists and those who use heterologous expression as a means to acquire large quantities of protein. This problem becomes significantly more challenging for membrane proteins.Membrane proteins are an elusive class of biomolecules that reside or closely associate with the lipid bilayers of cells and encompass transporters, receptors and channel pores, to name a few. They play an intrinsic role in maintaining proper cell function including signaling and selective uptake of extracellular ions and small molecules. G‐protein coupled receptors (GPCRs) constitute one of the largest groups of membrane proteins across the plant and animal kingdoms. Despite being of scientific and medical interest, GPCRs and many integral membrane proteins are notoriously difficult to isolate and work with because of their hydrophobicity, structural flexibility and instability in the absence of the cell membrane. Optimized experimental conditions often vary from protein to protein and necessitate a systematic approach to achieve. This typically requires labor and resource intensive efforts. The goal of this work is to uncover new insights into the role of post‐translational processing and protein localization in the cell. Using a combination of fluorescence microscopy and bioinformatics we formulated an approach to investigate the effects of N‐linked glycosylation on plasma membrane localization of GPCRs expressed in S. cerevisiae. We postulate a critical link between plasma membrane localization and expression of “ligand active” or structurally relevant GPCRs. Previous studies of the human adenosine A2A receptor (hA2AR) show it readily localizes to the plasma membrane during expression in yeast, while the human dopamine D2 receptor (hD2R) does not. A sequence comparison of the two reveals three putative N‐linked glycosylation sites at the N‐terminal extracellular region of hD2R which is lacking in hA2AR. Using a chimera of these two receptors prepared with a C‐terminal GFP tag, we show the chimera does not traffic to the plasma membrane similarly to full‐length hA2AR, suggesting N‐linked glycosylation strongly influences functional high‐level expression of GPCRs and possibly other membrane proteins in yeast.

Galectin‐9 activates platelet ITAM receptors glycoprotein VI and C‐type lectin‐like receptor‐2

BackgroundPlatelets are multifunctional cellular mediators in many physiological and pathophysiological processes such as thrombosis, angiogenesis, and inflammation. Several members of galectins, a family of carbohydrate‐binding proteins with a broad range of immunomodulatory actions, have been reported to activate platelets. ObjectiveIn this study, we investigated the role of galectin‐9 (Gal‐9) as a novel ligand for platelet glycoprotein VI (GPVI) and C‐type lectin‐like receptor 2 (CLEC‐2). MethodsPlatelet spreading, aggregation, and P‐selectin expression in response to Gal‐9 were measured in washed platelet suspensions via static adhesion assay, light transmission aggregometry, and flow cytometry, respectively. Solid‐phase binding assay and protein phosphorylation studies were utilized to validate the interaction between Gal‐9 and GPVI, and immunoprecipitation for detecting CLEC‐2 phosphorylation. Wild‐type (WT), GPVI‐knockout (Gp6−/−), and GPVI and CLEC‐2‐double knockout (Gp6−/−/Gp1ba‐Cre‐Clec1bfl/fl) mice were used. ResultsWe have shown that recombinant Gal‐9 stimulates aggregation in human and mouse washed platelets dose‐dependently. Platelets from both species adhere and spread on immobilized Gal‐9 and express P‐selectin. Gal‐9 competitively inhibited the binding of human recombinant D1 and D2 domains of GPVI to collagen. Gal‐9 stimulated tyrosine phosphorylation of CLEC‐2 and proteins known to lie downstream of GPVI and CLEC‐2 including spleen tyrosine kinase and linker of activated T cells in human platelets. GPVI‐deficient murine platelets exhibited significantly impaired aggregation in response to Gal‐9, which was further abrogated in GPVI and CLEC‐2‐double‐deficient platelets. ConclusionsWe have identified Gal‐9 as a novel platelet agonist that induces activation through interaction with GPVI and CLEC‐2.

Assessment of Factors that Affect Primary Stability of Minimally Invasive Implant (MII): An in vitro Study

Purpose: This study aimed to evaluate the factors affecting the primary stability of a new implant design based on minimally invasive implantation (MAGICORE® ; INNOBIOSURG Co. Ltd., Daejeon, Korea) using the evaluation index of Periotest value (PTV), implant stability quotient (ISQ), and implant stability test (IST).Materials and Methods: A total of 1056 implants were implanted in artificial bone, imitating human bones D1, D2, D3, and D4. The PTV, ISQ, and IST values of all implants were measured according to the manufacturer’s guidelines for each measuring instrument. To compare the factors affecting the stability of the implant for each measurement method, one-way ANOVA was performed, and posthoc analysis was performed using the Games–Howel test (p < .05). In addition, a stepwise multiple linear regression analysis was performed to evaluate the weight of each factor.Results: Implants with diameters greater than 5.0 mm showed significantly lower PTV values and higher ISQ and IST values. Implants with 11 mm length showed the lowest PTV and highest ISQ and IST values. A cuff size of 2 mm showed the highest implant stability among all the measurement methods. In this study, poor bone quality decreased the primary stability.Conclusion: As the cuff size increased, the stability decreased, and the larger the diameter of the implant, the longer the length and higher the primary stability. The bone quality and diameter of the implant contributed more to the stability of the implant than to the length of the implant and cuff size.

Ginger (Zingiber officinale) components as alternative for inhibition of the human dopamine receptor D2: a computational approach

ABSTRACT Human dopamine receptor D2 (DRD2) is one of the molecular targets for the drug development against some neurological disorders playing a vital role in the dopamine equilibrium in the brain. Atypical drugs like Risperidone, used against schizophrenia, with proven efficacy in reducing the extra pyramidal symptoms of this disease, act as agonist inhibitors of DRD2. However, these drugs are not capable of curing schizophrenia, there is still room for the drug development against this disease. We investigated here the ability of 12 components of the Ginger (Zingiber officinale) extract, known since ancient times to be effective against various diseases including neurological ones, for their molecular interactions with the human DRD2 receptor. Among all the amino acids participating in the binding process, we found that Trp386 plays a vital role, since it fixes the ligands by pi-stacking while Asp114 binds them through a H-bond. This knowledge is important since it supports the traditional use of this plant in natural medicine and also serves as a starting point for the design of drugs derived from these natural components.

Survey of Vitamin D Deficiency and Associated Comorbidities in Lahore

Vitamin D is recognized for its importance in bone health along with the reduced risk of cardiovascular, autoimmune and several chronic diseases. Objectives: Present study was designed to explore the prevalence of vitamin D deficiency and its association with comorbidities (Type 2 Diabetes, Thyroid Disease, Hypertension, Asthma, Heart Disease, Gastritisand Osteoporosis) among the subjects visiting outpatient departments (OPDs) of public hospitals in Lahore. Methods: Adult (&gt;18 years) male and female subjects (n = 200) participated in present cross-sectional study. Participating individuals were required to fill questionnaires which recorded their medical history and BMI. Blood samples were taken for laboratory evaluation of vitamin D3 deficiency. Data was analyzed for evaluation of diverse risk factors. Serum level of vitamin D3 (25-hydroxyvitamin D [25(OH)D3]) were measured using standard procedures of measurement using Human Vitamin D3 (VD3) ELISA Kit (MyBioSource, Inc. USA). Results: Vitamin D deficiency was more prevalent in females than in males. Of the 200 individuals, the highest number of patients suffered from Diabetes Myelitis Type-II (61%) followed by Thyroid Disease (38%). Whereas, the least number of vitamin D3 deficient subjects were suffering from Osteoporosis (5%).

A novel red fluorescence dopamine biosensor selectively detects dopamine in the presence of norepinephrine in vitro

Dopamine (DA) and norepinephrine (NE) are pivotal neuromodulators that regulate a broad range of brain functions, often in concert. Despite their physiological importance, untangling the relationship between DA and NE in the fine control of output function is currently challenging, primarily due to a lack of techniques to allow the observation of spatiotemporal dynamics with sufficiently high selectivity. Although genetically encoded fluorescent biosensors have been developed to detect DA, their poor selectivity prevents distinguishing DA from NE. Here, we report the development of a red fluorescent genetically encoded GPCR (G protein-coupled receptor)-activation reporter for DA termed ‘R-GenGAR-DA’. More specifically, a circular permutated red fluorescent protein (cpmApple) was replaced by the third intracellular loop of human DA receptor D1 (DRD1) followed by the screening of mutants within the linkers between DRD1 and cpmApple. We developed two variants: R-GenGAR-DA1.1, which brightened following DA stimulation, and R-GenGAR-DA1.2, which dimmed. R-GenGAR-DA1.2 demonstrated a reasonable dynamic range (ΔF/F0 = − 43%), DA affinity (EC50 = 0.92 µM) and high selectivity for DA over NE (66-fold) in HeLa cells. Taking advantage of the high selectivity of R-GenGAR-DA1.2, we monitored DA in presence of NE using dual-color fluorescence live imaging, combined with the green-NE biosensor GRABNE1m, which has high selectivity for NE over DA (> 350-fold) in HeLa cells and hippocampal neurons grown from primary culture. Thus, this is a first step toward the multiplex imaging of these neurotransmitters in, for example, freely moving animals, which will provide new opportunities to advance our understanding of the high spatiotemporal dynamics of DA and NE in normal and abnormal brain function.

Optimizing the Expression of Human Dopamine Receptors in Escherichia coli.

The human dopamine receptors D2S and D3 belong to the group of G protein-coupled receptors (GPCRs) and are important drug targets. Structural analyses and development of new receptor subtype specific drugs have been impeded by low expression yields or receptor instability. Fusing the T4 lysozyme into the intracellular loop 3 improves crystallization but complicates conformational studies. To circumvent these problems, we expressed the human D2S and D3 receptors in Escherichia coli using different N- and C-terminal fusion proteins and thermostabilizing mutations. We optimized expression times and used radioligand binding assays with whole cells and membrane homogenates to evaluate KD-values and the number of receptors in the cell membrane. We show that the presence but not the type of a C-terminal fusion protein is important. Bacteria expressing receptors capable of ligand binding can be selected using FACS analysis and a fluorescently labeled ligand. Improved receptor variants can thus be generated using error-prone PCR. Subsequent analysis of clones showed the distribution of mutations over the whole gene. Repeated cycles of PCR and FACS can be applied for selecting highly expressing receptor variants with high affinity ligand binding, which in the future can be used for analytical studies.