Fine-tuning GPCR-mediated neuromodulation by biasing signaling through different G protein subunits.

G-protein-coupled receptors (GPCRs) mediate neuromodulation through the activation of heterotrimeric G proteins (Gαßγ). Classical models depict that G protein activation leads to a one-to-one formation of Gα-GTP and Gßγ species. Each of these species propagates signaling by independently acting on effectors, but the mechanisms by which response fidelity is ensured by coordinating Gα and Gßγ responses remain unknown. Here, we reveal a paradigm of G protein regulation whereby the neuronal protein GINIP (Gα inhibitory interacting protein) biases inhibitory GPCR responses to favor Gßγ over Gα signaling. Tight binding of GINIP to Gαi-GTP precludes its association with effectors (adenylyl cyclase) and, simultaneously, with regulator-of-G-protein-signaling (RGS) proteins that accelerate deactivation. As a consequence, Gαi-GTP signaling is dampened, whereas Gßγ signaling is enhanced. We show that this mechanism is essential to prevent the imbalances of neurotransmission that underlie increased seizure susceptibility in mice. Our findings reveal an additional layer of regulation within a quintessential mechanism of signal transduction that sets the tone of neurotransmission.

The risk of DDH between breech and cephalic-delivered neonates using Graf ultrasonography.

PURPOSE: Developmental dysplasia of the hip (DDH) is one of the most common musculoskeletal disorder in infants. The most significant risk factors include female gender, breech presentation, left hip and family history. In this study, we utilized the Graf method at different time intervals to evaluate both breech-delivered and cephalic-born newborns. The objectives were to compare the incidence of DDH in cephalic and breech-delivered neonates and investigate whether the hip joints of neonates delivered in the breech position exhibit a distinct maturation pattern. MATERIAL AND METHODS: We studied prospectively 618 hip joints (309 newborns). Each hip joint was examined with the Graf method in four time periods as follows: Phase #1 (0-1 weeks), Phase #2 (1-4 weeks), Phase #3 (4-7 weeks), and Phase #4 (7-10 weeks). The α and ß angles for each hip joint were measured, and the hips were classified according to Graf classification. With our statistical analysis within the different phases, we were able to investigate potential variations in the maturation patterns between newborns delivered in the breech and cephalic delivery positions. RESULTS: A significant difference (at the 5% level) was observed in Phase 1 between breech and cephalic-delivered neonates (35.6-8.6%). This difference tended to decrease in next phases (13.6-1% in Phase 2, 2.5-0% in Phase 3 and 1.7-0% in Phase 4). A significant difference (at the 5% level) for cephalic-delivered neonates was also observed between Phase 1 and Phase 4 (8.5-0%), but the percentages were low. Additionally, the breech-delivered had extreme difference in incidence of DDH from Phase 1 to Phase 4 (35.6-11.9%, 2.5%, and 1.7%, respectively). CONCLUSION: It appears that there is an actual difference in the incidence of DDH between breech-delivered and cephalic-delivered neonates, although the difference may be less significant than previously considered. The majority of the breech-delivered neonates that were initially considered as pathological (Phase 1) are, in fact, healthy. This is ascertained in subsequent ultrasound examinations conducted in later phases (Phases 2-4), when the incidence of pathological cases decreases. This could be attributed to potential different maturation pattern between these groups.

Ultrasonographic screening for developmental dysplasia of the hip: the Graf method revisited.

Until the 1980s, the diagnosis of developmental dysplasia of the hip (DDH) was based on clinical examination and radiographic imaging. In 1980, Reinhard Graf developed his own ultrasonographic method for the examination of the infant hip joint. Graf's method evaluates the osseous and cartilaginous coverage of the femoral head by the acetabulum in the infantile hip joint by measuring the angles α and ß. The validity of Graf method is that with these measurements the hip joint is further classified by Graf classification into types I to IV that guide treatment. Currently, Graf method is considered the gold standard examination for the diagnosis of DDH in many European countries. This review article aims to discuss the incidence, risk factors and pathophysiology of DDH, and to emphasize on the Graf method for the evaluation, classification, prevention and further management of this entity.

Red Blood Cell Alloantibody Titration - Does the Titration Method Matter?

BACKGROUND: Red blood cell (RBC) alloantibody titration is a quasi-quantitative method to assess antibody concentration and is considered a useful means of estimating maternal alloimmunization during pregnancy. Traditionally, titration is performed using conventional tube test (CTT). The gel microcolumn agglutination-based method (GMA) has been proven reliable for many immunohematology tests. Our study compared CTT with GMA of two different, commercially available GMA systems for RBC alloantibody titration. METHODS: Serum samples with significant RBC-alloantibodies were evaluated in our study. Each sample was titrated concurrently with CTT, with ID-DiaMed-GmbH, Cressier, Switzerland (GMA1), and with DG Gel Coombs Diagnostic Grifols, Passeig Fluvial, Spain (GMA2). RESULTS: One hundred thirty-seven titration tests including 50 anti-D, 25 anti-Kell, 10 anti-E, 9 anti-Jka, 8 anti-c, 5 anti-Cw, 5 anti-Fya, 7 anti-M, 6 anti-Kpa, 3 anti-Lua, 1 anti-e, 3 anti-G, and 2 anti-Cha were performed and evaluated. Samples tested by CTT versus GMA1 and GMA2 generated mostly equal or higher titers by GMAs. The results of both comparisons were in good agreement (W = 0.91, p < 0.0001, and W = 0.92, p < 0.0001, respectively). For all antibody specificities, the mean absolute difference in titers ranged from 1 - 3 for both GMA1 and GMA2 versus CTT. Samples tested by GMA1 vs. GMA2 were in almost perfect agreement (W = 0.95, p < 0.0001). CONCLUSIONS: Although both GMAs were found slightly more sensitive than CTT for alloantibody titration, the differences were not significant and the agreement between all methods was very good, possibly indicating GMA as a suitable alternative to CTT in RBC antibody titration.

Specific inhibition of GPCR-independent G protein signaling by a rationally engineered protein.

Activation of heterotrimeric G proteins by cytoplasmic nonreceptor proteins is an alternative to the classical mechanism via G protein-coupled receptors (GPCRs). A subset of nonreceptor G protein activators is characterized by a conserved sequence named the Gα-binding and activating (GBA) motif, which confers guanine nucleotide exchange factor (GEF) activity in vitro and promotes G protein-dependent signaling in cells. GBA proteins have important roles in physiology and disease but remain greatly understudied. This is due, in part, to the lack of efficient tools that specifically disrupt GBA motif function in the context of the large multifunctional proteins in which they are embedded. This hindrance to the study of alternative mechanisms of G protein activation contrasts with the wealth of convenient chemical and genetic tools to manipulate GPCR-dependent activation. Here, we describe the rational design and implementation of a genetically encoded protein that specifically inhibits GBA motifs: GBA inhibitor (GBAi). GBAi was engineered by introducing modifications in Gαi that preclude coupling to every known major binding partner [GPCRs, Gßγ, effectors, guanine nucleotide dissociation inhibitors (GDIs), GTPase-activating proteins (GAPs), or the chaperone/GEF Ric-8A], while favoring high-affinity binding to all known GBA motifs. We demonstrate that GBAi does not interfere with canonical GPCR-G protein signaling but blocks GBA-dependent signaling in cancer cells. Furthermore, by implementing GBAi in vivo, we show that GBA-dependent signaling modulates phenotypes during Xenopus laevis embryonic development. In summary, GBAi is a selective, efficient, and convenient tool to dissect the biological processes controlled by a GPCR-independent mechanism of G protein activation mediated by cytoplasmic factors.

Glucopyranosylidene-spiro-iminothiazolidinone, a new bicyclic ring system: synthesis, derivatization, and evaluation for inhibition of glycogen phosphorylase by enzyme kinetic and crystallographic methods.

The reaction of thiourea with O-perbenzoylated C-(1-bromo-1-deoxy-ß-D-glucopyranosyl)formamide gave the new anomeric spirocycle 1R-1,5-anhydro-D-glucitol-spiro-[1,5]-2-imino-1,3-thiazolidin-4-one. Acylation and sulfonylation with the corresponding acyl chlorides (RCOCl or RSO2Cl where R=tBu, Ph, 4-Me-C6H4, 1- and 2-naphthyl) produced the corresponding 2-acylimino- and 2-sulfonylimino-thiazolidinones, respectively. Alkylation by MeI, allyl-bromide and BnBr produced mixtures of the respective N-alkylimino- and N,N'-dialkyl-imino-thiazolidinones, while reactions with 1,2-dibromoethane and 1,3-dibromopropane furnished spirocyclic 5,6-dihydro-imidazo[2,1-b]thiazolidin-3-one and 6,7-dihydro-5H-thiazolidino[3,2-a]pyrimidin-3-one, respectively. Removal of the O-benzoyl protecting groups by the Zemplén protocol led to test compounds most of which proved micromolar inhibitors of rabbit muscle glycogen phosphorylase b (RMGPb). Best inhibitors were the 2-benzoylimino- (Ki=9µM) and the 2-naphthoylimino-thiazolidinones (Ki=10 µM). Crystallographic studies of the unsubstituted spiro-thiazolidinone and the above most efficient inhibitors in complex with RMGPb confirmed the preference and inhibitory effect that aromatic (and especially 2-naphthyl) derivatives show for the catalytic site promoting the inactive conformation of the enzyme.

Developmental Dysplasia of the Hip: A Review.

Developmental dysplasia of the hip (DDH) is the most common musculoskeletal disorder of the infant age. Its incidence ranges from 0.06/1000 to 76.1/1000 live births and is more frequent in female infants. Breech position, family history and firstborn children are the main risk factors for DDH and this disorder is also associated with the presence of other congenital deformities. Anatomically, the acetabulum remains shallow and the femoral head grows in a wrong position. Clinical examination is important and tests such us Barlow and Ortolani give indications only for a part of the spectrum of this entity. Nowadays the sonographic examination is the most accurate option for the diagnosis. Graf classification categorizes the DDH cases in four types, from normal to dislocated hip, by description and measuring specific angles in sonographic examination. The wide usage of ultrasonography has decreased the non-diagnosed or neglected cases; treatment begins immediately in young age and is usually conservative with the usage of devices such as Pavlik harness and hip spica. To enhance the literature, we searched for published studies on DDH, to summarize the pathogenesis and the diagnosis and to discuss the treatment and outcome of the patients with this disorder.

The Correlation Between the Strength of the Shoulder and Trunk Muscular Systems in Elite Adolescent Water Polo Athletes.

Introduction Water polo is a competitive team sport played in the water between two teams of seven players each. Water polo players must have swimming speed, strong abdominal and back muscles, and strong shoulder muscles to cope with this sport's special conditions. In this study, we investigate the possible association of shoulder and trunk muscle systems in adolescent water polo athletes of high demands. Materials and methods The research included 42 water polo players aged 14-16, who train regularly for at least five years, six times a week, and participate in national championships and national teams. The athletes were evaluated on the strength and torque of these muscular systems using the isokinetic dynamometer Biodex System 4 Pro (Biodex Medical Systems, Inc, Shirley, NY). The correlation of the results was done using the statistical package SPSS 21. Results The correlations revealed statistically significant differences in trunk extension in combination with the shoulder external/internal rotation ratio. Also, most of the correlations occurred between the trunk and non-dominant limb of the athletes and, more often, in the female athletes. Furthermore, for the hand grip, the male athletes showed a greater difference in strength between the dominant and the non-dominant member than female athletes. Finally, the evaluation of the trunk extension/flexion ratio and external/internal rotation ratio for the shoulder joint showed that many athletes are outside the normal range and need targeted strengthening. Conclusion The negative correlation coefficient between trunk extension/flexion and shoulder external/internal rotation indicates that the trunk extension mechanism helps for better internal rotation of the shoulder. Therefore, water polo players should focus on the training of the stretching mechanism of the trunk and also give weight to achieving a balance between the competing muscular systems of the trunk and the shoulder. Thus, athletes can maximize their skills and, at the same time, protect themselves from injuries.

Platelets transfusion in Greece: Where, when, why? A national survey.

BACKGROUND: Platelet transfusion is among the most useful therapeutic tools in modern clinical settings which mean that ensuring an adequate supply is of paramount importance. AIM: The aim of our study was to record the use and wastage of platelet concentrates (PCs) in Greece, so as to come up with evidence-based interventions. METHODS: The study was conducted during May and June 2015. We evaluated the use of random-donor platelets (RDPs) and single-donor apheresis platelets (SDPs). We analyzed such parameters as hospital department and diagnosis, indication for transfusion, PCs' age at the time of transfusion, and wastage rate. RESULTS: We used data from 21 hospitals across the country. A total of 12,061 RDPs and 1189 SDPs were transfused, with an average of 4.84 (±2.72) and 1.12 (±2.73) units per episode, respectively. Most patients had been admitted to the internal medicine and hematology departments. The transfusions were mostly given prophylactically, usually in cases of acute leukemia, and mostly on the day before expiration. Wastage rate was 16.75% for RPDs and 2.70% for SDPs, primarily because of the expiration of the use-by date. CONCLUSIONS: This is the first national survey regarding platelet transfusion in Greece. Since most patients were admitted in internal medicine and hematology departments, we recommend that the staff of the abovementioned departments should undergo training on contemporary transfusion guidelines. Platelet discard rate could further be lowered through the centralization of inventory management along with the extension of the lifetime of PCs by means of emerging technologies.

The Gαi-GIV binding interface is a druggable protein-protein interaction.

Heterotrimeric G proteins are usually activated by the guanine-nucleotide exchange factor (GEF) activity of GPCRs. However, some non-receptor proteins are also GEFs. GIV (a.k.a Girdin) was the first non-receptor protein for which the GEF activity was ascribed to a well-defined protein sequence that directly binds Gαi. GIV expression promotes metastasis and disruption of its binding to Gαi blunts the pro-metastatic behavior of cancer cells. Although this suggests that inhibition of the Gαi-GIV interaction is a promising therapeutic strategy, protein-protein interactions (PPIs) are considered poorly "druggable" targets requiring case-by-case validation. Here, we set out to investigate whether Gαi-GIV is a druggable PPI. We tested a collection of >1,000 compounds on the Gαi-GIV PPI by in silico ligand screening and separately by a chemical high-throughput screening (HTS) assay. Two hits, ATA and NF023, obtained in both screens were confirmed in secondary HTS and low-throughput assays. The binding site of NF023, identified by NMR spectroscopy and biochemical assays, overlaps with the Gαi-GIV interface. Importantly, NF023 did not disrupt Gαi-Gßγ binding, indicating its specificity toward Gαi-GIV. This work establishes the Gαi-GIV PPI as a druggable target and sets the conceptual and technical framework for the discovery of novel inhibitors of this PPI.

A novel regulatory role of RGS4 in STAT5B activation, neurite outgrowth and neuronal differentiation.

The Regulator of G protein Signalling 4 (RGS4) is a multitask protein that interacts with and negatively modulates opioid receptor signalling. Previously, we showed that the δ-opioid receptor (δ-OR) forms a multiprotein signalling complex consisting of Gi/Go proteins and the Signal Transducer and Activator of Transcription 5B (STAT5B) that leads to neuronal differentiation and neurite outgrowth upon δ-ΟR activation. Here, we investigated whether RGS4 could participate in signalling pathways to regulate neurotropic events. We demonstrate that RGS4 interacts directly with STAT5B independently of δ-ΟR presence both in vitro and in living cells. This interaction involves the N-terminal portion of RGS4 and the DNA-binding SH3 domain of STAT5B. Expression of RGS4 in HEK293 cells expressing δ-OR and/or erythropoietin receptor results in inhibition of [D-Ser2, Leu5, Thr6]-enkephalin (DSLET)-and erythropoietin-dependent STAT5B phosphorylation and subsequent transcriptional activation. DSLET-dependent neurite outgrowth of neuroblastoma cells is also blocked by RGS4 expression, whereas primary cortical cultures of RGS4 knockout mice (RGS4-/-) exhibit enhanced neuronal sprouting after δ-OR activation. Additional studies in adult brain extracts from RGS4-/- mice revealed increased levels of p-STAT5B. Finally, neuronal progenitor cultures from RGS4-/- mice exhibit enhanced proliferation with concomitant increases in the mRNA levels of the anti-apoptotic STAT5B target genes bcl2 and bcl-xl. These observations suggest that RGS4 is implicated in opioid dependent neuronal differentiation and neurite outgrowth via a "non-canonical" signaling pathway regulating STAT5B-directed responses.

RGS2 and RGS4 proteins: New modulators of the κ-opioid receptor signaling.

Previous studies have shown that RGS4 associates with the C-termini of µ- and δ-opioid receptors in living cells and plays a key role in Gi/Go protein coupling selectivity and signalling of these receptors [12,20]. To deduce whether similar effects also occur for the κ-opioid receptor (κ-ΟR) and define the ability of members of the Regulators of G protein Signaling (RGS) of the B/R4 subfamily to interact with κ-ΟR subdomains we generated glutathione S-transferase fusion peptides encompassing the carboxyl-termini of κ-OR (κ-CT). Results from pull down experiments indicated that RGS2 and RGS4 directly interact within different domains of the κ-CT. Co-precipitation studies in living cells indicated that RGS2 and RGS4 associate with κ-ΟR constitutively and upon receptor activation and confer selectivity for coupling with a specific subset of G proteins. Expression of both members, RGS2 and/or RGS4, in 293F cells attenuated κ-agonist mediated-adenylyl cyclase inhibition and extracellular signal regulated kinase (ERK1,2) phosphorylation with a different amplitude in their modulatory effect in κ-ΟR signaling. Our findings demonstrate that RGS2 and RGS4 are new interacting partners that play key roles in G protein coupling to negatively regulate κ-ΟR signaling.

Selective interactions of spinophilin with the C-terminal domains of the δ- and µ-opioid receptors and G proteins differentially modulate opioid receptor signaling.

Previous studies have shown that the intracellular domains of opioid receptors serve as platforms for the formation of a multi-component signaling complex consisting of various interacting partners (Leontiadis et al., 2009, Cell Signal. 21, 1218-1228; Georganta et al., 2010, Neuropharmacology, 59(3), 139-148). In the present study we demonstrate that spinophilin a dendritic-spine enriched scaffold protein associates with δ- and µ-opioid receptors (δ-ΟR, µ-OR) constitutively in HEK293 an interaction that is altered upon agonist administration and enhanced upon forskolin treatment for both µ-OR and δ-ΟR. Spinophilin association with the opioid receptors is mediated via the third intracellular loop and a conserved region of the C-terminal tails. The portion of spinophilin responsible for interaction with the δ-OR and µ-OR is narrowed to a region encompassing amino acids 151-444. Spinophilin, RGS4, Gα and Gßγ subunits of G proteins form a multi-protein complex using specific regions of spinophilin and a conserved amino acid stretch of the C-terminal tails of both δ-µ-ORs. Expression of spinophilin in HEK293 cells potentiated DPDPE-mediated adenylyl-cyclase inhibition of δ-OR leaving unaffected the levels of cAMP accumulation mediated by the µ-OR. Moreover, measurements of extracellular signal regulated kinase (ERK1,2) phosphorylation indicated that the presence of spinophilin attenuated agonist-driven ERK1,2 phosphorylation mediated upon activation of the δ-OR but not the µ-OR. Collectively, these findings suggest that spinophilin associates with both δ- and µ-ΟR and G protein subunits in HEK293 cells participating in a multimeric signaling complex that displays a differential regulatory role in opioid receptor signaling.

Expression and membrane topology of Anopheles gambiae odorant receptors in lepidopteran insect cells.

A lepidopteran insect cell-based expression system has been employed to express three Anopheles gambiae odorant receptors (ORs), OR1 and OR2, which respond to components of human sweat, and OR7, the ortholog of Drosophila's OR83b, the heteromerization partner of all functional ORs in that system. With the aid of epitope tagging and specific antibodies, efficient expression of all ORs was demonstrated and intrinsic properties of the proteins were revealed. Moreover, analysis of the orientation of OR1 and OR2 on the cellular plasma membrane through the use of a novel 'topology screen' assay and FACS analysis demonstrates that, as was recently reported for the ORs in Drosophila melanogaster, mosquito ORs also have a topology different than their mammalian counterparts with their N-terminal ends located in the cytoplasm and their C-terminal ends facing outside the cell. These results set the stage for the production of mosquito ORs in quantities that should permit their detailed biochemical and structural characterization and the exploration of their functional properties.

Regulator of G protein signaling 4 confers selectivity to specific G proteins to modulate mu- and delta-opioid receptor signaling.

In vitro studies have shown that the Regulator of G protein Signaling 4 (RGS4) interacts with the C-termini of mu- and delta-opioid receptors (mu-OR, delta-OR) (Georgoussi et al., 2006, Cell. Signal.18, 771-782). Herein we demonstrate that RGS4 associates with these receptors in living cells and forms selective complexes with Gi/Go proteins in a receptor dependent manner. This interaction occurs within the predicted fourth intracellular loop of mu, delta-ORs as part of a signaling complex consisting of the opioid receptor, activated Galpha and RGS4. RGS4 is recruited to the plasma membrane upon opioid receptor stimulation. Expression of RGS4 in HEK293 cells attenuated agonist-mediated extracellular signal regulated kinase (ERK1,2) phosphorylation for both receptors and accelerated agonist-induced internalization of the delta-OR. RGS4 lacking its N-terminal domain failed to interact with both opioid receptors and to modulate opioid receptor signaling. Our findings demonstrate that RGS4 plays a key role in G protein coupling selectivity and signaling of the mu- and delta-OmicronRs.