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E-6375 (4-butoxy-2-[4-(2-cyanobenzoyl)-1-piperazinyl] pyrimidine hydrochloride) is a new intravenous general anaesthetic with an anaesthetic potency, in mice, comparable to propofol, or etomidate. Here, we examined the effect of E-6375 upon the GABAA receptor, a putative target of intravenous anaesthetic action. E-6375 reversibly enhanced GABA-evoked currents mediated by recombinant GABAA (α1β2γ2L) receptors expressed in Xenopus laevis oocytes, with little effect on NMDA- and kainate-evoked currents mediated by NR1a/NR2A and GluR1o/GluR2o glutamate receptors, respectively. E-6375 prolonged the decay of GABA-evoked miniature inhibitory postsynaptic currents recorded from rat Purkinje neurones demonstrating the anaesthetic also enhanced the activity of synaptic GABAA receptors. The GABA enhancing action of E-6375 on recombinant GABAA receptors was unaffected by the subtype of the α isoform (i.e. αxβ2γ2L; x=1–3) within the receptor, but was increased by the omission of the γ2L subunit. Receptors incorporating β2, or β3, subunits were more sensitive to modulation by E-6375 than those containing the β1 subunit. The selectivity of E-6375 was largely governed by the identity (serine or asparagine) of a single amino acid residue within the second transmembrane domain of the β-subunit. The various in vivo actions of general anaesthetics may be mediated by GABAA receptor isoforms that have a differential distribution within the CNS. The identification of agents, such as E-6375, that discriminate between GABAA receptor subtypes may augur the development of general anaesthetics with an improved therapeutic profile. [Copyright &y& Elsevier]

TBC1D3 is a primate-specific gene family that has expanded in the human lineage and has been implicated in neuronal progenitor proliferation and expansion of the frontal cortex. The gene family and its expression have been challenging to investigate because it is embedded in high-identity and highly variable segmental duplications. We sequenced and assembled the gene family using long-read sequencing data from 34 humans and 11 nonhuman primate species. Our analysis shows that this particular gene family has independently duplicated in at least five primate lineages, and the duplicated loci are enriched at sites of large-scale chromosomal rearrangements on Chromosome 17. We find that all human copy-number variation maps to two distinct clusters located at Chromosome 17q12 and that humans are highly structurally variable at this locus, differing by as many as 20 copies and ∼1 Mbp in length depending on haplotypes. We also show evidence of positive selection, as well as a significant change in the predicted human TBC1D3 protein sequence. Last, we find that, despite multiple duplications, human TBC1D3 expression is limited to a subset of copies and, most notably, from a single paralog group: TBC1D3-CDKL These observations may help explain why a gene potentially important in cortical development can be so variable in the human population., (© 2024 Guitart et al.; Published by Cold Spring Harbor Laboratory Press.)

Motivation: We are now in the era of being able to routinely generate highly contiguous (near telomere-to-telomere) genome assemblies of human and nonhuman species. Complex structural variation and regions of rapid evolutionary turnover are being discovered for the first time. Thus, efficient and informative visualization tools are needed to evaluate and directly observe structural differences between two or more genomes., Results: We developed SVbyEye, an open-source R package to visualize and annotate sequence-to-sequence alignments along with various functionalities to process alignments in PAF format. The tool facilitates the characterization of complex structural variants in the context of sequence homology helping resolve the mechanisms underlying their formation., Availability and Implementation: SVbyEye is available at https://github.com/daewoooo/SVbyEye., Competing Interests: Conflict of Interest: E.E.E. is a scientific advisory board (SAB) member of Variant Bio, Inc.

Introduction: In-hospital falls are multicausal in older hospitalized patients. Drugs with anticholinergic load and psychotropic effects can increase the risk of falling., Objective: This study aimed to determine the associations between fall risk-increasing drugs (FRIDs) and the anticholinergic risk score (ARS) with falls in hospitalized older hospitalized patients., Methods: This was a case‒control study of patients ≥65 years of age of either sex treated in four clinics in Colombia between 2018 and 2020. Each patient who suffered a fall during hospitalization was matched with four hospitalized patients who did not. Sociodemographic, clinical, and pharmacologic variables and the use of the ARS and FRIDs were evaluated. The risk associated with FRIDs was estimated using conditional logistic regression., Results: There were 250 patients and 1,000 controls (ratio of 1:4), with a mean age of 77.4 ± 7.4 years and a predominance of men ( n = 800, 64.0%). The majority of falls occurred during hospitalization ( n = 192 patients, 76.8%). Polypharmacy, calcium channel blockers, antiepileptics, antipsychotics, sodium-glucose cotransporter type 2 inhibitors, and nonsteroidal anti-inflammatory drugs were associated with falls during hospitalization. With an ARS score of 3, the probability of falling during the hospital stay increased (aOR: 2.34; 95% CI: 1.64-3.32)., Conclusion: There is an association between suffering a fall and the use of drugs with anticholinergic load or FRIDs in hospitalized adults more than 65 years of age in Colombia., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Machado-Duque, Camacho-Arteaga, Sabaté, Vidal-Guitart and Machado-Alba.)

Background: Drug provocation tests (DPT) are considered the gold standard procedure to ascertain the diagnosis of beta-lactam (BL) allergy. Regarding route of administration, current recommendations prioritize oral challenges, considering them safer, and reserving the intravenous route for drugs for which this is the only formulation., Objective: To compare in terms of tolerance and safety two protocols of BL DPT, using an oral protocol (OR-DPT) and an intravenous protocol (IV-DPT)., Methods: A descriptive, retrospective study was performed, including adult patients who underwent IV-DPT or OR-DPT for suspected immediate or delayed hypersensitivity to BL antibiotics, over a period of 4 years (between January 2018 and December 2021). Demographical data, index hypersensivity reactions' characteristics and tolerance to DPT were reviewed., Results: A total of 1036 patients underwent DPT, mean age of 56.8 (standard deviation, SD, 17.8) years, 655 were women (63.2%). Immediate drug hypersensitivity reactions (DHR) had occurred in 564 of patients (54.4%). OR-DPT were performed in 439 (42.4%) and IV-DPT in 597 (57.6%). The frequency of reactions during DPT, regardless of the route used, was low (3.6%): only 16 (3.6%) in OR-DPT and 21 (3.5%) in IV-DPT. From IV-DPT, 16 out 21 DHR during DPT were immediate compared with 4 out of 16 in OR-DPT. Adjusted relative risk of developing a hypersensitivity reaction during IV-DPT versus OR-DPT was 1.13 (95% confidence interval (CI)0.57-2.22)., Conclusion: The results suggest that OR-DPT and IV-DPT are both safe procedures when adequately performed. However, IV-DPT protocols showed a higher rate of immediate DHR during DPT probably due to the selection of basal high-risk patients to undergo IV-DPT. In conclusion, IV-DPT may be considered as an option for challenges in drug-allergy studies, entailing a precise administration., Competing Interests: Gustavo-Jorge Molina-Molina MD: The author report no competing interests. Manuel Gómez-Duque MD: The author report no competing interests. Xavier Vidal Guitart MD PhD: The author report no competing interests. Antònia Agustí Escasany MD PhD: The author report no competing interests. Moisés Labrador-Horrillo MD PhD: The author report no competing interests. Olga Luengo MD PhD: The author report no competing interests. Anna Sala-Cunill MD PhD: The author report no competing interests. Paula Galván Blasco MD: The author report no competing interests. Mar Guilarte MD PhD: The author report no competing interests. Victoria Cardona MD PhD: The author report no competing interests., (© 2024 The Authors.)

Single-nucleotide variants (SNVs) in segmental duplications (SDs) have not been systematically assessed because of the limitations of mapping short-read sequencing data 1,2 . Here we constructed 1:1 unambiguous alignments spanning high-identity SDs across 102 human haplotypes and compared the pattern of SNVs between unique and duplicated regions 3,4 . We find that human SNVs are elevated 60% in SDs compared to unique regions and estimate that at least 23% of this increase is due to interlocus gene conversion (IGC) with up to 4.3 megabase pairs of SD sequence converted on average per human haplotype. We develop a genome-wide map of IGC donors and acceptors, including 498 acceptor and 454 donor hotspots affecting the exons of about 800 protein-coding genes. These include 171 genes that have 'relocated' on average 1.61 megabase pairs in a subset of human haplotypes. Using a coalescent framework, we show that SD regions are slightly evolutionarily older when compared to unique sequences, probably owing to IGC. SNVs in SDs, however, show a distinct mutational spectrum: a 27.1% increase in transversions that convert cytosine to guanine or the reverse across all triplet contexts and a 7.6% reduction in the frequency of CpG-associated mutations when compared to unique DNA. We reason that these distinct mutational properties help to maintain an overall higher GC content of SD DNA compared to that of unique DNA, probably driven by GC-biased conversion between paralogous sequences 5,6 ., (© 2023. The Author(s).)

The clonal dynamics following hematopoietic stem progenitor cell (HSPC) transplantation with busulfan conditioning are of great interest to the development of HSPC gene therapies. Compared with total body irradiation (TBI), busulfan is less toxic and more clinically relevant. We used a genetic barcoded HSPC autologous transplantation model to investigate the impact of busulfan conditioning on hematopoietic reconstitution in rhesus macaques. Two animals received lower busulfan dose and demonstrated lower vector marking levels compared with the third animal given a higher busulfan dose, despite similar busulfan pharmacokinetic analysis. We observed uni-lineage clonal engraftment at 1 month post-transplant, replaced by multilineage clones by 2 to 3 months in all animals. The initial multilineage clones in the first two animals were replaced by a second multilineage wave at 9 months; this clonal pattern disappeared at 13 months in the first animal, though was maintained in the second animal. The third animal maintained stable multilineage clones from 3 months to the most recent time point. In addition, busulfan animals exhibit more rapid HSPC clonal mixing across bone marrow sites and less CD16 + NK-biased clonal expansion compared with TBI animals. Therefore, busulfan conditioning regimens can variably impact the marrow niche, resulting in differences in clonal patterns with implications for HSPC gene therapies., Competing Interests: The authors declare no competing interests.

A main rationale for the role of G protein-coupled receptor (GPCR) heteromers as targets for drug development is the putative ability of selective ligands for specific GPCRs to change their pharmacological properties upon GPCR heteromerization. The present study provides a proof of concept for this rationale by demonstrating that heteromerization of dopamine D 1 and D 3 receptors (D 1 R and D 3 R) influences the pharmacological properties of three structurally similar selective dopamine D 3 R ligands, the phenylpiperazine derivatives PG01042, PG01037 and VK4-116. By using D 1 R-D 3 R heteromer-disrupting peptides, it could be demonstrated that the three D 3 R ligands display different D 1 R-D 3 R heteromer-dependent pharmacological properties: PG01042, acting as G protein-biased agonist, counteracted D 1 R-mediated signaling in the D 1 R-D 3 R heteromer; PG01037, acting as a D 3 R antagonist cross-antagonized D 1 R-mediated signaling in the D 1 R-D 3 R heteromer; and VK4-116 specifically acted as a ß-arrestin-biased agonist in the D 1 R-D 3 R heteromer. Molecular dynamics simulations predicted potential molecular mechanisms mediating these qualitatively different pharmacological properties of the selective D 3 R ligands that are dependent on D 1 R-D 3 R heteromerization. The results of in vitro experiments were paralleled by qualitatively different pharmacological properties of the D 3 R ligands in vivo. The results supported the involvement of D 1 R-D 3 R heteromers in the locomotor activation by D 1 R agonists in reserpinized mice and L-DOPA-induced dyskinesia in rats, highlighting the D 1 R-D 3 R heteromer as a main pharmacological target for L-DOPA-induced dyskinesia in Parkinson's disease. More generally, the present study implies that when suspecting its pathogenetic role, a GPCR heteromer, and not its individual GPCR units, should be considered as main target for drug development., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2022. Published by Elsevier Ltd.)

The completion of a telomere-to-telomere human reference genome, T2T-CHM13, has resolved complex regions of the genome, including repetitive and homologous regions. Here, we present a high-resolution epigenetic study of previously unresolved sequences, representing entire acrocentric chromosome short arms, gene family expansions, and a diverse collection of repeat classes. This resource precisely maps CpG methylation (32.28 million CpGs), DNA accessibility, and short-read datasets (166,058 previously unresolved chromatin immunoprecipitation sequencing peaks) to provide evidence of activity across previously unidentified or corrected genes and reveals clinically relevant paralog-specific regulation. Probing CpG methylation across human centromeres from six diverse individuals generated an estimate of variability in kinetochore localization. This analysis provides a framework with which to investigate the most elusive regions of the human genome, granting insights into epigenetic regulation.

Despite their importance in disease and evolution, highly identical segmental duplications (SDs) are among the last regions of the human reference genome (GRCh38) to be fully sequenced. Using a complete telomere-to-telomere human genome (T2T-CHM13), we present a comprehensive view of human SD organization. SDs account for nearly one-third of the additional sequence, increasing the genome-wide estimate from 5.4 to 7.0% [218 million base pairs (Mbp)]. An analysis of 268 human genomes shows that 91% of the previously unresolved T2T-CHM13 SD sequence (68.3 Mbp) better represents human copy number variation. Comparing long-read assemblies from human ( n = 12) and nonhuman primate ( n = 5) genomes, we systematically reconstruct the evolution and structural haplotype diversity of biomedically relevant and duplicated genes. This analysis reveals patterns of structural heterozygosity and evolutionary differences in SD organization between humans and other primates.

Background and Objectives: Osteoporotic hip fracture is a relevant pathology due to its prevalence and social and health impact. The aim of this study is to explore the predictive validity of the CUPAX questionnaire on mortality, place of residence and post-fracture functionality., Materials and Methods: Prospective observational study. Two hundred and six patients older than 65 years were included, admitted after a hip fracture. The CUPAX questionnaire score was collected before fracture and one year later, and the place of residence and survival at hospital discharge, and after 6 and 12 months. The statistical analysis was carried out with the SAS® 9.4 and Stata® 13.1 programmes., Results: The median age of the sample was 87.0 years (80.1% women). The in-hospital and one-year mortality rate were 5.8% and 19.1%, respectively. Most of the patients were admitted from home (71.4%), and the most frequent discharge destination was a social health centre (48.2%). The percentage of retention of previous functional level in the total sample was 50%, being higher in the younger patients. The area under the curve ROC for mortality one year later was .697 (95% CI .626-.760) and .659 (95% CI .576-.741) for the discharge destination of patients admitted from home. Evaluation of functional retention one year after the fracture, identified three groups of patients based on the pre-fracture CUPAX value., Conclusions: These findings support the clinical utility of the CUPAX questionnaire as a predictive functional tool in elderly patients with hip fracture., (Copyright © 2021 Elsevier España, S.L.U. All rights reserved.)

Akathisia is an urgent need to move that is associated with treatment with dopamine receptor blocking agents (DRBAs) and with restless legs syndrome (RLS). The pathogenetic mechanism of akathisia has not been resolved. This article proposes that it involves an increased presynaptic dopaminergic transmission in the ventral striatum and concomitant strong activation of postsynaptic dopamine D 1 receptors, which form complexes (heteromers) with dopamine D 3 and adenosine A 1 receptors. It also proposes that in DRBA-induced akathisia, increased dopamine release depends on inactivation of autoreceptors, whereas in RLS it depends on a brain iron deficiency-induced down-regulation of striatal presynaptic A 1 receptors., Competing Interests: Disclosure The authors have nothing to disclose., (Published by Elsevier Inc.)

Aim: To assess cardiovascular (CV) events and all-cause mortality in type 2 diabetes mellitus (T2DM) patients treated with first-line monotherapies of non-insulin antidiabetic drugs (NIADs)., Methods: Longitudinal retrospective cohort study in the Catalan database SIDIAP (Information System for the Development of Research in Primary Care). T2DM patients ≥18 years newly prescribed first-line monotherapies during 2010-2015 were followed since their first prescription until the composite of major adverse CV events, MACE (myocardium infarction [MI], stroke and all-cause death), its components, heart failure (HF) and peripheral artery disease (PAD) or censoring. Cox proportional hazard models were used to estimate hazard ratios 95% confidence interval (HR [95%CI])., Results: Compared with metformin, the use of sulfonylureas, dipeptidyl peptidase-4 inhibitors (DPP-4 i) and meglitinides were significantly associated with higher risk for MACE (1.55 [1.42-1.68]); 1.49 [1.22-1.84] and 2.01 [1.29-3.12]) and all-cause mortality (1.67 [1.52-1.84], 1.65 [1.30-2.] and 2.08 [1.26-3.42]). Sulfonylureas users had increased risk of MI (1.38 [1.03-1.85]) stroke (1.31 [1.11-1.54]), HF (1.49 [1.28-1.72]) and PAD (1.24 [1.02-1.51]). Meglitinides users were at increased risks of MI, HR 2.03 (1.10-3.74)., Conclusion: In first-line monotherapies, compared with metformin, sulfonylureas were associated with increased risks in all the outcomes; DPP-4 i and repaglinide showed increased risks of MACE and mortality. Residual confounding cannot be ruled out., (Copyright © 2020 Primary Care Diabetes Europe. Published by Elsevier Ltd. All rights reserved.)

Background: Cardiovascular outcomes trials (CVOTs) have assessed the effects of glucagon-like peptide-1 receptor agonists (GLP-1 RAs) on major adverse cardiovascular events (MACE) and mortality in high cardiovascular (CV) risk populations. Observational research can provide complementary evidence about these effects in unselected populations., Aim: To systematically review retrospective observational cohort studies conducted in electronic healthcare databases (EHDs) assessing GLP-1 RAs´ effects on MACE and/or hospitalisation for heart failure (HHF) and/or all-cause mortality in Type 2 diabetes mellitus (T2DM) patients., Methods: We systematically searched studies meeting inclusion criteria, compared design, methods and population characteristics, assessed risk for bias and did a meta-analysis (MA) using a random-effects model to calculate overall hazard ratios (HRs) and 95% CI (confidence intervals)., Results: Sixteen studies included 285,436 T2DM patients exposed to GLP-1 RAs (exenatide bid, liraglutide, lixisenatide, long-acting exenatide), n ranged from 219 to 160,803 patients. Comparators included: no exposure, other antidiabetic medications (OADs), combined OADs, canagliflozin or multiple comparators. Ten studies estimated all-cause mortality, hazard ratios (HRs) ranged from 0.17 (95% CI 0.02-1.22) to 1.29 (95% CI 0.54-3.13). Thirteen studies assessed cardiovascular events and/or MACE; HRs ranged from 0.27 (95% CI 0.14-0.53) to 1.11 (95% CI 0.99-1.24). Eight studies assessed HHF, HRs ranged from 0.12 (95% CI 0.02-0.66) to 1.64 (95% CI 1.28-2.13). Excluding two studies because of temporal bias, we obtained pooled estimates for all-cause mortality: HR 0.63 (0.44-0.89), CV outcomes HR 0.84 (0.75-0.94) and HHF; HR 0.94 (0.78-1.14), (high between-study variability: I 2  = 83.35%; I 2  = 70.3%; and I 2  = 90.1%, respectively)., Conclusion: Pooled results of EHDs' studies assessing GLP-1 RAs effects favoured GLP-1 RAs for all-cause mortality and MACE while were neutral for HHF. Results should be interpreted cautiously because of studies' substantial heterogeneity and limitations of observational research., (© 2020 John Wiley & Sons Ltd.)

Background and Aim: Hip fractures in elderly patients are very frequent and are associated with high morbidity and mortality. We do not have validated instruments in Spanish that can faithfully assess functional capacity prior to fracture. The aim of this study was to develop and validate the CUPAX questionnaire in elderly patients with a hip fracture., Materials and Methods: Prospective and observational validation study. We included 215 patients older than 65 years, who were admitted to our centre after suffering a hip fracture. They were evaluated using the CUPAX questionnaire, Barthel Index and Parker Score. The statistical study was performed to corroborate the validity and reliability of the questionnaire., Results: The median age of the patient population was 84.0 years (75.3% women). The majority were patients who had suffered an extracapsular fracture; they had walking capacity and suffered a fall in their place of residence. The statistical analysis on the validity and reliability of the questionnaire obtained the following results: Cronbach's alpha coefficient showed excellent internal consistency (value of .94). Factor analysis showed 3 underlying factors. The interobserver intraclass correlation coefficient (ICC) was .82 (95% CI:.75-.87), and the intraobserver ICC was .96 (95% CI:.95-.97). Correlation with other functional scales was assessed using the Spearman correlation coefficient, which was .83 with the Barthel Index and .81 with the Parker score., Conclusions: These findings support the validity of the CUPAX questionnaire as a tool to measure the previous functional level in elderly patients affected by a hip fracture., (Copyright © 2019 Elsevier España, S.L.U. All rights reserved.)

Several studies found in vitro evidence for heteromerization of dopamine D 1 receptors (D1R) and D 3 receptors (D3R), and it has been postulated that functional D1R-D3R heteromers that are normally present in the ventral striatum mediate synergistic locomotor-activating effects of D1R and D3R agonists in rodents. Based also on results obtained in vitro, with mammalian transfected cells, it has been hypothesized that those behavioral effects depend on a D1R-D3R heteromer-mediated G protein-independent signaling. Here, we demonstrate the presence on D1R-D3R heteromers in the mouse ventral striatum by using a synthetic peptide that selectively destabilizes D1R-D3R heteromers. Parallel locomotor activity and ex vivo experiments in reserpinized mice and in vitro experiments in D1R-D3R mammalian transfected cells were performed to dissect the signaling mechanisms of D1R-D3R heteromers. Co-administration of D1R and D3R agonists in reserpinized mice produced synergistic locomotor activation and a selective synergistic AKT phosphorylation in the most ventromedial region of the striatum in the shell of the nucleus accumbens. Application of the destabilizing peptide in transfected cells and in the shell of the nucleus accumbens allowed demonstrating that both in vitro and in vivo co-activation of D3R induces a switch from G protein-dependent to G protein-independent D1R-mediated signaling determined by D1R-D3R heteromerization. The results therefore demonstrate that a biased G protein-independent signaling of D1R-D3R heteromers localized in the shell of the nucleus accumbens mediate the locomotor synergistic effects of D1R and D3R agonists in reserpinized mice.

Identifying non-addictive opioid medications is a high priority in medical sciences, but μ-opioid receptors mediate both the analgesic and addictive effects of opioids. We found a significant pharmacodynamic difference between morphine and methadone that is determined entirely by heteromerization of μ-opioid receptors with galanin Gal1 receptors, rendering a profound decrease in the potency of methadone. This was explained by methadone's weaker proficiency to activate the dopaminergic system as compared to morphine and predicted a dissociation of therapeutic versus euphoric effects of methadone, which was corroborated by a significantly lower incidence of self-report of "high" in methadone-maintained patients. These results suggest that μ-opioid-Gal1 receptor heteromers mediate the dopaminergic effects of opioids that may lead to a lower addictive liability of opioids with selective low potency for the μ-opioid-Gal1 receptor heteromer, exemplified by methadone.

Our working hypothesis is that a hypoadenosinergic state is a main pathogenetic factor that determines the sensory-motor symptoms and hyperarousal of restless legs syndrome (RLS). We have recently demonstrated that brain iron deficiency (BID) in rodents, a well-accepted animal model of RLS, is associated with a generalized downregulation of adenosine A 1 receptors (A1R) in the brain and with hypersensitivity of corticostriatal glutamatergic terminals. Here, we first review the experimental evidence for a pivotal role of adenosine and A1R in the control of striatal glutamatergic transmission and the rationale for targeting putative downregulated striatal A1R in RLS patients, which is supported by recent clinical results obtained with dipyridamole, an inhibitor of the nucleoside transporters ENT1 and ENT2. Second, we perform optogenetic-microdialysis experiments in rats to demonstrate that A1R determine the sensitivity of corticostriatal glutamatergic terminals and the ability of dipyridamole to counteract optogenetically-induced corticostriatal glutamate release in both animals with BID and controls. Thus, a frequency of optogenetic stimulation that was ineffective at inducing cortico-striatal glutamate release in control rats became effective with the local perfusion of a selective A1R antagonist. Furthermore, in animals with and without BID, the striatal application of dipyridamole blocked the optogenetic-induced glutamate release and decreased basal levels of glutamate, which was counteracted by the A1R antagonist. The results support the clinical application of ENT1 inhibitors in RLS., (2019 Published by Elsevier Inc.)

GATA2 deficiency is an inherited or sporadic genetic disorder characterized by distinct cellular deficiency, bone marrow failure, various infections, lymphedema, pulmonary alveolar proteinosis, and predisposition to myeloid malignancies resulting from heterozygous loss-of-function mutations in the GATA2 gene. How heterozygous GATA2 mutations affect human hematopoietic development or cause characteristic cellular deficiency and eventual hypoplastic myelodysplastic syndrome or leukemia is not fully understood. We used induced pluripotent stem cells (iPSCs) to study hematopoietic development in the setting of GATA2 deficiency. We performed hematopoietic differentiation using iPSC derived from patients with GATA2 deficiency and examined their ability to commit to mesoderm, hemogenic endothelial precursors (HEPs), hematopoietic stem progenitor cells, and natural killer (NK) cells. Patient-derived iPSC, either derived from fibroblasts/marrow stromal cells or peripheral blood mononuclear cells, did not show significant defects in committing to mesoderm, HEP, hematopoietic stem progenitor, or NK cells. However, HEP derived from GATA2 -mutant iPSC showed impaired maturation toward hematopoietic lineages. Hematopoietic differentiation was nearly abolished from homozygous GATA2 knockout (KO) iPSC lines and markedly reduced in heterozygous KO lines compared with isogenic controls. On the other hand, correction of the mutated GATA2 allele in patient-specific iPSC did not alter hematopoietic development consistently in our model. GATA2 deficiency usually manifests within the first decade of life. Newborn and infant hematopoiesis appears to be grossly intact; therefore, our iPSC model indeed may resemble the disease phenotype, suggesting that other genetic, epigenetic, or environmental factors may contribute to bone marrow failure in these patients following birth. However, heterogeneity of PSC-based models and limitations of in vitro differentiation protocol may limit the possibility to detect subtle cellular phenotypes.

Objectives: Recent animal models of restless legs syndrome (RLS) suggest that brain iron deficiency is associated with a hypoadenosinergic state, with downregulation of adenosine A 1 receptors (A1R) in the striatum and cortex. We hypothesized that an increase in extracellular adenosine induced by inhibitors of adenosine transporters, such as the non-selective ENT1/ENT2 inhibitor dipyridamole, would result in an improvement in RLS symptoms., Methods: In a prospective two-month open-label, non-placebo controlled clinical trial, 15 untreated idiopathic RLS patients began treatment with 100 mg dipyridamole (with uptitration to 400 mg if necessary). Multiple Suggested Immobilization Tests and polysomnography were performed at baseline and at eight weeks. Severity was assessed at four and eight weeks using the IRLS, and the CGI scales. The primary endpoint was therapeutic response (50% improvement in IRLS total score)., Results: Thirteen patients completed the study. IRLS score improved from a mean (±S.D.) of 23.4 ± 4.6 at baseline to 10.7 ± 4.5 at eight weeks. Six out of 13 patients were full responders and four were partial responders. The mean (±S.D.) effective dose of dipyridamole at eight weeks was 281.8 ± 57.5 mg/day. Sleep variables also improved, and the mean (±S.D.) periodic leg movement index decreased from 26.7 ± 7.2 to 4.3 ± 1.9. Dipyridamole was generally well tolerated. Main side effects were abdominal cramps, diarrhea, dizziness, and flushing., Conclusions: These preliminary results suggest that dipyridamole has significant therapeutic effects on both sensory and motor symptoms, as well as sleep. In addition, it provides evidence that hypoadenosinergic mechanisms play a central role in RLS., Classification of Evidence: The study provides class III evidence supporting the therapeutic effects of dipyridamole in RLS., (Copyright © 2018. Published by Elsevier B.V.)

The symptomatology of Restless Legs Syndrome (RLS) includes periodic leg movements during sleep (PLMS), dysesthesias, and hyperarousal. Alterations in the dopaminergic system, a presynaptic hyperdopaminergic state, seem to be involved in PLMS, while alterations in glutamatergic neurotransmission, a presynaptic hyperglutamatergic state, seem to be involved in hyperarousal and also PLMS. Brain iron deficiency (BID) is well-recognized as a main initial pathophysiological mechanism of RLS. BID in rodents have provided a pathogenetic model of RLS that recapitulates the biochemical alterations of the dopaminergic system of RLS, although without PLMS-like motor abnormalities. On the other hand, BID in rodents reproduces the circadian sleep architecture of RLS, indicating the model could provide clues for the hyperglutamatergic state in RLS. We recently showed that BID in rodents is associated with changes in adenosinergic transmission, with downregulation of adenosine A 1 receptors (A1R) as the most sensitive biochemical finding. It was hypothesized that A1R downregulation leads to hypersensitive striatal glutamatergic terminals and facilitation of striatal dopamine release. Hypersensitivity of striatal glutamatergic terminals was demonstrated by an optogenetic-microdialysis approach in the rodent with BID, indicating that it could represent a main pathogenetic factor that leads to PLMS in RLS. In fact, the dopaminergic agonists pramipexole and ropinirole and the α 2 δ ligand gabapentin, used in the initial symptomatic treatment of RLS, completely counteracted optogenetically-induced glutamate release from both normal and BID-induced hypersensitive corticostriatal glutamatergic terminals. It is a main tenet of this essay that, in RLS, a single alteration in the adenosinergic system, downregulation of A1R, disrupts the adenosine-dopamine-glutamate balance uniquely controlled by adenosine and dopamine receptor heteromers in the striatum and also the A1R-mediated inhibitory control of glutamatergic neurotransmission in the cortex and other non-striatal brain areas, which altogether determine both PLMS and hyperarousal. Since A1R agonists would be associated with severe cardiovascular effects, it was hypothesized that inhibitors of nucleoside equilibrative transporters, such as dipyridamole, by increasing the tonic A1R activation mediated by endogenous adenosine, could represent a new alternative therapeutic strategy for RLS. In fact, preliminary clinical data indicate that dipyridamole can significantly improve the symptomatology of RLS.

Objective: The first aim was to demonstrate a previously hypothesized increased sensitivity of corticostriatal glutamatergic terminals in the rodent with brain iron deficiency (BID), a pathogenetic model of restless legs syndrome (RLS). The second aim was to determine whether these putative hypersensitive terminals could constitute a significant target for drugs effective in RLS, including dopamine agonists (pramipexole and ropinirole) and α 2 δ ligands (gabapentin)., Methods: A recently introduced in vivo optogenetic-microdialysis approach was used, which allows the measurement of the extracellular concentration of glutamate upon local light-induced stimulation of corticostriatal glutamatergic terminals. The method also allows analysis of the effect of local perfusion of compounds within the same area being sampled for glutamate., Results: BID rats showed hypersensitivity of corticostriatal glutamatergic terminals (lower frequency of optogenetic stimulation to induce glutamate release). Both hypersensitive and control glutamatergic terminals were significant targets for locally perfused pramipexole, ropinirole, and gabapentin, which significantly counteracted optogenetically induced glutamate release. The use of selective antagonists demonstrated the involvement of dopamine D 4 and D 2 receptor subtypes in the effects of pramipexole., Interpretation: Hypersensitivity of corticostriatal glutamatergic terminals can constitute a main pathogenetic mechanism of RLS symptoms. Selective D 4 receptor agonists, by specifically targeting these terminals, should provide a new efficient treatment with fewer secondary effects. Ann Neurol 2017;82:951-960., (Published 2017. This article is a US Government work and is in the public domain in the USA.)

The initial goal of this study was to investigate alterations in adenosine A 2A receptor (A 2A R) density or function in a rat model of Huntington disease (HD) with reported insensitivity to an A 2A R antagonist. Unsuspected negative results led to the hypothesis of a low striatal adenosine tone and to the search for the mechanisms involved. Extracellular striatal concentrations of adenosine were measured with in vivo microdialysis in two rodent models of early neuropathological stages of HD disease, the Tg51 rat and the zQ175 knock-in mouse. In view of the crucial role of the equilibrative nucleoside transporter (ENT1) in determining extracellular content of adenosine, the binding properties of the ENT1 inhibitor [ 3 H]-S-(4-Nitrobenzyl)-6-thioinosine were evaluated in zQ175 mice and the differential expression and differential coexpression patterns of the ENT1 gene (SLC29A1) were analyzed in a large human cohort of HD disease and controls. Extracellular striatal levels of adenosine were significantly lower in both animal models as compared with control littermates and striatal ENT1 binding sites were significantly upregulated in zQ175 mice. ENT1 transcript was significantly upregulated in HD disease patients at an early neuropathological severity stage, but not those with a higher severity stage, relative to non-demented controls. ENT1 transcript was differentially coexpressed (gained correlations) with several other genes in HD disease subjects compared to the control group. The present study demonstrates that ENT1 and adenosine constitute biomarkers of the initial stages of neurodegeneration in HD disease and also predicts that ENT1 could constitute a new therapeutic target to delay the progression of the disease., Competing Interests: The authors declare no conflict of interest., (Published by Elsevier Inc.)