The BMP antagonist gremlin 1 contributes to the development of cortical excitatory neurons, motor balance and fear responses.

Bone morphogenetic protein (BMP) signaling is required for early forebrain development and cortical formation. How the endogenous modulators of BMP signaling regulate the structural and functional maturation of the developing brain remains unclear. Here, we show that expression of the BMP antagonist Grem1 marks committed layer V and VI glutamatergic neurons in the embryonic mouse brain. Lineage tracing of Grem1-expressing cells in the embryonic brain was examined by administration of tamoxifen to pregnant Grem1creERT; Rosa26LSLTdtomato mice at 13.5 days post coitum (dpc), followed by collection of embryos later in gestation. In addition, at 14.5 dpc, bulk mRNA-seq analysis of differentially expressed transcripts between FACS-sorted Grem1-positive and -negative cells was performed. We also generated Emx1-cre-mediated Grem1 conditional knockout mice (Emx1-Cre;Grem1flox/flox) in which the Grem1 gene was deleted specifically in the dorsal telencephalon. Grem1Emx1cKO animals had reduced cortical thickness, especially layers V and VI, and impaired motor balance and fear sensitivity compared with littermate controls. This study has revealed new roles for Grem1 in the structural and functional maturation of the developing cortex.

The spectrum of cochlear malformations in CHARGE syndrome and insights into the role of the CHD7 gene during embryogenesis of the inner ear.

PURPOSE: We reviewed the genotypes and the imaging appearances of cochleae in CHARGE patients from two large tertiary centres and analysed the observed cochlear anomalies, providing detailed anatomical description and a grading system. The goal was to gain insight into the spectrum of cochlear anomalies in CHARGE syndrome, and thus, in the role of the CHD7 gene in otic vesicle development. METHODS: We retrospectively reviewed CT and/or MR imaging of CHARGE patients referred to our institutions between 2005 and 2022. Cochlear morphology was analysed and, when abnormal, divided into 3 groups in order of progressive severity. Other radiological findings in the temporal bone were also recorded. Comparison with the existing classification system of cochlear malformation was also attempted. RESULTS: Cochlear morphology in our CHARGE cohort ranged from normal to extreme hypoplasia. The most common phenotype was cochlear hypoplasia in which the basal turn was relatively preserved, and the upper turns were underdeveloped. All patients in the cohort had absent or markedly hypoplastic semicircular canals and small, misshapen vestibules. Aside from a stenotic cochlear aperture (fossette) being associated with a hypoplastic or absent cochlear nerve, there was no consistent relationship between cochlear nerve status (normal, hypoplasia, or aplasia) and cochlear morphology. CONCLUSION: Cochlear morphology in CHARGE syndrome is variable. Whenever the cochlea was abnormal, it was almost invariably hypoplastic. This may shed light on the role of CHD7 in cochlear development. Accurate morphological description of the cochlea contributes to proper clinical diagnosis and is important for planning surgical treatment options.

The Reliability and Validity of a Portable Three-Dimensional Scanning System to Measure Leg Volume.

(1) Background: The study examined the reliability (test-retest, intra- and inter-day) and validity of a portable 3D scanning method when quantifying human leg volume. (2) Methods: Fifteen males volunteered to participate (age, 24.6 ± 2.0 years; stature, 178.9 ± 4.5 cm; body mass, 77.4 ± 6.5 kg; mean ± standard deviation). The volume of the lower and upper legs was examined using a water displacement method (the criterion) and two consecutive 3D scans. Measurements were taken at baseline, 1 h post-baseline (intra-day) and 24 h post-baseline (inter-day). Reliability and validity of the 3D scanning method was assessed using Bland-Altman limits of agreement and Pearson's product moment correlations. (3) Results: With respect to the test-retest reliability, the 3D scanning method had smaller systematic bias and narrower limits of agreement (±1%, and 3-5%, respectively) compared to the water displacement method (1-2% and 4-7%, respectively), when measuring lower and upper leg volume in humans. The correlation coefficients for all reliability comparisons (test-retest, intra-day, inter-day) would all be regarded as 'very strong' (all 0.94 or greater). (4) Conclusions: The study's results suggest that a 3D scanning method is a reliable and valid method to quantify leg volume.

Cardioprotection of Immature Heart by Simultaneous Activation of PKA and Epac: A Role for the Mitochondrial Permeability Transition Pore.

Metabolic and ionic changes during ischaemia predispose the heart to the damaging effects of reperfusion. Such changes and the resulting injury differ between immature and adult hearts. Therefore, cardioprotective strategies for adults must be tested in immature hearts. We have recently shown that the simultaneous activation of protein kinase A (PKA) and exchange protein activated by cAMP (Epac) confers marked cardioprotection in adult hearts. The aim of this study is to investigate the efficacy of this intervention in immature hearts and determine whether the mitochondrial permeability transition pore (MPTP) is involved. Isolated perfused Langendorff hearts from both adult and immature rats were exposed to global ischaemia and reperfusion injury (I/R) following control perfusion or perfusion after an equilibration period with activators of PKA and/or Epac. Functional outcome and reperfusion injury were measured and in parallel, mitochondria were isolated following 5 min of reperfusion to determine whether cardioprotective interventions involved changes in MPTP opening behaviour. Perfusion for 5 min preceding ischaemia of injury-matched adult and immature hearts with 5 µM 8-Br (8-Br-cAMP-AM), an activator of both PKA and Epac, led to significant reduction in post-reperfusion CK release and infarct size. Perfusion with this agent also led to a reduction in MPTP opening propensity in both adult and immature hearts. These data show that immature hearts are innately more resistant to I/R injury than adults, and that this is due to a reduced tendency of MPTP opening following reperfusion. Furthermore, simultaneous stimulation of PKA and Epac causes cardioprotection, which is additive to the innate resistance.

Inhibition of Cardiac RIP3 Mitigates Early Reperfusion Injury and Calcium-Induced Mitochondrial Swelling without Altering Necroptotic Signalling.

Receptor-interacting protein kinase 3 (RIP3) is a convergence point of multiple signalling pathways, including necroptosis, inflammation and oxidative stress; however, it is completely unknown whether it underlies acute myocardial ischemia/reperfusion (I/R) injury. Langendorff-perfused rat hearts subjected to 30 min ischemia followed by 10 min reperfusion exhibited compromised cardiac function which was not abrogated by pharmacological intervention of RIP3 inhibition. An immunoblotting analysis revealed that the detrimental effects of I/R were unlikely mediated by necroptotic cell death, since neither the canonical RIP3-MLKL pathway (mixed lineage kinase-like pseudokinase) nor the proposed non-canonical molecular axes involving CaMKIIδ-mPTP (calcium/calmodulin-dependent protein kinase IIδ-mitochondrial permeability transition pore), PGAM5-Drp1 (phosphoglycerate mutase 5-dynamin-related protein 1) and JNK-BNIP3 (c-Jun N-terminal kinase-BCL2-interacting protein 3) were activated. Similarly, we found no evidence of the involvement of NLRP3 inflammasome signalling (NOD-, LRR- and pyrin domain-containing protein 3) in such injury. RIP3 inhibition prevented the plasma membrane rupture and delayed mPTP opening which was associated with the modulation of xanthin oxidase (XO) and manganese superoxide dismutase (MnSOD). Taken together, this is the first study indicating that RIP3 regulates early reperfusion injury via oxidative stress- and mitochondrial activity-related effects, rather than cell loss due to necroptosis.

The Calculation, Thresholds and Reporting of Inter-Limb Strength Asymmetry: A Systematic Review.

The prevalence of inter-limb strength differences is well documented in the literature however, there are inconsistencies related to measurement and reporting, and the normative values and effects associated with inter-limb asymmetry. Therefore, the aims of this systematic review were to: 1) assess the appropriateness of existing indices for the calculation of asymmetry, 2) interrogate the evidence basis for literature reported thresholds used to define asymmetry and 3) summarise normative levels of inter-limb strength asymmetry and their effects on injury and performance. To conduct this systematic review, scientific databases (PubMed, Scopus, SPORTDiscus and Web of Science) were searched and a total of 3,594 articles were retrieved and assessed for eligibility and article quality. The robustness of each identified asymmetry index was assessed, and the evidence-basis of the identified asymmetry thresholds was appraised retrospectively using the references provided. Fifty-three articles were included in this review. Only four of the twelve identified indices were unaffected by the limitations associated with selecting a reference limb. Eighteen articles applied a threshold to original research to identify "abnormal" asymmetry, fifteen of which utilised a threshold between 10-15%, yet this threshold was not always supported by appropriate evidence. Asymmetry scores ranged between and within populations from approximate symmetry to asymmetries larger than 15%. When reporting the effects of strength asymmetries, increased injury risk and detriments to performance were often associated with larger asymmetry, however the evidence was inconsistent. Limitations of asymmetry indices should be recognised, particularly those that require selection of a reference limb. Failure to reference the origin of the evidence for an asymmetry threshold reinforces doubt over the use of arbitrary thresholds, such as 10-15%. Therefore, an individual approach to defining asymmetry may be necessary to refine robust calculation methods and to establish appropriate thresholds across various samples and methodologies that enable appropriate conclusions to be drawn.

Muscle-Tendon Unit Parameter Estimation of a Hill-Type Musculoskeletal Model Based on Experimentally Obtained Subject-Specific Torque Profiles.

The aim of this study was to generate a subject-specific musculoskeletal muscle model, based on isometric and isovelocity measurements of the whole lower extremity. A two-step optimization procedure is presented for optimizing the muscle-tendon parameters (MTPs) for isometric and isovelocity joint torque profiles. A significant improvement in the prediction of joint torque profiles for both the solely isometric and a combined isometric and dynamic method of optimization when compared to the standard scaling method of the AnyBody Modeling System (AMS) was observed. Depending on the specific purpose of the model, it may be worth considering whether the isometric-only would be sufficient, or the additional dynamic data are required for the combined approach.

Size-Dependent Ability of Liposomes to Accumulate in the Ischemic Myocardium and Protect the Heart.

Liposomes have the potential to be used for drug delivery. Meanwhile, liposome size may affect their accumulation in the target tissue. We investigated the myocardial accumulation of 2 populations of liposomes (∼70 and 110 nm diameter) during ischemia and their effect on ischemia/reperfusion injury. Isolated rat hearts were subjected to 30 minutes of low-flow ischemia with the liposomes, followed by 30 minutes of liposome-free reperfusion. The liposomes were loaded with the fluorescent dye Nile Red to assess their accumulation in the myocardium. The cardiac functional recovery during reperfusion was evaluated using force-velocity characteristics and coronary flow (CF). Reperfusion injury was evaluated by lactate dehydrogenase release. In addition, CF and contractility were assessed in hearts perfused normally with 70 nm liposomes. There was a 6- and 4-fold greater accumulation of the small liposomes in the myocardium and mitochondria, respectively, compared with the large liposomes. Importantly, even without any incorporated drugs, both populations of liposomes improved functional recovery and reduced lactate dehydrogenase release. However, the smaller liposomes showed significantly higher protective and vasodilatory effects during reperfusion than the larger particles. These liposomes also increased CF and contractility during normal perfusion. We suggest that the protective properties of the liposomes could be related to their membrane-stabilizing effect.

Consecutive Isoproterenol and Adenosine Treatment Confers Marked Protection against Reperfusion Injury in Adult but Not in Immature Heart: A Role for Glycogen.

Consecutive treatment of adult rat heart with isoproterenol and adenosine (Iso/Aden), known to consecutively activate PKA/PKC signaling, is cardioprotective against ischemia and reperfusion (I/R). Whether this is cardioprotective in an immature heart is unknown. Langendorff-perfused hearts from adult and immature (60 and 14 days old) male Wistar rats were exposed to 30 min ischemia and 120 min reperfusion, with or without prior perfusion with 5 nM Iso for 3 min followed by 30 µM Aden for 5 min. Changes in hemodynamics (developed pressure and coronary flow) and cardiac injury (Lactate Dehydrogenase (LDH) release and infarct size) were measured. Additional hearts were used to measure glycogen content. Iso induced a similar inotropic response in both age groups. Treatment with Iso/Aden resulted in a significant reduction in time to the onset of ischemic contracture in both age groups whilst time to peak contracture was significantly shorter only in immature hearts. Upon reperfusion, the intervention reduced cardiac injury and functional impairment in adults with no protection of immature heart. Immature hearts have significantly less glycogen content compared to adult. This work shows that Iso/Aden perfusion confers protection in an adult heart but not in an immature heart. It is likely that metabolic differences including glycogen content contribute to this difference.

Inhibition of AAK1 Kinase as a Novel Therapeutic Approach to Treat Neuropathic Pain.

To identify novel targets for neuropathic pain, 3097 mouse knockout lines were tested in acute and persistent pain behavior assays. One of the lines from this screen, which contained a null allele of the adapter protein-2 associated kinase 1 (AAK1) gene, had a normal response in acute pain assays (hot plate, phase I formalin), but a markedly reduced response to persistent pain in phase II formalin. AAK1 knockout mice also failed to develop tactile allodynia following the Chung procedure of spinal nerve ligation (SNL). Based on these findings, potent, small-molecule inhibitors of AAK1 were identified. Studies in mice showed that one such inhibitor, LP-935509, caused a reduced pain response in phase II formalin and reversed fully established pain behavior following the SNL procedure. Further studies showed that the inhibitor also reduced evoked pain responses in the rat chronic constriction injury (CCI) model and the rat streptozotocin model of diabetic peripheral neuropathy. Using a nonbrain-penetrant AAK1 inhibitor and local administration of an AAK1 inhibitor, the relevant pool of AAK1 for antineuropathic action was found to be in the spinal cord. Consistent with these results, AAK1 inhibitors dose-dependently reduced the increased spontaneous neural activity in the spinal cord caused by CCI and blocked the development of windup induced by repeated electrical stimulation of the paw. The mechanism of AAK1 antinociception was further investigated with inhibitors of α2 adrenergic and opioid receptors. These studies showed that α2 adrenergic receptor inhibitors, but not opioid receptor inhibitors, not only prevented AAK1 inhibitor antineuropathic action in behavioral assays, but also blocked the AAK1 inhibitor-induced reduction in spinal neural activity in the rat CCI model. Hence, AAK1 inhibitors are a novel therapeutic approach to neuropathic pain with activity in animal models that is mechanistically linked (behaviorally and electrophysiologically) to α2 adrenergic signaling, a pathway known to be antinociceptive in humans.

Structure activity relationship studies on chemically non-reactive glycine sulfonamide inhibitors of diacylglycerol lipase.

N-Benzylic-substituted glycine sulfonamides that reversibly inhibit diacylglycerol (DAG) lipases are reported. Detailed herein are the structure activity relationships, profiling characteristics and physico-chemical properties for the first reported series of DAG lipase (DAGL) inhibitors that function without covalent attachment to the enzyme. Highly potent examples are presented that represent valuable tool compounds for studying DAGL inhibition and constitute important leads for future medicinal chemistry efforts.

Movement velocity as a measure of exercise intensity in three lower limb exercises.

The purpose of this study was to investigate the relationship between movement velocity and relative load in three lower limbs exercises commonly used to develop strength: leg press, full squat and half squat. The percentage of one repetition maximum (%1RM) has typically been used as the main parameter to control resistance training; however, more recent research has proposed movement velocity as an alternative. Fifteen participants performed a load progression with a range of loads until they reached their 1RM. Maximum instantaneous velocity (Vmax) and mean propulsive velocity (MPV) of the knee extension phase of each exercise were assessed. For all exercises, a strong relationship between Vmax and the %1RM was found: leg press (r(2)adj = 0.96; 95% CI for slope is [-0.0244, -0.0258], P < 0.0001), full squat (r(2)adj = 0.94; 95% CI for slope is [-0.0144, -0.0139], P < 0.0001) and half squat (r(2)adj = 0.97; 95% CI for slope is [-0.0135, -0.00143], P < 0.0001); for MPV, leg press (r(2)adj = 0.96; 95% CI for slope is [-0.0169, -0.0175], P < 0.0001, full squat (r(2)adj = 0.95; 95% CI for slope is [-0.0136, -0.0128], P < 0.0001) and half squat (r(2)adj = 0.96; 95% CI for slope is [-0.0116, 0.0124], P < 0.0001). The 1RM was attained with a MPV and Vmax of 0.21 ± 0.06 m s(-1) and 0.63 ± 0.15 m s(-1), 0.29 ± 0.05 m s(-1) and 0.89 ± 0.17 m s(-1), 0.33 ± 0.05 m s(-1) and 0.95 ± 0.13 m s(-1) for leg press, full squat and half squat, respectively. Results indicate that it is possible to determine an exercise-specific %1RM by measuring movement velocity for that exercise.

Discovery of D1 Dopamine Receptor Positive Allosteric Modulators: Characterization of Pharmacology and Identification of Residues that Regulate Species Selectivity.

The present studies represent the first published report of a dopamine D1 positive allosteric modulator (PAM). D1 receptors have been proposed as a therapeutic target for the treatment of cognitive deficits associated with schizophrenia. However, the clinical utility of orthosteric agonist compounds is limited by cardiovascular side effects, poor pharmacokinetics, lack of D1 selectivity, and an inverted dose response. A number of these challenges may be overcome by utilization of a selective D1 PAM. The current studies describe two chemically distinct D1 PAMs: Compound A [1-((rel-1S,3R,6R)-6-(benzo[d][1,3]dioxol-5-yl)bicyclo[4.1.0]heptan-3-yl)-4-(2-bromo-5-chlorobenzyl)piperazine] and Compound B [rel-(9R,10R,12S)-N-(2,6-dichloro-3-methylphenyl)-12-methyl-9,10-dihydro-9,10-ethanoanthracene-12-carboxamide]. Compound A shows pure PAM activity, with an EC50 of 230 nM and agonist activity at the D2 receptor in D2-expressing human embryonic kidney cells. Compound B shows superior potency (EC50 of 43 nM) and selectivity for D1 versus D2 dopamine receptors. Unlike Compound A, Compound B is selective for human and nonhuman primate D1 receptors, but lacks activity at the rodent (rat and mouse) D1 receptors. Using molecular biology techniques, a single amino acid was identified at position 130, which mediates the species selectivity of Compound B. These data represent the first described D1-selective PAMs and define critical amino acids that regulate species selectivity.

The discovery of potent agonists for GPR88, an orphan GPCR, for the potential treatment of CNS disorders.

Modulating GPR88 activity is suggested to have therapeutic utility in the treatment of CNS disorders, such as schizophrenia. This Letter will describe the discovery and SAR development of a class of potent GPR88 agonists.

Design, synthesis, and evaluation of phenylglycinols and phenyl amines as agonists of GPR88.

Small molecule modulators of GPR88 activity (agonists, antagonists, or modulators) are of interest as potential agents for the treatment of a variety of psychiatric disorders including schizophrenia. A series of phenylglycinol and phenylamine analogs have been prepared and evaluated for their GPR88 agonist activity and pharmacokinetic (PK) properties.

The Role of the Neuroprotective Factor Npas4 in Cerebral Ischemia.

Stroke is one of the leading causes of death and adult disability in the world. Although many molecules have been documented to have a neuroprotective effect, the majority of these molecules failed to improve the neurological outcomes for patients with brain ischemia. It has been proposed that neuroprotection alone may, in fact, not be adequate for improving the prognosis of ischemic stroke. Neuroprotectants that can regulate other processes which occur in the brain during ischemia could potentially be targets for the development of effective therapeutic interventions in stroke. Neuronal Per-Arnt-Sim domain protein 4 (Npas4) is an activity-dependent transcription factor whose expression is induced in various brain insults, including cerebral ischemia. It has been shown that Npas4 plays an important role in protecting neurons against many types of neurodegenerative insult. Recently, it was demonstrated that Npas4 indeed has a neuroprotective role in ischemic stroke and that Npas4 might be involved in modulating the cell death pathway and inflammatory response. In this review, we summarize the current knowledge of the roles that Npas4 may play in neuroinflammation and ischemia. Understanding how ischemic lesion size in stroke may be reduced through modulation of Npas4-dependent apoptotic and inflammatory pathways could lead to the development of new stroke therapies.

Concise review: Preclinical studies on human cell-based therapy in rodent ischemic stroke models: where are we now after a decade?

Stroke, a debilitating brain insult, afflicts millions of individuals globally each year. In the last decade, researchers have investigated cell-based therapy as an alternative strategy to improve neurological outcome following stroke. This concise review critically examines preclinical reports using human adult and fetal stem/progenitor cells in rodent models of ischemic stroke. As we enter the second decade of study, we should aim to optimize our collective likelihood to translational success for stroke victims worldwide. We advocate international consensus recommendations be developed for future preclinical research.

Attempted symmetry affects dynamic gait stability in individuals with lower-limb amputation.

BACKGROUND: Gait in people with lower limb amputation (LLA) is typically asymmetrical. Reducing this asymmetry is often attempted to minimise the impact of secondary health issues. However, temporal-spatial asymmetry in gait of people with LLA has also been shown to underpin dynamic stability. RESEARCH QUESTION: The current study aimed to identify the effects of acute attempts to achieve temporal-spatial symmetry on the dynamic stability of people with unilateral transtibial amputation (UTA). The secondary aim of this study was to identify the corresponding biomechanical adaptations during attempted symmetrical gait. METHODS: Eleven people with UTA walked along a 15 m walkway in four different conditions: normal (NORM), attempted symmetrical step length and step frequency (SYMSL+SF) attempted symmetrical step length (SYMSL) and attempted symmetrical step frequency (SYMSF). Dynamic stability was measured using the backward (BW) and medio-lateral (ML) margins of stability (MoS). RESULTS: Results indicate that attempting SYMSF had a positive effect on gait stability in BW and ML directions, while attempting SYMSL had a potentially negative effect, although these results did not appear to be significant. The absence of clustering in principal component analysis, supported the lack of significant results, indicating no features differentiating between conditions of attempted symmetry. Conversely, there was clustering by limbs which were associated with differences in knee and ankle joint angles between the prosthetic and non-prosthetic limbs, and clustering by individuals highlighting the importance of patient-specific analysis. CONCLUSION: The data suggests that attempted symmetrical gait reduces asymmetry but also affects dynamic stability.

Upregulation of the neuronal Per-Arnt-Sim domain protein 4 (Npas4) in the rat corticolimbic system following focal cerebral ischemia.

The neuronal Per-Arnt-Sim domain protein 4 (Npas4) is an important transcriptional regulator of synaptic plasticity and cognition. The present study characterises the in vivo neuroanatomical expression pattern of the Npas4 protein in a rat model of focal cerebral ischemia. Animals were subjected to unilateral middle cerebral artery occlusion for 2 h, after which the spatiotemporal and neuronal profiles of Npas4 protein expression were analysed by immunohistochemistry at different time points post-reperfusion. Focal cerebral ischemia induced an early, transient and robust upregulation of Npas4 in a brain region-dependent manner involving predominantly principal neurons. Interestingly, we observed a unique differential induction of Npas4 protein expression in corticolimbic regions of the rat brain that are critically linked to cognition and emotion. These findings suggest that stroke-induced Npas4 upregulation may be involved in a transcriptional regulatory program within the corticolimbic circuitry following an ischemic insult.

Non-Syndromic Sensorineural Hearing Loss in Children.

Pediatric hearing loss is common with significant consequences in terms of language, communication, social and emotional development, and academic advancement. Radiological imaging provides useful information regarding hearing loss etiology, prognosis, therapeutic options, and potential surgical pitfalls. This review provides an overview of temporal bone imaging protocols, an outline of the classification of inner ear anomalies associated with sensorineural hearing loss and illustrates some of the more frequently encountered and/or important causes of non-syndromic hearing loss.