Improved DiOlistic labelling technique for neurons in situ: Detailed visualisation of dendritic spines and concurrent histochemical-detection in fixed tissue.

DiOlistic labelling is a robust, unbiased ballistic method that utilises lipophilic dyes to morphologically label neurons. While its efficacy on freshly dissected tissue specimens is well-documented, applying DiOlistic labelling to stored, fixed brain tissue and its use in polychromatic multi-marker studies poses significant technical challenges. Here, we present an improved, step-by-step protocol for DiOlistic labelling of dendrites and dendritic spines in fixed mouse tissue. Our protocol encompasses the five key stages: Tissue Preparation, Dye Bullet Preparation, DiOlistic Labelling, Confocal Imaging, and Image Analysis. This method ensures reliable and consistent labelling of dendritic spines in fixed mouse tissue, combined with increased throughput of samples and multi-parameter staining and visualisation of tissue, thereby offering a valuable approach for neuroscientific research.

TTLL1 and TTLL4 polyglutamylases are required for the neurodegenerative phenotypes in pcd mice.

Polyglutamylation is a dynamic posttranslational modification where glutamate residues are added to substrate proteins by 8 tubulin tyrosine ligase-like (TTLL) family members (writers) and removed by the 6 member Nna1/CCP family of carboxypeptidases (erasers). Genetic disruption of polyglutamylation leading to hyperglutamylation causes neurodegenerative phenotypes in humans and animal models; the best characterized being the Purkinje cell degeneration (pcd) mouse, a mutant of the gene encoding Nna1/CCP1, the prototypic eraser. Emphasizing the functional importance of the balance between glutamate addition and elimination, loss of TTLL1 prevents Purkinje cell degeneration in pcd. However, whether Ttll1 loss protects other vulnerable neurons in pcd, or if elimination of other TTLLs provides protection is largely unknown. Here using a mouse genetic rescue strategy, we characterized the contribution of Ttll1, 4, 5, 7, or 11 to the degenerative phenotypes in cerebellum, olfactory bulb and retinae of pcd mutants. Ttll1 deficiency attenuates Purkinje cell loss and function and reduces olfactory bulb mitral cell death and retinal photoreceptor degeneration. Moreover, degeneration of photoreceptors in pcd is preceded by impaired rhodopsin trafficking to the rod outer segment and likely represents the causal defect leading to degeneration as this too is rescued by elimination of TTLL1. Although TTLLs have similar catalytic properties on model substrates and several are highly expressed in Purkinje cells (e.g. TTLL5 and 7), besides TTLL1 only TTLL4 deficiency attenuated degeneration of Purkinje and mitral cells in pcd. Additionally, TTLL4 loss partially rescued photoreceptor degeneration and impaired rhodopsin trafficking. Despite their common properties, the polyglutamylation profile changes promoted by TTLL1 and TTLL4 deficiencies in pcd mice are very different. We also report that loss of anabolic TTLL5 synergizes with loss of catabolic Nna1/CCP1 to promote photoreceptor degeneration. Finally, male infertility in pcd is not rescued by loss of any Ttll. These data provide insight into the complexity of polyglutamate homeostasis and function in vivo and potential routes to ameliorate disorders caused by disrupted polyglutamylation.

Opposing effects of the purinergic P2X7 receptor on seizures in neurons and microglia in male mice.

BACKGROUND: The purinergic ATP-gated P2X7 receptor (P2X7R) is increasingly recognized to contribute to pathological neuroinflammation and brain hyperexcitability. P2X7R expression has been shown to be increased in the brain, including both microglia and neurons, in experimental models of epilepsy and patients. To date, the cell type-specific downstream effects of P2X7Rs during seizures remain, however, incompletely understood. METHODS: Effects of P2X7R signaling on seizures and epilepsy were analyzed in induced seizure models using male mice including the kainic acid model of status epilepticus and pentylenetetrazole model and in male and female mice in a genetic model of Dravet syndrome. RNA sequencing was used to analyze P2X7R downstream signaling during seizures. To investigate the cell type-specific role of the P2X7R during seizures and epilepsy, we generated mice lacking exon 2 of the P2rx7 gene in either microglia (P2rx7:Cx3cr1-Cre) or neurons (P2rx7:Thy-1-Cre). To investigate the protective potential of overexpressing P2X7R in GABAergic interneurons, P2X7Rs were overexpressed using adeno-associated virus transduction under the mDlx promoter. RESULTS: RNA sequencing of hippocampal tissue from wild-type and P2X7R knock-out mice identified both glial and neuronal genes, in particular genes involved in GABAergic signaling, under the control of the P2X7R following seizures. Mice with deleted P2rx7 in microglia displayed less severe acute seizures and developed a milder form of epilepsy, and microglia displayed an anti-inflammatory molecular profile. In contrast, mice lacking P2rx7 in neurons showed a more severe seizure phenotype when compared to epileptic wild-type mice. Analysis of single-cell expression data revealed that human P2RX7 expression is elevated in the hippocampus of patients with temporal lobe epilepsy in excitatory and inhibitory neurons. Functional studies determined that GABAergic interneurons display increased responses to P2X7R activation in experimental epilepsy. Finally, we show that viral transduction of P2X7R in GABAergic interneurons protects against evoked and spontaneous seizures in experimental temporal lobe epilepsy and in mice lacking Scn1a, a model of Dravet syndrome. CONCLUSIONS: Our results suggest a dual and opposing action of P2X7R in epilepsy and suggest P2X7R overexpression in GABAergic interneurons as a novel therapeutic strategy for acquired and, possibly, genetic forms of epilepsy.

Genetic diversity of United States Rambouillet, Katahdin and Dorper sheep.

BACKGROUND: Managing genetic diversity is critically important for maintaining species fitness. Excessive homozygosity caused by the loss of genetic diversity can have detrimental effects on the reproduction and production performance of a breed. Analysis of genetic diversity can facilitate the identification of signatures of selection which may contribute to the specific characteristics regarding the health, production and physical appearance of a breed or population. In this study, breeds with well-characterized traits such as fine wool production (Rambouillet, N = 745), parasite resistance (Katahdin, N = 581) and environmental hardiness (Dorper, N = 265) were evaluated for inbreeding, effective population size (Ne), runs of homozygosity (ROH) and Wright's fixation index (FST) outlier approach to identify differential signatures of selection at 36,113 autosomal single nucleotide polymorphisms (SNPs). RESULTS: Katahdin sheep had the largest current Ne at the most recent generation estimated with both the GONe and NeEstimator software. The most highly conserved ROH Island was identified in Rambouillet with a signature of selection on chromosome 6 containing 202 SNPs called in an ROH in 50 to 94% of the individuals. This region contained the DCAF16, LCORL and NCAPG genes that have been previously reported to be under selection and have biological roles related to milk production and growth traits. The outlier regions identified through the FST comparisons of Katahdin with Rambouillet and Dorper contained genes with known roles in milk production and mastitis resistance or susceptibility, and the FST comparisons of Rambouillet with Katahdin and Dorper identified genes related to wool growth, suggesting these traits have been under natural or artificial selection pressure in these populations. Genes involved in the cytokine-cytokine receptor interaction pathways were identified in all FST breed comparisons, which indicates the presence of allelic diversity between these breeds in genomic regions controlling cytokine signaling mechanisms. CONCLUSIONS: In this paper, we describe signatures of selection within diverse and economically important U.S. sheep breeds. The genes contained within these signatures are proposed for further study to understand their relevance to biological traits and improve understanding of breed diversity.

Pedigree diversity and implications for genetic selection of Katahdin sheep.

The Katahdin hair breed gained popularity in the United States as low input and prolific, with a propensity to exhibit parasite resistance. With the introduction of genomically enhanced estimated breeding values (GEBV) to the Katahdin genetic evaluation, defining the diversity present in the breed is pertinent. Utilizing pedigree records (n = 92,030) from 1984 to 2019 from the National Sheep Improvement Program, our objectives were to (i) estimate the completeness and quality of the pedigree, (ii) calculate diversity statistics for the whole pedigree and relevant reference subpopulations and (iii) assess the impact of current diversity on genomic selection. Reference 1 was Katahdins born from 2017 to 2019 (n = 23,494), while reference 2 was a subset with at least three generations of Katahdin ancestry (n = 9327). The completeness of the whole pedigree, and the pedigrees of reference 1 and reference 2, were above 50% through the fourth, fifth and seventh generation of ancestors, respectively. Effective population size (Ne) averaged 111 animals with a range from 42.2 to 451.0. The average generation interval was 2.9 years for the whole pedigree and reference 1, and 2.8 years for reference 2. The mean individual inbreeding and average relatedness coefficients were 1.62% and 0.91%, 1.74% and 0.90% and 2.94% and 1.46% for the whole pedigree, reference 1, and reference 2, respectively. There were over 300 effective founders in the whole pedigree and reference 1, with 169 in reference 2. Effective number of ancestors were over 150 for the whole pedigree and reference 1, while there were 67 for reference 2. Prediction accuracies increased as the reference population grew from 1k to 7.5k and plateaued at 15k animals. Given the large number of founders and ancestors contributing to the base genetic variation in the breed, the Ne is sufficient to maintain diversity while achieving progress with selection. Stable low rates of inbreeding and relatedness suggest that incorporating genetic conservation in breeding decisions is currently not of high priority. Current Ne suggests that with limited genotyping, high levels of accuracy for genomic prediction can be achieved. However, intense selection on GEBV may cause loss of genetic diversity long term.

Increased uptake of the P2X7 receptor radiotracer 18 F-JNJ-64413739 in the brain and peripheral organs according to the severity of status epilepticus in male mice.

OBJECTIVE: The P2X7 receptor (P2X7R) is an important contributor to neuroinflammation, responding to extracellularly released adenosine triphosphate. Expression of the P2X7R is increased in the brain in experimental and human epilepsy, and genetic or pharmacologic targeting of the receptor can reduce seizure frequency and severity in preclinical models. Experimentally induced seizures also increase levels of the P2X7R in blood. Here, we tested 18 F-JNJ-64413739, a positron emission tomography (PET) P2X7R antagonist, as a potential noninvasive biomarker of seizure-damage and epileptogenesis. METHODS: Status epilepticus was induced via an intra-amygdala microinjection of kainic acid. Static PET studies (30 min duration, initiated 30 min after tracer administration) were conducted 48 h after status epilepticus via an intravenous injection of 18 F-JNJ-64413739. PET images were coregistered with a brain magnetic resonance imaging atlas, tracer uptake was determined in the different brain regions and peripheral organs, and values were correlated to seizure severity during status epilepticus. 18 F-JNJ-64413739 was also applied to ex vivo human brain slices obtained following surgical resection for intractable temporal lobe epilepsy. RESULTS: P2X7R radiotracer uptake correlated strongly with seizure severity during status epilepticus in brain structures including the cerebellum and ipsi- and contralateral cortex, hippocampus, striatum, and thalamus. In addition, a correlation between radiotracer uptake and seizure severity was also evident in peripheral organs such as the heart and the liver. Finally, P2X7R radiotracer uptake was found elevated in brain sections from patients with temporal lobe epilepsy when compared to control. SIGNIFICANCE: Taken together, our data suggest that P2X7R-based PET imaging may help to identify seizure-induced neuropathology and temporal lobe epilepsy patients with increased P2X7R levels possibly benefitting from P2X7R-based treatments.

Assessment of Lab4P Probiotic Effects on Cognition in 3xTg-AD Alzheimer's Disease Model Mice and the SH-SY5Y Neuronal Cell Line.

Aging and metabolic syndrome are associated with neurodegenerative pathologies including Alzheimer's disease (AD) and there is growing interest in the prophylactic potential of probiotic bacteria in this area. In this study, we assessed the neuroprotective potential of the Lab4P probiotic consortium in both age and metabolically challenged 3xTg-AD mice and in human SH-SY5Y cell culture models of neurodegeneration. In mice, supplementation prevented disease-associated deteriorations in novel object recognition, hippocampal neurone spine density (particularly thin spines) and mRNA expression in hippocampal tissue implying an anti-inflammatory impact of the probiotic, more notably in the metabolically challenged setting. In differentiated human SH-SY5Y neurones challenged with ß-Amyloid, probiotic metabolites elicited a neuroprotective capability. Taken together, the results highlight Lab4P as a potential neuroprotective agent and provide compelling support for additional studies in animal models of other neurodegenerative conditions and human studies.

Rates of Abiotic MnII Oxidation by O2: Influence of Various Multidentate Ligands at High pH.

Oxidation of manganous manganese (MnII) is an important process driving manganese cycles in natural aquatic systems and leading to the formation of solid-phase MnIII,IV (hydr)oxide products. Previous research has shown that some simple ligands (e.g., phosphate, sulfate, chloride, fluoride) can bind with MnII to make it unreactive to oxidation by dissolved oxygen. However, there is little to no understanding of the role played by stronger, complex-forming ligands in MnII oxidation reactions. The objective of this study was to evaluate the rates of abiotic MnII oxidation by O2 in the presence of low concentrations of several complex-forming model ligands (pyrophosphate, tripolyphosphate, ethylenediaminetetraacetic acid, oxalate) in bicarbonate-carbonate buffered laboratory solutions of pH 9.42, 9.65, and 10.19. The influence of increasing ligand concentrations on observed autocatalytic profiles of MnII oxidation was investigated, and initial oxidation rates were linked quantitatively to the initial MnII speciation in experimental solutions. Observed rates of MnII oxidation decreased with increasing ligand concentration for all four ligands tested. However, the profiles observed with time and the magnitudes of decrease in initial oxidation rates were different for the different ligands. Likely explanations for these observations include the denticity of the tested ligands, the relative strength of the ligands to complex MnII versus MnIII, and the ability of some ligands to enhance the reduction of MnIII back to MnII on a time scale comparable to the forward homogeneous MnII oxidation reaction.

Polyadenylation of mRNA as a novel regulatory mechanism of gene expression in temporal lobe epilepsy.

Temporal lobe epilepsy is the most common and refractory form of epilepsy in adults. Gene expression within affected structures such as the hippocampus displays extensive dysregulation and is implicated as a central pathomechanism. Post-transcriptional mechanisms are increasingly recognized as determinants of the gene expression landscape, but key mechanisms remain unexplored. Here we show, for first time, that cytoplasmic mRNA polyadenylation, one of the post-transcriptional mechanisms regulating gene expression, undergoes widespread reorganization in temporal lobe epilepsy. In the hippocampus of mice subjected to status epilepticus and epilepsy, we report >25% of the transcriptome displays changes in their poly(A) tail length, with deadenylation disproportionately affecting genes previously associated with epilepsy. Suggesting cytoplasmic polyadenylation element binding proteins (CPEBs) being one of the main contributors to mRNA polyadenylation changes, transcripts targeted by CPEBs were particularly enriched among the gene pool undergoing poly(A) tail alterations during epilepsy. Transcripts bound by CPEB4 were over-represented among transcripts with poly(A) tail alterations and epilepsy-related genes and CPEB4 expression was found to be increased in mouse models of seizures and resected hippocampi from patients with drug-refractory temporal lobe epilepsy. Finally, supporting an adaptive function for CPEB4, deletion of Cpeb4 exacerbated seizure severity and neurodegeneration during status epilepticus and the development of epilepsy in mice. Together, these findings reveal an additional layer of gene expression regulation during epilepsy and point to novel targets for seizure control and disease-modification in epilepsy.

Buspirone for functional improvement after acute traumatic spinal cord injury: a propensity score-matched cohort study.

STUDY DESIGN: Retrospective analysis of treated inpatients compared to expected neurorecovery from a propensity score-matched national database cohort. OBJECTIVE: Evaluate the effectiveness of buspirone on clinical neurorecovery following traumatic SCI when started during acute inpatient rehabilitation. SETTING: University-based hospital in Boston, USA. METHODS: Chart review yielded thirty-one individuals with acute, traumatic SCI treated with buspirone during inpatient rehabilitation from 2011-2017. Propensity score matching to a cohort of individuals from the spinal cord injury model systems (SCIMS) national database was completed. Changes in upper extremity motor score (UEMS), lower extremity motor score (LEMS), American Spinal Injury Association Impairment Scale (AIS), neurological level of injury (NLI), and functional impairment measure (FIM) from admission to discharge and discharge to 1 year were computed and compared between matched pairs (buspirone and mean national SCIMs cohort). A local control cohort not treated with buspirone was similarly compared to a matched mean national SCIMs group to identify location-specific effects. RESULTS: From admission to discharge from inpatient rehabilitation, 95% confidence intervals of changes in UEMS (-2.43 to +2.78), LEMS (-1.02 to +6.02), AIS (-0.04 to +0.35), NLI (-0.42 to +1.08), and FIM (-4.42 to +6.40) were not significantly different between those individuals who received buspirone and their propensity-matched SCIMS cohort. Similarly, changes in these metrics were not significantly different at 1-year follow up. Buspirone group individuals with initial clinically complete SCI demonstrated a higher 1-year conversion rate to incomplete injury (6 out of 14; 42.9%) compared to the matched national SCIMS cohort (14 out of 70; 21.2%, p = 0.047) though this was not significantly different from non-buspirone local controls (p = 0.25). CONCLUSIONS: Retrospective analysis shows no statistically significant difference in gross markers of neurorecovery following acute traumatic SCI when buspirone is initiated indiscriminately during acute inpatient rehabilitation. In individuals with clinically complete SCI, findings suggest possible increased rates of 1-year conversion to incomplete injury.

Prosodic realizations of new, given, and corrective referents in the spontaneous speech of toddlers.

Our motivation was to examine how toddler (2;6) and adult speakers of American English prosodically realize information status categories. The aims were three-fold: 1) to analyze how adults phonologically make information status distinctions; 2) to examine how these same categories are signaled in toddlers' spontaneous speech; and 3) to analyze the three primary acoustic correlates of prosody (F0, intensity, and duration). During a spontaneous speech task designed as an interactive game, a set of target nouns was elicited as one of three types (new, given, corrective). Results show that toddlers primarily used H* across information status categories, with secondary preferences for deaccenting given information and for using L+H* for corrective information. Only duration distinguished information status, and duration, average pitch, and intensity differentiated pitch accent types for both adults and children. Discussion includes how pitch accent selection and input play a role in guiding prosodic realizations of information status.

Plasticity in Adult Mouse Visual Cortex Following Optic Nerve Injury.

Optic nerve (ON) injury is an established model of axonal injury which results in retrograde degeneration and death of retinal ganglion cells as well anterograde loss of transmission and Wallerian degeneration of the injured axons. While the local impact of ON crush has been extensively documented we know comparatively little about the functional changes that occur in higher visual structures such as primary visual cortex (V1). We explored the extent of adult cortical plasticity using ON crush in aged mice. V1 function of the contralateral hemisphere was assessed longitudinally by intrinsic signal imaging and 2-photon calcium imaging before and after ON crush. Functional imaging demonstrated an immediate shift in V1 ocular dominance towards the ipsilateral, intact eye, due to the expected almost complete loss of responses to contralateral eye stimulation. Surprisingly, within 2 weeks we observed a delayed increase in ipsilateral eye responses. Additionally, spontaneous activity in V1 was reduced, similar to the lesion projection zone after retinal lesions. The observed changes in V1 activity indicate that severe ON injury in adulthood evokes cortical plasticity not only cross-modally but also within the visual cortex; this plasticity may be best compared with that seen after retinal lesions.

Questions Can Answer Questions About Mechanisms of Preschoolers' Selective Word Learning.

This study examined how inferences about epistemic competence and generalized labeling errors influence children's selective word learning. Three- to 4-year-olds (N = 128) learned words from informants who asked questions about objects, mentioning either correct or incorrect labels. Such questions do not convey stark differences in informants' epistemic competence. Inaccurate labels, however, generate error signals that can lead to weaker encoding of novel information. Preschoolers retained novel labels from both informants but were slower to respond in the Inaccurate Labeler condition. When the test procedure was not sensitive to the strength of information encoding, children performed above chance in both conditions and their response times did not differ. These results suggest that epistemic-level inferences and error generalizations influence preschoolers' selective word learning concurrently.

Economic evaluations in paediatric dentistry clinical trials.

Economic evaluations play an important role in identifying the cost-effectiveness of alternative healthcare programmes, informing decisions surrounding funding and the allocation of resources. This paper outlines the basic principles of economic evaluation and how it can be conducted alongside a clinical trial. Furthermore, it considers the ways in which evidence from these studies can be used, and the challenges researchers are faced with when conducting economic evaluations in the field of children's oral health.

Population-based genetic testing of asymptomatic women for breast and ovarian cancer susceptibility.

PURPOSE: The identification of carriers of hereditary breast and ovarian cancer (HBOC) gene variants through family cancer history alone is suboptimal, and most population-based genetic testing studies have been limited to founder mutations in high-risk populations. Here, we determine the clinical utility of identifying actionable variants in a healthy cohort of women. METHODS: Germline DNA from a subset of healthy Australian women participating in the lifepool project was screened using an 11-gene custom sequencing panel. Women with clinically actionable results were invited to attend a familial cancer clinic (FCC) for post-test genetic counseling and confirmatory testing. Outcomes measured included the prevalence of pathogenic variants, and the uptake rate of genetic counseling, risk reduction surgery, and cascade testing. RESULTS: Thirty-eight of 5908 women (0.64%) carried a clinically actionable pathogenic variant. Forty-two percent of pathogenic variant carriers did not have a first-degree relative with breast or ovarian cancer and 89% pursued referral to an FCC. Forty-six percent (6/13) of eligible women pursued risk reduction surgery, and the uptake rate of cascade testing averaged 3.3 family members per index case. CONCLUSION: Within our cohort, HBOC genetic testing was well accepted, and the majority of high-risk gene carriers identified would not meet eligibility criteria for genetic testing based on their existing family history.

Intestinal failure-associated liver disease in adult patients.

PURPOSE OF REVIEW: The aim of this review is to give up-to-date information on intestinal failure-associated liver disease (IFALD) and how its investigation and management has evolved. Despite advances in treatment for patients with intestinal failure, IFALD remains a significant cause of mortality. RECENT FINDINGS: Liver biopsy remains as the gold standard for the diagnosis of IFALD, but its invasive nature has prompted assessment of noninvasive techniques. Risk factors for IFALD are both nonnutritional (e.g. sepsis) and nutritional. Strict protocols for the prevention of central venous catheter infections in patients with intestinal failure are well established, as is the optimization of the constituents of parenteral nutrition. Further research comparing the available lipid emulsions has become available. Novel approaches at maximizing intestinal absorption are discussed including glucagon-like peptide-2 analogues, as well as surgical approaches. SUMMARY: Although there are data on the novel investigative and therapeutic strategies for managing IFALD, further study is required to identify a suitable noninvasive technique for earlier diagnosis and then monitoring of IFALD. Further data are also required on the impact of novel therapies aimed at improving absorption and reducing parenteral nutrition load on IFALD occurrence and progression.

Pregnancy Outcomes Reported During the 13-Year TREAT Registry: A Descriptive Report.

OBJECTIVES: We described pregnancy outcomes in Crohn's disease (CD) patients enrolled in the TREAT Registry who received infliximab before, or during pregnancy and those not treated with infliximab or any biologic agent. METHODS: In the TREAT Registry (1999-2012), pregnancy outcomes were analyzed from maternal and paternal patients exposed to infliximab ≤365 days (gestational exposure), >365 days (pre-gestational exposure) of pregnancy outcome or without infliximab exposure (non-biologic exposed). "Healthy infants" were defined as those with no congenital abnormalities, neonatal complications (e.g., jaundice, prematurity, heart murmur, cortical vision/fine motor delay, cardiac failure, hemophilia, or torticollis), prolonged hospitalization, or those who received no special treatment. Disease activity and concomitant medications were also evaluated. RESULTS: Overall, 92.3% (324/351) of pregnancies had known outcomes. The majority of both maternal pregnancies (92.6, 91.2, and 87.8%) and partner outcomes (92.7, 93.8, and 91.7%) resulted in live births of healthy infants across gestational, pre-gestational, and non-biologic exposure groups, respectively. Among these, rates of neonatal complications were low for both maternal (6.2, 7.0, and 8.5%), and partner outcomes (4.9, 0, and 0%) in gestational, pre-gestational, and non-biologic exposure groups, respectively. Among maternal pregnancies, numerically higher rates of spontaneous abortions were observed for the gestational exposure group than for the pre-gestational or non-biologic exposed groups. CONCLUSIONS: The clinical condition of infants born to women with gestational infliximab exposure was similar to those without exposure. Although a lower live birth rate was reported among infliximab-exposed women, these patients had more severe CD and were more likely to have been exposed to immunosuppressives.

Eighteen-month-olds selectively generalize words from accurate speakers to novel contexts.

The present studies examine whether and how 18-month-olds use informants' accuracy to acquire novel labels for novel objects and generalize them to a new context. In Experiment 1, two speakers made statements about the labels of familiar objects. One used accurate labels and the other used inaccurate labels. One of these speakers then introduced novel labels for two novel objects. At test, toddlers saw those two novel objects and heard an unfamiliar voice say one of the labels provided by the speaker. Only toddlers who had heard the novel labels introduced by the accurate speaker looked at the appropriate novel object above chance. Experiment 2 explored possible mechanisms underlying this difference in generalization. Rather than making statements about familiar objects' labels, both speakers asked questions about the objects' labels, with one speaker using accurate labels and the other using inaccurate labels. Toddlers' generalization of novel labels for novel objects was at chance for both speakers, suggesting that toddlers do not simply associate hearing the accurate label with the reliability of the speaker. We discuss these results in terms of potential mechanisms by which children learn and generalize novel labels across contexts from speaker reliability.

Timing of initiating manual therapy and therapeutic exercises in the management of patients after hindfoot fractures: a randomized controlled trial.

STUDY DESIGN: Randomized clinical trial. BACKGROUND: Patients with fractures to the talus and calcaneus report decreased functional outcomes and develop long-term functional limitations. Although physical therapy is typically not initiated until six weeks after fixation, there's little research on the optimal time to initiate a formal physical therapy program. OBJECTIVES: To assess whether initiating physical therapy including range of motion (ROM) and manual therapy two weeks post-operatively (EARLY) vs. six weeks post-operatively (LATE) in patients with fixation for hindfoot fractures results in different clinical outcomes. METHODS: Fifty consecutive participants undergoing operative fixation of a hindfoot fracture were randomized to either EARLY or LATE physical therapy. Outcomes, including the American Orthopedic Foot and Ankle Society Hindfoot Scale (AOFAS), the Lower Extremity Functional Scale (LEFS), active ROM, swelling, and pain, were collected at three and six months and analyzed using linear mixed-modeling to examine change over time. Adverse events were tracked for 12 months after surgery. RESULTS: The EARLY group demonstrated significantly larger improvements for the AOFAS (p = .01) and the LEFS (p = .01) compared to the LATE group. Pairwise comparison of the LEFS favors the EARLY group at 6 months [7.5 (95%CI -.01 to 15.0), p = .05]. There were no differences between the groups with regard to ROM, pain, and swelling. The LATE group incurred increased adverse events in this study. CONCLUSION: Initiating early physical therapy may improve long-term outcomes and mitigate complications in patients after hindfoot fractures. LEVEL OF EVIDENCE: Therapy, level 2b.

Chronic in vivo nitric oxide deficiency impairs cardiac functional recovery after ischemia in female (but not male) mice.

Nitric oxide (NO) is an important regulator of cardiac function and plays a key role in ischemic cardioprotection. The role of chronic NO deficiency in coordinating ischemic vulnerability in female myocardium has not been established. The aim of this study was to determine the influence of chronic in vivo NO synthase inhibition in modulating ex vivo ischemia-reperfusion responses in female hearts (relative to males). Mice were subjected to l-NAME (l-NG-Nitroarginine-methyl-ester) treatment in vivo for 8weeks. Cardiac fibrotic, inflammatory and cardiomyocyte Ca2+ handling related gene expression changes were assessed. Hearts were Langendorff-perfused, subjected to 20min global ischemia with 45min reperfusion. In response to this moderate ex vivo ischemic insult, hearts derived from l-NAME treated female animals exhibited increased incidence of reperfusion arrhythmias, diastolic abnormality and reduced contractile recovery in reperfusion. This differential response was observed even though baseline performance of hearts from l-NAME treated animals was not different to vehicle controls, myocardial inflammatory and fibrotic indices were similar in males and females and the systolic blood pressure effect of l-NAME administration was equivalent in both sexes. Evaluation of a subgroup of mice with cardiomyocyte specific mineralocorticoid receptor deletion suggests involvement of this receptor in NO-deficiency mediated responses. To examine underlying pre-disposing mechanisms, expression of a panel of candidate genes encoding proteins involved in electromechanical homeostasis (particularly relevant to ischemic challenge) was evaluated in normoxic myocardial tissues from the l-NAME- and vehicle-treated animals. Analysis revealed that l-NAME treatment in females selectively regulated expression of genes related directly and indirectly to cardiomyocyte Ca2+ handling in a manner consistent with destabilization of Ca2+ homeostasis and arrhythmogenesis. Our investigation provides new insight into the role of sustained decrease in NO bioavailability in determining distinctive female cardiac vulnerability to ischemic challenge.