Strand-resolved mutagenicity of DNA damage and repair.

DNA base damage is a major source of oncogenic mutations1. Such damage can produce strand-phased mutation patterns and multiallelic variation through the process of lesion segregation2. Here we exploited these properties to reveal how strand-asymmetric processes, such as replication and transcription, shape DNA damage and repair. Despite distinct mechanisms of leading and lagging strand replication3,4, we observe identical fidelity and damage tolerance for both strands. For small alkylation adducts of DNA, our results support a model in which the same translesion polymerase is recruited on-the-fly to both replication strands, starkly contrasting the strand asymmetric tolerance of bulky UV-induced adducts5. The accumulation of multiple distinct mutations at the site of persistent lesions provides the means to quantify the relative efficiency of repair processes genome wide and at single-base resolution. At multiple scales, we show DNA damage-induced mutations are largely shaped by the influence of DNA accessibility on repair efficiency, rather than gradients of DNA damage. Finally, we reveal specific genomic conditions that can actively drive oncogenic mutagenesis by corrupting the fidelity of nucleotide excision repair. These results provide insight into how strand-asymmetric mechanisms underlie the formation, tolerance and repair of DNA damage, thereby shaping cancer genome evolution.

The Differential Translation Capabilities of the Human DHFR2 Gene Indicates a Developmental and Tissue-Specific Endogenous Protein of Low Abundance.

A functional role has been ascribed to the human dihydrofolate reductase 2 (DHFR2) gene based on the enzymatic activity of recombinant versions of the predicted translated protein. However, the in vivo function is still unclear. The high amino acid sequence identity (92%) between DHFR2 and its parental homolog, DHFR, makes analysis of the endogenous protein challenging. This paper describes a targeted mass spectrometry proteomics approach in several human cell lines and tissue types to identify DHFR2-specific peptides as evidence of its translation. We show definitive evidence that the DHFR2 activity in the mitochondria is in fact mediated by DHFR, and not DHFR2. Analysis of Ribo-seq data and an experimental assessment of ribosome association using a sucrose cushion showed that the two main Ensembl annotated mRNA isoforms of DHFR2, 201 and 202, are differentially associated with the ribosome. This indicates a functional role at both the RNA and protein level. However, we were unable to detect DHFR2 protein at a detectable level in most cell types examined despite various RNA isoforms of DHFR2 being relatively abundant. We did detect a DHFR2-specific peptide in embryonic heart, indicating that the protein may have a specific role during embryogenesis. We propose that the main functionality of the DHFR2 gene in adult cells is likely to arise at the RNA level.

TRPV1 and mast cell involvement in repeated variate stress (RVS) induced urinary bladder dysfunction in adult female mice.

The etiology of interstitial cystitis/bladder pain syndrome (IC/BPS) is unknown but likely multifactorial. IC/BPS symptoms can be exacerbated by psychological stress, but underlying mechanisms remain to be defined. TRPV1 channels, expressed on nerve fibers, have been implicated in bladder dysfunction and colonic hypersensitivity with stress in rodents. Histamine/H1R activation of TRPV1+ nerves increases bladder afferent fiber sensitivity to distension. TRPV1 channels are also expressed on mast cells, previously implicated in contributing to IC/BPS etiology and symptoms. We have examined the contribution of TRPV1 and mast cells to bladder dysfunction after repeated variate stress (RVS). RVS increased (p ≤ 0.05) serum and fecal corticosterone expression and induced anxiety-like behavior in wild type (WT) mice. Intravesical instillation of the selective TRPV1 antagonist capsazepine (CPZ) rescued RVS-induced bladder dysfunction in WT mice. Trpv1 knockout (KO) mice did not increase voiding frequency with RVS and did not exhibit increased serum corticosterone expression despite exhibiting anxiety-like behavior. Mast cell deficient mice (B6.Cg-Kitw-sh) failed to demonstrate RVS-induced increased voiding frequency or serum corticosterone expression whereas control (no stress) mast cell deficient mice had similar functional bladder capacity to WT mice. TRPV1 protein expression was significantly increased in the rostral lumbar (L1-L2) spinal cord and dorsal root ganglia in WT mice exposed to RVS but no changes were observed in lumbosacral (L6-S1) spinal segments or DRG. These studies demonstrated TRPV1 and mast cell involvement in RVS-induced increased voiding frequency and suggest that TRPV1 and mast cells may be useful targets to mitigate stress-induced urinary bladder dysfunction.

Lung Microbiome as a Treatable Trait in Chronic Respiratory Disorders.

Once thought to be a sterile environment, it is now established that lungs are populated by various microorganisms that participate in maintaining lung function and play an important role in shaping lung immune surveillance. Although our comprehension of the molecular and metabolic interactions between microbes and lung cells is still in its infancy, any event causing a persistent qualitative or quantitative variation in the composition of lung microbiome, termed "dysbiosis", has been virtually associated with many respiratory diseases. A deep understanding of the composition and function of the "healthy" lung microbiota and how dysbiosis can cause or participate in disease progression will be pivotal in finding specific therapies aimed at preventing diseases and restoring lung function. Here, we review lung microbiome dysbiosis in different lung pathologies and the mechanisms by which these bacteria can cause or contribute to the severity of the disease. Furthermore, we describe how different respiratory disorders can be caused by the same pathogen, and that the real pathogenetic mechanism is not only dependent by the presence and amount of the main pathogen but can be shaped by the interaction it can build with other bacteria, fungi, and viruses present in the lung. Understanding the nature of this bacteria crosstalk could further our understanding of each respiratory disease leading to the development of new therapeutic strategies.

Mutational analysis of the alpha subunit of eIF2B provides insights into the role of eIF2B bodies in translational control and VWM disease.

Eukaryotic initiation factor 2B (eIF2B) serves as a vital control point within protein synthesis and regulates translation initiation in response to cellular stress. Mutations within eIF2B result in the fatal disease, leukoencephalopathy with vanishing white matter (VWM). Previous biochemical studies on VWM mutations have illustrated that changes in the activity of eIF2B poorly correlate with disease severity. This suggests that there may be additional characteristics of eIF2B contributing to VWM pathogenesis. Here, we investigated whether the localization of eIF2B to eIF2B bodies was integral for function and whether this localization could provide insight into the pathogenesis of VWM. We demonstrate that the regulatory subunit, eIF2Bα, is required for the assembly of eIF2B bodies in yeast and that loss of eIF2B bodies correlates with an inability of cells to regulate eIF2B activity. Mutational analysis of eIF2Bα showed that missense mutations that disrupt the regulation of eIF2B similarly disrupt the assembly of eIF2B bodies. In contrast, when eIF2Bα mutations that impact the catalytic activity of eIF2B were analyzed, eIF2B bodies were absent and instead eIF2B localized to small foci, termed microfoci. Fluorescence recovery after photobleaching analysis highlighted that within these microfoci, eIF2 shuttles more slowly indicating that formation of eIF2B bodies correlates with full eIF2B activity. When eIF2Bα VWM mutations were analyzed, a diverse impact on localization was observed, which did not seem to correlate with eIF2B activity. These findings provide key insights into how the eIF2B body assembles and suggest that the body is a fundamental part of the translational regulation via eIF2α phosphorylation.

Insights into the genetic basis of retinal detachment.

Retinal detachment (RD) is a serious and common condition, but genetic studies to date have been hampered by the small size of the assembled cohorts. In the UK Biobank data set, where RD was ascertained by self-report or hospital records, genetic correlations between RD and high myopia or cataract operation were, respectively, 0.46 (SE = 0.08) and 0.44 (SE = 0.07). These correlations are consistent with known epidemiological associations. Through meta-analysis of genome-wide association studies using UK Biobank RD cases (N = 3 977) and two cohorts, each comprising ~1 000 clinically ascertained rhegmatogenous RD patients, we uncovered 11 genome-wide significant association signals. These are near or within ZC3H11B, BMP3, COL22A1, DLG5, PLCE1, EFEMP2, TYR, FAT3, TRIM29, COL2A1 and LOXL1. Replication in the 23andMe data set, where RD is self-reported by participants, firmly establishes six RD risk loci: FAT3, COL22A1, TYR, BMP3, ZC3H11B and PLCE1. Based on the genetic associations with eye traits described to date, the first two specifically impact risk of a RD, whereas the last four point to shared aetiologies with macular condition, myopia and glaucoma. Fine-mapping prioritized the lead common missense variant (TYR S192Y) as causal variant at the TYR locus and a small set of credible causal variants at the FAT3 locus. The larger study size presented here, enabled by resources linked to health records or self-report, provides novel insights into RD aetiology and underlying pathological pathways.

Propagation of Parkinson's disease by extracellular vesicle production and secretion.

Parkinson's disease (PD) is a common neurodegenerative condition affecting a significant number of individuals globally, resulting in the presentation of debilitating motor and non-motor symptoms, including bradykinesia, resting tremor, as well as mood and sleep disorders. The pathology of PD has been observed to spread through the central nervous system resulting in progressive brain degeneration and a poor prognosis. Aggregated forms of the protein α-synuclein, particularly intermediary aggregates, referred to as oligomers, or preformed fibrils, have been implicated as the causative agent in the degeneration of neuronal processes, including the dysfunction of axonal transport, mitochondrial activity, and ultimately cellular death. Extracellular vesicles (EVs) have been strongly implicated in the propagation of PD pathology. Current observations suggest that aggregated α-synuclein is transported between neurons via small EVs in a series of exocytosis and endocytosis cellular processes leading to the observed spread of neurotoxicity and cellular death. Despite some understanding of the role of EVs in neurodegeneration, the exact mechanism by which these lipidic particles participate in the progression of Parkinson's pathology is not entirely understood. Here we review the current understanding of the role of EVs in the propagation of PD and explore their potential as a therapeutic target.

A novel homozygous synonymous variant further expands the phenotypic spectrum of POLR3A-related pathologies.

Pathogenic biallelic variants in POL3RA have been associated with different disorders characterized by progressive neurological deterioration. These include the 4H leukodystrophy syndrome (hypomyelination, hypogonadotropic hypogonadism, and hypodontia) and adolescent-onset progressive spastic ataxia, as well as Wiedemann-Rautenstrauch syndrome (WRS), a recognizable neonatal progeroid syndrome. The phenotypic differences between these disorders are thought to occur mainly due to different functional effects of underlying POLR3A variants. Here we present the detailed clinical course of a 37-year-old woman in whom we identified a homozygous synonymous POLR3A variant c.3336G>A resulting in leaky splicing r.[3336ins192, =, 3243_3336del94]. She presented at birth with intrauterine growth retardation, lipodystrophy, muscular hypotonia, and several WRS-like facial features, albeit without sparse hair and prominent scalp veins. She had no signs of developmental delay or intellectual disability. Over the years, above characteristic facial features, she showed severe postnatal growth retardation, global lipodystrophy, joint contractures, thoracic hypoplasia, scoliosis, anodontia, spastic quadriplegia, bilateral hearing loss, aphonia, hypogonadotropic hypogonadism, and cerebellar peduncles hyperintensities in brain imaging. These manifestations partially overlap the clinical features of the previously reported POLR3A-associated disorders, mostly mimicking the WRS. Thus, our study expands the POLR3A-mediated phenotypic spectrum and suggests existence of a phenotypic continuum underlying biallelic POLR3A variants.

Sulfasalazine decreases astrogliosis-mediated seizure burden.

OBJECTIVE: Previously, we reported that inhibition of the astrocytic cystine/glutamate antiporter system xc- (SXC), using sulfasalazine (SAS), decreased evoked excitatory signaling in three distinct hyperexcitability models ex vivo. The current study expands on this work by evaluating the in vivo efficacy of SAS in decreasing astrogliosis-mediated seizure burden seen in the beta-1 integrin knockout (B1KO) model. METHODS: Video-EEG (electroencephalography) monitoring (24/7) was obtained using Biopac EEG acquisition hardware and software. EEG spectral analysis was performed using MATLAB. SAS was used at an equivalence of doses taken by Crohn's disease patients. Whole-cell patch-clamp recordings were made from cortical layer 2/3 pyramidal neurons. RESULTS: We report that 100% of B1KO mice that underwent 24/7 video-EEG monitoring developed spontaneous recurrent seizures and that intraperitoneal administration of SAS significantly reduced seizure frequency in B1KOs compared to B1KOs receiving sham saline. Spectral analysis found an acute reduction in EEG power following SAS treatment in B1KOs; however, this effect was not observed in nonepileptic control mice receiving SAS. Finally, whole-cell recordings from SXC knockout mice had hyperpolarized neurons and SXC-B1 double knockouts fired significantly less action potentials in response to current injection compared to B1KOs with SXC. SIGNIFICANCE: To devise effective strategies in finding relief for one-in-three patients with epilepsy who experience drug-resistant epilepsy we must continue to explore the mechanisms regulating glutamate homeostasis. This study explored the efficacy of targeting an astrocytic glutamate antiporter, SXC, as a novel antiepileptic drug (AED) target and further characterized a unique mouse model in which chronic astrogliosis is sufficient to induce spontaneous seizures and epilepsy. These findings may serve as a foundation to further assess the potential for SAS or inform the development of more potent and specific compounds that target SXC as a novel treatment for epilepsy.

Gut metabolite S-equol ameliorates hyperexcitability in entorhinal cortex neurons following Theiler murine encephalomyelitis virus-induced acute seizures.

OBJECTIVE: A growing body of evidence indicates a potential role for the gut-brain axis as a novel therapeutic target in treating seizures. The present study sought to characterize the gut microbiome in Theiler murine encephalomyelitis virus (TMEV)-induced seizures, and to evaluate the effect of microbial metabolite S-equol on neuronal physiology as well as TMEV-induced neuronal hyperexcitability ex vivo. METHODS: We infected C57BL/6J mice with TMEV and monitored the development of acute behavioral seizures 0-7 days postinfection (dpi). Fecal samples were collected at 5-7 dpi and processed for 16S sequencing, and bioinformatics were performed with QIIME2. Finally, we conducted whole-cell patch-clamp recordings in cortical neurons to investigate the effect of exogenous S-equol on cell intrinsic properties and neuronal hyperexcitability. RESULTS: We demonstrated that gut microbiota diversity is significantly altered in TMEV-infected mice at 5-7 dpi, exhibiting separation in beta diversity in TMEV-infected mice dependent on seizure phenotype, and lower abundance of genus Allobaculum in TMEV-infected mice regardless of seizure phenotype. In contrast, we identified specific loss of S-equol-producing genus Adlercreutzia as a microbial hallmark of seizure phenotype following TMEV infection. Electrophysiological recordings indicated that exogenous S-equol alters cortical neuronal physiology. We found that entorhinal cortex neurons are hyperexcitable in TMEV-infected mice, and exogenous application of microbial-derived S-equol ameliorated this TMEV-induced hyperexcitability. SIGNIFICANCE: Our study presents the first evidence of microbial-derived metabolite S-equol as a potential mechanism for alteration of TMEV-induced neuronal excitability. These findings provide new insight for the novel role of S-equol and the gut-brain axis in epilepsy treatment.

Models of spatiotemporal variation in rabbit abundance reveal management hot spots for an invasive species.

The European rabbit (Oryctolagus cuniculus) is a notorious economic and environmental pest species in its invasive range. To better understand the population and range dynamics of this species, 41 yr of abundance data have been collected from 116 unique sites across a broad range of climatic and environmental conditions in Australia. We analyzed this time series of abundance data to determine whether interannual variation in climatic conditions can be used to map historic, contemporary, and potential future fluctuations in rabbit abundance from regional to continental scales. We constructed a hierarchical Bayesian regression model of relative abundance that corrected for observation error and seasonal biases. The corrected abundances were regressed against environmental and disease variables in order to project high spatiotemporal resolution, continent-wide rabbit abundances. We show that rabbit abundance in Australia is highly variable in space and time, being driven primarily by internnual variation in temperature and precipitation in concert with the prevalence of a non-pathogenic virus. Moreover, we show that internnual variation in local spatial abundances can be mapped effectively at a continental scale using highly resolved spatiotemporal predictors, allowing "hot spots" of persistently high rabbit abundance to be identified. Importantly, cross-validated model performance was fair to excellent within and across distinct climate zones. Long-term monitoring data for invasive species can be used to map fine-scale spatiotemporal fluctuations in abundance patterns when accurately accounting for inherent sampling biases. Our analysis provides ecologists and pest managers with a clearer understanding of the determinants of rabbit abundance in Australia, offering an important new approach for predicting spatial abundance patterns of invasive species at the near-term temporal scales that are directly relevant to resource management.

TRPV4 blockade reduces voiding frequency, ATP release, and pelvic sensitivity in mice with chronic urothelial overexpression of NGF.

Transient receptor potential vanilloid family member 4 (TRPV4) transcript and protein expression increased in the urinary bladder and lumbosacral dorsal root ganglia of transgenic mice with chronic urothelial overexpression of nerve growth factor (NGF-OE). We evaluated the functional role of TRPV4 in bladder function with open-outlet cystometry, void spot assays, and natural voiding (Urovoid) assays with the TRPV4 antagonist HC-067047 (1 µM) or vehicle in NGF-OE and littermate wild-type (WT) mice. Blockade of TRPV4 at the level of the urinary bladder significantly (P ≤ 0.01) increased the intercontraction interval (2.2-fold) and void volume (2.6-fold) and decreased nonvoiding contractions (3.0-fold) in NGF-OE mice, with lesser effects (1.3-fold increase in the intercontraction interval and 1.3-fold increase in the void volume) in WT mice. Similar effects of TRPV4 blockade on bladder function in NGF-OE mice were demonstrated with natural voiding assays. Intravesical administration of HC-067047 (1 µM) significantly (P ≤ 0.01) reduced pelvic sensitivity in NGF-OE mice but was without effect in littermate WT mice. Blockade of urinary bladder TRPV4 or intravesical infusion of brefeldin A significantly (P ≤ 0.01) reduced (2-fold) luminal ATP release from the urinary bladder in NGF-OE and littermate WT mice. The results of the present study suggest that TRPV4 contributes to luminal ATP release from the urinary bladder and increased voiding frequency and pelvic sensitivity in NGF-OE mice.

Rabbit Hemorrhagic Disease Virus 2 (RHDV2; GI.2) Is Replacing Endemic Strains of RHDV in the Australian Landscape within 18 Months of Its Arrival.

Rabbit hemorrhagic disease virus 2 (RHDV2; Lagovirus GI.2) is a pathogenic calicivirus that affects European rabbits (Oryctolagus cuniculus) and various hare (Lepus) species. GI.2 was first detected in France in 2010 and subsequently caused epidemics in wild and domestic lagomorph populations throughout Europe. In May 2015, GI.2 was detected in Australia. Within 18 months of its initial detection, GI.2 had spread to all Australian states and territories and rapidly became the dominant circulating strain, replacing Rabbit hemorrhagic disease virus (RHDV/GI.1) in mainland Australia. Reconstruction of the evolutionary history of 127 Australian GI.2 isolates revealed that the virus arrived in Australia at least several months before its initial description and likely circulated unnoticed in wild rabbit populations in the east of the continent prior to its detection. GI.2 sequences isolated from five hares clustered with sequences from sympatric rabbit populations sampled contemporaneously, indicating multiple spillover events into hares rather than an adaptation of the Australian GI.2 to a new host. Since the presence of GI.2 in Australia may have wide-ranging consequences for rabbit biocontrol, particularly with the release of the novel biocontrol agent GI.1a/RHDVa-K5 in March 2017, ongoing surveillance is critical to understanding the interactions of the various lagoviruses in Australia and their impact on host populations.IMPORTANCE This study describes the spread and distribution of Rabbit hemorrhagic disease virus 2 (GI.2) in Australia since its first detection in May 2015. Within the first 18 months following its detection, RHDV2 spread from east to west across the continent and became the dominant strain in all mainland states of Australia. This has important implications for pest animal management and for owners of pet and farmed rabbits, as there currently is no effective vaccine available in Australia for GI.2. The closely related RHDV (GI.1) is used to control overabundant wild rabbits, a serious environmental and agricultural pest in this country, and it is currently unclear how the widespread circulation of GI.2 will impact ongoing targeted wild rabbit management operations.

Sulfasalazine decreases mouse cortical hyperexcitability.

OBJECTIVE: Currently prescribed antiepileptic drugs (AEDs) are ineffective in treating approximately 30% of epilepsy patients. Sulfasalazine (SAS) is an US Food and Drug Administration (FDA)-approved drug for the treatment of Crohn disease that has been shown to inhibit the cystine/glutamate antiporter system xc- (SXC) and decrease tumor-associated seizures. This study evaluates the effect of SAS on distinct pharmacologically induced network excitability and determines whether it can further decrease hyperexcitability when administered with currently prescribed AEDs. METHODS: Using in vitro cortical mouse brain slices, whole-cell patch-clamp recordings were made from layer 2/3 pyramidal neurons. Epileptiform activity was induced with bicuculline (bic), 4-aminopyridine (4-AP) and magnesium-free (Mg2+ -free) solution to determine the effect of SAS on epileptiform events. In addition, voltage-sensitive dye (VSD) recordings were performed to characterize the effect of SAS on the spatiotemporal spread of hyperexcitable network activity and compared to currently prescribed AEDs. RESULTS: SAS decreased evoked excitatory postsynaptic currents (eEPSCs) and increased the decay kinetics of evoked inhibitory postsynaptic currents (eIPSCs) in layer 2/3 pyramidal neurons. Although application of SAS to bic and Mg2+ -free-induced epileptiform activity caused a decrease in the duration of epileptiform events, SAS completely blocked 4-AP-induced epileptiform events. In VSD recordings, SAS decreased VSD optical signals induced by 4-AP. Co-application of SAS with the AED topiramate (TPM) caused a significantly further decrease in the spatiotemporal spread of VSD optical signals. SIGNIFICANCE: Taken together this study provides evidence that inhibition of SXC by SAS can decrease network hyperexcitability induced by three distinct pharmacologic agents in the superficial layers of the cortex. Furthermore, SAS provided additional suppression of 4-AP-induced network activity when administered with the currently prescribed AED TPM. These findings may serve as a foundation to assess the potential for SAS or other compounds that selectively target SXC as an adjuvant treatment for epilepsy.

Analyses of LMNA-negative juvenile progeroid cases confirms biallelic POLR3A mutations in Wiedemann-Rautenstrauch-like syndrome and expands the phenotypic spectrum of PYCR1 mutations.

Juvenile segmental progeroid syndromes are rare, heterogeneous disorders characterized by signs of premature aging affecting more than one tissue or organ starting in childhood. Hutchinson-Gilford progeria syndrome (HGPS), caused by a recurrent de novo synonymous LMNA mutation resulting in aberrant splicing and generation of a mutant product called progerin, is a prototypical example of such disorders. Here, we performed a joint collaborative study using massively parallel sequencing and targeted Sanger sequencing, aimed at delineating the underlying genetic cause of 14 previously undiagnosed, clinically heterogeneous, non-LMNA-associated juvenile progeroid patients. The molecular diagnosis was achieved in 11 of 14 cases (~ 79%). Furthermore, we firmly establish biallelic mutations in POLR3A as the genetic cause of a recognizable, neonatal, Wiedemann-Rautenstrauch-like progeroid syndrome. Thus, we suggest that POLR3A mutations are causal for a portion of under-diagnosed early-onset segmental progeroid syndromes. We additionally expand the clinical spectrum associated with PYCR1 mutations by showing that they can somewhat resemble HGPS in the first year of life. Moreover, our results lead to clinical reclassification in one single case. Our data emphasize the complex genetic and clinical heterogeneity underlying progeroid disorders.

Survey of plasma proteins in children with progeria pre-therapy and on-therapy with lonafarnib.

BackgroundHutchinson-Gilford progeria syndrome (HGPS) is an ultra-rare, fatal, segmental premature aging syndrome caused by the aberrant lamin A protein, progerin. The protein farnesyltransferase inhibitor, lonafarnib, ameliorates some aspects of cardiovascular and bone disease.MethodsWe performed a prospective longitudinal survey of plasma proteins in 24 children with HGPS (an estimated 10% of the world's population at the time) at baseline and on lonafarnib therapy, compared with age- and gender-matched controls using a multi-analyte, microsphere-based immunofluorescent assay.ResultsThe mean levels for 23/66 (34.8%) proteins were significantly lower and 7/66 (10.6%) were significantly higher in HGPS samples compared with those in controls (P≤0.05). Six proteins whose concentrations were initially lower normalized with lonafarnib therapy: interleukins 1α, 7, and 13, beta-2 microglobulin, C-reactive protein, and myoglobin. Alpha-2 macroglobulin, a protease inhibitor associated with stroke, was elevated at baseline and subsequently normalized with lonafarnib therapy.ConclusionThis is the first study to employ a multi-analyte array platform in HGPS. Novel potential biomarkers identified in this study should be further validated by correlations with clinical disease status, especially proteins associated with cardiovascular disease and those that normalized with lonafarnib therapy.

A novel somatic mutation achieves partial rescue in a child with Hutchinson-Gilford progeria syndrome.

BACKGROUND: Hutchinson-Gilford progeria syndrome (HGPS) is a fatal sporadic autosomal dominant premature ageing disease caused by single base mutations that optimise a cryptic splice site within exon 11 of the LMNA gene. The resultant disease-causing protein, progerin, acts as a dominant negative. Disease severity relies partly on progerin levels. METHODS AND RESULTS: We report a novel form of somatic mosaicism, where a child possessed two cell populations with different HGPS disease-producing mutations of the same nucleotide-one producing severe HGPS and one mild HGPS. The proband possessed an intermediate phenotype. The mosaicism was initially discovered when Sanger sequencing showed a c.1968+2T>A mutation in blood DNA and a c.1968+2T>C in DNA from cultured fibroblasts. Deep sequencing of DNA from the proband's blood revealed 4.7% c.1968+2T>C mutation, and 41.3% c.1968+2T>A mutation. CONCLUSIONS: We hypothesise that the germline mutation was c.1968+2T>A, but a rescue event occurred during early development, where the somatic mutation from A to C at 1968+2 provided a selective advantage. This type of mosaicism where a partial phenotypic rescue event results from a second but milder disease-causing mutation in the same nucleotide has not been previously characterised for any disease.

Association of Lonafarnib Treatment vs No Treatment With Mortality Rate in Patients With Hutchinson-Gilford Progeria Syndrome.

Importance: Hutchinson-Gilford progeria syndrome (HGPS) is an extremely rare fatal premature aging disease. There is no approved treatment. Objective: To evaluate the association of monotherapy using the protein farnesyltransferase inhibitor lonafarnib with mortality rate in children with HGPS. Design, Setting, and Participants: Cohort study comparing contemporaneous (birth date ≥1991) untreated patients with HGPS matched with treated patients by age, sex, and continent of residency using conditional Cox proportional hazards regression. Treatment cohorts included patients from 2 single-group, single-site clinical trials (ProLon1 [n = 27; completed] and ProLon2 [n = 36; ongoing]). Untreated patients originated from a separate natural history study (n = 103). The cutoff date for patient follow-up was January 1, 2018. Exposure: Treated patients received oral lonafarnib (150 mg/m2) twice daily. Untreated patients received no clinical trial medications. Main Outcomes and Measures: The primary outcome was mortality. The primary analysis compared treated patients from the first lonafarnib trial with matched untreated patients. A secondary analysis compared the combined cohorts from both lonafarnib trials with matched untreated patients. Results: Among untreated and treated patients (n = 258) from 6 continents, 123 (47.7%) were female; 141 (54.7%) had a known genotype, of which 125 (88.7%) were classic (c.1824C>T in LMNA). When identified (n = 73), the primary cause of death was heart failure (79.4%). The median treatment duration was 2.2 years. Median age at start of follow-up was 8.4 (interquartile range [IQR], 4.8-9.5) years in the first trial cohort and 6.5 (IQR, 3.7-9.0) years in the combined cohort. There was 1 death (3.7%) among 27 patients in the first trial group and there were 9 deaths (33.3%) among 27 patients in the matched untreated group. Treatment was associated with a lower mortality rate (hazard ratio, 0.12; 95% CI, 0.01-0.93; P = .04). In the combined cohort, there were 4 deaths (6.3%) among 63 patients in the treated group and 17 deaths (27.0%) among 63 patients in the matched untreated group (hazard ratio, 0.23; 95% CI, 0.06-0.90; P = .04). Conclusions and Relevance: Among patients with HGPS, lonafarnib monotherapy, compared with no treatment, was associated with a lower mortality rate after 2.2 years of follow-up. Study interpretation is limited by its observational design.

Nursing Student Collaborative With Meals on Wheels: Home Visits for At-Risk Senior Adults.

Nursing students at Washington State University Tri-Cities Campus are taking part in a program to provide care for homebound senior adults enrolled in home delivery meal services. Students perform home safety, functional, and cognitive assessments while learning the challenges of meeting nutritional needs of an at-risk senior adult population. This article outlines the program and its outcomes after two years of collaboration. Meals on Wheels is identified as an innovative placement for meeting nursing program outcomes and clinical practice hours. Areas for future growth include interdisciplinary collaboratives and partnering with local hospitals to study the program's impact on hospital readmission.

Altered phosphorylation, electrophysiology, and behavior on attenuation of PDE4B action in hippocampus.

BACKGROUND: PDE4 cyclic nucleotide phosphodiesterases regulate 3', 5' cAMP abundance in the CNS and thereby regulate PKA activity and phosphorylation of CREB, which has been implicated in learning and memory, depression and other functions. The PDE4 isoform PDE4B1 also interacts with the DISC1 protein, implicated in neural development and behavioral disorders. The cellular functions of PDE4B1 have been investigated extensively, but its function(s) in the intact organism remained unexplored. RESULTS: To specifically disrupt PDE4B1, we developed mice that express a PDE4B1-D564A transgene in the hippocampus and forebrain. The transgenic mice showed enhanced phosphorylation of CREB and ERK1/2 in hippocampus. Hippocampal neurogenesis was increased in the transgenic mice. Hippocampal electrophysiological studies showed increased baseline synaptic transmission and enhanced LTP in male transgenic mice. Behaviorally, male transgenic mice showed increased activity in prolonged open field testing, but neither male nor female transgenic mice showed detectable anxiety-like behavior or antidepressant effects in the elevated plus-maze, tail-suspension or forced-swim tests. Neither sex showed any significant differences in associative fear conditioning or showed any demonstrable abnormalities in pre-pulse inhibition. CONCLUSIONS: These data support the use of an isoform-selective approach to the study of PDE4B1 function in the CNS and suggest a probable role of PDE4B1 in synaptic plasticity and behavior. They also provide additional rationale and a refined approach to the development of small-molecule PDE4B1-selective inhibitors, which have potential functions in disorders of cognition, memory, mood and affect.