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.

Stress-induced symptom exacerbation: Stress increases voiding frequency, somatic sensitivity, and urinary bladder NGF and BDNF expression in mice with subthreshold cyclophosphamide (CYP).

Symptom exacerbation due to stress is prevalent in many disease states, including functional disorders of the urinary bladder (e.g., overactive bladder (OAB), interstitial cystitis/bladder pain syndrome (IC/BPS)); however, the mechanisms underlying the effects of stress on micturition reflex function are unclear. In this study we designed and evaluated a stress-induced symptom exacerbation (SISE) mouse model that demonstrates increased urinary frequency and somatic (pelvic and hindpaw) sensitivity. Cyclophosphamide (CYP) (35 mg/kg; i.p., every 48 hours for a total of 4 doses) or 7 days of repeated variate stress (RVS) did not alter urinary bladder function or somatic sensitivity; however, both CYP alone and RVS alone significantly (p ≤ 0.01) decreased weight gain and increased serum corticosterone. CYP treatment when combined with RVS for 7 days (CYP+RVS) significantly (p ≤ 0.01) increased serum corticosterone, urinary frequency and somatic sensitivity and decreased weight gain. CYP+RVS exposure in mice significantly (p ≤ 0.01) increased (2.6-fold) voiding frequency as we determined using conscious, open-outlet cystometry. CYP+RVS significantly (p ≤ 0.05) increased baseline, threshold, and peak micturition pressures. We also evaluated the expression of NGF, BDNF, CXC chemokines and IL-6 in urinary bladder in CYP alone, RVS alone and CYP+RVS mouse cohorts. Although all treatments or exposures increased urinary bladder NGF, BDNF, CXC and IL-6 content, CYP+RVS produced the largest increase in all inflammatory mediators examined. These results demonstrated that CYP alone or RVS alone creates a change in the inflammatory environment of the urinary bladder but does not result in a change in bladder function or somatic sensitivity until CYP is combined with RVS (CYP+RVS). The SISE model of CYP+RVS will be useful to develop testable hypotheses addressing underlying mechanisms where psychological stress exacerbates symptoms in functional bladder disorders leading to identification of targets and potential treatments.

Repeated variate stress increased voiding frequency and altered TrpV1 and TrpV4 transcript expression in lower urinary tract (LUT) pathways in female mice.

Psychological stress is associated with urinary bladder dysfunction (e.g., increased voiding frequency, urgency and pelvic pain); however, the mechanisms underlying the effects of stress on urinary bladder function are unknown. Transient receptor potential (TRP) channels (vanilloid family) may be potential targets for intervention due to their distribution in the LUT and role in pain. Here, we examine a model of repeated variate stress (RVS) of 2 week (wk) or 4 wk duration in female mice and its effects on bladder function, anxiety-like behavior, and TRPV transcript expression in urinary bladder and lumbosacral spinal cord and associated dorsal root ganglia (DRG). Using continuous infusion, open-outlet cystometry in conscious mice, RVS significantly (p ≤ 0.05) decreased infused volume and intermicturition interval. Bladder pressures (threshold, average, minimum, and maximum pressures) were unchanged with RVS. Quantitative PCR demonstrated significant (p ≤ 0.05) changes in TrpV1 and TrpV4 mRNA expression between control and RVS cohorts in the urothelium, lumbosacral spinal cord, and DRG. Future directions will examine the contribution of TRP channels on bladder function, somatic sensation and anxiety-like behavior following RVS.

Imatinib Mesylate Reduces Voiding Frequency in Female Mice With Acute Cyclophosphamide-Induced Cystitis.

Lamina propria interstitial cells that express the tyrosine kinase receptor, platelet-derived growth factor receptor alpha (PDGFRα) may play a role in urinary sensory signaling. Imatinib mesylate, also referred to as imatinib, is a tyrosine kinase inhibitor that can inhibit PDGFRα and has been widely used in urological research. We evaluated the functional effects of imatinib administration (via oral gavage or intravesical infusion) with two different experimental designs (prevention and treatment), in a cyclophosphamide (CYP)-induced cystitis (acute, intermediate, and chronic), male and female rodent model using conscious cystometry and somatic sensitivity testing. Imatinib significantly (0.0001 ≤ p ≤ 0.05) decreased voiding frequency and increased bladder capacity in acute CYP-induced cystitis, by the prevention (females) and treatment (females and males) designs. Imatinib was not effective in preventing or treating intermediate or chronic CYP-induced cystitis in either sex. Interestingly, in the prevention experiments, imatinib administration increased (0.0001 ≤ p ≤ 0.01) voiding frequency and decreased bladder capacity in control mice. However, in the treatment experiments, imatinib administration decreased (0.01 ≤ p ≤ 0.05) voiding frequency and increased bladder capacity in control mice. Bladder function improvements observed with imatinib treatment in acute CYP-induced cystitis mice remained and additionally improved with a second dose of imatinib 24 hours after CYP treatment. Imatinib administration did not affect pelvic somatic sensitivity in female mice with acute CYP-induced cystitis. Our studies suggest that (1) imatinib improves bladder function in mice with acute CYP-induced cystitis with a prevention and treatment design and (2) interstitial cells may be a useful target to improve bladder function in cystitis.

Imatinib Mesylate Reduces Neurotrophic Factors and pERK and pAKT Expression in Urinary Bladder of Female Mice With Cyclophosphamide-Induced Cystitis.

Imatinib mesylate is a tyrosine kinase inhibitor that inhibits platelet-derived growth factor receptor (PDGFR)-α, -ß, stem cell factor receptor (c-KIT), and BCR-ABL. PDGFRα is expressed in a subset of interstitial cells in the lamina propria (LP) and detrusor muscle of the urinary bladder. PDGFRα + interstitial cells may contribute to bladder dysfunction conditions such as interstitial cystitis/bladder pain syndrome (IC/BPS) or overactive bladder (OAB). We have previously demonstrated that imatinib prevention via oral gavage or treatment via intravesical infusion improves urinary bladder function in mice with acute (4 hour, h) cyclophosphamide (CYP)-induced cystitis. Here, we investigate potential underlying mechanisms mediating the bladder functional improvement by imatinib using a prevention or treatment experimental design. Using qRT-PCR and ELISAs, we examined inflammatory mediators (NGF, VEGF, BDNF, CCL2, IL-6) previously shown to affect bladder function in CYP-induced cystitis. We also examined the distribution of phosphorylated (p) ERK and pAKT expression in the LP with immunohistochemistry. Imatinib prevention significantly (0.0001 ≤ p ≤ 0.05) reduced expression for all mediators examined except NGF, whereas imatinib treatment was without effect. Imatinib prevention and treatment significantly (0.0001 ≤ p ≤ 0.05) reduced pERK and pAKT expression in the upper LP (U. LP) and deeper LP (D. LP) in female mice with 4 h CYP-induced cystitis. Although we have previously demonstrated that imatinib prevention or treatment improves bladder function in mice with cystitis, the current studies suggest that reductions in inflammatory mediators contribute to prevention benefits of imatinib but not the treatment benefits of imatinib. Differential effects of imatinib prevention or treatment on inflammatory mediators may be influenced by the route and frequency of imatinib administration and may also suggest other mechanisms (e.g., changes in transepithelial resistance of the urothelium) through which imatinib may affect urinary bladder function following CYP-induced cystitis.

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.

Effects of pharmacological neurotrophin receptor inhibition on bladder function in female mice with cyclophosphamide-induced cystitis.

Interstitial cystitis/bladder pain syndrome is a chronic inflammatory pelvic pain syndrome of unknown etiology characterized by a number of lower urinary tract symptoms, including increased urinary urgency and frequency, bladder discomfort, decreased bladder capacity, and pelvic pain. While its etiology remains unknown, a large body of evidence suggests a role for changes in neurotrophin signaling, particularly that of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Here, we evaluated the effects of pharmacological inhibition of the NGF receptor TrkA, BDNF receptor TrkB, and pan-neurotrophin receptor p75NTR on bladder function in acute (4-hour) and chronic (8-day) mouse models of cyclophosphamide (CYP)-induced cystitis. TrkA inhibition via ARRY-954 significantly increased intermicturition interval and bladder capacity in control and acute and chronic CYP-treatment conditions. TrkB inhibition via ANA-12 significantly increased intermicturition interval and bladder capacity in acute, but not chronic, CYP-treatment conditions. Interestingly, intermicturition interval and bladder capacity significantly increased following p75NTR inhibition via LM11A-31 in the acute CYP-treatment condition, but decreased in the chronic condition, potentially due to compensatory changes in neurotrophin signaling or increased urothelial barrier dysfunction in the chronic condition. Our findings demonstrate that these receptors represent additional potent therapeutic targets in mice with cystitis and may be useful in the treatment of interstitial cystitis and other inflammatory disorders of the bladder.

Relationship between vitamin D status and clinical outcomes in dogs with a cranial cruciate ligament rupture.

Cranial cruciate ligament rupture (CCLR) is one of the most common orthopaedic disorders diagnosed in dogs yet the factors which influence postoperative clinical outcomes are poorly understood. Low vitamin D status has been linked to poorer clinical outcomes in human patients undergoing elective orthopaedic surgery. The aim of this study was to examine the relationship between pre-operative vitamin D status, as defined by serum 25 hydroxyvitamin D (25(OH)D) concentrations, and initial disease severity and clinical outcomes in dogs undergoing surgical treatment for a CCLR. Serum 25(OH)D concentrations were measured in 44 dogs with a CCLR on the day before surgery. C-reactive protein concentrations were measured at a median time of 1 day post-surgery and the patient's clinical and radiographic response to CCLR surgical treatment was assessed at a median timepoint of 60 days post-surgery. Serum 25(OH)D concentrations in dogs with a CCLR was not significantly different to a population of healthy dogs (median 74.1 nmol/L and 88.40 nmol/L, respectively). There was no significant correlation between pre-operative serum 25(OH)D concentrations and length of pre-diagnosis clinical signs, pre-operative lameness scores or day 1 post-operative CRP concentrations. Thirty nine of the 44 dogs were re-examined at a median 60 days post-surgery. There was no relationship between the day 60 lameness scores and pre-operative serum 25(OH)D concentrations. In summary, we discovered that the vitamin D status of dogs with a CCLR was not significantly lower than healthy dogs and pre-operative serum 25(OH)D concentrations were not correlated to either pre-surgical disease severity or post-operative clinical outcomes.

Intrabladder PAC1 Receptor Antagonist, PACAP(6-38), Reduces Urinary Bladder Frequency and Pelvic Sensitivity in Mice Exposed to Repeated Variate Stress (RVS).

Stress causes symptom exacerbation in functional disorders of the urinary bladder. However, the potential mediators and underlying mechanisms of stress effects on micturition reflex function are unknown. We have characterized PACAP (Adcyap1) and PAC1 receptor (Adcyap1r1) signaling in stress-induced urinary bladder dysfunction in mice. We determined PACAP and PAC1 transcripts and protein expressions in the urinary bladder and lumbosacral dorsal root ganglia (DRG) and spinal cord in repeated variate stress (RVS) or control mouse (handling only) groups. RVS in mice significantly (p ≤ 0.01) increased serum corticosterone and urinary bladder NGF content and decreased weight gain. PACAP and PAC1 mRNA and protein were differentially regulated in lower urinary tract tissues with changes observed in lumbosacral DRG and spinal cord but not in urinary bladder. RVS exposure in mice significantly (p ≤ 0.01) increased (2.5-fold) voiding frequency as determined using conscious cystometry. Intrabladder administration of the PAC1 receptor antagonist, PACAP(6-38) (300 nM), significantly (p ≤ 0.01) increased infused volume (1.5-2.7-fold) to elicit a micturition event and increased the intercontraction interval (i.e., decreased voiding frequency) in mice exposed to RVS and in control mice, but changes were smaller in magnitude in control mice. We also evaluated the effect of PAC1 blockade at the level of the urinary bladder on pelvic sensitivity in RVS or control mouse groups using von Frey filament testing. Intrabladder administration of PACAP(6-38) (300 nM) significantly (p ≤ 0.01) reduced pelvic sensitivity following RVS. PACAP/receptor signaling in the CNS and PNS contributes to increased voiding frequency and pelvic sensitivity following RVS and may represent a potential target for therapeutic intervention.

Effects of surgery on free and total 25 hydroxyvitamin D concentrations in dogs.

BACKGROUND: It is unclear whether a low total 25(OH)D concentration is a cause or consequence of illnesses. To address this knowledge gap, studies measuring free and total 25(OH)D during the evolution and resolution of an inflammatory process are required. OBJECTIVES: Serum total and free 25(OH)D concentrations would transiently decline after cruciate surgery in dogs. ANIMALS: Seventeen client-owned dogs with a spontaneous cranial cruciate ligament rupture (CCLR). METHODS: A longitudinal cohort study involving the measurement of serum concentrations of total and free 25(OH)D, total calcium, creatinine, albumin, phosphate, C-reactive protein and plasma ionized calcium, at 1 day before and a median time of 1 and 60 days after surgical treatment of CCLR. RESULTS: Median serum concentrations of total 25(OH)D before surgery (80.3 nmoL/L [range, 43.5-137.3]) significantly declined immediately after surgery; (64.8 nmoL/L [range, 36.3-116.5] 1 day after surgery, P < .005) before increasing to become nonsignificantly different from concentrations before surgery at day 60 after surgery (median 78.0 nmoL/L [range, 24.2-115.8], P = .14). In contrast, median free 25(OH)D concentrations before surgery (7.6 pg/mL [range, 3.8-12.2]) significantly increased immediately after surgery (9.2 pg/mL [range, 5.2-15.7], P < .05) before declining to become nonsignificantly different from before surgery concentrations at day 60 after surgery (median 6.2 pg/mL [range, 4.0-15.8], P = .37). CONCLUSION AND CLINICAL IMPORTANCE: This study reveals the difficulties of assessing vitamin D status in dogs following elective surgery.

Glioma-induced peritumoral hyperexcitability in a pediatric glioma model.

Epileptic seizures are among the most common presenting symptom in patients with glioma. The etiology of glioma-related seizures is complex and not completely understood. Studies using adult glioma patient tissue and adult glioma mouse models, show that neurons adjacent to the tumor mass, peritumoral neurons, are hyperexcitable and contribute to seizures. Although it is established that there are phenotypic and genotypic distinctions in gliomas from adult and pediatric patients, it is unknown whether these established differences in pediatric glioma biology and the microenvironment in which these glioma cells harbor, the developing brain, differentially impacts surrounding neurons. In the present study, we examine the effect of patient-derived pediatric glioma cells on the function of peritumoral neurons using two pediatric glioma models. Pediatric glioma cells were intracranially injected into the cerebrum of postnatal days 2 and 3 (p2/3) mouse pups for 7 days. Electrophysiological recordings showed that cortical layer 2/3 peritumoral neurons exhibited significant differences in their intrinsic properties compared to those of sham control neurons. Peritumoral neurons fired significantly more action potentials in response to smaller current injection and exhibited a depolarization block in response to higher current injection. The threshold for eliciting an action potential and pharmacologically induced epileptiform activity was lower in peritumoral neurons compared to sham. Our findings suggest that pediatric glioma cells increase excitability in the developing peritumoral neurons by exhibiting early onset of depolarization block, which was not previously observed in adult glioma peritumoral neurons.

Potassium and glutamate transport is impaired in scar-forming tumor-associated astrocytes.

Unprovoked recurrent seizures are a serious comorbidity affecting most patients who suffer from glioma, a primary brain tumor composed of malignant glial cells. Cellular mechanisms contributing to the development of recurrent spontaneous seizures include the release of the excitatory neurotransmitter glutamate from glioma into extracellular space. Under physiological conditions, astrocytes express two high affinity glutamate transporters, Glt-1 and Glast, which are responsible for the removal of excess extracellular glutamate. In the context of neurological disease or brain injury, astrocytes become reactive which can negatively affect neuronal function, causing hyperexcitability and/or death. Using electrophysiology, immunohistochemistry, fluorescent in situ hybridization, and Western blot analysis in different orthotopic xenograft and allograft models of human and mouse gliomas, we find that peritumoral astrocytes exhibit astrocyte scar formation characterized by proliferation, cellular hypertrophy, process elongation, and increased GFAP and pSTAT3. Overall, peritumoral reactive astrocytes show a significant reduction in glutamate and potassium uptake, as well as decreased glutamine synthetase activity. A subset of peritumoral astrocytes displayed a depolarized resting membrane potential, further contributing to reduced potassium and glutamate homeostasis. These changes may contribute to the propagation of peritumoral neuronal hyperexcitability and excitotoxic death.

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.

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.

Cellular eIF2B subunit localization: implications for the integrated stress response and its control by small molecule drugs.

Eukaryotic initiation factor 2 (eIF2) is a G protein critical for translation. It is tightly regulated in the integrated stress response (ISR) via phosphorylation of eIF2α and the subsequent control of eukaryotic initiation factor 2B (eIF2B), a multisubunit guanine nucleotide exchange factor. Through studying the localization of eIF2B subunits, we identified cytoplasmic eIF2B bodies in mammalian cells. We highlight a relationship between body size and the eIF2B subunits localizing to them; larger bodies contain all subunits and smaller bodies contain predominantly catalytic subunits. eIF2 localizes to eIF2B bodies and shuttles within these bodies in a manner that correlates with eIF2B activity. On stress, eIF2α-P localizes predominately to larger bodies and results in a decreased shuttling of eIF2. Interestingly, drugs that inhibit the ISR can rescue eIF2 shuttling in a manner correlating to levels of eIF2α-P. In contrast, smaller bodies show increased eIF2 shuttling in response to stress, which is accompanied by the localization of eIF2Bδ to these bodies, suggesting the formation of a novel trimeric complex of eIF2B. This response is mimicked by ISR-inhibiting drugs, providing insight into their potential mechanism of action. This study provides evidence that the composition and function of mammalian eIF2B bodies are regulated by the ISR and the drugs that control it.

Perineuronal nets decrease membrane capacitance of peritumoral fast spiking interneurons in a model of epilepsy.

Brain tumor patients commonly present with epileptic seizures. We show that tumor-associated seizures are the consequence of impaired GABAergic inhibition due to an overall loss of peritumoral fast spiking interneurons (FSNs) concomitant with a significantly reduced firing rate of those that remain. The reduced firing is due to the degradation of perineuronal nets (PNNs) that surround FSNs. We show that PNNs decrease specific membrane capacitance of FSNs permitting them to fire action potentials at supra-physiological frequencies. Tumor-released proteolytic enzymes degrade PNNs, resulting in increased membrane capacitance, reduced firing, and hence decreased GABA release. These studies uncovered a hitherto unknown role of PNNs as an electrostatic insulator that reduces specific membrane capacitance, functionally akin to myelin sheaths around axons, thereby permitting FSNs to exceed physiological firing rates. Disruption of PNNs may similarly account for excitation-inhibition imbalances in other forms of epilepsy and PNN protection through proteolytic inhibition may provide therapeutic benefits.

Novel pathogenic mutations in C1QTNF5 support a dominant negative disease mechanism in late-onset retinal degeneration.

Late-onset retinal degeneration (L-ORD) is a rare autosomal dominant retinal dystrophy, characterised by extensive sub-retinal pigment epithelium (RPE) deposits, RPE atrophy, choroidal neovascularisation and photoreceptor cell death associated with severe visual loss. L-ORD shows striking phenotypic similarities to age-related macular degeneration (AMD), a common and genetically complex disorder, which can lead to misdiagnosis in the early stages. To date, a single missense mutation (S163R) in the C1QTNF5 gene, encoding C1q And Tumor Necrosis Factor Related Protein 5 (C1QTNF5) has been shown to cause L-ORD in a subset of affected families. Here, we describe the identification and characterisation of three novel pathogenic mutations in C1QTNF5 in order to elucidate disease mechanisms. In silico and in vitro characterisation show that these mutations perturb protein folding, assembly or polarity of secretion of C1QTNF5 and, importantly, all appear to destabilise the wildtype protein in co-transfection experiments in a human RPE cell line. This suggests that the heterozygous mutations in L-ORD show a dominant negative, rather than a haploinsufficient, disease mechanism. The function of C1QTNF5 remains unclear but this new insight into the pathogenetic basis of L-ORD has implications for future therapeutic strategies such as gene augmentation therapy.

Knowledge, Attitudes, and Beliefs Regarding Cardiovascular Disease in Women: The Women's Heart Alliance.

BACKGROUND: Cardiovascular disease (CVD) is the number 1 killer of women in the United States, yet few younger women are aware of this fact. CVD campaigns focus little attention on physicians and their roles in assessing risk. OBJECTIVES: In 2014, the Women's Heart Alliance (WHA) conducted a nationwide survey to determine barriers and opportunities for women and physicians with regard to CVD. METHODS: From September 18 to 26, 2014, a total of 1,011 U.S. women (age 25 to 60 years) were interviewed using the GfK ("Gesellschaft für Konsumforschung" Knowledge Panel). From May 6 to 12, 2014, the e-Rewards Inc. Physician and Healthcare Professional Panel surveyed 200 primary care physicians (PCPs) and 100 cardiologists. RESULTS: Overall, 45% of women were unaware that CVD is the number 1 killer of women; only 11% knew a woman who died from CVD. Overall, 45% of women reported it was common to cancel or postpone a physician appointment until losing weight. CVD was rated as the top concern by only 39% of PCPs, after weight and breast health. Only 22% of PCPs and 42% of cardiologists (p = 0.0477) felt extremely well prepared to assess CVD risk in women, while 42% and 40% felt well-prepared (p = NS), respectively. Few comprehensively implemented guidelines. CONCLUSIONS: CVD was rated as the top concern less frequently than weight issues by both women and physicians. Social stigma particularly regarding body weight appeared to be a barrier. Physicians reported limited training and use of guideline assessment, whereas most supported a campaign and improved physician education. Campaigns should make CVD "real" to U.S. women, countering stereotypes with facts and validated assessments. Both community women and physicians endorsed investment in women's CVD research and physician education.