Response of pollinator taxa to fire is consistent with historic fire regimes in the Sierra Nevada and mediated through floral richness†.

Many fire-prone forests are experiencing wildfires that burn outside the historical range of variation in extent and severity. These fires impact pollinators and the ecosystem services they provide, but how the effects of fire are mediated by burn severity in different habitats is not well understood. We used generalized linear mixed models in a Bayesian framework to model the abundance of pollinators as a function of burn severity, habitat, and floral resources in post-fire, mid-elevation, conifer forest, and meadow in the Sierra Nevada, California. Although most species-level effects were not significant, we found highly consistent negative impacts of burn severity in meadows where pollinators were most abundant, with only hummingbirds and some butterfly families responding positively to burn severity in meadows. Moderate-severity fire tended to increase the abundance of most pollinator taxa in upland forest habitat, indicating that even in large fires that burn primarily at high- and moderate-severity patches may be associated with improved habitat conditions for pollinator species in upland forest. Nearly all pollinator taxa responded positively to floral richness but not necessarily to floral abundance. Given that much of the Sierra Nevada is predicted to burn at high severity, limiting high-severity effects in meadow and upland habitats may help conserve pollinator communities whereas low- to moderate-severity fire may be needed in both systems.

Assessing the utility of common arguments used in expert review of in silico predictions as part of ICH M7 assessments.

Expert review of two predictions, made by complementary (quantitative) structure-activity relationship models, to an overall conclusion is a key component of using in silico tools to assess the mutagenic potential of impurities as part of the ICH M7 guideline. In lieu of a specified protocol, numerous publications have presented best practise guides, often indicating the occurrence of common prediction scenarios and the evidence required to resolve them. A semi-automated expert review tool has been implemented in Lhasa Limited's Nexus platform following collation of these common arguments and assignment to the associated prediction scenarios made by Derek Nexus and Sarah Nexus. Using datasets primarily donated by pharmaceutical companies, an automated analysis of the frequency these prediction scenarios occur, and the likelihood of the associated arguments assigning the correct resolution, could then be conducted. This article highlights that a relatively small number of common arguments may be used to accurately resolve many prediction scenarios to a single conclusion. The use of a standardised method of argumentation and assessment of evidence for a given impurity is proposed to improve the efficiency and consistency of expert review as part of an ICH M7 submission.

Estradiol- and Progesterone-Associated Changes in microRNA-Induced Silencing and Reduced Antiseizure Efficacy of an Antagomir in Female Mice.

About one-third of individuals living with epilepsy have treatment-resistant seizures. Alternative therapeutic strategies are thus urgently needed. One potential novel treatment target is miRNA-induced silencing, which is differentially regulated in epilepsy. Inhibitors (antagomirs) of specific microRNAs (miRNAs) have shown therapeutic promise in preclinical epilepsy studies; however, these studies were mainly conducted in male rodent models, and research into miRNA regulation in females and by female hormones in epilepsy is scarce. This is problematic because female sex and the menstrual cycle can affect the disease course of epilepsy and may, therefore, also alter the efficacy of potential miRNA-targeted treatments. Here, we used the proconvulsant miRNA miR-324-5p and its target, the potassium channel Kv4.2, as an example to test how miRNA-induced silencing and the efficacy of antagomirs in epilepsy are altered in female mice. We showed that Kv4.2 protein is reduced after seizures in female mice similar to male mice; however, in contrast to male mice, miRNA-induced silencing of Kv4.2 is unchanged, and miR-324-5p activity, as measured by the association with the RNA-induced silencing complex, is reduced in females after seizure. Moreover, an miR-324-5p antagomir does not consistently reduce seizure frequency or increase Kv4.2 in female mice. As a possible underlying mechanism, we found that miR-324-5p activity and the silencing of Kv4.2 in the brain were differentially correlated with plasma levels of 17ß-estradiol and progesterone. Our results suggest that hormonal fluctuations in sexually mature female mice influence miRNA-induced silencing and could alter the efficacy of potential future miRNA-based treatments for epilepsy in females.

Long-term consequences of neonatal encephalopathy in the hypothermia era: protocol for a follow-up cohort study at 9 years of age.

INTRODUCTION: Therapeutic hypothermia (TH) became the standard of care treatment for neonates with moderate and severe neonatal encephalopathy (NE) in most industrialized countries about 10 years ago. Although TH is effective in reducing mortality and the incidence of severe developmental disabilities, the recent literature converges in reporting frequent cognitive and behavioural difficulties at school entry in children with NE-TH. Although these challenges are deemed minor compared with cerebral palsy and intellectual disability, their impacts on a child's self-determination and family's well-being are quite significant. Therefore, the nature and extent of these difficulties need to be comprehensively described so that appropriate care can be offered. METHODS AND ANALYSIS: The current study will be the largest follow-up study of neonates with NE treated with TH to characterize their developmental outcomes and associated brain structural profiles at 9 years of age. Specifically, we will compare executive function, attention, social cognition, behaviour, anxiety, self-esteem, peer problems, brain volume, cortical features, white matter microstructure and myelination between children with NE-TH and matched peers without NE. Associations of perinatal risk factors and structural brain integrity with cognitive, behavioural and psycho-emotional deficits will be evaluated to inform about the potential aggravating and protective factors associated with function. ETHICS AND DISSEMINATION: This study is supported by the Canadian Institute of Health Research (202203PJT-480065-CHI-CFAC-168509), and received approval from the Pediatric Ethical Review Board of the McGill University Health Center (MP-37-2023-9320). The study findings will be disseminated in scientific journals and conferences and presented to parental associations and healthcare providers to inform best practices. TRIAL REGISTRATION NUMBER: NCT05756296.

Neurovascular Development in Pten and Tsc2 Mouse Mutants.

Hyperactivation of the mechanistic target of rapamycin (mTOR) signaling pathway is linked to more than a dozen neurologic diseases, causing a range of pathologies, including excess neuronal growth, disrupted neuronal migration, cortical dysplasia, epilepsy and autism. The mTOR pathway also regulates angiogenesis. For the present study, therefore, we queried whether loss of Pten or Tsc2, both mTOR negative regulators, alters brain vasculature in three mouse models: one with Pten loss restricted to hippocampal dentate granule cells [DGC-Pten knock-outs (KOs)], a second with widespread Pten loss from excitatory forebrain neurons (FB-Pten KOs) and a third with focal loss of Tsc2 from cortical excitatory neurons (f-Tsc2 KOs). Total hippocampal vessel length and volume per dentate gyrus were dramatically increased in DGC-Pten knock-outs. DGC-Pten knock-outs had larger dentate gyri overall, however, and when normalized to these larger structures, vessel density was preserved. In addition, tests of blood-brain barrier integrity did not reveal increased permeability. FB-Pten KOs recapitulated the findings in the more restricted DGC-Pten KOs, with increased vessel area, but preserved vessel density. FB-Pten KOs did, however, exhibit elevated levels of the angiogenic factor VegfA. In contrast to findings with Pten, focal loss of Tsc2 from cortical excitatory neurons produced a localized increase in vessel density. Together, these studies demonstrate that hypervascularization is not a consistent feature of mTOR hyperactivation models and suggest that loss of different mTOR pathway regulatory genes exert distinct effects on angiogenesis.

Food webs for three burn severities after wildfire in the Eldorado National Forest, California.

Wildfire dynamics are changing around the world and understanding their effects on ecological communities and landscapes is urgent and important. We report detailed food webs for unburned, low-to-moderate and high severity burned habitats three years post-fire in the Eldorado National Forest, California. The cumulative cross-habitat food web contains 3,084 ontogenetic stages (nodes) or plant parts comprising 849 species (including 107 primary producers, 634 invertebrates, 94 vertebrates). There were 178,655 trophic interactions between these nodes. We provide information on taxonomy, body size, biomass density and trophic interactions under each of the three burn conditions. We detail 19 sampling methods deployed across 27 sites (nine in each burn condition) used to estimate the richness, body size, abundance and biomass density estimates in the node lists. We provide the R code and raw data to estimate summarized node densities and assign trophic links.

Small mammal responses to fire severity mediated by vegetation characteristics and species traits.

The frequency of large, high-severity "mega-fires" has increased in recent decades, with numerous consequences for forest ecosystems. In particular, small mammal communities are vulnerable to post-fire shifts in resource availability and play critical roles in forest ecosystems. Inconsistencies in previous observations of small mammal community responses to fire severity underscore the importance of examining mechanisms regulating the effects of fire severity on post-fire recovery of small mammal communities. We compared small mammal abundance, diversity, and community structure among habitats that burned at different severities, and used vegetation characteristics and small mammal functional traits to predict community responses to fire severity three years after one mega-fire in the Sierra Nevada, California. Using a model-based fourth-corner analysis, we examined how interactions between vegetation variables and small mammal traits associated with their resource use were associated with post-fire small mammal community structure among fire severity categories. Small mammal abundance was similar across fire severity categories, but diversity decreased and community structure shifted as fire severity increased. Differences in small mammal communities were large only between unburned and high-severity sites. Three highly correlated fire-dependent vegetation variables affected by fire and the volume of soft coarse woody debris were associated with small mammal community structures. Furthermore, we found that interactions between vegetation variables and three small mammal traits (feeding guild, primary foraging mode, and primary nesting habit) predicted community structure across fire severity categories. We concluded that resource use was important in regulating small mammal recovery after the fire because vegetation provided required resources to small mammals as determined by their functional traits. Given the mechanistic nature of our analyses, these results may be applicable to other fire-prone forest systems, although it will be important to conduct studies across large biogeographic regions and over long post-fire time periods to assess generality.

Calculating qualified non-mutagenic impurity levels: Harmonization of approaches.

The presence of impurities in drugs is unavoidable. As impurities offer no direct benefit to the patient, it is critical that impurities do not compromise patient safety. Current guidelines on the derivation of acceptable impurity levels leave aspects of calculations open for interpretation, resulting in inconsistencies across industry and regulators. To understand current impurity qualification practices from a safety standpoint, regulatory expectations and the safety risk that impurities pose, the IQ DruSafe Impurities Working Group (WG) conducted a pharmaceutical industry-wide survey. Survey results highlighted areas that could benefit from harmonization, including nonclinical species/sex selection and the application of adjustment factors (i.e., body surface area). Recommendations for alignment on these topics is included in this publication. Additionally, the WG collated repeat-dose toxicity information for 181 starting materials and intermediates, reflective of pharmaceutical impurities, to understand the toxicological risks they generally pose in relation to the drug substance (DS) and the assumptions surrounding the calculation of qualified impurity levels. An evaluation of this dataset and the survey were used to harmonize how to calculate a safe limit for an impurity based on toxicology testing of the impurity when present within the DS.

GABAA Alpha 2,3 Modulation Improves Select Phenotypes in a Mouse Model of Fragile X Syndrome.

Fragile X syndrome (FXS) is the most common cause of inherited intellectual disability. FXS is caused by functional loss of the Fragile X Protein (FXP), also known as Fragile X Mental Retardation Protein (FMRP). In humans and animal models, loss of FXP leads to sensory hypersensitivity, increased susceptibility to seizures and cortical hyperactivity. Several components of the GABAergic system, the major inhibitory system in the brain, are dysregulated in FXS, and thus modulation of GABAergic transmission was suggested and tested as a treatment strategy. However, so far, clinical trials using broad spectrum GABAA or GABAB receptor-specific agonists have not yielded broad improvement of FXS phenotypes in humans. Here, we tested a more selective strategy in Fmr1 knockout (KO) mice using the experimental drug BAER-101, which is a selective GABAA α2/α3 agonist. Our results suggest that BAER-101 reduces hyperexcitability of cortical circuits, partially corrects increased frequency-specific baseline cortical EEG power, reduces susceptibility to audiogenic seizures and improves novel object memory. Other Fmr1 KO-specific phenotypes were not improved by the drug, such as increased hippocampal dendritic spine density, open field activity and marble burying. Overall, this work shows that BAER-101 improves select phenotypes in Fmr1 KO mice and encourages further studies into the efficacy of GABAA-receptor subunit-selective agonists for the treatment of FXS.

Use of less-than-lifetime (LTL) durational limits for nitrosamines: Case study of N-Nitrosodiethylamine (NDEA).

The ICH M7(R1) guideline describes a framework to assess the carcinogenic risk of mutagenic and carcinogenic pharmaceutical impurities following less-than-lifetime (LTL) exposures. This LTL framework is important as many pharmaceuticals are not administered for a patient's lifetime and as clinical trials typically involve LTL exposures. While there has been regulatory caution about applying LTL concepts to cohort of concern (COC) impurities such as N-nitrosamines, ICH M7 does not preclude this and indeed literature data suggests that the LTL framework will be protective of patient safety for N-nitrosamines. The goal was to investigate if applying the LTL framework in ICH M7 would control exposure to an acceptable excess cancer risk in humans. Using N-nitrosodiethylamine as a case study, empirical data correlating exposure duration (as a percentage of lifespan) and cancer incidence in rodent bioassays indicate that the LTL acceptable intake (AI) as derived using the ICH M7 framework would not exceed a negligible additional risk of cancer. Therefore, controlling N-nitrosamines to an LTL AI based on the ICH M7 framework is thus demonstrated to be protective for potential carcinogenic risk to patients over the exposure durations typical of clinical trials and many prescribed medicines.

In Silico Approaches In Carcinogenicity Hazard Assessment: Current Status and Future Needs.

Historically, identifying carcinogens has relied primarily on tumor studies in rodents, which require enormous resources in both money and time. In silico models have been developed for predicting rodent carcinogens but have not yet found general regulatory acceptance, in part due to the lack of a generally accepted protocol for performing such an assessment as well as limitations in predictive performance and scope. There remains a need for additional, improved in silico carcinogenicity models, especially ones that are more human-relevant, for use in research and regulatory decision-making. As part of an international effort to develop in silico toxicological protocols, a consortium of toxicologists, computational scientists, and regulatory scientists across several industries and governmental agencies evaluated the extent to which in silico models exist for each of the recently defined 10 key characteristics (KCs) of carcinogens. This position paper summarizes the current status of in silico tools for the assessment of each KC and identifies the data gaps that need to be addressed before a comprehensive in silico carcinogenicity protocol can be developed for regulatory use.

Practical Approaches and Knowledge Gaps in the Care for Children With Leukodystrophies.

Leukodystrophies are a group of neurodegenerative genetic disorders that affect approximately 1 in 7500 individuals. Despite therapeutic progress in individual leukodystrophies, guidelines in neurologic care are sparse and consensus among physicians and caregivers remains a challenge. At patient advocacy meetings hosted by Hunter's Hope from 2016-2018, multidisciplinary experts and caregivers met to conduct a literature review, identify knowledge gaps and summarize best practices regarding neurologic care. Stages of severity in leukodystrophies guided recommendations to address different levels of need based on a newly defined system of disease severity. Four core neurologic domains prioritized by families were identified and became the focus of this guideline: sleep, pain, seizures/epilepsy, and language/cognition. Based on clinical severity, the following categories were used: presymptomatic, early symptomatic, intermediate symptomatic, and advanced symptomatic. Across the leukodystrophies, neurologic care should be tailored to stages of severity while accounting for unique aspects of every disease and multiple knowledge gaps present. Standardized tools and surveys can help guide treatment but should not overburden families.

Experiences of Serving and Ex-Serving Members With the PTSD Coach Australia App: Mixed Methods Study.

BACKGROUND: PTSD Coach Australia is an app for serving and ex-serving defense members and was adapted for the Australian context in 2013 from PTSD Coach, which was created in the United States. OBJECTIVE: This study aimed to provide a user-centered evaluation of the app from the perspective of serving and ex-serving members of the Australian Defence Force. METHODS: Qualitative data were collected in response to questions to participants in 1 of 5 workshops (n=29) or in telephone interviews (n=24). Quantitative data were collected using the user version of Mobile Apps Rating Scale (uMARS). RESULTS: Analysis of the qualitative data demonstrated mixed support for the app. While some people found it extremely useful, especially as an adjunct to therapy, others pointed out limitations and cautioned against the app potentially triggering symptoms in people with PTSD. This perceived risk was usually found to stem from frustration with the app's functionality rather than its content. Participants spoke about the helpful and unhelpful aspects of the app and barriers to its use and made suggestions for improvement. Many participants encouraged its continued use and highlighted the need for it to be promoted more broadly, as many were not aware of it until they were invited to participate in this research. CONCLUSIONS: PTSD Coach Australia was seen in a positive light by some participants, but others thought it had too much text and the potential to trigger a traumatic response in users with PTSD. A need to update the app was also a common comment as was the need to increase awareness of the app's existence.

Clinicians' perceptions of PTSD Coach Australia.

INTRODUCTION: Applications or "mobile apps" are a potentially important source of assistance for serving and ex-serving Defence members with mental health problems. PTSD Coach Australia is a modified version of an application developed by the US Department of Veteran Affairs. Clinician perceptions of mobile apps are important as they influence the dissemination and adoption of apps. This study aimed to explore the perceptions of PTSD Coach Australia by clinicians with experience in assisting Defence members with mental health problems. METHOD: The study involved two samples of participants who were asked about their perceptions of PTSD Coach Australia. The first involved 33 clinicians who participated in one of five focus groups. The second comprised 30 clinicians who were individually interviewed by telephone. Qualitative responses to questions regarding PTSD Coach Australia were analysed to identify representative themes. Participants in the focus group sample also rated the app on the user version of the Mobile Apps Rating Scale (uMARS). RESULTS: On the uMARS, clinicians rated the mobile app's subjective quality as 'average' to 'good'. Participants generally saw the app as a useful to help track symptoms, improve engagement and help implement strategies between sessions. However, they also expressed concerns with the app not being user-friendly (e.g. too wordy, poor layout/navigation) and having technical issues (freezing or crashing on Android devices). DISCUSSION: PTSD Coach Australia is generally seen as being acceptable and useful by mental health clinicians. However, it is important to include their concerns in future developments of PTSD Coach Australia and similar mobile apps in order to maximize their utilisation in Defence members.

PI3K isoform-selective inhibition in neuron-specific PTEN-deficient mice rescues molecular defects and reduces epilepsy-associated phenotypes.

Epilepsy affects all ages, races, genders, and socioeconomic groups. In about one third of patients, epilepsy is uncontrolled with current medications, leaving a vast need for improved therapies. The causes of epilepsy are diverse and not always known but one gene mutated in a small subpopulation of patients is phosphatase and tensin homolog (PTEN). Moreover, focal cortical dysplasia, which constitutes a large fraction of refractory epilepsies, has been associated with signaling defects downstream of PTEN. So far, most preclinical attempts to reverse PTEN deficiency-associated neurological deficits have focused on mTOR, a signaling hub several steps downstream of PTEN. Phosphoinositide 3-kinases (PI3Ks), by contrast, are the direct enzymatic counteractors of PTEN, and thus may be alternative treatment targets. PI3K activity is mediated by four different PI3K catalytic isoforms. Studies in cancer, where PTEN is commonly mutated, have demonstrated that inhibition of only one isoform, p110ß, reduces progression of PTEN-deficient tumors. Importantly, inhibition of a single PI3K isoform leaves critical functions of general PI3K signaling throughout the body intact. Here, we show that this disease mechanism-targeted strategy borrowed from cancer research rescues or ameliorates neuronal phenotypes in male and female mice with neuron-specific PTEN deficiency. These phenotypes include cell signaling defects, protein synthesis aberrations, seizures, and cortical dysplasia. Of note, p110ß is also dysregulated and a promising treatment target in the intellectual disability Fragile X syndrome, pointing towards a shared biological mechanism that is therapeutically targetable in neurodevelopmental disorders of different etiologies. Overall, this work advocates for further assessment of p110ß inhibition not only in PTEN deficiency-associated neurodevelopmental diseases but also other brain disorders characterized by defects in the PI3K/mTOR pathway.

Skin sensitization in silico protocol.

The assessment of skin sensitization has evolved over the past few years to include in vitro assessments of key events along the adverse outcome pathway and opportunistically capitalize on the strengths of in silico methods to support a weight of evidence assessment without conducting a test in animals. While in silico methods vary greatly in their purpose and format; there is a need to standardize the underlying principles on which such models are developed and to make transparent the implications for the uncertainty in the overall assessment. In this contribution, the relationship between skin sensitization relevant effects, mechanisms, and endpoints are built into a hazard assessment framework. Based on the relevance of the mechanisms and effects as well as the strengths and limitations of the experimental systems used to identify them, rules and principles are defined for deriving skin sensitization in silico assessments. Further, the assignments of reliability and confidence scores that reflect the overall strength of the assessment are discussed. This skin sensitization protocol supports the implementation and acceptance of in silico approaches for the prediction of skin sensitization.

A new frontier in pelvic fracture pain control in the ED: Successful use of the pericapsular nerve group (PENG) block.

The pericapsular nerve group (PENG) block is a novel ultrasound-guided regional anesthesia technique derived from recent anatomic studies detailing the sensory innervation of the hip. Targeting these terminal sensory branches, the PENG block was originally developed as a potentially more effective block for perioperative hip fracture anesthesia, with the added benefit of preserving motor function. Subsequent research with higher volumes of local anesthetic demonstrated the successful utilization of PENG block for perioperative acetabular fractures. This raises the possibility that the PENG block may have a role in the Emergency Department (ED) where regional anesthesia options for pelvic fractures are lacking. Herein, we present the first description of PENG blocks successfully used for pelvic fractures in the ED setting.

MicroRNA inhibition upregulates hippocampal A-type potassium current and reduces seizure frequency in a mouse model of epilepsy.

Epilepsy is often associated with altered expression or function of ion channels. One example of such a channelopathy is the reduction of A-type potassium currents in the hippocampal CA1 region. The underlying mechanisms of reduced A-type channel function in epilepsy are unclear. Here, we show that inhibiting a single microRNA, miR-324-5p, which targets the pore-forming A-type potassium channel subunit Kv4.2, selectively increased A-type potassium currents in hippocampal CA1 pyramidal neurons in mice. Resting membrane potential, input resistance and other potassium currents were not altered. In a mouse model of acquired chronic epilepsy, inhibition of miR-324-5p reduced the frequency of spontaneous seizures and interictal epileptiform spikes supporting the physiological relevance of miR-324-5p-mediated control of A-type currents in regulating neuronal excitability. Mechanistic analyses demonstrated that microRNA-induced silencing of Kv4.2 mRNA is increased in epileptic mice leading to reduced Kv4.2 protein levels, which is mitigated by miR-324-5p inhibition. By contrast, other targets of miR-324-5p were unchanged. These results suggest a selective miR-324-5p-dependent mechanism in epilepsy regulating potassium channel function, hyperexcitability and seizures.

Isoform-selective phosphoinositide 3-kinase inhibition ameliorates a broad range of fragile X syndrome-associated deficits in a mouse model.

Defects in the phosphoinositide 3-kinase (PI3K) pathway are shared characteristics in several brain disorders, including the inherited intellectual disability and autism spectrum disorder, fragile X syndrome (FXS). PI3K signaling therefore could serve as a therapeutic target for FXS and other brain disorders. However, broad inhibition of such a central signal transduction pathway involved in essential cellular functions may produce deleterious side effects. Pharmacological strategies that selectively correct the overactive components of the PI3K pathway while leaving other parts of the pathway intact may overcome these challenges. Here, we provide the first evidence that disease mechanism-based PI3K isoform-specific inhibition may be a viable treatment option for FXS. FXS is caused by loss of the fragile X mental retardation protein (FMRP), which translationally represses specific messenger RNAs, including the PI3K catalytic isoform p110ß. FMRP deficiency increases p110ß protein levels and activity in FXS mouse models and in cells from subjects with FXS. Here, we show that a novel, brain-permeable p110ß-specific inhibitor, GSK2702926A, ameliorates FXS-associated phenotypes on molecular, cellular, behavioral, and cognitive levels in two different FMRP-deficient mouse models. Rescued phenotypes included increased PI3K downstream signaling, protein synthesis rates, and dendritic spine density, as well as impaired social interaction and higher-order cognition. Several p110ß-selective inhibitors, for example, a molecule from the same chemotype as GSK2702926A, are currently being evaluated in clinical trials to treat cancer. Our results suggest that repurposing p110ß inhibitors to treat cognitive and behavioral defects may be a promising disease-modifying strategy for FXS and other brain disorders.