RNA therapy for oncogenic NRAS-driven naevi induces apoptosis.

RAS proteins regulate cell division, differentiation and apoptosis via multiple downstream effector pathways. Oncogenic RAS variants are the commonest drivers in cancers, however they also drive many benign lesions predisposing to malignancy, such as melanocytic naevi, thyroid nodules, and colonic polyps. Reversal of these benign lesions could reduce cancer incidence, however the effects of oncogenic RAS have been notoriously difficult to target with downstream pathway inhibitors. Here we show effective suppression of oncogenic and currently undruggable NRASQ61K in primary cells from melanocytic naevi using siRNA targeted to the recurrent causal variant. This results in striking reduction in expression of ARL6IP1, a known inhibitor of endoplasmic reticulum stress-induced apoptosis not previously linked to NRAS. We go on to show that a single dose of siRNA in primary cells triggers an apoptotic cascade, in contrast to treatment with a MEK inhibitor. Protective packaging of the targeted siRNA into lipid nanoparticles permits successful delivery into a humanised mouse model of melanocytic naevi, and results in variant NRAS knockdown in vivo. These data show that RAS-induced protection from apoptosis is involved in persistence of NRAS-driven melanocytic naevi and anticipate that targeted siRNA could form the basis of clinical trials for RAS-driven benign tumours.

In vitro platform to model the function of ionocytes in the human airway epithelium.

BACKGROUND: Pulmonary ionocytes have been identified in the airway epithelium as a small population of ion transporting cells expressing high levels of CFTR (cystic fibrosis transmembrane conductance regulator), the gene mutated in cystic fibrosis. By providing an infinite source of airway epithelial cells (AECs), the use of human induced pluripotent stem cells (hiPSCs) could overcome some challenges of studying ionocytes. However, the production of AEC epithelia containing ionocytes from hiPSCs has proven difficult. Here, we present a platform to produce hiPSC-derived AECs (hiPSC-AECs) including ionocytes and investigate their role in the airway epithelium. METHODS: hiPSCs were differentiated into lung progenitors, which were expanded as 3D organoids and matured by air-liquid interface culture as polarised hiPSC-AEC epithelia. Using CRISPR/Cas9 technology, we generated a hiPSCs knockout (KO) for FOXI1, a transcription factor that is essential for ionocyte specification. Differences between FOXI1 KO hiPSC-AECs and their wild-type (WT) isogenic controls were investigated by assessing gene and protein expression, epithelial composition, cilia coverage and motility, pH and transepithelial barrier properties. RESULTS: Mature hiPSC-AEC epithelia contained basal cells, secretory cells, ciliated cells with motile cilia, pulmonary neuroendocrine cells (PNECs) and ionocytes. There was no difference between FOXI1 WT and KO hiPSCs in terms of their capacity to differentiate into airway progenitors. However, FOXI1 KO led to mature hiPSC-AEC epithelia without ionocytes with reduced capacity to produce ciliated cells. CONCLUSION: Our results suggest that ionocytes could have role beyond transepithelial ion transport by regulating epithelial properties and homeostasis in the airway epithelium.

Topical gene editing therapeutics using lipid nanoparticles: 'gene creams' for genetic skin diseases?

Patients living with inherited skin diseases have benefited from recent advances in DNA sequencing technologies that provide new or improved diagnostics. However, developing and delivering new treatments for the 'genodermatoses' remains challenging. The goal of creating topical preparations that can recover the inherent gene pathology remains largely aspirational. However, recent progress in two fields - the chemistry of topical delivery formulations (lipid nanoparticles) and the molecular biology of gene repair (CRISPR-Cas9, base and prime editing) - presents new opportunities to address this unmet need. In this review, we discuss how lipid nanoparticle delivery vehicles could be used to deliver gene-editing tools to formulate topical 'gene creams' suitable for the treatment of genodermatoses. We summarize the historical landscape of topical therapeutics and advances in gene editing that may herald an era of new therapies for patients with inherited skin disorders.

Molecular and functional correction of a deep intronic splicing mutation in CFTR by CRISPR-Cas9 gene editing.

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the CFTR gene. The 10th most common mutation, c.3178-2477C>T (3849+10kb C>T), involves a cryptic, intronic splice site. This mutation was corrected in CF primary cells homozygous for this mutation by delivering pairs of guide RNAs (gRNAs) with Cas9 protein in ribonucleoprotein (RNP) complexes that introduce double-strand breaks to flanking sites to excise the 3849+10kb C>T mutation, followed by DNA repair by the non-homologous end-joining pathway, which functions in all cells of the airway epithelium. RNP complexes were delivered to CF basal epithelial cell by a non-viral, receptor-targeted nanocomplex comprising a formulation of targeting peptides and lipids. Canonical CFTR mRNA splicing was, thus, restored leading to the restoration of CFTR protein expression with concomitant restoration of electrophysiological function in airway epithelial air-liquid interface cultures. Off-target editing was not detected by Sanger sequencing of in silico-selected genomic sites with the highest sequence similarities to the gRNAs, although more sensitive unbiased whole genome sequencing methods would be required for possible translational developments. This approach could potentially be used to correct aberrant splicing signals in several other CF mutations and other genetic disorders where deep-intronic mutations are pathogenic.

Improving Situational Awareness to Decrease Emergency ICU Transfers for Hospitalized Pediatric Cardiology Patients.

Introduction: Failure to recognize and mitigate critical patient deterioration remains a source of serious preventable harm to hospitalized pediatric cardiac patients. Emergency transfers (ETs) occur 10-20 times more often than code events outside the intensive care unit (ICU) and are associated with morbidity and mortality. This quality improvement project aimed to increase days between ETs and code events on an acute care cardiology unit (ACCU) from a baseline median of 17 and 32 days to ≥70 and 90 days within 12 months. Methods: Institutional leaders, cardiology-trained physicians and nurses, and trainees convened, utilizing the Institution for Healthcare Improvement model to achieve the project aims. Interventions implemented focused on improving situational awareness (SA), including a "Must Call List," evening rounds, a visual management board, and daily huddles. Outcome measures included calendar days between ETs and code events in the ACCU. Process measures tracked the utilization of interventions, and cardiac ICU length of stay was a balancing measure. Statistical process control chart methodology was utilized to analyze the impact of interventions. Results: Within the study period, we observed a centerline shift in primary outcome measures with an increase from 17 to 56 days between ETs and 32 to 62 days between code events in the ACCU, with sustained improvement. Intervention utilization ranged from 87% to 100%, and there was no observed special cause variation in our balancing measure. Conclusions: Interventions focused on improving SA in a particularly vulnerable patient population led to sustained improvement with reduced ETs and code events outside the ICU.

Expression of gain-of-function CFTR in cystic fibrosis airway cells restores epithelial function better than wild-type or codon-optimized CFTR.

Class Ia/b cystic fibrosis transmembrane regulator (CFTR) variants cause severe lung disease in 10% of cystic fibrosis (CF) patients and are untreatable with small-molecule pharmaceuticals. Genetic replacement of CFTR offers a cure, but its effectiveness is limited in vivo. We hypothesized that enhancing protein levels (using codon optimization) and/or activity (using gain-of-function variants) of CFTR would more effectively restore function to CF bronchial epithelial cells. Three different variants of the CFTR protein were tested: codon optimized (high codon adaptation index [hCAI]), a gain-of-function (GOF) variant (K978C), and a combination of both (hˆK978C). In human embryonic kidney (HEK293T) cells, initial results showed that hCAI and hˆK978C produced greater than 10-fold more CFTR protein and displayed ∼4-fold greater activity than wild-type (WT) CFTR. However, functionality was profoundly different in CF bronchial epithelial cells. Here, K978C CFTR more potently restored essential epithelial functions (anion transport, airway surface liquid height, and pH) than WT CFTR. hCAI and hˆK978C CFTRs had limited impact because of mislocalization in the cell. These data provide a proof of principle showing that GOF variants may be more effective than codon-optimized forms of CFTR for CF gene therapy.

Mental health-related limitations and political leadership in Germany: A multidisciplinary analysis of legal, psychiatric, and ethical frameworks.

In recent years, political events have reignited contentious debates about psychiatry and democratic governance. This discourse has largely centred around the ethics and morality of public commentary, particularly in relation to the American Psychiatric Association's Goldwater Rule. Yet, few studies have examined the practical implications of health-related limitations due to mental illness in national leadership and the constitutional and legal provisions that surround these issues, including voluntary or involuntary proceedings. Accordingly, this theoretical paper analyses these topics in a German context using the position at the head of the executive: the chancellorship. Germany was selected as a case example as the biggest democracy in Europe with modern legal frameworks representative of the post-World War Two era in European constitutionalism, and for its economic and political influence within the European Union. Throughout this paper, we do not speculate on the mental health of any individual (past or present), but instead explore jurisdictional mechanisms around health-related limitations in German high office. Consequently, we outline relevant constitutional and legal scenarios, and how short- or long-term medical incapacity may determine requisite responses and contingent complexities. This underpins our discussion, where we consider legal ambiguities, functional capacity, and ethical concerns in psychiatric practice.

Examining the economic costs of crime associated with psychopathic personality disorder: A reply to Verona and Joyner (2022).

In our article, "How much does that cost? Examining the economic costs of crime in North America attributable to people with psychopathic personality disorder" (Gatner et al., 2023, pp. 391-400), we estimated that psychopathic personality disorder (PPD) was associated with substantial crime costs, using a top-down approach of national costs in the United States and Canada. Verona and Joyner (2023) raised several concerns about our findings. Although we think some of their points help to map directions for future research, we disagree with others they raised related to the conceptualization of PPD, the problem of undetected crimes, and their concerns with putative national comparisons. We strongly welcome debate about the societal impacts of PPD in the hope that it spurs increased attention and innovation regarding the treatment and management of PPD. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

How much does that cost? Examining the economic costs of crime in North America attributable to people with psychopathic personality disorder.

Cost of illness research has established that mental disorders lead to significant social burden and massive financial costs. A significant gap exists for the economic burden of many personality disorders, including psychopathic personality disorder (PPD). In the current study, we used a top-down prevalence-based cost of illness approach to estimate bounded crime cost estimates of PPD in the United States and Canada. Three key model parameters (PPD prevalence, relative offending rate of individuals with PPD, and national costs of crime for each country) were informed by existing literature. Sensitivity analyses and Monte Carlo simulations were conducted to provide bounded and central tendency estimates of crime costs, respectively. The estimated PPD-related costs of crime ranged from $245.50 billion to $1,591.57 billion (simulated means = $512.83 to $964.23 billion) in the United States and $12.14 billion to $53.00 billion (simulated means = $25.33 to $32.10 billion) in Canada. These results suggest that PPD may be associated with a substantial economic burden as a result of crime in North America. Recommendations are discussed regarding the burden-treatment discrepancy for PPD, as the development of future effective treatment for the disorder may decrease its costly burden on health and justice systems. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Forecasting Stalking Recidivism Using the Guidelines for Stalking Assessment and Management (SAM).

We examined the long-term risk for stalking recidivism and the predictive validity of ratings made using the Guidelines for Stalking Assessment and Management (SAM) in 100 stalking offenders from a forensic clinic. Overall, 45 offenders were convicted of, charged with, or the subject of police investigation for stalking-related offenses during a potential time at risk that averaged 13.47 years. Survival analyses using the Cox proportional hazards model indicated that a composite score of the presence of SAM risk factors was significantly predictive of recidivism and had significant incremental validity relative to total scores on two scales commonly used in violence risk assessment, the Screening Version of the Hare Psychopathy Checklist-Revised (PCL:SV) and the Violence Risk Appraisal Guide (VRAG). Overall ratings of risk made using the SAM, however, were not significantly predictive of recidivism. We discuss the potential uses of the SAM in stalking risk assessment and provide recommendations for future research.

Morbidity and mortality in neonates with Down Syndrome based on gestational age.

BACKGROUND: Greater than 50% of neonates with Down Syndrome (DS) have perinatal complications that require admission to the neonatal intensive care unit (NICU) at birth. Previous studies have shown increased morbidity and mortality rates in neonates without DS delivered prior to 39 weeks of completed gestation. OBJECTIVE: To determine if an association exists between gestational age at delivery and adverse outcomes in neonates with DS. STUDY DESIGN: Neonates with DS admitted to a large, tertiary care center NICU from 2010 to 2020 were evaluated. Gestational age (GA) was stratified into 4 groups: <34 (preterm), 34-36 (late-preterm), 37-38 (early-term) and ≥39 (term + post-term) completed weeks. Fisher's exact tests were used to evaluate morbidity and mortality rates between groups. RESULT: Of the 314 neonates with DS, 10% (N = 31) were <34 weeks, 22% (N = 68) 34-36 weeks, 40% (N = 127) 37-38 weeks, and 28% (N = 88) ≥39 completed weeks at birth. Baseline characteristics were similar between groups. GA at birth <34 weeks was associated with a higher in-hospital mortality rate when compared to those born 37-38 (19% vs. 0%, P < 0.001) and ≥39 (19% vs. 3%, P = 0.01). Neonates with DS born <34 weeks had a higher likelihood of oxygen requirement at time of discharge compared to 34-36, 37-38, and ≥39 groups (P = 0.01; P < 0.001; P < 0.001 respectively). Neonates with DS < 34 weeks were more likely to develop necrotizing enterocolitis (P = 0.02) and require nitric oxide (P = 0.014) compared to neonates with DS ≥ 39. We observed no differences in the need for surgical interventions between groups aside from the rate of gastrostomy/jejunostomy tube placement between 34-36 weeks and 37-38 weeks GA. CONCLUSION: Neonates with DS born preterm (<34 weeks) represent a highly vulnerable subgroup. Multidisciplinary strategies are needed to address their higher rates of morbidity and mortality.

Quality Report: Postoperative Guideline Implementation Reduces Length of Stay after Fontan Procedure.

Introduction: Patients following the Fontan procedure have a physiology that results in prolonged pleural effusion, often delaying hospital discharge. The hospital length of stay (LOS) of patients following the Fontan procedure at our institution was significantly longer than the Society of Thoracic Surgery benchmark. This quality improvement project aimed to decrease hospital LOS in patients following the Fontan procedure from a baseline of 23 days to 7 days by January 1, 2021, and sustain indefinitely. Methods: We implemented standardized postoperative clinical practice guidelines in April 2020. We designed guidelines using previously published protocols. Key features included an ambulatory PleurX drain (BD, Franklin Lakes, N.J.), diuresis with fluid restriction, and pulmonary vasodilation with supplemental oxygen and sildenafil. All patients were discharged from the hospital with a PleurX drain in place. We compared clinical outcome variables before and after guideline implementation. As a balancing measure, we tracked 30-day readmissions. Results: One hundred seven patients underwent the Fontan procedure before guideline implementation from January 2015 to January 2020, with an average hospital LOS of 23 days. Postguideline implementation, 35 patients underwent the Fontan procedure from April 2020 to July 2022, with an average hospital LOS of 8 days in 2020, which further improved to an average hospital LOS of 7 days. There was no change in 30-day readmission after guideline implementation (24% pre versus 23% post; P = 0.86). Conclusion: Implementing clinical practice guidelines for patients following the Fontan procedure led to an over 50% reduction in hospital LOS without increasing 30-day readmission.

Leaf endophytic microbiomes of different almond cultivars grafted to the same rootstock.

AIMS: We compared the bacterial endophytic communities of three genetically different almond cultivars that were all grafted on the same type of rootstock, growing side by side within a commercial orchard. METHODS AND RESULTS: We examined the diversity of leaf bacterial endophytes using cultivation-independent techniques and assessed the relative abundance of bacterial families. Two of these three cultivars were dominated by Pseudomonadaceae, while the bacterial composition of the third cultivar consisted mainly of Streptococcaceae. CONCLUSIONS: The experimental set up allowed us to analyse the impact of the shoot cultivar on endophytes, minimizing the influence of rootstock, biogeography, and cultivation status. Our data suggest that the shoot cultivar can shape the leaf endophytic community composition of almond trees. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggest that the shoot cultivar controls the composition of the foliar bacterial endophytic community of almonds. Overall, our results could provide a first step to develop strategies for a more sustainable almond agriculture.

Landscape Topography and Regional Drought Alters Dust Microbiomes in the Sierra Nevada of California.

Dust provides an ecologically significant input of nutrients, especially in slowly eroding ecosystems where chemical weathering intensity limits nutrient inputs from underlying bedrock. In addition to nutrient inputs, incoming dust is a vector for dispersing dust-associated microorganisms. While little is known about dust-microbial dispersal, dust deposits may have transformative effects on ecosystems far from where the dust was emitted. Using molecular analyses, we examined spatiotemporal variation in incoming dust microbiomes along an elevational gradient within the Sierra Nevada of California. We sampled throughout two dry seasons and found that dust microbiomes differed by elevation across two summer dry seasons (2014 and 2015), which corresponded to competing droughts in dust source areas. Dust microbial taxa richness decreased with elevation and was inversely proportional to dust heterogeneity. Likewise, dust phosphorus content increased with elevation. At lower elevations, early season dust microbiomes were more diverse than those found later in the year. The relative abundances of microbial groups shifted during the summer dry season. Furthermore, mutualistic fungal diversity increased with elevation, which may have corresponded with the biogeography of their plant hosts. Although dust fungal pathogen diversity was equivalent across elevations, elevation and sampling month interactions for the relative abundance, diversity, and richness of fungal pathogens suggest that these pathogens differed temporally across elevations, with potential implications for humans and wildlife. This study shows that landscape topography and droughts in source locations may alter the composition and diversity of ecologically relevant dust-associated microorganisms.

Psychopathic Personality in Early Childhood: A Critical Comment on Lopez-Romero et al. (2021).

A recent article published in the Journal of Personality Disorders (López-Romero et al., 2021) described the identification of "putative psychopathic personality" in a school cohort of 3-6-year-old children from Spain. This comment offers cautionary considerations of the original article on scientific grounds and critical comments on policy grounds. We caution researchers, policymakers, attorneys, judges, and the general public about the dangers of using this label given present knowledge about the antecedents, early indicators, and stability of the adult disorder of psychopathic personality when assessed in childhood.

Lipid-peptide nanocomplexes for mRNA delivery in vitro and in vivo.

Despite recent advances in the field of mRNA therapy, the lack of safe and efficacious delivery vehicles with pharmaceutically developable properties remains a major limitation. Here, we describe the systematic optimisation of lipid-peptide nanocomplexes for the delivery of mRNA in two murine cancer cell types, B16-F10 melanoma and CT26 colon carcinoma as well as NCI-H358 human lung bronchoalveolar cells. Different combinations of lipids and peptides were screened from an original lipid-peptide nanocomplex formulation for improved luciferase mRNA transfection in vitro by a multi-factorial screening approach. This led to the identification of key structural elements within the nanocomplex associated with substantial improvements in mRNA transfection efficiency included alkyl tail length of the cationic lipid, the fusogenic phospholipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and cholesterol. The peptide component (K16GACYGLPHKFCG) was further improved by the inclusion of a linker, RVRR, that is cleavable by the endosomal enzymes cathepsin B and furin, and a hydrophobic motif (X-S-X) between the mRNA packaging (K16) and receptor targeting domains (CYGLPHKFCG). Nanocomplex transfections of a murine B16-F10 melanoma tumour supported the inclusion of cholesterol for optimal transfection in vivo as well as in vitro. In vitro transfections were also performed with mRNA encoding interleukin-15 as a potential immunotherapy agent and again, the optimised formulation with the key structural elements demonstrated significantly higher expression than the original formulation. Physicochemical characterisation of the nanocomplexes over time indicated that the optimal formulation retained biophysical properties such as size, charge and mRNA complexation efficiency for 14 days upon storage at 4 °C without the need for additional stabilising agents. In summary, we have developed an efficacious lipid-peptide nanocomplex with promising pharmaceutical development properties for the delivery of therapeutic mRNA.

Ecological and genomic responses of soil microbiomes to high-severity wildfire: linking community assembly to functional potential.

Increasing wildfire severity, which is common throughout the western United States, can have deleterious effects on plant regeneration and large impacts on carbon (C) and nitrogen (N) cycling rates. Soil microbes are pivotal in facilitating these elemental cycles, so understanding the impact of increasing fire severity on soil microbial communities is critical. Here, we assess the long-term impact of high-severity fires on the soil microbiome. We find that high-severity wildfires result in a multi-decadal (>25 y) recovery of the soil microbiome mediated by concomitant differences in aboveground vegetation, soil chemistry, and microbial assembly processes. Our results depict a distinct taxonomic and functional successional pattern of increasing selection in post-fire soil microbial communities. Changes in microbiome composition corresponded with changes in microbial functional potential, specifically altered C metabolism and enhanced N cycling potential, which related to rates of potential decomposition and inorganic N availability, respectively. Based on metagenome-assembled genomes, we show that bacterial genomes enriched in our earliest site (4 y since fire) harbor distinct traits such as a robust stress response and a high potential to degrade pyrogenic, polyaromatic C that allow them to thrive in post-fire environments. Taken together, these results provide a biological basis for previously reported process rate measurements and explain the temporal dynamics of post-fire biogeochemistry, which ultimately constrains ecosystem recovery.

"Examining the incremental and interactive effects of boldness with meanness and disinhibition within the triarchic model of psychopathy": Correction.

Reports an error in "Examining the incremental and interactive effects of boldness with meanness and disinhibition within the triarchic model of psychopathy" by Dylan T. Gatner, Kevin S. Douglas and Stephen D. Hart (Personality Disorders: Theory, Research, and Treatment, 2016[Jul], Vol 7[3], 259-268). In the original article, there were several errors in the reporting of data from the Social Emotional Questionnaire. In the "Outcome Measures" section of the Method, under the "Social and emotional functioning" heading, the Sociability subscale was incorrectly identified as the Social Conformity scale. The sentence referencing the data should have read, "However, the Antisocial Behavior (α = .39, MIC = .14) and Sociability (α = -.09, MIC = .02) subscales had poor internal consistency; the Sociability subscale was removed from our analyses." In the last paragraph of the "Research Question 2: Incremental Value of Boldness" section in the Results, the data for the Sociability scale have been replaced with the data for the Social Conformity scale. Additionally, the data reported for the Sociability scale in Tables 2 and 3 have been replaced with the data reported for the Social Conformity scale. (The following abstract of the original article appeared in record 2016-13473-001). The triarchic model of psychopathy (Patrick, Fowles, & Krueger, 2009) comprises 3 phenotypic domains: Meanness, Disinhibition, and Boldness. Ongoing controversy surrounds the relevance of Boldness in the conceptualization and assessment of psychopathy. In the current study, undergraduate students (N = 439) completed the Triarchic Psychopathy Measure (Patrick, 2010) to examine the association between Boldness and a host of theoretically relevant external criteria. Boldness was generally unrelated to either prosocial or harmful criteria. Boldness rarely provided incremental value above or interacted with Meanness and Disinhibition with respect to external criteria. Curvilinear effects of Boldness rarely emerged. The findings suggest that Boldness might not be a central construct in the definition of psychopathic personality disorder. Implications for the 5th edition of the Diagnostic and Statistical Manual of Mental Disorders (American Psychiatric Association, 2013) psychopathic specifier are discussed. (PsycInfo Database Record (c) 2022 APA, all rights reserved).