Micro-ultrasound for the detection of clinically significant prostate cancer in biopsy-naive men with negative MRI.

INTRODUCTION: Despite a negative magnetic resonance imaging (MRI), some patients may still harbor clinically significant prostate cancer (csPCa, Gleason grade group ≥2). High-resolution micro-ultrasound (microUS) is a novel imaging technology that could visualize csPCa that is missed by MRI. METHODS: This retrospective review included 1011 consecutive patients biopsied between September 2021 and July 2023 in Alberta, Canada. Among them were 103 biopsy-naive patients with negative MRI (Prostate Imaging Reporting & Data System [PI-RADS] ≤2) undergoing microUS-informed prostate biopsy (n=56) scored using Prostate Risk Identification Using Micro-ultrasound (PRI-MUS) or standard transrectal ultrasound prostate biopsy (n=47). The primary outcome was detection rate of csPCa stratified by biopsy technique and PRI-MUS score. RESULTS: MicroUS biopsy identified csPCa in 14/56 (25%) compared to standard biopsy in 8/47 (17%) (p=0.33). Patients with lesions PRI-MUS ≥3 had csPCa detected at a higher rate compared to patients with PRI-MUS ≤2 (42% vs. 16%, p=0.03). The csPCa detection rate was significantly different comparing patients with PSA density <0.15 and PRI-MUS ≤2 compared to patients with PSA density ≥0.15 and PRI-MUS ≥3 (14% vs. 60%, p=0.02). CONCLUSIONS: MicroUS may aid in the detection of csPCa for patients with negative MRI.

Urban socioeconomic variation influences the ecology and evolution of trophic interactions.

As urbanization expands, it is becoming increasingly important to understand how anthropogenic activity is affecting ecological and evolutionary processes. Few studies have examined how human social patterns within cities can modify eco-evolutionary dynamics. We tested how socioeconomic variation corresponds with changes in trophic interactions and natural selection on prey phenotypes using the classic interaction between goldenrod gall flies (Eurosta solidaginis) and their natural enemies: birds, beetles, and parasitoid wasps. We sampled galls from 84 sites across neighbourhoods with varying socioeconomic levels, and quantified the frequency of predation/parasitism on flies and natural selection by each enemy. We found that bird predation was higher in the highest income neighbourhoods, increasing the strength of selection for smaller galls. Wasp and beetle attack, but not their strength of selection, increased in lower income neighbourhoods. We show that socioeconomic variation in cities can have strong unintended consequences for the ecology and evolution of trophic interactions.

A metabolic signature for NADSYN1-dependent congenital NAD deficiency disorder.

Nicotinamide adenine dinucleotide (NAD) is essential for embryonic development. To date, biallelic loss-of-function variants in 3 genes encoding nonredundant enzymes of the NAD de novo synthesis pathway - KYNU, HAAO, and NADSYN1 - have been identified in humans with congenital malformations defined as congenital NAD deficiency disorder (CNDD). Here, we identified 13 further individuals with biallelic NADSYN1 variants predicted to be damaging, and phenotypes ranging from multiple severe malformations to the complete absence of malformation. Enzymatic assessment of variant deleteriousness in vitro revealed protein domain-specific perturbation, complemented by protein structure modeling in silico. We reproduced NADSYN1-dependent CNDD in mice and assessed various maternal NAD precursor supplementation strategies to prevent adverse pregnancy outcomes. While for Nadsyn1+/- mothers, any B3 vitamer was suitable to raise NAD, preventing embryo loss and malformation, Nadsyn1-/- mothers required supplementation with amidated NAD precursors (nicotinamide or nicotinamide mononucleotide) bypassing their metabolic block. The circulatory NAD metabolome in mice and humans before and after NAD precursor supplementation revealed a consistent metabolic signature with utility for patient identification. Our data collectively improve clinical diagnostics of NADSYN1-dependent CNDD, provide guidance for the therapeutic prevention of CNDD, and suggest an ongoing need to maintain NAD levels via amidated NAD precursor supplementation after birth.

Value of Incremental Biopsy Cores for Microultrasound Targeted Prostate Biopsies.

OBJECTIVE: To determine the optimal number of cores needed during microultrasound-informed prostate biopsy for the detection of clinically significant prostate cancer (csPCa, defined as Gleason Grade Group ≥2). METHODS: A retrospective review of 1011 consecutive patients between September 2021 and July 2023 at our institution were identified; 536 underwent microultrasound biopsy and 475 underwent magnetic resonance imaging (MRI)/ultrasound (US) targeted biopsy. Lesions were given a Prostate Risk Identification using Microultrasound (PRI-MUS) score, with lesions PRI-MUS ≥3 targeted. MRI lesions were scored with Prostate Imaging-Reporting and Data System (PI-RADS) and lesions PI-RADS ≥3 were targeted. The primary outcome is the detection of csPCa stratified by number of cores. RESULTS: One hundred thirty-eight patients underwent targeted biopsies for microultrasound only lesions, 182 for microultrasound and MRI lesions and 426 underwent MRI/US for MRI lesions. The first targeted core detected 78.0% (46/59), 77.8% (63/81), and 78.8% (216/274) of csPCa for microultrasound, microultrasound+MRI, and MRI/US, respectively. Comparing first to third core, there was not a significant difference in overall detection of csPCa by microultrasound, though MRI/US was significantly different (28.4% vs 36.4% P = .12, 32.5% vs 41.8% P = .06, 42.5% vs 53.9% P < .001 for microultrasound, microultrasound+MRI, and MRI/US, respectively). PI-RADS 3 and PRI-MUS 3 lesions had lower first core detection rates compared to PI-RADS 5 and PRI-MUS 5 lesions (44.4% vs 85.4% P = .01, 65.2% vs 81.4% P = .14, 60% vs 83.1% P = .07 for microultrasound, microultrasound+MRI, and MRI/US, respectively). CONCLUSION: A three-core targeted biopsy per microultrasound lesion improves detection rate of csPCa and should be considered to improve diagnostic accuracy.

Complication rates of ciprofloxacin alone vs. ciprofloxacin plus fosfomycin for transrectal prostate biopsy.

INTRODUCTION: Infectious complications after transrectal prostate biopsy have been increasing, driven in large part, by rates of antibiotic resistance to conventional prophylaxis, such as ciprofloxacin. This study was designed to compare conventional antibiotic prophylaxis (oral ciprofloxacin) with ciprofloxacin and fosfomycin combination therapy prior to biopsy. METHODS: This was a retrospective study looking at men between September 2021 and April 2023, who underwent transrectal prostate biopsy at several institutions in Alberta. The primary outcome was infectious complications within 30 days of prostate biopsy. Secondary outcomes included Clostridium difficile infections, urinary retention, gross hematuria, diarrhea, emergency room (ER) visits, hospital admissions, and intensive care unit (ICU) admissions. Data was collected on resistance patterns and pathogens isolated in culture. RESULTS: During the study period, 2168 underwent transrectal prostate biopsy. A total of 1216 men received ciprofloxacin alone and 877 received fosfomycin and ciprofloxacin. Infectious complications were significantly higher in the ciprofloxacin alone group (5.8% vs. 0.5%, p<0.0001). Thirty-day complications (7.2% vs. 2.1%, p<0.0001), 30-day ER visits (7.1% vs. 1.8%, p<0.0001), and 30-day hospitalizations (2.7% vs. 0.7%, p<0.001) were all higher in the ciprofloxacin alone group. The most commonly isolated pathogen was E. coli in 54/60 (90%). Ciprofloxacin resistance in the isolated pathogens was high, with 52/60 (87%) showing resistance to ciprofloxacin and 51/54 (94%) E. coli strains resistant. No difference was seen in retention, Clostridium difficile infections, bleeding, or diarrhea. CONCLUSIONS: The addition of fosfomycin for antibiotic prophylaxis prior to transrectal prostate biopsy was associated with significant improvement in infectious complications and healthcare utilization.

EndophilinA-dependent coupling between activity-induced calcium influx and synaptic autophagy is disrupted by a Parkinson-risk mutation.

Neuronal activity causes use-dependent decline in protein function. However, it is unclear how this is coupled to local quality control mechanisms. We show in Drosophila that the endocytic protein Endophilin-A (EndoA) connects activity-induced calcium influx to synaptic autophagy and neuronal survival in a Parkinson disease-relevant fashion. Mutations in the disordered loop, including a Parkinson disease-risk mutation, render EndoA insensitive to neuronal stimulation and affect protein dynamics: when EndoA is more flexible, its mobility in membrane nanodomains increases, making it available for autophagosome formation. Conversely, when EndoA is more rigid, its mobility reduces, blocking stimulation-induced autophagy. Balanced stimulation-induced autophagy is required for dopagminergic neuron survival, and a variant in the human ENDOA1 disordered loop conferring risk to Parkinson disease also blocks nanodomain protein mobility and autophagy both in vivo and in human-induced dopaminergic neurons. Thus, we reveal a mechanism that neurons use to connect neuronal activity to local autophagy and that is critical for neuronal survival.

Quantitative 3D analysis and visualization of cardiac fibrosis by microcomputed tomography.

Following myocardial infarction, damaged myocardium is replaced with a fibrotic scar that preserves cardiac structural integrity. Scar area measured from sample 2D images of serial heart sections does not faithfully measure the extent of fibrosis due to structural heterogeneity caused by tissue dynamics. Here, we present an X-ray microcomputed tomography (micro-CT) workflow that generates accurate volumetric quantification of scar and surviving myocardium in infarcted mouse hearts. This workflow could be applied to other fibrotic organs or hearts from other species. For complete details on the use and execution of this protocol, please refer to Janbandhu et al. (2021).

Hif-1a suppresses ROS-induced proliferation of cardiac fibroblasts following myocardial infarction.

We report that cardiac fibroblasts (CFs) and mesenchymal progenitors are more hypoxic than other cardiac interstitial populations, express more hypoxia-inducible factor 1α (HIF-1α), and exhibit increased glycolytic metabolism. CF-specific deletion of Hif-1a resulted in decreased HIF-1 target gene expression and increased mesenchymal progenitors in uninjured hearts and increased CF activation without proliferation following sham injury, as demonstrated using single-cell RNA sequencing (scRNA-seq). After myocardial infarction (MI), however, there was ∼50% increased CF proliferation and excessive scarring and contractile dysfunction, a scenario replicated in 3D engineered cardiac microtissues. CF proliferation was associated with higher reactive oxygen species (ROS) as occurred also in wild-type mice treated with the mitochondrial ROS generator MitoParaquat (MitoPQ). The mitochondrial-targeted antioxidant MitoTEMPO rescued Hif-1a mutant phenotypes. Thus, HIF-1α in CFs provides a critical braking mechanism against excessive post-ischemic CF activation and proliferation through regulation of mitochondrial ROS. CFs are potential cellular targets for designer antioxidant therapies in cardiovascular disease.

A structure of substrate-bound Synaptojanin1 provides new insights in its mechanism and the effect of disease mutations.

Synaptojanin1 (Synj1) is a phosphoinositide phosphatase, important in clathrin uncoating during endocytosis of presynaptic vesicles. It was identified as a potential drug target for Alzheimer's disease, Down syndrome, and TBC1D24-associated epilepsy, while also loss-of-function mutations in Synj1 are associated with epilepsy and Parkinson's disease. Despite its involvement in a range of disorders, structural, and detailed mechanistic information regarding the enzyme is lacking. Here, we report the crystal structure of the 5-phosphatase domain of Synj1. Moreover, we also present a structure of this domain bound to the substrate diC8-PI(3,4,5)P3, providing the first image of a 5-phosphatase with a trapped substrate in its active site. Together with an analysis of the contribution of the different inositide phosphate groups to catalysis, these structures provide new insights in the Synj1 mechanism. Finally, we analysed the effect of three clinical missense mutations (Y793C, R800C, Y849C) on catalysis, unveiling the molecular mechanisms underlying Synj1-associated disease.

Heterozygous loss of WBP11 function causes multiple congenital defects in humans and mice.

The genetic causes of multiple congenital anomalies are incompletely understood. Here, we report novel heterozygous predicted loss-of-function (LoF) and predicted damaging missense variants in the WW domain binding protein 11 (WBP11) gene in seven unrelated families with a variety of overlapping congenital malformations, including cardiac, vertebral, tracheo-esophageal, renal and limb defects. WBP11 encodes a component of the spliceosome with the ability to activate pre-messenger RNA splicing. We generated a Wbp11 null allele in mouse using CRISPR-Cas9 targeting. Wbp11 homozygous null embryos die prior to E8.5, indicating that Wbp11 is essential for development. Fewer Wbp11 heterozygous null mice are found than expected due to embryonic and postnatal death. Importantly, Wbp11 heterozygous null mice are small and exhibit defects in axial skeleton, kidneys and esophagus, similar to the affected individuals, supporting the role of WBP11 haploinsufficiency in the development of congenital malformations in humans. LoF WBP11 variants should be considered as a possible cause of VACTERL association as well as isolated Klippel-Feil syndrome, renal agenesis or esophageal atresia.

NAD deficiency due to environmental factors or gene-environment interactions causes congenital malformations and miscarriage in mice.

Causes for miscarriages and congenital malformations can be genetic, environmental, or a combination of both. Genetic variants, hypoxia, malnutrition, or other factors individually may not affect embryo development, however, they may do so collectively. Biallelic loss-of-function variants in HAAO or KYNU, two genes of the nicotinamide adenine dinucleotide (NAD) synthesis pathway, are causative of congenital malformation and miscarriage in humans and mice. The variants affect normal embryonic development by disrupting the synthesis of NAD, a key factor in multiple biological processes, from its dietary precursor tryptophan, resulting in NAD deficiency. This study demonstrates that congenital malformations caused by NAD deficiency can occur independent of genetic disruption of NAD biosynthesis. C57BL/6J wild-type mice had offspring exhibiting similar malformations when their supply of the NAD precursors tryptophan and vitamin B3 in the diet was restricted during pregnancy. When the dietary undersupply was combined with a maternal heterozygous variant in Haao, which alone does not cause NAD deficiency or malformations, the incidence of embryo loss and malformations was significantly higher, suggesting a gene-environment interaction. Maternal and embryonic NAD levels were deficient. Mild hypoxia as an additional factor exacerbated the embryo outcome. Our data show that NAD deficiency as a cause of embryo loss and congenital malformation is not restricted to the rare cases of biallelic mutations in NAD synthesis pathway genes. Instead, monoallelic genetic variants and environmental factors can result in similar outcomes. The results expand our understanding of the causes of congenital malformations and the importance of sufficient NAD precursor consumption during pregnancy.

Functional genomics and gene-environment interaction highlight the complexity of congenital heart disease caused by Notch pathway variants.

Congenital heart disease (CHD) is the most common birth defect and brings with it significant mortality and morbidity. The application of exome and genome sequencing has greatly improved the rate of genetic diagnosis for CHD but the cause in the majority of cases remains uncertain. It is clear that genetics, as well as environmental influences, play roles in the aetiology of CHD. Here we address both these aspects of causation with respect to the Notch signalling pathway. In our CHD cohort, variants in core Notch pathway genes account for 20% of those that cause disease, a rate that did not increase with the inclusion of genes of the broader Notch pathway and its regulators. This is reinforced by case-control burden analysis where variants in Notch pathway genes are enriched in CHD patients. This enrichment is due to variation in NOTCH1. Functional analysis of some novel missense NOTCH1 and DLL4 variants in cultured cells demonstrate reduced signalling activity, allowing variant reclassification. Although loss-of-function variants in DLL4 are known to cause Adams-Oliver syndrome, this is the first report of a hypomorphic DLL4 allele as a cause of isolated CHD. Finally, we demonstrate a gene-environment interaction in mouse embryos between Notch1 heterozygosity and low oxygen- or anti-arrhythmic drug-induced gestational hypoxia, resulting in an increased incidence of heart defects. This implies that exposure to environmental insults such as hypoxia could explain variable expressivity and penetrance of observed CHD in families carrying Notch pathway variants.

GTP Binding is Necessary for the Activation of a Toxic Mutant Isoform of the Essential GTPase ObgE.

Even though the Obg protein is essential for bacterial viability, the cellular functions of this universally conserved GTPase remain enigmatic. Moreover, the influence of GTP and GDP binding on the activity of this protein is largely unknown. Previously, we identified a mutant isoform of ObgE (the Obg protein of Escherichia coli) that triggers cell death. In this research we explore the biochemical requirements for the toxic effect of this mutant ObgE* isoform, using cell death as a readily accessible read-out for protein activity. Both the absence of the N-terminal domain and a decreased GTP binding affinity neutralize ObgE*-mediated toxicity. Moreover, a deletion in the region that connects the N-terminal domain to the G domain likewise abolishes toxicity. Taken together, these data indicate that GTP binding by ObgE* triggers a conformational change that is transmitted to the N-terminal domain to confer toxicity. We therefore conclude that ObgE*-GTP, but not ObgE*-GDP, is the active form of ObgE* that is detrimental to cell viability. Based on these data, we speculate that also for wild-type ObgE, GTP binding triggers conformational changes that affect the N-terminal domain and thereby control ObgE function.

Gene-environment interaction impacts on heart development and embryo survival.

Congenital heart disease (CHD) is the most common type of birth defect. In recent years, research has focussed on identifying the genetic causes of CHD. However, only a minority of CHD cases can be attributed to single gene mutations. In addition, studies have identified different environmental stressors that promote CHD, but the additive effect of genetic susceptibility and environmental factors is poorly understood. In this context, we have investigated the effects of short-term gestational hypoxia on mouse embryos genetically predisposed to heart defects. Exposure of mouse embryos heterozygous for Tbx1 or Fgfr1/Fgfr2 to hypoxia in utero increased the incidence and severity of heart defects while Nkx2-5+/- embryos died within 2 days of hypoxic exposure. We identified the molecular consequences of the interaction between Nkx2-5 and short-term gestational hypoxia, which suggest that reduced Nkx2-5 expression and a prolonged hypoxia-inducible factor 1α response together precipitate embryo death. Our study provides insight into the causes of embryo loss and variable penetrance of monogenic CHD, and raises the possibility that cases of foetal death and CHD in humans could be caused by similar gene-environment interactions.

NAD Deficiency, Congenital Malformations, and Niacin Supplementation.

BACKGROUND: Congenital malformations can be manifested as combinations of phenotypes that co-occur more often than expected by chance. In many such cases, it has proved difficult to identify a genetic cause. We sought the genetic cause of cardiac, vertebral, and renal defects, among others, in unrelated patients. METHODS: We used genomic sequencing to identify potentially pathogenic gene variants in families in which a person had multiple congenital malformations. We tested the function of the variant by using assays of in vitro enzyme activity and by quantifying metabolites in patient plasma. We engineered mouse models with similar variants using the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system. RESULTS: Variants were identified in two genes that encode enzymes of the kynurenine pathway, 3-hydroxyanthranilic acid 3,4-dioxygenase (HAAO) and kynureninase (KYNU). Three patients carried homozygous variants predicting loss-of-function changes in the HAAO or KYNU proteins (HAAO p.D162*, HAAO p.W186*, or KYNU p.V57Efs*21). Another patient carried heterozygous KYNU variants (p.Y156* and p.F349Kfs*4). The mutant enzymes had greatly reduced activity in vitro. Nicotinamide adenine dinucleotide (NAD) is synthesized de novo from tryptophan through the kynurenine pathway. The patients had reduced levels of circulating NAD. Defects similar to those in the patients developed in the embryos of Haao-null or Kynu-null mice owing to NAD deficiency. In null mice, the prevention of NAD deficiency during gestation averted defects. CONCLUSIONS: Disruption of NAD synthesis caused a deficiency of NAD and congenital malformations in humans and mice. Niacin supplementation during gestation prevented the malformations in mice. (Funded by the National Health and Medical Research Council of Australia and others.).

Too posh or too pushed?

I am a third-year student studying for a BSc in adult nursing. I was truly saddened by the Channel 4 documentary Undercover Angels. This was an inaccurate and deceitful portrayal of the nursing profession.