Systematic and scalable genome-wide essentiality mapping to identify nonessential genes in phages.

Phages are one of the key ecological drivers of microbial community dynamics, function, and evolution. Despite their importance in bacterial ecology and evolutionary processes, phage genes are poorly characterized, hampering their usage in a variety of biotechnological applications. Methods to characterize such genes, even those critical to the phage life cycle, are labor intensive and are generally phage specific. Here, we develop a systematic gene essentiality mapping method scalable to new phage-host combinations that facilitate the identification of nonessential genes. As a proof of concept, we use an arrayed genome-wide CRISPR interference (CRISPRi) assay to map gene essentiality landscape in the canonical coliphages λ and P1. Results from a single panel of CRISPRi probes largely recapitulate the essential gene roster determined from decades of genetic analysis for lambda and provide new insights into essential and nonessential loci in P1. We present evidence of how CRISPRi polarity can lead to false positive gene essentiality assignments and recommend caution towards interpreting CRISPRi data on gene essentiality when applied to less studied phages. Finally, we show that we can engineer phages by inserting DNA barcodes into newly identified inessential regions, which will empower processes of identification, quantification, and tracking of phages in diverse applications.

Arbuscular mycorrhiza convey significant plant carbon to a diverse hyphosphere microbial food web and mineral-associated organic matter.

Arbuscular mycorrhizal fungi (AMF) transport substantial plant carbon (C) that serves as a substrate for soil organisms, a precursor of soil organic matter (SOM), and a driver of soil microbial dynamics. Using two-chamber microcosms where an air gap isolated AMF from roots, we 13CO2-labeled Avena barbata for 6 wk and measured the C Rhizophagus intraradices transferred to SOM and hyphosphere microorganisms. NanoSIMS imaging revealed hyphae and roots had similar 13C enrichment. SOM density fractionation, 13C NMR, and IRMS showed AMF transferred 0.77 mg C g-1 of soil (increasing total C by 2% relative to non-mycorrhizal controls); 33% was found in occluded or mineral-associated pools. In the AMF hyphosphere, there was no overall change in community diversity but 36 bacterial ASVs significantly changed in relative abundance. With stable isotope probing (SIP)-enabled shotgun sequencing, we found taxa from the Solibacterales, Sphingobacteriales, Myxococcales, and Nitrososphaerales (ammonium oxidizing archaea) were highly enriched in AMF-imported 13C (> 20 atom%). Mapping sequences from 13C-SIP metagenomes to total ASVs showed at least 92 bacteria and archaea were significantly 13C-enriched. Our results illustrate the quantitative and ecological impact of hyphal C transport on the formation of potentially protective SOM pools and microbial roles in the AMF hyphosphere soil food web.

Measuring Dissolved Methane in Aquatic Ecosystems Using An Optical Spectroscopy Gas Analyzer.

Measuring greenhouse gas (GHG) fluxes and pools in ecosystems are becoming increasingly common in ecological studies due to their relevance to climate change. With it, the need for analytical platforms adaptable to measuring different pools and fluxes within research groups also grows. This study aims to develop a procedure to use portable optical spectroscopy-based gas analyzers, originally designed and marketed for gas flux measurements, to measure GHG concentrations in aqueous samples. The protocol involves the traditional headspace equilibration technique followed by the injection of a headspace gas subsample into a chamber connected through a closed loop to the inlet and outlet ports of the gas analyzer. The chamber is fabricated from a generic mason jar and simple laboratory supplies, and it is an ideal solution for samples that may require pre-injection dilution. Methane concentrations measured with the chamber are tightly correlated (r2 > 0.98) with concentrations determined separately through gas chromatography-flame ionization detection (GC-FID) on subsamples from the same vials. The procedure is particularly relevant for field studies in remote areas where chromatography equipment and supplies are not readily available, offering a practical, cheaper, and more efficient solution for measuring methane and other dissolved greenhouse gas concentrations in aquatic systems.

Effects of valproate on seizure-like activity in Drosophila melanogaster with a knockdown of Ube3a in different neuronal populations as a model of Angelman Syndrome.

Angelman Syndrome is a rare, genetically induced neurodevelopmental disorder. This disorder stems from a mutation or deletion of the maternal UBE3A gene. Characteristics of this disease include developmental delay, recurring seizures, and severe intellectual disabilities. We studied seizure activity in male Drosophila melanogaster with a knockdown of Ube3a in different neuronal populations (GABAergic, glutamatergic, mushroom body, and all neurons) and investigated the effects of the antiseizure medication (ASM) on seizure-like activity. Epileptiform activity was monitored in individual fruit flies using imaging chambers and mechanically induced seizures using a vortex assay. A positive control was also used: eas (easily shocked seizure phenotype). Seizure activity was analyzed for sums of seizure durations, number of seizures, and total time to return to normal activity. Ube3a knockdowns in GABAergic neurons elicited more seizure-like episodes than knockdowns in glutamatergic neurons and were on par with the positive control group and those with knockdowns in the mushroom bodies. We have established a method whereby valproate could be administered through food rather than through injections to effectively treat epileptiform activity. We demonstrated that if Ube3a is not knocked down pan-neuronally, Angelman Syndrome seizure-like activity can be studied using Drosophila melanogaster and therefore allows for high-throughput drug discovery.

Geochemical constraints on bacteriophage infectivity in terrestrial environments.

Lytic phages can be potent and selective inhibitors of microbial growth and can have profound impacts on microbiome composition and function. However, there is uncertainty about the biogeochemical conditions under which phage predation modulates microbial ecosystem function, particularly in terrestrial systems. Ionic strength is critical for infection of bacteria by many phages, but quantitative data is limited on the ion thresholds for phage infection that can be compared with environmental ion concentrations. Similarly, while carbon composition varies in the environment, we do not know how this variability influences the impact of phage predation on microbiome function. Here, we measured the half-maximal effective concentrations (EC50) of 80 different inorganic ions for the infection of E. coli with two canonical dsDNA and ssRNA phages, T4 and MS2, respectively. Many alkaline earth metals and alkali metals enabled lytic infection but the ionic strength thresholds varied for different ions between phages. Additionally, using a freshwater nitrate-reducing microbiome, we found that the ability of lytic phages to influence nitrate reduction end-products depended upon the carbon source as well as ionic strength. For all phage:host pairs, the ion EC50s for phage infection exceeded the ion concentrations found in many terrestrial freshwater systems. Thus, our findings support a model where phages most influence terrestrial microbial functional ecology in hot spots and hot moments such as metazoan guts, drought influenced soils, or biofilms where ion concentration is locally or transiently elevated and nutrients are available to support the growth of specific phage hosts.

Developing a Diagnostic Multivariable Prediction Model for Urinary Tract Cancer in Patients Referred with Haematuria: Results from the IDENTIFY Collaborative Study.

BACKGROUND: Patient factors associated with urinary tract cancer can be used to risk stratify patients referred with haematuria, prioritising those with a higher risk of cancer for prompt investigation. OBJECTIVE: To develop a prediction model for urinary tract cancer in patients referred with haematuria. DESIGN, SETTING, AND PARTICIPANTS: A prospective observational study was conducted in 10 282 patients from 110 hospitals across 26 countries, aged ≥16 yr and referred to secondary care with haematuria. Patients with a known or previous urological malignancy were excluded. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary outcomes were the presence or absence of urinary tract cancer (bladder cancer, upper tract urothelial cancer [UTUC], and renal cancer). Mixed-effect multivariable logistic regression was performed with site and country as random effects and clinically important patient-level candidate predictors, chosen a priori, as fixed effects. Predictors were selected primarily using clinical reasoning, in addition to backward stepwise selection. Calibration and discrimination were calculated, and bootstrap validation was performed to calculate optimism. RESULTS AND LIMITATIONS: The unadjusted prevalence was 17.2% (n = 1763) for bladder cancer, 1.20% (n = 123) for UTUC, and 1.00% (n = 103) for renal cancer. The final model included predictors of increased risk (visible haematuria, age, smoking history, male sex, and family history) and reduced risk (previous haematuria investigations, urinary tract infection, dysuria/suprapubic pain, anticoagulation, catheter use, and previous pelvic radiotherapy). The area under the receiver operating characteristic curve of the final model was 0.86 (95% confidence interval 0.85-0.87). The model is limited to patients without previous urological malignancy. CONCLUSIONS: This cancer prediction model is the first to consider established and novel urinary tract cancer diagnostic markers. It can be used in secondary care for risk stratifying patients and aid the clinician's decision-making process in prioritising patients for investigation. PATIENT SUMMARY: We have developed a tool that uses a person's characteristics to determine the risk of cancer if that person develops blood in the urine (haematuria). This can be used to help prioritise patients for further investigation.

De Garengeot's hernia: an unusual case on call.

De Garengeot's hernia is defined as a femoral hernia that contains the appendix. Owing to the extreme rarity of de Garengeot's hernia, it could represent a diagnostic challenge; however, it should remain in the differential diagnosis, particularly in this patient demographic. A female patient aged 96 years, who presented as an emergency with acute right iliac fossa pain, was found to have de Garengeot's hernia. The diagnosis was made intraoperatively, where she was treated with an appendicectomy and repair of the hernia defect. The incidence of de Garengeot's hernia is 0.5-5% and the incidence of appendicitis within a femoral hernia is extremely rare. Preoperative diagnosis is challenging to the treating surgeon, which requires a high index of suspicion and is usually made intraoperatively. There is no standard treatment; however, simple appendicectomy and hernia repair seems to be an accepted management.

A simple prioritisation system to improve the electronic handover.

The General Medical Council (GMC) states, "A well managed, thorough and organized handover is crucial for ensuring the quality and safety for patient care," and in their guidance on safe handover the British Medical Association (BMA) advised that "clinically unstable patients are known to the senior and covering clinicians; tasks should be prioritised; plans for further care are put in place; unstable patients are reviewed." The orthopaedic department at King's College Hospital, a busy major trauma centre in London, UK, has a significantly reduced workforce during the weekend. The general consensus was that the ward round was taking too long, giving the foundation year one (FY1) doctor very little time to commit to other ward jobs and reviewing unstable patients, making it a stressful and challenging environment. The electronic patient record (EPR), an electronic programme available on all Trust computers, is already a very reliable way to allow safe handover of information via a central electronic database. However it has limitations in clearly prioritising more unstable patients from those needing routine review. We created an easily identifiable traffic light coding system that could be simply incorporated into the electronic handover that was re-reviewed and finalised in order to improve the ability to prioritise patients for senior review. This in turn would directly impact the efficiency of the ward round and improve patient safety. Our immediate results demonstrated the efficiency of the ward round improved in all parameters: time to complete the ward round improved from 7.1% to 50%; prioritisation and ability to highlight the clinical urgency for patient review improved from 15.4% to 100%; and more notably, the clinician's impression of patient safety improved from 38.5% to 100%. Overall the introduction of an uncomplicated traffic light system provided an effective addition to the electronic handover structure aimed to allow patient prioritisation and improved efficiency during weekend hours.