Engineering Solutions to Improve the Removal of Fecal Indicator Bacteria by Bioinfiltration Systems during Intermittent Flow of Stormwater

Bioinfiltration systems facilitate the infiltration of urban stormwater into soil and reduce high flow events and flooding. Stormwater carries a myriad of pollutants including fecal indicator bacteria (FIB). Significant knowledge gaps exist about the ability of bioinfiltration systems to remove and retain FIB. The present study investigates the ability of model, simplified bioinfiltration systems containing quartz sand and iron oxide-coated quartz sand (IOCS) to remove two FIB (Enterococcus faecalis and Escherichia coli) suspended in synthetic stormwater with and without natural organic matter (NOM) as well as the potential for accumulated FIB to be remobilized during intermittent flow. The experiments were conducted in two phases: (1) the saturated columns packed with either sand or IOCS were contaminated by injecting stormwater with bacteria followed by injection of sterile stormwater and (2) the contaminated columns were subjected to intermittent infiltration of sterile stormwater preceded by a pause during which columns were either kept saturated or drained by gravity. During intermittent flow, fewer bacteria were released from the saturated column compared to the column drained by gravity: 12% of attached E. coli and 3% of attached Ent. faecalis were mobilized from the drained sand column compared to 3% of attached E. coli and 2% attached Ent. faecalis mobilized from the saturated sand column. Dry and wet cycles introduce moving air–water interfaces that can scour bacteria from grain surfaces. During intermittent flows, less than 0.2% of attached bacteria were mobilized from IOCS, which bound both bacteria irreversibly in the absence of NOM. Addition of NOM, however, increased bacterial mobilization from IOCS: 50% of attached E. coli and 8% of attached Ent. faecalis were released from IOCS columns during draining and rewetting. Results indicate that using geomedia such as IOCS that promote irreversible attachment of bacteria, and maintaining saturated condition, could minimize the mobilization of previous attached bacteria from bioinfiltration systems, although NOM may significantly decrease these benefits

Competition of Escherichia coli DNA Polymerases I, II and III with DNA Pol IV in Stressed Cells

Escherichia coli has five DNA polymerases, one of which, the low-fidelity Pol IV or DinB, is required for stress-induced mutagenesis in the well-studied Lac frameshift-reversion assay. Although normally present at ∼200 molecules per cell, Pol IV is recruited to acts of DNA double-strand-break repair, and causes mutagenesis, only when at least two cellular stress responses are activated: the SOS DNA-damage response, which upregulates DinB ∼10-fold, and the RpoS-controlled general-stress response, which upregulates Pol IV about 2-fold. DNA Pol III was also implicated but its role in mutagenesis was unclear. We sought in vivo evidence on the presence and interactions of multiple DNA polymerases during stress-induced mutagenesis. Using multiply mutant strains, we provide evidence of competition of DNA Pols I, II and III with Pol IV, implying that they are all present at sites of stress-induced mutagenesis. Previous data indicate that Pol V is also present. We show that the interactions of Pols I, II and III with Pol IV result neither from, first, induction of the SOS response when particular DNA polymerases are removed, nor second, from proofreading of DNA Pol IV errors by the editing functions of Pol I or Pol III. Third, we provide evidence that Pol III itself does not assist with but rather inhibits Pol IV-dependent mutagenesis. The data support the remaining hypothesis that during the acts of DNA double-strand-break (DSB) repair, shown previously to underlie stress-induced mutagenesis in the Lac system, there is competition of DNA polymerases I, II and III with DNA Pol IV for action at the primer terminus. Up-regulation of Pol IV, and possibly other stress-response-controlled factor(s), tilt the competition in favor of error-prone Pol IV at the expense of more accurate polymerases, thus producing stress-induced mutations. This mutagenesis assay reveals the DNA polymerases operating in DSB repair during stress and also provides a sensitive indicator for DNA polymerase competition and choice in vivo

Comparative cellular analysis of motor cortex in human, marmoset and mouse

The primary motor cortex (M1) is essential for voluntary fine-motor control and is functionally conserved across mammals1. Here, using high-throughput transcriptomic and epigenomic profiling of more than 450,000 single nuclei in humans, marmoset monkeys and mice, we demonstrate a broadly conserved cellular makeup of this region, with similarities that mirror evolutionary distance and are consistent between the transcriptome and epigenome. The core conserved molecular identities of neuronal and non-neuronal cell types allow us to generate a cross-species consensus classification of cell types, and to infer conserved properties of cell types across species. Despite the overall conservation, however, many species-dependent specializations are apparent, including differences in cell-type proportions, gene expression, DNA methylation and chromatin state. Few cell-type marker genes are conserved across species, revealing a short list of candidate genes and regulatory mechanisms that are responsible for conserved features of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic classification allows us to use patch-seq (a combination of whole-cell patch-clamp recordings, RNA sequencing and morphological characterization) to identify corticospinal Betz cells from layer 5 in non-human primates and humans, and to characterize their highly specialized physiology and anatomy. These findings highlight the robust molecular underpinnings of cell-type diversity in M1 across mammals, and point to the genes and regulatory pathways responsible for the functional identity of cell types and their species-specific adaptations

A transcriptomic and epigenomic cell atlas of the mouse primary motor cortex.

Single-cell transcriptomics can provide quantitative molecular signatures for large, unbiased samples of the diverse cell types in the brain1-3. With the proliferation of multi-omics datasets, a major challenge is to validate and integrate results into a biological understanding of cell-type organization. Here we generated transcriptomes and epigenomes from more than 500,000 individual cells in the mouse primary motor cortex, a structure that has an evolutionarily conserved role in locomotion. We developed computational and statistical methods to integrate multimodal data and quantitatively validate cell-type reproducibility. The resulting reference atlas-containing over 56 neuronal cell types that are highly replicable across analysis methods, sequencing technologies and modalities-is a comprehensive molecular and genomic account of the diverse neuronal and non-neuronal cell types in the mouse primary motor cortex. The atlas includes a population of excitatory neurons that resemble pyramidal cells in layer 4 in other cortical regions4. We further discovered thousands of concordant marker genes and gene regulatory elements for these cell types. Our results highlight the complex molecular regulation of cell types in the brain and will directly enable the design of reagents to target specific cell types in the mouse primary motor cortex for functional analysis

A multimodal cell census and atlas of the mammalian primary motor cortex

ABSTRACT We report the generation of a multimodal cell census and atlas of the mammalian primary motor cortex (MOp or M1) as the initial product of the BRAIN Initiative Cell Census Network (BICCN). This was achieved by coordinated large-scale analyses of single-cell transcriptomes, chromatin accessibility, DNA methylomes, spatially resolved single-cell transcriptomes, morphological and electrophysiological properties, and cellular resolution input-output mapping, integrated through cross-modal computational analysis. Together, our results advance the collective knowledge and understanding of brain cell type organization: First, our study reveals a unified molecular genetic landscape of cortical cell types that congruently integrates their transcriptome, open chromatin and DNA methylation maps. Second, cross-species analysis achieves a unified taxonomy of transcriptomic types and their hierarchical organization that are conserved from mouse to marmoset and human. Third, cross-modal analysis provides compelling evidence for the epigenomic, transcriptomic, and gene regulatory basis of neuronal phenotypes such as their physiological and anatomical properties, demonstrating the biological validity and genomic underpinning of neuron types and subtypes. Fourth, in situ single-cell transcriptomics provides a spatially-resolved cell type atlas of the motor cortex. Fifth, integrated transcriptomic, epigenomic and anatomical analyses reveal the correspondence between neural circuits and transcriptomic cell types. We further present an extensive genetic toolset for targeting and fate mapping glutamatergic projection neuron types toward linking their developmental trajectory to their circuit function. Together, our results establish a unified and mechanistic framework of neuronal cell type organization that integrates multi-layered molecular genetic and spatial information with multi-faceted phenotypic properties

Engineering Solutions to Improve the Removal of Fecal Indicator Bacteria by Bioinfiltration Systems during Intermittent Flow of Stormwater

Bioinfiltration systems facilitate the infiltration of urban stormwater into soil and reduce high flow events and flooding. Stormwater carries a myriad of pollutants including fecal indicator bacteria (FIB). Significant knowledge gaps exist about the ability of bioinfiltration systems to remove and retain FIB. The present study investigates the ability of model, simplified bioinfiltration systems containing quartz sand and iron oxide-coated quartz sand (IOCS) to remove two FIB (<i>Enterococcus faecalis</i> and <i>Escherichia coli</i>) suspended in synthetic stormwater with and without natural organic matter (NOM) as well as the potential for accumulated FIB to be remobilized during intermittent flow. The experiments were conducted in two phases: (1) the saturated columns packed with either sand or IOCS were contaminated by injecting stormwater with bacteria followed by injection of sterile stormwater and (2) the contaminated columns were subjected to intermittent infiltration of sterile stormwater preceded by a pause during which columns were either kept saturated or drained by gravity. During intermittent flow, fewer bacteria were released from the saturated column compared to the column drained by gravity: 12% of attached <i>E. coli</i> and 3% of attached <i>Ent. faecalis</i> were mobilized from the drained sand column compared to 3% of attached <i>E. coli</i> and 2% attached <i>Ent. faecalis</i> mobilized from the saturated sand column. Dry and wet cycles introduce moving air–water interfaces that can scour bacteria from grain surfaces. During intermittent flows, less than 0.2% of attached bacteria were mobilized from IOCS, which bound both bacteria irreversibly in the absence of NOM. Addition of NOM, however, increased bacterial mobilization from IOCS: 50% of attached <i>E. coli</i> and 8% of attached <i>Ent. faecalis</i> were released from IOCS columns during draining and rewetting. Results indicate that using geomedia such as IOCS that promote irreversible attachment of bacteria, and maintaining saturated condition, could minimize the mobilization of previous attached bacteria from bioinfiltration systems, although NOM may significantly decrease these benefits

Projecting health and economic impacts of Lassa vaccination campaigns in West Africa

AbstractBackgroundLassa fever is a zoonotic disease identified by the World Health Organization (WHO) as having pandemic potential. Estimates of the health-economic impacts of vaccination are needed to guide vaccine investments.MethodsWe estimated rates of Lassa virus (LASV) infection across West Africa, quantified the health-economic burden of disease and evaluated impacts of a series of vaccination campaigns. We also modelled the emergence of “Lassa-X” – a hypothetical pandemic LASV variant – and evaluated impacts of the 100 Days Mission, a pandemic response initiative aimed at delivering vaccines within 100 days of such a pathogen emerging.FindingsWe estimated 2.7M (95% uncertainty interval: 2.1M-3.4M) LASV infections, 23.7K (14.9K-34.6K) hospitalisations and 3.9K (1.3K-8.3K) deaths annually, resulting over ten years in 2.0M (793.8K-3.9M) disability-adjusted life-years (DALYs) and $1.6B ($805.1M-$2.8B) in societal costs (International dollars 2021). Reaching 80$128.2M ($67.2M-$231.9M) in discounted societal costs over ten years, thus averting approximately 11%-28% of DALYs in endemic countries. In the event of Lassa-X emerging, achieving 100 Days Mission vaccination targets averted approximately 22% of DALYs given a vaccine 70% effective against disease and 74% of DALYs given a vaccine 70% effective against both infection and disease.InterpretationVaccination will help alleviate Lassa fever’s health-economic burden. Investing in Lassa vaccination now may help prepare against potential future variants with pandemic potential.FundingCoalition for Epidemic Preparedness Innovations.Evidence before this studyLassa fever is a common but widely underreported emerging zoonotic disease endemic to West Africa. At least four Lassa vaccine candidates have begun clinical trials. We searched PubMed and preprint archives MedRxiv and BioRxiv up to 26 February 2024 for journal articles using the query (LASV OR Lassa) AND vaccin* AND (burden OR health-econ* OR econ* OR projection) without data or language restrictions. Several articles addressed Lassa vaccine candidates currently in development, including laboratory studies, experiments in animal models and one first-in-human phase 1 clinical trial. Reviews and editorials have discussed extensive gaps in Lassa fever surveillance, recent efforts to scale-up vaccine investment and challenges in designing efficient vaccination campaigns. However, no studies have attempted to estimate the potential impacts of Lassa vaccination on population health or economies. Estimates of Lassa fever burden and vaccine impact are needed to guide epidemiological study design and investment in Lassa vaccine development.Added value of this studyWe provide the first estimates of the health-economic burden of Lassa fever, synthesizing recent data on zoonotic risk, human-to-human transmission, clinical outcomes and economic costs associated with infection. We assess a series of vaccination campaigns and describe their projected impacts on population health and economies across the 15 countries of continental West Africa. We also consider a scenario describing the emergence of Lassa-X, a hypothetical novel Lassa-related virus with pandemic potential. We quantify health-economic impacts of reactive Lassa-X vaccination campaigns in line with the stated goals of the 100 Days Mission.Implications of all the available evidenceOur analysis predicts that vaccination campaigns predominantly targeting districts of Guinea, Liberia, Sierra Leone and Nigeria currently classified as endemic by WHO could substantially reduce the health-economic burden of Lassa fever. However, we further predict extensive underreported LASV infection in neighbouring regions, suggesting potential for significant benefits to expanding Lassa vaccination beyond WHO-classified endemic districts. In order to optimize vaccine rollout, improved surveillance is needed to better define populations at greatest risk of infection and severe disease throughout West Africa. Our study further demonstrates the critical health and economic benefits of achieving 100 Days Mission vaccination goals in response to a hypothetical pandemic variant of Lassa virus emerging. Although the probability of such a variant emerging is unknown, this analysis demonstrates potential for additional large-scale benefits to Lassa vaccine investment.</jats:sec

Identification and Prediction of Severe Hematologic Toxicity after CAR T-Cell Therapy Using Machine Learning-Based Time-Series Clustering

Severe hematologic toxicity is a significant complication associated with CAR T-cell therapy, leading to infections, transfusion dependency, and mortality. Using data from >400 CAR T-cell patients (pts), we hypothesized a time-series clustering-based approach could: i) automate the identification of pts with impaired absolute neutrophil count (ANC) recovery, ii) enable the identification of factors associated with ANC recovery, and iii) assess predictive models of hematologic toxicity after CAR T-cell therapy. Adults who received their first infusion of CAR T cells for hematologic malignancies with commercial or investigational products at our center (2013-2023) were included. ANC trajectories were clustered using non-supervised longitudinal k-means based on Euclidean distances. Sensitivity and specificity were computed based on the Youden criteria. 403 pts were included. The most common disease types were aggressive NHL (n = 161; 40%), indolent NHL (n = 82; 20%), ALL (n = 74; 18%), and MM/PCL (n = 44; 11%). The most common CAR T-cell products were investigational CD19 or CD20 CAR T-cell products (n = 174; 43%), axi-cel (n = 101; 25%), and liso-cel (n = 46; 11%). The longitudinal ANC data clustered into 4 distinct trajectories: 1) very good recovery (high nadir followed by rapid recovery), n = 294 (73%); 2) good recovery (low nadir followed by rapid recovery), n = 87 (22%); 3) poor recovery (low nadir followed by intermittent recovery), n = 13 (3%); 4) very poor recovery (aplastic phenotype), n = 9 (2%) (Figure 1). Pts with poor/very poor ANC recovery had significantly shorter overall survival than those with good/very good recovery (median 3 vs. 19 months, p < 0.001; Figure 2). 100-day mortality in pts with in very good/good vs. poor/very poor recovery was 62% vs. 11%, respectively (p < 0.001). In univariate logistic regression, predictors of poor/very poor recovery included disease type and inflammatory biomarkers (Table 1). Next, we assessed the ability of the CAR-HEMATOTOX score (high vs. low risk; Rejeski, Blood 2021) to predict poor/very poor ANC recovery. Due to missing data, day +0 rather than pre-lymphodepletion (LD) values were used for CRP and ferritin. The specificity and sensitivity of the CAR-HEMATOTOX were 31% and 100%, respectively. A logistic regression model using restricted cubic splines and including pre-LD ANC, peak CRP, and peak ferritin showed high discrimination (C-index: 0.91) with 74% specificity and 95% sensitivity. We introduce an automated and scalable framework that successfully identifies pts with the most severe hematologic toxicity after CAR T-cell therapy, and specifically those displaying an “aplastic” trajectory. We identified key factors associated with poor ANC recovery. A new model including peak inflammatory biomarkers showed improved performance compared to the CAR-HEMATOTOX score