An atlas of human vector-borne microbe interactions reveals pathogenicity mechanisms.

Vector-borne diseases are a leading cause of death worldwide and pose a substantial unmet medical need. Pathogens binding to host extracellular proteins (the "exoproteome") represents a crucial interface in the etiology of vector-borne disease. Here, we used bacterial selection to elucidate host-microbe interactions in high throughput (BASEHIT)-a technique enabling interrogation of microbial interactions with 3,324 human exoproteins-to profile the interactomes of 82 human-pathogen samples, including 30 strains of arthropod-borne pathogens and 8 strains of related non-vector-borne pathogens. The resulting atlas revealed 1,303 putative interactions, including hundreds of pairings with potential roles in pathogenesis, including cell invasion, tissue colonization, immune evasion, and host sensing. Subsequent functional investigations uncovered that Lyme disease spirochetes recognize epidermal growth factor as an environmental cue of transcriptional regulation and that conserved interactions between intracellular pathogens and thioredoxins facilitate cell invasion. In summary, this interactome atlas provides molecular-level insights into microbial pathogenesis and reveals potential host-directed targets for next-generation therapeutics.

Pre-treatment peripheral blood immunophenotyping and response to neoadjuvant chemotherapy in operable breast cancer.

BACKGROUND: Tumor immune infiltration and peripheral blood immune signatures have prognostic and predictive value in breast cancer. Whether distinct peripheral blood immune phenotypes are associated with response to neoadjuvant chemotherapy (NAC) remains understudied. METHODS: Peripheral blood mononuclear cells from 126 breast cancer patients enrolled in a prospective clinical trial (NCT02022202) were analyzed using Cytometry by time-of-flight with a panel of 29 immune cell surface protein markers. Kruskal-Wallis tests or Wilcoxon rank-sum tests were used to evaluate differences in immune cell subpopulations according to breast cancer subtype and response to NAC. RESULTS: There were 122 evaluable samples: 47 (38.5%) from patients with hormone receptor-positive, 39 (32%) triple-negative (TNBC), and 36 (29.5%) HER2-positive breast cancer. The relative abundances of pre-treatment peripheral blood T, B, myeloid, NK, and unclassified cells did not differ according to breast cancer subtype. In TNBC, higher pre-treatment myeloid cells were associated with lower pathologic complete response (pCR) rates. In hormone receptor-positive breast cancer, lower pre-treatment CD8 + naïve and CD4 + effector memory cells re-expressing CD45RA (TEMRA) T cells were associated with more extensive residual disease after NAC. In HER2 + breast cancer, the peripheral blood immune phenotype did not differ according to NAC response. CONCLUSIONS: Pre-treatment peripheral blood immune cell populations (myeloid in TNBC; CD8 + naïve T cells and CD4 + TEMRA cells in luminal breast cancer) were associated with response to NAC in early-stage TNBC and hormone receptor-positive breast cancers, but not in HER2 + breast cancer. TRIAL REGISTRATION: NCT02022202 . Registered 20 December 2013.

Introducing the portable AttentionTrip: An engaging tool for measuring the networks of attention.

BACKGROUND: Measurement of the efficacy of the networks of attention is a frequent component of research in cognitive and clinical neuroscience. Developed in 2002, the Attention Network Test (ANT), has become the most widely used tool for this purpose. NEW METHOD: In 2017 a more engaging, game-like tool based on the ANT, called the AttentionTrip was described. The network scores from five studies which used AttentionTrip are shown to be robust. NEWER METHOD: That version of AttentionTrip required a steering wheel and desk-top computer. Here we describe a new, portable version of the AttentionTrip that is administered using a hand-held tablet (iPad) RESULTS: Three samples of participants (total = 44) completed the portable version of AttentionTrip. The network scores generated using the portable AttentionTrip were also robust. Effect sizes compare favourably with those generated by the ANT and the desktop version. CONCLUSIONS: The findings support the use of the portable AttentionTrip as an alternative to the ANT when user engagement is important, such as when participants are prone to boredom, and when repeated administrations are required.

Residual disinfectant effectively suppresses Legionella species in drinking water distribution systems supplied by surface water in Minnesota, USA.

Seven public water systems in Minnesota, USA were analyzed from one to five times over a two-year period to assess temporal changes in the concentrations of total bacteria, Legionella spp., and Legionella pneumophila from source (i.e., raw water) through the water treatment process to the end water user. Bacterial biomass was collected by filtering large volumes of raw water (12 to 425 L, median: 38 L) or finished and tap water (27 to 1205 L, median: 448 L) using ultrafiltration membrane modules. Quantitative PCR (qPCR) was then used to enumerate all bacteria (16S rRNA gene fragments), all Legionella spp. (ssrA), and Legionella pneumophila (mip). Total coliforms, Escherichia coli, and L. pneumophila also were quantified in the water samples via cultivation. Median concentrations of total bacteria and Legionella spp. (ssrA) in raw water (8.5 and 4.3 log copies/L, respectively) decreased by about 2 log units during water treatment. The concentration of Legionella spp. (ssrA) in water collected from distribution systems inversely correlated with the total chlorine concentration for chloraminated systems significantly (p = 0.03). Although only 8 samples were collected from drinking water distribution systems using free chlorine as a residual disinfectant, these samples had significantly lower concentrations of Legionella spp. (ssrA) than samples collected from the chloraminated systems (p = 5 × 10-4). There was considerable incongruity between the results obtained via cultivation-independent (qPCR) and cultivation-dependent assays. Numerous samples were positive for L. pneumophila via cultivation, none of which tested positive for L. pneumophilia (mip) via qPCR. Conversely, a single sample tested positive for L. pneumophilia (mip) via qPCR, but this sample tested negative for L. pneumophilia via cultivation. Overall, the results suggest that conventional treatment is effective at reducing, but not eliminating, Legionella spp. from surface water supplies and that residual disinfection is effective at suppressing these organisms within drinking water distribution systems.

Radiation nanomedicines for cancer treatment: a scientific journey and view of the landscape.

BACKGROUND: Radiation nanomedicines are nanoparticles labeled with radionuclides that emit α- or ß-particles or Auger electrons for cancer treatment. We describe here our 15 years scientific journey studying locally-administered radiation nanomedicines for cancer treatment. We further present a view of the radiation nanomedicine landscape by reviewing research reported by other groups. MAIN BODY: Gold nanoparticles were studied initially for radiosensitization of breast cancer to X-radiation therapy. These nanoparticles were labeled with 111In to assess their biodistribution after intratumoural vs. intravenous injection. Intravenous injection was limited by high liver and spleen uptake and low tumour uptake, while intratumoural injection provided high tumour uptake but low normal tissue uptake. Further, [111In]In-labeled gold nanoparticles modified with trastuzumab and injected iintratumourally exhibited strong tumour growth inhibition in mice with subcutaneous HER2-positive human breast cancer xenografts. In subsequent studies, strong tumour growth inhibition in mice was achieved without normal tissue toxicity in mice with human breast cancer xenografts injected intratumourally with gold nanoparticles labeled with ß-particle emitting 177Lu and modified with panitumumab or trastuzumab to specifically bind EGFR or HER2, respectively. A nanoparticle depot (nanodepot) was designed to incorporate and deliver radiolabeled gold nanoparticles to tumours using brachytherapy needle insertion techniques. Treatment of mice with s.c. 4T1 murine mammary carcinoma tumours with a nanodepot incorporating [90Y]Y-labeled gold nanoparticles inserted into one tumour arrested tumour growth and caused an abscopal growth-inhibitory effect on a distant second tumour. Convection-enhanced delivery of [177Lu]Lu-AuNPs to orthotopic human glioblastoma multiforme (GBM) tumours in mice arrested tumour growth without normal tissue toxicity. Other groups have explored radiation nanomedicines for cancer treatment in preclinical animal tumour xenograft models using gold nanoparticles, liposomes, block copolymer micelles, dendrimers, carbon nanotubes, cellulose nanocrystals or iron oxide nanoparticles. These nanoparticles were labeled with radionuclides emitting Auger electrons (111In, 99mTc, 125I, 103Pd, 193mPt, 195mPt), ß-particles (177Lu, 186Re, 188Re, 90Y, 198Au, 131I) or α-particles (225Ac, 213Bi, 212Pb, 211At, 223Ra). These studies employed intravenous or intratumoural injection or convection enhanced delivery. Local administration of these radiation nanomedicines was most effective and minimized normal tissue toxicity. CONCLUSIONS: Radiation nanomedicines have shown great promise for treating cancer in preclinical studies. Local intratumoural administration avoids sequestration by the liver and spleen and is most effective for treating tumours, while minimizing normal tissue toxicity.

Bispecific radioimmunoconjugates exploit receptor heterogeneity for positron emission tomography of tumors expressing HER2 and/or EGFR.

HER2 heterogeneity is a challenge for molecular imaging or treating HER2-positive breast cancer (BC). EGFR is coexpressed in some tumors exhibiting HER2 heterogeneity. Bispecific radioimmunoconjugates (bsRICs) that bind HER2 and EGFR were constructed by linking trastuzumab Fab through polyethyleneglycol (PEG24) to EGF. We established s.c. tumors in NOD-SCID mice that homogeneously or heterogeneously expressed HER2 and/or EGFR by the inoculation of HER2-positive/EGFR-negative SK-OV-3 cells, EGFR-positive/HER2-negative MDA-MB-468 cells or mixtures of these cells. [64Cu]Cu-NOTA-trastuzumab Fab-PEG24-EGF were compared to [64Cu]Cu-NOTA-trastuzumab Fab or [64Cu]Cu-NOTA-EGF for the PET imaging of HER2 and/or EGFR-positive tumors. [64Cu]Cu-NOTA-trastuzumab Fab-PEG24-EGF bsRICs imaged tumors expressing HER2 or EGFR or heterogeneously expressing these receptors, while monospecific agents only imaged HER2-or EGFR-positive tumors. Our results indicate that bsRICs labeled with 64Cu are able to exploit receptor heterogeneity for tumor imaging. PET may select patients for radioimmunotherapy with bsRICs complexed to the ß-particle emitter, 177Lu or Auger electron-emitter, 111In in a theranostic approach.

Assessment of feto-placental oxygenation and perfusion in a rat model of placental insufficiency using T2* mapping and 3D dynamic contrast-enhanced MRI.

INTRODUCTION: Placental insufficiency may lead to preeclampsia and fetal growth restriction. There is no cure for placental insufficiency, emphasizing the need for monitoring fetal and placenta health. Current monitoring methods are limited, underscoring the necessity for imaging techniques to evaluate fetal-placental perfusion and oxygenation. This study aims to use MRI to evaluate placental oxygenation and perfusion in the reduced uterine perfusion pressure (RUPP) model of placental insufficiency. METHODS: Pregnant rats were randomized to RUPP (n = 11) or sham surgery (n = 8) on gestational day 14. On gestational day 19, rats imaged using a 7T MRI scanner to assess oxygenation and perfusion using T2* mapping and 3D-DCE MRI sequences, respectively. The effect of the RUPP on the feto-placental units were analyzed from the MRI images. RESULTS: RUPP surgery led to reduced oxygenation in the labyrinth (24.7 ± 1.8 ms vs. 28.0 ± 2.1 ms, P = 0.002) and junctional zone (7.0 ± 0.9 ms vs. 8.1 ± 1.1 ms, P = 0.04) of the placenta, as indicated by decreased T2* values. However, here were no significant differences in fetal organ oxygenation or placental perfusion between RUPP and sham animals. DISCUSSION: The reduced placental oxygenation without a corresponding decrease in perfusion suggests an adaptive response to placental ischemia. While acute reduction in placental perfusion may cause placental hypoxia, persistence of this condition could indicate chronic placental insufficiency after ischemic reperfusion injury. Thus, placental oxygenation may be a more reliable biomarker for assessing fetal condition than perfusion in hypertensive disorders of pregnancies including preeclampsia and FGR.

Understanding familial risk of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease that is the result of an accumulation of sequential genetic alterations. These genetic alterations can either be inherited, such as pathogenic germline variants that are associated with an increased risk of cancer, or acquired, such as somatic mutations that occur during the lifetime of an individual. Understanding the genetic basis of inherited risk of PDAC is essential to advancing patient care and outcomes through improved clinical surveillance, early detection initiatives, and targeted therapies. In this review we discuss factors associated with an increased risk of PDAC, the prevalence of genetic variants associated with an increased risk in patients with PDAC, estimates of PDAC risk in carriers of pathogenic germline variants in genes associated with an increased risk of PDAC. The role of common variants in pancreatic cancer risk will also be discussed.

A high-throughput method for quantifying Drosophila fecundity.

Measurements of Drosophila fecundity are used in a wide variety of studies, such as investigations of stem cell biology, nutrition, behavior, and toxicology. In addition, because fecundity assays are performed on live flies, they are suitable for longitudinal studies such as investigations of aging or prolonged chemical exposure. However, standard Drosophila fecundity assays have been difficult to perform in a high-throughput manner because experimental factors such as the physiological state of the flies and environmental cues must be carefully controlled to achieve consistent results. In addition, exposing flies to a large number of different experimental conditions (such as chemical additives in the diet) and manually counting the number of eggs laid to determine the impact on fecundity is time-consuming. We have overcome these challenges by combining a new multiwell fly culture strategy with a novel 3D-printed fly transfer device to rapidly and accurately transfer flies from one plate to another; the RoboCam, a low-cost, custom built robotic camera to capture images of the wells automatically; and an image segmentation pipeline to automatically identify and quantify eggs. We show that this method is compatible with robust and consistent egg laying throughout the assay period; and demonstrate that the automated pipeline for quantifying fecundity is very accurate (r2 = 0.98 for the correlation between the automated egg counts and the ground truth) In addition, we show that this method can be used to efficiently detect the effects on fecundity induced by dietary exposure to chemicals. Taken together, this strategy substantially increases the efficiency and reproducibility of high throughput egg laying assays that require exposing flies to multiple different media conditions.

An Automobile's Tail Lights: Sacrificing Safety for Playful Design?

OBJECTIVE: The counterintuitive "Union Jack"-inspired turn signals on versions of BMW's Mini vehicles was investigated to reveal potential impacts on human performance. BACKGROUND: When some Mini drivers indicate a change in direction, they do so with an oppositely oriented arrow. This conflict, between the task-irrelevant spatial shape and task-relevant location of the signal, mimics a "converse" spatial-Stroop effect that, in combination with the ubiquitous use of arrows on road signs, may be confusing. METHOD: Participants (n = 30) responded-via right and left keypresses-to the directions of road signs and turn signals in both pure and mixed blocks. Reaction times and accuracies were recorded to determine performance in each condition (compatible, neutral, incompatible). RESULTS: Performance suffered when the location and direction of the stimuli did not correspond. When responding to turn signals the cost to performance was especially salient in mixed blocks. Thus, when driving on roads where the meanings of arrows on road signs is important, turn signals pointing in a direction opposite from the directional intention indicated by the signals' location are likely to be confusing. CONCLUSION: The design of some Mini's "Union Jack" style taillights opposes well-established principles of cognitive functioning, caused confusion in our laboratory study and therefore may be a safety hazard-a possibility that ought to be explored in more realistic (e.g., driving simulator) situations. APPLICATION: BMW designers should consider universally adopting the neutral, "horizontal line," illumination style that is currently available in the aftermarket.

A magnetic separation method for isolating and characterizing the biomolecular corona of lipid nanoparticles.

Lipid nanoparticle (LNP) formulations are a proven method for the delivery of nucleic acids for gene therapy as exemplified by the worldwide rollout of LNP-based RNAi therapeutics and mRNA vaccines. However, targeting specific tissues or cells is still a major challenge. After LNP administration, LNPs interact with biological fluids (i.e., blood), components of which adsorb onto the LNP surface forming a layer of biomolecules termed the "biomolecular corona (BMC)" which affects LNP stability, biodistribution, and tissue tropism. The mechanisms by which the BMC influences tissue- and cell-specific targeting remains largely unknown, due to the technical challenges in isolating LNPs and their corona from complex biological media. In this study, we present a new technique that utilizes magnetic LNPs to isolate LNP-corona complexes from unbound proteins present in human serum. First, we developed a magnetic LNP formulation, containing >40 superparamagnetic iron oxide nanoparticles (IONPs)/LNP, the resulting LNPs containing iron oxide nanoparticles (IOLNPs) displayed a similar particle size and morphology as LNPs loaded with nucleic acids. We further demonstrated the isolation of the IOLNPs and their corresponding BMC from unbound proteins using a magnetic separation (MS) system. The BMC profile of LNP from the MS system was compared to size exclusion column chromatography and further analyzed via mass spectrometry, revealing differences in protein abundances. This new approach enabled a mild and versatile isolation of LNPs and its corona, while maintaining its structural integrity. The identification of the BMC associated with an intact LNP provides further insight into LNP interactions with biological fluids.

Changes in the Networks of Attention across the Lifespan: A Graphical Meta-Analysis.

Three Posnerian networks of attention (alerting, orienting, and executive control) have been distinguished on the bases of behavioural, neuropsychological, and neuroscientific evidence. Here, we examined the trajectories of these networks throughout the human lifespan using the various Attention Network Tests (ANTs), which were specifically developed to measure the efficacy of these networks. The ANT Database was used to identify relevant research, resulting in the inclusion of 36 publications. We conducted a graphical meta-analysis using network scores from each study, based on reaction time plotted as a function of age group. Evaluation of attentional networks from childhood to early adulthood suggests that the alerting network develops relatively quickly, and reaches near-adult level by the age of 12. The developmental pattern of the orienting network seems to depend on the information value of the spatial cues. Executive control network scores show a consistent decrease (improvement) with age in childhood. During adulthood (ages 19-75), changes in alerting depend on the modality of the warning signal, while a moderate increase in orienting scores was seen with increasing age. Whereas executive control scores, as measured in reaction time, increase (deterioration) from young adulthood into later adulthood an opposite trend is seen when scores are based on error rates.

mRNA delivery systems for cancer immunotherapy: Lipid nanoparticles and beyond.

mRNA-based vaccines are emerging as a promising alternative to standard cancer treatments and the conventional vaccines. Moreover, the FDA-approval of three nucleic acid based therapeutics (Onpattro, BNT162b2 and mRNA-1273) has further increased the interest and trust on this type of therapeutics. In order to achieve a significant therapeutic efficacy, the mRNA needs from a drug delivery system. In the last years, several delivery platforms have been explored, being the lipid nanoparticles (LNPs) the most well characterized and studied. A better understanding on how mRNA-based therapeutics operate (both the mRNA itself and the drug delivery system) will help to further improve their efficacy and safety. In this review, we will provide an overview of what mRNA cancer vaccines are and their mode of action and we will highlight the advantages and challenges of the different delivery platforms that are under investigation.

Leaf yield and mineral content of mizuna in response to cut-and-come-again harvest, substrate particle size, and fertilizer formulation in a simulated spaceflight environment.

The Veggie plant-growth unit deployed onboard the International Space Station (ISS) grows leafy vegetables to supplement crew diets. "Cut-and-come-again" harvests are tested to maximize vegetative yield while minimizing crew time. Water, oxygen, and fertilizer delivery to roots of leafy greens growing in microgravity have become the center of attention for Veggie. Current Veggie technology wicks water into particulate root substrates incorporating controlled-release fertilizer (CRF). Mizuna mustard (Brassica rapa) was grown under ISS-like environments in ground-based Veggie-analogue units comparing crop response to combinations of two different substrate particle sizes, two different fertilizer formulations, and three leaf-harvest times from each plant. Fine-particle porous ceramic substrate (Profile©) was compared with a 40:60 mix of fine-particle porous ceramic Profile© + coarse porous ceramic Turface© substrate. Identical 18-6-8 (NPK) CRF formulations consisting of [50% fast-release (T70) + 50% intermediate-release (T100) prills] vs. [50% fast-release (T70) + 50% slow-release (T180) prills] were incorporated into each substrate, and leaf tissues were harvested from each treatment combination at 28, 48, and 56 days after sowing. The combination of T100 CRF in 100% Profile© substrate gave the highest fresh mass (FM) and leaf area (LA) across harvests, whereas T180 CRF in 40% Profile© gave the lowest. Dry-mass (DM) yields varied with effects on leaf area. Tissue nitrogen (N) and potassium (K) specific contents declined across harvests for all treatment combinations but tended to be highest for T100 CRF/100% Profile©, and lowest for T180 CRF/40% Profile©. These major macronutrients were taken up faster by roots growing in small particle-size substrate incorporating intermediate-rate CRF, but also were depleted faster from the same treatment combination, suggesting it may not continue to be the best combination for additional harvests. Micronutrients did not decline in tissue specific content across treatment combinations, but manganese (Mn) accumulated in leaf tissue across treatments and apparently comes mainly from the ceramic substrate, regardless of particle size. Substrate leachate analysis following final harvest indicated that pH remained in the range for nominal availability of mineral nutrients for root uptake, but electro-conductivity measurements suggested depletion of fertilizer salts from root zones, especially from the treatment combination supporting the highest yields and major macronutrient contents. Although 100% Profile© was the better growth substrate for mizuna in combination with intermediate-rate CRF and three cut-and-come-again harvests in ground-based studies, mixed-particle-size substrates may be a better choice for plant growth under microgravity conditions, where capillary forces predominant and tend to cause saturation of a fine medium with water. Since there were no statistically significant interactions between substrate and fertilizer in this study, our ground-based findings for CRF choice should translate to the best substrate choice for microgravity, but if NASA wants to consider additional cut-and-come-again harvests from the same mizuna plants, more complex CRF formulations likely will have to be investigated.

Animal Feedlots and Domestic Wastewater Discharges are Likely Sources of N-Nitrosodimethylamine (NDMA) Precursors in Midwestern Watersheds.

N-nitrosodimethylamine (NDMA) precursor concentrations along four major rivers in Minnesota, USA were quantified and correlated with watershed land cover types, anthropogenic activity, and organic matter characteristics. River water samples (36 in total) were chloraminated under uniform formation conditions (UFC) before and after lime-softening treatment, and the resulting NDMA concentrations were quantified (NDMAUFC). Regarding land cover, NDMAUFC in raw river water exhibited weak positive correlations with urban land (ρ = 0.33, p = 0.05) and cropland coverage (ρ = 0.35, p = 0.04). For anthropogenic activity, NDMAUFC in raw river water positively correlated with the number of feedlots (ρ = 0.57), total weight of animals (ρ = 0.68), and total number of domestic wastewater treatment plants (WWTPs; ρ = 0.63) with p < 0.01. NDMAUFC positively correlated with region IV fluorescence intensity from fluorescence excitation-emission spectra (ρ = 0.70, p < 0.01). Lime softening of river water typically increased NDMAUFC and preferentially removed organic matter that fluoresces in region V, suggesting that the organic matter in this region decreases NDMAUFC by competing for available chloramines. Overall, animal feedlots, along with domestic WWTPs, are predominant sources of NDMA precursors in the studied watersheds, while croplands and urban runoff are of lesser importance.

Extracellular vesicle-mediated delivery of CRISPR/Cas9 ribonucleoprotein complex targeting proprotein convertase subtilisin-kexin type 9 (Pcsk9) in primary mouse hepatocytes.

The loss-of-function of the proprotein convertase subtilisin-kexin type 9 (Pcsk9) gene has been associated with significant reductions in plasma serum low-density lipoprotein cholesterol (LDL-C) levels. Both CRISPR/Cas9 and CRISPR-based editor-mediated Pcsk9 inactivation have successfully lowered plasma LDL-C and PCSK9 levels in preclinical models. Despite the promising preclinical results, these studies did not report how vehicle-mediated CRISPR delivery inactivating Pcsk9 affected low-density lipoprotein receptor recycling in vitro or ex vivo. Extracellular vesicles (EVs) have shown promise as a biocompatible delivery vehicle, and CRISPR/Cas9 ribonucleoprotein (RNP) has been demonstrated to mediate safe genome editing. Therefore, we investigated EV-mediated RNP targeting of the Pcsk9 gene ex vivo in primary mouse hepatocytes. We engineered EVs with the rapamycin-interacting heterodimer FK506-binding protein (FKBP12) to contain its binding partner, the T82L mutant FKBP12-rapamycin binding (FRB) domain, fused to the Cas9 protein. By integrating the vesicular stomatitis virus glycoprotein on the EV membrane, the engineered Cas9 EVs were used for intracellular CRISPR/Cas9 RNP delivery, achieving genome editing with an efficacy of ±28.1% in Cas9 stoplight reporter cells. Administration of Cas9 EVs in mouse hepatocytes successfully inactivated the Pcsk9 gene, leading to a reduction in Pcsk9 mRNA and increased uptake of the low-density lipoprotein receptor and LDL-C. These readouts can be used in future experiments to assess the efficacy of vehicle-mediated delivery of genome editing technologies targeting Pcsk9. The ex vivo data could be a step towards reducing animal testing and serve as a precursor to future in vivo studies for EV-mediated CRISPR/Cas9 RNP delivery targeting Pcsk9.

On-site filtration of large sample volumes improves the detection of opportunistic pathogens in drinking water distribution systems.

In this study, we compared conventional vacuum filtration of small volumes through disc membranes (effective sample volumes for potable water: 0.3-1.0 L) with filtration of high volumes using ultrafiltration (UF) modules (effective sample volumes for potable water: 10.6-84.5 L) for collecting bacterial biomass from raw, finished, and tap water at seven drinking water systems. Total bacteria, Legionella spp., Legionella pneumophila, Mycobacterium spp., and Mycobacterium avium complex in these samples were enumerated using both conventional quantitative PCR (qPCR) and viability qPCR (using propidium monoazide). In addition, PCR-amplified gene fragments were sequenced for microbial community analysis. The frequency of detection (FOD) of Legionella spp. in finished and tap water samples was much greater using UF modules (83% and 77%, respectively) than disc filters (24% and 33%, respectively). The FODs for Mycobacterium spp. in raw, finished, and tap water samples were also consistently greater using UF modules than disc filters. Furthermore, the number of observed operational taxonomic units and diversity index values for finished and tap water samples were often substantially greater when using UF modules as compared to disc filters. Conventional and viability qPCR yielded similar results, suggesting that membrane-compromised cells represented a minor fraction of total bacterial biomass. In conclusion, our research demonstrates that large-volume filtration using UF modules improved the detection of opportunistic pathogens at the low concentrations typically found in public drinking water systems and that the majority of bacteria in these systems appear to be viable in spite of disinfection with free chlorine and/or chloramine.IMPORTANCEOpportunistic pathogens, such as Legionella pneumophila, are a growing public health concern. In this study, we compared sample collection and enumeration methods on raw, finished, and tap water at seven water systems throughout the State of Minnesota, USA. The results showed that on-site filtration of large water volumes (i.e., 500-1,000 L) using ultrafiltration membrane modules improved the frequency of detection of relatively rare organisms, including opportunistic pathogens, compared to the common approach of filtering about 1 L using disc membranes. Furthermore, results from viability quantitative PCR (qPCR) with propidium monoazide were similar to conventional qPCR, suggesting that membrane-compromised cells represent an insignificant fraction of microorganisms. Results from these ultrafiltration membrane modules should lead to a better understanding of the microbial ecology of drinking water distribution systems and their potential to inoculate premise plumbing systems with opportunistic pathogens where conditions are more favorable for their growth.

Assessing the predictions from Posner's theory of phasic alertness using data from Los and Schut (2008).

Alertness has been construed as one of three fundamental components of attention. When generated by a warning signal, phasic changes in alertness ubiquitously decrease reaction time. But how does it do so? Based on earlier findings, in 1975, Posner proposed a theory of phasic alertness with two postulates: (i) phasic alertness does not affect the accumulation of information; (ii) phasic alertness accelerates when a response based on the accumulating information will be generated. When targets are continuously presented, this theory predicts that alertness will reduce reaction at the expense of an increase in errors-that is, generate a speed-accuracy trade-off. Los and Schut, Cognitive Psychology, 57, 20-55, (2008), while endorsing Posner's theory, claimed to have failed to replicated the tell-tale trade-off reported by Posner et al. Memory and Cognition, 1, 2-12, (1973, Experiment 1). The primary goal of this commentary was to use all the data from Los and Schut to see if the predicted speed-accuracy trade-off would be verified or not. With the increased power, it was confirmed that the conditions that benefited the most in reaction time from alertness also had higher error rates. It is noteworthy that recent studies have generated replications and extensions of the methods and findings of Posner et al; thus, it appears that the empirical pattern predicted by Posner's theory of phasic alertness is relatively robust.