A Genetic Locus in Elizabethkingia anophelis Associated with Elevated Vancomycin Resistance and Multiple Antibiotic Reduced Susceptibility.

The Gram-negative Elizabethkingia express multiple antibiotic resistance and cause severe opportunistic infections. Vancomycin is commonly used to treat Gram-positive infections and has also been used to treat Elizabethkingia infections, even though Gram-negative organisms possess a vancomycin permeability barrier. Elizabethkingia anophelis appeared relatively vancomycin-susceptible and challenge with this drug led to morphological changes indicating cell lysis. In stark contrast, vancomycin growth challenge revealed that E. anophelis populations refractory to vancomycin emerged. In addition, E. anophelis vancomycin-selected mutants arose at high frequencies and demonstrated elevated vancomycin resistance and reduced susceptibility to other antimicrobials. All mutants possessed a SNP in a gene (vsr1 = vancomycin-susceptibility regulator 1) encoding a PadR family transcriptional regulator located in the putative operon vsr1-ORF551, which is conserved in other Elizabethkingia spp as well. This is the first report linking a padR homologue (vsr1) to antimicrobial resistance in a Gram-negative organism. We provide evidence to support that vsr1 acts as a negative regulator of vsr1-ORF551 and that vsr1-ORF551 upregulation is observed in vancomycin-selected mutants. Vancomycin-selected mutants also demonstrated reduced cell length indicating that cell wall synthesis is affected. ORF551 is a membrane-spanning protein with a small phage shock protein conserved domain. We hypothesize that since vancomycin-resistance is a function of membrane permeability in Gram-negative organisms, it is likely that the antimicrobial resistance mechanism in the vancomycin-selected mutants involves altered drug permeability.

Addressing Challenges of Opportunistic Computed Tomography Bone Mineral Density Analysis.

Computed tomography (CT) offers advanced biomedical imaging of the body and is broadly utilized for clinical diagnosis. Traditionally, clinical CT scans have not been used for volumetric bone mineral density (vBMD) assessment; however, computational advances can now leverage clinically obtained CT data for the secondary analysis of bone, known as opportunistic CT analysis. Initial applications focused on using clinically acquired CT scans for secondary osteoporosis screening, but opportunistic CT analysis can also be applied to answer research questions related to vBMD changes in response to various disease states. There are several considerations for opportunistic CT analysis, including scan acquisition, contrast enhancement, the internal calibration technique, and bone segmentation, but there remains no consensus on applying these methods. These factors may influence vBMD measures and therefore the robustness of the opportunistic CT analysis. Further research and standardization efforts are needed to establish a consensus and optimize the application of opportunistic CT analysis for accurate and reliable assessment of vBMD in clinical and research settings. This review summarizes the current state of opportunistic CT analysis, highlighting its potential and addressing the associated challenges.

Neural network based integration of assays to assess pathogenic potential.

Limited data significantly hinders our capability of biothreat assessment of novel bacterial strains. Integration of data from additional sources that can provide context about the strain can address this challenge. Datasets from different sources, however, are generated with a specific objective and which makes integration challenging. Here, we developed a deep learning-based approach called the neural network embedding model (NNEM) that integrates data from conventional assays designed to classify species with new assays that interrogate hallmarks of pathogenicity for biothreat assessment. We used a dataset of metabolic characteristics from a de-identified set of known bacterial strains that the Special Bacteriology Reference Laboratory (SBRL) of the Centers for Disease Control and Prevention (CDC) has curated for use in species identification. The NNEM transformed results from SBRL assays into vectors to supplement unrelated pathogenicity assays from de-identified microbes. The enrichment resulted in a significant improvement in accuracy of 9% for biothreat. Importantly, the dataset used in our analysis is large, but noisy. Therefore, the performance of our system is expected to improve as additional types of pathogenicity assays are developed and deployed. The proposed NNEM strategy thus provides a generalizable framework for enrichment of datasets with previously collected assays indicative of species.

Internal calibration for opportunistic computed tomography muscle density analysis.

INTRODUCTION: Muscle weakness can lead to reduced physical function and quality of life. Computed tomography (CT) can be used to assess muscle health through measures of muscle cross-sectional area and density loss associated with fat infiltration. However, there are limited opportunities to measure muscle density in clinically acquired CT scans because a density calibration phantom, allowing for the conversion of CT Hounsfield units into density, is typically not included within the field-of-view. For bone density analysis, internal density calibration methods use regions of interest within the scan field-of-view to derive the relationship between Hounsfield units and bone density, but these methods have yet to be adapted for muscle density analysis. The objective of this study was to design and validate a CT internal calibration method for muscle density analysis. METHODOLOGY: We CT scanned 10 bovine muscle samples using two scan protocols and five scan positions within the scanner bore. The scans were calibrated using internal calibration and a reference phantom. We tested combinations of internal calibration regions of interest (e.g., air, blood, bone, muscle, adipose). RESULTS: We found that the internal calibration method using two regions of interest, air and adipose or blood, yielded accurate muscle density values (< 1% error) when compared with the reference phantom. The muscle density values derived from the internal and reference phantom calibration methods were highly correlated (R2 > 0.99). The coefficient of variation for muscle density across two scan protocols and five scan positions was significantly lower for internal calibration (mean = 0.33%) than for Hounsfield units (mean = 6.52%). There was no difference between coefficient of variation for the internal calibration and reference phantom methods. CONCLUSIONS: We have developed an internal calibration method to produce accurate and reliable muscle density measures from opportunistic computed tomography images without the need for calibration phantoms.

Nutrient and toxic elements in soils and plants across 10 urban community gardens: Comparing pXRF and ICP-based soil measurements.

Urban community gardens are becoming increasingly important to rehabilitate developed lands and combat the lack of access to fresh produce. Portable X-ray fluorescence (pXRF) offers a rapid, cost-effective method for assessing the elemental composition of soils but needs further study to determine its efficacy in urban agriculture. The objectives of this study were to evaluate if pXRF measurements of macronutrients (Ca, K, P), micronutrients (Cu, Mn, Zn), and toxic elements (As, Pb) generate results comparable with traditional soil analyses and if the soil measurements correlate with plant tissue concentrations at 10 community gardens across the eastern United States. From field-condition analyses of soils by pXRF and pseudototal digestions, we observed that both methods provide agreeable estimates of concentrations for some elements (Mn, Cu, Zn, Pb) but not for macronutrients (Ca, K, P). We hypothesize that low accuracy in pXRF measurements and macronutrients within silicates caused the poor agreement between the methods. Sieved and dried soil pXRF concentrations were in strong agreement with field-condition pXRF concentrations, suggesting rock removal and drying did not improve measurements. Our results highlight that pXRF can be an accurate and effective tool for screening for Mn, Cu, Zn, and Pb. Some elements, such as Pb in fruits; Mn, Cu, and Zn in leaves; and Zn and Pb in roots, could be estimated by soil pXRF or inductively coupled plasma-based analyses. Macronutrients were poorly estimated for fruits, leaves, and roots. Instead of soil concentrations, identifying genus-specific and garden-specific factors may be important for generating plant uptake predictive models.

"Think of It Like a Game": Older Adults' and Health Professionals' Perspectives on Interactive Exercise Technology Design.

Purpose: Interactive exercise technology (IET) is an effective and practical way to support physiotherapy for older adults. The purpose of this study was to use design thinking to collect feedback on the first iteration of an IET prototype from older adults and health professionals and to use that feedback to gain an understanding of their needs and values, with the goal of developing recommendations to inform the second iteration of the IET prototype. Method: This study was conducted using three steps of design thinking: (1) test, in which four focus groups were conducted, asking older adults and health professionals about their perspectives on an IET prototype; (2) empathize, in which the focus group discussions were recorded and transcribed and thematic content analysis was conducted; and (3) define, in which the needs and values of the participants were identified. Results: The participants were 19 health professionals and four older adults. Four themes, which represented the values that these groups held regarding IET design, were revealed: instruction, safety, accessibility, and motivation. Conclusions: Older adults and health professionals have specific needs for the design of IET, which should be considered in the development of future IET.

Objectif : la technologie des exercices interactifs (TEI) est un moyen efficace et pratique de soutenir la physiothérapie chez les aînés. La présente étude a fait appel à la réflexion conceptuelle pour colliger les commentaires d'aînés et de professionnels de la santé sur la première itération d'un prototype de TEI, s'inspirer de leurs commentaires pour comprendre leurs besoins et leurs valeurs et formuler des recommandations pour éclairer la deuxième itération du prototype de TEI. Méthodologie : la présente étude a été réalisée au moyen des trois étapes de la réflexion conceptuelle : 1) le test, dans le cadre duquel quatre groupes de travail ont été formés pour demander aux aînés et aux professionnels de la santé leurs points de vue sur un prototype de TEI; 2) l'empathie, visant l'enregistrement et la transcription les discussions des groupes de travail, suivis d'une analyse de contenu thématique et 3) la définition, afin de déterminer les besoins et les valeurs des participants. Résultats : les participants étaient 19 professionnels de la santé et quatre aînés. Quatre thèmes sont ressortis, représentatifs des valeurs de ces groupes à l'égard de la TEI : directives, sécurité, accessibilité et motivation. Conclusion : les aînés et leurs professionnels de la santé ont des besoins particuliers à l'égard de la conception de la TEI, dont il faudrait tenir compte dans l'élaboration de futures TEI.

Advancements in Osteoporosis Imaging, Screening, and Study of Disease Etiology.

PURPOSE OF REVIEW: The purpose of this review is to inform researchers and clinicians with the most recent imaging techniques that are employed (1) to opportunistically screen for osteoporosis and (2) to provide a better understanding into the disease etiology of osteoporosis. RECENT FINDINGS: Phantomless calibration techniques for computed tomography (CT) may pave the way for better opportunistic osteoporosis screening and the retroactive analysis of imaging data. Additionally, hardware advances are enabling new applications of dual-energy CT and cone-beam CT to the study of bone. Advances in MRI sequences are also improving imaging evaluation of bone properties. Finally, the application of image registration techniques is enabling new uses of imaging to investigate soft tissue-bone interactions as well as bone turnover. While DXA remains the most prominent imaging tool for osteoporosis diagnosis, new imaging techniques are becoming more widely available and providing additional information to inform clinical decision-making.

Apolipoprotein E4 Reduction with Antisense Oligonucleotides Decreases Neurodegeneration in a Tauopathy Model.

OBJECTIVE: Apolipoprotein E (ApoE) genotype is the strongest genetic risk factor for late-onset Alzheimer's disease, with the ε4 allele increasing risk in a dose-dependent fashion. In addition to ApoE4 playing a crucial role in amyloid-ß deposition, recent evidence suggests that it also plays an important role in tau pathology and tau-mediated neurodegeneration. It is not known, however, whether therapeutic reduction of ApoE4 would exert protective effects on tau-mediated neurodegeneration. METHODS: Herein, we used antisense oligonucleotides (ASOs) against human APOE to reduce ApoE4 levels in the P301S/ApoE4 mouse model of tauopathy. We treated P301S/ApoE4 mice with ApoE or control ASOs via intracerebroventricular injection at 6 and 7.5 months of age and performed brain pathological assessments at 9 months of age. RESULTS: Our results indicate that treatment with ApoE ASOs reduced ApoE4 protein levels by ~50%, significantly protected against tau pathology and associated neurodegeneration, decreased neuroinflammation, and preserved synaptic density. These data were also corroborated by a significant reduction in levels of neurofilament light chain (NfL) protein in plasma of ASO-treated mice. INTERPRETATION: We conclude that reducing ApoE4 levels should be explored further as a therapeutic approach for APOE4 carriers with tauopathy including Alzheimer's disease. ANN NEUROL 2021;89:952-966.

Division of the genus Chryseobacterium: Observation of discontinuities in amino acid identity values, a possible consequence of major extinction events, guides transfer of nine species to the genus Epilithonimonas, eleven species to the genus Kaistella, and three species to the genus Halpernia gen. nov., with description of Kaistella daneshvariae sp. nov. and Epilithonimonas vandammei sp. nov. derived from clinical specimens.

The genus Chryseobacterium in the family Weeksellaceae is known to be polyphyletic. Amino acid identity (AAI) values were calculated from whole-genome sequences of species of the genus Chryseobacterium, and their distribution was found to be multi-modal. These naturally-occurring non-continuities were leveraged to standardise genus assignment of these species. We speculate that this multi-modal distribution is a consequence of loss of biodiversity during major extinction events, leading to the concept that a bacterial genus corresponds to a set of species that diversified since the Permian extinction. Transfer of nine species (Chryseobacterium arachidiradicis, Chryseobacterium bovis, Chryseobacterium caeni, Chryseobacterium hispanicum, Chryseobacterium hominis, Chryseobacterium hungaricum,, Chryseobacterium pallidum and Chryseobacterium zeae) to the genus Epilithonimonas and eleven (Chryseobacterium anthropi, Chryseobacterium antarcticum, Chryseobacterium carnis, Chryseobacterium chaponense, Chryseobacterium haifense, Chryseobacterium jeonii, Chryseobacterium montanum, Chryseobacterium palustre, Chryseobacterium solincola, Chryseobacterium treverense and Chryseobacterium yonginense) to the genus Kaistella is proposed. Two novel species are described: Kaistella daneshvariae sp. nov. and Epilithonimonas vandammei sp. nov. Evidence is presented to support the assignment of Planobacterium taklimakanense to a genus apart from Chryseobacterium, to which Planobacterium salipaludis comb nov. also belongs. The novel genus Halpernia is proposed, to contain the type species Halpernia frigidisoli comb. nov., along with Halpernia humi comb. nov., and Halpernia marina comb. nov.

Lack of hepatic apoE does not influence early Aß deposition: observations from a new APOE knock-in model.

BACKGROUND: The apolipoprotein E (APOE) gene is the strongest genetic risk factor for late-onset Alzheimer disease (AD). ApoE is produced by both astrocytes and microglia in the brain, whereas hepatocytes produce the majority of apoE found in the periphery. Studies using APOE knock-in and transgenic mice have demonstrated a strong isoform-dependent effect of apoE on the accumulation of amyloid-ß (Aß) deposition in the brain in the form of both Aß-containing amyloid plaques and cerebral amyloid angiopathy. However, the specific contributions of different apoE pools to AD pathogenesis remain unknown. METHODS: We have begun to address these questions by generating new lines of APOE knock-in (APOE-KI) mice (ε2/ε2, ε3/ε3, and ε4/ε4) where the exons in the coding region of APOE are flanked by loxP sites, allowing for cell type-specific manipulation of gene expression. We assessed these mice both alone and after crossing them with mice with amyloid deposition in the brain. Using biochemical and histological methods. We also investigated how removal of APOE expression from hepatocytes affected cerebral amyloid deposition. RESULTS: As in other APOE knock-in mice, apoE protein was present predominantly in astrocytes in the brain under basal conditions and was also detected in reactive microglia surrounding amyloid plaques. Primary cultured astrocytes and microglia from the APOE-KI mice secreted apoE in lipoprotein particles of distinct size distribution upon native gel analysis with microglial particles being substantially smaller than the HDL-like particles secreted by astrocytes. Crossing of APP/PS1 transgenic mice to the different APOE-KI mice recapitulated the previously described isoform-specific effect (ε4 > ε3) on amyloid plaque and Aß accumulation. Deletion of APOE in hepatocytes did not alter brain apoE levels but did lead to a marked decrease in plasma apoE levels and changes in plasma lipid profile. Despite these changes in peripheral apoE and on plasma lipids, cerebral accumulation of amyloid plaques in APP/PS1 mice was not affected. CONCLUSIONS: Altogether, these new knock-in strains offer a novel and dynamic tool to study the role of APOE in AD pathogenesis in a spatially and temporally controlled manner.

Draft Genome Sequence of Kroppenstedtia sanguinis X0209T, a Clinical Isolate Recovered from Human Blood.

Kroppenstedtia sanguinis X0209T, a thermoactinomycete, was isolated from the blood of a patient in Sweden. We report on the draft genome sequence obtained with an Illumina MiSeq instrument. The assembled genome totaled 3.73 Mb and encoded 3,583 proteins. Putative genes for virulence, transposons, and biosynthetic gene clusters have been identified.

A Real-Time Multiplex PCR Assay for Detection of Elizabethkingia Species and Differentiation between Elizabethkingia anophelis and E. meningoseptica.

Nosocomial infections of Elizabethkingia species can have fatal outcomes if not identified and treated properly. The current diagnostic tools available require culture and isolation, which can extend the reporting time and delay treatment. Using comparative genomics, we developed an efficient multiplex real-time PCR for the simultaneous detection of all known species of Elizabethkingia, as well as differentiating the two most commonly reported species, Elizabethkingia anophelis and Elizabethkingia meningoseptica.

Tissue-specific localization of polyketide synthase and other associated genes in the lichen, Cladonia rangiferina, using laser microdissection.

The biosynthesis of two polyketides, atranorin and fumarprotocetraric acid, produced from a lichen-forming fungus, Cladonia rangiferina (L.) F. H. Wigg. was correlated with the expression of eight fungal genes (CrPKS1, CrPKS3, CrPKS16, Catalase (CAT), Sugar Transporter (MFsug), Dioxygenase (YQE1), C2H2 Transcription factor (C2H2), Transcription Factor PacC (PacC), which are thought to be involved in polyketide biosynthesis, and one algal gene, NAD-dependent deacetylase sirtuin 2 (AsNAD)), using laser microdissection (LMD). The differential gene expression levels within the thallus tissue layers demonstrate that the most active region for potential polyketide biosynthesis within the lichen is the outer apical region proximal to the photobiont but some expression also occurs in reproductive tissue. This is the first study using laser microdissection to explore gene expression of these nine genes and their location of expression; it provides a proof-of-concept for future experiments exploring tissue-specific gene expression within lichens; and it highlights the utility of LMD for use in lichen systems.

The draft genomes of Elizabethkingia anophelis of equine origin are genetically similar to three isolates from human clinical specimens.

We report the isolation and characterization of two Elizabethkingia anophelis strains (OSUVM-1 and OSUVM-2) isolated from sources associated with horses in Oklahoma. Both strains appeared susceptible to fluoroquinolones and demonstrated high MICs to all cell wall active antimicrobials including vancomycin, along with aminoglycosides, fusidic acid, chloramphenicol, and tetracycline. Typical of the Elizabethkingia, both draft genomes contained multiple copies of ß-lactamase genes as well as genes predicted to function in antimicrobial efflux. Phylogenetic analysis of the draft genomes revealed that OSUVM-1 and OSUVM-2 differ by only 6 SNPs and are in a clade with 3 strains of Elizabethkingia anophelis that were responsible for human infections. These findings therefore raise the possibility that Elizabethkingia might have the potential to move between humans and animals in a manner similar to known zoonotic pathogens.

Revisiting the taxonomy of the genus Elizabethkingia using whole-genome sequencing, optical mapping, and MALDI-TOF, along with proposal of three novel Elizabethkingia species: Elizabethkingia bruuniana sp. nov., Elizabethkingia ursingii sp. nov., and Elizabethkingia occulta sp. nov.

The genus Elizabethkingia is genetically heterogeneous, and the phenotypic similarities between recognized species pose challenges in correct identification of clinically derived isolates. In addition to the type species Elizabethkingia meningoseptica, and more recently proposed Elizabethkingia miricola, Elizabethkingia anophelis and Elizabethkingia endophytica, four genomospecies have long been recognized. By comparing historic DNA-DNA hybridization results with whole genome sequences, optical maps, and MALDI-TOF mass spectra on a large and diverse set of strains, we propose a comprehensive taxonomic revision of this genus. Genomospecies 1 and 2 contain the type strains E. anophelis and E. miricola, respectively. Genomospecies 3 and 4 are herein proposed as novel species named as Elizabethkingia bruuniana sp. nov. (type strain, G0146T = DSM 2975T = CCUG 69503T = CIP 111191T) and Elizabethkingia ursingii sp. nov. (type strain, G4122T = DSM 2974T = CCUG 69496T = CIP 111192T), respectively. Finally, the new species Elizabethkingia occulta sp. nov. (type strain G4070T = DSM 2976T = CCUG 69505T = CIP 111193T), is proposed.

Twelve Complete Reference Genomes of Clinical Isolates in the Capnocytophaga Genus.

We report here 1 near-complete genome sequence and 12 complete genome sequences for clinical Capnocytophaga isolates. Total read coverages ranged from 211× to 737×, and genome sizes ranged from 2.41 Mb to 3.10 Mb. These genomes will enable a more comprehensive taxonomic evaluation of the Capnocytophaga genus.

Complete Circularized Genome Sequences of Four Strains of Elizabethkingia anophelis, Including Two Novel Strains Isolated from Wild-Caught Anopheles sinensis.

We provide complete circularized genome sequences of two mosquito-derived Elizabethkingia anophelis strains with draft sequences currently in the public domain (R26 and Ag1), and two novel E. anophelis strains derived from a different mosquito species, Anopheles sinensis (AR4-6 and AR6-8). The genetic similarity of all four mosquito-derived strains is remarkable.

Evolutionary dynamics and genomic features of the Elizabethkingia anophelis 2015 to 2016 Wisconsin outbreak strain.

An atypically large outbreak of Elizabethkingia anophelis infections occurred in Wisconsin. Here we show that it was caused by a single strain with thirteen characteristic genomic regions. Strikingly, the outbreak isolates show an accelerated evolutionary rate and an atypical mutational spectrum. Six phylogenetic sub-clusters with distinctive temporal and geographic dynamics are revealed, and their last common ancestor existed approximately one year before the first recognized human infection. Unlike other E. anophelis, the outbreak strain had a disrupted DNA repair mutY gene caused by insertion of an integrative and conjugative element. This genomic change probably contributed to the high evolutionary rate of the outbreak strain and may have increased its adaptability, as many mutations in protein-coding genes occurred during the outbreak. This unique discovery of an outbreak caused by a naturally occurring mutator bacterial pathogen provides a dramatic example of the potential impact of pathogen evolutionary dynamics on infectious disease epidemiology.

Transcriptome analysis of the Brassica napus-Leptosphaeria maculans pathosystem identifies receptor, signaling and structural genes underlying plant resistance.

The hemibiotrophic fungal pathogen Leptosphaeria maculans is the causal agent of blackleg disease in Brassica napus (canola, oilseed rape) and causes significant loss of yield worldwide. While genetic resistance has been used to mitigate the disease by means of traditional breeding strategies, there is little knowledge about the genes that contribute to blackleg resistance. RNA sequencing and a streamlined bioinformatics pipeline identified unique genes and plant defense pathways specific to plant resistance in the B. napus-L. maculans LepR1-AvrLepR1 interaction over time. We complemented our temporal analyses by monitoring gene activity directly at the infection site using laser microdissection coupled to quantitative PCR. Finally, we characterized genes involved in plant resistance to blackleg in the Arabidopsis-L. maculans model pathosystem. Data reveal an accelerated activation of the plant transcriptome in resistant host cotyledons associated with transcripts coding for extracellular receptors and phytohormone signaling molecules. Functional characterization provides direct support for transcriptome data and positively identifies resistance regulators in the Brassicaceae. Spatial gradients of gene activity were identified in response to L. maculans proximal to the site of infection. This dataset provides unprecedented spatial and temporal resolution of the genes required for blackleg resistance and serves as a valuable resource for those interested in host-pathogen interactions.