In-Droplet Electromechanical Cell Lysis and Enhanced Enzymatic Assay Driven by Ion Concentration Polarization.

Droplets enable the encapsulation of cells for their analysis in isolated domains. The study of molecular signatures (including genes, proteins, and metabolites) from a few or single cells is critical for identifying key subpopulations. However, dealing with biological analytes at low concentrations requires long incubation times and amplification to achieve the requisite signal strength. Further, cell lysis requires additional chemical lysing agents or heat, which can interfere with assays. Here, we leverage ion concentration polarization (ICP) in droplets to rapidly lyse breast cancer cells within 2 s under a DC voltage bias of 30 V. Numerical simulations attribute cell lysis to an ICP-based electric field and shear stress. We further achieve up to 19-fold concentration enrichment of an enzymatic assay product resulting from cell lysis and a 3.8-fold increase in the reaction rate during enrichment. Our technique for sensitive in-droplet cell analysis provides scope for rapid, high-throughput detection of low-abundance intracellular analytes.

Pharmacist Identification of Older Patients' Priorities in a Home-Based Primary Care Program.

Background Patient Priorities Care (PPC) aims to identify and integrate patient goals and preferences into health care decision-making to provide more personalized care for multimorbid older individuals. Home-based primary care (HBPC) is a model of care delivery that supports aging in place. HBPC-integrated pharmacists can identify patient priorities and communicate with the team to ensure care is aligned with what matters most. Objectives Evaluate patients' perceptions of having priorities identification conversations with the pharmacist; identify the value domains represented by patients' health outcome goals. Setting HBPC program at a large family medicine practice where pharmacists are core members of the interdisciplinary team. Intervention Pharmacists led priorities identification conversations for patients newly enrolled in HBPC. Care preferences and health outcome goals were documented in the medical record and communicated during HBPC team meetings. Design This was a prospective, observational study of HBPC enrollees. After the priorities identification conversation, a three-question survey was administered to identify patients' perceptions of the conversation and interaction with the pharmacist. Health outcome goals and care preference statements were reviewed to determine with which value domain(s) they most aligned. Descriptive statistics were used for results analysis. Results Pharmacists led conversations with 30 participants. Average overall satisfaction with the conversation was 4.6 on a 5-point Likert scale (1 = least, 5 = most satisfied). Ninety-three percent of patients felt it was appropriate to have a pharmacist lead these conversations. Ninety-seven percent believed it was important/very important to discuss their values and goals with their health care team. The predominant value domains represented were Managing Health (43%) and Functioning (40%). Conclusion Patients were mostly satisfied with having PPC conversations and felt it was appropriate for a pharmacist to lead these conversations. Managing health conditions and preserving function were the most frequent value domains associated with patients' goals and care preferences.

Genomics accurately predicts antimicrobial resistance in Staphylococcus pseudintermedius collected as part of Vet-LIRN resistance monitoring.

Whole-genome sequencing (WGS) has changed our understanding of bacterial pathogens, aiding outbreak investigations and advancing our knowledge of their genetic features. However, there has been limited use of genomics to understand antimicrobial resistance of veterinary pathogens, which would help identify emerging resistance mechanisms and track their spread. The objectives of this study were to evaluate the correlation between resistance genotypes and phenotypes for Staphylococcus pseudintermedius, a major pathogen of companion animals, by comparing broth microdilution antimicrobial susceptibility testing and WGS. From 2017-2019, we conducted antimicrobial susceptibility testing and WGS on S. pseudintermedius isolates collected from dogs in the United States as a part of the Veterinary Laboratory Investigation and Response Network (Vet-LIRN) antimicrobial resistance monitoring program. Across thirteen antimicrobials in nine classes, resistance genotypes correlated with clinical resistance phenotypes 98.4 % of the time among a collection of 592 isolates. Our findings represent isolates from diverse lineages based on phylogenetic analyses, and these strong correlations are comparable to those from studies of several human pathogens such as Staphylococcus aureus and Salmonella enterica. We uncovered some important findings, including that 32.3 % of isolates had the mecA gene, which correlated with oxacillin resistance 97.0 % of the time. We also identified a novel rpoB mutation likely encoding rifampin resistance. These results show the value in using WGS to assess antimicrobial resistance in veterinary pathogens and to reveal putative new mechanisms of resistance.

Comparing RNA-Seq and microarray gene expression data in two zones of the Arabidopsis root apex relevant to spaceflight.

PREMISE OF THE STUDY: The root apex is an important region involved in environmental sensing, but comprises a very small part of the root. Obtaining root apex transcriptomes is therefore challenging when the samples are limited. The feasibility of using tiny root sections for transcriptome analysis was examined, comparing RNA sequencing (RNA-Seq) to microarrays in characterizing genes that are relevant to spaceflight. METHODS: Arabidopsis thaliana Columbia ecotype (Col-0) roots were sectioned into Zone 1 (0.5 mm; root cap and meristematic zone) and Zone 2 (1.5 mm; transition, elongation, and growth-terminating zone). Differential gene expression in each was compared. RESULTS: Both microarrays and RNA-Seq proved applicable to the small samples. A total of 4180 genes were differentially expressed (with fold changes of 2 or greater) between Zone 1 and Zone 2. In addition, 771 unique genes and 19 novel transcriptionally active regions were identified by RNA-Seq that were not detected in microarrays. However, microarrays detected spaceflight-relevant genes that were missed in RNA-Seq. DISCUSSION: Single root tip subsections can be used for transcriptome analysis using either RNA-Seq or microarrays. Both RNA-Seq and microarrays provided novel information. These data suggest that techniques for dealing with small, rare samples from spaceflight can be further enhanced, and that RNA-Seq may miss some spaceflight-relevant changes in gene expression.

Genetic dissection of the Arabidopsis spaceflight transcriptome: Are some responses dispensable for the physiological adaptation of plants to spaceflight?

Experimentation on the International Space Station has reached the stage where repeated and nuanced transcriptome studies are beginning to illuminate the structural and metabolic differences between plants grown in space compared to plants on the Earth. Genes that are important in establishing the spaceflight responses are being identified, their roles in spaceflight physiological adaptation are increasingly understood, and the fact that different genotypes adapt differently is recognized. However, the basic question of whether these spaceflight responses are actually required for survival has yet to be posed, and the fundamental notion that spaceflight responses may be non-adaptive has yet to be explored. Therefore the experiments presented here were designed to ask if portions of the plant spaceflight response can be genetically removed without causing loss of spaceflight survival and without causing increased stress responses. The CARA experiment compared the spaceflight transcriptome responses in the root tips of two Arabidopsis ecotypes, Col-0 and WS, as well as that of a PhyD mutant of Col-0. When grown with the ambient light of the ISS, phyD plants displayed a significantly reduced spaceflight transcriptome response compared to Col-0, suggesting that altering the activity of a single gene can actually improve spaceflight adaptation by reducing the transcriptome cost of physiological adaptation. The WS genotype showed an even simpler spaceflight transcriptome response in the ambient light of the ISS, more broadly indicating that the plant genotype can be manipulated to reduce the cost of spaceflight adaptation, as measured by transcriptional response. These differential genotypic responses suggest that genetic manipulation could further reduce, or perhaps eliminate the metabolic cost of spaceflight adaptation. When plants were germinated and then left in the dark on the ISS, the WS genotype actually mounted a larger transcriptome response than Col-0, suggesting that the in-space light environment affects physiological adaptation, which implies that manipulating the local habitat can also substantially impact the metabolic cost of spaceflight adaptation.

Second Generation Modifiers of Colistin Resistance Show Enhanced Activity and Lower Inherent Toxicity.

We recently reported a 2-aminoimidazole-based antibiotic adjuvant that reverses colistin resistance in two species of Gram-negative bacteria. Mechanistic studies in Acinetobacter baumannii demonstrated that this compound downregulated the PmrAB two-component system and abolished a lipid A modification that is required for colistin resistance. We now report the synthesis and evaluation of two separate libraries of substituted 2-aminoimidazole analogues based on this parent compound. From these libraries, a new small molecule was identified that lowers the minimum inhibitory concentration of colistin by up to 32-fold greater than the parent compound while also displaying less inherent bacterial effect, thereby minimizing the likelihood of resistance evolution.

Small-molecule suppression of ß-lactam resistance in multidrug-resistant gram-negative pathogens.

Recent efforts toward combating antibiotic resistance in bacteria have focused on Gram-positive bacteria; however, multidrug-resistant Gram-negative bacteria pose a significant risk to public health. An orthogonal approach to the development of new antibiotics is to develop adjuvant compounds that enhance the susceptibility of drug-resistant strains of bacteria to currently approved antibiotics. This paper describes the synthesis and biological activity of a library of aryl amide 2-aminoimidazoles based on a lead structure from an initial screen. A small molecule was identified from this library that is capable of lowering the minimum inhibitory concentration of ß-lactam antibiotics by up to 64-fold.

Oxidative stress tolerance in plants: novel interplay between auxin and reactive oxygen species signaling.

Biotic and abiotic stress conditions produce reactive oxygen species (ROS) in plants causing oxidative stress damage. At the same time, ROS have additional signaling roles in plant adaptation to the stress. It is not known how the two seemingly contrasting functional roles of ROS between oxidative damage to the cell and signaling for stress protection are balanced. Research suggests that the plant growth regulator auxin may be the connecting link regulating the level of ROS and directing its role in oxidative damage or signaling in plants under stress. The objective of this review is to highlight some of the recent research on how auxin's role is intertwined to that of ROS, more specifically H2O2, in plant adaptation to oxidative stress conditions.

Auxin and its transport play a role in plant tolerance to arsenite-induced oxidative stress in Arabidopsis thaliana.

The role of auxin in plant development is well known; however, its possible function in root response to abiotic stress is poorly understood. In this study, we demonstrate a novel role of auxin transport in plant tolerance to oxidative stress caused by arsenite. Plant response to arsenite [As(III)] was evaluated by measuring root growth and markers for stress on seedlings treated with control or As(III)-containing medium. Auxin transporter mutants aux1, pin1 and pin2 were significantly more sensitive to As(III) than the wild type (WT). Auxin transport inhibitors significantly reduced plant tolerance to As(III) in the WT, while exogenous supply of indole-3-acetic acid improved As(III) tolerance of aux1 and not that of WT. Uptake assays using H(3) -IAA showed As(III) affected auxin transport in WT roots. As(III) increased the levels of H2 O2 in WT but not in aux1, suggesting a positive role for auxin transport through AUX1 on plant tolerance to As(III) stress via reactive oxygen species (ROS)-mediated signalling. Compared to the WT, the mutant aux1 was significantly more sensitive to high-temperature stress and salinity, also suggesting auxin transport influences a common element shared by plant tolerance to arsenite, salinity and high-temperature stress.