Dilution reduces sample matrix effects for rapid, direct, and miniaturised phenotypic antibiotic susceptibility tests for bovine mastitis

The time-consuming nature of current methods for detecting antimicrobial resistance (AMR) to guide mastitis treatment and for surveillance, drives innovation towards faster, easier, and more portable technology. Rapid on-farm testing could guide antibiotic selection, reducing misuse that contributes to resistance. We identify challenges that arise when developing miniaturized antibiotic susceptibility tests (AST) for rapid on-farm use directly in milk. We experimentally studied three factors: sample matrix (specifically milk or spoiled milk); the commensal bacteria found in fresh bovine milk; and result time on the performance of miniaturised AST. Microfluidic “dip-and-test” devices made from microcapillary film (MCF) were able to monitor Gram-negative bacterial growth colourimetrically even in the presence of milk and yoghurt (used to simulate spoiled milk samples), as long as this sample matrix was diluted 1:5 or more in growth medium. Growth detection kinetics using resazurin was not changed by milk at final concentrations of 20% or lower, but a significant delay was seen with yoghurt above 10%. The minimum inhibitory concentration (MIC) for ciprofloxacin and gentamicin was increased in the presence of higher concentrations of milk and yoghurt. When diluted to 1% all observed MIC were within range, indicating dilution may be sufficient to avoid milk matrix interfering with microfluidic AST. We found a median commensal cell count of 6 × 105 CFU/mL across 40 healthy milk samples and tested if these bacteria could alter microfluidic AST. We found that false susceptibility may be observed at early endpoint times if testing some pathogen and commensal mixtures. However, such errors are only expected to occur when a susceptible commensal organism is present at higher cell density relative to the resistant pathogen, and this can be avoided by reading at later endpoints, leading to a trade-off between accuracy and time-to-result. We conclude that with further optimisation, and additional studies of Gram-positive organisms, it should be possible to obtain rapid results for microfluidic AST, but a trade-off is needed between time-to-result, sample dilution, and accuracy

Phytoplankton Phenology in the North Atlantic: Insights From Profiling Float Measurements

There was a typographical error in Equation (1) of our article, where the first term in the denominator should be 0.022 rather than 0.22. The fully corrected equation should be: (Formula presented.). The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated

Medication Utilization and the Impact of Continued Corticosteroid Use on Patient-reported Outcomes in Older Patients with Inflammatory Bowel Disease:

Older individuals with inflammatory bowel disease (IBD) require ongoing medications. We aimed to describe 1) medication use in older and younger IBD patients and 2) medication associations with patient reported outcomes (PRO’s) in older patients

Molecular testing for the clinical diagnosis of fibrolamellar carcinoma.

Fibrolamellar carcinoma has a distinctive morphology and immunophenotype, including cytokeratin 7 and CD68 co-expression. Despite the distinct findings, accurate diagnosis of fibrolamellar carcinoma continues to be a challenge. Recently, fibrolamellar carcinomas were found to harbor a characteristic somatic gene fusion, DNAJB1-PRKACA. A break-apart fluorescence in situ hybridization (FISH) assay was designed to detect this fusion event and to examine its diagnostic performance in a large, multicenter, multinational study. Cases initially classified as fibrolamellar carcinoma based on histological features were reviewed from 124 patients. Upon central review, 104 of the 124 cases were classified histologically as typical of fibrolamellar carcinoma, 12 cases as 'possible fibrolamellar carcinoma' and 8 cases as 'unlikely to be fibrolamellar carcinoma'. PRKACA FISH was positive for rearrangement in 102 of 103 (99%) typical fibrolamellar carcinomas, 9 of 12 'possible fibrolamellar carcinomas' and 0 of 8 cases 'unlikely to be fibrolamellar carcinomas'. Within the morphologically typical group of fibrolamellar carcinomas, two tumors with unusual FISH patterns were also identified. Both cases had the fusion gene DNAJB1-PRKACA, but one also had amplification of the fusion gene and one had heterozygous deletion of the normal PRKACA locus. In addition, 88 conventional hepatocellular carcinomas were evaluated with PRKACA FISH and all were negative. These findings demonstrate that FISH for the PRKACA rearrangement is a clinically useful tool to confirm the diagnosis of fibrolamellar carcinoma, with high sensitivity and specificity. A diagnosis of fibrolamellar carcinoma is more accurate when based on morphology plus confirmatory testing than when based on morphology alone

PiRamid: A compact Raspberry Pi imaging box to automate small-scale time-lapse digital analysis, suitable for laboratory and field use

Digital imaging permits the quantitation of many experiments, such as microbiological growth assays, but laboratory digital imaging systems can be expensive and too specialised. The Raspberry Pi camera platform makes automated, controlled imaging affordable with accessible customisation. When combined with open source software and open-source 3D printed hardware, the control over image quality and capture of this platform permits the rapid development of novel instrumentation. Here we present "PiRamid", a compact, portable, and inexpensive enclosure for autonomous imaging both in the laboratory and in the field. The modular three-piece 3D printed design makes it easy to incorporate different camera systems or lighting configurations (e.g., single wavelength LED for fluorescence). The enclosed design allows complete control of illumination unlike a conventional digital camera or smartphone, on a tripod or handheld, under ambient lighting. The stackable design permits rapid sample addition or camera focus adjustment, with a corresponding change in magnification and resolution. The entire unit is small enough to fit within a microbiological incubator, and cheap enough (∼£100) to scale out for larger parallel experiments. Simply, Python scripts fully automate illumination and image capture for small-scale experiments with an ∼110×85 mm area at 70-90 µm resolution. We demonstrate the versatility of PiRamid by capturing time-resolved, quantitative image data for a wide range of assays. Bacterial growth kinetics was captured for conventional microbiology (agar Petri dishes), 3D printed custom microbiology labware and microfluidic microbiology. To illustrate application beyond microbiology, we demonstrate time-lapse imaging of crystal growth and degradation of salad leaves. Minor modifications permit epi-illumination by addition of a LED ring to the camera module. We conclude that PiRamid permits inexpensive digital capture and quantitation of a wide range of experiments by time-lapse imaging to simplify both laboratory and field imaging

Rhodium(II) Proximity-Labeling Identifies a Novel Target Site on STAT3 for Inhibitors with Potent Anti-Leukemia Activity

Nearly 40 % of children with acute myeloid leukemia (AML) suffer relapse arising from chemoresistance, often involving upregulation of the oncoprotein STAT3 (signal transducer and activator of transcription 3). Herein, rhodium(II)-catalyzed, proximity-driven modification identifies the STAT3 coiled-coil domain (CCD) as a novel ligand-binding site, and we describe a new naphthalene sulfonamide inhibitor that targets the CCD, blocks STAT3 function, and halts its disease-promoting effects in vitro, in tumor growth models, and in a leukemia mouse model, validating this new therapeutic target for resistant AML

Peptidoglycan synthesis in Mycobacterium tuberculosis is organized into networks with varying drug susceptibility

Peptidoglycan (PG), a complex polymer composed of saccharide chains cross-linked by short peptides, is a critical component of the bacterial cell wall. PG synthesis has been extensively studied in model organisms but remains poorly understood in mycobacteria, a genus that includes the important human pathogen Mycobacterium tuberculosis (Mtb). The principle PG synthetic enzymes have similar and, at times, overlapping functions. To determine how these are functionally organized, we carried out whole-genome transposon mutagenesis screens in Mtb strains deleted for ponA1, ponA2, and ldtB, major PG synthetic enzymes. We identified distinct factors required to sustain bacterial growth in the absence of each of these enzymes. We find that even the homologs PonA1 and PonA2 have unique sets of genetic interactions, suggesting there are distinct PG synthesis pathways in Mtb. Either PonA1 or PonA2 is required for growth of Mtb, but both genetically interact with LdtB, which has its own distinct genetic network. We further provide evidence that each interaction network is differentially susceptible to antibiotics. Thus, Mtb uses alternative pathways to produce PG, each with its own biochemical characteristics and vulnerabilities

Immediate and Long-Lasting Impacts of the Mt. Pinatubo Eruption on Ocean Oxygen and Carbon Inventories

Large volcanic eruptions drive significant climate perturbations through major anomalies in radiative fluxes and the resulting widespread cooling of the surface and upper ocean. Recent studies suggest that these eruptions also drive important variability in air-sea carbon and oxygen fluxes. By simulating the Earth system using two initial-condition large ensembles, with and without the aerosol forcing associated with the Mt. Pinatubo eruption in June 1991, we isolate the impact of this volcanic event on physical and biogeochemical properties of the ocean. The Mt. Pinatubo eruption forced significant anomalies in surface fluxes and the ocean interior inventories of heat, oxygen, and carbon. Pinatubo-driven changes persist for multiple years in the upper ocean and permanently modify the ocean's heat, oxygen, and carbon inventories. Positive anomalies in oxygen concentrations emerge immediately post-eruption and penetrate into the deep ocean. In contrast, carbon anomalies intensify in the upper ocean over several years post-eruption, and are largely confined to the upper 150 m. In the tropics and northern high latitudes, the change in oxygen is dominated by surface cooling and subsequent ventilation to mid-depths, while the carbon anomaly is associated with solubility changes and eruption-generated El Niño—Southern Oscillation variability. We do not find significant impact of Pinatubo on oxygen or carbon fluxes in the Southern Ocean; but this may be due to Southern Hemisphere aerosol forcing being underestimated in Community Earth System Model 1 simulations