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The copy number of a plasmid is linked to its functionality, yet there have been few attempts to optimize higher-copy-number mutants for use across diverse origins of replication in different hosts. We use a high-throughput growth-coupled selection assay and a directed evolution approach to rapidly identify origin of replication mutations that influence copy number and screen for mutants that improve Agrobacterium-mediated transformation (AMT) efficiency. By introducing these mutations into binary vectors within the plasmid backbone used for AMT, we observe improved transient transformation of Nicotiana benthamiana in four diverse tested origins (pVS1, RK2, pSa and BBR1). For the best-performing origin, pVS1, we isolate higher-copy-number variants that increase stable transformation efficiencies by 60-100% in Arabidopsis thaliana and 390% in the oleaginous yeast Rhodosporidium toruloides. Our work provides an easily deployable framework to generate plasmid copy number variants that will enable greater precision in prokaryotic genetic engineering, in addition to improving AMT efficiency.

* Context.-Carbapenem-resistant Enterobacterales are disseminated worldwide and associated with infections with high rates of morbidity and mortality. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOF MS) is a useful tool for identification of pathogens directly from blood cultures in clinical microbiology laboratories. Furthermore, it has been applied for the detection of carbapenemase production, by evaluating carbapenem hydrolysis. Objective.-To determine meropenem hydrolysis to detect carbapenemase production directly from positive blood cultures, using logRQ to establish a quantitative measure of hydrolysis. Design.-We evaluated 100 Enterobacterales from positive blood cultures, with 81 carrying a carbapenemase gene ([bla.sub.KPC] [bla.sub.GES], [bla.sub.NDM-1], [bla.sub.IMP], [bla.sub.VIM], and [bla.sub.OXA-48]-like), as determined by real-time multiplex polymerase chain reaction with high-resolution melting (HRM-qPCR). Bacterial proteins extracted from positive blood culture bottles were incubated in a meropenem solution (2-4 hours) followed by centrifugation for MALDI-TOF MS analysis. The intensity of peaks of the hydrolyzed and nonhydrolyzed forms were used to calculate the logRQ value. Results.-Overall, sensitivity was 86.8% and specificity, 89.5%. Of note, sensitivity varied depending on enzyme type. For [bla.sub.KPC]-positive isolates, sensitivity was 97.9%, while it reduced significantly for [bla.sub.NDM-1] and [bla.sub.OXA-48]-like isolates: 62.5% (10 of 16) and 66 Indeed, logRQ was higher in [bla.sub.KPC]-positive isolates (0.371.97) than in [bla.sub.NDM-1] (-1.37 to 0.83) and [bla.sub.OXA-48]-like isolates (-1.08 to 1.79). Conclusions.-This is an inexpensive and rapid test to identify carbapenemase activity directly from blood culture bottles, which contributes to early adequate antimicrobial therapy and implementation of infection control measures. (Arch Pathol Lab Med. 2024;148:1145-1151; doi: 10.5858/arpa.2023-0199-OA), Bloodstream infections, including those caused by carbapenem-resistant Enterobacterales (CRE), are a subject of major concern, as mortality rates can be as high as 80%. (1-3) Furthermore, it is well established [...]

Background and Purpose: Decompressive surgery is a potentially life-saving treatment in patients with malignant space-occupying cerebellar infarction. However, there is only limited literature on functional outcomes and complications after surgery. Our aim was to establish markers which predict poor outcome. Methods: We retrospectively analyzed data of all patients who underwent surgery due to malignant swelling of a space-occupying cerebellar infarction in our hospital between 2005 and 2023. Statistical analyses were conducted on multiple parameters to identify predictors of poor functional outcome (mRS 4–6) 90 days after surgery. Complications during hospitalization were reviewed for each patient. Results: In total, 58 patients received decompressive surgery. The 90-day mortality rate was 27.6% (n = 16). A good functional outcome (mRS 0–3) 90 days after surgery was achieved in 24 patients (41.4%). Multivariable analysis revealed multiple factors associated with a poor outcome on day 90 (mRS 4–6): a higher premorbid mRS score (OR 2.715 [95% CI, 1.166–6.323]; p = 0.021), higher NIHSS score on admission (OR 1.088 [95% CI, 1.014, 1.168]; p = 0.019) and the presence of an additional brainstem infarction (OR 7.035, [95% CI, 1.255, 39.424], p = 0.027). Hyperactive delirium was associated with good clinical outcome (OR 0.020 [95%CI, 0.001–0.623]; p = 0.026). Aspiration pneumonia (n = 22, 37.9%), urinary tract infection (n = 15, 25.9%), and hyperactive delirium (n = 8, 13.8%) were the most common complications during hospitalization. Conclusions: Decompressive surgery is a safe, life-saving treatment for malignant space-occupying cerebellar infarction. Higher premorbid mRS, higher NIHSS score on admission and the presence of brainstem infarction are associated with a poor functional outcome.

Abstract BackgroundThe universal availability of smartphones has created new opportunities for innovative telemedicine applications in health care. The COVID-19 pandemic has heightened the demand for contactless health care services, making SARS-CoV-2 polymerase chain reaction (PCR) testing a crucial component of pandemic containment. ObjectiveThis feasibility study aimed to examine a comprehensive telemedicine approach for SARS-CoV-2 testing, focusing on the practicality, user satisfaction, and economic implications of self-sampling guided by a telemedicine platform. MethodsThe study process involved shipping self-sampling kits, providing instructions for at-home sample collection, processing biomaterials (swabs and capillary blood), communicating test results, and providing interoperable data for clinical routine and research through a medical mobile app. A total of 100 individuals were randomly assigned to either the conventional health care professional (HCP)–performed SARS-CoV-2 testing group (conventional testing group, CG) or the telemedicine-guided SARS-CoV-2 self-sampling approach (telemedicine group, TG). Feasibility of the TG approach, user satisfaction, user-centered outcomes, and economic aspects were assessed and compared between the groups. ResultsIn the TG group, 47 out of 49 (95%) individuals received a self-sampling kit via mail (76%) individuals successfully returned at least one sample for diagnostics. SARS-CoV-2 PCR tests were conducted in 95% () of TG cases compared with 88% () in the CG. Users in the TG reported high satisfaction levels with ease of use (5.2/7), interface satisfaction (5.2/7), and usefulness (4.3/7). A microcosting model indicated a slightly higher cost for the TG approach than the CG approach. The TG demonstrated the potential to facilitate interoperable data transmission by providing anonymized, standardized datasets for extraction using Health Level 7-Fast Healthcare Interoperability Resources. This supports the national COVID-19 Data Exchange Platform and facilitates epidemiological evaluation based on the German COVID Consensus dataset. ConclusionThese preliminary findings suggest that a telemedicine-based approach to SARS-CoV-2 testing is feasible and could be integrated into existing hospital data infrastructures. This model has the potential for broader application in medical care, offering a scalable solution that could improve user satisfaction and treatment quality in the future.