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Abstract Clonal transmission and horizontal gene transfer (HGT) contribute to the spread of vancomycin-resistant enterococci (VRE) in global healthcare. Our study investigated vesiduction, a HGT mechanism via membrane vesicles (MVs), for vanA and vanB genes that determine vancomycin resistance. We isolated MVs for VRE of different sequence types (STs) and analysed them by nanoparticle tracking analysis. Selected MV samples were subjected to DNA sequence analysis. In resistance transfer experiments, vancomycin-susceptible enterococci were exposed to MVs and bacterial supernatants of VRE. Compared to bacteria grown in lysogeny broth (MVs/LB), cultivation under vancomycin stress (MVs/VAN) resulted in increased particle concentrations of up to 139-fold (ST80). As a key finding, we could show that VRE isolates of ST80 and ST117 produced remarkably more vesicles at subinhibitory antibiotic concentrations (approx. 9.2 × 1011 particles/ml for ST80 and 2.4 × 1011 particles/ml for ST117) than enterococci of other STs (range between 1.8 × 1010 and 5.3 × 1010 particles/ml). In those MV samples, the respective resistance genes vanA and vanB were completely verifiable using sequence analysis. Nevertheless, no vancomycin resistance transfer via MVs to vancomycin-susceptible Enterococcus faecium was phenotypically detectable. However, our results outline the potential of future research on ST-specific MV properties, promising new insights into VRE mechanisms.

The roles companion animals have played in our lives has dramatically changed over the last few decades. At the same time, the terms we use to describe both the people and animals in these human-animal relationships have also changed. One example includes the use of the terms 'owner' or 'guardian' to refer to the human caretaker. While preferences by society appear to indicate increased interest in referring to companion animal caretakers as 'guardians', others have cautioned against its use or attempted to restrict it. Additionally, the use of animal welfare terminology has direct implications for how we conduct both welfare research and practice. Our paper examines the use of 'owner' and 'guardian' with respect to (1) the implications for changing terminology on the function, clarity and uniformity of their use, and (2) the ethical and welfare impact that coincides with each term's use. Our goal is to propose terminological considerations that could influence future welfare research, as well as help guide our interactions with companion animals.

The roles companion animals have played in our lives has dramatically changed over the last few decades. At the same time, the terms we use to describe both the people and animals in these human-animal relationships have also changed. One example includes the use of the terms ‘owner’ or ‘guardian’ to refer to the human caretaker. While preferences by society appear to indicate increased interest in referring to companion animal caretakers as ‘guardians’, others have cautioned against its use or attempted to restrict it. Additionally, the use of animal welfare terminology has direct implications for how we conduct both welfare research and practice. Our paper examines the use of ‘owner’ and ‘guardian’ with respect to (1) the implications for changing terminology on the function, clarity and uniformity of their use, and (2) the ethical and welfare impact that coincides with each term’s use. Our goal is to propose terminological considerations that could influence future welfare research, as well as help guide our interactions with companion animals.

Abstract In recent years, vibrational spectroscopic techniques based on Fourier transform infrared (FTIR) or Raman microspectroscopy have been suggested to fulfill the unmet need for microplastic particle detection and identification. Inter-system comparison of spectra from reference polymers enables assessing the reproducibility between instruments and advantages of emerging quantum cascade laser-based optical photothermal infrared (O-PTIR) spectroscopy. In our work, IR and Raman spectra of nine plastics, namely polyethylene, polypropylene, polyvinyl chloride, polyethylene terephthalate, polycarbonate, polystyrene, silicone, polylactide acid and polymethylmethacrylate were simultaneously acquired using an O-PTIR microscope in non-contact, reflection mode. Comprehensive band assignments were presented. We determined the agreement of O-PTIR with standalone attenuated total reflection FTIR and Raman spectrometers based on the hit quality index (HQI) and introduced a two-dimensional identification (2D-HQI) approach using both Raman- and IR-HQIs. Finally, microplastic particles were prepared as test samples from known materials by wet grinding, O-PTIR data were collected and subjected to the 2D-HQI identification approach. We concluded that this framework offers improved material identification of microplastic particles in environmental, nutritious and biological matrices.

We aimed to investigate whether a selective pre-PCR enrichment step improves test performance of RIDA®GENE EHEC/EPEC to detect diarrheagenic Escherichia coli from stool samples. Each of the 250 stool samples was analyzed for the presence of stx1/2 and eae both with and without pre-PCR enrichment in selective broth. In comparison to a reference method, sensitivities for stx1/2 and eae with and without pre-PCR enrichment were 84% (95%CI 70–93) and 89% (stx1/2, 95%CI 76–96), and 71% (95%CI 58–81) and 72% (eae, 95%CI 60–82), respectively. Specificity exceeded 97% for both methods and target genes. In summary, pre-PCR broth enrichment did not improve test performance.

Abstract Background Pimobendan is a widely used medication for the treatment of dogs with congestive heart failure (CHF) and preclinical degenerative mitral valve disease (DMVD) with cardiomegaly. The benefit of a treatment in dogs with preclinical DMVD but without cardiomegaly has not yet been elucidated. Some positive effects concerning life quality and a decrease in cardiac biomarkers could be verified. This study aimed to further investigate these results using a placebo-controlled double-blinded crossover design. Out of a total of 15 dogs, eight were allocated to sequence-group AB, in which dogs received pimobendan (A) during the first treatment period and placebo (B) during the second period. Accordingly, sequence-group BA was treated first with placebo followed by pimobendan. Each treatment period lasted six months and included a baseline investigation and follow-ups after 90 and 180 days. The investigations included a questionnaire completed by the owners, echocardiographic examination, and measurements of NT-proBNP, cTnI and lactate before and after a standardised submaximal exercise test. Results NT-proBNP values decreased significantly during the treatment period with pimobendan, and the post-exercise increase was attenuated at day 180. No significant treatment effects could be verified for cTnI and lactate, neither pre- nor post-exercise. Left ventricular size decreased under treatment, whereas no significant changes in left atrial size were detected. The owners described their dogs under treatment with pimobendan as being more active at day 90 (11/15) and day 180 (12/15). Those animals treated with placebo were described as being more active at day 90 (2/15) and day 180 (5/15). Conclusions Pimobendan had reducing effects on the concentrations of pre- and post-exercise cardiac biomarkers and the size of the left ventricle in dogs with DMVD ACVIM B1. Exercise testing in addition to an assessment of cardiac biomarkers might improve the decision when to initiate pimobendan treatment in dogs with DMVD.

PurposeHigh-grade gliomas (HGG) are still associated with a dismal prognosis. Prostate specific membrane antigen (PSMA) is discussed as a theranostic target for PSMA-directed radioligand therapy ([177Lu]Lu-PSMA RLT). Here, we report on the correlation of [68Ga]Ga-PSMA uptake with histological PSMA expression and on our preliminary experience with [177Lu]Lu-PSMA RLT in relapsing HGG.MethodsPatients with relapsing HGG underwent [68Ga]Ga-PSMA PET/MRI to evaluate eligibility for an individualized treatment approach with [177Lu]Lu-PSMA. Standard uptake values (SUV) for tumor and liver and respective tumor-to-background ratios (compared to the liver) (TBR) on [68Ga]Ga-PSMA PET/MRI were assessed. Eligibility criteria for [177Lu]Lu-PSMA therapy were exhaustion of all standard treatment options available and TBRmax>1.0. In 11 samples, immunohistochemical PSMA expression was determined, quantified using the H-score and correlated with uptake on [68Ga]Ga-PSMA PET/MRI.ResultsWe included 20 patients with a median age of 53 years (IQR 42-57). The median SUV on [68Ga]Ga-PSMA PET/MRI was 4.5 (3.7-6.2) for SUVmax and 1.4 (1.1-1.7) for SUVmean. The respective TBR was maximum 0.6 (0.4-0.8) and mean 0.3 (0.2-0.4). High TBRmax correlated with increased endothelial PSMA expression [H-score of 65 (62.5-77.5)]. Three patients (15%) presented a TBRmax>1.0 and qualified for [177Lu]Lu-PSMA RLT. No treatment related toxicity was observed.ConclusionOnly a minority of patients with relapsing HGG qualified for [177Lu]Lu-PSMA RLT. Our data demonstrates that PSMA expression in the neo-vasculature corresponds to PSMA uptake on [68Ga]Ga-PSMA PET/MRI and might be used as a screening tool for patient selection. Future prospective studies need to focus the debate on TBRmax thresholds as inclusion criteria for PSMA RLT.

In the course of efficient development and optimization of biotechnological processes, the need for methods to track morphological and compositional changes of single cells is increasing. So far, the material composition of cells is determined by chemical analysis of a pooled cell sample, which reflects the average composition of the taken cell collection. Conventional flow cytometry enables the analysis of individuals from a population. However, it cannot resolve such valuable information like morphological details and distribution of molecular compounds inside the cells. This gap is bridged by a combination of imaging flow cytometry and multispectral imaging. The potential of this Multispectral Imaging Flow Cytometry (MIFC) approach has been investigated and confirmed in the presented parameter study on the bioproduction of Astaxanthin (Ax) by the microalgae Haematococcus pluvialis (HP). As far as multispectral imaging in transmission mode, only three spectral channels (446 nm, 532 nm, 646 nm) were used to measure the amount of substance and the molecular distribution of the core components chlorophyll (Chl) and Ax. Both could be clearly separated from the phase-contrast information generated from the cellular structures and morphology. In general, the results from the MIFC method comply with the conventional measurements but extend them for details on the morphology and on compositional changes within the cultivated cell population during the cultivation process and in response to the applied stimuli.

Background and Purpose: Thrombo‐inflammation is a key feature of stroke pathophysiology and provides multiple candidate drug targets. Thrombin exerts coagulation‐independent actions via protease‐activated receptors (PAR), of which PAR1 has been implicated in stroke‐associated neuroinflammation. The role of PAR4 in this context is less clear. This study examined if the selective PAR4 antagonist ML354 provides neuroprotection in experimental stroke and explored the underlying mechanisms. Experimental Approach: Mouse primary cortical neurons were exposed to oxygen–glucose deprivation (OGD) and simulated reperfusion ± ML354. For comparison, functional Ca2+‐imaging was performed upon acute stimulation with a PAR4 activating peptide or glutamate. Male mice underwent sham operation or transient middle cerebral artery occlusion (tMCAO), with ML354 or vehicle treatment beginning at recanalization. A subset of mice received a platelet‐depleting antibody. Stroke size and functional outcomes were assessed. Abundance of target genes, proteins, and cell markers was determined in cultured cells and tissues by qPCR, immunoblotting, and immunofluorescence. Key Results: Stroke up‐regulated PAR4 expression in cortical neurons in vitro and in vivo. OGD augments spontaneous and PAR4‐mediated neuronal activity; ML354 suppresses OGD‐induced neuronal excitotoxicity and apoptosis. ML354 applied in vivo after tMCAO reduced infarct size, apoptotic markers, macrophage accumulation, and interleukin‐1β expression. Platelet depletion did not affect infarct size in mice with tMCAO ± ML354. Conclusions and Implications: Selective PAR4 inhibition during reperfusion improves infarct size and neurological function after experimental stroke by blunting neuronal excitability, apoptosis, and local inflammation. PAR4 antagonists may provide additional neuroprotective benefits in patients with acute stroke beyond their canonical antiplatelet action. [ABSTRACT FROM AUTHOR]

ObjectRecent studies demonstrated that gross total resection of brain metastases cannot always be achieved. Subtotal resection (STR) can result in an early recurrence and might affect patient survival. We initiated a prospective observational study to establish a MRI-based risk assessment for incomplete resection of brain metastases.MethodsAll patients in whom ≥1 brain metastasis was resected were prospectively included in this study (DRKS ID: DRKS00021224; Nov 2020 – Nov 2021). An interdisciplinary board of neurosurgeons and neuroradiologists evaluated the pre- and postoperative MRI (≤48h after surgery) for residual tumor. Extensive neuroradiological analyses were performed to identify risk factors for an unintended STR which were integrated into a regression tree analysis to determine the patients’ individual risk for a STR.ResultsWe included 150 patients (74 female; mean age: 61 years), in whom 165 brain metastases were resected. A STR was detected in 32 cases (19.4%) (median residual tumor volume: 1.36ml, median EORrel: 93.6%), of which 6 (3.6%) were intended STR (median residual tumor volume: 3.27ml, median EORrel: 67.3%) - mainly due to motor-eloquent location - and 26 (15.8%) were unintended STR (uSTR) (median residual tumor volume: 0.64ml, median EORrel: 94.7%). The following risk factors for an uSTR could be identified: subcortical metastasis ≥5mm distant from cortex, diffuse contrast agent enhancement, proximity to the ventricles, contact to falx/tentorium and non-transcortical approaches. Regression tree analysis revealed that the individual risk for an uSTR was mainly associated to the distance from the cortex (distance ≥5mm vs.

Abstract Background Segmental progeroid syndromes are a heterogeneous group of rare and often severe genetic disorders that have been studied since the twentieth century. These progeroid syndromes are defined as segmental because only some of the features observed during natural aging are accelerated. Methods Since 2015, the Molecular Genetics Laboratory in Marseille La Timone Hospital proposes molecular diagnosis of premature aging syndromes including laminopathies and related disorders upon NGS sequencing of a panel of 82 genes involved in these syndromes. We analyzed the results obtained in 4 years on 66 patients issued from France and abroad. Results Globally, pathogenic or likely pathogenic variants (ACMG class 5 or 4) were identified in about 1/4 of the cases; among these, 9 pathogenic variants were novel. On the other hand, the diagnostic yield of our panel was over 60% when the patients were addressed upon a nosologically specific clinical suspicion, excepted for connective tissue disorders, for which clinical diagnosis may be more challenging. Prenatal testing was proposed to 3 families. We additionally detected 16 variants of uncertain significance and reclassified 3 of them as benign upon segregation analysis in first degree relatives. Conclusions High throughput sequencing using the Laminopathies/ Premature Aging disorders panel allowed molecular diagnosis of rare disorders associated with premature aging features and genetic counseling for families, representing an interesting first-level analysis before whole genome sequencing may be proposed, as a future second step, by the National high throughput sequencing platforms (“Medicine France Genomics 2025” Plan), in families without molecular diagnosis.

ObjectiveThe utilization of fluorescein-guided biopsies and resection has been recently discussed as a suitable strategy to improve and expedite operative techniques for the resection of central nervous system (CNS) tumors. However, little is known about the optical properties of sodium fluorescein (NaFl) in human tumor tissue and their potential impact on ex vivo analyses involving fluorescence-based methods.MethodsTumor tissue was obtained from a study cohort of an observational study on the utilization of fluorescein-guided biopsy and resection (n=5). The optical properties of fluorescein-stained tissue were compared to the optical features of the dye in vitro and in control samples consisting of tumor tissue of high-grade glioma patients (n=3) without intravenous (i.v.) application of NaFl. The dye-exposed tumor tissues were used for optical measurements to confirm the detectability of NaFl emission ex vivo. The tissue samples were fixed in 4%PFA, immersed in 30% sucrose, embedded in Tissue-Tek OCT compound, and cut to 10 μm cryosections. Spatially resolved emission spectra from tumor samples were recorded on representative slides with a Confocal Laser Scanning Microscope FV1000 (Olympus GmbH, Hamburg, Germany) upon excitation with λexc = 488 nm.ResultsOptical measurements of fluorescein in 0.9% sodium chloride (NaCl) under in vitro conditions showed an absorption maximum of λmax abs = 479 nm as detected with spectrophotometer Specord 200 and an emission peak at λmax em = 538 nm recorded with the emCCD detection system of a custom-made microscope-based single particle setup using a 500 nm long-pass filter. Further measurements revealed pH- and concentration-dependent emission spectra of NaFl. Under ex vivo conditions, confocal laser scanning microscopy of fluorescein tumor samples revealed a slight bathochromic shift and a broadening of the emission band.ConclusionTumor uptake of NaFl leads to changes in the optical properties – a bathochromic shift and broadening of the emission band – possibly caused by the dye’s high pH sensitivity and concentration-dependent reabsorption acting as an inner filter of the dye’s emission, particularly in the short wavelength region of the emission spectrum where absorption and fluorescence overlap. Understanding the ex vivo optical properties of fluorescein is crucial for testing and validating its further applicability as an optical probe for intravital microscopy, immunofluorescence localization studies, and flow cytometry analysis.

Since March 2020, the COVID-19 pandemic forced hospitals worldwide to intensify their infection control measures to prevent health care-associated transmission of SARS-CoV-2. The correct use of personal protective equipment, especially the application of masks, was quickly identified as priority to reduce transmission with this pathogen. Here, we report a nosocomial cluster of methicillin-resistant Staphylococcus aureus (MRSA) that occurred during the COVID-19 pandemic in a gynecology/obstetrics department, despite these intensified contact precautions. Five MRSA originating from clinical samples after surgical intervention led to an outbreak investigation. Firstly, this included environmental sampling of the operation theatre (OT) and, secondly, a point prevalence screening of patients and health care workers (HCW). All detected MRSA were subjected to whole genome sequencing (WGS) and isolate relatedness was determined using core genome multilocus sequence typing (cgMLST). WGS revealed one MRSA cluster with genetically closely related five patient and two HCW isolates differing in a single cgMLST allele at maximum. The outbreak was terminated after implementation of infection control bundle strategies. Although contact precaution measures, which are also part of MRSA prevention bundle strategies, were intensified during the COVID-19 pandemic, this MRSA outbreak could take place. This illustrates the importance of adherence to classical infection prevention strategies.

MicroRNAs (miRNAs) are important regulators of gene expression. These small, non-coding RNAs post-transcriptionally silence messenger RNAs (mRNAs) in a sequence-specific manner. In this way, miRNAs control important regulatory functions, also in the retina. If dysregulated, these molecules are involved in several retinal pathologies. For example, several miRNAs have been linked to essential photoreceptor functions, including light sensitivity, synaptic transmission, and modulation of inflammatory responses. Mechanistic miRNA knockout and knockdown studies further linked their functions to degenerative retinal diseases. Of note, the type and timing of genetic manipulation before, during, or after retinal development, is important when studying specific miRNA knockout effects. Within this review, we focus on miR-124 and the miR-183/96/182 cluster, which have assigned functions in photoreceptors in health and disease. As a single miRNA can regulate hundreds of mRNAs, we will also discuss the experimental validation and manipulation approaches to study complex miRNA/mRNA regulatory networks. Revealing these networks is essential to understand retinal pathologies and to harness miRNAs as precise therapeutic and diagnostic tools to stabilize the photoreceptors’ transcriptomes and, thereby, function.

The receptor tyrosine kinase AXL (RTK-AXL) is implicated in therapy resistance and tumor progression in glioblastoma multiforme (GBM). Here, we investigated therapy-induced receptor modifications and how endogenous RTK-AXL expression and RTK-AXL inhibition contribute to therapy resistance in GBM. GBM cell lines U118MG and SF126 were exposed to temozolomide (TMZ) and radiation (RTX). Receptor modifications in response to therapy were investigated on protein and mRNA levels. TMZ-resistant and RTK-AXL overexpressing cell lines were exposed to increasing doses of TMZ and RTX, with and without RTK-AXL tyrosine kinase inhibitor (TKI). Colorimetric microtiter (MTT) assay and colony formation assay (CFA) were used to assess cell viability. Results showed that the RTK-AXL shedding product, C-terminal AXL (CT-AXL), rises in response to repeated TMZ doses and under hypoxia, acts as a surrogate marker for radio-resistance. Endogenous RTX-AXL overexpression leads to therapy resistance, whereas combination therapy of TZM and RTX with TKI R428 significantly increases therapeutic effects. This data proves the role of RTK-AXL in acquired and intrinsic therapy resistance. By demonstrating that therapy resistance may be overcome by combining AXL TKI with standard treatments, we have provided a rationale for future study designs investigating AXL TKIs in GBM.