The processing of staphylococcal biofilm images from Fluorescence and Confocal Microscopy to assess in vitro efficacy of antiseptic molecules

The in vitro efficacy of locally applied antiseptic molecules against staphylococcal biofilm is frequently assessed by a set of standard quantitative and semi-quantitative methods. The development of software for parametric image processing allowed to obtain parametric data also from microscopic images of biofilm dyed with a variety of dyes, especially with propidium iodine and SYTO-9, differentiating dead from live cells. In this work, using confocal/epifluorescent microscopy, we analyzed such major properties of staphylococcal biofilm in vitro as its thickness, cellular density and share of Live/Dead cells within its individual parts. We also scrutinized the impact of sample preparation and antiseptic introduction on the outcome obtained. As a result of our analyses we developed a revelatory method of assessment of the impact of antiseptic agents on staphylococcal biofilm in vitro, in which the microscopic images are processed with the use of ABE formula (Antiseptics Biofilm Eradication) which implements all the data and phenomena detected and revealed within the course of this study. We tested ABE with regard to polyhexanide, povidone-iodine and hypochlorous antiseptics and found a high correlation between this parameter and the results obtained by means of traditional techniques. Taking into account the fact that in vitro results of the efficacy of antiseptic agents against staphylococcal biofilm are frequently applied to back up their use in hospitals and ambulatory units, our work should be considered an important tool providing reliable, parametric data with this regard.

Role of the Sortase A in the Release of Cell-Wall Proteinase PrtS in the Growth Medium of Streptococcus thermophilus 4F44

Growth of the lactic acid bacterium Streptococcus thermophilus in milk depends on its capacity to hydrolyze proteins of this medium through its surface proteolytic activity. Thus, strains exhibiting the cell envelope proteinase (CEP) PrtS are able to grow in milk at high cellular density. Due to its LPNTG motif, which is possibly the substrate of the sortase A (SrtA), PrtS is anchored to the cell wall in most S. thermophilus strains. Conversely, a soluble extracellular PrtS activity has been reported in the strain 4F44. It corresponds, in fact, to a certain proportion of PrtS that is not anchored to the cell wall but rather is released in the growth medium. The main difference between PrtS of strain 4F44 (PrtS4F44) and other PrtS concerns the absence of a 32-residue imperfect duplication in the prodomain of the CEP, postulated as being required for the maturation and correct subsequent anchoring of PrtS. In fact, both mature (without the prodomain at the N-terminal extremity) and immature (with the prodomain) forms are found in the soluble PrtS4F44 form along with an intact LPNTG at their C-terminal extremity. Investigations we present in this work show that (i) the imperfect duplication is not implied in PrtS maturation; (ii) the maturase PrtM is irrelevant in PrtS maturation which is probably automaturated; and (iii) SrtA allows for the PrtS anchoring in S. thermophilus but the SrtA of strain 4F44 (SrtA4F44) displays an altered activity.

Correlation between ocular biometric parameters and corneal endothelium in a sample of young Egyptian adults

Background: To evaluate the normative values of corneal endothelial cell parameters within a group of healthy young Egyptian adults using specular microscopy and to examine any correlations between endothelial parameters and refractive or biometric parameters.Methods: In this cross-sectional study, specular microscopy was used to study the right eyes of 150 healthy young volunteers and evaluated endothelial cell parameters, including cellular density, hexagonality (HEX), and coefficient of variation (CV) at 15 different points on the back corneal surface, which were later grouped into the central zone and either four quadrants or three annular zones. The same eyes underwent refractive and biometric assessments.Results: Hundred fifty healthy adults were examined, and the age ranged from 20 to 30 years, with a median of 23 (interquartile range, 21?27) years. The mean ± standard deviation of central cell density was 2902.7 ± 270.7 cells/mm2. The superior paracentral area had the lowest mean density (2895.8 cells/mm2), but the highest mean HEX (67.7%), while the inferior peripheral area had the highest mean density (3100.5 cells/ mm2) but the lowest mean HEX (64%). The difference in cell density among the three annular zones was not statistically significant (P = 0.365). However, HEX and CV in the central and paracentral zones differed statistically significantly from those of the peripheral zone (P < 0.001 and P = 0.014, respectively). Weak but non-significant correlations were detected between endothelial cell density and all measured refractive and biometric parameters.Conclusions: The findings of this study provided useful normative biometric and specular data in a specific age group and a specific population, and could be useful in planning intraocular surgery in young Egyptian adults. However, future longitudinal studies with a larger sample could refine more endothelial cell parameter specifications over time.

Voxel-Based Analysis of the Relation of 3′-Deoxy-3′-[18F]fluorothymidine ([18F]FLT) PET and Diffusion-Weighted (DW) MR Signals in Subcutaneous Tumor Xenografts Does Not Reveal a Direct Spatial Relation of These Two Parameters

Purpose Multimodal molecular imaging allows a direct coregistration of different images, facilitating analysis of the spatial relation of various imaging parameters. Here, we further explored the relation of proliferation, as measured by [18F]FLT PET, and water diffusion, as an indicator of cellular density and cell death, as measured by diffusion-weighted (DW) MRI, in preclinical tumor models. We expected these parameters to be negatively related, as highly proliferative tissue should have a higher density of cells, hampering free water diffusion. Procedures Nude mice subcutaneously inoculated with either lung cancer cells (n = 11 A549 tumors, n = 20 H1975 tumors) or colorectal cancer cells (n = 13 Colo205 tumors) were imaged with [18F]FLT PET and DW-MRI using a multimodal bed, which was transferred from one instrument to the other within the same imaging session. Fiducial markers allowed coregistration of the images. An automatic post-processing was developed in MATLAB handling the spatial registration of DW-MRI (measured as apparent diffusion coefficient, ADC) and [18F]FLT image data and subsequent voxel-wise analysis of regions of interest (ROIs) in the tumor. Results Analyses were conducted on a total of 76 datasets, comprising a median of 2890 data points (ranging from 81 to 13,597). Scatterplots showing [18F]FLT vs. ADC values displayed various grades of relations (Pearson correlation coefficient (PCC) varied from − 0.58 to 0.49, median: -0.07). When relating PCC to tumor volume (median: 46 mm3, range: 3 mm3 to 584 mm3), lung tumors tended to have a more pronounced negative spatial relation of [18F]FLT and ADC with increasing tumor size. However, due to the low number of large tumors (> ~ 200 mm3), this conclusion has to be treated with caution. Conclusions A spatial relation of water diffusion, as measured by DW-MRI, and cellular proliferation, as measured by [18F]FLT PET, cannot be detected in the experimental datasets investigated in this study.

NIMG-58. CANONICAL CORRELATION ANALYSIS IN GLIOBLASTOMA REVEALS ASSOCIATIONS BETWEEN EXPRESSION OF RADIOMIC SIGNATURES AND GENOMICS

PURPOSE Understanding the molecular underpinnings of imaging signatures of glioblastoma can provide insights into the biologic basis of tumor formation and progression as well as in vivo surrogate markers of molecular events driving the tumor’s phenotype. Through machine learning (ML), this study demonstrates that distinct imaging subtypes of glioblastoma are related to specific molecular alterations. METHODS Pre-operative multi-parametric MRI (T1, T2, T1CE, T2-FLAIR, DSC-MRI, DTI-MRI) of 669 IDH-wildtype subjects with glioblastoma were retrospectively collected and radiomic features, including descriptors of morphology, intensity, histogram, and texture, were extracted. Imaging subtypes were identified by a feature selection and clustering approach. Genomic markers, obtained using next generation sequencing (NGS) panel of 27 key glioblastoma genes, were available in 358/669 patients. Canonical correlation analysis (CCA) was conducted within each imaging subtype between the selected imaging features and genetic variables to seek maximum correlations between combinations of variables in imaging and genomic sets, and hence elucidate the molecular drivers of respective subtypes. RESULTS Three distinct imaging subtypes were identified by clustering on 50 selected features, representing characteristics of morphology, tumor neo-angiogenesis (DSC-derived features), and cellular density (DTI-derived features). These subtypes yielded differentiable overall survival based on Kaplan-Meier analysis. The canonical coefficients of each subtype revealed the distinction of the underlying genomic characteristics: one exhibited frequently mutated [ARID2, NTRK1], another subtype showed increased frequency of mutation in [ATRX, EGFR, PIK3R1], while the third was associated with all these genes and [NF1, PIK3CA, RB1], additionally. CONCLUSION We derived three distinct radiomic MRI subtypes for glioblastoma that highly correlate with the patients' survival and molecular genetic characteristics. Investigating the relationship between imaging and genomic information may enable identification of molecularly- and phenotypically-consistent tumor subtypes, which would offer non-invasive approaches for characterizing heterogeneity of glioblastoma, further facilitating patient stratification and treatment planning.

Microscopic Quantification of Oxygen Consumption across Cortical Layers

The cerebral cortex is organized in cortical layers that differ in their cellular density, composition, and wiring. Cortical laminar architecture is also readily revealed by staining for cytochrome oxidase – the last enzyme in the respiratory electron transport chain located in the inner mitochondrial membrane. It has been hypothesized that a high-density band of cytochrome oxidase in cortical layer IV reflects higher oxygen consumption under baseline (unstimulated) conditions. Here, we tested the above hypothesis using direct measurements of the partial pressure of O 2 (pO 2 ) in cortical tissue by means of 2-photon phosphorescence lifetime microscopy (2PLM). We revisited our previously developed method for extraction of the cerebral metabolic rate of O 2 (CMRO 2 ) based on 2-photon pO 2 measurements around diving arterioles and applied this method to estimate baseline CMRO 2 in awake mice across cortical layers. To our surprise, our results revealed a decrease in baseline CMRO 2 from layer I to layer IV . This decrease of CMRO 2 with cortical depth was paralleled by an increase in tissue oxygenation. Higher baseline oxygenation and cytochrome density in layer IV may serve as an O 2 reserve during surges of neuronal activity or certain metabolically active brain states rather than baseline energy needs. Our study provides the first quantification of microscopically resolved CMRO 2 across cortical layers as a step towards informed interpretation and modeling of cortical-layer-specific Blood Oxygen Level Dependent (BOLD) fMRI signals.

Feasibility and utility of MRI and dynamic 18F-FDG-PET in an orthotopic organoid-based patient-derived mouse model of endometrial cancer

Background Pelvic magnetic resonance imaging (MRI) and whole-body positron emission tomography-computed tomography (PET-CT) play an important role at primary diagnostic work-up and in detecting recurrent disease in endometrial cancer (EC) patients, however the preclinical use of these imaging methods is currently limited. We demonstrate the feasibility and utility of MRI and dynamic 18F-fluorodeoxyglucose (FDG)-PET imaging for monitoring tumor progression and assessing chemotherapy response in an orthotopic organoid-based patient-derived xenograft (O-PDX) mouse model of EC. Methods 18 O-PDX mice (grade 3 endometrioid EC, stage IIIC1), selectively underwent weekly T2-weighted MRI (total scans = 32), diffusion-weighted MRI (DWI) (total scans = 9) and dynamic 18F-FDG-PET (total scans = 26) during tumor progression. MRI tumor volumes (vMRI), tumor apparent diffusion coefficient values (ADCmean) and metabolic tumor parameters from 18F-FDG-PET including maximum and mean standard uptake values (SUVmax/SUVmean), metabolic tumor volume (MTV), total lesion glycolysis (TLG) and metabolic rate of 18F-FDG (MRFDG) were calculated. Further, nine mice were included in a chemotherapy treatment study (treatment; n = 5, controls; n = 4) and tumor ADCmean-values were compared to changes in vMRI and cellular density from histology at endpoint. A Mann–Whitney test was used to evaluate differences between groups. Results Tumors with large tumor volumes (vMRI) had higher metabolic activity (MTV and TLG) in a clear linear relationship (r2 = 0.92 and 0.89, respectively). Non-invasive calculation of MRFDG from dynamic 18F-FDG-PET (mean MRFDG = 0.39 μmol/min) was feasible using an image-derived input function. Treated mice had higher tumor ADCmean (p = 0.03), lower vMRI (p = 0.03) and tumor cellular density (p = 0.02) than non-treated mice, all indicating treatment response. Conclusion Preclinical imaging mirroring clinical imaging methods in EC is highly feasible for monitoring tumor progression and treatment response in the present orthotopic organoid mouse model.

Red blood cell ATP release correlates with red blood cell hemolysis

The precise matching of blood flow to skeletal muscle during exercise remains an important area of investigation. Release of adenosine triphosphate (ATP) from red blood cells (RBCs) is postulated to mediate peripheral vascular tone in response to shear stress, hypoxia, and mechanical deformation. We tested the following hypotheses: 1) RBCs of different densities contain different quantities of ATP; 2) hypoxia is a stimulus for ATP release from RBCs; and 3) hypoxic ATP release from RBCs is related to RBC lysis. Human blood was drawn from male and female volunteers (n=11); the RBCs were isolated and washed. A Percoll gradient was used to separate RBCs by cellular density. Density groups were then re-suspended to 4% hematocrit and exposed to normoxia or hypoxia in a tonometer. Equilibrated samples were drawn and centrifuged; paired analyses of ATP (luminescence via a luciferase-catalyzed reaction) and hemolysis (Harboe spectrophotometric absorbance assay) were measured in the supernatant. ATP release was not different among low-density (LD) cells versus middle-density (MD) versus high-density (HD) cells. Similarly, hemoglobin (Hb) release was not different among the RBC subsets. No difference was found for either ATP release or Hb release following matched exposure to normoxic or hypoxic gas. [ATP] and [Hb] for all subsets combined were linearly correlated (r=0.59, p<0.001). With simultaneous probing for Hb and ATP in the supernatant of each sample, we conclude that ATP release from RBCs can be explained by hemolysis and that hypoxia per se does not stimulate either ATP release or Hb release from RBCs.

Loss of the ClpXP Protease Leads to Decreased Resistance to Cell-Envelope Targeting Antimicrobials in Bacillus anthracis Sterne

The ClpX ATPase is critical for resistance to cell envelope targeting antibiotics in Bacillus anthracis, however, it is unclear whether this is due to its function as an independent chaperone or as part of the ClpXP protease. In this study, we demonstrate that antibiotic resistance is due to formation of the ClpXP protease through construction of a ClpX complementation plasmid that is unable to interact with ClpP. Additionally, we genetically disrupted both clpP genes, clpP1 and clpP2, found in B. anthracis Sterne and find that the loss of either increases susceptibility to cell envelope targeting antimicrobials, although neither has as strong of a phenotype as loss of clpX and neither clpP gene is essential for virulence in a G. mellonella model of infection. Lastly, we looked at changes to cell envelope morphology that could contribute to increased antibiotic sensitivity. We find no difference in cell charge or cell lysis, although we do see increased hydrophobicity in the ΔclpX strain, decreased cellular density and slightly thinner cells walls. We also see significant cell division defects in ΔclpX, although only when cells are grown in the mammalian cell culture medium, RPMI. We conclude that the intrinsic resistance of B. anthracis to cell wall active antimicrobials is dependent on formation of the ClpXP protease and that this could be due, at least in part, to the role of ClpX in regulating cell envelope morphology.

Soma and Neurite Density MRI (SANDI) of the in-vivo mouse brain

Diffusion MRI (dMRI) provides unique insights into the neural tissue milieu by probing interaction of diffusing molecules and tissue microstructure. Most dMRI techniques focus on white matter tissues (WM) due to the relatively simpler modelling of diffusion in the more organized tracts; however, interest is growing in gray matter characterisations. The Soma and Neurite Density MRI (SANDI) methodology harnesses a model incorporating water diffusion in spherical objects (assumed to be associated with cell bodies) and in impermeable 'sticks' (representing neurites), which potentially enables the characterisation of cellular and neurite densities. Recognising the importance of rodents in animal models of development, aging, plasticity, and disease, we here sought to develop SANDI for preclinical imaging and provide a validation of the methodology by comparing its metrics with the Allen mouse brain atlas. SANDI was implemented on a 9.4T scanner equipped with a cryogenic coil, and experiments were carried out on N=6 mice. Pixelwise, ROI-based, and atlas comparisons were performed, and results were also compared to more standard Diffusion Kurtosis MRI (DKI) metrics. We further investigated effects of different pre-processing pipelines, specifically the comparison of magnitude and real-valued data, as well as different acceleration factors. Our findings reveal excellent reproducibility of the SANDI parameters, including the sphere and stick fraction as well as sphere size. More strikingly, we find a very good rank correlation between SANDI-driven soma fraction and Allen brain atlas contrast (which represents the cellular density in the mouse brain). Although some DKI parameters (FA, MD) correlated with some SANDI parameters in some ROIs, they did not correlate nearly as well as SANDI parameters with the Allen atlas, suggesting a much more specific nature of the SANDI parameters. We conclude that SANDI is a viable preclinical MRI technique that can greatly contribute to research on brain tissue microstructure.