Phase Contrast Research Articles

Tricuspid valve flow measurement using a deep learning framework for automated valve-tracking 2D phase contrast.

Tricuspid valve flow velocities are challenging to measure with cardiovascular MR, as the rapidly moving valvular plane prohibits direct flow evaluation, but they are vitally important to diastolic function evaluation. We developed an automated valve-tracking 2D method for measuring flow through the dynamic tricuspid valve. Nine healthy subjects and 2 patients were imaged. The approach uses a previously trained deep learning network, TVnet, to automatically track the tricuspid valve plane from long-axis cine images. Subsequently, the tracking information is used to acquire 2D phase contrast (PC) with a dynamic (moving) acquisition plane that tracks the valve. Direct diastolic net flows evaluated from the dynamic PC sequence were compared with flows from 2D-PC scans acquired in a static slice localized at the end-systolic valve position, and also ventricular stroke volumes (SVs) using both planimetry and 2D PC of the great vessels. The mean tricuspid valve systolic excursion was 17.8 ± 2.5 mm. The 2D valve-tracking PC net diastolic flow showed excellent correlation with SV by right-ventricle planimetry (bias ± 1.96 SD = -0.2 ± 10.4 mL, intraclass correlation coefficient [ICC] = 0.92) and aortic PC (-1.0 ± 13.8 mL, ICC = 0.87). In comparison, static tricuspid valve 2D PC also showed a strong correlation but had greater bias (p = 0.01) versus the right-ventricle SV (10.6 ± 16.1 mL, ICC = 0.61). In most (8 of 9) healthy subjects, trace regurgitation was measured at begin-systole. In one patient, valve-tracking PC displayed a high-velocity jet (380 cm/s) with maximal velocity agreeing with echocardiography. Automated valve-tracking 2D PC is a feasible route toward evaluation of tricuspid regurgitant velocities, potentially solving a major clinical challenge.

Experience in the production and clinical application of the cell-based medicinal product Easytense® for the repair of cartilage defects of the human knee

INTRODUCTION. The current cell-based cartilage repair methods, such as autologous chondrocyte transplantation, are not sufficiently effective, and the surgery is painful and traumatic. Therefore, there is a need for a more effective cell therapy product with a minimally invasive surgical procedure for its implantation into the patient.AIM. This study aimed to develop a manufacturing technology for the production of an autologous cell-based medicinal product (CBMP) comprising three-dimensional structures (3D-spheroids) based on chondrocytes isolated from the patient’s cartilage tissue, as well as to evaluate its clinical efficacy.MATERIALS AND METHODS. Autologous chondrocytes isolated from the patient’s cartilage biopsy were propagated in monolayer culture to obtain the required number of cells. Subsequently, the chondrocytes were cultivated on plates with a non-adhesive coating to form 3D spheroids. All CBMP production steps were performed under aseptic conditions in cell culture isolators. The authors used phase-contrast microscopy and immunohistochemical staining with specific fluorescence-labelled antibodies to characterise chondrocyte phenotypes at different stages of cultivation. Genetic stability was controlled by karyotyping. The efficacy of Easytense® was evaluated in a clinical trial using specialised functional tests and the Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) score. The primary efficacy endpoint was a change in the overall score on the Knee Injury and Osteoarthritis Outcome Score (KOOS).RESULTS. A manufacturing technology without using animal sera, growth factors, cytokines, or other additives was developed for the production of the autologous CBMP Easytense®. Karyological data confirmed that the chondrocytes retained genetic stability for 3 passages in monolayer culture. When cultured as 3D spheroids, the chondrocytes produced cartilage extracellular matrix proteins (type II collagen, aggrecan), thus acquiring the ability to repair damaged cartilage. The clinical trial demonstrated a statistically significant improvement in knee cartilage 12 months after the transplantation of 3D spheroids derived from autologous chondrocytes. The mean change in the overall KOOS score was 23.8±15.9.CONCLUSIONS. The clinical trial results indicate that Easytense® is highly effective for cartilage repair. Based on these results, the CBMP has been granted marketing authorisation and introduced into clinical practice in the Russian Federation. Easytense® has the potential to replace endoprosthetics and expensive surgeries abroad.

Comparative analysis of the effect of drugs lowering intraocular pressure on a primary culture of human corneal epithelium and A549 immortalized cell line

Background. Glaucoma as one of the most common eye diseases can be a comorbid condition of an epithelial corneal defect of various etiologies. Maintaining an optimal level of ophthalmotonus includes the prescription of antiglaucoma drugs, including benzalkonium chloride-preserved drugs.The aim of the study. To compare the effect of antiglaucoma drugs, as well as benzalkonium chloride (BC), on a primary culture of human corneal epithelium and A549 immortalized cell line.Methods. The effect of brimonidine, dorzolamide and timolol (1/100, 1/50, 1/20 and 1/10 dilutions; exposure 24 hours) on a monolayer of a human corneal epithelial primary culture and A549 immortalized cell line was assessed by structural changes in cells (phase-contrast microscopy) and MTT assay data. The cytotoxic effect of BC was studied in concentrations corresponding to its content in these dilutions of the antiglaucoma drug. Using a model of a linear defect in the monolayer of a corneal epithelial primary culture and A549 immortalized cell line, the effects of brimonidine, dorzolamide and timolol (1/100 and 1/20 dilutions; exposure 48 hours) were assessed by cell migration activity.Results. Among drugs (BC-free), dorzolamide (1/50, 1/20 and 1/10 dilutions) causes minor structural changes in human corneal epithelium and A549 immortalized cell line; timolol (1/100, 1/50, 1/20 and 1/10 dilutions) causes minor structural changes in A549 immortalized cell line. Structural changes in both types of cells, a decrease in their metabolic and migration activity occur under the influence of dorzolamide, brimonidine and timolol (BC-preserved) in 1/100, 1/50, 1/20 and 1/10 dilutions. BC at the studied concentrations exhibits a similar effect.Conclusion. The cytotoxic effect of antiglaucoma drugs is caused by the presence of BC in their composition. Despite similar morphofunctional changes in cells, A549 immortalized cell line is more resistant to the effects of drugs than the human corneal epithelial primary culture. When using it as a cellular model, it is advisable to change the experimental conditions (duration of exposure and concentration of the studied drug).

Short-wavelength-infrared upconversion edge enhancement imaging based on a Laguerre-Gaussian composite vortex filter

Edge-enhanced imaging by spiral phase contrast has proven instrumental in revealing phase or amplitude gradients of an object, with notable applications spanning feature extraction, target recognition, and biomedical fields. However, systems deploying spiral phase plates encounter limitations in phase mask modulation, hindering the characterization of the modulation function during image reconstruction. To address this need, we propose and demonstrate an innovative nonlinear reconstruction method using a Laguerre-Gaussian composite vortex filter, which modulates the spectrum of the target. The involved nonlinear process spectrally transforms the incident short-wavelength-infrared (SWIR) signal from 1550 to 864 nm, subsequently captured by a silicon charge-coupled device. Compared with conventional schemes, our novel filtering method effectively suppresses the diffraction noise, significantly enhancing image contrast and resolution. By loading specific phase holograms on the spatial light modulator, bright-field imaging, isotropic, amplitude-controlled anisotropic, and directional second-order edge-enhanced imaging are realized. Anticipated applications for the proposed SWIR edge-enhanced imaging system encompass domains such as artificial intelligence recognition, deep tissue medical diagnostics, and non-destructive defect inspection. These applications underscore the valuable potential of our cutting-edge methodology in furthering both scientific exploration and practical implementations.

A Lombard Variety of Sweet Pepper Regulating Senescence and Proliferation: The Voghera Pepper.

Aging and its related disorders are important issues nowadays and the first cause of this physio-pathological condition is the overproduction of ROS. Ascorbic acid is an antioxidant mediator and its anti-aging proprieties are well known. Our previous data demonstrated that Voghera sweet pepper (VP), a distinctive type of pepper cultivated in Italy, is particularly rich in ascorbic acid. Based on these data, the anti-aging effect mediated by extracts of the edible part of VP was evaluated on an in vitro model of both young and old Normal Human Diploid Fibroblasts (NHDF). Using phase contrast microscopy, we observed that VP may help cells in the maintenance of physiological morphology during aging. Cytofluorimetric analyses revealed that VP extracts led to an increase in DNA synthesis and percentage of living cells, linked to a consequent increase in mitotic events. This hypothesis is supported by the enhancement of PCNA expression levels observed in old, treated fibroblasts, corroborating the idea that this extract could recover a young phenotype in adult fibroblasts, confirmed by the study of p16 and p53 expression levels and TEM analyses. Based on these results, we may suppose that VP can lead to the partial recovery of "young-like" phenotypes in old fibroblasts.

3D imaging of SARS-CoV-2 infected hamster lungs by X-ray phase contrast tomography enables drug testing

X-ray Phase Contrast Tomography (XPCT) based on wavefield propagation has been established as a high resolution three-dimensional (3D) imaging modality, suitable to reconstruct the intricate structure of soft tissues, and the corresponding pathological alterations. However, for biomedical research, more is needed than 3D visualisation and rendering of the cytoarchitecture in a few selected cases. First, the throughput needs to be increased to cover a statistically relevant number of samples. Second, the cytoarchitecture has to be quantified in terms of morphometric parameters, independent of visual impression. Third, dimensionality reduction and classification are required for identification of effects and interpretation of results. To address these challenges, we here design and implement a novel integrated and high throughput XPCT imaging and analysis workflow for 3D histology, pathohistology and drug testing. Our approach uses semi-automated data acquisition, reconstruction and statistical quantification. We demonstrate its capability for the example of lung pathohistology in Covid-19. Using a small animal model, different Covid-19 drug candidates are administered after infection and tested in view of restoration of the physiological cytoarchitecture, specifically the alveolar morphology. To this end, we then use morphometric parameter determination followed by a dimensionality reduction and classification based on optimal transport. This approach allows efficient discrimination between physiological and pathological lung structure, thereby providing quantitative insights into the pathological progression and partial recovery due to drug treatment. Finally, we stress that the XPCT image chain implemented here only used synchrotron radiation for validation, while the data used for analysis was recorded with laboratory μ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\mu$$\\end{document}CT radiation, more easily accessible for pre-clinical research.

Tilting and Distortion in the Multiferroic Aurivillius Phase Bi6Ti3Fe1.5Mn0.5O18.

Aurivillius structured Bi6Ti3Fe1.5Mn0.5O18 (B6TFMO) has emerged as a rare room temperature multiferroic, exhibiting reversible magnetoelectric switching of ferroelectric domains under cycled magnetic fields. This layered oxide presents exceptional avenues for advancing data storage technologies owing to its distinctive ferroelectric and ferrimagnetic characteristics. Despite its immense potential, a comprehensive understanding of the underlying mechanisms driving multiferroic behavior remains elusive. Herein, we employ atomic resolution electron microscopy to elucidate the interplay of octahedral tilting and atomic-level structural distortions within B6TFMO, associating these phenomena with functional properties. Fundamental electronic features at varying bonding environments within this complex system are scrutinized using electron energy loss spectroscopy (EELS), revealing that the electronic nature of the Ti4+ cations within perovskite BO6 octahedra is influenced by position within the Aurivillius structure. Layer-by-layer EELS analysis shows an ascending crystal field splitting (Δ) trend from outer to center perovskite layers, with an average increase in Δ of 0.13 ± 0.06 eV. Density functional theory calculations, supported by atomic resolution polarization vector mapping of B-site cations, underscore the correlation between the evolving nature of Ti4+ cations, the extent of tetragonal distortion and ferroelectric behavior. Integrated differential phase contrast imaging unveils the position of light oxygen atoms in B6TFMO for the first time, exposing an escalating degree of octahedral tilting toward the center layers, which competes with the magnitude of BO6 tetragonal distortion. The observed octahedral tilting, influenced by B-site cation arrangement, is deemed crucial for juxtaposing magnetic cations and establishing long-range ferrimagnetic order in multiferroic B6TFMO.

Genomic and morphological characterization of Knufia obscura isolated from the Mars 2020 spacecraft assembly facility

Members of the family Trichomeriaceae, belonging to the Chaetothyriales order and the Ascomycota phylum, are known for their capability to inhabit hostile environments characterized by extreme temperatures, oligotrophic conditions, drought, or presence of toxic compounds. The genus Knufia encompasses many polyextremophilic species. In this report, the genomic and morphological features of the strain FJI-L2-BK-P2 presented, which was isolated from the Mars 2020 mission spacecraft assembly facility located at the Jet Propulsion Laboratory in Pasadena, California. The identification is based on sequence alignment for marker genes, multi-locus sequence analysis, and whole genome sequence phylogeny. The morphological features were studied using a diverse range of microscopic techniques (bright field, phase contrast, differential interference contrast and scanning electron microscopy). The phylogenetic marker genes of the strain FJI-L2-BK-P2 exhibited highest similarities with type strain of Knufia obscura (CBS 148926T) that was isolated from the gas tank of a car in Italy. To validate the species identity, whole genomes of both strains (FJI-L2-BK-P2 and CBS 148926T) were sequenced, annotated, and strain FJI-L2-BK-P2 was confirmed as K. obscura. The morphological analysis and description of the genomic characteristics of K. obscura FJI-L2-BK-P2 may contribute to refining the taxonomy of Knufia species. Key morphological features are reported in this K. obscura strain, resembling microsclerotia and chlamydospore-like propagules. These features known to be characteristic features in black fungi which could potentially facilitate their adaptation to harsh environments.

Toward Bioactive Hydrogels: A Tunable Approach via Nucleic Acid-Collagen Complexation

Abstract Purpose Nucleic acid-collagen complexes (NACCs) are unique biomaterials formed by binding short, monodisperse single-stranded DNA (ssDNA) with type I collagen. These complexes spontaneously generate microfibers and nanoparticles of varying sizes, offering a versatile platform with potential applications in tissue engineering and regenerative medicine. However, the detailed mechanisms behind the nucleic acid-driven assembly of collagen fibers still need to be established. We aim to understand the relationship between microscopic structure and bulk material properties and demonstrate that NACCs can be engineered as mechanically tunable systems. Methods We present a study to test NACCs with varying molar ratios of collagen to random ssDNA oligonucleotides. Our methods encompass the assessment of molecular interactions through infrared spectroscopy and the characterization of gelation and rheological behavior. We also include phase contrast, confocal reflectance, and transmission electron microscopy to provide complementary information on the 3D structural organization of the hydrogels. Results We report that adding DNA oligonucleotides within collagen robustly reinforces and rearranges the hydrogel network and accelerates gelation by triggering rapid fiber formation and spontaneous self-assembly. The elasticity of NACC hydrogels can be tailored according to the collagen-to-DNA molar ratio, ssDNA length, and collagen species. Conclusion Our findings hold significant implications for the design of mechanically tunable DNA-based hydrogel systems. The ability to manipulate hydrogel stiffness by tailoring DNA content and collagen concentration offers new avenues for fine tuning material properties, enhancing the versatility of bioactive hydrogels in diverse biomedical applications. Lay Summary This work is an example of forming fibers and gels with tunable elasticity that stems from the complexation of short-length nucleic acids (on the order of size of aptamers) and collagen, which can be potentially extended to a variety of functionalized hydrogel designs and tailored biomedical applications. Incorporating DNA induces mechanical changes in NACCs. Graphical Abstract

Recent developments in fly scan methods for phase and multi-contrast x-ray micro-CT based on amplitude modulated beams

Beam tracking and edge illumination are phase contrast imaging techniques that rely on amplitude modulated x-ray beams to generate sensitivity to refraction and scattering. While each technique has its advantage (“single shot” three-contrast imaging in beam tracking; the ability to work with relatively large pixels in edge illumination), they also share a common drawback, namely that the modulator shields parts of the sample and, thus, prevents those areas from contributing to the image (under-sampling). Sample stepping, by which frames are acquired with the sample in a different position relative to the modulator (sometimes referred to as “dithering”) can produce well-sampled images. However, in computed tomography (CT), stepping must be performed at each rotation angle, enforcing step-and-shoot acquisitions and leading to long scan times. To enable faster acquisitions, fly scan compatible scanning schemes based on “roto-translating” the sample in the modulated x-ray beam were recently developed. This article reviews these schemes and provides practical guidance for their implementation.

Exploring the effects of silver, silica-coated silver, and gold nanoparticles on lipid vesicles: Insights from LUVs and GUVs

Understanding the interaction between nanoparticles (NP) and lipid membranes is crucial for various biomedical applications. In this study, we investigated the effects of silver nanoparticles (AgNP), silica-coated silver nanoparticles (Ag@SiO2), and gold nanoparticles (AuNP) on 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid vesicles. The dynamic light scattering (DLS) and zeta potential measurements were employed to evaluate DOPC large unilamellar vesicles (LUVs) exposed to AgNP, AgNP@SiO2, or AuNP for 180 min. The DOPC giant unilamellar vesicles (GUVs) exposed to the same NP were evaluated by phase-contrast microscopy. The AgNP showed higher reactivity along the exposition time, leading the vesicles to rupture (LUVs and GUVs). The SiO2 coating from Ag@SiO2 decreases the rupture effects compared to uncovered AgNP, showing surface adsorption and, consequently, vesicle morphological changes. The AuNP showed a partial wrapping on the GUVs bilayer and permeation on the LUVs, however the integrity of the bilayer structure is maintained in both GUVs and LUVs. These findings highlight the varying effects of AgNP, Ag@SiO2, and AuNP on lipid vesicles, underscoring the importance of nanoparticle material and coating in their interactions with biological membranes.

Facilitated cell probing and hyperthermia treatment by fluorescent DNA engineered cobalt ferrite nanoparticles

BackgroundProbing a cell by fluorescence imaging is very convenient. We have observed that tagging of DNA with some organic dye molecules enhances fluorescence properties. This property of DNA can be utilised to enhance cell imaging quality to track the cell in better way for diagnosis as well as therapy. ResultsWe have shown internalisation of DNA functionalized cobalt-ferrite nanoparticles (CF-DNA NPs) with cells is better compared to free cobalt-ferrite nanoparticles (CF NPs). Therefore, CF NPs were functionalized by DNA (CF-DNA) and tagged them with a fluorophore like RITC for improvement of cell tracking by fluorescence imaging. Cell imaging and cytotoxicity effects were compared by treating the human mammary carcinoma cells (MDA MB-231) with CF NPs and CF-DNA NPs. An investigation was made on applicability of hyperthermia therapy for these particles under AC magnetic field. SignificancesA significant enhancement in fluorescence contrast of cancer cells was observed in case of CF-DNA NPs compared to CF NPs and both the particles show less cytotoxic effect for normal cells. A significant cell death and change in cell morphology after hyperthermia treatment was observed by phase contrast microscope and scanning electron microscope (SEM). Monitoring the treatment in a selective region is helped by this technique with the use of a very small quantity of theranostic material. This new treatment protocol may open a new avenue in cancer therapy.

#851 Effects of gliflozins on endothelial cells: in vitro and clinical evidence

Abstract Background and Aims Gliflozins are a class drugs widely used in diabetes as they act as sodium-glucose co-transporter type 2 inhibitors (SGLT2i). Irrespective from glycaemic control, SGLT2i are known to possess kidney and heart protective effects. The kidney- and heart-protective effects of gliflozins (SGLT2i) have received different explanations because of their efficacy in non-diabetic patients. Indeed, the non-glycemic effects of SGLT2i are puzzling because SGLT2, the molecular target of gliflozins, is mainly expressed in proximal tubule cells, with very low expression levels in other cell types. Though SGLT2 inhibitors might be nephroprotective because of a reduction of eGFR, this mechanism seems insufficient to explain heart protection. Emerging evidence suggests that SGLT2i have off-target effects in endothelial cells. In this study, we analyze this assumption by testing the effects of gliflozins on endothelial cells in vitro and verifying a differential effect of gliflozins on glomerular diseases involving primarily the endothelial compartment vs those involving podocytes or both. Methods In vitro studies have been conducted on EA.hy926 cells. A dose-response curve of gliflozins on mitochondrial activity and cell viability has been obtained using MTT staining. Morphological parameters were obtained using phase contrast microscopy, and the details of the plasma membranes were studied using Atomic Force microscopy. Patients with IgA nephropathy, minimal change disease or Focal Segmental Glomerulo Sclerosis FSGS, and with diabetic nephropathy (n = 12 per group). were studied for the percent change in albuminuria and creatinine four months after gliflozin treatment (empagliflozin or dapagliflozin). Three additional control groups (IgA, podocytopathies, diabetes) without gliflozin treatment (n = 12 per group) were also included. Results Gliflozin treatment induced a dose-response improvement in EA.hy926 cells' survival and restoration of cell-cell borders in AFM images. In patients, the albuminuria was reduced by a similar extent (−38 ± 6%) in all groups when treated with gliflozins. However, in diabetic patients and podocytopathies, this was associated with decreased eGFR (−6% and −8%), whereas in IgA nephropathy, the eGFR levels were increased after treatment (+3.8%). Conclusion In vitro, gliflozins show a direct endothelial protection effect. In vivo, part of the effects on proteinuria may be explained by a hemodynamic effect on the glomerular filtration rate in diseases affecting podocytes (diabetes, MCD, FSGS), as shown by the increase in creatinine. However, for diseases involving the endothelium (IgA nephropathy), the endothelial protection effect of gliflozins is demonstrated by the reduction in proteinuria and the protective effect on creatinine levels.

#2050 APRT deficiency: insights from in vitro modelling of DHA-induced kidney injury and identification of therapeutic target

Abstract Background and Aims Adenine phosphoribosyltransferase (APRT) deficiency is a rare autosomal recessive disorder of adenine metabolism that results in renal excretion of 2,8-dihydroxyadenine (DHA) in excessive amounts, leading to kidney stones and crystal nephropathy with associated inflammation and fibrosis. The effects of other kidney stone constituents, such as calcium oxalate and uric acid, have been thoroughly studied in cell culture models, both in monolayer and three-dimensional (3D) assays. However, the effect of DHA in cell culture models has not yet been investigated. This study aimed to establish a comprehensive cell culture model to investigate DHA crystal-induced kidney injury and to identify therapeutic targets for clinical intervention. Method Three lines of kidney cells (MDCK, HK-2, and HEK293) were utilized in monolayer and 3D assays, employing both liquid-liquid interface (LLI) and “on-top” of Matrigel culture models. DHA exposure, matching the quantities found in the urine of untreated patients with APRT deficiency, was used in conjunction with siRNA-mediated APRT gene knockdown to assess the changes in cellular phenotypes. Assessments included cell viability and migration assays, RT-PCR, western blotting, phase-contrast microscopy, and immunostaining. Paraffin-embedded kidney tissue samples from patients with APRT deficiency and healthy controls were obtained from the Landspitali National University Hospital Biobank (Icelandic IRB permit: VSN 21-117-V1) and phenotypically analyzed using immunohistochemistry (IHC) staining for collagen I (Col I), collagen III (Col III), and APRT. Results After 72 h of incubation with DHA at concentrations of 60, 120, 240, and 480 μg/mL, the viability of all cell lines decreased. Proliferation was evaluated using a scratch assay after treatment with DHA at two different concentrations (120 and 480 μg/mL) for 24 h, and all cell lines showed decreased migration at the highest concentration. Enhanced CD44 expression was observed in both HEK293 and MDCK cells with increasing DHA concentration. In a 3D environment “on-top” of Matrigel, MDCK cells maintained polarized structures despite accumulation of DHA and did not show an increased EMT phenotype compared with TGFβ-treated cells. HEK293 and HK-2 cells formed solid colonies with DHA accumulation within the colony. MDCK cells formed a polarized cell layer grown on Transwell polyester membranes and demonstrated trans-epithelial electrical resistance (TEER) in LLI, which decreased when the cells were treated with DHA at 120 and 480 μg/mL. APRT expression was significantly reduced in all cell lines after successful knockdown. Analysis of kidney tissue specimens using IHC showed increased expression of Col III in patients with APRT deficiency compared to healthy controls. Enhanced Col I expression was also observed in these patients, which, together with Col III, was consistent with increased fibrosis. Conclusion This study revealed that incubation with varying concentrations of DHA led to decreased viability and impaired migration in all cell lines tested. Increased CD44 expression suggests potential crystal binding to renal tubular epithelial cells when exposed to increasing DHA concentrations. In a 3D environment, MDCK cells maintained polarized structures with DHA accumulation without enhancement of the EMT phenotype. Treatment with DHA resulted in a decrease in TEER, indicating a defective barrier function. Increased expression of Col III and Col I was observed in kidney tissue samples from APRT-deficient patients, suggesting increased fibrosis. Overall, these findings highlight the impact of DHA on cell viability, proliferation, EMT, and barrier function, highlighting the role of DHA in kidney fibrosis in patients with APRT-deficiency.

Prediction of Nili-Ravi buffalo bull fertility through Fourier harmonic analysis of sperm

Fourier harmonic analysis (FHA) is a robust method for identification of minute changes in sperm nuclear shape that are indicative of reduced fertility. The current study was designed to develop a fertility prediction model for Nili-Ravi buffalo bulls through FHA of sperm. In experiment I, FHA technique was standardized, average sperm nuclear perimeter was measured and sperm nuclear shape plot of buffalo bull was constructed. Sperm of buffalo bulls (n = 10) were stained with YOYO-1 and Hoechst-33342 to differentiate live and dead, and digital images were captured using phase contrast and fluorescent microscopy. The images were analyzed by ImageJ software and 100 sperm/bull were evaluated. The results are described as mean ± SEM values of mean harmonic amplitude (mharm), skewness harmonic amplitude (skharm), kurtosis harmonic amplitude (kurharm) and variance harmonic amplitude (varharm) at Fourier frequencies 0–5 along with the cartesian and polar coordinate plots of buffalo bull sperm. In experiment II, a fertility prediction model was developed based on FHA of buffalo bull sperm. Semen samples of low (n = 6), medium (n = 3) and high (n = 8) fertility bulls were investigated for FHA of sperm and harmonic amplitudes (HA) were generated. Firstly, to determine if live and dead sperm population have unique nuclear shape distribution; the mean, skewness, kurtosis and variance HA 0–5 of 1700 live and 1294 dead spermatozoa of 17 bulls were evaluated. T-test signified a difference in the mharm0 (2.363 ± 0.01 vs. 2.439 ± 0.02), skharm0 (−0.0002 ± 0.07 vs. −0.266 ± 0.09), kurharm0 (−0.156 ± 0.07 vs. 0.260 ± 0.18), kurharm2 (0.142 ± 0.11 vs. 1.031 ± 0.32) and varharm4 (0.109 ± 0.00 vs. 0.082 ± 0.00) of live vs. dead sperm population (p < 0.05). Therefore, 100 live sperm/bull were further evaluated for mean, skewness, kurtosis and variance HA 0–5 values among high (n = 6) and low-fertility (n = 6) groups. Results of T-test showed higher values of mharm2 (0.739 ± 0.01 vs. 0.686 ± 0.00), mharm4 (0.105 ± 0.001 vs. 0.007 ± 0.001), and skharm0 (0.214 ± 0.109 vs. −0.244 ± 0.097) in high vs. low-fertility group (p < 0.05). In next step, five significantly different combinations of discriminant measures between high and low-fertility groups were obtained by discriminant analysis. In conclusion, mharm4, skharm0 and varharm2 correctly identified 91.7 % of bulls into their respective fertility groups, and upon cross validation the value of the canonical correlation was 0.928.

Delineating three-dimensional behavior of uveal melanoma cells under anchorage independent or dependent conditions

BackgroundAlthough rare, uveal melanoma (UM) is a life-threatening malignancy. Understanding its biology is necessary to improve disease outcome. Three-dimensional (3D) in vitro culture methods have emerged as tools that incorporate physical and spatial cues that better mimic tumor biology and in turn deliver more predictive preclinical data. Herein, we comprehensively characterize UM cells under different 3D culture settings as a suitable model to study tumor cell behavior and therapeutic intervention.MethodsSix UM cell lines were tested in two-dimensional (2D) and 3D-culture conditions. For 3D cultures, we used anchorage-dependent (AD) methods where cells were embedded or seeded on top of basement membrane extracts and anchorage-free (AF) methods where cells were seeded on agarose pre-coated plates, ultra-low attachment plates, and on hanging drops, with or without methylcellulose. Cultures were analyzed for multicellular tumor structures (MCTs) development by phase contrast and confocal imaging, and cell wellbeing was assessed based on viability, membrane integrity, vitality, apoptotic features, and DNA synthesis. Vascular endothelial growth factor (VEGF) production was evaluated under hypoxic conditions for cell function analysis.ResultsUM cells cultured following anchorage-free methods developed MCTs shaped as spheres. Regardless of their sizes and degree of compaction, these spheres displayed an outer ring of viable and proliferating cells, and a core with less proliferating and apoptotic cells. In contrast, UM cells maintained under anchorage-dependent conditions established several morphological adaptations. Some remained isolated and rounded, formed multi-size irregular aggregates, or adopted a 2D-like flat appearance. These cells invariably conserved their metabolic activity and conserved melanocytic markers (i.e., expression of Melan A/Mart-1 and HMB45). Notably, under hypoxia, cells maintained under 3D conditions secrete more VEGF compared to cells cultured under 2D conditions.ConclusionsUnder an anchorage-free environment, UM cells form sphere-like MCTs that acquire attributes reminiscent of abnormal vascularized solid tumors. UM cells behavior in anchorage-dependent manner exposed diverse cells populations in response to cues from an enriched extracellular matrix proteins (ECM) environment, highlighting the plasticity of UM cells. This study provides a 3D cell culture platform that is more predictive of the biology of UM. The integration of such platforms to explore mechanisms of ECM-mediated tumor resistance, metastatic abilities, and to test novel therapeutics (i.e., anti-angiogenics and immunomodulators) would benefit UM care.

#2974 Serial multiparametric magnetic resonance imaging in a prospective cohort of patients with acute kidney injury

Abstract Background and Aims Recovery from acute kidney injury (AKI) is traditionally measured using serum creatinine but this can often over-estimate the degree of renal recovery. It is known, however, that failure of recovery of serum creatinine 90 days after AKI strongly associates with subsequent long-term reductions in renal function making it an important clinical timepoint. Magnetic resonance imaging (MRI) is an imaging modality with promise to improve the understanding and characterisation of renal pathophysiology. In a single multiparametric MRI (mpMRI) scan, multiple measures can assess renal morphology, tissue microstructure (T1 relaxation time and diffusion-weighted-imaging (DWI)), oxygenation, perfusion and blood flow. We previously published the first study of renal mpMRI at the time of AKI and during follow up, this showed increased total kidney volume (TKV) and T1 as a measure of inflammation/fibrosis at the time of AKI which remained elevated in some participants at 3 and 12 months despite normalisation of serum creatinine. This study aimed to assess the degree of change between 30 and 90 days post AKI, as the first 90 days after AKI appear to be the important in terms of outcomes. Method Prospective observational study of 10 participants with AKI of all stages recruited at the time of AKI and followed up with monthly bloods and urine. MRI scans were collected on a Philips Ingenia 3T at day 30-60 and day 90. The 1-hour protocol included: T2-weighted scans for automated segmentation to compute total kidney volume (TKV), as well as T1 &amp; T2 mapping, DWI, ASL and phase contrast MRI for kidney perfusion and blood flow, and TRUST-MRI and BOLD R2* for kidney oxygenation, and MRE for stiffness. Results 10 participants were scanned at 2 timepoints, (mean day 45 for 1st scan, 95 for 2nd scan). Baseline characteristics are summarised in Table 1 along with key clinical measures in Table 2. 2 participants had CKD at baseline. Despite a range of peak creatinine values and AKI stages the majority of participants serum creatinine normalised to within 20% of baseline by 30 days (v3) (Fig. 1). Fig. 2 shows the TKV for AKI patients across the 2 visits as compared with healthy values. Despite normalisation of biochemistry, 5 participants had enlarged TKV. The predominant AKI aetiology for these patients was sepsis. Across all participants there is little dynamic change between visits. The participant with the small TKV across both visits had pre-existing CKD, as well as the highest peak creatine. The participant did not recover renal function by day 90 (Figs 1(j) and 2). Conclusion Despite recovery of biochemistry, at least half of this AKI cohort had abnormal MR measures at that persisted through one and three months after the AKI episode. There is a trend towards larger TKV in patients with AKI due to sepsis, which needs to be explored in a larger cohort. Analysis of more parameters as well as the addition of liquid biomarkers will enrich these preliminary findings.

Progressive Weakening of Granite by Piezoelectric Excitation of Quartz with Alternating Current

A promising solution to reduce energy usage and mitigate the wear of drilling and comminution tools during mining operations involves inducing vibrations within the piezoelectric phases dispersed in the structure of rocks using alternating current (AC). This paper presents experimental evidence of AC-induced weakening of Kuru granite, manifested as improvements in rock drillability and reductions of strength. Sievers’ J-miniature drill tests were used to assess surface drillability. The impact of AC treatment on the quasi-static strength of granite was assessed via three-point bending and indirect tension Brazilian disk tests. The influence of AC treatment on the dynamic tensile strength of the rock was determined using split Hopkinson bar tests, with the fragmentation process captured using in situ ultra-fast synchrotron X-ray phase contrast imaging. The quasi-static tests revealed no reduction in rock strength after the AC treatment. In contrast, reductions of 25% in hardness and 18% in dynamic tensile strength were observed. Fragmentation patterns differed between treated and non-treated rocks, with treated specimens exhibiting reduced macrocrack formation during loading.

#444 Novel MRI filtration fraction method to assess glomerular hemodynamics in diabetic kidney disease

Abstract Background and Aims Filtration fraction (FF) is the ratio of the glomerular filtration rate (GFR) to renal plasma flow (RPF) and is therefore an important measure of glomerular hemodynamics. The gold standard method to assess effective RPF is renal clearance of para-aminohippuric acid (PAH) but technical and labour-intensive difficulties limit its application [1]. Quicker and simpler assays of FF are needed, particularly to assess the renal hemodynamic dysfunction (hyper- or hypofiltration) and effects/mode of action of therapeutic interventions in chronic kidney disease (CKD). We assessed a novel magnetic resonance imaging (MRI) technique to measure FF in patients with diabetic kidney disease (DKD) and healthy controls (HC) [2]. Method This cross-sectional sub-study included 32 DKD subjects with measured GFR (mGFR) ≤ 60 ml/ min/1.73 m2 and stratified into the following GFR stages as per the Kidney Disease Improving Global Outcomes (KDIGO) classification: stage G3a (mGFR 45-59 ml/ min/1.73 m2, n = 3), G3b (mGFR 30-44 ml/ min/1.73 m2, n = 8), G4 (mGFR 15-29 ml/ min/1.73 m2, n = 15), G5 (&amp;lt;15 ml/ min/1.73 m2, n = 6), and 18 age-, gender-matched HC with mGFR ≥ 60 ml/min/1.73 m2. All subjects were 18-79 years old. mGFR was assessed using iohexol clearance. Renal blood flow (RBF) (ml/min) was measured by a 5-minute add-on phase contrast MRI of the renal artery of each kidney and then summed together. RBF was corrected for Body Surface Area. RPF and FF were calculated as below: ${\rm{RPF }} = {\rm{ RBF}} \times \ ( {1\ -{\rm{ Hematocrit}}} )$ ${\rm{FF}} = {\rm{mGFR}}/{\rm{RPF}}$ Results RBF and mGFR were highly correlated (R2 = 0.70, Fig. 1). FF was 17.1% for HC and decreased with increasing degrees of renal impairment (Fig. 2). While the difference between HC and G3a DKD group was not significant, there was highly significant difference between HC and all other DKD groups (p &amp;lt; 0.001). The standard deviation in the G3a group was relatively large with only 3 subjects in this group. Conclusion The phase contrast MRI scan can provide a quick and simple measurement of glomerular hemodynamics and therewith further insights into the pathophysiology of DKD and its progression. Given the limitations of the existing method for assessing renal plasma flow, this MRI method can facilitate the inclusion of filtration fraction in DKD clinical trials. Further, the described method could be used to identify hyperfiltration, a compensatory mechanism that can influence results in clinical trials of DKD.

A comprehensive study on the spatial and temporal variation of BTEX and asbestos in the northwest of Iran: Human risk assessment

Substances like asbestos and other air pollutants, such as BTEX (benzene, toluene, ethylbenzene, and xylene), are hazardous compounds due to their adverse effects on human health. This study aims to investigate the levels, seasonal variations, spatial distribution, potential sources, and associated health risks associated with BTEX compounds and asbestos fibers in the ambient air of Tabriz. Air samples were taken at 16 different locations during the 2020–2021 period. Glass containers with charcoal were used for sample collection, and the BTEX content was determined using the GC-FID method. Phase-contrast microscopy (PCM) analysis was conducted with a low-volume peripheral pump for asbestos fiber sampling. The results showed that the average concentration of ∑BTEX was 37.94 and 27.98 μg/m3 in autumn and spring, respectively. The same parameter was 2.26 and 1.68 f/L for asbestos in the autumn and winter, respectively. The contribution of BTEX to ozone formation potential (OFP) in the research area showed that xylene and toluene were the major contributors to ozone production in different seasons. The risk of exposure to benzene compounds was 24 × 10−4 in children and 55.9 × 10−4 in adults, while the risk of exposure to ethylbenzene was 3.78 × 10−4 in children and 3.25 × 10−4 in adults. The estimated lifetime cancer risk was found to be the highest for benzene, followed by ethylbenzene. The estimated cancer risk for benzene and ethylbenzene exceeded the threshold values set by EPA, which signals a significant carcinogenic risk due to exposure to these substances in the ambient air of Tabriz. According to the EPA guidelines, the low carcinogenicity risk levels are between 10−4 and 10−6. According to the findings for the exposure to asbestos fibers, the maximum values of excess cancer risk (ECR) and estimated lifetime cancer risk (ELCR) were observed in the 16–30 age range across all locations, suggesting increased exposure to asbestos fibers compared to other age groups.