Phase Contrast Research Articles

Optimizing Contrast in Automated 4D STEM Cryotomography.

4D STEM is an emerging approach to electron microscopy. While it was developed principally for high-resolution studies in materials science, the possibility to collect the entire transmitted flux makes it attractive for cryomicroscopy in application to life science and radiation-sensitive materials where dose efficiency is of utmost importance. We present a workflow to acquire tomographic tilt series of 4D STEM data sets using a segmented diode and an ultrafast pixelated detector, demonstrating the methods using a specimen of a T4 bacteriophage. Full integration with the SerialEM platform conveniently provides all the tools for grid navigation and automation of the data collection. Scripts are provided to convert the raw data to mrc format files and further to generate a variety of modes representing both scattering and phase contrasts, including incoherent and annular bright field, integrated center of mass, and parallax decomposition of a simulated integrated differential phase contrast. Principal component analysis of virtual annular detectors proves particularly useful, and axial contrast is improved by 3D deconvolution with an optimized point spread function. Contrast optimization enables visualization of irregular features such as DNA strands and thin filaments of the phage tails, which would be lost upon averaging or imposition of an inappropriate symmetry.

Multimodal 2D and 3D microscopic mapping of growth cartilage by computational imaging techniques – a short review including new research

Being able to image the microstructure of growth cartilage is important for understanding the onset and progression of diseases such as osteochondrosis and osteoarthritis, as well as for developing new treatments and implants. Studies of cartilage using conventional optical brightfield microscopy rely heavily on histological staining, where the added chemicals provide tissue-specific colours. Other microscopy contrast mechanisms include polarization, phase- and scattering contrast, enabling non-stained or ‘label-free’ imaging that significantly simplifies the sample preparation, thereby also reducing the risk of artefacts. Traditional high-performance microscopes tend to be both bulky and expensive. Computational imaging denotes a range of techniques where computers with dedicated algorithms are used as an integral part of the image formation process. Computational imaging offers many advantages like 3D measurements, aberration correction and quantitative phase contrast, often combined with comparably cheap and compact hardware. X-ray microscopy is also progressing rapidly, in certain ways trailing the development of optical microscopy. In this study, we first briefly review the structures of growth cartilage and relevant microscopy characterization techniques, with an emphasis on Fourier ptychographic microscopy (FPM) and advanced x-ray microscopies. We next demonstrate with our own results computational imaging through FPM and compare the images with hematoxylin eosin and saffron (HES)-stained histology. Zernike phase contrast, and the nonlinear optical microscopy techniques of second harmonic generation (SHG) and two-photon excitation fluorescence (TPEF) are explored. Furthermore, X-ray attenuation-, phase- and diffraction-contrast computed tomography (CT) images of the very same sample are presented for comparisons. Future perspectives on the links to artificial intelligence, dynamic studies and in vivo possibilities conclude the article.

Aqueductal CSF stroke volume is associated with the burden of perivascular space enlargement in chronic adult hydrocephalus

The inflow of CSF into perivascular spaces (PVS) in the brain is crucial for clearing waste molecules. Inefficiency in PVS flow leads to neurodegeneration. Failure of PVS flushing is associated with CSF flow impairment in the intracranial hydrodynamic condition of CSF hypo-pulsatility. However, enlarged PVS (ePVS), a finding indicative of PVS flow dysfunction, is also present in patients with derangement of CSF dynamics characterized by CSF hyper-pulsatility, which increases CSF flow. Intriguingly, two opposite intracranial hydrodynamic conditions would lead to the same result of impairing the PVS flushing. To investigate this issue, we assessed the subsistence of a dysfunctional interplay between CSF and PVS flows and, if the case, the mechanisms preventing a hyper-pulsatile brain from providing an effective PVS flushing. We analyzed the association between phase contrast MRI aqueductal CSF stroke volume (aqSV), a proxy of CSF pulsatility, and the burden of ePVS in chronic adult hydrocephalus, a disease involving a broad spectrum of intracranial hydrodynamics disturbances. In the 147 (85 males, 62 females) patients, the age at diagnosis ranged between 28 and 88 years (median 73 years). Ninety-seven patients had tri-ventriculomegaly and 50 tetra-ventriculomegaly. According to the extent of ePVS, 113 patients had a high ePVS burden, while 34 had a low ePVS burden. aqSV, which ranged between 0 and 562 μL (median 86 μL), was increased with respect to healthy subjects. Patients presenting with less ePVS burden had higher aqSV (p < 0.002, corrected for the multiple comparisons) than those with higher ePVS burden. The present study confirmed the association between CSF dynamics and PVS flow disturbances and demonstrated this association in intracranial hyper-pulsatility. Further studies should investigate the association between PVS flow failure and CSF hypo- and hyper-pulsatility as responsible/co-responsible for glymphatic failure in other neurodegenerative diseases, particularly in diseases in which CSF disturbances can be corrected, as in chronic adult hydrocephalus.

Lysophosphatidylcholine induces oxidative stress and calcium-mediated cell death in human blood platelets.

Platelets are essential component of circulation that plays a major role in hemostasis and thrombosis. During activation and its demise, platelets release platelet-derived microvesicles, with lysophosphatidylcholine (LPC) being a prominent component in their lipid composition. LPC, an oxidized low-density lipoprotein, is involved in cellular metabolism, but its higher level is implicated in pathologies like atherosclerosis, diabetes, and inflammatory disorders. Despite this, its impact on platelet function remains relatively unexplored. To address this, we studied LPC's effects on washed human platelets. A multimode plate reader was employed to measure reactive oxygen species and intracellular calcium using H2DCF-DA and Fluo-4-AM, respectively. Flow cytometry was utilized to measure phosphatidylserine expression, mitochondrial membrane potential (ΔΨm), and mitochondrial permeability transition pore (mPTP) formation using FITC-Annexin V, JC-1, and CoCl2/calcein-AM, respectively. Additionally, platelet morphology and its ultrastructure were observed via phase contrast and electron microscopy. Sonoclot and light transmission aggregometry were employed to examine fibrin formation and platelet aggregation, respectively. The findings demonstrate that LPC induced oxidative stress and increased intracellular calcium in platelets, resulting in increased phosphatidylserine expression and reduced ΔΨm. LPC triggered caspase-independent platelet death and mPTP opening via cytosolic and mitochondrial calcium, along with microvesiculation and reduced platelet counts. LPC increased the platelet's size, adopting a balloon-shaped morphology, causing membrane fragmentation and releasing its cellular contents, while inducing a pro-coagulant phenotype with increased fibrin formation and reduced integrin αIIbβ3 activation. Conclusively, this study reveals LPC-induced oxidative stress and calcium-mediated platelet death, necrotic in nature with pro-coagulant properties, potentially impacting inflammation and repair mechanisms during vascular injury.

Inline edge illumination X-ray phase contrast imaging through mask misalignment

Inline edge illumination X-ray phase contrast imaging through mask misalignment

Origin of giant enhancement of phase contrast in electron holography of modulation-doped [formula omitted]-type GaN

The electron optical phase contrast probed by electron holography at n-n+ GaN doping steps is found to exhibit a giant enhancement, in sharp contrast to the always smaller than expected phase contrast reported for p-n junctions. We unravel the physical origin of the giant enhancement by combining off-axis electron holography data with self-consistent electrostatic potential calculations. The predominant contribution to the phase contrast is shown to arise from the doping dependent screening length of the surface Fermi-level pinning, which is induced by FIB-implanted carbon point defects below the outer amorphous shell. The contribution of the built-in potential is negligible for modulation doping and only relevant for large built-in potentials at e.g. p-n junctions. This work provides a quantitative approach to so-called dead layers at TEM lamellas.

Insights into cellular interactions of characterized Mg–Al Layered Double Hydroxide on L929 cells

Layered double hydroxides are members of an anionic clay family, characterised by unique two-dimensional layered structures and lend versatility in various applications. These biocompatible compounds have the potential to get intercalated with biological compounds and physico-chemically adsorbed onto organic molecules. Thus, making them important candidates for pharmaceutical and biomedical purposes. This study aims to synthesise, characterise and investigate the cellular toxicity interactions of Mg–Al LDH towards the mouse fibroblast L929 cell line. The Mg–Al LDH was synthesized by a meticulous process of co-precipitation followed by the hydrothermal method to ensure a well-defined and stable structure for suitable biological application. Characterisation techniques like Dynamic Light Scattering, Zeta potential, Scanning Electron Microscopy, Fourier transform infrared, and X-ray diffraction analysis were employed to provide deeper insights into the physiochemical properties and structural integrity of the synthesized Mg–Al LDH. The investigation of cellular interactions with the L929 fibroblast cell line served to assess the biocompatibility and potential cytotoxic effects of Mg–Al LDH. This was observed by assessing the morphological changes and evaluating the cytotoxic effects of Mg–Al LDH by utilising various techniques like phase contrast microscopy, fluorescent staining, and Giemsa staining. The cellular metabolic activity was assessed by MTT assay, and the subcellular lysosomal alteration was examined using the fluorescent staining method by the acridine orange staining. The dose-dependent response observed in the cellular interaction underscores the importance of dosage considerations for potential biomedical applications. By elucidating the dose-response relationship, this study contributes valuable information for the safe and effective usage of LDH in biomedical contexts.

A promising eco-sustainable wound dressing based on cellulose extracted from Spartium junceum L. and impregnated with Glycyrrhiza glabra L extract: Design, production and biological properties

Glycyrrhiza glabra extract is widely known for its antioxidant and anti-inflammatory properties and can improve the wound healing process. The aim of this work was to shorten the time of the healing process by using an eco-sustainable wound dressing based on Spanish broom flexible cellulosic fabric by impregnation with G. glabra extract-loaded ethosomes. Chemical analysis of G. glabra extract was performed by LC-DAD-MS/MS and its encapsulation into ethosomes was obtained using the ethanol injection method. Lipid vesicles were characterized in terms of size, polydispersity index, entrapment efficiency, zeta potential, and stability. In vitro release studies, biocompatibility, and scratch test on 3T3 fibroblasts were performed. Moreover, the structure of Spanish broom dressing and its ability to absorb wound exudate was characterized by Synchrotron X-ray phase contrast microtomography (SR-PCmicroCT). Ethosomes showed a good entrapment efficiency, nanometric size, good stability over time and a slow release of polyphenols compared to the free extract, and were not cytotoxic. Lastly, the results revealed that Spanish broom wound dressing loaded with G. glabra ethosomes is able to accelerate wound closure by reducing wound healing time. To sum up, Spanish broom wound dressing could be a potential new green tool for biomedical applications.

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.

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).

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.

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

Estimating Flow Direction of Circle of Willis Using Dynamic Arterial Spin Labeling Magnetic Resonance Angiography.

The Circle of Willis (COW) is a crucial mechanism for cerebral collateral circulation. This proof-ofconcept study aims to develop and assess an analysis method to characterize the hemodynamics of the arterial segments in COW using arterial spin labeling (ASL) based non-contrast enhanced dynamic magnetic resonance angiography (dMRA). The developed analysis method uses a graph model, bootstrap strategy, and ensemble learning methodologies to determine the time-curve shift from ASL dMRA to estimate the flow direction within the COW. The performance of the method was assessed on 52 subjects, using the flow direction, either antegrade or retrograde, derived from 3D phase contrast (PC) MRI as the reference. A total of 340 arterial segments in COW were evaluated, among which 30 (8.8%) had retrograde flow according to 3D PC. The ASL dMRA-based flow direction estimation has an accuracy, sensitivity, and specificity of 95.47%, 80%, and 96.34%, respectively. Using ASL dMRA and the developed image analysis method to estimate the flow direction in COW is feasible. This study provides a new method to assess the hemodynamics of the COW, which could be useful for the diagnosis and study of cerebrovascular diseases. COW = Circle of Willis; ASL = arterial spin labeling; dMRA =dynamic magnetic resonance angiography; PC = phase contrast.

#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.