Phase Contrast Research Articles

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.

CMR provides comparable measurements of diastolic function as echocardiography

Clinical application of cardiac magnetic resonance (CMR) is expanding but CMR assessment of LV diastolic function is still being validated. The purpose of this study was to validate assessments of left ventricular (LV) diastolic dysfunction (DD) using CMR by comparing with transthoracic echocardiography (TTE) performed on the same day. Patients with suspected or diagnosed cardiomyopathy (n = 63) and healthy volunteers (n = 24) were prospectively recruited and included in the study. CMR diastolic parameters were measured on cine images and velocity-encoded phase contrast cine images and compared with corresponding parameters measured on TTE. A contextual correlation feature tracking method was developed to calculate the mitral annular velocity curve. LV DD was classified by CMR and TTE following 2016 guidelines. Overall DD classification was 78.1% concordant between CMR and TTE (p < 0.0001). The trans-mitral inflow parameters correlated well between the two modalities (E, r = 0.78; A, r = 0.90; E/A, r = 0.82; all p < 0.0001) while the remaining diastolic parameters showed moderate correlation (e’, r = 0.64; E/e’, r = 0.54; left atrial volume index (LAVi), r = 0.61; all p < 0.0001). Classification of LV diastolic function by CMR showed good concordance with standardized grades established for TTE. CMR-based LV diastolic function may be integrated in routine clinical practice.Name of the registry: Technical Development of Cardiovascular Magnetic Resonance Imaging. Trial registration number: NCT00027170. Date of registration: November 26, 2001. URL of trial registry record: https://clinicaltrials.gov/ct2/show/NCT00027170

Atomic-scale investigation on the evolution of T1 precipitates in an aged Al-Cu-Li-Mg-Ag alloy

The T1 (Al2CuLi) phase is one of the most effective strengthening nanoscale-precipitate in Al-Cu alloys with Li. However, its formation and evolution still need to be further clarified during aging due to the complex precipitation sequences. Here, a detailed investigation has been carried out on the atomic structural evolution of T1 precipitate in an aged Al-Cu-Li-Mg-Ag alloy using state-of-the-art Cs-corrected high-angle annular dark field (HAADF)- coupled with integrated differential phase contrast (iDPC)-scanning transmission electron microscopy (STEM) and energy-dispersive X-ray spectroscopy (EDXS) techniques. An intermediate T1’ phase between T1p and T1 phase, with a crystal structure and orientation relationship consistent with T1, but exhibiting different atomic occupancy and chemical composition was found. We observed the atomic structural transformation from T1p to T1’ phase (fcc→hcp), involving only 1/12<112>Al shear component. DFT calculation results validated our proposed structural models and the precipitation sequence. Besides, the distributions of minor solute elements (Ag, Mg, and Zn) in the precipitates exhibited significant differences. These findings may contribute to a further understanding of the nucleation mechanism of T1 precipitate.

Epithelial-mesenchymal Transition (EMT) and the Effect of Atorvastatin on it in ARPE-19 cells

Proliferative vitreoretinopathy (PVR) develops after an unsuccessful or complicated recovery from rhegmatogenous retinal detachment (RRD) surgery. Intraocular scar formation with the contribution of epithelial-mesenchymal transition (EMT) in RPE cells is prominent in the pathology of PVR. In the present study, the EMT process was experimentally induced in human retinal pigment epithelium (RPE; ARPE-19) cells, and the effect of atorvastatin on the process was studied. The mRNA and protein levels of mesenchymal markers actin alpha 2 (ACTA2) / alpha-smooth muscle actin (α-SMA) and fibronectin (FN), and epithelial markers occludin (OCLN) and zonula occludens-1 (ZO-1) were measured using quantitative real-time PCR (qRT-PCR) and western blot methods, respectively. In addition, α-SMA and FN were visualized using immunofluorescence staining. Cells were photographed under a phase contrast light microscope. Changes in the functionality of cells following the EMT process were studied using the IncuCyte scratch wound cell migration assay and the collagen cell invasion assay with confocal microscopy. The induction of EMT in ARPE-19 cells increased the expression of mesenchymal markers ACTA2/α-SMA and fibronectin and reduced the expression of epithelial marker OCLN both at mRNA and protein levels. The mRNA levels of ZO-1 were lower after EMT, as well. Increased levels of α-SMA and FN were confirmed by immunofluorescence staining. Atorvastatin further increased the mRNA levels of mesenchymal markers ACTA2 and FN as well as the protein levels of α-SMA and reduced the mRNA levels of epithelial markers OCLN and ZO-1 under the EMT process. EMT promoted wound closure and cell invasion into the 3D collagen matrix when compared to untreated control cells. These data present cellular changes upon the induction of the EMT process in ARPE-19 cells and the propensity of atorvastatin to complement the effect. More studies are needed to confirm the exact influence of the EMT process and atorvastatin treatment on the PVR development after RRD surgery.

Automated segmentation of liver and hepatic vessels on portal venous phase computed tomography images using a deep learning algorithm.

CT-image segmentation for liver and hepatic vessels can facilitate liver surgical planning. However, time-consuming process and inter-observer variations of manual segmentation have limited wider application in clinical practice. Our study aimed to propose an automated deep learning (DL) segmentation algorithm for liver and hepatic vessels on portal venous phase CT images. This retrospective study was performed to develop a coarse-to-fine DL-based algorithm that was trained, validated, and tested using private 413, 52, and 50 portal venous phase CT images, respectively. Additionally, the performance of the DL algorithm was extensively evaluated and compared with manual segmentation using an independent clinical dataset of preoperative contrast-enhanced CT images from 44 patients with hepatic focal lesions. The accuracy of DL-based segmentation was quantitatively evaluated using the Dice Similarity Coefficient (DSC) and complementary metrics [Normalized Surface Dice (NSD) and Hausdorff distance_95 (HD95) for liver segmentation, Recall and Precision for hepatic vessel segmentation]. The processing time for DL and manual segmentation was also compared. Our DL algorithm achieved accurate liver segmentation with DSC of 0.98, NSD of 0.92, and HD95 of 1.52mm. DL-segmentation of hepatic veins, portal veins, and inferior vena cava attained DSC of 0.86, 0.89, and 0.94, respectively. Compared with the manual approach, the DL algorithm significantly outperformed with better segmentation results for both liver and hepatic vessels, with higher accuracy of liver and hepatic vessel segmentation (all p<0.001) in independent 44 clinical data. In addition, the DL method significantly reduced the manual processing time of clinical postprocessing (p<0.001). The proposed DL algorithm potentially enabled accurate and rapid segmentation for liver and hepatic vessels using portal venous phase contrast CT images.

Characterization of spring and durum wheat using non-destructive synchrotron phase contrast X-ray microtomography during storage

Post-harvest losses during cereal grain storage are a big concern in both developing and developed countries, where spring and durum wheat are staple food grains. Varieties under these classes behave differently under storage, which affects their end storage life. High resolution imaging data of dry as well as spoiled seed are not available for any class of wheat; therefore, an attempt was made to generate 3D data for better understanding of seed structure and changes due to spoilage. Six wheat varieties (3 varieties for each class of wheat) were stored for 5 week at 17% moisture content (wb) before scanning. Seeds were also stored in a freezer (-18 °C) for further scanning to determine if any changes occur in the structure of seeds due to freezing. Spring varieties of wheat performed better than durum varieties and freezing did not affect seed structure. Data could also help plant breeders to develop varieties that do not easily spoil, adjust grain processing techniques, and develop post-harvest recommendations for other wheat varieties.

First Report of Tetranychus truncatus Ehara (Acari: Tetranychidae) Infesting Groundnut (Arachis hypogaea L.) in India through Morphological and Molecular Insight

Background: Red spider mite, Tetranychus truncatus Ehara (Acari: Tetranychidae) infests many agriculture and horticulture crops in India, but to date it had not been found damaging cultivated groundnut. During 2022-23, the incidence of this species was high in many groundnut growing regions of Tamil Nadu. Identification of the species is highly essential for effective management of the particular pest. Hence, the aim of this research was to identify the mite species infesting groundnut crop and describe the damage caused. Methods: Survey was carried out all over Tamil Nadu and samples of mite infested groundnut leaves were collected from field based on damage symptoms, observed and examined under Stereo zoom microscope, microslides were prepared using Hoyer’s medium and taxonomic characters were studied under phase contrast microscope. Molecular analysis was done by genomic DNA isolation, PCR amplification, sequencing and BLAST performed to check the identity with GenBank Sequences. ITS2 primer was used. Result: Red spider mite species, T. truncatus was identified first time in groundnut through both morphological characters and molecular tools. Female: Idiosomal setae longer than the interval between their longitudinal bases and gradually tapers distally. Diamond- shaped pattern of striae between e1 and f1 setal bases. Tarsus I with 4 tactile setae proximal to proximal duplex setae. Male: Aedeagus with tiny terminal knob, apparently truncate with a slight indentation on the dorsal margin.

Near‐Infrared Metalens Empowered Dual‐Mode High Resolution and Large FOV Microscope

AbstractThe spiral phase contrast microscope can clearly distinguish the morphological information of the low contrast objects (i.e., biological samples) owing to the isotropic edge‐enhancement effect, while the bright field microscope can image the overall morphology of amplitude objects. However, the imaging resolution, magnification, and field of view of conventional spiral phase contrast microscopes based on 4f filtering configuration are limited by the system's complexity. Here, compact dual‐mode microscopes working at near‐infrared using the engineered metalens are reported, which can be tuned between the spiral phase contrast imaging and bright field imaging by polarization control. The metalens combine the high‐resolution objective lens and polarization‐controlled phase filter into a single‐layer nanofins array. Two infinity‐corrected microscope systems are demonstrated to achieve subwavelength resolution (0.7λ), large magnification (58X), and large field of view (600 × 800 µm). Unstained onion epidermal is imaged by the microscope to show the dual‐mode imaging ability for the biological sample. Finally, a singlet dual‐mode microscope system is demonstrated to show the edge‐detection application for industrial standards. These results can open new opportunities in applications of biological imaging, industrial machine vision, and semiconductor inspection.

Impact of age and donor sites on bioactivities of tendon cells in autologous tenocyte implantation (OrthoATI™) for treatment of chronic tendinopathy

ObjectivesAutologous tenocyte implantation (OrthoATI™) therapy has demonstrated efficacy in treating patients with tendinopathy at various anatomical sites. This study evaluates the effect of patient age, gender, and tendon biopsy site on morphology, growth, and gene expression of autologous tendon cells used to treat chronic tendinopathy. MethodsPatients undergoing OrthoATI™ for tendinopathies between 2020 and 2022 were initially treated by biopsies taken from patella tendon (PT) or palmaris longus tendon (PL). The biopsies were sent to a Good Manufacturing Practice (GMP) cell laboratory where tendon cells were isolated, cultured, and expanded for four to six weeks. Cell morphology was assessed using phase contrast microscopy. Droplet digital PCR (ddPCR) was utilized for gene expression analysis. Dichotomous results were compared between groups using x2 or Fisher's exact tests with no adjustment for multiple comparisons. The nonparametric Mann–Whitney U and Kruskal–Wallis tests were utilized for the sex and age (<35y, 35-44y, 45-54y, >55y) analyses, respectively. All analyses were performed using IBM SPSS v27, and a two-tailed P-value of <0.05 was considered statistically significant. Results149 patients were included in the analysis. The PT was biopsied in 63 patients, and PL in 86 patients. There were no observer effects for age and gender between the PT and PL groups. There was no statistical significance between the PT and PL tendons for cell morphology, average cell population doubling time (PDT) (PT 83.9 vs PL 82.7 ​h, p ​= ​0.482), cellular yield (PT 16.2 vs PL 15.2 ​× ​106, p ​= ​0.099), and cell viability (PT 98.7 vs PL 99.0%, p ​= ​0.277). Additionally, ddPCR analyses showed no statistical significance found in tenogenic gene expression, including collagen type I (COL1, p ​= ​0.86), tenomodulin (TNMD, p ​= ​0.837) and scleraxis (SCX, p ​= ​0.331) between PT- and PL-derived tendon cells. An age stratification analysis found no effect on growth and gene expression. COL1 was found to be higher in males when compared to females (P ​< ​0.001), but otherwise no difference was seen in growth and gene expression in the gender analysis. No postbiopsy clinical complications were reported for either group. ConclusionThis study has shown that the growth and bioactivities of tendon cells from tendon biopsies for OrthoATI™ are not affected by tendon donor site and age. Level of evidenceIV.