Nuclear Area Research Articles

Clinical values of nuclear morphometric analysis in fibroepithelial lesions.

Fibroepithelial lesions (FELs) of the breast encompass a broad spectrum of lesions, ranging from commonly encountered fibroadenomas (FAs) to rare phyllodes tumors (PTs). Accurately diagnosing and grading these lesions is crucial for making management decisions, but it can be challenging due to their overlapping features and the subjective nature of histological assessment. Here, we evaluated the role of digital nuclear morphometric analysis in FEL diagnosis and prognosis. A digital nuclear morphometric analysis was conducted on 241 PTs and 59 FAs. Immunohistochemical staining for cytokeratin and Leukocyte common antigen (LCA) was used to exclude non-stromal components, and nuclear area, perimeters, calipers, circularity, and eccentricity in the stromal cells were quantified with QuPath software. The correlations of these features with FEL diagnosis and prognosis was assessed. All nuclear features, including area, perimeter, circularity, maximum caliper, minimum caliper and eccentricity, showed significant differences between FAs and benign PTs (p ≤ 0.002). Only nuclear area, perimeter, minimum caliper and eccentricity correlated significantly with PT grading (p ≤ 0.022). For differentiation of FAs from benign PTs, the model integrating all differential nuclear features demonstrated a specificity of 90% and sensitivity of 70%. For PT grading, the nuclear morphometric score showed a specificity of 78% and sensitivity of 96% for distinguishing benign/borderline from malignant PTs. In addition, a relationship of nuclear circularity was found with PT recurrence. The Kaplan-meier analysis, using the best cutoff determined by ROC curve, showed shorter event free survival in benign PTs with high circularity (chi-square = 4.650, p = 0.031). Our data suggested the digital nuclear morphometric analysis could have potentials to objectively differentiate different FELs and predict PT outcome. These findings could provide the evidence-based data to support the development of deep-learning based algorithm on nuclear morphometrics in FEL diagnosis.

Brazil's Uranium Mining Evolution: Domestic Developments and International Dynamics

This article provides an exploration of the historical trajectory of uranium mining in Brazil, providing insight into its emergence, development, and current status within the global nuclear landscape. From its early days, characterized by sporadic mining ventures to the establishment of strategic uranium reserves, Brazil's journey mirrors a complex interplay of domestic imperatives and international dynamics. However, despite Brazil's significant uranium resources and advances in nuclear technology, its narrative often remains overshadowed by other global nuclear actors. Thus, by delving into Brazil's historical engagement with uranium mining, this paper seeks to illuminate its evolving role and contributions to the nuclear domain. Furthermore, this study examines the impact of external influences, such as geopolitical pressures, the international price of uranium, and technological advancements, on Brazil's nuclear ambitions. Ultimately, it aims to enrich our understanding of Brazil's past, present, and future in the global nuclear area.

In bilico tra terra e acqua. Visioni e rotte di collisione su un tratto di fiume Po

Caorso Nuclear Power Plant area is extremely representative of the central Po River territorial system, which is characterized by a profound conflict between impactful infrastructures and semi-natural environments with high ecosystem value. In this context, the decommissioning of the Power Plant transcends the local scale, triggering reflections about the resurgence of the nature and the need to rethink our lifestyles and how they impact the planet.Starting from the outcomes of LOL2023 International Workshop by Politecnico di Milano, the article proposes methodological reasoning to investigate the potential that this decommissioning phase opens. In particular, the text investigates three design principles that can potentially be used to transform the Power Plant’s ‘dying’ energy landscape into an inviting habitat for a wide community of living beings, without, however, scarifying the spatial quality lent by design.

Multiplexed single-cell imaging reveals diverging subpopulations with distinct senescence phenotypes during long-term senescence induction.

Cellular senescence is a phenotypic state that contributes to the progression of age-related disease through secretion of pro-inflammatory factors known as the senescence associated secretory phenotype (SASP). Understanding the process by which healthy cells become senescent and develop SASP factors is critical for improving the identification of senescent cells and, ultimately, understanding tissue dysfunction. Here, we reveal how the duration of cellular stress modulates the SASP in distinct subpopulations of senescent cells. We used multiplex, single-cell imaging to build a proteomic map of senescence induction in human epithelial cells induced to senescence over the course of 31 days. We map how the expression of SASP proteins increases alongside other known senescence markers such as p53, p21, and p16 INK4a . The aggregated population of cells responded to etoposide with an accumulation of stress response factors over the first 11 days, followed by a plateau in most proteins. At the single-cell level, however, we identified two distinct senescence cell populations, one defined primarily by larger nuclear area and the second by higher protein concentrations. Trajectory inference suggested that cells took one of two discrete molecular paths from unperturbed healthy cells, through a common transitional subpopulation, and ending at the discrete terminal senescence phenotypes. Our results underscore the importance of using single-cell proteomics to identify the mechanistic pathways governing the transition from senescence induction to a mature state of senescence characterized by the SASP.

Assessment of the activity concentration of radon and its health hazard in surface water around the Rooppur Nuclear Power Plant (RNPP) site, Bangladesh

ABSTRACT Radon is a colourless, unscented, radioactive inert gas. As a gaseous element, Rn-222 can be effectively breathed in, leading to a health hazard. The main objective of this study is to measure the amount of Rn-222 concentration in surface water and its associated health hazards around the Rooppur Nuclear Power Plant (RNPP) construction site area in Bangladesh. Twenty water samples were collected each year (2022 and 2023) from different specific locations of the Padma River and investigated for radon concentration through a Durridge RAD7 detector with RAD H2O accessories. The range of radon activity in surface water measured from 0.070 ± 0.035 to 0.385 ± 0.211 BqL−1 with a mean activity of 0.194 ± 0.114 BqL−1 in the year 2022 and in the year 2023, the range of radon activity in surface water measured from 0.035 ± 0.070 to 0.455 ± 0.239 BqL−1 with an average activity of 0.228 ± 0.140 BqL−1. All the radon concentration results were below the recommended limits of WHO (100 BqL−1) and USEPA (11.1 BqL−1). The ranges of annual effective dose (AED) were from 0.1911 to 1.051 µSvy−1 and from 0.095 to 1.242 µSvy−1 in the years 2022 and 2023, respectively, which were lower than the AED recommended by WHO (0.1 mSvy−1). The mean values of excess lifetime cancer risk are 0.0021 × 10−3 and 0.0025 × 10−3 in the years 2022 and 2023, respectively, which were lower than the average standard value recommended by UNSCEAR (0.29 × 10−3). The result of this investigation can be applied as baseline data for radon concentration measurement in the under-construction nuclear power plant site area vicinity of Bangladesh.

Sex Chromatin in New Hampshire Cocks

Sex chromatin (SC) is defined as inactive X chromosome in the female somatic cells, it condensed as heterochromatic at interphase of the nucleus and appears like nuclear satellite projection, and only female cells contain SC in mammals. Different ages of cocks: immature (8,16 weeks old), growing (24 weeks old) and production age (32,48 weeks old) , were studied to detect the sex chromatin (SC) with it's various shapes :Sessile nodule(SS), Drum stick(DS), Tear drop(TD) and Club shape(CS) in the blood lymphocytes of blood smears prepared from WBCs of 45 New Hampshire(NH) strain cocks. Also SC measurements were detected which include: vertical (V) and horizontal (H) axes, SC area (SCA), nuclear area (NA), and the ratio(R) between these two areas. . Results show that SC is present in the male chicken blood lymphocytes in all ages. There is no significant difference in the incidence of the SC or its various measurements between ages, but the vertical axis was recorded a significant (P

Colorectal cancer histopathology image analysis: A comparative study of prognostic values of automatically extracted morphometric nuclear features in multispectral and red-blue-green imagery.

Multispectral imaging (MSI) has been utilized to predict the prognosis of colorectal cancer (CRC) patients, however, our understanding of the prognostic value of nuclear morphological parameters of bright-field MSI in CRC is still limited. This study was designed to compare the efficiency of MSI and standard red-green-blue (RGB) images in predicting the prognosis of CRC. We compared the efficiency of MS and conventional RGB images on the quantitative assessment of hematoxylin-eosin (HE) stained histopathology images. A pipeline was developed using a pixel-wise support vector machine (SVM) classifier for gland-stroma segmentation, and a marker-controlled watershed algorithm was used for nuclei segmentation. The correlation between extracted morphological parameters and the five-year disease-free survival (5-DFS) was analyzed. Forty-seven nuclear morphological parameters were extracted in total. Based on Kaplan-Meier analysis, eight features derived from MS images and seven featured derived from RGB images were significantly associated with 5-DFS, respectively. Compared with RGB images, MSI showed higher accuracy, precision, and Dice index in nuclei segmentation. Multivariate analysis indicated that both integrated parameters 1 (factors negatively correlated with CRC prognosis including nuclear number, circularity, eccentricity, major axis length) and 2 (factors positively correlated with CRC prognosis including nuclear average area, area perimeter, total area/total perimeter ratio, average area/perimeter ratio) in MS images were independent prognostic factors of 5-DFS, in contrast with only integrated parameter 1 (P<0.001) in RGB images. More importantly, the quantification of HE-stained MS images displayed higher accuracy in predicting 5-DFS compared with RGB images (76.9% vs 70.9%). Quantitative evaluation of HE-stained MS images could yield more information and better predictive performance for CRC prognosis than conventional RGB images, thereby contributing to precision oncology.

Common SYNE2 Genetic Variant Associated With Atrial Fibrillation Lowers Expression of Nesprin-2α1 With Downstream Effects on Nuclear and Electrophysiological Traits.

Atrial fibrillation GWAS (genome-wide association studies) identified significant associations for rs1152591 and linked variants in the SYNE2 gene encoding Nesprin-2, which connects the nuclear membrane with the cytoskeleton. Reporter gene vector transfection and CRISPR-Cas9 editing were used to identify the causal variant regulating the expression of SYNE2α1. After SYNE2 knockdown or SYNE2α1 overexpression in human stem cell-derived cardiomyocytes, nuclear phenotypes were assessed by imaging and atomic force microscopy. Gene expression was assessed by RNAseq and gene set enrichment analysis. Fura-2 AM staining assessed calcium transients. Optical mapping assessed action potential duration and conduction velocity. The risk allele of rs1152591 had lower promoter and enhancer activity and was significantly associated with lower expression of the short SYNE2α1 isoform in human stem cell-derived cardiomyocytes, without an effect on the expression of the full-length SYNE2 mRNA. SYNE2α1 overexpression had dominant negative effects on the nucleus with its overexpression or SYNE2 knockdown leading to increased nuclear area and decreased nuclear stiffness. Gene expression results from SYNE2α1 overexpression demonstrated both concordant and nonconcordant effects with SYNE2 knockdown. SYNE2α1 overexpression had a gain of function on electrophysiology, leading to significantly faster calcium reuptake and decreased assessed action potential duration, while SYNE2 knockdown showed both shortened assessed action potential duration and decreased conduction velocity. rs1152591 was identified as a causal atrial fibrillation variant, with the risk allele decreasing SYNE2α1 expression. Downstream effects of SYNE2α1 overexpression include changes in nuclear stiffness and electrophysiology, which may contribute to the mechanism for the risk allele's association with AF.

Oogenesis and Spermatogenesis of the Triploid Black Tiger Shrimp, Penaeus monodon

Triploidy (3n) induction of Penaeus monodon was performed separately by Australian and Thai groups of researchers 14 years ago, incidentally at the same period. The Australian group employed the chemical induction method, while the Thai group the cold shock one, and both groups obtained 3n P. monodon in their attempts. The success has led to several studies on the physiology of the 3n P. monodon, including growth, survival and reproductive functions. Both groups reported sterility of the 3n shrimp. The Australian group reported defected oogenesis and spermatogenesis of the 3n shrimp examined at subadult stage. Since the evaluation of the reproductive functions should be performed at the adult stage of the animals, we, therefore, performed in-depth studies on oogenesis and spermatogenesis of adult 3n P. monodon induced by cold shock. The studies include gross observation of the ovary, light (LM) and transmission electron (TEM) microscopy and image analysis of sex cells. The ovary of the 3n P. monodon females could not develop to full-maturation and their gonadosomatic index (GSI) was significantly lower than that of the 2n shrimp. Oogenesis of the 3n females proceeded from oogonia to cortical rod oocytes, but with significantly lower percentage than that of the 2n shrimp. The GSI of the 3n males was significantly lower than that of the 2n shrimp. Their spermatogenesis, however, proceeded normally from spermatogonia to spermatozoa, but with low density of spermatozoa in the seminiferous tubule and vas deferens, and none in the terminal ampoule. The average nuclear area of the 3n spermatozoa was significantly lower than that of the 2n shrimp as well. Altogether, these results suggest that 3n P. monodon induced by cold shock had defect in both oogenesis and spermatogenesis, probably more in quantity than in quality. HIGHLIGHTS Oogenesis and spermatogenesis of the triploid black tiger shrimp, monodon, induced by cold shock were studied. The study found abnormalities in the spermatogenesis, but not oogenesis, of the triploid P. monodon induced by cold shock. It may be concluded that triploidy condition of P. monodon induced by cold shock affects meiotic, but not mitotic activity of the sex cells. GRAPHICAL ABSTRACT

Distinguishing of Histopathological Staging Features of H-E Stained Human cSCC by Microscopical Multispectral Imaging.

Cutaneous squamous cell carcinoma (cSCC) is the second most common malignant skin tumor. Early and precise diagnosis of tumor staging is crucial for long-term outcomes. While pathological diagnosis has traditionally served as the gold standard, the assessment of differentiation levels heavily depends on subjective judgments. Therefore, how to improve the diagnosis accuracy and objectivity of pathologists has become an urgent problem to be solved. We used multispectral imaging (MSI) to enhance tumor classification. The hematoxylin and eosin (H&E) stained cSCC slides were from Shanghai Ruijin Hospital. Scale-invariant feature transform was applied to multispectral images for image stitching, while the adaptive threshold segmentation method and random forest segmentation method were used for image segmentation, respectively. Synthetic pseudo-color images effectively highlight tissue differences. Quantitative analysis confirms significant variation in the nuclear area between normal and cSCC tissues (p < 0.001), supported by an AUC of 1 in ROC analysis. The AUC within cSCC tissues is 0.57. Further study shows higher nuclear atypia in poorly differentiated cSCC tissues compared to well-differentiated cSCC (p < 0.001), also with an AUC of 1. Lastly, well differentiated cSCC tissues show more and larger keratin pearls. These results have shown that combined MSI with imaging processing techniques will improve H&E stained human cSCC diagnosis accuracy, and it will be well utilized to distinguish histopathological staging features.

Nuclear expansion and chromatin structure remodeling in mouse aging neurons

Abstract Aging, particularly in the brain, involves impairments in multiple cellular functions, many regulated at the nucleus. Chromatin structure plays a critical role in regulating gene expression and the maintenance of genomic stability. During differentiation, each cell type acquire their unique topology, this should be kept for a lifetime, but may deteriorate as we age. However, the effects of aging on the chromatin 3D structure of neurons remain largely unknown and much has been inferred from senescent cells. By combining chromosome conformation capture and microscopy techniques, we investigated cortical neurons of young and aged mice and discovered signs of neuronal nuclear expansion during neuronal aging, leading to increased distances between chromosomes. This expansion alters the topology of compartments, topologically associating domains (TADs) and chromatin loops. While larger TADs tend to dissociate, smaller TADs and loops exhibit strengthened interactions of chromatin in aged neurons. These topological changes impact the borders of TADs, resulting in weakening, this is parallel to Lamin-B weakening and nuclear envelope alterations. We attribute these alterations to changes in physical forces of an expanding nucleus, driving the distancing of chromosomes filling a growing nuclear area, affecting gene expression and topology, contributing to the functional declines observed during aging.

Changes in the liver of Djungarian hamsters under conditions of a three-month supply of water-soluble silicon of various concentrations

BACKGROUND: Silicon enters the human body through drinking water, air and food. Silicon nanoparticles used in the cosmetic, pharmaceutical and food industries are known to have biological activity. Taking into account the widespread prevalence of silicon compounds, the issue of the safety of its use is becoming more urgent. AIM: To study the effect of water-soluble silicon on the morphological structure of the liver of Djungarian hamsters for three months. MATERIALS AND METHODS: The experiment was carried out on Djungarian hamsters kept in normal vivarium conditions under natural light. The animals were divided into three groups: control, which received bottled drinking water; the first experimental group, which received the same water, but with the addition of sodium metasilicate nine-hydrate at a concentration of 10 mg/l in terms of silicon; the second experimental group, which also received the same water, but with the concentration of sodium metasilicate nine-hydrate doubled (up to 20 mg/l). After three months, the animals were removed from the experiment. Sections were processed by general histological (hematoxylin and eosin, Van Gieson method, toluidine blue), histochemical (monoamine oxidase-positive cells) methods. RESULTS: In the liver of hamsters from the experimental groups, changes in the micromorphological structure were revealed, such as an increase in the nuclear area, nuclear-cytoplasmic ratio of hepatocytes, and the diameter of sinusoidal capillaries. Moreover, more pronounced changes were observed in the liver of hamsters of the second experimental group, such as polymorphic cell infiltration of the portal tracts, an increase in the number of eosinophils, deformation of hepatocyte nuclei and the appearance of apoptotic bodies. A decrease in the area of mast cells and an increase in their number, as well as the number of monoamine oxidase-positive cells in the liver of hamsters of both experimental groups were recorded. CONCLUSIONS: An increase in the concentration of silicon supplied with drinking water in both cases is reflected in the micromorphological structure of the liver of hamsters. Moreover these changes are more pronounced in the liver of hamsters of the second experimental group.

Piperine Enhances the Anticancer Effects of Cisplatin on Tongue Squamous Cell Carcinoma Cell Line by Inducing Cell Apoptosis.

Oral squamous cell carcinoma is ranked as the predominant type of head and neck squamous cell carcinoma, comprising roughly 90% of all oral cancer cases. Natural products have proven to be highly valuable as complementary, or adjunctive in the treatment of cancer. Piperine, a natural compound derived from Piper nigrum, demonstrates anti-proliferative and anti-neoplastic effects across various types of cancer. This study focused on assessing the cytotoxic effect of piperine in conjunction with cisplatin within the OSCC cell line. In this in-vitro study, cultured OSCC cells were divided into four groups: a control group (untreated), a group exposed solely to piperine, a group exposed solely to cisplatin, and a group receiving both piperine and cisplatin. Cell viability was determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) assay technique. Additionally, flow cytometric analysis was employed to examine cell cycle progression and apoptosis. Assessment of reactive oxygen species activity, morphological changes, and nuclear area factor measurements were carried out. Expression of the apoptotic regulator Bax was assessed through western blotting analysis. Piperine has cytotoxic and apoptotic effects in a concentration-dependent manner. Piperine in combination with cisplatin exhibited a synergistic effect, resulting in more pronounced inhibition of cell viability in OSCC cells compared to using piperine and cisplatin alone. Piperine and cisplatin for 24 h induced apoptosis strongly by increasing Bax protein and ROS activity. Combining piperine with cisplatin demonstrated a greater effectiveness in triggering apoptosis in OSCC cells compared to using cisplatin alone, allowing for a reduction.

Enhancing terminal erythroid differentiation in human embryonic stem cells through TRIB3 overexpression

Tribbles pseudokinase 3 (TRIB3) expression significantly increases during terminal erythropoiesis in vivo. However, we found that TRIB3 expression remained relatively low during human embryonic stem cell (hESC) erythropoiesis, particularly in the late stage, where it is typically active. TRIB3 was expressed in megakaryocyte-erythrocyte progenitor cells and its low expression was necessary for megakaryocyte differentiation. Thus, we proposed that the high expression during late stage of erythropoiesis could be the clue for promotion of maturation of hESC-derived erythroid cells. To our knowledge, the role of TRIB3 in the late stage of erythropoiesis remains ambiguous. To address this, we generated inducible TRIB3 overexpression hESCs, named TRIB3tet-on OE H9, based on a Tet-On system. Then, we analyzed hemoglobin expression, condensed chromosomes, organelle clearance, and enucleation with or without doxycycline treatment. TRIB3tet-on OE H9 cells generated erythrocytes with a high proportion of orthochromatic erythroblast in flow cytometry, enhanced hemoglobin and related protein expression in Western blot, decreased nuclear area size, promoted enucleation rate, decreased lysosome and mitochondria number, more colocalization of LC3 with LAMP1 (lysosome marker) and TOM20 (mitochondria marker) and up-regulated mitophagy-related protein expression after treatment with 2 μg/mL doxycycline. Our results showed that TRIB3 overexpression during terminal erythropoiesis may promote the maturation of erythroid cells. Therefore, our study delineates the role of TRIB3 in terminal erythropoiesis, and reveals TRIB3 as a key regulator of UPS and downstream mitophagy by ensuring appropriate mitochondrial clearance during the compaction of chromatin.

Automated analysis of scattering-based light sheet microscopy images of anal squamous intraepithelial lesions.

We developed an algorithm for automatically analyzing scattering-based light sheet microscopy (sLSM) images of anal squamous intraepithelial lesions. We developed a method for automatically segmenting sLSM images for nuclei and calculating seven features: nuclear intensity, intensity slope as a function of depth, nuclear-to-nuclear distance, nuclear-to-cytoplasm ratio, cell density, nuclear area, and proportion of pixels corresponding to nuclei. 187 images from 80 anal biopsies were used for feature analysis and classifier development. The automated nuclear segmentation method provided reliable performance with the precision of 0.97 and recall of 0.91 when compared with the manual segmentation. Among the seven features, six showed statistically significant differences between high-grade squamous intraepithelial lesion (HSIL) and non-HSIL (non-dysplastic or low-grade squamous intraepithelial lesion, LSIL). A classifier using linear support vector machine (SVM) achieved promising performance in diagnosing HSIL versus non-HSIL: sensitivity of 90%, specificity of 70%, and area under the curve (AUC) of 0.89 for per-image diagnosis, and sensitivity of 90%, specificity of 80%, and AUC of 0.92 for per-biopsy diagnosis.

Effect of lamins and emerin on nuclear morphology and histological architecture in lung adenocarcinoma

Emerin and lamins not only influence nuclear morphology but are also involved in differentiation. We herein examined 82 resected cases of invasive lung adenocarcinoma using computer-assisted image analysis of nuclear morphology on Feulgen-stained and immunohistochemical sections of lamin A, B1, B2, and emerin (four proteins) to calculate the rank sum of the cell positivity rates for these four proteins. The rank sum of four proteins showed weak negative correlations with the nuclear area and perimeter and a weak positive correlation with the nuclear shape factor. Interestingly, the top three cases with the highest rank sum were papillary adenocarcinoma, and the bottom three cases were acinar adenocarcinomas containing cribriform patterns. We compared the rank sum for grading (differentiation: G1, G2, and G3) and predominant histological subtypes and found that the rank sum of G3 was lower than that of G1 and G2. Furthermore, the rank sum was lower for acinar adenocarcinoma with >20 % cribriform pattern (acinar+cribri) and solid adenocarcinoma than for lepidic and papillary adenocarcinoma. Individual examination of the four proteins revealed that emerin expression was lower in G3 than in G1, and lamin B2 expression was lower in G3 than in G1 and G2. Compared with lepidic adenocarcinoma, acinar+cribri showed significantly lower expression of all four proteins among histological subtypes. These data indicated that the expression of lamin A, B1, B2, and emerin was markedly decreased in poorly differentiated adenocarcinoma (i.e., G3), especially in acinar+cribri. Our data suggested that changes in these four proteins can not only affect nuclear morphology but also histological structure in lung adenocarcinoma.

Neurological recovery in rats with portocaval anastomosis-induced hepatic encephalopathy treated with leuprolide acetate, a GnRH agonist.

Hepatic encephalopathy (HE) is a neuropsychiatric complication of acute liver failure or chronic liver injury. Liver dysfunction impairs ammonia detoxification, allowing it to cross the blood-brain barrier (BBB) and disrupt brain function. The hippocampus becomes a crucial target during elevated ammonia levels, causing spatial memory impairment and decreased learning ability. Leuprolide acetate (LA), a GnRH agonist, has been implicated in neuroprotection and neuroregeneration in several regions of the central nervous system (CNS) including hippocampus. In this study, we aim to evaluate the effects of LA treatment on hippocampus of rats with HE induced by portocaval anastomosis (PCA) trough cognitive tests, histology analysis and expression of neuronal recovery marker proteins, such as neurofilament (NF200) and neurabin II, and astrocyte marker glial fibrillary acidic protein (GFAP). Rats were divided into three groups: SHAM, portocaval anastomosis with saline solution (PCA + SS) and portocaval anastomosis treated with LA (PCA + LA). To evaluate learning and spatial memory elevated T-maze (ETM) and Y-maze test (YMT) were respectively used. Results indicated that LA-treated rats performed significantly better in ETM and YMT than untreated rats. Histological analysis of hippocampus showed increased neuron density, nuclear area, and layer thickness in dentate gyrus of PCA + LA group compared to PCA + SS. Additionally, neurabin II and NF200 expression were higher in LA-treated rats, while GFAP expression was elevated in the PCA + SS group compared to control and PCA + LA groups. In conclusion, LA enhances hippocampal neuron recovery and reduces astrogliosis, suggesting its potential as a therapeutic intervention for attenuating hippocampal damage during HE.

Viscoelastic properties of colorectal liver metastases reflect tumour cell viability

BackgroundColorectal cancer is the third most common tumour entity in the world and up to 50% of the patients develop liver metastases (CRLM) within five years. To improve and personalize therapeutic strategies, new diagnostic tools are urgently needed. For instance, biomechanical tumour properties measured by magnetic resonance elastography (MRE) could be implemented as such a diagnostic tool. We postulate that ex vivo MRE combined with histological and radiological evaluation of CRLM could provide biomechanics-based diagnostic markers for cell viability in tumours.Methods34 CRLM specimens from patients who had undergone hepatic resection were studied using ex vivo MRE in a frequency range from 500 Hz to 5300 Hz with increments of 400 Hz. Single frequency evaluation of shear wave speed and wave penetration rate as proxies for stiffness and viscosity was performed, along with rheological model fitting based on the spring-pot model and powerlaw exponent α, ranging between 0 (complete solid behaviour) and 1 (complete fluid behaviour). For histological analysis, samples were stained with H&E and categorized according to the degree of regression. Quantitative histologic analysis was performed to analyse nucleus size, aspect ratio, and density. Radiological response was assessed according to RECIST-criteria.ResultsFive samples showed major response to chemotherapy, six samples partial response and 23 samples no response. For higher frequencies (> 2100 Hz), shear wave speed correlated significantly with the degree of regression (p ≤ 0.05) indicating stiffer properties with less viable tumour cells. Correspondingly, rheological analysis of α revealed more elastic-solid tissue properties at low cell viability and major response (α = 0.43 IQR 0.36, 0.47) than at higher cell viability and no response (α = 0.51 IQR 0.48, 0.55; p = 0.03). Quantitative histological analysis showed a decreased nuclear area and density as well as a higher nuclear aspect ratio in patients with major response to treatment compared to patients with no response (all p < 0.05).DiscussionOur results suggest that MRE could be useful in the characterization of biomechanical property changes associated with cell viability in CRLM. In the future, MRE could be applied in clinical diagnosis to support individually tailored therapy plans for patients with CRLM.

Abstract We007: High Throughput 3D-Printed Human Engineered Heart Tissues for Cardiac Disease Modeling

While cell models have been used to study cardiac diseases, there is currently no standardization or fully mature in vitro system. Obtaining a mature cardiomyocyte (CM) phenotype is necessary to create a physiologically relevant environment and accurately model and study cardiac diseases. Increased success in maturation of human induced pluripotent stem cell derived CMs (hiPS-CMs) has been achieved in 3D in vitro models in the form of engineered heart tissues (EHTs). Digital light processing (DLP) offers a high throughput and efficient method to fabricate hydrogels that recapitulate properties of native tissues. In this study, we use DLP-printed EHTs to seed and mature differentiated hiPS-CMs and test their potential for modeling pathological cardiac hypertrophy. To further encourage maturity of CMs, we utilized a low glucose maturation medium supplemented with fatty acids. DLP-printed EHTs displayed high reproducibility and increased CM maturity. To determine degree of maturity of our system, we performed RT-qPCR, western blot analysis, and immunofluorescence (IF) microscopy. We found increased expression of fatty-acid transport and beta oxidation genes and improved sarcomere alignment compared to 2D hiPS-CMs. To model pathological cardiac hypertrophy, we used two different approaches: 1. acute adrenergic agonism with isoproterenol and phenylephrine; 2. chronic culturing of EHTs in pathologically stiff conditions. To assess pathology, we conducted RT-qPCR, western blot, ELISA, IF microscopy, and functional analyses. We found that agonist-treated EHTs displayed increased pathology-associated gene expression compared to standard 2D hiPS-CMs. Both agonist-treated and stiff EHTs had increased secreted B-Type Natriuretic Peptide (BNP) and phosphorylation of proteins involved in pathological remodeling, including ERK and P38. Additionally, we observed increased nuclear area, increased calcium handling, and increased contractile force in the treatment groups. In conclusion, we have established a high throughput, reproducible, mature EHT platform that can be used to model pathological cardiac hypertrophy. This platform can be easily adopted by other laboratories to study a wide array of cardiac diseases.

CD4+ T cell-associated cytokines induce a chronic pro-inflammatory phenotype in induced pluripotent stem cell-derived midbrain astrocytes.

Astrocytes are mediators of homeostasis but contribute to neuroinflammation in Parkinson's disease (PD). Mounting evidence suggests involvement of peripheral immune cells in PD pathogenesis. Therefore, this study aimed to determine the potential role of peripheral immune secreted cytokines in modulating midbrain astrocyte reactivity. Human iPSC-derived midbrain astrocytes were exposed to 5% and 10% CD4+ T cell conditioned media (CD4CM) for 24 h, 72 h, and 7 days to assess chronic exposure. Additionally, astrocytes were exposed to the Th17 cell cytokine, IL-17A (10 ng/mL), alone and in combination with TNF-α (0.3 ng/mL) to assess potential synergistic effects of both cytokines at 24 h, 72 h, and 7 days. CD4CM induced acute and chronic alterations in midbrain astrocytes. Increased NFκB translocation to the nucleus, increased expression of the pro-inflammatory genes, IL-1β, CXCL10 at 24 h, C3, LCN2, IL-6 at 24 and 48 h, as well as an increase in their release of pro-inflammatory cytokines IL-6 and CXCL10 at both these time points were observed. A synergistic response to the combination of IL-17A and TNF-α on increasing inflammatory gene expression and cytokine release occurred. IL-17A and TNF-α increased intensity of S100β at 24 h, decreased nuclear area and increased circularity of astrocytes at 72 h. A synergistic effect on γH2AX intensity at 72 h and an increase in LDH release at 7 days was observed. Our results demonstrate that IL-17A and TNF-α act synergistically, enhancing midbrain astrocyte reactivity to a similar degree as CD4CM. This highlights the importance of the peripheral immune secreted cytokines in increasing the reactivity status of midbrain astrocytes, implicating their role in PD.