Optimizing Coreopsis tinctoria Flower Extraction and Inhibiting CML Activity: Box-Behnken Design.

BACKGROUND: Chronic myelogenous leukemia (CML) is an uncommon type of cancer of the bone marrow associated with high mortality. Although several effective therapies have been developed to reduce symptoms in patients with CML, many of these methods are associated with side effects. Coreopsis tinctoria Nutt. (C. tinctoria) is a natural medicinal material that possesses antioxidant and anticancer activities. Yet, its effect in treating leukemia has still not been fully explored. OBJECTIVE: To optimize the C. tinctoria flower extraction process and investigate whether these extracts can impair CML cell survival. METHODS: The extraction process of C. tinctoria was optimized by the Box-Behnken design response surface method. K562 cells were treated with different volumes (0, 10, 25, 50, and 100 µL) of C. tinctoria flower extracts. The effect of C. tinctoria extract on cell morphology and cell apoptosis was assessed by light microscopy, laser confocal microscopy, and flow cytometry. RESULTS: We established the following optimized C. tinctoria flower extraction conditions: temperature of 84.4 °C, extraction period of 10 mins, solid-liquid ratio of 1:65, and times 4. These conditions were applied for C. tinctoria flower extraction. Pre-incubation of extracts prepared under the aforementioned optimal conditions with K562 cells induced cell cytotoxicity and cell apoptosis. CONCLUSION: C. tinctoria flower extracts exert obvious anti-leukemia effects in vitro and may be a potential drug candidate for leukemia treatment.

Integrating Spatial and Morphological Characteristics into Melanoma Prognosis: A Computational Approach.

In this study, the prognostic value of cellular morphology and spatial configurations in melanoma has been examined, aiming to complement traditional prognostic indicators like mitotic activity and tumor thickness. Through a computational pipeline using machine learning and deep learning methods, we quantified nuclei sizes within different spatial regions and analyzed their prognostic significance using univariate and multivariate Cox models. Nuclei sizes in the invasive band demonstrated a significant hazard ratio (HR) of 1.1 (95% CI: 1.03, 1.18). Similarly, the nuclei sizes of tumor cells and Ki67 S100 co-positive cells in the invasive band achieved HRs of 1.07 (95% CI: 1.02, 1.13) and 1.09 (95% CI: 1.04, 1.16), respectively. Our findings reveal that nuclei sizes, particularly in the invasive band, are potentially prognostic factors. Correlation analyses further demonstrated a meaningful relationship between cellular morphology and tumor progression, notably showing that nuclei size within the invasive band correlates substantially with tumor thickness. These results suggest the potential of integrating spatial and morphological analyses into melanoma prognostication.

[Spatial Constraints of Rectangular Hydrogel Microgrooves Regulate the Morphology and Arrangement of Human Umbilical Vein Endothelial Cells]. / çŸ©å½¢æ°´å‡èƒ¶å¾®å‡¹æ§½çš„ç©ºé—´çº¦æŸè°ƒæŽ§äººè„é™è„‰å† çš®ç»†èƒžçš„å½¢æ€å’ŒæŽ’åˆ—.

Objective: To construct microscale rectangular hydrogel grooves and to investigate the morphology and alignment of human umbilical vein endothelial cells (HUVECs) under spatial constraints. Vascular endothelial cell morphology and alignment are important factors in vascular development and the maintenance of homeostasis. Methods: A 4-arm polyethylene glycol-acrylate (PEG-acrylate) hydrogel was used to fabricate rectangular microgrooves of the widths of 60 µm, 100 µm, and 140 µm. The sizes and the fibronectin (FN) adhesion of these hydrogel microgrooves were measured. HUVECs were seeded onto the FN-coated microgrooves, while the flat surface without micropatterns was used as the control. After 48 hours of incubation, the morphology and orientation of the cells were examined. The cytoskeleton was labelled with phalloidine and the orientation of the cytoskeleton in the hydrogel microgrooves was observed by laser confocal microscopy. Results: The hydrogel microgrooves constructed exhibited uniform and well-defined morphology, a complete structure, and clear edges, with the width deviation being less than 3.5%. The depth differences between the hydrogel microgrooves of different widths were small and the FN adhesion is uniform, providing a micro-patterned growth interface for cells. In the control group, the cells were arranged haphazardly in random orientations and the cell orientation angle was (46.9±1.8)°. In contrast, the cell orientation angle in the hydrogel microgrooves was significantly reduced (P<0.001). However, the cell orientation angles increased with the increase in hydrogel microgroove width. For the 60 µm, 100 µm, and 140 µm hydrogel microgrooves, the cell orientation angles were (16.4±2.8)°, (24.5±3.2)°, and (30.3±3.5)°, respectively. Compared to that of the control group (35.7%), the number of cells with orientation angles <30° increased significantly in the hydrogel microgrooves of different widths (P<0.001). However, as the width of the hydrogel microgrooves increased, the number of cells with orientation angles <30° gradually decreased (79.9%, 62.3%, 54.7%, respectively), while the number of cells with orientation angles between 60°-90° increased (P<0.001). The cell bodies in the microgrooves were smaller and more rounded in shape. The cells were aligned along the direction of the microgrooves and corresponding changes occurred in the arrangement of the cell cytoskeleton. In the control group, cytoskeletal filaments were aligned in random directions, presenting an orientation angle of (45.5±3.7)°. Cytoskeletal filaments were distributed evenly within various orientation angles. However, in the 60 µm, 100 µm, and 140 µm hydrogel microgrooves, the orientation angles of the cytoskeletal filaments were significantly decreased, measuring (14.4±3.1)°, (24.7±3.5)°, and (31.9±3.3)°, respectively. The number of cytoskeletal filaments with orientation angles <30° significantly increased in hydrogel microgrooves of different widths (P<0.001). However, as the width of the hydrogel microgrooves increased, the number of cytoskeletal filaments with orientation angles <30° gradually decreased, while the number of cytoskeletal filaments with orientation angles between 60°-90° gradually increased (P<0.001). Conclusion: Hydrogel microgrooves can regulate the morphology and orientation of HUVECs and mimic to a certain extent the in vivo microenvironment of vascular endothelial cells, providing an experimental model that bears better resemblance to human physiology for the study of the unique physiological functions of vascular endothelial cells. Nonetheless, the molecular mechanism of spatial constraints on the morphology and the assembly of vascular endothelial cell needs to be further investigated.

Understanding the Ultra-Rare Disease Autosomal Dominant Leukodystrophy: an Updated Review on Morpho-Functional Alterations Found in Experimental Models.

Autosomal dominant leukodystrophy (ADLD) is an ultra-rare, slowly progressive, and fatal neurodegenerative disorder associated with the loss of white matter in the central nervous system (CNS). Several years after its first clinical description, ADLD was found to be caused by coding and non-coding variants in the LMNB1 gene that cause its overexpression in at least the brain of patients. LMNB1 encodes for Lamin B1, a protein of the nuclear lamina. Lamin B1 regulates many cellular processes such as DNA replication, chromatin organization, and senescence. However, its functions have not been fully characterized yet. Nevertheless, Lamin B1 together with the other lamins that constitute the nuclear lamina has firstly the key role of maintaining the nuclear structure. Being the nucleus a dynamic system subject to both biochemical and mechanical regulation, it is conceivable that changes to its structural homeostasis might translate into functional alterations. Under this light, this review aims at describing the pieces of evidence that to date have been obtained regarding the effects of LMNB1 overexpression on cellular morphology and functionality. Moreover, we suggest that further investigation on ADLD morpho-functional consequences is essential to better understand this complex disease and, possibly, other neurological disorders affecting CNS myelination.

Elongated Riboflavin-Producing Shewanella oneidensis in a Hybrid Biofilm Boosts Extracellular Electron Transfer.

Shewanella oneidensis is able to carry out extracellular electron transfer (EET), although its EET efficiency is largely limited by low flavin concentrations, poor biofilm forming-ability, and weak biofilm conductivity. After identifying an important role for riboflavin (RF) in EET via in vitro experiments, the synthesis of RF is directed to 837.74 ± 11.42 µm in S. oneidensis. Molecular dynamics simulation reveals RF as a cofactor that binds strongly to the outer membrane cytochrome MtrC, which is correspondingly further overexpressed to enhance EET. Then the cell division inhibitor sulA, which dramatically enhanced the thickness and biomass of biofilm increased by 155% and 77%, respectively, is overexpressed. To reduce reaction overpotential due to biofilm thickness, a spider-web-like hybrid biofilm comprising RF, multiwalled carbon nanotubes (MWCNTs), and graphene oxide (GO) with adsorption-optimized elongated S. oneidensis, achieve a 77.83-fold increase in power (3736 mW m-2 ) relative to MR-1 and dramatically reduce the charge-transfer resistance and boosted biofilm electroactivity. This work provides an elegant paradigm to boost EET based on a synthetic biology strategy and materials science strategy, opens up further opportunities for other electrogenic bacteria.

Quantitative cell imaging approaches to metastatic state profiling.

Genetic heterogeneity of metastatic dissemination has proven challenging to identify exploitable markers of metastasis; this bottom-up approach has caused a stalemate between advances in metastasis and the late stage of the disease. Advancements in quantitative cellular imaging have allowed the detection of morphological phenotype changes specific to metastasis, the morphological changes connected to the underlying complex signaling pathways, and a robust readout of metastatic cell state. This review focuses on the recent machine and deep learning developments to gain detailed information about the metastatic cell state using light microscopy. We describe the latest studies using quantitative cell imaging approaches to identify cell appearance-based metastatic patterns. We discuss how quantitative cancer biologists can use these frameworks to work backward toward exploitable hidden drivers in the metastatic cascade and pioneering new Frontier drug discoveries specific for metastasis.

Evaluation of a Serum-Free Medium for Human Epithelial and Stromal Cell Culture.

Over the past decade, growing demand from many domains (research, cosmetics, pharmaceutical industries, etc.) has given rise to significant expansion of the number of in vitro cell cultures. Despite the widespread use of fetal bovine serum, many issues remain. Among them, the whole constitution of most serums remains unknown and is subject to significant variations. Furthermore, the presence of potential contamination and xenogeny elements is challenging for clinical applications, while limited production is an obstacle to the growing demand. To circumvent these issues, a Serum-Free Medium (SFM) has been developed to culture dermal and vesical fibroblasts and their corresponding epithelial cells, namely, keratinocytes and urothelial cells. To assess the impact of SFM on these cells, proliferation, clonogenic and metabolic assays have been compared over three passages to conditions associated with the use of a classic Fetal Bovine Serum-Containing Medium (FBSCM). The results showed that the SFM enabled fibroblast and epithelial cell proliferation while maintaining a morphology, cell size and metabolism similar to those of FBSCM. SFM has repeatedly been found to be better suited for epithelial cell proliferation and clonogenicity. Fibroblasts and epithelial cells also showed more significant mitochondrial metabolism in the SFM compared to the FBSCM condition. However, the SFM may need further optimization to improve fibroblast proliferation.

Substrate stiffness modulates migration and local intercellular membrane motion in pulmonary endothelial cell monolayers.

The pulmonary artery endothelium forms a semipermeable barrier that limits macromolecular flux through intercellular junctions. This barrier is maintained by an intrinsic forward protrusion of the interacting membranes between adjacent cells. However, the dynamic interactions of these membranes have been incompletely quantified. Here, we present a novel technique to quantify the motion of the peripheral membrane of the cells, called paracellular morphological fluctuations (PMFs), and to assess the impact of substrate stiffness on PMFs. Substrate stiffness impacted large-length scale morphological changes such as cell size and motion. Cell size was larger on stiffer substrates, whereas the speed of cell movement was decreased on hydrogels with stiffness either larger or smaller than 1.25 kPa, consistent with cells approaching a jammed state. Pulmonary artery endothelial cells moved fastest on 1.25 kPa hydrogel, a stiffness consistent with a healthy pulmonary artery. Unlike these large-length scale morphological changes, the baseline of PMFs was largely insensitive to the substrate stiffness on which the cells were cultured. Activation of store-operated calcium channels using thapsigargin treatment triggered a transient increase in PMFs beyond the control treatment. However, in hypocalcemic conditions, such an increase in PMFs was absent on 1.25 kPa hydrogel but was present on 30 kPa hydrogel-a stiffness consistent with that of a hypertensive pulmonary artery. These findings indicate that 1) PMFs occur in cultured endothelial cell clusters, irrespective of the substrate stiffness; 2) PMFs increase in response to calcium influx through store-operated calcium entry channels; and 3) stiffer substrate promotes PMFs through a mechanism that does not require calcium influx.

Mechanisms underlying the role of ankyrin-B in cardiac and neurological health and disease.

The ANK2 gene encodes for ankyrin-B (ANKB), one of 3 members of the ankyrin family of proteins, whose name is derived from the Greek word for anchor. ANKB was originally identified in the brain (B denotes "brain") but has become most widely known for its role in cardiomyocytes as a scaffolding protein for ion channels and transporters, as well as an interacting protein for structural and signaling proteins. Certain loss-of-function ANK2 variants are associated with a primarily cardiac-presenting autosomal-dominant condition with incomplete penetrance and variable expressivity characterized by a predisposition to supraventricular and ventricular arrhythmias, arrhythmogenic cardiomyopathy, congenital and adult-onset structural heart disease, and sudden death. Another independent group of ANK2 variants are associated with increased risk for distinct neurological phenotypes, including epilepsy and autism spectrum disorders. The mechanisms underlying ANKB's roles in cells in health and disease are not fully understood; however, several clues from a range of molecular and cell biological studies have emerged. Notably, ANKB exhibits several isoforms that have different cell-type-, tissue-, and developmental stage- expression profiles. Given the conservation within ankyrins across evolution, model organism studies have enabled the discovery of several ankyrin roles that could shed important light on ANKB protein-protein interactions in heart and brain cells related to the regulation of cellular polarity, organization, calcium homeostasis, and glucose and fat metabolism. Along with this accumulation of evidence suggesting a diversity of important ANKB cellular functions, there is an on-going debate on the role of ANKB in disease. We currently have limited understanding of how these cellular functions link to disease risk. To this end, this review will examine evidence for the cellular roles of ANKB and the potential contribution of ANKB functional variants to disease risk and presentation. This contribution will highlight the impact of ANKB dysfunction on cardiac and neuronal cells and the significance of understanding the role of ANKB variants in disease.

Palmitoylation of the small GTPase Cdc42 by DHHC5 modulates spine formation and gene transcription.

The small GTPase Cdc42 exists in the form of two alternatively spliced variants that are modified by hydrophobic chains: the ubiquitously expressed Cdc42-prenyl and a brain-specific isoform that can be palmitoylated, Cdc42-palm. Our previous work demonstrated that Cdc42-palm can be palmitoylated at two cysteine residues, Cys188 and Cys189, while Cys188 can also be prenylated. We showed that palmitoylation of Cys188 is essential for the plasma membrane localization of Cdc42-palm and is critically involved in Cdc42-mediated regulation of gene transcription and neuronal morphology. However, the abundance and regulation of this modification was not investigated. In the present study, we found that only a minor fraction of Cdc42 undergoes monopalmitoylation in neuroblastoma cells and in hippocampal neurons. In addition, we identified DHHC5 as one of the major palmitoyl acyltransferases that could physically interact with Cdc42-palm. We demonstrate that overexpression of dominant negative DHHC5 mutant decreased palmitoylation and plasma membrane localization of Cdc42-palm. In addition, knockdown of DHHC5 significantly reduced Cdc42-palm palmitoylation, leading to a decrease of Cdc42-mediated gene transcription and spine formation in hippocampal neurons. We also found that the expression of DHHC5 in the brain is developmentally regulated. Taken together, these findings suggest that DHHC5-mediated palmitoylation of Cdc42 represents an important mechanism for the regulation of Cdc42 functions in hippocampus.

Inactivation of Salmonella typhimurium SL1344 by Chlorogenic Acid and the Impairment of Cellular Integrity.

Chlorogenic acid (CGA) is an antibacterial agent that can be isolated from Eucommia ulmoides Oliver, a Chinese medicinal and edible plant food. The inhibitory effect of CGA on bacterial growth and stiffness of the outer membrane (OM) had been reported, while more evidence were required to elucidate its impairment of cell wall. In this study, the morphological and physiochemical changes of Salmonella cells under CGA treatment were investigated. Firstly, the minimum inhibitory concentration (MIC) of CGA against Salmonella was assayed. Later, the permeability of OM and activity of the proteins released were measured and observed to reveal the alteration of OM characteristic and cellular morphology. Finally, reactive oxygen species and cell membrane fluidity were analyzed, respectively, to elucidate how CGA damaged cell surface. The results showed that MIC of CGA against Salmonella was 6.25 mg/L. Under sub-lethal doses of CGA, the OM permeability and the release of soluble proteins were enhanced evidently, and Salmonella cells showed more deformed and shrunken, confirming the impairment of cellular integrity under CGA. Finally, the possible cause of cell surface damage was investigated. the fluidity of the membrane was increased upon CGA treatment, which may the possible cause of OM by CGA.

Characterization of a Novel Creeping Tartary Buckwheat (Fagopyrum tataricum) Mutant lazy1.

Gravity is known as an important environmental factor involved in the regulation of plant architecture. To identify genes related to the gravitropism of Tartary buckwheat, a creeping line was obtained and designated as lazy1 from the mutant bank by 60Co-γ ray radiation. Genetic analysis indicated that the creeping phenotype of lazy1 was attributed to a single recessive locus. As revealed by the horizontal and inverted suspension tests, lazy1 was completely lacking in shoot negative gravitropism. The creeping growth of lazy1 occurred at the early seedling stage, which could not be recovered by exogenous heteroauxin, hormodin, α-rhodofix, or gibberellin. Different from the well-organized and equivalent cell elongation of wild type (WT), lazy1 exhibited dilated, distorted, and abnormally arranged cells in the bending stem. However, no statistical difference of indole-3-acetic acid (IAA) levels was found between the far- and near-ground bending sides in lazy1, which suggests that the asymmetric cell elongation of lazy1 was not induced by auxin gradient. Whereas, lazy1 showed up-expressed gibberellin-regulated genes by quantitative real-time PCR (qRT-PCR) as well as significantly higher levels of gibberellin, suggesting that gibberellin might be partly involved in the regulation of creeping growth in lazy1. RNA sequencing (RNA-seq) identified a number of differentially expressed genes (DEGs) related to gravitropism at stages I (before bending), II (bending), and III (after bending) between WT and lazy1. Venn diagram indicated that only Pectate lyase 5 was down-expressed at stages I [Log2 fold change (Log2FC): -3.20], II (Log2FC: -4.97), and III (Log2FC: -1.23) in lazy1, compared with WT. Gene sequencing revealed that a fragment deletion occurred in the coding region of Pectate lyase 5, which induced the destruction of a pbH domain in Pectate lyase 5 of lazy1. qRT-PCR indicated that Pectate lyase 5 was extremely down-expressed in lazy1 at stage II (0.02-fold of WT). Meanwhile, lazy1 showed the affected expression of lignin- and cellulose-related genes and cumulatively abnormal levels of pectin, lignin, and cellulose. These results demonstrate the possibility that Pectate lyase 5 functions as the key gene that could mediate primary cell wall metabolism and get involved in the asymmetric cell elongation regulation of lazy1.

Detailed Morphologic Evaluation of Breast Papillary Lesions on Core Biopsy Is Critical for Accurate Classification and Clinical Management: The Experience of an Academic Institute.

BACKGROUND: Distinguishing between a breast intraductal papilloma and a papillary lesion with atypia or malignancy can be very challenging on core biopsy. There has been a long ongoing debate over whether or not it is necessary for breast papillary lesions diagnosed on core biopsies to be surgically excised, and the upgrading rate after excision varies. METHOD AND/OR RESULT: This study was carried out in a subspecialized academic pathology department, with well-formed criteria established among the faculty for the categorization of breast papillary lesions, with emphasis on the morphology evaluation of cellular features. A total of 320 breast core biopsies with follow-up excisions were identified. Of these, 286 cases had concordant results between the biopsy and excision, giving a concordance rate of 89.4%, with 98% concordance (143/146) in benign papilloma, 100% (111/111) in papillary carcinoma, and 51% (32/63) in papilloma with atypia. Of the upgraded cases, two were upgraded from benign to atypical, 11 from atypia to malignancy, and only one from benign to malignant. The overall average upgrading rate was 4.4% (14/320), with the critical upgrading (from benign to atypia or malignancy) rate of 0.94% (3/320). Downgrading was only identified in the group of papilloma with atypia, with 20 of 63 cases downgraded to benign papilloma on excision. CONCLUSION: Our study indicates that surgical excision may not be necessary for all papillary lesions after detailed evaluation of the morphology on core biopsies. Assessing the morphological features of the epithelial cells is critical for the accurate classification and clinical management of papillary lesions.

MORPHIOUS: an unsupervised machine learning workflow to detect the activation of microglia and astrocytes.

BACKGROUND: In conditions of brain injury and degeneration, defining microglial and astrocytic activation using cellular markers alone remains a challenging task. We developed the MORPHIOUS software package, an unsupervised machine learning workflow which can learn the morphologies of non-activated astrocytes and microglia, and from this information, infer clusters of microglial and astrocytic activation in brain tissue. METHODS: MORPHIOUS combines a one-class support vector machine with the density-based spatial clustering of applications with noise (DBSCAN) algorithm to identify clusters of microglial and astrocytic activation. Here, activation was triggered by permeabilizing the blood-brain barrier (BBB) in the mouse hippocampus using focused ultrasound (FUS). At 7 day post-treatment, MORPHIOUS was applied to evaluate microglial and astrocytic activation in histological tissue. MORPHIOUS was further evaluated on hippocampal sections of TgCRND8 mice, a model of amyloidosis that is prone to microglial and astrocytic activation. RESULTS: MORPHIOUS defined two classes of microglia, termed focal and proximal, that are spatially adjacent to the activating stimulus. Focal and proximal microglia demonstrated activity-associated features, including increased levels of ionized calcium-binding adapter molecule 1 expression, enlarged soma size, and deramification. MORPHIOUS further identified clusters of astrocytes characterized by activity-related changes in glial fibrillary acidic protein expression and branching. To validate these classifications following FUS, co-localization with activation markers were assessed. Focal and proximal microglia co-localized with the transforming growth factor beta 1, while proximal astrocytes co-localized with Nestin. In TgCRND8 mice, microglial and astrocytic activation clusters were found to correlate with amyloid-ß plaque load. Thus, by only referencing control microglial and astrocytic morphologies, MORPHIOUS identified regions of interest corresponding to microglial and astrocytic activation. CONCLUSIONS: Overall, our algorithm is a reliable and sensitive method for characterizing microglial and astrocytic activation following FUS-induced BBB permeability and in animal models of neurodegeneration.

Monocytes contribute to a pro-healing response in 40 µm diameter uniform-pore, precision-templated scaffolds.

Porous precision-templated scaffolds (PTS) with uniformly distributed 40 µm spherical pores have shown a remarkable ability in immunomodulating resident cells for tissue regeneration. While the pore size mediated pro-healing response observed only in 40 µm pore PTS has been attributed to selective macrophage polarization, monocyte recruitment and phenotype have largely been uncharacterized in regulating implant outcome. Here, we employ a double transgenic mouse model for myeloid characterization and a multifaceted phenotyping approach to quantify monocyte dynamics within subcutaneously implanted PTS. Within 40 µm PTS, myeloid cells were found to preferentially infiltrate into the scaffold. Additionally, macrophage receptor with collagenous structure (MARCO), an innate activation marker, was significantly upregulated within 40 µm PTS. When 40 µm PTS were implanted in monocyte-depleted mice, the transcription of MARCO was significantly decreased and an increase in pro-inflammatory inducible nitric oxide synthase (iNOS) and tumor necrosis factor alpha (TNFα) were observed. Typical of a foreign body response (FBR), 100 µm PTS significantly upregulated pro-inflammatory iNOS, secreted higher amounts of TNFα, and displayed a pore size dependent morphology compared to 40 µm PTS. Overall, these results identify a pore size dependent modulation of circulating monocytes and implicates MARCO expression as a defining subset of monocytes that appears to be responsible for regulating a pro-healing host response.

Cellular Substrates of Functional Network Integration and Memory in Temporal Lobe Epilepsy.

Temporal lobe epilepsy (TLE) patients are at risk of memory deficits, which have been linked to functional network disturbances, particularly of integration of the default mode network (DMN). However, the cellular substrates of functional network integration are unknown. We leverage a unique cross-scale dataset of drug-resistant TLE patients (n = 31), who underwent pseudo resting-state functional magnetic resonance imaging (fMRI), resting-state magnetoencephalography (MEG) and/or neuropsychological testing before neurosurgery. fMRI and MEG underwent atlas-based connectivity analyses. Functional network centrality of the lateral middle temporal gyrus, part of the DMN, was used as a measure of local network integration. Subsequently, non-pathological cortical tissue from this region was used for single cell morphological and electrophysiological patch-clamp analysis, assessing integration in terms of total dendritic length and action potential rise speed. As could be hypothesized, greater network centrality related to better memory performance. Moreover, greater network centrality correlated with more integrative properties at the cellular level across patients. We conclude that individual differences in cognitively relevant functional network integration of a DMN region are mirrored by differences in cellular integrative properties of this region in TLE patients. These findings connect previously separate scales of investigation, increasing translational insight into focal pathology and large-scale network disturbances in TLE.

C-MORE: A high-content single-cell morphology recognition methodology for liquid biopsies toward personalized cardiovascular medicine.

Cellular morphology has the capacity to serve as a surrogate for cellular state and functionality. However, primary cardiomyocytes, the standard model in cardiovascular research, are highly heterogeneous cells and therefore impose methodological challenges to analysis. Hence, we aimed to devise a robust methodology to deconvolute cardiomyocyte morphology on a single-cell level: C-MORE (cellular morphology recognition) is a workflow from bench to data analysis tailored for heterogeneous primary cells using our R package cmoRe. We demonstrate its utility in proof-of-principle applications such as modulation of canonical hypertrophy pathways and linkage of genotype-phenotype in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). In our pilot study, exposure of cardiomyocytes to blood plasma prior to versus after aortic valve replacement allows identification of a disease fingerprint and reflects partial reversibility following therapeutic intervention. C-MORE is a valuable tool for cardiovascular research with possible fields of application in basic research and personalized medicine.

Patient-specific microdosimetry: a proof of concept.

Microscopic energy deposition distributions from ionizing radiation vary depending on biological target size and are used to predict the biological effects of an irradiation. Ionizing radiation is thought to kill cells or inhibit the cell cycle mainly by damaging DNA in the cell nucleus. The size of cells and nuclei depends on tissue type, cell cycle, and malignancy, all of which vary between patients. The aim of this study was to develop methods to perform patient-specific microdosimetry, that being, determining microdosimetric quantities in volumes that correspond to the sizes of cells and nuclei observed in a patient's tissue. A histopathological sample extracted from a stage I lung adenocarcinoma patient was analyzed. A pouring simulation was used to generate a three-dimensional tissue model from cell and nucleus size information determined from the histopathological sample. Microdosimetric distributions includingf(y)andd(y)were determined forC60o,I192r,Y169bandI125in a patient-specific model containing a distribution of cell and nucleus sizes. Fixed radius models and a summation method were compared to the full patient-specific model to evaluate their suitability for fast determination of patient-specific microdosimetric parameters. In the summation method,f(y)from many fixed radii models are summed. Fixed radius models do not provide a close approximation of the full patient-specific modely¯fory¯dfor the lower energy sources investigated,Y169bandI125.The higher energy sources investigated,C60oandI192rare less sensitive to target size variation thanY169bandI125.The summation method yields the most accurate approximation of the full modeld(y)for all radioisotopes investigated. The use of a summation method allows for the computation of patient-specific microdosimetric distributions with the computing power of a personal computer. With appropriate biological inputs the microdosimetric distributions computed using these methods can yield a patient-specific relative biological effectiveness as part of a multiscale treatment planning approach.

Inhibitory Concentrations of Ciprofloxacin Induce an Adaptive Response Promoting the Intracellular Survival of Salmonella enterica Serovar Typhimurium.

Antimicrobial resistance (AMR) is a pressing global health crisis, which has been fueled by the sustained use of certain classes of antimicrobials, including fluoroquinolones. While the genetic mutations responsible for decreased fluoroquinolone (ciprofloxacin) susceptibility are known, the implications of ciprofloxacin exposure on bacterial growth, survival, and interactions with host cells are not well described. Aiming to understand the influence of inhibitory concentrations of ciprofloxacin in vitro, we subjected three clinical isolates of Salmonella enterica serovar Typhimurium to differing concentrations of ciprofloxacin, dependent on their MICs, and assessed the impact on bacterial growth, morphology, and transcription. We further investigated the differential morphology and transcription that occurred following ciprofloxacin exposure and measured the ability of ciprofloxacin-treated bacteria to invade and replicate in host cells. We found that ciprofloxacin-exposed S. Typhimurium is able to recover from inhibitory concentrations of ciprofloxacin and that the drug induces specific morphological and transcriptional signatures associated with the bacterial SOS response, DNA repair, and intracellular survival. In addition, ciprofloxacin-treated S. Typhimurium has increased capacity for intracellular replication in comparison to that of untreated organisms. These data suggest that S. Typhimurium undergoes an adaptive response under ciprofloxacin perturbation that promotes cellular survival, a consequence that may justify more measured use of ciprofloxacin for Salmonella infections. The combination of multiple experimental approaches provides new insights into the collateral effects that ciprofloxacin and other antimicrobials have on invasive bacterial pathogens. IMPORTANCE Antimicrobial resistance is a critical concern in global health. In particular, there is rising resistance to fluoroquinolones, such as ciprofloxacin, a first-line antimicrobial for many Gram-negative pathogens. We investigated the adaptive response of clinical isolates of Salmonella enterica serovar Typhimurium to ciprofloxacin, finding that the bacteria adapt in short timespans to high concentrations of ciprofloxacin in a way that promotes intracellular survival during early infection. Importantly, by studying three clinically relevant isolates, we were able to show that individual isolates respond differently to ciprofloxacin and that for each isolate, there was a heterogeneous response under ciprofloxacin treatment. The heterogeneity that arises from ciprofloxacin exposure may drive survival and proliferation of Salmonella during treatment and lead to drug resistance.

Visualization of Necroptotic Cell Death through Transmission Electron Microscopy.

Transmission electron microscopy (TEM) is an all-in-one tool to visualize the complex systems of any specimen that is 1 nm in size or smaller. The current chapter provides detailed guidelines for imaging morphological changes during programmed cell necrosis using TEM as a single-step methodology. In this protocol, a novel aldehyde dehydrogenase inhibitor is used to induce cell programmed necrosis in ovarian cancer cell lines (A2780 and SKOV3). This process is followed by gradient dehydration with ethanol, chemical fixation, sampled grid preparation, and staining with 0.75% uranyl formate. Following fixation and grid preparation, cells are imaged using TEM. The resulting images reveal morphological changes consistent with necrotic morphology, including swelling of cells and organelles, appearance of vacuoles, and plasma membrane rupture followed by leakage of cellular contents. The current approach allows a single-step methodology for characterization of cell-programmed necrosis in cells based on morphology.