Intelligent Image-Activated Cell Sorting.

A fundamental challenge of biology is to understand the vast heterogeneity of cells, particularly how cellular composition, structure, and morphology are linked to cellular physiology. Unfortunately, conventional technologies are limited in uncovering these relations. We present a machine-intelligence technology based on a radically different architecture that realizes real-time image-based intelligent cell sorting at an unprecedented rate. This technology, which we refer to as intelligent image-activated cell sorting, integrates high-throughput cell microscopy, focusing, and sorting on a hybrid software-hardware data-management infrastructure, enabling real-time automated operation for data acquisition, data processing, decision-making, and actuation. We use it to demonstrate real-time sorting of microalgal and blood cells based on intracellular protein localization and cell-cell interaction from large heterogeneous populations for studying photosynthesis and atherothrombosis, respectively. The technology is highly versatile and expected to enable machine-based scientific discovery in biological, pharmaceutical, and medical sciences.

[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.

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.

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.

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.

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.

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.

Partial loss of actin nucleator actin-related protein 2/3 activity triggers blebbing in primary T lymphocytes.

T lymphocytes utilize amoeboid migration to navigate effectively within complex microenvironments. The precise rearrangement of the actin cytoskeleton required for cellular forward propulsion is mediated by actin regulators, including the actin-related protein 2/3 (Arp2/3) complex, a macromolecular machine that nucleates branched actin filaments at the leading edge. The consequences of modulating Arp2/3 activity on the biophysical properties of the actomyosin cortex and downstream T cell function are incompletely understood. We report that even a moderate decrease of Arp3 levels in T cells profoundly affects actin cortex integrity. Reduction in total F-actin content leads to reduced cortical tension and disrupted lamellipodia formation. Instead, in Arp3-knockdown cells, the motility mode is dominated by blebbing migration characterized by transient, balloon-like protrusions at the leading edge. Although this migration mode seems to be compatible with interstitial migration in three-dimensional environments, diminished locomotion kinetics and impaired cytotoxicity interfere with optimal T cell function. These findings define the importance of finely tuned, Arp2/3-dependent mechanophysical membrane integrity in cytotoxic effector T lymphocyte activities.

Solidification Morphology and Bifurcation Predictions with the Maximum Entropy Production Rate Model.

The use of the principle of maximum entropy generation per unit volume is a new approach in materials science that has implications for understanding the morphological evolution during solid-liquid interface growth, including bifurcations with or without diffuseness. A review based on a pre-publication arXiv preprint is first presented. A detailed comparison with experimental observations indicates that the Maximum Entropy Production Rate-density model (MEPR) can correctly predict bifurcations for dilute alloys during solidification. The model predicts a critical diffuseness of the interface at which a plane-front or any other form of diffuse interface will become unstable. A further confidence test for the model is offered in this article by comparing the predicted liquid diffusion coefficients to those obtained experimentally. A comparison of the experimentally determined solute diffusion constant in dilute binary Pb-Sn alloys with those predicted by the various solidification instability models (1953-2011) is additionally discussed. A good predictability is noted for the MEPR model when the interface diffuseness is small. In comparison, the more traditional interface break-down models have low predictiveness.

Evaluation of the Removal of Potassium Cyanide and its Toxicity in Green Algae (Chlorella vulgaris).

To evaluate the removal of potassium cyanide (KCN) and its toxicity in algae, an initial comprehensive analysis was performed with Chlorella vulgaris. The algae showed potential removal capability for KCN, with the maximal removal rate of 61%. Moreover, effects of KCN on growth, cellular morphology and antioxidant defense system of C. vulgaris were evaluated. Cell number and chlorophyll a content decreased in most cases, with the maximal inhibition rates of 48% and 99%, respectively. The 100 mg L- 1 KCN seriously damaged the algal cell membrane. Additionally, activity of superoxide dismutase (SOD) was promoted by KCN exposure among 0.1-50 mg L- 1 and inhibited by 100 mg L- 1 KCN, while the malondialdehyde (MDA) content gradually decreased in C. vulgaris with increasing exposure concentration compared to the control. The present study reveals that C. vulgaris is useful in bio-treatment of cyanide-contaminated aquatic ecosystem, except in high concentrations which would cause overwhelming effects.

Resistance to anticancer drugs permanently alters electrophoretic mobility of cancer cell lines.

Electrophoretic mobility is a physical phenomenon defining the mobility of charged particles in a solution under applied electric field. As charged biological systems, living cells including both prokaryotes and eukaryotes have been assessed in terms of electrophoretic mobility to decipher their electrochemical structure. Moreover, determination of electrophoretic mobility of living cancer cells have promoted the advance exploration of the nature of the cancer cells and separation of cancer cells from normal ones under applied electric field. However, electrophoretic mobility of drug-resistant cells has not yet been examined. In the present study, we determined the electrophoretic mobility of drug-resistant cancer cell lines for both suspension and adherent cells and compared with those of drug-sensitive counterparts. We showed that resistance to anticancer drugs alters the electrophoretic mobility in a permanent manner, even lasting without any exposure to anticancer agents for a long time period. We also studied the cellular morphologies of adherent cells where the cellular invaginations and protrusions were increased in drug-resistant adherent cells, which could be direct cause of altered surface charge and electrophoretic mobility as a result. These findings could be helpful in terms of understanding the electrophysiological and physicochemical background of drug resistance in cancer cells and developing systems to separate drug-sensitive cells from drug-resistant ones.

Knockdown of DNA methyltransferase 3a alters gene expression and inhibits function of embryonic cardiomyocytes.

We previously found that in utero caffeine exposure causes down-regulation of DNA methyltransferases (DNMTs) in embryonic heart and results in impaired cardiac function in adulthood. To assess the role of DNMTs in these events, we investigated the effects of reduced DNMT expression on embryonic cardiomyocytes. siRNAs were used to knock down individual DNMT expression in primary cultures of mouse embryonic cardiomyocytes. Immunofluorescence staining was conducted to evaluate cell morphology. A video-based imaging assay and multielectrode array were used to assess cardiomyocyte contractility and electrophysiology, respectively. RNA-Seq and multiplex bisulfite sequencing were performed to examine gene expression and promoter methylation, respectively. At 72 h after transfection, reduced DNMT3a expression, but not DNMT1 or -3b, disrupted sarcomere assembly and decreased beating frequency, contractile movement, amplitude of field action potential, and cytosolic calcium signaling of cardiomyocytes. RNA-Seq analysis revealed that the DNMT3a-deficient cells had deactivated gene networks involved in calcium, endothelin-1, renin-angiotensin, and cardiac ß-adrenergic receptor signaling, which were not inhibited by DNMT3b siRNA. Moreover, decreased methylation levels were found in the promoters of Myh7, Myh7b, Tnni3, and Tnnt2, consistent with the up-regulation of these genes by DNMT3a siRNA. These data show that DNMT3a plays an important role in regulating embryonic cardiomyocyte gene expression, morphology and function.-Fang, X., Poulsen, R. R., Wang-Hu, J., Shi, O., Calvo, N. S., Simmons, C. S., Rivkees, S. A., Wendler, C. C. Knockdown of DNA methyltransferase 3a alters gene expression and inhibits function of embryonic cardiomyocytes.

Histological and biological comparisons between complete and incomplete discoid lateral meniscus.

The discoid lateral meniscus (DLM) is an anatomically abnormal meniscus that covers a greater area of the tibial plateau than the normal meniscus. The DLM is classified into two types: complete (CDLM) and incomplete (ICDLM) types. In this study, we investigated the histological and cell biological characteristics of CDLM and ICDLM. The number of blood vessels, proteoglycan deposition, and collagen distribution were assessed using meniscal tissues. Collagen production was also investigated in CDLM and ICDLM cells. The intercondylar region of the CDLM had a higher number of blood vessels than the inner region of the ICDLM. Safranin O staining density and type II collagen deposition in ICDLM were higher than those in CDLM. Type II collagen-positive cells were higher in ICLDM than in CDLM. CDLM cells showed slender fibroblastic morphology, while ICDLM cells were triangular chondrocytic in shape. This study demonstrated that the intercondylar region of the CDLM showed similar properties to the outer region of the meniscus. The inner region of the ICDLM, on the other hand, differed from the intercondylar region of the CDLM. Our results suggest that the intercondylar region of the CDLM may have a high healing potential like the outer meniscus.

Chronic intermittent ethanol exposure and withdrawal leads to adaptations in nucleus accumbens core postsynaptic density proteome and dendritic spines.

Alcohol use disorder is a chronic relapsing brain disease characterized by the loss of ability to control alcohol (ethanol) intake despite knowledge of detrimental health or personal consequences. Clinical and pre-clinical models provide strong evidence for chronic ethanol-associated alterations in glutamatergic signaling and impaired synaptic plasticity in the nucleus accumbens (NAc). However, the neural mechanisms that contribute to aberrant glutamatergic signaling in ethanol-dependent individuals in this critical brain structure remain unknown. Using an unbiased proteomic approach, we investigated the effects of chronic intermittent ethanol (CIE) exposure on neuroadaptations in postsynaptic density (PSD)-enriched proteins in the NAc of ethanol-dependent mice. Compared with controls, CIE exposure significantly changed expression levels of 50 proteins in the PSD-enriched fraction. Systems biology and functional annotation analyses demonstrated that the dysregulated proteins are expressed at tetrapartite synapses and critically regulate cellular morphology. To confirm this latter finding, the density and morphology of dendritic spines were examined in the NAc core of ethanol-dependent mice. We found that CIE exposure and withdrawal differentially altered dendrite diameter and dendritic spine density and morphology. Through the use of quantitative proteomics and functional annotation, these series of experiments demonstrate that ethanol dependence produces neuroadaptations in proteins that modify dendritic spine morphology. In addition, these studies identified novel PSD-related proteins that contribute to the neurobiological mechanisms of ethanol dependence that drive maladaptive structural plasticity of NAc neurons.

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.

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.

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.

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.

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.