Develop a 3D Neurological Disease Model of Human Cortical Glutamatergic Neurons Using Micropillar-Based Scaffolds

Establishing an effective three-dimensional (3D) in vitro culture system to better model human neurological diseases is desirable, since the human brain is a 3D structure. Here, we demonstrated the development of a polydimethylsiloxane (PDMS) pillar-based 3D scaffold that mimicked the 3D microenvironment of the brain. We utilized this scaffold for the growth of human cortical glutamatergic neurons that were differentiated from human pluripotent stem cells. In comparison with the 2D culture, we demonstrated that the developed 3D culture promoted the maturation of human cortical glutamatergic neurons by showing significantly more MAP2 and less Ki67 expression. Based on this 3D culture system, we further developed an in vitro disease-like model of traumatic brain injury (TBI), which showed a robust increase of glutamate-release from the neurons, in response to mechanical impacts, recapitulating the critical pathology of TBI. The increased glutamate-release from our 3D culture model was attenuated by the treatment of neural protective drugs, memantine or nimodipine. The established 3D in vitro human neural culture system and TBI-like model may be used to facilitate mechanistic studies and drug screening for neurotrauma or other neurological diseases

Exploring the molecular landscape of osteosarcoma through PTTG family genes using a detailed multi-level methodology

BackgroundOsteosarcoma (OS) poses a significant clinical challenge, necessitating a comprehensive exploration of its molecular underpinnings.MethodsThis study explored the roles of PTTG family genes (PTTG1, PTTG2, and PTTG3P) in OS, employing a multifaceted approach encompassing molecular experiments, including OS cell lines culturing, RT-qPCR, bisulfite and Whole Exome Sequencing (WES) and in silico experiments, including The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets-based validation, overall survival, gene enrichment, functional assays, and molecular docking analyses.ResultsOur findings reveal a consistent up-regulation of PTTG genes in OS cell lines, supported by RT-qPCR experiments and corroborated across various publically available expression datasets databases. Importantly, ROC curve analyses highlight their potential as diagnostic markers. Moving beyond expression profiles, we unveil the epigenetic landscape by demonstrating significant hypomethylation of CpG islands associated with PTTG genes in OS. The negative correlation between methylation status and mRNA expression emphasizes the regulatory role of promoter methylation in PTTG gene expression. Contrary to expectations, genetic mutations in PTTG genes are rare in OS, with only benign mutations observed. Moreover, functional assays also confirmed the oncogenic roles of the PTTG gene in the development of OS. Lastly, we also revealed that Calcitriol is the most appropriate drug that can be utilized to treat OS in the context of PTTG genes.ConclusionThe identification of PTTG genes as potential diagnostic markers and their association with epigenetic alterations opens new avenues for understanding OS pathogenesis and developing targeted therapies. As we navigate the complex landscape of OS, this study contributes essential insights that may pave the way for improved diagnostic and therapeutic strategies in its management

SEC24A deficiency lowers plasma cholesterol through reduced PCSK9 secretion.

The secretory pathway of eukaryotic cells packages cargo proteins into COPII-coated vesicles for transport from the endoplasmic reticulum (ER) to the Golgi. We now report that complete genetic deficiency for the COPII component SEC24A is compatible with normal survival and development in the mouse, despite the fundamental role of SEC24 in COPII vesicle formation and cargo recruitment. However, these animals exhibit markedly reduced plasma cholesterol, with mutations in Apoe and Ldlr epistatic to Sec24a, suggesting a receptor-mediated lipoprotein clearance mechanism. Consistent with these data, hepatic LDLR levels are up-regulated in SEC24A-deficient cells as a consequence of specific dependence of PCSK9, a negative regulator of LDLR, on SEC24A for efficient exit from the ER. Our findings also identify partial overlap in cargo selectivity between SEC24A and SEC24B, suggesting a previously unappreciated heterogeneity in the recruitment of secretory proteins to the COPII vesicles that extends to soluble as well as trans-membrane cargoes. DOI:http://dx.doi.org/10.7554/eLife.00444.001

Structure Analysis of Aerobic Granule from a Sequencing Batch Reactor for Organic Matter and Ammonia Nitrogen Removal

Aerobic granules were cultivated in a sequencing batch reactor (SBR). COD and ammonia nitrogen removal rate were 94% and 99%, respectively. The diameter, settling velocity and SVI10 of granules ranged from 2 to 5 mm, 80 to 110 m/h and about 40 mL/g, respectively. Freezing microtome images, DO concentration profiles by microelectrode, distribution of bacteria and EPS by confocal laser scanning microscopy (CLSM) show that the aerobic granules have a three-layer structure. Each layer has different thickness, character, bacteria, and DO transfer rate. A hypothesis for granule structure is proposed: the first layer, the surface of the granule, is composed mostly of heterotrophic organisms for organic matter removal, with a thickness range from 150 to 350 μm; the second layer, mostly composed of autotrophic organisms for ammonia nitrogen removal, with a thickness range from 250 to 450 μm; the third layer, located in the core of the granule, has mostly an inorganic composition and contains pores and channels

Epicardial Adipose Tissue–Derived Leptin Promotes Myocardial Injury in Metabolic Syndrome Rats Through PKC/NADPH Oxidase/ROS Pathway

Background The epicardial adipose tissue (EAT) of metabolic syndrome (MetS) is abnormally accumulated with dysfunctional secretion of adipokines, closely relating to cardiac dysfunction. The current study was designed to identify the effects of EAT‐derived leptin on the myocardium of MetS rats and explore the potential molecular mechanisms. Methods and Results A MetS rat model was established in 8‐week‐old Wistar rats by a 12‐week high‐fat diet. MetS rats exhibited increased leptin secretion from EAT, cardiac hypertrophy, and diastolic dysfunction with preserved systolic function. The myocardium of MetS rats had abnormal structure, increased oxidative stress injury, and higher inflammatory factor levels, especially the subepicardial myocardium, which was correlated with the EAT‐derived leptin level but not the serum leptin. The EAT was separated from each group of rats to prepare EAT‐conditioned medium. H9C2 rat cardiomyoblasts were treated with EAT‐conditioned medium or leptin, plus various inhibitors. EAT‐derived leptin from MetS rats promoted mitochondrial oxidative stress and dysfunction, induced mitochondrial pathway apoptosis, and inhibited cell viability in H9C2 cardiomyoblasts via the protein kinase C/reduced nicotinamide adenine dinucleotide phosphate oxidase/reactive oxygen species (PKC/NADPH oxidase/ROS) pathway. EAT‐derived leptin from MetS rats stimulated inflammation in H9C2 cardiomyocytes by promoting activator protein 1 nuclear translocation via the PKC/NADPH oxidase/ROS pathway. Leptin promoted the interaction between p‐p47phox and gp91phox in H9C2 cardiomyocytes via protein kinase C, activating nicotinamide adenine dinucleotide phosphate oxidase, increasing reactive oxygen species generation, and inhibiting cell viability. Conclusions EAT‐derived leptin induces MetS‐related myocardial injury through the following 2 cooperative ways via PKC/NADPH oxidase/ROS pathway: (1) inducing mitochondrial pathway apoptosis by promoting mitochondrial oxidative stress and dysfunction; and (2) stimulating inflammation by promoting activator protein 1 nuclear translocation

Accelerating Aerobic Sludge Granulation by Adding Dry Sewage Sludge Micropowder in Sequencing Batch Reactors

Micropowder (20–250 µm) made from ground dry waste sludge from a municipal sewage treatment plant was added in a sequencing batch reactor (R2), which was fed by synthetic wastewater with acetate as carbon source. Compared with the traditional SBR (R1), aerobic sludge granulation time was shortened 15 days in R2. Furthermore, filamentous bacteria in bulking sludge were controlled to accelerate aerobic granulation and form large granules. Correspondingly, the SVI decreased from 225 mL/g to 37 mL/g. X-ray Fluorescence (XRF) analysis demonstrated that Al and Si from the micropowder were accumulated in granules. A mechanism hypotheses for the acceleration of aerobic granulation by adding dry sludge micropowder is proposed: added micropowder acts as nuclei to induce bacterial attachment; dissolved matters from the micropowder increase abruptly the organic load for starved sludge to control overgrown filamentous bacteria as a framework for aggregation; increased friction from the movement of micropowder forces the filaments which extend outwards to shrink for shaping granules

Differences in Ectopic Pregnancy Rates between Fresh and Frozen Embryo Transfer after In Vitro Fertilization: A Large Retrospective Study

Ectopic pregnancy (EP) is increasingly found in women treated with in vitro fertilization and embryo transfer (IVF–ET). With the development of the freeze-all policy in reproductive medicine, it is controversial whether frozen embryo transfer (FET) could reduce the rate of EP. In this single-center, large-sample retrospective study, we analyzed 16,048 human chorionic gonadotrophin (hCG)-positive patients who underwent fresh embryo transfer (ET) or FET cycles between January 2013 and March 2022. Throughout the study, the total EP rate was 2.09% (336/16,048), 2.16% (82/3803) in the ET group, and 2.07% (254/12,245) in the FET group. After adjustment for age, infertility causes, and other confounding factors, logistic regression results showed no statistical difference in EP rates between FET and ET groups (odds ratio (OR) 0.93 (0.71–1.22), p > 0.05). However, among the 3808 patients who underwent fresh ET cycles, the OR for EP was significantly lower in the long agonist protocol group than in the gonadotropin-releasing hormone antagonist (GnRH-ant) protocol group (OR 0.45 (0.22–0.93), p < 0.05). Through a large retrospective study, we demonstrated a slightly lower EP rate in FET cycles than in fresh ET cycles, but there was no significant difference. The long agonist protocol in ET cycles had a significantly lower risk of EP than the GnRH-ant protocol

Characteristics, Cryoprotection Evaluation and In Vitro Release of BSA-Loaded Chitosan Nanoparticles

Chitosan nanoparticles (CS-NPs) are under increasing investigation for the delivery of therapeutic proteins, such as vaccines, interferons, and biologics. A large number of studies have been taken on the characteristics of CS-NPs, and very few of these studies have focused on the microstructure of protein-loaded NPs. In this study, we prepared the CS-NPs by an ionic gelation method, and bovine serum albumin (BSA) was used as a model protein. Dynamic high pressure microfluidization (DHPM) was utilized to post-treat the nanoparticles so as to improve the uniformity, repeatability and controllability. The BSA-loaded NPs were then characterized for particle size, Zeta potential, morphology, encapsulation efficiency (EE), loading capacity (LC), and subsequent release kinetics. To improve the long-term stability of NPs, trehalose, glucose, sucrose, and mannitol were selected respectively to investigate the performance as a cryoprotectant. Furthermore, trehalose was used to obtain re-dispersible lyophilized NPs that can significantly reduce the dosage of cryoprotectants. Multiple spectroscopic techniques were used to characterize BSA-loaded NPs, in order to explain the release process of the NPs in vitro. The experimental results indicated that CS and Tripolyphosphate pentasodium (TPP) spontaneously formed the basic skeleton of the NPs through electrostatic interactions. BSA was incorporated in the basic skeleton, adsorbed on the surface of the NPs (some of which were inlaid on the NPs), without any change in structure and function. The release profiles of the NPs showed high consistency with the multispectral results