A facile single-cell patterning strategy based on harbor-like microwell microfluidics.

Single-cell analysis is an effective method for conducting comprehensive heterogeneity studies ranging from cell phenotype to gene expression. The ability to arrange different cells in a predetermined pattern at single-cell resolution has a wide range of applications in cell-based analysis and plays an important role in facilitating interdisciplinary research by researchers in various fields. Most existing microfluidic microwell chips is a simple and straightforward method, which typically use small-sized microwells to accommodate single cells. However, this method imposes certain limitations on cells of various sizes, and the single-cell capture efficiency is relatively low without the assistance of external forces. Moreover, the microwells limit the spatiotemporal resolution of reagent replacement, as well as cell-to-cell communication. In this study, we propose a new strategy to prepare a single-cell array on a planar microchannel based on microfluidic flip microwells chip platform with large apertures (50 µm), shallow channels (50 µm), and deep microwells (50 µm). The combination of three configuration characteristics contributes to multi-cell trapping and a single-cell array within microwells, while the subsequent chip flipping accomplishes the transfer of the single-cell array to the opposite planar microchannel for cells adherence and growth. Further assisted by protein coating of bovine serum albumin and fibronectin on different layers, the single-cell capture efficiency in microwells is achieved at 92.1% ± 1%, while ultimately 85% ± 3.4% on planar microchannel. To verify the microfluidic flip microwells chip platform, the real-time and heterogeneous study of calcium release and apoptosis behaviours of single cells is carried out. To our knowledge, this is the first time that high-efficiency single-cell acquisition has been accomplished using a circular-well chip design that combines shallow channel, large aperture and deep microwell together. The chip is effective in avoiding the shearing force of high flow rates on cells, and the large apertures better allows cells to sedimentation. Therefore, this strategy owns the advantages of easy preparation and user-friendliness, which is especially valuable for researchers from different fields.

Integrated single-cell and spatial transcriptomic analysis reveals YBX1 drives immune regulation in GBM progression.

The RNA modification 5-methylcytosine (m5C) is widespread across various RNA types, significantly impacting RNA stability and translational efficiency. Accumulating evidence highlights its significant role within the tumorigenesis and progression of multiple malignancies. Nevertheless, the specific process through m5C is implicated in Glioblastoma (GBM) remains unclear. We conducted acomprehensive analysis of m5C expression distribution in single-cell GBM data. Our findings revealed elevated m5C scores in GBM single-cell data compared to the normal group. Additionally, multiple tumors exhibited significantly higher m5C scores than the normal group. Moreover, there was a positive correlation observed between the m5C score and inflammation score. m5C regulatory factor YBX1 exhibited a heightened expression in GBM, correlating closely with metastatic tendencies and an unfavorable prognosis across various cancer types. YBX1 has different biological functions in myeloid cells 1 and myeloid cells 2. YBX1 may act as immunosuppressive regulator by inhibiting the NF-κB pathway and inflammatory response in myeloid cells 1. YBX1 is essential for immune infiltrates, which creates a highly immunosuppressive tumor microenvironment by TNF signaling pathway in myeloid cells 2. YBX1+ neoplastic cells promote cell proliferation by NF-κB pathway. APOE mediates the interaction of YBX1+ myeloid cells and neoplastic cells by NF-κB.

Insight into deep learning for glioma IDH medical image analysis: A systematic review.

BACKGROUND: Deep learning techniques explain the enormous potential of medical image analysis, particularly in digital pathology. Concurrently, molecular markers have gained increasing significance over the past decade in the context of glioma patients, providing novel insights into diagnosis and more personalized treatment options. Deep learning combined with imaging and molecular analysis enables more accurate prognostication of patients, more accurate treatment plan proposals, and accurate biomarker (IDH) prediction for gliomas. This systematic study examines the development of deep learning techniques for IDH prediction using histopathology images, spanning the period from 2019 to 2023. METHOD: The study adhered to the PRISMA reporting requirements, and databases including PubMed, Google Scholar, Google Search, and preprint repositories (such as arXiv) were systematically queried for pertinent literature spanning the period from 2019 to the 30th of 2023. Search phrases related to deep learning, digital pathology, glioma, and IDH were collaboratively utilized. RESULTS: Fifteen papers meeting the inclusion criteria were included in the analysis. These criteria specifically encompassed studies utilizing deep learning for the analysis of hematoxylin and eosin images to determine the IDH status in patients with gliomas. CONCLUSIONS: When predicting the status of IDH, the classifier built on digital pathological images demonstrates exceptional performance. The study's predictive effectiveness is enhanced with the utilization of the appropriate deep learning model. However, external verification is necessary to showcase their resilience and universality. Larger sample sizes and multicenter samples are necessary for more comprehensive research to evaluate performance and confirm clinical advantages.

Predictive Panel for Immunotherapy in Low-Grade Glioma.

BACKGROUND: The main treatment of low-grade glioma (LGG) is still surgical resection followed by radiotherapy and/or chemotherapy, which has certain limitations, including side effects and drug resistance. Immunotherapy is a promising treatment for LGG, but it is generally hindered by the tumor microenvironment with the limited expression of tumor antigens. METHODS: We integrated RNA sequencing data sets and clinical information and conducted consistent cluster analysis to explore the most suitable patients for immune checkpoint therapy. Gene set enrichment analysis, UMAP analysis, mutation correlation analysis, TIMER analysis, and TIDE analysis were used to identify the immune characteristics of 3 immune subtypes and the feasibility of 5 antigens as immune checkpoint markers. RESULTS: We analyzed the isolation and mutation of homologous recombination repair genes (HRR) of the 3 immune subtypes, and the HRR genes of the 3 subtypes were obviously segregated. Among them, the IS2 subtype has a large number of HRR gene mutations, which increases the immunogenicity of tumors-this is consistent with the results of tumor mutation load analysis of 3 immune subtypes. Then we evaluated the immune cell infiltration of immune subtypes and found that IS2 and IS3 subtypes were rich in immune cells. It is worth noting that there are many Treg cells and NK cells in the IS1 subtype. In addition, when analyzing the immune checkpoint gene expression of the 3 subtypes, we found that they were upregulated most in IS2 subtypes compared with other subtypes. Then when we further confirmed the role of immune-related genes in LGG; through TIDE analysis and TISIDB analysis, we obtained 5 markers that can predict the efficacy of ICB in patients with LGG. In addition, we confirmed that they were associated with poor prognosis through survival analysis. CONCLUSIONS: We obtained 3 reliable immune subtypes, and patients with the IS2 subtype are suitable for immunotherapy, in which NAMPT, SLC11A1, TNC, VIM, and SPP1 are predictive panel markers for ICB in the LGG group. Our findings provide a rationale for immunotherapy selection and prediction of patient prognosis in LGG patients.

Protocol for high-throughput single-cell patterning using a reusable ultrathin metal microstencil.

Exploiting convenient strategies for single-cell preparation while maintaining a high throughput remains challenging. This protocol describes a simple workflow for high-throughput single-cell patterning using a reusable ultrathin metal microstencil (UTmS). We describe UTmS-chip design, fabrication, and quality characterization. We then detail the preparation of flat substrates and chip assembly for single-cell patterning, followed by culturing of cells on a chip. Finally, we describe the evaluation of single-cell patterning and the downstream applications for studying single-cell calcium release and apoptosis. For complete details on the use and execution of this protocol, please refer to Song et al. (2021).1.

Plant-specific histone deacetylases associate with ARGONAUTE4 to promote heterochromatin stabilization and plant heat tolerance.

High temperature causes devasting effects on many aspects of plant cells and thus enhancing plant heat tolerance is critical for crop production. Emerging studies have revealed the important roles of chromatin modifications in heat stress responses. However, how chromatin is regulated during heat stress remains unclear. We show that heat stress results in heterochromatin disruption coupled with histone hyperacetylation and DNA hypomethylation. Two plant-specific histone deacetylases HD2B and HD2C could promote DNA methylation and relieve the heat-induced heterochromatin decondensation. We noted that most DNA methylation regulated by HD2B and HD2C is lost upon heat stress. HD2B- and HD2C-regulated histone acetylation and DNA methylation are dispensable for heterochromatin maintenance under normal conditions, but critical for heterochromatin stabilization under heat stress. We further showed that HD2B and HD2C promoted DNA methylation through associating with ARGONAUTE4 in nucleoli and Cajal bodies, and facilitating its nuclear accumulation. Thus, HD2B and HD2C act both canonically and noncanonically to stabilize heterochromatin under heat stress. This study not only reveals a novel plant-specific crosstalk between histone deacetylases and key factor of DNA methylation pathway, but also uncovers their new roles in chromatic regulation of plant heat tolerance.

New insight on microglia activation in neurodegenerative diseases and therapeutics.

Microglia are immune cells within the central nervous system (CNS) closely linked to brain health and neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. In response to changes in the surrounding environment, microglia activate and change their state and function. Several factors, example for circadian rhythm disruption and the development of neurodegenerative diseases, influence microglia activation. In this review, we explore microglia's function and the associated neural mechanisms. We elucidate that circadian rhythms are essential factors influencing microglia activation and function. Circadian rhythm disruption affects microglia activation and, consequently, neurodegenerative diseases. In addition, we found that abnormal microglia activation is a common feature of neurodegenerative diseases and an essential factor of disease development. Here we highlight the importance of microglia activation in neurodegenerative diseases. Targeting microglia for neurodegenerative disease treatment is a promising direction. We introduce the progress of methods targeting microglia for the treatment of neurodegenerative diseases and summarize the progress of drugs developed with microglia as targets, hoping to provide new ideas for treating neurodegenerative diseases.

Bromodomain-containing factor GTE4 regulates Arabidopsis immune response.

BACKGROUND: Plants are continuously challenged with biotic stress from environmental pathogens, and precise regulation of defense responses is critical for plant survival. Defense systems require considerable amounts of energy and resources, impairing plant growth, and plant hormones controlling transcriptional regulation play essential roles in establishing the appropriate balance between defense response to pathogens and growth. Chromatin regulators modulating gene transcription are broadly involved in regulating stress-responsive genes. However, which chromatin factors are involved in coordinating hormone signaling and immune responses in plants, and their functional mechanisms, remains unclear. Here, we identified a role of bromodomain-containing protein GTE4 in negatively regulating defense responses in Arabidopsis thaliana. RESULTS: GTE4 mainly functions as activator of gene expression upon infection with Pseudomonas syringe. Genome-wide profiling of GTE4 occupancy shows that GTE4 tends to bind to active genes, including ribosome biogenesis related genes and maintains their high expression levels during pathogen infection. However, GTE4 is also able to repress gene expression. GTE4 binds to and represses jasmonate biosynthesis gene OPR3. Disruption of GTE4 results in overaccumulation of jasmonic acid (JA) and enhanced JA-responsive gene expression. Unexpectedly, over-accumulated JA content in gte4 mutant is coupled with downregulation of JA-mediated immune defense genes and upregulation of salicylic acid (SA)-mediated immune defense genes, and enhanced resistance to Pseudomonas, likely through a noncanonical pathway. CONCLUSIONS: Overall, we identified a new role of the chromatin factor GTE4 as negative regulator of plant immune response through inhibition of JA biosynthesis, which in turn noncanonically activates the defense system against Pseudomonas. These findings provide new knowledge of chromatic regulation of plant hormone signaling during defense responses.

[Development and Application of Medical Device Recall Event Tracking System].

A medical device recall event tracking system was designed, which can enable the users to obtain the recall, early warning and other information related to medical devices in time. The tracking system can timely obtain and release the recall information of medical devices, effectively improve the quality control of hospital medical devices, reduce the use risk of medical devices, and ensure the life safety of patients.

Membrane-cytoplasm translocation of annexin A4 is involved in the metastasis of colorectal carcinoma.

Annexin A4 (ANXA4) is a Ca2+- and phospholipid-binding protein that belongs to the annexin family, which is involved in the development of multiple tumour types via NF-κB signalling. In this study, we verified the high expression and membrane-cytoplasm translocation of ANXA4 in colorectal carcinoma (CRC). Calcium/calmodulin-dependent protein kinase II gamma (CAMK2γ) was found to be important for high ANXA4 expression in CRC, whereas carbonic anhydrase (CA1) promoted ANXA4 aggregation in the cell membrane. An increased Ca2+ concentration attenuated the small ubiquitin-like modifier (SUMO) modification of cytoplasmic ANXA4 and ANXA4 stabilization, and relatively high expression of ANXA4 promoted CRC tumorigenesis and epithelial-mesenchymal transition (EMT).

A reusable single-cell patterning strategy based on an ultrathin metal microstencil.

The ability to arrange distinct cells in specific, predefined patterns at single-cell resolution can have broad applications in cell-based assays and play an important role in facilitating interdisciplinary research for researchers in various fields. However, most existing methods for single-cell patterning are based on the complicated lithography-based microfabrication process, and require professional skills. Thus, exploiting convenient and universal strategies of single-cell preparation while maintaining high-throughput single-cell patterning remains a challenge. Here, we describe a simple approach for rapid and high-efficiency single-cell patterning using an ultrathin metal microstencil (UTmS) and common tools available in any laboratory. In this work, ultrathin steel microstencil plates with only 5 µm thickness could be fabricated with laser drilling and achieve single-cell prototyping on an arbitrary planar substrate under gravity-induced natural sedimentation without requiring additional fixation, reaction pools, and centrifugation procedures. In this method, the UTmS is reusable and single-cell occupancy could easily reach approximately 88% within 30 min on fibronectin-modified substrates under gravity-induced natural sedimentation, and no significant effect on cell viability was observed. To verify this method, the real-time and heterogeneous study of calcium release and apoptosis behaviors of single cells was carried out based on this new strategy. To our knowledge, it is the first time that a UTmS with 5 µm thickness is directly applied to facilitate the micropatterning of high-resolution single cells, which is valuable for researchers in different fields owing to its user-friendly operation.

Counting Bites and Recognizing Consumed Food from Videos for Passive Dietary Monitoring.

Assessing dietary intake in epidemiological studies are predominantly based on self-reports, which are subjective, inefficient, and also prone to error. Technological approaches are therefore emerging to provide objective dietary assessments. Using only egocentric dietary intake videos, this work aims to provide accurate estimation on individual dietary intake through recognizing consumed food items and counting the number of bites taken. This is different from previous studies that rely on inertial sensing to count bites, and also previous studies that only recognize visible food items but not consumed ones. As a subject may not consume all food items visible in a meal, recognizing those consumed food items is more valuable. A new dataset that has 1,022 dietary intake video clips was constructed to validate our concept of bite counting and consumed food item recognition from egocentric videos. 12 subjects participated and 52 meals were captured. A total of 66 unique food items, including food ingredients and drinks, were labelled in the dataset along with a total of 2,039 labelled bites. Deep neural networks were used to perform bite counting and food item recognition in an end-to-end manner. Experiments have shown that counting bites directly from video clips can reach 74.15% top-1 accuracy (classifying between 0-4 bites in 20-second clips), and a MSE value of 0.312 (when using regression). Our experiments on video-based food recognition also show that recognizing consumed food items is indeed harder than recognizing visible ones, with a drop of 25% in F1 score.

Photocatalytic kinetics and cyclic stability of photocatalysts Fe-complex/TiO2 in the synergistic degradation of phenolic pollutants and reduction of Cr(VI).

In this paper, the kinetic characteristics and cycle stability of Fe-complex/TiO2 in the process of degradation of phenolic pollutants and reduction of heavy metal Cr(VI) were studied systematically. First, the structural characteristics and photocatalytic activities of Fe(III)-(8-hydroxyquinoline-5-carboxylic acid)-TiO2 (Fe-HQC-TiO2) nanoparticle to degrade phenolic pollutants and reduce Cr(VI) simultaneously had been investigated. Compared with the single degradation, the efficiency of synergistic degradation/reduction had been improved and the degradation/reduction rate had been obviously accelerated. In particular, the cyclic stability of Fe-HQC-TiO2 photocatalyst decreased obviously when it was used to reduce Cr(VI) alone, but it could still keep above 90% after three cycles when it was used for reduction of Cr(VI) and degradation of phenol synergistically. Second, to Fe-HQS/TiO2 nanoparticle or Fe-HQS/TiO2 nanotube (HQS (8-hydroxyquinoline-5-sulfonic acid)), the synergistic degradation/reduction (2,4-dichlorophenol/Cr(VI)) efficiencies were always greater than those of a single degradation/reduction and the time was greatly reduced. All the results indicated that there were interactions between Cr(VI) and phenol or 2,4-dichlorophenol in the photocatalytic process. The possible mechanism of synergistic accelerated degradation of phenolic compounds and reduction of Cr(VI) was proposed by analyzing and testing the surface characteristics of photocatalyst and the properties of photocatalytic system during the synergistic degradation/reduction.

Corrigendum: Coagulation Factor X Regulated by CASC2c Recruited Macrophages and Induced M2 Polarization in Glioblastoma Multiforme.

[This corrects the article DOI: 10.3389/fimmu.2018.01557.].

Image-Based Food Classification and Volume Estimation for Dietary Assessment: A Review.

A daily dietary assessment method named 24-hour dietary recall has commonly been used in nutritional epidemiology studies to capture detailed information of the food eaten by the participants to help understand their dietary behaviour. However, in this self-reporting technique, the food types and the portion size reported highly depends on users' subjective judgement which may lead to a biased and inaccurate dietary analysis result. As a result, a variety of visual-based dietary assessment approaches have been proposed recently. While these methods show promises in tackling issues in nutritional epidemiology studies, several challenges and forthcoming opportunities, as detailed in this study, still exist. This study provides an overview of computing algorithms, mathematical models and methodologies used in the field of image-based dietary assessment. It also provides a comprehensive comparison of the state of the art approaches in food recognition and volume/weight estimation in terms of their processing speed, model accuracy, efficiency and constraints. It will be followed by a discussion on deep learning method and its efficacy in dietary assessment. After a comprehensive exploration, we found that integrated dietary assessment systems combining with different approaches could be the potential solution to tackling the challenges in accurate dietary intake assessment.

LRRC4 Suppresses E-Cadherin-Dependent Collective Cell Invasion and Metastasis in Epithelial Ovarian Cancer.

Epithelial ovarian cancer (EOC) is the most malignant gynecological carcinoma and is of a high incidence of death due to detection at late stages when metastasis already occurs. However, the mechanism underlying metastasis of EOC remains unclear. Analysis of the open database and experiments with immunochemistry showed that LRRC4 is lowly expressed in high-grade serous ovarian cancer (HGSC) cells and during EOC metastasis. The 3D cell culture system and the orthotopic ovarian xenograft model infected with LRRC4-containing adeno-associated virus serotype 9 (AAV9) were used to confirm collective invasion and metastasis of cells in vitro and in vivo. Phos-tag SDS-PAGE was used to detect the phosphorylation of LRRC4 and PIK3R1. A number of experiments with methods such as co-immunoprecipitation and immunoblotting were performed to explore the mechanism for the actions of LRRC4 and PIK3R1 in EOC metastasis. An inverse correlation between LRRC4 and E-cadherin expression was detected in the regions of invasion in primary EOC tissues and metastatic ascites. LRRC4 binds to the cSH2 domain of PIK3R1 and inhibits the activity of PIK3R1, without disrupting the physical interactions between PIK3R1 and PIK3CA. LRRC4 inhibits EOC metastasis by targeting E-cadherin-dependent collective cell invasion and does so by inhibiting the PIK3R1-mediated AKT/GSK3ß/ß-catenin signaling pathway. LRRC4 functions as a tumor suppressor gene to inhibit EOC collective invasion and metastasis in vitro and in vivo and does so by directly binding to the cSH2 domain of PIK3R1 to exert its regulatory function. Our findings provide a potential novel approach for metastasis prognosis and a new strategy for the treatment of EOC.

Electrochemiluminescence-Microscopy for microRNA Imaging in Single Cancer Cell Combined with Chemotherapy-Photothermal Therapy.

In this work, a new technology using ECL as a microscopy to parallel image miRNA-21 in single cancer cell was built. Phorbol-12-myristate-13-acetate (PMA) loaded gold nanocages (Au NCs) was closed with DNA gate which could be recognized and opened by miRNA-21 in HeLa cell. PMA was then released and further induced HeLa cells to produce reactive oxygen species (ROS; including O2-•, •OH and H2O2 etc.). With H2O2 as coreactant and luminol as ECL active material, ECL imaging of intracellular miRNA-21 in single HeLa cell was obtained by EMCCD. Moreover, ROS therapy and photothermal therapy (PPT) of Au NCs@PMA probe were also motivated by in situ miRNA-21 marker instead of the external source. The combined therapy leads to dramatically enhanced ability for cancer cell killing. Au NCs@PMA probe alone could not only achieve a high sensitivity and high resolution ECL-microscopy for imaging of intracellular miRNA-21 for the first time, but also realize the integrated diagnosis like ROS induced tumor damage and photothermal-induced intelligent therapy. This multifunctional platform is believed to be capable of playing an important role in future oncotherapy by the synergistic effects between chemotherapy and photothermal therapy.