Perivascular neurons instruct 3D vascular lattice formation via neurovascular contact.

The vasculature of the central nervous system is a 3D lattice composed of laminar vascular beds interconnected by penetrating vessels. The mechanisms controlling 3D lattice network formation remain largely unknown. Combining viral labeling, genetic marking, and single-cell profiling in the mouse retina, we discovered a perivascular neuronal subset, annotated as Fam19a4/Nts-positive retinal ganglion cells (Fam19a4/Nts-RGCs), directly contacting the vasculature with perisomatic endfeet. Developmental ablation of Fam19a4/Nts-RGCs led to disoriented growth of penetrating vessels near the ganglion cell layer (GCL), leading to a disorganized 3D vascular lattice. We identified enriched PIEZO2 expression in Fam19a4/Nts-RGCs. Piezo2 loss from all retinal neurons or Fam19a4/Nts-RGCs abolished the direct neurovascular contacts and phenocopied the Fam19a4/Nts-RGC ablation deficits. The defective vascular structure led to reduced capillary perfusion and sensitized the retina to ischemic insults. Furthermore, we uncovered a Piezo2-dependent perivascular granule cell subset for cerebellar vascular patterning, indicating neuronal Piezo2-dependent 3D vascular patterning in the brain.

LCDR regulates the integrity of lysosomal membrane by hnRNP K-stabilized LAPTM5 transcript and promotes cell survival.

Lysosome plays important roles in cellular homeostasis, and its dysregulation contributes to tumor growth and survival. However, the understanding of regulation and the underlying mechanism of lysosome in cancer survival is incomplete. Here, we reveal a role for a histone acetylation-regulated long noncoding RNA termed lysosome cell death regulator (LCDR) in lung cancer cell survival, in which its knockdown promotes apoptosis. Mechanistically, LCDR binds to heterogenous nuclear ribonucleoprotein K (hnRNP K) to regulate the stability of the lysosomal-associated protein transmembrane 5 (LAPTM5) transcript that maintains the integrity of the lysosomal membrane. Knockdown of LCDR, hnRNP K, or LAPTM5 promotes lysosomal membrane permeabilization and lysosomal cell death, thus consequently resulting in apoptosis. LAPTM5 overexpression or cathepsin B inhibitor partially restores the effects of this axis on lysosomal cell death in vitro and in vivo. Similarly, targeting LCDR significantly decreased tumor growth of patient-derived xenografts of lung adenocarcinoma (LUAD) and had significant cell death using nanoparticles (NPs)-mediated systematic short interfering RNA delivery. Moreover, LCDR/hnRNP K/LAPTM5 are up-regulated in LUAD tissues, and coexpression of this axis shows the increased diagnostic value for LUAD. Collectively, we identified a long noncoding RNA that regulates lysosome function at the posttranscriptional level. These findings shed light on LCDR/hnRNP K/LAPTM5 as potential therapeutic targets, and targeting lysosome is a promising strategy in cancer treatment.

The Efficacy of Recombinant Human Type III Collagen in the Treatment of Atrophic Scars and Its Impact on p38 MAPK Signaling Pathway Proteins.

Objective: To investigate the effects of recombinant human type III collagen on atrophic scars and its impact on the p38 mitogen-activated protein kinase (p38MAPK) signaling pathway. Methods: A total of 94 patients with atrophic scars admitted to our hospital from March 2020 to October 2022 were selected as subjects and evenly divided into a control group and an observation group. The control group (n = 47) received carbon dioxide fractional laser treatment, while the observation group (n = 47) was treated with recombinant human type III collagen dressings in addition to the laser treatment. Clinical efficacy, scar conditions, skin physiological parameters, serum levels of p38MAPK pathway-related proteins, and inflammatory markers were compared between the two groups. Results: The overall effective rate in the observation group was 95.74%, significantly higher than 74.47% in the control group (P < .05). Before treatment, there was no significant difference in Vancouver Scar Scale (VSS) scores, stratum corneum hydration, and transepidermal water loss between the two groups (P > .05). After treatment, the VSS score in the observation group was significantly lower than in the control group (P < .05). Similarly, prior to treatment, there were no significant differences in serum levels of mitogen-activated protein kinase 1 (MEK1), mitogen-activated protein kinase 2 (MEK2), extracellular signal-regulated kinase 1 (ERK1), and extracellular signal-regulated kinase 2 (ERK2), interleukin-10 (IL-10) and tumor necrosis factor-alpha (TNF-α) between the two groups (P > .05). After treatment, levels of MEK1, MEK2, ERK1, ERK2, IL-10, and TNF-α in the observation group were significantly lower than those in the control group (P < .05). Conclusion: Recombinant human type III collagen significantly improves the treatment of atrophic scars, effectively ameliorating scar conditions and skin physiology. It also regulates the p38MAPK signaling pathway and reduces inflammation.

Cudraflavone B induces human glioblastoma cells apoptosis via ER stress-induced autophagy.

BACKGROUND: Glioblastoma (GBM) is the most common malignant intracranial tumor with a low survival rate. However, only few drugs responsible for GBM therpies, hence new drug development for it is highly required. The natural product Cudraflavone B (CUB) has been reported to potentially kill a variety of tumor cells. Currently, its anit-cancer effect on GBM still remains unknown. Herein, we investigated whether CUB could affect the proliferation and apoptosis of GBM cells to show anti-GBM potential. RESULTS: CUB selectively inhibited cell viability and induced cell apoptosis by activating the endoplasmic reticulum stress (ER stress) related pathway, as well as harnessing the autophagy-related PI3K/mTOR/LC3B signaling pathway. Typical morphological changes of autophagy were also observed in CUB treated cells by microscope and scanning electron microscope (SEM) examination. 4-Phenylbutyric acid (4-PBA), an ER stress inhibitor, restored the CUB-caused alteration in signaling pathway and morphological change. CONCLUSIONS: Our finding suggests that CUB impaired cell growth and induced cell apoptosis of glioblastoma through ER stress and autophagy-related signaling pathways, and it might be an attractive drug for treatment of GBM.

The Effect of Bergenin on Isonicotinic Acid Hydrazide and Rifampicin-Induced Liver Injury Revealed by RNA Sequencing.

Bergenin (BER), a natural component of polyphenols, has a variety of pharmacological activities, especially in improving drug metabolism, reducing cholestasis, anti-oxidative stress and inhibiting inflammatory responses. The aim of this study was to investigate the effects of BER on liver injury induced by isonicotinic acid hydrazide (INH) and rifampicin (RIF) in mice. The mice model of liver injury was established with INH (100 mg/kg)+RIF (100 mg/kg), and then different doses of BER were used to intervene. The pathological morphology and biochemical indicators of mice were detected. Meanwhile, RNA sequencing was performed to screen the differentially expressed genes and signaling pathways. Finally, critical differentially expressed genes were verified by qRT-PCR and Western blot. RNA sequencing results showed that 707 genes were significantly changed in the INH+RIF group compared with the Control group, and 496 genes were significantly changed after the BER intervention. These differentially expressed genes were mainly enriched in the drug metabolism, bile acid metabolism, Nrf2 pathway and TLR4 pathway. The validation results of qRT-PCR and Western blot were consistent with the RNA sequencing. Therefore, BER alleviated INH+RIF-induced liver injury in mice. The mechanism of BER improving INH+RIF-induced liver injury was related to regulating drug metabolism enzymes, bile acid metabolism, Nrf2 pathway and TLR4 pathway.

Levels of Perfluoroalkyl Acids (PFAAs) in Human Serum, Hair and Nails in Guangdong Province, China: Implications for Exploring the Ideal Bio-Indicator.

The widespread human exposure to perfluoroalkyl acids (PFAAs) has led to increasing public concern. In this study, we present a comprehensive measurement of total fluorine (TF), extractable organic fluorine (EOF), identified organic fluorine (IOF, total concentration of identified PFAAs quantified as fluorine) and 11 target PFAAs in human serum (n = 60), hair (n = 49) and nails (n = 39) collected from non-occupation exposed volunteers in 10 cities of Guangdong Province, China. The results indicated that EOF was the major form of fluorine in serum, accounting for 70-80% of TF. The levels of IOF contributed less than 10% of EOF. Perfluorooctane sulfonic acid (PFOS) was found to be the dominant PFAA with mean concentration of 23 ng·mL-1 in serum, 35 ng·g-1 in hair and 33 ng·g-1 in nail, respectively. Short-chain PFAAs (C ≤ 10) were the predominant PFAAs in three matrices. Levels of PFOS, perfluorohexane sulfonic acid (PFHxS), perfluoroundecanoic acid (PFUdA) and perfluorotridecanoic acid (PFTrDA) in males are significantly higher than those in females (p < 0.01). Significant positive correlations were observed between nail and serum for PFOS (p < 0.01), perfluorooctanoic acid (PFOA) (p < 0.05) and PFHxS (p < 0.01), suggesting that human nails, a noninvasive sample, are a promising bio-indicator for PFAA risk assessment.

Crybb2 associates with Tmsb4X and is crucial for dendrite morphogenesis.

Dendrite morphogenesis is a complex but well-orchestrated process. Various studies reported the involvement of alteration in dendrite morphology in different brain disorders, including neuropsychiatric disorders. Initially, ßB2-crystallin (gene symbol: Crybb2/CRYBB2) has been described as a structural protein of the ocular lens. Mutations of the corresponding gene, Crybb2, lead to cataract. Recent studies in mice suggested that mutations in Crybb2 cause alterations in hippocampal morphology and function, albeit its function in hippocampal neuron development remained elusive. In the current study, we found that Crybb2 contributes to dendritogenesis in vitro and in vivo. Furthermore, screening of previous data on differential expression-arrays, we found Tmsb4X up-regulated in Crybb2 mutants mouse brain. Additionally, Tmsb4X was co-expressed with Crybb2 at actin-enriched cell ruffles. Over-expression of Tmsb4X in cultured hippocampal neurons inhibited dendritogenesis, which phenocopied Crybb2 knock-down. The current study uncovers a new function of Crybb2 in brain development, especially in dendritogenesis, and the possible interplay partner Tmsb4X involved in this process.

A review of the pharmacological action and mechanism of natural plant polysaccharides in depression.

Depression is a prevalent mental disorder. However, clinical treatment options primarily based on chemical drugs have demonstrated varying degrees of adverse reactions and drug resistance, including somnolence, nausea, and cognitive impairment. Therefore, the development of novel antidepressant medications that effectively reduce suffering and side effects has become a prominent area of research. Polysaccharides are bioactive compounds extracted from natural plants that possess diverse pharmacological activities and medicinal values. It has been discovered that polysaccharides can effectively mitigate depression symptoms. This paper provides an overview of the pharmacological action and mechanisms, intervention approaches, and experimental models regarding the antidepressant effects of polysaccharides derived from various natural sources. Additionally, we summarize the roles and potential mechanisms through which these polysaccharides prevent depression by regulating neurotransmitters, HPA axis, neurotrophic factors, neuroinflammation, oxidative stress, tryptophan metabolism, and gut microbiota. Natural plant polysaccharides hold promise as adjunctive antidepressants for prevention, reduction, and treatment of depression by exerting their therapeutic effects through multiple pathways and targets. Therefore, this review aims to provide scientific evidence for developing polysaccharide resources as effective antidepressant drugs.

Two-stage error detection to improve electron microscopy image mosaicking.

Large-scale electron microscopy (EM) has enabled the reconstruction of brain connectomes at the synaptic level by serially scanning over massive areas of sample sections. The acquired big EM data sets raise the great challenge of image mosaicking at high accuracy. Currently, it simply follows the conventional algorithms designed for natural images, which are usually composed of only a few tiles, using a single type of keypoint feature that would sacrifice speed for stronger performance. Even so, in the process of stitching hundreds of thousands of tiles for large EM data, errors are still inevitable and diverse. Moreover, there has not yet been an appropriate metric to quantitatively evaluate the stitching of biomedical EM images. Here we propose a two-stage error detection method to improve the EM image mosaicking. It firstly uses point-based error detection in combination with a hybrid feature framework to expedite the stitching computation while maintaining high accuracy. Following is the second detection of unresolved errors with a newly designed metric of EM stitched image quality assessment (EMSIQA). The novel detection-based mosaicking pipeline is tested on large EM data sets and proven to be more effective and as accurate when compared with existing methods.

Corrigendum: Learning the heterogeneous representation of brain's structure from serial SEM images using a masked autoencoder.

[This corrects the article DOI: 10.3389/fninf.2023.1118419.].

Learning the heterogeneous representation of brain's structure from serial SEM images using a masked autoencoder.

Introduction: The exorbitant cost of accurately annotating the large-scale serial scanning electron microscope (SEM) images as the ground truth for training has always been a great challenge for brain map reconstruction by deep learning methods in neural connectome studies. The representation ability of the model is strongly correlated with the number of such high-quality labels. Recently, the masked autoencoder (MAE) has been shown to effectively pre-train Vision Transformers (ViT) to improve their representational capabilities. Methods: In this paper, we investigated a self-pre-training paradigm for serial SEM images with MAE to implement downstream segmentation tasks. We randomly masked voxels in three-dimensional brain image patches and trained an autoencoder to reconstruct the neuronal structures. Results and discussion: We tested different pre-training and fine-tuning configurations on three different serial SEM datasets of mouse brains, including two public ones, SNEMI3D and MitoEM-R, and one acquired in our lab. A series of masking ratios were examined and the optimal ratio for pre-training efficiency was spotted for 3D segmentation. The MAE pre-training strategy significantly outperformed the supervised learning from scratch. Our work shows that the general framework of can be a unified approach for effective learning of the representation of heterogeneous neural structural features in serial SEM images to greatly facilitate brain connectome reconstruction.

Improving the Neural Segmentation of Blurry Serial SEM Images by Blind Deblurring.

Serial scanning electron microscopy (sSEM) has recently been developed to reconstruct complex largescale neural connectomes, through learning-based instance segmentation. However, blurry images are inevitable amid prolonged automated data acquisition due to imprecision in autofocusing and autostigmation, which impose a great challenge to accurate segmentation of the massive sSEM image data. Recently, learning-based methods, such as adversarial learning and supervised learning, have been proven to be effective for blind EM image deblurring. However, in practice, these methods suffer from the limited training dataset and the underrepresentation of high-resolution decoded features. Here, we propose a semisupervised learning guided progressive decoding network (SGPN) to exploit unlabeled blurry images for training and progressively enrich high-resolution feature representation. The proposed method outperforms the latest deblurring models on real SEM images with much less ground truth input. The improvement of the PSNR and SSIM is 1.04 dB and 0.086, respectively. We then trained segmentation models with deblurred datasets and demonstrated significant improvement in segmentation accuracy. The A-rand (Bogovic et al. 2013) decreased by 0.119 and 0.026, respectively, for 2D and 3D segmentation.

J-domain protein chaperone circuits in proteostasis and disease.

The J-domain proteins (JDP) form the largest protein family among cellular chaperones. In cooperation with the Hsp70 chaperone system, these co-chaperones orchestrate a plethora of distinct functions, including those that help maintain cellular proteostasis and development. JDPs evolved largely through the fusion of a J-domain with other protein subdomains. The highly conserved J-domain facilitates the binding and activation of Hsp70s. How JDPs (re)wire Hsp70 chaperone circuits and promote functional diversity remains insufficiently explained. Here, we discuss recent advances in our understanding of the JDP family with a focus on the regulation built around J-domains to ensure correct pairing and assembly of JDP-Hsp70 machineries that operate on different clientele under various cellular growth conditions.

The effects of taraxasterol on liver fibrosis revealed by RNA sequencing.

Effective treatment of liver fibrosis remains a challenging medical problem. Taraxasterol (TAR) has anti-inflammatory, anti-tumor and hepatoprotective effects. Studies have shown that TAR has good biological activity against liver injury induced by various factors. However, the anti-fibrotic effect of TAR and its mechanism are never clarified. The purpose of this study was to investigate the effects of TAR in liver fibrosis and to reveal its possible mechanism by RNA sequencing. Our results suggested that TAR attenuated CCl4-induced hepatocyte necrosis, inflammatory infiltration and ECM deposition. TAR inhibited the levels of ALT, AST, ALP, γ-GT, LN, HA, PC III and IV-C in serum and TNF-α, IL-6, IL-1ß and MDA in liver. In addition, TAR increased the activities of SOD and GSH-Px in liver. RNA sequencing analysis of liver tissues revealed that CCl4 and TAR significantly altered 4,155 genes and 2,675 genes, respectively. TAR reversed changes in ECM-related genes. More specifically, TAR mediated the expression of genes related to the activation of the Hippo pathway, while inhibiting the expression of genes related to the activation of HIF-1α, TGF-ß/Smad, and Wnt pathways. In the validation experiments, the qRT-PCR results showed that the expression levels of Yap1, Tead3, Hif1α, Vegfa, Tgfß1, Want3a, and Ctnnb1 mRNA were consistent with the RNA sequencing results. The Western blot results showed that TAR inhibited the levels of TGF-ß1 and p-Smad2. In addition, the results in vitro were consistent with those in vivo. Therefore, we concluded that TAR improved CCl4-induced liver fibrosis by regulating Hippo, HIF-1α, TGF-ß/Smad and Wnt pathways.

Novel receptor, mutation, vaccine, and establishment of coping mode for SARS-CoV-2: current status and future.

Since the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its resultant pneumonia in December 2019, the cumulative number of infected people worldwide has exceeded 670 million, with over 6.8 million deaths. Despite the marketing of multiple series of vaccines and the implementation of strict prevention and control measures in many countries, the spread and prevalence of SARS-CoV-2 have not been completely and effectively controlled. The latest research shows that in addition to angiotensin converting enzyme II (ACE2), dozens of protein molecules, including AXL, can act as host receptors for SARS-CoV-2 infecting human cells, and virus mutation and immune evasion never seem to stop. To sum up, this review summarizes and organizes the latest relevant literature, comprehensively reviews the genome characteristics of SARS-CoV-2 as well as receptor-based pathogenesis (including ACE2 and other new receptors), mutation and immune evasion, vaccine development and other aspects, and proposes a series of prevention and treatment opinions. It is expected to provide a theoretical basis for an in-depth understanding of the pathogenic mechanism of SARS-CoV-2 along with a research basis and new ideas for the diagnosis and classification, of COVID-19-related disease and for drug and vaccine research and development.

Combined effects of vitamin C and cold atmospheric plasma-conditioned media against glioblastoma via hydrogen peroxide.

Glioblastoma is the most lethal intracranial malignant tumor, for which the five-year overall survival rate is approximately 5%. Here we explored the therapeutic combination of vitamin C and plasma-conditioned medium on glioblastoma cells in culture and as subcutaneous or intracranial xenografts in mice. The combination treatment reduced cell viability and proliferation while promoting apoptosis, and the effects were significantly stronger than with either treatment on its own. Similar results were obtained in the two xenograft models. Vitamin C appeared to upregulate aquaporin-3 and enhance the uptake of extracellular H2O2, while the combination treatment increased intracellular levels of reactive oxygen species including H2O2 and activated the JNK signaling pathway. The cytotoxic effects of the combination treatment were partially reversed by the specific JNK signaling inhibitor SP600125. Our results suggest that the combination of vitamin C and plasma-conditioned medium has therapeutic potential against glioblastoma, and they provide mechanistic insights that may help investigate this and other potential therapies in greater depth.

A Convenient All-Cell Optical Imaging Method Compatible with Serial SEM for Brain Mapping.

The mammalian brain, with its complexity and intricacy, poses significant challenges for researchers aiming to understand its inner workings. Optical multilayer interference tomography (OMLIT) is a novel, promising imaging technique that enables the mapping and reconstruction of mesoscale all-cell brain atlases and is seamlessly compatible with tape-based serial scanning electron microscopy (SEM) for microscale mapping in the same tissue. However, currently, OMLIT suffers from imperfect coatings, leading to background noise and image contamination. In this study, we introduced a new imaging configuration using carbon spraying to eliminate the tape-coating step, resulting in reduced noise and enhanced imaging quality. We demonstrated the improved imaging quality and validated its applicability through a correlative light-electron imaging workflow. Our method successfully reconstructed all cells and vasculature within a large OMLIT dataset, enabling basic morphological classification and analysis. We also show that this approach can perform effectively on thicker sections, extending its applicability to sub-micron scale slices, saving sample preparation and imaging time, and increasing imaging throughput. Consequently, this method emerges as a promising candidate for high-speed, high-throughput brain tissue reconstruction and analysis. Our findings open new avenues for exploring the structure and function of the brain using OMLIT images.

Whole-transcriptome sequencing with ceRNA regulation network construction and verification in glioblastoma.

OBJECTIVES: To explore the key genes involved in the occurrence and development of glioblastoma (GBM) by analyzing whole-transcriptome sequencing and biologic data from GBM and normal cerebral cortex tissues and to search for important noncoding RNA (ncRNA) molecular markers based on the competitive endogenous RNA (ceRNA) network. METHODS: Ten GBM and normal cerebral cortex tissues were collected for full transcriptome sequencing, screened for differentially expressed (DE) mRNAs, miRNAs, lncRNAs, and circRNAs, and subjected to bioinformatic analysis. We constructed a Protein-Protein Interaction (PPI) network and a circRNA/lncRNA-miRNA-mRNA regulatory network and identified them using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Finally, The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) databases were used to validate and conduct a survival analysis of the target genes. RESULTS: A total of 5341 DEmRNAs, 259 DEmiRNAs, 3122 DElncRNAs, and 2135 DEcircRNAs were identified. Enrichment analysis showed that target genes regulated by DEmiRNA, DElncRNA, and DEcircRNA were closely related to chemical synaptic transmission and ion transmembrane transport. A PPI network analysis screened 10 hub genes that directly participate in tumor cell mitosis regulation. In addition, the ceRNA composite network showed that hsa-miR-296-5p and hsa-miR-874-5p were the central nodes of the network, and the reliability of relevant key molecules was successfully verified through RT-qPCR identification and the TCGA database. The CGGA database survival analysis produced 8 DEmRNAs closely related to GBM patient survival prognosis. CONCLUSIONS: This study revealed the important regulatory functions and molecular mechanisms of ncRNA molecules and identified hsa-miR-296-5p and hsa-miR-874-5p as key molecules in the ceRNA network. They may play an important role in GBM pathogenesis, treatment, and prognosis.

Using fixed fiduciary markers for stage drift correction.

To measure nanometric features with super-resolution requires that the stage, which holds the sample, be stable to nanometric precision. Herein we introduce a new method that uses conventional equipment, is low cost, and does not require intensive computation. Fiduciary markers of approximately 1 µm x 1 µm x 1 µm in x, y, and z dimensions are placed at regular intervals on the coverslip. These fiduciary markers are easy to put down, are completely stationary with respect to the coverslip, are bio-compatible, and do not interfere with fluorescence or intensity measurements. As the coverslip undergoes drift (or is purposely moved), the x-y center of the fiduciary markers can be readily tracked to 1 nanometer using a Gaussian fit. By focusing the light slightly out-of-focus, the z-axis can also be tracked to < 5 nm for dry samples and <17 nm for wet samples by looking at the diffraction rings. The process of tracking the fiduciary markers does not interfere with visible fluorescence because an infrared light emitting diode (IR-LED) (690 and 850 nm) is used, and the IR-light is separately detected using an inexpensive camera. The resulting motion of the coverslip can then be corrected for, either after-the-fact, or by using active stabilizers, to correct for the motion. We applied this method to watch kinesin walking with ≈ 8 nm steps.

Two-photon 3D FIONA of individual quantum dots in an aqueous environment.

We report the first two-photon (2P) microscopy of individual quantum dots (QDs) in an aqueous environment with both widefield and point-scan excitations at nanometer accuracy. Thiol-containing reductants suppress QD blinking and enable measurement of the 36 nm step size of individual Myosin V motors in vitro. We localize QDs with an accuracy of 2-3 nm in all three dimensions by using a 9 × 9 matrix excitation hologram and an array detector, which also increases the 3D scan imaging rate by 80-fold. With this 3D microscopy we validate the LamB receptor distribution on E. coli and the endocytosis of EGF-receptors in breast cancer cells.