Microarray Platforms Based on 3D Printing.

This paper introduces an innovative method for the fabrication and infusion of microwell arrays based on digital light processing (DLP) 3D printing. A low-cost DLP 3D printer is employed to fabricate microstructures rapidly with a broad dynamic range while maintaining high precision and fidelity. We constructed microwell arrays with varying diameters, from 200 to 2000 µm and multiple aspect ratios, in addition to microchannels with widths ranging from 45 to 1000 µm, proving the potential and flexibility of this fabrication method. The superimposition of parallel microchannels onto the microwell array, facilitated by positive or negative pressure, enabled the transfer of liquid to the microwells. Upon removal of the microchannel chip, a dispensed microdroplet array was obtained. This array can be modulated by adjusting the volume of the microwells and the inflow fluid. The filled microwell array allows chip-to-chip dispensing to the microreactor array through binding and centrifugation, facilitating multistep and multireagent assays. The 3D printing approach also enables the fabrication of intricate cavity designs, such as micropyramid arrays, which can be integrated with parallel microchannels to generate spheroid flowcells. This device demonstrated the ability to generate spheroids and manipulate their environment. We have successfully utilized precise modulation of spheroids size and performed parallel drug dose-response assays to evaluate its effectiveness. Furthermore, we managed to execute dynamic drug combinations based on a compact spheroids array, utilizing two orthogonal parallel microchannels. Our findings suggest that both the combination and temporal sequence of drug administration have a significant impact on therapeutic outcomes.

The Epidemiology of Tinea Capitis in Guangxi Province, China.

The aim of this study was to analyze the epidemiological characteristics and pathogen spectrum of tinea capitis in Guangxi, southern China. A multicenter prospective descriptive study was conducted in 8 hospitals across Guangxi. From January 2019 to July 2022, one hundred seventy-one (171) patients diagnosed with tinea capitis were included. Demographic data, risk factors, and fungal data were collected. If necessary, species were further identified by morphological or molecular sequencing in the central laboratory. Of the 171 cases of tinea capitis, 74.3% occurred in patients aged 2-8 years. Children with tinea capitis were mainly boys (59.6%) and were more likely than adults to have a history of animal contact (44.2% vs. 33.3%) and zoophilic dermatophyte infection (76.9% vs. 46.7%, P = 0.008). The adults were mainly female (53%) and were more likely than children to have a history of infection with anthropophilic organisms (53.3% vs. 18.9%). The causative agents of tinea capitis in Guangxi were diverse, and the most common pathogen was Microsporum canis (M. cani, n = 98, 62%), followed by Trichophyton mentagrophytes (T. mentagrophytes n = 18, 11.4%) and Trichophyton tonsurans (T. tonsurans n = 12, 7.6%). In addition, tinea capitis caused by Nannizzia incurvata (N. incurvata) and Trichophyton verrucosum (T. verrucosum) was detected in the study. Notably, the proportion of patients with kerion in the study was 41.5% (n = 71), and most of those patients were children (n = 68), especially neglected children living in the rural mountainous areas of Guangxi, where they were unable to receive timely diagnosis and appropriate treatment. In conclusion, the causative agents of tinea capitis in Guangxi, South China, are diverse, and the incidence of kerion is high, indicating that diagnosis and treatment modalities in the region remain grossly inadequate. Clinicians and policy-makers should collaborate to adopt public health strategies to control the disease.

Large-Scale Integration of Single-Cell RNA-Seq Data Reveals Astrocyte Diversity and Transcriptomic Modules across Six Central Nervous System Disorders.

The dysfunction of astrocytes in response to environmental factors contributes to many neurological diseases by impacting neuroinflammation responses, glutamate and ion homeostasis, and cholesterol and sphingolipid metabolism, which calls for comprehensive and high-resolution analysis. However, single-cell transcriptome analyses of astrocytes have been hampered by the sparseness of human brain specimens. Here, we demonstrate how large-scale integration of multi-omics data, including single-cell and spatial transcriptomic and proteomic data, overcomes these limitations. We created a single-cell transcriptomic dataset of human brains by integration, consensus annotation, and analyzing 302 publicly available single-cell RNA-sequencing (scRNA-seq) datasets, highlighting the power to resolve previously unidentifiable astrocyte subpopulations. The resulting dataset includes nearly one million cells that span a wide variety of diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), multiple sclerosis (MS), epilepsy (Epi), and chronic traumatic encephalopathy (CTE). We profiled the astrocytes at three levels, subtype compositions, regulatory modules, and cell-cell communications, and comprehensively depicted the heterogeneity of pathological astrocytes. We constructed seven transcriptomic modules that are involved in the onset and progress of disease development, such as the M2 ECM and M4 stress modules. We validated that the M2 ECM module could furnish potential markers for AD early diagnosis at both the transcriptome and protein levels. In order to accomplish a high-resolution, local identification of astrocyte subtypes, we also carried out a spatial transcriptome analysis of mouse brains using the integrated dataset as a reference. We found that astrocyte subtypes are regionally heterogeneous. We identified dynamic cell-cell interactions in different disorders and found that astrocytes participate in key signaling pathways, such as NRG3-ERBB4, in epilepsy. Our work supports the utility of large-scale integration of single-cell transcriptomic data, which offers new insights into underlying multiple CNS disease mechanisms where astrocytes are involved.

Investigation of hub genes and immune infiltration in androgenetic alopecia using bioinformatics analysis.

Background: Androgenetic alopecia (AGA) is a type of non-scarring hair loss. Current drugs for AGA are accompanied by adverse reactions and a high recurrence rate. Thus, the discovery of diagnostic biomarkers and therapeutic targets for AGA remains imperatively warranted. Methods: The GSE90594 dataset, which contained scalp skin biopsies from 14 male AGA cases and healthy volunteers, was used to identify the differentially expressed genes (DEGs). Functional enrichment analysis was subsequently performed. Next, the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database combined with the cytoHubba plugin of Cytoscape were used to obtain the key genes of AGA. Thereafter, the Cell-type Identification by Estimating Relative Subsets of RNA Transcripts (CIBERSORT) algorithm was performed to evaluate the relative abundance of immune cells between male AGA patients and healthy controls. The correlation between key genes and infiltrating immune cells was analyzed to obtain the significant immune-cell related genes (IRGs), then intersected with the DEGs between immortalized balding and non-balding human dermal papilla cells (DPCs) of the GSE93766 dataset as well as the DEGs obtained by the GSE90594 dataset, thus obtaining the hub genes of AGA. Finally, the hub genes were validated using GSE36169, which contained expression profiling of tissues biopsied from haired and bald scalps of five individuals with AGA. Results: A total of 234 DEGs were obtained from the GSE90594 dataset, which were mainly enriched in the extracellular matrix (ECM)-related pathways and immune-related activities. The STRING database and ten algorithms in the cytoHubba plugin of Cytoscape disclosed 21 key DEGs. The results of the CIBERSORT algorithm revealed the relative abundances of 20 kinds of immune cells between diseased and healthy individuals, and yielded 15 IRGs involved in the pathogenesis of AGA. Next, the intersection analysis identified four hub genes of AGA, comprising COL1A2, PCOLCE, ITGAX, and LOX. The GSE36169 dataset validated the expression pattern of hub genes in the haired scalp of AGA patients. Conclusions: We discovered that the hub genes identified are closely linked with the causative factors of AGA, which could be used as the viable diagnostic and therapeutic target in the clinical applications.

Identification of a Potentially Functional circRNA-miRNA-mRNA Regulatory Network in Melanocytes for Investigating Pathogenesis of Vitiligo.

CircRNAs have been reported to play essential roles in regulating immunity and inflammation, which may be an important regulatory factor in the development of vitiligo. However, the expression profile of circRNAs and their potential biological functions in vitiligo have not been reported so far. In our study we found there are 64 dysregulated circRNAs and 14 dysregulated miRNAs in the patients with vitiligo. Through the correlation analysis, we obtained 12 dysregulated circRNAs and 5 dysregulated miRNAs, forming 48 relationships in the circRNA-miRNA-mRNA regulatory network. Gene Ontology analysis indicated dysregulated circRNAs in vitiligo is closely related to the disorder of the metabolic pathway. The KEGG pathway of dysregulation of circRNAs mainly enriched in the biological processes such as ubiquitin mediated proteolysis, endocytosis and RNA degradation, and in Jak-STAT signaling pathway. Therefore, we found the circRNA-miRNA-mRNA regulatory network are involved in the regulation of numerous melanocyte functions, and these dysregulated circRNAs may closely related to the melanocyte metabolism. Our study provides a theoretical basis for studying the vitiligo pathogenesis from the perspective of circRNA-miRNA-mRNA network.

Comparison of a fractional 2940-nm Er:YAG laser and 5% amorolfine lacquer combination therapy versus a 5% amorolfine lacquer monotherapy for the treatment of onychomycosis: a randomized controlled trial.

Onychomycosis is a fungal infection of the nail. The aim of this randomized controlled clinical trial was to compare the efficacy of 2940-nm Er:YAG laser treatment combined with a 5% amorolfine lacquer versus amorolfine monotherapy for treating onychomycosis. In this study, patients with onychomycosis of the great toenail were randomly assigned to a combination therapy group and a monotherapy group. In the combination therapy group, the included toenails were treated with a fractional 2940-nm Er:YAG laser at weeks 1, 2, 3, 4, 8, and 12, combined with a 5% amorolfine lacquer twice a week for 12 weeks, while in the monotherapy group, the included toenails were treated with only a 5% amorolfine lacquer twice a week for 12 weeks. The onychomycosis severity index (OSI) score and the mycological clearance rate (MCR) of the included toenails were assessed at baseline, week 12, and week 24. At weeks 12 and 24, the great toenails with mild and moderate onychomycosis in the combination therapy group showed obvious improvement and a greater decrease in OSI than those in the monotherapy group. At week 24, the toenails with mild and moderate onychomycosis in the combination therapy group also showed a better MCR. For the toenails with severe onychomycosis, little improvement was observed in either group at week 12 or week 24. In conclusion, fractional 2940-nm Er:YAG laser treatment combined with a 5% amorolfine lacquer is more effective than amorolfine monotherapy in short-term improvement of onychomycosis.

Effect of Fonsecaea monophora on the Polarization of THP-1 Cells to Macrophages.

BACKGROUND: Chromoblastomycosis is a chronic, progressive fungal disease of the skin and subcutaneous tissue caused by a unique group of dematiaceous fungi. Fonsecaea monophora, a new species distinct from Fonsecaea pedrosoi strains, is the main pathogen responsible for chromoblastomycosis in south China. Macrophages can be polarized into two categories: classically activated and alternatively activated. OBJECTIVES: Little is known about the relationship between F. monophora and macrophage polarization. This study aimed to study the effect of F. monophora on the polarization of THP-1 cells to macrophages. METHODS: We established coculture systems of F. monophora and THP-1-derived macrophages in different activation states. RESULTS: F. monophora enhanced the phagocytosis by macrophages in the initially activated state and weakened the phagocytosis by classically activated macrophages without affecting that by alternatively activated macrophages. Classically activated macrophages had the strongest killing effect on F. monophora, while the initially activated macrophages had the weakest. The pathogen could not be rapidly cleared by any type of macrophage. F. monophora promoted the expression of proinflammatory cytokines and inhibited that of anti-inflammatory cytokines. CONCLUSIONS: F. monophora promoted the polarization of THP-1 cells to classically activated macrophages and inhibited that of THP-1 cells to alternatively activated macrophages.

An optimized Agrobacterium tumefaciens-mediated transformation system for random insertional mutagenesis in Fonsecaea monophora.

Chromoblastomycosis (CBM) is a chronic cutaneous or subcutaneous mycosis that is prevalent worldwide. Though CBM tends not to be fatal, it is difficult to treat and complications can include chronic, marked lesions, lymphatic damage, and neoplastic transformation. Fonsecaea monophora, as a new species segregated from Fonsecaea pedrosoi, is the predominant causative pathogen of CBM in southern China. However, research about F. monophora has been limited, which may be due to a lack of an effective genetic manipulation system for F. monophora. In this study, we successfully established a random insertional mutagenesis system by Agrobacterium tumefaciens-mediated transformation (ATMT) in F. monophora for the first time. In order to improve the efficiency of ATMT, various co-culture conditions were optimized, including: acetosyringone (AS) concentrations, co-culture duration, ratio of bacteria to conidia, and the A. tumefaciens strains. In addition, thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) was performed to identify the transferred DNA (T-DNA) flanking sequences of the F. monophora transformants. The valuable transformants obtained in this study will be used for research in the future.

Transcriptional profiling of macrophages infected with Fonsecaea monophora.

Fonsecaea monophora is a member of dematiaceous fungi capable of causing chromoblastomycosis through traumatic injury. However, little is known about the pathogenesis and early interactions between F. monophora and host. The aim of this study was to explore the potential mechanism of macrophages against F. monophora, especially the role of melanin during the pathogenic process. We carried out RNA sequencing based on the Illumina system. It showed that according to melanin contents, different strains of F. monophora induced different transcriptional profilings in macrophages. Functional analyses suggested the biological functions of differentially expressed genes were closely related to immune response, and the melanin might affect the interactions by regulating the MAPK signalling pathway of macrophages. Our results provide insights into the pathogenesis of infection by F. monophora conidia.