Grape SnRK2.7 Positively Regulates Drought Tolerance in Transgenic Arabidopsis.

In this study, we obtained and cloned VvSnRK2.7 by screening transcriptomic data to investigate the function of the grape sucrose non-fermenting kinase 2 (SnRK2) gene under stress conditions. A yeast two-hybrid (Y2H) assay was used to further screen for interaction proteins of VvSnRK2.7. Ultimately, VvSnRK2.7 was heterologously expressed in Arabidopsis thaliana, and the relative conductivity, MDA content, antioxidant enzyme activity, and sugar content of the transgenic plants were determined under drought treatment. In addition, the expression levels of VvSnRK2.7 in Arabidopsis were analyzed. The results showed that the VvSnRK2.7-EGFP fusion protein was mainly located in the cell membrane and nucleus of tobacco leaves. In addition, the VvSnRK2.7 protein had an interactive relationship with the VvbZIP protein during the Y2H assay. The expression levels of VvSnRK2.7 and the antioxidant enzyme activities and sugar contents of the transgenic lines were higher than those of the wild type under drought treatment. Moreover, the relative conductivity and MDA content were lower than those of the wild type. The results indicate that VvSnRK2.7 may activate the enzyme activity of the antioxidant enzyme system, maintain normal cellular physiological metabolism, stabilize the berry sugar metabolism pathway under drought stress, and promote sugar accumulation to improve plant resistance.

Femtosecond Laser-Induced Phase Transformation on Single-Crystal 6H-SiC.

Silicon carbide (SiC) is widely used in many research fields because of its excellent properties. The femtosecond laser has been proven to be an effective method for achieving high-quality and high-efficiency SiC micromachining. In this article, the ablation mechanism irradiated on different surfaces of 6H-SiC by a single pulse under different energies was investigated. The changes in material elements and the geometric spatial distribution of the ablation pit were analyzed using micro-Raman spectroscopy, Energy Dispersive Spectrum (EDS), and an optical microscope, respectively. Moreover, the thresholds for structural transformation and modification zones of 6H-SiC on different surfaces were calculated based on the diameter of the ablation pits created by a femtosecond laser at different single-pulse energies. Experimental results show that the transformation thresholds of the Si surface and the C surface are 5.60 J/cm2 and 6.40 J/cm2, corresponding to the modification thresholds of 2.26 J/cm2 and 2.42 J/cm2, respectively. The Raman and EDS results reveal that there are no phase transformations or material changes on different surfaces of 6H-SiC at low energy, however, decomposition and oxidation occur and then accumulate into dense new phase material under high-energy laser irradiation. We found that the distribution of structural phase transformation is uneven from the center of the spot to the edge. The content of this research reveals the internal evolution mechanism of high-quality laser processing of hard material 6H-SiC. We expect that this research will contribute to the further development of SiC-based MEMS devices.

Development of Cas13a-based therapy for cancer treatment.

Gene therapy has become a major focus of current biomedical research. CRISPR (Clustered Regularly Inter spaced Short Palindromic Repeats) systems have been extensively researched for disease treatment applications through genome editing specificity. Compared with Cas9 (CRISPR-associated proteins, Cas), a commonly used tool enzyme for genome editing, Cas13a exhibits RNA-dependent endonuclease activity, including collateral cleavage without obvious potential genetic risks. With its high specificity, Cas13a has significantly improved the sensitivity of viral diagnosis and shown potential to eliminate viruses. However, its efficacy in tumor therapy has not been determined. This review introduces the mechanism and research developments associated with the CRISPR-Cas13a system in tumor treatments and its potential to be used as a new tool for gene therapy. We hope more research would apply Cas13a-based therapy in cancer treatment in the future.

Correction to " Application of Terahertz Nondestructive Testing Technology in the Detection of Polyethylene Pipe Defects".

[This corrects the article DOI: 10.1021/acsomega.3c02701.].

Application of Terahertz Nondestructive Testing Technology in the Detection of Polyethylene Pipe Defects.

At present, polyethylene pipeline is widely used in urban gas projects, but a relatively mature and reliable nondestructive testing technology has not been formed. Therefore, it is urgent to develop a new nondestructive testing technology to meet the increasing demand for inspection of non-metallic pipes. The terahertz testing technology and related equipment have played an increasingly important role in the nondestructive testing of many nonmetallic structures, but they have not been applied to polyethylene (PE) pipes. In this work, terahertz time-domain spectroscopy was used to detect prefabricated defects inside the PE pipe specimens. The results show that the terahertz nondestructive testing technology can be used to detect common defects in nonblack PE pipes with a detection error of less than 10%. Higher-power terahertz devices can detect defects in black PE pipe, while lower-power terahertz devices cannot. Because the black PE pipe contains carbon and has a strong absorption of terahertz waves. The penetration of lower-power terahertz devices to the black PE pipe is not enough, resulting in a low resolution of the imaging. The results of this work may promote the progress of the nondestructive testing technology of nonmetallic pipelines.

MBGA-Net: A multi-branch graph adaptive network for individualized motor imagery EEG classification.

BACKGROUND AND OBJECTIVE: The development of deep learning has led to significant improvements in the decoding accuracy of Motor Imagery (MI) EEG signal classification. However, current models are inadequate in ensuring high levels of classification accuracy for an individual. Since MI EEG data is primarily used in medical rehabilitation and intelligent control, it is crucial to ensure that each individual's EEG signal is recognized with precision. METHODS: We propose a multi-branch graph adaptive network (MBGA-Net), which matches each individual EEG signal with a suitable time-frequency domain processing method based on spatio-temporal domain features. We then feed the signal into the relevant model branch using an adaptive technique. Through an enhanced attention mechanism and deep convolutional method with residual connectivity, each model branch more effectively harvests the features of the related format data. RESULTS: We validate the proposed model using the BCI Competition IV dataset 2a and dataset 2b. On dataset 2a, the average accuracy and kappa values are 87.49% and 0.83, respectively. The standard deviation of individual kappa values is only 0.08. For dataset 2b, the average classification accuracies obtained by feeding the data into the three branches of MBGA-Net are 85.71%, 85.83%, and 86.99%, respectively. CONCLUSIONS: The experimental results demonstrate that MBGA-Net could effectively perform the classification task of motor imagery EEG signals, and it exhibits strong generalization performance. The proposed adaptive matching technique enhances the classification accuracy of each individual, which is beneficial for the practical application of EEG classification.

Transcriptome and metabolites analysis of water-stressed grape berries at different growth stages.

Drought is one of the main abiotic factors affecting grape quality. However, the impacts of drought stress on sugar and related gene expression during grape berry ripening remain unclear. In this experiment, the grapes were subjected to different levels of continuous water stress from 45 to 120 days after flowering (DAA) to study the changes in berry sugar content and the expression of genes related to sugar metabolism under different water stresses. Data supported that glucose, fructose, sucrose, and soluble sugars increased from 45 DAA. Combined with previous research results, T1, T2, and Ct grape berries with 60 ~ 75 DAA and large differences in sucrose, fructose, glucose and soluble sugars compared with the Ct were selected for RNA sequencing (RNA-seq). Through transcriptome analysis, 4471 differentially expressed genes (DEGs) were screened, and 65 genes in photosynthesis, ABA signaling pathway and photosynthetic carbon metabolism pathway were analyzed further by qRT-PCR. At 60 DAA, the relative expression levels of CAB1R, PsbP, SNRK2, and PYL9 were significantly upregulated in response to water stress, while AHK1, At4g02290 were down-regulated. At 75 DAA, the relative expression levels of ELIP1, GoLS2, At4g02290, Chi5, SAPK, MAPKKK17, NHL6, KINB2, and AHK1 were upregulated. And CAB1R, PsbA, GoLS1, SnRK2, PYL9, and KINGL were significantly downregulated under moderate water stress. In addition, PsbA expression was down-regulated in response to water stress. These results will help us to fully understand the potential connections between glucose metabolism and gene expression in grapes under drought stress.

EEG emotion recognition using improved graph neural network with channel selection.

BACKGROUND AND OBJECTIVE: Emotion classification tasks based on electroencephalography (EEG) are an essential part of artificial intelligence, with promising applications in healthcare areas such as autism research and emotion detection in pregnant women. However, the complex data acquisition environment provides a variable number of EEG channels, which interferes with the model to simulate the process of information transfer in the human brain. Therefore, this paper proposes an improved graph convolution model with dynamic channel selection. METHODS: The proposed model combines the advantages of 1D convolution and graph convolution to capture the intra- and inter-channel EEG features, respectively. We add functional connectivity in the graph structure that helps to simulate the relationship between brain regions further. In addition, an adjustable scale of channel selection can be performed based on the attention distribution in the graph structure. RESULTS: We conducted various experiments on the DEAP-Twente, DEAP-Geneva, and SEED datasets and achieved average accuracies of 90.74%, 91%, and 90.22%, respectively, which exceeded most existing models. Meanwhile, with only 20% of the EEG channels retained, the models achieved average accuracies of 82.78%, 84%, and 83.93% on the above three datasets, respectively. CONCLUSIONS: The experimental results show that the proposed model can achieve effective emotion classification in complex dataset environments. Also, the proposed channel selection method is informative for reducing the cost of affective computing.

Service Reliability Test Method for Anticorrosion Coatings on the Compressor Outlet Pipelines of Natural Gas Stations.

Compressor outlets are subject to high temperatures and vibrations; when pipelines are subject to such conditions, degradation of the anticorrosive layer on the pipeline is likely. Fusion-bonded epoxy (FBE) powder coating is the most common type of anticorrosion coatings on compressor outlet pipelines. It is necessary to study the reliability of anticorrosive layers in compressor outlet pipelines. In this paper, a service reliability test method for the corrosion-resistant coatings of compressor outlet pipelines of natural gas stations is proposed. Testing involving the simultaneous exposure of the pipeline to high temperatures and vibrations is conducted to evaluate, on a compressed timescale, the applicability and service reliability of FBE coatings. The failure mechanism of FBE coatings exposed to high temperatures and vibrations is analyzed. It is found that, due to the influence of initial imperfections in the coatings, FBE anticorrosion coatings typically do not meet the standard requirements for use in compressor outlet pipelines. After simultaneous exposure to high temperatures and vibrations, the impact resistance, abrasion resistance, and bending resistance of the coatings are found not to meet the requirements for their intended applications. It is therefore suggested that FBE anticorrosion coatings be used with extreme caution in compressor outlet pipelines.

VaSUS2 confers cold tolerance in transgenic tomato and Arabidopsis by regulation of sucrose metabolism and ROS homeostasis.

KEY MESSAGE: VaSUS2 enhances cold tolerance of transgenic tomato and Arabidopsis by regulating sucrose metabolism and improving antioxidant enzymes activity. Sucrose synthetase (SUS) is a key enzyme of sugar metabolism, and plays an important role in response to abiotic stress in plant. However, the function of VaSUS2 remains unknown in cold tolerance. Here, the cloning and functional characterization of the plasma membrane-localized VaSUS2 gene isolated from Vitis amurensis was studied. The transcript level of VaSUS2 was up-regulated under cold stress in Vitis amurensis. Heterologous expression of VaSUS2 in tomato increased SUS activity, which promoted the accumulation of glucose and fructose under cold treatment. The transgenic tomato and Arabidopsis exhibited higher levels of antioxidant enzymes activity, lower relative electrolyte leakage (REL), malondialdehyde (MDA) and hydrogen peroxide (H2O2) content compared to wild type under cold stress. Importantly, the ability of scavenging reactive oxygen species (ROS) in transgenic plants was significantly improved. Moreover, yeast two-hybrid (Y2H) indicated that VaSnRK1 might be a potential interaction protein of VaSUS2. qRT-PCR showed that sucrose metabolism-related genes SlSUS, SlSPS and SlINV were significantly up-regulated in transgenic tomatoes. Meanwhile, the expression levels of antioxidant enzyme genes and cold-related genes CBF1, COR47 and ICE1 were up-regulated in transgenic plants. Taken together, these results suggested that VaSUS2 was involved in cold tolerance by increasing the levels of soluble sugars, improving the activity of antioxidant enzymes, and up-regulating the expression of cold-related genes in transgenic tomatoes and Arabidopsis.

Grape BES1 transcription factor gene VvBES1-3 confers salt tolerance in transgenic Arabidopsis.

BRI1-EMS-Suppressor 1 (BES1) regulates plant growth, development, and stress resistance, and plays a pivotal role in the brassinosteroid (BR) signal transduction pathway. In this study, a total of 12 BES1 genes were identified in the grape (Vitis vinifera) genome. Phylogenetic, structure, and motif sequence analyses of these genes provided insights into their evolutionary characteristics. Hormone-, stress-, and light-responsive and organ-specific cis-acting elements were identified in VvBES1 gene promoters. Microarray data analysis showed that VvBES1 family members exhibit diverse expression patterns in different organs. Quantitative real-time PCR (qRT-PCR) analysis showed that the expression levels of VvBES1 genes differed in response to BR, methyl jasmonate (MeJA), cold (4 °C), NaCl, and polyethylene glycol (PEG) treatments. The expression of VvBES1-3 was 29-fold higher under salt stress than control at 12 h. Moreover, VvBES1-3-overexpessing Arabidopsis thaliana plants showed lower malondialdehyde content, higher proline content, enhanced antioxidant enzyme (catalase, superoxide dismutase, peroxidase) activities, and higher salt-responsive gene expression levels than wild-type plants under salt stress, indicating that VvBES1-3 overexpression enhances salt stress tolerance in transgenic Arabidopsis. These results will contribute to further understanding the functions of BES1 transcription factors in the abiotic stress response.

Knockout of cryptochrome 1 disturbs the locomotor circadian rhythm and development of Plutella xylostella.

Cryptochrome 1 (CRY1) functions as a light-responsive photoreceptor, which is crucial for circadian rhythms. The identity and function of CRY1 in Plutella xylostella remain unknown. In this study, cry1 was cloned and identified in P. xylostella. Then, a cry1-knockout strain (Cry1-KO) of P. xylostella with a 2-bp deletion was established from the strain Geneva 88 (G88) using the CRISPR/Cas9 technology. No daily temporal oscillation of cry1 was observed in G88 and Cry1-KO, and cry1 mean daily transcription of Cry1-KO was lower than that of G88. Both G88 and Cry1-KO demonstrated rhythmic locomotion under the light/dark condition with Cry1-KO being more active than G88 in the daytime, whereas Cry1-KO completely lost rhythmicity under constant darkness. The developmental period of pre-adult of Cry1-KO was longer than that of G88; the lifespan of the Cry1-KO male adult was shorter than that of G88; the fecundity of Cry1-KO was lower than that of G88; and Cry1-KO showed lower intrinsic rate of increase (r), net reproduction rate (R0 ), finite increase rate (λ), and longer mean generation time (T) than G88. Our results indicate that cry1 is involved in the regulation of locomotor circadian rhythm and development in P. xylostella, providing a potential target gene for controlling the pest and a basis for further investigation on circadian rhythms in lepidopterans.

Existence of Classical Solutions for Nonlinear Elliptic Equations with Gradient Terms.

This paper deals with the existence of solutions of the elliptic equation with nonlinear gradient term -Δu=f(x,u,∇u) on Ω restricted by the boundary condition u|∂Ω=0, where Ω is a bounded domain in RN with sufficiently smooth boundary ∂Ω, N≥2, and f:Ω¯×R×RN→R is continuous. The existence results of classical solutions and positive solutions are obtained under some inequality conditions on the nonlinearity f(x,ξ,η) when |(ξ,η)| is small or large enough.

Genome-wide identification and expression analysis of PIN gene family under phytohormone and abiotic stresses in Vitis Vinifera L.

The auxin efflux transport proteins PIN-formed (PIN) has wide adaptability to hormone and abiotic stress, but the response mechanism of PINs in grape remains unclear. In this study, 12 members of VvPINs were identified and distributed on 8 chromosomes. The PIN genes of five species were divided into two subgroups, and the similarity of exons/introns and motifs of VvPIN genes were found in the same subgroup. Meanwhile, according to the examination of conserved motifs, the motif 3 included the conserved structure NPNTY. The promoter region of VvPIN gene family contained various cis-acting elements, which were related to light, abiotic stress, and hormones which are essential for growth and development. Additionally, VvPIN1, VvPIN9, and VvPIN11 proteins simultaneously interacted with the ARF, ABC, PINOID, GBF1, and VIT_08s0007g09010. The results of qRT-PCR revealed that the majority of the VvPINs were highly induced by NAA, GA3, ABA, MeJA, SA, NaCl, low-temperature (4 ℃), and PEG treatments, and the results were consistent with the prediction of the cis-acting elements. Moreover, the expression profile and quantitative real-time PCR (qRT-PCR) demonstrated that VvPIN genes were expressed in roots, stems, and leaves. The subcellular localization of VvPIN1 in Nicotiana benthamiana revealed that VvPIN1 was localized at the plasma membrane. Collectively, this study revealed that PIN genes could respond to various abiotic stresses, and provided a framework for regulating the expression of PIN genes to enhance the resistance of the grape. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01239-8.

Changes and response mechanism of sugar and organic acids in fruits under water deficit stress.

The content and the ratio of soluble sugars and organic acids in fruits are significant indicators for fruit quality. They are affected by multiple environmental factors, in which water-deficient is the most concern. Previous studies found that the content of soluble sugars and organic acids in fruit displayed great differences under varied water stress. It is important to clarify the mechanism of such difference and to provide researchers with systematic knowledge about the response to drought stress and the mechanism of sugar and acid changes in fruits, so that they can better carry out the study of fruit quality under drought stress. Therefore, the researchers studied dozens of research articles about the content of soluble sugar and organic acid, the activity of related metabolic enzymes, and the expression of related metabolic genes in fruits under water stress, and the stress response of plants to water stress. We found that after plants perceived and transmitted the signal of water deficit, the expression of genes related to the metabolism of soluble sugars and organic acids changed. It was then affected the synthesis of metabolic enzymes and changed their metabolic rate, ultimately leading to changes in soluble sugar and organic acid content. Based on the literature review, we described the pathway diagrams of sugar metabolism, organic acid metabolism, mainly malic acid, tartaric acid, and citric acid metabolism, and of the response to drought stress. From many aspects including plants' perception of water stress signal, signal conversion and transmission, induced gene expression, the changes in soluble sugar and the enzyme activities of organic acids, as well as the final sugar and acid content in fruits, this thesis summarized previous studies on the influence of water stress on soluble sugars and the metabolism of organic acids in fruits.

Predictors of Lung Adenocarcinoma With Leptomeningeal Metastases: A 2022 Targeted-Therapy-Assisted molGPA Model.

Objective: To explore prognostic indicators of lung adenocarcinoma with leptomeningeal metastases (LM) and provide an updated graded prognostic assessment model integrated with molecular alterations (molGPA). Methods: A cohort of 162 patients was enrolled from 202 patients with lung adenocarcinoma and LM. By randomly splitting data into the training (80%) and validation (20%) sets, the Cox regression and random survival forest methods were used on the training set to identify statistically significant variables and construct a prognostic model. The C-index of the model was calculated and compared with that of previous molGPA models. Results: The Cox regression and random forest models both identified four variables, which included KPS, LANO neurological assessment, TKI therapy line, and controlled primary tumor, as statistically significant predictors. A novel targeted-therapy-assisted molGPA model (2022) using the above four prognostic factors was developed to predict LM of lung adenocarcinoma. The C-indices of this prognostic model in the training and validation sets were higher than those of the lung-molGPA (2017) and molGPA (2019) models. Conclusions: The 2022 molGPA model, a substantial update of previous molGPA models with better prediction performance, may be useful in clinical decision making and stratification of future clinical trials.

Overexpression VaPYL9 improves cold tolerance in tomato by regulating key genes in hormone signaling and antioxidant enzyme.

BACKGROUND: Abscisic acid (ABA) has been reported in controlling plant growth and development, and particularly dominates a role in resistance to abiotic stress. The Pyrabactin Resistance1/PYR1-Like /Regulatory Components of ABA receptors (PYR1/PYL/RCAR) gene family, of which the PYL9 is a positive regulator related to stress response in ABA signaling transduction. Although the family has been identified in grape, detailed VaPYL9 function in cold stress remains unknown. RESULTS: In order to explore the cold tolerance mechanism in grape, VaPYL9 was cloned from Vitis amurensis. The subcellular localization showed that VaPYL9 was mainly expressed in the plasma membrane. Yeast two-hybrid (Y2H) showed VaPCMT might be a potential interaction protein of VaPYL9. Through the overexpression of VaPYL9 in tomatoes, results indicated transgenic plants had higher antioxidant enzyme activities and proline content, lower malondialdehyde (MDA) and H2O2 content, and improving the ability to scavenge reactive oxygen species than wild-type (WT). Additionally, ABA content and the ratio of ABA/IAA kept a higher level than WT. Quantitative real-time PCR (qRT-PCR) showed that VaPYL9, SlNCED3, SlABI5, and antioxidant enzyme genes (POD, SOD, CAT) were up-regulated in transgenic tomatoes. Transcriptome sequencing (RNA-seq) found that VaPYL9 overexpression caused the upregulation of key genes PYR/PYL, PYL4, MAPK17/18, and WRKY in transgenic tomatoes under cold stress. CONCLUSION: Overexpression VaPYL9 enhances cold resistance of transgenic tomatoes mediated by improving antioxidant enzymes activity, reducing membrane damages, and regulating key genes in plant hormones signaling and antioxidant enzymes.

PreS/2-21-Guided siRNA Nanoparticles Target to Inhibit Hepatitis B Virus Infection and Replication.

A viable therapy is needed to overcome the deadlock of the incurable chronic hepatitis B (CHB). The prolonged existence of covalently closed circular DNA (cccDNA) and integrated HBV DNA in the nucleus of hepatocytes is the root cause of CHB. As a result, it is critical to successfully suppress HBV DNA replication and eliminate cccDNA. RNA interference has been proven in recent research to silence the expression of target genes and thereby decrease HBV replication. However, siRNA is susceptible to be degraded by RNA enzymes in vivo, making it difficult to deliver successfully and lacking of tissue targeting. To exploit the advantages of siRNA technology while also overcoming its limitations, we designed a new strategy and prepared biomimetic nanoparticles that were directed by PreS/2-21 peptides and precisely loaded HBV siRNA. Experiments on these nanoparticles in vitro and in vivo revealed that they are tiny, stable, safe and highly targetable, with high inhibitory effects on HBV DNA, pgRNA, cccDNA, HBeAg and HBsAg. PreS/2-21-directed nanoparticles loaded with HBV gene therapy drugs are expected to be promising for the treatment of CHB.

Temperature-phase transcriptomics reveals that hormones and sugars in the phloem of grape participate in tolerance during cold acclimation.

KEY MESSAGE: Most of the upregulated genes contributed to the accumulation of soluble sugars and ABA in the phloem of 'Vitis amurensis' compared to 'Merlot' during cold acclimation. Extreme cold is one of the dominant abiotic factors affecting grape yield and quality. However, the changes in sugars, phytohormones, and gene expression in the branch phloem of different tolerant grape varieties during cold acclimation remain elusive. The data supported that with decreasing temperature, the contents of fructose, sucrose, and ABA in the phloem of Vitis amurensis (cold-tolerant, T) and 'Merlot' (cold-sensitive, S) increased during cold acclimation, and these indicators were higher in T than in S. Furthermore, the activities of sucrose synthetase, sucrose phosphate synthetase, and acid invertase peaked in the early phase of cold acclimation (approximately 5 °C) compared to other phases (approximately 28 °C, 0 °C, - 5 °C and - 10 °C). Moreover, the RNA sequencing results helped identify a total of 11,343 differentially expressed genes in the phloem of T and S, among which 4912 were upregulated and 6431 were downregulated. In the abscisic acid pathway, CRTISO, PSPY1-1, CYCP707A4-2, PYL4-1, PYL4-2, P2C08, SAPK2, TARAB1, and DBF3 were more highly expressed in T than in S. In the starch and sucrose metabolism pathway, HXK1, PGMP, GLGL1, SUS6, VCINV, BGL11, SSY1, GPS, BAM1 and BAM3 were also more highly expressed in T than in S. Moreover, the genes related to oxidative phosphorylation, such as NDHF, ND4, ND1, NAD7, NAD2, ATPB, YMF19, ATP9, PMA1 and AHA8, were upregulated in T. These results will be beneficial for understanding the potential differences in tolerance across two different cold-tolerant grapes with respect to sugar metabolism and gene expression.

Nanopore sequencing of cerebrospinal fluid of three patients with cryptococcal meningitis.

BACKGROUND: Cryptococcal meningitis (CM) has a high morbidity and mortality due to the low detection of Cryptococcus in cerebrospinal fluid (CSF) during the early stage of the disease with traditional methods. CASE PRESENTATION: In addition to the traditional methods of India ink staining and cryptococcal antigen (CrAg), we used nanopore sequencing and next-generation sequencing (NGS) to detect pathogenic DNA in CSF samples of three patients with CM. The CSF samples of all three patients were positive by India ink staining and CrAg. NGS also detected Cryptococcus in all three CSF samples. Nanopore sequencing detected Cryptococcus in two CSF samples. CONCLUSION: Nanopore sequencing may be useful in assisting with the clinical diagnosis of CM. Further research is needed to determine the sensitivity and specificity of nanopore sequencing of CSF.