Real-time Sequencing Research Articles

A Review of the Molecular Understanding of the Mpox Virus (MPXV): Genomics, Immune Evasion, and Therapeutic Targets

The Mpox virus (MPXV), a zoonotic pathogen from the Orthopoxvirus genus, has emerged as a significant global public health concern, especially after the unprecedented outbreak in 2022. This review synthesizes the MPXV’s molecular features, focusing on its genomic structure, replication mechanisms, immune evasion strategies, and implications for diagnostics and therapeutics. The study examines the virus’s genomic organization utilizing recent peer-reviewed literature, highlighting essential genes like OPG027 and D1L, which contribute to host adaptation, increased transmissibility, and immune evasion. Advances in molecular diagnostics, including real-time PCR and genome sequencing, are reviewed, emphasizing their critical role in outbreak monitoring and control. However, challenges persist, such as diagnostic limitations in resource-constrained settings and the lack of targeted vaccines and antivirals. This review discusses new antiviral candidates, confirmed through computational and in vitro techniques, identifying thymidine kinase and VP39 as key therapeutic targets. Emphasizing the need for genomic surveillance to track adaptive evolution, results show that particular mutations, such as in the OPG027 and D1L genes, increase the transmissibility and immune evasion of the MPXV. These molecular revelations highlight the urgent necessity for better diagnostics catered towards addressing present constraints and developing focused treatments that reduce the effect of the virus. This study emphasizes how these results underscore the need for combined public health plans to handle the changing MPXV epidemiology properly.

Comprehensive analysis of the multi-rings mitochondrial genome of Populus tomentosa

BackgroundPopulus tomentosa, known as Chinese white poplar, is indigenous and distributed across large areas of China, where it plays multiple important roles in forestry, agriculture, conservation, and urban horticulture. However, limited accessibility to the mitochondrial (mt) genome of P. tomentosa impedes phylogenetic and population genetic analyses and restricts functional gene research in Salicaceae family.ResultsSingle-molecule real-time (SMRT) sequencing technology was used to sequence, assemble, and annotate the mt genome of P. tomentosa. This genome has a complex structure composed of four circular molecules ranging from 153,004 to 330,873 base pairs (bp). Each of these four circular molecules contains unique gene sequences that constitute the mt genome of P. tomentosa. The mt genome comprises 69 functional genes, including 38 protein-coding genes (PCGs), 26 tRNA genes, and 5 rRNA genes. After removing duplications, 19 different tRNA coding genes remain, though only 10 amino acids can be recognized. The noncoding region constitutes 93.38% of the mt genome, comprising a large number of repetitive sequences, gene spacer regions, and insertion from chloroplast sequences. Specifically, 40 chloroplast-derived sequences, with a total length of 24,381 bp, were identified in P. tomentosa.ConclusionsIn the current study, the results provide mitochondrial genomic evidence for the maternal origin of P. tomentosa and enhance understanding of the gene dialog between organelle genomes, contributing to the conservation and utilization of the genetic resources of P. tomentosa.

Enhancing Neuron Activity Promotes Functional Recovery by Inhibiting Microglia-Mediated Synapse Elimination After Stroke.

Activating glutamatergic neurons in the ipsilesional motor cortex can promote functional recovery after stroke. However, the underlying molecular mechanisms remain unclear. Clarifying key molecular mechanisms involved in recovery could help understand the development of neuromodulation strategies after stroke. Adeno-associated virus 2/9-CamKIIa-hM3Dq-mCherry was injected into ipsilesional motor cortex by stereotaxic in the photothrombotic stroke model. Starting from the third day after the stroke, male mice were injected intraperitoneally with clozapine-N-oxide every day to activate excitatory neurons. C1q-blocking antibody and annexin V were used to inhibit C1q and exposed phosphatidylserine (EPS), respectively. The cylinder test and grid-walking test were performed to evaluate functional recovery. The potential molecular mechanisms of excitatory neuronal activation on microglia-mediated synaptic pruning after stroke by immunofluorescence, real-time polymerase chain reaction, Western blotting, and RNA sequencing. Activating excitatory neurons significantly promoted functional recovery and inhibited microglia-mediated synaptic pruning after stroke. Furthermore, it decreased EPS and C1q levels in synapses. On the contrary, inhibiting excitatory neurons aggravated functional defects, promoted microglia-mediated synaptic pruning, and increased EPS and C1q levels in synapses. Selective blocking of EPS repressed C1q tagging of synapses and microglia-mediated synaptic pruning and improved functional recovery. Meanwhile, blocking EPS markedly rescued synaptic density, and motor function deteriorated by chemogenetic inhibition. In addition, C1q-blocking antibody prevented phosphatidylserine engulfment by microglia. Together, these data provide mechanistic insight into microglia-mediated synapse pruning after neuronal activation after stroke and identify the role of C1q binding to EPS in stroke treatment during the repair phase.

The Complex Epigenetic Panorama in the Multipartite Genome of the Nitrogen-Fixing Bacterium Sinorhizobium meliloti.

In prokaryotes, DNA methylation plays roles in DNA repair, gene expression, cell cycle progression, and immune recognition of foreign DNA. Genome-wide methylation patterns can vary between strains, influencing phenotype, and gene transfer. However, broader evolutionary studies on bacterial epigenomic variation remain limited. In this study, we conducted an epigenomic analysis using single-molecule real-time sequencing on 21 strains of Sinorhizobium meliloti, a facultative plant nitrogen-fixing alphaproteobacterium. This species is notable for its multipartite genome structure, consisting of a chromosome, chromid, and megaplasmid, leading to significant genomic and phenotypic diversity. We identified 16 palindromic and nonpalindromic methylated DNA motifs, including N4-methylcytosine and N6-methyladenine modifications, and analyzed their associated methyltransferases. Some motifs were methylated across all strains, forming a core set of epigenomic signatures, while others exhibited variable methylation frequencies, indicating a dispensable (shell) epigenome. Additionally, we observed differences in methylation frequency between replicons and within coding sequences versus regulatory regions, suggesting that methylation patterns may reflect multipartite genome evolution and influence gene regulation. Overall, our findings reveal extensive epigenomic diversity in S. meliloti, with complex epigenomic signatures varying across replicons and genomic regions. These results enhance our understanding of multipartite genome evolution and highlight the potential role of epigenomic diversity in phenotypic variation.

Leptospirosis Risk Assessment in Rodent Populations and Environmental Reservoirs in Humanitarian Aid Settings in Thailand

Leptospirosis, a global zoonotic disease caused by Leptospira spp., presents high morbidity and mortality risks, especially in tropical regions like Thailand. Military personnel deployed in endemic areas, such as during the Cobra Gold Joint exercise, face heightened exposure. This study assessed Leptospira’s prevalence in rodents and environmental reservoirs at military training sites from 2017 to 2022. A surveillance program was conducted at Engineering Civil Assistance Program (ENCAP) training sites using real-time PCR, dark-field microscopy, and 16S rRNA gene sequencing to detect Leptospira in rodents and environmental samples. Results showed a 1.3% infection rate in rodents (15 of 1161), while Leptospira was detected in 10.2% of water samples (42 of 413) and 23.1% of soil samples (30 of 130). Diverse Leptospira interrogans strains circulated among rodents, and three groups of naturally circulating Leptospira strains were detected in environmental reservoirs. These findings underscore Leptospira’s survival and transmission potential within exercise sites, informing Force Health Protection (FHP) decisions. By integrating pre-exercise data on primary hosts and environmental reservoirs with historical local outbreak records and research on risk factors, this study identifies key areas for public health intervention and potential mitigation strategies.

ENPP2 promotes progression and lipid accumulation via AMPK/SREBP1/FAS pathway in chronic lymphocytic leukemia

BackgroundDisorders of lipid metabolism are critical factors in the progression of chronic lymphocytic leukemia (CLL). However, the characteristics of lipid metabolism and related regulatory mechanisms of CLL remain unclear.MethodsHence, we identified altered metabolites and aberrant lipid metabolism pathways in patients with CLL by ultra-high-performance liquid chromatography-mass spectrometry-based non-targeted lipidomics. A combination of transcriptomics and lipidomics was used to mine relevant target molecule and downstream signaling pathway. In vitro cellular assays, quantitative real-time polymerase chain reaction (qRT-PCR), western blot, fluorescent staining, RNA sequencing, and coimmunoprecipitation were used to monitor the molecular levels as well as to explore the underlying mechanisms.ResultsSignificant differences in the content of 52 lipid species were identified in CLL samples and healthy controls. Functional analysis revealed that alterations in glycerolipid metabolism, glycerophospholipid metabolism, sphingolipid metabolism, and metabolic pathways had the greatest impact on CLL. On the basis of the area under the curve value, a combination of three metabolites (phosphatidylcholine O-24:2_18:2, phosphatidylcholine O-35:3, and lysophosphatidylcholine 34:3) potentially served as a biomarker for the diagnosis of CLL. Furthermore, utilizing integrated lipidomic, transcriptomic, and molecular studies, we reveal that ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2) plays a crucial role in regulating oncogenic lipogenesis. ENPP2 expression was significantly elevated in patients with CLL compared with normal cells and was validated in an independent cohort. Moreover, ENPP2 knockdown and targeted inhibitor PF-8380 treatment exerted an antitumor effect by regulating cell viability, proliferation, apoptosis, cell cycle, and enhanced the drug sensitivity to ibrutinib. Mechanistically, ENPP2 inhibited AMP-activated protein kinase (AMPK) phosphorylation and promoted lipogenesis through the sterol regulatory element-binding transcription factor 1 (SREBP-1)/fatty acid synthase (FAS) signaling pathway to promote lipogenesis.ConclusionsTaken together, our findings unravel the lipid metabolism characteristics of CLL. Moreover, we demonstrate a previously unidentified role and mechanism of ENPP2 in regulation of lipid metabolism, providing a novel therapeutic target for CLL treatment.

Exploring a recurrent neural network (RNN) earned value based model for predicting lost labour value in construction projects

This study explores the application of deep learning techniques in forecasting labour losses in construction projects. By leveraging the predictive capabilities of ManHour Earned and Estimated ManHour values, this research aims to develop an accurate and reliable model for labour loss forecasting. Utilizing a dataset comprising 105 work items from industry practitioners who employed the Leonard and Moselhi methodology, this study investigates the efficacy of a Recurrent Neural Network (RNN) architecture with Long Short-Term Memory (LSTM) cells. The model's performance is optimized through backpropagation and hyperparameter tuning, focusing on key parameters such as learning rate, batch size, dropout rate, and number of epochs. To ensure the model's generalizability and mitigate overfitting, regularization strategies and performance monitoring are employed. The predictive potential of the model is evaluated through integration into a real-time sequence production job and deployment into an enabled environment. Results demonstrate the model's capacity for accurate labour loss forecasting, enabling proactive measures to enhance productivity and minimize unnecessary claims. Although this study focuses on building projects, its findings have broader implications for process and manufacturing engineering, where productivity losses and associated labour losses are significant concerns. The practical application of this research lies in its potential to assess and counter labour loss claims, disputes, warrants, and demands in construction projects, ultimately contributing to improved project management and reduced financial losses.

Characterization of Hepatitis B Virus Transcripts in Chronically HBV-Infected Chimpanzees and Patients Treated with ARC-520 siRNA Demonstrates Transcriptional Silencing of cccDNA.

Full-length hepatitis B virus (HBV) transcripts of chimpanzees and patients treated with multidose (MD) HBV siRNA ARC-520 and entecavir (ETV) were characterized by single-molecule real-time (SMRT) sequencing, identifying multiple types of transcripts with the potential to encode HBx, HBsAg, HBeAg, core, and polymerase, as well as transcripts likely to be derived from dimers of dslDNA, and these differed between HBeAg-positive (HBeAg+) and HBeAg-negative (HBeAg-) individuals. HBV transcripts from the last follow-up ~30 months post-ARC-520 treatment were categorized from one HBeAg+ (one of two previously highly viremic patients that became HBeAg- upon treatment and had greatly reduced cccDNA products) and four HBeAg- patients. The previously HBeAg+ patient received a biopsy that revealed that he had 3.4 copies/cell cccDNA (two to three orders of magnitude more cccDNA than HBeAg- chimpanzees) but expressed primarily truncated X and HBsAg from iDNA, like two patients that were HBeAg- at the start of the study and had one copy/cell cccDNA. No HBV transcripts were detected in two other HBeAg- patients that had ~0.3 copies/cell cccDNA, one of which had seroconverted for HBsAg. The paucity of cccDNA-derived transcripts in the presence of high cccDNA demonstrates the transcriptional silencing of HBV following MD siRNA treatment with ETV.

Comparative analysis of the human microbiome from four different regions of China and machine learning-based geographical inference.

The human microbiome, the community of microorganisms that reside on and inside the human body, is critically important for health and disease. However, it is influenced by various factors and may vary among individuals residing in distinct geographic regions. In this study, 220 samples, consisting of sterile swabs from palmar skin and oral and nasal cavities were collected from Chinese Han individuals living in Shanghai, Chifeng, Kunming, and Urumqi, representing the geographic regions of east, northeast, southwest, and northwest China. The full-length 16S rRNA gene of the microbiota in each sample was sequenced using the PacBio single-molecule real-time sequencing platform, followed by clustering the sequences into operational taxonomic units (OTUs). The analysis revealed significant differences in microbial communities among the four regions. Cutibacterium was the most abundant bacterium in palmar samples from Shanghai and Kunming, Psychrobacter in Chifeng samples, and Psychrobacillus in Urumqi samples. Additionally, Streptococcus and Staphylococcus were the dominant bacteria in the oral and nasal cavities. Individuals from the four regions could be distinguished and predicted based on a model constructed using the random forest algorithm, with the predictive effect of palmar microbiota being better than that of oral and nasal cavities. The prediction accuracy using hypervariable regions (V3-V4 and V4-V5) was comparable with that of using the entire 16S rRNA. Overall, our study highlights the distinctiveness of the human microbiome in individuals living in these four regions. Furthermore, the microbiome can serve as a biomarker for geographic origin inference, which has immense application value in forensic science.IMPORTANCEMicrobial communities in human hosts play a significant role in health and disease, varying in species, quantity, and composition due to factors such as gender, ethnicity, health status, lifestyle, and living environment. The characteristics of microbial composition at various body sites of individuals from different regions remain largely unexplored. This study utilized single-molecule real-time sequencing technology to detect the entire 16S rRNA gene of bacteria residing in the palmar skin, oral, and nasal cavities of Han individuals from four regions in China. The composition and structure of the bacteria at these three body sites were well characterized and found to differ regionally. The results elucidate the differences in bacterial communities colonizing these body sites across different regions and reveal the influence of geographical factors on human bacteria. These findings not only contribute to a deeper understanding of the diversity and geographical distribution of human bacteria but also enrich the microbiome data of the Asian population for further studies.

Construction of the time since deposition (TsD) model in saliva stains with 16S rRNA full-length sequencing technology and microbial markers.

Determining the time since deposition (TsD) and sex of saliva stains is crucial for revealing the time of the crime's occurrence and clarifying the nature of the crime. This process not only shortens the time required to solve the case but also helps narrow down the scope of investigation, thereby enhancing the efficiency of case resolution. Currently, the forensic study of the microbial composition in long-term saliva stains remains a relatively underexplored field. The purpose of this study was to explore the succession pattern of long-placed human saliva stains microbial communities and identify relevant microbial markers for estimating TsD and identifying the sex of the donor, in order to be an effective alternative tool for solving practical forensic cases. Therefore, in this study, saliva stains exposed to indoor environmental conditions for up to 140days were collected and 16S rRNA full-length sequencing was performed using single-molecule real-time sequencing technology based on the PacBio sequencing platform. The study reveals that after 140days of placement, the relative abundance of Firmicutes significantly decreased (p = 0.00304). At the genus level, the relative abundances of Streptococcus (p = 0.0008), Rothia (p = 0.0448), Gemella (p = 0.016), and Veillonella (p = 0.0208) also significantly decreased. Additionally, significant differences were found in the microbial communities between saliva stains from males and females (p = 0.00013). Then, we constructed a TsD estimating model for microbial community markers based on random forest, and the results showed that the mean absolute error was 9.59days, and the accuracy of sex classification model based on stepwise logistic regression model and 4 bacterial markers was 84.21%. This indicates that saliva stains that have been in place for a long time still retain significant forensic value, and microbial markers can be used to determine the time since deposition (TsD) of dried saliva stains as well as to identify the sex of the donor.

Improvement of mathematical methods for ensuring security based on real-time video sequence analysis

Purpose of research. Currently, mathematical methods of video sequence analysis represent a structured set of approaches to image recognition based on the difference in the glow of different image areas. Many of these values are described using mathematical dependencies, however, existing approaches work only for standard images obtained during video data processing. The purpose of this study is to develop a new approach to analyzing images obtained, including those using terahertz radiation, which has specific characteristics, both physical and mathematical.Methods. The following theoretical and empirical scientific methods were used in this study. Analysis (the analysis of the currently known mathematical methods of image processing in order to recognize images is carried out). Synthesis (a fundamentally new approach to security systems is proposed, which is a single system consisting of separate interconnected subsystems). Modeling (an information model of a security system based on ACS has been developed using a system for analyzing and recognizing potentially dangerous objects based on a real-time video stream).Mathematization (the image analysis system is described in the language of mathematical laws and formulas).Results. As a result of the research based on the analysis of modern materials, the concept of a security system based on real-time video sequence analysis with the use of advanced object scanning technologies is proposed in the future. As the main innovation, an improved Viola-Jones image analysis method is proposed using an additional set characterizing the feature space of objects in the terahertz radiation range.Conclusion. The use of high-frequency scanning technologies with intelligent object image recognition systems in real time will significantly reduce the risks of intruders entering protected facilities, as well as increase the safety of citizens with relatively low costs for the development and implementation of upgraded security systems.

Full-length transcriptome-referenced analysis reveals developmental and olfactory regulatory genes in Dermestes frischii.

Dermestes frischii Kugelann, 1792 is a storage pest worldwide, and is important for estimating the postmortem interval in forensic entomology. However, because of the lack of transcriptome and genome resources, population genetics and biological control studies on D. frischii have been hindered. Here, single-molecule real-time sequencing and next-generation sequencing were combined to generate the full-length transcriptome of the five developmental stages of D. frischii, namely egg, young larva, mature larva, pupa and adult. A total of 41,665 full-length non-chimeric sequences and 59,385 non-redundant transcripts were generated, of which 42,756 were annotated in public databases. Using the weighted gene co-expression network analysis, gene co-expression modules related to the five developmental stages were constructed and screened, and the genes in these modules were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. The expression patterns of the differentially expressed genes (DEGs) related to olfaction and insect hormone biosynthesis were also explored. Transcription of most odorant binding proteins was up-regulated in the adult stage, suggesting they are important for foraging in adults. Many genes encoding for the ecdysone-inducible protein were up-regulated in the pupal stage, may be mainly responsible for the tissue remodelling of metamorphosis. The results of the quantitative real-time polymerase chain reaction (qRT-PCR) were consistent with the RNA-seq results. This is the first full-length transcriptome sequencing of dermestids, and the data obtained here are vital for understanding the stage-specific development and olfactory system of D. frischii, providing valuable resources for storage pest and forensic research.

Revealing long-range heterogeneous organization of nucleoproteins with N6-methyladenine footprinting.

A major challenge in epigenetics is uncovering the dynamic distribution of nucleosomes and other DNA-binding proteins, which plays a crucial role in regulating cellular functions. Established approaches such as ATAC-seq, ChIP-seq, and CUT&RUN provide valuable insights but are limited by the ensemble nature of their data, masking the cellular and molecular heterogeneity that is often functionally significant. Recently, long-read sequencing technologies, particularly Single Molecule, Real-Time (SMRT/PacBio) sequencing, have introduced transformative capabilities, such as N6-methyladenine (6mA) footprinting. This technique enables the detection of joint binding events and long-range chromatin organization on individual DNA molecules spanning multiple kilobases. Despite the potential of 6mA footprinting, existing analytical tools for 6mA detection in SMRT sequencing data suffer from significant limitations in both performance and applicability, especially with the latest sequencing platform. To address these gaps, we developed a novel 6mA-calling pipeline based on polymerase kinetics analysis. Our approach significantly outperforms current tools in terms of accuracy and computational efficiency, setting a new benchmark for 6mA detection. Utilizing our optimized experimental and computational framework, we extensively mapped nucleosome positioning and transcription factor occupancy at the single-molecule level, revealing critical features of the transcription-associated epigenetic landscape. Additionally, our work established high-resolution, long-range binding events in mitochondrial DNA, revealing simultaneous loading of two sets of replication machinery onto the displacement loop (D-loop). Our study highlights the potential of 6mA footprinting in capturing the coordinate binding of nucleoproteins and unraveling heterogeneous epigenetic states with unprecedented resolution.

Development of gene-in-plasmid DNA reference materials certified by single-molecule counting.

The mole, the SI unit for measuring the amount of a substance, was redefined as a fixed number of entities. This definition enables straightforward quantification of substances by counting individual entities. Counting proves particularly effective for quantifying large and discrete biological entities such as DNA, proteins, viruses, and cells, which are challenging to quantify via traditional physical or chemical methods. In this study, we detail our approach to develop gene reference materials certified through single-molecule counting, which enables mole-traceable measurements. We quantified three plasmid DNA constructs, each carrying a specific gene of interest, via single-molecule counting. The resulting values were cross-validated via digital PCR and LC‒MS. Sequence impurities in the certified reference materials were quantified via single-molecule real-time sequencing, whereas fragment impurities were quantified via two-color digital PCR analysis. We precisely accounted for various sources of uncertainty, including measurement precision, weighing, homogeneity, and impurities, when estimating the total uncertainty of the reference materials. In conclusion, a practical format for gene-based DNA reference materials, a measurement method to achieve metrological traceability, and methods for quantifying fragments and sequence impurities were developed and implemented in this study. We anticipate that our gene-based DNA reference materials will serve as valuable higher-order standards for the calibration of other methods or reference materials for DNA quantification in a variety of bioanalytical applications.

Distribution of HPV Types in Cervical Cancer in Pakistan: Implications for Screening and Vaccination Programs.

HPV plays a key role in the development of cervical cancer. This study aims to investigate the prevalence of HPV genotypes in patients with Squamous cell carcinoma (SSC) and Adenocarcinoma (ADC) at NORI cancer Hospital Pakistan, with the aim of improving screening and prevention strategies. Cervical scrapings were collected from 129 diagnosed cervical cancer patients. HPV typing was performed using a real-time PCR assay and sequencing. Among the patients, 73.6% (90/129) were HPV positive. Proportion of HPV positivity was observed within each group. The highest incidence of HPV was observed in the 50-60 years age group (80.9%), followed by the 40-to-50-year group (75.8%). The positivity rate declined in the 60-to-70-year-old (63.6%) and further in the 70-80 years (62.5%). Eight different HPV subtypes were identified, with HPV 16 being the most prevalent (80.0%), followed by HPV 18 (9.5%), HPV 45 in 2 (2.1%), HPV 31 in 1 (1.1%), HPV 35 in 1 (1.1%), HPV 59 in 1 (1.1%), HPV 66 in 1 (1.1%), and HPV 89 in 1 (1.15). Histologically, 89.2% of the cases were Squamous cell carcinoma (SCC) and 10.8% were Adenocarcinomas. In SCC patients, HPV 16 was found in 80.9% of cases, while in Adenocarcinoma patients, HPV 16 was detected in 66.7% of cases. The prevalence of high-risk HPV types, both vaccine and non-vaccine, targeted in Pakistan highlights the urgent need for widespread screening and vaccination programs. Tailored public health strategies are essential to effectively reduce cervical cancer rates and mortality.

Comprehensive pathogen identification and antimicrobial resistance prediction from positive blood cultures using nanopore sequencing technology

BackgroundBlood cultures are essential for diagnosing bloodstream infections, but current phenotypic tests for antimicrobial resistance (AMR) provide limited information. Oxford Nanopore Technologies introduces nanopore sequencing with adaptive sampling, capable of real-time host genome depletion, yet its application directly from blood cultures remains unexplored. This study aimed to identify pathogens and predict AMR using nanopore sequencing.MethodsIn this cross-sectional genomic study, 458 positive blood cultures from bloodstream infection patients in central Taiwan were analyzed. Parallel experiments involved routine microbiologic tests and nanopore sequencing with a 15-h run. A bioinformatic pipeline was proposed to analyze the real-time sequencing reads. Subsequently, a comparative analysis was performed to evaluate the performance of species identification and AMR prediction.ResultsThe pipeline identified 76 species, with 88 Escherichia coli, 74 Klebsiella pneumoniae, 43 Staphylococcus aureus, and 9 Candida samples. Novel species were also discovered. Notably, precise species identification was achieved not only for monomicrobial infections but also for polymicrobial infections, which was detected in 23 samples and further confirmed by full-length 16S rRNA amplicon sequencing. Using a modified ResFinder database, AMR predictions showed a categorical agreement rate exceeding 90% (3799/4195) for monomicrobial infections, with minimal very major errors observed for K. pneumoniae (2/186, 1.1%) and S. aureus (1/90, 1.1%).ConclusionsNanopore sequencing with adaptive sampling can directly analyze positive blood cultures, facilitating pathogen detection, AMR prediction, and outbreak investigation. Integrating nanopore sequencing into clinical practices signifies a revolutionary advancement in managing bloodstream infections, offering an effective antimicrobial stewardship strategy, and improving patient outcomes.

Feasibility of fetal cardiac function and anatomy assessment by real-time spiral bSSFP MRI at 0.55T

BackgroundContemporary 0.55T MRI is promising for fetal MRI, due to the larger bore, reduced safety concerns, lower acoustic noise, and improved fast imaging capability. In this work, we explore improved fetal cardiac MRI (CMR) without relying on any synchronizing devices, prospective, or retrospective gating. PurposeTo determine the feasibility of real-time MRI evaluation of fetal cardiac function as well as cardiac and great vessel anatomies by using spiral balanced steady-state free precession (bSSFP) at 0.55T. MethodsA real-time spiral bSSFP pulse sequence for fetal CMR was implemented and optimized on a 0.55T whole-body MRI. Fetal CMR was prospectively performed between May 2022 and August 2023. The protocol included: 1) real-time images at standard cardiac views, for 10-20seconds/view and 40 to 43.6 ms/frame and 2) 4-9 stacks of slices at standard cardiac views that each cover the whole heart, with 15-30 slices/stack, and 2-5seconds/slice, at 320 to 349 ms/frame. Images were evaluated by a fetal cardiologist. Quantitative measurements of cardiothoracic area ratio and cardiac axis were compared with previous reports. Diagnostic accuracy was compared against postnatal echocardiographic findings. ResultsTwenty-nine participants were enrolled for 32 CMR exams, with mean maternal age 33.6±5.8 year (range 22-44 year) and mean gestational age 32.8±3.9 weeks (range 23-38 weeks). The proposed sequence enabled evaluation of the fetal heart in <30minutes in all cases (average 22minutes). Real-time MRI allowed easy adjustment of scan plan, automatic whole-heart volumetric sweeping, and flexible choice of reconstruction temporal resolution. For key cardiac anatomic features 60% were delineated well. Mean cardiothoracic area ratio and cardiac axis were 0.27±0.04 and 45.8±7.8. Diagnostic agreement with postnatal echocardiographic findings was 84%. ConclusionA spiral real-time bSSFP pulse sequence at 0.55T can provide both low framerate and high framerate fetal heart images without relying on maternal breath-hold, specialized gating devices, or cardiac gating. The low framerate images offer high diagnostic quality structural evaluations of the fetal heart, while the high framerate images capture fetal heart motion and may enable functional assessments.

Third generation sequencing transforming plant genome research: Current trends and challenges.

In recent years, third-generation sequencing (TGS) technologies have transformed genomics and transcriptomics research, providing novel opportunities for significant discoveries. The long-read sequencing platforms, with their unique advantages over next-generation sequencing (NGS), including a definitive protocol, reduced operational time, and real-time sequencing, possess the potential to transform plant genomics. TGS optimizes and enhances the efficiency of data analysis by removing the necessity for time-consuming assembly tools. The current review examines the development and application of bioinformatics tools for data analysis and annotation, driven by the rapid advancement of TGS platforms like Oxford Nanopore Technologies and Pacific Biosciences. Transcriptome analysis utilizing TGS has been extensively employed to elucidate complex plant transcriptomes and genomes, particularly those characterized by high frequencies of duplicated genomes and repetitive sequences. As a result, current methodologies that allow for generating transcriptomes and comprehensive whole-genome sequences of complex plant genomes employing tailored hybrid sequencing techniques that integrate NGS and TGS technologies have been emphasized herein. This paper, thus, articulates a vision for a future in which TGS effectively addresses the challenges faced in plant research, offering a comprehensive understanding of its advantages, applications, limitations, and promising prospects.

Cardiac and respiratory activities induce temporal changes in cerebral blood volume, balanced by a mirror CSF volume displacement in the spinal canal.

Understanding cerebrospinal fluid (CSF) dynamics is crucial for elucidating the pathogenesis and diagnosis of neurodegenerative diseases. The primary mechanisms driving CSF oscillations remain a topic of debate. This study investigates whether cerebral blood volume displacement (CBV), modulated by breathing and cardiac activity, is the predominant drivers of CSF oscillations. We examined 12 healthy volunteers (aged 20-34 years) using a clinical 3T MRI scanner to quantify cerebral blood flow at the intracranial level and CSF flow at the C2-C3 spinal level under free and deep breathing conditions, utilizing real-time phase-contrast sequences. We then obtained CBV and CSF volume displacement (CSFV) curves by integrating the flow rate signals. Cardiac and respiratory signals were recorded during acquisition to reconstruct cardiac-driven and breath-driven CBV and CSFV curves. During deep breathing, compared to free breathing, the total cerebral arterial flow rate decreased by 29 % (from 12.5 ml/s to 8.8 ml/s), and the duration of the cardiac cycle period shortened by 15 % (0.90 s to 0.77 s), leading to reductions of 37 % and 23 % in cardiac-driven CBV and CSFV amplitudes, respectively. Conversely, breath-driven CBV and CSFV amplitudes increased substantially by 207 % and 326 %, respectively. Notably, during free breathing, cardiac-driven CBV and CSFV were significantly greater than their breath-driven counterparts; however, during deep breathing, the amplitudes of cardiac-driven and breath-driven CBV and CSFV did not differ significantly. CBV and CSFV curves demonstrated strong coupled inverse oscillation under both breathing conditions, with consistent CSF inflow toward the intracranial compartment during inspiration. This study quantifies the contributions of cardiac and breathing activities to CBV and CSFV under varying breathing patterns, confirming that CBV changes, driven by cardiac and respiratory activities, are strongly inversely coupled with CSF oscillations. These findings enhance our understanding of CSF circulation mechanisms and offer potential diagnostic implications for neurodegenerative diseases.

Intestinal bacterium Bacillus siamensis M54 from Allomyrina dichotoma is a potential biocontrol agent against maize stalk rot

Maize stalk rot, caused by Fusarium spp., is a significant disease that adversely impacts the yield and quality of corn. Biological control plays a crucial role in managing numerous crop diseases, including maize stalk rot. Biocontrol agents are predominantly derived from soil and plant tissues, with limited reports on isolating highly efficient biocontrol agents from insects. In this study, 144 bacterial strains were isolated from the intestinal tract of third instar larvae of Allomyrina dichotoma. Through dual culture tests, twelve strains exhibiting strong antagonism against two maize stalk rot pathogens, F. graminearum and F. verticillioides, were identified. Among them, the M54 strain exhibited the most potent antagonistic effect against the two pathogenic fungi and was identified as Bacillus siamensis through 16S rRNA gene sequence analysis. The complete genome for M54 was assembled using PacBio single-molecule real-time (SMRT) and Illumina sequencing technologies. Whole genome phylogenetic analysis further confirmed M54 was B. siamensis. Microscopic examination revealed that M54 had the ability to inhibit the fungal spore germination and hyphal formation. Furthermore, M54 exhibited effective colonization in the maize rhizosphere and enhanced maize growth. It showed that treatment with M54 significantly suppressed lesion expansion induced by F. graminearum on maize stalks in the seedling and adult plant assays. Genomic analysis using antiSMASH revealed 11 gene clusters for secondary metabolite synthesis. This study provides a novel approach for isolating biocontrol agents to manage plant diseases and highlights B. siamensis M54 as a potential efficient biocontrol agent for maize stalk rot.