Species turnover and functional nestedness constitute the geographic patterns of stream diatoms in the Three Parallel Rivers region, China.

Unraveling biodiversity patterns and their driving processes is paramount in ecology and biogeography. However, there remains a limited understanding regarding the underlying mechanisms of community assembly, particularly in alpine streams where significant elevation gradients and habitat heterogeneity exist. We investigated the patterns and drivers of beta diversity, explicitly focusing on taxonomic and functional diversity, in the three parallel rivers region in China. We employed a beta diversity partitioning approach to examine the turnover and nestedness components of beta diversity and further deconstructed the diatom community into attached and unattached groups. Our results revealed distinct diversity patterns and drivers for taxonomic and functional beta diversity. Specifically, taxonomic beta diversity was mainly driven by the turnover component affected by spatial processes, whereas functional beta diversity was dominated by the nestedness component affected by environmental processes. Furthermore, our analysis of the division of the whole communities demonstrated that the varying responses of benthic diatoms with different attached abilities to environmental filtering, dispersal limitation, and directional flow were the essential reasons for shaping the biodiversity patterns of species turnover and functional nestedness in the alpine stream. Our findings suggested that partitioning beta diversity and dividing the entire community can more deeply infer underlying community assembly processes, thereby providing valuable insights into understanding biodiversity patterns, drivers, and conservation strategies.

Analysis of bacterial transcriptome and epitranscriptome using nanopore direct RNA sequencing.

Bacterial gene expression is a complex process involving extensive regulatory mechanisms. Along with growing interests in this field, Nanopore Direct RNA Sequencing (DRS) provides a promising platform for rapid and comprehensive characterization of bacterial RNA biology. However, the DRS of bacterial RNA is currently deficient in the yield of mRNA-mapping reads and has yet to be exploited for transcriptome-wide RNA modification mapping. Here, we showed that pre-processing of bacterial total RNA (size selection followed by ribosomal RNA depletion and polyadenylation) guaranteed high throughputs of sequencing data and considerably increased the amount of mRNA reads. This way, complex transcriptome architectures were reconstructed for Escherichia coli and Staphylococcus aureus and extended the boundaries of 225 known E. coli operons and 89 defined S. aureus operons. Utilizing unmodified in vitro-transcribed (IVT) RNA libraries as a negative control, several Nanopore-based computational tools globally detected putative modification sites in the E. coli and S. aureus transcriptomes. Combined with Next-Generation Sequencing-based N6-methyladenosine (m6A) detection methods, 75 high-confidence m6A candidates were identified in the E. coli protein-coding transcripts, while none were detected in S. aureus. Altogether, we demonstrated the potential of Nanopore DRS in systematic and convenient transcriptome and epitranscriptome analysis.

Adolescent co-exposure to environmental cadmium and high-fat diet induces cognitive decline via Larp7 m6A-mediated SIRT6 inhibition.

The effects and underlying mechanisms of adolescent exposure to combined environmental hazards on cognitive function remain unclear. Here, using a combined exposure model, we found significant cognitive decline, hippocampal neuronal damage, and neuronal senescence in mice exposed to cadmium (Cd) and high-fat diet (HFD) during adolescence. Furthermore, we observed a significant downregulation of Sirtuin 6 (SIRT6) expression in the hippocampi of co-exposed mice. UBCS039, a specific SIRT6 activator, markedly reversed the above adverse effects. Further investigation revealed that co-exposure obviously reduced the levels of La ribonucleoprotein 7 (LARP7), disrupted the interaction between LARP7 and SIRT6, ultimately decreasing SIRT6 expression in mouse hippocampal neuronal cells. Overexpression of Larp7 reversed the combined exposure-induced SIRT6 decrease and senescence in mouse hippocampal neuronal cells. Additionally, the results showed notably elevated levels of Larp7 m6A and YTH domain family protein 2 (YTHDF2) in mouse hippocampal neuronal cells treated with the combined hazards. Ythdf2 short interfering RNA, RNA immunoprecipitation, and RNA stability assays further demonstrated that YTHDF2 mediated the degradation of Larp7 mRNA under combined exposure. Collectively, adolescent co-exposure to Cd and HFD causes hippocampal senescence and cognitive decline in mice by inhibiting LARP7-mediated SIRT6 expression in an m6A-dependent manner.

Response of soil microbial community structure and function to the sewage leakage: A case study of a 25-year-old cesspool.

Providing many millions of rural households with decentralized sanitation facilities remains challenging. In undeveloped areas, cesspools have still been widely used due to technologically simple and low-cost. However, the influence of cesspools on the surrounding soil remains unclear. In this study, we investigated the influence of a 25-year-old household cesspool on soil physicochemical factors, microbial community composition and function, pathogens and antibiotic resistance genes (ARGs). Soil at the depth around the sewage liquid level (D70) was mostly disturbed where TOC, NO3-N and TP was increased to 16.8 g/kg, 18.2 mg/kg and 1.02 mg/kg respectively. Correspondingly, the element cycling genes of carbon fixation, methanotrophy, nitrogen fixation, ammonia oxidation, and nitrate reduction etc., were increased at D70. Notably, human derived pathogens such as Enterobacter, Salmonella, Pseudomonas aeruginosa, Klebsiella pneumoniae, Prevotella, and Vibrio were highly enriched by 5-10 folders in D70, indicating the potential health risk to human. Mantel tests suggested that EC, TP, pH, NH3-N and particularly NO3-N are important factors that influence the microbial community and element cycling genes in cesspool-affected soil. Overall, this study revealed the impact of household cesspool leakage on the surrounding soil and provided information for the selection and construction of basic sanitation facilities in poor regions.

Effects of Cadmium Stress on Tartary Buckwheat Seedlings.

Cadmium (Cd) is a naturally occurring toxic heavy metal that adversely affects plant germination, growth, and development. While the effects of Cd have been described on many crop species including rice, maize, wheat and barley, few studies are available on cadmium's effect on Tartary buckwheat which is a traditional grain in China. We examined nine genotypes and found that 30 µM of Cd reduced the root length in seedlings by between 4 and 44% and decreased the total biomass by 7 to 31%, compared with Cd-free controls. We identified a significant genotypic variation in sensitivity to Cd stress. Cd treatment decreased the total root length and the emergence and growth of lateral roots, and these changes were significantly greater in the Cd-sensitive genotypes than in tolerant genotypes. Cd resulted in greater wilting and discoloration in sensitive genotypes than in tolerant genotypes and caused more damage to the structure of root and leaf cells. Cd accumulated in the roots and shoots, but the concentrations in the sensitive genotypes were significantly greater than in the more tolerant genotypes. Cd treatment affected nutrient uptake, and the changes in the sensitive genotypes were greater than those in the tolerant genotypes, which could maintain their concentrations closer to the control levels. The induction of SOD, POD, and CAT activities in the roots and shoots was significantly greater in the tolerant genotypes than in the sensitive genotypes. We demonstrated that Cd stress reduced root and shoot growth, decreased plant biomass, disrupted nutrient uptake, altered cell structure, and managed Cd-induced oxidative stress differently in the sensitive and tolerant genotypes of Tartary buckwheat.

Functions of nonsuicidal self-injury and repeated nonsuicidal self-injury among adolescents: A moderating role of addictive features.

OBJECTIVE: The high prevalence and addictive features of nonsuicidal self-injury (NSSI) in adolescents have been documented, but the role of addictive features in the process from NSSI functions to behaviour remains unclear. The major aim of this study was to investigate the effect of addictive features on NSSI functions and the severity of repeated NSSI. METHODS: A total of 10,781 students from primary and middle schools in Chengdu and Karamay were invited to participate in the online cross-sectional survey, and 10,501 completed the survey. Two self-report questionnaires, the Ottawa Self-Injury Inventory (OSI) and the Adolescent Self-Harm Scale (ASHS), were used to collect data from all participants. RESULTS: Among the students, 23.45% and 6.64% reported having engaged in NSSI at least once or at least five times in the past year. Being a girl, being an only child, and being in a single-parent family were significantly associated with more severe NSSI. Addictive features have high value for predicting repeated NSSI. In addition to their significant independent/direct additive effects, addictive features mediated and moderated the relationship between NSSI functions and increased severity of NSSI in adolescents. DISCUSSION AND CONCLUSIONS: The findings suggest that addictive features play a critical role in the development of repeated NSSI in adolescents, which indicates that addiction models may partially explain the mechanism underlying increased severity of NSSI. This may enhance understanding of the reasons for repeated NSSI and inform interventions for repeated NSSI among adolescents.

Nanopore Current Events Magnifier (nanoCEM): a novel tool for visualizing current events at modification sites of nanopore sequencing.

Nanopore sequencing technologies have enabled the direct detection of base modifications in DNA or RNA molecules. Despite these advancements, the tools for visualizing electrical current, essential for analyzing base modifications, are often lacking in clarity and compatibility with diverse nanopore pipelines. Here, we present Nanopore Current Events Magnifier (nanoCEM, https://github.com/lrslab/nanoCEM), a Python command-line tool designed to facilitate the identification of DNA/RNA modification sites through enhanced visualization and statistical analysis. Compatible with the four preprocessing methods including 'f5c resquiggle', 'f5c eventalign', 'Tombo' and 'move table', nanoCEM is applicable to RNA and DNA analysis across multiple flow cell types. By utilizing rescaling techniques and calculating various statistical features, nanoCEM provides more accurate and comparable visualization of current events, allowing researchers to effectively observe differences between samples and showcase the modified sites.

Activation of lipophagy ameliorates cadmium-induced neural tube defects via reducing low density lipoprotein cholesterol levels in mouse placentas.

Neural tube defects (NTDs) represent a prevalent and severe category of congenital anomalies in humans. Cadmium (Cd) is an environmental teratogen known to cause fetal NTDs. However, its underlying mechanisms remain elusive. This study aims to investigate the therapeutic potential of lipophagy in the treatment of NTDs, providing valuable insights for future strategies targeting lipophagy activation as a means to mitigate NTDs.We successfully modeled NTDs by Cd exposure during pregnancy. RNA sequencing was employed to investigate the transcriptomic alterations and functional enrichment of differentially expressed genes in NTD placental tissues. Subsequently, pharmacological/genetic (Atg5-/- placentas) experiments confirmed that inducing placental lipophagy can alleviate Cd induced-NTDs. We found that Cd exposure caused NTDs. Further analyzed transcriptomic data from the placentas with NTDs which revealed significant downregulation of low-density lipoprotein receptor associated protein 1(Lrp1) gene expression responsible for positive regulation of low-density lipoprotein cholesterol (LDL-C) transport. Correspondingly, there was an increase in maternal serum/placenta/amniotic fluid LDL-C content. Subsequently, we have discovered that Cd exposure activated placental lipophagy. Pharmacological/genetic (Atg5-/- placentas) experiments confirmed that inducing placental lipophagy can alleviate Cd induced-NTDs. Furthermore, our findings demonstrate that activation of placental lipophagy effectively counteracts the Cd-induced elevation in LDL-C levels. Lipophagy serves to mitigate Cd-induced NTDs by reducing LDL-C levels within mouse placentas.

Daily process and key characteristics of phytoplankton bloom during a low-water level period in a large subtropical reservoir bay.

Reservoirs, heavily influenced by artificial management, often harbor phytoplankton assemblages dominated by cyanobacteria or dinoflagellates, triggering significant changes in aquatic ecosystems. However, due to limited sampling frequency and insufficient attention to species composition, the bloom processes and key characteristics of phytoplankton community structure have not been systematically elucidated. During the low-water level period when blooms are most likely to occur (June to September) in a tributary bay of the Three Gorges Reservoir, daily sampling was conducted to investigate phytoplankton community composition, identify significant environmental factors, and evaluate important structure characteristics of phytoplankton community. The results showed that Microcystis aeruginosa maintained a clear dominance for almost a month in stage 1, with low Shannon and evenness but a high dominance index. Phytoplankton total density and biomass decreased drastically in stage 2, but Microcystis aeruginosa still accounted for some proportion. The highest Shannon and evenness but the lowest dominance index occurred in stage 3. Peridiniopsis niei occurred massively in stage 4, but its dominant advantages lasted only one to two days. NH4-N was responsible for the dominance of Microcystis aeruginosa, while TP and PO4-P was responsible for the dominance of Peridiniopsis niei; however, precipitation contributed to their drastic decrease or disappearance to some extent. The TN : TP ratio could be considered as an important indicator to determine whether Microcystis aeruginosa or Peridiniopsis niei dominated the phytoplankton community. Throughout the study period, physiochemical factors explained more variation in phytoplankton data than meteorological and hydrological factors. Pairwise comparisons revealed an increase in average ß diversity with stage progression, with higher ß diversities based on abundance data than those based on presence/absence data. Repl had a greater effect on ß diversity differences based on presence/absence data, whereas RichDiff had a greater effect on ß diversity differences based on species abundance data. Co-occurrence networks for stage 1 showed the most complex structure, followed by stage 4, while the network for stage 3 was relatively sparse, although the overall community division remained compact. This study provides a useful attempt to explore the status and changes in phytoplankton community structure during the bloom process through high-resolution investigation.

Tackling assay interference associated with small molecules.

Biochemical and cell-based assays are essential to discovering and optimizing efficacious and safe drugs, agrochemicals and cosmetics. However, false assay readouts stemming from colloidal aggregation, chemical reactivity, chelation, light signal attenuation and emission, membrane disruption, and other interference mechanisms remain a considerable challenge in screening synthetic compounds and natural products. To address assay interference, a range of powerful experimental approaches are available and in silico methods are now gaining traction. This Review begins with an overview of the scope and limitations of experimental approaches for tackling assay interference. It then focuses on theoretical methods, discusses strategies for their integration with experimental approaches, and provides recommendations for best practices. The Review closes with a summary of the critical facts and an outlook on potential future developments.

Distinct differences of vertical phytoplankton community structure in mainstream and a tributary bay of the Three Gorges Reservoir, China.

The vertical distribution of phytoplankton plays a crucial role in shaping the dynamics and structure of aquatic communities. In highly dynamic reservoir systems, water level fluctuations significantly affect the physiochemical conditions and the phytoplankton community. However, the specific effects on the vertical characteristics of phytoplankton between the mainstream and the tributary bay of the reservoir remain unstudied. This study investigated the vertical aspects of phytoplankton density, biomass, α and ß diversity through monthly sampling over two years in the mainstream (Chang Jiang, CJ) and a tributary bay (Xiang Xi, XX) of the Three Gorges Reservoir in China. Phytoplankton density and biomass were significantly higher in XX, indicating an increased risk of algal blooms in the tributary. The phytoplankton community in CJ showed more stable species-environment relationships, a lower Shannon index and a higher evenness index, suggesting a relatively simple structure and a more uniform distribution of phytoplankton among different water layers. Conversely, XX showed greater differences between water layers (higher ß diversity), with significant negative correlations with water level and positive correlations with DO difference, dissolved silica (DSi) difference, and stratification. Peak phytoplankton density and biomass, as well as high ß diversity in XX, occurred during periods of decreased water levels with strong stratification in spring and summer. A structural equation model complemented by path analysis revealed that a decrease in water level could increase ß diversity either directly through internal processes with extended residence time or indirectly by modifying stratification and the vertical distribution of DSi in XX. Therefore, a proposed water quality management strategy for XX was to increase the water level or reduce ß diversity by implementing artificial mixing during stratification periods. Overall, this study lies in its comprehensive investigation of the vertical characteristics of the phytoplankton community in both the mainstream and the tributary bay of the Three Gorges Reservoir, elucidating the significant impact of water level fluctuations and providing insights for targeted water quality management strategies in the tributary bay to mitigate potential ecological impacts.

A Pilot Study on a Possible Mechanism behind Olfactory Dysfunction in Parkinson's Disease: The Association of TAAR1 Downregulation with Neuronal Loss and Inflammation along Olfactory Pathway.

Parkinson's disease (PD) is characterized not only by motor symptoms but also by non-motor dysfunctions, such as olfactory impairment; the cause is not fully understood. Our study suggests that neuronal loss and inflammation in brain regions along the olfactory pathway, such as the olfactory bulb (OB) and the piriform cortex (PC), may contribute to olfactory dysfunction in PD mice, which might be related to the downregulation of the trace amine-associated receptor 1 (TAAR1) in these areas. In the striatum, although only a decrease in mRNA level, but not in protein level, of TAAR1 was detected, bioinformatic analyses substantiated its correlation with PD. Moreover, we discovered that neuronal death and inflammation in the OB and the PC in PD mice might be regulated by TAAR through the Bcl-2/caspase3 pathway. This manifested as a decrease of anti-apoptotic protein Bcl-2 and an increase of the pro-apoptotic protein cleaved caspase3, or through regulating astrocytes activity, manifested as the increase of TAAR1 in astrocytes, which might lead to the decreased clearance of glutamate and consequent neurotoxicity. In summary, we have identified a possible mechanism to elucidate the olfactory dysfunction in PD, positing neuronal damage and inflammation due to apoptosis and astrocyte activity along the olfactory pathway in conjunction with the downregulation of TAAR1.

Mechanically Robust Hemostatic Hydrogel Membranes with Programmable Strain-Adaptive Microdomain Entanglement for Wound Treatment in Dynamic Tissues.

Supramolecular hydrogels emerge as a promising paradigm for sutureless wound management. However, their translation is still challenged by the insufficient mechanical robustness in the context of complex wounds in dynamic tissues. Herein, we report a tissue-adhesive supramolecular hydrogel membrane based on biocompatible precursors for dressing wounds in highly dynamic tissues, featuring robust mechanical resilience through programmable strain-adaptive entanglement among microdomains. Specifically, the hydrogels are synthesized by incorporating a long-chain polyurethane segment into a Schiff base-ligated short-chain oxidized cellulose/quaternized chitosan network via acylhydrazone bonding, which readily establishes interpenetrating entangled microdomains in dynamic cross-linked hydrogel matrices to enhance their tear and fatigue resistance against extreme mechanical stresses. After being placed onto dynamic tissues, the hydrogel dressing could efficiently absorb blood to achieve rapid hemostasis. Moreover, metal ions released from ruptured erythrocytes could be scavenged by the Schiff base linkers to form additional ionic bonds, which would trigger the cross-linking of the short-chain components and establish abundant crystalline microdomains, eventually leading to the in situ stiffening of the hydrogels to endure heavy mechanical loads. Benefiting from its hemostatic capacity and strain adaptable mechanical performance, this hydrogel wound dressing shows promise for the clinical management of various traumatic wounds.

Chromosome level genome assembly and transcriptome analysis of E11 cells infected by tilapia lake virus.

The E11 cell line, derived from striped snakehead fish (Channa striata), possesses a distinctive feature: it is persistently infected with a C-type retrovirus. Notably, it exhibits high permissiveness to piscine nodavirus and the emerging tilapia lake virus (TiLV). Despite its popularity in TiLV research, the absence of genome assembly for the E11 cell line and Channa striata has constrained research on host-virus interactions. This study aimed to fill this gap by sequencing, assembling, and annotating the E11 cell line genome. Our efforts yielded a 600.5 Mb genome including 24 chromosomes with a BUSCO score of 98.8%. In addition, the complete proviral DNA sequence of snakehead retrovirus (SnRV) was identified in the E11 cell genome. Comparative genomic analysis between the E11 cell line and another snakehead species Channa argus revealed the loss of many immune-related gene families in the E11 cell genome, indicating a compromised immune response. We also conducted transcriptome analysis of mock- and TiLV-infected E11 cells, unveiling new perspectives on virus-virus and host-virus interactions. The TiLV infection suppressed the high expression of SnRV in E11 cells, and activated some other endogenous retroviruses. The protein-coding gene comparison revealed a pronounced up-regulation of genes involved in immune response, alongside a down-regulation of genes associated with specific metabolic processes. In summary, the genome assembly and annotation of the E11 cell line provide valuable resources to understand the SnRV and facilitate further studies on nodavirus and TiLV. The RNA-seq profiles shed light on the cellular mechanisms employed by fish cells in response to viral challenges, potentially guiding the development of therapeutic strategies against TiLV in aquaculture. This study also provides the first insights into the viral transcriptome profiles of endogenous SnRV and evading TiLV, enhancing our understanding of host-virus interactions in fish.

Comprehensive genomic and plasmid characterization of multidrug-resistant bacterial strains by R10.4.1 nanopore sequencing.

The escalating prevalence of multidrug-resistant (MDR) bacteria pose a significant public health threat. Understanding the genomic features and deciphering the antibiotic resistance profiles of these pathogens is crucial for development of effective surveillance and treatment strategies. In this study, we employed the R10.4.1 nanopore sequencing technology, specifically through the use of the MinION platform, to analyze eight MDR bacterial strains originating from clinical, ecological and food sources. A single 72-hour sequencing run could yield approximately 12 million reads which covered a total of 34 gigabases (Gbp). The nanopore R10.4.1 data was processed using the Flye assembler, successfully assembling the genomes of eight bacterial strains and their 18 plasmids. Notably, the assemblies generated solely from R10.4.1 nanopore data closely matched those from next-generation sequencing data. Diverse antibiotic resistance patterns and specific resistance genes in the test strains were identified. Hospital strains that exhibited multidrug resistance were found to harbor various resistance genes that encode efflux pumps and extended-spectrum ß-lactamases. Environmental and food sources were found to display resistance profiles in a species-specific manner. The composition of structurally complex plasmids in the test strains could also be revealed by analysis of nanopore long reads, which also suggested evidence of horizontal transfer of plasmids between different bacterial species. These findings provide valuable insights into the genetic characteristics of MDR bacteria and demonstrating the practicality of nanopore sequencing technology for detecting of resistance elements in bacterial pathogens.

Genome constitution and evolution of Elymus atratus (Poaceae: Triticeae) inferred from cytogenetic and phylogenetic analysis.

BACKGROUND: Elymus atratus (Nevski) Hand.-Mazz. is perennial hexaploid wheatgrass. It was assigned to the genus Elymus L. sensu stricto based on morphological characters. Its genome constitution has not been disentangled yet. OBJECTIVE: To identify the genome constitution and origin of E. atratus. METHODS: In this study, genomic in situ hybridization and fluorescence in situ hybridization, and phylogenetic analysis based on the Acc1, DMC1 and matK sequences were performed. RESULTS: Genomic in situ hybridization and fluorescence in situ hybridization results reveal that E. atratus 2n = 6x = 42 is composed of 14 St genome chromosomes, 14 H genome chromosomes, and 14 Y genome chromosomes including two H-Y type translocation chromosomes, suggesting that the genome formula of E. atratus is StStYYHH. The phylogenetic analysis based on Acc1 and DMC1 sequences not only shows that the Y genome originated in a separate diploid, but also suggests that Pseudoroegneria (St), Hordeum (H), and a diploid species with Y genome were the potential donors of E. atratus. Data from chloroplast DNA showed that the maternal donor of E. atratus contains the St genome. CONCLUSION: Elymus atratus is an allohexaploid species with StYH genome, which may have originated through the hybridization between an allotetraploid Roegneria (StY) species as the maternal donor and a diploid Hordeum (H) species as the paternal donor.

Multigenerational paternal obesity enhances the susceptibility to male subfertility in offspring via Wt1 N6-methyladenosine modification.

There is strong evidence that obesity is a risk factor for poor semen quality. However, the effects of multigenerational paternal obesity on the susceptibility to cadmium (a reproductive toxicant)-induced spermatogenesis disorders in offspring remain unknown. Here, we show that, in mice, spermatogenesis and retinoic acid levels become progressively lower as the number of generations exposed to a high-fat diet increase. Furthermore, exposing several generations of mice to a high fat diet results in a decrease in the expression of Wt1, a transcription factor upstream of the enzymes that synthesize retinoic acid. These effects can be rescued by injecting adeno-associated virus 9-Wt1 into the mouse testes of the offspring. Additionally, multigenerational paternal high-fat diet progressively increases METTL3 and Wt1 N6-methyladenosine levels in the testes of offspring mice. Mechanistically, treating the fathers with STM2457, a METTL3 inhibitor, restores obesity-reduced sperm count, and decreases Wt1 N6-methyladenosine level in the mouse testes of the offspring. A case-controlled study shows that human donors who are overweight or obese exhibit elevated N6-methyladenosine levels in sperm and decreased sperm concentration. Collectively, these results indicate that multigenerational paternal obesity enhances the susceptibility of the offspring to spermatogenesis disorders by increasing METTL3-mediated Wt1 N6-methyladenosine modification.

Establishment and Characterization of an Epstein-Barr Virus-positive Cell Line from a Non-keratinizing Differentiated Primary Nasopharyngeal Carcinoma.

Nasopharyngeal carcinoma (NPC), a cancer that is etiologically associated with the Epstein-Barr virus (EBV), is endemic in Southern China and Southeast Asia. The scarcity of representative NPC cell lines owing to the frequent loss of EBV episomes following prolonged propagation and compromised authenticity of previous models underscores the critical need for new EBV-positive NPC models. Herein, we describe the establishment of a new EBV-positive NPC cell line, designated NPC268 from a primary non-keratinizing, differentiated NPC tissue. NPC268 can undergo productive lytic reactivation of EBV and is highly tumorigenic in immunodeficient mice. Whole-genome sequencing revealed close similarities with the tissue of origin, including large chromosomal rearrangements, while whole-genome bisulfite sequencing and RNA sequencing demonstrated a hypomethylated genome and enrichment in immune-related pathways, respectively. Drug screening of NPC268 together with six other NPC cell lines using 339 compounds, representing the largest high-throughput drug testing in NPC, revealed biomarkers associated with specific drug classes. NPC268 represents the first and only available EBV-positive non-keratinizing differentiated NPC model, and extensive genomic, methylomic, transcriptomic, and drug response data should facilitate research in EBV and NPC, where current models are limited. SIGNIFICANCE: NPC268 is the first and only EBV-positive cell line derived from a primary non-keratinizing, differentiated nasopharyngeal carcinoma, an understudied but important subtype in Southeast Asian countries. This model adds to the limited number of authentic EBV-positive lines globally that will facilitate mechanistic studies and drug development for NPC.

The prevention effect of pulsed electromagnetic fields treatment on senile osteoporosis in vivo via improving the inflammatory bone microenvironment.

This study aimed to assess PEMF in a rat model of senile osteoporosis and its relationship with NLRP3-mediated low-grade inflammation in the bone marrow microenvironment. A total of 24 Sprague Dawley (SD) rats were included in this study. Sixteen of them were 24-month natural-aged male SD rats, which were randomly distributed into the Aged group and the PEMF group (n = 8 per group). The remaining 8 3-month -old rats were used as the Young positive control group (n = 8). Rats in the PEMF group received 12 weeks of PEMF with 40 min/day, five days per week, while the other rats received placebo PEMF intervention. Bone mineral density/microarchitecture, serum levels of CTX-1 and P1CP, and NLRP3-related signaling genes and proteins in rat bone marrow were then analyzed. The 12-week of PEMF showed significant mitigation of aging-induced bone loss and bone microarchitecture deterioration, i.e. PEMF increased the bone mineral density of the proximal femur and L5 vertebral body and improved parameters of the proximal tibia and L4 vertebral body. Further analysis showed that PEMF reversed aging-induced bone turnover, specifically, decreased serum CTX-1 and elevated serum P1CP. Furthermore, PEMF also dramatically inhibited NLRP3-mediated low-grade inflammation in the bone marrow, i.e. PEMF inhibited the levels of NLRP3, proCaspase1, cleaved Caspase1, IL-1ß, and GSDMD-N. The study demonstrated that PEMF could mitigate the aging-induced bone loss and reverses the deterioration of bone microarchitecture probably through inhibiting NLRP3-mediated low-grade chronic inflammation to improve the inflammatory bone microenvironment in aged rats.

Utilization of nanopore direct RNA sequencing to analyze viral RNA modifications.

Modifications on viral RNAs (vRNAs), either genomic RNAs or RNA transcripts, have complex effects on the viral life cycle and cellular responses to viral infection. The advent of Oxford Nanopore Technologies Direct RNA Sequencing provides a new strategy for studying RNA modifications. To this end, multiple computational tools have been developed, but a systemic evaluation of their performance in mapping vRNA modifications is lacking. Here, 10 computational tools were tested using the Sindbis virus (SINV) RNAs isolated from infected mammalian (BHK-21) or mosquito (C6/36) cells, with in vitro-transcribed RNAs serving as modification-free control. Three single-mode approaches were shown to be inapplicable in the viral context, and three out of seven comparative methods required cutoff adjustments to reduce false-positive predictions. Utilizing optimized cutoffs, an integrated analysis of comparative tools suggested that the intersected predictions of Tombo_com and xPore were significantly enriched compared with the background. Consequently, a pipeline integrating Tombo_com and xPore was proposed for vRNA modification detection; the performance of which was supported by N6-methyladenosine prediction in severe acute respiratory syndrome coronavirus 2 RNAs using publicly available data. When applied to SINV RNAs, this pipeline revealed more intensive modifications in subgenomic RNAs than in genomic RNAs. Modified uridines were frequently identified, exhibiting substantive overlapping between vRNAs generated in different cell lines. On the other hand, the interpretation of other modifications remained unclear, underlining the limitations of the current computational tools despite their notable potential.IMPORTANCEComputational approaches utilizing Oxford Nanopore Technologies Direct RNA Sequencing data were almost exclusively designed to map eukaryotic epitranscriptomes. Therefore, extra caution must be exercised when using these tools to detect vRNA modifications, as in most cases, vRNA modification profiles should be regarded as unknown epitranscriptomes without prior knowledge. Here, we comprehensively evaluated the performance of 10 computational tools in detecting vRNA modification sites. All tested single-mode methods failed to differentiate native and in vitro-transcribed samples. Using optimized cutoff values, seven tested comparative tools generated very different predictions. An integrated analysis showed significant enrichment of Tombo_com and xPore predictions against the background. A pipeline for vRNA modification detection was proposed accordingly and applied to Sindbis virus RNAs. In conclusion, our study underscores the need for the careful application of computational tools to analyze viral epitranscriptomics. It also offers insights into alphaviral RNA modifications, although further validation is required.