Illumina HiSeq Research Articles

Genetic variation analysis of Guanling cattle based on whole-genome resequencing.

The objective of this study was to unravel the genetic traits of Guanling cattle, pinpoint genes advantageous for muscle growth, and lay a foundation for the preservation of genetic diversity and further analysis of regulation mechanism of important economic traits in local cattle breed. In this study, we sequenced the whole genome of 3 Guanling cattle in Guizhou province using the Illumina HiSeq cBo sequencing platform. And, high- multiplex polymerase chain reaction technology was employed to detect high-quality single nucleotide polymorphism (SNP) sites of other 55 Guanling cattle. Our study identified 166,411 non-synonymous SNPs (nsSNPs) and 42,423 insertions and deletions (indels). Through SNP annotation, gene function enrichment analysis, and comparing with Simmental, Angus, and Limousin cattle, we identified six genes (LEPR, AKAP9, SIX4, SPIDR, PRG4, FASN) which are potentially influential on meat quality traits, playing crucial roles in muscle growth, fat metabolism, and bodily support. We also examined polymorphisms at seven SNP sites in Guanling cattle and found that all seven were in Hardy-Weinberg equilibrium. These findings suggested that these gene sites are stable and widespread in the Guanling cattle population. Our research lays the groundwork for future genetic enhancement and variety identification of Guanling cattle.

Ectopic expression of a truncated NLR gene from wild Arachis enhances resistance to Fusarium oxysporum

Fusarium oxysporum causes devastating vascular wilt diseases in numerous crop species, resulting in substantial yield losses. The Arabidopsis thaliana-F. oxysporum f.sp. conglutinans (FOC) model system enables the identification of meaningful genotype–phenotype correlations and was applied in this study to evaluate the effects of overexpressing an NLR gene (AsTIR19) from Arachis stenosperma against pathogen infection. AsTIR19 overexpression (OE) lines exhibited enhanced resistance to FOC without any discernible phenotype penalties. To elucidate the underlying resistance mechanisms mediated by AsTIR19 overexpression, we conducted whole transcriptome sequencing of an AsTIR19-OE line and non-transgenic wild-type (WT) plants inoculated and non-inoculated with FOC using Illumina HiSeq4000. Comparative analysis revealed 778 differentially expressed genes (DEGs) attributed to transgene overexpression, while fungal inoculation induced 434 DEGs in the OE line, with many falling into defense-related Gene Ontology (GO) categories. GO and KEGG enrichment analysis showed that DEGs were enriched in the phenylpropanoid and flavonoid pathways in the OE plants. This comprehensive transcriptomic analysis underscores how AsTIR19 overexpression reprograms transcriptional networks, modulating the expression of stress-responsive genes across diverse metabolic pathways. These findings provide valuable insights into the molecular mechanisms underlying the role of this NLR gene under stress conditions, highlighting its potential to enhance resistance to Fusarium oxysporum.

The chloroplast genome sequence and phylogenetic analysis of Rubia alata Wall and Rubia ovatifolia Z. Ying Zhang. (Rubiaceae).

Rubia alata Wall (R. alata) and Rubia ovatifolia Z. Ying Zhang (R. ovatifolia) are unique medicinal plants native to China. Sequencing their chloroplast genomes is important for understanding species differentiation and establishing phylogenetic relationships. The chloroplast genomes of R. alata and R. ovatifolia were sequenced using the Illumina HiSeq platform. The chloroplast genome of R. alata is 154,973 base pairs (bp) in length, containing a large single-copy region (LSC) of 84,801bp, a small single-copy region (SSC) of 17,138bp, and a pair of inverted repeats (IRs) of the same length. The length of the chloroplast genome, LSC, SSC, and IR regions of R. ovatifolia is 26,517bp, 84,716bp, 17,116bp and 26,517bp, respectively. Codon usag e analysis revealed that R. alata had the highest frequency of Aspartic acid (Asp) (1650 occurrences) in protein-coding sequences (CDS), while R. ovatifolia showed the highest frequency of Tyrosine (Try) (1479 occurrences). Comparative analysis of chloroplast genomes across seven species from the genus Rubia identified the most divergent coding regions, including rps16, psbI-trns-CGA, and petN, while plastid rRNAs were the most conserved. Phylogenetic analysis showed R. alata clustering with R. cordifolia (66.3% support), and R. ovatifolia clustering with Rubia podantha (100% support). These findings enhance our understanding of the chloroplast genome structure in Rubia species and provide molecular information for the future development and utilization of R. alata and R. ovatifolia resources.

Microencapsulated Microbial Seed Coating Could Improve Soil Environment and Maize Grain Yield in Saline Soil

Soil salinization is one of the major challenges for modern agriculture, posing a great threat to soil health and food security. Field experiments were conducted to evaluate the effect of seed coating on soil environment and maize growth in saline soils. Three treatments were applied to maize seeds: coating with a microencapsulated microbial agent (ME), coating with microbial only (MB), and no coating (CK). High-throughput sequencing of soil bacterial and fungal 16S and ITS rRNA genes was performed using the Illumina HiSeq platform to analyze the effects of these treatments on soil bacterial and fungal diversity and community structure. Additionally, the influence of different treatments on endogenous hormones and yield of maize were investigated. It was found that the coating with a microencapsulated microbial agent led to decreases in pH and electrical conductivity (EC), while increasing the content of soil available phosphorus. This coating improved soil microbial diversity, significantly increasing the relative abundance of the main bacteria genera, Bacillus (34.9%), and the main fungal genera, Mortierella (190.4%). The treatment also significantly enhanced indole-3-acetic acid (IAA) by 51.2%, contributing to improvements in resistance to salt stress. The germination rate increased by 22.9%, the 100-grain weight increased by 12.7%, and grain yield increased by 14.3%. The use of the microencapsulated microbial agent effectively mitigated the adverse effects of salt stress on maize plants. This approach is beneficial for promoting sustainable agriculture in saline soils.

Chromosome-scale genome assembly of Apocynum pictum, a drought-tolerant medicinal plant from the Tarim Basin.

Apocynum pictum Schrenk is a semishrub of the Apocynaceae family with a wide distribution throughout the Tarim Basin that holds significant ecological, medicinal, and economic values. Here, we report the assembly of its chromosome-level reference genome using Nanopore long-read, Illumina HiSeq paired-end, and high-throughput chromosome conformation capture sequencing. The final assembly is 225.32 Mb in length with a scaffold N50 of 19.64 Mb. It contains 23,147 protein-coding genes across 11 chromosomes, 21,148 of which (91.36%) have protein functional annotations. Comparative genomics analysis revealed that A. pictum diverged from the closely related species Apocynum venetum approximately 2.2 million years ago and has not undergone additional polyploidizations after the core eudicot WGT-γ event. Karyotype evolution analysis was used to characterize interchromosomal rearrangements in representative Apocynaceae species and revealed that several A. pictum chromosomes were derived entirely from single chromosomes of the ancestral eudicot karyotype. Finally, we identified 50 members of the well-known stress-responsive WRKY transcription factor family and used transcriptomic data to document changes in their expression at 2 stages of drought stress, identifying a number of promising candidate genes. Overall, this study provides high-quality genomic resources for evolutionary and comparative genomics of the Apocynaceae, as well as initial molecular insights into the drought adaptation of this valuable desert plant.

New approach to agro-industrial solid and liquid waste management: Performance of an EGSB reactor at different hydraulic retention times for methane production

This study investigated the removal of agro-industrial wastes (5 g COD L⁻1 from coffee and 0.5 g COD L⁻1 from brewery wastewater, plus 1 g L⁻1 of coffee pulp and husk) in a continuous Expanded Granular Sludge Bed (EGSB) reactor at 35 °C. The effect of Hydraulic Retention Times (HRTs) of 72h, 48h, and 24h on CH₄ yield was examined using a mixed culture of cattle manure and granular sludge. Methane yields were 201, 124.5, and 113.8 mL CH₄ g⁻1 COD for the 1st, 2nd, and 3rd phases, respectively. Volatile fatty acids, particularly acetic acid, increased at lower HRTs. Sequencing of the 16S rRNA gene on the Illumina HiSeq platform revealed a syntrophic relationship between Syntrophorhabdus, Syntrophobacter, and Pseudomonas with methanogens Methanomassiliicoccus, Methanospirillum, and Methanobacterium, aiding in the removal of phenolic compounds. The study suggests that an HRT of 72h is optimal for maximizing CH₄ production in the EGSB reactor.

In Silico Identification of the Laccase-Encoding Gene in the Transcriptome of the Amazon River Prawn Macrobrachium amazonicum (Heller, 1862)

Background: Macrobrachium amazonicum is an opportunistic and omnivorous species that primarily feeds on plant material. Recent studies have shown that Endo-β-1,4-glucanase and Endo-β-1,4-mannanase are expressed in the transcriptome of adult specimens, while juveniles are capable of digesting nutrients from purified cellulose in their diet. In organisms that degrade raw plant material, laccase plays a key role in oxidizing phenolic compounds found in lignin, leading to its depolymerization and increasing access to cellulose and hemicellulose microfibrils. Objective: In this study, we conducted an in silico identification and characterization of the laccase-encoding gene, as this enzyme is linked to lignin biodegradation in herbivorous crustaceans. Methods: We analyzed the transcriptomes of the hepatopancreas from adult M. amazonicum, sequenced using the Illumina HiSeq 2500 platform. Subsequently, bioinformatics analyses were conducted to predict the conserved regions and active sites associated with laccase activity. Results: A complete open reading frame (ORF) of the laccase protein was identified in all datasets, comprising 609 amino acids. The top 40 similarity hits corresponded exclusively to crustaceans such as prawns, crayfish, and crabs (86.3–51.4%), while the highest divergence was observed in relation to fungi, plants, and bacteria. Three conserved domains were detected, along with the complete set of copper-binding centers (T1Cu, T2Cu, and T3Cu). A notable variable residue was methionine, suggesting a reduced redox potential in M. amazonicum laccase. Conclusion: These findings, combined with recent reports on the nutritional requirements of M. amazonicum, contribute to a deeper understanding of the digestive physiology of this species and offer valuable insights into its ability to utilize plant fibers as energy sources.

AMP-activated protein kinase mediates (-)-epigallocatechin-3-gallate (EGCG) to promote lipid synthesis in mastitis cows

Mastitis, a serious threat to the health and milk production function of dairy cows decreases milk quality. Blood from three healthy cows and three mastitis cows were collected in this study and their transcriptome was sequenced using the Illumina HiSeq platform. Differentially expressed genes (DEGs) were screened according to the |log2FoldChange| > 1 and P-value < 0.05 criteria. Pathway enrichment and functional annotation were performed through KEGG and GO analyses. Finally, the mechanism of the AMP-activated protein kinase (AMPK) mediation of (-)-epigallocatechin-3-gallate (EGCG) to promote lipid metabolism in mastitis cows was analyzed in bovine mammary epithelial cells (BMECs). Transcriptome analysis revealed a total of 825 DEGs, with 474 genes showing increased expression and 351 genes showing decreased expression. The KEGG analysis of DEGs revealed that they were mainly linked to tumour necrosis factor, nuclear factor-κB signalling pathway, and lipid metabolism-related signalling pathway, whereas GO functional annotation found that DEGs were enriched in threonine and methionine kinase activity, cellular metabolic processes, and cytoplasm. AMPK expression, which is involved in several lipid metabolism pathways, was downregulated in mastitis cows. The results of in vitro experiments showed that the inhibition of AMPK promoted the expression of lipid synthesis genes in lipopolysaccharide-induced BMECs and that EGCG could promote lipid synthesis by decreasing the expression of AMPK and downregulating the expression of inflammatory factors in inflammatory BMECs. In conclusion, our study demonstrated that AMPK mediated EGCG to inhabit of inflammatory responses and promote of lipid synthesis in inflammatory BMECs.

Pan-Chloroplast Genomes Reveal the Accession-Specific Marker for Gastrodia elata f. glauca.

Gastrodia elata rhizomes have been applied as traditional medicinal materials for thousands of years. In China, G. elata f. elata (red flower and stem, Ge), G. elata f. viridis (green, Gv), and G. elata f. glauca (black, Gg) represent the primary cultivars in artificial cultivation. Although the annual output of G. elata amounts to 150,000 tons, only 20% is Gg. The long production period, low yield, and high quality of Gg led to its extremely high market prices. However, an effective method to identify this crude drug based solely on its morphological or chemical characteristics is lacking. In this study, the complete chloroplast genomes of three G. elata variants were sequenced using the Illumina HiSeq 2500 platform. Another 21 chloroplast genomes from Gastrodia species, which have published in previous reports, were combined and analyzed together. Our results showed that larger genomic sizes, fewer long tandem repeats, and more simple sequence repeats were the major features of the Gg chloroplast genomes. Phylogenetic analysis showed that the Gg samples were separately clustered in a subclade. Moreover, an accession-specific marker was successfully developed and validated for distinguishing additional rhizome samples. Our study provides investigations of the taxonomic relationships of Gastrodia species. The molecular marker will be useful for differentiating Gastrodia products on the market.

Chloroplast genome sequencing and divergence analysis of 18 Pyrus species: insights into intron length polymorphisms and evolutionary processes.

Pears constitute an essential temperate crop and are primarily produced through interspecific hybridization owing to self-incompatibility that complicates their breeding history. To address this, we sequenced the complete chloroplast (cp) genomes of 18 Pyrus and one Malus species using the Illumina HiSeq4000 platform. The cp genomes ranged from 159,885 bp to 160,153bp and exhibited a conserved circular DNA structure with an average GC content of 36.5%. Each cp genome contained 127 genes, including 83 protein-coding, 36 tRNA, and 8 rRNA genes. Divergence analysis with mVISTA showed high conservation in the coding regions and notable variations in the non-coding regions. All species shared 17 intron-containing genes, with ycf3 and clpP each having two introns. Five intron-containing genes (ndhB, rpl2, rps12, trnA-UGC, and trnE-UUC) were located in the inverted repeat regions, while trnL-UAA was located in the large single-copy region, with conserved intron lengths across Pomoideae. We identified polymorphic intron sequences in the rpl22, petB, clpP, ndhA, and rps16 genes and designed primers for these regions. Notably, the two Pyrus ussuriensis accessions Doonggeullebae and Cheongdangrori showed intron-length polymorphisms despite being classified as the same species. Phylogenetic analysis of the cp genome sequences revealed two major clusters, indicating distinct maternal lineages and evolutionary origins. This study underscores the importance of cp gene polymorphisms in P. fauriei, P. calleryana, P. ussuriensis, and P. pyrifolia, providing valuable insights into Pyrus evolution as well as aiding in the conservation and breeding of pear germplasm.

Construction of competitive endogenous RNA network and identification of potential regulatory axis in vascular calcification.

Competitive endogenous RNAs (ceRNA) theory has been proved in numerous biological processes. Nevertheless, there is a lack of research applying the ceRNA theory to the study of vascular calcification (VC) in chronic kidney diseases (CKD). In the present study, a ceRNA network was constructed after conducting transcriptome sequencing of differentially expressed genes, followed by experimental validation to identify a new target for the diagnosis and treatment of vascular calcification. Total RNA was extracted from β-glycerophosphate (β-GP) cultured vascular smooth muscle cells (VSMCs) on Day 7. Illumina HiSeq platform was utilized to build sequencing libraries. GO and KEGG analysis was conducted to identify the function of the differentially expressed genes. Protein-protein interaction (PPI) network was constructed using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database. A ceRNA network was established based on TargetScan, miRDB, miRWALK, and miRanda database. Western blot and qRT-PCR were used to explore the expression level of protein and RNA, respectively. The direct binding sites were confirmed by dual-luciferase reporter assay. In total, 647 differentially expressed lncRNAs and 289 differentially expressed mRNAs were identified (|log2FC| ≥ 1, p < .05). The function of differentially expressed mRNAs was mainly enriched in negative regulation of osteoblast differentiation, regulation of RNA metabolic process, and other typical pathways. The ceRNA network was generated with a total of 107 interaction pairs. The lncRNA Prrc2c/miR-145-5p/Smad3 axis was considered a potential regulatory pathway within the ceRNA network. The regulatory relationship and targets of this ceRNA axis were validated via invitro experiments. For the first time, we found that lncRNA Prrc2c was highly expressed and promoted calcification of VSMCs. Luciferase reporter assay showed that lncRNA Prrc2c could bind miR-145-5p at site 1755-1761. Similarly, luciferase reporter assay showed that miR-145-5p inhibited Smad3 expression by binding to its 3'UTR. Our findings provide a comprehensive examination of the ceRNA networks in vascular smooth muscle cells (VSMCs) treated with high phosphorate. Specifically, we have identified the role of lncRNA Prrc2c in promoting VSMC calcification through the miR-145-5p/Smad3 axis.

Whole genome analysis of Pantoea species identified from sepsis patients in selected Ethiopian referral hospitals: emerging pathogens

BackgroundThe burden of sepsis worsens due to the continuation of emerging pathogens such as multidrug-resistant Pantoea species.MethodsA multicenter study was conducted between October 2019 and September 2020 at four hospitals located in central, southern, and northern parts of Ethiopia. A total of 1416 sepsis patients were recruited and blood cultures were performed. At each study site, positive cultures were characterized by their colony characteristics, gram stain, and conventional biochemical tests. All Pantoea species were identified using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI TOF) and subjected to whole genome sequencing (WGS) using Illumina HiSeq 2500. The phylogeny structure of Pantoea isolates was calculated using IQ-TREE v1.6.12 from single-nucleotide polymorphisms detected by Snippy v.4.6.0 and filtered by Gubbins v.2.3.4. Average nucleotide identity was estimated by using OrthoANI v.0.93.1 on Shovill v.1.1.0 assemblies. Antimicrobial resistance genes and plasmid replicons were detected using ARIBA v.2.14.6. Phylogenetic trees were visualized using iTOLv.6.5.2.ResultsMultiple Pantoea species include: P. dispersa (n = 19), P. septica (n = 1), and a novel Pantoea spp. (n = 1), were identified among sepsis patients. All P. dispersa isolates and the novel Pantoea species were isolated at Dessie Referral Hospital and displayed phylogenetic clonality, including the ubiquity of an IncM1 plasmid and identical antimicrobial resistance (AMR) gene profiles, encoding blaCTX−M−15, blaTEM−1D, blaSCO−1, and aac(3)-lla. The novel Pantoea spp. isolate harboured blaCTX−M−9 and blaTEM−1D and carried an IncN3 plasmid replicon. The P. septica was isolated at Tikur Anbessa Specialized Hospital in Addis Ababa and carried no detectable acquired AMR genes.ConclusionThe emerging Pantoea spp. carrying multiple AMR genes were identified from sepsis patients. Implementation of strong infection prevention strategies and building surveillance capacity with advanced bacteriology laboratories capable of identifying multidrug-resistant emerging pathogens is strongly recommended.

HLA-B allele frequencies and implications for pharmacogenetics in the Kuwaiti population.

We utilized the HLA-HD tool to extract, annotate, and analyse HLA-B alleles from the exome data of 561 Kuwaiti individuals, sequenced on the Illumina HiSeq platform. HLA typing was conducted using the HLA-HD tool with a reference panel from the IPD-IMGT/HLA database. The major HLA-B pharmacogenetic markers were obtained from the HLA Adverse Drug Reaction Database, focusing on alleles with significant ADR associations in published literature. The distribution of HLA-B alleles in the Kuwaiti population revealed that the most frequent alleles were HLA-B*50:01 (10.52%), HLA-B*51:01 (9.89%), HLA-B*08:01 (6.06%), HLA-B*52:01 (4.55%), HLA-B*18:01 (3.92%), and HLA-B*41:01 (3.65%). Notably, alleles HLA-B*13:01, HLA-B*13:02, HLA-B*15:02, HLA-B*15:13, HLA-B*35:02, HLA-B*35:05, HLA-B*38:01, HLA-B*40:02, HLA-B*44:03, HLA-B*51:01, HLA-B*57:01 and HLA-B*58:01 were identified with known associations to various ADRs. For example, HLA-B*51:01 was associated with clindamycin, phenobarbital, and phenytoin, and was found in 18% of individuals. Our study enriches the regional genetic landscape by delineating HLA-B allele variations within Kuwait and across the Arabian Peninsula. This genetic insight, along with the identification of markers previously linked to drug hypersensitivity, provides a foundation for future pharmacogenetic research and potential personalized medicine strategies in the region.

8696 Whole Exome Sequence Analysis in a Naïve Cohort of Congenital Hypopituitarism in a Single Center

Abstract Disclosure: F.Q. Aratani: None. D.P. Di Matteo: None. I.P. de Magalhães: None. P. Frudit: None. L.S. Motomia: None. S.K. Beeby: None. L.R. Barros: None. D.D. Bissegatto: None. L.R. Carvalho: None. Background: Deficiency of one or more pituitary hormones is defined as hypopituitarism. In the literature, molecular diagnosis (MD) is defined in 12% by the Sanger sequencing and large scale sequencing increased this number to 15%. Aim: To perform whole exome sequencing (WES) in a cohort of patients with congenital hypopituitarism (CH) naïve to MD approach. Patients: Nine patients with CH followed in a single Brazilian tertiary center were included. Methods: Libraries were prepared with kits from Agilent and sequenced on Hi-Seq (Illumina). The variants were called following GATK best practices, using the Haplotypecaller and genome version hg38. They were classified in the Franklin by genoox (https://franklin.genoox.com/clinical-db/home). WES that did not pass the platform's initial quality control and variants that did not reach an adequate confidence level (low/medium confidence: VAF &amp;lt; 50%) were excluded. The remaining variants were classified according to ACMG criteria. Results: Three among 9 patients presented allelic variants and aredescribed here. Case 1: A 22-year-old female presented height of 143 cm (-3,13 SDS) at the first visit without signs of puberty, had delayed bone age (DBA), hypoplastic adenohypophysis, interrupted pituitary stalk (IPS) and ectopic neurohypophysis (ENH) at the magnetic resonance imaging (MRI). Hormonal profile showed low E2, GH, FSH and LH. Twoheterozygous missense variants in the ALMS1 gene (c.5450G&amp;gt;A : p.Arg1817GIn; and ALMS1:c.4225G&amp;gt;C: p.Ala1409Pro) were seen in the exome; both classified as VUS according to ACMG criteria. This allelic variant is related to Alstrom Syndrome. Case 2: A 7-year-old female presented with short stature and was diagnosed with GH followed by LH/FSH, TSH and ACTH deficiencies. At MRI presented IPS and ENH. A heterozygous variant in the PROK2 gene c.364C&amp;gt;T p.R122 with a premature stop codon and truncated protein was present and classified as probably pathogenic according to ACMG criteria. This variant was already described with hypogonadotropic hypogonadism (HH) with or without anosmia. Case3: A 15-year-old male was referred with growth hormone deficiency (GHD), DBA, and HH. Growth hormone replacement was started followed by testosterone. Despite delayed puberty, the testes were normal in size. WES showed a variant c.629G&amp;gt;A:p.Trp210*, in hemizygosity in IGSF1 gene and was classified as probably pathogenic according to ACMG criteria. Loss-of-function mutations in IGSF1 may cause an X-linked syndrome of central hypothyroidism, macroorchidism and delayed puberty (delayed rise of testosterone, but normal timing of testicular growth). Conclusion: WES in a cohort of patients with congenital hypopituitarism naïve to molecular diagnosis revealed 33% (3/9) with positive genetic findings, two of them with clinical significance and family segregation according to the genetic inheritance. Presentation: 6/3/2024

Transcriptional response of primary hippocampal neurons following exposure to 3.0 GHz radiofrequency electromagnetic fields.

Exposure to radiofrequency (RF) electromagnetic fields (EMF) has been associated with the modulation of neuronal electrophysiology and synaptic plasticity. Given the potential of these changes to coincide with alterations in gene expression, this study investigated whether a transcriptional response would occur in neurons following exposure to RF-EMF, under both thermal and nonthermal conditions. Rat primary hippocampal neurons (PHNs) underwent either a single (one-time) or a multiple (3-times, once a day) exposures to RF-EMF (3.0 GHz, CW) at two different mean specific absorption rate (SAR) values of 0.57 W/kg or 5.91 W/kg, which induced a temperature change (ΔT °C) of approximately 0.3°C or 3.6°C, respectively. Alteration in transcription in the RF-EMF-exposed PHNs versus the sham counterparts was assessed at 0, 4, and 24 h postexposure via high-throughput RNA sequencing using Illumina HiSeq. 2000. A total of 20 differentially expressed genes (DEGs) exhibited significant upregulation due to RF-EMF exposure, observed only with the high SAR dose that induced a thermal rise. However, the expression of these DEGs was not significant at 24 h postexposure. Our findings confirmed a lack of nonthermal effects on gene expression under low RF-EMF exposure conditions as evaluated. Additionally, the results indicated a slight thermal effect of exposures at the dose nearing the standards threshold of 4 W/kg; however, the effect appeared to be transient. The study suggests that RF-EMF exposures at a level close to the standards threshold, despite inducing mild temperature elevations (i.e., 3-5°C above normal), would not trigger biologically critical cellular changes.

Two novel plasmids harbouring the multiresistance gene cfr in porcine Staphylococcus equorum

BackgroundThe emergence and transmission of the multidrug resistance gene cfr have raised public health concerns worldwide. ObjectivesMultidrug-resistant Staphylococcus equorum isolates can pose a threat to public health. In this study, we have characterised the whole-genome of one Staphylococcus equorum isolate harbouring two distinct cfr-carrying plasmids. MethodsAntimicrobial susceptibility testing was performed by broth microdilution. Genomic DNA was sequenced using both the Illumina HiSeq X Ten and Nanopore MinION platforms. De novo hybrid assembly was performed by Unicycler. Genomic data were assessed by in silico prediction and bioinformatic tools. ResultsStaphylococcus equorum isolate SN42 exhibited resistance or high MICs to linezolid, erythromycin, tetracycline, oxacillin, clindamycin, virginiamycin, tiamulin, chloramphenicol and florfenicol. It carried two cfr-harbouring plasmids: the RepA N-family plasmid pSN42–51 K and the Inc18-family plasmid pSN42–50 K. These two plasmids exhibited low structural similarities to the so far reported cfr-carrying plasmids. Both plasmids harboured an arsenic resistance operon, copper and cadmium resistance genes as well as the lincosamide-pleuromutilin-streptogramin A resistance gene lsa(B). In addition, plasmid pSN42–51 K carried two erm(B) genes for macrolide-lincosamide-streptogramin B resistance, the streptomycin resistance gene ant(6)-Ia as well as mercury resistance genes while pSN42–50 K was associated with the heavy metal translocating P-type ATPase gene hmtp. The co-carriage and co-existence of these antimicrobial resistance and heavy metal resistance genes increases the likelihood of co-selection of the cfr-carrying plasmids. ConclusionThis is the first report of S. equorum carrying two distinct cfr-carrying plasmids, underscoring the need for ongoing surveillance to address the potential dissemination of multi-drug resistance in bacteria from food-producing animals to ensure food safety and public health.

Complete mitochondrial genomes of two Phoretic mites (Parasitidae: Poecilochirus) on burying beetle: Genome characterization and phylogenetic analysis

A pair of intriguing phoretic instances are the burying beetle and Poecilochirus mites. Their interaction is environment-dependent and can be either mutualistic or antagonistic hostile depending on various biological and physical circumstances. In this study, we sequenced the complete mitogenomes of two Poecilochirus mite species, P. davydovae and P. mrciaki, which are associated with burying beetles, using Illumina HiSeq sequencing technology. Each mitochondrial genome comprised 37 typical genes, including 13 protein-coding genes (PCGs), 22 tRNA genes, two rRNA genes, and one control region (CR). The size of these mitogenomes were determined to be 14,489 bp and 14,517 bp, respectively. These two mitochondrial genomes contain a notable AT bias in their nucleotide composition, with positive AT and negative GC skews in the deflection index of mitochondrial genomes, and a unique incomplete stop codon T- in both protein-coding genes. The DHU arm of tRNA-S1 (AGN) only forms a simple loop structure, while the secondary structures of 21 tRNA genes in both species showed the conventional cloverleaf structures, like those of most metazoans. The phylogenetic tree of Gamasina was constructed using Maximum likelihood and Bayesian inference based on two rRNA genes and 13 PCGs. Based on the existing data, the Parasitidae forms a monophyletic, and Poecilochirus and Parasitus are closely related, possibly belonging to the same genus.

Endophytic bacteria with allelopathic potential regulate gene expression and metabolite production in host Casuarina equisetifolia.

Casuarina equisetifolia is a common protective forest in coastal areas. However, artificial C. equisetifolia forests cannot self-renew, mainly due to the accumulation of allelochemicals. Endophytic bacteria may alleviate the root growth inhibition caused by allelochemicals in C. equisetifolia seedlings. B. amyloliquefaciens and B. aryabhattai were endophytic bacteria with strong allelopathy in C. equisetifolia root. The allelopathy mechanism of these two endophytes and their interaction with C. equisetifolia remains to be studied. Whole-genome sequencing of B. amyloliquefaciens and B. aryabhattai isolated from the roots of allelochemical-accumulating C. equisetifolia was performed using Illumina Hiseq and PacBio single-molecule sequencing platforms. Sterile seedlings of C. equisetifolia were treated with either individual or mixed bacterial cultures through root drenching. Transcriptional and metabolomics analyses were conducted after 3 days of infection. Whole-genome sequencing of Bacillus aryabhattai and Bacillus amyloliquefaciens showed that the two strains contained various horizontal gene transfer elements such as insertion sequence, prophage and transposon. In addition, these two strains also contain numerous genes related to the synthesis and catabolism of allelochemicals. After these two strains of bacteria were individually or mixed infected with C. equisetifolia, metabolomics and transcriptomic analysis of C. equisetifolia showed the 11 important secondary metabolite biosynthesis among them alkaloids biosynthesis, phenylpropanoid and terpenes biosynthesis and related genes were putatively regulated. Correlation analysis revealed that 48 differentially expressed genes had strong positive correlations with 42 differential metabolites, and 48 differentially expressed genes had strong negative correlations with 36 differential metabolites. For example, CMBL gene showed positive correlations with the allelochemical (-)-Catechin gallate, while Bp10 gene showed negative correlations with (-)-Catechin gallate. The intergenerational accumulation of allelochemicals may induce horizontal gene transfer in endogenic bacteria of Casuarina equisetifolia root. Endophytic Bacillus plays an allelopathic role by assisting the host in regulating gene expression and the production and/or variety of allelochemicals. This comprehensive study sheds light on the intricate genetic and metabolic interactions between Bacillus endophytes and C. equisetifolia. These findings provide insights into endophyte-mediated allelopathy and its potential uses in plant biology and forest sustainability.

First report of potato virus H infecting tomato (Solanum lycopersicum) in China.

Potato virus H (PVH), belonging to the genus Carlavirus in the family Betaflexiviridae, was initially discovered in potato plants in Inner Mongolia, China (Li et al., 2013). Subsequently, it was documented to infect pepino, a perennial shrub of the Solanaceae family like potatoes (Abouelnasr et al., 2014). Tomato (Solanum lycopersicum L.), a major global crop, faces threats from various plant viruses. In an open field survey in Yunnan, China during July 2023, tomatoes (cultivar: Liangsi) showed typical virus symptoms: leaf yellowing, curling, mottling, and fruit with abnormal shape and color. Eleven symptomatic tomato samples were collected for high-throughput sequencing to identify the potential pathogen. RNA sequencing libraries were prepared using the TruSeq RNA sample prep kit (Illumina, San Diego, CA, USA), followed by RNA-seq sequencing on an Illumina HiSeq4000 platform (LC Sciences, USA). Approximately 77,928,560 paired-end reads (150-bp each) were generated. After quality control, 75,808,296 reads were retained and subjected to de novo assembly using Trinity (version 2.8.5). The assembled contigs, ranging from 198 nt to 15865 nt, were used as queries to search against the NCBI non-redundant protein sequence database (NR) or nucleotide sequence database (NT) to detect the potential pathogens using BLASTx and BLASTn program with a cutoff e-value of 10-5. As a consequence, certain contigs were assigned to 3 plant viruses, including PVH (the highest RdRp blastx identity to UAD82396.1: 97.8%), Capsicum chlorosis virus (CaCV, the highest RdRp blastx identity to APQ31267.1: 98.4%), and southern tomato virus (STV, the highest CP-RdRp fusion protein blastx identity to QOW17541.1: 99.74%). The presence of the identified 3 viruses was subsequently screened in the 11 tomato samples originally collected from the corresponding field. Notably, the specific detection primers for the PVH genome was designed from the newly assembled PVH genome (Forward primer: 5'- ATAGTTGTGCACTGTGTGCCTG-3'; Reverse primer: 5'-GCTTAAGGTTCTTAGCGTATTC-3'), targeting ~1.1kb. Consequently, PVH was detected in 3 out of 11 samples: 2 leaf samples and 1 fruit sample, with one leaf sample showing a single infection. The complete genome sequence of PVH in tomatoes (PVH-tomato) was successfully obtained by assembling nine overlapping regions spanning the entire PVH-tomato genome, following the RT-PCR and the 5' RACE and 3' RACE approaches, and deposited in NCBI nucleotide database with accession number OR397130.1Phylogenetic analysis based on the full genome sequences of PVH-tomato and other publicly available PVH isolates revealed that PVH-tomato was closely related to a PVH isolate found in potatoes in Yunnan (blastn similarity: 97.76%) (Fig. S1A). To test PVH-tomato infectivity and pathogenicity, four healthy Nicotiana benthamiana and four healthy tomato plants were mechanically inoculated with PVH-infected leaf sap; controls used sap from healthy plants. Three weeks post-inoculation, all N. benthamiana (4/4) and three tomato plants (3/4) were PVH-positive by RT-PCR. Symptoms were milder in N. benthamiana, and only two tomato plants (2/4) showed leaf curling. No PVH was detected in control samples (Figure S1B, S1C). Sanger sequencing confirmed the amplicons' expected length of 1093 bp. Previously, PVH was documented only in potato and pepino. This is the first report of tomatoes as natural PVH hosts and PVH infecting N. benthamiana under lab conditions.

Exosomal transcript cargo and functional correlation with HNSCC patients’ survival

BackgroundHPV status in a subset of HNSCC is linked with distinct treatment outcomes. Present investigation aims to elucidate the distinct clinicopathological features of HPV-positive and HPV-negative HNSCC and investigate their association with the HNSCC patient survival.Materials and methodsThe total RNA of exosomes from HPV-positive (93VU147T) and HPV-negative (OCT-1) HNSCC cells was isolated, and the transcripts were estimated using Illumina HiSeq X. The expression of altered transcripts and their clinical relevance were further analyzed using publicly available cancer transcriptome data from The Cancer Genome Atlas (TCGA).ResultsTranscriptomic analyses identified 3785 differentially exported transcripts (DETs) in HPV-positive exosomes compared to HPV-negative exosomes. DETs that regulate the protein machinery, cellular redox potential, and various neurological disorder-related pathways were over-represented in HPV-positive exosomes. TCGA database revealed the clinical relevance of altered transcripts. Among commonly exported abundant transcripts, SGK1 and MAD1L1 showed high expression, which has been correlated with poor survival in HNSCC patients. In the top 20 DETs of HPV-negative exosomes, high expression of FADS3, SGK3, and TESK2 correlated with poor survival of the HNSCC patients in the TCGA database.ConclusionOverall, our study demonstrates that HPV-positive and HPV-negative cells’ exosomes carried differential transcripts cargo that may be related to pathways associated with neurological disorders. Additionally, the altered transcripts identified have clinical relevance, correlating with patient survival in HNSCC, thereby highlighting their potential as biomarkers and as therapeutic targets.