Comparative genome analysis of programmed DNA elimination in nematodes.

Programmed DNA elimination is a developmentally regulated process leading to the reproducible loss of specific genomic sequences. DNA elimination occurs in unicellular ciliates and a variety of metazoans, including invertebrates and vertebrates. In metazoa, DNA elimination typically occurs in somatic cells during early development, leaving the germline genome intact. Reference genomes for metazoa that undergo DNA elimination are not available. Here, we generated germline and somatic reference genome sequences of the DNA eliminating pig parasitic nematode Ascaris suum and the horse parasite Parascaris univalens. In addition, we carried out in-depth analyses of DNA elimination in the parasitic nematode of humans, Ascaris lumbricoides, and the parasitic nematode of dogs, Toxocara canis. Our analysis of nematode DNA elimination reveals that in all species, repetitive sequences (that differ among the genera) and germline-expressed genes (approximately 1000-2000 or 5%-10% of the genes) are eliminated. Thirty-five percent of these eliminated genes are conserved among these nematodes, defining a core set of eliminated genes that are preferentially expressed during spermatogenesis. Our analysis supports the view that DNA elimination in nematodes silences germline-expressed genes. Over half of the chromosome break sites are conserved between Ascaris and Parascaris, whereas only 10% are conserved in the more divergent T. canis. Analysis of the chromosomal breakage regions suggests a sequence-independent mechanism for DNA breakage followed by telomere healing, with the formation of more accessible chromatin in the break regions prior to DNA elimination. Our genome assemblies and annotations also provide comprehensive resources for analysis of DNA elimination, parasitology research, and comparative nematode genome and epigenome studies.

Spatial distribution prediction of soil heavy metals based on sparse sampling and multi-source environmental data.

Predicting the precise spatial distribution of heavy metals in soil is crucial, especially in the fields of environmental management and remediation. However, achieving accurate spatial predictions of soil heavy metals becomes quite challenging when the number of soil sampling points is relatively limited. To address this challenge, this study proposes a hybrid approach, namely, Light Gradient Boosting Machine plus Ordinary Kriging (LGBK), for predicting the spatial distribution of soil heavy metals. A total of 137 soil samples were collected from the Shengli Coal-mine Base in Inner Mongolia, China, and their heavy metal concentrations were measured. Leveraging environmental covariates and soil heavy metal data, we constructed the predictive model. Experimental results demonstrate that, in comparison to traditional models, LGBK exhibits superior predictive performance. For copper (Cu), zinc (Zn), chromium (Cr), and arsenic (As), the coefficients of determination (R²) from the cross-validation results are 0.65, 0.52, 0.57, and 0.63, respectively. Moreover, the LGBK model excels in capturing intricate spatial features in heavy metal distribution. It accurately forecasts trends in heavy metal distribution that closely align with actual measurements. ENVIRONMENTAL IMPLICATION: This study introduces a novel method, LGBK, for predicting the spatial distribution of soil heavy metals. This method yields higher-precision predictions even with a limited number of sampling points. Furthermore, the study analyzes the spatial distribution characteristics of Cu, Zn, Cr, and As in the grassland coal-mine base, along with the key environmental factors influencing their spatial distribution. This research holds significant importance for the environmental regulation and remediation of heavy metal pollution.

RNA N6-methyladenosine modulates endothelial atherogenic responses to disturbed flow in mice.

Atherosclerosis preferentially occurs in atheroprone vasculature where human umbilical vein endothelial cells are exposed to disturbed flow. Disturbed flow is associated with vascular inflammation and focal distribution. Recent studies have revealed the involvement of epigenetic regulation in atherosclerosis progression. N6-methyladenosine (m6A) is the most prevalent internal modification of eukaryotic mRNA, but its function in endothelial atherogenic progression remains unclear. Here, we show that m6A mediates the epidermal growth factor receptor (EGFR) signaling pathway during EC activation to regulate the atherosclerotic process. Oscillatory stress (OS) reduced the expression of methyltransferase like 3 (METTL3), the primary m6A methyltransferase. Through m6A sequencing and functional studies, we determined that m6A mediates the mRNA decay of the vascular pathophysiology gene EGFR which leads to EC dysfunction. m6A modification of the EGFR 3' untranslated regions (3'UTR) accelerated its mRNA degradation. Double mutation of the EGFR 3'UTR abolished METTL3-induced luciferase activity. Adenovirus-mediated METTL3 overexpression significantly reduced EGFR activation and endothelial dysfunction in the presence of OS. Furthermore, thrombospondin-1 (TSP-1), an EGFR ligand, was specifically expressed in atheroprone regions without being affected by METTL3. Inhibition of the TSP-1/EGFR axis by using shRNA and AG1478 significantly ameliorated atherogenesis. Overall, our study revealed that METTL3 alleviates endothelial atherogenic progression through m6A-dependent stabilization of EGFR mRNA, highlighting the important role of RNA transcriptomics in atherosclerosis regulation.

Differential transcriptomic landscapes of multiple organs from SARS-CoV-2 early infected rhesus macaques.

SARS-CoV-2 infection causes complicated clinical manifestations with variable multi-organ injuries, however, the underlying mechanism, in particular immune responses in different organs, remains elusive. In this study, comprehensive transcriptomic alterations of 14 tissues from rhesus macaque infected with SARS-CoV-2 were analyzed. Compared to normal controls, SARS-CoV-2 infection resulted in dysregulation of genes involving diverse functions in various examined tissues/organs, with drastic transcriptomic changes in cerebral cortex and right ventricle. Intriguingly, cerebral cortex exhibited a hyperinflammatory state evidenced by significant upregulation of inflammation response-related genes. Meanwhile, expressions of coagulation, angiogenesis and fibrosis factors were also up-regulated in cerebral cortex. Based on our findings, neuropilin 1 (NRP1), a receptor of SARS-CoV-2, was significantly elevated in cerebral cortex post infection, accompanied by active immune response releasing inflammatory factors and signal transmission among tissues, which enhanced infection of the central nervous system (CNS) in a positive feedback way, leading to viral encephalitis. Overall, our study depicts a multi-tissue/organ transcriptomic landscapes of rhesus macaque with early infection of SARS-CoV-2, and provides important insights into the mechanistic basis for COVID-19-associated clinical complications.

Genomic Epidemiology of SARS-CoV-2 in Pakistan.

COVID-19 has swept globally and Pakistan is no exception. To investigate the initial introductions and transmissions of the SARS-CoV-2 in Pakistan, we performed the largest genomic epidemiology study of COVID-19 in Pakistan and generated 150 complete SARS-CoV-2 genome sequences from samples collected from March 16 to June 1, 2020. We identified a total of 347 mutated positions, 31 of which were over-represented in Pakistan. Meanwhile, we found over 1000 intra-host single-nucleotide variants (iSNVs). Several of them occurred concurrently, indicating possible interactions among them or coevolution. Some of the high-frequency iSNVs in Pakistan were not observed in the global population, suggesting strong purifying selections. The genomic epidemiology revealed five distinctive spreading clusters. The largest cluster consisted of 74 viruses which were derived from different geographic locations of Pakistan and formed a deep hierarchical structure, indicating an extensive and persistent nation-wide transmission of the virus that was probably attributed to a signature mutation (G8371T in ORF1ab) of this cluster. Furthermore, 28 putative international introductions were identified, several of which are consistent with the epidemiological investigations. In all, this study has inferred the possible pathways of introductions and transmissions of SARS-CoV-2 in Pakistan, which could aid ongoing and future viral surveillance and COVID-19 control.

Transcript profiles of Panax quinquefolius from flower, leaf and root bring new insights into genes related to ginsenosides biosynthesis and transcriptional regulation.

American ginseng (Panax quinquefolius L.) has been used for a wide range of therapeutic purposes in China. The major bioactive phytochemicals responsible for this plant's pharmacological features are ginsenosides. Thus far, little is known regarding the genes involved in ginsenosides biosynthesis in this species. As a non-model plant, information about its genomes is generally not available. In this study, we generated 6678 expressed sequence tags (ESTs) from the flower, leaf and root cDNA libraries of American ginseng. Assembly of ESTs resulted in 3349 unigenes including 534 contigs (with ESTs number ranging from 2 to 52) and 2815 singletons. By analyzing the predominant transcripts within specific tissues, a gene expression pattern was obtained in a tissue-specific manner. They were assigned according to the functional classification of unigenes to broad ranges of Gene Ontology categories which include biological processes, cellular components and molecular functions. Based on blastx search results, 24 unigenes representing candidates related to ginsenosides biosynthesis were identified. Cloning and characterization of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR, EC: 1.1.1.34), the rate-limiting enzyme in mevalonic acid pathway, demonstrated that it belonged to the plant HMGR family and was highly expressed in leaves. Putative transcription factors were detected in 63 unigenes, including zinc finger, WRKY, homeobox and MADS-box family proteins. Five hundred and eighty-eight simple sequence repeat motifs were identified, of which, dimer was the most abundant motif. These data will provide useful information on transcript profiles, gene discovery, transcriptional regulation, flower biogenesis and marker-assisted selections. The analysis and information from this study will greatly contribute to the improvement of this medicinal plant as well as of other species in the Araliaceae family, for the purpose of ensuring adequate drug resources.

A genetic variation map for chicken with 2.8 million single-nucleotide polymorphisms.

We describe a genetic variation map for the chicken genome containing 2.8 million single-nucleotide polymorphisms (SNPs). This map is based on a comparison of the sequences of three domestic chicken breeds (a broiler, a layer and a Chinese silkie) with that of their wild ancestor, red jungle fowl. Subsequent experiments indicate that at least 90% of the variant sites are true SNPs, and at least 70% are common SNPs that segregate in many domestic breeds. Mean nucleotide diversity is about five SNPs per kilobase for almost every possible comparison between red jungle fowl and domestic lines, between two different domestic lines, and within domestic lines--in contrast to the notion that domestic animals are highly inbred relative to their wild ancestors. In fact, most of the SNPs originated before domestication, and there is little evidence of selective sweeps for adaptive alleles on length scales greater than 100 kilobases.

Analysis of tarantula skeletal muscle protein sequences and identification of transcriptional isoforms.

BACKGROUND: Tarantula has been used as a model system for studying skeletal muscle structure and function, yet data on the genes expressed in tarantula muscle are lacking. RESULTS: We constructed a cDNA library from Aphonopelma sp. (Tarantula) skeletal muscle and got 2507 high-quality 5'ESTs (expressed sequence tags) from randomly picked clones. EST analysis showed 305 unigenes, among which 81 had more than 2 ESTs. Twenty abundant unigenes had matches to skeletal muscle-related genes including actin, myosin, tropomyosin, troponin-I, T and C, paramyosin, muscle LIM protein, muscle protein 20, a-actinin and tandem Ig/Fn motifs (found in giant sarcomere-related proteins). Matches to myosin light chain kinase and calponin were also identified. These results support the existence of both actin-linked and myosin-linked regulation in tarantula skeletal muscle. We have predicted full-length as well as partial cDNA sequences both experimentally and computationally for myosin heavy and light chains, actin, tropomyosin, and troponin-I, T and C, and have deduced the putative peptides. A preliminary analysis of the structural and functional properties was also carried out. Sequence similarities suggested multiple isoforms of most myofibrillar proteins, supporting the generality of multiple isoforms known from previous muscle sequence studies. This may be related to a mix of muscle fiber types. CONCLUSION: The present study serves as a basis for defining the transcriptome of tarantula skeletal muscle, for future in vitro expression of tarantula proteins, and for interpreting structural and functional observations in this model species.

Whole Genome Analyses of Chinese Population and De Novo Assembly of A Northern Han Genome.

To unravel the genetic mechanisms of disease and physiological traits, it requires comprehensive sequencing analysis of large sample size in Chinese populations. Here, we report the primary results of the Chinese Academy of Sciences Precision Medicine Initiative (CASPMI) project launched by the Chinese Academy of Sciences, including the de novo assembly of a northern Han reference genome (NH1.0) and whole genome analyses of 597 healthy people coming from most areas in China. Given the two existing reference genomes for Han Chinese (YH and HX1) were both from the south, we constructed NH1.0, a new reference genome from a northern individual, by combining the sequencing strategies of PacBio, 10× Genomics, and Bionano mapping. Using this integrated approach, we obtained an N50 scaffold size of 46.63 Mb for the NH1.0 genome and performed a comparative genome analysis of NH1.0 with YH and HX1. In order to generate a genomic variation map of Chinese populations, we performed the whole-genome sequencing of 597 participants and identified 24.85 million (M) single nucleotide variants (SNVs), 3.85 M small indels, and 106,382 structural variations. In the association analysis with collected phenotypes, we found that the T allele of rs1549293 in KAT8 significantly correlated with the waist circumference in northern Han males. Moreover, significant genetic diversity in MTHFR, TCN2, FADS1, and FADS2, which associate with circulating folate, vitamin B12, or lipid metabolism, was observed between northerners and southerners. Especially, for the homocysteine-increasing allele of rs1801133 (MTHFR 677T), we hypothesize that there exists a "comfort" zone for a high frequency of 677T between latitudes of 35-45 degree North. Taken together, our results provide a high-quality northern Han reference genome and novel population-specific data sets of genetic variants for use in the personalized and precision medicine.

The Genomes of Oryza sativa: a history of duplications.

We report improved whole-genome shotgun sequences for the genomes of indica and japonica rice, both with multimegabase contiguity, or almost 1,000-fold improvement over the drafts of 2002. Tested against a nonredundant collection of 19,079 full-length cDNAs, 97.7% of the genes are aligned, without fragmentation, to the mapped super-scaffolds of one or the other genome. We introduce a gene identification procedure for plants that does not rely on similarity to known genes to remove erroneous predictions resulting from transposable elements. Using the available EST data to adjust for residual errors in the predictions, the estimated gene count is at least 38,000-40,000. Only 2%-3% of the genes are unique to any one subspecies, comparable to the amount of sequence that might still be missing. Despite this lack of variation in gene content, there is enormous variation in the intergenic regions. At least a quarter of the two sequences could not be aligned, and where they could be aligned, single nucleotide polymorphism (SNP) rates varied from as little as 3.0 SNP/kb in the coding regions to 27.6 SNP/kb in the transposable elements. A more inclusive new approach for analyzing duplication history is introduced here. It reveals an ancient whole-genome duplication, a recent segmental duplication on Chromosomes 11 and 12, and massive ongoing individual gene duplications. We find 18 distinct pairs of duplicated segments that cover 65.7% of the genome; 17 of these pairs date back to a common time before the divergence of the grasses. More important, ongoing individual gene duplications provide a never-ending source of raw material for gene genesis and are major contributors to the differences between members of the grass family.

[Analysis of expressed sequence tags (EST) from Panax quinquefolium root].

To investigate the profile of gene expression in American ginseng (Panax quinquefolium L.) and discover its functional genes, for the first time, expressed sequence tags (EST) library of four-year-old American ginseng roots has been established. According to BLAST and Gene Ontology analysis, eleven genes, encoding cytochrome P450, glucosyltransferase, farnesyltransferase and cyclase family protein, are found to be associated with ginsenosides biosynthesis. Six other genes are obtained encoding auxin-regulated protein, auxin response factor 4 and auxin-repressed protein in the roots of American ginseng. In addition, thirteen expressed transcripts are stress-connected proteins and twelve expressed other transcripts are closely related to plant defense in four-year-old American ginseng roots. Furthermore, 62 genes no hit in BLAST and in Interproscan may be new genes. These results indicate EST is an useful tool for research on functional genomics of P. quinquefolium and it can be applied to the molecular modification of the ginsenosides biosynthetic pathway ultimately for improving the quality of American ginseng germplasm.

The C-terminal portion of the nucleocapsid protein demonstrates SARS-CoV antigenicity.

In order to develop clinical diagnostic tools for rapid detection of the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) and to identify candidate proteins for vaccine development, the C-terminal portion of the nucleocapsid (NC) gene was amplified using RT-PCR from the SARS-CoV genome, cloned into a yeast expression vector (pEGH), and expressed as a glutathione S-transferase (GST) and Hisx6 double-tagged fusion protein under the control of an inducible promoter. Western analysis on the purified protein confirmed the expression and purification of the NC fusion proteins from yeast. To determine its antigenicity, the fusion protein was challenged with serum samples from SARS patients and normal controls. The NC fusion protein demonstrated high antigenicity with high specificity, and therefore, it should have great potential in designing clinical diagnostic tools and provide useful information for vaccine development.

Transcriptome comparison between honey bee queen- and worker-destined larvae.

Caste differentiation in the female honey bee is one of the most intriguing polyphenism phenomena. This developmental switch depends on the differential expression of entire suites of the genes involved in the larval fate between the queens and workers. In this study, we compared the transcriptome differences between full-sister queen- (QL) and worker-destined larvae (WL) using high-throughput RNA-Seq. QL and WL at fourth (L4) and fifth instar (L5) were used to prepare four libraries and to generate 50,191,699 (QL4), 57,628,541 (WL4), 56,613,619 (QL5), and 58,626,829 (WL5) usable reads, which were assembled into groups of 7,952, 7,993, 7,971, and 8,023 genes, respectively. The transcriptome changes were investigated using the DEGs Package (DEGseq), which resulted in more than 4,500 differentially expressed genes (DEGs) between the castes. Eight of the DEGs were verified by quantitative real-time RT-PCR (qRT-PCR), and the results supported our sequencing data. All of the DEGs were analysed using Web Gene Ontology Annotation Plot (WEGO) and then mapped using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. These results suggest that over 70% of the DEGs in each instar were more highly expressed in QL than in WL, possibly suggesting that the QL genes had higher transcriptional activity than the WL genes during differentiation. The same gene set is active (but differentially expressed) in both castes, which in turn result in dimorphic females. The L4 stage is a very active gene expression period for both QL and WL before their pupal stage. The activity of the mTOR (a target of rapamycin) encoding gene in the mTOR signalling pathway is higher in QL4 than in WL4, and this difference was no longer present by the L5 feeding stage. The genes down-stream of mTOR maintained this change at the L5 stage. These results could contribute to an in-depth study of the candidate genes during honey bee caste differentiation and improve our current understanding of the polyphenism phenomenon in insects.

Complete genome sequence of bacteriophage T5.

The 121,752-bp genome sequence of bacteriophage T5 was determined; the linear, double-stranded DNA is nicked in one of the strands and has large direct terminal repeats of 10,139 bp (8.3%) at both ends. The genome structure is consistently arranged according to its lytic life cycle. Of the 168 potential open reading frames (ORFs), 61 were annotated; these annotated ORFs are mainly enzymes involved in phage DNA replication, repair, and nucleotide metabolism. At least five endonucleases that believed to help inducing nicks in T5 genomic DNA, and a DNA ligase gene was found to be split into two separate ORFs. Analysis of T5 early promoters suggests a probable motif AAA{3, 4 T}nTTGCTT{17, 18 n}TATAATA{12, 13 W}{10 R} for strong promoters that may strengthen the step modification of host RNA polymerase, and thus control transcription of phage DNA. The distinct protein domain profile and a mosaic genome structure suggest an origin from the common genetic pool.