A novel interpretable deep learning-based computational framework designed synthetic enhancers with broad cross-species activity.

Enhancers play a critical role in dynamically regulating spatial-temporal gene expression and establishing cell identity, underscoring the significance of designing them with specific properties for applications in biosynthetic engineering and gene therapy. Despite numerous high-throughput methods facilitating genome-wide enhancer identification, deciphering the sequence determinants of their activity remains challenging. Here, we present the DREAM (DNA cis-Regulatory Elements with controllable Activity design platforM) framework, a novel deep learning-based approach for synthetic enhancer design. Proficient in uncovering subtle and intricate patterns within extensive enhancer screening data, DREAM achieves cutting-edge sequence-based enhancer activity prediction and highlights critical sequence features implicating strong enhancer activity. Leveraging DREAM, we have engineered enhancers that surpass the potency of the strongest enhancer within the Drosophila genome by approximately 3.6-fold. Remarkably, these synthetic enhancers exhibited conserved functionality across species that have diverged more than billion years, indicating that DREAM was able to learn highly conserved enhancer regulatory grammar. Additionally, we designed silencers and cell line-specific enhancers using DREAM, demonstrating its versatility. Overall, our study not only introduces an interpretable approach for enhancer design but also lays out a general framework applicable to the design of other types of cis-regulatory elements.

Reticulocalbin 3 deficiency in alveolar epithelium attenuated LPS-induced ALI via NF-κB signaling.

Acute respiratory distress syndrome (ARDS) is characterized by acute lung injury (ALI) secondary to an excessive alveolar inflammatory response. Reticulocalbin 3 (Rcn3) is an endoplasmic reticulum (ER) lumen protein in the secretory pathway. We previously reported the indispensable role of Rcn3 in type II alveolar epithelial cells (AECIIs) during lung development and the lung injury repair process. In the present study, we further observed a marked induction of Rcn3 in the alveolar epithelium during LPS-induced ALI. In vitro alveolar epithelial (MLE-12) cells consistently exhibited a significant induction of Rcn3 accompanied with NF-κB activation in response to LPS exposure. We examined the role of Rcn3 in the alveolar inflammatory response by using mice with a selective deletion of Rcn3 in alveolar epithelial cells upon doxycycline administration. The Rcn3 deficiency significantly blunted the ALI and alveolar inflammation induced by intratracheal LPS instillation but not that induced by an intraperitoneal LPS injection (secondary insult); the alleviated ALI was accompanied by decreases in NF-κB activation and NLRP3 levels but not in GRP78 and cleaved caspase-3 levels. The studies conducted in MLE-12 cells consistently showed that Rcn3 knockdown blunted the activations of NF-κB signaling and NLRP3-dependent inflammasome upon LPS exposure. Collectively, these findings suggest a novel role for Rcn3 in regulating the alveolar inflammatory response to pulmonary infection via the NF-κB/NLRP3/inflammasome axis and shed additional light on the mechanism of ARDS/ALI.

Correlated Evolution of Large DNA Fragments in the 3D Genome of Arabidopsis thaliana.

In eukaryotes, the three-dimensional (3D) conformation of the genome is far from random, and this nonrandom chromatin organization is strongly correlated with gene expression and protein function, which are two critical determinants of the selective constraints and evolutionary rates of genes. However, whether genes and other elements that are located close to each other in the 3D genome evolve in a coordinated way has not been investigated in any organism. To address this question, we constructed chromatin interaction networks (CINs) in Arabidopsis thaliana based on high-throughput chromosome conformation capture data and demonstrated that adjacent large DNA fragments in the CIN indeed exhibit more similar levels of polymorphism and evolutionary rates than random fragment pairs. Using simulations that account for the linear distance between fragments, we proved that the 3D chromosomal organization plays a role in the observed correlated evolution. Spatially interacting fragments also exhibit more similar mutation rates and functional constraints in both coding and noncoding regions than the random expectations, indicating that the correlated evolution between 3D neighbors is a result of combined evolutionary forces. A collection of 39 genomic and epigenomic features can explain much of the variance in genetic diversity and evolutionary rates across the genome. Moreover, features that have a greater effect on the evolution of regional sequences tend to show higher similarity between neighboring fragments in the CIN, suggesting a pivotal role of epigenetic modifications and chromatin organization in determining the correlated evolution of large DNA fragments in the 3D genome.

Hypoosmotic stress induced tissue-specific immune responses of yellowfin seabream (Acanthopagrus latus) revealed by transcriptomic analysis.

Salinity is a limiting factor for many marine organisms, including fishes. The shift in the ambient salinity can cause osmotic stress and arouse immune responses in fish. In this study, yellowfin seabream (Acanthopagrus latus), a euryhaline marine teleost, was used to investigate immune responses of different tissues (gill, liver, and muscle) under hypoosmotic stress. Comparative transcriptomic and physiological analyses of three tissues were conducted after fish exposed to the fresh water (FW, salinity = 0 ppt), low-saline water (LW, salinity = 3 ppt), and brackish water (BW, salinity = 6 ppt) for 8 days. The results showed that hypoosmotic stress dramatically altered the gene expression of three tissues in yellowfin seabream; The investigation of differentially expressed genes (DEGs) related to osmoregulation and immune response indicated that T cell-mediate immunity pathways were essential to tackle such stress. In terms of tissues, gill was found to be the most sensitive tissue under hypoosmotic stress by enhancing of Na+K+-ATPase activity and preventing the loss of Na+ and K+; Liver, on the other hand, was under the most sever oxidative stress indicated by the fluctuation of SOD, CAT activities and the MDA content; In contrast, muscle had the least osmoregulation and immune related response. We also identified several potential candidate genes, which may serve as gene indicators to identify the stressor. Overall, this study provides preliminary mechanistic insights into hypoosmotic stress adaption of aquatic organism.

Chromatin Signature and Transcription Factor Binding Provide a Predictive Basis for Understanding Plant Gene Expression.

Chromatin accessibility and post-transcriptional histone modifications play important roles in gene expression regulation. However, little is known about the joint effect of multiple chromatin modifications on the gene expression level in plants, despite that the regulatory roles of individual histone marks such as H3K4me3 in gene expression have been well-documented. By using machine-learning methods, we systematically performed gene expression level prediction based on multiple chromatin modifications data in Arabidopsis and rice. We found that as few as four histone modifications were sufficient to yield good prediction performance, and H3K4me3 and H3K36me3 being the top two predictors with known functions related to transcriptional initiation and elongation, respectively. We demonstrated that the predictive powers differed between protein-coding and non-coding genes as well as between CpG-enriched and CpG-depleted genes. We also showed that the predictive model trained in one tissue or species could be applied to another tissue or species, suggesting shared underlying mechanisms. More interestingly, the gene expression levels of conserved orthologs are easier to predict than the species-specific genes. In addition, chromatin state of distal enhancers was moderately correlated to gene expression but was dispensable if given the chromatin features of the proximal regions of genes. We further extended the analysis to transcription factor (TF) binding data. Strikingly, the combinatorial effects of only a few TFs were roughly fit to gene expression levels in Arabidopsis. Overall, by using quantitative modeling, we provide a comprehensive and unbiased perspective on the epigenetic and TF-mediated regulation of gene expression in plants.

Identification and characterization of a new dwarf locus DS-4 encoding an Aux/IAA7 protein in Brassica napus.

KEY MESSAGE: A dominant dwarfing gene, ds - 4 , encodes an Aux/IAA protein that negatively regulates plant stature through an auxin signaling pathway. Dwarfism is an important agronomic trait affecting yield in many crop species. The molecular mechanisms underlying dwarfism in oilseed rape (Brassica napus) are poorly understood, restricting the progress of breeding dwarf varieties in this species. Here, we identified and characterized a new dwarf locus, DS-4, in B. napus. Next-generation sequencing-assisted genetic mapping and candidate gene analysis found that DS-4 encodes a nucleus-targeted auxin/indole-3-acetic acid (Aux/IAA) protein. A substitution (P87L) was found in the highly conserved degron motif of the Aux/IAA7 protein in the ds-4 mutant. This mutation co-segregated with the phenotype of individuals in the BC1F2 population. The P87L substitution was confirmed as the cause of the extreme dwarf phenotype by ectopic expression of the mutant allele BnaC05.iaa7 (equivalent to ds-4) in Arabidopsis. The P87L substitution blocked the interaction of BnaC05.iaa7 with TRANSPORT INHIBITOR RESPONSE 1 in the presence of auxin. The BnaC05.IAA7 gene is highly expressed in the cotyledons, hypocotyls, stems and leaves, but weakly in the roots and seeds of B. napus. Our findings provide new insights into the molecular mechanisms underlying dominant (gain-of-function) dwarfism in B. napus. Our identification of a distinct gene locus controlling plant height may help to improve lodging resistance in oilseed rape.

Sequence variation and functional analysis of a FRIGIDA orthologue (BnaA3.FRI) in Brassica napus.

BACKGROUND: Allelic variation at the FRIGIDA (FRI) locus is a major contributor to natural variation of flowering time and vernalization requirement in Arabidopsis thaliana. Dominant FRI inhibits flowering by activating the expression of the MADS box transcriptional repressor FLOWERING LOCUS C (FLC), which represses flowering prior to vernalization. Four FRI orthologues had been identified in the domesticated amphidiploid Brassica napus. Linkage and association studies had revealed that one of the FRI orthologues, BnaA3.FRI, contributes to flowering time variation and crop type differentiation. RESULTS: Sequence analyses indicated that three out of the four BnaFRI paralogues, BnaA3.FRI, BnaA10.FRI and BnaC3.FRI, contained a large number of polymorphic sites. Haplotype analysis in a panel of 174 B. napus accessions using PCR markers showed that all the three paralogues had a biased distribution of haplotypes in winter type oilseed rape (P < 0.01). Association analysis indicated that only BnaA3.FRI contributes to flowering time variation in B. napus. In addition, transgenic functional complementation demonstrated that mutations in the coding sequence of BnaA3.FRI lead to weak alleles, and subsequently to flowering time variation. CONCLUSION: This study for the first time provides a molecular basis for flowering time control by BnaA3.FRI in B. napus, and will facilitate predictive oilseed rape breeding to select varieties with favorable flowering time and better adaption to latitude and seasonal shifts due to changing climate.

Dissection of the genetic architecture of three seed-quality traits and consequences for breeding in Brassica napus.

Genome-wide association studies (GWASs) combining high-throughput genome resequencing and phenotyping can accelerate the dissection of genetic architecture and identification of genes for plant complex traits. In this study, we developed a rapeseed genomic variation map consisting of 4 542 011 SNPs and 628 666 INDELs. GWAS was performed for three seed-quality traits, including erucic acid content (EAC), glucosinolate content (GSC) and seed oil content (SOC) using 3.82 million polymorphisms in an association panel. Six, 49 and 17 loci were detected to be associated with EAC, GSC and SOC in multiple environments, respectively. The mean total contribution of these loci in each environment was 94.1% for EAC and 87.9% for GSC, notably higher than that for SOC (40.1%). A high correlation was observed between phenotypic variance and number of favourable alleles for associated loci, which will contribute to breeding improvement by pyramiding these loci. Furthermore, candidate genes were detected underlying associated loci, based on functional polymorphisms in gene regions where sequence variation was found to correlate with phenotypic variation. Our approach was validated by detection of well-characterized FAE1 genes at each of two major loci for EAC on chromosomes A8 and C3, along with MYB28 genes at each of three major loci for GSC on chromosomes A9, C2 and C9. Four novel candidate genes were detected by correlation between GSC and SOC and observed sequence variation, respectively. This study provides insights into the genetic architecture of three seed-quality traits, which would be useful for genetic improvement of B. napus.

High-density ddRAD linkage and yield-related QTL mapping delimits a chromosomal region responsible for oil content in rapeseed (Brassica napus L.).

Rapeseed (Brassica napus L.) is one of the most important oil crops almost all over the world. Seed-related traits, including oil content (OC), silique length (SL), seeds per silique (SS), and seed weight (SW), are primary targets for oil yield improvement. To dissect the genetic basis of these traits, 192 recombinant inbred lines (RILs) were derived from two parents with distinct oil content and silique length. High-density linkage map with a total length of 1610.4 cM were constructed using 1,329 double-digestion restriction site associated DNA (ddRAD) markers, 107 insertion/deletions (INDELs), and 90 well-distributed simple sequence repeats (SSRs) markers. A total of 37 consensus quantitative trait loci (QTLs) were detected for the four traits, with individual QTL explained 3.1-12.8% of the phenotypic variations. Interestingly, one OC consensus QTL (cqOCA10b) on chromosome A10 was consistently detected in all three environments, and explained 9.8% to 12.8% of the OC variation. The locus was further delimited into an approximately 614 kb genomic region, in which the flanking markers could be further evaluated for marker-assisted selection in rapeseed OC improvement and the candidate genes targeted for map-based cloning and genetic manipulation.

Metagenomic Analysis Manifesting Intrinsic Relatedness between Antibiotic Resistance Genes and Sulfate- and Iron-Reducing Microbes in Sediment Cores of the Pearl River Estuary.

Antibiotic resistance is an increasingly concerned hotspot of human health. Microbial determinants that may affect the profiles of antibiotic resistance genes (ARGs) in the environments need be explored. Here, sediment cores in the Pearl River Estuary (PRE) were analyzed using metagenomic approaches. ARGs were vertically stratified in the PRE sediment cores in terms of both diversity and absolute levels. Multidrug resistance genes could account for approximately 65.0% of the total ARGs, followed by sulfonamides (19.1%), aminoglycosides (5.9%), beta-lactams (4.5%), etc. ARGs related to aminoglycosides, lincosamides, macrolides, sulfonamides and tetracyclines were preferentially enriched in the surface layers of sediment cores. Sulfate-reducing microbes (SRMs) (e.g., Desulfocapsa and Desulfobulbus) and iron-reducing microbes (IRMs) (e.g., Pseudomonas and Sulfurospirillum) were consistently popular and dominant in the PRE sediment cores. The total levels of both SRMs and IRMs were significantly correlated with those of ARGs in the PRE sediment cores (p < 0.01). Network analysis showed that SRM and IRM genera (i.e., Pseudomonas, Shewanella, and Desulfovibrio) had the high co-occurrence with multiple ARG subtypes in the PRE sediment cores such as rsmA, mexK, and mexF. This study highlighted that anaerobic microbes could play significant roles in shaping vertical ARG distribution in the sediments of aquatic environments.

Functional genes and microorganisms controlling in situ methylmercury production and degradation in marine sediments: A case study in the Eastern China Coastal Seas.

Dominant microorganisms and functional genes, including hgcA, hgcB, merA, and merB, have been identified to be responsible for mercury (Hg) methylation or methylmercury (MeHg) demethylation. However, their in situ correlation with MeHg levels and the processes of Hg methylation and MeHg demethylation in coastal areas remains poorly understood. In this study, four functional genes related to Hg methylation and MeHg demethylation (hgcA, hgcB, merA, and merB) were all detected in the sediments of the Eastern China Coastal Seas (ECCSs) (representative coastal seas highly affected by human activities) using metagenomic approaches. HgcA was identified to be the key gene controlling the in situ net production of MeHg in the ECCSs. Based on metagenomic analysis and incubation experiments, sulfate-reducing bacteria were identified as the dominant microorganisms controlling Hg methylation in the ECCSs. In addition, hgcA gene was positively correlated with the MeHg content and Hg methylation rates, highlighting the potential roles of Hg methylation genes and microorganisms influenced by sediment physicochemical properties in MeHg cycling in the ECCSs. These findings highlighted the necessity of conducting similar studies in other natural systems for elucidating the molecular mechanisms underlying MeHg production in aquatic environments.

The Levels of Oxidized Phospholipids in High-Density Lipoprotein During the Course of Sepsis and Their Prognostic Value.

Purpose: To examine the levels of 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero phosphatidylcholine (POVPC) and 1-palmitoyl-2-glutaroyl-sn-glycero-phosphatidylcholine (PGPC) (the oxidized phosphatidylcholines) in HDL during the course of sepsis and to evaluate their prognostic value. Materials and Methods: This prospective cohort pilot study enrolled 25 septic patients and 10 healthy subjects from 2020 to 2021. The HDLs were extracted from patient plasmas at day 1, 3 and 7 after sepsis onset and from healthy plasmas (total 81 plasma samples). These HDLs were then subjected to examining POVPC and PGPC by using an ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) system. We further measured the levels of 38 plasma cytokines by Luminex and evaluated the correlation of HDL-POVPC level with these cytokines. Patients were further stratified into survivors and non-survivors to analyze the association of HDL-POVPC level with 28-day mortality. Results: Septic patients exhibited significant increase of HDL-POVPC at day 1, 3 and 7 after sepsis onset (POVPC-D1, p=0.0004; POVPC-D3, p=0.033; POVPC-D7, p=0.004, versus controls). HDL-PGPC was detected only in some septic patients (10 of 25) but not in healthy controls. Septic patients showed a significant change of the plasma cytokines profile. The correlation assay showed that IL-15 and IL-18 levels were positively correlated with HDL-POVPC level, while the macrophage-derived chemokine (MDC) level was negatively correlated with HDL-POVPC level. Furthermore, HDL-POVPC level in non-survivors was significantly increased versus survivors at day 1 and 3 (POVPC-D1, p=0.002; POVPC-D3, p=0.003). Area under ROC curves of POVPC-D1 and POVPC-D3 in predicting 28-day mortality were 0.828 and 0.851. POVPC-D1and POVPC-D3 were the independent risk factors for the death of septic patients (p=0.046 and 0.035). Conclusions: HDL-POVPC was persistently increased in the course of sepsis. POVPC-D1 and POVPC-D3 were significantly correlated with 28-mortality and might be valuable to predict poor prognosis.

Changes in Alpine Soil Bacterial Communities With Altitude and Slopes at Mount Shergyla, Tibetan Plateau: Diversity, Structure, and Influencing Factors.

The alpine ecosystem as one of the most representative terrestrial ecosystems has been highly concerned due to its susceptibility to anthropogenic impacts and climatic changes. However, the distribution pattern of alpine soil bacterial communities and related deterministic factors still remain to be explored. In this study, soils were collected from different altitudes and slope aspects of the Mount (Mt.) Shergyla, Tibetan Plateau, and were analyzed using 16S rRNA gene-based bioinformatics approaches. Acidobacteriota and Proteobacteria were identified consistently as the two predominant phyla in all soil samples, accounting for approximately 74% of the bacterial community. The alpha diversity of the soil bacterial community generally increased as the vegetation changed with the elevated altitude, but no significant differences in alpha diversity were observed between the two slopes. Beta diversity analysis of bacterial community showed that soil samples from the north slope were always differentiated obviously from the paired samples at the south slope with the same altitude. The whole network constituted by soil bacterial genera at the Mt. Shergyla was parsed into eight modules, and Elev-16S-573, Sericytochromatia, KD4-96, TK10, Pedomicrobium, and IMCC26256 genera were identified as the "hubs" in the largest module. The distance-based redundancy analysis (db-RDA) demonstrated that variations in soil bacterial community thereof with the altitude and slope aspects at the Mt. Shergyla were closely associated with environmental variables such as soil pH, soil water content, metal concentrations, etc. Our results suggest that environmental variables could serve as the deterministic factors for shaping the spatial pattern of soil bacterial community in the alpine ecosystems.

Selection and introgression facilitated the adaptation of Chinese native endangered cattle in extreme environments.

Although persistent efforts have identified and characterized a few candidate genes and related biological processes with potential functions in the adaptation of many species to extreme environments, few works have been conducted to determine the genomic basis of adaptation in endangered livestock breeds that have been living in extreme conditions for more than thousands of years. To fill this gap, we sequenced the whole genomes of nine individuals from three Chinese native endangered cattle breeds that are living in high-altitude or arid environments. Phylogenetic and evolutionary history analyses of these three and other six breeds showed that the genetic structure of the cattle populations is primarily related to geographic location. Interestingly, we identified pervasive introgression from the yak to Zhangmu cattle (ZMC) that cover several genes (e.g., NOS2, EGLN1 and EPAS1) involved in the hypoxia response and previously identified as positive selection genes in other species, which suggested that the adaptive introgression from yak may have contributed to the adaptation of ZMC to high-altitude environments. In addition, by contrasting the breeds in opposite living conditions, we revealed a set of candidate genes with various functions from hypoxia response, water metabolism, immune response and body shape change to embryo development and skeletal system development, etc., that may be related to high-altitude or arid adaptation. Our research provides new insights into the recovery and adaptation of endangered native cattle and other species in extreme environments and valuable resources for future research on animal husbandry to cope with climate change.

Ribavirin reduces mortality in enterovirus 71-infected mice by decreasing viral replication.

Enterovirus 71 (EV71) causes fatal encephalitis in young children. However, there is no effective antiviral drug available for infected patients. Ribavirin is currently used for the treatment of several RNA virus infections clinically, so its anti-EV71 efficacy was evaluated. In vitro results showed that ribavirin effectively reduced the viral yields (with an IC50 of 65 microg/mL) and virus-induced cytopathic effect in human and mouse cell lines. In vivo results showed that ribavirin reduced the mortality, morbidity, and subsequent paralysis sequelae in infected mice by decreasing viral loads in tissues. Thus, ribavirin could be a potential anti-EV71 drug.