Chromosome-level genome assembly of a high-altitude-adapted frog (Rana kukunoris) from the Tibetan plateau provides insight into amphibian genome evolution and adaptation.

BACKGROUND: The high-altitude-adapted frog Rana kukunoris, occurring on the Tibetan plateau, is an excellent model to study life history evolution and adaptation to harsh high-altitude environments. However, genomic resources for this species are still underdeveloped constraining attempts to investigate the underpinnings of adaptation. RESULTS: The R. kukunoris genome was assembled to a size of 4.83 Gb and the contig N50 was 1.80 Mb. The 6555 contigs were clustered and ordered into 12 pseudo-chromosomes covering ~ 93.07% of the assembled genome. In total, 32,304 genes were functionally annotated. Synteny analysis between the genomes of R. kukunoris and a low latitude species Rana temporaria showed a high degree of chromosome level synteny with one fusion event between chr11 and chr13 forming pseudo-chromosome 11 in R. kukunoris. Characterization of features of the R. kukunoris genome identified that 61.5% consisted of transposable elements and expansions of gene families related to cell nucleus structure and taste sense were identified. Ninety-five single-copy orthologous genes were identified as being under positive selection and had functions associated with the positive regulation of proteins in the catabolic process and negative regulation of developmental growth. These gene family expansions and positively selected genes indicate regions for further interrogation to understand adaptation to high altitude. CONCLUSIONS: Here, we reported a high-quality chromosome-level genome assembly of a high-altitude amphibian species using a combination of Illumina, PacBio and Hi-C sequencing technologies. This genome assembly provides a valuable resource for subsequent research on R. kukunoris genomics and amphibian genome evolution in general.

Soluble programmed death molecule 1 (sPD-1) as a predictor of interstitial lung disease in rheumatoid arthritis.

BACKGROUND: Previous studies have indicated that the programmed death molecule 1 (PD-1) signaling pathway may play a key role in rheumatoid arthritis (RA). However, the pathogenesis of rheumatoid arthritis-related interstitial lung disease (RA-ILD) is not clear. We examined the serum levels of soluble PD-1 in patients with RA and its relationship with RA-ILD. METHODS: Blood samples were obtained from 87 patients with RA (58 with ILD and 29 without ILD) and 45 healthy controls. Serum sPD-1 was measured by Enzyme-Linked Immunosorbent Assay. The pulmonary interstitial disease score was completed by a pulmonary physician and a radiologist through chest high-resolution computed tomography. Patients with RA-ILD were tested for lung function [e.g., forced vital capacity (FVC%), diffusing capacity of lungs for carbon monoxide (DLCO%)]. Associations between ILD and various markers, including sPD-1 and confounding factors, were investigated by logistic regression analysis. Diagnostic values of sPD-1 for the presence of ILD were investigated using receiver operating characteristic curve analysis. RESULTS: Serum sPD-1 levels were higher in RA patients with ILD than in RA patients without ILD and healthy controls (185.1 ± 109.0 pg/ml vs. 119.1 ± 77.5 pg/ml vs. 52.1 ± 21.7 pg/ml, P < 0.05). Serum sPD-1 levels were positively correlated with RF titer (P = 0.02, r = 0.249), anti-cyclic citrullinated peptide antibody status (P = 0.02, r = 0.243), and serum IgG levels (P < 0.001, r = 0.368), negatively associated with FVC% (P = 0.02, r = - 0.344), forced expiratory volume (FEV1%) (P = 0.01, r = - 0.354), total lung capacity (TLC%) (P = 0.046, r = - 0.302), and was independently associated with the presence of ILD in RA patients by multivariate logistic regression analysis. The sensitivity and specificity of sPD-1 levels for the detection of ILD in RA patients were 58.6% and 75.9%, respectively. The area under the curve was 0.689. CONCLUSION: Serum sPD-1 levels were increased in RA patients with ILD. Increased sPD-1 may be a valuable biomarker to predict the presence of ILD in patients with RA.

The Therapeutic Effects of Magnolia Officinalis Extraction on an Antibiotics-Induced Intestinal Dysbacteriosis in Mice.

Magnolia officinalis bark is a traditional Chinese medicine for gastrointestinal tract disorders. In this study, we explored the effects of M. officinalis extraction on intestinal flora to reveal its mechanism. Thirty SPF mice were divided into five groups: C (control), M (M. officinalis), A (antibiotics: cefradine and gentamicin sulfate), A&M (antibiotics + M. officinalis) and A&N (antibiotics + natural recovery). Faecal samples of all groups were collected and the taxonomic composition and diversity of bacteria was characterized using the 16S rRNA gene (16S). Alpha diversity showed gut bacteria diversity significantly decreased in the A group of mice but increased markedly after administration of M. officinalis extract. Beta diversity indicated that C, M and A&M shared similar bacterial community structure while A and A&N exhibited a different bacterial community. Furthermore, RDA combined with spearman correlation heatmap suggested the five physiological indicators (weight, fur, activity and feces) were highly correlated with bacterial community structure and diversity. Finally, functional categorization of the assigned OTUs was performed using the PICRUSt tool. The changes in PICRUSt inferred that function profile and metabolic pathways were observed in A and A&M, therefore the M. officinalis extract improved the intestinal flora of A&M and normalized its metabolic pathways gradually, improving mouse weight, fur quality, activity and feces qualities.

A circulating microRNA signature as noninvasive diagnostic and prognostic biomarkers for nonalcoholic steatohepatitis.

BACKGROUND: Noninvasive biomarkers are urgently needed for patients with nonalcoholic steatohepatitis (NASH) to assist in diagnosis, monitoring disease progression and assessing treatment response. Recently several exploratory studies showed that circulating level of microRNA is associated with NASH and correlated with disease severity. Although these data were encouraging, the application of circulating microRNA as biomarkers for patient screening and stratification need to be further assessed under well-controlled conditions. RESULTS: The expression of circulating microRNAs were profiled in diet-induced NASH progression and regression models to assess the diagnostic and prognostic values and the translatability between preclinical mouse model and men. Since these mice had same genetic background and were housed in the same conditions, there were minimal confounding factors. Histopathological lesions were analyzed at distinct disease progression stages along with microRNA measurement which allows longitudinal assessment of microRNA as NASH biomarkers. Next, differentially expressed microRNAs were identified and validated in an independent cohorts of animals. Thirdly, these microRNAs were examined in a NASH regression model to assess whether they would respond to NASH treatment. MicroRNA profiling in two independent cohorts of animals validated the up-regulation of 6 microRNAs (miR-122, miR-192, miR-21, miR-29a, miR-34a and miR-505) in NASH mice, which was designated as the circulating microRNA signature for NASH. The microRNA signature could accurately distinguish NASH mice from lean mice, and it responded to chow diet treatment in a NASH regression model. To further improve the performance of microRNA-based biomarker, a new composite biomarker was proposed, which consists of miR-192, miR-21, miR-505 and ALT. The new composite biomarker outperformed the microRNA signature in predicting NASH mice which had NAS > 3, and deserves further validations in large scale studies. CONCLUSION: The present study supported the translation of circulating microRNAs between preclinical models and humans in NASH pathogenesis and progression. The microRNA-based composite biomarker may be used for non-invasive diagnosis, clinical monitoring and assessing treatment response for NASH.

Large-scale identification of chemically induced mutations in Drosophila melanogaster.

Forward genetic screens using chemical mutagens have been successful in defining the function of thousands of genes in eukaryotic model organisms. The main drawback of this strategy is the time-consuming identification of the molecular lesions causative of the phenotypes of interest. With whole-genome sequencing (WGS), it is now possible to sequence hundreds of strains, but determining which mutations are causative among thousands of polymorphisms remains challenging. We have sequenced 394 mutant strains, generated in a chemical mutagenesis screen, for essential genes on the Drosophila X chromosome and describe strategies to reduce the number of candidate mutations from an average of -3500 to 35 single-nucleotide variants per chromosome. By combining WGS with a rough mapping method based on large duplications, we were able to map 274 (-70%) mutations. We show that these mutations are causative, using small 80-kb duplications that rescue lethality. Hence, our findings demonstrate that combining rough mapping with WGS dramatically expands the toolkit necessary for assigning function to genes.

The developmental transcriptome of Drosophila melanogaster.

Drosophila melanogaster is one of the most well studied genetic model organisms; nonetheless, its genome still contains unannotated coding and non-coding genes, transcripts, exons and RNA editing sites. Full discovery and annotation are pre-requisites for understanding how the regulation of transcription, splicing and RNA editing directs the development of this complex organism. Here we used RNA-Seq, tiling microarrays and cDNA sequencing to explore the transcriptome in 30 distinct developmental stages. We identified 111,195 new elements, including thousands of genes, coding and non-coding transcripts, exons, splicing and editing events, and inferred protein isoforms that previously eluded discovery using established experimental, prediction and conservation-based approaches. These data substantially expand the number of known transcribed elements in the Drosophila genome and provide a high-resolution view of transcriptome dynamics throughout development.

GATA3 controls the specification of prosensory domain and neuronal survival in the mouse cochlea.

HDR syndrome (also known as Barakat syndrome) is a developmental disorder characterized by hypoparathyroidism, sensorineural deafness and renal disease. Although genetic mapping and subsequent functional studies indicate that GATA3 haplo-insufficiency causes human HDR syndrome, the role of Gata3 in sensorineural deafness and auditory system development is largely unknown. In this study, we show that Gata3 is continuously expressed in the developing mouse inner ear. Conditional knockout of Gata3 in the developing inner ear disrupts the morphogenesis of mouse inner ear, resulting in a disorganized and shortened cochlear duct with significant fewer hair cells and supporting cells. Loss of Gata3 function leads to the failure in the specification of prosensory domain and subsequently, to increased cell death in the cochlear duct. Moreover, though the initial generation of cochleovestibular ganglion (CVG) cells is not affected in Gata3-null mice, spiral ganglion neurons (SGNs) are nearly depleted due to apoptosis. Our results demonstrate the essential role of Gata3 in specifying the prosensory domain in the cochlea and in regulating the survival of SGNs, thus identifying a molecular mechanism underlying human HDR syndrome.

Next-generation sequencing-based molecular diagnosis of 82 retinitis pigmentosa probands from Northern Ireland.

Retinitis pigmentosa (RP) is a group of inherited retinal disorders characterized by progressive photoreceptor degeneration. An accurate molecular diagnosis is essential for disease characterization and clinical prognoses. A retinal capture panel that enriches 186 known retinal disease genes, including 55 known RP genes, was developed. Targeted next-generation sequencing was performed for a cohort of 82 unrelated RP cases from Northern Ireland, including 46 simplex cases and 36 familial cases. Disease-causing mutations were identified in 49 probands, including 28 simplex cases and 21 familial cases, achieving a solving rate of 60 %. In total, 65 pathogenic mutations were found, and 29 of these were novel. Interestingly, the molecular information of 12 probands was neither consistent with their initial inheritance pattern nor clinical diagnosis. Further clinical reassessment resulted in a refinement of the clinical diagnosis in 11 patients. This is the first study to apply next-generation sequencing-based, comprehensive molecular diagnoses to a large number of RP probands from Northern Ireland. Our study shows that molecular information can aid clinical diagnosis, potentially changing treatment options, current family counseling and management.

Synthetic spike-in standards for RNA-seq experiments.

High-throughput sequencing of cDNA (RNA-seq) is a widely deployed transcriptome profiling and annotation technique, but questions about the performance of different protocols and platforms remain. We used a newly developed pool of 96 synthetic RNAs with various lengths, and GC content covering a 2(20) concentration range as spike-in controls to measure sensitivity, accuracy, and biases in RNA-seq experiments as well as to derive standard curves for quantifying the abundance of transcripts. We observed linearity between read density and RNA input over the entire detection range and excellent agreement between replicates, but we observed significantly larger imprecision than expected under pure Poisson sampling errors. We use the control RNAs to directly measure reproducible protocol-dependent biases due to GC content and transcript length as well as stereotypic heterogeneity in coverage across transcripts correlated with position relative to RNA termini and priming sequence bias. These effects lead to biased quantification for short transcripts and individual exons, which is a serious problem for measurements of isoform abundances, but that can partially be corrected using appropriate models of bias. By using the control RNAs, we derive limits for the discovery and detection of rare transcripts in RNA-seq experiments. By using data collected as part of the model organism and human Encyclopedia of DNA Elements projects (ENCODE and modENCODE), we demonstrate that external RNA controls are a useful resource for evaluating sensitivity and accuracy of RNA-seq experiments for transcriptome discovery and quantification. These quality metrics facilitate comparable analysis across different samples, protocols, and platforms.

Identification of TP53 as an acute lymphocytic leukemia susceptibility gene through exome sequencing.

Although acute lymphocytic leukemia (ALL) is the most common childhood cancer, genetic predisposition to ALL remains poorly understood. Whole-exome sequencing was performed in an extended kindred in which five individuals had been diagnosed with leukemia. Analysis revealed a nonsense variant of TP53 which has been previously reported in families with sarcomas and other typical Li Fraumeni syndrome-associated cancers but never in a familial leukemia kindred. This unexpected finding enabled identification of an appropriate sibling bone marrow donor and illustrates that exome sequencing will reveal atypical clinical presentations of even well-studied genes.

Biodegradation of phenol by using free and immobilized cells of Acinetobacter sp. BS8Y.

Strain BS8Y with high biodegradation activity and high tolerance of phenol was isolated from activated sludge in an insulating material plant of China. This strain was capable of removing 99.2% of the initial 600 mg/l phenol in liquid minimal medium within 24 h and tolerating phenol at concentrations of up to 1,200 mg/ml. DNA sequencing and homologous analysis of the 16S rRNA gene identified that the strain BS8Y belonged to an Acinetobacter species. Polyvinyl alcohol was used as gel matrix to immobilize the strain BS8Y. The factors affecting the phenol degradation by immobilized cells and the phenol removal efficiency of free and immobilized cells were investigated; the stability of the immobilized cells is also reported. The results show that the immobilized cells could tolerate a higher phenol level and protected the bacteria much more effectively against changes in temperature and pH. The phenol degradation efficiency was high at up to 96% within 30 h, with an initial concentration of 800 mg/l phenol, and the immobilized cells showed better performance than the suspended cells. Reusability tests revealed that the immobilized cells were stable enough even after reuse for ten times or storing at 4°C for 35 d. These results demonstrate that immobilized Acinetobacter sp. BS8Y possesses a good application potential in the treatment of phenol-containing wastewater.

Study on Safety Coefficient of Sedimentary Bauxite Strip Pillar under Valley Terrain.

The underground sedimentary bauxite ore body in Shanxi province has a shallow burial depth; the valley terrain caused stress concentration on a pillar which affected the pillar's safety and goaf stability. This paper proposed a pillar safety coefficient calculation method affected by the goaf structural parameters and the valley terrain, which was based on a pillar mechanics analysis under the valley terrain. The results show that the overlying valley terrain will cause stress concentration on the pillar, reducing the adequate bearing capacity and the pillar stability. The increase of the goaf span b and the height of the pillar h is extensively detrimental to pillar stability. Meanwhile, increasing the pillar burial depth would cause the pillar to weaken, but can effectively decrease the influence of the valley terrain. Furthermore, when the angle between the goaf strike and the valley strike ß < 50°, ß has a more significant impact on the stress concentration and safety coefficient. The stability of an underground sedimentary bauxite pillar is calculated by the method, the result complied with the actual situation.

CRISPR-surfaceome: An online tool for designing highly efficient sgRNAs targeting cell surface proteins.

CRISPR-based genome-editing tools have emerged as an efficient tool for functional genomics studies. Online tools and databases have been developed to facilitate the design and selection of CRISPR single guide RNA (sgRNA) for gene modifications. However, to the best of our knowledge, none of these tools or database are designated to cell surface proteins. In a previous study, we described the development and application of surfaceome CRISPR libraries targeting to cell surface proteins on human cells. Here, we present the design and construction of an online tool and database (https://crispr-surfaceome.siais.shanghaitech.edu.cn/home), named CRISPR-Surfaceome, for the design of highly efficient sgRNA targeting to the surface proteins on human cells. To show case and validate the efficiencies of sgRNAs designed by this online tool, we chose ICAM-1 gene for knockout studies and found that all the 10 designed ICAM-1 sgRNAs could efficiently generate knockout cells, with more than 80% gene disruption rates. These ICAM-1 knockout cells were found to be resistant to the infection of rhinovirus (RV), which utilizes ICAM-1 as the receptor. Therefore, CRISPR-Surfaceome can serve the research community for the functional genomics studies on cell surface proteins, such as identification of pathogen receptors and discovery of drug targets.

The mitochondrial genome and phylogenetic analysis of Rhacophorus rhodopus.

Classification of the genus Rhacophorus has been problematic. In particular there has been considerable controversy surrounding the phylogenetic relationships among Rhacophorus rhodopus, R. bipunctatus, and R. reinwardtii. To examine the relationship among these Rhacophorus species, we assembled the complete mitochondrial genome sequence of R. rhodopus. The R. rhodopus genome is 15,789 bp in length with 12 protein-coding genes (PCGs) (losing ND5), two ribosomal genes, 22 transfer RNA genes, and a control region (D-loop). Base composition of the overall sequence was 60.86% for A + T content and 39.14% for C + G content. Most of the PCGs used ATG as a start codon, except for the COX I gene, which used the ATA start codon. COX I and ND6 used AGG and ATP8 stop codons respectively, while ND3 and ND4L used the TAA stop codon. For the remaining seven genes, the stop codons was incomplete. In addition, both 5' and 3' of the control areas had distinct repeating regions. Based on three datasets and two methods (Bayesian inference (BI) and maximum likelihood (ML)), we reconstructed three phylogenetic trees to explore the taxonomic status of the species and the phylogenetic relationship among R. rhodopus, R. bipunctatus and R. reinwardtii. Our results indicated that these three species are non-monophyletic; thus, the phylogenetic relationship among them is complex and difficult to determine. Further, R. rhodopus is divided into three lineages from different parts of China. The two Rhacophorus samples showed very close phylogenetic relationship with R. rhodopus. Our results add to the mitochondrial genome database of amphibians and will help to disentangle the phylogenetic relationships within the Rhacophoridae.

Cloning, expression and characterization of the putative nuclear transport factor 2 (NTF2) gene from moss Conocephalum conicum(L.) Dum.

Biomacromolecules import into the nucleus is a complex progress which requires the participation of several cytosolic factors, and nuclear transport factor 2 (NTF2) is one of essential components in nuclear trafficking. Its main role is to transport RanGDP from cytoplasm to nucleus by interacting with FxFG nucleoporin repeats. In the study a putative new gene, designated as CcNTF2, was obtained from the moss (Conocephalum conicum) cDNA library we have constructed. The full-length cDNA sequence is 913 bp in size contains a 372 bp open reading frame (ORF) flanked by a 195 bp 5'-untranslated sequence and a long 346 bp 3'-non-coding region, encoding 123 amino acids of 13,575.3 Da. Part of the genomic sequence was also cloned and sequenced, which is 1,602 bp long and possesses two exons and one intron. Alignment analysis showed that the CcNTF2 protein is high conserved among plant NTF2 and shares 81% similarity with the ones from Arabidopsis thaliana and Brassica rapa. The expression of wild-type CcNTF2 was detected by immunoblotting of extraction of C. conicum and it indicated the putative protein is integral. Through functional expression of CcNTF2-green fluorescent protein (GFP) in tobacco, it was demonstrated that CcNTF2 can accumulate at the nuclear rim. Site-directed mutagenesis analysis confirmed CcNTF2 P71K has influence on the protein import into nucleus. In addition, overexpression of CcNTF2 P71K was observed to be deleterious for the plant cell. It is the first illumination of NTF2 in moss, and our study established the primary foundation for further research on moss NTF2.

Characterizing disease progression of nonalcoholic steatohepatitis in Leptin-deficient rats by integrated transcriptome analysis.

Nonalcoholic steatohepatitis (NASH) is an aggressive liver disease threatening human health, yet no medicine is developed to treat this disease. In this study, we first discovered that Leptin mutant rats (LepΔI14/ΔI14) exhibit characteristic NASH phenotypes including steatosis, lymphocyte infiltration, and ballooning after postnatal week 16. We then examined NASH progression by performing an integrated analysis of hepatic transcriptome in Leptin-deficient rats from postnatal 4 to 48 weeks. Initially, simple steatosis in LepΔI14/ΔI14 rats were observed with increased expression of the genes encoding for rate-limiting enzymes in lipid metabolism such as acetyl-CoA carboxylase and fatty acid synthase. When NASH phenotypes became well developed at postnatal week 16, we found gene expression changes in insulin resistance, inflammation, reactive oxygen species and endoplasmic reticulum stress. As NASH phenotypes further progressed with age, we observed elevated expression of cytokines and chemokines including C-C motif chemokine ligand 2, tumor necrosis factor ɑ, interleukin-6, and interleukin-1ß together with activation of the c-Jun N-terminal kinase and nuclear factor-κB pathways. Histologically, livers in LepΔI14/ΔI14 rats exhibited increased cell infiltration of MPO+ neutrophils, CD8+ T cells, CD68+ hepatic macrophages, and CCR2+ inflammatory monocyte-derived macrophages associated with macrophage polarization from M2 to M1. Subsequent cross-species comparison of transcriptomes in human, rat, and mouse NASH models indicated that Leptin-deficient rats bear more similarities to human NASH patients than previously established mouse NASH models. Taken together, our study suggests that LepΔI14/ΔI14 rats are a valuable pre-clinical rodent model to evaluate NASH drug safety and efficacy.

Characterization of complete mitochondrial genome of Hylarana guentheri (Anura: Ranidae) and its phylogenetic implication.

The Günther's frog (Hylarana guentheri) belongs to a member of the family Ranidae. We provide a complete mitogenome of H. guentheri and examine its phylogenetic position with other related species. Its mitogenome is a closed circular molecule 18,698 bp in length including 13 protein coding genes, 22 tRNA coding genes, two rRNA-coding genes, and a control region (CR) that are conserved in most Ranidae mitogenomes. The overall base composition of the H. guentheri mitogenome is 29.27% A, 30.45% T, 26.14% C, and 14.15% G, which is typical for Amphibious animals' mitochondrial genomes. The alignment of the Ranidae species control regions showed high levels of genetic variation and abundant AT content. Seven tandem repeats were found in the control region. Phylogenetic analysis with Bayesian inference and maximum likelihood based on 13 protein-coding genes indicated that H. guentheri is more closely related to Nidirana okinavana than to Babina subaspera and B. holsti. The complete mitogenome of H. guentheri provides a potentially useful resource for further exploration of the taxonomic status and phylogenetic relationships of Hylarana and related species.

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Large-scale estimation of forest biomass has received much attention. Constructing a stand-level biomass model is a method for estimating tree layer biomass. In this study, we constructed stand biomass models of Korean pine plantations based on aggregation method 1, aggregation method 2, adjustment method, and disaggregation method. The prediction precision of four additive methods was compared and analyzed to provide theoretical basis for biomass prediction of Korean pine plantations in Heilongjiang Province. Weighted functions were used to eliminate the heteroscedasticity of each model, with the leave-one-out cross validation (LOOCV) as the validation method. The results showed that the overall prediction ability of the adjustment method was slightly better than other methods. The specific prediction precision was ranked as adjustment method > aggregation method 1 > aggregation method 2 > disaggregation method. The prediction precision of four additive methods was not consistent when considering their prediction ability of different stand basal areas. When the stand basal area of Korean pine plantations was distributed in the interval of 0-10 or 50-60 m2·hm-2, the parameter estimation values of disaggregation method performed better. When the stand basal area was distributed in other intervals, the parameter estimation values of adjustment method was better.

The complete mitochondrial genome sequence of big-eyed Mountain keelback Pseudoxenodon macrops.

In this study, the complete mitochondrial genome of big-eyed mountain keelback Pseudoxenodon macrops was sequenced adopting Illumina high-throughput sequencing technology. The complete mitogenome of the species was 19,444 bp in length, including 13 protein-coding genes, 22 transfer RNA (tRNA) genes, two ribosomal RNA genes, and two non-coding control regions (CR). The overall base composition of mitogenome was 32.0% A, 25.5% T, 28.2% C, and 14.3% G. Most mitochondrial genes are encoded on the heavy strand, only ND6 and eight tRNA genes are on the light strand. We expect that the presented mitogenome can provide important data for future studies on phylogenetic relationship and population genetics of this species.