Abstract
The Silencing Mediator of Retinoid and Thyroid Hormone Receptors (SMRT) is a nuclear corepressor, regulating the transcriptional activity of many transcription factors critical for metabolic ...processes. While the importance of the role of SMRT in the adipocyte has been well-established, our comprehensive understanding of its in vivo function in the context of homeostatic maintenance is limited due to contradictory phenotypes yielded by prior generalized knockout mouse models. Multiple such models agree that SMRT deficiency leads to increased adiposity, although the effects of SMRT loss on glucose tolerance and insulin sensitivity have been variable. We therefore generated an adipocyte-specific SMRT knockout (adSMRT-/-) mouse to more clearly define the metabolic contributions of SMRT. In doing so, we found that SMRT deletion in the adipocyte does not cause obesity—even when mice are challenged with a high-fat diet. This suggests that adiposity phenotypes of previously described models were due to effects of SMRT loss beyond the adipocyte. However, an adipocyte-specific SMRT deficiency still led to dramatic effects on systemic glucose tolerance and adipocyte insulin sensitivity, impairing both. This metabolically deleterious outcome was coupled with a surprising immune phenotype, wherein most genes differentially expressed in the adipose tissue of adSMRT-/- mice were upregulated in pro-inflammatory pathways. Flow cytometry and conditioned media experiments demonstrated that secreted factors from knockout adipose tissue strongly informed resident macrophages to develop a pro-inflammatory, MMe (metabolically activated) phenotype. Together, these studies suggest a novel role for SMRT as an integrator of metabolic and inflammatory signals to maintain physiological homeostasis.
This study aimed to investigate the effects of different growth stages (booting period-SYK; initial flowering-SCK; full flowering-SSK) on the fermentation quality, microbial community, metabolic ...pathways and metabolomic characteristics of Italian ryegrass silage.
Single molecule real-time (SMRT) sequencing and ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) were used to analyze bacterial communities and metabolites, respectively.
After 60 d of fermentation, SYK had the lowest pH and the highest lactic acid content, which were significantly different from the other groups. The bacteria with the highest abundance in SYK, SCK and SSK groups were
(63.98%), Weissella minor (28.82%) and
(64.81%), respectively. In addition, among the main differential metabolites in different growth stages, the number of amino acids was the most, and the corresponding metabolic pathways were mainly amino acid metabolic pathways. The biosynthesis of phenylalanine, tyrosine and tryptophan was significantly enriched (p<0.01) at booting stage and full flowering stage. Purine metabolism and ABC transporter pathway were significantly enriched at the initial flowering (p<0.001).
had a negative correlation with xanthine and ganoderic acid F.
had a positive correlation with D-Mannose and ganoderic acid F.
had a positive correlation with xanthine, and
had a positive correlation with cinnamic acid, D-Mannose, 2-Hydroxycinnamic acid and uridine.
In conclusion, this study reveals the interaction mechanisms between ryegrass raw materials at different growth stages and epiphytic microorganisms during ensiling fermentation, providing new ideas for screening functional lactic acid bacteria, and laying a theoretical foundation for the production of safe and high-quality silage.
Summary
China is the origin and evolutionary centre of Oriental pears. Pyrus betuleafolia is a wild species native to China and distributed in the northern region, and it is widely used as rootstock. ...Here, we report the de novo assembly of the genome of P. betuleafolia‐Shanxi Duli using an integrated strategy that combines PacBio sequencing, BioNano mapping and chromosome conformation capture (Hi‐C) sequencing. The genome assembly size was 532.7 Mb, with a contig N50 of 1.57 Mb. A total of 59 552 protein‐coding genes and 247.4 Mb of repetitive sequences were annotated for this genome. The expansion genes in P. betuleafolia were significantly enriched in secondary metabolism, which may account for the organism's considerable environmental adaptability. An alignment analysis of orthologous genes showed that fruit size, sugar metabolism and transport, and photosynthetic efficiency were positively selected in Oriental pear during domestication. A total of 573 nucleotide‐binding site (NBS)‐type resistance gene analogues (RGAs) were identified in the P. betuleafolia genome, 150 of which are TIR‐NBS‐LRR (TNL)‐type genes, which represented the greatest number of TNL‐type genes among the published Rosaceae genomes and explained the strong disease resistance of this wild species. The study of flavour metabolism‐related genes showed that the anthocyanidin reductase (ANR) metabolic pathway affected the astringency of pear fruit and that sorbitol transporter (SOT) transmembrane transport may be the main factor affecting the accumulation of soluble organic matter. This high‐quality P. betuleafolia genome provides a valuable resource for the utilization of wild pear in fundamental pear studies and breeding.
Determining the sequence of DNA and RNA molecules has a huge impact on the understanding of cell biology and function. Recent advancements in next-generation short-read sequencing (NGS) technologies, ...drops in cost and a resolution down to the single-cell level shaped our current view on genome structure and function. Third-generation sequencing (TGS) methods further complete the knowledge about these processes based on long reads and the ability to analyze DNA or RNA at single molecule level. Long-read sequencing provides additional possibilities to study genome architecture and the composition of highly complex regions and to determine epigenetic modifications of nucleotide bases at a genome-wide level. We discuss the principles and advancements of long-read sequencing and its applications in genome biology.
The hyperpolymorphic HLA genes play important roles in disease and transplantation and act as genetic markers of migration and evolution. A panel of 107 B‐lymphoblastoid cell lines (B‐LCLs) was ...established in 1987 at the 10th International Histocompatibility Workshop as a resource for the immunogenetics community. These B‐LCLs are well characterised and represent diverse ethnicities and HLA haplotypes. Here we have applied Pacific Biosciences’ Single Molecule Real‐Time (SMRT) DNA sequencing to HLA type 126 B‐LCL, including the 107 International HLA and Immunogenetics Workshop (IHIW) cells, to ultra‐high resolution. Amplicon sequencing of full‐length HLA class I genes (HLA‐A, ‐B and ‐C) and partial length HLA class II genes (HLA‐DRB1, ‐DQB1 and ‐DPB1) was performed. We typed a total of 931 HLA alleles, 895 (96%) of which were consistent with the typing in the IPD‐IMGT/HLA Database (Release 3.27.0, January 20, 2017), with 595 (64%) typed at a higher resolution. Discrepant types, including novel alleles (n = 10) and changes in zygosity (n = 13), as well as previously unreported types (n = 34) were observed. In addition, patterns of linkage disequilibrium were distinguished by four‐field resolution typing of HLA‐B and HLA‐C. By improving and standardising the HLA typing of these B‐LCLs, we have ensured their continued usefulness as a resource for the immunogenetics community in the age of next generation DNA sequencing.
Intercropping is an important soil management practice for increasing orchard productivity and land-use efficiency because it has beneficial effects on soil microbial communities and soil properties. ...However, there is relatively little information available regarding the effects of different crops/grasses on soil microbial communities and soil metabolic products in apple orchards in arid and semi-arid regions. In this study, we showed the microbial communities of apple, intercropping plants, and sandy waste soil, using the third-generation PacBio SMRT long-read sequencing technology. Our results also revealed that the microbial communities and soil metabolic properties differed significantly between apple and the sandy waste soil and the intercropping plants. Intercropping could significantly enrich diverse microbial species, microbial nitrogen, and microbial carbon of soil. Moreover, intercropping with licorice showed better effects in recruiting beneficial microbes, compared to grass and pepper, significantly enriching species belonging to some well-known taxa with beneficial effects, including
Bacillus, Ensifer, Paenibacillus, Rhizobium
, and
Sphingomonas
. Thus, intercropping with licorice may improve apple tree growth and disease resistance. Furthermore,
Bradyrhizobium
and
Rubrobacter
were included among the keystone taxa of apple, whereas
Bacillus, Chitinophaga, Stenotrophobacter, Rubrobacter
, and
Luteimonas
were the keystone taxa of the intercropping plants. The results of our study suggest that intercropping with licorice is a viable option for increasing apple orchard productivity.
Long‐read sequencing can resolve regions of the genome that are inaccessible to short reads, and therefore are ideal for genome‐gap closure, solving structural rearrangements and sequencing through ...repetitive elements. Here we introduce the Xdrop technology: a novel microfluidic‐based system that allows for targeted enrichment of long DNA molecules starting from only a few nanograms of DNA. Xdrop is based on the isolation of long DNA fragments in millions of droplets, where the droplets containing a target sequence of interest are fluorescently labeled and sorted using flow cytometry. The final product from the Xdrop procedure is an enriched population of long DNA molecules that can be investigated by sequencing. To demonstrate the capability of Xdrop, we performed enrichment of the human papilloma virus 18 integrated into the genome of human HeLa cells. Analysis of the sequencing reads resolved three HPV18‐chr8 integrations at base‐pair resolution, and the captured fragments extended up to 30 kb into the human genome at the integration sites. Further, we enriched the complete TP53 locus in a leukemia cell line and could successfully phase coexisting mutations using PacBio sequencing. In summary, our results show that Xdrop is an efficient enrichment technology for studying complex genomic regions.
Here we introduce the Xdrop technology: a novel microfluidic‐based system that allows for targeted enrichment of long DNA molecules starting from only a few nanograms of DNA. Xdrop is based on the isolation of long DNA fragments in millions of droplets, where the droplets containing a target sequence of interest are fluorescently labeled and sorted using flow cytometry. The final product from the Xdrop procedure is an enriched population of long DNA molecules that can be investigated by sequencing.
Thalassemia is the most frequent recessive Mendelian inherited monogenic disease worldwide, and is characterized by the impaired synthesis of globin chains due to disease-causing variants in α- or ...β-globin genes. There are many conventional methods to diagnose thalassemia but all of them have limitations.
We present the case of a 37-year-old female with abnormal values of routine hematological indices who was admitted for genetic screening of thalassemia. Genomic DNA was extracted and used for genetic assays covering the known and potential novel genotypes in HBA and HBB genes using a suspension-array system, gap-polymerase chain reaction (Gap-PCR), PCR-reverse dot blot (PCR-RDB) and multiplex ligation-dependent probe amplification (MLPA). Finally, using long-read single-molecule real-time (SMRT) sequencing, we first confirmed the case with a novel 15.8 kb deletion located in the HBA gene (Chr16:163886–179768, GRch38/hg38).
Our results showed that long-read SMRT sequencing has great advantages in the detection of rare α-globin gene variants. This study may provide a reference protocol for the use of long-read SMRT sequencing for the detection of known and potential novel genotypes of thalassemia in the population and improve the accuracy of genetic counseling and prenatal diagnosis.
Candida glabrata is an opportunistic pathogen of humans, responsible for up to 30% of disseminated candidiasis. Adherence of C. glabrata to host cells is mediated by adhesin‐like proteins (ALPs), ...about half of which are encoded in the subtelomeres. We performed a de novo assembly of two C. glabrata strains, BG2 and BG3993, using long single‐molecule real‐time (SMRT) reads, and constructed high‐quality telomere‐to‐telomere assemblies of all 13 chromosomes to assess differences between C. glabrata strains. We documented variation between strains, and in agreement with earlier studies, found high (~0.5%–1%) frequencies of SNVs across the genome, including within subtelomeric regions. We documented changes in ALP gene structure and complement: there are large length differences in ALP genes in different strains, resulting from copy number variation in tandem repeats. We compared strains to characterize chromosome rearrangement events including within the poorly characterized subtelomeric regions. We show that rearrangements within the subtelomere regions all affect ALP‐encoding genes, and 14/16 involve just the most terminal ALP gene. We present evidence that these rearrangements are mediated by break‐induced replication. This study highlights the constrained nature of subtelomeric changes impacting ALP gene complement and subtelomere structure.
Candida glabrata subtelomeres are enriched for genes encoding adhesins and adhesin‐like proteins; most strain to strain
variation in gene complement is due to subtelomeric changes. We have used long read sequencing to assemble the
genomes, including subtelomeres, of three strains. We show that variation in subtelomere gene complement is limited,
and primarily due to translocation, duplication and deletion of the most telomeric gene for a subset of subtelomeres,
likely mediated by break‐induced replication.
The discovery of epigenetic bases has revolutionised the understanding of disease and development. Among the most studied epigenetic marks are cytosines covalently modified at the 5 position. In ...order to gain insight into their biological significance, the ability to determine their spatiotemporal distribution within the genome is essential. Techniques for sequencing on “next‐generation” platforms often involve harsh chemical treatments leading to sample degradation. Third‐generation sequencing promises to further revolutionise the field by providing long reads, enabling coverage of highly repetitive regions of the genome or structural variants considered unmappable by next generation sequencing technology. While the ability of third‐generation platforms to directly detect epigenetic modifications is continuously improving, at present chemical or enzymatic derivatisation presents the most convenient means of enhancing reliability. This Review presents techniques available for the detection of cytosine modifications on third‐generation platforms.
Third‐generation sequencing platforms provide fast, convenient, and often inexpensive means of DNA sequencing; however, direct detection of epigenetic bases is yet to be routinely achieved. Here, derivatisation and labelling techniques that enable profiling of cytosine modifications on these platforms are reviewed.
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