Down-Regulation of Mitochondrial Metabolism after Tendon Release Primes Lipid Accumulation in Rotator Cuff Muscle.

Atrophy and fat accumulation are debilitating aspects of muscle diseases and are rarely prevented. Using a vertical approach combining anatomic techniques with omics methodology in a tenotomy-induced sheep model of rotator cuff disease, we tested whether mitochondrial dysfunction is implicated in muscle wasting and perturbed lipid metabolism, speculating that both can be prevented by the stimulation of ß-oxidation with l-carnitine. The infraspinatus muscle lost 22% of its volume over the first 6 weeks after tenotomy before the area-percentage of lipid increased from 8% to 18% at week 16. Atrophy was associated with the down-regulation of mitochondrial transcripts and protein and a slow-to-fast shift in muscle composition. Correspondingly, amino acid levels were increased 2 weeks after tendon release, when the levels of high-energy phosphates and glycerophospholipids were lowered. l-Carnitine administration (0.9 g/kg per day) prevented atrophy over the first 2 weeks, and mitigated alterations of glutamate, glycerophospholipids, and carnitine levels in released muscle, but did not prevent the level decrease in high-energy phosphates or protein constituents of mitochondrial respiration, promoting the accumulation of longer lipids with an increasing saturation. We conclude that the early phase of infraspinatus muscle degeneration after tendon release involves the elimination of oxidative characteristics associated with an aberrant accumulation of lipid species but is largely unrelated to the prevention of atrophy with oral l-carnitine administration.

The haplotype-resolved chromosome pairs of a heterozygous diploid African cassava cultivar reveal novel pan-genome and allele-specific transcriptome features.

BACKGROUND: Cassava (Manihot esculenta) is an important clonally propagated food crop in tropical and subtropical regions worldwide. Genetic gain by molecular breeding has been limited, partially because cassava is a highly heterozygous crop with a repetitive and difficult-to-assemble genome. FINDINGS: Here we demonstrate that Pacific Biosciences high-fidelity (HiFi) sequencing reads, in combination with the assembler hifiasm, produced genome assemblies at near complete haplotype resolution with higher continuity and accuracy compared to conventional long sequencing reads. We present 2 chromosome-scale haploid genomes phased with Hi-C technology for the diploid African cassava variety TME204. With consensus accuracy >QV46, contig N50 >18 Mb, BUSCO completeness of 99%, and 35k phased gene loci, it is the most accurate, continuous, complete, and haplotype-resolved cassava genome assembly so far. Ab initio gene prediction with RNA-seq data and Iso-Seq transcripts identified abundant novel gene loci, with enriched functionality related to chromatin organization, meristem development, and cell responses. During tissue development, differentially expressed transcripts of different haplotype origins were enriched for different functionality. In each tissue, 20-30% of transcripts showed allele-specific expression (ASE) differences. ASE bias was often tissue specific and inconsistent across different tissues. Direction-shifting was observed in <2% of the ASE transcripts. Despite high gene synteny, the HiFi genome assembly revealed extensive chromosome rearrangements and abundant intra-genomic and inter-genomic divergent sequences, with large structural variations mostly related to LTR retrotransposons. We use the reference-quality assemblies to build a cassava pan-genome and demonstrate its importance in representing the genetic diversity of cassava for downstream reference-guided omics analysis and breeding. CONCLUSIONS: The phased and annotated chromosome pairs allow a systematic view of the heterozygous diploid genome organization in cassava with improved accuracy, completeness, and haplotype resolution. They will be a valuable resource for cassava breeding and research. Our study may also provide insights into developing cost-effective and efficient strategies for resolving complex genomes with high resolution, accuracy, and continuity.

De novo expression of gastrokines in pancreatic precursor lesions impede the development of pancreatic cancer.

Molecular events occurring in stepwise progression from pre-malignant lesions (pancreatic intraepithelial neoplasia; PanIN) to the development of pancreatic ductal adenocarcinoma (PDAC) are poorly understood. Thus, characterization of early PanIN lesions may reveal markers that can help in diagnosing PDAC at an early stage and allow understanding the pathology of the disease. We performed the molecular and histological assessment of patient-derived PanINs, tumor tissues and pancreas from mouse models with PDAC (KC mice that harbor K-RAS mutation in pancreatic tissue), where we noted marked upregulation of gastrokine (GKN) proteins. To further understand the role of gastrokine proteins in PDAC development, GKN-deficient KC mice were developed by intercrossing gastrokine-deficient mice with KC mice. Panc-02 (pancreatic cancer cells of mouse origin) were genetically modified to express GKN1 for further in vitro and in vivo analysis. Our results show that gastrokine proteins were absent in healthy pancreas and invasive cancer, while its expression was prominent in low-grade PanINs. We could detect these proteins in pancreatic juice and serum of KC mice. Furthermore, accelerated PanIN and tumor development were noted in gastrokine deficient KC mice. Loss of gastrokine 1 protein delayed apoptosis during carcinogenesis leading to the development of desmoplastic stroma while loss of gastrokine 2 increased the proliferation rate in precursor lesions. In summary, we identified gastrokine proteins in early pancreatic precursor lesions, where gastrokine proteins delay pancreatic carcinogenesis.

The RNA-binding protein Musashi controls axon compartment-specific synaptic connectivity through ptp69D mRNA poly(A)-tailing.

Synaptic targeting with subcellular specificity is essential for neural circuit assembly. Developing neurons use mechanisms to curb promiscuous synaptic connections and to direct synapse formation to defined subcellular compartments. How this selectivity is achieved molecularly remains enigmatic. Here, we discover a link between mRNA poly(A)-tailing and axon collateral branch-specific synaptic connectivity within the CNS. We reveal that the RNA-binding protein Musashi binds to the mRNA encoding the receptor protein tyrosine phosphatase Ptp69D, thereby increasing poly(A) tail length and Ptp69D protein levels. This regulation specifically promotes synaptic connectivity in one axon collateral characterized by a high degree of arborization and strong synaptogenic potential. In a different compartment of the same axon, Musashi prevents ectopic synaptogenesis, revealing antagonistic, compartment-specific functions. Moreover, Musashi-dependent Ptp69D regulation controls synaptic connectivity in the olfactory circuit. Thus, Musashi differentially shapes synaptic connectivity at the level of individual subcellular compartments and within different developmental and neuron type-specific contexts.

A comprehensive examination of Nanopore native RNA sequencing for characterization of complex transcriptomes.

A platform for highly parallel direct sequencing of native RNA strands was recently described by Oxford Nanopore Technologies, but despite initial efforts it remains crucial to further investigate the technology for quantification of complex transcriptomes. Here we undertake native RNA sequencing of polyA + RNA from two human cell lines, analysing ~5.2 million aligned native RNA reads. To enable informative comparisons, we also perform relevant ONT direct cDNA- and Illumina-sequencing. We find that while native RNA sequencing does enable some of the anticipated advantages, key unexpected aspects currently hamper its performance, most notably the quite frequent inability to obtain full-length transcripts from single reads, as well as difficulties to unambiguously infer their true transcript of origin. While characterising issues that need to be addressed when investigating more complex transcriptomes, our study highlights that with some defined improvements, native RNA sequencing could be an important addition to the mammalian transcriptomics toolbox.

Pain-Associated Transcriptome Changes in Synovium of Knee Osteoarthritis Patients.

Joint pain causes significant morbidity in osteoarthritis (OA). The aetiology of joint pain in OA is not well understood. The synovial membrane as an innervated joint structure represents a potential source of peripheral pain in OA. Here we analyse, using a hypothesis-free next generation RNA sequencing, the differences in protein-coding and non-coding transcriptomes in knee synovial tissues from OA patients with high knee pain (n = 5) compared with OA patients with low knee pain (n = 5), as evaluated by visual analogue scale (VAS). We conduct Gene Ontology and pathway analyses on differentially expressed mRNA genes. We identify new protein-coding, long non-coding RNA and microRNA candidates that can be associated with OA joint pain. Top enriched genes in painful OA knees encode neuronal proteins that are known to promote neuronal survival under cellular stress or participate in calcium-dependent synaptic exocytosis and modulation of GABA(γ-aminobutyric acid)ergic activity. Our study uncovers transcriptome changes associated with pain in synovial microenvironment of OA knees. This sets a firm ground for future mechanistic studies and drug discovery to alleviate joint pain in OA.

The transcriptome response of the ruminal methanogen Methanobrevibacter ruminantium strain M1 to the inhibitor lauric acid.

OBJECTIVE: Lauric acid (C12) is a medium-chain fatty acid that inhibits growth and production of the greenhouse gas methane by rumen methanogens such as Methanobrevibacter ruminantium. To understand the inhibitory mechanism of C12, a transcriptome analysis was performed in M. ruminantium strain M1 (DSM 1093) using RNA-Seq. RESULTS: Pure cell cultures in the exponential growth phase were treated with 0.4 mg/ml C12, dissolved in dimethyl sulfoxide (DMSO), for 1 h and transcriptomic changes were compared to DMSO-only treated cells (final DMSO concentration 0.2%). Exposure to C12 resulted in differential expression of 163 of the 2280 genes in the M1 genome (maximum log2-fold change 6.6). Remarkably, C12 hardly affected the expression of genes involved in methanogenesis. Instead, most affected genes encode cell-surface associated proteins (adhesion-like proteins, membrane-associated transporters and hydrogenases), and proteins involved in detoxification or DNA-repair processes. Enrichment analysis on the genes regulated in the C12-treated group showed a significant enrichment for categories 'cell surface' and 'mobile elements' (activated by C12), and for the categories 'regulation' and 'protein fate' (represssed). These results are useful to generate and test specific hypotheses on the mechanism how C12 affects rumen methanogens.