Skeletal muscle mitochondria adaptation to exercise training is mediated by molecular factors that are not fully understood. Mitochondria import over 1000 proteins encoded by the nuclear genome, but the RNA population resident within the organelle is generally thought to be exclusively encoded by the mitochondrial genome. However, recent in vitro evidence suggests that specific nuclear-encoded miRNAs and other noncoding RNAs (ncRNAs) can reside within the mitochondrial matrix. Whether these are present in mitochondria of skeletal muscle tissue, and whether this is affected by endurance training-a potent metabolic stimulus for mitochondrial adaptation-remains unknown. Rats underwent 4 weeks of moderate-intensity treadmill exercise training, then were humanely killed and tissues were collected for molecular profiling. Mitochondria from gastrocnemius skeletal muscle were isolated by immunoprecipitation, further purified, and then the resident RNA was sequenced to assess the mitochondrial transcriptome. Exercise training elicited typical transcriptomic responses and functional adaptations in skeletal muscle, including increased mitochondrial respiratory capacity. We identified 24 nuclear-encoded coding or noncoding RNAs in purified mitochondria, in addition to 50 nuclear-encoded miRNAs that met a specified abundance threshold. Although none were differentially expressed in the exercise vs. control group at FDR < 0.05, exploratory analyses suggested that the abundance of 3 miRNAs was altered (p < 0.05) in mitochondria isolated from trained compared with sedentary skeletal muscle. We report the presence of a specific population of nuclear-encoded RNAs in the mitochondria isolated from rat skeletal muscle tissue, which could play a role in regulating exercise adaptations and mitochondrial biology.

ABSTRACT Mitochondria are central to cellular function, particularly in metabolically active tissues such as skeletal muscle. Nuclear-encoded RNAs typically localise within the nucleus and cytosol but a small population may also translocate to subcellular compartments such as mitochondria. We aimed to investigate the nuclear-encoded RNAs that localise within the mitochondria of skeletal muscle cells and tissue. Intact mitochondria were isolated via immunoprecipitation (IP) followed by enzymatic treatments (RNase-A and proteinase-K) optimised to remove transcripts located exterior to mitochondria, making it amenable for high-throughput transcriptomic sequencing. Small-RNA sequencing libraries were successfully constructed from as little as 1.8 ng mitochondrial RNA input. Small-RNA sequencing of mitochondria from rat myoblasts revealed the enrichment of over 200 miRNAs. Whole-transcriptome RNA sequencing of enzymatically-purified mitochondria isolated by IP from skeletal muscle tissue showed a striking similarity in the degree of purity compared to mitoplast preparations which lack an outer mitochondrial membrane. In summary, we describe a novel, powerful sequencing approach applicable to animal and human tissues and cells that can facilitate the discovery of nuclear-encoded RNA transcripts localised within skeletal muscle mitochondria.

Initially thought to localise at the cytosol and nucleus only, emerging evidence indicates that miRNAs also localise within the mitochondria where they could regulate diverse pathological and physiological processes. Therefore, the aim of the current study was to use small RNA sequencing to profile and compare the entire population of miRNAs in human skeletal muscle of healthy males in whole-tissue and in isolated mitochondria at rest and in response to acute endurance exercise. Twelve healthy males (age 26(4) years, (mean(SD)) cycled for 60 min at 70% VO2peak and muscle biopsies were collected at rest, immediately after and 3 h after exercise. The mitochondria were isolated by immunoprecipitation, further purified, then the resident RNA was sequenced to assess the mitochondrial transcriptome. Small RNA sequencing revealed that mitochondria isolated from male skeletal muscle tissue contain a small and distinct population of miRNAs. Of the approximately 110 mature miRNAs detected in skeletal muscle mitochondria at each time-point, the canonical myo-miRs miR-1, miR-133 and miR-206 families constituted on average 45% of total mitochondria miRNA reads. However, none of these canonical myo-miRs were differentially expressed in mitochondria following endurance exercise. One miRNA, hsa-miR-146b-3p, was differentially expressed in both whole muscle tissue and mitochondria when adjusted for multiple testing (FDR <0.05). Future research is now required to investigate miRNA-mRNA interactions in the mitochondria of skeletal muscle tissue. ### Competing Interest Statement The authors have declared no competing interest.

The evolution of Australia’s distinctive marsupial fauna has long been linked to the onset of continent-wide aridity. However, how this profound climate change event affected the diversification of extant lineages is still hotly debated. Here, we assemble a DNA sequence dataset of Macropodoidea—the clade comprising kangaroos and their relatives—that incorporates a complete mitogenome for the Desert ‘rat-kangaroo’, Caloprymnus campestris . This enigmatic species went extinct nearly 90 years ago and is known from a handful of museum specimens. Caloprymnus is significant because it was the only macropodoid restricted to extreme desert environments, and therefore calibrates the group’s specialisation for increasingly arid conditions. Our robustly supported phylogenies nest Caloprymnus amongst the bettongs Aepyprymnus and Bettongia . Dated ancestral range estimations further reveal that the Caloprymnus - Bettongia lineage originated in nascent xeric settings during the middle to late Miocene, ~ 12 million years ago (Ma), but subsequently radiated into fragmenting mesic habitats after the Pliocene to mid-Pleistocene. This timeframe parallels the ancestral divergences of kangaroos in woodlands and forests, but predates their adaptive dispersal into proliferating dry shrublands and grasslands from the late Miocene to mid-Pleistocene, after ~ 7 Ma. We thus demonstrate that protracted changes in both climate and vegetation likely staged the emergence of modern arid zone macropodoids.

Understanding the molecular basis of plant heat tolerance helps to predict the consequences of a warming climate on plant performance, particularly in vulnerable environments. Our current understanding comes primarily from studies in Arabidopsis thaliana and selected crops exposed to short and intense heat stress. In this study we sought to characterise the molecular responses of an Australian alpine herb ( Wahlenbergia ceracea ) to growth under sustained moderate warming. We compared responses of pre-defined tolerant and sensitive lines, based on measures of photosynthetic stability, to growth under cool or warm temperatures to identify the pathways involved in thermal tolerance and acclimation. Under warmer growth temperatures, W. ceracea up-regulated genes involved in RNA metabolism, while down-regulating those involved in photosynthesis and pigment metabolism. In tolerant lines, genes related to photosystem II, light-dependent photosynthetic reactions, and chlorophyll metabolism were more strongly down-regulated. This suggests that the regulation of electron transport and its components may be involved in thermal acclimation. Our results also highlight the importance of hormonal gene networks, particularly ethylene, during longer-term moderate warming. In conclusion, our results point to post-transcriptional processes and the stabilisation of the electron transport chain as candidate mechanisms for thermal acclimation in W. ceracea . The study also revealed many non-orthologous temperature-responsive genes, whose characterization may enhance our understanding of physiological acclimation and have relevance for the biotechnological improvement of threatened species and crops.

The microbiota-gut-brain axis' role in Parkinson's disease (PD) pathophysiology, and how this differs from typical ageing, is poorly understood. Presently, gut-bacterial diversity, taxonomic abundance and metabolic bacterial pathways were compared across healthy young (n = 22, 18-35 years), healthy older (n = 33, 50-80 years), and PD groups (n = 18, 50-80 years) using shotgun sequencing and compositional data analysis. Asso-ciations between the gut-microbiome and PD symptoms, and between lifestyle factors (fibre intake, physical activity, and sleep) and the gut-microbiome were conducted. Alpha-diversity did not differ between PD partic-ipants and older adults, whilst beta-diversity differed between these groups. Lower abundance of Butyricimonas synergistica, a butyrate-producer, was associated with worse PD non-motor symptoms in the PD group. Regarding typical ageing, Bifidobacterium bifidum, was greater in the younger compared to older group, with no difference between the older and PD group. Abundance of metabolic pathways related to butyrate production did not differ among the groups, while other metabolic pathways differed among the three groups. Sleep efficiency was positively associated with Roseburia inulinivorans in the older group. These results highlight the relevance of gut-microbiota to PD and that reduced butyrate-production may be involved with PD pathophysiology. Future studies should account for lifestyle factors when investigating gut-microbiomes across ageing and in PD. This article is part of the Special Issue on "Microbiome & the Brain: Mechanisms & Maladies".

We present the genome of the living fossil, Wollemia nobilis, a southern hemisphere conifer morphologically unchanged since the Cretaceous. Presumed extinct until rediscovery in 1994, the Wollemi pine is critically endangered with less than 60 wild adults threatened by intensifying bushfires in the Blue Mountains of Australia. The 12 Gb genome is among the most contiguous large plant genomes assembled, with extremely low heterozygosity and unusual abundance of DNA transposons. Reduced representation and genome re-sequencing of individuals confirms a relictual population since the last major glacial/drying period in Australia, 120 ky BP. Small RNA and methylome sequencing reveal conservation of ancient silencing mechanisms despite the presence of thousands of active and abundant transposons, including some transferred horizontally to conifers from arthropods in the Jurassic. A retrotransposon burst 8-6 my BP coincided with population decline, possibly as an adaptation enhancing epigenetic diversity. Wollemia, like other conifers, is susceptible to Phytophthora, and a suite of defense genes, similar to those in loblolly pine, are targeted for silencing by sRNAs in leaves. The genome provides insight into the earliest seed plants, while enabling conservation efforts.

Salmonella infections across the globe are becoming more challenging to control due to the emergence of multidrug-resistant (MDR) strains. Lytic phages may be suitable alternatives for treating these multidrug-resistant Salmonella infections. Most Salmonella phages to date were collected from human-impacted environments. To further explore the Salmonella phage space, and to potentially identify phages with novel characteristics, we characterized Salmonella-specific phages isolated from the Penang National Park, a conserved rainforest. Four phages with a broad lytic spectrum (kills >5 Salmonella serovars) were further characterized; they have isometric heads and cone-shaped tails, and genomes of ~39,900 bp, encoding 49 CDSs. As the genomes share a <95% sequence similarity to known genomes, the phages were classified as a new species within the genus Kayfunavirus. Interestingly, the phages displayed obvious differences in their lytic spectrum and pH stability, despite having a high sequence similarity (~99% ANI). Subsequent analysis revealed that the phages differed in the nucleotide sequence in the tail spike proteins, tail tubular proteins, and portal proteins, suggesting that the SNPs were responsible for their differing phenotypes. Our findings highlight the diversity of novel Salmonella bacteriophages from rainforest regions, which can be explored as an antimicrobial agent against MDR-Salmonella strains.

The mitochondrial genome of the rare endemic New Guinean dasyurid Phascolosorex doriae (Thomas 1886) has been used to clarify relationships within ‘phascolosoricinae’. The mitochondrial genome has the typical gene arrangement seen in other marsupials. Molecular analyses using complete mitogenomes of other dasyurids resolve the red-bellied dasyure as sister to the narrow-striped dasyure Phascolosorex dorsalis and show that these two species diverged in the early Pliocene. The invasion of emergent New Guinean rainforest habitats (in the late Miocene) by the common ancestor of Ph. doriae, Ph. dorsalis and Neophascogale lorentzii represents one of three separate such invasions by dasyurid lineages.

The IL-2 family of cytokines act via receptor complexes that share the interleukin-2 receptor gamma common (IL-2Rγc) chain to play key roles in lymphopoiesis. Inactivating IL-2Rγc mutations results in severe combined immunodeficiency (SCID) in humans and other species. This study sought to generate an equivalent zebrafish SCID model. The zebrafish il2rga gene was targeted for genome editing using TALENs and presumed loss-of-function alleles analyzed with respect to immune cell development and impacts on intestinal microbiota and tumor immunity. Knockout of zebrafish Il-2rγc.a resulted in a SCID phenotype, including a significant reduction in T cells, with NK cells also impacted. This resulted in dysregulated intestinal microbiota and defective immunity to tumor xenotransplants. Collectively, this establishes a useful zebrafish SCID model.

The mitochondrial genome of the rare endemic New Guinean dasyurid Phascolosorex doriae (Thomas 1886) has been used to clarify relationships within ‘phascolosoricinae’. The mitochondrial genome has the typical gene arrangement seen in other marsupials. Molecular analyses using complete mitogenomes of other dasyurids resolve the red-bellied dasyure as sister to the narrow-striped dasyure Phascolosorex dorsalis and show that these two species diverged in the early Pliocene. The invasion of emergent New Guinean rainforest habitats (in the late Miocene) by the common ancestor of Ph. doriae, Ph. dorsalis and Neophascogale lorentzii represents one of three separate such invasions by dasyurid lineages.

We characterized the complete genome of a lytic Dickeya chrysanthemi bacteriophage, DchS19, which was isolated from a soil sample in Sungai Petani, Kedah, Malaysia. The phage, from the Autographviridae family, has a 39,149-bp double-stranded DNA genome containing 49 protein-coding genes and shares 94.65% average nucleotide identity with Erwinia phage pEp_SNUABM_12.

We characterized the complete genome sequence of the lytic Salmonella enterica bacteriophage PRF-SP1, isolated from Penang National Park, a conserved rainforest in northern Malaysia. The novel phage species from the Autographiviridae family has a 39,966-bp double-stranded DNA (dsDNA) genome containing 49 protein-encoding genes and shares 90.96% similarity with Escherichia phage DY1.

Labyrinthula spp. are saprobic, marine protists that also act as opportunistic pathogens and are the causative agents of seagrass wasting disease (SWD). Despite the threat of local- and large-scale SWD outbreaks, there are currently gaps in our understanding of the drivers of SWD, particularly surrounding Labyrinthula spp. virulence and ecology. Given these uncertainties, we investigated the Labyrinthula genus from a novel genomic perspective by presenting the first draft genome and predicted proteome of a pathogenic isolate Labyrinthula SR_Ha_C, generated from a hybrid assembly of Nanopore and Illumina sequences. Phylogenetic and cross-phyla comparisons revealed insights into the evolutionary history of Stramenopiles. Genome annotation showed evidence of glideosome-type machinery and an apicoplast protein typically found in protist pathogens and parasites. Proteins involved in Labyrinthula SR_Ha_C's actin-myosin mode of transport, as well as carbohydrate degradation were also prevalent. Further, CAZyme functional predictions revealed a repertoire of enzymes involved in breakdown of cell-wall and carbohydrate storage compounds common to seagrasses. The relatively low number of CAZymes annotated from the genome of Labyrinthula SR_Ha_C compared to other Labyrinthulea species may reflect the conservative annotation parameters, a specialized substrate affinity and the scarcity of characterized protist enzymes. Inherently, there is high probability for finding both unique and novel enzymes from Labyrinthula spp. This study provides resources for further exploration of Labyrinthula spp. ecology and evolution, and will hopefully be the catalyst for new hypothesis-driven SWD research revealing more details of molecular interactions between the Labyrinthula genus and its host substrate.

The mitochondrial genome of the rare endemic New Guinean dasyurid Phascolosorex doriae (Thomas 1886) has been used to clarify relationships within ‘phascolosoricinae’. The mitochondrial genome has the typical gene arrangement seen in other marsupials. Molecular analyses using complete mitogenomes of other dasyurids resolve the red-bellied dasyure as sister to the narrow-striped dasyure Phascolosorex dorsalis and show that these two species diverged in the early Pliocene. The invasion of emergent New Guinean rainforest habitats (in the late Miocene) by the common ancestor of Ph. doriae, Ph. dorsalis and Neophascogale lorentzii represents one of three separate such invasions by dasyurid lineages.

A Gram-negative, filamentous aerobic bacterium designated as strain Mgbs1T was isolated on 12 April 2017 from the subsurface soil and leaf litter substrate at the base of a Koompassia malaccensis tree in a tropical peat swamp forest in the northern regions of the state of Selangor, Malaysia (3° 39' 04.7' N 101° 17' 43.7'' E). Phylogenetic analyses based on the full 16S rRNA sequence revealed that strain Mgbs1T belongs to the genus Chitinophaga with the greatest sequence similarity to Chitinophaga terrae KP01T (97.65 %), Chitinophaga jiangningensis DSM27406T (97.58 %), and Chitinophaga dinghuensis DHOC24T (97.17 %). The major fatty acids of strain Mgbs1T (>10 %) are iso-C15 : 0, C16 : 1  ω5c and iso-C17 : 0 3-OH while the predominant respiratory quinone is menaquinone-7. Strain Mgbs1T has a complete genome size of 8.03 Mb, with a G+C content of 48.5 mol%. The DNA-DNA hybridization (DDH) score between strain Mgbs1T and C. jiangningensis DSM27406T was 15.9 %, while in silico DDH values of strain Mgbs1T against C. dinghuensis DHOC24T and C. terrae KP01T were 20.0 and 19.10% respectively. Concurrently, Average Nucleotide Identity (ANI) scores between strain Mgbs1T against all three reference strains are 73.2 %. Based on the phenotypic, chemotaxonomic, and phylogenetic consensus, strain Mgbs1T represents a novel species of the genus Chitinophaga, for which the name Chitinophaga extrema sp. nov. is proposed (=DSM 108835T=JCM 33276T).

The analysis of 13C-labeled lipids by mass spectrometry is challenging due to the complexity from labeling the large number of carbon atoms in lipids. To further add to the complexity, different adducts can be produced during electrospray ionization and in-source fragmentation, which can create complex overlapping isotope patterns that can only be resolved using high-resolution mass spectrometry. Co-elution of lipids even after chromatographic separation also adds to the potential for overlapping mass spectra. Here, we describe a procedure that enables full 13C-labeled patterns to be resolved in complex microalgal lipid extracts as well a procedure that provides structural labeling information. Mass resolving powers of 240000 full width half-maximum (fwhm) and fast targeted MS/MS allowed the differentiation of isotopologues, adducts, and unresolved lipid species after chromatographic separation. This enabled the percentage of 13C enrichment to be calculated for each individual lipid species over a time series in the microalgal lipidome. The application of tandem mass spectrometry (MS/MS) also allowed the degree of labeling within the headgroup vs acyl chains to be determined, further adding to the detail of information collected. This information is particularly useful for studying lipid synthesis and remodeling processes and can be extended to other biological systems.

Abstract Background The role of oral and gut microbiomes in the pathogenesis of schizophrenia has recently come to light with the advancement of sequencing technology. Recent studies have shown that schizophrenia patients have different gut and oral microbiome profiles than healthy people. The interaction of oral and gut microbiota with the brain is facilitated through the gut-brain axis. Several studies have profiled the gut microbiomes in the Malaysian population, however none has investigated its relationship with etiology of schizophrenia. The aim of this preliminary study is to therefore investigate the relationship between oral and gut microbiomes in pathogenesis of schizophrenia in the Malaysian population based on the total transcriptome profiles. Methods In this study, 15 schizophrenia patients and 15 healthy people (control) were recruited from the three major ethnic groups in Malaysia, namely Malay, Chinese and Indian. Throat swabs and stool samples were collected and subjected to total RNA extraction. Following this, ribosomal RNA depletion and cDNA conversion were performed prior to sequencing using Illumina platform. Bioinformatic tools will be used to analyse and compare the transcriptome profiles of microbiomes from schizophrenia and healthy subjects. Brain derived neurotrophic factor, serotonin and dopamine assays will be performed using their blood samples too. The results of the bioassays will be then correlated with the transcriptome profiles to identify possible mechanisms by which microbiota could promote schizophrenia Results The RNA sequencing results will reveal differences in gene expression profiles between the microbiomes of schizophrenic and healthy subjects. Based on the transcriptome profiles and bioassay results, the possible pathways and mechanisms by which these microbiota can cause schizophrenia will be identified. In addition, candidate biomarkers from the oral and/or gut microbiomes for diagnosis of schizophrenia can be identified. Discussion The first and second generations of schizophrenia treatments mostly are based on controlling the symptoms of the disease, whereas treatment based on microbiome dysbiosis correction can tackle the underlying mechanism leading to a precise treatment of the disease.The findings from this study will enable the identification of distinct schizophrenia associated bacteria, that may help in development of new treatments and novel diagnostics for schizophrenia via microbiome-targeted therapy including probiotics and prebiotic.

Empty fruit bunch (EFB) and palm oil mill effluent (POME) are the major wastes generated by the oil palm industry in Malaysia. The practice of EFB and POME digester sludge co-composting has shown positive results, both in mitigating otherwise environmentally damaging waste streams and producing a useful product (compost) from these streams. In this study, the bacterial ecosystems of 12-week-old EFB-POME co-compost and POME biogas sludge from Felda Maokil, Johor were analysed using 16S metagenome sequencing. Over ten phyla were detected, with Chloroflexi being the predominant phylum, representing approximately 53% of compost and 23% of the POME microbiome reads. The main bacterial lineage found in the compost and POME was Anaerolinaceae (Chloroflexi) with 30% and 18% of the total gene fragments, respectively. The significant differences between compost and POME communities were abundances of Syntrophobacter, Sulfuricurvum and Coprococcus. No methanogens were identified due to the bias in general 16S primers to eubacteria. The preponderance of anaerobic species in the compost and high abundance of secondary metabolite fermenting bacteria is due to an extended composting time, with anaerobic collapse of the pile due to the tropical heat. Predictive functional profiles of the metagenomes using 16S rRNA marker genes suggest that the presence of enzymes involved in degradation of polysaccharides such as glucoamylase, endoglucanase and arabinofuranosidase, all of which were strongly active in POME. Eubacterial species associated with cellulytic methanogenesis were present in both samples.

Complete genomes of xenobiotic-degrading microorganisms provide valuable resources for researchers to understand molecular mechanisms involved in bioremediation. Despite the well-known ability of Sphingomonas paucimobilis to degrade persistent xenobiotic compounds, a complete genome sequencing is lacking for this organism. In line with this, we report the first complete genome sequence of Sphingomonas paucimobilis (strain AIMST S2), an organophosphate and hydrocarbon-degrading bacterium isolated from oil-polluted soil at Kedah, Malaysia. The genome was derived from a hybrid assembly of short and long reads generated by Illumina HiSeq and MinION, respectively. The assembly resulted in a single contig of 4,005,505 bases which consisted of 3,612 CDS and 56 tRNAs. An array of genes involved in xenobiotic degradation and plant-growth promoters were identified, suggesting its' potential role as an effective microorganism in bioremediation and agriculture. Having reported the first complete genome of the species, this study will serve as a stepping stone for comparative genome analysis of Sphingomonas strains and other xenobiotic-degrading microorganisms as well as gene expression studies in organophosphate biodegradation.