Rapid national increases in the hospitalisation of Australian youth due to intentional self-harm between 2008 and 2019.

OBJECTIVE: Australian youth mental health services have received significant funding over the past 15 years. We analysed data on hospitalisation due to intentional self-harm to determine whether increased youth services were associated with reduction in a key indicator of youth population mental health. METHOD: Trends in national self-harm hospitalisation data from 2008 to 2019 for youth (<25 years) and adults (>25 years) were analysed using joinpoint regression. RESULTS: Rates of hospitalisation due to intentional self-harm increased significantly in both male (1.1% per annum, 95% CI [0.2%, 1.9%]) and female (3.0% per annum, 95% CI [0.9%, 5.1%]) youth aged <25 years between 2008 and 2019. Female youth had higher rates of hospitalisation than males, and there were average annual increases of 9.1% (95% CI [2.4%, 16.3%]) and 4.0% (95% CI [0.1%, 7.9%]), and absolute increases of 120% and 47.9%, in the rate of hospitalisation of females aged 0-14 and 15-19, respectively. In contrast, there was no overall change in adults (>25 years). CONCLUSIONS: Rates of hospitalisation due to intentional self-harm in Australian youth have increased despite significant investment in youth mental health services. This result could be attributable to several sociocultural factors and suggests a critical need for more hospital-based emergency youth mental health services.

Capturing chloroplast variation for molecular ecology studies: a simple next generation sequencing approach applied to a rainforest tree.

BACKGROUND: With high quantity and quality data production and low cost, next generation sequencing has the potential to provide new opportunities for plant phylogeographic studies on single and multiple species. Here we present an approach for in silicio chloroplast DNA assembly and single nucleotide polymorphism detection from short-read shotgun sequencing. The approach is simple and effective and can be implemented using standard bioinformatic tools. RESULTS: The chloroplast genome of Toona ciliata (Meliaceae), 159,514 base pairs long, was assembled from shotgun sequencing on the Illumina platform using de novo assembly of contigs. To evaluate its practicality, value and quality, we compared the short read assembly with an assembly completed using 454 data obtained after chloroplast DNA isolation. Sanger sequence verifications indicated that the Illumina dataset outperformed the longer read 454 data. Pooling of several individuals during preparation of the shotgun library enabled detection of informative chloroplast SNP markers. Following validation, we used the identified SNPs for a preliminary phylogeographic study of T. ciliata in Australia and to confirm low diversity across the distribution. CONCLUSIONS: Our approach provides a simple method for construction of whole chloroplast genomes from shotgun sequencing of whole genomic DNA using short-read data and no available closely related reference genome (e.g. from the same species or genus). The high coverage of Illumina sequence data also renders this method appropriate for multiplexing and SNP discovery and therefore a useful approach for landscape level studies of evolutionary ecology.

Stable internal reference genes for normalization of real-time RT-PCR in tobacco (Nicotiana tabacum) during development and abiotic stress.

Real-time RT-PCR is a powerful technique for the measurement of gene expression, but its accuracy depends on the stability of the internal reference gene(s) used for data normalization. Tobacco (Nicotiana tabacum) is an important model in studies of plant gene expression, but stable reference genes have not been well-studied in the tobacco system. We address this problem by analysing the expression stability of eight potential tobacco reference genes. Primers targeting each gene (18S rRNA, EF-1alpha, Ntubc2, alpha- and beta-tubulin, PP2A, L25 and actin) were developed and optimized. The expression of each gene was then measured by real-time PCR in a diverse set of 22 tobacco cDNA samples derived from developmentally distinct tissues and from plants exposed to several abiotic stresses. L25 and EF-1alpha demonstrated the highest expression stability, followed by Ntubc2. Measurement of L25 and EF-1alpha was sufficient for accurate normalization in either the developmental or stress-treated samples, but Ntubc2 was also required when considering the entire sample set. Analysis of a tobacco circadian gene (NTCP-23) verified these reference genes in an additional context, and all techniques were optimized to enable a high-throughput approach. These results provide a foundation for the more accurate and widespread use of real-time RT-PCR in tobacco.

Complete Chloroplast Genome of the Wollemi Pine (Wollemia nobilis): Structure and Evolution.

The Wollemi pine (Wollemia nobilis) is a rare Southern conifer with striking morphological similarity to fossil pines. A small population of W. nobilis was discovered in 1994 in a remote canyon system in the Wollemi National Park (near Sydney, Australia). This population contains fewer than 100 individuals and is critically endangered. Previous genetic studies of the Wollemi pine have investigated its evolutionary relationship with other pines in the family Araucariaceae, and have suggested that the Wollemi pine genome contains little or no variation. However, these studies were performed prior to the widespread use of genome sequencing, and their conclusions were based on a limited fraction of the Wollemi pine genome. In this study, we address this problem by determining the entire sequence of the W. nobilis chloroplast genome. A detailed analysis of the structure of the genome is presented, and the evolution of the genome is inferred by comparison with the chloroplast sequences of other members of the Araucariaceae and the related family Podocarpaceae. Pairwise alignments of whole genome sequences, and the presence of unique pseudogenes, gene duplications and insertions in W. nobilis and Araucariaceae, indicate that the W. nobilis chloroplast genome is most similar to that of its sister taxon Agathis. However, the W. nobilis genome contains an unusually high number of repetitive sequences, and these could be used in future studies to investigate and conserve any remnant genetic diversity in the Wollemi pine.

Selection and validation of reference genes for quantitative gene expression studies in Erythroxylum coca.

Real-time quantitative PCR is a powerful technique for the investigation of comparative gene expression, but its accuracy and reliability depend on the reference genes used as internal standards. Only genes that show a high level of expression stability are suitable for use as reference genes, and these must be identified on a case-by-case basis. Erythroxylum coca produces and accumulates high amounts of the pharmacologically active tropane alkaloid cocaine (especially in the leaves), and is an emerging model for the investigation of tropane alkaloid biosynthesis. The identification of stable internal reference genes for this species is important for its development as a model species, and would enable comparative analysis of candidate biosynthetic genes in the different tissues of the coca plant. In this study, we evaluated the expression stability of nine candidate reference genes in E. coca ( Ec6409, Ec10131, Ec11142, Actin, APT2, EF1α, TPB1, Pex4, Pp2aa3). The expression of these genes was measured in seven tissues (flowers, stems, roots and four developmental leaf stages) and the stability of expression was assessed using three algorithms (geNorm, NormFinder and BestKeeper). From our results we conclude that Ec10131 and TPB1 are the most appropriate internal reference genes in leaves (where the majority of cocaine is produced), while Ec10131 and Ec6409 are the most suitable internal reference genes across all of the tissues tested.

Transfer of plastid DNA to the nucleus is elevated during male gametogenesis in tobacco.

In eukaryotes, many genes were transferred to the nucleus from prokaryotic ancestors of the cytoplasmic organelles during endosymbiotic evolution. In plants, the transfer of genetic material from the plastid (chloroplast) and mitochondrion to the nucleus is a continuing process. The cellular location of a kanamycin resistance gene tailored for nuclear expression (35SneoSTLS2) was monitored in the progeny of reciprocal crosses of tobacco (Nicotiana tabacum) in which, at the start of the experiments, the reporter gene was confined either to the male or the female parental plastid genome. Among 146,000 progeny from crosses where the transplastomic parent was male, 13 transposition events were identified, whereas only one atypical transposition was identified in a screen of 273,000 transplastomic ovules. In a second experiment, a transplastomic beta-glucuronidase reporter gene, tailored to be expressed only in the nucleus, showed frequent stochastic expression that was confined to the cytoplasm in the somatic cells of several plant tissues. This gene was stably transferred in two out of 98,000 seedlings derived from a male transplastomic line crossed with a female wild type. These data demonstrate relocation of plastid DNA to the nucleus in both somatic and gametophytic tissue and reveal a large elevation of the frequency of transposition in the male germline. The results suggest a new explanation for the occurrence of uniparental inheritance in eukaryotes.

The fiber specificity of the cotton FSltp4 gene promoter is regulated by an AT-rich promoter region and the AT-hook transcription factor GhAT1.

Fiber-specific genes are expressed preferentially or exclusively in cotton (Gossypium spp.) fiber and are thought to have important functions in fiber development. The promoters of these genes are of interest because they control transcription in the fiber cell and may be used in the genetic manipulation of fiber quality. The promoter of a cotton lipid transfer protein gene, FSltp4, was isolated and shown to direct fiber-specific transcription of an abundant mRNA in cotton. In transgenic tobacco, this promoter was strongly active in leaf trichomes. Deletion analysis of the promoter identified an AT-rich 84 bp fiber specificity region (FSR) necessary for activity exclusively in the fiber cells. Cotton fiber proteins that bind the FSR were isolated using a yeast one-hybrid assay. One of these was a putative AT-hook transcription factor (GhAT1) containing two AT-hook motifs. GhAT1 was shown to be nuclear localized, and GhAT1 transcripts were found to be preferentially expressed in ovules and non-fiber tissues. Overexpression of GhAT1 strongly repressed the activity of the FSltp4 promoter in the trichomes of transgenic tobacco. These results suggest that GhAT1 assists in the specification of fiber cells by repressing FSltp4 in the non-fiber tissues of the cotton plant.