The second group (n = 16) contained most GCTs and a subset of SCOs, all of which mostly lacked identifiable genetic drivers. Outcome analysis demonstrated that the presence of chromosomal imbalances was significantly associated with reduced progression-free survival especially within the combined PITUI and SCO group (p = 0.031). In summary, we observed only subtle DNA methylation differences between posterior pituitary tumors, indicating that these tumors may be best classified as subtypes of a single entity. Nevertheless, our data indicate differences in mutation patterns and clinical outcome. For a clinically meaningful subclassification, we propose a combined histo-molecular approach into three subtypes: one subtype is defined by granular cell histology, scarcity of identifiable oncogenic mutations, and favorable outcome. The other two subtypes have either SCO or PITUI histology but are segregated by chromosomal copy number profile into a favorable group (no copy number changes) and a less favorable group . Both of the latter groups have recurrent MAPK/PI3K genetic alterations that represent potential therapeutic targets.Pituicytoma (PITUI), granular cell tumor (GCT), and spindle cell oncocytoma (SCO) are rare tumors of the posterior pituitary. Histologically, they may be challenging to distinguish and have been proposed to represent a histological spectrum of a single entity. We performed targeted next-generation sequencing, DNA methylation profiling, and copy number analysis on 47 tumors  to investigate molecular features and explore possibilities of clinically meaningful tumor subclassification. We detected two main epigenomic subgroups by unsupervised clustering of DNA methylation data, though the overall methylation differences were subtle. The largest group (The online version contains supplementary material available at 10.1007/s00401-021-02377-1. Spindle cell oncocytoma, pituicytoma, and granular cell tumor of the sellar region are rare pituicyte-derived neoplasms. They are listed as independent entities in the 2016 WHO Classification of Tumours of the Central Nervous System and are assigned as WHO grade I neoplasms  due to tGranular cell tumors are typically composed of cells with isomorphic, round to polygonal cell bodies, rather small and often eccentric nuclei, and abundant granular cytoplasm. Pituicytomas most closely resemble the majority of normal pituicytes in the posterior pituitary, which are spindled and elongated cells arranged in fascicles. The morphology of spindle cell oncocytomas is less clearly defined. These tumors vary in appearance and may show, among other patterns, spindled and/or epithelioid cells, with varying oncocytic changes and fascicular growth , 38.All three tumors originate in a very specific and confined region of the CNS, the posterior lobe, and the infundibulum of the pituitary gland, also called the neurohypophysis, which is formed during embryonic development from a protrusion of the developing diencephalon. In early gestation, it co-localizes with the early adenohypophysis, which derives from the oral ectoderm of Rathke’s pouch and together they form the pituitary gland in the sella turcica , 44, 59.NKX2-1 gene) which is a marker expressed in normal pituicytes, spindle cell oncocytomas, pituicytomas, and granular cell tumors of the sella  of tumor tissue. Mitotic activity was scored as: not present—0 mitotic figures; low—1 mitotic figure in 10 HPF; moderate—2 mitotic figures in 10 HPF; high—3 or more mitotic figures in HPF.H&E stained slides were available for evaluation of histological features from 46 of 47 tumors included in this study. For each case, two experienced pathologists (S.S. and D.C.) evaluated and graded a number of morphological features on H&E slides using a multihead microscope. Consensus was reached by discussion using the following scale: feature present in less than 1% of tumor cells (graded as “not present”), in 1–10% of tumor cells (“slight”), in 10–50% (“moderate”) of tumor cells, or in over 50% of tumor cells (“high”). The following features were graded in this way: fascicular growth of spindled cells, lobulated growth, granular cells , rounded to polygonal cells with distinct cell borders (epithelioid appearance), nuclear pleomorphism, prominent nucleoli, necrosis, and intraparenchymal sclerosis. In addition, we evaluated cell density by counting cells in an area of 1 mmImmunohistochemistry for mitochondrial antigen was performed using antibody clone 113-1 from BioGenex on whole formalin-fixed, paraffin-embedded tissue sections at 1:500 dilution following ER1 antigen retrieval for selected cases. Immunostaining was performed in a Leica BOND-III automated stainer with diaminobenzidine used as the chromogen, followed by hematoxylin counterstain.https://www.molecularneuropathology.org/mnp)  together with the cases of this series. The colors represent the three histological groups of posterior pituitary tumors and the main tumor classes of the brain tumor classifier. The tumors of this study all group together closely. The inset shows a higher zoom in the posterior pituitary tumors and indicates that all three main histological classes form a single group with no clear separation at the scale of this analysis. Two cases (Case 27 and 28) fall slightly to the side together with two cases of other groups. Both cases had prominent lymphoplasmacytic infiltration, likely contributing to this unusual clustering behavior  (TIF 22238 KB)Depiction of Hematoxylin and Eosin histology  and 400x ) for cases with unusual grouping in the large tSNE  or in the clustering analysis . In the histological review, cases 27 and 28 showed prominent lymphoplasmacytic infiltrates whereas cases 38 and 34 were remarkable for high cell density (TIF 33541 KB)Anti-mitochondrial-antigen staining and H&E stain of 4 spindle cell oncocytomas that clustered with the granular group  (TIF 79994 KB)tSNE representation with all posterior pituitary tumors of this series and additional normal control tissue, including samples from posterior pituitary lobe and infundibulum. Tumor and control samples group exceptionally close together (TIF 12436 KB)Summary copy number plots for the three posterior pituitary tumor DNA methylation groups. Granular group tumors and MAPK/PI3K group tumors show no highly recurrent cytogenetic alterations. Eleven of sixteen (69%) granular group tumors and 14 of 23 (60%) of MAPK/PI3K group tumors harbored no detectable chromosomal alterations (flat genome) with the remaining cases showing infrequent chromosomal gains or losses. Amplifications or focal deletions were not observed . In contrast, the small methylation group “CNV high” was characterized by numerous chromosomal gains and losses including loss of chromosome 1p in all four cases(c) (TIF 35106 KB)Kaplan-Meier analysis for progression free survival for cases of this series (a) excluding all cases where the initial tumor was not available for analysis or (b) excluding all cases that had a documented radiation therapy prior to sampling. In both subsets the presence of any copy number alterations was still significantly associated with worse outcome (TIF 18465 KB)Supplementary file7 (XLSX 12 KB)Supplementary file8 (XLSX 15 KB)Supplementary file9 (XLSX 15 KB)Supplementary file10 (XLSX 20 KB)Supplementary file11 (XLSX 12 KB)Supplementary file12 (XLSX 11 KB)Below is the link to the electronic supplementary material.


Vitis vinifera) using a novel pipeline developed to identify dicistronic transcripts from high-throughput RNA-sequencing data. We identified dicistronic tRNA–mRNA in leaf and berry samples from 22 commercial vineyards. Of the 124 tRNA genes that were expressed in both tissues, 18 tRNA were expressed forming part of 19 dicistronic tRNA–mRNAs. The presence and abundance of dicistronic molecules was tissue and geographic sub-region specific. In leaves, the expression patterns of dicistronic tRNA–mRNAs significantly correlated with tRNA expression, suggesting that their transcriptional regulation might be linked. We also found evidence of syntenic genomic arrangements of tRNAs and protein-coding genes between grapevine and Arabidopsis thaliana, and widespread prevalence of dicistronic tRNA–mRNA transcripts among vascular land plants but no evidence of these transcripts in non-vascular lineages. This suggests that the appearance of plant vasculature and tRNA–mRNA occurred concurrently during the evolution of land plants.Transfer RNAs (tRNA) are crucial adaptor molecules between messenger RNA (mRNA) and amino acids. Recent evidence in plants suggests that dicistronic tRNA-like structures also act as mobile signals for mRNA transcripts to move between distant tissues. Co-transcription is not a common feature in the plant nuclear genome and, in the few cases where polycistronic transcripts have been found, they include non-coding RNA species, such as small nucleolar RNAs and microRNAs. It is not known, however, the extent to which dicistronic transcripts of tRNA and mRNAs are expressed in field-grown plants, or the factors contributing to their expression. We analysed tRNA–mRNA dicistronic transcripts in the major horticultural crop grapevine ( Polycistronic transcripts are synthesized when multiple genes forming an operon are co-expressed from a single promoter. These transcripts are then translated into protein from two or more translation initiation sites. This strategy has been described as an efficient mechanism to coordinate gene expression1. Although polycistronic transcripts are less common in plants, several chloroplast genes are organized in clusters and are co-transcribed in polycistronic primary transcripts and subsequently processed to form mature RNAs2, reflecting their prokaryotic ancestry3. The majority of nuclear-encoded genes in plants are monocistronic with a few exceptions, such as certain classes of polycistronic microRNAs (miRNAs)4 and small nucleolar RNAs (snoRNAs), which are organized in genomic clusters and are transcribed from a common promoter5. These precursor transcripts are processed to mature snoRNA and miRNA molecules. There are also a few reports of dicistronic transcripts encoding genes that are not functionally related to each other such as tRNAs–snoRNA, snoRNA–miRNAs and tRNA–mRNA in some plant species7; however, the molecular and physiological significance of co-transcription for many of these transcripts is still poorly understood.Polycistronic mRNAs are RNA molecules that contain two or more open reading frames (ORFs). These are usually found in viruses, bacteria, archaea, protozoans and invertebratesArabidopsis thaliana and tobacco, has shed light on the function of dicistronic tRNAs-mRNAs7. Using transgenic lines, Zhang et al.7 demonstrated that tRNA-like structures (TLSs), when co-transcribed with mRNA transcripts, could act as mobility signals, triggering the systemic movement of the mRNA between roots and shoots. Notably, the mRNA components of the dicistronic transcripts were also shown to be translated into functional proteins. Endogenously produced tRNA–mRNA dicistronic transcripts have also been detected in A. thaliana suggesting that functional tRNA and tRNA-like structures could act as non-autonomous signals in plants able to deliver functional mRNAs to distantly located tissues. Beyond their canonical role in protein translation, tRNAs have been also demonstrated to function in other chemical transformations, for example, delivering amino acids during lipid modification and antibiotic biosynthesis8.Recent work in model plants, Vitis vinifera), the effect of growth environment on gene expression has been extensively studied10. Several studies have identified small non-coding RNAs (sRNAs) in grapevine that can influence development in response to environmental stimuli. Among these sRNAS, miRNAs respond to low temperature treatment11, application of exogenous gibberellin12 and viral infection13. In addition, studies have shown that miRNAs present tissue specificity in grapevine14. Bester et al.15 identified sRNA species in grapevine phloem. Notably, this study also showed the non-random manner in which tRNA-derived sRNAs originated15. A study looking at the effect of grafting in grapevine identified more than 3000 transcripts moving across graft junctions including transcripts for genes involved in the response to abiotic stress and signal transduction16. Zhang et al.7. confirmed that 11% of the mobile mRNA in V. vinifera from Yang et al. (2015)16 also had TLS motifs in their coding sequence or 3’ UTR.In grapevine (Dicistronic RNA Transcripts), a computational pipeline to detect dicistronic transcripts from short-read RNA-seq data that can be adapted for use in any organism. Using this pipeline, we analysed dicistronic tRNA–mRNA transcripts in commercial, field-grown grapevine and assessed the prevalence of these transcripts across the plant kingdom.We hypothesized that dicistronic tRNA–mRNA transcripts would be transcribed differentially between different grapevine tissues and in growing regions with different environments. As a first step towards identifying such transcripts, we present DiRT  and veraison (onset of ripening), respectively, from 22 vineyards (own-rooted) from the Barossa wine growing region, South Australia, Australia  Illumina reads for each leaf and berry sample  , defined by genomic boundaries that extend between the 5’ and 3’ untranslated regions. tRNA genes that overlap with the PCGs (5′ or 3′ untranslated regions or introns) were removed from further analysis since such reads could not be unambiguously assigned to either the tRNA or the PCG. Using DiRT, we detected 137 transcribed tRNA genes (read count ≥ 1) in both tissue samples (124 in leaf and 90 in berry) across all sub-regions. From the 137 tRNA genes transcribed, 77 were expressed in both tissues, 47 tRNAs were leaf specific and 13 tRNAs were berry specific. From the expressed tRNAs, 136 presented typical tRNA secondary structure with associated anticodon and just one tRNA from leaf was classified as undefined . In general, the numbers of expressed tRNAs are underrepresented in a standard short-read RNA-seq library due to tRNA secondary structure and post-transcriptional modifications limiting detection20. Although underrepresented, we evaluated the sensitivity of the pipeline to identify dicistronic tRNA–mRNA transcripts using a standard RNA-seq workflow as it permits expression analysis of monocistronic and dicistronic transcripts from the same sequenced library.The Genomic tRNA Database predicts 609 tRNA genes in the t-test, FDR < 0.05) than reads detected in the two closest introns  in intergenic regions than in the first two introns of dicistronic tRNA–mRNA pairs. Conversely, no significant difference in coverage was observed for tRNA–mRNA pairs deemed non-dicistronic and these transcripts had expected read coverage breakpoints in the intergenic region between tRNA and mRNA , with a median intergenic distance of 133 bp 7 or the mRNA has been demonstrated to be mobile 23  between dicistronic tRNAs and intergenic regions in leaves  through local Fisher discriminant analysis (LFDA). We observed that the expression patterns of PCGs occupies a unique eigen space, while dicistronic tRNAs and intergenic regions shared the eigen space occupied by all expressed tRNAs , ancient vascular plants  and Selaginella moellendorffii (lycophytes)), and non-vascular species (Physcomitrella patens (mosses) and Marchantia polymorpha (liverworts)). RNA-seq data for each representative vascular and non-vascular species was accessed from the short-read archive (SRA) and filtered based on organ samples that included vasculature tissue and sequencing criteria to ensure consistency across the libraries analysed (See Methods section). In total, 149 Illumina-based RNA-sequencing libraries from the nine vascular and non-vascular species were analysed using the DiRT pipeline . Across all vascular species analysed, 66 tRNA genes were detected to be co-transcribed with the neighbouring protein-coding gene, representing 20 distinct isoacceptor families (41 anticodons)  was observed to be co-transcribed with a PCG in all four flowering plants analysed.Dicistronic tRNA–mRNA transcripts were identified in all taxa presenting vascularization. In total, 139 dicistronic tRNA–mRNA transcripts were detected across eudicots, monocots, fern species and the lycophyte representative, ii Table . Corrobons) Fig. . We did Physcomitrella patens and Marchantia polymorpha that lack vascular tissue  of all expressed tRNAs in grapevine leaf and berry samples were putatively expressed in a dicistronic manner, with neighbouring protein-coding genes. We developed DiRT, a customised, computational pipeline to specifically detect dicistronic tRNA and mRNA candidates using stringent criteria. Using DiRT we were able to identify dicistronic transcripts in two different grapevine tissues (i.e. leaf and berry) sampled from commercial vineyards. Validation of the pipeline to accurately predict dicistronic candidates was confirmed in both tissues through RT-PCR detection and Sanger sequencing of dicistronic candidates in leaf samples.Vitis vinifera, 11 tRNA families have also found to be dicistronic in A. thaliana, and associated with transcripts demonstrated to be mobile between roots and shoots26. Among these, tRNA coding for GlyGCC and MetCAT were able to mobilise mRNA transcripts to different tissues as part of a fusion construct and translate into functional proteins in grafted A. thaliana plants, indicating that these tRNA were able to confer mobility to these transcripts7. This suggests a non-autonomous role for dicistronic tRNAs in delivering mRNA transcripts to distantly located tissues. A recent study also revealed that mobile RNA transcripts are enriched in the modified base 5-methylcytosine (m5C), indicating a role of RNA cytosine methylation in systemic RNA movement27. In plants, tRNA and mRNA m5C methylation is mediated by the methyltransferases DNMT2 and TRM4B29 and loss of these enzymes was demonstrated to impair transcript mobility27. Future studies will need to be undertaken to determine if the dicistronic tRNAs identified in this study also confer mRNA mobility and to assess the role of cytosine methylation in mRNA transport in grapevine.Interestingly, of the 12 tRNA isoacceptor families (representing 15 distinct anticodons) found to be dicistronic in A. thaliana and V. vinifera for the protein-coding gene and the adjacently co-transcribed tRNA genes. This indicates the genomic clustering of tRNA and protein-coding gene is, in some cases, evolutionarily conserved between both species. The dicistronic activity at these conserved loci may provide an explanation of why such syntenic clusters are conserved through evolution and suggests that these transcripts may have an important functional role.For four of the 19 dicistronic candidates, we also observed sequence conservation between Vitis vinifera genome, 18 were located fewer than 1000 base pairs from the co-transcribed upstream or downstream protein-coding gene (median distance 133 bp). Our findings suggest that tRNA genes and protein-coding genes need to be closely positioned in the genome in order to form dicistronic transcripts. Similar observations were obtained in A. thaliana, where the majority of the previously identified PCGs forming part of mobile dicistronic transcripts were located less than 200 bp from their partner tRNA7. Genomic proximity was found to be even closer between co-transcribed tRNA and snoRNA genes identified in higher plants such as A. thaliana, M. truncatula, P. trichocarpa, O. sativa and B. distachyon, in which the intergenic region ranged between 1 and 16 base pairs6.Of the 19 dicistronic tRNA genes identified in the 30. This suggests that passive diffusion of these transcripts through the phloem may contribute to their mobility. A significant proportion (11.4%) of these transcripts was subsequently shown to be dicistronically associated with tRNA7. However, when we assessed the expression levels of mRNA and tRNA that formed dicistronic transcripts in grapevine, we did not observe higher abundance of these transcripts in either tissue analysed. Thus, in our study, the expression level of the tRNA and mRNA was not a good indicator of the formation of dicistronic transcripts.Previous studies have indicated that a large proportion of mobile transcripts are also highly abundant31. Results from Kruszka et al.5 suggested that, in A. thaliana, dicistronic tRNA-snoRNA are transcribed by Pol III from the tRNA-gene promoter. However, Pol III transcribes genes shorter than 400 base pairs31 and the dicistronic transcripts identified in our study were considerably longer (between 1486 and 6002 bp) suggesting Pol III may not be co-transcribing these transcripts. A comparative analysis of flowering species showed a poly-T stretch immediately downstream of ≥90% of tRNA genes6. Additionally, Michaud et al6. reported that the few tRNAs lacking poly-Ts were capable of forming dicistronic transcript with snoRNAs. The authors hypothesized that the lack of the poly-T transcriptional termination signal could be a possible explanation for why these transcripts were transcribed as a single unit by Pol III. Sequence analysis of the upstream and downstream sequences of the dicistronic tRNAs identified in our study revealed canonical elements previously associated with transcription start and termination21. In particular, all dicistronic tRNA transcripts we identified had a poly-T termination signal suggesting that the transcriptional mechanism of these transcripts may be different from tRNA–snoRNAs. Another possibility is that the tRNA and mRNA are transcribed independently as monocistronic transcripts and are then ligated or spliced together to form a dicistronic transcript. Currently, the DiRT pipeline cannot distinguish between transcriptional read-though and post-transcriptional ligation of two independent transcripts. However, as both possibilities result in the same effective dicistronic molecule, detection is nevertheless important for understanding the physiological functions of these transcripts.In eukaryotes, tRNA and mRNA are transcribed by different types of RNA polymerase. RNA polymerase II (Pol II) transcribes protein-coding genes and RNA polymerase III (Pol III) for a variety of genes that generally encode for RNAs with catalytic activity such as tRNA15. Special effort has been put into deciphering the effect that the growing environment has on fruit quality traits associated with wine regionality33. However, the effect that the environment and tissue have on tRNA expression and on dicistronic transcript expression has not been previously described. Our results show that the expression patterns of dicistronic transcript-forming tRNA genes mimic those of all expressed tRNA ) were found to be organ specific and sensitive to regional environmental differences. The effect of organ and environment on PCG and tRNA-gene expression has been extensively studied in grapevineTaken collectively, our results suggest that environmentally induced dicistronic tRNA–mRNA expression is, at least partially, directed by the mechanisms regulating tRNA expression.Arabidopsis thaliana and tobacco, demonstrate tRNAs and closely related tRNA-like structures that are co-transcribed with mRNA have the ability to be systemically transported via the phloem vasculature. The translation of the mobile mRNA product into a functional protein indicates that such molecules have the potential to act as non-autonomous long-distance signals7. To provide a broader context for these transcripts in plants, we show here the first evidence for dicistronic tRNA–mRNA transcription in a commercially important crop species grown in field conditions. Furthermore, through a multi-species RNA-seq transcriptome analyses, we demonstrate wide prevalence of dicistronic tRNA–mRNA in land plants. Importantly, the detection of these transcripts in flowering angiosperms, ferns and lycophytes but absence in basal plant lineages such as liverworts and mosses indicates that the emergence of these co-transcribed molecules coincided with evolution of the vasculature trait in land plants. As the vascular system evolved in plants as means to translocate water, nutrients and other photoassimilates facilitating terrestrial colonization, the concurrent appearance of dicistronic tRNA–mRNA transcripts reinforces their potential role in long-distance communication. Thus, the evolution of vasculature may have led to plants acquiring a new role for tRNAs in mediating transport of large mRNA transcripts to distant organs as a means to integrate developmental and environmental cues.Prior work in the model species S. cucullata to 49 in S. moellendorfii. Such variation could reflect species-specific differences of tRNA-mediated mRNA transport though the vasculature, varying number of tRNA genes in the genome and/or differences in co-transcriptional machinery. Given the paucity of knowledge regarding tRNA–mRNA transcripts, biological differences between species are difficult to determine at this stage. Moreover, although we controlled for the expression of vascular genes and mined the range of high-quality datasets available publicly, we cannot rule out that subtle differences in tissue type and sampling conditions in each species analysed may have contributed to the variation of discistronic transcripts observed. For the purposes of this study, however, the results from the multi-species comparison served as qualitative measure of dicistronic tRNA–mRNA transcription in vascular versus non-vascular species rather than as quantitative study. Future work comparing syntenic regions of tRNA and mRNA genes between species and analysing the biological relevance of transported mRNAs will help in understanding species-specific functions of dicistronic transcripts.We detected a varying number of dicistronic tRNA–mRNA transcripts in the seven representative vascular species analysed ranging from two in It remains to be elucidated if tRNA–mRNA movement requires RNA-binding proteins for vascular transport, as is the case for viruses and viroids that require movement proteins for RNA transport. Future studies that use our in silico approach to survey expression patterns of these transcripts in an even wider range of model and non-model plant species and across a range of tissues will shed more light on expression dynamics; the source, intermediate and target cell types and signalling function of these transcripts.Vitis vinifera) cv. Shiraz from 22 commercial vineyards located in the Barossa wine zone . Vineyards were selected as part of a larger study of Barossa Terroir37. Vineyards were chosen to be representative of the climate, soil and management practices that are used in the different Barossa sub-regions. These sub-regions are the Eden Valley (EV) (3 vineyards), Northern Grounds (NG) (4 vineyards), Central Grounds (CG) (4 vineyards), Southern Grounds (SG) (3 vineyards), Eastern Edge (EE) (4 vineyards) and Western Ridge (WR) (4 vineyards).Tissues were sampled from own-rooted grapevines (38. Samples were snap-frozen in liquid nitrogen in the vineyard of collection. All samples of the same organ (leaf or berry) were ground using mortar and pestle under liquid nitrogen and pooled into a single sample per plant and stored at −80 °C.Leaves and berries were collected from nine plants in each of three rows in each vineyard  during the 2016 growing season. The first fully expanded leaf at budburst (E-L 7) was collected from three nodes per plant. Nine berries were collected at veraison (E-L 35) from three different bunches per plant (i.e. three berries per bunch). All samples were taken before dawn (between 10:00 pm and sunrise) to minimise variability associated with differences in plant water status39 using default settings. Alignment of trimmed reads was performed in HISAT2 v2.1.040 with default settings using the grapevine genome reference IGGP_12X, obtained from Ensembl Plants 45. BAM files from samples from the same vineyard were merged, sorted and indexed using SAMtools v1.841. Mapped reads were counted to genomic features using featureCounts v1.5.242, with the minimum mapping quality score for a read to be assigned to a feature was set to 10. The merged, sorted and indexed BAM files were then directly input into the R environment in order to identify the dicistronic tRNA–mRNA transcripts.Total RNA was extracted from each sample using the Spectrum Plant Total RNA kit (Sigma–Aldrich) following the manufacturer’s instructions and including DNAse treatment. Three samples per vineyard were generated by pooling 2 µg of total RNA from three plants from the same row in the vineyard for a total of 66 pools. Ribosome was depleted in 6 µg of RNA from each pool using the Dynabeads mRNA purification kit  following the manufacturer’s instructions. Ribosomal depleted RNA (25 ng per pool) was used as input for library preparation using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs Inc). Libraries were sequenced using Illumina NextSeq High Output 75 bp pair-end  at the Australian Genome Research Facility . Reads were trimmed and filtered using AdapterRemoval v2.2.1Dicistronic RNA Transcripts) is a custom pipeline implemented in the R environment and source codes are available at GitHub (https://github.com/CharlotteSai/DiRT). While the pipeline was developed for analysing grapevine RNA-seq data, it can be adapted for use in other species provided a genomic tRNA annotation is available. Firstly, protein-coding gene (PCG) information and coordinates were downloaded from Ensembl Plants (release 45) (http://plants.ensembl.org/Vitis_vinifera/Info/Index) and the chromosomal coordinates of tRNA genes were extracted from the Genomic tRNA database using tRNAscan-SE based on predicted structure analysis (http://gtrnadb.ucsc.edu/GtRNAdb2/genomes/eukaryota/Vvini/). We used BEDTools version 2.2543 to determine the relative location of PCGs in relation to all tRNA genes (both upstream and downstream of the tRNA). Predicted tRNAs overlapping with PCGs were discarded for further analysis as there is no intergenic region between them and such reads could not be unambiguously assigned to either the tRNA or the PCG. tRNAs expressed (i.e. above 1 raw read) in leaf and berry samples were identified using GenomicRanges44. To infer putative co-transcription, first we filtered the RNA-seq data for genomic regions where both tRNA loci and closest neighbouring gene were transcribed (Raw read ≥ 1 for tRNAs and raw read ≥ 10 in PCGs), independently of their DNA strand. A lower threshold of read coverage was applied for tRNAs due to underrepresentation in standard RNA-seq libraries20. Multi-mapped reads were used for mapping as there are over 600 predicted tRNA genes in V. vinifera often with multiple identical isodecoder sequences, which makes unique mapping of Illumina reads to individual transcribed tRNA loci challenging. In order to identify region-specific putative dicistronic transcripts, each Barossa sub-region was interrogated separately.DiRT . If the protein-coding gene contains just one intron, the t-test still is performed by comparing read coverage with the intergenic region. Of the 81 and 50 tRNA–mRNA pairs in leaf and berries, respectively, 18 mRNAs contained one intron in both tissues and the remaining mRNAs contained two or more introns. The complete set of p-values were adjusted using the Benjamini–Hochberg false discovery rate (FDR)45 and intergenic regions with higher mean coverage than both introns, and an FDR-adjusted p-value <0.05 were included for subsequent steps of the pipeline.The selected candidate tRNA–mRNA transcripts were then scanned for dicistronic transcripts. We demanded that, first; the sequencing coverage of the intergenic region must be significantly higher than the intron closest to the intergenic region and the second intron closest to intergenic region. To achieve this, reads for each base of the intergenic region, the closest intron and the second closest intron were counted by the coverage method from the tRNA–mRNA transcripts passing the first condition were further filtered for those with uninterrupted sequencing coverage spanning the tRNA, the intergenic region and the mRNA by selecting candidates with at least one count for every base in the intergenic region. This condition was implemented to make sure that at least one entire molecule of the dicistronic transcript had been potentially produced.46 for visual validation of continuous coverage. The candidates passing both of the t-tests and continuously coverage examination were deemed putative tRNA–mRNA dicistronic transcripts. Multidimensional scaling (MDS) were analysed in the R environment using the function plotMDS from the limma package47.Finally, dicistronic candidates with continuous coverage in the intergenic region were manually inspected using IGVGlyCCC_R) (+RT) and the reverse primer of the gene Elongation Factor 1-alpha (EF1a_R) as a positive control. Negative controls for the cDNA synthesis (−RT) in which reverse transcriptase enzyme was omitted were included for each of the dicistronic candidate. Resulting cDNA was diluted 1:10 and 2 µl was used for RT-PCR. The RT-PCR reaction was conducted using Kapa Taq PCR Kit  following the manufacturer’s instructions. The amplification program used was 95 °C for 3 min, 37 cycles at 95 °C for 30 sec followed by 60 °C for 30 sec and 72 °C for 50 sec and finally 72 °C per 2 min. For the candidate tRNAValCAC1.7-VIT_15s0046g02860 we used primers tRNAValCAC_F and Intergenic_tRNAValCAC-VIT_15s0046g02860_R (376 bp). For the candidate tRNAProTGG2.9-VIT_18s0001g09050 primers tRNAProTGG_F and intergenic_tRNAProTGG-VIT_18s0001g09050_R (172 bp) were used. For the candidate tRNAGlyCCC-VIT_19s0177g00220 we used primers VIT_19s0177g00220_F and intergenic_tRNAGlyCCC-VIT_19s0177g00220_R. Negative controls for the PCR reaction (-Ctr) contained all components for the reaction except the cDNA template. RT-PCR products were analysed by agarose gel electrophoresis and SYBR Safe DNA gel staining . RT-PCR products were purified using PCR Clean-up  following the manufacturer’s instructions. Sanger sequencing was performed at the Australian Genome Research Facility. Oligonucleotides used for RT-PCR are listed in Supplemental Table 48.Complementary DNA was synthesized from the same total RNA used for the RNA-seq using SuperScript IV first strand synthesis system  following the manufacturer’s instructions. Complementary DNA was synthesised using gene specific reverse primers that aligned to the second exon of the candidate gene , or the tRNA (tRNAlfda49 on the expression (logCPM) values of all tRNA, dicistronic tRNAs, intergenic region and the dicistronic genes. In order to determine whether primary mRNA or the tRNA drives the expression of candidate dicistronic tRNA–mRNA transcripts, we carried out Pearson correlation analyses between the expression of tRNA vs intergenic region and PCGs vs intergenic region for each dicistronic candidate identified. Pearson correlation analysis  was performed using the R function cor.test. Read counts of the intergenic regions were used as a proxy to define the expression of dicistronic transcripts. The rationale behind this lies in the assumption that reads mapping to the intergenic region can only be the result of the sequencing of a dicistronic RNA molecule, while reads mapping to tRNA genes and PCGs could result both from the expression of monocistronic and dicistronic transcripts (i.e. tRNA genes and PCGs pairs forming two independent RNA molecules or a single RNA molecules respectively) . Similarly, the regional expression levels of all expressed tRNAs were compared. Briefly, mapped reads for each selected genomic feature obtained from featureCounts, were analysed in the R environment for plotting the gene expression through heatmaps. The expression of all tRNA, dicistronic tRNAs, intergenic region and the dicistronic genes (logCPM) were plotted using the pheatmap. Local Fisher LFDA was performed in the R environment using the package ly) Fig. .18), BLASTP search from NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi), and from the Additional file 1 of Cramer et al.50. Protein information and gene ontology terms were obtained from UniProt (https://www.uniprot.org/uniprot). GO enrichment analysis was performed from Gene Ontology Consortium (http://geneontology.org/) with default settings.A bar plot was made to represent the distance (bp) between the tRNA and its proximal gene. Pairs of expressed tRNA–mRNA were split in two groups depending if they formed dicistronic or monocistronic transcripts. A non-overlapping sliding window approach (200 bp) was used to count the number of pairs of genes of each type. Expression of the dicistronic genes and dicistronic tRNA was assessed by plotting their expression values against the distribution of the total gene expression for each tissue from the RNA-seq data. Gene annotation for dicistronic genes was obtained from the V1 annotation of the 12× grapevine reference genome , and three species with archaic vasculatures  and (Selaginella moellendorffii (lycophytes), and two non-vascular plants (Physcomitrella patens (mosses) and Marchantia polymorpha (liverworts)). See Supplemental Table Marchantia polymorpha that do not contain true vasculature we included tissues that contain primitive vascular-like structures such as thallus, antheridiophore & archegoniophore. A minimum of three replicates per sample as denoted by the SRA metadata information were obtained. In order to reduce the experimental variables for the multi-species comparison, only wild-type plants and non-treated controls were included for downstream detection of tRNA–mRNA transcripts. The datasets were analysed using the DiRT pipeline as described previously.We collected short-read RNA-sequencing datasets (Illumina paired and unpaired reads) from 30 independent studies (149 accessions) accessed through the National Centre for Biotechnology Information-Sequence Read Archive (NCBI-SRA) for nine land-plant species  using gene codes from Arabidopsis thaliana (Supplemental Tables Identification of orthologous vascular genes in the lignin, cellulose and xylan pathways utilised a multi-step process. First, an OrthoFinder analysis was carried out as previously describedtrim_galore. Reads were then aligned to their respective species reference genome using HISAT2. After further processing of the aligned reads using the samtools package, read counts were calculated and normalized using TPMCalculator. The orthologous genes corresponding to the groups cellulose, xylan and lignin were then filtered from the dataset and their normalized expression was compared.For the expression analysis of the identified orthologous genes, RNA-seq datasets for the nine different plant species (Supplemental Table Supplemental File 1Supplemental Fig S1Supplemental Fig S2Supplemental Fig S3Supplemental Fig S4Supplemental Fig S5Supplemental Fig S6Supplemental Fig S7Supplemental Fig S8Supplementary table S1Supplementary table S2Supplementary table S3Supplementary table S4Supplementary table S5Supplementary table S6Supplementary table S7Supplementary table S8Supplementary table S9Supplementary table S10Supplementary table S11


ABSTRACT IMPACT: Approximately 15% of US adults have chronic kidney disease with over 700,000 of those in the end stages where treatment options are severely limited to dialysis or kidney transplant; the research presented here will help identify novel strategies that address oxidative imbalance and preserve renal function. OBJECTIVES/GOALS: Chronic Kidney Disease patients often develop secondary cardiovascular disease - Cardiorenal Syndrome Type 4. RNA sequencing data show increased apelin receptor expression in 5/6 nephrectomy rats. The Apelinergic (APJ) system is deemed beneficial in normal physiological systems. Here we explore links between stress and the APJ system in CRS4. METHODS/STUDY POPULATION: In preliminary studies performed in NRK cells, inflammatory cytokines, IL-1β and IL-6, caused increases in apelin receptor transcripts and decreased apelin transcripts, respectfully. The literature describes inflammatory processes that contribute to degradation of many organs  suggesting an oxidative imbalance. To investigate this imbalance within CRS4, three rat cell types’’ H9c2 cardiomyocytes, HII4E hepatocytes, and NRK renal epithelial cells” will be used to assess the role of exogenous apelin on pro- and anti-oxidant levels. Cells will be pretreated with apelin or vitamin E 48 hours prior to the addition of toxins or cytokines , to assess pro- and anti-oxidant protein levels via Western Blot. RESULTS/ANTICIPATED RESULTS: We anticipate with toxin or cytokine addition  in all cell types, an increase in protein levels for GPX’‘ a known measure of oxidative stress’‘ should be greater than the increases in antioxidants’‘ SOD1 and Catalase. After pre-treatment of vitamin E, GPX protein levels should decrease compared to toxin/cytokine control, while SOD1 and catalase protein levels increase; this coincides with vitamin E inducing antioxidant activity in animals and humans. The anticipated results for this study after exogenous apelin addition should reveal in all three cells types reduced levels of GPX and increased levels of SOD1 and Catalase comparable to the vitamin E treatment. DISCUSSION/SIGNIFICANCE OF FINDINGS: If addition of apelin causes an antioxidant response in all three cells types, this can build on evaluation of the APJ system as a therapeutic option for those with CKD and CRS4 to minimize both inflammatory and oxidative stress. With the data gathered here, we expect to recreate the results in a CKD rat model that highlights these same manifestations.


Eucalyptus globulus breeding populations to provide direct comparisons across the programs and indirect predictions in environments where pedigreed families had not been evaluated. Despite few known pedigree connections between the programs, genomic relationships provided the connectivity required to create a unified relationship matrix, H, which was used to compare pedigree-based and HBLUP models. Stem volume data from 48 sites spread across three regions of southern Australia and wood quality data across 20 sites provided comparisons of model accuracy. Genotyping proved valuable for correcting pedigree errors and HBLUP more precisely defines relationships within and among populations, with relationships among the genotyped individuals used to connect the pedigrees of the two programs. Cryptic relationships among the native range populations provided evidence of population structure and evidence of the origin of landrace populations. HBLUP across programs improved the prediction accuracy of parents and genotyped individuals and enabled breeding value predictions to be directly compared and inferred in regions where little to no testing has been undertaken. The impact of incorporating genetic groups in the estimation of H will further align traditional genetic evaluation pipelines with approaches that incorporate marker-derived relationships into prediction models.Single-step GBLUP (HBLUP) efficiently combines genomic, pedigree, and phenotypic information for holistic genetic analyses of disjunct breeding populations. We combined data from two independent multigenerational  Quantitative genetics has utilized pedigree-derived relationships to improve selection efficiencies of breeding programs for many years . Well-deGenetic markers such as single nucleotide polymorphisms (SNP) provide empirical rather than expected relationships by using estimates of allele similarity between pairs of individuals . GenotypG) are merged into the traditional pedigree-derived matrix of expected relationship coefficients (A) of the entire population (H) is then used in place of A in linear mixed models (LMM) to produce Best Linear Unbiased Predictions (BLUP) of genetic value. This “HBLUP” approach has been combined with efficient computing procedures , in some cases only for genotyped individuals  has on genetic parameter estimates and the accuracy of breeding value predictions. Replacement of A with H as the numerator relationship matrix (NRM) involves two types of changes to the information used in the analysis. Not only do genomic data inform the relationships amongst individuals in H, but the formulation of H applied thus far in tree breeding does not allow for races as genetic groups. Therefore, we aimed to disentangle the impact of including genomic information from that of removing race groups by comparing results from three types of models: ABLUP with race genetic groups , ABLUP without groups (ABLUP−race), and HBLUP.Most commercial tree improvement activities are practiced within the confines of an individual program. However, important exchanges of information and genetic material also occur amongst programs to support breeding and the establishment of planted forests. A major limitation to such exchanges exists when pedigrees have been coded differently and little connectivity is available between programs to provide unbiased comparisons among populations. This situation provided the stimulus for our work to quantify the impact that combining two distinct pedigrees into a joint relationship matrix  and EG2 (HVP Plantations). These commercial tree improvement programs progeny test full-sib families predominantly derived from first- and second-generation selections. Forty-eight field trials established between 1998 and 2015 provided phenotypic data from 126,467 full-sib progeny (1973 families), 7051 half-sib progeny (263 families), and 24,767 unpedigreed trees of commercial entries , the Green Triangle (GT), and Gippsland (GIPPS). EG1’s breeding program was focused in WA  with five trials in GT and none in GIPPS. EG2 trials were located predominantly in GT , with six trials in GIPPS and three in WA (The EG1 population of full-sib families was founded on 112 base-population trees and the EG2 population was founded on 83 base-population trees (Supplementary Table S1C describes populations). These “founders” are trees in native forests or international landraces that provided the seed for testing and domestication. Half the founders of the EG2 program originated from Strzelecki Ranges and Western Otways races, whereas the EG1 program is more strongly influenced by Furneaux, King Island, Portugal (landrace), and NE Tasmania. The classification of races follows the results of Inadequate interprogram relationships were available for joint analyses using pedigree derived relationships as only four founders were common to both programs. Pedigree-derived relationship coefficients amongst the two parent cohorts were rare, with two relationships of 0.25, 152 relationships of 0.125, and one expected relationship coefficient of 0.0625.Trials were primarily established in randomized incomplete-block designs  with four to eight replications of each family established in four- to five-trees in contiguous row-plots. Two EG1 trials were single-tree plot designs. Incomplete blocks were arranged into replicates that were generally contiguous to enable the resolution of spatial trend effects within each trial.Diameter at breast height (DBH) was measured with a tape for each tree at 3 years (7 sites), 4 years (9 sites), 5 years (29 sites), or 8 years  after planting. Tree height (HT) was measured with a hypsometer for each tree in the majority of trials, although a subset of 3–17% (mean 10%) of trees were assessed in 11 of the EG2 trials. In these cases, unmeasured HT data were predicted from the DBH–HT relationship established amongst measured trees for each trial. Stem volume (VOL) was calculated for each tree using DBH, HT (measured or predicted), and the region-specific volume function provided by each program. DBH of all stems forking below breast height was measured in 36 trials and in these cases, VOL was aggregated to the tree level before analysis.Given the expense of wood property assessments, a subset of trees in 20 of the 48 trials were selected for phenotyping (Supplementary Table S1D describes subset of populations assessed for wood properties). Basic density (DENS) data were collected for a subset of trees between 5 and 8 years. EG2 measured DENS directly by the displacement method using the outer 50 mm of wood cores extracted at breast height. EG1 used pilodyn penetration as an indirect measure of wood density . PilodynE. globulus marker panel consisted of 2579 SNP and small biallelic insertion/deletion (INDEL) markers within candidate genes associated with diameter, density, and cellulose yield that were identified using association analyses in previous studies  was formed for 238 genotyped individuals, the EG2 G matrix (EG2G) was formed for 144 genotyped individuals, and the joint G matrix (JOINTG) was formed amongst all 382 genotyped individuals. G was calculated in preGSf90 following the first method of Z is a matrix of centered genotype scores calculated as Z = M − 2 P; M is the n × m matrix of n genotypes and m markers scored 0 for homozygous reference allele, 1 for heterozygous, and 2 homozygous alternative allele, P is a matrix of frequency for the alternative allele, and jp is the reference allele frequency of the jth marker.Relationship matrices were produced using A was divided into four submatrices: A11 representing relationships amongst nongenotyped individuals, A22 representing relationships amongst genotyped individuals, and A12 and A21 representing relationships between genotyped nongenotyped individuals:The pedigree relationship matrix G and 22A H=AH was then resorted to align with the pedigree relationship matrix for comparisons with A. The section of H relating to genotyped individuals, their parents, and ancestors were compared with A in a preliminary step to highlight identity errors and mistakes in the documented pedigree. These errors were rectified in the pedigree and the process of creating the H−1 matrix was then repeated with the correct pedigree information.y is the vector of phenotypic values, X is the incidence matrix for fixed effects, b is the vector of fixed effects, Z is the incidence matrix for random effect, u is the vector of random effects with E(u) = 0, and e is the vector of residual effects expected to be independently normally distributed with E(e) = 0. The relationship matrix that differentiates ABLUP and HBLUP connects each individual in u, and this change in connectivity impacts genetic parameter estimates and the accuracy of breeding value predictions.All genetic analyses were conducted using ASReml v. 4.1 (adj) data that were used for preliminary cross-site analyses  in H matrices were greater than zero and provide further support for differentiation of races and population structure . The As expected, removing the race/landrace genetic groups from the EG2 pedigree caused significant increases in affected . The remded race  providin−race  for the ABLUP model when race effects were removed from the pedigree, which was slightly improved by using HBLUP instead of ABLUP+race models  based on core samples than the EG1 program (0.28 ± 0.03) based on penetrometer data, with an intermediate analysis . The pro9 ± 0.03 . Dominan+race models for the same parents (see Supplementary Table S2), resulting in generally higher estimates of +race due to the substantially smaller Overall, the greatest breeding value accuracy estimates  when parents were genotyped and accuracy increased by 44% (0.218 ± 0.029) when they were not genotyped. For EG2 parents that were unrepresented by progeny in WA, Prediction accuracy for parents of progeny tested in other regions improved markedly in the joint HBLUP analyses , although average a units) for EG2 in GIPPS, WA, and GT, respectively, when estimated with the joint HBLUP model (Supplementary Table S2) . However, average accuracy from joint HBLUP was 3–5% lower for EG2 progeny across regions due to the lower values of Accuracy of progeny EBVs was the least impacted by choice of relationship matrix. Average PEVs for progeny were marginally higher for EG1 (by 0.050 and 0.008 SDThe single-step GBLUP (HBLUP) approach was used to integrate genomic, pedigree, and phenotypic information and provide unbiased predictions of genetic merit for breeding programs with few pedigree connections. Genotyping the parents that were evaluated in separate progeny trial networks created sufficient connectivity between the programs to provide predictions of genetic merit for parents in regions where they were not evaluated and also improved the accuracy of predictions. Although the benefits of HBLUP to tree improvement have been demonstrated in a number of studies , this isA clear advantage of HBLUP is that relationships amongst genotyped individuals are quantified precisely, rather than represented by the expected average relationship derived from the pedigree. This advance has been well documented in tree breeding contexts , which generally resulted in larger values of H compared with A, and this is a subject of ongoing exploration and validation.Our results illustrate that HBLUP conferred little benefit to the prediction accuracy of parents and progeny within each program, possibly due to the very low proportion of genotyped individuals. PEV and HBLUP offers a clear advantage over ABLUP for predicting the value of genotyped individuals with no phenotype data and no pedigree linkages with individuals evaluated in trial networks. Breeding values were estimated for 52 EG1 individuals and 5 EG2 individuals that were genotyped but had no connections via pedigree. While accuracy estimates were lower than parental estimates, at around 0.4 and 0.5 for within-program and joint HBLUP, respectively, indirect estimates of breeding values were produced. Joint HBLUP enabled the prediction of a complete set of EBVs for EG1 parents, genotyped individuals with no phenotype data, and progeny in the GIPPS region where no progeny trials were established. Extending genomic prediction models to connect distinct pedigrees allows breeding programs to leverage information from one another and infer performance in environments where populations have not been evaluated. This study provides empirical evidence that may be used to promote collaboration among tree improvement programs to better characterize the genetic merit of individuals in environments where they have not been evaluated. The need for breeding value predictions in untested environments is expected to increase as forestry organizations examine options for adapting to climate change (Using genotype data to blend disconnected pedigrees and phenotype data from separate breeding programs into a unified analysis produces unbiased breeding values for direct comparisons between programs and indirect predictions of merit in environments where individuals may not have been evaluated. The joint HBLUP analysis significantly improved prediction error variance of parents and genotyped individuals, provided similar estimates of genetic parameters, more accurate EBV predictions, and offered the profound advantage of EBV prediction for genotyped individuals in regions they had not been evaluated.H will further align the traditional genetic evaluation pipelines that underpin tree breeding programs with approaches that incorporate marker-derived relationships into prediction models.Overall, the genotyping proved useful for correcting pedigree errors, more precisely defining relationships within and among populations, identifying the source of landrace populations, and integrating genotyped individuals with no phenotype or pedigree connections into the prediction framework. Understanding the impacts of incorporating genetic groups in the estimation of


All six patients with suspected CSF leak showed paraspinal regions of significantly greater [18F]FDG uptake compared to the corresponding areas in controls (P < 0.05). Two patients treated with local injections  on the site of abnormal PET/MRI findings reported temporary but significant improvement in symptoms. Our results suggest [18F]FDG PET/MRI is sensitive to abnormalities potentially due to suspected CSF leak, which are not necessarily visible on conventional MRI alone or by the standard-of-care imaging methods.A combination of magnetic resonance imaging (MRI), computed tomography (CT), and radionuclide cisternography are typically used to locate a cerebrospinal fluid (CSF) leak. However, the site of leakage cannot be determined, making treatment more difficult. Therefore, more sensitive imaging tools are needed. A whole-body [18F]fluorodeoxyglucose (FDG) positron emission tomography (PET)/MRI was conducted on six patients with suspected CSF leak and the resulting images were reviewed in comparison with those from six healthy controls. Paraspinal regions of focally increased uptake of [18F]FDG were quantified using maximum standardized uptake values (SUV The most characteristic symptom of a CSF leak is a persistent headache that worsens when upright and improves with recumbency (orthostatic headaches)2. When chronic, CSF leaks may present as “second half of the day headache” with patients consistently better in the morning after prolonged nocturnal recumbency followed by worsening symptoms as the day progresses with prolonged upright time3. Other symptoms include nausea, memory difficulties, cranial nerve dysfunction, neck and interscapular pain5. Hereditary disorders of connective tissue such as Marfan Syndrome, Ehlers-Danlos Syndrome, Neurofibromatosis type 1, and Polycystic kidney disease have been associated with spinal CSF leaks, suggesting these and other associated disorders of connective tissue may lead to abnormally vulnerable dura prone to CSF leak6. Among those who can identify a precipitating event, atraumatic activities such as stretching, or Valsalva maneuver are commonly involved9. Traumatic head/neck injury such as whiplash as well as iatrogenic dural tear during surgical interventions including lumbar puncture, durotomy, and skull base surgery are also important causes12. However, two thirds of patients with a spontaneous CSF leak can identify no precipitating events. The most common site of leak is along the spinal cord13. If the site of CSF leak is located, it is treated most commonly with an epidural injection of autologous blood  or an epidural fibrin patch, or surgery, depending on the case14.Cerebrospinal fluid (CSF) leak is a condition caused by drainage of CSF from the brain or the spinal cord through a hole in their surrounding structures, such as the skull and the dura. The prevalence rate of the CSF leak is estimated to be 1 in 50,000 individuals, but this relatively uncommon occurrence may be due to underdiagnosis15. Misdiagnosis is common, preventing proper treatment as the symptoms are often mistaken for those of other diseases, such as postural orthostatic tachycardia syndrome or new daily persistent headache16. The underlying sensitivity of magnetic resonance imaging (MRI) and computed tomography (CT) myelography remains unknown. While these two imaging modalities often are consistent in retrospective comparison17, this may reflect the common clinical scenario of using invasive CT myelography much more frequently in patients with positive MRI than in patients with negative MRI—attempting to localize the source of leak suggested by findings seen on the subset of positive MRI. In contrast, prospective studies of patients with orthostatic headaches suggest a potentially much lower sensitivity. In one study of 100 patients with orthostatic headaches suspected of having CSF leaks from a consortium of 11 hospitals in Japan, 70 had axial T2 spine MRI, finding 14 leaks, while 86 had MR myelograms, where only 3 leaks were noted18. For CT myelography, cases with slow or intermittent CSF leakage are very difficult to identify due to the insufficient contrast and resolution between CSF and background19. Previous studies estimate that 46–55% of these cases present no detectable signs of CSF leak on CT myelogram21. Thus, better non-invasive imaging techniques for detecting and localizing spinal CSF leaks are needed.Unfortunately, no one diagnostic test can consistently exclude the diagnosis of CSF leak or identify the site(s) of leakage22. We recently presented initial findings from our study23, and here we describe in detail our early experience of [18F]FDG PET/MRI of patients with chronic symptoms of suspected CSF leaks in comparison with healthy controls.In this report, we introduce the use of simultaneous [18F]fluorodeoxyglucose (FDG) positron emission tomography (PET)/MRI to locate CSF leaks. We hypothesized that [18F]FDG PET, if coupled with the high spatial and contrast resolution of MRI, could accurately identify inflammation around the sites of CSF leak due to its pro-inflammatory natureIn all six patients, we observed sites of abnormally increased focal uptake of 18F]FDG in paraspinal structures at various levels of the spine  as summarized in Table 8FFDG in max in lesions from the patient group showed a higher mean value than the corresponding areas from the control group in all tissue types . On the other hand, abnormalities on MRI that might be related to CSF leak were identified in only half of the patients. In a limited number of patients followed for the treatment outcome, the patients whose blood-patch treatment sites covered the abnormalities identified by our method achieved better symptomatic relief than the patient whose treatment marginally included the identified abnormalities.In this report, we introduce a novel [18F]FDG PET/MRI approach for detecting the sites of CSF leak. While other nuclear medicine approaches such as radioisotope cisternography have been used to identify CSF leaks22. This CSF-induced irritation is reflected clinically in studies of patients with chronic CSF leaks following accidental dural puncture, who report much higher rates of spinal pain at the site of CSF leak than controls without dural puncture25. Lesions with active inflammatory responses can have increased metabolism and energy requirements, becoming more glucose-avid than normal tissues26. [18F]FDG PET is a highly sensitive imaging modality of glucose metabolism, and has demonstrated its effectiveness in detection of such hypermetabolic inflammation29. Our results show abnormally increased uptake of [18F]FDG in the paraspinal region of all patients, supporting the feasibility of our approach for the detection of CSF leak. More abnormalities were found with [18F]FDG PET than 3T MRI, which suggests [18F]FDG PET might be better suited than MRI for the detection of early inflammatory changes due to CSF leak. However, our approach may not be feasible for detecting other etiologies without extradural CSF inflammation, such as CSF-venous fistular where CSF escapes the thecal sac directly into adjacent epidural veins.The proposed approach for the improved detection of CSF leak is based on the observation that extradural CSF may irritate and inflame tissues outside of the nervous system30 has demonstrated that seromas typically present a thin rim of mildly increased [18F]FDG uptake surrounding a central area of no [18F]FDG uptake in contrast to ‘solid’ or ‘thick-walled’ [18F]FDG avidity of more aggressive lesions such as soft tissue sarcomas.Unlike the conventional imaging methods for CSF leak diagnosis, our [18F]FDG PET approach is to find the paraspinal tissues potentially affected by CSF leak rather than the tear or puncture through which the CSF leaks. Therefore, using [18F]FDG PET in conjunction with an imaging modality that enables the investigation of the anatomic cause of the leak can create a synergistic effect in accurately specifying the site(s) of CSF leak. MRI is more advantageous than CT for accompanying [18F]FDG PET in that it can provide detailed anatomic examination with no further deposition of ionizing radiation to sensitive areas. The simultaneous acquisition of PET and MRI employed in this study also offers improved image co-registration compared to the conventional PET/CT that sequentially acquires PET and CT, rendering it susceptible to mis-registration due to patient motion between the two scans. Our PET/MRI approach has the additional benefit of potentially discriminating a benign post-operative seroma from an extraspinal collection of CSF. On MRI, they are difficult to differentiate because they are likely to appear isointense to each other. However, on [18F]FDG PET, the benign seroma is expected to have minimal or no [18F]FDG uptake, while the extraspinal CSF collection is significantly [18F]FDG avid, such as the one seen in Patient 6 Fig. . Indeed,32. Therefore, careful consideration of other etiologies should be considered when abnormal uptake of [18F]FDG is observed in the paraspinal regions. Our early results suggest it could be effective as an additional tool for locating the sites of CSF leak and target sites for epidural patching. Optimizing MRI sequences that can better complement [18F]FDG PET is also necessary to pinpoint the site of CSF leaks. Additionally, post-treatment scans in these same patients will be useful in determining whether a decrease in symptoms correlates with a decrease in [18F]FDG tracer uptake.Our study has a few limitations. More study subjects are needed to determine whether this [18F]FDG PET/MRI approach is overall more sensitive and specific to CSF leaks than other imaging modalities. Uptake of [18F]FDG is relatively non-specific and can be seen in other paraspinal inflammatory processes such as myositis and in musculoskeletal degenerative changes such as Baastrup’s diseaseIncreased [18F]FDG uptake can be observed on PET in the paraspinal tissues of patients with suspected CSF leak. Our results suggest [18F]FDG PET/MRI is sensitive to abnormalities potentially due to suspected CSF leak, which are not necessarily visible on the standard-of-care diagnostic imaging modalities.All experimental protocols in our prospective observational research were approved by the institutional review board (IRB) of the Research Compliance Office, Stanford University, U.S.A. (protocol number IRB-24972). All methods were conducted in accordance with relevant guidelines and regulations.For the patient group, 4 male and 2 female patients suspected of suffering from CSF leaks were recruited . All patients presented with orthostatic headache and previously received standard-of-care imaging methods (including CT myelogram), which did not identify sources of CSF leak. The events identified by the patients as a precipitating event were epidural steroid injection (Patient 1 and 3), traumatic injury (Patient 4 and 5), lumbar puncture (Patient 6), and none (Patient 2). Six healthy controls  were also imaged with the same [18F]FDG PET/MRI protocol used for patients to compare the [18F]FDG uptake between the patient and control groups. Prior to imaging, all participants signed an informed consent form regarding study participation and publishing acquired data and images. All data were acquired in compliance with the Health Insurance Portability and Accountability Act. The clinical trial registration number of our study is NCT03195270 (registration date: 07/22/2017), and the name of the associated clinical trial registry is ClinicalTrials.gov.34.All subjects were requested to fast for 4 hours before our imaging study to avoid non-specific elevation of the blood glucose level. One hour after a single 10-mCi injection of [18F]FDG, subjects were admitted to a SIGNA PET/MRI scanner  for imaging. We performed a whole-body PET/MRI scan of subjects, which consisted of 8 to 10 consecutive imaging stations. In each station, 4–8-min simultaneous PET/MRI scan was performed, making the total scan time to be 1 to 1.5 h depending on the patient’s height. Acquired PET and MRI raw data were reconstructed using the algorithms equipped in the scannerIn each station, we conducted following two MRI sequences: 3D axial T1-weighted spoiled gradient-recalled echo sequence with two-point Dixon fat–water separation  and 2D axial T2-weighted fast-spin-echo sequence with two-point Dixon fat–water separation . For one or two imaging stations where we suspected CSF leaks might occur based on the patient’s history, we additionally ran the following two sequences for high-resolution imaging: 3D axial double-echo-in-steady-state (DESS) with water-only excitation  and 3D coronal motion-sensitized-driven-equilibrium (MSDE) fast-spin-echo sequence (CUBE) with triple-echo Dixon fat–water separation . For MRI signal acquisition, a 16-channel head-neck coil, an integrated spine coil, and two 32-channel anterior body array coils were employed.max) of [18F]FDG was measured. We used image analysis software  for measuring SUVmax. The lesions on the PET images were categorized into 5 types: paraspinal muscle, interspinous ligament, osseous tissue, neuroforamina, and fluid collection. The SUVmax measurements in each lesion type were compared with the SUVmax of corresponding areas in the healthy controls using a two-sided Mann–Whitney U-test. We employed this nonparametric test because we could not assume the normality in the measurements from patients and the sample size was small. P-value of 0.05 was adopted as a significance level.Two radiologists reviewed patient PET and MRI images concurrently to find abnormalities that are likely to be related with patients’ symptoms. Abnormal [18F]FDG hotspots were identified as potential lesions and the maximum standardized uptake value  following the treatments in comparison with previous treatments, if possible. We compared the sites of the blood patches with the sites of PET/MRI abnormalities from our study to assess the relevance of detected abnormalities to the symptom based on the patient’s post-treatment evaluation.After our imaging study, three patients received epidural blood-patch treatments based on the information from the standard-of-care diagnostic methods, without using our imaging findings. Patient 2 had targeted fibrin epidural patches while Patients 4 and 6 had targeted epidural blood patches. When a leak site has been identified, fibrin patches may resolve the symptoms of some patients with CSF leak refractory to epidural patching with blood


Retinal bipolar cells survive even in the later stages of inherited retinal degenerations (IRDs) and so are attractive targets for optogenetic approaches to vision restoration. However, it is not known to what extent the remodelling that these cells undergo during degeneration affects their function. Specifically, it is unclear if they are free from metabolic stress, receptive to adeno-associated viral vectors, suitable for opsin-based optogenetic tools and able to propagate signals by releasing neurotransmitter.rd1/rd1 selectively expressing an enhanced yellow fluorescent protein (EYFP) as a marker in ON-bipolar cells. Subsequent mRNA extraction allowed Illumina® microarray comparison of gene expression in bipolar cells from degenerate to those of wild type retinae. Changes in four candidate genes were further investigated at the protein level using retinal immunohistochemistry over the course of degeneration.Fluorescence activated cell sorting (FACS) was performed to isolate labelled bipolar cells from dissociated retinae of litter-mates with or without the IRD mutation Pde6bA total of sixty differentially expressed transcripts reached statistical significance: these did not include any genes directly associated with native primary bipolar cell signalling, nor changes consistent with metabolic stress. Four significantly altered genes , implicated in synaptic remodelling, neurotransmitter release and viral vector entry had immunohistochemical staining colocalising with ON-bipolar cell markers and varying over the course of degeneration.Our findings suggest relatively few gene expression changes in the context of degeneration: that despite remodelling, bipolar cells are likely to remain viable targets for optogenetic vision restoration. In addition, several genes where changes were seen could provide a basis for investigations to enhance the efficacy of optogenetic therapies. •Bipolar cells are attractive targets for therapeutic optogenetics in IRDs.•This is the first cell specific transcriptomic analysis of bipolar cells in an IRD model.•Bipolar cells maintain expression of genes essential to act as targets for optogenetics.•Protein staining relating to four candidate genes  is confirmed using immunohistochemistry. ANOVAANalysis Of VArianceAAVAdeno Associated VirusEYFPEnhanced Yellow Fluorescent ProteinFACSFluorescence Activated Cell SortingFDRFalse Detection RateHSPGHeparin Sulphate ProteoglycanIHCImmunohistochemistryIRDsInherited Retinal Degenerations P40, P90, P120, P150 – Postnatal day 40, 90, 120, 150LTD/PLong Term Depression/PotentiationPKC αProtein Kinase C - αqPCRQuantitative Polymerase Chain Reaction1Advances in retinal gene therapy delivery methods, such as adeno-associated virus (AAV), have allowed retinal gene replacement to become a reality for patients suffering from certain inherited retinal degenerations (IRDs) . With thAAV delivered optogenetic tools have been shown to restore electrophysiological and behavioural responses to light in animal models of IRDs  by rendeWhile effective, the stimulation of retinal ganglion cells - especially by epiretinal prostheses - bypasses much of the early image processing carried out in the retina. This makes specific stimulation of cells higher in the retinal hierarchy, such as the bipolar cell, conceptually attractive. However, significant retinal remodelling does occur after the death of the photoreceptor , potentiTo date, it is not understood how changes specifically within the bipolar cells during degeneration will affect their long-term viability as optogenetic targets. Particularly, if they are free from metabolic stress, receptive to adeno-associated viral vectors, suitable for opsin based optogenetic tools and able to propagate their signal by releasing neurotransmitter in response to exogenous optogenetic stimulation. While studies of general gene expression changes in animal models of the degenerate retina exist in the literature , none haInvestigation of bipolar cells is particularly apposite for two reasons: first, the development of delivery tools to specifically target discrete retinal cell populations (such as cell specific promoters and AAV capsid tropism ) have maThe principal objective of this study was to confirm the continued expression of the principal components of both the ON-bipolar light signalling and other second messenger cascades during IRDs. Here we show that, despite remodelling, bipolar cells undergo remarkably limited transcriptomic changes in response to the loss of synaptic inputs from photoreceptors, even in the late stages of the disease in an animal model.wt/wt and Pde6brd1/rd1 retinae using immunohistochemistry. Together, these findings will be central to guiding investigations to effectively translate bipolar cell targeted optogenetic therapies into clinical use.The secondary aim of the study was to identify differentially expressed genes for further characterisation in both Pde6b22.1x/x”, supplementary methods) was used which:1.Expressed Cre recombinase under the control of the ON-bipolar cell specific promoter “L7” ,2.Were homozygous for “floxed” EYFP at the Rosa26 locus and therefore expressed EYFP in L7  cells.3.wt/wt or Pde6brd1/rd1 Were either Pde6bAll experiments involving animals were performed in accordance with the Animals for Scientific Procedures Act 1986, licence no. 30/3371 and approved by the University of Oxford animal welfare and ethical review body and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. A transgenic mouse line  according to the manufacturer's instructions   with 97.When isolating dissociated retinal cells in such a way, the large number of rod photoreceptor cells in the wild type retina as well as their invaginated bipolar cell synapses could potentially lead to rods being carried over with labelled bipolar cells into the isolate. Increasing the length of enzymatic dissociation can minimise rod contamination, yet such approaches must be weighed against the risk of damage to the isolated cells or RNA with extended papain dissociation times. These protocols not only require longer periods for cells in a ‘non-physiological’ state before mRNA extraction, but lead to greater loss of cell processes (dendrites and axons). These are especially relevant to bipolar cells given the dramatic changes to these parts of the cell during degeneration.Previous studies have attempted to control for rod carry over in different ways - for example, We specifically interrogated our samples (prior to gene array) for two known rod specific genes (Rho & Cnga1) in a small number of samples using qPCR to confirm relative sample purity . Gene ontology terms were used to group the genes into six broad functional groups  while re2.4wt/wt and Pde6brd1/rd1 mice  underwent cervical dislocation before immediate enucleation and processing of retinae for IHC (supplementary methods). Images were taken at a point three fields of view (at ×40 magnification) from the ora serata in three sections from one eye in each animal. Co-localised staining for the proteins of interest and a bipolar cell marker were taken as a semiquantitative index of protein expression levels in bipolar cells and were determined using Costes' method, described and validated previously , Pde6beviously . In brie33.1wt/wt or Pde6brd1/rd1). cDNA libraries were extracted from the resulting EYFP+ (ON-bipolar) cell isolates were processed and subjected to an Illumina® mouse gene array. To quantify contamination of these isolates with rod photoreceptors, qPCR for two rod specific genes  was performed to ensure that neither was detectable at a significant level in any bipolar cell isolate . Gene array analysis revealed sixty-six probes corresponding to sixty genes were shown to have differential expression between the degenerate and non-degenerate samples with a p value of <0.05  testing, Retinal ON-bipolar cells were isolated using FACS of dissociated retina from L7.Cre.EYFP transgenic mice which were additionally either wild type or homozygous for a clinically relevant IRD mutation  – this data driven approach was complemented by a manual, systematic, evaluation (including literature and gene ontology consortium annotation review) for each differentially expressed gene. This was used to group the potential candidates by function  and scor3.3wt/wt & Pde6brd1/rd1) at each timepoint  to give an impression of how protein levels may change over time.Antibody labelling for proteins encoded by each of the selected genes  showed co-localisation with classical ON bipolar markers (CHX10 or PKCa) at postnatal day 90 (P90) in our IHC study – although these proteins were typically not expressed exclusively within ON BCs , Fig. 5.There was a difference between genotypes in this index for all proteins excepting slf2 (Sulphatase 2) . In addition, differences in staining over time were seen in Srm2 (Shroom 2) & Slf2 . Whilst an interaction between genotype and time was seen in Srm2 and Cntn1 (Contactin 1) .  death, the retinal bipolar cells become the highest surviving cells of this neural hierarchy. As such, their stimulation may - in principle - enable more intra-retinal processing to be preserved at the synapses of the inner plexiform layer, presenting them as particularly attractive targets for optogenetic visual restoration.Targeting bipolar cells assumes that, within a degenerate retina, these cells retain levels of the second messengers required for light signalling. It also assumes such cells survive in a stable state without metabolic stress to allow effective propagation of this signal by synaptic neurotransmitter release and, importantly, are able to be transduced by AAV. Increasing evidence confirms that bipolar cells do undergo significant remodelling in the later stages of retinal degeneration with changes in morphology, synaptic connections, electrophysiological responses and receptor expression .4.2Here we show that, despite this remodelling, bipolar cells undergo remarkably limited transcriptomic changes in response to the loss of synaptic inputs from photoreceptors, even in the late stages of the disease. While gene array studies of whole retina may not have statistical power to detect very small changes in expression of single genes, our approach of limiting our comparison as far as possible to a single cell type in a clinically relevant disease model, will accentuate those changes that are biologically most relevant.Given the marked changes seen at the anatomical level over the whole retina during the neural remodelling of degeneration, it is perhaps surprising that we found such a small number of genes were differentially expressed in bipolar cells in this context . The absence of differential expression of genes related to the native bipolar cell light signalling cascade, second messaging in general or glutaminergic transmission  is partird1 model. Given that the rd1 mutation causes an IRD in humans similar in phenotype to that of the model, these findings are particularly interesting from a translational point of view (if they are reflected in human bipolar cells). In counterpoise however, the huge variety of causative mutations in human IRDs should still be borne in mind when extrapolating results.This finding of such stability at a gene expression level is particularly informative when seen in the light of studies where opsin based optogenetic tools  are targ4.3wt/wt and Pde6brd1/rd1 retinae. Staining of similar retinae at other timepoints over the course of degeneration , a systematic literature review for each gene could be used to group and prioritise those most promising for further characterisation at the protein level . Immunohistochemical staining at the timepoint corresponding to the gene array (P90) confirmed expression of all four proteins in bipolar cells of both Pde6bneration , Table 24.4The pattern of Shroom 2 staining  that we The Heparin Sulphate Proteoglycans (HSPG), from which Sulphatase 2 removes sulphate residues, are involved in  the binding of AAV in advance of its entry to the cell . ImportaEffective retinal optogenetic therapy requires neurotransmitter release from targeted cells; Annexin a7 is central to this process and interacts with PKCα, an enzyme known to regulate bipolar cells' light response kinetics  Hoque e   and in mThirdly, as this is the first study to investigate transcriptional changes in on-bipolar cells during retinal degeneration, a direct positive-control - a gene already known to be up or down regulated in this context – was lacking. Such positive controls provide a valuable technical validation of transcriptomic datasets but were not possible in this context.5Here we present the first comparison of gene expression in bipolar cells of degenerate and non-degenerate retinae. Our findings suggest relatively few changes in gene expression with degeneration, including genes essential to effective optogenetic bipolar light signalling. This suggests, that despite remodelling, bipolar cells are likely to remain viable and effective targets for optogenetic vision restoration and we highlight candidate genes where further investigation is likely to improve the translation of this important technique.Microarray data has been uploaded to the EMBL-EBI ArrayExpress platform and is available with the accession number E-MTAB-10357.10.13039/100010269Wellcome Trust (grant number 205151/Z/16/Z); The Woolf Fisher Trust; The 10.13039/501100001659German Research Foundation (DFG) (grant number LI2846/1-1) and The Biological Basic Sciences Research Council (grant number BB/M009998/1).This work was supported by The M.L. reports non-financial support from Optos, Genentech/Roche and Heidelberg Engineering, personal fees from Alimera Sciences, outside the submitted work.M.J.G., D.H., S.H., S.N.P., R.E.M. and M.W.H. have no relavent financial disclosures.