Taeniopygia guttata) revealed evolutionary modifications unique to songbirds, as well as transcriptional specializations in the song system . Here, we expand this approach to sodium, calcium, and chloride channels along with their modulatory subunits using comparative genomics and gene expression analysis encompassing microarrays and in situ hybridization.Vocal learning, the ability to learn to produce vocalizations through imitation, relies on specialized brain circuitry known in songbirds as the song system. While the connectivity and various physiological properties of this system have been characterized, the molecular genetic basis of neuronal excitability in song nuclei remains understudied. We have focused our efforts on examining voltage-gated ion channels to gain insight into electrophysiological and functional features of vocal nuclei. A previous investigation of potassium channel genes in zebra finches  in the zebra finch genome, several of which were previously unannotated. We determined 15 genes are missing relative to mammals, including several genes  linked to olfactory transduction. The majority of sodium and calcium but few chloride channels showed differential expression in the song system, among them SCN8A and CACNA1E in the direct motor pathway, and CACNG4 and RYR2 in the anterior forebrain pathway. In several cases, we noted a seemingly coordinated pattern across multiple nuclei  or sparse expression .The gene families examined are highly conserved between avian and mammalian lineages. Several cases of differential expression likely support high-frequency and burst firing in specific song nuclei, whereas cases of sparse patterns of expression may contribute to the unique electrophysiological signatures of distinct cell populations. These observations lay the groundwork for manipulations to determine how ion channels contribute to the neuronal excitability properties of vocal learning systems.The online version of this article (10.1186/s12864-019-5871-2) contains supplementary material, which is available to authorized users. Motor learning, the process by which motor skills are acquired and perfected through practice, requires fine-tuning of sensorimotor circuit elements to ultimately produce precise motor output. One remarkable example is vocal learning, a trait that allows individuals to learn their vocalizations through auditorily guided vocal practice. Vocal learning is demonstrated only by some mammals , and three groups of birds  . SongbirThe zebra finch song system is composed of the posterior direct motor pathway (DMP), necessary for song production , 15, andGiven their direct effects on membrane potential, cell excitability, and neuronal firing, ion channels genes are among the most important determinants of neuronal function –29. TheiWe identified 23 sodium, 38 calcium, and 33 chloride channel genes in the zebra finch genome - the majority with clear orthologs in mammals, indicating their broad conservation across vertebrates. We also established complements of ion channel genes that are differentially expressed within the major song system nuclei compared to the surround. These data represent the most comprehensive characterization of these ion channel gene families in a bird species to date. They also provide novel insights into how molecular specializations associated with neuronal excitability may have evolved to support a complex, learned vocal behavior.We identified the sets of sodium, calcium, and chloride channel genes in zebra finch using a modified strategy of that previously used for potassium channels . We firsWe next searched for the orthologous genes in zebra finch using the following pipeline: 1) Obtain from NCBI  the human records for the HGNC (HUGO Gene Nomenclature Committee) gene list; 2) Align the human models to the zebra finch genome to identify potential orthologous loci; (3) Verify the synteny of the top-scoring alignment; 4) Demonstrate that the zebra finch gene aligns preferentially to the corresponding human locus; and (5) Demonstrate that secondary alignments represent other family members. These steps were also applied to identify chicken orthologs  We queried the NCBI Entrez Gene database with the names of the 108 genes in the starting HGNC gene list, to retrieve the corresponding human gene records. All 108 human genes were found in NCBI. (2) We retrieved the human RefSeq nucleotide sequences of the longest isoform for each annotated gene and used these as queries to search the zebra finch genome  using t We querin = 61 genes, no symbols next to gene name and the “Zebra finch locus” column contains only Ensembl model(s)). We detected no cases of multiple top-scoring alignments with similar or different syntenies, thus we conclude that no novel paralogs and/or segmental duplications (e.g. of a cluster of syntenic genes) are present in the zebra finch genome for the gene families examined. In a few cases no Ensembl model was present but synteny of the top scoring finch locus was conserved with the human ortholog .Some sodium channel (SCNxA) genes are arranged in clusters; in such cases, BLAT alignments tend to span multiple family members, or identify another family member in a given cluster. For these genes, we compared the syntenies within and around the clusters as a group across species, and closely examined the number, orientation and alignments of the individual genes in each cluster before deciding orthology. Both clusters in this family were found conserved between finch and humans , falcon (outgroup to psitacopasseridae), chicken and/or quail , tinamou, ostrich, and emu , lizard, alligator and turtle , Xenopus , and platypus and opossum . We only used non-human orthologs that met two criteria: 1) conserved synteny and 2) aligned preferentially to the correct human locus. For 4 genes, this phylogenetic approach revealed an indirect relationship between the syntenies of the human query and the top scoring alignment in finch , or when the only significant alignment was to chrUn. For these genes, we next used the zebra finch Ensembl model to conduct searches of a newer zebra finch assembly  built exclusively from PacBio reads (Pacific Biosciences). This PacBio assembly has higher contiguity, fewer gaps , and often more genes and intergenic regions than the Sanger, 454, or Illumina assemblies . Brieflyn = 10; genes have a PacBio location instead of an Ensembl model in the “Zebra finch locus” column of Table In several other cases, no locus could be found through alignments of human or other species’ orthologs to taeGut1. For these genes, we searched the PacBio assembly with the human model and/or other species’ orthologs and confirmed synteny using BLAST to explore the surrounding scaffold sequence  We BLAST searched the finch PacBio assembly using models of syntenic genes from comparative species as queries. When the syntenic genes were found on the same scaffold, we conducted additional BLAST searches of short segments (1–2 kb) of their intergenic region at a time. We interpret cases in which syntenic genes were adjacent but no traces of the ion channel gene could be found intergenically as high likelihood cases of gene loss, noting again that finch PacBio scaffolds are gapless. (2) To better understand the origin of such losses, we systematically searched for the gene of interest and syntenic genes in all birds with an assembled genome deposited in NCBI, initially based on gene name searches in Entrez Gene. In cases where other avian species’ scaffolds contained syntenic genes but lacked a model for the gene of interest, we conducted BLAST searches of the intergenic region as described in Case 4. In cases where we could not find evidence for the gene in any bird species, we applied the same general strategy above to search for the gene of interest and its syntenic context in representative outgroups , to trace the most likely pattern of gene loss in groups ancestral to birds , we first obtained the transcript sequences of the orthologous chicken Ensembl models. As with zebra finch, we selected the longest transcript variant and concatenated split models. We then aligned the chicken and corresponding zebra finch models to taeGut1 using the UCSC Browser BLAT and exported the results as psl files. Using a custom Python (version 3.6.7) script [1: Table S1; frequency histogram in Additional file 1: Table S1).We also used an alignment-based method to evaluate the completeness of zebra finch models. Because chicken galGal6) had a higher coverage (82x) and contig N50 17,655,422) than taeGut1  and thus had more reliable and complete gene predictions, we used chicken models from Ensembl (release 95). RYR1 was excluded from this analysis as it is > 100 kb long, fragmented into at least 8 models across 8 different scaffolds that lack syntenic context in galGal5, and the only verified model in taeGut1 is highly partial and in a gappy region of chrUn. For all other genes with orthology-verified models in both zebra finch and chicken  script , we comp had a hiAdult male zebra finches were obtained from a commercial supplier  and acclimated in our animal facility in same-sex group cages for at least 2 weeks prior to the onset of experiments. The work described in this study was approved by the OHSU IACUC and is in accordance with NIH guidelines. To minimize the confound of song-induced gene expression, all birds were placed overnight in acoustic isolation chambers and monitored for singing for a period of 2 h after lights-on. Verified non-singing birds were sacrificed by decapitation and brains were rapidly dissected, blocked in TissueTec OCT , then flash-frozen in a slurry of isopropanol and crushed dry ice. Frozen brains were sectioned in the sagittal plane on a Leica CM1850 cryostat at 10 μm and melted onto charged microscope slides . Slides were stored at − 80 °C until use.All probes were derived from clones selected from the ESTIMA zebra finch cDNA library . All genWe followed the protocol by Carleton et al.  to generNon-radioactive in situ hybridizations to zebra finch sagittal brain sections were performed as in , using VTaeniopygia guttata 45 K oligo array and Area X samples were hybridized to the 20 K Agilent-019785 Custom Zebra Finch Microarray. RA was contrasted to ventrolateral arcopallium, Area X was contrasted to ventral striato-pallidum, and nXIIts was contrasted with the supra-spinal medullary nucleus (SSP). Detailed methods of sample preparation, mRNA isolation, array hybridization, and first-pass analyses are available for RA [We also evaluated differential brain gene expression of ion channels based on data from four previous microarray experiments where the song nucleus of interest and a nearby region were microdissected using laser capture microscopy. The HVC dataset was generated using spotted glass ESTIMA:Song collection “20 k” cDNA microarrays as detailed in  and HVC e for RA , Area X e for RA , and nXIe for RA .To determine the expression of ion channel and related genes in the zebra finch song system, these microarray datasets were reanalyzed as detailed in , 55. BriGenes that encode or modulate sodium, calcium, and chloride ion channels play important roles in determining the electrophysiological properties of brain circuits. In order to understand the potential contributions of these channels to functional circuits underlying vocal learning and production in songbirds, we started with a comprehensive analysis to define the full complement of these ion channel families in the zebra finch genome. Starting with a list of 108 ion channel genes previously identified in human plus one additional gene that lacks a human model (TPCN3), we used BLAT alignments and synteny analysis to identify orthologous loci in zebra finch and chicken. We identified 94  of these genes in zebra finch  channels (4 genes), Cav2  channels (3 genes), and Cav3 (T-type) channels (3 genes). All genes were found in zebra finch and chicken, with CACNA1A and CACNA1F in the PacBio assembly.Genes in this family encode the pore-forming α1 subunits that carry calcium across the plasma membrane . In mammCalcium channels are modified by non-conducting subunits, including α2 and δ. α2/δ genes are unique in that each gene codes for both the α2 and δ subunits: α2 is an extracellular glycoprotein that forms a disulfide linkage to the δ subunit, and the δ subunit keeps the complex tethered to the plasma membrane . All fouThese modulatory subunits are entirely intracellular and attached to the calcium channel complex through binding sites on the α1 subunit . All fouGamma subunits are glycoproteins with four transmembrane domains that associate with calcium channel complexes in the membrane . Of the This family encodes four α subunits (CATSPER1–4) and five auxiliary subunits  essential to sperm activation and motility , 71. In Other calcium channels may impact neuronal function by regulating internal calcium stores, including inositol 1,4,5-trisphosphate receptors (ITPR), ryanodine receptors (RYR), and two-pore channels (TPCN) , 73, 74.This family is composed of channels that open and conduct chloride ions in response to changes in membrane potential . MammalsThese channels are integrated into the membranes of organelles and participate in processes like membrane trafficking and cytoskeleton dynamics . They caSome anoctamin (ANO1 & ANO2) and bestrohpin (BEST2) genes encode channels that conduct chloride currents in response to surges in intracellular calcium, but the function of most other family members remains speculative , 77. MamANO7 lacks models in zebra finch and chicken, but traces were detected by aligning orthologs to the zebra finch and chicken PacBio assemblies. The synteny in these and other birds  is conserved with mammals and alligators. Given that only a few 3′ exons from orthologous models of this gene aligned to the chicken and zebra finch PacBio assemblies, combined with the lack of EST-based expression evidence, we conclude that ANO7 has likely become a pseudogene in some avian lineages, including oscines and galliformes.BEST4 is present and complete in galliforms (chicken) and psittaciformes (budgie), but appears pseudogenized in passerines, as only short segments of the 5′ and 3′ UTR regions were detected in zebra finch (PacBio), Bengalese finch, great tit, and starling. While BEST2 seems complete in chicken and songbirds like Tibetan tit and starling, we did not detect it at the corresponding locus in the zebra finch PacBio assembly. The upstream genes are in zebra finch similarly as in other birds, reptiles, and mammals, while the genes downstream of this locus reflect a synteny shared by Tibetan tit and starling that is presumably unique to songbirds. Because there are no BEST2 models and syntenic genes are isolated on short scaffolds in Bengalese and medium ground finch, we cannot distinguish between a loss in the finch lineage and a zebra finch-specific loss. Similar to CACNG6/7/8, BEST2 is located in a region of human chromosome 19 associated with extensive rearrangements and gene losses in birds .Mammals have several non-pore-forming accessory subunits with a broad functional repertoire that includes modulating chloride channels, cell adhesion, and tumor suppression . In humaLRRC8 channels are activated by cell swelling and may influence extracellular signaling by transporting neurotransmitters , 80. MamCystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel gated by ATP . Chloridn = 79 genes with an Ensembl model in both species). Because Ensembl chicken models are from a higher quality genome and are generally more complete than zebra finch models, this method revealed sequence blocks where the chicken model aligned but that are not present in the zebra finch Ensembl models , and a brainstem motor nucleus, nXIIts. Because our focus was on vocal control circuitry, not all brain areas of relevance for vocal learning (such as auditory forebrain) were assessed. This analysis encompassed 55 genes  that were part of the microarrays and/or for which cDNA clones were available in zebra finch. Summaries of the data are presented in Figs. The four alpha subunits with the highest brain expression in mammals (SCN1A/2A/3A/8A) showed varied patterns within the song system Fig. . SCN1A wSCN1B showed higher expression in all telencephalic nuclei, including NIf, as well as in DLM Fig. . In contv1s, CACNA1C was higher in Area X and nXIIts, while CACNA1D was lower in RA, Area X, and LMAN. Among Cav2s, CACNA1E was lower in RA  in HVC  identify previously undetected genes; (2) confirm with high confidence gene truncation; (3) define orthology based on synteny; (4) suggest a likely phylogenetic path to previously reported avian gene losses or truncations , 61. A lEach song nucleus examined differentially expresses a unique complement of ion channels, suggesting that diverse expression of these genes is fundamental to shaping excitability within the song system. With few exceptions, differential expression of individual genes was in the same direction across nuclei. Area X and HVC showed enhanced expression of more calcium channel genes than other song nuclei, suggesting a more prominent role of calcium currents in governing electrophysiological properties of the AFP. In contrast, chloride channel genes were mostly non-differential. Compared to other nuclei, nXIIts differentially expressed the fewest number of genes. We note that most genes that lacked brain expression evidence in zebra finch  are primarily expressed in non-neural tissues  in other organisms , 39.Some genes were strongly expressed in sparse cells and might contribute to defining the unique neuronal populations reported in Area X , 86, 87,Based on its known role in in mammals, the higher expression of SCN1A (Nav1.1) in all major song nuclei suggests an essential function of somatodendritic signal integration and action potential (AP) thresholding in the song system . SCN1A aβ subunits alter cell surface expression, gating properties, and voltage sensitivities of α subunits , and higSCN4B induces resurgent sodium current, an important adaptation for neurons that exhibit high-frequency firing , 105–109Calcium channels influence input processing in dendrites and signal transmission in synaptic terminals, which links them to plasticity mechanisms that serve learning and memory . Voltageh) channels as suggested by electrophysiology studies of HVC [T-type (Cav3) channels create low-threshold calcium potentials that are integral to driving rebound and burst firing in neurons , 127. Ths of HVC . HVC wass of HVC , 130. Res of HVC , 132. Ths of HVC . CACNA1I−/− mice are more resistant to seizure [CACNA1B, an N-type calcium channel, has a large single-channel conductance and is closely associated with the calcium-sensitive presynaptic vesicle release machinery –136. The seizure , possibl seizure . DependiThe modulatory α2δ (CACNA2D) and β (CACNB) subunits increase calcium currents as well as vesicle release probability , 142–145Most γ (CACNG) subunits are transmembrane AMPA receptor (AMPAR) regulatory proteins, or TARPs, which modulate gating and synaptic targeting of AMPARs , 148. A The high expression of multiple intracellular calcium channels suggests that mobilization of internal calcium stores is important for HVC. While the role of TPCNs in neural processing is unknown, inositol 1,2,5-trisphosphate (IP3) receptors (ITPRs) and ryanodine receptors (RYRs) are implicated in plasticity, learning, and memory . The higOf the gene families studied here, chloride channels are the least characterized and had a low incidence of differential expression. Considering their role in stabilizing the resting potential , 157, thPasseriformes appear to lack CLCA genes. In mammals, these calcium-activated proteases are expressed predominantly in intestine and airway epithelium, and only a few are expressed in brain . The absWe report here on the genomic identity and song system expression of three major ion channel families. The majority of these genes are conserved between birds and mammals, and the expression data paint a picture of abundant differential patterns, especially for sodium and calcium channel genes, in circuitry that supports the acquisition and production of learned vocalizations. We note that while changes in synaptic and intrinsic properties during the song learning period have been reported , 171, fuAdditional file 1:Table S1. Documents synteny analysis, chicken orthologs, model completeness, sequence recovery data, and EST evidence for ion channel genes in the zebra finch genome. Table S2. details evidence supporting gene loss conclusions. (XLSX 29 kb)Additional file 2:. Each box represents a step toward identifying an ortholog in zebra finch. Arrows connecting boxes indicate the most common workflows. Details of main pipeline (yellow) and all variations (Cases 1–6), including the use of zebra finch PacBio (pink) and other species (yellow), can be found in methods section. Dotted line indicates Case 4, where there is limited synteny information in taeGut1 and PacBio is required for verification (PDF 607 kb)Overview of ortholog identification pipelineAdditional file 3:Confirming orthology in the PacBio assembly using BLAST. Graphic summary of BLAST results demonstrating that SCN1B is present on gap-less PacBio scaffold MUGN01000920.1 with conserved exon structure and the conserved syntenic genes BCL3 and a fragment of RMB42  Frequency histogram of genes by zebra finch/human model length ratio. B) Frequency histogram of genes by percent of post-recovery length. (PDF 183 kb)Quantitative analysis of zebra finch model completenessAdditional file 5:. The zebra finch gene, CACNA2D2, is displayed using the UCSC Genome Browser. A zoomed-in region from within the green rectangle is shown in the bottom panel to highlight the detailed structure of alignments. This gene has three partial zebra finch Ensembl models (dark red track) in a region with numerous gaps (black Gap tack). Note the dips in sequence quality scores (light blue track) surrounding the gaps. Alignment of a more complete chicken model (magenta arrowheads) reveals additional sequence blocks that are missing from the zebra finch model, displayed in the SeqRecovery BED track highlighted in yellow. Non-zebra finch RefSeqs (dark blue tracks) provide further support for blocks of additional sequence recovered through alignments of the chicken model. (PDF 336 kb)Visualization of sequence recovery for a gene with split modelsAdditional file 6:. A taeGut1 BED track of the sequence recovered from aligning chicken models. (TXT 19 kb)Sequence recovery BED trackAdditional file 7:. Representative in situ hybridization photomicrographs of select ion channel genes in song nuclei RA, HVC, Area X, and DLM. All genes are differentially expressed except for CACNA2D1 and CACNG5 in RA, which appear to label sparse populations of cells. Area X panels show select genes with enhanced expression in sparse populations of cells. Camera lucida drawings indicating the location of these nuclei can be found in Fig. Expression of select ion channel genes in song nuclei


Chronic infection with Hepatitis B virus (HBV) is a major risk factor for the development of advanced liver disease including fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). The relative contribution of virological factors to disease progression has not been fully defined and tools aiding the deconvolution of complex patient virus profiles is an unmet clinical need. Variable viral mutant signatures develop within individual patients due to the low-fidelity replication of the viral polymerase creating ‘quasispecies’ populations. Here we present the first comprehensive survey of the diversity of HBV quasispecies through ultra-deep sequencing of the complete HBV genome across two distinct European and Asian patient populations. Seroconversion to the HBV e antigen (HBeAg) represents a critical clinical waymark in infected individuals. Using a machine learning approach, a model was developed to determine the viral variants that accurately classify HBeAg status. Serial surveys of patient quasispecies populations and advanced analytics will facilitate clinical decision support for chronic HBV infection and direct therapeutic strategies through improved patient stratification. This generates a viral ecosystem consisting of mutant swarms or ‘quasispecies’, a population of genetically distinct, but closely related viral variants5. HBV quasispecies, therefore, comprise a spectrum of viral variants with differing fitness, which allows for the rapid adaptation to selective pressures including host immune factors and antiviral agents7. Consequently, HBV variants have an impact (if positively selected to sufficient abundance) on disease pathogenesis, clinical progression, and response to therapeutic interventions11. Frequently reported estimates for infection prevalence (4%), absolute number of infected individuals (257 million), and annual mortality (887000) demonstrate the large global burden of disease attributable to HBV13. Together with hepatitis C virus (HCV), viral hepatitis represents the leading cause of hepatocellular carcinoma15.HBV is a relaxed circular, partially double-stranded DNA virus, with a unique genomic organization (four overlapping reading frames encoding 7 proteins in 3.2 kb) and replication mechanism. The low-fidelity reverse transcriptase  is associated with reduced viral replication and diminished long-term complications, for example, the risk of hepatocellular carcinoma18. Progression to seroconversion, either spontaneous or treatment-induced, may take years; contemporary studies have demonstrated differential rates of seroconversion associated with the duration and type of therapeutic regimen in addition to individual patient clinical profiles20. All virological factors associated with HbeAg seroconversion have not been elucidated and deeper knowledge of these may aid the definition of patients likely to seroconvert and direct appropriate therapeutic strategies.HBV e antigen is a secreted precore protein of Hepatitis B virus with an unclear viral function21. The plasma levels of these markers are used as proxies of viral activity in the liver; inferences about the spectrum of viral quasispecies in plasma reflecting those in hepatocytes has not been qualitatively assessed. Furthermore, HBeAg negative patients may have ongoing complex clinical profiles the virological basis of which has not been established.In addition to HBV DNA load, ALT levels, HBV surface antigen (HBsAg), the loss of plasma HBeAg and seroconversion to HBeAg represent valuable clinical waymarks of a functional curein vivo - this tolerance to infection is lost during the anti-HBe positive phase and immune-escape HBeAg-negative mutants may be selected22. Increased sequence diversity, associated with stochastic mutations, can result in changes in immune tolerance and reactivity with concomitant changes in selection pressure and evolution of the virus. The dynamic interplay and drivers of viral evolution are complex and difficult to resolve linearly; the use of advanced analytic tools may assist in the deconvolution of the prevailing virological factors that contribute to serocoversion.The progression of chronic hepatitis B is related to the prevailing viral and host immune activities. The tolerogenic activity of HBeAg facilitates the establishment of HBV infection 26, but associations between all variants in all HBV coding regions and clinical parameters has not been established. For example, the nG1896A (W28*) mutation in the precore and nA1762T/nG1764A double mutation in the basal core promoter have temporal correlations with seroconversion and have been shown to be associated with different clinical courses11. Next generation sequencing (NGS) technologies are optimal for uncovering the spectra of variants that exist within an infected individual and in the wider population for disease surveillance and healthcare planning27. Ultra-deep NGS enables the detection of viral variants at low allele frequency with much greater sensitivity and confidence. To date most studies have focused on particular regions of the HBV genome, e.g. HBs major hydrophilic region (MHR)28, core29, and reverse transcriptase (RT)30 or on whole genome sequencing9 at low coverage and, most recently, on single virions31. Here we present the first comprehensive survey of the diversity HBV quasispecies through ultra-deep sequencing of the complete HBV genome across two distinct patient populations and, in addition, explore quasispecies diversity between plasma and liver samples.Many studies have disclosed variants with statistical associations with clinical metrics in different regions of the HBV genome33 including applications where there is disease and treatment heterogeneity and for prescriptive analytics . The current paradigm for classification of HBV infected patients utilises a series of virological and biochemical factors to infer viral activity and liver damage, as well as histopathological analysis of liver biopsies for fibrosis scoring  encompasses a field of data-driven techniques for the study and construction of predictive algorithms that allow classification of factors/classes or estimation of quantitative traits from complex, multi-dimensional data (hundreds to thousands of co-variates) without 7 copies/ml).Patient samples were derived from two retrospective cohorts representing a single point in time for each patient; the first, a Western European cohort defined as Dataset A, the second, a Chinese cohort, defined as Dataset B, Table 35) and plasma were obtained from ten patients in accordance with the approved study protocol. For Dataset B the HBV infection of patients was confirmed by using commercial enzyme-linked immunosorbent assay kits  and quantified by using real-time fluorescence quantitative PCR. Patient liver biochemistry and virological metrics  at baseline was obtained from the respective electronic medical records.For Dataset A serum HBeAg was quantified in samples taken at baseline using the Elecsys® HBeAg assay . HBV infection of patients was assessed using commercial enzyme-linked immunosorbent assay kits. Paired liver biopsies  and by Guangzhou Kingmed Diagnostics . Multiplexed paired-end sequencing was performed on the Illumina MiSeq platform using the MiSeq v2 sequencing kit with 300 cycles for both sample populations. Demultiplexed FASTQ files were generated as an output. A complete description of workflow is provided in Supplementary Materials and Methods.python . All further data analysis, machine learning, phylogenetic and statistical analysis  and graphical plot generation was performed in R , The R Foundation for Statistical Computing) – a full list of software packages and methods are provided in Supplementary Materials and Methods.The sequencing analysis pipeline was implemented in https://www.ncbi.nlm.nih.gov/nuccore) with the following accessions: genotype A (AF090842); B (AB033554); C (AB033556); D (AF121240); and E (AB032431). FASTQ files were obtained from Illumina sequencing platform output for all samples. A full description of the sequencing analysis pipeline is provided in Supplementary Materials and Methods and Supplementary Fig. Nucleotide sequences for reference genotypes were obtained from NCBI  to limit effects on sample variant profile attributable to treatment methods. The input to the machine learning was a matrix of viral variant allele frequencies (0.01–0.99) and the associated HBeAg status (‘positive’ or ‘negative’) as defined by standard diagnostic tests. A random forest machine learning approach was employed to establish the variants that best classified HBeAg status. A series of test and training partitions and cross-validation steps were undertaken to optimize the model before testing against independent data excluded from model generation. Comparison of random forest models was based upon the following metrics: accuracy, balanced accuracy, sensitivity, specificity, and kappa values. For the combined dataset (Datasets A and B: n = 352 samples and n = 5533 unique variants) variants with near-zero variance were excluded leaving n = 432 variants. Full descriptions of the machine learning approach are provided in Supplementary Materials and Methods and Supplementary Fig. To investigate whether plasma samples sufficiently represent the hepatic quasispecies population paired liver biopsy and plasma samples were collected as part of Dataset A (n = 10). The frequency of HBV variants found in either the liver or plasma were compared to define systematic differences in the quasispecies populations. The majority of variants demonstrated small differences in allele frequency (1–2%), as shown in Fig. 21. Dataset B was derived exclusively from an ethnically Chinese (Han) patient cohort. After filtering Dataset B represented n = 207 patients of whom n = 170 were treatment naïve; a further n = 37 received either treatment with a nucleoside analogue or interferon therapy  represented a cohort of diverse ethnic background from Western Europe. The dataset consisted of five HBV genotypes (A-E); all samples were obtained from untreated patients. A graphical overview of Dataset A is presented in Fig. apy Fig. . Using tapy Fig. . The intper patient and per genotype was considered for Dataset A (genotypes A-E represented). In general, within a viral genotype most variants were found only once across all the patients, Fig. The average number of variants 36. Genomic variants in the reverse transcriptase/polymerase (RT/POL) gene, leading to amino acid substitutions conferring single and multi-drug resistance, were found in plasma samples of untreated patients in both datasets , paired plasma and liver samples (n = 10) variant profiles if present , and five reference genomes (genotypes A-E) for phylogenetic analysis. Genotypes B and C grouped together in the appropriate clades, Fig. Machine learning was used to define patterns of viral variant allele frequencies that demonstrated a strong association with HBeAg status and would act as robust classifiers and uncover novel virological factors. A model with a balanced accuracy of 1  was found using test data from Dataset A , Fig. Viral variant allele frequencies from Datasets A and B were combined (n = 352 samples and n = 2119 common variants). This represented a low overlap of shared variants across datasets (Jaccard index = 0.38). Random forest classification of HBeAg status utilised samples from treatment naïve patients from both cohorts with n = 100 HBeAg negative and n = 252 HBeAg positive samples. The most accurate models were achieved when variants with near-zero variance were excluded from combined dataset leaving n = 432 variants for machine learning. The best performing model had a balanced accuracy of 0.98  against test data derived from both data sets (0.9/0.1 ratio of train/test data), Fig. 27. We report the first study to undertake ultra-deep sequencing of the whole HBV genome in almost 400 patients from two distinct cohorts. Ultra-deep sequencing demonstrated that, in general, variants arising within a single patient represented unique or rare mis-sense mutations that occur at the level of detection for the platform used (>1%). A subset of variants were found exclusively or with high frequency within the distinct genotypes. Differences were evident between genotypes in terms of the heterogeneity of viral populations with genotype D samples revealing greater sequence diversity with many low frequency mutants within individuals and more variation between patients than genotype A; greater similarity was evident between genotypes B and C.Hepatitis B virus has a baseline mutation rate of 1–7 e-05 base mutations/year and this diversity is fundamental to an understanding of the associations with the course of clinical disease, mutational dynamics, evaluation of novel strains, susceptibility to therapeutic interventions and development of immunity37, to determine the correspondence of hepatic quasispecies with plasma populations in patients undergoing liver transplantation, no differences in Shannon entropy was found. Likewise, no significant differences have been found between multiple paired liver samples and plasma in HCV-infected patients38. Coffin, et al., reported location and disease phase-specific differences in variants39 although limited differences were found between liver, polymorphonuclear cells (PBMCs) and plasma over an extended period of analysis10. This corroboration of quasispecies populations in liver and plasma supports the further investigation of plasma biomarkers to evaluate the presence of disease-associated variants.The quasispecies populations of paired liver and plasma samples were analysed in a small patient cohort (n = 10). Few variants demonstrated a differential allele frequency greater than 10 percentage points between liver and plasma; we concluded that plasma samples represented an adequate proxy for inferring the diversity in the viral quasispecies population in chronically infected patients. In work by Nishijima and colleagues28, core29, and RT30, or small whole genome sequencing studies9. Using ultra-deep sequencing, with high coverage, of the complete HBV genome we employed a random forest algorithm40, an ensemble method that builds ‘forests’ of binary decision trees to improve the classification accuracy of weak predictors, to define HBeAg status in patients across genotypes and demographics. Although machine learning approaches have been applied recently for virus data43 these methods have been applied sporadically to HBV46. The analysis presented here demonstrates the utility of unbiased, data-driven approaches to reveal novel aspects of HBV biology. Harnessing the power of two cohorts ML defined patterns of viral variant allele frequencies that accurately classified HBeAg status that was not possible from a single dataset; this model contained both variants with known temporal associations with HBeAg seroconversion, confirming the validity of the approach, and novel variants that aided the discrimination of HBeAg status that would not have been discovered by traditional statistical approaches.Contemporary sequencing studies have focused on particular regions of the HBV genome, e.g. HBs MHR47 although these mutations are not present in all genotypes/sub-genotypes during seroconversion22. The ML models confirmed nG1896A as the most predictive mutation in classifying the HBeAg status of chronically infected HBV patients. Additionally, we note the strong contribution from nA1934T, nC2501T, nG1899A in HBeAg classification together with genotype-associated variants. The combined models included nC2501T, an intergenic variant found exclusively in genotype A in Dataset A at an allele frequency >0.91 in patients (n = 58) of both HBeAg classes. This variant is not associated with the six nucleotide insertion in genotype A. This highlights genotype specific discrimination in the combined model where the majority of samples were genotype B (n = 187). Similarly, the nA1934T precore/core variant was associated with genotype A, D and E samples and not found in genotype B and C samples in either dataset indicating another genotype partition in the combined model. The nA1934T mutation is reflected in a Thr12Ser amino acid substitution and is a mutation within the MHC class II restricted T-cell epitope (CD4 + Th epitope 1–20) in the core protein49 known to be associated with clinical reactivation during lamivudine treatment50 and HCC51. This suggests that these variants contribute to partitioning of genotypes with subsequent classification of HBeAg status by the frequency of nG1896A, nG1899A, nG1764A/A1764T mutations.HBe loss has a temporal correlation with elevated frequencies of a precore mutation at nG1896A and/or nG1764A/nA1764T basal core promotor mutations52. We are, however, encouraged by the performance of the general model in discriminating HBeAg status in the n = 37 patients receiving treatment from the Dataset B cohort which served as an independent test group. By including a diverse sample population for feature-selection it was possible to establish a general model for HBeAg classification with broad relevance to the clinical population. Furthermore, we characterize the incidence of resistance-associated mutants in naïve patients; previous work has shown the presence of these mutants in naïve patients53 and our work further supports the future requirement for baseline sequencing of infected individuals to tailor therapeutic regimens.This study identifies novel patterns of viral variants associated with existing HBeAg status, however, makes no inference about the mechanism by which this status was reached  or what the model means with respect to patient outcomes . Equally, we cannot comment on patient profiles that are not represented in the study population, e.g. HBeAg negative inactive carriers with low HBV DNA loads. The ML model was formed on the known HBeAg status as defined by standard diagnostic techniques, i.e. the development of an alternative diagnostic test was not the intent of this study. Although we present a classification model with high discriminative accuracy this does not translate directly to changes in clinical practice and decision support. To make such a model applicable we require prospective studies with serial sampling to capture patients in the process of seroconversion and follow treatment groups. Further, such a model requires to be calibrated to the population of interest  and be applied to a clear risk-sensitive decision point in the clinical setting (e.g. change in therapeutic regimen)54. Our study demonstrates that plasma HBV quasispecies adequately represent the viral populations within hepatocytes and that these profiles, when interrogated with machine learning approaches, can recapitulate classification of patients by clinical marker status in addition to revealing novel biology.The utility of ML approaches to clinical decision making in infectious diseases is not currently widely appreciated, however, the application of deep sequencing and ML analysis to identify data patterns could facilitate the targeting of specific therapeutic interventions to high risk groups, aid stratification of patients for more effective clinical trial design, link models to clinical decision support tools and, through incorporating patient demographic data, facilitate epidemiological and healthcare planning through a deeper understanding of the relationship between compound factorsSupplementary Materials and Methods


In Study 19, maintenance monotherapy with olaparib significantly prolonged progression-free survival vs placebo in patients with platinum-sensitive, recurrent high-grade serous ovarian cancer.Study 19 was a randomised, placebo-controlled, Phase II trial enrolling 265 patients who had received at least two platinum-based chemotherapy regimens and were in complete or partial response to their most recent regimen. Patients were randomised to olaparib  or placebo. We present long-term safety and final mature overall survival  data, from the last data cut-off (9 May 2016).P = 0.02138) irrespective of BRCA1/2 mutation status, although the predefined threshold for statistical significance was not met.Thirty-two patients (24%) received maintenance olaparib for over 2 years; 15 (11%) did so for over 6 years. No new tolerability signals were identified with long-term treatment and adverse events were generally low grade. The incidence of discontinuations due to adverse events was low (6%). An apparent OS advantage was observed with olaparib vs placebo (hazard ratio 0.73, 95% confidence interval 0.55‒0.95, BRCA1/2 mutation status and unprecedented long-term benefit with maintenance olaparib for a subset of platinum-sensitive, recurrent ovarian cancer patients.Study 19 showed a favourable final OS result irrespective of  Cumulative toxicities of chemotherapy and the emergence of drug resistance limit delivery and potential benefit of further treatment. Furthermore, the duration of benefit associated with ‘salvage’ chemotherapy for progression decreases with each subsequent line of treatment.9 There remains an unmet need for effective and well-tolerated long-term maintenance treatment options for patients with recurrent ovarian cancer to maintain quality of life and delay the need for further chemotherapy, particularly after response.Ovarian cancer is the most common cause of gynaecological cancer-related deaths and fifth leading cause of death from cancer in women.BRCA1/2 mutation (BRCAm) status.11Olaparib (Lynparza™), a poly(adenosine diphosphate–ribose) polymerase (PARP) inhibitor, is approved (tablet formulation) for treatment in the maintenance setting for patients with platinum-sensitive relapsed ovarian cancer, irrespective of P < 0.0001).5 The PFS prolongation was durable, with 32 of 136 patients (24%) who received olaparib being progression-free for >2 years.12 A pre-planned, retrospective analysis of patients in Study 19 demonstrated that BRCAm patients derived the greatest clinical benefit from olaparib ; however, a PFS advantage was also seen for BRCA wild-type (BRCAwt) patients .13 More recently, PFS data from the Phase III SOLO2 trial of olaparib tablets as maintenance monotherapy in patients with platinum-sensitive, relapsed ovarian cancer and a BRCAm confirmed a significant benefit for olaparib-treated patients compared with those who received placebo .6 Although the initial analysis of OS in Study 19 (data maturity: 38%) did not detect a treatment effect, previously reported updated data from an interim analysis  suggested an advantage in OS for patients receiving olaparib vs placebo .14We have previously reported data from Study 19 (NCT00753545), a Phase II trial that assessed the efficacy and safety of olaparib maintenance monotherapy in platinum-sensitive, recurrent high-grade serous ovarian cancer patients, and showed a significant improvement in PFS with maintenance olaparib vs placebo (hazard ratio (HR) 0.35, 95% confidence interval (CI) 0.25–0.49; 18 Furthermore, there was no detriment to health-related quality of life (HRQoL) while on treatment in either Study 19 or SOLO2.20 Given its clinical profile, long-term administration of olaparib as maintenance monotherapy in ovarian cancer patients is feasible and an attractive option. Here, we report the final protocol-defined OS analysis from Study 19, characterise those patients who have derived long-term benefit from olaparib and, in particular, assess the long-term safety and tolerability of olaparib treatment.Safety data from clinical trials in recurrent ovarian cancer patients have shown that olaparib (capsule) monotherapy is generally well tolerated, and data from SOLO2 suggest the new tablet formulation of olaparib has a similar tolerability profile.5 Known BRCAm status was not required but was established retrospectively by germline or tumour testing for patients with appropriate samples available who provided consent . Eligible patients were at least 18 years old, with recurrent ovarian, fallopian tube or primary peritoneal cancer with high-grade serous histology and were platinum sensitive. Patients had received two or more prior courses of platinum-based chemotherapy and were in complete or partial response to their most recent regimen  was administered orally and treatment continued until disease progression, if toxicities were manageable  were also conducted. Safety and tolerability were assessed throughout the study by recording adverse events , physical examination results, vital signs and laboratory findings.Tumour assessments were conducted every 12 weeks until week 60, and every 24 weeks thereafter, until objective disease progression or withdrawal of consent. Response Evaluation Criteria in Solid Tumours (RECIST) data were not collected after the primary DCO (30 June 2010). The primary endpoint of the study was PFS, for which data have been reported previously.α = 0.95%) was protocol defined and the analysis set for OS included all patients randomised to treatment. The results should be regarded as descriptive and P-values as nominal. OS, TFST and TSST were analysed using an adjusted Cox proportional hazards model , including 97 for whom BRCAm status was known at study entry; 136 patients were classified as BRCAm (74 olaparib vs 62 placebo). At the final DCO (9 May 2016), 14 olaparib-arm patients (10%) and 1 placebo-arm patient (1%) were continuing treatment.Of 265 patients randomised in Study 19, 136 were to olaparib and 129 to placebo . A separation in favour of olaparib treatment was seen in the Kaplan–Meier (KM) curves for the overall study population, BRCAm and BRCAwt subgroups as duration of follow-up increased , an exploratory restricted means OS analysis indicated a difference in survival of 6.1 months between treatment arms .The final DCO corresponded to 79% OS data maturity (210 deaths from 265 patients), with a median follow-up of 78.0 months. An apparent advantage in OS for patients randomised to olaparib maintenance monotherapy vs placebo in the total study population  in the placebo group subsequently received PARP inhibitors via other studies. We therefore conducted an exploratory post-hoc OS analysis of 103 olaparib-arm and 92 placebo-arm patients by excluding patients from sites where at least one patient had received subsequent PARP inhibitor treatment. At the final DCO, this resulted in an adjusted OS HR of 0.68  and TSST  were performed at the final DCO , 15 of 136 patients (11%) had received maintenance olaparib monotherapy for 6 years or more; eight of these had a Twenty-two of 32 olaparib-arm patients (69%) and all five placebo-arm patients who were on treatment for at least 2 years were receiving the full treatment dose (400 mg bid) immediately prior to discontinuation or the end of follow-up. Eight olaparib-arm patients (25%) were at a reduced dose of 200 mg bid, six of whom had their dose reduced prior to 2 years, with the remaining two (6%) receiving 100 mg bid, one of whom had their dose reduced prior to 2 years.The main reason for discontinuation of study treatment was disease progression  compliance with study treatment (assessed using capsule counts) was 96.9% (8.9) in the olaparib arm and 99.0% (3.4) in the placebo arm.BRCAm patients were similar to those in the overall population , fatigue/asthenia 63%), vomiting (35%) and diarrhoea (27%), consistent with those previously reported. No new safety findings were observed in the overall study population and safety findings for %, vomitiSince the last DCO (30 September 2015), three new SAEs were reported in two patients in the olaparib group .A smaller proportion of olaparib-arm patients reported AEs late in treatment (75%) compared with the full study duration (97%), although the prevalence of pruritus, urinary tract infection, dyspnoea and pain in extremity was increased Table . There wBRCAm, experienced AEs that resulted in death. One of these deaths had occurred at the time of the last DCO; this patient died due to AEs of haemorrhagic stroke and thrombocytopenia, deemed to be treatment related.14 The other death was due to previously reported AML; this AE occurred 14 days after discontinuation of study treatment and the patient, who had received olaparib for over 4 years, died approximately 1 year and 9 months later. This patient previously was reported to have a SAE of pancytopenia while on study treatment. This was the only case of AML reported in Study 19; two patients were reported with MDS, as described previously14 . In the investigators’ opinion, the vast majority of patients in both treatment groups died because of progression of their ovarian cancer (188 patients). Since the study began, two patients, both in the olaparib arm and both with a BRCAm see Supplementary 14 Overall, AEs leading to discontinuation of study treatment were reported for eight (6%) olaparib-arm and two (2%) placebo-arm patients  had dose interruptions (47 [35%] for AEs) and 59 (43%) had dose reductions (35 [26%] for AEs). In the placebo arm, 23 patients (18%) had dose interruptions (13 [10%] for AEs) and 29 patients (23%) had dose reductions . Prevalence plots for the common AEs show the proportion of patients at risk (receiving treatment or within their 30 day follow-up period) who experienced each AE during a specific month. This is irrespective of AE start date, so AEs with a long duration are represented over multiple months; note that when there are only a few patients on treatment the proportion experiencing an AE can appear to be high .Patients who experienced common AEs typically did so for the first time within a few months of the start of treatment and it was rare for common AEs to initially develop after >6 months on olaparib  in patients with platinum-sensitive, recurrent high-grade serous ovarian cancer in response to their most recent platinum-based chemotherapy regimen, representing the longest follow-up from any PARP inhibitor trial reported. Our findings suggest an OS advantage for patients receiving maintenance olaparib vs placebo, an effect not yet observed for any chemotherapy regimen or other maintenance therapies in the recurrent ovarian cancer setting.13 This is supported by an exploratory restricted means OS analysis, carried out to assess average life-expectancy over the whole survival curve, taking into account differences in hazard ratio over time. Although consistent with previously reported data showing that BRCAm patients had the greatest PFS benefit from olaparib,13 these long-term OS results indicate that a subset of patients within the BRCAwt group also experience durable long-term benefit from maintenance olaparib monotherapy. It is likely that these patients have homologous recombination repair (HRR) deficiencies (germline or somatic) resulting in synthetic lethality with exposure to olaparib. Approximately 50% of patients with high-grade serous ovarian cancer are thought to have a deficiency in HRR, opening up maintenance therapy for a significant number of women with recurrent ovarian cancer.25 The challenge is to identify which BRCAwt patients are most likely to benefit from a PARP inhibitor. It has been recently reported that a positive homologous recombination deficiency (HRD) score was associated with long-term response ( > 2 years) to olaparib.12 Additionally, a biomarker analysis of patients in Study 19 who remained on treatment for ≥ 6 years has shown at least 5 of 15 patients were classified as BRCAwt. This BRCAwt group included patients with other HRR pathway gene mutations or a positive HRD score, as well as a patient with no positive candidate predictive biomarker test results.23 Other baseline characteristics were broadly similar between the full study population and those on long-term treatment, although an increased proportion of patients on olaparib for at least 2 years had undergone secondary debulking surgery shortly prior to randomisation in Study 19. The long-term survivors in this study represent a highly selected subpopulation. Although it is likely that factors such as patient heterogeneity and the number of subsequent lines of treatment following progression may have contributed to the OS of patients, it should be noted that in this randomised trial, 15 (11%) patients were on olaparib for > 6 years, compared with only one (0.8%) placebo recipient. It remains to be established why these patients achieved such a durable response to olaparib.The apparent OS benefit seen with olaparib appears to be driven by long-term responders, with a separation in KM survival curves between treatment arms seen after approximately 3 years.26 This approach, excluding sites, was preferred to a similar method excluding just crossover patients as it was felt to lessen the potential confounding of removing only well-performing placebo patients and better retain the benefits of randomisation. As PARP inhibitors become more commonly utilised and widely available, the feasibility of showing a statistically significant OS benefit is decreasing; no future trials will be able to have as little crossover as Study 19 and, given the results of this and other maintenance studies with PARP inhibitors, placebo-controlled trials would no longer be ethical in this population.Following progression, OS may be influenced by subsequent therapy with platinum agents, bevacizumab and/or PARP inhibitors. Crossover was not permitted in our study; however, 13% of placebo-arm patients subsequently received a PARP inhibitor via other clinical trials. This could have caused confounding of the OS data and, consistent with this, we show an improved OS HR for olaparib vs placebo when excluding patients from sites where at least one patient received subsequent treatment with a PARP inhibitor, confirming similar findings from an earlier interim analysis.BRCAm and BRCAwt subgroups, consistent with previous analyses.14 A clear separation between the olaparib and placebo KM survival curves can be seen extending past 6 years of follow-up. Since no tumour assessments were carried out in Study 19 after the primary DCO (30 June 2010), TFST represents a reasonable long-term surrogate for PFS, which was analysed with a median of 5.6 months’ follow-up. TFST and TSST data are consistent with the observed advantage in PFS and OS for olaparib-arm patients and show a clinically meaningful increase in time between chemotherapy regimens, while also suggesting an extended efficacy benefit with olaparib persisting beyond the next line of therapy.27Exploratory data for TFST and TSST reported here show significant benefits with maintenance olaparib monotherapy compared with placebo for the overall population and for the BRCAm and BRCAwt subgroups, 10% of patients experienced a durable benefit from olaparib maintenance monotherapy for over 6 years. Almost 25% of patients received olaparib for at least 2 years (compared with 4% of placebo-arm patients), considerably longer than the expected median PFS of 4–6 months in this population.7 A higher proportion of patients who received olaparib long term had at least three prior lines of chemotherapy compared with those who received placebo. The rate of discontinuation of olaparib for any reason decreased after 2 years, suggesting patients who reach this milestone are likely to receive continued benefit from maintenance olaparib. It should be noted that patients continued treatment long-term despite taking 16 large capsules per day; a more patient-friendly formulation of olaparib, requiring only 4 tablets per day, is now available.11The long-term exposure seen in Study 19 is unprecedented for a PARP inhibitor; consistently across No new safety signals were identified since the previous analysis and data reported here indicate that the tolerability profile of olaparib is compatible with long-term treatment. Very few patients discontinued treatment due to AEs in Study 19; 6 and 2% in the olaparib and placebo arms, respectively. The majority of patients receiving olaparib long term were on full dose immediately prior to the end of treatment, which suggests enduring dose modifications are not required to maintain long-term tolerability.6 The MDS, AML and new primary malignancy AEs that occurred in Study 19 were unrelated to duration of exposure to olaparib.AEs of MDS and AML were rare in Study 19 and were reported in both treatment arms. The incidence in Study 19 was broadly consistent with that seen in SOLO2.18 the most common AEs in Study 19 were low-grade nausea, fatigue/asthenia and vomiting, with anaemia the most common haematological AE. These AEs usually occurred early and rarely first developed after >6 months on olaparib or placebo. Only one patient discontinued olaparib due to one of these common AEs (nausea). Prevalence data show that the proportion of patients who experienced vomiting or anaemia during any specific month was low. More patients at a given time experienced nausea and fatigue/asthenia, however, there was no increase in prevalence of these AEs with long-term treatment. Due to the increased length of time that patients spent on olaparib compared with placebo, it is expected that more AEs will be observed in the olaparib arm. Exposure-adjusted AE data . The generally low-grade, non-cumulative nature of the common AEs observed with olaparib in Study 19 supports the approach that they can be routinely managed by physicians through dose modifications when required and symptomatic treatment with standard procedures, such as antiemetics or occasional blood transfusions, although regular haematological monitoring is recommended for patients receiving olaparib.28These safety data should be considered in the context of toxicity profiles associated with palliative chemotherapy, which is commonly prescribed to patients at disease progression. There are many potential adverse effects (AEs) associated with chemotherapy, which can be cumulative, limiting the length of time patients can remain on treatment.19 makes olaparib a viable maintenance treatment option following response to platinum-based chemotherapy, which should be discussed with patients. Maintenance therapy with olaparib capsules prolonged PFS in Study 19,5 and results from the ongoing Phase III SOLO2 trial of maintenance therapy with the tablet formulation of olaparib also showed a significant increase in median PFS for BRCAm patients, compared with placebo.6 Long-term follow-up from SOLO2 and other ongoing trials will help further elucidate the efficacy and tolerability profiles of olaparib maintenance monotherapy and ultimately identify those patients with ovarian cancer most likely to receive long-term benefit from maintenance therapy with olaparib.Various factors should be considered when weighing the value of long-term maintenance therapy with a PARP inhibitor against platinum-based chemotherapy alone followed by further lines of treatment for recurrent disease at symptomatic progression. Maintenance olaparib may not be effective in all patients, and is often associated with low-grade intermittent toxicity that can usually be controlled with simple supportive measures. However, its efficacy and generally favourable long-term tolerability profile, coupled with previously reported data showing no detrimental impact on patients’ HRQoL during treatment,29 and patient preference data suggest that women with recurrent ovarian cancer would accept a shorter PFS in order to avoid severe adverse effects associated with chemotherapy .30 A delay in the time to subsequent chemotherapy is also likely to be of importance to patients, with TFST and TSST significantly prolonged with olaparib vs placebo in both Study 19 and SOLO2.6In addition, patient preference data showing the trade-off that women with recurrent ovarian cancer who are receiving maintenance PARP inhibitor therapy are willing to make in terms of gains in PFS and OS vs avoiding toxicity are also needed. Survey results indicate that most women with recurrent ovarian cancer required an increase in PFS of at least 5 months to make treatment worthwhile,BRCAm status, despite considerable crossover. This finding is supported by significant improvements in PFS, TFST and TSST with maintenance olaparib compared with placebo in BRCAm or BRCAwt women. Almost 25% of patients in Study 19 received olaparib for at least 2 years and over 10% continued on treatment for 6 years or more, demonstrating a prolonged, clinically meaningful benefit derived from olaparib maintenance therapy, which is unprecedented in patients with recurrent ovarian cancer. The capsule formulation of olaparib was well tolerated in Study 19; the majority of AEs in both the overall population and BRCAm subgroup were low grade and manageable with dose modifications or simple supportive treatments and no new tolerability signals were identified with long-term treatment. Taken together, these data support the use of olaparib maintenance monotherapy as long-term treatment for patients with platinum-sensitive, recurrent high-grade serous ovarian cancer with or without a BRCAm.The final analysis from Study 19 is the largest long-term survival follow-up data for a PARP inhibitor and suggests an OS advantage with olaparib maintenance monotherapy for patients with platinum-sensitive, recurrent high-grade serous ovarian cancer. Although the threshold for statistical significance was not met, a numerical OS advantage was seen, irrespective of Supplemental_revised clean


AUX1/LAX genes result in auxin related developmental defects and have been implicated in regulating key plant processes including root and lateral root development, root gravitropism, root hair development, vascular patterning, seed germination, apical hook formation, leaf morphogenesis, phyllotactic patterning, female gametophyte development and embryo development. Recently AUX1 has also been implicated in regulating plant responses to abiotic stresses. This review summarizes our current understanding of the developmental roles of AUX1/LAX gene family and will also briefly discuss the modelling approaches that are providing new insight into the role of auxin transport in plant development.Plant hormone auxin regulates several aspects of plant growth and development. Auxin is predominantly synthesized in the shoot apex and developing leaf primordia and from there it is transported to the target tissues e.g. roots. Auxin transport is polar in nature and is carrier-mediated. AUXIN1/LIKE-AUX1 (AUX1/LAX) family members are the major auxin influx carriers whereas PIN-FORMED (PIN) family and some members of the P-GLYCOPROTEIN/ATP-BINDING CASSETTE B4 (PGP/ABCB) family are major auxin efflux carriers. AUX1/LAX auxin influx carriers are multi-membrane spanning transmembrane proteins sharing similarity to amino acid permeases. Mutations in  Auxin is a key plant hormone that regulates several aspects of plant growth and development including plant tropic responses, embryo development, root development, shoot development, leaf development and phylotactic patterning and there are several good reviews highlighting the role of auxin in plant development .It is known for a long time that auxin transport is polar in nature. Following its synthesis in the leaf primordia and shoot apical meristem, auxin is transported downwards to its target tissues using either through the bulk flow in the phloem or through the polar auxin transport stream  gene family that also appears to be regulating auxin homeostasis. In addition, it has been suggested that nitrate transporter NRT1.1 can also be involved in auxin transport , gravity signal transmission  and gravity response (epidermis)  . Using a35S:CKX lines over expressing cytokinin degradation enzyme cytokinin dehydrogenase/oxidase show delayed root gravitropism. Interestingly, treatment with exogenous cytokinins does not affect root gravitropic response and so the authors suggest that proper concentration of endogenous cytokinins is crucial for gravity induced auxin redistribution. They also show that AUX1-YFP signal is reduced in these cytokinin depleted lines but the relocalization kinetics of PIN3 and PIN7 was comparable to wild type. It will be interesting to check if treatment with exogenous cytokinins can restore AUX1 signal in the cytokinin depleted lines to further understand the role of cytokinin in regulating auxin influx and root gravitropism.Recently, lax2 mutants have higher propensity of discontinuity in vascular strands in the cotyledon  and imbibed seeds have increased expression of auxin influx and efflux carriers compared to dormant seeds (Plant hormones such as Gibberellic Acid (GA) and Abscisic Acid (ABA) are known to regulate seed germination . GA promnt seeds .SNL1 (SWI-INDEPENDENT3 (SIN3)-LIKE1) and SNL2 result in increased speed of seed germination and faster radicle protrusion. They also showed that expression of several auxin synthesis, transport and responses genes were up regulated in snl1snl2 double mutants and these mutants accumulated significantly more IAA than wild type plants. This led snl1snl2 double mutant, many of their mutants had no obvious seed germination defects. However, they did find that aux1 mutants had weakly decreased germination of fresh seeds compared to controls. Further investigation showed that AUX1 levels were higher in the radicle tip of the snl1snl2 double mutants and AUX1 was a target for SNL mediated H3K9/18 histone deacetylation. Moreover, they also showed that the cell cycle genes CYCD1;1 and CYCD4;1 were upregulated in snl1snl2 double mutants, which is also in agreement with increased cell cycle activity in these mutants. In addition, auxin also can induce the expression of CYCD1;1 and CYCD4;1. Taken together, they showed that SNLs negatively regulate radicle protrusion and AUX1 plays a crucial role in SNL mediated seed germination. They further proposed a model, where during embryo development and seed maturation high SNL levels cause deacetylation of ABA hydrolysis genes causing high ABA levels inhibiting seed germination. During imbibition, the SNL levels go down that promotes expression of auxin synthesis and transport genes including AUX1. Increased auxin levels, in turn, switch on cell cycle genes that promote radicle growth.It is emerging now that histone modification may play a crucial role in regulating seed germination by regulating the expression of several auxin related genes. Recently, Apical hook protects the shoot apical meristem during seedling emergence from the soil . Light aArabidopsis  of hypocotyl. Among the auxin influx carriers, AUX1 and LAX3 have been implicated in regulating apical hook development in bidopsis . Vandenbembranes . In contechidna mutants, echidna mutants have defects in apical hook formation. They also have defects in differential auxin gradient formation in the apical hook. echidna mutants AUX1 trafficking to the plasma membrane is disrupted. In contrast, LAX3 or PIN3 trafficking is only marginally altered in echidna mutants. These findings suggest that not only there are distinct mechanisms for the trafficking of auxin influx and efflux carrier proteins to the plasma membrane, even the closely related AUX1 and LAX3 follow distinct trafficking pathways from the trans-Golgi network (TGN) to the plasma membrane in the hypocotyl.Though both AUX1 and LAX3   . GuanineARF-GEF) . In Arabarf1 and big mutants have defects in AUX1 trafficking and show hook developmental defects similar to aux1 mutants. In echidna mutants, ARF1 and BIG cannot localize to the TGN whereas in arf1 and big mutants, ECHIDNA localization is perturbed. They proposed that ECHIDNA and BIG facilitate recruitment of ARF1 to the TGN that then facilitates vesicle formation for AUX1 delivery to the plasma membrane, which is a prerequisite for the ethylene mediated hook development.Recently, During root development, cells arise linearly from a group of stem cells surrounding the quiescent center (QC). Stem cells divide asymmetrically to give rise to daughter cells that move away from the stem cell niche and differentiate . Auxin ivia an auxin–cytokinin module. In Arabidopsis, cytokinin signaling is mediated by a multi-step phosphorelay mechanism involving cytokinin receptors AHKs (Arabidopsis Histidine Kinases) AHPs (Arabidopsis histidine phosphotransfer proteins) and ARRs (Arabidopsis response regulators). Upon cytokinins perception, AHKs relay the signal to ARRs via AHPs. ARRs act as transcriptional regulators to regulate gene expression  turned out to have mutations in the AUX1 gene (Pro371Leu and Gly374Ser) in the extracellular loop between 9th and 10th transmembrane regions suggesting the role of AUX1 in cytokinin mediated root development. Indeed, aux1 mutants showed reduced root growth inhibition to cytokinin treatment but not lax1, lax2 or lax3. They further observed that the expression of the type-B response regulator ARR10 is auxin and AUX1 dependent and led them to propose that cytokinin and auxin regulate expression of ARR10 and AUX1 as part of an auto regulatory circuit.Cytokinins inhibit cell elongation and cell proliferation. via AUX1 in an ethylene dependent pathway.AUX1 has also been implicated in ABA mediated inhibition of root growth. ABA is known to inhibit root growth at high concentration and recently, AUX1 result in about 50% reduction in lateral root numbers  that accumulates in the lateral root primordia in phytochrome dependent fashion and regulates the plasma membrane abundance of LAX3 and PIN3 to reduce auxin levels in the overlaying cortex cells to reduce lateral root outgrowth.Light has been known to regulate root system architecture, but the mechanism has not been well understood. Recently, Adventitious roots are post embryonic roots and auxin has been known to regulate their formation . RecentlAuxin plays a critical role in root hair development . Auxin tRoot hair cell polarity has also been shown to be regulated by auxin . Root haRecently, Auxin mediated root hair elongation is a key adaptive response to low P . RecentlPIN based auxin efflux has been previously shown to define regions of fast and slow growing areas in leaf margins regulated by CUC (CUP-SHAPED COTYLEDON) transcription factors, that are known to regulate organ boundaries in plants . RecentlMore recently, Female gametophyte (megagametophyte) development begins with mega spore mother cell undergoing meiotic division and giving rise to four haploid cells. One of the haploid cell becomes a functional megaspore and undergoes three rounds of mitosis to produce seven-celled eight-nucleate highly polarized megagametophyte comprised of two synergid cells, one egg cell, one central cell and three antipodal cells. Auxin efflux carrier PIN1 was previously shown to play a role in regulating female gametophyte development . RecentlMONOPTEROS/AUXIN RESPONSE FACTOR5 result in severe embryonic defects  and maize (ZmAUX1) are involved in inflorescence development and root gravitropism. Inflorescence architecture is an important agronomic trait as it influences grain yield. Using a forward genetic approach, S. viridis, mutations in SvAUX1, result in major defects in inflorescence branch development leading to sparse panicle (spp) phenotypes. These mutants (spp1-1 and spp1-3) also show decreased plant height, reduced inflorescence branching and spikelet numbers and increased panicle length compared to the control plants.Recently, ZmPIN1 has been also implicated in maize inflorescence development  stress response. Cd stress induces the production of reactive oxygen species, which trigger cell death in plants. Auxin signaling is known to be involved in activating Cd-induced morphogenic defense responses in wheat, barley and Arabidopsis (OsAUX1/LAX genes (OsAUX1-5) are induced by Cd stress. Reporter analysis showed that OsAUX1 is distinctly induced under Cd stress in primary roots, lateral roots and root hairs and osaux1 mutants are more sensitive to Cd stress. Cd contents in the osaux1 mutant were not altered, but reactive oxygen species-mediated damage was enhanced, further increasing the sensitivity of the mutant to Cd stress. Taken together, their results indicated that OsAUX1 plays an important role in mediating plant responses to Cd stress. Arabidopsis, OsAUX1 is specifically expressed in root hair cell files, suggesting functional differences between monocots and dicots in regulating RH development.bidopsis . Yu et aMore recently, AtAUX1, BdAUX1 is expressed in roots displaying expression patterns in root protophloem, epidermis and columella. Additionally, authors observed that unlike AtAUX1, BdAUX1 is also expressed throughout the stele and in the outer cortex layers, encompassing the combined expression domains of AtAUX1, AtLAX2, and AtLAX3. Thus, possibly BdAUX1 have the combined functions in these tissues. Bdaux1 mutant roots are agravitropic and show longer root phenotypes due to increased mature cell lengths possibly due to higher free auxin content. Additionally, Bdaux1 mutants are also significantly thinner due to reduced cell file numbers in every tissue except xylem and phloem and due to smaller cell sizes radially. Bdaux1 mutants also show reduced root hair length and density. Unlike AtAUX1, Bdaux1 loss‐of‐function mutants are dwarfs with aberrant flower development, and consequently infertile suggesting a more crucial role for BdAUX1 in flower development.Recently, Casuarina glauca is facilitated by CgAUX1. More recently, Medicago is mediated by MtLAX2. They showed that MtLAX2 is auxin inducible and is expressed in the nodule primordia, vasculature of developing nodules and at the apex of matured nodules. Upon Rhizobium infection, mtlax2 mutants have fewer nodules and reduced DR5 activity at the infection sites clearly implicating the role of MtLAX2 in nodule development.MtLAX2 results in defects in root gravitropism, fewer lateral roots and shorter root hairs suggesting this to be a functional analog of AtAUX1 (MtLAX2 cannot rescue root gravitropic defects of Arabidopsis aux1 mutant when expressed under AtAUX1 promoter. This led In addition, f AtAUX1 . InteresArabidopsis and how auxin fluxes are established and maintained (Computer simulations and modelling approaches in the past decade have proven very useful in getting better understanding of the role of auxin transport in regulating auxin mediated developmental processes in intained . Increasintained . AuthorsMore recently, In the past two decades, there has been a significant increase in our understanding of molecular basis of auxin transport and roles of auxin transporters in plant development. Particularity, there has been a better understanding of auxin influx carriers and how they play crucial roles in almost all aspects of plant growth and development. More advanced computer models and high-resolution imaging and segmentation approaches have proved crucial in providing better understanding of auxin influx carriers in pattern formation especially how changes at the cellular scale affect organ-scale auxin patterns.via AUX1 but early events are not well understood as to how low P status is sensed by the plants. Further understanding of the early events will be crucial for our understanding of the root growth and development in changing environment and may help develop predictive models for future crop improvement programmes.Root development is very plastic and respond to their environment. Recently it has been shown that chromium inhibits primary root growth by regulating cell cycle genes. Chromium toxicity can cause major damage to crop yield. Genetic and physiological studies show a role for AUX1 in chromium mediated inhibition of root growth . SimilarAlternative splicing is another key area that has not been explored much in the plants but may be crucial for better understanding of the role of alternatively spliced transcripts in regulating and shaping plant development. It appears that alternative splicing in plants is more common than previously appreciated . With thRS and RB did the review of literature, prepared figures and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.