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Most head and neck squamous cell carcinoma (HNSCC) patients present with late-stage cancers, which are difficult to treat. Therefore, early diagnosis of high-risk premalignant lesions and incipient cancers is important. HNSCC is currently perceived as a single progression mechanism, resulting in immortal invasive cancers. However, we have found that ∼40% of primary oral SCCs are mortal in culture, and these have a better prognosis. About 60% of oral premalignancies (dysplasias) are also mortal. The mortal and immortal tumors are generated in vivo as judged by p53 mutations and loss of p16INK4A expression being found only in the original tumors from which the immortal cultures were derived. To investigate the relationships of dysplasias to SCCs, we did microarray analysis of primary cultures of 4 normal oral mucosa biopsies, 19 dysplasias, and 16 SCCs. Spectral clustering using the singular value decomposition and other bioinformatic techniques showed that development of mortal and immortal SCCs involves distinct transcriptional changes. Both SCC classes share most of the transcriptional changes found in their respective dysplasias but have additional changes. Moreover, high-risk dysplasias that subsequently progress to SCCs more closely resemble SCCs than nonprogressing dysplasias. This indicates for the first time that there are divergent mortal and immortal pathways for oral SCC development via intermediate dysplasias. We believe that this new information may lead to new ways of classifying HNSCC in relation to prognosis. (Cancer Res 2006; 66(15): 7405-13)

Supplementary Figure S1, Tables S1-S5 from Divergent Routes to Oral Cancer

Many dementias are propagated through the spread of “prion-like” misfolded proteins. This includes prion diseases themselves (such as Creutzfeldt-Jakob disease) and Alzheimer’s disease (AD), for which no treatments are available to slow or stop progression. The M 1 acetylcholine muscarinic receptor (M 1 receptor) is abundant in the brain, and its activity promotes cognitive function in preclinical models and in patients with AD. Here, we investigated whether activation of the M 1 receptor might slow the progression of neurodegeneration associated with prion-like misfolded protein in a mouse model of prion disease. Proteomic and transcriptomic analysis of the hippocampus revealed that this model had a molecular profile that was similar to that of human neurodegenerative diseases, including AD. Chronic enhancement of the activity of the M 1 receptor with the positive allosteric modulator (PAM) VU0486846 reduced the abundance of prion-induced molecular markers of neuroinflammation and mitochondrial dysregulation in the hippocampus and normalized the abundance of those associated with neurotransmission, including synaptic and postsynaptic signaling components. PAM treatment of prion-infected mice prolonged survival and maintained cognitive function. Thus, allosteric activation of M 1 receptors may reduce the severity of neurodegenerative diseases caused by the prion-like propagation of misfolded protein.

Many dementias are propagated through the spread of "prion-like" misfolded proteins. This includes prion diseases themselves (such as Creutzfeldt-Jakob disease) and Alzheimer's disease (AD), for which no treatments are available to slow or stop progression. The M

Introduction XYdisorders of sex development (DSD) result from variants in many different human genes but frequently have no detectable molecular cause. In approximately 25% of cases of XY DSD, the index case may have associated malformations. Genetic disorders of endoplasmic reticulum (ER) function are increasingly being recognised but have not been associated with DSD or pituitary disorders. Clinical case Three siblings (with unaffected non-consanguineous parents) were reviewed at the tertiary endocrine clinic. Child I was noted at birth to have cliteromegaly. Imaging and examination under anaesthetic revealed a normal vagina and uterus but gonads of indeterminate origin. She was 46,XY and basal endocrine investigations at the age of 4 years showed a low AMH for male but otherwise normal gonadal and thyroid function and normal IGF-1. She had a laparoscopic bilateral gonadectomy aged 5 years. Pathology demonstrated bilateral testicular tissue, with substantial fibrotic atrophic change and occasional placental alkaline phosphatase (PLAP) positive cells, suggestive of germ cell tumours. Aged 8 years she developed obesity and later hypertension. Child II was reviewed due to short stature and diagnosed with GH deficiency aged 2 years. She has normal adrenal and thyroid function and gonadotrophins. MRI demonstrated an ectopic posterior pituitary. Child III presented with perineal hypospadias, a small phallus, bilateral undescended testes and craniofacial abnormalities. Endocrine investigations revealed hypogonadotrophic hypogonadism, with no testosterone response to hCG stimulation, a low normal AMH and no response of LH or FSH on LHRH stimulation. He has panhypopituitarism with an ectopic posterior pituitary gland on MRI and is currently on treatment with GH, hydrocortisone and levothyroxine. His BP is on the 98th centile for age and height. Child I and Child III have mild developmental delay but are in mainstream school with additional educational support. High-throughput DNA sequencing revealed, in all three siblings, a heterozygous truncating variant in the SEC31A gene that encodes a component of the COPII-complex that coats the vesicles mediating ER to Golgi transport. CRISPR-Cas9 targeted knockout of the corresponding Sec31a region resulted in embryonic lethality in homozygous mice. mRNA phenotyping of ER-related genes demonstrated increased mRNA expression of ATF4 and CHOP in the affected children, genes encoding key ER stress-related proteins, associated with defective protein transport. Conclusions Dysregulation ofanterograde and retrograde COPII-coated-vesicle ER-Golgi transport is increasingly recognised to underlie human developmental disorders, including Craniolenticulosutural dysplasia (OMIM 607812) and Saul-Wilson syndrome (OMIM 618150). The de novo SEC31A nonsense variant in all three affected siblings, the ER stress response, plus reported developmental syndromes with dysfunction of this transport mechanism and evidence from the preclinical mouse model suggest that SEC31A might underlie a previously unrecognised clinical syndrome comprising DSD, endocrine abnormalities, dysmorphic features and developmental delay. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Transport of water, ions and signals from roots to leaves via the xylem vessels is essential for plant life and needs to be tightly regulated. The final composition of the transpiration stream before passage into the shoots is controlled by the xylem-adjacent cell layers, namely xylem parenchyma and pericycle, in the upper part of the root. To unravel regulatory networks in this strategically important location, we generated Arabidopsis lines expressing a nuclear tag under the control of the HKT1 promoter. HKT1 retrieves sodium from the xylem to prevent toxic levels in the shoot, and this function depends on its specific expression in upper root xylem-adjacent tissues. Based on FACS RNA-sequencing and INTACT ChIP-sequencing, we identified the gene repertoire that is preferentially expressed in the tagged cell types and discovered transcription factors experiencing cell-type specific loss of H3K27me3 demethylation. For one of these, ZAT6, we show that H3K27me3-demethylase REF6 is required for de-repression. Analysis of zat6 mutants revealed that ZAT6 activates a suite of cell-type specific downstream genes and restricts Na+ accumulation in the shoots. The combined Files open novel opportunities for ‘bottom-up’ causal dissection of cell-type specific regulatory networks that control root-to-shoot communication under environmental challenge.

The Malpighian (renal) tubule is capable of transporting fluid at remarkable rates. This review will focus on recent insights into the mechanisms by which these high rates are achieved and controlled, with particular reference to the tubules of Drosophila melanogaster, in which the combination of physiology and genetics has led to particularly rapid progress. Like many vertebrate epithelia, the Drosophila tubule has specialized cell types, with active cation transport confined to a large, metabolically active principal cell; whereas the smaller intercalated stellate cell controls chloride and water shunts to achieve net fluid secretion. Recently, the genes underlying many of these processes have been identified, functionally validated and localized within the tubule. The imminent arrival of new types of post-genomic data (notably single cell sequencing) will herald an exciting era of new discovery.

The most prevalent types of dementias, including Alzheimer’s disease, are those that are propagated via the spread of “prion-like” misfolded proteins. Despite considerable effort no treatments are available to slow or stop the progression of these dementias. Here we investigate the possibility that activation of the M1-muscarinic receptor (M1-receptor), which is highly expressed in the brain and that shows pro-cognitive properties, might present a novel disease modifying target. We demonstrate that the progression of murine prion disease, which we show here displays many of the pathological, behavioural and biochemical hallmarks of human neurodegenerative disease, is slowed and normal behaviour maintained by the activation of the M1-receptor with a highly tolerated positive allosteric modulator (VU846). This correlates with a reduction in both neuroinflammation and indicators of mitochondrial dysregulation, as well as a normalisation in the expression of markers associated with neurodegeneration and Alzheimer’s disease. Furthermore, VU846 preserves expression of synaptic proteins and post-synaptic signalling components that are altered in disease. We conclude that allosteric regulation of M1-receptors has the potential to reduce the severity of neurodegenerative diseases caused by the prion-like propagation of misfolded protein in a manner that extends life span and maintains normal behaviour.

SummaryPlant adaptation to a desert environment and its endemic heat stress is poorly understood at the molecular level. The naturally heat-tolerant Brassicaceae species Anastatica hierochuntica is an ideal extremophyte model to identify genetic adaptations that have evolved to allow plants to tolerate heat stress and thrive in deserts.We generated an A. hierochuntica reference transcriptome and pinpointed extremophyte adaptations by comparing Arabidopsis thaliana and A. hierochuntica transcriptome responses to heat and identifying positively selected genes in A. hierochuntica.The two species exhibit similar transcriptome adjustment in response to heat and the A. hierochuntica transcriptome does not exist in a constitutive heat “stress-ready” state. Furthermore, the A. hierochuntica global transcriptome as well as heat-responsive orthologs, display a lower basal and higher heat-induced expression than in A. thaliana. Genes positively selected in multiple extremophytes are associated with stomatal opening, nutrient acquisition, and UV-B induced DNA repair while those unique to A. hierochuntica are consistent with its photoperiod-insensitive, early-flowering phenotype.We suggest that evolution of a flexible transcriptome confers the ability to quickly react to extreme diurnal temperature fluctuations characteristic of a desert environment while positive selection of genes involved in stress tolerance and early flowering could facilitate an opportunistic desert lifestyle.

Cyanobacteria are photosynthetic prokaryotes being developed as sustainable platforms that use renewable resources (light, water, and air) for diverse applications in energy, food, environment, and medicine. Despite the attractive promise that cyanobacteria offer to industrial biotechnology, slow growth rates pose a major challenge in processes which typically require large amounts of biomass and are often toxic to the cells. Two-stage cultivation strategies are an attractive solution to prevent any undesired growth inhibition by de-coupling biomass accumulation (stage I) and the industrial process (stage II). In cyanobacteria, two-stage strategies involve costly transfer methods between stages I and II, and little work has been focussed on using the distinct growth and stationary phases of batch cultures to autoregulate stage transition. In the present study, we identified and characterised a growth phase-specific promoter, which can serve as an auto-inducible switch to regulate two-stage bioprocesses in cyanobacteria. First, growth phase-specific genes were identified from a new RNAseq dataset comparing two growth phases and six nutrient conditions in Synechocystis sp. PCC 6803, including two new transcriptomes for low Mg and low K. A type II NADH dehydrogenase (ndbA) showed robust induction when the cultures transitioned from exponential to stationary phase growth. Behaviour of a 600-bp promoter sequence (PndbA600) was then characterised in detail following the expression of PndbA600:GFP in Synechococcus sp. PCC 7002. Culture density and growth media analyses showed that PndbA600 activation was not dependent on increases in culture density per se but on N availability and on another activating factor present in the spent media of stationary phase cultures (Factor X). PndbA600 deactivation was dependent on the changes in culture density and in either N availability or Factor X. Electron transport inhibition studies revealed a photosynthesis-specific enhancement of active PndbA600 levels. Our findings are summarised in a model describing the environmental regulation of PndbA600, which can now inform the rational design of two-stage industrial processes in cyanobacteria.

Huntington disease \(HD) is an autosomal dominant neurodegenerative disorder caused by the expansion of a CAG repeat in the first exon of the _HTT_ gene. Affected individuals inherit more than 40 repeats and the CAG repeat is genetically unstable in both the germline and soma. Molecular diagnosis and genotyping of the CAG repeat is traditionally performed by estimation of PCR fragment size. However, this approach is complicated by the presence of an adjacent polymorphic CCG repeat and provides no information on the presence of variant repeats, flanking sequence variants or on the degree of somatic mosaicism. To overcome these limitations, we have developed an amplicon-sequencing protocol that allows the sequencing of hundreds of samples in a single MiSeq run. The composition of the _HTT_ exon one trinucleotide repeat locus can be determined from the MiSeq sequencing reads generated. With sufficient sequencing depth, such MiSeq data can also be used to quantify the degree of somatic mosaicism of the _HTT_ CAG repeat in the tissue analysed.

Metabolic reprogramming is a key hallmark of cancer, but less is known about metabolic plasticity of the same tumor at different sites. Here, we investigated the metabolic adaptation of leukemia in two different microenvironments, the bone marrow and the central nervous system (CNS). We identified a metabolic signature of fatty-acid synthesis in CNS leukemia, highlighting Stearoyl-CoA desaturase (SCD1) as a key player. In vivo SCD1 overexpression increases CNS disease, whilst genetic or pharmacological inhibition of SCD1 decreases CNS load. Overall, we demonstrated that leukemic cells dynamically rewire metabolic pathways to suit local conditions and that targeting these adaptations can be exploited therapeutically.

Myotonic dystrophy type 1 (DM1) is a multisystem disorder, caused by expansion of a CTG trinucleotide repeat in the 3'-untranslated region of the DMPK gene. The repeat expansion is somatically unstable and tends to increase in length with time, contributing to disease progression. In some individuals, the repeat array is interrupted by variant repeats such as CCG and CGG, stabilising the expansion and often leading to milder symptoms. We have characterised three families, each including one person with variant repeats that had arisen de novo on paternal transmission of the repeat expansion. Two individuals were identified for screening due to an unusual result in the laboratory diagnostic test, and the third due to exceptionally mild symptoms. The presence of variant repeats in all three expanded alleles was confirmed by restriction digestion of small pool PCR products, and allele structures were determined by PacBio sequencing. Each was different, but all contained CCG repeats close to the 3'-end of the repeat expansion. All other family members had inherited pure CTG repeats. The variant repeat-containing alleles were more stable in the blood than pure alleles of similar length, which may in part account for the mild symptoms observed in all three individuals. This emphasises the importance of somatic instability as a disease mechanism in DM1. Further, since patients with variant repeats may have unusually mild symptoms, identification of these individuals has important implications for genetic counselling and for patient stratification in DM1 clinical trials.

RationaleTransforming growth factor-beta (TGFβ) is tightly regulated at multiple levels, with regulation at the receptor level now recognized as a key determinant of the cellular response to this pleiotropic cytokine. TGFβ promotes saphenous vein graft neointima formation after coronary artery bypass graft (CABG) surgery, inducing smooth muscle cell (SMC) hyperplasia and fibrosis by signaling via activin receptor-like kinase 5(ALK5). However, the role of the alternate TGFβ receptor ALK1 remains completely unknown.ObjectiveTo define the receptor pathways activated by TGFβ in SMCs and their mechanistic importance during CABG neointima formation.Methods and resultsRadioligand co-IP assays revealed direct interactions between TGFβ, ALK5 and ALK1 in primary saphenous vein graft SMC (HSVSMC) from patients undergoing CABG. Knockdown and pharmacological inhibition of ALK5 or ALK1 in HSVSMC significantly attenuated TGFβ-induced phosphorylation of receptor-regulated (R)-Smads 2/3 and 1/5, respectively. Microarray profiling followed by qRT-PCR validation showed that TGFβ induced distinct transcriptional networks downstream of ALK5 or ALK1, associated with HSVSMC contractility and migration, respectively and confirmed using migration assays as well as qRT-PCR and western blot assays of contractile SMC markers. scRNAseq analysis of TGFβ-treated HSVSMC identified distinct subgroups of cells showing ALK5 or ALK1 transcriptional responses, while RNA velocity analyses indicated divergence in differentiation towards ALK5 or ALK1-dominant lineages. ALK1, ALK5 and their downstream effectors pSmad1/5 and pSmad2/3 were localized to αSMA+ neointimal SMCs in remodelled mouse vein grafts. Pharmacological inhibition or genetic ablation of Smad1/5 substantially reducing neointima formation following acute vascular injury. Notably, expression and activation of ALK1, ALK5 and their respective downstream R-Smads was already evident in hyperplastic saphenous veins prior to grafting.ConclusionsWhilst canonical TGFβ signaling via ALK5 promotes a contractile HSVSMC phenotype, transactivation of ALK1 by TGFβ induces neointima formation by driving cell migration. Restoring the balance between ALK1 and ALK5 in HSVSMC may represent a novel therapeutic strategy for vein graft failure.

Longitudinal proteomic analysis of bovine milk shows consistent changes over time across cows after intramammary challenge with Streptococcus uberis., Mastitis, inflammation of the mammary gland, is the most common and costly disease of dairy cattle in the western world. It is primarily caused by bacteria, with Streptococcus uberis as one of the most prevalent causative agents. To characterize the proteome during Streptococcus uberis mastitis, an experimentally induced model of intramammary infection was used. Milk whey samples obtained from 6 cows at 6 time points were processed using label-free relative quantitative proteomics. This proteomic analysis complements clinical, bacteriological and immunological studies as well as peptidomic and metabolomic analysis of the same challenge model. A total of 2552 non-redundant bovine peptides were identified, and from these, 570 bovine proteins were quantified. Hierarchical cluster analysis and principal component analysis showed clear clustering of results by stage of infection, with similarities between pre-infection and resolution stages (0 and 312 h post challenge), early infection stages (36 and 42 h post challenge) and late infection stages (57 and 81 h post challenge). Ingenuity pathway analysis identified upregulation of acute phase protein pathways over the course of infection, with dominance of different acute phase proteins at different time points based on differential expression analysis. Antimicrobial peptides, notably cathelicidins and peptidoglycan recognition protein, were upregulated at all time points post challenge and peaked at 57 h, which coincided with 10 000-fold decrease in average bacterial counts. The integration of clinical, bacteriological, immunological and quantitative proteomics and other-omic data provides a more detailed systems level view of the host response to mastitis than has been achieved previously.

Niche-adaptation of a bacterial pathogen hinges on the ability to recognize the complexity of signals from the environment and integrate that information with the regulation of genes critical for infection. Here we report the transcriptome of the attaching and effacing pathogen Citrobacter rodentium during infection of its natural murine host. Pathogen gene expression in vivo was heavily biased towards the virulence factor repertoire and was found to be co-ordinated uniquely in response to the host. Concordantly, we identified the host-specific induction of a metabolic pathway that overlapped with the regulation of virulence. The essential type 3 secretion system and an associated suite of distinct effectors were found to be modulated co-ordinately through a unique mechanism involving metabolism of microbiota-derived 1,2-propanediol, which dictated the ability to colonize the host effectively. This study provides novel insights into how host-specific metabolic adaptation acts as a cue to fine-tune virulence., Infection of mice with Citrobacter rodentium is a common model of infection with attaching-and-effacing pathogens. Here, Connolly et al. analyse the transcriptome of C. rodentium during mouse infection, showing host-induced coordinated upregulation of virulence factors and 1,2-propanediol metabolism.

Niche-adaptation of a bacterial pathogen hinges on the ability to recognize the complexity of signals from the environment and integrate that information with the regulation of genes critical for infection. Here we report the transcriptome of the attaching and effacing pathogen Citrobacter rodentium during infection of its natural murine host. Pathogen gene expression in vivo was heavily biased towards the virulence factor repertoire and was found to be co-ordinated uniquely in response to the host. Concordantly, we identified the host-specific induction of a metabolic pathway that overlapped with the regulation of virulence. The essential type 3 secretion system and an associated suite of distinct effectors were found to be modulated co-ordinately through a unique mechanism involving metabolism of microbiota-derived 1,2-propanediol, which dictated the ability to colonize the host effectively. This study provides novel insights into how host-specific metabolic adaptation acts as a cue to fine-tune virulence.

Salmonid fishes exhibit high levels of phenotypic and ecological variation and are thus ideal model systems for studying evolutionary processes of adaptive divergence and speciation. Furthermore, salmonids are of major interest in fisheries, aquaculture, and conservation research. Improving understanding of the genetic mechanisms underlying traits in these species would significantly progress research in these fields. Here we generate high quality de novo transcriptomes for four salmonid species: Atlantic salmon (Salmo salar), brown trout (Salmo trutta), Arctic charr (Salvelinus alpinus), and European whitefish (Coregonus lavaretus). All species except Atlantic salmon have no reference genome publicly available and few if any genomic studies to date.We used paired-end RNA-seq on Illumina to generate high coverage sequencing of multiple individuals, yielding between 180 and 210 M reads per species. After initial assembly, strict filtering was used to remove duplicated, redundant, and low confidence transcripts. The final assemblies consisted of 36,505 protein-coding transcripts for Atlantic salmon, 35,736 for brown trout, 33,126 for Arctic charr, and 33,697 for European whitefish and are made publicly available. Assembly completeness was assessed using three approaches, all of which supported high quality of the assemblies: 1) ~78% of Actinopterygian single-copy orthologs were successfully captured in our assemblies, 2) orthogroup inference identified high overlap in the protein sequences present across all four species (40% shared across all four and 84% shared by at least two), and 3) comparison with the published Atlantic salmon genome suggests that our assemblies represent well covered (~98%) protein-coding transcriptomes. Thorough comparison of the generated assemblies found that 84-90% of transcripts in each assembly were orthologous with at least one of the other three species. We also identified 34-37% of transcripts in each assembly as paralogs. We further compare completeness and annotation statistics of our new assemblies to available related species.New, high-confidence protein-coding transcriptomes were generated for four ecologically and economically important species of salmonids. This offers a high quality pipeline for such complex genomes, represents a valuable contribution to the existing genomic resources for these species and provides robust tools for future investigation of gene expression and sequence evolution in these and other salmonid species.

Dietary restriction (DR) is the most widely studied non-genetic intervention capable of extending lifespan across multiple taxa. Modulation of genes, primarily within the insulin/insulin-like growth factor signalling (IIS) and the mechanistic target of rapamycin (mTOR) signalling pathways also act to extend lifespan in model organisms. For example, mice lacking insulin receptor substrate-1 (IRS1) are long-lived and protected against several age-associated pathologies. However, it remains unclear how these particular interventions act mechanistically to produce their beneficial effects. Here, we investigated transcriptional responses in wild-type and IRS1 null mice fed an ad libitum diet (WTAL and KOAL) or fed a 30% DR diet (WTDR or KODR). Using an RNAseq approach we noted a high correlation coefficient of differentially expressed genes existed within the same tissue across WTDR and KOAL mice and many metabolic features were shared between these mice. Overall, we report that significant overlap exists in the tissue-specific transcriptional response between long-lived DR mice and IRS1 null mice. However, there was evidence of disconnect between transcriptional signatures and certain phenotypic measures between KOAL and KODR, in that additive effects on body mass were observed but at the transcriptional level DR induced a unique set of genes in these already long-lived mice.

Significance Light coordinates energy production, growth, and survival throughout plant development. In Arabidopsis, light stimulates transcriptional reprogramming during developmental transitions such as photomorphogenesis and flowering through the action of photoreceptors, transcription factors, and signaling components. Here we assign a function to a member of the zinc-finger homeodomain (ZFHD) transcription factor family in regulating light-induced development. Our findings reveal ZFHD10 to be a missing link in understanding how the recently discovered integrator of light and photoperiodic flowering, TANDEM ZINC-FINGER PLUS3 (TZP), controls the expression of growth-promoting transcriptional regulators via direct association with light-regulated promoter elements. Elucidating how such novel protein complexes coordinate gene expression will allow scientists and breeders to optimize plant growth and development in response to unfavorable environmental conditions., Integration of environmental signals and interactions among photoreceptors and transcriptional regulators is key in shaping plant development. TANDEM ZINC-FINGER PLUS3 (TZP) is an integrator of light and photoperiodic signaling that promotes flowering in Arabidopsis thaliana. Here we elucidate the molecular role of TZP as a positive regulator of hypocotyl elongation. We identify an interacting partner for TZP, the transcription factor ZINC-FINGER HOMEODOMAIN 10 (ZFHD10), and characterize its function in coregulating the expression of blue-light–dependent transcriptional regulators and growth-promoting genes. By employing a genome-wide approach, we reveal that ZFHD10 and TZP coassociate with promoter targets enriched in light-regulated elements. Furthermore, using a targeted approach, we show that ZFHD10 recruits TZP to the promoters of key coregulated genes. Our findings not only unveil the mechanism of TZP action in promoting hypocotyl elongation at the transcriptional level but also assign a function to an uncharacterized member of the ZFHD transcription factor family in promoting plant growth.

Integration of environmental signals and interactions among photoreceptors and transcriptional regulators is key in shaping plant development. TANDEM ZINC-FINGER PLUS3 (TZP) is an integrator of light and photoperiodic signaling that promotes flowering in Arabidopsis thaliana. Here we elucidate the molecular role of TZP as a positive regulator of hypocotyl elongation. We identify an interacting partner for TZP, the transcription factor ZINC-FINGER HOMEODOMAIN 10 (ZFHD10), and characterize its function in coregulating the expression of blue-light–dependent transcriptional regulators and growth-promoting genes. By employing a genome-wide approach, we reveal that ZFHD10 and TZP coassociate with promoter targets enriched in light-regulated elements. Furthermore, using a targeted approach, we show that ZFHD10 recruits TZP to the promoters of key coregulated genes. Our findings not only unveil the mechanism of TZP action in promoting hypocotyl elongation at the transcriptional level but also assign a function to an uncharacterized member of the ZFHD transcription factor family in promoting plant growth.

Background Salmonid fishes exhibit high levels of phenotypic and ecological variation and are thus ideal model systems for studying evolutionary processes of adaptive divergence and speciation. Furthermore, salmonids are of major interest in fisheries, aquaculture, and conservation research. Improving understanding of the genetic mechanisms underlying traits in these species would significantly progress research in these fields. Here we generate high quality de novo transcriptomes for four salmonid species: Atlantic salmon (Salmo salar), brown trout (Salmo trutta), Arctic charr (Salvelinus alpinus), and European whitefish (Coregonus lavaretus). All species except Atlantic salmon have no reference genome publicly available and few if any genomic studies to date. Results We used paired-end RNA-seq on Illumina to generate high coverage sequencing of multiple individuals, yielding between 180 and 210 M reads per species. After initial assembly, strict filtering was used to remove duplicated, redundant, and low confidence transcripts. The final assemblies consisted of 36,505 protein-coding transcripts for Atlantic salmon, 35,736 for brown trout, 33,126 for Arctic charr, and 33,697 for European whitefish and are made publicly available. Assembly completeness was assessed using three approaches, all of which supported high quality of the assemblies: 1) ~78% of Actinopterygian single-copy orthologs were successfully captured in our assemblies, 2) orthogroup inference identified high overlap in the protein sequences present across all four species (40% shared across all four and 84% shared by at least two), and 3) comparison with the published Atlantic salmon genome suggests that our assemblies represent well covered (~98%) protein-coding transcriptomes. Thorough comparison of the generated assemblies found that 84-90% of transcripts in each assembly were orthologous with at least one of the other three species. We also identified 34-37% of transcripts in each assembly as paralogs. We further compare completeness and annotation statistics of our new assemblies to available related species. Conclusion New, high-confidence protein-coding transcriptomes were generated for four ecologically and economically important species of salmonids. This offers a high quality pipeline for such complex genomes, represents a valuable contribution to the existing genomic resources for these species and provides robust tools for future investigation of gene expression and sequence evolution in these and other salmonid species. Electronic supplementary material The online version of this article (10.1186/s12864-017-4379-x) contains supplementary material, which is available to authorized users.

Metal tolerance proteins (MTPs) are plant members of the cation diffusion facilitator (CDF) transporter family involved in cellular metal homeostasis. Members of the CDF family are ubiquitously found in all living entities and show principal selectivity for Zn(2+), Mn(2+), and Fe(2+). Little is known regarding metal selectivity determinants of CDFs. We identified a novel cereal member of CDFs in barley, termed HvMTP1, that localizes to the vacuolar membrane. Unlike its close relative AtMTP1, which is highly selective for Zn(2+), HvMTP1 exhibits selectivity for both Zn(2+) and Co(2+) as assessed by its ability to suppress yeast mutant phenotypes for both metals. Expression of HvMTP1/AtMTP1 chimeras in yeast revealed a five-residue sequence within the AtMTP1 N-segment of the His-rich intracytoplasmic loop that confines specificity to Zn(2+). Furthermore, mutants of AtMTP1 generated through random mutagenesis revealed residues embedded within transmembrane domain 3 that additionally specify the high degree of Zn(2+) selectivity. We propose that the His-rich loop, which might play a role as a zinc chaperone, determines the identity of the metal ions that are transported. The residues within transmembrane domain 3 can also influence metal selectivity, possibly through conformational changes induced at the cation transport site located within the membrane or at the cytoplasmic C-terminal domain.

Epstein-Barr virus (EBV)-associated Burkitt's lymphoma is characterised by the deregulation of c-Myc expression and a restricted viral gene expression pattern in which the EBV nuclear antigen-1 (EBNA1) is the only viral protein to be consistently expressed. EBNA1 is required for viral genome propagation and segregation during latency. However, it has been much debated whether the protein plays a role in viral-associated tumourigenesis. We show that the lymphomas which arise in EµEBNA1 transgenic mice are unequivocally linked to EBNA1 expression and that both C-Myc and Mdm2 deregulation are central to this process. Tumour cell survival is supported by IL-2 and there is a skew towards CD8-positive T cells in the tumour environment, while the immune check-point protein PD-L1 is upregulated in the tumours. Additionally, several isoforms of Mdm2 are upregulated in the EµEBNA1 tumours, with increased phosphorylation at ser166, an expression pattern not seen in Eµc-Myc transgenic tumours. Concomitantly, E2F1, Xiap, Mta1, C-Fos and Stat1 are upregulated in the tumours. Using four independent inhibitors of Mdm2 we demonstrate that the EµEBNA1 tumour cells are dependant upon Mdm2 for survival (as they are upon c-Myc) and that Mdm2 inhibition is not accompanied by upregulation of p53, instead cell death is linked to loss of E2F1 expression, providing new insight into the underlying tumourigenic mechanism. This opens a new path to combat EBV-associated disease.

Research in evolutionary biology involving nonmodel organisms is rapidly shifting from using traditional molecular markers such as mtDNA and microsatellites to higher throughput SNP genotyping methodologies to address questions in population genetics, phylogenetics and genetic mapping. Restriction site associated DNA sequencing (RAD sequencing or RADseq) has become an established method for SNP genotyping on Illumina sequencing platforms. Here, we developed a protocol and adapters for double-digest RAD sequencing for Ion Torrent (Life Technologies; Ion Proton, Ion PGM) semiconductor sequencing. We sequenced thirteen genomic libraries of three different nonmodel vertebrate species on Ion Proton with PI chips: Arctic charr Salvelinus alpinus, European whitefish Coregonus lavaretus and common lizard Zootoca vivipara. This resulted in ~962 million single-end reads overall and a mean of ~74 million reads per library. We filtered the genomic data using Stacks, a bioinformatic tool to process RAD sequencing data. On average, we obtained ~11,000 polymorphic loci per library of 6-30 individuals. We validate our new method by technical and biological replication, by reconstructing phylogenetic relationships, and using a hybrid genetic cross to track genomic variants. Finally, we discuss the differences between using the different sequencing platforms in the context of RAD sequencing, assessing possible advantages and disadvantages. We show that our protocol can be used for Ion semiconductor sequencing platforms for the rapid and cost-effective generation of variable and reproducible genetic markers.

Human papillomavirus 16 (HPV16) is causative in human cancer. The E2 protein regulates transcription from and replication of the viral genome; the role of E2 in regulating the host genome has been less well studied. We have expressed HPV16 E2 (E2) stably in U2OS cells; these cells tolerate E2 expression well and gene expression analysis identified 74 genes showing differential expression specific to E2. Analysis of published gene expression data sets during cervical cancer progression identified 20 of the genes as being altered in a similar direction as the E2 specific genes. In addition, E2 altered the splicing of many genes implicated in cancer and cell motility. The E2 expressing cells showed no alteration in cell growth but were altered in cell motility, consistent with the E2 induced altered splicing predicted to affect this cellular function. The results present a model system for investigating E2 regulation of the host genome.

Intramammary infection leading to bovine mastitis is the leading disease problem affecting dairy cows and has marked effects on the milk produced by infected udder quarters. An experimental model of Streptococcus uberis mastitis has previously been investigated for clinical, immunological and pathophysiological alteration in milk, and has been the subject of peptidomic and quantitative proteomic investigation. The same sample set has now been investigated with a metabolomics approach using liquid chromatography and mass spectrometry. The analysis revealed over 3000 chromatographic peaks, of which 690 were putatively annotated with a metabolite. Hierarchical clustering analysis and principal component analysis demonstrated that metabolite changes due to S. uberis infection were maximal at 81 hours post challenge with metabolites in the milk from the resolution phase at 312 hours post challenge being closest to the pre-challenge samples. Metabolic pathway analysis revealed that the majority of the metabolites mapped to carbohydrate and nucleotide metabolism show a decreasing trend in concentration up to 81 hours post-challenge whereas an increasing trend was found in lipid metabolites and di-, tri- and tetra-peptides up to the same time point. The increase in these peptides coincides with an increase in larger peptides found in the previous peptidomic analysis and is likely to be due to protease degradation of milk proteins. Components of bile acid metabolism, linked to the FXR pathway regulating inflammation, were also increased. Metabolomic analysis of the response in milk during mastitis provides an essential component to the full understanding of the mammary gland's response to infection.

Funding was provided by a Kelvin Smith PhD Scholarship from the University of Glasgow (VM, PH, JR, and KAS), and Biotechnology and Biological Sciences Research Council David Phillips Research Fellowship (K.A.S.). Exposure to stress during early development can permanently influence an individual's physiology and behavior, and affect its subsequent health. The extent to which elevated glucocorticoids cause such long-term “programming” remains largely untested. Here, using the Japanese quail as our study species, we independently manipulated exposure to corticosterone during pre- and/or post-natal development and investigated the subsequent effects on global gene expression profiles within the hippocampus and hypothalamus upon adulthood. Our results showed that the changes in transcriptome profiles in response to corticosterone exposure clearly differed between the hippocampus and the hypothalamus. We also showed that these effects depended on the developmental timing of exposure and identified brain-region specific gene expression patterns that were either (1) similarly altered by corticosterone regardless of the developmental stage in which hormonal exposure occurred, or (2) specifically and uniquely altered by either pre-natal or post-natal exposure to corticosterone. Corticosterone-treated birds showed alterations in networks of genes which included known markers of the programming actions of early life adversity (e.g. brain-derived neurotrophic factor, and mineralocorticoid receptor within the hippocampus; corticotropin-releasing hormone and serotonin receptors in the hypothalamus). Altogether, these findings provide for the first time experimental support to the hypothesis that exposure to elevated glucocorticoids during development may be a key hormonal signaling pathway through which the long-term phenotypic effects associated with early life adversity emerge and potentially persist throughout the lifespan. These data also highlight that stressors might have different long-lasting impacts on the brain transcriptome depending on the developmental stage in which they are experienced; more work is now required to relate these mechanisms to organismal phenotypic differences. Publisher PDF

The FlyAtlas resource contains data on the expression of the genes of Drosophila melanogaster in different tissues (currently 25—17 adult and 8 larval) obtained by hybridization of messenger RNA to Affymetrix Drosophila Genome 2 microarrays. The microarray probe sets cover 13 250 Drosophila genes, detecting 12 533 in an unambiguous manner. The data underlying the original web application (http://flyatlas.org) have been restructured into a relational database and a Java servlet written to provide a new web interface, FlyAtlas 2 (http://flyatlas.gla.ac.uk/), which allows several additional queries. Users can retrieve data for individual genes or for groups of genes belonging to the same or related ontological categories. Assistance in selecting valid search terms is provided by an Ajax ‘autosuggest’ facility that polls the database as the user types. Searches can also focus on particular tissues, and data can be retrieved for the most highly expressed genes, for genes of a particular category with above-average expression or for genes with the greatest difference in expression between the larval and adult stages. A novel facility allows the database to be queried with a specific gene to find other genes with a similar pattern of expression across the different tissues.

A class of anti-virulence compounds, the salicylidene acylhydrazides, have been widely reported to block the function of the type three secretion system of several Gram-negative pathogens by a previously unknown mechanism. In this work, we provide the first identification of bacterial proteins that are targeted by this group of compounds. We provide evidence that their mode of action is likely to result from a synergistic effect arising from a perturbation of the function of several conserved proteins. We also examine the contribution of selected target proteins to the pathogenicity of Yersina pseudotuberculosis and to expression of virulence genes in Escherichia coli O157.

The diverse sequences of viral populations within individual hosts are the starting material for selection and subsequent evolution of RNA viruses such as foot-and-mouth disease virus (FMDV). Using next-generation sequencing (NGS) performed on a Genome Analyzer platform (Illumina), this study compared the viral populations within two bovine epithelial samples (foot lesions) from a single animal with the inoculum used to initiate experimental infection. Genomic sequences were determined in duplicate sequencing runs, and the consensus sequence of the inoculum determined by NGS was identical to that previously determined using the Sanger method. However, NGS revealed the fine polymorphic substructure of the viral population, from nucleotide variants present at just below 50% frequency to those present at fractions of 1%. Some of the higher-frequency polymorphisms identified encoded changes within codons associated with heparan sulfate binding and were present in both foot lesions, revealing intermediate stages in the evolution of a tissue culture-adapted virus replicating within a mammalian host. We identified 2,622, 1,434, and 1,703 polymorphisms in the inoculum and in the two foot lesions, respectively: most of the substitutions occurred in only a small fraction of the population and represented the progeny from recent cellular replication prior to onset of any selective pressures. We estimated the upper limit for the genome-wide mutation rate of the virus within a cell to be 7.8 × 10 −4 per nucleotide. The greater depth of detection achieved by NGS demonstrates that this method is a powerful and valuable tool for the dissection of FMDV populations within hosts.

The circadian oscillator in eukaryotes consists of several interlocking feedback loops through which the expression of clock genes is controlled. It is generally assumed that all plant cells contain essentially identical and cell-autonomous multiloop clocks. Here, we show that the circadian clock in the roots of mature Arabidopsis plants differs markedly from that in the shoots and that the root clock is synchronized by a photosynthesis-related signal from the shoot. Two of the feedback loops of the plant circadian clock are disengaged in roots, because two key clock components, the transcription factors CCA1 and LHY, are able to inhibit gene expression in shoots but not in roots. Thus, the plant clock is organ-specific but not organ-autonomous.

A key tenet of bone tissue engineering is the development of scaffold materials that can stimulate stem cell differentiation in the absence of chemical treatment to become osteoblasts without compromising material properties. At present, conventional implant materials fail owing to encapsulation by soft tissue, rather than direct bone bonding. Here, we demonstrate the use of nanoscale disorder to stimulate human mesenchymal stem cells (MSCs) to produce bone mineral in vitro, in the absence of osteogenic supplements. This approach has similar efficiency to that of cells cultured with osteogenic media. In addition, the current studies show that topographically treated MSCs have a distinct differentiation profile compared with those treated with osteogenic media, which has implications for cell therapies.

Nanotopographical material modification represents a possible way of producing surfaces that influence cellular adhesion for biomaterials purposes. Here, two low-adhesion surfaces are studied with human genome microarrays (120nm diameter pits produced by electron beam lithography and 11nm high columns produced by colloidal lithography). In order to present the large numbers of results produced in a succinct and easy to understand manner, two types of recent bioinformatics methods were used; iterative group analysis and Ingenuity pathway analysis. These methods allowed the easy comparison of the nanomaterials and showed large-scale changes in areas of extracellular matrix, cell signalling and inflammation. The results demonstrate that whilst modes of cellular response to low-adhesion materials are similar, the more adhesion is reduced, the further 'shut-down' of critical cellular activities is observed. We also feel that the analysis used could be of interest to biomaterials scientists looking for easy ways to display microarray data efficiently.

The ability of cells to alter their genomic regulation in response to mechanical conditioning or through changes in morphology and the organization of the interphase nuclei are key questions in cell biology. Here, two nanotopographies have been used as a model surfaces to change cell morphology in order to investigate spatial genomic changes within the nuclei of fibroblasts. Initially, centromeres for chromosome pairs were labeled and the average distance on different substrates calculated. Further to this, Affymetrix whole genome GeneChips were used to rank genomic changes in response to topography and plot the whereabouts on the chromosomes these changes were occurring. It was seen that as cell spreading was changed, so were the positions along the chromosomes that gene regulations were being observed. We hypothesize that as changes in cell and thus nuclear morphology occur, that this may alter the probability of transcription through opening or closing areas of the chromosomes to transcription factors.

The E2 protein of the carcinogen human papillomavirus 16 (HPV16) regulates replication and transcription of the viral genome in association with viral and cellular proteins. Our previous work demonstrated that E2 can regulate transcription from the host genome. E2 can activate transcription from adjacent promoters when located upstream using E2 DNA binding sequences and this activation is dependent upon the cellular protein Brd4; this report demonstrates that a Brd4 binding E2 mutant alters host genome expression differently from wild type E2. Of particular note is that highly down regulated genes are mostly not affected by failure to interact with Brd4 suggesting that the E2-Brd4 interaction is more responsible for the transcriptional activation of host genes rather than repression. Therefore failure to interact efficiently with Brd4, or altered levels of Brd4, would alter the ability of E2 to regulate the host genome and could contribute to determining the outcome of infection.

Most head and neck squamous cell carcinoma (HNSCC) patients present with late-stage cancers, which are difficult to treat. Therefore, early diagnosis of high-risk premalignant lesions and incipient cancers is important. HNSCC is currently perceived as a single progression mechanism, resulting in immortal invasive cancers. However, we have found that ∼40% of primary oral SCCs are mortal in culture, and these have a better prognosis. About 60% of oral premalignancies (dysplasias) are also mortal. The mortal and immortal tumors are generated in vivo as judged by p53 mutations and loss of p16INK4A expression being found only in the original tumors from which the immortal cultures were derived. To investigate the relationships of dysplasias to SCCs, we did microarray analysis of primary cultures of 4 normal oral mucosa biopsies, 19 dysplasias, and 16 SCCs. Spectral clustering using the singular value decomposition and other bioinformatic techniques showed that development of mortal and immortal SCCs involves distinct transcriptional changes. Both SCC classes share most of the transcriptional changes found in their respective dysplasias but have additional changes. Moreover, high-risk dysplasias that subsequently progress to SCCs more closely resemble SCCs than nonprogressing dysplasias. This indicates for the first time that there are divergent mortal and immortal pathways for oral SCC development via intermediate dysplasias. We believe that this new information may lead to new ways of classifying HNSCC in relation to prognosis. (Cancer Res 2006; 66(15): 7405-13)

The cAMP-specific phosphodiesterase PDE4D5 can interact with the signalling scaffold proteins RACK (receptors for activated C-kinase) 1 and beta-arrestin. Two-hybrid and co-immunoprecipitation analyses showed that RACK1 and beta-arrestin interact with PDE4D5 in a mutually exclusive manner. Overlay studies with PDE4D5 scanning peptide array libraries showed that RACK1 and beta-arrestin interact at overlapping sites within the unique N-terminal region of PDE4D5 and at distinct sites within the conserved PDE4 catalytic domain. Screening scanning alanine substitution peptide arrays, coupled with mutagenesis and truncation studies, allowed definition of RACK1 and beta-arrestin interaction sites. Modelled on the PDE4D catalytic domain, these form distinct well-defined surface-exposed patches on helices-15-16, for RACK1, and helix-17 for beta-arrestin. siRNA (small interfering RNA)-mediated knockdown of RACK1 in HEK-293 (human embryonic kidney) B2 cells increased beta-arrestin-scaffolded PDE4D5 approx. 5-fold, increased PDE4D5 recruited to the beta2AR (beta2-adrenergic receptor) upon isoproterenol challenge approx. 4-fold and severely attenuated (approx. 4-5 fold) both isoproterenol-stimulated PKA (protein kinase A) phosphorylation of the beta2AR and activation of ERK (extracellular-signal-regulated kinase). The ability of a catalytically inactive form of PDE4D5 to exert a dominant negative effect in amplifying isoproterenol-stimulated ERK activation was ablated by a mutation that blocked the interaction of PDE4D5 with beta-arrestin. In the present study, we show that the signalling scaffold proteins RACK1 and beta-arrestin compete to sequester distinct 'pools' of PDE4D5. In this fashion, alterations in the level of RACK1 expression may act to modulate signal transduction mediated by the beta2AR.