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Introduction: Triple Negative Breast Cancer (TNBC) is a heterogeneous disease, clinically defined for its aggressive tumour behaviour, metastatic ability and poor prognosis. Homeobox (HOX) genes are a family of homeodomain-containing transcription factors, which establish and maintain the identity and fate of cells and tissues during normal embryogenesis and organ development. A total of 39 HOX genes exist, located in four clusters (A-D) on different chromosomes. HOX genes are dysregulated in most cancers and they have an established role in driving key processes in breast carcinogenesis, including the regulation of the cell cycle, apoptosis, angiogenesis and metastasis. In this study, we evaluated the anti-tumour efficacy of a novel inhibitor of HOX protein function, HTL-001, which antagonizes the interactions between HOX proteins and their PBX cofactors. HTL-001, developed by HOX Therapeutics Ltd, is a synthetic peptide of 18 amino acids, comprised of a hexapeptide sequence, which resembles that on HOX proteins of paralogs 1-9, and a short C-terminal polyarginine sequence for cell penetration. We evaluated the efficacy of HTL-001 in vitro as a monotherapy in all cell lines representing all molecular classifications of breast cancer and in combination with chemotherapeutics for TNBC therapy. Most importantly, HTL-001 was examined in MDA-MB-231-BR cells, which have a propensity to metastasise to the brain, reflecting the disease course frequently observed in human patients. Methods: The differential expression of HOX and PBX genes in all subtypes of breast cancer was evaluated using bioinformatics analysis of TCGA and Oncomine data. The sensitivity to HTL-001 of breast cancer-derived cell lines of different molecular classifications, was determined using Cell Proliferation (MTS) Assay and FACS-based Annexin Assay. The anti-tumour effect of HTL-001 in combination with various chemotherapeutics was assessed and analysed for synergy using Bliss Analysis. The expression of genes previously shown to respond to HOX/PBX inhibition was measured from extracted RNA and protein using RT-qPCR and western blotting, respectively. The in vivo efficacy of HTL-001 will be investigated using a mouse flank tumour xenograft model with MDA-MB-231-BR cells, representative of TNBC. Results: TCGA and Oncomine data analysis showed TNBC tumours have a unique signature of HOX gene dysregulation, different to that of all other types of breast tumours, relative to normal breast tissues; most importantly upregulation in HOXB2 and HOXB3 genes. Targeting HOX/PBX dimers with HTL-001, induced apoptotic cell death in all breast cancer cell lines including; MCF-7, ZR-75-1, MDA-MB-231, BT-20 and SK-BR-3, with the highest sensitivity seen in brain seeking MDA-MB-231-BR cells. HTL-001 significantly upregulated the expression of key apoptotic and anti-survival genes including; AIF, cFOS, and DUSP1, resulting in the induction of apoptotic cell death in all breast cancer cell lines tested. Furthermore, we observed high levels of synergy between HTL-001 and the chemotherapeutic agents, Paclitaxel, Palbociclib and 5-Fluorouracil. Conclusion: HTL-001 is a novel inhibitor of HOX and PBX protein interactions capable of marked in vitro cytotoxicity in all types of breast cancer cells, particularly in TNBC. HTL-001 induced rapid apoptosis and triggered a downstream unique signature after target engagement. HTL-001 is currently the only therapy which targets HOX gene dysregulation and in doing so, may help address the current urgent unmet therapeutic need in TNBC through its unique mechanism of action and high levels of synergy with conventional anti-cancer agents. Citation Format: Einthavy Arunachalam, Guy R Simpson, Carla Sofia Möller-Levet, Nicola Annels, Richard Morgan, Timothy Robert Crook, Hardev Singh Pandha. Therapeutic targeting of HOX gene dysregulation in triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-10-15.

Aims Pre-B-cell Leukaemia (PBX) genes are important in organ development during embryogenesis. To date, four members of the PBX family (PBX1, PBX2, PBX3, PBX4) have been identified to be involved in human cancers, but little is known about their role in colorectal cancer (CRC). The aim of this study was to determine their differential expression, prognostic role and function in CRC. Methods Molecular and overall survival (OS) data from 614 patients with CRC were obtained from the National Cancer Institute, Tissue Cancer Genome Atlas (TCGA) database. To investigate the differential PBX gene mRNA expression, we performed a comparative cancer to normal computational analysis in edgeR. To determine PBXs prognostic value, we conducted Kaplan-Meier survival analysis and COX regression, selecting 10-year OS as primary outcome. Lastly, to explore the effect of PBX4 in CRC cell growth and angiogenesis, we performed gene expression modulation experiments using a PBX4-overexpressing plasmid-vector. Cell proliferation and VEGFA angiogenic factor expression were defined as primary and secondary in vitro outcomes respectively. Results Among PBXs only PBX4 was significantly upregulated showing a 4-fold increase in CRC vs normal colon (p Conclusions Comprehensive analysis of the PBX gene family identifies that PBX4 may function as a novel oncogene and may promote angiogenesis through VEGFA in CRC.

Aims Homeobox (HOX) proteins are emerging as promising biomarkers and targets for gene-therapy in cancer; however, their role in colorectal liver metastases (CRLM) is unknown. This study aims to investigate the role of HOXB9 as prognostic marker and potential therapeutic target in CRLM. Methods Two patient-cohorts were included: a) Patients with colorectal cancer (CRC) from the National Cancer Institute, Tissue Cancer Genome Atlas (TCGA) database (n = 614) and b) Institutional patient cohort who underwent liver resection for CRLM (n = 110) between 2007-2014. Primary outcome was 10-year overall survival (OS). COX regression and Kaplan-Meier survival analyses were performed including HOXB9 expression, demographics, clinicopathological and treatment-related variables. HOXB9 gene expression was modulated to assess its impact on CRC cell growth in vitro. Therefore, we conducted experimental studies using plasmid-vector and siRNA-interference to overexpress and knockdown HOXB9 respectively. Results Univariable TCGA analysis showed that HOXB9 did not predispose to poor OS (HR = 1, 95%CI:0.92-1.1, p = 0.620). On the contrary, univariable analysis in the CRLM patient cohort showed that high HOXB9 levels, right sided CRC, CRLM number≥4, CRLM diameter≥5cm, and intrahepatic recurrence were associated with significantly increased risk for worse OS. On multivariable models, only high HOXB9 expression (HR = 3.82, 95%CI:1.59-9.2, p = 0.003) and intrahepatic recurrence (HR = 4.28, 95%CI:1.88-9.72, p = 0.001) retained significance as independent prognostic factors after liver resection. Experimental studies showed that HOXB9 overexpression increased cell proliferation (p Conclusions HOXB9 demonstrates oncogenic properties and may serve as novel prognostic marker and potential target for gene-directed therapy in CRC/CRLM.

Murine norovirus (MNV) infection results in a late translation shutoff that is proposed to contribute to the attenuated and delayed innate immune response observed both in vitro and in vivo. Recently, we further demonstrated the activation of the α subunit of eukaryotic initiation factor 2 (eIF2α) kinase GCN2 during MNV infection, which has been previously linked to immunomodulation and resistance to inflammatory signaling during metabolic stress. While viral infection is usually associated with activation of double-stranded RNA (dsRNA) binding pattern recognition receptor PKR, we hypothesized that the establishment of a metabolic stress in infected cells is a proviral event, exploited by MNV to promote replication through weakening the activation of the innate immune response. In this study, we used multi-omics approaches to characterize cellular responses during MNV replication. We demonstrate the activation of pathways related to the integrated stress response, a known driver of anti-inflammatory phenotypes in macrophages. In particular, MNV infection causes an amino acid imbalance that is associated with GCN2 and ATF2 signaling. Importantly, this reprogramming lacks the features of a typical innate immune response, with the ATF/CHOP target GDF15 contributing to the lack of antiviral responses. We propose that MNV-induced metabolic stress supports the establishment of host tolerance to viral replication and propagation. IMPORTANCE During viral infection, host defenses are typically characterized by the secretion of proinflammatory autocrine and paracrine cytokines, potentiation of the interferon (IFN) response, and induction of the antiviral response via activation of JAK and Stat signaling. To avoid these and propagate, viruses have evolved strategies to evade or counteract host sensing. In this study, we demonstrate that murine norovirus controls the antiviral response by activating a metabolic stress response that activates the amino acid response and impairs inflammatory signaling. This highlights novel tools in the viral countermeasures arsenal and demonstrates the importance of the currently poorly understood metabolic reprogramming occurring during viral infections.

BACKGROUNDTwenty-four-hour rhythmicity in transcriptomes of tissues and organs is driven by local circadian oscillators, systemic factors, the central circadian pacemaker, and light-dark cycles. This rhythmicity is to some extent organ- and tissue-specific such that the sets of rhythmic transcripts or their timing are different across tissues/organs. Monitoring rhythmicity of tissues and organs holds promise for circadian medicine, but in humans most tissues and organs are not easily accessible. To investigate the extent to which rhythmicity in the human blood transcriptome reflects rhythmicity in tissues and organs, we compared the overlap and timing of rhythmic transcripts in human blood and rhythmic transcripts in 64 tissues/organs of the baboon.METHODSRhythmicity in the transcriptomes of humans and baboons were compared using set logic, circular cross-correlation, circular clustering, functional enrichment analyses and partial least squares regression.RESULTSOf the 759 orthologous genes that were rhythmic in human blood, 652 (86%) were also rhythmic in at least one baboon tissue. Most of these genes were associated with basic processes such as transcription and protein homeostasis. 109 (17%) of the 652 overlapping rhythmic genes were reported as rhythmic in only one baboon tissue or organ and several of these genes have tissue/organ-specific functions. Analysis of the alignment between baboon and human transcriptomes showed that in these diurnal species, rhythmicity is aligned with the onset, rather than midpoint or end of light period. In both species, the timing of rhythmic transcripts displayed prominent ‘night’ and ‘day’ clusters, with genes in the dark cluster associated with translation. The timing of human and baboon transcriptomes was significantly correlated in 25 tissue/organs with an average earlier timing of 3.21 h (SD 2.47 h) in human blood.CONCLUSIONSThe human blood transcriptome contains sets of rhythmic genes that overlap with rhythmic genes of tissues/organs, some of which are tissue/organ-specific, in the baboon. The rhythmic sets vary across tissues/organs but the timing of most rhythmic genes is similar across human blood and baboon tissues/organs. These results have implications for our understanding of the regulation of rhythmicity across tissues/organs and species and development of blood transcriptome-based biomarkers for rhythmicity in tissues and organs.

To rapidly respond and adapt to stresses, such as viral infections, cells have evolved several mechanisms, which include the activation of stress response pathways and the innate immune response. These stress responses result in the rapid inhibition of translation and condensation of stalled mRNAs, together with RNA-binding proteins and signalling components, into cytoplasmic biocondensates called stress granules. Increasing evidence suggests that stress granules contribute to antiviral defense and thus viruses need to evade these response pathways to propagate. In addition, the stress granule pathway is proposed to be dynamic and adaptable to specific stresses. We previously showed that Feline Calicivirus (FCV) impairs SGs assembly by cleaving the scaffolding protein G3BP1. We also observed that uninfected bystander cells assembled G3BP1-granules, suggesting a paracrine response trigged by the infection. We now present evidence that virus-free supernatant generated from infected cells can induce the formation of paracrine granules. They are different from canonical stress granules and exhibit specific kinetics of assembly-disassembly, protein and RNA composition and are linked to antiviral activity. We propose that this paracrine induction reflects a novel cellular defence mechanism to limit viral propagation and promote stress responses in bystander cells.Summary statementWe describe a novel type of paracrine induced RNA granules associated with viruses, highlighting how different stresses results in heterogeneous stress granule-like condensates with specific cellular functions.

Murine norovirus (MNV) infection results in a late translation shut-off, that is proposed to contribute to the attenuated and delayed innate immune response observed bothin vitroandin vivo.Recently, we further demonstrated the activation of the eIF2α kinase GCN2 during MNV infection, which has been previously linked to immunomodulation and resistance to inflammatory signalling during metabolic stress. While viral infection is usually associated with activation of dsRNA binding pattern recognition receptor PKR, we hypothesised that the establishment of a metabolic stress in infected cells is a proviral event, exploited by MNV to promote replication through weakening the activation of the innate immune response. In this study, we used multi-omics approaches to characterise cellular responses during MNV replication. We demonstrate the activation of pathways related to the integrated stress response, a known driver of anti-inflammatory phenotypes in macrophages. In particular, MNV infection causes an amino acid imbalance that is associated with GCN2 and ATF2 signalling. Importantly, this reprogramming lacks the features of a typical innate immune response, with the ATF/CHOP target GDF15 contributing to the lack of antiviral responses. We propose that MNV-induced metabolic stress supports the establishment of host tolerance to viral replication and propagation.ImportanceDuring viral infection, host defences are typically characterised by the secretion of pro-inflammatory autocrine and paracrine cytokines, potentiation of the IFN response and induction of the anti-viral response via activation of JAK and Stat signalling. To avoid these and propagate viruses have evolved strategies to evade or counteract host sensing. In this study, we demonstrate that murine norovirus controls the antiviral response by activating a metabolic stress response that activates the amino acid response and impairs inflammatory signalling. This highlights novel tools in the viral countermeasures tool-kit, and demonstrates the importance of the currently poorly understood metabolic reprogramming occurring during viral infections.

For the first time, we report the influence of vitamin D2 and vitamin D3 on genome-wide gene expression in whole blood from healthy women representing two ethnic groups, white European and South Asian. In this randomised placebo-controlled trial, participants were given daily physiological doses (15 μg) of either vitamin D2 or D3 for 12 weeks and changes in the transcriptome were compared relative to the transcriptome at baseline. While there was some overlap in the repertoire of differentially expressed genes after supplementation with each vitamin D source, most changes were specific to either vitamin D3 or vitamin D2, suggesting that each form of the vitamin may have different effects on human physiology. Notably, following vitamin D3 supplementation, the majority of changes in gene expression reflected a down-regulation in the activity of genes, many encoding components of the innate and adaptive immune systems. These are consistent with the emerging concept that vitamin D orchestrates a shift in the immune system towards a more tolerogenic status. The profound differences in gene expression after supplementation with vitamin D2 compared with vitamin D3 warrant a more intensive investigation of the biological effects of the two forms of vitamin D on human physiology. Significance statements This study suggests that the influence of vitamins D2 and D3 on human physiology may not be the same, as deduced from differences in gene expression within whole blood. South Asian participants were found to respond differently to vitamin D supplementation at the transcriptome level from white Europeans. The differentially expressed immune pathways identified in this study are consistent with vitamin D orchestrating a more tolerogenic immune status and this could be relevant in the context of the severity of immune response to viral infections such as COVID-19.

As a clinical setting in which local live biological therapy is already well established, non-muscle invasive bladder cancer (NMIBC) presents intriguing opportunities for oncolytic virotherapy. Coxsackievirus A21 (CVA21) is a novel intercellular adhesion molecule-1 (ICAM-1)-targeted immunotherapeutic virus. This study investigated CVA21-induced cytotoxicity in a panel of human bladder cancer cell lines, revealing a range of sensitivities largely correlating with expression of the viral receptor ICAM-1. CVA21 in combination with low doses of mitomycin-C enhanced CVA21 viral replication and oncolysis by increasing surface expression levels of ICAM-1. This was further confirmed using 300-μm precision slices of NMIBC where levels of virus protein expression and induction of apoptosis were enhanced with prior exposure to mitomycin-C. Given the importance of the immunogenicity of dying cancer cells for triggering tumor-specific responses and long-term therapeutic success, the ability of CVA21 to induce immunogenic cell death was investigated. CVA21 induced immunogenic apoptosis in bladder cancer cell lines, as evidenced by expression of the immunogenic cell death (ICD) determinant calreticulin, and HMGB-1 release and the ability to reject MB49 tumors in syngeneic mice after vaccination with MB49 cells undergoing CVA21 induced ICD. Such CVA21 immunotherapy could offer a potentially less toxic, more effective option for the treatment of bladder cancer. Keywords: bladder cancer, coxsackievirus A21, intercellular adhesion molecule-1

Studying circadian rhythms in most human tissues is hampered by difficulty in collecting serial samples. Here we reveal circadian rhythms in the transcriptome and metabolic pathways of human white adipose tissue. Subcutaneous adipose tissue was taken from seven healthy males under highly controlled ‘constant routine’ conditions. Five biopsies per participant were taken at six-hourly intervals for microarray analysis and in silico integrative metabolic modelling. We identified 837 transcripts exhibiting circadian expression profiles (2% of 41619 transcript targeting probes on the array), with clear separation of transcripts peaking in the morning (258 probes) and evening (579 probes). There was only partial overlap of our rhythmic transcripts with published animal adipose and human blood transcriptome data. Morning-peaking transcripts associated with regulation of gene expression, nitrogen compound metabolism, and nucleic acid biology; evening-peaking transcripts associated with organic acid metabolism, cofactor metabolism and redox activity. In silico pathway analysis further indicated circadian regulation of lipid and nucleic acid metabolism; it also predicted circadian variation in key metabolic pathways such as the citric acid cycle and branched chain amino acid degradation. In summary, in vivo circadian rhythms exist in multiple adipose metabolic pathways, including those involved in lipid metabolism, and core aspects of cellular biochemistry.

Significance Sleep disturbances are common in stress-related disorders but the nature of these sleep disturbances and how they relate to changes in the stress hormone corticosterone and changes in gene expression remained unknown. Here we demonstrate that in response to chronic mild stress, rapid–eye-movement sleep (REMS), a sleep state involved in emotion regulation and fear conditioning, changed first and more so than any other measured sleep characteristic. Transcriptomic profiles related to REMS continuity and theta oscillations overlapped with those for corticosterone, as well as with predictors for anhedonia, and were enriched for apoptotic pathways. These data highlight the central role of REMS in response to stress and warrant further investigation into REMS’s involvement in stress-related mental health disorders., One of sleep’s putative functions is mediation of adaptation to waking experiences. Chronic stress is a common waking experience; however, which specific aspect of sleep is most responsive, and how sleep changes relate to behavioral disturbances and molecular correlates remain unknown. We quantified sleep, physical, endocrine, and behavioral variables, as well as the brain and blood transcriptome in mice exposed to 9 weeks of unpredictable chronic mild stress (UCMS). Comparing 46 phenotypic variables revealed that rapid–eye-movement sleep (REMS), corticosterone regulation, and coat state were most responsive to UCMS. REMS theta oscillations were enhanced, whereas delta oscillations in non-REMS were unaffected. Transcripts affected by UCMS in the prefrontal cortex, hippocampus, hypothalamus, and blood were associated with inflammatory and immune responses. A machine-learning approach controlling for unspecific UCMS effects identified transcriptomic predictor sets for REMS parameters that were enriched in 193 pathways, including some involved in stem cells, immune response, and apoptosis and survival. Only three pathways were enriched in predictor sets for non-REMS. Transcriptomic predictor sets for variation in REMS continuity and theta activity shared many pathways with corticosterone regulation, in particular pathways implicated in apoptosis and survival, including mitochondrial apoptotic machinery. Predictor sets for REMS and anhedonia shared pathways involved in oxidative stress, cell proliferation, and apoptosis. These data identify REMS as a core and early element of the response to chronic stress, and identify apoptosis and survival pathways as a putative mechanism by which REMS may mediate the response to stressful waking experiences.

One of sleep’s putative functions is mediation of adaptation to waking experiences. Chronic stress is a common waking experience, however, which specific aspect of sleep is most responsive, and how sleep changes relate to behavioral disturbances and molecular correlates remain unknown. We quantified sleep, physical, endocrine and behavioral variables and the brain and blood transcriptome in mice exposed to nine weeks of unpredictable chronic mild stress (UCMS). Comparing 46 phenotypical variables revealed that rapid-eye-movement sleep (REMS), corticosterone regulation and coat state were most responsive to UCMS. REMS theta oscillations were enhanced whereas delta oscillations in non-REMS were unaffected. Transcripts affected by UCMS in the prefrontal cortex, hippocampus, hypothalamus and blood were associated with inflammatory and immune responses. A machine learning approach controlling for unspecific UCMS effects identified transcriptomic predictors for specific phenotypes and their overlap. Transcriptomic predictor sets for the inter-individual variation in REMS continuity and theta activity shared many pathways with corticosterone regulation and in particular pathways implicated in apoptosis, including mitochondrial pathways. Predictor sets for REMS and anhedonia, one of the behavioral changes following UCMS, shared pathways involved in oxidative stress, cell proliferation and apoptosis. RNA predictor sets for non-NREMS parameters showed no overlap with other phenotypes. These novel data identify REMS as a core and early element of the response to chronic stress, and identify apoptotic pathways as a putative mechanism by which REMS mediates adaptation to stressful waking experiences.Significance StatementSleep is responsive to experiences during wakefulness and is altered in stress-related disorders. Whether sleep changes primarily concern rapid-eye-movement sleep (REMS) or non-REM sleep, and how they correlate with stress hormones, behavioral and transcriptomic responses remained unknown. We demonstrate using unpredictable chronic (9-weeks) mild stress that REMS is the most responsive of all the measured sleep characteristics, and correlates with deficiency in corticosterone regulation. An unbiased machine learning, controlling for unspecific effects of stress, revealed that REMS correlated with RNA predictor sets enriched in apoptosis including mitochondrial pathways. Several pathways were shared with predictors of corticosterone and behavioral responses. This unbiased approach point to apoptosis as a molecular mechanism by which REMS mediates adaptation to an ecologically relevant waking experience.

HOX genes are vital for all aspects of mammalian growth and differentiation, and their dysregulated expression is related to ovarian carcinogenesis. The aim of the current study was to establish the prognostic value of HOX dysregulation as well as its role in platinum resistance. The potential to target HOX proteins through the HOX/PBX interaction was also explored in the context of platinum resistance. HOX gene expression was determined in ovarian cancer cell lines and primary EOCs by QPCR, and compared to expression in normal ovarian epithelium and fallopian tube tissue samples. Statistical analysis included one-way ANOVA and t-tests, using statistical software R and GraphPad. The analysis identified 36 of the 39 HOX genes as being overexpressed in high grade serous EOC compared to normal tissue. We detected a molecular HOX gene-signature that predicted poor outcome. Overexpression of HOXB4 and HOXB9 was identified in high grade serous cell lines after platinum resistance developed. Targeting the HOX/PBX dimer with the HXR9 peptide enhanced the cytotoxicity of cisplatin in platinum-resistant ovarian cancer. In conclusion, this study has shown the HOX genes are highly dysregulated in ovarian cancer with high expression of HOXA13, B6, C13, D1 and D13 being predictive of poor clinical outcome. Targeting the HOX/PBX dimer in platinum-resistant cancer represents a potentially new therapeutic option that should be further developed and tested in clinical trials.

Stress-induced adaptations require multiple levels of regulation in all organisms to repair cellular damage. In the present study we evaluated the genome-wide transcriptional and translational changes following heat stress exposure in the soil-dwelling model actinomycete bacterium, Streptomyces coelicolor. The combined analysis revealed an unprecedented level of translational control of gene expression, deduced through polysome profiling, in addition to transcriptional changes. Our data show little correlation between the transcriptome and ‘translatome’; while an obvious downward trend in genome wide transcription was observed, polysome associated transcripts following heat-shock showed an opposite upward trend. A handful of key protein players, including the major molecular chaperones and proteases were highly induced at both the transcriptional and translational level following heat-shock, a phenomenon known as ‘potentiation’. Many other transcripts encoding cold-shock proteins, ABC-transporter systems, multiple transcription factors were more highly polysome-associated following heat stress; interestingly, these protein families were not induced at the transcriptional level and therefore were not previously identified as part of the stress response. Thus, stress coping mechanisms at the level of gene expression in this bacterium go well beyond the induction of a relatively small number of molecular chaperones and proteases in order to ensure cellular survival at non-physiological temperatures.

Acute and chronic insufficient sleep are associated with adverse health outcomes and risk of accidents. There is therefore a need for biomarkers to monitor sleep debt status. None are currently available. We applied elastic net and ridge regression to transcriptome samples collected in 36 healthy young adults during acute total sleep deprivation and following 1 week of either chronic insufficient (

The power of the application of bioinformatics across multiple publicly available transcriptomic data sets was explored. Using 19 human and mouse circadian transcriptomic data sets, we found that NR1D1 and NR1D2 which encode heme-responsive nuclear receptors are the most rhythmic transcripts across sleep conditions and tissues suggesting that they are at the core of circadian rhythm generation. Analyzes of human transcriptomic data show that a core set of transcripts related to processes including immune function, glucocorticoid signalling, and lipid metabolism is rhythmically expressed independently of the sleep-wake cycle. We also identify key transcripts associated with transcription and translation that are disrupted by sleep manipulations, and through network analysis identify putative mechanisms underlying the adverse health outcomes associated with sleep disruption, such as diabetes and cancer. Comparative bioinformatics applied to existing and future data sets will be a powerful tool for the identification of core circadian- and sleep-dependent molecules.

Diagnosis and treatment of circadian rhythm sleep-wake disorders both require assessment of circadian phase of the brain’s circadian pacemaker. The gold-standard univariate method is based on collection of a 24-hr time series of plasma melatonin, a suprachiasmatic nucleus-driven pineal hormone. We developed and validated a multivariate whole-blood mRNA-based predictor of melatonin phase which requires few samples. Transcriptome data were collected under normal, sleep-deprivation and abnormal sleep-timing conditions to assess robustness of the predictor. Partial least square regression (PLSR), applied to the transcriptome, identified a set of 100 biomarkers primarily related to glucocorticoid signaling and immune function. Validation showed that PLSR-based predictors outperform published blood-derived circadian phase predictors. When given one sample as input, the R2 of predicted vs observed phase was 0.74, whereas for two samples taken 12 hr apart, R2 was 0.90. This blood transcriptome-based model enables assessment of circadian phase from a few samples. DOI: http://dx.doi.org/10.7554/eLife.20214.001

The AbsA1-AbsA2 two component signalling system ofStreptomyces coelicolorhas long been known to exert a powerful negative influence on the production of the antibiotics actinorhodin, undecylprodiginine and the Calcium-Dependent Antibiotic (CDA). Here we report the analysis of aΔabsA2deletion strain, which exhibits the classic precocious antibiotic hyper-production phenotype, and its complementation by an N-terminal triple-FLAG-tagged version of AbsA2. The complemented and non-complementedΔabsA2mutant strains were used in large-scale microarray-based time-course experiments to investigate the effect of deletingabsA2on gene expression and to identify thein vivoAbsA2 DNA-binding target sites using ChIP-on chip. We show that in addition to binding to the promoter regions ofredZandactII-orfIVAbsA2 binds to several previously unidentified sites within thecdabiosynthetic gene cluster within and/or upstream ofSCO3215-SCO3216,SCO3217,SCO3229-SCO3230, andSCO3226, and we relate the pattern of AbsA2 binding to the results of the transcriptomic study and antibiotic phenotypic assays. Interestingly, dual ‘biphasic’ ChIP peaks were observed with AbsA2 binding across the regulatory genesactII-orfIVandredZand theabsA2gene itself, while more conventional single promoter-proximal peaks were seen at the CDA biosynthetic genes suggesting a different mechanism of regulation of the former loci. Taken together the results shed light on the complex mechanism of regulation of antibiotic biosynthesis inStreptomyces coelicolorand the important role of AbsA2 in controlling the expression of three antibiotic biosynthetic gene clusters.

In PNAS, Braun et al. (1) describe the algorithm TimeSignature. We make the following observations. First, circadian time refers to the phase of internal biological clocks. Any algorithm assessing this should be validated against gold-standard markers of internal circadian phase. For example, the phase of the melatonin rhythm is considered a gold standard for the phase of the suprachiasmatic nucleus, the master circadian pacemaker in mammals. Braun et al. (1) validate TimeSignature against the clock time at which the sample was taken, which is not a marker of internal phase. It is unfortunate the authors do not discuss this major limitation and claim to assess circadian time. Others (e.g., refs. 2 and … [↵][1]1To whom correspondence should be addressed. Email: e.laing{at}surrey.ac.uk. [1]: #xref-corresp-1-1

HSV1 encodes an endoribonuclease termed virion host shutoff (vhs) that is produced late in infection and packaged into virions. Paradoxically, vhs is active against not only host but also virus transcripts, and is involved in host shutoff and the temporal expression of the virus transcriptome. Two other virus proteins—VP22 and VP16 –are proposed to regulate vhs to prevent uncontrolled and lethal mRNA degradation but their mechanism of action is unknown. We have performed dual transcriptomic analysis and single-cell mRNA FISH of human fibroblasts, a cell type where in the absence of VP22, HSV1 infection results in extreme translational shutoff. In Wt infection, host mRNAs exhibited a wide range of susceptibility to vhs ranging from resistance to 1000-fold reduction, a variation that was independent of their relative abundance or transcription rate. However, vhs endoribonuclease activity was not found to be overactive against any of the cell transcriptome in Δ22-infected cells but rather was delayed, while its activity against the virus transcriptome and in particular late mRNA was minimally enhanced. Intriguingly, immediate-early and early transcripts exhibited vhs-dependent nuclear retention later in Wt infection but late transcripts were cytoplasmic. However, in the absence of VP22, not only early but also late transcripts were retained in the nucleus by a vhs-dependent mechanism, a characteristic that extended to cellular transcripts that were not efficiently degraded by vhs. Moreover, the ability of VP22 to bind VP16 enhanced but was not fundamental to the rescue of vhs-induced nuclear retention of late transcripts. Hence, translational shutoff in HSV1 infection is primarily a result of vhs-induced nuclear retention and not degradation of infected cell mRNA. We have therefore revealed a new mechanism whereby vhs and its co-factors including VP22 elicit a temporal and spatial regulation of the infected cell transcriptome, thus co-ordinating efficient late protein production., Author summary Herpesviruses are large DNA viruses that replicate in the nucleus and express their genes by exploiting host cell mRNA biogenesis mechanisms including transcription, nuclear export, translation and turnover. As such, these viruses express multiple factors that enable the appropriation of cellular pathways for optimal virus production, and work in concert to shut off host gene expression and to overexpress virus genes in a well-described cascade that occurs in a temporal pattern of immediate-early, early and late proteins. We have analysed global and single cell changes in the host and virus transcriptome to uncover a novel mechanism by which the viral endoribonuclease, termed vhs, turns off early virus gene expression. This is achieved through the vhs-induced nuclear retention of the entire infected cell transcriptome at the onset of late gene expression. To enable the switch from early to late protein production the virus then requires a second factor called VP22 to specifically inhibit the nuclear retention of late transcripts allowing their translation in the cytoplasm. In this way, HSV1 elicits a temporal and spatial regulation of the infected cell transcriptome to co-ordinate efficient late protein production, a process that may be relevant to herpesviruses in general.

Background and purpose: Tumour hypoxia is associated with a poor prognosis in head and neck squamous cell carcinoma (HNSCC), however there is no accepted method for assessing hypoxia clinically. We aimed to conduct a technical validation of a hypoxia gene expression signature using the TaqMan Low Density Array (TLDA) platform to investigate if this approach reliably identified hypoxic tumours. Materials and methods: Tumour samples (n = 201) from 80 HNSCC patients were collected prospectively from two centres. Fifty-three patients received pimonidazole prior to surgery. TaqMan Low Density Array-Hypoxia Scores (TLDA-HS) were obtained by quantitative real-time PCR (qPCR) using a 25-gene signature and customised TLDA cards. Assay performance was assessed as coefficient of variation (CoV). Results: The assay was sensitive with linear reaction efficiencies across a 4log10 range of inputted cDNA (0.001-10 ng/μl). Intra- (CoV = 6.9%) and inter- (CoV = 2.0%) assay reproducibility were excellent. Intra-tumour heterogeneity was lower for TLDA-HS (23.2%) than for pimonidazole (67.2%) or single gene measurements of CA9 (62.2%), VEGFA (45.0%) or HIG2 (39.4%). TLDA-HS in HNSCC cell lines increased with decreasing pO2. TLDA-HS correlated with Affymetrix U133 Plus 2.0 microarray HS (p < 0.01) and positive pimonidazole scores (p = 0.005). Conclusions: Gene expression measurements of hypoxia using a 25-gene signature and TLDA cards are sensitive, reproducible and associated with lower intra-tumour heterogeneity than assaying individual genes or pimonidazole binding. The approach is suitable for further assessment of prognostic and predictive capability in clinical trial material. © 2012 Elsevier Ltd. All rights reserved.

A hypoxia-associated gene signature (metagene) was previously derived via in vivo data-mining. In this study, we aimed to investigate whether this approach could identify novel hypoxia regulated genes. From an initial list of nine genes, three were selected for further study (BCAR1, IGF2BP2 and SLCO1B3). Ten cell lines were exposed to hypoxia and interrogated for the expression of the three genes. All three genes were hypoxia inducible in at least one of the 10 cell lines with SLCO1B3 induced in seven. SLCO1B3 was studied further using chromatin immunoprecipitation and luciferase assays to investigate hypoxia inducible factor (HIF) dependent transcription. Two functional HIF response elements were identified within intron 1 of the gene. The functional importance of SLCO1B3 was studied by gene knockdown experiments followed by cell growth assays, flow cytometry and Western blotting. SLCO1B3 knockdown reduced cell size and 3-dimensional spheroid volume, which was associated with decreased activation of the mammalian target of rapamycin (mTOR) pathway. Finally, Oncomine analysis revealed that head and neck and colorectal tumours had higher levels of SLCO1B3 compared to normal tissue. Thus, the knowledge based approach for deriving gene signatures can identify novel biologically relevant genes. © 2013 Elsevier Ltd. All rights reserved.

Background: Degradation and chemical modification of RNA in formalin-fixed paraffin-embedded (FFPE) samples hamper their use in expression profiling studies. This study aimed to show that useful information can be obtained by Exon-array profiling archival FFPE tumour samples. Methods: Nineteen cervical squamous cell carcinoma (SCC) and 9 adenocarcinoma (AC) FFPE samples (10–16-year-old) were profiled using Affymetrix Exon arrays. The gene signature derived was tested on a fresh-frozen non-small cell lung cancer (NSCLC) series. Exploration of biological networks involved gene set enrichment analysis (GSEA). Differential gene expression was confirmed using Quantigene, a multiplex bead-based alternative to qRT–PCR. Results: In all, 1062 genes were higher in SCC vs AC, and 155 genes higher in AC. The 1217-gene signature correctly separated 58 NSCLC into SCC and AC. A gene network centered on hepatic nuclear factor and GATA6 was identified in AC, suggesting a role in glandular cell differentiation of the cervix. Quantigene analysis of the top 26 differentially expressed genes correctly partitioned cervix samples as SCC or AC. Conclusion: FFPE samples can be profiled using Exon arrays to derive gene expression signatures that are sufficiently robust to be applied to independent data sets, identify novel biology and design assays for independent platform validation.

Time course measurements are becoming a common type of experiment in the use of microarrays. The temporal order of the data and the varying length of sampling intervals are important and should be considered in clustering time-series. However, the shortness of gene expression time-series data limits the use of conventional statistical models and techniques for time-series analysis. To address this problem, this paper proposes the fuzzy short time-series (FSTS) clustering algorithm, which clusters profiles based on the similarity of their relative change of expression level and the corresponding temporal information. One of the major advantages of fuzzy clustering is that genes can belong to more than one group, revealing distinctive features of each gene's function and regulation. Several examples are provided to illustrate the performance of the proposed algorithm. In addition, we present the validation of the algorithm by clustering the genes which define the model profiles in Chu et al. (Science, 282 (1998) 699). The fuzzy c-means, k-means, average linkage hierarchical algorithm and random clustering are compared to the proposed FSTS algorithm. The performance is evaluated with a well-established cluster validity measure proving that the FSTS algorithm has a better performance than the compared algorithms in clustering similar rates of change of expression in successive unevenly distributed time points. Moreover, the FSTS algorithm was able to cluster in a biologically meaningful way the genes defining the model profiles.

Circadian organization of the mammalian transcriptome is achieved by rhythmic recruitment of key modifiers of chromatin structure and transcriptional and translational processes. These rhythmic processes, together with posttranslational modification, constitute circadian oscillators in the brain and peripheral tissues, which drive rhythms in physiology and behavior, including the sleep-wake cycle. In humans, sleep is normally timed to occur during the biological night, when body temperature is low and melatonin is synthesized. Desynchrony of sleep-wake timing and other circadian rhythms, such as occurs in shift work and jet lag, is associated with disruption of rhythmicity in physiology and endocrinology. However, to what extent mistimed sleep affects the molecular regulators of circadian rhythmicity remains to be established. Here, we show that mistimed sleep leads to a reduction of rhythmic transcripts in the human blood transcriptome from 6.4% at baseline to 1.0% during forced desynchrony of sleep and centrally driven circadian rhythms. Transcripts affected are key regulators of gene expression, including those associated with chromatin modification (methylases and acetylases), transcription (RNA polymerase II), translation (ribosomal proteins, initiation, and elongation factors), temperature-regulated transcription (cold inducible RNA-binding proteins), and core clock genes including CLOCK and ARNTL (BMAL1). We also estimated the separate contribution of sleep and circadian rhythmicity and found that the sleep-wake cycle coordinates the timing of transcription and translation in particular. The data show that mistimed sleep affects molecular processes at the core of circadian rhythm generation and imply that appropriate timing of sleep contributes significantly to the overall temporal organization of the human transcriptome.

Insufficient sleep and circadian rhythm disruption are associated with negative health outcomes, including obesity, cardiovascular disease, and cognitive impairment, but the mechanisms involved remain largely unexplored. Twenty-six participants were exposed to 1 wk of insufficient sleep (sleep-restriction condition 5.70 h, SEM = 0.03 sleep per 24 h) and 1 wk of sufficient sleep (control condition 8.50 h sleep, SEM = 0.11). Immediately following each condition, 10 whole-blood RNA samples were collected from each participant, while controlling for the effects of light, activity, and food, during a period of total sleep deprivation. Transcriptome analysis revealed that 711 genes were up- or down-regulated by insufficient sleep. Insufficient sleep also reduced the number of genes with a circadian expression profile from 1,855 to 1,481, reduced the circadian amplitude of these genes, and led to an increase in the number of genes that responded to subsequent total sleep deprivation from 122 to 856. Genes affected by insufficient sleep were associated with circadian rhythms (PER1, PER2, PER3, CRY2, CLOCK, NR1D1, NR1D2, RORA, DEC1, CSNK1E), sleep homeostasis (IL6, STAT3, KCNV2, CAMK2D), oxidative stress (PRDX2, PRDX5), and metabolism (SLC2A3, SLC2A5, GHRL, ABCA1). Biological processes affected included chromatin modification, gene-expression regulation, macromolecular metabolism, and inflammatory, immune and stress responses. Thus, insufficient sleep affects the human blood transcriptome, disrupts its circadian regulation, and intensifies the effects of acute total sleep deprivation. The identified biological processes may be involved with the negative effects of sleep loss on health, and highlight the interrelatedness of sleep homeostasis, circadian rhythmicity, and metabolism.

Gene expression time-series are discrete, noisy, short and usually unevenly sampled. Most of the existing methods used to compare expression profiles, operate directly on the time points. While modelling, the profiles can lead to more generalised, smooth characterisation of gene expressions. In this paper, a radial basis function neural network is employed to model gene expression time-series. The orthogonal least square method, used for selection of centres, is further combined with a width optimisation scheme. The experiments on a number of expression datasets have shown the advantages of the approach in terms of generalisation and approximation. The results on known datasets have indeed coincided with biological interpretations.

This paper introduces a novel approach for gene expression time-series modelling and clustering using neural networks and a shape similarity metric. The modelling of gene expressions by the Radial Basis Function (RBF) neural networks is proposed to produce a more general and smooth characterisation of the series. Furthermore, we identified that the use of the correlation coefficient of the derivative of the modelled profiles allows the comparison of profiles based on their shapes and the distributions of time points. The series are grouped into similarly shaped profiles using a correlation based fuzzy clustering algorithm. A well known dataset is used to demonstrate the proposed approach and a set of known genes are used as a benchmark to evaluate its performance. The results show the biological relevance and indicate that the proposed method is a useful technique for gene expression time-series analysis.

This paper proposes a new algorithm in the fuzzy-c-means family, which is designed to cluster time-series and is particularly suited for short time-series and those with unevenly spaced sampling points. Short time-series, which do not allow a conventional statistical model, and unevenly sampled time-series appear in many practical situations. The algorithm developed here is motivated by common experiments in molecular biology. Conventional clustering algorithms based on the Euclidean distance or the Pearson correlation coefficient are not able to include the temporal information in the distance metric. The temporal order of the data and the varying length of sampling intervals are important and should be considered in clustering time-series. The proposed short time-series (STS) distance is able to measure similarity of shapes which are formed by the relative change of amplitude and the corresponding temporal information. We develop a fuzzy time-series (FSTS) clustering algorithm by incorporating the STS distance into the standard fuzzy clustering scheme. An example is provided to demonstrate the performance of the proposed algorithm.

Despite its enormous promise to further our understanding of cellular processes involved in the regulation of gene expression, microarray technology generates data for which statistical pre-processing has become a necessity before any interpretation of data can begin. The process by which we distinguish (and remove) non-biological variation from biological variation is callednormalization. With a multitude of experimental designs, techniques and technologies influencing the acquisition of data, numerous approaches to normalization have been proposed in the literature. The purpose of this short review is not to add to the many suggestions that have been made, but to discuss some of the difficulties we encounter when analysing microarray data.

Purpose: Administering hypoxia modifying therapy to patients with head and neck squamous cell carcinoma (HNSCC) improves outcomes in those with identified hypoxic tumors. As no method for measuring tumor hypoxia has demonstrated clinical utility, this study aimed to validate use of a gene expression signature with a TaqMan Low Density Array (TLDA) platform. Experimental design: Tumor samples (n=201) from 80 HNSCC patients were collected prospectively from two centers. Fifty-three patients received pimonidazole prior to surgery. Hypoxia scores (TLDA HS) were obtained by quantitative real-time PCR (qPCR) using a 25-gene signature and customized TLDA cards. Assay performance was assessed as coefficient of variation (CoV). Relationships with outcome were determined by log-rank analysis. Results: The assay was sensitive with linear reaction efficiencies across a 4log10 range of inputted cDNA (0.001-10 ng/µl). Intra- (CoV=0.5%) and inter- (CoV=0.1%) assay reproducibility were excellent. Intra-tumor heterogeneity was lower for TLDA HS (23.2%) than for pimonidazole (67.2%) or single gene measurements of CA9 (62.2%), VEGFA (45.0%) or HIG2 (39.4%). TLDA HS in HNSCC cell lines increased with decreasing pO2 and was reduced but not completely abrogated in HIF1A silenced cells. TLDA HS correlated with Affymetrix U133 Plus 2.0 microarray HS (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-440. doi:1538-7445.AM2012-LB-440

A circular Self-Organising Map (SOM) based on a temporal metric has been proposed for clustering and characterising gene expressions. Expression profiles are first modelled with Radial Basis Functions. The co-expression coefficient, defined as the uncentred correlation of the differentiation of the models, is combined in a circular SOM for grouping and ordering the modelled expressions based on their temporal properties. In the proposed method the topology has been extended to temporal and cyclic ordering of the expressions. An example and a test on a microarray dataset are presented to demonstrate the advantages of the proposed method.