Extensive dynamic changes in the human transcriptome and its circadian organization during prolonged bed rest.

Physiological and molecular processes including the transcriptome change across the 24-h day, driven by molecular circadian clocks and behavioral and systemic factors. It is not known how the temporal organization of the human transcriptome responds to a long-lasting challenge. This may, however, provide insights into adaptation, disease, and recovery. We investigated the human 24-h time series transcriptome in 20 individuals during a 90-day constant bed rest protocol. We show that the protocol affected 91% of the transcriptome with 76% of the transcriptome still affected after 10 days of recovery. Dimensionality-reduction approaches revealed that many affected transcripts were associated with mRNA translation and immune function. The number, amplitude, and phase of rhythmic transcripts, including clock genes, varied significantly across the challenge. These findings of long-lasting changes in the temporal organization of the transcriptome have implications for understanding the mechanisms underlying health consequences of conditions such as microgravity and bed rest.

Circulating Adipocytokines and Insulin Like-Growth Factors and Their Modulation in Obesity-Associated Endometrial Cancer.

The rising global incidence of uterine cancer is linked to the escalating prevalence of obesity. Obesity results in alterations in adipocytokines and IGFs, driving cancer progression via inflammation, increased cell proliferation, and apoptosis inhibition, although the precise mechanisms are still unclear. This study examined a set of six markers, namely, adiponectin, leptin, IL6, TNFα, IGF1, and IGF2 and compared them between fifty age-matched endometrial cancer patients (study group) and non-cancer patients with benign gynaecological conditions (control group). We also assessed the relationship of these markers with obesity and explored the correlation between these markers and various tumour characteristics. In the cancer population, these markers were also assessed 24 h and 6 months post-surgery. Remarkably, low adiponectin levels were associated with a 35.8% increase in endometrial cancer risk. Interestingly, compared to control subjects where IGF levels decreased after menopause, post-menopausal women in the study group showed elevated IGF1 and IGF2 levels, suggesting a potential influence of endometrial cancer on the IGF system, particularly after menopause. Lastly, it is noteworthy that a discernible inverse relationship trend was observed in the levels of adipocytokines and IGFs 6 months post-surgery. This indicates that treatment for endometrial cancer may have a differential impact on adipocytokines and IGFs, potentially holding clinical significance that merits further investigation.

The dysfunctional immune response in renal cell carcinoma correlates with changes in the metabolic landscape of ccRCC during disease progression.

Renal cell carcinoma is an immunogenic tumour with a prominent dysfunctional immune cell infiltrate, unable to control tumour growth. Although tyrosine kinase inhibitors and immunotherapy have improved the outlook for some patients, many individuals are non-responders or relapse despite treatment. The hostile metabolic environment in RCC affects the ability of T-cells to maintain their own metabolic programme constraining T-cell immunity in RCC. We investigated the phenotype, function and metabolic capability of RCC TILs correlating this with clinicopathological features of the tumour and metabolic environment at the different disease stages. Flow cytometric analysis of freshly isolated TILs showed the emergence of exhausted T-cells in advanced disease based on their PD-1high and CD39 expression and reduced production of inflammatory cytokines upon in vitro stimulation. Exhausted T-cells from advanced stage disease also displayed an overall phenotype of metabolic insufficiency, characterized by mitochondrial alterations and defects in glucose uptake. Nanostring nCounter cancer metabolism assay on RNA obtained from 30 ccRCC cases revealed significant over-expression of metabolic genes even at early stage disease (pT1-2), while at pT3-4 and the locally advanced thrombi stages, there was an overall decrease in differentially expressed metabolic genes. Notably, the gene PPARGC1A was the most significantly down-regulated gene from pT1-2 to pT3-4 RCC which correlated with loss of mitochondrial function in tumour-infiltrating T-cells evident at this tumour stage. Down-regulation of PPARGC1A into stage pT3-4 may be the 'tipping-point' in RCC disease progression, modulating immune activity in ccRCC and potentially reducing the efficacy of immunotherapies in RCC and poorer patient outcomes.

A post-transcriptional regulatory landscape of aging in the female mouse hippocampus.

Aging is associated with substantial physiological changes and constitutes a major risk factor for neurological disorders including dementia. Alterations in gene expression upon aging have been extensively studied; however, an in-depth characterization of post-transcriptional regulatory events remains elusive. Here, we profiled the age-related changes of the transcriptome and translatome in the female mouse hippocampus by RNA sequencing of total RNA and polysome preparations at four ages (3-, 6-, 12-, 20-month-old); and we implemented a variety of bioinformatics approaches to unravel alterations in transcript abundance, alternative splicing, and polyadenylation site selection. We observed mostly well-coordinated transcriptome and translatome expression signatures across age including upregulation of transcripts related to immune system processes and neuroinflammation, though transcripts encoding ribonucleoproteins or associated with mitochondrial functions, calcium signaling and the cell-cycle displayed substantial discordant profiles, suggesting translational control associated with age-related deficits in hippocampal-dependent behavior. By contrast, alternative splicing was less preserved, increased with age and was associated with distinct functionally-related transcripts encoding proteins acting at synapses/dendrites, RNA-binding proteins; thereby predicting regulatory roles for RBM3 and CIRBP. Only minor changes in polyadenylation site selection were identified, indicating pivotal 3'-end selection in young adults compared to older groups. Overall, our study provides a comprehensive resource of age-associated post-transcriptional regulatory events in the mouse hippocampus, enabling further examination of the molecular features underlying age-associated neurological diseases.

Mistimed sleep and waking activity in humans disrupts glucocorticoid signalling transcripts and SP1, but not plasma cortisol rhythms.

Cortisol is a robust circadian signal that synchronises peripheral circadian clocks with the central clock in the suprachiasmatic nucleus via glucocorticoid receptors that regulate peripheral gene expression. Misalignment of the cortisol rhythm with the sleep-wake cycle, as occurs in shift work, is associated with negative health outcomes, but underlying molecular mechanisms remain largely unknown. We experimentally induced misalignment between the sleep-wake cycle and melatonin and cortisol rhythms in humans and measured time series blood transcriptomics while participants slept in-phase and out-of-phase with the central clock. The cortisol rhythm remained unchanged, but many glucocorticoid signalling transcripts were disrupted by mistimed sleep. To investigate which factors drive this dissociation between cortisol and its signalling pathways, we conducted bioinformatic and temporal coherence analyses. We found that glucocorticoid signalling transcripts affected by mistimed sleep were enriched for binding sites for the transcription factor SP1. Furthermore, changes in the timing of the rhythms of SP1 transcripts, a major regulator of transcription, and changes in the timing of rhythms in transcripts of the glucocorticoid signalling pathways were closely associated. Associations between the rhythmic changes in factors that affect SP1 expression and its activity, such as STAT3, EP300, HSP90AA1, and MAPK1, were also observed. We conclude that plasma cortisol rhythms incompletely reflect the impact of mistimed sleep on glucocorticoid signalling pathways and that sleep-wake driven changes in SP1 may mediate disruption of these pathways. These results aid understanding of mechanisms by which mistimed sleep affects health.

HOX and PBX gene dysregulation as a therapeutic target in glioblastoma multiforme.

BACKGROUND: Glioblastoma multiforme (GBM) is the most common high-grade malignant brain tumour in adults and arises from the glial cells in the brain. The prognosis of treated GBM remains very poor with 5-year survival rates of 5%, a figure which has not improved over the last few decades. Currently, there is a modest 14-month overall median survival in patients undergoing maximum safe resection plus adjuvant chemoradiotherapy. HOX gene dysregulation is now a widely recognised feature of many malignancies. METHODS: In this study we have focused on HOX gene dysregulation in GBM as a potential therapeutic target in a disease with high unmet need. RESULTS: We show significant dysregulation of these developmentally crucial genes and specifically that HOX genes A9, A10, C4 and D9 are strong candidates for biomarkers and treatment targets for GBM and GBM cancer stem cells. We evaluated a next generation therapeutic peptide, HTL-001, capable of targeting HOX gene over-expression in GBM by disrupting the interaction between HOX proteins and their co-factor, PBX. HTL-001 induced both caspase-dependent and -independent apoptosis in GBM cell lines. CONCLUSION: In vivo biodistribution studies confirmed that the peptide was able to cross the blood brain barrier. Systemic delivery of HTL-001 resulted in improved control of subcutaneous murine and human xenograft tumours and improved survival in a murine orthotopic model.

PBX4 functions as a potential novel oncopromoter in colorectal cancer: a comprehensive analysis of the PBX gene family.

Pre-B-cell leukaemia (PBX) is a transcription factor family (PBX1, PBX2, PBX3 and PBX4) that regulates important cellular functions and has been identified to be involved in human cancers. This study aimed to explore the expression of PBX genes and their clinical significance in colorectal cancer (CRC). We analysed the differential expression of PBX genes in CRC vs. normal tissue, using the Cancer Genome Atlas (TCGA) (https://portal.gdc.cancer.gov/) and ONCOMINE platform (https://www.oncomine.org/). The UALCAN (http://ualcan.path.uab.edu/) interactive OMICS web-server was used to evaluate the epigenetic regulation of PBX genes via their promoter methylation status. We found that only PBX4 was upregulated whereas PBX1 and PBX3 were downregulated (644 tumour vs. 51 normal samples) (P<0.001). The methylation status of PBX4 promoter appeared to be decreased (P=1.4e-07) whereas the methylation status of PBX1 and PBX3 promoters was increased (P=3.8e-04 and P=3.2e-07, respectively) in cancer vs. normal samples. To determine the prognostic value of PBXs, we conducted a Kaplan-Meier survival analysis and multivariable COX regression. We observed that high PBX4 expression was associated with increased risk for a worse overall survival (OS) in the TCGA CRC patient cohort (n=639), (HR 1.46, 95% CI 1.14-1.88, P=0.003) adjusted for age, gender, tumour location and metastases. We conducted in vitro gene expression modulation experiments to investigate the impact of PBX4 overexpression in CRC cell (HCT116) growth. Additionally, we evaluated the RNA expression of epithelial-mesenchymal transition (EMT) and angiogenesis markers. In vitro studies showed that PBX4 overexpression increased CRC cell proliferation (P<0.001) and upregulated the expression of EMT markers VIM, CDH1, CDH2, ZEB1, SNAI1 (P<0.05) and angiomarker VEGFA (P<0.0001). Lastly, through the Cistrome data browser (http://dbtoolkit.cistrome.org/) we investigated putative transcriptional regulators and we performed gene set enrichment analysis in Enrichr server (https://maayanlab.cloud/Enrichr/) to identify related biological processes. Nineteen factors were identified to be putative regulators of PBX4 and gene set enrichment analysis showed that biological processes related to cell cycle and cell proliferation were enriched (GO:0051726: CDK8, JUN, JUND, and IRF1, P=0.001). In conclusion, our study identified PBX4 as a potential novel oncopromoter in CRC and its overexpression was found to be associated with increased risk for worse survival rate.

Diurnal and circadian rhythmicity of the human blood transcriptome overlaps with organ- and tissue-specific expression of a non-human primate.

BACKGROUND: Twenty-four-hour rhythmicity in mammalian tissues and organs is driven by local circadian oscillators, systemic factors, the central circadian pacemaker and light-dark cycles. At the physiological level, the neural and endocrine systems synchronise gene expression in peripheral tissues and organs to the 24-h-day cycle, and disruption of such regulation has been shown to lead to pathological conditions. Thus, monitoring rhythmicity in tissues/organs holds promise for circadian medicine; however, most tissues and organs are not easily accessible in humans and alternative approaches to quantify circadian rhythmicity are needed. We investigated the overlap between rhythmic transcripts in human blood and transcripts shown to be rhythmic in 64 tissues/organs of the baboon, how these rhythms are aligned with light-dark cycles and each other, and whether timing of tissue-specific rhythmicity can be predicted from a blood sample. RESULTS: We compared rhythmicity in transcriptomic time series collected from humans and baboons using set logic, circular cross-correlation, circular clustering, functional enrichment analyses, and least squares regression. Of the 759 orthologous genes that were rhythmic in human blood, 652 (86%) were also rhythmic in at least one baboon tissue and most of these genes were associated with basic processes such as transcription and protein homeostasis. In total, 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. The timing of human and baboon rhythmic transcripts displayed prominent 'night' and 'day' clusters, with genes in the dark cluster associated with translation. Alignment between baboon rhythmic transcriptomes and the overlapping human blood transcriptome was significantly closer when light onset, rather than midpoint of light, or end of light period, was used as phase reference point. The timing of overlapping human and baboon rhythmic transcriptomes was significantly correlated in 25 tissue/organs with an average earlier timing of 3.21 h (SD 2.47 h) in human blood. CONCLUSIONS: The human blood transcriptome contains sets of rhythmic genes that overlap with rhythmic genes of tissues/organs in baboon. The rhythmic sets vary across tissues/organs, but the timing of most rhythmic genes is similar in human blood and baboon tissues/organs. These results have implications for development of blood transcriptome-based biomarkers for circadian rhythmicity in tissues and organs.

Vitamins D2 and D3 Have Overlapping But Different Effects on the Human Immune System Revealed Through Analysis of the Blood Transcriptome.

Vitamin D is best known for its role in maintaining bone health and calcium homeostasis. However, it also exerts a broad range of extra-skeletal effects on cellular physiology and on the immune system. Vitamins D2 and D3 share a high degree of structural similarity. Functional equivalence in their vitamin D-dependent effects on human physiology is usually assumed but has in fact not been well defined experimentally. In this study we seek to redress the gap in knowledge by undertaking an in-depth examination of changes in the human blood transcriptome following supplementation with physiological doses of vitamin D2 and D3. Our work extends a previously published randomized placebo-controlled trial that recruited healthy white European and South Asian women who were given 15 µg of vitamin D2 or D3 daily over 12 weeks in wintertime in the UK (Nov-Mar) by additionally determining changes in the blood transcriptome over the intervention period using microarrays. An integrated comparison of the results defines both the effect of vitamin D3 or D2 on gene expression, and any influence of ethnic background. An important aspect of this analysis was the focus on the changes in expression from baseline to the 12-week endpoint of treatment within each individual, harnessing the longitudinal design of the study. Whilst overlap in the repertoire of differentially expressed genes was present in the D2 or D3-dependent effects identified, most changes were specific to either one vitamin or the other. The data also pointed to the possibility of ethnic differences in the responses. Notably, following vitamin D3 supplementation, the majority of changes in gene expression reflected a down-regulation in the activity of genes, many encoding pathways of the innate and adaptive immune systems, potentially shifting the immune system to a more tolerogenic status. Surprisingly, gene expression associated with type I and type II interferon activity, critical to the innate response to bacterial and viral infections, differed following supplementation with either vitamin D2 or vitamin D3, with only vitamin D3 having a stimulatory effect. This study suggests that further investigation of the respective physiological roles of vitamin D2 and vitamin D3 is warranted.

HOXB9 Overexpression Promotes Colorectal Cancer Progression and Is Associated with Worse Survival in Liver Resection Patients for Colorectal Liver Metastases.

As is known, HOXB9 is an important factor affecting disease progression and overall survival (OS) in cancer. However, its role in colorectal cancer (CRC) remains unclear. We aimed to explore the role of HOXB9 in CRC progression and its association with OS in colorectal liver metastases (CRLM). We analysed differential HOXB9 expression in CRC using the Tissue Cancer Genome Atlas database (TCGA). We modulated HOXB9 expression in vitro to assess its impact on cell proliferation and epithelial-mesenchymal transition (EMT). Lastly, we explored the association of HOXB9 protein expression with OS, using an institutional patient cohort (n = 110) who underwent liver resection for CRLM. Furthermore, HOXB9 was upregulated in TCGA-CRC (n = 644) vs. normal tissue (n = 51) and its expression levels were elevated in KRAS mutations (p < 0.0001). In vitro, HOXB9 overexpression increased cell proliferation (p < 0.001) and upregulated the mRNA expression of EMT markers (VIM, CDH2, ZEB1, ZEB2, SNAI1 and SNAI2) while downregulated CDH1, (p < 0.05 for all comparisons). Conversely, HOXB9 silencing disrupted cell growth (p < 0.0001). High HOXB9 expression (HR = 3.82, 95% CI: 1.59-9.2, p = 0.003) was independently associated with worse OS in CRLM-HOXB9-expressing patients after liver resection. In conclusion, HOXB9 may be associated with worse OS in CRLM and may promote CRC progression, whereas HOXB9 silencing may inhibit CRC growth.

Novel stress granule-like structures are induced via a paracrine mechanism during viral infection.

To rapidly adapt to stresses such as 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 with RNA-binding proteins and signalling components into cytoplasmic biocondensates called stress granules (SGs). Increasing evidence suggests that SGs contribute to antiviral defence, and thus viruses need to evade these responses to propagate. We previously showed that feline calicivirus (FCV) impairs SG assembly by cleaving the scaffolding protein G3BP1. We also observed that uninfected bystander cells assembled G3BP1-positive granules, suggesting a paracrine response triggered by infection. We now present evidence that virus-free supernatant generated from infected cells can induce the formation of SG-like foci, which we name paracrine granules. They are linked to antiviral activity and exhibit specific kinetics of assembly-disassembly, and protein and RNA composition that are different from canonical SGs. We propose that this paracrine induction reflects a novel cellular defence mechanism to limit viral propagation and promote stress responses in bystander cells.

Murine Norovirus Infection Results in Anti-inflammatory Response Downstream of Amino Acid Depletion in Macrophages.

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.

Genome-wide analysis of the role of the antibiotic biosynthesis regulator AbsA2 in Streptomyces coelicolor A3(2).

The AbsA1-AbsA2 two component signalling system of Streptomyces coelicolor has 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 a ΔabsA2 deletion 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-complemented ΔabsA2 mutant strains were used in large-scale microarray-based time-course experiments to investigate the effect of deleting absA2 on gene expression and to identify the in vivo AbsA2 DNA-binding target sites using ChIP-on chip. We show that in addition to binding to the promoter regions of redZ and actII-orfIV AbsA2 binds to several previously unidentified sites within the cda biosynthetic gene cluster within and/or upstream of SCO3215-SCO3216, SCO3217, SCO3229-SCO3230, and SCO3226, 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 genes actII-orfIV and redZ and the absA2 gene 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 in Streptomyces coelicolor and the important role of AbsA2 in controlling the expression of three antibiotic biosynthetic gene clusters.

Circadian regulation in human white adipose tissue revealed by transcriptome and metabolic network analysis.

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.

REM sleep's unique associations with corticosterone regulation, apoptotic pathways, and behavior in chronic stress in mice.

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.

Identifying and validating blood mRNA biomarkers for acute and chronic insufficient sleep in humans: a machine learning approach.

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 (<6 hr) or sufficient sleep (~8.6 hr) to identify panels of mRNA biomarkers of sleep debt status. The size of identified panels ranged from 9 to 74 biomarkers. Panel performance, assessed by leave-one-subject-out cross-validation and independent validation, varied between sleep debt conditions. Using between-subject assessments based on one blood sample, the accuracy of classifying "acute sleep loss" was 92%, but only 57% for classifying "chronic sleep insufficiency." A reasonable accuracy for classifying "chronic sleep insufficiency" could only be achieved by a within-subject comparison of blood samples. Biomarkers for sleep debt status showed little overlap with previously identified biomarkers for circadian phase. Biomarkers for acute and chronic sleep loss also showed little overlap but were associated with common functions related to the cellular stress response, such as heat shock protein activity, the unfolded protein response, protein ubiquitination and endoplasmic reticulum-associated protein degradation, and apoptosis. This characteristic response of whole blood to sleep loss can further aid our understanding of how sleep insufficiencies negatively affect health. Further development of these novel biomarkers for research and clinical practice requires validation in other protocols and age groups.

Nuclear-cytoplasmic compartmentalization of the herpes simplex virus 1 infected cell transcriptome is co-ordinated by the viral endoribonuclease vhs and cofactors to facilitate the translation of late proteins.

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.