Syrian hamster convalescence from prototype SARS-CoV-2 confers measurable protection against the attenuated disease caused by the Omicron variant.

The mutation profile of the SARS-CoV-2 Omicron (lineage BA.1) variant posed a concern for naturally acquired and vaccine-induced immunity. We investigated the ability of prior infection with an early SARS-CoV-2 ancestral isolate (Australia/VIC01/2020, VIC01) to protect against disease caused by BA.1. We established that BA.1 infection in naïve Syrian hamsters resulted in a less severe disease than a comparable dose of the ancestral virus, with fewer clinical signs including less weight loss. We present data to show that these clinical observations were almost absent in convalescent hamsters challenged with the same dose of BA.1 50 days after an initial infection with ancestral virus. These data provide evidence that convalescent immunity against ancestral SARS-CoV-2 is protective against BA.1 in the Syrian hamster model of infection. Comparison with published pre-clinical and clinical data supports consistency of the model and its predictive value for the outcome in humans. Further, the ability to detect protection against the less severe disease caused by BA.1 demonstrates continued value of the Syrian hamster model for evaluation of BA.1-specific countermeasures.

Convalescence from prototype SARS-CoV-2 protects Syrian hamsters from disease caused by the Omicron variant

The mutation profile of the SARS-CoV-2 Omicron variant poses a concern for naturally acquired and vaccine-induced immunity. We investigated the ability of prior infection with an early SARS-CoV-2, 99.99% identical to Wuhan-Hu-1, to protect against disease caused by the Omicron variant. We established that infection with Omicron in naive Syrian hamsters resulted in a less severe disease than a comparable dose of prototype SARS-CoV-2 (Australia/VIC01/2020), with fewer clinical signs and less weight loss. We present data to show that these clinical observations were almost absent in convalescent hamsters challenged with the same dose of Omicron 50 days after an initial infection with Australia/VIC01/2020. The data provide evidence for immunity raised against prototype SARS-CoV-2 being protective against Omicron in the Syrian hamster model. Further investigation is required to conclusively determine whether Omicron is less pathogenic in Syrian hamsters and whether this is predictive of pathogenicity in humans.

ChAdOx1 nCoV-19 protection against SARS-CoV-2 in rhesus macaque and ferret challenge models.

Vaccines against SARS-CoV-2 are urgently required, but early development of vaccines against SARS-CoV-1 resulted in enhanced disease after vaccination. Careful assessment of this phenomena is warranted for vaccine development against SARS CoV-2. Here we report detailed immune profiling after ChAdOx1 nCoV-19 (AZD1222) and subsequent high dose challenge in two animal models of SARS-CoV-2 mediated disease. We demonstrate in rhesus macaques the lung pathology caused by SARS-CoV-2 mediated pneumonia is reduced by prior vaccination with ChAdOx1 nCoV-19 which induced neutralising antibody responses after a single intramuscular administration. In a second animal model, ferrets, ChAdOx1 nCoV-19 reduced both virus shedding and lung pathology. Antibody titre were boosted by a second dose. Data from these challenge models on the absence of enhanced disease and the detailed immune profiling, support the continued clinical evaluation of ChAdOx1 nCoV-19.

Comparison of rhesus and cynomolgus macaques as an infection model for COVID-19.

A novel coronavirus, SARS-CoV-2, has been identified as the causative agent of the current COVID-19 pandemic. Animal models, and in particular non-human primates, are essential to understand the pathogenesis of emerging diseases and to assess the safety and efficacy of novel vaccines and therapeutics. Here, we show that SARS-CoV-2 replicates in the upper and lower respiratory tract and causes pulmonary lesions in both rhesus and cynomolgus macaques. Immune responses against SARS-CoV-2 are also similar in both species and equivalent to those reported in milder infections and convalescent human patients. This finding is reiterated by our transcriptional analysis of respiratory samples revealing the global response to infection. We describe a new method for lung histopathology scoring that will provide a metric to enable clearer decision making for this key endpoint. In contrast to prior publications, in which rhesus are accepted to be the preferred study species, we provide convincing evidence that both macaque species authentically represent mild to moderate forms of COVID-19 observed in the majority of the human population and both species should be used to evaluate the safety and efficacy of interventions against SARS-CoV-2. Importantly, accessing cynomolgus macaques will greatly alleviate the pressures on current rhesus stocks.

Dose-dependent response to infection with SARS-CoV-2 in the ferret model and evidence of protective immunity.

There is a vital need for authentic COVID-19 animal models to enable the pre-clinical evaluation of candidate vaccines and therapeutics. Here we report a dose titration study of SARS-CoV-2 in the ferret model. After a high (5 × 106 pfu) and medium (5 × 104 pfu) dose of virus is delivered, intranasally, viral RNA shedding in the upper respiratory tract (URT) is observed in 6/6 animals, however, only 1/6 ferrets show similar signs after low dose (5 × 102 pfu) challenge. Following sequential culls pathological signs of mild multifocal bronchopneumonia in approximately 5-15% of the lung is seen on day 3, in high and medium dosed groups. Ferrets re-challenged, after virus shedding ceased, are fully protected from acute lung pathology. The endpoints of URT viral RNA replication & distinct lung pathology are observed most consistently in the high dose group. This ferret model of SARS-CoV-2 infection presents a mild clinical disease.

Prophylactic intranasal administration of a TLR2/6 agonist reduces upper respiratory tract viral shedding in a SARS-CoV-2 challenge ferret model.

BACKGROUND: The novel human coronavirus SARS-CoV-2 is a major ongoing global threat with huge economic burden. Like all respiratory viruses, SARS-CoV-2 initiates infection in the upper respiratory tract (URT). Infected individuals are often asymptomatic, yet highly infectious and readily transmit virus. A therapy that restricts initial replication in the URT has the potential to prevent progression of severe lower respiratory tract disease as well as limiting person-to-person transmission. METHODS: SARS-CoV-2 Victoria/01/2020 was passaged in Vero/hSLAM cells and virus titre determined by plaque assay. Challenge virus was delivered by intranasal instillation to female ferrets at 5.0 × 106 pfu/ml. Treatment groups received intranasal INNA-051, developed by Ena Respiratory. SARS-CoV-2 RNA was detected using the 2019-nCoV CDC RUO Kit and QuantStudio™ 7 Flex Real-Time PCR System. Histopathological analysis was performed using cut tissues stained with haematoxylin and eosin (H&E). FINDINGS: We show that prophylactic intra-nasal administration of the TLR2/6 agonist INNA-051 in a SARS-CoV-2 ferret infection model effectively reduces levels of viral RNA in the nose and throat. After 5 days post-exposure to SARS-CoV-2, INNA-051 significantly reduced virus in throat swabs (p=<0.0001) by up to a 24 fold (96% reduction) and in nasal wash (p=0.0107) up to a 15 fold (93% reduction) in comparison to untreated animals. INTERPRETATION: The results of our study support clinical development of a therapy based on prophylactic TLR2/6 innate immune activation in the URT, to reduce SARS-CoV-2 transmission and provide protection against COVID-19. FUNDING: This work was funded by Ena Respiratory, Melbourne, Australia.

Acellular Pertussis Vaccines Induce Anti-pertactin Bactericidal Antibodies Which Drives the Emergence of Pertactin-Negative Strains.

Despite high vaccination coverage, Bordetella pertussis the causative agent of whooping cough is still a health concern worldwide. A resurgence of pertussis cases has been reported, particularly in countries using acellular vaccines with waning immunity and pathogen adaptation thought to be responsible. A better understanding of protective immune responses is needed for the development of improved vaccines. In our study, B. pertussis strain B1917 variants presenting a single gene deletion were generated to analyze the role of vaccine components or candidate vaccine antigens as targets for bactericidal antibodies generated after acellular vaccination or natural infection. Our results show that acellular vaccination generates bactericidal antibodies that are only directed against pertactin. Serum bactericidal assay performed with convalescent samples show that disease induces bactericidal antibodies against Prn but against other antigen(s) as well. Four candidate vaccine antigens (CyaA, Vag8, BrkA, and TcfA) have been studied but were not targets for complement-mediated bactericidal antibodies after natural infection. We confirm that Vag8 and BrkA are involved in complement resistance and would be targeted by blocking antibodies. Our study suggests that the emergence and the widespread circulation of Prn-deficient strains is driven by acellular vaccination and the generation of bactericidal antibodies targeting Prn.

Prophylactic intranasal administration of a TLR2 agonist reduces upper respiratory tract viral shedding in a SARS-CoV-2 challenge ferret model

Respiratory viruses such as coronaviruses represent major ongoing global threats, causing epidemics and pandemics with huge economic burden. Rapid spread of virus through populations poses an enormous challenge for outbreak control. Like all respiratory viruses, the most recent novel human coronavirus SARS-CoV-2, initiates infection in the upper respiratory tract (URT). Infected individuals are often asymptomatic, yet highly infectious and readily transmit virus. A therapy that restricts initial replication in the URT has the potential to prevent progression of severe lower respiratory tract disease as well as limiting person-to-person transmission. We show that prophylactic intra-nasal administration of the TLR2/6 agonist INNA-051 in a SARS-CoV-2 ferret infection model effectively reduces levels of viral RNA in the nose and throat. The results of our study support clinical development of a therapy based on prophylactic TLR2/6 innate immune activation in the URT to reduce SARS-CoV-2 transmission and provide protection against COVID-19.

Comparison of Rhesus and Cynomolgus macaques as an authentic model for COVID-19.

A novel coronavirus, SARS-CoV-2, has been identified as the causative agent of the current COVID-19 pandemic. Animal models, and in particular non-human primates, are essential to understand the pathogenesis of emerging diseases and to the safety and efficacy of novel vaccines and therapeutics. Here, we show that SARS-CoV-2 replicates in the upper and lower respiratory tract and causes pulmonary lesions in both rhesus and cynomolgus macaques, resembling the mild clinical cases of COVID-19 in humans. Immune responses against SARS-CoV-2 were also similar in both species and equivalent to those reported in milder infections and convalescent human patients. Importantly, we have devised a new method for lung histopathology scoring that will provide a metric to enable clearer decision making for this key endpoint. In contrast to prior publications, in which rhesus are accepted to be the optimal study species, we provide convincing evidence that both macaque species authentically represent mild to moderate forms of COVID-19 observed in the majority of the human population and both species should be used to evaluate the safety and efficacy of novel and repurposed interventions against SARS-CoV-2. Accessing cynomolgus macaques will greatly alleviate the pressures on current rhesus stocks.

Dose-dependent response to infection with SARS-CoV-2 in the ferret model: evidence of protection to re-challenge

In December 2019 an outbreak of coronavirus disease (COVID-19) emerged in Wuhan, China. The causative agent was subsequently identified and named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which rapidly spread worldwide causing a pandemic. Currently there are no licensed vaccines or therapeutics available against SARS-CoV-2 but numerous candidate vaccines are in development and repurposed drugs are being tested in the clinic. There is a vital need for authentic COVID-19 animal models to further our understanding of pathogenesis and viral spread in addition to pre-clinical evaluation of candidate interventions. Here we report a dose titration study of SARS-CoV-2 to determine the most suitable infectious dose to use in the ferret model. We show that a high (5x106 pfu) and medium (5x104 pfu) dose of SARS-CoV-2 induces consistent upper respiratory tract (URT) viral RNA shedding in both groups of six challenged animals, whilst a low dose (5x102 pfu) resulted in only one of six displaying signs of URT viral RNA replication. The URT shedding lasted up to 21 days in the high dose animals with intermittent positive signal from day 14. Sequential culls revealed distinct pathological signs of mild multifocal bronchopneumonia in approximately 5-15% of the lung, observed on day 3 in high and medium dosed animals, with presence of mild broncho-interstitial pneumonia on day 7 onwards. No obvious elevated temperature or signs of coughing or dyspnoea were observed although animals did present with a consistent post-viral fatigue lasting from day 9-14 in the medium and high dose groups. After virus shedding ceased, re-challenged ferrets were shown to be fully protected from acute lung pathology. The endpoints of URT viral RNA replication in addition to distinct lung pathology and post viral fatigue were observed most consistently in the high dose group. This ferret model of SARS-CoV-2 infection presents a mild clinical disease (as displayed by 80% of patients infected with SARS-CoV-2). In addition, intermittent viral shedding on days 14-21 parallel observations reported in a minority of clinical cases.

PEITC-mediated inhibition of mRNA translation is associated with both inhibition of mTORC1 and increased eIF2α phosphorylation in established cell lines and primary human leukemia cells.

Increased mRNA translation drives carcinogenesis and is an attractive target for the development of new anti-cancer drugs. In this work, we investigated effects of phenethylisothiocyanate (PEITC), a phytochemical with chemopreventive and anti-cancer activity, on mRNA translation. PEITC rapidly inhibited global mRNA translation in human breast cancer-derived MCF7 cells and mouse embryonic fibroblasts (MEFs). In addition to the known inhibitory effects of PEITC on mTORC1 activity, we demonstrate that PEITC increased eIF2α phosphorylation. PEITC also increased formation of stress granules which are typically associated with eIF2α phosphorylation and accumulation of translationally stalled mRNAs. Analysis of genetically modified MEFs demonstrated that optimal inhibition of global mRNA translation by PEITC was dependent on eIF2α phosphorylation, but not mTORC1 inhibition. We extended this study into primary leukemic B cells derived from patients with chronic lymphocytic leukaemia (CLL). CLL cells were stimulated in vitro with anti-IgM to mimic binding of antigen, a major driver of this leukemia. In CLL cells, PEITC increased eIF2α phosphorylation, inhibited anti-IgM-induced mTORC1 activation and decreased both basal and anti-IgM-induced global mRNA translation. PEITC also inhibited transcription and translation of MYC mRNA and accumulation of the MYC oncoprotein, in anti-IgM-stimulated cells. Moreover, treatment of CLL cells with PEITC and the BTK kinase inhibitor ibrutinib decreased anti-IgM-induced translation and induced cell death to a greater extent than either agent alone. Therefore, PEITC can inhibit both global and mRNA specific translation (including MYC) via effects on multiple regulatory pathways. Inhibition of mRNA translation may contribute to the chemopreventive and anti-cancer effects of PEITC.

Natural product-derived antitumor compound phenethyl isothiocyanate inhibits mTORC1 activity via TSC2.

Phenethyl isothiocyanate (1) is a natural dietary phytochemical with cytostatic, cytotoxic, and antitumor activity. The effects of 1 were investigated on the activity of mTOR, a kinase that enhances the translation of many RNAs encoding proteins critical for cancer cell growth, including the angiogenesis regulator HIF1α. Compound 1 effectively blocked HIF1α RNA translation in MCF7 breast cancer cells, and this was associated with reduced phosphorylation of 4E-BP1 and p70 S6K, well-characterized downstream substrates of the mTOR-containing mTORC1 complex. Compound 1 also inhibited mTORC1 activity in mouse embryonic fibroblasts (MEFs). The 1-mediated inhibition of mTORC1 activity appeared to be independent of the upstream regulators PTEN, AKT, ERK1/2, and AMPK. By contrast, 1-mediated inhibition of mTORC1 activity was dependent on the presence of TSC2, part of a complex that regulates mTORC1 activity negatively. TSC2-deficient MEFs were resistant to 1-mediated inhibition of p70 S6K phosphorylation. TSC2-deficient MEFs were also partially resistant to 1-mediated growth inhibition. Overall, the present results confirm that 1 inhibits mTORC1 activity. This is dependent on the presence of TSC2, and inhibition of mTORC1 contributes to optimal 1-induced growth inhibition. Inhibition of RNA translation may be an important component of the antitumor effects of phenethyl isothiocyanate.

Differential induction of apoptosis in human breast cancer cell lines by phenethyl isothiocyanate, a glutathione depleting agent.

Phenethyl isothiocyanate (PEITC) is a naturally occurring electrophile which depletes intracellular glutathione (GSH) levels and triggers accumulation of reactive oxygen species (ROS). PEITC is of considerable interest as a potential chemopreventive/chemotherapeutic agent, and in this work, we have investigated the effects of PEITC on human breast cancer cell lines. Whereas PEITC readily induced apoptosis in MDA-MB-231 cells (associated with rapid activation of caspases 9 and 3, and decreased expression of BAX), MCF7 cells were relatively resistant to the apoptosis promoting effects of PEITC. The relative resistance of MCF7 cells was associated with high basal expression of NRF2, a transcription factor that coordinates cellular protective responses to oxidants and electrophiles and raised intracellular levels of GSH. This raised basal expression of NRF2 appeared to be a response to on-going production of ROS, since treatment with the antioxidant and GSH precursor N-acetylcysteine (NAC) reduced NRF2 expression. Moreover, pre-treatment of MDA-MB-231 cells with NAC rendered these cells relatively resistant to PEITC-induced apoptosis. In summary, our data confirm that PEITC may be an effective chemopreventive/therapeutic agents for breast cancer. However, differences in the basal expression of NRF2 and resultant changes in GSH levels may be an important determinant of sensitivity to PEITC-induced apoptosis.

Anti-angiogenic effects of dietary isothiocyanates: mechanisms of action and implications for human health.

Isothiocyanates (ITCs) are electrophilic compounds derived from plants and are thought to play a major role in the potential chemopreventive effects associated with high intake of cruciferous vegetables. ITCs are also being evaluated for chemotherapeutic activity in early phase clinical trials. In addition to their effects on carcinogen metabolism and cancer cell survival and proliferation, ITCs have been shown to effectively interfere with angiogenesis in vitro and in vivo. Angiogenesis is the development of a new blood supply from existing vasculature and is required for tumours to develop beyond a small size limit determined by the diffusion limit for oxygen. Inhibition of angiogenesis may play a key role in the potential chemopreventive/chemotherapeutic activity of ITCs. In this review we highlight recent data demonstrating that ITCs have anti-angiogenic activity and identify potential molecular targets for these effects, including hypoxia-inducible factor (HIF), nuclear factor κB (NF-κB), activator protein 1 (AP1) and tubulin. We also discuss these findings in light of the potential chemopreventive/chemotherapeutic effects of ITCs.

In vivo modulation of 4E binding protein 1 (4E-BP1) phosphorylation by watercress: a pilot study.

Dietary intake of isothiocyanates (ITC) has been associated with reduced cancer risk. The dietary phenethyl ITC (PEITC) has previously been shown to decrease the phosphorylation of the translation regulator 4E binding protein 1 (4E-BP1). Decreased 4E-BP1 phosphorylation has been linked to the inhibition of cancer cell survival and decreased activity of the transcription factor hypoxia-inducible factor (HIF), a key positive regulator of angiogenesis, and may therefore contribute to potential anti-cancer effects of PEITC. In the present study, we have investigated the in vitro and in vivo effects of watercress, which is a rich source of PEITC. We first demonstrated that, similar to PEITC, crude watercress extracts inhibited cancer cell growth and HIF activity in vitro. To examine the effects of dietary intake of watercress, we obtained plasma and peripheral blood mononuclear cells following the ingestion of an 80 g portion of watercress from healthy participants who had previously been treated for breast cancer. Analysis of PEITC in plasma samples from nine participants demonstrated a mean maximum plasma concentration of 297 nm following the ingestion of watercress. Flow cytometric analysis of 4E-BP1 phosphorylation in peripheral blood cells from four participants demonstrated significantly reduced 4E-BP1 phosphorylation at 6 and 8 h following the ingestion of watercress. Although further investigations with larger numbers of participants are required to confirm these findings, this pilot study suggests that flow cytometry may be a suitable approach to measure changes in 4E-BP1 phosphorylation following the ingestion of watercress, and that dietary intake of watercress may be sufficient to modulate this potential anti-cancer pathway.

Socioeconomic differences in physical disability at older age.

BACKGROUND: This study aims to investigate the association between socioeconomic position (SEP) and physical disability at older age, using a framework that incorporates education, social class and wealth. Wave One data from the English Longitudinal Study of Ageing are used. METHODS: Self-reported difficulties with activities of daily living, instrumental activities of daily living and motor skills were combined and categorised into "no disability", "mild disability" and "severe disability". The indicators of SEP used were wealth, education and social class. Multinomial regression was used to assess the associations between SEP and physical functioning reflecting the temporal relationship between education, social class and wealth. RESULTS: Men and women who had the highest levels of wealth, education and social class also had the lowest disability rates. The association was stronger in younger age groups and in men. The association of education with disability, which was found to be significant in the unadjusted models, was attenuated when adjusted for other factors such as occupation or wealth. This supports a temporal model of education feeding into occupation and then wealth. The association of SEP with disability was stronger for men and for men and women in the younger age group. CONCLUSIONS: Socioeconomic circumstances affect the prevalence and scale of physical disability even at older ages. In particular, wealth appears more important as a socioeconomic factor for physical disability than social class or education. Socioeconomic gradients in physical disability are greater for men than for women and for those in the younger age groups.

Cardiorespiratory risk factors as predictors of 40-year mortality in women and men.

OBJECTIVE: Most historical studies of cardiorespiratory risk factors as predictors of mortality have been based on men. This study examines whether they predict mortality over long periods in women and men. DESIGN: Prospective cohort study. SETTING: Participants were employees of the General Post Office. METHODS: Risk factor data were collected via clinical examination and questionnaire, 1966-7. Associations between cardiorespiratory risk factors and 40-year mortality were determined for 644 women and 1272 men aged 35-70 at examination. MAIN OUTCOME MEASURES: All-cause, cardiovascular (CVD), cancer and respiratory mortality. RESULTS: Associations between systolic blood pressure and all-cause and stroke mortality were equally strong for women and men, hazard ratio (95% confidence interval) 1.25 (1.1 to 1.4) and 1.18 (1.1 to 1.3); and 2.17 (1.7 to 2.8) and 1.69 (1.4 to 2.1), respectively. Cholesterol was higher in women and was associated with all-cause 1.22 (1.1 to 1.4) and CVD 1.39 (1.2 to 1.7) mortality, while associations between 2-hour glucose and all-cause 1.15 (1.1 to 1.2), coronary heart disease (CHD) 1.25 (1.1 to 1.4) and respiratory mortality 1.21 (1.0 to 1.5) were observed in men. Obesity was associated with stroke in women (2.42 (1.12 to 5.24)) and CHD in men (1.59 (1.02 to 2.49)), while ECG ischaemia was associated with CVD in both sexes. The strongest, most consistent predictor of mortality was smoking in women and poor lung function in men. However, evidence of sex differences in associations between the cardiorespiratory risk factors measured and mortality was sparse. CONCLUSIONS: Data from a 40-year follow-up period show remarkably persistent associations between risk factors and cardiorespiratory and all-cause mortality in women and men.

Inhibition of hypoxia inducible factor by phenethyl isothiocyanate.

Phenethyl isothiocyanate (PEITC), a natural dietary isothiocyanate, has anti-cancer activity in various in vitro and in vivo models. PEITC inhibits angiogenesis but the molecular mechanisms that underlie this effect are not known. We have now demonstrated that PEITC is an effective inhibitor of hypoxia inducible factor (HIF), a transcription factor that plays an important role in expression of pro-angiogenic factors. PEITC inhibited the activation of a HIF-dependent reporter construct following incubation of cells in hypoxia, or treatment with the hypoxia mimetic cobalt chloride. PEITC also interfered with the accumulation of HIF1alpha protein and induction of the endogenous HIF target genes, CAIX, GLUT1, BNIP3 and VEGF-A. The ability of PEITC to inhibit HIF activity was independent of the activity of prolyl hydroxylases, the Von-Hippel-Landau protein and the proteasome, all of which are required for the normal rapid turnover of HIF1alpha in normoxia. Decreased expression of HIF1alpha in PEITC treated cells was not associated with changes in the levels of HIF1alpha RNA suggesting that PEITC may inhibit HIF activity by decreasing translation of the HIF1alpha RNA. Consistent with this, PEITC decreased phosphorylation of the translation regulator 4E-BP1. Our data demonstrate that PEITC is an effective inhibitor of HIF activity. This may contribute to the anti-angiogenic and anti-cancer effects of PEITC.

Transcriptional regulation of plant senescence: from functional genomics to systems biology.

Leaf senescence is an active process that involves the increased expression of many hundreds of genes. Many putative transcription factors show enhanced transcription during leaf senescence in Arabidopsis and functional analysis of these should help to indicate their role in controlling gene expression during leaf senescence. In this paper, we describe the analysis of knockout insertion mutants in two different senescence-enhanced genes, one encodes a heat shock transcription factor and the other a zinc finger protein. Plants mutated in these genes show accelerated leaf senescence and reduced tolerance to drought stress, indicating that expression of these genes during senescence has a protective role to maintain viability during this essential developmental process. Analysis of gene expression changes in both mutants compared to the wild-type plants indicates an increased rate of senescence but does not show clearly the pathway that is dependent on these genes for expression. The complexities of signalling networks in plant stress and the plasticity of plant responses mean that the direct consequences of mutation are very difficult to define. The usefulness of this type of approach to address the burning question of how senescence is regulated is discussed, and an alternative approach aimed at a more global analysis of gene regulation using systems biology methods is described.