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T cell position in the tumor microenvironment determines the probability of target encounter and tumor killing. CD8+ T cell exclusion from the tumor parenchyma is associated with poor response to immunotherapy, and yet the biology that underpins this distinct pattern remains unclear. Here we show that the vascular destabilizing factor angiopoietin-2 (ANGPT2) causes compromised vascular integrity in the tumor periphery, leading to impaired T cell infiltration to the tumor core. The spatial regulation of ANGPT2 in whole tumor cross-sections was analyzed in conjunction with T cell distribution, vascular integrity, and response to immunotherapy in syngeneic murine melanoma models. T cell exclusion was associated with ANGPT2 upregulation and elevated vascular leakage at the periphery of human and murine melanomas. Both pharmacological and genetic blockade of ANGPT2 promoted CD8+ T cell infiltration into the tumor core, exerting antitumor effects. Importantly, the reversal of T cell exclusion following ANGPT2 blockade not only enhanced response to anti-PD-1 immune checkpoint blockade therapy in immunogenic, therapy responsive mouse melanomas, but it also rendered non-responsive tumors susceptible to immunotherapy. Therapeutic response after ANGPT2 blockade, driven by improved CD8+ T cell infiltration to the tumor core, coincided with spatial TIE2 signaling activation and increased vascular integrity at the tumor periphery where endothelial expression of adhesion molecules was reduced. These data highlight ANGPT2/TIE2 signaling as a key mediator of T cell exclusion and a promising target to potentiate immune checkpoint blockade efficacy in melanoma.

Background There are growing concerns about the impact of the COVID-19 pandemic on the mental health of older adults. We examined the effect of the pandemic on the risk of depression in older adults. Methods We analyzed data from the prospective cohort study of Korean older adults, which has been followed every 2 years. Among the 2308 participants who completed both the third and the fourth follow-up assessments, 58.4% completed their fourth follow-up before the outbreak of COVID-19 and the rest completed it during the pandemic. We conducted face-to-face diagnostic interviews using Mini International Neuropsychiatric Interview and used Geriatric Depression Scale. We performed generalized estimating equations and logistic regression analyses. Results The COVID-19 pandemic was associated with increased depressive symptoms in older adults [b (standard error) = 0.42 (0.20), p = 0.040] and a doubling of the risk for incident depressive disorder even in euthymic older adults without a history of depression (odds ratio = 2.44, 95% confidence interval 1.18–5.02, p = 0.016). Less social activities, which was associated with the risk of depressive disorder before the pandemic, was not associated with the risk of depressive disorder during the pandemic. However, less family gatherings, which was not associated with the risk of depressive disorder before the pandemic, was associated with the doubled risk of depressive disorder during the pandemic. Conclusions The COVID-19 pandemic significantly influences the risk of late-life depression in the community. Older adults with a lack of family gatherings may be particularly vulnerable.

Seizure is a common neurological presentation in patients visiting the emergency department (ED) that requires time for evaluation and observation. Timely decision and disposition standards for seizure patients need to be established to prevent overcrowding in the ED and achieve patients’ safety. Here, we conducted a retrospective cohort study to predict early seizure recurrence in the ED (ES-RED). We randomly assigned 688 patients to the derivation and validation cohorts (2:1 ratio). Prediction equations extracted routine clinical and laboratory information from EDs using logistic regression (Model 1) and machine learning (Model 2) methods. The prediction equations showed good predictive performance, the area under the receiver operating characteristics curve showing 0.808 in Model 1 [95% confidential interval (CI): 0.761–0.853] and 0.805 in Model 2 [95% CI: 0.747–0.857] in the derivation cohort. In the external validation, the models showed strong prediction performance of 0.739 [95% CI: 0.640–0.824] in Model 1 and 0.738 [95% CI: 0.645–0.819] in Model 2. Intriguingly, the lowest quartile group showed no ES-RED after 6 h. The ES-RED calculator, our proposed prediction equation, would provide strong evidence for safe and appropriate disposition of adult resolved seizure patients from EDs, reducing overcrowding and delays and improving patient safety.

ImportanceAlthough couples may share many risk factors for depressive disorders in their lifetime, whether these factors mediate the shared risk of depressive disorders has rarely been investigated.ObjectivesTo identify the shared risk factors for depressive disorder in couples and investigate their mediating roles in the shared risk of depressive disorders among older adult couples.Design, Setting, and ParticipantsThis nationwide, multicenter, community-based cohort study assessed 956 older adults from the Korean Longitudinal Study on Cognitive Aging and Dementia (KLOSCAD) and a cohort of their spouses (KLOSCAD-S) between January 1, 2019, to February 28, 2021.ExposuresDepressive disorders of the KLOSCAD participants.Main Outcomes and MeasuresThe mediating roles of shared factors in couples on the association between one spouse’s depressive disorder and the other’s risk of depressive disorders was examined using structural equation modeling.ResultsA total of 956 KLOSCAD participants (385 women [40.3%] and 571 men [59.7%]; mean [SD] age, 75.1 [5.0] years) and their spouses (571 women [59.7%] and 385 men [40.3%]; mean [SD] age, 73.9 [6.1] years) were included. The depressive disorders of the KLOSCAD participants were associated with an almost 4-fold higher risk of depressive disorders in their spouses in the KLOSCAD-S cohort (odds ratio, 3.89; 95% CI, 2.06-7.19; P P = .04; mediation proportion [MP] = 6.1%) and through chronic illness burden (β = 0.003; 95% CI, 0.000-0.006; P = .04; MP = 1.5%). Chronic medical illness burden (β = 0.025; 95% CI, 0.001-0.050; P = .04; MP = 12.6%) and presence of a cognitive disorder (β = 0.027; 95% CI, 0.003-0.051; P = .03; MP = 13.6%) mediated the association.Conclusions and RelevanceThe risk factors shared by older adult couples may mediate approximately one-third of the spousal risk of depressive disorders. Identification of and intervention in the shared risk factors of depression among older adult couples may reduce the risk of depressive disorders in the spouses of older adults with depression.

Combination treatment of BRAF inhibitor (BRAFi) and MEK inhibitor (MEKi) produces remarkable response rates and is expected to prevent ERK activation in BRAF-mutant melanoma. However, sporadic ERK reactivation occurs in melanoma models, and efficacy is severely limited by the emergence of drug resistance. The underlying molecular mechanisms that drive ERK reactivation and adaptation to BRAFi/MEKi remain elusive. Here, using single-cell imaging and optogenetic approaches, we show that the strength and duration of receptor tyrosine kinase (RTK) activation determines ERK reactivation and the development of BRAFi/MEKi-resistant cells. Single-cell data reveal that despite global RTK activation by the same external stimuli, only small subsets of melanoma cells induce effective RTK and ERK activation to develop BRAFi/MEKi resistance. Furthermore, by directly controlling RTK activity, we demonstrate that the kinetics of RTK activation determine ERK signaling dynamics and the percentage of drug-resistant cells. While these drug-resistant cells are rare during early stages of drug treatment, they eventually become the dominant population through sustained RTK-mediated ERK activation that drives cell proliferation. Thus, RTK activation in established drug-resistant cells causes effective ERK activation and cell proliferation. Importantly, limiting RTK signaling activation reverses the BRAFi/MEKi-resistant state. We show that targeting SHP2, a mediator of RTK signaling, effectively suppresses RTK-mediated ERK activation and cell proliferation in BRAFi/MEKi-resistant cells. Our results provide mechanistic insights into the contribution of heterogeneity in RTK activation kinetics to ERK reactivation and BRAFi/MEKi resistance, also highlighting SHP2 activity as a promising target to overcome drug resistance in BRAF-mutant melanoma. Citation Format: Sungsoo Kim, Richard Carvajal, Minah Kim, Hee Won Yang. Kinetics of RTK activation determine ERK signaling dynamics and resistance to BRAF and MEK inhibitors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3876.

T-cell position in the tumor microenvironment determines the probability of target encounter and tumor killing. CD8+ T-cell exclusion from the tumor parenchyma is associated with poor response to immunotherapy and yet the biology that underpins this distinct pattern remains unclear. Here we show that the vascular destabilizing factor angiopoietin-2 (ANGPT2) causes compromised vascular integrity in the tumor periphery, leading to impaired T-cell infiltration within tumor. We analyzed the spatial regulation of ANGPT2 in whole tumor cross-sections and its impact on CD8+ T-cell position, vascular integrity, and response to immunotherapy using syngeneic murine melanomas. We found that T-cell exclusion was associated with peritumoral expression of ANGPT2 and elevated vascular leakage in human and murine melanomas. Both pharmacological and genetic blockade of ANGPT2 promoted CD8+ T-cell infiltration to the tumor core, exerting antitumor effects. Importantly, the reversal of T-cell exclusion following ANGPT2 blockade not only enhanced response to anti-PD-1 therapy in immunogenic, therapy responsive mouse melanomas, but also rendered non-responsive tumors susceptible. Therapeutic response after ANGPT2 blockade, driven by improved CD8+ T-cell infiltration, coincided with TIE2 signaling activation and increased vascular integrity in the tumor periphery, indicating that maintenance of vascular integrity within tumors is necessary for robust response to immunotherapy. These data highlight ANGPT2/TIE2 signaling as a key mediator of T-cell exclusion and a promising target to potentiate checkpoint inhibitor efficacy in melanoma. Citation Format: Ha-Ram Park, Anahita Shiva, Portia Cummings, Richard Carvajal, Gavin Thurston, Joon-Yong An, Amanda W. Lund, Hee Won Yang, Minah Kim. Vascular regulation of T-cell exclusion in melanoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4588.

Although couples could share many risk factors of cognitive disorders in their lifetime, whether shared risk factors mediate the shared risk of cognitive disorders has rarely been investigated.To identify the risk factors of cognitive decline shared within couples and investigate their mediating roles in the shared risk of cognitive disorders and cognitive functions within couples.A prospective cohort study was launched in November 1, 2010, and 784 participants were followed up every 2 years until December 31, 2020. This nationwide, multicenter, community-based study included older couples from the Korean Longitudinal Study on Cognitive Aging and Dementia (KLOSCAD) and a cohort of their spouses (KLOSCAD-S).The cognitive disorder of a spouse was defined as mild cognitive impairment or dementia.The mediating roles of factors shared within couples on the association between one spouse's cognitive disorder and the other's risk of cognitive disorders was examined with structural equation modeling.Included were 784 KLOSCAD participants (307 women [39.2%] and 477 men [60.8%]; mean [SD] age, 74.8 [4.8] years) and their spouses (477 women [60.8%] and 307 men [39.2%]; mean [SD] age, 73.6 [6.2] years). The cognitive disorder of the KLOSCAD participants was associated with almost double the risk of cognitive disorder of their spouses in the KLOSCAD-S cohort (odds ratio, 1.74; 95% CI, 1.12-2.69; P = .01). History of head injury (β = 0.50; 95% CI, 0.09-0.90; P = .02) and age (β = 2.57; 95% CI, 1.37-3.76; P .001) mediated the association between cognitive disorder in the KLOSCAD participants and their spouses' risk of cognitive disorder. Physical inactivity mediated the association through major depressive disorder (β = 0.33, 95% CI, 0.09-0.57, P = .006 for physical inactivity; β = 0.28, 95% CI, 0.13-0.44, P .001 for major depressive disorder). These factors similarly mediated the association between spousal cognitive disorder and cognitive functions such as memory and executive function.These findings suggest that the risk factors shared within couples may mediate approximately three-quarters of the spousal risk of cognitive disorders. Identification of and intervention in the shared risk factors of dementia within couples may reduce the risk of cognitive disorders in the spouses of people with dementia.

Widespread ice shelf thinning has been recorded in the Amundsen Sea in recent decades, driven by basal melting and intrusions of relatively warm Circumpolar Deep Water (CDW) onto the continental shelf. The Dotson Ice Shelf (DIS) is located to the south of the Amundsen Sea polynya, and has a high basal melting rate because modified CDW (mCDW) fills the Dotson-Getz Trough (DGT) and reaches the base of the ice shelf. Here, hydrographic data in the DGT obtained during seven oceanographic surveys from 2007 to 2018 were used to study the interannual variation in mCDW volume and properties and their causes. Although mCDW volume showed relatively weak interannual variations at the continental shelf break, these variations intensified southward and reached a maximum in front of the DIS. There, the mCDW volume was ~8,000 km3 in 2007, rapidly decreased to 4,700 km3 in 2014 before rebounding to 7,300 km3 in 2018. We find that such interannual variability is coherent with local Ekman pumping integrated along the DGT modulated by the presence of sea ice, and complementing earlier theories involving shelf break winds only. The interannual variability in strength of the dominant south-southeast coastal wind modulates the amplitude of Ekman upwelling along the eastern boundary of the Amundsen Sea polynya during the austral summers of the surveyed years, apparently leading to change in the volume of mCDW along the DGT. We note a strong correlation between the wind variability and the longitudinal location of the Amundsen Sea Low.

The association between serum free hemoglobin (sfHb) level and white matter hyperintensity (WMH) volume is controversial. This study is to examine this association considering nonlinearity, sex dimorphism, and WMH type. We enrolled 704 older adults among the participants of the Korean Longitudinal Study on Cognitive Aging and Dementia and visitors to the Dementia Clinic of Seoul National University Bundang Hospital. We measured sfHb level in the venous blood and WMH volume (VWMH) using fluid-attenuated inversion recovery magnetic resonance images. The association between sfHb level and periventricular VWMH was linear in men (linear regression; β = − 0.18, p = 0.006) and U-shaped in women (restricted cubic spline; F = 6.82, p WMH in either sex. These findings were also observed in participants without anemia. To conclude, sfHb level is associated with periventricular VWMH in older adults of both sexes. Maintaining an optimal sfHb level may contribute to the prevention of WMH.

Objective This study evaluated the outcomes of ischemic stroke patients according to delirium motor subtype. Methods This study included patients who were admitted to the stroke unit between August 2017 and March 2019 and met the DSM-5 diagnostic criteria for delirium. Patients were assessed twice weekly throughout their delirium episodes using the Korean version of the Delirium Motor Subtype Scale (K-DMSS) and the Korean version of the Delirium Rating Scale-Revised-98 (K-DRS-98). The clinical characteristics and short-term outcomes of the patients were also assessed. Results A total of 943 stroke patients were included; the rate of incident delirium was 10.18%. Of the 95 delirium patients, 34 were classified as the hyperactive subtype, 30 as the mixed subtype, 25 as the hypoactive and six as no subtype. Among the subtype groups, the hypoactive subtype had the highest initial scores on the National Institutes of Health Stroke Scale (NIHSS; 6.72±4.75, p=0.02) and the modified Rankin Scale (mRS; 3.96±1.24, p

Objective: The effects of mood disorders on mortality may be mediated by their effects on the risk of dementia, and interventions to reduce the occurrence of dementia may reduce their overall mortality. This study aimed to investigate the direct effects of depressive and bipolar disorders on the 6-year risk of mortality and also their indirect effects on mortality due to their effect on the risk of dementia. Methods: A total of 5101 Koreans were selected from a community-based prospective cohort study, and 6-year risks of mortality and dementia in participants with depressive and bipolar disorders were estimated by Cox proportional hazard analysis. The direct and indirect effects of depressive and bipolar disorders on the risk of mortality were estimated using structural equation modeling. Results: The depressive and bipolar disorder groups showed 51% and 85% higher 6-year mortality, and 82% and 127% higher risk of dementia, respectively, compared to euthymic controls. The effects of depressive and bipolar disorders on mortality were mainly mediated by their effects on the risk of dementia in a structural equation model. The direct effects of each mood disorder on mortality were not significant. Conclusion: Both depressive and bipolar disorders increased the risks of mortality and dementia, and the effects of mood disorders on mortality were mainly mediated through dementia. As dementia occurs later in life than mood disorders, measures to prevent it may effectively reduce mortality in individuals with a history of mood disorders, as well as being more feasible than attempting to control other causes of death.

We describe a mammalian cell-based assay to identify coronavirus 3CL protease (3CLpro) inhibitors. This assay is based on rescuing protease-mediated cytotoxicity and does not require live virus. By enabling the facile testing of compounds across a range of 15 distantly related coronavirus 3CLpro enzymes, we identified compounds with broad 3CLpro-inhibitory activity. We also adapted the assay for use in compound screening and in doing so uncovered additional severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3CLpro inhibitors. We observed strong concordance between data emerging from this assay and those obtained from live-virus testing. The reported approach democratizes the testing of 3CLpro inhibitors by developing a simplified method for identifying coronavirus 3CLpro inhibitors that can be used by the majority of laboratories, rather than the few with extensive biosafety infrastructure. We identified two lead compounds, GC376 and compound 4, with broad activity against all 3CL proteases tested, including 3CLpro enzymes from understudied zoonotic coronaviruses. IMPORTANCE Multiple coronavirus pandemics have occurred over the last 2 decades. This has highlighted a need to be proactive in the development of therapeutics that can be readily deployed in the case of future coronavirus pandemics. We developed and validated a simplified cell-based assay for the identification of chemical inhibitors of 3CL proteases encoded by a wide range of coronaviruses. This assay is reporter free, does not require specialized biocontainment, and is optimized for performance in high-throughput screening. By testing reported 3CL protease inhibitors against a large collection of 3CL proteases with variable sequence similarity, we identified compounds with broad activity against 3CL proteases and uncovered structural insights into features that contribute to their broad activity. Furthermore, we demonstrated that this assay is suitable for identifying chemical inhibitors of proteases from families other than 3CL proteases.

Background: We evaluated whether the brain regional features could enhance the prediction of the occurrence of the post-stroke delirium along with consideration of clinical features in acute ischemic stroke patients. Method: We analyzed magnetic resonance imaging and clinical datasets prospectively collected from 1,024 acute ischemic stroke patients (620 men; mean age±SD, 70.2±11.8 years), who were admitted in stroke unit. Delirium occurrence in the patients was initially screened with Confusion Assessment Method and finally diagnosed using the criteria of the Diagnostic and Statistical Manual of Mental Disorders (fifth edition). The prediction performance of the clinical features was evaluated using the logistic regression analysis, and then, compared with the performance obtained after adding of the brain lesional features. Results: Post-stroke delirium occurred in 100 (9.8%) of the total patients. Independent features of the clinical features were old age (odds ratio [OR] for the delirium prediction: 1.07, 95% confidence interval [CI] 1.038-1.103), preexisting dementia (OR 0.423, CI 0.209-0.853) and atrial fibrillation OR 1.599, CI 0.938-2.726), neglect (OR 2.395, CI 1.152-4.976), visual defect (OR 2.209, CI 0.994-4.907), diplopia (OR 2.42, CI 1.021-5.738), dysuria (OR 2.404, 95% CI 1.155-5.000), Foley catheter insertion (OR 0.46, CI 0.24-0.881) and pneumonia (OR 1.954, CI 1.004-3.803). Right-sided hemispheric and mixed acute lesions (p=0.003), lesion locations in occipital lobe (OR 1.814, CI 0.923-3.564), cerebellum (OR 2.875, CI 1.32-6.258) and brainstem (OR 4.057, CI 1.714-9.601), and the presence of the periventricular white matter change (OR 1.321, CI 0.964-1.811) increased the risk for delirium. Sensitivity to predict the delirium of the regression model was enhanced from 10% obtained with the clinical features to 18% by adding the brain lesional features. Area under the curve of the receiver operating characteristics analysis was also enhanced from 0.827 with the clinical features to 0.847 after adding the brain lesions to the clinical features. Conclusion: The present study shows brain lesional characteristics that could enhance the prediction of the post-stroke delirium in acute ischemic stroke patients.

ImportanceThe association between social support and dementia risk has been debated. Most previous prospective studies have not differentiated the subtypes of social support.ObjectiveTo examine whether the association between social support and risk of dementia differs by subtype of social support and by sex.Design, Setting, and ParticipantsThis nationwide prospective cohort study included randomly sampled South Korean adults 60 years or older. The study was launched November 1, 2010, with follow-up every 2 years until November 30, 2020. The 5852 participants who completed the assessment for social support and were not diagnosed as having dementia, severe psychiatric disorders including major depressive disorder, or major neurological disorders at the baseline assessment were included in the analysis.ExposuresGeriatric psychiatrists administered the structured diagnostic interviews and physical examinations to every participant based on the Korean version of the Consortium to Establish a Registry for Alzheimer Disease (CERAD-K) Assessment Packet Clinical Assessment Battery.Main Outcomes and MeasuresBaseline levels of emotional and tangible support using the Medical Outcomes Survey Social Support Survey.ResultsAmong the 5852 participants (mean [SD] age, 69.8 [6.6] years; 3315 women [56.6%]; mean [SD] follow-up duration, 5.9 [2.4] years), 237 (4.0%) had incident all-cause dementia and 160 (2.7%) had incident Alzheimer disease (AD) subtype of dementia. Compared with women who reported having emotional support, those with low emotional support had almost a 2-fold higher incidence of all-cause dementia (18.4 [95% CI, 13.6-23.2] vs 10.7 [95% CI, 9.0-12.5] per 1000 person-years) and AD (14.4 [95% CI, 10.2-18.6] vs 7.8 [95% CI, 6.3-9.3] per 1000 person-years). Adjusted Cox proportional hazard analysis revealed that low emotional support was associated with increased risk of all-cause dementia (hazard ratio, 1.61 [95% CI, 1.10-2.36]; P = .02) and AD (hazard ratio, 1.66 [95% CI, 1.07-2.57]; P = .02) only in women. Low tangible support was not associated with a risk of all-cause dementia or AD regardless of sex.Conclusions and RelevanceThe findings of this cohort study suggest that older women with low emotional support constitute a population at risk for dementia. The level of emotional support should be included in risk assessments of dementia.

Recent rapid thinning of West Antarctic ice shelves are believed to be caused by intrusions of warm deep water that induce basal melting and seaward meltwater export. Dotson Ice Shelf has a high basal melt rate due to southward ocean heat transport in the Dotson-Getz Trough. We deployed three bottom-moored instrument arrays along the ice shelf calving front, obtaining continuous records of temperature, salinity, and current velocity throughout 2014 and 2015. Southward deep water velocities were highest along the eastern channel slope, while northward outflows of freshened ice shelf meltwater spread at intermediate depth above the western slope. Inflow warm water along the eastern slope into the sub-Dotson cavity reached a maximum of 182 MW m− 1 in Summer, 3.5 times larger than the autumn/winter values of 51 MW m− 1. The inflow correlated with the local ocean surface stress curl. At the western slope meltwater outflows were strongest during autumn and weakest in spring, following the warm influx along the eastern slope with a ~ 2–3 months delay. Ocean circulation near Dotson Ice Shelf, affected by sea ice distribution and wind, appears to be a significant control on the inflow of warm water and subsequent ice shelf melting on seasonal time-scales.

Background: The effects of hyperbaric oxygen therapy (HBOT) in sepsis remain unclear. This study evaluated its effects on acute liver injury and survival in a rat model. Methods: Cecal slurry peritonitis was induced in male rats, which were then randomly allocated into the HBOT and control groups. In the survival experiment, six 90 min HBOT sessions (2.6 atmospheres absolute 100% oxygen) were performed over 48 h, the survival rate was determined 14 days after sepsis induction. In the acute liver injury experiment, three HBOT sessions were performed, followed by liver and plasma harvesting, 24 h after sepsis induction. Serum levels of alanine aminotransferase (ALT), interleukin (IL)-6, and IL-10 were measured, and the hepatic injury scores were determined. Reactive oxygen species (ROS) generation was detected by 2&prime, 7&prime, dihydrodichlorofluorescein diacetate (H2DCF-DA) assay. Western blot assays assessed protein kinase B (Akt), phosphorylated-Akt (p-Akt), glycogen synthase kinase (GSK)-3&beta, phosphorylated-GSK-3&beta, and cleaved caspase-3 levels. Results: Survival in the HBOT group (57.1%) was significantly higher than that in the controls (12.5%, p = 0.029), whereas IL-6, IL-10, and ALT levels were significantly lower in the HBOT group. The ROS generation was significantly inhibited to a greater extent in the HBOT group than in the control group. Additionally, in the HBOT group, the p-Akt and p-GSK-3&beta, increased significantly and cleaved caspase-3 levels decreased significantly. Conclusions: HBOT showed a beneficial effect on acute liver injury and rat survival by enhancing the Akt signaling pathway and decreasing apoptosis.

Cell-cycle entry relies on an orderly progression of signaling events. To start, cells first activate the kinase cyclin D-CDK4/6, which leads to eventual inactivation of the retinoblastoma protein Rb. Hours later, cells inactivate APC/CCDH1 and cross the final commitment point. However, many cells with genetically deleted cyclin Ds, which activate and confer specificity to CDK4/6, can compensate and proliferate. Despite its importance in cancer, how this entry mechanism operates remains poorly characterized, and whether cells use this path under normal conditions remains unknown. Here, using single-cell microscopy, we demonstrate that cells with acutely inhibited CDK4/6 enter the cell cycle with a slowed and fluctuating cyclin E-CDK2 activity increase. Surprisingly, with low CDK4/6 activity, the order of APC/CCDH1 and Rb inactivation is reversed in both cell lines and wild-type mice. Finally, we show that as a consequence of this signaling inversion, Rb inactivation replaces APC/CCDH1 inactivation as the point of no return. Together, we elucidate the molecular steps that enable cell-cycle entry without CDK4/6 activity. Our findings not only have implications in cancer resistance, but also reveal temporal plasticity underlying the G1 regulatory circuit., How normal cells proliferate without CDK4 and CDK6, two cancer-driving kinases, remains unclear. Here, the authors show that without CDK4/6 activity, cells start the cell cycle with a different signaling order and commitment point, revealing unexpected flexibility in cell-cycle entry mechanisms.

We describe a mammalian cell-based assay capable of identifying coronavirus 3CL protease (3CLpro) inhibitors without requiring the use of live virus. By enabling the facile testing of compounds across a range of coronavirus 3CLpro enzymes, including the one from SARS-CoV-2, we are able to quickly identify compounds with broad or narrow spectra of activity. We further demonstrate the utility of our approach by performing a curated compound screen along with structure-activity profiling of a series of small molecules to identify compounds with antiviral activity. Throughout these studies, we observed concordance between data emerging from this assay and from live virus assays. By democratizing the testing of 3CL inhibitors to enable screening in the majority of laboratories rather than the few with extensive biosafety infrastructure, we hope to expedite the search for coronavirus 3CL protease inhibitors, to address the current epidemic and future ones that will inevitably arise.