Sweet corn phytoglycogen dendrimers as a lyoprotectant for dry-state protein storage.

Protein biotherapeutics typically require expensive cold-chain storage to maintain their fold and function. Packaging proteins in the dry state via lyophilization can reduce these cold-chain requirements. However, formulating proteins for lyophilization often requires extensive optimization of excipients that both maintain the protein folded state during freezing and drying (i.e., "cryoprotection" and "lyoprotection"), and form a cake to carry the dehydrated protein. Here we show that sweet corn phytoglycogens, which are glucose dendrimers, can act as both a protein lyoprotectant and a cake-forming agent. Phytoglycogen (PG) dendrimers from 16 different maize sources (PG1-16) were extracted via ethanol precipitation. PG size was generally consistent at ~70-100 nm for all variants, whereas the colloidal stability in water, protein contaminant level, and maximum density of cytocompatibility varied for PG1-16. 10 mg/mL PG1, 2, 9, 13, 15, and 16 maintained the activity of various proteins, including green fluorescent protein, lysozyme, ß-galactosidase, and horseradish peroxidase, over a broad range of concentrations, through multiple rounds of lyophilization. PG13 was identified as the lead excipient candidate as it demonstrated narrow dispersity, colloidal stability in phosphate-buffered saline, low protein contaminants, and cytocompatibility up to 10 mg/mL in NIH3T3 cell cultures. All dry protein-PG13 mixtures had a cake-like appearance and all frozen protein-PG13 mixtures had a Tg' of ~ -26°C. The lyoprotection and cake-forming properties of PG13 were density-dependent, requiring a minimum density of 5 mg/mL for maximum activity. Collectively these data establish PG dendrimers as a new class of excipient to formulate proteins in the dry state.

Mueller polarimetric imaging as a tool for detecting the effect of non-thermal plasma treatment on the skin.

Non-thermal plasma (NTP) is a promising technique studied for several medical applications such as wound healing or tumor reduction. The detection of microstructural variations in the skin is currently performed by histological methods, which are time-consuming and invasive. This study aims to show that full-field Mueller polarimetric imaging is suitable for fast and without-contact detection of skin microstructure modifications induced by plasma treatment. Defrosted pig skin is treated by NTP and analyzed by MPI within 30 minutes. NTP is shown to modify the linear phase retardance and the total depolarization. The tissue modifications are inhomogeneous and present distinct features at the center and the fringes of the plasma-treated area. According to control groups, tissue alterations are primarily caused by the local heating concomitant to plasma-skin interaction.

Implementing CAPABLE With Care Partners Through an Area Agency on Aging: Identifying Barriers and Facilitators Using the Consolidated Framework for Implementation Research.

BACKGROUND AND OBJECTIVES: Community Aging in Place, Advancing Better Living for Elders (CAPABLE) is an evidence-based intervention to promote aging in place. Although CAPABLE has been implemented in more than 40 community sites, wide variation in implementation exists. Guided by the Consolidated Framework for Implementation Research (CFIR), this study sought to determine key barriers and facilitators that may influence CAPABLE implementation with older adult and care partner dyads through an area agency on aging (AAA). RESEARCH DESIGN AND METHODS: A formative evaluation was completed using qualitative data from the pilot of a Hybrid Trial Type 1 study implementing CAPABLE in an AAA. Multiple sources of data were collected, including 2 focus groups, field notes, a tracking log, and meetings with CAPABLE interventionists. Data were analyzed using a framework method and validated through a negative case analysis approach in NVivo 12 Pro. RESULTS: Fourteen dyads enrolled in the pilot and 6 completed the CAPABLE intervention. Key themes aligned with 10 constructs from 5 domains of the CFIR. Facilitators included adaptability of the intervention, cost, networks and communication, and knowledge and belief of individuals. Barriers included intervention complexity, client needs and resources, and executing the planned process. DISCUSSION AND IMPLICATIONS: Results enhance understanding of contextual factors that can influence the implementation of CAPABLE with care partners. Strategies to overcome barriers include simplifying recruitment materials and targeting older adults with recent onset of disability. The CFIR is a valuable resource for planning and evaluation of the implementation of evidence-based interventions to promote aging in place.

Improving Inclusion for Children With Special Health Care Needs in Childcare Settings: Results From a Statewide Survey of Childcare Professionals.

INTRODUCTION: Families of children with special health care needs (CSHCN) routinely struggle to find suitable childcare. These families value childcare capable of supporting their child's needs, known as inclusive care, that requires trustworthy staff trained to safely care for CSHCN. METHOD: This study aimed to understand the training, experience, confidence, and perceived barriers to providing inclusive care for CSHCN from the perspective of childcare providers and directors. Childcare providers (n = 263) and directors (n = 418) responded to a statewide survey in Wisconsin in 2019. RESULTS: Results show that both childcare providers and directors perceive that the providers lack confidence in providing inclusive care. Lack of staffing, training, and expertise of childcare providers were the most frequently reported barriers to provide inclusive care. DISCUSSION: These findings confirm the need for additional education, training, resources, and research to create and maintain more inclusive childcare settings for CSHCN.

Prophylactic efficacy of Lactobacillus curvatus B67-derived postbiotic and quercetin, separately and combined, against Listeria monocytogenes and Salmonella enterica ser. Typhimurium on processed meat sausage.

This study investigated the antimicrobial and antibiofilm efficacy of separate and combined treatments of Lactobacillus curvatus B67-produced postbiotic and the polyphenolic flavanol quercetin against Listeria monocytogenes and Salmonella enterica ser. Typhimurium. The antimicrobial potentiality of the postbiotic was chiefly associated with organic acids (e.g., lactic and acetic acids). At sub-minimum inhibitory concentration (1/2 MIC), the postbiotic and quercetin effectively reduced the pathogenic biofilm cells on processed pork sausage and meat-processing surfaces (e.g., stainless-steel and rubber). Moreover, the postbiotic exhibited strong residual antimicrobial efficacy over diverse pH and temperature ranges. In addition, the combination of postbiotic with quercetin increased the leakage of pathogenic intracellular metabolites (e.g., nucleic acids and protein) and inhibited pathogenic biofilm formation on both biotic and abiotic surfaces. Therefore, this study confirmed that lactic acid bacteria-derived postbiotic and plant-derived quercetin could be used as potential alternative bioprotective agents in the meat processing industry.

Highly efficient and stable deep-blue organic light-emitting diode using phosphor-sensitized thermally activated delayed fluorescence.

Phosphorescent and thermally activated delayed fluorescence (TADF) blue organic light-emitting diodes (OLEDs) have been developed to overcome the low efficiency of fluorescent OLEDs. However, device instability, originating from triplet excitons and polarons, limits blue OLED applications. Here, we develop a phosphor-sensitized TADF emission system with TADF emitters to achieve high efficiency and long operational lifetime. Peripheral carbazole moieties are introduced in conventional multi-resonance-type emitters containing one boron atom. The triplet exciton density of the TADF emitter is reduced by facilitating reverse intersystem crossing, and the Förster resonant energy transfer rate from phosphor sensitizer is enhanced by high absorption coefficient of the emitters. The emitter exhibited an operational lifetime of 72.9 hours with Commission Internationale de L'Eclairage chromaticity coordinate y = 0.165, which was 6.6 times longer than those of devices using conventional TADF emitters.

Depolarization imaging for fast and non-invasive monitoring of cervical microstructure remodeling in vivo during pregnancy.

The cervix plays a crucial role in conception, maintenance of pregnancy, and childbirth. The mechanical properties of a pregnant woman's cervix change dramatically during gestation due to a remodeling of its microstructure, necessary for delivery. However, external factors can accelerate this process and lead to prematurity, the primary cause of perinatal mortality worldwide, due to the inefficiency of existing diagnostic methods. This study shows that polarized light is a powerful tool to probe the cervical microstructure during pregnancy. A wide-field multispectral polarimetric imaging system was fabricated to explore in vivo the cervix of full-term pregnant women. The polarimetric properties of the cervix change significantly with pregnancy progression. In particular, a set of several depolarization parameters (intrinsic and extrinsic) showed a strong linear correlation with gestational age in the red part of the visible spectral range. This trend can be attributed, among other things, to a decrease in collagen density and an increase in hydration of cervical connective tissue. Wide field depolarization imaging is a very promising tool for rapid and non-invasive analysis of cervical tissue in vivo to monitor the steady progression of pregnancy, providing the practitioner with useful information to improve the detection of preterm birth.

Antibiofilm efficacy of Leuconostoc mesenteroides J.27-derived postbiotic and food-grade essential oils against Vibrio parahaemolyticus, Pseudomonas aeruginosa, and Escherichia coli alone and in combination, and their application as a green preservative in the seafood industry.

Foodborne pathogen-mediated biofilms in food processing environments are severe threats to human lives. In the interest of human and environmental safety, natural substances with antimicrobial properties and generally regarded as safe (GRAS) status are the futuristic disinfectants of the food industry. In this study, the efficacy of bioactive, soluble products (metabolic by-products) from lactic acid bacteria (LAB) and plant-derived essential oils (EO) were investigated as biocidal agents. The postbiotic produced by kimchi-derived Leuconostoc mesenteroides J.27 isolate was analyzed for its metabolic components to reveal its antimicrobial potential against three pathogenic microorganisms (Vibrio parahaemolyticus, Pseudomonas aeruginosa, and Escherichia coli). Additionally, the efficacy of food-grade EO (eugenol and thymol, respectively) was also assessed in our study. Determination of the minimum inhibitory concentration (MIC) of postbiotic and EO against three tested pathogens revealed that the sub-MIC (0.5 MIC) of postbiotic and EO could efficiently inhibit the biofilm formation on both seafood (squid) and seafood-processing surfaces (rubber and low-density polyethylene plastic). Moreover, the polymerase chain reaction (PCR) analysis confirmed that the LAB J.27 isolate possesses bacteriocin- and enzyme-coding genes. The residual antibacterial activity of the produced postbiotic was maintained over a diverse pH range (pH 1-6) but was entirely abolished at neutral or higher pH values. However, the activity was unaffected by exposure to high temperatures (100 and 121 °C) and storage (30 days). Notably, the leakage of intracellular metabolites, damage to DNA, and the down-regulation of biofilm-associated gene expression in the pathogens increased significantly (p > 0.05) following the combination treatment of postbiotic with thymol compared to postbiotic with eugenol. Nonetheless, all in vitro results indicated the prospective use of combining Leu. mesenteroides J.27-derived postbiotic with both EO as a "green preservative" in the seafood industry to inhibit the formation of pathogenic microbial biofilms.

Efficacy of ficin and peroxyacetic acid against Salmonella enterica serovar Thompson biofilm on plastic, eggshell, and chicken skin.

Salmonella is the leading cause of zoonotic foodborne illnesses worldwide and a prevalent threat to the poultry industry. For controlling contamination, the use of chemical sanitizers in combination with biological compounds (e.g., enzymes) offers a solution to reduce the chemical residues. The current study investigated the biofilm reduction effects of a food-grade enzyme-ficin-and a common sanitizer-peroxyacetic acid (PAA)-against an emerging pathogen, Salmonella enterica ser. Thompson, on plastic, eggshell, and chicken skin surfaces. Results showed that PAA could kill S. Thompson, but ficin cannot. Maximum biofilm reduction was 3.7 log CFU/cm2 from plastic after individual treatment with PAA. However, sequential treatment of ficin and PAA led to biofilm reductions of 3.2, 5.0, and 6.5 log CFU/cm2 from chicken skin, eggshell, and plastic, respectively. Fourier-transform infrared spectroscopy and microscopic analysis confirmed that ficin increased PAA action, causing biofilm matrix destruction. Moreover, the quality of the food surfaces was only altered by 12.5 U/mL ficin and was not altered by PAA. This combined use of enzyme and sanitizer solved major safety issues and proved promising against S. Thompson-associated contaminations in poultry and poultry processing lines.

Are high medical costs incurred by people with disabilities excessive?: An empirical analysis of Korean National Health Insurance Data.

A crude comparison of medical costs between people with disabilities (PWD) and without disabilities (PWoD) shows a much higher expenditure among PWD and such results have been a cause for further stigmatization. This study aims to empirically analyze whether the medical costs for PWD are actually high when characteristics related to medical costs are adjusted. Ten percent of the total population was randomly selected from the Korean National Health Insurance (NHI) Database in 2016. A crude comparative analysis was performed to calculate the medical cost of PWD and PWoD. A subsequent multiple regression analysis was conducted to adjust factors affecting the medical costs such as socioeconomic status, disease, and health behavior-related characteristics. The medical cost for PWD was 3.6 times higher than that for PWoD by crude comparison. However, after multiple regression analysis, margin of difference decreased to 1.5 times although the cost for PWD remained higher. Substantial decrease in higher medical costs for PWD after multiple analyses compared to crude analysis implies that additional adjustment using variables such as disease severity, not available in the NHI database, may predict a further reduction in differences. Thus, it is difficult to determine that the medical expenditure for PWD is excessive.

Mueller polarimetric imaging for fast macroscopic mapping of microscopic collagen matrix remodeling by smooth muscle cells.

Smooth muscle cells (SMCs) are critical players in cardiovascular disease development and undergo complex phenotype switching during disease progression. However, SMC phenotype is difficult to assess and track in co-culture studies. To determine the contractility of SMCs embedded within collagen hydrogels, we performed polarized light imaging and subsequent analysis based on Mueller matrices. Measurements were made both in the absence and presence of endothelial cells (ECs) in order to establish the impact of EC-SMC communication on SMC contractility. The results demonstrated that Mueller polarimetric imaging is indeed an appropriate tool for assessing SMC activity which significantly modifies the hydrogel retardance in the presence of ECs. These findings are consistent with the idea that EC-SMC communication promotes a more contractile SMC phenotype. More broadly, our findings suggest that Mueller polarimetry can be a useful tool for studies of spatial heterogeneities in hydrogel remodeling by SMCs.

In Situ Formation of Proangiogenic Mesenchymal Stem Cell Spheroids in Hyaluronic Acid/Alginate Core-Shell Microcapsules.

Mesenchymal stem-cell (MSC)-based therapies have been recognized as promising strategies for the treatment of various injuries or diseases because of their unique characteristics, such as self-renewal, differentiation potential, and secretion of various bioactive molecules. However, MSC transplantation often results in low efficacy, including a cell viability loss and a low therapeutic activity. Alternatively, MSC spheroids have been studied to improve the viability and therapeutic activity of MSCs. Also, microencapsulation of cells can protect and retain the cells from harsh environments after transplantation. Here, MSC spheroids were formed in hyaluronic acid/alginate (HA@Alg) core-shell microcapsules and employed for neovascularization. A well-defined core-shell structure of HA@Alg microcapsules was produced by optimizing various electrospraying conditions. MSC spheroids could be spontaneously formed in the HA core of the microcapsules after 1 day of incubation. Enhanced secretion of various growth factors was found from MSC spheroids in HA@Alg. In vivo plug assay revealed the significant promotion of angiogenesis by MSC spheroids in HA@Alg compared to that by the controls (i.e., MSCs and MSC spheroids), which is likely because of the better retention of MSC spheroid forms in the microcapsules. Thus, the HA@Alg microcapsules embedding MSC spheroids will be greatly beneficial for various stem cell-based therapies.

Association between energy drink consumption, depression and suicide ideation in Korean adolescents.

BACKGROUND: Suicide is the leading cause of death among adolescents in South Korea. Negative influences of the abuse of energy drinks on mental health problems have emerged. AIMS: This study aimed to analyze the relationship between energy drink consumption, depression and suicide ideation. METHODS: We analyzed the data of 26,346 boys and 26,966 girls who participated in the 2015 Korean Youth Risk Behavior Web-Based Survey. The independent variable was the frequency of energy drink consumption per week. Experience of depression and suicide ideation were considered as the dependent variables. Multiple logistic regression was performed. RESULTS: An association was found between energy drink intake per week and depressive mood. Moreover, the most frequent energy drink intake (three times or more per week) group showed the highest association with depressive mood (odds ratios (ORs); boys: ORs = 1.40, 95% confidence interval (CI): 1.25-1.56; girls: ORs = 1.61, 95% CI: 1.44-1.79) and suicide ideation (boys: ORs = 1.46, 95% CI: 1.27-1.69; girls: ORs = 1.36, 95% CI: 1.20-1.55). CONCLUSION: We observed that consumption of excessive amounts of energy drinks is associated with depression and suicide ideation. Therefore, appropriate regulation or education is needed to prevent adolescents from experiencing adverse outcomes.

Functional Connectivity of the Striatum as a Neural Correlate of Symptom Severity in Patient with Obsessive-Compulsive Disorder.

OBJECTIVE: It is well established that the cortico-striato-thalamo-cortical (CSTC) circuit is implicated in the pathophysiology of obsessive- compulsive disorder (OCD). However, reports on corticostriatal functional connectivity (FC) in OCD have been inconsistent due to the structural and functional heterogeneity of the striatum. Therefore, in the present study, we investigated corticostriatal FC using a fine 12-seed striatal parcellation to overcome this heterogeneity and discover the neural correlates of symptoms in OCD patients. METHODS: We recruited 23 OCD patients and 23 healthy controls (HCs). Whole-brain FC based on striatal seeds was examined using resting-state functional magnetic resonance imaging data and compared across OCD patients and HCs. We conducted correlation analysis between FCs of striatal subregions with significant group differences and symptom severity scores on the Yale-Brown Obsessive Compulsive Scale (Y-BOCS), Hamilton Rating Scale for Depression, and Hamilton Rating Scale for Anxiety (HAM-A). RESULTS: Compared to HCs, patients demonstrated increased FC of the dorsal caudal putamen and ventral rostral putamen (VRP) with several cortical regions, such as the intracalcarine cortex, inferior frontal gyrus, supramarginal/angular gyrus (SMG/AG), and postcentral gyrus (PCG). Furthermore, FC between the VRP and SMG/AG and between the VRP and PCG was negatively correlated with scores on the Y-BOCS compulsive subscale and the HAM-A, respectively. CONCLUSION: These findings suggest that striatal subregions have strengthened FC with extensive cortical regions, which may reflect neural correlates of compulsive and anxious symptoms in OCD patients. These results contribute to an improved understanding of OCD pathophysiology by complementing the current evidence regarding striatal FC.

Anti-oxidant activity reinforced reduced graphene oxide/alginate microgels: Mesenchymal stem cell encapsulation and regeneration of infarcted hearts.

Mesenchymal stem cell (MSC) transplantation is promising for repairing heart tissues post myocardial infarction (MI). In particular, paracrine effects of the transplanted MSCs have been highlighted to play major roles in heart regeneration by secreting multiple growth factors and immune-modulatory cytokines. Nevertheless, its therapeutic efficacy still remains low, which is strongly associated with low viability and activity of the transplanted stem cells, because the transplanted MSCs are exposed to high shear stress during injection and harsh environments (e.g., high oxidative stress and host immune reactions) post injection. In this study, we aimed to develop novel injectable MSC-delivery microgel systems possessing high anti-oxidant activities. Specifically, we encapsulated MSCs in graphene oxide (GO)/alginate composite microgels by electrospraying. To further enhance the anti-oxidizing activities of the gels, we developed reduced MSC-embedded GO/alginate microgels (i.e., r(GO/alginate)), which have the potential to protect MSCs from the abovementioned harsh environments within MI tissues. Our in vitro studies demonstrated that the MSCs encapsulated in the r(GO/alginate) microgels showed increased viability under oxidative stress conditions with H2O2. Furthermore, cardiomyocytes (CMs), co-cultured with the encapsulated MSCs in transwells with H2O2 treatment, showed higher cell viability and cardiac maturation compared to monolayer cultured CMs, likely due to ROS scavenging by the gels and positive paracrine signals from the encapsulated MSCs. In vivo experiments with acute MI models demonstrated improved therapeutic efficacy of MSC delivery in r(GO/alginate) microgels, exhibiting significant decreases in the infarction area and the improvement of cardiac function. We believe that our novel MSC encapsulation system with GO, alginate, and mild reduction, which exhibits high cell protection capacity (e.g., anti-oxidant activity), will serve as an effective platform for the delivery of stem cells and other therapeutic cell types to treat various injuries and diseases, including MI.

Comprehensive mass spectrometry-guided phenotyping of plant specialized metabolites reveals metabolic diversity in the cosmopolitan plant family Rhamnaceae.

Plants produce a myriad of specialized metabolites to overcome their sessile habit and combat biotic as well as abiotic stresses. Evolution has shaped the diversity of specialized metabolites, which then drives many other aspects of plant biodiversity. However, until recently, large-scale studies investigating the diversity of specialized metabolites in an evolutionary context have been limited by the impossibility of identifying chemical structures of hundreds to thousands of compounds in a time-feasible manner. Here we introduce a workflow for large-scale, semi-automated annotation of specialized metabolites and apply it to over 1000 metabolites of the cosmopolitan plant family Rhamnaceae. We enhance the putative annotation coverage dramatically, from 2.5% based on spectral library matches alone to 42.6% of total MS/MS molecular features, extending annotations from well-known plant compound classes into dark plant metabolomics. To gain insights into substructural diversity within this plant family, we also extract patterns of co-occurring fragments and neutral losses, so-called Mass2Motifs, from the dataset; for example, only the Ziziphoid clade developed the triterpenoid biosynthetic pathway, whereas the Rhamnoid clade predominantly developed diversity in flavonoid glycosides, including 7-O-methyltransferase activity. Our workflow provides the foundations for the automated, high-throughput chemical identification of massive metabolite spaces, and we expect it to revolutionize our understanding of plant chemoevolutionary mechanisms.

Hydrogel Biomaterials for Stem Cell Microencapsulation.

Stem cell transplantation has been recognized as a promising strategy to induce the regeneration of injured and diseased tissues and sustain therapeutic molecules for prolonged periods in vivo. However, stem cell-based therapy is often ineffective due to low survival, poor engraftment, and a lack of site-specificity. Hydrogels can offer several advantages as cell delivery vehicles, including cell stabilization and the provision of tissue-like environments with specific cellular signals; however, the administration of bulk hydrogels is still not appropriate to obtain safe and effective outcomes. Hence, stem cell encapsulation in uniform micro-sized hydrogels and their transplantation in vivo have recently garnered great attention for minimally invasive administration and the enhancement of therapeutic activities of the transplanted stem cells. Several important methods for stem cell microencapsulation are described in this review. In addition, various natural and synthetic polymers, which have been employed for the microencapsulation of stem cells, are reviewed in this article.

Shape-Controlled Synthesis of Dumbbell-like Pt-Fe3O4-MnO x Nanoparticles by Governing the Reaction Kinetics.

The production of shape-controlled heterometallic nanoparticles (NPs) consisting of Pt and nonprecious metal oxides is crucial to demonstrate the composition-property relationship of NPs. Herein, we report a facile one-pot approach for the controlled synthesis of dumbbell-like Pt-Fe3O4-MnO x and dendritic Pt-MnO x NPs. The key to the success of this synthesis is in changing the quantity of Fe(CO)5 additive to control the reaction kinetics. In the absence of Fe(CO)5, dendritic Pt-MnO x NPs were synthesized through the assembly of small seed NPs. On the other hand, dumbbell-like Pt-Fe3O4-MnO x NPs were obtained in the presence of Fe(CO)5 through controlling the nucleation and growth of Fe and Mn on the Pt NPs, followed by air oxidation. Compared to a Pt/graphene oxide (GO) catalyst, dumbbell-like Pt-Fe3O4-MnO x NPs on GO showed an enhancement of specific activity toward the oxygen reduction reaction owing to the compressive-strain effect exerted on the Pt lattice.

Acylphloroglucinolated Catechin and Phenylethyl Isocoumarin Derivatives from Agrimonia pilosa.

Eight new compounds (1-8), including five acylphloroglucinolated catechins (1-5) and three phenylethyl isocoumarin glycosides (6-8), were isolated from Agrimonia pilosa along with six other known compounds (9-14). The new compounds were characterized structurally by NMR, MS, and ECD analyses. Compounds 4 and 5 were assigned as acylphloroglucinolated procyanidin derivatives, which are described for the first time from Nature. The absolute configuration of compound 8 was elucidated by computational analysis of its ECD spectrum. The isolated compounds were evaluated for their inhibitory activity against lipopolysaccharide-induced NO production in BV2 microglial cells.

Cdc6 protein obstructs apoptosome assembly and consequent cell death by forming stable complexes with activated Apaf-1 molecules.

Cdc6 is the bifunctional AAA+ ATPase that assembles prereplicative complexes on origins of replication and activates p21(CIP1)- or p27(KIP1)-bound Cdk2. During the G(1)-S transition, the Cdc6 gene essential for chromosomal replication is activated by the E2F transcriptional factor. Paradoxically, Apaf-1 encoding the central component of the apoptosome is also activated at the same time and by E2F. Consequently, genes for antipodal life and death are regulated in the same manner by the same transcriptional factor. Here we report a striking solution to this paradox. Besides performing prereplicative complex assembly and Cdk2 activation, Cdc6 obstructed apoptosome assembly by forming stable complexes very likely with a monomer of cytochrome c-activated Apaf-1 molecules. This function depended on its own ATPase domain but not on the cyclin-binding motif. In proliferating rodent fibroblasts, Cdc6 continued to block apoptosome assembly induced by a non-cytochrome c or some other mechanism, suppressing seemingly unintended apoptosis when promoting cell proliferation. Thus, Cdc6 is an AAA+ ATPase with three functions, all working for life.