Individual cell types in C. elegans age differently and activate distinct cell-protective responses.

Aging is characterized by a global decline in physiological function. However, by constructing a complete single-cell gene expression atlas, we find that Caenorhabditis elegans aging is not random in nature but instead is characterized by coordinated changes in functionally related metabolic, proteostasis, and stress-response genes in a cell-type-specific fashion, with downregulation of energy metabolism being the only nearly universal change. Similarly, the rates at which cells age differ significantly between cell types. In some cell types, aging is characterized by an increase in cell-to-cell variance, whereas in others, variance actually decreases. Remarkably, multiple resilience-enhancing transcription factors known to extend lifespan are activated across many cell types with age; we discovered new longevity candidates, such as GEI-3, among these. Together, our findings suggest that cells do not age passively but instead react strongly, and individualistically, to events that occur during aging. This atlas can be queried through a public interface.

LAMP2A, and other chaperone-mediated autophagy related proteins, do not decline with age in genetically heterogeneous UM-HET3 mice.

Chaperone-mediated autophagy (CMA) selectively degrades proteins that are crucial for glycolysis, fatty acid metabolism, and the progression of several age-associated diseases. Several previous studies, each of which evaluated males of a single inbred mouse or rat strain, have reported that CMA declines with age in many tissues, attributed to an age-related loss of LAMP2A, the primary and indispensable component of the CMA translocation complex. This has led to a paradigm in the field of CMA research, stating that the age-associated decline in LAMP2A in turn decreases CMA, contributing to the pathogenesis of late-life disease. We assessed LAMP2A levels and CMA substrate uptake in both sexes of the genetically heterogeneous UM-HET3 mouse stock, which is the current global standard for the evaluation of anti-aging interventions. We found no evidence for age-related changes in LAMP2A levels, CMA substrate uptake, or whole liver levels of CMA degradation targets, despite identifying sex differences in CMA.

The C. elegans Observatory: High-throughput exploration of behavioral aging.

Organisms undergo a variety of characteristic changes as they age, suggesting a substantial commonality in the mechanistic basis of aging. Experiments in model organisms have revealed a variety of cellular systems that impact lifespan, but technical challenges have prevented a comprehensive evaluation of how these components impact the trajectory of aging, and many components likely remain undiscovered. To facilitate the deeper exploration of aging trajectories at a sufficient scale to enable primary screening, we have created the Caenorhabditis elegans Observatory, an automated system for monitoring the behavior of group-housed C. elegans throughout their lifespans. One Observatory consists of a set of computers running custom software to control an incubator containing custom imaging and motion-control hardware. In its standard configuration, the Observatory cycles through trays of standard 6 cm plates, running four assays per day on up to 576 plates per incubator. High-speed image processing captures a range of behavioral metrics, including movement speed and stimulus-induced turning, and a data processing pipeline continuously computes summary statistics. The Observatory software includes a web interface that allows the user to input metadata and view graphs of the trajectory of behavioral aging as the experiment unfolds. Compared to the manual use of a plate-based C. elegans tracker, the Observatory reduces the effort required by close to two orders of magnitude. Within the Observatory, reducing the function of known lifespan genes with RNA interference (RNAi) gives the expected phenotypic changes, including extended motility in daf-2(RNAi) and progeria in hsf-1(RNAi). Lifespans scored manually from worms raised in conventional conditions match those scored from images captured by the Observatory. We have used the Observatory for a small candidate-gene screen and identified an extended youthful vigor phenotype for tank-1(RNAi) and a progeric phenotype for cdc-42(RNAi). By utilizing the Observatory, it is now feasible to conduct whole-genome screens for an aging-trajectory phenotype, thus greatly increasing our ability to discover and analyze new components of the aging program.

Diverse partial reprogramming strategies restore youthful gene expression and transiently suppress cell identity.

Partial pluripotent reprogramming can reverse features of aging in mammalian cells, but the impact on somatic identity and the necessity of individual reprogramming factors remain unknown. Here, we used single-cell genomics to map the identity trajectory induced by partial reprogramming in multiple murine cell types and dissected the influence of each factor by screening all Yamanaka Factor subsets with pooled single-cell screens. We found that partial reprogramming restored youthful expression in adipogenic and mesenchymal stem cells but also temporarily suppressed somatic identity programs. Our pooled screens revealed that many subsets of the Yamanaka Factors both restore youthful expression and suppress somatic identity, but these effects were not tightly entangled. We also found that a multipotent reprogramming strategy inspired by amphibian regeneration restored youthful expression in myogenic cells. Our results suggest that various sets of reprogramming factors can restore youthful expression with varying degrees of somatic identity suppression. A record of this paper's Transparent Peer Review process is included in the supplemental information.

Novel insights from a multiomics dissection of the Hayflick limit.

The process wherein dividing cells exhaust proliferative capacity and enter into replicative senescence has become a prominent model for cellular aging in vitro. Despite decades of study, this cellular state is not fully understood in culture and even much less so during aging. Here, we revisit Leonard Hayflick's original observation of replicative senescence in WI-38 human lung fibroblasts equipped with a battery of modern techniques including RNA-seq, single-cell RNA-seq, proteomics, metabolomics, and ATAC-seq. We find evidence that the transition to a senescent state manifests early, increases gradually, and corresponds to a concomitant global increase in DNA accessibility in nucleolar and lamin associated domains. Furthermore, we demonstrate that senescent WI-38 cells acquire a striking resemblance to myofibroblasts in a process similar to the epithelial to mesenchymal transition (EMT) that is regulated by t YAP1/TEAD1 and TGF-ß2. Lastly, we show that verteporfin inhibition of YAP1/TEAD1 activity in aged WI-38 cells robustly attenuates this gene expression program.

The naked truth: a comprehensive clarification and classification of current 'myths' in naked mole-rat biology.

The naked mole-rat (Heterocephalus glaber) has fascinated zoologists for at least half a century. It has also generated considerable biomedical interest not only because of its extraordinary longevity, but also because of unusual protective features (e.g. its tolerance of variable oxygen availability), which may be pertinent to several human disease states, including ischemia/reperfusion injury and neurodegeneration. A recent article entitled 'Surprisingly long survival of premature conclusions about naked mole-rat biology' described 28 'myths' which, those authors claimed, are a 'perpetuation of beautiful, but falsified, hypotheses' and impede our understanding of this enigmatic mammal. Here, we re-examine each of these 'myths' based on evidence published in the scientific literature. Following Braude et al., we argue that these 'myths' fall into four main categories: (i) 'myths' that would be better described as oversimplifications, some of which persist solely in the popular press; (ii) 'myths' that are based on incomplete understanding, where more evidence is clearly needed; (iii) 'myths' where the accumulation of evidence over the years has led to a revision in interpretation, but where there is no significant disagreement among scientists currently working in the field; (iv) 'myths' where there is a genuine difference in opinion among active researchers, based on alternative interpretations of the available evidence. The term 'myth' is particularly inappropriate when applied to competing, evidence-based hypotheses, which form part of the normal evolution of scientific knowledge. Here, we provide a comprehensive critical review of naked mole-rat biology and attempt to clarify some of these misconceptions.

Impact of a multifaceted intervention to improve emergency care on newborn and child health outcomes in Rwanda.

Implementing context-appropriate neonatal and paediatric advanced life support management interventions has increasingly been recommended as one of the approaches to reduce under-five mortality in resource-constrained settings like Rwanda. One such intervention is ETAT+, which stands for Emergency Triage, Assessment and Treatment plus Admission care for severely ill newborns and children. In 2013, ETAT+ was implemented in Rwandan district hospitals. We evaluated the impact of the ETAT+ intervention on newborn and child health outcomes. We used monthly time-series data from the DHIS2-enabled Rwanda Health Management Information System from 2012 to 2016 to examine neonatal and paediatric hospital mortality rates. Each hospital contributed data for 12 and 36 months before and after ETAT+ implementation, respectively. Using controlled interrupted time-series analysis and segmented regression model, we estimated longitudinal changes in neonatal and paediatric hospital mortality rates in intervention hospitals relative to matched concurrent control hospitals. We also studied changes in case fatality rate specifically for ETAT+-targeted conditions. Our study cohort consisted of 7 intervention hospitals and 14 matched control hospitals contributing 142 424 neonatal and paediatric hospital admissions. After controlling for secular trends and autocorrelations, we found that the ETAT+ implementation had no statistically significant impact on the rate of all-cause neonatal and paediatric hospital mortality in intervention hospitals relative to control hospitals. However, the case fatality rate for ETAT+-targeted neonatal conditions decreased immediately following implementation by 5% (95% confidence interval: -9.25, -0.77) and over time by 0.8% monthly (95% confidence interval: -1.36, -0.25) in intervention hospitals compared with control hospitals. Case fatality rate for ETAT+-targeted paediatric conditions did not decrease following the ETAT+ implementation. While ETAT+ focuses on improving the quality of hospital care for both newborns and children, we only found an impact on neonatal hospital mortality for ETAT+-targeted conditions that should be interpreted with caution given the relatively short pre-intervention period and potential regression to the mean.

Developing and implementing a model of equitable distribution of mentorship in districts with spatial inequities and maldistribution of human resources for maternal and newborn care in Rwanda.

BACKGROUND: The shortage of health care providers (HCPs) and inequity in their distribution along with the lack of sufficient and equal professional development opportunities in low-income countries contribute to the high mortality and morbidity of women and newborns. Strengthening skills and building the capacity of all HCPs involved in Maternal and Newborn Health (MNH) is essential to ensuring that mothers and newborns receive the required care in the period around birth. The Training, Support, and Access Model (TSAM) project identified onsite mentorship at primary care Health Centers (HCs) as an approach that could help reduce mortality and morbidity through capacity building of HCPs in Rwanda. This paper presents the results and lessons learnt through the design and implementation of a mentorship model and highlights some implications for future research. METHODS: The design phase started with an assessment of the status of training in HCs to inform the selection of Hospital-Based Mentors (HBMs). These HBMs took different courses to become mentors. A clear process was established for engaging all stakeholders and to ensure ownership of the model. Then the HBMs conducted monthly visits to all 68 TSAM assigned HCs for 18 months and were extended later in 43 HCs of South. Upon completion of 6 visits, mentees were requested to assist their peers who are not participating in the mentoring programme through a process of peer mentoring to ensure sustainability after the project ends. RESULTS: The onsite mentorship in HCs by the HBMs led to equal training of HCPs across all HCs regardless of the location of the HC. Research on this mentorship showed that the training improved the knowledge and self-efficacy of HCPs in managing postpartum haemorrhage (PPH) and newborn resuscitation. The lessons learned include that well trained midwives can conduct successful mentorships at lower levels in the healthcare system. The key challenge was the inconsistency of mentees due to a shortage of HCPs at the HC level. CONCLUSIONS: The initiation of onsite mentorship in HCs by HBMs with the support of the district health leaders resulted in consistent and equal mentoring at all HCs including those located in remote areas.

A genetic screen identifies new steps in oocyte maturation that enhance proteostasis in the immortal germ lineage.

Somatic cells age and die, but the germ-cell lineage is immortal. In Caenorhabditis elegans, germline immortality involves proteostasis renewal at the beginning of each new generation, when oocyte maturation signals from sperm trigger the clearance of carbonylated proteins and protein aggregates. Here, we explore the cell biology of this proteostasis renewal in the context of a whole-genome RNAi screen. Oocyte maturation signals are known to trigger protein-aggregate removal via lysosome acidification. Our findings suggest that lysosomes are acidified as a consequence of changes in endoplasmic reticulum activity that permit assembly of the lysosomal V-ATPase, which in turn allows lysosomes to clear the aggregates via microautophagy. We define two functions for mitochondria, both of which appear to be independent of ATP generation. Many genes from the screen also regulate lysosome acidification and age-dependent protein aggregation in the soma, suggesting a fundamental mechanistic link between proteostasis renewal in the germline and somatic longevity.

Mentors' perspectives on strengths and weaknesses of a novel clinical mentorship programme in Rwanda: a qualitative study.

OBJECTIVES: To identify mentors' perspectives on strengths and weaknesses of the Training, Support and Access Model for Maternal, Newborn and Child Health (TSAM-MNCH) clinical mentorship programme in Rwandan district hospitals. Understanding the perspectives of mentors involved in this programme can aid in the improvement of its implementation. DESIGN: The study used a qualitative approach with in-depth interviews. SETTING: Mentors of TSAM-MNCH clinical mentorship programme mentoring health professionals at district hospitals of Rwanda. PARTICIPANTS: 14 TSAM mentors who had at least completed six mentorship visits on a regular basis in three selected district hospitals. RESULTS: Mentors' accounts demonstrated an appreciation of the two mentoring structures which are interprofessional collaboration and training. These structures are highlighted as the strengths of the mentoring programme and they play a significant role in the successful implementation of the mentorship model. Inconsistency of mentoring activities and lack of resources emerged as major weaknesses of the clinical mentorship programme which could hinder the effectiveness of the mentoring scheme. CONCLUSION: The findings of this study highlight the strengths and weaknesses perceived by mentors of the TSAM-MNCH clinical mentorship programme, providing insights that can be used to improve its implementation. The study represents unique TSAM-MNCH structural settings, but its findings shed light on Rwandan health system issues that need to be further addressed to ensure better quality of care for mothers, newborns and children.

Split-wrmScarlet and split-sfGFP: tools for faster, easier fluorescent labeling of endogenous proteins in Caenorhabditis elegans.

We create and share a new red fluorophore, along with a set of strains, reagents and protocols, to make it faster and easier to label endogenous Caenorhabditis elegans proteins with fluorescent tags. CRISPR-mediated fluorescent labeling of C. elegans proteins is an invaluable tool, but it is much more difficult to insert fluorophore-size DNA segments than it is to make small gene edits. In principle, high-affinity asymmetrically split fluorescent proteins solve this problem in C. elegans: the small fragment can quickly and easily be fused to almost any protein of interest, and can be detected wherever the large fragment is expressed and complemented. However, there is currently only one available strain stably expressing the large fragment of a split fluorescent protein, restricting this solution to a single tissue (the germline) in the highly autofluorescent green channel. No available C. elegans lines express unbound large fragments of split red fluorescent proteins, and even state-of-the-art split red fluorescent proteins are dim compared to the canonical split-sfGFP protein. In this study, we engineer a bright, high-affinity new split red fluorophore, split-wrmScarlet. We generate transgenic C. elegans lines to allow easy single-color labeling in muscle or germline cells and dual-color labeling in somatic cells. We also describe a novel expression strategy for the germline, where traditional expression strategies struggle. We validate these strains by targeting split-wrmScarlet to several genes whose products label distinct organelles, and we provide a protocol for easy, cloning-free CRISPR/Cas9 editing. As the collection of split-FP strains for labeling in different tissues or organelles expands, we will post updates at doi.org/10.5281/zenodo.3993663.

Pertussis Infections Among Pregnant Women in the United States, 2012-2017.

BACKGROUND: Little is known about pertussis among pregnant women, a population at increased risk for severe morbidity from respiratory infections such as influenza. We used the Centers for Disease Control and Prevention's Enhanced Pertussis Surveillance (EPS) system to describe pertussis epidemiology among pregnant and nonpregnant women of childbearing age. METHODS: Pertussis cases in women aged 18-44 years with cough onset between 1 January 2012 and 31 December 2017 were identified in 7 EPS states. Surveillance data were collected through patient and provider interviews and immunization registries. Bridged-race, intercensal population data and live birth estimates were used as denominators. RESULTS: We identified 1582 pertussis cases among women aged 18-44 years; 5.1% (76/1499) of patients with a known pregnancy status were pregnant at cough onset. Of the pregnant patients with complete information, 81.7% (49/60) reported onset during the second or third trimester. The median ages of pregnant and nonpregnant patients were 29.0 and 33.0 years, respectively. Most pregnant and nonpregnant patients were White (78.3% vs. 86.4%, respectively; P = .09) and non-Hispanic (72.6% vs. 77.3%, respectively; P = .35). The average annual incidence of pertussis was 7.7/100000 among pregnancy women and 7/3/100000 among nonpregnant women. Compared to nonpregnant patients, more pregnant patients reported whoop (41.9% vs. 31.3%, respectively), posttussive vomiting (58.1% vs. 47.9%, respectively), and apnea (37.3% vs. 29.0%, respectively); however, these differences were not statistically significant (P values > .05 for all). A similar proportion of pregnant and nonpregnant patients reported ever having received Tdap (tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis vaccine; 31.6% vs. 32.7%, respectively; P = .84). CONCLUSIONS: Our analysis suggests that incidence of pertussis and clinical characteristics of disease are similar among pregnant and nonpregnant women. Continued monitoring is important to further define pertussis epidemiology in pregnant women.

Developing and implementing a novel mentorship model (4+ 1) for maternal, newborn and child health in Rwanda.

BACKGROUND: There are a number of factors that may contribute to high mortality and morbidity of women and newborns in low-income countries. These include a shortage of competent health care providers (HCP) and a lack of sufficient continuous professional development (CPD) opportunities. Strengthening the skills and building the capacity of HCP involved in the provision of maternal, newborn and child health (MNCH) is essential to ensure quality care for mothers, newborns and children. To address this challenge in Rwanda, mentorship of HCPs was identified as an approach that could help build capacity, improve the provision of care and accelerate the reduction in maternal and neonatal mortality and morbidity. In this paper, we describe the development and implementation of a novel mentorship model named Four plus One (4+ 1) for MNCH in Rwanda. METHODS: The mentorship model built on the basis of inter-professional collaboration (IPC) was developed in early 2017 through consultations with different key actors. The design phase included refresher courses in specific skills and training course on mentoring. Field visits were conducted in 10 hospitals from June 2017 to February 2020. Hospital management teams (MT) were involved in the development and implementation of this mentorship model to ensure ownership of the program. RESULTS: Upon completion of planned visits to each hospital, a total of 218 HCPs were involved in the process. Reports prepared by mentors upon each mentorship visit and compiled by Training Support and Access Model (TSAM) for MNCH'CPD team, highlighted the mothers and newborns who were saved by both mentors and mentees. Also, different logbooks of mentees showed how the capacity of staff was strengthened, thereby suggesting effectiveness of the model. Through different mentorship coordination meetings, the model was much appreciated by the MTs of hospitals, especially the IPC component of the model and confirmed the program 'effectiveness. CONCLUSION: The initiation of a mentorship model built on IPC together with the involvement of the leadership of the hospital may be the cause effect of reduction of specific mortality and improve MNCH in low resource settings even when there are a limited number of specialists in the health facilities.

Parental attitudes and decisions regarding MMR vaccination during an outbreak of measles among an undervaccinated Somali community in Minnesota.

Incidence of measles is increasing in the US, largely due to transmission among growing unvaccinated communities. To elucidate predictors of parental decision to obtain measles, mumps, and rubella (MMR) vaccine for unvaccinated children during a measles outbreak, we surveyed families among a vaccine-hesitant Somali community in Minnesota. The survey assessed attitudes and beliefs about MMR vaccine, motivators for vaccinating, and intention to vaccinate future children on time. Among 300 families surveyed, 95% vaccinated their child with MMR due to fear of measles. The predominating parental concern about MMR vaccine (71%) was a fallacious presumed connection between vaccination and autism. Only 41% of parents intended to vaccinate future children on time with MMR; parents who received recommendations for MMR vaccination from multiple sources were more likely than other parents to intend to do so. These findings support the importance of diverse outreach efforts to increase vaccine coverage among undervaccinated communities.

Application of Transcriptional Gene Modules to Analysis of Caenorhabditis elegans' Gene Expression Data.

Identification of co-expressed sets of genes (gene modules) is used widely for grouping functionally related genes during transcriptomic data analysis. An organism-wide atlas of high-quality gene modules would provide a powerful tool for unbiased detection of biological signals from gene expression data. Here, using a method based on independent component analysis we call DEXICA, we have defined and optimized 209 modules that broadly represent transcriptional wiring of the key experimental organism C. elegans These modules represent responses to changes in the environment (e.g., starvation, exposure to xenobiotics), genes regulated by transcriptions factors (e.g., ATFS-1, DAF-16), genes specific to tissues (e.g., neurons, muscle), genes that change during development, and other complex transcriptional responses to genetic, environmental and temporal perturbations. Interrogation of these modules reveals processes that are activated in long-lived mutants in cases where traditional analyses of differentially expressed genes fail to do so. Additionally, we show that modules can inform the strength of the association between a gene and an annotation (e.g., GO term). Analysis of "module-weighted annotations" improves on several aspects of traditional annotation-enrichment tests and can aid in functional interpretation of poorly annotated genes. We provide an online interactive resource with tutorials at http://genemodules.org/, in which users can find detailed information on each module, check genes for module-weighted annotations, and use both of these to analyze their own gene expression data (generated using any platform) or gene sets of interest.

Identifying Vaccine-associated Rash Illness Amidst a Large Measles Outbreak: Minnesota, 2017.

Characteristics of vaccine-associated rash illness (VARI) and confirmed measles cases were compared during a measles outbreak. Although some clinical differences were noted, measles exposure and identification of the vaccine strain were helpful for public health decision-making. Rapid, vaccine strain-specific diagnostic assays will more efficiently distinguish VARI from measles.

Stress Resistance Screen in a Human Primary Cell Line Identifies Small Molecules That Affect Aging Pathways and Extend Caenorhabditis elegans' Lifespan.

Increased resistance to environmental stress at the cellular level is correlated with the longevity of long-lived mutants and wild-animal species. Moreover, in experimental organisms, screens for increased stress resistance have yielded mutants that are long-lived. To find entry points for small molecules that might extend healthy longevity in humans, we screened ∼100,000 small molecules in a human primary-fibroblast cell line and identified a set that increased oxidative-stress resistance. Some of the hits fell into structurally related chemical groups, suggesting that they may act on common targets. Two small molecules increased C. elegans' stress resistance, and at least 9 extended their lifespan by ∼10-50%. We further evaluated a chalcone that produced relatively large effects on lifespan and were able to implicate the activity of two, stress-response regulators, NRF2/skn-1 and SESN/sesn-1, in its mechanism of action. Our findings suggest that screening for increased stress resistance in human cells can enrich for compounds with promising pro-longevity effects. Further characterization of these compounds may reveal new ways to extend healthy human lifespan.

X Chromosome Domain Architecture Regulates Caenorhabditis elegans Lifespan but Not Dosage Compensation.

Mechanisms establishing higher-order chromosome structures and their roles in gene regulation are elusive. We analyzed chromosome architecture during nematode X chromosome dosage compensation, which represses transcription via a dosage-compensation condensin complex (DCC) that binds hermaphrodite Xs and establishes megabase-sized topologically associating domains (TADs). We show that DCC binding at high-occupancy sites (rex sites) defines eight TAD boundaries. Single rex deletions disrupted boundaries, and single insertions created new boundaries, demonstrating that a rex site is necessary and sufficient to define DCC-dependent boundary locations. Deleting eight rex sites (8rexΔ) recapitulated TAD structure of DCC mutants, permitting analysis when chromosome-wide domain architecture was disrupted but most DCC binding remained. 8rexΔ animals exhibited no changes in X expression and lacked dosage-compensation mutant phenotypes. Hence, TAD boundaries are neither the cause nor the consequence of DCC-mediated gene repression. Abrogating TAD structure did, however, reduce thermotolerance, accelerate aging, and shorten lifespan, implicating chromosome architecture in stress responses and aging.