Adversity early in life can shape the reproductive potential of individuals through negative effects on health and life span. However, long‐lived populations with multiple reproductive events may present alternative life history strategies to optimize reproductive schedules and compensate for shorter life spans. Here, we quantify the effects of major hurricanes and density dependence as sources of early‐life ecological adversity on Cayo Santiago rhesus macaque female reproduction and decompose their effects onto the mean age‐specific fertility, reproductive pace, and lifetime reproductive success (LRS). Females experiencing major hurricanes exhibit a delayed reproductive debut but maintain the pace of reproduction past debut and show a higher mean fertility during prime reproductive ages, relative to unaffected females. Increasing density at birth is associated to a decrease in mean fertility and reproductive pace, but such association is absent at intermediate densities. When combined, our study reveals that hurricanes early in life predict a delay‐overshoot pattern in mean age‐specific fertility that supports the maintenance of LRS. In contrast to predictive adaptive response models of accelerated reproduction, this long‐lived population presents a novel reproductive strategy where females who experience major natural disasters early in life ultimately overcome their initial reproductive penalty with no major negative fitness outcomes. Density presents a more complex relation with reproduction that suggests females experiencing a population regulated at intermediate densities early in life will escape density dependence and show optimized reproductive schedules. Our results support hypotheses about life history trade‐offs in which adversity‐affected females ensure their future reproductive potential by allocating more energy to growth or maintenance processes at younger adult ages. The Cayo Santiago rhesus macaque population presents a novel reproductive strategy where females experiencing major natural disasters early in life overcome their initial reproductive penalty and exhibit no major negative fitness outcomes. Such strategy suggests that investing more energy into development and maintenance at younger ages allows long‐lived females experiencing early‐life ecological adversity to reproduce at a mean rate equivalent to that of a typical female cohort later in life. Density at birth presents a more complex relation with reproduction that suggests females experiencing a population regulated at intermediate densities early in life escape density‐dependent effects in reproduction. Adversity may decrease the reproductive pace of females by delaying reproductive debut. Here, optimizing reproductive success later in life may involve a lower frequency of birth skipping. Finally, we quantify whether females experiencing harsh environments early in life attain a different LRS relative to females not experiencing adversities. No reduction in LRS among females who experience adversity would suggest the evolution of demographic mechanisms for optimal reproductive success later in life.22.1Macaca mulatta), all of which descended from a group of 409 individuals released onto Cayo Santiago in 1938. Since establishment, the population has been maintained under seminatural conditions allowing for the natural formation of social groups, social rank, and mating seasons  is a 15.2‐ha island that serves as a research facility managed by the Caribbean Primate Research Center of the University of Puerto Rico for behavioral and noninvasive research. Located 1 km off the southeastern coast of Puerto Rico , the CSFS is inhabited by a population of free‐ranging rhesus macaques  to immaturity (<3 years of age). The gestation period was estimated by subtracting 165 days from the date of birth for each individual . Females were monitored until December 2020. Females culled or still alive in the population at the end of the monitoring period were right censored. For all reproductive metrics, we considered all live births born to each female, and thus attributed offspring fitness directly to the offspring, not mothers  for a total of 2108 reproductive histories (c) to address potential overfitting . These models are nonparametric extensions of generalized linear models that allow the evaluation of nonlinear relationships and thus are appropriate to model age‐specific reproductive metrics in primates. For this, we used a logit link function for the binary outcome of whether a female reproduced in a given year. We considered cohort type (born in a hurricane or nonhurricane year) as a fixed effect and age and density as smooth terms. We fitted a series of competing models that included cohort as both an additive effect, that is, intercepts vary but smooth terms are consistent across cohorts, and as an interactive effect, that is, intercepts and the smooth terms vary by cohort. We also included a random intercept of individual ID to account for unobserved traits of our focal females and repeated measurements. Considering all factor combinations resulted in a total of nine competing models that were evaluated using the Corrected Akaike's Information Criterion  = 102 and n(nonhurricane) = 288). Females removed from the population were not included in the analysis. To test the influence of hurricanes and density on LRS and given the right skewed distribution of LRS, we employed zero‐inflated regression models with negative binomial distribution evaluated with AICc. Prior to running the models, we determined the data followed a negative binomial error distribution by testing for overdispersion in the count portion of the model using the AER package in R  and were zero inflated by comparing model fit with the nonzero‐inflated analog using the pscl package . Those culled or alive at the end of the study period were right censored. Because the proportional hazards assumption was not met for the density variable, suggesting that the association between density early in life and mortality changes over time , regardless of their time of death , but mean fertility across cohort type was dependent on age. The data show that females in the hurricane cohort exhibited a lower probability of successfully reproducing during early ages , relative to the nonhurricane cohort females  in contrast to females from the nonhurricane cohort who debuted at a median age of 4.074 years . The model indicated that females in the hurricane cohort were more likely to give birth at later ages (relative to age class 3) 2.52 times that of females in the nonhurricane cohort, holding constant all other variables (SE: 8.67 ± 0.33). However, the odds of being among those with LRS = 0 decreased by 0.0007 for females belonging to the nonhurricane cohort, relative to the baseline measure  and eight offspring , respectively  and decreased the odds of having LRS = 0 by 0.00007 , we found no cohort type effects on LRS, but LRS increased with increasing density and age at death  and the nonhurricane cohort . The adversity‐affected strategy involved delaying reproductive debut. Harsh conditions early in life, especially those involving nutritional and psychosocial adversities, may delay reproductive debut by imposing constraints on development  can impose constraints on energy usage by females through a conflict in the need for growth and development between the mother and offspring  and very old ages (>16.5 years of age). This is important for our study as it suggests that the observed reproductive compensation of females may be possible due to such reduced mortality pressure among (prime) adult ages (>3.5–16.5 years of age). That is, as long as an early‐life adversity affected female makes it into adulthood (surviving the vulnerable young ages), she may be able to contribute to population growth without being exposed to strong mortality risk. In this way, our study reveals that density regulates this population by acting on females at multiple live stages.4.3Our study reveals an alternative life history strategy in this long‐lived species to that proposed by the PAR of accelerated reproduction following early‐life adversities. Female rhesus macaques experiencing early‐life ecological adversity suffered delays in reproduction but were able to compensate for it later in life. For long‐lived primates, life span is an important predictor of LRS ; Investigation ; Visualization ; Writing – original draft (lead); Writing – review & editing . Chris Sutherland: Investigation (supporting); Methodology (supporting); Writing – review & editing . Stephanie J. Gonzalez: Formal analysis ; Investigation (supporting); Visualization ; Writing – review & editing (supporting). Raisa Hernández‐Pacheco: Conceptualization (lead); Formal analysis ; Funding acquisition (lead); Investigation ; Methodology (lead); Project administration (lead); Resources (lead); Supervision (lead); Writing – original draft (supporting); Writing – review & editing .Supplementary MaterialClick here for additional data file.


Bilateral cataract is a significant cause of blindness in children in Ethiopia. This study aimed to identify the resources available for cataract surgery in children, and to assess current surgical practices, surgical output and factors affecting the outcome of surgery in Ethiopia.A Google Forms mobile phone questionnaire was emailed to nine ophthalmologists known to perform cataract surgery in young children (0–5 years).All nine responded. All but one had received either 12- or 3–5-month’s training in pediatric ophthalmology with hands-on surgical training. The other surgeon had received informal training from an experienced colleague and visiting ophthalmologists. The surgeons were based in seven health facilities: five in the capital (Addis Ababa) and eight in six public referral hospitals and one private center.Over 12 months (2017–2018) 508 children (592 eyes) aged 0–18 years (most < 15 years) were operated by these surgeons. 84 (17%) had bilateral cataract, and 424 (83%) had unilateral cataract mainly following trauma. A mean of 66 (range 18–145) eyes were operated per surgeon. Seventy-one additional children aged > 5 years were operated by other surgeons. There were substantially fewer surgeons per million population (nine for 115 million population) than recommended by the World Health Organization and they were unevenly distributed across the country.Methylcellulose and rigid intraocular lenses were generally available but less than 50% of facilities had a sharp vitrectomy cutter and cohesive viscoelastic. Mean travel time outside Addis Ababa to a facility offering pediatric cataract surgery was 10 h.Despite the high number of cases per surgeon, the output for bilateral cataracts was far lower than required. More well-equipped pediatric ophthalmology teams are urgently required, with deployment to under-served areas. The prevalence of blindness in children aged 0–15 years is higher in the sub-Saharan African (SSA) Global Burden of Diseases super-region (0.71/1000 children) than in other regions, due to greater exposure to risk factors and lower access to public health interventions and eye care services , and 10%To address blinding eye diseases in children in low and middle income countries, in 2002 the World Health Organization (WHO) recommended that one tertiary eye care facility for children be established for every 10 million population . RecommeIn high income countries good visual acuity outcomes following bilateral cataract surgery can be achieved, with at least 50% having an acuity of 6/18 or better after surgery . Good reIn view of the ongoing problem of cataract blindness in children and the variable outcomes in Ethiopia, we initiated the CHICOE project (Children’s Improvement in Cataract Outcome in Ethiopia). This first publication is authored by all nine ophthalmologists undertaking cataract surgery in young children (age 0–5 years) in Ethiopia, and provides a national picture of cataract surgery in children in terms of the training the ophthalmologists had received, the resources available, the volume of surgery and surgical techniques, and ophthalmologists’ views on the main reasons for poor outcomes and delay in surgery.In August 2018, the corresponding author, a pediatric ophthalmologist, emailed a two-part mobile phone survey questionnaire in Google Forms to all nine surgeons known to undertake cataract surgery in young children (aged 0–5 years) in Ethiopia. Part I consisted of 17 questions about the institution where the surgeon operates and their training in pediatric ophthalmology. Part II was a 20-question survey of surgical practices for cataract surgery on children aged 0–18 years, with some questions on surgical techniques on children aged 0–5 years. Questions included the number of eyes operated by each surgeon over the previous 12 months, the youngest age at which they implant an intraocular lens, and the availability of equipment and consumables. Surgeons were also invited to comment on factors they judged to have limited good outcomes of the surgery over the previous year, and the relative importance of different causes of delay in surgery.A third questionnaire consisting of 14 supplementary questions was developed based on the responses to the first two questionnaires. Each surgeon was asked if they were aware of any other surgeons or institutions in Ethiopia undertaking cataract surgery in children. They were asked what proportion of children age 0–18 years with cataract were operated by themselves, other participants in this study, or by other ophthalmologists.All nine surgeons believed to undertake cataract surgery in children aged 0–5 years responded to the questionnaire, and none were aware of any others operating on this age group. Surgeons in four of the centres reported that other surgeons performed surgery on older children (aged > 5 to 18 years).The surgeons were based in seven facilities: five government university teaching hospitals , one government non-teaching hospital (Bisidimo) and one private facility (Addis Ababa) Table , Fig. 1.A total of 508 children aged 0–18 years were operated for cataract by the participating surgeons between September 2017 and August 2018. The surgeons commented that they had extracted data from surgical logbooks, and majority were aged less than 15 years of age. 84 (17%) children had bilateral, and 424 (83%) had unilateral cataract, mainly following trauma. A total of 592 eyes were operated, giving a mean of 66 (range 18–145 standard deviation (SD) ±47) eyes per surgeon. At four institutions a further 71 older children aged > 5–18 years were operated on by other surgeons, including 12 operations by visiting pediatric ophthalmologists from high income countries  were available for > 80% of surgeries in six of the seven facilities and foldable IOLs were available for > 80% of cases in only two centers. The availability of other equipment and consumables varied, with handheld keratometry, Trypan blue and aphakic glasses never available in at least one facility Fig. A.Six of the nine surgeons had completed a one-year pediatric ophthalmology fellowship, but the opportunities for hands on surgery during this training varied Table . Two surA mean of 384 range 27–950) eyes had been operated for cataract in children aged 0–18 years by the nine surgeons throughout their careers. The youngest average age of children at which surgeons had implanted an IOL was 14 months (range 4–24 ± 8.0) and the mean age beyond which surgeons judged primary posterior capsulotomy not to be required was 7.4 (range 5–10 ± 1.9) years  with well trained staff and adequate resources for every 10 million population by the year 2020 [All but one of the surgeons had undergone formal training in cataract surgery for children, and the total number of cataract operations performed by each surgeon indicates extensive surgical experience. Recently there has been substantial support of fellowship training in pediatric ophthalmology in Ethiopia by two international non-government organizations, Orbis International and the Himalayan Cataract Project, with at least one established pediatric cataract surgeon now contributing to the training of newly appointed surgeons. This reflects what was reported in a baseline and follow up study of tertiary level child eye care facilities in SSA, which demonstrated that staffing and infrastructure had improved significantly between the two studies , 17.The method used for anterior capsulotomy gives an indication of the sophistication of the techniques surgeons were exposed to during training. In this study four surgeons never or almost never used CCCR even in older children and two performed can-opener capsulotomy in almost all cases. This is likely explained by the fact that most of the surgeons had been taught anterior capsulotomy using a vitrector rather than CCCR. In addition, because small incision cataract surgery is used for adults in Ethiopia which does not require CCCR, unlike phacoemulsification, they would not have gained skills in CCCR by operating on adults. The lack of capsulorrhexis forceps suitable for use through a small corneal incision may also have contributed to the relatively infrequent use of CCCR. There is a clear a need for further training with provision of relevant equipment, which all the surgeons would welcome. The recently proposed pediatric ophthalmology academic partnership between Canada and Ethiopia could help to address these gaps .Compared to national studies from high income countries, the number of cases operated per surgeon in our study was very high. For example, in a survey of US paediatric cataract surgeons in 2003, 86% operated on less than 21 eyes a year  comparedOur surgeons reported that two thirds of the children they operated on had unilateral cataract, mostly following trauma, which is a higher than in studies in high income countries (25–30%) . Other sSurgeons often cited “other parental commitments” as contributing to delayed surgery, which is likely to be exacerbated by uneven distribution of services and the long travel distances required to access them. This is consistent with other studies of inequalities in access to health care  and suggOur study has limitations as it was questionnaire based rather than observational and relied on data collected retrospectively and recall of surgeons’ experiences. Moreover, in a rapidly changing situation, this survey provides only a snapshot for the year 2017/2018. However, in the absence of other information about pediatric cataract surgical practice in Ethiopia, our study provides the first information on the limitations of these services.The number of CEHTFs is lower than the WHO recommendation, and the services that are available are poorly distributed across the country. It is, therefore, not surprising that late presentation is an important cause of poor outcomes. Many centers lack the equipment and consumables considered essential for high quality cataract surgery in young children.There is a need to increase the number and distribution of pediatric ophthalmologists and ensure they are adequately trained and equipped to provide high quality surgery. A minimum of five more CEHTFs with the full complement of competent allied eye health professionals are required to meet WHO recommendations. Advocacy with the Ministry of Health to address these challenges is very important, working in partnership with NGOs.We propose that this survey is followed by a retrospective review of the visual acuity outcome of bilateral cataract surgery in Ethiopian children to provide baseline data, followed by a prospective national study following the provision of additional resources.


Saccharomyces cerevisiae, where acetic acid can be either a physiological substrate or a stressor. This review will focus on acetic acid stress and its response in the context of cellular transport, pH homeostasis, metabolism and stress‐signalling pathways. This information has been integrated with the results obtained by multi‐omics, synthetic biology and metabolic engineering approaches aimed to identify major cellular players involved in acetic acid tolerance. In the production of biofuels and renewable chemicals from lignocellulosic biomass, the improvement of acetic acid tolerance is a key factor. In this view, how this knowledge could be used to contribute to the development and competitiveness of yeast cell factories for sustainable applications will be also discussed. Acetic acid stress represents a frequent challenge to counteract for yeast cells under several environmental conditions and industrial bioprocesses. The molecular mechanisms underlying its response have been mostly elucidated in the budding yeast  Acetic acid stress is a frequent challenge for budding yeast.Signalling pathways dissection and system‐wide approaches reveal a complex picture.Cell fitness and adaptation under acid stress conditions is environment dependent.Tolerance to acetic acid is a key factor in yeast‐based industrial biotechnology.There is no ‘magic bullet’: An integrated approach is advantageous to develop performing yeast cell factories. Saccharomyces cerevisiae.Multiple factors influenced acetic acid stress and tolerance in the budding yeast  On the other hand, extracellular acetic acid can be an environmental challenge and trigger adaptive responses at sublethal concentrations or alternatively be toxic and leading to cell death. In a physiological scenario where acetic acid derives from cellular metabolism, its accumulation occurs in parallel with glucose consumption and became maximal in stationary‐phase yeast cells. In a stress‐promoting context, the final toxicity for the cell is a combined function of extracellular acetic acid concentration, extracellular pH and consequent intracellular accumulation due to its uptake , it exists mainly in the undissociated form, which can freely diffuse through the plasma membrane with a rate that depends on the membrane solubility of the molecule , which pumps protons out of the cell, and the vacuolar H+‐ATPase, which pumps protons into the lumen of the vacuole  and mechanisms of toxicity  for a complete overview on acetic acid stress response. For acetic acid tolerance, a comprehensive picture focused on omics approaches is described in the work by Geng et al.  or necrotic‐like (160–200 mM) features, respectively . In a late phase of AA‐RCD, cytochrome c is degraded and mitochondrial dysfunction occurred with a decrease of the respiratory control index (RCI), a collapse of the mitochondrial membrane potential, a reduction in cytochrome c oxidase (COX) activity and in cytochromes a–a3 levels  is the main responsible of AA‐RCD resistance after low‐pH pre‐conditioning  biosynthesis and acetate degradation pathways (Qin et al., The ‘omics’ era has offered a new opportunity and perspective in the comprehension of biological processes and allowed the development of synthetic biology and metabolic engineering approaches. Also, the knowledge on acetic acid tolerance has been extended to a genomic point of view and important achievements based on the manipulation of genomes or metabolic fluxes have been reached to counteract acetic acid toxicity (Palma et al., Notwithstanding the positive effect on enhanced GSH accumulation, such multiple overexpression system may in themselves become the cause of cell stress, causing metabolic burden and unforeseen cells responses, as shown by recent results obtained with recombinant strains accumulating high levels of GSH (Raghavendran et al., 4Reflecting on the molecular mechanisms of acetic acid stress response in budding yeast gives the opportunity to get an integrated view connecting aspects related to cellular transport, pH homeostasis, metabolism and stress‐signalling pathways Figure . All thi


Debilitating diseases of the eye represent a large unmet medical need potentially addressable with stem cell‐based approaches. Over the past decade, the California Institute for Regenerative Medicine (CIRM) has funded and supported the translation, from early research concepts to human trials, of therapeutic stem cell approaches for dry age‐related macular degeneration, retinitis pigmentosa, and limbal stem cell deficiency. This article chronicles CIRM's journey in the ophthalmology field and discusses some key challenges and questions that were addressed along the way as well as questions that remain. https://www.freepik.com/premium-vector/eye-anatomy-vector_760161.htm.Cell‐based therapies comprised of or derived from stem/progenitor cells are being developed to treat debilitating eye diseases such as dry age‐related macular degeneration (AMD), retinitis pigmentosa (RP), and limbal stem cell deficiency (LSD). Shown are the routes of delivery and location within the eye of the specific cell therapies discussed in this article. Adapted from  Debilitating eye diseases represent a large unmet medical need potentially addressable by stem cell‐based approaches. This article describes the advances made over the past 10 years by California Institute for Regenerative Medicine‐supported grantees in developing and translating stem cell‐based therapies for dry age‐related macular degeneration, retinitis pigmentosa, and limbal stem cell deficiency. The approaches described are now under evaluation in the clinic or have obtained approval to begin a clinical trial.1Loss of vision caused by a debilitating eye disease has a major impact on an individual's mobility, independence, quality of life (QOL), and ability to function in the modern world.https://medlineplus.gov/genetics/condition/age‐related‐macular‐degeneration/#frequency). Although a number of antivascular endothelial growth factor therapies are available for the wet, exudative form of AMD, there are currently no approved therapies to treat the more common dry form (80%‐90% of cases) which destroys central vision needed for reading, driving, and recognizing faces, resulting in significant impact on QOL. Retinitis pigmentosa (RP) is the leading cause of inherited vision loss leading to blindness, affecting approximately 1/4000 individuals (https://medlineplus.gov/genetics/condition/retinitis-pigmentosa/#frequency). It is estimated that 1.5 million people worldwide are currently impacted and there are no approved therapies. RPE65‐mediated retinal dystrophy  is another rare inherited disorder causing progressive blindness. In 2017, the Food and Drug Administration (FDA) approved Luxturna (voretigene neparvovec), a gene therapy treatment for patients with confirmed biallelic RPE65 mutation‐associated retinal dystrophy. Luxturna is the first approved gene therapy for an inherited retinal disease and the first adeno‐associated virus vector gene therapy approved in the United States. Limbal stem cell deficiency (LSCD) is a rare, progressive corneal disease that is found throughout the world, has wide‐ranging etiology, and can culminate in blindness. Surgical tissue transplantation is the main treatment option, but is often unsuccessful.Eye diseases that cause progressive blindness include both age‐related and inherited retinal diseases as well as corneal diseases. Age‐related macular degeneration (AMD) is a leading cause of vision loss in people over 65 years and is currently estimated to affect 11 million Americans and 170 million individuals worldwide , together with its grantees, pioneered the development of cell‐based approaches for severe eye diseases including diseases of the retina such as dry AMD and RP as well as the corneal disease, LSCD. This article briefly describes the progress made from the bench to early clinical trials, beginning in the trail blazing years of the now burgeoning field of regenerative medicine. We highlight initial questions and what has been learned along the way as well as remaining questions and potential challenges as these programs progress in clinical development.2The first CIRM‐funded stem cell translational awards were launched in 2009 and 2010 and several focused on dry AMD, considered an ideal indication for pioneering a cell replacement approach. Briefly, dry AMD is a slowly progressing disease of the retina, driven by degeneration of the retinal pigment epithelium (RPE), a polarized monolayer of cells that underlies the photoreceptors (PRs) and is essential for maintaining PR viability and function. Loss of RPE leads to PR degeneration and eventual blindness. Early researchers reasoned that if damaged RPE could be replaced with healthy, human embryonic stem cell (hESC)‐derived RPE, disease progression could be arrested or possibly even reversed.But first, several important questions had to be addressed: Are hESC‐derived RPE cells functionally equivalent to natural RPE cells? Do different hESC lines differ in their capacity to differentiate into RPE and which line should be used to make transplantable RPE for use in humans? Can induced pluripotent stem cells (iPSCs) be used to make viable RPE? Early CIRM grantees addressed these questions and successfully derived transplantable RPE cells from a number of stem cell sources including various hESC and iPSC lines. These teams demonstrated that stem cell‐derived RPE is phenotypically and functionally comparable to primary human RPE based on morphology, gene and protein expression, metabolome, and rod outer segment phagocytosis, a key function of the RPE. They also showed that viable RPE can be derived using iPSC, suggesting that autologous approaches for treating AMD may be feasible.This early research relied on the spontaneous differentiation of hESC into RPE when cultured, a process that is slow and yields a low percentage of RPE.A key question was how to deliver stem cell‐derived RPE to the retina. What devices or tools could be used? Would the cells survive and persist? For RPE to perform their multiple essential functions, which include interacting with and supporting the PR and participating in the visual cycle,Mark Humayun, an early CIRM awardee and cofounder of the California Project to Cure Blindness (CPCB), addressed this concern by designing an implant composed of a polarized monolayer of hESC‐derived RPE (hESC‐RPE) on an ultrathin, synthetic parylene substrate designed to both provide a surface for RPE adhesion and to mimic the diffusion barrier characteristics of Bruch's membrane. In a comparative study in rats, the survival of hESC‐RPE after subretinal implantation was significantly improved when RPE cells were delivered as a polarized monolayer on parylene compared to as a cell suspension.A CIRM‐funded phase 1/2a first‐in‐human (FIH) study to test the CPCB hESC‐RPE implant was initiated in 2015 by Regenerative Patch Technologies, a company founded by the CPCB. Surgical delivery was achieved using a specifically designed tool into which the ultrathin composite implant could be folded for insertion through a small incision in the sclera and then positioned over the area of damage in the retina. Data from 16 patients enrolled in the study demonstrated that surgical delivery is feasible in an outpatient setting and that the implant could be successfully targeted to the area of GA.Follow‐up structural and functional analyses have been reported for five patients in cohort 1 of the study, comprising advanced AMD patients severely affected by GA. These patients had poor visual acuity at baseline and because the area of GA involved the fovea, their ability to visually fixate on an object was unstable or absent. Optical coherence tomography (OCT) imaging suggested that there was integration of the hESC‐RPE with PR in the retinal tissue of the recipient eye. Although patients with advanced GA are thought to have little potential for visual recovery, there were signs of improved visual function. None of the implanted eyes showed further progression of vision loss and one eye showed improvement in visual acuity. Two patients improved their ability to visually fixate, suggesting that the implanted hESC‐RPE monolayer supports visual function in the overlying, previously nonfunctional retina.2.1A number of alternative cell‐therapy approaches are being investigated for dry AMD using different cell types or different starting sources of cells to derive RPE and alternative/no scaffold to deliver the cells Table . All of 3CIRM began funding development of a stem cell therapy for RP in 2008. Briefly, RP is an inherited degenerative disease of the PRs, typically with earlier onset than AMD. The literature documents that RP can be caused by more than one hundred different mutations in multiple genes, many of them rod‐specific, making it challenging to address with a gene therapy approach. RP is characterized initially by loss of the rod PRs that are located in the peripheral retina and are responsible for low‐light vision. Clinically, this anatomical loss of rod PR manifests as loss of peripheral vision, diminished ability to see in low light conditions and difficulty seeing at night. Subsequently, because the more important cone PR that enable high‐resolution color vision rely upon cone survival factors released by the rods, loss of rods leads to loss of cones, eventually culminating in total blindness. Cell‐replacement of lost PR would require re‐establishing synapses and neural connections, events that are likely hard to accomplish. More feasible treatment strategies, at least in the near term, tend to focus on preserving cone PRs, either directly or by preserving the rods. Such strategies could in principle arrest disease progression, which would have a major impact on patients, particularly in earlier stages of disease. Beginning in 2011, CIRM has supported the translation and development of two novel therapeutic approaches for RP.3.1https://doi.org/10.1167/iovs.04-0511). Preclinical biodistribution and cell survival studies subsequently suggested that the mechanism of neuroprotection is primarily paracrine.One approach, pioneered by Henry Klassen, is transplantation of cultured, allogeneic retinal progenitor cells (RPCs) of fetal tissue origin into the eyes of patients with RP with the goal of preserving vision by achieving neuroprotection of PRs, particularly the more essential cones. A major advantage of this neuroprotective approach is that the potential to preserve or restore vision is independent of the specific RP mutation causing this heterogeneous disease. Based on studies using mouse RPC transplanted into retinal dystrophic mice, human RPC (hRPC) were initially thought to be protective via both a trophic mechanism as well as by differentiating into rod PR upon which cone survival depends . As an added benefit, it was recognized that intravitreal injection greatly facilitates delivery of the cells and allows for the option of retreatment. Preclinical safety studies in both large and small animal models demonstrated the feasibility and tolerability of intravitreal injection of hRPC as well as the absence of an immune response even following a repeat injection. Based on this work, an IND was filed and cleared by the FDA.https://iovs.arvojournals.org/article.aspx?articleid=2690954). Although not powered for efficacy, changes in best‐corrected visual acuity (BCVA) in treated eyes were found suggestive of a therapeutic benefit at the higher dose levels.With continued CIRM support, a FIH phase 1/2 trial sponsored by jCyte, a company cofounded by Henry Klassen, was initiated in 2015 to evaluate the safety and potential activity of a single dose of hRPC administered intravitreally in one eye of 28 adults with RP. This study demonstrated that intravitreal injection of hRPC was well tolerated at doses up to 3 million cells . Post hoc analysis of a subset of 37 patients representing a target patient population for a subsequent trial showed a statistically significant improvement in BCVA compared to sham control at the 6 million cell dose level, with corroborating improvements in secondary endpoints including low light mobility, contrast sensitivity, visual fields, and QOL . Based on the encouraging positive results from this phase 2b trial, a phase 3 trial is being planned.3.2For patients with RP, the ability to safely navigate activities of daily living can be severely impacted by the decreasing visual field and loss of light sensitivity despite relative preservation of visual acuity in the narrowing central field. Although BCVA is widely accepted as the gold standard endpoint for measuring visual function, it does not capture all aspects affecting QOL and is thought not to assess loss of peripheral vision or ability to see at night, both of which are impaired in RP patients.To more broadly evaluate the effects of hRPC therapy in RP patients, the jCyte team developed and validated a novel Low Light Mobility assay for inclusion in the phase 2b trial. This assay measures the lowest light level at which the patient can functionally navigate a maze. A similar multiluminance mobility test that assesses functional vision was used as the primary endpoint of the phase 3 clinical trial of voretigene neparvovec (Luxturna) and was the basis for its approval.RP investigators continue to tackle a number of questions related to the appropriate evaluation of therapies for this indication. Since patients with RP lose vision in both eyes, should treatment be administered to both eyes simultaneously or sequentially? What is the durability of the clinical effect? In the jCyte phase 2b study, effects were durable for at least 12 months, but it is anticipated that repeat dosing will be required and the final dosing regimen remains to be determined.3.3Beginning in 2015, CIRM funded late preclinical development of a complementary allogeneic neuroprotective strategy for RP that uses subretinal injection of CNS10‐NPC, a neural progenitor cell population derived from human fetal cortex. In rodent and non‐human primate models, subretinally delivered CNS10‐NPC migrate beyond the injection site to durably engraft as a layer between the RPE and PRs.ClinicalTrials.gov Identifier: NCT04284293). It is hoped that allogeneic NPC grafts will persist in the human eye and enable long‐term photoreceptor preservation. The blood retinal barrier (BRB) isolates the retina from circulating leukocytesA team led by Shaomei Wang and Clive Svendsen completed GMP manufacturing and IND‐enabling safety studies, refined the surgical delivery technique, and obtained FDA clearance of an IND to proceed with clinical development of CNS10‐NPC in RP. A CIRM‐supported phase 1 trial of CNS10‐NPC in patients with RP is now poised to initiate enrollment  that reside in the limbus, a region located at the edge of the cornea and conjunctiva.ClinicalTrials.gov Identifier: NCT03957954) is poised to begin.An alternative strategy is based on transplantation of ex vivo expanded, autologous LSC from the healthy contralateral eye. Clinical proof of concept for autologous LSC replacement therapy has been demonstrated,With CIRM support, Dr Deng addressed an additional roadblock. Understanding LSCD severity is important for selecting a patient's best treatment course. If sufficient LSC remain, an injured cornea may repair itself. With inadequate LSC, an injured cornea can neither repair nor maintain a corneal transplant on its own. Similarly, inadequate LSC in a patient's fellow eye renders it an ineligible donor for autologous transplant. In these cases, either allogeneic transplant with immune suppression or a corneal prosthesis would be indicated. A persistent challenge in the field, however, has been the lack of consistent and quantitative methods to both diagnose and accurately stage LSCD. LSCD diagnosis has relied on clinical symptoms, slit lamp examination, and impression cytology to assess the degree of corneal damage and abnormalities of the corneal surface such as neovascularization or conjunctivalization (as indicated by goblet cells). Unfortunately, reliance solely upon these findings can result in misdiagnosis and selection of an inappropriate therapeutic plan.To address this need, Dr Deng and her colleagues in the Cornea Society developed methods and consensus around LSCD diagnosis and staging.4.1The main treatment option for LSCD is a surgical approach using either allogeneic or autologous tissue for the graft. Graft failure due to rejection is a common complication in the case of allografts. Expanded autologous LSC (Holoclar) is approved for use in Europe but not in the United States (as indicated above). A number of alternative autologous approaches are in early stages of clinical development as well as an approach using cadaveric LSC Table .5Stem cell‐based therapies are showing promise for the treatment of previously intractable debilitating eye diseases. Over the past decade, CIRM funded research focused initially on (a) establishing the feasibility of generating transplantable therapeutic cells from stem cells; (b) assessing the “safe and efficacious” cell dose in preclinical and clinical studies; (c) investigations of cell delivery route; (d) GMP manufacturing of large numbers of cells for preclinical and clinical investigation as well as for potential commercialization; and (e) incorporation of new innovative clinical endpoints in the design of the clinical trials. Later, the first CIRM supported clinical trials in eye diseases demonstrated the feasibility of administering cell therapies to the eye in an outpatient setting, both to the retina as well as to the vitreous. Although longer term data are not yet available and a relatively small number of patients have been treated, there are suggestions that implanted cells survive, are functional, and persist for months with encouraging measurable visual improvement for the patients.Several regulatory challenges specific to cell‐based therapies needed to be overcome. These included demonstrating that the final cell product does not contain residual undifferentiated, potentially tumorigenic cells and does not induce tumors in preclinical animal models. Additional challenges included developing a potency assay and demonstrating manufactured lot consistency when the cellular product's mechanism of action is complex and not well understood. In some cases, a specifically designed delivery tool needed to be developed and tested in large animal preclinical models.The optimal route of delivery of a cell therapy for a retinal disease will likely depend on the mechanism of action. For a therapy that exerts a neuroprotective effect via a paracrine mechanism, intravitreal delivery appears to be the preferred route, allowing for diffusible factors to broadly access the retina. Intravitreal delivery has several additional advantages in that it is minimally invasive, allows for repeat injection and is routinely used in outpatient settings, including in the optometrist's office. In contrast, retinal cell replacement strategies may necessitate subretinal delivery, requiring specific tools and surgical expertise that may best be deployed at centers of excellence. A CIRM supported study has demonstrated that subretinal delivery is feasible and that durable graft retention can be achieved. The potential for long‐term clinical benefit thus could outweigh the risks.The need for immunosuppression remains an open question. Whether the eye is an immune privileged site remains to be determined through continued clinical investigation and immune monitoring. Nevertheless, surgical delivery of cells to the retina may temporarily breach the blood‐retinal barrier. To guard against possible immune rejection of transplanted cells, current clinical trial protocols include immunosuppression for a period of time. Important questions that remain to be resolved include how much immunosuppression is required and for how long, and whether systemic immunosuppression can be replaced with local immunosuppression.5.1The development of stem cell therapies for debilitating eye diseases progressed rapidly and measurably on several fronts during the past 5 years. Advancements include (a) the development of innovative tools and technologies that incorporate patients' QOL measures; (b) evolution of a regulatory paradigm that previously required a change of 13 letters on an eye chart in order to approve a therapy to acceptance of a mobility endpoint when appropriate; and (c) the advancement of gene therapy approaches for some rare eye diseases. New therapeutic modalities and genomics will expand the field further.As with all cell therapies, the development of a robust and scalable manufacturing process is critical to the final success of the therapy. The increasing adoption of expedited regulatory pathways, including regenerative medicine advanced therapy designation, has escalated the importance of defining critical quality attributes for each cell and gene product as early in the process as possible and of rapidly establishing a mature manufacturing process suitable for commercialization.The authors declared no potential conflicts of interest.All authors contributed significantly to the conception, design, writing, and review of the manuscript.


Tree regeneration is a key process for long‐term forest dynamics, determining changes in species composition and shaping successional trajectories. While tree regeneration is a highly stochastic process, tree regeneration studies often cover narrow environmental gradients only, focusing on specific forest types or species in distinct regions. Thus, the larger‐scale effects of temperature, water availability, and stand structure on tree regeneration are poorly understood.We investigated these effects in respect of tree recruitment (in‐growth) along wide environmental gradients using forest inventory data from Flanders (Belgium), northwestern Germany, and Switzerland covering more than 40 tree species. We employed generalized linear mixed models to capture the abundance of tree recruitment in response to basal area, stem density, shade casting ability of a forest stand as well as site‐specific degree‐day sum (temperature), water balance, and plant‐available water holding capacity. We grouped tree species to facilitate comparisons between species with different levels of tolerance to shade and drought.Basal area and shade casting ability of the overstory had generally a negative impact on tree recruitment, but the effects differed between levels of shade tolerance of tree recruitment in all study regions. Recruitment rates of very shade‐tolerant species were positively affected by shade casting ability. Stem density and summer warmth (degree‐day sum) had similar effects on all tree species and successional strategies. Water‐related variables revealed a high degree of uncertainty and did not allow for general conclusions. All variables had similar effects independent of the varying diameter thresholds for tree recruitment in the different data sets.Synthesis: Shade tolerance and stand structure are the main drivers of tree recruitment along wide environmental gradients in temperate forests. Higher temperature generally increases tree recruitment rates, but the role of water relations and drought tolerance remains uncertain for tree recruitment on cross‐regional scales. This study presents an analysis of tree recruitment in different regions with large environmental gradients. We show that stand structure, shade tolerance. And temperature are main drivers of tree recruitment. Furthermore, the effect of water relations is subject to a high degree of uncertainty. CurrentTree regeneration can be decomposed into several subprocesses, starting with seed production followed by seed dispersal, seed storage, germination, establishment of seedlings, growth of seedlings and saplings, and finally the recruitment of small trees that exceed a certain measurement threshold  threshold for the first time in a defined time interval, also called “in‐growth” in forest science. Tree recruitment can thus serve as a proxy for successful regeneration. Tree recruitment and its relationship to the environment and stand structure have been studied for various species in different regions  instead of the ecological properties of a species  of a forest stand as well as of degree‐day sum, water balance, and plant‐available soil water holding capacity on the recruitment rate? We expect that basal area, stem density, and shade casting ability determine the overall recruitment rate, while degree‐day sum, water balance, and plant‐available soil water holding capacity modulate the recruitment rate. Thus, effects of stand structure are expected to be stronger than climatic effects.Do these effects act differently on groups of species with varying levels of shade and drought tolerance? Thus, effects of stand structure are expected to differ between shade‐tolerant and shade‐intolerant tree species. Specifically, increased basal area along with high stem density and high values of SCA should have strong negative effects in general with shade‐tolerant tree species less affected than light‐demanding species. Furthermore, we expect low water availability to have negative effects on tree species with low drought tolerance and less negative or positive effects for species with high drought tolerance.What are the implications of using heterogeneous data sets for analyzing the abundance of recruitment in different regions and environmental settings? We expect different effects and levels of uncertainty between regions and data sets caused by the characteristics of each data set, for example, due to differences in forest types, environmental gradients, and sampling designs within each data set.In order to address these limitations of previous studies on tree recruitment, we aim to analyze (a) how tree recruitment is related to stand structure and environment, and (b) how it changes with different levels of shade tolerance and drought tolerance across a wide range of environmental and forest stand conditions. We group species from an ecological point of view that allows for assessing the recruitment of all species in four data sets covering managed and unmanaged forests in Flanders (Belgium), northwestern Germany, and Switzerland. Furthermore, we consistently use one unified method across all data sets to study patterns of tree recruitment at different environmental ranges. This includes the verification of results from previous studies on tree recruitment across different regions and the quantification of qualitatively known patterns regarding successional strategies defined by the shade and drought tolerance of European temperate tree species. Specifically, we address three research questions.22.1Analyzing tree recruitment across different regions Figure  and enviThe tree recruitment rates and variables related to stand structure were derived from forest inventories in Flanders (northern Belgium), northwestern Germany, and Switzerland Figure . The NatThe two Swiss data sets cover the largest environmental gradient in our study due to the large elevational gradient in this country. Zell et al.  present 2.2The annual recruitment rate was defined as the number of trees per ha and species that exceeded the calipering threshold (DBH threshold) for the first time between two consecutive inventories Table . Tree sp2/ha], stem density [ha−1], and shade casting ability (SCA) [value between 1 and 5] of the second census . All trees that already had been defined as recruitment trees were not considered for the calculation of these variables. SCA is a species‐specific trait related to the ability of a species to cast shade, ranging from 1 (very low) to 5 (very high). It is based on factors such as leaf size, leaf angle, leaf density, and phenological aspects  is based on soil depth, plant‐available water capacity, and coarse volumetric fraction. The calculation was adapted from Henne et al.  and defi2.3We defined six species groups according to their tolerance to shade and drought to differentiate between levels of shade tolerance and drought tolerance. These groups were subsequently treated as an attribute of a species, thus enabling the analysis of differences related to shade tolerance and drought tolerance without losing information  at the species level. The grouping was based on shade tolerance and drought tolerance values from the Functional Ecology of Trees (FET) data set  for CH FR, GER FR, and FLAN NFI to assess the implications of using different DBH thresholds between models. All variables except plot size and period length were transformed to be on the same scale and make the coefficients comparable  but high basal with high stem density describes stands with many smaller trees (mostly younger trees). It is important that the model can differentiate between such cases. Water balance and bucket size interact by determining the actual available water at a site. If one of these variables is low, the other variable may compensate and contributes to a higher level of water availability at a site. We did not include further interactions among stand structure or climate although they might lead to significant results because these interactions involve various processes and feedbacks that impede inferring on causal relationships.All observations were treated at the species level to account for species‐specific differences inside trait groups, but trait groups were used as grouping variable for the statistical analysis. For each plot observation, plot size [ha] and the period length defined by the number of years between two consecutive inventories were used to account for systematic offsets in the models.Y per plot i, year t, and species j followed a negative binomial distribution (NB) defined and parameterized by the mean μ and dispersion parameter ϕ according to chapter 14.2. in the function references of the Stan documentation  are the coefficients for each trait group g and variable m where Nvars is the number of variables. xi,t(m) is the observed value of the explanatory variables m, where the stand structure‐related variables  vary between each plot i and year t. The environmental variables degree‐day sum, water balance, and bucket size varied between plots i only. Random intercepts u0Ii,j were added for each ploI i and species j and random slopes uj(m) for each species j and variable m to account for species‐specific differences within trait groups. All random effects were assumed to be normally distributed:brms package , there were no deviations from the average interaction effect between trait groups.Estimates for basal area were negative, but distinctly stronger for the light‐demanding species of trait groups D2S1 and D5S1, with the exception for trait group D2S1 in the CH NFI data set. In addition, positive deviations from the mean were apparent for shade‐tolerant species of trait groups D3S4 and D2S5, again with an exception, this time for trait group D3S4. Stem density was positively associated with the recruitment rate for all trait groups in all data sets with almost no differences between trait groups, with the exception of the estimates for trait group D2S3 from the CH FR data set, which were negative , and the degree‐day sum influence tree recruitment rates similarly across the four study regions and along wide environmental gradients. Moreover, pooling multiple species based on their shade tolerance revealed consistent patterns of the relationship between recruitment and stand structure. Specifically, basal area along with species’ shade casting ability proved to be important stand characteristics for predicting differences in recruitment between successional strategies expressed by shade tolerance. A higher degree‐day sum was related to higher recruitment rates when assessing all trait groups, but regarding drought tolerance no evidence for differences between trait groups was found in our study. Aspects related to the water balance of a site turned out to be subject to large uncertainty. Overall, our results show that tree recruitment is primarily driven by stand attributes such as basal area and stem density as well as by the shade casting ability of the constituent species in the overstory. This was evident in all four data sets in spite of the large heterogeneity resulting from varying assessment methods and environmental ranges.Recruitment of trees into size classes of ≥4 cm DBH, as in our four data sets, represents a relatively late stage of forest regeneration. It integrates over sometimes long periods since germination and multiple environmental constraints act upon young trees of widely different size. This causes the statistical relationships between stand structure, climate, and tree recruitment to be manifold and complex. In the following, we discuss whether our models allow to infer on causal relationships for each variable and trait group and how underlying ecological processes explain the estimated effects.In addition, we compare our results to other studies. One of these studies is the one from Zell et al.  which we4.1Our findings regarding the relationship between basal area and tree recruitment are consistent with previous studies  a disturbance caused gaps in the stand and recruitment did not yet pass the diameter threshold to fill the gaps or (b) a few very large trees suppress the emergence of other trees. All stages between these two cases can be considered forest development stages as a function of stem density and basal area. Feldmann et al.  and Glat4.2We explain the positive effect of degree‐day sum by an increased turnover rate. If no other biotic or abiotic factors are limiting, all tree species profit from higher temperature because of higher photosynthesis rates that lead to increased growth rates. Nevertheless, the effect of degree‐day is difficult to link to specific processes without considering water‐related variables. Although we demonstrated a generally positive effect of the degree‐day sum on tree recruitment, which confirms the findings in Zell et al. , the eviWhat is the reason for climatic effects being so poorly represented in our study? One possible explanation is that trees of a species may experience ontogenetic shifts, that is, they are able to germinate and grow under certain climatic conditions but do not necessarily survive under the same climatic conditions until they reach a size class that is considered tree recruitment  revealed distinct effects of stand structure and the degree‐day sum on tree recruitment, these models were not able to account for water relations and could not elucidate whether different levels of drought tolerance affect the relationship between tree recruitment and climate. In recent decades, GLMMs were used successfully for analyzing growth and mortality  only show weak signals when analyzing tree recruitment with empirical models. All factors showed similar effects throughout all size classes represented in our data. Although temperature partly showed positive effects on tree recruitment, we could not disentangle the role of water relations in this respect. This has major implications for the projection of the development of forest ecosystems under future climate because the drivers of tree recruitment are only known on the local scale and remain poorly understood on larger spatial scales where variation in climate is high.Although data sets from different regions that were collected based on different sampling protocols are usually quite heterogeneous, we conclude that the long‐term monitoring of managed and unmanaged forests provides a valuable source of information for a better understanding of highly stochastic ecological processes such as tree recruitment ; Data curation (lead); Formal analysis (lead); Investigation (lead); Methodology (lead); Project administration (lead); Software (lead); Validation (lead); Visualization (lead); Writing‐original draft (lead); Writing‐review & editing (lead). Peter Meyer: Data curation (supporting); Resources ; Writing‐review & editing (supporting). Jonas Stillhard: Data curation (supporting); Resources ; Software (supporting); Writing‐review & editing (supporting). Emiel De Lombaerde: Resources (supporting); Writing‐review & editing (supporting). Jürgen Zell: Methodology (supporting); Writing‐review & editing (supporting). Golo Stadelmann: Data curation (supporting); Resources ; Writing‐review & editing (supporting). Harald Bugmann: Conceptualization (supporting); Formal analysis (supporting); Funding acquisition (lead); Investigation (supporting); Methodology (supporting); Project administration ; Resources ; Supervision (lead); Writing‐original draft (supporting); Writing‐review & editing (supporting). Christof Bigler: Conceptualization (supporting); Formal analysis (supporting); Investigation (supporting); Methodology (supporting); Supervision (lead); Validation (supporting); Visualization (supporting); Writing‐original draft (supporting); Writing‐review & editing .https://doi.org/10.5281/zenodo.5119919.This article has earned an Open Materials Badge for making publicly available the components of the research methodology needed to reproduce the reported procedure and analysis. In case of acceptance, we plan to make the R scripts available via Zenodo or Dryad. All materials are available at Appendix S1Click here for additional data file.Appendix S2Click here for additional data file.Appendix S3Click here for additional data file.


Narrative communication is often more persuasive for promoting health behaviour change than communication using facts and figures; the extent to which narrative persuasiveness is due to patients’ identification with the storyteller vs engagement with the story is unclear.To examine the relative impacts of patient engagement, age concordance and gender concordance on perceived persuasiveness of video‐recorded narrative clips about opioid tapering.Patient raters watched and rated 48 brief video‐recorded clips featuring 1 of 7 different storytellers describing their experiences with opioid tapering. The dependent variable was clips’ perceived persuasiveness for encouraging patients to consider opioid tapering. Independent variables were rater engagement with the clip, rater‐storyteller gender concordance and rater‐storyteller age concordance (<60 vs ≥60). Covariates were rater beliefs about opioids and opioid tapering, clip duration and clip theme. Mixed‐effects models accounted for raters viewing multiple clips and clips nested within storytellers.P < .001). Neither age concordance nor gender concordance significantly predicted perceived persuasiveness. The theme Problems with opioids also predicted perceived persuasiveness.In multivariable models, higher rater engagement with the clip was associated with higher perceived persuasiveness  and assessed the key study variables. For example, randomized clinical trials comparing narrative vs non‐narrative patient education videos have found that narrative videos resulted in significantly better blood pressure control among African American patients with uncontrolled hypertension.Understanding the impact of age and gender concordance on narrative persuasiveness is particularly important in health communication, where there is a need for brief, effective interventions that can be incorporated into clinical workflows to promote patient health behaviour change. Patient education videos are commonly used in clinical studies, and a systematic review of such videos found that videos delivering story‐based messages were more persuasive than non‐narrative videos.To examine the relative contribution of these factors, we analysed patient ratings of brief video clips showing patient stories that were collected to create a patient education video about opioid tapering. We focused on opioid tapering because reducing rates of opioid‐related harms (including overdose and opioid use disorder) is an urgent public health priority in the United States.22.1This study was part of a larger project to create a patient education video about opioid tapering. We recruited both compelling storytellers (who were featured in narrative video clips) and raters who evaluated those clips. Storytellers and raters were recruited in a 2‐phase process from the same population using identical recruitment procedures and eligibility criteria. All participants were adults at 13 primary care clinics in Northern California who reported moderate‐to‐severe chronic neck and/or back pain and had either tapered down or off long‐term prescription opioids (defined as at least one opioid dose per day for at least three months) within the past year. We recruited patients aged 35‐85 years because our prior research found very few patients younger than 35 who took long‐term opioids for chronic back or neck pain.2.1.1To identify compelling storytellers, in the first phase of recruitment we enrolled 21 participants who took part in 4 focus groups about opioid tapering (eighty‐nine percent of patients who were screened and eligible for inclusion agreed to participate).To select clips, researchers viewed all interviews and identified video segments that showed a storyteller recounting a brief, coherent story about their experiences related to opioid tapering. Consistent with the overall project goal, we did not select clips that explicitly discouraged opioid tapering or explicitly encouraged opioid dose escalation. All identified segments were edited into clips that were stored as separate digital files. Two examples of video clips rated in this study can be viewed in the Videos 2.1.2For the second phase, patient raters were recruited from the same clinics using the same methods and eligibility criteria as storytellers. We began recruiting raters when we had almost finished conducting focus groups. Participants who were unable to participate in focus groups were eligible to participate as raters. However, focus group participants were not eligible to be raters. In addition, rater recruitment was stratified by age (<60 vs ≥60 years) and gender. To our knowledge, no raters were acquainted with any of the storytellers.Each rater rated 24 randomly selected narrative clips, for a total of 1152 ratings . The 48 clips were first randomly divided into two groups (‘A’ and ‘B’) of 24 clips each. Raters were then scheduled to watch the video clips in a series of small groups. Each small group watched either the 24 clips in group ‘A’ or the 24 clips in group ‘B’. During each small group, raters were first informed of the study purpose and then watched and rated the 24 video clips on several Likert‐type items. Raters watched each clip and then immediately recorded their ratings for that clip using paper questionnaires. Rating all 24 clips took approximately 90 minutes; raters were given a break midway through to prevent fatigue. Raters were seated to ensure that they could not observe how others rated the clips. During each small group, raters viewed clips in a different random order (using a random order generated by the study biostatistician) to minimize potential order effects. Raters were assigned to small groups to ensure balanced recruitment for each gender‐by‐age category. Prior to viewing the clips, raters provided data on demographic characteristics , highest education attained, employment status, annual household income (US dollars), average pain severity, duration of chronic pain and opioid tapering status .Raters’ baseline questionnaires included the following covariates related to raters’ beliefs about opioids and opioid tapering (which could affect perceived persuasiveness). Raters’ attitudes about opioid tapering were measured using 1 item from the Prescribed Opioid Difficulties ScaleBaseline questionnaires also included dependent variable was perceived clip persuasiveness, which raters assessed after viewing each clip by answering the question, ‘After watching this clip, how willing do you think other people with chronic pain would be to try taking less opioid pain medication?’ . We designed this variable using this particular wording because the planned patient education video was intended to target patients who were candidates for tapering, and we thought that asking patients in varying stages of the tapering process about the clips’ perceived persuasiveness for others would produce more reliable and relevant information.Our independent variables were patient engagement with the clip, age concordance between rater and storyteller, and gender concordance between rater and storyteller. Raters assessed their engagement with each clip after viewing it by answering 5 items related to engagement  and clip theme. To evaluate clip theme, two investigators first viewed each clip and then coded whether the story in that clip included any of the following nine topics: reasons for tapering, opioid‐related risks, fears about tapering, benefits of tapering, communicating with clinicians, managing pain, managing opioids, getting through the day (ie completing daily activities while tapering) and support for tapering (ie support from family or friends). Disagreements were resolved by discussion. Topics were not mutually exclusive; the story in a single clip could be coded for more than one topic . The most common topics were managing pain (52%) and getting through the day (52%), followed by reasons for tapering (40%). The least common topics were fears about tapering (8%) and benefits of tapering (10%).Finally, covariates for 2.3Our sample size of 48 raters (12 per gender‐by‐age category) was chosen to achieve the aims of the larger study,When examining the effects of gender and age concordance, our sample was designed so that half of the ratings were from raters who had the same gender and/or age as the storyteller and half were from raters who did not. Using the same strategy as above, for an ICC of 0.7, the effective sample size is 33 per group and the power to detect a 0.5‐point difference in persuasiveness between the clips rated by age‐ or gender‐concordant raters and those rated by age or gender non‐concordant raters is at least 82%. The power would be >92% if the ICC was 0.5.2.4Given the large number of topics and the substantial proportion of clips that included multiple topics, we used latent class analysis to classify clips into homogenous classes, or themes, based on the combination of topics that each clip included. We performed a latent class analysis to simplify how we analysed clip content and reduce the chances of type I error. We examined models with two to five themes (classes) and selected the optimal number of themes based on the proportion of clips assigned to each class, interpretability of results and several goodness‐of‐fit criteria .We started by fitting separate linear mixed‐effects models to examine unadjusted (bivariate) associations between each independent variable  and our dependent variable (perceived persuasiveness).P < .2 in unadjusted analyses, one at a time, to the multivariable model containing our three independent variables and retained only those that were significantly associated with perceived persuasiveness in the final model. Finally, to evaluate the relative impact of storyteller vs story on perceived persuasiveness, we compared the ICCs for the storyteller vs the video clip to examine the proportion of total variance of the dependent variable explained by each random component of our mixed‐effects model.We also examined associations between covariates and our dependent variable. We first analysed each covariate separately in unadjusted linear mixed‐effects models controlling only for raters viewing multiple clips and for clips being nested within storytellers. We then added covariates with We used Mplus version 83As planned, we recruited 48 raters (12 for each gender‐by‐age category); 79% of patients who were screened and eligible for inclusion agreed to participate. Table Problems with opioids, Daily functioning and Opioid access. As conveyed in Table Problems with opioids theme were most likely to include the topics reasons for tapering, opioid risks and managing opioids; clips assigned to the Daily functioning theme were most likely to include the topics getting through the day and managing pain; and clips assigned to the Opioid access theme were most likely to include the topics communicating with clinicians and managing opioids. Of the 48 clips, 12 (25%) were assigned to Problems with opioids, 27 (56%) to Daily functioning and 9 (19%) to Opioid access. Assignment probabilities were excellent for all 3 themes .We chose the three‐class model as the best‐fitting latent class analysis model for the clip topics in our data. Table Table Problems with opioids and Daily functioning—were also associated with perceived persuasiveness (P < .2) in unadjusted analyses and so were considered for inclusion in the final model  were significantly associated with perceived persuasiveness in unadjusted analyses. Patient raters who endorsed a desire to taper or had already tapered rated clips as significantly more persuasive than raters who disagreed with a desire to taper. Two of the 3 themes—el Table .Problems with opioids—remained significantly associated with perceived persuasiveness and was retained in the final model. Clips with stories that discussed problems with opioids were perceived as significantly more persuasive  than clips assigned to either of the other two themes. Of the 3 independent variables, only patient engagement remained significantly associated with perceived persuasiveness  in the final model. The ICC was 6.5% for the clip level and 0% for the storyteller level in the empty model and 7.9% for the clip level and 0% for the storyteller level in the final mixed models.When added to the multivariable model containing all three independent variables, only one covariate—the clip theme 44.1This study examined factors correlated with perceived persuasiveness of video‐recorded narrative clips collected during a larger project on opioid tapering. Patients were involved in this study both as storytellers (in each clip) and as raters assessing perceived persuasiveness. We found that higher rater engagement with brief narrative clips was strongly and significantly associated with greater perceived persuasiveness. In contrast, age concordance and gender concordance between patient raters and storytellers were not significantly associated with clips’ perceived persuasiveness in multivariable models. These findings suggest that highly engaging stories or vignettes are likely persuasive to patients regardless of the storytellers’ age and gender or patients’ age and gender. One possible explanation of this finding is that, while a patient's identification with the storyteller is a key element of persuasion in narrative persuasion theory,Problems with opioids were more persuasive than other clips suggests that using stories that recount patients’ rationale for tapering, including concerns about opioid‐related risks, is a promising strategy for future interventions related to opioid tapering and, potentially, other interventions focused on behavioural cessation.One unexpected finding was that raters who agreed or strongly agreed that tapering was beneficial found clips to be much more persuasive than raters who disagreed that tapering was beneficial. An implication of this finding is that the video produced as part of the larger project may be more persuasive to patients who are at least open to the idea of tapering opioids vs patients who believe tapering will lead to worse pain control. This finding may relate to the consensus from prior reviews that it is particularly difficult to persuade patients to stop harmful or unwanted behaviour, compared with taking on or initiating healthy behaviour.any of the variance in perceived persuasiveness (ie ICC for storyteller was 0). Raters and storytellers were recruited from the same patient population using the same procedures, so one potential explanation for this finding is that persuasiveness was driven by raters’ identification with the clinical scenarios described in the clips or problems with opioids described by the storytellers rather than by the demographic characteristics of the storytellers. These findings indicate that narrative content also impacts the perceived persuasiveness of patient narratives.Notably, in this analysis of 48 clips featuring 7 different storytellers, storyteller identity did not independently explain Our study has some limitations. The small number of unique storytellers in the clips and sample size may have limited our ability to detect small effects or to reliably estimate the ICC for storyteller; however, our sample was balanced to optimize our ability to evaluate the effects of age and gender concordance. We could not examine racial concordance because most raters and storytellers identified as white. Our decision to measure whether raters perceived that clips were likely to be persuasive for others as the dependent variable rather than whether clips were persuasive to raters themselves was consistent with the larger project's goal, but measuring the latter as the dependent variable might have generated different results. The clips used in this study were shorter than the recorded and written narratives typically analysed in the studies of narrative transportation theory. However, patient engagement with stories and attitudes towards storytellers often form quickly, and ratings based on short video clips of the kind used in this study tend to be highly correlated with ratings based on longer recordings.4.2When viewing brief video‐recorded narrative clips about opioid tapering, greater patient engagement with the clip, but not age or gender concordance with the storyteller, was associated with greater perceived persuasiveness for promoting opioid tapering. Additional studies are needed in this area; however, our findings suggest that patient engagement with a story that is clinically relevant to the patient is likely more important for determining persuasiveness than are demographic similarities between patient and storyteller.Results from this study are relevant to clinicians, researchers and health educators planning education videos or interventions to promote health behaviour change. Videos and interventions that incorporate patient narratives that are clinically relevant and highly engaging to patients are likely to be persuasive regardless of storyteller demographics. Conversely, using different storytellers to target specific patient demographic groups may not substantially increase overall persuasiveness.No authors have any conflicts of interest to disclose.Henry conceived and designed the study, obtained funding, planned and interpreted the analysis results, wrote the first draft of the manuscript and approved the final submission. Feng planned the analysis, edited the manuscript for critical content and approved the final submission. Verba collected the data, edited the manuscript for critical content and approved the final submission. Kravitz interpreted the analysis results, edited the manuscript for critical content and approved the final submission. Iosif planned and conducted the analysis, interpreted the analysis results, edited the manuscript for critical content and approved the final submission.This study was approved by the University of California Davis Institutional Review Board (protocol number 897480).Video S1Click here for additional data file.Video S2Click here for additional data file.


The spreading of SARS‐CoV‐2 virus infection is still of great concern as well as clinical and social interest. The key to conquer this pandemic would be establishment of herd immunity by vaccination and of treatment, and I have discussed issues we are facing now. The emergence of variants of respiratory syndrome coronavirus 2 (SARS‐CoV‐2) and their rapid spread over the world has cast a shadow over potential victory enabled by novel and highly effective vaccines over this human tragedy. Coronavirus disease 2019 (COVID‐19) cases surpassed 100 million on January 27, 2021 according to the Center for Systems Science and Engineering at Johns Hopkins University, indicating that more than 1 in 80 humans on earth have already been infected. Some of the variants, especially the UK coronavirus variant (B.1.1.7), the South Africa variant (B.1.351), and the recently identified India variant (B.1.617), are more infectious and deadlier compared with SARS‐CoV‐2 before their appearance. The currently known major variants are characterized by mutations in the receptor‐binding domain of their spike‐protein ; the India variant has two characteristic mutations of E484Q and L452R that may impact vaccine efficacy. COVID‐19 is distinguished from other infectious diseases by its two‐faced clinical presentation: it is sometimes almost asymptomatic despite its transmission potential, or it may present with pneumonia that can lead to rapid disease progression and a fatal outcome.As diabetes and obesity are well known risk factors for the severity and mortality of COVID‐19, maintaining good glycemic control and appropriate body weight during the pandemic has become ever more important for patients and their physicians. While healthy eating and adequate physical activity are essential for patients with diabetes to maintain their glycemic control and body weight at all times, it has been reported that the containment measures for COVID‐19 negatively affect both the nutrition and exercise of patients with diabetes. We have reported that even under the mild containment measures imposed in Japan, patients with diabetes exhibited lifestyles altered toward a more sedentary mode, which caused a deterioration of glycemic control and weight gain, more seriously in the elderly populationTreatment of the disease and establishment of herd immunity are necessary to conquer the pandemic. Special interest is focused on the efficacy of currently available vaccines, most of which employ novel messenger ribonucleic acid and virus vector technologies, against the emerging variants of SARS‐CoV‐2. mRNA vaccines are created synthetically using a fragment of the RNA sequence of the virus surface protein and are taken up by muscle cells at the injection site with the possible addition of fibroblast and dendritic cells; the synthesized virus protein confers the subsequent immune response. The expected effect of vaccines is not only the prevention of infection but also the reduction or prevention of transmission of SARS‐CoV‐2 including variants.Vaccination not only protects against symptomatic illness but also mitigates the more severe manifestations of the disease, as well as the mortality rate. The mRNA vaccines show over 90% efficacy in preventing both symptomatic illness and severe disease of COVID‐19 in randomized clinical trials; in a real‐world setting, 7 days after the second dose, the Pfizer‐BioNTech mRNA vaccine showed over 90% efficacy for documented infections, symptomatic illness, and severe disease  in an analysis based on Israel’s mass‐vaccinationIt is not known whether the vaccines are similarly effective among patients having underlying diseases, especially those associated with an immunocompromised state such as diabetes, cancer, and immune disease. We found that in elderly Japanese patients with diabetes, although a single vaccination of Oka varicella zoster vaccine safely and successfully induced a humoral and cell‐mediated immune response, concurrent vaccination of Oka varicella zoster vaccine and 23‐valent pneumococcal polysaccharide vaccine (PPSV23) induced a reduced immune response to varicella zoster vaccineRegarding treatments, while there is no magic bullet, corticosteroids such as dexamethasone are established drugs for the amelioration of the cytokine storm sometimes seen in patients with COVID in intensive care units (ICU). A Janus kinase inhibitor, baricitinib, is recently drawing attention as an alternative anti‐inflammatory drug. Currently, the authorities recommend only limited drugs including the anti‐viral drug remdesivir, dexamethasone, and tocilizumab optionally for hospitalized patients in a severe state. Baricitinib is recommended only when corticosteroids cannot be used in combination with remdesivir; there is not enough evidence to justify the first line use of baricitinib. The results of an ongoing clinical trial, Adaptive COVID‐19 Treatment Trial 4 (ACTT‐4), evaluating the drug’s effects against dexamethasone is awaited. The interleukin‐6 (IL‐6) inhibitor, tocilizumab, a recombinant humanized monoclonal antibody against IL‐6 receptors, was reported to protect from fatality when used in addition to standard care such as corticosteroids and anti‐viral drugs such as remdesivir in patients with severe or critical COVID‐19 infection and elevated inflammatory markers. This finding was presented in the recent results from the open‐label, pragmatic Randomized Evaluation of COVID‐19 Therapy (RECOVERY) trialEarly diagnosis and the initiation of treatment as soon as possible are key to overcoming COVID‐19 infection. Recently, monoclonal antibodies developed for SARS‐CoV‐2 (to be used as combination therapy such as bamlanivimab and etesevimab or casirivimab and imdevimab) are being used in the treatment of patients with mild to moderate COVID‐19 who are not hospitalized but at high risk of disease progression and/or hospitalization, for example patients with diabetes, chronic kidney disease, and severe obesity. They are available only in limited countries and their application is restricted due to their administration by infusion. In this respect, the development of molnupiravir, an orally administered bioavailable prodrug of the ribonucleoside analog β‐d‐N4‐hydroxycytidine, is expected and is currently under phase 3 clinical trial for outpatients with COVID‐19.et al.Glycemic control before and during COVID‐19 infection in addition to the drugs used for hypertension and diabetes treatment are also significant factors affecting the severity and mortality of the disease. Glycemic control is an essential strategy for COVID‐19 treatment; poor glycemic control results in impaired immune defense and an abnormal immune response, which in COVID‐19 infection can lead to severe pneumonia and acute respiratory distress syndromeAgain, the key to the successful treatment of severe COVID‐19 infection is early diagnosis and prompt initiation of treatment. In this respect, patients with diabetes should have better access to medical services than they do presently. Moreover, if herd immunity and effective treatment are not established, those who are at highest risk from infection of COVID‐19 would have to continue in voluntary lock‐down indefinitely. The efficacies of the vaccines and treatments in certain groups such as patients with underlying diseases including diabetes, require verification.Y Hamamoto received speaker fees from Novo Nordisk Pharma Ltd and received a research grant from Sumitomo‐Dainippon Pharma Co., Ltd and Nippon Boehringer Ingelheim.


Human impact is noticeable around the globe, indicating that a new era might have begun: the Anthropocene. Continuing human activities, including land‐use changes, introduction of non‐native species and rapid climate change, are altering the distributions of countless species, often giving rise to human‐mediated hybridization events. While the interbreeding of different populations or species can have detrimental effects, such as genetic extinction, it can be beneficial in terms of adaptive introgression or an increase in genetic diversity. In this paper, I first review the different mechanisms and outcomes of anthropogenic hybridization based on literature from the last five years (2016–2020). The most common mechanisms leading to the interbreeding of previously isolated taxa include habitat change (51% of the studies) and introduction of non‐native species . These human‐induced hybridization events most often result in introgression (80%). The high incidence of genetic exchange between the hybridizing taxa indicates that the application of a genic view of speciation (and introgression) can provide crucial insights on how to address hybridization events in the Anthropocene. This perspective considers the genome as a dynamic collection of genetic loci with distinct evolutionary histories, giving rise to a heterogenous genomic landscape in terms of genetic differentiation and introgression. First, understanding this genomic landscape can lead to a better selection of diagnostic genetic markers to characterize hybrid populations. Second, describing how introgression patterns vary across the genome can help to predict the likelihood of negative processes, such as demographic and genetic swamping, as well as positive outcomes, such as adaptive introgression. It is especially important to not only quantify how much genetic material introgressed, but also what has been exchanged. Third, comparing introgression patterns in pre‐Anthropocene hybridization events with current human‐induced cases might provide novel insights into the likelihood of genetic swamping or species collapse during an anthropogenic hybridization event. However, this comparative approach remains to be tested before it can be applied in practice. Finally, the genic view of introgression can be combined with conservation genomic studies to determine the legal status of hybrids and take appropriate measures to manage anthropogenic hybridization events. The interplay between evolutionary and conservation genomics will result in the constant exchange of ideas between these fields which will not only improve our knowledge on the origin of species, but also how to conserve and protect them. The exact start of the Anthropocene has been a matter of debate and its onset has been connected to different events, such as the rise of deforestation and agriculture  to identify the most common mechanisms and outcomes of human‐mediated hybridization events  with transgressive phenotypes could expand into more harsh environments with higher food availability  and grizzly bears (U. arctos) in the Arctic  in Italy  that originated from the interbreeding of two Italian species (S. aethnensis and S. chrysanthemifolius) in British gardens at the turn of the 18th century  and variegate darters (E. variatum) are replacing the now endangered candy darter  and hybrids show higher population growth rates than the parental taxa, genetic swamping might occur in which the parental taxa are replaced by hybrids  and the invasive Madagascar ragwort (S. madagascariensis). Hybridization rates between these species are very high, but hybrids are not viable. As the invasive plant increases in abundance, the native plant continues to waste reproductive effort and will start declining in numbers  that could lead to the decline of the latter species  and hybridization rates are high, parental taxa will waste reproductive effort when hybridizing. This situation can lead to demographic swamping where parental taxa decline due to decreased population growth rates  which focuses on reproductive isolation between diverging lineages Mayr, . This coThis new perspective is nicely illustrated by recent studies that showed how genetic differentiation between diverging lineages is heterogeneously distributed across the genome, often concentrated in a few genomic regions, so‐called “islands of differentiation” , second‐generation hybrids (F2), backcrosses, etc. For example, a mixture of different generational hybrids indicates the formation of a hybrid swarm, whereas the absence of backcrosses and second‐generation hybrids suggests selection against hybrids. Most conservation genetic studies use a set of variable genetic markers  to quantify the genetic make‐up of different individuals using software packages such as The use of a few genetic markers works well to identify early‐generation hybrids and backcrosses, but can run into several issues  and American black ducks (Anas rubripes) might lead to genetic extinction of the latter species  and Japanese sika (C. nippon). The RADseq data were able to identify more advanced backcrosses compared to the microsatellites, leading to a more fine‐grained picture of introgression dynamics between these species.These two issues—setting a realistic admixture threshold and the presence of homozygous markers—can often be solved by adding more markers. Indeed, genomic studies can more confidently identify hybrids and later‐generation backcrosses, leading to more sound conclusions about the hybridization dynamics  and twaite shad  up to the third generation. Similar marker sets and sequencing protocols have been developed for other study systems  and tundra bean goose (A. f. serrirostris) resulted in a largely homogeneous genomic landscape, but a few genomic islands of differentiation seem to prevent these taxa from merging  adapted to high levels of water pollution through introgressive hybridization with the non‐native Atlantic killifish (F. heteroclitus). And a study on hybridization between the local moth species Helicoverpa zea and the invasive H. armigera revealed that an insecticide‐resistant locus introgressed into the local species and increased in frequency  in the United States which lists and protects vulnerable species and subspecies of plants and animals  in the San Francisco Estuary  to theoretical considerations, such as patterns of introgression, ancient hybridization events, and the reticulated nature of evolution.From a conservation perspective, the unpredictability of (adaptive) introgression dynamics (Taylor & Larson, None declared.