Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).The Osteosarcoma Surveillance Study was initiated in the United States in 2003 to monitor for a potential association between the osteoporosis treatment teriparatide and osteosarcoma. Osteosarcoma occurs at a background incidence rate of approximately 2.5 cases per million per year in US adults aged 40 years or older. For this study, incident cases of osteosarcoma diagnosed between January 1, 2003, and December 31, 2016, were identified through participating cancer registries in the United States. Information on prior exposure to medications and possible risk factors was obtained by self‐report (or proxy report) in telephone interviews. Exposure information was verified through medical record abstraction for a sample of patients. A standardized incidence ratio was estimated to compare the observed and expected numbers of osteosarcoma patients with a prior history of teriparatide treatment. Interviews were completed for 24% (1173) of patients diagnosed with osteosarcoma between 2003 and 2016; three reports of teriparatide use before diagnosis were identified. Based on the background incidence rate, the expected number of osteosarcoma cases among patients treated with teriparatide was 4.17. Given the three observed cases, the standardized incidence ratio was 0.72 . Demographic characteristics were similar for interviewed and noninterviewed patients. Agreement was >90% between self‐reported and chart‐recorded exposure to osteoporosis medications. Mean age of interviewed patients was 61 years; 53% of patients were male, 84% were white, and 5% were Hispanic. The prevalence of suspected risk factors for development of osteosarcoma among the osteosarcoma cohort was 19% for history of radiation and 4% for history of Paget's disease of bone. These findings showed that the incidence of osteosarcoma associated with teriparatide use during the 15‐year surveillance period was no different than would be expected based on the background incidence rate of osteosarcoma. © 2020 The Authors.  The 15‐year US Osteosarcoma Surveillance Study was initiated in 2003 to evaluate a potential association between the recombinant human parathyroid hormone analog teriparatide  and osteosarcoma in humans. The study was established under a postmarketing commitment to the US Food and Drug Administration (FDA) following the November 2002 approval of teriparatide. In the United States, teriparatide is indicated for the treatment of postmenopausal women with osteoporosis at high risk for fracture, for the increase of bone mass in men with primary or hypogonadal osteoporosis at high risk for fracture, and for the treatment of men and women with osteoporosis associated with sustained systemic glucocorticoid therapy at high risk for fracture. However, subsequent studies demonstrated a “no‐effect” dose in rats and no bone evidence of bone tumor in a long‐term study of cynomolgus monkeys. On the basis of preclinical evidence, the US prescribing information for teriparatide includes a warning about a potential risk of osteosarcoma and precautions against use of the product for patients with risk factors for osteosarcoma . The current label recommends that lifetime treatment with teriparatide should be limited to a maximum duration of 2 years, whereas the average duration of use in patients ≥65 years has been reported to be 10 months.Preclinical evidence suggested a potential association between teriparatide and osteosarcoma. Specifically, in preclinical rat studies, a dose‐dependent increase in the risk of osteosarcoma incidence was observed after administration of teriparatide.1 Howe but potential risk factors include Paget's disease of the bone and prior radiation treatment administered to the bones. Nevertheless, most osteosarcomas are diagnosed in patients without these risk factors. As estimated from National Cancer Institute Surveillance, Epidemiology, and End Results (NCI‐SEER) data, osteosarcoma occurs at a background incidence rate of approximately 2.5 cases per million per year in US adults aged ≥40 years.Osteosarcoma is a primary malignant bone tumor characterized by the production of osseous matrix by neoplastic cells. Little is known about the etiology of osteosarcoma in humans,6, 7 a Forteo Patient Registry in the Unites States, and two population‐based observational cohort studies. This US case‐series study started 90 days after the first marketed use of teriparatide and had a duration of 15 years. The objectives were to identify newly diagnosed cases of osteosarcoma among men and women aged ≥40 years and to determine incident osteosarcoma cases, if any, with a history of teriparatide treatment.In addition to the US case‐series study, the surveillance program for teriparatide includes four other components: a companion case‐series surveillance study in Europe (completed in 2014),13 a F Briefly, the study used a case‐series design to identify incident cases of osteosarcoma from participating cancer registries in the US. Patients diagnosed with osteosarcoma were identified through state, regional, or comprehensive cancer treatment center cancer registries. Information on medical history and antecedent exposures, including teriparatide and other medications, was collected through a structured telephone interview with patients or their proxies. Responses were validated by medical record review in a random sample. The observed number of patients with osteosarcoma who had a confirmed exposure to teriparatide was compared with the number of exposed osteosarcoma cases expected to be identified by the cancer registries using a standardized incidence ratio (SIR) and corresponding 90% confidence interval (CI).The methodology and interim results for this study have been described in detail previously.15 BriA total of 30 US cancer registries participated in the study, including population‐based registries with regional (n = 1) or state‐level catchment areas (n = 26) and hospital‐based registries affiliated with an oncology referral center (n = 3). Participating registries were asked to use prespecified International Classification of Diseases for Oncology, 3rd edition  codes to identify patients with osteosarcoma during the study period (April 2004–October 2018). Registries collect pertinent tumor‐related information  as part of their standard case‐ascertainment process, and they capture diagnoses from both inpatient and outpatient treatment facilities. Because reporting of incident cancer cases to cancer registries is legally mandated, these registries were ideal for identifying a cancer population.Eligible patients had been diagnosed with osteosarcoma on or after January 1, 2003, and had a primary residence in the United States. The case definition for study inclusion was meeting all of the following criteria: (i) patient aged ≥40 years at the time of diagnosis; (ii) diagnosis reported from a participating cancer registry; (iii) diagnosis of osteosarcoma based on at least one of 12 ICD‐O‐3 oncology codes for osteosarcoma or one of five other ICD‐O‐3 oncology codes for which the primary tumor site was bone. additional cancers were included if diagnosed with one of five ICD‐O‐3 morphology codes with a primary tumor site listed as bone , and demographics. If the patient was deceased or unable to participate in the interview, a proxy familiar with the patient's medical history was interviewed. Capture of self‐reported exposure to teriparatide or other similar medications  was facilitated using a structured, deductive question series. All instances of self‐reported teriparatide treatment or daily injections for any reason underwent review and adjudication to investigate possible exposure to teriparatide. Data collection was initiated in July 2004 for patients diagnosed on or after January 1, 2003. Beginning in September 2008, in an effort to improve the response rate for the telephone interviews, patients or proxies participating in the interviews received $25 compensation for their time.Self‐reported information provided during the telephone interview was validated among a randomly sampled subset of patients to assess agreement between some self‐reported telephone interview responses by patient or proxy and abstracted medical record data. A minimum of 10% of all patients interviewed or 25 patients annually (whichever was greater) were selected for validation. Eligible patients must have returned a Health Insurance Portability and Accountability Act (HIPAA) release form and listed a primary care physician seen before the qualifying cancer diagnosis. Trained medical record abstractors contacted the treating physician, obtained the records, and abstracted the information. The abstractors provided the completed abstraction forms for data entry and analysis of the level of agreement between self‐reported information and the medical record. for additional details on the analyses.Cancer case counts were summarized separately for patients with an ICD‐O‐3 code indicating osteosarcoma and for patients diagnosed with one of the five additional ICD‐O‐3 codes for which the primary tumor site was bone. Descriptive analyses were conducted to summarize osteoporosis history and treatments, including teriparatide, demographics, medical and family history, and lifestyle and environmental exposures. Prior teriparatide exposure was derived from interview data. See Andrews and colleagues15 forThe observed number of patients with osteosarcoma and a confirmed exposure to teriparatide was compared with the number of exposed osteosarcoma cases expected to be identified by the cancer registries using a SIR. The expected number of osteosarcoma cases among patients treated with teriparatide was estimated based on the background rate of osteosarcoma in the United States , age‐ and sex‐adjusted to the teriparatide‐treated population, and the estimated number of cumulative person‐years at risk among patients treated with teriparatide in the United States. The estimate was further refined to account for the osteosarcoma cases that the study identified and the number of patients interviewed. The analysis years (2003–2016) were restricted to diagnosis years, with more complete case ascertainment and reporting from participating cancer registries, and excluded diagnosis year 2017. Precision of the SIR estimator was evaluated using exact 90% CIs around the ratio, produced by the following formulas:D = observed number patients with osteosarcoma reporting teriparatide use; E = the expected number of osteosarcoma cases among teriparatide users; χα,υ2 = the α percentile of the chi‐square distribution with υ degrees of freedom, with SIR lower bound, and SIR upper bound.where Sensitivity analyses were conducted to examine changes in the SIR by varying the observed and expected number of osteosarcoma cases among teriparatide users. The observed number of cases was increased by including the five additional ICD‐O‐3 codes  for which the primary site was bone. The expected number of osteosarcoma cases among teriparatide users was recalculated for a variety of plausible alternative values for the data components from which the number was derived: (i) the background incidence rate of osteosarcoma, (ii) the estimated person‐years at risk, and (iii) the estimated interview rate obtained in the surveillance study.The study was approved by the RTI International institutional review board and local institutional review boards and other committees affiliated with the cancer registries.A total of 3808 patients diagnosed with osteosarcoma from 2003 through 2016 were identified by the 30 cancer registries that contributed data during the study; 4940 patients were expected in the United States for that period, thus 77% of the expected osteosarcoma patients were identified by the participating registries. Of the 3808 patients identified by the registries, 2549 were reported to the study center and met all necessary requirements to be contacted for an interview. A total of 1173 (31%) patients or their proxies were interviewed; therefore, based on the expected total of all osteosarcoma cases (4940 expected), 24% of patients or their proxies were interviewed , chondroblastic osteosarcoma , and fibroblastic osteosarcoma . The anatomical site of the tumor varied, but the lower extremities predominated, with nearly one‐half of cases occurring in the legs (33%) or the pelvic region (14%). The next most prevalent sites included the skull or face region (16%), connective or soft tissue (10%), and the upper limbs (9%).Among the 1173 patients with osteosarcoma who were interviewed or whose proxies were interviewed, more than one‐half were men (53%), most were white (84%), and 5% were Hispanic. The mean age at the time of diagnosis of osteosarcoma was 61 years . More than one‐half of patients (918/1173) were living when their osteosarcoma was reported Table . The mosAmong the interviews completed for patients diagnosed with osteosarcoma, 144 respondents (12%) reported history of osteoporosis and 12 (1%) reported possible prior use of teriparatide. After additional follow‐up  to confirm exposure to teriparatide, three exposures were considered valid , eight were confirmed to be incorrect (because of patient or proxy confusion with the name of the medication), and the remaining one patient reported teriparatide use that was confirmed by their healthcare provider to have been prescribed after osteosarcoma diagnosis. Of the three exposures considered valid, one was observed approximately 8 years after starting teriparatide, another approximately 3 months after initial exposure, and the third approximately 2 years after starting teriparatide. All had diagnosis of ICD‐O3 histology code “Osteosarcoma, NOS” and the site of tumor was different for each case.n = 83) developed osteosarcoma in the exact site of the radiation therapy, and an additional 36% (n = 82) developed osteosarcoma in the same region of the body as the radiation therapy. The most commonly reported cancers in patient family histories were breast cancer (23%), brain cancer (8%), and leukemia (6%). Only 52 patients (4%) reported an immediate blood relative with prior osteosarcoma that started in the bone.Among all patients diagnosed with osteosarcoma who had completed interviews, 181 patients (15%) had a prior injury or infection in the same bone as the site of their bone cancer. More than one‐quarter of patients  reported history of another type of cancer before the osteosarcoma diagnosis, 226 (19%) reported history of X‐ray or radiation treatment for prior conditions, and 127 (11%) reported history of chemotherapy for prior conditions. Paget's disease of the bone was reported by 46 patients (4%). Of patients who had radiation therapy before developing osteosarcoma, 37%  reported that they had smoked at least 100 cigarettes in their lifetime, and the majority of patients (63%) consumed alcohol in the year before osteosarcoma diagnosis. Among the environmental exposures assessed over each patient's lifetime, pesticides were the most prevalent given farm proximity (24%) or manufacturing (5%). Petrochemicals (12%) and nuclear facility proximity (7%) were less common, and only 80 patients (7%) reported prior occupations with radiation exposure .Among patients treated from 2003 through the end of 2016, 5,432,764 person‐years of risk were estimated. A background incidence rate of osteosarcoma of 3.2 cases per million per year—derived from the NCI‐SEER rate (2.5 per million population per year) and age‐ and sex‐adjusted to the teriparatide‐treated population—and study interview rate of 24% were estimated. Given these parameters, the expected number of cases of osteosarcoma exposed to teriparatide treatment is 4.17 cases. With three observed cases, the SIR is 0.72 .The following alternative values of the components for the expected numbers of osteosarcoma cases were considered in five scenarios for the sensitivity analyses of the SIR. First, along with including five additional ICD‐O‐3 codes, induction periods of 1, 2, and 3 years were included. Second, a lower background incidence rate of osteosarcoma of 2.5 cases per million per year was assumed . Third, the estimated person‐years at risk were decreased by 25% . Fourth, a lower estimated interview rate in the study—20% of cases instead of 24%—was assumed . Fifth, a combination of the aforementioned values was assumed that included a decrease in the estimated person‐years at risk by 25%, an interview rate of 20% of cases , and background incidence of 2.5 cases per million per year .From the recalculated observed and expected numbers of osteosarcoma cases, a series of SIRs (with CIs) for the alternative values of the data components was generated. Table For the validation component of the study, 1009 patients (or their proxies) who completed the interview and signed and returned a medical release form were considered “selection eligible” for the medical record abstraction component of the study. A random sample of 482 cases was selected; in addition, eight of the 13 cases for which possible teriparatide use was reported during the interview  were included in the validation sample . Of these 490 selected cases, medical records were abstracted for 351 patients. For 139 patients, records could not be obtained or contained insufficient information.Agreement was high between answers provided during the telephone interview and data abstracted from the patient's medical records: 96% or higher agreement for osteoporosis medication use , 85% agreement for history of osteoporosis, 89% or higher agreement for history of radiation or chemotherapy treatment or history of prior cancer, and 97% agreement for history of Paget's disease.Among patients with osteosarcoma, there were no notable differences in the distributions for age at diagnosis , sex, or ethnicity between all patients identified by participating registries and patients interviewed ; an additional case was identified in the similar case group defined by the five additional ICD‐O‐3 codes. The histological type for all three cases was the most common overall  and location of the tumor varied for each exposed case. Although 53% of osteosarcoma cases identified were male, two out of three exposed cases were female. This is not unexpected given that approximately 90% of Forteo users are female (data on file).The SIR for the three observed cases in the osteosarcoma case group was 0.72 , and the lower bound of the 90% CI for the SIR did not exceed the null value of 1.0. Even with inclusion of the additional case in the SIR calculation from the patients identified by diagnosis with one of the five additional ICD‐O‐3 codes, the lower bound of the SIR did not exceed 1. Therefore, diagnostic misclassification is unlikely to explain the lack of an association observed in this study. In a post hoc subgroup analysis among females, the SIR was 0.61 . For statistical evidence of an association between teriparatide treatment and osteosarcoma, the lower bound of the 90% CI for the SIR would need to exceed 1.0. Nine cases reporting prior teriparatide use would need to have been observed during the same period for the lower bound of the 90% CI to exceed 1.0. The study had statistical power to detect a potential threefold increased risk, had this existed, or one additional case of osteosarcoma per 156,000 teriparatide‐treated patients per year.This long‐term case‐series study of adult patients aged ≥40 years with osteosarcoma provides additional information relating to the demographics, tumor characteristics, and potential risk factors for a large, population‐based sample of patients with a rare disease. The most common morphology was osteosarcoma not otherwise specified, and the most common tumor site was the lower extremities, consistent with clinical expectations and the European component of this study. In addition, approximately 19% of patients interviewed reported having received radiation therapy before developing osteosarcoma; of these, 37% developed osteosarcoma in the exact site of the radiation therapy and an additional 36% developed osteosarcoma in the same region of the body as the radiation therapy. Patients diagnosed with osteosarcoma and interviewed in this study reflected the age, sex, and ethnicity of all patients identified by participating registries. In addition, tumor type and site were similar. White patients were slightly overrepresented in the interviewed population. These results can be generalized to the US population.http://www.encepp.eu/encepp/viewResource.htm;jsessionid?id=8541). None of these 112 patients had a record of teriparatide use. However, given the small study size, identification of a teriparatide‐exposed patient would have occurred only if teriparatide were associated with a large increased risk of osteosarcoma. Two other, US‐based studies included a population‐based comparative study linking pharmacy claims data from Medicare with cancer registry data from 26 states and a population‐based comparative study linking pharmacy claims from a large commercial data source with cancer registry data from 29 states. Analyses from these studies were recently completed, and results are forthcoming. Finally, in a US study, the Forteo Patient Registry, as of the final annual linkage of data from more than 75,000 registered teriparatide‐exposed patients with 42 state cancer registries, no incident cases of osteosarcoma have been identified (data on file).Other noninterventional studies have examined the potential relationship between teriparatide and osteosarcoma as part of an osteosarcoma surveillance program; findings from those studies to date are consistent with findings from this study. These studies include a 10‐year case‐finding surveillance study conducted in the five Nordic countries that identified 129 cases of osteosarcoma, for which 112 patient medical records were abstracted . Interviewing all identified patients was not feasible, and there was heterogeneity in access to patients among registries because of different state and institutional‐level research approval requirements and different mechanisms in place to protect patient privacy. It is possible that these factors limited generalizability and increased possible selection bias; however, the study cohort was evaluated, and interviewed patients were found to be generally similar to all eligible patients identified by participating registries with respect to demographic and tumor‐specific characteristics. Residual information bias may have resulted from exposure misclassification. This bias may be caused by inaccurate recall by patients or inaccuracies as a result of the time elapsed from diagnosis to study interview or from reliance on proxy interviews—38% of interviewees were adult proxies. However, misclassification of teriparatide exposure was expected to be limited owing to its unique drug delivery system (daily injections) and storage requirements (refrigeration). In addition, validation using medical record data demonstrated high agreement (>85%) between interview responses and patient records. Although it is well known that induction and latency periods exist between exposure to a carcinogen and development of cancer, the expected number of osteosarcoma cases among exposed patients was determined assuming no induction period. However, assuming longer periods of latency up to 3 years did not affect the interpretation of the study findings and only one case was diagnosed >3 years after starting teriparatide (~8 years), so considering a longer induction period would serve only to further lower the SIR . Furthermore, uncertainty may exist regarding estimated expected values that were not accounted for in CI calculations. Finally, the measure obtained from this analysis is limited to the age‐ and sex‐adjusted SIR, which may suffer from residual confounding because of differences other than age and sex between patients exposed to teriparatide and the US cancer registry population. Given approximately 90% of teriparatide users are female whereas approximately 53% of osteosarcoma cases identified by the registries were male, the study cohort may not be representative of the population treated with teriparatide in terms of sex distribution.In conclusion, this study found that the incidence of osteosarcoma associated with teriparatide use during the 15‐year surveillance period was no different than would be expected based on the background incidence rate of osteosarcoma. Three patients diagnosed with osteosarcoma reported prior teriparatide exposure; this count was below the estimated expected given estimated inputs for person‐years of risk since drug launch, background osteosarcoma incidence rates, and study interview rate. The study had the statistical ability to detect a threefold increase in risk, which, for an outcome so rare, would result in one additional osteosarcoma diagnosis per 156,000 teriparatide‐treated patients per year.This study was performed under a research contract between RTI Health Solutions and Eli Lilly and Company and was funded by Eli Lilly and Company. Elizabeth Andrews, Alicia Gilsenan, David Harris, David McSorley, and Kirk Midkiff are or were salaried employees of RTI Health Solutions. Nicole Kellier‐Steele is a salaried employee of Eli Lilly and Company. The contract between RTI Health Solutions and Eli Lilly and Company assures independent publication rights for RTI Health Solutions. The authors have no other conflict of interest to declare.Supplemental Appendix Results for Patients With Other Diagnostic CodesClick here for additional data file.


Acute myocardial infarction causes lethal injury to cardiomyocytes during both ischaemia and reperfusion (IR). It is important to define the precise mechanisms by which they die in order to develop strategies to protect the heart from IR injury. Necrosis is known to play a major role in myocardial IR injury. There is also evidence for significant myocardial death by other pathways such as apoptosis, although this has been challenged. Mitochondria play a central role in both of these pathways of cell death, as either a causal mechanism is the case of mitochondrial permeability transition leading to necrosis, or as part of the signalling pathway in mitochondrial cytochrome c release and apoptosis. Autophagy may impact this process by removing dysfunctional proteins or even entire mitochondria through a process called mitophagy. More recently, roles for other programmed mechanisms of cell death such as necroptosis and pyroptosis have been described, and inhibitors of these pathways have been shown to be cardioprotective. In this review, we discuss both mitochondrial and mitochondrial‐independent pathways of the major modes of cell death, their role in IR injury and their potential to be targeted as part of a cardioprotective strategy. This article is part of a special Issue entitled ‘Mitochondria as targets of acute cardioprotection’ and emerged as part of the discussions of the European Union (EU)‐CARDIOPROTECTION Cooperation in Science and Technology (COST) Action, CA16225. In this review, we focus on cell death pathways occurring in cardiomyocytes. Clearly, mitochondria are central to the function of cardiomyocytes, occupying nearly 40% of the cytosolic volume,2m). ATP is further depleted by the F0F1ATPase running in reverse, expending ATP in a futile attempt to maintain ΔΨm. Eventually, the sarcolemmal ion pumps fail and the cell swells in a process called ‘oncosis’, which is defined as a pre‐lethal stage following cellular injury.During myocardial ischaemia, oxygen is rapidly depleted, causing mitochondrial respiration to cease. Anaerobic metabolism is activated within seconds of flow cessation, but is unable to provide sufficient ATP for maintaining sarcolemmal ion gradients and mitochondrial membrane potential , leading to mitochondrial Ca2+ overload and opening of the mitochondrial permeability transition pore (MPTP).m, and shortly afterwards, ATP stores are depleted, ion pumps cease functioning, and the cells die through a process of oncosis and necrosis.Mitochondria play a critical role in the process of myocardial IR injury. In particular, upon reperfusion, when the supply of oxygen to the cardiac cells is re‐introduced and mitochondrial respiration recommences. The mitochondrial substrate succinate, having accumulated during ischaemia, provides a powerful source of electrons which produce oxygen radicals by reverse electron transport via complex I, resulting in oxidative stress (excess reactive oxygen species or ROS).It may be possible to target necrosis during the early stages of infarction in order to protect the heart from IR injury. Mice lacking either the MPTP or the MCU have smaller infarct sizes following IR.3Apoptosis is a form of cell death that can be distinguished microscopically from oncosis by cell shrinkage, chromatin condensation and distinctive blebbing (budding) of the plasma membrane. Apoptosis can occur via intrinsic or extrinsic mechanisms but both result in mitochondrial outer membrane permeabilization (MOMP), mitochondrial cytochrome c release, caspase activation, DNA fragmentation and cell blebbing.Early studies detected evidence of apoptosis along with necrotic cell death following myocardial IR.In contrast to the above, other studies have argued against the significant role of apoptosis in IR‐induced cell death, based on the fact that there is minimal expression of most proteins required for the apoptotic program in adult cardiomyocytes.2+ overload leading to MPTP opening may contribute to apoptosis by increasing MOMP and cytochrome c release.Mitochondria play an important role in the execution of apoptosis. These organelles are the major contributors of ROS and the major target for ROS‐induced damage. The mitochondrial apoptosis pathway is triggered by mitochondrial swelling and outer mitochondrial membrane rupture, thus favouring the release of pro‐apoptotic factors such as cytochrome c and SMAC/Diablo from the intermembrane space into the cytosol.Another important mechanism for mitochondrial quality control is mitochondrial fusion and fission. Mitochondrial fission is considered to be the prerequisite for the occurrence of mitophagy and several studies revealed the causal relationship between mitochondrial fission and the induction of apoptosis.42+ dyshomeostasis, increased oxidative stress and incomplete digestion of mitochondrial DNA. These events play a role in triggering the formation of inflammasomes within cells.Mitochondrial damage negatively affects cardiomyocyte function via disruption of oxidative phosphorylation, CaMitophagy can be considered a beneficial cellular process that enhances cell viability following stressful stimuli by eliminating dysfunctional mitochondria. Mitophagy is therefore essential for cardiomyocyte survival.,The results of early studies of the role of autophagy in IR‐induced cardiomyocyte death were somewhat contradictory, indicating that autophagy could be cyto‐protective, but could also direct cells towards apoptosis.5−/− mice, it has been suggested that cyclophilin D, an important regulatory component of the MPTP, might be involved in necroptosis signalling2+‐calmodulin‐dependent protein kinase), serving as an upstream regulator of the MPTP, is likely to trigger this critical event in IR‐induced necro(pto)sis.Necroptosis, a regulated mode of cell death with a necrotic appearance, has been identified in various cardiac pathologies, including myocardial IR  and in vivo rat hearts subject to IR, as evidenced by increased levels of RIP1, RIP3 and MLKL.Subcellular analyses have further revealed that RIP3 co‐localizes with mitochondriaFrom the above discussion, it is evident that the extent of mitochondrial participation in necroptosis in cardiomyocytes or other cell types is not clear. It has been suggested that the contribution of mitochondria to necroptosis may be secondary or context‐dependent.6Pyroptosis, meaning ‘fire’ and ‘falling’, is a pro‐inflammatory cell‐death program occurring after cytosolic receptor‐mediated recognition of pathogen‐associated molecular patterns (PAMPs), or host‐derived, danger signals such as damage‐associated molecular patterns (DAMPs). Some well‐characterized DAMPs include glucose‐regulated proteins (GRPs), high‐mobility group box 1 (HMGB‐1), IL‐1β, S100 family proteins and some heat shock proteins (HSPs). Interaction of these proteins with cellular pattern recognition receptors can lead to the assembly of the intracellular NLRP3 inflammasome complex. The assembled NLRP3 inflammasome elicits auto‐proteolytic cleavage and activation of caspase‐1, which mediates the cleavage/maturation of the pro‐inflammatory cytokines, pro‐IL‐18 and pro‐IL‐1β, and cleaves/activates gasdermin‐D (GSDMD), releasing its N‐terminal fragment.vicious cycle that expands the region of ischaemic damage. Evidence for a role of inflammasome/pyroptosis in acute myocardial IR injury comes from several studies.−/− mice in an ex vivo Langendorff perfused heart model of IR.The DAMPs released after cell death can induce further injury, thus causing a The mechanisms of myocardial IR damage by DAMPs may involve binding to receptor for AGE  or to TLR4 (toll‐like receptor 4), thus activating NFκB and exacerbating myocardial damage. Indeed, among pro‐inflammatory genes, NFκB promotes the transcription of components of the NLRP3 inflammasome in cardiac cells.Despite the fact that IR‐damaged myocardium releases a combination of priming and triggering factors of the NLRP3 inflammasome, it has been proposed that the NLRP3 inflammasome in the heart is not sufficient to respond to a trigger signal in the absence of a priming.+ efflux‐dependent way. Furthermore, the serine threonine kinase NIMA‐related kinase 7 (NEK7) is a promoter of NLRP3 inflammasome assembly downstream of ROS and K+ efflux.Although little is known about the role of mitochondria in pyroptosis occurring in the myocardium specifically, there is evidence for mitochondrial involvement in other cell types. In fact, mitochondrial dysfunction, leading to oxidative stress and mitochondrial DNA (mtDNA) release, is emerging as a key mechanism in triggering NLRP3 assembly and activation in several conditions and indeed has been proposed as the universal trigger for NLRP3 activation.Recent evidence has shown that autophagy is necessary to reduce myocardial damage after acute myocardial infarction, thus confirming that the autophagic process can limit the activation of the NLRP3 inflammasome by removing damaged mitochondria and that impaired mitophagy may contribute to adverse cardiac remodelling in myocardial infarction.Arguing against an important role for NLRP3 inflammasome in cardiac injury is a work of Sandanger et al who reported that NLRP3 inflammasome activation is cardioprotective during myocardial IR.7Although the involvement of mitochondria in extrinsic and intrinsic apoptotic pathways is undeniable, and their role in necroptosis and pyroptosis is being elucidated, the degree to which mitochondria are involved in other, more newly recognized, forms of cell death has just begun to be disentangled. In this respect, ‘parthanatos’ entails cell death characterized by excessive activation of poly(ADP‐ribose) polymerase‐1 (PARP‐1).‘Ferroptosis’ is a form of programmed cell death dependent on iron. Ferroptosis seems to converge on the BCL2‐family protein member BID, promoting its translocation to the mitochondria where it causes profound mitochondrial fragmentation and dysfunction.8Although chemical inhibitors of specific cell death pathways are very effective in limiting infarct size after IR in the experimental setting, few studies have investigated these drugs in patients experiencing cardiac IR.9An important limitation in all investigations of cell death pathways is the methodology used to evaluate cell death in cardiomyocytes Table . TUNEL sIn summary, mitochondria seem to be a convergent point between various regulated cell death processes. In some cases, such as MPTP‐mediated necrosis, apoptosis and parthanatos, their participation is clear, whereas in other cases, such as necroptosis, the extent of their involvement might be context‐dependent. In either case, increasing evidence points to a crosstalk between the diverse pathways of death, with mitochondria likely to be a central node of all such pathways. They are therefore a key target for maintaining the health of cardiomyocytes during IR and thereby protecting the heart from injury.The authors confirm that there are no conflicts of interest.Each author drafted and critically revised a section the paper; SD critically revised the entire manuscript; and all authors approved the entire submitted and final versions.


STF, are often interpreted as evidence for alleles that contribute to local adaptation. However, theoretical studies have shown that in spatially structured populations the spread of beneficial mutations with spatially uniform fitness effects can also induce transient genetic differentiation. In recent years, numerous empirical studies have suggested that such species‐wide, or global, adaptation makes a substantial contribution to molecular evolution. In this perspective, we discuss how commonly such global adaptation may influence the genome‐wide distribution of STF and generate genetic differentiation patterns, which could be mistaken for local adaptation. To illustrate this, we use forward‐in‐time population genetic simulations assuming parameters for the rate and strength of beneficial mutations consistent with estimates from natural populations. We demonstrate that the spread of globally beneficial mutations in parapatric populations may frequently generate STF outliers, which could be misinterpreted as evidence for local adaptation. The spread of beneficial mutations causes selective sweeps at flanking sites, so in some cases, the effects of global versus local adaptation may be distinguished by examining patterns of nucleotide diversity within and between populations in addition to STF. However, when local adaptation has been only recently established, it may be much more difficult to distinguish from global adaptation, due to less accumulation of linkage disequilibrium at flanking sites. Through our discussion, we conclude that a large fraction of STF outliers that are presumed to arise from local adaptation may instead be due to global adaptation.Spatially varying selection promotes variance in allele frequencies, increasing genetic differentiation between the demes of a metapopulation. For that reason, outliers in the genome‐wide distribution of summary statistics measuring genetic differentiation, such as  STF outlier test. In this paper, we show that such patterns of genetic differentiation between local populations may also commonly occur because of adaptation to global conditions, where a single mutation sweeps across the range of a species. This can occur either when populations are observed partway through the sweep process or when recombination during the sweep leaves a spatially variable pattern in the frequency of hitchhiking alleles. The spatial genetic pattern that results from either mechanism is similar to the pattern expected under local adaptation, with high values of STF. Although the ways that global adaptation can generate such extreme patterns in STF were previously recognized, we show that this global adaptation process likely happens often enough to be a significant source of false positives in searches for the genetic basis of local adaptation.Local adaptation occurs when a population of organisms adapts to the conditions that occur locally and is commonly observed in spatially heterogeneous environments. Finding the genes responsible for such local adaptation is a common goal for both understanding the process of evolution and for managing critical populations. One way to identify the genetic basis of local adaptation is to search for genes that are most different between local populations, using a so‐called Determining the genetic basis of local adaptation is of primary interest to evolutionary biologists, as it provides insights into how natural selection shapes genetic variation. The genetic basis of local adaptation has increasingly been studied using various types of “genome scans,” that is, methods which are applied across the genome to detect genetic patterns that are expected under the process of local adaptation (reviewed in Haasl and Payseur STF (or its derivatives), which measures the variance in allele frequencies among the demes of a metapopulation. Because long‐term local adaptation promotes variance in allele frequency among demes, regions of the genome subject to spatially varying selection should have STF values that appear extreme in the genome‐wide distribution  alleles on the haplotype may also end up at high frequencies. Selective sweeps will, therefore, homogenize genetic variation among populations and typically cause a reduction in nucleotide diversity within and between populations Barton  and can n Bierne —we referSTF outliers to be driven by global adaptation is well recognized, it is unclear how often we should expect this to be a problem for scans using STF to detect local adaptation. To answer that question, one may ask how common are such global selective sweeps? The best information on the process of global adaptation in eukaryotes comes from Drosophila melanogaster. An estimate of the number of incomplete selective sweeps in that species can be obtained as follows: it is estimated that D. melanogaster and Drosophila simulans began to diverge around 14 million generations ago  in D. melanogaster has been estimated in numerous studies to be ∼0.5  and selection coefficients for beneficial mutations (as), expected fixation times can be calculated  under panmixia. Taken together, these calculations suggest that at any point in time, D. melanogaster is subject to approximately 22,200/280 = 80 incomplete selective sweeps for alleles that will ultimately reach fixation. This calculation assumes a panmictic population, but population structure is ubiquitous in the natural world. Because population structure prolongs fixation times relative to panmixia  may fix due to the force of migration from demes where they are favored. Alternatively, local extinctions could cause alleles that were once spatially restricted in their distribution to fix through the remnants of the species range. We currently do not know how much between‐species divergence is generated by global adaptation or by local adaptation.Drosophila calculation presented above, some incomplete global sweeps may induce ephemeral genetic differentiation and be detected when scanning the genome for local adaptation . The simulated genome consisted of 500 Mbp of simulated sequence, with 25 Mbp of functional sites. The number of functional sites was chosen to approximately reflect the total number of nonsynonymous sites and sites in the untranslated regions of protein‐coding genes in D. melanogaster. Advantageous mutations occurred at functional sites with fitness effects and rates similar to those that have been estimated for D. melanogaster , we found there was an average of 13 STF outliers for every 10,000 analysis windows in a low migration case (emN = 1) and three outliers in a high migration case (emN = 10). Note that the distribution of fitness effects for beneficial mutations is not well understood for natural populations and a given value of α can arise under various combinations of selection parameters (Supporting Information Appendix S1). For example, a high frequency of weakly beneficial mutations may yield the same adaptive substitution rate as a comparatively small number of strongly selected mutations. Indeed, Figure α, but that these exhibit quite different numbers of STF outliers , we found very little evidence that global adaptation impacted the landscape of STF, as under any of the parameter combinations we tested the number of outliers observed was not greater than expected under neutrality  and between (XYd) populations in genomic regions surrounding STF outliers using simulations of parapatric populations. To examine the effects of the lag‐type of STF outlier, we sampled simulated populations when a globally beneficial mutation was at 50% frequency across the metapopulation. To examine the effects of the Bierne type, we sampled simulated populations when a globally beneficial mutation had swept to fixation (frequency greater than 99% across the metapopulation). In addition, we simulated long‐term local adaptation due to alleles with spatially antagonistic fitness effects. Figure STF outliers that arose in four different ways.We examined the patterns of STF outlier of the “lag type,” within‐population nucleotide diversity (Wπ) near selected sites is reduced, but between population genetic diversity (XYd) is not systematically affected.Figure STF outlier, that is, after a globally beneficial mutation has swept to fixation , so recently established spatially antagonistic alleles may not exhibit that particular signal. Additionally, we have focused on local adaptation via spatially antagonistic alleles; if local adaptation were instead driven by conditionally beneficial alleles, these would likely only be detected using genome scans when they have recently swept to high frequencies where they are favored, but not yet spread into areas where they are neutral. Thus, conditionally beneficial alleles or recently established spatially antagonistic ones would resemble the “lag” type of STF peak observed under global adaptation, that is, with no increase in XYd. Note that Wπ is not a particularly useful diagnostic for local adaptation as either increased or decreased levels relative to background can occur depending on the relative strength of migration and selection , Martin et al. .Environments vary over space, so geographically distant populations may be subject to distinct biotic and abiotic environments. Studies may intentionally sample geographically distant populations to maximize the extent by which local adaptation shapes patterns of genetic differentiation. However, increasing the distance between sampled demes  would increase the chance that global adaptation induces The confounding effects of global adaptation we have discussed in this perspective arise because populations may be sampled along a linear array that is parallel to a possible path taken by a globally sweeping mutation. This would be analogous to sampling along a coastline or a river valley, where a single axis determines both the spatial patterning of gene flow and environmental change. Although we did not explicitly study genotype–environment association analyses , however, so alleles with more modest effects on fitness were perhaps missed by their approach. In populations with smaller sizes, global adaptation may have a limited influence on the landscape of genetic variability  for a two‐deme metapopulation.Figure S7 The pattern of STF, Wπ, and XYd around individual STF outliers observed when a globally beneficial mutation was at 100% frequency  for a two‐deme metapopulation.Figure S8 The pattern of STF, Wπ, and XYd around individual STF outliers observed when a spatially antagonistic allele has been segregating for 5eN generations in a two‐deme metapopulation .Figure S9 The pattern of STF, Wπ, and XYd around individual STF outliers observed under neutrality in a two‐deme metapopulation.Figure A1.1 The number of incomplete selective sweeps and the proportion of adaptive substitutions (α) under exponential DFEs for advantageous mutations.Click here for additional data file.


Compartmentalization and spatial organization of biochemical reactions are essential for the establishment of complex metabolic pathways inside synthetic cells. Phospholipid and fatty acid membranes are the most natural candidates for this purpose, but also polymers have shown great potential as enclosures of artificial cell mimics. Herein, we report on the formation of giant vesicles in a size range of 1 μm–100 μm using amphiphilic elastin‐like polypeptides. The peptide vesicles can accommodate cell‐free gene expression reactions, which is demonstrated by the transcription of a fluorescent RNA aptamer and the production of a fluorescent protein. Importantly, gene expression inside the vesicles leads to a strong growth of their size—up to an order of magnitude in volume in several cases—which is driven by changes in osmotic pressure, resulting in fusion events and uptake of membrane peptides from the environment. Encapsulation of RNA transcription or cell‐free protein expression reactions inside vesicles made from elastin‐like polypeptide membranes promotes strong growth of the vesicles, which is driven by the generated osmotic pressure accompanied by multiple fusion events. Compared to other membranous compartments, the vesicles display extraordinary stability, which allows growth over several orders of magnitude in volume. One of the most prominent goals of bottom‐up synthetic biology is the creation of synthetic cells.Compartmentalization is an essential prerequisite for the remaining properties, and has been achieved using a wide variety of different approaches.Several synthetic cell models already displayed at least some of the desired properties listed above. For instance, Szostak and co‐workers reported on fatty acid vesicles—serving as primordial cell models—, which were able to grow and divide upon external feeding with fatty acids.in vesiculo production of mixed membranes would be even more challenging than for homogeneous membranes.More closely mimicking lipid synthesis in natural cells, Scott et al. established parts of the complex phospholipid synthesis pathway inside of liposomes. To this end, all the required enzymes were encoded on DNA templates and produced inside of the liposomes via cell‐free gene expression.n (shorthand notation: Xn) is derived from tropoelastin, where X is any natural amino acid except proline and n is the number of pentapeptide repeats. Depending on the amino acid used for X the peptide displays different hydrophobicity.Other membrane‐forming molecules such as amphiphilic block co‐polymers or polypeptides represent an interesting alternative to phospholipids.1b, 14We have recently shown that ELPs can form ≈200 nm sized vesicular structures for the compartmentalization of biochemical reactions such as transcription of RNA aptamers and the expression of fluorescent proteins.Transcription of RNA aptamers inside the vesicles resulted in mixed growth behaviors, in which some vesicles started to shrink at one point, while others continued to grow. By contrast, when expressing the fluorescent protein YPet, 97 % of the vesicles continually increased in size; for the remaining two vesicles no clear shrinkage or growth could be observed. Our experiments hence demonstrate biochemically driven growth of peptide‐based synthetic cellular structures, which could set the stage for competition and selection dynamics emerging among such compartments.5Q5)2‐F20 (in shorthand notation) as the amphiphilic membrane component  to dilute residual IS. The OS contained an isotonic amount of glucose as well as a low percentage of Triton X‐100 (0.01 %). Due to the higher density of the IS, the vesicles sedimented at the bottom of a microscopy sample chamber and could thus be easily observed for several hours. If an osmotic shock is applied the vesicles vanish .The solvent evaporation method resulted in two populations of vesicles of different sizes, namely small vesicles (SV) with radii far below 1 μm and larger vesicles with sizes spanning two to three orders of magnitude, which we will collectively refer to as giant vesicles (GV). In order to characterize the size of the vesicles across these scales, we utilized transmission electron microscopy (TEM), dynamic light scattering (DLS) as well as light microscopy (LM). For the SVs, a mean radius of 0.03 μm ±0.01 μm was determined using TEM Figure , where tm) to the OS to facilitate vesicle growth through their incorporation into the membrane. We found that in the absence of external ELPs, the observed vesicles were generally smaller and less abundant. Furthermore, the vesicles were not stable during the experiments and tended to shrink.We next studied the capability of the GVs for fusion and growth. We speculated that—similarly as previously observed with fatty acid vesiclesThe initial vesicle population was highly polydisperse in size, which likely resulted in large variations in the contents of the compartments generated via fusion of the two types of vesicles. This in turn was expected to result in a broad distribution of transcriptional activities among the vesicles.The observed growth of the vesicles is consistent with our expectation that compartmentalized RNA polymerization is accompanied by an increasing osmotic pressure in the vesicles.In order to understand the dynamics of vesicle growth and shrinkage, we have to consider the interplay of compartmentalized RNA polymerization, the incorporation of externally provided ELPs into the membrane and water influx. As shown in Figure Our experimental observations further suggest that the availability of a supply of ELPs  in their immediate vicinity determines whether vesicles will grow or shrink. Some vesicles are found to repeatedly grow and shrink, and finally even disappear . Figures In another set of experiments, we encapsulated a bacterial cell extract‐based protein expression system (TX‐TL) mixed with sucrose solution and a plasmid encoding the yellow fluorescent protein YPet into the GVs (Supporting Information section 1.2). The OS contained glucose, ELPs, and kanamycin to inhibit translation outside of the GVs by potentially present non‐encapsulated TX‐TL components. The vesicle fluorescence Figures ; S11 andin vesiculo production of biopolymers and osmotically driven growth. In contrast to the TX mix, the much more complex cell extract contains nearly the complete proteome of BL21 rosetta E. coli cell, in which case the osmotic pressure of the vesicle will be influenced by a more complex network of biochemical reactions. Only very few cases of shrinking vesicles were observed, but most vesicles enter a plateau phase of constant volume and constant YPet fluorescence after completion of the TX‐TL reaction, which indicates that an osmotic equilibrium has been attained between the inner and the outer solution.As in the TX case the correlation between YPet fluorescence and volume is approximately linear Figure , which aIn conclusion we have demonstrated the generation of cell‐sized peptide vesicles, which upon encapsulation of cell‐free transcription and protein expression reactions exhibit volume changes over at least an order of magnitude in several cases. The size changes appear to be caused by a combination of fusion events and osmotically driven growth, when fed with membrane components from the outside. Vesicle growth promoted by internal bio‐polymerization reactions is thus much more pronounced than previously observed for other membrane systems, which may be related to the high permeability of the ELP membranes for water combined with their considerable mechanical stability.It is conceivable that usage of more complex membrane compositions will facilitate the implementation of other cell‐like behaviors such as compartmental division and reproduction. In fact, we already observed occasional budding events in our experiments , which may be taken as precursors for such processes. As the growth of our peptide vesicles is coupled to internal gene expression activity, our results also may lay the ground for a competition between different peptide compartments based on the efficiency of the compartmentalized reactions.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


This integrative review aimed to determine the features of effective physical activity programs for culturally and linguistically diverse (CALD) older adults.We searched for relevant articles in MEDLINE, PubMed, Google Scholar, Scopus and CINAHL. Articles were selected for evaluation if they included CALD older adults and implemented physical activity programs with culturally specific design features. Consistent with the Whittemore and Knafl integrative review methodology, we used the Downs and Black Checklist, Mixed Methods Appraisal Tool and the McMaster University appraisal tool for quantitative studies to assess the quality of included articles.Seven articles were included in this review. Effective community‐based exercise or physical activity programs for CALD populations commonly featured close‐to‐home delivery, native language instruction and adaptations of culturally familiar activities.The provision of culturally appropriate community‐based physical activity programs appears to support and encourage engagement among CALD older adults. Protocols and guidelines designed to encourage and increase CALD older adults’ participation in physical activities should ensure that culturally familiar activities are provided at a convenient and accessible location and in the person's own language.Essential aspects for ensuring successful participation by CALD older adults in physical activity programs include providing the program at a convenient and accessible location and in an older person's native language, and ensuring activities are culturally familiar.1There is strong evidence to suggest regular physical activity positively impacts older adults’ health and well‐being. Regular physical exercise in older adult populations has been shown to decrease the risk for falls, osteoporotic changes and muscular atrophy, improve sleep and blood pressure, and enhance mood and general well‐being.1.1What are the common characteristics of community‐based physical activity programs that were effective for CALD older adults?What culturally appropriate recommendations can be made for physical activity programs or interventions undertaken in CALD older adult populations?This integrative review aimed to explore community‐based physical activity programs undertaken among CALD older adult populations. It specifically sought answers to the following questions:2Research of all methodology types was included in this integrative review. We assumed the inclusion of research using diverse methodologies would contribute to a comprehensive understanding of features of effective community‐based physical activity programs for CALD populations. Further, this inclusive approach assumed that exercise and active participation programs targeting CALD older adults may use complex designs. To ensure a rigorous review process, we systematically followed the Whittemore and Knafl2.1Following the Whittemore and Knafl framework,2.2Published in a peer‐reviewed journal written in English or translated English version;Participants were identified in the studies as culturally and linguistically diverse or ‘CALD’;Participants were identified as ‘older adults’ or equivalent, based on a chronological age of ≥ 65 years ;The intervention had a physical activity or exercise program component undertaken in community‐based settings such as local community centres, organisations or groups.The exercise/activity program included a cultural adaptation or culture‐specific design; andCulturally specific program design outcomes were formally evaluated.Inclusion criteria for article selection were as follows:2.3Articles that reported participants as indigenous populations (or African American in the USA) were excluded as they are not CALD populations. This methodological decision was based on the definition of CALD and non‐CALD that previous researchers used. In a literature review, O’Driscoll and colleaguesPrograms undertaken in non–community‐based settings such as hospitals and aged care facilities were excluded from this review.2.4One author (ID) screened the abstracts of identified articles against the inclusion and exclusion criteria. Next, full‐text versions of relevant articles (n = 60) were read and screened against the inclusion and exclusion criteria. In total, 17 articles were identified for potential selection and independently reviewed by two authors (JM and SN) who were external to the literature retrieval process. These authors disagreed on at least one inclusion criterion for five of these articles, which were then screened independently by two other authors (VW and JS‐R). Consensus regarding article selection was reached through discussion. Seven articles that reported results for six studies were selected for this integrative review. Two articles from the same study2.5Consensus on the appropriate critical appraisal tools for this integrative review occurred at the beginning of the process. Randomised controlled trials2.6The Whitmore and KnaflA preliminary data synthesis was conducted to compare mutual features among the articles, and identify the most common program features. Two independent authors analysed the data, and consensus was reached regarding the cultural adaptation features that contributed to the effectiveness of the reported programs for CALD participants. Conclusions were then drawn subject to verification of data and rechecking against the original studies. Articles with low quality scores from the appraisal were treated with caution in terms of integration and contribution to the main results.33.1In total, we selected seven articles,3.2All of the reviewed articles that utilised culturally specific interventions were noted to have effective outcomes particularly with program adherence. Five articles reported further evaluation methods, such as preassessment of participants’ acceptance and understanding of the outcome measures,3.2.1The programs reported in the selected studies were strategically designed for delivery in locations with high immigrant populations. Therefore, they were deemed geographically close to home and convenient for CALD participants to attend. In addition, geographically focused recruitment strategies were reported in four articles.3.2.2Removing potential communication barriers was an integral feature designed to promote the success of the physical activity or exercise program for CALD participants. All articles described aspects of instruction or conversation in participants’ native languages; however, there were some variations. Bilingual staff or group facilitators3.2.3Most of the reviewed interventions embedded culturally familiar activities in the program design. For example, Tai Chi was used as the main intervention for participants who were solely ChineseAll included articles reported using multimodal strategies to recruit participants and retain engagement in the intervention. Such strategies were reported to help build rapport with participants and keep participants engaged throughout the duration and in all aspects of the study. For example, some studies recognised the importance of native language even at the point of evaluation4This integrative review identified the strategic use of close neighbourhood venues, native language instruction and culturally familiar activities as key features of effective physical activity programs for CALD older adults. These three program design features highlighted the importance of geographic, cultural accessibility, ease of communication and sense of belonging through performing familiar physical leisure activities for health benefits to participants. Across all reported studies, cultural adaptations in the interventions for specific CALD populations were evident to varying degrees, including the choice of exercise themes and activities, outcome measures, recruitment methods, program staff and program review methods. In particular, the use of bilingual staff and facilitators who were culturally and ethnically similar to program participants was reported as an effective method. Older immigrants’ feelings of belonging among co‐ethnic peoples have been reported in other studies of participation.The included articles reported bespoke intervention programsProgram adherence was the common evaluation method used in the included articles to report program effectiveness for CALD participants. However, adherence is only a proxy measure. Good practice for the evaluation of the effectiveness of cultural adaptations of a program was evident in articles that reported using individual/group interviews to obtain participants’ views on cultural appropriateness and acceptability. The best example was the involvement of participants in a semi‐structured focus group following the intervention program,The effective program features synthesised from this integrative review were loosely aligned with the Pen‐3 framework, which situates cultural needs as a core value to explain cultural influences on health outcomes.There is limited research involving CALD older adult populations that aims to understand factors that promote their uptake of physical activity or exercise programs at a level that reaches physical activity guidelines for older adults. Importantly, previous research has explored participants’ perceptions of what influenced group exercise participation among CALD populations. For example, Belza et al4.1As with any review that utilised secondary data, there were limitations in terms of the relevance and generalisability of the findings. This review focused on interventions implemented among CALD populations only and did not explore features of interventions and outcomes with non‐CALD groups. Future research should consider a comparative exploration of program strategies with non‐CALD populations. It is possible that some research related to community‐based physical activity programs for CALD older adults may have not been indexed correctly in electronic databases, and therefore, some relevant studies may have been overlooked in this review.5This integrative review sought to identify factors that made community‐based physical activity and exercise programs effective for CALD older adults in their host societies. The synthesised results showed programs that used ‘close‐to‐home’ venues, native language instruction and culturally familiar activities were deemed effective, as evaluated by adherence rates and participants’ subjective experiences. There is a need for studies on physical activity and exercise among CALD older adult populations to evaluate the effectiveness of culturally tailored program features from participants’ perspectives and experiences, rather than relying on proxy measures of effectiveness such as program adherence. The concept of co‐design strategies and interventions with older people is also an emerging methodology that would be useful for optimising CALD groups’ engagement in physical activity and exercises. It is beyond the scope of this integrative review to recommend best practice guidelines. However, the common features of effective programs identified in this review could inform the adaptation of existing community programs to make them culturally appropriate for specific ethnic groups.


Subsurface chlorophyll maximum (SCM) layers are prevalent throughout the Arctic Ocean under stratified conditions and are observed both in the wake of retreating sea ice and in thermally stratified waters. The importance of these layers on the overall productivity of Arctic pelagic ecosystems has been a source of debate. In this study, we consider the three principal factors that govern productivity within SCMs: the shape of the chlorophyll profile, the photophysiological characteristics of phytoplankton and the availability of light in the layer. Using the information on the biological and optical parameters describing the vertical structure of chlorophyll, phytoplankton absorption and photosynthesis–irradiance response curves, a spectrally resolved model of primary production is used to identify the set of conditions under which SCMs are important contributors to water-column productivity. Sensitivity analysis revealed systematic errors in the estimation of primary production when the vertical distribution of chlorophyll was not taken into account, with estimates of water-column production using a non-uniform profile being up to 97% higher than those computed using a uniform one. The relative errors were shown to be functions of the parameters describing the shape of the biomass profile and the light available at the SCM to support photosynthesis. Given that SCM productivity is believed to be largely supported by new nutrients, it is likely that the relative contribution of SCMs to new production would be significantly higher than that to gross primary production. We discuss the biogeochemical and ecological implications of these findings and the potential role of new ocean sensors and autonomous underwater vehicles in furthering the study of SCMs in such highly heterogeneous and remote marine ecosystems.This article is part of the theme issue ‘The changing Arctic Ocean: consequences for biological communities, biogeochemical processes and ecosystem functioning'. Here and elsewhere in the paper ‘chlorophyll’ refers to chlorophyll-a (and the sum of chlorophyll-a and its derivatives when such information is available from HPLC measurements). The overall importance of these chlorophyll layers to annual water-column production in Arctic waters has been debated in the literature  and Beaufort [0.5 mg C (mg chl)−1 h−1 for all sampling depths] Seas [αB had a mean value of 0.034 mg C (mg chl)−1 h−1−1 h−1αB value was higher than those reported for the Chukchi [0.017 mg C (mg chl)−1 h−1 for surface assemblages and 0.025 mg C (mg chl)−1 h−1 for SCM] and Beaufort [0.017 mg C (mg chl)−1 h−1] Seas [kE averaged 68 The PE parameters also showed significant variability in this sector of the Arctic Ocean . The avehs] Seas ,36 but t−1] Seas ,36. The ort Seas , which min vivo absorption spectrum of phytoplankton was remarkably similar across the three datasets  (αB(λ)] in the primary production model.The average shape of the or Seas) . The Grein situ bio-optical data and a vertically and spectrally resolved production model to identify the key parameters that are critical for accurate assessments of SCM productivity. For our initial model set-up, average PE parameters were assigned based on measurements made on natural Atlantic Arctic assemblages: αB were assigned values of 1.74 [mg C (mg chl)−1 h−1] and 0.034 [mg C (mg chl)−1 h−1 (µmol quanta m−2 s−1)−1], respectively. We first examined the difference between computed rates of daily primary production (DPP) when we held the surface concentration of chlorophyll constant with depth (uniform profile) and when we allowed chlorophyll to vary with depth using profile parameters obtained from fitting the nonlinear Gaussian function to Arctic field data (non-uniform profile)  is situated in the upper part of the photic zone (i.e. low m/zpz), the resulting biomass-normalized production rate (PB) as defined by the curvilinear PE response curve (equation (2.5)) approaches the plateau PB will be governed by the quasi-linear region of the curve leading to estimates of PB that are much lower than m/zpz, and again errors are highest in the upper portion of the water column where light is saturating and thus PB. The inverse is true for errors in the initial slope, αB: when the initial slope is overestimated (αB + 1 s.d.) or underestimated (αB − 1 s.d.) we find the largest errors occur when most of the biomass is contained within the light-limited region of the euphotic zone (high m/zpz).To examine how uncertainty in our assignment of the PE parameters may impact estimates of primary production for regions characterized by a diverse range of profile shapes we adjusted the PE parameters by ±1 s.d., which accounts for a large fraction of the seasonal and geographical variability in our Atlantic Arctic dataset, and also encompassed the average values obtained from other geographical sectors of the Arctic Ocean . Errors zm to zp , which i−1 h−1 is often used [−1 h−1 obtained in this study. Part of the difficulty in trying to capture the seasonal variability in the photophysiological properties of Arctic assemblages in primary production models is that there is no consensus on how to assign the PE parameters in an operational manner. Temperature [14C productivity experiments conducted at sea, there is also the potential of acquiring information on the photosynthetic characteristics of marine phytoplankton in a non-intrusive manner and over wider spatio-temporal scales using single turnover active chlorophyll fluorometry [Clearly our choice of photosynthetic parameters directly impacts our estimates of marine primary production. Yet in several remote sensing studies of the Arctic Ocean a single ten used  and is cperature ,38, nutrperature  and diatperature  have allorometry , althougorometry ,40,41.mz/σ), as our shape index. Note that in order to achieve the best nonlinear fit, mz may be assigned a value above the sea surface and as a result some mz/σ values were slightly negative. As an index of the relative contribution of SCMs to integrated primary production, we computed the per cent difference between model estimates when we include the profile parameters and when the chlorophyll was assumed to be uniformly distributed with depth. When we plot the relative change in primary production against mz/σ  result from the peak being situated deeper in the water-column (higher mz) (figure 9b), but as the peak width is narrower, a large fraction of the chlorophyll biomass is still contained within the euphotic zone. The presence of deeper and more prominent peaks leads to the SCM accounting for a significant fraction of the integrated primary production as indicated by the large difference between the estimates using uniform and non-uniform profiles and relative errors reaching a maximum of 98%.mz/σ values the strong relationship between our index of SCM contribution to integrated primary production and profile shape breaks down. There are two scenarios that lead to this result. First is the situation where a thin chlorophyll layer is present (c), which leads to anomalously high mz/σ values. Even though these profiles exhibited some of the highest concentrations in subsurface chlorophyll and highest rates of primary production per unit volume, when integrated over the water column, their impact on integrated rates of primary production was not as high as subsurface peaks that were broader but less intense. The other situation was profiles which had a very small bump superimposed on a uniform background concentration (c). Although both of these cases are relatively rare in our dataset, it should be noted that the presence of thin subsurface chlorophyll layers can be a common occurrence in temperate shelf seas [Another interesting feature of  present c, which ntration c. Althouelf seas  and the −1, which can be considered an extreme case when surface waters are heavily influenced by terrigenous sources, estimates of integrated primary production decreased by as much as 83% and this was a strong function of the depth of the chlorophyll peak  reads. As one of the smallest photosynthetic eukaryotic organisms in the Arctic Ocean, it has been suggested that Micromonas may be used as a sentinel of a changing Arctic Ocean [SCMs have also been shown to be important ecological niches that host diverse phytoplankton assemblages. Within these layers, the growth of larger cells (e.g. diatoms) can be sustained by the subsurface nutrient reservoir while at the same time these algae are able to harvest sufficient light despite being prone to high pigment packaging ,22. Evenic Ocean ,54. Thus4.Here, we show that the importance of the relative contribution of SCMs to water-column productivity is strongly controlled by the shape of the chlorophyll profile and that a failure to account for the presence of SCMs can lead to a significant underestimate in water-column primary production, with relative errors approaching 100%. This highlights the importance of capturing detailed information on the vertical structure of SCMs and demonstrates that spatio-temporal averaging of chlorophyll profiles could lead to an underestimation of the contribution of SCMs to Arctic primary production. A variety of autonomous platforms that are able to record the vertical distribution of chlorophyll fluorescence at a high depth resolution will provide an unprecedented view of the geographical extent of SCMs and their structural diversity and provide invaluable insight into the environmental factors that control their properties and distribution.Previous modelling studies have shown that errors introduced by ignoring the presence of SCMs when computing annual rates of primary production over ocean basins or provinces are modest (<10%), and that the impact of the vertical structure of chlorophyll on estimates of integrated rates can be considered of secondary importance ,5 when c


Confronting the challenge of the outbreak of COVID‐19 should sharpen our focus on global drug access as a key issue in antiviral therapy testing. The testing and adoption of effective therapies for novel coronaviruses are hampered by the challenge of conducting controlled studies during a state of emergency. The access to direct antiviral drugs, such as ribavirin, that have an existing inventory and reliable supply chain may be a priority consideration for therapies developed for the 2019‐nCoV infection outbreaks and any strain variants that may emerge. On the basis of the direct antiviral activity of ribavirin against 2019‐nCoV in vitro and evidence for potency enhancement strategies developed during the prior SARS and MERS outbreaks, ribavirin may significantly impact our ability to end the lingering outbreaks in China and slow outbreaks in other countries. The apparent COVID‐19 pandemic provides an opportunity to follow dosage guidelines for treatment with ribavirin, test new therapeutic concepts, and conduct controlled testing to apply the scientific rigor required to address the controversy around this mainstay of antiviral therapy. The multimodal anti‐viral mechanisms of action of ribavirin are described.Treatment of novel coronaviruses with ribavirin during SARS and MERS outbreaks are detailed.Challenges in evaluation of drug efficacy from viral outbreaks are described.Registered clinical trials to evaluate ribavirin for COVID‐19 are listed. However, novel coronaviruses (nCoV) that crossover from zoonotic hosts possess unknown sensitivities to treatments and are a principal source of pandemic risk. The clinical effectiveness of treatments from the frontlines of an outbreak can be most informative; however, the options in such an environment are limited by the shallow global pharmacopoeia of general antiviral medicines.In this review, we critically evaluate the studies that underlie the inconclusive benefit of ribavirin for the treatment of prior nCoV outbreak strains and characterize the primary sources of the controversy. On the one hand, there persist issues of global access and medication affordability, its efficacy in general clinical practice, multimodal mechanisms of direct antiviral activity, and indirect activity of the immune system. On the other hand, there are challenges of conducting controlled clinical studies in an outbreak environment, the limitations of retrospective studies, and the absence of nCoV cases showing acute resolution of infection after treatment as well as in vitro testing data of activity against 2019‐nCoV.2The pathology of COVID‐19 resembles that of the 2013 MERS‐CoV and 2003 SARS‐CoV infections such that the extrapolation of treatment guidance from those prior clinical experiences can provide guidance for the current outbreak of 2019‐nCoV.As strain isolates of the 2019‐nCoV are distributed for laboratory testing in cell‐based and animal model systems, recommendations for treatment may be ascribed. The first 2019‐nCoV viral strain submitted for laboratory testing was 2019BetaCoV/Wuhan/WIV04/20192 (WIV04), which was isolated from the lung fluid of one patient in a cohort of seven, six of whom worked in the proximity of the Wuhan seafood market.Indeed, as a single agent and due to its cost and availability in China, a chloroquine phosphate multicenter trial was possible, and this drug showed signals of apparent efficacy against 2019‐nCoV.With the rapid transmission of 2019‐nCoV and our limited understanding of viral evolution during this process, the guidance on drug usage and testing must remain extensive. Forthcoming laboratory‐based test results on new strains and those obtained by using different models may change the order of sensitivity to available treatments for emergent strain variants. The spread of 2019‐nCoV globally is a factor that can also influence individual‐ and population‐level treatment outcomes with different therapies.3Ribavirin is a guanosine analog that interferes with the replication of RNA and DNA viruses. However, the antiviral activity of ribavirin is not limited to interference with polymerases, that is, the structure of ribavirin also interferes with RNA capping that relies on natural guanosine to prevent RNA degradation. Moreover, to further promote the destabilization of viral RNA, ribavirin inhibits natural guanosine generation by directly inhibiting inosine monophosphate dehydrogenase in a pathway that is vital for the production of the guanine precursor to guanosine.Even when treatment incompletely blocks the virus from replicating, viral nucleic acid replication in the presence of ribavirin occurs with reduced fidelity, leading to the introduction of random mutations that can reduce the viability of the virus.The indirect antiviral properties of ribavirin as mediated by the immune system were first observed in the treatment of patients with hepatitis whose symptoms improved without a reduction in the viral load.As a very mature drug, with significant pharmacological research behind it, the pharmacokinetics and bioavailability data for ribavirin are available to inform dosing both as a single agent and as a part of combination therapies.The mean bioavailability of a 400‐mg dose of ribavirin is 51.8% ± 21.8% after an IV loading dose of 150 mg. Using a three‐compartment model for PK analysis, the mean gamma phase half‐life is 37.0 ± 14.2 hours. Ribavirin is rapidly absorbed and has a T‐max after the oral administration of 1 hour after the first dose, 1.7 hours after the second dose, and 3 hours after the multiple‐dose. The route of ribavirin elimination is renal.Box 1For people living in highly populated areas in regions affected by the 2019‐nCoV pandemic, there is the creation of a high‐risk environment due to the high density of multiple strains of viruses, including 2019‐nCoV. Environments that are outside the traditional medical setting require a new approach to treatment and prevention, and they represent a new aspect of nCoV pandemic control: treatment within large‐scale and high‐density quarantines of infected and noninfected individuals. To prevent either the emergence of new 2019‐nCoV strains or the spread of other viruses, the treatment of mild cases in these areas with antiviral therapy is a high priority for local and global health professionals.1)Broad activity toward conventional and novel viruses of DNA and RNA types.2)Multiple mechanisms of direct antiviral action.3)Random mutagenesis of viruses to promote T cell response.4)Indirect mechanism of action via Th1 polarization.5)Tolerable and well‐characterized side effect profile.6)Mature clinical experience and comprehensive demographic characterization.7)Accessibility.8)Affordability.We propose the usage of ribavirin in this environment for the following reasons:The cost for manufacturing ribavirin is US $0.20 to $2.10 per gram.4Ribavirin has a well‐established history of usage in emergency clinical management plans for nCoV, in which the greatest benefit has been reported with early administration upon presentation with pneumonia and before sepsis or organ system failure.The global clinical experience with ribavirin delivery for the treatment of nCoV started with SARS‐CoV, for which ribavirin was initially indicated on the basis of the pathological similarity of SARS‐CoV to the acute respiratory syndrome, which requires a typical administration of ribavirin and corticosteroid.Subsequently, in the Canadian SARS‐CoV outbreak, ribavirin was administered early with corticosteroids, and no conclusive results of efficacy could be established, despite viral and symptom flare‐up in a portion of patients after treatment cessation.Although the reports of the lower dose ribavirin treatment schedule used in Hong Kong in 2003 did not include descriptions of adverse events, the Canadian experience with higher dosing that year provided a greater insight into the adverse effects of both ribavirin and corticosteroids. Ribavirin usage was associated with hemolysis in 76% of patients, defined as a 1.5‐fold increase in bilirubin or a decrease in haptoglobin. In 49% of patients, a 2 g/dL decrease of hemoglobin was observed. In addition, some indication of liver toxicity was indicated on the basis of elevated transaminases, defined as a 1.5‐fold increase in aspartate aminotransferase or alanine aminotransferase in 40% of patients. Acute toxicity led to the discontinuation of ribavirin in 18% of patients.To further complicate the evaluation of high‐dose ribavirin monotherapy is the possibility that corticosteroids may have delayed viral clearance, prolonging infections while reducing the symptomatic inflammatory cytokines.Upon recognition of the potential efficacy of lopinavir/ritonavir against SARS‐nCoV in vitro in 2003, the protease inhibitor lopinavir/ritonavir was combined with ribavirin. A study of 41 SARS‐CoV patients showed a favorable clinical response with lopinavir/ritonavir and ribavirin when compared with historical outcomes with ribavirin and corticosteroids.Challenges in the evaluation of ribavirin activity in patients during the previous SARS‐nCoV and MERS‐nCoV outbreaks continue to leave family doctors who reside in areas of outbreak without clear answers regarding the benefit of ribavirin. Although the drug has significant activity against coronaviruses in laboratory testing, the dose required to achieve that activity in patients may not have been known in prior practices without limiting toxicities.The coronavirus encodes RNA replication proofreading machinery that can partially resist one mechanism of action of nucleoside analogs, placing additional importance on our ability to determine therapeutic doses of ribavirin.5In the pursuit of better treatment of MERS‐CoV, multiple assay cell lines were used to test for antiviral activity against the strain hCoV‐EMC/2012, yielding insights into ribavirin.2, group 2: 0.333‐0.833 mL/sec/m2, and group 3: <0.333 mL/sec/m2 or on dialysis. After receiving an initial 2000‐mg loading dose, the 10‐day ribavirin schedules for each group were as follows: group 1: 1200 mg every 8 hours for 4 days and then 600 mg every 8 hours for 4 to 6 days; group 2: 600 mg every 8 hours for 4 days and then 200 mg every 6 hours for 4 to 6 days; group 3: 200 mg every 6 hours for 4 days and then 200 mg every 12 hours for 4 to 6 days.During the outbreak of MERS‐CoV, ribavirin was paired with either IFN‐α2b or IFN‐α2a to engage two independent mechanisms of antiviral activity. This combination was synergistic in laboratory tests, reducing the therapeutic threshold for ribavirin to block viral replication.This treatment combination for MERS‐CoV was deployed for the limited number of cases in Korea.6The government initially recommended the use of ribavirin in 2019‐nCoV pneumonia diagnosis in cases of China, based on Treatment Plan Edition 5, such that upon the diagnosis of pneumonia, a 4‐g oral loading dose should be delivered, followed by a 1.2‐g oral dose every 8 hours.Although significant effort and resources are contributed to the research and development of nCoV treatments, in times of outbreak, care and preparation are required to apply a scientific approach to quantify the therapeutic benefit of medicines that are already available during such viral outbreaks. On the basis of these prior clinical experiences and others, controlled studies are underway to evaluate the available courses of therapy for COVID‐19. Among the possible studies that should be completed are drug combinations that use the widely available ribavirin.The public good requires that patients can access life‐saving treatments for infectious diseases in an affordable and timely fashion. Access to medical products and establishing their therapeutic benefit are both essential to meet this obligation. Effective clinical treatments prepared from the existing pharmacopoeia can save many lives and achieve the greatest benefit for the public while facing the challenge of 2019‐nCoV and future nCoV strains.7The wide availability and low cost of ribavirin support its potential to significantly impact the treatment of nCoV infections. The challenges in the evaluation of ribavirin efficacy from 2003 during SARS and the 2013 MERS outbreaks led to a summary evaluation of its utility as controversial in the treatment of COVID‐19 patients. A large number of clinical studies and retrospective analyses that will come from the 2019‐nCoV outbreak will put the controversy of ribavirin efficacy in a broader context. For ribavirin and myriad other treatments, both the clinical results and quality of evidence will reveal the challenges that face frontline physicians who treat patients in a medical setting and evaluate prophylaxis for novel high‐risk environments formed by large quarantined populations. The critical need for treatment and patient care in outbreak settings, on the frontlines of nCoV outbreaks, will place stress on any medical system and clinical research mechanism. However, controlled clinical studies are underway to permit a prospective evaluation of efficacy, and the government Treatment Plan Edition 5 and revised and prescribed usage guidelines distributed in China will assist in the comparability of multicenter experiences in retrospective analyses. The efforts of clinical research professionals will help both in this outbreak and future outbreaks of nCoV, which will possess unknown sensitivities to our antiviral pharmacopoeia.The authors disclose that their affiliated organizations will support clinical trials with ribavirin for the treatment of novel coronavirus infections.