Chondrosarcomas are rare malignant cartilaginous tumor affecting adult and elderly patient. Pelvic and long bones are the most common location. We differentiate conventional chondrosarcoma which arises do novo from preexisting normal bone (primary chondrosarcoma) or within a preexisting lesion such as enchondromas or osteochondromas (secondary chondrosarcoma), Other rare subtypes of chondrosarcoma include clear cell chondrosarcoma, dedifferentiated chondrosarcoma, and mesenchymal chondrosarcoma, which will be considered separately. Although there are diverse clinical presentations depending on the anatomic extend, radiographic features of chondrosarcoma are very characteristic comprising frequently a combination of bone expansion and heterogeneous calcifications. We report a case of a 56-year-old male suffering from fixed mass adhering to the right pubic bone. MRI views showed a lytic lesion of right superior pubic rami, surgical biopsy was in favor of chondrosarcomas, then an en bloc resection was performed following a Pfannenstiel approach without any recurrence after three years of follow-up. •Chondrosarcomas are rare bone tumor of adult above the fourth decades.•They affect principally pelvis, proximal femur and proximal humerus.•Symptoms are essentially pain, rarely palpable mass; However pelvic location can cause gynecological, bowel or bladder problems.•Treatment involves radical resection because of chondrosarcomas resistance to chemotherapy and radiotherapy. Surgical resection has been considered the only curative treatment because of non-response to the chemotherapy and radiotherapy; however, pubic location is very difficult to remove because of depth and complicated anatomy of this region. Survival in chondrosarcoma has remained unchanged over the last years .The aim of this article is to describe the particularity of chondrosarcoma in the Enneking type III location, its symptomatology and surgical management through Pfannenstiel approach.This work was reported in line with the SCARE criteria .2The case involved a 56-year-old male without any personal or familial diseases, was worried by swelling around his right pubic region progressing during one-year, clinical exam noted a painful fixed mass adhering to the pubic bone measuring about 3 cm long and 2 cm wide without any inflammatory signs. Pelvic x-ray  and CT-sMRI images showed the lytic lesion of right pecten pubis with a low intensity in T1, and heterogeneous characteristics in T2-weighted sequences, exerting a mass effect on the bladder which was by elsewhere homogeneous and with thin wall. On the other hand, adjacent acetabular bone and hip joint were not affected .Fig. 2MRSurgical biopsy was performed through direct anterior approach under fluoroscopic control, macroscopic view of the tumor revealed a white cartilaginous constituent and histopathologic study was in favor of chondrosarcoma grade I.On the way to analyze tumor extension, we realized thoracoabdominal CT-scan and a whole-body scintigraphy without any pathological findings. Therefore, an en bloc resection of pubic rami and ischiatic branch was completed through a Pfannenstiel approach with careful soft tissue dissection, hemostasis and pulling laterally of femoral neurovascular bundle. Bone resection was started at the level of the contralateral pubic symphysis, followed by right pecten pubis osteotomy in a propre periacetabular zone cheeked with fluoroscopy, then homolateral ischial tuberosity that was the last osteotomy allowing total tumor removal. Finally surgical mesh was implanted to avoid herniation . Post-opTotal weight bearing was started after three months, and patient was regularly checked clinically and by imaging based on multiple X-rays at 3, 6 months and every year associated with annual pelvic CT-scan. All these exams were normal without detection of any recurrence after three-year follow-up.3Chondrosarcomas are the third most common primary bone tumor after myeloma and osteosarcoma. They occur particularly after 50 years old, with an identical gender distribution, their estimated incidence in the United States population is 1 in 200,000 per year [Chondrosarcomas originate frequently from pelvic girdles and metaphyseal surface of long bones, particularly proximal femur and humerus; however, they can arise at any skeletal site. Conventional chondrosarcomas comprise the largest group, representing 90% of all chondrosarcomas, they can arises do novo from preexisting normal bone (primary chondrosarcoma) or within a preexisting lesion (secondary chondrosarcoma). These secondary tumors are very rare, they have many predisposing factors and diseases like Maffuci syndrome, Ollier's disease in which malignant transformation occurs in 5% of osteochondromas either in multiple or solitary forms ,4. OtherConventional chondrosarcomas are also subclassified by their location in central (intraosseous), periosteal, or peripheral chondrosarcomas .Histologically, chondrosarcomas are made of pure hyaline cartilage differentiation, but they may have myxoid and calcification component. Microscopic appearance varies depending on the histologic grade of the neoplasm which is an important predictor factor of local recurrence and metastasis; therefore, chondrosarcomas are divided into three grades based upon their histopathology findings; grade I define a low-grade with moderate hyperchromatic nuclei, uniform similar to enchondroma, followed by grade II characterized by greater nuclear atypia, then grade III in which tumors are more pleomorphic and atypical than grade II. The fourth group of chondrosarcomas, considered as grade IV; called dedifferentiated chondrosarcoma is a pleomorphic tumor without significant cartilaginous matrix and makes up 10% of all chondrosarcomas .Low grade chondrosarcomas are often surgically cured because are poorly vascularized and there is no tumor cells dissemination. Therefore, in late grades the presence of blood vessels represents a potential risk for pulmonary micrometastases but also for the poor response to chemotherapy .Chondrosarcomas are often asymptomatic but becomes symptomatic when are of large size or in case of anterior pelvis location, they are often manifested by atypical signs, vascular compression, urethral or bladder compression and gynecological disturbances or sciatica in cases of ischium location . Plain xSurgical biopsy and histopathologic analyze confirm the diagnosis of chondrosarcoma, they have usually a typical macroscopic appearance composed of white hyaline cartilage with gritty white calcification. After malignancy finding, a complementary of biologic and radiologic assessment must be completed to evaluate metastatic extension such as bone scintigraphy, chest and abdominal CT-scan.The en bloc resection of pelvic tumors has been classified into 3 types according to Enneking and Dunham; type I represents resection of the ilium; type II involves resection of the acetabulum and type III involves resection of the ilio and ischiopubic branch .A number of surgical approaches to the anterior pelvis allowing Enneking type III resection exist, the most used is ilioinguinal approach, as published in a similar case by RĂZVAN ENE and col . In addiChemotherapy is normally not efficient in conventional chondrosarcoma, but may have a role in dedifferentiated grade, on the other hand, radiotherapy is used for tumors where surgical resection is impossible or limited. Also, there is a new therapeutic method with microwave-induced hyperthermia (MWA) that is still in evaluation and improvement, this procedure helps to reduce tumor proper margins and economize skeletal resection .4Chondrosarcoma of the pubic ramus have many challenging facts, such as complex dissection procedure, because of its specific location neighboring major neurovascular structures and pelvic organs, also limited reconstruction options after ischiopubic bone resection that must be strengthened by surgical mesh. On the other hand, tumors are often asymptomatic and diagnosis is always late making complete surgical resection very hazardous.The study is exempt from ethnical approval in our institution.This is a case report and the patient give us informed consent for publication.All authors disclose that this manuscript didn't received no specific grant from any funding agency.Abderrahim Zaizi and Abdelhay Rabah make substantial contributions to acquisition of data, conception and design, and analysis and interpretation of data.AS. Bouabid and Mostapha Boussouga participate in revising it critically for important intellectual content and give final approval of the version to be submitted.This case report doesn't need to be registered because is not first-in-man.This case report doesn’t need to be registered because is not first-in-man.Abderrahim Zaizi and Abdelhay Rabah are the guarantor of this publication.Patient give us informed consent for publication.Written informed consent was obtained from the patient for publication of this case report and accompanying images.Not commissioned, externally peer reviewed.All authors report no conflicts of interest.


Numerous cellular processes, including toxic protein aggregation and oxidative stress, have been studied extensively as potential mechanisms underlying neurodegeneration. However, limited therapeutic efficacy targeting these processes has prompted other mechanisms to be explored. Previous research has emphasized a link between cellular senescence and neurodegeneration, where senescence induced by excess DNA damage and deficient DNA repair results in structural and functional changes that ultimately contribute to brain dysfunction and increased vulnerability for neurodegeneration. Specific DNA repair proteins, such as breast cancer type 1, have been associated with both stress-induced senescence and neurodegenerative diseases, however, specific mechanisms remain unclear. Therefore, this review explores DNA damage-induced senescence in the brain as a driver of neurodegeneration, with particular focus on breast cancer type 1, and its potential contribution to sex-specific differences associated with neurodegenerative disease. DNA damage and deficient DNA repair are well established features of neurodegenerative diseases. This review highlights how the dysregulation of a DNA repair protein encoded by breast cancer type 1 promotes the onset of cellular senescence and contributes to brain dysfunction as it drives neurodegeneration.  Throughout life, the brain undergoes many changes that can impact cognition and overall brain health. With normal ageing, the brain experiences brain atrophy and white matter degeneration due to a myriad of factors that can negatively affect cognition, such as neuroinflammation, oxidative stress, hypoperfusion and myelin loss.,,,,,When investigating potential causative mechanisms underlying neurodegeneration, abnormal protein aggregation has been a predominant area of study. Several neurodegenerative diseases are characterized primarily by toxic protein aggregation that contributes to cognitive decline.,Researchers have documented evidence suggesting that the accumulation of DNA double-stranded breaks and deficient DNA repair may precede pathology present in several neurodegenerative diseases.,,,,,Defective DNA repair and DNA damage accumulation are well established features of neurodegeneration. As part of the DNA damage response, cells have several repair pathways available to repair different lesions. For single-stranded breaks, lesions caused by base alterations without a helical distortion are repaired via base excision repair, while those with a helical distortion are repaired via nucleotide excision repair.,,It has been reported that DNA damage affects neurons in a region-specific manner, suggesting differential neuronal vulnerability throughout the brain with age.,,,,Under certain circumstances, a cell can be driven to undergo cellular senescence or apoptosis.,in vitro have been shown to adopt a senescent-like phenotype in response to DNA damage, and are characterized by increased levels of pro-inflammatory cytokines, senescence-associated beta-galactosidase, oxidative damage, and double-stranded break accumulation.Owing to senescence being a state of permanent cell cycle arrest, it is more often associated with mitotic cells. As a result, senescence in post-mitotic cells, such as neurons, has been studied to a much lesser extent. However, studies have speculated that neurons indeed undergo senescence and exhibit several markers and phenotypes characteristic of senescence.,,Other cell types in the brain have also been documented to undergo senescence or adopt a senescent-like phenotype with age an9d neurodegeneration,,In addition to the onset of senescence in the brain, cellular senescence has been implicated in several neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, Down Syndrome and multiple sclerosis,,,With increasing evidence supporting neurodegeneration driven by DNA repair deficiency potentially through DNA damage-induced senescence in the brain, several DNA repair proteins have been studied. SIRT6 (sirtuin 6), a histone deacetylase, is involved in several DNA repair pathways, including double-stranded break repair, base excision repair and nucleotide excision repair.Knowing the toxicity of double-stranded breaks and how compromised genomic integrity can induce DNA-damage induced senescence in the brain, BRCA1 is of particular interest. A study by Suberbielle et al.BRCA1 has been widely recognized for conferring increased susceptibility for breast and ovarian cancer in individuals carrying a mutation in the gene. A prospective cohort study reported the cumulative risk of breast cancer by age 80 to be 72% for BRCA1 mutation carriers, whereas that of ovarian cancer was reported to be 44%.BRCA1 mutations not only have a substantial increased risk for breast and ovarian cancer, but an increased risk for other cancers, such as pancreatic and cervical cancer as well. Underlying the reputation of BRCA1 in breast and ovarian cancer, BRCA1 primarily functions in the nuclear compartment as a tumour suppressor, and participates in various cellular processes, including cell-cycle checkpoints, DNA repair, antioxidant responses and gene silencing  receptors that triggered proteasomal degradation of BRCA1 levels.,BRCA1 expression did not appear dysregulated in motoneurons, but an upregulation of BRCA1 was identified in microglia.As previously mentioned, mice deficient of BRCA1 in the brain exhibited increased neuronal double-stranded breaks and identifiable cognitive deficits, both of which were exacerbated in an Alzheimer’s disease mouse model.,,Although neurons are post-mitotic, cell cycle re-entry can occur following defective DNA repair. In fact, re-initiation and dysregulation of the cell cycle is a recognized feature of neurodegenerative disorders,In a study investigating how the clinical presentation of Alzheimer’s disease is influenced by DNA damage and cell cycle dysregulation, researchers found that more severe manifestations of Alzheimer’s disease were associated with reduced DNA repair, in addition to increased cell-cycle progression and cell death in the hippocampus.It is unclear how BRCA1 dysregulation contributes more broadly to neurodegeneration as it seems differentially dysregulated across multiple neurodegenerative diseases. Although BRCA1 expression can stimulate apoptosis by initiating cell cycle re-entry in neurons associated with Alzheimer’s disease,,,,Further adding to the complexity of the pathophysiology associated with neurodegeneration, sex differences have been reported in the incidence, progression and outcomes of neurodegenerative diseases.,,Although specific mechanisms underlying these sex differences are currently unclear, several mechanisms have been proposed, such as differences in microglial function and oxidative stressors between males and females. Differences in immune presence that impact levels of neuroinflammation have been reported, where elevated levels of oestrogen appear to confer an enhanced immune response in females, exhibiting neuroprotective effects by reducing activated glial populations and attenuating neuroinflammation.,BRCA1 mutation carriers. In a recent study, oestrogen metabolites were shown to mediate senescence of hypothalamic astrocytes in culture, exhibiting an increase in DNA damage and senescence-associated markers.Although it is unclear how causative these mechanisms may be, hormonal differences between males and females are consistently presented as contributors of sex-specific differences, with oestrogen harbouring neuroprotective effects.Oestrogen signalling is also involved in learning and memory, where it has been reported to elicit opposing effects in cognition that differ based on which brain regions are affected. For instance, higher levels of oestrogen seemed beneficial towards hippocampal-associated tasks, but disadvantageous towards striatum-associated tasks.Together, these studies demonstrate both neurotoxic and neuroprotective effects of oestrogen in the brain, with loss of BRCA1 contributing to oestrogen neurotoxicity. Although neuroprotective effects of oestrogen have been studied in depth, the continuous sex differences associated with neurodegeneration suggests other pathways may be at work to drive worse outcomes in females. With the capacity to induce cellular senescence and drive tissue dysfunction, DNA damage accumulation and the role of BRCA1 in oestrogen metabolite-induced DNA damage would be an interesting avenue to explore in its ability to initiate stress-induced senescence, and how that contributes not only to neurodegenerative pathology but also sex-specific differences.Cellular senescence is becoming increasingly recognized as a feature of neurodegeneration as a process that promotes chronic tissue dysfunction and functional decline. Stress-induced senescence, in particular, has been documented to occur following excess DNA damage and defective DNA repair, and has been observed to even precede toxic protein aggregation in several neurodegenerative diseases. Evidently, the onset of senescence within various cell types in the brain interfere with proper structure and function, contributing to impaired cognition over time. Given the functional importance of BRCA1 in the brain as a key DNA repair protein in maintaining genomic stability, several studies have affirmed its association to stress-induced senescence and neurodegeneration. Together these studies emphasize a need for future research involving BRCA1, as it will not only provide further insights into DNA damage-induced senescence, but also introduce novel therapeutic avenues against neurodegenerative diseases and associated sex-specific differences.The authors report no competing interests.This review article did not involve the generation of new data.


Human cardiac tissue engineering holds great promise for early detection of drug-related cardiac toxicity and arrhythmogenicity during drug discovery and development. We describe shortcomings of the current drug development pathway, recent advances in the development of cardiac tissue constructs as drug testing platforms, and the challenges remaining in their widespread adoption.Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been used to develop a variety of constructs including cardiac spheroids, microtissues, strips, rings, and chambers. Several ambitious studies have used these constructs to test a significant number of drugs, and while most have shown proper negative inotropic and arrhythmogenic responses, few have been able to demonstrate positive inotropy, indicative of relative hPSC-CM immaturity.Several engineered human cardiac tissue platforms have demonstrated native cardiac physiology and proper drug responses. Future studies addressing hPSC-CM immaturity and inclusion of patient-specific cell lines will further advance the utility of such models for in vitro drug development. The current drug development pathway is exorbitantly expensive and time-consuming. It is estimated that between 2009 and 2018, the median cost to bring a new drug to market was $985.3 million, including capitalized research and development investment costs , 5. Addiether-á-go-go related gene (hERG) potassium channel, as this channel plays a major role in cardiac repolarization and hERG-blocking compounds frequently cause ventricular arrhythmias [The shortcomings of the canonical drug development process are perhaps most evident when considering cardiac effects, as side effects such as cardiac toxicity and arrhythmogenicity are the most common reasons for late-stage drug failure or drug withdrawal , 8. To ahythmias , 10. Whihythmias , 11. Whihythmias . Thus, tThe human heart is the ideal platform for drug testing; however, the limited availability and inadequate ex vivo viability of primary samples prevents their widespread use. Consequently, the development of human pluripotent stem cells  and hPSC-derived cardiomyocytes (hPSC-CMs) holds great promise for advancing drug development platforms –16. To dHuman engineered cardiac tissues can be broadly defined as multicellular aggregates made from hPSC-CMs, often accompanied by other cell types, with or without the presence of extracellular matrix protein scaffolds. Such engineered constructs more closely mimic the native myocardium by recapitulating key cell–cell and cell–matrix biology that has been shown to further advance hPSC-CM maturation and facilitate key measures of cardiac function such as force production and voltage propagation –25. ThesSpheroidsAt the smallest end of the scale are cardiac spheroids, also often referred to as cardiac organoids. Cardiac spheroids are small hPSC-CM aggregates that are formed by hanging-drop  or self-Cardiac spheroids have been used extensively to develop drug testing platforms. In an earlier study, it was shown that cardiac spheroids generated from hiPSC-CMs were similarly able to model doxorubicin-mediated cardiotoxicity as compared to spheroids made from primary human cardiomyocytes . DemonstUsing cardiac spheroids, it is also possible to model disease states. To model cardiac fibrosis, spheroids generated from hESC-CMs and hESC-derived mesenchymal stem cells were treated with transforming growth factor beta (TGF-β) . It was While the use of cardiac spheroids for drug screening is advantageous given their relative accessibility and high-throughput nature, these culture systems do not promote uniaxially aligned contractile machinery, and functional outputs are often limited to cell viability with some insight into contractility and arrhythmogenicity by measurement of spheroid deflection. However, it is possible for spheroids to serve as building blocks for higher order tissues, as was demonstrated with bioprinting of spheroids into larger cardiac rings . UltimatMicrotissuesTo increase functional readout capacity while retaining the high-throughput benefits of spheroid culture systems, many have developed what we here call microtissues, where a similarly small number of cells, sometimes in a hydrogel scaffold, are self-assembled onto manufactured posts, such that they form geometries similar to the cardiac strips and rings presented in the following sections –41. It wPossibly the most useful application of microtissues or other smaller cardiac platforms is as an intermediate screen between high-throughput two-dimensional in vitro experiments and animal studies. This paradigm was demonstrated in a study screening for pro-proliferative compounds using 96-well microtissues, where an initial pool of approximately 5,000 compounds were screened in two-dimensional hiPSC-CMs for their ability to induce cellular proliferation •. Of thiSheetsCardiac sheets consist of one or multiple layers of hPSC-CMs and are particularly useful for detecting arrhythmogenicity. Using fluorescent voltage or calcium-sensitive dyes or genetically encoded sensors, it is possible to model arrhythmic risk of drug compounds by visualizing conduction speeds and re-entry waves as was done in two-dimensional cell sheets . This plTissue StripsCardiac strips are perhaps the most commonly thought of hEHT platform along with spheroids and are made from hPSC-CMs embedded in a hydrogel that is cast into a mold where it solidifies and subsequently compacts and begins beating spontaneously . CardiacTo date, cardiac strips have been used to test a wide panel of compounds with and without known cardiac effects. A panel of eleven compounds was tested on cardiac strips, demonstrating that these tissues were able to faithfully reproduce positive and negative inotropic effects when compared to human atrial trabeculae, but the relative immaturity of the hiPSC-CMs was evident due to limited observed beta-adrenergic effects . The benChronic electrical stimulation of cardiac strips has been demonstrated to improve tissue maturity and promote positive force-frequency relationships, thus improving the accuracy of drug responses and the ability to model positive inotropy •. SimilaTissue RingsCardiac rings are similar to cardiac strips save for their shape, where cardiac rings are cast in circular molds before being transferred to isometric or elastomeric supports . Given tChambersAt the opposite end of the spectrum from cardiac spheroids are cardiac chambers, which resemble a miniaturized ventricle , 63. TheOne such platform  is generated by casting hESC-CMs in a hydrogel around a balloon catheter, which is removed after tissue compaction . This inCompared to other cardiac tissue platforms, cardiac chambers are limited in terms of the increased resources and technical expertise required, ultimately resulting in a lower-throughput platform. However, with the addition of additional maturation techniques such as electrical stimulation and anisotropic cell sheet patterning , such moAs a category, engineered cardiac tissues have come to include any cell culture platform facilitating multicellular, three-dimensional culture of synchronously contracting hPSC-CMs. As such, these platforms take many forms that vary widely in geometry and scale, the inclusion of non-myocytes, and the presence of scaffold proteins. Broadly, those on the smaller scale such as spheroids and microtissues more easily facilitate high-throughput cardiotoxicity screening while retaining some aspects of cardiac function, whereas larger platforms including cardiac sheets, strips, rings, and chambers are more suited to lower-throughput assessment of drug effect on cardiac function, as they more closely resemble native cardiac tissue and enable measurement of voltage propagation and force generation Fig. . In the Despite wide-spread excitement surrounding engineered cardiac platforms for drug development and several commercialization efforts underway, several hurdles remain. Compared to adult myocardium, or even neonatal myocardium, engineered cardiac constructs present a very immature phenotype, potentially limiting their physiological relevance. In early stages after differentiation with no intervention, hPSC-CMs display only fetal transcriptomes , ion chaA significant difficulty that has become apparent when working with engineered tissues is controlling variability and demonstrating reproducibility. This variability arises, in part, from hPSC-CM batch-to-batch variability and the different protocols used by different institutions for hPSC-CM differentiation and tissue generation, both of which are inherently human processes where results may vary simply by the hands performing the experiment. To remove human sources of variability, many are turning toward automation of tissue generation and analysis, which will also increase platform throughput , 57, 77.Conversely, this variability can be seen as a facet of hPSC and tissue engineering that has not yet been fully taken advantage of. There are numerous studies describing the development of hPSC lines harboring cardiomyopathy-associated mutations that could be further used to screen disease-specific drug candidates in engineered cardiac constructs . AdditioA significant limitation to translatability is that most platforms lack the biological complexity needed to fully reproduce native myocardium, necessitating the continued reliance on animal models for preclinical testing. At their simplest, cardiac constructs contain only hPSC-CMs, while others have included additional cell types including fibroblasts, endothelial cells, and mesenchymal cells, which have been shown to improve tissue quality and maturity. A biological element often missing from engineered cardiac constructs is vasculature, a key component needed to accurately model drug delivery. Strategies to vascularize constructs consist of co-culturing with endothelial cells and addition of angiogenic factors, three-dimensional bioprinting, or microfluidic systems , 82. TheLastly, it is worth noting that the vast majority of studies described in this review evaluated only compounds with known effects in human patients. As such, further studies are needed to demonstrate the true predictive capabilities of engineered cardiac tissues for clinical trial success. However, some pioneering studies have used engineered constructs to explore novel antifibrotic agents , performIn order to increase the efficiency with which new drugs are discovered and brought to clinical trials, it is necessary to improve the human in vitro models used, to increase their biological relevance and enable the field to move away from a reliance on animal models as the gold standard. To address concerns of cardiac toxicity, great efforts have been put toward the development of engineered cardiac tissues from hPSC-CMs. These platforms range in shape and scale from cardiac spheroids and microtissues, to sheets, strips, and rings, to chambers emulating an entire ventricle, all of which present their own advantages and limitations. This review has highlighted recent advances made in the development of cardiac tissue engineering for drug screening platforms. While lacking functional maturity and structural complexity in some regards, these constructs hold merit as drug screening platforms with powerful predictive capabilities that, as they stand, can provide value to early stages of the drug screening pipeline. With continued advancements in tissue maturity, automation, and throughput, it is our prediction that cardiac tissue engineering will continue to gain favor in the pharmaceutical industry.


Lastly, recent clinical evidence was presented linking urolithin A, a natural mitophagy activator, to improved mitochondrial and cellular health. The virtual panel discussed how stimulation of mitochondrial function via biological pathways, such as mitophagy and NAD+ augmentation, could improve cellular function and muscle health, potentially impacting clinical signs of AACD and overall healthy aging.Aging is a primary risk factor for the progressive loss of function, disease onset, and increased vulnerability to negative health-related outcomes. These clinical manifestations arise in part from declines in mitochondrial, metabolic, and other processes considered to be hallmarks of aging. Collectively, these changes can be defined as age-associated cellular decline (AACD) and are often associated with fatigue, reduced strength, and low physical activity. This manuscript summarizes a recent Gerontological Society of America Annual Scientific Meeting symposium that explored mechanisms, clinical signs, and emerging cellular nutrition interventions for AACD. The session opened by highlighting results of an expert consensus that developed an initial framework to identify self-reported symptoms and observable signs of AACD in adults aged >50 years. Next, findings from the multi-ethnic molecular determinants of sarcopenia study were discussed, showing impaired mitochondrial bioenergetic capacity and NAD Geroscience has proposed that the underlying biological mechanisms of aging are central to the global increase in susceptibility to disease and disability that occurs with aging. Strong correlations have been revealed between biological mechanisms and some clinical phenotypes that are typical of aging, particularly declines in autophagy, mitochondrial dysfunction, and cellular senescence . StudyinRecently, experts convened at the virtual 2020 Annual Scientific Meeting of the Gerontological Society of America to review recent developments to target the origins of cellular decline, understand the modifiable signs and risks of accelerated aging and cellular decline, discuss data showing how mitochondrial dysfunction in older adults impairs muscle bioenergetics and function, and share emerging evidence on nutritional interventions to mitigate age-related mitochondrial dysfunction and improve health. As muscle tissues have a high abundance of easily accessible mitochondria and are of clinical significance to overall altered mitochondrial morphology, special attention was given to the common changes in skeletal muscle—from mechanisms to symptoms and performance —tightly In a landmark article, López-Otín and colleagues  first prThe medical model has traditionally focused on identifying and treating specific disease states and individual organ failure exhibited by older persons. This is related to but distinct from a more biologically driven approach of detecting cellular dysfunction, which often overlaps in different organs and predisposes the development of multiple age-related conditions. Moving beyond the traditional disease-specific focus to better promote healthy aging involves developing tools to better characterize the signs and symptoms of age-associated cellular decline (AACD) to facilitate early identification of those at risk of accelerated aging. To further this change in clinical approach, two interdisciplinary expert panels were recently convened to propose a novel framework to classify risk factors, early indicators, and clinical differentiators of AACD for the purpose of identifying signs, symptoms, and markers indicative of AACD .What are the early signs and symptoms of AACD?How could early age-associated decline be differentiated from other conditions with similar symptoms?What are the risk factors for AACD?Describe the profile of individuals at high risk of AACD?Which of the features of AACD are clinically relevant and important triggers for intervention? A group of international experts with clinical and research expertise was convened through a virtual workshop for decision-making in this area. A consensus was established using the modified nominal group technique ,6. Brief+) levels through perturbed NAD+ biosynthesis and salvage in sarcopenic muscle cells [Skeletal muscle is important for overall health, and loss of muscle mass or strength is a powerful predictor of poor health outcomes . A recenle cells .+ can lead to a loss of mitochondrial homeostasis [+ levels have been shown to be downregulated by approximately 30% among individuals with sarcopenia [+, whereas repletion with precursors, such as nicotinamide riboside, has shown a potential to reverse this process in experimental models [+ repletion through nicotinamide riboside oral supplementation in both young and aged mice has indeed shown the potential to increase muscle stem cell activity and regenerative capacity and to enhance muscle strength and performance in the context of aging [+ salvage pathway is partially impaired in sarcopenia since two rate-limiting enzymes are decreased in this condition, but de novo and exogenous pathways are intact and can likely be leveraged for therapeutic interventions. Nicotinamide riboside has been shown to be safe and efficacious to increase NAD+ levels in human blood and muscle in the context of aging [A reduction in NADeostasis . NAD+ lercopenia . Mitochol models ,12,13. Nof aging . Interesof aging ,16,17, aof aging .In humans, urolithins have been shown to be end-products or gut microbial metabolites of both ellagitannins and ellagic acid, which are complex polyphenols naturally contained in walnuts and fruits, such as pomegranates and red berries . UrolithEllagitannins are first hydrolyzed under the acidic conditions of the stomach into ellagic acid, which is then further metabolized by the microflora into urolithins via loss of one of its two lactones and by successful removal of hydroxyl groups . HoweverWith regard to safety, two rodent toxicological studies have evaluated the effects of high-dose ellagitannin supplementation at levels up to 4800 mg/kg of punicalagin (the most abundant ellagitannin in pomegranate) per day ,26. The Caenorhabditis elegans), exposure to urolithin A activated mitochondrial biogenesis [Research investigating the mechanism of action in regard to potential effects of urolithins on mitochondrial function and sarcopenia following dietary supplementation is emerging. In older worms  were also investigated from skeletal muscle biopsies and blood samples at baseline and after 28 days of each dosing regimen. Dosing of urolithin A at 250 mg showed no significant improvement in biomarkers; however, dose-dependent decreases in acylcarnitine levels were demonstrated in the 500- and 1000-mg groups [GABARAPL1, fatty acid-binding protein FABP3) [A single phase I (two-part) clinical study of urolithin A was reported in the scientific literature . Part 1 g groups . Acylcarg groups . The dirn FABP3) . These r+ levels, which is important for mitochondrial biogenesis. Recent clinical data demonstrate urolithin A’s ability to activate cellular mitophagy and improve mitochondrial health in the skeletal muscle of healthy older adults. Additional research is underway on both of these cellular nutrients to understand their potential to impact cellular and clinical outcomes during aging.The scientific literature clearly demonstrates that ubiquitous cellular changes drive many age-associated chronic disease states. Targeting these specific pathways may have the potential to effectively alter the aging trajectory. Certain nutrients, including nicotinamide riboside and urolithin A, have been identified as promising ways to improve mitochondrial health, with each targeting different cellular pathways. Although the typical diet may provide low amounts of these nutrients, insufficient intakes are likely obtained due to their low abundance in commonly consumed foods and interindividual differences in their metabolization by the gut microbiome. Supplemental nicotinamide riboside has been shown to effectively boost NAD


Asthma has become a global health issue, suffering more than 300 million people in the world, which is a heterogeneous disease, usually characterized by chronic airway inflammation and airway hyperreactivity. Combination of inhaled corticosteroids (ICS) and long acting β-agonists (LABA) can relieve asthma symptoms and reduce the frequency of exacerbations, especially for patients with refractory asthma, but there are limited treatment options for people who do not gain control on combination ICS/LABA. The increase in ICS dose generally provides little additional benefit, and there is an increased risk of side effects. Therefore, therapeutic interventions integrating the use of different agents that focus on different targets are needed to overcome this set of diseases. Some findings suggest autophagy is closely correlated with the severity of asthma through eosinophilic inflammation, and its modulation may provide novel therapeutic approaches for severe allergic asthma. The chinese herbal medicine (CHM) have been demonstrated clinically as potent therapeutic interventions for asthma. Moreover some reports have found that the bioactive components isolated from CHM could modulate autophagy, and exhibit potent Anti-inflammatory activity. These findings have implied the potential for CHMs in asthma or allergic inflammation therapy via the modulation of autophagy. In this review, we discuss the basic pathomechanisms underpinning asthma, and the potential role of CHMs in treating asthma with modulating autophagy. Asthma has become a global health issue affecting approximately 4% of the world population. The number of patients suffering from the disorder is expected to reach 400 million worldwide by 2025 . AccordiAutophagy, a cellular self-engulfing process employed for the removal of unwanted organelles and misfolded proteins, plays a critical role in the pathogenesis of a variety of inflammatory disorders, including respiratory illness diseases like asthma . IntriguAsthma is a heterogeneous disease with several underlying disease processes, usually characterized by chronic airway inflammation, airway hyperreactivity and airway remodeling. The classic allergic airway inflammation is caused by submucosal infiltration of activated T-lymphocytes, eosinophils, neutrophils, epithelial cells, macrophages and mast cells . Thus, iThe extensive infiltration of eosinophils into the airway is a hallmark of allergic asthma. The numbers of eosinophils in peripheral blood, bronchoalveolar lavage fluid (BALF), and epithelium were not only associated with lung function but also associated with the severity of asthma . EosinopMany researchers have reported that there is a subtype of severe asthma with persistent symptoms and high risk of exacerbation. IL-17 cytokines released by Th17 cells mediate severe asthma inflammation via augmentation of expression of GR-β, which inhibits the action of glucocorticoid receptor GR-α Autophagy is a highly conserved homeostatic mechanism for cell survival under conditions of stress, and is widely implicated as an important pathway in many biological processes and diseases . The norAllergic asthma is mediated by TH2 cells and type two innate lymphoid cells (ILC2) for production of inflammatory cytokines IL-5 and IL-13 . Airway However, in pulmonary dendritic cells, asthma could reduce the numbers of LC3 foci. Atg5 deficency mice were found to have no decrease in AHR showing a steroid resistant phenotype with increase concentrations of IL-1 and IL-23 further contributing to TH17 neutrophilic polarity . In macr+ lymphocytes’ differentiation into Th17 cells , a natural compounds, taking as efficiency source of autophagy modulators come into our sight. Since autophagy pathway underlines a broad range of pathological conditions in asthma, the successful therapeutic outcomes of using autophagy modulators suggest the need for intensively investigating the pharmaceutical potential of compounds with autophagy-adjusting ability. Recent research findings not only shed light on the potential novel applications and formulation of CHMs via regulation of autophagy possibly being an important mechanism underlying the therapeutic effect of CHMs in treating disease, but also highlight that the natural autophagic compounds and extracts from CHM are of interest due to their potential new therapeutic application in diseases. While a number of autophagy regulators, such as Oyaksungisan, Shensuyin, Ginseng, Turmeric and Ginger have been reported, natural autophagic compounds from CHMs are of interested because of their potential new therapeutic applications .CHM is an ancient yet still active medicinal system widely used in China. A major therapeutic approach of CHM is the use of a mixture of herbs, called the Traditional Medicine Formulation. The mixture of herbs exerts therapeutic actions and modulates other effects, with the principal herbal constituents providing the main therapeutic actions.Glycyrrhizae Radix et Rhizoma, Zingiberis Rhizoma Crudus, and Zizyphi Fructus (2O2-induced apoptosis and anti-inflammation effects (Oyaksungisan (OY) is a traditional herbal medication formula consisting of twelve herbs: Ephedra Herb, Citrus Unshiu Peel, Lindera Root, Cnidii Rhizoma, Angelica Dahurica Root, Batryticatus Bombyx, Aurantii Fructus Immaturus, Platycodon Root, Zingiberis Rhizoma,  Fructus . Some of Fructus . OY can  Fructus . Numerou effects . OY cont effects . NF-κB s effects . These iPingchuanning decoction is a well-known traditional Chinese medicine for the treatment of airway inflammatory diseases. Pingchuanning decoction had an intervention effect on alleviation of airway inflammation, relief of bronchial smooth muscle spasm, and inhibition of airway remodeling in asthma . PingchuGlycyrrhizae Radix et Rhizoma, Citri Unshius Pericarpium, Zingiberis Rhizoma Crudus, and Ziziyphi Fructus. Recent studies have reported that SSE can modulate asthma reactions and pulmonary damage via inhibition of the expression of cyclooxygenase 2 (COX-2), of inflammatory cytokines and of the activation of NF-κB , a traditional herbal formula, has been widely used to treat cough and fever . The herof NF-κB . SSE hasof NF-κB . Thus SSFor the past several thousands of years, Traditional Herbal Formulation has been used to prevent and treat a number of disorders. But to find the directed association between scientific proof and molecular analysis of CHM, a growing number of studies of CHM in disease have focused on molecules extracted and purified from individual herbs. At present, a number of individual herbs have been reported to be beneficial to curing disease via modulating autophagy, such as Radix Ginseng (Ren Shen), Ridix Sophorae Flavescentis (Ku Shen) and others.Ginseng has been prescribed for maintaining the bioenergetics balance, as suggested by Chinese herbalists, for breathlessness and diabetes. Rb1, Rg1, Rg3, Rh1, Re, and Rd are bioactive ginsenosides from Ren Shen that were revealed by pharmacological studies . The traRidix Sophorae Flavescentis (Ku Shen), documented in editions of the pharmacopoeia of the People’s Republic of China, has had more than 200 compounds isolated and extracted. Modern pharmacological studies have shown that matrine is the most active of these components with multiple pharmacological effects, including anti-tumor, antiviral, and anti-inflammation, as well as anti-asthmatic activities . The maiAt present, there are occurring a lot of discoveries of natural autophagy regulators from CHM with potential use in therapeutic applications in cancer and neurodegenerative disease models. Although little direct linkage between the traditional herbs-induced autophagy and its anti-asthmatic effects has been reported, owing to the significant role of autophagy in immunomodulation, the involvement of such a process cannot be disregarded.Glycyrrhiza uralensis, is one of the most frequently used drugs in traditional Chinese medicine. A large number of prescriptions contain licorice as a significant component present in Shang-Han-Lun  is a common herbal remedy in the field of traditional medicine. The root of skullcaps, known as Radix Scutellariae, is the source of the Chinese medicine Huang Qin . It has nea pigs . Currentnea pigs . AlthougTurmeric (Jiang Huang Su) possesses a wide spectrum of biological and pharmacological activities . CurcumiArtemisia asiatica Nakai (Ai Cao), which belongs to the family Compositae, specifically the leaves of this plant, is known for its anti-inflammatory, anti-microbial, anti-tumor, anti-oxidative, neuroprotective, and gastroprotective properties, has been documented to be effective in various disease conditions . EupatilPicrorhiza kurroa (Hu Huang Lian) is a small perennial herb belonging to the family scrophulariaceae, and is a well-known herb of traditional medicine with anti-oxidant, anti-inflammatory, and immunomodulatory activities . The rhiGinkgo biloba (Yin Xing) has been used in traditional Chinese medicine for about 1,000 years. Ginkgo biloba extract (GBE) is collected from the dried green leaves of the plant . PharmacFructus evodiae (Wu Zhu Yu) has been used frequently as a traditional medicine against inflammatory diseases in China and Japan. Evodiamine (EVO) and rucarpine are compounds extracted from Fructus evodiae, which were shown to have inhibitory effects on TNF-α and IL-4 protein expression in RBL-2H3 cells induced by IgE-antigen complex . These rGinger (Sheng Jiang) has been used in traditional medicine for the treatment of respiratory diseases such as asthma . PharmalBronchial inflammation and airway remolding are common pathological features shared by asthma. However airway remolding the major difficult in treatment accompaning decreasing of lung function characteristiced smooth muscles rewalling, globet cell hyperplasia and mucus hypersecretion, which is induced extensively, though not exclusively. These asthma therapeutic compounds listed here were reported mostly by their anti-inflammation features along with relaxation of airway smooth muscle (ASM), attenuating airway hyperresponsiveness and inhibiting inflammatory cell infiltration. Another facet to consider for asthma therapy with these proposed potential traditional medicines is their polypharmacological nature. For example, they are also autophagy modulators in cancer or neurodisease, reinforcing that these CHMs potentialy simultaneously target autophagy passway focused on anti-inflammation and futhur anti-mucus hypersecretion via interaction with inflammation cytokines.Above all, CHM is one of the main lines of complementary and alternative therapy of bronchial asthma, as it is the third most popular choice of both adults and children suffering from this condition. An increasing number of CHMs have been discovered as autophagy modulators. Such autophagic-regulatory effects are potentially therapeutic for allergic inflammatory diseases such as asthma. Although we have found that many autophagy modulators isolated from anti-asthmatic-CHMs were possibly related to new mechanisms and functions of these plants in the treatment of asthma diseases, we are still far away from translating these traditional herbs or formula into clinical applications in asthma therapy. The cell type-specific property of autophagy should be taken into account, which may otherwise minimize the efficacy of the applied herbs. Moreover, the functional consequences of autophagy induction on these cells vary under different disease conditions. Therefore, detailed and systematic investigations concerning the interaction between CHMs and autophagy-related disorders in a comprehensive molecular approach are needed. Positive findings in these areas could widen the scope of CHM applications by suggesting novel intervention strategies, which have not been mentioned in the traditional Chinese pharmacopeia.Since the molecular mechanisms and functions of many autophagy modulators isolated from CHM have been intensively studied, we will work in the future to provide a direct link from traditional use to new pharmacological applications associated with autophagy in asthma treatment with these plants.in vivo and invtro to reveal the polypharmacological approach of natural products taget the diseases with complex pathologies such as asthma.Taken to note, as the certein mechanisms of either of the CHMs list in the paper modulates autophagy in response to asthma related stimulis may vary depending on the type of cells or the cell metabolism and/or environment Thus we need to further studing asthma therapeutic drugs or autophagy modulators conducted on asthma models


Other papers critically investigate the significance of methods used to detect developmental senescence, or “canonical” and “non-canonical” roles of components of the apoptosis machinery.In the history of developmental biology, apoptosis and senescence will foreseeably share at least four characteristics. Firstly, both have made their way into this field after detours. Thus, dead cells were admittedly discovered early within embryos of various vertebrate classes, well before the term apoptosis came into use . ConceptThis Research Topic includes 20 articles written by 118 authors and reviewed by 41 referees, who in their entirety came from 25 different countries and six continents. The following section presents the focus and key findings of each article.Wanner et al.). This overview starts by addressing zygotes and blastocysts, which are later revisited under in vitro conditions . In the opposite direction, the chronological spectrum even covers transitions to ageing processes. Further main topics are epigenetic modifications and the ambivalence of several tumor suppressors  which “play antagonistic roles by increasing fitness and decreasing mortality early in life but contribute to deleterious effects and pathologies later in life” or, in short, which reveal “antagonistic pleiotropy” .We open our Research Topic with a review on senescence and apoptosis as the architects of mammalian development  inhibitor p21 are reliable in vivo markers of cellular senescence. The authors focus on retinal and limb development, but also outline other organ systems. Possible interrelationships between apoptosis and senescence are additionally considered. Given that ß-galactosidase activity overlaps with areas of cell death and, like p21, is also present in newly differentiated retinal neurons, both “senescence markers” may be less specific than previously thought. Ultimately, methodological concerns highlighted here underscore the recent postulate that proper detection of senescence must be based on experiments using at least three independent markers  or p21 . Furthermore, it is demonstrated that senescent cells in the floor plate signaling centre . Using the Nestin-Cre Mcl-1 conditional knockout (Mcl-1 CKO) mouse line, the authors detected apoptotic waves which, in the spinal cord, proceeded ventrodorsally and rostrocaudally from embryonic day 9 onwards. Consequently, Mcl-1 does not contribute to neural tube formation, but to the differentiation of neuronal precursor cells (NPC) into immature neurons. Breeding Mcl-1 CKO mice with Bax null mice further demonstrated “that Mcl-1 promotes NPC survival primarily by inhibiting the activation of Bax, but that Bax is not the sole pro-apoptotic target of Mcl-1 during embryonic neurogenesis” .The third paper in this series examines the anti-apoptotic role of the Bcl2 protein Mcl-1 (Myeloid cell leukemia-1) in the embryonic spinal cord, brainstem and forebrain .Following this, Li et al.). It thus continues earlier work documenting the occurrence of increased apoptosis in the nervous system, head and limbs of ethanol-exposed embryonic mice . Specifically, the vegetable-derived isothiocyanate sulforaphane “protects against ethanol-induced apoptosis (…) through diminishing ethanol-induced hypermethylation at the promoters of the genes encoding the inhibitor of apoptosis proteins” .The fifth paper raises the question of how ethanol-induced apoptosis can be prevented in human neural crest cells .Next, and face . The autKim et al. demonstrate that hypoxia-responsive oxygen nanobubbles prevent hypoxia-induced pathological increases of apoptosis in the cap and bell stages of mouse tooth germs. It can therefore be assumed that hypoxia-responsive oxygen nanobubbles will be suitable for regenerative approaches in the dental field. Whether, in tooth germs, hypoxia-inducible factor-1  cells including tooth germs, but also to the topic of regeneration. Undoubtedly, “dental pulp stem cells (…) have attracted considerable attention as promising cells for regenerative endodontics (…) and systemic regenerative medicine” (Maeda).The subsequent paper by Wu et al.). Caffeine exposure interferes with the expression of genes modulating apoptosis, proliferation, and differentiation of myogenic progenitors in differentiating somites as well as in the chest wall. Resembling the case of ethanol-treated human neural crest cells , increased apoptosis affected caffeine-treated myogenic progenitors. Unexpectedly, however, significantly increased proliferation rates were additionally recorded under the influence of caffeine. All pathological changes were suppressible by the administration of retinoic acid antagonists.The following group of three articles is dedicated to trunk and limb development. We begin with a study on the teratogenic effects of caffeine in chicken embryos . Apoptosis seems to dominate the onset of interdigital tissue remodeling, but is later coincident with “destructive developmental cell senescence”, which, however, is difficult to distinguish from other lysosomal pathway-mediated processes. A second type of (true?) senescence observed during digit formation shows no association with apoptosis, but is most likely related to tendon differentiation . Overall, the authors are certainly right to warn against overly rapid classification attempts in cases of cellular degradation pathway confluence.The second article illustrates that at least two different mechanisms are involved in the degradation of superfluous cells during digit formation . The authors show that cell death in the ANZ can be triggered either by inhibition of FGF or WNT signaling, or by activation of BMP signaling. Surprisingly, however, FGF signaling induces the expression of the pro-apoptotic genes Dickkopf (Dkk) and Bmp4. Furthermore, FGF8 in the apical ectodermal ridge and BMP4 in the anterior border of the limb are inhibited by BMP4. If, on the other hand, BMP4 is blocked by NOGGIN, Dkk expression in the ANZ gets reduced. Double treatment experiments support the hypothesis that during BMP4 activation, FGF signaling may represent the final step in the molecular cascade leading to early cell death in the ANZ .The article closing the limb topic deals with the “anterior necrotic zone” (ANZ) which, in chicken embryos, promotes digit number reduction . In our thematic context, ribosomopathies are attractive, among other aspects, because they can trigger apoptosis or senescence via p53  show lower survival rates due to the failure of their buoyancy system. Now, the authors report that animals living in captivity  exhibit massively increased apoptosis rates. In line with this, changes in the expression levels of cell death-associated genes are demonstrated .Three other papers explore senescence and/or apoptosis at particularly early developmental stages. At the outset, in vitro bovine embryo development . In pre-implantational embryos, protective senescence initially gains regulatory significance. Later during morula and blastula stages, apoptosis is in the foreground, whereby “embryonic quality is normally associated with lower apoptotic rates”. Main parts of this work provide practical guidance on how to assess embryo quality using structural criteria and molecular markers for measuring senescence and apoptosis.Next is a review on the occurrence of senescence and apoptosis during Yu et al. address the question on how to predict bovine blastocyst quality. 42% of the blastocysts examined show cellular fragments in the perivitelline space before transfer, and 37% of these fragments are TUNEL-negative, i.e. non-apoptotic. Furthermore, neither correlative nor causal relationships were found between the occurrence of perivitelline fragments and blastocyst quality. The authors therefore conclude “that apoptosis and fragmentation are methods to remove abnormal cells from the embryo, and thereby protect the developmental competence of the embryo”.Correspondingly, de Mera-Rodríguez et al.), we extend the scope of our Research Topic one last time to the postnatal developmental period, this time using the skin as an example. Specifically, Chou et al. intend to establish a mouse model that recapitulates key pathological features of human diseases associated with dysfunctions of the tumor suppressor gene Wwox. They show that Wwox deficiency leads to decreases in the proliferation and differentiation of keratinocytes as well as to increased apoptosis rates in Wwox−/− mouse epidermis, primary keratinocyte cultures and Wwox knockdown human HaCaT cells. Most probably, Wwox deficiency is mediated by the downregulation of prosurvival MEK/ERK signaling.As with the retina  which contain nucleic acids, protein lipids, mitochondria, and plasma membrane, among others. Possible developmental and/or regenerative functions of ApoEVs include the apoptosis-induced activation of compensatory proliferation and/or differentiation processes as well as the elimination of senescent cells. Interestingly, senescent cells, owing to their senescence-associated secretory phenotype (SASP), display fascinating parallels to ApoEVs by releasing cytokines, growth factors or DNA fragments into their environment.The two concluding articles of this Research Topic focus on possible links between apoptosis, senescence, development and regeneration. Firstly, Guerin et al. Typical subjects of discussion are functions of BMPs in the limbs or those of Jun-N terminal kinase (JNK) in the brain. Furthermore, the importance of apoptosis for cell signaling and patterning mechanisms is being investigated. Overall, the authors conclude that, despite numerous commonalities, apoptosis-induced regeneration by no means simply recapitulates apoptosis-induced developmental mechanisms.Molecular similarities between apoptosis-induced regenerative and developmental processes are also explored in the final review prepared by Taken together, the results of this Research Topic first of all show that numerous previously undescribed cases of ontogenetic cell death still await discovery. Furthermore, there is increasing evidence that developmental events may not be propelled by one type of cell death alone, but that several different types of cell death may be involved, either simultaneously or successively. We conclude from this that, even in developmental contexts where, for example, apoptosis safely fulfils specific functions, we should nevertheless explore the remaining spectrum of cell death forms. This need is all the more acute in cases where the functions of ontogenetic cell death are still unclear, or where experimental suppression of programmed cell death entails inconclusive results. Especially in the latter instance, switching between several types of cell death might be part of the embryo’s “survival repertoire”. Understanding such possible cell death transitions might also help to better appreciate similarities and differences between developmental and regenerative processes.With the discovery of programmed senescence, the situation initially became even more complicated for developmental biologists interested in programmed cell death. That is why it is all the more imperative now to meticulously document examples of the complementary, simultaneous and/or successive occurrence of both processes in well-defined developmental contexts. We are convinced that without such systematic investigations it will hardly be possible to gain experimentally validated insights into the functional interactions of programmed cell death and senescence. To achieve these objectives, as in the case of programmed cell death, it will undoubtedly be necessary to solve numerous methodological problems that currently hamper the reliable diagnosis of senescent cells. Conversely, our ever-increasing knowledge about the regulatory potential of secretory active senescent cells will certainly inspire developmental biologists to take a fresh look at the developmentally relevant functions of ApoEvs as well as at the non-canonical functions of components of the apoptosis machinery.Finally, we are left with the pleasant duty to express our sincere thanks to all those who helped to realize this Research Topic. We were particularly impressed by the constant willingness of both our authors and reviewers to contribute ambitiously to the optimization of submitted manuscripts through precise, critical and self-critical comments, questions and responses. Unwanted by all, the Corona pandemic has also left its marks on our project, as much through months of laboratory closures as through severe diseases affecting families and friends. It was therefore all the more encouraging to learn how much trust and tolerance still prevail in the scientific community. Especially the latter experience would never have become reality without the always excellent cooperation with the Frontiers Editorial Team.


Resilience in the face of major life stressors is changeable over time and with experience. Accordingly, differing sets of neurobiological factors may contribute to an adaptive stress response before, during, and after the stressor. Longitudinal studies are therefore particularly effective in answering questions about the determinants of resilience. Here we provide an overview of the rapidly-growing body of longitudinal neuroimaging research on stress resilience. Despite lingering gaps and limitations, these studies are beginning to reveal individual differences in neural circuit structure and function that appear protective against the emergence of future psychopathology following a major life stressor. Here we outline a neural circuit model of resilience to trauma. Specifically, pre-trauma biomarkers of resilience show that an ability to modulate activity within threat and salience networks predicts fewer stress-related symptoms. In contrast, early post-trauma biomarkers of subsequent resilience or recovery show a more complex pattern, spanning a number of major circuits including attention and cognitive control networks as well as primary sensory cortices. This novel synthesis suggests stress resilience may be scaffolded by stable individual differences in the processing of threat cues, and further buttressed by post-trauma adaptations to the stressor that encompass multiple mechanisms and circuits. More attention and resources supporting this work will inform the targets and timing of mechanistic resilience-boosting interventions. Resilience has been defined as the set of complex and dynamic processes that allow individuals to maintain psychological well-being in the face of adversity , 2. By tthe development of translatable biomarkers [to] facilitate the study of stress responses, resilience, and vulnerability across both human and animal studies [”. To facilitate the development of a longitudinal model of neural contributors to resilience, here we review the emerging body of literature employing neuroimaging in longitudinal cohort studies to predict psychological well-being following trauma exposure. For the purposes of the review, trauma is defined as an event meeting DSM-5 criterion A for trauma- and stressor-related disorders [Neuroimaging data collected from before a traumatic event, during the peri-trauma, and during the post-trauma period can show the full sequence of neural features and adaptations that lead to recovery. Understanding neural circuit contributors to an adaptive response to trauma is critical to our ability to create new interventions that will improve and accelerate recovery. This is in line with current research priorities of the National Institute of Mental Health, which underscores the need for “ studies ”. To faisorders  such thaWe conducted a literature review of neuroimaging studies from January 1995 to May 2021, using the following search terms: trauma OR PTSD OR “posttraumatic stress disorder” AND prospective OR longitudinal OR cohort OR predict AND MRI OR neuroimaging OR “magnetic resonance imaging” OR “neural correlates”. This search was conducted in Pubmed, Web of Science, and Scopus. We included all unique studies that had a longitudinal component such that neuroimaging was conducted either at one or multiple timepoints, with subsequent assessments of trauma-related symptoms including posttraumatic stress disorder (PTSD) or depression, and excluded findings from drug or intervention studies. Table We discuss the findings following general principles outlined by Williams et al. , in termBox 1 Canonical neural circuits contributing to post-trauma resilienceWe organize our discussion of neural features contributing to resilience around an established framework relating neural circuits to psychopathology . We alsoThreat and Salience: amygdala, hippocampus, insula, and both dorsal and ventral portions of the prefrontal cortex, including the dorsal medial prefrontal cortex (dmPFC), dorsal anterior cingulate cortex (dACC), and the ventral mPFC (vmPFC) and rostral ACC (rACC)Reward: ventral striatum and projections to the orbitofrontal cortex (OFC) and vmPFCDefault mode: mPFC, angular gyrus, posterior cingulate/precuneus, medial temporal lobe, and ventrolateral PFC (vlPFC)Attention: Medial superior frontal cortices (msPFC), aI (auditory), superior/inferior posterior parietal corticesCognitive control: dorsolateral prefrontal cortex (dlPFC), ACC, precentral gyrus (PCG), dorsal parietal cortex (DPC)Longitudinal studies have proved to be a powerful tool in identifying aspects of neural structure and function that contribute to resilience. One particularly important goal has been to search for traits that exist prior to a trauma, which confer protection from chronic stress-related symptoms following the trauma. Due to challenges in predicting who will be exposed to trauma and when, it has been difficult to study pre-existing neural traits of resilience. However, an emerging literature across both military-related and civilian longitudinal studies has captured pre-trauma structural and functional neural features that predict changes in later brain function and adaptive natural recovery from stress-related symptoms after trauma. These studies provide key insights into neural resilience and how the brain changes and recovers after exposure to psychological stress.The majority of research on risk and resilience factors for trauma-related psychopathology links the psychological response to trauma with individual differences in the structure and function of areas associated with threat processing, which has some overlap with the salience network. The areas discussed can be categorized by those involved in threat inhibition , versus those involved in threat response and salience detection .The amygdala is widely studied in trauma research due to its role in responding to threatening stimuli and the expression of fear . It hasIndividuals with less amygdala reactivity may also show less sympathetic output when new threats are encountered. For example, among medical students scanned prior to a stressful ED internship, less connectivity between the amygdala and a key downstream noradrenergic output region the locus coeruleus (LC)  measuredSimilarly, the ventromedial prefrontal cortex (vmPFC) and its connections with the amygdala or hippocampus may be important contributors to adaptive threat responses. The vmPFC implements top-down control over fear-related regions like the amygdala, and helps to adaptively modulate threat responses . A recenThe hippocampus is another region likely to aid in appropriate regulation of physiological arousal responses to threat, known to be important for contextualizing memories and experiences . ClassicThe salience network has been implicated in the monitoring of affective environmental cues. Structural and functional differences in distinct subregions of the ACC may denote specific PTSD resilience factors. Studies of combat exposed veterans and their unexposed twins have found that less resting state dACC and mid-cingulate activity , as wellRegardless of symptom presentation, regions involved in threat detection and response, including the amygdala and insula , 31, 34 A pattern of conserved hippocampal volume and function from pre- to post-exposure to stress has also been associated with resilience. Hippocampal volume appears to decrease as a function of stress exposure; for example, individuals who experienced a greater number of stressful life events demonstrated larger decreases in left parahippocampal and right hippocampal volumes . Thus, iThe uncinate fasciculus (Uf) is the primary white matter tract connecting the vmPFC and orbitofrontal cortex to the amygdala, and is involved in emotional processing . StandarVery little work has focused on the reward circuit pre- to post- trauma exposure. One study found that differences in nucleus accumbens (NAc) activation pre-deployment did not predict resilience, but that at the post-deployment assessment, more resilient individuals showed greater NAc response to reward compared to individuals experiencing worse post-trauma PTSD symptoms . AdditioThe anterior prefrontal cortex (aPFC) and in particular the frontopolar cortex is thought to be involved in action selection and reasoning , 39. KocPotential resilience or protective factors prior to a trauma Fig.  include It is important to note that while being a “resilient” individual with a dampened threat and salience response or greater threat inhibition may be adaptive in times of safety and security, these same traits may be harmful in situations where responding to threat cues is vital for survival, like in the case of a soldier on the battlefield. Trait-like, lower reactivity to threatening cues may not keep a person safe in times of real danger. Resilience may therefore be reflected in the adaptive regulation of circuits that modulate autonomic and emotional arousal, but not defined by any specific level of regional reactivity.Most civilian trauma neuroimaging research occurs in the aftermath of trauma, during peri-trauma and in the years following. The main focus of this research has been to establish structural and functional differences or changes that predict the future emergence of chronic trauma-related symptoms or resilience. As many trauma-exposed individuals seek acute treatment for injuries or related medical issues, the peri-traumatic period is a key time window for early assessment of stress resilience and for the deployment of early interventions to boost resilience and recovery.The majority of peri- and post-trauma neuroimaging findings implicate the structure and function of areas involved in threat inhibition  and those involved in threat response and salience detection .Consistent with pre-trauma findings, amygdala reactivity to threat cues acutely post-trauma correlates negatively with later resilience. For example, in participants recruited from the emergency department (ED), who were scanned 1-month post-trauma, amygdala reactivity to negative emotional stimuli negatively correlated with resilience, as measured by fewer PTSD symptoms months later . While tOther areas of overlap between the threat and salience network include the dACC and insula. Within these areas, increased reactivity and structural integrity are associated with resilience. A pattern of larger insula, dACC, and rACC volume assessed shortly after trauma predicts fewer future PTSD symptoms after head injury trauma , and motThe amygdala is regulated by the vmPFC, and longitudinal findings support that an increase of vmPFC morphometry or activity is important for later resilience. Among PTSD patients scanned at varying times since trauma, a longitudinal increase in sgACC over 6–9 months predicted natural recovery over the same timespan . SimilarHippocampal activation and structural integrity overall appear to be predictive of resilience. Studies of hippocampal structure suggest that greater hippocampal volume correlates with greater resilience to trauma, with larger hippocampal volumes early post-trauma among those who demonstrated the fewest future PTSD symptoms –54. HoweConnectivity within and to the threat network appears highly important to the response to the trauma, with many studies finding amygdala FC to be involved in fear learning and symptom severity following trauma. Amygdala-PFC, -PCC, -dACC, -superior temporal gyrus, and -insula functional connectivity have all been implicated in recovery from trauma-related psychopathology. Amygdala-PFC connectivity, and PFC sub areas, has previously been supported as a resilience factor . The vmPThese findings support proposed models suggesting that hippocampus and vmPFC modulation of amygdala activity early post-trauma supports resilience and recovery. On a basic level, less amygdala reactivity, greater hippocampus, and vmPFC activity and volume, and greater functional and structural connectivity between these regions and the amygdala early post-trauma correlate with better PTSD recovery. However, evidence across the collected studies suggests that functional correlations are task dependent, with vmPFC engagement across repeated presentations of threat correlating with quicker recovery, whereas the vmPFC response to threatening versus neutral social cues was associated with greater future symptom maintenance. Similarly, in the hippocampus, engagement during inhibition is associated with less future PTSD symptom symptoms, but greater response to threat cues during conditioning correlates only with trait resilience. Therefore, a multifaceted approach is necessary to fully understand these three areas of the threat network. Methods for assessing resilience more broadly than PTSD symptoms and recovery are necessary.In the peri-trauma period, again, a very limited set of studies have focused on reward circuits and resilience. One longitudinal study in cancer survivors did find that GMV in the right orbitofrontal cortex 3–15 months post-surgery and two years later was significantly larger in resilient survivors and non-trauma controls compared to those with PTSD , which iDefault mode network (DMN) activation is observed while at rest and during spontaneous reflection , 71, 72.While DMN engagement and threat-network connectivity predict acute trauma-related psychopathology, they may lead to later recovery and long-term resilience. For example, reactivity in the DMN to trauma-related pictures two months post-trauma was negatively correlated with early PTSD resilience, but positively related to resilience 2 years later . ParticiThe attention network is involved in directing and maintaining perceptual resources as a function of task demands , 85. EmeThe cognitive control network is involved in working memory and selective attention , 87, 88.Alterations in the cognitive control network may also be related to its reciprocal projections with the thalamus. Traumatic events involving a head injury have been associated with decreased thalamus-frontoparietal control network FC , and FA Sensory areas showing associations with longitudinal resilience include the occipital cortex and fusiform gyrus, involved in vision, and precentral cortex, involved in relaying and regulating sensory input. Vision related activation within the first year post-trauma appears to negatively correlate with resilience. Peritraumatic dissociation, a known risk factor for later chronic PTSD, depression, and chronic pain –94, posiPotential resilience protective factors post-trauma include increased vmPFC structure, reactivity, and connectivity, larger hippocampal volumes and activation, decreased amygdala reactivity, greater salience network activation and structure, and greater reward circuit/orbitofrontal cortex GMV Fig. . A majorPeri-trauma and post-trauma studies to date are limited by the timing of scans, both in how early individuals are scanned post-trauma and later, longitudinal follow-ups. Neuroimaging studies face logistical constraints, in that most studies are unable to schedule individuals for a scan until weeks after a trauma occurs. In addition, most studies only evaluate trauma-related psychopathology or perform a second scan within the first year following trauma. This prevents evaluation of factors that may predict which individuals display long-term recovery and resilience compared to those with chronic trauma-related psychopathology. Studies that evaluate changes from peri-trauma to years post-trauma within these networks, and how they correlate with resilience over time, are necessary. Further, it is currently difficult to form conclusions regarding the influence of trauma type on findings, as many studies recruit individuals who have been exposed to all different kinds of trauma. While some studies are limited to motor vehicle collisions, a particular natural disaster, or military deployment, for example, the mix of time points that data is collected at and differences in neuroimaging data collected make it difficult to directly compare findings. Finally, while post-trauma longitudinal findings have been made within each of the described networks, the threat network and DMN have had the most findings, and the reward network the least. While the reported studies here reflect the importance of the threat network and DMN in particular in understanding resilience, they also elucidate gaps in the field regarding other network areas.Several PTSD symptom clusters have a common neural correlate pattern. Specifically, intrusions, alterations in cognition and mood and altered arousal and reactivity symptoms are often associated with deficits in corticolimbic inhibition . The finGiven these findings, it follows that less vmPFC or dACC engagement before or shortly after a traumatic experience might be protective against developing both chronic dissociation and posttraumatic stress symptoms. To our knowledge, this has yet to be directly tested. However, a recent longitudinal treatment study in adolescents supports this conclusion. Specifically, this study demonstrated that improvements in dissociation post treatment were associated with decreased dACC and amygdala activation during an emotionally provocative task .Taken together, some studies point toward more threat inhibition network activity and some point toward less as the profile of resilience. How do we begin to reconcile these seemingly opposite patterns of resilience? First, this evidence suggests there are different neural features at early timepoints that appear protective against different potential outcomes. More corticolimbic inhibition associated with threat inhibition and emotion regulation may protect against intrusions, alterations in mood and hyperarousal. However, too much corticolimbic inhibition may interfere with emotional learning , 105 andExposure to stress and traumatic events is thought to be particularly impactful during childhood, because of rapid brain changes that occur as part of brain maturation . NeuroimEarly stress and trauma seem to negatively impact threat and salience network structure and function. For example, early life stress predicted smaller bilateral hippocampal and amygdala volumes , with soWhile there is more developmental trauma research needed in networks other than threat and salience, a few findings have been published. For example, one observed that in a 10-year longitudinal study following children into adulthood, early stress exposure at the beginning of the study predicted less dlPFC engagement in error monitoring in adulthood , which iOne important consideration for longitudinal studies in children or adolescents is the timing of traumatic events and its interaction with developmental period. The brain does not develop linearly, instead, there are specific sensitive periods for maturation of different cortical and subcortical structures . The intA second challenge for developmental research aiming to define predictors for PTSD risk is the need for large cohort studies which prospectively assess neural structure and function and trauma exposures over development. Only one prospective study to date has collected MRI data prior to trauma exposure and onset of PTSD when the cohort happened to be exposed to the Boston marathon terrorist attack . LongituA final consideration for longitudinal developmental studies is related to the long-term consequences of stress and trauma and related brain alterations. It is postulated that traumatic events during development could shape brain structure and function to be adaptive in the current situation , but it Longitudinal neuroimaging studies of trauma and related mental health consequences are beginning to outline a consistent set of factors contributing to trauma resilience. The overall pattern across networks and timepoints is illustrated in Fig. These conclusions, however, must be tempered by the fact that this is a relatively new literature, with a number of limitations. Studies of the pre-trauma period are particularly sparse, with very small samples and power-related limits on more exploratory analyses. Future work in larger samples needs to address neural circuit function across the brain, looking at relationships between pre-trauma circuit-level connectivity and psychological outcomes post-trauma, as well as changes in these circuits following trauma. Such studies may very well reveal pre-trauma resilience factors outside the threat and salience networks. Additionally, biomarkers of resilience will only become useful in prognosis and precision medicine approaches with further work to establish measurement variability, reliability, and expected normative values. This has been a major stumbling block for efforts to integrate neuroscience and psychiatry, and must become a key priority area for further research.With these limitations in mind, we can leverage current findings to guide resilience-boosting interventions targeting different time windows and subgroups. For example, a recent study took a random selection of first-year college students to receive an intervention designed to increase their resilience through training in mindfulness, goal-building, and resilience skills . For thi


We conducted a systematic review and network meta-analysis to identify which human immunodeficiency virus (HIV) self-testing (HIVST) distribution strategies are most effective.We abstracted data from randomized controlled trials and observational studies published between 4 June 2006 and 4 June 2019.We included 33 studies, yielding 6 HIVST distribution strategies. All distribution strategies increased testing uptake compared to standard testing: in sub-Saharan Africa, partner HIVST distribution ranked highest (78% probability); in North America, Asia, and the Pacific regions, web-based distribution ranked highest (93% probability), and facility based distribution ranked second in all settings. Across HIVST distribution strategies HIV positivity and linkage was similar to standard testing.A range of HIVST distribution strategies are effective in increasing HIV testing. HIVST distribution by sexual partners, web-based distribution, as well as health facility distribution strategies should be considered for implementation to expand the reach of HIV testing services. We conducted a systematic review and network meta-analysis to assess effectiveness of human immunodeficiency virus self-test (HIVST) distribution strategies. HIVST distribution by sexual partners in sub-Saharan Africa and web-based in North America, Asia and the Pacific region resulted in the highest uptake. Knowledge of human immunodeficiency virus (HIV) status is the gateway to HIV treatment and prevention services. The gap in HIV testing and diagnosis remains a critical barrier to meet global goals, particularly for certain populations including men, young people and key populations. HIV self-testing (HIVST), a World Health Organization (WHO)-recommended HIV testing approach, has been shown to be safe, accurate, and acceptable , 2. It cHIVST is currently being scaled up globally alongside other HIV testing approaches including traditional (standard) rapid HIV testing conducted by healthcare workers (HCWs) or trained lay providers at health facilities or in the community , 5. HIVSNetwork meta-analyses offer a complementary methodology for comparing heterogenous implementation strategies: within networks, the effects of multiple interventions can be compared and direct comparisons can be used to generate indirect effect estimates; meta-regression can be utilized to account for heterogeneity, and distribution strategies can be ranked to identify which models are most effective . We therhttps://airtable.com/), reviewed by a second author, and discrepancies were resolved by a third author. Risk of bias in randomized controlled trials was assessed across 5 domains according to the Cochrane risk of bias tool [We searched MEDLINE, EMBASE, Cochrane, Web of Science, Global Health, Social Policy and Practice, Health Management Information Consortium, EBSCO, CINAHL Plus, Sociological Abstracts, and PsycINFO databases for randomized controlled trials (RCTs) conducted between 1 January 2006 and 4 June 2019, with additional searches of clinical trial registries, and major HIV conferences up until 31 July 2019 . Abstracias tool .We conducted and reported analyses according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines for pairwise and network meta-analyses (NMAs) , 12. We To address intransitivity in the network meta-analysis , 2 netwTo explore the heterogeneity of population types included in the network and the impact of this on network estimates, we conducted sensitivity analyses where female sex-workers were excluded  and conducted metaregression by gender group. The meta and gemtc packages in R programming software were used for all analyses .Searches yielded 14 254 citations of which 24 RCTs and 9 observational studies were included in the review . CharactNineteen studies were conducted in sub-Saharan Africa: Malawi , Kenya , South AAll RCTs were judged as high risk of bias primarily due to self-reported outcomes and lack of blinding of patients and study personnel, as well as lack of blinding of outcome assessors . The majSix direct comparisons contributed to this network meta-analysis , with thRanking probabilities  demonstrWe conducted meta-regression including population type as a covariate , After aEight studies and 3 HIVST distribution strategies contributed to the network meta-analysis of uptake of HIV testing  in NorthSix direct comparisons contributed to pairwise meta-analysis of the effect of distribution strategy on HIV positivity in Sub-Saharan Africa . DistribFive direct comparisons contributed to pairwise meta-analysis of the effect of HIV distribution strategy on HIV positivity in North America, Asia, and the Pacific region . All  resulted in the highest uptake of HIV testing compared to all other HIVST distribution strategies, including HCW facility-based, HCW community-based and secondary distribution by peers. Across North America, Asia, and the Pacific region, where studies primarily focused on MSM and TGW, web-based tools with subsequent mail delivery of HIVSTs showed the highest uptake compared to facility-based HIVST distribution by HCWs. HIVST distribution by HCWs at health facilities ranked second in all settings. In pairwise meta-analyses, we found that there was little or no difference in HIV positivity or linkage with HIVST distribution strategies compared to standard testing across regions and populations.Secondary distribution strategies by partners or peers can leverage existing sexual and social networks to access marginalized groups . The higIn North America, Asia, and the Pacific region, our review found that web-based mail delivery of HIVST to primarily MSM and TGW populations was commonly used and improved uptake of HIV testing. The success of this intervention suggests that convenience and confidentiality—which are identified desirable features of self tests , 6—are vWe found that even when offered by HCWs in healthcare facilities, HIVST can increase HIV testing. Although this approach is less focused on reaching underserved groups who do not routinely attend health facilities, the fact that this strategy showed better uptake than traditional health worker administered facility-based testing across a wide variety of population groups implies that in routine service delivery settings, providing the option of a different testing modality can increase testing and enhance reach among those who do attend health facilities but do not routinely test.The effects of community based HIVST distribution campaigns on HIV testing uptake were modest in comparison to other distribution strategies: 4 studies from Malawi and Zambia employed lay health workers and community volunteers to distribute HIVST in general communities; these strategies showed lower uptake than others, possibly indicating already well established HIV testing programs and high coverage of testing and treatment. These studies did, however, show benefits in frequently missed subgroups such as men and young people , 29, 53,The effects of individual distribution strategies on HIV positivity rates varied by study design, with all RCTs showing no difference in positivity rates between HIVST distribution strategies and traditional HIV testing, and cohort studies showing either no difference or higher positivity rates in a few instances. Similarly, compared to traditional HIV testing by HCWs, there appeared to be no difference in linkage between HIVST and traditional HIV testing by HCWs in the health facility or community.This analysis was limited by overall few studies contributing to each strategy, resulting in weak networks in the network meta-analysis and insufficient data to draw conclusions on optimum strategies for increasing HIV positivity rates and linkage in pairwise comparisons. In addition, we included unadjusted estimates from observational studies. Despite these limitations, the use of an NMA approach allowed for comparisons across strategies to increase uptake that were not directly assessed by pair-wise meta-analysis. Although NMAs have traditionally been applied to clinical drug efficacy trials, this analysis shows that the utility of these methods extends to implementation strategies if accompanied by careful examination of heterogeneity.In this review a range of HIVST distribution models were found to be effective in increasing HIV testing uptake and achieve positivity and linkage similar to standard HIV testing methods. Promising models include secondary distribution of HIV self-tests through sexual partners of HIV-positive individuals and clinic attendees in sub-Saharan Africa and web-based mail distribution to MSM in North America, Asia, and the Pacific region. Facility-based HIVST distribution may additionally be considered to improve efficiency and testing coverage in health facilities.Clinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at ciab029_suppl_Supplementary_MaterialsClick here for additional data file.