G protein-coupled receptors (GPCRs) and their associated proteins represent one of the most diverse cellular signaling systems involved in both physiological and pathophysiological processes. Aging represents perhaps the most complex biological process in humans and involves a progressive degradation of systemic integrity and physiological resilience. This is in part mediated by age-related aberrations in energy metabolism, mitochondrial function, protein folding and sorting, inflammatory activity and genomic stability. Indeed, an increased rate of unrepaired DNA damage is considered to be one of the ‘hallmarks’ of aging. Over the last two decades our appreciation of the complexity of GPCR signaling systems has expanded their functional signaling repertoire. One such example of this is the incipient role of GPCRs and GPCR-interacting proteins in DNA damage and repair mechanisms. Emerging data now suggest that GPCRs could function as stress sensors for intracellular damage, e.g., oxidative stress. Given this role of GPCRs in the DNA damage response process, coupled to the effective history of drug targeting of these receptors, this suggests that one important future activity of GPCR therapeutics is the rational control of DNA damage repair systems. With the knowledge gained about mechanisms underlying health and disease, as well as improved living standards and sanitization, there has been a major increase in the global average lifespan . The worTransmembrane heptahelical GPCRs represent perhaps the most studied and effective drug targets to date. Their near ubiquitous role in physiological processes, coupled to their capacity to recognize a wide diversity of impinging molecules, makes them ideal targets for pharmacotherapeutic design ,11. As aAs life proceeds through the individual’s aging process, both endogenous ) and environmental  stressors are constantly attacking DNA, causing structural damage . UnrepaiSystemic aging in humans is strongly associated with the accumulation of deleterious molecular perturbations that negatively affect the functionality of almost all cells, tissues and organs. This progressive and stochastic accumulation of molecular perturbations induces significant cellular signaling dysfunctions that affect multiple processes related to energy metabolism, cell survival, genomic instability  and aberrant cellular replication.The molecular control of the aging process has long been associated with the highly-conserved insulinotropic receptor signaling system. This metabolic system controls the effective uptake and metabolism of glucose as the primary energy source in the majority of higher organisms. The crucial role of this signaling system in aging has been evidenced by the demonstration of lifespan extension, in species ranging from nematodes to mice, by mutations affecting insulin receptor signaling ,24,25,26The inexorable generation of systemic metabolic dysfunction (linked to insulinotropic system aberration) induces a global imbalance between ROS and endogenous antioxidant pathways. This systemic perturbation results in an increased susceptibility of lipids, proteins and nucleic acids to oxidative radical attack and the creation of sustained oxidative damage . The HarStress-induced DNA damage is a routine process in cells; this damage can occur at the level of whole chromosome structures, as well as to exposed single- or double-strand entities. Chromosomal DNA stability is provided by nucleoprotein-DNA structures termed telomeres . MammaliFurthering the associations between metabolic dysfunction, aberrant DDR and advanced aging phenotypes, several classical DDR-associated diseases  are linked to dysglycemic states and insulin resistance ,68,69,70It has recently been demonstrated that the cellular clock and circadian rhythm are disrupted in the aging process . CircadiIn these initial sections (1 and 2), we have outlined how the seemingly impenetrably complex process of aging, with its strong association with DDR events, may be more effectively understood using signaling network-based concepts. Forming the first level of synergy between the GPCR and DDR systems, we have also detailed how the currently expanding range of GPCR signaling modalities also seems to operate at a network level. At the second level of GPCR-DDR synergy, we have also demonstrated that both of these systems interconnect via the observed metabolic dysfunctions in the aging process. At a third synergistic level, both GPCR and DDR systems, via the alteration of energy metabolism, conspire to accelerate aging pathologies via accumulated oxidative damage. In the final fourth level of GPCR-DDR synergy, we have shown that these two systems converge via their common roles in both circadian clock and metabolic regulation to create a coherent and pervasive role of GPCR-DDR functionality in the aging process. In the following sections, we shall further refine these observations and illustrate them with specific exemplary findings.The GPCR superfamily represents perhaps the most diverse group of transmembrane proteins in the human proteome . GPCRs h2-adrenergic receptor (β2AR), leads to increased DNA damage, p53 degradation and the promotion of apoptosis [2AR could be biased to signal through a more ‘non-β-arrestin’ signaling mode, DNA damage repair could be promoted. Implicit with the additional complexity of GPCR signaling repertoires, it has been demonstrated that these additional GPCR transduction mechanisms are facilitated and specified by the creation of stable multiprotein complexes with the receptor [GPCRs provide a simple, but highly flexible, mechanism to facilitate the signal transfer of the ‘message’ of the extracellular stimulator  to the intracellular milieu. Hence, the stimulated receptor entrains characteristic cell signaling cascade responses to generate a productive cellular response to the external input . These vpoptosis ,112. Thereceptor ,107. TheIn addition to the recent introduction of non-G protein signaling to the functional repertoire of GPCR activity, new theories associated with the enlarged variety and cellular spatial nature of receptor activity are redefining our future concepts of therapeutic development. From their initial discovery, GPCRs were classically considered to be only ligand responsive when expressed on the cell surface plasma membrane. In contrast to this plasma membrane expression, a large majority of the total cellular amount of receptor protein was thought to be held in a cytosolic ‘reserve’ as nascent GPCRs ready to replenish the ‘actively signaling’ plasma membrane forms. This classical view of receptor pharmacology is still valid, especially for rapid extracellular stimulator-based G protein activation. There is now considerable evidence however demonstrating that GPCRs can also signal from intracellular membranes such as endosomes, mitochondria, endoplasmic reticulum, Golgi apparatus and the nucleus ,114. ThiA considerable proportion of the global mechanistic process of aging is driven by a degradation of metabolic function resulting in elevated oxidative stress and DNA damage. In recent years, it has been demonstrated that there is a complex neuroendocrine control network of inter-connected GPCR systems that regulate ‘neurometabolic’ activity. This convergence of GPCR-based systems bridges the functional domains of endocrine and neuronal systems in health and disease ,119,120.Metabolically-driven aging is characterized by the accumulation of adverse changes in cells over time that attenuates global homeostatic energy control and augments the risk of developing nearly all diseases . In addiIn addition to telomeric degradation, additional stressors have been shown to engender cellular senescence, e.g., certain DNA lesions and ROS attack ,137, botWhile the aging process and the accumulation of age-related damage seem inevitable facts of metabolic life, the strong involvement of GPCR-associated signaling cascades at many levels of this process provides a potentially important and effective drug-based mechanism for amelioration and/or retardation of this process ,148,149.2AX lesions [The GPCR heptahelical core still remains the primary target of therapeutic drug development, but it is clear from considerable research that the functionality of this core transmembrane protein is heavily modulated by accessory protein interactions in addition to the standard G protein associations. These accessory protein interactions have been shown to control receptor dimerization, linkage to non-receptor signaling adaptors and associations with other complex receptor systems ,160,161. lesions  and enha lesions .Protein arginine methylation regulates diverse functions in eukaryotic cells, including gene expression, the DDR and circadian rhythms. Protein arginine methyltransferase 5 (PRMT5) has been shown to interact directly with and effect the methylation of the dopamine D2 receptor (D2R). This would therefore represent a potential new signaling pathway with which novel pharmacological agents could modulate GPCR signaling by changing the methylation status of key cell signaling associated with DDR responses . In addiThe chemokine receptor, CXCR4, has been strongly associated with the modulation of DDR-related activity and cell cycle control in the context of oncology. Small peptide antagonists, potentially acting via non-G protein-dependent signaling pathways, have been shown to possess anti-neoplastic activity via the activation of ‘mitotic catastrophe’, an event associated with a premature or inappropriate cellular entry into mitosis . The expThe primary metabolite lactate was originally considered to be a biomedical waste product of metabolism. Lactate has however been shown to possess important positive signaling roles, especially in the central nervous system (CNS). In the CNS, lactate is released by astrocytes in response to neuronal activation, after which it is taken up by neurons, oxidized to pyruvate and used for synthesizing acetyl-CoA to feed oxidative phosphorylation . The dis2AX, the phosphorylated form of histone 2AX [While a considerable degree of DNA damage can be induced during the aging process via ROS attack, the long-term exposure to solar ultraviolet radiation can also contribute to age-related genomic frailty. Recent research has shown that both melanocortin 1 (MC1R), as well as endothelin B (ENDBR) receptors play important roles in the constitutive regulation of melanocytes and their response to solar ultraviolet radiation . Ligand-tone 2AX .Emerging data have demonstrated the importance of maintaining effective aortic vascular compliance during the metabolic aging process . The the2ARs) promoted dephosphorylation of β-arrestin2 and its suppression of NF-kappaB (NF-κB) activation. NF-κB activation in response to UV-induced DNA damage is vital to maintain an effective DDR response [2AR, induces an Akt-1-mediated activation of the E3 ubiquitin ligase, MDM2. This β-arrestin-dependent activation of MDM2 promotes the direct binding of this ligase to p53, thus promoting its degradation resulting in a detrimental effect upon the integrity of DDR systems, finally leading to increases in nuclear γ-H2AX adducts [2AR, e.g., epinephrine and norepinephrine, to trigger GPCR-β-arrestin-mediated pro-DNA damage effects led to subsequent testing of these findings in murine models of stress, in which an elevated catecholamine drive would be present. Using an underwater trauma model of stress, Sood et al. [2AR, β-arrestin1 and p53 with MDM2, thus creating a pro-DNA damage state in the CNS. Reinforcing this finding, Hara et al. [2AR system; for example, a simple pro-DNA damage β-arrestin1-p53-MDM2 signaling paradigm has been demonstrated for the previously mentioned MC1R [Human β-arrestins comprise a small family of cytosolic proteins originally studied for their role in the desensitization and intracellular trafficking of GPCRs. Despite this humble beginning, the β-arrestins (β-arrestin1 (ARRB1) and β-arrestin2 (ARRB2)) have emerged as key regulators of multiple signaling pathways involved in aging. By acting as cellular scaffolding proteins that link vital signaling pathway entities to GPCRs, β-arrestins can exert homeostatic and ligand-responsive allostatic control of intermediary cell metabolic events and long-term cellular functionality . As mentresponse . Subsequ adducts . The appd et al.  found tha et al.  demonstrned MC1R . These dned MC1R .As we have discussed, stressful stimulation of GPCR systems, e.g., via circulating norepinephrine, can lead to pro-DNA damaging events. Therefore, the molecular mechanisms that control the sensitivity/activity of GPCRs may also be an important nexus for controlling age-related DNA damage. In response to ligand stimulation, the vast majority of GPCRs are reflexively ‘cut-off’ from generating further G protein-dependent signals via a ‘desensitization’ of the receptor. This tachyphylactic response is typically mediated by the phosphorylation of the receptor by heterologous desensitization (via second messenger-dependent protein kinases such as protein kinase A) and/or homologous desensitization (via a selective phosphorylation through a G protein-coupled receptor kinase (GRK)). Within seconds of receptor stimulation, these kinases phosphorylate serine and threonine residues within the intracellular domains of GPCRs, thereby uncoupling the receptors from heterotrimeric G proteins ,193,194.GRKs belong to a coherent family of associated proteins that all share at least a similar kinase activity. The GRK superfamily of related proteins can be subdivided into three main groups based on sequence homology: (i) rhodopsin kinase or visual GRK subfamily (GRK1 and GRK7); (ii) the β-adrenergic receptor kinases subfamily (GRK2/GRK3); (iii) the GRK4 subfamily . These kinases share certain characteristics, but are distinct enzymes with specific regulatory properties. GRK2, 3, 5 and 6 are ubiquitously expressed in mammalian tissues, whereas GRK1/7 (retina) and 4  demonstrate more tissue-specific expression patterns ,200,201.2AX lesion inclusion in central nervous cortex tissue in experimental mice [As previously mentioned, many GRK-interacting proteins mediate other significant signaling functions. One of these proteins that possesses an important role in controlling DDR is the GRK-interacting transcript 2 (GIT2). GIT2 is a widely-expressed ADP-ribosylation factor GTPase-activating protein (Arf-GAP) ,208,209.tal mice . In addital mice . This dotal mice . These dtal mice . The demtal mice ,14,213 ftal mice . In thistal mice . In GIT2tal mice . The theThe regulation of GPCR activity is highly complex and well controlled, with multiple layers of interconnected signaling pathways activated upon receptor stimulation that feedback to modulate receptor signaling. The most studied GPCR signal ‘conditioning’ mechanisms are mediated by GRKs and β-arrestins; however, an extra level of control is common to many GPCRs, as well, i.e., that exerted by the regulator of G protein-signaling (RGS) proteins . RGS proPresently, there are thought to be at least twenty canonical RGS protein versions found in mammals . These mIt is evident from the growing body of literature concerning the functional and effective intersections between the GPCR and DDR systems that future research into this paradigm will hopefully yield actionable therapeutic strategies to mitigate age-associated DNA damage and the age-related disorders this damage triggers. So far, we have shown that GPCRs themselves, β-arrestins, GRKs and their interacting proteins, as well as RGS proteins can play important regulatory roles in DNA-management processes. As we have stated before, however, the true functional spectrum of GPCR-system associated proteins, including ones likely to affect the stoichiometry of GPCR receptorsome structures, has yet to be conclusively mapped. Therefore, in this final section, we shall discuss the role(s) of other non-canonical GPCR-interacting factors that also control the functional intersection of GPCR and DDR signaling systems.The availability of cellular nutrients and prevalent metabolic energy levels are functionally detected by signaling mechanisms that involve the mTORC1  kinase. In response to the presence or absence of these stimuli, mTORC1 can control cell growth and viability. The cellular ability to maintain energy homeostasis is tightly linked to a cells’ capacity to maintain DNA integrity and stability. To this end, the catalytic activity of mTORC1 can be inhibited by the absence of sufficient nutrients or via the sensation of cellular stressors through the responsive overexpression of REDD1 (regulated in development and DNA damage responses) . REDD1 wFanconi anemia (FA) is a rare genetic disease resulting in impaired responses to DNA damage. Among FA patients, the majority develop cancer, most often acute myelogenous leukemia, and 90% develop bone marrow failure (the inability to produce blood cells) by the age of 40. Over two-thirds of FA patients present with congenital defects including: short stature; abnormalities of the skin, arms, head, eyes, kidneys, ears, developmental disabilities and infertility . FA is t®.The poly(ADP-ribose) polymerase (PARP1) protein is directly involved in the BER DDR pathway. PARP1 catalyzes the poly(ADP-ribosyl)ation of a number of acceptor proteins involved in the regulation of chromatin architecture, as well as DNA metabolism. This poly(ADP-ribosyl)ation tracks DNA damages and represents a crucial step in the sensory signaling pathway leading to the repair of DNA strand breaks ,234,235.Further to the role of PARP1 in receptor-mediated protection of CNS DNA, it has been shown in human neuronal cells that the aging keystone GIT2 forms active complexes with both PARP1 and PARP2 in response to DNA-damaging stress caused by cisplatin treatment or ionizing radiation. The interaction of GIT2 served to enhance the signaling activity of PARP1 in these cells and likely contributed to the DNA-protecting activity of the GIT2 protein .We have previously indicated that with respect to age-related cardiovascular pathophysiologies, the Ang II ligand-receptor system is an important player in this paradigm . HoweverIn recent years, ever stronger connections have been observed between the functional realms of GPCR and DDR systems. Both signaling systems comprise a highly important and organized set of interacting proteins that together connect and coordinate physiological responses to multiple stressors experienced during an organism’s lifespan. To illustrate this important functional intersection in an unbiased manner, we employed latent semantic analysis ,248 of bModel 1′ signaling. However, considerable emerging data suggest that actively signaling GPCRs are not solely associated with the plasma membrane. Instead, GPCR signaling can also emanate from various intracellular membrane structures and can display distinct signaling features such as diverse receptorsome structures, altered lipid environments or differential ‘stimulator’ sensitivities [Model 1) still holds true, it is now evident that GPCRs can be activated at intracellular membranes through intracellularly-synthesized stimulators, as well as membrane-permeable or even endocytosed receptor ligands [Model 2′ signaling. It is interesting to note that the majority of our receptor activation theories (as well as drug design strategies) have been entirely based upon the Model 1 concept. In addition, our molecular and structural appreciation of GPCR activation has also been driven from a Model 1-biased standpoint. One widely investigated aspect of Model 1 signaling is the well-characterized rhodopsin-like receptor transmembrane helix 3 Asp-Arg-Tyr (DRY) motif [Model 2 signaling? If so, then perhaps a re-adjustment of our concepts of receptor activation and ligand sensitivity will be important to potentially exploit the presence of these intracellular Model 2 receptors. A more thorough study of such Model 2 receptors may represent an important resource to identify potential GPCR sensors of damage that can then synergize productively with the DDR machinery to reduce age/metabolism-dependent DNA damage.With a more advanced understanding of the therapeutically-tractable points of intersection between these two systems, it might be possible to create a novel series of drug-based strategies rationally to regulate genomic stability and the aging process. These multifunctional GPCR-DDR controllers may likely demonstrate the capacity to retard the onset of major debilitating age-associated diseases. It has been demonstrated over several decades that perhaps the most effective mechanism of drug development lies in the exploitation of GPCR signaling systems. Research into the nuances of GPCR signaling have revealed the potential for new avenues of therapeutic discovery based on the selective regulation of GPCR signaling. Historically, some of the earliest theories of GPCR signaling considered that the receptor exists in a simple two-state equilibrium between “on” or “off” states distinguished by their ability to trigger downstream responses . Furthertivities ,256,257. ligands  to allow ligands . In thisY) motif  that is Y) motif . Does thTaken together, our aggregated findings suggest that multiple components of the GPCR signaling system can modulate the activity of signaling proteins directly or indirectly involved in DNA damage and/or repair. As such, GPCR signaling systems may represent multifunctional sensors for DNA damaging insults, and their rational exploitation via novel drug design may facilitate our ability to augment DNA repair processes therapeutically. Thus, GPCR systems may have long evolved side-by-side with emerging DDR systems to act as sensors, and ameliorative effectors, for intracellular DNA damage and age-related stresses.


Frail older adults are commonly prescribed antidepressants. Yet, there is little evidence to determine the efficacy and safety of antidepressants to treat depression with concomitant frailty. To better understand this issue, we examined the efficacy and safety of second-generation antidepressants for the treatment of older adults with depression and then considered implications for frailty.Due to the absence of therapeutic studies of frail older adults with depression, we conducted a systematic review and meta-analysis of double-blind, randomized controlled trials that compared antidepressants versus placebo for adults with depression, age 65 years or older. We searched PubMed/MEDLINE, Cochrane Library, reference lists from meta-analyses/studies, hand searches of publication lists, and related articles on PubMed. Outcomes included rates of response, remission, and adverse events. After evaluating the data, we applied a frailty-informed framework to consider how the evidence could be applied to frailty.n = 2704). Subjects had moderate to severe depression. For older adults with depression, there was no statistically significant difference in response or remission to second-generation antidepressants compared to placebo. Response occurred in 45.3% of subjects receiving an antidepressant compared to 40.5% receiving placebo . Remission occurred in 33.1% with antidepressant versus 31.3% with placebo  . There were more withdrawals due to adverse events with antidepressants, 13% versus 5.8% .Nine trials were included in the meta-analysis (Subjects in the meta-analysis did not have obvious characteristics of frailty. Using framework questions to consider the implications of frailty, we hypothesize that, like older adults, frail individuals with depression may not respond to antidepressants. Further, observational studies suggest that those who are frail may be less responsive to antidepressants compared to the non-frail. Given the vulnerability of frailty, adverse events may be more burdensome.Second-generation antidepressants have uncertain benefit for older adults with depression and cause more adverse events compared to placebo. Until further research clarifies benefit, careful consideration of antidepressant prescribing with frailty is warranted. Frailty is associated with an increased risk of being diagnosed with depression –3. FurthOur original goal was to assess the efficacy and safety of antidepressants for the treatment of frail older adults with depression but without dementia. However, we found no double-blind, randomized, controlled trials (DBRCTs) that specifically evaluated the use of antidepressants in the frail population. Thus, we evaluated the efficacy and safety of antidepressants for older adults with depression. Due to the convention that a person aged 65 years or older is often referred to as ‘elderly’, . We subsWe performed a systematic review and meta-analysis in accordance with PRISMA  guidelines .Funding was provided by a Knowledge Synthesis grant (FRA 2015-A-04) from the Canadian Frailty Network and the Nova Scotia Department of Health and Wellness, as well as in-kind contributions from Dalhousie University Continuing Professional Development, the Canadian Agency for Drugs and Technologies in Health (CADTH), and Sobeys National Pharmacy Group. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Due to the significant placebo response of antidepressants in clinical trials, we considered only DBRCTs of adults 65 years or older. We included studies of subjects who had been diagnosed with depression according to Diagnostic and Statistical Manual of Mental Disorders (DSM) criteria or achieving a pre-specified rating on a validated depression scale. We focused on second-generation antidepressants, as these are most commonly prescribed for older adults, and included trials of SSRIs, serotonin-norepinephrine reuptake inhibitors (SNRIs), and noradrenergic and specific serotonergic antidepressants (NaSSAs).We reviewed publications of DBRCTs, meta-analyses, and systematic reviews that provided response and remission data. As the response to antidepressants with dementia may differ from those without dementia,  we aimedWe searched PubMed/MEDLINE, Cochrane Library, and reference lists from meta-analyses/studies, hand searches of publication lists, and related articles featured on PubMed. We used the search terms (antidepressants AND depression) with specified limits of meta-analysis; systematic review; randomized controlled trial; humans; English language; and aged: 65+ years that were published between January 1, 1996 and March 1, 2016. Studies were downloaded and screened using Covidence online software . Two invThree members of the review team extracted and/or verified data from each study . Data was double checked by LM and discrepancies resolved through discussion. We used a Microsoft Excel™ extraction sheet to catalog information including: study purpose; design/year/setting; drug/comparator mean dose; participant numbers; trial duration; study withdrawals; inclusion/exclusion criteria; assessment procedures/rating scales; baseline demographics; statistical adjustments; study results for primary/secondary outcomes; adverse events; withdrawals; mortality; subgroup analyses; reviewer conclusions; and risk of bias criteria.Two authors  assessed study quality and risk of bias in individual studies using GRADE criteria  and Coch2 statistic.We used RevMan™ 5.3 to calculate pooled effect sizes and 95% confidence intervals using a random-effects model. Heterogeneity was assessed using the Ip ≤ 0.05 and calculated number needed to treat/number needed to harm (NNT/NNH) by applying the pooled estimate of relative risk (RR) to the pooled event rate in the placebo group.We used an alpha level for statistical significance of Primary outcomes were response and remission of depression. We defined response as a decrease of 50% or greater in depression scores or a Clinical Global Impression-Improvement (CGI-I) Scale score ≤ 2 at the final visit, which rates improvement in symptoms as “better” or “much better.” Remission was determined using the definitions in the included studies based on the following scores: Hamilton Depression Rating Scale (HDRS)-24 ≤ 10, HDRS-17 ≤ 7, or Montgomery-Åsberg Depression Rating Scale (MADRS) ≤ 11 or ≤ 12. If more than one scale was reported, we gave priority to HDRS, as was done in an earlier meta-analysis .We pooled data for specific adverse events and only analyzed adverse events for which there were sufficient data to conduct a meta-analysis of results, which included withdrawals due to adverse events, dry mouth, dizziness, constipation, headache, diarrhea, nausea, somnolence, insomnia, and fatigue.Summary results are presented using relative and absolute risk statistics and 95% confidence intervals. Concluding statements were derived from team consensus based on meta-analysis findings, assessment of study quality, and application to frailty based on the guiding questions of the framework.Initially, 1619 abstracts were identified. Of these, 1530 were excluded as they were either duplicates, reports that addressed other topics/populations, or not randomized controlled trials of second-generation antidepressants. Abstracts or published articles for the remaining 89 clinical trials were obtained and thoroughly reviewed. Of 89 potential trials, 80 were excluded for reasons listed in Fig. Nine DBRCTs met our inclusion criteria Table  14–22].–22.14–22p-values for multiple comparisons.Blinding and allocation concealment were often inadequately described. Five of the nine studies used intention-to-treat analysis (ITT), , 21, 22 All studies, except one small study,  were funThe mean age of participants ranged from 71 to 82 years . Remission occurred in 33.1% of those given an antidepressant versus 31.3% with placebo   or remission  with antidepressants compared to placebo.Since older adults with depression and concomitant dementia might have a different response to antidepressants compared to those without dementia,  we examip = 0.001; I2 = 63%; NNH 11, 95% CI:7–30). Seven studies (N = 2444 subjects) recorded withdrawals due to adverse events, which occurred more frequently with second-generation antidepressants compared to placebo, 13% versus 5.8% . Other adverse events occurring more frequently in the antidepressant group were fatigue ; constipation ; and dizziness .Reporting of adverse events was inconsistent. Nevertheless, we combined frequently reported events in the meta-analysis. The most common adverse event was nausea, with a rate of 14% for second-generation antidepressants and 6.8% for placebo  but not in the first 12 weeks .4.Have potential harms been sufficiently considered?In this meta-analysis, study duration ranged from 8 to 12 weeks, a reasonable timeframe to achieve benefit. However, none of the studies addressed the sustainability of response nor the likelihood of developing adverse effects as frailty increases over time. In one 12-week study that had a 12-week extension,  falls weSince the frail are vulnerable, medication adverse effects may impact their quality of life and health status to a greater extent compared to healthier adults. Thus, both potential risks and benefits related to treatment need to be equally considered in the context of frailty.5.Is there further evidence pertaining to frail populations?The harms reported in this meta-analysis appear to be minor. However, nausea, fatigue, constipation, and dizziness were more frequent with antidepressants compared to placebo, which may be burdensome for frail older adults who are less able to tolerate perturbations in health due to decreased reserve. Notably, rates of withdrawal due to adverse events in subjects receiving antidepressants was twice the rate for those receiving placebo. In addition, frail older adults with multiple co-morbidities are at risk of polypharmacy. As the number of medications increase, so too does the potential for adverse medication events related to antidepressants.Although frail patients may not be specifically enrolled in randomized controlled trials, other categories of evidence can shed light on frailty response. Here, we consider two sources: (1) examination of medical conditions that are significantly associated with frailty; and (2) observational studies.We deliberated on two medical conditions that are associated with high rates of frailty—heart failure and Parkinson’s disease.p < 0.001) [With heart failure, a systematic review and meta-analysis showed that the overall estimated prevalence of frailty was 44.5%   includedWe examined five meta-analyses of antidepressants for depression in Parkinson’s disease –53. ThreIn our meta-analysis, the two studies that enrolled subjects with depression and concomitant medical conditions, likewise, showed no statistically significant benefit from antidepressants , 15.2 = 8.3, df = 2, P = .016). Analogously, in a longitudinal study [P = .005). Finally, using data from the Nordic Research on Ageing (NORA), [Although randomized trials are the best way to determine medication efficacy, observational studies may suggest potential associations. Three observational studies support the hypothesis that those who are frail may be less responsive to antidepressants compared to the non-frail. A multi-site naturalistic prospective cohort study from the Netherlands of 378 subjects over age 60  found thal study  of 189 p (NORA),  Brown fon = 5). In contrast, when the Tedeschini meta-analysis employed an age threshold of > 55 years, a statistically significant benefit for antidepressants was found, which raised the possibility that there may be less response to antidepressants in later life, according to the authors. With the addition of four trials, [In our meta-analysis of older adults with depression, there was no statistically significant response or remission for second-generation antidepressants compared to placebo. Our results are similar to a meta-analysis by Tedeschini,  who also trials, , 18, 20 Our review highlights the absence of clinical trials for frail older adults with depression. Subjects enrolled in our meta-analysis of older adults did not have obvious characteristics of frailty. Yet, treatment with antidepressants appears to be common in the frail population, as evidenced by the high rate of antidepressant use among long-term care residents, where there is a high prevalence of frailty , 5.So, what can be theorized about the expected response to antidepressants for frail older adults with depression? If non-frail older adults with depression do not exhibit response or remission to antidepressants, frail adults would, likewise, not be expected to respond. In fact, preliminary data from longitudinal cohort studies imply that frail individuals respond less favorably to antidepressants compared to older adults who are not frail, which could be related to several factors. First, although major depression can co-exist with frailty, there is overlap between symptoms of depression and the characteristics of frailty, as described above , 57. An Frailty is associated with increased potential for adverse events –25. As sThis review has several limitations. The focus on adults above age 65 could be viewed as an arbitrary cut-off. However, 65 years is generally accepted as a transition into older age,  However,Use antidepressants judiciously, with awareness of the limited evidence for efficacy in adults age ≥ 65 years and, by association, older adults with frailty.Attentively consider the potential for adverse effects with frailty.Reflect on patient circumstances and carefully assess whether changes in mood could be situational and/or related to frailty; make a concerted effort to provide the kind of support that can benefit frail older adults, such as home and situational supports.Consider that, due to varied populations and heterogeneous response, antidepressants may be beneficial for some older adults who are frail.If antidepressants are trialed, adequate dose and treatment time should be assured before judging efficacy. On the other hand, continuing antidepressants when they are not effective contributes to polypharmacy. Therefore, when treatment is initiated, attentively assess treatment response with the aim of stopping medications when no benefit is observed. When making treatment decisions, consider the high placebo response.Routinely re-evaluate whether antidepressants should be continued when they are used for prolonged periods of time.Reconsider guidelines that recommend screening for depression in frail populations (such as in nursing homes), as this approach likely contributes to the over-diagnosis of depression and polypharmacy.Considering the possibility that a frail patient may have limited response to antidepressants and is more at risk for adverse events, we recommend the following:To conclude, many authors conjecture that “late-life depression remains underdiagnosed and inadequately treated.” , 61, 62 Our findings contribute to the growing discourse of uncertain clinical benefit from antidepressants and high risk of bias in antidepressant trials , 64. Som


Dear Editor,Deciphering the role of any protein in a cell requires knowledge of the structure, subcellular localization, and the topological orientation of the protein within the relevant cells. The Sigma-1 receptor (S1R) largely localized to the endoplasmic reticulum (ER) serves as a pluripotent intracellular signaling molecule with diverse roles in the cell including ion channel modulation, stress signaling, and transcriptional regulation  as our neuronal target where S1R is abundantly expressed Below is the link to the electronic supplementary material.


Ontogeny of higher organisms as well the regulation of tissue homeostasis in adult individuals requires a fine-balanced interplay of regulating factors that individually trigger the fate of particular cells to either stay undifferentiated or to differentiate towards distinct tissue specific lineages. In some cases, these factors act synergistically to promote certain cellular responses, whereas in other tissues the same factors antagonize each other. However, the molecular basis of this obvious dual signaling activity is still only poorly understood. Bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs) are two major signal protein families that have a lot in common: They are both highly preserved between different species, involved in essential cellular functions, and their ligands vastly outnumber their receptors, making extensive signal regulation necessary. In this review we discuss where and how BMP and FGF signaling cross paths. The compiled data reflect that both factors synchronously act in many tissues, and that antagonism and synergism both exist in a context-dependent manner. Therefore, by challenging a generalization of the connection between these two pathways a new chapter in BMP FGF signaling research will be introduced. Canonical signaling cascades activated by bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs) both play crucial parts in essential cell and tissue functions, from early development to repair, maintenance and homeostasis of adult tissue. Hence it is not very surprising that BMPs and FGFs mechanistically interact with each other, and that their mostly divergent roles often result in a functional antagonism. Investigating the BMP-FGF interplay hence touches on many areas of interest in research, and insights into the underlying molecular interactions may represent fundamental biological processes in complex organisms.The main focus of this review is on the inter-connections between BMP and FGF signaling. Their individual properties and their regulation will only be introduced shortly, as they have already been described in great detail elsewhere.  For Caenorhabditis elegans SMA  and Drosophila MAD (“Mothers Against Decapentaplegic” proteins) proteins. R-SMADs belong to one of two subgroups, and each type I receptor (with the exception of activin-like kinase 1 (ALK1)) activates SMADS of just one subgroup: Most BMP/GDF receptors phosphorylate SMADs 1, 5 and 8, whereas the TGFβ type I (ALK5) and the activin type I receptors phosphorylate SMADs 2 and 3. Activated R-SMADs form heterotrimers with one so-called common-mediator (Co-SMAD) SMAD4 protein, and translocate into the nucleus to exert their influence on gene expression with the help of co-factors and histone modifying enzymes. Apart from this canonical signaling pathway, BMP and TGFβ receptors have also been reported to activate other signaling proteins, including mitogen-activated protein kinases (MAPKs), such as p38 and Erk, as well as phosphoinositide 3-kinase (PI3K) [BMPs and growth and differentiation factors (GDFs) are dimeric growth factors forming a subgroup of the transforming growth factor β (TGFβ) family, with other subgroups, such as the eponymous “true” TGFβs (isotypes 1–3), the activins, and others like anti muellerian hormon (AMH) and GDF15 . An actie (PI3K) ,7.It appears plausible that a signaling system with high receptor promiscuity and limited downstream events like this one can only generate so many distinct functions if it is extensively regulated. Diverse regulatory mechanisms exist at any point in the signaling cascade. Intrinsically, receptor specificity and ligand concentration determine, such as active signaling complexes can form. A multitude of extracellular modulators, e.g., Noggin, Follistatin, Chordin, and others, prevent receptor-ligand interaction by sequestering ligands and masking their receptor-binding epitopes ,8, and sIn the adult organism, FGF signaling is active in most tissues and fulfills vital functions both in the cellular and in the tissue metabolism. Looking at the level of individual cells, FGFs are linked to proliferation, migration, and/or differentiation, which translates into the regulation of tissue homeostasis and repair . These mThe FGF family comprises 22 members, 18 of which get secreted and are able to bind to one or more of the four FGF receptors (FGFR) 1–4. FGF ligands are monomeric factors whose close association to extracellular matrix components, in particular to heparan sulfate proteoglycans (HSPGs), determine their auto- or paracrine nature. HSPGs not only limit the distribution of FGFs, but also act as stabilizers of ligand-binding to the respective receptors [FGF receptors are tyrosine kinases with a single transmembrane domain that dimerize and become autophosphorylated upon ligand binding. They exist as various isoforms derived from alternative splicing events, adding further complexity to the FGF signaling system. Once activated, FGFRs trigger four main pathways within the cell, comprising mitogen-activated protein (MAP) kinases, phosphoinositide 3-kinase (PI3K), signal transducers and activators of transcription (STATs) and phospholipase Cγ (PLCγ). The adaptor protein FGFR substrate 2α (FRS2α) directly binds to the FGFR and triggers signaling via MAP kinase and PI3K cascades via growth factor receptor-bound protein 2 (GRB2) . STATs 1As is the case in BMP signaling, various growth factor functions are governed via a small number of receptors. Hence, signal transduction has to be tightly and variably regulated. At the cell surface, the system gains plasticity by the high number of different FGF ligands, alternative splicing and glycosylation status of the FGF receptors, and the necessity for co-receptors, such as HPSGs or Klotho proteins, as has been mentioned above. FGF ligands also experience short half-lifes, due to free cysteines. Consequently, not every cell capable of FGF signaling can respond to every ligand, and signaling becomes cell-type specific ,13,14. RIntracellularly, most regulatory proteins only address one or two of the four activated pathways. For instance, binding of GRB14 to the activated FGFR1 only inhibits the activation of PLCγ. If in turn FRS2α and/or GRB2 are targeted, for example by ubiquitination via CBL (an E3 ubiquitin ligase) or by dephosphorylation via Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2), only the MAP and PI3 kinase pathways are impaired [Both BMP and FGF signaling systems contain a number of ligands that by far exceeds the number of their receptors, and both fulfill multiple functions in various tissues. Subsequently, both systems need to be extensively regulated and fine-tuned by distinct spatial and temporal expression patterns and a variety of regulatory proteins, as has been briefly addressed above.A graphical depiction of general pathway components of both pathways is shown below .We must remain aware that any biological effect observed is only the sum product of a whole concert of interaction partners, and that connections between single events within one of these pathways are not always as straightforward as we would like them to be. Receptor hyper-activation does not necessarily lead to an elevated biological response to its ligand ; highly Cell fate determination and axis formation are often the result of growth factor gradient formation, or the integration of different gradients, of morphogens, such as BMPs and FGFs. In this context, BMP-FGF antagonism has been described as “constituting a common module” , and exaGata6, a Nanog-suppressor) [Tbx6, as well as a high migratory potential in descendants of the primitive streak and thereby determines mesoderm, the origin of the musculoskeletal system and others [The central roles of TGFβ family members and FGFs in early development already become evident at the very first stages of cell fate determination. For example, once the fertilized egg develops into a hollow sphere (the blastocyst), the inner cell mass ICM (which gives rise to the embryo itself) can be distinguished from the outer cell layer (trophoblast). Pluripotency factors Oct4 and Nanog within the ICM induce FGF4 expression to elevate FGFR2-mediated MAPK signaling, which in turn leads to the ICM’s separation into the epiblast (expressing Nanog and FGF4) and the primitive endoderm (expressing FGFR2 and its downstream target pressor) . The epipressor) . FGF8-acd others .Apart from the three germ layers, the early embryo also develops three distinct axes, the anterioposterior (AP) one along which the germ layers originally form, the dorsoventral (DV) axis, and the left-right (L/R) axis. FGF signaling (most prominently the FGFs 4 and 8) and TGFβ/BMP signaling  are involved in the formation of all three axes: Along the AP axis, extraembryonal BMP2/4 signaling via the type I receptor BMPRIA helps to determine the posterior primitive streak within the embryonal tissue ,21. The The appendicular skeleton. The skeleton forms mainly from mesodermal tissue, although parts of the skull and teeth originate from ectodermal precursors. Signaling events in osteochondral development has been most extensively studied in the context of appendicular skeleton formation, which starts with the appearance of limb buds in the lateral plate mesoderm. Their outgrowth is driven by FGFs  and Gremlin-1, a BMP inhibitor. Distally, expression of BMPs signaling via BMPRIA  limits FGF signaling and limb bud outgrowth ,23,24,25Histologically, the formation of limb bones is described by a number of steps in a process called endochondral ossification: (1) Mesenchymal cell condensation, (2) chondrocyte proliferation, (3) column formation (pre-hypertrophy), (4) hypertrophy, and (5) vascularization and mineralization. While most chondrocytes in the skeletal anlagen become hypertrophic and are replaced by bone forming cells, there are two regions where cartilage persists after birth: The epiphysis or growth plate, whose chondrogenic core keeps proliferating and undergoing hypertrophy for the sake of longitudinal growth until early adulthood and the joints, where non-proliferative cartilage resides on top of the bones, flanking the joint space, avoiding hypertrophy through life . Twist2 promoter, which is active in early osteoblastic stages: Both gene deletions lead to the same phenotype, namely reduced hypertrophic zones [Understandably, the molecular regulatory processes governing these steps are quite complex, including the influence of a multitude of growth factors, such as BMPs and FGFs, as well as Ihh, Wnt ligands, parathyroid hormone related protein PTHrP, and TGFβs; which regulate key transcription factors like Sox9 (cartilage) and Runx2 (bone), as well as various homeobox genes ,27,28. Sic zones . In contic zones . Thus, iic zones ,39. NotaCranium. Some parts of the skull, including but not limited to the cranial base, develop via endochondral ossification as well. These structures are paraxial mesoderm-derived and are collectively referred to as the chondrocranium. As in long bones, there are cartilage sites that persist after birth to function in the same way as growth plates, called synchondroses, and the steps of bone formation, as well as the molecular regulation is highly similar to long bone and growth plate mechanisms ,40. Skull parts forming via membraneous ossification are neural crest-derived. They encompass the facial bones (viscerocranium) and the membraneous neurocranium, entailing most of the calvaria . To alloThe mechanisms involved in membraneous ossification, suture formation, and suture maintenance are quite similar if compared to later stages of endochondral ossification: For example, Runx2 is an essential driver of osteoblast differentiation in membraneous ossification as well , and mesAs BMPs and FGFs are quite ubiquitously expressed and involved in essential cell functions, such as proliferation and differentiation, they meet in many other tissues as well. Skeletal development, as described above, is a very prominent example, but far from being the only one.Teeth, another mineralized tissue, develop by close interaction of the oral epithelium and the underlying neural crest-derived mesenchyme. The integration of its four core signaling pathways, Wnts, hedgehogs, BMPs (mainly 2 and 4), and FGFs , is largely independent of the surrounding tissues . Due to Eyes and Skin. Other ectodermal tissues, such as skin and eyes, likewise rely on FGF-BMP interaction in their development. For example, the optical lens is induced both by FGF and BMP7 signaling  and bothSkin originates from ectodermal progenitors due to (Wnt-mediated) BMP4 signaling in antagonism with the FGFs that are linked to neural development. While both BMPs and FGFs continue to be expressed at the sites of epidermal and dermal development, they are not described as key players in skin maturation or skin cell differentiation ,55. Central Nervous System. For ectodermal progenitors, neural differentiation is the alternative route to epidermal differentiation, and according to the default model, it is largely driven by the absence of BMP4 signaling. This is enforced by a number of BMP inhibitors, such as Noggin, Follistatin and Chordin, rather than by the presence of an inductive agent ,57. WhilKidneys and Heart. On the mesodermal side, FGF-BMP interaction can be observed for example in nephrogenesis and cardiogenesis. In nephrogenesis, once the uteretic bud forms from the intermediate mesoderm, bud outgrowth and branching is initiated by FGF7 and FGF10 from the adjacent mesenchyme, signaling via FGFR2 expressed in the outgrowing tip. BMP4 is one of the negative regulators of this outgrowth. Down the road of kidney development, other members of these growth factor families, namely BMP7, FGF9 and FGF10 synergize in promoting the maintenance and proliferation of nephron progenitors . In the Lung and Gut. The gastrointestinal tract, encompassing thyroid, stomach, intestine, lung, liver, and pancreas, develops along the AP axis in close communication between mesoderm and endoderm. Among the key factors, FGFs and BMPs, expressed in the mesoderm, help to determine the endodermal cell fates along the axis . For exaIn order to investigate FGF- and/or BMP-related disorders, knock-in or knock-out models provide the basis to understand the specific role of these factors in the native developmental processes, as well as in maintenance of any given tissue.Mutations in FGF ligands and receptors affect a plethora of tissues in both mice and men. Due to the pleiotropic functions of FGFs, mutations in a single FGF family member can have widely differing consequences, ranging from early embryonic lethality over (severe) neuronal or cardiac deficiencies to short legs or long hair . In termIn contrast to FGF signaling derailing, mutations in BMP signaling components rarely affect cancer progression, as their physiological functions are mostly linked to differentiation processes or apoptosis, rather than proliferation or cell motility. A vast literature search by Skovrlj et al. yielded no evidence of BMP2 causing tumors de novo , althougJust like for FGFs, mutations in BMP ligands and their receptors have widely differing consequences, depending on the affected component and/or the specific mutation. Loss-of-function of central BMP ligands or receptors can result in (early) embryonic lethality, while non-lethal phenotypes range from severe and progressive disorders, such as pulmonary hypertension or fibrodysplasia ossificans progressiva, via eye malformations to irregularities in single finger joints .The wide range of phenotypes caused by members either of the FGF or the BMP family is quite startling. Based on their omnipresence and developmental importance, one would assume that mutations that affect one of them should not be compatible with life . However, the reason why most mutations in a single ligand or receptor cause non-lethal phenotypes or syndromes lies in their vast promiscuity and excessive regulation. These are also the reason why different mutations in the same protein can sometimes produce bafflingly distinct phenotypes. In FGFR3, a P250R mutation causes the closure of a specific cranial suture, sometimes accompanied by hand malformations and deafness, while an A391E mutation causes craniosynostosis accompanied by a skin condition, and a G380R mutation neither affects cranial sutures nor skin or hearing ability upon birth, but causes achondroplasia resulting in dwarfism (OMIM 134934). Even more discrete phenotypes arise upon specific mutations in the BMP ligand GDF5, sometimes only affecting one or few determined finger joints .While we do not understand why some specific mutations cause such specific disorders, the focus on shared phenotypes between FGF and BMP family members nonetheless helps to shed light on their molecular connections.Syndromes rooted in either BMP of FGF signaling mutations are mostly exclusive in their phenotypes, but do sometimes overlap in bone/cartilage malformations, such as craniosynostosis.Craniosynostosis is described to originate from enhanced bone formation rather than altered proliferation, as is the case in growth plate deficiencies ,82. WhilIn contrast to the midline sutures, the coronal suture is unique in its embryonic origin, emerging between the neural crest-derived frontal bone and mesodermal parietal bone. As mutations in the neural-crest related osteoinductive Twist1 or site-specific expression of a constitutive active BMP type I receptor (BMPRIA) in cranial neural crest both also cause premature suture closing ,84,87, tWitnessing the underlying mutations, it seems that both elevated BMP and/or elevated FGF signaling may cause premature suture closing. This indicates BMP-FGF synergism in the context of craniosynostosis. Indeed, it has been described that FGFR2 and FGF2 suppress the expression of the BMP inhibitor Noggin, whose expression is vital for patent cranial sutures. In turn, Noggin overexpression in skulls of healthy mice prevents natural suture fusion ; and appThe inter-connections between FGF and BMP signaling, which have been briefly mentioned in the preceded sections are summarized in With the BMP and FGF families having as many members and functions as they have, their molecular interaction with each other can hardly be generalized for all developmental stages and tissues. Most observations are context-dependent, and many examples of that have been detailed above. One is BMP2/FGF2 synergism in ossification, and underlying mechanisms entail influencing members of each other’s signaling pathway, as well as regulating shared target genes: BMP2 and FGF2 positively regulate each other’s expression in murine bone ,39, and Only a few interactions have been encountered multiple times and can be considered more general: For example, Erk and other MAP kinases are known to phosphorylate SMAD1/5 proteins in their linker region, hindering their translocation into the nucleus and promoting their degradation. The kinases JNK, p38 and Erk may also cause proteosomal degradation of SMAD4 ,99.All examples described here have in common that they study BMP-FGF connections in terms of their effectors , and/or in terms of transcriptional changes of selected genes. If and how BMPs and FGFs influence each other on the cell surface remains mainly hypothetical, although they share important binding partners in the extracellular matrix (ECM). Most FGFs and many (SMAD1/5/8-inducing) BMP ligands are paracrine factors because of their ECM association, and heparan sulfates are the mediators see .Heparan sulfates (HS) represent a heterogeneous group of sulfated disaccharide chains, anchored to the cell membrane or the extracellular matrix (ECM)  via HS proteoglycans (HSPGs) . As statIf interactions between members of the FGF and BMP families are as diverse and context-dependent as described above, how do we approach finding more general connections between them?To identify common mechanisms, combining models that reduce the number of influencing parameters seems to be a good start. In drosophila melanogaster, the growth factor families are much smaller, providing the complexity of an entire organism without that wide range of ligands and receptors. Cell culture models also provide a reduced set of ligands and receptors; and make spatiotemporal parameters both optional and controllable.50 values) for the induction of different BMP4 targets: In a cross-inhibitory positive feedback loop, un-specified intermediates in both FGF and BMP signaling cross-inhibit each other, affecting downstream targets in both signaling cascades in dependence of their inhibitory effectiveness, their abundance, and their influence on said targets [Further, mathematical models are a great tool to compare what is possible to what is observed. Before the nature of morphogens was even understood, such models already were potent tools to describe morphogen gradients as basis for the formation of complex anatomy . A more  targets .Certainly, there are examples where downstream components of both pathways are  antagonizing, as exemplified in the inactivation of R-SMADs by Erk-mediated linker-phosphorylation, or in the inhibition of bHLH factors downstream of FGF2 by ID proteins downstream of BMP4 . HoweverIn this context, it is noteworthy that canonical signal transduction via SMADs does not include signal amplification by repeated phosphorylation steps, as is the case in MAPK signaling. On the contrary, SMADs, the agents that both transport and execute the signal inside of the nucleus, are also the ones directly activated by the receptor. Combined with the fast and constitutive dephosphorylation of SMADs in activated cells, this results in a linear, proportional relation of receptor activation and SMAD phosphorylation. Thus, BMP ligand-induced receptor activation does not “switch on” a response that is amplified and hence stabilized inside the cell, as is the case for FGF ligand-induced signals, but rather elicits a readily reversible activated cell state in close relation to BMP receptor activity . These vAn interesting additional possibility is the integration at the cell surface, as has been discussed above. This area of interaction has hardly been explored, but there are indications that it would deserve to be. Surely, one indicator is the observation that heparan sulfates play such a pivotal role in gradient formation of both BMPs and FGFs that their influence on morphogen crosstalk is far more likely than not. Another is the recent description of muscle-specific kinase MuSK as a BMP co-receptor . MuSK is


The western blot measurement of apoptosis-protein and cancer stem cell (CSC) marked-protein demonstrated that UA led to activation-induced tumor cell death and Bmi1 siRNA resulted in inhibition of cancer stem cells. Overall, these results indicate that Bmi1 as a regulating gene for cancer stem cell is an effective target for cancer treatment using siRNA and co-delivery of UA and Bmi1 siRNA using folate-targeted liposomes is a promising strategy for improved anti-tumor effect.Overexpression of Bmi1 gene is an important feature of cancer stem cell in various human tumors. Therefore, Bmi1 gene can be a potential target for small interfering RNA (siRNA) mediated cancer therapy. Ursolic acid (UA) as a natural product plays a pivotal role in anti-tumor field, although its performance is limited by low bioavailability and poor hydrophilicity. A folate receptor-targeted cationic liposome system was designed for the purpose of investigating the relationship between Bmil siRNA and UA. The folate receptor-targeted cationic liposomes co-delivering UA and Bmi1 siRNA (FA-UA/siRNA-L) were fabricated by electrostatic interaction between folate UA liposome (FA-UA-L) and Bmi1 siRNA. Tumor growth is inhibited by FA-UA/siRNA-L  It is widely acknowledged that small interfering RNA (siRNA) plays a key role in gene therapy and can be potentially applied in clinical practice. Recently, the use of siRNA to down-regulate the expression of oncogenes has become an attractive strategy to inhibit the proliferation of tumor  targeting multidrug resistant (MDR) cancer cells enhanced the anti-tumor efficacy of DOX , a pentacyclic triterpenoid carboxylic acid, can be derived from many plants and constitutes the main components of some Chinese herbal medicine. Recently, there have been increasing interests in the biological functions of UA, such as anti-inflammation, anti-oxidative and anti-tumor activities . The purpose of this study was to explore the relationship between UA and Bmi1 siRNA and to detect the property of folate-target cationic liposome. UA was encapsulated into liposomes and its solubility was improved, Bmi1 siRNA was absorbed on the surface of liposomes, which enhanced the stability of the siRNA. In addition, functionalizing the liposomes with folate-targeted ligands increased the active tumor-targeting effect. The performance of FA-UA/siRNA-L was evaluated UA and folate-PEG-CHEMS were obtained from the pharmacy school of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China (purity >95%). 1, 2-dioleoyl-3-trimethylammonium-propane  (DOTAP) was purchased from Avanti Polar Lipids Inc. . Monomethoxy polyethylene glycol 2000-distearoyl phosphatidylethanolamine (MPEG-DSPE2000) was obtained from Lipoid GmbH (Germany). Cholesterol (CHOL) was purchased from Acros Organics. Calcein was obtained from Aladdin Chemistry Co. Ltd. Folic acid was purchased from Sinopharm Chemical Reagent Co., Ltd. RPMI 1640 media were purchased from Sigma-Aldrich Corp. . Methyl thiazolyl-diphenyl-tetrazolium bromide (MTT). Fetal bovine serum (FBS) was purchased from Zhejiang Tianhang Biological Technology Co., Ltd., Hangzhou, China. Female Kunming mice  and female Balb/c nude mice  were obtained from Beijing Huafukang Bioscience Technology Co.  2009-0004). Cy3-labeled Bmi1 siRNA were synthesized by Ribobio Co., Ltd . The Bmi1 siRNA sequences for mice were sense strand 5'-CCA GAC CAC UCC UGA ACA UTT-3' and anti-sense strand 5'-AUG UUC AGG AGU GGU CUG GTT-3', and for human were sense strand 5'-CCA GAC CAC UAC UGA AUA UAA-3' and anti-sense strand 5'-UUA UAU UCA GUA GUG GUC UGG UU-3'.2 incubators.KB and LO2 cells were purchased from the China Center for Type Culture Collection of Wuhan University . The cells were cultured with high glucose or DMEM folate-free RPMI 1640 medium with streptomycin, penicillin, and 10% FBS solution in 37 °C and 5% CO2O to form FA-UA/siRNA-L.The liposomes were prepared using thin-film dispersed hydration method according to the previous study  at room temperature for 10 min. The liposomes collected in the supernatant were lysed in methanol and the released UA was determined by HPLC with a UV wavelength of 210 nm. The entrapment efficiency was calculated as the ratio of amount of liposome-encapsulated UA to amount of UA initially added  conform to the specification. Each sample was detected 3 times at room temperature and the average values and standard deviations were calculated.Atomic Force Microscope (AFM) was used to determine the shape of liposomes. Mica was used to disperse the liposome solution at a volume of 5 μL. After 10 min incubation, the surface of mica was rinsed with DI water 4 times and dried with nitrogen. Then, the condensated samples were determined by Atomic Force Microscope .KB cells were seeded in 12-well plate with folate-free RPMI 1640 medium and cultured for 24 h. In order to block the folate receptor, 1 mM free folate was first added to the medium. After washing the KB cells grown in the plates with the medium for 3 times, Calcein/siRNA-L (Cy3 labeled) or FA- Calcein/siRNA-L (Cy3 labeled) was added and incubated with the cell for 1 h at 37 °C. Then, the KB cells were washed by PBS 5 times and 4% paraformaldehyde solution was used to fix the cells for 15 min. The cell nucleus was stained with 4,6-Diamidino-2-phenylindole (DAPI) solution and the uptake of FR-targeted liposomes was determined by a fluorescence microscope .To determine the gene silencing efficiency, the cells were transfected respectively with FA-UA/siRNA-L and UA/siRNA-L. The KB cells were collected after incubating for 48 h and washed with PBS 5 times. Then, total RNA extraction was applied with Trizol reagent. Reverse Transcription System  was used to synthesize cDNA. The CFX96 Real-Time PCR detection system with SYBR Green was used to measure Bmi1 mRNA.5 per well) were seeded in 4-well plate and treated with related liposomes and then incubated in CelLytic M Cell Lysis Reagent  for 30 min on ice. BCA assay was used to quantify and extract protein. The supernatant was collected after centrifugation at 12,000 rpm (Eppendorf 5415 D). Cell lysates were separated on a 10% polyacrylamide gel and transferred to a PVDF membrane. The membrane was blocked for 1 h in 5% skim milk and then incubated with monoclonal antibody against Bmi1 , CD133  , Caspase 9 , or tubulin  overnight. The membrane was then washed in TBST (PBS with 0.1% Tween-20) 3 times and then incubated for 1 h with secondary antibody. Then the membrane was washed 4 times and developed by an enhanced chemiluminescence system according to the manufacturer’s instructions .The KB cells  was added in the culture medium. After another 24 h incubation, the proportion of living cells were measured by the colorimetrical method according to the user’s manual.The cytotoxic effect of related formulations against the tumor cells was evaluated by MTT assay. KB cells were first seeded in 96-well plates at a density of 8 × 10To determine whether FA-UA/siRNA-L is stable in blood circulation, FA-UA/siRNA-L was incubated with serum  and transfected to KB cells for 1 h. Then, the KB cells were washed by PBS 5 times after the incubation and 4% paraformaldehyde solution was used to fix the cells for 15 min. The cell nucleus was stained with 4,6-Diamidino-2-phenylindole (DAPI) solution and detected by a fluorescence microscope.3 (the mice were fed with folate-free diet after the tumor implanted), saline, free UA, UA-L, FA-siRNA-L, FA-UA-L or FA-UA/siRNA-L was given intravenously through a tail vein five times every other day. The injection dose of each formulation was 4.5 mg/kg for UA and 170 μg/kg for siRNA. During the administration, the tumor volume and body weight of nude mice were measured regularly every 2 days. Tumor tissue and blood were collected after mice were sacrificed. Blood was used to detect serological markers  and tumor tissue was homogenized for H&E assay and western blot assay.Subcutaneous KB tumor-bearing balb/c nude mice  that obtained from Huafukang technology corporation  was used to evaluate the anti-tumor effect of FA-UA/siRNA-L. When the tumor size reaches to 50–100 mmCells were suspended in soft agar and growth medium in 6-well plates at a density of 5000 cells per well. After 2–3 weeks, colonies (6 cells per treatment) were counted under the microscope in 5 fields per well and photographed.t-test and one-way ANOVA with Dunnett’s post-test. Statistical significant difference was defined as *p < .05, **p < .01 and ***p < .001.GraphPad Prism 6 software was used to perform the statistical analysis. Multiple groups are compared by One-way ANOVA with Dunnett’s post-test. Comparison between groups was performed by Student’s Figure S1A), the chemical structure of folate ligands , siRNA sequence of Bmi1  and the chemical structure of UA  were shown in Figure S1. The folate ligand structure was determined by mass spectrum. The centered distribution curve was at 4166 Da, consistent with the calculated molecular weight of 4158 Da . These results revealed that the folate ligand was successfully added onto the liposomes.The schematic diagram of FA-UA/siRNA-L (Table S1). The image of AFM revealed that the shape and size of liposomes were uniform . This observation indicates that FA-UA/siRNA-L was stable in blood circulation and could be used further for in vivo anti-tumor study.Before the evaluation of anti-tumor effect in vivo, we determined the stability of FA-UA/siRNA-L in serum in vitro. FA-UA/siRNA-L was incubated with serum  and transfected to KB cells for 1 h. As shown in 3, each formulation was administrated 5 times on every other day by tail vein injection. The mice were sacrificed after 25 days and tumor tissue was collected for detection of volume and biochemical. From the result, we could see that free UA group (1300 mm3) and UA-L group (1156 mm3) showed smaller tumors after the mice sacrificed at 25 days compared with the saline group (2254 mm3) (3) and FA-siRNA-L group (1080 mm3) showed an even smaller tumor and stronger tumor growth inhibition (3) demonstrated very significant tumor growth inhibition compared to other groups (p = .03) between F-UA/siRNA-L and FA-UA-L group. These results suggested that co-delivery of UA and Bmi1 siRNA by folate ligand modified liposomes could enhance the anti-tumor effect.To evaluate the anti-tumor effect of FA-UA/siRNA-L, human KB tumor xenograft nude mice were used. Various formulations loaded with UA and/or Bmi1 siRNA were divided into 6 groups . The mice of each group had freedom of movement and were in good spirits. They had a good appetite for food and water, and excretion was normal. After the growth of subcutaneous tumor attained to around 50–100 mm254 mm3) . Tumor g254 mm3) . Comparehibition . The trer groups . As the th day. However, the weight resumed slowly after the injection of the 4th day. For further safety evaluations of liposomes, biochemical index of AST, ALT (hepatic function index), BUN and CR  were tested after injection of FA-UA/siRNA-L, FA-L, and saline. As shown in Figure S4A, both ALT and AST levels were significantly higher after injections of FA-L and FA-UA/siRNA-L injection compared to injection of saline. The AST/ALT ratios in the FA-L and FA-UA/siRNA-L groups were significantly lower compared to the saline group. These results suggest that the hepatic toxicity induced by liposomes was a mild and short-term effect. There were no significant differences in Blood Urea Nitrogen (BUN) and CR levels among the three groups, indicating low renal toxicity of liposomes . Then, heart, kidney and liver tissues were taken out for H&E staining and no significant toxicity was found after injection of FA-UA/siRNA-L . All these findings show that FA-UA/siRNA-L had little and reversible systematic toxicity.The body weight of mice was monitored to detect the systemic toxicity of FA-UA/siRNA-L. The mice were weighed every 4 days and the data was documented. As shown in Many reports have elaborated the role of Bmi1 as an important regulator connected to self-renewal and maintenance of stem cells in many tissues  in vivo and in vitro ). Anchorage-independent colony formation is a widely used method to evaluate Superior tumorigenesis or tumor initiating capability of CSC. Compared with non-siRNA treatment groups, siRNA treatment groups formed less colonies in soft agar, suggesting that Bmi1siR treatment significantly inhibited the colony formation ability of KB cells, indicating that the proliferation of CSCs were repressed .In the current study, we continued to apply this mature folate-targeted system and for the first time examined the synergistic anti-tumor effect of Bmi1 siRNA with native compounds (UA). FA-UA/siRNA-L showed the best anti-tumor effect Figure S7). Most studies of co-delivery systems have been designed for the purpose of enhancing the anti-tumor effect of first-line clinic chemotherapy drugs. The application of native compounds with potential anti-tumor effects has been rarely reported for combination with gene therapy. This study successfully combined gene therapy with native compounds and achieved the therapeutic goal.Although many reports suggested that the down-regulation of Bmi1 could block cell cycle and might cause apoptosis, these effects were far less impactful than tumor self-renewal  were developed and evaluated


There has been evidence demonstrating that China has had a persistently low and below-replacement level fertility since early 1990s, causing concerns of a rapidly aging population and sustainability of the Chinese economy. To avoid adverse effects of excessively low fertility, the Chinese government has recently changed its family planning policy from "one-child policy" to "two-child policy." Nonetheless, the effectiveness of the newly initiated two-child policy is questionable if women's average desired number of children or desired fertility for their lifetime is below the threshold fertility allowed by the two-child policy. Therefore, this study argues that it would be interesting and pertinent to know women's fertility desires under the circumstances of no policy restrictions and understand major factors that may affect their desired fertility. Based on a multi-stage stratified cluster sampling survey with 2,516 women respondents in rural Shaanxi, this study tries to estimate desired fertility of rural women and evaluate the impact of important socioeconomic factors on their desired fertility. The results of this study reveal that the average lifetime desired fertility for rural women of childbearing age in Shaanxi is about 1.71, below the total fertility rate at the replacement level. The findings of this study suggest that women's marriage age, the pecuniary costs of having children, women's income forgone for having children, and social security benefits available for rural residents at retirement age, are significantly and negatively related to desired fertility. However, rural women's cultural views towards fertility are significantly but positively related to their desired fertility. This study further confirms that China has entered an era of low fertility, and thus, any policy restrictions on fertility may no longer be necessary. Instead, government programs which support childbearing and childrearing are needed to prevent excessive low fertility and rapid aging of the population. Historically, China had a very high fertility rate due to its cultural tradition of family's obsession with male heirs to extend family lineage and endorsement of early marriage, resulting in a huge and increasing population size . Shortlywan-xi-shao) campaign, advocating later marriage, longer spacing, and fewer children ).Other studies have contended that social norms or cultural values are influential to regulate fertility, and they are thus used to explain the difference of fertility behavior among societies e.g., –35). Non. Non35])In order to understand individuals' incentives and constraints in fertility decisions, Becker's seminal contribution to economic analysis of fertility  and subsOn the cost side of having children, there are direct and indirect costs of childbearing and childrearing. The direct costs include monetary expenditure on both healthcare of the mother and the child during childbearing and children’s cost of living  before a certain age. The indirect cost of children generally refers to the "opportunity costs" of childbearing and childrearing, mainly including women's education and income forgone through time spent on both childbearing and childrearing .On the benefit or return side, there are also direct and indirect utilities of having children. Viewing children as consumption goods, having children, like the consumption of many other goods, directly increases the utility of parents . ChildreHowever, it has been argued that the direct economic utility of having children is often low and the indirect utility is usually embedded in social beliefs, for example, "marriage is for procreation" or that "children make a marriage" , 43. GivWith such an approach, various socioeconomic forces that may influence individual's desired fertility or actual choice of fertility can be classified into two categories: factors that affect the costs of having children and factors that represent the benefits of having children. Of course, the applicability of economic theory and approaches, as Werding has pointed out, is based on if choice is technically possible and if required freedom of choice is permitted . TherefoFor thousands of years, China was largely an agricultural society. Its large scale industrialization and urbanization is only a recent phenomenon. As China’s industrialization process started to accelerate over the last couple of decades, the weight of economic consideration on an individual's fertility decision has significantly increased due to tremendous changes in socioeconomic contexts.Before the economic reform in 1978, China was notably less developed. Although there was no clear policy restriction on individuals’ fertility decisions before early 1970s, the knowledge of modern means of fertility control were limited and they were not widely available to individuals in a context of nationwide poverty and economic crises. Even if couples exercised conscious planning on their family size, their desired fertility may not be realized due to lack of effective contraceptive methods.Significant changes in the context of fertility occurred in 1979 when the one-child policy was introduced. Under the policy, couples in China did not have the freedom of choice in terms of fertility decision. However, various modern means of contraception, including intrauterine device (IUD) insertions for women, sterilization, and abortion, have become widely available since the introduction of the one-child policy . Now, wiFirstly, policy restriction on fertility has driven up both the direct cost and indirect cost of children. Since the enforcement of the one-child policy in China, most urban couples only have one child and rural couples, on average, have about two children. Families with fewer children pay closer attention to not only the quality of children but also the personal safety of them , 46. ConSecondly, the rapid industrialization and economic development provides women with much more opportunities for employment and education, thus increasing the opportunity costs of having children. In late 1970s, at the beginning of the economic reform, rural residents mainly worked in small-scale farming. As industrialization and urbanization accelerated, many rural residents started to work in other industries and earn a much higher wage than in the agriculture sector , with inyang-er-fang-lao). Such a belief has changed in urban China because urban residents have been receiving pension from either the state-run enterprises or collective welfare programs for a few decades. As Cheng & Maxim pointed out, urban pension programs have been one of the most important factors causing the decline of China's urban fertility )Hypothesis 3: women's level of education is either positively or negatively related to their desired fertility.It refers to the total number of siblings of a woman respondent and her husband. Studies have found that parental fertility positively affects children’s fertility e.g. –64) and  and 64])Hypothesis 4: number of siblings is positively related to women's desired fertility.In existing literature, scholars have argued that the relationship between income and number of children could be either positive or negative. From the point of view that individuals could obtain direct pleasure from having and raising children , the posHypothesis 5: women's income is negatively related to their desired fertility.In the economic theory of fertility, the costs of children are argued to be one of the major factors that affect the demand for children . EmpiricHypothesis 6: direct costs of child are negatively related to women's desired fertility.It refers to women's views on traditional culture towards fertility. In traditional culture, social beliefs or norms towards fertility are generally considered an important factor on peoples’ fertility decision . As mentHypothesis 7: women who are more influenced by traditional cultural beliefs or social norms towards fertility have a higher desired fertility.It means the social security coverage available to the elderly in survey regions. In societies in which there is no social security program to cover the elderly at advanced age, individuals may depend mainly on their grown-up children to provide them with financial support and personal care . NonetheHypothesis 8: social security coverage for the elderly is negatively related to women’s desired fertility.st of May to 31st of October in 2015. The targeted population was married women of childbearing age (20–49) with a rural household identification (nong-cun-hu-kou) in rural Shaanxi during the survey. Shaanxi is geographically and culturally divided into 3 areas, North Shaanxi (shan-bei), Guan-zhong Plain (guan-zhong), and South Shaanxi (shan-nan) ): an100 Yuan increase in social security payment would lead to 0.37 reduction of desired fertility on average.P<0.05), and of those moderately influenced is about 1.165 times that of the weakly influenced . Although traditional cultural influence is still statistically significant and positively related to women’s desired fertility in the multivariate analysis, we would like to point out that the IRR values for both the deeply influenced and the weakly influenced were greatly reduced when we move from the univariate analysis to the multivariate analysis.The results of the multivariate analysis also suggest that the degree of traditional cultural influence on women has a significant impact on desired fertility. The desired fertility of those deeply influenced is 1.183 times that of the weakly influenced  and their impacts on the effects of other three variables , we estimated three additional models by adding three key economic variables to the multivariate regression model with age, age at first marriage, education, number of siblings, and traditional culture one by one (results not shown). Our results demonstrate that the results of three key variables remain almost unchanged, that the significances of both “age” and “education” were mostly absorbed by these two key variables “social security” and “direct cost of child”, and that the reduction of the influence of traditional culture on desired fertility was due to adding women’s income, direct costs of child and social security coverage for the elderly together.Historically, rural women had a much higher fertility rate in China and rural fertility was the main source for the increase of Chinese population, even in the period of stringent family planning policy to purposely control population growth. Nevertheless, this study has found that on average, rural women's desired fertility in Shaanxi, China in 2015 is only about 1.71, assuming no policy restriction on fertility. Such a number is not only below the replacement fertility level of 2.1, but also below 2.01, the average desired fertility reported by Mo's earlier study based on a nationwide survey of rural women in 2002 in China , and 1.8In the future, as the Chinese economy further develops, rural women would be more and more present in the labor market and have higher income, more pension benefits and increased costs of having children. That is, these three forces that are negatively related to women’s desired fertility would be on a rise. At the same time, as older generation of women exceeds childbearing age, women in childbearing age would have less siblings and weaker influence of traditional culture towards fertility. Thus, the two forces that are positively related to women’s desired fertility would wither in the future. If such a trend continues, there would be a further decline in fertility in rural Shaanxi. Our empirical evidence along with other national and regional studies e.g., , 74, 75), 7574, 7Beside its implications to policy-making, we would like to acknowledge that the findings of this study are based on the survey in Shaanxi province alone and cannot be used to make a broad generalization to the national level. Nonetheless, the results of this study may represent a general trend in desired fertility in rural China since the country as a whole shares a centralized policy-making in economic development and family planning, and has a similar pattern of social economic changes. It is expected that what is happening now in Shaanxi with regarding to rural fertility may have occurred in more developed regions such as coastal provinces and would be happening in less developed areas than Shaanxi. For the purpose of making comparisons between provinces in China, to the best of our knowledge, very few studies have been found on socioeconomic determinants of rural fertility in other provinces or at the national level. It is necessary to conduct new studies to understand more about fertility change in China.


Mobile edge computing is proposed as a promising computing paradigm to relieve the excessive burden of data centers and mobile networks, which is induced by the rapid growth of Internet of Things (IoT). This work introduces the cloud-assisted multi-cloudlet framework to provision scalable services in cloudlet-based mobile edge computing. Due to the constrained computation resources of cloudlets and limited communication resources of wireless access points (APs), IoT sensors with identical computation offloading decisions interact with each other. To optimize the processing delay and energy consumption of computation tasks, theoretic analysis of the computation offloading decision problem of IoT sensors is presented in this paper. In more detail, the computation offloading decision problem of IoT sensors is formulated as a computation offloading game and the condition of Nash equilibrium is derived by introducing the tool of a potential game. By exploiting the finite improvement property of the game, the Computation Offloading Decision (COD) algorithm is designed to provide decentralized computation offloading strategies for IoT sensors. Simulation results demonstrate that the COD algorithm can significantly reduce the system cost compared with the random-selection algorithm and the cloud-first algorithm. Furthermore, the COD algorithm can scale well with increasing IoT sensors. With the rapid emergence and sharp growth of Internet of Things (IoT), enormous computation and communication requirements are induced, exceeding the capacity of current data centers and mobile networks ,2,3,4. MIn this paper, the cloud-assisted multi-cloudlet framework is introduced, as shown in This paper investigates the computation offloading decision problem of IoT sensors in the cloud-assisted multi-cloudlet framework. Many existing works have focused on computation offloading problems, including computation offloading problems of mobile devices ,21,22,23In this work, the computation offloading decision problem of IoT sensors is analyzed based on game theory. In more detail, the computation offloading decision problem of IoT sensors is formulated as a computation offloading game. To analyze the property of this game, the tool of potential game is introduced. Based on the definition of potential game, the condition of Nash equilibrium is derived, and the Computation Offloading Decision (COD) algorithm is further designed by leveraging the finite improvement property of potential game. In the COD algorithm, the improvement range is optimized in each improvement iteration, such that the computation complexity of the algorithm is significantly reduced. We implement extensive simulations to evaluate the COD algorithm. Simulation results demonstrate that the COD algorithm can effectively reduce the system cost , compared with the random-selection algorithm and the cloud-first algorithm. Furthermore, the COD algorithm can scale well with increasing IoT sensors.This paper is organized as follows. K cloudlets, to which IoT sensors can offload the computation tasks through wireless access points (APs). Since processing delay and energy consumption are two important factors that influence the performance of IoT sensors, this work considers these two issues as the metrics of QoS. In the cloudlet-based mobile edge computing framework, computation resources of each cloudlet are limited. In addition, IoT sensors connected to the identical wireless APs interfere with each other. Therefore, the performance of one sensor can be affected by the other sensors with identical computation offloading decisions. As both the computation process and the communication process play a significant role in computation offloading of IoT sensors, analysis of these two processes is provided as follows.This section presents the system model. As shown in n has a computation task K cloudlets, then n is offloaded to cloudlet i, and n offloads the computation task to the cloud. Denote by n.In the cloud-assisted cloudlet-based mobile edge computing framework, each IoT sensor n can obtain when offloading computation task to the cloudlet. Then, the computation rate of sensor n can be represented asAs scalable computation resources can be provided by the cloud, when the computation task is offloaded to the cloud, the computation rate of each sensor can be guaranteed, denoted as n isThus, the computation delay of sensor n can achieve when capturing this channel, and As shown in n can be summarized asAs computation and communication resources of cloudlets are constrained, when cloudlets have dense population, IoT sensors can connect to the Internet core via the base station and further offload computation tasks to the cloud. With the development of cellular network, especially the rapid emergence and deployment of 5G network, sufficient spectrum is provided and extreme low latency is guaranteed. Thus, interference between IoT sensors are not considered when transmitting via the base station. According to the above analysis, the communication delay of sensor n in the cellular network, and Here, Processing delay and energy consumption are two important QoS metrics of mobile applications. Processing delay consists of computation delay and communication delay, which is given asscanning energy transmission energy maintaining energy During the computation offloading of IoT sensors, energy consumption only contains the energy consumed to offload computation tasks to cloudlets or to the cloud. When offloading computation tasks to cloudlets, IoT sensors transmit data blocks via wireless APs. This work adopts the energy model similar to the energy model in . The eneScanning energy n. Therefore, the energy consumption when offloading to cloudlets  algorithmInput:   Computation tasks of sensors Output:   Computation offloading strategy of sensors Initialization      Each sensor initially offloads its computation task to the cloud, 1:n when offloading the computation task to the cloud, Compute the cost of each sensor Equation .2:for each improvement iteration tdo3:for each sensor do 4:n when offloading the computation task to each cloudlet according to   obtain the computation and communication resources allocated to sensor 5:  compute the better strategy set quations  and 14)ρnt based6:  choose the improvement decision 7:end for 8:if the better strategy set of all sensors then 9:n with   each sensor 10:if sensor n wins the contention then  11:   12:end if  13:else 14:break;  15:end if 16:end forIt is indicated in Definition 2 that the potential function decreases when each player changes its strategy to reduce its cost function. In addition, the computation offloading game of IoT sensors has a finite improvement property. As in each improvement iteration sensors with larger N IoT sensors, each of which has a computation task In this section, extensive simulations are conducted to evaluate the COD algorithm. Consider the simulation scenario in which there are ation in  as the e 2.4 GHz . The upl 2.4 GHz ,34. The  2.4 GHz , as showThe performance of the COD algorithm is compared with the random-selection algorithm and the cloud-first algorithm. In the random-selection algorithm, each IoT sensor randomly selects a computation offloading decision among the cloudlets and the cloud. In the cloud-first algorithm, all IoT sensors offload the computation tasks to the cloud.Defining the system cost as the average cost of all IoT sensors,As shown in When the sensor number is small, the cloudlets can provide sufficient computation resources and the wireless APs have abundant communication resources, offloading computation tasks to cloudlets yields lower costs than offloading to the cloud. In the COD algorithm and the random-selection algorithm, part of IoT sensors offload computation tasks to cloudlets, thus the system costs are lower than the cloud-first algorithm. As sensor number increases, the computation resources of cloudlets and the communication resources of wireless APs become scarce. The system costs of the COD algorithm and the random-selection algorithm increase rapidly with increasing sensor number. In the COD algorithm, the IoT sensor with the largest improvement range is endowed with the highest priority to update the computation offloading strategy in each iteration. Thus, the system cost of the COD algorithm increases slower than the random-selection algorithm, and finally approaches the result of the cloud-first algorithm.NK). Therefore, the computation complexity of the COD algorithm approaches O(N2K), and the COD algorithm scales well with increasing sensor number.To evaluate the scalability of the COD algorithm, simulations are further implemented to record the number of improvement iterations with different sensor numbers. The results are shown in n = 100). The results are illustrated as This part evaluates the influence of different parameters on the results of the COD algorithm. Simulations are conducted to record the number of sensors offloading to the cloud , thus this algorithm can scale well with increasing IoT sensors.This work has investigated the computation offloading decision problem of IoT sensors in the cloud-assisted multi-cloudlet framework. Theoretic analysis has been presented and the condition of Nash equilibrium has been derived based on the potential game. By exploiting the finite property of the computation offloading game of IoT sensors, the COD algorithm has been proposed. Simulation results have demonstrated that the COD algorithm can effectively reduce the system cost compared with the random-selection algorithm and the cloud-first algorithm. In addition, the complexity of the COD algorithm is O(