Regiella insecticola on the life table parameters of Sitobion avenae. The results showed that R. insecticola can decrease the intrinsic rate of increase (r), the finite rate of increase (λ) and birth rate and can increase the mean generation times (T) of S. avenae clones, suggesting that R. insecticola may decelerate the normal development of the hosts. No significant differences of these parameters were observed between the examined Sitobion avenae clones, and the symbiont treatment by genotype interaction affected only the net reproduction rate R0, pre-adult duration and total longevity but not the other parameters. Additionally, a population projection showed that R. insecticola decelerated the growth of the S. avenae clones. The evocable effects of R. insecticola on the S. avenae clones may have significant ramifications for the control of S. avenae populations under field/natural conditions.Many insects harbor heritable endosymbionts, whether obligatory or facultative, and the role of facultative endosymbionts in shaping the phenotype of these species has become increasingly important. However, little is known about whether micro-injected endosymbionts can have any effects on aphid clones, which was measured using various ecological parameters. We examined the effects between symbiotic treatments and the vital life history traits generated by  Buchnera aphidicola, which belongs to the class γ-proteobacterium. B. aphidicola synthesizes essential amino acids and other nutrients that are ingested in very small quantities from restricted diets, such as plant phloem, for their host aphids23Regiella insecticola, Hamiltonella defensa and Serratia symbiotica belong to the class γ-proteobacterium, and Rickettsia and Spiroplasma belong to the class α-proteobacterium56789101112Many insect species harbor endosymbionts, which are indispensable to their survival and common reproduction. Because the hosts and their endosymbionts share a common fate, endosymbionts can often profoundly affect the ecology and evolution of their hosts. Among them, the endosymbionts in aphids are ideal targets for studying their role in ecological fitnessAcyrthosiphon pisum (Harris), including resistance to heat shock . However, a key component of this knowledge is the life table. The life table analysis is the most reliable method for evaluating the survival and reproduction of a populationr) represents the reproductive potential of a population in relation to its capacity to increaseR0) has been used to summarize the mean number of offspring that an individual can produce during its lifetime, with the mean generation time (T) defined as the period that a population needs to increase its size R0-foldBuchnera, on which aphid performance and fecundity dependsR. insecticola on hosts, it is necessary to obtain S. avenae clones with R. insecticola inoculated via micro-injection, which would have the same genetic background as symbiont-free aphids. Additionally, different aphid species or different genetic backgrounds within one species in the field can harbor various endosymbionts, and it is not clear whether the genotypes of the hosts could impact the ecological effects of endosymbionts. Therefore, the study of the interactive effects between micro-injected endosymbionts and the genotypes of the hosts and the ecological effects of endosymbionts on the aphid population is warranted.Secondary endosymbionts do not present in all aphid individuals, suggesting they are not generally required for the survival and reproduction of aphids. However, endosymbionts can significantly affect host aphid ecological fitness and behavior. In the past few decades, secondary endosymbionts have been found to confer benefits on the pea aphid R. insecticola on different S. avenae clones based on the life table data. The results that we present here might provide new insight for the future systematic study of endosymbionts and its ramifications in suppressing S. avenae populations.The objectives of the study are to investigate the ecological effects of R. insecticola significantly impacted the population parameters . Clone nested in ‘R. insecticola’ had no impact on r (P = 0.091), T (P = 0.236) and λ (P = 0.090), but significantly impacted R0(P = 0.001).A two-way nested analysis of variance was used to analyze the effect of endosymbionts on the population parameters in the different genotypes of aphid host . R. inseF = 27.418, P < 0.001 and F = 28.216, P < 0.001 for r and λ, respectively) than the controls Linyi-NA (Linyi clone) and Neixiang-NA (Neixiang clone). There were no differences between the treatments  . However, the mean generation times (T) of treatments Linyi-1, Linyi-2, Neixiang-1 and Neixiang-2 were significantly longer  than that of the controls Linyi-NA and Neixiang-NA, and no differences were found between the treatments  (P = 0.119).The population parameters of these original aphid clones (controls) were compared to those corresponding to infected clones (treatments) . Body flR. insecticola clearly affected the birth rate (P < 0.001), pre-adult duration (P < 0.001) and total longevity (P = 0.001). Clone nested in ‘R. insecticola’ had no impact on birth rate (P = 0.091) but had a significant impact on pre-adult duration and total longevity .A two-way nested analysis of variance was used to analyze the effect of endosymbionts on fitness traits in the different genotypes of the aphid host . R. inseR. insecticola had a remarkable effect on birth rate, and birth rates in the treatments Linyi-1, Linyi-2, Neixiang-1 and Neixiang-2 were significantly lower  than that in the controls Linyi-NA and Neixiang-NA. No differences in birth rate were found between the treatments  (P = 0.163). The pre-adult duration of the treatments Linyi-1, Linyi-2, Neixiang-1 and Neixiang-2  were clearly longer than that of the controls Linyi-NA (7.03 ± 0.126) and Neixiang-NA (6.62 ± 0.950). In addition, the total longevity of the treatments Linyi-1, Linyi-2, Neixiang-1 and Neixiang-2  were clearly higher than that of the control Linyi-NA (22.29 ± 0.756).Fitness traits  of the controls clones were compared to those corresponding to infected clones (treatments) . R. insest instar nymphs, are shown in th, 15th, 16th and 16th day, respectively, and the corresponding controls Linyi-NA and Neixiang-NA both occurred on the 14th day. This showed that the outbreak times of the treatments were delayed compared to those of the controls, and it suggested that R. insecticola might postpone the timing of the adult populations in the studied S. avenae clones.The simulated population growth and stage structure of the adult stages for 30 days, beginning with an initial population of 10 pairs of 1A. pisum, Aphis craccivora and Myzus persicae) have been extensively studied in recent decades56789183132333435H. defensa can protect A. pisum from its natural enemies11618et al.H. defensa has little effect on the resistance to the natural enemies in S. avenae, indicating that secondary endosymbionts might, to some extent, have different effects on related species. Therefore, it is necessary to study the effects of the secondary symbionts on different aphid clones using the life table.The diverse infections and functions of secondary endosymbionts of aphids , though the interaction did affect the net reproductive rate (R0), pre-adult duration and total longevity. For a comprehensive understanding of the treatment by genotype interaction, more studies are needed.A linear two-way nested analysis of variance (ANOVA) showed that the treatment by genotype interaction did not affect aphid fitness , the total developmental time of nymphs (DT5), and 10 d fecundity. However, their research did not focus on the population parameters. In life table research, the population parameters represent the intrinsic rate of increase r, the net reproductive rate R0, the mean generation times T and the finite rate of increase λ. In our study, the main life table parameters researched were the population parameters.Understanding the entire life history plays a significant role in evaluating the population parameters that are influenced by secondary endosymbionts, and the key component of this analysis is the life table, which can provide a comprehensive description of the development, survival, and fecundity of a populationr) of the treatments was significantly lower than that of the controls. The intrinsic rate of increase is a key demographic parameter that has been used to summarize the reproductive potential of an animal population in relation to its capacity to increaseR. insecticola on the potential growth of S. avenae clones is intriguing. This notion was confirmed by the mean generation times (T) of the treatments, which were shorter than that of the controls. Oliver et al.H. defensa shortened the mean generation time (T) of A. pisum. Although R. insecticola might decelerate the potential growth of S. avenae populations, the finite rates of increase (λ) were all over 1 between the treatments and the controls, indicating that the studied S. avenae population increased continuously. The net reproductive rates (R0 > 1) between the treatments and the controls also confirmed this observation. However, the finite rates of increase (λ) of the treatments with the secondary symbionts were remarkably less than that of the controls, suggesting that the daily increase of S. avenae clones in the treatment groups was significantly lower than that of the controls. This was confirmed by the birth rates of the treatment groups, which were significantly lower than that of the controls. In conclusion, R. insecticola may reduce the potential growth of S. avenae clones, and this is consistent with the findings of Wang et al.The intrinsic rate of increase , finite rates of increase (λ), mean generation time (T) and birth rate demonstrated that R. insecticola infection had a negative effect on the natural development of S. avenae clones, and the impact levels differed among different genetic backgrounds. The difference between genotypes might be due to variations in host nutrients. For example, the effect of nitrogen content and protein quality might affect the fecundity of herbivores383940S. symbiotica, H. defense and Rickettsia, need to be examined individually, and (3) more donor and recipient clones or species could be used, as only 2 aphid clones were used as donors and 2 aphid clones were recipients in the current study. Such efforts would provide us with a comprehensive perspective that would aid our understanding of the complex symbiotic relationships between aphids and bacterial endosymbionts.The intrinsic rates of increase , pre-adult duration and total longevity. In addition, the significant reduction of fitness traits  and the clear increase of the mean generation times (T) indicated that R. insecticola might have a negative effect on the potential population growth of S. avenae.In conclusion, the findings of this study indicate that the interaction effects between R. insecticola were collected in Neixiang , Henan Province and in Linyi , Shandong Province in May 2014. Collected clones were genotyped at four microsatellite loci  as described in the R. insecticola donor clones were collected in April 2014 in two quadrats, at a distance of 300 m from each other, in Yangpingguan , Shaanxi. They were named Yangpingguan 1 and Yangpingguan 2. Each clone was derived from a single parthenogenetic S. avenae female, and the endosymbionts of the four clones were detected. The primers are shown in the Triticum aestivum L.) seedlings (variety ‘1376’) that were changed at ten-day intervalsAphid clones (considered genotypes in this paper) used in this study were established from individuals collected from winter wheat plants on private farm land, with the permission of the owners. Clones without R. insecticola into either 2nd or 3rd instar S. avenae individuals. To do this, the Yangpingguan clones 1 and 2 (from Shaanxi) that hosted R. insecticola were chosen as the donor clones. Body fluids from the Yangpingguan clones 1 and 2 were transferred into the Linyi (from Shandong) and the Neixiang clones (from Henan), respectively, resulting in the creation of four new clones . Therefore, the Linyi-1 and Linyi-2 clones and the Neixiang-1 and Neixiang-2 clones have the same genetic background as the Linyi (Shandong) and the Neixiang clones (Shandong), respectively. The surviving aphids  after the injection were transferred to fresh wheat plants (one aphid per plant). Twelve hours later, the dead aphids were discarded, and the remaining surviving aphids were reared till they became adults and produce nymphs. Next, one single offspring individual from each clone that tested “positive” was subjected to PCR using PCR primers described in Sandström et al.Taq DNA polymerase mix, and the following cycle parameters: 94 °C for 7 min and 35 cycles of 94 °C for 30 s, 57 °C for 45 s and 72 °C for 30 s. The resulting PCR products were separated in a 1% agarose gel and visualized under UV after gel staining with ethidium bromide for the presence of R. insecticola and were then placed on T. aestivum and allowed to reproduce for several days. At this time, adults were removed and 5 individuals were tested again for the presence of R. insecticola. One positive clone was then used to conduct the following study. To ensure that R. insecticola densities in S. avenae produced clones approached equilibrium, the original data of the life table were collected after a minimum of 10 generationsThe micro-injection technique advocated by Chen & PurcellS. avenae clones (unpublished data). Experiments were performed using three groups for each individual clone, with a total of 30 aphids in each group. Newly emerged 1st instar nymphs were individually transferred within 24 h to a 9-cm Petri dish containing a fresh wheat leaf blade to start the experiment. The collection of life table data was initiated approximately 24 h later. Each aphid was observed daily at the same time until all individuals died. All offspring were removed daily, and the wheat leaves were replaced every 2 days. Molting and mortality events, as well as the number of produced nymphs, were recorded.Linyi-1 and Linyi-2 clones were compared with the Linyi clone control as they all shared a common genetic background. This clone was named Linyi-NA for convenience. Similarly, Neixiang-1 and Neixiang-2 clones were compared with the Neixiang clone control, and the control was named Neixiang-NA for convenience. A preliminary experiment showed that the control clone (no injection) and the negative control (injection of water) showed no difference in the growth and development of the studied r) was estimated using an interactive bisection method and the following Euler–Lotka equation  was calculated as follows  to analyze the effects of ‘R. insecticola’ and clone  nested in ‘R. insecticola’. We used Tukey’s procedureP < 0.05. Mean values (±standard errors of the mean) were calculated and used in the above analyses. If needed, data were transformed to meet the required assumptions of normality and homoscedasticity. Graphs were generated using SigmaPlot 12.5.A 2 × 3 factorial randomized in complete blocks with 30 replications was deployed to test How to cite this article: Luo, C. et al. Ecological impact of a secondary bacterial symbiont on the clones of Sitobion avenae (Fabricius) (Hemiptera: Aphididae). Sci. Rep.7, 40754; doi: 10.1038/srep40754 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


N-acetylcysteine, and the recovery of cell viability was observed. The increase in ROS production in response to 6-shogaol was associated with cell death. Similarly, glucose uptake decreased with incremental concentrations of 6-shogaol, and an increase in the expression of mTOR-p and Akt-p proteins was observed; PTEN was active in all the treatments with 6-shogaol. Thus, the results suggest that cells activate uncontrolled signaling pathways, such as phosphoinositide 3-kinase (PI3K)/Akt/mTOR, among other alternative mechanisms of metabolic modulation and of survival in order to counteract the pro-oxidant effect of 6-shogaol and the decrease in glucose uptake. Interestingly, a differential response was observed when non-cancerous cells were treated with 6-shogaol. Ginger is a plant that is native to southern China. In the last decade and research on the components of ginger has significantly increased; of these components, 6-shogaol exhibits the greatest potential antitumor capacity. However, the molecular mechanism through which 6-shogaol exerts its effects has not yet been elucidated. In this study, the effect of 6-shogaol on tumor cells that were derived from human fibrosarcoma (HT1080) was evaluated. Cell viability was determined by a -2,5-diphenyltetrazolium bromide) MTT assay testing different concentrations of 6-shogaol (2.5–150 μM). Subsequently, the effect of 6-shogaol on reactive oxygen species (ROS) production, glucose uptake, and protein expression of the signaling pathway phosphatase and tensin homolog/ protein kinase B /mammalian target of rapamycin (PTEN/Akt/mTOR) was measured. 6-Shogaol reduced the viability of the tumor cells and caused an increase in ROS production, which was attenuated with the addition of  Zingiber officinale Roscoe, Zingiberaceae) has been used for thousands of years as a spice, and it has been considered to be an important ingredient in traditional Chinese medicine for the treatment of certain diseases, such as diabetes, cardiovascular diseases, rheumatism, and cancer [Ginger  of 6-shogaol in HT1080 cells was 52.8 μM. Low concentrations of 6-shogaol exhibited effects on the cell viability of the HT1080 tumor model. From 30 μM, a clear decrease in cell viability was observed, even below 80%. No cells survived at 150 μM 6-shogaol. Fibroblasts that were derived from human periodontal ligament (HPdLF) exhibited greater resistance to treatment with 6-shogaol as compared to the HT1080 cells (p = 0.0058). HPdLF decreased its viability from 70 μM 6-shogaol (6-Shogaol (2.5–150 μM) decreases HT1080 cell viability in a dose-dependent manner. The half maximal inhibitory concentration (IC-shogaol A.p = 0.0011) than fibroblasts that were derived from the periodontal ligament . The fibroblasts did not show alterations following treatment with 6-shogaol. Importantly, it can be concluded that, relative to ROS production in response to treatment with 6-shogaol, a different effect is observed between the tumor and non-tumor cells (p = 0.001 HT1080) (p = 0.0029 HPdLF). This allowed us to conclude that the increase in ROS production was due to the effect of 6-shogaol.To evaluate cell dynamics in response to treatment with 6-shogaol, concentrations of 30–70 μM were used, which resulted in a significant decrease in cell viability when applied in the tumor model. An increase in dose-dependent ROS production was observed in HT1080 cells, which was significant at 50 μM 6-shogaol (or cells C. When tp = 0.0091). Therefore, we concluded that cell death generated by 6-shogaol is associated with an increase in ROS production.Finally, the cell viability of the tumor model was evaluated under treatment with 6-shogaol (30–70 μM) and joint treatment with NAC (5 mM). Cells only treated with 6-shogaol showed decreased viability at all concentrations D, whereap = 0.0114), which can be explained by the high metabolic activity and high energy demand of tumoral cells for the maintenance of their proliferation [The assay results showed that basal glucose uptake by the tumoral cell model was greater than for normal fibroblasts , however, from 60 μM 6-shogaol, a progressive decrease in glucose uptake was observed with increasing 6-shogaol concentration , there was a decrease in the expression of these proteins A. It was observed that 6-shogaol (10 and 50 μM) induced phosphorylation of the mTOR protein, whereas PTEN was not observed to be phosphorylated at any of its three activation sites; this suggests that PTEN could be in its active (dephosphorylated) form B.p = 0.0091)  C.The differential effects of 6-shogaol on normal and tumor cell lines have already been described, however the cause of this compound having this differential effect is still unknown. It is thought that the effect is related to the increased production of ROS induced by 6-shogaol, which acts as a pro-oxidant; it is likely that this effect is more effectively neutralized by non-tumor cells through more efficient endogenous antioxidant mechanisms. In addition, it can be considered that tumor cells already have a high amount of intracellular ROS, which can make them more sensitive to this type of insult, which compromises their viability.It should be considered that 6-shogaol is not the only volatile compound with biological activities against cancer that is present in ginger. It is also known that 6-gingerol has similar antitumoral properties, but to a lesser degree .The gingerols are present at higher concentrations in fresh root. The main isoform of gingerol has a double bond in carbon 6, and it is called 6-gingerol A. 6-GingIt has been shown that 6-gingerol inhibits the progression of skin cancer induced by phorbol ester in addition to inhibiting metastasis in breast cancer (MDA-MB-231), induced apoptosis in prostate cancer (LNCaP), and it could inhibit tumor progression in melanomas and kidney cancer in murine models; there are few reports in relation to other types of cancer .Shogaols B are anaSimilarly, it has been reported that 6-shogaol has greater anti-inflammatory capacity than 6-gingerol, which inhibits the production of prostaglandin E2 (PGE2) as well as the expression of proinflammatory cytokines, such as interleukin 1β, tumor necrosis factor (TNF-α), p38, and nuclear factor kappa β (NF-kB). In addition, it inhibits the release of nitric oxide and the expression of the enzyme nitric oxide synthase in mouse microglia cells (BV-2) treated with lipopolysaccharide (LPS), primary cultures of glia, and in vivo models of neuroinflammation . There a50 of 29.6 and 22.1 μM, respectively, when treated with 6-shogaol for 48 h [50 between the tumor lines might be due to the histological differences of each of the cell lines, which affects their differential sensitivity to the compound. The cytotoxic effects of 6-shogaol have been described in several tumor lines and using different concentrations, such as in the case of HT-29 cells of colorectal cancer, whose viability was reduced by 50% when treated with 60 μM and they were completely inviable upon treatment with 80 μM . Other tfor 48 h . The difp = 0.0001) coincide with reports that were described in human gingival fibroblasts (HGF-1), where a 10% decrease in cell viability was observed when treated with upwards of 80 μM 6-shogaol [In the present work, the significant differences in viability that were observed between non-tumor cells (HPdLF) and the HT1080 line in response to 6-shogaol treatment . However, the mechanism of 6-shogaol uptaken by cells is unknown .Studies of the SAR (structure–activity relationship) have shown that the free hydroxyl group of 6-gingerol contributes to its antitumor activity against breast cancer, and that the length of the aliphatic side chain increases this activity, since this chain loses activity when its length is reduced, as shown in the tumor models . HoweverIn tumor cells, the presence of complex alterations, such as disruption of some metabolic pathways and the permanent activation of signaling pathways involved in survival, as well as the increase in nutrient consumption, can lead not only to a higher production of ROS, but also to a decrease in the ability to neutralize them effectively; therefore, the tumor cell exhibits a higher production of ROS. To counteract the effect of increased ROS on viability, recent studies have indicated that tumor cells develop alternative mechanisms of activation of antioxidant pathways, which are not sufficient when cells undergo additional pro-oxidant stimuli (6-shogaol). It is well known that increased ROS production in tumor cells induces the uncontrolled activation of the PI3K signaling pathway (PI3K/Akt/mTOR), in comparison with the response of normal cells. Consequently, the RAS pathway is activated, which leads to metabolic modulation and survival. Additionally, the resulting hyperactivation of HIF-α is also mediated by factors, such as VEGF, modular angiogenesis, and increased proliferation and cell migration, among others .Similar results to those that were observed in the present work have also been described in other tumor models, such as mouse kidney epithelial cells (TMCK-1) and mesangial cells (MC) . These aSimilarly, Akimoto et al. 2015 demonstrated that the pro-oxidant capacity of 6-shogaol and its effect on the reduction of cell viability can be maintained with the treatment of 10 mM NAC in a cellular model of PANC-1 (pancreatic tumor cells) . It has The effect of 6-shogaol on the increase of glucose uptake in non-tumor cell models was described for the first time in adipocytes (3T3-L1) and myotubules (C2C12), demonstrating that this compound generated a greater uptake in comparison with 6-gingerol and zingerone . This efp = 0.0114); this can be explained by its high metabolic activity and its high energy demands for the maintenance of its proliferation rate (10). Additionally, the effect is characteristic of the so-called Warburg effect, which, with its modifications, has been described in recent literature reports as aerobic lactate genesis, characterized by a metabolic reprogramming that leads to an increase in glucose uptake, higher production of lactate, and increased translocation of GLUT-1 to the cell membrane [The basal glucose uptake that was determined in both cell models showed that the tumor model captures more glucose than the non-tumor model  exhibited a significant reduction in glucose uptake. It is already known that glucose competes with ascorbic acid for GLUT-1. However, more studies are needed that characterize GLUT transporter activity in this type of cell .Otherwise, the canonical concept of the role of Akt as a protein that promotes proliferation, survival, and cell growth is based on downstream Akt inducing the activation of proteins, such as mTOR, which promotes cell growth in addition to the inactivation of several proapoptotic factors such as BAD, procaspase-9, and transcription factor FKHR (forkhead). Additionally, the activation of Akt promotes the increase of transcription factors that increase the expression of anti-apoptotic genes, such as CREB, as well as direct phosphorylation of NF-kB and HIF-1α.Another mechanism through which Akt promotes survival is through the inactivation of the p53 tumor suppressor gene . TherefoIt has recently been described that the activation of Akt might be related to an increase in glucose in the medium, a process that induces ROS production and is characteristic of hyperglycemia mechanisms, where, if glucose is unable to enter the cell, either by decreasing the translocation of GLUT-1 or by any other mechanism, its extracellular accumulation will allow for the activation of Akt and the induction of premature senescence .Premature cellular senescence is an irreversible state of cell growth arrest that is characterized by a set of physicochemical changes and cellular functions . CurrentWhile considering the results that were obtained and the information outlined in previous paragraphs, it is very likely that 6-shogaol induces premature senescence through increased ROS production, and that this is related to the activation of Akt; similarly, the recently described mechanisms suggest that the resulting decrease in glucose uptake makes the cells more sensitive to the induction of ROS-mediated apoptosis, even if the order in which they are triggered is not yet clear. Undoubtedly, research in this field includes a large number of possibilities that should be explored.2. 6-Shogaol was obtained from Sigma-Aldrich  [The fibrosarcoma cell line (HT1080) was obtained from American Type Culture Collection (ATCC-CCL-12), and the human periodontal-derived ligament fibroblast (HPdLF) line from Lonza Ref. CC-7049 . Both cells lines were maintained in complete DMEM Dulbecco’s modified Eagle medium that was supplemented with 10% fetal bovine serum  and a 1% antibiotic–antimycotic cocktail, and at 37 °C and 5% COMO, USA) .2. Upon conclusion of the treatment, the medium containing 6-shogaol was replaced with 100 µL of fresh DMEM and then incubated with 10 µL of MTT reagent for 2 h at 37 °C, followed by 10 µL detergent reagent at room temperature overnight. Viability was measured at 570 nm while using a FLUOstar microplate reader . Cell viability was assessed while using a MTT assay . Briefly2− [2. The cells were then incubated with 1.5 µL of DHE dye for 30 min. at 37 °C and 5% CO2. Mean intensity fluorescence was measured at 535/610 nm using a FLUOstar microplate reader . Hydrogen peroxide was used as a positive control. N-acetylcysteine was used as negative control in co-treatment with 6-shogaol to measure the recovery from death induced by ROS.A dihydroethidium (DHE) probe was used to quantify the levels of superoxide anion O2− . Cells wN-amino)-2-deoxyglucose) probe was used to quantify glucose uptake. The cells were treated as previously described. Briefly, the cells were trypsinized, centrifugated, and resuspended in 200 µL of staining buffer (1× PBS and 50 µM 2-NBDG). After 30 min. of incubation, cells were washed twice with 200 µL of 1× PBS. Mean intensity fluorescence of glucose uptake was measured with a green laser (430 nm) while using a Flow Cytometer Guava easyCyte  [A 2-NBDG (2-(Germany) .2, 1% Triton X-100, and protease and phosphatase inhibitor cocktail). Equal concentrations of total cell lysate were resolved by 10% SDS-PAGE and then transferred to a polyvinylidene fluoride membrane (PVDF). Nonspecific binding sites were blocked with BSA (10%), followed by an incubation with primary antibodies for anti-mTOR, anti-phospho-mTOR-S2448, anti-PTEN, anti-phospho-PTEN-S380/T382/383, anti-Akt, and anti-phospho-Akt-Ser473. Protein complexes were detected with peroxidase-conjugated secondary antibodies and enhanced with chemiluminescence reagent  [A total of 150,000 cells were harvested in lysis buffer , 10 mM MgCl Canada) . The fil2. After the treatments were complete, cells were fixed with 1.6% formaldehyde for 10 min. Subsequently, cells were permeabilized with methanol for 1 h. The treatments were replaced with two changes of 600 µL staining medium (1× PBS and 2% BSA) and then incubated with a different antibody. The following human antibodies were from Cell Signaling: anti-mTOR (#2972), anti-phospho-mTOR-S2448 (#2971), anti-PTEN (#9552), anti-phospho-PTEN-S380/T382/383 (#9554), anti-Akt (#9272), anti-phospho-Akt-Ser473 (#9271), anti-GAPDH (14C10), and rabbit mAB (#2118) . Anti-β-actin was from ABCAM reference (#ab8227)  and was used as the loading control. The secondary antibodies used were either anti-mouse Alexa 488 (1:400) and anti-rabbit Alexa 488 (1:400). Mean intensity fluorescence was measured with a green laser (430 nm) while using Flow Cytometer Guava easyCyte . Incubations with only secondary antibody were used as the negative controls [A total of 50,000 cells/well were seeded in a 24-well plate. The cells were treated for 24 h with 10–50 µM 6-shogaol concentrations at 37 °C and 5% COp < 0.05 (*), p < 0.01 (**), and p < 0.001 (***). The results were subject to statistical analysis while using GraphPad Prism 7  [Data are presented as the mean ± SD of at least three independent experiments and they were analyzed by two-way ANOVA followed by a post hoc Dunnett’s test. Data were classified at different levels of significance corresponding to CA, USA) .6-Shogaol showed different effects when used to treat either HT1080 tumor cells or fibroblasts. The decrease in HT1080 viability was associated with the increase in ROS that was caused by treatment with 6-shogaol, unlike fibroblasts, which showed no significant changes. The increase in ROS was attenuated by NAC. It is suggested that in the tumor model (HT1080), 6-shogaol induces premature senescence through increased ROS production, the activation of Akt/mTOR, and the decrease in glucose uptake. Taken together, these effects can make the cells more sensitive to the induction of apoptosis by mechanisms that have not yet been completely described. In future, it will be necessary to evaluate other proteins of the signaling pathway (PI3K/Akt/mTOR) to check the modulation efficacy of pathway activation in inducing apoptosis.


As the aging population increases, large changes have occurred among household structures in Japan. Half of older adults lived with their children’s families in 1980. Now around 60 percent of them live by themselves: 27% in single households and 31% as older couples alone. Older adults in single households are said to be at higher risk of social isolation. A Japanese white paper reported that they had scanty of social interactions compared to other types of households. This study examines differences in the social relationships and health statuses among household types by gender and explores the risk factors of social isolation. Nationally representative 2012 Japanese Social Survey data were used for analyses; a subsample comprised participants aged 60 to 74 years. A series of ANCOVAs were conducted. The distribution of the gender and household types were single male 105 (10.0%), married male 387 (36.8%), single female 180 (17.1%), and married female 381 (36.2%); the main effects were being female and married. An interaction effect between them  was observed. Neighborhood relationships were better among females and married participants. Married participants were more active in community meetings, social participation, and volunteering. However, no difference was observed in social network size. Thus, network size alone was not related to social isolation, but being active in social relationships and the quality of relationships influenced social isolation and well-being. Being married and female may facilitate higher quality relationships and may lead to activity and buffer social isolation.


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Many therapeutically active molecules are non-soluble in aqueous systems, chemically and biologically fragile or present severe side effects. Lipid-based nanoparticle (LBNP) systems represent one of the most promising colloidal carriers for bioactive organic molecules. Their current application in oncology has revolutionized cancer treatment by improving the antitumor activity of several chemotherapeutic agents. LBNPs advantages include high temporal and thermal stability, high loading capacity, ease of preparation, low production costs, and large-scale industrial production since they can be prepared from natural sources. Moreover, the association of chemotherapeutic agents with lipid nanoparticles reduces active therapeutic dose and toxicity, decreases drug resistance and increases drug levels in tumor tissue by decreasing them in healthy tissue. LBNPs have been extensively assayed in in vitro cancer therapy but also in vivo, with promising results in some clinical trials. This review summarizes the types of LBNPs that have been developed in recent years and the main results when applied in cancer treatment, including essential assays in patients. Cancer nanotechnology as a way of anticancer drug delivery has been developed as a promising cancer treatment . NanoparLipid-based nanoparticles (LBNPs) such as liposomes, solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) have received great attention in drug discovery and cancer treatment. These nanoparticles can transport hydrophobic and hydrophilic molecules, display very low or no toxicity, and increase the time of drug action by means of a prolonged half-life and a controlled release of the drug . Lipid n®, a liposome formulation encapsulating DOX, was one of the first anticancer drug nanosystems commercially used.Many antitumor agents, such as cisplatin, irinotecan (IRI), paclitaxel (PTX), doxorubicin (DOX) oxaliplatin, daunorubicin, cytarabine or vincristine, have been studied in nanoformulations, and some of them have been analyzed in clinical trials and/or are commercially available for clinical use in patients . In factThis review highlights the main contributions to LBNPs that have been developed in recent years and applied in cancer treatment, including essential assays in patients.Liposomes are the most studied delivery systems due to the biocompatibility and biodegradability that they present. The main components of these nanoparticles are phospholipids, which are organized in a bilayer structure due to their amphipathic properties. In presence of water, they form vesicles, improving the solubility and stability of anticancer drugs once they are loaded into their structure. They are capable of encapsulating either hydrophobic or hydrophilic drugs . In addiIt is well known that there are two general ways to achieve nanocarrier vectorization. One of them is passive targeting, which occurs when liposomes only enter the tumor cell by molecular movement through the cellular membrane. The other is active targeting, which involves structurally modified liposomes holding antibodies that recognize tumor cells . A third3O4 cores are increasingly being used to functionalize different types of nanoparticles , which can be degraded by sonication, leading to drug release. On the other hand, liposomes modified with PEG and anacardic acid have been developed, and these were employed to encapsulate docetaxel, improving the stability of this anticancer drug [The synthesis and development of new liposomes have been extensively studied in recent years. In fact, Fearticles . In 2014lesterol . Moreovelesterol , such ascer drug .Yan et al.  develope0.75Sr0.25MnO3 and iron oxide to combine self-controlled hyperthermia and chemotherapy. PTX was encapsulated in this type of hybrid liposome. In vitro and in vivo biological studies demonstrated the biocompatibility and therapeutic efficacy of this new magnetic liposome [Gogoi et al.  exploredliposome . In 2018liposome  designedliposome . In anotliposome . Studiesliposome . A notewliposome , who encSLNs represent a relatively new colloidal drug delivery system, composed of physiological lipids that remain in a solid state at both room and body temperature. These particles are in the size range of 50–1000 nm. The solid lipid used forms a matrix material for drug encapsulation and include mono-, di- or triglycerides, fatty acids and complex glyceride mixtures. This matrix is stabilized by a mixture of surfactants or polymers. SLNs have significant advantages, such as site-specific targeting, physical stability over a long period, possibility of controlled release of both lipophilic and hydrophilic drugs, protection of labile drugs, low cost, ease of preparation and nontoxic. Furthermore, in reference to toxicity, SLNs have exceptionally low toxicity effects against human granulocytes. All these outstanding advantages make them an important candidate for drug delivery systems . In contGiven their unique properties, SLNs have been extensively studied for drug delivery of active anticancer compounds, incorporating them into the SLN formulation in order to improve the oral bioavailability of drugs, protect labile anticancer drugs, and also to reduce side effects by decreasing the dosage by effective targeting to the site of action. The main contributions reported in the last year show that SLNs can be used for encapsulated anticancer drugs. For example, in 2018, Pindiprolu et al.  developeNLCs represent a second generation of lipid-based nanocarriers, developed from SLN, which comprise a combination of solid and liquid lipids. This system was developed in order to overcome the limitations of SLNs; hence, NLCs have higher drug loading capacity, and could also avoid drug expulsion during storage by avoiding lipid crystallization due to the presence of liquid lipids in the NLC formulation. While SLNs are composed of solid lipids, NLCs are a mixture of solid and liquid lipids, such as glyceryl tricaprylate, ethyl oleate, isopropyl myristate and glyceryl dioleate. The mean particle sizes are highly similar to SLNs, generally in the range of 10–1000 nm, and are affected by the nature of the containing lipids and the manufacturing process . The maiAmong the contributions reported in recent years, a first relevant example is presented by fluvastatin, which, when combined with lipoic acid and ellagic acid in a NLC, could be used as a candidate for prostate cancer therapy due to the effects of the combination with respect to cell death in comparison to free drugs . AnotherNanoparticles constitute nano-sized carriers (5–200 nm)  whose vaActive targeting is one of the most promising applications for NPs, since they make it possible to take advantage of the specific characteristics and specific profile of each tumor niche to direct treatments to it by functionalizing the NPs with antibodies, tumor-specific antigens (TSA), microRNAs or siRNA, which, together with the properties of certain nanomaterials used in their synthesis, such as pH and/or temperature dependent degradation, response to light, magnetic or ultrasound stimuli, make it possible to release its load specifically at the tumor site, decreasing systemic toxicity enormously with respect to traditional treatment ,46,47.Currently, NPs have acquired a dual profile, being able to help in the diagnosis of disease in addition to granting targeted and specific therapy of disease (teragnosis) . In addi® or Abraxane®) have already been approved for BreC treatment [Lipid-Based NPs (LBNPs) constitute a broad and diverse group of nanoparticles that are particularly relevant in BreC treatment. However, despite their variety, liposomes are so extensively used due to their high degree of biocompatibility and their ability to encapsulate a wide range of cargos. LBNPs are currently being used in several trials, and some of them  is the fifth most common cancer and the third most common cause of cancer-related death worldwide ,42. Only5β1 . Additiong cells . In adding cells . Preliming cells  showed ang cells  designedng cells . Jiang eng cells  designedng cells , which eng cells , compris188Re)-liposome in combination with radiotherapy to evaluate its effectiveness in BE-3  tumor-bearing mice. This system determined an increased tumor growth inhibition, with no alterations of WBC, hemogram profile or weight loss. Another interesting approach, developed by Feng et al. [On the other hand, for esophageal cancer (EC), the seventh most frequent cancer worldwide ,42, someg et al. , was theMDR-P-gp overexpression) to IRI by reducing the IC50 of the free drug 6 and 9.5 times at 72 and 96 h of exposure, respectively.Colorectal cancer represents a serious health problem due to its high mortality (it is the second most common cause of cancer-related death) and the continuous increase in its incidence in recent years ,42. LBNPThere are no feasible screening tests for early detection of pancreatic cancer (PaC). Therefore, PaC is often detected in advanced stages, when surgery cannot be applied. However, the failure of pancreatic cancer treatment is not only caused by the advanced state of the tumor , but also by the difficulty the drugs have in penetrating deeply, given the nature of the tumor stroma. Nanotechnology offers some therapeutic strategies to improve the prognosis of these patients. Chirio et al.  prepared®, benefitting its accumulation and permanence in plasma [Liver cancer (LivC) therapies are often limited by poor drug physicochemical properties. In fact, chemotherapy and targeted drugs such as sorafenib induce a minimal impact in patient survival. In addition, radiotherapy is generally ineffective. Combining drugs with some nanoplatforms has been proposed as a strategy to increase overall treatment efficacy and patient survival. In this context, 5-FU-loaded cubosomes and PTX-loaded NLCs have been used in LivC treatment. On the one hand, 5-FU-loaded cubosomes prevent the drug from its rapid enzymatic degradation, increasing its accumulation in the liver. On the other hand, PTX-loaded NLCs improve the effect of the commercial formulation Intaxeln plasma ,76. In an plasma . Furthern plasma  and Qu en plasma  developen plasma  designedn plasma  or CUR-gn plasma  have shon plasma .6-(4-bromothenyl)guanine (O6BTG) derivative/N9-modified liposomes capable of inhibiting the expression of the MGMT gene in SMA-497 and ZH-161 cells. Furthermore, the intravenous application of O6BTG derivative liposomes and TMZ in a GBM intracranial model using SMA-497 cells resulted in a reduction of tumor growth and in an increase in survival rates in comparison with other treatments. In conclusion, the results of some clinical trials reinforce the potential application of these new therapeutic strategies in GBM. In fact, Myocet®, a non-PEG-DOX liposome, was used in pediatric patients with high-grade glioma  after TMZ-based chemotherapy  [Glioblastoma multiforme (GBM) is the most aggressive and frequent type of malignant brain tumor (50% of glioma cases), with a very low 5-year survival rate (10%) . The curl trial) . With thl trial) .Lung cancer (LuC) is the most common cancer and the primary cause of cancer-related death worldwide in both men and women ,42. Desp© with cyclophosphamide (CM) or vinorelbine (MV) in patients with metastatic BreC has also been developed [Breast cancer, the primary cause of cancer-related death in women ,42, is ceveloped .On the other hand, niosomes have become one of the most interesting LBNPs in BreC treatment, which has led to the development of various systems to facilitate the vehiculization of gingerol , tamoxifOther LBNPs used in BreC research include SLNs. Yu et al.  proposedCurrently, NEs, liposomes and solid-lipid NPs (SLNs) are the main LBNPs being tested as new therapeutic strategies in prostate cancer (PrC). Recently, Ahmad et al.  developep53, MALT1, KRAS, HGAL, ERBB2, PIK3CA, AKT) [Drug resistance (DR) is an essential aspect that limits the effectiveness and application of cancer treatment . A distiCA, AKT) ,129. In CA, AKT)  synthesiFinally, the epithelial-mesenchymal transition (EMT), by which epithelial cells lose typical markers of differentiation (mainly E-cadherin) and begin to express markers of mesenchymal cell differentiation (N-cadherin), may be relevant in cancer resistance. This transition has been linked to processes such as cancer metastasis. Therefore, it is suspected that EMT could induce cancer stem cell (CSC) characteristics by increasing MDR phenomena ,131. In ®, Abraxane®, nal-IRI and Myocet®, have been approved in clinic for the treatment of cancer. This makes LBNPs a very promising group for the treatment of cancer in the future.LBNPs constitute a diverse and extensive group that has been used in the treatment of numerous pathologies, mainly in cancer. Liposomes are the most widely used LBNPs, due to their great biocompatibility and versatility; however, recently, SLNs and NLCs have been gaining great attention. Even so, research is not focused solely on these NPs, and there are many publications focused on new strategies using LBNPs to treat different types of cancers. Some of these have even taken the leap to the next level and have begun their path in clinical trials, and what is more, some examples, such as Doxil


The attitudes of care staff toward the sexuality of residents with dementia they care for is assumed to influence the residents’ expression of their sexuality in the way they want. This paper examines the effect of organizational factors, person-centered care, and the culture of the organization on the attitudes of care staff toward the sexuality of residents with dementia in residential care facilities (RCF) .N = 187) participated. Using a survey, we gathered information on demographics and care-staff careers, attitudes toward resident sexuality, the culture of the organization, person-centered care, and knowledge of resident sexuality. Ordinary least square (OLS) hierarchical analyses were performed to analyze results.Care staff in different functions at six RCF organizations (Care staff attitudes were found to be positively affected by person-centered care, and marginally positively affected by a supportive culture in the organization, Moreover, knowledge of resident sexuality positively affected care staff ‘attitudes toward resident sexuality, and the presence of policy regarding resident sexuality affected them negatively .Despite different study limitations, these results give a first insight in a broad perspective on care staff attitudes toward resident sexuality. In addition to improving knowledge of the care staff, enhancing person-centered care and a supportive culture in the organization will improve care-staff attitudes toward resident sexuality. Positive intimate and sexual experiences are found to influence health and quality of life (QoL) positively in the elderly –3. HowevA small body of research exists on caregiver attitudes toward the sexuality of residents with dementia, based on the assumption that a more open or positive attitude positively influences the expression of sexuality by residents . AlthougSeveral factors at the level of the individual caregiver and their careers were reported to influence their attitudes toward resident sexuality. First, age was an influence. Older employees had more positive attitudes toward sexuality of residents with dementia than their younger colleagues . This diIn addition to these individual factors and career characteristics (e.g. participation in a training program), previous literature has emphasized the importance of factors at the level of the organization , 13, 14.In addition to the organizational culture, in recent years the concept of person-centered care has generally gained popularity in dementia care. In this concept, care is meant to be holistic and empowering, and should aim at increasing resident QoL , 22. ThiThe aim of this study is to examine in a broad perspective the attitudes of care staff toward the sexuality of residents with dementia, by examining the possible influence of organizational factors on care staff attitudes. In this study, organizational factors are operationalized as person-centered care and the culture of the organization. Next to the organizational factors, the role of individual factors, knowledge of resident sexuality, and some characteristics of care staff career (e.g. years of tenure and current function) over the possible effect on the main target variable “attitude” are included.The data for this study were collected at psychogeriatric care units of RCFs located in the south of the Netherlands. The RCFs participated in an academic collaborative network with the aim of connecting research and practice to elderly care. The participating RCFs varied in size between 590 and 1000 residents. An admission into a RCF becomes inevitable when the cognitive and physical impairments of patients with dementia rise to severity levels that make care at home with help of a private and formal care network impossible. In the Netherlands, this highly intensive care is provided in psychogeriatric care units. These are protected living environments, where approximately six to ten persons with moderate to severe dementia reside in a closed unit.Employees with both direct and indirect contact with residents of such psychogeriatric care units were recruited. Direct caregivers are employees with a vocational level of education, who work together in a care team that belongs to a specific psychogeriatric unit and so provide direct daily care to residents with dementia. However, Dutch psychogeriatric care is organized in a multidisciplinary way. This means that several professionals with different expertise and tasks, and mostly higher levels of education, are indirectly involved in the care of residents . Although RCF care staff other than direct caregivers are less involved in daily care, they are mostly responsible for policy, guidelines, and treatment or care advice for the direct caregivers.Initially, a convenience sample of 191 employees participated in the study. One minor (< 18 years of age) was excluded because of considerations concerning the ethical review. Two respondents were excluded because they barely filled in the questionnaire (< 10% of the questionnaire). To maximize the sample size in analyses, we included all cases for which 80% of the items on the dependent variable, attitude toward resident sexuality, were completed. This resulted in a final sample size of 187.n = 179, 95.7%). Mean age was 40.8 years (range 18–64), and they had an average of 16 years of tenure within care (range 1–43). Most of the participants completed an average vocational education and worked as direct caregivers.The participant characteristics are shown in Table The Tilburg University psychological ethics committee granted ethical approval (Reg. No. EC 2015.60), and approval of the executive and ethical boards of the participating organizations was also obtained.Hard-copy surveys were distributed after scheduled (team) meetings. A convenience sample was gathered through the selection of the meetings, in which all employers were represented. Approximately ten members of care staff attended these meetings and care staff with different functions were present during most meetings. Participation was voluntary and participants could withdraw from the study at any time. The participants received an information letter, an informed-consent form, and the survey for immediate completion. The informed consent form and anonymous questionnaires were stored separately. One author (TR) or a student assistant was present during data collection to answer all questions. The author and assistant aligned their answers in frequent discussions about the data-collection process.The survey questionnaire was divided into five sections; one includes the main outcome measure , and four include the covariates. To estimate reliability of the measures based on the sample, we used Cronbach’s alpha test for internal consistency. Values between .7 and .9 were considered acceptable .The Dutch version of the aging sexual knowledge and attitudes scale (AKSAS) , 25 was The attitude subsection consists of 25 items, rated in a seven-point Likert scale from 1  to 7 , it included such items as “Aged people have little interest in sexuality.” Ten items were reversed after completion of data collection. An example of such an item is “Masturbation is an acceptable sexual activity for older males.” Total scores were calculated ranging from 25 (most positive attitude) to 175 (least positive attitude) for analysis, as used before .For this subsection, the developers of the scale found high internal consistency and reliability (Cronbach’s α = .88) and sufficient content validity , based on the judgment of ten experts . ReliabiPerson-centeredness is defined as a holistic view of residents, and in this maintaining personhood, despite increasing cognitive and physical impairments . The perThe FOCUS Questionnaire  was usedThe four cultural orientations are measured from a descriptive and an evaluative perspective. The complete questionnaire consists of 54 items that are distributed over eight different variables (four orientations measured from two perspectives). The descriptive perspective measures directly observable behavior and consists of 25 items rated in a six-point scale . An example of an item is “How often is constructive criticism accepted?” The evaluative part measures the perception of employees regarding typical characteristics of the organization and consists of 29 items, rated on a six-point scale . An example of an item is “How typical is mutual understanding?”Reliability  was assessed for the eight different variables separately . The intThe subsection of the Dutch version of the ASKAS  was alsoGender , age (in years), level of education ), tenure within care (in years), and current function  were assessed. Second, participation in training in handling resident sexuality  was assessed. Finally, care organization (labeled as A through F) and the reported presence of policy concerning resident sexuality (yes/no) were included.Before data collection, a power analyses was performed to find an appropriate sample size, using the program G*power . To reacT-tests were used for two categories: policy regarding sexuality within an organization (yes/no). Analysis of variances (ANOVA) was used for more than two categories: level of education, employer, function, and sexual education. Differences among continuous independent variables  were assessed using linear regression analysis.Descriptive analyses were performed to assess participant characteristics. Means and standard deviations were assessed for continuous measures, percentages for categorical measures. More descriptive statistics were estimated to assess preliminary differences in attitudes regarding residents’ sexuality between groups of employees. p values. The .1 of p level is considered marginally significant, owing to the large number of variables in the model.Hierarchical OLS regression analyses were conducted to examine the effects on and the addition to the variance explained in the dependent variable: attitude toward resident sexuality. Knowledge of resident sexuality was entered in Model 1. Because of the expected interrelation between knowledge and attitude, the control variables age, level of education, and years of tenure were included in Model 2 (gender was excluded because of the overrepresentation of women). Employment , policy regarding resident sexuality, current function, and training were included in Model 3. Finally, in the last model, our main independent variables of interest, person-centered care and the eight variables measuring organizational culture, were entered (Model 4). Dummy variables were constructed for four variables with categorical measures: level of education , employer (Organization B is the reference category), function (caregivers is reference category), and training (none is reference category). Values of .1, .05, and .01 on α level were used to test for significance of the M) and standard deviations (SD)) have demonstrated that differences in attitude toward resident sexuality were found between groups of employees based on levels of education F  = 11.36, p < .01. Employees with high levels of education reported more positive attitudes than other employees; function F  = 10.88, p < .01. Therapists reported more positive attitudes than direct caregivers and other employees. Finally, attitudes were significantly influenced by the presence or absence of policy t (165) = − 3.79, p < .01. Employees, who reported that policy regarding resident sexuality was not present, were found to have a more positive attitude than employees who did report that policy was present in their RCF organization. Descriptive results from analyses between care staff of different employers cannot be reported owing to inequality in group sizes. No single effects were found for age, years of tenure, and if employees received training concerning resident sexuality .Descriptive analyses  24.39, p < .01), and this effect remains in all subsequent models (yet changes slightly in magnitude). More knowledge of resident sexuality goes together with a positive attitude toward resident sexuality. Model 2 shows that care staff with a ‘high vocational level’  are found to have more positive attitudes toward resident sexuality than the ‘average vocational level’, the reference category. At first, the highest educated caregivers were also found to enhance more negative attitudes but this result diminishes as more variables are included. We found no effects of age on caregiver attitudes and years of tenure. In Model 3, significantly more negative attitudes were found in care staff of Employers B and F than Employer D, the reference category. This effect, however, diminished in Model 4, in which the culture of the organization and person-centered-care are included in the model. The presence of policy regarding resident sexuality  was significantly and positively associated with the attitudes of care staff in Models 3 and 4.Complete results for all models of the effects on care staff attitudes toward resident sexuality are presented in Table F  = 2.94, p < .01. In this final model, person-centered care and the eight variables on the culture of the organization explained an additional 5% of the variance in attitudes. Person-centered care was found to significantly predict differences in attitudes . Employees reporting to provide more person-centered care, report more positive attitudes with regard to resident sexuality. Of the six included variables measuring organizational culture, only the descriptive measurement of the support orientation was marginally significant in predicting differences in attitudes . Employees reporting more supportive behaviors, policies, and procedures reported more positive attitudes toward resident sexuality.In the final model (Model 4) 44% of the variance in attitudes toward resident sexuality was explained by all of the variables together, Person-centered care was found to have a significant effect on the attitudes of care staff towards the sexuality of residents with dementia. Employees of care organizations, who feel they provide more person-centered care, have more positive attitudes toward the sexuality of residents. The person-centered care paradigm advocates that residents are viewed in a holistic and empowering way , 22. TheRegarding the culture of the organization, only the descriptive part of the support orientation, including observable supportive behavior, procedures, and policy in the organization, marginally affected care staff attitudes toward resident sexuality. Care staff that report their care organization to be observably more supportive had more positive attitudes toward the sexuality of residents with dementia. Our results are in line with the assumption made by Roach (2004), who noted the influence of the culture of an organization on care staff attitudes.Results also demonstrated that the presence of policy had a significant impact on attitudes of care staff. When reporting the absence of a policy considering resident sexuality in their organization, they had a more positive attitude toward resident sexuality. This result is in contrast with previous research, in which the presence of policy was perceived as having a positive influence on care staff attitudes , 20. ThiAlso contrary to previous research , our samFinally, we found that knowledge of the sexuality of residents influenced care staff attitudes. This variable was included as a control variable in this study, although the results confirm previous research  that theThis study is characterized by several strengths and limitations. A first strength is that this study is, to our knowledge, the first attempt to assess the effect of organizational factors on care staff attitudes toward sexuality of residents with dementia. Although assumptions were made in previous research, an actual assessment has not yet been undertaken , 14, 20.A second strength lies in the data collection. The completion of the questionnaires was planned after scheduled (team) meetings. Remaining participant questions could be answered before, during, and after completion. Most questions were related to textual and lingual ambiguities; none were related to completing the knowledge question. During some team meetings, discussion arose on the topic of resident sexuality. The topic raised thoughts and concerns among care staff, as they potentially reevaluated their knowledge, attitude, and skills in responding to the sexual needs of residents with dementia.This study also has limitations. First, although validated instruments are used, person-centered care and organizational culture are considered difficult to operationalize. For example, the definitions of both constructs are subject to different definitions.Second, the way data were collected yielded a limitation, because care staff might have felt peer pressure to participate. Although both the author and trained student assistant emphasized that participation was voluntary and withdrawal was possible at any time, the care staff still chose to participate.Third, we encountered limitations with the questionnaires. First, the way measures are granted in the knowledge section of the ASKAS questionnaire prompted a discussion among the authors. An incorrect answer was granted a “higher” score than the “I don’t know” option, which seems counterintuitive. It means that respondents who admit to not knowing the answer are perceived as having less knowledge of resident sexuality than respondents who gave an incorrect answer. However, this questionnaire has been well studied and validated , and wasA fourth limitation lies in the study’s rather small scale. Only one region in the Netherlands was selected, which is a limitation with regard to generalization. Moreover, in this region of the Netherlands, most residents are Christian (Catholic or Protestant), which is why we did not include religion as an individual factor .Finally, the gender distribution was a limitation, because women were overrepresented in the sample. A gender effect on the attitudes could not be explored owing to this distribution. However, this distribution is representative of the actual situation in clinical practice, and this was also the experience of researchers who conducted previous studies , 10.Next to known benefits , 22, theHowever, neither implication is straightforward in its practical implementation. Both imply a profound change on the organizational level , such asFurthermore, greater knowledge of resident sexuality was specifically found to benefit care staff attitudes toward resident sexuality. The way to improve this knowledge needs more detailed consideration, because participation in education in resident sexuality did not significantly influence these attitudes in our study. In previous research, greater knowledge was found as a result of a training program .To further close the lacuna in research, the assumed influence of the attitude toward resident sexuality on the actual behavior of care staff needs further investigation. The behavior of care staff was found to be important, because they influence the possibility of residents and their possible partners expressing sexuality as they want. Spouses of residents mentioned, in qualitative research, that the behavior of care staff (both direct and indirect care staff) was important to their experiences of intimacy and sexuality . HoweverFinally, replication of this study, in another cultural setting, could add detail to the image that is presented here, especially as future generations of the elderly enter RCFs, and will probably demand more facilitation with regard to their intimate and sexual needs . MoreoveTo establish a broader understanding of the attitudes of the care staff toward the sexuality of residents with dementia, it is important to know more about the organizational factors (person-centered care and the culture of the organization) that might influence factors these attitudes, next to factors on an individual level . The aim of this study was to determine the effect of these two organizational factors.Person-centered care was found to influence the attitudes and a supportive culture of the care organization was found to marginally influence care staff attitudes toward the sexuality of residents with dementia that they care for positively.Moreover, knowledge of the sexuality of residents influenced care staff attitudes positively and the known presence of policy regarding resident sexuality influenced the attitudes negatively, which contradicted results from previous research.


ACR) in first morning void (FMV) urine samples collected over three days is the recommended method for measuring and monitoring albuminuria in adults in the clinical setting. Such a guideline is not available for toddlers and young children. We tested several urine collection strategies for albuminuria measurement in toddlers in a prospective observational study.Urinary albumin:creatinine ratio (UAC) and UACR were compared using only the first measurement and using all three measurements per time point. In addition, these were compared with published CV of adults.Both a FMV and a random daytime urine sample were collected on three consecutive days at week 0, 4, and 8 in toddlers aged 12–48 months. Intra-individual coefficients of variation (CV) of urinary albumin . CV of UAC was similar to values published for adults. However, UACR CV was considerably higher in toddlers.A total of 80 toddlers  were included. Intra-individual CV of FMV samples appeared lower than with random samples. The intra-individual CV in UThese data show that—in analogy with adult data—multiple first morning void urine samples should be preferred to single or random urine samples for establishing and monitoring albuminuria in toddlers. Further studies are needed to investigate why creatinine correction for differences in urine dilution is less effective in children. Measurement of urine albumin is important, both in clinical practice and in epidemiological studies and clinical trials. To accurately quantify albuminuria in the adult population, a 24-hour urine collection is considered the gold standard. This method is rather cumbersome and has thus been replaced in clinical practice by alternatives such as collection of a first morning void (FMV) or a random daytime urine sample. To correct for urine concentration in these samples, albumin concentration is divided by creatinine concentration, where the latter is assumed to be excreted at a constant rate over 24 hours.ACR, represented by the coefficient of variation (CV), is lower in FMV than in random samples. Furthermore, FMV performs at least as good as a 24-hour urine collection, so that a first morning void urine sample constitutes a good alternative to a 24-hour urine collection.[To validate these alternative albuminuria measurements against the 24-hour urine albuminuria measurement, several studies have been performed. These stHowever, there is still no consensus on how to optimally measure and monitor albuminuria over time in toddlers. Because a 24-hour urine collection is even more cumbersome in young children than in adults and is not feasible for regular controls, it is very important to have reliable alternatives that can be easily implemented in clinical practice.Therefore, in this study we have compared various urine collection techniques to measure and monitor albuminuria in healthy toddlers. The results are compared with previously reported intra-individual CV’s of albuminuria and creatinine measurements in adults.In this observational prospective cohort study, healthy toddlers (12–48 months of age) were recruited from May 2015 until October 2016 via well-baby clinics and children’s daycare centers in Groningen, Drenthe and Friesland (three Northern provinces in the Netherlands). Children with previously diagnosed kidney disease were excluded from the study. Both parents or the legal guardian of the child signed for informed consent. The study was approved by the medical ethical committee of the University Medical Center of Groningen (UMCG) (METc reference number 2014–512) and was conducted according the principles of the declaration of Helsinki.Data was collected on three consecutive days at three different time points: 0, 4 and 8 weeks. Data consisted of three first morning voids (FMV), three random daytime samples and a questionnaire at each time point . All wer® device, which is a validated tool for urine collection in both children who are continent for urine and children who are not.[AC) was measured with the Roche Modular P using the immunoturbimetric assay and expressed in mg/L. Urinary creatinine concentration (UCR) was measured with the Roche Modular P using creatinase to sarcosine oxidase based colorimetric method and expressed in g/L. To correct UAC for urine dilution, urinary albumin:creatinine ratio (UACR) was assessed. Urine samples were tested for leukocytes with urinary dipsticks (Combur-test® strips), in order to exclude an intercurrent urinary tract infection.Urine was collected with the PeeSpot are not. It consi are not.,5 Tubes Before starting the study, we calculated that a study population of 100 subjects would provide 80% power in detecting a CV between 14% and 24% (difference of 5% relative to 19%), assuming a SD of the within subject CV of 20%. However, in an interim analysis halfway the study, we discovered that the SD of the within subject CV was considerably lower (17.5%). Therefore, we decided to include 80 participants.AC and UACR are represented by geometric mean and sample standard deviation. Geometric mean was used as albuminuria has a non-parametric distribution. In order to assess the intra-individual variability of albuminuria, individual standard deviation and individual geometric mean were assessed to calculate coefficients of variation (CV) of albuminuria per individual with the following formula: AC ≥ 20 mg/L or UACR ≥ 30 mg/g).[Descriptive characteristics of the study population were reported as mean ± standard deviation. U30 mg/g). DifferenOf the 95 children of whom informed consent was signed, a total of 80 toddlers (mean age 26.6 months (SD 10.8), 53% male) completed follow-up and were included in this analysis. The remaining 15 children were lost to follow-up. The main characteristics of the study population are shown in AC and UACR) than random sample collections, although the differences did not always reach statistical significance. In addition, AC and UACR) compared to a single collection per time point. The lowest median intra-individual CV was observed when UAC was measured in FMV on three consecutive days (38.3%). When comparing the CV’s of albumin concentration with albumin:creatinine ratio, UACR consistently showed a higher CV. This was the case both with a single urine sample per time point or three urine samples per time point and with either FMV or random samples.AC ≥ 20mg/l or UACR ≥ 30mg/mg) as well as using the geometric mean of the three consecutive samples (geometric mean of 3 consecutive samples within time point UAC ≥ 20mg/l or UACR ≥ 30mg/mg). It is clear from the results in AC) is very low (around 2%) using the adult guideline, and is higher (around 5%) using the geometric mean. Interestingly, creatinine correction gives very high prevalences (17.5 to 33.8%) when using both the adult guideline and the geometric mean.In clinical practice, urine albumin excretion is classified in normo-, micro, and macroalbuminuria, or recenAC and UCR in this population were similar. However, UACR CV in our cohort was much higher.We compared the intra-individual CV found in this study with results from a previous study in adults. In this In this study we compared for the first time various urine collection strategies to optimally measure and monitor albuminuria in toddlers. As expected, albuminuria CV’s are lower when measured in first morning void compared to random samples, and when done with 3 consecutive urine collections versus one collection. This supports implementation of multiple first morning void urine samples for establishing and monitoring albuminuria in toddlers, conform to adult guidelines.ACR was used as compared to UAC. This could be due to a larger variability of urine creatinine in children. However, we did not observe a difference in urinary creatinine CV’s between adults and toddlers. We therefore do not have a clear explanation why creatinine correction does not work in toddlers, other than that urine creatinine measurement itself is flawed in children.However, we also identified differences with the adult population. In adults, urine albumin correction for urine creatinine  helps to correct for differences in urine dilution and reduces variability in the measurements in first morning void or random urine samples. In adults this indeed leads to a more precise assessment of albuminuria and a smaller intra-individual variability in albuminuria. Surprisingly, in our study we observed higher CV’s when UACR ≥ 30mg/mg) also increases substantially as compared to the prevalence defined on the basis of UAC (≥ 20mg/l). The most likely explanation for this is the fact that children have a lower muscle mass, which accounts for an overall lower urinary creatinine excretion and therefore higher physiologic values of UACR. Indeed, multiple studies reported that UACR is higher in younger children.[AC and UACR in FMV samples and compare these results with 24-hour urine albumin excretion to establish optimal age-dependent reference values for UACR. In addition, the urine creatinine measurement itself in children should be validated and compared with adults to find an explanation why UACR correction does not work in children. At this stage, we recommend to interpret UACR in young children with extreme caution.Another difference we encountered with the adult population is that when using urine albumin correction for creatinine in children, the prevalence of microalbuminuria Click here for additional data file.