It is most likely that the need for long-term care increases considerably in the next decades due to demographic shifts. Thus, we aimed at identifying the preferences for characteristics of professional long-term caregivers among old age individuals in Germany.Data were gathered from a population-based survey of the German population aged 65 and above in 2015 (n = 1006).It was important for individuals in old age that long-term caregivers were ‘empathetic, kind’ (99.3%), ‘punctual, reliable’ (98.2%), have an ‘orderly appearance’ (96.4%), work in a ‘small team’ (92.5%) and have ‘enough time’ (91.5%). Moreover, while most of the individuals (76.5%) reported high preferences for ‘German language’, the preferences were lower for ‘same cultural background’ (54.2%) and ‘same gender’ (35.7%). In multiple logistic regressions, preferences for ‘same gender’ were positively associated with being female [OR 8.3 (5.6–12.1)], living with partner or spouse [OR 1.4 (1.0–1.9)], and being born abroad [OR 1.8 (1.1–3.1)]. Preferences for ‘German language’ were positively associated with being female [OR: 1.5 (1.1–2.1)]. Preferences for ‘same cultural background’ were positively associated with age [OR 1.0 (1.0–1.1)], living with partner or spouse [OR 1.4 (1.0–1.9)], and East Germany [OR 1.9 (1.3–2.7)]. Preferences for ‘orderly appearance’, ‘empathetic, kind’, ‘punctual, reliable’ and ‘small nursing team’ were all not significantly associated with included independent variables, whereas preferences for ‘enough time’ were positively associated with being female [OR 1.9 (1.1–3.5)], living with partner or spouse [OR 1.9 (1.1–3.4)], education , not providing care for family/friends [OR 1.9 (1.1–3.3)], and involvement in the issue of need for care [OR 1.3 (1.1–1.6)].Our data suggest that it is important to almost every individual aged 65 and above in Germany that professional long-term caregivers are (i) empathetic, kind, and understanding, (ii) punctual and reliable, (iii) have enough time , (iv) and have an orderly appearance. Furthermore, several factors such as gender or region were found to be associated with preferences for characteristics of caregivers. By knowing these factors, nursing services can tailor their activities to the needs of care-recipients.The online version of this article (doi:10.1186/s13104-017-2717-3) contains supplementary material, which is available to authorized users. It is most likely that the number as well as the proportion of old age individuals will considerably increase in the upcoming decades . Since aWhile it is well known that individuals prefer to be cared for by family members at home as long as possible —which isHence, we aimed at examining which factors are associated with preferences for characteristics of professional long-term caregivers among individuals in old age (65 years and above). Previous studies found that, besides professional caring skills, soft skills such as empathy or sensitivity are important caregiver characteristics for care-recipients –11. WithThis knowledge is important for caregivers as well as nursing services. Knowing the factors which are important for care-recipients might help to reduce the gap between care-recipients’ expectations and reality in professional caregiving at home or nursing home facilities. Our study focused on individuals aged 65 and above as these individuals are at risk of needing long-term care in the near future. Moreover, it was demonstrated that these individuals are well-informed about different aspects of long-term care .Trained staff interviewed 1006 individuals aged 65 years and over living in private households with conventional telephone connection by phone  in 2015 , 15. FieWhen the respondents agreed to complete the interview, oral informed consent was given. Oral consent is common in survey research in Germany. The ethical guidelines of the International Code of Marketing and Social Research Practise by the International Chamber of Commerce and the European Society for Opinion and Marketing Research were followed. Drawing on expert interviews  as well In order to improve the questionnaire different pretests were done including evaluation conversations and a pilot study. First, evaluation conversations were carried out with experts, guided interviews and telephone interviews with participants not included in the current study. Subsequently, a pretest was done under real life conditions (n = 31) with the target-population. Furthermore, the trained staff from USUMA received a glossary where the items and the underlying intentions were explained in detail.… they have the same gender (short (notation used in Table … they have very good German language skills (short: German language).… they share the same cultural background .… they show an orderly appearance (short: orderly appearance).… they are empathetic, kind, understanding .… they are punctual and reliable .… they have enough time  (short: enough time).… the nursing team is small .The preferences for characteristics of professional caregivers were quantified as follows: Regarding the characteristics of professional caregivers irrespective of care provided at home or in a nursing home, it is important to me that … :The dependent variables were dichotomized  to reflect high preferences versus low preferences. For the sake of readability, the terms “importance” and “preferences” have been used interchangeably in our study because we assume that these factors are highly correlated.Socioeconomic variables were used as follows: age, sex, whether they have children or not (Ref.: no children), whether they were born in Germany or not (Ref.: not born in Germany), West and East Germany (Ref.: East Germany), living situation ), status of health insurance , and education ).Moreover, individuals were asked whether they have ever provided informal care for family or friends (Ref.: no). As for health status, self-rated health (from 1 “very bad” to 5 “very good”) and level of care (Ref.: no) were included as independent variables. Recipients are classified into three levels of care (depending on the care required) based on an assessment by a nurse or a physician of the medical service of the German statutory health insurance system. The need of care was dichotomized . Furthermore, the involvement in the issue of need for care (“How much have you thought about the issue of ‘need for care’”) was assessed, ranging from 1 (“very little”) to 5 (“very much”).Bivariate associations between preferences  and independent variables were analyzed using t-tests, Chi square and Fisher’s exact tests, as appropriate. Logistic regressions were used to investigate the relationship between our numerous independent variables and the preference outcomes. Eight logistic regressions models were computed. Outcome variables were (preferences for…): (i) Same gender, (ii) German language, (iii) Same cultural background, (iv) Orderly appearance, (v) Empathetic, kind, (vi) Punctual, reliable, (vii) Enough time, (viii) Small team. In each model, independent variables were age, sex, living situation, region, education, place of birth, having children, status of health insurance, provided care for family/friends, level of care, self-rated health, and involvement in the issue of need for care.In additional analysis, we used penalized maximum likelihood logistic regression , 18. It Table Preferences for ‘same gender’ were significantly associated with age, gender, living situation, education, status of health insurance, need of care, and self-rated health. Preferences for ‘German language’ were significantly associated with gender, education, having children, status of health insurance, and self-rated health. Preferences for ‘same cultural background’ were significantly associated with age, region, having children, provided care for family/friends, self-rated health, and involvement in the issue of need for care. Neither preferences for ‘orderly appearance’, nor preferences for ‘empathetic, kind’, nor preferences for ‘punctual, reliable’ were significantly associated with included predictors. Preferences for ‘enough time’ were significantly associated with age, education, and involvement in the issue of need for care. Preferences for ‘small team’ were significantly associated with education.Results of multiple logistic regressions are depicted in Table Preferences for ‘same gender’ were positively associated with being female [OR 8.3 (5.6–12.1)], living with partner or spouse [OR 1.4 (1.0–1.9)], and being born abroad [OR 1.8 (1.1–3.1)]. Preferences for ‘German language’ were positively associated with being female [OR 1.5 (1.1–2.1)]. Preferences for ‘same cultural background’ were positively associated with age [OR 1.0 (1.0–1.1)], living with partner or spouse [OR 1.4 (1.0–1.9)], and East Germany [OR 1.9 (1.3–2.7)]. While preferences for ‘orderly appearance’, ‘empathetic, kind’, ‘punctual, reliable’ as well as ‘small nursing team’ were all not significantly associated with included independent variables, preferences for ‘enough time’ were positively associated with being female [OR 1.9 (1.1–3.5)], living with partner or spouse [OR 1.9 (1.1–3.4)], education , not providing care for family/friends [OR 1.9 (1.1–3.3)], and involvement in the issue of need for care [OR 1.3 (1.1–1.6)].Since some of the cells formed by the outcome and categorical predictor variable have no observations, we repeated our estimates with penalized maximum likelihood logistic regressions (instead of logistic regressions) in additional analysis. In terms of effect sizes and significance, our findings were comparable to those from the main logistic regressions (results not shown but available upon request).By using a large, population-based survey in individuals aged 65 and above in Germany, we aimed at investigating which factors are associated with characteristics of professional long-term caregivers among individuals in old age. More specifically, the predictors of preferences for characteristics of caregivers were examined.The preferences were highest for ‘empathetic, kind’ (99.3%), ‘punctual, reliable’ (98.2%), ‘orderly appearance’ (96.4%), ‘small team’ (92.5%) and ‘enough time’ (91.5%). Moreover, most of the individuals (76.5%) reported high preferences for ‘German language’, whereas the preferences were markedly lower for ‘same cultural background’ (54.2%) and ‘same gender’ (35.7%).In multiple logistic regressions, preferences for ‘same gender’ were positively associated with being female, living with partner or spouse, and being born abroad. Preferences for ‘German language’ were positively associated with being female. Preferences for ‘same cultural background’ were positively associated with age, living with partner or spouse, and East Germany. Preferences for ‘orderly appearance’, ‘empathetic, kind’, ‘punctual, reliable’ and ‘small nursing team’ were all not significantly associated with included independent variables, whereas preferences for ‘enough time’ were positively associated with being female, living with partner or spouse, education, not providing care for family/friends, and involvement in the issue of need for care.As already found in the literature , 10, sofBeing female was positively associated with preferences for ‘same gender’ and preferences for ‘German language’. This is also in line with previous studies reporting that women prefer a general practitioner of the same gender –25. MoreLiving with partner was positively associated with preferences for ‘same gender’ and preferences for ‘same cultural background’. Consequently, individuals living with partner might be more afraid of care provided by the opposite gender. This might be explained by the fact that individuals in old age living with partner could not imagine that other individuals apart from their wife or husband provide assistance with basic activities of daily living such as toileting or bathing.In addition, preferences for ‘same cultural background’ were positively associated with age and East Germany. These associations might reflect differences in cultural values –30. DiffThe non-significant associations between ‘orderly appearance’, ‘empathetic, kind’, ‘punctual, reliable’ as well as ‘small nursing team’ and included predictors might be mainly explained by the fact that nearly every individual wishes to have them in the future. Thus, these preferences for characteristics of professional caregivers might be generally seen as basic (human) needs.The positive association between preferences for ‘enough time’  and being female, higher education as well as living with partner or spouse might be explained by the greater need for social interactions in these groups , 33.Furthermore, while the positive association between involvement in the issue of need for care and preferences for ‘enough time’ might be explained by the fact that a high involvement in this issue is associated with a higher preference for soft skills of caregivers, the positive association between not providing care and the preferences for ‘enough time’ was quite unexpected and might be explained by unobserved factors associated with providing informal care . In It should be highlighted that our data were derived from a large, population-based sample among individuals in old age. Moreover, numerous important independent and dependent variables were captured. For example, adjusting for numerous potential confounders, we provide novel evidence that region (West and East Germany) is differentially associated with preferences for ‘same cultural background’. However, our study also has some limitations. This is a cross-sectional study. Therefore, temporal relationships cannot be determined. Future studies aimed at examining the long-term impact of predictors on long-term care preferences are needed. In addition, upcoming studies should validate the instruments used in our study. In addition, the self-reported data might suffer some degree of inaccuracy.Our data suggest that it is important to almost every individual aged 65 and above in Germany that professional long-term caregivers are (i) empathetic, kind, and understanding, (ii) punctual and reliable, (iii) have enough time , (iv) and have an orderly appearance. Furthermore, high preferences for skills in German language were reported. Moreover, it is important to them to be cared for in a small team. Characteristics such as having the same cultural background or having the same gender are less important. Furthermore, several factors such as gender or region were found to be associated with characteristics of caregivers in nursing home facilities. By knowing these factors, nursing services can tailor their activities to the needs of care-recipients. Reducing the gap between caregivers’ needs and reality might in turn help to increase the satisfaction of care-recipients in nursing home facilities.


Our results indicated that acute IMI exposure moderately induce apoptosis in the brain but caused severe histopathological lesions, inflammation, and oxidative stress in the gills, liver, and brain of the common carp.Imidacloprid (IMI) is a neonicotinoid that is widely used for the protection of crops and carnivores from insects and parasites, respectively. It is well known that imidacloprid exposure has a harmful effect on several organisms. However, there is little information about imidacloprid toxicity in aquatic animals, particularly fish. Thus, in the current study, we assessed the histopathological changes; activation of iNOS, 8-OHdG and TNF-α; and expression levels of caspase 3, iNOS, CYP1A and MT1 genes in the common carp exposed to imidacloprid. For this purpose, fish were exposed to either a low dose (140 mg/L) or a high dose (280 mg/L) of imidacloprid for 24 h, 48 h, 72 h and 96 h. After IMI exposure, we detected hyperplasia of secondary lamellar cells and mucous cell hyperplasia in the gills, as well as hydropic degeneration in hepatocytes and necrosis in the liver. Moreover, 8-OHdG, iNOS and TNF-α activation was found particularly in the gills and liver but also moderately in the brain. Transcriptional analysis showed that caspase 3 expression was altered low dose and high doses of IMI for 72 h and 96 h exposure ( Long-teSeveral environmental pollutants such as pesticides can cause programmed cell death (PCD) or apoptosis in animals and humans ,28. ApopMetallothioneins (MTs) can bind to zinc and copper. MTs have been shown to eliminate the adverse effects of exposure to toxic elements, including heavy metals and pesticides, and to protect organisms from several stress conditions such as oxidative stress . PesticiSince there are no available studies on IMI toxicity in the common carp, we investigated the potential toxic effects of IMI and assessed inflammation, oxidative stress, DNA damage and apoptosis associated with IMI in this animal model. For this purpose, we first evaluated the histopathological changes due to IMI toxicity by histopathological examination of gills, liver and brain. Subsequently, the activation of iNOS, 8-OHdG and TNF-α was assessed by immunofluorescence (IF) in the brain. Lastly, we used RT-qPCR to measure mRNA levels of caspase 3, iNOS, CYP1A and MT1 genes in the brain.22.19H10CIN5O2 , was purchased from Sigma-Aldrich (Germany). The molecular structure of imidacloprid is shown in Imidacloprid (IMI), C2.2Common carps were obtained from Atatürk University, Faculty of Fisheries and the Inland Water Fish Breeding and Research Center. This study was performed in accordance with the approved ethical rules of Atatürk University. Fish were fed for 45 days in a stock pond to achieve acclimatization to their environmental conditions. After the adaptation period, twelve fish were put in each water aquarium (volume 70 l). The fish were exposed to the IMI LD50 for periods of 24 h, 48 h, 72 h and 96 h. The tested fish had an average weight of 88 ± 1.8 g and length of 12.5 ± 0.4 cm. The physico-chemical properties of the aquarium water were the following: O2 = 8.3 ± 0.1 ppm, pH = 7.6 ± 0.2, SO4-2 = 10.32 ppm, CO3–2 = 124 ppm, HCO3- = 145.6 ppm, NO3- = 3.45 ppm, NO2- = trace, conductivity = 276 ms/cm, total hardness = 119 ppm of CaCO3, and temperature (24–330C) and water was changed every day. The fish were divided into six groups. Two aquaria were prepared for each group. Each aquarium contained 12 fish (n = 72). The fish were exposed to the chemical for periods of 96 h in static aquarium systems. The fish in groups I and II were the control. The fish in groups III and IV were administered doses of 280 mg/L (1/5 LD50), whereas those in groups V and VI were given a dose of 140 mg/L (1/10 LD50) of imidacloprid . These d2.3Tissue samples were stored for 1 day to be fixed in a 10% buffered formalin solution. Routine histopathological alcohol and xylene series for samples was performed in Shandon Citadel 2000 (USA) tissue system. After the routine histopathology process, all samples were poured into paraffin for blocking and prepared microtome sections in 5 μm by using rotary microtome (Leicia RM 2255). All sections were stained with routine Haematoxylin eosin staining for histopathological evaluation. Slides were examined under the light microscopy (Olympus BX51 with DP72 camera attachment).2.42O2 solution was dropped on slides for ten minutes to inactivate endogenous peroxidase activity. Then the slides were immersed in antigen retrieval solution  (pH 6.0) and heated in microwave for 15 min to unmasked antigens. After waiting for cooling, sections were incubated with protein block solution  to prevent nonspecific bindings for 15 min. A solution  was used for antibody dilution. Sections were incubated with diluated antibodies  at room temperature for 30 min. The seconder polyclonal fluorescence antibody goat pAB to Rb IgG (FITCH)  was used for sections incubated with iNOS and TNFα. The seconder monoclonal fluorescence antibody pAB to Ms IgG (FITCH)  was used for sections incubated with 8-OHdG. Finally all sections were covered with lamellas by using glycerin diluated 1/9 with distilled water for evaluation under fluorescence microscopy system (Zeiss Scope A1 with Axio cam ICc5 camera attachment system).After deparaffinization, 3% H2.5Cyprinus Carpio) using the Oligo 6.0 and Primer 5.0 design programs. Primer sequences and the corresponding PCR conditions are listed in −ΔΔCT method [For the assessment of mRNA expression of caspase 3, iNOS, CYP1A and MT1, real-time PCR was used with a CFX96 TouchTM Real-Time PCR Detection System . Total RNA was isolated from brain tissue using TRIZOL reagent  according to the manufacturer’s instructions. The RNA samples were treated with RNase-free DNase , and then cDNA synthesis was performed using a QuantiTect Reverse Transcription kit  starting from 1 μg of the treated RNA according to the manufacturer’s instructions. One microgram of each cDNA was used as a template for amplification using SYBR Green Master Mix  and gene-specific primers. Real-time PCR primers  were designed based on the sequences of Common Carp . Differences in measured parameters among the groups were analyzed with a nonparametric test . Dual comparisons between groups exhibiting significant values were evaluated with a Mann-Whitney U test (p < 0.05).One-way ANOVA followed by Tukey’s post hoc test was used to analyze the mRNA levels of caspase 3, iNOS, CYP1A and MT1 in treated and control groups. The analysis was performed using the GraphPad Prism software version 7.0 . RT-qPCR results were expressed as the mean ± SEM (standard error of the mean). Differences were considered to be statistically significant with p values of p < 0.05, 33.1p < 0.05). The most obvious lesions in liver and gills at both doses were detected at the 96 h exposure.Tissues obtained from the control groups showed normal features A,D,G. No3.2p > 0.05). Positive signals for 8-OHdG were observed in gill epithelial cells . However, iNOS positivity was observed in hepatocytes (p < 0.05). Lastly, 2.5D immunopositivities for TNF-α, iNOS and 8-OHdG were observed, as shown in Fig. S1B. C. D)Weak positive signals were observed for all the biomarkers in tissues obtained from the control groups Figs. A, D, G  al cells B, hepatoal cells E and neual cells H in the al cells C, F, I ial cells B, C . However, when treating with the low dose of IMI for 96 h and the high dose of IMI for 96 h, 72 h, the caspase 3 gene expression was up-regulated compared to that of the control group . However, 96 h IMI exposure with the low and high dose was significantly up-regulated CYP1A gene expression  and the high dose  of IMI  was performed to assess the activation of iNOS, 8-OHdG and TNF-α in IMI-exposed gills, liver and brain of the common carp. IF is more sensitive than immunohistochemistry (IHC) and histopathological examination . In the p < 0.05). Previous studies have shown that imidacloprid intoxication induces the caspase 3-dependent apoptosis pathway in the brain of honey bees and neonatal rats [Danio rerio) [In the present study, caspase 3 mRNA expression was not significantly up-regulated in a time-dependent manner in the brain of the imidacloprid-exposed common carp; only the exposure to the low and high dose of imidacloprid for 96 h promoted caspase 3 expression  . Mesnageo rerio) . Furthero rerio) . There ap < 0.05, p < 0.01 and p < 0.001). This result confirmed the immunopositivity of iNOS and indicated that IMI exposure triggers oxidative stress and inflammation in the brain tissue.Duzguner and Erdogan (2012) observed that iNOS synthesis was increased in the liver and central nervous system of rats exposed to IMI and that IMI toxicity induced oxidative stress and inflammation in rat livers and brains . Lanore Danio rerio) embryos [p > 0.05) except with the low and high doses of IMI for 96 h in the brain (p < 0.05). Therefore, acute IMI toxicity may not significantly affect CYP1A expression in the brain of the common carp.Cytochrome P450 (CYP) enzymes are important modulators of chemical detoxification of herbicides, pesticides, and carcinogens in animals ,62. In a embryos . Li et a embryos . In thisp < 0.05 and p < 0.01), whereas the other doses and exposure time points did not induce any change. Therefore, acute IMI toxicity does not promote the induction of MT1. The up-regulation of MT1 in the brain exposed to a high dose of IMI for 72 h and 96 h indicates that IMI toxicity leads to oxidative stress at these time points. In addition, chronic IMI toxicity may induce oxidative stress-dependent MT1 synthesis in the brain of the common carp. In the future, it will be necessary to investigate the MT1 response to chronic IMI toxicity in the common carp.MTs play a role in the regulation and detoxification of metals. In addition, several other factors such as antibiotic use, freezing, anoxia and pesticide contamination can lead to the induction of MTs ,68. InduIn conclusion, acute IMI toxicity caused histopathological lesions in the gills and liver; activation of 8-OHdG, iNOS and TNF-α in the gills, liver and brain; significant up-regulation of the iNOS gene; and a modest up-regulation of the caspase 3, CYP1A and MT1 genes in the brain. Based on these results, we concluded that inflammation and oxidative stress are induced by IMI exposure in the common carp. Therefore, the assessment of fish mortality in aquatic ecosystems will be facilitated by histopathological examinations. The use of pesticides should not be left to the initiative of farmers. Furthermore, it should not be forgotten that all pesticides have been tested and evaluated because of their risk of synergistically cause further and permanent damage; for this reason, a pesticide atlas should be developed for aquatic animals.


Haematococcus pluvialis is a commercial microalga, that produces abundant levels of astaxanthin under stress conditions. Acetate and Fe2+ are reported to be important for astaxanthin accumulation in H. pluvialis. In order to study the synergistic effects of high light stress and these two factors, we obtained transcriptomes for four groups: high light irradiation (HL), addition of 25 mM acetate under high light (HA), addition of 20 μM Fe2+ under high light (HF) and normal green growing cells (HG). Among the total clean reads of the four groups, 156,992 unigenes were found, of which 48.88% were annotated in at least one database . The statistics for DEGs  showed that there were more than 10 thousand DEGs caused by high light and 1800–1900 DEGs caused by acetate or Fe2+. The results of DEG analysis by GO and KEGG enrichments showed that, under the high light condition, the expression of genes related to many pathways had changed, such as the pathway for carotenoid biosynthesis, fatty acid elongation, photosynthesis-antenna proteins, carbon fixation in photosynthetic organisms and so on. Addition of acetate under high light significantly promoted the expression of key genes related to the pathways for carotenoid biosynthesis and fatty acid elongation. Furthermore, acetate could obviously inhibit the expression of genes related to the pathway for photosynthesis-antenna proteins. For addition of Fe2+, the genes related to photosynthesis-antenna proteins were promoted significantly and there was no obvious change in the gene expressions related to carotenoid and fatty acid synthesis. Haematococcus pluvialis is a unicellular freshwater microalga that is rich in astaxanthin. The red ketocarotenoid astaxanthin  is the most powerful biological antioxidant and is highly demanded in health care and aquaculture, with the latest research showing that astaxanthin may play roles in anti-depression and preventing UVA-induced skin photoaging [H. pluvialis are under stress conditions, such as nutrient deprivation, high irradiation, or high salt concentration, astaxanthin is produced in the form of fatty acyl mono-or diesters [otoaging ,2,3,4. Aotoaging ,6,7. WheH. pluvialis is now the best source of natural astaxanthin, constituting up to 4% of cellular dry weight [H. pluvialis can be divided into four stages: I, vegetative cell growth; II, encystment (vegetative to immature cyst cells); III, maturation (immature to mature cyst cells); and IV, germination (mature cyst to vegetative cells) [H. pluvialis by optimization of induction conditions have been reported. Jeon found that 30 mM acetate and 170 μEm−2·s−1 light intensity was good for astaxanthin accumulation in H. pluvialis [2+ than by the addition of acetate alone in H. pluvialis, from which he concluded that oxidative stress was involved in the posttranslational activation of carotenoid biosynthesis in acetate-induced cyst cells [2+ to enhance the production of astaxanthin [2+ as variables to explore the molecular mechanism of astaxanthin production in the red-cell stage of H. pluvialis by transcriptome analysis.y weight ,9,10. The cells) . Astaxanluvialis . Kobayasst cells . Hong suaxanthin . FollowiH. pluvialis, the accumulation of astaxanthin is correlated with increasing fatty acid biosynthesis under stress conditions [H. pluvialis cultured under high irradiance [H. pluvialis, the lipid content is close to 35% of the dry weight under stress conditions and the content of polyunsaturated fatty acids (PUFAs) is closed to 50% of the neutral lipid [H. pluvialis could also produce fatty acids as a byproduct which could be used in human health supplements.Moreover, it has been reported that microalgae can produce fatty acids with unusual chain lengths and a high degree of unsaturation, which are not found in significant amounts in nature elsewhere. Prominent examples are the polyunsaturated fatty acids (PUFAs) eicosapentaenoic acid  or docosahexaenoic acid , ω-3 fatty acids, that are commercially produced as human health supplements ,16. In Hnditions . Gwak inradiance . In H. pal lipid . This suH. pluvialis were sequenced using Illumina Hiseq2000 . Through transcriptome analysis of H. pluvialis, we attempt to find the specific changes in metabolic pathways of algal cells grown with acetate and Fe2+ under high light, which may lay a foundation for insight into the astaxanthin synthesis mechanism in H. pluvialis and also provide guidance for production of astaxanthin in industrial farming.RNA sequencing (RNA-seq) is a newly developed deep-sequencing technology for transcriptome analysis, which has been a key technique for biological inquiry for decades . In the H. pluvialis, fifty to sixty-five million raw reads of each sample were obtained. To guarantee the quality of data used for the analyses, stringent parameters were used to measure the quality of reads and all reads with undetermined information greater than 10% were removed. After this filtering, about sixty million clean reads of each sample were generated, with more than 95% bases showing quality greater than Q20, while the GC percentage was around 60% (k = 25) technique was used to measure the nucleotide sequence of reads. As a no-reference genome project, these clean reads were assembled into contigs using overlap information until the contigs could not be further extended. After a series of subsequent steps, 156,992 unigenes (>200 bp) were generated, with an N50 of 1620 bp and N90 of 456 bp. The size of the unigenes varied from 201 bp to 35,138 bp with a mean length of 1047 bp and a total length of 164,437,994 bp , NT (NCBI nucleotide sequences), KO (KEGG Ortholog), SwissProt , PFAM (Protein family), KOG (Clusters of Orthologous Groups of proteins), and GO (Gene Ontology). As a whole, there were 76,744 unigenes (48.88%) annotated in at least one database, 5918 unigenes (3.76%) were annotated in all of the databases. Specifically, 55,835 unigenes (35.56%), 60,846 unigenes (38.75%), 58,643 unigenes (37.35%), 23,632 unigenes (15.05%) and 21,569 unigenes (13.73%) were annotated in NR, GO, PFAM, KO and KOG, respectively , Cellular Component (CC) and Molecular Function (MF). All of the 60,846 unigenes annotated in the GO database were classified into 56 sub-categories. The top three sub-categories for BP were cellular process, metabolic process and single-organism process, for which the numbers of genes were 35,416 , 32,315 (53.11%) and 26,630 (43.77%) respectively (some of the unigenes could annotated in more than one category). The top three sub-categories of CC were cell part, cell and organelle, for which the numbers of genes were 20,557 (33.79%), 20,577 (33.79%) and 7693 (12.64%) respectively. The top three sub-categories of MF were binding, catalytic activity and transporter activity, for which the numbers of genes were 32,522 (53.45%), 27,253 (44.79%) and 3974 (6.53%) respectively .According to the annotations in the KOG database, 21,569 annotated unigenes were subdivided to 25 groups. The top KOG category was “Posttranslational modification, protein turnover, chaperones” , followed by “General function prediction only” . The smallest groups were “Cell motility”  and “Extracellular structures”  .The 23,632 unigenes annotated using the KO database were classified into five pathways of “hierarchy 1.” These are Cellular Processes, Environmental Information Processing, Genetic Information Processing and Organismal Systems. As for the number of unigenes in “hierarchy 2,” the largest number was 2513 unigenes (10.63%) in the pathway for “Translation,” followed by 1899 unigenes (8.03%) in the pathway for “Carbohydrate metabolism” .H. pluvialis, six comparison groups of DEGs were set as follows: HLvsHG—high light treatment group (HL) compared with green growth group (HG); HAvsHG—addition of acetate together with high light treatment (HA) compared with green growth group; HAvsHL—addition of acetate together with high light treatment compared to only high light treatment; HFvsHG—addition of Fe2+ together with high light treatment (HF) compared with green growth group; HFvsHL—addition of Fe2+ together with high light treatment compared to only high light treatment; HAvsHF—acetate treatment compared with Fe2+ treatment. The DEG numbers for each comparison group are shown in 2+, caused a pronounced change in gene expression and that the effect of acetate was greater than that of Fe2+.To investigate the effect of a different inducing factor of p value < 0.05) in the DEGs responding to high light irradiation. The main categories were in the “Biological Process” domain, including “metabolic process,” “single-organism process” and “single-organism cellular process.” The main categories in the “Molecular Function” domain were “hydrolase activity, acting on glycosyl bonds,” “hydrolase activity, hydrolyzing O-glycosyl compounds” and “phosphorus-oxygen lyase activity.” There was only one category in the “Cellular Component” domain, which was “ribonucleotide-diphosphate reductase complex”  in 22 KEGG terms. Among these, numerous genes were involved in nucleotide metabolism, such as “Amino sugar and nucleotide sugar metabolism,” “DNA replication,” “Purine metabolism,” “Pyrimidine metabolism.” Another large DEG group was enriched in lipid-related pathways, such as “Fatty acid elongation,” “Fatty acid degradation,” “Folate biosynthesis,” “Ascorbate and aldarate metabolism,” “alpha-Linolenic acid metabolism,” “One carbon pool by folate.” The 8846 down-regulated DEGs were significantly enriched in 19 KEGG terms, which were related to metabolism  and biosynthesis . In summary, the genes related to fatty acid pathways and the carotenoid pathway are changed under high light condition. To identify the biological pathways that were active under high light irradiation, the up-regulated and down-regulated DEGs were mapped to canonical signaling pathways in the KEGG database. The 8243 up-regulated DEGs were significantly enriched  DEGs in the comparison of HAvsHG (acetate treatment group under high light condition compared with green growth group) and 1824 (1121 up-regulated and 703 down-regulated) DEGs in the comparison of HAvsHL (acetate treatment group compared with only high light irradiation group). GO enrichment analysis showed that 78 GO terms were significantly enriched in the comparison of HAvsHG. The main categories were in “Biological Process” and “Molecular Function” domain, including “single-organism process,” “single-organism cellular process,” “single-organism cellular process” and “catalytic activity.” There were also some categories related to fatty acid, such as “fatty acid biosynthetic process” and “fatty acid metabolic process.” There were only five significant terms in the “Cellular Component” domain, these were viral envelope, viral membrane, exodeoxyribonuclease VII complex, obsolete flagellum, nematocyst. The top 3 significantly enriched terms were “cyclic nucleotide biosynthetic process,” “cyclic nucleotide metabolic process”  and “phosphorus-oxygen lyase activity” (Molecular Function) . FurtherUsing the KEGG database to analyze the DEGs in the HAvsHL comparison, the up-regulated genes were significantly enriched in 18 KEGG terms, including “Carbon fixation in photosynthetic organisms,” “Fatty acid elongation,” “Taurine and hypotaurine metabolism,” “Biosynthesis of unsaturated fatty acids,” “Carotenoid biosynthesis,” “Linoleic acid metabolism” and so on. The down-regulated genes were significantly enriched in 11 KEGG terms, including “DNA replication,” “Amino sugar and nucleotide sugar metabolism,” “Oxidative phosphorylation,” “Photosynthesis,” “Photosynthesis-antenna proteins” and so on.There were 21,271  DEGs in the HFvsHG comparison and 1900 (1182 up-regulated and 718 down-regulated) DEGs in HFvsHL comparison. The 21,271 DEGs in HFvsHG were significantly enriched in 43 GO terms, in which the main categories were in “Biological Process” and “Molecular Function” domain, including “metabolic process,” “catalytic activity,” “organic substance metabolic process,” “single-organism process,” “primary metabolic process” and no categories were in the “Cellular Component” domain. The top 5 significantly enriched categories were “cyclic nucleotide biosynthetic process,” “nucleoside phosphate biosynthetic process,” “nucleotide biosynthetic process,” “cyclic nucleotide metabolic process”  and “phosphorus-oxygen lyase activity” (Molecular Function) . The 190Using the KEGG database to analyze the DEGs in the HFvsHL comparison, the up-regulated genes were significantly enriched in 6 KEGG terms, including “Photosynthesis,” “Photosynthesis-antenna proteins,” “Ribosome,” “Glyoxylate and dicarboxylate metabolism,” “Carbon fixation in photosynthetic organisms” and “Flavonoid biosynthesis.” The down-regulated genes were significantly enriched in 5 KEGG terms, including “Ribosome,” “Oxidative phosphorylation,” “Tryptophan metabolism,” “Fatty acid elongation,” “Porphyrin and chlorophyll metabolism”.H. pluvialis under high light stress. The 4864 (2844 up-regulated and 2020 down-regulated) DEGs in the HAvsHF comparison were analyzed using the KEGG database. The 2844 up-regulated DEGs were significantly enriched in 17 KEGG terms, such as “Fatty acid elongation,” “Fatty acid biosynthesis,” “Linoleic acid metabolism,” “Glycerolipid metabolism,” “Biosynthesis of unsaturated fatty acids,” which related to lipid substances and “Carotenoid biosynthesis,” which related to astaxanthin synthesis. The 2020 down-regulated DEGs were significantly enriched in 12 KEGG terms, in which the main terms were related to DNA replication  and photosynthesis . All the above shows that acetate promotes the up-regulated expression of genes related to lipid and carotenoid pathways in H. pluvialis. As for Fe2+, the expression of genes that related to photosynthesis were significantly promoted compared with the inhibition effect of sodium acetate.The above DEG analyses indicated that the two salts had different influences on 2+) on H. pluvialis, based on the DEG analysis in KEGG, three specific pathways were chosen: “carotenoid biosynthesis,” “fatty acid biosynthesis” and “photosynthesis-antenna proteins”.In order to explore the specific effects of the three induction treatments , lycopene β-cyclase (lcyB), prolycopene isomerase (crtISO), carotene epsilon-monooxygenase (LUT1), zeaxanthin epoxidase (ZEP), beta-carotene hydroxylase (crtZ), β-ring hydroxylase (LUT5), carotenoid cleavage dioxygenase 8 (CCD8). In HAvsHL, the 7 DEGs related to carotenoid biosynthesis were annotated as 3 genes: CCD8, crtZ and lycopene epsilon-cyclase (lcyE) and there were no DEGs in HFvsHL  to β-carotene, including PDS, crtISO, lcyB, were down-regulated under high light in this study. The results indicate that not all the genes involved in astaxanthin synthesis were up-regulated under the stress condition, maybe due to the stress duration, stress condition or other reasons. LcyE is an important gene in the lycopene to lutein synthesis pathway and it was down-regulated in HAvsHL, which would decrease competition for the precursor of β-carotene. ZEP converts zeaxanthin to violaxanthin under low-light condition, which would further generate abscisate. ZEP was down-regulated in HLvsHG, which means that the conversion of zeaxanthin to violaxanthin was strongly inhibited under high light. The last gene CCD8 is related to the conversion of β-carotene to strigol  were annotated as 8 genes: phytoene desaturase  and the second to form long-chain fatty acids (n > 16) in the endoplasmic reticulum (KCS) and the other three were mitochondrial trans-2-enoyl-CoA reductase (MECR), very-long-chain (3R)-3-hydroxyacyl-CoA dehydratase (PHS1) and palmitoyl-protein thioesterase (PPT), respectively  can be divided into two pathways, one to form palmitic acid in mitochondria, (0 < eticulum . There wectively . In the LHCA1–5) encode binding proteins in light-harvesting complex I and those encoding light-harvesting complex II chlorophyll a/b binding proteins 1–7 (LHCB1–7) encode binding proteins in light-harvesting complex II. In HLvsHG, the significantly up-regulated genes were LHCA1, LHCA3 and LHCB5. More up-regulated genes were found in HFvsHL, including LHCA1, LHCA3, LHCA4, LHCB2, LHCB3, LHCB5, LHCB6 and LHCB7. But in HAvsHL, all of the genes related to photosynthesis-antenna proteins were down-regulated  is shown in egulated . These rcrtZ, lcyB, crtISO and ZEP. The results of qRT-PCR showed that crtZ was significantly up-regulated in three experimental groups  relative to the control group (HG). The level increased more than 10 fold in HAvsHL and HFvsHL and 6 fold in HLvsHG. CrtISO and LcyB were down-regulated in three experimental groups, decreasing 1.5–7.5 fold. ZEP was significantly down-regulated in the HAvsHG group (7.5 fold)  . OverallH. pluvialis that in the case of three genes—crtZ, ZDS, PDS—involved in the carotenoid biosynthesis pathway, crtZ was up-regulated after induction with both salicylic acid and jasmonic acid [BKT or crtZ), phytoene synthase (PSY) and phytoene desaturase (PDS) were all up-regulated under high irradiance [crtZ was affected by high light and was significantly promoted by acetate. It can be seen that β-carotene hydroxylase (crtZ) played a fundamental role in the astaxanthin biosynthesis pathway and many factors can promote its up-regulated expression, such as high light, acetate and specific hormones. High light impacted the expression of more genes involved in the carotenoid biosynthesis pathway, including PDS, crtISO, LcyB, LUT1, LUT5 and ZEP, than the other two induction conditions, which shows that high light is the main driver for changes in expression of genes related to carotenoid biosynthesis. Acetate further promotes astaxanthin biosynthesis by enhancing the expression of crtZ and inhibiting the expression of LcyE. With regard to lipids, using a comparison of lipidomes and transcriptomes, Gwak found that synthesis of the storage lipid, triacylglycerol (TAG), was substantially stimulated under high irradiance and that it was regulated by expression of de novo fatty acid biosynthesis at the gene level [KCS, MECR). From all of the above, it can be seen that there was a similar effect of high light and acetate on the expression of genes involved in the carotenoid pathway and fatty acid pathway. There may be some link between the two processes where affecting one also affects the other. As for Fe2+, Kobayashi pointed out that Fe2+ promoted carotenoid formation by providing oxidative stress in acetate-induced cyst cells (through the method of inhibiting by potassium iodide) and the effect of Fe2+ could be substituted by active oxygen species [2+ were related to photosynthesis, especially the photosynthesis - antenna proteins. The effect of Fe2+ on H. pluvialis was not directly on the genes involved in astaxanthin biosynthesis but rather indirectly by promoting oxidative stress, affecting the genes related to photosynthesis and thereby promoting astaxanthin accumulation.In recent years, transcriptome analysis has become a hot tool for studying the level of gene transcription . It has nic acid . Using tradiance . In our ne level . Here, t species . Here, tH. pluvialis HOUC9 was stored in the Laboratory of Phycology at the Ocean University of China. 1000 mL conical flasks were used to culture 600 mL of algal cells in modified BBM  medium, with a 12 h:12 h light/dark cycle at 22 °C. The light intensity used for the green growth period was 30 μEm−2·s−1. When the cell concentration reached to (4–5) × 105 cells/mL, induction treatments were initiated. 2+ as inducing salt. The experiments are grouped as follows: group HL was cultured under high light intensity (195 μEm−2·s−1) without any addition of inducing salt; group HA had acetate added to a total concentration of 2 g/L under the same high light intensity as group HL; group HF had Fe2+ added to a total concentration of 20 μM under the same high light intensity as group HL; group HG was the control group grown under normal light intensity (30 μEm−2·s−1) without addition of an inducing salt. Each group was comprised of three biological repeats and were cultured for 48 h. The algal cells were then collected and frozen in liquid nitrogen for RNA extraction. The inductions were performed in 250 mL conical flasks with 50 mL inducing medium added to 50 mL of algal culture. The induction medium contained either acetate or Fe® Plant RNA Kit . Then, four different methods were used to detect the quality and quantity of the total RNA. First, agarose gel electrophoresis was used to determine the RNA content. Second, a Nanodrop  spectrophotometer OD260/280 ratio, was used to measure its purity. Then, the concentration of the RNA was quantified using a Qubit fluorimeter . Finally, an Agilent 2100 bioanalyzer  was used to determine the integrity of the RNA accurately.Total RNA of all samples was extracted using the EasyPureAfter the samples were characterized, the eukaryotic mRNA was enriched using Oligo(dT) magnetic beads. The mRNA was cut into short fragments using fragment Buffer and then used as the template for single-strand cDNA synthesis, after adding 6-bp random hexamers. The second-strand cDNA synthesis was performed by adding buffer, dNTPs, DNA polymerase I and RNase H. After purification of the double-stranded cDNA using AMPure XP beads, the ends were repaired and poly (A) was linked to the 3′-cDNA. Sequencing adapters, which contained a recognition site, were added to the end of the poly (A) sequence. After amplifying the double-stranded cDNA, the PCR amplification product was purified by AMPure XP beads. Finally, the cDNA library was constructed and the preliminary initial quantitation of the library was performed by Qubit 2.0 . Then, the cDNA concentration was diluted to 1.5 ng/µL. Subsequently, the insert size was checked with an Agilent 2100. After ascertaining that the insert size met the requirement, the effective concentration of the library was accurately quantified by Q-PCR (Effective concentration of library >2 nM) to guarantee the quality of the library. After detection of the libraries of all samples, the different libraries were pooled according to the effective concentration and the required amount of data and then sequencing was performed by Illumina HiSeq™ .phred software. If the sequence error rate was expressed in e, the base-quality value of Illumina HiSeq™ was expressed in Qphred, then the Qphred = −10Log10 (e). And the distribution of the sequence error rate can reflect the quality of the sequenced data. Q10, Q20, Q30, Q40 represent an accuracy of 90%, 99%, 99.9%, 99.99%, respectively. The low-quality reads were filtered out,  and the remaining clean reads were retained [The raw data were obtained and the base quality was tested using Qk = 25) dictionary, selecting seed k-mer and extending on both sides. Chrysalis was used to form components through the cluster of overlapped contigs and every component became a collection of possible characterization of variable shear isoform or homologous genes. And each component had the corresponding de Bruijn graph. Butterfly was used to output full-length transcripts of variable shear isoforms and comb transcripts that corresponded to paralogous genes through simplifying each component’s de Bruijn graph. After these three steps, the assembled results of TRINITY.fasta were finally obtained.For transcriptome without reference sequence, the clean reads were de novo assembled by using Trinity  to obtaihttps://code.google.com/p/corset-project/).Then Corset was used to hierarchically cluster transcripts by expression patterns and the number of reads which had been blasted to transcripts . After hH. pluvialis and the similarity of gene sequences between H. pluvialis and related species. By GO annotation, the annotated genes were classified to the next terms of the three GO categories . By KO annotation, the annotated genes could be classified according to their KEGG metabolic pathway.Gene functional annotation was performed using seven databases, including Nr, Nt, Pfam, KOG/COG, SwissProt, KEGG and GO. By using Blast to search the Nr database, we could find functional information for genes of p-value) as threshold value to screen out DEGs in different comparison groups, which had been set to 6 groups: HLvsHG, HAvsHG, HAvsHL, HFvsHG, HFvsHL and HAvsHF. Then two comparison combinations of the Venn diagram: HLvsHG-HAvsHG-HFvsHG and HAvsHL-HFvsHL, were set to show the number of total genes and specific genes between those comparison groups. As for the expression level of DEGs, cluster analysis was used to determine the expression patterns of DEGs under different experimental conditions based on the FPKM (expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced) values. It is the most common method of gene expression level estimation [http://www.geneontology.org/) was used to map the DEGs to the term types and calculate the number of every term type. With the termed GOseq, the significantly enriched terms were found by comparing them to the whole genome background [For biologically repeated samples, DESeq  was usedtimation . Then thckground . KEGG isckground . KEGG enn is the number of DEGs in N. M is the number of genes annotated to a certain pathway among all the genes and m is the number of DEGs in M. Those pathways with FDR  ≤ 0.05 were defined as the significant enrichment pathways for DEGs. In this paper, FDR was a corrected p-value obtained by the BH method using KOBAS (2.0) [N stands for the number of genes that can be annotated by pathways and AS (2.0) .crtZ, crtISO, lcyB, ZEP) were chosen for a validity check of the transcriptome result, using real-time fluorescence quantitative PCR (qRT-PCR). The sample was prepared together with the transcriptome experiment and was kept in a −80 °C refrigerator for RNA extraction. The RNA was extracted using RNA kits (Takara) and the synthesis of cDNA was performed using a PrimeScript II 1st Strand cDNA Synthesis Kit . Oligo dT primer and random 6-mer primers were both used as RT-primers [Based on the transcriptome result, genes related to carotenoid pathway  was used as the reference gene. All primers were designed using the Primer Premier 5.0 software  and are listed in −CtΔΔ (RQ) method. Then a log function (logRQ) was used to analyze the qRT-PCR results. The qRT-PCR was performed using a SYBR Premix Ex Taq™ II Kit  with a 20 μL reaction system containing 10 μL 2× SYBR green Mastermix , 1 μL each of the forward and reverse primers, 1 μL cDNA and 7 μL ddH are different under different induction conditions. The results indicated that acetate and Fe2+ have different mechanisms for inducing astaxanthin production in H. pluvialis. High light is the direct factor for the changes in gene expression during the induction period. Acetate could further enhance expression of the genes involved in carotenoid and fatty acid synthesis. Fe2+ mainly influences the genes involved in photosynthesis and especially promoted the expression of genes related to photosystem I and photosystem II. Taken together, these results obtained through application of RNA-seq provide a foundation and orientation for future studies on H. pluvialis. Based on the DEGs involved in carotenoid metabolism, the expression pattern of 9 genes


Electronic cigarettes (e-cigarettes) were the most commonly used tobacco product among U.S. middle school and high school students in 2016 , sex, or race/ethnicity , 4,049 ever users, including 1,281 current users, were included in the analysis. Data were weighted to account for the complex survey design and adjusted for nonresponse. Point estimates, 95% confidence intervals, and population totals corresponding to reasons for use were computed among ever and current e-cigarette users, both overall and by school level, sex, race/ethnicity, and current use of other tobacco products. Chi-square tests were used to assess statistically significant (p<0.05) differences across groups.Among U.S. middle and high school e-cigarette ever users in 2016, the most commonly selected reasons for using e-cigarettes were “friend or family member used them” (39.0%), “they are available in flavors, such as mint, candy, fruit, or chocolate” (31.0%), and “they are less harmful than other forms of tobacco such as cigarettes” (17.1%). Reasons for use varied by school level and sex. For example, “friend or family member used them” was more commonly selected by middle school students (43.7%) than high school students (37.5%), and by females (46.7%) than males (32.2%) . “They aAmong U.S. middle and high school students who reported using e-cigarettes  during the past 30 days, the most commonly selected reasons for e-cigarette use were “they are available in flavors, such as mint, candy, fruit, or chocolate” , “friend or family member used them” , and “they are less harmful than other forms of tobacco, such as cigarettes”  .Among U.S. middle and high school students who had ever used e-cigarettes in 2016, the most commonly selected reasons for e-cigarette use were “friend or family member used them,” “they are available in flavors, such as mint, candy, fruit, or chocolate,” and “they are less harmful than other forms of tobacco, such as cigarettes.” Regardless of whether users reported using e-cigarettes exclusively or with other tobacco products during the past 30 days, these reasons remained the most commonly selected reasons for e-cigarette use. The availability of flavors, use by a friend or family member, and belief that e-cigarettes are less harmful than other forms of tobacco might be important factors for initiation or maintenance of e-cigarette use among middle school and high school students. Although percentages reported here are lower, the findings from this study are consistent with those of previous studies reporting that availability of flavors is among the most prominently cited reasons for youths’ e-cigarette use  were the most commonly used tobacco product among U.S. middle school and high school students in 2016. The Surgeon General concluded that e-cigarettes can contain harmful and potentially harmful constituents, including nicotine. Nicotine exposure during adolescence can cause addiction and can harm the developing adolescent brain.Among student respondents to the National Youth Tobacco Survey reporting ever using e-cigarettes in 2016, the most commonly selected reasons for use were used by “friend or family member” (39%), availability of “flavors such as mint, candy, fruit, or chocolate” (31%), and the belief that “they are less harmful than other forms of tobacco such as cigarettes” (17%). The least commonly selected reasons were “they are easier to get than other tobacco products, such as cigarettes” (5%), “they cost less than other tobacco products such as cigarettes” (3%), and “famous people on TV or in movies use them” (2%). Efforts to prevent middle school and high school students from initiating the use of any tobacco product, including e-cigarettes, are important to reduce tobacco product use among U.S. youths. Regulation of the manufacturing, distribution, and marketing of tobacco products by the Food and Drug Administration, along with sustained implementation of comprehensive tobacco control and prevention strategies, could reduce e-cigarette use and initiation by middle school and high school students.


Arabidopsis thaliana in AP generation and shaping, we show that the plant AP does depend on similar Kv-like transport systems to those of the animal signal. We demonstrate that the outward-rectifying potassium-selective channel GORK limits the AP amplitude and duration, while the weakly-rectifying channel AKT2 affects membrane excitability. By computational modelling of plant APs, we reveal that the GORK activity not only determines the length of an AP but also the steepness of its rise and the maximal amplitude. Thus, outward-rectifying potassium channels contribute to both the repolarisation phase and the initial depolarisation phase of the signal. Additionally, from modelling considerations we provide indications that plant APs might be accompanied by potassium waves, which prime the excitability of the green cable.Fast responses to an external threat depend on the rapid transmission of signals through a plant. Action potentials (APs) are proposed as such signals. Plant APs share similarities with their animal counterparts; they are proposed to depend on the activity of voltage-gated ion channels. Nonetheless, despite their demonstrated role in (a)biotic stress responses, the identities of the associated voltage-gated channels and transporters remain undefined in higher plants. By demonstrating the role of two potassium-selective channels in  Fast and ubiquitous signals are vital in the response of an organism to any external threat, challenge or stimulus. In animals, electrical signals such as action potentials (APs) transmit information from one part of the body to another via nerve cell axons connected by synapses. Plants also show electrical responses, occurring after stimuli such as abrupt changes in temperature and light intensity, the action of depolarising agents , wounding and the application of electrical currents ,2. The sIn short, VPs are long-term—in the minute range. The depolarisations are characterised by an irregular shape, which can be accompanied in many cases, by additional depolarisation voltage spikes. They are induced by local damage, for instance, local burning or wounding. As they can pass through dead tissues, they are thought to be secondary effects of hydraulic and/or chemical signals. Variation potential intensity and duration are proportional to the intensity of the damaging stimulus, and signals decrease with increasing distance from the damaged zone.In contrast, action potentials (APs) are induced by non-damaging stress such as cooling, touch or electric currents. They only propagate through living tissues and share similar characteristics to those of animals APs, namely (i) an apparent all-or-nothing character, (ii) a constant amplitude and duration under control conditions and (iii) self-propagation ,6,7. Com+-ATPase activity and an anion efflux, which results in depolarisation of the cell. A subsequent potassium efflux has been proposed to repolarise the cell. So far though, the only molecular players that have been identified in plant AP initiation are from the glutamate receptors channel family: GLR3.2, GLR3.3 and GLR3.6 ). An open channel (AKT2-NN) could result in higher apoplastic [K+], and an inward rectifier (AKT2-KS), in lower apoplastic [K+]. AKT2 channels could therefore have an effect on membrane excitability by controlling both the electrical membrane properties and the apoplastic potassium concentration.In animal cells, background non-rectifying potassium channels regulate membrane excitability by clamping the membrane voltage to the potassium equilibrium potential and decreasing cell excitability . The Araon steps ,40. We e channel ,41,42,43In this study we used an established, albeit coarse technique to induce APs in Arabidopsis. We required a reliable, AP-inducing stimulus that would allow consistent and repeatable measurements, necessary for screening for excitability in different mutant lines. Therefore, we followed a procedure that has been shown in a previous publication  to provoKv channels , which would down-regulate GORK activity and increase excitability. The elimination of AKT2 would eliminate this effect and would, as observed, reduce excitability. The conversion of AKT2 into a pure K+-uptake channel  would favour the sequestration of K+ by the cells and thus reduce apoplastic [K+]. This would, again as observed, further reduce excitability.It is thus tempting to speculate that plant APs are not of pure electric nature but instead are a combination of an electrical component and one or more chemical components, with potassium being critical. This concept would explain the slow speed of APs in plants, which then would be limited by the diffusion of the chemical component(s). Interestingly, this concept would also explain the observations in the experiments with AKT2 . As specAKT2 )/(1 + exp[−0.1 × (V + 70 mV)/25 mV]) ≈ gBG × (V + 70 mV) [BG = gBG/CM = 1 × s−1. (ii) Because plant outward-rectifying K+ channels do not show any sign of C- or N-type inactivation even under prolonged stimulation, they can be described in a first approximation by a two-state model (open and closed [K = exp(V/25 mV – 3.0) × s−1 and dK = exp(−0.6 × V/25 mV – 2.0) × s−1, respectively. The Nernst equilibrium for K+ was EK = −70 mV and the K+-conductance-dependent parameter was φK = gK/CM varied between 31.7 × s−1  and 0 × s−1 (no K+ channels). (iii) A depolarisation-activated, slowly inactivating anion (or Ca2+) channel was described by a four-state model , with activation and deactivation rate constants of aA = exp(V/25 mV – 1.0) × s−1 and dA = exp(−1.4 × V/25 mV + 1.4) × s−1, and inactivation and recovery rate constants of inA = exp(0.2 × V/25 mV – 3.0) × s−1 and reA = exp(−3.0 × V/25 mV – 6.0) × s−1, respectively. The Nernst equilibrium for the depolarising ion was EA = +40 mV and the conductance-dependent parameter was φA = gA/CM = 120 × s−1. Simulations were carried out numerically as described in the x-axis (matrix index n) and 5001 time-points (matrix index m) with time interval of Δt = 10 ms and α = gx/CM × Δt/(Δx)2 = 0.02. Action potentials were induced by a stimulus n = 1 for 499 time-intervals Δt .To qualitatively illustrate the contribution of outward-rectifying K cable’  , with thd closed ), with a


P < 0.001). Glycaemic control status did not significantly differ between the T2D patients with parental FH and those with sibling FH. Compared with patients with paternal FH, patients with maternal FH had a higher risk of poor glycaemic control . Stratified analyses showed that a FH of diabetes was significantly associated with poor glycaemic control among T2D patients with a low education level (P < 0.05). In the <60 years old, overweight, and low level of physical activity groups, patients with a maternal history of diabetes showed a higher risk of poor glycaemic control than those without a FH (P < 0.05). In conclusion, FH of diabetes, especially a maternal history, had an independently adverse effect on the glycaemic control of T2D patients.To investigate the association of familial history (FH) of diabetes with the glycaemic control status of patients with type 2 diabetes (T2D), a cross-sectional study using stratified cluster sampling was conducted with 20,340 diabetic patients in Jiangsu, China. In total, 21.3% of the subjects reported a FH of diabetes. Patients with a FH of diabetes showed a higher risk of poor glycaemic control (59.7%) than those without a diabetic FH (49.8%), with an odds ratio (OR) of 1.366 ( Diabetes, as one of the NCDs of most concern, was shown to have an overall prevalence of 11.6% in China in 20101. It is an understatement to say that the current prevalence of diabetes merely outweighs the figure in 1980, as it is greater by ten-fold6. While diabetes and its complications pose threats to patients’ health, the associated medical expenditures inevitably cause a large financial burden for the patients, families, and society as a whole8.In recent decades, non-communicable diseases (NCDs) have contributed substantially to the increased mortality rate and have massively increased the global burden of disease13 and with a higher incidence of diabetes in the general population17. People diagnosed with early-onset type 2 diabetes (T2D) were found to face more difficulties in managing their glycaemic control and to be more likely to experience microvascular complications22. However, the association between a FH of diabetes and glycaemic control of patients with diabetes remains controversial26.A familial history (FH) of diabetes has extensively been reported to be associated with an earlier onset of diabetes among patients29. However, the mechanisms underlying the relationships are poorly understood. An improved understanding of the role of diabetic FH in the development of diabetes may help explain the association between a FH of diabetes and glycaemic control of T2D patients. Using the number of affected first-degree relatives (FDRs) and second-degree relatives (SDRs), Valdez R. et al. found that the odds of developing diabetes among individuals with moderate  and high familial risk (at least one FDR and two SDRs or two FDRs) were 2.3 and 5.5 times higher, respectively, than the odds among those with an average risk (none or only one SDR)30. However, few investigations have investigated the relevance of a FH of diabetes and glycaemic control status in the Chinese population. In the present study, we examined the relationship between a FH of diabetes and the glycaemic control of T2D patients using a combined evaluation of fasting plasma glucose (FPG) and haemoglobin A1c (HbA1c).Several factors, including the maternally determined environment , genomic imprinting, and nutritional/lifestyle influences, can all contribute to the incidence of diabetes and hyperglycaemiaA cross-sectional study using stratified cluster sampling was conducted in 65 townships of three areas , and a total of 39,564 patients with T2D were targeted. Townships operating solely within the health care programme of the National Basic Public Health Service 2012) were included. From the 44 selected townships, 23,240 of the T2D patients were recruited for this investigation. According to the criteria established by the American Diabetes Association (ADA) in 2010, T2D patients were defined as having FPG levels ≥7.0 mmol/L or a self-reported T2D history without type 1 diabetes. A total of 20,340 subjects consented to complete a standardized epidemiology questionnaire and physical examinations. All examinations were performed within two months (from Dec 2013 to Jan 2014). FPG and HbA1c measurements were collected from 20,015 and 19,997 of the T2D patients, respectively, and 19,992 of the T2D patients had both FPG and HbA1c data available. General characteristics of the participants are shown in Supplementary Table 2 were inThe study protocol was approved by the Ethics Board of the Jiangsu Provincial Centers for Disease Control and Prevention (No. 2013026). All patients were well informed about the study and provided written consent before participating; all methods were performed in accordance with the relevant guidelines and regulations.The investigators were trained and qualified in a standardized manner. The training contained all the relevant information and procedures for the questionnaire, anthropometric measurements, blood sample collection and processing, and quality control. The investigators were qualified after the training and prior to the formal investigation. Personal information on the T2D patients, including demographic characteristics, medical history, and lifestyle factors, was collected using a standardized questionnaire.2).Anthropometric measurements, including height and body weight, were obtained by trained technicians. Body weight and height were measured twice in individuals ensuring that heavy clothes and shoes were removed before measurements, and the figures were rounded to the nearest 0.1 kg and 0.1 cm, respectively. Body mass index (BMI) was calculated as weight (kg)/height squared .31 (uncontrolled) for HbA1c. In addition, an evaluation of HbA1c and FPG combined was used to assess glycaemic control status. The level of glycaemic control was defined as “good”, “common” and “poor” when the criteria of HbA1c <7% (53 mmol/mol) and FPG <7 mmol/L were both met, when either criterion was met, and when neither was met, respectively.The outcome assessment indices for the glycaemic control of T2D were categorized as <7 mmol/L (controlled) and ≥7 mmol/L (uncontrolled) for FPG and <7% (controlled) and ≥7%2 were defined as normal weight, overweight, and obese, respectively32. Duration of T2D was defined as the time between the survey and the time the patient was diagnosed with T2D. Physical activity included both dynamic behaviour and static behaviour. Dynamic behaviours represented physical activities at work, transportation, and leisure time. The intensity of activities was defined as vigorous or moderate during work and leisure time according to the duration and amount. The level of participants’ physical activity per day was then assessed by estimating the metabolic equivalent (MET). By its definition, the MET is a ratio of the working metabolic rate to a standard resting metabolic rate of 1.0 (4.184 kJ) · kg−1 · h−1, and 1 MET was considered the resting metabolic rate obtained in a sedentary setting33. The weekly time participants engaged in dynamic behaviours was divided by 7 to generate the average time of daily activity. For the purpose of rapid scoring, the following MET levels were assigned to each class of activity: sleep = 1 MET; static activity = 1.1 METs; moderate activity = 4 METs, and vigorous activity = 8 METs35. In the stratified analysis, age (year) was categorized into <60 and ≥60 years old, and physical activity was classified as low (MET < 31.22) or high (MET ≥ 31.22) based on the median MET (31.22).Patients’ educational levels were classified into 4 categories as follows: “without formal education”, “primary”, “middle school”, and “high school and above”. Smoking habits were analysed by “yes” or “no” responses to the question, “Have you ever smoked 100 cigarettes in the past”? Drinking was defined as consuming at least once alcoholic drink per month, and drinking habits were categorized into “current”, “former” and “no drinking”. Subjects with a BMI of 18.5 to 23.9, 24 to 27.9, and ≥28 kg/mAccording to whether the patients’ father, mother, sibling, grandparents/maternal grandparents, uncles, and/or aunts were reported to have diabetes, the FH of diabetes was generally categorized into “parental”, “sibling”, and “other relatives”. Based on the combination of parental FH, sibling FH, and other relatives FH, patients were categorized into eight groups, and their average HbA1c and FPG level and glycaemic control status are shown in Supplementary Table χ2) test was used for the comparison of categorized variables. Student’s t-test and analysis of variance (ANOVA) were used to compare the normally distributed continuous variables, and Mann-Whitney U test was used to compare abnormally distributed data. Logistic regression analyses were performed, and the relative risk was estimated as the odds ratio (OR) and 95% confidence interval (CI). Stratified analyses were conducted by age, gender, education, BMI, antidiabetic treatment, and physical activity. Bonferroni correction was performed for multiple comparison tests. A two-tailed P value < 0.05 was considered to indicate statistically significance. All statistical analyses were performed using IBM SPSS Statistics 15.0  and Stata 12.0 .The mean (SD range) was calculated for continuous variables with a normal distribution, whereas those with a skewed distribution were described as the median [inter-quartile range (IQR)]. A chi-square (The average age of the subjects was 63.35 ± 9.86 (years), and 39.2% of the subjects were male. Overall, 32.0% of T2D patients had a FPG level considered controlled (<7 mmol/L), and 41.9% were controlled based on HbA1c (<7%). With regard to glycaemic control according to both FPG & HbA1c, 25.7% of the patients achieved a good level, 22.4% were in the common level, and the remaining 51.9% were poorly controlled. A total of 917 patients were excluded for missing data regarding their FH of diabetes, and an additional 200 patients were excluded for missing information about details of their FH. As a result, 21.3% of the total 19,075 patients self-reported a FH of diabetes, and 18,875 individuals provided detailed information about their FH, in which the percentage of familial diabetes from their mother, father, and both parents represented 5.9%, 2.6%, and 0.8%, respectively (Supplementary Table P < 0.001).The demographic characteristics and FH of diabetes were listed according to glycaemic control status as determined by FPG and HbA1c levels  based on HbA1c (33.9% vs. 44.0%), FPG (24.1% vs. 34.0%), and HbA1c & FPG (17.7% vs. 27.8%) , and the proportion of those with “good” glycaemic control based on HbA1c & FPG  also increased across the three groups (Ptrend < 0.01). By contrast, no significant difference in FPG or HbA1c levels was observed between the three groups (P > 0.05).The FPG and HbA1c levels and proportion of glycaemic control in terms of FPG, HbA1c and HbA1c & FPG did not significantly differ between the “parental and sibling”, “parental only”, and “sibling only” groups , “parental only” and “sibling only” histories of diabetes remained significantly associated with a higher risk of poor glycaemic control (adjusted P < 0.05). In addition, compared with patients without FH, patients with a “maternal” history had a higher risk of “poor” glycaemic control , and the association remained significant (P = 0.003) after Bonferroni correction . Furthermore, patients with a “sibling only” and “maternal” history also had a higher risk of “common” glycaemic control, with ORs (95% CI) of 1.384 (1.180–1.623)  and 1.343 (1.041–1.732), respectively.Subjects with a FH of diabetes showed a significantly higher risk of poor glycaemic control  after adjusting for the covariates compared with those without a FH (Table P = 0.039) after Bonferroni correction  (Table P > 0.1), and there was no significant association between FH of diabetes and “common” glycaemic control among T2D patients with parental and sibling FH of diabetes.Compared with a “paternal” history of diabetes, a “maternal” history of diabetes resulted in a higher risk of poor glycaemic control , and the correlation remained significant  , aged <60 years OR 1.653) and engaged in a low level of physical activity (OR 1.660), with  4) Fig. . However and engaP < 0.05).The homogeneity test showed that the association between a FH of diabetes (vs. without FH of diabetes) and “poor” glycaemic control was heterogeneous among patients with different education levels, as was the association between a maternal history of diabetes (vs. no FH of diabetes) and “poor” control among BMI and gender groups , 34% among Danish T2D patients, and 35% among Italian T2D patients24. A longitudinal study of patients of multiple ethnicities25 reported that serum FPG and HbA1c remained significantly higher in T2D patients with a maternal FH of diabetes but not a paternal FH after adjusting for age, diabetes duration, and treatment type, although gender and BMI, which appeared to have an important influence on glycaemic control, were not adjusted for in that study. By contrast, a cross-sectional study of 2,113 T2D patients in Italy observed no significant influence of parental diabetes on glycaemic control23. Meanwhile, our current study also observed that T2D patients with a sibling who had diabetes tended to have a higher risk of “poor” or “common” glycaemic control based on the combined FPG & HbA1c index.Previously, studies have observed only a single glycaemic control index, either FPG or HbA1c, and have mainly investigated the influence of parental FH of diabetes. A study of 359 urban African-American T2D patients indicated a barely significant impact of a FH of diabetes on HbA1c level41. These differential effects between paternal and maternal history of diabetes may contribute to the influences of maternally determined environments , genomic imprinting, and nutritional/lifestyle factors on diabetes29. Genomic imprinting can be inherited maternally rather than paternally or vice versa in several regions of the diabetes-related genome or in molecular modifications of DNA in germ-line cells43.The results of this study suggest that maternal FH, rather than paternal FH, has a strong negative association with glycaemic control. Similarly, previous studies have indicated a stronger maternal-offspring correlation in terms of T2D compared with the paternal-offspring correlation44. In this study, the stratified analysis indicated that among overweight patients, maternal FH was a risk factor for poor glycaemic control. Boule, N. G. et al. reported that “high intensity exercise shows benefits with HbA1c reduction”45. Similarly, this study observed that in patients with a low level of physical activity (MET <31.22), but not in patients with a high level of physical activity (MET ≥ 31.22), maternal FH was a risk factor of poor glycaemic control. Meanwhile, community-based lifestyle modification programmes have been shown to be effective not only in reducing important risk factors for diabetes but also in improving blood glucose control47. These findings imply the importance of losing weight and improving physical activity for glycaemic control in T2D patients with a FH of diabetes.Previous studies have reported that overweight and obesity were risk factors for glycaemic control in patients with diabetesAccording to the findings of the current study, for community-based T2D programmes, a FH of diabetes should be routinely evaluated. This information may help determine the risk factors for poor glycaemic control in susceptible patients and may facilitate the process of designing effective family-based risk assessment and risk reduction strategies.48. Some limitations also existed in this study. The ‘sibling’ category might also include biologically unrelated siblings who were not identified by self-reporting; however, the proportion of siblings in this category would be low. In addition, a cross-sectional design was used, and thus causal inferences could not be made for the association of FH of diabetes with glycaemic control.Our study has several strengths. First, this study used cluster sampling to survey representative T2D patients from a community-based diabetes management system and had a large sample size  to detect the influence of FH of diabetes on glycaemic control of T2D patients; these factors enabled higher statistical power and stable relevant results. Second, FPG, HbA1c and a combination of FPG and HbA1c were jointly used to assess glycaemic control, and these indices provided a comprehensive estimation of diabetes control among T2D patients. Third, the association between FH of diabetes considering various relatives and glycaemic control provided further insight into aggregate familial effect on diabetes incidence and hyperglycaemiaThe present study contributes novel evidence regarding the significant difference in the association between parental history of diabetes and glycaemic control, and low education levels and overweight could further the risks presented by a FH of diabetes. These findings have important implications for clinical interventions targeted to patients with a familial history of diabetes, particularly a maternal history.Supplemental Material (tables)


The purpose of this research was to examine the validity of an adult attachment style questionnaire, to understand the relationships between the type of attachment style in relation to self-perceived stress and social support, and to investigate the influence of gender, ethnicity and religion on the above constructs. The participants were 308 university students from Malaysia. A demographic questionnaire and three self-report inventories were administrated in this study. The data indicated that the Relationship Scales Questionnaire (RSQ) is a multidimensional construct with nine factors: “dismissing,” “preoccupied with romance,” “preoccupied with close relationships,” “fearful,” “preoccupied with dependency,” “secure emotional,” “comfortable depending,” “preoccupied with mistrust” and “mutual secure.” Different attachment styles were positively or negatively correlated at a significant level with perceived stress and social support. Attachment styles were explained by 20 and 33% of the total variance in self-perceived stress and perceived social support, respectively. There were significant gender, ethnic and religious differences in attachment styles, perceived stress and social support. Attachment theory focuses on parent-child bonding and postulates that attachment to a caregiver is part of the fundamental survival system. The original theory of Bowlby  defined The four-category model of attachment of Bartholomew and Horowitz  is basedHowever, the current conceptualizations show attachment theory as a culture-sensitive framework influenced by observational instruments and middle-class assumptions in Western cultures. The question remains whether similar results would be obtained in rural eco-social environments in non-Western cultures . Therefosecure base for exploration in a culture, allowing a person to pursue personal aspirations in a safe and effective way . Indeed, many studies have shown that people with high levels of secure attachment appraise stressful events more optimistically. They attain higher scores on the measures of intimacy, trust, prosocial behavior and relationship satisfaction  and 21.85 (SD = 1.31), respectively. The sample incorporated various ethnic groups that include Malay (n = 152), Chinese (n = 91), and others (n = 65). Also, the sample incorporated three religions; Islam (n = 210), Christian (n = 19), and Buddhist (n = 79). Participants were selected from those taking a psychology course in the university; they were given extra-credits as an incentive to participate in the study. They were from various departments in the university and from various years of study. After informed consent was acquired, participants completed a questionnaire containing sections on demographic information, attachment style, perceived stress, and social support.The participants were 308 undergraduate students  of a public university in Malaysia. The means (and standard deviations) of age for males and females were 22.35  the Relationship Scales Questionnaire (RSQ), (2) the Chinese College Stress Scale (CCSS), and (3) the Multiple Perceived Social Support Scale (MPSSS).Relationship Scales Questionnaire , academic hassles , and negative life events . Items are completed on a Likert scale ranging from 1 (strongly low) to 4 (strongly high). Using Cronbach’s alpha, the internal consistency estimates were .84 for the academic hassles subscale, .88 for personal hassles, and .83 for negative life events. The entire 30-item CCSS had an internal-consistency estimate of .92.Multiple Perceived Social Support Scale . The MSPSS produces three scores. The MSPSS’s reliability and validity were confirmed in several studies  = 4.252, p < .0001. “Preoccupied with mistrust” had significant positive relationships with perceived stress, but there was a significant negative relationship between “mutual secure” style and perceived stress. Findings showed that attachment styles explained 20% of the variance in social support, R = .578, 2R = .335, F  = 15.234, p < .0001. “Secure emotional” and “mutual” secure styles had significant positive relationships with social support, but there was also a significant negative relationship between “preoccupied with mistrust” style and social support  in predicting perceived stress and social support in this sample. Findings revealed that attachment styles accounted for 20% of the variance in perceived stress, R = .259, 2R = .067, F  = 5.616, p < .001. Results affirmed that gender, religion and ethnicity could also explain 5% of the variance for perceived stress, R = .234, 2R = .055, F  = 2.672, p < .05. Results indicated that gender, religion and ethnicity explained 4% of the variance for perceived social support; R = .220, 2R = .048, F  = 4.050, p = .008.The fourth hypothesis of this study is that gender, ethnicity and religion are significantly related to attachment style, perceived stress and social support. Initially, the multiple regression models were computed to evaluate the possible roles of gender, religion and ethnicity as independent variables in predicting these constructs. Findings proved that gender, religion and ethnicity explained 6% of the variance for attachment style, t (302) = 4.32, p < .0001, and “preoccupied with dependency,” t (303) = 3.06, p < .002, than females; and females had a significantly higher “secure emotional” style, t (303) = –2.07, p < .039, than males. There were significant ethnic differences in “dismissing,” F  = 7.84, p < .001; “preoccupied with close relationships,” F  = 3.08, p < .04; “secure emotional,” F  = 4.24, p < .015; and “comfortable depending,” F  = 6.09, p < .003, attachment styles. Moreover, there were significant ethnic differences in perceived stress, F  = 4.06, p < .019, family support, F  = 6.29, p = .002; and the total perceived social support, F  = 4.24, p < .015. Findings indicated significant religious differences in “dismissing,” F  = 8.72, p < .0001; and “comfortable depending” styles, F  = 7.01, p < .001; family support, F  = 5.48, p < .005, and total perceived support, F  = 3.63, p < .02.Additionally, a t-test for independent groups was conducted to evaluate the effect of gender, and two ANOVAs were calculated for religion and ethnicity differences in the aforementioned independent variables Table . FindingPost-hoc LSD tests indicated that Chinese and Other participants had a significantly higher “dismissing” style than Malays. Chinese participants had significantly higher “preoccupied with close relationships” style than Malay and Others. Other participants had significantly higher “secure emotional” attachment than the Malays and Chinese, and Malays and Others had significantly higher “comfortable depending” style than Others. Malay and Chinese participants had significantly higher perceived stress than Others. Other participants had significantly higher family support and total perceived support than Malay and Chinese participants. Muslim participants had significantly lower “dismissing” attachment than Buddhist and Christian participants. Muslim and Christian participants had significantly lower “comfortable depending” style than Buddhist participants. But Buddhist participants had significantly lower family support and total perceived support than Muslims and Christians.Additionally, to examine possible gender differences and age groups’ interaction, a multivariate analysis of variance (MANOVA) was run with gender, ethnicity and religion and their interactions as independent variables and the attachment style, perceived stress and social support as dependent variables. There were no significant interactions effects between gender, ethnicity and religion for attachment style, perceived stress and social support.With respect to our first hypothesis, the results from this study collected in a Malaysian sample confirmed that attachment style is a multidimensional construct. We obtained nine factors in which were labeled “dismissing,” “preoccupied with romance,” “preoccupied with close relationships,” “fearful,” “preoccupied with dependency;” “secure emotional,” “comfortable depending,” “preoccupied with mistrust” and “mutual secure.” This finding is in line with many cross-cultural differences in adult attachment style which highlights the possible roles of cultural factors such as child rearing, values, psychological well- being, social competence and social functions of relationships in the emergence of attachment style, all of which manifest themselves in different forms across different cultures . As MalaHowever, friendliness may be an important cultural construct that exists across subcultures within Malaysia and this is indicated by their emotional orientations toward social and interpersonal relationships. This tendency is reflected as “preoccupied with romance,” “preoccupied with close relationships,” “preoccupied with dependency,” “secure emotional,” “comfortable depending” and “preoccupied with mistrust” in the present study. The above attachment styles could express the importance of emotions for Malaysian young adults in different social settings and various types of relationships. On the other hand, this capability for emotionality in the Malaysian people is often defined by subcultural and traditional forces across their experiences within their families, friendships, work settings and social networks. Consequently, there are a few distinguished schemas and cognitive working models for these attachment styles, and they might transmit from childhood to adulthood by individual socialization within families and social institutions. However, this conceptualization needs further exploration in both clinical and non-clinical samples by techniques such as schema analysis and qualitative research.The present results with respect to the second hypothesis affirmed that perceived stress was significantly and positively correlated with “dismissing,” “preoccupied with romance,” “preoccupied with close relationships” and “fearful” attachment styles. In contrast, social support and its subscales were negatively related with “dismissing,” “fearful,” “preoccupied with dependency” and “preoccupied with mistrust” styles. Moreover, there was a significant negative relationship between social support from friends and “preoccupied with romance” style. In addition, social support was positively associated with “secure emotional” and “mutual secure” styles. These findings are consistent with previous speculations and attachment theories’ predictions, and they are in line with previous studies that related attachment relationships to perceived stress and supportive seeking behaviors , 1994b.Findings with respect to the third hypothesis supported the role of attachment style in relationship to perceived stress and social support. Results revealed that “secure emotional,” “preoccupied with mistrust” and “mutual secure” styles explained 20% of the variance in perceived stress. The “secure emotional,” “preoccupied with mistrust,” and “mutual secure” styles summation explained 33% of the variance in perceived social support. These findings help to understand the role of attachment styles on perception of social stressors and negative emotions . TherefoWith respect to the fourth hypothesis, our findings showed that gender, religion and ethnicity are related to attachment style, perceived stress and social support. Males had significantly higher “dismissing” and “preoccupied with dependency” styles than females, and females had significantly higher “secure emotional” style than males. Findings showed significant ethnic differences in “dismissing,” “preoccupied with close relationships,” “secure emotional” and “comfortable depending” attachment styles, perceived stress, family support, and total social support. Findings indicated significant religious differences in “dismissing” and “comfortable depending” styles, family support and total support. In agreement with the present findings, previous studies showed similar gender differences in attachment style during childhood and similar relationships with family environments . HoweverThe current research adds to the body of psychological literature by contributing to knowledge with regard to the effects of culture, gender, ethnicity and religion on attachment styles. We showed relationships between attachment style, perceived stress and social support in a youth sample. Nevertheless, this study is somewhat limited because it relies mainly on survey data, application of exploratory factor analysis, and the recruitment of a specific sample of undergraduate students. Future studies might use confirmatory factor analysis (CFA) and may determine whether a 9-factor structure is the best conceptualization in Malaysian culture. Future research might also apply experimental and longitudinal designs with observational instruments to examine these constructs across different cultural samples, both in clinical and non-clinical populations.Authors wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.