The Cd-contaminated areas are mainly located in west areas of the Pearl River Delta. Cd concentrations in all vegetables were lower than the national standard of Safe vegetables (0.2 mg kg−1). 88% of vegetable samples met the standard of No-Polluted vegetables (0.05 mg kg−1). The Cd concentration in vegetables was mainly influenced by the interactions of total Cd concentration in soils, soil pH and vegetable species. The fit lines of soil-to-plant transfer factors and total Cd concentration in soils for various vegetable species were best described by the exponential equation  concentration levels in soils and in leaf vegetables across the Pearl River Delta (PRD) area; and reveal the transfer characteristics of Cadmium (Cd) from soils to leaf vegetable species on a regional scale. 170 paired vegetables and corresponding surface soil samples in the study area were collected for calculating the transfer factors of Cadmium (Cd) from soils to vegetables. This investigation revealed that in the study area Cd concentration in soils was lower (mean value 0.158 mg kg Cadmium is non-essential element to biota. It is known to be toxic for plants as well as animals to a much higher extent and at lower concentrations than e.g. Zn, Pb, or Cu. It especially affects humans because of their longevity and the accumulation of Cd in their organs by eating Cd-contaminated food −1 in China −1 in the Netherlands and Switzerland, 0.5–1 mg kg−1 in Austria −1 in Taiwan Soil Cd is naturally derived from parent materials because of chemical weathering, and as a contaminant in many areas of anthropogenic activities such as mining, smelting, composts, phosphate fertilizer application, waste disposal, and vehicle exhausts −1 for non-environmental pollution vegetable – so called “No-Polluted vegetable” −1 in leaf vegetables for food security – so called “Safe vegetable” Because soil Cd is easily accumulated by plants, soil-plant-human transfer of Cd has been considered as a major pathway of human exposure to soil Cd Although undoubtedly soil Cd is the primary source of vegetable Cd, and total soil Cd concentration was commonly used for soil environmental quality estimation, its usefulness to predict soil-to-plant transfer was often questioned since the phytoavailability of Cd in soils 2. The vegetable production was 1129.2×104 tons in 2011. The leaf vegetable is the dominant consumption for Guangdong people, the planting area is about 33% of the total planting area 4 tons In the PRD area, the vegetable planting area is about 4830 kmIn this study, we conducted a systemic environmental quality survey of the vegetable soil and various vegetables in the PRD area. The aim of this study was to: (1) obtain the information on concentrations and spatial patterns of Cd in soils and leaf vegetables across the PRD area; (2) investigate the relationship between soil Cd and vegetable Cd on a regional scale; (3) reveal the transfer characteristic of Cd from soil to various leaf vegetable species.This study was carried out on collective-owned lands, and the owners of the lands gave us permission to conduct the study on these sites. The field studies did not involve endangered or protected species.2 of land area  (n = 31), Chinese flowering cabbage (Brassica campestris L. ssp. chinensis) (n = 37), Leaf mustard (Brassica juncea Coss) (n = 16), Romaine lettuce (Lactuca sativa L. var. longifolia) (n = 31), Chinese lettuce (Lactuca sativa L. var. asparagina) (n = 15), Cauliflower (Brassica oleracea L.var. botrytis L.) (n = 7), Water spinach (Ipomaea aquatic Forssk) (n = 7), Celery (Apium graveolens) (n = 6), Chinese chives (Allium tuberosum) (n = 5), Spinach (Spinacia oleracea) (n = 5), Amaranth (Amaranthus mangostanus L.) (n = 5), watercress  (n = 5).Both surface soil samples and vegetable samples used in this study were collected from locations shown in Vegetables and field soils at a soil depth of 0–15 cm which was the root concentrated layer in this study were collected when the vegetable were suitable for harvest. Each vegetable and soil sample consisted of five subsamples, and combined and mixed well. The fresh vegetable samples were put in clean plastic bags and immediately transported to the laboratory for sample treatment.The vegetable samples were cleaned with tap water and Milli-Q water, and then the edible parts (including leaves and steams) were separated, weighed (Fresh weight). The washed samples were then dried in an oven at 60°C, and their dry weights (DW) were recorded. The dry vegetable samples were ground to pass through a 250 µm sieve in a steel grinder. Soil samples were air-dried at room temperature (25°C) and ground to pass through a 2 mm nylon sieve. The fine vegetable and soil sample powders were stored in polythene zip bags.4, 2.5 ml HCl, and 2.5 ml HNO3. Total Cd concentrations in soils were determined by Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES) . The recovered soil Cd concentration of the National Research Center for GeoAnalysis soil standard reference materials (SRM)  was 0.081 mg kg−1 (n = 32). The analytic precision was 2.4% for soil Cd.The sieved soil samples were ground further to pass through a 150 µm nylon sieve. The prepared soil samples were digested to dryness using an acid mixture of 10 ml HF, 5 ml HClO2 solution (pH = 7.0), the mixture was shaken for one hour on 25°C. the suspension was filtered immediately after shaking. The Cd concentration in the filtrate solutions were measured by Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES) .Seventy five soil samples were analyzed for exchangeable Cd using the first step of Tessier sequential extraction3, and diluted to 25 mL with deionized water. Cd concentrations in vegetables were determined by Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES) . A plant standard reference material  was used in order to control the determination quality. The recovered Cd concentration was 0.059 mg kg−1 (n = 10). The analytic precision 3.5% for vegetable Cd. The Cd concentration in vegetable samples was expressed on a fresh weight basis.The ground vegetable samples were ashed in a muffle furnace for 16 h at 500°C, dissolved in 0.5 M HNOThe pH of soil was measured by taking 10 g of sample into 25 ml of deionized water vegetableC is Cd concentration (FW) in the edible parts of vegetables, and soilC is the total Cd concentration (DW) in the soil where the vegetable was grown.The transfer factors (TFs) of Cd from soil to vegetable (edible part) were calculated using the following equation The data statistical analysis was performed using the Minitab 16 statistical software . Spatial interpolation was performed using ordinary kriging. For the low-density sampling, the ordinary kriging estimate can be thought of simply as an optimally weighted average of the data −1 in the PRD area (−1 and geometric standard deviations (GSD) of 1.39 were used to represent the central tendency and variations of the data. The present GM value was much higher than its background concentration (0.04 mg kg−1) of soil in Guangdong province −1 in the reclaimed tidal flat soil and 1.4–1.8 mg kg−1 in some Cd-contaminated vegetable soils in the PRD area Total Cd concentrations in soils range from 0.012 to 1.18 mg kgPRD area . These vPRD area , therefo−1−1 in the Zhongshan city, followed by 0.291 mg kg-1 in the Foshan city, 0.226 mg kg-1 in the Guangzhou City, and 0.204 mg kg-1 in the Zhuhai City , 150 vegetable samples met the standard of No-Polluted vegetables (0.05 mg kg−1). Twenty vegetable samples were in between Safe vegetables and No-polluted vegetables, of which14 vegetable sample sites were in the Guangzhou.The present results show that in the PRD area the range of Cd concentration in all vegetable samples was 0.002–0.082 mg kg mg kg−1 . In this−1)> Huizhou (0.026 mg kg−1)> Dongguan (0.024 mg kg−1)> Zhongshan (0.022 mg kg−1)> Foshan (0.017 mg kg−1)> Jiangmen (0.014 mg kg−1)> Zhuhai (0.008 mg kg−1)  . This ormg kg−1) .−1 to 0.031 mg kg−1  and between transfer factor and vegetable Cd (r2 = 0.435)  . Our res= 0.435) , suggestThe soil-to-vegetable transfer factors (TFs) reflected the ability of vegetables to take up soil metals. TFs varied significantly with plant species In this study, the TFs of Cd from soil to vegetable for the 12 vegetable species varied from 0.045 in Water spinach to 0.753 in Amaranth . The TF b) for five kinds of main vegetables:The regression equations between the TF values and total Cd concentrations in soils can be described by power model (y  =  ax−1), the TFs decreased sharply; with the gradual increasing of soil total Cd concentration, TFs decreased only slowly, till to a stable value. Moreover, the 95% confidence interval of regression lines showed that there were a larger error range at the low Cd concentration interval of soils (0–0.1 or 0.2 mg kg−1) for the various leaf vegetable species, and when soil Cd concentrations were greater than 0.1 or 0.2 mg kg−1, the TFs of various vegetables would be reasonable with a lower error range for the assessment of environmental risk (For main leaf vegetables, the transfer factors of Cd decreased with increasing total Cd concentrations , suggesttal risk . As a reThe present results showed that the total Cd concentration in soils is safe for planting leaf vegetables in the study area. All vegetable samples met the national standard of safe vegetables, and 88% of which met the standard of no-polluted vegetables. Total Cd concentrations in soils and soil pH played an important role to Cd absorption of vegetables, but there were no obviously linear correlations. There was no spatial correlation between total Cd concentrations in soils and Cd concentrations in vegetables. For a givenl vegetable species the regression model between the TF value and the corresponding total Cd concentrations in soils could be used to estimate the transfer abilities of Cd from soil to vegetable at a given total Cd concentration in soils.


No standard technique for performing the assay currently exists. The impact of methodological heterogeneity was determined.Reduced ex vivo to evaluate the effect of variables identified via literature review on tumor necrosis factor-α (TNFα) release. These included sample handling, stimulation technique, and incubation conditions. Reporting convention was additionally assessed.Clinical experimental study set in a research laboratory. Venous blood from 5 to 10 healthy volunteers/experiment  was stimulated P < 0.01). Sample handling prior to stimulation  also caused significant alterations in TNFα release. Considerable interindividual variation was observed . Normalization by monocyte count and pretreatment with a cyclooxygenase inhibitor (indomethacin 10 μM) reduced the coefficient of variation from 47.17% to 32.09%.Measured TNFα release was significantly altered by source of LPS, concentration of LPS employed, duration and temperature of incubation prior to supernatant aspiration, and predilution of blood (repeated measures ANOVA, all ex vivo LPS-stimulated whole blood cytokine release assay. A standardized validated technique is required. The advent of trials of immunoadjuvant agents renders this a clinical imperative.Inconsistency in interlaboratory methodology and reporting impairs interpretation, comparability, and reproducibility of the  Late mortality in critically ill patients with severe sepsis, or following trauma, burn or major surgery, is now thought due to the predominance of anti-inflammatory processes. Whether excessive, sustained, or secondary to a failure of the immune system to recover from their functional consequences, the net effect is one of immunosuppression characterized by susceptibility to opportunistic infection and acquisition of secondary sepsis ,2. Theraex vivo lipopolysaccharide (LPS)-stimulated whole blood (WB) tumor necrosis factor α (TNFα) release  expression ,7 and ex release –10. Whil release , the samex vivo cytokine release assay, and result reporting convention, determining their impact on measured TNFα and hence quantification of immune competence.This study sought to identify key inter-laboratory differences in sample handling, technical performance of the WB Eighteen healthy volunteers  were recruited, 5 to 10 individuals participating in each experiment dependent on the variable evaluated. All samples were obtained and processed in a clinical research laboratory. Ethical approval for the protocol was provided by the University College London Research Ethics Committee (project ID: 4332/001).Health Volunteer Blood: Venous blood was collected and stored in lithium heparin , ethylenediaminetetraacetic acid (EDTA), or sodium citrate  containing tubes prior to stimulation.Salmonella abortus equi S-form [TLRgrade], Enzo Life Sciences, Farmingdale, NY), after Kox et al.  in 15 mL polypropylene tubes  and stimulated for 6 h  with 1 ng/mL LPS , LPS concentration , duration of stimulation , anticoagulant employed , incubation conditions , sample dilution , and sample handling . In addition, the effect of pretreating the blood with a non-steroidal anti-inflammatory drug  was evaluated. Where not supplied in solution LPS was reconstituted in sterile deionized water and vortexed extensively prior to use.Variables: On the basis of the results of the literature review the core technique was modified to evaluate the effect of LPS type . Differential leucocyte counts were performed via a Sysmex XE2100 flow cytometric analyzer by The Doctors Laboratory .t tests. Statistical significance was set at P < 0.05. All analyses were conducted in Prism 6 (GraphPad Software).Data points represent the average of three technical repeats, each assayed in duplicate. The D’Agostino–Pearson test was used to test for normality. Differences between categorical variables were tested for using either repeated measures one-way or two-way analysis of variance  with Tukey multiple comparisons test, or paired P < 0.001, one-way RM-ANOVA, Fig. S. minnesota causing significantly greater TNFα release than the 3 “smooth”-LPS sources . Cytokine release was further altered by the use of alternate anticoagulants  being significantly lower when EDTA was employed than either LH or Na Cit . Stimulation with either increasing doses of LPS (S. abortus equi), or for variable periods of time, predictably led to differential concentrations of TNFα in the supernatant when aspirated; 1 ng/mL and incubation time >4 h leading to maximal or equivalent responses  from different bacterial sources elicited significantly different concentrations of TNFα , increasing time leading to lower release and 20°C storage eliciting significantly greater release than either 4°C or 37°C . No significant interaction was observed between the two variables (P = 0.2). Predilution of blood, for ease of technical performance and to enhance supernatant yield, as expected, leads to a fall in TNFα concentration per unit supernatant with an increased ratio of media to WB .Variations in sample handling prior to LPS stimulation caused discrepancies in assayed TNFα. Both time to LPS addition and storage temperature significantly effected supernatant TNFα concentration  having measured concentrations ranging from 1,024 to 4,649 pg/mL . Normalization of these values by monocyte count further reduced the co-efficient of variation . While intra-individual variation in serially assessed TNFα release was additionally noted, the statistical significance of this was not tested .Considerable interindividual variation in LPS-stimulated TNFα release was observed, the supernatant of heparinized blood from 10 volunteers stimulated with 1 ng/mL Ex vivo LPS-stimulated WB TNFα release is a biomarker of monocyte function and reflective of global immune competence, persistent reduction being predictive of adverse outcomes, and its restoration an indicator of clinical improvement (rovement ,9,10,13.rovement –18, impaSelection of LPS source, LPS concentration, duration and temperature of incubation, anticoagulant and decision to dilute the blood varied extensively between laboratories, and were all demonstrated to significantly alter the assay result. Sample handling prior to stimulation, rarely reported in manuscripts, was additionally observed to contribute variance—both time to LPS addition and WB storage temperature— impacting subsequent TNFα release. Reporting inconsistencies further obfuscate results, studies presenting either concentration of TNFα alone  or expreex vivo WB assay (These results are not unexpected. It has long been known that varying the LPS dose or duration of incubation alters the readout of the WB assay ,15. Nor WB assay ,20. EstaWB assay . It is vex vivo WB stimulation assay, instead seeking to identify key sources of methodological variance and highlight their implications. Participants were healthy volunteers, a “core” method was selected and only one method of TNFα measurement was performed. Response variation may have differed had an alternate system and population been employed. In particular, the importance of normalizing TNFα release to monocyte count—more variable in acute illness—to accurately gauge immune competence may have been underestimated. The blood collection tubes employed were sterile and from one batch, yet not guaranteed to be pyrogen free. Additional technical factors not elaborated here may also impact measured TNFα release including the method of LPS extraction, purification and solubilization, and the testing of either fresh or frozen supernatants. Future work may address these deficiencies and should seek to formulate and validate the predictive and diagnostic value of a standardized assay—in conjunction with additional metrics of immunosuppression—as a clinical imperative.It should be noted that this study was not designed to define a “gold-standard” method of undertaking the


The Purpose of this study was to evaluate the sexual function after partial penectomy for penile carcinoma patients. Between January 2010 and May 2013, patients treated with partial penectomy at our institution were prospectively enrolled in this study. Sexual function (IIEF-15), age, body mass index, penile length in the flaccid state after partial penectomy (PL), treatment, having a partner and psychological factors (SAS scores and SDS scores) were assessed. Univariate and multivariate linear regression analyses were performed. 43 patients were included in our study. The median age was 56 years, and the median PL was 4 cm. The preoperative IIEF-15, SAS, SDS scores were significantly different from the postoperative scores. There was no statistically significant difference between the patients treated with partial penectomy and partial penectomy+ lymphadenectomy on IIEF-15 scores. Age was negatively associated with erectile function, sexual desire, and overall satisfaction; PL was positively associated with intercourse satisfaction; SAS score was negatively associated with erectile function, orgasmic function, sexual desire, and intercourse satisfaction. Our preliminary findings suggest that the sexual function after partial penectomy was significantly reduced. The sexual function was negatively affected by age and anxiety but positively affected by PL. Penile carcinoma is a rare malignant disease with a significantly higher incidence in some developing countries. Partial penectomy is a common treatment for penile carcinoma. After partial penectomy, vaginal penetration is frequently possible. According to Romero’s study, 55.6% of patients reported erection of the penile allowed vaginal penetration after partial penectomyBetween January 2010 and May 2013, penile carcinoma patients at our institution were prospectively enrolled in this study. The patients met the following selection criteria were eligible for study: 1) patients with penile carcinoma require partial penectomy. 2) Patients with regular sexual activity before surgery. Patients were excluded from our study if they had been treated with total penectomy, been treated with radiotherapy or chemotherapy, developed recurrent or metastasis disease. Laparoscopic bilateral inguinal lymphadenectomy (LBIL) was performed for patients with T2, T3 or T1G3 disease. These patients underwent LBIL within 3 months after partial penectomy. The methods were carried out in accordance with approved guidelines. The study was approved by the Ethics Review Board of Xiangya Hospital. The informed consent was obtained from all patients.The patients were followed up at outpatient every 3 months for 2 years after partial penectomy (or LBIL). The age, BMI, PL, treatment , having a partner, psychological factors, and sexual function were selected as monitored parameters. Sexual function was assessed with the IIEF-15 before partial penectomy and at the 6 month after partial penectomy (or LBIL)3SPSS17.0 software was applied for statistical analysis. We compared the demographic characteristics of partial penectomy patients with partial penectomy + LBIL patients using t-test and chisquare test. We compared the IIEF-15 scores of partial penectomy patients with partial penectomy + LBIL patients using t-test. We compared the preoperative IIEF-15, SAS and SDS scores with the postoperative scores using t-test. The level of statistical significance was set at p < 0.05. We used linear regression analyses to explore associations between each IIEF-15 term and age, BMI, PL, treatment, partner, SAS score, and SDS score. After first performing univariate regression analyses, variables with a p value < 0.15 were entered in the multivariate model, p values ≤ 0.5 were considered to reflect statistical significance.2, and the median PL was 4 cm. Of the 43 patients, 34 patients having a partner, 35 patients were performed LBIL after partial penectomy.Of 51 patients potentially eligible for the study, 8 were excluded: 4 patients treated with radiotherapy or chemotherapy after partial penectomy; 2 patients developed recurrent disease; 1 patient refused to complete questionnaires; 1 patient lost follow-up after partial penectomy. The remaining 43 patients fulfilled the inclusion and exclusion criteria for the study. The median age of patients was 56 years, the median BMI was 24 g/mAfter partial penectomy, 21 (48.8%) patients reported erectile function that “most times” or “always” allowed sexual intercourse. 12 patients reported erection of the penile “sometimes” or “a few times” hard enough for penetration. 10 patients reported having “no sexual activity” or “almost never”. On multivariate analysis, age and SAS score were negatively associated with erectile function. .When had sexual stimulation or intercourse after partial penectomy, 28 patients reported “most times” or “always” ejaculate or have the feeling of orgasm, 5 patients reported “sometimes” or “a few times” ejaculate or orgasm, 10 patients reported having “no intercourse”, or “almost never” ejaculate or orgasm. On multivariate analysis, SAS score was negatively associated with orgasmic function. .26 patients reported they “most times” or “always” had felt sexual desire after surgery and with a “high” or “very high” level of desire, 12 patients reported “sometimes” or “a few times” had felt sexual desire, 5 patients reported “almost never” had felt sexual desire. On multivariate analysis, age and SAS score were negatively associated with sexual desire. .12 patients reported they had more than seven attempts sexual intercourse one month after surgery. 19 patients reported they had three to six attempts sexual intercourse one month. 12 patients reported they had “one to two” or “not attempts” sexual intercourse. When attempted sexual intercourse, 16 patients “most times” or “always” felt satisfied, 16 patients “sometimes” or “a few times” felt satisfied, 11 patients “never” felt satisfied or “not attempt intercourse”. 10 patients reported their sexual intercourse “highly” or “very highly” enjoyable. 19 patients reported their sexual intercourse “fairly enjoyable”. 14 patients reported their sexual intercourse “no enjoyment” or they “no intercourse”. On multivariate analysis, SAS score was negatively associated with intercourse satisfaction; PL was positively associated with intercourse satisfaction. .7 patients reported they were “very or moderately satisfied” with their overall sex life or sexual relationship after surgery. 28 patients reported they were “about equally satisfied and dissatisfied” or “moderately dissatisfied”. 8 patients reported they were “very dissatisfied”. On multivariate analysis, age was negatively associated with overall satisfaction. .While penile carcinoma is a rare malignant disease, it has significant physiological and psychological effects on the patient. A variety of treatment modalities exist for patients with penile carcinoma, which dependent on the tumor location, size and stage. Aggressive therapy with partial penectomy is a common treatment for penile carcinoma patients. Although there is a reduction in the length of penis, several studies have reported that the remaining penis may still become erect and vaginal penetration is frequently possibleThe psychological states were also getting worse. According to SAS/SDS scores after surgery, 25 patients (58%) were anxiety and 17 patients (39%) were depression. Few studies investigating psychological states after partial penectomy, and their results were controversial. In the study of Ficarra, the General Health Questionnaire showed impaired well being in 37.5% of patients after partial penectomyFew studies had evaluated the effect of inguinal lymphadenectomy on sexual function for penile carcinoma patients. Intraoperative nerve damage and postoperative lymphedema are both likely to affect sexual function. In Kieffer study, patients treated with lymphadenectomy were not significantly different from those without lymphadenectomy on sexual functionWe found that the sexual function of penile carcinoma patients after partial penectomy was negatively affected by age and SAS score. As mentioned above, the morbidity of anxiety and depression were significantly increased after partial penectomy, more than half of the patients had experienced psychological problems. As psychological states were significantly related to sexual function, multidisciplinary follow up with psychologists is necessary.Intercourse satisfaction was positively influenced by PL in our study. Longer penile length after partial penectomy was benefit for sexual function. The incision site is marked at 2 cm from the tumor in our study when performed partial penectomy. In Agrawa study, the specimens were evaluated histologically for grade and for proximal microscopic extensions beyond the visible penile tumor margin, by examining serial proximal 5 mm sectionsPartner support is an important factor that helped patients to overcome the difficulties after partial penectomy11Although this is a relatively large sample size prospectively study compared to earlier studies, only 43 patients were included in our study. More subgroup analysis was not allowed, because the sample size was not sufficient. Another limitation was no control group was included in our study, which might induce selection bias. Further large-scale studies with control group should be performed to investigate the sexual function of penile carcinoma patients.In conclusion, our preliminary findings suggest that the sexual function after partial penectomy was significantly reduced. The sexual function was negatively affected by age and anxiety but positively affected by PL.How to cite this article: Yu, C. et al. Sexual Function after Partial Penectomy: A Prospectively Study From China. Sci. Rep. 6, 21862; doi: 10.1038/srep21862 (2016).


Together with plaques deposition, the hyperphosphorylation of the microtubules associated protein tau and the formation of intraneuronal neurofibrillary tangles are a typical neuropathological feature in AD brains. Cellular dysfunctions involving specific subcellular compartments, such as mitochondria and endoplasmic reticulum (ER), are emerging as crucial players in the pathogenesis of AD, as well as increased oxidative stress and dysregulation of calcium homeostasis. Specifically, dysregulation of intracellular calcium homeostasis has been suggested as a common proximal cause of neural dysfunction in AD. Aberrant calcium signaling has been considered a phenomenon mainly related to the dysfunction of intracellular calcium stores, which can occur in both neuronal and nonneuronal cells. This review reports the most recent findings on cellular mechanisms involved in the pathogenesis of AD, with main focus on the control of calcium homeostasis at both cytosolic and mitochondrial level.Alzheimer's Disease (AD) is a neurodegenerative disorder characterized by progressive neuronal loss. AD is associated with aberrant processing of the amyloid precursor protein, which leads to the deposition of amyloid- Alzheimer's Disease (AD) is the most common type of dementia affecting millions of people. According to Alzheimer's Disease International (ADI), as of 2015 people suffering from dementia worldwide accounted for estimated 46.8 million. Approximately 70% of these cases were attributed to AD. This amount will increase to an estimated 74.7 million in 2030 and 131.5 million in 2050, with a parallel rise of healthcare costs. As a matter of fact, global costs of dementia have increased from US$ 604 billion in 2010 to US$ 818 billion in 2015, for a 35.4% increase. The incidence rate for AD grows exponentially with age, with the main onset time observed in people aged over 60, in particular between the age of 70 and 80 [β (Aβ) peptides and the intracellular accumulation of neurofibrillary tangles (NFTs). Aβ peptides are cleaved products of APP obtained via sequential proteolysis by two membrane-bound endoproteases, aspartyl β-secretase and presenilin-dependent secretase (γ-secretase) [α-secretase to produce nontoxic fragments, which are thought to antagonize Aβ peptides generation [β is a protein consisting of 39–43 amino acids, and it mainly exists in two isoforms: soluble Aβ1–40 (~80–90%) and insoluble Aβ1–42 (~5–10%) [β1–40, Aβ1–42 seems to be the main pathological isoform [β globular oligomers can form along a new aggregation pathway independent of Aβ fibril formation. These globular Aβ oligomers have been found in the brain of patients affected by AD and APP transgenic mice, and they bind specifically to neurons and affect synaptic plasticity, as demonstrated by Barghorn and coworkers [β oligomers has also been supported by evidence showing that they can bind to glutamate receptors (both ionotropic and metabotropic), thereby impairing glutamatergic neurotransmission [β can be involved in important physiological processes, such as synapse activity and behavior [AD is a progressive neurodegenerative disorder leading to severe cognitive, memory, and behavioral impairment . The majcretase) , 14. APPneration . Aβ is a(~5–10%) , 16. In  isoform . Interesoworkers . The dissmission , 20. It behavior , 22.β-mediated caspases activation, Aβ-mediated oxidative stress, chronic oxidative stress, and reduced insulin-like growth factor 1-mediated oxidative stress [β oligomers, it promotes excitotoxicity. Additionally, tau phosphorylation can modulate DNA integrity and global changes in transcriptional events [As for NFTs, it has been found that their major constituent is the protein tau. Tau is the predominant microtubule-associated protein found in mammalian brain . During e stress . Over thl events .β plaques and NFTs, often referred to as “positive features” [β plaques are mainly distributed. Aβ-deposition renders cells more likely to develop inflammatory responses that involve the production of neuronal and glial cytokines belonging to the Tumor Necrosis Factor-α (TNF-α) superfamily [β-induced toxicity [ in vitro experiments conducted using the differentiated human neuroblastoma cell line SH-SY5Y demonstrated that the nonsteroidal anti-inflammatory derivative CHF5074 abrogates neurotoxic effects of both Aβ25–35 and TRAIL [Aeatures” , occur ieatures” , 13. In eatures” –40. Groweatures” , focusinerfamily . Interestoxicity . In thisnd TRAIL , suggestAD patients show symptoms that can be divided into two main categories: cognitive and psychiatric. Cognitive symptoms include loss of long term memory, aphasia, apraxia, and agnosia, while psychiatric symptoms include personality changes, depression, and hallucinations . AD is Calcium can be considered a ubiquitous intracellular messenger within cells acting as a regulator in multiple physiological functions. As a divalent cation, calcium can bind to several proteins, receptors, and ion channels. All of these properties are of great importance within neurons, where continuous firing of action potentials leads to calcium cycling, and it implies an influx through the calcium channels at the plasma membrane level, intracellular buffering, and an efflux through the calcium plasma membrane transporters. This cycling involves several subcellular compartments and proteins. In particular, two organelles play a major role in calcium buffering, namely, endoplasmic reticulum (ER) and mitochondria, whereas ATPase calcium pump and NCX are the two main systems involved in calcium efflux through the plasma membrane . Perturb in vitro evidence suggests that neuroprotection could be mediated by the restoration of calcium homeostasis. Different calcium channel blockers have been reported to be effective in preventing long- and short-term memory impairment induced by Aβ25–35  has been described to attenuate Aβ25–35-induced death in SH-SY5Y cells and to regulate Aβ-induced signal cascade in neurons [There is a large body of evidence documenting a connection between calcium homeostasis disruption and the development of neurodegenerative diseases such as Alzheimer's . The inv peptide ) and in  neurons –60. Howe neurons –63.β, calcium, and AD has been postulated by Arispe and coworkers [β oligomers can form calcium-permeable channels in membranes. It seems that energy deficits can promote this association, consistently with the observation that neurons with low cytosolic ATP levels showed a pronounced vulnerability to Aβ-induced toxicity [β plaques, thanks to the high concentration of Aβ oligomers found in these areas [β does not seem to be necessarily sustained by extracellular calcium influx. By using the human neuroblastoma SH-SY5Y cell line, Jensen and coworkers [β1–42 fragment can occur in the absence of extracellular calcium. Such observation supports the role of calcium release from the ER [β1–42 when the fragment was applied to permeabilized cells deficient in IP3 receptors (IP3R). Notably, this effect could underpin an additional direct effect of Aβ1–42 upon the ER and a mechanism for induction of toxicity by intracellular Aβ1–42 [β-induced calcium release from ER, as described by Ferreiro and coworkers [β1–40 or to Aβ25–35 peptides, the authors observed an increase in cytosolic calcium levels that was counteracted by either xestospongin C or dantrolene, pharmacological inhibitors of IP3R and RyR, respectively. Once calcium has been mobilized, it can initiate a cascade of events promoting free radicals generation, cytochrome c release from mitochondria, and activation of caspases, culminating in apoptotic cell death [β1–40 or Aβ25–35 peptides. These findings lend support to the hypothesis that intracellular calcium deregulation induced by ER stress may be critical in the neurodegenerative processes triggered by Aβ peptide. Furthermore, the role of SERCA has been also investigated in the context of the FAD. Specifically, it has been proposed that SERCA activity is physiologically regulated by the interaction with presenilin [β fragment. The finding that the modulation of SERCA activity would alter Aβ production may entail a possible role of the SERCA in the pathogenesis of AD [At cellular level, it is well documented that abnormal amyloid metabolism induces an upregulation of neuronal calcium signaling, firstly resulting in a decline of memory and then leading to apoptosis , 64, 65.oworkers , who sugtoxicity . In linetoxicity , 69, supse areas . Tau prose areas . Within oworkers  interestm the ER  to the g Aβ1–42 . As a maoworkers , 74. Expll death , 74. It esenilin , the memis of AD .β oligomers can disrupt neuronal glutamate uptake and promote long-term synaptic depression (LTD), a form of synaptic plasticity. In particular, the elegant study by Li and coworkers [β oligomers from several sources, including human brain extracts, facilitated electrically evoked LTD in the mouse hippocampal CA1 region, involving both metabotropic and ionotropic glutamate receptors, and high extracellular glutamate levels. Accordingly, neuronal synaptic glutamate uptake was significantly decreased by Aβ. It is interesting to note that Aβ-facilitated LTD was mimicked by the action of the glutamate reuptake inhibitor DL-threo-beta-benzyloxyaspartate (TBOA), confirming that Aβ oligomers ability to perturb synaptic plasticity may rely upon glutamate recycling alteration at the synaptic level. In this regard, a dramatic reduction in the expression of two members of the EAAT family, EAAT1 and EAAT2, has been described at both gene and protein levels in hippocampus and gyrus frontalis medialis of AD patients [α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptors. Considering that both NMDA and AMPA receptors are known to mediate long-term potentiation [The alteration of the glutamatergic system may be another important factor causing calcium imbalance in AD. Once released at glutamatergic synapses, glutamate is cleared from the extracellular space by the activity of the high affinity sodium-dependent glutamate transporters  , which roworkers  showed tpatients . Interespatients . Such upntiation , 85, thentiation .β may imbalance calcium homeostasis in brain endothelial cells with an increase in oxidative stress [β alters ER ability to buffer calcium, and it enhances the mitochondrial and cytosolic response to ATP-stimulated ER calcium release. Although these responses are compensated after a longer exposure to Aβ, the early increase in oxidant levels and the concomitant decrease of antioxidant defenses induce deleterious effects on endothelial cells that undergo apoptosis, contributing to the cerebrovascular impairment observed in AD [Although the observed alterations in intracellular calcium homeostasis in neurons significantly contribute to the pathogenesis of AD, more recent findings suggest that calcium dysregulation occurring in other cell types that support neuronal activity may contribute to degenerative processes . In thise stress . Using red in AD .β interaction with the Calcium Sensing Receptor (CaSR) [β interaction induces a downregulation of CaSR, leading the neighboring neurons to oversecrete de novo synthesized Aβ as well as nitric oxide (NO) and the toxic peroxynitrite (ONOO−) [β and CaSR in human astrocytes may activate a signaling able to stimulate de novo production and secretion of vascular endothelial growth factor (VEGF) [Astrocytes are also emerging as active players in AD , as highr (CaSR) . The CaSr (CaSR) , where tr (CaSR) . They sh (ONOO−) , 93. Recr (VEGF) , whose er (VEGF) –97.In general, the available literature suggests that the prolonged intracellular calcium elevation occurring within brain cells may be a crucial early event in AD pathogenesis, even though the mechanisms have not been fully explained.In terms of proteins contributing to the calcium homeostasis in the brain, particular attention should be focused on NCX. NCX is a transporter that can move sodium across the membrane in exchange for calcium, operating in either calcium-efflux/sodium-influx mode (forward mode) or calcium-influx/sodium-efflux mode (reverse mode) depending upon the electrochemical ion gradients . Three Nβ could interact with the hydrophobic surface of NCX, leading to an altered activity of the transporter [β oligomers with the plasma membrane could be per se responsible for the alteration of NCX transport properties [β, supporting the hypothesis that the NCX activity modulation can be connected to a direct interaction with Aβ; furthermore, in all synaptic terminals containing Aβ, NCX1-3 expression is upregulated [β-induced alteration in calcium homeostasis. But, the different pattern observed in NCX isoforms expression can underpin a specific role for each isoform within the neurodegenerative process accompanying AD. In this regard, a specific alteration has been demonstrated for NCX3 isoform, leading to inactivation. NCX proteins can be inactivated by specific calpain 1 operated cleavage, and this can produce an increase of intracellular calcium levels contributing to the neurodegenerative calcium overload [β increases calpain-mediated cleavage of NCX3, resulting in a decreased NCX3 activity [The role of NCX isoforms in the pathogenesis of AD is still under investigation. In 1991 Colvin and coworkers , measurinsporter ; howeveroperties . The piooperties . The anaoperties . Interesegulated . It couloverload , 106. Inactivity . Interesactivity  suggestsactivity .β may enter mitochondria, which generate reactive oxygen species leading to oxidative damage, thereby affecting mitochondrial function. That is why the upregulation of mitochondrial genes could be a compensatory response to mitochondrial dysfunction induced by mutant APP or Aβ [Mitochondria are essential organelles for both cell survival and death, as they produce the largest part of cellular energy in the form of ATP and they play an active role in apoptosis induction , 110. MiPP or Aβ , 116.In healthy neurons synaptic activity can be influenced by mitochondrial dynamics, such as fission and fusion events . A numbeβ, recently there has been an increasing interest on the role of the hyperphosphorylated form of tau. Hyperphosphorylation can decrease tau binding to microtubules, thereby affecting their stability and axonal transport of organelles, including mitochondria [β can cause an excessive mitochondrial fragmentation and both mitochondrial and synaptic deficiencies, leading to neuronal damage and cognitive decline [β and tau has been further investigated in a recent study by Quintanilla and colleagues who demonstrated that, in aging neuronal cultures, phosphorylated tau potentiates Aβ-induced mitochondrial dysfunction by affecting mitochondrial membrane potential and increasing oxidative stress [β [β plaques. Only triple transgenic mice, combining both pathologies, at early age (8 months old) showed a reduction of the mitochondrial membrane potential, while at the age of 12 months they showed the strongest defects on oxidative phosphorylation, synthesis of ATP, and reactive oxygen species formation, emphasizing synergistic and age-associated effects of Aβ and tau in perishing mitochondria [β and that hyperphosphorylation of tau can enhance the Aβ-induced mitochondrial neuronal damage. Notably, mitochondria are also involved in the maintenance of cellular activities through the contact they establish with ER [γ-secretase complex which processes APP to produce Aβ [β overproduction by γ-secretase activity leading to FAD [β in subcellular fractions isolated from mouse brain. They found that a large amount of Aβ was produced at mitochondria-ER contact sites. They postulated that the enhanced Aβ production may perturb mitochondria and mitochondria-ER contact site functions, leading to neurodegeneration and, therefore, to AD [σ1 receptor (σ1R) [σ1R promotes calcium transport into mitochondria from the ER by interacting with the IP3R [β results in the increase of MAMs-associated proteins expression and of the amount of contact points between ER and mitochondria in different AD models  [Although it was common to focus primarily on Achondria , 31. Recchondria  in SH-SYchondria . An imbachondria  who demo decline . Regardle stress . In a pre stress , signifitress [β . Moreovechondria . Globall with ER , 128. Mi with ER , 129. Th with ER , componeoduce Aβ . A largeg to FAD , 133. Reg to FAD . Howeverg to FAD . This ing to FAD . A receng to FAD  supportse, to AD . As a mae, to AD , 136. Ine, to AD . In partor (σ1R) . These por (σ1R) , whereasthe IP3R . Their kthe IP3R . FurtherD brain) . In turnD brain) , 50, 67. in situ electron microscopy immunocytochemistry in rat neocortex and hippocampus, we observed a large population of neuronal and astrocytic mitochondria expressing NCX1–3 [β shows, through a proteomic analysis, that one-third of the deregulated proteins in different AD mouse models is made up of mitochondrial proteins involved in oxidative phosphorylation [M), also called “ΔΨM flickering,” which is normally induced by cyclosporine. It is worth noting that mNCX blockade with CGP-37157 suppresses flickering in control cells, thus recreating a condition similar to the one observed in AD. The role of mNCX in AD is also supported by the work of Chin and colleagues [β potentiates the increase in cytosolic calcium concentration evoked by nicotine in dissociated rat basal forebrain neurons in a CGP-37157-sensitive way.Intracellular calcium dysregulation is a central event in neurodegeneration; it involves plasma membrane transporters and also intracellular organelles, such as mitochondria, thereby creating an endless futile cycle that can have several consequences on neuronal survival . The excg NCX1–3 , 27 (Figg NCX1–3 , 140. Thg NCX1–3 . One of rylation . Hence, rylation , 140. Thrylation . Thus, trylation . An incrrylation , 100. Asrylation , 100. Thrylation . In an irylation  reportedlleagues , who obsDysregulation of intracellular calcium homeostasis has been suggested as a proximal cause of cellular dysfunction during AD, and in this context calcium imbalance has been considered a phenomenon mainly related to the dysfunction of subcellular organelles, such as mitochondria. Functional impairment of calcium-related proteins may play a major role in the pathogenesis of AD. One of the main regulators of intracellular calcium levels, NCX, is emerging as a transporter possibly involved in the nervous system pathophysiology, although its involvement in AD is still poorly investigated. Recent studies conducted by our group , 100 sho


PsCHS, PsF3′H, PsDFR, and PsANS, which expressed at a significantly higher level in the purple spot than in the white non-spot. We further validated the digital expression data using quantitative real-time PCR. Our result uncovered transcriptome variance between the spot and non-spot of tree peony flower, and revealed that the co-expression of four anthocyanin structural genes was responsible for spot pigment in tree peony. The data will further help to unravel the genetic mechanism of peony flower spot formation.The pigmented cells in defined region of a petal constitute the petal spots. Petal spots attract pollinators and are found in many angiosperm families. Several cultivars of tree peony contain a single red or purple spot at the base of petal that makes the flower more attractive for the ornamental market. So far, the understanding of the molecular mechanism of spot formation is inadequate. In this study, we sequenced the transcriptome of the purple spot and the white non-spot of tree peony flower. We assembled and annotated 67,892 unigenes. Comparative analyses of the two transcriptomes showed 1,573 differentially expressed genes, among which 933 were up-regulated, and 640 were down-regulated in the purple spot. Subsequently, we examined four anthocyanin structural genes, including  Paeonia suffruticosa Andr.) – a woody shrub of the genus Paeonia and family Paeoniaceae – is a popular ornamental flower owing to its distinct petal colors. In some peony cultivars, the pigmented cells on the corolla form petal spots and show more intense colors as compared with the background. In China, more than 1000 tree peony cultivars are mainly divided into four geographical groups including Xibei, Zhongyuan, Jiangnan, and Xinan groups , three bHLH regulators , and four MYB regulators  , chalcone isomerase (CHI), flavonoid 3-hydroxylase (F3H), and flavonoid 3′-hydroxylase (F3′H), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS), and UDP flavonoid glucosyl transferase (UFGT) are the well-characterized genes from the anthocyanin biosynthetic pathway . AnthocytMYB114) . In sometMYB114) , like intMYB114) . Recentl Mimulus , LhMYB12n lilies , 2014, ahidaceae . Tree pePrevious studies of anthocyanin have provided unique insights into the molecular mechanisms of many non-model plant species. In the absence of complete genome sequence (as in tree peony), transcriptomic analysis is an effective method for gaining insights into differentially expressed genes (DEGs). Transcriptome sequencing of the spotted tissues and non-spotted tissues of peony will provide useful insights into the genetics of spot formation. Therefore, we sequenced the transcriptome of petal spot and background of the tree peony flower using Illumina HiSeq2000 platform. We then compared the two transcriptomes and filtered the DEGs. Our main objective was to annotate and analyze the DEGs to identify the candidate genes involved in spot development.Paeonia suffruticosa Andr. cv. “Jinrong” plants were grown under field conditions in Luoyang Academy of Agriculture and Forestry Sciences . As is described by Total RNA was extracted using the modified CTAB method, and was then purified with the RNeasy Plant Mini Kit according to manufacturer’s protocols. The RNA-integrity number (RIN) of each sample was determined using an Agilent 2100 Bioanalyzer. mRNA was enriched from 20 μg total RNA using magnetic beads with Oligo (dT), and was cut into random fragments using fragmentation buffer. First-strand cDNA was synthesized using short fragments as templates and random hexamer primers, followed by synthesis of the second-strand cDNA with dNTPs, RNase H and DNA polymerase I. The double-strand cDNA was purified via magnetic beads, subjected to an end repair process by adding a single nucleotide A (adenine) to the 3′ ends and ligating with sequencing adaptors. The suitable fragments were selected and enriched with PCR amplification as templates. The libraries were validated using Agilent 2100 Bioanalyzer and sequenced using an Illumina HiSeq2000 sequencing platform. The preparation of libraries and sequencing projects were performed at Encode Genomics Bio-Technology Co., LTD. . The transcriptome datasets were deposited at the NCBI database under accession numbers SRR1825648 and SRR1825653.E-value of less than 1e-5. Blast2GO . Quantitative RT-PCR was performed using SYBR Premix Ex Taq (Takara) on ABI 7500 system. Primers used are listed in Supplementary Table PCR data . The relT method . The staFigure 1). The anthocyanin content of the spot was significantly different from that of the petal. HPLC analysis revealed that highly content of cyanidin-3-O-glucoside was accumulated in the spot (1.83 ± 0.07 mg/g), but was barely detectable in the non-spot extraction.The “Jinrong” cultivar of the Xibei group – with a purple spot and white petal – shows significantly demarked spot because of its white background  were between 200 and 500 nt in length; 11,873 unigenes (17.49%) were between 500 and 1000 nt; and 13,159 unigenes (19.38%) were longer than 1000 nt .Sequencing projects generated 6.95 G and 7.46 G raw data from spot and non-spot libraries, respectively. E-value of less than 1e-5. Our results showed that 28,039 unigenes were annotated in public databases, accounting for 41.3% of total unigenes. We annotated 27,917 (41.1%), 17,341(25.5%), and 20,800 (30.6%) unigenes to nr, Swiss-Prot, and GO databases, respectively. About 7,980 unigenes (11.8%) and 5,770 (8.5%) unigenes had hits in the COG and KEGG databases, respectively.All unigenes were annotated by querying against the public databases, including non-redundant protein database (nr), Swiss-Prot, Gene Ontology (GO), KEGG, and the database of Cluster of Orthologous Groups of proteins database (COG). The best hit was selected from the hits with an Figure 3). Among these, 933 unigenes were significantly up-regulated while 640 unigenes were down-regulated. We assigned 988 of the 1,573 DEGs to three main GO categories including “molecular functions,” “biological processes,” and “cellular components” . Among them, 631 unigenes were grouped in the category “cellular components,” 771 unigenes in “molecular function,” and 792 unigenes in “biological processes.” In the “cellular component” category, “cell part” (505 unigenes), “cell” (498 unigenes), “organelle” (391 unigenes), and “membrane” (312 unigenes) were the most abundant groups. Under the “molecular function” category, the unigenes were most abundant in the “catalytic activity” (538 unigenes) and “binding” (434 unigenes) classes. In the “biological processes” category, the “metabolic process” (645 unigenes) and “cellular process” (526 unigenes) contained more DEGs than others.Comparative transcriptome profiling of the purple spot and non-spot tissues yielded 1,573 DEGs . Among the five main categories, “metabolism” was the largest category containing 122 unigenes, followed by “genetic information processing” (29 unigenes), “environmental information processing” (23 unigenes), “organismal systems” (5 unigenes), and “cellular processes” (5 unigenes). Since, the spot pigment was correlated with anthocyanin accumulation; we were interested in the anthocyanin biosynthesis pathway. We identified three pathways including “phenylpropanoid biosynthesis” , “flavonoid biosynthesis” , and “flavone and flavonol biosynthesis”  in the metabolism category, which were related to color development. Moreover, there were seven glucide metabolic pathways, including “starch and sucrose metabolism ,” “glycosaminoglycan degradation ,” “amino sugar and nucleotide sugar metabolism ,” “pentose and glucuronate interconversions ,” “glycolysis/gluconeogenesis ,” “fructose and mannose metabolism ,” and “galactose metabolism (4 DEGs ko00052).” All of these metabolic pathways may relate to the synthesis of substrates for anthocyanin biosynthesis.We further analyzed the unigenes by searching the KEGG database where out of the 1,573 DEGs, 179 unigenes were assigned to five main categories including 79 pathways (Table 1). Among them, c38856.graph_c0 (KT758291), c50492.graph_c0 (KT758292), and c56659.graph_c0 (KT758293) encode F3′H, DFR, and ANS, respectively. They were the same genes with that reported previously in tree peony . Among these four MYBs, nine bHLHs, and one WD40 were up-regulated in spot. Phylogenetic analysis revealed that c60620.graph_c0 (KT758294) was closely related to VvMYBA1 and VvMYBA2, both of which regulated anthocyanin biosynthesis in grape .In the anthocyanin biosynthesis pathway, structural genes are controlled by transcription factors from MYB, bHLH, and WD40 families . We idenPsCHS, PsF3′H, PsDFR, and PsANS were expressed at a significantly higher level in the red spot than in the white non-spot. HPLC analysis showed that the anthocyanin content was consistent with color variance between the spot and non-spot. Anthocyanins are tightly linked to color variation in the flower. In petal spot, the color cue is usually determined by anthocyanin accumulation. Cyanidin-based glycosides accumulate abundantly at the basal petal in tree peony and result in spot formation  and “flavone and flavonol biosynthesis” (ko00944) pathways relevant to pigment development were annotated. Moreover, we also annotated seven glucide metabolic pathways that may synthesize substrate for anthocyanin. Anthocyanins and their co-pigments are responsible for color formation in flowers. The pathways involved in the production of both anthocyanins and their co-pigments were described in transcriptomic analysis of plants like a juncea , peach fa juncea , grape ha juncea , herbacea juncea , and Saﬄa juncea . Our resClarkia gracilis  to yield pelargonidin, cyanidin, and delphinidin pigments . Plant cgracilis , Litchi sis Sonn , Magnolieri Pamp , and toreri Pamp . Our traNEGAN in monkey flowers (PeMYB11 in Orchidaceae (C. gracilis and pansy, a spot similar with tree peony was present in petal, researches revealed that the precise spatio-temporal expression of DFR2, F3′H1 in spot, and DFR1 and F3′5′H1 genes in background lead to spot formation in C. gracilis (VwF3′5′H, VwDFR, and VwANS was significantly increased in cyanic blotches of pansy, but which is the critical regulatory genes was still unclear (Flowers color is usually determined by activation of anthocyanin pathway. MYB-bHLH-WD40 were the key regulatory genes responsible for flower color patterns. Spots are common pigmentation pattern in flower, and some arbitrary and variable spots were caused by transposable elements insertion in anthocyanin structural or regulatory genes . Recentl flowers  and PeMYhidaceae . Tree pegracilis , while t unclear . As is rThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


AbstractThereusoppia species group includes species with and without a scent pad, which is a histologically and morphologically characterized male secondary sexual structure on the dorsal surface of the forewing. To assess the hypothesis that these structures are lost evolutionarily, but not regained (Dollo’s Law), the taxonomy of this species group is revised. Thereuslomalargasp. n., and Thereusbrockisp. n., are described. Diagnostic traits, especially male secondary structures, within the Thereusoppia species group are illustrated. Distributional and biological information is summarized for each species. Three species have been reared, and the caterpillars eat Loranthaceae. An inferred phylogeny is consistent with the hypothesis that scent pads in the Thereusoppia species group have been lost evolutionarily twice , and not re-gained.The  Eumaeini. Evolutionary losses appear to occur when a species is allopatric with its closest relative (Thereus Hübner (Lycaenidae: Eumaeini) possesses a variety of male secondary sexual structures, for which reason we are beginning to revise the genus systematically. The four primary secondary sexual structures in Thereus are forewing scent patches, hindwing scent patches, forewing scent pads, and abdominal brush organs  and Thereusoppia , is distinguished from the remainder of the genus by the presence of scent patches near the costa of the dorsal hindwing and on the inner margin of the ventral forewing .Another two oup Figs , 12, 13.pia Figs –24. For Thereusoppia species group lack scent pads on the dorsal surface of the forewing and two possess scent pads , this result would be consistent with the re-evolution of a scent pad, which would represent the first such documented case in the Eumaeini.Two species of the ads Figs . Given pads Figs , we predPageBreakThereus and to provide a brief overview of the biology of the genus. Another is to delimit the Thereusoppia species group and to provide names for the two unnamed species in this group. These names are needed for the phylogenetic analysis and for the publications of the life history of one of them. The third purpose is to propose a preliminary phylogenetic hypothesis for the Thereusoppia species group to assess whether male scent pad re-evolution is likely to have occurred in this species group.One purpose of this paper is to illustrate the proposed traits that characterize Thereusoppia species group is based on an analysis of variation among 121 pinned specimens from various museum and private collections, as noted below. Species accounts include notes, when relevant, on nomenclature, history, morphological variation, elevation, seasonality, and behavior. Males and females were associated by similarity of ventral wing pattern and distribution. In one species, rearing confirmed the association in a species with sexually dimorphic wing patterns. Geographic distributions of the new species are mapped. Labels on holotypes are recorded verbatim with brackets used for information not explicitly noted on the labels and for descriptions of the labels. Otherwise, months are abbreviated by their first three letters in English. Citations for original descriptions can be found in The species level taxonomy of the Thereusoppia. Standard references for morphological terminology are Eumaeini; Morphological characters for the phylogenetic analyses are utilized because DNA sequences are currently available only for Thereusoppia species group (Table Thereuscithonius (Godart), which shares small teeth on the dorsal tip of the penis with two species in the Thereusoppia species group , which shares a relatively straight vinculum strut in lateral aspect and a slender “crescent” shaped dorsal cornutus with the Thereusoppia species group  omitted. Characters were then mapped on trees with WinClada software (To assess scent pad evolution in the software  with theSpecimens cited in this study are deposited in the following collections (abbreviations where available from Evenhuis (2013)). The Natural History Museum [formerly British Museum ], London, United KingdomBMNH.PageBreak Instituto Alexander von Humboldt, Villa de Leyva, Boyacá, ColombiaIAVH. Museo de Entomología de la Universidad del Valle, Cali, ColombiaMUSENUV. Museo de Historia Natural, Universidad Nacional Mayor de San Marcos, Lima, PeruMUSM. Private Collection of Robert C. Busby, Andover, MA, USARCB. Entomology Research Museum, Department of Entomology, University of California, Riverside, California, USAUCRC. National Museum of Natural History, Smithsonian Institution, Washington, DC, USAUSNM.Taxon classificationAnimaliaLepidopteraLycaenidaeHübner, [1819]Papiliolausus CramerThereus by (1) a pair of sclerotized invaginations on the membrane attached to the ventro-lateral sides of the papillae anales , suggesting that this species is the phylogenetic sister to the remainder of the genus. Interestingly, it is the only Thereus species that appears to be a subtropical endemic. During this study, we found an instance in which sexual dimorphism of antennal nudum length was four segments—not five or greater—so the generic diagnosis is modified accordingly.les Figs , (2) a ples Figs , and (3)les Figs . The secThereus contained 27 species, but more than a decade later, 31 were listed, of which nine were undescribed and two had questionable generic placement , Thereuswojtusiaki Bálint, Thereusorasus , and an undescribed species are montane endemics, and Thereuspseudarcula is subtropical. The widespread Thereuscithonius occurs in a great variety of habitats, from wet forest to very dry deciduous forest and from sea level to 2,000 m elevation.Most species inhabit wet lowland forest, with only a few exceptions. Loranthaceae (mistletoe). As noted in this paper, males of some species set up mating territories in the morning before 09:30 hours while others set up territories in the early afternoon.Heredia and Robbins (in prep.) summarize the food plant records for the genus, almost all of which belong to the plant family Diagnosis. The four members of the Thereusoppia species group possess the proposed synapomorphies of Thereus and are distinguished by a convex inner margin of the forewing  Two of the four species have a scent pad on the dorsal surface of the forewing located at the basal origin of veins r M1 Figs . Scent p M1 Figs . Scent ps-M1 occurs in all four species of the Thereusoppia group  androconia are iridescent in Thereusorasus and Thereuslomalarga  A scent patch on the dorsal surface of the hindwing centered at the base of cell roup Figs , but in rga Figs . The andcki Figs . There apia Fig. . Superfiosmaitia  and LathLathecla , but notEumaeini, but not in other Thereus or Rekoa. In Thereusoppia and Thereusbrocki, there are also erect piliform setae that attach to the inner margin  A scent patch on the ventral surface of the forewing located between the inner margin and the cubital vein Figs . Again, gin Fig. . The tip A scent cki Fig. . Superfidorigini : 403, whThereus species have a pair of dorsal and a pair of ventral brush organs  All ans Figs . Brush o segment . They ha segment . The onl Thereus .Male genitalia . Male behavior is recorded for Thereuslomalarga and Thereusoppia.Taxon classificationAnimaliaLepidopteraLycaenidaeThereusorasus differs from other members of the Thereusoppia group by having a gray ventral ground color, not brown  with Theclaorasus  because they share a similar ventral wing pattern and occur in the same habitats and have the same distribution. We have examined both types.Thereusorasus is an uncommon species that is recorded from montane habitats from central Mexico (Colima and Veracruz) to those of western Panama (Chiriquí) at elevations from 1100 to 1800 m.Struthanthuscondensatus Kuijt (Loranthaceae). An eclosed adult female and its pupal case are deposited in UCRC. The mistletoe plant was growing on coffee and was identified by Kuijt.Greg Ballmer collected a larva on 30 Aug 1988 at El Jabalí, 13 mi NE Comala, Colima, Mexico, at 1100–1200 m. The caterpillar was eating Taxon classificationAnimaliaLepidopteraLycaenidaeRobbins, Heredia & Busbysp. n.http://zoobank.org/39501F5C-16C9-437D-874F-74722CEF1AB6Holotype: ♂ . Costa Rica. 1♀ Turrialba, 2,000 ft, 13 Jul 1965 (USNM). Panama. Canal Area. Paraíso, Cerro Luisa, 4 Feb 1979 (2♂ USNM), 16 Feb 1979 (1♂ USNM), 1 Mar 1979 (1♂ USNM), 4 Mar 1979 (1♂ USNM), 10 Mar 1979 (1♂ USNM). Pedro Miguel, Chiva Chiva Road, 14 Jan 1979 PageBreak(1♀ USNM). Panama Province. Cerro Campana. 1500 ft/500m. 3 Jan 1965 (1♀), 26 Jan 1966 (1♀ USNM), 28 Jan 1980 (1♀ USNM). 850 m. 23 Feb 1979 (1♀ USNM). Chiriquí Province. Potrerillos. 3600 ft. 27 Dec 1965 (1♀USNM), 28 Dec 1965 (2♀ USNM), 27 Dec 1965 (1♀USNM), 1 Jan 1966 (2♀ USNM), 28 Jan 1966 (2♀ USNM), 29 Jan 1966 (1♀ USNM), 2 Feb 1966 (1♀ USNM), 19 Feb 1966 (1♀ USNM), 5 Mar 1966 (1♀ USNM). Colombia. Valle del Cauca, Cali. Pance, 3000 ft, 14 Jan 1985 (1♀ USNM)). Loma Larga, 1200m. 3°19'N/76°34'W. 15 May 2009 (1♂ MUSENUV).18 Nov 2010 . 5 Dec 2010 (1♂ MUSENUV). 10 Dec 2010 (1♀ MUSENUV). 13 Jan 2011 . 15 Mar 2011 (1♀ MUSENUV). 17 Mar 2011 (1♀ MUSENUV). 19 Mar 2011 (1♀ MUSENUV). 31 Mar 2011 (1♀ MUSENUV). 1 Apr 2011 (1♂&1♀ MUSENUV). 2 Apr 2011 (1♂ MUSENUV). 5 Apr 2011 (1♀ MUSENUV). 6 Apr 2011 . 24 Apr 2011 . 21 May 2011 . 29 May 2011 (1♂ MUSENUV). 30 May 2011 . 1 Jun 2011 . 15 Jun 2011 (1♂ MUSENUV). 17 Jun 2011 (1♂ MUSENUV). 21 Jun 2011 . 23 Jun 2011 (1♂ MUSENUV). 27 Jun 2011 (1♀ MUSENUV). 28 Jun 2011 . 14 Jul 11 (1♂ MUSENUV). 23 Jul 2011 (1♂ MUSENUV). 17 Dec 2011 (1♀ MUSENUV). 31 Dec 2011 . 20 Feb 2012 (1♂ MUSENUV). 23 Feb 2012 (1♀ MUSENUV). 3 Mar 2012 (1♂ MUSENUV). 4 Mar 2012 (1♂ MUSENUV). 6 Mar 2012 (1♂ MUSENUV). 12 Mar 2012 (1♂ MUSENUV). 3 Apr 2012 (1♂ MUSENUV). 15 Apr 2012 (1♂ MUSENUV). 19 Apr 2012 (1♂ MUSENUV). 22 Apr 2012 (1♀ MUSENUV). 29 Aug 2012 (1♂ MUSENUV). 25 Jun 2014 (1♀ MUSENUV). 4 Jul 2014 (1♂ MUSENUV). 3 Jul 2014 (1♂ MUSENUV). Ecuador. Pichincha, 10 km Celica-Sardinas Road, 0°11.6'N, 79°00.8'W, 550-775 m, 27 May 2008, (1♀ RCB); 7 km Pacto-Guayabillas Road, 0°09.0'N, 78°48.9'W, 1600m, 18 Jun 2014, (2♀ RCB); 5 km Nanegal- García Moreno Road, 0°09.2'N, 78°39.4'W, 1375–1700m 21 Jan 2015, (1♀ RCB); 24 May 2008, (1♀ RCB).This species is named for Loma Larga, a housing development on the outskirts of Parque Nacional Natural Farallones de Cali. Loma Larga has had an ecological and conservation policy for about 15 years that has designated a substantial plot of land for natural forest regeneration Fig.  in contrFig. . The typThereuslomalarga belongs to Thereus because it possesses the synapomorphies of the genus , Canal Area, Panama (vouchers in USNM). The longitude on one specimen is incorrectly labeled 79°38'W.Six males displayed territorial hilltopping behavior from 09:00–09:30 hours during the dry season  at the top of a small tree on the southwest edge of the summit of Cerro Luisa, Paraíso  Kuijt (Loranthaceae) growing on Miconiaminutiflora (Bonpl.) DC. Details of the life history will be published elsewhere (Heredia and Robbins in prep.).Thereuslomalarga is a peculiar butterfly in that adult females are far more frequently encountered—at least by butterfly collectors—than are adult males. For example, all collected adults from Costa Rica, Colombia, and Ecuador are females. Among collected adults in Panama, females have been found from sea level in the Canal Area to Cerro Campana  to Potrerillos at 1,100 m (Chiriquí Province). In contrast, adult males have been collected only at the top of one small tree on Cerro Luisa in the Canal Area in the dry season in 1979. All other males, including the holotype, were reared from caterpillars. Among 44 reared individuals at the type locality, 27 are males, so the sex ratio among immatures is not biased towards females.Thereuslomalarga are “non-descript small gray hairstreaks”, and other specimens may be found in museum collections.No museum specimens other than those in the type series have been seen by the authors. However, females of Taxon classificationAnimaliaLepidopteraLycaenidaeThereusoppia is distinguished from Thereuslomalarga and Thereusbrocki by lacking a scent pad on the dorsal forewing , in contrast to the early morning territorial behavior of Thereuslomalarga.Territorial male behavior was observed at Ciudad Valles, SLP, Mexico in the early afternoon (vouchers in Thereusoppia were found nectaring on Cordia (Boraginaceae) flowers at two localities in Veracruz, Mexico (vouchers in RCB).Adults of Struthanthusorbicularis (Kunth) Blume (Loranthaceae) at Sendero Carobonal, Santa Rosa, Area de Conservación Guanacaste, Guanacaste, Costa Rica, latitude 10.77594, longitude -85.65799. An adult male  emerged 16 May 1993. As an associated comment on the web site, “red-brown pupa with white markings laterally so that it looks just like a bird turd, sitting on the top of a mistletoe leaf in middle of large plant ; host tree was leafless.” The leafless host tree is the reason that we consider the mistletoe plant on which the pupa was found to be the caterpillar food plant.From PageBreakTaxon classificationAnimaliaLepidopteraLycaenidaeRobbins, Heredia & Busbysp. n.http://zoobank.org/458AA4B0-A519-40D3-90BA-9FC7F286DBCEHolotype: ♂ .(1♀).  1♀ Fig. . Napo, 1Colombia. 1♀. Vaupés, Mitú, 28 Jun 1972 (USNM). Peru. 1♂ San Martin, Juanjuí, 7°11'S,77°44'W, 300–400 m, Nov 2011 . 1♀. Huánuco, Tingo María, 800 m, 24 Jun 1982 (USNM).. This species is named for James Brock of Tucson, Arizona. He collected the holotype and has made numerous contributions to the knowledge and enjoyment of butterflies. The name is a masculine noun in the genitive case.01°20'S,77°55'W, but incorrectly placed in Napo Province). The entrance to this locality is approximately 25 km (measured by a car odometer) from Puyo on the western side of the Puyo-Tena Rd. New metal road markers have been erected which place the entrance between km 26 and km 27. The trees in this location have been selectively logged for decades, and we do not believe much forest remains.The type locality has been a well-known collecting site for 40 years at about 975 m elevation , we have not had an opportunity to examine it. It is identified as Thereusbrocki because it has the shortened forewing discal cell of the holotype and the same male wing secondary sexual traits, except that the erect piliform setae are not visible in the image. For this reason, we exclude it from the type series.The holotype and the Peruvian male are the only males in collections, so far as we are aware. The type locality has been a “famous” collecting locality for decades, as noted, so it is somewhat unexpected that the holotype remains the only known Ecuadorian male. It would appear that adult males of Thereusorasus  (Thecla)type locality: Guatemalaechinita  (Thecla)= type locality: Mexico (VER)PageBreakThereuslomalarga Robbins, Heredia & Busby, sp. n.type locality: ColombiaThereusoppia  (Thecla)type locality: Mexico (VER)Thereusbrocki Robbins, Heredia & Busby, sp. n.type locality: EcuadorK resulted in one tree, also with the same topology as the most parsimonious equal weight tree. When the data were analyzed with the scent pad omitted (Character 12), the tree topology was the same. In accord with Based on morphological characters Table  coded inSystematics. The genus Thereus has been described six times, probably because the genus contains species that possess a variety of androconial organs, wing patterns, wing shapes, and wing sizes. However, Thereus is clearly characterized by synapomorphies of the male genitalia, female genitalia, and antennae, as illustrated in this paper. Further, a third of the species have been reared, and with the exception of an undescribed, morphologically aberrant species, all use Loranthaceae as a caterpillar food plant. A genus that is morphologically and biologically homogenous, such as Thereus, conveys information, which makes it a useful and predictive classification. For example, it allows us to hypothesize that all Thereus species that have not yet been reared, including Thereusbrocki, eat Loranthaceae.Thereuslomalarga and Thereusbrocki are very similar  of these and related species. More generally, it may be the best way to document and study the biology of Thereus species.Thereusoppia and Thereusbrocki )) or )) would be consistent with the evolutionary loss and re-gain of the scent pad. However, according to the phylogenetic results in this paper, in which the scent pad character was omitted, the scent pad was unambiguously lost twice evolutionarily and not regained. In each case, the species that lost the scent pad is allopatric with its sister species. This result is consistent with previous findings (findings  and more


Functional connectivity MRI (fcMRI) studies of individuals currently diagnosed with major depressive disorder (MDD) document hyperconnectivities within the default mode network (DMN) and between the DMN and salience networks (SN) with regions of the cognitive control network (CCN). Studies of individuals in the remitted state are needed to address whether effects derive from trait, and not state or chronic burden features of MDD.fcMRI data from two 3.0 Tesla GE scanners were collected from 30 unmedicated (47% medication naïve) youth  with remitted MDD  and compared with data from 23 healthy controls (HCs) using four bilateral seeds in the DMN and SN (posterior cingulate cortex (PCC), subgenual anterior cingulate (sgACC), and amygdala), followed by voxel-based comparisons of the whole brain.Compared to HCs, rMDD youth exhibited hyperconnectivities from both PCC and sgACC seeds with lateral, parietal, and frontal regions of the CCN, extending to the dorsal medial wall. A factor analysis reduced extracted data and a PCC factor was inversely correlated with rumination among rMDD youth. Two factors from the sgACC hyperconnectivity clusters were related to performance in cognitive control on a Go/NoGo task, one positively and one inversely.Findings document hyperconnectivities of the DMN and SN with the CCN (BA 8/10), which were related to rumination and sustained attention. Given these cognitive markers are known predictors of response and relapse, hyperconnectivities may increase relapse risk or represent compensatory mechanisms. Studying individuals with a history of major depressive disorder (MDD) who are currently in the remitted state allows for a unique examination of potential trait-based mechanisms of depression and depression relapse e.g., . As suchOne method for understanding trait-based markers for MDD involves studying network function through measurements of network connectivity. Resting state fMRI has emerged as an approach for the identification of brain-based biomarkers, particularly in the detection of variations in network connectivity deriving from clinical features In contrast, a task positive network includes regions that increase in activation during attention to demanding tasks Resting state fMRI examines intrinsic network functioning by capturing temporal correlations between brain regions in the blood oxygen level-dependent (BOLD) signal, offering a reliable Increased connectivity within and between networks is hypothesized to contribute to the tendency of depressed individuals to attend to internal stimuli and to return inadvertently to internal thoughts at the expense of external tasks Thus, a critical unanswered question is whether abnormal neural connectivities represent trait markers of vulnerability to illness and recurrence that are not related to state or chronic illness scars. To address this issue, we examined functional connectivity among youth in a remitted state. This design mitigates several potential confounds including current mood state, illness severity, increased scar due to repeated episodes, and the effects of current medication. We hypothesized that hyperconnectivities would be observed within and between regions of the DMN and SN with regions of the CCN among unmedicated youth with rMDD compared to Healthy Controls (HCs), reflecting potential trait-based risk for relapse, or conversely resilience. Finally, given preliminary research correlating DMN functional connectivity with repetitive negative thoughts such as rumination e.g.,  as well The current study was approved by the University of Michigan (UM) and the University of Illinois at Chicago (UIC) Institutional Review Boards and all participants signed consent. Participants were recruited using flyers and multiple forms of posting on the internet. All participants completed an identical assessment protocol, including the Diagnostic Interview for Genetic Studies DIGS; , the HamSelf-report rumination was collected using the Ruminative Response Scale RRS; . Forty iThis task  using T2*-weighted single shot reverse spiral sequence with the following parameters: 90 degree flip, field-of-view 20, matrix size = 64×64, slice thickness = 4 mm, 30 ms echo time, 29 slices. Eyes-open, resting scans at UIC were collected over eight minutes on a 3.0 T GE Discovery scanner  using parallel imaging with ASSET and T2* gradient-echo axial EPI with the following parameters: 90 degree flip, field-of-view 22, matrix size = 64×64, slice thickness = 3 mm, 22.2 ms echo time, 44 slices. At both sites, high-resolution anatomic T1 scans were obtained for spatial normalization and motion was minimized with foam pads, a visual tracking line (UIC only) and/or cross (UIC and UM) on the display, and by conveying the importance of staying still to participants, with TRs of 2000 ms and 240 TRs total. Site effects of acquisition parameters and scanner were evaluated and are reported in Supplementary Material .http://www.fil.ion.ucl.ac.uk/spm/doc/) and motion detection algorithms were applied using FSL (http://fsl.fmrib.ox.ac.uk/fsl/fslwiki/). Coregistration of structural images to functional images was followed by spatial normalization of the coregistered T1-spgr to the Montreal Neurological Institute (MNI) template. The resulting normalization matrix was then applied to the slice-time-corrected, physiologically corrected, time series data. These normalized T2* time-series data were spatially smoothed with a 5 mm Gaussian kernel resulting in T2* images with isotropic voxels, 2 mm on a side.Several steps were taken to reduce potential sources of noise and artifact. Slice timing was completed with SPM8  for group comparisons with a corrected p value of .05. Images are displayed on an averaged brain anatomy derived from the current sample.Correlation coefficients were calculated between mean time course for seed regions and all other voxels of the brain, resulting in a 3-dimensional correlation coefficient image (r image). These r images were transformed to z scores using a Fisher transformation. Resulting z images were used in 2-sample Student Importantly, movement was addressed using regression of white matter signal as recommended in the recent literature Factor analysis is a statistical technique that can be used to discover which variables form coherent subsets that are somewhat independent. Factors are hypothesized to reflect underlying processes resulting in correlations between variables. Factor analysis differs from principal components analysis in that factor analysis examines shared variance only and attempts are made to estimate and eliminate variance that derives from error Across groups, the left PCC seed was correlated with regions encompassing the DMN including the medial prefrontal cortex (PFC), posterior and superior temporal gyri, and bilateral hippocampus. Across groups, left sgACC activation was significantly correlated with nearby areas including the orbital frontal cortex, thalamus, hippocampus, and the PCC. Across groups, left amygdala activation was significantly correlated with surrounding bilateral amygdaloid, hippocampal, and uncal regions. Results from exploratory factor analysis suggested a two factor solution for the PCC seed and a three factor solution for the sgACC seed. PCC Factor 1 had higher loadings on six of eight clusters, as indicated in p<.01). For the parametric Go/No-Go, rMDD youth exhibited greater impulse control problems , but did not differ in Go Accuracy  or Go Response Time . Among youth with rMDD, Go Response Time was inversely correlated with rumination . Also among rMDD youth, the PCC Factor 2  was inversely correlated with rumination . These factors did not significantly affect between-group results for the PCC and sgACC seeds.The current results address an understudied, yet very important set of questions regarding the dissociation of state, trait, compensatory, and scar features of MDD. To begin unravelling these questions, we obtained resting state data from a homogeneous sample of carefully characterized, unmedicated youth in a remitted state, allowing us to study MDD course in the absence of state and chronic burden features that may have confounded previous results. This is the first study of young adults with rMDD and we demonstrate hyperconnectivity of both the DMN and SN with portions of the right superior and middle frontal gyri – a central node in the CCN. Furthermore, observed hyperconnectivities were related to two known predictors of relapse: rumination (inverse correlation) and sustained attention (positive correlation).Self-report rumination has previously been documented to correlate with sgACC-PCC connectivity during task among actively depressed adults consequence of depressive illness and sadness, as opposed to a potential risk or protective factor for future MDEs. In fact, a recent investigation of actively depressed, unmedicated adolescents found that higher levels of rumination were associated with reduced connectivity between the sgACC and the medial frontal gyrus increased connectivity between these same regions may strengthen the adolescent's ability to resist rumination and support attentional control, thereby promoting and potentially sustaining remission. Remitted youth whose CCN is overly-engaged may more effectively downregulate mind-wandering, self-referential thought supported by the DMN.The sgACC has long been implicated as a dysfunctional node in acute MDD and has been correlated with negative mood states, treatment response, and “treatment refractoriness” Our results extend and contextualize findings of a proposed abnormal ‘dorsal nexus’ in MDD It is also worth noting, however, that the primary factors (for both the PCC and sgACC seeds) accounting for the majority of factor variance across networks were not related to clinical features of illness or functioning in the current study – our results may reflect trait or scar biomarkers. A trait biomarker would represent a characteristic observable prior to illness and initially independent of illness, whereas a scar biomarker is a result of illness and represents a failure to achieve complete inter-episode recovery. The second and third factors, accounting for less variance, and more likely to be sample-specific, were related to cognitive markers of illness. Hyperconnectivities of the DMN and SN with portions of the CCN may result in increased neural resources directed towards emotion regulation among vulnerable individuals. Developmental hyperconnectivities may be protective to an extent, but may not always be sufficient for mounting adaptive responses to the increased adversity and stressors experienced by this population at-risk for multiple MDEs and comorbidities. We are currently following this sample longitudinally and will be able to identify predictors of both relapse and resilience.We note several limitations of our study. First, the current data cannot discriminate between network abnormalities that render an individual vulnerable to the first-onset of MDD versus scar consequences of illness, normal maturation processes, or even protective compensatory mechanisms. In this context, prospective studies of high-risk cohorts represent an important direction for future research. Second, despite the strength of recruiting a medication-free sample, we could not examine how previous medication use or therapy exposure may have affected current results. As a related point, because our participants have had relatively fewer MDEs and have been well for relatively extended periods of time, they may represent a more mild severity or course of illness. To capture developmental trajectories that contribute to resiliency and risk in MDD, future longitudinal research can examine whether excessive coupling of intrinsic networks predicts first-onset of depression or relapse as adolescents transition into early adulthood. In addition, anatomical specificity is of concern when conducting seed-based analyses, particularly in regions such as the PCC In sum, this study provides evidence of brain-based traits associated with MDD course that can be observed outside of a MDE, early in the course of illness. We believe the late adolescent-early adulthood transition represents a unique window for observing mechanisms of MDD, as stability of networks has been established developmentally, but opportunities remain for secondary prevention prior to the initiation of chronic illness. Thus, understanding the continuum of functional to dysfunctional connectivity patterns in the human brain is critical in elucidating the developmental psychopathology of MDD. Increased coupling between networks in our early course, remitted sample suggests that hyperconnectivities can be evaluated as developmental trait or resiliency factors, as targets for treatment, or as potential outcomes of early illness.Figure S1Participants with movement are easily identifiable by movement deviation. Mean movement deviations among the Healthy Control and remitted Major Depressive group in the x, y, and z planes.(TIF)Click here for additional data file.Figure S2Extracted connectivity bar graphs by cluster, site, and group. rMDD = remitted Major Depressive Disorder; HC = Healthy Control; UM = University of Michigan; UIC = University of Illinois by Chicago. Tailarach coordinates corresponding to each numbered cluster: Clusters 1–7 are connectivities with the left PCC seed, Clusters 8–24 are connectivities with the left sgACC seed, Clusters 25–28 are connectivities with the left amygdala: 1 = −41, 35, 12; 2 = −6, −63, 49; 3 = 12, 45, 14; 4 = 17, −9, 3; 5 = 26, 32, 42; 6 = 27, 24, 20; 7 = 43, −36, 30; 8 = −22, −38, 0; 9 = −24, −30, 16; 9 = −24, 43, 13; 10 = −24, 43, 13; 11 = −31, −83, −33; 12 = −36, 16, 45; 13 = −43, −65, 35; 14 = −54, −54, −1; 15 = −57, −15, −19; 16 = −1, −15, 6; 17 = 8, 32, 49; 18 = 10, 63, 19; 19 = 15, −69, −39; 20 = 29, −71, −39; 21 = 31, −36, 49; 22 = 36, 24, 41; 23 = 43, 14, −3; 24 = 4, 45, 39; 25 = −18, −9, −27; 26 = 19, 12, 21; 27 = 19, 24, 19; 28 = 48, −19, 39.(TIF)Click here for additional data file.File S1Supplementary material containing supporting tables.(DOCX)Click here for additional data file.