Epinephelus akaara. In this study, E. akaara juveniles were fed diets containing few (0% corn starch), moderate (18% corn starch), and high (30% corn starch) levels of dietary carbohydrate. After an 8-week feeding trial, E. akaara fed 30% dietary carbohydrates exhibited poor growth performance compared with those fed 0% and 18% dietary carbohydrates (P > 0.05). Genes related to the immune system, including IL8, TLR9, CXCR4, CCL4, and NFκB inhibitor alpha, were over-expressed in E. akaara fed the highest level of carbohydrate (30%). This general over-expression could indicate activation of inflammatory processes in the liver. The liver transcriptome data of E. akaara reported here indicate that high carbohydrate level of diet can lead to poor growth and inflammatory immune response in E. akaara.As the cheapest energy source, carbohydrates are used in fish feeds to improve physical quality and reduce catabolism of proteins and lipids. The liver is the primary organ for metabolism and is also an important site of immune regulation. Here, we investigated the effect of different dietary carbohydrate levels on growth and health by evaluating the liver transcriptome of  Oncorhynchus mykiss), Atlantic salmon , European eel (Anguilla anguilla), Atlantic cod (Gadus morhua), and different carp species 7. If sufficient carbohydrate content is not provided in the diet, other nutrients such as proteins and lipid can be used for energy8; This could lead to an increase in cost and nutritional imbalances. In addition, post-prandial prolonged hyperglycemia occurs when a high level of carbohydrates is consumed14. An imbalance carbohydrates in the diet may make the fish under metabolic stress15 and have negative effects on nutrient retention, growth, metabolism, and health16. Thus, it is important to supply an appropriate level of carbohydrates in aqua-feeds.Carbohydrates are the cheapest energy sources and the major compounds that make up organisms. Some studies have indicated that certain levels of carbohydrates can improve feed utilization and protein retention in rainbow trout  infection in rainbow trout. Similarly, when inappropriate feed supplied, the liver also can function as an immunocompetent organ22. A growing body of research has focused on the relationship between carbohydrate supplementation and immune function. For example, carbohydrates can regulate the production of proinflammatory cytokines to enhance the endurance performance and attenuated stress hormone response23.The liver is the primary target organ for metabolism. Nutrients are absorbed into the body through the small intestine and then delivered to liver. The critical metabolic functions of the liver often eclipse its role as an important organ for immune regulation26. Herbivorous and omnivorous fish can utilize as much as 45% carbohydrate content in diet29, but carnivorous fish show significantly poorer growth when fed diets with 30% carbohydrate levels compared to those fed moderate carbohydrate levels , such as Atlantic salmon5 and cobia (Rachycentron canadum)30. Epinephelus akaara  is an important marine carnivorous fish with a high market value in Asia, To date, few nutrient requirements have been investigated in E. akaara, except for appropriate level of proteins31 and lipids32. To obtain the appropriate formulation of fish feed, it is an essential pre-requisite to understand the capacity of fish to utilize carbohydrates8. High-throughput sequencing can provide an unprecedented view of global gene expression and detailed molecular information on responses to nutrition metabolism. Therefore, we here focused on the liver transcriptome of E. akaara fed diets containing few (0%), moderate (18%), and high (30%) levels of carbohydrates using high-throughput sequencing to identify genes responsible for growth and immune system alterations.Carbohydrate utilization differs among fish speciesE. akaara was measured by percentage weight gain (PWG). In this study, the final body weight (g/fish) in each group was 29.30 ± 3.32, 28.54 ± 2.87, and 24.02 ± 0.92, respectively; the PWG (%) were 276.16 ± 41.99, 266.26 ± 28.33, and 208.69 ± 12.68 in the C1, C2, and C3 groups, respectively. PWG generally decreased with increasing dietary carbohydrate (P > 0.05), and fish fed 30% carbohydrates exhibited the lowest PWG.The growth performance of P < 0.05), IGFBP4, and IGFBP6 (P > 0.05) were decreased in the C2 and C3 groups compared to the C1 group, whereas IGFBP1 (P < 0.05), IGFBP5, and IGFBP7 (P > 0.05) were upregulated in the C3 group to levels comparable to the C1 and C2 groups , 88.10% , 85.33%  in C1, C2, and C3 groups, respectively. There were 94% sequences up to the quality score of Q30, which describes quality score logarithmically linked to error probabilities . The sequencing results showed that these data were appropriate for analysis (Table −5) with the NT, NR, gene, and string were used, and 27,327, 27,851, 31,234 contigs in each group featured a corresponding annotation .The KEGG database was used to obtain more information to predict the unigene functions; 38,938 genes ware classified into 113 KEGG pathways. The KEGG pathway analysis was also used to identify genes observed to be differentially expressed in the C1-C2, C2-C3, and C1-C3 pair groups fed few (C1), moderate (C2), or high (C3) levels of carbohydrates. The numbers of differentially-expressed genes in each group  were 20,499, 35,984, and 37,203, respectively  on growth and health by evaluating the liver transcriptome in E. akaara.As the cheapest energy sources, carbohydrates are used in fish feeds to improve physical quality and reduce catabolism of proteins and lipidsE. akaara fed a diet with 30% carbohydrate content exhibited poor growth performance (PWG = 208.69%) compared with those fed 0% (PWG = 276.16%) and 18% (PWG = 266.26%) carbohydrate content. These results indicated that PWG decreased with an increase in dietary carbohydrate levels. Various mechanisms could account for growth, including endocrine system change34. As in mammals, there are two major molecular targets, growth hormone (GH) and insulin-like growth factor (IGF), in the potential endocrine regulation of growth in the teleost38. They both belong to the GH/IGF-system, which also consists of multiple subtypes of GH receptors (GHRs) and insulin-like growth factor binding protein (IGFBP)39.In the present study, 45. Studies of coho salmon, gilthead sea bream (Sparus aurata), and tilapia have indicated that the levels of hepatic GHR and IGF mRNA are positively correlated with body growth rate50. In accord with these findings, E. akaara fed high levels of carbohydrates had the lowest PWG value and the lowest expression of IGF1 and GHR genes  compared to the other two groups. Elsewise, expression of the IGFBP 2, 3, 4, and 6 genes was reduced in the C3 group, The expression of multiple IGFBPs genes is regulated by different levels of carbohydrate, but little is known about their function in liver. And the mechanism is still not clear. These findings may reinforce the need for additional research about the relationship between IGFBPs gene expression and growth performance in fish.In addition to IGF and GHR, IGFBPs are the main members of the GH/IGF-system. These IGFBPs are traditionally thought to function as carrier proteins and regulate circulating IGF turnover, transport, and distribution. And some studies have shown that IGFBPs are ubiquitously expressed across numerous tissues with autocrine or paracrine effects in salmonids and modulate IGF activities in target tissuesThe liver is an important organ for metabolism and immune function. Significant numbers of natural killer receptor-positive (NKR+) cells and macrophages reside in the healthy liver, which can detect foreign substances and produce inflammatory mediators. In the present study, most genes involved in innate and adaptive immune system were up-regulated following increased dietary carbohydrate levels, suggesting that high levels of carbohydrates can invoke an immune response.55, and, if the harmful antigens are present in the liver, it causes inflammatory-related genes to be over-expressed immediately. In our study, IL8 was more highly expressed in the group fed high levels of carbohydrates compared to the C1 and C2 groups, which had a low constitutive expression  induces the T-cell receptor (TCR) to generate an activation signal in T lymphocytes58. PRRs responded to many exogenous pathogens and/or endogenous serious signals, by recognizing highly conserved structures, such as pathogen associated molecular patterns (PAMPs) and danger/damage associated molecular patterns (DAMPs). Following recognition by PRRs, downstream signaling pathways induce expression of genes of inflammatory molecules that can activate the cytokine signaling pathway to regulate innate and adaptive immunity59. The genes belonging to these three pathways had significantly higher expression in the group fed high levels of carbohydrates, suggesting that an imbalance in carbohydrates signals the liver to produce immunocompetence through the innate and adaptive immune system.Toll-like receptors (TLRs), inhibitor of nuclear factor kappa-B kinase subunit alpha (IKKA), caspase-8, proto-oncogene c-Fos (Fos), interferon regulatory factor 3 (IRF3), tumor necrosis factor (TNF), and tumor necrosis factor receptor (TNFR) belong to Toll-like receptor signaling pathway, NOD-like receptor signaling pathway, and RIG-I-like receptor signaling pathway. These three pathways are cytoplasmic pattern recognition receptors (PRRs), which are involved in the recognition of viruses by the innate immune system65. However, Sahlmann et al.66 found that the gene expression changes could be detected in third day, which is earlier than signs of inflammation in histological evaluation. In this study, we got the liver transcriptome data of E. akaara after 8 weeks of rearing experiment and it showed that the fish consuming high level of carbohydrate may increase inflammation response. Then, we did a short feeding trial with C2 and C3 experimental diets to make up the defect of functional verification in original experiment. After 2 weeks, although there was no difference in histological section of liver tissue, the immune response related genes were higher expression in C3 group than C2 group by RT-qPCR. It suggested that the high level of carbohydrate had the tendency to give rise to immune reaction in E. akaara. On the other hand, we used 3 fishes of each replicating group per treatment as the RNA templet to confirmed the sequencing data with RT-qPCR. They were consistent with each other. These results all confirmed that our data and conclusion is credible.In Atlantic salmon, another sea carnivorous fish, numerous studies reported plant feedstuffs could lead to the changes of histomorphological and immune response related genes expression in intestine at least four weeksE. akaara reported here indicate that high carbohydrate level of diet can lead to poor growth and inflammatory immune response in E. akaara. The up-regulation of a large number of genes in immune system pathway revealed the fish maybe experience inflammatory response which may be related with a decrease in growth rate. The prominent effects caused by diet composition on immune function parameters underline the importance of nutritional factors in the defense system of E. akaara.The liver transcriptome data of This study was implemented at the Zhejiang province Key Lab of Mariculture and Enhancement in Zhoushan, China. Before the experiments, the juvenile fish were temporarily fed a commercial diet. At the beginning of the experiment, fish  were weighed and sorted into 9 cages (20 fish/cage). The experimental system consisted of 9 net cages . All net cages were placed in one large concrete tank (13.0 × 4.0 × 1.5 m). A recirculating water system  was used during the experimental period. Three replicate groups of fish were used for each experiment diet. Three semi-purified isoproteic (48%) and isolipidic (9%) diets were formulated with different levels of corn starch . All fish were fed to apparent satiation twice daily at 8:30 and 16:00, and the feeding trial lasted for 8 weeks.During the experimental period, dissolved oxygen (DO), salinity, and temperature were 6.2 ± 0.1 mg/L, 25.1 ± 0.9 g/L and 27.5 ± 2.3 °C, respectively. At the end of the feeding trial, three fish in each cage were dissected, livers were immediately frozen in liquid nitrogen, and then they were stored at −80 °C until RNA extraction for sequencing. The experiment was implemented at the Zhejiang province Key Lab of Mariculture and Enhancement of Zhejiang Ocean University . All experimental procedures were approved by the Local Animal Care and Use Committee , and the study was carried out in accordance with regulations for the administration of affairs concerning experimental animals of China .Total RNA was extracted from livers of each group by homogenization in 1 ml TRIZol (Invitrogen) following the manufacturer’s instructions. The concentration and quality of total RNA were determined by electrophoresis . And total RNA was used for paired-end RNA sequencing. Library construction and sequencing were performed on an Illumina HiSeq. 2000 sequencer according to manufacturer’s specifications.67. Transcripts shorter than 200 bp were removed from the subsequent analysis68. The assembly sequencing results evaluated used bowtie-1.0.169, which mapped the raw reads to the assembled transcriptome. Transcripts from the previous step were translated in all six possible open reading frames (ORFs) with TransDecoder tool, which was used to predict the ORF of the RNA sequencing assembly sequence. Proper translation was defined as one that gave the longest amino acid sequence70 according to the hidden Markov model (HMM).Raw reads were pre-processed by discarding reads with adaptors, with unknown sequence (N) proportion greater than 5%, and those of low quality . Then, raw reads were assembled by Trinity software using default parametershttp://trinotate.sourceforge.net/) through searching against the Uniprot Knowledgebase and Swiss-Prot. In addition, we ran HMMER, signalIP, and tmHMM in Trinotate to identify protein domains, predict signal peptides, and transmembrane regions, respectively.The functional annotations of predicted amino acid sequences were performed using Trinotate  pathways were assigned to the assembled transcripts using the online KEGG Automatic Annotation Server (KAAS) (http://www.genome.jp/kegg/kaas/). The bi-directional best-hit (BBH) method was used to obtain KEGG orthology assignments.Gene Ontology (GO) annotations of transcripts were obtained by searching against the non-redundant (nr) database using the Blast2GO program73 was used to compare RNA-seq data and identify differentially expressed genes. And the input of DEGseq is uniquely mapped reads from RNA-seq data with a gene annotation of the corresponding transcript expression values provided by RPKM74. A set of 20 genes which were significantly affected by high carbohydrate food were quantified by RT-qPCR to validate sequencing performance. Primers were designed using Primer premier 6 software , following manufacturer’s instructions. Amplifications were carried out in triplicate in a final volume of 20 μl containing 2 μl cDNA (1/10 dilution), 0.5 μM of each primer and 10 μl SYBR Green Supermix (Takara). The RT-qPCR profiles contained an initial activation step at 95 °C for 10 min, followed by 40 cycles: 15 s at 95 °C, 15 s at the specific primer pair annealing temperature  for Windows.Data on initial body weight were used to calculate percentage weight gain (PWG). PWG (%) = 100% × /initial body weight. All measured values were presented as the mean ± standard deviation (SD). All data were tested for homogeneity (Levene’s test) and normal distribution (Kolmogorov-Smirnov tests), and necessary data were transformed before analysis. When data did not present variance homogeneity and normal distribution, Spearman’s correlation was used to determine the relationship between the response data and dietary carbohydrate. Values were regarded as statistically significant at


An increased release of serotonin secreted by ileal NETs is thought to be the major factor causing the carcinoid syndrome. However, in acutely arising carcinoid crisis also other vasoactive factors may lead to hazardous fluctuations in blood pressure and bronchial constriction. In rare cases, systemic vasoconstriction can be observed, probably caused by catecholamines or similar acting substances. Here, we report a fatal case of fulminant systemic vasoconstriction possibly caused by catecholamines in a patient with metastasized ileal NET. The vasospasm was detected by CT-angiography, and hemodynamic monitoring revealed a high systemic vascular resistance. Epinephrine, norepinephrine, and chromogranin A levels in plasma were elevated as was the urinary 5-hydroxyindoleacetic acid (5-HIAA). The cause of death was heart failure due to severe circulatory insufficiency. The progression of the tumor disease was confirmed by autopsy. Neuroendocrine tumors (NET) of the gastroenteropancreatic system are low-grade malignant neoplasms derived from local neuroendocrine cells .Their clinical presentation largely depends on primary tumor site and whether or not significant amounts of hormones are produced and secreted. The predominantly secreted hormonally active substances, such as polypeptides, biogenic amines, and prostaglandins, vary, depending on tumor localization –5. NETs An excessive release of these active substances caused by different triggers like surgery/anaesthesia, interventional diagnostic and therapy, radionuclide therapy, medication, and examination can lead to carcinoid crisis characterized by dramatic blood pressure fluctuation, arrhythmias, bronchospasm, and mental disturbances –13. CommHere, we report a fatal case of a metastasized syndromic NET of the terminal ileum with systemic vasospasm resulting in secondary low-output syndrome and consecutive multiorgan failure. CT-angiography, advanced hemodynamic monitoring, and determination of catecholamines, chromogranin A in plasma, and 5-hydroxyindoleacetic acid (5-HIAA) in urine were performed. The cause of death was determined post-mortem by autopsy.In 2011, a liver biopsy of hepatic lesions of a 51-year-old female revealed liver metastases from a well-differentiated small intestine NET .A DOTA-TOC-PET/CT demonstrated metastatic disease with a suspected ileal primary; see An excess of vasoactive amines secreted by the NET was now regarded as the most likely reason for clinical deterioration. Massively elevated chromogranin A in plasma and, notably, elevated catecholamine plasma levels (in pg/ml) supported this hypothesis: epinephrine 2318.3 (50–100 pg/ml), norepinephrine 1339.3 (<400 pg/mL), metanephrine 170.2 (<90 pg/mL), normetanephrine 349,5  (<180 pg/mL), and chromogranin A  896 (11/2011), 717 (06/2012), 1.120 (01/2013), and 16.310 6 days before and 33.580 6 days after ICU admission. In contrast, the 5-HIAA levels in urine were elevated but remained stable: 66.2 mg/12 h on day 6 after admission and 153 mg/24 h 452 days before admission (1–10 mg/24 h); see Octreotide dose was increased and administered intravenously combined with orally administered cyproheptadine. Treatment attempts with nimodipine, phentolamine, and dobutamine aiming at reducing SVRI and cardiac afterload and thereby improving the CI were largely ineffective. Respiratory insufficiency worsened but intubation and mandatory ventilation were not performed in agreement with the family due to the underlying progressive malignancy. The patient finally died 7 days after ICU admission.At autopsy the affected organs included the heart , lungs (disseminated metastases and perivascular), stomach, pancreas, soft tissues, thyroid gland, ovaries, and bones. Histologically, a partial treatment response was found. The final TNM UICC classification was aT3 (m), aN1 (2/2), aM1 , aL1, aV1, and aPn1; grading G1. The cause of death was determined as heart failure due to severe circulatory insufficiency.The dramatic deterioration in our patient was caused by a progressive cardiac failure with increased vascular resistance and low cardiac index leading to a secondary low-output syndrome.The massive increase of chromogranin A suggested a carcinoid crisis, although presenting with an atypical systemic vasoconstriction instead of more commonly found vasodilatation and might have possibly been triggered by the preceding PRRT.Excluding cardiac valvular lesions, the main symptoms of the typical carcinoid syndrome with cutaneous flushing, venous telangiectasia, diarrhoea, and bronchospasm were present for about 2 years in our patient. Functional midgut NETs mainly secret serotonin leading to the typical carcinoid syndrome. Depending on the location of the NET  other hormones can predominate in systemic circulation resulting in an atypical carcinoid syndrome with variant clinical symptoms . CarcinoStill, there are differing opinions regarding octreotide pretreatment against carcinoid crisis before invasive procedures. A retrospective study evaluating the perioperative high-dose octreotide infusion showed a minimized incidence of carcinoid crisis . In contμg/24 h continuously iv was also ineffective (recommended dose for carcinoid crisis 50–600 μg/day iv). There are reports of the successful administration of 150 μg/h iv continuously over several days or of a bolus dose of up to 1,5 mg iv [Pharmacological interventions to overcome the systemic vasoconstriction ultimately failed, despite the use of potent vasodilators. Treatment with nimodipine applied in the early phase of the ICU course seemed to be more effective compared to phentolamine. Intravenous octreotide applied in a dose of 500 ,5 mg iv .Both sepsis and previous PRRT might have served as a trigger but despite effective sepsis therapy the patient's condition worsened. Furthermore, a high SVRI along with a low CI strongly indicates the development of a cardiogenic shock; see The renal function was normal before PRRT with a creatinine of 0.8 mg/dL and slightly impaired after ICU admission . In the long term, PRRT can worsen renal function . We postMyocardial tumor infiltration may have additionally contributed to the development of cardiac failure, but the cardiac metastases with a maximum diameter of only 1.5 cm were not pronounced enough to explain the finding sufficiently. Cardiac metastases of a midgut NET are rare but may occur . InteresIn our case, only a minimal increase of 5-HIAA levels in urine was observed and this might indicate a less pronounced serotonin excess. However, the value was derived from a 12-hour period urine collection and an oliguric kidney injury was present; see It has suggested that serotonin may lead to ergotism, a similar condition of systemic vasoconstriction . SerotonMidgut NETs are able to excrete catecholamines . A singlBy immunohistochemically staining of catecholamine-synthesizing enzymes it has been shown that the enzyme pathway for catecholamines in midgut NETs is similar to that in pheochromocytomas .Serotonin is mainly assumed to be responsible for the carcinoid crisis. Based on the clinical presentation of our patient with a concomitant excess of catecholamines, we suggest that catecholamines at last contributed to the occurrence of the observed generalized systemic vasoconstriction, ultimately leading to acute heart failure.


Understanding the dynamics of malaria transmission in diverse endemic settings is key for designing and implementing locally adapted and sustainable control and elimination strategies. A parasitological and epidemiological survey was conducted in September–October 2012, as a baseline underlying a 3-year population-based longitudinal cohort study. The aim was to characterize malaria transmission patterns in two contrasting ecological rural sites in the Peruvian Amazon, Lupuna (LUP), a riverine environment, and Cahuide (CAH), associated with road-linked deforestation.After a full population census, 1941 individuals 3 years and older  were interviewed, clinically examined and had a blood sample taken for the detection of malaria parasites by microscopy and PCR. Species-specific parasite prevalence was estimated overall and by site. Multivariate logistic regression models assessed risk factors for parasite infection by PCR, while SaTScan detected spatial clusters of PCR-positive individuals within each site. In addition, data from routine malaria surveillance in the period 2009–2012 were obtained.Plasmodium vivax (6.2% vs. 3.9%) and for Plasmodium falciparum (2.6% vs. 1.2%). Among PCR-confirmed infections, asymptomatic (Asy) parasite carriers were always more common than symptomatic (Sy) infections for P. vivax (Asy/Sy ratio: 2/1 in LUP and 3.7/1 in CAH) and for P. falciparum (Asy/Sy ratio: 1.3/1 in LUP and 4/1 in CAH). Sub-patent (Spat) infections also predominated over patent (Pat) infections for both species: P. vivax (Spat/Pat ratio: 2.8/1 in LUP and 3.7/1 in CAH) and P. falciparum malaria (Spat/Pat ratio: 1.9/1 in LUP and 26/0 in CAH). For CAH, age, gender and living in a household without electricity were significantly associated with P. vivax infection, while only age and living in a household with electricity was associated with P. falciparum infection. For LUP, only household overcrowding was associated with P. falciparum infection. The spatial analysis only identified well-defined clusters of P. vivax and P. falciparum infected individuals in CAH. Reported malaria incidence indicated that malaria transmission has long occurred in LUP with primarily seasonal patterns, and confirmed a malaria outbreak in CAH since May 2012.Parasite prevalence by PCR was higher in CAH than in LUP for This parasitological and epidemiological baseline assessment demonstrates that malaria transmission and parasite prevalence is heterogeneous in the Peruvian Amazon, and influenced by local socio-demographics and ecological contexts. Riverine and road construction/deforestation contexts must be taken into account in order to carry out effective anti-malaria control and elimination efforts.The online version of this article (doi:10.1186/s12936-017-1957-y) contains supplementary material, which is available to authorized users. Plasmodium falciparum. It is thought that these interventions contributed to a reduction of microscopically-confirmed malaria cases in Loreto, from 54,291 cases in 2005 to 10,504 and 11,793 cases in 2010 and 2011, respectively [Malaria remains an important public health problem in Peru, especially in the Amazonian department of Loreto where most malaria occurs –4. Folloectively , and to ectively .Anopheles darlingi  [Routine malaria control activities in the Peruvian Amazon are based on passive case detection (PCD) using light microscopy (LM) , 15 on p Amazon) . Collect Amazon) –25.Plasmodium vivax and P. falciparum parasitaemias contribute to the maintenance of malaria hypoendemicity in the Amazon region drives the work of our Amazonia International Center of Excellence in Malaria Research (Amazonia ICEMR), which was established in 2010 as one of ten such centers supported by the National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health (NIH) [P. vivax and P. falciparum prevalence by LM and PCR at the baseline enrollment surveys in September–October 2012 are also discussed and those estimates were related to data from routine malaria surveillance and implemented control efforts to better understand the dynamics of malaria transmission in both sites.The hypothesis that asymptomatic th (NIH) . The Amath (NIH) . This paEthics clearance for the study was obtained from the Ethics Review Board of the Universidad Peruana Cayetano Heredia, Lima, Peru (SIDISI code # 5739) and from the University of California San Diego Human Subjects Protection Program (Project # 100765). Permissions were received from health and local authorities after explaining the purpose and procedures of the study. Signed informed consent was obtained prior the study enrollment to participation and blood sampling by all adults and the parents of all participating children <18 years. In addition to parental/guardian consent, children older than 7 years provided a signed informed assent. All the methods were carried out in accordance with approved guidelines.A census and an epidemiological survey were conducted as baseline activities of a 3-year population-based, longitudinal cohort study in the Lupuna (LUP) and Cahuide (CAH) sites in the northeastern Peruvian Amazon Department of Loreto. The study population included individuals aged 3 years and older who resided in study sites and agreed to participate in the cohort providing a written signed consent.Anopheles darlingi is the primary malaria vector in the area [LUP site is located on the outskirts of Iquitos district, 10 km from downtown (latitude 03°44.591′S longitude 73°19.615′W)  for 3 days , plus primaquine (PQ) for 7 days (0.5 mg/kg/day); P. falciparum malaria was treated with mefloquine (MQ) (12.5 mg/kg/day for 2 days) plus artesunate (AS) (4 mg/kg/day for 3 days), according to Peruvian national policy.ire year . As in mA complete census of the population in each village was conducted in July–August 2012, collecting individual data on socio-demographics , and on previous malaria episodes. Each house was identified with a unique number and geo-referenced using a handheld Global Positioning System (GPS) device , and household characteristics collected . Each individual subject was given a seven-digit unique code number combining the village, household and individual code.Individuals aged 3 years and older available at the time of the baseline survey (September–October 2012) were enrolled in the study area after consenting. Whenever selected individuals were absent, the household was revisited up to four times in a period of 30 days to maximize subject participation. Each participant had the axillary temperature taken, history of fever or any other malaria symptoms were recorded, and a finger-prick blood sample collected for immediate microscopy (thick and thin blood smears) and on filter paper  for later molecular tests. Filter paper dried blood samples were individually stored at 4 °C with desiccant until processed. Subjects with microscopically-confirmed infections were referred to health posts for treatment according to Peruvian national guidelines.Additionally, routine surveillance data, namely reported malaria episodes by species in Iquitos and San Juan district from 2009 to 2012 were obtained from the Regional Directorate of Health, Loreto-Ministry of Health in Iquitos. Peruvian policy mandates weekly notification of confirmed malaria cases (by standard LM) at health facilities and data aggregation at district and department level. Since malaria surveillance data could be reliably disaggregated only to the district level but not to the village level, a retrospective review of all registered individuals detected by PCD between January 2009 and December 2012 was carried out in all health facilities near the study sites. Any other surveillance in addition to routine PCD conducted in CAH and LUP in the 12 months before the survey was recorded.Thick and thin smears were stained for 10 min with a 10% Giemsa solution, and parasite density was calculated by counting the number of asexual parasites for 200 white blood cells (WBC) in the thick smear, assuming a concentration of 8000 WBCs/μl. Slides were read on site, then a day later by an expert at microscopy at the reference laboratory in Iquitos. A slide was declared negative if no malaria parasite was found after examining 100 fields . Quality2 pieces for DNA extraction using QIAamp DNA Microkit of QIAGEN following the manufacturer instructions. A quantitative real-time polymerase chain reaction (qPCR) method targeting the 18s rRNA gene region was used for molecular diagnosis, following the protocol reported by Mangold et al. [P. vivax from P. falciparum.Filter paper blood spots were cut into ~6 mmd et al.  Primers Census and survey data were doubly entered and cross-checked in Access . Data analysis was performed using R v.2.15 software . Baseline characteristics between sites were compared using the Chi squared test. Species-specific parasite prevalence by LM and by PCR were estimated overall and by site, and their two-sided 95% confidence intervals (CI) were calculated using the Wilson score method.Plasmodium spp.) infection was an individual with a positive PCR result regardless of symptoms. Cases were further classified as patent (detectable by LM) or sub-patent (not detectable by LM). A symptomatic infection was defined as an individual with confirmation of parasites in the blood sample at any level who also had fever or history of fever, headache, chills or general discomfort in the previous 7 days. Uni- and multivariate mixed-effects logistic regression models, with fixed effects and a random intercept that accounted for individual clustering at household, were used to determine risk factors for species-specific malaria infection in each site The following potential risk factors were assessed: age, gender, time living in the village, education level, forest-related job as main economical activity (including the intensity of these activities during the week), trip in the past month to another endemic area, the history of malaria in the past year, household overcrowding , low household bed net coverage (<80% of beds covered with a net in a household), housing structure , and the availability of electricity. Factors with p values <0.1 for the Wald test in the univariate analysis were considered for inclusion in the multivariate model. Using manual backward, final models retained all factors that were significantly associated with malaria infection . Interactions were systematically checked for up to order two. Likelihood ratio tests (LRTs) were used to assess statistical differences between nested models.The case definition for malaria parasite  was identified as the most likely cluster. The relative risk (RR) reported for each identified cluster was the estimated risk within the cluster divided by the estimated risk outside the cluster [The QGIS software QGIS v.2.16  was used to map all surveyed households and to classify them according to the number of household members with malaria infections identified by PCR. The SaTScan software v.9.3  was used to identify spatial clustering of households with malaria infections in each site, using the following characteristics: pure spatial analysis, Poisson probability model , latitud cluster .A total of 487 households (HHs) and 2447 individuals were identified during the census in July–August 2012, distributed over the two sites as follows: LUP  and CAH . In addition, the distribution by village was: LT , SR  and SP  for LUP, and CT , LH  and DA  for CAH (Table p = 0.008). Males slightly outnumbered females  without differences between sites. The proportion of participants aged >10 years old who have resided in the site ten or more years was significantly higher in LUP (85.0%) than in CAH (41.3%) (p < 0.001). Among adults 18 years and older, the proportion of individuals who had completed secondary education level was higher in LUP (47.0%) than in CAH (32.8%) (p < 0.001). The most commonly reported occupation among adults was farmers, followed by housewives, indoor laborers/employees and traders with similar proportions in both sites; security guards (p = 0.002) and loggers (p < 0.001) were more frequent in CAH than in LUP. Participants reporting having experienced malaria in their lifetime were more common in LUP (73.4%) than in CAH (67.0%) (p = 0.003). However, this was not the case when only recent episodes were considered, with more participants reporting have had malaria episodes within the past 12 months in CAH (48.9%) than in LUP (14.1%) (p < 0.001).The cohort study enrolled 1941 of the 2447 censused inhabitants (79.3%) in 448 HH (92.0%) available at the time of the baseline survey. Of these 1941 individuals, 829 (42.7%) lived in LUP and 1117 (57.3%) in CAH. Socio-demographic characteristics and history of past malaria episodes by site and by village are presented in Table p < 0.001). In both sites, most individuals lived in houses with wooden walls and roofs composed of palm leaf; concrete (brick and cement) and tin were also common building materials, especially in the LT village of LUP (p < 0.001) . Most participants had electricity at home in CAH (55.7%), primarily those living along the road and in the DA village (p < 0.001); however this electrical service mainly exclusively benefited participants living in LT village in LUP (p < 0.001). The use of potable water for drinking and food preparation was reported by only a small proportion of individuals in LUP (15.6%) and CAH (30.7%), while firewood was reported as the main fuel used for cooking by most participants (>80%) in both sites. Moreover, most participants in both sites reported having precarious or lack of sanitation facilities, as well as lack of trash disposal systems. The majority (97.0%) of participants in CAH reported that their houses were sprayed indoors with insecticide in the past 3 months, while only 12.4% reported this in LUP (p < 0.001). Household bed net coverage was high in both sites; the predominant material of bed nets reported by participants varied across sites. In all villages of LUP, most bed nets (about 90%) were LLINs. In CAH, about half of subjects reported having LLINs and half reported using non-impregnated insecticide nets made of tocuyo (non-processed cotton) or nylon.Table P. vivax, 2 P. falciparum infections), and by PCR 7.2%  (Table (p < 0.05). Of the total 38 P. vivax infections by LM, 30 were found in CAH (16 with parasitaemia <100 parasites/μl), and only 8 in LUP (one with parasitaemia <100 parasites/μl); while the two microscopically confirmed P. falciparum infections were found in LUP (one with parasitaemia <100 parasites/μl). The presence of gametocytes was recorded in 15 (50.0%) and 3 (37.5%) of the total microscopically confirmed P. vivax infections in CAH and LUP, respectively. Gametocytes were not observed in microscopically confirmed P. falciparum infections. Parasite prevalence by PCR was higher in CAH than in LUP for P. vivax  and for P. falciparum . Considering PCR-confirmed infections, asymptomatic (Asy) parasite carriers were always more common than symptomatic (Sy) infections for P. vivax (Asy/Sy ratio: 2/1 in LUP and 3.7/1 in CAH) and for P. falciparum (Asy/Sy ratio: 1.3/1 in LUP and 4/1 in CAH). Similarly, sub-patent (Spat) infections also predominated over patent (Pat) infections for both species: P. vivax (Spat/Pat ratio: 2.8/1 in LUP and 3.7/1 in CAH) and P. falciparum malaria (Spat/Pat ratio: 1.9/1 in LUP and 26/0 in CAH).All 1941 individuals enrolled in the cohort had samples obtained for malaria diagnosis by LM at the time of the baseline survey; most of these  had available sample for PCR analysis. By LM, the overall parasite prevalence was 2.1% . No significant factors were found for P. vivax infection . Moreover, the lack of electricity increased the odds of infection in household members by a factor of 1.9  compared with individuals living in households that had available this public service. Considering P. falciparum, the odds of infection confirmed by PCR significantly increased at younger age (test for trend p < 0.001), with individuals <15 years being four times more likely to have P. falciparum infections than adults >40 years . In this case, the availability of electricity was associated with highest risk of P. falciparum in household members  compared with individuals living in households without electricity. No association was found between P. vivax and P. falciparum infection with regard to time of residence in the village, education level, occupation, trip in the past month to another endemic area, the history of malaria in the past year, and housing characteristics of study participants.Given the relatively small number of infections detected by LM, uni- and multivariate mixed-effects logistic regression analyses were carried out only for confirmed infections by PCR for each site. In final models the estimates of the random-effects coefficients confirmed the influence of clustered sampled data at household level. In LUP, household overcrowding  was the only factor significantly associated with I 1.0–24.). No sigP. vivax and P. falciparum infections were identified using SaTScan  was in the southern part of CT village . For P. falciparum, the most likely cluster adjusted for age and electricity availability was the closest area to the fluvial port in CT . With a radius of 99 meters, the cluster included 61 individuals  in 14 households   without adjustment for covariates.Figures can Fig. , mainly age Fig. a, includal) Fig. b. This cP. vivax, 7 P. falciparum) represented about 15% of the total 880 reported cases in Iquitos district. Regarding malaria intervention efforts, deliveries of LLINs by GFATM’s PAMAFRO malaria project in 2008 and 2010 were the main malaria control intervention in LUP during the evaluated period. On the other hand, malaria cases in CAH between 2009 and 2011 occurred with stochastic patterns , and did not follow the same trends of the malaria incidence than San Juan district during that period  from CAH villages accounted for about 35% of the total 3617 reported cases in San Juan district in 2012.The retrospective analysis of reported malaria cases between 2009 and 2012 showed that the malaria incidence in LUP varied across years, with some periods (mainly in year 2009) not following the same trends of malaria incidence compared to the Iquitos district Fig. a. Despitiod Fig. b. The raP. falciparum and P. vivax.The baseline analysis presented here of a population-based, longitudinal malaria cohort study in two contrasting ecological villages in the Peruvian Amazon indicates substantial prevalence of malaria parasitaemia in the region and is an important benchmark for ongoing longitudinal studies. The riverine rural site, Lupuna (LUP), is home to a fairly stable population . Here, the combined analysis of survey findings and routine reported malaria incidence suggests that malaria transmission has long occurred with primarily seasonal patterns. In contrast, Cahuide (CAH), a rural site with road-driven deforestation and a more transient population (about 60%), has experienced a notable and concerning increase of cases since May 2012 that compelled the regional government to carry out intensive outbreak control measures until August 2012 (1 month before the survey). Low parasite rates were observed in both sites during the survey, with high predominance of asymptomatic and sub-patent infections over symptomatic and patent infections. Moreover, the site-specific analysis of malaria prevalence and reported incidence suggested a high degree of heterogeneity at the micro-geographic level and over time for both Community surveys for determining malaria parasite carriage are commonly used to estimate malaria transmission in endemic areas , 33. BecP. vivax and P. falciparum. This was observed here to have occurred despite the intensive response from RHDL-MoH to a severe malaria outbreak from May to August 2012. These interpretations are further supported by a recently published entomological study of our Amazonian ICEMR that found that the highest monthly entomological inoculation rates (EIRs) are associated with seasonal transmission in both sites, with an important factor being seasonally high river levels that enable mosquito breeding [An. darlingi  and higher human biting rates (HBRs) in LUP between February and June (maximum EIR in April) in successive years 2011 and 2012; while for CAH, the highest monthly EIR (2.52) with also confirmed P. vivax- and P. falciparum-infected An. darlingi occurred during the 2012 malaria outbreak. Interestingly, despite decreased HBR levels leading to a much lower estimated EIR in CAH in October 2012 (period of baseline survey), the entomological study found a high infection rate of collected mosquitoes (1.47%) which indicated onward malaria transmission in the area [The richness of these datasets is important to consider with regard to how future analyses will guide anti-malaria interventions. In riverine, rural LUP, malaria prevalence by PCR may indicate the seasonal-time dependent parasite carriage level in an endemic area  with no major recent changes in the population socio-demographics and in local control measures. In contrast, in road/deforestation and riverine rural CAH, prevalence of parasite carriers (with confirmed gametocytaemia by LM in some of them) reflects the parasite reservoir that enables persistence of malaria transmission by both breeding . Indeed,the area , and thethe area  followinthe area .P. vivax and P. falciparum—were asymptomatic (64–85%) and sub-microscopic (75–77%), despite differences in malaria prevalence and variations in malaria transmission over time. A limitation of such cross-sectional surveys is that they might not correctly estimate the proportion of infections that later might become symptomatic or have higher parasite density levels (as detectable by LM) [As previously described elsewhere in the Peruvian Amazon –21, the e by LM) , 34. Fewe by LM) , 22. A 6e by LM) , 34. Anoe by LM) , 27, offP. vivax and P. falciparum. Household overcrowding, the only factor found to be significantly associated with P. falciparum infection in LUP, has also been reported in several studies conducted in areas where transmission is exclusively due to P. falciparum, or co-endemic with P. vivax [P. falciparum infection and other proxy variables for socio-economic status in LUP, it is important to note that overcrowding can also reflect poor economic household conditions. The complex association between poverty and malaria is well known, which likely operates in both directions [Considering the site-specific socio-demographics, household characteristics and ecological conditions in LUP and CAH, risk factor and spatial analyses of malaria infections were performed separately for each site. The low number of infections in LUP detected during the survey in low transmission season would primarily explain the difficulty in identifying malaria risk factors and spatial clusters of high transmission in the site for both P. vivax , 36. TheP. vivax , leadingrections , 39: pooP. vivax and P. falciparum malaria. This outbreak likely included both: parasite carriers that remained despite the response to a first severe outbreak occurred between May and August 2012, and new infections by either similar or different parasite populations than those of the first outbreak. The increased risk for P. vivax in males and individuals aged >15 years together with the confirmed infection of mosquitoes in the site [An. darlingi mosquitoes bite mainly outdoors [Regardless of how the relationship of species-specific malaria with potential risk factors in CAH might be explained, it must be considered that our parasitological baseline survey probably captured the incipient onset of a second local outbreak of the site  suggestsoutdoors , and by outdoors , 40.P. vivax infections and consequently suggest that transmission is not entirely outdoors; the electricity availability as risk factor for P. falciparum may be related with an increase of evening outdoor activities  of household members [P. falciparum species in CAH  may explain the significant relationship of P. falciparum infections with younger age groups [P. falciparum infections had low parasite densities non-detectable by LM [P. vivax decreased significantly faster with age than that of P. falciparum, suggesting that acquiring clinical immunity is different species-specific, as has been seen in other co-endemic countries such as Papua New Guinea [The differences in malaria risk between species with respect to the availability of electricity at household can be explained by the different connotations that this public service can have. While the lack of electricity may reflect poor socio-economic status associated with increased  members . On the e groups –44, as wle by LM . The resw Guinea , Thailanw Guinea , Vanuatuw Guinea , Ethiopiw Guinea , and Braw Guinea , 49. TheP. vivax and P. falciparum only in the CAH site. Since the cluster located on the south of CAH for P. vivax was found to be associated with an increased presence of male individuals, age groups older than 15 years and the lack of electricity at households, an adjusted spatial analysis for these covariates was performed resulting in the disappearance of the cluster. The observation indicating that the aggregation of P. vivax infections was mainly determined by the aggregation of groups with high-risk than by specific ecological conditions within the site has also been reported in other villages of the Peruvian Amazon [An. darlingi, and the high mobility of infected individuals through the unique and highly accessible road could further increase the dispersal of parasites. Conversely, the cluster for P. falciparum remained in the closest area to the river even after being adjusted for age and electricity availability . Its highest prevalence may be explained by the unprecedented floods from Itaya River in 2012 resulting in large and multiple breeding sites in the area, but also by the presence of a fluvial port to which individuals (resident and non-residents) arrive after being exposed to malaria due to travel-related economical activities in other endemic riverine villages.The high heterogeneity in malaria transmission in the Peruvian Amazon creates opportunities for targeted interventions . Howevern Amazon . Local mParasitological measures (by LM and PCR), and individual and household characteristics collected during the baseline survey of a 3-year population-based malaria cohort study in the Amazonian Lupuna and Cahuide villages, together with contextual data obtained from malaria surveillance systems and health facilities, allowed for a good characterization of their population and malaria transmission patterns. On a population basis in both sites, low parasite rates were observed, with findings that asymptomatic and sub-patent infections predominated, compared to symptomatic and patent infections. However, the site-specific analysis of malaria prevalence and reported incidence showed substantial heterogeneity in malaria transmission at the micro-geographical level and over time, likely influenced by the site-specific socio-demographics, household characteristics, and ecological conditions in the villages. Riverine and road construction/deforestation contexts must be taken into account in order to carry out effective anti-malaria control and elimination efforts.Additional file 1: Table S1. Baseline socio-demographic characteristics of study participants by villages. Table S2. Baseline household characteristics of study participants by villages.Additional file 2: Figure S1. Weekly reported malaria cases in Cahuide (B) in 2012, and main control interventions implemented. (1) Mass screening and treatment (MS&T) and indoor spraying with residual insecticide with >90% of household coverage;  MS&T; (4) distribution of LLINs.


Cerebral vasospasm (CVS) is the most common neurological complication after aneurysmal subarachnoid hemorrhage (aSAH) and associated with poor functional outcome and mortality. Reports on incidence and predictors of CVS in Chinese patients with aSAH were scarce. We aimed to estimate the incidence and predictors of angiographic vasospasm (AV), symptomatic vasospasm (SV), and cerebral infarction in Chinese patients with aSAH.We retrospectively reviewed the medical records of 542 consecutive aSAH patients admitted to neurosurgery department of the First Affiliated Hospital of Xinjiang Medical University in Urumqi city of China between January 1, 2011 and December 31, 2015. AV, SV and cerebral infarction were defined based on clinical data and neuroimaging findings. Univariate and multivariate analyses were performed to identify predictors of AV, SV or cerebral infarction.9/L on admission and being current or former smokers were independent risk factors of cerebral infarction. Leukocyte count ≥11× 109/L on admission and aneurysm size ≥ 10 mm were independent risk factors of SV. Serum glucose level ≥7.0 mmol/L on admission was an independent risk factor of AV.343 (63.3%) patients fulfilled the inclusion and exclusion criteria. Of them, 182(53.1%) developed AV, 99 (28.9%) developed SV, and 87 (25.4%) developed cerebral infarction. A history of hypertension, poor modified Fisher grade (3–4) and poor Hunt-Hess grade (4–5) on admission were common risk factors for AV, SV and cerebral infarction. Patients from Uyghur ethnic group or other minorities were less likely to develop AV, SV or cerebral infarction, compared to those from Han ethic group after adjustment of other potential confounders. Additionally, age ≥53 years, leukocyte count ≥11× 10Risk factors of different definitions of CVS were diverse in Chinese patients with aSAH; however, risk factors of SV and cerebral infarction seem to be similar. We recommend early and aggressive therapy in these patients at-risk of CVS. Non-traumatic subarachnoid hemorrhage is a neurosurgical emergency characterized by the extravasation of blood into the spaces covering the central nervous system that are filled with cerebrospinal fluid. The leading cause of non-traumatic subarachnoid hemorrhage is rupture of an intracranial aneurysm, which accounts for about 80 percent of total cases . CerebraWe retrospectively reviewed the demographic and clinical data from patients admitted to the Department of Neurosurgery at Xinjiang Medical University First Affiliated Hospital  with confirmed aSAH between January 1, 2011 and December 31, 2015. aSAH was determined by clinical symptoms and neuroimaging findings . Lumbar puncture was performed when angiography could not confirm subarachnoid hemorrhage. Exclusion criteria included secondary subarachnoid hemorrhage from trauma, arteriovenous malformations, or other causes and age less than 15 years. Patients were only included when they met the following criteria: they were admitted to neurosurgery center within two days after aSAH onset, and received surgical clipping and/or endovascular coiling within three days after admission, and had repeated DSA performed after aneurysm treatment, and had CT scan performed on admission and repeated several times within six weeks after aSAH.Patients with aSAH were treated following a standardized institutional protocol. Upon admission, all patients were treated with intravenous fluids to maintain euvolemia. Oral nimodipine was administered to all patients (60 mg every four hours) for 14 days from the time of admission. For those who were not able to take oral nimodipine, intravenous nimodipine was administered. Selection of treatment with surgical clipping or endovascular coiling resulted from a consensus reached between the treating neurosurgeon and the interventional neuro-radiologist after analyzing risks and chances of success of both therapeutic modalities on each case. All patients underwent DSA or CTA, and CT scan on admission. Based on clinical symptoms and neuroimaging findings on admission, the clinical condition of aSAH patients was graded according to Hunt and Hess  and the SV was defined as the development of new focal neurological signs within 21 days after aSAH onset, including worsening headaches, stiff neck and insidious onset of confusion, or decline in level of consciousness, or focal deficits not clinically or radiographically attributable to other causes . AV was defined as moderate-to-sever arterial narrowing on DSA and not attributable to atherosclerosis, catheter-induced spasm or vessel hypoplasia. The degree of narrowing was determined by comparing the diameter of an affected artery to its pre-narrowed segment or to the most distal segment of the parental vessel. Narrowing was classified as moderate when the decrease was between 25% and 50% and severe when the vessel was narrowed by more than 50% . AV, SV,9/L, 7.0 mmol/L and 2.26 mmol/L, respectively.Other variables potentially associated with AV, SV (both transient and permanent), and cerebral infarctions were collected. Those variables included basic demographic information , size and number of aneurysm, Hunt-Hess grades and modified fisher grades on admission, aneurysm treatment  and admission laboratory findings . The age variable was dichotomized into “≥ 53 years” and “< 53 years” . Aneurysm size was determined by measuring the maximal inner diameter on DSA and categorized into two groups: “size ≥ 10 mm” (at least one aneurysm size ≥ 10 mm for multiple aneurysms) and “size < 10 mm” . The Hunt-Hess and Modified Fisher grades on admission were dichotomized into good-grade status (Modified Fisher grade “0–2” or Hunt-Hess grade “1–3”) and poor-grade status (Modified Fisher grade “3–4” or Hunt-Hess grade “4–5”). Admission leukocyte count, serum glucose, and triglyceride were dichotomized clinically at the level of 11× 10The Xinjiang Medical University First Affiliated Hospital Ethics Committee reviewed and approved the analysis. All private information was kept confidential and used for population analysis only.2 tests. The relative difference between outcomes was expressed as an odds ratios (ORs) with 95% confidence interval (CI). Binary logistic regression with stepwise forward selection was then used to adjust for variables identified in univariate analysis to be significantly different between the outcomes. The probability value for choice of predictive variables in logistic regression was set at 0.05. Adjusted ORs (aORs) with 95% CI were given. Independence of variables was tested using the likelihood ratio test on reduced models. Significance was set at P < 0.05 for all analyses.All statistical analyses were performed in SPSS version 17 . Univariate analysis was performed separately using AV, or SV, or cerebral infarction as the dependent variable. The demographic, clinical, radiographic, treatment-related, and in-hospital complication variables described above were independent variables. Categorical variables were analyzed using χA total of 678 patients with subarachnoid hemorrhage were admitted to the Department of Neurosurgery at Xinjiang Medical University First Affiliated Hospital between January 1, 2011 and December 31, 2015. Of them, 542 patients were diagnosed with aSAH. A total of 343 aSAH patients (63.3%) met the inclusion and exclusion criteria, and were included in the final analysis . Demogra9/L, and serum glucose level ≥7.0 mmol/L on admission were risk factors of AV, or SV, or cerebral infarction. Additionally, a history of hypertension and being current or former smokers were risk factors for SV or cerebral infarction. Aneurysm size ≥ 10 mm was risk factor for AV or SV , poor Hunt-Hess grade (4–5), leukocyte count level ≥11× 10AV or SV . NotablyIn multivariate analysis, ethnicity, history of hypertension, modified Fisher grade, and Hunt-Hess grade were associated with development of AV, or SV, or cerebral infarction. In addition, age, leukocyte count and history of cigarette smoking were associated with development of cerebral infarction. Leukocyte count and aneurysm size were associated with development of SV. Serum glucose level was associated with development of AV . Gender,To our knowledge, this is the largest study that investigates the incidence and predictors of AV, SV and cerebral infarction among Chinese patients with aSAH. The estimated incidence of AV, SV and cerebral infarction among patients with aSAH in our study is 53.1%, 28.9% and 25.4%, respectively. These findings are in alignment with the observed incidence between lowest and highest incidences reported by other studies , 22–26. History of hypertension was associated with AV, SV, and cerebral infarction in our study. This is consistent with findings from another study conducted among Japanese aSAH patients . HoweverNotably, we found patients from Uyghur ethnic group or other minorities were less likely to develop AV, SV or cerebral infarction, compared to those from Han ethic group. In previous studies, incidence of SV , cerebra9/L and current or former smokers before aSAH onset were independent risk factors of cerebral infarction in Chinese patients with aSAH. Similarly, most of previous studies have demonstrated a positive association of leukocytosis, age and history of smoking with the development of SV and cerebral infarction [Additionally, age ≥53 years, leukocyte count ≥11× 10farction ,40–43. Ffarction . This isThis study has several limitations. Firstly, the design of our study is retrospective, and we only included patients with repeated neuroimaging findings, which may introduce selection bias. These patients without repeated neuroimaging findings are more likely to be aged ≥53 years . As such9/L on admission and being current or former smokers were independent risk factors of cerebral infarction. Leukocyte count ≥11× 109/L and aneurysm size ≥ 10 mm on admission were independent risk factors of SV. Serum glucose level ≥7.0 mmol/L on admission was independent risk factor of AV. We recommend early and aggressive therapy in these at-risk populations to prevent poor outcome in patients with aSAH. However, the associations observed in this study may not be applicable in general to Chinese patients with aSAH since nearly 37% of patients were excluded due to various reasons.Our study demonstrated that a history of hypertension, poor modified Fisher grade (3–4), poor Hunt-Hess grade (4–5) and Han ethnicity were associated with increased risk of AV, SV, and cerebral infarction in Chinese patients with aSAH. Additionally, age ≥53 years, leukocyte count ≥11× 10S1 Table(DOCX)Click here for additional data file.


MC4R) gene polymorphisms can affect the total energy intake and the consumption of fat, protein and carbohydrates. Our study aims at investigating the association of the MC4R polymorphism with appetite and food intake among Chinese children.Recent studies indicated that eating behaviors are under genetic influence, and the melanocortin 4 receptor  and a self-designed questionnaire measuring food intake were performed. The FBAT and PBAT software packages were used.A family-based association study was conducted among 151 Chinese trios whose offsprings were overweight/obese children aged 9–15 years. The rs12970134 near P = 0.014). After adjusting for age, gender and standardized BMI, rs12970134 was significantly associated with food responsiveness (FR) among children , and with satiety responsiveness (SR) in trios (P = -0.026). The polymorphism was associated with beverage intake , but not significantly associated with vegetable, fruit or meat intake (P>0.050). We further found a significant mediation effect among the rs12970134, FR and beverage intake .The family-based association analysis showed that there was a significant association between rs12970134 and obesity (Z = 2.449, MC4R was associated with appetite and beverage intake, and food responsiveness could mediate the effect of rs12970134 on beverage intake in overweight and obese Chinese children population. Further studies are needed to uncover the genetic basis for eating behaviors, which could lead to develop and implement effective interventional strategies early in life.Our study is the first to report that rs12970134 near  The prevalence of childhood obesity has experienced a continuous increase worldwide. As reported in 2013, 23·8% of boys and 22·6% of girls were overweight/obese in developed countries, and 12.9% of boys and 13.4% of girls were overweight/obese in developing countries [MC4R) gene was the second gene validated by the genome-wide association study (GWAS) for obesity [MC4R polymorphisms  have been found to be associated with obesity or BMI, including rs12970134 [MC4R gene and was in strong linkage disequilibrium (LD) with rs17782313  and rs571312 , which could be used as the proxy of the other two polymorphisms. Polymorphisms near MC4R not only play an important role in the predisposition to obesity, but also affect eating behaviors directly. It was reported that the risk alleles of polymorphisms near MC4R were associated with higher total energy intake [MC4R were also found to influence appetite, measured by Children Eating Behavior Questionnaire (CEBQ). Two studies have used CEBQ to evaluate the effect of polymorphisms near MC4R on appetite [P = 0.04) and a lower score of “satiety responsiveness” subscale (P = 0.02), as compared to children with TT genotype [It has long been appreciated that obesity is highly heritable . More re obesity . In tota12970134 ,9, rs17712970134 ,11, rs57y intake , and couy intake , proteiny intake  and carby intake . Beside appetite ,17, bothMC4R and eating behaviors mostly relied on the case-control study design [MC4R with obesity, but not the associations with eating behaviors [Previous studies focusing on the polymorphisms near y design ,15,17. Iehaviors . The famehaviors . Besidesehaviors –22, and ehaviors –25.MC4R could affect different aspects of eating behaviors, there was no evidence on their association with beverage intake, and no studies have reported their association with appetite among Chinese children. Our hypothesis was that rs12970134 near MC4R could affect appetite and further influence the food intake in overweight/obese Chinese children. Therefore, we conducted a family-based association study among 151 Chinese trios whose offsprings were overweight/obese children. The objectives of this study were: 1) To investigate the association between rs12970134 and appetite measured by CEBQ; 2) To identify the association between rs12970134 and beverage intake; and 3) To explore whether the appetite plays a mediator role between rs12970134 and beverage intake in overweight/obese Chinese children.Although studies have unraveled that the polymorphisms near th percentile were defined as obese, whereas those with BMI between 85th and 95th percentile were defined as overweight. All the individuals were recruited with their voluntary participation. Written informed consent was provided by all participants. The study was approved by the Ethic Committee of Peking University Health Science Center.A family-based association study was conducted among 151 Chinese parent-offspring trio families , whose offsprings were overweight or obese children aged 9–15 years. Each trio consisted of 3 individuals: the father, mother and child. The study subjects came from the Nuclear Family Study of Childhood Obesity (NFSCO), recruited from 7 elementary schools and 3 middle schools in Chaoyang District and Changping District (both urban regions) of Beijing, China. We followed a family-based study design which was a special design for transmission/disequilibrium test (TDT). Family-based studies had the advantages of avoiding the influence of population substructure . The unihttp://www.who.int/childgrowth/standards/bmi_for_age/en/).Fasting venous blood samples were taken for DNA extraction. Parents’ age, gender, height and weight were reported by themselves and the children’s were reported by their parents. BMI was calculated accordingly. It has been shown that self-reported figural scales had a high predictive ability for identifying overweight/obese subjects, and can be used with confidence as proxies of BMI . The sexChildren’s appetite was evaluated using the Children’s Eating Behavior Questionnaire (CEBQ) completed by their parents. This questionnaire was first designed in 2001 and consisted of 35 items measuring 8 dimensions of eating behavior, and used a 5-point Likert-type scale . The queFood intake including vegetable intake, fruit intake, meat intake and beverage intake was investigated using a self-designed questionnaire and completed by the subjects themselves. This questionnaire was designed according to Block Kids Food Screener (BKFS), which was developed to evaluate dietary intake of nutrients and food groups in youth aged 2–17 years by NutritionQuest  in 2007 , and latwww.sequenom.com). The genotyping call rate of rs12970134 was 99.56%. All the experiments were done by investigators who were blind to the phenotypes.The rs12970134 polymorphism was genotyped using genomic DNAs extracted from blood leukocytes by the phenol-chloroform extraction method. Genotyping was conducted on MassARRAY System . Primers, including a pair of amplification primers and an extension primer, were designed with Sequenom MassArray Assay Design Suite. A multiplex polymerase chain reaction was performed, and unincorporated double stranded nucleotide triphosphate bases were dephosphorylated with shrimp alkaline phosphatase followed by primer extension. The purified primer extension reaction was spotted on to a 384-element silicon chip  and analyzed in the Matrix assisted laser desorption ionization time of flight mass Spectrometry . The resulting spectra were processed with MassArray Typer (Sequenom) (www.biostat.harvard.edu/fbat/fbat.htm). Linear regression analyses were used to analyze the association between rs12970134 and dietary variables within the offsprings. Multiple approaches have been developed and different types of phenotypes could be analyzed including dichotomous (affection status), quantitative and censored  [P values could be computed on the basis of both asymptotic theory and permutation tests [www.hsph.harvard.edu/clange/default.htm). For both linear regression method and PBAT method, covariates of age, gender and BMI-sds were adjusted for stepwise. Quanto software  was used to conduct power analysis. The polymorphism was analyzed under the additive model. A two-sided P<0.05 was considered as nominally significant.Quality control (QC) of the genotype was performed prior to association analysis by testing Mendelian inheritance consistency and Hardy-Weinberg equilibrium proportions. Regular statistical analyses were performed using SPSS  version 18.0 software . Descriptive values were given as mean ± standard deviation or percentages, and the statistical differences in demographic and dietary characteristics between obese and overweight subjects were tested with t-tests (continuous variables) or Pearson Chi-square tests . Family-based associations between obesity and rs12970134 were tested in the family sample using family-based association tests  (FBAT vef onset) . For thef onset)  was usedf onset)  and PDT f onset) , PBAT waon tests . We usedX, Y and M, and the coefficients in the model were represented by a, b, c and c’. The first regression model used c as the coefficient of X in association with Y; the second model used a as the coefficient of X in association with M; the third model put M and X as independent variables simultaneously, and used b and c’ as the coefficients in association with Y.The mediation analysis was based on the model brought forward by Baron, et al. . Accordi2 and 25.0 kg/m2, for fathers and mothers respectively. The 151 recruited children aged 9–15 years with the average age of 11.7 years, and 68.2% of them were boys. Among them, 98 children were obese and 53 were overweight, and the average BMI of the offsprings was 26.6 kg/m2. We observed no difference between overweight and obese children in age, gender or height (P>0.05).The descriptive information on age, gender and anthropometrics is provided in P = 0.035; 3.7±0.9 vs 3.5±0.6, P = 0.030), but no significant difference was found in satiety responsiveness (P = 0.664). In terms of food intake, the vegetable intake and the beverage intake among obese children were significantly higher than that of overweight children . Obese children also reported slightly more meat intake and fruit intake than overweight children, but no significant differences were observed (P>0.05).P = 0.175) and among parents (P = 0.267).The QC analyses found no discrepancies in Mendelian inheritance. The risk allele  frequency of rs12970134 polymorphism was 21.2% in children and 19.2% in parents, and the genotype distribution were in Hardy-Weinberg equilibrium both among children (P = 0.159). However, we then used only obese children (N = 98) as probands, and FBAT analyses showed that there was significant positive association between rs12970134 and obesity , revealing that the risk allele  of rs12970134 could increase the risk of childhood obesity. Further, we tested the continuous variable BMI-sds, but the association between rs12970134 and BMI-sds was not significant by PBAT analyses among 151 trios (P = 0.790), or by linear regression among 151 children .We first conducted FBAT analyses using the 151 overweight and obese children as probands, no significant association was found between rs12970134 and ‘overweight + obesity’  and without , indicating that the rs12970134 A allele increased children’s food responsiveness level, but the result was not validated in PBAT analyses (P>0.05). There was marginal negative association between rs12970134 and SR score among children , and the association was significant using PBAT analyses (P = -0.026), revealing that children with the rs12970134 A allele could be hard to have satiety responsiveness. EF was not significantly associated with rs12970134 in any of the analyses (P>0.05).The association between rs12970134 and FR score was significant among children, both with BMI-sds as a covariate  and in trios PBAT analysis (P = 0.043), indicating that the A allele of rs12970134 could increase the beverage intake among overweight and obese children.Four aspects of food intake were analyzed: vegetable, fruit, meat and beverage intake. As illustrated in 1age + q1gender + r1 BMI-sds + e1; (2) M = ax + p2age+ q2gender + r2 BMI-sds + e2; (3) Y = c’X + bM + p3age+ q3gender + r3 BMI-sds + e3. Significant mediation effect of FR was found on the association between rs12970134 and beverage intake , although the mediation effect was incomplete . Further calculation (ab/c × 100%) reflected that the mediation effect of FR could explain 9.5% of the total association between rs12970134 and beverage intake.Mediation analysis was conducted among 151 children. Since rs12970134 was associated with both FR and beverage intake, we used a mediation model to examine whether rs1297134 could mediate the effect of rs12970134 and beverage intake. MC4R and obesity, and found its association with childhood appetite (food responsiveness and satiety responsiveness) and beverage intake in overweight/obese children. To our knowledge, this study is the first to report that the MC4R rs12970134 polymorphism was associated with appetite and beverage intake, and food responsiveness could mediate the effect of rs12970134 on beverage intake in overweight and obese Chinese population.In this study, we conducted a family-based association study among 151 Chinese trios whose offsprings were overweight/obese children aged 9–15 years. We validated the association between the polymorphism rs12970134 near MC4R gene is located on chromosome 18q22 and encodes for the melanocortin 4 receptor, which is a G protein-coupled receptor and is critically involved in energy balance regulation [MC4R is in multiple regions of the brain including thalamus and hypothalamus, and can influence the leptin-melanocortin signaling system [MC4R expressed in the paraventricular nucleus has a critical role in the mechanisms of food intake [MC4R knockout mouse demonstrated the characteristics of being obesity, hyperphagia, and hyperinsulinemia [MC4R, with the majority (145 variants) of them differentially expressed in brain, and 1 variant differentially expressed in testis. It was reported by Yoon, et al. that there was significant coexpression of MC4R and the dopamine D2 receptor in the hypothalamic area, and also in the bed nucleus of the stria terminalis (BNST) area which was an important site for food reward [gulation . The expg system . MC4R exd intake , and theulinemia . Genotypd reward .MC4R gene but was an intergenic variant located in-between the gene MC4R and LOC342784. For a long time, rs12970134 was referred as an ‘MC4R variant/polymorphism’ or a ‘variant/polymorphism near MC4R’, but the link between rs12970134 with the gene LOC342784 was ignored. We used the online tool SCAN  to retrieve genes with variants in LD with rs12970134, and found that rs12970134 was not in LD with MC4R, but in strong LD with the gene LOC342784 (r2 = 1). Actually, other polymorphisms near MC4R had the same situation. Rs12970134, rs17782313 and rs571312 were in complete LD with LOC342784 (r2 = 1), rs17700144 was in strong LD with LOC342784 (r2 = 0.867), and all the four SNPs were not in LD with MC4R. The gene LOC342784 was a pseudo gene and currently we had no clues about its function, or whether or not it had function. Future genotype-phenotype association studies should be cautious about the SNP location and linkage in/near MC4R and LOC342784, and future functional researches should pay more attention on the gene LOC342784.An important point should be noted that the rs12970134 polymorphism was not located in the P = 1.7×10−9), with homozygotes for the risk allele of rs12970134 have 2 cm increased waist circumference [−6), and was positively associated with BMI  and weight . In this study, the association between rs12970134 and obesity was studied in a Chinese family-based association analysis. As to the allele frequency, the rs12970134 risk allele  frequency was 0.2119 in the current study, which was similar as compared to 0.2195 in the CHB population.Rs12970134 was reported to be associated with obesity and obesity-related traits. In 2008, rs12970134 was reported by a GWAS to be significantly associated with waist circumference  and positively associated with enjoyment of food subscale (P = 0.03). Besides, there was a marginal association of 17782313 with food responsiveness subscale (P = 0.09). Vega, et al. reported that rs17782313 was associated with higher scores of uncontrolled eating in Chilean adults [P = 0.02) [P = 0.04) and a lower score of “satiety responsiveness” (P = 0.02). Our result on “satiety responsiveness” was in consistency with previous studies, but we did not find significant association between the polymorphism near MC4R and the “enjoyment of food” subscale. Another instrument to measure eating behaviors regarding cognition and motivation is the Three Factors Eating Questionnaire (TFEQ), which contains fifty-one items and three dimensions (factors) including disinhibition, restraint and hunger [P = 0.02) [MC4R mutation carriers with obesity and 33 overweight non-carriers found a positive association between the score of disinhibition and MC4R mutation (P = 0.007), but it was argued that the difference in weight status was not well controlled and could be a potential confounder [MC4R were conducted among French or German population, but no significant results were reported [We found no published literatures reporting the association between , et al.  first ren adults . Obregón = 0.02) . Ho-Urrid hunger . Stutzma = 0.02) . Anothernfounder . Three oreported –54. The reported , and thereported .AlthoughMC4R. McCaffery, et al. reported that rs571312 was associated with higher caloric intake in white adults  [P = 0.028), total fat (P = 0.008) and protein (P = 0.003)[MC4R and food intake, either in terms of total energy intake or macro/micronutrient intake, as reviewed by Valette, et al. in 2013 [MC4R rs17782313 C allele showed a significant trend towards higher percentages of snacking, in the population of French obese children (P = 0.01), in Swiss obese adults (P = 0.04), and in Finnish adolescents as well (P = 0.04). We found no published literatures reporting the association of MC4R polymorphisms with beverage intake.Different aspects of food intake have been investigated for the association with polymorphisms near  0.0194) . In 2008 = 0.003), while f in 2013 . Another in 2013  in 2009,P value of the mediation analysis, both possibilities should be interpreted carefully.Regarding the mediation analysis, our hypothesis was that appetite (i.e. food responsiveness) was the mediator of the SNP association with food intake (i.e. beverage intake). In fact, the other hypothesis that the beverage intake served as the mediator was also possible, but we did not find evidence of population studies supporting the hypothesis. Due to the cross-sectional nature of the current study, the causation of appetite and food intake could not be determined, therefore we could not rule out the possibility that rs12970134 affected appetite through the beverage intake. Also, due to the marginal MC4R knock-out mice, Cone, et al. found that animals heterozygous for the melanocortin-4 receptor were intermediate to wild type and MC4R null mice in obesity-related phenotypes including growth rate, serum leptin concentrations, linear growth, and fasting serum insulin concentrations [As for the genetic model, we chose to use the additive model in this study. In fact, different genetic models have been used in previous studies, including the dominant model , the rectrations . The ‘geMC4R, nor did us test the appetite or food intake in animal models. Further studies were needed for understanding the mechanism of the polymorphisms in/near MC4R. Secondly, the food intake in this study was self-reported instead of measured objectively, and we chose several kinds of food intake instead of evaluating food intake systematically. The total caloric intake was not investigated, which could provide more information of the SNP effect on food intake. Thirdly, as children’s eating behaviors were largely influenced by the home environment and their parents’ attitude and behaviors, those information should be investigated in future studies in order to fully assess the genetic effect on eating behaviors. Fourthly, we did not replicate the findings in another independent population, and the sample size of this study was not large. Using the Quanto software, we conducted the power analysis on the beverage intake variable, and the parameters and estimated power were as followings: Using the parent-offspring study design for the continuous variable, with effect allele frequency of 0.2, the Mean±SD of beverage intake of 4.7±6.0 servings/week, and the effect of rs12970134 on beverage intake of 3.8 (unstandardized β), under the additive genetic model, at a two-sided significance level of P<0.05, the analysis showed that 151 trios could reach the power of 90.6%.Several limitations of this study should be noted. First of all, we did not test gene expression or cytokines data of P<0.0018 Bonferroni criteria, the association between SNP and beverage intake in children was still significant after multiple-testing correction. However, the SNP association with FR or SR had only marginal P values, which should be interpreted with caution. In our study, the SNP was not associated with BMI-sds in the children dataset. Since mediation analyses had the prerequisite that the association between the independent variable and the dependent variable should be significant, we could not analyze the mediation effect of eating behavior on the SNP-adiposity association. Further studies could focus on the hypothesis that rs12970134 could possibly increase adiposity through affecting eating behaviors.Considering the study design in the current study, we could only conclude that there was SNP association with eating behavior in overweight and obese Chinese children population, but whether or not the association existed in the general population could not be decided. Besides, when testing the association between rs12970134 and eating behaviors, 7 behaviors were tested and 4 statistical models were performed for each behavior, which resulted in the multiple-testing problem. Using a MC4R was associated with appetite and beverage intake in overweight and obese Chinese children, and appetite could mediate the effect of the polymorphism near MC4R on beverage intake. Uncovering the genetic basis for eating behaviors could lead to develop and implement effective interventional strategies early in life, which could help avoid the health risks related to childhood obesity.In conclusion, we found the polymorphism near S1 File(XLS)Click here for additional data file.


Paeonia lactiflora is a herbaceous flower in the family Paeoniaceae with both hypocotyl and epicotyl dormant seeds. We used high-throughput transcriptome sequencing on two different developmental stages of P. lactiflora seeds to identify seed dormancy and germination-related genes. We performed de novo assembly and annotated a total of 123,577 unigenes, which encoded 24,688 putative proteins with 47 GO categories. A total of 10,714 unigenes were annotated in the KEGG database, and 258 pathways were involved in the annotations. A total of 1795 genes were differentially expressed in the functional enrichment analysis. The key genes for seed germination and dormancy, such as GAI1 and ARF, were confirmed by quantitative reverse transcription-polymerase chain reaction analysis. This is the first report of sequencing the P. lactiflora seed transcriptome. Our results provide fundamental frame work and technical support for further selective breeding and cultivation of Paeonia. Our transcriptomic data also serves as the basis for future genetics and genomics research on Paeonia and its closely related species. Paeonia lactiflora is a herbaceous perennial flower plant in the family Paeoniaceae, which is native to Central and Eastern Asia and widely grown in China. Paeonia lactiflora is one of horticulturally important flower species. It has been primarily grown for in use in the horticultural industry as home garden plants and is also cultivated as commercially cut flower. Furthermore, P. lactiflora, as a temperament plant species, is highly cold resistant and can normally grow and bloom under temperature −46.5°C. Therefore, P. lactiflora plants have not only become the main source of peonies for the cut flower business but they are also valuable cold-resistant genetic resources for breeding and cultivation [tivation .P. lactiflora, for example, abscisic acid (ABA) has been identified to be one of the major endogenous physiological factors to inhibit seed germination and root growth [Plant seeds usually experience dormancy where they are unable to germinate in a specified period of time. Seed dormancy is a very important mechanism to inhibit germination during unsuitable ecological conditions, for example, low temperature . It has t growth .Paeonia lactiflora seeds display both hypocotyl and epicotyl dormancy and time from sowing to fully germination takes six to seven months under natural conditions. Hypocotyls of P. lactiflora start to elongate and stimulate the root growth when temperature goes down in later fall after sowing. After experiencing long winter, the dormancy of epicotyl is broken and starts to grow during spring [Paeonia lactiflora breeding and cultivation. Due to these dual and sequential dormancy scenarios in P. lactiflora, understanding the hypocotyl dormancy is fundamental to tackle the epicotyl dormancy and further to unravel the dormancy of P. lactiflora [P. lactiflora. However, molecular mechanism of seed dormancy in P. lactiflora remains unexplored.g spring . Furtherctiflora . PreviouArabidopsis, numerous genes with various functions have been identified and shown differential expression between dormant seeds and dormancy-releasing seeds [P. lactiflora, which display dual and long dormancy process, here, we utilized “-omics” approach to obtain transcriptomes of two different germination stages of peony seeds. We extracted total RNA, sequenced the RNA using the Illumina/HiSeq™2000/MiSeq™ platform, assembled de novo, and annotated unigenes [P. lactiflora genes that were differentially expressed by seed hypocotyls during germination and analyzed their functions and the mechanism of differential expression. We ultimately aim to identify the key seed germination and dormancy genes to improve the breeding process.Previous studies have demonstrated that many genes regulate seed dormancy and germination and especially genes involving in ABA and gibberellic acid (GA) pathway –11. Usinng seeds , 13. To unigenes . We idenP. lactiflora seeds before germination of down-hypocotyl (PDB) and after germination of up-hypocotyl (PDA), we obtained a total of 120,181,964 unigene sequences. 48%  and 5.2%  unigene sequences were present in RNAs of PDB and PDA, respectively. A total of 123,577 contigs were obtained from sequence assembly, and 30% of the contigs have the length larger than 2000 bases.Based on RNA-seq data from stratification 0 day and stratification 40 days of ARF), ABA-responsive element-binding factors (ABF), brassinosteroid insensitive 1 (BRI1), and transcription factor TGA (TGA). “Binding”  was the second most prevalent category and included ARF and BRI1, followed by “metabolic processes” , including ARF, pyrabactin resistance/pyrabactin resistance-like (PYR/PYL), protein phosphatase 2C (PP2C), ethylene-insensitive protein 2 (EIN2), ABF, and TGA. Our annotation results show only a small proportion of the P. lactiflora unigenes with GO categories assigned, possibly due to the large number of uninformative gene descriptions of the plant protein hits. These classification results show the overall gene expression profile of P. lactiflora seeds.The GO classification based on sequence homology revealed that 24,688 of the assembled unigenes were categorized into 47 functional groups . “CellulCOI1), glycine-rich RNA-binding proteins (GRP2), and other genes, followed by “posttranslational modification, protein turnover, and chaperones” , including S-phase kinase-associated protein 1(SKP1), inhibitors of invertases (INH), and other genes. The next most prevalent category was “translation, ribosomal structure, and biogenesis” , including ribosomal protein L27b (RPL27b), ribosomal protein L13aa (RPL13aa), and other genes. The three rare groups were “unnamed proteins” , “cell motility” , and “extracellular structures” .The KOG classification of 31,215 nonredundant hits indicated that 11,855 unigenes were clustered into 26 functional categories . “GeneraThe KEGG analysis of unigenes showed that 10,714 unigenes were assigned to 258 pathways . The uniP. lactiflora transcriptomes, accounting for 13.56% of all contigs. SSR types were abundant, including mononucleotide repeat to hexanucleotide repeat , Swiss-Prot, KEGG, and KOG databases, we obtained that 30,803 unigenes contain CDS and encoded proteins. The lengths of predicted CDS are shown in q value < 0.005 and │log2 (fold change)│ > 1. The statistical analysis results of the DEGs in the two samples are shown in The results of comparison of the two samples were quantitatively analyzed. According to the magnitude of gene expression, namely, expected number of fragments per kilobase of transcript sequence per million base pairs sequenced (FPKM), we calculated the multiple of gene expression difference between different samples. DEGs were defined with a threshold We identified DEGs that were significantly enriched in GO entries and identified their biological processes, cellular components, and molecular functions at three levels of gene function . We enri“Oxidation-reduction processes” was the most significantly enriched biological process GO term for DEGs, accounting for 13.99%. “Cell periphery” was the most significantly enriched GO term in cellular components, accounting for 7.85%. “Carbohydrate binding” was the most significantly enriched GO term in molecular functions, accounting for 2.39%.In organisms, different genes are coordinated to exercise their biological functions. The most important biochemical metabolic pathways and signal transduction pathways of differentially expressed genes can be identified by the DEGs. The KEGG is the largest public pathway database . We perfAs the results of the analysis shown in GAI1, ARF, and BRI1, which were upregulated in seeds, and JAZ, which was downregulated  under field conditions. The male parent was “Fen Yulou,” and the female parent was “Fen Yunu.” We harvested the seeds annually every August and used two different seed developmental stages (0 and 40 days) as material. We extracted total RNA from seeds in the two stages and performed an RNA-seq transcriptome analysis.P. lactiflora seed transcriptome [Beijing Biological Information Technology Ltd.  extracted total RNA, according to the manufacturer's instructions, which was stored at −80°C. Total RNA was extracted from seeds using the RNAprep pure Plant Kit ; each sample contains about ten seeds. We used the Illumina HiSeq™2000/MiSeq™ sequencer to sequence the criptome . The totN ratio > 2% was used to remove the low-quality reads and obtain the clean reads. All subsequent analyses were based on the clean reads.The image data file obtained by sequencing was converted into raw reads using CASAVA base calling. Joint reads were removed from raw reads, and an P. lactiflora has no reference genome, we carried out de novo assembly using Trinity. First, the contigs were assembled using the overlapping area of the reads. Second, connected to the contig assembly sequence into the ends cannot be extended again (unigene). The unigenes were compared with the Nr, NCBI nonredundant nucleotide sequences (Nt), Swiss-Prot, KEGG, and KOG databases to determine the direction of the unigenes.Because The GO functional classification of the unigenes was performed using Blast2GO , and theP value was adjusted using q value. q value < 0.005 and │log2 (fold change)│ > 1 were set as the threshold for significantly differential expression.Prior to differential gene expression analysis, for each sequenced library, the read counts were adjusted by edgeR program package through one scaling normalized factor. Differential expression analysis of two samples was performed using the DEGseq (2010) R package. The analytic formula isN is the number of genes with pathway annotations in all genes; n is the number of differentially expressed genes in N; M is the number of genes annotated for a particular pathway in all genes; m is the number of differentially expressed genes for a particular pathway.In the formula, GO enrichment analysis of the DEGs was implemented by the GOseq R package-based Wallenius noncentral hypergeometric distribution , which chttp://www.genome.jp/kegg/). We used KOBAS [KEGG  is a dated KOBAS  softwarehttp://pgrc.ipk-gatersleben.de/misa/misa.html), and primer for each SSR was designed using Primer3 (http://primer3.sourceforge.net/releases.php). The standard used for a SSR was 10 single-nucleotide repeats, six dinucleotide repeats, and three, four, five, and six nucleotides repeated at least five times [SSRs of the transcriptome were identified using MISA  software to obtain the nucleotide and amino acid sequences of this portion of the gene. The CDS results predicted by Estscan software are shown in Supplementary File 2.The highest score from the BLAST  alignment results was used to determine the CDS of the unigene, using the Nr, Swiss-Prot, KEGG, and Genes databases, in that order, to compare the unigenes. The transcripts were extracted from the comparative result of open-reading frame- (ORF-) encoding box information, and the standard table-coding region sequences were translated into amino acid sequences (5′-3′ order). The results obtained by comparing the known protein database with blast are shown in Supplementary File 1 available online at μl containing 2 μl cDNA, 10 μl 26SYBR premix Ex taq™ , 0.4 μl each of 10 mM forward and reverse primers, and 7.2 μl RNase-free water. The thermal cycling conditions were 95°C for 5 min, 45 cycles at 95°C for 5 s for denaturation, and 56°C for 25 s for annealing and extension.Four unigenes involved in plant hormone signal transduction were chosen for validation by qRT-PCR. The reference gene selected in this experiment was actin (Gene Bank query number is gi: 48927617). The primers were designed with Primer Premier 5.0 software . Total RSupplementary file 1: The result of alignment. Supplementary file 2: The result of prediction.