Episyrphus balteatus and Eupeodes corollae (Diptera: Muscomorpha: Syrphidae) are important natural aphid predators. We obtained mitochondrial genome sequences from these two species using methods of PCR amplification and sequencing. The complete Episyrphus mitochondrial genome is 16,175 bp long while the incomplete one of Eupeodes is 15,326 bp long. All 37 typical mitochondrial genes are present in both species and arranged in ancestral positions and directions. The two mitochondrial genomes showed a biased A/T usage versus G/C. The cox1, cox2, cox3, cob and nad1 showed relatively low level of nucleotide diversity among protein-coding genes, while the trnM was the most conserved one without any nucleotide variation in stem regions within Muscomorpha. Phylogenetic relationships among the major lineages of Muscomorpha were reconstructed using a complete set of mitochondrial genes. Bayesian and maximum likelihood analyses generated congruent topologies. Our results supported the monophyly of five species within the Syrphidae (Syrphoidea). The Platypezoidea was sister to all other species of Muscomorpha in our phylogeny. Our study demonstrated the power of the complete mitochondrial gene set for phylogenetic analysis in Muscomorpha.The hoverflies  The mitochondrion is a fundamental eukaryotic organelle. Energy production via oxidative phosphorylation is its most-studied function, though it also functions in apoptosis and cell aging132527891011Muscomorpha (Diptera: Brachycera), an infraorder of Brachycera, is a large and diverse group of flies, containing the bulk of the Brachycera. It includes a number of the most common flies, such as the housefly, the fruit fly, and the blowfly. The Muscomorpha can be separated into two sections, the Aschiza and the Schizophora9131414Hoverflies, also called flower flies, compose the insect family Syrphidae. This family contains almost 6,000 described species in 200 genera, and is nearly worldwide in distribution1719172218Episyrphus balteatus (Syrphidae) is a relatively small hoverfly (9–12 mm), widespread throughout the Palearctic ecozone, endemic to Europe, North Asia, and North Africa. Its color patterns may appear wasp-like to animals, such as birds, protecting it from predationEupeodes corollae (Syrphidae) is another common hoverfly species. The adults are often migratory with a worldwide distribution. It has been tested as a biological control agent for aphids and scale insects in greenhouses. However, the larval flies preferred the fruit in the experiment, consuming more fruit than aphidsThe marmalade hoverfly Ocyptamus sativus, Simosyrphus grandicornis and an unknown genus sp. Syrphidae (accession KM244713)916Episyrphus and Eupeodes, representing the first mitochondrial genomes reported from the genera Episyrphus and Eupeodes. Furthermore, we compared genome features and investigated phylogenetic relationships within Muscomorpha using complete mitochondrial genomes from GenBank along with our two newly sequenced mitochondrial genomes   (The complete U351241) . The parU379658) . ParticuNo gene rearrangement was observed in our analyses: (1) as compared with the putative ancestral insect arrangementEpisyrphus has 255 bp of intergenic nucleotides, in 22 different locations, with intergenic spacer lengths ranging from 1 to 60 bp. Seven pairs of genes overlap each other, with overlap lengths ranging from 1 to 7 bp. Eight pairs of genes directly adjacent one another including the pairs of rrnL-trnV and trnV-rrnS. The mitochondrial genome of Eupeodes has 230 bp of intergenic nucleotides, in 19 locations, with intergenic spacer lengths from 2 to 47 bp. Nine pairs of genes overlap each other, with overlap lengths ranging from 1 to 7 bp. Nine pairs of genes directly adjacent one another including the pairs of rrnL-trnV and trnV-rrnS. In both species, the longest intergenic spacer was located between trnK and trnD, followed by the one located between trnE and trnF. The longest overlapping regions were located between atp8 and atp6, nad4 and nad4l. The intergenic and overlapping region of these two species are similar to most other insect mitochondrial genomes with no gene rearrangement occurred, compared with those with frequent gene rearrangementEach of the 37 typical mitochondrial genes is present in both species. The mitochondrial genome of Episyrphus and Eupeodes mitochondrial genomes show a very strong bias in nucleotide composition (A + T% > G + C%), which is typical of insect mitochondrial genomes. The 16 Muscomorpha species we analyzed have PCG A + T contents between 71.10%  and 78.97% (Ocyptamus sativus); The mitochondrial genomes of Episyrphus and Eupeodes have A + T contents of 78.85% and 78.83%, respectively . All GC-skews are positive, indicating that the PCGs contain a higher percentage of T and C than A and G nucleotides , ATT (Ile), TTT (Phe), and ATA (Met) are the most frequent codons, as is typical of other insect mitochondrial genomes3033Mitochondrial genome codon usage in  A and T  as in ot A and T . In the cox1, cox2, cox3, cob and nad1 showed relatively low level, five genes of atp6, nad6, nad4, nad4l and nad5 showed medial level, whereas three genes of atp8, nad2 and nad6 showed the highest level of nucleotide diversity  to 72 bp (trnV). The Eupeodes mitochondrial genome contains 1,479 bp within tRNA genes, at an A + T content of 80.12%. Individual tRNAs range in length from 64 bp (trnR) to 72 bp (trnV).The Episyrphus and Eupeodes, all tRNA genes fold into the canonical clover-leaf structure. The dihydrouridine (DHU) arm of all the tRNAs is a large loop, instead of a conserved stem-and-loop structure; however, this is typical of metazoan mitochondrial genomesSecondary structure models of the tRNA genes in the two newly sequenced mitochondrial genomes were predicted using the Mitos WebServertrnM was the most conserved one without any nucleotide variation in stem regions, followed by trnV and trnE with three site mutations. The trnC showed the highest number of site mutation on stem regions (17 sites), followed by the trnH (16 sites)  .Episyrphus and Eupeodes, respectively. Among the six mismatched base pairs in Episyrphus, five are U-U pairs, located in the AA and TΨC stems; the other is an A-A pair, located in the A-A stem. Eupeodes has four U-U pairs, located in the AA and TΨC stems, and an A-A pair, located in the A-A stem.Base pairs other than canonical A-Us and C-Gs are occasionally used in our tRNAs, based on predicted tRNA secondary structures. We found six and five mismatched base pairs in the tRNAs from rrnL and rrnS, are 1,338 bp long, with an A + T content of 84.61%; and 804 bp long, with an A + T content of 83.96%, respectively, in Episyrphus. In Eupeodes, rrnL is 1334 bp long, with an A + T content of 84.78%; rrnS is 795 bp long, with an A + T content of 83.14% ))). Syrphoidea and Opomyzoidea formed a lineage, and then sister to the other species of Schizophora. The Opomyzoidea was traditionally considered as a superfamily of Schizophora, which was proved to be a monophyletic groupFergusobia (Tylenchida: Neotylenchidae) nematodes. The novel life history of the species from Fergusoninidae might affect the evolutionary pattern of their mitochondrial genomes9131415We reconstructed phylogenies within the Muscomorpha using the nucleotide sequences of the 37 mitochondrial genes. Bayesian and maximum likelihood (ML) methods estimated congruent topologies . Our ana11The specimens were collected from Sichuan Province, China. Specimens were initially preserved in 100% ethanol in the field when collected, and then stored at −80 °C prior to DNA extraction. Whole genomic DNA was extracted from the legs and thorax of the specimens using a DNeasy tissue kit , following the manufacturer’s protocols.Initially we used a previously designed set of universal primers for insect mitochondrial genomes1http://mitos.bioinf.uni-leipzig.de/index.py)916Mitochondrion DNA sequences were assembled using Lasergene software . The tRNA genes were initially identified using the Mitos WebServer  were used as outgroups because of the close relationship between Tabanomorpha and MuscomorphaWe used 14 Muscomorpha species with published mitochondrial genomes, and our two newly sequenced mitochondrial genomes for phylogenetic analyses . The 16 MAFFT version 7.205, which implements consistency-based algorithms, was used for the alignment of protein-coding and RNA genesData partitioning, and the ability to apply specific models to different partitions, is ideal for analyzing mitochondrial genomesWe constructed phylogenies among the Muscomorpha with the Bayesian inference method (BI) using Mrbayes version 3.2.5How to cite this article: Pu, D.-q. et al. Mitochondrial genomes of the hoverflies Episyrphus balteatus and Eupeodes corollae (Diptera: Syrphidae), with a phylogenetic analysis of Muscomorpha. Sci. Rep.7, 44300; doi: 10.1038/srep44300 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Purpose. Liver dysfunction and failure are severe complications of sepsis and result in poor outcome and increased mortality. The underlying pathologic mechanisms of hepatocyte dysfunction and necrosis during sepsis are only incompletely understood. Here, we investigated whether procalcitonin, a biomarker of sepsis, modulates liver cell function and viability. Materials and Methods. Employing a previously characterized and patented biosensor system evaluating hepatocyte toxicity in vitro, human hepatocellular carcinoma cells (HepG2/C3A) were exposed to 0.01–50 ng/mL procalcitonin for 2 × 72 h and evaluated for proliferation, necrosis, metabolic activity, cellular integrity, microalbumin synthesis, and detoxification capacity. Acetaminophen served as positive control. For further standardization, procalcitonin effects were confirmed in a cellular toxicology assay panel employing L929 fibroblasts. Data were analyzed using ANOVA/Tukey's test. Results. Already at concentrations as low as 0.25 ng/mL, procalcitonin induced HepG2/C3A necrosis (P < 0.05) and reduced metabolic activity, cellular integrity, synthesis, and detoxification capacity . Comparable effects were obtained employing L929 fibroblasts. Conclusion. We provide evidence for procalcitonin to directly impair function and viability of human hepatocytes and exert general cytotoxicity in vitro. Therapeutical targeting of procalcitonin could thus display a novel approach to reduce incidence of liver dysfunction and failure during sepsis and lower morbidity and mortality of septic patients. Sepsis is one of the leading causes of death in critically ill patients admitted to intensive care units . High mo itself to play a role in sepsis pathology -2H-tetrazolium hydroxide (XTT) which is metabolically reduced in viable cells by mitochondrial dehydrogenase to a water-soluble formazan product detectable by absorbance readings , 17. LDHFor the determination of microalbumin synthesis, hepatocytes were incubated with procalcitonin for two times 72 h and supernatants were collected, centrifuged, and stored at −80°C pending analysis. Microalbumin was then determined nephelometrically  . InductiMurine fibroblasts were grown in Dulbecco's modified Eagle's Medium supplemented with 5% fetal calf serum, 1% of glutamine solution (PAA), and 1% of antibiotics solution according to 9935 ISO Norm. Effects of 0.01–100 ng/mL procalcitonin on proliferation, vitality, and mitochondrial dehydrogenase were investigated as mentioned above.P value of ≤0.05.Data was analyzed employing GraphPad Prism Software version 7.01  using One-Way ANOVA followed by Tukey's test for correction of multiple comparisons. The results are expressed as mean and standard deviation (SD). Differences were considered significant at a P < 0.001; P < 0.05; P < 0.001, P < 0.001, Incubation of hepatocytes with procalcitonin ranging from 0.01 to 50 ng/mL dose dependently inhibited hepatocyte proliferation over the time-course of 72 h with significant effects starting at 2.5 ng/mL . In add itself to induce hepatocyte dysfunction and impairment of cellular integrity. This suggests procalcitonin as part of a vicious circle of sterile inflammation and liver cell necrosis. Future studies may thus address whether procalcitonin can be a potential target for the treatment of acute liver failure, the importance of inflammatory mediators, or whether the interplay of hepatocytes and procalcitonin is independent of infection.Interestingly, procalcitonin's ability to identify bacteraemia does not apply to patients suffering from liver failure. Hepatocyte necrosis correlates with high levels of procalcitonin irrespective of infection and authors concluded that the massive inflammation associated with hepatocyte necrosis may have triggered excess procalcitonin release . During Sepsis is associated with vascular barrier dysfunction, fluid extravasation, and rapid induction of microcirculatory disturbance. Our previous study identified procalcitonin to induce, within a few hours, endothelial cell dysfunction and endothelial cell death . Liver fIn conclusion, our experiments provide evidence for procalcitonin to directly impair function and viability of human hepatocytes and exert general cytotoxicity in vitro. The mechanisms for the hepatotoxicity of procalcitonin are unclear and need further investigations in suitable in vitro and in vivo models.


Local understandings of malaria and use of preventive measures-are critical factors in sustained control of malaria. This study assessed caretakers’ knowledge on malaria, use of Long Lasting Insecticide Treated Nets (LLINs) and care-seeking behavior for their children’s illness in different malaria transmission settings of Ethiopia.Data were collected from 709 caretakers of children of 2–9 years of age during in 2016. A standard questionnaire was used to assess caretakers’ perceptions of malaria, use of LLIN and care seeking behavior for febrile illness of children aged 2–9 years.p < 0.05). Majority, 72.2% (512), of the caretakers knew that sleeping under LLIN could prevent malaria. Overall knowledge on malaria (mean = 51.2%) was very low with significant variations by localities, altitude and levels of malaria transmission, being low in high altitude and low in transmission areas (p < 0.05). Four hundred ninety-one  of the children slept under LLIN in the previous night. Of malaria related knowledge items, only knowledge of LLIN was associated with net use; non-use of LLN was higher among caretakers who did not know the role of LLIN . Of course, attributing causation of malaria to stagnant water discouraged use of net (p = 0.021). Of febrile children (n = 122), only 50 (41.0%) sought care with only 17 (34.0%) seeking the care promptly. There was no significant link between knowledge of malaria and care seeking behavior (p > 0.05). However, knowledge of malaria had some level of influence on treatment source preference where caretakers with greater knowledge preferred pharmacy as source of care.The caretakers recognized malaria mostly by chills , fever  and headache . Overall, only 66.4% (471) of the caretakers knew that mosquito bite caused malaria and that it was quite heterogeneous by localities (ranging from 26.1% to 89.4%) and altitude (The findings demonstrated that caretakers’ understanding of malaria was unsatisfactory with marked heterogeneity by localities. The present evidence suggests that knowledge is not sufficient enough to drive LLIN use and care seeking. Yet, context-specific health education interventions are important besides ensuring access to necessary preventive tools. Malaria is one of the oldest mosquito-borne diseases . It is cIn accordance with the global malaria elimination program 2016–2030) [6–2030 1, 18, EthIn the context of elimination targets, it is essential to continuously monitor local understandings and perceptions that influence utilization of malaria preventive measures for evidence-based programing , 20. HowThe study was conducted mainly in sentinel sites established for malaria surveillance in Ethiopia , 27. Then = 763) to assess their perceptions of malaria. Hence, this analysis focused on caretakers’ perceptions of malaria and its preventive measures, and examined its association with use of LLIN and care seeking among children 2–9 years of age. In this case, caretakers were mostly parents (either mothers or fathers), and in few cases, sisters, brothers or other close relatives were presented as caretakers.The data were collected in June 2016 as part of a larger community based cross sectional study conducted to quantify malaria endemicity levels in Ethiopia. The larger study was proposed to involve 1040 children aged 2–9 years and finally, 763 children were enrolled in the study. As part of the study, the caretakers of each child were enrolled in the study (Study participants (i.e. caretakers accompanying their children) were sampled from ten PHCUs. The detailed sampling process was implemented as follows. The sample size (1040 children with their caretakers) was equally allocated to each PHCU. Additionally, two satellite health posts, which were part of the respective PHCU, were randomly selected. Equal number of children along with the respective caretakers was considered from each of the selected health post. Within the selected health posts, the list of households with eligible children (aged 2–9 years) was obtained from family the register. Simple random sampling was used to select eligible households using the list obtained from health post level. Then, caretakers were invited to participate in the study through local administrators, community volunteers and community based health workers, mainly health extension workers. Caretakers were invited to come to the nearest health facility . Upon arrival, caretakers were given detailed information about the study and signed written consent form in expression of agreement to participate to the study.A standard questionnaire, adapted from literatures, was used to assess caretakers’ knowledge on malaria and their children’s use of LLIN and care-seeking behaviors , 30. NinCaretakers provided information about experience of fever within two weeks recall period immediately preceding the survey. Prompt care-seeking behavior was defined as seeking care from health facilities within 24 h of onset of fever. In addition, caretakers were asked about LLIN ownership (i.e. proportion of households in the study with at least one LLIN) and LLIN use the previous night preceding the survey. Households who owned at least one LLIN for every two people of the household members were defined as having sufficient access to LLIN within the households.Plasmodium parasite; P.falciparum, P. vivax, P. ovale and P. malariaeduring all stages of infections process, and was performed as recommended by the manufacturer [Serological Markers (SM) of exposure to infections was used to define malaria transmission levels by localities. The test was based on the binding of specific antibodies present in the human plasma to antigens immobilized on a 96-well Enzyme Immuno Assay (EIA) plate. The test detects antibodies  to the four species of facturer . Detailefacturer . Based oThe data were collected through questionnaire interviewers administered by trained and experienced interviewers. The questionnaire was translated into the local language (Afan Oromo) and back translated into English to check for consistency and appropriateness. The Afan Oromo version was pre-tested in a similar setting. Data collection process was closely supervised by the investigators. Caretakers were interviewed at the nearest health facility .p-value less than 0.05 were used to declare statistically significant association.The data were analyzed using SPSS software package version 21.0. Descriptive statistics such as mean and frequency distribution were used to present the findings. Key findings were sorted by altitude, malaria transmission levels and study sites. Chi-square test was used to assess the association between knowledge of malaria with some selected factors such as study sites, altitude and malaria transmission levels. In addition, Spearman (r) test was used to examine the relationship between knowledge of causality and prevention strategies. One-way ANOVA was used to compare mean knowledge score by selected background factors. Logistic regression was used to assess the association between malaria related knowledge and use of LLIN/care-seeking behavior for fever. A 95% confidence interval and Overall, 778 caretakers were invited for participation, and finally, 709 91.3%) caretakers were enrolled in the study. Table .3% caretIn this study, 702 (99.0%) of the caretakers reported that they knew malaria. Table p = 0.001, 73.5% at low, 61.4% at mid and 64.9% at high altitude) and localities (p = 0.001). Likewise, knowledge of mosquito bite as route of malaria transmission was the highest in Metehara (89.4%), Bulbula (85.9%) and Goda Dhera (73.6%). In contrast, knowledge of mosquito bite was extremely low in some study sites such as Dembi (26.1%), Kersa (36.5%) and Shebe Sombo (43.9%). Knowledge of mosquito bite did not significantly vary by malaria transmission levels (p > 0.05). In fact, the proportion of caretakers who knew mosquito bite as route of malaria transmission was the highest in high malaria transmission areas  and the lowest in low transmission areas . In moderate transmission areas, 71.4% (152/213) of the caretakers were aware that mosquito bites cause malaria. Moreover, a considerable proportion of caretakers associated malaria with different conditions such as stagnant water (9.4%), poor sanitation and hygiene (9.4%), and cold weather (6.3%). The analysis revealed that twenty different misperceptions on causations of malaria were recorded even though some of these misconceptions were less frequent or mentioned by few caretakers (Table Two-third (66.4%) of the caretakers directly linked causation of malaria to mosquito bite with significant variations by altitude  of the caretakers mentioned that sleeping under insecticide treated mosquito net prevents malaria. In addition, valid malaria preventive strategies such as cleaning home surroundings and draining stagnant water were reported by 275 38.8%) and 113 (15.9%) of the caretakers, respectively. However, a significant proportion of caretakers also believed that practices such as using toilet, keeping personal hygiene, using clean water for drinking, use of onion, eating hot pepper, and not eating fruits such as mango, avocado, drinking bile etc. prevent malaria (Table 8.8% and p > 0.05) whereas knowledge of indoor residual spraying and environmental management significantly varied by altitude (p < 0.001). Consequently, the proportion of caretakers who knew that IRS prevents malaria was consistently low at all ranges of altitude. Of course, the difference was not significant; it was relatively better in areas of low altitude (13.5%). In contrast, the proportion of caretakers who knew environmental management as core malaria preventive measure was significantly the highest in mid altitude areas and the lowest in low altitude areas. Moreover, knowledge of these core malaria preventive measures were quite heterogeneous and significantly varied (p < 0.001) by localities. For instance, knowledge of LLIN was as low as 26.1% (in Dembi) and as high as 90.6%% (in Metehara)  and knowledge of IRS . However, knowledge of mosquito bite was negatively correlated with knowledge of environmental management . Likewise, knowledge of LLIN and environmental management was negatively correlated with each other . On the other hand, knowledge of IRS positively went with knowledge of LLIN , but negatively went with knowledge of environmental management .This study indicated that caretakers’ knowledge on causal factors and prevention rules of malaria was weak and incoherent. Knowledge of mosquito bite as a route of malaria transmission was moderately positively correlated with knowledge of LLIN (p < 0.005) higher in areas with high altitude. In low altitude areas, only 18.5% (51/275) and 56.0% (154/275) of the caretakers mentioned pregnant women and children under the age of five as vulnerable group to malaria, respectively. On the other hand, in mid altitude areas, 21.7% (78/360) and 55.0% (198/360) of the caretakers mentioned pregnant women and children under age five, respectively.Over half, 408 (57.5%), of the caretakers identified that children under the age of five were most vulnerable to acquire malaria infections. Nevertheless, only 158 (22.3%) of the caretakers mentioned that pregnant women were one of the vulnerable groups to malaria infections. Overall, only 128 (18.1%) of the caretakers mentioned that pregnant women and children under the age of five years were most at risk to malaria infections. On the other hand, 93 (13.1%) of the caretakers stated that they did not know specific groups at risk of or vulnerable to malaria infection. The proportion of caretakers who knew that pregnant women  and children under the age of five years  are vulnerable to malaria infection was significantly (P = 0.001). With respect to localities, the highest mean score was recorded in Goro  followed by Goda Dhera . The lowest mean knowledge score was documented in Dembi (mean = 29.5%) and Kersa (mean = 37.4%). Mean knowledge score was significantly low in low malaria transmission areas . Caretakers who resided in areas of moderate  and high  malaria transmission scored higher mean knowledge (p = 0.001). Moreover, mean knowledge scores were 52.7 , 49.2  and 55.9  in low, mid and high altitude areas, respectively (p = 0.019).Overall mean knowledge score was quite low  with significant variations by localities (p > 0.05). In fact, the rate of access to LLIN was the lowest (32.3%) in high malaria transmission areas whereas 42.7% and 44.1% of the households had sufficient LLIN in low and moderate transmission areas, respectively. Likewise, 43.6% (120/275) 39.4% (142/360) and 50.0% (37/74) of the households had sufficient access to LLIN in low, mid and high altitude areas, respectively.Six hundred fifty eight  of the caretakers reported that they had at least one LLIN in their houses. However, only 299 (42.2%) of the households had sufficient nets for every member of the household (i.e. 1 net for every 2 people). The rate of access to LLIN significantly varied by study sites (ranging from 13.0% to 65.2%). However, it did not significantly vary by altitude and level of malaria transmission (p = 0.003). On the other hand, net use was significantly lower among children whose caretakers directly attributed causation of malaria to stagnant water . However, knowledge of mosquito bite and overall knowledge of malaria did not have associations with net use among children (p > 0.05). Of course, even though significant association did not exist, increased overall knowledge aligned with increased probability of net use among children  of the caretakers reported that their children (aged 2–9 years old) slept under LLIN the previous night. Table p = 0.001), altitude (p = 0.001) and malaria transmission levels (p = 0.007). Accordingly, the prevalence varied from 0.0% (in Dembi) to 34.1% (in Metehara). And, in terms of altitude, the highest prevalence of fever was recorded in low altitude areas , followed by high altitude areas , and the lowest prevalence of fever was recorded in mid altitude areas . The prevalence of fever was also the highest in high malaria transmission areas (30.8%), followed by low transmission areas (16.7%). In moderate and low malaria transmission settings, the two weeks’ prevalence of fever was 14.1% (30/213) and 16.7% (72/431), respectively.The study indicated that the two weeks’ prevalence of fever was 17.2% (122/709) with very high heterogeneity by study sites (n = 122), only 50 (41.0%) sought care from any source, with no significant differences by study sites, altitude and malaria transmission levels (P > 0.05). Of those who sought care, only 17 (34.0%) sought the care promptly (within 24 h) after onset of fever, with no substantial variability by study sites, altitude and transmission levels (p > 0.05). The prevalence of prompt care-seeking was 33.3% (10/30), 35.3% (6/17) and 1 (33.3%) in low, mid and high altitude areas, respectively. Likewise, 37.0% (10/27), 42.9% (6/14), and 11.1% (1/11) sought prompt care in low, moderate and high transmission areas, respectively. Figure Of those children with fever ( only 50 1.0% sougp = 0.001). Similarly, care-seeking behavior was higher among households with no or insufficient LLIN as compared to households with sufficient LLINs .Table p < 0.05). Accordingly, the probability of using private clinic was increased with increased malaria endemicity levels whereas the probability of visiting government health facilities and pharmacies decreased as the malaria endemicity levels increased. In low transmission areas, 40.7% (7/27) of the caretakers consulted pharmacies and  visited government health facilities. Private clinics were consulted by 25.9% (7/27). In contrast, in high transmission areas, 66.7% (3/9) of the febrile children visited private clinics and the remaining visited government health facilities. In areas where malaria transmission was moderate, 50 (7/14), 42.9% (6/14) and 7.1% (1/14) of the febrile children visited government facilities, private clinics and pharmacies, respectively. Figure r = 0.759, p = 0.044). However, preference of private clinics and government health facilities did not have significant association with overall malaria knowledge of caretakers.More than third  of the persons who sought care consulted private clinics, and 32.0% 16/50) visited government health centers. Pharmacy and health post accounted for 24% (12/50) and 6% (3/50), respectively. Figure /50 visitn = 50), 29 (58.0%) said that their children were tested for blood examination. The proportion of subjects who received blood test was significantly higher in government health facilities  and in private clinics  (p = 0.001). And those who consulted pharmacy did not receive any test.Of those who reported visiting health facilities for fever  in the study population and it was close to the national target (i.e. 100% coverage) . The covUse of LLINs is one of the most recommended malaria vector control strategies, especially in tropical areas –56. ConsThis study indicated that caretakers’ care-seeking behavior for febrile illness of their children was very poor and low as compared to earlier studies , 65–70. This study might have some limitations. Overall knowledge was computed by summing up most important items to produce a composite score. Composite score is easier to interpret and facilitate understanding. However, it assumes that all items have equal contributions which may not be the case. Consequently, it does not allow for differentiation of high and low scores on a specific knowledge item. Nevertheless, item-based analysis was performed for most important items which could help to complement the limitation of using composite score. In addition, the findings of this study might have been affected by response bias due to the fact that caretakers were interviewed in health facilities. This is because facility based studies produce more positive responses by caretakers. Moreover, the data were collected mainly from sentinel surveillance sites in Ethiopia which may not represent all malaria transmission settings in the county.This study revealed that local understanding of malaria was unsatisfactory and very limited in some localities with arrays of misconceptions on causalities and prevention strategies. On the other hand, access to and use of LLIN were low and far behind set targets in the studied communities. Additionally, caretakers’ habit of prompt care-seeking for febrile illness of their children was not optimal. Furthermore, significant proportions of caretakers, especially in areas with high transmission, preferred private health facilities for treatment of malaria where the practice of test and treat is basically low. This further increases surveillance and monitoring difficulties which is a key in elimination program. Ideally, higher knowledge about a disease and its prevention practices  produces positive protective behavior. However, the evidence in this study depicted that only knowledge of the benefits of LLIN positively mediated use of LLIN. In fact, some beliefs such as attributing malaria to stagnant water discouraged use of LLIN. Moreover, there was no significant link between caretakers’ knowledge of malaria and care-seeking behavior, but level of knowledge may affect treatment source preference for malaria. Lack of proper connection between malaria related knowledge and desired malaria preventive practices suggests that knowledge was not sufficient to drive use of LLIN and care-seeking. This may suggest that caretakers’ knowledge may not be influential in terms of use of LLIN and care seeking at household level. Nevertheless, it is not possible to underestimate the role of knowledge in mediating malaria preventive behavior since, without adequate knowledge, people may be unaware of the health problems and the need to take preventive actions. Thus, despite lack of strong linkage between knowledge of malaria and use of LLIN and care-seeking, it important to build and maintain broader understandings of malaria and its preventive strategies  and the importance of prompt care-seeking. Thus, well-planned and locally sensitive ongoing public educational interventions are essential to educate and help communities to improve and sustain malaria related knowledge and desired protective behavior while addressing external factors such as access to LLIN.


Prenatal androgen exposure has been suggested to play a role in polycystic ovary syndrome. Given the limited information on what maternal characteristics influence maternal testosterone levels, and the even less explored routes by which female fetus androgen exposure would occur, the aim of this study was to investigate the impact of maternal age, BMI, weight gain, depressed mood and aromatase SNPs on testosterone levels in maternal serum and amniotic fluid of female fetuses.n = 216) obtained in gestational weeks 35–39, and pre-labor amniotic fluid samples from female fetuses (n = 56), taken at planned Caesarean section or in conjunction with amniotomy for induction of labor, were analyzed. Maternal serum testosterone and amniotic fluid testosterone and cortisol were measured by tandem mass spectrometry.Blood samples from pregnant women (P < 0.001), self-rated depression  and weight gain  were independent explanatory factors for the maternal total testosterone levels. Maternal age , weight gain  and amniotic fluid cortisol levels  were independent explanatory factors of amniotic fluid testosterone in female fetuses, explaining 64.3% of the variability in amniotic fluid testosterone.Multiparity (β = −0.28, Young maternal age and excessive maternal weight gain may increase the prenatal androgen exposure of female fetuses. Further studies are needed to explore this finding. The impact of prenatal testosterone exposure has been a focus of interest in the last decades. Testosterone plays an important role in the organization and sexual differentiation of the brain during early fetal development, and exposure to high levels of testosterone during critical periods of fetal life promotes behavioral masculinization in a variety of mammals . Most ofHowever, increased levels of testosterone during fetal life are also suggested as one of the potential causes of PCOS development. Sheep , monkeysMaternal serum testosterone concentrations increase by 70% during pregnancy , 9 and pCYP19A1), is about 130 kb long, and located on chromosome 15q21. CYP19A1 tissue-specific expression is regulated by the 5′ flanking region of the gene, which includes multiple tissue-specific promoters  cohort. The BASIC study investigates biological correlates of mood and anxiety disorders during pregnancy and in the postpartum period , 30, 31.BASIC participants with EPDS score ≥ 13 in gestational week 32, and a random sample of women with EPDS scores < 13 at gestational week 32 were invited for this sub-study to the BASIC project. The participants visited the research laboratory at the Department of Women’s and Children’s Health around gestational week 38  between January 2010 and May 2013. The visits were scheduled between 08:00 h and 15:00 h, with the majority starting either at 09:00 h or at 13:00 h. A detailed medical history was completed, and obstetric outcomes were later retrieved from the medical records. Information on maternal BMI and maternal weight gain was obtained from the standardized antenatal medical records. Weight gain was defined as the difference between weight in gestational week 36 and weight at first antenatal booking, and this information was only available in 150 women. A venous blood sample was drawn, which was immediately centrifuged and stored in −70°C. Out of a total of 234 women who participated in the study, serum for this study was available from 216 women.For the genetic analyses, non-Scandinavian ethnicity was an exclusion criterion. Genotyping was successfully completed in 201 women out of 215 women of Scandinavian ethnicity in study population 1.n = 20) and samples from male fetuses (n = 600), leaving 509 samples from female fetuses. In order to minimize the impact of labor stress (n = 56). Remaining samples were excluded because they had been obtained during labor. Caesarean section samples were taken in conjunction with the amniotomy, and all samples were immediately stored on dry ice and transferred to −70°C within one hour. Information on maternal and fetal characteristics was derived from BASIC questionnaires and medical records.By June 2015, 1129 amniotic fluid samples were available in the BASIC biobank. From these, we excluded samples from twin pregnancies  coupled with tandem mass spectrometry  ). The sample preparation (100 µL of serum or 500 µL of amniotic fluid) involves liquid extraction together with derivatization into methoxyamine prior to the analysis  . The anaanalysis . The qua2R) were 0.998 and 0.999 for testosterone and cortisol, respectively. The limits of quantifications were 0.17 nmol/L for testosterone and 0.14 nmol/L for cortisol.Duplicate analyses of each sample were performed and the average values were reported (CV <6%). The linearity of the method was evaluated over a range of concentrations (0.17–104 nmol/L for testosterone and 0.14–1379 nmol/L for cortisol) and correlation coefficients . Total coefficient of variation (CV) for SHBG was 3% at 38–109 nmol/L. Serum albumin was determined using BCP reagent from Abbott Diagnostics on an Architect 16000 (Abbott Laboratories). Total CV for serum albumin was 2% at 40.6–79.8 µmol/L. All analyses were performed at the accredited laboratory of Department of Clinical Chemistry, Uppsala University hospital. Two subjects were excluded due to insufficient sample volume. The Vermeulen method was used to estimate serum concentrations of bioavailable testosterone, by use of the Mazer spreadsheet , and calCYP19A1) rs28757184, rs56658716, rs2236722, rs700518 and rs6493497 single nucleotide polymorphisms (SNPs) were performed using the Kbioscience Allele-Specific Polymorphism assay (KASP) based on competitive allele-specific PCR and bi-allelic scoring of the SNP. No-template control samples were included to enable the detection of contamination or non-specific amplification. Genotype and allele frequencies are reported in Supplementary Table 1 (see section on r2) and potential haplotype blocks were estimated with the EM algorithm using SNP and Variation Suite 7 (GoldenHelix). The SNPs showed low linkage disequilibrium (r2) between each other, and no haplotypes blocks were detected.DNA was extracted from blood using the silica-based Kleargene DNA extraction method. Genotyping analyses of aromatase . Spearman correlations were used to assess the bivariate correlations, and group comparisons were made with the Kruskal–Wallis test or Mann–Whitney P values <0.05 were considered statistically significant.Multivariable linear regression analyses were performed to investigate the effect of age, BMI in early pregnancy, weight gain and depressive symptoms on total maternal testosterone levels. Natural logarithm transformed total testosterone levels were normally distributed and were used in the adjusted regression models to meet the assumption of randomly distributed residuals. Besides the variables of interest, inclusion of variables in the final models was based on significant associations in the bivariate analyses. Thus, the predictor variables for maternal testosterone included age, BMI, parity and EPDS scores in gestational week 32, with addition of gestational weight gain in a second model. Similarly, the predictor variables for amniotic fluid testosterone included maternal age, parity, BMI, gestational weight gain (dichotomized), assisted reproduction, gestational age and amniotic fluid cortisol levels. Gestational weight gain was dichotomized as above or below 1 standard deviation score (determined in the entire BASIC cohort), which equaled a weight gain > 18.9 kg at the end of pregnancy. Based on the literature, polycystic ovary syndrome was included in all models .Maternal total and bioavailable testosterone were significantly negatively correlated with maternal age, and positively correlated with maternal BMI at first antenatal visit, gestational weight gain and self-rated depression scores in gestational week 17 and 32 . PrimipaIn the multivariable linear regression models, maternal age and multiparity were independent negative explanatory variables of total testosterone, whereas BMI and self-rated depressive symptoms were independent positive explanatory variables . AdditioCYP19A1) gene in maternal blood DNA showed that rs700518 had borderline significant effect on maternal total testosterone levels . When considering the sex of the child, a similar pattern on total testosterone levels was observed in mothers of male children only, where there was a significant effect by rs700815 genotype on total testosterone levels . Mothers of male children, carrying the CC genotype, had higher levels than heterozygous CT genotype . None of the four other SNPs within the aromatase gene were associated with testosterone levels, data not shown.Genotyping analysis of the aromatase (Supplementary Table 2). In addition, the amniotic fluid testosterone was higher in women with a weight gain exceeding one S.D. from the cohort mean (4.96 nmol/L (IQR: 2.62–15.9) vs 1.96 nmol/L (IQR: 1.07–3.12), P < 0.05, Supplementary Table 2).According to the bivariate analyses, amniotic fluid testosterone was significantly negatively correlated with maternal age but positively correlated with gestational age and cortisol levels . SimilarIn the multivariable linear regression model maternal age, maternal weight gain and amniotic fluid cortisol levels remained independent explanatory variables of amniotic fluid testosterone, explaining 64.3% of the variability . The sigThe present study suggests that maternal weight gain, parity and depressed mood influence total testosterone levels in pregnant women. Additionally, in mothers carrying a male fetus, the rs700518 genotype for the aromatase enzyme had a significant effect on maternal testosterone levels. Finally, this study identified that a significant part of the variance in amniotic fluid testosterone levels of female fetuses can be explained by maternal age, maternal weight gain and fetal cortisol levels.de novo synthesis of testosterone. Meanwhile, as women with PCOS have higher testosterone levels during pregnancy, the complete suppression of ovarian activity during pregnancy may be questioned , The Medical Faculty of Uppsala University (CH), Swedish Research Council; 2015-4870 (JB), 2013-2339 (ISP), 523-2014-2342 (AS), 2015-00495 (EC) Marianne and Marcus Wallenberg Foundation (AS), The county council of Sörmland (OA), Marta Lundqvist Foundation  and Swedish Society of Medicine . E.C. is a Marie Skłodowska Curie fellow and received funds called the EU FP7-People-Cofund (INCA 600398).I S P, T K-K, A S, O A, C H and R C were involved in the conception and design of the study. I S P, T K-K, A S and C H participated in the acquisition of data. K U, J B, E C, L S-F, I S P, T K-K and C H did the data analyses and R C, A S and O A contributed to the interpretation. I S P, T K-K, L S-F and C H drafted the article, and all authors revised the manuscript critically for important intellectual content. All authors have approved the final version of the manuscript.


Gancao had no diuretic effect, but could impair Yuanhua’s diuretic effect through up-regulating renal AQP 2. Glycyrrhetinic acid (GRA) in Gancao could up-regulate AQP 2 and counteract the AQP 2 regulation effect of Yuanhuacine (YHC) and Ginkwanin (GKW) in Yuanhua. Network pharmacology method suggested that YHC, GKW and GRA could bind to MEK1/FGFR1 protein and influence ERK-MAPK pathway, which was verified by Western blotting. This study supports TCM theory and reminds that more attention should be paid to the safety and efficacy problems induced by improper combination between herbs. Moreover, we suggested that promising diuretics with less side effects can be developed from Chinese Medicines such as Yuanhua.As recorded in Traditional Chinese Medicine (TCM) theory, Gancao (Glycyrrhizae Radix et Rhizoma) could weaken the pharmacological effect or increase the toxicity of Yuanhua (Genkwa Flos). However, the theory has been suspected due to lack of evidence. Here, we investigate whether Gancao could weaken Yuanhua’s diuretic effect, if so, which chemicals and which targets may be involved. Results showed that Yuanhua exerted diuretic effect through down-regulating renal AQP 2, without electrolyte disturbances such as K The safety and efficacy problems are still the main barriers in modern Chinese Medicine development. Today, with the modernization of the Chinese herbal formulas4, it is hypothesized that the safety or efficacy problems can be induced by combination of different types of herbs. As warned by the theory of “eighteen incompatible medicaments” (EIM), the most representative cases of improper herbal combination in TCM, certain herbs could interact with each other and cause impaired curative effect or enhanced toxicity5. However, this theory has not been well elucidated, the evidences and mechanisms still needs further explanation. Yuanhua  and Gancao  are one of the herbal pairs, and the incompatibility between them has been questioned by some masters of TCM.The safety and efficacy of traditional Chinese Medicine (TCM) have been concerned and questioned excessively for many yearsDaphne genkwa Sieb. et Zucc. (Thymelaeaceae family), is a famous herbal in TCM since the age of Shennong’s Herbal Classics (Shen Nong Ben Cao Jing)6. It was described as a diuretic medicine, and was used to treat diseases caused by retained morbid fluid, such as hydrothorax, ascites, edema, asthma and arthritic9, although its diuretic activity still needs more detailed demonstration. Modern pharmacological researches have widely uncovered its anti-cancer11, anti-inflammation12, and anti-oxidant13 activities. At least two types of chemical compounds in YH are believed to be responsible for its activities: flavonoids and Daphnane diterpene esters15.YH, the dried flower buds of Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Batalin, or Glycyrrhiza glabra L. , is the most frequently used herbal medicine in TCM, composing most Chinese herbal formulas16. GC can cause water retention if taken at high dose (above the upper limit of the Chinese Pharmacopeia). This effect may partially explain why GC is incompatible with YH. However, TCM doctors will not use GC at such high dose.GC, made of dried roots and rhizomes of Since YH and GC have both been increasingly used or developed by doctors and scientists, there’s possibility that YH and GC could be used in combination. And more possibly, the chemical compounds of YH and GC could be mixed together by using other herbs, because chemicals in different herbs could be overlapped. Therefore, it’s also urgent to figure out the chemical essence of the incompatibility between YH and GC.In a word, this issue promoted us to investigate if GC could weaken YH’s diuretic efficacy under clinical doses, and if so, which chemical compounds could explain it, and what targets/pathways may be involved. The summary of this study has been showed in Fig. 17, and the 8-hour procedure was proved suitable for evaluating diuretic effect of YH.To monitor urine excretion of mouse, mouse were loaded with normal saline (NS) and weighted 0 to 8 hours after water-loading, the accumulated weight loss was calculated as diuresis index (DI) to reflect urine output. The water-loading diuresis model made it possible to monitor urine output in a convenient wayYH at high dose (YH-h) and low dose (YH-l) considerably increased DI between 2 to 6 hours, whereas hydrochlorothiazide (HCTZ) increased DI from 2 to 8 hours, which shows that they have diuretic effect. GC high dose (GC-h), GC low dose (GC-l), YH plus GC at high dose (YG-h) or low dose (YG-l) did not influence DI, which showed that although GC had no diuretic effect, but it can counteract the diuretic effect of YH Fig. .Figure 2To elucidate the key process involved in YH’s diuretic effect and GC’s interference, we investigated some key factors involving in urine formation, which is summarized in Supplementary Fig. P < 0.01). While YH, GC and YG did not influence Ccr  can increase Ccr compared with normal group (N). HCTZ considerably reduced Ccr, which showed that HCTZ can decrease GFR  and aldosterone (ALD) level, the main two hormones regulating body water and electrolytes balance, again with negative results , and transported to the membrane. During the transportation, phosphorylation or dephosphorylation process at Ser261 (p261-AQP 2) and Ser269 sites (p269-AQP 2) are also essential according to literature25. AVP and its receptor AVPV2R play critical roles in AQP 2 regulation27.The AQP proteins are membrane proteins mediating high-efficiency cross-membrane water transportAccording to DI curve, 4 hours after water loading is the best time point to further study the diuretic mechanism. Animals were sacrificed at this time point and kidneys of high dose groups were subjected to AQPs measurement by western blotting. As shown in Fig. in vitro.The next step of this study is to investigate chemicals related with the AQP 2 regulation activity in GC and YH. The chemical compounds in YH and GC are complicated, it is hard to survey how many compound can regulate renal AQP 2. To simplify the situation, we select the most abundant chemicals that can be detected in blood after administered YH and GC, and use mouse renal collecting duct cells mIMCD3 to evaluated their activity on AQP 2 expression 28, YHC is one of the most detectable diterpene-esters in animal plasma, and GKW, Apigenin (APG), Luteolin-5-O-glucoside (LUTG) are the most abundant flavones detectable in blood after oral administration of YH. GRA is considered the main active compound in GC, which is metabolized from Glycyrrhizic acid (GA) in the intestine before absorption after oral administration of GC29.According to our previous reportResults showed that YHC and GKW but not APG or LUTG dose-dependently decreased total AQP 2 expression in mIMCD3 cells Fig. . In contin-vivo study to confirm the diuresis activities of the two compounds as well as their regulation effects on renal total AQP 2 level. The designation basis of the doses were as follow: For YHC, since we have previously tested its toxicity on mice at 10 mg/Kg/day dose, so we designed 1, 0.5, and 0.1 mg/Kg/day dose in this study. And for GKW, in reference to the dose of HCTZ and Tolvaptan, 5 mg/Kg/day was used as the middle dose, and 20 and 1 mg/Kg/day were used as high/low doses respectively. GA was also used alone or combined with YHC and GKW, the dose was set at 100 mg/Kg/day according to literatures19. We choose GA rather than GRA because GA is the main form of triterpenoid saponin in GC, and it can be metabolized into the circulation form as GRA in the intestine. Results showed that YHC of 1 mg/Kg/day (YHC-h group) significantly promoted urine output from 1 to 4 hours after NS loading (P < 0.05); YHC of 0.5 mg/Kg/day (YHC-m group) only increased DI at the 4 hour point; and YHC of 0.1 mg/Kg/day (YHC-l group) did not change DI , while GA and GAYHC-m group increased AQP 2 levels. When compared to YHC-m or GKW-m group, GAYHC-m and GAGKW-m groups both increased AQP 2 levels  and FGFR1 are upstream factors in ERK-MAPK signaling pathway, and it is all connected with YHC, GKW and GRA. This pathway drew our attention since it is related to AQP 2 expression. As reported, AQP 2 expression in collecting duct is dominated by several pathways, the total targets include more than 15 signaling molecules  increased AQP 2 expression, while YHC (8 μM) and GKW (25 μM) both decreased AQP 2 expression. When ERK-MAPK signaling pathway was inhibited by U0126 (10 mΜ), AQP 2 protein expresion level also decreased, which proved the important role of ERK-MAPK signaling in AQP 2 regulation Fig. . However34. These researches have reminded medical scientists and clinical doctors to re-assess potential problems induced by herbal-herbal or herbal-drug interactions.With concerns about TCM safety and efficiency, the EIM researches have been widely conducted these years, aiming to uncover the mechanisms behind those incompatible herbal pairs, and providing clues for deeper understanding of TCM theories+ loss, which has been observed as side effect of most clinical diuretics including loop diuretics, thiazides and potassium-sparing diuretics. Among the crucial factors influencing urine excretion, renal Aquaporin (AQP) 2 protein was proved to be the key target in YH’s diuretic activity and GC’s counteraction effect. Among the multi-components in YH and GC, YHC, GKW and GRA can be detected in blood and can up or down-regulate AQP 2 expression in mIMCD3 cells. In water loading mice model, YHC and GKW can also promote urine excretion and down-regulate renal AQP 2, while GA can up-regulate AQP 2 and abolish the diuresis effect of YHC or GKW. Furthermore, network pharmacology strategy revealed that all three components can interact with proteins in ERK-MAPK pathway, which is involved in AQP 2 regulation. Western blot analysis further verified that p-ERK1/2 and its downstream p-CREB levels can be regulated by these compounds and further lead to changes in AQP 2 expression. In conclusion, at clinical related doses, GC could counteract YH’s diuretic effect through regulating renal AQP 2 protein expression. GRA (GA) in GC and YHC, GKW in YH are responsible for the AQP2 regulation effect, and ERK-MAPK signaling pathway is also involved.In this study, we focused on the diuretic effect of YH when administered with or without GC in diuretic mice model, where GC counteract YH’s diuretic effect. YH increased urine excretion without electrolyte disturbance such as K+ loss or hyperkalemia. Since these diseases are usually complicated with electrolyte disturbances, diuretics like furosemide may further worsen the electrolyte disturbances and have big limitations35. Recently, tolvaptan, a novel diuretic targeting AQP 2, has shown superior efficacy in treating ascites. However, it’s also related to hyponatremia and hepatotoxicity37. Therefore, the development of diuretics with high safety is urgently needed. In this study, our data shows that YH could exert diuretic effect through modulating AQP 2, but did not influence electrolyte reabsorption and therefore may not lead to electrolyte disturbances, which suggest YH shows advantage to the currently used diuretic drugs and may be developed as a valuable and promising herbal diuretic medicine.In clinical practice, doctors usually use diuretic medicines such as spironolactone, HCTZ and furosemide in treating edema, ascites, and hydrothorax. These diuretics target on urine ion reabsorption and may cause electrolyte disturbances such as K38, while diterpenes like YHC are relatively rare and its hepatotoxicity has been known. This study reveals that GKW and YHC both contribute to YH’s diuretic activity, and GKW may be a safer and more critical active compounds. With high safety and high content, GKW shows a light prospect in diuretic drug development. The renal AQPs have been studied in the diuresis mechanism of herbal diuretics like Fuling and Zhuling40. This study shows that AQPs may be a key target for the development of diuresis-related herbs. GA was also reported to up-regulate renal AQP 2 in vivo, and the effect was related to the mineralocorticoid receptor19, an ion reabsorption regulator. So it can be supposed that GRA could also regulate AQP 2 by osmotic pressure (hypertonicity) in vivo.Diterpene esters and flavones are two main type of active compounds in YH. GKW is one of the most abundant flavones in YH, with a content of up to 0.12% in crude drug42. Previously, we found that GA could accelerate the dissolving process of weak polar compounds from herbal pieces when YH and GC were boiled together, the acceleration would probably change the efficacy of YH43. In this study, YH and GC were extracted separately before combination, avoiding any interaction happened in boiling procedure. It is also noted that YH was extracted by ethanol, because YH can be taken in powder form in TCM, such as in the famous “Shizao decoction”. GC was extracted by water, in consistent with its application form. What’s more, the doses in the experiments were all designed according to the Chinese Pharmacopeia, making the study consistent with actual situations.Our research group have been focused on the study of YH and GC, seeking probable disadvantages caused by their combinationin-silico strategies. The simplification makes the study of high efficiency but many physiological evidences may be ignored, so lots of work still need to be done for other compounds and for different application situations, such as in tumor treatment, which have been implied by the network pharmacology data. What’s more, advanced research method can also be applied to discover deeper relationships between YH and GC based on the results of this study.The herbal-herbal interactions exist widely in TCM formulas, involving multi-compounds and multi-targets as well as complicated application situation. This study only focused on the traditional curative effect of YH, the diuresis effect, to study the disturbance of GC on YH, and the compounds studied in these herbs were also limited to relatively high content compounds. The related targets of these compounds were also screened and simplified by 45. In this study, involving compounds was complicated, and pathways were numerous. However, this method largely accelerated the screening of compounds and pathways, proves itself suitable for herbal-herbal or herbal-drug interactions research. Data showed more than 30 targets and more than 49 pathways probably involved in YH-GC interaction. Among the pathways, mainly two aspects stand out- several tumors and metabolism pathways. As for metabolism, GC’s influence on cytochrome P450 enzymes (CYPs) have drawn attentions from scientists46. And for tumors, according to Fig. 48 and can be found in collecting duct cells of adult kidneys49. FGFR1 and MEK1 are both part of ERK-MAPK pathway in collecting duct cells, this information is critical for us to link YHC, GKW or GRA with AQP 2 through ERK-MAPK pathway. Although YHC, GKW and GRA all target ERK-MAPK pathway, their regulations on p-ERK1/2 levels are entirely different. We did not measure all factor levels in this pathway, and the detailed mechanism still remains unknown, but literatures have indicate that GRA could up or down-regulate ERK-MAPK pathway in different situations51.The network pharmacology method has been increasingly engaged in TCM researches, due to its focuses on the multi-component and multi-target features of TCMFinally, we show the possibility in which impaired efficacy can be induced by specific combination of herbs. This possibility gives support to the EIM theory of TCM, indicating that when Yuanhua was used to treat diseases caused by retained morbid fluid including edema, hydrothorax, and ascites, Gancao should not be used in combination. The study reminds that more attention should be paid to the safety and efficacy problems induced by improper combination of modern drugs, natural products and herbs. Also, our work gives an example of systemic herbal-herbal interaction research, from whole body to organs, to cells and molecule targets, step by step. Moreover, it’s revealed that Yuanhua and its active component GKW shows a light prospect in diuretic drug development.Daphne genkwa Sieb. et Zucc.) and Glycyrrhizae Radix et Rhizoma , and the voucher specimen of YH (No. 110216) and GC (No. 110701) were deposited at the Herbarium in Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of Chinese Medicine. The UPLC-TOF-MS profile of YH was acceptable with several chemicals identified by the method established by our laboratory  and used in the experiments after 7 days of acclimatization. All experimental protocols including the care and handling of mice were approved by the Animal Ethics Committee of Center for Drug Safety Evaluation and Research of Nanjing University of Chinese Medicine . The methods were carried out in accordance with the Guiding Principles for the Care and Use of Experimental Animals from the committee in accordance with the internationally accepted standard guidelines for use of animals. The mice were kept in a temperature controlled environment (22 ± 2 °C), 55% ± 5% relative humidity with a 12-h: 12-h light-dark cycle and fed with standard chow.ICR mice were divided randomly into 9 groups with equal number (n = 15): The normal group (N), model group , HCTZ group, YH high dose group (YH-h), YH low dose group (YH-l), GC high dose group (GC-h), GC low dose group (GC-l), YH high dose plus GC high dose group (YG-h) and YH low dose plus GC low dose group (YG-l). The N and M groups of mice were orally administrated by syringe feeding with NS , the other groups were orally administrated with HCTZ (7.5 mg/kg/day), YH high or low dose (0.28 or 0.14 g crude drug/kg/day), GC high or low dose (1.5 or 0.75 g crude drug/kg/day), YG high or low dose. All high doses were equal to the upper limits of the Chinese Pharmacopeia. HCTZ was used as the reference drug to verify the diuretic activity tests. It was suspended in NS prior to administration. In another independent experiment, animals were treated like above, and YHC of 1, 0.5 and 0.1 mg/Kg/day doses  as well as GKW of 20, 5, and 1 mg/Kg/day doses  were examined in the model. HCTZ (7.5 mg/kg/day), GA (100 mg/kg/day) and its combination with YHC-m and GKW-m doses were also examined for comparison.53. Drinking water was revoked and the mice were immediately weighted every hour for 8 hours. Mice were placed individually in a small stainless-steel cage on a glass dish and urine was gathered and combined every 10 minutes. In one experiment, at the time point of 4-hour after water-loading, animals were sacrificed and plasma was collected. High dose groups of YH, GC, YG and the N, M, HCTZ groups, as well as YHC, GKW and GA groups were selected, and animal kidneys were removed and cut into pieces, snap frozen in liquid nitrogen and kept at −80 °C.The administration of drugs was implemented once a day for 3 days. On the fourth day all mice except for N group were weighted and intraperitoneal injected with NS of 15% body weight54. Total amount of secreted electrolytes were also standardized with urine Cr. Given that bodies generate and excrete Cr at a relatively constant speed, the total amount of excreted urine Cr is relatively constant. So the amount of urine electrolytes excretion can be standardized by Cr excretion amount. Data was calculated as electrolytes/Cr concentration ratio56. We adopt plasma Cr clearance (Ccr) as estimated GFR57, the Ccr was calculated as urine Cr multiplied with urine volume and divided with plasma Cr.After water-loading, mice urinated, and their weight loss were measured every hour, DI representing urine output for each mouse was calculated as the percentage of weight loss in total loaded water. The data were expressed as mean with standard deviation for each group. Another parameter, urine osmolality was measured to reflect urine concentrating ability, and additionally, UCF was calculated to indicate the water reabsorption percentage. Based on the fact that plasma Cr is filtered to the primary urine without a concentration change, then the primary urine is reabsorbed without Cr reabsorption, so the UCF can be calculated as urine/plasma Cr concentration ratio+, K+, Cl− concentration were measured with automatic biochemical analyzer . Reagent kits for Cl− concentration was based on Mercury Thiocyanate method; the Cr, Na+ and K+ concentration were based on enzyme methods, and were used according to the manuals. Urine osmolality was measured by Osmometer .Plasma AVP and ALD were measured with ELISA kits, all the kits were obtained from Sangon Biotech Shanghai Co., Ltd. (China).Urine Cr, plasma Cr and urine Na2 and 95% air. Before treatment, the cells were planted into 6 well plates until adhering to the bottom. Then culture medium was replaced and drug containing medium was added, incubating for 24 hours and cell number was multiplied. After rinsing with phosphate buffered saline for 3 times, cells were kept in −80 °C refrigerator.The mIMCD3 cells (mouse inner medullary collecting duct cells) were purchased from ATCC, cultured with RPMI 1640 medium containing 10% fetal bovine serum, incubated in 37 °C provided with 5% CO30. (http://59.78.96.61/pharmmapper/index.php), target set was limited in human proteins, and the other parameters were kept as default. The top 30 targets with higher statistical “Fit” number was chosen, further, their Uniprot accession number were submitted to Molecule Annotation System  to search possible pathways against KEGG database in human spices31. In final results, the top 30 possible pathways with P value less than 0.01 was showed by Cytoscape software (version 3.3.0).Three dimensional confirmation file of YHC, GKW and GRA were downloaded at the site of PubChem Compound (NCBI), with SDF file format, their CID numbers are shown above. These files were imported into PharmMapper Server58, and the receptors were downloaded from RSCB PDB database (http://www.rcsb.org/pdb/home/home.do), with ID number of “1s9j” for MEK1 protein and “3c4f” for FGFR1 protein. The receptors were prepared by deleting water and ligand, adding polar Hydrogens and “Kollman” charges with AutoDockTools (version 1.5.6). Docking was completed in AutoDock Vina59, parameters were all kept as default according to software manuals.Next, molecular docking study was conducted to show how small molecules interact with selected targets. The ligand molecule file (MOL2 file) for YHC, GKW and GRA were downloaded from TCMSP databaseFor each kidney sample, about 100 mg tissue was extracted with RIPA lysis-buffer. For mIMCD3 cells, 150 μL RIPA was added per well. Protein concentrations were measured by Bradford protein assay, and about 50 μg proteins were loaded on a 12% or 15% of SDS-polyacrylamide gel for separation and then transferred onto polyvinylidene fluoride membrane. Membranes were blocked with 5% bovine serum albumin in a solution  for 2 hours, then incubated overnight at 4 °C with primary antibodies. After eight 5-min washing with TBST, the blots were incubated with secondary antibody at room temperature for 1 hour. Washing as above, the targeted band was visualized using the enhanced chemi-luminescence system. Polyclonal antibodies to AQP 1 and 3 were obtained from Santa Cruz Biotechnology (USA), polyclonal antibodies to AQP 2 phospho S269 was obtained from GeneTex (USA), polyclonal antibodies to AQP 2 phospho S256 and phospho S261, AQP 4 and AVPV2R, were from Abcam (USA), and polyclonal antibody to AQP 2, ERK1/2, p-ERK1/2, CREB, p-CREB and β-tubulin was purchased from Cell Signaling Technology (USA). Secondary antibody was from Proteintech (USA).P < 0.05 was considered as statistically significant, and when P < 0.01, the P value is claimed in the main text. The blotting bands were visualized and quantifed via densitometry using ChemiDoc XRS+ system and inner software. The bar chart and blotting images are combined with GraphPad Prism 6 software. Schematics are drawn by Adobe Illustrator software.All data is expressed as means ± standard deviation. After confirming the normality of the data, statistical significance of differences was evaluated by one-way analysis of variance (ANOVA) followed by Dunnett’s T3 test (SPSS11.5). The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.Supplementary Information


Particulate matter (PM) modulates the expression of autophagy; however, the role of selective autophagy by PM remains unclear. The objective of this study was to determine the underlying mechanisms in protein oxidation and degradation caused by PM. Human epithelial A549 cells were exposed to diesel exhaust particles (DEPs), urban dust (UD), and carbon black . Cell survival and proliferation were significantly reduced by DEPs and UD in A549 cells. First, benzo(a)pyrene diolepoxide (BPDE) protein adduct was caused by DEPs at 150 μg/ml. Methionine oxidation (MetO) of human albumin proteins was induced by DEPs, UD, and CB; however, the protein repair mechanism that converts MetO back to methionine by methionine sulfoxide reductases A (MSRA) and B3 (MSRB3) was activated by DEPs and inhibited by UD, suggesting that oxidized protein was accumulating in cells. As to the degradation of oxidized proteins, proteasome and autophagy activation was induced by CB with ubiquitin accumulation, whereas proteasome and autophagy activation was induced by DEPs without ubiquitin accumulation. The results suggest that CB-induced protein degradation may be via an ubiquitin-dependent autophagy pathway, whereas DEP-induced protein degradation may be via an ubiquitin-independent autophagy pathway. A distinct proteotoxic effect may depend on the physicochemistry of PM. Air pollution consists of numerous reactive oxygen species (ROS), which are associated with the development of cardiopulmonary disease24S-MetO and R-MetO. The peptide MetO enzymes, methionine sulfoxide reductase A (MSRA) and MSRB, are able to catalytically reverse S-MetO and R-MetO, respectively, allowing maintenance of the protein’s function in some casesNotably, ROS can damage surrounding tissues and result in the formation of highly reactive organic molecules, which are able to non-enzymatically modify proteins and that target specific peptide residues7Approximately 90% of damaged proteins are degraded into small peptides by the ubiquitin-proteasome pathway14Beside the ubiquitin-proteasome pathway, autophagy is also an essential pathway to degrade oxidized proteins. Autophagy is a regulated cellular mechanism for degrading proteins that is mediated by lysosomal-dependent processing. The autophagosome then fuses with and delivers its contents to the lysosome. Lysosomal enzymes subsequently facilitate degradation to regenerate metabolic precursor molecules. Increasing numbers of studies have demonstrated that autophagy expression is associated with air pollution16The objectives of this study were: (1) to discover the potency of protein oxidation caused by different types of particulate air pollution; (2) to investigate the MSR repair system for methionine oxidation caused by particles; (3) to understand the regulation of ubiquitin-proteasome pathways after particle exposure; and (4) to examine selective autophagy mediated by particles.The SRM of DEPs (representing diesel-related traffic particles) and UD (representing ambient urban particles) were used to examine protein oxidation and degradation in A549 cells, whereas CB (nearly pure carbon element) was used in this study as a negative control. Spherical CB and DEP and amorphous UD aggregates were the dominant form observed by FE-SEM . The sizp < 0.05) (p < 0.05), particularly DEPs. The results show that these particles activated ERK phosphorylation with 24 h of exposure, leading to apoptosis. The survival and proliferation of A549 cells after 20 μg/ml CB, DEP, and UD exposure were further assessed using a colony formation assay. As shown in p < 0.05). Notably, the growth rate of DEP-treated A549 cells was slower than those treated with CB and UD.To investigate the particle toxicity among CB, DEPs, and UD, cell viability, p-ERK1/2, and colony formation were determined in A549 cells. First, we found that a significant reduction of cell viability occurred with DEPs at 50 and 150 μg/ml and UD at 150 μg/ml ( < 0.05) . There wp < 0.05), whereas there was no significant alteration in BPDE protein oxidation caused by CB or UD. Methionine oxidation caused by the particles was examined because methionine can reduce BPDE oxidation. Significant oxidation of five peptides was observed for CB (82% for LVRPEVDVMCTAFHDNEETFLK and 83% for SHCIAEVENDEMPADLPSLAADFVESK), DEPs , and UD  compared to the HSA control (p < 0.05). Distinct effects of CB, DEPs, and UD on MSRA and MSRB were observed in A549 cells (p < 0.05), but UD significantly reduced MSRA and MSRB levels after exposure (p < 0.05). Exposure of A549 cells to CB had no significant effects on MSRA or MSRB3 alterations. We observed that activation of MSRA and MSRB production occurred with DEPs, whereas MSRA and MSRB3 levels were suppressed by UD. These results indicated that DEPs and UD had distinct effects on the protein repair mechanism of converting MetO back to methionine by MSRA and MSRB3.Protein oxidation caused by CB, DEPs, and UD was first assessed by BPDE protein oxidation . The BPD49 cells . MSRA anp < 0.05), particularly DEPs. Interestingly, we observed that CB significantly increased ubiquitin expression compared to the control (p < 0.05), whereas LC3B I to II conversion significantly occurred by DEP exposure at 150 μg/ml (p < 0.05). There was no significant effect of UD on LC3B conversion.To determine if protein degradation occurred after exposure to these particles, proteasomes and ubiquitin were first investigated.  control , whereas control . We obseProtein oxidation in the lung environment has been linked to several lung diseases such as chronic obstructive pulmonary disease (COPD), which is considered to play an important role in regulating the pathophysiology of lung diseases19Ambient PM presents unique and often distinct physicochemical properties, as the physiochemical properties change from generation to secondary formation/interactions, which contribute to particle toxicity. In the present study, three types of PM were used to determine the potency of protein oxidation and degradation, and these were also used for a mechanical studyAlterations of cell survival and proliferation caused by CB, DEPs, and UD were first examined. We observed that DEPs had higher particle toxicity than CB or UD, which is consistent with a previous report27S-MetO and MSRB which reduces R-MetOROS are reactive molecular species with an unpaired electron in their outer orbitNext, we investigated regulation of protein degradation pathways when an overload of oxidized protein accumulated in cells caused by the particles. Biologically, intracellular proteins are mainly degraded by the ubiquitin-proteasome pathwayUpon encountering oxidative stress, proteins are extensively oxidized by ROS, and these oxidized proteins are degraded by proteasome and autophagic proteolytic systems for bulk degradation. Deng and colleagues showed that ambient PM regulated the autophagy-related proteins, Atg5 and Beclin 1, which are associated with particle-driven oxidative stressNotably, taken together with the expression of ubiquitin, we observed that CB exposure was associated with ubiquitin-dependent autophagy, whereas DEPs were associated with ubiquitin-independent autophagy even at the lowest concentration. The effects of lower dose of DEP could be represented in a real-world exposure. Oxidized proteins are tagged with ubiquitin signals that are subsequently sensed by ubiquitin binding domain-containing receptors to deliver them to proteasomes or autophagosomes during autophagyThis study showed a proteotoxic effect, of regulation by the MSR repair system, the proteasome-ubiquitin pathway, and selective autophagy, after exposure of A549 cells to CB, DEPs, and UD. Based on our findings, we propose the distinct proteotoxicity of CB, DEPs, and UD. The hypothetical pathways of protein oxidation and degradation in response to exposure to CB, DEP, and UD are shown in Diesel exhaust particles (DEPs) and urban dust (UD) as Standard Reference Materials (SRM) 2975 and 1649b, respectively, were obtained from the National Institute of Standards and Technology . Carbon black (CB), with an average diameter of 65 nm , was selected as a control particle. All of the other reagents were obtained from Sigma  if a source is not explicitly stated.Inspect™ FE-SEM  and an EDX microanalysis were used to investigate morphological and elemental features of the particles. Preparation of the FE-SEM samples was previously described5 cells/ml and incubated for 24 h. Cells were cultured in RPMI medium containing 10% fetal bovine serum, penicillin, and streptomycin and were incubated at 37 °C with 95% humidity and 5% CO2. Particles were freshly prepared in serum-free RPMI medium at mass concentrations of 0, 20, 50, and 150 μg/ml. Cells were then incubated at 37 °C for 24 h in a humidified atmosphere with 5% CO2.Human lung epithelial A549 cells obtained from the American Type Culture Collection  were seeded in surface-treated, 96-well transwells  at a density of 10A sulforhodamine B (SRB) colorimetric assay was used to examine cell viability according a previous methodThe protocol of the colony formation assay was described previouslyhttp://www.matrixscience.com). The search parameters specified variable modification for deamidation (NQ), oxidation (M), and methylation (K) and a fixed modification for carbamidomethylation (C). The maximum mass tolerance was set to 10 ppm for precursor ions and 0.05 Da for fragmented ions. The degree of oxidation was calculated based on the peak area [ × 100%]. The Swiss-Pdb Viewer vers. 4.1.0  was used to analyze the peptides oxidized in HSA oxidized by the particlesMethionine oxidation by particles was previously reported10g and 2 °C. Supernatants were collected to determine phosphorylated extracellular signal-regulated kinase (ERK)1/2 (p-ERK1/2) , benzo(a)pyrene diolepoxide (BPDE) protein adduct , MSRA , and MSRB3 (Uscn Life Science) and proteasome activity (Abcam) using an ELISA according to the manufacturer’s instructions.Cells were collected form the plates and stored at −20 °C overnight in 1x PBS after particle exposure. After two freeze-thaw cycles to break up cell membranes, cell lysates were centrifuged at 5000 xWestern blot analyses were performed as described previouslyp < 0.05.All experiments were performed in quintuplicate. Data are expressed as the mean ± standard deviation (SD). A one-way analysis of variance (ANOVA) with Tukey’s post-hoc test was used for comparisons among multiple values. Statistical analyses were performed using GraphPad vers. 5 for Windows. The level of significance was set to How to cite this article: Lai, C.-H. et al. Protein oxidation and degradation caused by particulate matter. Sci. Rep. 6, 33727; doi: 10.1038/srep33727 (2016).


This study aims to estimate the association between visual impairment and low vision and sleep duration and poor sleep quality in a national sample of older adults in South Africa. A national population-based cross-sectional Study of Global Ageing and Adults Health (SAGE) wave 1 was conducted in 2008 with a sample of 3840 individuals aged 50 years or older in South Africa. The interviewer-administered questionnaire assessed socio-demographic characteristics, health variables, sleep duration, quality, visual impairment, and vision. Results indicate that 10.0% of the sample reported short sleep duration (≤5 h), 46.6% long sleep (≥9 h), 9.3% poor sleep quality, 8.4% self-reported and visual impairment (near and/or far vision); and 43.2% measured low vision (near and/or far vision)  and 7.5% low vision . In fully adjusted logistic regression models, self-reported visual impairment was associated with short sleep duration and poor sleep quality, separately and together. Low vision was only associated with long sleep duration and poor sleep quality in unadjusted models. Self-reported visual impairment was related to both short sleep duration and poor sleep quality. Population data on sleep patterns may want to include visual impairment measures. SimilarOnly few studies investigated the correlation between visual impairment and sleep duration and poor sleep quality. In a national survey in the USA, visual impairment was related to both short (<6 h) and long >8 h) sleep durations . Among K h sleep To our knowledge, there have been no large population-based studies assessing the association between visual impairment and sleep duration and poor sleep quality in Africa. Our hypothesis is that visual impairment is associated with both short and long sleep durations and with poor sleep quality. The aim of this study was to investigate the association between visual impairment, low vision, sleep duration and poor sleep quality in a nationally representative sample of older South Africans., multi-ancestry (Coloured), Indian or Asian African, and White African). At the household level, trained nurses using a structured questionnaire interviewed all persons aged 50 years and older and health measurements were conducted; proxy respondents were identified for selected persons who were unable to complete the interview [A cross-sectional population-based “Study of Global Ageing and Adult Health (SAGE)” involving a sample of 3840 persons (aged 50 years or older) was conducted in South Africa in 2008. A two-staged probability sampling strategy was used in order to provide precise national and sub-national estimates across strata, i.e., provincial level, geographic type , and population group  Far vision: “In the last 30 days, how much difficulty did you have in seeing and recognizing a person you know across the road ?” . (2) NeaUsing a tumbling “E” log MAR chart, visual acuity was assessed for both near and far vision in each eye . MeasureTwo questions were posed about self-reported hours of sleep the previous night and the night before the previous night: “How many hours (and minutes) did you sleep?” . The nocPoor sleep quality was assessed based on the question: “Overall in the last 30 days, how much of a problem did you have with sleeping, such as falling asleep, waking up frequently during the night or waking up too early in the morning?” (Response options ranged from 1 = none to 5 = extreme/cannot do) . Poor slEducational Level was assessed by asking participants, “How many years of school, including higher education, have you completed?” The response option was the number of years.Economic or Wealth Status of a given household was estimated based on a list of household assets, and subsequently, wealth quintiles were created from these . Chronic Diseases—such as angina, diabetes, and stroke—were measured by self-report, as having been diagnosed by a health care provider. Obesity, defined as having a body mass index (BMI) of 30.0 or higher kg divided by height metre squared , was meaBlood pressure (systolic and diastolic) was assessed “three times on the right arm/wrist of the seated respondent using an automated recording device ” . Of the The World Mental Health Survey version of the “Composite International Diagnostic Interview” was used to assess symptom-based depression in the past 12 months . The depThe “World Health Organization (WHO) Disability Assessment Schedule (WHODAS-II)” was used to assess health, functioning, and disability in the past 30 days . To estip-values of less than 5% were used to indicate statistical significance. Both the reported p-values and the 95% confidence intervals are adjusted for the complex sample design of the study.Using STATA software version 13.0  data were analysed taking into account the complex sampling design. Pearson correlation was administered to assess correlations between the different vision measures. Associations between confounding variables  ,8 visualp < 0.001) and low vision   , 44.1% men and 55.9% women and the most prominent population group was Back African (74.0%). Ten percent of the sample reported short sleep duration (≤5 h), 46.6% long sleep (≥9 h), 9.3% poor sleep quality, 8.4% self-reported visual impairment (near and/or far vision), 43.2% low vision (near and/or far vision) , and 7.5% low vision (near and/or far vision) . The proportion of poor sleep quality, visual impairment, and low vision was higher in older individuals, women and those with lower education. There was a correlation between visual impairment and low vision   see .In fully adjusted logistic regression models, visual impairment was associated with short sleep duration and poor sleep quality, separately and together. Low vision  was only in an unadjusted model associated with long sleep and low vision (0.01–0.125) was only in an unadjusted model associated with poor sleep quality see . FurtherThis study examined the association between visual impairment, low vision, sleep duration, and poor sleep quality in a nationally representative sample of older South Africans. Previous studies concentrated on studying these variables in high-income countries ; the studies found that visual impairment was associated with the prevalence of short and long sleep duration and poor sleep quality ,9,10,11.This study also had limitations. Since this was a cross-sectional study, no causative conclusions can be drawn between independent and outcome variables. Certain health variables such as sleep duration, certain chronic conditions, and visual impairment were assessed by self-report. However, Klein et al.  found a This study showed that self-reported visual impairment was associated with short sleep duration and poor sleep quality in a sample of older adults in South Africa. Health care providers should be aware of the possibility of sleep disorders in patients with visual impairment.


JAK2, c.1849G > T , leading to constitutive activation of JAK-STAT signaling pathway. Familial clustering is also observed occasionally, but high-penetrance predisposition genes to PV have remained unidentified.Polycythemia vera (PV), characterized by massive production of erythrocytes, is one of the myeloproliferative neoplasms. Most patients carry a somatic gain-of-function mutation in ZXDC, c.1931C > G (p.Pro644Arg) in ATN1, and c.701G > A (p.Arg234Gln) in LRRC3. We also observed a rare, predicted benign germline variant c.2912C > G (p.Ala971Gly) in BCORL1 in all four patients. Somatic mutations in BCORL1 have been reported in myeloid malignancies. We further screened the variants in eight PV patients in six other Finnish families, but no other carriers were found.We studied the predisposition to PV by exome sequencing (three cases) in a Finnish PV family with four patients. The 12 shared variants  predicted damaging in silico and absent in an additional control set of over 500 Finns were further validated by Sanger sequencing in a fourth affected family member. Three novel predisposition candidate variants were identified: c.1254C > G (p.Phe418Leu) in Exome sequencing provides a powerful tool for the identification of novel variants, and understanding the familial predisposition of diseases. This is the first report on Finnish familial PV cases, and we identified three novel candidate variants that may predispose to the disease.The online version of this article (doi:10.1186/s40246-017-0102-x) contains supplementary material, which is available to authorized users. Myeloproliferative neoplasms (MPNs) are a group of hematological malignancies with enhanced proliferation of myeloid cells due to an acquired mutation of a single hematopoietic stem cell (HSC) resulting in clonal progeny , 2. ExpaJAK2), c.1849G > T   were excluded. To exclude common variants, we further filtered the variant set against the Exome Aggregation Consortium  data , settingBCORL1 cDNA were validated by direct Sanger sequencing. Also, the predisposing germline variant rs10974944 in JAK2 was checked. DNA was extracted from FFPE blocks with a standard phenol-chloroform method or with NucleoSpin® DNA FFPE XS kit . Primers were designed using Primer3Plus software (http://www.bioinformatics.nl/primer3plus). The DreamTaq™ DNA Polymerase  or Invitrogen™, Platinum™, SuperFi™ DNA Polymerase (Thermo Scientific) were used in PCR reactions, and PCR products were purified with A’SAP PCR clean-up method . BigDye Terminator v3.1 sequencing reaction was used in the DNA sequencing, and capillary electrophoresis was performed on an ABI3730xl DNA Analyzer  at the Institute for Molecular Medicine Finland (FIMM). The results were analyzed manually using FinchTV v.1.4.0 .The shared variants found in the exome data and For cDNA sequencing, RNA was extracted from index case’s whole blood sample with NucleoSpin® RNA kit (Macherey-Nagel), and reverse-transcripted to cDNA with Promega M-MLV Reverse Transcriptase (Thermo Scientific) according to manufacturers’ protocols.Here, we have studied a Finnish family with four PV cases with the aim of identifying novel PV-predisposing germline variants. Germline DNA exomes from three affected family members and the peripheral blood DNA exome of the index case were sequenced. The average coverage at each base was 58 reads and 88% of the captured regions had a minimum coverage of four reads. We filtered the called variants based on their predicted, damaging effect on the gene product, and their presence in an in-house control set of 542 Finns. We removed the variants that occurred in any of the 542 Finnish controls , leaving us with 12 shared variants; 1 splice site and 11 possibly pathogenic missense variants (Table ZXDC (ENST00000389709); c.1931C > G (p.Pro644Arg) in ATN1 (ENST00000356654); and rs148872771, c.701G > A (p.Arg234Gln) in LRRC3 (ENST00000291592). We screened the three variants in lymphoma patient 1.19, who carried the variant in LRRC3. Also, we checked for the three variants in the germlines of eight PV patients from six other Finnish families, but the variants were not observed. It may still be possible, however, that some of the three variants or other variants in these genes may play a role in PV development. We identified also one rare benign  missense SNP, rs144332650, c.2912C > G (p.Ala971Gly), and in BCORL1 (ENST00000540052) in all four PV patients in the family. Mutations in BCORL1 have been associated with the leukemogenic process in AML [From these variants predicted as damaging, 1.10 carried three rare single-nucleotide variants (SNVs): c.1254C > G (p.Phe418Leu) in s in AML –32. PV ptrans-activator (CIITA), which leads to CIITA binding to promoter elements involved in constitutive MHC class II expression [ZXDC may contribute to carcinogenesis and malignant progression of tumors by participating in the suppression of MHC class II genes. The mutated site of ZXDC identified in our study is the first amino acid of the ninth zinc finger repeat. Zinc fingers are necessary for full activity of cooperation with CIITA [ZXDC gene function is unknown. ZXDC is enriched in myeloid lineages and has been observed to regulate transcription of key genes during myeloid cell differentiation [http://nar.oxfordjournals.org/content/early/2015/10/26/nar.gkv1101.abstract, www.proteinatlas.org). Solely based on gene function, ZXDC variant would be the most attractive predisposition candidate of the three, but further studies are warranted.Zinc-finger X-linked duplicated family member C (ZXDC) belongs to the ZXD family of transcription factors, which has been observed to regulate transcription of major histocompatibility complex (MHC) class I and II genes in antigen presenting cells . In addipression , 34. By pression . Thus, dth CIITA . Beyond ntiation . During www.proteinatlas.org). ATN1 contains glutamine-repeats, and two arginine-glutamic acid dipeptide-like repeats (RE-repeats) [Atrophin-1 (ATN1) is a nuclear transcriptional corepressor, and aberrant form of ATN1 is associated with neurodegenerative diseases such as dentatorubral-pallidoluysian atrophy (DRPLA), and cancer in humans . It is urepeats) . In neurrepeats) . In normwww.proteinatlas.org). One lymphoma patient of the family with DNA available carried the rare variant rs148872771 in LRRC3, too, which may indicate the particular variant not being responsible for PV predisposition exclusively.Little is known about the function of Leucine-rich repeat containing 3 (LRRC3) gene product, but it is widely expressed in different tissues, including the bone marrow. Most malignancies display moderate cytoplasmic-positive staining, and the strongest expression is observed in colorectal cancers , whereas 1.9 was heterozygous (CG genotype). Also, all eight PV patients from the six other families carried the risk variant: two of them were heterozygous, and six were homozygous. The SNP in TERT, rs2736100, was also checked from the patients. All the PV patients in the studied family were homozygous for the risk variant (CC).The SNP in ATG2B and GSKIP has been shown to predispose to familial myeloid malignancies [Germline duplication of gnancies . A possiJAK2V617F, in index case’s blood sample. Loss of heterozygosity (LOH) in the 12 damaging gene variants detected by exome sequencing was looked for in the index case’s blood sample, but only the variants c.582A > G (p.Ile194Met) in SLC24A2 and c.3929C > T  in CSMD1, in addition to JAK2V617F, showed clear LOH. In known MPN-associated genes, the index case carried two missense variants, c.680C > T (p.Thr227Met) in FLT3 and c.5162 T > G (p.Leu1721Trp) in TET2 (Illumina TruSight® Myeloid Sequencing Panel), predicted as possibly damaging by PolyPhen-2 and SIFT. In addition, we identified possibly damaging missense variants c.3263C > T (p.Ser1088Phe) and c.1235C > T  in FANCA, which is one of the genes associated with other myeloid malignancies [We identified the most frequent somatic variation in PV patients, gnancies .BCORL1 gene is located on the X chromosome. The index case being a woman, we checked which of the alleles was expressed; the BCORL1 variant or the wild-type. By Sanger sequencing the cDNA, we identified the expression of BCORL1 variant.Identification of predisposing genes and mutations is important for families with PV susceptibility and acknowledging family history is essential in order to unveil the genetic background. New hereditary gene defects may lead to screening and genetic counseling of family members and may improve the diagnosis and treatment thus affecting the quality of life. Also, the identification of specific gene mutations gives the possibility to screen individuals at higher risk. This is the first report on Finnish familial PV cases, and we identified three candidate predisposition variants by exome-sequencing. We would like to present these genes as candidates for PV susceptibility and for further validation by the research community.