Aedes aegypti is found in close association with human habitations, often leading to high population densities of mosquitoes in urban settings. In the Peruvian Amazon, this vector has been expanding to rural communities over the last 10–15 years, but to date, the population genetic structure of Ae. aegypti in this region has not been characterized. To investigate the relationship between Ae. aegypti gene flow and human transportation networks, we characterized mosquito population structure using a panel of 8 microsatellite markers and linked results to various potential mechanisms for long-distance dispersal. Adult and immature Ae. aegypti  were collected from Iquitos city and from six neighboring riverine communities, i.e., Nauta, Indiana, Mazan, Barrio Florida, Tamshiaco, and Aucayo. FST statistics indicate significant, but low to moderate differentiation for the majority of study site pairs. Population structure of Ae. aegypti is not correlated with the geographic distance between towns, suggesting that human transportation networks provide a reasonable explanation for the high levels of population mixing. Our results indicate that Ae. aegypti gene flow among sub-populations is greatest between locations with heavy boat traffic, such as Iquitos-Tamshiaco and Iquitos-Indiana-Mazan, and lowest between locations with little or no boat/road traffic between them such as Barrio Florida-Iquitos. Bayesian clustering analysis showed ancestral admixture among three genetic clusters; no single cluster was exclusive to any site. Our results are consistent with the hypothesis that human transportation networks, particularly riverways, are responsible for the geographic spread of Ae. aegypti in the Peruvian Amazon. Our findings are applicable to other regions of the world characterized by networks of urban islands connected by fluvial transport routes.In the Americas, as in much of the rest of the world, the dengue virus vector  Aedes aegypti, the primary mosquito vector of dengue, is a highly invasive species that is expanding from urban to peri-urban and rural areas throughout the Americas. Previous studies documented the role of human transportation networks in Ae. aegypti long-distance dispersal. We examined whether patterns of Ae. aegypti gene flow are consistent with this observation. Mosquitoes were collected from seven locations, including the large Amazonian city of Iquitos, Peru, and six neighboring rural communities, and their genetic relatedness was compared using 8 microsatellite markers. Our results showed ample gene flow among mosquito populations in this region, with greater gene flow observed among sites that are connected by fluvial routes. These findings are consistent with the hypothesis that human transportation networks, especially via boats, are a primary contributing factor to the spread of Ae. aegypti in the Peruvian Amazon. Anthropogenic activities such as trade and transportation contribute to the unintentional spread of invasive organisms across the globe , 2, resuAedes aegypti, is the primary vector of dengue, urban yellow fever, and Zika viruses, is an important vector of chikungunya virus [Ae. aegypti most likely was transported to the Americas via ships used for the transport of slaves and goods in the 15th-19th centuries [Ae. aegypti was first reported in Peru in 1852, but was declared eradicated in 1958 following the success of a large-scale Pan American Health Organization yellow fever control program [Ae. aegypti (in 1984) and dengue (in 1990) reestablishment in Peru [Ae. aegypti mosquitoes have been expanding geographically from urban to peri-urban and rural areas throughout Peru and the rest of Latin America [The invasive mosquito, ya virus –8, and iya virus . Thoughtenturies –12. Ae.  program . The Ama in Peru , 15. In  America –18.Ae. aegypti dispersal can occur in one of two ways: the slower, active dispersal of flying adult females in search of bloodmeals or oviposition sites, or the faster, passive human-mediated dispersal, by which humans unintentionally transport mosquitoes via vehicle traffic . The latter can involve transport of eggs, larvae, pupae, or adults, all of which have been documented in vehicles, particularly in boats [Ae. aegypti infestation on different vehicles commonly used for trade and transportation, including large barges, medium-sized barges, and buses [in boats , 19–22. nd buses , 24. ChaAe. aegypti population genetic structure at global scales [Ae. aegypti genetic differentiation across various spatial scales is likely due to its limited flight range , hetnditions , 38, 39.aterways , 32. The ecology , yet litPermission for this study was granted by the Loreto Regional Health Department, and the study protocol was approved by the NAMRU-6 Institutional Review Board in compliance with all applicable federal regulations governing the protection of human subjects (protocol number NAMRU6.2012.0039). In addition, the Emory University Institutional Review Board determined that this study does not represent human subjects based research.Ae. aegypti, because the region’s inhabitants are dependent on both fluvial and terrestrial routes for trade and transportation.Accessible only by plane or boat, Iquitos is surrounded by other, smaller settlements that are primarily connected to one another via river networks. The only significant source of terrestrial transit is the 95km Iquitos-Nauta Highway, connecting Iquitos  to the smaller city of Nauta  . Once thFig 1and Table 1summarize characteristics associated with each town. . Indiana and Mazan are only 1.2 km apart and our data indicated that mosquito populations in those two locations were genetically indistinguishable (FST = 0.032). The Euclidean distances between pairs of towns ranged from approximately 15km (Aucayo and Tamshiaco) to 125km (Nauta-Indiana/Mazan). (S1 Table demonstrates Euclidean pairwise distances between towns.)Mosquitoes were collected from the city of Iquitos, and the neighboring towns of Nauta , Indiana-Mazan , Barrio Florida (pop: 728), Tamshiaco , and Aucayo (pop: 806). ntum GIS  using shntum GIS .) After lsewhere . Within Fig 1). The location Iquitos A (Puerto Masusa) serves as a major hub of fluvial transit and predominantly harbors large barges, which carry cargo and passengers throughout the Peruvian Amazon distances up to ~500km. Iquitos B (Huequito) is a secondary fluvial port, primarily harboring medium-sized barges that also carry cargo and passengers, but that travel more locally (up to distances of ~250km) [We collected mosquitoes from multiple locations in Iquitos to determine whether mosquitoes in port areas were more closely related to those in the surrounding towns ( ~250km) . We also2O, and submitted to the University of California Davis College of Agriculture and Environmental Sciences Genomics Facility for fragment analysis on an ABI 3730 XL capillary electrophoresis sequencer (Life Technology Corp.). GS600 LIZ size standard (Life Technology Corp.) was included with each sample to determine the size of individual peaks. ABI Peak Scanner software  was used to visualize resulting chromatograms. After identifying fragments, alleles were assigned using the MsatAllele package in R [Methods used for genotyping mosquitoes with microsatellite markers are described by Wong et al . Genomicage in R , 50.IS, for each site and genetic locus in Arlequin v3.5.2.2 [We used the microsatellite data to calculate observed and expected heterozygosity values, in addition to inbreeding coefficients, Fv3.5.2.2 . We alsoST was calculated in Arlequin v3.5.2.2  [ST analysis on all nine sampling locations to understand the degree of genetic differentiation among all sample locations including Iquitos. We then conducted a second pairwise FST analysis combining Iquitos samples to understand degree of connectivity between the city center and the five villages outside the city. Mantel tests conducted in the package ade4 in R [ST values used to generate isolation by distance plots are shown in F < 0.05) . We firsde4 in R , 52 wereAe. aegypti infestation in different vehicle types with the frequency of travel between Iquitos and surrounding towns. We calculated infestation probabilities from data collected in 2013 across six different vehicle types common in Iquitos including: large and medium size barges, water taxis, speed boats, buses, and taxis [Ae. aegypti across multiple months . Simultaneous with entomological surveys, we interviewed vehicle drivers to determine the frequency of travel between Iquitos and surrounding towns for each vehicle type to estimate the approximate number of vehicles traveling to each town. The Propagule Pressure Index (PrPI) is calculated as follows:We also developed a “Propagule Pressure Index,” combining the probability of nd taxis . Our res= Σj = 1,n)PrPI Where:γi = probability of vehicle infestationθi = number of trips from Iquitos to surrounding townsi represents individual vehicles of a certain typej represents different vehicle types Ae. aegypti infestation for each vehicle type. Values of PrPI were plotted against FST to visually explore the relationship between these two variables. However, no statistical tests were conducted due to low sample sizes—pairwise calculations of PrPI were only available between Iquitos and other towns, leaving a total of five observations.PrPI therefore represents the number of trips made between pairs of locations, weighted by the probability of Table 1; Aucayo, Barrio Florida, Indiana-Mazan, and Tamshiaco were counted as separated populations, but Iquitos sites were given hierarchical consideration. The total variation observed was attributed to differences between individuals within populations, and among populations. Accordingly, we calculated pairwise F statistic analogues characterizing the variation among individuals and populations .We also estimated the genetic divergence of the populations by an analysis of molecular variance (AMOVA) in Alrequin v3.5.2.2 . The geoAe. aegypti with the program Structure v. 2.3.4 [We investigated the regional population structure of v. 2.3.4 . StructuIn Structure, we used an admixture model with uncorrelated allele frequencies to avoid the risk of overestimating the number of populations . We started simulations with K = 10, to allow for the possibility of more genetic clusters than sampling locations , and then ran simulations for K values of 10 through 1. For each K, we ran 10 simulations to ensure consistency between runs, and used the log likelihood  and DeltIS > 0.50) among four loci  (Table 2). These comparison were distributed across five sites including Barrio Florida (B07), Tamshiaco (B07), Iquitos A (AC1 and AG2), Iquitos C (B07 and H08), and Iquitos D (AC1 and B07). At a site-level, significant and low to moderate levels of heterozygous deficits were observed in Nauta, Tamshiaco, and all sites within Iquitos (FIS range among these sites: 0.19629 to 0.37096).Of 72 tests, 45 (62.5%) loci from all populations were found to be in Hardy-Weinberg equilibrium. The majority of deviations were from Nauta and Tamshiaco; when those populations were excluded, 73.2% of loci (41 of 56) were in Hardy-Weinberg equilibrium.After applying a Sidak correction for multiple comparisons (p < 0.00071) for all populations, significant heterozygous deficits were detected in eight comparisons . Mosquitoes from Barrio Florida and Nauta were significantly differentiated from all other sampling locations, with FST values ranging from 0.022 to 0.11. Barrio Florida mosquitoes had the highest FST values observed for all study sites (FST > 0.05 in 4 out of 8 pairwise comparisons). We also found significant genetic differentiation for 7 out of 8 pairwise comparisons for all other sites  except for Iquitos D. Iquitos samples generally showed a lower degree genetic differentiation both when comparing sites within Iquitos and sites outside of Iquitos. Notably, mosquitoes collected from Iquitos A and B  had lower FST values than mosquitoes collected from the interior of Iquitos.Pairwise FST) and geographic distance for the three standard distance models . We were unable to statistically evaluate the relationship between PrPI and genetic distances because of insufficient transportation network data.Our analysis of isolation by distance showed no clear relationship between genetic distance . That is, the DeltaK statistic was highest for k = 3 groups (DeltaK = 9.5) in comparison with all other possible numbers of groups . Structure results showed a clear pattern of genetic admixture from all the sample populations, suggesting steady gene flow within this region . Of 339 individual mosquitoes, 181 (53.4%) showed dominant haplotype membership associated with a single genetic group . Dominant haplotype membership (> 0.8) to the same group  was observed among 84.4% of mosquitoes collected from Indiana/Mazan, 87.5% of mosquitos from Iquitos B, 75% of mosquitoes from Aucayo, and 64.5% of individuals from Barrio Florida. Mosquitoes collected from Tamshiaco and Nauta showed the greatest degree of ancestral admixture, with each of the three genetic groups represented approximately equally.Results from the DeltaK analysis of Structure output indicated that there were most likely three genetic clusters  suggest mosquito mobility between human settlements. The pattern of mosquito gene flow also mirrors human transportation patterns: FST values for a regional port (Iquitos B) were overall lower than FST values collected from the interior of Iquitos (Iquitos C and D), implying more population mixing between Iquitos port mosquito populations and surrounding towns than between Iquitos interior populations and surrounding towns. Barrio Florida populations, in contrast, were the most genetically isolated compared to other towns. Iquitos is the major regional transportation hub, with frequent visits of barges and other types of boats from surrounding areas, whereas Barrio Florida is a small town (pop: ~800), receiving most of its traffic from vehicles unlikely to be infested with Ae. aegypti  [Our results support the hypothesis that r taxis) .Ae. aegypti gene flow, have changed since mosquito samples were collected in 2008. More recent samples and better transportation data would be required to assess this possibility. Our limited data on human transport patterns also meant that we were only able to evaluate the Propagule Pressure Index by comparisons between Iquitos and surrounding towns. To that end, in some locations our sample sizes were low, particularly in Aucayo. Further, some scholars have argued that the allozyme methods to estimate population subdivision  are coarse measures of gene flow, and in some instances lead to erroneous conclusions [Ae. aegypti dispersal throughout the riverine landscape characteristic of this region. Fine-scale landscape genetics methods may be of particular interest to identify barriers to and pathways that facilitate dispersal [Some limitations should be noted when interpreting our data. It is possible that patterns of human traffic, and by extension, clusions . Still, ispersal .Ae. aegypti spreads farther along rivers than terrestrial routes [Ae. aegypti spread is facilitated by certain vehicle types, large barges in particular  [Ae. aegypti population genetics provides additional evidence to support the hypothesis that boats are major drivers of the passive transport of Ae. aegypti mosquitoes over long distances (between cities > 5 km apart). In other regions, fluvial traffic has also been implicated as a driver of Ae. aegypti spread [Our previous studies in the Peruvian Amazon have shown that l routes , that Aeehicles) , and thaehicles) . When coi spread , but theAe. aegypti genetic variation and geographic distance did not reveal a pattern of isolation by distance. At this spatial scale (sampling locations < 100km apart), network distance might be the best predictor of genetic mixing. Although both field ecology [Ae. aegypti spread, to our knowledge, none of these studies linked ecological evidence with transportation and population genetics data . The novel Propagule Pressure Index, PrPI, integrates these data by taking into account heterogeneity of Ae. aegypti infestation by vehicle type, in addition to the frequency of traffic between two locations. We were unable to statistically test for isolation by distance using PrPI because of limited sample sizes, however, the isolation by distance plot  is suggestive of a negative correlation between genetic and network distance. In other words, populations closer to one another in network space (as measured by PrPI) are likely to be more genetically related. Future studies with larger sample sizes are required to evaluate its utility.Consistent with other findings from Peru , our com ecology , 17 and  ecology , 60, 61 Ae. aegypti genetic admixture of port mosquitoes and recently urbanized mosquitoes were similar, while interior mosquitoes had a different pattern of population membership. Within cities, mosquito population structure may be a function of availability of immature habitat, mosquito movement, or vector control programs that have differentially impacted various neighborhoods within the city. Perhaps, for example, insecticide application focused in the interior parts of the city act as barriers to population mixing, ultimately resulting in differing patterns of population group membership. This vector control explanation has been proposed in other settings [Within Iquitos, settings .Ae. aegypti population structure in the Amazon has many other implications for dengue control programs. Our data suggest that Ae. aegypti are continually introduced to smaller surrounding communities from locations such as Iquitos that have stable and spatially diverse population structure. For example, the spread of insecticide resistance or vector competence genes would occur but could potentially lead to significant spatial heterogeneity. Significant spatial variation in vector competence has been reported previously [Indeed, eviously , so undeAe. aegypti gene flow impact arboviral transmission dynamics? Dengue in the Peruvian Amazon has been characterized by transmission in large population centers with most cases observed from outlying communities assumed to be associated with frequent travel to these commercial hubs. The distribution of Ae. aegypti is driven by urbanization along highways, but infestation in communities along rivers is far less consistent spatially [Ae. aegypti in these communities is driven in part by repeated introductions as well as by local ecological characteristics such as water storage, management of containers, and microclimate. Over the last 20 years Ae. aegypti has spread to smaller communities, with significant implications for vector control programs which are already stretched to cover Iquitos and other larger cities. Dengue outbreaks are known to occur in several of the small riverine towns near Iquitos. Although these outbreaks are typically small and fleeting, the resources necessary for sustained control of Ae. aegypti  are limited. Clearly, transmission in these smaller communities could lead to infections in travelers (and mosquitoes) that could then carry virus to other locations, thus serving as a mechanism of viral spread at broader scales.An extension of our results is to ask, how does patially . We hypoS1 FigThe DeltaK statistic captures the degree of change between the log probability of the data for consecutive values of K populations. Results showed the most likely number of groups to be K = 3 genetic clusters.(TIFF)Click here for additional data file.S1 TableAe. aegypti infestation.The number of visits between site pairs for large and medium barges were weighted according to the probability of their infestation . Our previous research demonstrated a 71% probability of infestation among large barges, 35% probability of infestation among medium barges, and 12.5% infestation among combis (taxis). PrPI is calculated by summing the number of visits by large and medium barges and combis, weighted for the probability of (DOCX)Click here for additional data file.S2 TableIquitos was counted as a single population because transportation data between different sites within Iquitos and surrounding towns was not available.(DOCX)Click here for additional data file.


Knockdown of Septin4 significantly increased PDGF-BB-induced HAVSMCs proliferation, migration and phenotypic transformation, while overexpression of Septin4 had the opposite effects. Mechanically, co-immunoprecipitation results demonstrated that Septin4 was a novel interacting protein of STAT3 and SIRT1. Septin4 formed a complex with SIRT1-STAT3, enhancing the interaction between SIRT1 and STAT3, ensuing promoting SIRT1-regulated STAT3-K685 deacetylation and STAT3-Y705 dephosphorylation, which inhibited PDGF-BB-induced HAVSMCs proliferation, migration and phenotype transformation. Therefore, our findings provide novel insights into the prevention and treatment of atherosclerosis.SIRT1 and STAT3 are key to human aortic vascular smooth muscle cells (HAVSMCs) proliferation, migration and phenotypic transformation, but the regulatory mechanism of SIRT1-STAT3 in this process is still unclear. Septin4 is a cytoskeleton-related protein that regulates oxidative stress-vascular endothelial injury. However, the role and underlying mechanism of Septin4 in atherosclerosis remains unknown. Here, we revealed the role and mechanism of Septin4 in regulating SIRT1-STAT3 in atherosclerosis. We determined that the expression of Septin4 were markedly increased in Apoe Despite the improvement of treatment methods, the incidence and mortality of atherosclerosis are still increasing The constant recognition of the influence of proteins post-translational modification on the HAVSMCs has opened up new horizons of studies involving in atherosclerosis. Septin4 is a member of GTP-binding proteins, belonging to the Septins family genes that are essential for cell separation, apoptosis, vesicle trafficking, tumor suppression and other cell processes Signal transducer and activator of transcription 3 (STAT3) is a transcription activator, which enters the nucleus and binds to promoter sequences of target genes to promote their transcription -/- mice, and PDGF-BB-induced proliferation, migration and phenotypic transformation in HAVSMCs. Septin4 knockdown significantly promoted PDGF-BB-induced proliferation, migration and phenotype transformation of HAVSMCs, while Septin4 overexpression remarkably reduced this phenomenon. Mechanically, co-immunoprecipitation identified that Septin4 is a novel interacting protein of STAT3 and SIRT1, forming a complex with SIRT1-STAT3, ensuing promoting the interaction between SIRT1 and STAT3. In addition, Septin4 promotes SIRT1-regulated STAT3-K685 acetylation and STAT3-Y705 phosphorylation reductions in PDGF-BB-induced HAVSMCs model.Here, we firstly showed that Septin4 is significantly increased during the development of atherosclerosis in Apoe-/- (n=8) and ApoE+/+ (n=8) mice (8-10 weeks) were purchased form Vitalriver company and housed in individually ventilated cages with 12 hours light/dark cycle and controlled temperature (20 °C-22 °C). High-fat diet (containing 21% fat and 0.15% cholesterol) for ApoE-/- and ApoE+/+ mice were performed for 8 weeks to induce atherosclerosis model . Hematoxylin and eosin (H&E) staining and Western-blot were performed for mice in each group. Aortic root vascular tissue specimens from mice were fixed with 4% formalin (4h), paraffin embedded and sectioned at 5-µm. After xylene dewaxing and rehydration by graded ethyl alcohol, the sections underwent H&E staining. All animal handling complied with animal welfare regulations of China Medical University. The Animal Subjects Committee of China Medical University approved the animal study protocol. The investigation conforms to the guide for the care and use of laboratory animals published by the US National Institutes of Health .Specific pathogen-free (SPF) male ApoE2 at 37˚C. HAVSMCs were passaged 4-6 times before use. Plasmids encoding the full-length human Septin4 (Shanghai Genechem) was cloned to Flag tagged destination vectors. Transfection was performed with Lipofectamine 3000  as directed by the manufacturer. Control- and Septin4-siRNAs were provided by RIBOBIO (China). Septin4 knockdown was performed with the jetPRIME transfection reagent . Three target sequences were assessed for excluding off-target effects. Septin4 knockdown efficiency was confirmed by immunoblot.HAVSMCs were provided by Cambrex  and maintained in H-Dulbecco's modified Eagle medium (H-DMEM)  containing 10% fetal bovine serum (FBS) (HyClone) in a humid environment with 5% COSeptin4 siRNA-1: GGACCGGAAACTTCTTGGT;Septin4 siRNA-2: GGAGATCACTAAGCATGCA;Septin4 siRNA-3: TGGCAGAATACATTGATCA.Antibodies to polyclone rabbit anti-Septin4 , monoclonal rabbit anti-Flag , polyclone rabbit anti-STAT3-K685 , polyclone rabbit anti-STAT3-Y705 , polyclonal rabbit anti-STAT3 , polyclonal rabbit anti-SIRT1 , polyclonal rabbit anti-SM22α , polyclonal rabbit anti-α-SM-actin , monoclonal rabbit anti-MMP9 , polyclonal rabbit anti-MMP2 , polyclonal rabbit anti-PCNA  and monoclonal mouse anti-GAPDH  were purchased commercially. Protein A/G immunoprecipitation magnetic beads was obtained from Biotool and used for immunoprecipitation.For the purpose of co-immunoprecipitation, the cells were washed twice with a newly prepared protease inhibitor and dissolved with a marker solution buffer. The lysates were incubated with antibody (3 hours) and protein A/G immunoprecipitation beads  . The immune complex was detected by SDS-PAGE.Western blot was used co-immunoprecipitation buffer for cell lysis. The protein was extracted by centrifugation . BCA protein detection kit  was used for total protein quantification. The same amount of protein was separated by SDS = page and transferred to PVDF membrane by electricity. Then, 5% bovine serum albumin (BSA) was sealed in Tris buffer tween (TBST) under environmental conditions (1 hours), and then incubated overnight (4 °C) with antibodies. GAPDH was used as a loading control. Image J v1.46  was employed for analysis.3 cells/well in H-DMEM containing 10% FBS and underwent transfection with control and Flag-Septin4 plasmids, respectively, or control and Septin4-siRNA, respectively. PDGF-BB administration was used for 24 hours after starvation in serum-free medium for 24 hours, and 100 µl of CCK-8 reagent was added per well for 1.5 h. Absorbance was determined at 450 nm on a Bio-Rad microplate reader .Cell Counting Kit-8  was employed to assess cell viability. HAVSMCs were seeded into a 96-well plate at 3x103 cells were implanted into the upper cavity of the 24 well plate. PDGF-BB administration was used for 24 hours after starvation in serum-free medium for 24 hours. The top chambers were used serum-free H-DMEM, however, the lower ones contained with 10% FBS. After incubation at 37 °C overnight, non-migrating cells were removed with cotton swabs. The migrants were fixed with frozen methanol, stained with crystal violet, and 5 random areas were counted.Cell migration was assessed in transwell plates . A total of 5x10Phalloidin  staining of HAVSMCs was performed after fixation with 4% formalin (20 min) and permeabilization with 0.5% Triton X-100 (30 min), as directed by the manufacturer. Cell morphology and actin filaments were observed under a fluorescence microscope (Olympus).P values were adjusted for multiple comparisons when applicable. All data were analyzed by SPSS 22.0 , and P<0.05 was considered statistically significant.Data are mean ± standard deviation (SD). Homogeneity of variance was evaluated by the F test (group pair) or Brown-Forsythe test (multiple groups). The Shapiro-Wilk test was performed for assessing data normality. Student's t-test and Welch t test were employed to assess data of group pairs with normal and skewed distributions, respectively (two groups). ANOVA and indicated non-parametric tests were performed to compare multiple groups. One-way ANOVA and two-way ANOVA were performed for comparing groups for single and two factors, respectively. in vivo, high-fat diet was used to induce atherosclerosis in Apoe-/- mice. The results showed that compared with Apoe+/+ mice, the expression of Septin4 was significantly increased in Apoe-/- mice . In addition, we found that the vascular tissues were significantly hypertrophy during the development of atherosclerosis in Apoe-/- mice .Firstly, in order to detect Septin4 expression in atherosclerosis model in vitro model, PDGF-BB was used to induce HAVSMCs proliferation, migration and phenotypic transformation. The results showed that with the increase of PDGF-BB concentration, the expression level of Septin4 increased gradually . In addition, HAVSMCs had obvious increase in proliferation, migration and phenotypic transformation with the increase of PDGF-BB concentration .Next, in order to detect Septin4 expression In vivo and in vitro results suggested that Septin4 may be involved in the regulation of atherosclerosis and HAVSMCs proliferation, migration and phenotypic transformation.Figure ), while knockdown of Septin4 significantly aggravated PDGF-BB-induced HAVSMCs proliferation  and migration  .In order to clarify the role of Septin4 in PDGF-BB-induced HAVSMCs proliferation and migration, overexpression and knockdown Septin4 were performed in PDGF-BB-induced HAVSMCs model. CCK8 and transwell experiments showed that overexpression of Septin4 significantly relieved PDGF-BB-induced HAVSMCs proliferation  and migration  . While, knockdown of Septin4 had the opposite effects .In addition, overexpression of Septin4 significantly decreased PDGF-BB-induced proliferation and migration makers PCNA, MMP2 and MMP9 expression . While, knockdown of Septin4 had the opposite effects  .In order to further clarify role of Septin4 in HAVSMCs phenotypic transformation, overexpression and knockdown Septin4 were performed in PDGF-BB-induced HAVSMCs phenotypic transformation. FITC-phalloidin showed that overexpression of Septin4 significantly antagonized PDGF-BB-induced HAVSMCs phenotypic transformation  . While, knockdown of Septin4 further reduced the expression of PDGF-BB-induced α-SM-actin and SM22α .In addition, overexpression of Septin4 significantly stabilized the expression of PDGF-BB-induced HAVSMCs contraction phenotype makers α-SM-actin and SM22α  and SIRT1  by endogenous co-immunoprecipitation assays. In addition, the interaction between Septin4 and SIRT1 was significantly enhanced in PDGF-BB-induced HAVSMCs .To further explored the molecular mechanism of Septin4-regulated PDGF-BB-induced HAVSMCs proliferation, migration and phenotypic transformation, co-immunoprecipitation assays were performed to determine the interacting proteins of Septin4 in HAVSMCs. The results showed that Septin4 is a novel interacting protein of STAT3 , while knockdown Septin4 significantly decreased the interaction between SIRT1 and STAT3 .As SIRT1 and STAT3 play important roles in HAVSMCs proliferation, migration and phenotype transformation The above results suggested that Septin4 forms a complex with SIRT1-STAT3 , promoting the interaction between SIRT1 and STAT3.Figure ), while knockdown Septin4 significantly enhanced the level of STAT3 acetylation and phosphorylation .As STAT3 acetylation and phosphorylation were the key to promote HAVSMCs proliferation, migration and phenotypic transformation Figure ), while knockdown Septin4 significantly enhanced the level of STAT3-K685 acetylation and STAT3-Y705 phosphorylation .Previous studies have shown that SIRT1 regulates acetylation and phosphorylation of STAT3 mainly acting on STAT3-K685 and STAT3-Y705, respectively Figure ). While, knockdown of Septin4 had the opposite effects .Finally, we validated this mechanism in the PDGF-BB-induced HAVSMCs model. The results showed that overexpression of Septin4 significantly alleviated PDGF-BB-induced expression of STAT3-K685 acetylation and STAT3-Y705 phosphorylation (Taken altogether, our graphical abstract reveals that Septin4 formed a complex with SIRT1-STAT3, enhancing the interaction between SIRT1 and STAT3, ensuing promoting SIRT1-regulated STAT3-K685 acetylation and STAT3-Y705 phosphorylation reductions, which inhibited PDGF-BB-induced HAVSMCs proliferation, migration and phenotype transformation.1, expression of Septin4 were markedly increased in Apoe-/- atherosclerosis mice and PDGF-BB-induced HAVSMCs. Knockdown of Septin4 significantly increased PDGF-BB-induced HAVSMCs proliferation, migration and phenotypic transformation, while overexpression of Septin4 had the opposite effects. 2, Septin4 was a novel interacting protein of STAT3 and SIRT1, which formed a complex with SIRT1-STAT3, ensuing promoting for the interaction between SIRT1 and STAT3. 3, STAT3-K685 acetylation and STAT3-Y705 phosphorylation are of critical importance in the regulation of STAT3 by Septin4 during atherosclerosis. Our study firstly identified the role of Septin4 and the mechanism of Septin4-SIRT1-STAT3 complex in the proliferation, migration and phenotypic transformation of HAVSMCs, providing new ideas for the therapeutic strategies of atherosclerosis.Atherosclerosis is mainly caused by the proliferation and migration of HAVSMCs, and the phenotype transformation is the core to control the proliferation and migration of HAVSMCs Septin4 was considered as an apoptosis-related protein, playing an important role in the process of various organ damage SIRT1 and STAT3 play key roles in the proliferation and migration of HAVSMCs Our study firstly clarified the key role of Septin4 in inhibiting proliferation, migration and phenotype transformation of HAVSMCs by regulating SIRT1-STAT3, which provides a theoretical basis for exploring new therapeutic strategies for atherosclerosis. It is meaningful to construct the knockout and transgenic mice of Septin4 and to explore its role in atherosclerosis in the future. It will also be significant to explore the role and mechanism of Septin4 in other cardiovascular diseases.In conclusion, Septin4 formed a complex with SIRT1-STAT3, enhancing the interaction between SIRT1 and STAT3, ensuing promoting SIRT1-regulated STAT3-K685 deacetylation and STAT3-Y705 dephosphorylation, which inhibited PDGF-BB-induced HAVSMCs proliferation, migration and phenotype transformation.


The Galway sheep population is the only native Irish sheep breed and this livestock genetic resource is currently categorised as ‘at-risk’. In the present study, comparative population genomics analyses of Galway sheep and other sheep populations of European origin were used to investigate the microevolution and recent genetic history of the breed. These analyses support the hypothesis that British Leicester sheep were used in the formation of the Galway. When compared to conventional and endangered breeds, the Galway breed was intermediate in effective population size, genomic inbreeding and runs of homozygosity. This indicates that, although the Galway breed is declining, it is still relatively genetically diverse and that conservation and management plans informed by genomic information may aid its recovery. The Galway breed also exhibited distinct genomic signatures of artificial or natural selection when compared to other breeds, which highlighted candidate genes that may be involved in production and health traits. Sheep were domesticated more than 10,000 years ago and have since been bred for a variety of uses including meat, milk and wool production . During The local sheep breeds on the periphery of Northern Europe are recognised as heritage livestock populations that should be conserved and represent important sources of novel genetic diversity accumulated over centuries of microevolution and adaptation to marginal agroecological environments . In thisThe Galway breed is thought to have originated as a composite of indigenous and imported sheep populations, present in Ireland in the mid-19th century, through the breeding endeavours at that time, which were concerned mainly with improved wool production . Sheep bAs a local breed with a low census population size, the main threat to the long-term survival of the Galway breed is replacement by more productive commercial breeds, which would further reduce the population size, reduce genetic diversity and increase inbreeding. Other challenges faced by threatened local livestock breeds include poor animal husbandry and management, deliberate or inadvertent crossbreeding and geographical isolation, which increases the risk of extinction . In receTo provide information that may be relevant to genetic conservation of the Galway sheep breed, we performed high-resolution population genomics analyses in conjunction with 21 comparator breeds of European origin. These analyses included multivariate analyses of genomic diversity, phylogenetic network graph reconstruction, evaluation of genetic structure and inbreeding, modelling of historical effective population sizes and functional analyses of artificial and natural selection across the Galway sheep genome.The Galway and Irish Suffolk sheep DNA samples used for the current survey were generated from peripheral blood samples collected in standard heparinised Vacutainer blood collection tubes . High-quality genomic DNA was then purified from 200 µl of blood from each animal using standard laboratory methods . The 49 ® OvineSNP50 BeadChip . This included populations previously examined and known to be more closely related due to their shared European origins .Medium-density SNP data were obtained from the International Sheep Genomics Consortium Sheep HapMap Project and consisted of 2,819 sheep from 74 breeds genotyped for 49,034 evenly spaced SNPs using the IlluminaBeadChip . To focu origins . These c origins . Figure The initial data set had already been filtered to remove SNPs with <0.99 call rate, assay abnormality, MAF <0.01, discordant genotypes and inheritance problems . The corPrincipal component analysis (PCA) was performed using 47,412 genome-wide SNPs and SMARTPCA from the EIGENSOFT software package (version 4.2) . The numFST values  phylogenetic trees with ancestry graphs were generated for the core and extended sample data sets using 47,412 genome-wide SNPs and the TreeMix (version 1.12) software package. For the core sample set, the Italian Comisana breed (COM)  was usedK = 2 to 11. The simple prior approach described by K-value for the number of ancestral populations was estimated using a series of fastSTRUCTURE runs with pre-defined K-values that were examined using the chooseK.py script   as descry script . Outputsy script .Ne) trends were modelled with genome-wide SNP linkage disequilibrium data from 47,412 genome-wide SNPs for the core sample set using the SNeP software tool (version 1.1)   implemenion 1.1) . Graphs  ggplot2 .F) estimated from SNP heterozygosity data was performed using 47,412 genome-wide SNPs and the PLINK v1.07 –het command  are continuous tracts of homozygosity that most likely arise due to inbreeding and can be identified through surveys of genome-wide SNP data in populations . Individ ggplot2 .FST), the directional change in selected allele frequency (ΔSAF) and cross-population extended haplotype homozygosity (XP-EHH) tests into one composite statistic for each SNP in a population genomics data set  method  was useddata set . For the® Pathway Analysis  was then used to perform an overrepresentation enrichment analysis with this gene set to identify canonical pathways and functional processes of biological importance. The total gene content of Ensembl release 85 version of the OAR3.1 ovine genome assembly , which inhabits a small island, the breed exhibits moderate genetic differentiation from other European breeds. The Galway breed exhibited relatively low pairwise FST values with the New Zealand Romney (ROM: 0.110), Australian Merino (MER: 0.118) and Scottish Texel (STX: 0.119) breeds. This is unsurprising because the Romney, Merino and Texel breeds are known to have shared origins with the Galway breed (The PCA results are supported by the interpopulation weighted ay breed .The ML phylogeny and ancestry graph in K) ranging from 2 to 11 are visualised to explain the structure in the data and to maximise the marginal likelihood. These results demonstrate that the 11 breeds can be considered discrete populations, thereby supporting interpretation of sheep breeds as separate genetic units  separates the isolated Soay sheep population (SOA) from the other breeds. The second split (K = 3) then differentiates the Finnish Landrace (FIN) from the remaining breeds. At K = 9 the Galway breed emerges as a distinct cluster and this genetic component is also apparent in the New Zealand Romney breed (ROM). With K = 11 each breed emerges as a distinct genetic cluster. However, some individual animals show evidence of prior crossbreeding or historical admixture, which is indicated by bars that exhibit varying colour proportions. Based on these results, some individual Galway animals exhibit 10% or more admixture with other sheep breeds, particularly the Border Leicester (BRL), Scottish Texel (STX) and Scottish Blackface (SBF). The observed signature of a Galway genomic component in the New Zealand Romney breed (ROM) is supported by the relatively low pairwise FST value for these breeds, the TreeMix results (The colours on  results .Ne) for the range of conventional and at-risk sheep breeds . Inspection of Ne for the 11 breeds analysed decline towards the present. However, the GAL breed are intermediate between the breeds with large census populations  and at-risk breeds with relatively small census populations  breeds. In addition, the most recent modelled Ne value for the GAL breed is 184 animals 13 generations ago, which is comparable to some of the breeds . These modelled Ne values, which are based on linkage disequilibrium, may be underestimates due to the physical linkage between many SNPs  values estimated for individual animals across all breeds range up to 0.389 for a single Dorset Horn (DSH) animal  and the WIL (0.299) and the two breeds with the lowest median F values were the MER (0.045) and the SBF (0.060). The other breeds exhibited intermediate median F values: BRL (0.243), DSH (0.169), FIN (0.087), GAL (0.127), ISF (0.185), ROM (0.086) and STX (0.111). These results provide a window on the different population histories for the breeds. For example, Soay sheep (SOA) have existed as a relatively small and isolated population on the island of Soay for hundreds of years while the Wiltshire breed (WIL) has recently experienced a dramatic decline in census population and is considered at risk by the FAO . An analysis of all pairwise breed comparisons using the non-parametric Wilcoxon rank sum test (with Bonferroni correction) was then performed  were observed for inbreeding coefficients estimated using ROH (FROH)  is likely due to their longer geographical isolation and a consequence of early historical inbreeding that produced ROH tracts, which have broken down due to recombination  indicating that this statistic is useful for identifying animals that should not be prioritised for conservation programmes. With regards to historical inbreeding in the Galway breed (GAL), inbreeding coefficients have previously been calculated using pedigree information for the population in 1969 , which has been observed previously . In term® OvineSNP50 BeadChip data are available as part of the International Sheep Genomics Consortium Ovine SNP50 HapMap Dataset (www.sheephapmap.org/download.php).The Galway sheep (GAL) and additional sheep breed IlluminaAnimal biological sample collection was conducted under license issued in accordance with Irish and European Union legislation  as described previously . All aniDEM, DH, MM and JH conceived and designed the project. DH, MM, DAM, ES and JH organised sample collection and genotyping. GM, SB, IAR, IWR, SP, MD and CC performed the analyses. GM and DEM wrote the manuscript and all authors reviewed and approved the final manuscript.This work was supported by Department of Agriculture, Food and the Marine (DAFM) funding under the Genetic Resources for Food and Agriculture scheme (grant no: 09/GR/06); an Investigator Programme Grant from Science Foundation Ireland (SFI/08/IN.1/B2038); a Research Stimulus Grant from DAFM (RSF 06 406); a European Union Framework 7 Project Grant (KBBE-211602-MACROSYS); the Brazilian Science Without Borders Programme (CAPES grant no. BEX-13070-13-4); and the UCD MSc Programme in Evolutionary Biology.The authors IWR and SP are employed by IdentiGEN, Ltd. All other authors declare no competing interests and that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Potamon ibericum (Brachyura: Potamidae) in the drainages of the western Caucasus biodiversity hot spot  to infer spatial genetic structure and potential refugia for a freshwater crab in this region. These areas have traditionally considered as a refugium due to the presence of Tertiary relict species. We integrated population genetic data and historical demographic analysis from cytochrome oxidase subunit I sequences and paleoclimatic data from species distribution modeling (SDM). The results revealed the lack of phylogeographic structure and provided evidence for demographic expansion. The SDM presented a rather homogenous and large refugium that extended from northeast Turkey to Colchis during the last glacial period. In contrast to these findings, previous phylogeographic study on P. ibericum of the eastern Caucasus biodiversity hot spot  identified multiple independent refugia. By combining these results, we explain the significance of this important western Palearctic hot spot of biological diversity in shaping the geographic distribution of intraspecific genetic diversity in a freshwater taxon.Refugia are critical for the maintenance of biodiversity during the periods of Quaternary climatic oscillations. The long‐term persistence of refugial populations in a large continuous refugium has resulted in a homogenous pattern of genetic structure among populations, while highly structured evolutionary lineages characterize the restriction of refugial populations to smaller subrefugia. These mechanisms have resulted in the identification of hot spots of biodiversity within putative glacial refugia. We studied phylogeography of  ThFor many temperate species, hot spots of intraspecific genetic diversity have been identified within putative glacial refugia. Hot spots, areas where diversity has evolved  and used the MUSCLE algorithm in MEGA 6  under accession numbers MK570620–28.Our studied area included eight localities across Georgia, covering the major geographic range of n Figure . These ln Figure  and COH1n Figure  followinn Figure . The amp2.2h), haplotype diversity (Hd), nucleotide diversity (π), and number of segregating sites (S) in DnaSP v.5.10 . Analysis of molecular variance (AMOVA) was performed to test for the genetic partitioning of the two main drainages  by computing among drainage differences (ΦCT) and 1,000 permutations to test for significance in Arlequin v.3.5.2.2  and the estimate of genetic differentiation for phylogenetically ordered alleles (NST) with 10,000 permutations in SPADS 1.0  and reconstructed a phylogenetic tree with Bayesian Inference (BI) by using MrBayes 3.2.2 . The initial 25% of trees were discarded as burn‐in and the remaining trees were used to construct the Bayesian consensus tree. The resulting tree was viewed and edited in FigTree v.1.4.2  in BEAST 2.4.7  including the Last Glacial Maximum (LGM) scenario. The Community Climate System Model (CCSM4) and the earth system model (MIROC‐ESM) were used as the general atmospheric circulation models for this scenario. All the data were downloaded as raster format with 30 arc second for current layers and 2.5 min spatial resolution for the LGM. All the variables were extracted from WorldClim dataset  analysis  , low mean nucleotide diversity (0.00074), and shallow evolutionary divergence (mean uncorrected p‐distance = 0.00007) were observed in Potamon ibericum within the studied area. We found only nine unique haplotypes among 75 COI sequences . The lack of phylogeographic structure was approved from phylogeographic structure analysis .Overall, moderate haplotype diversity  and Tajima's D  tests support an excessive number of rare haplotypes as a result of a recent population expansion . A Bayesian skyline plot showed an increase in the mtDNA effective population size toward the present with a marked expansion which began at about 25,000 years BP  and when the evidence of recent and rapid demographic expansion in the region is pronounced, it would not be implausible to observe genetic homogeneity across a relatively small and homogeneous landscape like the Colchis and western Caucasus. A thorough genetic sampling would unravel true haplotype diversity across this region.Although we found private haplotypes  in four localities, including Dzirula, Dzama, Aragvi, and Ilto, the overall lack of geographic and population genetic structure was confirmed in the studied area. Most of the haplotypes are present in more than one population Figure c, which P. ibericum along the eastern and southeastern coasts of the Black Sea and in Colchis  is an area between the western Lesser Caucasus and northeast Turkey which, based on paleontological data, harbored a homogeneous refugium during the LGM  suggests a Hyrcanian origin of populations in this region. Our findings together with previous study on P. ibericumsuggest a pattern of “refugia‐within‐refugia” in the Hyrcanian regions of the Caucasus biodiversity hot spot, in contrast to a large homogenous refugium in the Colchis region. In order to clarify the phylogeographic breaks and the spatial distribution of evolutionary lineages of potamids in the Caucasus biodiversity hot spot, we recommend that future studies pursue a more thorough investigation of the drainages of Azerbaijan and incorporate variable nuclear markers.The interaction between climatic fluctuations during the Pleistocene and landscape features of the Caucasus biodiversity hot spot have initiated the microevolutionary processes that shaped the current phylogeographic structure of All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.The authors declare that they have no conflict of interest.E.P., R.N., A.K., and C.D.S. conceived the study; R.N. and A.K. carried out the fieldwork; S.S.F. performed the SDM analysis; F.S. performed genetic laboratory work. E.P. performed genetic analysis and drafted the manuscript. All authors corrected and approved the final version of the manuscript. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Emitter clogging is a major problem in microirrigation systems, which may result from the isolated or combined effects of physical, chemical, and biological agents. Clogging caused by suspended solid particles is the most common plugging form of emitters. Water quality and emitter geometry are key aspects in clogging processes. Any suitable test procedure to assess the sensitivity of drippers to clogging should take into account the predominant factors that influence clogging and must reproduce the field conditions. This research set out to assess the performance and suitability of a laboratory clogging test procedure in order to validate a methodology and to provide scientific results that may support the standardization of a clogging test method. The evaluated methodology has been used by the IRSTEA laboratory since 1974  and its contents are currently being discussed by the ISO TC23/SC18 committee. The aim is to define a standardized testing protocol to evaluate the sensitivity of emitters to clogging due to solid particles. Replications analyzing the clogging resistance of four models of emitting pipes were carried out in a laboratory. The clogging test procedure enabled an accurate assessment of the combinations of concentration and size of particles that caused clogging in each model of dripper. However, a significant variability in degree of clogging was identified when the results of replications for each model of dripper were compared. Several requirements, concerns, and improvements related to the clogging test protocol were discussed. Clogging of emitters is a serious problem affecting microirrigation systems , 2. The Methodologies to evaluate or compare the resistance of emitters to clogging may be useful to select the appropriate emitter design for a given set of water characteristics and further recommend a consistent filtration method and grade once the emitters' performance is known. Understanding some of the clogging processes is essential to defining a set of testing protocols required to compare or assess emitter sensitivity to clogging. A testing procedure must include the predominant factors that influence clogging while accelerating the drippers ageing process. The facilities and methodologies to be used must provide results of high repeatability and reproducibility in order to ensure that a given material presents similar results when evaluated by different laboratories.This research set out to assess a methodology developed by the laboratory of IRSTEA-PReSTI (France) since 1974. Its contents are currently being discussed under ISO TC23/SC18-Irrigation techniques (document ISO DTR 21540), in order to define a standardized testing protocol to evaluate emitter sensitivity to clogging due to solid/mineral particles.Experiments were carried out at the Irrigation Laboratory of Luiz de Queiroz College of Agriculture (ESALQ-USP), Piracicaba, SP, Brazil. Four models of emitting pipes accounting for nonpressure compensating flat drippers were tested under a steady 98.1 kPa pressure . The emi3 h−1, and maximum pressure head = 46 m) injects water and suspended particles from the bottom of the tank to a manifold, which consists of symmetric bifurcations connected to 8 parallel emitting pipe segments of 5 m length  that conveys water back to the tank. The flow velocity into the gutter was relatively high and ensured that all particles were dragged back to the tank. Finally, a screen filter with size equal to 595 μm was installed at the pump outlet in order to trap contaminant debris larger than those added to the tank.The experiments were undertaken at the testing bench shown in m length . The pipμS cm−1.The soil mixture employed during the clogging tests was prepared based on requirements defined by . The mixμS cm−1; (Stage 2) pH=9.32, EC=113.4 μS cm−1; (Stage 3) pH=9.36, EC=107.8 μS cm−1; (Stage 4) pH=9.69, EC=213.1 μS cm−1 . The disinfection procedure aimed to eliminate or reduce the microorganism population that could promote biofilm growth at the initial stages of clogging tests. Secondly, the distilled water was added to the tank and the initial discharge of emitters was measured. Finally, solid particles corresponding to each testing level were added to the tank.The clogging test procedure established by IRSTEA consists of 4 stages varying in the size and concentration of particles added to the water tank . The parThe discharge from 26 drippers of each model was measured every day after 8 h of operation. A precision scale  and a stopwatch were used to determine the flow rate of emitters by weighing the amount of water collected during 30 min. The water temperature and water pH were measured daily using a mercury thermometer and a pH meter, respectively.Three replications were undertaken under similar testing conditions. At the beginning of each replication, the testing bench was cleaned and disinfected (see previous section), and the emitting pipes were replaced with new ones.DC . If DC is higher than 25%, the group of emitters is considered to be clogged. Emitter sensitivity to clogging was evaluated based on the discharge decrease of emitters during the tests. The emitters were considered clogged when their discharge decreased by more than 25% compared with the initial discharge –13. For DC is the degree of clogging (%); qi is the current discharge of emitter i (L h−1); n is the number of emitters whose discharge has been monitored (dimensionless); −1) operated under the testing pressure.Images of clogged emitters were obtained using a Leica M125C stereo microscope. At the end of each replication, emitters were manually opened using a scalpel. Causes of clogging were suggested based on the visual inspection of the labyrinths.μm. The only geometrical difference between both models was the labyrinth depth (a difference of 0.1 mm) and it did not influence dripper sensitivity to clogging. Previous studies had found that clogging resistance capability is more influenced by the geometric design of the flow path than by the cross-sectional area or flow rate of emitters [The results obtained during the experiments are shown in emitters .μm size and 500-ppm concentration. Although that model presents a simpler labyrinth design when compared to the design of B1 and B2, the filtration grid at the flow path inlet and/or the design of the flow path of the model A2 resulted in an efficient mechanism to prevent blockage of drippers. In addition, A2 has the largest dimensions of flow path among the evaluated models of drippers , which reflects particle nature and is diversely influenced by pH and concentration of salts in the aqueous media [ζ deviates from zero, the potential for aggregation becomes lower [In us media , 17. As es lower .The other clogging behavior is related to the accumulation of relatively large particles  at the labyrinths' inlet and at channel corners . The depClogging due to the accumulation of relatively large particles at the labyrinth inlet and at channel corners is also observed in the other models of drippers investigated during the experiments and seems to be the most common type of plugging. Figures Finally, drippers B1 and B2 presented barbs across the flow path resulting from molding imperfections . The depIn each replication, four dripper models were evaluated simultaneously under the same testing conditions. The clogging resistance performance varied according to the model of dripper. The results are consistent with the literature and clearly indicate that emitter design is a key aspect in terms of clogging resistance performance , 22, 23.Comparing the results from three replications, each dripper model always clogged at the same test stage . This inDC) was identified when comparing the results of replications within each model. On a daily timescale, the highest deviations between maximum and minimum values of DC were 16.0, 31.7, and 67.6% for the corresponding models B1, B2, and A1 . Chlorine is widely used for disinfecting water due to its stability and effectiveness . ContinuThe influence of chemical agents of clogging should also be avoided considering that the clogging test procedure aims to evaluate sensitivity to solid particles. Although distilled water was used during the experiments, a significant number of chemical elements might have been solubilized from the soil compound, which was added to the water. The precipitation of ions is affected by several factors, although water pH seems to be the most important. When water pH is maintained lower than 7, most clogging problems related to precipitation of ions are mitigated . An adjuAnother important aspect around the proposed clogging test protocol relates to the mixture of particles. The first concern is related to the solid shape and dynamics of these particles when transported by fluid flow. Suspended particles in irrigation waters are of various shapes and present smooth edges because of wearing processes occurring in the nature. This is the reason why particles are sieved from in-field samples and may present some variability. Standardized commercial compounds could be used to prepare the mixture of particles, but such compounds account for sharp particle edges due to their production process  and usually have higher density than natural particles. The flow dynamics of spherical or smooth-angled shape suspended particles within pipes or labyrinths are quite different from sharp-angled particles. The second concern refers to the mineralogy and chemical composition of soil, since both characteristics may influence particle aggregation phenomena. Probably the nature of clay particles added to the soil compound should be standardized, since aggregation potential relies upon the nature of particles , 17. FinThe clogging test procedure does not allow for separation between clogging caused by concentration and the size of particles. When a test progresses from one stage to the next, the previous water mixture remains in the tank and larger particles are added to the mixture. Moreover, samples of emitting pipes are not replaced from one stage to the next; there is therefore a residual effect that prevents us from differentiating between clogging caused by the concentration and size of particles. The protocol considers that clogging severity is more influenced by particles size range than concentration because when drippers are not clogged at a given stage, they are not considered to be sensitive to the size of particles used at that stage. The idea that clogging rate is more affected by particle size than concentration is also supported by previous research . AssuminFinally, the use of pumps whose impeller and housing are made of cast iron is not recommended. Depending on water and solid particles characteristics, iron particles may detach from inner parts of the pump due to corrosion or abrasion. Such particles may escape filtration and reach the emitters resulting in their clogging or the development of ferro-bacteria biofilm. The use of pumps consisting of impeller and housing made of stainless steel is strongly advised for further researches. In addition, a methodology for real time monitoring of the concentration of particles within the tank or in the pipes would be helpful to monitor the testing conditions.Finally, the number of drippers that should be monitored in a clogging test protocol in order to improve result reliability remains unanswered.The clogging test procedure enabled an accurate assessment of the combinations of concentration and size of particles that caused clogging in each model of dripper.A significant variability in degree of clogging was identified when comparing the results of replications for one model of dripper. The excessive variability might be explained by noncontrolled variables that influenced each model of dripper differently.The experiments demonstrated that the results of clogging tests are quite sensitive and may change significantly due to minor details related to the methodology or testing facilities. Additional requirements and details related to the clogging test protocol and its facilities must be more accurately defined to ensure the repeatability and reproducibility of results.Recalling that the objective of such test is to give trends in sensitivity to clogging of one dripper model, due to its design and its manufacturing quality that may not be revealed by standard hydraulic test (ISO 9261), research is still needed to refine understanding of the influence (or not) of some parameters: pH, conductivity, and particles zeta potential.


Picea jezoensis is completely dependent on coarse woody debris for seedling establishment. To understand the fine-scale spatial genetic structure (FSGS) of this species, a 5-ha plot was established in central Hokkaido, and 531 individual trees were categorized into four life-stages  on the basis of age and size. The FSGS of the established seedlings and later growth stages was investigated using 11 nuclear simple sequence repeat loci. A STRUCTURE analysis of seedlings and saplings established on fallen logs revealed that genetically related individuals were spatially localized between adjacent logs. We also found a significant FSGS in early life-stages based on a decline in the kinship coefficient calculated between individuals over shorter to longer spatial distances. Furthermore, the estimation of dispersal kernels indicated the frequent occurrence of short-distance seed dispersal. These results indicated that genetically related seedlings and saplings regenerated on the same or nearby fallen logs. In contrast to the results for the early stages, mature-stage trees showed no significant FSGS. We ran a simulation to examine the hypothesis that the FSGS could be eliminated by demographic thinning during life history processes. We calculated values for simulated offspring generated under three sets of conditions; i.e., by removing (i) inbred individuals, (ii) randomly chosen individuals, and (iii) all individuals on the specific fallen logs. However, the results for the FSGS were significant for all simulated data sets. This indicated that inbreeding depression, stochastic loss, or eradication of establishment sites by local disturbances alone could not explain the lack of FSGS among mature-stage trees. Therefore, it is possible that the colonization history of mature trees present on the study site might differ from that of the current offspring.Conifers in northern forests, such as fir and spruce, preferably regenerate on coarse woody debris, including fallen logs, stumps, and snags. In northern Japan, the sub-boreal conifer species  Picea, Abies, and Tsuga. These coniferous species successfully regenerate at safe sites, which are most often decaying logs and areas of coarse woody debris (CWD)  , and somis (CWD) , the Pacis (CWD) , and Easis (CWD) . CWD is is (CWD)  and feweis (CWD) . Previouis (CWD) . HoweverAbies sachalinensis, a CWD-dependent regeneration species, A. sachalinensis. In contrast to A. sachalinensis, which mainly regenerates on CWD but can also regenerate directly on forest soil  within wild populations; i.e., the non-random spatial distribution of genotypes , is a coest soil , the esteedlings . Furtherall size . Consequall size . Okada ee-stages . HoweverSp is the reciprocal of the neighborhood size under certain conditions  strong restriction of safe-sites decreases the number of mother trees contributing to offspring and results in a strong FSGS at the early demographic stages and (2) accordingly, the FSGS at the early demographic stage should be weaker at the later demographic stages. Finally, we examined the factors affecting the FSGS, such as inbreeding depression, stochastic loss of individuals, and the eradication of establishment sites by local disturbances using simulation studies.In the present study, we estimated the FSGS in Fraxinus mandshurica var. japonica  was established at the fluvial terrace of the Iwanazawa Forest Reserve, University of Tokyo Hokkaido Forest (UTHF) in central Hokkaido , Japan. The FSGS of co-occurring tree species; namely, japonica , Cercidiaponicum , and Betwicziana  were invP. jezoensis plants and identified four growth stages in the study plot: mature, juvenile, sapling, and seedling. We observed all 123 mature P. jezoensis individuals with a diameter at breast height (dbh) ≥20 cm, and all 39 juveniles with a dbh ≥5 cm but <20 cm. Individual trees with a dbh <5 cm were differentiated into two categories by age as determined by the number of bud scale vestiges. For saplings and seedlings, we selected half of the individuals established on fallen logs in this study, so that 190 saplings aged ≥3 years and 179 seedlings aged <3 years were included in this study. The locations of the areas of CWD and individual trees were identified using a digital compass , and their needle leaf tissues were collected for DNA analysis.In this study site, there were 112 fallen logs the diameters of which were larger than 20 cm at the base. Fallen logs were regarded as a major component of CWD in this forest. We measured individual GC1, GD1, GG3 . The following 11 microsatellite loci, GD1, GG3 , PaGB3, 3, PgGB5 , EATC1E0EATC2G05 , Pj4, Pjand Pj24  were anaFIS was also calculated considering the null alleles using INEst , ranging from 1 to 10. To determine the most likely value for K, we first used the methods suggested in the original description of STRUCTURE, which involves comparing the log probability to the data (ln Pr [X|K]) for each run to evaluate the genetic structure. Then, we calculated ΔK   for all lated ΔK  to deterpriate K  for the Fij (coancestry) /(1–Qm), where Qij is the probability of identity in state between two randomly chosen genes from i and j, and Qm is the average probability of identity by state between two randomly chosen individuals from the reference population. Assuming that the reference population is the parental generation, Fij is expected to have values derived from the pedigree information  , in which spatial distances were permuted randomly among pairs of individuals. Furthermore, to compare the intensity of the FSGS among the four stages, we calculated Sp, a parameter indicating FSGS intensity, using the formula Sp = bF/(F1–1). In this formula, bF is the slope of Fij against the logarithm of the distance between individuals, and F1 is the average kinship coefficient among individuals in the first distance class  in this study. The value for bF was estimated based on the regression of all spatial distances between individuals, and the significance level was calculated using 1000 permutation tests. All the FSGS-related calculations were performed using SPAGEDI ver. 1.4  . In the ll-sibs) . For thever. 1.4 .Fis) as a kinship coefficient between genes within each individual  To examine the deaths of random offspring, we randomly extracted 123 individuals from the offspring data without replacement and repeated this procedure 10 times to generate 10 different datasets. This randomization procedure was conducted using Visual Basic for Applications. In the first and second simulations, the number (123) of surviving offspring was the same as that of mature trees. (3) To determine the effects of local disturbances such as flooding, which may eradicate all offspring on specific CWD, we generated six datasets by excluding all offspring on CWD with (i) the largest number of offspring; (ii) the largest and second largest number of offspring; (iii) the largest, second, and third largest number of offspring; (iv) the largest, second, third, and fourth largest number of offspring; (v) the largest, second, third, fourth, and fifth largest number of offspring; and (vi) more than eight offspring, resulting in 250, 232, 216, 200, 186, and 122 surviving individual data sets, respectively. The number of datasets from (vi) was approximately the same as that of the mature trees. We calculated test statistics for the FSGS of the simulated offspring datasets , to examine whether the FSGS was significant in each dataset. In these simulation-based analyses, all the statistical calculations for the FSGS of actual mature trees, and actual and simulated offspring were determined using the same methods as previously mentioned for the examination of FSGS at each growth stage, but without the reference population.Finally, to examine whether demographic thinning by inbreeding depression, stochastic loss, or both during the transition from adolescent to mature stage affected the FSGS among mature trees, we simulated seedling and sapling deaths under three sets of conditions. We first combined all 179 seedlings and 190 saplings into a dataset of 369 offspring. We then generated the following datasets. (1) To examine the deaths of inbred offspring; i.e., death by inbreeding depression, we generated a set consisting of 123 offspring with low HS) of each growth stage ranged from 0.743 to 0.751, and the allelic richness  ranged from 14.01 to 15.19 (Table FIS) was the highest and lowest in the seedling and mature stages (0.241 and 0.113), respectively. This was also observed for the FIS values  estimated using the Bayesian approach taking into account the null alleles . The 11 loci were polymorphic in mature trees, with 3 to 46 alleles per locus and mean HE values of 0.743 , which all had high null allele frequencies, ranging from 0.090 to 0.185. Therefore, these loci were excluded from the calculations of Fij and Fis. There was no significant linkage disequilibrium between any pair of loci analyzed.The genetic diversity  between successive K values , saplings (-0.013), and seedlings (-0.011) were all significant , but were not significant for mature trees  were much lower than those for juveniles (0.009), saplings (0.014), and seedlings (0.012). This indicated that the FSGS was stronger in the adolescents than in the mature trees.Correlograms of the average Fis) at all four growth stages was significantly high  was the highest, and the values then decreased with life history for saplings , juveniles , and mature trees .The average value of the individual inbreeding coefficient (a), shape parameter (b), average dispersal distance (d), and immigration rate (m) were calculated to be 81.816, 3.097, and 59.9 m and 86.6%, respectively, for pollen dispersal in the seedling stage. Furthermore, the corresponding values for seed dispersal were 3.823, 0.571, and 40.6 m and 74.4%, respectively, for the seedling stage , the F1 and bF values remained significantly high . For the sets of offspring simulated under the two sets of conditions related to random death and eradication of all the offspring on specific CWD, the average Fis, F1, and bF values for all the data sets were significantly high . Furthermore, the Sp values (0.004–0.010) for all the sets of simulated offspring were higher than those of mature trees (0.001).Fine-scale spatial genetic structure values were significant for seedlings and saplings observed on the study site, and for all the sets of simulated offspring under the three conditions, except for mature trees , indicating that the genetic clustering of adolescents was stronger than that of mature trees  values (0.046–0.061) for adolescents were much lower than those expected for half-sibs (0.125). Moreover, the average Fij values for seedlings and saplings for Cluster 1 in the STRUCTURE analysis  were also much smaller than the kinship coefficients expected for half-sibs. The significant FSGS and low F1 values for adolescents indicated that spatially limited seed dispersal should generate a half-sib family structure around seed parents, but this structure may be weakened by the overlapping of seed shadows.The STRUCTURE analysis indicated that adjacent areas of CWD accommodated genetically related progeny at the center of the study plot Figures . This mies Table . AlthougA. sachalinensis, at the same study site, for which the average distance of seed dispersal was 23.7 m  for A. sachalinensis in the same plot . The stronger FSGS for P. jezoensis may be due to its stronger dependency on CWD for establishment than that of A. sachalinensis.Even in the case of anemochory, the seed shadow of each mother tree would create a spatial genetic structure during the initial regeneration process . The pros 23.7 m . It is pame plot , the disFis value was high at the seedling stage and decreased for the later growth stages  in this study was much lower than that of other tree species that relay on wind-pollination and wind-dispersal of seeds (0.002–0.011)   , and tha (0.003) .P. jezoensis. In conclusion, we cannot attribute the decline in the FSGS throughout the life history of P. jezoensis to demographic thinning alone. It is probable that the regeneration processes responsible for each generation have changed significantly over long periods. Thus, not only demographic thinning but also other factors, such as differences in colonization histories between the mature and offspring populations, and seeds derived from multiple sources, should be considered in explaining the low Sp values and lack of FSGS in mature P. jezoensis populations.However, according to the results of the simulations under the conditions of death by inbreeding depression, stochastic loss, and local disturbance, the FSGS values for all simulated offspring datasets were significant  also suggests that the substantial number of areas of CWD accumulated after the initial establishment and regeneration of the younger mature trees. In addition, an artificial disturbance appears to have occurred at the study site in the past. The distribution of juveniles reflected the track of an abandoned forest railway and the present-day forest road. As there are large broadleaf trees growing at the center of the study plot, it seems that few areas of CWD are available for the regeneration of the present juveniles. The limited area available for regeneration may lead to a weak but significant FSGS in the juvenile stage in spite of its small sample size (Figure P. jezoensis (Figures e Figure . Recentlezoensis . In factP. jezoensis. Moreover, our FSGS simulations suggested that present mature individuals may have been established by different disturbance regimes compared to the other demographic stages. It might be one of the characteristics of CWD-dependent tree species that different genetic variations could be created through spatiotemporal differences in the production of areas of CWD.In conclusion, the number and distribution of establishment sites, such as CWD, may be attributable to the FSGS in each generation of KK, CL, and SG designed the study and interpreted the results. KK was responsible for the laboratory work. KK and AN analyzed the data. All authors contributed to the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Direct-to-consumer (DTC) genetic testing is a controversial issue although Korean Government is considering to expand DTC genetic testing. Preventing the exaggeration and abusing of DTC genetic testing is an important task considering the early history of DTC genetic testing in Korea. And the DTC genetic testing performance or method has been rarely reported to the scientific and/or medical community and reliability of DTC genetic testing needs to be assessed. Law enforcement needs to improve these issues. Also principle of transparency needs to be applied. Direct-to-consumer (DTC) genetic testing is a method of marketing genetic tests to consumers without the direct involvement of a health care provider . DTC genThere is a high chance of exaggerated advertisements and abuses of DTC genetic testing. For example, DRD4*7R allele was reported as associated with novelty seeking . PersonsMost important concern is whether commercial genetic services’ predictive value is sufficient to meet the standards for clinical use. The clinical utility of a genetic test should be an essential criterion for deciding to offer this test to a person or a group of persons .Reliability has been one of the major issues from the beginning even in United States. Dr. Craig Ventor reported the discrepancies of the interpretation of the same individuals between 23andMe and Navigenics . GAO  for predictive models among genome-wide association study (GWAS) results from the relevant literatures. In case of single SNPs for complex disease or trait, its predictive performances would be almost meaningless because odds ratio of most validated SNPs are below 1.5 except few SNPs such as Diseases . That isnot all of them. However, when applying to Korean population, the odds ratio of the used SNPs in the predictive model is critical to calculate the relative risk of the individual consumer. But the magnitudes of relative risks of the each validated SNPs between Caucasian and Korean subjects do not show same values. Therefore, the established models for each trait need to be validated for Korean patients prospectively at least. But the most company-run or company-plan-to-run traits show lack of these evidences. It is not possible to judge or estimate which company supports better predictive models or result. This situation drives to go to marketing with exaggerations and abuse of tests or price competition rather than competing to improve the quality of prediction.In case of using multiple SNPs, Some SNPs come from the study of Korean subjects, but many SNPs depend on the results from Caucasian subjects. Considering the experiences from GWAS studies among Korean subjects, most validated SNPs from Caucasian subjects without studying Korean subjects are likely to be replicated among Korean subjects although The current DTC genetic testing needs more objective evidences. If they have a scientific basis from Korean population, even poor performances can have a chance to improve because they know which part needs to be updated or changed. But companies extremely rarely show the method and performance of their predictive model from Korean population.They demand to the government authorities to allow disease prediction services as DTC genetic testing including cancer, diabetes and heart disease without disclosing their models and performances to the scientific or medical societies. If it is allowed, it will cause the profound confusion or chaos to the consumers, medical institutions, and health insurance system and the huge waste of medical resources. Currently the facility and human resources in DTC genetic testing company are not regulated by law. There is no penalty to their rejection to proficiency test by law. For improving the situation with the above mentioned direction, the legal coercion is inevitable. Amendment of Bioethics and Safety Act or making a law equivalent to Clinical Laboratory Improvement Act (CLIA) in United States for appropriate regulation is a prerequisite step.The predictive models of DTC genetic testing need the validation before implementation. But practically not all of the tests can be validated or achieved to the certain standard.transparently for the right choices by the consumers instead can be considered. The relevant information includes the explanation of the whole process of the tests, the references of the method they used, disclosing the implication and limitation of the results, how to deliver the report to consumers etc. And the company should consider the possible outcomes after getting trait(s) risk and provide the report guidelines not to cause any harm or negative effect to the consumers including psychological distress or waste of medical resources.If so, disclosing all the relevant information New genomic technologies and knowledges expand our view and their applications would improve human health. Medical diagnosis and treatment is shifting to genetic based precision medicine. Its progress is strictly guided by evidence-based medicine (EBM). In contrast, if DTC genetic testing with lack of equivalent level of EBM is not regulated, our society will pay a lot.