The MiSeq platform was used for sequencing of key genes and some of their subtypes in the ADRB2 signaling pathway in lung tissues, and target gene expression was normalized and calculated according to reads per kilobase million.phenylethanolamine N-methyltransferase (PNMT), ADRB2, AC, AKAP and EPAC subtypes , and various phosphodiesterase (PDE) subtypes , with persistently high expression of AC6, PDE4B, and AKAP maintained throughout the lung-developmental process, PNMT, ADRB2, AC(4/6), PDE4B, and AKAPwere highly expressed at the neonatal stage.At different lung-developmental stages, we observed expression of AC6, PDE4B, and AKAP showed persistently high expression during the entire lung-developmental process, which provides a reference for the development and utilization of key gene subtypes in this pathway.During normal lung development in rhesus monkeys, key genes associated with ADRB2–G protein–AC signaling and some of their subtypes are almost all expressed at the neonatal stage, suggesting that this signaling pathway plays a role in this developmental stage. Additionally,  ADRB2 agonists are mainly used to treat neonatal wet lung syndrome , bronchoADRB2 is the main subtype of βAR in human lung. ADRB2 is a downstream effector of the adrenergic signaling pathway and specifically binds adrenaline synthesized by phenylethanolamine N-methyltransferase (PNMT) catalysis of noradrenaline . The binThere are 11 families and 30 subtypes of PDE in humans, of which PDE4, PDE7(A and B), and PDE8 (A and B) have high specificity for cAMP , 11. PDEIn this study, we measured and analyzed spatiotemporal changes in key genes intimately associated with the ADRB2–Gs–AC signaling pathway and some of their subtypes during lung development in rhesus monkeys, particularly during the neonatal stage. The findings provide a theoretical basis for rational drug use in the neonatal stage and references for the development and utilization of some key gene subtypes in this pathway.n = 1; and Day 70, n = 1), the middle of embryonic development , late embryonic development , the neonatal stage , and adolescence . All animal experiments complied with the ARRIVE guidelines ; however, ADRB2 expression gradually increased with age  revealed that ngs Fig. e. Moreovngs Fig. f. AC7 wault Fig. g. AC9 waage Fig. h.PDE3B showed an increasing expression trend in late pregnancy, neonatal period and adulthood and EPAC1 expression were stable during all developmental stages  was mainly expressed in the lung in adulthood  were mainly expressed in the lung in addition to the intestinal tract , suggesting that the respiratory tract is connected to the external environment after birth, and that various natural stimuli require dynamic adaptations (such as airway relaxation). Adrenaline expression is an important method of bodily adaptation; therefore, PNMT expression might represent a preparatory mechanism for increasing adrenaline. These findings suggest that a role of elevated PNMT expression might be to prepare the fetus for birth maintaining its health at the neonatal stage.PNMT is a rate-limiting and essential enzyme that catalyzes the methylation of noradrenaline to adrenaline , and theADRB2 and PNMT expression showed persistent and progressive increases in the late gestational and neonatal stages; however, in contrast to PNMT, ADRB2 expression increased with age and showed differences in expression only in the liver before and after delivery. Moreover, ADRB2 expression in the lungs at the neonatal stage was not significantly different, whereas significant differences were present at the adolescence stage. This confirms that the density of ADRB2 receptor is related to age, reaching adult levels at school age [Both hool age . ADRB2 ihool age . These fhool age . FurtherAC8, AKAP4/5) associated with the ADRB2–Gs–AC signaling pathway were expressed in the lungs at the neonatal stage. These results indicated that this signaling pathway might play a role in airway dilation during the neonatal stage.The results showed that all subtypes  in cultured hASM cells [We speculated that AC6 might play an important role in this signaling pathway. Previous studies report that transcripts of all AC subtypes, except  in hASM . Xu et aSM cells , and ShaSM cells . These f2+ fluxes affecting contractility. PDE3 inhibitor is important in preventing mast cells predominantly in the ASM layer degranulation. Therefore, it is an acute bronchodilator in humans [PDE3 and PDE4 were expressed during the entire lung-development process. We speculate that the application of double PDE3/4 inhibitors may be feasible. Moreover, we found that PDE4B was highly expressed during the entire lung-development process. Previous studies proposed that PDE4B and PDE4D played critical roles in airway cells [PDE3 and PDE4 are the two major cAMP-hydrolyzing enzymes in ASM. PDE3 is an enzyme hydrolyzing both cAMP and cGMP, but the rate of hydrolyzing cAMP is 10 times that of hydrolyzing cGMP. PDE3 and PDE4 can regulate different cAMP pools because they are located in different parts of the ASM , 25. PDEn humans –29. Howen humans , 28. Desn humans –32. Somen humans . In thisay cells , 35, PDEay cells , 37. We AKAP were highly expressed during the entire lung-development process. Previous studies have shown that there are protein and/or mRNA expressions of AKAP subtypes in human ASM. Especially, AKAP12 and EZR were highly expressed [8/9/12/13, and EZR) play an important role in this pathway, especially EZR. Further studies are needed to confirm the expression of AKAPs subtypes in ASM.AKAP has been shown to regulate intracellular cAMP localization and regulate ADRB2 signaling in human ASM . We founxpressed . EZR(alsxpressed . We specHowever, our study has some limitations. First, because our samples were limited, we did not perform reverse transcription polymerase chain reaction analysis to confirm expression levels, western blotting analysis to detect protein levels, or immunohistochemical analysis to determine protein localization. We focused solely on the expression of key genes in this signaling pathway in the entire lung and did not measure the expression and activity in specific cell types in ASM; therefore, this requires further study. Second, the sample size of rhesus monkeys in this study was low. Moreover, there might be species-specific differences in the expression of various subtypes. Therefore, it remains unclear whether these results truly reflect human lung development.We compared the expression levels of key genes associated with the ADRB2 signaling pathway at different developmental stages in the lungs of rhesus monkeys. We found that almost all key genes of this classical signaling pathway are expressed at the neonatal stage, which provides references for correct application of therapeutics for diseases associated with this signaling pathway in neonates. Furthermore, our findings that the expression of specific subtypes dominated during lung development provide novel insights that will promote the development of novel strategies for treating respiratory diseases.


Rhododendron spp. across different elevations of Hehuanshan and Taipingshan forests in Taiwan. The isolation frequency and diversity of foliar endophytes were higher at low elevations than at high elevations. The foliar endophyte community structure differed as a function of host family and forest vegetation type. Elevation, mean annual temperature, and precipitation were significantly correlated with the community structure. Fungal growth rate was correlated with the endophyte abundance, which indicates that fast-growing fungi might have a competitive advantage when coexisting with other fungi in a plant host.Fungal endophytes inhabit plant tissues without causing disease symptoms. They are highly diverse and distributed globally in all plants that have been investigated. Host, geographic, and environmental effects on endophyte communities have been reported in several studies, but the direct effect of fungal growth rate on endophyte composition has not been tested. To understand the relationship between foliar endophyte composition and fungal growth and to examine the effect of host, elevation, and climatic factors on the foliar endophyte communities, this study examined the foliar endophyte communities of representative gymnosperms and  Fungal endophytes are fungi that inhabit plants without causing disease symptoms. Several studies have reported the beneficial roles of symbiotic endophytes. For instance, endophytes can improve plant resistance to pathogens, herbivores, heat, and drought ,2,3,4. ADiversification, dispersal, selection, and drift are the major processes of microbial community assembly, and each of them could affect the spatial or temporal patterns of a community . Spore dA recent study showed that the early colonization of endophytes in plants is an opportunistic process  though fTaiwan is a biodiversity hot spot located at the boundary of subtropical and tropical areas of the western Pacific coast. The unique geographic locality, climatic condition, and steep elevation gradients result in diverse vegetation types: from tropical forests at low elevation to subalpine zones at mountain tops above 3600 m . The verRhododendron species around. Since no single plant species can grow across ca. 2500 m elevational differences, at least one dominant gymnosperm species was paired with a nearby Rhododendron species at each site. Three trees of each species were sampled as replicates. The middle elevation area of Hehuanshan was not sampled due to the severe human destruction in the area. Occasionally, more than one gymnosperm species, or less than three trees of one species, were collected from a site according to changes in the forest vegetation along the trails and their availability. In total, thirty-five trees were sampled in March and April of 2018. Since the sampling processing after sampling was time-consuming, the sampling could not be done on the same day. However, all trees were sampled in spring to reduce the variations caused by sampling times. Three, ca. 30 cm long branches with leaves were collected from each tree, put into a Ziploc bag and transferred to the lab for processing within 72 h. Leaf branches of Rhododendron spp. were collected near the top of the shrubs; leaf branches of gymnosperms were cut at ca. 4 to 5 m above ground. The detailed information of sampling sites and trees is listed in Healthy leaves were collected from two montane areas in central and northeast Taiwan, Hehuanshan and Taipingshan, respectively . For eacRhododendron spp., three leaves were selected from each of the three branches/tree, rinsed with tap water, and cut into 2 mm2 segments. For gymnosperms, leaves are not countable because of the needle or scale-like structure. Instead, three short branches about 10 cm long were selected from each of the three branches/tree and rinsed with tap water. All leaves from the 10 cm branches were cut into ca. 2 mm2 segments. The leaf segments from each tree were then pooled together and soaked in 95% ethanol for 10 s, 0.5% NaOCl for 2 min, and 70% ethanol for 2 min. After surface sterilization, fifty leaf segments from each tree were randomly selected and placed on a 2% malt extract agar (MEA) slant in individual 1.5 mL microtubes. The resultant 50 MEA slants with leaf segments from a single tree were then stored in a Ziploc bag and incubated at room temperature (between 22–30 °C) for 2 months. Each week, the slants were examined for fungal growth and, once observed, a small piece of mycelia was collected for DNA extraction (see below) and cultured onto two plates (6 cm in diameter) of 2% MEA. In total, 484 fungal endophytes were isolated from the 1750 leaf segments (50 leaf segments/tree × 35 trees). After a few months of growth, the cultured agar was cut into 8 mm cubes, stacked in a sterile 2 mL cryotube, and filled with double-autoclaved distilled water for preservation and further studies. The isolation frequency was calculated as the percent of isolates from each sample tree and was ln-transformed for normality.Endophytes were isolated from surface-sterilized leaves. For TM Plant Tissue PCR Kit . Mycelia were first disrupted with Zirconium oxide beads  in 100 µL of extraction buffer shaken at 30 times per second for 1 min using a TissueLyser II . After incubation at 95 °C for 10 min, 100 μL of dilution solution was added and gently mixed. The genomic DNA was stored at −20 °C until PCR amplification. ITS1F/LR3 primers  [TM PCR ReadyMix, and 7.4 μL of sterile distilled water. To confirm PCR success, PCR products were loaded in a 1% agarose gel in 1X TAE buffer with 5% HealthView nucleic acid stain  and run for 25 min at 100V. Gels were visualized using a G:Box Mini with GeneSys software . If the amplification with ITS1F/LR3 primers was unsuccessful, ITS1F/ITS4 primers (ITS4: 5′-TCCTCCGCTTATTGATATGC-3′) [Genomic DNA was extracted from fungal mycelia using the REDExtract-N-AmpAGAC-3′) ,23 were ATGC-3′)  were useThe PCR products were diluted with 5 μL of sterile distilled water and cleaned with 1 μL of ExoSAP-IT PCR Product Cleanup Reagent  at 37 °C for 1 h followed by another incubation at 80 °C for 15 min. For each sample, 6 μL of cleaned PCR product and 1 μL of 10 μM primer were sent to the DNA Sequencing Core, College of Medicine, National Taiwan University for Sanger sequencing by ABI 3730 xl DNA Analyzer  with BigDye Terminator v3 Cycle Sequencing Kits . Each isolate was sequenced bidirectionally, and both sequences were assembled with Phred and Phrap ,26 in MeAll sequences were clustered into operational taxonomic units (OTUs) using the “sanger_otu_cultering_workflow” on the Mobyle SNAP Workbench ,30. The To determine the phylogenetic placement of each isolate at class level, all sequences were submitted to the Tree-Based Alignment Selector toolkit (T-BAS) ,34 for aRepresentative isolates of the 94 OTUs were individually cultured on a 6 cm plate of 2% MEA. Mycelia of most isolates covered the whole plate within 4 to 10 days. Slow-growing isolates were grown until the radius of colony reached 1 cm before transfer. A 4 mm agar disc containing mycelia cut from the edge of colony was placed on a 9 cm plate of 2% MEA. The colony radius was measured 7 days after transfer . The radMultiple regression models were built to analyze the effects of host family and elevation on isolation frequency and diversity. Effect of host was tested at family level because host specificity of endophytes is mostly confined to the family level, not genus or species levels . SamplesTo test if the leaf endophyte composition of vegetation correlated with environmental factors, fungal class dissimilarity and environmental dissimilarity were calculated and their correlation tested with a Mantel test. The Morisita–Horn index was used to estimate the dissimilarity of class-level composition between vegetation types. Environmental dissimilarity was calculated as the Euclidean distance of factor scores from principal component analyses that included the annual mean temperature, mean diurnal temperature range, annual precipitation, precipitation seasonality, and elevation. In addition, multiple regression on distant matrices (MRM) was used to test the correlation between the community composition of foliar endophytes and climatic factors. To examine the correlation between fungal growth and the composition of leaf endophyte communities, a bootstrap Kolmogorov–Smirnov test (K-S test) was used to test the similarity between fungal growth distributions and OTU abundance in a sample tree. The fungal colony radius on day 7 represented the fungal growth of an OTU; the number of isolates of each OTU represented the OTU abundance. Sample trees with more than one OTU were selected for analysis. In each tree, singleton OTUs were excluded from the analyses.All the statistical analyses and the climatic data acquisition were conducted in R version 3.5.2 using vegan, ecodist, Matching, and raster packages.This study isolated 484 endophytes from 1750 leaf segments of 35 host plants at 6 sample sites in Hehuanshan and Taipingshan. The overall isolation frequency was 27.7%, which was calculated as the percentage of the total number of isolates to the total number of leaf segments. OTU clustering based on 95% sequence similarity resulted in 94 OTUs . Phyloge2 = 0.8073, F = 17.6, p < 0.001). Individually, host family and elevation were significant factors, while montane area was not  = 12.8, p < 0.001). The effects of host family, elevation, and area were tested by ANOVA, and the results showed that leaf endophyte diversity differed as functions of host family and elevation . Additionally, the dissimilarity of class-level composition between vegetation types was significantly correlated with the environmental dissimilarity (p = 0.014) and precipitation seasonality (p = 0.009). These results suggest that environmental factors could shape the endophyte community in different vegetation types.The phylogenetic composition at the class level reflected the different compositions of leaf endophyte communities between forest vegetations . Fisher’milarity . MRM indThe distributions of fungal growth and OTU abundance in sample trees are shown in Rhododendron spp. across different elevations in the Hehuanshan and Taipingshan forests of Taiwan. Both isolation frequency and diversity decreased as the elevation increased. Community structures differed as a function of host family and forest vegetation. The preliminary phylogenetic analysis indicated different phylogenetic compositions between forest vegetation types. Environmental factors were also correlated with community structure and composition. In addition, fungal growth was a critical factor for leaf endophyte community composition.This study examined the leaf endophyte diversity of representative gymnosperms and Pinus ponderosa along an elevation gradient at the southwestern United States shows a trend for higher abundances of foliar endophytes at low elevations [Communities changing with elevation has been reported in macroorganisms, while the diversity of some microorganisms has no clear association with the elevation gradient ,39,40. Sevations ; an illuevations . In the Endophytes are horizontally transmitted between plants through spores dispersed in the air . While eVegetation integrates the effects of host and environment at a specific elevation. In general, temperature decreases and precipitation increases along with the elevation, though precipitation may differ by area. This study was unable to separate the effect of elevation from precipitation or temperature due to the collinearity between elevation and the climatic factors, but the results still indicated the integrated effects of environmental factors on leaf endophyte communities. In addition, the changes in the climatic factors alter the plant composition in the forest; thus, forest vegetation and elevation are highly correlated ,49. HoweRecently, high-throughput sequencing has been widely used in microbial ecology to study the environmental samples. Johnston et al.  comparedAn endophyte community consists of multiple fungal species from a regional species pool, and the host and environment are external filters that affect endophyte community assembly . After fThis study examined the effects of host, elevation, and climatic factors on leaf endophyte communities. Vegetation integrated the effects of these factors and was related to endophyte compositions. The intrinsic fungal growth rate was correlated with OTU abundance in the communities, which indicated that fast-growing fungi might have a competitive advantage when coexisting with other fungi in a plant host. These findings add information to our understanding of endophyte communities in Taiwan and provide evidence for the importance of host, environment and fungal growth in endophyte community assembly.


Supplemental Digital Content is available in the text. 2). Associations were in the same direction but marginally weaker for venular tortuosity and diameter. This study assessing the retinal microvasculature at scale has shown clear associations between retinal vessel morphometry, BP, and arterial stiffness index. These observations further our understanding of the preclinical disease processes and interplay between microvascular and macrovascular disease.To examine the baseline associations of retinal vessel morphometry with blood pressure (BP) and arterial stiffness in United Kingdom Biobank. The United Kingdom Biobank included 68 550 participants aged 40 to 69 years who underwent nonmydriatic retinal imaging, BP, and arterial stiffness index assessment. A fully automated image analysis program  provided measures of retinal vessel diameter and tortuosity. The associations between retinal vessel morphology and cardiovascular disease risk factors/outcomes were examined using multilevel linear regression to provide absolute differences in vessel diameter and percentage differences in tortuosity , adjusted for age, sex, ethnicity, clinic, body mass index, smoking, and deprivation index. Greater arteriolar tortuosity was associated with higher systolic BP , higher mean arterial pressure, 1.3%; 0.9, 1.7% per 10 mmHg, and higher pulse pressure . Narrower arterioles were associated with higher systolic BP , mean arterial pressure , PP , and arterial stiffness index (−0.12 µm; −0.14, −0.09 µm per ms/m It has long been thought that the microvasculature may play a pivotal role in the pathogenesis of CVD.2 Optimal vascular architecture achieves the most efficient blood flow with minimum energy allowing for maximum vascular diffusion.3 Therefore, alterations in geometry of the retinal vasculature may reflect a state of dysfunction of the microvasculature more generally and provide a noninvasive marker of lifetime CVD risk factor load4 and potentially predict disease development.5Heart disease remains a leading cause of death internationally,8 the onset of hypertension9 and with an increased risk of incident CVD events and mortality.2 Evidence is emerging to suggest an association between tortuosity and an increased risk of stroke,10 and a growing body of evidence has suggested associations with arterial stiffness, indicating systemic effects.11 To date, limited research has been undertaken to assess the association of retinal vessel tortuosity with BP, and the direction of associations observed with vessel tortuosity have been inconsistent.13 Previous smaller studies have shown an increase in vessel tortuosity (both arteriolar and venular tortuosity) with higher BP,8 while others have shown an association in the opposite direction or no association at all.14Retinal arteriolar narrowing has consistently been associated with higher blood pressure (BP)15 using a deep learning approach has provided some encouraging evidence on the potential value of risk prediction utilizing retinal imaging at scale. While retinal vessels seem to be key areas of interest, understanding how these models predict risk still remains a substantial problem of the deep learning approach. Retinal vessel morphometry assessment provides insight into the nature of vessel changes, furthering our understanding of potential mechanistic pathways. We examined the association of retinal vessel morphometry 16 with blood pressure (BP) and arterial stiffness in United Kingdom Biobank (UKBB).With recent advances in technology, a study by Poplin et alhttps://www.ukbiobank.ac.uk/scientists-3/. UKBB recruited >500 000 people aged 40 to 69 years, between 2006 and 2010, from across the United Kingdom. A subset of 68 550 participants from 6 UKBB centers had retinal images captured. The Cohort profile: design and methods in the eye and vision consortium of UKBB has recently been published.17 The eye and vision substudy has a very similar profile to the main UKBB study. Nonmydriatic fundus cameras (Topcon 3D OCT-1000 Mk 2) with a 45° field-of-view were used to capture color images. Images were saved in PNG format with a resolution of 2048×1536 pixels.Anonymized data will be made available by the UKBB on request 18 and the average of the measures used in the analyses. A question on medication for BP was used to define antihypertensive medication usage. Hypertension was defined as having systolic BP≥140 mm Hg, or diastolic BP≥90 mm Hg, or self-reported use of antihypertensive medication. Mean arterial pressure (MAP) was calculated as [(2×diastolic BP)+systolic BP]/3. Mean pulse pressure (PP) was calculated as systolic BP minus diastolic BP. History of myocardial infarction, stroke, and diabetes mellitus were determined by self-report. The HbA1c assay was performed using 5 Bio-Rad Variant II Turbo analyzers. Analysis of serum biomarkers utilized 10 immunoassay analyzers .Age, sex, and other sociodemographic characteristics  were collected by questionnaire. Height was measured to the nearest centimeter (cm) using a Seca 202 height measure, and a Tanita BC-418 body composition analyzer was used to measure weight to the nearest 0.1 kg. Body mass index (BMI) was calculated as weight (kilograms) divided by height squared (square meters). Two BP and heart rate measurements were taken with an Omron 705 IT electronic BP monitor, while seated at least 1-minute apart16 In brief, the automated system obtained thousands of measures of diameter and tortuosity from the whole retinal image.20 These measures were summarized using mean diameter and tortuosity, weighted by vessel segment length, for arterioles and venules separately for each image. The following image processing modules were all validated on a subset of 4692 retinal images from a random sample of 2346 UKBB participants: including vessel segmentation, image quality score, optic disc detection, vessel width measurement, tortuosity measurement, arteriolar venular recognition.21 The performance of the Arteriole/Venule recognition had detection rates of up to 96% for arterioles and 98% for venules when the automated probability of arteriole or venule was set to a cutoff of 0.8. An automated assessment of image quality was also made based on the segmented vasculature.21 The algorithm achieved a sensitivity of 95.3% and a specificity of 91.1% for the detection of inadequate images.20 A model eye was used to quantify the magnification characteristics of the Topcon 3D OCT-1000 Mk 2 fundus camera, allowing pixel dimensions of vessel diameter to be converted to real size.22Retinal images were processed using an automated computerized system .23 Participant’s height (meters) was divided by the time between the peaks of the pulse waveform to obtain the Arterial Stiffness Index (ASI) in meters per second.Arterial stiffness was measured noninvasively using the pulse waveform obtained at the finger with an infra-red sensor .The UKBB study was approved by the Northwest Region NHS research ethics committee.P for interaction were <0.01 given the large sample size of UKBB.Stata 15.0 IC  was used to analyze the data. Retinal vessel diameters were normal distributed, measures of tortuosity were positively skewed, and therefore log-transformed. Multilevel linear regression models adjusting for age, sex, ethnicity, and UKBB center as fixed effects, with a random effect for person to allow for repeated measures of vessel indices within the same person (model 1), were used to examine associations of cardiometabolic risk factors with retinal vessel outcomes. Model 2 extended model 1 with further adjustment for smoking, Townsend deprivation index, and BMI. Model 3 included the same factors as model 2 with additional adjustment for total cholesterol, triglycerides, and HbA1c and exclusion participants with a history of myocardial infarction, stroke, diabetes mellitus, on treatment for hypertension or who answered unknown or declined to answer. Data missing on categorical variables were included as an additional category for each variable, to minimize data loss. Associations with the log-transformed tortuosity were exponentiated to give percentage differences in tortuosity and absolute differences (in microns) in vessel width per specified increase with CVD risk markers. Associations between retinal markers and each CVD risk factor were examined for interactions with sex; interactions were considered statistically significant if the A total of 54 714 participants (79.8% of UKBB participants who underwent fundus imaging) were included in these analyses  rise in arteriolar tortuosity. Associations observed were unaffected by additional adjustments in model 2 or after removal of participants with history of CVD and with adjustment for total cholesterol, triglycerides, and HbA1c (model 3—Table S1 in the online-only Data Supplement). Increased retinal venular tortuosity was associated with higher systolic BP, diastolic BP, MAP, PP, and ASI (model 1). For example, a 10 mmHg rise in PP was associated with a 1.1% (0.8%–1.3%) rise in venular tortuosity. Adjustment for factors in model 2 marginally attenuated the association of venular tortuosity with systolic BP and PP, but each association remained highly statistically significant. However, associations were no longer significant for MAP or ASI. Removal of participants with history of CVD  and adjustment for total cholesterol, triglycerides, and hbA1c did not alter these associations but the association with diastolic BP was no longer statistically significant. The association of increased tortuosity (both arteriolar and venular tortuosity) with higher systolic BP remained after adjustment for retinal diameters.Increased arteriolar tortuosity was associated with higher systolic BP, diastolic BP, MAP and PP, after adjustment Table , model 1P=8.59×10−23) decrease in arteriolar diameter, which would equate approximately to 0.33 µm narrowing per SD increase in ASI. Adjustments in model 2 had no impact on the association of narrower arterioles with higher systolic BP, diastolic BP, MAP, PP, heart rate, and ASI. Additional adjustment for systolic BP had little impact on the association of ASI with arteriolar diameter. The associations with CVD risk factors were unaffected by removal of those with history of CVD or adjustment for total cholesterol, triglycerides, and HbA1c . Narrower venules were also associated with higher systolic BP, diastolic BP, MAP, and PP. Wider venules were associated with increased ASI and heart rate . None of the associations were in opposing directions and the results are presented for men and women combined.In this study, the first to examine the retinal microvasculature at scale using fully automated software (with over 3.5 million vessel segments from over 50 000 participants), we have shown novel associations between retinal microvascular tortuosity with BP and ASI and confirmatory associations with diameters. Each significant risk factor expressed in deciles Figure  showed sP as small as 1×10−300. The retinal microvasculature abnormalities observed with increased BP have not been so clear until now . Disagreements between studies may be because of smaller sample sizes together with measurement error , where there is less certainty over the presence or absence of underlying associations.The key findings from the present study were that increased retinal tortuosity (arteriolar and venular) showed strong graded associations with higher BP, MAP, and PP, with w Figure . The Eur24While tortuosity (arteriolar and venular) was higher overall among women compared with men, in the present study, the association between CVD risk markers and tortuosity was evident in both sexes (and independent of further adjustment for height). Overall, the association of increased tortuosity (both arteriolar and venular tortuosity) with higher BP in the present study was independent of retinal diameters. This suggests that tortuosity reflects different structural vascular changes from those shown for diameters and may provide additional value to CVD risk prediction tools beyond the current testing of diameters.10 In UKBB, there was no evidence of an association between self-reported stroke and arteriolar tortuosity. This difference may be related to differences in age (UKBB participants are relatively younger) and case ascertainment of stroke. In UKBB, presence of stroke was based on self-report, while in the other studies it was based on clinical information from health records.10 Conversely, in the present study, increased venular tortuosity was strongly associated with stroke for which some evidence has been reported previously.10 The discrepancies overall in findings from earlier studies with tortuosity may relate to the limited sample sizes, characteristics of the population  or the time placement of the study. For example, treatment has improved over time and therefore it may be harder to observe changes associated with a history of events in more recent studies compared with studies from 10 to 20 years ago. Given the strong graded association of CVD risk factors with tortuosity (both arteriolar and venular) in the present study of over 50 000 participants and the replication of the findings observed in EPIC,8 the associations between tortuosity and CVD risk factors are now very clear warranting verification in a large longitudinal follow-up study, to confirm causality.The association of retinal tortuosity with stroke has not been well studied, although 2 studies have shown a more tortuous retinal microvascular network with prevalent stroke.25 and meta-analysis,9 supporting the finding of the present study .8 The impact of hypertension treatment duration could not be assessed, so we are unsure of the impact of treatment duration on the association between retinal measures and BP, though we did note that removing those on BP treatment did not alter the associations observed. The PWV device used a nonreference method to estimate PWV, which is not as accurate as other approaches and may have led to an underestimation of the association, particularly given that pulse pressure shows stronger associations than PWV in this study. However, reassuringly, the association is in the expected direction and a P of 2.9×10−24 is highly unlikely to be a chance finding. This lends weight to the null association observed between PWV and vessel tortuosityThe strength of this study includes its large sample size of over 50 000 participants. The QUARTZ software is fully automated, incorporates convolutional neural network technology, and utilizes information from all vessels extracted within an image, providing precise measurement. A potential limitation is the use of the entire retinal image compared with a section of the image used by other grading systems; however, given our findings are consistent with previous literature, this is unlikely to be a major issue. The study limitations include the cross-sectional study design, meaning the issue of causality cannot be resolved until further follow-up. Although the UKBB is not a representative sample of the United Kingdom population, which is an issue if one were attempting to determine prevalence of outcomes or genetic associations in particular.This study assessing the retinal microvasculature at scale has shown clear associations between retinal vessel morphometry, BP, and ASI. These observations further our understanding of the preclinical disease processes and interplay between microvascular and macrovascular disease.http://www.ukbiobankeyeconsortium.org.uk/people).The project was funded by a grant from the British Heart Foundation (PG/15/101/31889). This research has been conducted using the United Kingdom Biobank Resource under Application Number 522. The list of UK Biobank Eye and Vision Consortium members is available from the consortium website (None.


The available arable land is unable to fulfill the food production need of rapidly the exponentially growing human population in the world. Pesticides are one of those different measures taken to meet this demand. As a plant growth regulator to block gibberellin, paclobutrazol (PBZ) is used excessively throughout the world to promote early fruit setting, and to increase seed setting which might be harmful because PBZ is a very stable compound; therefore, it can bioaccumulate into the food chain of an ecosystem. In the present study, we discovered unexpected effects of PBZ on zebrafish larvae and adult behaviors by challenging them with low dose exposure. Zebrafish larvae aged 4 days post-fertilization (dpf) were exposed for 24 h at 10 µg/L (0.01 ppm) and 100 µg/L (0.1 ppm) of PBZ, respectively, and adults were incubated at 100 µg/L (0.1 ppm) and 1000 µg/L (1 ppm) concentrations of PBZ, respectively, for fourteen days. After incubation, the locomotor activity, burst, and rotation movement for the larvae; and multiple behavioral tests such as novel tank exploration, mirror biting, shoaling, predator avoidance, and social interaction for adult zebrafish were evaluated. Brain tissues of the adult fish were dissected and subjected to biochemical analyses of the antioxidant response, oxidative stress, superoxide dismutase (SOD), and neurotransmitter levels. Zebrafish larvae exposed to PBZ exhibited locomotion hyperactivity with a high burst movement and swimming pattern. In adult zebrafish, PBZ resulted in anxiolytic exploratory behavior, while no significant results were found in social interaction, shoal making, and predator avoidance behaviors. Interestingly, high dose PBZ exposure significantly compromised the innate aggressive behavior of the adult fish. Biochemical assays for oxidative stress, antioxidant response, and superoxide dismutase (SOD) showed significant reductions in their relative contents. In conclusion, for the first time, our behavior assays revealed that chronic PBZ exposure induced behavioral alterations in both larvae and the adult zebrafish. Because PBZ is a widely-used plant growth regulator, we suggest that it is necessary to conduct more thorough tests for its biosafety and bioaccumulation. Pesticides are considered as one of the major factors in the development of agricultural yield in the 20th century . OrganicDanio rerio) [Sebastiscus marmoratus) have been reported in previous studies [The level of PBZ contamination in the soil is estimated to be 150 mg/kg, and the concentration in the groundwater is estimated to be 4.2 µg/L (0.0042 ppm) and the surface water of the river is 119.6 ng/L (ppb) . Chemicao rerio) . Impairm studies ,12. Furt studies ,14. Far  studies . Keeping studies . Therefo studies .Zebrafish is a widespread model for research in pharmacological and other chemical compounds owing to their salient features and behavior phenotypes as well as well-matched concurrence with rodent model experiments. A comprehensive set of behavioral test panel is available for zebrafish. They are most frequently studied for analyzing the underlying mechanism of the pharmacological compounds that alter behavior with many other findings, which can be translated in compiling results and data in large amounts leading to find a cure that can be translated for mammals and human disorders. The acclimation ability of zebrafish to a new environment makes it a good model for behavior and neuroscience studies ,18. The Earlier studies reported that low dose PBZ has an acute toxic effect on antioxidant enzymes in fish  and thisIn this study, we evaluated the locomotor activity in zebrafish larvae after exposure of 10 µg/L (0.01 ppm) and 100 µg/L (0.1 ppm) of PBZ as acute toxicity test. For chronic toxicity tests, we evaluated the behavioral changes in adult zebrafish after exposed to 100 µg/L (0.1 ppm) and 1000 µg/L (1 ppm) of PBZ. The PBZ dosage used in this study is closed to the environmental concentration been reported in groundwater and surface water of the river [The locomotor activity is one of the major activity controlled by the brain and muscle, any impairment to the locomotion activity of the zebrafish directly indicate the side effects the tested chemicals have caused on to the model organism, in our case, the locomotor activity of larvae was measured by the considering three major endpoints like total distance traveled, burst movement and rotatory movement by each group. The total distance traveled in 10 µg/L (0.01 ppm) PBZ-treated group reduced in both dark and light periods. On the other hand, 100 µg/L (0.1 ppm) PBZ treated group of larvae covered the higher distance in the dark period and lower in the light period as compared to the control group B,C. WhenNext, we measured the burst movement in zebrafish larvae. Burst movement is a spontaneous movement of zebrafish larvae in response to stimuli and can be used as an indicator to evaluate how larvae fish copy with environmental challenges. Generally, during positive phototaxis (testing arena is dark), a rate of burst initiations elevated. The previous study discovered that ventromedial serotonergic neurons played an important role in zebrafish larvae burst swimming . In addiKeeping in mind the circular shape of the 48-well plate used in locomotion test, fish rotation or its rotatory movement were also calculated. Rotation reflects motor aspects of zebrafish swimming and their movement orientations . The rotThe exploratory behavior in the novel environment for adult zebrafish was analyzed after fish were continuously exposed to 100 µg/L (0.1 ppm) or 1000 µg/L (1 ppm) PBZ for 14 days. The fish were individually transferred into the novel tank, and their locomotor activity was recorded for the first minute immediately after animals were transferred to the testing tank. Then fish exploratory activity toward a novel environment was video recorded at 4–5, 9–10, 14–15, 19–20, 24–25, and 29–30 min for comparison C. The viMirror biting assay is an efficient and simple method to analyze the aggressive behavior of fish. Aggressive behavior was evaluated by placing a mirror to the back sidewall of the tank and counting how the high frequency of the tested fish interact with their mirror images. After PBZ exposure, zebrafish were transferred to the assay instrument, and videos were recorded for five minutes after one minute of acclimation. The entry of the fish most towards the mirror would be considered an opponent and most away from the mirror as avoidance. Less mirror biting interesting was observed in fish exposed to 1ppm PBZ showing marked lower mirror biting percentage A and dur(Amatitlania nigrofasciata) into a tank separated into two parts by using a transparent separator [The predator avoidance is an innate behavior of zebrafish when it engages its predator. To test whether chronic exposure to PBZ can alter such innate behavior, we placed the predator fish convict cichlid eparator . The tesZebrafish has been reported to display active social interaction and shoaling behavior , thus prShoaling is an innate behavior of fish, swim together in order to reduce anxiety and the risk being captured by the predators. Zebrafish is a fish species live in a group and swim together to minimize the anxiety upon the engagement of predator . This inBased on larvae and adult fish tests, our behavioral assay clearly demonstrated PBZ indeed can induce behavioral alteration in both larvae and adult fish even when the exposed dose is relatively low. Why PBZ exposure can trigger such behavioral abnormality in zebrafish is an intriguing question for us. To probe this question, we attempted to evaluate the possible underlying mechanism by performing biochemical assay targeting some crucial biomarkers in the brain. Hyperactive in larvae, reduced innate aggressive and anxiolytic behavior can be correlated with the neurotransmitters and some oxidative stress enzymes in the brain. By ELISA (enzyme-linked immunosorbent assay) measurement, our data showed acetylcholine A and dopTo improve the productivity of the crops, enhancing the morphological, chemical, and physical features for economic benefits, pesticides are used at a very large scale by cultivators/farmers. PBZ is one such farming additive of triazole family used as a growth inhibitor, an inducer of morphological modification of leaves, and increase root density in plants. PBZ specifically inhibits the ent-kaurene oxidase enzyme activity to block gibberellins biosynthesis in plants . GibberePBZ is a very stable compound and its high mobility provides its potential for bioaccumulation in the ecological system. Being a very stable compound, the mobility of PBZ is a serious concern in soil and underground water ,14. TherCompared to embryonic toxicity, the potential toxicity of PBZ is seldom be addressed at the adult stage. Although some toxicity studies have been performed on adult zebrafish from triazole family, indicating, altered behavioral responses such as hypoactivity, prominently at 100 µM but variable at lower doses; concluding that relatively higher doses are required to induce deformities and impair locomotion activities and oxidative respiration in early developmental stages of zebrafish . The locThe important outputs of our results are we provided solid evidence at both behavioral and biochemical levels showing the exposure of environment relevant level of PBZ indeed can induce behavior alterations at both larvae and adult fish for the first time. The zebrafish larvae showed hyperactivity as treated with the lowest dose of 10 µg/L and 100 µg/L of PBZ . Furthermore, the higher burst movement and rotation suggested that PBZ treatment alter the swimming pattern. The chronic exposure to PBZ in adult zebrafish can induce anxiolytic behavioral effects as it spent more time and higher traveled distance on the top of the tank, and the similar nicotine-related anxiolytic effect was also reported by Levin et al. . Based oA receptors [Our findings revealed that zebrafish chronically treated with PBZ at 1 ppm level displayed significant aggression reductions. Fontana et al. reported that aggressive behavior is coupled with positive allosteric modulators of GABAeceptors , so the eceptors .In the light of this study, we can confirm our hypothesis that PBZ exposure induces anxiolytic behavioral changes as both treated group to have higher time in top duration, lower number of entries in the top explaining fish remain on the top, which is robust in higher concentration. Finally, the total distance in the top is higher in both treatment groups explaining they mostly swim on the top also observed aggressive response deficit. If aggressiveness deficit it results in serious problem of resource utilization like nesting and foraging site and mate disputes. Furthermore, the current toxicity levels of PBZ in water bodies responsible to decrease the exploratory behavior, lead the fish to unbalance in its habitats  at Chung Yuan Christian University  for proper use of zebrafish. All studies were carried out in strict accordance with the guidelines of the IACUC. One female and two males were transferred into a spawning tray and separated them by placing a transparent separator between them. After 24 h, the separator was removed and embryos were collected, sanitized, and placed at 28 °C after a few hours. All the dead embryos were removed every day until day three.Zebrafish AB strain stock was housed in a system connected with a recirculating aquatic system at 26.5 °C with a 10/14 dark-light cycle and fed two times a day with artemia according to the breeders in an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) approved animal facility. Zebrafish embryos aged at 4 dpf (days post-fertilization) were placed into a petri dish and exposed to different concentration of 10 µg/L and 100 µg/L of PBZ . Wild-type adult zebrafish aged at 4–6 months with mixed genders were incubated in 10L containers with either 100 µg/L (0.1 ppm) or 1000 µg/L (1 ppm) PBZ. During the 14 days incubation with PBZ, adult fish were routinely fed and the water of the whole tank was changed every after two days to maintain the constant concentration of PBZ exposure.The zebrafish larvae were shifted to clear 48-well plates and put 800 µL of fish water in each well. After 30 min of acclimation time, the locomotor activity of zebrafish larvae aged at 120 hpf was recorded for an hour and twenty minutes using 10-min alternative light  and dark cycle (infrared light). The infrared light remained on throughout the recording session. However, the visible light was switched on after every 10 min for the light cycle, and then switched off for the dark period. Later we did the behavioral assay such as total distance covered, turning frequency, and bursts during every 10 mints of the dark and light cycle using the ZebraBox instrument equipped with infrared camera and their tracking extension software system according to a previous protocol ,40,41. AAfter 14 days of incubation, the behavior assay was immediately done according to the protocol listed in After performing all behavioral assay, the adult fish were sacrificed with a high concentration of tricaine (MS-222) solution (200 mg/L). For each biochemical assay, the fish brain tissues were collected and combine three brain tissues of zebrafish to prepare an independent homogenate. Phosphate buffer saline (PBS) at pH 7.2 was added to each sample then centrifuged at 15,000 rpm at 4 °C for 15 min. Supernatant was separated and store at −80 °C for further analysis. A BCA Protein Assay Kit  was used to determine the concentration of total protein. We used a microplate reader  at 562 nm to analyze the color formation during BCA.For measuring neurotransmitter content in the brain tissues, brains from three fish were pooled into one sample and four duplicates per group were performed to ensure consistency. The relative contents ACh (acetylcholine), AChE (acetylcholine esterase), GABA (γ-aminobutyric acid), dopamine, serotonin, and cortisol were measured using sandwich-based ELISA (enzyme-linked immunosorbent assay) kits  following protocol they mentioned. Shortly, 45 µL of dilution buffer and 5 µL of brain homogenate was placed on to the specific antibody-coated ELISA plate. Then, 100 µL of the working reagent solution mix was added into each well. After 15 min of incubation, the absorbance was analyzed at 450 nm using a microplate reader .The reactive oxygen species (ROS) were analyzed using the ELISA kit  by following the protocol provided by the company. The absorbance values were determined at 450 nm. The production of superoxide dismutase (SOD), the strong antioxidant enzyme in the brain was determined using the ELISA kit . The color formation was analyzed by a microplate reader at a wavelength of 450 nm.p < 0.05 regarded as statistically significant.The statistical analysis and data visualization were carried out with GraphPad Prism . The comparison was made using one-way ANOVA, parametric, or non-parametric test, depending on data normality for significance determination. The data shown are presented with mean ± SEM with To sum up: this is the first study to demonstrate the behavioral alterations in both larvae and adult zebrafish after chronic exposure of relatively low concentration of PBZ by conducting a battery of zebrafish behavioral assays. In the zebrafish larvae, the alterations were shown in their hyper-locomotor activity and reduced photomotor response. Meanwhile, loss of aggressiveness and predator avoidance behavior, followed by anxiolytic exploratory behavior were exhibited by the adult zebrafish after chronically treated with PBZ. In addition, several abnormal levels in neurotransmitters or stress hormones were also displayed by the PBZ treated-fish, which might elucidate their altered behaviors (summarized in


Anopheles is a diverse genus of mosquitoes comprising over 500 described species, including all known human malaria vectors. While a limited number of key vector species have been studied in detail, the goal of malaria elimination calls for surveillance of all potential vector species. Here, we develop a multilocus amplicon sequencing approach that targets 62 highly variable loci in the Anopheles genome and two conserved loci in the Plasmodium mitochondrion, simultaneously revealing both the mosquito species and whether that mosquito carries malaria parasites. We also develop a cheap, nondestructive, and high-throughput DNA extraction workflow that provides template DNA from single mosquitoes for the multiplex PCR, which means specimens producing unexpected results can be returned to for morphological examination. Over 1000 individual mosquitoes can be sequenced in a single MiSeq run, and we demonstrate the panel’s power to assign species identity using sequencing data for 40 species from Africa, Southeast Asia, and South America. We also show that the approach can be used to resolve geographic population structure within An. gambiae and An. coluzzii populations, as the population structure determined based on these 62 loci from over 1000 mosquitoes closely mirrors that revealed through whole genome sequencing. The end-to-end approach is quick, inexpensive, robust, and accurate, which makes it a promising technique for very large-scale mosquito genetic surveillance and vector control. HoweverAnopheles. Targeted amplicon sequencing approaches can be used across multiple regions of the genome to give a more comprehensive view into species assignment and appropriate targets can be selected when comparative genomics data are available . Most regions fell inside exons, sometimes overlapping UTRs or spanning short introns. Regions spanning introns demonstrated high levels of sequence variability even on the level of populations, suggesting potential to reveal more fine-grained population-scale variation. Additional annotations using the repeat library  were used to remove regions overlapping with microsatellites and transposable elements. Next, we evaluated sequence variation of the potential amplicon regions and excluded those where fewer than 10 distinct sequences were found for the 10 to 21 genomes.The obtained regions were annotated using the AgamP3  gene set 8 from VectorBase ; those that were highly conserved, that is, retained alignments in at least one of the three outgroup species (n = 27 available); and those located on the An. gambiae X chromosome, while also being found in most other species (n = 6 available). The remaining 536 amplicons were sequentially added for design prioritizing those that were likely to reveal the highest number of distinct sequences across species, and then by coordinates in the AgamP3 genome to spread targets across all chromosome arms.Primer design was performed using an in-house modified version of mprimer . The proWe also incorporated several different conservation requirements for the primers themselves (rather than the full amplicon sequence) to ensure the greatest chance of successful primer binding across highly divergent species. The sequence for each amplicon target region was provided as an input FASTA file for each of the following: masking at all sites that differed between species, masking at any site that had a diverged site in two or more of the reference genomes, and no masking. Primer pairs were selected for each target region using these three masks sequentially—first allowing no variants in primers, then allowing for variants in a single aligned genome, and finally fully relaxing these restrictions. For each input file, mprimer was run using the following parameters: primer min_gc 20, primer opt_gc 50, primer max_gc 80, primer min_tm 50, primer opt_tm 58, primer max_tm 68, product_min_size 190 bp, product_ max_size 250 bp, penalty allowance 10. In the final design, degenerate bases were used in 23 primers in cases of polymorphisms found in two or more genomes.An. gambiae genome, the resulting 62 amplicons are distributed across the autosomes and the X chromosome, with 17 that fall within exons, 22 that span introns, and 23 that are intergenic  X-linked, 14 (of 22) variable, and nine (of 27) conserved amplicons. In the tergenic . Comparav.0.9.13 .Plasmodium infection status and species identity, two additional primer pairs were selected based on the mitochondrial genome alignments of five Plasmodium species that infect humans: P. falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi. Both amplicons overlapped several ribosomal RNA fragment genes .In order to assess nt genes : for P1—Anopheles nuclear genome and two targeting the Plasmodium mitochondrial genome. Mitochondrial markers were selected for Plasmodium detection because the copy number of Plasmodium mitochondria inside a mosquito is likely to be much higher than for the Plasmodium nuclear genome and should increase the sensitivity of parasite detection in what is likely to be majority mosquito DNA.Thus, the final version of the multiplex PCR panel included 64 pairs of primers, with 62 targeting the O-Methyl base exchanged for the penultimate DNA base, used in order to minimise potential primer dimers (To enable sample barcoding (described below) coupled with Illumina library preparation, a common Illumina adapter sequence motif was added to each primer sequence: 5′-ACACTCTTTCCCTA CACGACGCTCTTCCGATCT-[amplicon-specific forward]-3′, 5′-TCG GCATTCCTGCTGAACCGCTCTTCCGATCT-[amplicon-specific reverse]-3′, as well as a 2′-r dimers .2.3We evaluated the performance of the panel to amplify target regions and resolve species relationships by testing the panel on DNA from many previously identified species originating from three continents. From Africa, we included 89 specimens representing 24 species of wild-caught mosquitoes from Gabon that were identified morphologically with further PCR-based species attribution where possible . From SoAnopheles species  without2.4.2Samples were amplified using a bespoke miniaturised high-throughput amplicon sequencing protocol. This is a highly multiplexed two stage PCR, with the first 64-plex “Target Amplification PCR” employing bipartite primers (as previously described in the “Reactions were set up using a modified TTP Labtech Mosquito HV (incorporating on-deck cooling) for a 5 μl final volume in a 4titude FrameStar 384-well plate, with 1 μl of purified extract or 1:10 diluted crude lysate as input. Qiagen Multiplex mastermix (P/N 206145) was used based on its previously observed superior performance at the Sanger Institute on different samples in multiplex PCR compared to high-fidelity polymerases (data not shown). No effect of the master mix concentration in the range of 1×–1.25× on amplification efficiency was found for unpurified or purified lysate samples (data not shown). Plates were heat-sealed using a Bio-Rad PX1 heat sealer and peelable foil seals.O-Methyl modifications at the penultimate 3′ base to inhibit primer-dimer formation. The average final primer concentration was 300 pM with the exact primer concentration determined by the independent processes for mosquito , and the reactions are mixed thoroughly to rehydrate and disperse these primers into the reaction. Because the Taq present in the reactions has already been activated, in order to inhibit any off-target product formation during this process the transfer is performed at 4°C using the chilled decks of a modified TTP Labtech Mosquito HV, the new plates are heat sealed in the same manner as previously described and the second PCR is started immediately afterwards with the plates transferred from the chilled deck to the preheated PCR block.The cycling conditions for the second PCR are as follows: 95°C hold ; 95°C for 20 s (denaturation), 62°C for 15 s , 72°C for 20 s (extension), repeated for 31 cycles; 72°C for 3 min  followed by 4°C hold. These conditions have been carefully chosen to ensure that the carryover first stage primers no longer significantly participate in the second PCR therefore obviating the need to perform any sort of purification between the first and second PCRs . Furthermore, the cycling conditions achieve plateau by effective exhaustion of the available barcoding primers over a wide range of input, therefore the total yields per PCR are normalised to approximately the same level irrespective of the initial DNA concentration.2.4.3As the PCR stages have already incorporated Illumina-compatible flowcell adaptor sequences and sequencing read motifs, no further library preparation is required. In addition, as the PCR design achieves a large degree of normalisation across targets and samples, the sequencing library can be made by pooling PCRs in a simple equivolume manner. We do this by removing the plate seal and inverting the PCR plate over a Clickbio VBLOK200 reservoir, followed by gentle centrifugation. An aliquot of the plate pool is then cleaned up with two successive 0.75× volumes of AMPure XP beads, the eluted library pool is checked for expected sizing using an Agilent Bioanalyser with a High Sensitivity chip and then finally the library pool is quantified by qPCR using Kapa’s Illumina library quantification kit. After denaturation and dilution to 16 pM in HT1 buffer as per the Illumina MiSeq System Denature and Dilute Libraries Guide, the library is sequenced on an Illumina MiSeq using a 300 cycle v2 kit using a total of 316 cycles (2 x 150 bp paired end + 2 × 8 cycles for barcode reading).2.4.4bold  and specificity  is an important consideration. Here, we implemented and tested two pipelines using different software— v.2.6.4  and dadav.1.10.0  .The data set used here included mosquitoes collected from Africa, Southeast Asia and South America, as well as target sequences extracted from reference genomes—a total of 161 samples belonging to 57 species from four subgenera . The performance of both sets of thresholds was evaluated by excessive species splits between clusters and multiple species falling into a single cluster. The set of Ag1000g-derived thresholds proved to have higher species resolution with similar level of splits - presumably due to wider species sampling compared to sequenced mosquito data set, so it was used in the downstream analysis . The resulting species labelling data set included 9589 of 10057 sequences available for both reference genomes and sequenced samples, and represented 56 of 57 species.In order to predict the species of each sample based on clustering results, we generated a “species labelling data set” comprising sequences with reliable species predictions. In this data set, we included all predicted amplicon sequences generated from reference genomes as we consider their species and homology predictions to be robust. For the mosquitoes we sequenced here, we introduced a filter to ensure that only consistent data were included in the species labelling reference data set. In doing this, we did not question the original species labels that were given to samples based on morphology and, in some cases, a single molecular marker. Instead, we focused on internal coherence of the sequencing data—each species should be represented by a set of relatively similar sequences across the samples representing that species. We analysed within-species distances across all amplicons and identified eight samples that were too distant from other samples of that species. For one of the species, An. coluzzii because 75 of 80 alleles detected in the sample are found in the “species labelling data set” assigned to An. coluzzii, yet 70 of those alleles are also found in the set of alleles assigned to An. gambiae.For the purpose of species identification, the species labelling data set was clustered together with the remaining sequences. Each cluster for each amplicon was labelled with the set of reference data set species whose sequences fell within that cluster . The spefasttree v.2.1.10  dimensionality reduction technique, using the software umap-learn v.0.3.10 (D) and pairwise population Hudson’s FST were estimated as implemented in scikit-allel v.1.2.1.The overall population structure for the combined data set including all v.0.3.10 . For the33.1Anopheles DNA extracts we used were generated using common proprietary tissue lysis and DNA extraction kits. However, due to the potential scale of an amplicon based approach in screening thousands of samples, and the likely discovery of new species that will ensue, we developed an alternative approach to simultaneously protect the morphology of samples so that they can be returned to post sequencing, and also to minimise the per sample extraction costs and required labour. We tested a set of nondestructive DNA extraction protocols with custom lysis buffers, and selected a buffer that resulted in good DNA yields and which, when diluted, could be used directly in PCR without further purification. PCR products and amplicon sequencing results generated using this custom DNA extraction approach were comparable in quality with results obtained from samples prepared using commonly used proprietary DNA extraction kits , with P2 amplifying more efficiently than P1  and processed those using the dada2 pipeline. The sequences of an additional 28 samples from 23 species  were extracted from published genome assemblies  were heterozygous and another 175 (3%) were multiallelic. Heterozygosity level  was high3.4Anopheles species to assist with accurate identification of known species, and phylogenetic placement of previously undescribed species. To demonstrate that the panel based assignments surpass what is possible with single marker based assignments, we sequenced two conventional molecular species ID markers, COI and ITS2  and revealed a few mis-labelled samples .An. gambiae and An. coluzzii. We tested both sets of thresholds and found the Ag1000g-derived option more informative, as it resolved more species and did not excessively split them into several clusters  based on amplicon sequencing data combined with reference genomes  and (ii)clusters .An. gambiae and An. coluzzii , An. coustani, An. tenebrosus, An. ziemanni (Coustani group), and An. brohieri, An. demeilloni, An. hancocki . All of these groups showed evidence of introgression according to COI, ITS2 and amplicon sequencing. Moving forwards, both clustering and phylogenetic trees could be used to identify closest relatives for the new unidentified samples.Species prediction requires a reference data set with trusted species labels. Here, we created one using a combination of reference genome sequences and quality-filtered amplicon sequence data for wild-caught specimens, retaining 9589 of 10057 (96%) sequences representing 56 of 57 sampled species. The species assignment for each of our samples was based on the most frequently encountered species label across all sequences available for a given sample. This approach resulted in reliable species resolution for most of the species groups and complexes . The separation of those three populations was also supported by F-statistics  with four samples sequenced, had only 4 ± 3 heterozygous amplicons in any sample among the 39 ± 7 amplicons recovered, and only 26 SNPs across 7196 bp of all amplicons alignment. In fact, the diversity for An. barbirostris was comparable to An. gambiae, where for five sequenced samples 57 ± 2 amplicons were recovered, 27 ± 5 were heterozygous, and 382 SNPs were discovered across 9556 bp all amplicons alignment. Thus, the within-species variation can be recovered persistently across species in both Africa and South-East Asia and for both Cellia and Anopheles subgenera.Amplicon sequencing informativeness was retained even when fewer amplicons were recovered and the combined amplicon length never dropped below 7kbp for any sequenced species, except y sample  and in s4Plasmodium within sampled mosquitoes for the entire Anopheles genus, many of which act as vectors of human malaria. Much of our efforts were focused on automating and reducing the cost of sample processing, such as using a simplified DNA extraction that does not rely on pre-existing kits or product purification, and implementing a multiplex PCR that can be paired with one of the most widespread sequencing technologies, Illumina MiSeq. A number of optimisation and validation experiments also proved that the detection of Plasmodium infections within a mosquito was reliable to a certain infection titre  by BLASTing the amplicon data against Plasmodium spp. mitochondrial genomes. Apart from microscopy and ELISA, a widely used method to detect parasites in mosquitoes is PCR, often using nested primers targeting the18S rRNA gene (Plasmodium species are addressed using qPCR, where a multitude of assays have been developed—mostly targeting nuclear 18S rRNA and using various qPCR techniques (Plasmodium infections in laboratory-infected mosquitoes.At this stage, we have primarily tested the ability of the RNA gene  and alteRNA gene . The quachniques . For valchniques  that is chniques . DespitePlasmodium (Anopheles species composition, population structure, and infection status as we drive malaria towards elimination.Malaria genomic surveillance is important for both parasites and vectors, with research examining genetic diversity, population structure, and the genetic basis of insecticide resistance in mosquitoes  and drugasmodium . We hopeSupplementary Information


To compare the surgical outcomes of autogenous spinous process with iliac bone graft in managing single segment lumbar pyogenic spondylitis (PS) after posterior debridement and instrumentation. We performed a retrospective study for adult patients with single level lumbar PS. 60 patients with single segment lumbar PS underwent one-stage posterior debridement, autogenous bone graft and instrumentations. The patients were divided into Group A (autogenous iliac bone) and Group B (autogenous spinous process). Preoperative Charlson comorbidity index (CCI) was analyzed to assess the comorbidity. Low back pain was evaluated using the visual analog scale (VAS). Neurological status was assessed with the American Spinal Injury Association (ASIA) scale. Clinical infection index including the C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) was also reviewed. Moreover, fusion and changes of sagittal alignment were investigated radiologically. There was a significantly longer operative time, hospital stay and greater blood loss in group A. The VAS scores improved significantly at each follow-up interval and post-operative VAS score was significantly lower in group B. At the last follow-up, ESR and CRP returned to normal for all patients. There was at least one grade level improvement in ASIA score. No statistical difference in corrected rate, loss of sagittal angle and lumbar lordosis was found between the two groups. There was no significant difference in fusion rate, mean fusion time and complications between the two groups. Compared with iliac bone graft, the autogenous spinous process bone graft can be less invasive and painful for the single segment lumbar PS. One-stage posterior debridement, autogenous spinous process bone graft and instrumentation can provide satisfactory results for appropriate cases. However, spinal instability, neurological compression or recurrent infection are deemed as potential surgical indications2.Pyogenic spondylitis (PS) encompasses a broad range of clinical entities, which is rare but severe, even potentially life-threatening. Although the pathophysiology of PS is not fully clear, it commonly arises from a hematogenous spread of bacteria. Therefore, conservative treatment with immobilization and systemic administration of antibiotics will be first recommended for patients with PS3. In addition, we considered the autologous spinous process, which could be achieved in the same operation area with lower donor site morbidity and less invasion, can be used as an ideal interbody fusion material for PS with posterior surgery.The purpose of surgical treatment for PS is sufficient decompression and radical debridement, followed by an anterior fusion. After extensive debridement of the infected tissue, structural bone or cage grafting would be performed as structural grafts to repair the bone defects. However, conventional autologous bone graft is often limited due to extensive bone loss and may be complicated by donor site morbidity. Posterior approach is becoming more popular for dealing with anterior vertebral column pathologies including infection, which can stabilize the spine and correct kyphosis, even has a lower incidence of complications than anterior surgeryIn summary, this study aimed to compare the clinical and imaging outcomes of PS treated by one-stage posterior debridement with autogenous spinous process or autologous iliac bone strut as structural grafts.http://www.chictr.org.cn). All methods were carried out in accordance with relevant guidelines and regulations (Declaration of Helsinki). Between January 2011 and January 2016, 60 consecutive patients (24 men and 36 women) with single segment lumbar PS underwent one-stage posterior debridement, autogenous bone grafts fusion, and instrumentations in the study. In group A, 33 cases obtained autogenous iliac bone graft for reconstruction. In group B, 27 cases used previously obtained specially formed autologous spinous process (grind the cortex bone of the upper and lower surface) in the operation area for reconstruction  in prone position. Pedicle screws  were implanted into two or four segments adjacent to the lesion segment. If the upper third of the vertebral body and pedicles were not affected, screws were also inserted into the diseased vertebra.After performing semi-laminectomy or whole laminectomy of the decompression segment, we acquired the spinous process of the unaffected segment completely and cleared the necrotic tissues with curette and nucleus pulposus forceps. The affected intervertebral space was repeatedly rinsed with a large amount of normal saline and filled with gelfoam containing sensitive or empirical antibiotics (for cases with negative culture). Different methods were used for bone grafting: (1) Group A: obtained a tricortical iliac bone, trimmed to a suitable size, and implanted it into the bone defects. (2) Group B: previously obtained autologous spinous process was grinded the cortex bone and clipped into a suitable supporting bone graft. Then the autologous spinous process together with the bone granule were inserted into the anterior bone defects. When hemostasis and neural decompression were meticulously confirmed, the dura was covered with gelatin sponge, and a negative pressure drainage was placed before suturing the incision for each patient. The pus, granulomatous tissue and intervertebral disc were pathologically examined and cultured after operation.After surgery, all the patients received initially 4–6 weeks of intravenous antibiotics therapy , followed by oral antibiotics until CRP and ESR were normal.CRP and ESR values were chosen to evaluate the infection status. The visual analogue scale (VAS) score was used to assess low back pain and American Spinal Injury Association (ASIA) impairment scale was used to evaluate the neurological function before operation and during the follow-up periods.4. The angle of lumbar lordosis was measured by the intersection of lines drawn parallel to the inferior endplate of T12 vertebrae and the superior endplate of S1 vertebrae. The Cobb angle formed between the upper endplate of the vertebra above the infected vertebra and the lower endplate of the vertebra below the infected vertebra in the sagittal plane was used to evaluate the correction effect of local fusion segment kyphosis.Lumbar X-ray or 3D reconstruction CT images was performed for all patients 3 day, 3 months, 6 months, 12–30 months after surgery. The same two authors evaluated the fusion condition according to the images. Solid bone fusion was confirmed if there was continuous trabeculae between the graft and adjacent vertebral body without radiolucency, mottling, collapse of the graft, or motion beyond 3°The Student's t test, Mann–Whitney U test and Fisher’s exact test were used for statistical analysis with IBM SPSS software package, version 26.0 . P < 0.05 was considered statistically significant.The vertebral levels involved in our patient population ranged from L1-2 to L5-S1. The average follow-up time was 20.5 ± 6.0 months in group A and 18.4 ± 4.1 months in group B . Both groups had similar CCI distribution. No recurrence was observed during the whole follow-up. According to intraoperative findings, the intervertebral space is filled with necrotic intervertebral disc, abscess and sequestra. The necrotic intervertebral disc was fragile and easily scraped off , Enterobacter cloacae (7 cases), Acinetobacter baumannii (3 cases) and others (9 cases).On admission, 11 patients in group A (33.3%) and 8 patients in group B (29.6%) suffered fever. The bacterial culture of the intraoperative specimen or blood was positive in 41 patients  including Complications including major vessel injury, dural tear, nerve root injury, and pulmonary embolism were not observed in the further follow-up. There was no abscess or fistula after removing the irrigation and drainage tubes.Compared with Group A, the operative time and hospitalization duration were shorter, and the operative blood loss was less in group B (P < 0.05), however, no statistical difference between the two groups was found in preoperative CRP or ESR (P > 0.05). Levels of CRP and ESR in both treatment groups continued to decline during follow-up. Autologous iliac bone and spinous process graft had no effect on CRP or ESR levels. What’s more, the post-operative VAS scores of group B were lower (P < 0.05). While at the end of follow-up, the VAS score, CRP and ESR were all significantly improved in both groups (P < 0.05). The clinical results of the two groups were shown in Table The comparison of the fusion rate, average fusion time, lumbar lordosis and sagittal angle results between group A and group B were summarized in Table The fusion rate in group A and group B were 93.9% and 88.9%. And the mean fusion time in group A and group B were 6.8 ± 1.4 (range 5–9) and 7.9 ± 2.2 (range 5–12) months, respectively. Two patients  diagnosed with methicillin-resistant S. aureus (MRSA) in group A experienced no fusion at 24-month follow-up. Those numbers come to 11.1% (3/27) in group B. The fusion rate and average fusion time have no difference between the two groups (P > 0.05).At the final follow-up, ASIA score of each patient had improved by at least one grade. ASIA grade in group A changed from C to D in 9 cases and from D to E in 24 cases. In group B, ASIA grade changed from C to D in 8 cases, from C to E in 2 cases, and from D to E in 17 cases. At the end of follow-up, there was no significant difference in postoperative neurological function between the two groups Table .Table 3CRestoration of a stable anterior spinal column is essential for normal spinal biomechanics and infection healing. Therefore, bone grafting is a vital procedure in surgical treatment of PS, which is not only for bone fusion, but also for structural reconstruction. The present study is the first to report the efficacy of autologous spinous process used as bone graft in managing PS through posterior approach.6, posterior8, and combined anterior–posterior approaches10. The choice of the appropriate surgical procedure is often determined on surgeons’ preference, neurological deficits and predicted spinal instability13. However, recent literature suggested that posterior only approach may be associated with fewer complications compared with the combined approach including anterior debridement and posterior instrumentation. By a posterior surgery, the spinal deformity can be corrected and the anterior column can be supported, which also helps to promote fusion14. In addition, the lumbar stenosis, which is a common disease in the aged, can be cured at the same time. Zhang et al. reported that single-stage posterior fusion was effective in the management of mono-segmental lumbar or lumbosacral PS, which was compatible with our findings in this study15.There are several surgical approaches for the treatment of PS, including anterior16. Using local autograft as a substitute for iliac bone strut has theoretical inferiorities in that there exists a smaller volume and a relatively higher proportion of cortical to cancellous bone, which inevitably contains lower osteogenic cells and a lesser amount of trabecular area for performing the osteogenic and osteoconductive properties of autograft17. However, iliac bone grafting has obvious disadvantages of neurovascular injury, persistent pain and potential infection of the donor site18. Furthermore, because the majority patients with PS are elderly and suffer osteoporosis, there is a risk of hematoma and instability19.Bone graft materials had a significant influence on fusion. The autologous bone grafting has been widely used in traditional surgery for PS. The iliac bone strut was the most commonly used graft due to its high ratio of bony fusion, which was considered as the “gold criteria”. Past studies have reported that the single-level lumbar fusion rate with iliac bone strut was as high as 90–100%Our study has revealed that use of a local bone graft technique (spinous process) showed the similar fusion rate and fusion time as that of iliac bone strut with less surgical time and fewer complications. Furthermore, we found the autologous spinous process bone graft has some advantages over iliac bone strut graft, which are embodied in the following aspects: (1) the surgeons have to spend extra time on some surgical procedures such as making another incision, harvesting iliac bone, hemostasis and suturing. However, in group B, they just need to removed cortical part of the spinous process to make bone grafts. What’s more, the decompression, debridement, deformity correction and intervertebral grafting and instruments can be performed simultaneously in one incision. Obviously, this will save surgical time and reduce operation time and blood loss. (2) Patients in whom tricortical full-thickness iliac graft was harvested had a quite high prevalence of donor site morbidity, such as persistent pain of the donor site which corresponds to our result that the post-operative VAS score was significantly higher in group A. However, using the autogenous spinous process grafting may relieve the postoperative pain, shorten the hospital stay and reduce hospitalization expense, which is in line with the concept of enhanced recovery after surgery. (3) Compared with iliac crest, autologous spinous process bone grafts also have a better supporting biomechanical properties and abundance sources. Multiple spinous process bone grafts can be taken according to the defects without increasing extra trauma.In our study, we innovatively removed cortical part of the spinous process to make bone grafts, which could be performed simultaneously in one incision with debridement, decompression, deformity correction and intervertebral bone grafting and instrument just through the posterior only approach. More importantly, this study showed the fusion rate of spinous process bone graft was similar to that of iliac bone strut graft. This may be associated with the following reasons: (1) most of the cortical bone of the spinous process where contact with the end plates was removed before grafting. This may increase the fusion rate of the grafting. (2) With the firmly fixed instruments, spinous process bone grafts only need to bear little load, which is beneficial for the fusion between the graft and the vertebral body. (3) Besides the spinous process bone graft , the bone granule  harvested during posterior decompression were also packed into the intervertebral space as much as possible. Widening the graft area may be helpful for fusion. (4) The complete removal of all infected and necrotic tissue allows for extensive contact between the graft and adjacent vertebral cancellous bone, which may facilitate fusion.The present study had several limitations. First, this study is a retrospective study with a small sample size. According to our results, there are statistical differences among the four indexes (P < 0.05): post-operative VAS score for low back pain, operative duration, operation blood loss and hospital stay. Using the mean value and standard deviation of the four indexes, powers (1-β error probability) and effect sizes of the four indexes calculated in the G-Power software were 0.976, 0.998, 0.998 and 0.912 and 1.03, 1.30, 1.32 and 0.87 respectively. However, larger randomized control trials are needed to analyze the outcomes of autogenous spinous process bone graft in the setting of lumbar pyogenic spondylodiscitis. A prospective study with a more homogenous populations might have provided more rigid efficacies and limitations of this surgical strategy. Moreover, the case series was limited because we excluded patients who obtained allogeneic bone or cage insertion without bone grafting from the pelvis treatments. Whether the autogenous spinous process bone graft was more applicable than allogeneic bone or cage in the treatment of lumbar PS remained uncertain and should be studied in the future work.In conclusion, compared with iliac bone graft, autologous spinous process bone grafting had similar fusion rate but less invasion for single segment of lumbar PS. These two grafting methods showed similar clinical outcomes for patients with an adjacent small abscess in the anterior spine and mild vertebral destruction, those with epidural abscesses around the dura, and lumbar canal stenosis. One-stage posterior debridement, autogenous spinous process bone graft and instrumentation can alleviate low back pain, correct kyphotic deformity and improve nerve function for proper cases.


Head and neck squamous cell cancer (HNSCC) is the sixth most common cancer in the world; its pathogenic mechanism remains to be further clarified.  Robust rank aggregation (RRA) analysis was utilized to identify the metasignature dysregulated genes, which were then used for potential drug prediction. Weighted gene coexpression network analysis (WGCNA) was performed on all metasignature genes to find hub genes. DNA methylation analysis, GSEA, functional annotation, and immunocyte infiltration analysis were then performed on hub genes to investigate their potential role in HNSCC. ITM2A, GALNTL1, FAM107A, MFAP4, PGM5, and OGN) were selected . All six genes were downregulated in tumor tissue (FDR < 0.01) and were related to the clinical stage and prognosis of HNSCC in different degrees. Methylation analysis showed that the dysregulation of ITM2A, GALNTL1, FAM107A, and MFAP4 may be caused by hypermethylation. Moreover, the expression level of all 6 hub genes was positively associated with immune cell infiltration, and the result of GSEA showed that all hub genes may be involved in the process of immunoregulation.  A total of 862 metasignature genes were identified, and 6 potential drugs were selected based on these genes. Based on the result of WGCNA, six hub genes ( All identified hub genes could be potential biomarkers for HNSCC and provide a new insight into the diagnosis and treatment of head and neck tumors. Head and neck squamous cell carcinoma (HNSC) is the sixth most common cancer in the world . WorldwiIn the past twenty years, with the application of microarray and next-generation sequencing technologies, a great number of novel diagnostic or therapeutic biomarkers have been identified in HNSCC . HoweverWGCNA  is an efMMP1 (FDR = 4.77e − 53), MMP10 (FDR = 8.25e − 40), PTHLH (FDR = 1.48e − 38), MMP3 (FDR = 5.38e − 38), and SPP1 (FDR = 2.79e − 37) while TMPRSS11B (FDR = 1.53e − 36), MAL (FDR = 8.50e − 35), CRISP3 (FDR = 1.60e − 34), CRNN (FDR = 4.32e − 33), and KRT4 (FDR = 1.24e − 30) were the most significant downregulated genes. The chromosomal locations and expression patterns of the top 100 DEGs are visualized in The workflow of our study is shown in We select the top 300 DEGs to perform GO and KEGG enrichment analyses. Among the KEGG pathway database, we can find that these DEGs were enriched in multiple cancer-related pathways like focal adhesion, PI3K-Akt signal pathway, pathway in cancer, small-cell lung cancer, transcriptional misregulation in cancer, and chemical carcinogenesis . FurtherAccording to our screen criteria, 6 small molecule drugs  were identified . Their 2p value < 0.05, and MM p value < 0.05), 6 genes , which were also significantly correlated with each other  were upregulated in the T1-T2 stage and downregulated in the T3-T4 stage, which is consistent with the result in WGCNA. However, there is no correlation between hub genes and tumor N stage  was significantly associated with poor overall survival  may be caused by hypermethylation. To find the key methylation site of hub genes, we also used MethSurv to explore the prognostic role of these methylation sites . A total of 15 methylation sites were found to be important prognostic factors for HNSCC  families. Its family members have been proved to play a vital role in the progression, invasion, and metastasis of HNSCC . The mos cancers . To furtAfter identifying the robust DEGs in HNSCC, we try to use the expression pattern of these genes to predict the potential small molecule drugs. The CMap database was used, and six small molecule drugs were selected because they can reverse the expression pattern of metasignature DEGs. Among all these drugs, four of which have been studied in HNSCC previously. For instance, thiostrepton has been reported to affect the proliferation, apoptosis, and radiosensitivity in head and neck cancer , 16. LevITM2A, GALNTL1, OGN, FAM107A, MFAP4, and PGM5) according to our selection criteria. We used other independent databases to validate the expression pattern and clinical relationship of these hub genes. The result showed that all hub genes were downregulated in tumor tissue and were negatively correlated with tumor T stage. Furthermore, compared with tumor tissue, these 6 hub genes were also downregulated in dysplasia tissue. The ROC curve indicated that these genes may help us better identify the HNSCC. Besides, four genes  also performed well in the prognosis prediction of HNSCC. Interestingly, all 6 hub genes were seldom explored in HNSCC previously. ITM2A, a family member of BRICHOS, has been reported to be downregulated in both breast and ovarian cancers which may affect the proliferation and autophagy process of tumors [PGM5 was identified as a diagnostic and prognostic biomarker in liver and colorectal cancers [PGM5 was showed to inhibit the proliferation and metastasis of tumors [OGN was also reported to inhibit the process of EMT through the EGFR/Akt pathway [To identify the hub genes among all 862 metasignature DEGs, WGCNA was utilized to construct a coexpression network. Finally, we identified 6 hub genes  may be significantly associated with hypermethylation in multiple methylation sites. Because DNA methylation is a reversible process, targeted therapies for the unique methylation site of the tumor are promising. To further screen out methylation sites with research potential, we also performed survival analysis and found that hypermethylation of 15 methylation sites in FAM107A, GALNTL1, and MFAP4 was significantly associated with poor overall survival. All selected hub genes and their methylation conditions may help us better judge the state of HNSCC (inert or invasive), so as to develop a more appropriate treatment strategy.As we all know, hypermethylation is an important cause of the downregulation of gene expression. A recent study showed that hypermethylation may lead to the low expression of l cancer , which iOGN can promote the infiltration of CD8+ T cells thus inhibiting the formation of new blood vessels in colorectal cancer [ITM2A, MFAP4) in immunoregulation [A great number of previous researches have revealed that the infiltration of immune cells in the tumor microenvironment could largely affect the development of cancer cells , 26. Thel cancer . Some stgulation , 29. HowITM2A, GALNTL1, OGN, FAM107A, MFAP4, and PGM5) in the blue module were selected. All hub genes were significantly downregulated in tumor tissue of HNSCC. The expression pattern of four hub genes  may be caused by hypermethylation. All six hub genes may play a role in immunological regulation in the microenvironment of HNSCC which need more experiment to verify.In conclusion, by utilizing the RRA method, we identified a series of robust DEGs in HNSCC. Based on WGCNA, 6 hub genes  Included datasets must provide the gene expression profile of HNSCC and corresponding normal tissue control. (2) Each group of one dataset should contain at least 5 samples. (3) The platform of each study should contain more than 8000 genes. Finally, a total of 25 studies were included in our research, and among which, 24 independent studies were used for RRA analysis; one dataset (GSE30784) with gene expression data of dysplasia tissue was used for further validation and exploration. Detailed information of included GEO datasets is shown in p value < 0.05). The up- or downregulated genes were arranged from large to small according to the absolute value of logFC. The “RobustRankAggreg” package in R was created for comparison of ranked gene lists and identification of metasignature genes. The result of RRA can help us identify more robust genes from different studies, and the detailed method of the RRA method has been described by previous articles [p value of the output result was subjected to Bonferroni correction, and mRNA with adjusted p value < 0.05 was considered significantly dysregulated. Furthermore, “OmicCircos” R package was utilized to visualize the expression patterns of the top 100 metasignature DEGs in each included study .R package “GEOquery” was used to directly obtain series matrix files, sample phenotype data, and corresponding platform information from the GEO database. We used “limma” R package to normalize the data and obtain DEGs of each study (articles . In the http://david.abcc.ncifcrf.gov/) to annotate the top 300 metasignature genes. GO and KEGG enrichment analyses were performed by using the prediction tool on the website. Bubble charts were used to visualize the top 20 terms of enrichment results.We used DAVID Bioinformatics Resources 6.8  can help us to predict the potential drugs which can reverse the expression of specific genes. In this study, we input the top 300 metasignature genes (165 upregulated and 135 downregulated genes) into the online tool of CMap for gene set enrichment analysis. Each small molecule will be assigned an enrichment score between -1 and 1. The lower the enrichment score, the better the drug effect to reverse the state of HNSCC cells. In our study, drugs with p value < 0.01 and the enrichment score < −0.7 were considered potential small molecules. We also used PubChem (http://www.pubchem.ncbi.nlm.gov) to visualize the 2D structure of selected small molecules.The Connectivity Map (CMap)  databaseβ = 7, R2 = 0.9). All metasignature genes were distributed in different gene modules according to the value of the TOM matrix. Here, we set the minimal module size as 15 and cut height as 0.5. The module with a significant correlation with clinical characteristics was selected. GO and KEGG enrichment analyses were performed on the clinical-related modules. We selected the hub gene according to the value of GS and MM .A total of 862 metasignature genes were included for WGCNA with expression data from GSE65858. We construct a gene coexpression network for all metasignature DEGs; “WGCNA” R package was applied to explore the relationship between each coexpression module and clinical phenotype. A correlation matrix was constructed which was subsequently transformed to a TOM matrix based on the soft threshold  was used appropriately to test the result of the comparison. Furthermore, we also plot the ROC curves to assess hub genes' diagnostic value; the area under the ROC curve (AUC) was calculated by the “pROC” R package. Survival analysis was also performed on all hub genes by using GEPIA . The median is considered to be the cutoff for high and low expression of hub genes.We used the TCGA database at first to validate the diagnostic role of hub genes and the relationship between hub genes and clinical characteristics. We also used an independent dataset (GSE30748) to explore the hub genes' expression levels between dysplasia tissue and tumor tissue. The Student http://bioinfo.hrbmu.edu.cn/diseasemeth/), which is a website focusing on collecting methylation data from various tumor tissue. We compare the mean value of methylation between HNSCC and corresponding normal tissue. Furthermore, we also used MEXPRESS [http://mexpress.be) to explore the association between the expression level of hub genes and the methylation level of the corresponding methylation site. Those methylation sites that are negatively correlated with gene expression are defined as candidate sites. To further screen potential key methylation sites, we also conducted survival analyses on these candidate sites by using MethSurv [https://biit.cs.ut.ee/methsurv/).In order to further explore the reason for the dysregulation of hub gens, we performed methylation analysis on all hub genes based on DiseaseMeth 2.0  (http://MEXPRESS  (http://MethSurv  (https:/https://cistrome.shinyapps.io/timer/), an online tool based on the TCGA database, to evaluate the infiltration score for six kinds of important immune cells . The Pearson correlation coefficient between hub genes and the infiltration score were then calculated.To explore the association between immune cell infiltration and expression level of hub genes, we used TIMER  (https:/http://software.broadinstitute.org/gsea/msigdb/index.jsp).According to the mean expression value of 6 hub genes, all HNSCC samples in the TCGA database were divided into high expression groups and low expression groups. GSEA analysis was performed and visualized by using the “clusterprofiler” R package. The KEGG gene set was directly downloaded from MSigDB (