Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is the most common craniofacial birth defect. Its etiology is complex and it has a lifelong influence on affected individuals. Despite many studies, the pathogenic gene alleles are not completely clear. Here, we recruited a Chinese NSCL/P family and explored the candidate causative variants in this pedigree.We performed whole‐exome sequencing on two patients and two unaffected subjects of this family. Variants were screened based on bioinformatics analysis to identify the potential etiological alleles. Species conservation analysis, mutation function prediction, and homology protein modeling were also performed to preliminarily evaluate the influence of the mutations.GLI2 as candidate causative variants. All of these three mutations were predicted to be deleterious, and they affect amino acids that are conserved in many species. The mutation c.2684C > T was predicted to affect the structure of the GLI2 protein.We identified three rare mutations that are located on a single chromatid  in GLI2 variants play a role in the pathogenesis of NSCL/P, and the three rare missense mutations combined are probably the potential disease‐causing variants in this family.Our results further demonstrate that  Understanding and written informed consent was obtained from the subjects themselves or from their guardians.2.2A Hui Chinese family from Urumqi, Xinjiang with NSCL/P of unknown etiology was recruited at Peking University School and Hospital of Stomatology. We also recruited an independent cohort of 31 NSCL/P‐unaffected subjects to verify the allele frequency. All of the subjects were interviewed and clinically assessed by at least two experienced oral and maxillofacial surgeons. Full clinical checkups were completed to identify any further anomalies such as congenital heart disease, cyclopism, polydactyly, and so on, which would suggest an underlying syndrome. Additional demographic information was obtained through a detailed questionnaire, including gender, age, nationality, and maternal exposure history of smoking, drinking, medicine, radiation, poisons, and chemicals during the first trimester of pregnancy.We collected 2–4 ml peripheral blood samples from each accessible participant. The buccal swabs from the maternal grandparents of the proband were carefully acquired and reserved in TIANGEN Sample Preservation Buffer . Genomic DNA was extracted using the QIAamp DNA Blood Mini Kit  or TIANGEN Hi‐Swab DNA Kit  according to the manufacturer's instructions. The quality and quantity of the DNA samples were assessed using a NanoDrop 8000  and agarose gel electrophoresis.2.3https://software.broadinstitute.org/gatk/). Repeat reads were removed using Picard Tools (http://broadinstitute.github.io/picard/). Local realignment and base quality recalibration were conducted using the GATK. Sequencing depth, target coverage, and capture specificity were calculated based on the alignment. Subsequently, single nucleotide polymorphisms (SNPs) and insertions/deletions (InDels) were detected and screened using HaplotypeCaller (GATK v3.3.0). SnpEff  was then used to perform annotation and prediction.DNA libraries with enrichment of exonic sequences were constructed for the proband, as well as the elder sister and parents of the proband. High‐throughput sequencing of the libraries was performed, and raw sequencing reads were generated using the BGISEQ‐500 platform   as well as variants in non‐coding DNA sequences, including the 3'‐UTR, 5'‐UTR, downstream, upstream, intergenic, intron, synonymous, stop‐retained, and noncoding exon variants. Next, a Mendelian inheritance model was used to narrow down the candidates. Only heterozygous variants present in all individuals with cleft and not present in any individuals without cleft were retained. Then, the MAF of each retained variant was verified in several public databases by handwork, including Ensembl (http://asia.ensembl.org/index.html), ExAC (http://exac.broadinstitute.org), and NCBI dbSNP (https://www.ncbi.nlm.nih.gov/snp/), and those with high frequency were screened out. In addition, since we used the allele positions to select variants that accord with the dominant inheritance model, we checked and excluded variants with the same allele positions but with inconsistent variant forms in subjects D1 and D2, such as c.859A > G (NM_001146181.2) in D1 and c.859A > AGC (NM_001146181.2) in D2. Finally, the literature was reviewed for candidate variants with potential significant relationships to the phenotype and possible deleterious effects on craniofacial structures.After annotation, a screening process was performed to identify the candidate gene mutations. First, we excluded variants with a minor allele frequency (MAF) ≥ 0.5% in the 1000 Genome Project database (2.5GLI2 (NM_005270.4) mutation we identified, which has not been reported in the databases, we further performed Sanger sequencing for validation of the MAF on 31 unaffected Chinese subjects. Polymerase chain reaction (PCR) primers were designed  using Primer Premier 5.0. The reaction mixture in a 50‐μl volume was made using 0.5 μl TaKaRa LA Taq (5 U/μl), 25 μl 2 × GC Buffer I, 8 μl dNTP Mixture (2.5 mM each), 1 μl DNA template (80 ng/μl), 1 μl forward primer (10 mM), 1 μl reverse primer (10 mM), and 13.5 μl ddH2O. Since the PCR target sequence is GC‐rich, we applied hot‐start PCR and touch‐down PCR. The reaction procedures were as follows: 95°C for 15 min, the first 15 cycles of 95°C for 1 min, 70–56°C for 30 s, and 72°C for 3 min, and the last 16 cycles of 95°C for 1 min, 58°C for 30 s, and 72°C for 3 min. The Sanger sequencing data were analyzed using Chromas v1.0.0.1 software.We performed Sanger sequencing for validation of three potential causal variants on the family members. For the novel 2.6Escherichia coli competent cells. The bacteria were plated on ampicillin‐containing Luria‐Bertani (LB) solid medium. After incubating at 37°C for 14 hr, monoclonal colonies were selected. Sanger sequencing was performed on the cloned DNA sequences to identify the arrangement of the alleles of the three variants on the chromatid.The PCR procedure was repeated for DNA samples of subjects D1 and D2, as well as the father of D2. PCR products were purified by gel extraction using an AxyPrep DNA Gel Extraction Kit  after agarose gel electrophoresis. Then, the 2,831‐bp DNA target sequence was ligated to a TOPO TA vector  for 1 hr at room temperature. Next, the products were transformed into 2.7http://www.uniprot.org/) and ClustalX V2.1. To predict the potential functional effects of the identified variants, in silico tools such as SIFT , PROVEAN , PolyPhen‐2 , and MutationAssessor  were applied. Additionally, the website‐based domain prediction tool SMART (Simple Modular Architecture Research Tool) (http://smart.embl-heidelberg.de) was used to predict domains for GLI2 . SWISS‐MODEL (https://www.swissmodel.expasy.org/) was used for the molecular simulation  and two elder sisters of the proband D1 were not affected with cleft lip/palate. One of the elder sisters died from unknown causes in infancy. The grandfather, grandmother, as well as uncles, and aunts from both the paternal and maternal sides to whom we had no access for physical examinations claimed to be not affected with cleft lip/palate through telephone. The photographs of the father and mother of D2 taken by themselves and mailed to us were presented in Supporting Information Figure During the pregnancy, D2 was not exposed to diseases, smoking, alcohol, radioactivity, or chemical teratogens, and there was no supplementation with folic acid, iron, or vitamin B6.3.2We performed WES on the two patients D1 and D2) as well as two unaffected family members (C1 and C2) in this NSCL/P hereditary family. These samples yielded 21.55 Gb data of mappable targeted exome sequences on average, with a mean sequencing depth of 229.03‐fold. In total, 97.63% of the exonic regions were covered at least 20‐fold, indicating high sequencing quality , predicting p.Ala895Val, p.Gln1450His, and p.Ser1541Tyr respectively as candidate pathogenic factors in the family. Among the three mutations, M1: c.2684C > T (NM_005270.4) is novel and has not been reported in any database previously, while M2: c.4350G > T (NM_005270.4) and M3: c.4622C > A (NM_005270.4) have already been reported to be very rare with MAFs of 0.4‰ and 0.6‰ in the 1,000 Genomes database, respectively  and unaffected subjects (C1 and C2) of the family by Sanger sequencing  and five clones showed mutant alleles in all of the three loci , a consistent result indicating that in the genome of patient D1, the three mutations of GLI2 are located on a single chromatid. In D2, there were four clones with WT alleles in all three loci  and four clones with mutant alleles in all three loci . This indicates that in the genome of patient D2, the three GLI2 mutations are also located on a single chromatid. In the father of D2, five clones presented WT alleles in all three loci  and three clones showed mutant alleles in all three loci , indicating that the three mutations of GLI2 are also located on a single chromatid in the genome of the father of D2. It suggests that all of the three mutant alleles of the GLI2 gene were delivered from the father of the subject D2 to the subject D2, and then passed down to the subject D1.  were 0.02, 0.00, and 0.01 for the mutations M1 , M2 (c.4350G > T_p.Gln1450His), and M3 (c.4622C > A_p.Ser1541Tyr), respectively. The PROVEAN scores (deleterious score ≤ −2.5) were −3.43, −3.42, and −3.42 for the mutations M1, M2, and M3, respectively. The PolyPhen‐2 scores (deleterious score ≥ 0.909) were 0.911, 0.972, and 0.998 for the mutations M1, M2, and M3, respectively. The MutationAssessor scores (deleterious score ≥ 1.9) were 2.58, 2.54, and 3.015 for the mutations M1, M2, and M3, respectively  affects an amino acid located in the phosphorylation cluster and around acetylation sites and sumoylation sites, which are located in the C‐terminal to DNA‐binding domain that comprises five tandem C2H2 zinc‐fingers. The mutations M2 (p.Gln1450His) and M3 (p.Ser1541Tyr) lie in the C‐terminal activation domain of the GLI2 protein  and have already been reported to be rare variants. We demonstrated that the three mutant alleles are located on a single chromatid, giving rise to a mutant protein with replaced amino acids in all three sites. The three sites, with valine , histidine (His), and tyrosine (Tyr) replacing alanine (Ala), glutamine (Gln), and serine (Ser) at the 895th, 1450th, and 1541th residues, respectively, are all conserved in a multiple sequence alignment of multiple species. In addition, homology models and in silico predictions indicated that these mutations may alter the normal structure and function of the protein and are therefore potential causes of the phenotype in this family.In this study, we performed WES in a Chinese family with inherited NSCL/P. After screening and Sanger sequencing, three missense mutations in the GLI2 mutations were de novo in patient D2. However, when we tried our best to get DNA samples of the parents of D2, we found that the father of D2, who had claimed to be not affected with cleft, had mutant alleles in all of the three loci of the GLI2 gene. It suggested that the three mutations were delivered from the father of D2 to the subject D2, and then to the subject D1, and the effects of the three mutations might be incompletely penetrant, as reported in some previous genetic studies of clefting  and the son (D1) are affected with NSCL/P, while the others are not. We firstly inferred that the three orbicularis oris muscle . The truncated protein, which retains its DNA‐binding domain, then translocates to the nucleus to repress target gene expression (Hui & Angers, GLI2 mutations leading to NSCL/P are not fully understood. Further demonstrations are needed through animal models and cellular/molecular experiments. Besides, the genetic contribution rates of these variants also remain to be explored in a large sporadic population.Recent studies have shown that the primary cilium, a microtubule‐based organelle that bulges out of the surface of quiescent cells, has a prominent role in modulating vertebrate SHH signaling (Eggenschwiler & Anderson, GLI2 by WES in a Chinese NSCL/P hereditary family. Preliminary studies such as functional prediction, conservation analysis, and homology modeling suggested that the variants c.2684C > T_p.Ala895Val, c.4350G > T_p.Gln1450His, and c.4622C > A_p.Ser1541Tyr potentially underlie NSCL/P in this pedigree. Our results further add to the increasing evidence that GLI2 variants play a role in the pathogenesis of NSCL/P.In conclusion, we identified three candidate missense mutations in the coding region of The authors have declared no competing interests.P. Meng and H. Zhao conducted PCR and DNA cloning, analyzed data, and drafted the paper. W. Huang, Y. Zhang, and F. Chen analyzed data. W. Zhong, M. Zhang, and P. Jia collected human subjects. Z. Zhou and Gulibaha Maimaitili examined the affected patients. J. Zhang and J. Lin designed the study. Click here for additional data file.


Evidence focused on exposure to ambient carbon monoxide (CO) and the risk of hospitalizations for cardiovascular diseases (CVD) is lacking in developing countries. This study aimed to examine the effect of CO exposure on hospitalizations for CVD in Beijing, China.A total of 460,938 hospitalizations for cardiovascular diseases were obtained from electronic hospitalization summary reports from 2013 to 2017. A time-stratified case-crossover design was conducted to investigate the association between CO exposure and hospitalizations for total and cause-specific CVD, including coronary heart disease (CHD), atrial fibrillation (AF), and heart failure (HF). Stratified analysis was also conducted by age group (18–64 years and ≥ 65 years) and sex.0–1 day). For a 1 mg/m3 increase in a 2-day moving average CO concentration, an increase of 2.8% [95% confidence interval (CI): 2.2 to 3.3%] and 3.0% (95% CI: 2.4 to 3.6%) in daily hospital admissions for CVD and CHD were estimated, respectively. This association was robust after adjusting for other copollutants and did not vary by age group and sex.Linear exposure-response curves for the association between ambient CO exposure and hospitalizations for CVD was observed. Ambient CO was positively associated with hospitalizations for total CVD and CHD. However, the observed increased risk was not statistically significant for hospitalizations for AF and HF. The strongest effect of CO concentration was observed on the current- and previous-day of exposure (lag Ambient CO exposure increased the risk of hospitalizations for CVD, especially for CHD in Beijing. Further studies are warranted to explore the association between ambient CO and hospitalizations for AF and HF. All analyses were performed using Stata 14 , with the “xtpoisson” command to fit the conditional Poisson regression, “mkspline” command to build the nature cubic spline, “pca” command to conduct PAC, and “testparm” commend to perform the Wald-test. P value < 0.05 was deemed statistically significant.The relative risk (RR) was calculated from the conditional Poisson regression model . During the study period, the daily 24-h mean pollution concentration was 76.9 μg/m3 for PM2.5, 15.3 μg/m3 for SO2, and 49.7 μg/m3 for NO2, and the 8-h maximum mean concentration for O3 was 95.7 μg/m3. The daily mean ambient temperature was 13.9 °C, relative humidity was 24.4% and air pressure was 1016.7 hPa , NO2 (r = 0.820) as well as SO2 (r = 0.670), but the correlation between CO and O3 was negative and weak (r = − 0.379). Additionally, the correlations between CO and each weather condition were relatively weak.The pairwise correlation among different air pollution concentrations and weather conditions during the study period is provided in Table 3 increment) with varying lag patterns on the percentage change of daily hospital admission for CVD, CHD, AF and HF. The effect of ambient CO gradually decreased from lag 0 to lag 5 day. We found that ambient CO was positively and significantly associated with the percentage change in daily hospital admissions for CVD at lag 0 and lag 1 day, but negative and significant associations were observed at lag 4 and lag 5 day. The largest effect was observed on lag 0–1 day. Therefore, the following analyses only focused on the cumulative delay effects of CO on lag 0–1 day. The lag patterns for CHD were consistent with CVD. These associations between CO exposure and AF as well as HF, however, were not significantly detected on any lag day .Fig. 0–1 day and daily hospitalizations for total and cause-specific CVD. We did not find any apparent nonlinear exposure-response relationships for daily hospitalization for CVD , CHD , AF  or HF .Figure 0–1 day) of CO concentrations on daily hospital admissions for total and cause-specific CVD. In the single pollutant model, for a 1 mg/m3 increase in the 2-day moving average CO concentrations (lag 0–1), we observed significant increases of 2.8% (95% CI: 2.2 to 3.3%) in the daily hospital admission for CVD, 3.0% (95% CI: 2.4 to 3.6%) for CHD, 1.4% (95% CI: -1.4 to 4.2%) for AF and 1.2% (− 0.4 to 2.7%) for HF. In the multipollutant models where copollutants were adjusted by PCA, the significant associations observed in the single-pollutant models weakened but remained significant for CVD and CHD. Additionally, a week association was observed between ambient CO and hospital admissions for AF.Table 0–1 day) and hospital admission for total and cause-specific CVD, stratified by age group, and sex. No effect modifications were found to be significant .Figure 0–1 day. Our findings were robust after controlling for other pollutants in the multipollutant models.In the present study, we used a time-stratified case-crossover design with conditional Poisson model to investigate the associations of ambient CO and hospitalizations for CVD in Beijing from 2013 to 2017. We found that short-term exposure to ambient CO was significantly associated with an increased risk of hospital admissions for CVD and CHD, but not for AF and HF. The largest estimate effects were observed on the lag Bell, et al. found that a 1 mg/m3 increase in the same-day daily 1-h maximum CO was associated with a 1.0% (95% CI: 0.8 to 1.1%) increase in the risk of CVD admissions in 126 United States urban counties from 1999 to 2005 [Barnett, et al. conducted a case-crossover study in seven Australian and New Zealand cities and combined the estimates across these cities using a random-effects meta-analysis. They found that a 0.9 mg/m3 increase in CO significantly increased the risk of hospital admissions for cardiovascular disease and ischemic heart disease by 2.2% (95% CI: 0.9 to 3.8%) and 2.3% (95% CI: 0.9 to 3.4%), respectively, in the elderly [3 increase in CO concentrations on the same day was significantly associated with a 9.4% (95% CI: 5.1 to 14.0%) increase in total cardiovascular hospital admissions [2 [Bell, et al’ study, this association was attenuated after adjusting with same-day NO2 [2.5, NO2, SO2 and O3 [Increasingly, scientific evidence has indicated that outdoor CO exposure is related to CVD morbidity and mortality , 32–34.  to 2005 . Barnett elderly . A study-day NO2 . To redu2 and O3 , 14.2, O3, and NO2) has become a serious environmental problem. Ambient CO is primarily produced by incomplete combustion of carbon-containing fuels. The major source of CO in Beijing is generated from vehicle emissions. However, the adverse health effects of CO exposure on cardiovascular hospital admissions is understudied in China. Our findings were supported by a recent multicity analysis conducted in 14 large Chinese cities that reported a 0.8 mg/m3 increase in CO concentrations on lag 2 day was significantly associated with a 1.1% (95% CI: 0.4 to 1.8%) increase in acute myocardial infarction admissions [0 and lag 1 day, and a maximum CO exposure effect on lag 0–1 day. Another recent study conducted in Beijing showed similar findings compared to our results [4 and lag 5 day, which indicated a presence of “harvesting effect” in the current analysis. Similarly, a time-series study conducted in Beijing in 2009–2010 also found that ambient CO had a negative effect on CVD mortality at lag 3 and lag 4 day [In China, urbanization and economic development have greatly increased, especially in large cities such as Beijing and Shanghai. Gaseous air pollution (CO, SOmissions . Moreovemissions  and in tmissions . Our resmissions , 14, 35  results . Notablyag 4 day , which w3 CO concentrations [The exposure-response relationship was approximately linear and there was no noticeable threshold effect below which CO exposure had no effects on cardiovascular hospitalizations in the current analysis, which was in accordance with other findings in previous studies , 13. Howtrations , which w3 increment in CO concentrations [We also found that the association between short-term CO exposure and hospital admissions varied by cause-specific CVD. The adverse effect was obvious and robust for daily hospitalizations for CHD, but not for AF or HF. Given the distinct differences in the underlying mechanisms through which that CO exposure triggers CHD compared with AF and HF , 37, 38,trations . Howevertrations ; thus thtrations . Moreovetrations  and 0.9%trations , respectIdentifying subpopulations who are the most susceptible is critically important for policy-maker to promote a targeted reduction in ambient CO . A few sAs previously shown, inhaled CO can bind to hemoglobin and form carboxyhemoglobin which decreases the oxygen-carrying capacity of hemoglobin and causes cellular hypoxia . HoweverOur study had several strengths. First, cardiovascular hospital admission data were obtained from an established monitoring system in Beijing; thus, misclassifications in cardiovascular outcomes were less likely to occur. Second, we explored the potential association between ambient CO and cause-specific CVD admissions. However, the limitations of our study also need to be noted. Similar to other studies exploring the impact of air pollutants on health outcomes, we need to carefully interpret and infer the cause-effect relationship between carbon monoxide exposure and hospitalizations for CVD due to the ecological design of the present study. We also used ambient CO data aggregated from fixed-site air quality monitoring stations to represent individual-level exposure, so measurement bias may exist.Our findings have elucidated that short-term exposure to ambient CO significantly increased the risk of hospitalizations for total CVD, especially for CHD, in Beijing. Further study is necessary to identify the relationship between ambient CO and hospital admissions for AF and HF.


To evaluate the safety and effectiveness of femtosecond laser-assisted in situ keratomileusis (LASIK) in the treatment of residual myopia and astigmatism following femtosecond laser-enabled keratoplasty (FLEK).  Retrospective case review.  Chart review of all patients with prior FLEK who subsequently underwent femto-LASIK surgery after full suture removal was performed at the Gavin Herbert Eye Institute at the University of California, Irvine. A total of 14 eyes in 13 patients met this criterion, and their comprehensive examinations performed at standard intervals were reviewed. Main outcome measures include uncorrected distance visual acuity (UDVA) and corrected distance visual acuity (CDVA) expressed as the logarithm of the minimum angle of resolution (logMAR), manifest refractive astigmatism, and spherical equivalent. P = 0.002) with no loss of CDVA . All 14 eyes showed significant improvement in manifest refractive astigmatism (4.71 ± 1.77 to 2.18 ± 1.45 diopters (D), P = 0.003) and spherical equivalent . There were no flap or graft complications as a result of femto-LASIK.  From the preoperative visit to the 3 month visit, all 14 eyes significantly improved in UDVA (logMAR, 0.93 ± 0.23 to 0.44 ± 0.32,  Our findings suggest that femto-LASIK on eyes with prior FLEK is safe and effective in improving visual acuity and reducing residual astigmatism. In penetrating keratoplasty (PK), a variety of perioperative factors influences the refractive outcome, which makes it challenging to achieve a desirable and predictable result . These iAfter the graft has healed and stabilized, a variety of corneal and lenticular surgeries may be performed to neutralize refractive error . IntraocThe advent of femtosecond (FS) laser improved PK surgeries by creating precise and consistent incisions identical in host and donor corneas . ComparePatients were included in this study if they had a prior FLEK with clear and stable grafts after full suture removal and a subsequent LASIK surgery from September 2008 to July 2016 at the Gavin Herbert Eye Institute, University of California, Irvine. These patients were also included regardless of other corneal or lenticular surgeries that may have occurred in between FLEK and femto-LASIK surgeries, including cataract extraction and intraocular lens placement or intraocular lens exchange and repositioning.Preoperatively, each patient was screened for refractive stability and for retinal pathologies, glaucoma, and other ocular diseases that could affect visual potential. These complete ophthalmic examinations included measurements of uncorrected distance visual acuity (UDVA) and corrected distance visual acuity (CDVA) with a Snellen chart, intraocular pressure, manifest refraction, and corneal topography with the Pentacam , ATLAS , Orbscan , and the IOLMaster . Refractive stability was ensured postsuture removal by at least three serial refraction and topographies prior to a final decision on laser refractive correction. Graft health was primarily determined by endothelial cell analysis using specular microscopy with a CellChek XL . Complete ophthalmic examinations were also performed routinely at postoperative months 1, 3, 12, and beyond, up to 3 years. Given the visits that occurred 1 to 3 years postoperatively varied in time and number, the patient's last visit during that time period was chosen as the “1 yr+” visit. The “Final” visit represents the last follow-up visit that occurred within those 3 years. Data collection at routine follow-up visits included UDVA, CDVA, and manifest refraction. Corneal topography was also assessed when available. Institutional Review Board approval was obtained at the University of California, Irvine, for this retrospective study.All FLEK and femto-LASIK surgeries were performed at Gavin Herbert Eye Institute at University of California, Irvine. All surgeries followed standard protocol. Two surgeons (RFS and MF) performed the surgeries using the same techniques. Patients reviewed and signed informed consent forms prior to each surgery.μm from the anterior corneal surface. The final cut extends from the anterior corneal surface into the stroma at an angle of 30° towards the periphery. The anterior diameter of the cut in each host cornea was set at 8, 8.5, or 9 mm, and the graft size was accordingly set at an identical diameter. Standard postoperative drops included moxifloxacin and prednisolone acetate 1%, tapered per surgeon preference. Postoperative healing of wound was ensured prior to full suture removal, which occurred postoperatively between 3 and 26 months.FLEK surgeries were performed with the IntraLase FS laser . Identical zig-zag patterns were used to create full-thickness cuts in host and donor corneas. Three separate cuts were created to intersect contiguously in order to form a full-thickness cut from the corneal surface to the anterior chamber. The first cut extends from the posterior corneal surface in the anterior chamber into the stroma at an angle of 30° towards the periphery. The second cut consists of a lamellar ring with a width of 0.5 mm at a depth of 320 μm, along with a superior hinge. No flap crossed the graft-host junction. After separating and lifting the flap, the stroma was ablated with a VISX Star S4 excimer laser . The ablation was set for a conventional treatment based on manifest refraction. If femto-LASIK retreatment was needed, the flap was relifted and the stromal bed was reablated. Postoperatively, patients were prescribed moxifloxacin and prednisolone acetate 1%, tapered per surgeon preference.In all cases, the surgeon who performed the patient's FLEK also subsequently treated the patient with femto-LASIK surgery. A 150-kHz FS laser  was used to create all flaps. Flaps were created within the diameter of the PK graft at an intended thickness of 120 t test analyses in Microsoft Excel . A P value less than 0.05 was considered statistically significant.For statistical analysis, Snellen values from UDVA and CDVA were converted to logarithm of the minimum angle of resolution (logMAR) scale. Outcome measurements of UDVA, CDVA, manifest astigmatism, and spherical equivalent were compared preoperatively and postoperatively using paired and unpaired Patient characteristics are presented in P=0.003). This improvement remained stable over time, with a mean UDVA of 0.44 ± 0.32, or 20/55, at postoperative month 3; 0.22 ± 0.12, or 20/33, at postoperative month 12; and 0.19 ± 0.12, or 20/31, at the last postoperative visit over 1 year. Overall, the mean UDVA at the final visit was 0.35 ± 0.29, or 20/45 (P < 0.0001). The preoperative UDVA ranged from 20/80 to 20/400. At final visit, 9 of 14 eyes (64.3%) had a UDVA of 20/40 or better and 12 of 14 eyes (85.7%) had a UDVA of 20/70 or better.Figures P=0.47). Mean CDVA values were at 0.18 ± 0.23, or 20/30, at postoperative month 3; 0.14 ± 0.21, or 20/28, at postoperative month 12; 0.07 ± 0.09, or 20/23, at the last postoperative visit over 1 year; and 0.21 ± 0.23, or 20/32, at final visit (P=0.42). The preoperative CDVA ranged from 20/20 to 20/80. At final examination, 6 eyes (42.9%) improved by 1 or more Snellen lines, 5 eyes (35.7%) maintained their preoperative CDVA, and 3 eyes (21.4%) lost 1 or more Snellen lines.The results for CDVA showed no significant improvements postoperatively, but remained stable throughout the entire study period from 0.26 ± 0.19, or 20/36, preoperatively to 0.29 ± 0.26, or 20/39, at postoperative month 1 (P=0.007). This remained stable throughout the entire study period. Mean refractive cylinder was 2.18 ± 1.45 D at postoperative month 3, 1.38 ± 1.22 D at postoperative month 12, and 1.63 ± 1.09 D at the last postoperative visit over 1 year. Taken together, the mean refractive cylinder at final visit was 1.34 ± 0.99 D (P < 0.0001). Complete cylindrical correction was achieved in 1 eye from a preoperative refractive astigmatism of 2.5 D. In another eye, refractive astigmatism worsened by 0.25 D, but visual acuity improved so that overall spherical equivalent was significantly improved. Cylindrical regression of 1.00 D from postoperative month 1 occurred in 1 eye, but was still an improvement at 3.25 D at final visit compared to 6.00 D preoperatively.P=0.005). This remained stable over time with a mean of −0.48 ± 0.83 D at postoperative month 3, −0.22 ± 1.23 D at postoperative month 12, and −0.63 ± 1.11 D at the last postoperative visit over 1 year. At final visit, the mean refractive spherical equivalent was −0.46 ± 0.98 D (P=0.004), and 11 of 14 eyes (78.6%) were within ±1.0 D.All fourteen eyes were myopic preoperatively, with a mean refractive spherical equivalent of −2.57 ± 2.45 D. No complications arose during femto-LASIK surgery, including wound dehiscences, flap dislocations, epithelial ingrowth, flap striae, buttonholes, ectasia, or corneal edema. There were also no eyes that experienced graft infection, rejection, or failure, postoperatively. Four eyes (28.6%) underwent femto-LASIK retreatment for treatment of additional cylinder. Prior to retreatment, all four eyes had residual refractive astigmatisms of more than 1 D at postoperative month 3 of the primary femto-LASIK. After retreatment, all eyes improved favorably and without complications. Visual parameters after enhancements are included in the primary outcome results.In this retrospective case series, femto-LASIK has shown to be safe and effective in improving visual acuity and reducing residual astigmatism in eyes with prior FLEK. UDVA, manifest refractive astigmatism, and spherical equivalent all significantly improved by postoperative month 1 and remained stable through the final visit. While there appear to be no studies on femto-LASIK post-FLEK, there are many studies on LASIK after conventional PK with blade trephination. All of these studies have shown significant improvements in visual acuity, refractive astigmatism, and spherical equivalent, with minimal to no complications , 18–23.Photorefractive keratectomy was not considered in these cases due to the potential risk of haze formation, and the surgeons are not in favor of using mitomycin C on ocular surfaces and corneas that are already compromised. The use of a femtosecond laser flap allows excellent visualization of the flap and perfect centration prior to initiating the laser cut. The risk of flap dislocations and complications has been shown to be significantly reduced with femto-flaps. For these reasons, femto-LASIK is always preferred to PRK in the post-PKP patient in our practice.The results of this unique study compare favorably with the aforementioned studies. At final examination, UDVA was 20/40 or better in 64.3% of our cases versus 29% , 54% 2222, 70% , and 86%As this was a retrospective study, follow-up data were limited given that those who were most satisfied with their improved vision often did not return as frequently as others. Furthermore, the refraction of corneal grafts can continue to change beyond 12 months; hence, longer follow-up is needed to study the long-term stability of these refractive outcomes. While this study's results suggest that femto-LASIK after FLEK is effective in improving visual acuity and reducing residual astigmatism, direct differences in surgical outcomes between femto-LASIK after FLEK and femto-LASIK after conventional PK are still unknown. However, the previous studies have shown that FLEK may have advantages postoperatively in healing, tissue alignment, and biomechanical strength compared to PKs performed with manual blade trephination –17. FLEKIn this study, four eyes needed femto-LASIK enhancement within the first year after primary femto-LASIK. These were eyes that had preoperative cylinders high enough to require two treatments due to the limitations from laser correction. As such, these patients were told they would require at least two treatments prior to their primary LASIK operations. After retreatment, these eyes improved favorably with reduced residual refractive astigmatisms. Femto-LASIK retreatment is not unique to eyes with prior FLEK, as retreatment in post-PK corneas is known to be more common than in corneas that have not undergone any previous operations , 20, 23.With regards to safety outcomes evaluated in this study, performing femto-LASIK surgery on eyes with prior FLEK was not associated with significantly adverse complications. Most severe complications involve damage to the transplant or to the graft-host wound interface. These adverse outcomes were not reported for any of the eyes reviewed in this study since the diameter of the femtosecond flaps was programmed to be smaller than the diameter of the graft, avoiding the graft-host junction , 34.In conclusion, femto-LASIK appears to be a safe and effective surgical option to correct residual refractive error from a prior FLEK. This study is unique given the dearth of studies monitoring the performance of femto-LASIK on corneal transplants done with FS laser. The results of this study showed statistically significant improvement in UDVA, manifest refractive astigmatism, and spherical equivalent in eyes with prior FLEK. CDVA remained stable, and no surgical complications were reported. Furthermore, our results with femto-LASIK after FLEK compare favorably to results from similar studies on femto-LASIK after conventional PK. Future studies comparing direct differences between these two combinations are warranted.


Transcriptomic profiles can improve our understanding of the phenotypic molecular basis of biological research, and many statistical methods have been proposed to identify differentially expressed genes (DEGs) under two or more conditions with RNA-seq data. However, statistical analyses with RNA-seq data are often limited by small sample sizes, and global variance estimates of RNA expression levels have been utilized as prior distributions for gene-specific variance estimates, making it difficult to generalize the methods to more complicated settings. We herein proposed a Bartlett-Adjusted Likelihood-based LInear mixed model approach (BALLI) to analyze more complicated RNA-seq data. The proposed method estimates the technical and biological variances with a linear mixed-effects model, with and without adjusting small sample bias using Bartlkett’s corrections.We conducted extensive simulations to compare the performance of BALLI with those of existing approaches . Results from the simulation studies showed that BALLI correctly controlled the type-1 error rates at various nominal significance levels and produced better statistical power and precision estimates than those of other competing methods in various scenarios. Furthermore, BALLI was robust to variation of library size. It was also successfully applied to Holstein milk yield data, illustrating its practical value.BALLI is statistically more efficient and valid than existing methods, and we conclude that it is useful for identifying DEGs in RNA-seq analysis.The online version of this article (10.1186/s12864-019-5851-6) contains supplementary material, which is available to authorized users. Transcriptomic profiles can improve our understanding of the phenotypic molecular basis of biological research, and many attempts have been made to identify differentially expressed genes (DEGs) by microarray analysis. However, microarray analysis often suffers from many systematic errors, such as hybridization and dye-based detection bias, hampering the detection of true DEGs , 2. ReceRNA-seq quantifies the numbers of reads aligned to particular transcripts or genes, and various approaches have been proposed to manage RNA-seq data . There aNegative-binomial distributions for read counts and normal distributions for log-transformation of counts per million (CPM) successfully describe distributions of RNA-seq data. However, RNA-seq is relatively expensive compared with microarray, and thus, further adjustment has been made to handle the problem of small sample size. If the sample size is small, the estimated variance can have large standard errors, and thus, multiple methods that incorporate prior knowledge have been proposed. For example, variances of read counts are assumed to be positively related to their means, and their relationships can be estimated by comparing the means and variances of read counts for all genes. This can often be utilized to shrink variance parameters , 12. edgExisting methods shrink the variance estimate of each gene toward global variance estimates or use variance estimates based on the relationships between means and variances. Such assumptions are often very useful if the sample sizes are small. However, there are multiple factors that can distort these relationships, and if they are violated, the performance of existing approaches can be affected. For example, the quality of data is highly dependent on the preparation steps, and unexpected noise, such as noise from different storage periods or sequencing organization of samples, can occur during preparation steps. Moreover, read counts of cancer tissues are more heterogeneous than those of normal tissues, and biological variances can be affected by disease status . Thus, thttp://cran.r-project.org) or http://healthstat.snu.ac.kr /software/balli/.In this article, we present new methods for identifying DEGs with RNA-seq data, namely, BALLI and LLI. Statistical analyses with log-transformed read counts are often more powerful than other existing methods and are relatively insensitive to various errors , 17. ThuM different groups, and the averages of the expressed read counts for each gene were compared among these groups. For case-control studies, M = 2. We assumed that there were nm subjects in group m and denoted the total sample size by N; thus, we got N = n1 +  …  + nM. We defined dummy variables for subject i in group m byWe assumed that there were A design matrix for group variables is defined byn1 subjects were in group 1; the second n2 subjects were in group 2; and so on. The effect of continuous variables can also be tested, and then some or all of the columns of the design matrix, X, become their realization. We assumed that expressed read counts were observed for G genes and were denoted by rgi for gene g of subject i . Then, the library size for subject i, Ri, was equivalent to cpm function in the edgeR R package. If we denoted the normalized Ri with the trimmed mean of the M-value X and Here, Dandg, Mg, Pg, νg, and τg depend on the structure of Vg, and counterparts to general Vg s are shown in Additional file The forms of DThe log-likelihood function for our final model is given byP = Vg−1 − Vg−1tVg−1)tVg−1, the profile log-likelihood of Vg is a function of Here, optimize function in R [Σg, b. Then, , where ion in R . It alwa, which maximizes the likelihood under the null hypothesis. Under the null hypothesis, if we let P0 = Vg−1 − Vg−1Z(ZtVg−1Z)ZtVg−1, the profile log-likelihood of The Bartlett-adjusted LRT requires Vg0 and denoted it as We considered two real datasets consisting of samples from unrelated Nigerian people and Holstein cattle, respectively. Nigerian subjects participated in the International HapMap Project and comprised 29 male and 40 female participants . The reaψg = 1 and N was greater than or equal to 12. LLI also successfully controlled the nominal type-1 error rates if N was greater than or equal to 20. However, if N was less than 20, p values by LLI were inflated. edgeR showed the least performance, and the estimated type-1 error rates were always inflated at 0.05, 0.01, and 0.005 nominal significance levels. Interestingly, DESeq2 tended to be conservative at 0.1 and 0.05 but liberal at 0.01 and 0.005 nominal significance levels. Thus, we could conclude that the proposed linear mixed model with Bartlett’s correction reasonably controlled the type-1 error, and Bartlett’s correction was required if the sample size was less than 20.We applied the proposed linear mixed models to the simulated data based on Nigerian individuals’ RNA-seq data and calculated empirical type-1 error rates and statistical powers with these models. The data were then compared with DESeq2 (v1.20.0), edgeR v3.22.3), and voom (v3.36.5). Table .22.3, anN = 12, 16, 20, 24, 28, 40, 64, or 68. Figure p values are less than 0.1. According to Fig. N was less than 40  and DESeq2 (0.399). The estimated precision of BALLI was 0.940, and those of voom and DESeq2 were 0.930 and 0.907, respectively. If N = 40 and δ = 0.5σ, the estimated power and precision of BALLI were 0.342 and 0.943, which were higher than those of DESeq2  and voom .Figure Our method was also applied to simulation data based on RNA-seq data from Holstein cows. The results were similar to those of the simulation data based on Nigerian people’s data. Additional file P values obtained from LLI and edgeR tended to be inflated. DESeq2 generally showed deflation of type-1 error rates at a 0.1 nominal significance level. Second, we considered the effects of library size variance on statistical analyses. Data with unequal library sizes among subjects were generated by negative binomial distribution whose mean parameters (agi) were the product of mean estimates, under the equal library size assumption, and random numbers from U, where u = 0.2, 0.4, 0.6,  0.8, or 1, and dispersion parameters were estimated from u became smaller, the library size had larger variances. Figure u. Figure u. The estimated type-1 error rates of LLI were affected by sample size. If N was larger than or equal to 40, LLI controlled the type-1 error rates the most correctly and was not affected by the library size variation. BALLI was slightly conservative, but the amount remained constant. Results at the 0.005 significance level are provided in Additional file RNA-seq data are generally known to follow negative binomial distribution, and we conducted simulation studies with RNA-seq data generated from negative binomial distributions. First, we assumed that library sizes were the same among subjects. The overall trend of the estimated type-1 error rate was similar to that of simulation studies based on real RNA-seq data. Estimated type-1 error rates by voom and BALLI usually maintained the nominal significance levels Table . P valueu. BALLI usually had the best estimated power and precision, as was observed in simulation studies based on real RNA-seq data. For example, when u = 0.2, N = 28, and δ = 0.5σ, the estimated power by BALLI was 0.438, whereas those for DESeq2 and voom were 0.317 and 0.352, respectively  were significantly validated. According to Seo et al. (2016), however, no DEGs, including the nine genes, were found at an FDR 0.1 significance level by DESeq2, voom, and their methods owing to the lack of statistical power [TOX4 gene between the high and low milk yield groups at the FDR-adjusted 0.1 significance level, but other methods did not detect any significant genes. The FDR-adjusted p value of TOX4 by BALLI was 0.1267, which was much smaller than those of DESeq2, edgeR, and voom. Table p values for the nine genes, including TOX4. P values for most of the nine genes obtained by LLI and BALLI were smaller than those obtained by other methods. The nine genes were not significantly affected by parity or lactation period , DESeq2 (27.21 s) and edgeR (33.25 s) ; otherwise, it is better to use BALLI.In this article, we suggested new methods, designated BALLI and LLI, for identifying DEGs with RNA-seq data. We assumed that log-cpm values of read counts asymptotically followed normal distributions, and the linear mixed-effects model with Bartlett’s correction was proposed. The proposed methods were compared with existing methods, such as DESeq2, edgeR, and voom, with extensive simulation studies. According to our results, negative-binomial-based approaches often failed to preserve the nominal type-1 error rates. For example, Furthermore, we evaluated the effects of library size variations on statistical analyses. We found that library size variance could affect the estimated type-1 error rates, and the effect was the largest for voom. Library sizes are affected by multiple factors, such as the amount of mRNA and the sequencing instrument, which can generate substantial variation among library sizes for subjects. Our simulation studies showed that BALLI was robust with regard to library size variation in samples of various sizes and was a reasonable choice if large library size variance was observed.μ + ϕ with first order approximation. In addition, voom considered log-cpm value as a response and assumed that they were normally distributed. However, our simulation studies revealed the superiority of the proposed methods compared with voom, which was found to be attributable to their different variance structures. For the proposed methods, 1/μ + ϕ was derived from the first order approximation of the negative binomial distribution and thus may be a natural assumption for RNA-seq data. Furthermore, for 1/μ + ϕ, ϕ obviously indicates the overdispersion parameter, and biological and technical variances can be estimated with BALLI. However, voom assumes ϕ/μ, and the amount attributable to biological or technical variances cannot be clearly defined.The proposed methods assumed that log-cpm values of read counts asymptotically followed a normal distribution and that their variances were approximately equal to 1/μ + ϕ and had the most generalized variance parameter space. Incorporation of φ = 1 yielded BALLI and LLI, and ϕ = 0 yielded voom. We found that BALLI was the most efficient in the considered scenarios; however, in real data analyses, various factors affected variance structure. For example, subjects with different ethnicities can cause φ to be larger than 1, and thus, a better model may differ according to RNA-seq data. φ and ϕ can be estimated with the proposed linear mixed model by implementing only a simple modification, and thus, we can choose the best model using AIC or LRTs. The selected models can then be utilized to identify DEGs. This model was implemented as an R package and can be downloaded from CRAN (http://cran.r-project.org) or http://healthstat.snu.ac.kr/software/balli/. Furthermore, in contrast to methods based on a generalized linear mixed model such as MACAU [We also suggested the most flexible and general linear mixed model for log-cpm. The proposed model assumed that the variance of log-cpm was φ/as MACAU , the proWith simulation studies for various scenarios, we showed that the proposed methods were usually the most efficient. However, Bartlett’s correction seems inappropriate when N ≤ 6 The forms of CAdditional file 2:Steps of generating simulation data. (DOCX 18 kb)Additional file 3:N = 4, 6, 8, 28, 40, 64, and 68 based on Nigerian people’s data. (DOCX 21 kb)Estimated type-1 error rates with simulation data for Additional file 4:N = 4, 6, 8, 12, 16 and 20 based on Holstein cow’s data. (DOCX 20 kb)Estimated type-1 error rates with simulation data for Additional file 5:N = 12, 16 and 20 based on Holstein cow’s data. (DOCX 197 kb)Estimated powers and precisions with simulation data when δ = 0.5σ or 1σ and Additional file 6:N = 4, 6, 8, 28, 40, 64, and 68 based on simulated data from negative binomial distribution. (DOCX 21 kb)Estimated type-1 error rates with simulation data for Additional file 7:N = 12, 16, 20, 24, 28, 40, 64, or 68 at the 0.005 nominal significance level. (DOCX 371 kb)Effect of varying library sizes on the type-1 error rates when u = 0.2, 0.4, 0.6, 0.8, and 1 and Additional file 8:N = 12, 16, 20, 24, 28, 40, 64, or 68. (DOCX 593 kb)Effect of varying library sizes on the statistical power and precision when u = 1, 0.8, 0.6, or 0.4, δ = 1σ and Additional file 9:Analysis of covariables, parity and lactation period, for true nine DEGs of Holstein cow's data. (DOCX 18 kb)Additional file 10:Computation time for each method when analyzing Holstein cow’s data. (DOCX 14 kb)


Blackwater fever (BWF) is a rare but serious complication of malaria that is a consequence of antimalarial treatment. Its prevalence seems to have increased. Its diagnosis is based on clinical symptoms and urine color. We report on 4 BWF cases admitted to the infectious diseases department of Zinder National Hospital.  Four patients were hospitalized in September 2017 for a hepatorenal syndrome of jaundice, port wine-colored urine, renal failure, and hepatic cytolysis following antimalarial treatment with quinine salts. Quinine treatment was stopped and treatment was continued with injectable artemether. Three patients underwent extra-renal purification. Their evolution was favorable. One patient died less than 24 hours after admission.  A rare and severe complication, blackwater fever must be considered for patients under antimalarial treatment who present with jaundice, abdominal pain, and acute renal insufficiency with port wine-colored urine. Rapid diagnosis and management in an intensive care unit are crucial for improving the prognosis. Blackwater fever (BWF) is an acute intravascular hemolysis that results from antimalarial drug use –3. It clGlucose-6-phosphate dehydrogenase deficiency (G6PD) also is frequently associated with this syndrome; however, its role is not clearly demonstrated , 6. MorePlasmodium falciparum antigen in the blood. This prompted the initiation of an antimalarial treatment based on quinine injection at a rate of 25 mg/kg/day, which was discontinued after 3 days. Treatment was resumed 4 days later after the reappearance of symptoms. On the third day after resumption of treatment, corresponding to 10 days after the onset of symptoms, the patient was referred to Zinder National Hospital following a clinical aggravation marked by impaired consciousness. On arrival, clinical examination revealed a fever at 39°C, impaired consciousness with a Glasgow score of 11, mucous membrane pallor, conjunctival jaundice , positive result reflecting the presence of jaundice , tachypn3, normochromic anaemia with a hemoglobin level of 4 g/dl, and a normal platelet count of 194,000/mm3. The direct Coombs test was negative. Urea: 74.9 mmol/L, serum creatinine: 559.6 μmol/L, proteinuria: 0.56 g/24 h, ASAT: 334 IU/L, ALAT: 185 IU/L, CRP: 92 mg/L, blood ionogram: 128 mEq/L of sodium and 5 mEq/L of potassium, TP: 100%, INR: 0.99, TCA: 29.5, TQ: 12 sec, and a blood bilirubin level of 200 mg/L predominantly unconjugated. Abdominal ultrasound showed normal-sized kidneys, discrete renal dedifferentiation, and vesicular sludge. This presentation required dialysis for uremic syndrome. The patient received 3 hemodialysis sessions with transfusion of erythrocyte concentrates. The antimalarial treatment was continued with an intramuscular artemether derivative. Clinical status and renal function normalized after 7 days. The patient was discharged cured.The haemodynamic state was stable with a blood pressure of 120/80 mm Hg. Urine was Coca-Cola colored , which lP. falciparum. The hemoglobin concentration was 5 g/dl, and the blood glucose level was 3.9 mmol/L. Urinary catheter insertion revealed port wine-colored urine, which prompted the BWF diagnosis. The urine test strip indicated hemoglobinuria at 2 plus; bilirubinuria 3 plus; proteinuria 2 plus; absence of urobilinogens, red blood cells, and leukocytes; creatinemia of 450 μmol/L; and urea of 31.39 mmol; and quinine discontinuation was followed by treatment with intramuscularly injected artemether at a dose of 3.2 mg/kg daily on day 1 and 1.6 mg/kg daily from day 2 to day 5. The patient had three sessions of extrarenal purification and transfusion with 4,450 ml bags of blood. Evolution was favorable with clinical improvement and recovery of renal function after 5 days.A 35-year-old woman with no particular history was hospitalized for a headache, fever, jaundice , and impPlasmodium falciparum trophozoites per mm3. The hemoglobin concentration was 10.3 g/dl, and blood glucose was 9.4 mmol/L. Quinine treatment at a dose of 25 mg/kg per 24 hours was instituted in the emergency department. Obnubilation-type consciousness disorders and conjunctival jaundice appeared after 12 hours of hospitalization. Even though the patient was afebrile, the diagnosis of BWF was evoked due to the port wine color of his urine. The emergency bioassay showed a creatinemia of 720 μmol, urea of 28 mmol, and hemoglobin of 5.5 g/dl. Quinine was immediately stopped, in favor of intramuscularly injected artemether prescribed at a dose of 3.2 mg/kg daily on day 1 and 1.6 mg/kg daily from day 2 to day 5. Unfortunately, the patient only received the first dose because he died less than 24 hours after admission of shock in coma and with anuria.A 42-year-old man with no particular history was hospitalized for drowsiness, 38.9°C fever and a heart rate of 140 beats/minute. He weighed 55 kg. The thick peripheral blood smear was positive with 17,400 P. falciparum. Hemoglobin concentration was 9.0 g/dl, blood glucose was 5.6 mmol/L, and serum creatinemia was 1500 μmol/L. The patient had received a first dose of quinine at a dose of 25 mg/kg/24 hours the previous day. An oligoanuria with port wine-colored urine was noted. Quinine was discontinued and replaced with intramuscularly injected artemether at a dose of 3.2 mg/kg daily on day 1 and 1.6 mg/kg daily from day 2 to day 5. The patient had 4 hemodialysis sessions. The evolution was favorable with progressive improvement of renal function.A 38-year-old man with no particular history was hospitalized for fever and jaundice. At admission, his temperature was 38.5°C with a Glasgow score of 11/15, a weight of 62 kg, a respiratory rate of 28 cycles/minute, and a heart rate of 100 beats/minute. The thick peripheral blood smear was negative, but the rapid diagnostic test was positive for P. falciparum was also reported by many authors [Plasmodium vivax. In 2016, Baber et al. [Plasmodium knowlesi, and Madhuri et al. [Plasmodium malariae.BWF is found in areas where malaria is endemic , 11. Sev authors . However authors  publishei et al.  had descTreatment with quinine was the triggering factor in our patients as is noted by most authors , 12–15. Plasmodium falciparum, causing acute hemolysis [BWF pathophysiology is complex and not yet well known, but according to some authors, the disease is secondary to double red blood cell sensitivity to antimalarial drugs and emolysis , 2. Intremolysis . The relemolysis . The exiemolysis . This anClinically, the syndrome is well characterized . PresentThe very high lethality observed initially has today significantly decreased in hospitals , 17. CurBWF treatment is based on the discontinuation of quinine or other drugs that may trigger hemolysis, the use of artemisinin derivatives when they are not incriminated, blood transfusion, intravenous fluids, electrolytic re-equilibration, and, frequently, extrarenal purification , 19, 20.Limitations of this study are technical. Indeed some explorations are not available in our context, including the search for G6PD deficiency and drug concentration dosages in the blood or urine.BWF is a serious and rare complication of malaria and its treatment. It should be remembered in a patient undergoing malaria treatment admitted with jaundice, abdominal pain, and acute renal failure with port-colored urine. The disease seems to be more frequent in periods of high endemicity (rainy season in the Sahel). Treatment often requires extra-renal cleansing which is essential. Quinine incriminated in our patients is not the only drug involved in the occurrence of BWF. Several other drugs  used as an alternative to this are also reported in the literature.


GDNF, and the “rearranged during transfection” proto-oncogene, RET, in kidney and urinary tract development, variations in these genes are proposed to be candidates for medullary sponge kidney. Hyperparathyroidism is observed in a few patients with medullary sponge kidney, but the exact pathogenesis of this association is unknown. This case report highlights the coexistence of these two conditions associated with RET polymorphism, which contributes toward the understanding of the pathogenesis of medullary sponge kidney.Medullary sponge kidney is a rare renal malformation, which usually manifests as nephrocalcinosis, renal tubular acidosis, and recurrent urinary tract infections. Medullary sponge kidney is often associated with renal developmental anomalies and tumors, and its exact pathogenesis is not yet clearly explained. Given the key role of the interaction of glial cell line-derived neurotrophic factor gene, GDNF and RET genes were determined by Sanger sequencing. Two RET polymorphisms were found in our patient, one was nonsynonymous c.2071G>A  located in exon 11, the other was synonymous c.2712C>G.  located in exon 15.A 52-year-old Chinese woman with recurrent renal stones presented to our hospital. Subsequently she was diagnosed as having medullary sponge kidney and tertiary hyperparathyroidism and underwent parathyroidectomy. Genomic DNA was isolated from lymphocytes and the RET G691S/S904S polymorphism in this patient, but additional studies are required to explore the role of the RET gene in medullary sponge kidney with hyperparathyroidism.We demonstrated a case of medullary sponge kidney combined with tertiary hyperparathyroidism, which contributes to further understanding of the pathogenesis of this disease. Besides, we also found  GDNF and RET genes, which play a key role in kidney and urinary tract development, maybe reasonable candidates for MSK. Hyperparathyroidism is observed in a few patients with MSK, but the exact pathogenesis of this association is unknown [GDNF and RET gene variations, which contributes to further understanding of the pathogenesis of this disease.Medullary sponge kidney (MSK) is a nephropathy which usually manifests as nephrolithiasis, renal tubular acidosis, concentration defects, medullary cystic dilatations, and recurrent urinary tract infections. Its incidence is 1/20,000 to 1/5000 in the general population, 3 to 5% in patients with kidney stones, and up to 20% in patients with recurrent kidney stones , 5. Thus unknown . In the 2CP) 17.8 mmol/L, creatinine 249 μmol/L, serum parathyroid hormone (PTH) 1147 pg/ml  of her kidney showed MSK and polycystic kidney disease  37.5 °C, pulse (P) 82/minute, respiratory rate (R) 20/minute, blood pressure (BP) 130/80 mmHg, weight (W) 51 kg, and height (H) 154 cm. Respiratory movement and cardiac examination were normal. On abdominal examination no masses or tenderness were noted on both light and deep palpation. Her liver and spleen were not palpable. A sensory and motor system examination did not reveal any abnormality. Her neurological reflexes were normal. A routine urine examination showed: white blood cell 87/hpf (WBC2+), red blood cell 34/hpf (RBC2+), and pH 7. 0. A routine blood test showed: hemoglobin (Hb) 95 g/L. Her serum potassium was 3.2 mmol/L, calcium 2.92 mmol/L, phosphate 1.3 mmol/L, carbon dioxide combining power , red blood cell 27/hpf (RBC1+), and pH 7. 5. A routine blood test showed: Hb 88 g/L. Her serum potassium was 3.1 mmol/L, calcium 2.83 mmol/L, phosphorus 1.21 mmol/L, COnum Fig. . Parathynum Fig. . After s2CP 15.6 mmol/L, serum creatinine 766 μmol/L, and PTH 766.4 pg/ml  and pH 7.0. A routine blood test showed: Hb 76 g/L, potassium 4.0 mmol/L, calcium 2.09 mmol/L, phosphorus 2.37 mmol/L, CORET proto-oncogene are also present in 97% of individuals with multiple endocrine neoplasia (MEN) 2A (MEN2A). In order to explore the possible genetic mechanisms, we analyzed the GDNF and RET genes in this patient. After obtaining informed consent from our patient, genomic DNA was extracted from peripheral blood leukocytes by standard phenol–chloroform procedures. All 20 exons and flanking splice sites of the GDNF and RET genes were amplified by polymerase chain reaction (PCR) with the primers listed in Table RET polymorphisms were found in this patient, one was nonsynonymous c.2071G>A  located in exon 11 . Fabris et al.  showed tet al. . Some pret al. , 6, but et al. . In our GDNF and RET genes, which play a key role in the kidney–urinary tract development and nephrogenesis, might be the reasonable candidates for MSK. Torregrossa et al. [GDNF and RET genes of 55 patients with sporadic MSK and found eight patients with MSK had heterozygous variations of GDNF gene c.-45G>C, c.-27 + 18G>A. RET encodes a receptor tyrosine kinase, expressed primarily in neural crest and urogenital precursor cells. It is a developmentally important gene, required for kidney morphogenesis, maturation of peripheral nervous system lineages, and for differentiation of spermatogonia. In RET knockout mice, the loss of the RET protein results in anomalies of renal embryogenesis, which indicates that the RET protein is required for the development of the urinary excretory system. Primary hyperparathyroidism (PHPT) may occur as part of a complex syndrome or as an isolated disorder. Syndromic PHPT includes MEN types 1 to 4 (MEN1 to MEN4). RET gene mutations were also found in 97% of patients with MEN2A, who have an increased risk for parathyroid adenoma or hyperplasia, medullary carcinoma of the thyroid, and pheochromocytoma [et al. [RET C634Y mutation. In addition, sporadic forms of parathyroid tumors may arise because of somatic gene abnormalities. To date, reports have shown that more than 10% of patients with hyperparathyroidism may have a germline mutation involving the MEN1, RET, cell division cycle 73 (CDC73), calcium-sensing receptor (CASR), cyclin-dependent kinase inhibitor (CDNK1B), or PTH genes. Thakker [RET gene polymorphisms G691S (exon 11) and S904S (exon 15) were identified, but no GDNF variants were found. It was shown that the nonsynonymous RET G691S polymorphism is able to increase downstream signaling compared with RET wild type [RET pathogenic genes in MTC is enhanced by the presence of G691S, suggesting a possible modifier role of this nonsynonymous RET polymorphism [RET are in linkage disequilibrium with each other. Robledo et al. [RET in 104 patients with MEN2A, and found the homozygous for these polymorphisms were, on average, 10 years younger at the time of diagnosis compared with heterozygous and wild-type homozygous, indicating that the G691S/S904S variants of RET have a modifier effect on the age at onset of MEN2A. However, the exact mechanism of RET G691S/S904S polymorphism in the pathogenesis of MSK and hyperparathyroidism still remains to be uncovered.As MSK is considered a congenital disease, it is speculated that the a et al.  analyzed [et al.  reported Thakker  suggesteild type , 13. Mormorphism , 15. Premorphism  have shoo et al.  analyzedet al. [Considering the pivotal role of incomplete dRTA in MSK, Fabris et al.  recommenet al. . The proRET G691S/S904S polymorphism in this patient, but additional studies are required to explore the exact mechanism of the RET gene in MSK with hyperparathyroidism.We demonstrated a case of MSK combined with tertiary hyperparathyroidism, which further expands the pathophysiology of this disease. Early recognition of MSK symptoms has important clinical implications for the prevention of further progress of the disease. Besides, we also found


Plicosepalus is an important genus of the Loranthaceae family, and it is a semiparasitic plant grown in Saudi Arabia, traditionally used as a cure for diabetes and cancer in human and for increasing lactation in cattle. A flavonoid quercetin (P1), (-)-catechin (P2), and a flavane gallate 2S,3R-3,3′,4′,5,7-pentahydroxyflavane-5-O-gallate (P3) were isolated from the methanol extract of the aerial parts of P. curviflorus (PCME). The PCME and the isolated compounds were subjected to pharmacological assays to estimate peroxisome proliferator-activated receptors PPARα and PPARγ agonistic, anti-inflammatory, cytotoxic, and antimicrobial activities. Results proved for the first time the dual PPAR activation effect of the PCME and catechin (P2), in addition to the promising anti-inflammatory activity of the flavonoid quercetin (P1). Interestingly, both PCME and isolated compounds showed potent antioxidant activities while no antimicrobial effect against certain microbial strains had been reported from the extract and the isolated compounds. Based on the pharmacological importance of these compounds, an HPTLC validated method was developed for the simultaneous estimation of these compounds in PCME. It was found to furnish a compact and sharp band of compounds P1, P2, and P3 at Rf = 0.34, 0.47, and 0.65, respectively, using dichloromethane, methanol, and formic acid ) as the mobile phase. Compounds P1, P2, and P3 were found to be 11.06, 10.9, 6.96 μg/mg, respectively, in PCME. The proposed HPTLC method offers a sensitive, precise, and specific analytical tool for the quantification of quercetin, catechin, and flavane gallates in P. curviflorus. Flavonoids and flavane gallates are naturally occurring secondary metabolites in the plant species. They present in good amounts in dietary supplements and functional food. Quercetin is one of the important bioflavonoids present in many ethnic plants. Quercetin has importance in terms of ethnopharmacology due to its use as anti-inflammatory, antioxidant, anticancer, antiobesity, and antiatherosclerotic activities , 2.The flavane gallates, catechins, and their derivatives have attracted attention in terms of beneficial effects on human health, due to their high availability, less toxic effect, and low cost . PreviouPlicosepalus curviflorus is one of the two known species grown in Saudi Arabia and belongs to Loranthaceae family. This parasitic plant found to be growing in East Africa, North East Africa, Saudi Arabia, and Yemen [P. curviflorus showed the presence of flavonoids, flavane gallates, sterols, and terpenoids [P. curviflorus growing in Saudi Arabia, we isolated the quercetin (P1), catechin (P2), and flavane gallate 2S, 3R-3,3′,4′,5,7-pentahydroxyflavane-5-O-gallate (P3)   from theα and PPARγ agnostic, anti-inflammatory, cytotoxic, and antimicrobial effects to verify its traditional uses and try to refer activities to the isolated compounds.Consequently, the present study was focused on further pharmacological investigations of the plant extract and isolated compounds in order to explore their peroxisome proliferator-activated receptors PPARThe HPTLC (high-performance thin-layer chromatography) has been widely used recently in the quality control of herbs and their formulations due to its low operation cost, high sample throughput, and small mobile phase requirement. Different wavelengths of light which observed with specific types of analyses can give a complete profile of the plant extract. It is widely used for the identification, stability, dissolution, purity testing, or content uniformity of crude extracts of the plant .P. curviflorus and its isolated compounds  motivated the authors for concurrent analysis of biomarkers P1, P2, and P3 in the methanol extract of P. curviflorus by a validated HPTLC method.The good pharmacological activities shown by the methanol extract of All the spectroscopic techniques used were the same as reported earlier .P. curviflorus were collected from Hijaz, Saudi Arabia, and identified by a plant taxonomist of Pharmacy College, King Saud University. A voucher specimen (No. 127) has been placed in the herbarium of Pharmacognosy Department, King Saud University.The aerial parts of P1, P2, and P3 has been described in our previous published article on the plant [The aerial parts of the plant (500 g) were ground after shade-dried and successfully extracted with methanol (2 × 1.5 L) at room temperature. Isolation of compounds he plant .P1, P2, and P3 in P. curviflorus methanol extract (PCME) were performed on (20 × 10 cm) precoated silica gel F254 HPTLC plates. During the experiment, the microliter syringe adjusted with the Automatic TLC Sampler-4 was used for spotting the samples and the extract on a HPTLC plate at a rate of 160 nL/sec. The plate was developed under certain condition using GAMAC Automated Developing Chamber-2 (ADC-2). In order to improve resolution and separation of different compounds existing in P. curviflorus, a number of trials for best mobile phases combinations were carried out. We found that dichloromethane : methanol : formic acid in the ratio of 90 : 9.5 : 0.5 (v/v/v) is the appropriate mobile phase mixture which was chosen to carry out the analysis. The following chamber saturation condition (at 25 ± 2°C and 60 ± 5% humidity) using a twin-trough glass chamber (20 × 10 cm) was prepared for the development of the plate. Then, the developed plate was dried and scanned by GAMAG TLC Scanner-3 at λ = 460 nm (wavelength) in the mode of absorbance.The HPTLC analyses of compounds µg/mL) were prepared from the serial dilution of each standard P1, P2, and P3 stock solutions (1 mg/mL) dissolved in methanol. In order to get a linearity range of 100–1200 ng/band, 10 μL of each concentration of P1, P2, and P3 were spotted on the HPTLC plate.Seven different concentrations  for LOD (limit of detection), LOQ (limit of quantification), precision, linearity range, robustness, recovery, and accuracy.The standard addition method was used to determine accuracy. Compounds 1–3 (200 ng/band) were spiked with the extra 0, 50, 100, and 150% of P1, P2, and P3, and the solutions were reanalyzed in six replicates (n = 6) by the proposed method. The percent relative standard deviation (%RSD) and recovery took place.P1, P2, and P3 were prepared by duplicate analyses (n=6) for calculation of interday and intraday precision of the proposed method. Intraday assays were repeated on three different days for determination of the interday precision.Three different concentration values 400, 600, and 800 ng/band of compounds Small deliberate changes were made to the mobile phase composition, mobile phase volume, and duration of mobile phase saturation for analyzing robustness.The results were determined in terms of %RSD and SD of peak areas after three times repetition of the experiment at 300 ng/band. Different amounts of dichloromethane : methanol : formic acid (v/v/v) were prepared for the chromatography mobile phase. Activation of plates took place for 30 minutes at 110°C before chromatography process.S) of the calibration curve and the standard deviation (SD) of the response are parameters used to identify the LOQ and the LOD according to the formula: LOQ = 10 (SD/S) and LOD = 3.3 (SD/S). Taking into consideration that LOQ is the lowest amount that can be determined at a distinct level of accuracy or precision and the LOD is the minimum amount of an analyte that may be calculated from the assay background at a distinct level of confidence.The slope . The achieved results were indicated as mean ± SD; differences were considered significant at PPARα and PPARγ, respectively.A reporter gene assay was carried out as described previously . BrieflyμM of ABAP. The area under the curve (AUC) of fluorescence units and time were two parameters used for indication of the percent decrease in oxidative stress. Percent decrease in oxidative stress = 100 − ((AUC sample/AUC control) × 100). Quercetin was used as a positive control.Human hepatoma cell line (HepG2) was the cell line of choice in this study according to the previously described method . At a deμg/mL) and followed by 24-hour incubation as well. The nitrite level in cell supernatant was measured by Griess reagent. The dose response curves gave indication for IC50 values. Parthenolide was used as positive control.Mouse macrophage (RAW264.7) cells were seeded in the wells of 96-well plates followed by 24 hrs for confluency according to the method described previously , 14. The5 cells per well were seeded with human chondrosarcoma cells (SW1353) transfected with NF-κB luciferase plasmid construct. After incubation for 24 h, cells were treated with the test samples for half an hour followed by PMA (70 ng/mL) for 8 h. Luciferase assay kit  was used for determination of luciferase activity, and parthenolide was used as the positive control in this test.According to the method described earlier , 13, thein vitro cytotoxic study. Test samples were added on the cell lines with variable concentrations, followed by 48 h incubation. According to the Borenfreund method, the cell viability was determined at the end of incubation [Four human cancer cell lines  and two noncancerous kidney cell lines (VERO and LLC-PK1) were measured in the cubation , using dP1, P2, and P3 was assessed against Gram-negative Enterobacter xiangfangensis (CP017183.1), Escherichia fergusonii (CU928158.2), and Pseudomonas aeruginosa (NR-117678.1) and Gram-positive Staphylococcus aureus (CP011526.1) and Bacillus licheniformis (KX785171.1) using well diffusion and broth microdilution techniques as described by Berghe and Vlietinck [µL of the PCME, P1, P2, and P3. Amikacin was used as the positive control. The clear area which was free of microbial growth was measured to detect the diameter of the zone of inhibition (mean of triplicates ± SD), and the least concentration of the PCME and P1, P2, and P3 that did not show any visible growth of the bacteria in broth culture (MIC) was calculated as well.The antimicrobial activity of PCME and isolated compounds lietinck . The bacPlicosepalus comprises two species in Saudi Arabia. Previous work on this genus reported antiviral, antidiabetic, and cytotoxic activities. Little phytochemical and biological attention was drawn on the plant, and few previous studies detected only the hypoglycemic and cytotoxic activities of P. curviflorus extract and compounds [P1, P2, and P3 were obtained from PCME after subjecting it to a series of silica gel and Sephadex LH-20 column chromatographic separations [P1, P2, and P3 were subjected to different biological investigations for concluding the complete information about its active constitutes.Genus ompounds , 17. In arations . The PCMP2  was exhibited by PCME and P2 . Severaldulators . The duaterature .P1 and P3, showed no PPAR agonistic activity. However, this is the first report of the PPAR agonistic activity of the PCME and P2.The other two isolates, namely, P1, P2, and P3 showed decrease in oxidative stress. The largest decrease in oxidative stress was exhibited by the PCME (66% decrease at 500 µg/mL), which may be attributed to its constituents . All three constituents P1, P2, and P3 showed decrease in oxidative stress by 60%, 52%, and 57%, respectively, at 250 µg/mL. This indicates that the significant antioxidant effect of the PCME seems to be due to its high phenolic content. Flavonoids are polyphenolic substances with free-radical scavenging properties that can inhibit oxidative enzymes and demonstrate anti-inflammatory action [From the results shown in P2 showed iNOS inhibition while P3 did not show any. P1 was the most active iNOS inhabitant with IC50 of 25 µg/mL.The other targets related to anti-inflammatory activity were only weakly to moderately affected by the PCME, such as that shown by inhibition of the nuclear transcription factor NF-kB and inducible nitric oxide synthase (iNOS) . InteresP1, P2, and P3 from the P. curviflorus were not cytotoxic although PCME showed weak activity against all four cancerous cell lines as well as the two normal cell lines with the IC50 values ranging from 70–92 μg/mL  and Pseudomonas aeruginosa (ATCC 27853) with MIC of 6.2 and 6.6 µg/mL, respectively, followed by the activity of PCME against Bacillus cereus (ATCC 14579) and Pseudomonas aeruginosa (ATCC 27853) with MIC of 7.6 and 8.4 µg/mL, respectively, while P2 showed the lowest activity and P3 showed any noticeable inhibition.Several studies have reported that flavonoids and flavane gallates suppress the activity of the pathogenic microorganisms –22. In tamikacin , the refP1, P2,and P3. Sharp and compact peaks of P1, P2, and P3 took place at Rf = 0.34, 0.47, and 0.65, respectively (r2) for P1, P2, and P3 were found as Y = 2.725X + 122.44/0.993, Y = 1.675X + 755.87/0.993, and Y = 52.008X + 211.51/0.9972, respectively, in the linearity range 100–1200 ng/spot. The limit of detection (LOD) and limit of quantification (LOQ) were found as 31.04/94.06, 29.23/88.58, and 12.33/37.36, respectively, for P1, P2, and P3  were detected as 1.15–1.20/1.11–1.20, 1.17–1.27/1.12–1.21, and 1.27–1.33/1.271.32 respectively, for P1, P2, and P3 which exhibited good precision of the proposed method  was found to be the most effective mobile phase for the estimation ectively , with goectively . 20 mL aectively . The reg, and P3 . The acc, and P3 . The recectively . The %RSd method . The robP1, P2, and P3 in PCME. These biologically active markers were found to be present in PCME (μg/mg of the dried weight of extracts) of biomarkers P1, P2, and P3 in PCME were found in the following order: 11.06 > 10.9 > 6.96 μg/mg , 2S,3R-3,3′,4′,5,7-pentahydroxyflavane-5-O-gallate (P3), and (-)-catechin (P2) in P. curviflorus. Hence, we are privileged to report this maiden research on the screening of antidiabetic, cardiovascular, anti-inflammatory, and cytotoxic biomarkers in the methanol extract of the plant.Different analytical techniques such as HPLC, HPTLC had been reported for the quantitative analysis of quercetin in many plant extracts such as detection of quercetin by the HPTLC method in the aerial parts of a indica  and Thesulnea L. . To our P1, P2, and P3 have been isolated from the methanolic extract of the leaves of P. curviflorus. All compounds and the extract showed significant antioxidant activities. Catechin (P2) and the methanol extract (PCME) revealed dual PPAR activation effect, while compound P1 possesses the most potential anti-inflammatory activity. The maiden HPTLC method was developed for the simultaneous analysis of the biomarker compounds quercetin (P1), (-)-catechin (P2), and 2S,3R-3,3′,4′,5,7-pentahydroxyflavane-5-O-gallate (P3) in the methanol extract of P. curviflorus (PCME). The proposed HPTLC method can be further applied for the analysis of these compounds P1, P2, and P3 in different plant extracts, biological samples, and herbal formulations as well as for the in-process quality control.Compounds


Bland–Altman analysis indicated the 95% limits of agreement (−0.26 to 1.57) without any evidence of systemic difference. Lin's ρc represented a weak concordance between PWVβ and aPWV . There was no difference in PWVβ between N and O, whereas aPWV was higher in O: 4.81 ± 0.42 m/s than in N: 4.65 ± 0.32 m/s (p < 0.001). The difference, Δ0.16 m/s, 95% CI , was significant, t(121) = −3.76, p < 0.001, with a medium-sized effect. PWVβ (ultrasound) and aPWV (oscillometry) show a level of disagreement that includes clinically important discrepancies. A discrimination between normal and altered vascular function was possible with aPWV but not with PWVβ.Vascular alterations may lead to manifest cardiovascular disease in future life. There is a tremendous time delay between the onset and obvious clinical appearance of vascular alterations. Pulse wave velocity (PWV) is one subclinical parameter to detect vascular alterations at a very early stage. Different techniques exist to measure PWV non-invasively as a vascular parameter—all with their own technique-inherent advantages, challenges, and pitfalls. The aim of this study was to compare two techniques to measure PWV, to assess their agreement, and interchangeability. In 780 (♀ = 49.4%) healthy children and adolescents (mean age: 11.61 ± 2.11 years), PWV was obtained with two different techniques. Ultrasound-measured local PWV (PWVβ) at the carotid artery was graphically compared by a Bland–Altman plot with aortic PWV (aPWV), measured oscillometrically on the brachial artery. Reproducibility was assessed with the concordance correlation coefficient by Lin (ρc). Furthermore, participants were categorized by BMI as normal weight (N) or overweight/obese (O) to identify differences in PWVβ and aPWV caused by an increased BMI. Mean PWVβ was lower (4.01 ± 0.44 m/s) than mean aPWV (4.67 ± 0.34 m/s). The two methods differ by mean Δ0.66 ± 0.47 m/s (95% CI: 0.62 to 0.69 m/s;  Cardiovascular disease (CVD) is the most common cause of death in Germany and other industrialized nations . Among cThe measurement of PWV is a simple and reproducible method to examine vascular stiffness . Therefon = 780 (♀ = 49.4%) apparently healthy school children, aged 8–17 years. The project took part in a school setting. Written informed consent was obtained from children and/or parents. Children with chronic conditions like congenital heart defects were not involved in the study. The study protocol was approved by the ethical board of the Technical University of Munich (project number 5490/12). Study rationale, design, and further hemodynamic results are published elsewhere . Body height was measured to the nearest 0.1 cm with a stadiometer . Standard deviation scores (SDS) for BMI were calculated and corresponding weight categories were defined according to German reference values .BP was measured oscillometrically  on the left arm, after participants rested for 10 min in supine position. According to German reference values , BP was z-score BMI SDS ≤1.282 indicating normal weight or underweight (N) vs. BMI SDS >1.282 representing overweight or obesity (O)], according to German reference values , the movement of the arterial wall during the cardiac cycle from diastole to systole is tracked with two digital tracking gates, placed at the CCA near and far wall to automatically follow wall motion and calculate diameter changes during heart cycles  Figure . The metaPWV at the A. brachialis was assessed with the Mobil-O-Graph . The measurement is similar to an automated BP measurement. Depending on the upper arm circumference, an appropriate BP cuff (five different sizes) was chosen for each participant and attached on the left arm. The ARCSolver method consists of a three-level algorithm. First, single pressure waves are verified for plausibility. Second, a comparison of each single pressure wave is performed to detect artefacts. Third, a filtered, averaged pulse wave is derived and used to calculate the central aortic pulse wave  and hemot-test, Pearson correlation coefficient r, coefficient of variation, interclass correlation coefficient) used in the context of assessing concordance between two alternative methods (All parameters (continuous variables) are expressed as mean ± standard deviation (SD) or as frequencies and percentages (non-continuous variables). The two methods were graphically compared by a Bland–Altman plot , 21. The methods . It migh methods  but is l methods .t-test was performed to identify differences in aPWV and PWVβ by an increased BMI (>90th percentile). Data were analyzed using IBM SPSS statistical software for Windows, version 23 . Level of significance was defined as p < 0.05; all tests were two-sided.The independent z-score of BMI was 0.04 ± 1.1, indicating a study population with BMI values very close to the German reference population . Mean age was 11.61 ± 2.11 years. Mean BMI was 18.6 ± 3.4; pulation ; 86.8% oInterobserver variability for PWVβ was 1.37%, calculated by coefficient of variation (CV). The oscillometric measurement of aPWV, which is similar to a standard blood pressure measurement, is an operator-independent method , 28, 29.Mean SBP was 116.29 ± 9.71 mmHg, and mean DBP was 68.09 ± 8.16 mmHg; 64.2% of all participants had normotensive or high normotensive blood pressure values; 16.8% had a single hypertensive blood pressure measurement, and 19% had an ISH measurement. The descriptive data are given in p < 0.001. A scatter plot of PWVβ and aPWV is given in Mean values for PWVβ (ultrasound) were lower (4.01 ± 0.44 m/s) than aPWV . Results of the two methods differ by 0.66 ± 0.47 m/s , The Bland–Altman analysis  indicaten = 677). In this subgroup, concordance between the two measurements is even lower .As obesity and overweight might influence PWV, Lin's concordance correlation coefficient was re-evaluated with the subgroup of non-overweight participants (p = 0.781) or peripheral blood pressure . Height  and central systolic blood pressure  were higher in overweight children. SDS of peripheral blood pressure values indicate possible influences of height or age on this parameter.There was no substantial difference between normal weight (N) and overweight (O) participants regarding age (p = 0.378. But aPWV was higher in overweight participants [(O) 4.81 ± 0.42 m/s] compared to normal weight participants [(N) 4.65 ± 0.32 m/s], p < 0.001 (t(121) = −3.76, p < 0.001, with a medium-sized effect . Along the arterial tree, elastic properties of the vessel vary within each region. Proximal arteries are more elastic whereas stiffness increases in distal arteries. Majesky stated that “[…] different vessels, or even different segments of the same vessel, are composed of smooth muscle cell populations that arise from distinct sources of progenitors, each with its own unique lineage and developmental history” . This vaAnother explanation could be that the technique to obtain PWVβ is less sensitive for subtle changes caused by earliest vascular alterations. The question remains, if vascular alterations are stable and predictive for future adverse cardiovascular phenotypes, which could already be demonstrated for other vascular parameters like flow-mediated dilatation (FMD) . TherefoIn conclusion, PWVβ measured by ultrasound and aPWV obtained oscillometrically do not provide similar information and cannot be compared interchangeably. The level of disagreement is not only statistically significant but also clinically relevant: The range of PWVβ is from 2.91 to 5.92 m/s in girls (age 8–17) and 2.25 to 5.71 m/s in boys (age 8–17), and for aPWV, it is 3.62 to 5.45 m/s in girls (age 8–17), and 3.79 to 6.08 m/s in boys (age 8–17). The mean differences between PWVβ and aPWV is 0.61 m/s, a difference in speed that is higher than one SD in this population (mean PWVβ ± SD: 4.01 ± 0.44 m/s and mean aPWV: 4.67 ± 0.34 m/s). In adults, an increase in aPWV of 1 SD is associated with a 1.35 higher risk for coronary heart disease, 1.54 higher risk for stroke, and 1.45 higher risk for CVD . TherefoIn clinical routine, the sonographic PWV measurement is an interesting alternative to other methods. In addition to IMT and other vascular parameters, carotid PWVβ could be measured in only one examination, with one device. However, the ultrasound technique for PWVβ is possibly less sensitive in detecting earliest vascular changes.Ethics Commission, Medical Faculty, Technical University of Munich Projet number 5490/12.JE: study concept, data analysis, and writing the manuscript. HW: study concept, data aquisition, and editing the manuscript. MM: data aquisition and reviewing the manuscript. KP: data analysis and reviewing the manuscript. RO: study concept and reviewing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.