In addition, subjects carrying the minor AA genotype at rs12654812 ) have higher susceptibility to nephrolithiasis . Among nephrolithiasis patients, subjects with GG at rs7627468 ) have lower pH level in urine (p = 0.0088). Importantly, rs7627468 is associated with the expressions of IQCB1 and EAF2. rs12654812 could influence the expression of RGS14 itself, MXD3, and FGFR4. In summary, this study successfully validated the genetic roles of rs1256328 and rs12654812 in human nephrolithiasis.Nephrolithiasis is a common disease affecting almost all populations, with an increasing prevalence over the past decades. Previous studies revealed several functional polymorphisms associated with the pathogenesis of nephrolithiasis. However, data on Asian populations are limited. In this study, three candidate polymorphisms were selected from previous studies to investigate the correlations with nephrolithiasis in a Taiwanese population. In total, 454 nephrolithiasis patients were recruited from Kaohsiung Medical University Hospital, with SNP frequency for 1513 subjects of general population from the Taiwan Biobank (TWB) as a genotypic reference. Results revealed that subjects with minor TT genotype at rs1256328 ( In the United States, the prevalence of kidney stones was estimated to have increased from 3.8% in the 1970s to 8.8% in the 21st century3. In Asia, the prevalence of nephrolithiasis is 1–5%4.Kidney stone disease (nephrolithiasis) affects almost all populations worldwide with an increasing prevalence over the past few decades. The prevalence rate of nephrolithiasis was reported to be 5~15% in developed countries, but was as high as 20~25% in the Middle East6. Furthermore, nephrolithiasis is a disease of high recurrence risk with a relapse rate of 50% within 5~10 years and 75% at 20 years8. Continuous recurrence of kidney stone may lead to persistent renal obstruction and even permanent renal damage6. As a result, nephrolithiasis significantly influences patients’ quality of life and places a heavy burden on society and healthcare systems.Although many patients are persistently asymptomatic, some patients occasionally pass stones and experience hematuria. Paroxysmal renal colic may also occur and cause variable levels of pain9. Furthermore, candidate gene association studies attempted to assess the roles of several genes involved in stone formation10. Previous studies indicated that genetic variances of the vitamin D receptor (VDR), osteopontin (OPN), and calcium-sensing receptor (CASR) genes were highly associated with nephrolithiasis17. The TRPV5 and CLCN5 genes were also proven to be important in the pathogenesis of nephrolithiasis19. In addition, genetic polymorphisms of calcium channel, ORAI1 , were reporetd to associate with the development of nephrolithiasis20ITPKC (rs2607420) is also associated with the estimated creatinine clearance in nephrolithiasis patients21.Many factors were suggested to elevate risks of nephrolithiasis, such as environmental exposure, low fluid intake, dietary intake, hypercalciuria, genetics, and a family’s health history. However, the etiology of nephrolithiasis remains unclear. A previous study of paired twins reported a higher concordance rate of the disease in monozygotic twins than that in dizygotic twins, suggesting that genetic factors may play a pivotal role in its etiology25. The first GWAS on idiopathic calcium oxalate urolithiasis was published in 2009 that identified a member of the claudin gene family (CLDN14) as a risk locus22. Three loci at 5q35.3, 7p14.3, and 13q14.1 were reported to associate with kidney stones with an accumulative effect in a Japanese population24. Recently, Oddsson et al. indicated that four loci were associated with nephrolithiasis in an Icelandic population25, but this association has not been confirmed in any Asian population yet. The purpose of this study was to investigate whether the selected polymorphisms from the previous GWAS report25 are associated with nephrolithiasis in a Taiwanese population.Recently, some regions were found to be associated with nephrolithiasis by genome-wide association studies (GWASs) or meta-analysis of GWASs in various populationsIn this study, nephrolithiasis patients were recruited from Kaohsiung Medical University Hospital . Genotypic statistics of general population was obtained from the Taiwan Biobank (TWB) project. We investigated the susceptibility variants with risk of nephrolithiasis in the Taiwanese population. Furthermore, associations of genetic variants with the stone frequency, stone number, clinical biochemical data, and estimated glomerular filtration rate (eGFR) were analyzed.We investigated the regional prevalence of nephrolithiasis through the analysis of longitudinal health insurance data from the National Health Insurance Research Database. The prevalence of nephrolithiasis in Taiwan varies by geographical region. The prevalence of nephrolithiasis in 2010 was 3.9%, 5.95%, 4.62%, and 3.78% in northern, central, southern, and eastern Taiwan, respectively Fig. . In thisALPL) and rs12654812 (RGS14) revealed significant differences in genotypic distributions, even after correction with SNP numbers . rs12654812 (RGS14) also revealed a borderline significant difference in its allelic distribution (p = 0.0346). However, the significance did not persist after applying Bonferroni correction. For rs7627468 (CASR), no significant difference was observed. On the other hand, we also investigated associations of these two SNPs (rs1256328 and rs12654812) with nephrolithiasis susceptibility under different genetic models (Tables ALPL) and rs12654812 (RGS14), the most significant associations were observed under a recessive model , with estimated ORs of 2.03 and 1.91, respectively. These results indicate that subjects with the minor TT genotype at rs1256328 (ALPL) or the minor AA genotype at rs12654812 (RGS14) had higher susceptibility to nephrolithiasis, compared to those with major alleles in these two SNPs  or G allele at rs12654812 (RGS14)).To investigate associations of polymorphisms with nephrolithiasis susceptibility, we compared the genotype and allele distributions between groups of nephrolithiasis patients and general populations for each SNP  were associated with the risk of multiple stones or recurrence of nephrolithiasis. As shown in Supplementary Table CASR) was highly associated with the urine pH, before or after adjustment with gender and age (Table CASR) than in the AG or AA genotype groups . In addition, the rs12654812 (RGS14) AA genotype was potentially correlated with an increased UCa/UCr compared to the AG or GG genotypes in nephrolithiasis patients . After the subset analysis, we found that the CASR SNP (rs7627468) was associated with the UCa/UCr (p = 0.0170)  and Cockcroft-Gault (C-G) methods, widely used tools to predict renal function of nephrolithiasis patients. We tested the relationship between genetic polymorphisms and renal function. Among the SNPs we tested, no SNP was significantly associated with values of renal function calculated by either the MDRD or CG method (Supplementary Table IQCB1 (IQ motif containing B1) gene in the thyroid (p = 2.2 × 10−7) and the EAF2 (ELL-associated factor 2) gene in the esophagus-mucosa (p = 4.1 × 10−5)  itself and is very likely to influence expressions of MXD3 (MAX dimerization protein 3) in a variety of tissues. A significantly associated eQTL for rs12654812 was also identified for FGFR4 (encoding fibroblast growth factor receptor 4) in the tibial nerve and F12 (encoding coagulation factor XII) in esophagus mucosa. However, no significant eQTLs were found for rs1256328.From data of the GTEx portal, we found that rs7627468 could influence the expression of the ALPL associated with higher susceptibility of nephrolithiasis in a Taiwanese population, which is consistent with a previous study in Icelandic population25. ALPL encodes tissue-nonspecific alkaline phosphatase (TNSALP), which is essential for the normal development of bones and teeth. TNSALP hydrolyses phosphate substrates such as pyrophosphate (PPi) and phosphorylated glycoproteins like osteopontin, and releases inorganic phosphate, thereby promoting appropriate calcification26. Animal study reported decreased bone mineralization in ALPL-knockout mice due to lower activity of phosphatase27. Furthermore, ALPL gene mutations were found in patients with hypophosphatasia, characterized by elevated urinary PPi excretion28. PPi was reported as a potent inhibitor of hydroxyapatite crystallization, which binds to the surface of basic calcium phosphate crystals and blocks subsequent crystal growth31. TNSALP is expressed in proximal tubules of the kidneys28 and frees phosphate through PPi hydrolysis26, which may further lead to kidney stone formation. Moreover, a previous study indicated associations of kidney stone diseases with other genes involved in phosphate regulation, including VDR, klotho (KL), and sodium-hydrogen antiporter 3 regulator 1 (NHERF1)35, although rs1256328 (ALPL) had no significant eQTL effects on these genes. Since GTEx portal only collects limited cell types, and much evidence has suggested an important role of ALPL in the pathophysiology of nephrolithiasis via phosphate regulatory pathways. Therefore, further studies are required to elucidate the physiological effects of rs1256328 in human nephrolithiasis.In this study, we showed that the TT genotype of rs1256328 in RGS14 was significantly associated with nephrolithiasis susceptibility in a Taiwanese population, which is also consistent with a previous study on an Icelandic population25. Association of rs12654812 with nephrolithiasis was validated in a Japanese population36. However, studies in Chinese population were not totally consistence with each other38. RGS14 encodes a member of the regulators of G-protein. Previous study on knockout mice indicated that RGS14 may regulate Ca2+ and affect downstream signals in neurons39. Similar cis-eQTL effects of rs12654812 on RGS14 expression were observed in nervous tissue in our study. However, this effect was not observed in kidney tissue. Since sample size of kidney tissue is limited by the current release of GTex, further studies are required to investigate the effects of rs12654812 on RGS14 expression in kidney tissue. Finally, we identified a significant eQTL in the tibial nerve for rs12654812 with FGFR4  variant on CASR predisposes patients to primary hypercalciuria and nephrolithiasis44. Furthermore, Ala986Ser in exon 7 of CASR was repeatedly reported to be associated with a risk of kidney stones and serum calcium concentrations in different ancestries47. Our previous study also showed a significant association between CASR (rs17251221) and stone multiplicity in nephrolithiasis patients13. In addition, urinary acidification and urinary volume are two important factors associated with kidney stones48. For example, TRPV5−/−knockout mice demonstrated robust hypercalciuria and hyperphosphaturia without calcium-phosphate stone precipitation49. In the same study, researchers discovered that activation of the renal CASR can promote H+ excretion and downregulate aquaporin 2 leading to urinary acidification and polyuria49. These beneficial adaptations promoted the excretion of large amounts of soluble calcium, which was crucial to preventing the formation of kidney stones. This evidence indicates that CASR may be involved in kidney stone formation not only via controlling the calcium concentration, but also via regulating the urine pH. Furthermore, rs7627468 was associated with IQCB1 expression in the thyroid, which encodes the nephrocystin protein that interacts with calmodulin50. rs7627468 was associated with EAF2 expression in the esophageal mucosa. However, the physiological functions of EAF2 in renal tissues are still unclear.RGS14 protein may restrict Cain silico, further experiments are required to validate the effects of the SNPs on other genes and to elucidate the detailed subsequent mechanisms of the development of nephrolithiasis.There are some limitations in this study. Data of the controls from the Taiwan Biobank were accessed through the Taiwan View website, which provides only summarized genotype counts, but not genotypes of individual subjects. For nephrolithiasis patients, important confounding factors, such as lifestyle, disease duration, and drug treatment, were unavailable, and we were therefore unable to adjust for the data. Furthermore, the modest sample size of nephrolithiasis patients collected in this study might have reduced the power of the statistical testing. Finally, although we observed eQTL effects of rs7627468 and rs12654812 ALPL and RGS14, rs1256328 and rs12654812, with susceptibility to nephrolithiasis in a Taiwanese population. Our study further indicated that rs7627468 in CASR was associated with pH values and the calcium/creatinine ratio in urine of nephrolithiasis patients.In summary, this study successfully replicated an association of SNPs in To investigate the prevalence of nephrolithiasis in Taiwan, we extracted calcium nephrolithiasis patients from the subset database  of the National Health Insurance Research Database (NHIRD) using ICD-9-CM code 592.X. LHID database consists of medical claims data for 1,000,000 subjects randomly selected from the entire NHIRD (n = 23.5 million).Declaration of Helsinki, and the design of the study and final report were performed with approval of the institutional review board of KMUH. We received informed consent from all subjects before any data were collected. Subjects were excluded if they had a history of renal tubular acidosis, renal failure, gout, primary or secondary hyperparathyroidism, chronic diarrhea, or cancer. In total, 454 calcium nephrolithiasis patients were diagnosed by a qualified urologist and were recruited. Each patient’s echographic and radiographic examinations of renal stones were retrieved along with clinical laboratory data, such as gender, age, and family history of nephrolithiasis. Blood and urine samples were collected at a random time point to measure total calcium, uric acid, and creatinine levels. All urinary parameters were corrected using urinary creatinine levels. Renal stone samples were obtained by either surgical intervention or spontaneous stone passage. Moreover, for the stone number analysis, patients with stone numbers exceeding one were allocated to the multiple-stone group, while patients with a single stone were grouped together. Patients without clear evidence for classifying them into either the multiple-stone or single-stone group were excluded from the stone number analysis.Patients who fulfilled the diagnostic criteria for nephrolithiasis were recruited at Kaohsiung Medical University Hospital (KMUH). Our study conformed to the The past medical history of the stone episode was retrospectively traced back as far as the patient’s medical record was available. Patients with only one episode were placed in the non-recurrent group; those with two or more symptomatic episodes within 6 months were allocated to the recurrent group. Since the medical history was retrospectively retrieved, patients without clear evidence for classifying them into either the single or recurrent group were excluded from the recurrence analysis.51. Among them, blood samples from 1517 subjects were sequenced. Four subjects exited the project afterwards, with 1513 subjects remaining. In this current study, SNPs frequencies for these 1513 subjects were obtained through the Taiwan View website (https://taiwanview.twbiobank.org.tw/index) as genotypic references of general population.Taiwan Biobank is a prospective cohort of Taiwanese general population, in which cancer patients and subjects with nationalities other than Taiwan were excludedPeripheral venous blood was collected from patients and processed the same day. Blood was centrifuged at 3000 rpm for 10 min at 4 °C to separate serum and cells. The buffy coat containing peripheral blood mononuclear cells (PBMCs) was extracted and washed with red blood cell (RBC) lysis. Samples were mixed with a cell lysis buffer for several days after lysing the RBCs in lysis buffer. Proteins were precipitated using a protein precipitation solution. Finally, 95% isopropanol and 80% alcohol were used to isolate total genomic DNA.et al.25. Based on a GWAS, Oddsson et al. reported four SNPs associated with kidney stones in a European population. Among these SNPs, rs199565725 (CLDN14) was excluded from the study, because no frequency data were found in a search of the Taiwan View website. Furthermore, the minor allele frequency of this SNP and any other SNPs in high linkage (r2 ≧ 0.8) to rs199565725 are of low frequency (<0.05) in Asian population and Taiwanese population, according to HaploReg v4.1 and Taiwan Biobank, respectively. Therefore, three SNPs were analyzed in this study (Table The SNPs investigated in this study were selected from a paper by Oddsson 2) test. Quantitative variables are expressed as the mean ± standard deviation (SD). A t-test or analysis of variance (ANOVA) was used to compare the means of continuous variables  among different genotypes in nephrolithiasis patients. An analysis of covariance (ANCOVA) was applied to adjust for age and gender. The likelihood ratio test was used to analyze the association between biochemical data and each genotype, and each p value was adjusted for age and gender. Multiple testing was adjusted for SNP numbers using the Bonferroni correction. A significance level of 0.016 (0.05/3) was used. All statistical analyses were implemented using SAS 9.3 statistical software  for Windows. Regarding to Expression Quantitative Trait Locus (eQTL) analysis, we accessed the GTEx portal to investigate the effects of the SNPs on gene expressions in different tissues52.Statistical differences between patients and controls, multiple- and single-stone episodes, and between the recurrence and non-recurrence groups in genotype and allele frequencies were analyzed by a Chi-squared (χSupplementary Information


Veillonella, Treponema, Filifactor, Fretibacterium, Peptostreptococcaceae_[XI][G-6], Peptostreptococcaceae_[XI][G-5], Bacteroidetes_[G-5], Bacteroidetes_[G-3], Peptostreptococcaceae_[XI][G-4], Peptostreptococcaceae_[XI][G-2] significantly increased both in buccal and subgingival plaque samples in periodontitis subjects (ChP and AgP) compared with HP. Moreover, the results based on the Unweighted UniFrac distance showed that buccal and subgingival plaque samples from the same individuals show higher community divergence than same habitats from different subject samples. This study demonstrated that the microbiome of buccal mucosa can be influenced by periodontitis. However, subgingival and buccal mucosa microbiome seem to be characterized by species-specific colonization patterns. This pilot study provides a glimpse at the changes of subgingival and buccal mucosa associated with periodontitis from a holistic view. Further studies should be taken to illuminate the interplay between these detected changes and periodontitis development.Periodontal microorganisms not only colonize subgingival pockets, but also are detected on various mucous membranes in patients with periodontitis. The object of this pilot study was, using the next-generation sequencing of 16S RNA gene, to characterize the microbiota in two oral habitats  in patients with different forms of periodontitis. Thirty-two buccal swab samples and 113 subgingival samples were obtained from eleven subjects with chronic periodontitis (ChP), twelve subjects with aggressive periodontitis (AgP), and nine periodontally healthy individuals (HP). Using Miseq Sequencing of 16S rRNA gene, we found that the subgingival and buccal mucosa microbiome of ChP and AgP patients both differed from HP. Meanwhile,  The bacteria colonizing the hard and soft tissues of the oral cavity profoundly influence oral health and disease. Microbiologic studies have identified over 700 species of microorganisms in oral cavity  is recognized as a group of periodontal diseases with faster destruction of connective tissue attachment that are different from chronic periodontitis (ChP)  of bacterial 16S rRNA gene makes it possible to broaden our knowledge of the complexity of the microbiome, which has the advantage of detecting the composition of microbial communities in unprecedented depth. Several studies using NGS were to assess the composition of the polymicrobial periodontal community  in patients with ChP or AgP, and to compare them with samples of subjects with periodontal health (HP).From January 2017 to July 2017, eleven patients with ChP and twelve patients with AgP were recruited from the Department of Periodontology, Peking University School and Hospital of Stomatology, and nine HP subjects were selected as controls. The clinical inclusion criteria for all participants were that: (1) they agreed to join the research and signed an informed consent, (2) they were free of systemic disease and not pregnant or lactating, (3) they had not received any kind of periodontal treatment in the past half year, nor had taken antibiotics within the past 3 months, (4) without removable or fixed denture in month, (5) without salivary glands disease, (6) without aggressive caries, untreated pulp disease or periapical problems, (7) they were non-smoker. The clinical inclusion criteria for periodontally healthy individuals and the diagnostic criteria for ChP and AgP have been previously described in detail  were carried out by one practitioner (WH). His reliability was calibrated as described by Shi . All enrolled individuals gave written informed consent.Buccal and subgingival plaque samples were collected 1 week after the full-mouth periodontal examination. All participants were requested to refrain from food for 8 h and oral hygiene (brushing or flossing the teeth) for 12 h before sampling. Samples from all subjects were collected and stored at −80°C for subsequent processing.The buccal swab sample was collected before the subgingival plaque sample. Buccal swab sample was obtained from mucosa of both cheeks with a sterile cotton swab and placed in a separate sterile 1.5 ml Eppendorf microcentrifuge tube containing 1 ml TE buffer . All subgingival plaque samples were collected from each participant from mesiobuccal sites of molars. For each patient with periodontitis, three to five samples were collected from the mesiobuccal site of molars with PD ≥ 4 mm. For each HP subject, two subgingival plaque samples were collected from the mesiobuccal site of molars with PD ≤ 3 mm without AL and BOP. The samples were obtained and processed was based on our previous research  following the manufacturer's instructions, with an extra lysozyme treatment  for bacterial cell lysis. The final concentration was measured with a NanoDrop ND-1000 spectrophotometer  and monitored on 1% agarose gels. The results showed that the A260:A280 ratios were 1.8–2.1 and the DNA concentrations were all more than 20 ng/μL. Based on the quantity and the quality of the DNA extracted, samples were diluted to 1 ng/μL using sterile water and stored at −80°C until use.PCR amplification of the 16S ribosomal RNA gene V4-V5 region was performed using primers 338F  and 806R , where barcode is an eight-base sequence unique to each sample. The PCR cycling conditions were used: initial temperature of 95°C for 3 min, followed by 25 cycles consisting of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and elongation at 72°C for 45 s and a final extension step at 72°C for 10 min. PCR reactions were performed in triplicate 20 μL mixture containing 4 μL of 5 × FastPfu Buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL of FastPfu Polymerase, and 10 ng of template DNA.PCR products were separated by 2% agarose gels electrophoresis, and purified using the AxyPrep DNA Gel Extraction Kit  according to the manufacturer's instructions and quantified with a QuantiFluor™ -ST Handheld Fluorometer with UV/Blue Channel .http://drive5.com/uparse/). The taxonomy of each 16S rRNA gene sequence was analyzed by Ribosomal Database Project Classifier tool (http://rdp.cme.msu.edu/) against the Human Oral Microbiome Database using a default confidence threshold of 0.7 .Purified amplicons were pooled in equimolar and paired-end sequenced (2 × 250) on an Illumina MiSeq platform at Majorbio Bio-Pharm Technology . Raw sequencing data was filtered and trimmed by using QIIME (version 1.17), and classified into operational taxonomic units (OTUs) with 97% similarity cutoff using UPARSE  or Mann–Whitney U-test (BI). After rarefying the OTU table, two metrics were calculated for the evaluation of alpha diversity: abundance-based-coverage (ACE) estimates the species abundance; and the diversity of the sample microbiota was estimated by the Shannon index. The Mann–Whitney U test was used to compare significant differences of the alpha diversity indexes between the different groups (p < 0.05). For evaluating the similarity of microbial community structure among all samples, a principal coordinates analysis (PCoA) was performed on the OTU level. Analysis of similarities was calculated to compare the intra- and inter-group similarities based on the Unweighted UniFrac distance, a phylogenetic measures of beta diversity which was calculated by QIIME. The Mann–Whitney U-test was used to analyze the differences between the Unweighted UniFrac distances among groups and the results were visualized by constructing a scatter diagram and box plot. PCoA analysis, scatter diagram and box plot and ternary plots were performed using R 3.2.5. The taxonomy compositions and abundances of different samples were analyzed and visualized by GraphPad PRISM® software (version 4.0). To compare the taxa in buccal and subgingival plaque samples between HP, AgP, and ChP, the Mann–Whitney U-test was used. The Mann–Whitney test, Student's t-test were performed using SPSS 20.0.Normality tests were conducted in each group of data. The mean clinical parameters were then compared via a paired Student's Thirty-two subjects were recruited in the study, including twenty-three patients with periodontitis: eleven of them were diagnosed as ChP, twelve of them were diagnosed as AgP, and other nine subjects were defined as HP. Consequently, 32 buccal swab samples (showed as HP/ChP/AgP_B), and 113 subgingival samples (showed as HP_Sub: 1-2 sample(s) per/subject, ChP/AgP_Sub: 3-5 samples per/patient, for a total of 16 samples belonged to HP_Sub, 46 samples belonged to ChP_Sub and 51 samples belonged to AgP_Sub) were collected. The demographic and clinical metrics of subjects on baseline are detailed in In the HP_B, ChP_B, and AgP_B groups, the five most abundant of the 12 phyla were Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, and Fusobacteria, which represented more than 95% of the total sequences. In samples of subgingival plaque, HP_Sub, ChP_Sub, and AgP_Sub, the aforementioned five phyla also constituted the majority of the sequences, but the abundance of TM7, Spirochaetes, and Synergistetes increased, especially in ChP_Sub and AgP_Sub .Streptococcus, Haemophilus, Gemella, Neisseria, and Veillonella. These constituted more than 60% of the total sequences. In subgingival plaque samples, Streptococcus, Prevotella, Neisseria, Fusobacterium, Actinomyces, Capnocytophaga, Veillonella, and Leptotrichia represented more than 50% of the total sequences  were conducted based on the OTU abundances. The ChP_B and AgP_B samples clustered together and could not be distinguished from each other, while the HP_B samples appeared more dispersive . As for We analyzed our data in phyla level using ternary plots based on three main status: the intraoral environment of periodontal healthy status (HP), and the intraoral environment of periodontitis (ChP and AgP). According to the most abundant OTUs , HP_B haThe Unweighted UniFrac distances were calculated and compared between groups to investigate the similarity of all samples . The disU-test. There were significant differences between all groups at several levels in both buccal and subgingival plaque sample  compared to HP group   significantly increased when compared with HP group. Among them, The PCoA showed all subgingival plaque samples clustered tighter with each other compared with buccal samples. These results agree with those described by Gohler, each oral habitat appears to be preferentially populated by different and somewhat unique sets of microbes  were maximal in each group. The oral microbiota of hard and soft tissues appeared to be characterized by species-specific colonization patterns, which is in accordance with previous study by Mager et al. They reported differences in the bacterial profiles of 40 oral cultivable species on various oral habitats in healthy individuals and found that the profiles of soft tissues were more similar to each other when compared to those from subgingival plaques  in patients with ChP or AgP, and to compare them with samples of subjects with periodontal health (HP). We found that buccal and subgingival microbial communities in healthy samples and periodontitis largely differed, with higher abundance in periodontitis. Some genera had significant difference between health and periodontitis both in buccal and subgingival plaque samples. While buccal mucosa and subgingival pockets to be characterized by species-specific colonization patterns.In our study, we found that the subgingival and buccal mucosa microbiome in health and periodontitis largely differed, with higher bacterial abundance in periodontitis. In addition, we found 10 genera in both buccal and subgingival plaque samples of periodontitis groups (ChP and AgP) significantly increased when compared with HP group. Moreover, the results based on the Unweighted UniFrac distance showed that buccal and subgingival plaque samples from the same individuals show higher community divergence than same habitats from different subject samples, which corroborated a notion that individual oral habitats are characterized by species-specific colonization patterns. Within the limitation of the relatively small sample size, this pilot study provides a glimpse at the changes in the microbiota of subgingival and buccal mucosa associated with periodontitis from a holistic view.WH and XW: conception and design. YW, MS, MZ, and WH: recruitment of patients and collection of oral samples. MS, YW, CW, and YN: analysis and interpretation of data. YW and MS: manuscript preparation. YW, MS, MZ, WH, and XW: manuscript revisions.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.


The objective of the study was to establish the effect of component share in mixtures and harvest date on concentrations of selected heavy metals in the green fodder of field pea, oat, and their mixtures. The research hypothesis assumed that the cultivation of peas and oats in pure sowing, and in mixtures will also allow to choose combinations from which the forage will have the lowest content of heavy metals. Field research was conducted at the Zawady Experimental Farm  which belongs to Siedlce University of Natural Sciences and Humanities. Two factors were tested in the study: I—component share in the mixture: field pea—pure stand 100%, oat—pure stand 100%, field pea 75% + oat 25%, field pea 50% + oat 50%, field pea 25% + oat 75%; II—harvest date: field pea flowering stage, field pea flat pod stage. Plant material was sampled to determine the following elements: Cu, Zn, Cd, Pb, Cr, and Ni. The results of the study demonstrated that field pea grown in pure stand had the highest copper and zinc contents, and the lowest chromium and nickel contents. Field pea mixed with oat significantly reduced heavy metal content in green fodder. Cadmium and lead contents in the green fodder of field pea/oat mixtures were too low to be determined by means of the spectrometer Perkin Elmer Optima 8300. Regular checks of heavy metal contents are recommended in spite of their low amounts in the green fodder of field pea/oat mixtures. Green fodder produced from legume/cereal mixtures is a valuable source of dairy cattle feed, providing it has a beneficial chemical composition, including a low concentration of heavy metals , and the harvested area was 20 m2 (4 m × 5 m). Two factors were tested in the study: I—component share in the mixture: field pea—pure stand 100%, oat—pure stand 100%, field pea 75% + oat 25%, field pea 50% + oat 50%, field pea 25% + oat 75%; II—harvest date: field pea flowering stage, field pea flat green pod stage. The species proportion in a mixture was established in relation to the number of seeds planted in pure stands, that is 560 grains of oat and 120 grains of field pea m2. The following sowing rates were used: field pea 170 kg ּha−1, oat 180 kg ּha−1, field pea 128 kgּ ha−1 + oat 45 kg ּha−1, field pea 85 kg ּha−1 + oat 90 kg ּha−1, field pea 43 kg ּha−1 + oat 135 kg ּha−1.Field research was conducted in the years 2010 to 2012 at the Zawady Experimental Farm  which belongs to Siedlce University of Natural Sciences and Humanities. The experimental soil was Albic Luvisol (Arenic). The concentration of elements in the soil was as follows: P 5.21 mg 100 g−1 P in the form of granular triple superphosphate and 99.6 kg ּha−1 K in the form of 60% potassium salt, depended on soil chemical composition. In spring, nitrogen fertiliser, at the rate of 30 kg ּha−1 N in the form of ammonium nitrate, was applied preplant to all the plots, excluding the units assigned to field pea grown in pure stand. At the stage of stem elongation, additional N was applied (50 kg ּha−1 for oat and 30 kg ּha−1 for field pea/oat mixtures). Field pea and oat seeds were planted in early April as described for the first experimental factor. Plants were harvested in late June and early July. During harvest of mixtures, fresh matter samples were taken from each plot to determine microelements. Cu, Zn, Cd, Pb, Cr, and Ni contents were determined by means of inductively coupled plasma optical emission spectrometry (ICP-OES) using the spectrometer Perkin Elmer Optima 8300.In all the study years, the mixtures were preceded by winter triticale. Phosphorus and potassium fertilisers were applied in autumn and their rates, 35.2 kg ּhaEach characteristic tested was subjected to analysis of variance suitable for the split-block design. When significant sources of variation were confirmed, their means were separated using Tukey test. Calculations were performed in MS Excel 12.0.The years of the study were characterised by significantly changeable weather conditions Table . In 2010−1 d.m.) was recorded in field pea grown in pure stand, it being the lowest in oat (3.811 mg ּkg−1 d.m.). Also, Woźniak and Soroka (Copper content in the green matter of field pea/oat mixtures was significantly affected by the experimental factors and their interaction Table . The higd Soroka , Jareckid Soroka , and Alid Soroka  observedd Soroka . Plants d Soroka . In the d Soroka  who repod Soroka  as well d Soroka  demonstrd Soroka , Demirezd Soroka , and Mucd Soroka  have claStatistical analysis demonstrated a significant effect of the experimental factors and their interaction on zinc content in the green fodder of field pea/oat mixtures Table . Zinc plLead and cadmium contents were insignificantly affected by the experimental factors and their concentrations in the green fodder of field pea/oat mixtures were too low to be determined by means of the emission spectrometer Perkin Elmer Optima 8300. Thus, they did not exceed the standards set for green fodder in the Official Journal of the European Union . It was Statistical analysis confirmed a significant effect of the experimental factors and their interaction on chromium content in the green fodder of field pea/oat mixtures Table . The lowNickel content in the green fodder of field pea mixed with oat was significantly affected by the experimental factors and their interaction Table . The lowField pea grown in pure stand had the highest copper and zinc contents, and the lowest chromium and nickel contents. Field pea mixed with oat contributed to a significant decline in the green fodder content of heavy metals.Field pea/oat mixtures harvested at the stage of field pea flowering contained more copper but less zinc, chromium, and nickel compared with mixtures harvested at the stage of field pea flat green pod.Cadmium and lead contents in the green fodder of field pea mixed with oat were insignificantly affected by the experimental factors, and were too low to be determined by means of the emission spectrometer Perkin Elmer Optima 8300.Green fodder of field pea mixed with oat, whether harvested at the flowering stage or the stage of field pea flat green pod, was safe for cattle because it did not contain excessive amounts of heavy metals.Despite a low heavy metal content in the green fodder of field pea mixed with oat, continual monitoring of these elements is recommended.


Depleted uranium (DU) is oxidized readily due to its chemical activities, which limits its applications in nuclear industry. TiN film has been applied widely due to its good mechanical properties and its excellent corrosion resistance. In this work, TiN protection films were deposited on DU by direct current magnetron sputtering (DCMS) and high power pulsed magnetron sputtering (HPPMS), respectively. The surface morphology and microstructures were investigated by atomic force microscope (AFM), scanning electron microscopy (SEM), and grazing incidence X-ray diffraction (GIXRD). The hardness and Young’s modulus were determined by nano-Indenter. The wear behavior and adhesion was analyzed by pin-on-disc tests and scratch adhesion tests and the corrosion resistance was evaluated by electrochemical measurements. The results show that the TiN films that were deposited by HPPMS outperformed TiN film deposited by DCMS, with improvements on surface roughness, mechanical properties, wear behavior, adhesion strength, and corrosion resistance, thanks to its much denser columnar grain growth structure and preferred orientation of (111) plane with the lowest strain energy. Besides, the process of Ti interlayer deposition by HPPMS can enhance the film properties to an extent as compared to DCMS, which is attributed to the enhanced ion bombardment during the HPPMS. However, it is chemically active and susceptible to corrosion, especially in salty, humid, and high-temperature environments ,2. Surfa, C+, N+ ,8 ion-imTiN films have been commonly used for protective purposes on different kinds of substrate materials due to its chemical stability and excellent mechanical properties ,11,12,13Efforts have been attempted to deposit TiN film on DU under low temperature by various methods including the conventional direct current magnetron sputtering (DCMS) and arc ion plating (AIP) techniques . However−2. This technology enriched in metal ion plasma, could deposit dense structures and offer virtually defect free films at a relatively low temperature [High power pulsed magnetron sputtering (HPPMS) is a novel technique in which the power provides the target with pulses of high power densities of a few kW cmperature ,17.In this work, HPPMS technique was utilized to deposit dense TiN films on uranium for the first time. TiN films were deposited on DU by DCMS and HPPMS. The surface roughness, morphology, nano-hardness, adhesion strength, and electrochemical properties of these films were characterized in order to study the differences between conventional DCMS and HPPMS.The Ti/TiN films were deposited on DU substrate and silicon (100) wafers, the later ones were prepared for phase and morphology analysis. The DU samples with a size of Ф15 mm × 3 mm were grinded using SiC water papers from 500# to 1000# progressively, mechanically polished to minor, and ultrasonically cleaned in acetone and ethanol, respectively, and then put into a vacuum chamber immediately when the substrates dried.−4 Pa and the chamber temperature was fixed to 180 °C. Prior to deposition, the DU samples were sputtered by Ar+ ions  for 30 min to clean the surface contaminations and partially remove the native oxide layer. After pre-sputtering, the Ti interlayer of approximately 100~300 nm thick was pre-deposited on the DU substrates to reduce the stress at the substrate interface and to enhance the adhesion between the film and the substrate. During deposition, the TiN films were deposited on the substrate with thickness of 4~5 μm.The base pressure was lower than 5 × 10Three deposition modes were designed to compare the structure and properties of the films deposited by HPPMS and DCMS, in order to investigate how the high power pulse introduced into magnetron sputtering process influences performance of the TiN films. Lc).The surface and cross-sectional morphology of the samples were observed by scanning electron microscopy . The phase structure of the TiN films was characterized by grazing incidence X-ray diffraction  with Cu Kα radiation and the incident angle of 0.5°, where the scan range was from 30° to 80°. The Hardness and Young’s modulus of the deposited films were measured by a nano-Indenter  with a Berkovich head. The test was applied with a load of 1 mN and depth of 90 nm. Then, the surface roughness of the TiN films was measured by an atomic force microscope  fitted to the indenter. The tribological test of the deposited films was performed on a ball-on-disc tribometer . In this test, SiN balls (diameter of 6 mm) were selected as friction pairs. The balls were under a constant normal load of 1 N, while the discs were circumrotated at a certain speed to generate abrasion of the films  as reference. The scan rate of potentiodynamic polarization was 0.2 mV/s.The corrosion behavior of DU and TiN coated DU samples were studied by potentiodynamic polarization techniques on an electrochemical workstation . All of the electrochemical measurements were conducted using a conventional three-electrode electrochemical cell in aerated neutral NaCl solution with 50 μg/g ClThe surface morphologies of TiN film by different deposition modes are shown in Ra) of the samples was calculated accordingly  that the surface energy controlled the growth of the TiN film and the (200) preferred orientation favors [+ ion bombardment increased the adatom mobility and diffusivity, thereby improving the strain energy, which facilitated the preferred orientation of (111) [GIXRD measurements were performed on TiN films and the corresponding XRD patterns are shown in n favors . In the of (111) .H = 20.56 GPa) was higher as compared to that of DCMS film (H = 15.75 GPa), and the hardness of HP + DC film (H = 22.09 GPa) was between them. The Young’s modulus exhibits a similar tendency. The enhanced hardness was attributed to both a dense microstructure without inter-columnar voids that were prepared during film growth in HPPMS discharge [Hardness and Young’s modulus values of the film deposited by different modes are listed in ischarge  and the ischarge .μ) of TiN film. It can be identified that the DCMS TiN has the highest wear coefficient (μ = 0.56). The film by HP + DC mode exhibits a reduced friction coefficient (μ = 0.42). While, the HPPMS deposited one has a further improved wear resistant (μ = 0.34) when compared to the two modes described above.The wear behavior of the TiN films was evaluated by pin-on-disc tests. The wear tracks of TiN films by three modes were observed by optical microscopy (OM). Deep and wide grooves with some obvious debris were formed on the film by DCMS mode a, which The HP + DC mode provides high energy ion bombardment during the Ti interlayer growth, which shows a certain improvement in surface roughness, porosity, hardness, and adhesion strength, as compared to DCMS mode. Further, HPPMS-deposited TiN films show superior performance in structure and mechanical performance, therefore resulting in enhanced tribology resistance.To observe and quantify the adhesion strength of TiN film by different deposition modes, progressive micro-scratch tests were performed under the same condition . The optical micrographs of scratches TiN film by different modes after scratch test are shown in −6 A/cm2. TiN films could prevent DU from rapid corrosion, as demonstrated by curve b, c, d. Especially, all of the TiN films were characterized by a passive region. However, the current density (icorr) of HPPMS TiN (2.6 × 10−8 A/cm2) was lower by an order of magnitude than that of HP + DC TiN (2.7 × 10−7 A/cm2) and two orders of magnitude than that of DCMS TiN (5.1 × 10−7 A/cm2), respectively. As reported, the electrochemical resistance of film is strongly related to the microstructure and surface morphology [rphology ,27. The In this study, TiN film has been deposited on DU under low temperature by direct current magnetron sputtering (DCMS) and high power pulsed magnetron sputtering (HPPMS), respectively.Lc 16 N, and an improved corrosion resistance. Besides, the process of Ti interlayer deposition by HPPMS can enhance the film properties as compared to DCMS, which is attributed to the enhanced ion bombardment during the HPPMS.The experimental results shown in this work demonstrate that the HPPMS deposited TiN film exhibited a compact and smooth surface as compared to DCMS that was deposited TiN. The DCMS deposited TiN films exhibited porous columnar structure, which corresponds to zone I in the well-known structure zone model (SZM); while, the HPPMS counterpart shows competitive texture growth, which corresponds to zone T in SZM. The preferred orientation of DCMS produced TiN films changed from (200) to (111) with HPPMS. The HPPMS fabricated TiN film reveals a high hardness of 22.09 GPa, Young’s modulus of 220.21, low friction coefficient of 0.34, high adhesion of In summary, the results that are shown in this work prove that the TiN film deposited by HPPMS on DU could significantly improves its mechanical, tribological and corrosion performance and therefore serves as a promising surface protective coating for DU.


Meanwhile, for the sake of illuminating strain rate and temperature effect mechanisms, glass yarn samples were complementally tested at four different strain rates  and varying temperatures  utilizing an Instron drop-weight impact system. In addition, quasi-static properties of GFRP and glass yarn are supplemented as references. The stress–strain responses at varying strain rates and elevated temperatures are discussed. A Weibull statistics model is used to quantify the degree of variability in tensile strength and to obtain Weibull parameters for engineering applications.Unidirectional glass fiber reinforced polymer (GFRP) is tested at four initial strain rates (25, 50, 100 and 200 s With decreasing manufacturing costs, recently, they have won the attention of engineers involved in the construction of civil structures [etc. and vary temperature conditions during their service life. Under such conditions, the mechanical properties of FRP involving Young’s Modulus, tensile strength, toughness, etc. may suffer great changes [Fiber reinforced polymer (FRP) composites have many merits, such as high stiffness/weight and strength/weight ratios, advanced fatigue and corrosion resistances, ructures . Structu changes ,3,4,5,6.−1, servo-hydraulic high speed machines [4 s−1 is the split Hopkinson bar, which was first introduced by Kolsky [A variety of testing techniques and procedures have been developed for gaining insight into the dynamic responses of materials under different strain rates and different methods are suited for different ranges of strain rate. The conventional screw drive load frame  is used machines  or drop-machines  can be uy Kolsky . With thet al. [et al. [et al. [−1 by means of a servo-hydraulic testing apparatus. A significant increase of the tensile strength was observed with increasing strain rate. Ochola et al. [−3 and 450 s−1. The experimental results reported that the dynamic material strength of GFRP increases with increasing strain rates.The tensile behaviors of carbon fiber-reinforced polymers (CFRP) under different strain rates were studied by several researchers ,11,12,13et al.  found thet al. . It show [et al.  determin [et al.  studied a et al.  investiget al. [et al. [et al. [−1 and temperatures from −25 to 100 °C by Ou and Zhu [−1.Temperature effects on GFRP are another important issue that needs further investigation in order to adopt the use of such composites in strengthening structural reinforced concrete (RC) members and their connections when exposed to elevated temperatures and harsh environment. Hawileh et al.  experime [et al.  evaluate [et al.  conducte and Zhu  to inves−1, and only few experimental results are accessible on the dynamic tensile behavior of GFRP at elevated temperatures and harsh environment. Furthermore, as the main load-bearing body of GFRP, glass fibers play a significant role in GFRP subjected to an impact load. Therefore, revealing the variation of tensile properties of fibers at different strain rates is the key to gaining a deep insight into the mechanical behavior of GFRP under impact loads and then optimizing structural design.Although many efforts have been carried out on the strain rate and temperature effects on the mechanical properties of GFRP, very limited information is available on intermediate strain rate ranging from 25 to 200 sThe primary objective of this study is to investigate the effects of intermediate strain rates and temperatures on the mechanical properties of glass fiber bundle (yarn) and unidirectional GFRP laminate samples. In the next section, the experimental procedure and test results are presented and discussed. Then, comparison of the obtained values of tensile strength of GFRP specimens at different strain rates with the values found in literature is displayed. In the fourth section, the results of Weibull statistical analysis based on experimental results are discussed.2 by taking into account the linear density of the material and dividing it by its bulk density, following the same method in the reference [i.e., the glass yarn and epoxy resin, were listed in The glass unidirectional fiber fabric made by Nanjing Hitech Composites Co., Ltd.  was used in this study, which had been fabricated with individual yarns (5.3 yarns/cm), as shown in eference . Epoxy rIndividual yarn was extracted carefully from the glass fabric for single yarn tests. In order to reduce stress concentration and to improve the load transfer mechanism between the specimens and steel wedges, thin aluminum sheets (10 mm × 5 mm × 0.5 mm) were roughhewed by toothed steel plate and glued at both ends of the single yarn specimens using the same epoxy. When the epoxy was fully cured, the final glass yarn specimen was constructed by cutting off the redundant length at both sides using an electric scissor as shown in GFRP were manufactured by Vacuum Assisted Resin Transfer Molding (VARTM) , thus thversus time were recorded simultaneously by a high speed data acquisition system at a sampling rate of 1 MHz with 14-bit resolution. In addition, the CEAST Visual Impact software (INSTRON (China) Co., Ltd., Shanghai, China) is used for system management and data processing . An environmental chamber can be heated by electric resistance wire and cooled by liquid nitrogen, giving operating temperatures ranging from −50 to 100 °C. In this work, four different impact velocities  were chosen to obtain the strain rates of 40, 80, 120 and 160 s−1 for specimens at room temperature (25 °C). In addition, four different temperatures, i.e., 25, 50, 75 and 100 °C were selected for the study of temperature effect under an impact velocity of 1 m/s (strain rate = 40 s−1). Ten samples were tested for each strain rate and temperature.Dynamic tensile tests of glass yarn under various strain rates and temperatures were conducted on a state-of-the-art drop-weight impact system  as shown in −1, respectively. At the initial strain rate of 25 s−1, six different temperatures  were selected for temperature effect testing. The automatically controlled environmental chamber was set to the required temperature and maintained for 30 min to guarantee that the test specimens reached a uniform temperature. Ten replicates were tested for each condition to reduce the influence of random error. A high-speed digital acquisition card  was used to collect force and displacement data at a sampling rate of 500 kHz. A Phantom v7.3 high speed digital camera  was used to capture the deformation and failure behavior of GFRP specimens with a sampling rate of 20,000 fps.In addition, dynamic tensile tests of GFRP specimens under various strain rates and temperatures were performed using an MTS high rate servo-hydraulic testing machine with an environmental chamber in Arizona State University  (MTS Systems (China) Co., Ltd., Shanghai, China)  with a lvs. strain curves of glass yarn samples at different strain rates. The high-frequency oscillations in the stress curves obtained under dynamic loadings were caused by the system ringing phenomenon [vs. strain curves of GFRP samples at different strain rates. Note that the actual strain rates during dynamic testing was remarkably different from the initial strain rates, which is attributed to the fact that the reaction force of the test specimen slowed down the stroke, especially at higher loading speeds when the hydraulic pressures were relative lower. In the subsequent discussion of this paper, the initial strain rates will be used to facilitate analysis. Meanwhile, it is also worth noting that the curves were similar with the strain–stress curve of mild steel because the stress fluctuated after a small elastic deformation until the final failure at 25 and 50 s−1. This is because the yarns of GFRP samples do not fail simultaneously during the loading, as shown in −1.enomenon ,26. In a−1) to dynamic loading (40 s−1). In addition, tensile strength maintains growth with increasing strain rate, while toughness abruptly decreases at the strain rate of 160 s−1 due to the diminution of strain. Specifically, tensile strength and toughness increase as much as 88.0% and 474.3% from 919 ± 102 MPa and 7.0 ± 1.1 MPa to 1729 ± 67 MPa and 40.2 ± 4.5 MPa, respectively, when the strain rate increases from 1/600 to 40 s−1. After that, tensile strength goes on to increase nearly 22.0% when the strain rate increases from 40 to 160 s−1, while toughness increases about 26.4% over a strain rate range of 40–120 s−1 and then nearly drops by 10.2% between 120 and 160 s−1. The initial significant enhancement in strength and toughness can be explained as: the friction between adjacent fibers always plays an important role due to random and misaligned breakage of filaments in the yarn under tension and the contact force among adjacent filaments increases with increasing strain rate, resulting in the increase of dynamic frictional force (friction coefficient is assumed to be constant). Therefore, tensile strength and energy-consumption are improved noticeably.The effects on mechanical properties of GFRP can be summarized based on the stress–strain curves under different strain rates. −1, while the variation of its toughness is similar to the tensile strength of glass yarn. Concretely speaking, tensile strength increases nearly 49.1% when the strain rate increases from 1/600 to 200 s−1. By contrast, toughness increases remarkably from 15.5 ± 3.7 MP a at a strain rate of 1/600 s−1 to 32.5 ± 7.8 MPa at 25 s−1, enhanced by 109.7%, and then increases about 34.2% over the strain rate range of 25–200 s−1. Those kinds of strengthening and toughening mechanisms are different from glass yarn and could be described as: (i) there is not enough time to initiate internal defects in the material at higher strain rates. Therefore, more energy is needed for damage initiation and propagation, which leads to a higher level of tensile strength under high strain rates, and (ii) more damage is involved with increasing strain rate.For GFRP, the tensile strength linearly increases over the strain rate range of 1/600–200 s−1 together with the longitudinal strain (εyy) distributions measured by DIC are shown in t = 0.3 ms), a relatively uniform strain distribution is obtained. As the load increases (t = 2.0 ms), tensile strain starts to localize near the top and bottom edges of area of interest (AOI), which is represented by the red zone. This may imply the stress concentration due to the clamps and associate with the non-linear behavior observed in the stress-strain curve. The inhomogeneity of the strain fields may be attributed to multiple aspects such as the presence of random defects, variability in the stiffness of basalt yarns and potential eccentric loads. The localized zones continue to grow with increasing load (t = 4.0 ms) and ultimately spread out over the entire AOI prior to failure (t = 5.0 ms). Non-uniform strain fields are also observed in the specimens tested at other strain rates, as shown in In order to further study the tensile behavior and failure pattern of GFRP, digital image correlation (DIC) analysis was performed on the specimens tested under room temperature. The stress–strain curve of a GFRP tested at 25 s−1), the damage area was limited to a small region; however, under higher strain rate (200 s−1), the damage path covers the entire gauge section, indicating more materials of the specimen are involved in energy dissipation. This phenomenon was also observed by Shokrieh [Shokrieh  in a rel−1. The effect of temperature on tensile strength and toughness in terms of average values and standard deviations are summarized in etc. Heating will undoubtedly weaken the chemical bond and van der Waals force and then lower the tensile strength of fibers. Furthermore, maximum strain over temperature range of 25–75 °C has no obvious change, so toughness also decreases. However, when heated to 100 °C, tensile strength of glass yarn rebounds 19.2% at the base of value at 75 °C, mainly due to the fact that the frictional coefficient of the fiber surface increases because of volatilization of sizing and water at 100 °C, resulting in the increase of frictional force between fibers. At the same time, toughness gets a sharp increase (about 44.5%), which could be attributed to the obvious increases in both tensile strength and maximum strain.Tg) is reached. This softening effect would weaken the interfaces between fibers and matrix, and decrease the stiffness of epoxy matrix during loading. For GFRP, tensile strength shows almost no change (within 3%) when temperature increases from −25 to 50 °C. However, a sharp decrease (about 18.9%) is observed over the temperature range of 50–100 °C, which is caused by the softening of the resin matrix when glass transition temperature of epoxy resin  is reached. This would weaken the interfaces between fibers and matrices and decrease the resistance of matrices during deformation. Reference [eference  also foueference .et al. [In addition, some researchers  proposedet al.  confirmeet al.  found thm is the Weibull modulus or shape parameter. The cumulative probability density, P, is estimated as: N is the total number of tests and i is the current test number.The determination of the statistics and probability distributions of the random variables characterizing material properties plays an important role in the development of probabilistic-based design specifications. The selection of the probability distribution assigned to characterize the material property data will have a significant effect on the computed reliability. Assuming distinct distributions for the material properties may result in calculated probabilities of failure that vary by over an order of magnitude. This can be attributed to the lower tail behavior of different cumulative distribution functions, which has become acquainted as the tail-sensitivity problem in structural reliability . The Weim is related to the variation of strength among the tested specimens. For glass yarn, in general, the results obtained from higher strain rate have a lower shape parameter than the counterpart. This manifests that yarn subjected to higher strain rate is usually characterized by a more random breakage process.The Weibull parameters extracted from tensile strength data of glass yarn and GFRP samples are presented in (1)−1) to dynamic loading (40 s−1), but toughness decreases about 10.2% when the strain rate changes from 120 to 160 s−1 due to the diminution of strain. For GFRP, the tensile strength linearly increases nearly 49.1% over the strain rate range of 1/600–200 s−1, and toughness increases remarkably (about 109.7%) during a transition from quasi-static loading (1/600 s−1) to dynamic loading (25 s−1).There is an apparent dependence of the dynamics tensile properties of glass yarn and GFRP on the strain rate. For the glass yarn, tensile strength and toughness increase as much as 88.0% and 474.3% during a transition from quasi-static loading (1/600 s(2)The mechanical properties of glass yarn and GFRP are also dependent on the temperature. For glass yarn, tensile strength and toughness decrease nearly 25.3% and 31.1% when temperature increases from 25 to 75 °C. However, when heated to 100 °C, tensile strength and toughness of glass yarn rebound due to the augment of frictional force between fibers. For GFRP, tensile strength shows almost no change (within 3%) when temperature increases from −25 to 50 ° C, but decreases sharply (about 18.9%) over the temperature range of 50–100 °C because of the softening of the resin matrix when Tg of epoxy resin is reached.(3)The failure patterns of the GFRP specimens are closely related to the loading conditions. At low strain rates, the fracture surface is limited in a small region, while, with increasing strain rate, the damage path covers the entire gauge section where extensive debonding between fibers and matrix was also observed, which leads to an increase in tensile strength and energy absorption. In addition, the components of GFRP specimen are more separated after failure at higher temperatures.(4)The scale parameter (This experimental study focuses on the tensile characterization and failure pattern of glass yarn and GFRP samples under different loading conditions. The strain rate and temperature effects on the mechanical properties and fracture morphologies are investigated and discussed comparatively. The following conclusions can be reached:


Clostridium difficile-associated diarrhea (CDAD) is a major public health threat that results in increased length of stay, hospital readmissions, deaths, and economic burden. CDAD treatment is often guided by severity of disease. Although various tools exist to determine CDAD severity, real-world data evaluating the use of such tools in treatment algorithms are sparse.C. difficile recurrence. Patient and clinical data was retrospectively gathered to evaluate the utility and outcomes of the treatment pathway.A local CDAD treatment pathway was developed independently to guide fidaxomicin prescribing at wellStar Health System (WellStar) and at Lee Health (LH) and Sarasota Memorial Hospital (SMH). Each algorithm was designed locally by the stewardship pharmacist and was utilized to identify patients at high risk for There were 262 patients that received fidaxomicin at these three hospitals during the study time period. Only 30% at WellStar and 20% at LH or SMH met the study criteria and adhered to the pathway requirements. After completion of fidaxomicin, 30-day recurrence rates at WellStar was 0 and at LH and SMH 7%. Clinical cure rates were 83% in WellStar and 93% in LH and SMH.The results from these two pathways show positive outcomes for the use of fidaxomicin in patients at high risk for CDAD recurrence. This data supports the potential utility of fidaxomicin against CDAD. Clostridium difficile associated diarrhea (CDAD) constitute a substantial and increasing burden to patients and healthcare facilities in the United States (US) and throughout the world. In the US, it has been estimated that there may be over 450,000 incident infections annually, with 83,000 of these being first recurrences, and over 29,000 cases leading to mortality [C. difficile imposes numerous burdens on resources including the need for private rooms, isolation supplies, enhanced environmental cleaning, and vigilant hand hygiene, resulting in an estimated excess cost of as much as $4.8 billion to US acute care facilities, in addition to severe negative impact on patients’ quality of life [Diarrheal illness and complications caused by ortality . Of thesHistorically, treatments for CDAD included vancomycin and metronidazole, with the choice of therapy usually dependent on disease severity; tigecycline can also be used in cases of CDAD colitis . UpdatedC. difficile growth as well as sporulation through RNA transcription inhibition, and whose narrow spectrum minimizes alterations of the colonic microbiota compared to existing agents [Increasing incidence of treatment failure with traditional agents (i.e. metronidazole and vancomycin) prompted research into and development of new molecules. Fidaxomicin, an oral macrocyclic antimicrobial that inhibits g agents , was appg agents , 8. A reg agents .Despite its demonstrated efficacy and effectiveness, however, it has been difficult to precisely define fidaxomicin’s place in the CDAD armamentarium, due to unclear definitions of risk factors for recurrence or severe/complicated CDAD, lack of clinical data in multiple recurrences or complicated CDAD, and high economic cost . For maxC. difficile disease scoring systems exist including a disease severity measure in the IDSA guidelines [C. difficile infections; however, there is a lack of data evaluating the implementation of these tools in a real world setting. This study describes the local implementation and outcomes associated with two different fidaxomicin treatment pathways.Various idelines , the ATLidelines , and theidelines . The ATLidelines . The D’Aidelines . Due to C. difficile recurrence. Patient and clinical data was retrospectively gathered to evaluate the utility of the treatment pathway.A local CDAD treatment pathway was developed independently to guide fidaxomicin prescribing at WellStar Health System (WellStar) in Georgia and at Lee Health (LH) and Sarasota Memorial Hospital (SMH) in Florida. Each algorithm was designed locally by the stewardship pharmacist and was utilized to identify patients at high risk for C. difficile PCR result from clinical microbiology testing with Xpert C. difficile/Epi  and a clinical diagnosis consistent with CDAD between 12/1/12 and 3/31/14 from 5 of the hospitals in the WellStar system were included in this study. Patients over age 18 were included if it was their initial episode or first recurrence of CDAD with laboratory confirmed, non NAP1 strains with less than 24 h of enteral (PO) vancomycin or parenteral (IV) or PO metronidazole and at least one of the following conditions: age > 65 with severe disease, ulcerative colitis/Crohn’s disease, creatinine clearance (CrCl) < 30 mL/min or end-stage renal disease (ESRD), active malignancy, concomitant systemic antibiotic therapy, or immunocompromised status , or immunosuppressive agents .WellStar is a not-for-profit system of eleven hospitals serving NW metropolitan Atlanta. Patients with a positive C. difficile result based on Xpert C. difficile/Epi  were included in the study.LH is an integrated delivery network consisting of four acute care hospitals and two specialty hospitals with a total of 1423 beds in Fort Myers and Cape Coral, FL. Patients with a positive C. difficile/Epi C. diff Quik Chek Complete®  with reflex PCR using Xpert C. difficile/Epi .SMH is an 806-bed regional medical center, including a network of outpatient, long-term care, and rehabilitation centers, serving approximately 800,000 patients each year. CDAD was assessed at SMH using Xpert LH and SMH evaluated the impact of a treatment pathway targeting fidaxomicin for patients based on ATLAS Score and CDAD episode between 1/5/15 and 11/2/15  maintained throughout the treatment course and 2 days afterward or until hospital discharge, documentation of clinical resolution in the medical record, no requirement for additional CDAD therapy (including the need for a switch in CDAD therapy) on or before Day 14.Descriptive statistics were used to describe the patient population and the outcomes among patients adhering to the treatment pathway. Continuous variables were reported as means and standard deviations. Categorical variables were reported as frequency and percent.The institutional review board at each participating institution approved the study.There were 120 patients who received fidaxomicin at a participating WellStar institution within the study timeframe, but only 36 (30%) met inclusion/exclusion criteria. Exclusions were due to NAP1 strain , multiple recurrences , prior CDAD treatment , and absent/missing PCR   of patients and the CDAD was present within 24 h of admission for 15 (42%) of the patients. Patients received an average of 8.4 ± 3.6 days of fidaxomicin. Most patients (83%) were considered clinical cure based on resolution of diarrhea (37%), clinical documentation (23%) and/or no additional CDAD treatment (50%). There were no recurrences at 30 or 60 days and there were five readmissions, but none were related to CDAD.There were 142 patients who received fidaxomicin during the study period, but only 28 (20%) met study criteria. Patients were excluded due to receiving greater than 24 h of vancomycin or metronidazole , not meeting ATLAS pathway criteria , receiving less than 3 days of fidaxomicin , no ID consult , or having fulminant disease . Of the eligible patient population, the average age was 82 years . Half were male and the majority was Caucasian . Many were admitted from another care facility  and the majority had a previous hospitalization . The patients included had several comorbidities including cardiovascular disease (54%), diabetes (27%) and moderate to severe renal disease (18%). The majority  of patients had an ATLAS score ≥ 5.This was the first episode for 20 (71%) and the majority  had CDAD within 24 h of admission. Patients received an average of 7.9 ± 5.2 days of fidaxomicin. Most patients  were considered to have a clinical cure, and two (6%) had recurrence at 30 days. Six (19%) patients were readmitted within 30 days, of whom three (50%) were related to CDAD. All three of these patients had at least one previous CDAD episode prior to the index event.The results from these two pathways show positive outcomes for the use of fidaxomicin in patients at high risk for CDAD recurrence. This data supports the benefits of fidaxomicin against CDAD. Although these two pathways used different criteria to identify patients at high risk, most of the patients included were older and had multiple comorbidities. Most patients presented with an initial CDAD episode  and first CDAD recurrent episodes  and resulted in similar known recurrence rates . After cC. difficile-infection has become more prevalent and is a leading cause of morbidity and mortality in hospitalized patients [These results are consistent with that of other CDAD studies , 15. C. patients . Due to patients , 19, 20.patients . In ordeA limitation to these studies was poor adherence or exclusions to the CDAD treatment pathways , which resulted in a smaller than expected sample size. In addition, these are retrospective studies and data is limited to what is collected in the normal course of care and entered into the patient’s clinical record. Nonetheless, these pathways allowed for evaluation of fidaxomicin use and demonstrate the difficulties of adherence and evaluation of institution-specific pathways across multiple institutions. CDAD scoring tools can aid in identifying severity of disease and allow for modification of treatment regimens to optimize patient outcomes.


Enterovesicular fistulas (EVFs) are abnormal connections between the colon and the urinary bladder. They are estimated to account for one in every 3,000 surgical hospital admissions and are rarely associated with long-standing Crohn’s colitis. We present an interesting case of a 93-year-old man with a long-standing history of Crohn’s colitis on mesalamine, whose mechanical fall at home lead to the discovery of a colovesicular fistula with invading urothelium concerning for squamous cell carcinoma. Enterovesicular fistulas (EVFs) are abnormal connections between the colon and the urinary bladder. They are estimated to account for one in every 3,000 surgical hospital admissions with the most common etiology being diverticulitis (65%-79% of cases), followed by malignancy 10%-20%), and, much less commonly, long-standing Crohn’s colitis (5%-7%) [0%-20%, aA 93-year-old male presented to the emergency department (ED) after experiencing a witnessed mechanical fall at home. In the weeks leading up to the fall, the patient had been treated for a urinary tract infection (UTI) with a course of antibiotics. After the initial diagnosis of UTI, the patient experienced over eight episodes of watery, non-bloody diarrhea a day. The patient’s past medical history was significant for longstanding Crohn’s colitis, complete heart block, hypertension, and a seizure disorder. At the time of presentation, the patient was only taking his home medications, which were atorvastatin 40 mg daily, ezetimibe 10 mg daily, lisinopril 40 mg daily, amlodipine 5 mg daily, phenytoin 300 mg daily, acetaminophen 650 mg daily, vitamin B1 100 mg daily, vitamin D3 1000 units daily, and mesalamine 2.4 grams daily.While in the ED, the patient had a temperature of 100.4 degrees Fahrenheit. Physical exam was significant only for mild suprapubic tenderness. On laboratory studies, the patient had an elevated white blood cell count of 11,200 uL and acute kidney injury (AKI) with a blood urea nitrogen and creatinine of 32 mg/dL and 1.7 mg/dL (baseline creatinine of 1.0 mg/dL), respectively. A Foley catheter was placed and subsequent drainage was significant for feculent material. Urine cultures grew over 100,000 colonies of Escherichia coli, over 1000 colonies of Enterococcus casseliflavus, and over 1000 colonies of Enterococcus enterodurans. The patient’s blood cultures remained negative throughout his hospitalization. He was started on ceftriaxone and metronidazole for his urinary infection and once his AKI improved, he underwent computerized tomography (CT) abdomen and pelvis with oral and rectal contrast. The CT scan showed evidence of a sigmoid EVF with intraluminal gas and diffuse wall thickening of the bladder (Figure Our patient’s sigmoid EVF was likely in the setting of long-standing Crohn’s colitis based on the granulomatous tissue found on fistula biopsy. EVFs are uncommon in Crohn’s colitis and emerge due to abscess formation or bowel perforation from the development of aphthous ulcers . Though Moreover, our patient’s fistula had a peculiar finding on histology: nests of urothelium with atypia and high mitotic activity. Many of the urothelial nests contained a lumen, suggesting that they were probably submucosal glands of the bladder lined by atypical urothelium. Focal areas of squamous differentiation were also present. It is unclear as to the origin of these cellular nests, as on cystoscopy, the patient did not have any indication of malignancy. A review of available literature showed that vesicocolonic fistula formation secondary to primary bladder malignancy has been previously reported though it is incredibly rare, with only two published cases on the subject -6. In ouOperative management is the treatment of choice for EVFs of both malignant and nonmalignant origin. It was previously believed that foregoing operative repair of EVFs predisposes patients to urosepsis or uremia, with up to 75% of affected patients dying from septic complications -9. For tEVFs are a common cause of surgical hospital admissions and are most frequently seen in the setting of diverticulitis, malignancy, and, rarely, in long-standing Crohn’s colitis. While operative management is still considered the treatment of choice for EVFs, previously described complication rates without surgical correction have recently been called into question. In some cases, where the fistula is minimally symptomatic and of nonmalignant nature, expectant management is a treatment option.


RTEL1), a telomere length‐related gene, is closely linked to cancer and age‐related diseases. The aim of this study was to investigate the association between genetic polymorphisms in the RTEL1 gene and coronary heart disease (CHD) risk.Regulator of telomere elongation helicase 1  and the recessive model . In addition, the haplotype “Grs6010620Trs6010621Trs4809324” of RTEL1 was associated with a 0.03‐fold decreased risk of CHD once the results were adjusted for age and gender .In the allele model, using the chi‐square test, we found that the patients with the “G” allele of rs6010620 and the “C” allele of rs4809324 in the RTEL1 may have a protective role against CHD risk.Our findings have demonstrated that the genetic variants of  Written informed consent was obtained from all participants after a full explanation of the study. All samples were coded to protect donor identity. The experimental protocol was implemented in accordance with the approved guidelines.2.2This study included 596 CHD patients and 603 healthy controls. All subjects in our study were recruited from Haikou People's Hospital, Hainan, China. Patients were unrelated subjects ages 19–83 years old. The controls were recruited from routine healthy examinations in the same hospital. Patients were diagnosed with CHD using standard coronary angiography. Coronary angiography had to reveal 50% narrowing of the lumina of at least one of the major coronary arteries for a patient to be included in the study. Subjects with myocardial infarction, stable angina, and unstable angina were classified as CHD subjects. Non‐CHD controls have no congenital heart disease, familial hypercholesterolemia, end‐stage renal disease, or known vasculitides.2.3RTEL1 with minor allele frequencies (MAF) of >0.05 were identified in an association analysis of a Beijing Chinese population. The selected SNPs were reported to be associated with CHD and other cardiovascular disease risk. The SNP was found within an intronic region and was unlikely to possess any functional significance, according to the RegulomeDB. The GTEx result for this SNP shows that it is not known to be associated with gene expression in the most relevant tissue ; however, the SNP and the associated variants in LD are known as eQTLs in artery tissue  was used to extract genomic DNA from whole‐blood samples. DNA samples were stored at −80°C prior to analysis. DNA concentrations were measured using a NanoDrop 2000 . Agena MassARRAY Assay Design 4.0 software was used to design a multiplexed SNP MassEXTEND assay, and SNP genotyping was performed using the Agena MassARRAY RS1000 with manufacturer protocols. The PCR primers for each SNP are shown in 2.4http://www.nhgg.org/analysis/) were used to estimate pairwise linkage disequilibrium (LD), haplotype construction, and genetic association at polymorphism loci . Allele and genotype frequencies were determined using direct counts. Hardy–Weinberg equilibrium values for each SNP were determined using an exact test to compare the expected frequencies of genotypes in controls. Allele and genotype frequencies in CHD patients and controls were calculated using chi‐squared and Fisher's exact tests. Associations between SNPs and the risk of steroid‐induced CHD were tested in genetic models using PLINK software (Version 1.07). Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using unconditional logistic regression analysis with adjustment for gender and age .A total of 596 CHD cases  and 603 controls  were included in the study. The clinical characteristics of the cases and controls are shown in Table 3.2RTEL1 locus were genotyped in CHD patients and healthy controls .Five SNPs within the ls Table . Using c3.3RTEL1 gene conferred a decreased risk of CHD in the codominant model  and the recessive model .As is shown in Table 3.4RTEL1 genes. RTEL1 genes rs6089953, rs6010620, and rs6010621 had very strong linkage disequilibria   Table .4RTEL1 gene and the risk of CHD in a Chinese Han population. We found that rs6010620 and rs4809324 were associated with a decreased risk of CHD. In addition, the “Grs6010620Trs6010621Trs4809324” RTEL1 haplotype was associated with a decreased risk of CHD. Our findings have shed new light on the RTEL1 polymorphisms that may contribute to the protection of CHD. Additionally, eQTL analysis based on the GTEx database  indicated that the rs6010620 polymorphisms may alter the expression level of RTEL1 in coronary artery tissues , an essential DNA helicase, is located in 20q13.3 and includes 40 exons. RTEL1 disassembles a variety of DNA secondary structures to facilitate DNA replication, repair, and recombination processes, thereby helping to maintain telomere integrity (Vannier, Sarek, & Boulton, Regulator of Telomere Elongation Helicase 1 (RTEL1 genomic locus in several cancers such as breast, lung, esophagus, gastric, and colorectal cancer (Muleris, Almeida, Gerbault‐Seureau, Malfoy, & Dutrillaux, RTEL1 could support cell growth by participating in Wnt/β‐catenin signaling, which suggests that RTEL1 may be considered to be an oncogene (Wu, Sandhu, Nabi, & Hao, RTEL1 variants have been associated with the decreased risk of several brain cancers and age‐related disease including glioma, astrocytoma, glioblastomas, and congenital dyskeratosis. In our study, we investigated six SNPs in RTEL1: rs6089953, rs6010620, rs6010621, rs2297440, rs4809324, and rs2297441. Among these SNPs, Ding Y et al., (Ding et al., RTEL1 gene and stroke risk, and the result showed that the rs6010620, rs6010621, and rs6089953 were associated with an increased risk of stroke. However, Olivier, Charbonnel, Amiard, White, and Gallego (RAD51 and RTEL1 gene could compensate telomere loss and protect cell stability when telomere was absent. And another study indicated that the presence of rs6010620 and rs2297440 resulted in a decreased risk of astrocytoma (Jin et al., RETL1 polymorphisms rs6010620 and rs4809324, and CHD risk. More studies should investigate these SNPs using more clinical data with bigger samples. This result may provide a new data to facilitate earlier diagnosis and promote early prevention, and shed light on the new candidate genes and new ideas for the study of subsequent occurrence mechanism of CHD. However, some potential limitations in our current study should be considered when analyzing the results. Our study only conducted preliminary basic research. Moreover, further functional studies and larger population‐based prospective studies are required to fully understand the genetic factors underlying CHD.Furthermore, previous studies have revealed overexpression of the  Gallego  showed R5RTEL1 are associated with CHD in a Chinese Han population. These SNPs may serve as prognostic biomarkers for CHD in the Chinese Han population.Our results indicate that the rs6010620 and the rs4809324 polymorphisms in The authors have no conflicts of interest to report. Click here for additional data file.