Intrahepatic cholangiocarcinoma (ICC) is a heterogeneous group of cancers of the intrahepatic biliary tract. However, few studies have evaluated integrin expression according to an ICC subgroup. We immunohistochemically investigated α6β4 (β4) and αvβ6 (β6) integrin expressions in 48 ICCs, and evaluated their relationship with clinical and pathological parameters and ligand expression, as well as transforming growth factor (TGF)-β1. β4 and β6 expressions were detected in 46 (96%) and 35 (73%) ICC cases, respectively. We classified ICC into negative, low , or high  integrin expression groups. β4 and β6 integrin levels were higher in the non-peripheral central localization type ICC than in the peripheral localization type; they were also higher in the periductal-infiltrating or intraductal-growth types than in the mass-forming type ICC; lastly, they were higher in the well-differentiated type than in the poorly-differentiated type ICC. High expression was related to bile duct invasion. In addition, β4 and β6 expressions were associated with mucin production and the expression of cytoplasmic epithelial membrane antigen, laminin-5, and tenascin-C. TGF-β1 was correlated with β6 expression and poor overall survival. These results suggest that integrin expression is associated with subclassification and clinicopathological features of ICC through the coincident expression of their ligands and TGF-β1. Intrahepatic cholangiocarcinoma (ICC) is the second most common cancer arising in the liver; it comprises 15% of primary liver cancers. Surgical resection is the only potentially curative treatment in patients with ICC, but its resectability remains low (15% at the time of diagnosis), and the outcome of patients after resection has hardly improved, with a median survival time of fewer than three years . ICC is ICC is a heterogeneous group of cancers of the intrahepatic biliary tract. Several ICC subclassifications have been proposed based on localization, disease extent, gross morphology, histology, biology, and cellular origin, as well as molecular classification . AccordiIntegrins are cell surface receptors that connect the cytoskeleton to the extracellular matrix (ECM) and regulate cell adhesion and movement. Integrins comprise a family of transmembrane glycoproteins composed of α and β subunits, forming 24 αβ heterodimeric members that are widely distributed and interact with many ligands, including tenascin and laminin. Integrins are known to be important at every stage of cancer, including tumor cell migration, invasion, proliferation, and survival; they also contribute to tumor progression and metastasis .The α6β4 (β4) integrin is a biliary-type integrin that is expressed on normal and proliferating biliary epithelia and cholangiocarcinomas, but not on hepatocytes and hepatocellular carcinoma (HCC) . β4 inteThe αvβ6 (β6) integrin is a receptor for the extracellular matrix proteins, including fibronectin, vitronectin, and tenascin. It is expressed exclusively on epithelial cells, and typically only during tissue remodeling, which occurs in inflammation and cancer. However, β6 is not expressed in normal adult epithelia . β6 is rIn our previous study, we evaluated the expressions of the αvβ6, α6β4, and α3β1 integrins and their ligands, fibronectin and laminin, in cholangiolocellular carcinoma (CLC), cholangiocarcinoma, and HCC. Our results revealed the significant down-regulation of the αvβ6, α6β4, and α3β1 integrins in CLC in contrast to their high expressions in cholangiocarcinoma, as well as the distinct immunolocalization of fibronectin and laminin in CLC . These rHowever, few studies have reported the expression of these integrins and ligands in ICC subgroups. The present study immunohistochemically investigated the expressions of β4 and β6 integrins and their ligands, laminin-5 and tenascin-C, in 48 ICCs, and evaluated their relationships with clinical and pathological parameters. Furthermore, we investigated the transforming growth factor (TGF)-β1, which is known to be activated by the β6 integrin and induce cancer-associated myofibroblasts that are positive for α-smooth muscle actin (SMA) ,20. The Positive immunohistochemical staining for β4 and β6 integrins were observed in 46 (96%) and 35 (73%) of 48 ICCs, respectively. The localization of β4 and β6 expressions were classified as cell membrane, basal lamina, or cytoplasmic patterns A–C 18]..18]. In p = 0.033 and 0.0092, respectively)  , and lower in the poorly-differentiated type than in the well-differentiated types . High expression was related to bile duct invasion . β4 and β6 integrin expressions were both high in 10 cases; β4 only was high in nine cases, β6 only was high in two cases, and there was a negative to low expression of both in 27 cases. β4 or β6 integrin expressions were not significantly correlated with overall survival  A; higherctively) B; higherp = 0.045)  . p = 0.018 and 0.16, respectively)   in normal bile ducts are closely related to duct size. Smaller bile ducts show a luminal membranous pattern, while larger ducts show a cytoplasmic pattern . In the ctively) C. Alcianctively) D. p < 0.001) (p = 0.016)  B, but no= 0.016) A, but no= 0.016) C. β4 was= 0.016) D. p = 0.019), and were also higher in the infiltrative-growth types than in the expansive-growth types (p = 0.045). High expression was related to hepatic vein invasion (p = 0.034) and lymph node metastasis (p = 0.029). Tenascin-C expression levels were significantly higher in the infiltrative-growth types than in the expansive growth types (p = 0.031)  .p = 0.015), while tenascin-C expression was not correlated (p = 0.090) (The expression level of laminin-5 was significantly correlated with OS (= 0.090) .α-SMA was observed in the stroma of ICC , but was not correlated with SMA . Moreover, TGF-β1 expression was associated with portal vein invasion (p = 0.022), intrahepatic metastasis (p = 0.049), and OS rate (p = 0.0062), but was not associated with lymph node metastasis (p = 0.31) in patients with ICC , MAPK, and c-MET, in which mutations in KRAS and BRAF resulted in a poor prognosis. However, the relationship between this molecular classification and previously described ICC subclassifications remains to be fully elucidated. In addition, genetic heterogeneity, including population level and intratumoral level, makes the identification of targeted genes and targeted therapy for ICC more complex [Using an integrated genomic analysis, Sia et al. recently identified inflammation and proliferation types among 119 cases of ICC . The pro complex ,25. MoleITGB4 and ITGB6 mRNA levels in cholangiocarcinoma cell lines were high in HuCCT1; however, almost no expression of either molecule was observed in HuH28, which is known to grow slowly [The present study also showed that high β4 and β6 integrin expressions were related to the infiltrative growth and bile duct invasion of ICC. Additionally, β6-positive ICC, including the low expression group, was significantly associated with lymph node metastasis, indicating the close relationship between integrin expression and clinicopathological features in ICC, although integrin β4 and β6 expression levels were not significantly correlated with OS in the present study. The number of cases examined seems insufficient for survival analysis because metastasis, invasive growth, and histological grade are known as prognostic factors of ICC. In a study of α6β1 and α6β4, Ding et al. reported that α6 overexpression was significantly correlated with larger tumors, multiple nodules, microvascular/bile duct invasion, and lymphatic metastasis, and an lower OS rate in ICC patients; they indicated that high α6 expression enhanced the activation of extracellular signal-regulated kinases (ERK1/2) and protein kinase B (AKT) signals, inducing ICC cell metastasis and invasion . Li et aw slowly ,26. Partw slowly ,28,29,30w slowly ,32,33.Laminin-5, a ligand of β4, is an ECM protein belonging to the laminin family that is mainly expressed in the basement membrane and has been reported in HCC as well as other cancers. Its expression is significantly correlated with cancer metastasis and poor prognosis . TenasciTGF-β1 expression was correlated with β6 integrin expression as reported thus far. TGF-β1 is known to be activated by β6, stimulate myofibroblast proliferation, and promote fibrosis in cancer . HoweverTaken together, the results of the present study showed, for the first time, the different levels of β4 and β6 integrin expression in ICC subgroups, and suggested that integrin expression is associated with the subclassification and clinicopathological features of ICC through the coincident expression of laminin-5 and tenascin-C, as well as of TGF-β1.Tissue samples from 48 patients with ICC were obtained by surgical resection at Tokyo Medical and Dental University Hospital between 2007–2014. Tissue samples were fixed in a 10% formalin solution, and embedded in paraffin for histological diagnosis and immunohistochemistry analysis. The patients included 36 men and 12 women ranging from 39 to 84 years of age . Five patients were positive for the serum hepatitis B surface antigen (HBsAg), and seven were positive for the anti-hepatitis C virus (HCV) antibody; one was positive for both, and 35 were negative for both. The largest tumor diameters ranged from 18 mm to 220 mm . CLCs and mixed cholangiohepatocellular carcinomas were excluded from the analysis. ICC was grossly classified into three groups: mass-forming (MF), periductal-infiltrating (PI), and intraductal-growth (IG), as well as mixed types (MF+PI and IG+PI). Based on surgical findings and macroscopic examination, the tumors were defined as peripheral localization types involving the septal and interlobular bile ducts, or as non-peripheral central localization types involving the first to third-order branches of the right and left hepatic bile ducts. The histological differentiation grades for ICC were assigned according to World Health Organization classifications [Patient follow-up was completed on 31 March 2017. The median follow-up period was 32.2 months (range: 1–109 months). All of the patients that were followed at Tokyo Medical and Dental University Hospital were monitored postoperatively using chest-computed tomography (CT) and abdominal contrast-enhanced CT every 1 to 12 months, depending on the postoperative time. OS was defined as the period from surgery to death or the last observation point.This study was approved by the ethics committee of Tokyo Medical and Dental University .2O2 in distilled water for 5 min. The slides were incubated with primary antibodies for 30 min at room temperature, followed by incubation with the secondary antibody and detection using an EnVision FLEX  according to the manufacturer’s protocol, and counterstained with hematoxylin.Immunohistochemistry analysis was performed using monoclonal and polyclonal antibodies . ParaffiImmunofluorescence staining for β6 integrin and tenascin-C was performed on formalin-fixed, paraffin-embedded tissue sections (4 μm thick) of selected cases with representative histopathological types and immunohistochemical results. Tissue sections were blocked using the streptavidin/biotin blocking kit  for 15 min at room temperature, respectively, and incubated with anti-β6 antibody for 60 min at room temperature. The slides were washed with 0.1% Tween phosphate-buffered saline (PBS), and incubated with the biotinylated anti-mouse IgG (Vector). After being washed with 0.1% Tween PBS, the slides were incubated with the DyLight 488-labeled streptavidin (Vector) (green color), and diluted 1:100 in PBS for 5 min at room temperature. Furthermore, serial tissue sections of the same selected cases were stained for immunofluorescence analysis of tenascin-C, using an anti-tenascin antibody as the primary antibody, and DyLight 549-labeled streptavidin (Vector) for red immunofluorescence detection.For double immunofluorescence of β4 integrin and laminin-5, rabbit polyclonal anti-β4 antibody was used as the primary antibody, and biotinylated anti-rabbit IgG (Vector) was used as the secondary antibody in the first layer of the staining with green DyLight 488-labeling, and mouse monoclonal anti-laminin-5 antibody was used as the primary antibody and biotinylated anti-mouse IgG (Vector) was used as the secondary antibody in the second layer with red DyLight 549-labeling; these were then applied on the same tissue sections. The fixation, antigen retrieval, blocking, and washing steps were conducted as described above. Immunofluorescence was visualized using the Keyence BZ-X710 microscope .The intensity of β4 and β6 integrin expressions was semi-quantitatively scored as 0 (negative), 1 (weak), 2 (moderate), or 3 (strong). The percentages of cells or areas positive for β4 or β6 were semi-quantitatively scored into five categories: 0 (<1%), 1 (1–25%), 2 (26–50%), 3 (51–75%), or 4 (76–100%). The relationships between β4 or β6 integrin expressions and clinicopathological parameters were evaluated by using a final score, which was calculated by multiplying the scores of staining intensities by the scores for the percentages of positive cells. Based on the final score, the cases were grouped as negative (a score of 0), low (1–4), and high (5–12). Mucin production was assessed by using Alcian blue staining (pH 2.5), and defined as positive for >10% positivity within glandular lumens or cytoplasm. Epithelial membrane antigen (EMA) expression patterns were categorized as dominantly luminal or cytoplasmic in the positive areas. The percentages of positive cells and stromal areas for laminin-5, tenascin-C, TGF-β1, and α-SMA were scored into five categories: 0 (<1%), 1 (1–25%), 2 (26–50%), 3 (51–75%), and 4 (76–100%). Negative was defined as category 0, focally positive was defined as category 1, and definitely positive was defined as >25% (more than two in the category) of tumor cells or areas positive for cytoplasmic and stromal staining.χ2 and Student’s t-tests. Survival analysis based on the Kaplan–Meier method and log-rank tests were used to assess the survival data. The correlations between TGF-β1 and α-SMA expressions and β6 integrin expression in ICC were analyzed based on Spearman’s rank correlation coefficient. Data are presented as means ± standard deviations. p values < 0.05 were considered significant.The correlations among the clinicopathological findings; mucin production; EMA, laminin-5 and tenascin-C expressions; β4 and β6 integrin expression, and laminin-5, tenascin-C, and TGF-β1 expression in ICC were assessed using Fisher’s exact or The present study showed, for the first time, the different levels of β4 and β6 integrin expression in ICC subgroups, and suggested that integrin expression is associated with the subclassification and clinicopathological features of ICC through the coincident expression of laminin-5 and tenascin-C, as well as of TGF-β1.


Claudins are a family of more than 20 subtypes, but our knowledge about expression and localization of each subtype in the cochlea is limited.Claudins are major components of tight junctions, which form the paracellular barrier between the cochlear luminal and abluminal fluid compartments that supports the large transepithelial voltage difference and the large concentration differences of KCldn-16, − 17 and − 18) were not detected. Claudins that increased expression during development include Cldn-9, − 13, − 14, − 15, and -19v2, while Cldn-6 decreased. Those that do not change expression level during postnatal development include Cldn-1, − 2, − 3, − 4, − 5, − 7, − 8, −10v1, −10v2, − 11, − 12, −19v1, − 20, − 22, and − 23. Our investigation revealed unique localization of some claudins. In particular, Cldn-13 expression rapidly increases during early development and is mainly expressed in bone but only minimally in the lateral wall (including stria vascularis) and in the medial region (including the organ of Corti). No statistically significant changes in expression of Cldn-11, − 13, or − 14 were found in the cochlea of Slc26a4−/− mice compared to Slc26a4+/− mice.We examined by quantitative RT-PCR the expression of the mRNA of 24 claudin isoforms in mouse cochlea during postnatal development and localized the expression in separated fractions of the cochlea. Transcripts of 21 claudin isoforms were detected at all ages, while 3 isoforms  contains supplementary material, which is available to authorized users. Tight junctions are structures consisting of proteins that join epithelial and endothelial cells to form continuous sheets and tubules which separate two liquid compartments. They consist of claudins , occludiClaudins are a family of more than 20 subtypes . The speCldn-11 knockout mice [Cldn-14 led to degeneration of a different cochlear structure, the sensory organ of Corti, and was associated with the human hereditary deafness DFNB29 [Cldn-1 to Cldn-18 using immunohistochemistry, but their study was limited by a lack of antibodies for some claudins. We localized in the current study transcript expression of most of the claudin isoforms in multiple tissue fractions of the cochlea, including the outer layer of cochlear bone.Claudins are known to be critical for normal hearing , 7. A maout mice . In conts DFNB29 . It is ts DFNB29 . Kitajirs DFNB29  examinedCldn-13, observed in the present study to be predominantly expressed in the outer bone fraction, would be altered by deletion of the Slc26a4 gene in the mouse model.One of the most common hereditary deafness genes is pendrin (SLC26A4), which has been surprisingly shown to exert its strongest effects on cochlear function by expression in the endolymphatic sac during early development . Lack ofSlc26a4 knockout on claudin isoform expression.The aim in the present study was to determine a) expression of claudin transcripts during early development, b) localization of the claudin isoforms among the cochlear regions and c) the potential effects of Slc26a4+/− and Slc26a4−/− mice were obtained from a colony at Kansas State university and the heterozygous mice served as controls. Animals were deeply anesthetized with sodium pentobarbital (100 mg/kg i.p.). Temporal bones were removed from both male and female mice and whole cochleae were collected from age-sex matched littermates of Slc26a4+/− and Slc26a4−/−. Expression of claudins was determined on RNA isolated from a) whole cochlea, b) cochlear lateral wall tissues, c) cochlear medial fraction tissues, and d) outer cochlear bone. Lateral wall tissues were further microdissected into spiral ligament and stria vascularis fractions, while the medial fraction was further microdissected into organ of Corti and modiolus fractions. All procedures involving animals were approved by the Institutional Animal Care and Use Committee of Kansas State University (protocol 2925).Total RNA was isolated from these tissues using the RNeasy Micro Kit  and care was taken that RNA was extracted from all cell types. Recombinant bovine DNase I, Grade 1  was used to remove residual DNA. The quality and quantity of 18S rRNA were determined by using the RNA 6000 Nano Kit  with a Bioanalyzer  and a spectrophotometer . The amount of RNA in each sample was calculated as the average of the results of the Bioanalyzer and NanoDrop assays.Cldn-1 to − 23 (excluding Cldn-21) were designed using software Primer3 (http://primer3.sourceforge.net/). The sequences of primers are documented in Table Primer pairs for mouse P values of < 0.05 were considered as significant differences; analyses were performed with SigmaStat for Windows Version 4.0 software.Data are given as means ± standard deviation (SD) or ± standard error of the mean (SEM), as reported in Results. N values refer to the number of cochleae Figs.  and 4 orSlc26a4+/−) mice at three different ages after birth: P2 and P6 before the onset of endocochlear potential generation and hearing in mice, and at P15, after acquisition of hearing. Transcripts of 21 claudin isoforms were detected at all ages, while 3 isoforms  were not detected  lateral wall (exclusive of outer bone), 2) medial region, and 3) outer bone. These fractions were assayed for 12 claudin isoforms: Cldn-5, − 6, − 7, −10v1, −10v2, − 13, − 15, −19v1, −19v2, − 20, − 22, and − 23. All of these claudins were detected both in the lateral wall and the medial region fraction. Cldn-19v1 and Cldn-19v2 were expressed most strongly in the medial region.The cochlear tissues expressing these claudins were resolved in two subsequent experimental series. In the first series Fig. , cochleaCldn-13 was expressed virtually exclusively in the outer bone fraction, in spite of the statistically significant difference in the minimal expression in the two soft tissues. Wongdee et al. examined claudin expression in skull and tibia bone [Cldn-5, − 11, − 14, − 15 and − 16. The expression was limited to the cells lining the bone (periostieum), suggesting a function of claudin other than tight junction formation. They, however, did not test bone for the presence of Cldn-13. Johnson et al. reported Cldn-13 expression in G1E cells, a proerythroblastic cell line [Cldn-13 was identified in a stress induced erythropoiesis pathway that is mainly expressed in tissues associated with haematopoietic function [Cldn-13 in cochlear outer bone might originate from the associated bone marrow, which develops during the early postnatal period [Slc26a4−/− mice exhibit delayed bone marrow maturation between P6 and P15 [Cldn-13 does not have a human homolog [Interestingly, bia bone  and deteell line  and Cldnfunction . It is tl period . In supp and P15 . Mouse CSlc26a4+/− mice were subdivided further into four micro-dissected fractions: 1) the stria vascularis and 2) spiral ligament fractions were separated from the lateral wall; 3) the organ of Corti and the 4) modiolus were separated from the medial structures. Claudins in the lateral wall and medial fractions that gave high expression signals (claudin mRNA/18S > 4.5) in the first experimental series  and modiolus (neurons). By contrast, the other six isoforms did not show statistically significant differences between the epithelial fractions and their respective adjacent non-epithelial fractions. Non-significant comparisons are not shown in Fig. In the second series, cochleae of adult  ies Fig.  were anaies Fig. . The epiSlc26a4 gene deletion on inner ear developmental expression of claudins. Developmental expression of the three isoforms demonstrated a dramatic postnatal increase in Cldn-13 that was not characteristic of the other two claudins, consistent with the notion that Cldn-13 is not regulated by a mechanism common to the claudins highly expressed in the epithelial tissues. We examined RNA from whole cochleae from age- and sex-matched littermates of Slc26a4+/− and Slc26a4−/− and analyzed by two-way ANOVA 1) Cldn-11, which is expressed in basal cells of stria vascularis [Cldn-13, which is expressed in cochlear outer bone (this report), and 3) Cldn-14, which is expressed in organ of Corti and is responsible for human hereditary deafness DFNB29. The results of analysis (Fig. Three claudins were selected to investigate the possible effect of scularis , and whoCldn-21” was not included in this study. The nomenclature has varied and developed since 2001 and was not identified in mouse at the time of this study [Cldn-21 [+-selective paracellular transport pathway [“is study . The mou[Cldn-21 , 26 has Cldn-5, − 6 and − 15 was not detected by Kitajiri et al. [Cldn-5 and -15 are expressed in endothelial cells, not epithelial cells [Cldn-5 and Cldn-15 in stria vascularis nor spiral ligament, both highly vascularized tissues. We found Cldn-6 expression, but it gradually decreased during early development. Consistent with our observation, Kitajiri et al. [Cldn-6 in the adult cochlea.Some of our data differ from previous observations: expression of i et al. , but weral cells . By contal cells  reportedi et al.  did not Cldn-7, Cldn-13, Cldn-19 to − 23 in the cochlea, but the results of our study showed regional localization of transcripts of these genes in the cochlea and developmental changes in two of them. We observed that Cldn-13 is expressed in bone and that its expression increased rapidly during early postnatal development. Most of the claudins were expressed in stria vascularis and organ of Corti, tissue fractions rich in tight junctions. However, this study suggests a novel function of Cldn-13 in the cochlea, which may be linked to cochlear bone marrow maturation.We analyzed 24 claudins in structures of the inner ear. Previous studies did not show the presence and localization of


Many thermophiles thriving in a natural high-temperature environment remain uncultivated, and their ecophysiological functions in the biogeochemical cycle remain unclear. In the present study, we performed long-term continuous cultivation at 65°C and 70°C using a microbial mat sample, collected from a subsurface geothermal stream, as the inoculum, and reconstructed the whole genome of the maintained populations using metagenomics. Some metagenome-assembled genomes (MAGs), affiliated into phylum-level bacterial and archaeal clades without cultivated representatives, contained genes involved in nitrogen metabolism including nitrification and denitrification. Our results show genetic components and their potential interactions for the biogeochemical nitrogen cycle in a subsurface geothermal environment. Phylogenetically diverse, uncultivated thermophiles are present in natural high-temperature environments, such as terrestrial hot springs and deep-sea hydrothermal vents, as suggested by 16S rRNA gene analyses , 16, 32.4+ (>100 μM) and Fe2+ (>70 μM) (Candidatus (Ca.) “Nitrosocaldus”, “Aigarchaeota”, and “Acetothermia” in the stream and inoculated mat sample  , 24, 33.t sample . The MAGt sample . Althouganalysis . Geochem 2 years . These f50 value of 10,355 bp. Binning using longer contigs (≥2.5 kbp) and manual curation resulted in a total of 41 MAGs  to the environmental clone Papm3A43 recovered from deep-sea hydrothermal fluid , hydroxylamine oxidoreductase (Hao), and nitrite oxidoreductase (Nxr), assimilatory and dissimilatory nitrate reduction by nitrate and nitrite reductases (Nap/Nar/Nas/Nir), denitrification by nitric oxide reductase (Nor) and nitrous oxide reductase (Nos), and nitrogen fixation by nitrogenase (Nif), we focused on genes for the key subunits of the above enzymes .amoABC genes were found in the “Nitrosocaldus” MAGs (HR04 and HR05), but not in any of the other MAGs or unbinned contigs. These amo genes have also been detected in enrichment cultures of “Nitrosocaldus yellowstonii” , and were also absent in the previously reported MAGs and SAGs of deeper-branching thaumarchaeotic lineages than “Nitrosocaldus”  operated at 70°C for more than 4 years  including “Aigarchaeota”, “Fischerbacteria”, and “Dadabacteria” contained nosZ. Thus, they may produce N2 from N2O, which may be produced abiotically from NH2OH and NO  may be primary producers in the microcosm as suggested previously , which was in media at a final concentration of approximately 100 μM to simulate the chemical composition of geothermal groundwater at the sampling site, may be another potential energy source for the population. The “Fischerbacteria” and Chlorobi MAGs (HR11 and HR21) contained a homolog for Cyc2  oxidation in several iron oxidizers, such as Acidithiobacillus spp., Ferriphaselus spp., Gallionella spp., and Mariprofundus spp. (rbcSL in the Calvin cycle for carbon fixation was found in HR11. These results imply that the bacterium derived from the “Fischerbacteria” MAG is potentially an iron-oxidizing autotroph and has a role as a primary producer in the microcosm.In addition to nitrogen metabolism, other potential metabolic functions of MAGs, such as carbon fixation and energy acquisition, are summarized in 0 μM 12; . The ammfor Cyc2 , an outedus spp. , 19. MorThe present study revealed the unique physiological functions and ecological roles of diverse, previously uncultivated thermophiles in the nitrogen cycle in a subsurface geothermal environment. These results provide important information for the isolation of novel MAG-derived thermophiles, and will contribute to the characterization and elucidation of their ecophysiologies. Further cultivation-based metagenomics for a number of high-temperature environments with different pH, salinities, redox potentials, and energy sources may reveal uncultivated thermophiles and unidentified thermophilic microbial ecosystems.


Callosobruchus chinensis and Callosobruchus maculatus). Breeding bruchid-resistant mungbean is a major goal in mungbean breeding programs. Recently, we demonstrated in mungbean (Vigna radiata) accession V2802 that VrPGIP2, which encodes a polygalacturonase inhibiting protein (PGIP), is the Br locus responsible for resistance to C. chinensis and C. maculatus. In this study, mapping in mungbean accession V2709 using a BC11F2 population of 355 individuals revealed that a single major quantitative trait locus, which controlled resistance to both C. chinensis and C. maculatus, was located in a 237.35 Kb region of mungbean chromosome 5 that contained eight annotated genes, including VrPGIP1 (LOC106760236) and VrPGIP2 (LOC106760237). VrPGIP1 and VrPGIP2 are located next to each other and are only 27.56 Kb apart. Sequencing VrPGIP1 and VrPGIP2 in “V2709” revealed new alleles for both VrPGIP1 and VrPGIP2, named VrPGIP1-1 and VrPGIP2-2, respectively. VrPGIP2-2 has one single nucleotide polymorphism (SNP) at position 554 of wild type VrPGIP2. This SNP is a guanine to cystine substitution and causes a proline to arginine change at residue 185 in the VrPGIP2 of “V2709”. VrPGIP1-1 has 43 SNPs compared with wild type and “V2802”, and 20 cause amino acid changes in VrPGIP1. One change is threonine to proline at residue 185 in VrPGIP1, which is the same as in VrPGIP2. Sequence alignments of VrPGIP2 and VrPGIP1 from “V2709” with common bean (Phaseolus vulgaris) PGIP2 revealed that residue 185 in VrPGIP2 and VrPGIP1 contributes to the secondary structures of proteins that affect interactions between PGIP and polygalacturonase, and that some amino acid changes in VrPGIP1 also affect interactions between PGIP and polygalacturonase. Thus, tightly linked VrPGIP1 and VrPGIP2 are the likely genes at the Br locus that confer bruchid resistance in mungbean “V2709”.Nearly all mungbean cultivars are completely susceptible to seed bruchids ( Vigna radiata (L.) Wilczek] is an important legume crop of Asia. It is cultivated on ~6 million hectares, mainly in Asia  and cowpea weevil  are the major bruchid species that feed on seeds of mungbean and other food legumes, including cowpea (Vigna unguiculata L.), black gram [Vigna mungo (L.) Hepper], Bambara groundnut [Vigna subterranea (L.) Verdc.], azuki bean [Vigna angularis (Willd.) Ohwi and Ohashi], soybean [Glycine max (L.) Merr.], chickpea (Cicer arietinum L.) and pigeon pea [Cajanus cajan (L.) Millsp.]. Although C. chinensis and C. maculatus originated in Asia and Africa, respectively, they are currently present in almost every continent owing to international seed/grain trading. Generally, bruchids are controlled by chemical fumigation and dusting. However, these chemicals are not only harmful to humans and the environment, but also increase production costs. Additionally, chemical control is not practical for small-landholder farmers. Thus, a major goal in mungbean breeding programs is to develop bruchid-resistant cultivar(s)  are serious pests that damage legume seeds after harvest. Bruchids infest seeds in the field and after harvest, and the latter can results in total loss of a seed lot within 3–4 months  accession TC1966 is completely resistant to C. chinensis and C. maculatus. Talekar and Lin  markers Bng110 and Bng143. The Br gene is only 0.2 cM away from the Bng143. Chotechung et al.  was probably responsible for the bruchid resistance. They also showed that “V2802”, “V1128”, “V2817”, and “TC1966” had the same VrPGIP2 allele.Kitamura et al.  showed ta et al.  demonstrIshimoto  fine mapg et al.  found thg et al.  narrowedVrPGIP2 sequence, did not reveal a polymorphism between “V2709” and bruchid-susceptible mungbean “Kamphaeng Saen 1”  fine map the Br locus in “V2709”, (ii) identify candidate gene(s) for the Br locus in “V2709” and (iii) identify mutation(s) responsible for the resistance.In this study, we report the identification of new alleles for bruchid resistance in mungbean accession 11F2 mapping population was developed from “KPS1” and “V2709”  method  of 1- to 3-day-old bruchids were introduced into the box, kept for 7 day for egg laying, and then removed from the box. Parental seeds were tested and replicated five times. The seeds were maintained at 28°C and 70% relative humidity. Then, 60 day after insect introduction, the numbers of damaged seeds were counted and the percentages calculated.Cultures of a et al.  with min11F2 plants were classified into two classes; plants with 0–80%-damaged seeds were classified as resistant, which included the homozygous resistant genotype  and heterozygous resistant genotype , while plants with 81–100%-damaged seeds were classified as the homozygous susceptible genotype  test was conducted to determine a 3 (resistance):1 (susceptible) goodness of fit using software R-program 2.0.10  was 0.001.The QTL for bruchid resistance was mapped using the inclusive composite interval mapping (ICIM) method /Br_03940_R13 (5′-CTCAACTTGGTTAATGATGCTTA-3′), and in “V2709” using the primer pair Br_03940_F1 (5′-CGACTAGCACCGGAAATTA-3′)/Br_03940_R14 (5′- ACACTGCACACGTGTCAAA−3′). PCR amplification and sequencing protocols were the same as those for VrPGIP2. NCBI nucleotide accession numbers for VrPGIP1 and VrPGIP2 are MF398961 and MF398954, respectively, for “KPS1”, MF496138 and MF398959, respectively, for “V2709”, and MF398960 and MF398955, respectively, for “V2802”.g et al. . VrPGIP1VrPGIP1 and VrPGIP2 from “KPS1”, “V2709”, and “V2802” were aligned using ClustalW , which is the only PGIP that has a known crystal structure  [CaPGIP1 (XP_004504732.1) and CaPGIP2 (XP_012572409.1)], common bean (Phaseolus vulgaris L.) , soybean (G. max Merr.)  and Medicago truncatula  were subjected to phylogenic analysis using software Phylogeny.fr .VrPGIP1 and VrPGIP2 sequences and PGIP sequences of chickpea  between the percentages of damaged seeds caused by the two bruchid species, suggesting that the resistance to C. chinensis and to C. maculatus in “V2709” is controlled by the same gene(s). The frequency distribution of the percentage of damaged seeds was nearly discontinuous and bimodal :1 (susceptible) ratio . This result contrasted previous findings reported by Somta et al.  and a single major QTL for C. maculatus (qBrm5.1) were detected by ICIM  from “V2709” decreased the seed damage caused by bruchids. Because qBrc5.1 and qBrm5.1 were at the same position and conferred similar genetic effects for the resistance, we considered them as the same locus and named it “qBr5.1”.A single major QTL for is qBrc5. and a siIM Table . qBrc5.1qBr5.1 was between markers VRID5 and VrBr-SSR037, covering ~237.35 Kb , in which there were eight annotated protein-encoding genes, including VrPGIP1 (LOC106760236) and VrPGIP2 (LOC106760237), based on the annotation by NCBI  between the two mungbean accessions. The SNPs at positions 958, 995 and 1,003 caused amino acid changes in VrPGIP2 at positions 320, 332, and 335 in “V2802” .n Figure . The nucn Figure . There wn Figure . We desiVrPGIP1 was annotated by Kang et al. (VrPGIP1 (LOC106760236) is intronless, has 1,011 nucleotides and encodes a 336-amino acid protein, which is the same as VrPGIP2. We conducted Sanger sequencing of this gene in “KPS1”, “V2709”, and “V2802”, and successfully obtained the ORF sequences for “KPS1” and “V2802” but we only obtained a continuous 960-bp sequence (including start codon) for “V2709”. We obtained the full sequence of VrPGIP1 from our in-house whole genome sequence (WGS) data of “V2709”. However, 100% of the partial VrPGIP1 sequence of “V2709” obtained from the Sanger sequencing was identical to the full VrPGIP1 sequence generated from the WGS (data not shown). This indicated the high accuracy of the VrPGIP1 sequence in “V2709” generated by the WGS. A sequence alignment of VrPGIP2 genes from “V2709”, “V2802”, “KPS1” and the reference sequence (“VC1973A”) showed that the sequences from “KPS1”, “VC1973A”, and “V2802” were identical and that there were 43 SNPs between “V2709” and “KPS1”  of bruchids that degrade pectin in the seeds, producing an energy source for the bruchids and helping other digestive enzymes to gain access to their substrates in bruchid food  covered ~237.35 Kb  with eight protein-encoding genes, including VrPGIP1 and VrPGIP2 . The VrPGIP2 encoded by VrPGIP2-2 possessed an amino acid change at the position 185, as compared with the wild type protein    is common and it is far from the QTLs in both studies. Although the RP marker that is closely linked to a QTL reported by Hong et al. . Three forms of PGs were predicted from LOC106759697. A BLASTN algorithm-based search revealed that the gene was also most similar to a gene from azuki bean  that encodes a RESPONSIVE TO DEHYDRATION22 (RD22)-like protein of the BURP family. The RD22 protein is responsive to abiotic stresses, such as drought and salinity  with durable resistance to bruchids by pyramiding or rotating the VrPGIP1 and VrPGIP2 resistance genes.In addition to g et al. . BURP dog et al. . Based og et al. , g39185 AnK and AlK carried out DNA marker analysis and data analysis. PrS, AnK, TY, and PeS prepared plant materials and conducted insect resistance evaluation. JC, XY, and XC performed DNA sequencing, and analyzed sequencing results. PrS initiated and coordinated the study, and analyzed the results. AnK, JC, PrS, XC, and PeS wrote the manuscript. All authors approved 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.


Loosening of screws is a common problem in orthopedic and maxillofacial surgery. Modifying the implant surface to improve the mechanical strength of screws has been tried and reported. We developed screws coated with fibroblast growth factor-2 (FGF-2)−apatite composite layers. We then showed, in a percutaneous external fixation model, that this composite layer had the ability to hold and release FGF-2 slowly, thereby reducing the risk of pin tract infection of the percutaneous external fixation. The objective of the current study was to clarify the effect of FGF-2−apatite composite layers on titanium screws on bone formation around the screw.We analyzed samples of previously performed animal experiments. The screws were coated with FGF-2−apatite composite layers by immersing them in supersaturated calcium phosphate solutions containing FGF-2. Then, the uncoated, apatite-coated, and FGF-2−apatite composite layer-coated screws were implanted percutaneously in rabbits. Finally, using inflammation-free histological sections, we histomorphometrically assessed them for the presence of bone formation. Weibull plot analysis was then applied to the data.-4 for screws coated with the FGF-2−apatite composite layers versus 0.05 for screws in the absence of FGF-2.On average, screws coated with FGF-2−apatite composite layers showed a significantly higher bone apposition rate than the uncoated or apatite-coated screws. Although the difference in the average bone apposition rate was small, the FGF-2−apatite composite layers produced a significant, marked reduction in the incidence of impaired bone formation around the screw compared with the incidence in the absence of FGF-2 (uncoated and apatite-coated screws). The probability of resulting in a bone apposition rate equal to or less than 63.75%, for example, is 3.5 × 10FGF-2-apatite composite layer coating significantly reduced the risk of impaired bone apposition to the screw. Thus, it is feasible to use titanium screws coated with FGF-2−apatite composite layers as internal fixation screws to decrease the risk of loosening. Loosening of screws is a severe clinical problem in orthopedic and maxillofacial surgery, and it could be exacerbated in patients with compromised bone quality –3. Screw2−strontium−CaSiO3−biopolymer composite, acid-etching, zinc-modified Ca−Si ceramic, and CaTiSiO5 ceramic  gives a straight line with a slope of m. In this study, σ is the bone apposition rate, and S is the probability of resulting in a bone apposition rate at or less than σ. The measured σ values were arranged in ascending order. S was derived from the average rank method using Sj = j/(N + 1), where j is an order number of an individual σ value, and N is the total number of measured σ values.where t test was used to test statistically significant differences in the length of the bone−screw interface line, the screw’s peripheral line, the bone apposition rate and the slope of the regression line of the Weibull plot. The F test was used to test a statistically significant difference in dispersion in the frequency histogram. A value of p < 0.05 was considered to indicate statistical significance for each analysis.Student’s p = 0.017)  while there was no significant difference in the length of screw’s peripheral line between FGF(+) (49.2 ± 9.4 mm) and FGF(−) (46.8 ± 10.1 mm) (p = 0.30) (Table p < 0.0001). Thus, qualitatively, the FGF(+) group resulted in a lower incidence of impaired bone formation around screws than the FGF(−) group.The bone apposition rate was significantly higher for FGF(+) (88.6 ± 4.4%) than for FGF(−) 83.0 ± 9.5%) (17) Fig. . The bon.0 ± 9.5%p = 7.3 × 10−34). Using the regression lines, the risk of impaired bone formation was calculated at some threshold vales of bone apposition rate which were selected in an arbitrary manner (Table −4 and 0.05 for FGF(+) and FGF(−), respectively. In the same manner, if impaired bone formation is defined as a state at ≤68.82% of the bone apposition rate, the probabilities of impaired bone formation are 0.002 and 0.1 for FGF(+) and FGF(−), respectively. Thus, the risk of impaired bone formation is remarkably lower for FGF(+) than for FGF(−).We used Weibull plot analysis to analyze the risk of impaired bone formation quantitatively Fig. . The WeiFGF(+) had a significantly higher bone apposition rate and lower incidence of impaired bone formation around the screws than FGF(−) in inflammation-free histological sections obtained by percutaneous implantation of external fixation screws. Because there were individual differences in bone-forming ability among the animals with an implanted screw, we recorded and compared the ranges of the bone apposition rates for the FGF(+) and FGF(−) groups Fig. .p = 0.017)  than for FGF(−), with significant difference (17) Fig. . The dif17) Fig. . The freThe sharp contrast in the broadness of the frequency distribution suggested that a small number of animals with less-strong bone-forming ability were randomly assigned to both the FGF(+) and FGF(−) groups. In addition, FGF-2 enhanced the bone formation of only those animals with less-strong bone-forming ability in the FGF(+) group. FGF-2 is known to exhibit a bell-shaped dose response, where an excess of FGF-2 has less or no enhancement effect on bone formation , 14. AniS) of resulting in a bone apposition rate equal to or less than a specific value of σ. For example, if the specific values are set at 68.82, 63.75, and 50%, the probabilities of resulting in a bone apposition rate equal to or less than 68.82, 63.75, and 50% are 0.1, 0.05, and 0.005, respectively, for FGF(−). For FGF(+), the probabilities for the same specific values are 0.002, 3.5 × 10−4, and 1.4 × 10−6  increases when the specific value of the bone apposition rate decreases. The thresholds of ≤68.82%, ≤63.75%, and ≤50% correspond to bottom 0.5%, 5%, and 10%, respectively, of FGF(−) samples in the rank of bone apposition rate. Thus, FGF(+) was more reliable than FGF(−) in terms of preventing impaired bone apposition regardless of the individual’s bone-forming ability. The decreased risk is not a result of the apatite, but rather the presence of FGF-2 in the FGF-2−apatite composite layer, as the slopes for Ti and 25F0 (apatite-coated Ti) were similar and significantly lower than those for 25F0.5, 25F1, 25F2, and 37F4  than for FGF(−), as revealed by the Weibull plot analysis Table . The WeiIt is feasible to apply the FGF-2−apatite composite layer to internal fixation screws, which have varied stability at the screw–bone interface depending on the quantity, quality, and regeneration of bone surrounding the screw –32. The In the present study, the use of the Weibull plot analysis provided new insights into the effect of FGF-2 on bone formation. The previously reported effects of FGF-2 on increasing bone formation were equivocal. Some authors reported increased effects, whereas others reported no effect or only a limited effect –40. All Limitations of the present study are that (i) the FGF-2−apatite composite layers that were prepared under different conditions are involved in the analysis, (ii) the number of inflammation-free histological sections in each group was small and varied greatly, and (iii) no pull-out strength data are presented.−4/0.05) with the use of FGF−apatite composite layer coating compared with uncoated and apatite-coated screws. Therefore, FGF-2−apatite composite layers are feasible for coating internal fixation screws, as it is known that bone quality, quantity, and formation around the screw is crucial to prevent loosening of internal fixation screws. Further study is required.Histomorphometrical analysis of inflammation-free histological sections obtained using percutaneous implantation of external fixation screws revealed that, on average, screws coated with FGF-2−apatite composite layers showed a slightly higher bone apposition rate, with a significant difference from the uncoated and apatite-coated screws. However, FGF-2−apatite composite layers have a much more marked effect on reducing the incidence of impaired bone apposition than on enhancing the bone apposition rate. The Weibull plot analysis revealed that the risk of resulting in less than 63.75%, for example, in bone apposition rate was reduced by 1/142 times (3.5 × 10Animal studies that include implanting internal fixation screws, measuring local bone formation, and determining the pull-out strength of the screw are required in combination with the Weibull plot analysis. These studies could further clarify whether the FGF-2−apatite composite layers reduce the risk of screw loosening.


The high-throughput sequencing of pear “Dangshan Su” × “Yali”  and pear “Dangshan Su” × “Wonhwang”  found that the expressions of these two miRNAs (pyr-1809 and pyr-novel-miR-144-3p) were significantly different; their corresponding target genes encode two kinds of laccase (Pbr018935.1 and Pbr003857.1). qRT-PCR results showed that these two enzymes are involved in the formation of lignin and stone cells and the existence of these two miRNAs has a negative effect on them. It was concluded that the effect of pollination on the development of stone cells may affect the synthesis of lignin, through the regulation of laccase controlled by miRNAs, and ultimately affect the formation of stone cell and fruit quality. Capra hircus, Salmo salar, and Plutella xylostella  [∗ (passenger strand) is often degraded [ Prunus persica. In Populus trichocarpa [MicroRNAs (miRNAs) are small, endogenous, noncoding 20–25 nucleotide (nt) small RNAs , 2 that se.org/) , 9. Plandegraded –6. A numdegraded –12, secodegraded , 14. Kajdegraded  reporteddegraded  validatechocarpa , ptr-miRPyrus bretschneideri cv. Dangshan Su is a Chinese pear variety, widely grown across China and other Asian countries [ountries . It is sountries . In receountries .P. bretschneideri Rehd.) as male parent, whose fruit lignin and stone cell content are high and quality is poor, while the fruits with low content of lignin and stone cell have better quality, using “Wonhwang” (P. pyrifolia Nakai) as the male parent. Thus, in this study, we used “Yali” and “Wonhwang” to pollinate “Dangshan Su” and built “Dangshan Su” microRNA expression profiles under different pollen donators using Solexa high-throughput sequencing technology. With bioinformatics method, the xenia phenomenon effect on “Dangshan Su” microRNA expression levels was analyzed to predict differently expressed microRNAs and their corresponding target genes. Furthermore, the lignin metabolism-related enzyme genes and its corresponding microRNA expression changes were determined using Poly (T) method and real-time fluorescent quantitative PCR (qRT-PCR) assay at different developmental stages of “Dangshan Su” × “Yali” and “Dangshan Su” × “Wonhwang”, respectively. The above researches can lay a foundation for the regulation of lignin and stone cell formation and improve the quality of pear fruit by exploring the related miRNA which control the lignin synthesis and stone cell development, which also provide a certain theoretical basis and technical methods for subsequent research on xenia affecting fruit quality by regulating miRNA expression in which way. These results also could provide an important theoretical basis for the screening of suitable “Dangshan Su” pollinated varieties and the improvement of “Dangshan Su” fruit quality.In recent years, it has been reported that miRNAs were widely involved in the regulation of pear fruit development and fruit quality, via sugar and acid metabolism and hormone signaling . And no P. bretschneideri Rehd.) and “Wonhwang” (P. pyrifolia Nakai) male parents. Pollens were collected from “Yali” and “Wonhwang” flower androecia and used to manually pollinate “Dangshan Su,” respectively. At the end of pollination processing, flowers were covered with bags for seven days. Pear fruits of different male parent pollination were collected after pollination at 23 days after flowering and the samples were taken every 8 days  and stored at −80°C in our laboratory at Anhui Agricultural University, China.Fruits were obtained from 50-year-old pear trees grown on an orchard localized at Dangshan County, Anhui, China. The “Dangshan Su” female parents were fertilized with pollens from “Yali”  following the manufacturer's instructions. All RNA samples were examined for protein contamination (A260/A280 ratios) and reagent contamination (A260/A230 ratios) by using a Nanodrop ND 1000 spectrophotometer . Before library construction, the RNAs of the “DangshanSu” × “Yali” (DangsuA) or “DangshanSu” × “Wonhwang” (DangsuB) were pooled together to form sample groups, respectively.For library construction, total RNA was firstly resolved on denatured 15% polyacrylamide gels and 18–30 nt gel fragments were excised. A pair of adaptors was ligated to both ends of the eluted small RNA fragments. Subsequently, the adapter-ligated small RNAs were reversely transcribed into cDNA with Super-Script II Reverse Transcriptase (Invitrogen) and amplified by PCR. Finally, the resulting library was quantified and sequenced by using the Illumina HiSeq 2000 platform .For the raw data, all low quality reads, such as reads with adapter contaminants or poly A sequences, were filtered. After sequencing, the sequences that are longer than 30 nt or shorter than 18 nt and without adapters were removed, the remaining ones were used for subsequent analysis.http://www.sanger.ac.uk/Software/Rfm/). Reads aligned in the genome, excluding those matching tRNAs, rRNAs, snRNA, and snoRNAs, were used for further analysis. Mature miRNAs and their precursors were retrieved from miRBase  [The clean sequencing data were mapped into two genome and Rfam database v11 (ase.org) , 25.The remaining reads were applied to predicting novel miRNAs and quantitative analysis using the miRDeep2 with the parameters for plants described in miREvo. To assess the reliability of predicted novel microRNAs, their MFEI (minimum folding free energy index) were calculated to compare with the known microRNAs –28.The frequency of microRNAs from different libraries was normalized by total clean reads of microRNAs in each sample group. If the normalized read count of a given microRNA is zero, the expression value was modified to 1 for further analysis.http://peargenome.njau.edu.cn/) [The expression differences between small RNA libraries of both samples were determined using EdgeR package with R language. Then, tools named “TAPIR” and “TargetFinder” were used for the microRNAs target prediction, which was evaluated with a better prediction performance . The taredu.cn/) .p value < 0.05, comparing total transcripts. This method firstly maps all target gene candidates to GO terms in the database (http://www.geneontology.org/), calculating gene numbers for each term, then using hypergeometric test to find significantly enriched GO terms in target gene candidates comparing to the reference gene background. The calculating formula isN is the number of all genes with GO annotation; n is the number of target gene candidates in N; M is the number of all genes that are annotated to a certain GO term; and m is the number of target gene candidates in M. We used the Bonferroni Correction for the p value to obtain a corrected p value. GO terms with corrected p value ≤ 0.05 are defined as significantly enriched in target gene candidates.In order to understand the function of the differentially expressed genes, hyper geometric distribution test was carried out to identify GO functions and KEGG pathways in which differentially expressed genes are significantly enriched N is the number of all genes with KEGG annotation, n is the number of target gene candidates in N, M is the number of all genes annotated to a certain pathway, and m is the number of target gene candidates in M. Genes with FDR ≤ 0.05 are considered as significantly enriched in target gene candidates. The KEGG analysis could reveal the main pathways which the target gene candidates are involved in.KEGG calculating formula is the same as that in GO analysis. Here And differentially expressed genes hierarchically clustered to represent the expression patterns by using ward method with Euclidean distance as a measurement of similarity. All above analyses were programmed in python 2.7.5 with numpy (1.9.2), scipy (0.15.1), and matplotlib (1.4.3).https://doi.org/10.1155/2017/2794040). qRT-PCR analysis on cDNA samples at different developmental stages from two pollinated pear varieties was carried out with three repeats. Relative gene expression levels were calculated by the 2−ΔΔCT method [μL of 2x TransStar® Tip Green qPCR SuperMix, 2 μL of template cDNA, 0.4 μL of forward and Universal miRNA qPCR Primer, 0.4 μL of Passive Reference Dye (50x), and water to 20 μL. PCR amplification was carried out as follows: 50°C for 2 min, 94°C for 34 s, followed by 40 cycles of 94°C for 5 s, and 60°C for 30 s.To identify significantly expressed miRNAs between “Dangshan Su” × “Yali” and “Dangshan Su” × “Wonhwang”, miRNA quantification from fruit samples at 15 d, 23 d, 31 d, 47 d, 55 d, 63 d, and 145 d after flowering (DAF) was performed using Poly (T) RT-PCR. To produce miRNA fused with Poly (T) cDNA, 0.5 mg total RNA was used for the reverse transcription with miRNA mature sequence specific Poly (A) RT primers according to the Poly (T) RT-PCR protocol . The 5s To examine the expression of target genes in “Dangshan Su” × “Yali” and “Dangshan Su” × “Wonhwang,” fruit samples at 15 d, 23 d, 31 d, 47 d, 55 d, 63 d, and 145 d after flowering (DAF) were used. Total RNAs from samples were extracted using the Trizol reagent (Invitrogen) according to the manufacturer's instructions. The DNase-treated RNA was reverse-transcribed using M-MLV reverse transcriptase (Invitrogen). Tubulin gene (GenBank: AB239680.1) was used as an internal reference with primers synthesized by Sangon Biotech Co., Ltd. . qRT-PCR was carried out as above mentioned. Reaction system contained 10 μL of SYBR Premix Ex Taq II (2x), 1 μL of template cDNA, 0.5 μL of forward and reverse primer, and water to 20 μL. PCR amplification was carried out using the following program: 50°C for 2 min, 95°C for 30 s, followed by 40 cycles of 95°C for 15 s, 60°C for 20 s, and 72°C for 20 s.Primers  for qRT-PCR were designed using Primer Express 3.0 software (Applied Biosystems), and theStone cell content was measured as described by Cai et al.  Pulp  and “Dangshan Su” × “Wonhwang” (DangsuB), small RNA libraries were constructed for DangsuA and DangsuB, respectively. Using deep sequencing, a total of 10,830,189 and 9,925,992 reads were generated from DangsuA and DangsuB, respectively. After removing adapter sequences and low-quality reads, 10,809,068 (99.81%) and 9,906,978 (99.81%) clean reads from each sample group were maintained for analysis .http://rfam.janelia.org/). Using “blastn” program with command “blastn-short task,” we separated the small RNAs that matched other noncoding sequences, such as ribosomal RNA (rRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), and transfer RNA (tRNA). The annotation of these small RNAs is listed in  Citrus trifoliate [ Medicago truncatula [All these reads from both samples were annotated to small RNA sequences deposited in the Rfam11.0 database , generating 53.72% and 54.05% genome-matched reads within DangsuA and DangsuB libraries, respectively , we had to compare our measured sRNA sequences with miRNA precursor/miRNA sequences from other published plants within the miRBase21 database, in order to display sequences and quantity of miRNA families from the samples tested, regardless of species.After normalizing the miRNA expression levels of both DangsuA and DangsuB, 617 types of the known microRNAs were identified. Out of these microRNAs, 451 are present in both sample groups and other 71 species; 82 and 84 miRNAs are specifically expressed in DangsuA and DangsuB and distributed among 44 and 42 species, respectively . Malus domestica and Glycine max, each with 32 matches, followed by Prunus persica with 31, Arabidopsis thaliana with 28, and Vitis vinifera with 27 matches, respectively . Compared with other RNAs, MFEI of novel microRNAs larger than 0.85 are relatively more credible  is characterized to help in microRNA identification by ARGONAUTEI. Conservative small molecule RNA was subjected to base preferences analysis to initially determine the reliability of the resulting sequence. The results showed that most of the first base of known and novel microRNAs were U, and the known microRNAs had substantially equal nucleotide contents for A, C, and G, while the novel ones had relatively more C nucleotide content but relatively less of the G base .Expression pattern analysis of these microRNAs indicated that the expression quantity of some microRNAs in DangsuB  was highp value < 0.05).To check out if there are significant differences in expression levels between two samples, we performed the microRNA expressed statistics in DangsuA and DangsuB using microRNA edgeR (R package). With volcano plot analysis , the ove6.2 and 23.3 times higher than those in DangsuA. There are some microRNAs, such as pyr-miR3329 and pyr-novel-miR-10-3p, with the significantly reduced expression . These results suggested that difference in pollination should be able to significantly alter some microRNAs expression in “Dangshan Su.”As showed in According to the known interactions between miRNAs and target genes, it was found that 2–8 nucleotides in the miRNA 5′ end were completely complementary with UTR region in target mRNA 3′ end , 39. ThiAmong 68 differentially expressed microRNAs, 641 target genes were obtained and classified into different gene families which were involved in transcriptional regulation of stress response, signal transduction, growth, and transmembrane transport in plants . Regulatory network from the differentially expressed microRNA target genes and their corresponding mapping , was divGene Ontology (GO) is an international standardized classification system for gene function, which supplies a set of controlled vocabularies to comprehensively describe the property of genes and gene products. There are three ontologies in GO: molecular function, cellular component, and biological process. The basic unit of GO is GO term, each of which belongs to one type of ontology.q value ≤ 0.05) were related to molecular function (80%), a small amount with biological process (20%), and no cellular component was detected . That means in the different pollination of “Dangshan Su,” the effect of male parent to “Dangshan Su” stone cell formation and quality was altered by the microRNA expression quantity of target genes which has the molecular regulation functions and biological processes and is not related to “Dangshan Su” cellular components.GO enrichment analysis can predict target gene candidates of novel miRNAs, as well as their biological function. The result could reveal the functions significantly related to predicted target gene candidates of novel miRNAs. As shown in KEGG pathway analysis is also used for the target gene candidates. In organisms, genes usually interact with each other to play different roles in certain biological function. The analysis based on pathways could facilitate the understanding of biological functions of genes. KEGG is the major public pathway-related database . KEGG pap value ≤ 0.05, the significant enrichment of KEGG pathways mainly has plant pathogen interactions (Ko04626), organic selenium compound metabolized (Ko00450), sulfur metabolism (Ko00920), single stick biosynthesis (Ko00902), other polysaccharides degradation (Ko00511), purine metabolism (Ko00230), and so on.KEGG pathway analysis of the differentially expressed microRNA target genes  suggested that 68 target genes were enriched into 52 KEGG pathways. When the parameter  HCT which is encoding a key enzyme of lignin metabolism [ Populus tomentosa can have effective influence on the tree structure and the lignin content [ Populus tomentosa [Stone cells are formed by secondary thickening and lignin deposition of parenchyma cell wall , 41. Thetabolism . Rubinel content . Lu et aomentosa .Through the differentially expressed microRNA GO enrichment and KEGG pathway analysis, we discovered that the expression level of pyr-novel-miR-144-3p in DangsuB was significantly higher than that in DangsuA. Through target genes prediction of this microRNA, 6 target genes  were obtained. Pbr028952.1 encodes for an ATP enzyme. Both Pbr002903.1 and Pbr001412.1 play the function of transport substrates via transmembrane. Pbr016259.1 encodes for an acidic amino acid ligase. Pbr018935.1, as well as Pbr003857.1, encodes for a laccase  which is involved in the last key step of lignin metabolic pathways. Therefore, we proposed that via different pollination on “Dangshan Su,” the different males could affect pyr-novel-miR-144-3p expression, which subsequently regulated the laccase expression within lignin monomer polymerization process and thus controlled lignin and stone cells synthesis in pear fruit; this result is consistent with Wu and Niu , 23. PbrLAC35 and PbrLAC57 in “Dangshan Su,” we used NCBI BLAST to retrieve the genes which has 75% more similarity with PbrLAC35 and PbrLAC57 amino acid sequence in other species. The online analysis tools like ExPAsy (http://www.expasy.org/) and WoLF PSORT (http://www.genscript.com/wolf-psort.html) were used to screen essential information of amino acids, such as length, molecular weight, isoelectric point, and subcellular localization of proteins  (bootstrap = 1000) to build composite evolutionary trees of different species. As showed in  PbrLAC35 and PbrLAC57 at the different developmental stages of “Dangshan Su” with different pollination. It can be found that the expression of pyr-novel-miR-144-3p in DangsuA at different times after the flowering was significantly higher than that in DangsuB  in lignin metabolic pathways. The results were consistent with previous conclusion from the target gene prediction.In DangsuB, the expression trends of both Populus tomentosa [PbrLAC57 and PbrLAC35), which was consistent with the previous reports that a microRNA could regulate multiple target genes and the same target gene is regulated by multiple microRNA [According to previous reports, ptr-miR397a can significantly lower the expression of laccase in lignin synthesis regulation inomentosa , 21. qRTmicroRNA . PbrLAC57 was maximal at 23 d in DangsuA and at 47 d in DangsuB. Therefore, we inferred that due to the impact of xenia phenomenon, PbrLAC57 expression trend was variable in both DangsuA and DangsuB and is expressed in the early and late fruit development stage of DangsuA, while it is expressed in the mid stage of DangsuB. Moreover the DangsuA lignin content was higher than DangsuB at the development of early and late stage, while DangsuA lignin content was less than in DangsuB at the mid-stage. Thus, laccase PbrLAC57 may be a key regulatory enzyme for catalyzing lignin monomer coupling polymerization into lignin polymer.“Dangshan Su” pear by different pollination had the same changing trend of stone cell content, increasing at first, reaching a small peak at 47 d, and then down-up again to reach maximum at 63 d, then decreasing. At 47 d and 63 d, DangsuA stone cells content was less than DangsuB, while in other times, they were higher than DangsuB. Thus it can be seen that stone cell contents were significantly related to lignin contents with a correlation coefficient of 0.871 and 0.981 in DangsuA and DangsuB, respectively. PbrLAC35 and PbrLAC57 expressions have significant difference in different developmental stages of different pollination; (3) PbrLAC35 and PbrLAC57, through expression changing, cause catalyzing of lignin monomer coupling polymerization reaction change and lignin content change; and (4) the change of lignin content results in the content change of stone cells formed by lignin deposition and achieves the effect of pear fruit quality by changing the stone cell contents.According to the previous results in this study, we hypothesized that different pollination types could regulate “Dangshan Su” fruit quality through the following steps : (1) difAt the same time, xenia cannot regulate lignin biosynthesis through direct effects on lignin key enzyme genes in the phenylpropanoid metabolism of the metabolic pathway upstream. Instead, it can be indirect regulation through microRNAs, which are regulators of the related laccases, to affect the synthesis of lignin and the formation of stone cells in order to control the quality of pear. This is also consistent with the principle of the evolution of species. In the critical period of lignin synthesis, the regulation can precisely catalyze the laccases for lignin monomer polymerization, which not only can accurately promote or inhibit the synthesis of lignin, but also can avoid the regulation of the upstream related gene expression and the impact of the entire secondary metabolic pathways.We found that xenia phenomenon, instead of direct regulation, indirectly controlled lignin biosynthesis via the microRNAs which target the key genes in the phenylpropanoid metabolism, such as laccase genes. Moreover, it is found the formation of stone cells was significantly related to the lignin synthesis, which could further affect pear quality.These Supplementary Materials contained the detailed data of the primer sequences used in this study, those known and novel microRNAs, differentially expressed microRNAs, the target genes of the differentially expressed microRNAs and which corresponding GO function enrichment analysis and KEGG pathway analysis in “Dangshan su” Pear of different male parent pollination.


Our aim was to evaluate the quality of published guidelines on musculoskeletal ultrasound (MSK-US) for adults.Between June and July 2016, we conducted an online search for MSK-US guidelines, which were evaluated by four independent readers blinded to each other using the AGREE II tool. A fifth independent reviewer calculated scores per each domain and agreement between reviewers’ scores using the intraclass correlation coefficient (ICC).Five guidelines were included in this appraisal. They were published between 2001 and 2014. Our appraisal showed intermediate results, with four out of five guidelines scoring “average” as overall quality. Domain 1 (scope and purpose) achieved the highest result . Domain 6  had the lowest score . Interobserver agreement was very good for all the evaluated guidelines (ICC ranged between 0.932 and 0.956).Overall, quality of MSK-US guidelines ranges from low to average when evaluated using the AGREE II tool. The editorial independence domain was the most critical, thus deserving more attention when developing future guidelines.• Four of five guidelines on MSK-US had an average quality level.• Scope/purpose had the highest score (71.1% ± 18.7%).• Editorial independence had the lowest score (26.3% ± 19.3%).• Interobserver agreement was very good (ranges: 0.932–0.956).The online version of this article (doi:10.1007/s13244-017-0563-4) contains supplementary material, which is available to authorised users. Ultrasound (US) is currently considered the preferred first-line imaging modality for investigating several musculoskeletal disorders , 2. ReceAt the same time, the widespread use of MSK-US has raised the need for “evidence-based” recommendations to help physicians in choosing when US is more indicated in the daily routine and how to perform a technically correct US examination. In fact, appropriate use of US examinations is fundamental for increasing the diagnostic accuracy as well as to reduce the number of unnecessary examinations , 10. ForDifferent quality appraisal tools have been developed for the evaluation of guidelines. Among them, the Appraisal of Guidelines for Research & Evaluation version II (AGREE II) has been reported to be a reliable, internationally used, and validated tool .The European Network for the Assessment of Imaging in Medicine (EuroAIM), a joint initiative of European Institute for Biomedical Imaging Research, was initiated with the aim to increase the evidence for the rational use of medical imaging , 20. RecThe aim of this study was to evaluate the quality of current guidelines on MSK-US for adults using the AGREE II quality assessment tool.Between June and July 2016, we performed a literature search for MSK-US guidelines using PubMed, EMBASE, Google, and the Wiley Online Library, with the following keywords: “musculoskeletal ultrasound”, “musculoskeletal ultrasonography”, “Guidelines”, “Indications”, “Official Positions” and their expansions. Once guidelines were retrieved, their references were screened for further papers to include. We excluded from the results of our search those papers that were not primarily focused on MSK-US, such as guidelines about imaging of specific disorders in which US was described in the setting of a more comprehensive evaluation. Inclusion criteria were as follows: full manuscript available in English; guidelines issued by national or international medical bodies; guidelines mainly focused on the evaluation of musculoskeletal disorders by means of US; guidelines might contain recommendation on the MSK-US anatomy technique; guidelines must focus mainly on the adult population (age > 18 years).Guideline quality evaluation was made using the AGREE II instrument via the official website dedicated online platform . The AGRhttp://www.agreetrust.org/about-the-agree-enterprise/introduction-to-agree-ii/scoring-the-agree-ii/].Four independent reviewers  with 4 to 14 years’ experience in MSK-US and scientific research gave a score to each guideline. Reviewers were selected by the EuroAIM working group among the members of the US subcommittee of the ESSR. All reviewers were previously trained to use the AGREE II scoring system through the user manual, which is available on the online platform; in addition, reviewers were asked to complete two online training tools specifically developed to assist users in effectively applying the instrument. According to the instruction tool, each item was rated on a 7-point scale, which ranges from 1  to 7 . Final domain scores were calculated by summing up scores of all items within the domain and by scaling the total as a percentage of the maximum possible score for that domain [available via For analysis purposes, the evaluations performed by each reviewer were averaged; the average of each domain is reported in the results. Agreement between reviewers’ scores was calculated using the intraclass correlation coefficient (ICC), defined as follows: <0.20, poor; 0.21–0.40, fair; 0.41–0.60, moderate; 0.61–0.80, good; 0.81–1.00, very good. As for previous studies, the overall quality of each guidelines was evaluated using a threshold of 60% for the final score of each domain , 23. HigOur strategy of literature search identified 206 papers, 5 of which met our inclusion criteria , 11–14. average level of quality, having 3 or 4 domains with a score higher than 60%. Among these, “Clinical indications for musculoskeletal ultrasound” by Klauser et al. [low level of quality, with only two domains scoring >60% and a total score of 35.9% ± 20.6%. The paper “Musculoskeletal ultrasound: technical guidelines” by Martinoli [Table r et al.  on behalr et al.  on behalartinoli  on behalartinoli  had the Domain scores ranged between 8.3%  and 87.Total mean score of domain 1 (“Scope and Purpose”) was 71.1% with moderate variability (SD = 18.7%). The ESSR “Clinical Indications for Musculoskeletal Ultrasound”  had a “vTotal mean score of domain 2 (“Stakeholder Involvement”) was 57.5% with the lowest variability (SD = 9.1%). Within this domain, AIUM guidelines  had the Domain 3 (“Rigor of Development”) was the one with the second lowest mean score (32.7%) and, overall, the higher variability (SD = 25.5%). This variability is related to the considerable difference between the guideline with the highest domain score , the overall mean score was “acceptable” with a mean score of 73.9%. Quality scores variability was 3.6% SD. The ESSR “Clinical indications for musculoskeletal ultrasound”  was the Domain 5 (“Applicability”) had a mean quality score of 46.3% with moderate variability (SD = 9.3%). Guideline scores varied from the maximum value of 63.5% (acceptable) from the ACR–AIUM–SPR–SRU  conjoineOverall, the domain with the lowest score was domain 6 , with a mean score of 26.3%. This domain had the second larger variability, with a SD of almost 20%. As for the other domains, this variability is influenced by the difference between the highest and lowest domain score. The ESSR “Clinical indications for musculoskeletal ultrasound”  had the Interobserver variability ranges were 0.956  for the ESSR “Musculoskeletal ultrasound: technical guidelines” ; 0.932  was reached only three times. When considering the within-domain scores, variability was somehow smaller, with domain 2 (stakeholder involvement) showing the lowest SD.One of the reasons for these results may be that four guidelines –14 are mDomains with the highest quality were “clarity of presentation” (domain 4) and “scope and purpose” (domain 1), as both scored over 70%. Although less evident in our appraisal, this is comparable to previous papers that evaluated other guideline with the AGREE II tool, regardless of the topic –26. The Editorial independence (domain 6) scored “very low” in all guidelines, except for the paper ESSR “Clinical indications for musculoskeletal ultrasound”, whose final score was “low”. Therefore, this was the domain with the poorest score 26.3%). A similar situation was reported by Armstrong et al. in a recent appraisal of osteoporosis guidelines, with domain 6 presenting with the lowest score (45%) . The sco.3%. A siAccording to the AGREE II instrument, a substantial increase in the overall quality score of MSK-US was observed from 2001 to 2012, but not so from 2012 to 2014. Compared to EULAR guidelines  who founImportantly, interobserver agreement was very good for all the evaluated guidelines; hence, all reviewers were quite unanimous about the conferred scores. In fact, all reviewers’ comments were somehow uniform, a finding that further confirm the reviewers’ confidence with the AGREE II appraisal system.This study has limitations, some of them being intrinsic to the AGREE II system. In fact, this tool is not aimed at evaluating all aspects of a guideline. First, AGREE II does not evaluate the degree of consistency between guideline recommendation and the reported evidence . Also, AIn conclusion, our study showed that overall quality of MSK-US guidelines is intermediate according to the AGREE II evaluation. Domain 6  was the most critical considering the overall score; therefore, emphasis should be given to these aspects, to produce recommendations with a lower risk of bias.ESM 1(DOCX 20 kb)ESM 2(DOCX 20 kb)ESM 3(DOCX 20 kb)ESM 4(DOCX 20 kb)ESM 5(DOCX 20 kb)


The six-minute walk test (6MWT) is considered to be a simple and inexpensive tool for the assessment of functional tolerance of submaximal effort. The aim of this work was 1) to background the nonlinear nature of the energy expenditure process due to physical activity, 2) to compare the results/scores of the submaximal treadmill exercise test and those of 6MWT in pulmonary patients and 3) to develop nonlinear mathematical models relating the two.The study group included patients with the COPD. All patients were subjected to a submaximal exercise test and a 6MWT. To develop an optimal mathematical solution and compare the results of the exercise test and the 6MWT, the least squares and genetic algorithms were employed to estimate parameters of polynomial expansion and piecewise linear models.Mathematical analysis enabled to construct nonlinear models for estimating the MET result of submaximal exercise test based on average walk velocity (or distance) in the 6MWT.Submaximal effort tolerance in COPD patients can be effectively estimated from new, rehabilitation-oriented, nonlinear models based on the generalized MET concept and the 6MWT. Even though the double labeled water method is considered the most general technique to estimate energy expenditure due to physical activity , it is cFrom a clinical point of view it is essential to prescribe an “optimal” level of training loads in patients with cardiopulmonary diseases. For this purpose submaximal exercise test (ET) is usually performed. It defines the amount of effort that a patient can safely perform, and allows to define exercise heart rate limit for each patient. Exercise test result is expressed in Watts or METs. The most commonly used ET protocol for patients with cardiopulmonary dysfunction is the modified Bruce treadmill protocol, where the result is expressed in METs.One MET is defined as 1 kcal/kg/hour and is roughly equivalent to the energy cost of sitting quietly. A MET unit is also defined as oxygen uptake in ml/kg/min with one MET equal to the oxygen cost of sitting quietly, equivalent to some 3.5 ml/kg/min . Howeverr < 0.91) with maximal oxygen uptake derived from a cardiopulmonary exercise test in a well-defined group of patients vi − treadmill velocity at the ith stage of test, i = 1, …, S + 1 [km/h] = 1000/60 [m/min] = 50/3 [m/min]S − number of stages completed,δD − distance walked during the (S + 1)th stage (uncompleted)δD < Ts,S+1vS+1 50/3 [m]δTs,S+1 − walk time at the (S + 1)th stage (uncompleted)δTs,S+1 < Ts,S+1 [min]M in METs (via VO2max) can now be related with the walk distance D as in (v = D/T [m/min] = (3/50) D/T [km/h], giving M = MD(D) or M = Mv(v), respectively (instead of M = MT(T)). In the 6MWT environment, featuring vM6 = D/6 [m/min] = D/100 [km/h], they will be substituted with M = MM6(10−2D) or, equivalently, M = MM6(v).The walk timeExample: Modified Bruce (mBruce) protocol to be used hereinafter. For the mBruce protocol we have Ts,i = Ts = 3 [min], i = 1, …, S, so thatδD = (50/3)δTs,S+1vS+1 [m], δTs,S+1 = T − STs [min] and vi [m/min] = (3/50) [km/h], i = 1, …, S + 1, as specified (for particular stages) in the mBruce protocol. Now, a table for the mBruce protocol could be supplemented with e.g. distance-related data in meters (or velocity-related data in km/h) but we refrain from recalling the supplemented table for space-saving reasons. Rather, we exemplify the calculations for one specific treadmill score, e.g. T = 10.5 [min], M = 5.95 [MET], for which we have S = 3, v1 = v2 = v3 = 2.7 [km/h], v4 = 4.0 [km/h], δTs,4 = 1.5 [min], δD = 100 [m], D = 505 [m], v = (3/50)D/T [km/h] = 2.886 [km/h] and, reffering to the 6MWT, vM6 = (D/100) [km/h] = 5.05 [km/h]. Comparing the values of v and vM6, the example clearly supports the well-known fact that the 6MWT is less exhausting than the treadmill test.Applications of the simple, affordable and reproducible 6MWT have been presented in such a plethora of publications that any collection of references could by no means be considered complete. We recall a selected series of recent 6MWT-related contributions covering various medical fields –33, in aM with the average patient’s velocity v = vM6 in the context of the 6MWT was proposed by Connors and Hilling , the abscissa velocity scale in km/h will be equivalent to scaling it with distance 10−2D in meters. The first mathematical model relating the energy expenditure  Hilling M1=(1.76M1 = f1(v) was sometimes used in the rehabilitation practice in the form , , M1 = f1D = φ(M), with the resting unity MET voiding. A similar nonlinear reverse characteristic, without a model however, was considered by Beatty et al. T being analytically least squares (LS) estimated using a linear regression formulation. Note that for v = 0 we have the resting MET equal to 1.(A deterministic part of) the polynomial expansion model can be expressed asn-segment piecewise linear model can be presented asai, i = 1, …, n, bj, j = 2, …, n, are the unknown parameters and Vi, i = 1, …, n, are the unknown change points, compare T could be estimated numerically using the least squares (LS) method. Unfortunately, the estimation problem is highly nonlinear with respect to the parameters V1, …, Vn, so that the numerical LS minimization algorithm is often stuck in a local minimum. A remedy is to use a genetic algorithm (GA) which is much likely to converge to a global minimum. Luckily, the ranges of possible changes in Vis can be easily “guessed” by an expert designer, so this can facilitate the GA procedure. Nonetheless, we have succeeded in the development of a combined LS/GA minimization procedure which is effectively used here.Note that, due to the linear spline property of the segments, the Remark 1. In order to discriminate between the above specified model outputs and results/scores measured from the tests we will denote the estimated model outputs with M, possibly adding the appropriate subindexes wherever necessary.M of treadmill exercise tests, measured in METs vs. results of the 6MWT, expressed in velocity vM6 [km/h] (or distance 10−2D [m]) are plotted for men (population Nm = 159), women (Nw = 140) and the whole COPD patient population (Np = Nm + Nw = 299) in Figs M vs. vM6 suggest nonlinear (stochastic) models. The figures also contain the corresponding outputs K = 3 and the marked standard deviation ranges (results for K = 5 are quite comparable). For negative comparison, the output l = 1, …, N, is the number of consecutive samples of the result M and its modeled value N is the total number of samples, with N being equal to Nm, Nw or Np for the populations of men, women and all the COPD patients, respectively.Results n models  for K = ar model  is only K to 9 (or higher odd) leads, unsurprisingly, to an increase in RMSE due to rather highly disturbed measurements M(l).It is worth mentioning that increasing the model order RMSE as high as 3.3969.For negative comparison, the (very poor) linear model  applied n = 4 and the marked standard deviation ranges. Below you can find the sets of the optimal LS/GA parameter estimates In a similar way, the modeling and simulation work was performed for the three population cases as above and the piecewise linear model . In addir models  for n = Unsurprisingly, RMSE values while using models  and 9)  9) are qhttps://en.wikipedia.org/wiki/Model_selection. In our case, we choose a popular model validation technology which is cross-validation (CV) , inion (CV) . LOO CV RMSEs obtained above in this Section. Also, Figs R are very high for both cases. This confirms the validity of the presented polynomial expansion and piecewise linear models.As seen from n = 4 linear segments. Why linear and not e.g. cubic splines? Just for simplicity. Note that we optimize the locations of change points Vis (in addition to parameter estimates) and this corresponds with ‘optimization’ of their MET switch points i = 1, …, n, that can be used in the patients’ division into rehabilitation classes. And yet, final selections of Mi) division into cardiopulmonary rehabilitation groups typically encountered in the therapeutic society, also in Poland:Clearly, the linear model  or 7) i i7) is uM(1) = 2.304, M(2) = 4.094 and M(3) = 7.782. Those are not quite in agreement with the group division presented above, which might suggest some corrections to the classification  to its VO2max score or, ‘almost’ equivalently to the (sub)maximal effort tolerance M in METs. Abstracting from our 6MWT rehabilitation-oriented application, this is nothing but our model . . T [min]T = 0, which is the resting condition, we should have the oxygen uptake of some 3.5 [ml/kg/min] and this is the value the offset term should be approximately equal to. But for the two linear models we have the offset terms close to 3.5. This means that the two models were constructed for populations of 1) severe sick (women) patients mainly and 2) young men of low effort tolerance mainly. This is in contrast to the linear model with the offset term equal to −3.9, which suggests that the population of low sick or low medium (women) patients was mainly investigated.Apparently, the above linear models are unacceptable as the phenomenon is nonlinear, in general. Well, get the first model which is nonlinear. However, its offset term is 14.76, which is a disaster because for Now, treadmill exercise test procedures are equipped with specific, typically nonlinear models but still they may not be population-related. The treadmill exercise test procedures need banks of nonlinear reference models designed for various patient populations and called e.g. from keyboard for each individual patient under test, depending on various conditions including gender, type/class of sickness, possibly age range and others. This might be involving and time-consuming in general and that is why we have sought for the simple 6MWT substitute herein.Still, we believe the concept of developing the banks of nonlinear models for treadmill exercise test procedures could be attractive sooner or later. The intimation of such an idea is the second goal of this paper. To this end, we can make generally available (upon request) all our programming packages necessary for construction and simulation of models  and 9)..9). The Remark 2. Similar banks of nonlinear models for treadmill exercise test procedures could be developed for athlete management software, in particular for junior athletes in their preliminary selection process.Remark 3. Even though many VO2max or MET-involving models are single input, with the input variable being either walk time or velocity or distance, the time has come to thoroughly introduce the second input variable into the treadmill exercise test model, that is the grade. Such a more complex and general model is out of scope of this study and it will be a subject of our future research.Apart from the above-mentioned, general need for treadmill exercise test procedures to incorporate banks of ‘reference’ nonlinear models (rather than one ‘average’ model), right the same holds for the 6MWT score modeled by Eqs  or 9). . 9). OurRemark 4. Note that e.g. a 6MWT bank for COPD patient populations should strictly ‘cooperate’ with the corresponding COPD bank within the mBruce procedure. Unfortunately, this ‘compatibility rule’ is currently rather seldom observed during treadmill exercise tests, that is e.g. COPD patients are tested against some nonlinear reference model of VO2max vs. T, being likely a sort of an ‘average’ characteristic for cardiopulmonary patients.Having backgrounded the energy expenditure process due to physical activity, we justified the nonlinear relationship between VO2max and power output during a submaximal exercise test. This has led to the proposal of two classes of nonlinear models, namely polynomial expansion and piecewise linear models. The models can be used to estimate submaximal effort tolerance in patients with COPD by means of the cost-saving 6MWT, without the use of cardiorespiratory analysis. The models’ LS fit to data was successfully assessed by means of the LOO cross-validation technique. The above constitutes an original modeling methodology for estimation of submaximal effort tolerance, which is the main achievement of the paper. Our new nonlinear estimation algorithms have been finally applied for piecewise linear models in rehabilitation of COPD patients, improving their assignment rules to four rehabilitation classes. The second achievement of the paper has been the proposal to use our nonlinear estimation methodology in treadmill exercise test procedures in order to construct a bank of (nonlinear) ‘reference’ models cooperating compatibly with various patient populations under various patient management environments. This challenging, expert system-oriented future research direction will be followed in our works. Another interesting future research topic could be an extension of our methodology to self-paced treadmill exercise tests, combined with 6MWT, compare . Yet anoS1 Table(TXT)Click here for additional data file.S2 Table(TXT)Click here for additional data file.


Gupta S, Agarwal R, Aggarwal KC, et al. Complementary feeding at 4 versus 6 months of age for preterm infants born at less than 34 weeks of gestation: a randomised, open-label, multicentre trial. Lancet Glob Health 5: e501–11—2017; In this Article, the last sentence of the Procedures section should have read “Z scores for weight, length, head circumference, weight for length, and body–mass index at subsequent timepoints,  were calculated using WHO-MGRS growth standards.” In Figure 1, the box describing patients who did not receive allocated intervention in the 6 month group should have included “1 did not start complementary feeding until 12 months” and the box describing patients who did not receive allocated intervention in the 4 month group should have included “1 did not start complementary feeding before death, which occurred between 5 and 6 months”. These corrections have been made as of April 10, 2018.