Vigna angularis var. angularis) is a dietary legume crop in East Asia. The presumed progenitor (Vigna angularis var. nipponensis) is widely found in East Asia, suggesting speciation and domestication in these temperate climate regions. Here, we report a draft genome sequence of adzuki bean. The genome assembly covers 75% of the estimated genome and was mapped to 11 pseudo-chromosomes. Gene prediction revealed 26,857 high confidence protein-coding genes evidenced by RNAseq of different tissues. Comparative gene expression analysis with V. radiata showed that the tissue specificity of orthologous genes was highly conserved. Additional re-sequencing of wild adzuki bean, V. angularis var. nipponensis, and V. nepalensis, was performed to analyze the variations between cultivated and wild adzuki bean. The determined divergence time of adzuki bean and the wild species predated archaeology-based domestication time. The present genome assembly will accelerate the genomics-assisted breeding of adzuki bean.Adzuki bean ( Vigna angularis var. angularis) is a diploid legume crop (2n = 2x = 22) with an estimated genome size of 538 mega bases (Mb)Vigna in the Ceratotropis subgenus, under the papilionoid subfamily of the FabaceaeV. angularis var. nipponensis, V. nakashimae, and V. nepalensis, are widely distributed across East Asia and Himalayan countriesAdzuki bean  of V. angularis var. angularis has a diploid genome. Based on flow cytometry analysis the genome size was estimated to be 612 Mb  and the wild species V. nakashimae (IT178530). GBS short reads were mapped to genomic regions flanked by ApeKI restriction sites using Bowtie2 softwareTo assemble the pseudo-molecules, we implemented the genotyping by sequencing (GBS) methodde novo assembly using Trinity softwareE-value 1e-10 , and introns to the gene models of P. vulgaris, G. max, and V. radiata. The density plot showed consistent distributions of CDS and intron length among the three legume genomes. However, the proportion of short genes (~250 bp) was higher in V. angularis  retrotransposons5621We surveyed the repetitive sequences within the scaffold sequences to examine the abundance and distribution of transposable elements, which are known as major drivers of genome evolutionV. angularis var. angularis, we sequenced wild relatives of adzuki bean, V. nepalensis (AusTRCF85148) and V. angularis var. nipponensis (IT241912) (Vigna nakashimae (IT178530), which was generated in our previous studyHuman selection activities on crops have inflicted major effects on crop genomes, which ultimately result in domestication syndrome, which is marked by loss of seed shattering, minimization of seed dormancy, and an increase in both seed size and numberT241912) . Single V. nakashimae, which showed a much larger genome size than V. angularis var. angularis and the rest of the wild adzuki beans.The 22-base k-mer frequency analysis of these short read sequences revealed a variation of genome size among wild adzuki beans . This isV. angularis var. nipponensis had 667,097 SNPs compared with the reference genome and the SNP frequency to total mapped regions (SNPs per kb) was 1.82. 75,476 of the identified SNPs are located within coding regions and 3,840 of those SNPs could potentially cause non-synonymous protein sequence changes in 1,421 gene products. We also identified 97, 932 insertions and deletions (INDELs) of which 14,033 were in coding regions.We mapped the short read sequences onto our adzuki bean reference genome using Burrows-Wheeler Aligner software 25. V. anV. nepalensis showed much higher polymorphism than V. angularis var. nipponensis as demonstrated by the presence of 3,511,378 SNPs (10.24 SNPs per kb). The number of SNPs in coding region was 433,210, and there were 18,034 non-synonymous SNPs that affected 6,464 genes. A total of 410,232 INDELs were identified and 71,337 were within coding regions.V. nakashimae showed 3,342,795 SNPs and 624,856 intragenic SNPs. However, the ratio of aligned reads to total reads and the predicted number of INDELs were notably lower than those of other wild adzuki beans. This could be due to the difficulty of read mapping between the reference species (V. angularis) and the highly diverged species (V. nakashimae) showing notable genome size discrepancy  in the Ka/Ks calculation. We observed one consistent peak at Ka/Ks value of 0.2 suggesting purifying selection between cultivated and three wild adzuki beans (Ka/Ks < 1)V. angularis var. angularis x V.angularis var. nipponensis and V. angularis var. angularis x V. nepalensis were highly similar consistently showing three peaks . In both comparisons, a total of 307, 152, and 75 genes were commonly found within the three peaks, respectively  markers using MISA softwareG. max is a warm-season model legume crop closely related to V. angularis var. angularis showing co-linearity of most of the gene content , between Phaseolus and Vigna−9V. angularis, V. radiata, and P. vulgaris . Thus, this method could be used as an alternative to the longer sequencing reads or mate pair library with larger insert size solving the assembly problem. In addition, we anchored super-scaffolds onto the genetic map constructed using the GBS-based genotypes of F4 recombinant inbred lines of V. angularis var. angularis and V. nakashimae. Due to the segregation distortion, relatively small amounts of GBS-derived SNPs were used for constructing the genetic map. The possible cause of this segregation distortion in this population is the genome size difference of parental lines, which may result in ectopic recombination , and wild adzuki bean accession  were provided by the Rural Development Administration (RDA) Genebank Information Center in Korea. The other wild adzuki bean  was sourced from the Australian Collections of Plant Genetic Resources in Australia.The cultivated adzuki bean accession, Gyeongwon . Values for nuclear DNA content are estimated by comparing fluorescence intensities of the nuclei of the testing sample with those of an appropriate internal DNA standard such as nuclei from Chicken Red blood cells (2.5 pg/2C), Glycine max (2.45 pg./2C), Oryza sativa cv. Nipponbare (0.96 pg/2C), or Arabidopaiathaliana (0.36 pg/2C). For each measurement, the propidium iodide fluorescence area signals (FL2-A) from 1000 nuclei were collected and analyzed by CellQuest software .We also estimated the genome size by the flowcytometry analysis using procedure modified from Arumuganathan and Earle V. nakahsimae using the option k = 84 and q = 20 for “abyss-pe” command. The traces of repetitive sequences were searched using LTR-harvesthttp://transposonpsi.sourceforge.net/) with default parameters. The captured repetitive sequence blocks were annotated by LTR-digestG. max TE classification based on sequence homologyFor genome assembly of cultivated adzuki bean, we used the Illumina HiSeq 2000 using types of sequencing libraries such as two fragment libraries and two 5 kb and one 10 kb mate pair libraries. One additional single linear library was sequenced using the Roche GS-FLX+ . The assde novo assembled using Trinity software with default parameterde novo assembly and redundant sequences were removed by the CD-HIT-EST algorithm in CD-HIT softwareG. max protein sequences, and the complete protein sequences of Arabidopsis from Uniprot as evidence for the homology based gene prediction. For ab initio gene prediction, we used AUGUSTUS softwareV. radiata) training set. The resulting protein sequences were annotated using InterProScan5Adzuki bean gene contents were predicted following MAKER pipelineV. angularis var. angularis and V. nakashimae were classified based on the TF classification rules described in Lang et alA. thaliana, G. max, M. truncatula, C. cajan, C. arietinum, B. distachyon, Z. mays, O. sativa).Using the Pfam annotations from InterProScan result, transcription factors (TF) of adzuki bean proteins from V. angularis var. angularis specific hmm profiles of LRR and NBS using the HMMER software package with default parameter for “hmmbuild” commandV. angularis-specific hmm profiles, proteins with NBS and/or LRR domain were identified using HMMER software package with default parameter for “hmmscan” command. The putative functions of NBS-LRR were annotated using the blast result against Uniprot databaseUsing PfamP. vulgaris, V. radiata, and V. angularis. The pairwise synteny relationships, and the Ks values of gene pairs were calculated using MCSCANX softwareV. radiata and P. vulgaris were used as links for scaffolding. We retrieved every link that could be deduced from synteny blocks and concatenated each other to build consensus links (We further linked the scaffolds based on the gene order conservation (synteny) of us links . The ori4 lines derived from the cross between Gyeongwon and V. nakashimae (IT178530) using genotyping by sequencing (GBS)de novo assembled scaffolds. The short reads were mapped onto the ApeKI flanking sequences using software Bowtie24 lines were supplied to JoinMap 4, and we constructed 11 linkage groups using regression mapping algorithm with Kosambi mapping function.To construct a genetic map of adzuki bean, we genotyped 133 FV. nakashimae, V. nepalensis, and V. angularis var. nipponensis were aligned to our reference genome of V. angularis var. angularis using the BWA-MEM algorithm of BWA software with default optionshttp://picard.sourceforge.net) and GenomeAnalysisTK (3.1–1 version) to optimize the sequence alignmentThe sequence reads of P. vulgaris, V. radiata, V. angularis var. angularis, V. angularis var. nipponensis, V. nakashimae, and V. nepalensis based on Bayesian Markov Chain Monte Carlo (MCMC) analysis using the 60 orthologous loci by *BEAST of the software package BEAST version 1.8P. vulgaris, V. radiata, V. angularis, and M. truncatula. Even though we didn't include the M. truncatula for species tree construction, we used it to narrow down the confident orthologs. Among the retrieved orthologs, 60 highly conserved ones were chosen, that showed the low relative standard deviation (RSD < 0.00001) of protein length to have clear speciation signal. To this confident ortholog set, we also added the corresponding orthologs of V. nepalensis, V. angularis var. nipponensis, and V. nakashimae. The coding sequences of the orthologous loci were aligned using Prank software with –translation optionVigna and Phaseolus of previous estimationWe constructed the species tree among the closely related diploid warm season legume species, such as http://plantgenomics.snu.ac.kr. This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession JRFV00000000.The adzuki bean genome information such as genome assembly, gene prediction and annotation, genetic markers, and other related files of this study can be searched and downloaded from Supplementary informationSupplementary Table S6Supplementary Table S7Supplementary Table S9Supplementary Table S11Supplementary Table S12Supplementary Table S13Supplementary Table S17Supplementary Table S18Supplementary Table S19Supplementary Table S22


Brittle1 (OsBT1) gene, which is expressed specifically in the developing endosperm. The osbt1 mutant showed a white-core endosperm and a significantly lower grain weight than the wild-type. The formation and development of compound starch granules in osbt1 was obviously defective: the amyloplast was disintegrated at early developmental stages and the starch granules were disperse and not compound in the endosperm cells in the centre region of osbt1 seeds. The total starch content and amylose content was decreased and the physicochemical properties of starch were altered. Moreover, the degree of polymerization (DP) of amylopectin in osbt1 was remarkably different from that of wild-type. Map-based cloning of OsBT1 indicated that it encodes a putatively ADP-glucose transporter. OsBT1 coded protein localizes in the amyloplast envelope membrane. Furthermore, the expression of starch synthesis related genes was also altered in the osbt1 mutant. These findings indicate that OsBT1 plays an important role in starch synthesis and the formation of compound starch granules.Starch is the main storage carbohydrate in higher plants. Although several enzymes and regulators for starch biosynthesis have been characterized, a complete regulatory network for starch synthesis in cereal seeds remains elusive. Here, we report the identification and characterization of the rice  Oryza sativa L.) is one of the most important staple foods for more than half of the world’s population. Starch synthesis and accumulation occur in the amyloplasts that exist in tubers, cotyledons, and endosperms, and is also an important determinant for crop yield1Starch is the main storage carbohydrate in higher plants and not only is the primary source of calories in human diet but also has great value in industrial applications. Because its starch content, rice  cause a shrunken endosperm, due to a significant reduction in starch synthesisGBSSI (Waxy) gene produce an amylose-free endosperm9SSIIIa leads to abnormal amylose content, amylopectin structure, and starch granule properties11ISA1 produces a sugary endosperm and a dramatic alteration of polyglucan structure from amylopectin to phytoglycogen13BEIIb in the amylose-extender (ae) mutant generates a chalkiness endosperm and alterations in the structure of amylopectin and in the gelatinization properties of starch granulesStarch comprises two major components, amylose and amylopectin. A series of key enzymes involved in starch biosynthesis have been identified. ADP-glucose (ADPG) is the substrate for starch synthesis, which is converted from glucose-1-phosphate by ADP-glucose pyrophosphorylase (AGPase). Granule-bound starch synthase I (GBSSI), which is encoded by the via interaction with a bHLH proteinFLO6, encoding a CBM48 domain-containing protein, is involved in compound granule formation and starch synthesis putatively through interaction with ISA1Grain Incomplete Filling 1 (GIF1) encodes a cell-wall invertase that regulates sucrose unloading, and the mutation of GIF1 leads to significantly reduced starch content and grain weightOsAlaAT1 gene, encoding an alanine-aminotransferase, also displays altered amylose content and amylopectin structureRice Starch Regulator (RSR1), encoding an AP2/EREBP family transcription factor, negatively regulates the expression of type I starch synthesis genes, and RSR1 deficiency results in enhanced expression of starch synthesis genes in seedsBesides these key enzyme genes, several regulators are known to indirectly regulate starch biosynthesis in rice endosperm. For instance, FLO2, a tetratricopeptide (TPR) domain-containing protein, regulates the expression of genes involved in the production of storage starch and storage proteins in the endosperm et al. first proposed BT1 proteins as ADPG transporters in cereal endosperms23252627283031As the substrate for starch synthesis, ADPG needs to be transported into amyloplasts from the cytosol by an ADPG transporter located in the envelope membrane of amyloplasts. Sullivan osbt1. Map-based cloning showed that OsBT1 encodes an ADPG transporter which locates in the amyloplast envelope membrane and functions in the regulation of starch biosynthesis and compound starch granule formation during rice seed development.In this study, we characterize a white-core endosperm mutant, osbt1. The endosperm of osbt1 was opaque and presented a white-core in the centre region of the seed, while the periphery region was translucent compared to that of wild-type  were prepared to compare the formation of compound starch granules in endosperm cells between ild-type . The resild-type . Howevere mutant . By contloplasts .osbt1, and the central endosperm cells of wild-type were filled with about a dozen of polyhedral starch granules and the space between the starch granules was gradually occupied during seed development, to eventually form normal compound starch granules  analysis was further carried out to observe the development of compound starch granules. The amyloplasts in the endosperm peripheral cells of both wild-type and granules . Howeverrm cells , which wrm cells .osbt1 mutant. The total starch and amylose contents were significantly decreased in osbt1 compared to wild-type , peak (Tp), and conclusion (Tc) temperatures of gelatinization of endosperm starch in the osbt1 mutant were all about 3 °C lower than those in the wild-type  with NJ-11 (indica). The mutated locus was first mapped to a short arm in chromosome 2 between the simple sequence repeat (SSR) markers RM12669 and RM12679. Based on three SSR markers and three newly-developed insertion/deletion (Indel) markers, the osbt1 locus was further narrowed down to a 39.5-kb region between markers Ind3 and Ind10 by using a total of 1,222 recessive homozygous individuals. Seven open reading frames (ORFs) were included in this region (Os02g0202400) in the mutant, which lead to a six amino acid increase in the encoded protein compared to the wild-type sequence  web portal, the result showed that the OsBT1 protein cannot be normally folded due to six amino acids added in osbt1  generated in a wild-type genetic background which showed reduced expression of OsBT1 . The results showed that OsBT1 was specifically expressed in seeds and that its relative expression level gradually increased after fertilization, with a maximum expression level at 21 DAF  was generated and transformed into the Kitaake rice cultivar. Laser scanning confocal microscope showed that OsBT1-GFP was exclusively localized at the amyloplast envelope membranes in endosperm cells , and five amylopectin synthesis related genes  were lower at 6 DAF, but were elevated at 9 to 15 DAF compared to wild-type. The expression levels of OsAGPL1 and OsBEIIb were higher in osbt1 than in the wild-type, but no significant differences were detected at 6 or 9 DAF. OsAGPS1 expression was higher in osbt1 than in the wild-type at the early grain filling stage (6–12 DAF), but maintained a similar expression level at 15 DAF. The expression pattern of the OsBE1 gene was opposite to OsAGPS1, which was lower than in the wild-type at the early grain filling stage (6–12 DAF), but higher at 15 DAF 1218202133flo1-flo3, ae, pdil1-1, gpa1 to gpa3, osagpl2–3, flo6, ssg4, osalaat1, flo7, ssg6, pfp, and ss3a/ss4b)16171819353637383940414243444546isa1/sugary1, osagpl2, osagps2, and pho1)814dull1, dull3, and waxy)1049Chalk5 was also identified to influence grain chalkiness in riceStarch biosynthesis is a complex metabolic process and the molecular regulatory machinery remains largely unknown. Characterization of new rice endosperm-defective mutants is useful to further elucidate the starch synthesis process. Previous studies have identified at least 29 endosperm-defective mutants, including six chalky endosperm mutants OsbZIP58, was inactivated, the expression of OsAGPS2b, OsAGPL2, OsSSI, OsSSIIIa, OsSSIVb, OsBEIIb, and OsISA2 was distinctly up-regulated, while that of OsAGPL3, OsPHO1, Wx, and SBE1 was down-regulated, leading to a reduced content of total starch and amylose. Also, the fine structure of amylopectin is altered in the osbzip58 mutantChalk5, which encodes a vacuolar H+-translocating pyrophosphatase influencing grain chalkiness in rice, is over-expressed, the transcript levels of eight genes involved in starch metabolism are significant elevated in seeds at 5 DAF, implying a feedback regulation of Chalk5 expression in controlling grain chalkinessosbt1 3031OsBT1 gene was identified using a forward genetics approach. Subcellular localization analysis showed that OsBT1 is located to the envelope membrane of amyloplasts. Thus, we proposed that OsBT1 is the ADPG transporter in rice endosperm cells. To validate our hypothesis, we carried out import studies using [14C] ADPG into Escherichia coli cells harboring recombinant OsBT1, however this assumption was also recently confirmed by Cakir et al.As the predominantly product of photosynthesis, sucrose occurs in source leaves, which is then transported into endosperm cells to be used as carbon source for starch synthesis. Sucrose is divided and converted into ADPG, G6P, and Glc and then imported into amyloplasts by their corresponding transporters . The gluOsBT1 has an essential role in starch synthesis and compound starch granule formation. Ongoing efforts are still needed to identify new regulatory steps of starch biosynthesis as means to aid breeders and biotechnologists to improve quality in crops.In summary, our results indicate that osbt1 mutant was generated from Oryza sativa japonica cv. Nipponbare induced by tissue culture in vitro. An F2 population was produced by crossing the osbt1 mutant with an indica rice cv. Nanjing11 (NJ-11) for gene mapping. The rice plants were grown under natural conditions at China National Rice Research Institute experimental field plots, in Hangzhou, China.The osbt1 mutant was cut transversely with the back of a knife, and the ruptured transverse surface was coated with gold to prepare samples. The ruptured transverse surface was observed by scanning electron microscope (SEM) which was performed as described previously using a HITACHI S-3400N scanning electron microscope (http://www.hitachi-hitec.com) osbt1 endosperms at 9 DAF were used to make samples of semi-thin sections. Samples were treated as described by Peng et al.2-KI for 5 s and subsequently examined under a light microscope . For the ultrastructure observation of amyloplasts, developing seeds (6–12 DAF) were fixed over 12 h in 0.2 M phosphate buffer (PH 7.2) with 2.5% glutaraldehyde. Samples were treated as described by Takemoto et al.http://www.rmcproducts.com). A transmission electron microscope  was used for observation.The brown rice of wild-type and the http://www.megazyme.com/). Amylose content was determined as described by Liu et al.osbt1 mutant and wild-type were measured following the method described by Kang et al.Pseudomonas amyloderamosa isoamylase (Megazyme) and then analyzed by capillary electrophoresis . For the gelatinization temperature analysis, 5 mg of rice powder was placed in an aluminum sample cup, mixed with 10 μl of distilled water, and sealed, and then the samples were analyzed by a differential scanning calorimeter . The heating rate was 10 °C min−1 over a temperature range of 35 °C to 100 °C. The swelling modes of endosperm starch in urea solution were measured according to a previous reportThe total starch content of the rice flour was measured according to the manufacturer’s protocol, with a starch assay kit . Gene expression was measured by quantitative real-time PCR (qRT-PCR) using the Ubiquitin gene (GenBank accession AF184280) as reference. The qRT-PCR primers used to evaluate the expression of starch synthesis-related genes have been described previously−ΔΔCT method.Total RNA from roots, stems, leaves, leaf sheaths, panicles, and developing seeds at 6, 9, 12, 15, 18, 21, 24 DAF were extracted using the RNAprep pure Plant kit . 2 μg of total RNA was reverse-transcribed by priming with oligo(dT18) in a 40 μl reaction based on the PrimeScript Reverse Transcriptase kit  were introduced into the Agrobacterium strain EHA105 and used to infiltrate N. benthamiana leaves. Plants were then incubated at 28 °C for 2 days before examination according to protocols described previouslyN. benthamiana protoplasts were isolated as described previouslyOsBT1 coding sequence without a termination codon was also cloned in-frame in front of GFP in the pCAMBIA1305-GFP vector to generate the 1305-OsBT1-GFP construct under the control of the maize UBIQUTIN1 promoter. The 1305-OsBT1-GFP plasmid was transformed into an indica rice cv. Kitaake. Endosperms from T1 transgenic seeds were used for observation.The How to cite this article: Li, S. et al. OsBT1 encodes an ADP-glucose transporter involved in starch synthesis and compound granule formation in rice endosperm. Sci. Rep.7, 40124; doi: 10.1038/srep40124 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Wide-angle X-ray scattering and electron microscopy provided measured data which quantify microstructural arrangements associated with stiffness. The present study measures stiffness parallel to the lamellae of the cornea which approximately doubles with an increase in strain rate from 0.5 to 5%/min, while the underlying stromal matrix provides a stiffness 2–3 orders of magnitude lower than the lamellae. The model has been simultaneously calibrated to within 3% error across three age groups ranging from 50 to 95 years and three strain rates across the two loading scenarios. Age and strain-rate-dependent material coefficients allow numerical simulation under varying loading scenarios for an individual patient with material stiffness approximated by their age. This present study addresses a significant gap in numerical representation of the cornea and has great potential in daily clinical practice for the planning and optimisation of corrective procedures and in preclinical optimisation of diagnostic procedures.A constitutive model based on the continuum mechanics theory has been developed which represents interlamellar cohesion, regional variation of collagen fibril density, 3D anisotropy and both age-related viscoelastic and hyperelastic stiffening behaviour of the human cornea. Experimental data gathered from a number of previous studies on 48  The ocular vessel consists of the cornea, sclera and corneoscleral limbal junction. The vessel protects the internal contents of the eye and maintains the eye’s general shape which is necessary for clear vision. The transparent cornea, at the anterior segment of the eye, provides two-thirds of the eye’s optical power Fatt , and thiThe topography of the cornea is determined by the balanced state between the forces acting upon it and its mechanical stiffness, which is defined by cornea’s geometry, thickness and the material stiffness. While the geometry and thickness, and their contribution to overall mechanical stiffness, are easy to determine, the material stiffness is much more difficult to quantify as it is dependent on the microstructure of the stroma, the main load carrying layer of the cornea. The stroma is comprised of over 200 lamellae Freegard , each ofTo date, there have been a significant number of studies which have progressed the numerical representation of the cornea in its quasi-static state. These have included Alastrue et al. , Pandolfex vivo human data. The research builds on a recent study by Whitford et al. . Geometric modelling was performed using bespoke software that provides geometry, which can be imported into finite element solvers as an orphan mesh. Finite element solver Abaqus/Standard 6.13  was used. Abaqus is well known for its ability to analyse nonlinear problems. However, its ability to provide state-of-the-art representation of biological material properties, and both regional and local variation of these properties, is limited. Thus, Abaqus was used in conjunction with bespoke subroutines (SDVINI & UMAT) written in FORTRAN to implement the constitutive model described above.The integral of Eq. Subroutine SDVINI was used to provide initial, reference-configuration and location-based conditions such as fibril density representation. These location-based properties are defined individually for each integration point.These are implemented into the numerical simulation using the UMAT subroutine as demonstrated in the Abaqus User Subroutines Reference Guide Abaqus . UMAT isD . The cornea inflation models consisted of three layers and 26 rings of elements. The shape of elements, and their arrangement, was chosen to provide uniform element sizes and consistent approximation of geometry. Trials of these models indicated analysis periods of 5–10 mins depending on variations in materials definitions. Thickness values were assumed to be rotationally symmetric; central corneal thickness (CCT) of 545 olzapfel , the remCharacteristic experimental data have been obtained from 48 fresh human donor corneas. Data include 36 corneas tested under inflation and 12 corneas tested under shear. The corneas tested under inflation were divided into two groups: 23 corneas tested with 37.5 mmHg/min pressure rate and 13 corneas tested with 3.75 mmHg/min rate , e.g. Bao et al. . The perex vivo experimental studies is required. While there is potential inconsistencies between in vivo and ex vivo material behaviour which cannot be ignored, models calibrated from ex vivo data have demonstrated the clinical suitability of these methods, for example (Joda et al. In the absence of in vivo material properties, derivation of material relationships from By addressing a significant gap in viscoelastic representation of the cornea, modelling which includes these properties can now be used to develop and improve treatment procedures based on characteristic behaviour across the population and for an individual patient with material stiffness approximated by their age. In addition, the study has provided valuable information of the magnitude and dissipation periods of viscoelastic effects in the cornea; comibined here with the 3D anisotropic, age-related and regional variations of hyperelastic behaviour, this model is intended to contribute to future in vivo biomechanical analysis.


In this first issue of the Netherlands Heart Journal in 2017, I would like to introduce to you a new editorial board, consisting of an editor-in-chief and five deputy editors. It is an Amsterdam-based editorial board to facilitate close interaction between the board members. It is the ambition of the new editorial board to further improve the journal’s current impact factor as an index of its quality and success. The attractiveness of the journal for authors may be enhanced by offering a rapid review system, which requires close collaboration between the associate editors and the national editorial and international advisory board. Moreover, the Netherlands Heart Journal is an open access, peer review journal offering services without payment for authors in contrast with numerous other open access journals.Professor Jan J. Piek Fig.  completeYigal Pinto Fig.  was traiThe main research interest of the Pinto group  is to identify novel mechanisms that can help treat or diagnose cardiomyopathies, such as genetic forms of DCM. His group focuses on RNA biology in relation to genetic variants that are related to heart failure and translates findings to biomarkers or therapeutic targets.Robbert de Winter Fig.  studied The main research interest of his research group relates to acute coronary syndromes, diagnostics, risk prediction and outcome in STEMI and NSTEMI patients and biomarker studies. In addition, there is increased involvement in structural and congenital heart disease both in percutaneous procedures and in research projects.Ron Peters Fig.  had all cum laude), and defended his PhD thesis at that same institution in 2001. He was a research fellow with Michael R. Rosen MD  and with José Jalife . After his training as a cardiologist (2008) he subspecialised as a clinical electrophysiologist (2010), both at the AMC. The research interests of his group consist of mechanisms and treatment of atrial fibrillation, closely intertwined with the thoracoscopic atrial fibrillation ablation program and the Laboratory of Experimental Cardiology. Furthermore, his group publishes on arrhythmias in adult congenital heart disease.Joris de Groot Fig.  was bornJolanda van der Velden Fig.  studied The editorial board would like to take the opportunity to thank the previous board members and in particular Professor E.E. van der Wall, who served as editor-in-chief for almost three decades, initially as the editor-in-chief of the Nederlands Tijdschrift voor Cardiologie followed by the journal Cardiologie and finally the Netherlands Heart Journal. These efforts led to the current position of the Netherlands Heart Journal among the top of the 60 national society journals of the ESC-related countries. The editorial board acknowledges the support of the Netherlands Society of Cardiology in its endeavour to further improve the quality of the Netherlands Heart Journal.


The PDF and HTML versions of the paper have been modified accordingly.This article was originally published under a Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0), but has now been made available under a Creative Commons Attribution 4.0 International License (


However, 68Ga has several shortcomings as a radiolabel including short half-life and non-ideal energies, and this has motivated consideration of 18F-labelled analogs. 18F-PSMA-1007 was selected among several 18F-PSMA-ligand candidate compounds because it demonstrated high labelling yields, outstanding tumor uptake and fast, non-urinary background clearance. Here, we describe the properties of 18F-PSMA-1007 in human volunteers and patients.The prostate-specific membrane antigen (PSMA) targeted positron-emitting-tomography (PET) tracer 18F-PSMA-1007 was determined in three healthy volunteers who underwent whole-body PET-scans and concomitant blood and urine sampling. Following this, ten patients with high-risk prostate cancer underwent 18F-PSMA-1007 PET/CT (1 h and 3 h p.i.) and normal organ biodistribution and tumor uptakes were examined. Eight patients underwent prostatectomy with extended pelvic lymphadenectomy. Uptake in intra-prostatic lesions and lymph node metastases were correlated with final histopathology, including PSMA immunostaining.Radiation dosimetry of 18F-PSMA-1007 behaves similar to other PSMA-PET agents as well as to other 18F-labelled PET-tracers. In comparison to other PSMA-targeting PET-tracers, 18F-PSMA-1007 has reduced urinary clearance enabling excellent assessment of the prostate. Similar to 18F-DCFPyL and with slightly slower clearance kinetics than PSMA-11, favorable tumor-to-background ratios are observed 2–3 h after injection. In eight patients, diagnostic findings were successfully validated by histopathology. 18F-PSMA-1007 PET/CT detected 18 of 19 lymph node metastases in the pelvis, including nodes as small as 1 mm in diameter.With an effective dose of approximately 4.4–5.5 mSv per 200–250 MBq examination, 18F-PSMA-1007 performs at least comparably to 68Ga-PSMA-11, but its longer half-life combined with its superior energy characteristics and non-urinary excretion overcomes some practical limitations of 68Ga-labelled PSMA-targeted tracers.The online version of this article (doi:10.1007/s00259-016-3573-4) contains supplementary material, which is available to authorized users. In two studies  sites of biochemical recurrence (BCR) were localized in 90 % of patients including those with modest elevations in prostate specific antigen (PSA) F-Py-TFP [18F-PSMA-1007. The chemical structure of 18F-PSMA-1007 is presented in Fig. The non-radioactive reference compound F-Py-TFP  for coupF-Py-TFP . A direcAll imaging was performed on a Biograph mCT Flow scanner . PET was acquired in 3-D mode (matrix 200 × 200) using FlowMotion (Siemens). The emission data was corrected for randoms, scatter and decay. Reconstruction was performed with an ordered subset expectation maximization (OSEM) algorithm with two iterations/21 subsets and Gauss-filtered to a transaxial resolution of 5 mm at full-width at half-maximum (FWHM); Attenuation correction was performed using the unenhanced low-dose CT data. The CT-scans were reconstructed to a slice thickness of 5 mm, increment of 3–4 mm, soft tissue reconstruction kernel (B30), using CareDose (Siemens).18F-PSMA-1007 PET/CT with scans obtained at multiple time points up to 6 h p.i. The healthy subjects were imaged in three blocks. Block 1 began at PET-1 (start 5 min p.i.) and extended to PET-7 (ending 140 min p.i.), block 2 began at PET-8 (start 180 min p.i.) and extended to PET-9 (240–270 min p.i.) and block 3 began at PET-10 (440–480 min p.i.). A non-enhanced low-dose CT  for attenuation correction was performed at the beginning of each block, followed by serial emission scans without moving the volunteers in between.To determine initial dosimetry, three healthy volunteers  underwent n = 10) gave written informed consent to receive 18F-PSMA-1007 following guidelines of the German Pharmaceuticals Act §13(2b). All patients had newly diagnosed high-risk prostate cancer  with a median age of 65 years (range 55–77) and underwent 18F-PSMA-1007 PET/CT imaging 1 and 3 h post injection. The attenuation correction CTs were also performed 1 and 3 h post tracer injection. All men were treated clinically with a multimodal approach and eight men underwent radical prostatectomy with extended pelvic lymphadenectomy. Two patients who were found to have metastatic disease did not undergo prostatectomy. The data were analyzed retrospectively with approval of the local ethics committee. Detailed patient characteristics and final histopathological evaluation are provided in Table All patients n = 0 gave wrThe dosimetry analysis was performed using the QDOSE dosimetry software suite . Automatic rigid co-registration and, if necessary, additional manual correction, were performed for all PET and CT data-sets. Kidneys, liver, spleen, whole heart, upper and lower large intestine, parotid glands, submandibular glands and urinary bladder were segmented into volumes of interest (VOIs) using a percentage of maximum threshold between 20 and 30 % and the corresponding CT as guidance. Afterwards the time activity curves (TACs) were calculated for all organs. The TACs for red marrow were derived from venous blood samples, and the red marrow dose was calculated as described previously , 23. TheThe cumulative activity Ã between time 0 and the first measured time point was calculated assuming a linear increase from 0 to the first measured activity. The Ã between the first measured time point and the last measured time point was integrated numerically using trapezoidal approximation. The Ã from the last measured time point to infinity was integrated using the fitted function. The total body Ã was calculated based on the injected activity assuming only physical decay neglecting the voided urine. The Ã of the remainder of the body was then automatically calculated by subtracting all source organs from the total body activity. All source organ residence times were calculated by dividing the Ã by the injected activity.Absorbed and effective dose calculations were performed using the ICRP endorsed IDAC 1.0 package  which isDuring the dosimetry evaluation, urine was collected at the following intervals: 0–2 h, 2–4 h, and 4–6 h. Venous blood samples were obtained at 2, 5, 10, 15, 30, 45, 60, 80, 120, 180, 240, and 360 min post injection. After the whole blood was sampled, the remaining volume was centrifuged and serum was extracted. Serum activities were measured in a well counter and corrected for decay. Activity concentrations in blood vs. serum were compared using the individually determined  hematocrit. Total blood volume was estimated from size, weight, and hematocrit.mean and SUVmax at 1 h and 3 h post injection. For calculation of the standardized uptake value (SUV), circular regions of interest were drawn around the area with focally increased uptake in transaxial slices and automatically adapted to a three-dimensional VOI with e.soft software (Siemens) at a 40 % isocontour. Primary tumors and lymph node metastases were evaluated separately. The normal bladder (after voiding), background (pelvic fat), blood, brain, salivary and lacrimal glands, lung, liver, spleen, pancreas, small intestine, and kidneys were evaluated with a 2–3 cm sphere placed inside the organ parenchyma.The tracer biodistribution in patients was quantified by SUVAnalyses of prostatectomy specimens were performed under the supervision of dedicated uropathologists, using International Society of Urological Pathology nomenclature . PatholoAll patients and healthy volunteers tolerated the examination well. No drug-related pharmacological effects or physiologic responses occurred. All observed parameters  remained normal and unchanged during and after the examination. No patient reported subjective symptoms.18F-labelled PSMA-ligands DCFPyL (column 5), DCFBC (column 6), the 68Ga-labelled compounds PSMA-11  and PSMA-617 (column 4). In addition, the mean absorbed doses for the submandibular glands were 0.075 mGy/MBq, parotid glands 0.09 mGy/MBq and prostate gland 0.045 mGy/MBq. The intra-individual dose ratios are very similar for all PSMA-targeted agents; in comparison to 18F-DCFPyl, the effective dose of 18F-PSMA-1007 is slightly higher, while lower clearance through the urinary route reduces dose to the bladder wall.Maximum intensity projection (MIP) images of the serially performed whole-body PET-scans of one healthy volunteer are depicted in Fig. mean 2, perfusion dependent background <1) in the axillary lymph nodes was observed. Taking into account his normal PSA-value and an increased CRP and BSR (blood sedimentation rate), this finding implies the potential of false-positive uptake in inflammatory activated lymph nodes. The time-activity-curve of the normal prostate tissue in the healthy volunteers is provided in Fig. In healthy subjects, the blood pool contained mean 76, 22, 12 and 8 % of the injected dose at 2 min, 1 h, 2 h and 3 h p.i., respectively. Approximately 95 % of the blood pool activity was found in the serum; the blood and serum curves converge with more complete clearance. It is possible that there was a minimal and reversible diffusion of the agent into blood cells; however, irreversible binding to blood cells can be safely excluded. Clearance via the urinary tract was minimal and in average only 1.2, 0.7 and 0.5 % of the injected activity were eliminated in the urine during the 0–2, 2–4 and 4–6 h intervals, respectively. In one healthy volunteer, symmetric tracer uptake . Overall, 18F-PSMA-1007 PET/CT detected 18 of 19 lymph node metastases in the pelvis including a node with a diameter as small as 1 mm. In contrast to one healthy subject who was suspicious for unspecific uptake in inflammatory lymph nodes, there were no false-positive findings in the patient cohort (specificity 100 %). Similarly, the peak intra-prostatic uptake correlated with the main tumor mass  was comparable  allowing for excellent curve fitting. One source of uncertainty is the limited number of healthy subjects (n = 3). Nonetheless, since the normal organ biodistribution of 18F-PSMA-1007 has proven highly similar to other PSMA-targeted radiotracers, such as PSMA-11, PSMA-617, DCFBC and DCFPyL [18F-DCFPyL, in which it was shown that the dosimetry in normal organs was reliably assessed independently of the presence of tumor [Compared to other similar agents, the effective dose of d DCFPyL , 29, 30,of tumor .max of 18F-PSMA-1007 in tumor tissue, parotid glands, liver, kidneys and spleen can be compared to data for PSMA-11 [68Ga-labelled PSMA-ligands were also performed at our institution with similar imaging protocols, cross-calibrated scanners and analysis software. In contrast, 18F-DCFBC and 18F-DCFPyL have been evaluated with different scanners and imaging protocols which makes the comparison less accurate. However, even the comparison with PSMA-11 and PSMA-617 has limitations. The low patient numbers might introduce errors, especially on the highly variable inter-individual tumor uptake. In addition, due to the higher positron energy and increased spill-out-effects, 68Ga quantification might be systematically underestimated in small lesions. Furthermore, the patient selection differs: PSMA-617 was evaluated in a heterogeneous group of patients [177Lu-PSMA-617 dosimetry [68Ga-labelled PET-tracer. 18F-PSMA-1007 shares the slightly slower tracer kinetics of PSMA-617, although this is less crucial due to the longer half-life of 18F in comparison to 68Ga. Due to its structural similarity to PSMA-617, it has been suggested that it could be an ideal diagnostic surrogate for patients considering 177Lu-PSMA-617 therapy [18F-PSMA-1007 in visceral organs is negligible with regard to radiation burden and is also of limited clinical impact as visceral metastases occur late in the course of the disease. More importantly the increased uptake in tumor tissue compared to other tracers improves tumor-to-background ratios making it easier to detect small lymph node metastases. It remains to be seen whether patients with advanced castration-resistant PCa will have higher liver background which may interfere with the detection of metastases in comparison to the actual reference compound 68Ga-PSMA-11. One clear advantage for local staging is that 18F-PSMA-1007 is temporarily retained in the kidney parenchyma. For 18F-PSMA-1007, clearance via the urinary tract was only 1.2 % injected activity during 0–2 h and another 0.7 % 2–4 h p.i.; in comparison, 11 % of 18F-DCFPyl is eliminated during the first 2 h via the urinary tract and another 5 % until 3 h p.i. [max 40 (68Ga-PSMA-617) and SUVmax 100 (68Ga-PSMA-11) has been reported for the 68Ga-containing compounds, respectively [max 5 for 18F-PSMA-1007 Below is the link to the electronic supplementary material.


There is a lack of evidence concerning associations between novel parameters of arterial stiffness as cardiovascular risk markers and traditional structural and functional vascular biomarkers in a population-based Caucasian cohort. We examined these associations in the second follow-up of the Swiss Cohort Study on Air Pollution and Lung and Heart Diseases in Adults (SAPALDIA 3).Arterial stiffness was measured oscillometrically by pulse wave analysis to derive the cardio-ankle vascular index (CAVI), brachial-ankle (baPWV) and aortic pulse wave velocity (aPWV), and amplitude of the forward and backward wave. Carotid ultrasonography was used to measure carotid intima-media thickness (cIMT) and carotid lumen diameter (LD), and to derive a distensibility coefficient (DC). We used multivariable linear regression models adjusted for several potential confounders for 2,733 people aged 50–81 years.p < 0.001). All parameters were significantly associated with LD (p < 0.001), with aPWV and the amplitude of the forward wave explaining the highest proportion of variance (2%). Only CAVI and baPWV were significant predictors of DC (p < 0.001), explaining more than 0.3% of the DC variance.CAVI, aPWV and the amplitude of the forward and backward wave were significant predictors of cIMT (We demonstrated that novel non-invasive oscillometric arterial stiffness parameters are differentially associated with specific established structural and functional local stiffness parameters. Longitudinal studies are needed to follow-up on these cross-sectional findings and to evaluate their relevance for clinical phenotypes. Cardiovascular diseases (CVDs) are among the main causes of death globally, accounting for one third of all deaths . ArteriaPulse wave velocity (PWV) measurements, such as the cardio-ankle vascular index (CAVI) or brachial-ankle PWV (baPWV), are commonly used as surrogate measures of arterial stiffness . Novel pNovel parameters of arterial stiffness based on PWV measurement, such as CAVI or baPWV, and PWA parameters, such as estimated aPWV and wave reflection characteristics (amplitude of the forward and backward wave), have not yet been studied in a Caucasian population-based cohort. In particular, there is lack of evidence regarding their association with traditional structural (carotid intima-media thickness (cIMT), lumen diameter (LD)) and functional (distensibility coefficient (DC)) vascular biomarkers. Therefore, we examined whether these novel biomarkers of arterial stiffness are associated with traditional and well-established structural and functional markers of local stiffness in a cohort of older adults.http://www.sapaldia.ch).Data were derived from the Swiss Cohort Study on Air Pollution and Lung and Heart Diseases in Adults (SAPALDIA), an ongoing multi-center cohort study initiated in 1991 among randomly selected adults  . The preIn SAPALDIA 3, arterial stiffness was measured non-invasively by baPWV and CAVI using a VaSera VS-1500N vascular screening system . The degree of reproducibility between two measurements within a 90-day period is high in the study population, with a low mean coefficient of variation of 3.9% for baPWV and 4.4% for CAVI; high intraclass-correlation coefficients of 0.9 and 0.8, respectively; and low mean differences in percent of the respective mean for baPWV (0.9%) and CAVI (1.7%) [On the basis of these peripheral recordings, PWA was performed retrospectively using the ARCSolver algorithm  in order to derive aPWV for 1,267 participants as a surrogate marker of aortic stiffness, as well as central systolic and diastolic blood pressure and the amplitude of the forward and backward wave. PWA analysis was performed on a subsample of the study population  that showed no statistically significant differences from the rest of the study sample  in the main covariates of interest . The ARCSolver method is a mathematical procedure for calculating aortic stiffness and central hemodynamic measures from peripheral pulse waves and blood pressure measurements at the brachial artery using a common occlusive cuff . We have2)/(PP× dLD2), where deltaLD is the systolic-diastolic lumen diameter difference, dLD the diastolic lumen diameter, and PP the pulse pressure [Following a standardized protocol, bilateral ultrasound B-mode scans of the common carotid artery were taken by trained sonographers using the UF-870 ultrasound system . Previous analyses of this study population have proven excellent reproducibility of structural parameters, with a mean coefficient of variation of 3.98% and an intraclass correlation coefficient of 0.89 for cIMT and 1.6% and 0.97 for LD, respectively, as well as good reproducibility of functional indices  . Within pressure .p <0.05 was defined as statistical significance.Unless stated otherwise, data are expressed as mean (standard deviation (SD)). We used Pearson’s correlation coefficients for bivariate analyses. In multivariable linear regression models, the associations of either cIMT, LD or DC as outcomes with either CAVI, baPWV, aPWV or amplitude of the forward or backward wave as main predictors were adjusted for age, sex, body mass index, heart rate, mean arterial pressure ((2 x DBP)+SBP)/3, doctor diagnosed cardiovascular disease assessed by questionnaire , doctor diagnosed diabetes mellitus, and pack-years of smoking. All statistical analyses were performed using the statistical software STATA  and p < 0.001).CAVI was moderately and positively associated with cIMT r = 0.36) and LD (r = 0.33), and negatively associated with DC (r = -0.43) in bivariate analyses Figs –3. baPWV6 and LD p < 0.001) (p ≤ 0.001), with aPWV and the amplitude of the forward wave accounting for the highest proportion of variance compared to the other PWA predictors (PWA covariate R-squared 2%). Only CAVI and baPWV were significant predictors of DC in the multivariable regression models , accounting for 0.3% of the DC variance. aPWV was the strongest predictor of cIMT according to standardized beta coefficients (0.94), followed by age (0.35), and also the strongest predictor of LD (0.66), followed by sex (0.41) (data for age and sex not shown). Age was the strongest predictor of cIMT in the other models (standardized beta coefficients 0.37–0.42). Sex was the strongest predictor of LD in the other models (standardized beta coefficients 0.31–0.42). Age was the strongest predictor of DC in all models (standardized beta coefficients 0.42–0.50). The results of sensitivity analyses excluding participants with CVD were not materially different.Multivariable regression analyses, adjusted for several potential confounders, revealed that CAVI, aPWV, and the amplitude of the forward and backward wave were significant predictors of cIMT  . aPWV anCross-sectional results from a Swiss population of older adults show that novel non-invasive oscillometric arterial stiffness and wave reflection parameters are significantly associated with established structural and functional local stiffness parameters. aPWV had the strongest association with cIMT and LD. Only CAVI and baPWV were significantly associated with DC in multivariable regression models adjusted for several potential confounders. To the best of our knowledge, this is the first epidemiological study comparing the novel arterial stiffness parameters, CAVI, baPWV, estimated aPWV, and amplitude of the forward and backward wave, based on their associations with established local stiffness and structural parameters used for cardiovascular risk assessment and prediction.A one unit increase in CAVI was associated with a cIMT increase of 0.01 mm; a 1 m/s increase in baPWV and aPWV was associated with a cIMT increase of 0.002 mm and 0.08 mm, respectively; and a 1 mmHg increase of the forward and backward wave was associated with a cIMT increase of 0.005 and 0.004 mm, respectively. Given that the risk for future cardiovascular events increases by 16% per 0.1 mm increment in cIMT, according to a large meta-analysis of 16 cohorts , every iThe level of evidence concerning the usefulness of carotid ultrasonography-based measurements of cIMT for primary and secondary CVD prevention has recently been classified with the highest grade, A . HoweverOscillometrically measured arterial stiffness and wave reflection parameters are regarded as easily applicable tissue biomarkers and as cumulative measures of several cardiovascular risk factors and their influence on the vasculature with ageing . Both CAaPWV estimated from peripheral blood pressure measurement, using a transfer function like the ARCSolver algorithm, has shown good agreement with invasively measured aPWV—the gold-standard for aortic stiffness measurement . FurtherStandardized beta regression coefficients give the relative strength of the various predictors within a model. Our analyses showed that aPWV accounts for a certain amount of variance of cIMT and LD beyond the common underlying age factor, according to the beta coefficients of the multivariable regression analyses. Age is usually the strongest predictor of structural and functional markers of arterial stiffness , which wThis is the first population-based Caucasian cohort comparing the novel non-invasive arterial stiffness indices CAVI and baPWV with traditional markers of structural and functional local stiffness. So far, CAVI and baPWV have mainly been used among Asian populations, reducing the possibility of extrapolating the evidence to other ethnicities . FurtherWe demonstrated that novel non-invasive oscillometric arterial stiffness parameters are associated with established structural and functional local stiffness parameters. These parameters should not be used interchangeably, as these cross-sectional data point to a different predictive value of each of the parameters analyzed. Longitudinal studies are needed to follow-up on these cross-sectional findings and to evaluate their relevance for clinical phenotypes. Since arterial stiffness integrates the biological burden of various risk factors on the vasculature over a long period of time and is a non-invasive, easily applicable measure, it might have the potential to improve cardiovascular risk assessment in daily clinical practice.S1 Fig(TIF)Click here for additional data file.S2 FigScatter plots with linear prediction line (linear fit) and 95% confidence interval (95% CI). r denotes Pearson’s correlation coefficient.(TIF)Click here for additional data file.S1 File(DOC)Click here for additional data file.


International Journal of Molecular Sciences have established the Best Paper Award to recognize the most outstanding articles published in the areas of molecular biology, molecular physics and chemistry that have been published in the International Journal of Molecular Sciences. The prizes have been awarded annually since 2012 [The Editors of the nce 2012 ,4,5.International Journal of Molecular Science Best Paper Award” for 2017. Nominations, chosen from all papers published in 2016, were made by the Editorial Board. The awards are issued to reviews and research articles separately. Following a review process by the Editorial Board, three top-voted research articles and the three top-voted reviews as follows, in no particular order, have won “International Journal of Molecular Science Best Paper Award” for 2017:We are pleased to announce the “A RNA–DNA Hybrid Aptamer for Nanoparticle-Based Prostate Tumor Targeted Drug DeliveryJohn C. Leach, Andrew Wang, Kaiming Ye and Sha JinInt. J. Mol. Sci.2016, 17(3), 380; doi:10.3390/ijms17030380http://www.mdpi.com/1422-0067/17/3/380Available online: + prostate cancer cells, resulting in the deaths of most of the cells. This report demonstrated that the drug delivery platform yields statistically significant effectiveness, being more cytotoxic to the targeted cells as opposed to the non-targeted cells . A super paramagnetic iron oxide nanoparticle conjugated with aptamer-doxorubicin (SPIO-Apt-Dox) has been fabricated and employed as a targeted drug delivery agent. The experimental results indicate that the aptamer anti-tumor agent inhibits nonspecific uptake of membrane-permeable doxorubicin to the non-target cells, leading to reduced untargeted cytotoxicity and endocytic uptake. In the meantime, it has specificity to targeted PSMAed cells .Relevance of MicroRNA200 Family and MicroRNA205 for Epithelial to Mesenchymal Transition and Clinical Outcome in Biliary Tract Cancer PatientsRomana Urbas, Christian Mayr, Eckhard Klieser, Julia Fuereder, Doris Bach, Stefan Stättner, Florian Primavesi, Tarkan Jaeger, Stefanie Stanzer, Anna Lena Ress, Magdalena Löffelberger, Andrej Wagner, Frieder Berr, Markus Ritter, Martin Pichler, Daniel Neureiter and Tobias KiesslichInt. J. Mol. Sci.2016, 17(12), 2053; doi:10.3390/ijms17122053http://www.mdpi.com/1422-0067/17/12/2053Available online: Biliary tract cancer (BTC) is a very aggressive kind of cancer characterized by late diagnosis of disease, still limited therapeutic options and bad outcome. Hence, there is an urgent need for new therapeutic approaches and targets. A commonly observed histopathological characteristic of BTC is an intensive epithelial-to-mesenchyml transiton (EMT)—a key event in tumor progression und metastasis and potential reason for the dismal outcome of BTC. It is known that EMT is essentially regulated by microRNAs (miRs). Therefore, we started a comprehensive molecular-pathological in-vitro and in-situ expression investigation of the anti-EMT miR200 family  and miR205 as well as of the EMT-related proteins E-Cadherin and Vimentin in a panel of BTC cell lines and clinical specimens. The results revealed a significant association of miR expression with (i) the expression patterns of E-Cadherin and Vimentin; (ii) clinicopathological characteristics; and (iii) survival data. The detailed statistical survival analysis could demonstrate that expression levels of the miR200 family members (but not miR205) as well as of E-Cadherin and Vimentin could serve as positive and negative prognostic markers in cases with BTC and possibly as therapeutic targets in the future .Comparative Study of Green Sub- and Supercritical Processes to Obtain Carnosic Acid and Carnosol-Enriched Rosemary Extracts with in Vitro Anti-Proliferative Activity on Colon Cancer CellsAndrea del Pilar Sánchez-Camargo, Virginia García-Cañas, Miguel Herrero, Alejandro Cifuentes and Elena IbáñezInt. J. Mol. Sci.2016, 17(12), 2046; doi:10.3390/ijms17122046http://www.mdpi.com/1422-0067/17/12/2046Available online: In the last few years, our laboratory has been intensively working on the Foodomics evaluation of rosemary extracts with in-vitro antiproliferative activity against colon cancer cell lines. During our studies, two compounds were detected as the main cause of the observed bioactivity (carnosic acid and carnosol). In the present work, we evaluated the potentiality of four different green extraction processes to obtain enriched fractions of these two phenolic diterpenes; these processes were all based on the use of compressed fluids’ technologies, employing green solvents and following the most recent trend of intensifying or integrating processes. Moreover, an in-depth chemical characterization of the most active extracts  suggested that other compounds, such as rosmaridiphenol and safficinolide, could also play a role as additional contributors to the observed strong anti-proliferative activity of carnosic acid and carnosol enriched extracts .Telomeres, NAFLD and Chronic Liver DiseaseBenedetta Donati and Luca ValentiInt. J. Mol. Sci.2016, 17(3), 383; doi:10.3390/ijms17030383http://www.mdpi.com/1422-0067/17/3/383Available online: In the Review article entitled “Telomeres, NAFLD and Chronic Liver Disease”, we survey the literature supporting a role of telomeres homeostasis and Telomerase complex activity in the pathogenesis of non-alcoholic fatty liver disease (NAFLD), now the most frequent cause of liver damage worldwide, focusing on its link to the progression of chronic liver disease and cancer.We describe telomeres shortening as a genetic tract that can be inherited through generations predisposed to organ failure and often connected to the occurrence of mutations in Telomerase complex genes. Indeed, experimental evidence links telomere impairment to degenerative and chronic conditions such as fibrosis, defining telomere shortening, a hallmark of cirrhosis. Furthermore, we summarize the different mechanisms of telomeres re-elongation in the development of hepatocellular carcinoma (HCC) including Telomerase reverse transcriptase (TERT) reactivation, the increased expression of the other proteins involved in telomere regulation and alternative lengthening of telomere (ALT). We highlighted that TERT also influences liver carcinogenesis by other pathways independent of telomeres regulation. In conclusion, we propose a mechanism accounting for the predisposing effect of telomeres length deregulation and TERT germline mutations on the pathogenesis of NAFLD-HCC, a condition strongly influenced by genetic factors. Finally, we speculate on the possible use of drugs targeting the telomere machinery in clinical practice for the prevention and treatment of severe liver disease related to NAFLD and other causes of liver damage .The Functions of Metallothionein and ZIP and ZnT Transporters: An Overview and PerspectiveTomoki Kimura and Taiho KambeInt. J. Mol. Sci.2016, 17(3), 336; doi:10.3390/ijms17030336http://www.mdpi.com/1422-0067/17/3/336Available online: The field of zinc metabolism and homeostasis has undergone a dramatic expansion. In recent decades, an increasing amount of evidence has uncovered critical roles of a number of proteins in zinc metabolism and homeostasis through influxing, chelating, sequestrating, coordinating, releasing, and effluxing zinc. Metallothioneins (MT) and Zrt- and Irt-like proteins (ZIP) and Zn transporters (ZnT) are the proteins primarily involved in these processes, and thus play crucial roles in maintaining the cellular zinc homeostasis. This review paper summarizes our understanding of the physiological and biological functions of MTs and ZIP and ZnT transporters focusing on recent progresses. We hope it provides a better understanding of zinc biology as well as an understanding of the roles of zinc in health and disease .o-Quinones in Catecholamine Metabolism and Eumelanin BiosynthesisReactivities of Quinone Methides versus Manickam SugumaranInt. J. Mol. Sci.2016, 17(9), 1576; doi:10.3390/ijms17091576http://www.mdpi.com/1422-0067/17/9/1576Available online: N-acyldopamine quinone methides and quinone methide imine amides play a vital role in the crosslinking and hence hardening reactions. Finally, the biological activities of dopa/dehydrodopyl peptides found in numerous marine organisms are often exhibited through transient quinone methide intermediates. Our laboratory also identified three enzymes associated with quinone methide metabolism. Thus, this review highlights the importance of quinone methides as key transient intermediates of numerous catecholamine metabolisms (Catecholamines form the building blocks of melanin pigment in animals and the exoskelatal pigment in insects. Tyrosine and dopa serve as the precursor for melanogenesis and a related process, called sclerotization in insects. My laboratory is actively involved in studying the metabolic fate of catecholamines and enzymes associated with the process. The overall aim of our research is to establish the importance of quinone methides in the biochemistry of catecholamine metabolism involved in melanin biosynthesis, insect cuticular sclerotization, and bioadhesive/antibacterial activities of tunicates and mussels. In this article, the detailed biochemistry of quinone methides formed during the biotransformation of different catecholamines is discussed. In melanin biosynthesis, quinone methides are generated as key intermediates of dopachrome conversion to dihydroxyindoles. In addition, they are also involved at the later stages of indole polymerization. In insect cuticular sclerotization, quinone methide derivatives such as abolisms .International Journal of Molecular Science and the scientific research field. On behalf of the International Journal of Molecular Science Editorial Board, we would like to congratulate these teams for their excellent work. In recognition of their accomplishment, they will receive the privilege of publishing an additional research article or review paper free of charge in open access format in the International Journal of Molecular Science, after the usual peer-review procedure.We believe that these six exceptional papers are valuable contributions to the International Journal of Molecular Science, and thank the International Journal of Molecular Science Editorial Board for voting and helping with this “Best Paper Award”.We would like to take this opportunity to thank all the nominated research groups of the above exceptional papers for their contributions to the International Journal of Molecular Science is committed to meeting the needs of the molecular research community by providing useful and timely reviews of all manuscripts submitted, and providing an open access journal for your results. Please consider submitting your work to the International Journal of Molecular Science, and we look forward to announcing your paper as an International Journal of Molecular Science Best Paper in the future.The Editorial Board and Editorial Staff at the International Journal of Molecular Science Editorial Board.


Thoracic endovascular aortic repair (TEVAR) has been shown to lead to increased aortic stiffness. The aim of this study was to investigate the effect of stent graft type and stent graft length on aortic stiffness in a controlled, experimental setting.1); after deployment of a 100-mm long stent graft (t2); and after distal extension through deployment of a second 100-mm long stent graft (t3). Four different types of stent grafts  were evaluated.Twenty porcine thoracic aortas were connected to a pulsatile mock loop system. Intraluminal pressure was recorded at two sites in order to measure pulse wave velocity (PWV) for each aorta: before stent graft deployment , and by a mean 1.4 m/s or 23.0% of baseline PWV after distal extension of the stent graft (P<0.001). Univariable regression analysis showed a significant correlation between aortic PWV and extent of stent graft coverage, (P<0.001), but no significant effect of baseline aortic length, baseline aortic PWV, or stent graft type on the percentual increase in PWV at tIn this experimental set-up, aortic stiffness increased significantly after stent graft deployment with each of the four types of stent graft, with the increase in aortic stiffness depending on the extent of stent graft coverage. Thoracic endovascular aortic repair (TEVAR) is the first choice of treatment for patients with thoracic aortic diseases and suitable anatomy . UnderstIt is currently unknown which factors are related to the increase in aortic stiffness after TEVAR. The material of aortic stent grafts can be up to a 100 times stiffer than native aortic tissue , 5. HoweThe experimental set-up that was used has been described previously . For the1). The aorta was disconnected from the circuit and a 100-mm long stent graft with a diameter of 26 mm was deployed at the level where the aorta had a diameter of ±23 mm, to reach a 10% degree of oversizing. The aorta was then reconnected and intraluminal pressures were recorded for five cycles (t2). The stent graft was then extended with a second 100-mm long stent graft with a diameter of 22–24 mm and a 4 cm overlap, as is most commonly done in clinical practice. Intraluminal pressures were again recorded for five cycles (t3). Average aortic pulse wave velocity (PWV) at t1, t2, and t3 was calculated by dividing the distance between ascending aorta and celiac trunk by the average difference in time between the pressure peaks over the five recorded cycles  is characterized by a continuous sinusoidal-shaped nitinol stent; Type 2  is made of Z-shaped nitinol stents with a woven polyester graft and one longitudinal nitinol bar. Type 3  is made of Z-shaped nitinol stents with a woven polyester graft and external fixation barbs. Type 4  is made of Z-shaped nitinol stents with woven polyester graft. A description of the stent grafts used in the experiments is shown in Four different stent graft types of comparable sizes were used; each stent graft type was tested in five different aortas. Stent graft type 1 (Conformable Gore1 vs t2 vs t3) on aortic PWV, and the effect of baseline aortic length and PWV and stent graft type on the percentual increase in PWV at t2 and t3. Statistical significance was assumed at P<0.05.IBM SPSS Statistics version 22.0 was used for statistical analysis. Normality of data was tested with the Shapiro-Wilk test. Univariable regression analysis was performed to test the effect of extent of stent graft coverage (t2), and 7.9 m/s (±1.5 m/s) after distal extension of the stent graft (t3). This corresponded to an 8.9% (±8.8%) increase in PWV at t2 (P<0.001) and a 23.0% (±17.4%) increase at t3 (P<0.001).Mean aortic length (n = 20) was 384.3 mm (± 28.44 mm) at baseline, and did not differ between the different stent graft groups (P = 0.294). Mean aortic PWV was 6.4 m/s (±0.7 m/s) at baseline, 7.0 m/s (±0.9 m/s) after deployment of a 100-mm proximal stent graft . Baseline PWV increased by 21.3% (±12.2%) with stent graft type 1, 11.7% (±9.4%) with stent graft type 2, by 30.1% (±20.6%) with stent graft type 3, and by 27.9% (±23.3%) with stent graft type 4 at t3. The differences in PWV increase at t3 did not reach statistical significance (P = 0.462).Baseline PWV increased by 8.0% (±5.4%) with stent graft type 1, by 4.7% (±8.8%) with stent graft type 2, by 8.4% (±8.3%) with stent graft type 3, and by 14.5% (±11.6%) with stent graft type 4 at t2  or on the increase in PWV at t3 (P = 0.548 and P = 0.610). Full details on the data underlying these findings can be found as supporting information Univariable regression analysis showed a significant correlation between aortic PWV and extent of stent graft coverage, (P<0.001). There was no statistically significant effect of baseline aortic length or baseline aortic PWV on PWV increase at tThe interaction between native aorta and aortic stent grafts is not fully understood and may depend on stent graft design , 10. We Increased aortic PWV is widely accepted as a predictor of cardiovascular mortality, because it is a marker of concomitant atherosclerotic disease. However, there are also independent pathophysiological consequences of increased aortic stiffness. A stiffer aorta has a reduced capacity for storage, causing the pulse wave to reach the resistance of the peripheral circulation at a higher velocity, increasing the amount of pulse wave reflections and cardiac afterload . FurtherApparently, the most commonly used types of graft fabric and shapes of stent rings have at least similar stiffening effects. None of the currently used stent grafts have the capacity for longitudinal expansion. Developing a stent graft that does preserve the longitudinal distensibility of the native aorta while providing enough strength is a challenge that may be worth exploring. Aortic radial pulsatility may be preserved after stent graft deployment with the right amount of oversizing , thanks Experimental set-ups have inherent limitations. No formal quality control of the porcine aortic specimens was performed, so the presence of connective tissue disorders or microscopic atherosclerosis could not be excluded, and may have confounded our results. Porcine tissue is more elastic than very aged  or aneurAortic stiffness increased significantly after stent graft deployment in this experimental study. The increase in aortic stiffness depended on the length of the aorta that was covered by stent graft but not on the type of stent graft.S1 Table(XLSX)Click here for additional data file.


We should not forget that the journal has appeared many years ago, when information sources were limited – its appearance was well received due to the information provided. The appearance of “Mr. Google” generated, as in many others domains, changes of the community’s attitude towards it. Many critical opinions have emerged because the journal was not able to support those who were in need of promotion, due to the lack of quotations. The editorial staff made admirable efforts, but if we make an effort to publish an article, we should get the right amount of support for it. Obtaining a quotation for the journal is a long and difficult process that requires access to a “club” of periodical publications and involves certain rules for publishing.To summarize, there are some mandatory conditions:• international editorial board• representative graphic design• respecting the date of the appearance – quarterly• articles written only in English.Perhaps many of us do not know that, in order to publish in a journal with international recognition, one has to pay an important amount of money - hundreds of euros or dollars. But, more than that, the journal reflects the national scientific level of our specialty and, in order to be appreciated as closer to reality as possible, it must be read by colleagues from other countries, not only by us, the Romanians. We advocate to publish the journal in an international language. The Board of the Society chose the English language. Knowing the results of the community work, being aware of its value is difficult, requires continuity, consistency and time. The journal is a reflection of what we do and where we stand, with good and bad things. This is what the Board of the Society is trying to do now, by assuming these principles and by taking this decision. I think we made a wise decision that will pay back on the long run. It is not in our advantage to continue in the same manner and the result can be seen.Marketing is an important process that should be known everywhere. This means an effort made mainly by specialists, not only by us, in marketing. We also have to send our review to different places  and to different personalities. This is something to be evaluated in time, depending on the evolution of our success with the “Romanian Journal of Ophthalmology”.


STJ stiffness was also correlated with the composite spasticity index (CSI), implying that this index had an advantage in reflecting the mechanism of valgus deformity and should be considered as a necessary measurement of foot valgus in CP children. The present method for quantification of STJ stiffness could improve the accuracy in the diagnosis and classification of foot deformity and may help increase the understanding of the biomechanical factors in foot deformity rehabilitation.Relaxed calcaneal stance position (RCSP) is an important index in the correctional treatment of foot valgus deformities for cerebral palsy (CP) children. However, patients with similar RCSP showed diverse outcomes when accepting similar treatment, as the corrective resistance of subtalar joint (STJ) could be quite different. This study aimed to investigate the relationship between STJ stiffness and RCSP in different loading conditions. 38 valgus feet of 19 CP subjects were included in the study. A reposition force was applied beneath the STJ and pushed the foot from pronated position to neutral position. The STJ stiffness was calculated as the slope of the line fitting the force-displacement data. Correlations between the STJ stiffness, RCSP, and composite spasticity index (CSI) were analyzed. The spearman correlation coefficient indicated that STJ stiffness had no correlation with RCSPs, yet it had negative correlation with the change of RCSP under difference loading conditions (ΔRCSP Planovalgus is the most common foot deformity in cerebral palsy (CP) children –4. It isRelaxed calcaneal stance position (RCSP), radiographs, hindfoot valgus angle, and valgus index are commonly used methods to assess the foot valgus severity , 15–17. Subtalar joint (STJ) stiffness is a biomechanical index that reflects the corrective resistance of the foot planovalgus. Clinicians have found that patients with similar morphological valgus could have very different corrective resistance –24. TreaThis study aimed to investigate the relationship between the STJ stiffness and the morphological characteristics of valgus foot (RCSP) in CP children. Since there was no exact definition of STJ stiffness, the STJ stiffness was defined as the resistance in response to a reposition force, which was widely used for valgus deformity correction in CP children. To compare the STJ stiffness with RCSP from the perspective of foot deformity pathology, the correlations between the composite spasticity index (CSI) and the valgus parameters (STJ stiffness and RCSP) were also investigated.A total of 19 diplegic CP children  participated in this study. The inclusion criteria were as follows: (a) the spasticity was lower than level III according to the modified Ashworth scale ; (b) valThe STJ stiffness was measured with the device designed in our previous study  26]. To. To26]. α in The subject was positioned in relaxed standing. The RCSP was defined as the angle between the calcaneal bisection line and the line perpendicular to the ground   < 0.05 indicate a considerable correlation. A two-way random average measure intraclass correlation coefficient ) was used to assess the repeatability of the methodology [Spearman correlation matrices of STJ stiffness, RCSPs, and CSI were calculated by correlation analysis with the SPSS software . A correlation coefficient different from 0 and a significant level  was 0.999, 0.998, and 0.998 N2, respectively. The STJ stiffness-CSI data was shown in The force-displacement data of the reposition process in one foot was shown in 1w) was 15.91° ± 4.49°, under half-weight-bearing condition (mean RCSP0.5w) it was 11.06° ± 4.64°, and under non-weight-bearing condition (mean RCSP0w) it was 1.92° ± 4.61°. The RCSP-CSI data of 38 feet under three loading conditions were shown in Figures 1w and RCSP0.5w (ΔRCSP1w−0.5w), between RCSP1w and RCSP0w (ΔRCSP1w−0w), and between RCSP0.5w and RCSP0w (ΔRCSP0.5w−0w) were calculated , and 10° < RCSP0.5w ≤ 20° was classified as moderate valgus group (group 2) [0.5w increasing, mean STJ decreased and ΔRCSP1w−0.5w decreased, while ΔRCSP1w−0w and ΔRCSP0.5w−0w increased.The feet were further divided into two groups. 0° < RCSPgroup 2) . The mea1w−0.5w but has negative correlations with ΔRCSP1w−0w and ΔRCSP0.5w−0w.The correlations between STJ stiffness, RCSPs, and ΔRCSPs were shown in r = 0.343∗; p = 0.035). RCSP1w, RCSP0.5w, and RCSP0w were correlated with ΔRCSP1w−0w and ΔRCSP0.5w−0w. ΔRCSP1w−0.5w was not correlated with any other variables. Furthermore, the correlations between CSI subindexes  and the valgus indexes were shown in r = 0.335∗; p = 0.040) and clonus . Tendon jerk was not correlated with any other variables. ICC was 0.995 and the lower and upper bounds of the 95% confidence interval were 0.991 and 0.997, which indicate desirable repeatability of the methodology.A Spearman correlation matrix of all 8 variables was shown in r = 0.343; p = 0.035), whereas it had no correlation with RCSPs . Since CSI is a reliable measure of lower extremity spasticity, which is one of the critical factors of foot valgus in CP children, the findings implied that STJ stiffness could be more directly related to the gait and foot function than RCSP.This study investigated the relationship between indexes of the foot valgus in spastic CP children, the STJ stiffness, and RCSPs. The character of STJ to resist deformation was contributed by talocalcaneal joint, talonavicular joint, and the related connective tissues. The STJ stiffness plays an important role in foot kinetics, especially during weight-bearing activities. However, there was no exact definition of STJ stiffness previously. In the present study, the STJ stiffness was defined as the stiffness in response to an external force with a horizontal angle of 40 degrees, which was a widely used reposition force for valgus deformity in CP children. In the present study, CSI had a significant correlation with STJ stiffness  had no significant relationship with STJ stiffness. Although some patients' RCSPs are similar, their joint mechanical properties relevant to the reposition ability may be diverse  [RCSP was chosen as the morphological parameter to indicate the degree of foot deformity in this study, because the morphology-based indexes  have high correlations between each other , 18, andibility) . The prep = 0.014) and ΔRCSP0.5w−0w . The correlation factors were small, because ΔRCSP reflected the calcaneal deformation in response to the vertical force on pelma, yet STJ stiffness reflected the calcaneal deformation in response to the reposition force.Although RCSP is a morphological index under a loading condition, ΔRCSP could indicate the morphological change under different loadings, which means that ΔRCSP could partially reflect the ankle stiffness in the coronal plane of the heel. The results indicated that STJ stiffness had significant negative correlations with ΔRCSP1w−0w . STJ stiffness was also correlated with the composite spasticity index (CSI), implying that this index had an advantage in reflecting the mechanism of valgus deformity and should be considered as a necessary measurement of foot valgus in CP children. The present method for quantification of STJ stiffness could improve the accuracy in the diagnosis and classification of foot deformity and may help increase the understanding of the biomechanical factors in foot deformity rehabilitation.The present study quantified two indexes of the foot valgus in 38 feet of spastic CP children, the STJ stiffness and RCSPs (in three loading conditions). STJ stiffness had no correlation with RCSPs, yet it had negative correlation with the change of RCSP under different loading conditions (ΔRCSP


Myrtus communis and Pistacia lentiscus. To do so, we collected open-pollinated progeny from selected maternal plants, calculated mating system parameters using microsatellite genotyping and conducted sowing experiments under greenhouse and field conditions. Our results showed that some progeny fitness components were negatively affected by the high correlated paternity of maternal plants. In Myrtus communis, high correlated paternity had a negative effect on the proportion and timing of seedling emergence in the natural field conditions and in the greenhouse sowing experiment, respectively. In Pistacia lentiscus, seedling emergence time under field conditions was also negatively influenced by high correlated paternity and a progeny survival analysis in the field experiment showed greater mortality of seedlings from maternal plants with high correlated paternity. Overall, we found effects of correlated paternity on the progeny performance of Myrtus communis, a self-compatible species. Further, we also detected effects of correlated paternity on the progeny emergence time and survival in Pistacia lentiscus, an obligate outcrossed species. This study represents one of the few existing empirical examples which highlight the influence that correlated paternity may exert on progeny performance in multiple stages during early seedling growth.Anthropogenic habitat deterioration can promote changes in plant mating systems that subsequently may affect progeny performance, thereby conditioning plant recruitment for the next generation. However, very few studies yet tested mating system parameters other than outcrossing rates; and the direct effects of the genetic diversity of the pollen received by maternal plants (i.e. correlated paternity) has often been overlooked. In this study, we investigated the relation between correlated paternity and progeny performance in two common Mediterranean shrubs,  Mating patterns are fundamental for determining the amount and distribution of genetic variation within and between populations of plant species , 2. AnthHowever, although numerous studies aimed at evaluating variation in mating systems within species , 15, onlPistacia lentiscus L. (Anacardiaceae) and Myrtus communis L. (Myrtaceae) . Even though their life-history traits are similar in a number of ways (e.g. both are long-lived and their seeds are dispersed mainly by birds), they differ in their breeding and pollen dispersal systems. Myrtus has a mixed-mating system with hermaphroditic flowers pollinated by insects, while Pistacia is a wind-pollinated dioecious species (and thus an obligate outcrosser). The mating systems of both species have been extensively studied. For instance, Albaladejo et al. ).For each species, we compared seedling performance between experiments (greenhouse vs. field) using the non-parametric Wilcoxon paired test. Correlations between seedling height and seedling total dry biomass, as well as between shoot and root dry biomass were made using Spearman’s correlations. We reported data as means ± SE.rp) and progeny performance measurements using generalized linear models (GLMs) in R 3.0.2 . We conducted all analyses at maternal plant level and used a gamma distribution with a log link function for modelling the response variables. To check whether rp was correlated with other mating system variables we performed Spearman’s correlation tests and observed no significant correlations between correlated paternity and any other genetic parameter  neither in Myrtus nor in Pistacia (Myrtus fruit) and weighed it to the nearest 0.1 mg.We evaluated the relationships between the correlated paternity  and Pistacia (n = 283). Microsatellite markers were highly polymorphic in both species with a total of 57 and 40 different alleles identified across all maternal plants for Myrtus and Pistacia, respectively. Linkage disequilibrium tests showed that all pairs of loci in the two species were independent and null allele presence was not significant at any loci within any species.We genotyped open-pollinated progeny from 18 maternal plants of Myrtus maternal plants had high levels of homozygosity (mean HL = 0.386 ± 0.035) and correlated paternity (mean rp = 0.395 ± 0.045)  and correlated paternity (mean rp = 0.060 ± 0.006). Both parameters presented a large variation among Myrtus maternal plants: HL ranged from 0.18 to 0.69 and rp from 0.10 to 0.87. In Pistacia maternal plants, we also found high variability in HL levels, ranging from 0.00 to 0.49 but rp values were more stable among maternal plants, ranging from 0.03 to 0.11. As expected in a dioecious species, estimated outcrossing rate (tm) in Pistacia was very close to one .Overall, ± 0.045) . On the e to one . The mea to 0.98 . Myrtus Myrtus and 49.17% (± 1.73) in Pistacia. Myrtus seedlings took on average 20.04 (± 0.40) days to emerge (emergence time was 14–36 days after sowing), while Pistacia seedlings took 18.90 (± 0.40) days for emergence (range of 14–44 days) (Myrtus and 16.38% in Pistacia). Approximately one month after the start of the experiment, Pistacia seedlings had a mean height of 26.9 (± 0.8) mm and Myrtus seedlings a mean of 16.7 (± 0.8) mm (Pistacia seedlings and 257.5 (± 9.3) mm for Myrtus seedlings. Pistacia seedlings had a final mean dry biomass of 2.87 (± 0.04) g and Myrtus seedlings 2.39 (± 0.04) g . Shoot and root dry biomass were also highly correlated in both species .The mean percentage of seedling emergence was 53.33% (± 2.26) in 44 days) . Seedlin 0.8) mm . At the  0.04) g . In bothMyrtus ‘proportion of seedling emergence’, ‘seedling growth’ or ‘seedling biomass’ but had a significant positive effect on ‘seedling emergence time’  (The influence of correlated paternity on seedling performance in the greenhouse environment is summarized in ce time’ . Correlaiomass’) .Myrtus and in the first measurement of seedling height (‘seedling height after 36 days’) in both species . However, field conditions had a strongly significant negative effect on Myrtus seedling emergence . Both Myrtus and Pistacia seedlings took longer to emerge in the field than in the greenhouse  (Myrtus seedlings in the field took on average 39.19 (± 0.95) days to emerge, whereas Pistacia seedlings emerged slightly earlier, 34.24 (± 0.60) days.ctively) . Myrtus Pistacia (only 40 seedlings survived to the end of the experiment) and due to the lack of surviving Myrtus seedlings. Myrtus seedling mortality rate rose dramatically from 12.38% at day 29 to approximately 100% at day 78 and was clearly associated with the measured volumetric water content in the ground . Our study is one of the few to date that has investigated whether the pollen diversity naturally received by maternal plants–here assessed by the correlated paternity–has an influence on the subsequent progeny performance. To accomplish our goal, we integrated mating system analysis with data on progeny performance both in greenhouse and field-sowing experiments.Myrtus. Under greenhouse conditions, we observed that high correlated paternity was associated to a delay in seedling emergence. In Mediterranean environments early emergence is of critical importance in determining subsequent seedling growth ).rp independently of tm or HL results is observed), which agrees with findings in other studies . NevertPistacia, no significant impact of correlated paternity on progeny performance was evident in the greenhouse conditions. Pistacia is an obligate outcrossed species so it is not surprising that it has low levels of correlated paternity. Thus, the inherent pollination biology of Pistacia, (leading to inherent low levels of correlated paternity) is diluting the potential trends that we found in Myrtus.In Pistacia seedling performance. For instance, and even though abiotic factors  may be a major cause of the low seedling establishment in the Mediterranean [However, under field conditions we were able to detect the influence of correlated paternity on erranean , 44, 51,erranean , our surerranean , 53.Pistacia emergence time, a fundamental characteristic in the Mediterranean, as mentioned before. In fact, several Pistacia characteristics such as early seedling emergence make this species competent and able to survive efficiently in the Mediterranean climate [Pistacia seedlings (as compared to Myrtus) in the field coincided with more benign conditions that resembled those in the greenhouse, which may have led to the similar percentage of emergence observed in the two sowing experiments for this plant. After the first summer, only 8% of Pistacia seedlings survived in the field experiment, a low survival rate but higher than that of Myrtus (in which no seedlings survived to the end of the experiment). Summer water deficit is the main factor of stress and mortality for seedlings in Mediterranean woody plants [Myrtus seedling mortality was already 48.57% after just two months of the experiment (approximately at the end of April), by which time the measured volumetric water content in the ground had fallen dramatically. Plant species that do not form persistent soil seed banks (such as Myrtus or Pistacia) depend on a short temporal window after dispersal or on rare rainfall events to be able to recruit. Bearing in mind these conditions, the probability of natural seedling establishment in these two species–and especially in Myrtus–is extremely limited [Under field conditions, we also observed the influence of correlated paternity in delaying  climate . Early e climate . The timy plants , 44, 51. limited .In this study, seedlings assessed for progeny fitness were not genotyped, which have been ideally the best approach in order to obtain direct fitness–mating system associations. However, we rather used family mating system estimates to detect associations between correlated paternity and progeny fitness. We considered this approach in order to avoid bias in the estimation of the mating system parameters, because genotyping only emerged seedlings could have biased upwardly the mating system estimates (considering that the percentage of emerged seeds in the greenhouse was ~ 50% for both species and in the field experiment the percentage was even lower). Bearing this in mind, it is fundamental to reckon that in this study the detected effects between mating system parameters and the fitness variables are indirect.Our results on progeny fitness-correlated paternity associations found multiple non-significant effects and thus, we recognize that our results might lack significance once a multiple correction is applied. However, we considered that our study present novel results which could promote the forthcoming of new researches on the effects correlated paternity in other plant species and future meta-analysis studies.Myrtus, a self-compatible species. Even so, in Pistacia (outcrossed species) the negative consequences of high correlated paternity on progeny emergence time and survival under field conditions were also detected. We conclude that our findings, in combination with a handful of recent empirical studies, suggest that mating pattern parameters–other than outcrossing rates–influence progeny performance in multiple stages during early seedling growth.We found that the correlated paternity has an impact on different aspects of progeny performance. In this study, we report a strong influence of correlated paternity on the progeny performance of S1 TablePearson’s correlation coefficients shown.(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 TableData are given as means ± SE.(PDF)Click here for additional data file.