Gracilis Muscle

We added scorings for Yonghesuchus sangbiensis, based upon our original observations of the holotype of this taxon (IVPP V 12378), to the morphological cladistic data set of Nesbitt [2 ]. We also added a new character to the data set, resulting in a modified data matrix composed of 413 characters and 78 taxa (following the a priori pruning of the following operational taxonomic units [OTUs] that were also excluded by Nesbitt [2 ]: Archosaurus rossicus, Prestosuchus chiniquensis, UFRGS 0156 T, UFRGS 0152 T, Lewisuchus admixtus and Pseudolagosuchus major). In addition, several scorings for Gracilisuchus stipanicicorum and Turfanosuchus dabanensis were modified based upon new direct observations and interpretations of the relevant specimens. Additional file 1 provides details of and justifications for all rescorings and Additional file 2 is the data matrix. This data matrix is also available at TreeBASE (http://purl.org/phylo/treebase/phylows/study/TB2:S15917).
The data matrix was analysed under equally weighted parsimony using TNT 1.1 [21 (link)]. A heuristic search with 100 replicates of Wagner trees (with random addition sequence) followed by TBR branch-swapping (holding 10 trees per replicate) was performed. The best trees obtained from the replicates were subjected to a final round of TBR branch swapping. Zero length branches in any of the recovered MPTs were collapsed (rule 1 of Coddington & Scharff [22 (link)]). Characters 32, 52, 75, 121, 137, 139, 156, 168, 188, 223, 247, 258, 269, 271, 291, 297, 328, 356 and 399 were treated as additive (ordered) following Nesbitt [2 ], as was the new character 413. Decay indices (=Bremer support values) were calculated and a bootstrap resampling analysis, using 10,000 pseudoreplicates, was performed reporting both absolute and GC (i.e. difference between the frequencies of recovery in pseudoreplicates of the original group and the most frequently recovered contradictory group) frequencies. Templeton tests (T-t) were conducted using PAUP* 4.0 [23 ] to determine the significance of alternative phylogenetic topologies, with a 5% threshold for significance (p-value ≤ 0.05 = significantly less parsimonious [S]; p-value > 0.05 = non-significant [NS]).
Nesbitt & Butler [20 (link)] included the enigmatic archosaurs Erpetosuchus granti and Parringtonia gracilis within the data matrix of Nesbitt [2 ]. They recovered E. granti and P. gracilis within a monophyletic Erpetosuchidae; however, this clade acted as a wildcard taxon that substantially reduced phylogenetic consensus within Archosauria. Erpetosuchidae was recovered by Nesbitt & Butler [20 (link)] within a major polytomy that also included Avemetatarsalia, Ornithosuchidae, Aetosauria + Revueltosaurus, Ticinosuchus + Paracrocodylomorpha, G. stipanicicorum and T. dabanensis. It is noteworthy that Erpetosuchidae was found as the sister taxon of T. dabanensis in some of the most parsimonious trees recovered by Nesbitt & Butler [20 (link)]. As a result, we conducted a second analysis where we added E. granti and P. gracilis to the data matrix. Character scorings for the two erpetosuchids follow Nesbitt & Butler [20 (link)], with the exception of the scorings for the new character added here. This data matrix was therefore composed of 413 characters and 80 taxa, and was analysed under the same search criteria described above. This modified data matrix is supplied as Additional file 3, and is also available at TreeBASE as submission S15917 (http://iczn.org/code).
There is some disagreement over the identification and interpretation of the element described as the astragalus of T. dabanensis by Wu & Russell [12 (link)], with Nesbitt [2 ] scoring all astragalus characters as uncertain for T. dabanensis. To reflect this uncertainty, we reran both of the analyses described above, but rescored all astragalus characters (characters 354–370) as uncertain for T. dabanensis.

To evaluate the measuring method we used subjects selected from an already existent study of rehabilitation and muscle atrophy after ACL-reconstruction with semitendinosus and gracilis tendon graft. The Ethics Committee at the Karolinska Institutet approved the design of the study, and the patients gave their informed consent of the planned procedures. For our reliability study we included the first 31 examined patients (22 men and 9 women). The median age of these patients was 27 years with a range from 16 to 45 years. All the CT-examinations included in this study were performed before surgery.
Axial CT images were acquired at three levels. At the level of, as well as 50 mm and 150 mm above the knee joint with the patients in a supine position. For assessing the reproducibility it was, according to our opinion, enough to evaluate the level of 150 mm above the knee joint which is best suited for evaluation of muscle CSA of the levels examined. The scans were performed by a Philips Tomoscan SR 7000 (single slice helical CT- scanner, 100 kV and 75 mAs) for 26 patients and with a Siemens Volume Zoom (4 slice MDCT-scanner, 120 kV and 40 mAs) for 5 patients. The use of two different CT-scanners was due to change of equipment at our department during the study period. Slice thickness in all images was 10 mm. The images were saved as DICOM-images in the departments PACS-system for later analysis.
The images were analyzed by two investigators (MLW and SS) independently using NIH ImageJ version 1.38× software http://rsbweb.nih.gov/ij/ packages. All images were analyzed by both investigators at two times with a minimum of 3 weeks between the two readings.
Both the leg with the ACL-injury and the contralateral leg were analyzed. The muscles identified and measured were: quadriceps, sartorius, gracilis, semimembranosus, semitendinosus and biceps femoris. No attempt was made to separate the different parts of quadriceps (vastus medialis, vastus intermedius, vastus lateralis and rectus femoris) or the two heads of biceps femoris (caput longum and caput breve). Even when analyzing anatomical dissection in cadaver studies it is not always possible to separate the different parts of e.g. quadriceps [11 (link)]. On most of the images a small part of the muscles of the adductor group was also present but not measured.
CSA of the individual muscles was measured by outlining the borders of the muscles with the polygon selection tool. This was made after adjusting the image to level 50 and window width to 400 to obtain as good visual discrimination between adipose tissue and muscle as possible. CSA was measured as the area inside the borders with attenuation values from 1 to 101 Hounsfield units (HU) (figure 1). When outlining the borders we tried to avoid nerves and vessels as they have attenuation values within the chosen limits.
Apart from CSA the mean attenuation of the individual muscles was also measured. For some subjects the distribution of attenuation values between -29 HU to 150 HU was also registered to test the validity of the chosen limits of attenuation (figure 2). In this case a line was drawn just inside the border of the muscle to avoid volume averaging at the border affecting attenuation values.
To improve the speed of the process we used the ability of ImageJ to use self-defined macros that reduced the amount of clicking necessary for each measurement.

For the construction of resveratrol-producing strains, Pc4CL (P14912.1) from Petroselinum crispum, VvSTS (P28343.2) from Vitis vinifera, RtPAL/TAL (P11544) from Rhodotorula toruloides (Synonyms R. gracilis), AtCPR1 (NP_194183.1) and AtC4H (NP_180607.1) from Arabidopsis thaliana were codon-optimized for expression in S. cerevisiae and synthesized by Sangon Biotech (Shanghai, China). SeACS^L641P (MP052228.1) and selective marker cassettes (HIS3, LEU2 and URA3) were also synthesized by Sangon Biotech (Shanghai, China). Integration homologous arms (about 500 bp), ScACC1, ScARO4, ScARO7, and ScARO2 were amplified from the genome of BY4742. LYS2 including its homologous arms was amplified from the genome of W303-1A. EcAROL was amplified from the genome of E. coli. Mutants of ACC1, ARO4 and ARO7 were created by overlap PCR according to the previous report. Expression cassettes with promoters, terminators and genes were assembled to plasmid G418 using Minerva Super Fusion Cloning Kit (Yuheng Biotech, Suzhou, China). The plasmids pCas with specific gRNA sequences used for CRISPR/Cas9 editing were obtained according to the standard Quick-Change Site-Directed Mutagenesis protocol. The gRNA sequences of sites cit2, lpp1, pha2, and dpp1 were designed using online tool E-CRISP [37 (link)], and the gRNA sequences of other sites were reported in the previous study [38 (link)]. Recombinant plasmids were confirmed by DNA sequencing. The detailed information of plasmids (Additional file 1: Table S2) and the primers (Additional file 1: Table S3) used in our study were listed in supplementary information.

Healthy male subjects (aged 18–32) undergoing surgery of the knee for anterior cruciate ligament (ACL) reconstruction using hamstring autografts were recruited from outpatient clinics of two hospitals: Erasmus Medical Center and Medisch Centrum Haaglanden. Inclusion criteria included age, sex, and the amount of routine exercise. Subjects eligible for reconstructive ACL surgery were mobile, had full range of knee motion, minimal to no knee swelling and had physiotherapy until the surgery.
A total of seven biopsies were taken from six different leg muscles (Figure 1A). To study molecular differences within the muscle, two biopsies from the middle and distal sides of the semitendinosus muscle (STM and STD, respectively) were collected. During the surgery, the tendons of the gracilis (GR) and semitendinosus muscles were used to reconstruct the ACL, and biopsies from these muscles were taken directly from the graft after harvesting the autografts at the beginning of the operation. After the ACL construction, biopsies from gastrocnemius lateralis (GL) rectus femoris (RF), vastus lateralis (VL), and vastus medialis (VM) muscles were taken by percutaneous biopsy (modified Bergstrom, 1975 (link)) using a minimally invasive biopsy needle. All biopsies were immediately frozen in liquid nitrogen and were kept at –80 °C.
The study was approved by the local Medical Ethical Review Board of The Hague Zuid-West and the Erasmus Medical Centre and conducted in accordance with the ethical standards stated in the 1964 Declaration of Helsinki and its later amendments (ABR number: NL54081.098.16). All subjects provided written informed consent prior to participation.