Kelfizine

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.

Primers and Taqman^® probes were designed to target the B. longum subsp. infantis sialidase gene (locus tag ‘Blon2348’ from strain ATCC 15697, NCBI Reference Sequence: NC_011593.1) and the B. longum subsp. longum sugar kinase gene (locus tag ‘BL0274’ from strain NCC 2705, NCBI Reference Sequence: NC_004307.2) using ‘primer 3’ (Untergasser et al., 2012 (link)). Primers and probes were obtained from IDT (Singapore) and are described in Table 1. The primer/probe combinations were tested for reaction efficiency and specificity using genomic DNA (gDNA) purified from bifidobacterial type cultures (the gold standard cultures for species) of species reported to be detected in infant feces: B. adolescentis (DSM 20083^T), B. animalis subsp. lactis (DSM 10140^T), B. angulatum DSM 20098^T, B. bifidum (DSM 20456^T), B. breve (ATCC 15700^T), B. catenulatum (DSM 20224^T), B. dentium (ATCC 27534^T), B. longum subsp. infantis (DSM 20088^T), B. longum subsp. longum (ATCC 15707^T), B. pseudocatenulatum (DSM 20438^T), and B. pseudolongum (ATCC 25526^T), (Grönlund et al., 2011 (link); Makino et al., 2013 (link); Huda et al., 2014 (link); Bäckhed et al., 2015 (link); Vazquez-Gutierrez et al., 2015 (link); Martin et al., 2016 (link)). A Life Technologies ViiA™7 real time PCR system and MicroAmp Fast optical 96-well or 384-well plates with optical adhesive film (Applied Biosystems, Carlsbad, CA, USA) were used. All reactions were carried out in a final volume of 15 μl containing 1 × TaqMan^® Fast PCR mastermix (Applied Biosystems), 300 nM of each primer and 100 nM TaqMan^® probe. For specificity testing, template DNA was diluted to 5 ng∕μl, and 2 ng was added to each reaction. The thermocycling profile consisted of an initial activation of the polymerase at 95 °C for 30 s, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. Fluorescence levels were measured after the 60 °C annealing/extension step. Standard curves (to measure reaction efficiency) were generated using gDNA extracted from bifidobacterial strains Bifidobacterium longum subsp. longum (ATCC 15707^T) and Bifidobacterium longum subsp. infantis (DSM 20088^T) using the bead-beating phenol/chloroform/ethanol protocol described previously (Tannock et al., 2013 (link)). The standard DNA was quantified spectrophotometrically using a NanoDrop 1,000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) and diluted in 10-fold steps from 5 × 10⁶ to 5 × 10¹ genomes/reaction, calculated using target gene copies per genome obtained from genome sequence information (NCBI). All reactions were carried out in duplicate and were run twice on separate plates. No-template controls were also included on each plate. Reactions in which duplicate Ct values varied by more than 0.5 Ct’s were also repeated.

A bidirectional BLAST search was used to compare type strains of B. infantis and B. longum to identify subspecies-specific genes. Candidate genes were screened against B. infantis EVC001 ATCC SD7035, and Blon_0915 was found to be present in both the type strain of B. infantis and EVC001 ATCC SD7035 and other closely related strains of B. infantis but not among other Bifidobacterium species. BLASTN searches confirmed these findings, indicating that Blon_0915 had little sequence homology among other Bifidobacterium sequences in the NCBI database. Primer3 (41 (link), 42 (link)) was used to identify primer pairs with high efficiency and specificity for B. infantis. In comparison to the B. longum group primers used here (Table 5), primers Blon_0915F and Blon_0915R, when coupled with Blon_0915P, did not produce false amplification from infants who were not previously fed B. infantis. This was true even when tested in fecal samples from infants natively colonized by B. longum, which was independently verified by using genus-specific Bifidobacterium and B. longum group-specific qPCR primer sets (Fig. S2). The TaqMan reaction was carried out using the manufacturer’s instructions (Thermo, Fisher Scientific; Waltham, MA, USA), which included a preincubation step for 2 min at 50°C and then 10 min at 95°C, followed by 40 cycles of a two-step PCR for 15 s at 95°C and 60 s at 60°C for Blon_0915 primers; other primer/probe chemistries are outlined in Table 5.

Sequences were assembled with MetaSPADES genome assembler v3.13.0 (Nurk et al., 2017), with a range of k-mer sizes (21–127) and the assemblies were filtered to exclude sequences of less than 500 bp in length. Assembly performance was analyzed with QUAST v.5.0.0,^{66 (link)} using the default parameters. Open reading frames (ORFs) were predicted with Prodigal v2.6.3^{67 (link)} in metagenomic mode and then filtered to obtain ORFs with start or stop codons, yielding a mean of 53.085 (± 24.138) genes per sample. Genes from all samples were clustered with CD-HIT v4.8.1.^{68 (link)} with the following criteria: 90% alignment coverage and 95% gene sequence identity, to generate a non-redundant de novo gene set. A count matrix for each sample was generated from the non-redundant de novo gene set with NGLess v.1.0.0,^{65 (link)} retaining only primary mapped reads with a minimum match size of 45 nt displaying at least 95% alignment, with a dist1 for multiple mapping reads. Pathogenic factors were identified with PathoFact software, using default parameters.^{69 (link)} Carbohydrate-active enzyme (CAZy) was assessed with dbcan2.^{70 (link)} In short, a triple annotation was performed with (i) HMMer against the dbCAN HMM database, (ii) DIAMOND search against the CAZy pre-annotated CAZyme sequence database, and (iii) eCAMI run against the CAZyme database; CAZy terms annotated with at least two methods were retained for downstream analysis.
For taxonomic analysis, the filtered reads were aligned with single-copy marker genes present in almost all bacteria, viruses, and archaea. The relative abundances of the taxa identified were calculated with the MetaPhlAn3 v.3 pipeline.^{71 (link)} The pipeline used is depicted in Figure S1. Bifidobacterium longum subsp. infantis (B. infantis) and B. longum subsp. longum were detected with PanPhlAn software^{72 (link)} applied to the B. longum pangenome (14 B. infantis and 30 B. longum reference genomes) (Table S2). In short, B. infantis HMO cluster genes (Blon_2331–Blon_2361) were selected from the B. longum (species) pangenome (Sela et al., 2008), together with the B. longum subsp. longum-specific genes (araA and araD),^{34 (link)} to assess the presence of these subspecies in the metagenomes for Kenyan and Swedish infants. We excluded 18 samples from Swedish infants (18% of the sample set) and two samples (2% of the sample set) from Kenyan infants from the analysis (below the default PanPhlAn threshold for the presence of B. longum genes).
External dataset. A four-month-old infant microbiome gene set catalog was obtained from the GIGAdb website (gigadb.org/dataset/100145)^{35 (link)} and compared with the non-redundant de novo gene set.

Surface electromyography (EMG) signals were recorded from (1) musculus (m.) brachioradialis, (2) m. biceps brachii, (3) m. triceps brachii caput laterale, (4) m. triceps brachii caput longum, (5) m. deltoideus pars anterior (6) m. deltoideus pars posterior, and (7) m. pectoralis major. However, in line with previous findings, it was the latter three muscles (i.e., shoulder actuators: pectoralis, anterior, and posterior deltoid) that were primarily engaged in the delayed reach task of our setup, hence only these three muscles were used for statistical analyses. We used surface EMG electrodes (Bagnoli DE-2.1, Delsys) that have contact dimensions 10.0 × 1.0 mm with 10 mm interelectrode spacing. Before attaching the electrodes, the skin was cleaned using alcohol swabs. The electrodes were coated with conductive gel and placed on the peak of the belly of the studied muscles in the direction of the muscle fibers. All electrodes were attached with double-sided tape and further secured using surgical tape. One ground electrode (Dermatrode HE-R Reference Electrode type 00200–3400, American Imex), with a diameter of 5.08 cm, was placed on the processus spinosus of the C7 region. The EMG signals were analog bandpass filtered through the EMG system (20-450 Hz) and sampled at 1 kHz.

After 21 days of seeding, the culture medium of the 24-well plates was replaced with fresh antibiotic-free DMEM. Subsequently, PRL2022 cells (final concentration 10⁸ cells/mL) were added to Caco-2/HT29-MTX cell monolayer, as previously described (Serafini et al., 2013 (link); Fontana et al., 2022 (link)). The 24-well plates were subsequently incubated at 5% CO₂ at 37°C. After 4 h of incubation, bacterial cells and human cell lines were separately recovered in RNA later and preserved at −80°C until processing.
Specifically, for this trial, B. longum subsp. longum PRL2022 was grown in MRS broth under anaerobic conditions at 37°C. Once the exponential growth phase (0.6 < OD_600nm < 0.8) was reached, bifidobacterial cells were enumerated by using the Thoma cell counting chamber (Herka), possibly diluted to reach a final concentration of 1 × 10⁸ cells/mL, washed in PBS, resuspended in 400 μL of antibiotic-free DMEM, and seeded on Caco-2/HT29-MTX cell monolayers. The strain PRL2022 resuspended in DMEM and maintained under the same incubation conditions of the 24-well plates without any contact with human cell lines was used as bacterial control sample, while Caco-2/HT29-MTX cell monolayers without any bifidobacterial seeding were used as human cell line control sample. All experiments were carried out in triplicate.