Tumor necrosis factor (TNF) and, possibly, lymphotoxin alpha (LTα) signaling contribute to inflammation and rheumatoid arthritis (RA) pathogenesis. Pateclizumab  is a humanized monoclonal antibody that blocks and depletes anti-LTα. This phase 2, randomized, head-to-head, active- and placebo-controlled trial examined the safety and efficacy of pateclizumab compared to adalimumab in RA patients with an inadequate response to disease-modifying antirheumatic drugs (DMARD-IR).n = 214) with active RA  on oral DMARDs were randomized (2:2:1) to receive pateclizumab 360 mg, adalimumab 40 mg, or placebo subcutaneously every 2 weeks. The primary endpoint, 4-variable, 28-joint disease activity score erythrocyte sedimentation rate (DAS28(4)-ESR) response, was evaluated at 12 weeks using an analysis of covariance (ANCOVA) model with adjustments for concomitant DMARD use and geographic region. Secondary efficacy endpoints included American College of Rheumatology (ACR) 20, ACR50, and ACR70 responses at Day 85. Pharmacokinetics, pharmacodynamics, and immunogenicity of pateclizumab were assessed.Patients (Pateclizumab reduced the DAS28(4)-ESR response (−1.89) at 12 weeks, however, this did not reach statistical significance compared to placebo (−1.54), while adalimumab (−2.52) differed significantly from both placebo and pateclizumab. Pateclizumab 12-week ACR20, ACR50 and ACR70 response rates  suggested clinical activity but were not statistically significant compared to placebo rates . CXCL13 serum levels decreased significantly following pateclizumab and adalimumab administration, demonstrating pharmacological target engagement by both drugs. Overall, adverse events (AEs) were comparable among all cohorts. Infections were the most common AE, occurring with comparable frequency in all groups. Serious AEs occurred in 0% of pateclizumab, 5.9% of adalimumab, and 2.3% of placebo patients, with serious infection in 2.3% of adalimumab patients and none in pateclizumab and placebo patients.Pateclizumab had a good safety profile in patients inadequately responsive to DMARDs, but no statistically significant improvement in RA signs and symptoms after 12 weeks of treatment. Adalimumab demonstrated efficacy and safety comparable to published results in this head-to-head comparison in DMARD-IR RA patients.NCT01225393, Registered 18 October 2010.ClinicalTrials.gov  The risk of disease progression in rheumatoid arthritis (RA) occurs in patients who do not respond adequately to available treatment options . Despitede novo lymphoid aggregates. These ectopic lymphoid aggregates are instigated and maintained by lymphotoxin (LT)-mediated pathways. Lymphotoxin alpha (LTα) and beta (LTβ) are two related TNF superfamily (TNFSF) members produced predominately by activated cells of the innate and adaptive immune response. LTα exists as a secreted homotrimeric molecule (LTα3) that signals via TNF receptor 1 (TNFR1) and TNFR2 to promote inflammation; whereas LTα complexed with LTβ (LTα1β2), on the surface of T and B cells, signals through the LTβ receptor (LTβR) [Synovitis in RA develops as a result of infiltration of innate and adaptive immune cells causing a significant inflammatory response and cytokine release, including, but not limited to tumor necrosis factor alpha (TNF-α). During chronic inflammation these cellular infiltrates organize into r (LTβR) . Lymphoir (LTβR) ,6. B celr (LTβR) . Moreover (LTβR) . In compr (LTβR) ,9.3-TNFR interactions, LTα1β2-LTβR interactions, specifically depletes LTα1β2-expressing lymphocytes [Given the role lymphotoxin, TNF-α, and Th cells may play in inflammation and RA pathogenesis, and the additional role soluble LTα3 may play in disease pathogenesis -15, we gphocytes . In a phphocytes . Here, wThe ALTARA study was conducted in 47 centers in 10 countries, including sites in the United States, Europe, and Latin America. The study was conducted in compliance with the Declaration of Helsinki and the International Conference on Harmonization Good Clinical Practice Guidelines. It was approved by the Comités de Ética e Investigación en Salud del Centro de Especialidades Médicas del Sureste S.A. de C.V. (Unidad de Investigacion Biomedica del Cem). All patients provided written informed consent before performance of any study procedures.Pateclizumab was administered as subcutaneous (SC) injections, 360 mg every other week. This study regimen was selected based on the following considerations: 1) the total exposure was, on average, 60% higher than the 3 mg/kg biweekly SC doses evaluated in the pateclizumab phase I study; 2) this regimen was expected to result in a maximal pharmacological effect as suggested by plateaued reductions in serum CXCL13 level in all dose groups at 1-mg/kg or higher doses; and 3) this regimen has 2.2- to 4.5-fold exposure safety coverage by the highest exposure level assessed in the phase I study . An SC bEligible patients were ≥18 years of age, had a diagnosis of RA for ≥6 months (meeting the American College of Rheumatology (ACR) 1987 revised criteria for RA ), and haMycobacterium tuberculosis; clinically significant infection(s); and a history of malignancy, with the exception of adequately treated nonmetastatic basal cell or squamous cell cancer of the skin or cervical carcinoma in situ.Key exclusion criteria included the following: previous biologic therapy for RA; evidence of hematopoietic disorders at screening or within 3 months; creatinine >1.5 × upper limit of normal (ULN), aspartate aminotransferase (AST) or alanine aminotransferase (ALT) levels 1.5 × ULN; positive QuantiFeron  results This was a phase 2, randomized, double-blind, active- and placebo-controlled, parallel-group study. Patients were randomized 2:2:1 to receive one of the following treatments: pateclizumab 360 mg, adalimumab 40 mg, or placebo. The study drug was administered SC once every other week for 10 weeks (baseline (Week 0) and Weeks 2, 4, 6, 8, and 10). We were unable to construct a placebo syringe corresponding to the manufactured syringe supplied with adalimumab. As a result, unblinded medical personnel administered the allocated treatment while maintaining a strict blind for the patients and medical personnel associated with safety, joint, and efficacy evaluations.Patients continued on a stable background regimen of RA therapy, which included antimalarial agents, nonsteroidal anti-inflammatory drugs, acetaminophen, and/or oral corticosteroids . Patients continued on either methotrexate or leflunomide at protocol-defined stable doses throughout the study. Intra-articular corticosteroids were prohibited in the 6 weeks prior to screening; they were allowed in one joint during the study, but were discouraged between Weeks 10 and 12. For all injected joints, the treated joint was censored from the efficacy analysis from the time of injection up to a maximum of 12 weeks.The primary objective was to compare the efficacy of pateclizumab to that of adalimumab by assessing the change from baseline in the 4-variable, 28-joint disease activity score erythrocyte sedimentation rate (DAS-ESR(4)) on Day 85 (Week 12).Secondary objectives included the durability of the pateclizumab treatment response, safety of the treatment over 24 weeks, and the efficacy of adalimumab over 12 weeks. The secondary efficacy endpoints analyzed throughout the study included the following: the ACR20, ACR50, and ACR70 response rates, and the proportions of patients achieving disease remission, according to the 3-variable disease activity score in 28 joints (DAS28)  using thSafety was evaluated by nature, severity, and drug relation of adverse events (AEs), graded according to National Cancer Institute Common Toxicity Criteria for Adverse Events (NCI-CTCAE v3.0) and reported by incidence. Vital signs, physical examination findings, concomitant medications, and laboratory and pregnancy test results were collected every 2 weeks during the study. Incidence of anti-therapeutic antibodies (ATA) to pateclizumab in serum was evaluated using a bridging enzyme-linked immunosorbent assay (ELISA) on Days 57, 85, and 155 or early termination.For pharmacokinetic (PK) assessment, serum samples were obtained at predose Day 1; at 4 hours and at 3 and 6 days after the first dose; predose on Days 15, 29, 57, and 71; 3 and 14 days after final dose, and during drug washout for up to 12 weeks. The PK samples were analyzed using a validated ELISA with a lower limit of quantification of 100 ng/mL pateclizumab.For ATA assessment, serum samples were obtained at predose and multiple postdose time points  and assessed using a bridging ELISA . The relCXCL13 was measured as a pharmacodynamic biomarker, as identified in the phase 1 study ,22. SeruP values for comparing treatment differences in ACR20/50/70 responses were based on the Cochran-Mantel-Haenszel test with adjustment for stratification factors of geographic region and concomitant DMARD use. The P values were not corrected for multiple comparisons.All efficacy analyses were performed using the modified intent-to-treat (mITT) populations; all safety analyses were performed using the as-treated population. The primary efficacy endpoint of change of DAS28(4)-ESR at Day 85 was assessed using an analysis of covariance (ANCOVA) model with adjustment of concomitant DMARD use, geographic region, and baseline value. The secondary efficacy endpoints include ACR20, ACR50, and ACR70 responses at Day 85. Any missing ACR score components were imputed with the last-observation-carried-forward (LOCF) method. The Overall, 214 patients were randomized to receive study treatment. Of these, 180 received study treatment and completed the study for efficacy evaluations (mITT). The 34 patients withdrawn prior to the completion of the study included 13 patients treated with pateclizumab, 11 treated with adalimumab, and 10 treated with placebo. Reasons for early withdrawal included AE (n = 7), patient decision (n = 17), sponsor decision (n = 8), and loss to follow-up from the pateclizumab cohort (n = 2). An additional 11 patients did not meet eligibility criteria. Thus, of the 214 patients, 170 who received a study treatment were included in efficacy and safety evaluations.Randomization achieved treatment groups that were well balanced for baseline demographic and clinical characteristics Table . The majP <0.01) whereas the difference between the pateclizumab and placebo groups was not (P >0.05).Pateclizumab had minimal clinical activity by DAS28(4)-ESR but this did not reach statistical significance compared to placebo at Week 12. In contrast, adalimumab demonstrated a robust treatment effect at Week 12. The mean changes from baseline in DAS28(4)-ESR at Day 85 were −1.89, −2.52, and −1.54 for the pateclizumab, adalimumab, and placebo groups, respectively (Table P <0.01), but the difference between pateclizumab and placebo was not (P >0.05). Pateclizumab failed to reach statistical significance for other key secondary efficacy endpoints, including ACR20 and ACR70. Pateclizumab was significantly different from placebo for two components of the composite ACR score: swollen joint count (P <0.04) and physician’s global visual analog scale (VAS) (P <0.01). Adalimumab significantly differed from placebo in each of the ACR components. In addition, SF-36 individual and component summary scores also showed a statistically significant difference between adalimumab and pateclizumab but not between pateclizumab and placebo.The ACR50 response was achieved at Day 85 in 26 (33.3%), 43 (57.3%), and 9 (24.3%) patients from the pateclizumab, adalimumab, and placebo groups, respectively : five patients who received adalimumab and one patient who received placebo. Of the six SAEs, two (pulmonary tuberculosis (TB) and lymphopenia, both in the adalimumab group) were judged by the investigator to be related to the study drug. No patients were withdrawn from treatment due to an SAE.AEs in the pateclizumab cohort were comparable in frequency to the placebo rates Table . In the The most frequent (≥3%) treatment-emergent AEs are listed in Table trough,ss) was 21.3 ± 12.0 μg/mL. The mean trough concentration ratio of Day 85 (14 days after the last dose) to Day 15 (14 days after the first dose) was 1.79, indicating a mild exposure accumulation. The observed PK profile was well aligned with the profile predicted by a population PK model built using the PK data from the pateclizumab phase I study  observed serum concentration was 41.1 ± 18.4 μg/mL, and occurred on Day 74 (3 days following the sixth dose). The observed steady-state average trough concentration . Adalimumab also decreased CXCL13 levels significantly , as expected [Pre- and postdose levels of serum CXCL13 were evaluated as a biomarker for target modulation. The three treatment groups did not significantly differ in mean (± SD) CXCL13 baseline levels (pg/mL). Serum CXCL13 levels decreased rapidly and significantly in patients treated with pateclizumab Figure , demonstexpected . Serum CAmong the 85 patients who were treated with pateclizumab, four patients were positive for ATA, and all positive ATA signals were detected at a single time point on Day 155, approximately 3 months after the final pateclizumab dose.To our knowledge, ALTARA is the first phase 2 study directly comparing the safety and efficacy of an investigational agent, pateclizumab, with an approved anti-TNF product for the treatment of DMARD-IR patients on background DMARDs. Efficacy was directly compared in a head-to-head study for 3 months of treatment, which is considered a sufficient duration of treatment for such comparisons ,24. SincPateclizumab had a trend for clinical effects on the signs and symptoms of rheumatoid arthritis after 3 months of treatment in patients with active disease and an inadequate response to DMARDs but the differences from placebo responses were not statistically significant. Pateclizumab had an acceptable safety profile, with no SAEs and no increase in infectious AEs compared to placebo likely related to its limited immunosuppressant effects. Injection-site AEs with pateclizumab were uncommon, typically low grade in severity, and comparable to those with placebo.The pateclizumab dose regimen evaluated in this study provided expected and sufficient exposure during the treatment period as judged by a rapid and sustained decrease in serum CXCL13 levels at all times out to 85 days. The 40 to 50% inhibition of serum CXCL13 levels in the pateclizumab-treated group in this study confirmed that the exposure of 360 mg (approximately 5 mg/kg based on a median body weight of 72 kg) SC biweekly doses is sufficient to produce maximal downstream pharmacological effect in blocking the LTα pathway. However, although such a pharmacodynamic effect was maximized and similar to the CXCL13 reduction observed in the adalimumab-treated cohort, pateclizumab treatment was not associated with significant clinical benefit.1β2-LTβR interaction, was evaluated in two clinical trials and found not to be efficacious for the treatment of the signs and symptoms of RA. In a 14-week, dose-ranging phase 2b trial in DMARD-IR patients (N = 391) [Despite the reports of elevated levels of LTα and LTβ expression in the synovium of RA patients ,6,28, asN = 391)  and in a3-TNFR interactions and LTα1β2-LTβR interactions, with pateclizumab on background DMARDs is insufficient to produce a significant reduction in the inflammatory process in RA or to provide superior efficacy over adalimumab, an anti-TNF agent that is commonly used for DMARD-IR RA patients.Our findings suggest that targeting lymphotoxin signaling, LTα


To facilitate quantification of cerebellum cerebral blood flow (CBF), studies were performed to systematically optimize arterial spin labeling (ASL) parameters for measuring cerebellum perfusion, segment cerebellum to obtain separate CBF values for grey matter (GM) and white matter (WM), and compare FAIR ASST to PICORE. Cerebellum GM and WM CBF were measured with optimized ASL parameters using FAIR ASST and PICORE in five subjects. Influence of volume averaging in voxels on cerebellar grey and white matter boundaries was minimized by high-probability threshold masks. Cerebellar CBF values determined by FAIR ASST were 43.8 ± 5.1 mL/100 g/min for GM and 27.6 ± 4.5 mL/100 g/min for WM. Quantitative perfusion studies indicated that CBF in cerebellum GM is 1.6 times greater than that in cerebellum WM. Compared to PICORE, FAIR ASST produced similar CBF estimations but less subtraction error and lower temporal, spatial, and intersubject variability. These are important advantages for detecting group and/or condition differences in CBF values. The cerebellum is important for motor control, attention, language, and emotion , 2 and iAs a completely noninvasive perfusion imaging technique, arterial spin labeling (ASL) has been useful for both clinical –10 and rFAIR ASST , 17, by Ten healthy male adults  took part in four studies in three sessions to optimize the ASL sequences for the cerebellum: three subjects for the first study and session, four for the second study and session, and another three for the last two studies in the third session (see “ASL Optimization Studies” below). Five healthy male adults  participated in the subsequent quantitative cerebellum perfusion study. Since caffeine is a vasoconstrictor, all subjects refrained from caffeine-containing coffee, tea, and soft drinks at least 8 hours before the studies. Subjects were instructed to avoid any movements of arms or legs and keep eyes closed but remain awake. Subjects' heads were tightly restrained with foam padding. Studies were performed at approximately the same time in the early afternoon to avoid potential effects of circadian rhythms. Informed consent was obtained from all subjects prior to the studies, according to a protocol approved by the local Institutional Review Board.z direction and 40 mT/m in the x and y directions, maximum slew rate 200 T/m/s). The body coil was used for transmission, and the Siemens 12-channel phased array head coil was used for reception.Studies were performed using a 3T Siemens Trio TIM whole-body scanner with 60 cm diameter magnet bore and SQ gradients  [TI2) by using echo planar imaging (EPI) as the readout. All saturation modules consisted of a 90-degree RF pulse followed by a spoiling gradient. The total duration of one saturation module was about 11 ms. Depending on the thickness of the saturation slab, an appropriate RF saturation pulse was automatically selected from a set of sinc RF pulses optimized for various thicknesses.For both FAIR ASST and PICORE sequences , a 15.36 mm slab . Both seb [TI1) . After aThe FAIR ASST , 17 sequT1-weighted high-resolution anatomic imaging sequence MPRAGE, and ASL scans with an EPI readout.The Siemens coregistration tool AutoAlign was used to provide consistent slice orientation and position with respect to a standard head atlas across subjects and sessions. Axial ASL imaging slices were positioned to have the first inferior slice cover the lower edge of the cerebellum, oriented parallel to the anterior-posterior direction . Due to 3, matrix size = 256 × 256 × 160, resolution = 0.9 × 0.9 × 1 mm3, bandwidth = 160 Hz/pixel, GRAPPA iPAT factor = 2 with 24 reference lines, partial Fourier (PF) = 7/8, slice oversampling = 10%, slice orientation = sagittal, phase encoding direction = anterior to posterior, and total imaging time = 4 minutes and 38 seconds.The MPRAGE sequence used the following parameters: TR/TE/TI = 2250/4/900 ms, flip angle = 9°, field of view (FOV) = 230 × 230 × 160 mmTI2 value, to estimate arterial transit time (ATT) and bolus duration, the latter for properly setting the TI1 value for the following experiments  and varied postlabeling delay times to identify the proper delay to avoid intravascular artifacts, (3) a multiple-TR perfusion study to determine if a short TR significantly affects the refreshment of arterial blood at the labeling site, and (4) a study varying the number of inferior saturation pulses to find the minimum number necessary for effective suppression of residual labeled blood spins.The following ASL studies were performed to optimize FAIR ASST parameters for measuring cerebellar CBF: (1) a series of perfusion experiments, each with a different eriments , (2) a pTI2 perfusion study employed the following MRI parameters: TR/TE = 4000/9.2 ms, FOV = 230 × 230 mm2, matrix size = 66 × 66, in-plane resolution = 3.48 × 3.48 mm2, number of imaging slices = 12, slice thickness/gap = 5 mm/1 mm, imaging slab size = 71 mm, pairs of label and control measurements = 30, iPAT GRAPPA factor = 2 with 24 reference lines, and partial Fourier (PF) = 7/8. Imaging slices were acquired in ascending order. The slab sizes for the superior saturation, imaging section inversion, and spatially confined selective inversion were equal to 100 mm, 91 mm, and 271 mm, respectively. For each subject, the experiment was conducted twelve times using randomly ordered TI2, values of 50, 300, 600, 900, 1200, 1500, 1800, 2100, 2400, 2700, 3000, and 3300 ms. The inferior saturation pulse train was turned off. Total imaging time for ASL acquisitions was about 48 minutes.The variable TI2 values, except as follows. Parameters for perfusion studies using varied postlabeling delay times were TR = 3000 ms, inferior saturation pulse interval/thickness = 25 ms/20 mm, TI1 value/postlabeling times = 800/{200,400,600,800,1000,1200,1400} ms (randomized for each subject), inferior saturation pulse train lasting for the entire postlabeling delay period, and total ASL imaging time of about 21 minutes. In the perfusion studies exploring the effect of TR, parameters were TR = {2.5,3.0,3.5} s (randomized for each subject), TI1/TI2 = 800 ms/1800 ms, inferior saturation pulse interval/thickness/number = 25 ms/20 mm/40, and total imaging time of about 9 minutes. In the study evaluating the number of inferior saturations, parameters were TR = 3 s, TI1/TI2 = 800 ms/1800 ms, and inferior saturation pulse interval/thickness = 25 ms/20 mm. The number of inferior saturation pulses was varied from 0 to 40 in increments of 10, and the order of the imaging scans was randomized for each subject. The total imaging time for evaluating the sufficient number of inferior saturations was about 15 minutes.The rest of the optimization studies used the same ASL parameters as those in the studies with varied 2, matrix size = 72 × 72, slice thickness/gap = 3.5/0.7 mm, slice number = 16, in-plane resolution = 2.5 × 2.5 mm2, slice thickness/gap = 3.5/0.7 mm, TR/TE = 2500/12 ms, pairs of label and control measurements = 90, iPAT GRAPPA factor = 2 with 24 reference lines, partial Fourier (PF) = 7/8, acquisition order = ascending (foot to head), TI1/TI2 = 800 ms/1800 ms, inferior saturation interval/thickness/number = 25 ms/20 mm/20, superior saturation slab thickness = 100 mm, selective inversion slab = 86.5 mm, and spatially confined inversion slab = 266.5 mm. For PICORE, a 100 mm slab, approximately the same as the effective inferior labeling slab size applied in FAIR ASST experiments, was used for proximal inversion, with a 10 mm gap between the imaging slab and the inversion slab. For each ASL scan, two proton density (M0) images were acquired, one before and one after each ASL series, by using the same sequence with TR = 8 s, and the average M0 image was used in subsequent model fits [The following ASL parameters were used for quantitative cerebellum perfusion studies: FOV = 180 × 180 mmTI2 values.Image processing operations, such as motion correction and coregistration, were performed with SPM2 . Four-parameter iterative nonlinear least squares model fitting was performed using scripts implemented in Matlab 7.1  for perfusion signals from studies using multiple varied M0 images in each series were averaged to obtain a mean M0 image.Each ASL imaging series was first evaluated for subject motion, and whenever the translational motion was larger than 1 mm or the rotation around any axis was larger than 1°, motion correction was performed using trilinear interpolation. Pairs of labeling and control images with motions larger than 2 mm in translation or 2° in rotation around any axis were excluded from further processing or analysis. A mean image of the ASL series was generated for later use in coregistration. Each ASL label-control image series was processed by pairwise subtraction to generate a perfusion-weighted imaging series, and images within the series were averaged to produce a mean perfusion-weighted image. The two M(t) is the measured ASL difference signal between label and control images in a specified ROI or voxel at inversion time t, Δt is the arterial transit time, τ is the bolus duration, CBF is the cerebral blood flow, Mb0 is the fully relaxed magnetization of the blood, Tb1 is the longitudinal relaxation time of the arterial blood, and α is the labeling efficiency, assumed to be 0.95 for the hyperbolic secant pulse used in this study [An iterative nonlinear least-squares model fitting was performed to fit ASL signals in the defined ROIs to the three-phase, single blood compartment model :(1)ΔM [The mean perfusion-weighted image and the mean nt model , 28, 29: be 0.9) .Individual whole-brain GM and WM tissue masks were automatically generated using SPM with a probability threshold of 0.75. To obtain GM and WM masks constrained to cerebellum, a mask for cerebellum was manually traced from the high-resolution anatomic image for each subject, and a Boolean operation was then performed between the hand-drawn cerebellar ROIs and the probability-based GM and WM segmentation maps from SPM. GM and WM segmentation masks constrained to cerebellum obtained from that operation were coregistered to the mean image of the ASL scan series. The cerebellar GM and WM masks occasionally exhibited some nonbinary voxel values due to interpolation, and a few of these intermediate-valued voxels extended slightly beyond the cerebellum. In these cases, a threshold  was used to further conservatively limit the masks, based on individual inspection.For each subject, overall cerebellum GM and WM CBF values were estimated for the two PASL methods. Group mean CBF and standard deviation were calculated for cerebellum GM and WM. Interslice, spatial, and temporal variabilities were evaluated by computing the coefficient of variance (C.V.), or standard deviation divided by mean, expressed as percentage. Interslice variability was defined as the ratio between the standard deviation and the mean of slice mean CBF values. Spatial variability was calculated for all voxels within cerebellum GM and WM. The temporal variability was calculated by using the mean perfusion signals of four imaging slices near transverse sinuses.t-tests; P < 0.05 was used as the threshold of statistically significant difference.Comparisons of GM CBF, WM CBF and perfusion signal variability between the two PASL methods were performed using two-tailed paired TI2 values indicated that the estimated bolus durations (ca. 800–1500 ms) and arterial transit times (ca. 550–1000 ms) varied from slice to slice in the cerebellum  from the value of 40.35 ± 8.5 mL/100 g/min that was obtained using PICORE  from the slightly lower value of 23.7 ± 7.5 mL/100 g/min from PICORE . CBF measured by FAIR ASST gave lower intersubject ; a slight correlation of CBF and M0 voxel intensity for GM (R2 = 0.404) disappeared when the 9.6% of GM voxels with intensities less than 800 were ignored (R2 = 0.029). The correlation of CBF and M0 voxel intensity for WM was only slightly affected by this threshold . The GM voxels with intensity < 800 were located almost entirely at the superficial cerebellar boundaries (M0 voxel intensities were slightly skewed to high intensity for WM and to low intensity for GM, suggesting some averaging of intensities on the grey-white matter boundary (To check if signal reduction and image distortions arising from magnetic susceptibility gradients near the cerebellum  might beere made  and Pearundaries , more suboundary .Reliable perfusion measurements depend upon the proper selection of ASL parameters , 32, 33.3 voxels [3 voxels [3 voxels [3 voxels [3 voxels in this study is lower than those obtained using traditional FAIR [Several previous quantitative measurements of cerebellar CBF by PET , SPECT , and ASL3 voxels  and of 53 voxels  agree we3 voxels  and the 3 voxels , SPECT [3 voxels –37, and 3 voxels  and othe3 voxels , 18, 19,3 voxels , 17 in Fnal FAIR , 20–22; nal FAIR , our aveAlthough it has been suggested that EPI-based ASL sequences for obtaining cerebellar perfusion might exhibit signal reduction and image distortions arising from magnetic susceptibility gradients near the cerebellum , these pThese studies were performed on young healthy adults in the age range 25–40 years. For elderly subjects and patients, the blood velocity is lower , 40, whiBecause the superior inflow of venous blood is suppressed at the outset in FAIR ASST, confounding venous inflow effects are minimized, resulting in perfusion images with more stable ASL signals, as indicated quantitatively by the temporal stability analysis . The amoUsing properly selected ASL parameters based on the results of ASL optimization studies, CBF values for cerebellum GM and WM were measured using FAIR ASST and PICORE. Results indicated that FAIR ASST is preferable to PICORE, giving similar CBF estimations but with lower intersubject and spatial variability, less subtraction error, and greater temporal stability. These are important advantages, which should make FAIR ASST more sensitive and specific than PICORE in detecting abnormal CBF values in different conditions and/or subject groups.


Improvement of current GVHD prophylactic therapies remains an important goal in the allo-HSCT. We have described a novel prophylaxis regimen in a single institution trial. The Chinese Bone Marrow Transplant Cooperative Group (CBMTCG) initiated a phase II multicenter study.The study was designed as a prospective, single arm phase II open-label, multicenter clinical trial. The primary endpoint was improvement of aGVHD by 25% over historical control (40%) in Chinese patients. 508 patients were enrolled. All of the patients received cyclosporine A (CsA), methotrexate (MTX) and mycophenolate mofetil (MMF) (0.5-1.0 g daily for 30 days) as GVHD prophylaxis regimen.The primary endpoint was met with cumulative incidences of grades 2 to 4 and grades 3 to 4 aGVHD of 23.2% and 10.3%, respectively. Incidence for cGVHD was 67.4%. The non-relapse mortality (NRM) rate was 18.4% at 2 years. The probabilities of leukemia free survival (LFS) for non-advanced stage and advanced stage patients at 2 years were 69.7% and 44.8% respectively (p = 0.000). Recipient age ≥ 40 years, advanced stage and Busulfan-Fludarabine(BuFlu) conditioning regimen were identified as major risk factors for aGVHD. Recipient age ≥ 40 years, BuFlu conditioning regimens, female donor/male recipient and prior aGVHD were associated with cGVHD. Despite lower RM , patients with grade 2–4 aGVHD had higher NRM and worse OS and LFS compared to patients with grade 0–1 aGVHD. In contrast, patients with cGVHD had better OS and LFS and lower RM compared to patients without cGVHD.The novel GVHD regimen decreased the risk for aGVHD by 42% without improving the risk for cGVHD compared to historical controls. Development of aGVHD was associated with worse OS and LFS as well as higher NRM. In contrast, cGVHD was associated with improved OS and LFS likely attributed to a GVL effect. Despite the use of prophylaxis regimens, Graft-versus-host disease (GVHD) remains a major cause for mortality and morbidity with allogeneic hematopoietic stem cell \transplantations . It is also the primary cause of death in 16% and 18% of deaths after HLA-match sibling and unrelated donor allo-HSCT respectively -[6]. AnaWe have previously described a combination prophylaxis regimen consisting of cyclosporine A (CsA), methotrexate (MTX) and a low-dose, short-course mycophenolate mofetil (MMF) (0.5 daily for 30 days) in a single institution trial for a cohort of 100 patients with hematologic malignancies who underwent HLA-matched sibling allo-HSCT. The rationale behind a regimen designed with a short course of MMF was to primarily improve aGVHD without substantially impacting the incidence of cGVHD because of the associated beneficial GVL effect. Although cGVHD is an undesired complication of BMT, we hypothesized that this strategy would lead to reduced leukemia relapse to an extent which would result in an overall survival net benefit across all patients. We did indeed report a substantial decrease in the risk for aGVHD in our initial study . In ordeThe trial was designed as a prospective, open-label, multicenter clinical protocol and was conducted by the Chinese Bone Marrow Transplant Cooperative Group (CBMTCG), a cooperative transplant group consisting of seven Chinese transplant centers: the Peking University Institute of Hematology, n = 264; the First Affiliated Hospital of Guangxi Medical University, n = 81; the First Affiliated Hospital of Xinjiang Medical University, n = 50; Changzheng Hospital, the Second Military Medical University, n = 10; Kunming General Hospital of Chengdu Command, n = 55; Nanfang Hospital Southern Medical University, n = 28; First Affiliated Hospital Chongqing Medical University, n = 20. Patients with hematologic malignancies in need of an allo-HSCT who had an HLA-identical sibling donor were eligible. Additional eligibility criteria included: 1) Age:15 to 65 years old; 2) Medically suitable to tolerate a myeloablative (MA) conditioning regimen; 3) Eastern Cooperative Oncology Group (ECOG) performance status ≤ 2; 4) Bilirubin ≤ 2 mg/dL, 5) creatinine < 1.5 times the upper limit of normal, 6) preserved heart and lung function; 7) Negative infectious evaluation . Between August 2007 and October 2010, 508 patients were enrolled and completed treatment. The protocol was approved by the Institutional Review Board of each center, and prior treatment written informed consent was obtained from both patients and donors. All participating institutions and investigators had subscribed to the principles and conduct of the WMA Declaration of Helsinki - Ethical Principles for Medical Research Involving Human Subjects. Patient characteristics are summarized in Table The protocol permitted use of three myeloablative conditioning regimens at investigators discretion: 1) BuCy: Busulfan (12–16 mg/kg) and cyclophosphamide (120 mg/kg) was given to 347 patients. 2) BuFlu: Busulfan (12–16 mg/kg) combined with Fludarabine (250 mg or 150 mg/m2) was given to 134 patients and Fludarabine (250 mg) combined with Melphalan (140 mg/m2) was given to one patient. 3) TBICy: TBI (7.7-10.0Gy) and cyclophosphamide (120 mg/kg) was given to 26 patients.6 CD34cells per kilogram of recipient weight. The median numbers of mononuclear cells (MNC) and CD34 cells infused were 9.11 (2.93-17.6) × 108/kg and 4.25 (1.15-16.6) × 106/kg, respectively. 2) PB + BM: 250 patients were grafted with a combination PBSC and bone marrow stem cells (BMSC). PBSC were harvested after donors were treated with G-CSF at a dose of 5–10 ug/kg/d subcutaneously for 4 consecutive days and bone marrow was harvested on the following day by standard technique using general anesthesia. The ratio of CD34 cells numbers of PB to BM was 2–4:1. The median numbers of MNCs and CD34 cells infused were 7.48(3.14-12.53) × 108/kg and 2.34 (0.40-7.45) × 106/kg, respectively. This combination grafting procedure is commonly used in China and based on publications previously describing improved outcomes . Re. Re9/L oFor our Chinese population treated in China, historically grade 2–4 aGVHD is reported to be around 40% -9]. Our. Our9]. Cumulative incidences were estimated for engraftment, aGVHD, cGVHD, non-relapse mortality (NRM), relapse mortality (RM) and relapse in order to evaluate competing risks. The competing risk for engraftment was death without engraftment; the competing risk for GVHD was death without GVHD and graft rejection; relapse was a competing risk for NRM; and NRM was a competing risk for relapse. The time of GVHD occurrence was defined from day 1 after graft infusion to the onset of any grade of GVHD; aGVHD was censored at day 100 after HSCT and cGVHD was censored at the last follow-up visit. The worst stage of the GVHD was assessed as the degree of the GVHD reported. The probabilities of overall survival (OS) and LFS were estimated by the Kaplan-Meier method . Potenti9 cells/L was 14 (7–24) days. During the follow-up period, 496 patients (97.6%) exhibited platelet engraftment, and the median time to reach a platelet count above 20 × 109 cells/ L was 13 (6–124) days.503 (99.0%) patients achieved sustained myeloid engraftment. The median time to reaching an ANC above 0.5 × 10At 100 days after transplantation, the cumulative incidence was 23.2%  for grade 2 to 4 aGVHD, and 10.3%  for grade 3 to 4 aGVHD . Probabilities for OS and LFS in all patients at 2 years were 68.0% (95% CI =63.3%-75.8%) and 66.0% (95% CI =63.3%-75.8%) respectively. Probabilities for OS in patients transplanted at non-advanced stage and advanced stage at 2 years were 71.4% (95% CI =65.9%-77.0%) and 41.8% (95% CI =20.9%-62.6%) (p = 0.000), respectively. The probabilities of LFS in non-advanced stage and advanced stage patients at 2 years were 69.7% (95% CI =64.7%-74.8%) and 44.8% (95% CI =30.7%-58.9%)(p = 0.000), respectively.Sixty-nine patients relapsed after transplantation, reaching a cumulative relapse of 17.7%  at 2 years. The NRM rate was 4.3% (95% CI = 3.4% - 5.2%) and 18.4% (95% CI = 16.3%-20.5%) at 100 days and 2 years, respectively. 53 out of 131 patients died of leukemia relapse. Seventy-eight patients died from other causes than relapse. The most frequent cause of death was infection, specifically, pneumonia. Severe GVHD was determined as cause of death in nineteen patients.In order to analyze the impact of GVHD on the overall clinical outcomes of our patients, we compared outcomes for patients with and without acute and/or chronic GVHD Table . AlthougAlthough GVHD has been recognized more than fifty years as a complication of allo-HSCT, current prophylactic therapies remain insufficient and a high medical need to improve outcomes remains -21]. Th. Th21]. Several studies have identified risk factors for GVHD over the past 3 decades ,26]-[33-[3326]-. AnotherWe observed cumulative incidences of cGVHD and extensive cGVHD at 2 years at 67.4% and 45.1%, respectively. Compared to some historic studies (Table Although we found in our study grade 2–4 aGVHD to be associated with lower rates of relapse mortality, this also resulted in worse OS and LFS mainly due to increased NRM. In contrast, we found that cGVHD was associated with better OS and LFS outcomes which we explain with concurrently decreased rates of relapse mortality Table . NeverthIn summary, although due to the study design a direct comparison of the low dose short course MMF containing regimen to CsA/MTX alone for GVHD prophylaxis could not be performed, we demonstrated a substantial decrease for the risk of aGVHD development. In contrast, the incidence for cGVHD could not be improved when compared to historical results in Chinese patients. Our results also suggest that Chinese patients may have slight variations in risk factors for developing GVHD.allo-HCST: Allogeneic hematopoietic cell transplantationGVHD: Graft-versus-host diseaseaGVHD: Acute GVHDcGVHD: Chronic GVHDCsA: CyclosporineMTX: MethotrexateMMF: Mycophenolate mofetilHLA: Human leukocyte antigenLFS: Leukemia free survivalOS: Overall survivalCR: Complete remissionTBI: Total body irradiationG-CSF: Granulocyte colony-stimulating factorMNC: Mononuclear cellsNRM: Non-relapse mortalityRM: Relapse mortalityThe authors declare that they have no competing interests.YRL and YHC designed the research, interpreted the data and wrote the manuscript; DMH, MJ, QL, LL, JH, PS, QCL, ZMZ and KYL performed the study and contributed to writing the manuscript; XJH is the principal investigator, designed the research, interpreted the data, and wrote the manuscript. All authors read and approved the final manuscript.


In previous studies of transverse sinus (TS) hypoplasia, discrepancies between TS diameter measured by magnetic resonance venography (MRV) and contrast T1-weighted magnetic resonance (contrast T1) were observed. To investigate these discrepancies, and considering that TS hypoplasia is associated with neurological disorders, we performed a post hoc analysis of prospectively collected data from 3 case–control studies on transient global amnesia (TGA), transient monocular blindness (TMB), and panic disorders while retaining the original inclusion and exclusion criteria. Magnetic resonance (MR) imaging of 131 subjects was reviewed to evaluate TS diameter and the location and degree of venous flow stenosis and obstruction.P < 0.001), which was significantly larger in patients than controls. Compensatory dilatation of contralateral TS diameter was only observed with MRV, not with contrast T1 imaging.MRV without contrast revealed that TS hypoplasia was observed in 69 subjects, whom we classified into 2 subgroups according to the concordance with contrast T1 observations: concordance indicated anatomically small TS (30 subjects), and discrepancy indicated that the MRV diagnosis is in fact flow-related and that TS is not anatomically small (39 subjects). The latter subgroup was associated with at least 1 site of venous compression/stenosis in the internal jugular vein (IJV) or the left brachiocephalic vein (BCV) (The clinical implication of these results is that using MRV only, IJV/BCV compression/stenosis may be misdiagnosed as TS hypoplasia. And contralateral TS have no compensatory dilatation in its diameter in contrast T1 imaging, just compensatory increased flow volume. Of note is that these studies used either angiography or magnetic resonance venography (MRV) to evaluate TS flow, but none used contrast T1-weighted magnetic resonance imaging (contrast T1) to assess actual TS lumen shape and size.Anatomical asymmetry of transverse sinuses (TS) is a common finding and was considered a normal variant, as unilateral hypoplasia or aplasia is observed in 20% to 39% of cases.8 and transient monocular blindness (TMB),9 we noted that there was not only a discrepancy in TS diameter measured by MRV and contrast T1, but also the discrepancy with correlating the internal jugular vein (IJV) stenosis/compression. This observation was consistent with another previous study.10 We found that some subjects displayed TS asymmetry on both MRV and contrast T1 8 TMB,9 and panic disorder.11We retrospectively reviewed the MR images of subjects enrolled from our previous prospective studies of venous outflow impairment in TGA,11The detailed research methods and population composition of the TGA, TMB, and panic disorder patients and their age- and gender-matched controls can be found in the original reports.12 Age- and gender-matched individuals for the control group were recruited prospectively from people receiving physical check-ups and who had no history of neurologic signs or symptoms.8TGA patients were prospectively recruited from the Neurology Department of Taipei Veterans General Hospital. All patients were examined by a neurologist, and TGA was diagnosed according to criteria modified and validated by Hodge and Warlow.9Patients diagnosed with TMB were recruited from consecutive outpatients of the Neurology Department of Taipei Veterans General Hospital and from referrals for cerebrovascular survey by ophthalmologists or other physicians. All patients were examined by 1 neurologist and were questioned about the characteristics of their transient loss of vision using a standardized questionnaire. The control group was selected from individuals receiving physical check-ups who had no carotid stenosis and no history of visual problems.13 Patients who were unwilling to participate or had history of hypertension, diabetes, smoking, stroke, ischemic heart disease, congestive heart disease, arrhythmia, pulmonary diseases, carotid or intracranial artery stenosis, and malignancies were not included.11For panic disorder, patients were consecutively referred by the psychological clinics of Taipei Veterans General Hospital (Dr. Hong) and were diagnosed according to the Diagnostic and Statistical Manual of Mental Disorders, 4th edition (DSM-IV-TR) criteria.Overall, 131 patients were included in our study.Examinations of all patients and controls were performed with contrast-enhanced MR imaging using a 1.5 T MR imaging scanner .The protocol included TRICKS, contrast-enhanced axial T1-weighted magnetic resonance imaging (contrast T1), and MRV. The MRV used was a phase contrast based contrast-free angiography image, acquired with Inhance 3D Velocity . We used the following MR sequences: Inhance 3D Velocity MRV: sagittal plane repetition time (TR)/echo time (TE)/flip angle (FA): 11.7/4.5/10°, matrix size: 320 × 256, field of view (FOV): 24 cm × 21.6 cm, slice thickness: 1.4 mm with interpolation to 0.7 mm slice interval using a parallel imaging technique, acceleration factor: velocity encoding: 25 cm/s. TRICKS: coronal plane TR/TE/FA: 3.1/1.1/30°, matrix size: 320 × 192, FOV: 34 cm × 30.6 cm, slice thickness: 3.2 mm with interpolation to 0.8 mm slice interval. Contrast-enhanced T1 spoiled gradient recalled (SPGR) acquisition in the steady-state MR sequence: axial plane TR/TE/FA: 8.6/2.5/15°, matrix size: 320 × 256, FOV: 24 cm × 18 cm, slice thickness: 3 mm. Only those patients and controls who had complete studies with MR imaging were included in this study.10 grade 0 = normal round or ovoid appearance; grade 1 = mild flattening; grade 2 = moderate flattening; grade 3 = severe flattening or not visualized , middle IJV (at C3–5 level), and lower IJV (at C6–T2), using contrast T1. IJV compression/stenosis was evaluated according to the following criteria by Zaharchuk et al level, m8 .TS size measurements were presented as median and interquartile range (IQR). We compared the frequency of TS hypoplasia among different groups of patients and controls using Fisher exact test or a Chi-squared test, and conducted post hoc analysis of pairwise comparisons using the Bonferroni correction. Continuous data were compared using the nonparametric Kruskal–Wallis test, and pairwise comparisons were conducted using the Wilcoxon rank-sum test with the Bonferroni correction. Two-sided A total of 131 subjects  were enrolled in our studies of TGA, TMB, and panic disorders during the years 2008 to 2012.P = 0.005) and a post hoc analysis showed a significant difference in frequency between TGA patients and controls (P = 0.001) and between TMB patients and controls (P < 0.001)  and with at least 1 site of venous compression/stenosis in the IJV or BCV   . Though there was no significant difference in TS diameter between the 2 types of hypoplasia in MRV imaging . Using contrast T1 images .For contralateral TS diameters, using MRV imaging  than in controls . Using contrast T1, we also found no evidence for compensatory dilatation of contralateral TS diameter, which indicates that it is flow volume, and not TS diameter, that is compensatory increased.


Previous studies with broiler have shown dietary supplementation with multi-enzyme complex containing non-starch polysaccharides (NSP) degrading enzymes and phytase is efficient in releasing phosphorus (P), calcium (Ca), energy and amino acids from corn-soybean meal diets or corn-sorghum diets, hence compensating considerable levels of nutrients in formulation. Notwithstanding, such potentials have not been well defined in duck nutrition. Giving China being the largest duck producing country, we conducted this study to establish adequate specifications of major nutrients along with multi-enzyme complex to meat duck from day-old to slaughter, focusing on performance, utilization of nutrients and bone mineralization. Five dietary treatments were: Positive control : the nutrients concentration of diet for 1 to 14 d of age were apparent metabolizable energy(AME) 2,800 kcal/kg, crude protein (CP)19.39%, Ca 0.85%, available phosphorus (avP) 0.42%; for 15 to 35 d of age these parameters were AME 2,900 kcal/kg, CP 16.47%,Ca 0.76%,avP 0.38%; Negative control 1, the AME and digestible amino acids (DAA) were reduced by 70 kcal/kg and 2.0%, avP and Ca by 1.0 g/kg from PC diet; Negative control 2, the down-spec from PC diet was AME 100 kcal/kg, DAA 2.5%, avP 1.5 g/kg and Ca 1.2 g/kg; The enzyme complex was added at the same dosage  on NC1 (T3) and NC2 (T5) diets.P < 0.05) body weight ( d 14 and 35), feed intake (d 35), tibia ash, Ca and P contents (d 14 and 35), and the utilization of nutrients (P < 0.05). The supplementation with the enzyme complex to the NC diets restored growth rate, utilization of nutrients and bone mineralization to the level of the PC diet, and increased AME by 60 kcal/kg and 117 kcal/kg, respectively for the NC1 and NC2 diets.Comparing with the ducks fed on T1, T3 and T5 diets, the birds fed on NC2 diet showed the lowest (These results suggest that down-spec AME by 100 kcal/kg, DAA by 2.5%, avP by 1.5 g/kg and Ca by 1.2 g/kg caused detrimental effects on duck performance compared with those fed on the PC diet, and these performance losses can be compensated by the addition of the multiple-enzyme complex. Previous studies demonstrated single or multiple enzyme complex such as carbohydrases and phytase can improve utilization of dietary nutrients, thus decrease the cost of feeds in poultry production . CarbohyFrancesch and Geraert  reportedThe experimental protocol used in this study was approved by the Institutional Animal Care and Use Committee of Sichuan Agricultural University.Penicillium funiculosum and Schizosaccharomyces pombe, Adisseo France SAS). The liquid enzyme was applied after feed pelleting at a dose of 200 mL/1,000 kg feed to provide a minimum 1,100 visco-units of endo-β-1,4-xylanase, 100 units of endo-1,3(4)-β-glucanase, and 500 phytase units (FTU)/kg feeds. There were five dietary treatments replicated 7 times each of 16 ducks per replicate and allocated at random by blocks, according to the location in the experimental house. The feeding program consisted of 2 diets, starter diet supplied from d 1 to d 14, followed by a grower diet from d 15 to d 35  diet was formulated to be adequate or to exceed requirements of NRC (1994) , NC1  were placed into individual metabolic cages (50 cm × 50 cm) in a room with temperature maintained at 22-25°C with continuous lighting. At day 39, each duckling was surgically fitted with a retainer ring  and fed Five hundred and sixty one-day-old male Cherry Valley ducks were used and distributed into 35 cages of 16 ducks per cage. All ducks were reared in cages (2.2  m × 1.2 m × 0.9 m) in a temperature and humidity controlled room with a 24 h constant light schedule and had free access to water and feed throughout the experimental period.At the age of d 14 and d 35, birds and feed consumption were weighed by cage, body weight (BW) gain, feed intake (FI), feed to gain ratio (F/G) were then calculated. Seven ducklings per treatment (1 bird per pen) were randomly selected for blood sampling via the jugular vein. Serum was obtained by centrifuge at 3,000 rpm, 4°C for 10 min. All serum samples were stored -20°C. Contents of Ca, P and alkaline phosphatase (ALP) activity were measured by automated hematology analyzer . Once blood was collected, birds were euthanized by cervical dislocation. Their left tibia was removed and stored at -20°C. After boiling/extraction, tibia ash, Ca and P contents were determined, according to the procedures of AOAC .Diet and dried excreta samples were ground to pass through 0.5 mm screen using a mill grinder. Duplicate proximate analyses were performed on diets and excreta samples. Dry matter(DM) analysis of samples was conducted by drying the samples in an oven at 105°C for 24 h. Energy content of the samples was determined by the adiabatic oxygen bomb calorimeter. Nitrogen, ester extract (EE), ash, Ca and total P contents of the samples were determined according to AOAC .P ≤ 0.05.To establish differences between PC and reduced nutrient diets, with and without enzyme supplementation, data were analyzed as a randomized complete block design with a one-way ANOVA using the GLM procedures of SAS . To deteP < 0.05) BW (d 14 and 35) and FI (d 35) compared to those fed on PC, NC1 with the enzyme and NC2 with the enzyme. The addition of the enzyme complex significantly increased (P < 0.05) BW and FI at days 14 and 35 to the level of PC diet, but no effect on F/G. Feed to gain ratio in NC1, NC1 with enzyme, NC2 and NC2 with enzyme diets was marked higher (P < 0.05) than that of the PC diet. Interaction between reduced dietary nutrient level and enzyme supplementation was detected (P <0.05) for BW at 14 d of age and FI during the whole experiment. These results suggest that NC1 nutrient levels  were able to meet the growth requirement of the meat ducks; but NC2 nutrient level  showed a negative effect on performance. Xie et al. [The results of growth performance are shown in Table e et al.  studied e et al. ,26.TableP < 0.05), respectively. This is consistent with the previous findings by Avila [The supplementation with multiple-enzyme complex to the diets of chickens has been shown to liberate metabolizable energy from the otherwise unavailable sugars from NSP and P of the phytate molecule,15. Mengby Avila , who repby Avila .P < 0.01) availability of CP and EE compared with the PC diet. Feeding NC2 diet reduced (P < 0.01) availability of DM, energy, CP, EE, ash, Ca and P compared to feeding PC and NC1 diet. The addition of the enzyme to the NC diets significantly improved (P < 0.05) energy, CP, EE, ash, Ca and P availability. Likewise, the enzyme addition to the NC2 diet significantly improved (P < 0.05) energy, DM, CP, EE, ash and P retention, but didn’t restore to the level of the PC (P < 0.05). Supplementing the enzyme complex to the NC1 diet increased AME by 60 kcal/kg, and in NC2 diet by 117 kcal/kg. Meng et al. [The effects of enzyme supplementation on nutrition utilization are summarized in Table d P < 0.0 energy, g et al. ,29. Carbg et al. . This stg et al. , who demP < 0.05) compared with PC diet (Table P < 0.05). The interaction of diets and enzyme supplementation was significant (P < 0.05) on serum P content. The effects of the enzyme supplementation on bone mineralization are summarized in Table P < 0.05), but not P. The enzyme supplementation to NC1 diet significantly improved tibia ash, P, Ca concentrations (P < 0.05) to the level of the PC group. At days 14 and 35, tibia ash, P, Ca concentrations of birds fed NC2 diet were lower (P < 0.05) compared with those on the PC diet. The enzyme addition to NC2 diet markedly improved these parameters (P < 0.05) to the level of the PC group, with an exception that tibia ash and P concentration at 14 d did not reach the level of the PC diet. Diet × enzyme interactions were detected for 35 d tibia ash and Ca concentration (P <0.05).At the age of 35 d, serum P content of the birds fed on NC2 diet decreased by 26.5% . Viveros et al. ,32.Bone mineral concentration may be more sensitive than performance in estimating mineral deficiency. The requirement of avP and Ca for maximum P, Ca and ash deposition in tibia was greater than for adequate growth performance ,26. SimiIn conclusion, results of the present experiment indicate that the lower nutrient density diets resulted in a reduction in growth performance, nutrient utilization and bone mineralization of meat ducklings which can be restored by supplementation of exogenous enzyme complex, and that the multi-enzyme complex has the potential to compensate AME 100 kcal/kg, DAA 2.5%, av P 1.5 g/kg and Ca 1.2 g/kg in basal diet consisting in corn, soybean meal and by-products.


Stichodacyla haddoni (shCP). A plasmid in which shCP cDNA is driven by a heat-inducible promoter was linearized and electroporated into 2.5×108 protoplasts of Nannochloropsis oculata. Following regeneration and cultivation on an f/2 medium plate for two weeks, we observed 26 colonies that displayed a slightly dark green coloration. After individually subculturing and performing five hours of heat shock at 42°C, a dark brown color was mosaically displayed in five of these colonies, indicating that both untransformed and transformed cells were mixed together in each colony. To obtain a uniform expression of shCP throughout the whole colony, we continuously isolated each transformed cell that exhibited brown coloration and subcultured it on a fresh plate, resulting in the generation of five transgenic lines of N. oculata which stably harbored the shCP gene for at least 22 months, as confirmed by PCR detection and observation by the naked eye. As shown by Western blot, exogenous shCP protein was expressed in these transgenic microalgae. Since shCP protein is biodegradable and originates from a marine organism, both environmental and food safety concerns have been eliminated, making this novel shCP reporter gene a simple, but effective and ecologically safe, marker for screening and isolating transgenic microalgae.Among the methods used to screen transgenic microalgae, antibiotics selection has raised environmental and food safety concerns, while the observation of fluorescence proteins could be influenced by the endogenous fluorescence of host chloroplasts. As an alternative, this study isolated the purple chromoprotein (CP) from  General as Nit1  and Arg7t1 [Arg7 , which r AphVIII –7, whiched cells , such ased cells , 10 or bed cells .Cre/lox [FLP/FRT [R/RS systems [Cre/lox recombination system requires retransformation treatment which is both labor-intensive and time-consuming [Most selective marker genes have some disadvantages. In the case of auxotrophic selection markers, it is argued that auxotrophic mutant strains are required, making this marker suitable for only haploid genome algae . With flCre/lox , FLP/FRT[FLP/FRT  and R/RS systems , have be systems –26. Howe systems , 28. SSR systems , 26, 29.onsuming , 30, andonsuming , 29, 31.onsuming  knocked Stichodactyla haddoni, termed shCP [Hecteractis crispa. This protein possesses a maximum absorption wavelength peak (λmax) at 574 nm, resulting in a purple color. Microalgae are photosynthetic microorganisms, and some species have been utilized in aquaculture as essential nutritional feed for the production of farmed organisms and other commercially important aquaculture species [Nannochloropsis oculata is the most frequently used microalga in aquaculture applications [N. oculata is an ovoid-shaped marine unicellular microalga that grows in a wide range of conditions that vary by pH, temperature and salinity. It also possesses a high rate of biomass production and a high content of unsaturated fatty acids [N. oculata becomes a potentially attractive bioreactor for biofuel production and live feed for fish and shellfish larvae [N. oculata, enabling the identification and isolation of transgenic microalgae by direct visual detection without the necessity of either auxotrophic mutants or complicated fluorescence equipment.We previously isolated a cDNA fragment encoding a novel GFP-like chromoprotein from the carpet anemone med shCP . The shC species . Nannochications –38. N. oty acids , which aty acids . Furtherh larvae . In thisHSP70A::RBCS2 promoter that can be induced by light and heat shock [N. oculata protoplast cells. After cultivation of all treated algal cells (2.5 × 108 cells) for two weeks on an f/2 medium plate, 94 colonies were grown  were cultured on an f/2 medium plate for two weeks. A total of 384 colonies were grown on the plate  and Nannochloropsis sp.  thes and pH –46. TherChen and Melis  reportedAccording to Bhattacharya et al. , the hubN. oculata. However, it should further study whether chromophores may work as well in diploid organisms or organelles having multiple copies of the genome.In this study, the shCP cDNA-encoded purple chromophore protein shCP was used to identify microalgae transformants by simple visual detection of clones that present a dark brown color, eliminating the need for fluorescent microscopy equipment. Moreover, shCP cDNA is a safe selection marker gene since antibiotics, herbicides and drug-resistant chemicals are not needed to distinguish the transformed cells. Instead, the safety of shCP protein encoded by shCP cDNA for commercial use is ensured because it originates from marine anemones, which are edible and biodegradable proteins. Thus, the use of a chromophore as a selection marker appears to work well in haploid cells such as S. haddoni and transferred it into the microalga N. oculata. Although both anemone and microalga are eukaryotes, they have their own codon preference. When we used E-CAI  software to calculate the CAI value of the shCP gene, it was found to be 0.256. This value is defined as a low translation level, indicating that the shCP gene may not yield efficient expression in microalgae. Nevertheless, as shown in It has been reported that optimizing the codon usage of transferred genes before gene transfer can improve foreign protein expression and reduce exogenous gene silencing . SeveralNheI cutting site and a 7-bp Kozak sequence are included) and antisense primer 5’-GAATTCTCAATTTGCTTTTTCAGGAAGATCACTGTA-3’ (an EcoRI cutting site is included). The PCR reaction was performed under the following conditions: 1 cycle of denaturation at 94°C for 5 min, 25 cycles of denaturation at 94°C for 30 sec and annealing at 58°C for 30 sec and extension at 72°C for 45 sec, followed by 1 cycle of final extension at 72°C for 5 min. The resultant 703-bp shCP PCR product, which was extracted from a 1.5% agarose gel after electrophoresis, was cloned into plasmid pGEM-T easy. The resultant plasmid was digested with NheI and EcoRI to obtain the shCP fragment. The shCP fragment was then inserted into plasmid phr-LFB-DsRed [HSP70A plus RBCS2, which was obtained by cutting NheI and EcoRV from pCB740 vector, an artificial promoter from C. reinhardtii which can improve transgene expression in algae [NheI and EcoRI, an inducible promoter, HSP70A::RBCS2, and backbone were preserved, but the DsRed fragment was removed. The final expression plasmid, phr-shCP, was generated. This plasmid was linearized by SacII for transformation.Plasmid pET-15b-shCP, which was first constructed by Chiang et al. , served FB-DsRed . This exin algae . After tN. oculata NIES-2146 were obtained from the National Institute for Environmental Studies. The cells were cultured in f/2 medium under continuous light with a photon flux density of 85 μmol photons m−2 s−1 at 28°C. Agar plates were prepared with 0.8% agar (Sigma) in f/2 medium [2 medium  with seaN. oculata protoplasts was followed by Li and Tsai [N. oculata cells were grown in f/2 medium at mid-log phase (∼2 × 106 cells/mL). Then 5 × 108 cells were collected and the pellet was washed twice with 1 ml of sterilized seawater, followed by resuspension in 500 μL of synthetic gastric juice (pH 2.0) with 5% pepsin (Sigma) to digest microalgae cell walls [2, 0.01 mM HEPES, 0.2 mM Mannitol and 0.2 mM Sorbitol [The procedure to produce and Tsai  with somll walls  and, finSorbitol  and finaN. oculata followed procedures previously described by Li and Tsai [N. oculata protoplasts (5 × 108 cells) in a volume of 200 μL were resuspended in the electroporation buffer. Half of them (2.5 × 108 cells) were taken and added to 10 μg of linearized plasmid phr-shCP which was used for each electroporation within one hr. Electroporation was performed with an electroporator in a 1-mm cuvette . The electroporator was adjusted to 2,000V voltage, 25 μs pulse length, and 10 pulse time. After electroporation, cells were transferred to a 15 mL glass tube which contained 5 mL f/2 medium and incubated in low light overnight. Then, all cells (2.5 × 108 cells) were plated on an f/2 agar plate for two weeks, followed by counting single algal cells exhibiting green or slightly dark green coloration. Those algal cells that displayed a slightly dark green color were individually selected, given a name and continuously subcultured on a fresh f/2 agar plate for two successive weeks. They were then treated with heat-shock at 42°C for five hrs. After heat-shock treatment, colonies were cultured for one more week. Afterwards, cells that displayed a dark brown color were selected for further confirmation by DNA analysis. These steps were carried out twice under the same conditions.The transformation of and Tsai . BrieflyN. oculata followed the description of [8) were harvested and resuspended in 350 μL hexadecyltrimethylammonium bromide buffer by the following protocol. After the mixture was incubated at 65°C for 30 min, the lysate was mixed with 1 volume of phenol/chloroform and left to stand 5 min at room temperature, followed by centrifugation at 13,000 x g at 25°C for 10 min and extraction of aqueous layer to a new Eppendorf. Finally, the genomic DNA was precipitated by 1/10 volume of sodium acetate and 2 volumes of 99.5% ethanol in -20°C for at least 90 min, centrifuged at 13,000 x g at 4°C for 10 min, washed with 70% ethanol to remove salt, and centrifuged again at 13,000 x g at 4°C for 5 min. Finally, the pellet was dried for 3 min to remove additional ethanol and then resuspended in 25 μL of distilled deionized water.The extraction of genomic DNA of ption of  with modThe existence of plasmid phr-shCP in microalgae was determined by PCR analysis using genomic DNAs as templates to amplify the coding region of shCP cDNA when the forward primer 5’-ATGGCCGGTTTGTTGAAAGAAA-3’ and the reverse primer 5’-TCAATTTGCTTTTTCAGGAAGATCA-3’ were used. Each PCR consisted of 50 μL of solution containing 20 ng of template, 10 μmol of each primer, 25 mM of each dNTP, and 5 units of Taq enzyme in a 10 X PCR buffer . Amplification was performed with 35 cycles of denaturation at 94°C for 1 min, annealing at 62°C for 1 min, and extension at 72°C for 80 sec, followed by 7 min extension at 72°C. The PCR products were subjected to electrophoresis on a 0.8% agarose gel .The putative transgenic clones of microalgae were also observed by a stereological image system to visualize the transgenic clones containing phr-shCP. Images were captured with an Applied Precision Spectris optical sectioning microscope system equipped with a stereo investigator and neurolucida microscopy, an Olympus Plan Apo 1006 oil immersion objective, and Qimaging digital camera. Using stereological image software, the brightness and contrast were adjusted, setting the area outside of cells as background.th round of replating, we picked up ten single colonies and cultured them separately on the plate. After cultivation for three weeks, algal cells were harvested from these ten separate single colonies, transferred into a 15 mL glass tube containing 5 mL f/2 medium, and continuously cultivated for three weeks without shaking. After that, all algal cells were inoculated into 50 mL of fresh f/2 medium and cultivated for another three weeks. Then, all algal cells were continuously enriched in 100 mL of fresh f/2 medium for one week. Finally, all algal cells were inoculated into 500 mL of fresh f/2 medium for three weeks. Total proteins were extracted from these cultured N. oculata using the protocol described by Mayfield and Schultz [E. coli system and purified by His GraviTrap .When transgenic line CP10 was subcultured at the 12 Schultz  with somS1 File(DOCX)Click here for additional data file.S1 Fig8 algal cells were cultured on f/2 medium plate for two weeks. (A) A total of 94 colonies were grown. Most of them exhibited a green coloration. However, 26 colonies displayed a slightly dark green color. (B) These 26 colonies were individually selected, given a name and continuously subcultured on a fresh f/2 medium plate. After two weeks of cultivation, each colony grew normally, and they were ready for further heat-shock treatment.After electroporation, a total of 2.5 x 10(DOCX)Click here for additional data file.S2 Fig8 algal cells were cultured on f/2 medium plate for two weeks. A total of 384 colonies were grown. Most of them exhibited a green coloration. However, 15 colonies displayed a slightly dark green color. (B) These 15 colonies were individually selected, given a name and continuously subcultured on a fresh f/2 medium plate. After two weeks of cultivation, each colony grew normally, and they were ready for further heat-shock treatment.After electroporation, a total of 2.5 x 10(DOCX)Click here for additional data file.S3 FigN. oculata were grown on f/2 medium plates for two weeks, followed by heat-shock at 42°C for 5 hr. After cultivation for one week, a dark brown coloration presented by reporter chromoprotein was observed in some cells in each colony, as indicated by red arrows. The wild-type colony served as a negative control. (B) The putative transformants of N. oculata were picked up and cultivated on the second round of replating (G2), the third round of replating (G3) and so on until the fifteenth round of replating (G15) within 17 months. In contrast to the green coloration presented in the wild-type strain after heat-shock treatment (HS), a dark brown coloration was exhibited in the whole colony of transgenic strains CP2nd 1, CP2nd 4∼5 and CP2nd7∼14. “Stable” indicates that foreign protein could be continuously expressed beyond the 15th round of replating.(A) After electroporation with plasmid phr-shCP, 15 colonies of (DOCX)Click here for additional data file.S4 FigN. oculata. After transformation, all microalgal cells were cultured, and the putative clones were selected to determine the existence of the shCP gene in N. oculata. The wild-type strain served as a negative control, whereas plasmid phr-shCP served as a positive control. The expected molecular size of phr-shCP after PCR amplification was 0.684 kb. The small subunit 18S ribosomal RNA (18s) served as the internal control.Plasmid phr-shCP was used to transform (DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Geobacillus sp. Y412MC52 was isolated from Obsidian Hot Spring, Yellowstone National Park, Montana, USA under permit from the National Park Service. The genome was sequenced, assembled, and annotated by the DOE Joint Genome Institute and deposited at the NCBI in December 2011 (CP002835). Based on 16S rRNA genes and average nucleotide identity, Geobacillus sp. Y412MC52 and the related Geobacillus sp. Y412MC61 appear to be members of a new species of Geobacillus. The genome of Geobacillus sp. Y412MC52 consists of one circular chromosome of 3,628,883 bp, an average G + C content of 52 % and one circular plasmid of 45,057 bp and an average G + C content of 45 %. Y412MC52 possesses arabinan, arabinoglucuronoxylan, and aromatic acid degradation clusters for degradation of hemicellulose from biomass. Transport and utilization clusters are also present for other carbohydrates including starch, cellobiose, and α- and β-galactooligosaccharides.The online version of this article (doi:10.1186/s40793-015-0075-0) contains supplementary material, which is available to authorized users. Thermus aquaticus [Thermus brockianus [Acidothermus cellulolyticus [Identification of new organisms that produce biomass-degrading enzymes is of considerable interest. Commercial uses for these enzymes include paper manufacturing, brewing, biomass deconstruction and the production of animal feeds –3. Hot squaticus , 5, Therockianus , and Aciolyticus  that posolyticus . As partGeobacillus species were the most common aerobic organisms isolated during the cultivation of most hot springs samples. Geobacillus species were originally classified as members of the genus Bacillus, but were subsequently reclassified as a separate genus based on 16S rRNA gene sequence analysis, lipid and fatty acid analysis, phenotypic characterization, and DNA—DNA hybridization experiments [Geobacillus species have been isolated from a number of extreme environments including high-temperature oilfields [Geobacillus species to thrive in these varied and often hostile environments suggests that these species possess enzymes suitable for applications in challenging industrial environments. We therefore sequenced a number of these Geobacillus isolates including strains Y41MC52, Y41MC61, C56-T3, and Y4.1MC1 [eriments . Geobaciilfields , a corroilfields , Africanilfields  and Russilfields  hot spriilfields , and theilfields , yet theilfields ., The ab Y4.1MC1  to identGeobacillus sp. Y412MC52 and Geobacillus sp. Y412MC61 are two thermophilic organisms isolated from Obsidian Hot Spring, Yellowstone National Park, Montana, USA (44.6100594° latitude and −110.4388217° longitude) under a sampling permit from the National Park Service. The hot spring possesses a pH of 6.37 and a temperature range of 42–90 °C. The organisms were isolated from a sample of hot spring water by enrichment and plating on YTP-2 medium [Bacillus Genetic Stock Center as GSCID: 96A11 (MC52) and GSCID: 96A12 (MC61). Both cultures are routinely grown in YTP-2 medium media and maintained on YTP-2 agar plates. MC52, is a Gram-positive, rod-shaped facultative anaerobe . Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute. A summary of the project information and its association with MIGS identifiers is shown in Table Y412MC52 was selected for sequencing on the basis of its biotechnological potential as part of the U.S. Department of Energy Genomic Science program (formerly Genomics:GTL). The genome sequence is deposited in the Genomes On Line Database , 24  using a combination of Sanger, Illumina and 454 technologies [The genome of nologies . An Illunologies , and thenologies –30 in thGenes were identified using Prodigal  as part Geobacillus sp. Y412MC52 consists of one circular chromosome of 3,628,883 bp (Table The genome of Geobacillus species. The comparison of the MC52 genome to the other genomes (Table Geobacillus sp. C56-T3 (98.3 %). These values are above the species cutoff value of 98.2 % to 99.0 % [Average Nucleotide Identity (ANI) calculations  were usees Table  confirmso 99.0 %  indicatio 99.0 %  shows onG. stearothermophilus [G. stearothermophilus [MC52 possesses a 45-gene arabinan and xylan degradation cluster that allows degradation of hemicellulose components of biomass (GYMC52_1817 through GYMC52_1867). The cluster contains one secreted xylanase (GYMC52_1825) and one secreted arabinase (GYMC52_1858), in agreement with the experimental results. The organization of the xylan degradation portion of the cluster matches the glucuronic acid utilization cluster described for mophilus . The aramophilus , lackingmophilus . These aGeobacillus strains, including Geobacillus sp. Y412MC61 (GYMC61_2740 through GYMC52_2750), Geobacillus sp. Y4.1MC1 (GY4MC1_2192 through GY4MC1_2202), and Geobacillus sp. C56-YS93 (Geoth_2276 through Geoth_2288). The cluster does not code for a secreted protease or peptidase, but contains an annotated five-gene ABC peptide transporter system and two intracellular peptidases.The smaller arabinan cluster in MC52 is the result of an 11-gene insert (GYMC52_1870 through GYMC52_1880) coding for a peptide utilization cluster that replaces part of the arabinan cluster. This peptide utilization cluster is found in only a few Geobacillus strain Y412MC52 possesses a 54.4 Kb, 73-gene insert that codes for 47 phage genes identified using phast [Geobacillus phage E2 (GenBank NC_009552) [Geobacillus strain C56-YS93 also isolated from Obsidian Hot Spring, indicating the phage may have a limited range of hosts in the hot spring.ng phast  phage id_009552) , isolatePaenibacillus lautus Y412MC10 [Geobacillus thermoglucosidans C56-YS93 (manuscript submitted) and Geobacillus sp. Y412MC52 and Y412MC61. Especially of interest is the isolation of both low G + C  and high G + C  xylanolytic Geobacillus species from the same hot spring sample. This suggests that the high and low G + C Geobacillus species may occupy separate ecological niches that allow each strain to thrive in the same site. Based on the genomic analysis, Geobacillus sp. Y412MC52 appears to utilize only some biomass components such as xylan, arabinoglucuronoxylan, and the arabinan component of arabinogalactan. MC52 shows no genes coding for utilization of other biomass components such as cellulose, mannan, glucomannan, galactomannan, chitin, or pectin, confirming experimental observations. The limited range of substrates suggests MC52 functions as part of a microbial consortium in degrading biomass. The presence of aromatic acid metabolic clusters and the lack of mannan-utilization clusters suggest the organism has a preference for utilization of hemicellulose derived from grassy plants rather than woody plants.Obsidian Hot Spring is home to a wide variety of organisms, including Y412MC10 , GeobaciGeobacillus sp. Y412MC52 and the related Geobacillus sp. Y412MC61 appear to be members of a new species of Geobacillus. The presence of multiple 16S rRNA genes in Geobacillus species as well as the small differences observed in 16S rRNA gene sequences makes assignment of strains to new or existing species difficult. Utilization of recN sequences [Geobacillus species.Based on 16S rRNA genes and average nucleotide identity, equences  has been


Glycine max grown in Ottawa, Canada, were previously characterized and placed in a novel group within the genus Bradyrhizobium. To verify their taxonomic status, these strains were further characterized using a polyphasic approach. All strains possessed identical 16S rRNA gene sequences that were 99.79 % similar to the closest relative, Bradyrhizobium liaoningense LMG 18230T. Phylogenetic analysis of concatenated atpD, glnII, recA, gyrB, rpoB and dnaK genes divided the 16 strains into three multilocus sequence types that were placed in a highly supported lineage distinct from named species of the genus Bradyrhizobium consistent with results of DNA–DNA hybridization. Based on analysis of symbiosis gene sequences (nodC and nifH), all novel strains were placed in a phylogenetic group with five species of the genus Bradyrhizobium that nodulate soybeans. The combination of phenotypic characteristics from several tests including carbon and nitrogen source utilization and antibiotic resistance could be used to differentiate representative strains from recognized species of the genus Bradyrhizobium. Novel strain OO99T elicits effective nodules on Glycine max, Glycine soja and Macroptilium atropurpureum, partially effective nodules on Desmodium canadense and Vigna unguiculata, and ineffective nodules on Amphicarpaea bracteata and Phaseolus vulgaris. Based on the data presented, we conclude that our strains represent a novel species for which the name Bradyrhizobium ottawaense sp. nov. is proposed, with OO99T ( = LMG 26739T = HAMBI 3284T) as the type strain. The DNA G+C content is 62.6 mol%.Sixteen strains of symbiotic bacteria from root nodules of  Glycine max (L.) Merrill] is an economically important grain legume that can form a nitrogen-fixing association with species of soil bacteria belonging to the genus Bradyrhizobium. In a previous study  had rod-shaped cells, subpolar flagella and a cell size  that is consistent with the characteristics of the genus Bradyrhizobium  were obtained from root nodules of soybeans grown at a field site in Ottawa, Ontario  and purehizobium  with bachizobium   and symbiotic (nodC and nifH) genes were generated for the 16 novel strains of the genus Bradyrhizobium as well as for reference taxa not available in public databases. Preparation of genomic DNA, amplification, primers, nucleotide sequencing, sequence alignment and editing was as described previously (dnaK sequence of ‘radyrhizobium retamaeB’ Ro19 required designing a second forward sequencing primer (FdnaK_SC1: GAGCAGCAGATCCGGATTCA) in order to obtain the 3′ end of the sequence. GenBank accession numbers of nucleotide sequences are given in Tables S1 and S2.Almost full-length 16S rRNA gene sequences were generated using primers 16Sa and 16Sb  and sequsd of split frequencies fell below 0.01 and the potential scale reduction factor statistics were approaching 1.0 . All 16 novel strains had identical 16S rRNA gene sequences and were placed in the phylogenetic group represented by B. japonicum. The sequence similarities of novel strains varied between 98.44 (Bradyrhizobium denitrificans) and 99.79 % (Bradyrhizobium liaoningense) relative to type strains of the 15 species in the B. japonicum group (Table S3).Consistent with a previous report  that were placed with high confidence (>95 % posterior probability) in a lineage that was distinct from named species of the genus Bradyrhizobium. Relative to OO99T, strains OO85 and OM9 had sequence similarities for the six concatenated housekeeping genes of >99.60 % whereas those for 19 named species varied between 88.35 and 95.42 % (Tables S3).MLSA of at least five housekeeping genes is used as a reliable method to define phylogenetic relationships and to identify novel lineages within the genus T, OM9 and OO85 and reference taxa using four random primers  and amplification methods described by upgma and the Dice coefficient implemented in GelCompare II software version 5.10 (Applied Maths). The three representative strains were readily distinguished and were placed in a single cluster separate from reference taxa , consistent with their assignment to distinct STs, based on MLSA of protein encoding genes.To further analyse genetic differences between novel strains, we generated random amplified polymorphic DNA (RAPD) fingerprints for representative strains OO99T, OM9 and OO85) and relatives, Bradyrhizobium yuanmingense LMG 21827T and B. liaoningense LMG 18230T. DNA–DNA hybridization values (Table S4) among the three novel strains varied between 73 % and 88 % whereas values for these strains hybridized with B. liaoningense LMG 18230T and B. yuanmingense LMG 21827T were between 23 % and 38 %. These data are consistent with the value of 70 % DNA–DNA relatedness that is considered the threshold for novel species definition Bradyrhizobium.DNA–DNA hybridization experiments were performed as described by nod and nif) can provide useful information on the host range and specificity of symbiotic bacteria . Analyses of nifH gene sequences  provided almost identical results.Analysis of accessory genes encoding symbiotic functions  on Glycine max, Glycine soja and Macroptilium atropurpureum, partially effective nodules (fix±) on Desmodium canadense and Vigna unguiculata, and ineffective nodules (fix-) on Amphicarpaea bracteata and Phaseolus vulgaris.Plant tests were carried out using Leonard jars as described by T was characteristic of the genus Bradyrhizobium with fatty acids 16 : 0 (12.83 %) and summed feature 8 (76.99 %) predominating  were common to all strains tested whereas seven  were detected in only some strains.For analysis of fatty acids, strains were grown on YEM agar at 28 °C. Bacteria were harvested and fatty acids extracted as described by Phenotypic characteristics were further investigated using a variety of tests. Utilization of carbon and nitrogen sources was tested using YNB and YCB (Becton Dickinson) basal liquid media as described by −1): erythromycin , penicillin G sodium salt , kanamycin sulphate , tetracycline , chloramphenicol , cefuroxime sodium salt . Bacterial growth was recorded after 7 days at 28 °C. Tests for bacterial growth on YEM agar at 10, 28 and 37 °C, at pH 5 and pH 10 and in the presence of 1 % (w/v) NaCl were done as described by Bradyrhizobium.Tests of intrinsic antibiotic resistance were done according to ottawaense sp. nov.Based on data for genotypic and phenotypic analyses presented in this study, we propose that the novel group of 16 strains represent a novel species, named Bradyrhizobium ottawaense .Bradyrhizobium d-mannose, d-xylose, l-arabinose, d-glucose, d-mannitol, maltose, l-rhamnose, l-proline, l-glutamic acid and l-threonine but not lactose, trehalose, succinic acid, l-sorbose, aspartic acid, glycine or dl-α-alanine. Resistance (µg ml−1) to antibiotics for representative strain OO99T: kanamycin (100), tetracycline (20), chloramphenicol (100), erythromycin (100), cefuroxime (<5), penicillin (<10). Summed feature 8 (18 : 1ω6c and/or 18 :1ω7c) and 16 : 0 are predominant fatty acids. Strain OO99T elicits effective nodules on Glycine max, Glycine soja and Macroptilium atropurpureum, partially effective nodules on Desmodium canadense and Vigna unguiculata, and ineffective nodules on Amphicarpaea bracteata and Phaseolus vulgaris.Cells are motile with subpolar flagella, Gram-stain-negative, aerobic, non-spore-forming rods (approx. 1.9 µm long and 0.8 µm wide). Colonies are circular, convex, translucent, beige, <1 mm diameter after 7 days at 28 °C on YEM agar medium. Mean generation time approx. 12–13 h in YEM broth at 28 °C. Produce an alkaline reaction on YEM agar. Growth occurs at pH 5–10 . Growth is optimal at 28 °C but no growth occurs at 10 or 37 °C or in the presence of 1 % (w/v) NaCl. Utilizes T ( = LMG 26739T = HAMBI 3284T), was isolated from an effective nodule of Glycine max in Ottawa, Ontario, Canada. The DNA G+C content of the type strain is 62.6 mol%.The type strain, OO99