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Summary Eucalyptus are of tremendous economic importance being the most planted hardwoods worldwide for pulp and paper, timber and bioenergy. The recent release of the Eucalyptus grandis genome sequence pointed out many new candidate genes potentially involved in secondary growth, wood formation or lineage‐specific biosynthetic pathways. Their functional characterization is, however, hindered by the tedious, time‐consuming and inefficient transformation systems available hitherto for eucalypts. To overcome this limitation, we developed a fast, reliable and efficient protocol to obtain and easily detect co‐transformed E. grandis hairy roots using fluorescent markers, with an average efficiency of 62%. We set up conditions both to cultivate excised roots in vitro and to harden composite plants and verified that hairy root morphology and vascular system anatomy were similar to wild‐type ones. We further demonstrated that co‐transformed hairy roots are suitable for medium‐throughput functional studies enabling, for instance, protein subcellular localization, gene expression patterns through RT‐qPCR and promoter expression, as well as the modulation of endogenous gene expression. Down‐regulation of the Eucalyptus cinnamoyl‐CoA reductase1 (EgCCR1) gene, encoding a key enzyme in lignin biosynthesis, led to transgenic roots with reduced lignin levels and thinner cell walls. This gene was used as a proof of concept to demonstrate that the function of genes involved in secondary cell wall biosynthesis and wood formation can be elucidated in transgenic hairy roots using histochemical, transcriptomic and biochemical approaches. The method described here is timely because it will accelerate gene mining of the genome for both basic research and industry purposes.

Background: Dental cellulitis management is no longer a simple procedure, as more and more patients are needing long-time hospitalization, several surgeries and intensive care follow-up. This prospective study seeks to highlight criteria that can split patients with severe odontogenic infection into two groups: those with simple evolution and those for whom complex management is necessary. Methods: In this observational study, all patients considered with a severe odontogenic infection (which necessitated hospital admission, intravenous antibiotics and general anaesthesia) were enrolled between January 2004 and December 2014 from Clermont-Ferrand University Hospital (France). They were split into two groups: those who needed one surgical intervention with tooth extraction and collection drainage combined with probabilistic antibiotic to treat infection and those who need several surgeries, intensive care unit follow-up or tracheotomy to achieve healing. Results: 653 patients were included, of which 611 (94%) had one surgery, 42 (6%) had more than one surgery before healing. Penicillin allergy (p < 0.001), psychiatric disorders (p = 0.005), oropharyngeal oedema (p = 0.008), floor oedema (p = 0.004), fever (p = 0.04) and trismus (p = 0.018) on admission were the most relevant predictors of complex evolution. A conditional inference tree (CTREE) illustrated the association of prognostic factors and the need of multiple surgery. Conclusions: Besides clinical symptoms of severity, complications of severe odontogenic infection are predicted by measurables and objectives criteria as penicillin allergy, mandibular molar, C-reactive protein level, psychiatric disorders and alcohol abuse. Their specific association potentialize the risks. IRB number: CE-CIC-GREN-12-08.

ObjectiveWe used the postoperative recurrence model to better understand the role of adherent and invasive Escherichia coli (AIEC) bacteria in Crohn’s disease (CD), taking advantage of a well-characterised postoperative cohort.DesignFrom a prospective, multicentre cohort of operated patients with CD, AIEC identification was performed within the surgical specimen (M0) (N=181 patients) and the neoterminal ileum (n=119 patients/181) during colonoscopy performed 6 months after surgery (M6). Endoscopic postoperative recurrence was graded using Rutgeerts’ index. The mucosa-associated microbiota was analysed by 16S sequencing at M0 and M6. Relative risks or ORs were adjusted on potential confounders.ResultsAIEC prevalence was twofold higher within the neoterminal ileum at M6 (30.3%) than within the surgical specimen (14.9%) (p<0.001). AIEC within the neoterminal ileum at M6 was associated with higher rate of early ileal lesions (i1) (41.6% vs 17.1%; aRR 3.49 (95% CI 1.01 to 12.04), p=0.048) or ileal lesions (i2b+i3) (38.2% vs 17.1%; aRR 3.45 (95% CI 1.06 to 11.30), p=0.040) compared with no lesion (i0). AIEC within the surgical specimen was predictive of higher risk of i2b-endoscopic postoperative recurrence (POR) (aOR 2.54 (95% CI 1.01 to 6.44), p=0.049) and severe endoscopic POR (aOR 3.36 (95% CI 1.25 to 9.06), p=0.017). While only 5.0% (6/119) of the patients were AIEC-positive at both M0 and M6, 43.7% (52/119), patients with history of positive test for AIEC (M0 or M6) had higher risk of ileal endoscopic POR (aOR 2.32 (95% CI 1.01 to 5.39), p=0.048)), i2b-endoscopic postoperative recurrence (aOR 2.41 (95% CI 1.01 to 5.74); p=0.048) and severe endoscopic postoperative (aOR=3.84 (95% CI 1.32 to 11.18), p=0.013). AIEC colonisation was associated with a specific microbiota signature including increased abundance of Ruminococcus gnavus.ConclusionBased on the postoperative recurrence model, our data support the idea that AIEC are involved in the early steps of ileal CD.Trial registration number NCT03458195.

Eucalypts are the most planted hardwoods worldwide. The availability of the Eucalyptus grandis genome highlighted many genes awaiting functional characterization, lagging behind because of the lack of efficient genetic transformation protocols. In order to efficiently generate knock-out mutants to study the function of eucalypts genes, we implemented the powerful CRISPR/Cas9 gene editing technology with the hairy roots transformation system. As proofs-of-concept, we targeted two wood-related genes: Cinnamoyl-CoA Reductase1 (CCR1), a key lignin biosynthetic gene and IAA9A an auxin dependent transcription factor of Aux/IAA family. Almost all transgenic hairy roots were edited but the allele-editing rates and spectra varied greatly depending on the gene targeted. Most edition events generated truncated proteins, the prevalent edition types were small deletions but large deletions were also quite frequent. By using a combination of FT-IR spectroscopy and multivariate analysis (partial least square analysis (PLS-DA)), we showed that the CCR1-edited lines, which were clearly separated from the controls. The most discriminant wave-numbers were attributed to lignin. Histochemical analyses further confirmed the decreased lignification and the presence of collapsed vessels in CCR1-edited lines, which are characteristics of CCR1 deficiency. Although the efficiency of editing could be improved, the method described here is already a powerful tool to functionally characterize eucalypts genes for both basic research and industry purposes.

A multi-tiered transcriptional network regulates xylem differentiation and secondary cell wall (SCW) formation in plants, with evidence of both conserved and lineage-specific SCW network architecture. We aimed to elucidate the roles of selected R2R3-MYB transcription factors (TFs) linked to Eucalyptus wood formation by identifying genome-wide TF binding sites and direct target genes through an improved DAP-seq protocol combined with machine learning for target gene assignment (DAP-seq-ML). We applied this to five TFs including a well-studied SCW master regulator (EgrMYB2; homolog of AtMYB83), a repressor of lignification (EgrMYB1; homolog of AtMYB4), a TF affecting SCW thickness and vessel density (EgrMYB137; homolog of PtrMYB074) and two TFs with unclear roles in SCW regulation (EgrMYB135 and EgrMYB122). Each DAP-seq TF peak set (average 12,613 peaks) was enriched for canonical R2R3-MYB binding motifs. To improve the reliability of target gene assignment to peaks, a random forest classifier was developed from Arabidopsis DAP-seq, RNA-seq, chromatin, and conserved noncoding sequence data which demonstrated significantly higher precision and recall to the baseline method of assigning genes to proximal peaks. EgrMYB1, EgrMYB2 and EgrMYB137 predicted targets showed clear enrichment for SCW-related biological processes. As validation, EgrMYB137 overexpression in transgenic Eucalyptus hairy roots increased xylem lignification, while its dominant repression in transgenic Arabidopsis and Populus reduced xylem lignification, stunted growth, and caused downregulation of SCW genes. EgrMYB137 targets overlapped significantly with those of EgrMYB2, suggesting partial functional redundancy. Our results show that DAP-seq-ML identified biologically relevant R2R3-MYB targets supported by the finding that EgrMYB137 promotes SCW lignification in planta.Key messageWe combined DAP-seq with machine learning to identify biologically relevant gene targets for five Eucalyptus R2R3-MYB transcription factors linked to wood formation, additionally demonstrating that EgrMYB137 promotes lignification in planta.

Comparative phylogenetic analyses of the R2R3-MYB transcription factor family revealed that five subgroups were preferentially found in woody species and were totally absent from Brassicaceae and monocots (Soler et al., 2015). Here, we analyzed one of these subgroups (WPS-I) for which no gene had been yet characterized. Most members of WPS-I are preferentially expressed in the vascular cambium, the secondary meristem responsible for tree radial growth. We focused on , which is the most specifically and highly expressed in vascular tissues, and showed that it behaves as a transcriptional activator in yeast. Then, we functionally characterized in both transgenic and poplar plants overexpressing either the native or the dominant repression form (fused to the Ethylene-responsive element binding factor-associated Amphiphilic Repression motif, EAR). The transgenic lines had no phenotype whereas the poplar lines overexpressing exhibited a substantial increase in the levels of the flavonoid catechin and of some salicinoid phenolic glycosides (salicortin, salireposide, and tremulacin), in agreement with the increase of the transcript levels of landmark biosynthetic genes. A change in the lignin structure (increase in the syringyl vs. guaiacyl, S/G ratio) was also observed. Poplar lines overexpressing the dominant repression form did not show a strict opposite phenotype. The level of catechin was reduced, but the levels of the salicinoid phenolic glycosides and the S/G ratio remained unchanged. In addition, they showed a reduction in soluble oligolignols containing sinapyl -hydroxybenzoate accompanied by a mild reduction of the insoluble lignin content. Altogether, these results suggest that , and more largely members of the WPS-I group, could control in cambium and in the first layers of differentiating xylem the biosynthesis of some phenylpropanoid-derived secondary metabolites including lignin.

T cell clonal expansions are present in the inflamed mucosa of patients with Crohn’s disease (CD) and may be implicated in postoperative recurrence after ileocolonic resection.MethodsT cell receptor (TCR) analysis was performed in 57 patients included in a prospective multicentre cohort. Endoscopic recurrence was defined by a Rutgeerts score >i0. DNA and mRNA were extracted from biopsies collected from the surgical specimen and endoscopy, and analysed by high throughput sequencing and microarray, respectively.ResultsTCR repertoire in the mucosa of patients with CD displayed diverse clonal expansions. Active smokers at time of surgery had a significantly increased proportion of clonal expansions as compared with non-smokers (25.9%vs17.9%, p=0.02). The percentage of high frequency clones in the surgical specimen was significantly higher in patients with recurrence and correlated with postoperative endoscopic recurrence (area under the curve (AUC) 0.69, 95% CI 0.54 to 0.83). All patients with clonality above 26.8% (18/57) had an endoscopic recurrence. These patients with a high clonality were more frequently smokers than patients with a low clonality (61% vs 23%, p=0.005). The persistence of a similar TCR repertoire at postoperative endoscopy was associated with smoking and disease recurrence. Patients with high clonality showed increased expression of genes associated with CD8 T cells and reduced expression of inflammation-related genes. Expanded clones were found predominantly in the CD8 T cell compartment.ConclusionClonal T cell expansions are implicated in postoperative endoscopic recurrence. CD patients with increased proportion of clonal T cell expansions in the ileal mucosa represent a subgroup associated with smoking and where pathogenesis appears as T cell driven.Trial registration numberNCT03458195.

Eucalypts are the most planted trees worldwide, but most of them are frost sensitive. Overexpressing transcription factors for CRT-repeat binding factors (CBFs) in transgenic Eucalyptus confer cold resistance both in leaves and stems. While wood plays crucial roles in trees and is affected by environmental cues, its potential role in adaptation to cold stress has been neglected. Here, we addressed this question by investigating the changes occurring in wood in response to the overexpression of two CBFs, taking advantage of available transgenic Eucalyptus lines. We performed histological, biochemical, and transcriptomic analyses on xylem samples. CBF ectopic expression led to a reduction of both primary and secondary growth, and triggered changes in xylem architecture with smaller and more frequent vessels and fibers exhibiting reduced lumens. In addition, lignin content and syringyl/guaiacyl (S/G) ratio increased. Consistently, many genes of the phenylpropanoid and lignin branch pathway were upregulated. Most of the features of xylem remodeling induced by CBF overexpression are reminiscent of those observed after long exposure of Eucalyptus trees to chilling temperatures. Altogether, these results suggest that CBF plays a central role in the cross-talk between response to cold and wood formation and that the remodeling of wood is part of the adaptive strategies to face cold stress.

Abstract Background Retroperitoneal schwannomas are extremely rare, as they account for only 3% of retroperitoneal tumors. Clinical symptoms are nonspecific and of late onset, meaning that these tumors are diagnosed at an advanced stage. Surgical resection is required for histological diagnosis and to prevent possible malignant transformation. Celioscopy offers numerous benefits, reducing postoperative pain and speeding up the patient’s return to autonomy, but it can pose a real challenge due to the size of these lesions. Case presentation We report a case of laparoscopic resection of a very large right-sided retroperitoneal schwannoma, with a particular histological form. Conclusion Surgical resection in a single unit remains the golden rule, and a laparoscopy can be proposed when the diagnosis is beyond doubt. The large size of the retroperitoneal melanotic schwannomas, a common feature, increases surgical difficulties but is not a contraindication to this approach.

Videofluoroscopic Swallow studies (VFSS) are useful radiological examinations to explore swallowing disorders but which require ionizing radiation. The aim of our study was to evaluate the comparability of pediatric VFSS at 15 frames per second (fps) with 30 fps. Fifty-five loops including 190 swallowings of VFSS at 30 fps performed on 32 consecutive pediatric patients in a University Hospital Center were retrospectively modified by a software to delete one image out of two to obtain secondary loops with a frame rate of 15 fps. An otorhinolaryngologist-phonatrician and a radiologist reviewed all swallowings blindly and randomly using the penetration and aspiration scale (PAS). In case of discordance, they concluded a consensual interpretation. Fifteen girls and seventeen boys were included. The median age was 4 years and 8 months (range = 4 months–16 yr.). 144 swallowings were normal. Swallowing disorder was confirmed in 46 swallowings, (23 supraglottic penetrations and 23 aspirations). Considering each swallowing at 15 fps, sensitivity and specificity were, respectively, 93% (CI 0.82–0.98) and 98% (CI 0.94–0.99). The Cohen’Kappa coefficient between each interpretation at 15 and 30 fps was “almost perfect” (κ = 0.95; CI 0.88–0.99). Considering each loop, conclusion was identical. Reducing frame rate at 15 fps during pediatric VFSS seemed to be acceptable with comparable diagnostic performances without clinical impact compared to 30 fps, while being an efficient way to reduce the ionizing radiation exposition in children. We would suggest reconsidering the possibility of using VFSS with a 15 fps in a pediatric population.