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Background: The aim of this study was to assess the performance and tolerance of an innovative disposable instrument delivering resorbable clips (I-Clip, Sofradim, France) intended for mesh fixation in inguinal, incisional and umbilical hernias of the abdominal wall. The fixation device was designed to be resorbable in 1 year, with reduced trauma to the underlying tissues or the mesh, and with initial mechanical properties equivalent to those of conventional metal staples.Methods: The study involved 105 patients with inguinal, umbilical or incisional hernias enrolled from 11 centres. Inguinal totally extra peritoneal (TEP) or trans abdomino pre-peritoneal (TAPP) repair was performed with Parietex mesh, incisional or umbilical hernias were treated via the intraperitoneal route with Parietex composite. I-Clips were used for mesh fixation in both indications according to the surgeon's habits. Efficacy was the principal assessment criteria evaluated by two parameters: quality of fixation evaluated subjectively at the time of procedure and recurrence rate according to the follow up at 1, 6 and 12 months. Pain evaluated by the patients using a visual analogue scale (VAS) was the principal secondary assessment criteria. Other tolerance criteria were also evaluated during surgery and follow up.Results: The surgeons' evaluation of the fixation quality was assessed as good to very good in 100% of ventral hernias and good to very good in 85-92% of inguinal hernias. At 1 month, 90% of patients (94/104) were totally pain-free (VAS score: 0) and only ten patients reported low pain (VAS scores: 0.3-3.1). At 1 year, the pain described by those ten patients finally disappeared, 98% of patients (102/104) were totally pain-free. The rate of minor complications not related to the device concerned 5% of the patients at 1 month, which was reduced to 2% at one year and no recurrence or mesh sepsis was observed.Conclusions: The ease of use of this device, combined with the absence of recurrence related to the investigated device and the good pain-free outcome in this group of patients confirmed the effectiveness and tolerance of the resorbable fixation concept of I-Clip(TM). [ABSTRACT FROM AUTHOR]

Small ribonucleic acids (RNAs) have been shown to play important roles in cross-kingdom communication, notably in plant-pathogen relationships. Plant micro RNAs (miRNAs)-one class of small RNAs-were even shown to regulate gene expression in the gut microbiota. Plant miRNAs could also affect the rhizosphere microbiota. Here we looked for plant miRNAs in the rhizosphere of model plants, and if these miRNAs could affect the rhizosphere microbiota. We first show that plant miRNAs were present in the rhizosphere of Arabidopsis thaliana and Brachypodium distachyon . These plant miRNAs were also found in or on bacteria extracted from the rhizosphere. We then looked at the effect these plants miRNAs could have on two typical rhizosphere bacteria, Variovorax paradoxus and Bacillus mycoides . The two bacteria took up a fluorescent synthetic miRNA but only V. paradoxus shifted its transcriptome when confronted to a mixture of six plant miRNAs. V. paradoxus also changed its transcriptome when it was grown in the rhizosphere of Arabidopsis that overexpressed a miRNA in its roots. As there were differences in the response of the two isolates used, we looked for shifts in the larger microbial community. We observed shifts in the rhizosphere bacterial communities of Arabidopsis mutants that were impaired in their small RNA pathways, or overexpressed specific miRNAs. We also found differences in the growth and community composition of a simplified soil microbial community when exposed in vitro to a mixture of plant miRNAs. Our results support the addition of miRNAs to the plant tools shaping rhizosphere microbial assembly., Competing Interests: The authors report no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Primary transcripts of microRNAs (pri-miRNAs) were initially defined as long non-coding RNAs that host miRNAs further processed by the microRNA processor complex. A few years ago, however, it was discovered in plants that pri-miRNAs actually contain functional open reading frames (sORFs) that translate into small peptides called miPEPs, for microRNA-encoded peptides. Initially detected in Arabidopsis thaliana and Medicago truncatula , recent studies have revealed the presence of miPEPs in other pri-miRNAs as well as in other species ranging from various plant species to animals. This suggests that miPEP numbers remain largely underestimated and that they could be a common signature of pri-miRNAs. Here we present the most recent advances in miPEPs research and discuss how their discovery has broadened our vision of the regulation of gene expression by miRNAs, and how miPEPs could be interesting tools in sustainable agriculture or the treatment of certain human diseases.

The current agriculture main challenge is to maintain food production while facing multiple threats such as increasing world population, temperature increase, lack of agrochemicals due to health issues and uprising of weeds resistant to herbicides. Developing novel, alternative, and safe methods is hence of paramount importance. Here, we show that complementary peptides (cPEPs) from any gene can be designed to target specifically plant coding genes. External application of synthetic peptides increases the abundance of the targeted protein, leading to related phenotypes. Moreover, we provide evidence that cPEPs can be powerful tools in agronomy to improve plant traits, such as growth, resistance to pathogen or heat stress, without the needs of genetic approaches. Finally, by combining their activity they can also be used to reduce weed growth., (© 2023. The Author(s).)

Recent studies have shown that hundreds of small proteins were occulted when protein-coding genes were annotated. These proteins, called alternative proteins, have failed to be annotated notably due to the short length of their open reading frame (less than 100 codons) or the enforced rule establishing that messenger RNAs (mRNAs) are monocistronic. Several alternative proteins were shown to be biologically active molecules and seem to be involved in a wide range of biological functions. However, genome-wide exploration of the alternative proteome is still limited to a few species. In the present article, we describe a deep peptidomics workflow which enabled the identification of 401 alternative proteins in Drosophila melanogaster . Subcellular localization, protein domains, and short linear motifs were predicted for 235 of the alternative proteins identified and point toward specific functions of these small proteins. Several alternative proteins had approximated abundances higher than their canonical counterparts, suggesting that these alternative proteins are actually the main products of their corresponding genes. Finally, we observed 14 alternative proteins with developmentally regulated expression patterns and 10 induced upon the heat-shock treatment of embryos, demonstrating stage or stress-specific production of alternative proteins., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Fabre, Choteau, Duboé, Pichereaux, Montigny, Korona, Deery, Camus, Brun, Burlet-Schiltz, Russell, Combier, Lilley and Plaza.)

MicroRNAs (miRNAs) are small regulatory non-coding RNAs, resulting from the cleavage of long primary transcripts (pri-miRNAs) in the nucleus by the Microprocessor complex generating precursors (pre-miRNAs) that are then exported to the cytoplasm and processed into mature miRNAs. Some miRNAs are hosted in pri-miRNAs annotated as long non-coding RNAs (lncRNAs) and defined as MIRHGs (for miRNA Host Genes). However, several lnc pri-miRNAs contain translatable small open reading frames (smORFs). If smORFs present within lncRNAs can encode functional small peptides, they can also constitute cis -regulatory elements involved in lncRNA decay. Here, we investigated the possible involvement of smORFs in the regulation of lnc pri-miRNAs in Human and Drosophila , focusing on pri-miRNAs previously shown to contain translatable smORFs. We show that smORFs regulate the expression levels of human pri-miR-155 and pri-miR-497, and Drosophila pri-miR-8 and pri-miR-14, and also affect the expression and activity of their associated miRNAs. This smORF-dependent regulation is independent of the nucleotidic and amino acidic sequences of the smORFs and is sensitive to the ribosome-stalling drug cycloheximide, suggesting the involvement of translational events. This study identifies smORFs as new cis -acting elements involved in the regulation of pri-miRNAs and miRNAs expression, in both Human and Drosophila melanogaster .

MicroRNAs (miRNAs) are transcribed as long primary transcripts (pri-miRNAs) by RNA polymerase II. Plant pri-miRNAs encode regulatory peptides called miPEPs, which specifically enhance the transcription of the pri-miRNA from which they originate. However, paradoxically, whereas miPEPs have been identified in different plant species, they are poorly conserved, raising the question of the mechanisms underlying their specificity. To address this point, we identify and re-annotate multiple Arabidopsis thaliana pri-miRNAs in order to identify ORF encoding miPEPs. The study of several identified miPEPs in different species show that non-conserved miPEPs are only active in their plant of origin, whereas conserved ones are active in different species. Finally, we find that miPEP activity relies on the presence of its own miORF, explaining both the lack of selection pressure on miPEP sequence and the ability for non-conserved peptides to play a similar role, i.e., to activate the expression of their corresponding miRNA., Competing Interests: Declaration of interests J.P.C. is a founder and a scientific advisor of Micropep Technologies, which aims to commercialize peptides in agronomy. In this study, Micropep Technologies has participated in the identification of Arabidopsis pri-miRNAs., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Recent genome-wide studies of many species reveal the existence of a myriad of RNAs differing in size, coding potential and function. Among these are the long non-coding RNAs, some of them producing functional small peptides via the translation of short ORFs. It now appears that any kind of RNA presumably has a potential to encode small peptides. Accordingly, our team recently discovered that plant primary transcripts of microRNAs (pri-miRs) produce small regulatory peptides (miPEPs) involved in auto-regulatory feedback loops enhancing their cognate microRNA expression which in turn controls plant development. Here we investigate whether this regulatory feedback loop is present in Drosophila melanogaster., Results: We perform a survey of ribosome profiling data and reveal that many pri-miRNAs exhibit ribosome translation marks. Focusing on miR-8, we show that pri-miR-8 can produce a miPEP-8. Functional assays performed in Drosophila reveal that miPEP-8 affects development when overexpressed or knocked down. Combining genetic and molecular approaches as well as genome-wide transcriptomic analyses, we show that miR-8 expression is independent of miPEP-8 activity and that miPEP-8 acts in parallel to miR-8 to regulate the expression of hundreds of genes., Conclusion: Taken together, these results reveal that several Drosophila pri-miRs exhibit translation potential. Contrasting with the mechanism described in plants, these data shed light on the function of yet undescribed primary-microRNA-encoded peptides in Drosophila and their regulatory potential on genome expression.

Some miRNAs are located in RNA precursors (pri-miRNAs) annotated as long non-coding (lncRNAs) due to absence of long open reading frames (ORFs). However, recent studies have shown that some lnc pri-miRNAs encode peptides called miPEPs (miRNA-encoded peptides). Initially discovered in plants, three miPEPs have also been identified in humans. Herein, we found that a dozen human pri-miRNAs potentially encode miPEPs, as revealed by ribosome profiling and proteomic databases survey. So far, the only known function of plant miPEPs is to enhance the transcription of their own pri-miRNAs, thereby increasing the level and activity of their associated miRNAs and downregulating the expression of their target genes. To date, in humans, only miPEP133 was shown to promote a positive autoregulatory loop. We investigated whether other human miPEPs are also involved in regulating the expression of their miRNAs by studying miPEP155, encoded by the lnc MIR155HG, miPEP497, a sORF-encoded peptide within lnc MIR497HG, and miPEP200a, encoded by the pri-miRNA of miR-200a/miR-200b. We show that overexpression of these miPEPs is unable to impact the expression/activity of their own pri-miRNA/miRNAs in humans, indicating that the positive feedback regulation observed with plant miPEPs and human miPEP133 is not a general rule of human miPEP function.

The importance of microorganisms in plant development, nutrition, and stress resistance is unquestioned and has led to a more holistic approach of plant-microbe interactions, under the holobiont concept. The structure of the plant microbiota is often described as host driven, especially in the rhizosphere, where microbial communities are shaped by diverse rhizodeposits. Gradually, this anthropogenic vision is fading and being replaced by the idea that plants and microorganisms co-shape the plant microbiota. Through coevolution, plants and microbes have developed cross-kingdom communication channels. Here, we propose that miRNAs are crucial mediators of plant-microbe interactions and microbiota shaping in the rhizosphere. Moreover, we suggest, as an alternative to generally unsuccessful strategies based on microbial inoculants, miRNAs as a promising tool for novel holobiont engineering., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Short open reading frame (sORF)-encoded polypeptides (SEPs) have recently emerged as key regulators of major cellular processes. Computational methods for the annotation of sORFs combined with transcriptomics and ribosome profiling approaches predicted the existence of tens of thousands of SEPs across the kingdom of life. Although, we still lack unambiguous evidence for most of them. The method of choice to validate the expression of SEPs is mass spectrometry (MS)-based peptidomics. Peptides are less abundant than proteins, which tends to hinder their detection. Therefore, optimization and enrichment methods are necessary to validate the existence of SEPs. In this article, we discuss the challenges for the detection of SEPs by MS and recent developments of biochemical approaches applied to the study of these peptides. We detail the advances made in the different key steps of a typical peptidomics workflow and highlight possible alternatives that have not been explored yet., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

MiPEPs are short natural peptides encoded by microRNAs in plants. Exogenous application of miPEPs increases the expression of their corresponding miRNA and, consequently, induces consistent phenotypical changes. Therefore, miPEPs carry huge potential in agronomy as gene regulators that do not require genome manipulation. However, to this end, it is necessary to know their mode of action, including where they act and how they enter the plants. Here, after analyzing the effect of Arabidopsis thaliana miPEP165a on root and aerial part development, we followed the internalization of fluorescent-labelled miPEP165a into roots and compared its uptake into endocytosis-altered mutants to that observed in wild-type plants treated or not with endocytosis inhibitors. The results show that entry of miPEP165a involves both a passive diffusion at the root apex and endocytosis-associated internalization in the differentiation and mature zones. Moreover, miPEP165a is unable to enter the central cylinder and does not migrate from the roots to the aerial part of the plant, suggesting that miPEPs have no systemic effect., Competing Interests: Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Figure S1. Effect of miPEP165a and importance of its stability. Figure S2. Physiochemical properties of miPEP165a. Figure S3. Quantification of miPEP165a-FAM uptake in Arabidopsis roots. Figure S4. MβCD impairs the miPEP165a-FAM entry in the Arabidopsis root cap/meristematic zone.

Root colonization by arbuscular mycorrhizal (AM) fungi is a complex and finely tuned process. Previous studies have shown that, among other plant hormones, auxin plays a role in this process but the specific involvement of Aux/IAAs, the key regulators of auxin responses, is still unknown. In this study, we addressed the role of the tomato Sl-IAA27 during AM symbiosis by using Sl-IAA27-RNAi and pSL-IAA27::GUS stable tomato lines. The data show that Sl-IAA27 expression is up-regulated by the AM fungus and that silencing of Sl-IAA27 has a negative impact on AM colonization. Sl-IAA27-silencing resulted in down-regulation of three genes involved in strigolactone synthesis, NSP1, D27 and MAX1, and treatment of Sl-IAA27-silenced plants with the strigolactone analog GR24 complemented their mycorrhizal defect phenotype. Overall, the study identified an Aux/IAA gene as a new component of the signaling pathway controlling AM fungal colonization in tomato. This gene is proposed to control strigolactone biosynthesis via the regulation of NSP1., (No claim to original US government works New Phytologist © 2016 New Phytologist Trust.)

Arbuscular mycorrhizal (AM) symbiosis associates most plants with fungi of the phylum Glomeromycota. The fungus penetrates into roots and forms within cortical cell branched structures called arbuscules for nutrient exchange. We discovered that miR171b has a mismatched cleavage site and is unable to downregulate the miR171 family target gene, LOM1 (LOST MERISTEMS 1). This mismatched cleavage site is conserved among plants that establish AM symbiosis, but not in non-mycotrophic plants. Unlike other members of the miR171 family, miR171b stimulates AM symbiosis and is expressed specifically in root cells that contain arbuscules. MiR171b protects LOM1 from negative regulation by other miR171 family members. These findings uncover a unique mechanism of positive post-transcriptional regulation of gene expression by miRNAs and demonstrate its relevance for the establishment of AM symbiosis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Arbuscular mycorrhizal (AM) symbiosis is an intimate and ancient symbiosis found between most of terrestrial plants and fungi from the Glomeromycota family. Later during evolution, the establishment of the nodulation between legume plants and soil bacteria known as rhizobia, involved several genes of the signaling pathway previously implicated for AM symbiosis. For the past years, the identification of the genes belonging to this Common Symbiotic Signaling Pathway have been mostly done on nodulation. Among the different genes already well identified as required for nodulation, we focused our attention on the involvement of Nodule Inception ( NIN ) in AM symbiosis. We show here that NIN expression is induced during AM symbiosis, and that the Medicago truncatula nin mutant is less colonized than the wild-type M. truncatula strain. Moreover, nin mutant displays a defect in the ability to be infected by the fungus Rhizophagus irregularis . This work brings a new evidence of the common genes involved in overlapping signaling pathways of both nodulation and in AM symbiosis.

Contents 22 I. 22 II. 24 III. 25 IV. 27 V. 29 VI. 10 31 References 32 SUMMARY: Plants have evolved a remarkable faculty of adaptation to deal with various and changing environmental conditions. In this context, the roots have taken over nutritional aspects and the root system architecture can be modulated in response to nutrient availability or biotic interactions with soil microorganisms. This adaptability requires a fine tuning of gene expression. Indeed, root specification and development are highly complex processes requiring gene regulatory networks involved in hormonal regulations and cell identity. Among the different molecular partners governing root development, microRNAs (miRNAs) are key players for the fast regulation of gene expression. miRNAs are small RNAs involved in most developmental processes and are required for the normal growth of organisms, by the negative regulation of key genes, such as transcription factors and hormone receptors. Here, we review the known roles of miRNAs in root specification and development, from the embryonic roots to the establishment of root symbioses, highlighting the major roles of miRNAs in these processes., (© 2016 European Union New Phytologist © 2016 New Phytologist Trust.)

MicroRNAs (miRNAs) are small regulatory RNA molecules that inhibit the expression of specific target genes by binding to and cleaving their messenger RNAs or otherwise inhibiting their translation into proteins. miRNAs are transcribed as much larger primary transcripts (pri-miRNAs), the function of which is not fully understood. Here we show that plant pri-miRNAs contain short open reading frame sequences that encode regulatory peptides. The pri-miR171b of Medicago truncatula and the pri-miR165a of Arabidopsis thaliana produce peptides, which we term miPEP171b and miPEP165a, respectively, that enhance the accumulation of their corresponding mature miRNAs, resulting in downregulation of target genes involved in root development. The mechanism of miRNA-encoded peptide (miPEP) action involves increasing transcription of the pri-miRNA. Five other pri-miRNAs of A. thaliana and M. truncatula encode active miPEPs, suggesting that miPEPs are widespread throughout the plant kingdom. Synthetic miPEP171b and miPEP165a peptides applied to plants specifically trigger the accumulation of miR171b and miR165a, leading to reduction of lateral root development and stimulation of main root growth, respectively, suggesting that miPEPs might have agronomical applications.

MicroRNAs (miRNAs) are short RNA molecules negatively regulating the expression of many important genes in plants and animals. We have recently shown that plant primary transcripts of miRNAs encode peptides (miPEPs) able to increase specifically the transcription of their associated miRNA.(1) We discuss here the possibility of using miPEPs as a new tool for functional analysis of single members of miRNA families in plants, including in non-model plants, that could avoid transgenic transformation and minimize artifactual interpretation. We also raise several fundamental and crucial questions that need to be address for a deeper understanding of the cellular and molecular mechanisms underlining the regulatory activity of miPEPs.

We used Agrobacterium-mediated insertional mutagenesis to identify genes in the ectomycorrhizal fungus Hebeloma cylindrosporum that are essential for efficient mycorrhiza formation. One of the mutants presented a dramatically reduced ability to form ectomycorrhizas when grown in the presence of Pinus pinaster. It failed to form mycorrhizas in the presence of glucose at 0.5 g liter(-1), a condition favorable for mycorrhiza formation by the wild-type strain. However, it formed few mycorrhizas when glucose was replaced by fructose or when glucose concentration was increased to 1 g liter(-1). Scanning electron microscopy examination of these mycorrhizas revealed that this mutant was unable to differentiate true fungal sheath and Hartig net. Molecular analyses showed that the single-copy disrupting T-DNA was integrated 6,884 bp downstream from the start codon, of an open reading frame potentially encoding a 3,096-amino-acid-long protein. This gene, which we named HcMycE1, has orthologs in numerous fungi as well as different other eukaryotic microorganisms. RNAi inactivation of HcMycE1 in the wild-type strain also led to a mycorrhizal defect, demonstrating that the nonmycorrhizal phenotype of the mutant was due to mutagenic T-DNA integration in HcMycE1. In the wild-type strain colonizing P. pinaster roots, HcMycE1 was transiently upregulated before symbiotic structure differentiation. Together with the inability of the mutant to differentiate these structures, this suggests that HcMycE1 plays a crucial role upstream of the fungal sheath and Hartig net differentiation. This study provides the first characterization of a fungal mutant altered in mycorrhizal ability.

Background: Legume roots show a remarkable plasticity to adapt their architecture to biotic and abiotic constraints, including symbiotic interactions. However, global analysis of miRNA regulation in roots is limited, and a global view of the evolution of miRNA-mediated diversification in different ecotypes is lacking., Results: In the model legume Medicago truncatula, we analyze the small RNA transcriptome of roots submitted to symbiotic and pathogenic interactions. Genome mapping and a computational pipeline identify 416 miRNA candidates, including known and novel variants of 78 miRNA families present in miRBase. Stringent criteria of pre-miRNA prediction yield 52 new mtr-miRNAs, including 27 miRtrons. Analyzing miRNA precursor polymorphisms in 26 M. truncatula ecotypes identifies higher sequence polymorphism in conserved rather than Medicago-specific miRNA precursors. An average of 19 targets, mainly involved in environmental responses and signalling, is predicted per novel miRNA. We identify miRNAs responsive to bacterial and fungal pathogens or symbionts as well as their related Nod and Myc-LCO symbiotic signals. Network analyses reveal modules of new and conserved co-expressed miRNAs that regulate distinct sets of targets, highlighting potential miRNA-regulated biological pathways relevant to pathogenic and symbiotic interactions., Conclusions: We identify 52 novel genuine miRNAs and large plasticity of the root miRNAome in response to the environment, and also in response to purified Myc/Nod signaling molecules. The new miRNAs identified and their sequence variation across M. truncatula ecotypes may be crucial to understand the adaptation of root growth to the soil environment, notably in the agriculturally important legume crops.

Most land plant species live in symbiosis with arbuscular mycorrhizal fungi. These fungi differentiate essential functional structures called arbuscules in root cortical cells from which mineral nutrients are released to the plant. We investigated the role of microRNA393 (miR393), an miRNA that targets several auxin receptors, in arbuscular mycorrhizal root colonization. Expression of the precursors of the miR393 was down-regulated during mycorrhization in three different plant species: Solanum lycopersicum, Medicago truncatula, and Oryza sativa. Treatment of S. lycopersicum, M. truncatula, and O. sativa roots with concentrations of synthetic auxin analogs that did not affect root development stimulated mycorrhization, particularly arbuscule formation. DR5-GUS, a reporter for auxin response, was preferentially expressed in root cells containing arbuscules. Finally, overexpression of miR393 in root tissues resulted in down-regulation of auxin receptor genes (transport inhibitor response1 and auxin-related F box) and underdeveloped arbuscules in all three plant species. These results support the conclusion that miR393 is a negative regulator of arbuscule formation by hampering auxin perception in arbuscule-containing cells., (© 2014 American Society of Plant Biologists. All Rights Reserved.)

Background: The aim of this study was to assess long-term quality of life (QoL) over a period of 6 years in women with breast cancer (BC) who underwent sentinel lymph node biopsy (SLNB), axillary lymph node dissection (ALND), or SLNB followed by ALND., Methods: The European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC-QLQ)-C30 and the EORTC-QLQ-BR-23 questionnaires were used to assess QoL before surgery, just after surgery, 6, 12 and 72 months later. The longitudinal effect of surgical modalities on QoL was assessed with a mixed model analysis of variance for repeated measurements., Results: Five hundred and eighteen BC patients were initially included. The median follow-up was 6 years. During the follow-up, 61 patients died. None of the patients of the SLNB group developed lymphedema during follow-up and the relapse rate was similar in the different groups (P=0.62). Before surgery, global health status (P=0.52) and arm symptoms (BRAS) (P=0.99) QoL scores were similar whatever the surgical procedure. The BRAS score (P=0.0001) was better in the SLNB group 72 months after surgery. Moreover, during follow-up, patients treated with SLNB had lower arm symptoms scores than ALND patients and there was no difference for arm symptoms between patients treated with ALND and those treated with SLNB followed by complementary ALND., Conclusion: Long-term follow-up showed that SLNB was associated with less morbidity than ALND.

Aux/IAA genes play a pivotal role in auxin transcriptional regulation. Their functions were mainly studied in Arabidopsis through analysis of gain-of-function mutants. In the tomato, the Solanaceae reference species, different studies on Sl-IAA down-regulated lines showed specific role for Sl-IAA genes. Our recent work revealed that the Sl-IAA 27 gene displays a distinct behavior compared with most Aux/IAA genes, being down-regulated by auxin. Interestingly, the silencing of Sl-IAA27 leads to altered chlorophyll accumulation in leaves, reduced fertilization, altered fruit development and altered root formation. Here we report that IAA27 could be a key auxin signaling gene involved in AM in tomato and also in Medicago model plant. Indeed both Sl-IAA27 and its closest homolog in Medicago truncatula, Mt-IAA27, are overexpressed in mycorrhized roots. These data are in line with the putative role of auxin in arbuscular mycorrhization.

Nodulation and arbuscular mycorrhization require the activation of plant host symbiotic programs by Nod factors, and Myc-LCOs and COs, respectively. The pathways involved in the perception and downstream signaling of these signals include common and distinct components. Among the distinct components, NSP1, a GRAS transcription factor, has been considered for years to be specifically involved in nodulation. Here, we analyzed the degree of conservation of the NSP1 sequence in arbuscular mycorrhizal (AM) host and non-AM host plants and carefully examined the ability of Medicago truncatula nsp1 mutants to respond to Myc-LCOs and to be colonized by an arbuscular mycorrhizal fungus. In AM-host plants, the selection pressure on NSP1 is stronger than in non-AM host ones. The response to Myc-LCOs and the frequency of mycorrhizal colonization are significantly reduced in the nsp1 mutants. Our results reveal that NSP1, previously described for its involvement in the Nod factor signaling pathway, is also involved in the Myc-LCO signaling pathway. They bring additional evidence on the evolutionary relatedness between nodulation and mycorrhization., (No claim to original French goverment works New Phytologist © 2013 New Phytologist Trust.)

The root system is crucial for acquisition of resources from the soil. In legumes, the efficiency of mineral and water uptake by the roots may be reinforced due to establishment of symbiotic relationships with mycorrhizal fungi and interactions with soil rhizobia. Here, we investigated the role of miR396 in regulating the architecture of the root system and in symbiotic interactions in the model legume Medicago truncatula. Analyses with promoter-GUS fusions suggested that the mtr-miR396a and miR396b genes are highly expressed in root tips, preferentially in the transition zone, and display distinct expression profiles during lateral root and nodule development. Transgenic roots of composite plants that over-express the miR396b precursor showed lower expression of six growth-regulating factor genes (MtGRF) and two bHLH79-like target genes, as well as reduced growth and mycorrhizal associations. miR396 inactivation by mimicry caused contrasting tendencies, with increased target expression, higher root biomass and more efficient colonization by arbuscular mycorrhizal fungi. In contrast to MtbHLH79, repression of three GRF targets by RNA interference severely impaired root growth. Early activation of mtr-miR396b, concomitant with post-transcriptional repression of MtGRF5 expression, was also observed in response to exogenous brassinosteroids. Growth limitation in miR396 over-expressing roots correlated with a reduction in cell-cycle gene expression and the number of dividing cells in the root apical meristem. These results link the miR396 network to the regulation of root growth and mycorrhizal associations in plants., (© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.)

Most land plants live symbiotically with arbuscular mycorrhizal fungi. Establishment of this symbiosis requires signals produced by both partners: strigolactones in root exudates stimulate pre-symbiotic growth of the fungus, which releases lipochito-oligosaccharides (Myc-LCOs) that prepare the plant for symbiosis. Here, we have investigated the events downstream of this early signaling in the roots. We report that expression of miR171h, a microRNA that targets NSP2, is up-regulated in the elongation zone of the root during colonization by Rhizophagus irregularis (formerly Glomus intraradices) and in response to Myc-LCOs. Fungal colonization was much reduced by over-expressing miR171h in roots, mimicking the phenotype of nsp2 mutants. Conversely, in plants expressing an NSP2 mRNA resistant to miR171h cleavage, fungal colonization was much increased and extended into the elongation zone of the roots. Finally, phylogenetic analyses revealed that miR171h regulation of NSP2 is probably conserved among mycotrophic plants. Our findings suggest a regulatory mechanism, triggered by Myc-LCOs, that prevents over-colonization of roots by arbuscular mycorrhizal fungi by a mechanism involving miRNA-mediated negative regulation of NSP2., (© 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.)

Strigolactones (SLs) have been proposed as a new group of plant hormones, inhibiting shoot branching, and as signaling molecules for plant interactions. Here, we present evidence for effects of SLs on root development. The analysis of mutants flawed in SLs synthesis or signaling suggested that the absence of SLs enhances lateral root formation. In accordance, roots grown in the presence of GR24, a synthetic bioactive SL, showed reduced number of lateral roots in WT and in max3-11 and max4-1 mutants, deficient in SL synthesis. The GR24-induced reduction in lateral roots was not apparent in the SL signaling mutant max2-1. Moreover, GR24 led to increased root-hair length in WT and in max3-11 and max4-1 mutants, but not in max2-1. SLs effect on lateral root formation and root-hair elongation may suggest a role for SLs in the regulation of root development; perhaps, as a response to growth conditions.

Purpose: This prospective multicenter study explored different definitions of time to deterioration (TTD) in quality of life (QoL) scores, according to different cutoffs of the minimal clinically important difference (MCID) as a modality for longitudinal QoL assessment in breast cancer patients., Methods: QoL was assessed using the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire C30 and BR-23 before surgery, after surgery, and 6 and 12 months later. The global health score, arm symptoms score (BRAS), and breast symptoms score were analyzed. For a given baseline score, QoL was considered to have deteriorated if this score decreased by ≥5 points at any time point after baseline. Analyses were repeated using an MCID of 10 points and taking the score after surgery as the reference score (to explore the occurrence of response shift). TTD was calculated using the Kaplan-Meier method and Cox regression was used to identify independent factors associated with TTD., Results: Two hundred thirty-five patients underwent axillary lymph node dissection (ALND), 222 underwent sentinel lymph node biopsy (SLNB), and 61 underwent SLNB plus ALND. Patients who underwent SLNB had a significantly longer TTD for the BRAS dimension than those who underwent ALND. Cox multivariate analyses showed that treatment using SLNB and age >59 years were independently associated with longer TTD for the BRAS, whereas surgery elsewhere than at the Centre Georges François Leclerc was associated with a shorter TTD., Conclusion: Exploration of different definitions of TTD in QoL provides meaningful longitudinal QoL results for clinicians.

LEOPARD syndrome (LS), a disorder with multiple developmental abnormalities, is mainly due to mutations that impair the activity of the tyrosine phosphatase SHP2 (PTPN11). How these alterations cause the disease remains unknown. We report here that fibroblasts isolated from LS patients displayed stronger epidermal growth factor (EGF)-induced phosphorylation of both AKT and glycogen synthase kinase 3beta (GSK-3beta) than fibroblasts from control patients. Similar results were obtained in HEK293 cells expressing LS mutants of SHP2. We found that the GAB1/phosphoinositide 3-kinase (PI3K) complex was more abundant in fibroblasts from LS than control subjects and that both AKT and GSK-3beta hyperphosphorylation were prevented by reducing GAB1 expression or by overexpressing a GAB1 mutant unable to bind to PI3K. Consistently, purified recombinant LS mutants failed to dephosphorylate GAB1 PI3K-binding sites. These mutants induced PI3K-dependent increase in cell size in a model of chicken embryo cardiac explants and in transcriptional activity of the atrial natriuretic factor (ANF) gene in neonate rat cardiomyocytes. In conclusion, SHP2 mutations causing LS facilitate EGF-induced PI3K/AKT/GSK-3beta stimulation through impaired GAB1 dephosphorylation, resulting in deregulation of a novel signaling pathway that could be involved in LS pathology.

Background: This prospective multicenter study assessed and compared the impact of different surgical procedures on quality of life (QoL) in breast cancer patients., Patients and Methods: The EORTC QLQ-C30 and the EORTC QLQ-BR-23 questionnaires were used to assess global health status (GHS), arm (BRAS) and breast (BRBS) symptom scales, before surgery, just after surgery and 6 and 12 months later. The Kruskal-Wallis test with the Bonferroni correction was used to compare scores. A mixed model analysis of variance for repeated measurements was then applied to assess the longitudinal effect of surgical modalities on QoL., Results: Before surgery, GHS (P = 0.7807) and BRAS (P = 0.7688) QoL scores were similar whatever the surgical procedure: sentinel node biopsy (SLNB), axillary node dissection (ALND) or SLNB + ALND. As compared with other surgical groups, GHS 75.91 [standard deviation (SD) = 17.44, P = 0.041] and BRAS 11.39 (SD = 15.36, P < 0.0001) were better in the SLNB group 12 months after surgery. Whatever the type of surgery, GHS decreased after surgery (P < 0.0001), but increased 6 months later (P = 0.0016). BRAS symptoms increased just after surgery (P = 0.0329) and until 6 months (P < 0.0001) before decreasing (P < 0.0001)., Conclusions: SLNB improved GHS and BRAS QoL in breast cancer patients. However, surgeons must be cautious, SLNB with ALND results in a poorer QoL.

In an attempt to get a marker gene suitable for genetical transformation of the ectomycorrhizal fungus Hebeloma cylindrosporum, the gene Hc.Sdh (R) that confers carboxin-resistance was isolated from a UV mutant of this fungus. It encodes a mutant allele of the Fe-S subunit of the succinate dehydrogenase gene that carries a single amino acid substitution known to confer carboxin-resistance. This gene was successfully used as the selective marker to transform, via Agrobacterium tumefaciens, monokaryotic and dikaryotic strains of H. cylindrosporum. We also successfully transformed hygromycin-resistant insertional mutants. Transformation yielded mitotically stable carboxin-resistant mycelia. This procedure produced transformants, the growth of which was not affected by 2 microg l(-1) carboxin, whereas wild-type strains were unable to grow in the presence of 0.1 microg l(-1) of this fungicide. This makes the carboxin-resistance cassette much more discriminating than the hygromycin-resistance one. PCR amplification and Southern blot hybridisation indicated that more than 90% of the tested carboxin-resistant mycelia contained the Hc.Sdh (R) cassette, usually as a single copy. The AGL-1 strain of A. tumefaciens was a much less efficient donor than LBA 1126; the former yielded ca. 0-30% transformation frequency, depending on fungal strain and resistance cassette used, whereas the latter yielded ca. 60-95%.

Mechanisms regulating legume root nodule development are still poorly understood, and very few regulatory genes have been cloned and characterized. Here, we describe EFD (for ethylene response factor required for nodule differentiation), a gene that is upregulated during nodulation in Medicago truncatula. The EFD transcription factor belongs to the ethylene response factor (ERF) group V, which contains ERN1, 2, and 3, three ERFs involved in Nod factor signaling. The role of EFD in the regulation of nodulation was examined through the characterization of a null deletion mutant (efd-1), RNA interference, and overexpression studies. These studies revealed that EFD is a negative regulator of root nodulation and infection by Rhizobium and that EFD is required for the formation of functional nitrogen-fixing nodules. EFD appears to be involved in the plant and bacteroid differentiation processes taking place beneath the nodule meristem. We also showed that EFD activated Mt RR4, a cytokinin primary response gene that encodes a type-A response regulator. We propose that EFD induction of Mt RR4 leads to the inhibition of cytokinin signaling, with two consequences: the suppression of new nodule initiation and the activation of differentiation as cells leave the nodule meristem. Our work thus reveals a key regulator linking early and late stages of nodulation and suggests that the regulation of the cytokinin pathway is important both for nodule initiation and development.

Nod factors are key bacterial signaling molecules regulating the symbiotic interaction between bacteria known as rhizobia and leguminous plants. Studying plant host genes whose expression is affected by Nod factors has given insights into early symbiotic signaling and development. Here, we used a double supernodulating mutant line that shows increased sensitivity to Nod factors to study the Nod factor-regulated transcriptome. Using microarrays containing more than 16,000 70-mer oligonucleotide probes, we identified 643 Nod-factor-regulated genes, including 225 new Nod-factor-upregulated genes encoding many potential regulators. Among the genes found to be Nod factor upregulated, we identified and characterized MtRALFL1 and MtDVL1, which code for two small putative peptide regulators of 135 and 53 amino acids, respectively. Expression analysis confirmed that these genes are upregulated during initial phases of nodulation. Overexpression of MtRALFL1 and MtDVL1 in Medicago truncatula roots resulted in a marked reduction in the number of nodules formed and in a strong increase in the number of aborted infection threads. In addition, abnormal nodule development was observed when MtRALFL1 was overexpressed. This work provides evidence for the involvement of new putative small-peptide regulators during nodulation.

Legume root architecture is characterized by the development of two de novo meristems, leading to the formation of lateral roots or symbiotic nitrogen-fixing nodules. Organogenesis involves networks of transcription factors, the encoding mRNAs of which are frequently targets of microRNA (miRNA) regulation. Most plant miRNAs, in contrast with animal miRNAs, are encoded as single entities in an miRNA precursor. In the model legume Medicago truncatula, we have identified the MtMIR166a precursor containing tandem copies of MIR166 in a single transcriptional unit. These miRNAs post-transcriptionally regulate a new family of transcription factors associated with nodule development, the class-III homeodomain-leucine zipper (HD-ZIP III) genes. In situ expression analysis revealed that these target genes are spatially co-expressed with MIR166 in vascular bundles, and in apical regions of roots and nodules. Overexpression of the tandem miRNA precursor correlated with MIR166 accumulation and the downregulation of several class-III HD-ZIP genes, indicating its functionality. MIR166 overexpression reduced the number of symbiotic nodules and lateral roots, and induced ectopic development of vascular bundles in these transgenic roots. Hence, plant polycistronic miRNA precursors, although rare, can be processed, and MIR166-mediated post-transcriptional regulation is a new regulatory pathway involved in the regulation of legume root architecture.

MtHAP2-1 is a CCAAT-binding transcription factor from the model legume Medicago truncatula. We previously showed that MtHAP2-1 expression is regulated both spatially and temporally by microRNA169. Here we present a novel regulatory mechanism controlling MtHAP2-1 expression. Alternative splicing of an intron in the MtHAP2-1 5'leader sequence (LS) becomes predominant during the development of root nodules, leading to the production of a small peptide, uORF1p. Our results indicate that binding of uORF1p to MtHAP2-1 5'LS mRNA leads to reduced accumulation of the MtHAP2-1 transcript and may contribute to spatial restriction of MtHAP2-1 expression within the nodule. We propose that miR169 and uORF1p play essential, sequential, and nonredundant roles in regulating MtHAP2-1 expression. Importantly, in contrast to previously described cis-acting uORFs, uORF1p is able to act in trans to down-regulate gene expression. Our work thus contributes to a better understanding of the action of upstream ORFs (uORFs) in the regulation of gene expression.

We show here that MtMMPL1, a Medicago truncatula nodulin gene previously identified by transcriptomics, represents a novel and specific marker for root and nodule infection by Sinorhizobium meliloti. This was established by determining the spatial pattern of MtMMPL1 expression and evaluating gene activation in the context of various plant and bacterial symbiotic mutant interactions. The MtMMPL1 protein is the first nodulin shown to belong to the large matrix metalloendoproteinase (MMP) family. While plant MMPs are poorly documented, they are well characterized in animals as playing a key role in a number of normal and pathological processes involving the remodeling of the extracellular matrix. MtMMPL1 represents a novel MMP variant, with a substitution of a key amino acid residue within the predicted active site, found exclusively in expressed sequence tags corresponding to legume MMP homologs. An RNA interference approach revealed that decreasing MtMMPL1 expression leads to an accumulation of rhizobia within infection threads, whose diameter is often significantly enlarged. Conversely, MtMMPL1 ectopic overexpression under the control of a constitutive (35S) promoter led to numerous abortive infections and an overall decrease in the number of nodules. We discuss possible roles of MtMMPL1 during Rhizobium infection.

In the model legume Medicago truncatula, we identified a new transcription factor of the CCAAT-binding family, MtHAP2-1, for which RNA interference (RNAi) and in situ hybridization experiments indicate a key role during nodule development, possibly by controlling nodule meristem function. We could also show that MtHAP2-1 is regulated by microRNA169, whose overexpression leads to the same nodule developmental block as MtHAP2-1 RNAi constructs. The complementary expression pattern of miR169 and MtHAP2-1 and the phenotype of miR169-resistant MtHAP2-1 nodules strongly suggest, in addition, that the miR169-mediated restriction of MtHAP2-1 expression to the nodule meristematic zone is essential for the differentiation of nodule cells.

Polyethylene glycol-mediated transformation of protoplasts was used as a method for insertional mutagenesis to obtain mutants of the ectomycorrhizal fungus Hebeloma cylindrosporum impaired in symbiotic ability. Following restriction enzyme-mediated integration or conventional plasmid insertion, a library of 1,725 hygromycin-resistant monokaryotic transformants was generated and screened for the symbiotic defect, using Pinus pinaster seedlings as host plants. A total of 51 transformants displaying a dramatically reduced mycorrhizal ability were identified. Among them, 29 were nonmycorrhizal (myc-), but only 10 of them had integrated one or several copies of the transforming plasmid in their genome. Light and scanning electron microscopy observations of pine roots inoculated with myc- mutants suggested that we selected mutants blocked at early stages of interaction between partners or at the stage of Hartig net formation. Myc- mutants with plasmid insertions were crossed with a compatible wild-type monokaryon and allowed to fruit. Monokaryotic progenies were obtained in three independent crosses and were analyzed for symbiotic activity and plasmid insertion. In all three progenies, a 1:1 myc-:myc+ segregation ratio was observed, suggesting that each myc- phenotype resulted from a single gene mutation. However, for none of the three mutants, the myc- phenotype segregated with any of the plasmid insertions. Our results support the idea that master genes, the products of which are essential for symbiosis establishment, do exist in ectomycorrhizal fungi.

The basidiomycete Hebeloma cylindrosporum has been extensively studied with respect to mycorrhiza differentiation and metabolism and also to population dynamics. Its life cycle can be reproduced in vitro and it can be genetically transformed. Combined biochemical, cytological, genetical and molecular approaches led to the characterisation of mutant strains affected in mycorrhiza formation. These studies demonstrated the role of fungal auxin as a signal molecule in mycorrhiza formation and should allow the characterisation of essential fungal genes necessary to achieve a compatible symbiotic interaction. Random sequencing of cDNAs has identified numerous key functional genes which allowed dissection of essential nitrogen assimilation pathways. H. cylindrosporum also proved to be a remarkable model species to uncover the dynamics of natural populations of ectomycorrhizal fungi and the way in which they respond and adapt to anthropogenic disturbance of the forest ecosystem. Although studies on mycorrhiza differentiation and functioning and those on the population dynamics of H. cylindrosporum have been carried out independently, they are likely to converge in a renewed molecular ecophysiology which will envisage how ectomycorrhizal symbiosis functions under varying field conditions. Contents Summary 481 I. Introduction 482 II. Taxonomy, distribution, autecology, and host range of H. cylindrosporum 482 III. The Hebeloma cylindrosporum toolbox 483 IV. Mycorrhiza differentiation 486 V. Nutritional interactions 488 VI. Genetic diversity and dynamics of H. cylindrosporum populations in P. pinaster forest ecosystems 491 VII. Future directions 494 Acknowledgements 494 References 494.

We transformed haploid mycelium of Hebeloma cylindrosporum via Agrobacterium tumefaciens and optimised the procedure to develop a new tool for insertional mutagenesis in this fungus. Southern blot analysis of 83 randomly selected transformants showed that they all contained plasmid inserts. Each of them showed a unique hybridisation pattern, suggesting that integration was random in the fungal genome. Sixty percent of transformants obtained in the presence of bacteria pre-treated with acetosyringone integrated a single transferred DNA copy. Thermal asymmetric interlaced polymerase chain reaction allowed us to recover the left border and the right border junctions in 85% and 15% of transformants analysed, respectively. Results show that A. tumefaciens-mediated transformation may be a powerful tool for insertional mutagenesis in H. cylindrosporum.