Targeted mutation of barley (1,3;1,4)-β-glucan synthases reveals complex relationships between the storage and cell wall polysaccharide content

Barley (Hordeum vulgare L) grain is comparatively rich in (1,3;1,4)-β-glucan, a source of fermentable dietary fibre that protects against various human health conditions. However, low grain (1,3;1,4)-β-glucan content is preferred for brewing and distilling. We took a reverse genetics approach, using CRISPR/Cas9 to generate mutations in members of the Cellulose synthase-like (Csl) gene superfamily that encode known (HvCslF6 and HvCslH1) and putative (HvCslF3 and HvCslF9) (1,3;1,4)-β-glucan synthases. Resultant mutations ranged from single amino acid (aa) substitutions to frameshift mutations causing premature stop codons, and led to specific differences in grain morphology, composition and (1,3;1,4)-β-glucan content. (1,3;1,4)-β-Glucan was absent in the grain of cslf6 knockout lines, whereas cslf9 knockout lines had similar (1,3;1,4)-β-glucan content to wild-type (WT). However, cslf9 mutants showed changes in the abundance of other cell-wall-related monosaccharides compared with WT. Thousand grain weight (TGW), grain length, width and surface area were altered in cslf6 knockouts, and to a lesser extent TGW in cslf9 knockouts. cslf3 and cslh1 mutants had no effect on grain (1,3;1,4)-β-glucan content. Our data indicate that multiple members of the CslF/H family fulfil important functions during grain development but, with the exception of HvCslF6, do not impact the abundance of (1,3;1,4)-β-glucan in mature grain.</p

Following the Formation of Synaptonemal Complex Formation in Wheat and Barley by High-Resolution Microscopy

International audienceWheat and barley have large genomes of 15 Gb and 5.1 Gb, respectively, which is much larger than the human genome (3.3 Gb). The release of their respective genomes has been a tremendous advance the understanding of the genome organization and the ability for deeper functional analysis in particular meiosis. Meiosis is the cell division required during sexual reproduction. One major event of meiosis is called recombination, or the formation of crossing over, a tight link between homologous chromosomes, ensuring gene exchange and faithful chromosome segregation. Recombination is a major driver of genetic diversity but in these large genome crops, the vast majority of these events is constrained at the end of their chromosomes. It is estimated that in barley, about 30% of the genes are located within the poor recombining centromeric regions, making important traits, such as resistance to pest and disease for example, difficult to access. Increasing recombination in these crops has the potential to speed up breeding program and requires a good understand of the meiotic mechanism. However, most research on recombination in plant has been carried in Arabidopsis thaliana which despite many of the advantages it brings for plant research, has a small genome and more spread out of recombination compare to barley or wheat. Advance in microscopy and cytological procedures have emerged in the last few years, allowing to follow meiotic events in these crops. This protocol provides the steps required for cytological preparation of barley and wheat pollen mother cells for light microscopy, highlighting some of the differences between the two cereals

Rapidly Aggravated Dissecting Flap by Angiography during Percutaneous Stent Placement for Acute Isolated Superior Mesenteric Artery Dissection

Acutely aggravated dissecting flap and consequent occlusion of the superior mesenteric artery (SMA) by simple contrast passage during initial angiography for percutaneous stent placement is a uncommon event, which usually is not reported. After analysis of many factors that underlie development of such complications, we present herein one case of successful treatment of isolated SMA dissection and its complications with favorable outcomes during 25 months follow-up after percutaneous stent placement

Acute thrombosis of the superior mesenteric artery in a 39-year-old woman with protein-S deficiency: a case report

<p>Abstract</p> <p>Introduction</p> <p>Acute thromboembolic occlusion of the superior mesenteric artery is a condition with an unfavorable prognosis. Treatment of this condition is focused on early diagnosis, surgical or intravascular restoration of blood flow to the ischemic intestine, surgical resection of the necrotic bowel and supportive intensive care. In this report, we describe a case of a 39-year-old woman who developed a small bowel infarct because of an acute thrombotic occlusion of the superior mesenteric artery, also involving the splenic artery.</p> <p>Case presentation</p> <p>A 39-year-old Caucasian woman presented with acute abdominal pain and signs of intestinal occlusion. The patient was given an abdominal computed tomography scan and ultrasonography in association with Doppler ultrasonography, highlighting a thrombosis of the celiac trunk, of the superior mesenteric artery, and of the splenic artery. She immediately underwent an explorative laparotomy, and revascularization was performed by thromboendarterectomy with a Fogarty catheter. In the following postoperative days, she was given a scheduled second and third look, evidencing necrotic jejunal and ileal handles. During all the surgical procedures, we performed intraoperative Doppler ultrasound of the superior mesenteric artery and celiac trunk to control the arterial flow without evidence of a new thrombosis.</p> <p>Conclusion</p> <p>Acute mesenteric ischemia is a rare abdominal emergency that is characterized by a high mortality rate. Generally, acute mesenteric ischemia is due to an impaired blood supply to the intestine caused by thromboembolic phenomena. These phenomena may be associated with a variety of congenital prothrombotic disorders. A prompt diagnosis is a prerequisite for successful treatment. The treatment of choice remains laparotomy and thromboendarterectomy, although some prefer an endovascular approach. A second-look laparotomy could be required to evaluate viable intestinal handles. Some authors support a laparoscopic second-look. The possibility of evaluating the arteriotomy, during a repeated laparotomy with a Doppler ultrasound, is crucial to show a new thrombosis. Although the prognosis of acute mesenteric ischemia due to an acute arterial mesenteric thrombosis remains poor, a prompt diagnosis, aggressive surgical treatment and supportive intensive care unit could improve the outcome for patients with this condition.</p

A randomized controlled trial of tea tree oil (5%) body wash versus standard body wash to prevent colonization with methicillin-resistant Staphylococcus aureus (MRSA) in critically ill adults: research protocol

<p>Abstract</p> <p>Background</p> <p>Over the past ten years MRSA has become endemic in hospitals and is associated with increased healthcare costs. Critically ill patients are most at risk, in part because of the number of invasive therapies that they require in the intensive care unit (ICU). Washing with 5% tea tree oil (TTO) has been shown to be effective in removing MRSA on the skin. However, to date, no trials have evaluated the potential of TTO body wash to prevent MRSA colonization or infection. In addition, detecting MRSA by usual culture methods is slow. A faster method using a PCR assay has been developed in the laboratory, but requires evaluation in a large number of patients.</p> <p>Methods/Design</p> <p>This study protocol describes the design of a multicentre, phase II/III prospective open-label randomized controlled clinical trial to evaluate whether a concentration of 5% TTO is effective in preventing MRSA colonization in comparison with a standard body wash (Johnsons Baby Softwash) in the ICU. In addition we will evaluate the cost-effectiveness of TTO body wash and assess the effectiveness of the PCR assay in detecting MRSA in critically ill patients. On admission to intensive care, swabs from the nose and groin will be taken to screen for MRSA as per current practice. Patients will be randomly assigned to be washed with the standard body wash or TTO body wash. On discharge from the unit, swabs will be taken again to identify whether there is a difference in MRSA colonization between the two groups.</p> <p>Discussion</p> <p>If TTO body wash is found to be effective, widespread implementation of such a simple colonization prevention tool has the potential to impact on patient outcomes, healthcare resource use and patient confidence both nationally and internationally.</p> <p>Trial Registration</p> <p>[ISRCTN65190967]</p

A highly mutagenised barley (cv. Golden Promise) TILLING population coupled with strategies for screening-by-sequencing

Background:We developed and characterised a highly mutagenised TILLING population of the barley (Hordeum vulgare) cultivar Golden Promise. Golden Promise is the 'reference' genotype for barley transformation and a primary objective of using this cultivar was to be able to genetically complement observed mutations directly in order to prove gene function. Importantly, a reference genome assembly of Golden Promise has also recently been developed. As our primary interest was to identify mutations in genes involved in meiosis and recombination, to characterise the population we focused on a set of 46 genes from the literature that are possible meiosis gene candidates. Results:Sequencing 20 plants from the population using whole exome capture revealed that the mutation density in this population is high (one mutation every 154 kb), and consequently even in this small number of plants we identified several interesting mutations. We also recorded some issues with seed availability and germination. We subsequently designed and applied a simple two-dimensional pooling strategy to identify mutations in varying numbers of specific target genes by Illumina short read pooled-amplicon sequencing and subsequent deconvolution. In parallel we assembled a collection of semi-sterile mutants from the population and used a custom exome capture array targeting the 46 candidate meiotic genes to identify potentially causal mutations. Conclusions:We developed a highly mutagenised barley TILLING population in the transformation competent cultivar Golden Promise. We used novel and cost-efficient screening approaches to successfully identify a broad range of potentially deleterious variants that were subsequently validated by Sanger sequencing. These resources combined with a high-quality genome reference sequence opens new possibilities for efficient functional gene validation.Miriam Schreiber, Abdellah Barakate, Nicola Uzrek, Malcolm Macaulay, Adeline Sourdille, Jenny Morris, Pete E. Hedley, Luke Ramsay and Robbie Waug

Endophytic actinomycetes from spontaneous plants of Algerian Sahara: indole-3-acetic acid production and tomato plants growth promoting activity

Twenty-seven endophytic actinomycete strains were isolated from five spontaneous plants well adapted to the poor sandy soil and arid climatic conditions of the Algerian Sahara. Morphological and chemotaxonomical analysis indicated that twenty-two isolates belonged to the Streptomyces genus and the remaining five were non- Streptomyces. All endophytic strains were screened for their ability to produce indole-3-acetic acid (IAA) in vitro on a chemically defined medium. Eighteen strains were able to produce IAA and the maximum production occurred with the Streptomyces sp. PT2 strain. The IAA produced was further extracted, partially purified and confirmed by thin layer chromatography (TLC) analysis. The 16S rDNA sequence analysis and phylogenetic studies indicated that strain PT2 was closely related to Streptomyces enissocaecilis NRRL B 16365T, Streptomyces rochei NBRC 12908T and Streptomyces plicatus NBRC 13071T, with 99.52 % similarity. The production of IAA was affected by cultural conditions such as temperature, pH, incubation period and L-tryptophan concentration. The highest level of IAA production (127 lg/ml) was obtained by cultivating the Streptomyces sp. PT2 strain in yeast extract-tryptone broth supplemented with 5 mg L-tryptophan/ ml at pH 7 and incubated on a rotary shaker (200 rpm) at 30°C for 5 days. Twenty-four-hour treatment of tomato cv. Marmande seeds with the supernatant culture of Streptomyces sp. PT2 that contained the crude IAA showed the maximum effect in promoting seed germination and root elongation