Effects of the different waxy proteins on starch biosynthesis, starch physicochemical properties and Chinese noodle quality in wheat.

Noodles are an important food in Asia. Wheat starch is the most important component in Chinese noodles. Loss of the waxy genes leads to lower activity of starch synthesis enzymes and decreased amylose content that further affects starch properties and noodle quality. To study the effects of different waxy (Wx) protein subunits on starch biosynthesis and processing quality, the high-yielding wheat cultivar Jimai 22 was treated with the mutagen ethyl methane sulfonate (EMS) to produce a population of Wx lines and chosen 7 Wx protein combinations. The amylose content increased but swelling power decreased as the number of Wx proteins increased. Both GBSS activity and gene expression were the lowest for the waxy mutant, followed by the mutants with 1 Wx protein. The combinations of these mutant alleles lead to reductions in both RNA expression and protein levels. Noodles made from materials with 2 Wx protein subunits had the highest score, which agreed with peak viscosity. The influence of the Wx-B1 protein on amylose synthesis and noodle quality was the highest, whereas the influence of Wx-A1 protein was the lowest. Mutants with lower amylose content caused by the absence of 1 subunit, especially the Wx-B1 subunit, had superior noodle quality. Additionally, the identified mutant lines can be used as intermediate materials to improve wheat quality. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01292-x.

Role of miRNAs and alternative mRNA 3'-end cleavage and polyadenylation of their mRNA targets in cardiomyocyte hypertrophy.

miRNAs play critical roles in heart disease. In addition to differential miRNA expression, miRNA-mediated control is also affected by variable miRNA processing or alternative 3'-end cleavage and polyadenylation (APA) of their mRNA targets. To what extent these phenomena play a role in the heart remains unclear. We sought to explore miRNA processing and mRNA APA in cardiomyocytes, and whether these change during cardiac hypertrophy. Thoracic aortic constriction (TAC) was performed to induce hypertrophy in C57BL/6J mice. RNA extracted from cardiomyocytes of sham-treated, pre-hypertrophic (2 days post-TAC), and hypertrophic (7 days post-TAC) mice was subjected to small RNA- and poly(A)-test sequencing (PAT-Seq). Differential expression analysis matched expectations; nevertheless we identified ~400 mRNAs and hundreds of noncoding RNA loci as altered with hypertrophy for the first time. Although multiple processing variants were observed for many miRNAs, there was little change in their relative proportions during hypertrophy. PAT-Seq mapped ~48,000 mRNA 3'-ends, identifying novel 3' untranslated regions (3'UTRs) for over 7000 genes. Importantly, hypertrophy was associated with marked changes in APA with a net shift from distal to more proximal mRNA 3'-ends, which is predicted to decrease overall miRNA repression strength. We independently validated several examples of 3'UTR proportion change and showed that alternative 3'UTRs associate with differences in mRNA translation. Our work suggests that APA contributes to altered gene expression with the development of cardiomyocyte hypertrophy and provides a rich resource for a systems-level understanding of miRNA-mediated regulation in physiological and pathological states of the heart.

Highly predictive SNP markers for efficient selection of the wheat leaf rust resistance gene Lr16.

BACKGROUND: Lr16 is a widely deployed leaf rust resistance gene in wheat (Triticum aestivum L.) that is highly effective against the North American Puccinia triticina population when pyramided with the gene Lr34. Lr16 is a seedling leaf rust resistance gene conditioning an incompatible interaction with a distinct necrotic ring surrounding the uredinium. Lr16 was previously mapped to the telomeric region of the short arm of wheat chromosome 2B. The goals of this study were to develop numerous single nucleotide polymorphism (SNP) markers for the Lr16 region and identify diagnostic gene-specific SNP marker assays for marker-assisted selection (MAS). RESULTS: Forty-three SNP markers were developed and mapped on chromosome 2BS tightly linked with the resistance gene Lr16 across four mapping populations representing a total of 1528 gametes. Kompetitive Allele Specific PCR (KASP) assays were designed for all identified SNPs. Resistance gene analogs (RGAs) linked with the Lr16 locus were identified and RGA-based SNP markers were developed. The diagnostic potential of the SNPs co-segregating with Lr16 was evaluated in a diverse set of 133 cultivars and breeding lines. Six SNP markers were consistent with the Lr16 phenotype and are accurately predictive of Lr16 for all wheat lines/cultivars in the panel. CONCLUSIONS: Lr16 was mapped relative to SNP markers in four populations. Six SNP markers exhibited high quality clustering in the KASP assay and are suitable for MAS of Lr16 in wheat breeding programs.

Protective antibodies against Clostridium difficile are present in intravenous immunoglobulin and are retained in humans following its administration.

The prevalence of serum antibodies against Clostridium difficile (CD) toxins A and B in healthy populations have prompted interest in evaluating the therapeutic activity of intravenous immunoglobulin (IVIg) in individuals experiencing severe or recurrent C. difficile infection (CDI). Despite some promising case reports, a definitive clinical role for IVIg in CDI remains unclear. Contradictory results may be attributed to a lack of consensus regarding optimal dose, timing of administration and patient selection as well as variability in specific antibody content between commercial preparations. The purpose of this study was to investigate retrospectively the efficacy of three commercial preparations of IVIg for treating severe or recurrent CDI. In subsequent mechanistic studies using protein microarray and toxin neutralization assays, all IVIg preparations were analysed for specific binding and neutralizing antibodies (NAb) to CD antigens in vitro and the presence of anti-toxin NAbs in vivo following IVIg infusion. A therapeutic response to IVIg was observed in 41% (10 of 17) of the CDI patients. Significant variability in multi-isotype specific antibodies to a 7-plex panel of CD antigens and toxin neutralization efficacies were observed between IVIg preparations and also in patient sera before and after IVIg administration. These results extend our current understanding of population immunity to CD and support the inclusion of surface layer proteins and binary toxin antigens in CD vaccines. Future strategies could enhance IVIg treatment response rates by using protein microarray to preselect donor plasma/serum with the highest levels of anti-CD antibodies and/or anti-toxin neutralizing capacities prior to fractionation.

Identification of candidate genes, regions and markers for pre-harvest sprouting resistance in wheat (Triticum aestivum L.).

BACKGROUND: Pre-harvest sprouting (PHS) of wheat grain leads to a reduction in grain yield and quality. The availability of markers for marker-assisted selection (MAS) of PHS resistance will serve to enhance breeding selection and advancement of lines for cultivar development. The aim of this study was to identify candidate regions and develop molecular markers for PHS resistance in wheat. This was achieved via high density mapping of single nucleotide polymorphism (SNP) markers from an Illumina 90 K Infinium Custom Beadchip in a doubled haploid (DH) population derived from a RL4452/'AC Domain' cross and subsequent detection of quantitative trait loci (QTL) for PHS related traits (falling number [FN], germination index [GI] and sprouting index [SI]). SNP marker sequences flanking QTL were used to locate colinear regions in Brachypodium and rice, and identify genic markers associated with PHS resistance that can be utilized for MAS in wheat. RESULTS: A linkage map spanning 2569.4 cM was constructed with a total of 12,201 SNP, simple sequence repeat (SSR), diversity arrays technology (DArT) and expressed sequence tag (EST) markers. QTL analyses using Multiple Interval Mapping (MIM) identified four QTL for PHS resistance traits on chromosomes 3B, 4A, 7B and 7D. Sequences of SNPs flanking these QTL were subject to a BLASTN search on the International Wheat Genome Sequencing Consortium (IWGSC) database (http://wheat-urgi.versailles.inra.fr/Seq-Repository). Best survey sequence hits were subject to a BLASTN search on Gramene (www.gramene.org) against both Brachypodium and rice databases, and candidate genes and regions for PHS resistance were identified. A total of 18 SNP flanking sequences on chromosomes 3B, 4A, 7B and 7D were converted to KASP markers and validated with matching genotype calls of Infinium SNP data. CONCLUSIONS: Our study identified candidate genes involved in abscissic acid (ABA) and gibberellin (GA) metabolism, and flowering time in four genomic regions of Brachypodium and rice respectively, in addition to 18 KASP markers for PHS resistance in wheat. These markers can be deployed in future genetic studies of PHS resistance and might also be useful in the evaluation of PHS in germplasm and breeding material.

Genetic variation of seed dormancy in wheat (Triticum aestivum L.) is mediated by transcriptional regulation of abscisic acid metabolism and signaling.

Abscisic acid (ABA) regulates seed dormancy and therefore preharvest sprouting (PHS) in wheat. This study investigated the contribution of transcriptional regulation of ABA metabolism and signaling genes to genetic variation in dormancy of wheat seeds. Our results showed that genetic variation in seed dormancy is highly correlated with ABA content (r > 0.86), which, in turn, was closely associated with the expression levels of ABA biosynthesis genes, TaNCED1 (r = 0.78) and TaNCED2 (r = 0.67). A relatively lower correlation was observed between ABA content and the expression levels of ABA catabolism genes, TaCYP707A1 (r = 0.51) and TaCYP707A2 (r = 0.57). The expression level of TaABI5 exhibited strong associations with the levels of ABA (r = 0.8) and seed dormancy (r > 0.9), indicating the importance of seed ABA sensitivity in mediating genetic variation in dormancy. Furthermore, high positive correlations were prevalent between the expression patterns of TaABI5 and TaNCED1 (r = 0.91) or TaNCED2 (r = 0.82). Overall, our results implicated the significance of TaNCEDs and TaABI5 in regulating genetic variation in ABA level and sensitivity and thereby seed dormancy, highlighting the potential use of these genes to develop molecular markers for incorporating PHS resistance in wheat.

Genetics and mapping of seedling resistance to Ug99 stem rust in Canadian wheat cultivars 'Peace' and 'AC Cadillac'.

Stem rust (caused by Puccinia graminis Pers.:Pers. f. sp. tritici Eriks. & E. Henn.) has re-emerged as a threat to wheat production with the evolution of new pathogen races, namely TTKSK (Ug99) and its variants, in Africa. Deployment of resistant wheat cultivars has provided long-term control of stem rust. Identification of new resistance genes will contribute to future cultivars with broad resistance to stem rust. The related Canadian cultivars Peace and AC Cadillac show resistance to Ug99 at the seedling stage and in the field. The purpose of this study was to elucidate the inheritance and genetically map resistance to Ug99 in these two cultivars. Two populations were produced, an F(2:3) population from LMPG/AC Cadillac and a doubled haploid (DH) population from RL6071/Peace. Both populations showed segregation at the seedling stage for a single stem rust resistance (Sr) gene, temporarily named SrCad. SrCad was mapped to chromosome 6DS in both populations with microsatellite markers and a marker (FSD_RSA) that is tightly linked to the common bunt resistance gene Bt10. FSD_RSA was the closest marker to SrCad (≈ 1.6 cM). Evaluation of the RL6071/Peace DH population and a second DH population, AC Karma/87E03-S2B1, in Kenya showed that the combination of SrCad and leaf rust resistance gene Lr34 provided a high level of resistance to Ug99-type races in the field, whereas in the absence of Lr34 SrCad conferred moderate resistance. A survey confirmed that SrCad is the basis for all of the seedling resistance to Ug99 in Canadian wheat cultivars. While further study is needed to determine the relationship between SrCad and other Sr genes on chromosome 6DS, SrCad represents a valuable genetic resource for producing stem rust resistant wheat cultivars.

Integration of full divertor detachment with improved core confinement for tokamak fusion plasmas.

Divertor detachment offers a promising solution to the challenge of plasma-wall interactions for steady-state operation of fusion reactors. Here, we demonstrate the excellent compatibility of actively controlled full divertor detachment with a high-performance (ßN ~ 3, H98 ~ 1.5) core plasma, using high-ßp (poloidal beta, ßp > 2) scenario characterized by a sustained core internal transport barrier (ITB) and a modest edge transport barrier (ETB) in DIII-D tokamak. The high-ßp high-confinement scenario facilitates divertor detachment which, in turn, promotes the development of an even stronger ITB at large radius with a weaker ETB. This self-organized synergy between ITB and ETB, leads to a net gain in energy confinement, in contrast to the net confinement loss caused by divertor detachment in standard H-modes. These results show the potential of integrating excellent core plasma performance with an efficient divertor solution, an essential step towards steady-state operation of reactor-grade plasmas.

An introgression on wheat chromosome 4DL in RL6077 (Thatcher*6/PI 250413) confers adult plant resistance to stripe rust and leaf rust (Lr67).

Adult plant resistance (APR) to leaf rust and stripe rust derived from the wheat (Triticum aestivum L.) line PI250413 was previously identified in RL6077 (=Thatcher*6/PI250413). The leaf rust resistance gene in RL6077 is phenotypically similar to Lr34 which is located on chromosome 7D. It was previously hypothesized that the gene in RL6077 could be Lr34 translocated to another chromosome. Hybrids between RL6077 and Thatcher and between RL6077 and 7DS and 7DL ditelocentric stocks were examined for first meiotic metaphase pairing. RL6077 formed chain quadrivalents and trivalents relative to Thatcher and Chinese Spring; however both 7D telocentrics paired only as heteromorphic bivalents and never with the multivalents. Thus, chromosome 7D is not involved in any translocation carried by RL6077. A genome-wide scan of SSR markers detected an introgression from chromosome 4D of PI250413 transferred to RL6077 through five cycles of backcrossing to Thatcher. Haplotype analysis of lines from crosses of Thatcher × RL6077 and RL6058 (Thatcher*6/PI58548) × RL6077 showed highly significant associations between introgressed markers (including SSR marker cfd71) and leaf rust resistance. In a separate RL6077-derived population, APR to stripe rust was also tightly linked with cfd71 on chromosome 4DL. An allele survey of linked SSR markers cfd71 and cfd23 on a set of 247 wheat lines from diverse origins indicated that these markers can be used to select for the donor segment in most wheat backgrounds. Comparison of RL6077 with Thatcher in field trials showed no effect of the APR gene on important agronomic or quality traits. Since no other known Lr genes exist on chromosome 4DL, the APR gene in RL6077 has been assigned the name Lr67.

Mutagenesis and functional characterization of the RNA and protein components of the toxIN abortive infection and toxin-antitoxin locus of Erwinia.

Bacteria are constantly challenged by bacteriophage (phage) infection and have developed multiple adaptive resistance mechanisms. These mechanisms include the abortive infection systems, which promote "altruistic suicide" of an infected cell, protecting the clonal population. A cryptic plasmid of Erwinia carotovora subsp. atroseptica, pECA1039, has been shown to encode an abortive infection system. This highly effective system is active across multiple genera of gram-negative bacteria and against a spectrum of phages. Designated ToxIN, this two-component abortive infection system acts as a toxin-antitoxin module. ToxIN is the first member of a new type III class of protein-RNA toxin-antitoxin modules, of which there are multiple homologues cross-genera. We characterized in more detail the abortive infection phenotype of ToxIN using a suite of Erwinia phages and performed mutagenesis of the ToxI and ToxN components. We determined the minimal ToxI RNA sequence in the native operon that is both necessary and sufficient for abortive infection and to counteract the toxicity of ToxN. Furthermore, site-directed mutagenesis of ToxN revealed key conserved amino acids in this defining member of the new group of toxic proteins. The mechanism of phage activation of the ToxIN system was investigated and was shown to have no effect on the levels of the ToxN protein. Finally, evidence of negative autoregulation of the toxIN operon, a common feature of toxin-antitoxin systems, is presented. This work on the components of the ToxIN system suggests that there is very tight toxin regulation prior to suicide activation by incoming phage.

A cumulative meta-analysis of the effects of individual physical activity interventions targeting healthy adults.

Despite a large and increasing evidence base on physical activity interventions, the high rates of physical inactivity and associated chronic diseases are continuing to increase globally. The purpose of this cumulative meta-analysis was to investigate the evolution of randomized controlled trial evidence of individual-level physical activity interventions to asses if new trials are contributing novel evidence to the field. Through a two-staged search process, primary studies examining the effects of interventions targeted at increasing physical activity within healthy adult populations were pooled and selected from eligible systematic reviews. Cumulative meta-analyses were performed on effect sizes immediately post-intervention (n = 62), and for long-term behaviour change (≥12-month post-baseline; n = 27). Sufficiency and stability of the evidence was assessed through application of pre-published indicators. Meta-analyses suggest overall positive intervention effects on physical activity. The evidence base for effectiveness immediately post-intervention reached levels of sufficiency and stability in 2007; and for long-term follow-up in 2011. In the time since, intervention effectiveness has not substantially changed, and further trials are unlikely to change the direction and magnitude of effect. Substantial evidence exists demonstrating that physical activity interventions can modify individual behaviour in controlled settings. Researchers are urged to shift focus towards investigating the optimization, implementation, sustainability and cost-effectiveness of interventions.

Relationship between QTL for grain shape, grain weight, test weight, milling yield, and plant height in the spring wheat cross RL4452/'AC Domain'.

Kernel morphology characteristics of wheat are complex and quantitatively inherited. A doubled haploid (DH) population of the cross RL4452/'AC Domain' was used to study the genetic basis of seed shape. Quantitative trait loci (QTL) analyses were conducted on a total of 18 traits: 14 grain shape traits, flour yield (Fyd), and three agronomic traits (Plant height [Plht], 1000 Grain weight [Gwt], Test weight [Twt]), using data from trial locations at Glenlea, Brandon, and Morden in Manitoba, Canada, between 1999 and 2004. Kernel shape was studied through digital image analysis with an Acurum® grain analyzer. Plht, Gwt, Twt, Fyd, and grain shape QTL were correlated with each other and QTL analysis revealed that QTL for these traits often mapped to the same genetic locations. The most significant QTL for the grain shape traits were located on chromosomes 4B and 4D, each accounting for up to 24.4% and 53.3% of the total phenotypic variation, respectively. In addition, the most significant QTL for Plht, Gwt, and Twt were all detected on chromosome 4D at the Rht-D1 locus. Rht-D1b decreased Plht, Gwt, Twt, and kernel width relative to the Rht-D1a allele. A narrow genetic interval on chromosome 4B contained significant QTL for grain shape, Gwt, and Plht. The 'AC Domain' allele reduced Plht, Gwt, kernel length and width traits, but had no detectable effect on Twt. The data indicated that this variation was inconsistent with segregation at Rht-B1. Numerous QTL were identified that control these traits in this population.

Multivalent Fcγ-receptor engagement by a hexameric Fc-fusion protein triggers Fcγ-receptor internalisation and modulation of Fcγ-receptor functions.

Engagement of Fcγ-receptors triggers a range of downstream signalling events resulting in a diverse array of immune functions. As a result, blockade of Fc-mediated function is an important strategy for the control of several autoimmune and inflammatory conditions. We have generated a hexameric-Fc fusion protein (hexameric-Fc) and tested the consequences of multi-valent Fcγ-receptor engagement in in vitro and in vivo systems. In vitro engagement of hexameric-Fc with FcγRs showed complex binding interactions that altered with receptor density and triggered the internalisation and degradation of Fcγ-receptors. This caused a disruption of Fc-binding and phagocytosis. In vivo, in a mouse ITP model we observed a short half-life of hexameric-Fc but were nevertheless able to observe inhibition of platelet phagocytosis several days after hexameric-Fc dosing. In cynomolgus monkeys, we again observed a short half-life, but were able to demonstrate effective FcγR blockade. These findings demonstrate the ability of multi-valent Fc-based therapeutics to interfere with FcγR function and a potential mechanism through which they could have a sustained effect; the internalisation and degradation of FcγRs.

Effect of nitrous oxide on cerebral blood velocity while reclining and standing.

Cerebral blood velocity (CBV) (measured with transcranial Doppler, TCD) and other physiological and rating scale indices were measured before, during, and after inhaling a mixture of 40% nitrous oxide/oxygen and 40% nitrogen/oxygen, given during two separate visits in 7 normal male volunteers. During nitrous oxide/oxygen but not nitrogen/oxygen inhalation, CBV and euphoria increased significantly with minimal changes in other physiological indices except an increase in pulse rate after nitrous oxide/oxygen. In another 7 male volunteers, similar measurements were obtained while lying down and standing up during nitrous oxide/oxygen mixture and nitrogen/oxygen mixture inhalations, given during two separate visits. Nitrous oxide/oxygen inhalation produced significant CBV increase, which showed nonsignificant decrease of no clinical significance during standing up. There were no significant postural changes in blood pressure. Standing up during nitrous oxide/oxygen inhalation was associated with significant though mild dizziness.

Changes in cerebral blood velocity after intravenous diazepam.

Cerebral blood velocity (CBV) was measured with transcranial Doppler in 6 normal right-handed male volunteers before and for 50 min after an intravenous injection of 0.1 mg/kg of diazepam and normal saline during 2 separate visits to the laboratory. Blood pressure, pulse rate, end tidal levels of carbon dioxide and mood changes were quantified before and after the injections. Diazepam injection was associated with significant increases in fatigue and sleepiness. There were no significant changes in end tidal carbon dioxide, respiration, pulse rate, and blood pressure after the injection. Postdiazepam CBV was significantly lower following diazepam compared to CBV following placebo.

Changes in middle cerebral artery velocity after marijuana.

Velocity of blood flow in the middle cerebral artery was measured with transcranial Doppler flowmeter before, during, and 1 hr after smoking a marijuana cigarette and a placebo cigarette during two separate visits to the laboratory. Ten healthy, right-handed male volunteers with a history of marijuana smoking took part in the study. The participants were drug-free for a minimum of 3 mo before the project. During the experiment, blood pressure, pulse rate, and end tidal levels of carbon dioxide were continually monitored. Marijuana smoking was associated with a significant increase in middle cerebral artery velocity. Although marijuana smoking was associated with increased pulse rate, the changes in blood velocity and pulse rate followed different time courses. Marijuana smoking was not associated with significant changes in blood pressure or end tidal carbon dioxide.

Cerebral vasodilation and vasoconstriction associated with acute anxiety.

A randomized, between-groups, repeated measures design was used to evaluate changes in cerebral blood flow (CBF), rating scales, and physiologic indices under resting conditions, during 5% CO2 inhalation in combination with epinephrine or saline infusions, in generalized anxiety disorder patients and controls. Subjects were divided into those with decreased anxiety and mild and more severe anxiety increase. The first group was found to have most pronounced CBF increase during CO2 inhalation, with the second group showing less marked increase, and the last group the least increase. In animals, sympathetic activation limits hypercapnic cerebral vasodilation. Thus, the restricted hypercapnic cerebral vasodilation during severe anxiety may be mediated through cervical sympathetic fibers, which innervate cerebral vessels.

Depersonalization after marijuana smoking.

Depersonalization and other behavioral and physiological indices were monitored before and after the administration of high- and low-potency marijuana cigarettes and a placebo cigarette in 35 physically and mentally healthy normal volunteers. The cigarettes were administered under double-blind conditions during three visits to the laboratory separated by a minimum of 1 week. Marijuana smoking, but not placebo smoking, was associated with significant depersonalization that was maximal 30 min after smoking the high-potency cigarettes. Other behavioral changes induced by marijuana included disintegration of time sense, sensation of "high," increased state anxiety, tension, anger, and confusion. Respiration, pulse rate, and systolic blood pressure also increased after marijuana smoking. Multiple regression identified temporal disintegration as the most significant predictor of depersonalization.

Regional cerebral blood flow after marijuana smoking.

Regional CBF was measured with the 133Xe inhalation technique before and thrice after smoking marijuana of two strengths and placebo in 20 physically and mentally healthy male volunteers with a previous history of exposure to marijuana. They were drug-free at the time of the study. Blood pressure, pulse rate, end-tidal carbon dioxide, end-tidal carbon monoxide, and forehead skin perfusion were quantified during the CBF measurements. Blood samples were drawn for quantification of plasma levels of delta 9-tetrahydrocannabinol (THC) before and during the 2 h after smoking marijuana or placebo. Drug-induced intoxication and changes in mood were quantified with rating scales. Marijuana smoking was associated with bilateral CBF increase, which was maximal 30 min later. Greater CBF increases were seen in the frontal region and right hemisphere. No significant CBF changes were seen after placebo. Pulse rate and respiration increased significantly after marijuana but not placebo. Both marijuana and placebo smoking were associated with increased end-tidal carbon monoxide. CBF increase in both hemispheres correlated significantly with degree of intoxication, plasma levels of THC, and pulse rate.

Co-expression of human protein disulphide isomerase (PDI) can increase the yield of an antibody Fab' fragment expressed in Escherichia coli.

Secretion to the periplasm of Escherichia coli enables production of many eukaryotic extracellular proteins in a soluble form. The complex disulphide bond arrangement of such proteins is probably a major factor in determining the low yield of correctly folded product observed in many cases. Here we show that co-expression of human protein disulphide isomerase increased the yield of a monoclonal antibody Fab' fragment in the periplasm of E. coli.