Mutational and biophysical robustness in a prestabilized monobody.

The fibronectin type III (FN3) monobody domain is a promising non-antibody scaffold, which features a less complex architecture than an antibody while maintaining analogous binding loops. We previously developed FN3Con, a hyperstable monobody derivative with diagnostic and therapeutic potential. Prestabilization of the scaffold mitigates the stability-function trade-off commonly associated with evolving a protein domain toward biological activity. Here, we aimed to examine if the FN3Con monobody could take on antibody-like binding to therapeutic targets, while retaining its extreme stability. We targeted the first of the Adnectin derivative of monobodies to reach clinical trials, which was engineered by directed evolution for binding to the therapeutic target VEGFR2; however, this function was gained at the expense of large losses in thermostability and increased oligomerization. In order to mitigate these losses, we grafted the binding loops from Adnectin-anti-VEGFR2 (CT-322) onto the prestabilized FN3Con scaffold to produce a domain that successfully bound with high affinity to the therapeutic target VEGFR2. This FN3Con-anti-VEGFR2 construct also maintains high thermostability, including remarkable long-term stability, retaining binding activity after 2 years of storage at 36 °C. Further investigations into buffer excipients doubled the presence of monomeric monobody in accelerated stability trials. These data suggest that loop grafting onto a prestabilized scaffold is a viable strategy for the development of monobody domains with desirable biophysical characteristics and that FN3Con is therefore well-suited to applications such as the evolution of multiple paratopes or shelf-stable diagnostics and therapeutics.

Overgrowth mutants determine the causal role of gibberellin GA2oxidaseA13 in Rht12 dwarfism of wheat.

The induced dwarf mutant Rht12 was previously shown to have agronomic potential to replace the conventional DELLA mutants Rht-B1b/Rht-D1b in wheat. The Rht12 dwarfing gene is not associated with reduced coleoptile length (unlike the DELLA mutants) and it is dominant, characteristics which are shared with the previously characterized dwarfing genes Rht18 and Rht14. Using the Rht18/Rht14 model, a gibberellin (GA) 2-oxidase gene was identified in the Rht12 region on chromosome 5A. A screen for suppressor mutants in the Rht12 background identified tall overgrowth individuals that were shown to contain loss-of-function mutations in GA2oxidaseA13, demonstrating the role of this gene in the Rht12 dwarf phenotype. It was concluded that Rht12, Rht18, and Rht14 share the same height-reducing mechanism through the increased expression of GA 2-oxidase genes. Some of the overgrowth mutants generated in this study were semi-dwarf and taller than the original Rht12 dwarf, providing breeders with new sources of agronomically useful dwarfism.

Rht18 Semidwarfism in Wheat Is Due to Increased GA 2-oxidaseA9 Expression and Reduced GA Content.

Semidwarfing genes have improved crop yield by reducing height, improving lodging resistance, and allowing plants to allocate more assimilates to grain growth. In wheat (Triticum aestivum), the Rht18 semidwarfing gene was identified and deployed in durum wheat before it was transferred into bread wheat, where it was shown to have agronomic potential. Rht18, a dominant and gibberellin (GA) responsive mutant, is genetically and functionally distinct from the widely used GA-insensitive semidwarfing genes Rht-B1b and Rht-D1b In this study, the Rht18 gene was identified by mutagenizing the semidwarf durum cultivar Icaro (Rht18) and generating mutants with a range of tall phenotypes. Isolating and sequencing chromosome 6A of these "overgrowth" mutants showed that they contained independent mutations in the coding region of GA2oxA9GA2oxA9 is predicted to encode a GA 2-oxidase that metabolizes GA biosynthetic intermediates into inactive products, effectively reducing the amount of bioactive GA (GA1). Functional analysis of the GA2oxA9 protein demonstrated that GA2oxA9 converts the intermediate GA12 to the inactive metabolite GA110 Furthermore, Rht18 showed higher expression of GA2oxA9 and lower GA content compared with its tall parent. These data indicate that the increased expression of GA2oxA9 in Rht18 results in a reduction of both bioactive GA content and plant height. This study describes a height-reducing mechanism that can generate new genetic diversity for semidwarfism in wheat by combining increased expression with mutations of specific amino acid residues in GA2oxA9.

Adjuvant therapies in venous leg ulcer management: A scoping review.

Compression therapy is the current evidence-based approach to manage venous leg ulcers (VLU); however, adherence is a major barrier to successful treatment. Combination approaches may relieve the burden of treatment by shortening the time to ulcer healing. This scoping review conducted by Australian researchers aimed to establish the evidence of effectiveness of various adjuvant methods on wound healing and recurrence. Randomized Controlled Trials (RCTs), and Systematic Reviews (SR) and Meta-Analyses (MA) on VLU management approaches published from January 2015 to December 2018 were included in this review. The articles included in the scoping review were grouped according to the management approaches, including (1) pharmaceutical interventions, (2) surgical interventions, (3) topical agents, (4) the use of devices, and (5) other, such as physiotherapy and psychological interventions. Results of this scoping review indicate that there is a limited high-quality evidence of effectiveness in most adjuvant therapies on wound healing and recurrence. Given the low-quality evidence observed in this scoping review for adjuvant treatments, the implication for practice is that current management guidelines be followed. Further rigorous studies have the potential to produce better quality evidence. Quality of evidence can be improved by ensuring large sample sizes of a single etiology wounds, standardizing reporting outcomes, and maintaining detailed and evidence-based protocols in physiological or psychological interventions.

Differential coupling of gibberellin responses by Rht-B1c suppressor alleles and Rht-B1b in wheat highlights a unique role for the DELLA N-terminus in dormancy.

During the Green Revolution, substantial increases in wheat (Triticum aestivum) yields were realized, at least in part, through the introduction of the Reduced height (Rht)-B1b and Rht-D1b semi-dwarfing alleles. In contrast to Rht-B1b and Rht-D1b, the Rht-B1c allele is characterized by extreme dwarfism and exceptionally strong dormancy. Recently, 35 intragenic Rht-B1c suppressor alleles were created in the spring wheat cultivar Maringá, and termed overgrowth (ovg) alleles. Here, 14 ovg alleles with agronomically relevant plant heights were reproducibly classified into nine tall and five semi-dwarf alleles. These alleles differentially affected grain dormancy, internode elongation rate, and coleoptile and leaf lengths. The stability of these ovg effects was demonstrated for three ovg alleles in different genetic backgrounds and environments. Importantly, two semi-dwarf ovg alleles increased dormancy, which correlated with improved pre-harvest sprouting (PHS) resistance. Since no negative effects on grain yield or quality were observed, these semi-dwarf ovg alleles are valuable for breeding to achieve adequate height reduction and protection of grain quality in regions prone to PHS. Furthermore, this research highlights a unique role for the first 70 amino acids of the DELLA protein, encoded by the Rht-1 genes, in grain dormancy.

EARLY FLOWERING3 Regulates Flowering in Spring Barley by Mediating Gibberellin Production and FLOWERING LOCUS T Expression.

EARLY FLOWERING3 (ELF3) is a circadian clock gene that contributes to photoperiod-dependent flowering in plants, with loss-of-function mutants in barley (Hordeum vulgare), legumes, and Arabidopsis thaliana flowering early under noninductive short-day (SD) photoperiods. The barley elf3 mutant displays increased expression of FLOWERING LOCUS T1 (FT1); however, it remains unclear whether this is the only factor responsible for the early flowering phenotype. We show that the early flowering and vegetative growth phenotypes of the barley elf3 mutant are strongly dependent on gibberellin (GA) biosynthesis. Expression of the central GA biosynthesis gene, GA20oxidase2, and production of the bioactive GA, GA1, were significantly increased in elf3 leaves under SDs, relative to the wild type. Inhibition of GA biosynthesis suppressed the early flowering of elf3 under SDs independently of FT1 and was associated with altered expression of floral identity genes at the developing apex. GA is also required for normal flowering of spring barley under inductive photoperiods, with chemical and genetic attenuation of the GA biosynthesis and signaling pathways suppressing inflorescence development under long-day conditions. These findings illustrate that GA is an important floral promoting signal in barley and that ELF3 suppresses flowering under noninductive photoperiods by blocking GA production and FT1 expression.

The use of SNP hybridisation arrays and cytogenetics to characterise deletions of chromosome 4B in hexaploid wheat (Triticum aestivum L.).

KEY MESSAGE: Many deletions of the wheat Della ( Rht - B1 ) gene and its flanking regions were isolated in a simple phenotypic screen, and characterised by modified analysis of SNP hybridisation data and cytogenetics. In a dwarf wheat suppressor screen, many tall 'revertants' were isolated following mutagenesis of a severely dwarfed (Rht-B1c) hexaploid wheat. About 150 lines were identified as putative deletions of Rht-B1c, based on the PCR analysis. Southern blot hybridisation established that most of them lacked the Rht-B1 gene, but retained the homoeologues Rht-A1 and Rht-D1. PCR assays were developed for orthologues of two genes that flank Rht-1/Della in the genomes of the model species Brachypodium and rice. Deletion of the B-genome-specific homoeologues of these two genes was confirmed in the Rht-B1 deletion lines, indicating loss of more than a single gene. SNP chip hybridisation analysis established the extents of deletion in these lines. Based on the synteny with Brachypodium chromosomes 1 and 4 g, and rice chromosomes 3g and 11g, notional deletion maps were established. The deletions ranged from interstitial deletions of 4BS through to loss of all 4BS markers. There were also instances, where all 4BS and 4BL markers were lost, and these lines had poor fertility and narrow stems and leaves. Cytogenetic studies on selected lines confirmed the loss of portions of 4BS in lines that lacked most or all 4BS markers. They also confirmed that lines lacking both 4BS and 4BL markers were nullisomics for 4B. These nested deletion lines share a common genetic background and will have applications in assigning markers to regions of 4BS as well as to 4BL. The potential for this type of analysis in other regions of the wheat genome is discussed.

New species and other new records of the family Mycetophilidae (Insecta, Diptera) from Morocco.

Twelve species in nine genera of Mycetophilidae are newly recorded from Morocco and from North Africa. Five species are described as new to science: Rymosiaebejerisp. nov., Leiaarcanasp. nov., Megophthalmidiaamsemlilisp. nov., Mycomyamirasp. nov., and Phthiniasnibbypinsaesp. nov. Three species are newly recorded from Gibraltar.

Inhibition of tiller bud outgrowth in the tin mutant of wheat is associated with precocious internode development.

Tillering (branching) is a major yield component and, therefore, a target for improving the yield of crops. However, tillering is regulated by complex interactions of endogenous and environmental signals, and the knowledge required to achieve optimal tiller number through genetic and agronomic means is still lacking. Regulatory mechanisms may be revealed through physiological and molecular characterization of naturally occurring and induced tillering mutants in the major crops. Here we characterize a reduced tillering (tin, for tiller inhibition) mutant of wheat (Triticum aestivum). The reduced tillering in tin is due to early cessation of tiller bud outgrowth during the transition of the shoot apex from the vegetative to the reproductive stage. There was no observed difference in the development of the main stem shoot apex between tin and the wild type. However, tin initiated internode development earlier and, unlike the wild type, the basal internodes in tin were solid rather than hollow. We hypothesize that tin represents a novel type of reduced tillering mutant associated with precocious internode elongation that diverts sucrose (Suc) away from developing tillers. Consistent with this hypothesis, we have observed upregulation of a gene induced by Suc starvation, downregulation of a Suc-inducible gene, and a reduced Suc content in dormant tin buds. The increased expression of the wheat Dormancy-associated (DRM1-like) and Teosinte Branched1 (TB1-like) genes and the reduced expression of cell cycle genes also indicate bud dormancy in tin. These results highlight the significance of Suc in shoot branching and the possibility of optimizing tillering by manipulating the timing of internode elongation.

'Overgrowth' mutants in barley and wheat: new alleles and phenotypes of the 'Green Revolution' DELLA gene.

A suppressor screen using dwarf mutants of barley (Hordeum vulgare L.) led to the isolation of 'overgrowth' derivatives, which retained the original dwarfing gene but grew at a faster rate because of a new mutation. The new mutations were in the Slender1 (Sln1) gene (11/13 cases), which encodes the DELLA protein central to gibberellin (GA) signalling, showed 100% genetic linkage to Sln1 (1/13), or were in the Spindly1 (Spy1) gene (1/13), which encodes another protein involved in GA signalling. The overgrowth mutants were characterized by increased GA signalling, although the extent still depended on the background GA biosynthesis capacity, GA receptor function, and DELLA activity. A comparison between two GA responses, α-amylase production and leaf growth rate, revealed degrees of specificity for both the overgrowth allele and the GA response under consideration. Many overgrowth mutants were also isolated in a dwarf line of bread wheat (Triticum aestivum L.) and 19 new alleles were identified in the Rht-B1 gene, one of the 'Green Revolution' semi-dwarfing genes and the orthologue of Sln1. The sites of amino acid substitutions in the DELLA proteins of both species provide insight into DELLA function, and included examples where identical but independent substitutions were observed. In both species, the starting lines were too dwarfed to be directly useful in breeding programmes, but new overgrowth derivatives with semidwarf heights have now been characterized. The variation they exhibit in GA-influenced traits identifies novel alleles with perfect markers that are of potential use in breeding.

Molecular characterization of Rht-1 dwarfing genes in hexaploid wheat.

The introduction of the Reduced height (Rht)-B1b and Rht-D1b semidwarfing genes led to impressive increases in wheat (Triticum aestivum) yields during the Green Revolution. The reduction in stem elongation in varieties containing these alleles is caused by a limited response to the phytohormone gibberellin (GA), resulting in improved resistance to stem lodging and yield benefits through an increase in grain number. Rht-B1 and Rht-D1 encode DELLA proteins, which act to repress GA-responsive growth, and their mutant alleles Rht-B1b and Rht-D1b are thought to confer dwarfism by producing more active forms of these growth repressors. While no semidwarfing alleles of Rht-A1 have been identified, we show that this gene is expressed at comparable levels to the other homeologs and represents a potential target for producing novel dwarfing alleles. In this study, we have characterized additional dwarfing mutations in Rht-B1 and Rht-D1. We show that the severe dwarfism conferred by Rht-B1c is caused by an intragenic insertion, which results in an in-frame 90-bp insertion in the transcript and a predicted 30-amino acid insertion within the highly conserved amino-terminal DELLA domain. In contrast, the extreme dwarfism of Rht-D1c is due to overexpression of the semidwarfing Rht-D1b allele, caused by an increase in gene copy number. We show also that the semidwarfing alleles Rht-B1d and Rht-B1e introduce premature stop codons within the amino-terminal coding region. Yeast two-hybrid assays indicate that these newly characterized mutations in Rht-B1 and Rht-D1 confer "GA-insensitive" dwarfism by producing DELLA proteins that do not bind the GA receptor GA INSENSITIVE DWARF1, potentially compromising their targeted degradation.

Further interceptions of the Neotropical fungus gnat Sciophilafractinervis Edwards, 1940 (Diptera, Mycetophilidae) in Britain with comments and observations on its biology and spread.

From 2020 onwards, several specimens of the Neotropical fungus gnat Sciophilafractinervis (Edwards, 1940) have been intercepted by Fera Science Ltd. on or near plant material in the United Kingdom originating from nurseries and glasshouses at four separate locations: Preston, Lancashire; Chichester, West Sussex; East Riding of Yorkshire; and Middlesbrough, North Yorkshire, as well as a single interception from the Netherlands. Gnat interceptions were associated with a wide range of plant species: Ficusbenjamina, Ficuselastica, Dracaenareflexavar.angustifolia, Origanumvulgare, Rosmarinusofficinalis, Thymusvulgaris, Impatienshawkeri (Impatiens New Guinea hybrids), Chrysanthemum, as well as Fragariavesca (var. Lusa). The species does not appear to be doing any damage to affected plants with growing conditions likely promoting multiplication under protection. The larvae likely feed on spores (generally saprophytic) adhering to webs they erect on the soil and around the base of plants with spores likely originating from the growing medium and plants. Their spread is likely facilitated by movement of the growing medium or plant material where pupae are suspended in the lower stem or leaf axils. An account of the various interceptions is provided, as well as images of the different life-stages with a brief discussion of Sciophilacincticornis Edwards, 1940, its relationship with Sciophilafractinervis and further evidence of instability in vein R2+3 in the Sciophila genus.

New faunistic records of the families Bolitophilidae and Keroplatidae (Diptera, Sciaroidea) from Morocco.

The family Bolitophilidae is recorded for the first time from Morocco with one species Bolitophila (Bolitophila) saundersii (Curtis, 1836). Ten new species are added to the Moroccan fauna of Keroplatidae, known until now by only two species, raising the number of species currently known in Morocco to 12.

Polymer End Group Control through a Decarboxylative Cobalt-Mediated Radical Polymerization: New Avenues for Synthesizing Peptide, Protein, and Nanomaterial Conjugates.

Cobalt-mediated radical polymerizations (CMRPs) have been initiated by the radical decarboxylation of tetrachlorophthalimide activated esters. This allows for the controlled radical polymerization of activated monomers across a broad temperature range with a single cobalt species, with the incorporation of polymer end groups derived from simple carboxylic acid derivatives and termination with an organozinc reagent. This method has been applied to the synthesis of a polymer/graphene conjugate and a water-soluble protein/polymer conjugate, demonstrating the first examples of CMRP in graphene and protein conjugation.

Catalogue of the Diptera (Insecta) of Morocco- an annotated checklist, with distributions and a bibliography.

The faunistic knowledge of the Diptera of Morocco recorded from 1787 to 2021 is summarized and updated in this first catalogue of Moroccan Diptera species. A total of 3057 species, classified into 948 genera and 93 families (21 Nematocera and 72 Brachycera), are listed. Taxa (superfamily, family, genus and species) have been updated according to current interpretations, based on reviews in the literature, the expertise of authors and contributors, and recently conducted fieldwork. Data to compile this catalogue were primarily gathered from the literature. In total, 1225 references were consulted and some information was also obtained from online databases. Each family was reviewed and the checklist updated by the respective taxon expert(s), including the number of species that can be expected for that family in Morocco. For each valid species, synonyms known to have been used for published records from Morocco are listed under the currently accepted name. Where available, distribution within Morocco is also included. One new combination is proposed: Assuaniamelanoleuca (Séguy, 1941), comb. nov. (Chloropidae).

Zooplankton variability in the Strait of Georgia, Canada, and relationships with the marine survivals of Chinook and Coho salmon.

The Strait of Georgia, Canada, has complex interactions among natural and human pressures that confound understanding of changes in this system. We report on the interannual variability in biomass of 12 zooplankton taxonomic groups in the deep (bottom depths greater than 50 m) central and northern Strait of Georgia from 1996 to 2018, and their relationships with 10 physical variables. Total zooplankton biomass was dominated (76%) by large-sized crustaceans (euphausiids, large and medium size calanoid copepods, amphipods). The annual anomaly of total zooplankton biomass was highest in the late 1990s, lowest in the mid-2000s, and generally above its climatological (1996-2010) average after 2011, although many individual groups had different patterns. Two latent trends (derived from dynamic factor analyses) described the variability of annual biomass anomalies underlying all zooplankton groups: a U-shaped trend with its minimum in the mid-2000s, and a declining trend from 2001 to 2011. Two latent trends also described the physical variables. The variability represented by these four latent trends clustered into two periods: 1996-2006, with generally declining zooplankton biomass and increasing salinities, and 2007-2018, with increasing zooplankton biomass and decreasing salinities. ARIMA modelling showed sea surface salinity at Entrance Island in the middle Strait of Georgia, the Pacific Decadal Oscillation, and the peak date of the spring phytoplankton bloom were significantly related to the two latent zooplankton trends. ARIMA models comparing zooplankton and physical variables with the marine survivals of four salmon populations which enter the Strait as juveniles (Chinook: Cowichan River, Puntledge River, Harrison River; Coho: Big Qualicum River) all included zooplankton groups consistent with known salmon prey; prominent among the physical variables were sea surface salinity and variables representing the flow from the Fraser River. These regressions explained (adjR2) 38 to 85% of the annual variability in marine survival rates of these salmon populations over the study time period. Although sea temperature was important in some relationships between zooplankton biomass and salmon marine survival, salinity was a more frequent and more important variable, consistent with its influence on the hydrodynamics of the Strait of Georgia system.

Manipulation of plant innate immunity and gibberellin as factor of compatibility in the mutualistic association of barley roots with Piriformospora indica.

Fungi of the order Sebacinales (Basidiomycota) are involved in a wide spectrum of mutualistic symbioses with various plants, thereby exhibiting unique potential for biocontrol strategies. Piriformospora indica, a model organism of this fungal order, is able to increase the biomass and grain yield of crop plants, and induces local and systemic resistance to fungal diseases and tolerance to abiotic stress. To elucidate the molecular basis for root colonization, we characterized the interaction of P. indica with barley roots by combining global gene expression profiling, metabolic profiling, and genetic studies. At the metabolic level, we show that fungal colonization reduces the availability of free sugars and amino acids to the root tip. At the transcriptional level, consecutive interaction stages covering pre-penetration-associated events and progressing through to root colonization showed differential regulation of signal perception and transduction components, secondary metabolism, and genes associated with membrane transport. Moreover, we observed stage-specific up-regulation of genes involved in phytohormone metabolism, mainly encompassing gibberellin, auxin and abscisic acid, but salicylic acid-associated gene expression was suppressed. The changes in hormone homoeostasis were accompanied with a general suppression of the plant innate immune system. Further genetic studies showed reduced fungal colonization in mutants that are impaired in gibberellin synthesis as well as perception, and implicate gibberellin as a modulator of the root's basal defence. Our data further reveal the complexity of compatibility mechanisms in host-microbe interactions, and identify gibberellin signaling as potential target for successful fungi.

Fine mapping and syntenic integration of the semi-dwarfing gene sdw3 of barley.

The barley mutant allele sdw3 confers a gibberellin-insensitive, semi-dwarf phenotype with potential for breeding of new semi-dwarfed barley cultivars. Towards map-based cloning, sdw3 was delimited by high-resolution genetic mapping to a 0.04 cM interval in a "cold spot" of recombination of the proximal region of the short arm of barley chromosome 2H. Extensive synteny between the barley Sdw3 locus (Hvu_sdw3) and the orthologous regions (Osa_sdw3, Sbi_sdw3, Bsy_sdw3) of three other grass species (Oryza sativa, Sorghum bicolor, Brachypodium sylvaticum) allowed for efficient synteny-based marker saturation in the target interval. Comparative sequence analysis revealed colinearity for 23 out of the 38, 35, and 29 genes identified in Brachypodium, rice, and Sorghum, respectively. Markers co-segregating with Hvu_sdw3 were generated from two of these genes. Initial attempts at chromosome walking in barley were performed with seven orthologous gene probes which were delimiting physical distances of 223, 123, and 127 kb in Brachypodium, rice, and Sorghum, respectively. Six non-overlapping small bacterial artificial chromosome (BAC) clone contigs (cumulative length of 670 kb) were obtained, which indicated a considerably larger physical size of Hvu_sdw3. Low-pass sequencing of selected BAC clones from these barley contigs exhibited a substantially lower gene frequency per physical distance and the presence of additional non-colinear genes. Four candidate genes for sdw3 were identified within barley BAC sequences that either co-segregated with the gene sdw3 or were located adjacent to these co-segregating genes. Identification of genic sequences in the sdw3 context provides tools for marker-assisted selection. Eventual identification of the actual gene will contribute new information for a basic understanding of the mechanisms underlying growth regulation in barley.

Development and Differentiation in Monobodies Based on the Fibronectin Type 3 Domain.

As a non-antibody scaffold, monobodies based on the fibronectin type III (FN3) domain overcome antibody size and complexity while maintaining analogous binding loops. However, antibodies and their derivatives remain the gold standard for the design of new therapeutics. In response, clinical-stage therapeutic proteins based on the FN3 domain are beginning to use native fibronectin function as a point of differentiation. The small and simple structure of monomeric monobodies confers increased tissue distribution and reduced half-life, whilst the absence of disulphide bonds improves stability in cytosolic environments. Where multi-specificity is challenging with an antibody format that is prone to mis-pairing between chains, multiple FN3 domains in the fibronectin assembly already interact with a large number of molecules. As such, multiple monobodies engineered for interaction with therapeutic targets are being combined in a similar beads-on-a-string assembly which improves both efficacy and pharmacokinetics. Furthermore, full length fibronectin is able to fold into multiple conformations as part of its natural function and a greater understanding of how mechanical forces allow for the transition between states will lead to advanced applications that truly differentiate the FN3 domain as a therapeutic scaffold.

New faunistic records of the family Mycetophilidae (Insecta, Diptera) from Morocco.

A total of 54 species of Mycetophilidae are recorded for the first time in Morocco, of which 38 species are new to North Africa. A first checklist of Moroccan Mycetophilidae is appended, containing 64 species in 25 genera.