A point mutation in the kinase domain of CRK10 leads to xylem vessel collapse and activation of defence responses in Arabidopsis.

Cysteine-rich receptor-like kinases (CRKs) are a large family of plasma membrane-bound receptors ubiquitous in higher plants. However, despite their prominence, their biological roles have remained largely elusive so far. In this study we report the characterization of an Arabidopsis mutant named crk10-A397T in which alanine 397 has been replaced by a threonine in the αC helix of the kinase domain of CRK10, known to be a crucial regulatory module in mammalian kinases. The crk10-A397T mutant is a dwarf that displays collapsed xylem vessels in the root and hypocotyl, whereas the vasculature of the inflorescence develops normally. In situ phosphorylation assays with His-tagged wild type and crk10-A397T versions of the CRK10 kinase domain revealed that both alleles are active kinases capable of autophosphorylation, with the newly introduced threonine acting as an additional phosphorylation site in crk10-A397T. Transcriptomic analysis of wild type and crk10-A397T mutant hypocotyls revealed that biotic and abiotic stress-responsive genes are constitutively up-regulated in the mutant, and a root-infection assay with the vascular pathogen Fusarium oxysporum demonstrated that the mutant has enhanced resistance to this pathogen compared with wild type plants. Taken together our results suggest that crk10-A397T is a gain-of-function allele of CRK10, the first such mutant to have been identified for a CRK in Arabidopsis.

Novel Thermal Imaging Method for Rapid Screening of Drug-Polymer Miscibility for Solid Dispersion Based Formulation Development.

This study aimed to develop a rapid, simple, and inexpensive screening method for selecting the best polymeric candidates possessing high active pharmaceutical ingredient (API) miscibility during the early stages of formulation development of solid dispersion based pharmaceutical products. A new thermal imaging based method, thermal analysis by structural characterization (TASC), was used as a thermoptometric tool in conjunction with data analysis software to detect the melting point depression and postmelting dissolution of felodipine particles screened over thin spin-coated films of ten polymers commonly used in the pharmaceutical field. On the polymeric substrates the drug showed different degrees of melting point reduction, reflecting their different levels of polymer-drug miscibility. Using TASC to detect melting point depression is significantly (20-40 times) faster than the conventional DSC method without loss of the sensitivity of detection. The quantity of the material required for the screening is less than 1/1000th of the material used in conventional DSC tests, which significantly reduce the material wastage. Isothermal TASC tests and IR imaging confirmed the occurrence of thermal dissolution of the drug in the polymer for more miscible pairs. The real-time stability tests validate the accuracy of the polymer-drug miscibility screening results. These results demonstrate TASC as a promising screening tool for rapidly selecting the polymeric excipients for pharmaceutical formulations development.

Release of cell wall phenolic esters during hydrothermal pretreatment of rice husk and rice straw.

BACKGROUND: Rice husk and rice straw represent promising sources of biomass for production of renewable fuels and chemicals. For efficient utilisation, lignocellulosic components must first be pretreated to enable efficient enzymatic saccharification and subsequent fermentation. Existing pretreatments create breakdown products such as sugar-derived furans, and lignin-derived phenolics that inhibit enzymes and fermenting organisms. Alkali pretreatments have also been shown to release significant levels of simple, free phenolics such as ferulic acid that are normally esterified to cell wall polysaccharides in the intact plant. These phenolics have recently been found to have considerable inhibitory properties. The aim of this research has been to establish the extent to which such free phenolic acids are also released during hydrothermal pretreatment of rice straw (RS) and rice husk (RH). RESULTS: RS and RH were subjected to hydrothermal pretreatments over a wide range of severities (1.57-5.45). FTIR analysis showed that the pretreatments hydrolysed and solubilised hemicellulosic moieties, leading to an enrichment of lignin and crystalline cellulose in the insoluble residue. The residues also lost the capacity for UV autofluorescence at pH 7 or pH 10, indicating the breakdown or release of cell wall phenolics. Saponification of raw RS and RH enabled identification and quantification of substantial levels of simple phenolics including ferulic acid (tFA), coumaric acid (pCA) and several diferulic acids (DiFAs) including 8-O-4'-DiFA, 8,5'-DiFA and 5,5'-DiFA. RH had higher levels of pCA and lower levels of tFA and DiFAs compared with RS. Assessment of the pretreatment liquors revealed that pretreatment-liberated phenolics present were not free but remained as phenolic esters (at mM concentrations) that could be readily freed by saponification. Many were lost, presumably through degradation, at the higher severities. CONCLUSION: Differences in lignin, tFA, DiFAs and pCA between RS and RH reflect differences in cell wall physiology, and probably contribute to the higher recalcitrance of RH compared with RS. Hydrothermal pretreatments, unlike alkali pretreatments, release cinnamic acid components as esters. The potential for pretreatment-liberated phenolic esters to be inhibitory to fermenting microorganisms is not known. However, the present study shows that they are found at concentrations that could be significantly inhibitory if released as free forms by enzyme activity.

Development of a Simple Mechanical Screening Method for Predicting the Feedability of a Pharmaceutical FDM 3D Printing Filament.

PURPOSE: The filament-based feeding mechanism employed by the majority of fused deposition modelling (FDM) 3D printers dictates that the materials must have very specific mechanical characteristics. Without a suitable mechanical profile, the filament can cause blockages in the printer. The purpose of this study was to develop a method to screen the mechanical properties of pharmaceutically-relevant, hot-melt extruded filaments to predetermine their suitability for FDM. METHODS: A texture analyzer was used to simulate the forces a filament is subjected to inside the printer. The texture analyzer produced a force-distance curve referred to as the flexibility profile. Principal Component Analysis and Correlation Analysis statistical methods were then used to compare the flexibility profiles of commercial filaments to in-house made filaments. RESULTS: Principal component analysis showed clearly separated clustering of filaments that suffer from mechanical defects versus filaments which are suitable for printing. Correlation scores likewise showed significantly greater values with feedable filaments than their mechanically deficient counterparts. CONCLUSION: The screening method developed in this study showed, with statistical significance and reproducibility, the ability to predetermine the feedability of extruded filaments into an FDM printer.

Dissecting the complex regulation of lodging resistance in Brassica napus.

Lodging continues to be a major cause of yield loss in important crop species such as Brassica napus. Understanding the genetic regulation of lodging resistance is therefore of key interest to breeders worldwide. Current strategies aimed at minimising lodging risk involve the incorporation of dwarfing genes or the application of plant growth regulators. However, despite these efforts, lodging continues to be a persistent problem and it is therefore of high interest that novel, complimentary strategies for lodging control are implemented. One approach would be to focus on understanding the genetic properties underlying stem mechanical strength. With this in mind, we screened a training genetic diversity panel of B. napus accession for variation in stem mechanical strength and related traits. Using Associative Transcriptomics, we identified molecular markers for a suite of valuable traits. Using an independent test genetic diversity panel, we show that the methods employed are robust for identification of predictive markers. Furthermore, based on conserved synteny with Arabidopsis thaliana, we are able to provide a biological context to the marker associations detected and provide evidence for a role in pectin methylesterification in contributing to stem mechanical strength in Brassicaceae.

Feedstock selection for polymer and chemical production: feedstock-specific recalcitrance.

Plant cell wall materials derived from a range of waste biomass sources have great potential as a source of sustainable alternatives to petrochemicals. Perhaps the most straightforward way of realising this potential would be to hydrolyse the most efficiently fermentable polymers into their constituent sugars and use yeast to ferment these into useful chemicals. However, it also makes sense to pre-extract components which have a greater value in polymeric form. This is particularly true for non-cellulosic polymers, which are rich in poorly-fermentable pentose sugars. Liquid hot water (LHW) pretreatment can be used to extract non-cellulosic carbohydrates in a cost-effective manner, leaving a cellulose-rich substrate which is easier to hydrolyse using commercial cellulases. However, inherent differences in the plant cell wall structure and composition mean that some biomass sources may be more suitable for exploitation than others. Here, we examine eight different feedstocks (two each from hardwood, softwood, cereal straws and dicotyledonous crops), expose them to 26 different LHW pretreatment conditions and hydrolyse the entire pretreated slurry with a commercial cellulase. This enables side-by-side comparisons, in terms of saccharification yield, of the feedstocks. The results clearly demonstrate considerable differences in suitability between the feedstocks, in relation to the quantity of products released and the processes needed to obtain them.

A rapid-screening approach to detect and quantify microplastics based on fluorescent tagging with Nile Red.

A new approach is presented for analysis of microplastics in environmental samples, based on selective fluorescent staining using Nile Red (NR), followed by density-based extraction and filtration. The dye adsorbs onto plastic surfaces and renders them fluorescent when irradiated with blue light. Fluorescence emission is detected using simple photography through an orange filter. Image-analysis allows fluorescent particles to be identified and counted. Magnified images can be recorded and tiled to cover the whole filter area, allowing particles down to a few micrometres to be detected. The solvatochromic nature of Nile Red also offers the possibility of plastic categorisation based on surface polarity characteristics of identified particles. This article details the development of this staining method and its initial cross-validation by comparison with infrared (IR) microscopy. Microplastics of different sizes could be detected and counted in marine sediment samples. The fluorescence staining identified the same particles as those found by scanning a filter area with IR-microscopy.

CX3CR1+ Cell-Mediated Salmonella Exclusion Protects the Intestinal Mucosa during the Initial Stage of Infection.

During Salmonella Typhimurium infection, intestinal CX3CR1+ cells can either extend transepithelial cellular processes to sample luminal bacteria or, very early after infection, migrate into the intestinal lumen to capture bacteria. However, until now, the biological relevance of the intraluminal migration of CX3CR1+ cells remained to be determined. We addressed this by using a combination of mouse strains differing in their ability to carry out CX3CR1-mediated sampling and intraluminal migration. We observed that the number of S. Typhimurium traversing the epithelium did not differ between sampling-competent/migration-competent C57BL/6 and sampling-deficient/migration-competent BALB/c mice. In contrast, in sampling-deficient/migration-deficient CX3CR1-/- mice the numbers of S. Typhimurium penetrating the epithelium were significantly higher. However, in these mice the number of invading S. Typhimurium was significantly reduced after the adoptive transfer of CX3CR1+ cells directly into the intestinal lumen, consistent with intraluminal CX3CR1+ cells preventing S. Typhimurium from infecting the host. This interpretation was also supported by a higher bacterial fecal load in CX3CR1+/gfp compared with CX3CR1gfp/gfp mice following oral infection. Furthermore, by using real-time in vivo imaging we observed that CX3CR1+ cells migrated into the lumen moving through paracellular channels within the epithelium. Also, we reported that the absence of CX3CR1-mediated sampling did not affect Ab responses to a noninvasive S. Typhimurium strain that specifically targeted the CX3CR1-mediated entry route. These data showed that the rapidly deployed CX3CR1+ cell-based mechanism of immune exclusion is a defense mechanism against pathogens that complements the mucous and secretory IgA Ab-mediated system in the protection of intestinal mucosal surface.

Pectin extraction from pomegranate peels with citric acid.

Pectins were extracted from pomegranate peels with citric acid, according to a central composite design with three variables: pH (2-4), temperature (70-90°C), and extraction time (40-150min). Fourier transform infrared (FTIR) spectroscopy was used to follow changes in material composition during the main steps of pectin extraction, and also to determine the degree of methyl esterification and galacturonic acid content of pectins produced under different conditions. Harsh conditions enhanced the extraction yield and the galacturonic acid contents, but decreased the degree of methoxylation. The optimum extraction conditions, defined as those predicted to result in a yield of galacturonic acid higher than 8g/100g while keeping a minimum degree of methoxylation of 54% were: 88°C, 120min, pH 2.5. Close agreement was found between experimental and predicted values at the extraction conditions defined as optimum.

Development of pectin films with pomegranate juice and citric acid.

The influence of pomegranate juice (PJ, replacing water as solvent) and citric acid (CA) on properties of pectin films was studied. PJ provided the films with a bright red color, and acted as a plasticizer. Increasing PJ/water ratio from 0/100 to 100/0 resulted in enhanced elongation (from 2% to 20%), decreased strength (from 10 to <2 MPa) and modulus (from 93 to <10 MPa), increased water vapor permeability (WVP, from 3 to 9 g.mm.kPa(-1).h(-1).m(-2)), and decreased insoluble matter (IM, from 35% to 24%). Although a crosslinking effect by CA was not confirmed, it has been suggested to occur from its effects on films. CA noticeably increased IM (from <10% to almost 40%); moreover, when measured on a dry film basis, the CA effects presented a noticeable tendency to increases strength and modulus, and to decrease WVP. The red color density was decreased by CA, suggesting a destabilization of anthocyanins.