BdGT43B2 functions in xylan biosynthesis and is essential for seedling survival in Brachypodium distachyon.

Xylan is the predominant hemicellulose in the primary cell walls of grasses, but its synthesis and interactions with other wall polysaccharides are complex and incompletely understood. To probe xylan biosynthesis, we generated CRISPR/Cas9 knockout and amiRNA knockdown lines of BdGT43B2, an ortholog of the wheat TaGT43-4 xylan synthase scaffolding protein in the IRX14 clade, in Brachypodium distachyon. Knockout of BdGT43B2 caused stunting and premature death in Brachypodium seedlings. Immunofluorescence labeling of xylans was greatly reduced in homozygous knockout BdGT43B2 mutants, whereas cellulose labeling was unchanged or slightly increased. Biochemical analysis showed reductions in digestible xylan in knockout mutant walls, and cell size was smaller in knockout leaves. BdGT43B2 knockdown plants appeared morphologically normal as adults, but showed slight reductions in seedling growth and small decreases in xylose content in isolated cell walls. Immunofluorescence labeling of xylan and cellulose staining was both reduced in BdGT43B2 knockdown plants. Together, these data indicate that BdGT43B2 functions in the synthesis of a form of xylan that is required for seedling growth and survival in Brachypodium distachyon

Persistent Growth of a Human Plasma-Derived Hepatitis C Virus Genotype 1b Isolate in Cell Culture

HCV (hepatitis C virus) research, including therapeutics and vaccine development, has been hampered by the lack of suitable tissue culture models. Development of cell culture systems for the growth of the most drug-resistant HCV genotype (1b) as well as natural isolates has remained a challenge. Transfection of cultured cells with adenovirus-associated RNAI (VA RNAI), a known interferon (IFN) antagonist and inhibitor of dsRNA-mediated antiviral pathways, enhanced the growth of plasma-derived HCV genotype 1b. Furthermore, persistent viral growth was achieved after passaging through IFN-α/β-deficient VeroE6 cells for 2 years. Persistently infected cells were maintained in culture for an additional 4 years, and the virus rescued from these cells induced strong cytopathic effect (CPE). Using a CPE-based assay, we measured inhibition of viral production by anti-HCV specific inhibitors, including 2′-C-Methyl-D-Adenosine, demonstrating its utility for the evaluation of HCV antivirals. This virus constitutes a novel tool for the study of one of the most relevant strains of HCV, genotype 1b, which will now be available for HCV life cycle research and useful for the development of new therapeutics

BdCESA7, BdCESA8, and BdPMT Utility Promoter Constructs for Targeted Expression to Secondary Cell-Wall-Forming Cells of Grasses.

Utility vectors with promoters that confer desired spatial and temporal expression patterns are useful tools for studying gene and cellular function and for industrial applications. To target the expression of DNA sequences of interest to cells forming plant secondary cell walls, which generate most of the vegetative biomass, upstream regulatory sequences of the Brachypodium distachyon lignin biosynthetic gene BdPMT and the cellulose synthase genes BdCESA7 and BdCESA8 were isolated and cloned into binary vectors designed for Agrobacterium-mediated transformation of monocots. Expression patterns were assessed using the β-glucuronidase gene GUSPlus and X-glucuronide staining. All three promoters showed strong expression levels in stem tissue at the base of internodes where cell wall deposition is most active, in both vascular bundle xylem vessels and tracheids, and in interfascicular tissues, with expression less pronounced in developmentally older tissues. In leaves, BdCESA7 and BdCESA8 promoter-driven expression was strongest in leaf veins, leaf margins, and trichomes; relatively weaker and patchy expression was observed in the epidermis. BdPMT promoter-driven expression was similar to the BdCESA promoters expression patterns, including strong expression in trichomes. The intensity and extent of GUS staining varied considerably between transgenic lines, suggesting that positional effects influenced promoter activity. Introducing the BdPMT and BdCESA8 Open Reading Frames into BdPMT and BdCESA8 utility promoter binary vectors, respectively, and transforming those constructs into Brachypodium pmt and cesa8 loss-of-function mutants resulted in rescue of the corresponding mutant phenotypes. This work therefore validates the functionality of these utility promoter binary vectors for use in Brachypodium and likely other grass species. The identification, in Bdcesa8-1 T-DNA mutant stems, of an 80% reduction in crystalline cellulose levels confirms that the BdCESA8 gene is a secondary-cell-wall-forming cellulose synthase

Misregulation of p-coumaroyl-CoA:monolignol transferase in Brachypodium distachyon

International audienc

p-Coumaroyl-CoA:monolignol transferase (PMT) acts specifically in the lignin biosynthetic pathway in Brachypodium distachyon

Grass lignins contain substantial amounts of p-coumarate (pCA) that acylate the side-chains of the phenylpropanoid polymer backbone. An acyltransferase, named p-coumaroyl-CoA:monolignol transferase (OsPMT), that could acylate monolignols with pCA in vitro was recently identified from rice. In planta, such monolignol-pCA conjugates become incorporated into lignin via oxidative radical coupling, thereby generating the observed pCA appendages; however p-coumarates also acylate arabinoxylans in grasses. To test the authenticity of PMT as a lignin biosynthetic pathway enzyme, we examined Brachypodium distachyon plants with altered BdPMT gene function. Using newly developed cell wall analytical methods, we determined that the transferase was involved specifically in monolignol acylation. A sodium azide-generated Bdpmt-1 missense mutant had no (<0.5%) residual pCA on lignin, and BdPMT RNAi plants had levels as low as 10% of wild-type, whereas the amounts of pCA acylating arabinosyl units on arabinoxylans in these PMT mutant plants remained unchanged. pCA acylation of lignin from BdPMT-overexpressing plants was found to be more than three-fold higher than that of wild-type, but again the level on arabinosyl units remained unchanged. Taken together, these data are consistent with a defined role for grass PMT genes in encoding BAHD (BEAT, AHCT, HCBT, and DAT) acyltransferases that specifically acylate monolignols with pCA and produce monolignol p-coumarate conjugates that are used for lignification in planta

The Cancer Financial Experience (CAFÉ) study: randomized controlled trial of a financial navigation intervention to address cancer-related financial hardship.

BackgroundThere is an urgent need for evidence on how interventions can prevent or mitigate cancer-related financial hardship. Our objectives are to compare self-reported financial hardship, quality of life, and health services use between patients receiving a financial navigation intervention versus a comparison group at 12 months follow-up, and to assess patient-level factors associated with dose received of a financial navigation intervention.MethodsThe Cancer Financial Experience (CAFÉ) study is a multi-site randomized controlled trial (RCT) with individual-level randomization. Participants will be offered either brief (one financial navigation cycle, Arm 2) or extended (three financial navigation cycles, Arm 3) financial navigation. The intervention period for both Arms 2 and 3 is 6 months. The comparison group (Arm 1) will receive enhanced usual care. The setting for the CAFÉ study is the medical oncology and radiation oncology clinics at two integrated health systems in the Pacific Northwest. Inclusion criteria includes age 18 or older with a recent cancer diagnosis and visit to a study clinic as identified through administrative data. Outcomes will be assessed at 12-month follow-up. Primary outcomes are self-reported financial distress and health-related quality of life. Secondary outcomes are delayed or foregone care; receipt of medical financial assistance; and account delinquency. A mixed methods exploratory analysis will investigate factors associated with total intervention dose received.DiscussionThe CAFÉ study will provide much-needed early trial evidence on the impact of financial navigation in reducing cancer-related financial hardship. It is theory-informed, clinic-based, aligned with patient preferences, and has been developed following preliminary qualitative studies and stakeholder input. By design, it will provide prospective evidence on the potential benefits of financial navigation on patient-relevant cancer outcomes. The CAFÉ trial's strengths include its broad inclusion criteria, its equity-focused sampling plan, its novel intervention developed in partnership with clinical and operations stakeholders, and mixed methods secondary analyses related to intervention dose offered and dose received. The resulting analytic dataset will allow for rich mixed methods analysis and provide critical information related to implementation of the intervention should it prove effective.Trial registrationClinicalTrials.gov NCT05018000 .&nbsp;August 23, 2021