Coat Protein Research Articles

Cowpea lipid transfer protein 1 regulates plant defense by inhibiting the cysteine protease of cowpea mosaic virus

Many virus genomes encode proteases that facilitate infection. The molecular mechanism of plant recognition of viral proteases is largely unexplored. Using the system of Vigna unguiculata and cowpea mosaic virus (CPMV), we identified a cowpea lipid transfer protein (LTP1) which interacts with CPMV-encoded 24KPro, a cysteine protease, but not with the enzymatically inactive mutant 24KPro(C166A). Biochemical assays showed that LTP1 inhibited 24KPro proteolytic cleavage of the coat protein precursor large coat protein-small coat protein. Transient overexpression of LTP1 in cowpea reduced CPMV infection, whereas RNA interference-mediated LTP1 silencing increased CPMV accumulation in cowpea. LTP1 is mainly localized in the apoplast of uninfected plant cells, and after CPMV infection, most of the LTP1 is relocated to intracellular compartments, including chloroplast. Moreover, in stable LTP1-transgenic Nicotiana benthamiana plants, LTP1 repressed soybean mosaic virus (SMV) nuclear inclusion a protease activity, and accumulation of SMV was significantly reduced. We propose that cowpea LTP1 suppresses CPMV and SMV accumulation by directly inhibiting viral cysteine protease activity.

Transient Expression Vector Construction, Subcellular Localisation, and Evaluation of Antiviral Potential of Flagellin BP8-2.

This study used the DNA of Bacillus amyloliquefaciens Ba168 as a template to amplify the flagellin BP8-2 gene and ligate it into the fusion expression vector pCAMBIA1300-35S-EGFP after digestion for the construction of the expression vector pCAMBIA1300-EGFP-BP8-2. Next, using Nicotiana benthamiana as receptor material, transient expression was carried out under the mediation of Agrobacterium tumefaciens C58C1. Finally, the transient expression and subcellular localisation of flagellin BP8-2 protein were analysed using the imaging of co-transformed GFP under laser confocal microscopy. The results showed that flagellin BP8-2 was localised in the cell membrane and nucleus, and the RT-PCR results showed that the BP8-2 gene could be stably expressed in tobacco leaf cells. Furthermore, there was stronger antiviral activity against tobacco mosaic virus (TMV) infection in Nicotiana glutinosa than in BP8-2 and ningnanmycin, with an inhibitory effect of 75.91%, protective effect of 77.45%, and curative effect of 68.15%. TMV movement and coat protein expression were suppressed, and there was a high expression of PR-1a, PAL, and NPR1 in BP8-2-treated tobacco leaf. These results suggest that flagellin BP8-2 inhibits TMV by inducing resistance. Moreover, BP8-2 has low toxicity and is easily biodegradable and eco-friendly. These results further enrich our understanding of the antiviral mechanisms of proteins and provide alternatives for controlling viral diseases in agriculture.

Mechanistic divergences of endocytic clathrin-coated vesicle formation in mammals, yeasts and plants.

Clathrin-coated vesicles (CCVs), generated by clathrin-mediated endocytosis (CME), are essential eukaryotic trafficking organelles that transport extracellular and plasma membrane-bound materials into the cell. In this Review, we explore mechanisms of CME in mammals, yeasts and plants, and highlight recent advances in the characterization of endocytosis in plants. Plants separated from mammals and yeast over 1.5 billion years ago, and plant cells have distinct biophysical parameters that can influence CME, such as extreme turgor pressure. Plants can therefore provide a wider perspective on fundamental processes in eukaryotic cells. We compare key mechanisms that drive CCV formation and explore what these mechanisms might reveal about the core principles of endocytosis across the tree of life. Fascinatingly, CME in plants appears to more closely resemble that in mammalian cells than that in yeasts, despite plants being evolutionarily further from mammals than yeast. Endocytic initiation appears to be highly conserved across these three systems, requiring similar protein domains and regulatory processes. Clathrin coat proteins and their honeycomb lattice structures are also highly conserved. However, major differences are found in membrane-bending mechanisms. Unlike in mammals or yeast, plant endocytosis occurs independently of actin, highlighting that mechanistic assumptions about CME across different systems should be made with caution.

NDV targets the invasion pathway in malaria parasite through cell surface sialic acid interaction.

Merozoites utilize sialic acids on the red blood cell (RBC) cell surface to rapidly adhere to and invade the RBCs. Newcastle disease virus (NDV) displays a strong affinity toward membrane-bound sialic acids. Incubation of NDV with the malaria parasites dose-dependently reduces its cellular viability. The antiplasmodial activity of NDV is specific, as incubation with Japanese encephalitis virus, duck enteritis virus, infectious bronchitis virus, and influenza virus did not affect the parasite propagation. Interestingly, NDV is reducing more than 80% invasion when RBCs are pretreated with the virus. Removal of the RBC surface proteins or the NDV coat proteins results in disruption of the virus binding to RBC. It suggests the involvement of specific protein: ligand interaction in virus binding. We established that the virus engages with the parasitized RBCs (PRBCs) through its hemagglutinin neuraminidase (HN) protein by recognizing sialic acid-containing glycoproteins on the cell surface. Blocking of the HN protein with free sialic acid or anti-HN antibodies abolished the virus binding as well as its ability to reduce parasite growth. Interestingly, the purified HN from the virus alone could inhibit the parasite's growth in a dose-dependent manner. NDV binds strongly to knobless murine parasite strain Plasmodium yoelii and restricted the parasite growth in mice. Furthermore, the virus was found to preferentially target the PRBCs compared to normal erythrocytes. Immunolocalization studies reveal that NDV is localized on the plasma membrane as well as weakly inside the PRBC. NDV causes neither any infection nor aggregation of the human RBCs. Our findings suggest that NDV is a potential candidate for developing targeted drug delivery platforms for the Plasmodium-infected RBCs.

Compromised COPII vesicle trafficking leads to glycogenic hepatopathy.

Being a vital cellular process, coat protein complex II (COPII) vesicle trafficking has been found to play a crucial role in liver metabolism. However, its functions and the underlying mechanisms in systemic metabolic homeostasis have not been fully understood. Here, with a newly identified gene trap zebrafish line (sec31anju221), we show that compromised COPII vesicle trafficking leads to biphasic abnormal hepatic metabolism. During the larval stage, deficiency of COPII-mediated trafficking leads to activation of the unfolded protein response and the development of hepatic steatosis. By using epistasis analysis, we found that the eIF2α-ATF4 pathway serves as the primary effector for liver steatosis. In adult sec31anju221 fish, the hepatosteatosis was reversed and the phenotype switched to glycogenic hepatopathy. Proteomic profiling and biochemical assays indicate that sec31anju221 fish are in a state of hypothyroidism. Moreover, our study shows that thyroid hormone treatment alleviates the metabolic defects. This study provides insights into processes of liver diseases associated with vesicle trafficking impairments and expands our understanding of the pathological interplay between thyroid and liver.

Design, Synthesis, and Antiviral and Fungicidal Activities of 4-Oxo-4H-quinolin-1-yl Acylhydrazone Derivatives.

To discover novel antiviral agents, based on the high antiviral activity of (4-oxo-4H-quinolin-1-yl)-acetic acid hydrazide (C), a series of 4-oxo-4H-quinoline acylhydrazone derivatives were designed, synthesized, and first evaluated for their antiviral and fungicidal activities. Most acylhydrazone derivatives exhibited moderate to good antiviral activities in vivo. The inactive, curative, and protective activities of compounds 4 (51.2, 47.6, and 46.3%), 11 (49.6, 43.0, and 45.2% at 500 mg/L), and 17 (47.1, 49.2, and 44.1%) were higher than those of ribavirin (39.2, 38.0, and 40.8%) at 500 mg/L. Molecular docking showed that compound 4 exhibited a stronger affinity to TMV coat protein (TMV-CP) than ribavirin, with a binding energy (-6.89 kcal/mol) slightly lower than that of ribavirin (-6.08 kcal/mol). Microscale thermophoresis showed that compound 4 (K d = 0.142 ± 0.060 μM) exhibited a strong binding ability to TMV-CP, superior to that of ribavirin (K d = 0.512 ± 0.257 μM). The results of transmission electron microscopy showed that compound 4 hindered the self-assembly and growth of TMV. The antifungal activities of most compounds were moderate at 50 mg/L, among which compounds 12 and 21 exhibited a 72.1 and 76.5% inhibitory rate against Physalospora piricola, respectively. Meanwhile, compound 16 exhibited a 60% inhibitory rate against Cercospora arachidicola Hori at 50 mg/L.

Genomic Analysis of a Novel Torradovirus "Rehmannia Torradovirus Virus": Two Distinct Variants Infecting Rehmannia glutinosa.

Rehmannia glutinosa, a crucial medicinal plant native to China, is extensively cultivated across East Asia. We used high-throughput sequencing to identify viruses infecting R. glutinosa with mosaic, leaf yellowing, and necrotic symptoms. A novel Torradovirus, which we tentatively named "Rehmannia torradovirus virus" (ReTV), was identified. The complete sequences were obtained through reverse-transcription polymerase chain reaction (RT-PCR), 5' and 3' rapid amplification of cDNA ends, and Sanger sequencing. The amino acid sequence alignment between the ReTV-52 isolate and known Torradovirus species in the Pro-Pol and coat protein regions were 51.3-73.3% and 37.1-68.1%, respectively. Meanwhile, the amino acid sequence alignment between the ReTV-8 isolate and known Torradovirus species in the Pro-Pol and coat protein regions were 52.7-72.8% and 36.8-67.5%, respectively. The sequence analysis classified ten ReTV strains into two variants. The ReTV-52 genome has two RNA segments of 6939 and 4569 nucleotides, while that of ReTV-8 consists of two RNA segments containing 6889 and 4662 nucleotides. Sequence comparisons and phylogenetic analysis showed ReTV strains clustered within the Torradovirus, exhibiting the closet relation to the squash chlorotic leaf spot virus. The RT-PCR results showed a 100% ReTV detection rate in all 60 R. glutinosa samples. Therefore, ReTV should be classified as a novel Torradovirus species. ReTV is potentially dangerous to R. glutinosa, and necessitating monitoring this virus in the field.

The Influence of Chitosan Derivatives in Combination with Bacillus subtilis Bacteria on the Development of Systemic Resistance in Potato Plants with Viral Infection and Drought.

Viral diseases of potatoes are among the main problems causing deterioration in the quality of tubers and loss of yield. The growth and development of potato plants largely depend on soil moisture. Prevention strategies require comprehensive protection against pathogens and abiotic stresses, including modeling the beneficial microbiome of agroecosystems combining microorganisms and immunostimulants. Chitosan and its derivatives have great potential for use in agricultural engineering due to their ability to induce plant immune responses. The effect of chitosan conjugate with caffeic acid (ChCA) in combination with Bacillus subtilis 47 on the transcriptional activity of PR protein genes and changes in the proteome of potato plants during potato virus Y (PVY) infection and drought was studied. The mechanisms of increasing the resistance of potato plants to PVY and lack of moisture are associated with the activation of transcription of genes encoding PR proteins: the main protective protein (PR-1), chitinase (PR-3), thaumatin-like protein (PR-5), protease inhibitor (PR-6), peroxidase (PR-9), and ribonuclease (PR-10), as well as qualitative and quantitative changes in the plant proteome. The revealed activation of the expression of marker genes of systemic acquired resistance and induced systemic resistance under the influence of combined treatment with B. subtilis and chitosan conjugate indicate that, in potato plants, the formation of resistance to viral infection in drought conditions proceeds synergistically. By two-dimensional electrophoresis of S. tuberosum leaf proteins followed by MALDI-TOF analysis, 10 proteins were identified, the content and composition of which differed depending on the experiment variant. In infected plants treated with ChCA, the synthesis of proteinaceous RNase P 1 and oxygen-evolving enhancer protein 2 was enhanced in conditions of normal humidity, and 20 kDa chaperonin and TMV resistance protein N-like was enhanced in conditions of lack of moisture. The virus coat proteins were detected, which intensively accumulated in the leaves of plants infected with potato Y-virus. ChCA treatment reduced the content of these proteins in the leaves, and in plants treated with ChCA in combination with Bacillus subtilis, viral proteins were not detected at all, both in conditions of normal humidity and lack of moisture, which suggests the promising use of chitosan derivatives in combination with B. subtilis bacteria in the regulation of plant resistance.

SEC16A Variants Predispose to Chronic Pancreatitis by Impairing ER-to-Golgi Transport and Inducing ER Stress.

Chronic pancreatitis (CP) is a complex disease with genetic and environmental factors at play. Through trio exome sequencing, a de novo SEC16A frameshift variant in a Chinese teenage CP patient is identified. Subsequent targeted next-generation sequencing of the SEC16A gene in 1,061 Chinese CP patients and 1,196 controls reveals a higher allele frequency of rare nonsynonymous SEC16A variants in patients (4.90%vs 2.93%; odds ratio [OR], 1.71; 95% confidence interval [CI], 1.26-2.33). Similar enrichments are noted in a French cohort (OR, 2.74; 95% CI, 1.67-4.50) and in a biobank meta-analysis (OR, 1.16; 95% CI, 1.04-1.31). Notably, Chinese CP patients with SEC16A variants exhibit a median onset age 5 years earlier than those without (40.0vs 45.0; p=0.012). Functional studies using three CRISPR/Cas9-edited HEK293T cell lines show that loss-of-function SEC16A variants disrupt coat protein complex II (COPII) formation, impede secretory protein vesicles trafficking, and induce endoplasmic reticulum (ER) stress due to protein overload. Sec16a+/- mice, which demonstrate impaired zymogen secretion and exacerbated ER stress compared to Sec16a+/+, are further generated. In cerulein-stimulated pancreatitis models, Sec16a+/- mice display heightened pancreatic inflammation and fibrosis compared to wild-type mice. These findings implicate a novel pathogenic mechanism predisposing to CP.

Electroless Deposition of Noble Metals on Rod-Shape Plant Viruses in Various Aqueous Metal Precursor Solutions.

The challenge of synthesizing noble metal nanostructures sustainably has encouraged researchers to explore biological routes for nanostructure production, such as biotemplating. Plant viruses with rod-shape morphology, such as tobacco mosaic virus (TMV) and barley stripe mosaic virus (BSMV), offer promising biotemplates to produce metal nanorods. TMV and BSMV can be incubated in aqueous metal precursor solutions to mineralize metals on the coat proteins (CPs) of the viruses. Previous studies have primarily examined palladium (Pd) mineralization on TMV and BSMV using Na2PdCl4 as the Pd precursor. There is limited scientific literature on the effect of using alternative Pd precursor solutions besides Na2PdCl4 such as K2PdCl4 and PdCl2 to mineralize Pd on TMV and BSMV. Past attempts at mineralizing other noble metals such as platinum (Pt) and gold (Au) required an initial layer of Pd to be deposited on the TMV and BSMV biotemplates. In this study, we aimed to expand the understanding of using alternative Pd precursor solutions to mineralize Pd on TMV and BSMV. Additionally, the deposition of Pt and Au onto TMV and BSMV without the need for an initial Pd mineralization layer was achieved using alternative Pt and Au precursors, including K2PtCl4 and AuCl3, respectively. Pd, Pt, and Au were successfully deposited on TMV and BSMV by incubation in aqueous solutions of Na2PdCl4, K2PdCl4, PdCl2, K2PtCl4, and AuCl3. Kinetic studies were also conducted using ultraviolet-visible (UV-vis) spectroscopy to examine the rates at which Pd, Pt, and Au precursor ions were reduced during the mineralization process, mimicking their adsorption onto TMV and BSMV CPs. BSMV adsorbed noble metal precursor ions faster than TMV as determined by UV-vis spectroscopy. While palladium nanorods (PdNRs) offer high electrical conductivity desirable for electronic applications, Pd-coated TMV and BSMV may face limitations due to their organic cores, potentially compromising conductivity. To address this, one approach is to convert the organic core into conductive amorphous carbon through thermal annealing. In this study, in situ transmission electron microscopy was utilized to thermally anneal Pd-TMV2Cys, thereby transforming them into PdNRs with amorphous carbon cores.

Design, synthesis, and biological activities of arecoline derivatives containing 1,3,4-oxadiazole structure

Pesticides play an important role in the development of agriculture, as they can prevent and control crop diseases and pests, improve crop yield and quality. However, the abuse and improper use of pesticides can lead to negative impacts such as environmental pollution and pest resistance issues. There is an urgent need to develop green, safe, and efficient pesticides. In this work, natural product arecoline was selected as parent structure, a series of arecoline derivatives were designed, synthesized, and systematically investigated antiviral activities against tobacco mosaic virus (TMV). These compounds were found to have good to excellent anti-TMV activities for the first time. The antiviral activities of 4a, 4 h, 4 l, 4p, 6a, 6c, and 6f are higher than that of ningnanmycin. Compounds 4 h (EC50 value 146 µg/mL) and 4p (EC50 value 161 µg/mL) with simple structures and excellent activities emerged as new antiviral candidates. We chose 4 h to further investigate the antiviral mechanism, which revealed that it can cause virus fragmentation by acting on the viral coat protein (CP). We further validated this result through molecular docking. These compounds also displayed broad-spectrum fungicidal activities against 8 plant pathogenic fungi. This work lays the theoretical foundation for the application of arecoline derivatives in the agricultural field.

Discovery of novel Trichoderma-based bioactive compounds for controlling potato virus Y based on molecular docking and molecular dynamics simulation techniques

BackgroundAlthough potato virus Y (PVY) is the most serious virus-infecting potato plants worldwide, the losses concurred by it remain unmanageable due to the lack of efficient anti-PVY agents. Hence, the objective of this study was to assess the antiviral properties of secondary metabolite compounds obtained from culture filtrates of four Trichoderma spp. isolates. The assessment was conducted using computational methods, including molecular docking, absorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis, as well as molecular dynamics simulations. The aim was to develop novel and effective agents for combating PVY.ResultsThe GC–MS analysis of the studied Trichoderma spp. secondary metabolites revealed 24 compounds with relative amounts exceeding 10%. Molecular docking was then performed using MOE software to evaluate the activity of these compounds against the PVY protein coat (PDB-ID: 6HXX). Ningnanmycin and ribavirin, known plant virus inhibitors, were employed as reference ligands for comparison. Among the compounds tested, C9, C10, C13, and C19 exhibited superior docking scores, root mean square deviation (RMSD) values, and binding modes compared to the reference ligands. In addition, these compounds successfully passed the ADMET analysis. Further investigation focused on compounds C13 and C19, which underwent in-depth analysis through MDs for 100 ns. The MDs trajectories demonstrated that both complexes exhibited favorable stability, compactness, and binding modes throughout the simulation period. However, the C19/PVY-CP complex outperformed the C13 complex in all calculated parameters such as RMSD, root mean square fluctuation (RMSF), radius of gyration (RoG), solvent-accessible surface area (SASA), and intermolecular hydrogen bonds. Interestingly, these findings aligned with the results obtained from the docking analysis, indicating that C9 and C10 possess high potential against PVY, as they exhibited binding modes like that of C19.ConclusionThese promising outcomes provide a solid foundation for considering the potential use of compounds C9, C10, C13, and C19 as antiviral agents. Further experimental validation and in-depth studies are warranted to assess the efficacy and safety of these compounds and their potential as antiviral therapeutics. To our knowledge, this is the first report to study the biological activities of the Trichoderma-based bioactive compounds against PVY using computational techniques.Graphical abstract

Structural basis for coupling of the WASH subunit FAM21 with the endosomal SNX27–Retromer complex

Endosomal membrane trafficking is mediated by specific protein coats and formation of actin-rich membrane domains. The Retromer complex coordinates with sorting nexin (SNX) cargo adaptors including SNX27, and the SNX27-Retromer assembly interacts with the Wiskott-Aldrich syndrome protein and SCAR homolog (WASH) complex which nucleates actin filaments establishing the endosomal recycling domain. Crystal structures, modeling, biochemical, and cellular validation reveal how the FAM21 subunit of WASH interacts with both Retromer and SNX27. FAM21 binds the FERM domain of SNX27 using acidic-Asp-Leu-Phe (aDLF) motifs similar to those found in the SNX1 and SNX2 subunits of the ESCPE-1 complex. Overlapping FAM21 repeats and a specific Pro-Leu containing motif bind three distinct sites on Retromer involving both the VPS35 and VPS29 subunits. Mutation of the major VPS35-binding site does not prevent cargo recycling; however, it partially reduces endosomal WASH association indicating that a network of redundant interactions promote endosomal activity of the WASH complex. These studies establish the molecular basis for how SNX27-Retromer is coupled to the WASH complex via overlapping and multiplexed motif-based interactions required for the dynamic assembly of endosomal membrane recycling domains.

Reverse Transcription-polymerase Chain Reaction (RT-PCR) Based Detection and Identification of Monopartite Bean Common Mosaic Virus in Mungbean

Background: Bean common mosaic virus (BCMV) which belongs to the genus Potyvirus and the family Potyviridae, causes significant yield losses in legumes across the globe. Traditional methods for BCMV detection and diagnosis, such as visual inspection and serological assays, are often time-consuming, labour-intensive and may lack the sensitivity required for early detection. Advances in molecular techniques have made RT-PCR a powerful tool for the accurate detection of BCMV-infected plants. Methods: Three pairs of specific primers that flank the BCMV coat protein and polyprotein gene were designed using the Primer 3web tool to amplify fragments approximately ranging from 191 to 205 bp. The RT-PCR protocol was then standardized, including the optimization of annealing temperatures for all three primers, followed by the sequencing of the amplified PCR products. The, specificity and sensitivity of primer pair BCMV2 were then established. Result: Three pairs of specific primers were designed and RT-PCR protocol was standardized. The sequenced PCR products showed 98.03% to 99.50 % nucleotide similarity with the corresponding sequences of BCMV isolates used in primer designing. Further, the BCMV2 primer pair specifically amplified the target amplicon (200 bp) from the suspected leaf sample and no amplicon was observed when tested against other pathotypes of potyvirus such as BCMNV, BYMV, CABMV, SMV and PeMoV. The designed primer was sensitive enough to detect 0.05% of target cDNA. Additionally, the RT-PCR protocol was validated using field samples collected from Hyderabad, India. ELISA in combination with RT-PCR using the developed specific BCMV primer pair will ensure a foolproof, sensitive and rapid, procedure for diagnosis of BCMV in the quarantine processing of imported germplasm.

Structural Simplification of Podophyllotoxin: Discovery of γ-Butyrolactone Derivatives as Novel Antiviral Agents for Plant Protection.

Natural products are a valuable resource for the discovery of novel crop protection agents. A series of γ-butyrolactone derivatives, derived from the simplification of podophyllotoxin's structure, were synthesized and assessed for their efficacy against tobacco mosaic virus (TMV). Several derivatives exhibited notable antiviral properties, with compound 3g demonstrating the most potent in vivo anti-TMV activity. At 500 μg/mL, compound 3g achieved an inactivation effect of 87.8%, a protective effect of 71.7%, and a curative effect of 67.7%, surpassing the effectiveness of the commercial plant virucides ningnanmycin and ribavirin. Notably, the syn-diastereomer (syn-3g) exhibited superior antiviral activity compared to the anti-diastereomer (anti-3g). Mechanistic studies revealed that syn-3g could bind to the TMV coat protein and interfere with the self-assembly process of TMV particles. These findings indicate that compound 3g, with its simple chemical structure, could be a potential candidate for the development of novel antiviral agents for crop protection.

Virus-Based Separation of Rare Earth Elements.

The utilization of biomaterials for the separation of rare earth elements (REEs) has attracted considerable interest due to their inherent advantages, including diverse molecular structures for selective binding and the use of eco-friendly materials for sustainable systems. We present a pioneering methodology for developing a safe virus to selectively bind REEs and facilitate their release through pH modulation. We engineered the major coat protein of M13 bacteriophage (phage) to incorporate a lanthanide-binding peptide. The engineered lanthanide-binding phage (LBPh), presenting ∼3300 copies of the peptide, serves as an effective biological template for REE separation. Our findings demonstrate the LBPh's preferential binding for heavy REEs over light REEs. Moreover, the LBPh exhibits remarkable robustness with excellent recyclability and stability across multiple cycles of separations. This study underscores the potential of genetically integrating virus templates with selective binding motifs for REE separation, offering a promising avenue for environmentally friendly and energy-efficient separation processes.

An In Vitro Model of the Blood-Brain Barrier for the Investigation and Isolation of the Key Drivers of Barriergenesis.

The blood-brain barrier (BBB) tightly regulates substance transport between the bloodstream and the brain. Models for the study of the physiological processes affecting the BBB, as well as predicting the permeability of therapeutic substances for neurological and neurovascular pathologies, are highly desirable. Existing models, such as Transwell utilizing-models, do not mimic the extracellular environment of the BBB with their stiff, semipermeable, non-biodegradable membranes. To help overcome this, we engineered electrospun membranes from poly L-lactic acid in combination with a nanometric coating of poly(ethyl acrylate) (PEA) that drives fibrillogenesis of fibronectin, facilitating the synergistic presentation of both growth factors and integrin binding sites. Compared to commercial semi-porous membranes, these membranes significantly improve the expression of BBB-related proteins in brain endothelial cells. PEA-coated membranes in combination with different growth factors and extracellular protein coatings reveal nerve growth factor (NGF) and fibroblast growth factor (FGF-2) caused formation of better barriers in vitro. This BBB model offers a robust platform for studying key biochemical factors influencing barrier formation that marries the simplicity of the Transwell model with the highly tunable electrospun PEA-fibronectin membranes. This enables the generation of high-throughput drug permeability models without the need of complicated co-culture conditions.

Investigation of Tissue-Specific Distribution and Genetic Variation of Alfalfa Mosaic Virus and Chinese Artichoke Mosaic Virus in Chinese Artichoke (Stachys affinis miq.).

The Chinese artichoke (Stachys affinis syn. S. sieboldii) is a widely cultivated crop, and its rhizome is used as a medicinal vegetable. To investigate the causes of viral diseases in Chinese artichokes, the infection rates of four virus species infecting Chinese artichoke were investigated. Since the Chinese artichoke propagates through its tuber, this study aimed to determine whether viral transmission to the progeny is possible through the tuber, by identifying the virus present in the tuber and investigating its accumulation. First, reverse transcription polymerase chain reaction analysis was performed to detect viruses using total RNA extracted from the flowers, leaves, and tubers of Chinese artichoke plants. Alfalfa mosaic virus (AMV) and Chinese artichoke mosaic virus (ChAMV) had high infectivity in Chinese artichoke and most plants were simultaneously infected with AMV and ChAMV. These viruses were present in all tissues, but their detection frequency and accumulation rates varied across different tissues of the Chinese artichoke. Also, we sequenced the coat protein (CP) genes of AMV and ChAMV to investigate genetic variations of virus between the leaf and tuber. It provides information on CP gene sequences and genetic diversity of isolates identified from new hosts of AMV and ChAMV. This study offers valuable insights into the distribution and spread of the ChAMV and AMV within Chinese artichoke plants, which have implications for the management and control of viral infections in crops.

First report of Triticum mosaic virus infecting oat crop in the United States.

Triticum mosaic virus (TriMV, genus Poacevirus, family Potyviridae) was first reported in 2006 (Seifers et al. 2008) to infect wheat, and since then, it has been established as a constraint for US wheat production (Byamukama et al. 2013). In the field, TriMV often exists as a coinfection with wheat streak mosaic virus (WSMV), and these two viruses interact synergistically to produce severe symptoms and greater yield loss (Byamukama et al. 2012; Tatineni et al. 2022). Both TriMV and WSMV are transmitted by wheat curl mites (Aceria tosichella Keifer) (McMechan et al. 2014). Wheat is the primary host reported for TriMV in the field, but Seifers et al. (2010) established oat, rye, barley, and several other cereals and grasses as hosts under controlled conditions. However, there are no documented cases of TriMV infecting oats in the field. Between 10-25 June, 2023, a total of 273 field oat plants showing foliar yellowing, yellow flecking, and streaking symptoms were collected from four different fields in Nebraska (Big Springs: 41.1029° N, 102.1451° W; Mead: 41.2292° N, 96.4938° W; Odell: 40.0459° N, 96.7984° W; Stumf: 40.5048° N, 101.4223° W). Total RNA was extracted using the MagMax Plant RNA Isolation kit (Thermo Fisher Scientific) and the KingFisher Flex Magnetic Particle Processor (Thermo Fisher Scientific) (Mondal et al. 2023). Sample RNA was assayed with a single-step multiplex reverse transcription polymerase chain reaction (RT-PCR) to determine presence of WSMV and TriMV. Out of 273 symptomatic oat plants, 254 (93.04%) tested positive for at least one virus. Out of total positive samples, 238 were positive for WSMV (93.70 %), 12 plants tested positive for both TriMV and WSMV (4.70%), and 4 plants were infected with TriMV alone (1.60%). As a secondary confirmation, amplified fragments from the TriMV single infection were gel purified using a gel extraction kit (QIAquick) and sequenced (Eurofins Genomics). The nucleotide sequences were analysed using the BlastN program, compiled, and edited in the BioEdit software (Hall 1999). Sequences were deposited in the NCBI GenBank database (accession number PP475806). Nucleotide BLAST searches of the target coat protein (CP) gene showed > 98% identity to the corresponding sequences in TriMV accession MK318274. For further validation, virus inoculum was prepared by grinding field-collected plant material from plants with only TriMV present in 20 mM sodium phosphate buffer, pH 7.0, and then mechanically inoculating two-week-old oats (cv. Shaw n=8) and wheat (cv. Sattler, n=8) plants. Three weeks post-inoculation, all the eight wheat plants exhibited mild yellowing and streaking symptoms, while oat plants did not show obvious foliar symptoms. All wheat and oat plants were further tested positive with DAC-ELISA (antibodies produced against TriMV CP at the USDA-ARS facility in Lincoln, NE) and with RT-PCR. The specific attribution of these symptoms to TriMV in oats is not possible as none produced prominent symptoms. Asymptomatic oat infection from symptomatic field-collected oat samples could be due to oat cultivar differences. Although the prevalence of TriMV in wheat has been established across the Great Plains of the United States, to our knowledge, this is the first report of TriMV infection in US oat fields. Our finding warrant further investigation into the incidence and impact of the virus in oat crop and its potential for serving as a asymptomatic virus reservoir.

Detection of exogenous siRNA inside sweet corn bundle sheath cells and the RNAi dynamics in the early stage of Maize dwarf mosaic virus infection

Maize dwarf mosaic virus (MDMV) is one of the most serious viruses of sweet corn. Utilising the process of RNA interference, the exogenous introduction of small RNA molecules mimicking virus-derived small interfering RNA (siRNA) into the plant prior to infection triggers the antiviral RNA silencing effect, thereby promoting more effective antiviral protection. Hence, a treatment with MDMV-derived small RNA was applied to sweet corn plants one day before MDMV virus inoculation. ALEXA FLUOR®488 fluorophore-bound exogenous siRNA was successfully detected inside intact sweet corn cells using confocal fluorescence microscopy. Furthermore, it was demonstrated that the exogenous siRNA treatment led to a notable upregulation of the AGO1, AGO2b, AGO10b, AGO18a, DCL1, DCL3a, DCL4, RDR1, and MOP1 genes within 24 h of the treatment. Overall, exogenous siRNA treatment resulted in better virus control of infected sweet corn plants, as indicated by the lower viral RNA and coat protein levels compared to the infected group without pre-treatment.