Phytohormone Priming of Tomato Plants Evoke Differential Behavior in Rhizoctonia solani During Infection, With Salicylate Priming Imparting Greater Tolerance Than Jasmonate

In the field of phytohormone defense, the general perception is that salicylate (SA)-mediated defense is induced against biotrophic pathogens while jasmonate (JA)-mediated defense functions against necrotrophic pathogens. Our goals were to observe the behavior of the necrotrophic pathogen Rhizoctonia solani in the vicinity, on the surface, and within the host tissue after priming the host with SA or JA, and to see if priming with these phytohormones would affect the host defense differently upon infection. It was observed for the first time, that R. solani could not only distinguish between JA versus SA-primed tomato plants from a distance, but surprisingly avoided SA-primed plants more than JA-primed plants. To corroborate these findings, early infection events were monitored and compared through microscopy, Scanning Electron Microscopy, and Confocal Laser Scanning Microscopy using transformed R. solani expressing green fluorescence protein gene (gfp). Different histochemical and physiological parameters were compared between the unprimed control, JA-primed, and SA-primed plants after infection. The expression of a total of fifteen genes, including the appressoria-related gene of the pathogen and twelve marker genes functioning in the SA and JA signaling pathways, were monitored over a time course during early infection stages. R. solani being traditionally designated as a necrotroph, the major unexpected observations were that Salicylate priming offered better tolerance than Jasmonate priming and that it was mediated through the activation of SA-mediated defense during the initial phase of infection, followed by JA-mediated defense in the later phase. Hence, the present scenario of biphasic SA-JA defense cascades during R. solani infection, with SA priming imparting maximum tolerance, indicate a possible hemibiotrophic pathosystem that needs to be investigated further.

Neuron-Recognizable Characteristics of Peptides Recombined Using A Neuronal Binding Domain of Botulinum Neurotoxin

Recombinant peptides were designed using the C-terminal domain (receptor binding domain, RBD) and its subdomain (peptide A2) of a heavy chain of botulinum neurotoxin A-type 1 (BoNT/A1), which can bind to the luminal domain of synaptic vesicle glycoprotein 2C (SV2C-LD). Peptide A2- or RBD-containing recombinant peptides linked to an enhanced green fluorescence protein (EGFP) were prepared by expression in Escherichia coli. A pull-down assay using SV2C-LD-covered resins showed that the recombinant peptides for CDC5328 BoNT/A1, referred to EGFP-A2ʹ and EGFP-RBDʹ, exhibited ≥ 2.0-times stronger binding affinity to SV2C-LD than those for the wild-type BoNT/A1. Using bio-layer interferometry, an equilibrium dissociation rate constant (KD) of EGFP-RBDʹ to SV2C-LD was determined to be 5.45 mM, which is 33.87- and 15.67-times smaller than the KD values for EGFP and EGFP-A2ʹ, respectively. Based on confocal laser fluorescence micrometric analysis, the adsorption/absorption of EGFP-RBDʹ to/in differentiated PC-12 cells was 2.49- and 1.29-times faster than those of EGFP and EGFP-A2ʹ, respectively. Consequently, the recombinant peptides acquired reasonable neuron-specific binding/internalizing ability through the recruitment of RBDʹ. In conclusion, RBDs of BoNTs are versatile protein domains that can be used to mark neural systems and treat a range of disorders in neural systems.

Assessment of the Gastrointestinal Fate of Bacterial Cellulose and Its Toxicological Effects After Repeated-dose Oral Administration

BackgroundBacterial cellulose (BC) is a nanofibrillar polysaccharide produced by certain acetic acid bacteria. BC may be used in food, pharma and many other applications. However, detailed studies of the oral toxicology of BC are limited. Controversial data is published regarding this topic, specially when it comes to answering the question on whether cellulose is absorbed at the intestine.MethodsFollowing the European Food Safety Authority guidelines (EFSA), this work presents the results of a 21-day repeated dose oral toxicity of BC in male and female Wistar Han rats (Wistar rats). In parallel, microcrystalline cellulose Avicel LM310 (commercially available as a food additive) was used. Wistar rats were subjected to daily oral gavage of 0.75 mL of an aqueous suspension 1% (m/v) BC or of its counterpart of plant origin, Avicel LM310. Rats not submitted to gavage were included in the experiment as controls. Clinical observations, such as body weight measurements, food consumption and ophthalmologic evaluations were performed during the assay. After occision, serum chemistry, necropsic examination and histopathological analyses of the liver, kidneys, spleen and small and large intestines were performed. The presence of BC fibers along the gastrointestinal tract was assessed histologically using a Green Fluorescence Protein coupled to a Cellulose Carbohydrate Binding Module (GFP-CBM) from Clostridium cellulolyticum.ResultsNo adverse clinical observations related to BC administration were noticed, nor appreciable differences in the toxicological endpoints evaluated were detected. No evidence of BC persorption was found. Particularly, no BC was detected in the Peyer´s patches or in the mesenteric lymphatic nodules. Moreover, the histopathological analyses revealed that the global architecture and morphology of the organs and tissues was preserved, among the different experimental groups, with no significant pathological changes among them. Regarding serum biochemistry, no significant differences were recorded, for both sexes.ConclusionsThese results demonstrate that BC nanofibers can be considered safe and, as the vegetal cellulose, can be used as a food additive.

O-OGC07 Endoscopically derived oesophageal adenocarcinoma organoids to assess potential response to neo-adjuvant therapy and virotherapy

Background Organoids are 3D models that retain the architecture and function of the organ from which they are derived. Culture of oesophageal adenocarcinoma organoids from individual’s standard endoscopic biopsies to assess response to therapy could dramatically alter the neo-adjuvant treatment paradigm, giving clarity over who will benefit from therapy, including novel treatment methodologies. Immune checkpoint blockade (ICB) has been shown to be effective in oesophageal adenocarcinoma. Combining Oncolytic Virotherapy with ICB could enhance the action of ICB alone, through selective infection of tumour cells accompanied by immunogenic cell death, with release of neo-tumour antigens and alteration of the tumour microenvironment. Methods This study uses organoids derived from endoscopic biopsies to assess the viability of an oncolytic herpes simplex virus in the treatment of oesophageal adenocarcinoma. Samples were taken at staging endoscopy using standard biopsy forceps. Tissue specimens were dissociated using the Miltenyi tumour dissociation kit before being suspended in Matrigel in conditioned media. Media was changed every 48 hours with domes being split every 7-10 days. After >6 passages organoids were incubated with an oncolytic herpes simplex virus lacking ICP 34.5 and 47. Growth was monitored, and green fluorescence protein expression measured using the Incucyte SX5 Live Cell Analysis system. Results Organoids were successfully established and cultured beyond 6 passages for patients with oesophageal adenocarcinoma. Organoids incubated with an oncolytic herpes simplex virus demonstrated significantly reduced growth compared to untreated organoids with increased expression of green fluorescence protein indicating viral infection. Conclusions We have demonstrated a successful methodology to culture Oesophageal adenocarcinoma organoids from endoscopic biopsies. Further work to determine their responses to standard chemotherapy used in the perioperative phase will help to assess their potential for providing bespoke therapy in the future. Oncolytic herpes simplex virus is able to infect and cause lysis of OAC organoids supporting its potential use in driving an increased inflammatory tumour microenvironment which could be combined with immune checkpoint blockade to induce durable responses for patients.

The Feasibility of Pressurised Intraperitoneal Aerosolised Virotherapy (PIPAV) to Administer Oncolytic Adenoviruses

Background: The prognosis of patients with peritoneal metastases is poor. Treatment options are limited because systemically delivered chemotherapy is not usually effective in this type of disease. Pressurised intraperitoneal aerosolised chemotherapy (PIPAC) is a recently developed alternative technology for delivering intraperitoneal chemotherapy, potentially enhancing treatment efficacy. Here, we assess the feasibility of pressurised intraperitoneal aerosolised virotherapy (PIPAV) to deliver a different class of anticancer agents, oncolytic adenoviruses, in vitro and in vivo. Methods: Adenoviral vectors expressing reporter genes green fluorescence protein (Ad5.GFP) or firefly luciferase (Ad5.Luc) were subject to pressurised aerosolisation. The ability of the virus to survive PIPAV was assessed in vitro and in vivo by monitoring reporter gene activity. Wistar rats subjected to PIPAV were assessed for any adverse procedure related events. Results: In vitro transduction assays demonstrated that Ad5 retained viability following pressurised aerosolisation and could transduce permissive cells equally effectively as non-aerosolised control vector. PIPAV was well tolerated in rats, although minimal transduction was observed following intraperitoneal administration. Conclusions: PIPAV appears viable and well tolerated, though in vivo efficacy requires further optimisation.

Membrane Vesicles of Pectobacterium as an Effective Protein Secretion System

Bacteria of genus Pectobacterium are Gram-negative rods of the family Pectobacteriaceae. They are the causative agent of soft rot diseases of crops and ornamental plants. However, their virulence mechanisms are not yet fully elucidated. Membrane vesicles (MVs) are universally released by bacteria and are believed to play an important role in the pathogenicity and survival of bacteria in the environment. Our study investigates the role of MVs in the virulence of Pectobacterium. The results indicate that the morphology and MVs production depend on growth medium composition. In polygalacturonic acid (PGA) supplemented media, Pectobacterium produces large MVs (100–300 nm) and small vesicles below 100 nm. Proteomic analyses revealed the presence of pectate degrading enzymes in the MVs. The pectate plate test and enzymatic assay proved that those enzymes are active and able to degrade pectates. What is more, the pathogenicity test indicated that the MVs derived from Pectobacterium were able to induce maceration of Zantedeschia sp. leaves. We also show that the MVs of β-lactamase producing strains were able to suppress ampicillin activity and permit the growth of susceptible bacteria. Those findings indicate that the MVs of Pectobacterium play an important role in host-pathogen interactions and niche competition with other bacteria. Our research also sheds some light on the mechanism of MVs production. We demonstrate that the MVs production in Pectobacterium strains, which overexpress a green fluorescence protein (GFP), is higher than in wild-type strains. Moreover, proteomic analysis revealed that the GFP was present in the MVs. Therefore, it is possible that protein sequestration into MVs might not be strictly limited to periplasmic proteins. Our research highlights the importance of MVs production as a mechanism of cargo delivery in Pectobacterium and an effective secretion system.

Susceptibility of Dog, Hamster, and Mouse Cells to the Replication-Competent Adenovirus 11p E1/E3 Green Fluorescence Protein Vector Has Implications for the Selection of Animal Vaccine Models

Human adenovirus (Ad)-vectored vaccines require viruses that can internalize into host cells and express the vaccine antigen. Evaluation of the expressed antigen in animal cells is a critical step in preclinical trials of viral vaccines. Due to the species specificity of Ads, it is difficult to find a suitable animal model. Thus, in this study, we compared the efficacy of Ad 11 prototype (Ad11p)-mediated green fluorescence protein (GFP) expression in cell lines of dog (MDCK), hamster (CHO), and mouse (McCoy and C127). Although these cell lines did not express the known primary cellular receptors for Ad11p virus infection (i.e., CD46), Ad11pE1GFP could infect and express GFP with various efficacies. For instance, it manifested relatively higher GFP expression in MDCK than in CHO, McCoy, and C127. However, infection leading to efficient viral release was not observed in any of the studied cell lines. The apparent differences were attributed to particularities of mouse and hamster cell lines, which might have led to the repression of viral DNA synthesis and to the low level of GFP expression mediated by Ad11pe3GFP. Moreover, our results revealed that undetectable hexon protein hampered the assembly of virus particles in CHO and MDCK cells. Ad11p differed from Ad5 in the ability for viral DNA synthesis when infecting CHO cells. Although a defective Ad has been successfully developed for SARS-CoV-2 vaccines in clinical applications, it has been difficult to generate one that can be used as an oral SARS-CoV-2 vaccine. Fortunately, our replication-competent Ad 11p vector might solve this problem. Regarding the use of Ad-vector candidates for vaccine purposes, this study demonstrates the selection of animal cell lines and determination of suitable virus doses in in vitro experiments.

Correlative Localization Analysis Between mRNA and Enhanced Green Fluorescence Protein-Fused Protein by a Single-Molecule Fluorescence in situ Hybridization Using an egfp Probe in Aspergillus oryzae

Although subcellular localization analysis of proteins fused with enhanced green fluorescence protein (EGFP) has been widely conducted in filamentous fungi, little is known about the localization of messenger RNAs (mRNAs) encoding the EGFP-fused proteins. In this study, we performed single-molecule fluorescence in situ hybridization (smFISH) using an egfp probe to simultaneously visualize EGFP-fused proteins and their mRNAs in the hyphal cells of the filamentous fungus Aspergillus oryzae. We investigated the subcellular localization of mRNAs encoding cytoplasmic EGFP, an actin marker protein Lifeact tagged with EGFP, and several EGFP-fused proteins AoSec22, AoSnc1, AoVam3, and AoUapC that localize to the endoplasmic reticulum (ER), the apical vesicle cluster Spitzenkörper, vacuolar membrane, and plasma membrane, respectively. Visualization of these mRNAs by smFISH demonstrated that each mRNA exhibited distinct localization patterns likely depending on the mRNA sequence. In particular, we revealed that mRNAs encoding Lifeact-EGFP, EGFP-AoSec22, EGFP-AoVam3, and AoUapC-EGFP, but not cytoplasmic EGFP and EGFP-AoSnc1, were preferentially localized at the apical cell, suggesting certain mechanisms to regulate the existence of these transcripts among hyphal regions. Our findings provide the distinct localization information of each mRNA in the hyphal cells of A. oryzae.

Microplate reader operating procedure v1

Use Tecan Spark® multimode microplate reader to measure absorbance or fluorescence intensity of green fluorescence protein.

Rescuing eGFP-Tagged Canine Distemper Virus for 40 Serial Passages Separately in Ribavirin- and Non-Treated Cells: Comparative Analysis of Viral Mutation Profiles

Due to lacking a proofreading mechanism in their RNA-dependent RNA polymerases (RdRp), RNA viruses generally possess high mutation frequencies, making them evolve rapidly to form viral quasispecies during serial passages in cells, especially treated with mutagens, like ribavirin. Canine distemper virus (CDV) belongs to the genus Morbillivirus. Its L protein functions as an RdRp during viral replication. In this study, a recombinant enhanced green fluorescence protein-tagged CDV (rCDV-eGFP) was rescued from its cDNA clone, followed by viral identification and characterization at passage-7 (P7). This recombinant was independently subjected to extra 40 serial passages (P8 to 47) in ribavirin- and non-treated cells. Two viral progenies, undergoing passages in ribavirin- and non-treated VDS cells, were named rCDV-eGFP-R and -N, respectively. Both progenies were simultaneously subjected to next-generation sequencing (NGS) at P47 for comparing their quasispecies diversities with each other. The rCDV-eGFP-R and -N showed 62 and 23 single-nucleotide mutations (SNMs) in individual antigenomes, respectively, suggesting that the ribavirin conferred a mutagenic effect on the rCDV-eGFP-R. The spectrum of 62 SNMs contained 26 missense and 36 silent mutations, and that of 23 SNMs was composed of 17 missense and 6 silent mutations. Neither the rCDV-eGFP-R nor -N exhibited nonsense mutation in individual antigenomes. We speculate that the rCDV-eGFP-R may contain at least one P47 sub-progeny characterized by high-fidelity replication in cells. If such a sub-progeny can be purified from the mutant swarm, its L protein would elucidate a molecular mechanism of CDV high-fidelity replication.