Mobility of kinetochore proteins measured by FRAP analysis in living cells

The kinetochore is essential for faithful chromosome segregation during mitosis and is assembled through dynamic processes involving numerous kinetochore proteins. Various experimental strategies have been used to understand kinetochore assembly processes. Fluorescence recovery after photobleaching (FRAP) analysis is also a useful strategy for revealing the dynamics of kinetochore assembly. In this study, we introduced fluorescence protein-tagged kinetochore protein cDNAs into each endogenous locus and performed FRAP analyses in chicken DT40 cells. Centromeric protein (CENP)-C was highly mobile in interphase, but immobile during mitosis. CENP-C mutants lacking the CENP-A-binding domain became mobile during mitosis. In contrast to CENP-C, CENP-T and CENP-H were immobile during both interphase and mitosis. The mobility of Dsn1, which is a component of the Mis12 complex and directly binds to CENP-C, depended on CENP-C mobility during mitosis. Thus, our FRAP assays provide dynamic aspects of how the kinetochore is assembled.

An "OFF-ON-OFF" fluorescence protein-labeling probe for real-time visualization of the degradation of short-lived proteins in cellular systems

The ability to monitor proteolytic pathways that remove unwanted and damaged proteins from cells is essential for understanding the multiple processes used to maintain cellular homeostasis. In this study, we...

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.

Novel LysM motifs for antigen display on lactobacilli for mucosal immunization

We characterized two LysM domains of Limosilactobacillus fermentum, belonging to proteins Acglu (GenBank: KPH22907.1) and Pgb (GenBank: KPH22047.1) and bacterium like particles (BLP) derived from the immunomodulatory strain Lacticaseibacillus rhamnosus IBL027 (BLPs027) as an antigen display platform. The fluorescence protein Venus fused to the novel LysM domains could bind to the peptidoglycan shell of lactobacilli and resisted harsh conditions such as high NaCl and urea concentrations. Acglu with five LysM domains was a better anchor than Pgb baring only one domain. Six-week-old BALB/c mice were nasally immunized with the complex Venus-Acglu-BLPs027 at days 0, 14 and 28. The levels of specific serum IgG, IgG1 and IgG2a and the levels of total immunoglobulins (IgT) and IgA in broncho-alveolar lavage (BAL) were evaluated ten days after the last boosting. Venus-Acglu-BLPs027, nasally administered, significantly increased specific BAL IgT and IgA, and serum IgG levels. In addition, spleen cells of mice immunized with Venus-Acglu-BLPs027 secreted TNF-α, IFN-γ and IL-4 when stimulated ex vivo in a dose-dependent manner. We constructed a Gateway compatible destination vector to easily fuse the selected LysM domain to proteins of interest for antigen display to develop mucosal subunit vaccines.

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.

Fast and reliable sgRNA efficiency testing using HIReff

CRISPR/Cas9 is the method of choice for gene editing like the endogenous knock-in of sequences in order to investigate protein function, abundance or intracellular localization. One of the crucial steps in the preparation of CRISPR/Cas9-mediated knock-ins is the design of sgRNAs, which need to be tested carefully in order to minimize off-target binding and reach highest cleavage efficiency. Usually, sgRNA is evaluated via mismatch cleavage assays, like Surveyor or T7 endonuclease 1 assay. We demonstrate that these methods are often highly cost- and time-intensive with a low sensitivity and high fail rate. As an alternative, we present a new HITI-based sgRNA efficiency (HIReff) test to precisely evaluate sgRNA efficiency. HIReff is based on a sophisticated integration vector with on-site generation of a linear donor fragment that allows a comparably easy read-out via fluorescence signal and integrates several internal controls. Next to a quantifiable sgRNA assessment, HIReff provides additional information on the 'gene/protein to be studied' abundance, subcellular localization and promoter activity and allows derivation of fluorescence protein labeled clonal lines. We highlight benefits of HIReff in comparison to commonly used enzyme-based assays and demonstrate improved practicability and high sensitivity, while being less time-, labor- and cost-intensive at the same time. Our results suggest HIReff as a fast and easy-to-use alternative for sgRNA efficiency testing.