In cultured cells the baculovirus P10 protein forms two independent intracellular structures that play separate roles in occlusion body maturation and their release by nuclear disintegration.

P10 is a small, abundant baculovirus protein that accumulates to high levels in the very late stages of the infection cycle. It is associated with a number of intracellular structures and implicated in diverse processes from occlusion body maturation to nuclear stability and lysis. However, studies have also shown that it is non-essential for virus replication, at least in cell culture. Here, we describe the use of serial block-face scanning electron microscopy to achieve high-resolution 3D characterisation of P10 structures within Trichoplusia ni TN-368 cells infected with Autographa californica multiple nucleopolyhedrovirus. This has enabled unparalleled visualisation of P10 and determined the independent formation of dynamic perinuclear and nuclear vermiform fibrous structures. Our 3D data confirm the sequence of ultrastructural changes that create a perinuclear cage from thin angular fibrils within the cytoplasm. Over the course of infection in cultured cells, the cage remodels to form a large polarised P10 mass and we suggest that these changes are critical for nuclear lysis to release occlusion bodies. In contrast, nuclear P10 forms a discrete vermiform structure that was observed in close spatial association with both electron dense spacers and occlusion bodies; supporting a previously suggested role for P10 and electron dense spacers in the maturation of occlusion bodies. We also demonstrate that P10 hyper-expression is critical for function. Decreasing levels of p10 expression, achieved by manipulation of promoter length, correlated with reduced P10 production, a lack of formation of P10 structures and a concomitant decrease in nuclear lysis.

Superinfection exclusion in alphabaculovirus infections is concomitant with actin reorganization.

UNLABELLED: Superinfection exclusion is the ability of an established virus to interfere with a second virus infection. This effect was studied in vitro during lepidopteran-specific nucleopolyhedrovirus (genus Alphabaculovirus, family Baculoviridae) infection. Homologous interference was detected in Sf9 cells sequentially infected with two genotypes of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), each one expressing a different fluorescent protein. This was a progressive process in which a sharp decrease in the signs of infection caused by the second virus was observed, affecting not only the number of coinfected cells observed, but also the level of protein expression due to the second virus infection. Superinfection exclusion was concurrent with reorganization of cytoplasmic actin to F-actin in the nucleus, followed by budded virus production (16 to 20 h postinfection). Disruption of actin filaments by cell treatment with cytochalasin D resulted in a successful second infection. Protection against heterologous nucleopolyhedrovirus infection was also demonstrated, as productive infection of Sf9 cells by Spodoptera frugiperda nucleopolyhedrovirus (SfMNPV) was inhibited by prior infection with AcMNPV, and vice versa. Finally, coinfected cells were observed following inoculation with mixtures of these two phylogenetically distant nucleopolyhedroviruses--AcMNPV and SfMNPV--but at a frequency lower than predicted, suggesting interspecific virus interference during infection or replication. The temporal window of infection is likely necessary to maintain genotypic diversity that favors virus survival but also permits dual infection by heterospecific alphabaculoviruses. IMPORTANCE: Infection of a cell by more than one virus particle implies sharing of cell resources. We show that multiple infection, by closely related or distantly related baculoviruses, is possible only during a brief window of time that allows additional virus particles to enter an infected cell over a period of ca. 16 h but then blocks multiple infections as newly generated virus particles begin to leave the infected cell. This temporal window has two important consequences. First, it allows multiple genotypes to almost simultaneously infect cells within the host, thus generating genetically diverse virus particles for transmission. Second, it provides a mechanism by which different viruses replicating in the same cell nucleus can exchange genetic material, so that the progeny viruses may be a mosaic of genes from each of the parental viruses. This opens a completely new avenue of research into the evolution of these insect pathogens.

Expression and plasma membrane localization of the mammalian B-cell receptor complex in transgenic Nicotiana tabacum.

The B-cell antigen receptor (BCR), displayed on the plasma membrane of mature B cells of the mammalian immune system, is a multimeric complex consisting of a membrane-bound immunoglobulin (mIg) noncovalently associated with the Igα/Igß heterodimer. In this study, we engineered transgenic tobacco plants expressing all four chains of the BCR. ELISA, Western blotting and confocal microscopy demonstrated that the BCR was correctly assembled in plants, predominantly in the plasma membrane, and that the noncovalent link was detergent sensitive. This is the first example of a noncovalently assembled plasma membrane-retained heterologous receptor in plants. In B cells of the mammalian immune system, following antigen binding to mIg, BCR is internalized and tyrosine residues on Igα and Igß are phosphorylated activating a signaling cascade through interaction with protein kinases that ultimately leads to the initiation of gene expression. Expression of the BCR may therefore be an important tool for the study of plant endocytosis and the identification of previously unknown plant tyrosine kinases. The specificity and diversity of the antibody repertoire, coupled to the signal transduction capability of the Igα/Igß heterodimer, also indicates that plants expressing BCR may in future be developed as environmental biosensors.

Prognostic significance of the neutrophil-to-lymphocyte ratio and platelet-to-lymphocyte ratio for advanced non-small cell lung cancer patients with high PD-L1 tumor expression receiving pembrolizumab.

BACKGROUND: We investigated the association of peripheral blood inflammatory markers with overall survival (OS) in pembrolizumab treated advanced non-small cell lung cancer (aNSCLC) patients with programmed death ligand 1 (PD-L1) expression ≥50%. Clinical risk factors for development of immune-related adverse events (irAE) were also explored. METHODS: aNSCLC patients with high PD-L1 expression receiving pembrolizumab monotherapy outside of clinical trials were identified retrospectively. All patients were treated at one of six British Columbia Cancer clinics between August 2017 and June 2019. Patients were dichotomized using baseline neutrophil-to-lymphocyte ratio (NLR,

Association of age with differences in immune related adverse events and survival of patients with advanced nonsmall cell lung cancer receiving pembrolizumab or nivolumab.

OBJECTIVES: To explore the association of age with development of immune related adverse events (irAE) and survival in patients with advanced nonsmall cell lung cancer (aNSCLC) receiving programmed cell death 1 antibodies (PD-1 Ab) outside of clinical trials. METHODS: A multicenter retrospective study of PD-1 Ab prescription for patients with aNSCLC between 06/2015-11/2018 at BC Cancer. Multivariable (MVA) logistic regression identified baseline variables associated with irAE manifested within 3 months of PD-1 Ab initiation. Overall survival (OS) analyzed in a propensity-score matched cohort and survival outcomes compared between age groups by stratified log-rank. Six-week landmark analysis was performed and OS compared between patients with interrupted versus continuous treatment by log-rank. RESULTS: Of 527 patients, 40.6% were age ≤ 64 years, 40.6% were 65-74 years, and 18.8% were ≥ 75 years. In MVA, ECOG performance status 2/3 (p = .034), squamous histology (p = .031), and nivolumab therapy (vs. pembrolizumab, p = .012) were associated with increased odds of irAE by 3 months of treatment. Across age groups no difference existed in any grade irAE (p = .98), hospitalization (p = 1.0), or corticosteroids use (p = .51). The propensity score-matched survival analysis comprised 77 patients from each age group; all covariates were balanced. OS did not differ significantly by age in the matched cohort (p = .17). Treatment interruption due to irAE at 6 weeks was more common in patient ≥75 years (vs. <75, p = .055) and correlated with lower OS (p = .002). CONCLUSION: In this cohort of patients with aNSCLC treated in routine clinical practice with PD-1 Ab, immune-toxicity and observed survival were similar amongst age groups.

Pembrolizumab for advanced nonsmall cell lung cancer: Efficacy and safety in everyday clinical practice.

OBJECTIVES: While pembrolizumab improves overall survival (OS) in a subset of advanced nonsmall cell lung cancer (aNSCLC) patients (pts) in clinical trials, individuals with poor Eastern Cooperative Oncology Group performance status (ECOG PS) were excluded. Furthermore, some studies have identified a potential link between improved pt outcomes and development of immune related adverse events (irAE.) In a large provincial cohort, we studied the efficacy and safety of pembrolizumab for poor ECOG PS pts and whether irAE correlate with improved OS. MATERIALS AND METHODS: aNSCLC pts treated with pembrolizumab between 06/2015 and 08/2018 at BC Cancer were retrospectively identified. Kaplan-Meier curves of OS from initiation of pembrolizumab were plotted. 3-, 6-, and 9- month landmark Kaplan-Meier analysis was performed and log-rank tests used to determine an association of irAE subtypes with OS. Multivariable logistic regression identified variables associated with grade ≥3 irAE within 3 months of pembrolizumab initiation. RESULTS: Of 190 pts, 74.2% were treatment naïve and 92.6% had PD-L1 expression ≥ 50%. Median OS in the 1st line and ≥2nd line settings were 24.3 months (95% CI, 9.7-not reached, NR) and 13.4 months (95% CI, 8.1-NR), respectively. Pts with ECOG PS 2/3 had lower median OS than if ECOG PS 0/1 (5.8 months vs. 16.7 months, p < 0.0001). In multivariable analysis, the odds of grade ≥ 3 irAE within 3 months was 6.3 fold higher if ECOG PS 2/3 versus 0/1 (p = 0.05). Development of pneumonitis at the 9 month landmark weakly correlated with decreased OS (p = 0.09). CONCLUSION: In the studied cohort, ECOG PS 2/3 pts had a significantly lower OS and greater odds of experiencing high-grade irAE than if ECOG PS 0/1. Development of irAE did not result in improved OS. Randomized trials to determine benefit of pembrolizumab for poor ECOG PS pts are needed.

Anchorage to the cytosolic face of the endoplasmic reticulum membrane: a new strategy to stabilize a cytosolic recombinant antigen in plants.

The levels of accumulation of recombinant vaccines in transgenic plants are protein specific and strongly influenced by the subcellular compartment of destination. The human immunodeficiency virus protein Nef (negative factor), a promising target for the development of an antiviral vaccine, is a cytosolic protein that accumulates to low levels in transgenic tobacco and is even more unstable when introduced into the secretory pathway, probably because of folding defects in the non-cytosolic environment. To improve Nef accumulation, a new strategy was developed to anchor the molecule to the cytosolic face of the endoplasmic reticulum (ER) membrane. For this purpose, the Nef sequence was fused to the C-terminal domain of mammalian ER cytochrome b5, a long-lived, tail-anchored (TA) protein. This consistently increased Nef accumulation by more than threefold in many independent transgenic tobacco plants. Real-time polymerase chain reaction of mRNA levels and protein pulse-chase analysis indicated that the increase was not caused by higher transcript levels but by enhanced protein stability. Subcellular fractionation and immunocytochemistry indicated that Nef-TA accumulated on the ER membrane. Over-expression of mammalian or plant ER cytochrome b5 caused the formation of stacked membrane structures, as observed previously in similar experiments performed in mammalian cells; however, Nef-TA did not alter membrane organization in tobacco cells. Finally, Nef could be removed in vitro by its tail-anchor, taking advantage of an engineered thrombin cleavage site. These results open up the way to use tail-anchors to improve foreign protein stability in the plant cytosol without perturbing cellular functions.

Fluorescent protein fusions to a human immunodeficiency virus monoclonal antibody reveal its intracellular transport through the plant endomembrane system.

SUMMARY: In order to further understand the production and intracellular trafficking of pharmaceutical proteins in plants, the light and heavy chains (LC and HC) of the human immunodeficiency virus neutralizing monoclonal antibody 2G12 were fused to fluorescent proteins [Venus and monomeric red fluorescent protein (mRFP)] to enable the visualization of their passage through the plant cell. Co-expression of LC and HC with various markers of the endomembrane system demonstrated that LC fusions were found in mobile punctate structures, which are likely to be pre-vacuolar compartments (PVCs) as a proportion of the LC fusions were found to be located in the vacuole. In addition, apoplast labelling was also observed with a 2G12LC-RFP fusion. The HC fusion expressed alone was found only in the endoplasmic reticulum (ER). When the LC and HC fusions were expressed together, they were found to co-locate to larger punctate structures, which were morphologically distinct from any observed on expression of LC or HC alone. These structures appeared to be in close association with the ER and their labelling partially overlapped with PVC marker fluorescence, but no increase in apoplast labelling was observed. Co-immunoprecipitation data demonstrated that the presence of the fluorescent proteins did not affect the assembly of the antibody, and also showed the association of BiP with the antibody chains. The antigen-binding activity of the Venus-fused 2G12 antibody was confirmed by enzyme-linked immunosorbent assay.

Retention and mobility of the mammalian lamin B receptor in the plant nuclear envelope.

BACKGROUND INFORMATION: In a previous study, we showed that GFP (green fluorescent protein) fused to the N-terminal 238 amino acids of the mammalian LBR (lamin B receptor) localized to the NE (nuclear envelope) when expressed in the plant Nicotiana tabacum. The protein was located in the NE during interphase and migrated with nuclear membranes during cell division. Targeting and retention of inner NE proteins requires several mechanisms: signals that direct movement through the nuclear pore complex, presence of a transmembrane domain or domains and retention by interaction with nuclear or nuclear-membrane constituents. RESULTS: Binding mutants of LBR-GFP were produced to investigate the mechanisms for the retention of LBR in the NE. FRAP (fluorescence recovery after photobleaching) analysis of mutant and wild-type constructs was employed to examine the retention of LBR-GFP in the plant NE. wtLBR-GFP (wild-type LBR-GFP) was shown to have significantly lower mobility in the NE than the lamin-binding domain deletion mutant, which showed increased mobility in the NE and was also localized to the endoplasmic reticulum and punctate structures in some cells. Modification of the chromatin-binding domain resulted in the localization of the protein in nuclear inclusions, in which it was immobile. CONCLUSIONS: As expression of truncated LBR-GFP in plant cells results in altered targeting and retention compared with wtLBR-GFP, we conclude that plant cells can recognize the INE (inner NE)-targeting motif of LBR. The altered mobility of the truncated protein suggests that not only do plant cells recognize this signal, but also have nuclear proteins that interact weakly with LBR.

Overview of the Baculovirus Expression System.

This unit provides information on the replication cycle of insect baculovirus to provide an understanding of how this virus has been adapted for use as an expression vector for recombinant proteins in insect cells. We provide an overview of the virus structure and its unique bi-phasic replication cycle, which has been exploited in developing the virus as an expression vector. We also review the development of the baculovirus expression vector system (BEVS), from the mid-1980s to the present day in which the BEVS is now an established tool for the production of a range of recombinant proteins and multi-protein complexes including virus-like particles. We describe advances made to the BEVS to allow the rapid and easy production of recombinant viruses and developments to improve protein yield. We finish by describing the application of recombinant BacMam as vectors for the delivery of genes into mammalian and human cells. © 2018 by John Wiley & Sons, Inc.

Protein Production Using the Baculovirus Expression System.

Baculovirus expression systems are well established as an easy and reliable way to produce high quality recombinant proteins. Baculoviruses can also be used to transduce mammalian cells, termed 'BacMam', with considerable potential in biomedical applications. This chapter explains the process of making a recombinant baculovirus, encompassing production of a recombinant virus by homologous recombination in insect cells, followed by amplification and titration of the virus-all steps needed before commencing gene expression and protein production. We also cover the use of small-scale test expression to provide an initial indication of quality and protein yield. Whereas proteins expressed at high levels can be directly scaled up, more challenging proteins may require optimization of cell lines, growth conditions, or harvest times. Scale-up and purification approaches are discussed, focusing on working with large shake cultures and use of the Wave bioreactor. © 2018 by John Wiley & Sons, Inc.

The non-targeted effects of radiation are perpetuated by exosomes.

Exosomes contain cargo material from endosomes, cytosol, plasma membrane and microRNA molecules, they are released by a number of non-cancer and cancer cells into both the extracellular microenvironment and body fluids such as blood plasma. Recently we demonstrated radiation-induced non-targeted effects [NTE: genomic instability (GI) and bystander effects (BE)] are partially mediated by exosomes, particularly the RNA content. However the mechanistic role of exosomes in NTE is yet to be fully understood. The present study used MCF7 cells to characterise the longevity of exosome-induced activity in the progeny of irradiated and unirradiated bystander cells. Exosomes extracted from conditioned media of irradiated and bystander progeny were added to unirradiated cells. Analysis was carried out at 1 and 20/24 population doublings following medium/exosome transfer for DNA/chromosomal damage. Results confirmed exosomes play a significant role in mediating NTE of ionising radiation (IR). This effect was remarkably persistent, observed >20 doublings post-irradiation in the progeny of bystander cells. Additionally, cell progeny undergoing a BE were themselves capable of inducing BE in other cells via exosomes they released. Furthermore we investigated the role of exosome cargo. Culture media from cells exposed to 2 Gy X-rays was subjected to ultracentrifugation and four inoculants prepared, (a) supernatants with exosomes removed, and pellets with (b) exosome proteins denatured, (c) RNA degraded, and (d) a combination of protein-RNA inactivation. These were added to separate populations of unirradiated cells. The BE was partially inhibited when either exosome protein or exosome RNA were inactivated separately, whilst combined RNA-protein inhibition significantly reduced or eliminated the BE. These results demonstrate that exosomes are associated with long-lived signalling of the NTE of IR. Both RNA and protein molecules of exosomes work in a synergistic manner to initiate NTE, spread these effects to naïve cells, and perpetuate GI in the affected cells.

The plant nuclear envelope: new prospects for a poorly understood structure.

The nuclear envelope (NE) is one of the least characterized cellular structures in plant cells. In particular, knowledge of its dynamic behaviour during the cell cycle and of its protein composition is limited. This review summarizes current views on the plant NE and highlights fundamental differences with other organisms. We also introduce the power of new technology available to investigate the NE and how this has already begun to revolutionize our knowledge of the biology of the plant NE. Contents Summary 227 I. Introduction 227 II. The membranes of the nuclear envelope 228 III. Functions of the nuclear envelope 231 IV. Proteins associated with the nuclear envelope 236 V. New tools for studying the nuclear envelope 239 VI. Conclusions and future prospects 241 Acknowledgements 242 References 242.

Host cell processes to accomplish mechanical and non-circulative virus transmission.

Mechanical vector-less transmission of viruses, as well as vector-mediated non-circulative virus transmission, where the virus attaches only to the exterior of the vector during the passage to a new host, are apparently simple processes: the viruses are carried along with the wind, the food or by the vector to a new host. We discuss here, using the examples of the non-circulatively transmitted Cauliflower mosaic virus that binds to its aphid vector's exterior mouthparts, and that of the mechanically (during feeding activity) transmitted Autographa californica multicapsid nucleopolyhedrovirus, that transmission of these viruses is not so simple as previously thought. Rather, these viruses prepare their transmission carefully and long before the actual acquisition event. Host-virus interactions play a pivotal and specialised role in the future encounter with the vector or the new host. This ensures optimal propagation and enlarges the tremendous bottleneck transmission presents for viruses and other pathogens.

The effect of genetic background and dose on non-targeted effects of radiation.

PURPOSE: This work investigates the hypothesis that genetic background plays a significant role in the signalling mechanisms underlying induction and perpetuation of genomic instability following radiation exposure. MATERIALS AND METHODS: Bone marrow from two strains of mice (CBA and C57) were exposed to a range of X-ray doses (0, 0.01, 0.1, 1 and 3 Gy). Different cellular signalling endpoints: Apoptosis, cytokine levels and calcium flux, were evaluated at 2 h, 24 h and 7 d post-irradiation to assess immediate and delayed effects. RESULTS: In CBA (radiosensitive) elevated apoptosis levels were observed at 24 h post X-irradiation, and transforming growth factor-ß (TGF-ß) levels which increased with time and dose. C57 showed a higher background level of apoptosis, and sustained apoptotic levels 7 days after radiation exposure. Levels of tumor necrosis factor-α (TNF-α were increased in C57 at day 7 for higher X-ray doses. TGF-ß levels were higher in CBA, whilst C57 exhibited a greater TNF-α response. Calcium flux was induced in reporter cells on exposure to conditioned media from both strains. CONCLUSIONS: These results show genetic and dose specific differences in radiation-induced signalling in the initiation and perpetuation of the instability process, which have potential implications on evaluation of non-targeted effects in radiation risk assessment.

No influence of serotonin levels in foetal bovine sera on radiation-induced bystander effects and genomic instability.

PURPOSE: The aim of this study was to contribute to an inter-laboratory investigation within the Non-Targeted Effects of Ionising Radiation Integrated project (NOTE) (2006-2010) to investigate the role of serum serotonin concentration on radiation-induced bystander effects using our successful experimental design. Two sera of high and low serotonin levels were tested alongside standard serum used in our laboratory. MATERIALS AND METHODS: Primary Human Fibroblast 19 (HF19) cells were sham/irradiated with 0.5 Gy alpha-particles, in medium supplemented with serum of different levels of serotonin. Filtered medium was transferred to unirradiated HF19 bystander cells. Cells from irradiated and bystander populations were harvested for chromosomal analysis at early and delayed times post-irradiation/media transfer to assess initial damaging effects and induction of delayed chromosomal instability respectively. RESULTS: Chromosomal damage was elevated to significant levels (p = ≤ 0.005) above respective controls in both cell populations in all groups. Induction of delayed chromosomal instability was significantly observed in all groups at delayed time post irradiation/medium transfer. Furthermore, induction of bystander effects at early and delayed times was not significantly different between groups. CONCLUSIONS: No effect of serotonin on the induction of either bystander effects of genomic instability was observed using this experimental system.

Possible role of exosomes containing RNA in mediating nontargeted effect of ionizing radiation.

Communication between irradiated and un-irradiated (bystander) cells can cause damage in cells that are not directly targeted by ionizing radiation, a process known as the bystander effect. Bystander effects can also lead to chromosomal/genomic instability within the progeny of bystander cells, similar to the progeny of directly irradiated cells. The factors that mediate this cellular communication can be transferred between cells via gap junctions or released into the extracellular media following irradiation, but their nature has not been fully characterized. In this study we tested the hypothesis that the bystander effect mediator contains an RNA molecule that may be carried by exosomes. MCF7 cells were irradiated with 2 Gy of X rays and the extracellular media was harvested. RNase treatment abrogated the ability of the media to induce early and late chromosomal damage in bystander cells. Furthermore, treatment of bystander cells with exosomes isolated from this media increased the levels of genomic damage. These results suggest that the bystander effect, and genomic instability, are at least in part mediated by exosomes and implicate a role for RNA.

The first 238 amino acids of the human lamin B receptor are targeted to the nuclear envelope in plants.

In plants, the nuclear envelope (NE) is one of the least characterized cellular structures. In particular, little is known about its dynamics during the cell cycle. This is due to the absence of specific markers for in vivo studies. To generate such an in vivo marker, the suitability of the human lamin B receptor (LBR) was tested. When the first 238 amino acids of the LBR, fused to the green fluorescent protein (GFP), were expressed in tobacco plants, fluorescence accumulated only at the NE of leaf epidermal cells. This was confirmed by electron microscopy. The protein was shown to be membrane-integral by phase separation. Distribution of fluorescence was compared with two ER markers, GFP-calnexin and GFP-HDEL. While co-localization of all three markers was noted at the NE, only LBR-GFP was specific to the NE, while the other two also showed fluorescence of the cortical ER. These results suggest that common targeting mechanisms to those in animals and fungi exist in plants to direct and locate proteins to the NE. This chimaeric construct is the first available fluorescent integral membrane protein marker to be targeted exclusively to the plant NE and it provides a novel opportunity to investigate the dynamics of this membrane system in vivo. With it, the cell cycle was followed in tobacco BY-2 cells stably expressing the fusion protein. The interphase labelling of the NE altered in metaphase into an ER-like meshwork, suggesting the dispersal of the NE to ER as in animal cells. Finally, the meshwork of fluorescent membranes was lost and new fluorescent NE formed around the daughter nuclei.

BY-2 cells: culture and transformation for live cell imaging.

Tobacco Bright Yellow-2 (BY-2) suspension cells are a widely used biological material for studying plant cell morphology and physiology. These cells are easy to transform and maintain in culture and tolerate transformation with fluorescent proteins such as the green fluorescent protein and its derivatives. These, by the addition of plant or mammalian targeting sequences, can be directed to specific subcellular locations for the study of cell dynamics in vivo. This unit describes the production of BY-2 cell stable transformants via an Agrobacterium based method to permit the visualisation of cellular components in vivo by epifluorescence or confocal microscopy.