Novel bi- and trifunctional inhibitors of tumor-associated proteolytic systems

Serine proteases, cysteine proteases, and matrix metalloproteinases (MMPs) are involved in cancer cell invasion and metastasis. Recently, a recombinant bifunctional inhibitor (chCysuPA(19-31)) directed against cysteine proteases and the urokinasetype plasminogen activator (uPA)/plasmin serine protease system was generated by introducing the uPA receptor (uPAR)binding site of uPA into chicken cystatin (chCysWT). In the present study, we designed and recombinantly produced multifunctional inhibitors also targeting MMPs. The inhibitors comprise the Nterminal inhibitory domain of human TIMP-1 (tissue inhibitor of matrix metalloproteinase-1) or TIMP-3, fused to chCysuPA(19-31) or chCysWT. As demonstrated by various techniques, these fusion proteins effectively interfere with all three targeted protease systems. In in vitro Matrigel invasion assays, the addition of recombinant inhibitors strongly reduced invasion of ovarian cancer cells (OVMZ-6\#8). Additionally, OVMZ 6\#8 cells were stably transfected with expression plasmids encoding the various inhibitors. Synthesis and secretion of the inhibitors was verified by a newly developed ELISA, which selectively detects the recombinant proteins. Invasive capacity of inhibitorproducing cells was significantly reduced compared to vectortransfected control cells. Thus, these novel, compact, and smallsize inhibitors directed against up to three different tumorassociated proteolytic systems may represent promising agents for prevention of tumor cell migration and metastasis

Tracing baculovirus AcMNPV infection using a real time method based on ANCHORTM DNA labeling technology

Many steps in the baculovirus life cycle, from initial ingestion to the subsequent infection of all larval cells, remain largely unknown; primarily because it has hitherto not been possible to follow individual genomes and their lineages. Use of ANCHORTM technology allows a high intensity fluorescent labelling of DNA. When applied to a virus genome, it is possible to follow individual particles, and the overall course of infection. This technology has been adapted to enable labelling of the baculovirus Autographa californica Multiple NucleoPolyhedroVirus genome, as a first step to its application to other baculoviruses. AcMNPV was modified by inserting the two components of ANCHORTM: a specific DNA-binding protein fused to a fluorescent reporter, and the corresponding DNA recognition sequence. The resulting modified virus was stable, infectious, and replicated correctly in Spodoptera frugiperda 9 (Sf9) cells and in vivo. Both budded viruses and occlusion bodies were clearly distinguishable, and infecting cells or larvae allowed the infection process to be monitored in living cells or tissues. The level of fluorescence in the culture medium of infected cells in vitro showed a good correlation with the number of infectious budded viruses. A cassette that can be used in other baculoviruses has been designed. Altogether our results introduce for the first time the generation of autofluorescent baculovirus and their application to follow infection dynamics directly in living cells or tissues

Phosphorylation induces structural changes in the Autographa californica nucleopolyhedrovirus P10 protein

Baculoviruses encode a variety of auxiliary proteins that are not essential for viral replication but provide them with a selective advantage in nature. P10 is a 10 kDa auxiliary protein produced in the very-late phase of gene transcription by Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The P10 protein forms cytoskeletal-like structures in the host cell that associate with microtubules varying from filamentous forms in the cytoplasm to aggregated peri-nuclear tubules that form a cage-like structure around the nucleus. These P10 structures may have a role in the release of occlusion bodies (OBs) and thus mediate horizontal transmission of the virus between insect hosts. Here it is demonstrated, using mass spectrometric analysis, that the C-terminus of P10 is phosphorylated during virus infection of cells in culture. Analysis of the P10 mutants encoded by recombinant baculoviruses in which putative phosphorylation residues were mutated to alanine showed that serine 93 is a site of phosphorylation. Confocal microscopy examination of the serine 93 mutant structures revealed an aberrant formation of the peri-nuclear tubules. Thus, phosphorylation of serine 93 may induce aggregation of filaments to form tubules. Together, these data suggest that the phosphorylation of serine 93 affects P10 structural conformation. IMPORTANCE The baculovirus P10 protein has been researched intensively since it was first observed in 1969, but its role during the viral infection remains unclear. It is conserved in the alphabaculoviruses and expressed at high levels during virus infection. Producing large amounts of a protein is wasteful for the virus unless it is advantageous for survival of its progeny and therefore P10 presents an enigma. As P10 polymerises to form organised cytoskeletal structures that co-localise with the host cell microtubules, the structural relationship of the protein with the host cell may present a key to help understand the function and importance of this protein. This study addresses the importance of the structural changes in P10 during infection and how they may be governed by phosphorylation. The P10 structures affected by phosphorylation are closely associated with the viral progeny and thus, potentially, be responsible for its dissemination and survival

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

Effects of a programme of vigorous physical activity during secondary school physical education on academic performance, fitness, cognition, mental health and the brain of adolescents (Fit to Study): Study protocol for a cluster randomised trial

Background. Early adolescence is a period of dynamic neurobiological change. Converging lines of research suggest that regular physical activity (PA) and improved aerobic fitness have the potential to stimulate positive brain changes, improve cognitive function and boost academic attainment in this age group, but high-quality studies are needed to substantiate these findings. The primary aim of the Fit to Study trial is to investigate whether short infusions of vigorous PA (VPA) delivered during secondary school physical education (PE) can improve attainment in maths, as described in a protocol published by NatCen Social Research. The present protocol concerns the trial’s secondary outcome measures, which are variables thought to moderate or mediate the relationship between PA and attainment, including the effect of the intervention on cardiorespiratory fitness, cognitive performance, mental health and brain structure and function. Method. The Fit to Study project is a cluster-randomised controlled trial that includes Year 8 pupils (aged 12–13) from secondary state schools in South/Mid-England. Schools were randomised into an intervention condition in which PE teachers delivered an additional 10 min of VPA per PE lesson for one academic year, or a ‘PE as usual’ control condition. Intervention and control groups were stratified according to whether schools were single-sex or co-educational. Assessments take place at baseline (end of Year 7, aged 11–12) and after 12 months (Year 8). Secondary outcomes are cardiorespiratory fitness, objective PA during PE, cognitive performance and mental health. The study also includes exploratory measures of daytime sleepiness, attitudes towards daily PA and PE enjoyment. A sub-set of pupils from a sub-set of schools will also take part in a brain imaging sub-study, which is embedded in the trial. Discussion. The Fit to Study trial could advance our understanding of the complex relationships between PA and aerobic fitness, the brain, cognitive performance, mental health and academic attainment during adolescence. Further, it will add to our understanding of whether school PE is an effective setting to increase VPA and fitness, which could inform future PA interventions and education policy

Identification and Preliminary Characterization of a Chitinase Gene in the Autographa californica Nuclear Polyhedrosis Virus Genome

AbstractA functional chitinase gene (chiA) has been identified in the genome of the Autographa californica nuclear polyhedrosis virus (AcMNPV). It is expressed in the late phase of virus replication in insect cells. High levels of both endo- and exochitinase activity were detected by 12 hr p.i. and remained stable throughout infection. An AcMNPV chiA protein-specific antibody was prepared using recombinant material prepared in bacteria. This was used to demonstrate that a product of approximately 58 kDa was synthesised in virus-infected cells. Immunofluorescence analysis of virus-infected cells showed that most chitinase was located in the cytoplasm. Primer extension analysis of mRNA from AcMNPV-infected cells confirmed that transcription initiated from a baculovirus late start site (TAAG), 14 nucleotides upstream from the putative translation initiation codon. The predicted protein sequence of the AcMNPV chiA shares extensive sequence similarity with chitinases from bacteria and, in particular, the Serratia marcescens chitinase A (60.5% identical residues). Phylogenetic analyses indicate that AcMNPV, or an ancestral baculovirus, acquired the chitinase gene from a bacterium via horizontal gene transfer

Improved baculovirus vectors for transduction and gene expression in human pancreatic islet cells

Pancreatic islet transplantation is a promising treatment for type 1 diabetes mellitus offering improved glycaemic control by restoring insulin production. Improved human pancreatic islet isolation has led to higher islet transplantation success. However, as many as 50% of islets are lost after transplantation due to immune responses and cellular injury. Gene therapy presents a novel strategy to protect pancreatic islets for improved survival post-transplantation. To date, most of the vectors used in clinical trials and gene therapy studies have been derived from mammalian viruses such as adeno-associated or retrovirus. However, baculovirus BacMam vectors provide an attractive and safe alternative. Here a novel BacMam was constructed containing a frameshift mutation within fp25, which results in virus stocks with higher infectious titres. This improved in vitro transduction when compared to control BacMams. Additionally, incorporating a truncated vesicular stomatitis virus G protein increased transduction efficacy and production of EGFP and BCL2 in human kidney (HK-2) and pancreatic islet β cells (EndoC βH3). Lastly, we have shown that our optimized BacMam vector can deliver and express egfp in intact pancreatic islet cells from human cadaveric donors. These results confirm that BacMam vectors are a viable choice for providing delivery of transgenes to pancreatic islet cells

Safety and Immunogenicity of H5N1 Influenza Vaccine Based on Baculovirus Surface Display System of Bombyx mori

Avian influenza virus (H5N1) has caused serious infections in human beings. This virus has the potential to emerge as a pandemic threat in humans. Effective vaccines against H5N1 virus are needed. A recombinant Bombyx mori baculovirus, Bmg64HA, was constructed for the expression of HA protein of H5N1 influenza virus displaying on the viral envelope surface. The HA protein accounted for approximately 3% of the total viral proteins in silkworm pupae infected with the recombinant virus. Using a series of separation and purification methods, pure Bmgp64HA virus was isolated from these silkworm pupae bioreactors. Aluminum hydroxide adjuvant was used for an H5N1 influenza vaccine. Immunization with this vaccine at doses of 2 mg/kg and 0.67 mg/kg was carried out to induce the production of neutralizing antibodies, which protected monkeys against influenza virus infection. At these doses, the vaccine induced 1:40 antibody titers in 50% and 67% of the monkeys, respectively. The results of safety evaluation indicated that the vaccine did not cause any toxicity at the dosage as large as 3.2 mg/kg in cynomolgus monkeys and 1.6 mg/kg in mice. The results of dose safety evaluation of vaccine indicated that the safe dose of the vaccine were higher than 0.375 mg/kg in rats and 3.2 mg/kg in cynomolgus monkeys. Our work showed the vaccine may be a candidate for a highly effective, cheap, and safe influenza vaccine for use in humans