Abstract Autographa californica M nucleopolyhedrovirus (AcMNPV) open reading frame 109 ( ac109 ) is conserved in all known baculovirus genomes, suggesting a crucial role in virus replication. ...Although viruses lacking ac109 have been previously characterized, the phenotypes differ from production of non-infectious virions to lack of virion production. To re-examine ac109 function, we constructed a recombinant AcMNPV bacmid, AcBAC109KO, with a deletion in ac109 . We did not detect infectious budded virus after transfection of AcBAC109KO DNA into cells. In the nucleus, nucleocapsids had envelopment defects and polyhedra lacked virions. DNA synthesis and gene expression between AcBAC109KO and a control virus were similar. However, lower levels of non-infectious budded virus were detected from AcBAC109KO DNA-transfected cells compared to the parental virus using Q-PCR to detect viral DNA or by immunoblotting to detect a budded virus protein. Therefore, deletion of ac109 affects envelopment of nucleocapsids in the nucleus and the production of infectious budded virus.
Palmitoylation of tetraspanins affects protein–protein interactions, suggesting a key role in the assembly of the tetraspanin web. Since palmitoylation occurs on intracellular cysteine residues, we ...examined whether mutating these residues in the human tetraspanin CD81 would affect the association of CD81 with other surface membrane proteins. Mutation of at least six of the eight juxtamembrane cysteines was required to completely eliminate detectable CD81 palmitoylation, indicating that several sites can be palmitoylated. Interestingly, these mutated proteins exhibited reduced cell surface detection by antibody compared to wild-type CD81, but this was not due to differences in the level of protein expression, trafficking to the cell surface, protein stability, or anti-CD81 antibody binding affinity. Instead, the mutant CD81 proteins appeared to be partially hidden from detection by anti-CD81 antibody, presumably due to altered interactions with other proteins at the cell surface. Associations with the known CD81-interacting proteins CD9 and EWI-2 were also impaired with the mutant CD81 proteins. Taken together, these findings indicate that mutation of juxtamembrane cysteines alters the interaction of CD81 with other proteins, either because of reduced palmitoylation, structural alterations in the mutant proteins, or a combination of both factors, and this affects the CD81 microenvironment on the cell surface.
Background
Immune markers in the peripheral blood of melanoma patients provide useful information for clinical management although there is poor consensus on circulating cells which could putatively ...reflect the disease activity and play a prognostic role. Here, we investigated both dendritic cells (DCs) and T-regulatory cells (Tregs).
Methods
The number of DC subsets as myeloid (m) and plasmacytoid was measured by flowcytometry in 113 melanoma patients in different clinical stages and correlated with the disease activity to evaluate the recurrence free survival (RFS) calculated as difference between baseline and post-surgical values in relation to the criteria for the melanoma staging, as primary tumor removal, sentinel lymph node biopsy and completion of lymph node dissection.
Results
Circulating mDC levels were significantly lower in metastatic melanoma than in other stages and inversely correlated to Treg values while both populations were similarly expressed in inactive disease at stage I-III. Furthermore, the levels of these cells after melanoma removal were apparently related to the disease activity since their persistent defect reflected high risk of recurrence and reduced the RFS.
Conclusions
This work highlighted the role of immune cell measurement for the management of melanoma activity and the identification of patients at potential risk of recurrence based on the mDC ratio.
•The effect of the LIPSS process on the surface chemical properties and consequently on SEY is investigated.•Femtosecond Laser-Induced Periodic Surface Structures (LIPSS) are used to reduce SEY in ...copper.•Copper treated with LIPSS shows a low surface debris density and is therefore less critical for ultra-high vacuum applications in particle accelerators.
The electron-cloud phenomenon is one cause of beam instabilities in high intensity positive particle accelerators. Among the proposed techniques to mitigate or control this detrimental effect, micro-/nano-geometrical modifications of vacuum chamber surfaces are promising to reduce the number of emitted secondary electrons. Femtosecond laser surface structuring readily allows the fabrication of Laser Induced Periodic Surface Structures (LIPSS) and is utilized in several fields, but has not yet been tested for secondary electron emission reduction. In this study, such treatment is carried out on copper samples using linearly and circularly polarized femtosecond laser pulses. The influence of the formed surface textures on the secondary electron yield (SEY) is studied. We investigate the morphological properties as well as the chemical composition by means of SEM, AFM, Raman and XPS analyses. Surface modification with linearly polarized light is more effective than using circularly polarized light, leading to a significant SEY reduction. Even though the SEY maximum is only reduced to a value of ~1.7 compared to standard laser-induced surface roughening approaches, the femtosecond-LIPSS process enables to limit material ablation as well as the production of undesired dust, and drastically reduces the number of redeposited nanoparticles at the surface, which are detrimental for applications in particle accelerators. Moreover, conditioning tests reveal that LIPSS processed Cu can reach SEY values below unity at electron irradiation doses above 10−3 C/mm2.
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Abstract The Autographa californica M nucleopolyhedrovirus (Ac M NPV) viral fibroblast growth factor (vFGF) has functional parallels to cellular FGFs. Deletion of the Ac M NPV v fgf has no obvious ...phenotype in cell culture but delays the time of insect death. Here, we determined vFGF production during virus infection. vFGF was detected at 24 hours post infection and through the remainder of the infection cycle. Since vFGF is thought to be a secreted membrane-binding protein and virions acquire an envelope derived from the cell membrane, we examined virions for the presence of vFGF using microscopy, flow cytometry, and affinity chromatography. We found that vFGF associated with virions. Furthermore, budded virus carrying vFGF had more affinity to heparin than vFGF-deficient budded virus, consistent with the affinity of FGFs for heparan sulfate proteoglycans. Although the function of virion-associated vFGF is not clear, we found that virion-associated vFGF stimulated cell motility and affected virus attachment.
The enzymes of the shikimate pathway represent potential molecular targets for the development of non-toxic antimicrobial agents and anti-parasite drugs. One of the most promising of these enzymes is ...shikimate kinase (EC 2.7.1.71), which is responsible for the fifth step in the shikimate pathway. This enzyme phosphorylates shikimic acid to yield shikimate-3-phosphate, using ATP as a substrate. In this work, the conformational dynamics of the shikimate kinase from Mycobacterium tuberculosis was investigated in its apostate in solution. For this study, the enzyme was subjected to a gradient of temperatures from 15°C to 45°C in the presence or absence of deuterium oxide, and the amide H/D exchange was monitored using ESI-mass spectrometry. We observed: i) the phosphate binding domain in the apo-enzyme is fairly rigid and largely protected from solvent access, even at relatively high temperatures; ii) the shikimate binding domain is highly flexible, as indicated by the tendency of the apo-enzyme to exhibit large conformational changes to permit LID closure after the shikimate binding; iii) the nucleotide binding domain is initially conformationally rigid, which seems to favour the initial orientation of ADP/ATP, but becomes highly flexible at temperatures above 30°C, which may permit domain rotation; iv) part of the LID domain, including the phosphate binding site, is partially rigid, while another part is highly flexible and accessible to the solvent.
Abstract The fibroblast growth factor ( vfgf ) gene encoded by Autographa californica M nucleopolyhedrovirus (Ac M NPV) has been shown to share functional properties with cellular fgfs ; it is a ...secreted protein, binds heparin, and stimulates motility of insect cells. We previously reported that viruses containing or lacking vfgf produced similar yields of budded virus and had similar kinetics of viral DNA and protein syntheses in cultured cells. In this study, we characterized these viruses in two permissive hosts, Spodoptera frugiperda and Trichoplusia ni , using two insect developmental stages and two infection routes, by feeding and intrahemocoelic injection. In addition, we constructed an Ac M NPV bacmid overexpressing vfgf under polyhedrin promoter control and characterized it in both cell culture and insects. Deletion of vfgf had no effect on the infectivity of Ac M NPV. However, lack of vfgf delayed the time of death in two host species when the virus was delivered by feeding but not by intrahemocoelic injection. The virus overexpressing vfgf produced less budded virus than the control virus in cultured cells. In insect bioassays, the infectivity of this virus was greater than that of the parental virus in both insect species and significantly accelerated time of death of both hosts tested. Our results suggest that the Ac M NPV vfgf may play a role in dissemination of virus infection from the midgut in the insect species tested.
The treatment of patients with brain-spread renal cell carcinoma (RCC) is an unmet clinical need, although more recent therapeutic strategies have significantly improved RCC patients’ life ...expectancy. Our multicenter, retrospective, observational study investigated a real-world cohort of patients with brain metastases (BM) from RCC (BMRCC).
A total of 226 patients with histological diagnosis of RCC and radiological evidence of BM from 22 Italian institutions were enrolled. Univariate and multivariate models were performed to investigate the impact of clinicopathological features and multimodal treatments on both overall survival (OS) from the BM diagnosis and intracranial progression-free survival (iPFS).
The median OS from the BM diagnosis was 18.8 months (interquartile range: 6.2-43 months). Multivariate analysis confirmed the following as positive independent prognostic factors: a Karnofsky Performance Status >70% hazard ratio (HR) = 0.49, 95% confidence interval (CI) 0.26-0.92, P = 0.0026 and a single BM (HR = 0.51, 95% CI 0.31-0.86, P = 0. 0310); in contrast, the following were confirmed as worse prognosis factors: progressive extracranial disease (HR = 1.66, 95% CI 1.003-2.74, P = 0.00181) and only one line of systemic therapy after the BM occurrence (HR = 2.98, 95% CI 1.62-5.49, P = 0.029). Subgroup analyses showed no difference in iPFS according to the type of the first systemic treatment immunotherapy (IT) or targeted therapy (TT) carried out after the BM diagnosis (HR = 1.033, 95% CI 0.565-1.889, P = 0.16), and revealed that external radiation therapy (eRT) significantly prolonged iPFS when combined with IT (10.7 months, 95% CI 4.9-48 months, P = 0.0321) and not when combined with TT (9.01 months, 95% CI 2.7-21.2 months, P = 0.59).
Our results suggest a potential additive effect in terms of iPFS for eRT combined with IT and encourage a more intensive multimodal therapeutic strategy in a multidisciplinary context to improve the survival of BMRCC patients.
•The treatment of patients with BM from RCC is an unmet clinical need.•Data from prospective studies on the effectiveness of multimodal treatments for RCC patients with BM are limited.•This real-life study investigated prognostic factors and treatment strategies in 226 patients with BMRCC.•The BMRCC study suggests a potential additive effect in terms of iPFS for eRT combined with IT.•The results of the BMRCC study encourage a more intensive multimodal therapeutic strategy for patients with BMRCC.
Membrane-based virus filtration is used in biotechnological processes due to advantages such as easy scale-up but is currently lacking standardized validation protocols. Thus, ultrafiltration (UF) of ...a rod-shaped virus was assessed to contribute to the body of knowledge regarding the characterization of virus filtration. A recombinant baculovirus of
Autographa californica M nucleopolyhedrovirus (Ac
MNPV), vHSGFP, expressing
egfp was filtered using polyethersulfone membranes ranging from 30 to 1000 kDa molecular weight cut-off (MWCO) and filtration parameters were previously assessed for their potential to affect virus stability. Ac
MNPV was concentrated 20-fold in the retentate using 100–1000 kDa membranes. However, evidence for a superposition of cake formation and pore plugging through viruses and other solution components (likely proteins) causing partially irreversible fouling of membranes dependent on the MWCO was observed. Results support UF using a 300 kDa MWCO to concentrate Ac
MNPV with minimized membrane fouling.
The
Autographa californica M nucleopolyhedrovirus (Ac
MNPV) encodes a gene (open reading frame 32) with homology to vertebrate and invertebrate fibroblast growth factors (
fgfs), key regulators of ...developmental processes affecting the growth, differentiation, and migration of many cell types. We studied the temporal regulation of the Ac
MNPV
fgf,
vfgf, by Northern (RNA) blot hybridization;
vfgf was transcribed as a 0.6-kb mRNA at early times but as part of a 1.4-kb bicistronic mRNA at late times. The product of
vfgf, vFGF, exhibited a number of characteristics that have also been demonstrated for other FGF homologs. vFGF had strong affinity to heparin, a property important for FGF signaling via an FGF receptor. vFGF was secreted into the extracellular fluid when expressed in insect cells, suggesting that it acts as an extracellular ligand. Finally, vFGF was able to stimulate migration of several different types of insect cells. We discuss how this activity may be important for its function during virus infection.
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