Accelerated and Improved Quantification of Lymphocytic Choriomeningitis Virus (LCMV) Titers by Flow Cytometry

Lymphocytic choriomeningitis virus (LCMV), a natural murine pathogen, is a member of the Arenavirus family, may cause atypical meningitis in humans, and has been utilized extensively as a model pathogen for the study of virus-induced disease and immune responses. Historically, viral titers have been quantified by a standard plaque assay, but for non-cytopathic viruses including LCMV this requires lengthy incubation, so results cannot be obtained rapidly. Additionally, due to specific technical constraints of the plaque assay including the visual detection format, it has an element of subjectivity along with limited sensitivity. In this study, we describe the development of a FACS-based assay that utilizes detection of LCMV nucleoprotein (NP) expression in infected cells to determine viral titers, and that exhibits several advantages over the standard plaque assay. We show that the LCMV-NP FACS assay is an objective and reproducible detection method that requires smaller sample volumes, exhibits a ∼20-fold increase in sensitivity to and produces results three times faster than the plaque assay. Importantly, when applied to models of acute and chronic LCMV infection, the LCMV-NP FACS assay revealed the presence of infectious virus in samples that were determined to be negative by plaque assay. Therefore, this technique represents an accelerated, enhanced and objective alternative method for detection of infectious LCMV that is amenable to adaptation for other viral infections as well as high throughput diagnostic platforms

Biochemical evidence for an alternate pathway in N-linked glycoprotein biosynthesis

Asparagine-linked glycosylation is a complex protein modification conserved among all three domains of life. Herein we report the in vitro analysis of N-linked glycosylation from the methanogenic archaeon Methanococcus voltae. Using a suite of synthetic and semisynthetic substrates, we show that AglK initiates N-linked glycosylation in M. voltae through the formation of α-linked dolichyl monophosphate N-acetylglucosamine, which contrasts with the polyprenyl diphosphate intermediates that feature in both eukaryotes and bacteria. Notably, AglK has high sequence homology to dolichyl phosphate β-glucosyltransferases, including Alg5 in eukaryotes, suggesting a common evolutionary origin. The combined action of the first two enzymes, AglK and AglC, afforded an α-linked dolichyl monophosphate glycan that serves as a competent substrate for the archaeal oligosaccharyl transferase AglB. These studies provide what is to our knowledge the first biochemical evidence revealing that, despite the apparent similarity of the overall pathways, there are actually two general strategies to achieve N-linked glycoproteins across the domains of life.National Institutes of Health (U.S.) (Grant GM039334

Altering α-dystroglycan receptor affinity of LCMV pseudotyped lentivirus yields unique cell and tissue tropism

BACKGROUND: The envelope glycoprotein of lymphocytic choriomeningitis virus (LCMV) can efficiently pseudotype lentiviral vectors. Some strains of LCMV exploit high affinity interactions with α-dystroglycan (α-DG) to bind to cell surfaces and subsequently fuse in low pH endosomes. LCMV strains with low α-DG affinity utilize an unknown receptor and display unique tissue tropisms. We pseudotyped non-primate feline immunodeficiency virus (FIV) vectors using LCMV derived glycoproteins with high or low affinity to α-DG and evaluated their properties in vitro and in vivo. METHODS: We pseudotyped FIV with the LCMV WE54 strain envelope glycoprotein and also engineered a point mutation in the WE54 envelope glycoprotein (L260F) to diminish α-DG affinity and direct binding to alternate receptors. We hypothesized that this change would alter in vivo tissue tropism and enhance gene transfer to neonatal animals. RESULTS: In mice, hepatic α- and β-DG expression was greatest at the late gestational and neonatal time points. When displayed on the surface of the FIV lentivirus the WE54 L260F mutant glycoprotein bound weakly to immobilized α-DG. Additionally, LCMV WE54 pseudotyped FIV vector transduction was neutralized by pre-incubation with soluble α-DG, while the mutant glycoprotein pseudotyped vector was not. In vivo gene transfer in adult mice with either envelope yielded low transduction efficiencies in hepatocytes following intravenous delivery. In marked contrast, neonatal gene transfer with the LCMV envelopes, and notably with the FIV-L260F vector, conferred abundant liver and lower level cardiomyocyte transduction as detected by luciferase assays, bioluminescent imaging, and β-galactosidase staining. CONCLUSIONS: These results suggest that a developmentally regulated receptor for LCMV is expressed abundantly in neonatal mice. LCMV pseudotyped vectors may have applications for neonatal gene transfer. ABBREVIATIONS: Armstrong 53b (Arm53b); baculovirus Autographa californica GP64 (GP64); charge-coupled device (CCD); dystroglycan (DG); feline immunodeficiency virus (FIV); glycoprotein precursor (GP-C); firefly luciferase (Luc); lymphocytic choriomeningitis virus (LCMV); nuclear targeted β-galactosidase (ntLacZ); optical density (OD); PBS/0.1% (w/v) Tween-20 (PBST); relative light units (RLU); Rous sarcoma virus (RSV); transducing units per milliliter (TU/ml); vesicular stomatitis virus (VSV-G); wheat germ agglutinin (WGA); 50% reduction in binding (C50)

Altering α-dystroglycan receptor affinity of LCMV pseudotyped lentivirus yields unique cell and tissue tropism

BACKGROUND: The envelope glycoprotein of lymphocytic choriomeningitis virus (LCMV) can efficiently pseudotype lentiviral vectors. Some strains of LCMV exploit high affinity interactions with α-dystroglycan (α-DG) to bind to cell surfaces and subsequently fuse in low pH endosomes. LCMV strains with low α-DG affinity utilize an unknown receptor and display unique tissue tropisms. We pseudotyped non-primate feline immunodeficiency virus (FIV) vectors using LCMV derived glycoproteins with high or low affinity to α-DG and evaluated their properties in vitro and in vivo. METHODS: We pseudotyped FIV with the LCMV WE54 strain envelope glycoprotein and also engineered a point mutation in the WE54 envelope glycoprotein (L260F) to diminish α-DG affinity and direct binding to alternate receptors. We hypothesized that this change would alter in vivo tissue tropism and enhance gene transfer to neonatal animals. RESULTS: In mice, hepatic α- and β-DG expression was greatest at the late gestational and neonatal time points. When displayed on the surface of the FIV lentivirus the WE54 L260F mutant glycoprotein bound weakly to immobilized α-DG. Additionally, LCMV WE54 pseudotyped FIV vector transduction was neutralized by pre-incubation with soluble α-DG, while the mutant glycoprotein pseudotyped vector was not. In vivo gene transfer in adult mice with either envelope yielded low transduction efficiencies in hepatocytes following intravenous delivery. In marked contrast, neonatal gene transfer with the LCMV envelopes, and notably with the FIV-L260F vector, conferred abundant liver and lower level cardiomyocyte transduction as detected by luciferase assays, bioluminescent imaging, and β-galactosidase staining. CONCLUSIONS: These results suggest that a developmentally regulated receptor for LCMV is expressed abundantly in neonatal mice. LCMV pseudotyped vectors may have applications for neonatal gene transfer. ABBREVIATIONS: Armstrong 53b (Arm53b); baculovirus Autographa californica GP64 (GP64); charge-coupled device (CCD); dystroglycan (DG); feline immunodeficiency virus (FIV); glycoprotein precursor (GP-C); firefly luciferase (Luc); lymphocytic choriomeningitis virus (LCMV); nuclear targeted β-galactosidase (ntLacZ); optical density (OD); PBS/0.1% (w/v) Tween-20 (PBST); relative light units (RLU); Rous sarcoma virus (RSV); transducing units per milliliter (TU/ml); vesicular stomatitis virus (VSV-G); wheat germ agglutinin (WGA); 50% reduction in binding (C50)

Lesson learned from early and long-term results of 327 cases of coexisting surgical abdominal diseases and aortic aneurysms treated in open and endovascular surgery

Patients with abdominal aortic aneurysm (AAA) frequently have other abdominal pathologies of surgical interest (other diseases, OD). Out of 1,375 elective open aortic replacements for AAA, 315 cases with OD were subdivided in Group 1 (82 patients with “clean wound” OD) and Group 2 (233 patients with “clean-contaminated wound” OD). The results of the sub-groups in which OD was treated at the same time as AAA were analysed (1a, 66 cases and 2a, 86 cases) and compared with OD not treated at the same time as AAA (1b, 16 cases and 2b, 147 cases). EVAR was done in 12 patients with a infrarenal AAA and concomitant abdominal disease. In this group post-operative complications occured in two patients (endoleaks) and no sign of endograft infection was developed. Mean follow-up was 36 months. Mortality was 0% in Group 1a, 1b, 2b and 5.8% in Group 2a. In Group 1a there were one haemoperitoneum, one ischaemic colitis and one graft infection. In Group 1b there were 4 nefrectomies for renal carcinoma and three emergency hernia repairs within 18 months from AAA operation. In Group 2a the follow-up was uneventful. In Group 2b there was no acute complication of OD and 57.2% of patients were subsequently operated for OD. In the EVAR group the 30-day and late mortality rates were 0 and 25%, respectively and all deaths were cancer-related. Contemporary correction of OD in open surgery for AAA should be performed in clean wound cases, while clean-contaminated operations can be done only in selected cases. EVAR is a valid alternative technique to open vascular surgery for the concomitant treatment of aortic aneurysms and abdominal pathologies

Problematic Stabilizing Films in Petroleum Emulsions: Shear Rheological Response of Viscoelastic Asphaltene Films and the Effect on Drop Coalescence

Adsorption of asphaltenes at the water-oil interface contributes to the stability of petroleum emulsions by forming a networked film that can hinder drop-drop coalescence. The interfacial microstructure can either be liquid-like or solid-like, depending on (i) initial bulk concentration of asphaltenes, (ii) interfacial aging time, and (iii) solvent aromaticity. Two techniques--interfacial shear rheology and integrated thin film drainage apparatus--provided equivalent interface aging conditions, enabling direct correlation of the interfacial rheology and droplet stability. The shear rheological properties of the asphaltene film were found to be critical to the stability of contacting drops. With a viscous dominant interfacial microstructure, the coalescence time for two drops in intimate contact was rapid, on the order of seconds. However, as the elastic contribution develops and the film microstructure begins to be dominated by elasticity, the two drops in contact do not coalescence. Such step-change transition in coalescence is thought to be related to the high shear yield stress (~10(4) Pa), which is a function of the film shear yield point and the film thickness (as measured by quartz crystal microbalance), and the increased elastic stiffness of the film that prevents mobility and rupture of the asphaltene film, which when in a solid-like state provides an energy barrier against drop coalescence

A Systematic Survey of Mini-Proteins in Bacteria and Archaea

BACKGROUND: Mini-proteins, defined as polypeptides containing no more than 100 amino acids, are ubiquitous in prokaryotes and eukaryotes. They play significant roles in various biological processes, and their regulatory functions gradually attract the attentions of scientists. However, the functions of the majority of mini-proteins are still largely unknown due to the constraints of experimental methods and bioinformatic analysis. METHODOLOGY/PRINCIPAL FINDINGS: In this article, we extracted a total of 180,879 mini-proteins from the annotations of 532 sequenced genomes, including 491 strains of Bacteria and 41 strains of Archaea. The average proportion of mini-proteins among all genomic proteins is approximately 10.99%, but different strains exhibit remarkable fluctuations. These mini-proteins display two notable characteristics. First, the majority are species-specific proteins with an average proportion of 58.79% among six representative phyla. Second, an even larger proportion (70.03% among all strains) is hypothetical proteins. However, a fraction of highly conserved hypothetical proteins potentially play crucial roles in organisms. Among mini-proteins with known functions, it seems that regulatory and metabolic proteins are more abundant than essential structural proteins. Furthermore, domains in mini-proteins seem to have greater distributions in Bacteria than Eukarya. Analysis of the evolutionary progression of these domains reveals that they have diverged to new patterns from a single ancestor. CONCLUSIONS/SIGNIFICANCE: Mini-proteins are ubiquitous in bacterial and archaeal species and play significant roles in various functions. The number of mini-proteins in each genome displays remarkable fluctuation, likely resulting from the differential selective pressures that reflect the respective life-styles of the organisms. The answers to many questions surrounding mini-proteins remain elusive and need to be resolved experimentally