Fabrication of 3D cell-laden hydrogel microstructures through photo-mold patterning.

Native tissues are characterized by spatially organized three-dimensional (3D) microscaled units which functionally define cells-cells and cells-extracellular matrix interactions. The ability to engineer biomimetic constructs mimicking these 3D microarchitectures is subject to the control over cell distribution and organization. In the present study we introduce a novel protocol to generate 3D cell laden hydrogel micropatterns with defined size and shape. The method, named photo-mold patterning (PMP), combines hydrogel micromolding within polydimethylsiloxane (PDMS) stamps and photopolymerization through a recently introduced biocompatible ultraviolet (UVA) activated photoinitiator (VA-086). Exploiting PDMS micromolds as geometrical constraints for two methacrylated prepolymers (polyethylene glycol diacrylate and gelatin methacrylate), micrometrically resolved structures were obtained within a 3 min exposure to a low cost and commercially available UVA LED. The PMP was validated both on a continuous cell line (human umbilical vein endothelial cells expressing green fluorescent protein, HUVEC GFP) and on primary human bone marrow stromal cells (BMSCs). HUVEC GFP and BMSCs were exposed to 1.5% w/v VA-086 and UVA light (1 W, 385 nm, distance from sample = 5 cm). Photocrosslinking conditions applied during the PMP did not negatively affect cells viability or specific metabolic activity. Quantitative analyses demonstrated the potentiality of PMP to uniformly embed viable cells within 3D microgels, creating biocompatible and favorable environments for cell proliferation and spreading during a seven days' culture. PMP can thus be considered as a promising and cost effective tool for designing spatially accurate in vitro models and, in perspective, functional constructs.

Air quality biomonitoring: assessment of air pollution genotoxicity in the Province of Novara (North Italy) by using Trifolium repens L. and molecular markers.

Mixed air pollutants are considered a major cause of DNA damage in living species. In this study Trifolium repens L. cv Regal was used as a bioindicator to assess the genotoxicity of air stressors in the Italian province of Novara. Two on-site biomonitoring experiments were performed during the spring and autumn of 2004. Test plants were exposed at 19 monitoring sites distributed homogeneously throughout the province, and each experiment lasted for a period of 6 weeks. Genotoxicity was evaluated with Amplified Fragment Length Polymorphism (AFLP) molecular markers. The results show the predominantly rural central-west region of the Novara Province to have the worst air quality with regard to genotoxicity. Analyses of geomorphology, land use and climatic factors suggest that the compromised air quality in the region could be attributed to wind strength and direction, transporting pollution from vehicular traffic on the A4 highway and from the urban/industrialized centres of Novara and Vercelli. Plant growth, changes in plant photochemical efficiency and the presence of ozone related leaf injuries were also measured to better interpret the results of genotoxicity. Statistical analyses show that although climatic factors such as light intensity and temperature influence plant growth, they do not contribute to atmospheric stressor-induced DNA damage. Further analyses indicated that, as expected, a mixture of genotoxic and non-genotoxic pollutants coexist in the Novara Province troposphere, and that the elevated ozone concentrations experienced during the study may have contributed to the DNA damage in the tested plants by enhancing genotoxicity via interaction with other air stressors.

Isolation and partial characterization of an antiviral, RC-183, from the edible mushroom Rozites caperata.

A protein of 10,425 Da was purified from the edible mushroom Rozites caperata and shown to inhibit herpes simplex virus types 1 and 2 replication with an IC50 value of < or = 5 microM. The protein designated RC-183 also significantly reduced the severity of HSV-1 induced ocular disease in a murine model of keratitis, indicating in vivo efficacy. HSV mutants lacking ribonucleotide reductase and thymidine kinase were also inhibited, suggesting the mechanism does not involve these viral enzymes. Antiviral activity was also seen against varicella zoster virus, influenza A virus, and respiratory syncytial virus, but not against adenovirus type VI, coxsackie viruses A9 and B5, or human immunodeficiency virus. Characterization of RC-183 by mass spectroscopy, sequencing, and other methods suggests it is composed of a peptide (12 or 13 mer) coupled to ubiquitin via an isopeptide bond between the c-terminal glycine of ubiquitin and the epsilon amino group of a lysine residue in the peptide. The peptide sequence did not match any known sequence. Thus, RC-183 is a novel antiviral that may have clinical utility or serve as a lead compound for further development. Determining the mechanism of action may lead to identification of novel steps in viral replication.

Echovirus 11 infections of newborns with mortality during the 1979 enterovirus season in Milwaukee, Wis.

Echovirus serotype 11 (ECHO-11) was implicated in three neonatal deaths during an enterovirus outbreak from July through October 1979 in Milwaukee. The deaths followed congenital infections acquired in the community during late pregnancy. Two of the three ECHO-11 and one Coxsackie B4 deaths of infants occurred after cesarean section deliveries. Of 225 confirmed echovirus infections, 30 to 45 percent occurred in infants under 60 days old, 54 to 67 percent in the first year of life, and 13 to 25 percent in the over-10 age groups. In 13 cases with onset of symptoms in the first week of life. 8 (including the 4 fatalities) were acquired congenitally; 6 of the 8 were associated with ECHO-11, 2 with ECHO-7, and 1 with Coxsackie B4. ECHO-7 and 30 other predominant strains were isolated during the outbreak, but none was associated with mortality or severe disease in neonates. At a Milwaukee hospital, a temporal association was observed between echovirus infection, particularly ECHO-11, and increased numbers of stillbirths.

Plaque formation in chick embryo fibroblast cells by Chlamydia isolated from avian and mammalian sources.

Fifteen strains of chlamydial agents, 2 Chlamydia trachomatis and 13 Chlamydia psittaci, were tested for plaque formation on chick embryo fibroblast cells. C. trachomatis strains did not form plaques; 12 strains of C. psittaci did form plaques and 1 strain did not. The distribution of plaque sizes with C. psittaci strains was studied and it was found that the 12 strains could be tentatively grouped into three plaquing types: small (<0.50 mm), intermediate (0.50 to 0.90 mm), and large (>0.90 mm). Small plaques were predominantly associated with strains M56, ET, CP3, CT4, EBA, CS1, and CT2. Intermediate size plaques were predominantly associated with strains NJ1, VT1, and H81. Large plaques were predominantly associated with strains WC and OG. Plaque size appears to be related to virulence of Chlamydia for laboratory animals.

The mechanism of genetic resistance of chick embryo cells to infection by Rous sarcoma virus-Bryan strain (BS-RSV).

The early steps of Rous sarcoma virus-Bryan strain (BS-RSV) infection of genetically resistant and susceptible chick embryo cells were investigated. The methods used were the uptake of virus by cells grown in monolayer, and the ability of infected cells to form foci of transformed cells after plating on resistant and susceptible assay plates. It was found that the initial attachment of virus to either genetic type of cells did not differ appreciably. Attachment on susceptible cells was immediately followed by penetration, which was 70% complete after 1 hour. Attachment on resistant cells was not followed by penetration of the cell membrane. It was concluded that the genetic block of resistance to infection of chick embryo cells by BS-RSV occurs at the step of viral penetration of the cell membrane.