Construction and growth properties of bovine herpesvirus type 5 recombinants defective in the glycoprotein E or thymidine kinase gene or both

Bovine herpesvirus type 5 (BoHV-5) is an important pathogen of cattle in South America. We describe here the construction and characterization of deletion mutants defective in the glycoprotein E (gE) or thymidine kinase (TK) gene or both (gE/TK) from a highly neurovirulent and well-characterized Brazilian BoHV-5 strain (SV507/99). A gE-deleted recombinant virus (BoHV-5 gE∆) was first generated in which the entire gE open reading frame was replaced with a chimeric green fluorescent protein gene. A TK-deleted recombinant virus (BoHV-5 TK∆) was then generated in which most of the TK open reading frame sequences were deleted and replaced with a chimeric β-galactosidase gene. Subsequently, using the BoHV-5 gE∆ virus as backbone, a double gene-deleted (TK plus gE) BoHV-5 recombinant (BoHV-5 gE/TK∆) was generated. The deletion of the gE and TK genes was confirmed by immunoblotting and PCR, respectively. In Madin Darby bovine kidney (MDBK) cells, the mutants lacking gE (BoHV-5 gE∆) and TK + gE (BoHV-5 gE/TK∆) produced small plaques while the TK-deleted BoHV-5 produced wild-type-sized plaques. The growth kinetics and virus yields in MDBK cells for all three recombinants (BoHV-5 gE∆, BoHV-5 TK∆ and BoHV-5 gE/TK∆) were similar to those of the parental virus. It is our belief that the dual gene-deleted recombinant (BoHV-5 gE/TK∆) produced on the background of a highly neurovirulent Brazilian BoHV-5 strain may have potential application in a vaccine against BoHV-5.

Construction of a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen and mouse 4-1BBL genes and its effect on dendritic cells

Our aim was to construct a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen (tPSMA) and mouse 4-1BBL genes and to determine its effect on dendritic cells (DCs) generated from bone marrow suspensions harvested from C57BL/6 mice for which the effect of 4-1BBL on DCs is not clear, especially during DCs processing tumor-associated antigen. Replication deficient adenovirus AdMaxTM Expression System was used to construct recombinant adenovirus Ad-tPSMA-internal ribosome entry site-mouse 4-1BBL (Ad-tPSMA-IRES-m4-1BBL) and Ad-enhanced green fluorescent protein. Day 7 proliferating DC aggregates generated from C57BL/6 mice were collected as immature DCs and further mature DCs were obtained by lipopolysaccharide activated immature DCs. After DCs were exposed to the recombinant adenovirus with 250 multiplicity of infection, the expression of tPSMA and m4-1BBL proteins were detected by Western blot, and the apoptosis and phenotype of DCs were analyzed by flow cytometry. Cytokines (IL-6 and IL-12) in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Proliferation of T cells was detected by allogeneic mixed lymphocyte reactions. The tPSMA and m4-1BBL proteins were expressed correctly. The apoptosis rate of DCs transfected with Ad-tPSMA-IRES-m4-1BBL was 14.6%, lower than that of control DCs. The expression of co-stimulatory molecules [CD80 (81.6 ± 5.4%) and CD86 (80.13 ± 2.81%)] up-regulated in Ad-tPSMA-IRES-m4-1BBL-pulsed DCs, and the level of IL-6 (3960.2 ± 50.54 pg/mL) and IL-12 (249.57 ± 12.51 pg/mL) production in Ad-tPSMA-IRES-m4-1BBL-transduced DCs were significantly higher (P < 0.05) than those in control DCs. Ad-tPSMA-IRES-m4-1BBL induced higher T-cell proliferation (OD450 = 0.614 ± 0.018), indicating that this recombinant adenovirus can effectively enhance the activity of DCs.

Elevated levels of plasma osteoprotegerin are associated with all-cause mortality risk and atherosclerosis in patients with stages 3 to 5 chronic kidney disease

Osteoprotegerin (OPG) regulates bone mass by inhibiting osteoclast differentiation and activation, and plays a role in vascular calcification. We evaluated the relationship between osteoprotegerin levels and inflammatory markers, atherosclerosis, and mortality in patients with stages 3-5 chronic kidney disease. A total of 145 subjects (median age 61 years, 61% men; 36 patients on hemodialysis, 55 patients on peritoneal dialysis, and 54 patients with stages 3-5 chronic kidney disease) were studied. Clinical characteristics, markers of mineral metabolism (including fibroblast growth factor-23 [FGF-23]) and inflammation (high-sensitivity C-reactive protein [hsCRP] and interleukin-6 [IL-6]), and the intima-media thickness (IMT) in the common carotid arteries were measured at baseline. Cardiac function was assessed by color tissue Doppler echocardiography. After 36 months follow-up, the survival rate by Kaplan-Meier analysis was significantly different according to OPG levels (χ2=14.33; P=0.002). Increased OPG levels were positively associated with IL-6 (r=0.38, P<0.001), FGF-23 (r=0.26, P<0.001) and hsCRP (r=0.0.24, P=0.003). In addition, OPG was positively associated with troponin I (r=0.54, P<0.001) and IMT (r=0.39, P<0.0001). Finally, in Cox analysis, only OPG (HR=1.07, 95%CI=1.02-1.13) and hsCRP (HR=1.02, 95%CI=1.01-1.04) were independently associated with increased risk of death. These results suggested that elevated levels of serum OPG might be associated with atherosclerosis and all-cause mortality in patients with chronic kidney disease.

Empirical studies on software quality construction: Exploring human factors and organizational influences

Software quality has become an important research subject, not only in the Information and Communication Technology spheres, but also in other industries at large where software is applied. Software quality is not a happenstance; it is defined, planned and created into the software product throughout the Software Development Life Cycle. The research objective of this study is to investigate the roles of human and organizational factors that influence software quality construction. The study employs the Straussian grounded theory. The empirical data has been collected from 13 software companies, and the data includes 40 interviews. The results of the study suggest that tools, infrastructure and other resources have a positive impact on software quality, but human factors involved in the software development processes will determine the quality of the products developed. On the other hand, methods of development were found to bring little effect on software quality. The research suggests that software quality is an information-intensive process whereby organizational structures, mode of operation, and information flow within the company variably affect software quality. The results also suggest that software development managers influence the productivity of developers and the quality of the software products. Several challenges of software testing that affect software quality are also brought to light. The findings of this research are expected to benefit the academic community and software practitioners by providing an insight into the issues pertaining to software quality construction undertakings.

Challenges of user-driven innovations on late stages of innovation process: evidence from ICT companies

The goal of this thesis is to study user-driven innovations and user involvement throughout the innovation process in context of B2B companies. Significant emphasis in the analysis put onto the late stages of innovation process and commercialization of innovations. Thesis includes detailed review of theoretical concepts and underlying frameworks of innovation process, lead users and user-driven innovations. The empirical part of the thesis consist of interviews of the four companies from ICT industry, followed by the comprehensive analysis and comparison of the results. The presented findings indicate common challenges, which ICT companies face, when shifting towards innovation by users paradigm. Linkages and connections among current situation and theoretical frameworks presented in the discussion part of the thesis allow to draw practical managerial implications. The results of the research emphasize valuable insights and challenges of user interactions within innovation process as well as output and participation related benefits for the companies and users. The research points out current state of the user involvement techniques and tools used for user interactions as well as suggests the possibilities for improvement in the future.

Volatile compounds in noni (Morinda citrifolia L.) at two ripening stages

The volatile components of noni at two ripening stages were isolated by headspace solid-phase microextraction using 65 µm Polydimethylsiloxane-Divinylbenzene (PDMS/DVB) fibers and analyzed using GC/MS. Both maturation stages had several compounds in common. Ninety-six compounds were identified, from which octanoic acid ( 70% of total extract) and hexanoic acid ( 8% of total extract) were found to be the major constituents. Due to noni maturation, octanoic acid, decanoic acid and 2E-nonenal decreased their concentrations, while some esters (methyl hexanoate, methyl octanoate, ethyl octanoate and methyl 4E-decenoate), which their fruity odor notes, increased their contents. Two unsaturated esters, reported for the first time in this fruit, 3-methyl-3-buten-1-yl hexanoate and 3-methyl-3-buten-1-yl octanoate, significantly decreased their concentration in the ripe to over-ripe fruits.

Chemical characterization of leaves of organically grown carrot Dacus carota L.) in various stages of development for use as food

In Brazil, street markets and vegetable distributors discard vegetable leaves and stems, including those of carrot (Dacus carota L.). Seeking to reduce the waste of vegetable parts, this study characterized chemically the leaves of organically grown carrot in three stages of development to determine the best time for their removal and consumption as food. The leaves were dehydrated in an oven at 70 °C for 43 hours and analyzed for chemical composition, antioxidant activity, chlorophyll content, fatty acid composition, and also calcium (Ca), sodium (Na), potassium (K), magnesium (Mg), manganese (Mn), iron (Fe), zinc (Zn), and copper (Cu) contents. The analyses indicated 100 days of development as the ideal stage for the removal and consumption of carrot leaves with good antioxidant activity requiring only 63.78 ± 0.5 mg.L-1 methanol leaf extract to inhibit 50% of the concentration of the free radical DPPH (2,2-diphenyl-1picrilidrazil), and total protein and alpha-linolenic acid (18:3 n-3/LNA) contents of 18.23% ± 2.8 and 876.55 ± 20.62 mg.100 g-1 of dry matter, respectively.

Changes in enzymes, phenolic compounds, tannins, and vitamin C in various stages of jambolan (Syzygium cumini Lamark) development

The physiological state of a fruit is closely related to ripening and climatic conditions during the growing period when the fruit undergo changes in color, texture, and flavor. The ripening of the fruit can involve a complex series of biochemical reactions with alteration in enzymes activities, phenols, tannins, and ascorbic acid. The activity of enzymes (carboximethylcellulase, polygalacturonase, and pectinlyase), the total concentration of phenolic compounds, condensed tannins, and vitamin C in five stages of maturation were studied. Significant changes were observed between the maturity stages. The phenolic compounds were higher at green stage (705.01 ± 7.41); tannins were higher at green/purple stage (699.45 ± 0.22). The results showed that the ascorbic acid levels of the pulp varied significantly from 50.81 ± 1.43 to 6.61 ± 1.04 mg.100 g-1 during maturation. The specific activity of pectin lyase was higher at green stage (1531.90 ± 5.83). The specific activity of polygalacturonase was higher at mature stage (1.83 ± 0.0018). The specific activity of carboximetilcelulose was higher at ripe mature stage (4.61 ± 0.0024). The low ascorbic acid content found in jambolan fruit indicates that this fruit is not a rich source of this nutrient; however, other characteristics can make jambolan products fit for human consumption.

Construction of 2D transparent micromodels in polyester resin with porosity similar to carrots

Microscopic visualization, especially in transparent micromodels, can provide valuable information to understand the transport phenomena at pore scale in different process occurring in porous materials (food, timber, soils, etc.). Micromodels studies focus mainly on the observation of multi-phase flow, which presents a greater proximity to reality. The aim of this study was to study the process of flexography and its application in the manufacture of polyester resin transparent micromodels and its application to carrots. Materials used to implement a flexo station for micromodels construction were thermoregulated water bath, exposure chamber to UV light, photosensitive substance (photopolymer), RTV silicone polyester resin, and glass plates. In this paper, data on size distribution of a particular kind of carrot we used, and a transparent micromodel with square cross-section as well as a Log-normal pore size distribution with pore radii ranging from 10 to 110 µm (average of 22 µm and micromodel size of 10 × 10 cm) were built. Finally, it stresses that it has successfully implemented the protocol processing 2D polyester resin transparent micromodels.

Physicochemical changes in cubiu fruits (Solanum sessiliflorum Dunal) at different ripening stages

Cubiu shrubs (Solanum sessiliflorum Dunal) have drawn the attention of researchers for their biological versatility (preferential heliophilous or facultative ombrophilous shrubs), their capacity to grow in upland or lowland areas, and the good technological quality of their fruits for the food industry. The aim of this study was to verify physicochemical changes in cubiu fruits during maturation. The fruits were harvested from the experimental station of olericulture of the Instituto Nacional de Pesquisas da Amazônia (INPA), Brazil. The analyses were performed in whole cubiu fruits (peel, pulp, and placenta) at four traditional ripening stages (green, turning, ripe, and fully ripe) for the determination of weight, moisture, total solids, total carotenoids, proteins, lipids, and ash. Cubiu fruits showed large weight variation, with amodal distribution. The ripe stage was critical to maintain moisture, and from that stage on, water loss became evident. The lipids increased steadily over the four ripening stages, maintaining, however, insignificant calorie content. Total carotenoids, proteins, and ash reached the maximum level at the fully ripe stage. With the exception of weight and moisture, all physicochemical changes exhibited the same general behavior, i.e. they increased as the fruits ripened at the four investigated stages.

Prevalent fatty acids in cashew nuts obtained from conventional and organic cultivation in different stages of processing

Brazil is one of the three largest producers of fruits in the world, and among those fruit trees, the cashew tree stands out due to the high nutritional and commercial value of its products. During its fruit processing, there are losses in some compounds and few studies address this issue. Over the last decade the conventional system of food production has been substituted for the organic cultivation system, which is a promising alternative source of income given the global demand for healthy food. Therefore, this research aimed to characterize and quantify the prevalent fatty acids found in cashew nuts obtained from conventional and organic cultivation during various stages of processing. The prevalent fatty acids found were palmitic, linoleic, oleic, and stearic acid. The average of these fatty acids were 6.93 ± 0.55; 16.99 ± 0.61; 67.62 ± 1.00 and 8.42 ± 0.55 g/100 g, respectively. There was no reduction in the palmitic, oleic and stearic fatty acid contents during processing. Very little difference was observed between the nuts obtained from conventional and organic cultivation, indicating that the method of cultivation used has little or no influence on the content of cashew nut fatty acids.

Chemical and physical characterization of mume fruit collected from different locations and at different maturity stages in São Paulo State

Prunus mume is widely studied due to its health benefits regarding increase of blood fluidity and consequent improvement of the cardiovascular system and the prevention or even the fight against different types of cancer. However, in Brazil this culture is found only among oriental descendants. The present study aimed to characterize mume fruit collected from three different locations in the State of São Paulo regarding general aspects such as pH, total titratable acidity (TTA), total soluble solids (TTS), pectin content and yield of pulp and chemical characteristics: total phenolic compounds (TPC) and antioxidant capacity. Mume fruit were collected unripe and analyzed until maturation about 88 days after flowering. Fruit collected in Botucatu came from a commercial mume fruit producer and had average weight of 16.9 g, while in fruit from other locations weight varied from 5.7-6.9 g. TSS ranged from 9.5 to 10.0 Brix, total solids was 10.2-12.2% and pH showed values between 2.5 and 2.7 for all locations. TTA expressed in citric acid decreased from 4.0-5.7 g (100g- 1) at unripe stage to 2.0-3.8 g (100g- 1) in mature-stage fruit. Pectin content decreased from 11.2 to 10.8% during fruit maturation, TPC content was 147-226 mg catechin (g- 1) on a dry matter basis and the antioxidant capacity was 96-169 µMol Trolox (g- 1) on a dry matter basis or 21-34 µMol Trolox (g- 1) on a wet matter basis.