A meta-analysis of hairy vetch as a previous cover crop for maize.

Background: The use of hairy vetch (Vicia villosa Roth.) as cover crop is increasing worldwide. Hairy vetch can contribute as a nitrogen (N) source with potential to impact subsequent high N demanding cereals such as maize (Zea mays L.). Contrasting literature results emphasize the need for a global synthesis analysis to quantify changes in maize yield after hairy vetch. Objectives: A meta-analysis was conducted to i) quantify maize yield response to hairy vetch as previous crop, ii) explore hairy vetch influence on fertilized and non-N fertilized maize yields, and iii) assess the tillage and environment factors on maize yield response to hairy vetch. Methods: The global systematic search yielded 23 publications selected by the following criteria, i) hairy vetch dry matter at the end of the season, ii) maize grain yield, and iii) experimental design with (Mzhv) and without (Mzcontrol) hairy vetch treatments. Information such as N fertilization for maize, N accumulation in hairy vetch, organic matter, and tillage before maize sowing were recorded. Hairy vetch effects (effect size) were expressed as a ratio (percentage of grain yield variation in Mzhv/Mzcontrol). Results: Under non-N fertilization (n = 9), results revealed hairy vetch had mostly a positive effect, ranging from 13 to 45% (n = 6). In contrast, N-fertilized maize (n = 20) showed a high chance of neutral effects (n = 12), moderate probability of positive yield impact (7 to 38%, n = 6), and a low likelihood of negative effects (-32 and -17%, n = 2). Notably, maize yields improved by 21-25% when the N accumulation in hairy vetch ranged from 95 to 150 kg ha-1 and N rate from 0 to 120 kg ha-1. Non-N-fertilized maize exhibited a 14% increase in response in no-till systems and a 31% increase with conventional tillage. Conclusion: This study summarizes potential benefits of hairy vetch preceding maize. Yet, the heterogeneous outcomes deserve further exploration in terms of environment and management factors.

The Possible Role of Matrix Metalloprotienase-2 in the Relapse in Patients with Stage II Colon Cancer Treated by Curative Surgery.

AIMS: To determine the association of micro-metastatic matrix metalloproteinase-2 (MMP-2) expression,  the absolute lymphocyte count (ALC)) and outcome in stage II colon cancer. MATERIALS AND METHODS: A single centre, prospective observational study, one month post-surgery blood for ALC, circulating tumour cell (CTC) detection and a bone marrow biopsy for micro-metastasis detection were obtained.  CTCs were detected using differential gel centrifugation and immunocytochemistry with anti-CEA and anti-MMP-2, the bone marrow biopsy for the detection of micro-metastasis was processed as for CTCs . At each follow-up  ALC and CTC counts were determined. Bone marrow sampling was repeated if the ALC decreased by >10%, at relapse or at the end of the study period. Three MRD subgroups were defined, Group I MRD negative, Group II only positive for micro-metastasis and   Group III in which CTCs were detected. RESULTS: One hundred and eighty one patients  participated; 105 (58%) patients formed Group 1, 36 (20%) formed Group II  and 40 (22%)  formed Group III for a median follow-up of 4 years .  Of Group I 3/105 (3%), Group II 16/36 (44%) and Group III 34/40 (84%) patients relapsed. The ALC was significantly higher in Groups I and II, the expression of MMP-2 and MMP-2 score in Group II was significantly lower than in Group III patients. A low ALC was associated with a higher expression of MMP-2 in the micro-metastasis and presence of CTCs. CONCLUSIONS: Patients with stable ALCs did not relapse; decreasing ALCs were associated with increasing MMP-2 scores, the appearance of CTCs and relapse.

Self-collected saliva for SARS-CoV-2 detection: A prospective study in the emergency room.

Current diagnostic standards involve severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in nasopharyngeal swabs (NPS), but saliva is an attractive and noninvasive option for diagnosis. The objectives were to determine the performance of saliva in comparison with NPS for detecting SARS-CoV-2 and to compare the optimized home brew reverse-transcription polymerase chain reaction (RT-PCR) with a commercial RT-PCR. Paired NPS and saliva specimens were prospectively collected and tested by RT-PCR from patients presenting at an emergency room with signs and symptoms compatible with coronavirus disease-2019. A total of 348 samples from 174 patients were tested by RT-PCR assays. Among 174 patients with symptoms, 63 (36%) were SARS-CoV-2 positive in NPS using the optimized home-brew PCR. Of these 63 patients, 61 (98%) were also positive in saliva. An additional positive SARS-CoV-2 saliva was detected in a patient with pneumonia. Kappa Cohen's coefficient agreement between NPS and saliva was 0.96 (95% confidence interval [CI], 0.90-0.99). Median Ct values in NPS versus saliva were 18.88 (interquartile range [IQR], 15.60-23.58; range, 11.97-38.10) versus 26.10 (IQR, 22.75-30.06; range, 13.78-39.22), respectively (p < .0001). The optimized home-brew RT-PCR demonstrated higher analytical and clinical sensitivity compared with the commercial RT-PCR assay. A high sensitivity (98%) and agreement (kappa 0.96) in saliva samples compared to NPS was demonstrated when using an optimized home-brew PCR even when the viral load in saliva was lower than in NPS. This noninvasive sample is easy to collect, requires less consumable and avoids discomfort to patients. Importantly, self-collection of saliva can diminish exposure to healthcare personnel.

Differential response of pineal microglia to surgical versus pharmacological stimuli.

Microglial cells are one of the interstitial elements of the pineal gland (PG). We recently reported the pattern of microglia colonization and activation, and microglia-Pax6+ cell interactions during normal pineal ontogeny. Here, we describe the dynamics of microglia-Pax6+ cell associations and interactions after surgical or pharmacological manipulation. In adult rats, the superior cervical ganglia (SCG) were exposed, and either bilaterally excised (SCGx) or decentralized (SCGd). In the SCGx PGs, the density of Iba1+ microglia increased after surgery and returned to sham baseline levels 13 days later. Pineal microglia also responded to SCGd, a more subtle denervation. The number of clustered Iba1+ /PCNA+ /ED1+ microglia was higher 4 days after both surgeries compared to the sham-operated group. However, the number of Pax6+ /PCNA- cells and the percentage of Pax6+ cells contacted by and/or phagocytosed by microglia increased significantly only after SCGx. Separate groups of rats were treated with either bacterial lipopolysaccharides (LPS) or doxycycline (DOX) to activate or inhibit pineal microglia, respectively. Peripheral LPS administration caused an increase in the number of clustered Iba1+ /PCNA+ /ED1+ microglial cells, and in the percentage of Pax6+ cells associated with and/or engulfed by microglia. In the LPS-treated PGs, we also noted an increase in the number of PCNA+ cells that were Iba1- within the microglial cell clusters. The density of Pax6+ cells did not change after LPS treatment. DOX administration did not influence the parameters analyzed. These data suggest that pineal microglia are highly receptive cells capable of rapidly responding in a differential manner to surgical and pharmacological stimuli.

A single dose of the novel chimeric subunit vaccine E2-CD154 confers early full protection against classical swine fever virus.

Classical swine fever is an economically important, highly contagious disease of swine worldwide. Subunit vaccines are a suitable alternative for the control of classical swine fever. However, such vaccines have as the main drawback the relatively long period of time required to induce a protective response, which hampers their use under outbreak conditions. In this work, a lentivirus-based gene delivery system is used to obtain a stable recombinant HEK 293 cell line for the expression of E2-CSFV antigen fused to porcine CD154 as immunostimulant molecule. The E2-CD154 chimeric protein was secreted into the medium by HEK293 cells in a concentration around 50mg/L in suspension culture conditions using spinner bottles. The E2-CD154 immunized animals were able to overcome the challenge with a high virulent CSF virus strain performed 7days after a unique dose of the vaccine without clinical manifestations of the disease. Specific anti-CSFV neutralizing antibodies and IFN-γ were induced 8days after challenge equivalent to 14days post-vaccination. The present work constitutes the first report of a subunit vaccine able to confer complete protection by the end of the first week after a single vaccination. These results suggest that the E2-CD154 antigen could be potentially used under outbreak conditions to stop CSFV spread and for eradication programs in CSF enzootic areas.

Cellular Basis of Pineal Gland Development: Emerging Role of Microglia as Phenotype Regulator.

The adult pineal gland is composed of pinealocytes, astrocytes, microglia, and other interstitial cells that have been described in detail. However, factors that contribute to pineal development have not been fully elucidated, nor have pineal cell lineages been well characterized. We applied systematic double, triple and quadruple labeling of cell-specific markers on prenatal, postnatal and mature rat pineal gland tissue combined with confocal microscopy to provide a comprehensive view of the cellular dynamics and cell lineages that contribute to pineal gland development. The pineal gland begins as an evagination of neuroepithelium in the roof of the third ventricle. The pineal primordium initially consists of radially aligned Pax6+ precursor cells that express vimentin and divide at the ventricular lumen. After the tubular neuroepithelium fuses, the distribution of Pax6+ cells transitions to include rosette-like structures and later, dispersed cells. In the developing gland all dividing cells express Pax6, indicating that Pax6+ precursor cells generate pinealocytes and some interstitial cells. The density of Pax6+ cells decreases across pineal development as a result of cellular differentiation and microglial phagocytosis, but Pax6+ cells remain in the adult gland as a distinct population. Microglial colonization begins after pineal recess formation. Microglial phagocytosis of Pax6+ cells is not common at early stages but increases as microglia colonize the gland. In the postnatal gland microglia affiliate with Tuj1+ nerve fibers, IB4+ blood vessels, and Pax6+ cells. We demonstrate that microglia engulf Pax6+ cells, nerve fibers, and blood vessel-related elements, but not pinealocytes. We conclude that microglia play a role in pineal gland formation and homeostasis by regulating the precursor cell population, remodeling blood vessels and pruning sympathetic nerve fibers.

Energy expenditure and intake comparisons in Chilean children 4-5 years attending day-care centres / Comparación entre el gasto y el consumo de energía en niños chilenos de 4-5 años asistentes a jardines infantiles

Introduction: the doubly labelled water (DLW) method has an accuracy of 1% and within-subject precision of 5-8%, depending on subject’s age and environments issues. Energy intake assessment is prone to errors (>15- 20%) depending in the method utilized. Objective: to quantify DLW methodology errors in four to five year olds that could affect the comparison with energy intake. Methods: energy expenditure (TEE, by DLW), was assessed during 14 days in 18 preschool children, who attended eight hours daily to day-care centres. Energy intake was determined by a combined method: food weighing during weekdays and recall after leaving the Centre (17h to sleep time) plus 24 h recall, during the weekend. Several assumptions affecting DLW total error were assessed to determine their influence in the comparison to energy intake (i.e. background variability, space ratio, proportion of water subject to fractionation, food quotient value). Results: the individual mean energy expenditure was 1 373 ± 177 kcal and the energy intake (1 409 ± 161 kcal). The overall difference between intake and expenditure was 42.9 kcal/day (limits of agreement + 259.1 to -112.3 kcal/day). TEE measurement error only explained a minor quantity (2.4%), between both measurements, and the observed mean isotope dilution space was 1.030 ± 0.010 confirming the value utilized in adults studies. Conclusions: energy expenditure data is similar to other studies in preschool children. The small difference found between energy intake and expenditure may be attributed to the applied energy intake methodology, the homogeneous diet at care centres during the week-days and the lower DLW methodology error (AU)

Introducción: el método del agua doblemente marcada (ADM) tiene una precisión del 1% y en un mismo sujeto es de 5-8%, dependiendo de la edad y el entorno del sujeto. La evaluación de la ingesta energética es propensa a errores (> 15-20%), dependiendo del método utilizado. Objetivo: cuantificar los errores metodológicos del ADM en niños de 4-5 años que podrían afectar la comparación con la ingesta de energía. Métodos: el gasto de energía (GTE, por ADM), se evaluó durante 14 días en 18 preescolares, asistentes a guarderías infantiles. La ingesta energética se determinó mediante un método combinado: pesaje de alimentos durante los días de la semana y registro después de salir del centro (17 horas en adelante), además de un recordatorio de 24 horas, durante un día del fin de semana. Resultados: el promedio individual del gasto energético total fue 1373 ± 177 kcal y la ingesta de energía (1.409 ± 161 kcal). La diferencia global entre la ingesta y el gasto fue 42,9 kcal/día. El error de medición del GET explicó una variación del 2,4%, entre ambas mediciones, y el espacio de dilución de isótopos fue 1030 ± 0.010, confirmando el valor utilizado en los estudios de adultos. Conclusiones: los datos de GET fueron similares a otros estudios realizados en niños en edad preescolar. La pequeña diferencia encontrada entre la ingesta y el gasto energético se puede atribuir a la metodología de la ingesta de energía aplicada, la dieta homogénea en los centros de atención, durante los días de la semana, y el bajo error metodológico del ADM (AU)

ENERGY EXPENDITURE AND INTAKE COMPARISONS IN CHILEAN CHILDREN 4-5 YEARS ATTENDING DAY-CARE CENTRES.

INTRODUCTION: the doubly labelled water (DLW) method has an accuracy of 1% and within-subject precision of 5-8%, depending on subject's age and environments issues. Energy intake assessment is prone to errors (>15- 20%) depending in the method utilized. OBJECTIVE: to quantify DLW methodology errors in four to five year olds that could affect the comparison with energy intake. METHODS: energy expenditure (TEE, by DLW), was assessed during 14 days in 18 preschool children, who attended eight hours daily to day-care centres. Energy intake was determined by a combined method: food weighing during weekdays and recall after leaving the Centre (17h to sleep time) plus 24 h recall, during the weekend. Several assumptions affecting DLW total error were assessed to determine their influence in the comparison to energy intake (i.e. background variability, space ratio, proportion of water subject to fractionation, food quotient value). RESULTS: the individual mean energy expenditure was 1 373 ± 177 kcal and the energy intake (1 409 ± 161 kcal). The overall difference between intake and expenditure was 42.9 kcal/day (limits of agreement + 259.1 to -112.3 kcal/day). TEE measurement error only explained a minor quantity (2.4%), between both measurements, and the observed mean isotope dilution space was 1.030 ± 0.010 confirming the value utilized in adults studies. CONCLUSIONS: energy expenditure data is similar to other studies in preschool children. The small difference found between energy intake and expenditure may be attributed to the applied energy intake methodology, the homogeneous diet at care centres during the week-days and the lower DLW methodology error.

Introducción: el método del agua doblemente marcada (ADM) tiene una precisión del 1% y en un mismo sujeto es de 5-8%, dependiendo de la edad y el entorno del sujeto. La evaluación de la ingesta energética es propensa a errores (> 15-20%), dependiendo del método utilizado. Objetivo: cuantificar los errores metodológicos del ADM en niños de 4-5 años que podrían afectar la comparación con la ingesta de energía. Métodos: el gasto de energía (GTE, por ADM), se evaluó durante 14 días en 18 preescolares, asistentes a guarderías infantiles. La ingesta energética se determinó mediante un método combinado: pesaje de alimentos durante los días de la semana y registro después de salir del centro (17 horas en adelante), además de un recordatorio de 24 horas, durante un día del fin de semana. Resultados: el promedio individual del gasto energético total fue 1373 ± 177 kcal y la ingesta de energía (1409 ± 161 kcal). La diferencia global entre la ingesta y el gasto fue 42,9 kcal/día. El error de medición del GET explicó una variación del 2,4%, entre ambas mediciones, y el espacio de dilución de isótopos fue 1030 ± 0.010, confirmando el valor utilizado en los estudios de adultos. Conclusiones: los datos de GET fueron similares a otros estudios realizados en niños en edad preescolar. La pequeña diferencia encontrada entre la ingesta y el gasto energético se puede atribuir a la metodología de la ingesta de energía aplicada, la dieta homogénea en los centros de atención, durante los días de la semana, y el bajo error metodológico del ADM.

Influence of adhesive systems on microtensile bond strength of resin-based endodontic sealers to the root dentin.

OBJECTIVE: The aim of this study was to determine the microtensile bond strength to root dentin of AH Plus™ and EndoREZ® with Clearfil Liner Bond 2V and Optibond Solo™ Plus adhesive systems. STUDY DESIGN: The coronal and middle thirds of six single rooted bovine teeth was split longitudinally in a mesio-distal direction. The two halves were joined with AH Plus or EndoREZ, with and without the use of Clearfil Liner Bond 2V and Optibond Solo™ Plus adhesive systems. Build-ups were vertically sectioned into quadrangular (≈1mmx1mm) compound bars and subjected to tensile tests at a constant crosshead speed (1 mm/min) until debonding. RESULTS: Optibond® Solo Plus™ in combination with AH Plus™ and EndoREZ® showed the highest mean microtensile bond strength values, in both coronal and middle thirds. The lowest results were seen in the groups where no dentine adhesive was applied, and in those where the self-etching adhesive Clearfil Liner Bond 2V was used. CONCLUSION: The microtensile bond strength to root dentin of AH Plus™ and EndoREZ may be increased with the use of a total-etch adhesive. Key words:Adhesive systems, AH Plus, EndoREZ, microtensile bond strength, root dentin.

Evolution of vegetation and soil nutrients after uranium mining in Los Ratones mine (Cáceres, Spain).

The evolution of vegetation structure following mine rehabilitation is rather scarce in the literature. The concentration of long-lived radionuclides of the (238)U series might have harmful effects on living organisms. We studied soil properties and the natural vegetation occurring along a gradient in Los Ratones, an area rehabilitated after uranium mining located in Cáceres, Spain. Soil and vegetation were sampled seasonally and physical and chemical properties of soil were analysed, including natural isotopes of (238)U, (230)Th, (226)Ra and (210)Pb. Species richness, diversity, evenness and plant cover were estimated and correlated in relation to soil physical and chemical variables. The location of the sampling sites along a gradient had a strong explanatory effect on the herbaceous species, as well as the presence of shrubs and trees. Seasonal effects of the four natural isotopes were observed in species richness, species diversity and plant cover; these effects were directly related to the pH values in the soil, this being the soil property that most influences the plant distribution. Vegetation in the studied area resembles that of the surroundings, thus proving that the rehabilitation carried out in Los Ratones mine was successful in terms of understorey cover recovery.

Procedure for microbial identification based on Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry from screening-positive urine samples.

Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry is a widely used proteomic technique in clinical microbiology laboratories, and enables microbial identification directly from clinical samples. This study seeks to establish a protocol for bacterial identification from monomicrobial urine samples that have tested positive in the screening with Sysmex UF-1000i (Sysmex Corporation, Kobe, Japan). Sysmex UF-1000i counts ≥1 × 10(7) bacteria/mL indicate a sufficient bacterial concentration to allow direct identification from urine, with 87.5% sensitivity. Microbial identification from urine with Sysmex UF-1000i counts between 1 × 10(5) and 1 × 10(7) bacteria/ml requires preincubation to obtain the adequate amount of bacteria needed for analysis, and 91.7% sensitivity thus being achieved.

Sequence characterization, in silico mapping and cytosine methylation analysis of markers linked to apospory in Paspalum notatum.

In previous studies we reported the identification of several AFLP, RAPD and RFLP molecular markers linked to apospory in Paspalum notatum. The objective of this work was to sequence these markers, obtain their flanking regions by chromosome walking and perform an in silico mapping analysis in rice and maize. The methylation status of two apospory-related sequences was also assessed using methylation-sensitive RFLP experiments. Fourteen molecular markers were analyzed and several protein-coding sequences were identified. Copy number estimates and RFLP linkage analysis showed that the sequence PnMAI3 displayed 2-4 copies per genome and linkage to apospory. Extension of this marker by chromosome walking revealed an additional protein-coding sequence mapping in silico in the apospory-syntenic regions of rice and maize. Approximately 5 kb corresponding to different markers were characterized through the global sequencing procedure. A more refined analysis based on sequence information indicated synteny with segments of chromosomes 2 and 12 of rice and chromosomes 3 and 5 of maize. Two loci associated with apomixis locus were tested in methylation-sensitive RFLP experiments using genomic DNA extracted from leaves. Although both target sequences were methylated no methylation polymorphisms associated with the mode of reproduction were detected.

Human αIFN co-formulated with milk derived E2-CSFV protein induce early full protection in vaccinated pigs.

Subunit vaccines are a suitable alternative for the control of classical swine fever. However, such vaccines have as the main drawback the relatively long period of time required to induce a protective response, which hampers their use under outbreak conditions. In this work, type I interferon is used as an immunostimulating molecule in order to increase the immunogenicity of a vaccine candidate based on the E2-CSFV antigen produced in goat milk. Pigs vaccinated with E2-CSFV antigen co-formulated with recombinant human alpha interferon were protected against clinical signs and viremia as early as 7 days post-vaccination. It was also demonstrated that interferon stimulates a response of specific anti-CSFV neutralizing antibodies. The present work constitutes the first report of a subunit vaccine able to confer complete protection by the end of the first week after vaccination. These results suggest that the E2-CSFV antigen combined with type I interferons could be potentially used under outbreak conditions to stop CSFV spread and for eradication programs in CSF enzootic areas.

Early onset and long lasting protection in pigs provided by a classical swine fever E2-vaccine candidate produced in the milk of goats.

For vaccination against classical swine fever virus (CSFV), it is strongly desirable to induce a rapid and long lasting protection. At present, only live attenuated CSFV vaccines have shown early onset of protection, differing with the recombinant subunit-based vaccines reported so far. Recently, a new vaccine formulation based on E2 envelope viral glycoprotein produced in the milk of goats (E2his) has been shown to induce a highly protective response in pigs against CSFV infection. Pigs immunized with a single dose of this vaccine candidate, formulated as a water-in oil emulsion, elicited an effective response against CSF as early as 7 days post-vaccination. No severe CSF clinical signs were observed and no animals died although the challenge dose was 10(5)PDL(50) of a highly pathogenic CSFV strain. Noticeably, this response completely prevented CSFV infection in pigs when they were challenged under the same conditions 2 weeks after a single dose of the recombinant E2his vaccine formulation. A schedule consisting of a primary immunization with the same vaccine candidate, followed by a booster dose 2 weeks later induced a highly protective response against CSFV infection for as long as 9 months post-vaccination. These promising results demonstrate by far the feasibility of using the E2his-based vaccine in regional programs for preventing and controlling CSF.

Thermal stress treatment does not affect the stability and protective capacity of goat milk derived E2-marker vaccine formulation against CSFV.

Classical swine fever virus produces a huge mortality in infected herds during recurrent outbreaks, predominantly in tropical and subtropical areas. In this scenario, it is common that cold-chain related issues affect the efficacy of virus attenuated-derived vaccines, which are frequently used in eradication programs. In the present work, the stability and protective capacity of a recombinant vaccine preparation, based on goat milk derived E2 glycoprotein extracellular domain, were both analyzed after incubation at 4 degrees C or 37 degrees C for 1 week. Differences in the viscosity and in the homodimeric form of the antigen were observed after comparing physicochemical properties of stressed and not stressed vaccine formulations. However, these differences did not affect the immunogenicity and protective capacity of such preparations. Noticeably, pigs immunized with the E2-based vaccine subjected to thermal stress became totally protected from the viral infection, after a challenge with 10(5) PLD(50) of a high virulent classical swine fever strain. This result supports the practical value of this vaccine preparation mostly for those regions in which cold-chain related failures tend to affect the protective capability of conventional virus attenuated vaccines.

Biochemical and structural characterization of RHDV capsid protein variants produced in Pichia pastoris: advantages for immunization strategies and vaccine implementation.

Rabbit hemorrhagic disease virus (RHDV) VP60 capsid protein was recently expressed at approximately 1.5 gL(-1) associated with the disruption pellet of Pichia pastoris, thus requiring an additional process of extraction by solubilization. Consequently, the expression of a soluble variant of VP60 was undertaken in order to attain an easier approach for vaccine production. The VP60 gene was cloned without secretion signal under the transcriptional control of the AOX1 yeast promoter. The antigen obtained was intracellular and soluble at approximately 480 mg L(-1). Its characterization by size-exclusion HPLC, ultracentrifugation, and electron microscopy, showed the presence of high molecular weight structures similar in mass, size and buoyant density to native RHDV. The antigenic profile was similar to that from authentic virions as determined with monoclonal antibodies directed against RHDV conformational epitopes. These analyses, conducted on VP60 obtained insoluble in P. pastoris revealed the formation of protein aggregates rather than the presence of ordered multimeric structures. An immunization trial was conducted in which the soluble VP60 was employed by subcutaneous (s.c.) injection either purified by a single chromatographic step or contained within raw disruption supernatant, emulsified in Montanide 888. The insoluble variant was administered as a yeast extract powder by oral and s.c. routes. The earliest IgG response, titers and persistence of antibodies were studied by competition ELISA and hemagglutination inhibition (HI) assays. All rabbits immunized with the yeast-derived antigens developed a strong RHDV-specific response (including the "RHDVa" subtype) that lasted over one year after the primary immunization. Early HI titers up to 1/40 960 were generated. The immune response was similar to that induced by VP60 from Sf9 cells and superior to the response elicited with inactivated RHDV. Overall it was found that the soluble VP60 multimers, safely and easily produced in P. pastoris, are a valuable candidate for the rational implementation of a low-cost, scalable subunit vaccine against RHDV.

Classical swine fever virus E2 glycoprotein antigen produced in adenovirally transduced PK-15 cells confers complete protection in pigs upon viral challenge.

E2 is the major envelope glycoprotein present on the outer surface of the classical swine fever virus (CSFV). It is exposed as a homodimer originated by disulfide linkage and represents an important target for the induction of neutralizing immune responses against the viral infection. The E2his glycoprotein nucleotide sequence used in this work contains the CSFV E2 extracellular domain preceded by the tissue plasminogen signal peptide and a hexa-histidine tag in the 3' terminus. The recombinant antigen was produced at a range of 120-150 microg/mL in the culture media of epithelial kidney pig cells, transduced with a replication defective adenoviral vector (Ad-E2his) generated by means of cloning the E2his sequence in the vector genome. The glycoprotein was obtained from clarified culture media as a homodimer of 110 kDa with purity over 95% after a single affinity chromatography step in Ni-NTA Agarose column. The E2his characterization by lectin-specific binding assay showed the presence of N-linked oligosaccharides of both hybrid and complex types. The protective capacity of E2his was demonstrated in two immunization and challenge experiments in pigs using doses of 15 or 30 microg of the glycoprotein, emulsified in Freund's adjuvant. The intramuscular immunization followed by a unique boost three weeks later, elicited high titers of neutralizing antibodies between the second and the fourth week after the primary vaccination. The immunized animals were fully protected from the viral infection after challenge with 10(5) PLD(50) of homologous CSFV "Margarita" strain administered by intramuscular injection. Consequently, no clinical signs of the disease or viral isolation from lymphocytes were detected in the vaccinated pigs. These results suggest that the E2his antigen produced in mammalian cells may be a feasible vaccine candidate for CSF prevention.

Highly protective E2-CSFV vaccine candidate produced in the mammary gland of adenoviral transduced goats.

Classical swine fever virus is the etiological agent of the most economically important highly contagious disease of swine worldwide. E2 is the major envelope glycoprotein present as a homodimer on the outer surface of the virus and represents an important target for the induction of neutralizing immune response against the viral infection. The E2 extracellular domain was expressed in the milk of adenoviral transduced goats at the highest level about 1.2g/L. The recombinant glycoprotein was purified from clarified serum milk by a single metal chelate affinity chromatography step, as a homodimer of approximately 100kDa and purity over 98%. Glycosylation analysis showed the presence of oligomannoside, hybrid and complex type N-glycans, attached to the recombinant E2. The capacity of goat milk-derived E2 antigen to protect pigs from both classical swine fever clinical signs and viral infection was assessed in a vaccination and challenge trial. The immunized pigs became protected after challenge with 10(5) LD(50) of a highly pathogenic CSFV strain. In the context of veterinary vaccines, this expression system has the advantages that the recombinant antigen could be harvested in about 48h after adenoviral transduction with expression levels in the range of g/L. This approach may turn into a scalable expression system for the assessment and production of veterinary vaccines.

Comparison of regional bond strength in root thirds among fiber-reinforced posts luted with different cements.

AIM: To compare regional bond strength in different thirds of the root canal, among glass fiber-reinforced (FRC) endodontic posts luted with different cements, using the push-out test. MATERIAL AND METHODS: Sixty extracted human anterior teeth were endodontically treated with gutta-percha and AH Plus sealer. The crown portion was removed, and a dowel space was prepared. Prepared teeth were randomly assigned to one of six groups (n = 10) for luting glass FRC Postec posts, with one of the six cement systems (Ketac Cem Aplicap, Relyx Unicem Aplicap, Variolink II/Excite DCS, Panavia F/ED Primer, C&B cement/All-Bond 2, and Multilink/Multilink Primer A/B), using an alignment technique. Specimens were embedded in resin, and each root was sectioned into six 1-mm thick serial slices. A push-out test was performed to measure regional bond strengths and to identify the type of failure. RESULTS: The highest bond strength values were found in the cervical third and the lowest in the apical third. Highest values were obtained using Variolink II, Panavia F, and Multilink resin cements followed by C&B resin cement and Relyx Unicem ionomer resin cement; Ketac-cem ionomer cement showed the lowest value. CONCLUSION: Highest bond strength values were obtained in the cervical third and with resin cements.

High expression level of recombinant human erythropoietin in the milk of non-transgenic goats.

The high degree of structural conservation of erythropoietin between species, make it, especially, difficult to produce this protein growth factor in the milk of transgenic animals. Here, we show that through the direct transduction of the mammary epithelium, it is possible to produce high levels of recombinant human erythropoietin in the milk of non-transgenic goats without causing harm to the animals. The efficiency of viral transduction was improved through a temporal disruption of tight-junctions with EGTA allowing for the expression of human erythropoietin at levels of up to 2g/L in milk. The human erythropoietin was purified from the milk using a multi-step protocol involving milk clarification, two precipitation steps and two affinity chromatographies, with a yield of about 70% and purity over 98%. However, the human erythropoietin expressed in milk was underglycosylated, which seems to be the main cause for its low in vivo hematopoietic activity. Nonetheless, these results demonstrate that through the direct transduction of the mammary epithelium it is possible to produce potentially toxic proteins in milk, at levels high enough for their purification and biological characterization.