Picoliter-scale protein microarrays by laser direct write

We demonstrate the accurate picoliter-scale dispensing of active proteins using a novel laser transfer technique. Droplets of protein solution are dispensed onto functionalized glass slides and into plastic microwells, activating as small as 50-microm diameter areas on these surfaces. Protein microarrays fabricated by laser transfer were assayed using standard fluorescent labeling techniques to demonstrate successful protein and antigen binding. These results indicate that laser transfer does not damage the active site of the dispensed protein and that this technique can be used to successfully fabricate a functioning protein microarray. Also, as a result of the efficient nature of the process, material usage is reduced by two to four orders of magnitude compared to conventional pin dispensing methods for protein spotting.     

Serum hemorphin‐7 levels are decreased in obesity

Objective:

Hemorphin peptides exhibit biological activities that interfere with the endorphin system, the inflammatory response, and blood‐pressure control. VV‐hemorphin‐7 and LVV‐hemorphin‐7 peptides exert a hypotensive effect, in particular, by inhibiting the renin–angiotensin system. Furthermore, levels of circulating hemorphin‐7 peptides have been found to be decreased in diseases such as type 1 and type 2 diabetes.

Design and Methods:

Because type 2 diabetes and obesity share common features, such as insulin resistance, microinflammation, high glomerular‐filtration rate (GFR), and cardiovascular risk, we evaluated serum VV‐hemorphin‐7 like immunoreactivity (VVH7‐i.r.) levels, using an enzyme‐linked immunosorbent assay method, on a group of 54 obese subjects without diabetes or hypertension, compared with a group of 33 healthy normal‐weight subjects.

Results:

Circulating VVH7‐i.r. levels were significantly decreased in the obese group compared with the control group (1.98 ± 0.19 vs. 4.86 ± 0.54 µmol/l, respectively, P < 0.01), and a significant negative correlation between VVH7‐i.r. and diastolic blood pressure (DBP) was found in obese patients (r = ?0.35, P = 0.011). There was no significant correlation between VVH7‐i.r. level and insulin resistance, metabolic syndrome, or GFR.

Conclusions:

The decreased serum hemorphin‐7 found in obese subjects, as in diabetes, may contribute to the development of hypertension and to the cardiovascular risk associated with these metabolic diseases.

     

An example of molecular co-evolution: reactive oxygen species (ROS) and ROS scavenger levels in Schistosoma mansoni/Biomphalaria glabrata interactions

The co-evolution between hosts and parasites involves huge reciprocal selective pressures on both protagonists. However, relatively few reports have evaluated the impact of these reciprocal pressures on the molecular determinants at the core of the relevant interaction, such as the factors influencing parasitic virulence and host resistance. Here, we address this question in a host-parasite model that allows co-evolution to be monitored in the field: the interaction between the mollusc, Biomphalaria glabrata, and its trematode parasite, Schistosoma mansoni. Reactive oxygen species (ROS) produced by the haemocytes of B. glabrata are known to play a crucial role in killing S. mansoni. Therefore, the parasite must defend itself against oxidative damage caused by ROS using ROS scavengers in order to survive. In this context, ROS and ROS scavengers are involved in a co-evolutionary arms race, and their respective production levels by sympatric host and parasite could be expected to be closely related. Here, we test this hypothesis by comparing host oxidant and parasite antioxidant capabilities between two S. mansoni/B. glabrata populations that have co-evolved independently. As expected, our findings show a clear link between the oxidant and antioxidant levels, presumably resulting from sympatric co-evolution. We believe this work provides the first supporting evidence of the Red Queen Hypothesis of reciprocal evolution for functional traits at the field-level in a model involving a host and a eukaryotic parasite.     

Partial deletion of 4p16 band in a ring chromosome and wolf syndrome

Summary A new case of ring chromosome 4 in a 2-day-old female child with multiple malformations is described. By means of the GTG-banding technique, a karyotype 46,XX,r(4), (p16q35) was determined. The characteristics of the child's karyotype and the relationship with the structure of the chromosome, especially the location of the deletion that produces the syndrome, are compared with previous reports.     

Genetic diversity in Spanish donkey breeds using microsatellite DNA markers

Genetic diversity at 13 equine microsatellite loci was compared in five endangered Spanish donkey breeds: Andaluza, Catalana, Mallorquina, Encartaciones and Zamorano-Leonesa. All of the equine microsatellites used in this study were amplified and were polymorphic in the domestic donkey breeds with the exception of HMS1, which was monomorphic, and ASB2, which failed to amplify. Allele number, frequency distributions and mean heterozygosities were very similar among the Spanish donkey breeds. The unbiased expected heterozygosity (H_E) over all the populations varied between 0.637 and 0.684 in this study. The low G_ST value showed that only 3.6% of the diversity was between breeds (P < 0.01). Significant deviations from Hardy-Weinberg equilibrium were shown for a number of locus-population combinations, except HMS5 that showed agreement in all analysed populations. The cumulative exclusion probability (PE) was 0.999 in each breed, suggesting that the loci would be suitable for donkey parentage testing. The constructed dendrogram from the D_A distance matrix showed little differentiation between Spanish breeds, but great differentiation between them and the Moroccan ass and also with the horse, used as an outgroup. These results confirm the potential use of equine microsatellite loci as a tool for genetic studies in domestic donkey populations, which could also be useful for conservation plans.     

A polygalacturonase-inhibiting protein from grapevine reduces the symptoms of the endopolygalacturonase BcPG2 from Botrytis cinerea in Nicotiana benthamiana leaves without any evidence for in vitro interaction

Six endopolygalacturonases from Botrytis cinerea (BcPG1 to BcPG6) as well as mutated forms of BcPG1 and BcPG2 were expressed transiently in leaves of Nicotiana benthamiana using agroinfiltration. Expression of BcPG1, BcPG2, BcPG4, BcPG5, and mutant BcPG1-D203A caused symptoms, whereas BcPG3, BcPG6, and mutant BcPG2-D192A caused no symptoms. Expression of BcPG2 caused the most severe symptoms, including wilting and necrosis. BcPG2 previously has been shown to be essential for B. cinerea virulence. The in vivo effect of this enzyme and the inhibition by a polygalacturonase-inhibiting protein (PGIP) was examined by coexpressing Bcpg2 and the Vvpgipl gene from Vitis vinifera in N. benthamiana. Coinfiltration resulted in a substantial reduction of the symptoms inflicted by the activity of BcPG2 in planta, as evidenced by quantifying the variable chlorophyll fluorescence yield. In vitro, however, no interaction between pure VvPGIP1 and pure BcPG2 was detected. Specifically, VvPGIP1 neither inhibited BcPG2 activity nor altered the degradation profile of polygalacturonic acid by BcPG2. Furthermore, using surface plasmon resonance technology, no physical interaction between VvPGIP1 and BcPG2 was detected in vitro. The data suggest that the in planta environment provided a context to support the interaction between BcPG2 and VvPGIP1, leading to a reduction in symptom development, whereas neither of the in vitro assays detected any interaction between these proteins.     

Morpho-physiology and maize grain yield under periodic soil flooding in successive selection cycles

This study was carried out to evaluate maize plants of different recurrent selection cycles of the variety (Zea mays L.—Saracura-BRS-4154) regarded to genetic gains, morphophysiology, and grain yield, achieved over the selection cycles under intermittent flooding of the soil. This variety has the capacity to survive and produce in temporarily flooded soils and was developed by the National Maize and Sorghum Research Center (EMBRAPA). The experiment was conducted in greenhouse by using ten alternating selection cycles (Cycle 1–18) and BR 107 a variety known for its susceptibility to flooding. The flooding initiated at six-leaf stage by applying 50 mm of water three times a week. At flowering, the following parameters were evaluated: rate of leaf photosynthesis, stomatal conductance, intercellular CO₂ concentration, transpiration rate, photosynthetically active leaf area, root porosity, relative chlorophyll content, grain yield, harvest index, ear length, and interval between male and female flowering. Yield as a function of root porosity and photosynthesis were also evaluated. An index was created in this study, in order to help the discussion of the characteristics evaluated, it was called “Relative Tolerance Value—RTV”, only gaseous exchange measurements was not included in this index. By the way, it was observed throughout the selection cycles an increase in all gaseous exchange parameters, being the cycle 18, the one which presented the greatest averages. RTV for leaf area showed the greatest values for cycles 7 and 18, whereas root porosity, chlorophyll relative content, and harvest index, the greater RTV values were found in cycles 17 and 18. The largest grain yield RTV was observed in cycle 7, followed by cycles 13, 15, and 18. Flooding resulted in longer Anthesis-Silking Interval, especially for the first cycles. At flooding condition, grain yield was strongly related to root porosity (R ² = 0.66). These results showed that the selection cycles of “Saracura” maize improved some morphophysiologic characters, which favor their survival in flooded environments, also resulting in higher productivity.     

A comparison of soil carbon pools and profiles in wetlands in Costa Rica and Ohio

Wetlands are large carbon pools and play important roles in global carbon cycles as natural carbon sinks. This study analyzes the variation of total soil carbon with depth in two temperate (Ohio) and three tropical (humid and dry) wetlands in Costa Rica and compares their total soil C pool to determine C accumulation in wetland soils. The temperate wetlands had significantly greater (P < 0.01) C pools (17.6 kg C m⁻²) than did the wetlands located in tropical climates (9.7 kg C m⁻²) in the top 24 cm of soil. Carbon profiles showed a rapid decrease of concentrations with soil depth in the tropical sites, whereas in the temperate wetlands they tended to increase with depth, up to a maximum at 18–24 cm, after which they started decreasing. The two wetlands in Ohio had about ten times the mean total C concentration of adjacent upland soils (e.g., 161 g C kg⁻¹ were measured in a central Ohio isolated forested wetland, and 17 g C kg⁻¹ in an adjacent upland site), and their soil C pools were significantly higher (P < 0.01). Among the five wetland study sites, three main wetland types were identified – isolated forested, riverine flow-through, and slow-flow slough. In the top 24 cm of soil, isolated forested wetlands had the greatest pool (10.8 kg C m⁻²), significantly higher (P < 0.05) than the other two types (7.9 kg C m⁻² in the riverine flow-though wetlands and 8.0 kg C m⁻² in a slowly flowing slough), indicating that the type of organic matter entering into the system and the type of wetland may be key factors in defining its soil C pool. A riverine flow-through wetland in Ohio showed a significantly higher C pool (P < 0.05) in the permanently flooded location (18.5 kg C m⁻²) than in the edge location with fluctuating hydrology, where the soil is intermittently flooded (14.6 kg C m⁻²).