The effect of equilibration time on survival and development rates of mouse pronuclear-stage embryos vitrified in solid surface (SSV) and convential straws: in vitro and in vivo evaluations

The objective of this study was to improve the efficiency of cryopreservation of pronuclear-stage (PN) mouse embryos. A novel vitrification technique (solid surface vitrification, SSV) was compared with a convential one in straws both for cryosurvival and obtaining progeny from cryopreserved PN mouse embryos. In the SSV method, 15-20 PN embryos were exposed to vitrification solutions for approximately 20 sec after equilibration, and then they were dropped in 2 microl drops onto a pre-cooled (-150 to -180 degrees C) metal surface. In the straws method, groups of 5-10 PN embryos were loaded in a single straw after equilibration. In experiment I, it was compared the effect of the vitrification solutions alone, without vitrification. No reduction was detected in survival, cleavage and blastocysts rates and the lowest development rate was obtained from hatched blastocyst for 20 min equilibration (24.5%). In experiment II, SSV method resulted in significantly higher survival and cleavage rates than that of in-straw vitrified 15-20 min group (87% vs. 60%, 83% vs. 67%, respectively; P < 0.05). There were no statistical differences among any of the blastocyts groups. However, there was a statistical difference in hatched blastocysts between 15 to 5, 10, and 20 min (P < 0.05). In experiment III, it was found no major effect among equilibration time periods in toxicity groups according to the mean cell number of blastocysts developed from PN embryos. But, there was a significant differences between 15 min SSV and 10 min in straw vitrified according to the mean cell number of blastocysts developed from PN embryos following vitrification (P < 0.05). The good results were obtained from 15 min equilibration group for SSV and 10 min equilibration group for straw vitrification. In the last experiment, embryo transfer after vitrification and toxicity was investigated. There were significant differences between SSV and straw just on the rate of pups born (30% and 20.5% respectively; P < 0.05). In conclusion, vitrification of PN mouse embryos by SSV can result in high rates of in vitro development to expanded and hatched blastocyst stage and in vivo development to live pups.     

Evidence of septic system failure determined by a bacterial biochemical fingerprinting method

AIMS: To provide evidence of septic system failure by comparing two faecal indicator bacteria, enterococci and Escherichia coli, from defective septic tanks and adjacent creeks. METHODS AND RESULTS: A biochemical fingerprinting method was used to type and compare enterococci and E. coli strains from 39 septic tanks with creek water samples. Phenotypic diversity of enterococci (0.5 +/- 0.3) and E. coli (0.5 +/- 0.3) in septic tanks were significantly lower than those found in water samples (0.8 +/- 0.1, P < 0.0001 for enterococci and 0.9 +/- 0.1, P < 0.0001 for E. coli). Among 1072 enterococci isolates tested from septic tanks, 203 biochemical phenotypes (BPTs) were found of which 98 BPTs from 33 septic tanks were identical to several water samples. Similarly, among 621 E. coli isolates tested from septic tanks, 159 BPTs were found of which 53 BPTs from 26 septic tanks were also identical to water samples. The number of the latter bacteria was significantly (P = 0.01) higher in water samples collected from downstream compared with that of upstream in the study area. A high similarity between the populations of both indicator bacteria was also found between defective septic tanks and downstream water samples further indicating the contamination of both creeks by defective septic systems. CONCLUSIONS: Biochemical fingerprinting of faecal indicator bacteria is a useful and rapid method to provide direct evidence for septic system failure. Combination of both faecal indicator bacteria (enterococci and E. coli) provides a better judgement of the performance of a septic system. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first to provide direct evidence of septic system failure by identifying the presence of specific bacterial types in septic tanks and surface waters. Based on our findings, we suggest that the performance evaluation of a septic system should be accompanied by direct analysis of faecal indicator bacteria.     

Application of response surface methodology in medium optimization for spore production of <Emphasis Type="Italic">Coniothyrium minitans</Emphasis> in solid-state fermentation

Summary Spore production of Coniothyrium minitans was optimized by using response surface methodology (RSM), which is a powerful mathematical approach widely applied in the optimization of fermentation process. In the first step of optimization, with Plackett–Burman design, soluble starch, urea and KH²PO⁴ were found to be the important factors affecting C. minitans spore production significantly. In the second step, a 2³ full factorial central composite design and RSM were applied to determine the optimal concentration of each significant variable. A second-order polynomial was determined by the multiple regression analysis of the experimental data. The optimum values for the critical components for the maximum were obtained as follows: soluble starch 0.643 (36.43 g. l⁻¹), urea −0.544 (3.91 g l⁻¹) and KH²PO⁴ 0.049 (1.02 g l⁻¹) with a predicted value of maximum spore production of 9.94 × 10⁹ spores/g IDM. Under the optimal conditions, the practical spore production was 1.04 × 10¹⁰ spores/g IDM. The determination coefficient (R²) was 0.923, which ensure an adequate credibility of the model.     

Soil seed banks of two montane riparian areas: implications for restoration

Understanding the role of seed banks can be important for designing restoration projects. Using the seedling emergence method, we investigated the soil seed banks of two montane, deciduous riparian forest ecosystems of southeastern Arizona. We contrasted the seed banks and extant vegetation of Ramsey Canyon, which is the site of riparian restoration activities, with that of Garden Canyon, which has been less affected by human land uses. Fewer plant species were found at Ramsey Canyon than Garden Canyon, for both the seed bank and extant vegetation, and the vegetation at Ramsey Canyon (seed bank and extant) had consistently drier wetland indicator scores. As well, vegetation patterns within sampling zones (channel margins and adjacent riparian forests) differed between canyons. At Garden Canyon channel margins, the seed bank and extant vegetation had relatively high similarity, with herbaceous wetland perennial species dominating. Extant vegetation in the floodplain riparian forest zone at Garden Canyon had a drier wetland indicator score than the seed bank, suggesting that the floodplains are storing seeds dispersed from wetter fluvial surfaces. Vegetation patterns for Ramsey Canyon channel margins were similar to those for Garden Canyon floodplains. Vegetation patterns in the Ramsey Canyon riparian forest zone were indicative of non-flooded conditions with an abundance of upland species in the soil seed bank and extant vegetation. Channel geomorphology measurements indicated that much of the riparian forest zone at Ramsey Canyon is functionally a terrace, a condition that may be a legacy of channel erosion from historic land uses. Steep, erodible channel slopes may contribute to the low seed bank germinant density at Ramsey Canyon channel margins, and narrower flood-prone area may explain the greater terrestrialization of the vegetation in both sampling zones. We recommend testing the use of donor soils from more diverse stream reaches to restore biodiversity levels at Ramsey Canyon, following restoration activities such as channel-widening. Seed banks from Garden Canyon, for example, although predominantly consisting of herbaceous perennials, would supply species with a range of moisture tolerances, life spans, and growth forms. We also recommend that restorationists take care not to harm seed banks exposed during removal of introduced species; at Ramsey Canyon, soil seed banks were equally diverse in areas with high and low cover of the introduced Vinca major (a legacy of Ramsey Canyon land use).     

Orthogonal site-specific protein modification by engineering reversible thiol protection mechanisms

Covalent modification is an important strategy for introducing new functions into proteins. As engineered proteins become more sophisticated, it is often desirable to introduce multiple, modifications involving several different functionalities in a site-specific manner. Such orthogonal labeling schemes require independent labeling of differentially reactive nucleophilic amino acid side chains. We have developed two protein-mediated protection schemes that permit independent labeling of multiple thiols. These schemes exploit metal coordination or disulfide bond formation to reversibly protect cysteines in a Cys(2)His(2) zinc finger domain. We constructed a variety of N- and C-terminal fusions of these domains with maltose-binding protein, which were labeled with two or three different fluorophores. Multiple modifications were made by reacting an unprotected cysteine in MBP first, deprotecting the zinc finger, and then reacting the zinc finger cysteines. The fusion proteins were orthogonally labeled with two different fluorophores, which exhibited intramolecular fluorescene resonance energy transfer (FRET). These conjugates showed up to a threefold ratiometric change in emission intensities in response to maltose binding. We also demonstrated that the metal- and redox-mediated protection methods can be combined to produce triple independent modifications, and prepared a protein labeled with three different fluorophores that exhibited a FRET relay. Finally, labeled glucose-binding protein was covalently patterned on glass slides using thiol-mediated immobilization chemistries. Together, these experiments demonstrated that reversible thiol protection schemes provide a rapid, straightforward method for producing multiple, site-specific modifications.     

Minimal surface as a model of beta-sheets

The purpose of this article is to present arguments based on experimental data that the beta-sheet structures in proteins are the result of the tendency to minimize surface areas. Thus, we propose the model that all beta-sheet structures are almost minimal surfaces, namely, their mean curvatures are nearly zero. To support this model, we chose 1740 disjoint beta-sheets with less than 10 strands from the all beta-protein class in a nonredundant 40% Structural Classification of Proteins (SCOP) database and applied the least-squares method to fit the minimal surface catenoid (and in some rare cases, the plane) to the beta-sheet structures. The fitting errors were extremely small: The error of 1729 beta-sheets with catenoid minimal surface is 0.90 +/- 0.55 A and the error of the remaining 11 flat sheets with the plane is 0.64 +/- 0.46 A. The fact that the commonly used models for some beta-sheet surfaces (i.e., the hyperboloid and strophoid) have very small mean curvatures (< 0.05) supports our model. Moreover, we showed that this model also includes the isotropically stressed configuration model proposed by Salemme, in which the intrastrand tendency of the individual chains to twist or coil is in equilibrium with the tendency of the interstrand hydrogen bonding to resist twisting of the sheet as a whole. As an application we used our model to quantify the two principal independent modes in the flexibility of beta-sheets, that is, the bending parameter of beta-sheets and the inclined angle of beta-strands in a sheet.     

Plastic changes of leaf mass per area and leaf nitrogen content in response to canopy openings in saplings of eight deciduous broad-leaved tree species

Leaf nitrogen content per area (N_area) is a good indicator of assimilative capacity of leaves of deciduous broad-leaved trees. This study examined the degrees of increase in N_area in response to canopy openings as leaf mass per area (LMA) and leaf nitrogen content per mass (N_mass) in saplings of eight deciduous broad-leaved tree species in Hokkaido, northern Japan. Five of the species were well-branched species with a large number of small leaves (lateral-growth type), and the other three species were less-branched species with a small number of large leaves (vertical-growth type). The degrees of increase in N_area were compared between the two crown types. In closed-canopy conditions, leaves of the vertical-growth species tended to have a lower LMA and higher N_mass than those of the lateral-growth species, which resulted in similar N_area for both. LMA increased in canopy openings in the eight species, and the degrees of increase were not largely different between the lateral- and vertical-growth species. On the contrary, N_mass was unchanged in canopy openings in the eight species. As a result, N_area of each species increased in canopy openings in proportion to the increase in LMA, and the degrees of increase in N_area were similar in the lateral- and vertical-growth species. Therefore, this study showed that the degrees of increase in N_area were not correlated with the crown architecture (i.e., the lateral- and vertical-growth types).     

Surprising Effects of Synaptic Excitation

Typically, excitatory synaptic coupling is thought of as an influence that accelerates and propagates firing in neuronal networks. This paper reviews recent results explaining how, contrary to these expectations, the presence of excitatory synaptic coupling can drastically slow oscillations in a network and how localized, sustained activity can arise in a network with purely excitatory coupling, without sustained inputs. These two effects stem from interactions of excitatory coupling with two different forms of intrinsic neuronal dynamics, and both serve to highlight the fact that the influence of synaptic coupling in a network depends strongly on the intrinsic properties of cells in the network.This work was partially supported by the National Science Foundation, under award DMS-0414023     

Protection against malaria induced by chirally modified Plasmodium falciparum's MSP-1 42 pseudopeptides

The C-terminal portion of the Plasmodium falciparum blood stage MSP-1 antigen plays a key role in invasion of human erythrocytes. The MSP-1(1282-1301) non-polymorphic 1585 peptide, from the processed MSP-1(42) fragment, is poorly immunogenic and highly alpha-helical [Angew. Chem. Int. Ed. 40 (2001) 4654]. Assessing the alpha-carbon asymmetry and its implication in the host immune response is proposed in this work to overcome the 1585 peptide's immunological properties. Accordingly, the effect of incorporating single D-amino acids and psi-[CH(2)-NH] isoster bonds into the 1585 peptide was examined both at the immunogenic and 3D-structure levels. Therefore, specific binding to RBCs is promoted by site-directed chiral modifications on the native peptide as well as by simultaneously combining specific D-substitutions with psi-[CH(2)-NH] isoster bonds transforming this molecule into a high specific HLAbeta1*1101 allele binder. D-analog pseudopeptide immunized animals induced antibodies selectively recognizing a recombinant as well as native MSP-1(42) and MSP-1(33) fragments. Protection and low parasitemia levels were induced in Aotus monkeys immunized with the EVLYL(dK)PLAGVYRSLKKQLE analog. Peptide alpha-carbon chiral transformation is therefore an important target for structural modulation and, consequently, represents a novel approach towards designing multi-component subunit-based malarial vaccines.