An efficient protocol for the complete incorporation of methyl-protonated alanine in perdeuterated protein

A strategy for the introduction of (¹H,¹³C-methyl)-alanine into perdeuterated proteins is described. Specific protonation of alanine methyl groups to a level of 95% can be achieved by overexpressing proteins in M9/D₂O based bacterial growth medium supplemented with 800 mg/l of 2-[²H], 3-[¹³C] l-alanine. However, though simple, this approach results in undesired, non-specific background labeling due to isotope scrambling via different amino acid metabolic pathways. Following a careful analysis of known metabolic pathways we found that co-addition of perdeuterated forms of α-ketoisovalerate-d₇, succinate-d₄ and l-isoleucine-d₁₀ with labeled l-alanine, reduces undesired background labeling to <1%. When combined with recently developed methyl TROSY experiments, this methyl-specific labeling protocol permits the acquisition of excellent quality correlation spectra of alanine methyl groups in high molecular weight proteins. Our cost effective strategy offers a significant enhancement in the level of incorporation of methyl-labeled alanine in overexpressed proteins over previously reported methods. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Purification and Characterization of Cardiac Tropomyosins

A new procedure was developed to purify tropomyosin. The procedure was an adaptation of that described for purification of myosin. By eliminating troponin before precipitating with (NH₄), SO₄ it was possible to obtain pure tropomyosin from the same preparation from which myosin was purified. When tropomyosin was subjected to isoelectrofocusing two tropomyosins were present, having similar isoelectric points of pH 5, 4 and 5.6; two tropomyosin subunits were resolved in the presence of 6 M urea. The two subunits had very similar isoelectric points, pH 4.7 and 5.0. According to Ouchterlony analyses the tropomyosins from canine skeletal and cardiac tissue were immunologically identical when incubated with goat γG antitropomyosin (cardiac).     

A scale down process for the development of large volume cryopreservation

The process of ice formation and propagation during cryopreservation impacts on the post-thaw outcome for a sample. Two processes, either network solidification or progressive solidification, can dominate the water–ice phase transition with network solidification typically present in small sample cryo-straws or cryo-vials. Progressive solidification is more often observed in larger volumes or environmental freezing. These different ice phase progressions could have a significant impact on cryopreservation in scale-up and larger volume cryo-banking protocols necessitating their study when considering cell therapy applications.This study determines the impact of these different processes on alginate encapsulated liver spheroids (ELS) as a model system during cryopreservation, and develops a method to replicate these differences in an economical manner.It was found in the current studies that progressive solidification resulted in fewer, but proportionally more viable cells 24 h post-thaw compared with network solidification. The differences between the groups diminished at later time points post-thaw as cells recovered the ability to undertake cell division, with no statistically significant differences seen by either 48 h or 72 h in recovery cultures.Thus progressive solidification itself should not prove a significant hurdle in the search for successful cryopreservation in large volumes. However, some small but significant differences were noted in total viable cell recoveries and functional assessments between samples cooled with either progressive or network solidification, and these require further investigation.     

Liquidus Tracking: Large scale preservation of encapsulated 3-D cell cultures using a vitrification machine

Currently, cryo-banking of multicellular structures such as organoids, especially in large volumes at clinical scale >1 L, remains elusive for reasons such as insufficient dehydration and cryoprotectant additive (CPA¹) penetration, slow cooling and warming rates and devitrification processes. Here we introduce the concept of Liquidus Tracking (LT) using a semi-automated process for liquid volumes of up to 450 ml including 130 ml of alginate encapsulated liver cells (AELC) that archived controlled and reversible vitrification with minimized toxicity.First a CPA solution with optimal properties for LT was developed by employing different small scale test systems. Combining sugars such as glucose and raffinose with Me₂SO improved post-exposure (at +0.5 °C) viabilities from 6% ±3.6 for Me₂SO alone up to 58% ±6.1 and 65% ±14.2 respectively (p < 0.01). Other permeating CPAs (e.g. ethylene glycol, propylene glycol, methanol) were investigated as partial replacements for Me₂SO. A mixture of Me₂SO, ethylene glycol and glucose (ratio 4:2:1– termed LTdeg) supported glass-forming tendencies with appropriate low viscosities and toxicities required for LT. When running the full LT process, using Me₂SO alone, no viable cells were recovered; using LTdeg, viable recoveries were improved to 40% ±8 (p<0.001%). Further refinements of improved mixing technique further improved recovery after LT. Recoveries of specific liver cell functions such as synthesis of albumin and alpha-fetoprotein (AFP) were retained in post thaw cultures.In summary: By developing a low-toxicity CPA solution of low viscosity (LTdeg) suitable for LT and by improving the stirring system, post-warming viability of AELC of up to 90% and a AFP secretion of 89% were reached. Results show that it may be possible to develop LT as a suitable cryogenic preservation process for different cell therapy products at large scale.     

SNP genotypes reveal breed substructure,selection signatures and highly inbred regions in Piétrain pigs

The Piétrain pig originates from the Belgian village Piétrain some time between 1920 and 1950. Owing to its superior conformation, the Piétrain has spread worldwide since the 1960s. As initial population sizes were limited and close inbreeding was commonplace, the breed’s genetic diversity has been questioned. Therefore, this study examines Piétrain breed substructure, diversity and selection signatures using SNP data in comparison with Duroc, Landrace and Large White populations. Principal component analysis indicated three subpopulations, and F_ST analysis showed that US Piétrains differ most from European Piétrains. Average inbreeding based on runs of homozygosity (ROH) segments larger than 4 Mb ranged between 16.7 and 20.9%. The highest chromosomal inbreeding levels were found on SSC8 (42.7%). ROH islands were found on SSC8, SSC15 and SSC18 in all Piétrain populations, but numerous population-specific ROH islands were also detected. Moreover, a large ROH island on SSC8 (34–126 Mb) appears nearly fixed in all Piétrain populations, with a unique genotype. Chromosomal ROH patterns were similar between Piétrain populations. This study shows that Piétrain populations are genetically diverging, with at least three genetically distinct populations worldwide. Increasing genetic diversity in local Piétrain populations by introgression from other Piétrain populations seems to be only limited. Moreover, a unique 90 Mb region on SSC8 appeared largely fixed in the Piétrain breed, indicating that fixation was already present before the 1960s. We believe that strong selection and inbreeding during breed formation fixed these genomic regions in Piétrains. Finally, we hypothesize that independent coat color selection may have led to large ROH pattern similarities on SSC8 between unrelated pig breeds.     

Construction and Characterization of a Novel Bacmid AcBac-Syn Based on a Synthesized Baculovirus Genome

Virologica Sinica - Baculoviruses are large DNA viruses which have been widely used as expression vectors and biological insecticides. Homologous recombination and Bac-to-Bac system have been the...     

Patterns of herbivore species richness in Kenya and current ecoclimatic stability

The increased attention to biodiversity worldwide has stimulatedinterest in understanding biophysical factors associated with indicators ofbiodiversity such as species richness. Although levels of biodiversity may seemto be equivalent in different areas, high species richness may be caused byaccumulation of species over a long time in places where environmentalconditions remained stable and predictable. The advanced very high resolutionradiometer (AVHRR)–normalized difference vegetation index (NDVI) has beenestablished to be a good proxy for studying interannual climate variability aswell as regional drought condition. In this study, I examined the relationshipbetween large herbivore species richness and AVHRR–NDVI derivedclimatic-variability indices, interannual average NDVI and coefficient ofvariation of NDVI at a regional spatial scale in Kenya. Regions with a relativelylow coefficient of variation of NDVI and high interannual average NDVIcharacterize current ecoclimatic stability. By contrast, a high coefficient ofvariation of NDVI and relatively low interannual average NDVI characterizeecoclimatic instability (drought risk). Statistical analyses revealed that a highinterannual average NDVI increases species richness, whereas a high coefficient ofvariation of NDVI lowers species richness. This indicates that maximum numbers ofspecies are found in regions with current ecoclimatic stability. Understandingsuch relationships can help in explaining spatial distribution of speciesrichness and predicting global changes resulting from human impacts on theenvironment.     

Temporal and spatial variation in an index of biological integrity for the middle Wabash River, Indiana

Few studies have addressed within-year temporal variation of IBI scores. We compared index of biotic integrity (IBI) scores for two summer sampling events from a large river during 25 annual periods. The results indicated that IBI scores calculated from June samples were not significantly different from July samples. Spatial autocorrelation was present, such that sites that were closer together produced similar IBI scores and could not be considered independent. Temporal autocorrelation was present, but was not strong. Lower quality sites (low IBI score) did not have higher variation than higher quality sites. Our results show that a single sample of fishes by boat electrofisher during the summer in a large river such as the Wabash River can produce a repeatable estimate of IBI score. Thus, repeated or additional sampling within the summer season to improve the quality of the evaluation is not warranted. Handling editor: J. Trexler     

Projecting future fire activity in Amazonia

Fires are major disturbances for ecosystems in Amazonia. They affect vegetation succession, alter nutrients and carbon cycling, and modify the composition of the atmosphere. Fires in this region are strongly related to land‐use, land‐cover and climate conditions. Because these factors are all expected to change in the future, it is reasonable to expect that fire activity will also change. Models are needed to quantitatively estimate the magnitude of these potential changes. Here we present a new fire model developed by relating satellite information on fires to corresponding statistics on climate, land‐use and land‐cover. The model is first shown to reproduce the main contemporary large‐scale features of fire patterns in Amazonia. To estimate potential changes in fire activity in the future, we then applied the model to two alternative scenarios of development of the region. We find that in both scenarios, substantial changes in the frequency and spatial patterns of fires are expected unless steps are taken to mitigate fire activity.     

H/D exchange mass spectrometry and statistical coupling analysis reveal a role for allostery in a ferredoxin-dependent bifurcating transhydrogenase catalytic cycle

Recent investigations into ferredoxin-dependent transhydrogenases, a class of enzymes responsible for electron transport, have highlighted the biological importance of flavin-based electron bifurcation (FBEB). FBEB generates biomolecules with very low reduction potential by coupling the oxidation of an electron donor with intermediate potential to the reduction of high and low potential molecules. Bifurcating systems can generate biomolecules with very low reduction potentials, such as reduced ferredoxin (Fd), from species such as NADPH. Metabolic systems that use bifurcation are more efficient and confer a competitive advantage for the organisms that harbor them. Structural models are now available for two NADH-dependent ferredoxin-NADP⁺ oxidoreductase (Nfn) complexes. These models, together with spectroscopic studies, have provided considerable insight into the catalytic process of FBEB. However, much about the mechanism and regulation of these multi-subunit proteins remains unclear. Using hydrogen/deuterium exchange mass spectrometry (HDX-MS) and statistical coupling analysis (SCA), we identified specific pathways of communication within the model FBEB system, Nfn from Pyrococus furiosus, under conditions at each step of the catalytic cycle. HDX-MS revealed evidence for allosteric coupling across protein subunits upon nucleotide and ferredoxin binding. SCA uncovered a network of co-evolving residues that can provide connectivity across the complex. Together, the HDX-MS and SCA data show that protein allostery occurs across the ensemble of iron?sulfur cofactors and ligand binding sites using specific pathways that connect domains allowing them to function as dynamically coordinated units.