The impact of the megafauna extinctions on savanna woody cover in South America

Has land surface cover in South America been impacted by the loss of most large herbivores following the severe Pleistocene and Early Holocene megafauna extinctions on this continent? Here, we estimate how mean savanna woody biomass may have changed in the Americas following these extinctions by creating an empirical model to understand how large herbivores impact savanna woody biomass. To create this empirical model, we combine a large recently published dataset of savanna woody cover from Lehmann et al. (2014) (n = 2154 plots) with estimates of mammals ranges and weights from the IUCN database. We evaluate how variables such as number of megaherbivores (mammal species ≥ 1000 kg), log10 sum species weights, and total number of mammal species predict changes to woody cover by using both ordinary least squares regression analysis (OLS) and simultaneous auto‐regressive (SAR) analysis to control for spatial autocorrelation. Both number of megaherbivores and log10 sum species weights, which both disproportionately weight for megaherbivores, significantly explained much (～ 5–13%) variance in woody cover, but the third variable weighting all animals equally, did not. We then combined these biotic variables with abiotic variables such as temperature, precipitation, and fire frequency to create a model predicting 36% of the variance of savanna woody cover. We used this model combined with estimated range maps of extinct South American megafauna to estimate that had those South American megafauna not gone extinct, total savanna woody cover in South America could possibly have decreased by ～ 29% and that savannas would likely have been more open like current African savannas.     

Thermal performance of quartz capillaries for vitrification

In this paper we report the thermal behavior of a new approach for vitrification. Thermal performance of traditional open pulled straws is compared with a new technique based on the combined use of quartz capillaries with slush nitrogen. This new method of vitrification achieved ultrafast cooling rates of 250,000 °C/min. As a result, a much lower concentration of cryoprotectant was needed to reach vitrification. In fact, a cryoprotectant solution typically used in oocyte slow freezing protocols was shown to remain transparent after cooling to liquid nitrogen temperatures indicating apparent “vitrification”. This approach offers a new and very promising technique for vitrification of cells using low levels of cryoprotectants.     

Inhibition of cobalamin-dependent methionine synthase by substituted benzo-fused heterocycles

The cobalamin-dependent cytosolic enzyme, methionine synthase (EC.2.1.1.13), catalyzes the remethylation of homocysteine to methionine using 5-methyltetrahydrofolate as the methyl donor. The products of this remethylation--methionine and tetrahydrofolate--participate in the active methionine and folate pathways. Impaired methionine synthase activity has been implicated in the pathogenesis of anaemias, cancer and neurological disorders. Although the need for potent and specific inhibitors of methionine synthase has been recognized, there is a lack of such agents. In this study, we designed, synthesized and evaluated the inhibitory activity of a series of substituted benzimidazoles and small benzothiadiazoles. Kinetic analysis revealed that the benzimidazoles act as competitive inhibitors of the rat liver methionine synthase, whilst the most active benzothiadiazole (IC(50) = 80 microm) exhibited characteristics of uncompetitive inhibition. A model of the methyltetrahydrofolate-binding site of the rat liver methionine synthase was constructed; docking experiments were designed to elucidate, in greater detail, the binding mode and reveal structural requirements for the design of inhibitors of methionine synthase. Our results indicate that the potency of the tested compounds is related to a planar region of the inhibitor that can be positioned in the centre of the active site, the presence of a nitro functional group and two or three probable hydrogen-bonding interactions.     

Validation of XP-C pathogenic variations in archival material from a live XP patient

Xeroderma pigmentosum (XP) genetic complementation group C (XP-C) is the most common form of the disease worldwide. Thirty-four distinct genetic defects have been identified in 45 XP-C patients. Further identification of such defects and the frequency of their occurrence offers the potential of generating diagnostic and prognostic molecular screening panels. Archival material (such as formalin-fixed paraffin embedded skin) may be useful for the identification of novel genetic variations and for documenting the frequency of individual genetic defects in patients who are no longer available for study. However, the use of archival material precludes direct analysis of changes in the mRNA resulting from genomic changes. The serendipitous reacquisition of an XP individual in whom genetic defects were previously characterized in archival material allowed confirmation of the defects as well as a direct analysis of the consequences of these defects on mRNA, mRNA expression and on cellular phenotypes.     

Re-assessment of the potential impact of physiologically relevant pH changes on the hydration properties of the isolated mammalian corneal stroma

The pH sensitivity of the swelling of the mammalian corneal stroma was reinvestigated to assess whether or not there were detectable differences in the hydration properties of this collagen-keratocyte matrix within a physiologically relevant range (as opposed to extremes of acid or alkaline pH) and at a physiologically relevant temperature. From recent post-mortem eyes of adult cows, square (8 x 8 mm) samples of corneal stroma were prepared and incubated in an isotonic, buffered (HEPES etc.), mixed salts solution with added glucose at 37 degrees C. The time-dependent changes in wet mass were assessed over 24 h. The rate and magnitude of stromal swelling were different within the range of pH 6.5-8.5. The wet mass of stromal samples increased almost 2-fold within 1 h, and then at lesser rates to realise 3.25-3.75-fold and 4-5-fold increases in wet mass by 9 h and 24 h respectively. The maximum increases were observed at pH 7.25-7.5, with most of the effect being the result of differences in the initial rate of swelling. The discontinuous swelling and the pH effect on the rates of swelling were also evident when the data were fitted to a previous kinetic model (Elliott et al., J. Physiol. (Lond.) 298 (1980) 453-470). It is concluded that pH changes in the physiological range can have a small but reproducible impact on the swelling kinetics of the isolated mammalian corneal stroma ex vivo.     

Influence of initial fixation protocol on the appearance of primary cilia on the rabbit corneal endothelial cell apical surface as assessed by scanning electron microscopy

The objective was to examine primary cilia at the apical surface of corneal endothelial cells after using different fixatives. Female albino rabbits (2 kg) were euthanised at 15:00 h and the corneas fixed immediately (usually with an isotonic 2% glutaraldehyde-cacodylate fixative) either after dissection, by application fixative at 4 degrees C, by immersion of the eyeball in fixative at room temperature (RT), or by application of an isotonic or a hypertonic (Karnovsky-type) fixative at RT. Images at 2000x were taken from the central corneal region, and number and length of primary cilia assessed. The length was the same regardless of method (overall average of 1.67+/-0.70 microm), but the incidence of primary cilia was hypertonic fixative (87% of cells) >cold drop fixation (71%), >whole globe immersion (68%) >dissect then fix methods (67%) >RT drop fixation (34%). The first four methods however yielded cells with unacceptable artefacts (especially distortion). More details should be provided of the primary fixation method used.     

Hepatitis delta virus RNA encoding the large delta antigen cannot sustain replication due to rapid accumulation of mutations associated with RNA editing

Hepatitis delta virus (HDV) contains two RNA species (HDV-S and HDV-L), which encode the small and large forms of hepatitis delta antigens (S- and L-HDAg), respectively. HDV-L RNA is a result of an RNA editing event occurring at an amber/W site of HDV-S RNA. RNA editing must be regulated to prevent premature and excessive accumulation of HDV-L RNA in the viral life cycle. In this study, we used an RNA transfection procedure to study the replication abilities of HDV-L and HDV-S RNA. While HDV-S led to robust RNA replication, HDV-L could not replicate even after 6 days following transfection. The failure of HDV-L to replicate was not due to insufficient amounts of S-HDAg, as identical results were obtained in a cell line that stably overexpresses S-HDAg. Also, it was not due to possible inhibition by L-HDAg, as HDV-S RNA replication was not affected when both HDV-L and HDV-S RNA were cotransfected. Further, when L-HDAg expression from HDV-L RNA was abolished by site-directed mutagenesis, the mutant HDV-L RNA also failed to replicate. Unexpectedly, when the kinetics of RNA replication was examined daily, HDV-L was found to replicate at a low level at the early time points (1 to 2 days posttransfection) but then lose this capability at later time points. Sequence analysis of the replicated HDV-L RNA at day 1 posttransfection showed that it had undergone multiple nucleotide changes, particularly in the region near the putative promoter region of HDV RNA replication. In contrast, very few mutations were found in HDV-S RNA. These results suggest that the editing at the amber/W site triggers a series of additional mutations which rapidly reduce the replication efficiency of the resultant HDV genome and thus help regulate the amount of HDV-L RNA in infected cells. They also explain why L-HDAg is not produced early in HDV infection, despite the fact that HDV-L RNA is present in the virion.     

A descriptive and quantitative study of the keratocytes of the corneal stroma of albino rabbits using transmission electron microscopy

The present morphometric study was designed to assess the dimensions and shape of keratocytes and their nuclei by transmission electron microscopy, and to assess these features in relation to the stromal lamellae. Corneas from 10 albino rabbits were fixed in 2% glutaraldehyde in cacodylate buffer (pH 7.4, 300 mOsm/kg) and embedded in Spurr's epoxy resin. Both transverse and coronal thin sections through the corneal stroma were prepared. The stromal lamellae had an average thickness of 2.45+/-1.15 microm. The average cell thickness of the keratocytes was 1.34+/-0.46 microm (range 0.49-4.76 microm), with the apparent cell thickness being related to the average anterior-posterior thickness of the adjacent lamellae (r = 0.424, P = 0.001)). The relative length and thickness of the cell nucleus, in transverse section, was measured to be 0.65+/-0.13 and 0.76+/-0.10 of the cell body section respectively. As assessed by planimetry, the area of the keratocyte cell body viewed in coronal section was 292+/-118 microm2, with a nucleus-to-cytoplasm ratio of 0.437+/-0.295. The electron micrographs confirmed the presence of gap junctions between keratocyte cell processes, and the occasional presence of centrioles in the cells. Some keratocyte processes were observed to extend from one face of the lamellae to the other, suggesting anterior-to-posterior cell communication. These studies indicate that the keratocyte cell thickness is influenced by the physical pressure exerted by adjacent stromal lamellae. The cell nucleus, while a dominant feature in transverse section, has a normal size in relation to the cell cytoplasm when viewed in coronal section.