Modification of calf lens crystallins as determined by Gel electrophoresis

Summary Variations in size and charge of calf lens proteins, particularly gamma crystallins, were studied by polyacrylamide gel electrophoresis. Exposure of gamma crystallins to near-UV light in the presence of L-tryptophan produces species of higher electrophoretic mobility and higher retardation. Treatment with urea and sulfonation also produced changes in the retardation co-efficient. The increase of retardation co-efficient of gamma crystallin is interpreted to be a result of conformational changes. Gamma crystallins are particularly sensitive to photo-modification, and this process may be associated with age-related changes in the lens.Supported by research grants from the U.S.P.H.S. EY #00459, a FIGHT-FOR-SIGHT Postdoctoral Research Fellowship (FIGHT-FOR-SIGHT, INC., N.Y.C.) and The Rochester Eye and Human Parts Bank, Inc.     

Attenuation of SARS‐CoV‐2 replication and associated inflammation by concomitant targeting of viral and host cap 2'‐O‐ribose methyltransferases

The SARS‐CoV‐2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2''‐O‐ribose cap needed for viral immune escape. We find that the host cap 2''‐O‐ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS‐CoV‐2 replication. Using in silico target‐based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti‐SARS‐CoV‐2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co‐substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID‐19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection‐induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID‐19.     

Permeabilization and lysis induced by bacteriocins and its effect on aldehyde formation by Lactococcus lactis

Permeabilization induced by lacticin 3147, lactococcins A, B and M, enterocin AS-48 and nisin, bacteriocins described as cell membrane-pore forming and lytic agents, enhanced in all cases aldehyde formation by Lactococcus lactis IFPL730. Nevertheless, the conversion of isoleucine into 2-methylbutyraldehyde depended not only on the degree of permeabilization but also on the bacteriocin that caused the cell membrane damage. The highest values of 2-methylbutyraldehyde corresponded to cell suspensions containing lacticin 3147 and lactococcins, treatments that provoked further lysis in addition to induced permeabilization.     

Afforestation effects on SOC in former cropland: oak and spruce chronosequences resampled after 13 years

Chronosequences are commonly used to assess soil organic carbon (SOC) sequestration after land‐use change, but SOC dynamics predicted by this space‐for‐time substitution approach have rarely been validated by resampling. We conducted a combined chronosequence/resampling study in a former cropland area (Vestskoven) afforested with oak (Quercus robur) and Norway spruce (Picea abies) over the past 40 years. The aims of this study were (i) to compare present and previous chronosequence trends in forest floor and top mineral soil (0–25 cm) C stocks; (ii) to compare chronosequence estimates with current rates of C stock change based on resampling at the stand level; (iii) to estimate SOC changes in the subsoil (25–50 cm); and (iv) to assess the influence of two tree species on SOC dynamics. The two chronosequence trajectories for forest floor C stocks revealed consistently higher rates of C sequestration in spruce than oak. The chronosequence trajectory was validated by resampling and current rates of forest floor C sequestration decreased with stand age. Chronosequence trends in topsoil SOC in 2011 did not differ significantly from those reported in 1998, however, there was a shift from a negative rate (1998: ?0.3 Mg C ha^?1 yr^?1) to no change in 2011. In contrast SOC stocks in the subsoil increased with stand age, however, not significantly (P = 0.1), suggesting different C dynamics in and below the former plough layer. Current rates of C change estimated by repeated sampling decreased with stand age in forest floors but increased in the topsoil. The contrasting temporal change in forest floor and mineral soil C sequestration rates indicate a shift in C source‐sink strength after approximately 40 years. We conclude that afforestation of former cropland within the temperate region may induce soil C loss during the first decades followed by a recovery phase of yet unknown duration.     

Cryotops versus open-pulled straws (OPS) as carriers for the cryopreservation of bovine oocytes: effects on spindle and chromosome configuration and embryo development

Two experiments were designed to assess the effectiveness of cryopreserving bovine MII oocytes using cryotops as the carrier system for vitrification. In the first experiment, we examined the developmental competence of oocytes after: (i) vitrification in open-pulled straws (OPS method); or (ii) vitrification in <0.1 μl medium droplet on the surface of a specially constructed fine polypropylene strip attached to a plastic handle (Cryotop method). In the second experiment, warmed oocytes that had been vitrified in OPS or cryotops were fixed to analyze spindle and chromosome configuration. In all experiments both cow and calf oocytes were used. Significantly different fertilization rates were observed between the vitrification groups: 31.5% and 20.2% for the cow and calf oocytes vitrified in OPS, respectively, versus 46.1% and 46.4% for the oocytes vitrified using cryotops. After in vitro fertilization, 3.8% of the calf oocytes and 5.3% of the cow oocytes developed to the blastocyst stage. All blastocysts from vitrified oocytes resulted from the Cryotop method. A significantly lower percentage of the OPS-vitrified calf oocytes showed a normal spindle configuration (37.8%) compared to control fresh oocytes (69.9%), while normal spindle and chromosome configurations were observed in a significantly higher proportion of the cryotop-vitrified calf oocytes (60.2%). For the cow oocytes, 60.6% in the OPS group and 60.3% in the Cryotop group exhibited a normal morphology after warming. These findings suggest the cryotop system is a more efficient carrier for vitrification than OPS for the cryopreservation of bovine oocytes.     

Plasmodium falciparum expresses a multidrug resistance-associated protein

Plasmodium falciparum proteins that efflux toxic metabolic products such as oxidised glutathione (GSSG) are possible targets for anti-malarial drug development. Proteins capable of transporting GSSG and glutathione conjugates include the multidrug resistance-associated transporters (MRPs). A gene, PFA0590w, encoding a MRP homologue, has been identified in P. falciparum. Here we show the presence of full-length mRNA (5.5 kb) of this PfMRP in trophozoites by RT-PCR and Northern blotting. A polyclonal anti-PfMRP antibody generated against two unique, hydrophilic peptides in the predicted sequence produced a strong immunoreactive protein band of 210-215 kDa on Western blots of schizonts of chloroquine-sensitive and chloroquine-resistant strains, confirming expression of PfMRP protein. Using confocal microscopy the protein was seen to be localised at the edge of the schizonts with no obvious staining of the food vacuole. We suggest that PfMRP may act as the GSSG transporter in the parasite plasma membrane.     

Arabidopsis DEMETER-LIKE proteins DML2 and DML3 are required for appropriate distribution of DNA methylation marks

Cytosine DNA methylation is a stable epigenetic mark for maintenance of gene silencing across cellular divisions, but it is a reversible modification. Genetic and biochemical studies have revealed that the Arabidopsis DNA glycosylase domain-containing proteins ROS1 (REPRESSOR OF SILENCING 1) and DME (DEMETER) initiate erasure of 5-methylcytosine through a base excision repair process. The Arabidopsis genome encodes two paralogs of ROS1 and DME, referred to as DEMETER-LIKE proteins DML2 and DML3. We have found that DML2 and DML3 are 5-methylcytosine DNA glycosylases that are expressed in a wide range of plant organs. We analyzed the distribution of methylation marks at two methylated loci in wild-type and dml mutant plants. Mutations in DML2 and/or DML3 lead to hypermethylation of cytosine residues that are unmethylated or weakly methylated in wild-type plants. In contrast, sites that are heavily methylated in wild-type plants are hypomethylated in mutants. These results suggest that DML2 and DML3 are required not only for removing DNA methylation marks from improperly-methylated cytosines, but also for maintenance of high methylation levels in properly targeted sites.     

Impact of climatic variation on populations of pine processionary moth Thaumetopoea pityocampa in a core area of its distribution

• 1 There is growing appreciation of climatic effects on insect population dynamics at the margins of distribution limits. Climatic effects might be less important and/or involve different drivers and processes near the centre of distributions.
• 2 We evaluated the effects of interannual variation in temperatures, radiation and precipitation on populations of pine processionary moth Thaumetopoea pityocampa in Central–South Portugal, a low altitude area with a relatively mild Mediterranean climate near the centre of the north–south range of the species.
• 3 We tested for effects of climate on mortality of young larvae, growth rates, final larval mass and fecundity.
• 4 Results indicated high mortality of early instars associated with low minimum and maximum daily temperatures and low precipitation. Low minimum temperatures were further associated with high parasitism by the larval parasitoid Phryxe caudata (Rondani) (Diptera, Tachinidae). Furthermore, larval growth rates were higher with high solar radiation during December and January, which was itself negatively related to precipitation and air temperature. Slow larval growth rates led to lower final mass at pupation in the spring, and smaller egg masses and smaller initial colony sizes during the next autumn.
• 5 Thus climatic factors, and temperature in particular, apparently contributed to population dynamics of T. pityocampa in the core of its distribution, as well as at its northern limits. The most specific climatic parameters of importance, however, and the connections between climate, physiology and insect demographics in the core area were clearly different from northern areas.

     

Acceleration of cyanobacterial dominance in north temperate‐subarctic lakes during the Anthropocene

Increases in atmospheric temperature and nutrients from land are thought to be promoting the expansion of harmful cyanobacteria in lakes worldwide, yet to date there has been no quantitative synthesis of long‐term trends. To test whether cyanobacteria have increased in abundance over the past ~ 200 years and evaluate the relative influence of potential causal mechanisms, we synthesised 108 highly resolved sedimentary time series and 18 decadal‐scale monitoring records from north temperate‐subarctic lakes. We demonstrate that: (1) cyanobacteria have increased significantly since c. 1800 ce , (2) they have increased disproportionately relative to other phytoplankton, and (3) cyanobacteria increased more rapidly post c. 1945 ce . Variation among lakes in the rates of increase was explained best by nutrient concentration (phosphorus and nitrogen), and temperature was of secondary importance. Although cyanobacterial biomass has declined in some managed lakes with reduced nutrient influx, the larger spatio‐temporal scale of sedimentary records show continued increases in cyanobacteria throughout the north temperate‐subarctic regions.