Effect of Ca2+ and Mg2+ upon the reassociation byEscherichia coli of material released by ethylenediaminetetraacetate

Parameters involved in reconstitution of the outer membrane permeability described by Brunner, Caputo, and Treick [3] were examined. The most efficient reconstitution was obtained when divalent cations accompanied the addition of exogenous outer membrane material. Studies indicated that the effectiveness of Ca²⁺ or Mg²⁺ to promote reassociation of outer membrane material, and subsequent protection against actinomycin D, was dependent upon the strain ofEscherichia coli. More specifically, the data suggest that the effectiveness of the different divalent cations in promoting reassociation was determined by the relative amounts of F1 and F2 fractions released by ethylenediaminetetraacetate (EDTA). Reconstitution was shown by cell survival to be as high as 25% and dependent upon the total amount of material released from the outer membrane by EDTA. Between 50 and 80% of the bound material could be removed from the cells by subsequent EDTA treatment.     

Relative position of trypsin banded homologous chromosomes in human (female) metaphase figures.

"Generalized distances" between centromeres were statistically analyzed (chi2 test) on 50 normal female trypsin-banded metaphase figures. This study revealed that the homologous chromosomes of the pairs 13, 17, 14, and 21 lie closer together than would be expected by a reference distribution, and this in a statistically significant way. The same relative position was demonstrated for the chromosome groups 13-14, 13-21, 14-21, 15-22, and 14-22. Evidences were collected that also showed that homologous chromosomes of the pairs 1, 19, and 20 and the chromosome groups 15-21, 13-15, and 18-20 tend to lie closer together. Giving a functional interpretation to the phenomenon of non-random distribution of chromosomes in metaphase figures, it may be suggested that the chromosomes 13, 14, and 21 are involved in the organization of the human nucleolar organizers, more frequently than the other D- and G-group chromosomes.     

Raman studies of the conformation of the basic pancreatic trypsin inhibitor.

The vibrational Raman spectra of the basic pancreatic trypsin inhibitor in aqueous solution, as lyophilized powder and in a single crystal and presented. The thermal stability of this protein is demonstrated by the fact that minor alterations in the spectrum, mainly in the amide III band near 1260 cm-1, occur in the solution spectrum only at temperatures above 75 degrees C. No significant spectral changes appear when the pH value of the solution is varied in the range from 1.5 to 8.7. The distinct differences of the powder spectrum compared to that of the solution, show that lyophilization causes appreciable conformational changes both in the main-chain and in the side-chains. A difference in main chain conformation of the basic pancreatic trypsin inhibitor in single crystal and in solution is suggested by different amide III frequencies.     

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.     

Monoacylglycerol Lipases Act as Evolutionarily Conserved Regulators of Non-oxidative Ethanol Metabolism

Fatty acid ethyl esters (FAEEs) are non-oxidative metabolites of ethanol that accumulate in human tissues upon ethanol intake. Although FAEEs are considered as toxic metabolites causing cellular dysfunction and tissue damage, the enzymology of FAEE metabolism remains poorly understood. In this study, we used a biochemical screen in Saccharomyces cerevisiae to identify and characterize putative hydrolases involved in FAEE catabolism. We found that Yju3p, the functional orthologue of mammalian monoacylglycerol lipase (MGL), contributes >90% of cellular FAEE hydrolase activity, and its loss leads to the accumulation of FAEE. Heterologous expression of mammalian MGL in yju3Δ mutants restored cellular FAEE hydrolase activity and FAEE catabolism. Moreover, overexpression or pharmacological inhibition of MGL in mouse AML-12 hepatocytes decreased or increased FAEE levels, respectively. FAEEs were transiently incorporated into lipid droplets (LDs) and both Yju3p and MGL co-localized with these organelles. We conclude that the storage of FAEE in inert LDs and their mobilization by LD-resident FAEE hydrolases facilitate a controlled metabolism of these potentially toxic lipid metabolites.     

Evolutionary history of contagious asexuality in Daphnia pulex

Asexual taxa are short-lived, suggesting that transitions to asexuality represent evolutionary dead-ends. However, with high rates of clonal origin and coexistence of asexuals and sexuals via selective asymmetries, asexuality may persist in the long term as a result of a dynamic equilibrium between clonal origin and extinction. Few such systems have been studied in detail. Here, we investigate the evolutionary history of asexual lineages of Daphnia pulex, which are derived from sexual relatives via the inheritance of a dominant female-limited meiosis-suppressing locus and inhabit ponds throughout northeastern North America (NA). Our extensive sampling and subsequent phylogenetic analysis using mitochondrial sequence data reveals a young and genetically diverse asexual assemblage, reflecting high rates of clonal origin due to the contagious nature of asexuality. Yet, asexuality is restricted to two phylogroups (B and C) with historical and/or present associations with northeastern NA and is absent from a northwestern phylogroup (A), supporting a recent northeastern origin of asexuality in this species. Furthermore, macrogeographic patterns of genetic variability indicate that phylogroups B and C recolonized northeastern NA from opposite directions, yet their presently overlapping geographic distributions are similarly divided into an eastern asexual and a western sexual region. We attribute these patterns to a recent contagious spread of asexuality from a northeastern source. If environment-mediated selective asymmetries play no significant role in determining the outcome of competitive interactions between sexuals and asexuals, regions of contact may be setting the stage for continued asexual conquests.     

A new peptide ligand for targeting human carbonic anhydrase IX, identified through the phage display technology

Carbonic anhydrase IX (CAIX) is a transmembrane enzyme found to be overexpressed in various tumors and associated with tumor hypoxia. Ligands binding this target may be used to visualize hypoxia, tumor manifestation or treat tumors by endoradiotherapy.

Methods

Phage display was performed with a 12 amino acid phage display library by panning against a recombinant extracellular domain of human carbonic anhydrase IX. The identified peptide CaIX-P1 was chemically synthesized and tested in vitro on various cell lines and in vivo in Balb/c nu/nu mice carrying subcutaneously transplanted tumors. Binding, kinetic and competition studies were performed on the CAIX positive human renal cell carcinoma cell line SKRC 52, the CAIX negative human renal cell carcinoma cell line CaKi 2, the human colorectal carcinoma cell line HCT 116 and on human umbilical vein endothelial cells (HUVEC). Organ distribution studies were carried out in mice, carrying SKRC 52 tumors. RNA expression of CAIX in HCT 116 and HUVEC cells was investigated by quantitative real time PCR.

Results

In vitro binding experiments of ¹²⁵I-labeled-CaIX-P1 revealed an increased uptake of the radioligand in the CAIX positive renal cell carcinoma cell line SKRC 52. Binding of the radioligand in the colorectal carcinoma cell line HCT 116 increased with increasing cell density and correlated with the mRNA expression of CAIX. Radioligand uptake was inhibited up to 90% by the unlabeled CaIX-P1 peptide, but not by the negative control peptide octreotide at the same concentration. No binding was demonstrated in CAIX negative CaKi 2 and HUVEC cells. Organ distribution studies revealed a higher accumulation in SKRC 52 tumors than in heart, spleen, liver, muscle, intestinum and brain, but a lower uptake compared to blood and kidney.

Conclusions

These data indicate that CaIX-P1 is a promising candidate for the development of new ligands targeting human carbonic anhydrase IX.     

Morphological and molecular investigations of Tubulinosema ratisbonensis gen. nov., sp. nov. (Microsporidia: Tubulinosematidae fam. nov.), a parasite infecting a laboratory colony of Drosophila melanogaster (Diptera: Drosophilidae)

A new species of microsporidia from Drosophila melanogaster was investigated by light and electron microscopy and by ribosomal RNA (rRNA) sequencing. This microsporidium and the previously described Nosema kingi and Nosema acridophagus have been transferred to the new genus Tubulinosema gen. nov. with the following characters: nuclei are in diplokaryotic arrangement during the life cycle. All stages are in direct contact with the host cell cytoplasm, slightly anisofilar polar tube with the last coils being smaller in diameter arranged in one or two rows on both sides of the diplokaryon and small tubuli on the surface of late meronts. Spores are oval or slightly pyriform. Thick endospore wall, thinner over anchoring disc. This new genus and the genus Brachiola have been placed in a new family Tubulinosematidae fam. nov. Phylogenetic analysis of small subunit rRNA sequences by different methods placed Tubulinosema spp. in one clade with the genus Brachiola forming its sister clade, which is distant from the clade containing the true Nosema spp. including Nosema bombycis.