Nutrient conservation strategies in native Andean‐Patagonian forests

Abstract. Nutrient conservation in vegetation affects rates of litter decomposition and soil nutrient availability. Although resorption has been traditionally considered one of the most important plant strategies to conserve nutrients in temperate forests, long leaf life‐span and low nutrient requirements have been postulated as better indicators. We aimed at identifying nutrient conservation strategies within characteristic functional groups of NW Patagonian forests on Andisols. We analysed C‐, N‐, P‐, K‐ and lignin‐concentrations in mature and senescent leaves of ten native woody species within the functional groups: broad‐leaved deciduous species, broad‐leaved evergreens and conifers. We also examined mycorrhizal associations in all species. Nutrient concentration in mature leaves and N‐ resorption were higher in broad‐leaved deciduous species than in the other two functional groups. Conifers had low mature leaf nutrient concentrations, low N‐resorption and high lignin/N ratios in senescent leaves. P‐ and K‐resorptions did not differ among functional groups. Broad‐leaved evergreens exhibited a species‐dependent response. Nitrogen in mature leaves was positively correlated with both N resorption and soil N‐fertility. Despite the high P‐retention capacity of Andisols, N appeared to be the more limiting nutrient, with most species being proficient in resorbing N but not P. The presence of endomycorrhizae in all conifers and the broad‐leaved evergreen Maytenus boaria, ectomycorrhizae in all Nothofagus species (four deciduous, one evergreen), and cluster roots in the broad‐leaved evergreen Lomatia hirsuta, would be possibly explaining why P is less limiting than N in these forests.     

Genome-wide CRISPR Screens Reveal Host Factors Critical for SARS-CoV-2 Infection

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Dimer–monomer equilibrium of human thymidylate synthase monitored by fluorescence resonance energy transfer

An ad hoc bioconjugation/fluorescence resonance energy transfer (FRET) assay has been designed to spectroscopically monitor the quaternary state of human thymidylate synthase dimeric protein. The approach enables the chemoselective engineering of allosteric residues while preserving the native protein functions through reversible masking of residues within the catalytic site, and is therefore suitable for activity/oligomerization dual assay screenings. It is applied to tag the two subunits of human thymidylate synthase at cysteines 43 and 43′ with an excitation energy donor/acceptor pair. The dimer–monomer equilibrium of the enzyme is then characterized through steady‐state fluorescence determination of the intersubunit resonance energy transfer efficiency.     

From Regional to National Clouds: TV Coverage in the Czech Republic

Media, and particularly TV media, have a great impact on the general public. In recent years, spatial patterns of information and the relevance of intangible geographies have become increasingly important. Gatekeeping plays a critical role in the selection of information that is transformed into media. Therefore, gatekeeping, through national media, also co-forms the generation of mental maps. In this paper, correspondence analysis (a statistical method) combined with cloud lines (a new visual analytics technique) is used to analyze how individual major regional events in one of the post-communist countries, the Czech Republic, penetrate into the media on a national scale. Although national news should minimize distortions about regions, this assumption has not been verified by our research. Impressions presented by the media of selected regions that were markedly influenced by one or several events in those regions demonstrate that gatekeepers, especially news reporters, functioned as a filter by selecting only a few specific, and in many cases, unusual events for dissemination.     

Mycobacterium tuberculosis ClpX Interacts with FtsZ and Interferes with FtsZ Assembly

FtsZ assembly at the midcell division site in the form of a Z-ring is crucial for initiation of the cell division process in eubacteria. It is largely unknown how this process is regulated in the human pathogen Mycobacterium tuberculosis. Here we show that the expression of clpX was upregulated upon macrophage infection and exposure to cephalexin antibiotic, the conditions where FtsZ-ring assembly is delayed. Independently, we show using pull-down, solid-phase binding, bacterial two-hybrid and mycobacterial protein fragment complementation assays, that M. tuberculosis FtsZ interacts with ClpX, the substrate recognition domain of the ClpXP protease. Incubation of FtsZ with ClpX increased the critical concentration of GTP-dependent polymerization of FtsZ. Immunoblotting revealed that the intracellular ratio of ClpX to FtsZ in wild type M. tuberculosis is approximately 1∶2. Overproduction of ClpX increased cell length and modulated the localization of FtsZ at midcell sites; however, intracellular FtsZ levels were unaffected. A ClpX-CFP fusion protein localized to the cell poles and midcell sites and colocalized with the FtsZ-YFP protein. ClpX also interacted with FtsZ mutant proteins defective for binding to and hydrolyzing GTP and possibly for interactions with other proteins. Taken together, our results suggest that M. tuberculosis ClpX interacts stoichiometrically with FtsZ protomers, independent of its nucleotide-bound state and negatively regulates FtsZ activities, hence cell division.     

Ca2+ phospholipid-dependent and independent phosphorylation of synthetic peptide substrates by protein kinase C

Several synthetic peptides reproducing fragments of protamines have been used as model substrates for Ca2+/phospholipid-dependent protein kinase C, tested both in the absence of any effector (basal conditions) and upon activation by either Ca2+ and phosphatidylserine (or diacylglycerol) or limited proteolysis. Only the peptide Arg4-Tyr-Gly-Ser-Arg6-Tyr [Ga(52-65)] shares the unique property of protamines of being readily phosphorylated even under basal conditions. Optimal activity in the absence of effectors is observed with Tris/HCl buffer pH 7.5; Pipes and Hepes are less effective at pH 7.5, and at pH 6.5 basal phosphorylation is reduced. Under the best conditions for basal phosphorylation of Ga(52-65), its derivative with ornithine replaced for arginine and those corresponding to its C-terminal fragments Gly-Ser-Arg6-Tyr [Ga(57-65)] and Gly-Ser-Arg3 [Ga(57-61)], as well as the peptides Pro-Arg5-Ser2-Arg-Pro-Val-Arg [Th(1-12)], Arg4-Tyr-Arg2-Ser-Thr-Val-Ala [Th(13-23)] and Arg2-Leu-Ser2-Leu-Arg-Ala are not significantly affected though all of them, like histones, are more or less readily phosphorylated upon activation of protein kinase C by Ca2+/phosphatidylserine. The peptide Ser2-Arg-Pro-Val-Arg [Th(7-12)] however, corresponding to the C-terminal part of Th(1-12), is not phosphorylated even in the presence of activators. Limited proteolysis can roughly mimic the Ca2+/phosphatidylserine effect inducing however different extents of activation depending on the nature of the peptide substrates. Our results support the following two conclusions. Basal phosphorylation by protein kinase C in the absence of any effector requires peptide substrates whose target residue(s) are included between two extended arginyl blocks and is also dependent on pH and nature of the buffer. Peptides having extended clusters of either arginyl or ornithyl residues on the C-terminal side of serine are also readily phosphorylated, but they need activation of protein kinase by either Ca2+/phosphatidylserine or limited proteolysis. The same is true of peptides having basic residues only on the N-terminal side, or even on both sides but in limited number.     

Mammalian DNA polymerase alpha: a replication-competent holoenzyme form from calf thymus

Calf thymus DNA polymerase alpha, like the replication-specific DNA polymerase III holoenzyme of Escherichia coli, can be isolated as a distinct complex. A specific multiprotein form of the polymerase alpha, a form designated replication-competent (RC) holoenzyme, consists of a complex of a polymerase-primase core and at least six other polypeptides. The RC holoenzyme can efficiently replicate several naturally occurring templates, including the genomic DNA of the porcine circovirus (PCV). The DNA of this virion consists of a single-stranded circle with a defined replication origin, and its replication requires the cellular DNA replication machinery. It might therefore provide an invaluable opportunity to investigate chromosomal replication mechanisms, analogous to the way that studies on E. coli bacteriophage DNA replication elucidated host DNA replication mechanisms. Calf RC holoenzyme alpha selectively initiates PCV DNA replication in vitro at a site that possibly represents a consensus sequence of cellular DNA replication origins. The cell-free PCV replication system will be exploited for the in vitro dissection and reconstitution of the RC holoenzyme and the functional analysis of its component polypeptides.