High‐Throughput DNA sequencing of ancient wood

Reconstructing the colonization and demographic dynamics that gave rise to extant forests is essential to forecasts of forest responses to environmental changes. Classical approaches to map how population of trees changed through space and time largely rely on pollen distribution patterns, with only a limited number of studies exploiting DNA molecules preserved in wooden tree archaeological and subfossil remains. Here, we advance such analyses by applying high‐throughput (HTS) DNA sequencing to wood archaeological and subfossil material for the first time, using a comprehensive sample of 167 European white oak waterlogged remains spanning a large temporal (from 550 to 9,800 years) and geographical range across Europe. The successful characterization of the endogenous DNA and exogenous microbial DNA of 140 (~83%) samples helped the identification of environmental conditions favouring long‐term DNA preservation in wood remains, and started to unveil the first trends in the DNA decay process in wood material. Additionally, the maternally inherited chloroplast haplotypes of 21 samples from three periods of forest human‐induced use (Neolithic, Bronze Age and Middle Ages) were found to be consistent with those of modern populations growing in the same geographic areas. Our work paves the way for further studies aiming at using ancient DNA preserved in wood to reconstruct the micro‐evolutionary response of trees to climate change and human forest management.     

The N‐Terminal Sequences of Four Major Hydrogenosomal Proteins Are Not Essential for Import into Hydrogenosomes of Trichomonas vaginalis

The human pathogen Trichomonas vaginalis harbors hydrogenosomes, organelles of mitochondrial origin that generate ATP through hydrogen‐producing fermentations. They contain neither genome nor translation machinery, but approximately 500 proteins that are imported from the cytosol. In contrast to well‐studied organelles like Saccharomyces mitochondria, very little is known about how proteins are transported across the two membranes enclosing the hydrogenosomal matrix. Recent studies indicate that—in addition to N‐terminal transit peptides—internal targeting signals might be more common in hydrogenosomes than in mitochondria. To further characterize the extent to which N‐terminal and internal motifs mediate hydrogenosomal protein targeting, we transfected Trichomonas with 24 hemagglutinin (HA) tag fusion constructs, encompassing 13 different hydrogenosomal and cytosolic proteins of the parasite. Hydrogenosomal targeting of these proteins was analyzed by subcellular fractionation and independently by immunofluorescent localization. The investigated proteins include some of the most abundant hydrogenosomal proteins, such as pyruvate ferredoxin oxidoreductase (PFO), which possesses an amino‐terminal targeting signal that is processed on import into hydrogenosomes, but is shown here not to be required for import into hydrogenosomes. Our results demonstrate that the deletion of N‐terminal signals of hydrogenosomal precursors generally has little, if any, influence upon import into hydrogenosomes. Although the necessary and sufficient signals for hydrogenosomal import recognition appear complex, targeting to the organelle is still highly specific, as demonstrated by the finding that six HA‐tagged glycolytic enzymes, highly expressed under the same promoter as other constructs studied here, localized exclusively to the cytosol and did not associate with hydrogenosomes.     

LuxS quorum sensing: more than just a numbers game

Quorum sensing is a process of bacterial cell-to-cell communication involving the production and detection of extracellular signaling molecules called autoinducers. Quorum sensing allows populations of bacteria to collectively control gene expression, and thus synchronize group behavior. Processes controlled by quorum sensing are typically ones that are unproductive unless many bacteria act together. Most autoinducers enable intraspecies communication; however, a recently discovered autoinducer AI-2 has been proposed to serve as a 'universal signal' for interspecies communication. Studies suggest that AI-2 encodes information in addition to specifics about cell number.     

Generation of a double-fluorescent double-selectable Cre/loxP indicator vector for monitoring of intracellular recombination events

Here we describe the generation of a double-fluorescent Cre/loxP indicator system. This protocol involves (i) all cloning steps to generate the plasmid vector (3-5 months); (ii) a guide to prepare high-efficiency transformation competent E. coli; (iii) generation of double-fluorescent reporter cell lines (3-4 weeks); and (iv) the functional testing of the indicator cell lines by application of cell-permeable Cre recombinase. The indicator is designed to monitor recombination events by switching the fluorescence light from red to green. The red fluorescence, indicating the nonrecombined state, is accompanied by the expression of a resistance gene against the antibiotic blasticidin. Appearance of green fluorescence concomitantly with the activation of puromycin-acetyltransferase monitors the recombination of the indicator construct by the Cre recombinase. In summary, we have developed a plasmid vector allowing a fast, stable and straightforward generation of transgenic clones. The expression of red fluorescent protein enables the selection of positive clones upon transfection and significantly shortens the time for identification of stable clones. This feature and the option to select for recombined cells by puromycin application are advantages compared with other alternative methods. Moreover, we developed a method utilizing cell-permeable Cre protein to validate the transgenic clones. Ultimately, this powerful methodology facilitates Cre/loxP-based applications such as cell lineage tracking or monitoring of cell fusion.     

A simple reverse genetics method to generate recombinant coronaviruses

Engineering recombinant viruses is a pre‐eminent tool for deciphering the biology of emerging viral pathogens such as the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). However, the large size of coronavirus genomes renders the current reverse genetics methods challenging. Here, we describe a simple method based on “infectious subgenomic amplicons” (ISA) technology to generate recombinant infectious coronaviruses with no need for reconstruction of the complete genomic cDNA and apply this method to SARS‐CoV‐2 and also to the feline enteric coronavirus. In both cases we rescue wild‐type viruses with biological characteristics similar to original strains. Specific mutations and fluorescent red reporter genes can be readily incorporated into the SARS‐CoV‐2 genome enabling the generation of a genomic variants and fluorescent reporter strains for in vivo experiments, serological diagnosis, and antiviral assays. The swiftness and simplicity of the ISA method has the potential to facilitate the advance of coronavirus reverse genetics studies, to explore the molecular biological properties of the SARS‐CoV‐2 variants, and to accelerate the development of effective therapeutic reagents.     

Architecture and function of metallopeptidase catalytic domains

The cleavage of peptide bonds by metallopeptidases (MPs) is essential for life. These ubiquitous enzymes participate in all major physiological processes, and so their deregulation leads to diseases ranging from cancer and metastasis, inflammation, and microbial infection to neurological insults and cardiovascular disorders. MPs cleave their substrates without a covalent intermediate in a single‐step reaction involving a solvent molecule, a general base/acid, and a mono‐ or dinuclear catalytic metal site. Most monometallic MPs comprise a short metal‐binding motif (HEXXH), which includes two metal‐binding histidines and a general base/acid glutamate, and they are grouped into the zincin tribe of MPs. The latter divides mainly into the gluzincin and metzincin clans. Metzincins consist of globular ～130–270‐residue catalytic domains, which are usually preceded by N‐terminal pro‐segments, typically required for folding and latency maintenance. The catalytic domains are often followed by C‐terminal domains for substrate recognition and other protein–protein interactions, anchoring to membranes, oligomerization, and compartmentalization. Metzincin catalytic domains consist of a structurally conserved N‐terminal subdomain spanning a five‐stranded β‐sheet, a backing helix, and an active‐site helix. The latter contains most of the metal‐binding motif, which is here characteristically extended to HEXXHXXGXX(H,D). Downstream C‐terminal subdomains are generally shorter, differ more among metzincins, and mainly share a conserved loop—the Met‐turn—and a C‐terminal helix. The accumulated structural data from more than 300 deposited structures of the 12 currently characterized metzincin families reviewed here provide detailed knowledge of the molecular features of their catalytic domains, help in our understanding of their working mechanisms, and form the basis for the design of novel drugs.     

Stimulating action of methyl 12, 12, 12-trifluorofarnesoate on in vitro juvenile hormone III biosynthesis in blattella germanica

Methyl 12, 12, 12-trifluorofarnesoate (MTFF) at a dose of 10 μM, stimulated in vitro juvenile hormone (JH) release in corpora allata (CA) from 6-day-old, freshly ecdysed, and 8-day-old (period of ootheca transport) adult virgin females of Blattella germanica. In addition, MTFF also induced intraglandular accumulation of JH and MF in treated CA. Trifluorofarnesoic acid (TFFA) and trifluorofarnesol (TFF) exhibited the same properties, although to a lesser extent than MTFF. The detection of MTFF in TFFA-treated CA suggested that TFFA and TFF were biotransformed into MTFF by the CA enzymatic system and that this ester might be responsible for the activity observed. Equivalent experiments carried out with farnesoic acid (FA) resulted in a more significant stimulation of JH production. This is not surprising, because exogenous FA is readily epoxidized at C10-C11 double bond and methylated to afford JH. Conversely, analytical data have shown that the C6-C7 double bond of MTFF is epoxidized by the CA enzymatic system, whereas that at C10-C11 remains practically unaltered.