Genetic bottlenecks and population passages cause profound fitness differences in RNA viruses.

Repeated clone-to-clone (genetic bottleneck) passages of an RNA phage and vesicular stomatitis virus have been shown previously to result in loss of fitness due to Muller's ratchet. We now demonstrate that Muller's ratchet also operates when genetic bottleneck passages are carried out at 37 rather than 32 degrees C. Thus, these fitness losses do not depend on growth of temperature-sensitive (ts) mutants at lowered temperatures. We also demonstrate that during repeated genetic bottleneck passages, accumulation of deleterious mutations does occur in a stepwise (ratchet-like) manner as originally proposed by Muller. One selected clone which had undergone significant loss of fitness after only 20 genetic bottleneck passages was passaged again in clone-to-clone series. Additional large losses of fitness were observed in five of nine independent bottleneck series; the relative fitnesses of the other four series remained close to the starting fitness. In sharp contrast, when the same selected clone was transferred 20 more times as large populations (10(5) to 10(6) PFU transferred at each passage), significant increases in fitness were observed in all eight passage series. Finally, we selected several clones which had undergone extreme losses of fitness during 20 bottleneck passages. When these low-fitness clones were passaged many times as large virus populations, they always regained very high relative fitness. We conclude that transfer of large populations of RNA viruses regularly selects those genomes within the quasispecies population which have the highest relative fitness, whereas bottleneck transfers have a high probability of leading to loss of fitness by random isolation of genomes carrying debilitating mutations. Both phenomena arise from, and underscore, the extreme mutability and variability of RNA viruses.     

Fine structure of the spermatozoa of Crassostrea gigas (Mollusca,Bivalvia)

We describe sperm ultrastructure and acrosome differentiation during spermiogenesis in Crassostrea gigas (Mollusca Bivalvia). The sperm cell is a uniflagellated cell of the primitive type. The head region contains a rounded or conical nucleus surmounted by small acrosome. This organelle consists of a membrane-bound acrosomal granule, the contents of which have a homogeneous density, except in the anterior region, which is positive for PTA. The acrosome also surrounds the perforatorium, which includes oriented fibrillar elements: this is the axial body. The middle piece contains four mitochondria encircling two perpendicular centrioles. The distal centriole is provided with a system of mechanical fixation to the plasma membrane, consisting of nine fibers in radial arrangement. The tail flagellum, about 50 m?m long, contains the usual microtubular axoneme. © 1993 Wiley-Liss, Inc.     

Analysis of parameters about useful life extension in 70 tools and methods related to eco-design and circular economy

One of the approaches followed by the circular economy (CE) to achieve sustainability through design is product life extension. Extending the life of products to make them useful for as long as possible is a means to reduce waste production and materials consumption, as well as the related impacts. For designers, conceptualizing products in a way that allows them to be used for longer is a challenge, and assessing how well they extend their lifespan can be helpful when it comes to choosing the best proposal. In this paper, 70 tools and methods related to eco-design and circular economy are studied to determine how many of them consider parameters related to life extension and which can be applied in the early stages of design. The results of the analysis show that most of the existing tools and methods are applicable to developed products, and only a few of them take into account parameters related to extending the useful life. Of the 70 tools and methods, only 14 include some parameter related to life extension and are applicable to concepts. CE toolkit, Eco-design PILOT, CE Designer, Circularity Assessment tool, Circularity Potential Indicator and Circular Design Tools take into consideration eight or more parameters to assess life extension in concepts. This will help designers select the most appropriate and will indicate the need for more complete tools to consider useful life extension in the early stages of design and thus enhance the selection of more sustainable products.     

Study of localization of the protein-synthesizing machinery along actin filament bundles.

Indirect immunofluorescent microscopy was used to study the distribution of elongation factor 2 (eEF-2) in fixed human skin diploid and mouse embryo fibroblasts. It was found earlier that some of the eEF-2 ribosomes and initiation factor 2 (eIF-2) are co-localized with a part of the actin microfilament bundles in these cells (Gavrilova et al., 1987; Shestakova et al., 1991). Here it has been shown that inhibition of protein synthesis either by inactivation of eEF-2 itself with diphtheria toxin or by inactivation of ribosomes with ricin does not abolish the distribution of eEF-2 along the actin microfilament bundles. At the same time, the disassembly of actin microfilaments by cytochalasin D results also in the disappearance of eEF-2-carrying threads. This means that the eEF-2-carrying threads do not exist per se, and that the organization of eEF-2 in visible "filaments" depends upon the integrity of the actin cytoskeleton.     

SruI restriction endonuclease from Selenomonas ruminantium

Abstract Sru I, specific restriction endonuclease, has been characterized from Selenomonas ruminantium isolated from the rumen of fallow deer. Results from the study demonstrate that S. ruminantium 18D possesses a type II restriction endonuclease, which recognizes the sequence 5'-TTT↓AAA-3'. The recognition sequence of Sru I was identified using digestions on pBR322, pBR328, pUC18, M13mp18RF, pACYC184 and λDNA. The cleavage patterns obtained were compared with computer-derived data. Sru I recognises the palindromic hexanucleotide sequence and cleaves DNA after the third T in the sequence, producing blunt ends. The purification and characterization of restriction endonuclease Sru I presented here is the first described for Selenomonas ruminantium spp. and demonstrates that this microorganism pocesses a DNA-cleaving enzyme with the same specificity as Dra I or Aha III.     

Enterocin 226NWC, a bacteriocin produced by Enterococcus faecalis 226, active against Listeria monocytogenes

F. VILLANI, G. SALZANO, E. SORRENTINO, O. PEPE, P. MARINO AND S. COPPOLA. 1993. Enterococcus faecalis 226, isolated from natural whey cultures utilized as starters in the manufacture of mozzarella cheese from water-buffalo milk, produces a bacteriocin designated enterocin 226NWC. The bacteriocin was isolated from culture supernatant fluids of the producer strain and was active against strains of the same species and Listeria monocytogenes, but not against useful lactic acid bacteria. Enterocin 226NWC is a protein with an apparent molecular weight of about 5800; it is relatively heat-stable and has a bactericidal mode of action. Listeria monocytogenes, growing in the presence of the enterocin 226NWC producer strain in broth and in reconstituted skim milk, was inhibited.     

A novel tomato spotted wilt virus isolate encoding a noncanonical NSm C118F substitution associated with Sw-5 tomato gene resistance breaking

The nonstructural protein NSm of tomato spotted wilt virus (TSWV) has been identified as the avirulence determinant of the tomato single dominant Sw-5 resistance gene. Although Sw-5 effectiveness has been shown for most TSWV isolates, the emergence of resistance-breaking (RB) isolates has been observed. It is strongly associated with two point mutations (C118Y or T120N) in the NSm viral protein. TSWV-like symptoms were observed in tomato crop cultivars (+Sw-5) in the Baja California peninsula, Mexico, and molecular methods confirmed the presence of TSWV. Sequence analysis of the NSm 118–120 motif and three-dimensional protein modelling exhibited a noncanonical C118F substitution in seven isolates, suggesting that this substitution could emulate the C118Y-related RB phenotype. Furthermore, phylogenetic and molecular analysis of the full-length genome (TSWV-MX) revealed its reassortment-related evolution and confirmed that putative RB-related features are restricted to the NSm protein. Biological and mutational NSm 118 residue assays in tomato (+Sw-5) confirmed the RB nature of TSWV-MX isolate, and the F118 residue plays a critical role in the RB phenotype. The discovery of a novel TSWV-RB Mexican isolate with the presence of C118F substitution highlights a not previously described viral adaptation in the genus Orthotospovirus, and hence, the necessity of further crop monitoring to alert the establishment of novel RB isolates in cultivated tomatoes.     

RosettaDDGPrediction for high-throughput mutational scans: From stability to binding

Reliable prediction of free energy changes upon amino acid substitutions (ΔΔGs) is crucial to investigate their impact on protein stability and protein–protein interaction. Advances in experimental mutational scans allow high-throughput studies thanks to multiplex techniques. On the other hand, genomics initiatives provide a large amount of data on disease-related variants that can benefit from analyses with structure-based methods. Therefore, the computational field should keep the same pace and provide new tools for fast and accurate high-throughput ΔΔG calculations. In this context, the Rosetta modeling suite implements effective approaches to predict folding/unfolding ΔΔGs in a protein monomer upon amino acid substitutions and calculate the changes in binding free energy in protein complexes. However, their application can be challenging to users without extensive experience with Rosetta. Furthermore, Rosetta protocols for ΔΔG prediction are designed considering one variant at a time, making the setup of high-throughput screenings cumbersome. For these reasons, we devised RosettaDDGPrediction, a customizable Python wrapper designed to run free energy calculations on a set of amino acid substitutions using Rosetta protocols with little intervention from the user. Moreover, RosettaDDGPrediction assists with checking completed runs and aggregates raw data for multiple variants, as well as generates publication-ready graphics. We showed the potential of the tool in four case studies, including variants of uncertain significance in childhood cancer, proteins with known experimental unfolding ΔΔGs values, interactions between target proteins and disordered motifs, and phosphomimetics. RosettaDDGPrediction is available, free of charge and under GNU General Public License v3.0, at https://github.com/ELELAB/RosettaDDGPrediction .     

Molecular analysis of a microaerobically induced operon required for hydrogenase synthesis in Rhizobium leguminosarum biovar viciae

The nucleotide sequence (6138 bp) of a microaerobically inducible region (hupV/VI) from the Rhizobium leguminosarum bv. viciae hydrogenase gene cluster has been determined. Six genes, arranged as a single operon, were identified, and designated hypA, B, F, C, D and E based on the sequence similarities of all of them, except hypF, to genes from the hydrogenase pleiotropic operon (hyp) from Escherichia coli. The gene products from hypBFCDE were identified by in vivo expression analysis in E. coli, and their molecular sizes were consistent with those predicted from the nucleotide sequence. Transposon Tn5 insertions into hypB, hypF, hypD and hypE resulted in R. leguminosarum mutants that lacked any hydrogenase activity in symbiosis with peas, but still were able to synthesize the polypeptide for the hydrogenase large subunit. The gene products HypA, HypB, HypF and HypD contained CX₂C motifs characteristic of metal-binding proteins. In addition, HypB bore a long histidine-rich stretch of amino acids near the N-terminus, suggesting a possible role in nickel binding for this protein. The gene product HypF, which was translationally coupled to HypB, presented two cysteine motifs (CX₂CX₈₁CX₂C) with a capacity to form zinc finger-like structures in the N-terminal third of the protein. A role in nickel metabolism in relation to hydrogenase synthesis is postulated for proteins HypB and HypF.     

Integrating sample similarities into latent class analysis: a tree-structured shrinkage approach

This paper is concerned with using multivariate binary observations to estimate the probabilities of unobserved classes with scientific meanings. We focus on the setting where additional information about sample similarities is available and represented by a rooted weighted tree. Every leaf in the given tree contains multiple samples. Shorter distances over the tree between the leaves indicate a priori higher similarity in class probability vectors. We propose a novel data integrative extension to classical latent class models with tree-structured shrinkage. The proposed approach enables (1) borrowing of information across leaves, (2) estimating data-driven leaf groups with distinct vectors of class probabilities, and (3) individual-level probabilistic class assignment given the observed multivariate binary measurements. We derive and implement a scalable posterior inference algorithm in a variational Bayes framework. Extensive simulations show more accurate estimation of class probabilities than alternatives that suboptimally use the additional sample similarity information. A zoonotic infectious disease application is used to illustrate the proposed approach. The paper concludes by a brief discussion on model limitations and extensions.