In vivo,in vitro and in silico: an open space for the development of microbe-based applications of synthetic biology

Living systems are studied using three complementary approaches: living cells, cell-free systems and computer-mediated modelling. Progresses in understanding, allowing researchers to create novel chassis and industrial processes rest on a cycle that combines in vivo, in vitro and in silico studies. This design–build–test–learn iteration loop cycle between experiments and analyses combines together physiology, genetics, biochemistry and bioinformatics in a way that keeps going forward. Because computer-aided approaches are not directly constrained by the material nature of the entities of interest, we illustrate here how this virtuous cycle allows researchers to explore chemistry which is foreign to that present in extant life, from whole chassis to novel metabolic cycles. Particular emphasis is placed on the importance of evolution.     

Honey, I shrunk the DNA: DNA length as a probe for nucleic-acid enzyme activity

The replication, recombination, and repair of DNA are processes essential for the maintenance of genomic information and require the activity of numerous enzymes that catalyze the polymerization or digestion of DNA. This review will discuss how differences in elastic properties between single- and double-stranded DNA can be used as a probe to study the dynamics of these enzymes at the single-molecule level.     

Temporal dynamics and linkage disequilibrium in natural Caenorhabditis elegans populations

Caenorhabditis elegans is a major laboratory model system yet a newcomer to the field of population genetics, and relatively little is known of its biology in the wild. Recent studies of natural populations at a single time point revealed strong spatial population structure and suggested that these populations may be very dynamic. We have therefore studied several natural C. elegans populations over time and genotyped them at polymorphic microsatellite loci. While some populations appear to be genetically stable over the course of observation, others seem to go extinct, with full replacement of multilocus genotypes upon regrowth. The frequency of heterozygotes indicates that outcrossing occurs at a mean frequency of 1.7% and is variable between populations. However, in genetically stable populations, linkage disequilibrium between different chromosomes can be maintained over several years at a level much higher than expected from the heterozygote frequency. C. elegans seems to follow metapopulation dynamics, and the maintenance of linkage disequilibrium despite a low yet significant level of outcrossing suggests that selection may act against the progeny of outcrossings.     

Characterization of reemerging chikungunya virus

An unprecedented epidemic of chikungunya virus (CHIKV) infection recently started in countries of the Indian Ocean area, causing an acute and painful syndrome with strong fever, asthenia, skin rash, polyarthritis, and lethal cases of encephalitis. The basis for chikungunya disease and the tropism of CHIKV remain unknown. Here, we describe the replication characteristics of recent clinical CHIKV strains. Human epithelial and endothelial cells, primary fibroblasts and, to a lesser extent, monocyte-derived macrophages, were susceptible to infection and allowed viral production. In contrast, CHIKV did not replicate in lymphoid and monocytoid cell lines, primary lymphocytes and monocytes, or monocyte-derived dendritic cells. CHIKV replication was cytopathic and associated with an induction of apoptosis in infected cells. Chloroquine, bafilomycin-A1, and short hairpin RNAs against dynamin-2 inhibited viral production, indicating that viral entry occurs through pH-dependent endocytosis. CHIKV was highly sensitive to the antiviral activity of type I and II interferons. These results provide a general insight into the interaction between CHIKV and its mammalian host.     

Repeatable inter‐individual variation in the thermal sensitivity of metabolic rate

Assessing whether trait variations among individuals are consistent over time and among environmental conditions is crucial to understand evolutionary responses to new selective pressures such as climate change. According to the universal thermal dependence hypothesis, thermal sensitivity of metabolic rate should not vary strongly and consistently among organisms, implying limited evolutionary response for metabolic traits under climate change. However, this hypothesis has been rarely tested at an individual level, leaving a gap in our understanding of climate change impacts on metabolic responses and their potential evolution. Using the amphipod Gammarus fossarum, we investigated the variability and repeatability of individual metabolic thermal reaction norms over time. We found large variations in both the thermal sensitivity (i.e. slope) and expression level (i.e. intercept) of individual metabolic reaction norms. Moreover, differences among individuals were consistent over time, and therefore repeatable. Inter‐individual variations in body mass resulted in a high repeatability of metabolic expression level but had no significant effect on the repeatability of thermal sensitivity. Overall, our results highlight that inter‐individual variability and repeatability of thermal reaction norms can be substantial. We conclude that these consistent differences among individuals should not be overlooked when apprehending the ecological and evolutionary effects of climate change.     

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.     

Shape matters: Lifecycle of cooperative patches promotes cooperation in bulky populations

Natural cooperative systems take many forms, ranging from one‐dimensional cyanobacteria arrays to fractal‐like biofilms. We use in silico experimental systems to study a previously overlooked factor in the evolution of cooperation, physical shape of the population. We compare the emergence and maintenance of cooperation in populations of digital organisms that inhabit bulky (100 × 100 cells) or slender (4 × 2500) toroidal grids. Although more isolated subpopulations of secretors in a slender population could be expected to favor cooperation, we find the opposite: secretion evolves to higher levels in bulky populations. We identify the mechanistic explanation for the shape effect by analyzing the lifecycle and dynamics of cooperator patches, from their emergence and growth, to invasion by noncooperators and extinction. Because they are constrained by the population shape, the cooperator patches expand less in slender than in bulky populations, leading to fewer cooperators, less public good secretion, and generally lower cooperation. The patch dynamics and mechanisms of shape effect are robust across several digital cooperation systems and independent of the underlying basis for cooperation (public good secretion or a cooperation game). Our results urge for a greater consideration of population shape in the study of the evolution of cooperation across experimental and modeling systems.     

Hepatic Proliferation and Angiogenesis Markers Are Increased after Portal Deprivation in Rats: A Study of Molecular,Histological and Radiological Changes

MethodsWe conducted an experimental study comparing portacaval shunt (PCS), total portal vein ligation (PVL), and sham (S) operated rats. Each group were either sacrificed at 6 weeks (early) or 6 months (late). Arterial liver perfusion was studied in vivo using CT, and histopathological changes were noted. Liver mRNA levels were quantified by RT-QPCR for markers of inflammation (Il10, Tnfa), proliferation (Il6st, Mki67, Hgf, Hnf4a), angiogenesis: (Vegfa, Vegfr 1, 2 and 3; Pgf), oxidative stress (Nos2, and 3, Hif1a), and fibrosis (Tgfb). PCS and PVL were compared to the S group.ResultsPeriportal fibrosis and arterial proliferation was observed in late PCS and PVL groups. CT imaging demonstrated increased arterial liver perfusion in the PCS group. RT-QPCR showed increased inflammatory markers in PCS and PVL early groups. Tnfa and Il10 were increased in PCS and PVL late groups respectively. All proliferative markers increased in the PCS, and Hnf4a in the PVL early groups. Mki67 and Hnf4a were increased in the PCS late group. Nos3 was increased in the early and late PCS groups, and Hif1a was decreased in the PVL groups. Markers of angiogenesis were all increased in the early PCS group, and Vegfr3 and Pgf in the late PCS group. Only Vegfr3 was increased in the PVL groups. Tgf was increased in the PCS groups.ConclusionsPortal deprivation in rats induces a sustained increase in intrahepatic markers of inflammation, angiogenesis, proliferation, and fibrosis.     

Ecological assembly rules in plant communities—approaches,patterns and prospects

Understanding how communities of living organisms assemble has been a central question in ecology since the early days of the discipline. Disentangling the different processes involved in community assembly is not only interesting in itself but also crucial for an understanding of how communities will behave under future environmental scenarios. The traditional concept of assembly rules reflects the notion that species do not co‐occur randomly but are restricted in their co‐occurrence by interspecific competition. This concept can be redefined in a more general framework where the co‐occurrence of species is a product of chance, historical patterns of speciation and migration, dispersal, abiotic environmental factors, and biotic interactions, with none of these processes being mutually exclusive. Here we present a survey and meta‐analyses of 59 papers that compare observed patterns in plant communities with null models simulating random patterns of species assembly. According to the type of data under study and the different methods that are applied to detect community assembly, we distinguish four main types of approach in the published literature: species co‐occurrence, niche limitation, guild proportionality and limiting similarity. Results from our meta‐analyses suggest that non‐random co‐occurrence of plant species is not a widespread phenomenon. However, whether this finding reflects the individualistic nature of plant communities or is caused by methodological shortcomings associated with the studies considered cannot be discerned from the available metadata. We advocate that more thorough surveys be conducted using a set of standardized methods to test for the existence of assembly rules in data sets spanning larger biological and geographical scales than have been considered until now. We underpin this general advice with guidelines that should be considered in future assembly rules research. This will enable us to draw more accurate and general conclusions about the non‐random aspect of assembly in plant communities.