Where to Dig for Fossils: Combining Climate-Envelope,Taphonomy and Discovery Models

Fossils represent invaluable data to reconstruct the past history of life, yet fossil-rich sites are often rare and difficult to find. The traditional fossil-hunting approach focuses on small areas and has not yet taken advantage of modelling techniques commonly used in ecology to account for an organism’s past distributions. We propose a new method to assist finding fossils at continental scales based on modelling the past distribution of species, the geological suitability of fossil preservation and the likelihood of fossil discovery in the field, and apply it to several genera of Australian megafauna that went extinct in the Late Quaternary. Our models predicted higher fossil potentials for independent sites than for randomly selected locations (mean Kolmogorov-Smirnov statistic = 0.66). We demonstrate the utility of accounting for the distribution history of fossil taxa when trying to find the most suitable areas to look for fossils. For some genera, the probability of finding fossils based on simple climate-envelope models was higher than the probability based on models incorporating current conditions associated with fossil preservation and discovery as predictors. However, combining the outputs from climate-envelope, preservation, and discovery models resulted in the most accurate predictions of potential fossil sites at a continental scale. We proposed potential areas to discover new fossils of Diprotodon, Zygomaturus, Protemnodon, Thylacoleo, and Genyornis, and provide guidelines on how to apply our approach to assist fossil hunting in other continents and geological settings.     

Synergistic and Antagonistic Effects of Combined Subzero Temperature and High Pressure on Inactivation of Escherichia coli

The combined effects of subzero temperature and high pressure on the inactivation of Escherichia coli K12TG1 were investigated. Cells of this bacterial strain were exposed to high pressure (50 to 450 MPa, 10-min holding time) at two temperatures (−20°C without freezing and 25°C) and three water activity levels (a_w) (0.850, 0.992, and ca. 1.000) achieved with the addition of glycerol. There was a synergistic interaction between subzero temperature and high pressure in their effects on microbial inactivation. Indeed, to achieve the same inactivation rate, the pressures required at −20°C (in the liquid state) were more than 100 MPa less than those required at 25°C, at pressures in the range of 100 to 300 MPa with an a_w of 0.992. However, at pressures greater than 300 MPa, this trend was reversed, and subzero temperature counteracted the inactivation effect of pressure. When the amount of water in the bacterial suspension was increased, the synergistic effect was enhanced. Conversely, when the a_w was decreased by the addition of solute to the bacterial suspension, the baroprotective effect of subzero temperature increased sharply. These results support the argument that water compression is involved in the antimicrobial effect of high pressure. From a thermodynamic point of view, the mechanical energy transferred to the cell during the pressure treatment can be characterized by the change in volume of the system. The amount of mechanical energy transferred to the cell system is strongly related to cell compressibility, which depends on the water quantity in the cytoplasm.     

Divergence thresholds and divergent biodiversity estimates: can metabarcoding reliably describe zooplankton communities?

DNA metabarcoding is a promising method for describing communities and estimating biodiversity. This approach uses high‐throughput sequencing of targeted markers to identify species in a complex sample. By convention, sequences are clustered at a predefined sequence divergence threshold (often 3%) into operational taxonomic units (OTUs) that serve as a proxy for species. However, variable levels of interspecific marker variation across taxonomic groups make clustering sequences from a phylogenetically diverse dataset into OTUs at a uniform threshold problematic. In this study, we use mock zooplankton communities to evaluate the accuracy of species richness estimates when following conventional protocols to cluster hypervariable sequences of the V4 region of the small subunit ribosomal RNA gene (18S) into OTUs. By including individually tagged single specimens and “populations” of various species in our communities, we examine the impact of intra‐ and interspecific diversity on OTU clustering. Communities consisting of single individuals per species generated a correspondence of 59–84% between OTU number and species richness at a 3% divergence threshold. However, when multiple individuals per species were included, the correspondence between OTU number and species richness dropped to 31–63%. Our results suggest that intraspecific variation in this marker can often exceed 3%, such that a single species does not always correspond to one OTU. We advocate the need to apply group‐specific divergence thresholds when analyzing complex and taxonomically diverse communities, but also encourage the development of additional filtering steps that allow identification of artifactual rRNA gene sequences or pseudogenes that may generate spurious OTUs.     

A Monoacylglycerol Lipase from Mycobacterium smegmatis Involved in Bacterial Cell Interaction

MSMEG_0220 from Mycobacterium smegmatis, the ortholog of the Rv0183 gene from M. tuberculosis, recently identified and characterized as encoding a monoacylglycerol lipase, was cloned and expressed in Escherichia coli. The recombinant protein (rMSMEG_0220), which exhibits 68% amino acid sequence identity with Rv0183, showed the same substrate specificity and similar patterns of pH-dependent activity and stability as the M. tuberculosis enzyme. rMSMEG_0220 was found to hydrolyze long-chain monoacylglycerol with a specific activity of 143 ± 6 U mg⁻¹. Like Rv0183 in M. tuberculosis, MSMEG_0220 was found to be located in the cell wall. To assess the in vivo role of the homologous proteins, an MSMEG_0220 disrupted mutant of M. smegmatis (MsΔ0220) was produced. An intriguing change in the colony morphology and in the cell interaction, which were partly restored in the complemented mutant containing either an active (ComMsΔ0220) or an inactive (ComMsΔ0220S111A) enzyme, was observed. Growth studies performed in media supplemented with monoolein showed that the ability of both MsΔ0220 and ComMsΔ0220S111A to grow in the presence of this lipid was impaired. Moreover, studies of the antimicrobial susceptibility of the MsΔ0220 strain showed that this mutant is more sensitive to rifampin and more resistant to isoniazid than the wild-type strain, pointing to a critical structural role of this enzyme in mycobacterial physiology, in addition to its function in the hydrolysis of exogenous lipids.Tuberculosis, which is caused by Mycobacterium tuberculosis, is a major public health issue worldwide. Because of the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains and the high incidence of HIV and tuberculosis coinfection (16), it is becoming increasingly difficult to combat the spread of this disease, and the global health burden of tuberculosis is extremely heavy. The reasons for the persistence of the tubercle bacillus include not only its ability to enter into a state of dormancy in its host for decades, evading the immune system by forming structures called granulomas (17), but also its unique and complex cell wall composed of specific lipids (8). These characteristics are thought to be good focus points for drug development. In granulomas, during the nonreplicative stage, the bacteria have been found to accumulate lipids in the form of intracellular lipid inclusion bodies (LIBs) (13). These lipids are composed mainly of triacylglycerols (TAG) (9, 13) and may originate from the lipolysis of host lipids and/or fatty acid uptake. In fact, M. tuberculosis in the granuloma center can even accumulate lipids originating from the degradation of immune cells (20). In addition, it has been reported that M. tuberculosis internalized by foamy macrophages accumulated LIBs when it joined cell lipid droplets composed of neutral lipids (32). Lipid storage may provide the bacillus with energy via the β-oxidation pathway followed by the glyoxylate cycle, during the chronic phase and the reactivation step (3, 17). These lipids may also supply precursors for the synthesis of bacterial cell membrane lipids, which play a key role in the pathogenicity of M. tuberculosis (4, 23). To investigate the molecular basis of the virulence and pathogenicity of M. tuberculosis, it was therefore proposed to study the lipid metabolism and cell wall remodeling processes in this bacterium.The enzymes involved in the lipid degradation processes induced by this bacterium have attracted considerable attention during the last few years. Based on the complete M. tuberculosis H37Rv genome sequence (6), several open reading frames (ORFs) encoding proteins potentially involved in the lipid metabolism of this strain have been identified, among which are the two lipases from M. tuberculosis that have been purified and characterized so far. Deb et al. identified an enzyme, Rv3097c (LipY), belonging to the hormone-sensitive lipase family, which is able to hydrolyze long-chain TAG (10). A study of LIB mobilization in a lipY-deficient mutant has shown that LipY was involved in TAG hydrolysis under nutriment-deprived conditions (10). LipY may therefore be involved in the degradation of TAG stored during the dormant stage and the subsequent reactivation of the pathogen. In addition, electron microscopy immunolabeling studies of LipY clearly showed that the enzyme had a cell surface localization, thus in direct contact with the host immune system (28). The last identified lipase to date is a monoacylglycerol lipase annotated Rv0183 (7). Like LipY, Rv0183 is located in the cell wall, but its exact physiological function has not yet been elucidated. One hypothesis could be that, like some mammalian cells (e.g., adipocytes), M. tuberculosis expresses several lipolytic enzymes sequentially involved in the lipolysis of TAG (37). The Rv0183 enzyme is conserved in M. bovis (Mb0189) and M. leprae (ML2603), as well as in M. smegmatis (MSMEG_0220), a nonpathogenic mycobacterium which provides a useful model organism and a surrogate host for molecular analysis of M. tuberculosis (19). In order to decipher the cellular role of Rv0183 in M. tuberculosis H37Rv and its contribution to the lipid metabolism of this bacterium, biochemical studies were performed on the homologue MSMEG_0220. For this purpose, the MSMEG_0220 gene from M. smegmatis, encoding a protein showing 68% amino acid sequence identity with Rv0183, was cloned, and the recombinant MSMEG_0220 enzyme (rMSMEG_0220) was produced in Escherichia coli, purified, and biochemically characterized. An M. smegmatis mutant with an MSMEG_0220 disrupted gene was produced to investigate the physiological role of MSMEG_0220.     

Lipomannan and lipoarabinomannan from a clinical isolate of Mycobacterium kansasii: novel structural features and apoptosis-inducing properties

Although Mycobacterium kansasii has emerged as an important pathogen frequently encountered in immunocompromised patients, little is known about the mechanisms of M. kansasii pathogenicity. Lipoarabinomannan (LAM), a major mycobacterial cell wall lipoglycan, is an important virulence factor for many mycobacteria, as it modulates the host immune response. Therefore, the detailed structures of the of M. kansasii LAM (KanLAM), as well as of its biosynthetic precursor lipomannan (KanLM), were determined in a clinical strain isolated from a human immunodeficiency virus-positive patient. Structural analyses revealed that these lipoglycans possess important differences as compared with those from other mycobacterial species. KanLAM carries a mannooligosaccharide cap but is devoid of the inositol phosphate cap present in Mycobacterium smegmatis. Characterization of the mannan core of KanLM and KanLAM demonstrated the following occurrences: 1) alpha1,2-oligo-mannopyranosyl side chains, contrasting with the single mannopyranosyl residues substituting the mannan core in all the other structures reported so far; and 2) 5-methylthiopentose residues that were described to substitute the arabinan moiety from Mycobacterium tuberculosis LAM. With respect to the arabinan domain of KanLAM, succinyl groups were found to substitute the C-3 position on 5-arabinofuranosyl residues, reported to be linked to the C-2 of the 3,5-arabinofuranose in Mycobacterium bovis bacillus calmette-guerin LAM. Because M. kansasii has been reported to induce apoptosis, we examined the possibility of the M. kansasii lipoglycans to induce apoptosis of THP-1 cells. Our results indicate that, in contrast to KanLAM, KanLM was a potent apoptosis-inducing factor. This work underlines the diversity of LAM structures among various pathogenic mycobacterial species and also provides evidence of LM being a potential virulence factor in M. kansasii infections by inducing apoptosis.     

A hydroponic culture system for growing<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plantlets under sterile conditions

We developed a hydroponic cultivation system for growingArabidopsis plantlets under sterile, controlled environmental conditions. The system consists of a piece of stainless-steel wire cloth (125 μm mesh size) that is fixed between 2 flat rings and held in place by 3 legs, placed in a commercially-available glass jar, and covered by the original glass lid or a sheet of sterilized cellophane. Sterilized seeds were distributed evenly across the mesh piece, the size of which allowed root growth and kept the seeds in place. After 3 weeks of cultivation, shoot and root tissues were easily harvested without mechanical damage. Proteome and metabolite analyses were performed on root and shoot tissues and demonstrated excellent reproducibility, indicating that the system is advantageous when biological variation is minimized. Induction experiments can be performed by transferring the apparatus (with plants) to a new jar containing a different nutrient solution. The apparatus is reusable and can easily be sterilized by autoclaving or dry heat. The system can be adapted to other small-seed plants by varying the mesh size.     

Viral proteomics: global evaluation of viruses and their interaction with the host

Viruses constantly adapt to and modulate the host environment during replication and propagation. Both DNA and RNA viruses encode multifunctional proteins that interact with and modify host cell proteins. While viral genomes were the first complete sequences known, the corresponding proteomes are only now elucidated, with some surprising results. Even more daunting is the task to globally monitor the impact of viral infection on the proteome of the host cell and many technical hurdles must still be overcome in order to facilitate robust and reproducible measurements. Further complicating the picture is the dynamic nature of proteins, including post-translational modifications, enzymatic cleavage and activation or destruction by proteolytic events. Nevertheless, several promising studies have been published using high-throughput methods directly measuring protein abundance. Particularly, quantitative or semiquantitative mass spectrometry-based analysis of viral and cellular proteomes are now being used to characterize viruses and their host interaction. In addition, the full set of interactions between viral and host proteins, the interactome, is beginning to emerge, with often unexpected interactions that need to be carefully validated. In this review, we will discuss two major areas of viral proteomics: first, virion proteomics (such as the protein characterization of viral particles) and second, proteoviromics, including the viral protein interactomics and the quantitative analysis of host cell proteome during viral infection.