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Nonalcoholic fatty liver disease, recently renamed metabolic dysfunction- associated steatotic liver disease (MASLD), is a progressive metabolic disorder that begins with aberrant triglyceride accumulation in the liver and can lead to cirrhosis and cancer. A common variant in the gene PNPLA3, encoding the protein PNPLA3- I148M, is the strongest known genetic risk factor for MASLD. Despite its discovery 20 y ago, the function of PNPLA3, and now the role of PNPLA3- I148M, remain unclear. In this study, we sought to dissect the biogenesis of PNPLA3 and PNPLA3- I148M and characterize changes induced by endogenous expression of the disease- causing variant. Contrary to bioinformatic predictions and prior studies with overexpressed proteins, we demonstrate here that PNPLA3 and PNPLA3- I148M are not endoplasmic reticulum- resident transmembrane proteins. To identify their intracellular associations, we generated a paired set of isogenic human hepatoma cells expressing PNPLA3 and PNPLA3- I148M at endogenous levels. Both proteins were enriched in lipid droplet, Golgi, and endosomal fractions. Purified PNPLA3 and PNPLA3- I148M proteins associated with phosphoinositides commonly found in these compartments. Despite a similar fractionation pattern as the wild- type variant, PNPLA3- I148M induced morphological changes in the Golgi apparatus, including increased lipid droplet--Golgi contact sites, which were also observed in I148M- expressing primary human patient hepatocytes. In addition to lipid droplet accumulation, PNPLA3- I148M expression caused significant proteomic and transcriptomic changes that resembled all stages of liver disease. Cumulatively, we validate an endogenous human cellular system for investigating PNPLA3- I148M biology and identify the Golgi apparatus as a central hub of PNPLA3- I148M- driven cellular change. [ABSTRACT FROM AUTHOR]

Gram-negative bacteria have an outer membrane that serves as a barrier to noxious agents in the environment. This protective function is dependent on lipopolysaccharide, a large glycolipid located in the outer leaflet of the outer membrane. Lipopolysaccharide is synthesized at the cytoplasmic membrane and must be transported to the cell surface. To understand this transport process, we reconstituted membrane-to-membrane movement of lipopolysaccharide by incorporating purified inner and outer membrane transport complexes into separate proteoliposomes. Transport involved stable association between the inner and outer membrane proteoliposomes. Our results support a model in which lipopolysaccharide molecules are pushed one after the other in a PEZ dispenser–like manner across a protein bridge that connects the inner and outer membranes. [ABSTRACT FROM AUTHOR]

Gram-negative bacteria possess an outer membrane (OM) containing lipopolysaccharide (LPS). Proper assembly of the OM not only prevents certain antibiotics from entering the cell, but also allows others to be pumped out. To assemble this barrier, the seven-protein lipopolysaccharide transport (Lpt) system extracts LPS from the outer leaflet of the inner membrane (IM), transports it across the periplasm and inserts it selectively into the outer leaflet of the OM. As LPS is important, if not essential, in most Gram-negative bacteria, the LPS biosynthesis and biogenesis pathways are attractive targets in the development of new classes of antibiotics. The accompanying paper (Simpson BW, May JM, Sherman DJ, Kahne D, Ruiz N. 2015 Phil. Trans. R. Soc. B 370, 20150029. (doi:10.1098/rstb.2015.0029)) reviewed the biosynthesis of LPS and its extraction from the IM. This paper will trace its journey across the periplasm and insertion into the OM. [ABSTRACT FROM AUTHOR]

Background: The complexity of genome-scale metabolic models makes them quite difficult for human users to read, since they contain thousands of reactions that must be included for accurate computer simulation. Interestingly, hidden similarities between groups of reactions can be discovered, and generalized to reveal higher-level patterns. Results: The web-based navigation system Mimoza allows a human expert to explore metabolic network models in a semantically zoomable manner: The most general view represents the compartments of the model; the next view shows the generalized versions of reactions and metabolites in each compartment; and the most detailed view represents the initial network with the generalization-based layout (where similar metabolites and reactions are placed next to each other). It allows a human expert to grasp the general structure of the network and analyze it in a top-down manner Conclusions: Mimoza can be installed standalone, or used on-line at http://mimoza.bordeaux.inria.fr/, or installed in a Galaxy server for use in workflows. Mimoza views can be embedded in web pages, or downloaded as COMBINE archives. [ABSTRACT FROM AUTHOR]

The cell surface of Gram-negative bacteria contains lipopolysaccharides (LPS), which provide a barrier against the entry of many antibiotics. LPS assembly involves a multiprotein LPS transport (Lpt) complex that spans from the cytoplasm to the outer membrane. In this complex, an unusual ATP-binding cassette transporter is thought to power the extraction of LPS from the outer leaflet of the cytoplasmic membrane and its transport across the cell envelope. We introduce changes into the nucleotide-binding domain, LptB, that inactivate transporter function in vivo. We characterize these residues using biochemical experiments combined with high-resolution crystal structures of LptB pre- and post- ATP hydrolysis and suggest a role for an active site residue in phosphate exit. We also identify a conserved residue that is not required for ATPase activity but is essential for interaction with the transmembrane components. Our studies establish the essentiality of ATP hydrolysis by LptB to power LPS transport in cells and suggest strategies to inhibit transporter function away from the LptB active site. [ABSTRACT FROM AUTHOR]

The complex process of genome-scale metabolic network reconstruction involves semi-automatic reaction inference, analysis, and refinement through curation by human experts. Unfortunately, decisions by experts are hampered by the complexity of the network, which can mask errors in the inferred network. In order to aid an expert in making sense out of the thousands of reactions in the organism's metabolism, we developed a method for knowledge-based generalization that provides a higher-level view of the network, highlighting the particularities and essential structure, while hiding the details. In this study, we show the application of this generalization method to 1,286 metabolic networks of organisms in Path2Models that describe fatty acid metabolism. We compare the generalised networks and show that we successfully highlight the aspects that are important for their curation and comparison. [ABSTRACT FROM AUTHOR]

Abstract: The theory of hybrid systems allows us to model biological functions with many interactive processes, describe complexity and hierarchy levels, and consider behavior law changes. However, we need to develop an implementation to simulate these models. The BioRica framework allows a non-ambiguous implementation, and, as shown here, the QSS method (Quantized States Systems) helps us to approach complex systems in a more efficient way. This method allows us to numerically solve stiff differential equations by separately choosing the temporal partition for each sub-model and variable, depending on how fast it changes over time. With that, one obtains more accurate solutions and decreases the number of computations compared to classic methods. Moreover, QSS does not need to store trajectories and interpolate when mode transitions occur between partition times. Herein, we exhibit a translation from BioRica to QSS models, which preserves the semantics. We implement QSS method with BioRica, and illustrate with applications in Biology, the Tyson model of cell cycle, and examples in Engineering. [Copyright &y& Elsevier]

Abstract: The presence of lipopolysaccharide (LPS) in the outer leaflet of the outer membrane (OM) of Gram-negative bacteria creates a permeability barrier that prevents the entry of most currently available antibiotics. The seven lipopolysaccharide transport (Lpt) proteins involved in transporting and assembling this glycolipid are essential for growth and division in Escherichia coli; therefore, inhibiting their functions leads to cell death. LptB, the ATPase that provides energy for LPS transport and assembly, forms a complex with three other inner membrane (IM) components, LptC, F, and G. We demonstrate that inhibitors of pure LptB can also inhibit the full IM complex, LptBFGC, purified in detergent. We also compare inhibition of LptB and the LptBFGC complex with the antibiotic activity of these compounds. Our long-term goal is to develop tools to study inhibitors of LPS biogenesis that could serve as potentiators by disrupting the OM permeability barrier, facilitating entry of clinically used antibiotics not normally used to treat Gram-negative infections, or that can serve as antibiotics themselves. [Copyright &y& Elsevier]

Abstract: The Génolevures online database (URL: http://www.genolevures.org) stores and provides the data and results obtained by the Génolevures Consortium through several campaigns of genome annotation of the yeasts in the Saccharomycotina subphylum (hemiascomycetes). This database is dedicated to large-scale comparison of these genomes, storing not only the different chromosomal elements detected in the sequences, but also the logical relations between them. The database is divided into a public part, accessible to anyone through Internet, and a private part where the Consortium members make genome annotations with our Magus annotation system; this system is used to annotate several related genomes in parallel. The public database is widely consulted and offers structured data, organized using a REST web site architecture that allows for automated requests. The implementation of the database, as well as its associated tools and methods, is evolving to cope with the influx of genome sequences produced by Next Generation Sequencing (NGS). [Copyright &y& Elsevier]

Abstract: Many biological and biomedical laboratory assays require the use of antibodies and antibody fragments that strongly bind to their cell surface targets. Conventional binding assays, such as the enzyme-linked immunosorbent assay (ELISA) and flow cytometry, have many challenges, including capital equipment requirements, labor intensiveness, and large reagent and sample consumption. Although these techniques are successful in mainstream biology, there is an unmet need for a tool to quickly ascertain the relative binding capabilities of antibodies/antibody fragments to cell surface targets on the benchtop at low cost. We describe a novel cell capture assay that enables several candidate antibodies to be evaluated quickly as to their relative binding efficacies to their cell surface targets. We used chimeric rituximab and murine anti-CD20 monoclonal antibodies as cell capture agents on a functionalized microscope slide surface to assess their relative binding affinities based on how well they capture CD20-expressing mammalian cells. We found that these antibodies’ concentration-dependent cell capture profiles correlate with their relative binding affinities. A key observation of this assay involved understanding how differences in capture surfaces affect the assay results. This approach can find utility when an antibody or antibody fragment against a known cell line needs to be selected for targeting studies. [Copyright &y& Elsevier]

Background: The search for enriched features has become widely used to characterize a set of genes or proteins. A key aspect of this technique is its ability to identify correlations amongst heterogeneous data such as Gene Ontology annotations, gene expression data and genome location of genes. Despite the rapid growth of available data, very little has been proposed in terms of formalization and optimization. Additionally, current methods mainly ignore the structure of the data which causes results redundancy. For example, when searching for enrichment in GO terms, genes can be annotated with multiple GO terms and should be propagated to the more general terms in the Gene Ontology. Consequently, the gene sets often overlap partially or totally, and this causes the reported enriched GO terms to be both numerous and redundant, hence, overwhelming the researcher with non-pertinent information. This situation is not unique, it arises whenever some hierarchical clustering is performed (e.g. based on the gene expression profiles), the extreme case being when genes that are neighbors on the chromosomes are considered. Results: We present a generic framework to efficiently identify the most pertinent overrepresented features in a set of genes. We propose a formal representation of gene sets based on the theory of partially ordered sets (posets), and give a formal definition of target set pertinence. Algorithms and compact representations of target sets are provided for the generation and the evaluation of the pertinent target sets. The relevance of our method is illustrated through the search for enriched GO annotations in the proteins involved in a multiprotein complex. The results obtained demonstrate the gain in terms of pertinence (up to 64% redundancy removed), space requirements (up to 73% less storage) and efficiency (up to 98% less comparisons). Conclusion: The generic framework presented in this article provides a formal approach to adequately represent available data and efficiently search for pertinent over-represented features in a set of genes or proteins. The formalism and the pertinence definition can be directly used by most of the methods and tools currently available for feature enrichment analysis. [ABSTRACT FROM AUTHOR]

The proteasome is the central component of the main cellular protein degradation pathway. During the past four decades, the critical function of the proteasome in numerous physiological processes has been revealed, and proteasome activity has been linked to various human diseases. The proteasome prevents the accumulation of misfolded proteins, controls the cell cycle, and regulates the immune response, to name a few important roles for this macromolecular "machine." As a therapeutic target, proteasome inhibitors have been approved for the treatment of multiple myeloma and mantle cell lymphoma. However, inability to sufficiently inhibit proteasome activity at tolerated doses has hampered efforts to expand the scope of proteasome inhibitor-based therapies. With emerging new modalities in myeloma, it might seem challenging to develop additional proteasome-based therapies. However, the constant development of new applications for proteasome inhibitors and deeper insights into the intricacies of protein homeostasis suggest that proteasome inhibitors might have novel therapeutic applications. Herein, we summarize the latest advances in proteasome inhibitor development and discuss the future of proteasome inhibitors and other proteasome-based therapies in combating human diseases. [ABSTRACT FROM AUTHOR]

Building models is essential for understanding the functions and dynamics of microbial communities. Metabolic models built on genome‐scale metabolic network reconstructions (GENREs) are especially relevant as a means to decipher the complex interactions occurring among species. Model reconstruction increasingly relies on metagenomics, which permits direct characterisation of naturally occurring communities that may contain organisms that cannot be isolated or cultured. In this review, we provide an overview of the field of metabolic modelling and its increasing reliance on and synergy with metagenomics and bioinformatics. We survey the means of assigning functions and reconstructing metabolic networks from (meta‐)genomes, and present the variety and mathematical fundamentals of metabolic models that foster the understanding of microbial dynamics. We emphasise the characterisation of interactions and the scaling of model construction to large communities, two important bottlenecks in the applicability of these models. We give an overview of the current state of the art in metagenome sequencing and bioinformatics analysis, focusing on the reconstruction of genomes in microbial communities. Metagenomics benefits tremendously from third‐generation sequencing, and we discuss the opportunities of long‐read sequencing, strain‐level characterisation and eukaryotic metagenomics. We aim at providing algorithmic and mathematical support, together with tool and application resources, that permit bridging the gap between metagenomics and metabolic modelling. [ABSTRACT FROM AUTHOR]

In respose to de Marco and colleagues ‘Minimum Information for Protein Functional Evaluation,’ I observe that they run the risk of imposing a Procrustean solution to the problem of rigorously reporting experimental results involving recombinant proteins. While they rightly identify a need for experimental reporting standards defined by their scientific community, they wrongly assign this role to ‘Minimum information’ guidelines. In my response I clarify the role and development processes of minimum information standards, and relate a similar experience that we had in the ProteomeBinders Consortium, that led to a distinction between minimum information guidelines for describing a binder and its efficiency as a molecular tool, and the need to define community standards for experimental binder assessment. [Copyright &y& Elsevier]

A correction to the article "Family Relationships: Should Consensus Reign?--Consensus Clustering for Protein Families" that was published in a 2007 issue is presented.

The cell surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS). The network of charges and sugars provided by the dense packing of LPS molecules in the outer leaflet of the outer membrane interferes with the entry of hydrophobic compounds into the cell, including many antibiotics. In addition, LPS can be recognized by the immune system and plays a crucial role in many interactions between bacteria and their animal hosts. LPS is synthesized in the inner membrane of Gram-negative bacteria, so it must be transported across their cell envelope to assemble at the cell surface. Over the past two decades, much of the research on LPS biogenesis has focused on the discovery and understanding of Lpt, a multi-protein complex that spans the cell envelope and functions to transport LPS from the inner membrane to the outer membrane. This paper focuses on the early steps of the transport of LPS by the Lpt machinery: the extraction of LPS from the inner membrane. The accompanying paper (May JM, Sherman DJ, Simpson BW, Ruiz N, Kahne D. 2015 Phil. Trans. R. Soc. B 370, 20150027. (doi:10.1098/rstb.2015.0027)) describes the subsequent steps as LPS travels through the periplasm and the outer membrane to its final destination at the cell surface. [ABSTRACT FROM AUTHOR]

Novobiocin is an orally active antibiotic that inhibits DNA gyrase by binding the ATP-binding site in the ATPase subunit. Although effective against Grampositive pathogens, novobiocin has limited activity against Gram-negative organisms due to the presence of the lipopolysaccharide-containing outer membrane, which acts as a permeability barrier. Using a novobiocin-sensitive Escherichia coli strain with a leaky outer membrane, we identified a mutant with increased resistance to novobiocin. Unexpectedly, the mutation that increases novobiocin resistance was not found to alter gyrase, but the ATPase that powers lipopolysaccharide (LPS) transport. Co-crystal structures, biochemical, and genetic evidence show novobiocin directly binds this ATPase. Novobiocin does not bind the ATP binding site but rather the interface between the ATPase subunits and the transmembrane subunits of the LPS transporter. This interaction increases the activity of the LPS transporter, which in turn alters the permeability of the outer membrane. We propose that novobiocin will be a useful tool for understanding how ATP hydrolysis is coupled to LPS transport. [ABSTRACT FROM AUTHOR]

Oenococcus oeni is a lactic acid bacteria species encountered particularly in wine, where it achieves the malolactic fermentation. Molecular typing methods have previously revealed that the species is made of several genetic groups of strains, some being specific to certain types of wines, ciders or regions. Here, we describe 36 recently released O. oeni genomes and the phylogenomic analysis of these 36 plus 14 previously reported genomes. We also report three genome sequences of the sister species Oenococcus kitaharae that were used for phylogenomic reconstructions. Phylogenomic and population structure analyses performed revealed that the 50 O. oeni genomes delineate two major groups of 12 and 37 strains, respectively, named A and B, plus a putative group C, consisting of a single strain. A study on the orthologs and single nucleotide polymorphism contents of the genetic groups revealed that the domestication of some strains to products such as cider, wine, or champagne, is reflected at the genetic level. While group A strains proved to be predominant in wine and to form subgroups adapted to specific types of wine such as champagne, group B strains were found in wine and cider. The strain from putative group C was isolated from cider and genetically closer to group B strains. The results suggest that ancestral O. oeni strains were adapted to low-ethanol containing environments such as overripe fruits, and that they were domesticated to cider and wine, with group A strains being naturally selected in a process of further domestication to specific wines such as champagne. [ABSTRACT FROM AUTHOR]

Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are intracellular receptors that control innate immunity and otherbiotic interactions inanimalsandplants. NLRs havebeencharacterized inplantandanimal lineages, but in fungi, thisgenefamily has not been systematically described. There is however previous indications of the involvement of NLR-like genes in nonself recognition and programmed cell death in fungi. We have analyzed 198 fungal genomes for the presence of NLRs and have annotated a total of 5,616 NLR candidates. We describe their phylogenetic distribution, domain organization, and evolution. Fungal NLRs are characterized by a great diversity of domain organizations, suggesting frequently occurring combinatorial assortments of different effector, NODand repeat domains. The repeat domains are of theWD, ANK, and TPR type; no LRR motifs were found. As previously documented for WD-repeat domains of fungal NLRs, TPR, and ANK repeats evolve under positive selection and show highly conserved repeats and repeat length polymorphism, suggesting the possibility of concerted evolution of these repeats. We identify novel effector domains not previously found associated withNLRs, whereas others are related to effector domains of plant or animals NLRs. In particular, we show that the HET domain found in fungal NLRs may be related to Toll/interleukin-1 receptor domains found in animal and plant immune receptors. This description of fungalNLR repertoires reveals both similarities and differences with plant and animals NLR collections, highlights the importance of domain reassortment and repeat evolution and provides a novel entry point to explore the evolution of NLRs in eukaryotes. [ABSTRACT FROM AUTHOR]

In order to describe the dynamic behavior of a complex biological system, it is useful to combine models integrating processes at different levels and with temporal dependencies. Such combinations are necessary for modeling acclimatization, a phenomenon where changes in environmental conditions can induce drastic changes in the behavior of a biological system. In this article we formalize the use of hybrid systems as a tool to model this kind of biological behavior. A modeling scheme called strong switches is proposed. It allows one to take into account both minor adjustments to the coefficients of a continuous model, and, more interestingly, large-scale changes to the structure of the model. We illustrate the proposed methodology with two applications: acclimatization in wine fermentation kinetics, and acclimatization of osteo-adipo differentiation system linking stimulus signals to bone mass. [ABSTRACT FROM AUTHOR]

Abstract: In order to treat osteoporosis and other bone mass disorders it is necessary to understand the regulatory processes that control the cell fate decisions responsible for going from bone precursor cells to bone tissue. Many processes interact to regulate cell division, differentiation and apoptosis. There are models for these basic processes, but not for their interactions. In this work we use the theory of switched systems, reuse and composition of validated models to describe the cell fate decisions leading to bone and fat formation. We describe the differentiation of osteo-adipo progenitor cells by composing its model with differentiation stimuli. We use the activation of the Wnt pathway as stimulus to osteoblast lineage, including regulation of cell division and apoptosis. This model is our first step to simulate physiological responses in silico to treatments for bone mass disorders. [Copyright &y& Elsevier]