New tools for discovering and characterizing microbial diversity

To discover and characterize microbial diversity, approaches based on new sequencing technologies, novel isolation techniques, microfluidics, and metagenomics among others are being used. These approaches have contributed to discovery of novel genes from environmental samples, to massive characterization of functional and phylogenetic genes and to isolation of members of formerly uncultured yet ubiquitous groups like Verrucomicrobia, Acidobacteria, OP10, and methanogenic Archaea. Cheaper sequencing is key in this process by making available applications that were previously restricted to big research centers, complementing previously available methodologies and potentially replacing some of them. The new tools are reshaping the way we study the environment, increasing the resolution at which microbial communities, their complexities and dynamics, can be studied to reveal their genetic potential and their functional diversity.     

Chemical biology tools for imaging-based analysis of organelle membranes and lipids

Membrane biology studies have revealed that in addition to providing structural support for compartment formation and membrane protein function, subcellular biomembranes are also critically involved in many biological events. To facilitate our understanding of the functions, biophysical properties and structural dynamics of organelle membranes, various exciting chemical biology tools have recently emerged. This short review aims to describe the latest molecular probes for organelle membrane studies. In particular, we will feature chemical strategies to visualize and quantitatively analyze the dynamic propeties of organelle membranes and lipids and discuss current limitations and potential future directions of this challenging research area.     

Analytical and numerical tools for diffusion-based movement models

I present a general diffusion-based modeling framework for the analysis of animal movements in heterogeneous landscapes, including terms representing advection, mortality, and edge-mediated behavior. I use adjoint operator theory to develop mathematical machinery for the assessment of a number of biologically relevant quantities, such as occupancy times, hitting probabilities, quasi-stationary distributions, the backwards equation, and conditional probability densities. I derive finite-element approximations, which can be used to obtain numerical solutions in domains which do not allow for an analytical treatment. As an example, I model the movements of the butterfly Melitaea cinxia in an island consisting of a set of habitat patches and the intervening matrix habitat. I illustrate the behavior of the model and the mathematical theory by examining the effects of a hypothetical movement barrier and advection caused by prevailing wind conditions.     

Using secondary outcome to sharpen bounds for treatment harm rate in characterizing heterogeneity

In a clinical trial, statistical reports are typically concerned about the mean difference in two groups. Now there is increasing interest in the heterogeneity of the treatment effect, which has important implications in treatment evaluation and selection. The treatment harm rate (THR), which is defined by the proportion of people who has a worse outcome on the treatment compared to the control, was used to characterize the heterogeneity. Since THR involves the joint distribution of the two potential outcomes, it cannot be identified without further assumptions even in the randomized trials. We can only derive the simple bounds with the observed data. But the simple bounds are usually too wide. In this paper, we use a secondary outcome that satisfies the monotonicity assumption to tighten the bounds. It is shown that the bounds we derive cannot be wider than the simple bounds. We also construct some simulation studies to assess the performance of our bounds in finite sample. The results show that a secondary outcome, which is more closely related to the primary outcome, can lead to narrower bounds. Finally, we illustrate the application of the proposed bounds in a randomized clinical trial of determining whether the intensive glycemia could reduce the risk of development or progression of diabetic retinopathy.     

Analytical tools for unravelling the metabolism of gas-fermenting Clostridia

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The (in)complete organelle genome: exploring the use and nonuse of available technologies for characterizing mitochondrial and plastid chromosomes

Not long ago, scientists paid dearly in time, money and skill for every nucleotide that they sequenced. Today, DNA sequencing technologies epitomize the slogan ‘faster, easier, cheaper and more’, and in many ways, sequencing an entire genome has become routine, even for the smallest laboratory groups. This is especially true for mitochondrial and plastid genomes. Given their relatively small sizes and high copy numbers per cell, organelle DNAs are currently among the most highly sequenced kind of chromosome. But accurately characterizing an organelle genome and the information it encodes can require much more than DNA sequencing and bioinformatics analyses. Organelle genomes can be surprisingly complex and can exhibit convoluted and unconventional modes of gene expression. Unravelling this complexity can demand a wide assortment of experiments, from pulsed‐field gel electrophoresis to Southern and Northern blots to RNA analyses. Here, we show that it is exactly these types of ‘complementary’ analyses that are often lacking from contemporary organelle genome papers, particularly short ‘genome announcement’ articles. Consequently, crucial and interesting features of organelle chromosomes are going undescribed, which could ultimately lead to a poor understanding and even a misrepresentation of these genomes and the genes they express. High‐throughput sequencing and bioinformatics have made it easy to sequence and assemble entire chromosomes, but they should not be used as a substitute for or at the expense of other types of genomic characterization methods.     

Interrelation between the natural antioxidant content and lipid viscosity in normal organelle membranes

Two systems regulating lipid peroxidation (LP) natural antioxidants and structural organization were studied in the liver mitochondria, microsomes and nuclear membranes, using chemiluminescent and spin labelling techniques. Mitochondrial lipids were most protected against LP and were characterized by the maximum content of effective antioxidants and high viscosity. Nuclear lipids were the least protected against the systems studied and had lower antioxidant efficiency and viscosity. Direct correlation between the content of natural antioxidants and lipid viscosity was established.     

Evidence for organelle origin

A selection of evaluations from Faculty of 1000 covering the Methanococcus maripaludis genome; recombination in animal mitochondria; modulating cell cycle progression; gene silencing by microRNA directed methylation; yeast DNA breakage and repair.     

Lipid rafts,microdomain heterogeneity and inter‐organelle contacts: Impacts on membrane preparation for proteomic studies

In recent years, there has been considerable interest in mapping the protein content of isolated organelles using mass spectrometry. However, many subcellular compartments are highly dynamic with diverse and intricate architectures that are not always preserved during membrane isolation procedures. Furthermore, lateral heterogeneities in intra‐membrane lipid and protein concentrations underlie the formation of membrane microdomains, trafficking vesicles and inter‐membrane contacts. These complexities in membrane organisation have important consequences for the design of membrane preparation strategies and test the very concept of organelle purity. We illustrate how some of these biological considerations are relevant to membrane preparation and assess the numerous potential pitfalls in attempting to purify organelles from mammalian cells.     

Mitochondrial heterogeneity during staurosporine-induced apoptosis in HL60 cells: analysis at the single cell and single organelle level

BACKGROUND: Apoptosis is a complex phenomenon during which several events occur. A growing interest exists on the role and functionality of mitochondria during this type of cell death. The responsibility of modifications in mitochondrial membrane potential (Delta Psi) in triggering apoptosis is under investigation. METHODS: We evaluated Delta Psi changes in HL60 cells treated with staurosporine (STS). Flow cytometry and confocal microscopy have been used to analyze samples stained with two Delta Psi-sensitive probes, JC-1 and MitoTrackertrade mark Red CMXRos. RESULTS: At the cellular level, we found heterogeneic behavior. Indeed, after STS treatment, some cells displayed typical markers of apoptosis and a collapse in Delta Psi. Others were apoptotic with no changes in Delta Psi, others changed Delta Psi without being apoptotic, and others were healthy. The same heterogeneic response to STS was found at the single organelle level. In a given cell, some mitochondria were depolarized whereas others were not. CONCLUSION: In this model of apoptosis, changes in Delta Psi can be different among cells of the same type and among different organelles of the same cell. The collapse in Delta Psi is thus a heterogeneic phenomenon that seems to be an ancillary event following the irreversible phase of the apoptotic process.     

Semi-automated analysis of organelle movement and membrane content: understanding rab-motor complex transport function

Organelle motility is an essential cellular function that is regulated by molecular motors, and their adaptors and activators. Here we established a new method that allows more direct investigation of the function of these peripheral membrane proteins in organelle motility than is possible by analysis of the organelle movement alone. This method uses multi-channel time-lapse microscopy to record the movement of organelles and associated fluorescent proteins, and automatic organelle tracking, to compare organelle movement parameters with the association of membrane proteins. This approach allowed large-scale, unbiased analysis of the contribution of organelle-associated proteins and cytoskeleton tracks in motility. Using this strategy, we addressed the role of membrane recruitment of Rab GTPases and effectors in organelle dynamics, using the melanosome as a model. We found that Rab27a and Rab32/38 were mainly recruited to sub-populations of slow-moving/static and fast-moving melanosomes, respectively. The correlation of Rab27a recruitment with slow movement/docking was dependent on the effector melanophilin. Meanwhile, using cytoskeleton-disrupting drugs, we observed that this speed:Rab content relationship corresponded to a decreased frequency of microtubule (MT)-based transport and an increased frequency of actin-dependent slow movement/docking. Overall, our data indicate the ability of Rab27a and effector recruitment to switch melanosomes from MT- to actin-based tethering and suggest that a network of Rab signalling may integrate melanosome biogenesis and transport.     

Advances,challenges and tools in characterizing bacterial serine,threonine and tyrosine kinases and phosphorylation target sites.

Introduction: The cellular response to infection by bacterial pathogens involves a complex and highly regulated series of pathways that carry messages to various parts of the cell. These messages are transferred using post-translational modifications including phosphorylation by kinases. Understanding the host’s signaling pathways is valuable in identifying potential treatment targets, but the bacterial signaling pathways and host-pathogen crosstalk are equally important to the development of therapeutics.

Areas covered: This review summarizes some of the recent findings related to the bacterial phosphoproteome and especially serine/threonine/tyrosine sites, including methods and considerations for identifying novel phosphosites. We also consider the bioinformatics tools that have been developed to sift through the large volume of data in these studies and connect them to biologically relevant knowledge about pathways and function. Literature databases used include PubMed and Google Scholar from April 2018 to December 2018.

Expert opinion: While the field has developed significantly in the past decade of research, high-quality experimental sequence data remains the limiting factor to future research into bacterial phosphoproteomics. As more proteomes are explored, it will be easier to tailor tools and techniques to prokaryotes. It will be necessary to consider the phosphoproteome in the broader biological context, through interdisciplinary collaborations.     

A role for calmodulin in organelle membrane tubulation.

Membrane tubules of uniform diameter (60-80 nm) and variable lengths have been seen to extend from the main bodies of the Golgi complex, trans Golgi network (TGN), and endosomes. In the case of endosomes, these tubules appear to mediate membrane and receptor recycling events. Brefeldin A (BFA) is a potent drug that completely blocks coated vesicle formation from the Golgi complex and TGN, but at the same time causes the enhanced formation of membrane tubules from these same organelles. Recently, experiments have shown that calmodulin antagonists inhibit the transport of receptors out of endosomes, perhaps by inhibiting the formation of recycling tubules. Using the potent calmodulin-specific antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide (W-13), and N-(4-aminobutyl)-5-chloro-1-naphthalenesulfonamide (C-1), we found that the recycling of transferrin from endosomes to the cell surface was significantly inhibited, resulting in the formation of enlarged endosomal vacuoles. In addition, these same calmodulin antagonists also potently inhibited the formation of BFA-stimulated membrane tubules from the Golgi complex, TGN, and endosomes. In the case of the Golgi complex, failure to form tubules resulted in the inhibition of BFA-stimulated retrograde transport to the endoplasmic reticulum. These results suggest that calmodulin is a general regulator of membrane tubulation and is capable of influencing the morphology of several organelles.