Comparative biology: beyond sequence analysis

Comparative analysis is a fundamental tool in biology. Conservation among species greatly assists the detection and characterization of functional elements, whereas inter-species differences are probably the best indicators of biological adaptation. Traditionally, comparative approaches were applied to the analysis of genomic sequences. With the growing availability of functional genomic data, comparative paradigms are now being extended also to the study of other functional attributes, most notably the gene expression. Here we review recent works applying comparative analysis to large-scale gene expression datasets and discuss the central principles and challenges of such approaches.     

Stochastic emergence of repeating cortical motifs in spontaneous membrane potential fluctuations in vivo

It was recently discovered that subthreshold membrane potential fluctuations of cortical neurons can precisely repeat during spontaneous activity, seconds to minutes apart, both in brain slices and in anesthetized animals. These repeats, also called cortical motifs, were suggested to reflect a replay of sequential neuronal firing patterns. We searched for motifs in spontaneous activity, recorded from the rat barrel cortex and from the cat striate cortex of anesthetized animals, and found numerous repeating patterns of high similarity and repetition rates. To test their significance, various statistics were compared between physiological data and three different types of stochastic surrogate data that preserve dynamical characteristics of the recorded data. We found no evidence for the existence of deterministically generated cortical motifs. Rather, the stochastic properties of cortical motifs suggest that they appear by chance, as a result of the constraints imposed by the coarse dynamics of subthreshold ongoing activity.     

Front-to-Rear Membrane Tension Gradient in Rapidly Moving Cells

Membrane tension is becoming recognized as an important mechanical regulator of motile cell behavior. Although membrane-tension measurements have been performed in various cell types, the tension distribution along the plasma membrane of motile cells has been largely unexplored. Here, we present an experimental study of the distribution of tension in the plasma membrane of rapidly moving fish epithelial keratocytes. We find that during steady movement the apparent membrane tension is ∼30% higher at the leading edge than at the trailing edge. Similar tension differences between the front and the rear of the cell are found in keratocyte fragments that lack a cell body. This front-to-rear tension variation likely reflects a tension gradient developed in the plasma membrane along the direction of movement due to viscous friction between the membrane and the cytoskeleton-attached protein anchors embedded in the membrane matrix. Theoretical modeling allows us to estimate the area density of these membrane anchors. Overall, our results indicate that even though membrane tension equilibrates rapidly and mechanically couples local boundary dynamics over cellular scales, steady-state variations in tension can exist in the plasma membranes of moving cells.     

Synthetic peptides mimicking the binding site of human acetylcholinesterase for its inhibitor fasciculin 2

Molecules capable of mimicking protein binding and/or functional sites present useful tools for a range of biomedical applications, including the inhibition of protein–ligand interactions. Such mimics of protein binding sites can currently be generated through structure‐based design and chemical synthesis. Computational protein design could be further used to optimize protein binding site mimetics through rationally designed mutations that improve intermolecular interactions or peptide stability. Here, as a model for the study, we chose an interaction between human acetylcholinesterase (hAChE) and its inhibitor fasciculin‐2 (Fas) because the structure and function of this complex is well understood. Structure‐based design of mimics of the hAChE binding site for Fas yielded a peptide that binds to Fas at micromolar concentrations. Replacement of hAChE residues known to be essential for its interaction with Fas with alanine, in this peptide, resulted in almost complete loss of binding to Fas. Computational optimization of the hAChE mimetic peptide yielded a variant with slightly improved affinity to Fas, indicating that more rounds of computational optimization will be required to obtain peptide variants with greatly improved affinity for Fas. CD spectra in the absence and presence of Fas point to conformational changes in the peptide upon binding to Fas. Furthermore, binding of the optimized hAChE mimetic peptide to Fas could be inhibited by hAChE, providing evidence for a hAChE‐specific peptide–Fas interaction. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Photoinactivation of Candida albicans by Its Own Endogenous Porphyrins

The possibility of photoeradicating the prokaryotic microorganism Candida albicans by enhancing its endogenous porphyrin production and accumulation was investigated in this study. Induction of porphyrin synthesis was performed by the addition of δ-aminolevulinic acid (ALA), or its hydrophobic derivative ALA methyl ester (m-ALA). Photoinactivation of C. albicans was performed under blue light (407–420 nm) illumination. A decrease in viability of about 1.6 or 2.1 orders of magnitudes was obtained with a light dose of 36 J/cm² for an initial concentration of 100-mg/ml ALA or m-ALA, respectively. Endogenous porphyrins extracted from the cells showed that cultures incubated with m-ALA accumulated a relatively higher amount of endogenous porphyrins than ALA, indicating better transport through the yeast cell barriers. When a combination of miconazole and ketoconazole (antifungal agents) is given at a sub-inhibitory concentration (0.5 μg/ml each) with an inducer, a 2.1 or 3.2 orders of magnitude decrease in viability is caused with ALA or with m-ALA, respectively, upon illumination. Fluorescence intensities of the accumulated porphyrins as demonstrated by FACS indicate that the combination of the two azole drugs and an inducer cause a relatively high amount of endogenous porphyrins. Although the additive action of both azole drugs allow better penetration of the inducer, especially m-ALA photoeradication remained limited because of an acidic pH generated in the presence of the inducer. The acidic pH is probably the cause for the inefficiency of the photodynamic treatment. More hydrophobic inducers than m-ALA and less acidic must be investigated to improve the photodynamic treatment by endogenous-induced porphyrins.     

Autoantibodies Against an Extracellular Peptide of the GluR3 Subtype of AMPA Receptors Activate Both Homomeric and Heteromeric AMPA Receptor Channels

Autoantibodies to the GluR3-subtype of AMPA/glutamate receptors are found in the sera and cerebrospinal fluid of some individuals with epilepsy. They could possibly play a role in the pathophysiology of epilepsy since anti-GluR3 sera display glutamatergic agonist activity. We have investigated here the ability of affinity-purified antibodies (Abs) directed against the immunogenic peptide GluR3B (amino-acid 372–395) to interact with and activate recombinant GluR3-receptor channels expressed by Xenopus oocytes. We report here that the affinity-purified anti-GluR3B Abs directly activate GluR3-containing homomeric and heteromeric AMPA receptor complexes without the requirement of neuronal, glial or blood ancillary molecules. We present some of the properties of the purified anti-GluR3B Abs and discuss the possible physiological or pathological consequences of their activation of glutamate receptors.     

Molecular identification, phylogenetic status, and geographic distribution of Culicoides oxystoma (Diptera: Ceratopogonidae) in Israel

Culicoides oxystoma (Diptera: Ceratopogonidae) is an important vector species, reported mainly from Asia, with high potential to transmit viral diseases affecting livestock. In Japan, many arboviruses have been isolated from C. oxystoma, suggesting it as a key player in the epidemiology of several Culicoides-borne diseases. Over the years, C. oxystoma has also been reported in the Middle East region, including Israel. In this region, however, C. oxystoma cannot be easily distinguished morphologically from its sibling species included in the Culicoides schultzei complex. We therefore used genomic data for species identification and phylogeny resolution. Phylogenetic analyses based on internal transcribed spacer 1 (ITS-1) of ribosomal DNA and the mitochondrial gene encoding cytochrome oxidase subunit I (COI) showed that C. oxystoma from Israel is closely related to C. oxystoma from Japan. Using differential probing PCR, we showed that C. oxystoma is distributed all over the country, especially in Mediterranean climate regions. Culicoides oxystoma is less common or even absent in arid regions, while the other genetic cluster of C. schultzei complex was found only in the east of the country (mostly arid and semiarid regions). The molecular finding of C. oxystoma in wide geographical regions, together with its high proportion in the general Culicoides population and its vectoring potential, imply that it may be an important vector species in the Middle East.     

A systematic approach to pair secretory cargo receptors with their cargo suggests a mechanism for cargo selection by Erv14

The endoplasmic reticulum (ER) is the site of synthesis of secreted and membrane proteins. To exit the ER, proteins are packaged into COPII vesicles through direct interaction with the COPII coat or aided by specific cargo receptors. Despite the fundamental role of such cargo receptors in protein traffic, only a few have been identified; their cargo spectrum is unknown and the signals they recognize remain poorly understood. We present here an approach we term "PAIRS" (pairing analysis of cargo receptors), which combines systematic genetic manipulations of yeast with automated microscopy screening, to map the spectrum of cargo for a known receptor or to uncover a novel receptor for a particular cargo. Using PAIRS we followed the fate of ～150 cargos on the background of mutations in nine putative cargo receptors and identified novel cargo for most of these receptors. Deletion of the Erv14 cargo receptor affected the widest range of cargo. Erv14 substrates have a wide array of functions and structures; however, they are all membrane-spanning proteins of the late secretory pathway or plasma membrane. Proteins residing in these organelles have longer transmembrane domains (TMDs). Detailed examination of one cargo supported the hypothesis that Erv14 dependency reflects the length rather than the sequence of the TMD. The PAIRS approach allowed us to uncover new cargo for known cargo receptors and to obtain an unbiased look at specificity in cargo selection. Obtaining the spectrum of cargo for a cargo receptor allows a novel perspective on its mode of action. The rules that appear to guide Erv14 substrate recognition suggest that sorting of membrane proteins at multiple points in the secretory pathway could depend on the physical properties of TMDs. Such a mechanism would allow diverse proteins to utilize a few receptors without the constraints of evolving location-specific sorting motifs.     

Cytokine release and expression induced by OmpA proteins from the Gram-negative anaerobes, Porphyromonas asaccharolytica and Bacteroides fragilis

OmpA proteins from Gram-negative anaerobes Porphyromonas asaccharolytica and Bacteroides fragilis induced release and expression of IL-1alpha, tumor necrosis factor (TNF)-alpha, IFN-gamma, IL-6, and IL-10 from murine splenocytes in vitro in a dose-dependent fashion. The release of the cytokines induced by B. fragilis Bf-OmpA was at much lower levels compared with P. asaccharolytica Omp-PA; Bf-OmpA did not induce release of IL-10. Omp-PA and Bf-OmpA were able to upregulate mRNA expression of the tested cytokines. The results obtained with refolded Bf-OmpA were similar to those with native Bf-OmpA. The data presented in this research demonstrate for the first time that Omps from anaerobic bacteria can induce the release of cytokines, suggesting that Omp-PA and Bf-OmpA may play important roles in the pathogenic processes of these bacteria.