Regulation of procollagen synthesis during the development of chick embryo calvaria. Correlation with procollagen mRNA content

During the embryonic development of chick calvaria (membranous cranial bones), the relative rate of procollagen synthesis increased from about 12% of total protein synthesis on Day 10 to about 65% on Day 17. This increase is due to a 1.7-fold increase in the absolute rate of procollagen synthesis and a 3-fold decrease in the synthesis of noncollagenous proteins. The increase in procollagen synthesis is directly proportional to an increase in procollagen mRNA content per cell as measured either by cell-free translation or by hybridization with complementary DNA. The results indicate that translational control of procollagen mRNA does not play a substantial role during calvaria development and that the specialization in the synthesis of this protein is largely due to the loss or inactivation of mRNAs for noncollagenous proteins.     

Detecting Subtle Plasma Membrane Perturbation in Living Cells Using Second Harmonic Generation Imaging

The requirement of center asymmetry for the creation of second harmonic generation (SHG) signals makes it an attractive technique for visualizing changes in interfacial layers such as the plasma membrane of biological cells. In this article, we explore the use of lipophilic SHG probes to detect minute perturbations in the plasma membrane. Three candidate probes, Di-4-ANEPPDHQ (Di-4), FM4-64, and all-trans-retinol, were evaluated for SHG effectiveness in Jurkat cells. Di-4 proved superior with both strong SHG signal and limited bleaching artifacts. To test whether rapid changes in membrane symmetry could be detected using SHG, we exposed cells to nanosecond-pulsed electric fields, which are believed to cause formation of nanopores in the plasma membrane. Upon nanosecond-pulsed electric fields exposure, we observed an instantaneous drop of ∼50% in SHG signal from the anodic pole of the cell. When compared to the simultaneously acquired fluorescence signals, it appears that the signal change was not due to the probe diffusing out of the membrane or changes in membrane potential or fluidity. We hypothesize that this loss in SHG signal is due to disruption in the interfacial nature of the membrane. The results show that SHG imaging has great potential as a tool for measuring rapid and subtle plasma membrane disturbance in living cells.As the epicenter for many cellular functions, understanding the dynamics of the plasma membrane is important to monitoring biological phenomena. External forces acting upon the plasma membrane (e.g., electric, mechanical) have been shown to cause rapid disturbances, often resulting in dramatic changes in cell physiology (1–3). To understand this interaction, a minimally invasive, highly sensitive imaging technique that enables monitoring the structure of the plasma membrane is needed. Lipophilic dyes, which embed themselves into lipid membranes, are sensitive to the surrounding electric field and, therefore, report changes in membrane fluidity as well as voltage due to the capacitive nature of the membranes (4,5). This sensitivity is typically detected as a shift in the fluorescence emission spectrum. Localization of the fluorescence signal to only the plasma membrane is difficult because the probes also label internal membrane structures. Thus, to overcome this lack of spatial selectivity, second harmonic generation (SHG) has been used as an alternative to fluorescence for membrane imaging (6,7).In SHG, a second-order nonlinear polarization is induced by electronic disruption of a probe molecule from the electromagnetic field of the incident laser beam. This polarization generates oscillating dipole moments that reradiate light at twice the energy of the excitation beam. The induction of this dipole is sensitive to the static electric field surrounding the probe and the steady-state molecular polarization of the probe molecule. These properties make SHG probes useful for monitoring changes in biological membranes.First, as the voltage potential across the membrane changes, the static electric field around the probe also shifts, making the probe sensitive to these variations (7). Several SHG probes have, therefore, been employed to monitor plasma membrane potential (7,8).Second, because the dipole is affected by the steady-state molecular polarization of the probe itself, a SHG signal is only produced in materials that lack a center of inversion symmetry. In the centrosymmetric case, any emitted radiation is cancelled out by destructive interference. The properties of an interfacial environment, such as a cellular plasma membrane, not only provide the necessary asymmetry, but cause the polarized lipophilic dyes to be aligned in respect to the interface, instead of being randomly distributed as they would in a bulk environment. This alignment allows the generation of a coherent SHG signal from the plasma membrane while the rest of the cell remains nearly signal-free (6,7).We investigated whether the alignment sensitivity of the SHG response could be used to detect minute changes in the organization of the plasma membrane. Jurkat clone E6-1 human T-lymphocytes with a spherical morphology were selected for optimum signal clarity and cultured as directed by American Type Culture Collection (ATCC, Manassas, VA) with 1 I.U./mL penicillin and 0.1 μg/mL streptomycin. Cells were added to 35-mm poly-L-lysine-coated glass-bottomed dishes (MatTek, Ashland, MA) and incubated for 1 h in growth media to allow adherence. Before loading, the cells were rinsed with a buffer consisting of 135 mM NaCl, 5 mM KCl, 2 mM MgCl2, 10 mM HEPES, 10 mM glucose, 2 mM CaCl2, pH 7.4, 290–310 mOsm. SHG probes, Di-4-ANEPPDHQ (Di-4) (5 μM final concentration), FM4-64 (15 μM) or ATR (100 μM, 1 mg/mL BSA) were added to the buffer solution and incubated for 1 h. Cellular imaging was performed in the labeling buffer to limit diffusion of the probe molecules out of the cell membranes.A Ti:sapphire oscillator at 980 nm (Coherent Chameleon, 130 fs, 80 MHz, ∼15 mW at the sample; Coherent Laser, Santa Clara, CA) was coupled through the scan head of a modified model No. TCS SP5 II (Leica Geosystems, Norcross, GA) for SHG and multiphoton-excited fluorescence imaging (40×, water, 1.1 NA) using resonant scanning. SHG signal was collected in transmission by a photomultiplier tube after 680-nm shortpass and 485/25-nm bandpass filters; simultaneous fluorescence signal was collected in the epi-direction by two non-descanned photomultiplier tubes with 540/60-nm and 650/60-nm bandpass filters.Three SHG probes previously used to monitor voltage or membrane order in living cells were tested (8–10). Although ATR is reported to be effective in monitoring membrane voltage, we obtained nearly no SHG signal, despite successful loading as indicated by the fluorescence signal (Fig. 1). When FM4-64 and Di-4 were loaded to similar fluorescence intensities, nearly equivalent SHG signal was collected. Di-4 did appear to have a greater internalization of the dye. However, after the first frame, the FM4-64 signal dropped considerably (Fig. 1 b), an observation reported as a membrane voltage-independent bleaching effect (8). This drop in signal recovered after excitation was blocked for several seconds, but quantification of the response was difficult. Di-4 did not suffer as dramatic a drop in signal upon excitation, and still had sufficient SHG signal/noise after several seconds, so it was used in all further experiments.Open in a separate windowFigure 1(a) Fluorescence (top) and SHG (bottom) images for the three probes. (b) Signal/noise for the fluorescence and SHG for the initial frame and shortly after beginning acquisition. Error bars represent the mean ± SE (n = 10). Scale bar is 10 μm.To test whether Di-4 would report a rapid change in membrane organization, we applied a single nanosecond-duration pulsed electric field (nsPEF) to the labeled cell. These ultrabrief, high-intensity (MV/m) pulses differ from longer (μs-ms), lower-intensity (kV/m) pulses traditionally associated with electroporation in induced cellular response (3,11,12). Through selective uptake of small ions (Ca²⁺, Ti⁺) with limited uptake of propidium iodide, nsPEF have been previously postulated to cause nanopores (<2 nm diameter) in the plasma membrane. In contrast with a previous study observing poration resulting from traditional electroporation (13), the brevity of this apparently novel cellular insult allows for the decoupling of the mechanical effects of the pulse on the membrane from the electrical effects of the pulse itself. A single pulse, generated by a custom pulse generator, was delivered to the cells using a pair of 125-μm diameter tungsten electrodes, separated edge-to-edge by 150 μm, as previously described in Ibey et al. (14). For maximum visualization of changes in the SHG signal, a half-wave plate was placed before the scan head to align the polarization of the laser such that the brightest signals from the plasma membrane were at the poles facing the electrodes.The Di-4 SHG signal in response to a single 16.7 kV/cm, 600-ns nsPEF is shown in Fig. 2. Before the pulse, the intensity of the SHG signal is high at each of these poles. Immediately after the pulse, the SHG intensity drops by ∼50% on the side of the cell facing the anodic electrode, whereas little intensity is lost at the other pole. This response is plotted in Fig. 2 (pulse applied at 2 s), where it is apparent that the response is near instantaneous with little recovery in signal in the 5 s postexposure. The SHG response matches the previously observed effect of this stimulus, where ion uptake displayed a polar dependence and persisted for a number of minutes (11,12). Images taken 5 min after an nsPEF exposure are also shown in Fig. 2. These images confirm the eventual recovery of the cell and the corresponding return of SHG signal to preexposure levels.Open in a separate windowFigure 2(a) SHG images showing drop in signal on the anodic (or A-pole) of the cell. (b) Time trace of SHG response with the electrical pulse applied at 2 s that shows a near-instantaneous drop in the SHG signal at the anodic pole of the cell. (c) SHG image preexposure, immediately postexposure, and then 5-min postexposure showing recovery of the SHG signal.To decouple membrane disturbance from environmental changes around the membrane, we compared the SHG response to the simultaneously acquired fluorescence signal. Because fluorescence is not subject to the strict orientation requirement of SHG, the plasma membrane fluorescence signal provides an indication of the membrane fluidity and/or potential. Despite the dramatic shift in SHG intensity on the anodic pole upon the electrical pulse exposure (Fig. 3 a), the fluorescence channels display little response from the equivalent membrane sections with the exception of photobleaching and a slight increase in signal in both emission bands on the anodic side (Fig. 3, b and c). The shading in these graphs represents the mean ± SE for six cells. Although this slight increase may indicate that a small amount of dye is simply diffusing in or out of the membrane upon exposure, the fluorescence response is not as rapid or as lasting as the SHG response. Change in membrane fluidity or voltage can also be quantized using these fluorescence signals and a value known as the generalized polarization (GP) (4),GP=I515−570−I620−680I515−570+I620−680.(1)As with the raw intensity of the individual signals, the GP value for the membrane (Fig. 3 d) shows no significant shift, indicating that the membrane is likely not transitioning between a more raft- and fluidlike state. Thus, it seems likely that the dye was initially aligned in the tightly-packed ordered membrane so that the probes were able to generate a SHG photon. As shown in Fig. 3 e, we postulate that upon electrical pulse exposure, the membrane was disrupted by the formation with nanopores giving the probe molecules the flexibility to disorient within the membrane. The resulting alignment of the probes is more isotropic in nature, thereby limiting the probes probability of producing a SHG photon. The fluorescence signal remained, however, indicating that the probes remained active in the membrane.Open in a separate windowFigure 3(a) Average SHG signal showing the dramatic drop in signal on the anodic pole at the pulse application (2 s). (b and c) Simultaneous TPF signals showing nearly no instantaneous change at the pulse application. (d) GP showing no observable changes in the membrane potential or fluidity after the pulse. (Shaded areas) Fit to the mean ± SE for each trace (n = 6). (e) Conceptualization of the hypothesized membrane disruption underlying the observed change in SHG response.Thus, by taking advantage of the selection criteria of SHG, we were able to successfully use the SHG probe, Di-4, to monitor rapid disruption of the plasma membrane. Because SHG can only be generated when the probes are aligned in the plasma membrane, the SHG signal diminishes significantly upon disruption. The simultaneous collection of the multiphoton-excited fluorescence signal was advantageous in that it demonstrated that the probes did not simply diffuse out of the membrane, did not appear to be energetically disrupted by the electric pulse, and showed that the membrane changes were not simply a change in lipid order. We believe that this technique holds tremendous potential for use in the study of how external stimuli interact with and change the orientation of biological membranes. Such knowledge may allow for further understanding of how manipulation of cells and biological systems can be achieved using external stimuli.     

From Dinosaurs to Modern Bird Diversity: Extending the Time Scale of Adaptive Radiation

What explains why some groups of organisms, like birds, are so species rich? And what explains their extraordinary ecological diversity, ranging from large, flightless birds to small migratory species that fly thousand of kilometers every year? These and similar questions have spurred great interest in adaptive radiation, the diversification of ecological traits in a rapidly speciating group of organisms. Although the initial formulation of modern concepts of adaptive radiation arose from consideration of the fossil record, rigorous attempts to identify adaptive radiation in the fossil record are still uncommon. Moreover, most studies of adaptive radiation concern groups that are less than 50 million years old. Thus, it is unclear how important adaptive radiation is over temporal scales that span much larger portions of the history of life. In this issue, Benson et al. test the idea of a “deep-time” adaptive radiation in dinosaurs, compiling and using one of the most comprehensive phylogenetic and body-size datasets for fossils. Using recent phylogenetic statistical methods, they find that in most clades of dinosaurs there is a strong signal of an “early burst” in body-size evolution, a predicted pattern of adaptive radiation in which rapid trait evolution happens early in a group''s history and then slows down. They also find that body-size evolution did not slow down in the lineage leading to birds, hinting at why birds survived to the present day and diversified. This paper represents one of the most convincing attempts at understanding deep-time adaptive radiations.

“It is strikingly noticeable from the fossil record and from its results in the world around us that some time after a rather distinctive new adaptive type has developed it often becomes highly diversified.” – G. G. Simpson ([1], pp. 222–223)

George Gaylord Simpson was the father of modern concepts of adaptive radiation—the diversification of ecological traits in a rapidly speciating group of organisms (Figure 1; [2]). He considered adaptive radiation to be the source of much of the diversity of living organisms on planet earth, in terms of species number, ecology, and body form [1]–[3]. Yet more than 60 years after Simpson''s seminal work, the exact role of adaptive radiation in generating life''s extraordinary diversity is still an open and fundamental question in evolutionary biology [3],[4].Open in a separate windowFigure 1An example of adaptive radiation and early bursts in rates of speciation and phenotypic evolution.(a) The adaptive radiation of the modern bird clade Vanginae, which shows early rapid speciation, morphological diversity, and diversity in foraging behavior and diet [15],[32]. (b) Hypothetical curve of speciation rates through time that would be expected in adaptive radiation. The exponential decline in speciation rates shows that there was an “early burst” of speciation at the beginning of the clade''s history. (c) Hypothetical curve of rates of phenotypic evolution through time that would be expected in adaptive radiation, also showing an early burst of evolution with high initial rates. Part (a) is reproduced from [32] with permission (under CC-BY) from the Royal Society and the original authors.To address this question, researchers have looked for signatures of past adaptive radiation in the patterns of diversity in nature. In particular, it has been suggested that groups that have undergone adaptive radiation should show an “early-burst” signal in both rates of lineage diversification and phenotypic evolution through time—a pattern in which rates of speciation and phenotypic evolution are fast early in the history of groups and then decelerate over time (Figure 1; [3]–[5]). These predictions arise from the idea that clades should multiply and diversify rapidly in species number, ecology, and phenotype in an adaptive radiation and that rates of this diversification should decrease later as niches are successively occupied [2].Early bursts have been sought in both fossils and phylogenies. Few fossil studies have discussed their results in the context of adaptive radiation (but see [6]), but they often have found rapid rises in both taxonomic and morphological diversity early in the history of various groups [7], ranging from marine invertebrates [8] to terrestrial mammals [9]. However, fossils often lack the phylogeny needed to model how evolution has proceeded [7]. On the other hand, studies that test for early bursts in currently existing (extant) species typically use phylogenies, which allow us to model past evolution in groups with few or no fossils [5]. Phylogenies have most often been used to test early bursts in speciation (see, e.g., [10]). However, such tests may be misled by past extinction, which will decay the statistical signal of rapid, early diversification [11]. Furthermore, diverse evolutionary scenarios beyond adaptive radiation can give rise to early bursts in speciation [12]. By contrast, studies of phenotypic diversification may be more robust to extinction [13] and they test the distinguishing feature that separates adaptive from nonadaptive radiation [2],[12].Thus, studies of adaptive radiation in extant organisms increasingly have focused on phylogenetic tests of the early-burst model of phenotypic evolution. Some studies show strong support for this prediction in both birds [14],[15] and lizards [5],[16]. However, the most extensive study to date showed almost no support for the early-burst model. In this study, Harmon et al. [17] examined body size in 49 (and shape in 39) diverse groups of animals, including invertebrates, fishes, amphibians, reptiles, birds, and mammals. They found strong support for the early-burst model in only two of these 88 total datasets.This result raises an important question: if adaptive radiation explains most of life''s diversity [1], how is it possible that there is so little phylogenetic evidence for early bursts of phenotypic evolution? One possibility is that early bursts are hard to detect. This can be due to low statistical power in the most commonly employed tests [18]. It may also be due to a lack of precision in the way “early burst” is defined (and thus tested), as the ecological theory of adaptive radiation suggests that the rate of phenotypic evolution will decrease as species diversity increases in a group, not just over time [14],[16]. Indeed, recent studies [14],[16] detected a decline in rates with species diversity in clades that were also in the Harmon et al. [17] study, yet for which no decline over time was detected.A second possible reason for why early-burst patterns are uncommon is more fundamental: the patterns of phenotypic diversity that result from adaptive radiation may be different at large time scales. Many of the best examples of adaptive radiation are in groups that are relatively young, including Darwin''s finches (2.3 million years old [myr]; [19]) and Lake Malawi and Victoria cichlids (2.3 myr; [20]), whereas most groups that are examined for early bursts in phenotypic evolution are much older (e.g., 47 of 49 in Harmon et al. [17]; mean ± sd = 23.8±29.2 myr). So there may be an inherent difference between what unfolds over the relatively short time scales emphasized by Schluter [2] and what one sees at macroevolutionary time scales (see [21] for an in-depth discussion of this idea as it relates to speciation).The time scale over which adaptive radiations unfold has been little explored. As a result, the link between extant diversity and major extinct radiations remains unclear. Simpson [1] believed that adaptive radiation played out at the population level, but that it should manifest itself at larger scales as well—up to phyla (e.g., chordates, arthropods). He suggested that we should see signals of adaptive radiations in large, old clades because they are effectively small-scale adaptive radiation writ large [1]. Under this view, we should see the signal of adaptive radiation even in groups that diversified over vast time scales, particularly if adaptive radiation is as important for explaining life''s diversity as Simpson [1] thought it was.Part of the reason why potential adaptive radiations at deep time scales remain poorly understood is that studies either focus on fossils or phylogenies, but rarely both. In this issue, Benson et al. [22] combine these two types of data to address whether dinosaurs show signs that they adaptively radiated. Unlike most other studies, the temporal scale of the current study is very large—in this case, over 170 million years throughout the Mesozoic era, starting at 240 million years ago in the Triassic period. This characteristic allowed Benson et al. to shed light on deep-time adaptive radiation.The authors estimated body mass from fossils by using measurements of the circumference of the stylopodium shaft (the largest bone of the arm or leg, such as the femur), which shows a consistent scaling relationship with body mass in extant reptiles and mammals [23]. They then combined published phylogenies to obtain a composite phylogeny for the species in their body-size dataset. The authors finally conducted two types of tests of the rate of body-size evolution—tests of early bursts in phenotypic evolution that are the same as those of Harmon et al. [17], as well as an additional less commonly used test that estimates whether differences between estimated body size at adjacent phylogenetic nodes decreases over time.Benson et al. [22] found two striking results. First, in both of their analyses, the early-burst model was strongly supported for most clades of dinosaurs. This early burst began in the Triassic period, indicating that diversification in body size in dinosaurs began before the Triassic-Jurassic mass extinction event would have opened competition-free ecological space (as commonly hypothesized; [24],[25]). Rather, the authors [22] suggest that a key innovation led to this rise in dinosaurs, though it is not clear what this innovation was [26]. In general, though, the finding of an early burst in body-size evolution in most dinosaurs—if a consequence of adaptive evolution—suggests that adaptive radiation may play out over large evolutionary time scales, not just on the short time scales typical of the most well-studied cases of extant groups.Second, one clade—Maniraptora, which is the clade in which modern-day birds are nested—was the only part of the dinosaur phylogeny that did not show such a strong early burst in body-size evolution. Instead, this clade fit a model to a single adaptive peak—an optimum body size, if you will—but also maintained high rates of undirected body-size evolution throughout their history. Benson et al. [22] suggest that this last result connects deep-time adaptive radiation in the dinosaurs, which quickly exhausted the possibility of phenotypic space, with the current radiation in extant birds, which survived to the present day because their constant, high rate of evolution meant that they were constantly undergoing ecological innovation. This gives a glimpse into why modern birds have so many species (an order of magnitude higher than the nonavian dinosaurs) and so much ecological diversity.The use of fossils allowed Benson et al. [22] to address deep-time radiation in dinosaurs and its consequence on present-day bird diversity. Nevertheless, the promise of using fossils to understand adaptive radiation has its limits. The paleontological dataset presented here is exceptional, yet still insufficient to explore major components of adaptive radiations like actual ecological diversification. As in many paleontological studies, Benson et al. used body-size data to represent ecology because body size is one of the few variables that is available for most species. But it is unclear how important body size really is for ecological diversification and niche filling, because body size is important for nearly every aspect of organismal function. Consequently, evolutionary change in body size can result not only from the competition that drives adaptive radiation, but also from predation pressure, reproductive character displacement, and physiological advantages of particular body sizes in a given environment, among other reasons [27].Despite the broad coverage of extinct species presented in Benson et al. [22], the data were insufficient to study another major part of adaptive radiation: early bursts of lineage diversification. While new approaches are becoming available to study diversification with phylogenies containing extinct species [28],[29] or with incomplete fossil data [30], these approaches are limited when many taxa are known from only single occurrences. This is the case in the Benson et al. dataset, and more generally in most fossil datasets.Given that few fossils exist for many extant groups, a major goal for future studies will be the incorporation of incomplete fossil information into analyses primarily focused on traits and clades for which mostly neontological data are available. For example, Slater et al. [31] developed an approach to include fossil information in analyses of phenotypic evolution. They showed that adding just a few fossils (12 fossils in a study of a 135-species clade) drastically increased the power and accuracy of their analyses of extant taxa. Thus, the combination of fossil data and those based on currently living species is important for future studies, as are new approaches that allow analyzing early bursts of lineage diversification along with phenotypic evolution in fossils.So what answers do Benson et al. [22] bring to Simpson''s original question of the importance of adaptive radiation for explaining diversity on earth? The authors present an intriguing and unconventional link between adaptive radiation and the diversity of modern-day birds. They argue that bird diversification was possible because the dinosaur lineage leading to birds did not exhaust niche space, potentially thanks to small body sizes; in contrast, other dinosaur groups adaptively radiated, filled niche space, and thus could not produce the ecological innovation that may have been necessary to survive the Cretaceous-Paleogene mass extinction. This intriguing hypothesis suggests an important role for the relative starting points of successive adaptive radiations in explaining current diversity, giving a new spin to the pivotal question raised by Simpson more than 60 years ago.     

BAC-based upgrading and physical integration of a genetic SNP map in Atlantic salmon

A better understanding of the genotype–phenotype correlation of Atlantic salmon is of key importance for a whole range of production, life history and conservation biology issues attached to this species. High-density linkage maps integrated with physical maps and covering the complete genome are needed to identify economically important genes and to study the genome architecture. Linkage maps of moderate density and a physical bacterial artificial chromosome (BAC) fingerprint map for the Atlantic salmon have already been generated. Here, we describe a strategy to combine the linkage mapping with the physical integration of newly identified single nucleotide polymorphisms (SNPs). We resequenced 284 BAC-ends by PCR in 14 individuals and detected 180 putative SNPs. After successful validation of 152 sequence variations, genotyping and genetic mapping were performed in eight salmon families comprising 376 individuals. Among these, 110 SNPs were positioned on a previously constructed linkage map containing SNPs derived from expressed sequence tag (EST) sequences. Tracing the SNP markers back to the BACs enabled the integration of the genetic and physical maps by assigning 73 BAC contigs to Atlantic salmon linkage groups.     

Prevention of intestinal Campylobacter jejuni colonization in broilers by combinations of in‐feed organic acids

Aim: We have tested the effect of various combinations of formic acid and sorbate on Campylobacter jejuni colonization in broiler chickens to reduce the colonization of this zoonotic pathogen in broiler chicken flocks. Methods and Results: Chickens were offered feed supplemented with different concentrations and combinations of formic acid and/or potassium sorbate. We found little or no effect on the Camp. jejuni colonization levels in chickens that were given feed supplemented with formic acid alone. A combination of 1·5% formic acid and 0·1% sorbate reduced the colonization of Camp. jejuni significantly, while a concentration of 2·0% formic acid in combination with 0·1% sorbate prevented Camp. jejuni colonization in chickens. This inhibition was replicated in two independent trials with a combination of three different Camp. jejuni strains. Conclusions: Our results show a novel and promising intervention strategy to reduce the incidence of Camp. jejuni in poultry products and to obtain safer food. Significance and Impact of the Study: To ensure food safety, a reduction of the carcass contamination with Camp. jejuni through reduced colonization of this pathogen in broiler chicken flocks is important. A range of organic acids as additives in feed and drinking water have already been evaluated for this purpose. However, no studies have yet shown a complete inhibition of Camp. jejuni colonization in broiler chickens.     

Analysis of covariance patterns in gene expression data and FT-IR spectra

The aim of this study was to detect and interpret correlation patterns in several large data matrices from the same biological system using Partial Least Squares Regression (PLSR) in order to get information on the system under investigation. To do this, DNA microarray data and Fourier Transform Infrared (FT-IR) spectra from a designed study where Campylobacter jejuni was exposed to environmental stress conditions, were used. The experimental design included variation in atmospheric conditions, temperature and time. PLSR was first used to analyse each of the two data types separately in order to explore the effect of the experimental parameters on the data. The results showed that both the gene expression and FT-IR spectra were affected by the variations in atmosphere, temperature and time, but that the effect was different for the two types of data. When the DNA microarray data and FT-IR spectra were linked together by PLSR, covariation due to temperature was seen. Both specific genes and ranges in the FT-IR spectra that were connected to the variation in temperature were detected. Some of these are possibly connected to properties of the cell wall of the bacteria. The results in this study show the potential of PLSR for investigation of covariance structures in biological data. By doing this, valuable information about the biological system can be detected and interpreted. It was also shown that the use of FT-IR spectroscopy provided important information about the stress responses in the bacteria, information that was not detected from the DNA microarray data.     

The major autolysin Acm2 from Lactobacillus plantarum undergoes cytoplasmic O-glycosylation

The major autolysin Acm2 from the probiotic strain Lactobacillus plantarum WCFS1 contains high proportions of alanine, serine, and threonine in its N-terminal so-called AST domain. It has been suggested that this extracellular protein might be glycosylated, but this has not been experimentally verified. We used high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS) to study the possible occurrence of glycans on peptides generated from lactobacillary surface proteins by protease treatment. This approach yielded five glycopeptides in various glycoforms, all derived from the AST domain of Acm2. All five glycopeptides contained the hydroxy-amino acids serine and threonine, suggesting that Acm2 is O-glycosylated. By using lectin blotting with succinylated wheat germ agglutinin, and by comparing the wild-type strain with an Acm2-negative derivative (NZ3557), we found that the attached N-acetylhexosamines are most likely N-acetylglucosamines (GlcNAc). NZ3557 was further used as a genetic background to express an Acm2 variant lacking its secretion signal, resulting in intracellular expression of Acm2. We show that this intracellular version of Acm2 is also glycosylated, indicating that the GlcNAc modification is an intracellular process.     

Contrasting patterns of gene diversity between microsatellites and mitochondrial SNPs in farm and wild Atlantic salmon

Levels of genetic variability at 12 microsatellite loci and 19 single nucleotide polymorphisms in mitochondrial DNA were studied in four farm strains and four wild populations of Atlantic salmon. Within populations, the farm strains showed significantly lower allelic richness and expected heterozygosity than wild populations at the 12 microsatellite loci, but a significantly higher genetic variability with respect to observed number of haplotypes and haplotype diversity in mtDNA. Significant differences in allele- and haplotype-frequencies were observed between farm strains and wild populations, as well as between different farm strains and between different wild populations. The large genetic differentiation at mitochondrial DNA between wild populations (F_ST = 0.24), suggests that the farm strains attained a high mitochondrial genetic variability when created from different wild populations seven generations ago. A large proportion of this variability remains despite an expected lower effective population size for mitochondrial than nuclear DNA. This is best explained by the particular mating schemes in the breeding programmes, with 2–4 females per male. Our observations suggest that for some genetic polymorphisms farm populations might currently hold equal or higher genetic variability than wild populations, but lower overall genetic variability. In the short-term, genetic interactions between escaped farm salmon and wild salmon might increase genetic variability in wild populations, for some, but not most, genetic polymorphisms. In the long term, further losses of genetic variability in farm populations are expected for all genetic polymorphisms, and genetic variability in wild populations will be reduced if escapes of farm salmon continue.     

Identification and Characterization of a Novel Human Methyltransferase Modulating Hsp70 Protein Function through Lysine Methylation

Hsp70 proteins constitute an evolutionarily conserved protein family of ATP-dependent molecular chaperones involved in a wide range of biological processes. Mammalian Hsp70 proteins are subject to various post-translational modifications, including methylation, but for most of these, a functional role has not been attributed. In this study, we identified the methyltransferase METTL21A as the enzyme responsible for trimethylation of a conserved lysine residue found in several human Hsp70 (HSPA) proteins. This enzyme, denoted by us as HSPA lysine (K) methyltransferase (HSPA-KMT), was found to catalyze trimethylation of various Hsp70 family members both in vitro and in vivo, and the reaction was stimulated by ATP. Furthermore, we show that HSPA-KMT exclusively methylates 70-kDa proteins in mammalian protein extracts, demonstrating that it is a highly specific enzyme. Finally, we show that trimethylation of HSPA8 (Hsc70) has functional consequences, as it alters the affinity of the chaperone for both the monomeric and fibrillar forms of the Parkinson disease-associated protein α-synuclein.