Origin of an Alternative Genetic Code in the Extremely Small and GC–Rich Genome of a Bacterial Symbiont

The genetic code relates nucleotide sequence to amino acid sequence and is shared across all organisms, with the rare exceptions of lineages in which one or a few codons have acquired novel assignments. Recoding of UGA from stop to tryptophan has evolved independently in certain reduced bacterial genomes, including those of the mycoplasmas and some mitochondria. Small genomes typically exhibit low guanine plus cytosine (GC) content, and this bias in base composition has been proposed to drive UGA Stop to Tryptophan (Stop→Trp) recoding. Using a combination of genome sequencing and high-throughput proteomics, we show that an α-Proteobacterial symbiont of cicadas has the unprecedented combination of an extremely small genome (144 kb), a GC–biased base composition (58.4%), and a coding reassignment of UGA Stop→Trp. Although it is not clear why this tiny genome lacks the low GC content typical of other small bacterial genomes, these observations support a role of genome reduction rather than base composition as a driver of codon reassignment.     

Two Sides of the Same Coin? Consistency in Aggression to Conspecifics and Predators in a Female Songbird

Different forms of aggression have traditionally been treated separately according to function or context (e.g. aggression towards a conspecific versus a predator). However, recent work on individual consistency in behavior predicts that different forms of aggression may be correlated across contexts, suggesting a lack of independence. For nesting birds, aggression towards both conspecifics and nest predators can affect reproductive success, yet the relationship between these behaviors, especially in females, is not known. Here we examine free-living female dark-eyed juncos (Junco hyemalis) and compare their aggressive responses towards three types of simulated intruders near the nest: a same-sex conspecific, an opposite-sex conspecific, and a nest predator. We also examine differences in the strength of response that might relate to the immediacy of the perceived threat the intruder poses for the female or her offspring. We found greater aggression directed towards a predator than a same-sex intruder, and towards a same-sex than an opposite-sex intruder, consistent with a predator being a more immediate threat than a same-sex intruder, followed by an opposite-sex intruder. We also found positive relationships across individuals between responses to a same-sex intruder and a simulated predator, and between responses to a same-sex and an opposite-sex intruder, indicating that individual females are consistent in their relative level of aggression across contexts. If correlated behaviors are mediated by related mechanisms, then different forms of aggression may be expressions of the same behavioral tendency and constrained from evolving independently.     

School’s Out: Seasonal Variation in the Movement Patterns of School Children

School children are core groups in the transmission of many common infectious diseases, and are likely to play a key role in the spatial dispersal of disease across multiple scales. However, there is currently little detailed information about the spatial movements of this epidemiologically important age group. To address this knowledge gap, we collaborated with eight secondary schools to conduct a survey of movement patterns of school pupils in primary and secondary schools in the United Kingdom. We found evidence of a significant change in behaviour between term time and holidays, with term time weekdays characterised by predominately local movements, and holidays seeing much broader variation in travel patterns. Studies that use mathematical models to examine epidemic transmission and control often use adult commuting data as a proxy for population movements. We show that while these data share some features with the movement patterns reported by school children, there are some crucial differences between the movements of children and adult commuters during both term-time and holidays.     

Localization of a Wide-Ranging Panel of Antigens in the Rat Retina by Immunohistochemistry: Comparison of Davidson��s Solution and Formalin as Fixatives

The preferred fixative for whole eyes is Davidson’s solution, which provides optimal tissue preservation while avoiding retinal detachment. Hitherto, the compatibility of Davidson’s solution with immunohistochemistry has been largely untested. The goal of the present study was to compare the immunolabeling patterns of a wide-ranging panel of commercially available, previously validated antibodies in formalin- and Davidson’s-fixed retinas. Immunohistochemistry was performed in normal pigmented rat eyes and, to facilitate localization of inducible proteins, eyes injected with the bacterial toxin lipopolysaccharide or subjected to laser-induced photoreceptor damage. Specificity of labeling was judged by the morphology and distribution of immunopositive cells, by the absence of signal in appropriate controls, and by comparison with expected staining patterns. Retinas fixed in formalin displayed only adequate morphological integrity but were highly compatible with all 39 antibodies evaluated. Retinas fixed in Davidson’s solution displayed morphological integrity superior to those fixed in formalin. Generally, the cellular and subcellular patterns and intensities of immunoreactivities obtained with each fixative were identical; however, Davidson’s fixative was less compatible with certain antibodies, such as the neurotransmitter γ-aminobutyric acid, the microglial marker iba1, the macroglial stress protein nestin, and the small heat shock proteins Hsp27 and αB-crystallin, shortfalls that somewhat temper enthusiasm concerning its use.     

Stem Cell Proliferation and Quiescence—Two Sides of the Same Coin

The kinetics of label uptake and dilution in dividing stem cells, e.g., using Bromodeoxyuridine (BrdU) as a labeling substance, are a common way to assess the cellular turnover of all hematopoietic stem cells (HSCs) in vivo. The assumption that HSCs form a homogeneous population of cells which regularly undergo cell division has recently been challenged by new experimental results. For a consistent functional explanation of heterogeneity among HSCs, we propose a concept in which stem cells flexibly and reversibly adapt their cycling state according to systemic needs. Applying a mathematical model analysis, we demonstrate that different experimentally observed label dilution kinetics are consistently explained by the proposed model. The dynamically stabilized equilibrium between quiescent and activated cells leads to a biphasic label dilution kinetic in which an initial and pronounced decline of label retaining cells is attributed to faster turnover of activated cells, whereas a secondary, decelerated decline results from the slow turnover of quiescent cells. These results, which support our previous model prediction of a reversible activation/deactivation of HSCs, are also consistent with recent findings that use GFP-conjugated histones as a label instead of BrdU. Based on our findings we interpret HSC organization as an adaptive and regulated process in which the slow activation of quiescent cells and their possible return into quiescence after division are sufficient to explain the simultaneous occurrence of self-renewal and differentiation. Furthermore, we suggest an experimental strategy which is suited to demonstrate that the repopulation ability among the population of label retaining cells changes during the course of dilution.     

Salinity and Bacterial Diversity: To What Extent Does the Concentration of Salt Affect the Bacterial Community in a Saline Soil?

In this study, the evaluation of soil characteristics was coupled with a pyrosequencing analysis of the V2-V3 16S rRNA gene region in order to investigate the bacterial community structure and diversity in the A horizon of a natural saline soil located in Sicily (Italy). The main aim of the research was to assess the organisation and diversity of microbial taxa using a spatial scale that revealed physical and chemical heterogeneity of the habitat under investigation. The results provided information on the type of distribution of different bacterial groups as a function of spatial gradients of soil salinity and pH. The analysis of bacterial 16S rRNA showed differences in bacterial composition and diversity due to a variable salt concentration in the soil. The bacterial community showed a statistically significant spatial variability. Some bacterial phyla appeared spread in the whole area, whatever the salinity gradient. It emerged therefore that a patchy saline soil can not contain just a single microbial community selected to withstand extreme osmotic phenomena, but many communities that can be variously correlated to one or more environmental parameters. Sequences have been deposited to the SRA database and can be accessed on ID Project PRJNA241061.     

Localization and synthesis of α-fetoprotein in the chicken

Summary The localization and sites of synthesis of -fetoprotein in chick embryos throughout development have been investigated using the combined techniques of immunofluorescence microscopy and tissue culture in the presence of radiolabelled amino acids, followed by immunoautoradiographic analysis.Alpha-fetoprotein is present in a range of embryonic tissues and especially concentrated in the yolk sac, liver and connective tissue. Analysis of culture fluids revealed that the yolk sac is the major site of -fetoprotein synthesis with smaller, but significant quantities being produced by the liver.These results are discussed in relation to mammalian -fetoprotein, and the merits of the chick embryo for studies on the biological function of AFP are considered.Supported by an award from the Science Research Council, to whom grateful acknowledgement is made     

For the Insect Pathogen Photorhabdus luminescens, Which End of a Nematode Is Out?

The nematode Heterorhabditis bacteriophora is the vector for transmitting the entomopathogenic bacterium Photorhabdus luminescens between insect larvae. The dauer juvenile (DJ) stage nematode selectively retains P. luminescens in its intestine until it releases the bacteria into the hemocoel of an insect host. We report the results of studying the transmission of the bacteria by its nematode vector. Cells of P. luminescens labeled with green fluorescent protein preferentially colonized a region of the DJ intestine immediately behind the basal bulb, extending for various distances toward the anus. Incubation of DJ nematodes in vitro in insect hemolymph induced regurgitation of the bacteria. Following a 30-min lag, the bacteria migrated in a gradual and staggered movement toward and ultimately exited the mouth. This regurgitation reaction was induced by a low-molecular-weight, heat- and protease-stable, anionic component present in arthropod hemolymph and in supernatants from insect cell cultures. Nematodes anesthetized with levamisole or treated with the antihelmenthic agent ivermectin did not release their bacteria into hemolymph. The ability to visualize P. luminescens in the DJ nematode intestine provides the first clues to the mechanism of release of the bacteria during infection of insect larvae. This and the partial characterization of a component of hemolymph triggering release of the bacteria render this fascinating example of both a mutualistic symbiosis and disease transmission amenable to future genetic and molecular study.     

Longitudinal Spatial Patterns of Bacterial Production and Respiration in a Large River–Estuary: Implications for Ecosystem Carbon Consumption

Rivers and estuaries transport organic carbon (C) from terrestrial and freshwater ecosystems to the marine environment. During this transit, bacteria actively utilize and transform organic C, but few studies have measured detailed spatial variation in rates of bacterial respiration (BR) and production (BP). We measured BP at 39 stations and BR at 12 stations at monthly intervals along a 200-km reach of the tidal Hudson River. We observed strong repeatable spatial patterns for both BP and BR, with rates declining in the downstream direction. Bacterial Production had much greater dynamic range of spatial variation than BR. We used the detailed seasonal and spatial data on BP and BR to measure the total C demand of bacteria at several scales. We calculated volumetric and areal rates for 12 sections of the Hudson, as well as the total C utilization. Volumetric BR averaged 20 g-C-m^–3 y^–1, but it was highest in the most upstream section at 30 g C m^–3 y^–1. Areal rates averaged over the entire river were 174 g C m^–2 y^–1, but they were 318 g C m^–2 y^–1 in the deepest section of the river, indicating the importance of morphometric variation. Total bacterial C demand increased downriver with increasing total volume. Overall, bacteria in the freshwater section of the river consumed approximately 18–25.5 × 10⁹ g C y^–1, about 20% of the total organic C load.     

Metabolic Interplay between the Asian Citrus Psyllid and Its Profftella Symbiont: An Achilles’ Heel of the Citrus Greening Insect Vector

‘Candidatus Liberibacter asiaticus’ (CLas), the bacterial pathogen associated with citrus greening disease, is transmitted by Diaphorina citri, the Asian citrus psyllid. Interactions among D. citri and its microbial endosymbionts, including ‘Candidatus Profftella armatura’, are likely to impact transmission of CLas. We used quantitative mass spectrometry to compare the proteomes of CLas(+) and CLas(-) populations of D. citri, and found that proteins involved in polyketide biosynthesis by the endosymbiont Profftella were up-regulated in CLas(+) insects. Mass spectrometry analysis of the Profftella polyketide diaphorin in D. citri metabolite extracts revealed the presence of a novel diaphorin-related polyketide and the ratio of these two polyketides was changed in CLas(+) insects. Insect proteins differentially expressed between CLas(+) and CLas(-) D. citri included defense and immunity proteins, proteins involved in energy storage and utilization, and proteins involved in endocytosis, cellular adhesion, and cytoskeletal remodeling which are associated with microbial invasion of host cells. Insight into the metabolic interdependence between the insect vector, its endosymbionts, and the citrus greening pathogen reveals novel opportunities for control of this disease, which is currently having a devastating impact on citrus production worldwide.     

A Multi Size-Level Assessment of Benthic Marine Communities in a Coastal Environment: Are They Different Sides of the Same Coin?

Organism body size has been demonstrated to be a discriminating element in shaping the response of living beings to environmental factors, thus playing a fundamental role in community structuring. Despite the importance of studies elucidating relations among communities of different size levels in ecosystems, the attempts that have been made in this sense are still very scarce and a reliable approach for these research still has to be defined. We characterized the benthic communities of bacteria, microbial eukaryotes, meiofauna and macrofauna in a coastal environment, encompassing a 10000-fold gradient in body size, testing and discussing a mixed approach of molecular fingerprinting for microbes and morphological observations for meio- and macrofauna. We found no correlation among structures of the different size-level communities: this suggests that community composition at one size-level could have no (or very low) influence on the community composition at other size-levels. Moreover, each community responds in a different way to the environmental parameters and with a degree of sensitivity which seems to increase with organism size. Therefore, our data indicate that the characterization of all the different size levels is clearly a necessity in order to study the dynamics really acting in a system.     

Spitzenk?rper,Exocyst, and Polarisome Components in Candida albicans Hyphae Show Different Patterns of Localization and Have Distinct Dynamic Properties

During the extreme polarized growth of fungal hyphae, secretory vesicles are thought to accumulate in a subapical region called the Spitzenkörper. The human fungal pathogen Candida albicans can grow in a budding yeast or hyphal form. When it grows as hyphae, Mlc1 accumulates in a subapical spot suggestive of a Spitzenkörper-like structure, while the polarisome components Spa2 and Bud6 localize to a surface crescent. Here we show that the vesicle-associated protein Sec4 also localizes to a spot, confirming that secretory vesicles accumulate in the putative C. albicans Spitzenkörper. In contrast, exocyst components localize to a surface crescent. Using a combination of fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) experiments and cytochalasin A to disrupt actin cables, we showed that Spitzenkörper-located proteins are highly dynamic. In contrast, exocyst and polarisome components are stably located at the cell surface. It is thought that in Saccharomyces cerevisiae exocyst components are transported to the cell surface on secretory vesicles along actin cables. If each vesicle carried its own complement of exocyst components, then it would be expected that exocyst components would be as dynamic as Sec4 and would have the same pattern of localization. This is not what we observe in C. albicans. We propose a model in which a stream of vesicles arrives at the tip and accumulates in the Spitzenkörper before onward delivery to the plasma membrane mediated by exocyst and polarisome components that are more stable residents of the cell surface.Polarized growth of fungi requires that a supply of secretory vesicles is delivered along cytoskeletal tracks to the site of cell expansion (for reviews, see references 13, 29, 30, and 31). Fusion of these membrane-bound vesicles with the plasma membrane allows the necessary expansion of the plasma membrane and releases the enzymes and raw materials for the synthesis of new cell wall material and the remodeling necessary to allow this newly synthesized material to be inserted into the existing cell wall. The process of polarized growth has been extensively studied in the budding yeast Saccharomyces cerevisiae and provides a model for studying the process in other fungi (for a review, see reference 20). Post-Golgi vesicles travel to sites of polarized growth along actin cables (23). Actin cables are nucleated at sites of polarized growth by the formin Bni1 facilitated by a multiprotein complex called the polarisome, which consists of Spa2, Bud6, and Pea1(5, 22, 24, 27). The motive force for vesicle transport is provided by Myo2, a class V myosin, complexed to its regulatory light chain Mlc1 (22, 26). At the plasma membrane, secretory vesicles dock with a second multiprotein complex called the exocyst before fusion with the plasma membrane (14, 15, 32, 33), mediated by v-SNARES on the vesicle and t-SNARES on the membrane. The exocyst is an octomeric complex composed of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84 (21). It is thought that Sec3 and a fraction of the Exo70 pool are localized at sites of polarized growth independently of the actin cytoskeleton (3, 6). The other exocyst subunits and the remainder of the Exo70 pool are thought to be transported to sites of polarized growth on secretory vesicles, where together with Sec3 and Exo70 they form the exocyst complex (3). Secretory vesicles exit the Golgi apparatus, travel toward sites of polarized growth, and dock with the exocyst by use of the Rab-type GTPase Sec4 in its GTP-bound form, which is activated by its GEF, Sec2 (12, 19, 35, 36). In the S. cerevisiae cell cycle, polarized growth is initially directed toward the bud tip in young buds (17). Growth subsequently becomes isotropic in larger buds before being directed toward the mother bud neck during cytokinesis at the end of the cell cycle. Accordingly, polarisome and exocyst components localize to the tips of young buds (7, 27, 28).The rate of hyphal tip extension is much greater than that of the growth of a yeast or pseudohyphal bud. In rich yeast extract-peptone-dextrose (YEPD) medium, Candida albicans hyphae extend at the rate of 0.25 μm min⁻¹, compared to 0.0625 μm min⁻¹ in yeast buds and 0.125 μm min⁻¹ in pseudohyphal cells (P. Sudbery unpublished observations). In hyphae of filamentous fungi, a structure called a Spitzenkörper is present at the tip, which is rich in secretory vesicles (8, 9, 11, 29, 34). It is believed that the Spitzenkörper acts as a vesicle supply center (VSC) (1). This model proposes that the Spitzenkörper is maintained at a fixed distance from the hyphal tip. Vesicles radiate out in equal directions to fuse with the plasma membrane, so that more vesicles per unit area fuse with the hyphal tip itself than with other parts of the hyphae. Mathematical modeling shows that this explains the distinctive shape of hyphal tips.In order to investigate the mechanism of polarized growth in the hyphae of Candida albicans, we previously determined the localization of Mlc1-yellow fluorescent protein (YFP) and the polarisome components Bud6-YFP and Spa2-YFP (4). We found that in hyphae, polarisome components localized to a surface crescent, as they did in young yeast buds and the tips of elongated pseudohyphal buds. However, in hyphae Mlc1-YFP localized to a bright spot, which at least in some hyphae was clearly inside the tip, rather than at the surface, and which appeared spherical in three-dimensional reconstructions. We concluded that this represented a Spitzenkörper. In some hyphae Mlc1-YFP also localized to a surface crescent, similar to the pattern displayed by polarisome components. This observation suggested that the Spitzenkörper and polarisome were separate structures, both of which were present at hyphal tips, but that only the polarisome was present at the bud tips of pseudohyphae and yeast. Moreover, the dual localization of Mlc1-YFP to a crescent and a spot suggested that Mlc1 may be present in both structures.While S. cerevisiae has proved to be an excellent model to investigate the molecular genetics of polarized growth, it is less optimal to study the spatial organization of the molecular components because polarized growth of the bud is restricted to a short period after bud emergence when the nascent bud is small. Thus, there has been little effort to investigate the fine detail of the spatial organization of the different components of the polarization machinery beyond noting that they localize to sites of polarized growth. In this study we exploited the opportunities afforded by the continuous polarized growth of C. albicans hyphae to clarify the relationship between the Spitzenkörper, polarisome, and exocyst, which cooperate to mediate the extreme polarized growth of hyphae. We show that the vesicle-associated marker Sec4 also localizes to a Spitzenkörper-like structure, confirming the existence of a vesicle-rich area corresponding to a Spitzenkörper at the hyphal tip. We show that exocyst components such as Sec3, Sec6, Sec8, Exo70, and Exo84 localize to a surface crescent, so the exocyst, like the polarisome, is also a spatially separate structure from the Spitzenkörper. We used three independent strategies to investigate the dynamic properties of these structures. Fluorescence recovery after photobleaching (FRAP) was used to measure the rate at which new proteins arrived at the tip. Fluorescence loss in photobleaching (FLIP) was used to measure the rate at which proteins exited the tip. Cytochalasin A was used to disrupt actin cables, allowing the persistence of proteins at the tip to be measured after the supply of new proteins was blocked. In each case we found that Spitzenkörper components Sec4, Sec2, and Mlc1 were highly dynamic, while the polarisome component Spa2 was stable. Intriguingly, exocyst components showed intermediate dynamic properties, suggesting that they are delivered to the tip on vesicles but that not all vesicles carry a complement of exocyst components. We suggest that these data are consistent with a model in which a stream of vesicles arrives at the tip and accumulates in the Spitzenkörper before onward delivery to the plasma membrane mediated by exocyst and polarisome components that are more stable residents of the cell surface.     

Feedforward Inhibition and Synaptic Scaling – Two Sides of the Same Coin?

Feedforward inhibition and synaptic scaling are important adaptive processes that control the total input a neuron can receive from its afferents. While often studied in isolation, the two have been reported to co-occur in various brain regions. The functional implications of their interactions remain unclear, however. Based on a probabilistic modeling approach, we show here that fast feedforward inhibition and synaptic scaling interact synergistically during unsupervised learning. In technical terms, we model the input to a neural circuit using a normalized mixture model with Poisson noise. We demonstrate analytically and numerically that, in the presence of lateral inhibition introducing competition between different neurons, Hebbian plasticity and synaptic scaling approximate the optimal maximum likelihood solutions for this model. Our results suggest that, beyond its conventional use as a mechanism to remove undesired pattern variations, input normalization can make typical neural interaction and learning rules optimal on the stimulus subspace defined through feedforward inhibition. Furthermore, learning within this subspace is more efficient in practice, as it helps avoid locally optimal solutions. Our results suggest a close connection between feedforward inhibition and synaptic scaling which may have important functional implications for general cortical processing.