Sequence variations of Env signal peptide alleles in different clinical stages of HIV infection

The human immunodeficiency virus has been shown to increase its infectivity throughout the course of infection. This virus selection property has been associated with genome mutations and recombinations among virus variants, causing amino acid residue alterations in important viral proteins. In order to explore the contribution of Env signal peptide (Env-sp) to Env glycoprotein expression and its possible relationship to increased virus infectivity observed at late stages of infection, we characterized Env-sp sequences derived from twelve patients at “early” and “late” stages of HIV infection without antiretroviral therapy use. In spite of the remarkable overall similarity between both stages, we observed the deletion of a sequence of neutral and basic residues at the Env-sp amino terminus in virus from early stage specimens and the insertion of basic residues in the hydrophobic region on late-stage viral isolates. The Env-sp sequence alterations may have viral adaptive functions during HIV infection.     

A unified framework for connectionist systems

Pattern classification using connectionist (i.e., neural network) models is viewed within a statistical framework. A connectionist network's subjective beliefs about its statistical environment are derived. This belief structure is the network's subjective probability distribution. Stimulus classification is interpreted as computing the most probable response for a given stimulus with respect to the subjective probability distribution. Given the subjective probability distribution, learning algorithms can be analyzed and designed using maximum likelihood estimation techniques, and statistical tests can be developed to evaluate and compare network architectures. The framework is applicable to many connectionist networks including those of Hopfield (1982, 1984), Cohen and Grossberg (1983), Anderson et al. (1977), and Rumelhart et al. (1986b).     

Heterogeneity in imprinted methylation patterns of pluripotent embryonic germ cells derived from pre-migratory mouse germ cells

Pluripotent stem cells, termed embryonic germ (EG) cells, have been generated from both human and mouse primordial germ cells (PGCs). Like embryonic stem (ES) cells, EG cells have the potential to differentiate into all germ layer derivatives and may also be important for any future clinical applications. The development of PGCs in vivo is accompanied by major epigenetic changes including DNA demethylation and imprint erasure. We have investigated the DNA methylation pattern of several imprinted genes and repetitive elements in mouse EG cell lines before and after differentiation. Analysed cell lines were derived soon after PGC specification, “early”, in comparison with EG cells derived after PGC colonisation of the genital ridge, “late” and embryonic stem (ES) cell lines, derived from the inner cell mass (ICM). Early EG cell lines showed strikingly heterogeneous DNA methylation patterns, in contrast to the uniformity of methylation pattern seen in somatic cells (control), late EG cell and ES cell lines. We also observed that all analysed XX cell lines exhibited less methylation than XY. We suggest that this heterogeneity may reflect the changes in DNA methylation taking place in the germ cell lineage soon after specification.     

Temperature and pH-Dependent Supramolecular Self-Assembly of Amelogenin Molecules: A Dynamic Light-Scattering Analysis

Evidence for the molecular self-assembly of amelogenin proteins to form quasi-spherical particles (“nanospheres”) in solution, bothin vitroandin vivo,has recently been documented. A particle-size distribution analysis of dynamic light-scattering data was undertaken to investigate the influence of temperature on this molecular self-assembly process at three different pH's. The long-term objective was to correlate these observations to the unusual physiochemical characteristics of the protein, to improve understanding of the molecular mechanisms involved in the generation of amelogenin “nanospheres” and understanding of their putative relation to amelogenin functionin vivo. We analyzed data using two different algorithms: Dynamics and DynaLS. It was found that at pH 8, in a temperature range between 5 and 25°C, the size of the recombinant amelogenin nanospheres is monodisperse, giving rise to particles of 15–18 nm in hydrodynamic radius. However, heterogeneous distribution of particle size was observed at temperature ranges between 27 and 35°C, becoming monodisperse again with larger particles (60–70 nm) after the temperature was elevated to 37–40°C. We interpret these results to suggest that amelogenin molecular self-association possesses a second stage assembly process at temperatures of 30–35°C, creating larger entities which apparently are structured and stable at 37–40°C. The effect of pH on the size of amelogenin “aggregates” was much more noticeable at 37°C compared to that at 25°C. This observation suggests that at physiological temperature (i.e., 37°C) amelogenin molecular self-assembly is extremely sensitive to pH changes. This finding supports the notion that local pH changes in the microenvironment of the enamel extracellular matrix may play critical roles in controlling the structural organization of the organic matrix framework.     

Distribution of plasma membrane rosettes and kinetics of cellulose formation in xylem development of higher plants

Summary Germ roots of several higher plants—maize (Zea mays), mung bean (Vigna radiata) and cress (Lepidium sativum)—were freeze-fractured without cryoprotection in order to confirm and extend the informations on frequency and distribution of plasma membrane particle complexes with respect to cellulose formation. In all three objects the PF of developing xylem elements showed rosette accumulations in the regions of wall thickenings. The rosette-distribution pattern ranges from random in a young stage, to more grouped in a probable intermediate stage to strictly localized in later stages. The frequency of rosettes increases from stage to stage.In all three objects the EF of developing xylem elements is relatively poor in particles. Observations of terminal globules were rare and undistinct. This leads to the assumption that rosettes on the PF and terminal globules on the EF are not part of the same complex.A comparison of the number and distribution of microtubules underlying the xylem wall thickenings with rosette frequency and distribution leads to the conclusion that there seem to be no direct connections between these two structures. Microtubules may be involved in grouping of rosettes, thus indirectly orienting microfibril deposition. Calculations based on the observed rosette frequencies and the amount of wall material formed indicate that in xylem development 1,000 nm elementary fibril per rosette per minute may be formed and that the active phase of one rosette may be about 10 minutes.Abbreviations EF exoplasmic fracture face - PF protoplasmic fracture face     

Onset of rosette formation during spontaneous neural differentiation of hESC and hiPSC colonies

In vitro neural differentiation of human embryonic stem cells (hESCs) is an advantageous system for studying early neural development. The process of early neural differentiation in hESCs begins by initiation of primitive neuroectoderm, which is manifested by rosette formation, with consecutive differentiation into neural progenitors and early glial-like cells. In this study, we examined the involvement of early neural markers – OTX2, PAX6, Sox1, Nestin, NR2F1, NR2F2, and IRX2 – in the onset of rosette formation, during spontaneous neural differentiation of hESC and human induced pluripotent stem cell (hiPSC) colonies. This is in contrast to the conventional way of studying rosette formation, which involves induction of neuronal differentiation and the utilization of embryoid bodies. Here we show that OTX2 is highly expressed at the onset of rosette formation, when rosettes comprise no more than 3–5 cells, and that its expression precedes that of established markers of early neuronal differentiation. Importantly, the rise of OTX2 expression in these cells coincides with the down-regulation of the pluripotency marker OCT4. Lastly, we show that cells derived from rosettes that emerge during spontaneous differentiation of hESCs or hiPSCs are capable of differentiating into dopaminergic neurons in vitro, and into mature-appearing pyramidal and serotonergic neurons weeks after being injected into the motor cortex of NOD-SCID mice.     

Fixation probabilities in evolutionary game dynamics with a two-strategy game in finite diploid populations

Fixation processes in evolutionary game dynamics in finite diploid populations are investigated. Traditionally, frequency dependent evolutionary dynamics is modeled as deterministic replicator dynamics. This implies that the infinite size of the population is assumed implicitly. In nature, however, population sizes are finite. Recently, stochastic processes in finite populations have been introduced in order to study finite size effects in evolutionary game dynamics. One of the most significant studies on evolutionary dynamics in finite populations was carried out by Nowak et al. which describes “one-third law” [Nowak, et al., 2004. Emergence of cooperation and evolutionary stability in finite populations. Nature 428, 646-650]. It states that under weak selection, if the fitness of strategy α is greater than that of strategy β when α has a frequency , strategy α fixates in a β-population with selective advantage. In their study, it is assumed that the inheritance of strategies is asexual, i.e. the population is haploid. In this study, we apply their framework to a diploid population that plays a two-strategy game with two ESSs (a bistable game). The fixation probability of a mutant allele in this diploid population is derived. A “three-tenth law” for a completely recessive mutant allele and a “two-fifth law” for a completely dominant mutant allele are found; other cases are also discussed.     

Epigenomic communication systems in humans and honey bees: From molecules to behavior

A 2010 Nature editorial entitled “Time for the Epigenome” trumpets the appearance of the International Human Epigenome Consortium and likens it to Biology's equivalent of the Large Hadron Collider. It strongly endorses the viewpoint that selective modifications of “marks” on DNA and histones constitute the crucial codes of life, a proposition which is hotly contested (Ptashne et al., in 2010). This proposition reflects the current mindset that DNA and histone modifications are the prime movers in gene regulation during evolution. This claim is perplexing, since the well characterized organisms, Drosophila melanogaster and Caenorhabditis elegans, lack methylated DNA “marks” and the DNA methytransferase enzymology. Despite their complete absence, D. melanogaster nevertheless has extensive gene regulatory networks which drive sophisticated development, gastrulation, migration of germ cells and yield a nervous system with significant neural attributes. In stark contrast, the honey bee Apis mellifera deploys its human-type DNA methyltransferase enzymology to “mark” its DNA and it too has sophisticated development. What roles therefore is DNA methylation playing in different animals? The honey bee brings a fresh perspective to this question. Its combinatorial chemistry of pheromones, tergal and cuticular exudates provide an exquisite communication system between thousands of individuals. The development of queen and worker is strictly controlled by differential feeding of royal jelly and their adult behaviors are accompanied by epigenomic changes. Their interfaces with different “environments” are extensive, allowing an evaluation of the roles of epigenomes in behavior in a natural environment, in the space of a few weeks, and at requisite levels of experimental rigor.     

Varicella-zoster virus infects human embryonic stem cell-derived neurons and neurospheres but not pluripotent embryonic stem cells or early progenitors

Pluripotent human stem cells are a powerful tool for the generation of differentiated cells that can be used for the study of human disease. We recently demonstrated that neurons derived from pluripotent human embryonic stem cells (hESC) can be infected by the highly host-restricted human alphaherpesvirus varicella-zoster virus (VZV), permitting the interaction of VZV with neurons to be readily evaluated in culture. In the present study, we examine whether pluripotent hESC and neural progenitors at intermediate stages of differentiation are permissive for VZV infection. We demonstrate here that VZV infection is blocked in naïve hESC. A block to VZV replication is also seen when a bacterial artificial chromosome (BAC) containing the VZV genome is transfected into hESC. In contrast, related alphaherpesviruses herpes simplex virus 1 (HSV-1) and pseudorabies virus (PrV) productively infect naïve hESC in a cell-free manner, and PrV replicates from a BAC transfected into hESC. Neurons differentiate from hESC via neural progenitor intermediates, as is the case in the embryo. The first in vitro stage at which permissiveness of hESC-derived neural precursors to VZV replication is observed is upon formation of “neurospheres,” immediately after detachment from the inductive stromal feeder layer. These findings suggest that hESC may be useful in deciphering the yet enigmatic mechanisms of specificity of VZV infection and replication.     

Calcium trafficking integrates endoplasmic reticulum function with mitochondrial bioenergetics

Calcium homeostasis is central to all cellular functions and has been studied for decades. Calcium acts as a critical second messenger for both extracellular and intracellular signaling and is fundamental in cell life and death decisions (Berridge et al., 2000) [1]. The calcium gradient in the cell is coupled with an inherent ability of the divalent cation to reversibly bind multiple target biological molecules to generate an extremely versatile signaling system [2]. Calcium signals are used by the cell to control diverse processes such as development, neurotransmitter release, muscle contraction, metabolism, autophagy and cell death. “Cellular calcium overload” is detrimental to cellular health, resulting in massive activation of proteases and phospholipases leading to cell death (Pinton et al., 2008) [3]. Historically, cell death associated with calcium ion perturbations has been primarily recognized as necrosis. Recent evidence clearly associates changes in calcium ion concentrations with more sophisticated forms of cellular demise, including apoptosis (Kruman et al., 1998; Tombal et al., 1999; Lynch et al., 2000; Orrenius et al., 2003) , ,  and . Although the endoplasmic reticulum (ER) serves as the primary calcium store in the metazoan cell, dynamic calcium release to the cytosol, mitochondria, nuclei and other organelles orchestrate diverse coordinated responses. Most evidence supports that calcium transport from the ER to mitochondria plays a significant role in regulating cellular bioenergetics, production of reactive oxygen species, induction of autophagy and apoptosis. Recently, molecular identities that mediate calcium traffic between the ER and mitochondria have been discovered (Mallilankaraman et al., 2012a; Mallilankaraman et al., 2012b; Sancak et al., 2013)[8–10]. The next questions are how they are regulated for exquisite tight control of ER–mitochondrial calcium dynamics. This review attempts to summarize recent advances in the role of calcium in regulation of ER and mitochondrial function. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.     

Calcareous nannofossils and foraminifers herald the Messinian Salinity Crisis: The Pollenzo section (Alba, Cuneo; NW Italy)

During the Messinian, the Mediterranean area experienced fast and prominent paleoenvironmental changes, culminating in the so-called Messinian Salinity Crisis, with the deposition of the evaporitic series. This work investigates the micropaleontological assemblages in the pre-evaporitic sediments of the Sant’Agata Fossili Marls (SAF) of the Pollenzo section (Cuneo area, North Western Italy). A semiquantitative analysis is carried out on the upper part of the marly and pelitic sediments of the SAF underlying the first gypsum bed, ascribed to the Vena del Gesso Fm. (VDF). The studied interval belongs to the planktonic foraminifer Globorotalia conomiozea Zone and “non distinctive Zone” of Iaccarino and to the calcareous nannofossil MNN11b/c Zone of Raffi et al. (1998, 2003) ( [Raffi et al., 1998] and [Raffi et al., 2003]). Decrease of diversity and abundance of the foraminifer and calcareous nannofossil assemblages is recorded 12 m below the VDG and clearly reflects environmental stress. From bottom to top, six paleoecological events are recorded: (1) the first peak abundance of “small” Reticulofenestra and the last recovery (LR) of planktonic foraminifers; (2) the peak abundance of Pontosphaera japonica and the last recovery of warm water taxa Discoaster spp.; (3) the last recovery of benthic foraminifers; (4) the co-occurring peak abundances of Helicosphaera carteri and Sphenolithus abies, and the last recovery of warm water taxa Amaurolithus spp.; (5) the second peak of “small” Reticulofenestra; (6) the definitive disappearance of calcareous nannofossils. These paleoecological events describe a progressive isolation of the basin from the world ocean and increasingly stressed environment (LR planktonic foraminifers; LR Discoaster spp.), increasing dysoxic to anoxic conditions at the sea floor (LR benthic foraminifers), shallowing of the water column (peak of H. carteri), increasing salinity in surface waters (peak of S. abies), and enhanced nutrient concentration in surface waters (peak of “small” Reticulofenestra); these are related to paleoenvironmental changes predating gypsum deposition at Pollenzo and affecting the whole Mediterranean basin.     

Comparative models for human apolipoprotein A-I bound to lipid in discoidal high-density lipoprotein particles

We have constructed a series of models for apolipoprotein A-I (apo A-I) bound to discoidal high-density lipoprotein (HDL) particles, based upon the molecular belt model [Segrest, J. P., et al. (1999) J. Biol. Chem. 274, 31755-31758] and helical hairpin models [Rogers, D. P., et al. (1998) Biochemistry 37, 11714-11725], and compared these with picket fence models [Phillips, J. C., et al. (1997) Biophys. J. 73, 2337-2346]. Molecular belt models for discoidal HDL particles with differing diameters are presented, illustrating that the belt model can explain the discrete changes in HDL particle size observed experimentally. Hairpin models are discussed for the binding of apo A-I to discoidal HDL particles with diameters identical to those for the molecular belt model. Two models are presented for the binding of three monomers of apo A-I to a 150 A diameter discoidal HDL particle. In one model, two monomers of apo A-I bind to the exterior of the HDL particle in an antiparallel belt, with a third monomer of apo A-I bound to the disk in a hairpin conformation. In the second model, all three monomers of apo A-I are bound to the discoidal HDL particle in a hairpin conformation. Previously published experimental data for each model are reviewed, with FRET favoring either the belt or hairpin models over the picket fence models for HDL particles with diameters of 105 A. Naturally occurring mutations appear to favor the belt model for the 105 A particles, while the 150 A HDL particles favor the presence of at least one hairpin.     

Cytoskeleton‐associated proteins are enriched in human embryonic‐stem cell‐derived neuroectodermal spheres

The ability to generate neural lineages from human embryonic stem cells (hESCs) in a controlled manner would further investigation of human neurogenesis and development of potential cell therapeutic applications to treat neurological diseases; however, generating such neural stem cells (NSCs) remains a challenge. In an attempt to characterize the cellular mechanisms involved in hESC differentiation into neuroprogenitor cells, we performed 2‐DE using protein extracts from hESC‐derived embryoid bodies (EBs) and neuroectodermal spheres (NESs) bearing neuroprogenitors. Of 47 differentially expressed protein spots, 28 nonredundant spots were shown to be upregulated in the NESs; these protein spots included neurogenesis‐related proteins (TAF1, SEPT2, NPH3, and CRABP), as expected. Interestingly, 6 of these 28 protein spots were cytoskeleton‐associated proteins (CSAP) such as Fascin‐1, Cofilin‐1, and Stathmin‐1. Western‐blot analyses confirmed the increased levels of these proteins in the NESs. Furthermore, immunostaining analysis showed that both Fascin‐1 and Stathmin‐1 were preferentially expressed in the inner rims of neural rosettes, which are characteristic features of neuroprogenitors in culture. We also confirmed prominent expression of Fascin‐1 in (sub‐)ventricular zone in E15.5 mouse fetal brain. Our results suggest that, in addition to the induction of those genes involved in neural development, hESC differentiation into the NES is associated with a marked reorganization of the cellular cytoskeleton.     

Plasma-membrane rosettes in root hairs of Equisetum hyemale

Particle arrangement in the plasma membrane during cell wall formation was investigated by means of the double-replica technique in root hairs of Equisetum hyemale. Particle density in the protoplasmic fracture face of the plasma membrane was higher than in the extraplasmic fracture face. Apart from randomly distributed particles, particle rosettes were visible in the PF face of the plasma membrane. The rosettes consisted of six particles arranged in a circle and had an outer diameter of approx. 26 nm. No gradient in the number of rosettes was found, which agrees with micrifibril deposition taking place over the whole hair. The particle rosettes were found individually, which might indicate that they spin out thin microfibrils as found in higher-plant cell walls. Indeed microfibril width in these walls, measured in shadowed preparations, is 8.5±1.5 nm. It is suggested that the rosettes are involved in microfibril synthesis. Non-turgid cells lacked microfibril imprints in the plasma membrane and no particle rosettes were present on their PF face. Fixation with glutaraldehyde caused, probably as a result of plasmolysis, the microfibril imprints to disappear together with the particle rosettes. The PF face of the plasma membrane of non-turgid hairs sometimes showed domains in which the intramembrane particles were aggregated in a hexagonal pattern. Microfibril orientation during deposition will be discussed.Abbreviations EF extraplasmic fracture face - PF protoplasmic fracture face     

Tip cell growth and the frequency and distribution of particle rosettes in the plasmalemma: Experimental studies inFunaria protonema cells

Summary ComparingFunaria protonema tip cells of different age and of experimentally modified growth rate (by changing the light-dark-regime, by application of colchicine and of D₂O and by plasmolysis) we found that the site and intensity of growth are related closely to the distribution and frequency of particle rosettes in the PF of the plasma membrane. The results confirm previous suggestions that the rosettes are involved in cellulose fibril formation and that they have a rather short life time (about 10–15 minutes,Reiss et al. 1984). The appearance of rosettes seems to depend on the exocytosis of Golgi vesicle containing wall matrix material. Morphometric calculations suggest that each Golgi vesicle may incorporate one rosette into the plasmalemma in caulonema tip cells.     

DNA Sequence-Directed Organization of Chromatin: Structure-Based Computational Analysis of Nucleosome-Binding Sequences

The folding of DNA on the nucleosome core particle governs many fundamental issues in eukaryotic molecular biology. In this study, an updated set of sequence-dependent empirical “energy” functions, derived from the structures of other protein-bound DNA molecules, is used to investigate the extent to which the architecture of nucleosomal DNA is dictated by its underlying sequence. The potentials are used to estimate the cost of deforming a collection of sequences known to bind or resist uptake in nucleosomes along various left-handed superhelical pathways and to deduce the features of sequence contributing to a particular structural form. The deformation scores reflect the choice of template, the deviations of structural parameters at each step of the nucleosome-bound DNA from their intrinsic values, and the sequence-dependent “deformability” of a given dimer. The correspondence between the computed scores and binding propensities points to a subtle interplay between DNA sequence and nucleosomal folding, e.g., sequences with periodically spaced pyrimidine-purine steps deform at low cost along a kinked template whereas sequences that resist deformation prefer a smoother spatial pathway. Successful prediction of the known settings of some of the best-resolved nucleosome-positioning sequences, however, requires a template with “kink-and-slide” steps like those found in high-resolution nucleosome structures.     

Converted neural cells: induced to a cure?

Many neurodegenerative disorders such as Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS) and others often occur as a result of progressive loss of structure or function of neurons. Recently, many groups were able to generate neural cells, either differentiated from induced pluripotent stem cells (iPSCs) or converted from somatic cells. Advances in converted neural cells have opened a new era to ease applications for modeling diseases and screening drugs. In addition, the converted neural cells also hold the promise for cell replacement therapy (Kikuchi et al., 2011; Krencik et al., 2011; Kriks et al., 2011; Nori et al., 2011; Rhee et al., 2011; Schwartz et al., 2012). Here we will mainly discuss most recent progress on using converted functional neural cells to treat neurological diseases and highlight potential clinical challenges and future perspectives.     

Space radiation enhancement linked to geomagnetic disturbances.

Space radiation dosimetry measurements have been made on board the Space Shuttle. A newly developed active detector called "Real-time Radiation Monitoring Device (RRMD)" was used (Doke et al., 1995; Hayashi et al., 1995). The RRMD results indicate that low Linear Energy Transfer (LET) particles steadily penetrate around the South Atlantic Anomaly (SAA) without clear enhancement of dose equivalent and some daily periodic enhancements of dose equivalent due to high LET particles are seen at the lower geomagnetic cutoff regions (Doke et al., 1996). We also have been analyzing the space weather during the experiment, and found that the anomalous high-energy particle enhancement was linked to geomagnetic disturbance due to the high speed solar wind from a coronal hole. Additional analysis and other experiments are necessary for clarification of these phenomena. If a penetration of high-energy particles into the low altitude occurs by common geomagnetic disturbances, the prediction of geomagnetic activity becomes more important in the next Space Station's era.