Differential analysis of Bacillus anthracis after pX01 plasmid curing and comprehensive data on Bacillus anthracis infection in macrophages and glial cells

Bacillus anthracis is a gram-positive bacterial organism responsible for anthrax. This organism has two pathogenic plasmids: pX01 and pX02. The genetic function of pX01, which comprises about 198 kb, is not known, except for a region called the pathogenic island, which contains three genes-pag, lef, and cya-that code for three toxic proteins. A 2-D difference gel electrophoresis (2-D DIGE) system was used to verify the existence of proteins controlled by the pX01 plasmid, and protein regulation data were obtained using DeCyder software. A total of 1728 proteins were identified in the wild-type strain of this organism and 1684 in the pX01 plasmid. Twenty-seven of these proteins disappeared and eight appeared when the pX01 plasmid was removed. An additional 52 proteins were downregulated and 15 were upregulated when this plasmid was removed. A total of 102 proteins have been identified using the MALDI-TOF method of analysis, including 49 whose functions are unknown. Among these, 31 participate in metabolic processes, two in cellular processes, 15 in the processing of genetic information, and five in the processing of extracellular information. Another seven proteins participate in bacterial virulence and pathogenesis. We investigated the functions of these proteins in other bacteria, particularly the B. anthracis derivative H9041. Bacterial growth differed between pX01+/pX02+ B. anthracis and its pX01-/pX02+ derivative as did the cytotoxicity of macrophages infected by pX01+/pX02+ B. anthracis and the pX01-pX02+ derivative. We also found that S100B protein levels increased in the host infected with pX01+/pX02+ B. anthracis or its pX01-/pX02+ derivative. These data suggest that the pX01 plasmid plays a key role in the regulation of protein functions in B. anthracis.     

The effects of plasma-derived extracellular vesicles on cumulus expansion and oocyte maturation in mice

Cumulus cell expansion is required for the ovulation of a fertilizable oocyte. Extracellular vesicles (EVs) are bilayer-lipid membrane vesicles that may be found in a variety of bodily fluids and play an important role in biological processes. This study aimed to examine the effects of plasma-derived EVs on cumulus expansion and in vitro maturation (IVM) of the oocyte. EVswere isolated using ultracentrifugation from the plasma of female mice. The morphology and size of EVs were analyzed by transmission electron microscopy (TEM) and dynamic light scattering (DLS). Western blotting allowed us to identify CD63, CD81, CD9, and HSP70 protein markers of EVs; the expression of the genes related to cumulus cell expansion, including hyaluronan synthase 2 (Has2) and prostaglandinendoperoxide synthase 2 (Ptgs2), were assessed using real-time polymerase chain reaction. Plasma-derived EVs labeled with Dil dye were successfully incorporated with cumulus cells during IVM. Plasma-derived EVs significantly induced cumulus expansion and maturation of oocytes. The percentage of oocytes that reached the MII stage was significantly greater in the EVs treatment group compared with other groups. Although treatment with epidermal growth factor (EGF) significantly increased cumulus expansion in cumulus-oocyte complexes (COCs), the impact was less than that seen with plasma-derived EVs. Furthermore, EVs generated from plasma substantially enhanced Has2 and Ptgs2 mRNA expression in the cumulus-oocyte complex. This research indicates that EVs derived from plasma are capable of promoting cumulus expansion and oocyte maturation.     

Automatic determination of the handedness of single-particle maps of macromolecules solved by CryoEM

Single-Particle Analysis by Cryo-Electron Microscopy is a well-established technique to elucidate the three-dimensional (3D) structure of biological macromolecules. The orientation of the acquired projection images must be initially estimated without any reference to the final structure. In this step, algorithms may find a mirrored version of all the orientations resulting in a mirrored 3D map. It is as compatible with the acquired images as its unmirrored version from the image processing point of view, only that it is not biologically plausible.In this article, we introduce HaPi (Handedness Pipeline), the first method to automatically determine the hand of electron density maps of macromolecules solved by CryoEM. HaPi is built by training two 3D convolutional neural networks. The first determines α-helices in a map, and the second determines whether the α-helix is left-handed or right-handed. A consensus strategy defines the overall map hand. The pipeline is trained on simulated and experimental data. The handedness can be detected only for maps whose resolution is better than 5 Å. HaPi can identify the hand in 89% of new simulated maps correctly. Moreover, we evaluated all the maps deposited at the Electron Microscopy Data Bank and 11 structures uploaded with the incorrect hand were identified.     

Albumin uptake and distribution in the zebrafish liver as observed via correlative imaging

Although liver transport routes have been extensively studied in rodents, live imaging under in situ and in vivo conditions of large volumes is still proven to be difficult. In this study, we took advantage of the optical transparency of zebrafish and their small size to explore their usefulness for correlative imaging studies and liver transport experimentations. First, we assessed the micro-architecture of the zebrafish liver and compared its fine structure to the rodent and humans’ literature. Next, we investigated the transport routes and cellular distribution of albumin using combined and correlative microscopy approaches. These methods permitted us to track the injected proteins at different time points through the process of liver uptake and clearance of albumin.We demonstrate strong structural and functional resemblance between the zebrafish liver and its rodents and humans’ counterparts. In as short as 5?min post-injection, albumin rapidly accumulated within the LSECs. Furthermore, albumin entered the space of Disse where it initially accumulated then subsequently was taken up by the hepatocytes. We propose the zebrafish as a viable alternative experimental model for hepatic transport studies, allowing swift multimodal imaging and direct quantification on the hepatic distribution of supramolecular complexes of interest.     

Pseudomonas aeruginosa β-carbonic anhydrase,psCA1, is required for calcium deposition and contributes to virulence

Calcification of soft tissue leads to serious diseases and has been associated with bacterial chronic infections. However, the origin and the molecular mechanisms of calcification remain unclear. Here we hypothesized that a human pathogen Pseudomonas aeruginosa deposits extracellular calcium, a process requiring carbonic anhydrases (CAs). Transmission electron microscopy confirmed the formation of 0.1-0.2 μm deposits by P. aeruginosa PAO1 growing at 5 mM CaCl₂, and X-ray elemental analysis confirmed they contain calcium. Quantitative analysis of deposited calcium showed that PAO1 deposits 0.35 and 0.75 mM calcium/mg protein when grown at 5 mM and 10 mM CaCl₂, correspondingly. Fluorescent microscopy indicated that deposition initiates at the cell surface. We have previously characterized three PAO1 β-class CAs: psCA1, psCA2, and psCA3 that hydrate CO₂ to HCO₃⁻, among which psCA1 showed the highest catalytic activity (Lotlikar et. al. 2013). According to immunoblot and RT-qPCR, growth at elevated calcium levels increases the expression of psCA1. Analyses of the deletion mutants lacking one, two or all three psCA genes, determined that psCA1 plays a major role in calcium deposition and contributes to the pathogen’s virulence. In-silico modeling of the PAO1 β-class CAs identified four amino acids that differ in psCA1 compared to psCA2, and psCA3 (T59, A61A, A101, and A108), and these differences may play a role in catalytic rate and thus calcium deposition. A series of inhibitors were tested against the recombinant psCA1, among which aminobenzene sulfonamide (ABS) and acetazolamide (AAZ), which inhibited psCA1 catalytic activity with K_Is of 19 nM and 37 nM, correspondingly. The addition of ABS and AAZ to growing PAO1 reduced calcium deposition by 41 and 78, respectively. Hence, for the first time, we showed that the β-CA psCA1 in P. aeruginosa contributes to virulence likely by enabling calcium salt deposition, which can be partially controlled by inhibiting its catalytic activity.     

Sequence- and seed-structure-dependent polymorphic fibrils of alpha-synuclein

Synucleinopathies comprise a diverse group of neurodegenerative diseases including Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy. These share a common pathological feature, the deposition of alpha-synuclein (a-syn) in neurons or oligodendroglia. A-syn is highly conserved in vertebrates, but the primary sequence of mouse a-syn differs from that of human at seven positions. However, structural differences of their aggregates remain to be fully characterized. In this study, we found that human and mouse a-syn aggregated in vitro formed morphologically distinct amyloid fibrils exhibiting twisted and straight structures, respectively. Furthermore, we identified different protease-resistant core regions, long and short, in human and mouse a-syn aggregates. Interestingly, among the seven unconserved amino acids, only A53T substitution, one of the familial PD mutations, was responsible for structural conversion to the straight-type. Finally, we checked whether the structural differences are transmissible by seeding and found that human a-syn seeded with A53T aggregates formed straight-type fibrils with short protease-resistant cores. These results suggest that a-syn aggregates form sequence-dependent polymorphic fibrils upon spontaneous aggregation but become seed structure-dependent upon seeding.     

Remodelling of the nuclear periphery during muscle cell differentiation in vitro

We have examined the composition and ultrastructure of the nuclear periphery during in vitro myogenesis of the rat myoblast cell line, L6E9. Immunofluorescence labelling and immunoblotting showed that lamins A/C and B were all present in undifferentiated cells, but that they increased significantly before extensive cell fusion had occurred, with lamins A/C increasing proportionately more. Electron microscopic observations were consistent with these results, showing an increase in the prominence of the lamina during differentiation. On the other hand, immunofluorescence labelling suggested that the P1 antigen began to disappear from the nuclear periphery as the cells were fusing, after the increase in lamin quantity, and was no longer detectable in multinucleated cells. Unexpectedly, however, P1 was readily detected in isolated nuclei, whether prepared from myoblast or differentiated cultures, as well as in both myoblast and myotube nuclear matrices. It appears probable, therefore, that the fading of P1 labelling is due to masking of the epitope by a soluble factor recruited to the nuclear periphery as cells differentiate. These data, together with evidence that the genome is substantially rearranged during L6E9 myogenesis [Chaly and Munro, 1996], suggest that L6E9 cells are a useful model system in which to study the interrelationship of nuclear envelope organization, chromatin spatial order, and nuclear function. © 1996 Wiley-Liss, Inc.     

In vivo observations of timecourse and distribution of morphological dynamics in Xenopus retinotectal axon arbors

Changes in neuronal structure can contribute to the plasticity of neuronal connections in the developing and mature nervous system. However, the expectation that they would occur slowly precluded many from considering structural changes as a mechanism underlying synaptic plasticity that occurs over a period of minutes to hours. We took time-lapse confocal images of retinotectal axon arbors to determine the timecourse, magnitude, and distribution of changes in axon arbor structure within living Xenopus tadpoles. Images of axons were collected at intervals of 3 min, 30 min, and 2 h over total observation periods up to 8 h. Branch additions and retractions in arbors imaged at 3- or 30-min intervals were confined to shorter branches. Sites of additions and retractions were distributed throughout the arbor. The average lifetime of branches was about 10 min. Branches of up to 10 μm could be added to the arbor within a single 3-min observation interval. Observations of arbors at 3-min intervals showed rapid changes in the structure of branchtips, including transitions from lamellar growth cones to more streamlined tips, growth cone collapse, and re-extension. Simple branchtips were motile and appeared capable of exploratory behavior when viewed in time-lapse movies. In arbors imaged at 2-h intervals over a total of 8 h, morphological changes included longer branches, tens of microns in length. An average of 50% of the total branch length in the arbor was remodeled within 8 h. The data indicate that the elaboration of the arbor occurs by the random addition of branches throughout the arbor, followed by the selective stabilization of a small fraction of the new branches and the retraction of the majority of branches. Stabilized branches can then elongate and support the addition of more branches. These data show that structural changes in presynaptic axons can occur very rapidly even in complex arbors and can therefore play a role in forms of neuronal plasticity that operate on a timescale of minutes. © 1996 John Wiley & Sons, Inc.     

Comparison of the subcellular distribution of G-proteins in hepatocytes in situ and in primary cultures

The subcellular localization of the heterotrimeric G-proteins in hepatocytes in situ was compared to that in hepatocytes in primary culture. The ability of various ligands to activate adenylyl cyclase (AC) in membrane preparations was also investigated. In hepatocytes in situ the G proteins were mainly localized at the plasma membrane while in hepatocytes in culture they were predominantly cytoplasmic. The localization of the G-proteins in hepatocytes in situ correlates with their role in signal transduction. In homogenates prepared from the cultured cells, ligands which stimulate AC via G_sα were without effect, which was consistent with the localization of G_sα in the cytoplasmic and nuclear compartments. The “relocalization” of the G proteins to the cytoplasm when cells are cultured suggests that transmembrane signalling may be regulated by cell differentiation and cell-cell and cell-extracellular matrix interactions. © 1996 Wiley-Liss, Inc.