Plasmodium circumsporozoite protein promotes the development of the liver stages of the parasite

The liver stages of malaria are clinically silent but have a central role in the Plasmodium life cycle. Liver stages of the parasite containing thousands of merozoites grow inside hepatocytes for several days without triggering an inflammatory response. We show here that Plasmodium uses a PEXEL/VTS motif to introduce the circumsporozoite (CS) protein into the hepatocyte cytoplasm and a nuclear localization signal (NLS) to enter its nucleus. CS outcompetes NFkappaB nuclear import, thus downregulating the expression of many genes controlled by NFkappaB, including those involved in inflammation. CS also influences the expression of over one thousand host genes involved in diverse metabolic processes to create a favorable niche for the parasite growth. The presence of CS in the hepatocyte enhances parasite growth of the liver stages in vitro and in vivo. These findings have far reaching implications for drug and vaccine development against the liver stages of the malaria parasite.     

Modeling the onset and propagation of trabecular bone microdamage during low-cycle fatigue

Relatively small amounts of microdamage have been suggested to have a major effect on the mechanical properties of bone. A significant reduction in mechanical properties (e.g. modulus) can occur even before the appearance of microcracks. This study uses a novel non-linear microdamaging finite-element (FE) algorithm to simulate the low-cycle fatigue behavior of high-density trabecular bone. We aimed to investigate if diffuse microdamage accumulation and concomitant modulus reduction, without the need for complete trabecular strut fracture, may be an underlining mechanism for low-cycle fatigue failure (defined as a 30% reduction in apparent modulus). A microCT constructed FE model was subjected to a single cycle monotonic compression test, and constant and variable amplitude loading scenarios to study the initiation and accumulation of low-cycle fatigue microdamage. Microcrack initiation was simulated using four damage criteria: 30%, 40%, 50% and 60% reduction in bone element modulus (el-MR). Evaluation of structural (apparent) damage using the four different tissue level damage criteria resulted in specimen fatigue failure at 72, 316, 969 and 1518 cycles for the 30%, 40%, 50% and 60% el-MR models, respectively. Simulations based on the 50% el-MR model were consistent with previously published experimental findings. A strong, significant non-linear, power law relationship was found between cycles to failure (N) and effective strain (Deltasigma/E(0)): N=1.394x10(-25)(Deltasigma/E(0))(-12.17), r(2)=0.97, p<0.0001. The results suggest that microdamage and microcrack propagation, without the need for complete trabecular strut fracture, are mechanisms for high-density trabecular bone failure. Furthermore, the model is consistent with previous numerical fatigue simulations indicating that microdamage to a small number of trabeculae results in relatively large specimen modulus reductions and rapid failure.     

Renal oxalosis in free-ranging green turtles Chelonia mydas

Eighteen green turtles Chelonia mydas recovered from the Atlantic and Gulf coasts of Florida and Tortuguero National Park, Costa Rica, were diagnosed with renal oxalosis by histopathological examination. Affected sea turtles included 14 adults and 4 immature animals, which comprised 26% (18/69) of green turtle necropsy cases available for review. Calcium oxalate deposition ranged from small to moderate amounts and was associated with granuloma formation and destruction of renal tubules. All affected turtles died from traumatic events or health problems unrelated to renal oxalosis; however, 1 immature turtle had notable associated renal injury. Crystal composition was confirmed by infrared and scanning electron microscopy and energy dispersive X-ray analysis. The source of calcium oxalate is unknown and is presumed to be of dietary origin.     

Identification of regional mechanical anisotropy in soft tissue analogs

In a previous work (Raghupathy and Barocas, 2010, "Generalized Anisotropic Inverse Mechanics for Soft Tissues,"J. Biomech. Eng., 132(8), pp. 081006), a generalized anisotropic inverse mechanics method applicable to soft tissues was presented and tested against simulated data. Here we demonstrate the ability of the method to identify regional differences in anisotropy from full-field displacements and boundary forces obtained from biaxial extension tests on soft tissue analogs. Tissue heterogeneity was evaluated by partitioning the domain into homogeneous subdomains. Tests on elastomer samples demonstrated the performance of the method on isotropic materials with uniform and nonuniform properties. Tests on fibroblast-remodeled collagen cruciforms indicated a strong correlation between local structural anisotropy (measured by polarized light microscopy) and the evaluated local mechanical anisotropy. The results demonstrate the potential to quantify regional anisotropic material behavior on an intact tissue sample.     

CENP-C is a structural platform for kinetochore assembly

Centromeres provide a region of chromatin upon which kinetochores are assembled in mitosis. Centromeric protein C (CENP-C) is a core component of this centromeric chromatin that, when depleted, prevents the proper formation of both centromeres and kinetochores. CENP-C localizes to centromeres throughout the cell cycle via its C-terminal part, whereas its N-terminal part appears necessary for recruitment of some but not all components of the Mis12 complex of the kinetochore. We now find that all kinetochore proteins belonging to the KMN (KNL1/Spc105, the Mis12 complex, and the Ndc80 complex) network bind to the N-terminal part of Drosophila CENP-C. Moreover, we show that the Mis12 complex component Nnf1 interacts directly with CENP-C in vitro. To test whether CENP-C's N-terminal part was sufficient to recruit KMN proteins, we targeted it to the centrosome by fusing it to a domain of Plk4 kinase. The Mis12 and Ndc80 complexes and Spc105 protein were then all recruited to centrosomes at the expense of centromeres, leading to mitotic abnormalities typical of cells with defective kinetochores. Thus, the N-terminal part of Drosophila CENP-C is sufficient to recruit core kinetochore components and acts as the principal linkage between centromere and kinetochore during mitosis.     

Positive Feedback Mechanisms among Local Ca Releases,NCX, and ICaL Ignite Pacemaker Action Potentials

Recent data suggest that cardiac pacemaker cell function is determined by numerous time-, voltage-, and Ca-dependent interactions of cell membrane electrogenic proteins (M-clock) and intracellular Ca cycling proteins (Ca-clock), forming a coupled-clock system. Many aspects of the coupled-clock system, however, remain underexplored. The key players of the system are Ca release channels (ryanodine receptors), generating local Ca releases (LCRs) from sarcoplasmic reticulum, electrogenic Na/Ca exchanger (NCX) current, and L-type Ca current (I_CaL). We combined numerical model simulations with experimental simultaneous recordings of action potentials (APs) and Ca to gain further insight into the complex interactions within the system. Our simulations revealed a positive feedback mechanism, dubbed AP ignition, which accelerates the diastolic depolarization (DD) to reach AP threshold. The ignition phase begins when LCRs begin to occur and the magnitude of inward NCX current begins to increase. The NCX current, together with funny current and T-type Ca current accelerates DD, bringing the membrane potential to I_CaL activation threshold. During the ignition phase, I_CaL-mediated Ca influx generates more LCRs via Ca-induced Ca release that further activates inward NCX current, creating a positive feedback. Simultaneous recordings of membrane potential and confocal Ca images support the model prediction of the positive feedback among LCRs and I_CaL, as diastolic LCRs begin to occur below and continue within the voltage range of I_CaL activation. The ignition phase onset (identified within the fine DD structure) begins when DD starts to notably accelerate (～0.15 V/s) above the recording noise. Moreover, the timing of the ignition onset closely predicted the duration of each AP cycle in the basal state, in the presence of autonomic receptor stimulation, and in response to specific inhibition of either the M-clock or Ca-clock, thus indicating general importance of the new coupling mechanism for regulation of the pacemaker cell cycle duration, and ultimately the heart rate.     

96 Study of the stacking of DNA homoassociates by the extended cluster approach

Metropolis Monte Carlo method based on the extended cluster approach (Danilov, Dailidonis, van Mourik, & Fruchtl, 2011a, 2011b; Dailidonis, Danilov, Früchtl, & van Mourik, 2011) is used to investigate adenine–adenine (AA), guanine–guanine (GG), thymine–thymine (TT), and cytosine–cytosine (CC) homoassociates in a cluster consisting of 400 water molecules. The starting structures taken were AA N(7) amino symmetric, TT N(3) – O(4) symmetric, GG N(1) – O(6) symmetric, and CC N(3) amino symmetric base pairs. A water spherical cluster with the density of water at room temperature and a radius sphere equal 13.9 Å was used, which corresponds to the most difficult conditions for the formation of stacks (see Abraham, 1982). In spite of such initial conditions, it is shown that during the simulation, each base pair is transformed into a more favorable stacked configuration. The results obtained allow to observe the whole process of convergence for the first time (for more information, visit the Website http://biophys.in.ua/).     

Novel Clostridium thermocellum Type I Cohesin-Dockerin Complexes Reveal a Single Binding Mode

Protein-protein interactions play a pivotal role in a large number of biological processes exemplified by the assembly of the cellulosome. Integration of cellulosomal components occurs through the binding of type I cohesin modules located in a non-catalytic molecular scaffold to type I dockerin modules located at the C terminus of cellulosomal enzymes. The majority of type I dockerins display internal symmetry reflected by the presence of two essentially identical cohesin-binding surfaces. Here we report the crystal structures of two novel Clostridium thermocellum type I cohesin-dockerin complexes (CohOlpC-Doc124A and CohOlpA-Doc918). The data revealed that the two dockerins, Doc918 and Doc124A, are unusual because they lack the structural symmetry required to support a dual binding mode. Thus, in both cases, cohesin recognition is dominated by residues located at positions 11, 12, and 19 of one of the dockerin binding surfaces. The alternative binding mode is not possible (Doc918) or highly limited (Doc124A) because residues that assume the critical interacting positions, when dockerins are reoriented by 180°, make steric clashes with the cohesin. In common with a third dockerin (Doc258) that also presents a single binding mode, Doc124A directs the appended cellulase, Cel124A, to the surface of C. thermocellum and not to cellulosomes because it binds preferentially to type I cohesins located at the cell envelope. Although there are a few exceptions, such as Doc918 described here, these data suggest that there is considerable selective pressure for the evolution of a dual binding mode in type I dockerins that direct enzymes into cellulosomes.     

New Approaches for Absolute Quantification of Stable‐Isotope‐Labeled Peptide Standards for Targeted Proteomics Based on a UV Active Tag

Targeted proteomics depends on the availability of stable isotope labeled (SIL) peptide standards, which for absolute protein quantification need to be absolutely quantified. In the present study, three new approaches for absolute quantification of SIL peptides are developed. All approaches rely on a quantification tag (Qtag) with a specific UV absorption. The Qtag is attached to the peptide during synthesis and is removed by tryptic digestion under standard proteomics workflow conditions. While one quantification method (method A) is designed to allow the fast and economic production of absolutely quantified SIL peptides, two other methods (methods B and C) are developed to enable the straightforward re‐quantification of SIL peptides after reconstitution to control and monitor known problems related to peptide solubility, precipitation, and adhesion to vials. All methods yield consistent results when compared to each other and when compared to quantification by amino acid analysis. The precise quantitation methods are used to characterize the in vivo specificity of the H3 specific histone methyltransferase EZH2.     

Characterization of Cyt2Bc toxin from Bacillus thuringiensis subsp. medellin

We cloned and sequenced a new cytolysin gene from Bacillus thuringiensis subsp. medellin. Three IS240-like insertion sequence elements and the previously cloned cyt1Ab and p21 genes were found in the vicinity of the cytolysin gene. The cytolysin gene encodes a protein 29.7 kDa in size that is 91.5% identical to Cyt2Ba from Bacillus thuringiensis subsp. israelensis and has been designated Cyt2Bc. Inclusions containing Cyt2Bc were purified from the crystal-negative strain SPL407 of B. thuringiensis. Cyt2Bc reacted weakly with antibodies directed against Cyt2Ba and was not recognized by an antiserum directed against the reference cytolysin Cyt1Aa. Cyt2Bc was hemolytic only upon activation with trypsin and had only one-third to one-fifth of the activity of Cyt2Ba, depending on the activation time. Cyt2Bc was also mosquitocidal against Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus, including strains resistant to the Bacillus sphaericus binary toxin. Its toxicity was half of that of Cyt2Ba on all mosquito species except resistant C. quinquefasciatus.