Site-specific labeling of ‘second generation’ annexin V with 99mTc(CO)3 for improved imaging of apoptosis in vivo

In this study ‘second generation’ AnxV was specifically labeled with ^99mTc in three different ways outside the binding region of the protein to obtain an improved target-to-background activity ratio. The compounds were tested in vitro and in vivo in normal mice and in a model of hepatic apoptosis (anti-Fas mAb). The apoptosis binding was most prominent for the HIS-tagged ‘second generation’ AnxV labeled with ^99mTc(CO)₃ in comparison to ^99mTc-HYNIC-cys-AnxV and ^99mTc(CO)₃-DTPA-cys-AnxV.     

Improved inhibition of the histone acetyltransferase PCAF by an anacardic acid derivative

Several lines of evidence indicate that histone acetyltransferases (HATs) are novel drug targets for treatment of diseases like, for example, cancer and inflammation. The natural product anacardic acid is a starting point for development of small molecule inhibitors of the histone acetyltransferase (HAT) p300/CBP associated factor (PCAF). In order to optimize the inhibitory potency, a binding model for PCAF inhibition by anacardic acid was proposed and new anacardic acid derivatives were designed. Ten new derivatives were synthesized using a novel synthetic route. One compound showed a twofold improved inhibitory potency for the PCAF HAT activity and a twofold improved inhibition of histone acetylation in HEP G2 cells.     

Personality and the emergence of the pace-of-life syndrome concept at the population level

The pace-of-life syndrome (POLS) hypothesis specifies that closely related species or populations experiencing different ecological conditions should differ in a suite of metabolic, hormonal and immunity traits that have coevolved with the life-history particularities related to these conditions. Surprisingly, two important dimensions of the POLS concept have been neglected: (i) despite increasing evidence for numerous connections between behavioural, physiological and life-history traits, behaviours have rarely been considered in the POLS yet; (ii) the POLS could easily be applied to the study of covariation among traits between individuals within a population. In this paper, we propose that consistent behavioural differences among individuals, or personality, covary with life history and physiological differences at the within-population, interpopulation and interspecific levels. We discuss how the POLS provides a heuristic framework in which personality studies can be integrated to address how variation in personality traits is maintained within populations.     

Hepatitis C Virus Transmission Bottlenecks Analyzed by Deep Sequencing

Hepatitis C virus (HCV) replication in infected patients produces large and diverse viral populations, which give rise to drug-resistant and immune escape variants. Here, we analyzed HCV populations during transmission and diversification in longitudinal and cross-sectional samples using 454/Roche pyrosequencing, in total analyzing 174,185 sequence reads. To sample diversity, four locations in the HCV genome were analyzed, ranging from high diversity (the envelope hypervariable region 1 [HVR1]) to almost no diversity (the 5′ untranslated region [UTR]). For three longitudinal samples for which early time points were available, we found that only 1 to 4 viral variants were present, suggesting that productive infection was initiated by a very small number of HCV particles. Sequence diversity accumulated subsequently, with the 5′ UTR showing almost no diversification while the envelope HVR1 showed >100 variants in some subjects. Calculation of the transmission probability for only a single variant, taking into account the measured population structure within patients, confirmed initial infection by one or a few viral particles. These findings provide the most detailed sequence-based analysis of HCV transmission bottlenecks to date. The analytical methods described here are broadly applicable to studies of viral diversity using deep sequencing.Hepatitis C virus (HCV) is a positive-strand enveloped RNA virus of the flavivirus family. HCV infects ∼170 million people worldwide with a high rate of persistence (1, 2) and is a major etiological agent of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. The current standard of therapy is the combined use of pegylated alpha interferon (IFN-α) and ribavirin (9), although there are substantial limitations due to toxicity and resistance profiles (47). Recent development of various small-molecule inhibitors that specifically target HCV offer some promise (13), but challenges still remain because the size and diversity of viral populations promote rapid development of drug resistance (28, 42). In an infected individual, serum HCV RNA levels can reach 10 to 100 million IU/ml (40). The viral RNA polymerase is estimated to make 1 error per 10,000 to 100,000 bp copied (22), but the viral genome is only 9,600 bases, resulting in diversification of the viral population, so that most viral genomes differ in sequence from the population consensus (16, 20, 21). Thus, when antiviral pressure is exerted on a viral population, sequence variants with reduced sensitivity may expand in the presence of the selective pressure (30, 41) and cause resistance (37). Consistent with this, differential sequence diversity in HCV populations has been linked to clinical outcome (7, 8).The size and complexity of HCV populations has made their analysis challenging. However, new deep-sequencing and bioinformatics methods are well suited to analyzing this problem. Using the 454/Roche technology, it is possible to generate more than 10⁸ bases of DNA sequence in a single 1-day run, albeit in fragments 200 to 500 bases in length (24). In addition, many samples can be multiplexed in single experiments using DNA barcodes introduced in amplification primers to tag each sample (3, 12, 45, 46), allowing many viral sequences to be characterized in a single experiment.Here, we analyze HCV diversity by pyrosequencing a series of representative viral regions contained within PCR amplicons, and we use methods from the environmental microbiology field for data processing and analysis. In both virology and environmental microbiology, populations of interest commonly consist of many related but nonidentical sequences (e.g., viral lineages with related sequences or bacteria harboring related 16S rRNA gene sequences). Assembly of short pyrosequence reads into longer scaffolds is quite difficult in such a setting, because the related sequences present in the population can be assembled in many different ways. Complicated data-processing methods yield at best complex probabilistic models of variants likely to be present in the population (6). For this reason, in studies of bacterial 16S DNA from uncultured communities, many groups have used simplified analysis of single 16S amplicons that query short regions of the 16S rRNA gene (5, 11, 14, 23, 25, 38, 44). Extensive simulations and practical applications show that analysis of such “sequence tags” can disclose biologically meaningful clusters and gradients in collections of samples. Here, we apply a similar approach, using sequence tags for several regions of the HCV genome. This approach has the disadvantage of losing linkage information between amplicons, but it does allow the efficient analysis of large numbers of viral variants over many samples.A major challenge, however, is distinguishing variations authentically present in viral populations from artifactual mutations introduced as a result of the isolation procedure or sequencing error. Sequence recovery involves PCR steps that can result in base pair substitutions or artifactual chimera formation. The 454/Roche method, like any sequencing method, has a characteristic error rate and particularly elevated error rates at homopolymer runs (24). In this study, we took advantage of improved methods for error control using the PyroNoise program of Quince and colleagues, which was first used for analysis of 16S rRNA gene sequences (29). The PyroNoise program preclusters the raw light intensity data generated during pyrosequencing by the 454/Roche method, which removes most homopolymer errors. In reconstruction experiments, Quince and colleagues showed that 454/Roche sequence analysis of artificially constructed mock 16S rRNA gene communities yielded greatly inflated numbers of sequence types due to error, but preclustering using PyroNoise reduced the diversity to values much closer to the correct value. Here, we used a two-stage clustering method to remove noise. In the first stage, raw light intensity data were preclustered with PyroNoise (29); then, in the second stage, after interpretation of the sequence as base calls, sequences were clustered at 98.5% identity. The second step allowed us to take advantage of redundancy in the reads to improve sequence quality, though distinguishing genuine low-level variations in the viral populations from error is a challenge.We determined 174,185 high-quality HCV sequence reads to characterize (i) longitudinal variation in HCV populations following transmission, (ii) differences in HCV variation between HCV-monoinfected and HIV-HCV-coinfected subjects, and (iii) variation in a control HCV genome cloned in a bacterial plasmid to quantify variation arising during the isolation and analytical procedure. We developed amplicons to characterize four regions of the HCV genome (Fig. ​(Fig.1A)1A) and found that HCV sequence diversity ranged from almost nonexistent to extreme depending on the region of the viral genome studied. Using the deep-sequencing data, we estimate that only one or a few viral variants seeded initial infection, but after that, viral variants could expand to >100 in a single individual. Thus, these data specify the numbers of particles seeding productive infection and provide a general framework for the use of deep-sequencing data to characterize the structures of viral populations.Open in a separate windowFIG. 1.HCV genome and characteristics of three subjects studied longitudinally during acute HCV infection. (A) The HCV genome and the positions of amplicons studied. The amplicons are numbered 1 to 4 from left to right, and a letter is used to indicate the direction of sequence determination. For the E1E2 HVR1 (3) and E2 (4) amplicons, two slightly different primers were used in each direction in an effort to maximize the diversity of recovered sequence variants, and these are indicated by the two bars. (B) HCV load and ALT levels for patients 1 to 3 during acute HCV infection. The x axis shows the number of weeks after clinical presentation, which for these patients was close in time to initial infection. Further patient characteristics were as follows: patient 1, injecting drug user, anti-HCV negative on 18 June 2001, first ALT flare (ALT, 677) on 6 July 2001, anti-HCV positive on 11 October 2001; patient 2, possible medical exposure, anti-HCV negative on 16 May 2001, initial ALT flare (ALT, 467) on 9 January 2004, anti-HCV positive on 22 April 2004; patient 3, injecting drug user, anti-HCV negative on 31 January 2006 (slightly abnormal ALT, 73), initial ALT flare (ALT, 640) on 10 April 2006, anti-HCV positive on 11 April 2006.     

Structural Characterization of the E2 Domain of APL-1, a Caenorhabditis elegans Homolog of Human Amyloid Precursor Protein, and Its Heparin Binding Site

The amyloid β-peptide deposit found in the brain tissue of patients with Alzheimer disease is derived from a large heparin-binding protein precursor APP. The biological function of APP and its homologs is not precisely known. Here we report the x-ray structure of the E2 domain of APL-1, an APP homolog in Caenorhabditis elegans, and compare it to the human APP structure. We also describe the structure of APL-1 E2 in complex with sucrose octasulfate, a highly negatively charged disaccharide, which reveals an unexpected binding pocket between the two halves of E2. Based on the crystal structure, we are able to map, using site-directed mutagenesis, a surface groove on E2 to which heparin may bind. Our biochemical data also indicate that the affinity of E2 for heparin is influenced by pH: at pH 5, the binding appears to be much stronger than that at neutral pH. This property is likely caused by histidine residues in the vicinity of the mapped heparin binding site and could be important for the proposed adhesive function of APL-1.     

Inositol 1,4,5‐trisphosphate receptor 1 degradation in mouse eggs and impact on [Ca2+]i oscillations

The initiation of normal embryo development depends on the completion of all events of egg activation. In all species to date, egg activation requires an increase(s) in the intracellular concentration of calcium ([Ca²⁺]_i), which is almost entirely mediated by inositol 1,4,5‐trisphosphate receptor 1 (IP₃R1). In mammalian eggs, fertilization‐induced [Ca²⁺]_i responses exhibit a periodic pattern that are called [Ca²⁺]_i oscillations. These [Ca²⁺]_i oscillations are robust at the beginning of fertilization, which occurs at the second metaphase of meiosis, but wane as zygotes approach the pronuclear stage, time after which in the mouse oscillations cease altogether. Underlying this change in frequency are cellular and biochemical changes associated with egg activation, including degradation of IP₃R1, progression through the cell cycle, and reorganization of intracellular organelles. In this study, we investigated the system requirements for IP₃R1 degradation and examined the impact of the IP₃R1 levels on the pattern of [Ca²⁺]_i oscillations. Using microinjection of IP₃ and of its analogs and conditions that prevent the development of [Ca²⁺]_i oscillations, we show that IP₃R1 degradation requires uniform and persistently elevated levels of IP₃. We also established that progressive degradation of the IP₃R1 results in [Ca²⁺]_i oscillations with diminished periodicity while a near complete depletion of IP₃R1s precludes the initiation of [Ca²⁺]_i oscillations. These results provide insights into the mechanism involved in the generation of [Ca²⁺]_i oscillations in mouse eggs. J. Cell. Physiol. 222:238–247, 2010. © 2009 Wiley‐Liss, Inc.     

Unconventional secretion of Acb1 is mediated by autophagosomes

Starving Dictyostelium discoideum cells secrete AcbA, an acyl coenzyme A–binding protein (ACBP) that lacks a conventional signal sequence for entering the endoplasmic reticulum (ER). Secretion of AcbA in D. discoideum requires the Golgi-associated protein GRASP. In this study, we report that starvation-induced secretion of Acb1, the Saccharomyces cerevisiae ACBP orthologue, also requires GRASP (Grh1). This highlights the conserved function of GRASP in unconventional secretion. Although genes required for ER to Golgi or Golgi to cell surface transport are not required for Acb1 secretion in yeast, this process involves autophagy genes and the plasma membrane t-SNARE, Sso1. Inhibiting transport to vacuoles does not affect Acb1 secretion. In sum, our experiments reveal a unique secretory pathway where autophagosomes containing Acb1 evade fusion with the vacuole to prevent cargo degradation. We propose that these autophagosome intermediates fuse with recycling endosomes instead to form multivesicular body carriers that then fuse with the plasma membrane to release cargo.