Presence and isolation of 2 lipoproteins immunologically related to apolipoprotein A I in human serum

Very high density lipoproteins d : 1.23--1.25 g/ml (VHDL2) have been isolated from human serum by preparative ultracentrifugation. They contain 80 per cent proteins and 20 per cent lipids. Lipids are mainly phospholipids (80 per cent). The proportion of lysolecithin (50 per cent) is higher than that of lecithin (40 per cent). The quantity of cholesterol is low, the free cholesterol: total cholesterol ratio is 0.35. VHDL2 consisted principally in lipoprotein D and two lipoproteins immunologically apparented to apolipoprotein A I, called LP A I1 and LP A I2. The LP A I1 has a molecular weight slightly higher and a hydrated density lower than that of LP AI2. Our experiments suggest that LP A I1 exists in the serum before ultracentrifugation while LP A I 2 comes from HDL degradation during ultracentrifugation. The immunological heterogeneity of apo A I forming different protein-lipid complexes is discussed.     

Structure and modeling studies of the carboxy-terminus region of human tropoelastin.

Elastin macromolecular assembly is a highly complex mechanism involving many steps including coacervation, cross-linking, and probably other (not known) phenomena. In past studies, it has been proposed that the C-terminal part of tropoelastin is also involved in this process and may play a key role in tropoelastin interactions with other proteins of the final elastic fibres scaffold. Presented here are the results of the biophysical studies (biospectroscopy, bioinformatics) of the C-terminal domain of tropoelastin. We report the detailed structures adopted by the oxidized (native) and reduced forms of the free synthetic peptide with sequence encoded by exon 36 of human tropoelastin (GGACLGKACGRKRK) and propose a dynamical interpretation of which structures may be involved in interactions with other extra-cellular matrix proteins. We also suggest that these structures may be retrieved in other proteins sharing a consensus sequence; however no definitive conclusion can be drawn here on a possible structure-function relationship.     

Synthesis of gatifloxacin derivatives and their biological activities against Mycobacterium leprae and Mycobacterium tuberculosis

Novel 3′-piperazinyl derivatives of the 8-hydrogeno and 8-methoxy-6-fluoro-1-cyclopropyl-4-quinolone-3-carboxylic acid scaffolds were designed, synthesized and characterized by ¹H, ¹³C and ¹⁹F NMR, and HRMS. The activity of these derivatives against pathogenic mycobacteria (M. leprae and M. tuberculosis), wild-type (WT) strains or strains harboring mutations implicated in quinolone resistance, were determined by measuring drug concentrations inhibiting cell growth (MIC) and/or DNA supercoiling by DNA gyrase (IC₅₀), or inducing 25% DNA cleavage by DNA gyrase (CC₂₅). Compound 4 (with a methoxy in R₈ and a secondary carbamate in R₃′) and compound 5 (with a hydrogen in R₈ and an ethyl ester in R₃′) displayed biological activities close to those of ofloxacin but inferior to those of gatifloxacin and moxifloxacin against M. tuberculosis and M. leprae WT DNA gyrases, whereas all of the compounds were less active in inhibiting M. tuberculosis growth and M. leprae mutant DNA gyrases. Since R₃′ substitutions have been poorly investigated previously, our results may help to design new quinolone derivatives in the future.     

Eimeria tenella ROP kinase EtROP1 induces G0/G1 cell cycle arrest and inhibits host cell apoptosis

Coccidia are obligate intracellular protozoan parasites responsible for human and veterinary diseases. Eimeria tenella, the aetiologic agent of caecal coccidiosis, is a major pathogen of chickens. In Toxoplasma gondii, some kinases from the rhoptry compartment (ROP) are key virulence factors. ROP kinases hijack and modulate many cellular functions and pathways, allowing T. gondii survival and development. E. tenella's kinome comprises 28 putative members of the ROP kinase family; most of them are predicted, as pseudokinases and their functions have never been characterised. One of the predicted kinase, EtROP1, was identified in the rhoptry proteome of E. tenella sporozoites. Here, we demonstrated that EtROP1 is active, and the N‐terminal extension is necessary for its catalytic kinase activity. Ectopic expression of EtROP1 followed by co‐immunoprecipitation identified cellular p53 as EtROP1 partner. Further characterisation confirmed the interaction and the phosphorylation of p53 by EtROP1. E. tenella infection or overexpression of EtROP1 resulted both in inhibition of host cell apoptosis and G0/G1 cell cycle arrest. This work functionally described the first ROP kinase from E. tenella and its noncanonical structure. Our study provides the first mechanistic insight into host cell apoptosis inhibition by E. tenella. EtROP1 appears as a new candidate for coccidiosis control.     

Merging Children’s Oncology Group Data with an External Administrative Database Using Indirect Patient Identifiers: A Report from the Children’s Oncology Group

Purpose

Clinical trials data from National Cancer Institute (NCI)-funded cooperative oncology group trials could be enhanced by merging with external data sources. Merging without direct patient identifiers would provide additional patient privacy protections. We sought to develop and validate a matching algorithm that uses only indirect patient identifiers.

Methods

We merged the data from two Phase III Children’s Oncology Group (COG) trials for de novo acute myeloid leukemia (AML) with the Pediatric Health Information Systems (PHIS). We developed a stepwise matching algorithm that used indirect identifiers including treatment site, gender, birth year, birth month, enrollment year and enrollment month. Results from the stepwise algorithm were compared against the direct merge method that used date of birth, treatment site, and gender. The indirect merge algorithm was developed on AAML0531 and validated on AAML1031.

Results

Of 415 patients enrolled on the AAML0531 trial at PHIS centers, we successfully matched 378 (91.1%) patients using the indirect stepwise algorithm. Comparison to the direct merge result suggested that 362 (95.7%) matches identified by the indirect merge algorithm were concordant with the direct merge result. When validating the indirect stepwise algorithm using the AAML1031 trial, we successfully matched 157 out of 165 patients (95.2%) and 150 (95.5%) of the indirectly merged matches were concordant with the directly merged matches.

Conclusions

These data demonstrate that patients enrolled on COG clinical trials can be successfully merged with PHIS administrative data using a stepwise algorithm based on indirect patient identifiers. The merged data sets can be used as a platform for comparative effectiveness and cost effectiveness studies.     

A Phase I Double Blind,Placebo-Controlled,Randomized Study of the Safety and Immunogenicity of an Adjuvanted HIV-1 Gag-Pol-Nef Fusion Protein and Adenovirus 35 Gag-RT-Int-Nef Vaccine in Healthy HIV-Uninfected African Adults

Background

Sequential prime-boost or co-administration of HIV vaccine candidates based on an adjuvanted clade B p24, RT, Nef, p17 fusion protein (F4/AS01) plus a non-replicating adenovirus 35 expressing clade A Gag, RT, Int and Nef (Ad35-GRIN) may lead to a unique immune profile, inducing both strong T-cell and antibody responses.

Methods

In a phase 1, double-blind, placebo-controlled trial, 146 healthy adult volunteers were randomized to one of four regimens: heterologous prime-boost with two doses of F4/AS01_E or F4/AS01_B followed by Ad35-GRIN; Ad35-GRIN followed by two doses of F4/AS01_B; or three co-administrations of Ad35-GRIN and F4/AS01_B. T cell and antibody responses were measured.

Results

The vaccines were generally well-tolerated, and did not cause serious adverse events. The response rate, by IFN-γ ELISPOT, was greater when Ad35-GRIN was the priming vaccine and in the co-administration groups. F4/AS01 induced CD4+ T-cells expressing primarily CD40L and IL2 +/- TNF-α, while Ad35-GRIN induced predominantly CD8+ T-cells expressing IFN-γ +/- IL2 or TNF-α. Viral inhibition was induced after Ad35-GRIN vaccination, regardless of the regimen. Strong F4-specific antibody responses were induced. Immune responses persisted at least a year after the last vaccination. The complementary response profiles, characteristic of each vaccine, were both expressed after co-administration.

Conclusion

Co-administration of an adjuvanted protein and an adenovirus vector showed an acceptable safety and reactogenicity profile and resulted in strong, multifunctional and complementary HIV-specific immune responses.

Trial Registration

ClinicalTrials.gov NCT01264445     

Bayesian characterization of uncertainty in species interaction strengths

Considerable effort has been devoted to the estimation of species interaction strengths. This effort has focused primarily on statistical significance testing and obtaining point estimates of parameters that contribute to interaction strength magnitudes, leaving the characterization of uncertainty associated with those estimates unconsidered. We consider a means of characterizing the uncertainty of a generalist predator’s interaction strengths by formulating an observational method for estimating a predator’s prey-specific per capita attack rates as a Bayesian statistical model. This formulation permits the explicit incorporation of multiple sources of uncertainty. A key insight is the informative nature of several so-called non-informative priors that have been used in modeling the sparse data typical of predator feeding surveys. We introduce to ecology a new neutral prior and provide evidence for its superior performance. We use a case study to consider the attack rates in a New Zealand intertidal whelk predator, and we illustrate not only that Bayesian point estimates can be made to correspond with those obtained by frequentist approaches, but also that estimation uncertainty as described by 95% intervals is more useful and biologically realistic using the Bayesian method. In particular, unlike in bootstrap confidence intervals, the lower bounds of the Bayesian posterior intervals for attack rates do not include zero when a predator–prey interaction is in fact observed. We conclude that the Bayesian framework provides a straightforward, probabilistic characterization of interaction strength uncertainty, enabling future considerations of both the deterministic and stochastic drivers of interaction strength and their impact on food webs.     

Requirements for the valid quantification of immunostains on tissue microarray materials using image analysis

Antibody‐based proteomics applied to tissue microarray (TMA) technology provides a very efficient means of visualizing and locating antigen expression in large collections of normal and pathological tissue samples. To characterize antigen expression on TMAs, the use of image analysis methods avoids the effects of human subjectivity evidenced in manual microscopical analysis. Thus, these methods have the potential to significantly enhance both precision and reproducibility. Although some commercial systems include tools for the quantitative evaluation of immunohistochemistry‐stained images, there exists no clear agreement on best practices to allow for correct and reproducible quantification results. Our study focuses on practical aspects regarding (i) image acquisition (ii) segmentation of staining and counterstaining areas and (iii) extraction of quantitative features. We illustrate our findings using a commercial system to quantify different immunohistochemistry markers targeting proteins with different expression patterns (cytoplasmic, nuclear or membranous) in colon cancer or brain tumor TMAs. Our investigations led us to identify several steps that we consider essential for standardizing computer‐assisted immunostaining quantification experiments. In addition, we propose a data normalization process based on reference materials to be able to compare measurements between studies involving different TMAs. In conclusion, we recommend certain critical prerequisites that commercial or in‐house systems should satisfy in order to permit valid immunostaining quantification.     

Ribosome biogenesis is temperature‐dependent and delayed in Escherichia coli lacking the chaperones DnaK or DnaJ†

In Escherichia coli strains carrying null mutations in either the dnaK or dnaJ genes, the late stages of 30S and 50S ribosomal subunit biogenesis are slowed down in a temperature‐dependent manner. At high temperature (44°C), 32S and 45S particles (precursors to 50S subunits) and 21S particles (precursors to 30S subunits) accumulate. The latter are shown by 3′5′ rapid amplification of cDNA ends analysis to contain unprocessed or partially processed 16S ribosomal RNA at the 5′ end, but the 3′ end was never processed. This implies that maturation of 16S ribosomal RNA starts at the 5′‐terminus, and that the 3′‐terminus is only trimmed at a later step. At normal temperatures (30°C?37°C), ribosome assembly in both mutants is not arrested but is significantly delayed, as shown by pulse‐chase analysis. Assembly defects are partially compensated by an overexpression of other heat‐shock proteins, which occurs in the absence of their negative regulator DnaK, or by a plasmid‐driven overexpression of GroES/GroEL, suggesting the involvement of a network of chaperones in ribosome biogenesis.