Site-specific PEGylation at histidine tags

The efficacy of protein-based medicines can be compromised by their rapid clearance from the blood circulatory system. Achieving optimal pharmacokinetics is a key requirement for the successful development of safe protein-based medicines. Protein PEGylation is a clinically proven strategy to increase the circulation half-life of protein-based medicines. One limitation of PEGylation is that there are few strategies that achieve site-specific conjugation of PEG to the protein. Here, we describe the covalent conjugation of PEG site-specifically to a polyhistidine tag (His-tag) on a protein. His-tag site-specific PEGylation was achieved with a domain antibody (dAb) that had a 6-histidine His-tag on the C-terminus (dAb-His(6)) and interferon α-2a (IFN) that had an 8-histidine His-tag on the N-terminus (His(8)-IFN). The site of PEGylation at the His-tag for both dAb-His(6)-PEG and PEG-His(8)-IFN was confirmed by digestion, chromatographic, and mass-spectral studies. A methionine was also inserted directly after the N-terminal His-tag in IFN to give His(8)Met-IFN. Cyanogen bromide digestion studies of PEG-His(8)Met-IFN were also consistent with PEGylation at the His-tag. By using increased stoichiometries of the PEGylation reagent, it was possible to conjugate two separate PEG molecules to the His-tag of both the dAb and IFN proteins. Stability studies followed by in vitro evaluation confirmed that these PEGylated proteins retained their biological activity. In vivo PK studies showed that all of the His-tag PEGylated samples displayed extended circulation half-lives. Together, our results indicate that site-specific, covalent PEG conjugation at a His-tag can be achieved and biological activity maintained with therapeutically relevant proteins.     

A multi-element psychosocial intervention for early psychosis (GET UP PIANO TRIAL) conducted in a catchment area of 10 million inhabitants: study protocol for a pragmatic cluster randomized controlled trial

ABSTRACT: BACKGROUND: Multi-element interventions for first-episode psychosis (FEP) are promising, but have mostly been conducted in non-epidemiologically representative samples, thereby raising the risk of underestimating the complexities involved in treating FEP in 'real-world' services. METHODS/DESIGN: The Psychosis early Intervention and Assessment of Needs and Outcome (PIANO) trial is part of a larger research program (Genetics, Endophenotypes and Treatment: Understanding early Psychosis - GET UP) which aims to compare, at 9?months, the effectiveness of a multi-component psychosocial intervention versus treatment as usual (TAU) in a large epidemiologically based cohort of patients with FEP and their family members recruited from all public community mental health centers (CMHCs) located in two entire regions of Italy (Veneto and Emilia Romagna), and in the cities of Florence, Milan and Bolzano. The GET UP PIANO trial has a pragmatic cluster randomized controlled design. The randomized units (clusters) are the CMHCs, and the units of observation are the centers' patients and their family members. Patients in the experimental group will receive TAU plus: 1) cognitive behavioral therapy sessions, 2) psycho-educational sessions for family members, and 3) case management. Patient enrolment will take place over a 1-year period. Several psychopathological, psychological, functioning, and service use variables will be assessed at baseline and follow-up. The primary outcomes are: 1) change from baseline to follow-up in positive and negative symptoms' severity and subjective appraisal; 2) relapse occurrences between baseline and follow-up, that is, episodes resulting in admission and/or any case-note records of re-emergence of positive psychotic symptoms. The expected number of recruited patients is about 400, and that of relatives about 300. Owing to the implementation of the intervention at the CMHC level, the blinding of patients, clinicians, and raters is not possible, but every effort will be made to preserve the independency of the raters. We expect that this study will generate evidence on the best treatments for FEP, and will identify barriers that may hinder its feasibility in 'real-world' clinical settings, patient/family conditions that may render this intervention ineffective or inappropriate, and clinical, psychological, environmental, and service organization predictors of treatment effectiveness, compliance, and service satisfaction. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT01436331.     

Preclinical research in Rett syndrome: setting the foundation for translational success

In September of 2011, the National Institute of Neurological Disorders and Stroke (NINDS), the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), the International Rett Syndrome Foundation (IRSF) and the Rett Syndrome Research Trust (RSRT) convened a workshop involving a broad cross-section of basic scientists, clinicians and representatives from the National Institutes of Health (NIH), the US Food and Drug Administration (FDA), the pharmaceutical industry and private foundations to assess the state of the art in animal studies of Rett syndrome (RTT). The aim of the workshop was to identify crucial knowledge gaps and to suggest scientific priorities and best practices for the use of animal models in preclinical evaluation of potential new RTT therapeutics. This review summarizes outcomes from the workshop and extensive follow-up discussions among participants, and includes: (1) a comprehensive summary of the physiological and behavioral phenotypes of RTT mouse models to date, and areas in which further phenotypic analyses are required to enhance the utility of these models for translational studies; (2) discussion of the impact of genetic differences among mouse models, and methodological differences among laboratories, on the expression and analysis, respectively, of phenotypic traits; and (3) definitions of the standards that the community of RTT researchers can implement for rigorous preclinical study design and transparent reporting to ensure that decisions to initiate costly clinical trials are grounded in reliable preclinical data.     

A genome-wide collection of Mos1 transposon insertion mutants for the C. elegans research community

Methods that use homologous recombination to engineer the genome of C. elegans commonly use strains carrying specific insertions of the heterologous transposon Mos1. A large collection of known Mos1 insertion alleles would therefore be of general interest to the C. elegans research community. We describe here the optimization of a semi-automated methodology for the construction of a substantial collection of Mos1 insertion mutant strains. At peak production, more than 5,000 strains were generated per month. These strains were then subject to molecular analysis, and more than 13,300 Mos1 insertions characterized. In addition to targeting directly more than 4,700 genes, these alleles represent the potential starting point for the engineered deletion of essentially all C. elegans genes and the modification of more than 40% of them. This collection of mutants, generated under the auspices of the European NEMAGENETAG consortium, is publicly available and represents an important research resource.     

Thermodynamic and spectroscopic investigation on the role of Met residues in Cu(II) binding to the non-octarepeat site of the human prion protein

Among the common features shared by neurodegenerative diseases there is the central role played by specific proteins or peptides which accumulate in neurons as insoluble plaques or tangles, containing abnormal amounts of redox-active metal ions, like copper and iron. In the case of transmissible spongiform encephalopathies (TSE), the involved protein is known as "prion protein" (PrP(C)) since "prions" (proteinaceous and infectious) are the agents which make TSE transmissible. It is widely accepted that PrP(C), in its wild-type form, can bind up to six Cu(II) ions, four of them in the so-called "octarepeat domain" and the others in the "fifth (non-octarepeat) binding-site". The latter domain contains two His residues, acting as anchoring sites for Cu(II) ions, and other potential binding residues, such as Lys and Met. While it is widely accepted that Lys residues do not take part in complex-formation, the role of methionines is still debated. In order to shed light on this issue, some peptides have been synthesized, either directly mimicking the sequence of the second half of the fifth binding site of human-PrP(C) (apo-form) or analogues where Met residues have been substituted by n-leucine. In addition, a series of short peptides, containing both His and Met residues in different relative positions, have been investigated, for the sake of comparison. Spectroscopic results, including NMR spectra of systems containing Ni(II) as a probe for the paramagnetic Cu(II) ion, agree on the exclusion of any direct interaction between the sulphur atom of Met residues and the Cu(II) ion already bound to His-imidazole side-chains. However, thermodynamic data show that Met-109 somewhat contributes to stability of complex species and this can be attributed to different electronic and steric effects.     

"Cheek-to-cheek" urinary proteome profiling via combinatorial peptide ligand libraries: A novel, unexpected elution system

A new method is here reported for facile elution of the human urinary proteome after being captured with combinatorial peptide ligand libraries (CPLL, ProteoMiner). It consists in challenging the beads with 100 mM Tris, pH 7.4, or with 100 mM Lys, pH 7.4 or even better with a mixture of Lys, Arg, Asp and Glu (150 mM final concentration). These elutions permit recovery of species in a native form, for monitoring any biological activity of the eluted species, while avoiding the noxious presence of sodium dodecyl sulphate (SDS), reported as the best eluant so far from CPLL beads. SDS, albeit permitting quantitative recovery from the beads, has to be removed from the sample prior to mass spectrometry analysis. This unorthodox elution, which most likely will work only for urine samples, seems to be due to the fact that bile salts and urinary pigments are massively adsorbed by the beads, thus masking the hydrophobic binding sites of aromatic and non-aromatic amino acids. The binding thus occurs mostly via ionic and hydrogen bond interactions via the “Grand Catchers” Arg, Lys, His, which can then be easily challenged by positively charged species, such a Tris, free Lys and free Arg in the eluant as well as by negatively charged compounds, such as Glu and Asp. When eluting with the four-amino acid mix, at least 3300 spots can be visualized in a 2D map.     

Hand-held echocardiography: added value in clinical cardiological assessment

In the present article we review the main published data on the application of Tissue Doppler Imaging (TDI) to stress echocardiography for the detection of myocardial ischemia. TDI has been applied to stress echocardiography in order to overcome the limitations of visual analysis for myocardial ischemia. The introduction of a new technology for clinical routine use should pass through the different phases of scientific assessment from feasibility studies to large multicenter studies, from efficacy to effectiveness studies. Nonetheless the pro-technology bias plays a major role in medicine and expensive and sophisticated techniques are accepted before their real usefulness and incremental value to the available ones is assessed. Apparently, TDI is not exempted by this approach : its applications are not substantiated by strong and sound results. Nonetheless, conventional stress echocardiography for myocardial ischemia detection is heavily criticized on the basis of its subjectivity. Stress echocardiography has a long lasting history and the evidence collected over 20 years positioned it as an established tool for the detection and prognostication of coronary artery disease. The quantitative assessment of myocardial ischemia remains a scientific challenge and a clinical goal but time has not come for these newer ultrasonographic techniques which should be restricted to research laboratories.     

Intrinsic competition between the parasitoids Eretmocerus mundus and Encarsia formosa in Bemisia tabaci

Immature stages of Eretmocerus mundus Mercet and Encarsia formosa Gahan (both Hymenoptera: Aphelinidae) compete in larvae of their host, Bemisia tabaci (Gennadius) (Homoptera: Aleyrodidae). Laboratory tests were carried out on excised tree tobacco leaves, exposing B. tabaci nymphs to one of the parasitoid species alone or to both species, one after the other, to obtain multi‐parasitization. Parasitization by E. mundus and E. formosa was allowed on specific host stages in order to obtain interactions between different immature stages of the two parasitoids (eggs, and first, second, and third instars). The outcome from each multi‐parasitization treatment was verified by analysing data on parasitoid adult emergence. Observations under a stereomicroscope and dissections of multi‐parasitized hosts were also performed in order to demonstrate any factors potentially determining the outcome of competition. Eretmocerus mundus clearly prevailed over E. formosa when multi‐parasitism occurred. A higher percentage of adults emerging from multi‐parasitized hosts belonged to this parasitoid species (68.0–88.9% depending on the treatment). The lowest percent emergence by E. mundus (68.0%) and total percent emergence of parasitoid adults (52.2%) were obtained when E. mundus first instars interacted with hosts parasitized by E. formosa third instars. Observations and dissections showed that first‐instar E. mundus induced mortality in E. formosa immatures at penetration into the hosts, although they encountered greater difficulty in exploiting hosts inside which E. formosa had reached the third stage of development. In contrast, development of E. formosa immatures was not immediately inhibited if parasitization took place on hosts inside which E. mundus larvae had already penetrated. In this case, however, E. mundus also prevailed over E. formosa (72.5% of the emerged adults). Implications for the use of these parasitoid species against B. tabaci in biological control programmes are discussed.