Challenges and opportunities in the purification of recombinant tagged proteins

The purification of recombinant proteins by affinity chromatography is one of the most efficient strategies due to the high recovery yields and purity achieved. However, this is dependent on the availability of specific affinity adsorbents for each particular target protein. The diversity of proteins to be purified augments the complexity and number of specific affinity adsorbents needed, and therefore generic platforms for the purification of recombinant proteins are appealing strategies. This justifies why genetically encoded affinity tags became so popular for recombinant protein purification, as these systems only require specific ligands for the capture of the fusion protein through a pre-defined affinity tag tail. There is a wide range of available affinity pairs “tag-ligand” combining biological or structural affinity ligands with the respective binding tags. This review gives a general overview of the well-established “tag-ligand” systems available for fusion protein purification and also explores current unconventional strategies under development.     

Biochemical and energy dynamics throughout the reproductive cycle of the striped venus Chamelea gallina (Mollusca,Bivalvia)

The striped venus Chamelea gallina is an important commercial bivalve species in Europe. However, large inter-annual fluctuations in stock abundance and periodic recruitment failure threaten the biological and economic sustainability of this fishery. This study aimed to improve the knowledge of the reproductive cycle and reproductive strategies of this species from the Algarve coast (southern Portugal) in order to contribute to the establishment of management measures and to assess its potential for aquaculture. The reproductive cycle of C. gallina followed a seasonal cycle, significantly influenced by sea surface temperature and food availability. Gametogenesis took place in winter, coinciding with the phytoplankton bloom. Spawning occurred during summer, followed by a short period of sexual inactivity in autumn. Condition index did not reflect the reproductive cycle of C. gallina and generally, followed the same trend of chlorophyll a. Glycogen was positively correlated with gonadal index and chlorophyll a. High total lipid values were recorded throughout gonad ripeness and spawning, but decreased at the end of the spawning and in the rest period. The extended spawning period of C. gallina will allow larvae to be obtained for much of the year by artificial spawning of wild broodstock.     

Epidemiology of Dengue Disease in the Philippines (2000–2011): A Systematic Literature Review

This literature analysis describes the available dengue epidemiology data in the Philippines between 2000 and 2011. Of 253 relevant data sources identified, 34, including additional epidemiology data provided by the National Epidemiology Center, Department of Health, Philippines, were reviewed. There were 14 publications in peer reviewed journals, and 17 surveillance reports/sources, which provided variable information from the passive reporting system and show broad trends in dengue incidence, including age group predominance and disease severity. The peer reviewed studies focused on clinical severity of cases, some revealed data on circulating serotypes and genotypes and on the seroepidemiology of dengue including incidence rates for infection and apparent disease. Gaps in the data were identified, and include the absence incidence rates stratified by age, dengue serotype and genotype distribution, disease severity data, sex distribution data, and seroprevalence data.

Protocol registration

PROSPERO CRD42012002292     

Changes in JC Virus-Specific T Cell Responses during Natalizumab Treatment and in Natalizumab-Associated Progressive Multifocal Leukoencephalopathy

Progressive multifocal leukoencephalopathy (PML) induced by JC virus (JCV) is a risk for natalizumab-treated multiple sclerosis (MS) patients. Here we characterize the JCV-specific T cell responses in healthy donors and natalizumab-treated MS patients to reveal functional differences that may account for the development of natalizumab-associated PML. CD4 and CD8 T cell responses specific for all JCV proteins were readily identified in MS patients and healthy volunteers. The magnitude and quality of responses to JCV and cytomegalovirus (CMV) did not change from baseline through several months of natalizumab therapy. However, the frequency of T cells producing IL-10 upon mitogenic stimulation transiently increased after the first dose. In addition, MS patients with natalizumab-associated PML were distinguished from all other subjects in that they either had no detectable JCV-specific T cell response or had JCV-specific CD4 T cell responses uniquely dominated by IL-10 production. Additionally, IL-10 levels were higher in the CSF of individuals with recently diagnosed PML. Thus, natalizumab-treated MS patients with PML have absent or aberrant JCV-specific T cell responses compared with non-PML patients, and changes in T cell-mediated control of JCV replication may be a risk factor for developing PML. Our data suggest further approaches to improved monitoring, treatment and prevention of PML in natalizumab-treated patients.     

The interferon type I signature towards prediction of non-response to rituximab in rheumatoid arthritis patients

Introduction

B cell depletion therapy is efficacious in rheumatoid arthritis (RA) patients failing on tumor necrosis factor (TNF) blocking agents. However, approximately 40% to 50% of rituximab (RTX) treated RA patients have a poor response. We investigated whether baseline gene expression levels can discriminate between clinical non-responders and responders to RTX.

Methods

In 14 consecutive RA patients starting on RTX (test cohort), gene expression profiling on whole peripheral blood RNA was performed by Illumina^® HumanHT beadchip microarrays. Supervised cluster analysis was used to identify genes expressed differentially at baseline between responders and non-responders based on both a difference in 28 joints disease activity score (ΔDAS28 < 1.2) and European League against Rheumatism (EULAR) response criteria after six months RTX. Genes of interest were measured by quantitative real-time PCR and tested for their predictive value using receiver operating characteristics (ROC) curves in an independent validation cohort (n = 26).

Results

Genome-wide microarray analysis revealed a marked variation in the peripheral blood cells between RA patients before the start of RTX treatment. Here, we demonstrated that only a cluster consisting of interferon (IFN) type I network genes, represented by a set of IFN type I response genes (IRGs), that is, LY6E, HERC5, IFI44L, ISG15, MxA, MxB, EPSTI1 and RSAD2, was associated with ΔDAS28 and EULAR response outcome (P = 0.0074 and P = 0.0599, respectively). Based on the eight IRGs an IFN-score was calculated that reached an area under the curve (AUC) of 0.82 to separate non-responders from responders in an independent validation cohort of 26 patients using Receiver Operator Characteristics (ROC) curves analysis according to ΔDAS28 < 1.2 criteria. Advanced classifier analysis yielded a three IRG-set that reached an AUC of 87%. Comparable findings applied to EULAR non-response criteria.

Conclusions

This study demonstrates clinical utility for the use of baseline IRG expression levels as a predictive biomarker for non-response to RTX in RA.     

Inhibiting citrullination in rheumatoid arthritis: taking fuel from the fire

Citrullination is a post-translational modification catalysed by peptidylarginine deiminase and is a common feature of inflammation. The presence of anti-citrullinated protein/peptide antibodies (ACPA), however, is unique to rheumatoid arthritis. Several lines of evidence suggest that ACPA are important in the pathogenesis of rheumatoid arthritis. A popular hypothesis for this pathogenesis is a two-hit model. The first hit gives rise to ACPA, and the second hit, an unrelated episode of synovial inflammation accompanied by citrullination, is perpetuated by the pre-existing antibodies. This model suggests that reducing citrullination might ameliorate disease. Recent findings indicate that citrullination closely correlates with inflammation, and that glucocorticoids decrease peptidylarginine deiminase expression independent of their other anti-inflammatory effects.     

Evolution of outcrossing in experimental populations of Caenorhabditis elegans

Caenorhabditis elegans can reproduce exclusively by self-fertilization. Yet, males can be maintained in laboratory populations, a phenomenon that continues to puzzle biologists. In this study we evaluated the role of males in facilitating adaptation to novel environments. For this, we contrasted the evolution of a fitness component exclusive to outcrossing in experimental populations of different mating systems. We introgressed a modifier of outcrossing into a hybrid population derived from several wild isolates to transform the wild-type androdioecious mating system into a dioecious mating system. By genotyping 375 single-nucleotide polymorphisms we show that the two populations had similar standing genetic diversity available for adaptation, despite the occurrence of selection during their derivation. We then performed replicated experimental evolution under the two mating systems from starting conditions of either high or low levels of diversity, under defined environmental conditions of discrete non-overlapping generations, constant density at high population sizes (N = 10⁴), no obvious spatial structure and abundant food resources. During 100 generations measurements of sex ratios and male competitive performance showed: 1) adaptation to the novel environment; 2) directional selection on male frequency under androdioecy; 3) optimal outcrossing rates of 0.5 under androdioecy; 4) the existence of initial inbreeding depression; and finally 5) that the strength of directional selection on male competitive performance does not depend on male frequencies. Taken together, these results suggest that androdioecious males are maintained at intermediate frequencies because outcrossing is adaptive.     

Improved in vitro rooting of Prunus dulcis Mill. cultivars

A highly reproducible system was developed for efficient rooting of cultivars Boa Casta (BC) and Peneda and a BC seedling-derived clone (BC VII) of almond (Prunus dulcis Mill.). Twenty-four accessions derived from the clone BC VII and subjected to various in vitro culture treatments were screened. The long induction pre-treatment (LIP, 5 d), the brief induction pre-treatment (BIP, 16 h) and the hormonal shock by short dipping in hormone solution (1 min), were tested. BIP was the only that allowed rooting of cultivars. In BC VII, it induced high rooting frequencies (47–100 %) when using a solution of 0.4 mM indole-3-butyric acid solidified with 2 g dm⁻³ gellam gum for 16-h. The response to the auxin type was variable depending on the cultivar and the root induction pre-treatment used. Root number was significantly different between the two cultivars and BC VII. Root length was significantly higher when using 0.005 mM IBA in LIP but this concentration induced apical necrosis. The improved acclimatization procedure for up to 4 weeks increased the survival to 45 %. The initiation and development of adventitious roots were proved to be asynchronous.     

Colonization history, ecological shifts and diversification in the evolution of endemic Galápagos weevils

Mitochondrial DNA sequence data were obtained for eight species of flightless Galapaganus endemic weevils and one winged close relative in order to study their colonization history and modes of diversification in the Galápagos Archipelago. Contrary to most other insular radiations, the phylogeny estimates we recovered for Galapaganus do not follow the progression rule of island biogeography. The penalized likelihood age estimates of colonization of the archipelago exceed the age of the emerged islands and underscore the potential role of now sunken seamounts for the early evolution of Galapaganus . The phylogeny proposes one intra-island origin for Galapaganus endemics, but monophyly tests suggest a larger contribution of in-situ speciation on older islands. Generalist habitat preferences were reconstructed as ancestral while shifts to highland habitats were reconstructed as having evolved independently on different islands. Magnitudes and patterns of diversification rate were found to differ between older and younger islands. Our analyses reveal that the colonization sequence of islands and timing of colonization of Galapaganus could be linked with the geological and volcanic history of the islands in a rather complex scenario. Even though most islands appear to have been colonized soon after their emergence, there are notable deviations from the pattern of sequential colonization expected under the progression rule when considering only the extant emerged islands. Patterns of diversification rate variation on older and younger islands correspond to the volcanic activity or remnants of such activity, while the pattern of independent evolution of restricted habitat preferences in different islands suggests that habitat shifts could also have contributed to species diversity in Galapaganus .