One universal common endpoint in mouse models of amyotrophic lateral sclerosis

There is no consensus among research laboratories around the world on the criteria that define endpoint in studies involving rodent models of amyotrophic lateral sclerosis (ALS). Data from 4 nutrition intervention studies using 162 G93A mice, a model of ALS, were analyzed to determine if differences exist between the following endpoint criteria: CS 4 (functional paralysis of both hindlimbs), CS 4+ (CS 4 in addition to the earliest age of body weight loss, body condition deterioration or righting reflex), and CS 5 (CS 4 plus righting reflex >20 s). The age (d; mean ± SD) at which mice reached endpoint was recorded as the unit of measurement. Mice reached CS 4 at 123.9±10.3 d, CS 4+ at 126.6±9.8 d and CS 5 at 127.6±9.8 d, all significantly different from each other (P<0.001). There was a significant positive correlation between CS 4 and CS 5 (r = 0.95, P<0.001), CS 4 and CS 4+ (r = 0.96, P<0.001), and CS 4+ and CS 5 (r = 0.98, P<0.001), with the Bland-Altman plot showing an acceptable bias between all endpoints. Logrank tests showed that mice reached CS 4 24% and 34% faster than CS 4+ (P = 0.046) and CS 5 (P = 0.006), respectively. Adopting CS 4 as endpoint would spare a mouse an average of 4 days (P<0.001) from further neuromuscular disability and poor quality of life compared to CS 5. Alternatively, CS 5 provides information regarding proprioception and severe motor neuron death, both could be important parameters in establishing the efficacy of specific treatments. Converging ethics and discovery, would adopting CS 4 as endpoint compromise the acquisition of insight about the effects of interventions in animal models of ALS?     

Human disturbance influences reproductive success and growth rate in California sea lions (Zalophus californianus)

The environment is currently undergoing changes at both global (e.g., climate change) and local (e.g., tourism, pollution, habitat modification) scales that have the capacity to affect the viability of animal and plant populations. Many of these changes, such as human disturbance, have an anthropogenic origin and therefore may be mitigated by management action. To do so requires an understanding of the impact of human activities and changing environmental conditions on population dynamics. We investigated the influence of human activity on important life history parameters (reproductive rate, and body condition, and growth rate of neonate pups) for California sea lions (Zalophus californianus) in the Gulf of California, Mexico. Increased human presence was associated with lower reproductive rates, which translated into reduced long-term population growth rates and suggested that human activities are a disturbance that could lead to population declines. We also observed higher body growth rates in pups with increased exposure to humans. Increased growth rates in pups may reflect a density dependent response to declining reproductive rates (e.g., decreased competition for resources). Our results highlight the potentially complex changes in life history parameters that may result from human disturbance, and their implication for population dynamics. We recommend careful monitoring of human activities in the Gulf of California and emphasize the importance of management strategies that explicitly consider the potential impact of human activities such as ecotourism on vertebrate populations.     

A "mesmer"izing new approach to site-directed mutagenesis in large transformation-ready constructs: Mutagenesis via Serial Small Mismatch Recombineering

Creating designer mutations in large genes is a challenge. Size limitations imposed by site-directed mutagenesis (SDM), coupled with the paucity of unique restriction enzyme sites, make subsequent cloning of these constructs extremely difficult. "Mutagenesis via Serial Small Mismatch Recombineering" (MSSMR) combines sequential recombineering steps with SDM to create seamless, pre-specified mutations as small as a single base pair. We demonstrate the simultaneous cloning of wild type and mutant constructs of a > 30 kb gene directly into attB transformation vectors. No post-transformation manipulations are required, and because the technique relies on recombineering methods, addition of undesired mutations via PCR is minimized.     

An unexpected recurrent transmission of Rift Valley fever virus in cattle in a temperate and mountainous area of Madagascar

Rift Valley fever is an acute, zoonotic viral disease of domestic ruminants, caused by a phlebovirus (Bunyaviridae family). A large outbreak occurred in Madagascar in 2008-2009. The goal of the present study was to evaluate the point prevalence of antibodies against Rift Valley Fever Virus (RVFV) in cattle in the Anjozorobe district, located in the wet and temperate highland region of Madagascar and yet heavily affected by the disease, and analyse environmental and trade factors potentially linked to RVFV transmission. A serological study was performed in 2009 in 894 bovines. For each bovine, the following variables were recorded: age, location of the night pen, minimum distance from the pen to the nearest water point and the forest, nearest water point type, and herd replacement practices. The serological data were analyzed using a generalized linear mixed model. The overall anti-RVFV IgG seroprevalence rate was 28% [CI95% 25-31]. Age was statistically linked to prevalence (p = 10(-4)), being consistent with a recurrent RVFV circulation. Distance from the night pen to the nearest water point was a protective factor (p = 5.10(-3)), which would be compatible with a substantial part of the virus transmission being carried out by nocturnal mosquito vectors. However, water point type did not influence the risk of infection: several mosquito species are probably involved. Cattle belonging to owners who purchase animals to renew the herd were significantly more likely to have seroconverted than others (p = 0.04): cattle trade may contribute to the introduction of the virus in this area. The minimum distance of the night pen to the forest was not linked to the prevalence. This is the first evidence of a recurrent transmission of RVFV in such an ecosystem that associates a wet, temperate climate, high altitude, paddy fields, and vicinity to a dense rain forest. Persistence mechanisms need to be further investigated.     

Tet1 and Tet2 regulate 5-hydroxymethylcytosine production and cell lineage specification in mouse embryonic stem cells

TET family enzymes convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. Here, we show that Tet1 and Tet2 are Oct4-regulated enzymes that together sustain 5hmC in mouse embryonic stem cells (ESCs) and are induced concomitantly with 5hmC during reprogramming of fibroblasts to induced pluripotent stem cells. ESCs depleted of Tet1 by RNAi show diminished expression of the Nodal antagonist Lefty1 and display hyperactive Nodal signaling and skewed differentiation into the endoderm-mesoderm lineage in embryoid bodies in?vitro. In Fgf4- and heparin-supplemented culture conditions, Tet1-depleted ESCs activate the trophoblast stem cell lineage determinant Elf5 and can colonize the placenta in midgestation embryo chimeras. Consistent with these findings, Tet1-depleted ESCs?form aggressive hemorrhagic teratomas with increased endoderm, reduced neuroectoderm, and ectopic appearance of trophoblastic giant cells. Thus, 5hmC is an epigenetic modification associated with the pluripotent state, and Tet1 functions to regulate the lineage differentiation potential of ESCs.     

Integrated dataset of screening hits against multiple neglected disease pathogens

New chemical entities are desperately needed that overcome the limitations of existing drugs for neglected diseases. Screening a diverse library of 10,000 drug-like compounds against 7 neglected disease pathogens resulted in an integrated dataset of 744 hits. We discuss the prioritization of these hits for each pathogen and the strong correlation observed between compounds active against more than two pathogens and mammalian cell toxicity. Our work suggests that the efficiency of early drug discovery for neglected diseases can be enhanced through a collaborative, multi-pathogen approach.     

Selection of a CXCR4 antagonist from a human heavy chain CDR3-derived phage library

Phage display technology is a powerful selection approach to identify strong and specific binders to a large variety of targets. In this study, we compared the efficacy of a phage library displaying human heavy chain complementarity determining region 3 (HCDR3) repertoires with a set of conventional random peptide libraries for the identification of CXCR4 antagonists using a peptide corresponding to the second extracellular loop of the receptor CXCR4 as target. A total of 11 selection campaigns on this target did not result in any specific ligand from the random peptide libraries. In contrast, a single selection campaign with an HCDR3 library derived from the IgM repertoire of a nonimmunized donor resulted in nine specific peptides with lengths ranging from 10 to 19 residues. Four of these HCDR3 sequences interacted with native receptor and the most frequently isolated peptide displayed an affinity of 5.6 μm and acted as a CXCR4 antagonist (IC(50) = 23 μm). To comprehend the basis of the highly efficient HCDR3 library selection, its biochemical properties were investigated. The HCDR3 length varied from 3 to 21 residues and displayed a biased amino acid content with a predominant proportion of Tyr, Gly, Ser and Asp. Repetitive and conserved motifs were observed in the majority of the HCDR3 sequences. The strength and efficacy of the HCDR3 libraries reside in the combination of multiple size peptides and a naturally biased sequence variation. Therefore, HCDR3 libraries represent a powerful and versatile alternative to fully randomized peptide libraries, in particular for difficult targets.     

Methodological reflections on using pilot data from fracture patients to develop a qualitative study

Background

Qualitative studies are particularly valued for their exploratory nature but, like other research methods, they do require careful planning to ensure rigorous study design. Our objective was to undertake a pilot study to inform the development of a larger qualitative study.

Results

We conducted a series of brief interviews with out-patients in a hospital setting. The interviews were designed to elicit superficial information about whether (and how) post-fracture osteoporosis investigation and/or treatment were being initiated among patients receiving treatment or follow-up for a current or recent fracture. We used thematic analysis to identify key themes in the data that related to the broader research questions.We analysed data obtained from 11 out of a total of 12 interviews conducted. Participants were male and female, aged 19-83 years of age (median age 57 years). Participants attended 2-8 medical appointments to seek treatment and follow up for a current or recent fracture. The following four overarching themes emerged from thematic analysis of the data: fracture event, referral pathway, osteoporosis investigation and/or treatment, and communication by health practitioners and staff.

Conclusions

This pilot study was necessarily tentative and exploratory in nature, but provided a helpful snapshot of some typical experiences in the public health system following fracture. Several themes emerged for consideration in the design of the main study.Despite its critics, theoretical sampling and saturation continue to provide sustainable methods for ensuring that relevant themes and categories are covered in sufficient depth and breadth, appropriate to the needs of the study.

     

Oligodendrocyte progenitor cells proliferate and survive in an immature state following treatment with an axolemma-enriched fraction

The ability of an AEF (axolemma-enriched fraction) to influence the proliferation, survival and differentiation of OPC (oligodendrocyte progenitor cells) was evaluated. Following addition of AEF to cultured OPC, the AEF associated with the outer surface of OPC so that subsequent metabolic events were likely mediated by direct AEF-OPC contact. Addition of AEF to the cultured OPC resulted in a dose- and time-dependent increase in proliferation that was partially dependent on Akt (protein kinase B) and MAPK (mitogen-activated protein kinase) activation. The major mitogen in an AEF-SE (soluble 2.0 M NaCl extract of the AEF) was identified as aFGF (acidic fibroblast growth factor) and accounted for 50% of the mitogenicity. The remaining 50% of the mitogenicity had properties consistent with bFGF (basic fibroblast growth factor) but was not unequivocally identified. Under conditions that limit the survival of OPC in culture, AEF treatment prolonged the survival of the OPC. Antigenic and morphological examination of the AEF-treated OPC indicated that the AEF treatment helped the OPC survive in a more immature state. The potential downstream metabolic pathways potentially activated in OPC by AEF and the consequences of these activated pathways are discussed. The results of these studies are consistent with the view that direct contact of axons with OPC stimulates their proliferation and survival while preventing their differentiation.     

Fast and easy protocol for the purification of recombinant S-layer protein for synthetic biology applications

A goal of synthetic biology is to make biological systems easier to engineer. One of the aims is to design, with nanometer-scale precision, biomaterials with well-defined properties. The surface-layer protein SbpA forms 2D arrays naturally on the cell surface of Lysinibacillus sphaericus, but also as the purified protein in solution upon the addition of divalent cations. The high propensity of SbpA to form crystalline arrays, which can be simply controlled by divalent cations, and the possibility to genetically alter the protein, make SbpA an attractive molecule for synthetic biology. To be a useful tool, however, it is important that a simple protocol can be used to produce recombinant wild-type and modified SbpA in large quantities and in a biologically active form. The present study addresses this requirement by introducing a mild and non-denaturing purification protocol to produce milligram quantities of recombinant, active SbpA.