High affinity single-chain variable fragments are specific and versatile targeting motifs for extracellular vesicles

Exosomes are extracellular vesicles that mediate cell-to-cell communication by transferring biological cargo, such as DNA, RNA and proteins. Through genetic engineering of exosome-producing cells or manipulation of purified exosomes, it is possible to load exosomes with therapeutic molecules and target them to specific cells via the display of targeting moieties on their surface. This provides an opportunity to exploit a naturally-occurring biological process for therapeutic purposes. In this study, we explored the potential of single chain variable fragments (scFv) as targeting domains to achieve delivery of exosomes to cells expressing a cognate antigen. We generated exosomes targeting the Her2 receptor and, by varying the affinity of the scFvs and the Her2 expression level on recipient cells, we determined that both a high-affinity anti-Her2-scFv (KD ≤ 1 nM) and cells expressing a high level (≥106 copies per cell) of Her2 were optimally required to enable selective uptake. We also demonstrate that targeting exosomes to cells via a specific cell surface receptor can alter their intracellular trafficking route, providing opportunities to influence the efficiency of delivery and fate of intracellular cargo. These experiments provide solid data to support the wider application of exosomes displaying antibody fragments as vehicles for the targeted delivery of therapeutic molecules

Do red deer stags (Cervus elaphus) use roar fundamental frequency (F0) to assess rivals?

It is well established that in humans, male voices are disproportionately lower pitched than female voices, and recent studies suggest that this dimorphism in fundamental frequency (F0) results from both intrasexual (male competition) and intersexual (female mate choice) selection for lower pitched voices in men. However, comparative investigations indicate that sexual dimorphism in F0 is not universal in terrestrial mammals. In the highly polygynous and sexually dimorphic Scottish red deer Cervus elaphus scoticus, more successful males give sexually-selected calls (roars) with higher minimum F0s, suggesting that high, rather than low F0s advertise quality in this subspecies. While playback experiments demonstrated that oestrous females prefer higher pitched roars, the potential role of roar F0 in male competition remains untested. Here we examined the response of rutting red deer stags to playbacks of re-synthesized male roars with different median F0s. Our results show that stags’ responses (latencies and durations of attention, vocal and approach responses) were not affected by the F0 of the roar. This suggests that intrasexual selection is unlikely to strongly influence the evolution of roar F0 in Scottish red deer stags, and illustrates how the F0 of terrestrial mammal vocal sexual signals may be subject to different selection pressures across species. Further investigations on species characterized by different F0 profiles are needed to provide a comparative background for evolutionary interpretations of sex differences in mammalian vocalizations

Characterisation of recombinant rat TRPM2 and a TRPM2-like conductance in cultured rat striatal neurons

Original article can be found at: http://www.sciencedirect.com/science/journal/00283908 Copyright Elsevier Ltd. DOI: 10.1016/j.neuropharm.2005.08.021 [Full text of this article is not available in the UHRA]TRPM2, a member of the TRP ion channel family, is expressed both in the brain and immune cells of the monocyte lineage. Functionally, it is unique in its activation by intracellular ADP-ribose and both oxidative and nitrosative stress. To date studies of this channel have concentrated on human recombinant channels and rodent native preparations. This provides the potential for cross-species complications in the interpretation of native tissue observations based on recombinant channel phenotype. Consequently, we have cloned and heterologously expressed rat TRPM2 (rTRPM2) in HEK293 cells. We find that, like hTRPM2, it responds to intracellular ADP-ribose in a manner dependent on extracellular Ca2+. At the single channel level rTRPM2 is a slow gating, large conductance (84 pS) channel that rapidly runs down in isolated membrane patches. Pharmacologically, rTRPM2 is rapidly and irreversibly blocked by clotrimazole (10 μM), thus resembling hTRPM2 but not the TRPM2-like current of the rat-derived insulinoma CRI-G1, which exhibits reversible inhibition by this agent. We show that cultured rat striatal neurones exhibit an ADP-ribose-activated conductance at both the whole cell and single channel level. Pharmacologically this neuronal current can be irreversibly inhibited by clotrimazole. It is also sensitive to removal of extracellular Ca2+, suggesting that it is mediated by TRPM2-containing channels. These data provide a functional characterisation of heterologously expressed rTRPM2 and demonstrate that, in addition to the previous descriptions in immune cells, microglia and insulinomas, a TRPM2-like conductance can be found in neurones derived from the rodent CNS.Peer reviewe

Holistic needs assessment and care plans for women with gynaecological cancer: do they improve cancer-specific health-related quality of life? A randomised controlled trial using mixed methods

Holistic needs assessment (HNA) and care planning is proposed to address unmet needs of people treated for cancer. We tested whether HNA and care planning by an allied health professional improved cancer-specific quality of life for women following curative treatment for stage I-III gynaecological cancer. Methods Consecutive women were invited to participate in a randomised controlled study (HNA and care planning vs. usual care) at a UK cancer centre. Data were collected by questionnaire at baseline, three and six months. The outcomes were six month change in EORTC-QLQ-C30 global score (primary), and in EORTC sub-scales, generic quality of life, self-efficacy (secondary). The study was blinded for data management and analysis. Differences in outcomes were compared between groups. Health service utilisation and Quality Adjusted Life Years (from SF-6) were gathered for a cost-effectiveness analysis. Thematic analysis was used to interpret data from an exit interview. Results 150 women consented (75 per group), ten undertook interviews. For 124 participants (61 intervention, 63 controls) with complete data, no statistically significant differences were seen between groups in the primary end-point. The majority of those interviewed reported important personal gains they attributed to the intervention which reflected trends to improvement seen in EORTC functional and symptom scales. Economic analysis suggests a 62% probability of cost-effectiveness at a £30,000/QALY threshold. Conclusion: Care plan development with an allied health professional is cost-effective, acceptable and useful for some women treated for stage 1-111 gynaecological cancer. We recommend its introduction early in the pathway to support person-centred care

Holistic needs assessment and care plans for women with gynaecological cancer: do they improve cancer-specific health-related quality of life? A randomised controlled trial using mixed methods

ObjectivesHolistic needs assessment (HNA) and care planning are proposed to address unmet needs of people treated for cancer. We tested whether HNA and care planning by an allied health professional improved cancer-specific quality of life for women following curative treatment for stage I–III gynaecological cancer.MethodsConsecutive women were invited to participate in a randomised controlled study (HNA and care planning vs usual care) at a UK cancer centre. Data were collected by questionnaire at baseline, 3 and 6 months. The outcomes were 6-month change in European Organisation for Research and Treatment of Cancer (EORTC) Quality of Life Questionnaire-C30 (version 3), global score (primary) and, in EORTC subscales, generic quality of life and self-efficacy (secondary). The study was blinded for data management and analysis. Differences in outcomes were compared between groups. Health service utilisation and quality-adjusted life years (QALY) (from Short Form-6) were gathered for a cost-effectiveness analysis. Thematic analysis was used to interpret data from an exit interview.Results150 women consented (75 per group); 10 undertook interviews. For 124 participants (61 intervention, 63 controls) with complete data, no statistically significant differences were seen between groups in the primary endpoint. The majority of those interviewed reported important personal gains they attributed to the intervention, which reflected trends to improvement seen in EORTC functional and symptom scales. Economic analysis suggests a 62% probability of cost-effectiveness at a £30 000/QALY threshold.ConclusionCare plan development with an allied health professional is cost-effective, acceptable and useful for some women treated for stage I–III gynaecological cancer. We recommend its introduction early in the pathway to support person-centred care.</jats:sec

From peptides to proteins: coiled-coil tetramers to single-chain 4-helix bundles

The design of completely synthetic proteins from first principles—de novo protein design—is challenging. This is because, despite recent advances in computational protein–structure prediction and design, we do not understand fully the sequence-to-structure relationships for protein folding, assembly, and stabilization. Antiparallel 4-helix bundles are amongst the most studied scaffolds for de novo protein design. We set out to re-examine this target, and to determine clear sequence-to-structure relationships, or design rules, for the structure. Our aim was to determine a common and robust sequence background for designing multiple de novo 4-helix bundles. In turn, this could be used in chemical and synthetic biology to direct protein–protein interactions and as scaffolds for functional protein design. Our approach starts by analyzing known antiparallel 4-helix coiled-coil structures to deduce design rules. In terms of the heptad repeat, abcdefg—i.e., the sequence signature of many helical bundles—the key features that we identify are: a = Leu, d = Ile, e = Ala, g = Gln, and the use of complementary charged residues at b and c. Next, we implement these rules in the rational design of synthetic peptides to form antiparallel homo- and heterotetramers. Finally, we use the sequence of the homotetramer to derive in one step a single-chain 4-helix-bundle protein for recombinant production in E. coli. All of the assembled designs are confirmed in aqueous solution using biophysical methods, and ultimately by determining high-resolution X-ray crystal structures. Our route from peptides to proteins provides an understanding of the role of each residue in each design