Deletional Analysis of the rod Photoreceptor Cell Peripherin/RDS Carboxy-Terminal Region

The C-terminal region of peripherin/rds contains three predicted α-helical domains. One of these domains, corresponding to amino acids 311-322, form an amphiphilic α-helix previously shown to promote membrane fusion. The present studies were conducted to determine how the additional α-helical regions of the peripherin/rds C-terminus affect complex formation with rom-1, glycosylation, intracellular localization and membrane fusion properties. Bovine peripherin/rds and rom-1 were epitope tagged with an amino-terminal FLAG-tag or amino-terminal hemagglutinin (HA)-tag, respectively, and cloned into the pCI-neo expression vector for transient transfection into COS cells. Similarly, four C-terminal peripherin/rds truncation mutants (Δ1, Δ2, Δ3 and Δ4), corresponding to deletions of -19, -29, -39 and -59 amino acids were designed to disrupt the α-helical domains. Immunofluorescence microscopy and enzymatic digestions demonstrated that full-length peripherin/rds and the four C-terminal deletion mutants were localized to intracellular membranes and were all Endo-H sensitive. Western blotting and immunoprecipitation studies showed that the FLAG-tagged bovine peripherin/rds (full-length) was expressed as a 76 kDa dimer, which associates with HA-tagged rom-1 to form a higher order complex. The deletion mutants were also able to associate with rom-1. However, when analyzed using non-denaturing tricine electrophoresis, full-length peripherin/rds and the Δ1, Δ2 and Δ3 mutants formed homo-oligomeric complexes, while the Δ4 mutant appeared to form only homodimers suggesting a region upstream of amino acid 300 may be involved in C-terminal interactions. Membrane fusion was then evaluated using fluorescence resonance energy transfer (RET) techniques. Intracellular COS cell membranes containing full-length peripherin/rds fused with rod outer segment plasma membrane vesicles. This fusion was inhibited with the addition of a synthetic peptide (PP-5) corresponding to the fusion domain of peripherin/rds. In contrast, fusion was negligible with any of the C-terminal truncation mutants. Collectively, these results suggest that in addition to the fusion domain, other regions of the peripherin/rds C-terminus are required for fusion. Most interesting is the observation that the last 19 amino acids, a region downstream of the fusion peptide that is deleted in the Δ1 mutant, appear to be necessary for fusion. This region corresponds to the epitope for anti-peripherin/rds monoclonal antibody 2B6, which is shown to partially inhibit peripherin/rds mediated membrane fusion. © 2002 Elsevier Science Ltd

Deletional Analysis of the Rod Photoreceptor Cell Peripherin/RDS Carboxy-Terminal Region

The C-terminal region of peripherin/rds contains three predicted α-helical domains. One of these domains, corresponding to amino acids 311–322, form an amphiphilic α-helix previously shown to promote membrane fusion. The present studies were conducted to determine how the additional α-helical regions of the peripherin/rds C-terminus affect complex formation with rom-1, glycosylation, intracellular localization and membrane fusion properties. Bovine peripherin/rds and rom-1 were epitope tagged with an amino-terminal FLAG-tag or amino-terminal hemagglutinin (HA)-tag, respectively, and cloned into the pCI-neo expression vector for transient transfection into COS cells. Similarly, four C-terminal peripherin/rds truncation mutants (Δ1, Δ2, Δ3 and Δ4), corresponding to deletions of −19, −29, −39 and −59 amino acids were designed to disrupt the α-helical domains. Immunofluorescence microscopy and enzymatic digestions demonstrated that full-length peripherin/rds and the four C-terminal deletion mutants were localized to intracellular membranes and were all Endo-H sensitive. Western blotting and immunoprecipitation studies showed that the FLAG-tagged bovine peripherin/rds (full-length) was expressed as a 76 kDa dimer, which associates with HA-tagged rom-1 to form a higher order complex. The deletion mutants were also able to associate with rom-1. However, when analyzed using non-denaturing tricine electrophoresis, full-length peripherin/rds and the Δ1, Δ2 and Δ3 mutants formed homo-oligomeric complexes, while the Δ4 mutant appeared to form only homodimers suggesting a region upstream of amino acid 300 may be involved in C-terminal interactions. Membrane fusion was then evaluated using fluorescence resonance energy transfer (RET) techniques. Intracellular COS cell membranes containing full-length peripherin/rds fused with rod outer segment plasma membrane vesicles. This fusion was inhibited with the addition of a synthetic peptide (PP-5) corresponding to the fusion domain of peripherin/rds. In contrast, fusion was negligible with any of the C-terminal truncation mutants. Collectively, these results suggest that in addition to the fusion domain, other regions of the peripherin/rds C-terminus are required for fusion. Most interesting is the observation that the last 19 amino acids, a region downstream of the fusion peptide that is deleted in the Δ1 mutant, appear to be necessary for fusion. This region corresponds to the epitope for anti-peripherin/ rds monoclonal antibody 2B6, which is shown to partially inhibit peripherin/rds mediated membrane fusion

Steps towards collective sustainability in biomedical research

The optimism surrounding multistakeholder research initiatives does not match the clear view of policies that are needed to exploit the potential of these collaborations. Here we propose some action items that stem from the integration between research advancements with the perspectives of patient-advocacy organizations, academia, and industry

ROM-1 Potentiates Photoreceptor Specific Membrane Fusion Processes

Photoreceptor outer segment (OS) renewal requires a series of tightly regulated membrane fusion events which are mediated by a fusion complex containing protein and lipid components. The best characterized of these components, is a unique photoreceptor specific tetraspanin, peripherin/rds (P/rds, a.k.a., peripherin-2, Rds and Prph). In these studies we investigated the role of peripherin\u27s non-glycosylated homolog, ROM-1, in OS fusion using a COS cell heterologous expression system and a well characterized cell free fusion assay system. Membranes isolated from COS-7 cells transfected with either FLAG-tagged P/rds or HA-tagged ROM-1 or both proteins were assayed for their ability to merge with fluorescently labeled OS plasma membrane (PM). Such membrane merger is one measure of membrane fusogenicity. The highest percent fusion was observed when the proteins were co-expressed. Furthermore detailed analysis of the fusion kinetics between fluorescently labeled PM and proteo-liposomes containing either, pure P/rds, pure ROM-1 or the ROM-1-P/rds complex clearly demonstrated that optimal fusion requires an ROM-1/P/rds complex. Proteo-liposomes composed of ROM-1 alone were not fusogenic. Peptide competition studies suggest that optimization of fusion may be due to the formation of a fusion competent peripherin/rds C-terminus in the presence of ROM-1. These studies provide further support for the hypothesis that a P/rds dependent membrane fusion complex is involved in photoreceptor renewal processes. © 2006 Elsevier Ltd. All rights reserved

ROM-1 Potentiates Photoreceptor Specific Membrane Fusion Processes

Photoreceptor outer segment (OS) renewal requires a series of tightly regulated membrane fusion events which are mediated by a fusion complex containing protein and lipid components. The best characterized of these components, is a unique photoreceptor specific tetraspanin, peripherin/rds (P/rds, a.k.a., peripherin-2, Rds and Prph). In these studies we investigated the role of peripherin’s non-glycosylated homolog, ROM-1, in OS fusion using a COS cell heterologous expression system and a well characterized cell free fusion assay system. Membranes isolated from COS-7 cells transfected with either FLAG-tagged P/rds or HA-tagged ROM-1 or both proteins were assayed for their ability to merge with fluorescently labeled OS plasma membrane (PM). Such membrane merger is one measure of membrane fusogenicity. The highest percent fusion was observed when the proteins were co-expressed. Furthermore detailed analysis of the fusion kinetics between fluorescently labeled PM and proteo-liposomes containing either, pure P/rds, pure ROM-1 or the ROM-1-P/rds complex clearly demonstrated that optimal fusion requires an ROM-P/rds1 complex. Proteo-liposomes composed of ROM-1 alone were not fusogenic. Peptide competition studies suggest that optimization of fusion may be due to the formation of a fusion competent peripherin/rds C-terminus in the presence of ROM-1. These studies provide further support for the hypothesis that a P/rds dependent membrane fusion complex is involved in photoreceptor renewal processes

Strong interlayer coupling in van der Waals heterostructures built from single-layer chalcogenides

Semiconductor heterostructures are the fundamental platform for many important device applications such as lasers, light-emitting diodes, solar cells and high-electron-mobility transistors. Analogous to traditional heterostructures, layered transition metal dichalcogenide (TMDC) heterostructures can be designed and built by assembling individual single-layers into functional multilayer structures, but in principle with atomically sharp interfaces, no interdiffusion of atoms, digitally controlled layered components and no lattice parameter constraints. Nonetheless, the optoelectronic behavior of this new type of van der Waals (vdW) semiconductor heterostructure is unknown at the single-layer limit. Specifically, it is experimentally unknown whether the optical transitions will be spatially direct or indirect in such hetero-bilayers. Here, we investigate artificial semiconductor heterostructures built from single layer WSe2 and MoS2 building blocks. We observe a large Stokes-like shift of ~100 meV between the photoluminescence peak and the lowest absorption peak that is consistent with a type II band alignment with spatially direct absorption but spatially indirect emission. Notably, the photoluminescence intensity of this spatially indirect transition is strong, suggesting strong interlayer coupling of charge carriers. The coupling at the hetero-interface can be readily tuned by inserting hexagonal BN (h-BN) dielectric layers into the vdW gap. The generic nature of this interlayer coupling consequently provides a new degree of freedom in band engineering and is expected to yield a new family of semiconductor heterostructures having tunable optoelectronic properties with customized composite layers.Comment: http://www.pnas.org/content/early/2014/04/10/1405435111.abstrac

Efficacité de biofilms de bactéries As-oxydantes pour l'étape de traitement biologique d'eaux potabilisables arséniées

L'arsenic est un métalloïde toxique dont la présence, relativement fréquente, dans les eaux et les sols est liée soit au fond géochimique, soit aux activités humaines. En ce qui concerne les eaux destinées à la consommation, la législation impose une concentration maximale en arsenic de 10 µg.L-1. Les effets nocifs de l'arsenic sur la santé humaine rendent nécessaire le développement de technologies efficaces et peu couteuse pour éliminer cet élément des eaux potables, ainsi que dans les aquifères pollués et dans les effluents miniers (Wang et Zhao, 2009). Une unité de traitement biologique d'eaux potabilisable faiblement arséniée (As< 50µg/L), couplée à une unité de piégeage de l'As en sortie du bioréacteur, a été mise en œuvre sur un site réel afin d'étudier la robustesse du bioprocédé. Un bioréacteur contenant de la pouzzolane (matériau utilisé dans les traitements d'eaux) a été préalablement ensemencé par une souche bactérienne As(III) oxydante autotrophe (Thiomonas arsenivorans) (Battaglia-Brunet et al., 2002, Michon et al., 2010 ; Wan et al., 2010) puis alimenté par l'eau issue du forage à température ambiante (15-17°C) avec un fonctionnement discontinu (asservissement de l'alimentation du bioréacteur à la pompe du forage d'alimentation en eau). Le suivi du développement du biofilm As(III) oxydant au cours du traitement biologique a été réalisé par la recherche des gènes codant pour l'ARNr 16S (diversité bactérienne totale) et ceux codant pour une arsénite oxydase (aoxB) (diversité des bactéries As(III)-oxydantes). Ce suivi a montré une colonisation rapide et stable du support minéral par des bactéries endogènes de l'eau à traiter. Le rendement d'oxydation de l'étape d'oxydation biologique est compris entre 54 et 100 % avec des temps de séjour de 30 minutes à 7 minutes qui sont comparables à des temps de séjour de techniques classiques de traitement. Les concentrations résiduelles en As en sortie du procédé complet (oxydation biologique + piégeage) sont inférieures à 1 µg/L, et qui sont donc très encourageants pour une application industrielle

Forced labor risk is pervasive in the US land-based food supply

Social risk assessments and case studies of labor conditions in food production primarily focus on specific subpopulations, regions, and commodities. To date, research has not systematically assessed labor conditions against international standards across diverse, complex food products. Here, we combine data on production, trade, labor intensity, and qualitative risk coding to quantitatively assess the risk of forced labor embedded in the U.S. land-based food supply, building upon our previous assessment of fruits and vegetables. We demonstrate that animal-based proteins, processed fruits and vegetables, and discretionary foods are major contributors to forced labor risk and that 62% of total forced labor risk stems from domestic production or processing. Our findings reveal the widespread risk of forced labor present in the US food supply and the necessity of collaborative action across all countries – high, middle, and low income - to eliminate reliance on labor exploitation