How a Diverse Research Ecosystem Has Generated New Rehabilitation Technologies: Review of NIDILRR’s Rehabilitation Engineering Research Centers

Over 50 million United States citizens (1 in 6 people in the US) have a developmental, acquired, or degenerative disability. The average US citizen can expect to live 20% of his or her life with a disability. Rehabilitation technologies play a major role in improving the quality of life for people with a disability, yet widespread and highly challenging needs remain. Within the US, a major effort aimed at the creation and evaluation of rehabilitation technology has been the Rehabilitation Engineering Research Centers (RERCs) sponsored by the National Institute on Disability, Independent Living, and Rehabilitation Research. As envisioned at their conception by a panel of the National Academy of Science in 1970, these centers were intended to take a “total approach to rehabilitation”, combining medicine, engineering, and related science, to improve the quality of life of individuals with a disability. Here, we review the scope, achievements, and ongoing projects of an unbiased sample of 19 currently active or recently terminated RERCs. Specifically, for each center, we briefly explain the needs it targets, summarize key historical advances, identify emerging innovations, and consider future directions. Our assessment from this review is that the RERC program indeed involves a multidisciplinary approach, with 36 professional fields involved, although 70% of research and development staff are in engineering fields, 23% in clinical fields, and only 7% in basic science fields; significantly, 11% of the professional staff have a disability related to their research. We observe that the RERC program has substantially diversified the scope of its work since the 1970’s, addressing more types of disabilities using more technologies, and, in particular, often now focusing on information technologies. RERC work also now often views users as integrated into an interdependent society through technologies that both people with and without disabilities co-use (such as the internet, wireless communication, and architecture). In addition, RERC research has evolved to view users as able at improving outcomes through learning, exercise, and plasticity (rather than being static), which can be optimally timed. We provide examples of rehabilitation technology innovation produced by the RERCs that illustrate this increasingly diversifying scope and evolving perspective. We conclude by discussing growth opportunities and possible future directions of the RERC program

Conserved determinants of lentiviral genome dimerization

Abstract Background Retroviruses selectively package two copies of their unspliced genomes by what appears to be a dimerization-dependent RNA packaging mechanism. Dimerization of human immunodeficiency virus Type-1 (HIV-1) genomes is initiated by “kissing” interactions between GC-rich palindromic loop residues of a conserved hairpin (DIS), and is indirectly promoted by long-range base pairing between residues overlapping the gag start codon (AUG) and an upstream Unique 5′ element (U5). The DIS and U5:AUG structures are phylogenetically conserved among divergent retroviruses, suggesting conserved functions. However, some studies suggest that the DIS of HIV-2 does not participate in dimerization, and that U5:AUG pairing inhibits, rather than promotes, genome dimerization. We prepared RNAs corresponding to native and mutant forms of the 5′ leaders of HIV-1 (NL4-3 strain), HIV-2 (ROD strain), and two divergent strains of simian immunodeficiency virus (SIV; cpz-TAN1 and -US strains), and probed for potential roles of the DIS and U5:AUG base pairing on intrinsic and NC-dependent dimerization by mutagenesis, gel electrophoresis, and NMR spectroscopy. Results Dimeric forms of the native HIV-2 and SIV leaders were only detectable using running buffers that contained Mg2+, indicating that these dimers are more labile than that of the HIV-1 leader. Mutations designed to promote U5:AUG base pairing promoted dimerization of the HIV-2 and SIV RNAs, whereas mutations that prevented U5:AUG pairing inhibited dimerization. Chimeric HIV-2 and SIV leader RNAs containing the dimer-promoting loop of HIV-1 (DIS) exhibited HIV-1 leader-like dimerization properties, whereas an HIV-1NL4-3 mutant containing the SIVcpzTAN1 DIS loop behaved like the SIVcpzTAN1 leader. The cognate NC proteins exhibited varying abilities to promote dimerization of the retroviral leader RNAs, but none were able to convert labile dimers to non-labile dimers. Conclusions The finding that U5:AUG formation promotes dimerization of the full-length HIV-1, HIV-2, SIVcpzUS, and SIVcpzTAN1 5′ leaders suggests that these retroviruses utilize a common RNA structural switch mechanism to modulate function. Differences in native and NC-dependent dimerization propensity and lability are due to variations in the compositions of the DIS loop residues rather than other sequences within the leader RNAs. Although NC is a well-known RNA chaperone, its role in dimerization has the hallmarks of a classical riboswitch.http://deepblue.lib.umich.edu/bitstream/2027.42/113284/1/12977_2015_Article_209.pd

Suppression of the Macrophage Proteasome by Ethanol Impairs MHC Class I Antigen Processing and Presentation

<div><p>Alcohol binge-drinking (acute ethanol consumption) is immunosuppressive and alters both the innate and adaptive arms of the immune system. Antigen presentation by macrophages (and other antigen presenting cells) represents an important function of the innate immune system that, in part, determines the outcome of the host immune response. Ethanol has been shown to suppress antigen presentation in antigen presenting cells though mechanisms of this impairment are not well understood. The constitutive and immunoproteasomes are important components of the cellular proteolytic machinery responsible for the initial steps critical to the generation of MHC Class I peptides for antigen presentation. In this study, we used an <i>in-vitro</i> cell culture model of acute alcohol exposure to study the effect of ethanol on the proteasome function in RAW 264.7 cells. Additionally, primary murine peritoneal macrophages obtained by peritoneal lavage from C57BL/6 mice were used to confirm our cell culture findings. We demonstrate that ethanol impairs proteasome function in peritoneal macrophages through suppression of chymotrypsin-like (Cht-L) proteasome activity as well as composition of the immunoproteasome subunit LMP7. Using primary murine peritoneal macrophages, we have further demonstrated that, ethanol-induced impairment of the proteasome function suppresses processing of antigenic proteins and peptides by the macrophage and in turn suppresses the presentation of these antigens to cells of adaptive immunity. The results of this study provide an important mechanism to explain the immunosuppressive effects of acute ethanol exposure.</p> </div

Ethanol suppresses 20S Cht-L proteasome activity in murine primary peritoneal macrophages. A.

<p>Murine peritoneal macrophages (2.5×10⁵ cells/ml) were treated with 50 mM or 100 mM EtOH for 24 h or 48 h. 0 mM (control) cells received no treatment. Cell lysates obtained at the indicated time points were analyzed for 20S Cht-L proteasome activity by fluorigenic assay as described in Methods. <b>B.</b> Peritoneal macrophages were treated with MG132 (5 uM), as a positive control of proteasome suppression. Cell lysates were obtained at the indicated time points and analyzed as described in Panel A. <b>C.</b> Ethanol-treated peritoneal macrophages were stimulated with heat-killed <i>Kp</i>, 6 hours after initial EtOH exposure. Cell lysates obtained at indicated time points were analyzed for proteasome activity as described in panel A. <b>D.</b> MG132-treated peritoneal macrophages were stimulated with <i>Kp</i> as described in Panel C. Cell lysates were obtained at the indicated time points and analyzed for proteasome activity as in Panel A. Proteasome activity data plotted as mean relative fluorescence units (R.F.U.) ± SEM (three replicates per experiment) and is representative of three independent experiments. Higher R.F.U. values indicate higher proteasome activity. *p<0.05 vs. control at the respective time point. **p<0.05 vs. control and 50 mM EtOH at the respective time points.</p

Ethanol does not alter steady-state protein levels of constitutive proteasome subunit beta 5 (PSMB5), but suppresses steady-state protein levels of immunoproteasome subunit LMP7.

<p><b>A.</b> Murine unstimulated RAW 264.7 cells (2×10⁵ cells/ml) were treated with 50 mM (lane 3) or 100 mM (Lane 4) ethanol (EtOH) for 48 h. Cells were also treated with MG132 (0.5 uM) (lane 2) as a positive control of proteasome inhibition. 0 mM (control) cells received no treatment (lane1). Cell lysates were obtained 48 h post EtOH exposure. The lysates of triplicate samples were pooled for gel loading, analyzed by 15% SDS-PAGE and immunoblotted with anti-PSMB5 antibody as described in the methods (upper panel). The membrane was stripped and reprobed with anti-beta-actin antibody to ensure equal protein loading as described in the methods section (lower panel). The bar graph represents densitometry quantitation of PSMB5 levels between treatment groups and is represented as INT/mm². The depicted gel blot is representative of two independent experiments. Each band of the blot represents the pooled lysates from three independently treated samples. <b>B.</b> Unstimulated RAW 264.7 cells (2×10⁵ cells/ml) were treated with 50 mM (lane 3) or 100 mM (lane 4) EtOH for 24 h (Left panel) or 48 h (Right Panel). Cells were also treated with MG132 (0.5 uM) (lanes 2) as a positive control of proteasome inhibition. 0 mM (control) cells received no treatment (lane1). Cell lysates were obtained 24 h or 48 h post-EtOH exposure. The lysates of triplicate samples were pooled for gel loading, analyzed by 15% SDS-PAGE and immunoblotted with anti-LMP7 antibody as described in the methods (24 h and 48 h upper panels). The membrane was stripped and reprobed with anti-beta-actin antibody to ensure equal protein loading as described in the methods section (24 h and 48 h lower panel). The bar graphs beneath each immunoblot represent densitometry quantitation of LMP7 levels between treatment groups at 24 h and 48 h respectively and is represented as INT/mm². The depicted gel blots are representative of two independent experiments. Each band of the blot represents the pooled lysates from three independently treated samples.</p

20S chymotrypsin-like (Cht-L) proteasome activity is suppressed in ethanol-treated RAW 264.7 cells. A.

<p>Murine RAW 264.7 (2×10⁵ cells/ml) cells were treated with 50 mM or 100 mM ethanol (EtOH) for 24 h or 48 h. 0 mM (control) cells received no treatment. Cell lysates obtained at the indicated time points were analyzed for 20S Cht-L proteasome activity by fluorigenic assay as described in Methods. <b>B.</b> RAW 264.7 cells were treated with MG132 (0.5 uM) as a positive control of proteasome suppression. Cell lysates were analyzed at the indicated time points for proteasome activity as described in panel A. <b>C.</b> Ethanol-treated RAW 264.7 cells were stimulated with heat-killed <i>K.pneumoniae</i> (<i>Kp</i>), 6 hours after initial EtOH exposure. Cell lysates obtained at the indicated time points were analyzed as described above in panel A. <b>D.</b> RAW 264.7 cells were treated with MG132 (0.5 uM) followed by challenge with <i>Kp</i> as described in panel C. Cells were lysed at the indicated time points and analyzed for proteasome activity as described above. Proteasome activity data was plotted as mean relative fluorescence units (R.F.U.) ± SEM (three replicates per experiment) and is representative of three independent experiments. Higher R.F.U. values indicate higher proteasome activity. *p<0.05 vs. control (0 mM) at the respective time point. **p<0.05 vs. control (0 mM) and 50 mM EtOH at the respective time point.</p

Ovalbumin antigen MHC-Class I processing and presentation is suppressed in ethanol-treated murine primary peritoneal macrophages.

<p><b>A.</b> Schematic representation of experimental setup as described in Methods. Briefly, murine peritoneal macrophages were treated 50 mM or 100 mM EtOH for 24, 48 or 72 h. Proteasome inhibitors MG132 (5 uM) or lactacystin (10 uM) were used as positive controls of proteasome inhibition. EtOH-exposed cells were treated with full-length chicken ovalbumin (2 mg/ml) for 2 hours. Macrophages washed with PBS and co-incubated with OT-1 mouse CD8+ cytotoxic T lymphocytes (Ratio 2∶1) for 18 h. Cell supernatants were collected and analyzed for cytokine IL-2. <b>B.</b> CD8+ T cell derived IL-2 levels from proteasome inhibitor-treated peritoneal macrophage groups is shown. <b>C.</b> CD8+ T cell derived IL-2 levels from 24 h EtOH-treated peritoneal macrophage groups is shown. <b>D.</b> CD8+ T cell derived IL-2 levels from 48 h EtOH-treated peritoneal macrophage groups is shown. <b>E.</b> CD8+ T cell derived IL-2 levels from 72 h EtOH-treated peritoneal macrophage groups. Data plotted is mean levels of cytokine IL-2 (pg/ml) ± SEM (three replicates per experiment) and is representative of three independent experiments. *p<0.05 vs. control (0 mM) at respective time point. **p<0.05 vs. control (0 mM) and 50 mM EtOH at respective time point.</p

Antigen presentation of C-terminal extended SIINFEKLTE is suppressed in ethanol-treated murine primary peritoneal macrophages.

<p>Experimental setup is the same as detailed in Fig. 4A, except that instead of full length OVA the C-terminal extended SIINTEKLTE peptide was used. <b>A.</b> CD8+ T cell-derived IL-2 levels from 24 h EtOH-treated peritoneal macrophage groups is shown. <b>B.</b> CD8+ T cell-derived IL-2 levels from 48 h EtOH-treated macrophage groups is shown. Data plotted is mean levels of cytokine IL-2 (pg/ml) ± SEM (three replicates per experiment) and is representative of three independent experiments. **p<0.05 vs. control and 50 mM EtOH at respective time point.</p

A review of the ecological value of Cusuco National Park: an urgent call for conservation action in a highly threatened Mesoamerican cloud forest

Cloud forests are amongst the most biologically unique, yet threatened, ecosystems in Mesoamerica. We summarize the ecological value and conservation status of a well-studied cloud forest site: Cusuco National Park (CNP), a 23,440 ha protected area in the Merendón mountains, northwest Honduras. We show CNP to have exceptional biodiversity; of 966 taxa identified to a species-level to date, 362 (37.5%) are Mesoamerican endemics, 67 are red-listed by the IUCN, and at least 49 are micro-endemics known only from the Merendón range. CNP also provides key ecosystem services including provision of drinking water and downstream flood mitigation, as well as carbon sequestration, with an estimated stock of 3.5 million megagrams of carbon in 2000. Despite its ecological importance, CNP faces multiple environmental threats and associated stresses, including deforestation (1,759 ha since 2000 equating to 7% of total forest area), poaching (7% loss of mammal relative abundance per year), amphibian declines due to chytridiomycosis (70% of species threatened or near-threatened), and climate change (a mean 2.6 °C increase in temperature and 112 mm decrease in rainfall by 2100). Despite conservation actions, including community ranger patrols, captive-breeding programmes, and ecotourism initiatives, environmental degradation of CNP continues. Further action is urgently required, including reinforcement and expansion of ranger programmes, greater stakeholder engagement, community education programmes, development of alternative livelihood projects, and legislative enforcement and prosecution. Without a thorough and rapid response to understand and mitigate illegal activities, the extirpation and extinction of species and the loss of vital ecosystem services are inevitable in the coming decades