Deep Multilayer Brain Proteomics Identifies Molecular Networks in Alzheimer\u27s Disease Progression

Alzheimer\u27s disease (AD) displays a long asymptomatic stage before dementia. We characterize AD stage-associated molecular networks by profiling 14,513 proteins and 34,173 phosphosites in the human brain with mass spectrometry, highlighting 173 protein changes in 17 pathways. The altered proteins are validated in two independent cohorts, showing partial RNA dependency. Comparisons of brain tissue and cerebrospinal fluid proteomes reveal biomarker candidates. Combining with 5xFAD mouse analysis, we determine 15 Aβ-correlated proteins (e.g., MDK, NTN1, SMOC1, SLIT2, and HTRA1). 5xFAD shows a proteomic signature similar to symptomatic AD but exhibits activation of autophagy and interferon response and lacks human-specific deleterious events, such as downregulation of neurotrophic factors and synaptic proteins. Multi-omics integration prioritizes AD-related molecules and pathways, including amyloid cascade, inflammation, complement, WNT signaling, TGF-β and BMP signaling, lipid metabolism, iron homeostasis, and membrane transport. Some Aβ-correlated proteins are colocalized with amyloid plaques. Thus, the multilayer omics approach identifies protein networks during AD progression

Conditional mutagenesis in vivo reveals cell type- and infection stage-specific requirements for LANA in chronic MHV68 infection.

Gammaherpesvirus (GHV) pathogenesis is a complex process that involves productive viral replication, dissemination to tissues that harbor lifelong latent infection, and reactivation from latency back into a productive replication cycle. Traditional loss-of-function mutagenesis approaches in mice using murine gammaherpesvirus 68 (MHV68), a model that allows for examination of GHV pathogenesis in vivo, have been invaluable for defining requirements for specific viral gene products in GHV infection. But these approaches are insufficient to fully reveal how viral gene products contribute when the encoded protein facilitates multiple processes in the infectious cycle and when these functions vary over time and from one host tissue to another. To address this complexity, we developed an MHV68 genetic platform that enables cell-type-specific and inducible viral gene deletion in vivo. We employed this system to re-evaluate functions of the MHV68 latency-associated nuclear antigen (mLANA), a protein with roles in both viral replication and latency. Cre-mediated deletion in mice of loxP-flanked ORF73 demonstrated the necessity of mLANA in B cells for MHV68 latency establishment. Impaired latency during the transition from draining lymph nodes to blood following mLANA deletion also was observed, supporting the hypothesis that B cells are a major conduit for viral dissemination. Ablation of mLANA in infected germinal center (GC) B cells severely impaired viral latency, indicating the importance of viral passage through the GC for latency establishment. Finally, induced ablation of mLANA during latency resulted in complete loss of affected viral genomes, indicating that mLANA is critically important for maintenance of viral genomes during stable latency. Collectively, these experiments provide new insights into LANA homolog functions in GHV colonization of the host and highlight the potential of a new MHV68 genetic platform to foster a more complete understanding of viral gene functions at discrete stages of GHV pathogenesis

O73.loxP exhibits efficient acute replication in lungs of mice.

<p>C57BL/6 (A) or CD19^Cre/+ (B) mice were infected IN with 1000 PFU of the indicated viruses. Mice were sacrificed on day 7 post-infection, and viral titers in lung homogenates were determined by plaque assay. Each dot represents one mouse. Error bars represent standard error of the means. * denotes p < 0.05 in a two-tailed student’s t-test.</p

Derivation and validation of O73.loxP MHV68.

<p>(A) Schematic depicting the insertion of <i>loxP</i> sites flanking <i>ORF73</i> in the MHV68 genome and its deletion in the presence of Cre recombinase. (B) 3T3 fibroblasts were infected with WT MHV68 or O73.loxP at an MOI of 5 PFU/cell (single-step, left panel) or 0.05 PFU/cell (multi-step, right panel). Viral titers were determined by plaque assay at the indicated times post-infection. Results are means of triplicate samples. Error bars represent standard deviations. (C and D) 3T3 fibroblasts that encode Cre-ERT2 were treated with vehicle (NOT induced) or 4-hydroxytamoxifen (Cre induced) to induce Cre activity 24 h prior to infection. Treated cells were infected with the indicated viruses at an MOI of 0.05 PFU/cell. (C) Total DNA was isolated on day 4 post-infection, and PCR was performed as illustrated in the schematic to detect the intact or deleted <i>ORF73</i> locus or the distal <i>ORF59</i> locus as a control. (D) Cells were lysed on day 4 post-infection, and proteins were resolved by SDS-PAGE. Immunoblot analyses were preformed using antibodies to detect the indicated proteins. Cellular β-actin serves as a loading control.</p

Curriculum Design and Implementation of the Emergency Medicine Chief Resident Incubator

Background: Chief residents receive minimal formal training in preparation for their administrative responsibilities. There is a lack of professional development programs specifically designed for chief residents. Objective: In 2015, Academic Life in Emergency Medicine designed and implemented an annual, year-long, training program and virtual community of practice for chief residents in emergency medicine (EM). This study describes the curriculum design process and reports measures of learner engagement during the first two cycles of the curriculum. Methods: Kern’s Six-Step Approach for curriculum development informed key decisions in the design and implementation of the Chief Resident Incubator. The resultant curriculum was created using constructivist social learning theory, with specific objectives that emphasized the needs for a virtual community of practice, longitudinal content delivery, mentorship for participants, and the facilitation of multicenter digital scholarship. The 12-month curriculum included 11 key administrative or professional development domains, delivered using a combination of digital communications platforms. Primary outcomes measures included markers of learner engagement with the online curriculum, recognized as modified Kirkpatrick Level One outcomes for digital learning. Results: An average of 206 chief residents annually enrolled in the first two years of the curriculum, with an overall participation by 33% (75/227) of the allopathic EM residency programs in the United States (U.S.). There was a high level of learner engagement, with an average 13,414 messages posted per year. There were also 42 small group teaching sessions held online, which included 39 faculty and 149 chief residents. The monthly e-newsletter had a 50.7% open rate. Digital scholarship totaled 23 online publications in two years, with 67 chief resident co-authors and 21 faculty co-authors. Conclusions: The Chief Resident Incubator is a virtual community of practice that provides longitudinal training and mentorship for EM chief residents. This incubator conceptual framework may be used to design similar professional development curricula across various health professions using an online digital platform

O73.loxP establishes latency in spleens of C57BL/6 mice following IN or IP inoculation.

<p>C57BL/6 mice were infected IN (A and B) or IP (C and D) with 1000 PFU of the indicated virus. Mice were sacrificed on days 16–18 post-infection. (A and C) Single-cell suspensions of spleen cells were serially diluted, and frequencies of cells harboring MHV68 genomes were determined using a limiting-dilution PCR analysis. (B and D) Reactivation frequencies were determined by <i>ex vivo</i> plating of serially diluted cells on an indicator monolayer. Cytopathic effect was scored 2–3 weeks post-plating. Groups of 3–5 mice were pooled for each infection and analysis. Results are means of 2–3 independent infections. Error bars represent standard error of the means.</p

Reciprocal frequencies of ORF50⁺ and ORF73⁺ cells for vehicle or tamoxifen-treated Cre-ERT2 or WT mice infected with O73.loxP or FRT MHV68.

<p>Reciprocal frequencies of ORF50⁺ and ORF73⁺ cells for vehicle or tamoxifen-treated Cre-ERT2 or WT mice infected with O73.loxP or FRT MHV68.</p

Frequency of viral-genome positive cells.

<p>Frequency of viral-genome positive cells.</p

Cre-mediated deletion of <i>ORF73</i> yields a reduction in previously established MHV68 latency.

<p>(A) Schematic depicting the working model and experimental strategy for selective Cre-mediated deletion of ORF73 during MHV68 latency maintenance. (B) WT or Hemizygous Cre-ERT2 transgenic mice were infected IN with 1000 PFU of the indicated virus. On days 23–28 post-infection, mice were injected IP with vehicle or tamoxifen (2 mg/dose; one dose per day for 5 consecutive days) to induce Cre-ERT2 nuclear translocation. Mice were sacrificed on day 42 post-infection. Single-cell suspensions of splenocytes were serially diluted, and frequencies of cells harboring MHV68 genomes were determined using a limiting-dilution PCR analysis to detect either <i>ORF50</i> or <i>ORF73</i> genomic loci. Note: <i>ORF73</i> primer pairs overlap the deletion junction. Therefore, the target sequence is lost upon Cre-mediated deletion of <i>ORF73</i>. Results are means of 2 independent infections. Error bars represent standard error of the means. CTRL = mice treated with corn oil; TAM = mice treated with tamoxifen.</p

Infection of CD19^Cre/+ mice with O73.loxP results in <i>ORF73</i> deletion and impaired splenic latency and reactivation.

<p>CD19^Cre/+ mice were infected IN (A-C) or IP (D-I) with 1000 PFU of the indicated virus. Mice were sacrificed on days 16–18 post-infection. (A, D, and G) Single-cell suspensions of splenocytes (A and D) or PECs (G) were serially diluted, and frequencies of cells harboring MHV68 genomes were determined using a limiting-dilution PCR analysis. (B, E and H) Reactivation frequencies were determined by <i>ex vivo</i> plating of serially-diluted cells on an indicator monolayer. Cytopathic effect was scored 2–3 weeks post-plating. Groups of 3–5 mice were pooled for each infection and analysis. Results are means of 2–3 independent infections. Error bars represent standard error of the means. (C, F, and I) Total DNA was isolated from spleens (C and F) or PECs (I) at the time of harvest. PCR was performed as illustrated in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006865#ppat.1006865.g001" target="_blank">Fig 1</a> to detect the indicated viral loci or cellular <i>GAPDH</i> as a control.</p