Synthetic recording and in situ readout of lineage information in single cells

Reconstructing the lineage relationships and dynamic event histories of individual cells within their native spatial context is a long-standing challenge in biology. Many biological processes of interest occur in optically opaque or physically inaccessible contexts, necessitating approaches other than direct imaging. Here, we describe a new synthetic system that enables cells to record lineage information and event histories in the genome in a format that can be subsequently read out in single cells in situ. This system, termed Memory by Engineered Mutagenesis with Optical In situ Readout (MEMOIR), is based on a set of barcoded recording elements termed scratchpads. The state of a given scratchpad can be irreversibly altered by Cas9-based targeted mutagenesis, and read out in single cells through multiplexed single-molecule RNA fluorescence hybridization (smFISH). To demonstrate a proof of principle of MEMOIR, we engineered mouse embryonic stem (ES) cells to contain multiple scratchpads and other recording components. In these cells, scratchpads were altered in a progressive and stochastic fashion as cells proliferated. Analysis of the final states of scratchpads in single cells in situ enabled reconstruction of the lineage trees of cell colonies. Combining analysis of endogenous gene expression with lineage reconstruction in the same cells further allowed inference of the dynamic rates at which ES cells switch between two gene expression states. Finally, using simulations, we showed how parallel MEMOIR systems operating in the same cell can enable recording and readout of dynamic cellular event histories. MEMOIR thus provides a versatile platform for information recording and in situ, single cell readout across diverse biological systems

Availability and quality of paraffin blocks identified in pathology archives: A multi-institutional study by the Shared Pathology Informatics Network (SPIN)

BACKGROUND: Shared Pathology Informatics Network (SPIN) is a tissue resource initiative that utilizes clinical reports of the vast amount of paraffin-embedded tissues routinely stored by medical centers. SPIN has an informatics component (sending tissue-related queries to multiple institutions via the internet) and a service component (providing histopathologically annotated tissue specimens for medical research). This paper examines if tissue blocks, identified by localized computer searches at participating institutions, can be retrieved in adequate quantity and quality to support medical researchers. METHODS: Four centers evaluated pathology reports (1990–2005) for common and rare tumors to determine the percentage of cases where suitable tissue blocks with tumor were available. Each site generated a list of 100 common tumor cases (25 cases each of breast adenocarcinoma, colonic adenocarcinoma, lung squamous carcinoma, and prostate adenocarcinoma) and 100 rare tumor cases (25 cases each of adrenal cortical carcinoma, gastro-intestinal stromal tumor [GIST], adenoid cystic carcinoma, and mycosis fungoides) using a combination of Tumor Registry, laboratory information system (LIS) and/or SPIN-related tools. Pathologists identified the slides/blocks with tumor and noted first 3 slides with largest tumor and availability of the corresponding block. RESULTS: Common tumors cases (n = 400), the institutional retrieval rates (all blocks) were 83% (A), 95% (B), 80% (C), and 98% (D). Retrieval rate (tumor blocks) from all centers for common tumors was 73% with mean largest tumor size of 1.49 cm; retrieval (tumor blocks) was highest-lung (84%) and lowest-prostate (54%). Rare tumors cases (n = 400), each institution's retrieval rates (all blocks) were 78% (A), 73% (B), 67% (C), and 84% (D). Retrieval rate (tumor blocks) from all centers for rare tumors was 66% with mean largest tumor size of 1.56 cm; retrieval (tumor blocks) was highest for GIST (72%) and lowest for adenoid cystic carcinoma (58%). CONCLUSION: Assessment shows availability and quality of archival tissue blocks that are retrievable and associated electronic data that can be of value for researchers. This study serves to compliment the data from which uniform use of the SPIN query tools by all four centers will be measured to assure and highlight the usefulness of archival material for obtaining tumor tissues for research

Safety and Immunogenicity of an HIV-1 Gag DNA Vaccine with or without IL-12 and/or IL-15 Plasmid Cytokine Adjuvant in Healthy, HIV-1 Uninfected Adults

DNA vaccines are a promising approach to vaccination since they circumvent the problem of vector-induced immunity. DNA plasmid cytokine adjuvants have been shown to augment immune responses in small animals and in macaques.We performed two first in human HIV vaccine trials in the US, Brazil and Thailand of an RNA-optimized truncated HIV-1 gag gene (p37) DNA derived from strain HXB2 administered either alone or in combination with dose-escalation of IL-12 or IL-15 plasmid cytokine adjuvants. Vaccinations with both the HIV immunogen and cytokine adjuvant were generally well-tolerated and no significant vaccine-related adverse events were identified. A small number of subjects developed asymptomatic low titer antibodies to IL-12 or IL-15. Cellular immunogenicity following 3 and 4 vaccinations was poor, with response rates to gag of 4.9%/8.7% among vaccinees receiving gag DNA alone, 0%/11.5% among those receiving gag DNA+IL-15, and no responders among those receiving DNA+high dose (1500 ug) IL-12 DNA. However, after three doses, 44.4% (4/9) of vaccinees receiving gag DNA and intermediate dose (500 ug) of IL-12 DNA demonstrated a detectable cellular immune response.This combination of HIV gag DNA with plasmid cytokine adjuvants was well tolerated. There were minimal responses to HIV gag DNA alone, and no apparent augmentation with either IL-12 or IL-15 plasmid cytokine adjuvants. Despite the promise of DNA vaccines, newer formulations or methods of delivery will be required to increase their immunogenicity.Clinicaltrials.gov NCT00115960 NCT00111605

Elicitation of Neutralizing Antibodies Directed against CD4-Induced Epitope(s) Using a CD4 Mimetic Cross-Linked to a HIV-1 Envelope Glycoprotein

The identification of HIV-1 envelope glycoprotein (Env) structures that can generate broadly neutralizing antibodies (BNAbs) is pivotal to the development of a successful vaccine against HIV-1 aimed at eliciting effective humoral immune responses. To that end, the production of novel Env structure(s) that might induce BNAbs by presentation of conserved epitopes, which are otherwise occluded, is critical. Here, we focus on a structure that stabilizes Env in a conformation representative of its primary (CD4) receptor-bound state, thereby exposing highly conserved “CD4 induced” (CD4i) epitope(s) known to be important for co-receptor binding and subsequent virus infection. A CD4-mimetic miniprotein, miniCD4 (M64U1-SH), was produced and covalently complexed to recombinant, trimeric gp140 envelope glycoprotein (gp140) using site-specific disulfide linkages. The resulting gp140-miniCD4 (gp140-S-S-M64U1) complex was recognized by CD4i antibodies and the HIV-1 co-receptor, CCR5. The gp140-miniCD4 complex elicited the highest titers of CD4i binding antibodies as well as enhanced neutralizing antibodies against Tier 1 viruses as compared to gp140 protein alone following immunization of rabbits. Neutralization against HIV-27312/V434M and additional serum mapping confirm the specific elicitation of antibodies directed to the CD4i epitope(s). These results demonstrate the utility of structure-based approach in improving immunogenic response against specific region, such as the CD4i epitope(s) here, and its potential role in vaccine application