Seminal Plasma-Derived Extracellular-Vesicle Fractions from HIV-Infected Men Exhibit Unique MicroRNA Signatures and Induce a Proinflammatory Response in Cells Isolated from the Female Reproductive Tract.

The continuing spread of HIV/AIDS is predominantly fueled by sexual exposure to HIV-contaminated semen. Seminal plasma (SP), the liquid portion of semen, harbors a variety of factors that may favor HIV transmission by facilitating viral entry into host cells, eliciting the production of proinflammatory cytokines, and enhancing the translocation of HIV across the genital epithelium. One important and abundant class of factors in SP is extracellular vesicles (EVs), which, in general, are important intercellular signal transducers. Although numerous studies have characterized blood plasma-derived EVs from both uninfected and HIV-infected individuals, little is known about the properties of EVs from the semen of HIV-infected individuals. We report here that fractionated SP enriched for EVs from HIV-infected men induces potent transcriptional responses in epithelial and stromal cells that interface with the luminal contents of the female reproductive tract. Semen EV fractions from acutely infected individuals induced a more proinflammatory signature than those from uninfected individuals. This was not associated with any observable differences in the surface phenotypes of the vesicles. However, microRNA (miRNA) expression profiling analysis revealed that EV fractions from infected individuals exhibit a broader and more diverse profile than those from uninfected individuals. Taken together, our data suggest that SP EVs from HIV-infected individuals exhibit unique miRNA signatures and exert potent proinflammatory transcriptional changes in cells of the female reproductive tract, which may facilitate HIV transmission.IMPORTANCE Seminal plasma (SP), the major vehicle for HIV, can modulate HIV transmission risk through a variety of mechanisms. Extracellular vesicles (EVs) are extremely abundant in semen, and because they play a key role in intercellular communication pathways and immune regulation, they may impact the likelihood of HIV transmission. However, little is known about the properties and signaling effects of SP-derived EVs in the context of HIV transmission. Here, we conduct a phenotypic, transcriptomic, and functional characterization of SP and SP-derived EVs from uninfected and HIV-infected men. We find that both SP and its associated EVs elicit potent proinflammatory transcriptional responses in cells that line the genital tract. EVs from HIV-infected men exhibit a more diverse repertoire of miRNAs than EVs from uninfected men. Our findings suggest that EVs from the semen of HIV-infected men may significantly impact the likelihood of HIV transmission through multiple mechanisms.

A cooled CCD camera-based protocol provides an effective solution for in vitro monitoring of luciferase.

Luciferase assay has become an increasingly important technique to monitor a wide range of biological processes. However, the mainstay protocols require a luminometer to acquire and process the data, therefore limiting its application to specialized research labs. To overcome this limitation, we have developed an alternative protocol that utilizes a commonly available cooled charge-coupled device (CCCD), instead of a luminometer for data acquiring and processing. By measuring activities of different luciferases, we characterized their substrate specificity, assay linearity, signal-to-noise levels, and fold-changes via CCCD. Next, we defined the assay parameters that are critical for appropriate use of CCCD for different luciferases. To demonstrate the usefulness in cultured mammalian cells, we conducted a case study to examine NFκB gene activation in response to inflammatory signals in human embryonic kidney cells (HEK293 cells). We found that data collected by CCCD camera was equivalent to those acquired by luminometer, thus validating the assay protocol. In comparison, The CCCD-based protocol is readily amenable to live-cell and high-throughput applications, offering fast simultaneous data acquisition and visual and quantitative data presentation. In conclusion, the CCCD-based protocol provides a useful alternative for monitoring luciferase reporters. The wide availability of CCCD will enable more researchers to use luciferases to monitor and quantify biological processes.

Live visualization and quantification of pathway signaling with dual fluorescent and bioluminescent reporters.

Despite their fundamental importance, the dynamics of signaling pathways in living cells remain challenging to study, due to a lack of non-invasive tools for temporal assessment of signal transduction in desired cell models. Here we report a dual-reporter strategy that enables researchers to monitor signal transduction in mammalian cells in real-time, both temporally and quantitatively. This is achieved by co-expressing green fluorescent protein and firefly luciferase in response to signaling stimuli. To display the versatility of this approach, we constructed and assessed eight unique signaling pathway reporters. We further validated the system by establishing stable NF-κB pathway reporter cell lines. Using these stable cell lines, we monitored the activity of NF-κB-mediated inflammatory pathway in real-time, both visually and quantitatively. Live visualization has the power to reveal individual cell responses and is compatible with single cell analysis, In addition, we provide evidence that this system is readily amenable to a high-throughput format. Together, our findings demonstrate the potential of the dual reporter system, which significantly improves the capacity to study signal transduction pathways in mammalian cells.

Replication study: Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET.

As part of the Reproducibility Project: Cancer Biology we published a Registered Report (Lesnik et al., 2016) that described how we intended to replicate selected experiments from the paper 'Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET' (Peinado et al., 2012). Here we report the results. We regenerated tumor cells stably expressing a short hairpin to reduce Met expression (shMet) using the same highly metastatic mouse melanoma cell line (B16-F10) as the original study, which efficiently downregulated Met in B16F10 cells similar to the original study (Supplementary Figure 5A; Peinado et al., 2012). Exosomes from control cells expressed Met, which was reduced in exosomes from shMet cells; however, we were unable to reliably detect phosphorylated Met in exosomes. We tested the effect of exosome-dependent Met signaling on primary tumor growth and metastasis. Similar to the results in the original study, we did not find a statistically significant change in primary tumor growth. Measuring lung and femur metastases, we found a small increase in metastatic burden with exosomes from control cells that was diminished when Met expression was reduced; however, while the effects were in the same direction as the original study (Figure 4E; Peinado et al., 2012), they were not statistically significant. Differences between the original study and this replication attempt, such as level of knockdown efficiency, cell line genetic drift, sample sizes, study endpoints, and variability of observed metastatic burden, are factors that might have influenced the outcomes. Finally, we report meta-analyses for each result.

Viral Cre-LoxP tools aid genome engineering in mammalian cells.

BACKGROUND: Targeted nucleases have transformed genome editing technology, providing more efficient methods to make targeted changes in mammalian genome. In parallel, there is an increasing demand of Cre-LoxP technology for complex genome manipulation such as large deletion, addition, gene fusion and conditional removal of gene sequences at the target site. However, an efficient and easy-to-use Cre-recombinase delivery system remains lacking. RESULTS: We designed and constructed two sets of expression vectors for Cre-recombinase using two highly efficient viral systems, the integrative lentivirus and non-integrative adeno associated virus. We demonstrate the effectiveness of those methods in Cre-delivery into stably-engineered HEK293 cells harboring LoxP-floxed red fluorescent protein (RFP) and puromycin (Puro) resistant reporters. The delivered Cre recombinase effectively excised the floxed RFP-Puro either directly or conditionally, therefore validating the function of these molecular tools. Given the convenient options of two selections markers, these viral-based systems offer a robust and easy-to-use tool for advanced genome editing, expanding complicated genome engineering to a variety of cell types and conditions. CONCLUSIONS: We have developed and functionally validated two viral-based Cre-recombinase delivery systems for efficient genome manipulation in various mammalian cells. The ease of gene delivery with the built-in reporters and inducible element enables live cell monitoring, drug selection and temporal knockout, broadening applications of genome editing.