Identification of a nucleoside analog active against adenosine kinase-expressing plasma cell malignancies.

Primary effusion lymphoma (PEL) is a largely incurable malignancy of B cell origin with plasmacytic differentiation. Here, we report the identification of a highly effective inhibitor of PEL. This compound, 6-ethylthioinosine (6-ETI), is a nucleoside analog with toxicity to PEL in vitro and in vivo, but not to other lymphoma cell lines tested. We developed and performed resistome analysis, an unbiased approach based on RNA sequencing of resistant subclones, to discover the molecular mechanisms of sensitivity. We found different adenosine kinase-inactivating (ADK-inactivating) alterations in all resistant clones and determined that ADK is required to phosphorylate and activate 6-ETI. Further, we observed that 6-ETI induces ATP depletion and cell death accompanied by S phase arrest and DNA damage only in ADK-expressing cells. Immunohistochemistry for ADK served as a biomarker approach to identify 6-ETI-sensitive tumors, which we documented for other lymphoid malignancies with plasmacytic features. Notably, multiple myeloma (MM) expresses high levels of ADK, and 6-ETI was toxic to MM cell lines and primary specimens and had a robust antitumor effect in a disseminated MM mouse model. Several nucleoside analogs are effective in treating leukemias and T cell lymphomas, and 6-ETI may fill this niche for the treatment of PEL, plasmablastic lymphoma, MM, and other ADK-expressing cancers.

Systemic expression of Kaposi sarcoma herpesvirus (KSHV) Vflip in endothelial cells leads to a profound proinflammatory phenotype and myeloid lineage remodeling in vivo.

KSHV is the causative agent of Kaposi sarcoma (KS), a spindle-shaped endothelial cell neoplasm accompanied by an inflammatory infiltrate. To evaluate the role of KSHV vFLIP in the pathogenesis of KS, we constructed mice with inducible expression of vFLIP in endothelial cells. Abnormal cells with endothelial marker expression and fusiform appearance were observed in several tissues reminiscent of the spindle cells found in KS. Serum cytokines displayed a profound perturbation similar to that described in KSHV inflammatory cytokine syndrome (KICS), a recently described clinical condition characterized by elevated IL6 and IL10. An increased myeloid component with suppressive immune phenotype was found, which may contribute to functional changes in the microenvironment and cellular heterogeneity as observed in KS. These mice represent the first in vivo demonstration that vFLIP is capable of inducing vascular abnormalities and changes in host microenvironment with important implications for understanding the pathogenesis and treating KSHV-associated diseases.

Solar thermal polymerase chain reaction for smartphone-assisted molecular diagnostics.

Nucleic acid-based diagnostic techniques such as polymerase chain reaction (PCR) are used extensively in medical diagnostics due to their high sensitivity, specificity and quantification capability. In settings with limited infrastructure and unreliable electricity, however, access to such devices is often limited due to the highly specialized and energy-intensive nature of the thermal cycling process required for nucleic acid amplification. Here we integrate solar heating with microfluidics to eliminate thermal cycling power requirements as well as create a simple device infrastructure for PCR. Tests are completed in less than 30 min, and power consumption is reduced to 80 mW, enabling a standard 5.5 Wh iPhone battery to provide 70 h of power to this system. Additionally, we demonstrate a complete sample-to-answer diagnostic strategy by analyzing human skin biopsies infected with Kaposi's Sarcoma herpesvirus (KSHV/HHV-8) through the combination of solar thermal PCR, HotSHOT DNA extraction and smartphone-based fluorescence detection. We believe that exploiting the ubiquity of solar thermal energy as demonstrated here could facilitate broad availability of nucleic acid-based diagnostics in resource-limited areas.

A new set of small, extrachromosomal expression vectors for Dictyostelium discoideum.

A new set of extrachromosomal Dictyostelium expression vectors is presented that can be modified according to the experimental needs with minimal cloning efforts. To achieve this, the vector consists of four functional regions that are separated by unique restriction sites, (1) an Escherichia coli replication region, and regions for (2) replication, (3) selection and (4) protein expression in Dictyostelium. Each region was trimmed down to its smallest possible size. A basic expression vector can be constructed from these modules with a size of only 6.8 kb. By exchanging modules, a large number of vectors with different properties can be constructed. The resulting set of vectors allows most basic expression needs, such as immuno blotting, protein purification, visualization of protein localization and identification of protein-protein interactions. In addition, two genes can be simultaneously expressed on one vector, which yields far more synchronous levels of expression than when expressing two genes on separate plasmids.