Mycobacterium tuberculosis Rv3802c Encodes a Phospholipase/Thioesterase and Is Inhibited by the Antimycobacterial Agent Tetrahydrolipstatin

The cell wall of M. tuberculosis is central to its success as a pathogen. Mycolic acids are key components of this cell wall. The genes involved in joining the α and mero mycolates are located in a cluster, beginning with Rv3799c and extending at least until Rv3804c. The role of each enzyme encoded by these five genes is fairly well understood, except for Rv3802c. Rv3802 is one of seven putative cutinases encoded by the genome of M. tuberculosis. In phytopathogens, cutinases hydrolyze the waxy layer of plants, cutin. In a strictly mammalian pathogen, such as M. tuberculosis, it is likely that these proteins perform a different function. Of the seven, we chose to focus on Rv3802c because of its location in a mycolic acid synthesis gene cluster, its putative essentiality, its ubiquitous presence in actinomycetes, and its conservation in the minimal genome of Mycobacterium leprae. We expressed Rv3802 in Escherichia coli and purified the enzymatically active form. We probed its activities and inhibitors characterizing those relevant to its possible role in mycolic acid biosynthesis. In addition to its reported phospholipase A activity, Rv3802 has significant thioesterase activity, and it is inhibited by tetrahydrolipstatin (THL). THL is a described anti-tuberculous compound with an unknown mechanism, but it reportedly targets cell wall synthesis. Taken together, these data circumstantially support a role for Rv3802 in mycolic acid synthesis and, as the cell wall is integral to M. tuberculosis pathogenesis, identification of a novel cell wall enzyme and its inhibition has therapeutic and diagnostic implications

Genetic modification of primary human B cells to model high-grade lymphoma

Abstract: Sequencing studies of diffuse large B cell lymphoma (DLBCL) have identified hundreds of recurrently altered genes. However, it remains largely unknown whether and how these mutations may contribute to lymphomagenesis, either individually or in combination. Existing strategies to address this problem predominantly utilize cell lines, which are limited by their initial characteristics and subsequent adaptions to prolonged in vitro culture. Here, we describe a co-culture system that enables the ex vivo expansion and viral transduction of primary human germinal center B cells. Incorporation of CRISPR/Cas9 technology enables high-throughput functional interrogation of genes recurrently mutated in DLBCL. Using a backbone of BCL2 with either BCL6 or MYC, we identify co-operating genetic alterations that promote growth or even full transformation into synthetically engineered DLBCL models. The resulting tumors can be expanded and sequentially transplanted in vivo, providing a scalable platform to test putative cancer genes and to create mutation-directed, bespoke lymphoma models

Modeling Nasal Physiology Changes Due to Septal Perforations

OBJECTIVE: To use computational fluid dynamics (CFD) technology to help providers understand 1) how septal perforations may alter nasal physiology and 2) how these alterations are influenced by perforation size and location. STUDY DESIGN: Computer simulation study SETTING: Facial Plastic and Reconstructive Surgery clinic SUBJECTS AND METHODS: With the aid of medical imaging and modeling software, septal perforations of 1 and 2 cm in anterior, posterior, and superior locations were virtually created in a nasal cavity digital model. CFD techniques were used to analyze airflow, nasal resistance, air conditioning, and wall shear stress. RESULTS: Bilateral nasal resistance was not significantly altered by a septal perforation. Airflow allocation changed, with more air flowing through the lower-resistance nasal cavity. This effect was greater for anterior and posterior perforations than for the superior location. At the perforation sites, there was less localized heat and moisture flux and wall shear stress in superior perforations compared to those in anterior or posterior locations. For anterior perforations, a larger size produced higher wall shear and velocity, while in posterior perforations a smaller size produced higher wall shear and velocity. CONCLUSIONS: Septal perforations may alter nasal physiology. In the subject studied, airflow allocation to each side was changed as air was shunted through the perforation to the lower-resistance nasal cavity. Anterior and posterior perforations caused larger effects than those in a superior location. Increasing the size of anterior perforations and decreasing the size of posterior perforations enhanced alterations in wall shear and velocity at the perforation