Rapid deployment of inexpensive open-source orbital shakers in support of high-throughput screening.

Open-source projects continue to grow in popularity alongside open-source educational resources, software, and hardware tools. The impact of this increased availability of open-source technologies is that end users are empowered to have greater control over the tools that they work with. This trend extends in the life science laboratory space, where new open-source projects are routinely being published that allow users to build and modify scientific equipment specifically tailored to their needs, often at a reduced cost from equivalent commercial offerings. Recently, we identified a need for a compact orbital shaker that would be usable in temperature and humidity-controlled incubators to support the development and execution of a high-throughput suspension cell-based assay. Based on the requirements provided by staff biologists, an open-source project known as the DIYbio orbital shaker was identified on Thingiverse, then quickly prototyped and tested. The initial orbital shaker prototype based on the DIYbio design underwent an iterative prototyping and design process that proved to be straightforward due to the open-source nature of the project. The result of these efforts has been the successful initial deployment of ten shakers as of August 2021. This afforded us the scalability and efficacy needed to complete a large-scale screening campaign in less time and at less cost than if we purchased larger, less adaptable orbital shakers. Lessons learned from prototyping, modifying, validating, deploying and maintaining laboratory devices based on an open-source design in support of a full-scale drug discovery high-throughput screening effort are described within this manuscript.

A mycobacterial enzyme essential for cell division synergizes with resuscitation-promoting factor.

The final stage of bacterial cell division requires the activity of one or more enzymes capable of degrading the layers of peptidoglycan connecting two recently developed daughter cells. Although this is a key step in cell division and is required by all peptidoglycan-containing bacteria, little is known about how these potentially lethal enzymes are regulated. It is likely that regulation is mediated, at least partly, through protein-protein interactions. Two lytic transglycosylases of mycobacteria, known as resuscitation-promoting factor B and E (RpfB and RpfE), have previously been shown to interact with the peptidoglycan-hydrolyzing endopeptidase, Rpf-interacting protein A (RipA). These proteins may form a complex at the septum of dividing bacteria. To investigate the function of this potential complex, we generated depletion strains in M. smegmatis. Here we show that, while depletion of rpfB has no effect on viability or morphology, ripA depletion results in a marked decrease in growth and formation of long, branched chains. These growth and morphological defects could be functionally complemented by the M. tuberculosis ripA orthologue (rv1477), but not by another ripA-like orthologue (rv1478). Depletion of ripA also resulted in increased susceptibility to the cell wall-targeting beta-lactams. Furthermore, we demonstrate that RipA has hydrolytic activity towards several cell wall substrates and synergizes with RpfB. These data reveal the unusual essentiality of a peptidoglycan hydrolase and suggest a novel protein-protein interaction as one way of regulating its activity.

Lopinavir/Ritonavir Impairs Physical Strength in Association with Reduced Igf1 Expression in Skeletal Muscle of Older Mice.

BACKGROUND: Late-middle age HIV patients are prone to fatigue despite effective viral control by antiretroviral therapies. Rodent models to recapitulate this phenotype are still not available. HYPOTHESIS: Drug treatment may compromise muscle strength and physical performance more in older individuals with pre-existing metabolic disorders than normal young ones. METHODS: Kaletra was given to overweight male mice at late-middle age and normal young adults; both on a rodent diet containing 30% fat calorie. Body composition and grip strength were measured at baseline and after drug treatment. Rota-rod running, insulin and glucose tolerance were measured at the end of the experiment. Drug effect on metabolic activity and spontaneous movements were assessed using the metabolic cage system. Representative muscle and fat tissue were analyzed for protein and mRNA expression. Selected findings were tested using murine C2C12 myotubes. RESULTS: Kaletra reduced grip strength in both young and older mice but impaired rotarod performance only in the old. Spontaneous movements were also reduced in Kaletra-treated old mice. Kaletra reduced IGF-1 expression in all muscle groups tested for the old and in cultured myotubes but to a less extent in the muscle of young animals. Reduced IGF-1 expression correlated with increased expression of muscle-specific atrogene MAFbx and MuRF1. Kaletra also increased abdominal fat mass markedly in the old animals and to a less extend in the young. CONCLUSION: Long-term Kaletra intake aggravated abdominal obesity and impaired muscle strength. This effect was worse in older animals than in normal young adults.

A partner for the resuscitation-promoting factors of Mycobacterium tuberculosis.

Many cases of active tuberculosis are thought to result from the reactivation of dormant Mycobacterium tuberculosis from a prior infection, yet remarkably little is known about the mechanism by which these non-sporulating bacteria reactivate. A family of extracellular bacterial proteins, known as resuscitation-promoting factors (Rpfs), has previously been shown to stimulate growth of dormant mycobacteria. While Rpf proteins are clearly peptidoglycan glycosidases, the mechanism and role of Rpf in mediating reactivation remains unclear. Here we use a yeast two-hybrid screen to identify potential binding partners of RpfB and report the interaction between RpfB and a putative mycobacterial endopeptidase, which we named Rpf-interacting protein A (RipA). This interaction was confirmed by in vitro and in vivo co-precipitation assays. The interacting domains map to the C-termini of both proteins, near predicted enzymatic domains. We show that RipA is a secreted, cell-associated protein, found in the same cellular compartment as RpfB. Both RipA and RpfB localize to the septa of actively growing bacteria by fluorescence microscopy. Finally, we demonstrate that RipA is capable of digesting cell wall material and is indeed a peptidoglycan hydrolase. The interaction between these two peptidoglycan hydrolases at the septum suggests a role for the complex in cell division, possibly during reactivation.