A Single Nucleotide Change in the polC DNA Polymerase III in Clostridium thermocellum Is Sufficient To Create a Hypermutator Phenotype.

Clostridium thermocellum is a thermophilic, anaerobic bacterium that natively ferments cellulose to ethanol and is a candidate for cellulosic biofuel production. Recently, we identified a hypermutator strain of C. thermocellum with a C669Y mutation in the polC gene, which encodes a DNA polymerase III enzyme. Here, we reintroduced this mutation using recently developed CRISPR tools to demonstrate that this mutation is sufficient to recreate the hypermutator phenotype. The resulting strain shows an approximately 30-fold increase in the mutation rate. This mutation is hypothesized to function by interfering with metal ion coordination in the PHP (polymerase and histidinol phosphatase) domain, which is responsible for proofreading. The ability to selectively increase the mutation rate in C. thermocellum is a useful tool for future directed evolution experiments. IMPORTANCE Cellulosic biofuels are a promising approach to decarbonize the heavy-duty-transportation sector. A longstanding barrier to cost-effective cellulosic biofuel production is the recalcitrance of cellulose to solubilization. Native cellulose-consuming organisms, such as Clostridium thermocellum, are promising candidates for cellulosic biofuel production; however, they often need to be genetically modified to improve product formation. One approach is adaptive laboratory evolution. Our findings demonstrate a way to increase the mutation rate in this industrially relevant organism, which can reduce the time needed for adaptive evolution experiments.

Modern operative nuances for the management of eloquent high-grade gliomas.

INTRODUCTION: Despite advancements in the treatment of high-grade gliomas (HGG), the rate of tumor recurrence is high and survival rate for the patient is low. Gross total resection has shown increased survival but the location of the tumor in the eloquent brain poses significant risk of morbidity. In this report, we focus on modern surgical nuances for resection of tumors located in the eloquent brain. EVIDENCE ACQUISITION: Research of the literature was conducted using the following search terms: surgical resection of gliomas, high-grade gliomas, and the role of vascular encasement - from 1986-2018. An institutional experience from the first author of this paper was also reviewed for selection of our illustrative cases. EVIDENCE SYNTHESIS: Gross total resection remains the mainstay of therapy for high-grade gliomas. The resection of the peritumoral FLAIR, when possible, has been associated with increased survival but also has the potential to cause increased morbidity. In the eloquent brain, the resection of the tumor itself is possible if attention is given to the interface of the tumor and brain, or if a safe pseudo-interface is created by the surgeon. Tumor-seeding to the ventricular system needs to be avoided. Devascularization, dissection away from the brain, and retractorless brain surgery are key to successful surgical outcomes. Management of the venous and arterial invasion/encasement are also outlined in this report. Technical aspects are discussed with corresponding videos. CONCLUSIONS: High-grade gliomas involving eloquent brain areas require a tailored treatment plan. While the medical treatment is undergoing quick evolution, gross total resection still remains one of the key milestones of treatment for improved survival. Surgical techniques play key role. We propose that encasement and/or the invasion of arteries and veins, should be considered equally as important as the eloquent brain when contemplating the resection of gliomas.