Antibody fragments such as Fabs possess properties that can enhance protein and RNA crystallization and therefore can facilitate macromolecular structure determination. In particular, Fab BL3–6 binds to an AAACA RNA pentaloop closed by a GC pair with ∼100 nM affinity. The Fab and hairpin have served as a portable module for RNA crystallization. The potential for general application make it desirable to adjust the properties of this crystallization module in a manner that facilitates its use for RNA structure determination, such as ease of purification, surface entropy or binding affinity. In this work, we used both in vitro RNA selection and phage display selection to alter the epitope and paratope sides of the binding interface, respectively, for improved binding affinity. We identified a 5′-GNGACCC-3′ consensus motif in the RNA and S97N mutation in complimentarity determining region L3 of the Fab that independently impart about an order of magnitude improvement in affinity, resulting from new hydrogen bonding interactions. Using a model RNA, these modifications facilitated crystallization under a wider range of conditions and improved diffraction. The improved features of the Fab–RNA module may facilitate its use as an affinity tag for RNA purification and imaging and as a chaperone for RNA crystallography.

KRAS is the most commonly mutated oncogene in human cancer, with particularly high mutation frequencies in pancreatic cancers, colorectal cancers, and lung cancers [Ostrem, J. M., and Shokat, K. M. (2016) Nat. Rev. Drug Discovery 15, 771–785]. The high prevalence of KRAS mutations and its essential role in many cancers make it a potentially attractive drug target; however, it has been difficult to create small molecule inhibitors of mutant K-Ras proteins. Here, we identified a putative small molecule binding site on K-RasG12D using computational analyses of the protein structure and then used a combination of computational and biochemical approaches to discover small molecules that may bind to this pocket, which we have termed the P110 site, due to its adjacency to proline 110. We confirmed that one compound, named K-Ras allosteric ligand KAL-21404358, bound to K-RasG12D, as measured by microscale thermophoresis, a thermal shift assay, and nuclear magnetic resonance spectroscopy. KAL-21404358 did not bind to four mutants in the P110 site, supporting our hypothesis that KAL-21404358 binds to the P110 site of K-RasG12D. This compound impaired the interaction of K-RasG12D with B-Raf and disrupted the RAF-MEK-ERK and PI3K-AKT signaling pathways. We synthesized additional compounds, based on the KAL-21404358 scaffold with more potent binding and greater aqueous solubility. In summary, these findings suggest that the P110 site is a potential site for binding of small molecule allosteric inhibitors of K-RasG12D.

Study Design. Retrospective cohort. Objective. To compare robotics and navigation for minimally invasive elective lumbar fusion in terms of radiation exposure and time demand. Summary of Background Data. Although various studies have been conducted to demonstrate the benefits of both navigation and robotics over fluoroscopy in terms of radiation exposure, literature is lacking in studies comparing robotics versus navigation. Materials and Methods. Patients who underwent elective one-level or two-level minimally invasive transforaminal lumbar interbody fusion (TLIF) by a single surgeon using navigation (Stryker SpineMask) or robotics (ExcelsiusGPS) were included (navigation 2017–2019, robotics 2019–2021, resulting in prospective cohorts of consecutive patients for each modality). All surgeries had the intraoperative computed tomography workflow. The two cohorts were compared for radiation exposure [fluoroscopy time and radiation dose: image capture, surgical procedure, and overall) and time demand (time for setup and image capture, operative time, and total operating room (OR) time]. Results. A total of 244 patients (robotics 111, navigation 133) were included. The two cohorts were similar in terms of baseline demographics, primary/revision surgeries, and fusion levels. For one-level TLIF, total fluoroscopy time, total radiation dose, and % of radiation for surgical procedure were significantly less with robotics compared with navigation (20 vs. 25 s, P<0.001; 38 vs. 42 mGy, P=0.05; 58% vs. 65%, P=0.021). Although time for setup and image capture was significantly less with robotics (22 vs. 25 min, P<0.001) and operative time was significantly greater with robotics (103 vs. 93 min, P<0.001), there was no significant difference in the total OR time (145 vs. 141 min, P=0.25). Similar findings were seen for two-level TLIF as well. Conclusion. Robotics for minimally invasive TLIF, compared with navigation, leads to a significant reduction in radiation exposure both to the surgeon and patient, with no significant difference in the total OR time.

Study Design: Literature review. Objectives: To review the evidence for surface-based navigation in minimally-invasive spine surgery (MIS), provide an outline for its workflow, and present a wide range of MIS case examples in which surface-based navigation may be advantageous. Methods: A comprehensive review of the literature and compilation of findings related to surface-based navigation in MIS was performed. Workflow and case examples utilizing surface-based navigation were described. Results: The nascent literature regarding surface-based intraoperative navigation (ION) in spine surgery is encouraging and initial studies have shown that surface-based navigation can allow for accurate pedicle screw placement and decreased operative time, fluoroscopy time, and radiation exposure when compared to traditional fluoroscopic imaging. Surface-based navigation may be particularly useful in MIS cervical and lumbar decompressions and MIS lumbar instrumentation cases. Conclusions: Overall, it is possible that surface-based ION will become a mainstay in the armamentarium of enabling technologies utilized by minimally-invasive spine surgeons, but further studies are needed assessing its accuracy, complications, and cost-effectiveness.

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