In Bacteroidetes, one of the dominant phyla of the mammalian gut, active uptake of large nutrients across the outer membrane is mediated by SusCD protein complexes via a "pedal bin" transport ...mechanism. However, many features of SusCD function in glycan uptake remain unclear, including ligand binding, the role of the SusD lid and the size limit for substrate transport. Here we characterise the β2,6 fructo-oligosaccharide (FOS) importing SusCD from Bacteroides thetaiotaomicron (Bt1762-Bt1763) to shed light on SusCD function. Co-crystal structures reveal residues involved in glycan recognition and suggest that the large binding cavity can accommodate several substrate molecules, each up to ~2.5 kDa in size, a finding supported by native mass spectrometry and isothermal titration calorimetry. Mutational studies in vivo provide functional insights into the key structural features of the SusCD apparatus and cryo-EM of the intact dimeric SusCD complex reveals several distinct states of the transporter, directly visualising the dynamics of the pedal bin transport mechanism.
Objectives/Hypothesis: To develop a technique for computer enhanced robotic transoral supraglottic partial laryngectomy in the canine model.
Study Design: Surgical procedure on the larynx in a canine ...model with a commercially available surgical robot.
Methods: With use of the da Vinci Surgical Robot (Intuitive Surgical, Inc., Sunnyvale, CA), the supraglottic partial laryngectomy was performed on a mongrel dog that had been orotracheally intubated using general anesthesia. The videoscope and the 8 mm end‐effectors of the robotic system were introduced through three ports, transorally. The surgical procedure was performed remotely from the robotic system console. The procedure was documented with still and video photography.
Results: Supraglottic partial laryngectomy was successfully performed using the da Vinci Surgical Robot, with 8 mm instrumentation. The robotic system allowed for celerity and accuracy secondary to findings specific to the surgical approach, including excellent hemostasis, superb visualization of the operative field with expeditious identification of laryngeal submucosal soft tissue and skeletal landmarks, and multiplanar transection of tissues. In addition, the use of the robotic system also was found to have technical advantages inherent in robotic surgery, including the use of “wristed” instrumentation, tremor abolition, motion scaling, and three‐dimensional vision.
Conclusions: The da Vinci Surgical Robot allowed for successful robotic transoral supraglottic partial laryngectomy in the canine model.
•Transoral surgery is FDA approved for removal of oropharynx cancer.•Credentialing and quality assurance can standardize surgical clinical trials.•Our results may apply to other surgical disciplines ...and clinical trials.
Understanding the role of transoral surgery in oropharyngeal cancer (OPC) requires prospective, randomized multi-institutional data. Meticulous evaluation of surgeon expertise and surgical quality assurance (QA) will be critical to the validity of such trials. We describe a novel surgeon credentialing and QA process developed to support the ECOG-ACRIN Cancer Research Group E3311 (E3311) and report outcomes related to QA.
E3311 was a phase II randomized clinical trial of transoral surgery followed by low- or standard-dose, risk-adjusted post-operative therapy with stage III-IVa (AJCC 7th edition) HPV-associated OPC. In order to be credentialed to accrue to this trial, surgeons were required to demonstrate active hospital credentials and technique-specific surgical expertise with ≥20 cases of transoral resection for OPC. In addition, 10 paired operative and surgical pathology reports from the preceding 24 months were reviewed by an expert panel. Ongoing QA required <10% rate of positive margins, low oropharyngeal bleeding rates, and accrual of at least one patient per 12 months. Otherwise surgeons were placed on hold and not permitted to accrue until re-credentialed using a new series of transoral resections.
120 surgeons trained in transoral minimally invasive surgery applied for credentialing for E3311 and after peer-review, 87 (73%) were approved from 59 centers. During QA on E3311, positive final pathologic margins were reported in 19 (3.8%) patients. Grade III/IV and grade V oropharyngeal bleeding was reported in 29 (5.9%) and 1 (0.2%) of patients.
We provide proof of concept that a comprehensive credentialing process can support multicenter transoral head and neck surgical oncology trials, with low incidence of positive margins and *grade III/V oropharyngeal bleeding.
MYH9 encodes non-muscle myosin heavy chain IIA (NMMHCIIA), the predominant force-generating ATPase in non-muscle cells. Several lines of evidence implicate a role for MYH9 in podocytopathies. ...However, NMMHCIIA's function in podocytes remains unknown. To better understand this function, we performed immuno-precipitation followed by mass-spectrometry proteomics to identify proteins interacting with the NMMHCIIA-enriched actin-myosin complexes. Computational analyses revealed that these proteins belong to functional networks including regulators of cytoskeletal organization, metabolism and networks regulated by the HIV-1 gene nef. We further characterized the subcellular localization of NMMHCIIA within podocytes in vivo, and found it to be present within the podocyte major foot processes. Finally, we tested the effect of loss of MYH9 expression in podocytes in vitro, and found that it was necessary for cytoskeletal organization. Our results provide the first survey of NMMHCIIA-enriched actin-myosin-interacting proteins within the podocyte, demonstrating the important role of NMMHCIIA in organizing the elaborate cytoskeleton structure of podocytes. Our characterization of NMMHCIIA's functions goes beyond the podocyte, providing important insights into its general molecular role.
Objectives/Hypothesis: The trend toward minimally invasive surgery has led to the development and mastery of endoscopic and laparoscopic surgical techniques. These minimally invasive approaches, ...which only two decades ago were either novel or experimental, are now mainstream. More recently, robot‐assisted surgery has evolved as an adjunct to open and endoscopic techniques. Surgical robots are now approved by the United States Food and Drug Administration for a variety of thoracic and abdominal/pelvic surgical procedures. The purpose of this study is to demonstrate the technical feasibility of robot‐assisted microlaryngeal surgery.
Study Design: Experimental surgical manipulation of the larynx in an airway mannequin with a surgical robot.
Methods: A variety of laryngoscopes and mouthgags, coupled with the daVinci Surgical Robot's (Intuitive Surgical, Sunnyvale, CA) 0‐degree and 30‐degree, two‐dimensional and three‐dimensional endoscopes, were utilized to optimize visualization of the larynx in an airway mannequin. Five millimeter and 8 mm microinstruments compatible with the daVinci robot were utilized to manipulate different elements of the larynx. Experiments were recorded with both still and video photography.
Results: The endoscope and robotic arms of the daVinci robot are well suited to airway surgery.
Conclusions: Robot‐assisted laryngeal surgery can be performed with currently available technology. The potential for fine manipulation of tissues, increased freedom of instrument movement, and endolaryngeal suturing may increase the precision of endoscopic laryngeal microsurgery and offers the potential to increase the variety of laryngeal procedures that can be performed endoscopically.
Objectives/Hypothesis: Robotic surgery has significant potential in pharyngeal and microlaryngeal surgery. We demonstrate the use of a surgical robot in pharyngeal and microlaryngeal surgery in a ...cadaver.
Study Design: Six experimental surgical dissections, modeled after commonly performed pharyngeal and microlaryngeal procedures, were performed in a cadaver with a commercially available surgical robot in an operating room suite to demonstrate proof of concept.
Methods: Using the daVinci Surgical Robot (Intuitive Surgical, Sunnyvale, CA), surgical procedures were performed on an edentulous, female cadaver. The procedures included 1) bilateral true vocal cord stripping, 2) rotation of a mucosal flap from the epiglottis to the anterior commissure, 3) partial vocal cordectomy, 4) arytenoidectomy, 5) partial epiglottectomy and thyrohyoid dissection and 6) partial resection of the base of tongue with primary closure. All procedures were timed and documented with still and video photography.
Results: The daVinci Surgical Robot, with currently available instruments, enabled performance of several laryngeal and pharyngeal surgical procedures on a cadaver. Laryngeal and pharyngeal exposure was excellent, instruments movement was unimpeded, tissue handling was delicate and precise, and endolaryngeal suturing was relatively easily performed. The duration of the different robotic cadaver dissections was comparable to procedure duration using conventional techniques.
Conclusions: Using the daVinci Surgical Robot, six different pharyngeal and microlaryngeal dissections were successfully performed in a cadaver. The recent development of surgical robotics has a potential role in pharyngeal and microlaryngeal surgery. Surgical robots offer the ability to manipulate instruments at their distal ends with increased freedom of movement, scaled movement, tremor buffering, and under stereoscopic three‐dimensional visualization. Surgical robots may increase the precision with which we perform currently described procedures; additionally, surgical robots may advance the field of endoscopic laryngeal and pharyngeal surgery.
Background.
Pharmacokinetically guided (PK‐guided) versus body surface area‐based 5‐fluorouracil (5‐FU) dosing results in higher response rates and better tolerability. A paucity of data exists on ...PK‐guided 5‐FU dosing in the community setting.
Patients and Methods.
Seventy colorectal cancer patients, from one academic and five community cancer centers, received the mFOLFOX6 regimen (5‐FU 2,400 mg/m2 over 46 hours every 2 weeks) with or without bevacizumab at cycle 1. The 5‐FU continuous‐infusion dose was adjusted for cycles 2–4 using a PK‐guided algorithm to achieve a literature‐based target area under the concentration‐time curve (AUC). The primary objective was to demonstrate that PK‐guided 5‐FU dosing improves the ability to achieve a target AUC within four cycles of therapy. The secondary objective was to demonstrate reduced incidence of 5‐FU‐related toxicities.
Results.
At cycles 1 and 4, 27.7% and 46.8% of patients achieved the target AUC (20–25 mg × hour/L), respectively (odds ratio OR: 2.20; p = .046). Significantly more patients were within range at cycle 4 compared with a literature rate of 20% (p < .0001). Patients had significantly higher odds of not being underdosed at cycle 4 versus cycle 1 (OR: 2.29; p = .037). The odds of a patient being within range increased by 30% at each subsequent cycle (OR: 1.30; p = .03). Less grade 3/4 mucositis and diarrhea were observed compared with historical data (1.9% vs 16% and 5.6% vs 12%, respectively); however, rates of grade 3/4 neutropenia were similar (33% vs 25%–50%).
Conclusion.
PK‐guided 5‐FU dosing resulted in significantly fewer underdosed patients and less gastrointestinal toxicity and allows for the application of personalized colorectal cancer therapy in the community setting.
A paucity of data exists on pharmacokinetically guided 5 fluorouracil (5‐FU) dosing in the community setting. This multicenter study demonstrated the practicality of using a simple methodology to guide 5‐FU dosing in the community setting and resulted in significantly fewer underdosed patients and less gastrointestinal toxicity.
Lipid nanoparticles (LNPs) are advanced core-shell particles for messenger RNA (mRNA) based therapies that are made of polyethylene glycol (PEG) lipid, distearoylphosphatidylcholine (DSPC), cationic ...ionizable lipid (CIL), cholesterol (chol), and mRNA. Yet the mechanism of pH-dependent response that is believed to cause endosomal release of LNPs is not well understood. Here, we show that eGFP (enhanced green fluorescent protein) protein expression in the mouse liver mediated by the ionizable lipids DLin-MC3-DMA (MC3), DLin-KC2-DMA (KC2), and DLinDMA (DD) ranks MC3 ≥ KC2 > DD despite similar delivery of mRNA per cell in all cell fractions isolated. We hypothesize that the three CIL-LNPs react differently to pH changes and hence study the structure of CIL/chol bulk phases in water. Using synchrotron X-ray scattering a sequence of ordered CIL/chol mesophases with lowering pH values are observed. These phases show isotropic inverse micellar, cubic Fd3m inverse micellar, inverse hexagonal Formula: see text and bicontinuous cubic Pn3m symmetry. If polyadenylic acid, as mRNA surrogate, is added to CIL/chol, excess lipid coexists with a condensed nucleic acid lipid Formula: see text phase. The next-neighbor distance in the excess phase shows a discontinuity at the Fd3m inverse micellar to inverse hexagonal Formula: see text transition occurring at pH 6 with distinctly larger spacing and hydration for DD vs. MC3 and KC2. In mRNA LNPs, DD showed larger internal spacing, as well as retarded onset and reduced level of DD-LNP-mediated eGFP expression in vitro compared to MC3 and KC2. Our data suggest that the pH-driven Fd3m-Formula: see text transition in bulk phases is a hallmark of CIL-specific differences in mRNA LNP efficacy.
The proteasome is a multicatalytic proteinase complex responsible for the degradation of most intracellular proteins, including proteins crucial to cell cycle regulation and programmed cell death, or ...apoptosis. In preclinical cancer models, proteasome inhibitors induce apoptosis, have in vivo antitumor efficacy, and sensitize malignant cells and tumors to the proapoptotic effects of conventional chemotherapeutics and radiation therapy. Interestingly, transformed cells display greater susceptibility to proteasome inhibition than nonmalignant cells. Therefore, proteasome inhibition holds promise as a novel approach to the treatment of cancer. Inhibitors of the proteasome impact on cells in part through down-regulation of nuclear factor kappaB, but also through modulation of cell cycle proteins and other pro- and antiapoptotic pathways. Bortezomib (VELCADE; formerly PS-341), the first such inhibitor to undergo clinical testing, has demonstrated impressive antitumor activity and manageable toxicities in Phase I and II trials both as a single agent, and in combination with other drugs. It has been approved recently by the Food and Drug Administration for therapy of patients with multiple myeloma who have received at least two prior regimens and progressed on the last of these. Ongoing preclinical evaluations of the mechanisms that underlie the antitumor effects of proteasome inhibitors, and clinical trials in a variety of tumor types, will allow additional refinement of the role these agents will play in cancer therapy. Below we discuss the rationale behind targeting the proteasome for cancer therapy, and review the preclinical and clinical data on proteasome inhibitors alone, and in combination with conventional chemotherapeutics.
To achieve clinical validation of cutoff values for newborn screening by tandem mass spectrometry through a worldwide collaborative effort.
Cumulative percentiles of amino acids and acylcarnitines in ...dried blood spots of approximately 25–30 million normal newborns and 10,742 deidentified true positive cases are compared to assign clinical significance, which is achieved when the median of a disorder range is, and usually markedly outside, either the 99th or the 1st percentile of the normal population. The cutoff target ranges of analytes and ratios are then defined as the interval between selected percentiles of the two populations. When overlaps occur, adjustments are made to maximize sensitivity and specificity taking all available factors into consideration.
As of December 1, 2010, 130 sites in 45 countries have uploaded a total of 25,114 percentile data points, 565,232 analyte results of true positive cases with 64 conditions, and 5,341 cutoff values. The average rate of submission of true positive cases between December 1, 2008, and December 1, 2010, was 5.1 cases/day. This cumulative evidence generated 91 high and 23 low cutoff target ranges. The overall proportion of cutoff values within the respective target range was 42% (2,269/5,341).
An unprecedented level of cooperation and collaboration has allowed the objective definition of cutoff target ranges for 114 markers to be applied to newborn screening of rare metabolic disorders.
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