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Background: In the last decade there have been significant advances in diagnosing and classifying adult acute myeloid leukemia (AML) based on genomic profiling, enabling risk-stratification and targeted therapies. In 2017 the US FDA approved the first gene mutation targeted therapies for AML with multiple additional targeted therapies since approved or in development. Given the typical acuity of AML at initial presentation however and the current turnaround time for next-generation sequencing (NGS) assays, most patients will start definitive initial therapy before all potentially targetable mutations are known. There is, therefore, a significant need for a fast molecular genotyping test to determine eligibility for personalized therapy in AML. The NCI Myeloid Assay (NMA) is a comprehensive targeted NGS assay on the Ion Torrent Genexus System, a fully automated platform that provides a rapid turnaround time from specimen receipt to clinical reporting. NMA utilizes Thermo Fisher Scientific's Oncomine Myeloid Assay GX and appears ideally suited for use in upcoming AML targeted therapy trials but has yet to be extensively tested in a cohort of AML patient diagnostic samples and compared to a standard targeted "myeloid panel" NGS assay platform (s-NGS). Methods: DNA samples (n=173) extracted from pretreatment bone marrow and/or peripheral blood of adult patients (n=112) diagnosed with de novo AML or high-risk myelodysplastic syndrome (MDS), were blindly tested in parallel using the NMA and s-NGS assays. For the NMA assay, 27.75ng of DNA was put into the Genexus System. All runs, controls, and samples were first analyzed for sequencing quality using established quality control (QC) metrics to assess pass/fail status. For all samples that passed QC metrics, variant results generated by the Ion Torrent Genexus pipeline were manually reviewed prior to being called true positive variants. For the s-NGS, using the ArcherDx Myeloid VariantPlex assay, a DNA input of 50ng was used for library preparation on a dual pre- and post-PCR separated automated liquid-handling workflow. Resulting libraries were sequencing on the Novaseq 6000 (Illumina) and the data analyzed using the Archer Analysis software and filtered as previously described (PMID: 34258102). Results from the two assays were compared for mutations with a variant allele fraction (VAF) >5% occurring in genes of interest in small molecule targeted clinical trials including: FLT3, IDH1, IDH2, JAK2, KIT, NPM1, NRAS, KRAS, and TP53. For FLT3-ITD comparison, the presence or absence of a call by the assay was used. Results: Utilizing a 5% VAF reporting threshold, a total of 171 and 174 variants were detected by NMA and s-NGS assays, respectively. A high rate of concordance was observed between the assays, with NMA detecting 96% of s-NGS variants and s-NGS detecting 95% of NMA variants. The VAF of detected single nucleotide variants was highly correlated (r=0.9848, P We investigated the discordant calls (n=15 total in 11 patients). One patient was correctly identified as having an NRAS p.Gly12 mutation by both approaches, but the resulting mutation was incorrectly annotated by the s-NGS pipeline. Samples from two patients (including one with both blood and marrow tested) were correctly identified as being FLT3 tyrosine kinase domain mutated by both sequencing approaches, although only the major of two missense variants identified by s-NGS was reported by the NMA pipeline. None of these patients, however, would be misclassified. The remaining 11 discordant calls were false negatives (including 6 variants detected by s-NGS but not by NMA). All of these "edge case" variants were detectable by lowering the VAF reporting threshold below 5% (Figure 1B). Conclusions: NMA is an automated sample-to-results workflow that can identify myeloid disorder-associated genomic variants in less than 48 hours from library preparation to clinical reporting. We show that NMA is highly concordant with a standard DNA NGS assay for detecting mutations within recurrently mutated AML genes. Accurate rapid genotyping is required for assignment to initial treatment with targeted therapy, and this technology may be a valuable tool for upcoming clinical trials for patients with myeloid malignancies. Figure 1 Figure 1. Disclosures Zhang: Thermo Fisher Scientific: Current Employment. Sedova: Thermo Fisher Scientific: Current Employment. Huang: Thermo Fisher Scientific: Current Employment. Mittal: Thermo Fisher Scientific: Current Employment. Hatch: Thermo Fisher Scientific: Current Employment. Ni: Thermo Fisher Scientific: Current Employment. Kaznadzey: Thermo Fisher Scientific: Current Employment. Sadis: Thermo Fisher Scientific: Current Employment. Smith: Thermo Fisher Scientific: Current Employment. Williams: Illumina: Other: CRADA. Hourigan: Sellas: Research Funding.

Hexokinase-II (HK-II) catalyzes the first step of glycolysis and also functions as a protective molecule; however, its role in protective autophagy has not been determined. Results showed that inhibition of HK-II diminished, while overexpression of HK-II potentiated, autophagy induced by glucose deprivation in cardiomyocyte and noncardiomyocyte cells. Immunoprecipitation studies revealed that HK-II binds to and inhibits the autophagy suppressor, mTOR complex 1 (TORC1), and that this binding was increased by glucose deprivation. The TOS motif, a scaffold sequence responsible for binding TORC1 substrates, is present in HK-II, and mutating it blocked its ability to bind to TORC1 and regulate protective autophagy. The transition from glycolysis to autophagy appears to be regulated by a decrease in glucose-6 phosphate. We suggest that HK-II binds TORC1 as a decoy substrate and provides a previously unrecognized mechanism for switching cells from a metabolic economy, based on plentiful energy, to one of conservation, under starvation.

Influenza remains a major global health issue and the effectiveness of current vaccines and antiviral drugs is limited by the continual evolution of influenza viruses. Therefore, identifying novel prophylactic or therapeutic treatments that induce appropriate innate immune responses to protect against influenza infection would represent an important advance in efforts to limit the impact of influenza. Cellular pattern recognition receptors (PRRs) recognize conserved structures expressed by pathogens to trigger intracellular signaling cascades, promoting expression of proinflammatory molecules and innate immunity. Therefore, a number of approaches have been developed to target specific PRRs in an effort to stimulate innate immunity and reduce disease in a variety of settings, including during influenza infections. Herein, we discuss progress in immunomodulation strategies designed to target cell-associated PRRs of the innate immune system, thereby, modifying innate responses to IAV infection and/or augmenting immune responses to influenza vaccines.

The Ebola virus (EBOV) envelope glycoprotein (GP) is the primary target of protective immunity. Mature GP consists of two disulfide-linked subunits, GP1 and membrane-bound GP2. GP is highly glycosylated with both N- and O-linked carbohydrates. We measured the influences of GP glycosylation on antigenicity, immunogenicity, and protection by testing DNA vaccines comprised of GP genes with deleted N-linked glycosylation sites or with deletions in the central hypervariable mucin region. We showed that mutation of one of the two N-linked GP2 glycosylation sites was highly detrimental to the antigenicity and immunogenicity of GP. Our data indicate that this is likely due to the inability of GP2 and GP1 to dimerize at the cell surface and suggest that glycosylation at this site is required for achieving the conformational integrity of GP2 and GP1. In contrast, mutation of two N-linked sites on GP1, which flank previously defined protective antibody epitopes on GP, may enhance immunogenicity, possibly by unmasking epitopes. We further showed that although deleting the mucin region apparently had no effect on antigenicity in vitro, it negatively impacted the elicitation of protective immunity in mice. In addition, we confirmed the presence of previously identified B-cell and T-cell epitopes in GP but show that when analyzed individually none of them were neither absolutely required nor sufficient for protective immunity to EBOV. Finally, we identified other potential regions of GP that may contain relevant antibody or T-cell epitopes.

Purpose The purpose of this study was to determine the accuracy and reproducibility of intra-articular step-off and gap displacements measured on plain radiographs using a standard cadaver model. Methods Twenty-two physicians, in a blinded randomized fashion using a standard technique, examined the radiographs of 12 unique combinations of step and gap displacement created by a 3-part intra-articular osteotomy of the distal radius. Observer accuracy, inter- and intraobserver agreement, and tolerance limits were calculated. Results The results of this study suggest that observers, independent of skill level, may measure step-off and gap displacements accurately to within .62 ± .53 mm (95% confidence interval=.59–65). The accuracy of measurement was influenced by the quality of the radiograph. Intraclass correlation coefficient scores showed "substantial" (.78) to "almost perfect" (.81) inter- and intraobserver agreement. Conclusions These data can aid in the interpretation of clinical studies of acute distal radius fractures that are based on plain radiography.

Background: This study was performed to determine if the accuracy of screw placement was improved with use of the Herbert-Whipple cannulated screw compared with use of the AO/ASIF cannulated screw and also to evaluate the functional results in patients with an acute displaced fracture of the waist of the scaphoid treated with open reduction and internal fixation with a cannulated screw. Methods: We retrospectively reviewed the results for thirty-five patients in whom an acute displaced fracture of the waist of the scaphoid had been treated with internal fixation with use of a cannulated screw. The patients were divided into two groups; Group 1 consisted of nineteen patients managed with a 3.5-millimeter cannulated AO/ASIF screw from 1990 through 1997, and Group 2 consisted of sixteen patients managed with a Herbert-Whipple screw from 1993 through 1997. Results: There were no clinical or radiographic differences between the two groups. The average time to union (and standard deviation), confirmed with tomography, was 4.2 ± 1.2 months for Group 1 and 4.0 ± 1.2 months for Group 2. Both screws significantly improved the alignment of the scaphoid and decreased carpal collapse (p < 0.01). Importantly, the use of either cannulated screw improved the height-to-length ratio and the lateral intrascaphoid angle, which were correlated with an increase in the range of motion of the wrist (r = 0.584 and 0.625). In addition, both screws allowed for accurate placement in the central portion of the proximal pole. Regardless of the type of screw used, the time to union increased with increasing age of the patient (r = 0.665) and with increasing initial displacement of the fracture (r = 0.541). Within both groups, the time to union was longer for the patients who smoked (p < 0.01). Conclusions: Within both groups, cannulated screw fixation maintained the corrected fracture alignment and promoted healing and return of function. Our study shows cannulated screws to be a safe and effective method of treatment.

Congenital absence of the scaphoid without associated thumb or radial hypoplasia is a rare condition. This report is the third case presented in the literature of this condition. The case presented is of a patient who presented initially with wrist pain. Radiographs revealed a congenitally absent scaphoid and examination revealed no evidence of thumb hypoplasia. A brief review of the literature on congenital absence of the scaphoid is discussed.

Backgound: Hexokinase II binds to mitochondria and promotes cell survival. Results: Akt phosphorylates HK-II but not the threonine 473 mutant. The phosphomimetic T473D mutant decreases its dissociation from mitochondria induced by G-6P and increases cell viability against stress. Conclusion: Akt phosphorylates HK-II at Thr-473, resulting in increased mitochondrial HK-II and cell protection. Significance: The Akt-HK-II signaling nexus is important in cell survival., Hexokinase II (HK-II) is an enzyme that catalyzes the first step in glycolysis and localizes not only in the cytosol but also at mitochondria. Akt, activated by insulin-like growth factor 1 (IGF-1) treatment in neonatal rat ventricular myocytes, translocates to mitochondria and increases mitochondrial HK-II binding. Expression of an HK-II-dissociating peptide diminished IGF-1-induced increases in mitochondrial HK-II as well as protection against hydrogen peroxide treatment, suggesting an important role of mitochondrial HK-II in IGF-1/Akt-mediated protection. We hypothesized, on the basis of an Akt phosphorylation consensus sequence present in HK-II, that Thr-473 is the target of Akt kinase activity. Indeed, recombinant kinase-active Akt robustly phosphorylates WT HK-II, but not Thr-473 mutants. Phosphomimetic (T473D)HK-II, but not non-phosphorylatable (T473A)HK-II, constitutively increased mitochondrial binding compared with WT HK-II and concomitantly confers greater protection against hydrogen peroxide. Glucose 6-phosphate (G-6P), a product of the catalytic activity of HK-II, is well known to dissociate HK-II from mitochondria. Addition of G-6P to isolated mitochondria dose-dependently dissociates WT HK-II, and this response is inhibited significantly in mitochondria isolated from cardiomyocytes expressing T473D HK-II. Pretreatment with IGF-1 also inhibits G-6P-induced overexpressed or endogenous HK-II dissociation, and this response was blocked by Akt inhibition. These results show that Akt phosphorylation of HK-II at Thr-473 is responsible for the Akt-mediated increase in HK-II binding to mitochondria. This increase is, at least in part, due to the decreased sensitivity to G-6P-induced dissociation. Thus, phosphorylation-mediated regulation of mitochondrial HK-II would be a critical component of the protective effect of Akt.

In 1984, in an effort to address the silicone wear particle problem, titanium implants were developed for the scaphoid, lunate, and trapeziometacarpal joint. The design of these implants closely resembled their silicone counterparts, though some modifications were made to accommodate the properties of unalloyed titanium and enhance their stability. Carpal bone implants act as articulating spacers to help maintain the relationship of adjacent carpal bones after local resection procedures. Their use allows carpal stabilization procedures and provides functional mobility with good strength and pain relief. Their surgical application began in 1985. The 10-year clinical experience seems very promising to date.