Research Spotlight

Rios, J. J., K. Denton, J. Russell, J. Kozlitina, C. R. Ferreira, A. F. Lewanda, J. E. Mayfield, E. Moresco, S. Ludwig, M. Tang, X. Li, S. Lyon, A. Khanshour, N. Paria, A. Khalid, Y. Li, X. Xie, J. Q. Feng, Q. Xu, Y. Lu, R. E. Hammer, C. A. Wise and B. Beutler (2021). “Germline Saturation Mutagenesis Induces Skeletal Phenotypes in Mice.” J Bone Miner Res 36(8): 1548-1565.

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Proper embryonic and postnatal skeletal development require coordination of myriad complex molecular mechanisms. Disruption of these processes, through genetic mutation, contributes to variation in skeletal development. We developed a high-throughput N-ethyl-N-nitrosourea (ENU)-induced saturation mutagenesis skeletal screening approach in mice to identify genes required for proper skeletal development. Here, we report initial results from live-animal X-ray and dual-energy X-ray absorptiometry (DXA) imaging of 27,607 G3 mice from 806 pedigrees, testing the effects of 32,198 coding/splicing mutations in 13,020 genes. A total of 39.7% of all autosomal genes were severely damaged or destroyed by mutations tested twice or more in the homozygous state. Results from our study demonstrate the feasibility of in vivo mutagenesis to identify mouse models of skeletal disease. Furthermore, our study demonstrates how ENU mutagenesis provides opportunities to create and characterize putative hypomorphic mutations in developmentally essential genes. Finally, we present a viable mouse model and case report of recessive skeletal disease caused by mutations in FAM20B. Results from this study, including engineered mouse models, are made publicly available via the online Mutagenetix database.

Mostafavi, D., M. M. Methani, W. Piedra-Cascón, A. Zandinejad and M. Revilla-León (2021). “Influence of the Rinsing Postprocessing Procedures on the Manufacturing Accuracy of Vat-Polymerized Dental Model Material.” J Prosthodont 30(7): 610-616.

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PURPOSE: To evaluate the influence of rinsing solvents, namely isopropyl alcohol (IPA) and tripropylene glycol monomethyl ether (TPM), and rinsing times (5-, 7-, 9-, and 11-minutes) for the postprocessing procedures on the manufacturing accuracy of an additively manufactured dental model resin material. MATERIAL AND METHODS: The standard tessellation language (STL file) of the digital design of a bar (15 mm × 4 mm × 3 mm) was obtained. A resin dental material (E-Model Light; Envisiontec, Dearborn, MI) and a 3D printer (VIDA HD; Envisiontec) was selected to manufacture all the specimens using the STL file following the recommended printing parameters at a room temperature of 23 °C. Two groups were generated based on the rinsing solvent used on the postprocessing procedures, namely isopropyl alcohol (IPA-group) and tripropylene glycol monomethyl ether (TPM-group). Each group was further divided into 4 subgroups (IPA-1 to IPA-4 and TPM-1 to TPM-4) depending on the rinsing time performed (5-, 7-, 9-, and 11-minutes). Twenty specimens per subgroup were fabricated. The dimensions (length, width, and height) of all the specimens were measured using a low force digital caliper (Absolute Low Force Caliper Series 573; Mitutoyo, Takatsu-ku, Kawasaki, Kanagawa). Each measurement was performed 3 times and the mean value determined. The volume of each specimen was calculated using the formula V = l × w × h. Shapiro-Wilk test revealed that the data were not normally distributed. Data were analyzed using Kruskal-Wallis (α = 0.05), followed by pairwise Mann-Whitney U tests (α = 0.0018). RESULTS: The IPA groups obtained significantly lower trueness and precision values compared with TPM groups (p < 0.0018). Among the IPA groups, IPA-1 subgroup obtained the highest trueness and precision values compared to the rest of the IPA subgroups. The TPM-1 and TPM-2 subgroups obtained the highest trueness and prevision values among the TPM group and among all the groups tested. No significant difference was found between the TMP-1 and TPM-2 subgroups (p > 0.0018). CONCLUSIONS: None of the manufacturing workflows tested were able to manufacture a perfect match of the bar virtual design dimensions. TPM solvent group obtained higher trueness and precision values compared to the IPA solvent group. The IPA-1 subgroup that replicated the manufacturer´s recommendations obtained the highest manufacturing accuracy among the IPA subgroup. TPM solvent used in a rinsing ultrasonic bath between 3 and 4 minutes followed by a second ultrasonic clean bath between 2 and 3 minutes of the just printed vat polymerized dental model specimens obtained the highest manufacturing accuracy values.

Serra-Pastor, B., N. Bustamante-Hernández, A. Fons-Font, M. Fernanda Solá-Ruíz, M. Revilla-León and R. Agustín-Panadero (2021). “Periodontal Behavior and Patient Satisfaction of Anterior Teeth Restored with Single Zirconia Crowns Using a Biologically Oriented Preparation Technique: A 6-Year Prospective Clinical Study.” J Clin Med 10(16).

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OBJECTIVES: The aim of this study was to analyze the behavior of the periodontal tissues around teeth in the anterior region when restored with zirconia single crowns, using a biologically oriented preparation technique (BOPT), over a 6-year follow-up. METHODS: The study investigated tooth-supported single crowns in the anterior region that were fabricated with a zirconia core and feldspathic ceramic covering, in 34 patients. Follow-up analysis took place annually for 6 years, assessing periodontal responses by evaluating the following variables: plaque index (PI); probing depth (PD); gingival index (GI); gingival thickness adjacent to the restoration; and stability of the gingival margin (MS). Any (biological and mechanical) complications were also recorded, as well as the patients’ satisfaction with the treatment. RESULTS: After 6 years’ follow-up, a low mean plaque index was obtained, probing depth was stable, and gingival thickness and margin stability had increased. Complications (biological and mechanical) did not present a statistically significant incidence and a crown survival rate of 97.2% was achieved. Patients’ satisfaction obtained a mean VAS score of 9.04 under 10. CONCLUSION: Teeth that are prepared with BOPT in the anterior region present good periodontal behavior around the restored teeth, particularly in terms of the stability of the gingival margin and increased gingival thickness. Single crowns prepared with BOPT obtain an excellent clinical survival rate, as well as a high score in patients’ satisfaction after 6 years.

Zhang, W., A. S. Bhagwath, Z. Ramzan, T. A. Williams, I. Subramaniyan, V. Edpuganti, R. R. Kallem, K. B. Dunbar, P. Ding, K. Gong, S. A. Geurkink, M. S. Beg, J. Kim, Q. Zhang, A. A. Habib, S. H. Choi, R. Lapsiwala, G. Bhagwath, J. E. Dowell, S. D. Melton, C. Jie, W. C. Putnam, T. H. Pham and D. H. Wang (2021). “Itraconazole Exerts Its Antitumor Effect in Esophageal Cancer By Suppressing the HER2/AKT Signaling Pathway.” Mol Cancer Ther Aug 10. [Epub ahead of print].

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Itraconazole, an FDA-approved antifungal, has antitumor activity against a variety of cancers. We sought to determine the effects of itraconazole on esophageal cancer and elucidate its mechanism of action. Itraconazole inhibited cell proliferation and induced G(1)-phase cell-cycle arrest in esophageal squamous cell carcinoma and adenocarcinoma cell lines. Using an unbiased kinase array, we found that itraconazole downregulated protein kinase AKT phosphorylation in OE33 esophageal adenocarcinoma cells. Itraconazole also decreased phosphorylation of downstream ribosomal protein S6, transcriptional expression of the upstream receptor tyrosine kinase HER2, and phosphorylation of upstream PI3K in esophageal cancer cells. Lapatinib, a tyrosine kinase inhibitor that targets HER2, and siRNA-mediated knockdown of HER2 similarly suppressed cancer cell growth in vitro. Itraconazole significantly inhibited growth of OE33-derived flank xenografts in mice with detectable levels of itraconazole and its primary metabolite, hydroxyitraconazole, in esophagi and tumors. HER2 total protein and phosphorylation of AKT and S6 proteins were decreased in xenografts from itraconazole-treated mice compared to xenografts from placebo-treated mice. In an early phase I clinical trial (NCT02749513) in patients with esophageal cancer, itraconazole decreased HER2 total protein expression and phosphorylation of AKT and S6 proteins in tumors. These data demonstrate that itraconazole has potent antitumor properties in esophageal cancer, partially through blockade of HER2/AKT signaling.

Lawler, P. R., E. C. Goligher, J. S. Berger, M. D. Neal, B. J. McVerry, J. C. Nicolau, M. N. Gong, M. Carrier, R. S. Rosenson, H. R. Reynolds, A. F. Turgeon, J. Escobedo, D. T. Huang, C. A. Bradbury, B. L. Houston, L. Z. Kornblith, A. Kumar, S. R. Kahn, M. Cushman, Z. McQuilten, A. S. Slutsky, K. S. Kim, A. C. Gordon, B. A. Kirwan, M. M. Brooks, A. M. Higgins, R. J. Lewis, E. Lorenzi, S. M. Berry, L. R. Berry, A. W. Aday, F. Al-Beidh, D. Annane, Y. M. Arabi, D. Aryal, L. Baumann Kreuziger, A. Beane, Z. Bhimani, S. Bihari, H. H. Billett, L. Bond, M. Bonten, F. Brunkhorst, M. Buxton, A. Buzgau, L. A. Castellucci, S. Chekuri, J. T. Chen, A. C. Cheng, T. Chkhikvadze, B. Coiffard, T. W. Costantini, S. de Brouwer, L. P. G. Derde, M. A. Detry, A. Duggal, V. Džavík, M. B. Effron, L. J. Estcourt, B. M. Everett, D. A. Fergusson, M. Fitzgerald, R. A. Fowler, J. P. Galanaud, B. T. Galen, S. Gandotra, S. García-Madrona, T. D. Girard, L. C. Godoy, A. L. Goodman, H. Goossens, C. Green, Y. Y. Greenstein, P. L. Gross, N. M. Hamburg, R. Haniffa, G. Hanna, N. Hanna, S. M. Hegde, C. M. Hendrickson, R. D. Hite, A. A. Hindenburg, A. A. Hope, J. M. Horowitz, C. M. Horvat, K. Hudock, B. J. Hunt, M. Husain, R. C. Hyzy, V. N. Iyer, J. R. Jacobson, D. Jayakumar, N. M. Keller, A. Khan, Y. Kim, A. L. Kindzelski, A. J. King, M. M. Knudson, A. E. Kornblith, V. Krishnan, M. E. Kutcher, M. A. Laffan, F. Lamontagne, G. Le Gal, C. M. Leeper, E. S. Leifer, G. Lim, F. G. Lima, K. Linstrum, E. Litton, J. Lopez-Sendon, J. L. Lopez-Sendon Moreno, S. A. Lother, S. Malhotra, M. Marcos, A. Saud Marinez, J. C. Marshall, N. Marten, M. A. Matthay, D. F. McAuley, E. G. McDonald, A. McGlothlin, S. P. McGuinness, S. Middeldorp, S. K. Montgomery, S. C. Moore, R. Morillo Guerrero, P. R. Mouncey, S. Murthy, G. B. Nair, R. Nair, A. D. Nichol, B. Nunez-Garcia, A. Pandey, P. K. Park, R. L. Parke, J. C. Parker, S. Parnia, J. D. Paul, Y. S. Pérez González, M. Pompilio, M. E. Prekker, J. G. Quigley, N. S. Rost, K. Rowan, F. O. Santos, M. Santos, M. Olombrada Santos, L. Satterwhite, C. T. Saunders, R. E. G. Schutgens, C. W. Seymour, D. M. Siegal, D. G. Silva, Jr., M. Shankar-Hari, J. P. Sheehan, A. B. Singhal, D. Solvason, S. J. Stanworth, T. Tritschler, A. M. Turner, W. van Bentum-Puijk, F. L. van de Veerdonk, S. van Diepen, G. Vazquez-Grande, L. Wahid, V. Wareham, B. J. Wells, R. J. Widmer, J. G. Wilson, E. Yuriditsky, F. G. Zampieri, D. C. Angus, C. J. McArthur, S. A. Webb, M. E. Farkouh, J. S. Hochman and R. Zarychanski (2021). “Therapeutic Anticoagulation with Heparin in Noncritically Ill Patients with Covid-19.” N Engl J Med 385(9): 790-802.

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BACKGROUND: Thrombosis and inflammation may contribute to the risk of death and complications among patients with coronavirus disease 2019 (Covid-19). We hypothesized that therapeutic-dose anticoagulation may improve outcomes in noncritically ill patients who are hospitalized with Covid-19. METHODS: In this open-label, adaptive, multiplatform, controlled trial, we randomly assigned patients who were hospitalized with Covid-19 and who were not critically ill (which was defined as an absence of critical care-level organ support at enrollment) to receive pragmatically defined regimens of either therapeutic-dose anticoagulation with heparin or usual-care pharmacologic thromboprophylaxis. The primary outcome was organ support-free days, evaluated on an ordinal scale that combined in-hospital death (assigned a value of -1) and the number of days free of cardiovascular or respiratory organ support up to day 21 among patients who survived to hospital discharge. This outcome was evaluated with the use of a Bayesian statistical model for all patients and according to the baseline d-dimer level. RESULTS: The trial was stopped when prespecified criteria for the superiority of therapeutic-dose anticoagulation were met. Among 2219 patients in the final analysis, the probability that therapeutic-dose anticoagulation increased organ support-free days as compared with usual-care thromboprophylaxis was 98.6% (adjusted odds ratio, 1.27; 95% credible interval, 1.03 to 1.58). The adjusted absolute between-group difference in survival until hospital discharge without organ support favoring therapeutic-dose anticoagulation was 4.0 percentage points (95% credible interval, 0.5 to 7.2). The final probability of the superiority of therapeutic-dose anticoagulation over usual-care thromboprophylaxis was 97.3% in the high d-dimer cohort, 92.9% in the low d-dimer cohort, and 97.3% in the unknown d-dimer cohort. Major bleeding occurred in 1.9% of the patients receiving therapeutic-dose anticoagulation and in 0.9% of those receiving thromboprophylaxis. CONCLUSIONS: In noncritically ill patients with Covid-19, an initial strategy of therapeutic-dose anticoagulation with heparin increased the probability of survival to hospital discharge with reduced use of cardiovascular or respiratory organ support as compared with usual-care thromboprophylaxis. (ATTACC, ACTIV-4a, and REMAP-CAP ClinicalTrials.gov numbers, NCT04372589, NCT04505774, NCT04359277, and NCT02735707.).