Selective disruption of an oncogenic mutant allele by CRISPR/Cas9 induces efficient tumor regression

Approximately 15% of non-small cell lung cancer cases are associated with a mutation in the epidermal growth factor receptor (EGFR) gene, which plays a critical role in tumor progression. With the goal of treating mutated EGFR-mediated lung cancer, we demonstrate the use of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system to discriminate between the oncogenic mutant and wild-type EGFR alleles and eliminate the carcinogenic mutant EGFR allele with high accuracy. We targeted an EGFR oncogene harboring a single-nucleotide missense mutation (CTG > CGG) that generates a protospacer-adjacent motif sequence recognized by the CRISPR/Cas9 derived from Streptococcus pyogenes. Co-delivery of Cas9 and an EGFR mutation-specific single-guide RNA via adenovirus resulted in precise disruption at the oncogenic mutation site with high specificity. Furthermore, this CRISPR/Cas9-mediated mutant allele disruption led to significantly enhanced cancer cell killing and reduced tumor size in a xenograft mouse model of human lung cancer. Taken together, these results indicate that targeting an oncogenic mutation using CRISPR/Cas9 offers a powerful surgical strategy to disrupt oncogenic mutations to treat cancers; similar strategies could be used to treat other mutation-associated diseases.

Clinical Characteristics of Patients Diagnosed With Odontogenic Rhinosinusitis After Dental Implants

Background and Objectives With the ongoing development of intraoral surgical treatment and invasive dental treatments such as implants, odontogenic rhinosinusitis (ORS) is on the rise. ORS related to dental implants accounts for 8% to 37% of cases. The purpose of this study is to define the characteristics of patients with ORS related to dental implants. Methods From 2015 to 2019, the medical records of 15 patients who developed maxillary sinus disease after receiving dental implants were retrospectively analyzed among patients who visited the ear nose and throat and dentistry departments. We reviewed the chief complaint, assessment, diagnosis, treatment and prognosis of these patients. Results Of the 15 patients, all were diagnosed with ORS. One patient with a post-operative cheek cyst, 1 with fungal sinusitis, 1 with an inverted papilloma, 1 with chronic rhinosinusitis, and 1 with a radicular cyst were diagnosed after surgery. Endoscopic sinus surgery was performed in 14 patients and 2 patients underwent a combined operation. One patient improved after medical treatment. The follow-up period was about 8.6 months. No recurrence was found in any of the patients. Conclusion If an implant problem is suspected based on history-taking and physical examination, active consultation with dentistry is needed to diagnose ORS

Feasibility of Using Phase Change Materials to Control the Heat of Hydration in Massive Concrete Structures

This paper presents experimental results that can be applied to select a possible phase change material (PCM), such as a latent heat material (LHM), to control the hydration heat in mass concrete structures. Five experimental tests (microconduction, simplified adiabatic temperature rise, heat, and compressive strength tests) were conducted to select the most desirable LHM out of seven types of inorganic PCM used in cement mortar and to determine the most suitable mix design. The results of these experimental tests were used to assess the feasibility of using PCM to reduce hydration heat in mass concrete that was examined. The experimental results show that cement mortar containing barium- [Ba(OH)2·8H2O] based PCM has the lowest amount of total hydration heat of the cement pastes. The barium-based PCM provides good latent heat properties that help to prevent volume change and microcracks caused by thermal stress in mass concrete

Identification of Domains Directing Specificity of Coupling to G-proteins for the Melanocortin MC3 and MC4 Receptors

The melanocortin receptors, MC3R and MC4R, are G protein-coupled receptors that are involved in regulating energy homeostasis. Using a luciferase reporter gene under the transcriptional control of a cAMP-responsive element (CRE), the coupling efficiency of the MC4R and MC3R to G-proteins was previously shown to be different. MC4R exhibited only 30-50% of the maximum activity induced by MC3R. To assess the role of the different MC3R and MC4R domains in G-protein coupling, several chimeric MC3R/MC4R receptors were constructed. The relative luciferase activities, which were assessed after transfecting the chimeric receptors into HEK 293T cells, showed that the i3 (3rd intracellular) loop domain has an essential role in the differential signaling of MC3R and MC4R. To reveal which amino acid residue was involved in the MC4R-specific signaling in the i3 loop, a series of mutant MC4Rs was constructed. Reporter gene analysis showed that single mutations of Arg220 to Ala and Thr232 to either Val or Ala increased the relative luciferase activities, which suggests that these specific amino acids, Arg220 and Thr232, in the i3 loop of MC4R play crucial roles in G-protein coupling and the subtype-specific signaling pathways. An examination of the inositol phosphate (IP) levels in the cells transfected with either MC3R or MC4R after being exposed to the melanocortin peptides revealed significant stimulation of IP production by MC3R but no detectable increase in IP production was observed by MC4R. Furthermore, none of the MC4R mutants displayed melanocortin peptide-stimulated IP production. Overall, this study demonstrated that MC3R and MC4R have distinct signaling in either the cAMP- or the inositol phospholipid-mediated pathway with different conformational requirements

Effects of HA and NA glycosylation pattern changes on the transmission of avian influenza A(H7N9) virus in guinea pigs

AbstractAvian influenza H7N9 virus has posed a concern of potential human-to-human transmission by resulting in seasonal virus-like human infection cases. To address the issue of sustained human infection with the H7N9 virus, here we investigated the effects of hemagglutinin (HA) and neuraminidase (NA) N-linked glycosylation (NLG) patterns on influenza virus transmission in a guinea pig model. Based on the NLG signatures identified in the HA and NA genetic sequences of H7N9 viruses, we generated NLG mutant viruses using either HA or NA gene of a H7N9 virus, A/Anhui/01/2013, by reverse genetics on the 2009 pandemic H1N1 virus backbone. For the H7 HA NLG mutant viruses, NLG pattern changes appeared to reduce viral transmissibility in guinea pigs. Intriguingly, however, the NLG changes in the N9 NA protein, such as a removal from residue 42 or 66 or an addition at residue 266, increased transmissibility of the mutant viruses by more than 33%, 50%, and 16%, respectively, compared with a parental N9 virus. Given the effects of HA-NA NLG changes with regard to viral transmission, we then generated the HA-NA NLG mutant viruses harboring the H7 HA of double NLG addition and the N9 NA of various NLG patterns. As seen in the HA NLG mutants above, the double NLG-added H7 HA decreased viral transmissibility. However, when the NA NLG changes occurred by a removal of residue 66 and an addition at 266 were additionally accompanied, the HA-NA NLG mutant virus recovered the transmissibility of its parental virus. These demonstrate the effects of specific HA-NA NLG changes on the H7N9 virus transmission by highlighting the importance of a HA-NA functional balance

Pediatric Radiofrequency Catheter Ablation: Results of Initial 100 Consecutive Cases Including Congenital Heart Anomalies

Radiofrequency catheter ablation (RFCA) has recently become a management option for pediatric tachycardia. We reviewed the records of a total of 100 patients (aged 10 months to 19 yr) who had undergone RFCA, from March 2000 to June 2004. Types of arrhythmia (age, acute success rate) were as follows: atrioventricular reentrant tachycardia (AVRT, 9.0±3.7 yr, 66/67), atrioventricular nodal reentrant tachycardia (AVNRT, 13±2.5 yr, 16/16), ectopic atrial tachycardia (6.4±3.3 yr, 5/5), junctional ectopic tachycardia (10 month, 1/1), ventricular tachycardia (12±4.9 yr, 6/6), postsurgical intraatrial reentrant tachycardia (15.6±4.1 yr, 2/3), twin node tachycardia (4 yr, 0/1), and His bundle ablation (9 yr, 1/1). The age of AVNRT was older than that of AVRT (p=0.002). Associated cardiac disease was detected in 17 patients, including 6 univentricular patients, and 3 Ebstein's anomaly patients. RFCA for multiple accessory pathways required longer fluoroscopic times than did the single accessory pathway (53.9±4.8 vs. 36.2±24.1 min; p=0.03), and was associated with a higher recurrence rate (3/9 vs. 3/53; p=0.03). Regardless of the presence or absence of cardiac diseases, the overall acute success rate was 97% without major complications, the recurrence rate was 8.2%, and the final success rate was 97%. This experience confirmed the efficacy and safety of RFCA in the management of tachycardia in children