Targeting mutant KRAS with CRISPR-Cas9 controls tumor growth.

KRAS is the most frequently mutated oncogene in human tumors, and its activating mutations represent important therapeutic targets. The combination of Cas9 and guide RNA from the CRISPR-Cas system recognizes a specific DNA sequence and makes a double-strand break, which enables editing of the relevant genes. Here, we harnessed CRISPR to specifically target mutant KRAS alleles in cancer cells. We screened guide RNAs using a reporter system and validated them in cancer cells after lentiviral delivery of Cas9 and guide RNA. The survival, proliferation, and tumorigenicity of cancer cells in vitro and the growth of tumors in vivo were determined after delivery of Cas9 and guide RNA. We identified guide RNAs that efficiently target mutant KRAS without significant alterations of the wild-type allele. Doxycycline-inducible expression of this guide RNA in KRAS-mutant cancer cells transduced with a lentiviral vector encoding Cas9 disrupted the mutant KRAS gene, leading to inhibition of cancer cell proliferation both in vitro and in vivo. Intra-tumoral injection of lentivirus and adeno-associated virus expressing Cas9 and sgRNA suppressed tumor growth in vivo, albeit incompletely, in immunodeficient mice. Expression of Cas9 and the guide RNA in cells containing wild-type KRAS did not alter cell survival or proliferation either in vitro and in vivo. Our study provides a proof-of-concept that CRISPR can be utilized to target driver mutations of cancers in vitro and in vivo.

In vivo high-throughput profiling of CRISPR-Cpf1 activity.

CRISPR from Prevotella and Francisella 1 (Cpf1) is an effector endonuclease of the class 2 CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) gene editing system. We developed a method for evaluating Cpf1 activity, based on target sequence composition in mammalian cells, in a high-throughput manner. A library of >11,000 target sequence and guide RNA pairs was delivered into human cells using lentiviral vectors. Subsequent delivery of Cpf1 into this cell library induced insertions and deletions (indels) at the integrated synthetic target sequences, which allowed en masse evaluation of Cpf1 activity by using deep sequencing. With this approach, we determined protospacer-adjacent motif sequences of two Cpf1 nucleases, one from Acidaminococcus sp. BV3L6 (hereafter referred to as AsCpf1) and the other from Lachnospiraceae bacterium ND2006 (hereafter referred to as LbCpf1). We also defined target-sequence-dependent activity profiles of AsCpf1, which enabled the development of a web tool that predicts the indel frequencies for given target sequences (http://big.hanyang.ac.kr/cindel). Both the Cpf1 characterization profile and the in vivo high-throughput evaluation method will greatly facilitate Cpf1-based genome editing.

Adductor Canal Blocks With Bupivacaine and Magnesium After Same-day Discharge Total Knee Arthroplasty Improve Postoperative Pain Relief and Decrease Opioid Consumption: A Prospective Randomized Controlled Trial.

OBJECTIVES: Adequate pain management is a critical component of facilitating same-day discharge for total knee arthroplasty (TKA). Adductor canal blocks (ACB) have been shown to be an effective technique for managing pain after TKA. The objective of this study was to investigate the impact of adding magnesium to local anesthetic in ACB on postoperative pain, opioid consumption, nausea, and overall patient satisfaction. MATERIALS AND METHODS: A sample of 119 adults undergoing elective unilateral TKA were included. Patients were randomly assigned to receive ACB with magnesium and bupivacaine (n=56) or with bupivacaine only (n=63). Primary outcomes were total opioid consumption in the first 48 hours after surgery and pain scores. Secondary outcomes were the incidence of nausea in the first 48 hours after surgery and total overall satisfaction. RESULTS: Opioid consumption decreased significantly in the Mg group compared with the no-Mg group over the first 24 hours (33.2±3.0 vs. 21.3±2.4, P=0.003), the second 24 hours (35.4±2.7 vs. 27.3±2.3, P=0.026), and the first 48 hours total after surgery (68.6±5.1 vs. 48.6±4.3, P=0.004). Pain scores were reduced in the Mg group (24 h: 5.1±2.3 vs. 3.5±2.0, P=0.000; 48 h: 5.1±1.6 vs. 3.9±1.6, P=0.000). Secondary outcomes showed no difference in the incidence of nausea over the first 48 hours and overall satisfaction. CONCLUSION: The addition of magnesium to local anesthetic in ACB decreases pain scores and opioid consumption, without increasing nausea, when compared with ACB with local anesthetic alone.

Recurrent mitral paravalvular leak: benefits of leak site repair compared to re-replacement.

BACKGROUND: Mitral paravalvular leak (PVL) recurrence after surgical correction has not been well demonstrated. The aims of this study were to evaluate the long-term results of surgical mitral PVL correction, including recurrent PVL, and to elucidate the factors - including surgical technique - that affect the risk of recurrent PVL. METHODS: Eighty-six patients who underwent surgical treatment for mitral PVL were enrolled in this study. Thirty-six patients underwent leak site repair (MVP group), and 50 patients underwent re-replacement (MVR group). Leak site repair was the preferred method and was performed whenever possible. The mean follow-up duration was 58.6±44.1 months (0.1-156.5 months). RESULTS: Operative mortality occurred in 7 patients (8.1%). There were no significant differences in operative mortality or postoperative complications between the groups. Overall survival rates at 5 and 10 years were 67.9% and 48.3%, respectively, without intergroup differences. Recurrent PVL without any evidence of infective endocarditis was found in 25 patients (29.1%). Five- and 10-year PVL-free rates were 69.9% and 18.3%, respectively. The mortality rate of reoperation for recurrent PVL was 35.2% (6/17). The risk factors of recurrent PVL were the MVR group (hazard ratio: 2.865, 95% CI: 1.077-7.619) and presence of extensive dehiscence (>25% of annulus: 2.861, 95% CI: 1.163-7.038). CONCLUSIONS: Recurrent PVL was not infrequent after surgical correction of mitral PVL, and reoperation may be a high-risk procedure. Leak site repair, if it could be performed, would be a good surgical option for mitral PVL because re-replacement was a risk factor for recurrence of PVL.

Proteomics analysis of the non-muscle myosin heavy chain IIa-enriched actin-myosin complex reveals multiple functions within the podocyte.

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.

Suicide mortality of suicide attempt patients discharged from emergency room, nonsuicidal psychiatric patients discharged from emergency room, admitted suicide attempt patients, and admitted nonsuicidal psychiatric patients.

The suicide mortality rate and risk factors for suicide completion of patients who presented to an emergency room (ER) for suicide attempt and were discharged without psychiatric admission, patients who presented to an ER for psychiatric problems other than suicide attempt and were discharged without psychiatric admission, psychiatric inpatients admitted for suicide attempt, and psychiatric inpatients admitted for other reasons were examined. The records of 3,897 patients who were treated at a general hospital in Seoul, Korea, from July 2003 to December 2006 were reviewed. Forty-three of the 3,897 subjects died by suicide during the 2.5-year observation period. Compared to the general Korean population, the suicide mortality rate was 82-fold higher for suicide attempt patients, admitted; 54-fold higher for suicide attempt patients, discharged; 21-fold higher for nonsuicidal patients, admitted; and 11-fold higher for nonsuicidal patients, discharged. In all four groups, diagnosis of a depressive disorder and suicide attempt at presentation were each significant independent risk factors for suicide completion. These results highlight the need for suicide prevention strategies for depressed patients who present to the ER or are admitted to a psychiatric ward after a suicide attempt.