Impact of the FilmArray Rapid Multiplex PCR Assay on Clinical Outcomes of Patients with Bacteremia.

Bacteremia is a serious disease with a reported mortality of 30%. Appropriate antibiotic use with a prompt blood culture can improve patient survival. However, when bacterial identification tests based on conventional biochemical properties are used, it takes 2 to 3 days from positive blood culture conversion to reporting the results, which makes early intervention difficult. Recently, FilmArray (FA) multiplex PCR panel for blood culture identification was introduced to the clinical setting. In this study, we investigated the clinical impact of the FA system on decision making for treating septic diseases and its association with patients' survival. Our hospital introduced the FA multiplex PCR panel in July 2018. In this study, blood-culture-positive cases submitted between January and October 2018 were unbiasedly included, and clinical outcomes before and after the introduction of FA were compared. The outcomes included (i) the duration of use of broad-spectrum antibiotics, (ii) the time until the start of anti-MRSA therapy to MRSA bacteremia, and (iii) sixty-day overall survival. In addition, multivariate analysis was used to identify prognostic factors. In the FA group, overall, 122 (87.8%) microorganisms were concordantly retrieved with the FA identification panel. The duration of ABPC/SBT use and the start-up time of anti-MRSA therapy to MRSA bacteremia were significantly shorter in the FA group. Sixty-day overall survival was significantly improved by utilizing FA compared with the control group. In addition, multivariate analysis identified Pitt score, Charlson score, and utilization of FA as prognostic factors. In conclusion, FA can lead to the prompt bacterial identification of bacteremia and its effective treatment, thus significantly improving survival in patients with bacteremia.

[The Pharmaceuticals and Medical Devices Agency's Approach to Facilitate Risk Communication and Its Challenges].

　The issue of drug lag in Japan has been rapidly reduced in recent years, and newly approved drugs now become available on Japanese and international markets at the same time. In this context, the risk management plan (RMP) system was introduced in 2012. RMPs describe important safety concerns recognized by Japan's Pharmaceuticals and Medical Devices Agency (PMDA) and marketing authorization holders (MAHs), as well as safety measures that MAHs request healthcare professionals (HCPs) to follow. The publication of RMPs is expected to support the sharing of drug risk management among HCPs during the postmarketing phase. In addition, to encourage risk communication between HCPs and patients, the PMDA website provides drug guides for patients and other information to promote proper understanding of drugs by patients and their families and enable them to identify serious adverse drug reactions at an early stage. However, the results of surveys conducted by the PMDA in FY2014 and FY2015 revealed low levels of awareness of RMPs and drug guides for patients in hospitals and other healthcare institutions. The surveys also showed that information regarding the proper use of drugs from MAHs and the PMDA was not incorporated into practice at healthcare institutions, resulting in the repeated release of identical safety alerts. To facilitate the increased utilization of risk communication tools, the PMDA has been providing and disseminating these tools through its website. This study addresses those efforts and the associated challenges.

Fyn Accelerates M Phase Progression by Promoting the Assembly of Mitotic Spindle Microtubules.

The mitotic spindle is the major piece of cellular machinery essential for faithful chromosome segregation. Whereas Fyn, a member of Src-family kinases, is known to be localized to the meiotic and mitotic spindle microtubules, the role of Fyn in mitotic spindle formation has not yet been completely elucidated. In this study, we studied the role of Fyn in spindle formation and effects on M-phase progression. Re-expression of Fyn induced increases in the fluorescence intensity of mitotic spindle microtubules in SYF cells having triple knock-out mutations of c-Src, c-Yes, and Fyn. Cold treatment results showed that Fyn increases the maximum length of microtubules in HeLa S3 cells in a manner dependent on Fyn kinase activity. Complete depolymerization of microtubules under cold treatment and the following release into 37 °C revealed that the increase in the microtubule length in Fyn-expressing cells may be attributed to the promotion of microtubule polymerization. After cold treatment, Fyn promotes the accumulation of EB1, which is a plus-end tracking protein and facilitates microtubule growth, in a manner dependent on the kinase activity. Furthermore, Fyn accelerates the M phase progression of cells from nocodazole arrest. These results suggest that Fyn facilitates mitotic spindle formation through the increase in microtubule polymerization, resulting in the acceleration of M-phase progression.

c-Src but not Fyn promotes proper spindle orientation in early prometaphase.

Src family tyrosine kinases (SFKs) participate in mitotic signal transduction events, including mitotic entry, cleavage furrow ingression, and cytokinesis abscission. Although SFKs have been shown to associate with the mitotic spindle, the role of SFKs in mitotic spindle formation remains unclear. Here, we show that c-Src promotes proper spindle orientation in early prometaphase. Src localizes close to spindle poles in a manner independent of Src kinase activity. Three-dimensional analyses showed that Src inhibition induced spindle misorientation, exhibiting a tilting spindle in early prometaphase. Spindle misorientation is frequently seen in SYF cells, which harbor triple knock-out mutations of c-Src, c-Yes, and Fyn, and reintroduction of c-Src but not Fyn into SYF cells rescued spindle misorientation. Spindle misorientation was also observed upon Src inhibition under conditions in which Aurora B was inhibited. Inducible expression of c-Src promoted a properly oriented bipolar spindle, which was suppressed by Src inhibition. Aster formation was severely inhibited in SYF cells upon Aurora B inhibition, which was rescued by reintroduction of c-Src into SYF cells. Furthermore, reintroduction of c-Src facilitated microtubule regrowth from cold-induced depolymerization and accelerated M phase progression. These results suggest that c-Src is involved in spindle orientation through centrosome-mediated aster formation.

Enrichment of cell populations in metaphase, anaphase, and telophase by synchronization using nocodazole and blebbistatin: a novel method suitable for examining dynamic changes in proteins during mitotic progression.

Mitosis is a continuous process to separate replicated chromosomes into two daughter cells through prophase, metaphase, anaphase, and telophase. Although a number of methods have been established to synchronize cells at different phases of the cell cycle, it is difficult to synchronize cells at the specific phases, anaphase and telophase, during mitosis because of the short duration of anaphase. Here, we show that HeLa S3 cells in anaphase and in telophase are successfully enriched by treatment with a combination of low concentrations of the microtubule-depolymerizing agent nocodazole and the myosin II inhibitor blebbistatin. After 9-h release from thymidine block at G1/S phase, addition of nocodazole at 20 ng/ml but not 40 ng/ml ensures rapid release from the nocodazole arrest. Subsequently, the cells are cultured in the presence of 50 µM blebbistatin for 20 and 50 min to enrich cells in anaphase and telophase, respectively. Western blot analysis verifies down-regulation of phospho-histone H3-Ser10, phospho-Aurora A/B/C, and cyclin B1 during M-phase progression. Furthermore, we show how the electrophoretic mobility shifts of the Src-family kinases c-Yes and c-Src can change in each phase of mitosis. These results provide a useful synchronization method for biochemically examining protein dynamics during M-phase progression.