Targeting miR-223 in neutrophils enhances the clearance of Staphylococcus aureus in infected wounds

Abstract Argonaute 2 bound mature microRNA (Ago2‐miRNA) complexes are key regulators of the wound inflammatory response and function in the translational processing of target mRNAs. In this study, we identified four wound inflammation‐related Ago2‐miRNAs (miR‐139‐5p, miR‐142‐3p, miR‐142‐5p, and miR‐223) and show that miR‐223 is critical for infection control. miR‐223Y/− mice exhibited delayed sterile healing with prolonged neutrophil activation and interleukin‐6 expression, and markedly improved repair of Staphylococcus aureus‐infected wounds. We also showed that the expression of miR‐223 was regulated by CCAAT/enhancer binding protein alpha in human neutrophils after exposure to S. aureus peptides. Treatment with miR‐223Y/−‐derived neutrophils, or miR‐223 antisense oligodeoxynucleotides in S. aureus‐infected wild‐type wounds markedly improved the healing of these otherwise chronic, slow healing wounds. This study reveals how miR‐223 regulates the bactericidal capacity of neutrophils at wound sites and indicates that targeting miR‐223 might be of therapeutic benefit for infected wounds in the clinic

オウヨウセイブツカガクブ1ネンセイ ヲ タイショウ トシタ ショクイクトライアル ノ ジッシ ト ヒョウカ ジュギョウサンカシャ ヘノ アンケート ニヨル ケントウ

応用生物科学部1年生を対象に,食育トライアル授業として,食の生産,安全管理,健康管理などに関する10回の講義と2回の農業体験(種まき,収穫作業等)を実施し,学生の授業への出席状況及び授業前後のアンケートによって,プログラムの内容および学生の食育に対する知識,意欲等の変化を評価した。応用生物科学部全学科の学生に授業への参加を呼びかけた結果,授業の出席者は27名であり,栄養科学科の学生が20名と最も多く,参加学生の学科構成に偏りがみられた。授業の初回に行ったアンケートの回答者は24名,最終回に行ったアンケートの回答者は11名であった。授業後のアンケートでは,「食育活動への興味は深まったか」「知りたい情報は得られたか」「参加後,農業と食の関連に対する考えが変わったか」「授業として開講された場合受講するか」という問いに対し,それぞれ73～91%が「はい」と回答し,「授業への総合的な満足度」についても73%が「(非常に)満足である」と回答した。「農業」に原点をおいた食育トライアル授業によって,食べ物の成長を通した気づきと感動,生産者への感謝の気持ち,環境への配慮,栄養と健康との関連知識の習得等に寄与することができたと考えられる。今後は授業への参加者数を増やし,結果の一般化可能性について検討するとともに,授業の実現に向けた人的・物的資源の問題について検討する必要がある。We aimed to evaluate changes in both knowledge of dietary education (Shokuiku) and in motivation, in first-year students of the Faculty of Applied Biosciences who attended a voluntary Shokuiku program in 2008. The program comprised 10 lectures on food production, safety management, and health management, as well as 2 farming practicum classes (seed sowing, harvesting, etc.). Twenty of the 27 students who attended the courses belonged to the Department of Nutritional Sciences. We received responses from 24 and 11 students to questionnaires administered before and after the program, respectively. We obtained a "yes" answer 73-91% of the time in response to the following questions : 1) Was your interest in dietary education activities enhanced? 2) Were you able to obtain the information you sought? 3) After attending these courses, did your understanding of the relationship between farming and diet change? and 4) Would you take this course if it were officially offered as a class? Seventy-three percent of students replied that they were very satisfied with the courses. By placing "farming" as the central principle, these courses contributed to food awareness and appreciation, feelings of gratitude toward producers, consideration for the environment, and knowledge acquisition about the relationship between nutrition and health. Future studies should address the generalizability by examining a larger pool of participants. In addition, other issues such as human and material resources should be considered in realization of the Shokuiku program curriculum

Why Are Outcomes Different for Registry Patients Enrolled Prospectively and Retrospectively? Insights from the Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF).

Background: Retrospective and prospective observational studies are designed to reflect real-world evidence on clinical practice, but can yield conflicting results. The GARFIELD-AF Registry includes both methods of enrolment and allows analysis of differences in patient characteristics and outcomes that may result. Methods and Results: Patients with atrial fibrillation (AF) and ≥1 risk factor for stroke at diagnosis of AF were recruited either retrospectively (n = 5069) or prospectively (n = 5501) from 19 countries and then followed prospectively. The retrospectively enrolled cohort comprised patients with established AF (for a least 6, and up to 24 months before enrolment), who were identified retrospectively (and baseline and partial follow-up data were collected from the emedical records) and then followed prospectively between 0-18 months (such that the total time of follow-up was 24 months; data collection Dec-2009 and Oct-2010). In the prospectively enrolled cohort, patients with newly diagnosed AF (≤6 weeks after diagnosis) were recruited between Mar-2010 and Oct-2011 and were followed for 24 months after enrolment. Differences between the cohorts were observed in clinical characteristics, including type of AF, stroke prevention strategies, and event rates. More patients in the retrospectively identified cohort received vitamin K antagonists (62.1% vs. 53.2%) and fewer received non-vitamin K oral anticoagulants (1.8% vs . 4.2%). All-cause mortality rates per 100 person-years during the prospective follow-up (starting the first study visit up to 1 year) were significantly lower in the retrospective than prospectively identified cohort (3.04 [95% CI 2.51 to 3.67] vs . 4.05 [95% CI 3.53 to 4.63]; p = 0.016). Conclusions: Interpretations of data from registries that aim to evaluate the characteristics and outcomes of patients with AF must take account of differences in registry design and the impact of recall bias and survivorship bias that is incurred with retrospective enrolment. Clinical Trial Registration: - URL: http://www.clinicaltrials.gov . Unique identifier for GARFIELD-AF (NCT01090362)

Risk profiles and one-year outcomes of patients with newly diagnosed atrial fibrillation in India: Insights from the GARFIELD-AF Registry.

BACKGROUND: The Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF) is an ongoing prospective noninterventional registry, which is providing important information on the baseline characteristics, treatment patterns, and 1-year outcomes in patients with newly diagnosed non-valvular atrial fibrillation (NVAF). This report describes data from Indian patients recruited in this registry. METHODS AND RESULTS: A total of 52,014 patients with newly diagnosed AF were enrolled globally; of these, 1388 patients were recruited from 26 sites within India (2012-2016). In India, the mean age was 65.8 years at diagnosis of NVAF. Hypertension was the most prevalent risk factor for AF, present in 68.5% of patients from India and in 76.3% of patients globally (P < 0.001). Diabetes and coronary artery disease (CAD) were prevalent in 36.2% and 28.1% of patients as compared with global prevalence of 22.2% and 21.6%, respectively (P < 0.001 for both). Antiplatelet therapy was the most common antithrombotic treatment in India. With increasing stroke risk, however, patients were more likely to receive oral anticoagulant therapy [mainly vitamin K antagonist (VKA)], but average international normalized ratio (INR) was lower among Indian patients [median INR value 1.6 (interquartile range {IQR}: 1.3-2.3) versus 2.3 (IQR 1.8-2.8) (P < 0.001)]. Compared with other countries, patients from India had markedly higher rates of all-cause mortality [7.68 per 100 person-years (95% confidence interval 6.32-9.35) vs 4.34 (4.16-4.53), P < 0.0001], while rates of stroke/systemic embolism and major bleeding were lower after 1 year of follow-up. CONCLUSION: Compared to previously published registries from India, the GARFIELD-AF registry describes clinical profiles and outcomes in Indian patients with AF of a different etiology. The registry data show that compared to the rest of the world, Indian AF patients are younger in age and have more diabetes and CAD. Patients with a higher stroke risk are more likely to receive anticoagulation therapy with VKA but are underdosed compared with the global average in the GARFIELD-AF. CLINICAL TRIAL REGISTRATION-URL: http://www.clinicaltrials.gov. Unique identifier: NCT01090362

Applicability of Preoperative Nuclear Morphometry to Evaluating Risk for Cervical Lymph Node Metastasis in Oral Squamous Cell Carcinoma

Background: We previously reported the utility of preoperative nuclear morphometry for evaluating risk for cervical lymph node metastases in tongue squamous cell carcinoma. The risk for lymph node metastasis in oral squamous cell carcinoma, however, is known to differ depending on the anatomical site of the primary tumor, such as the tongue, gingiva, mouth floor, and buccal mucosa. In this study, we evaluated the applicability of this morphometric technique to evaluating the risk for cervical lymph node metastasis in oral squamous cell carcinoma. Methods: A digital image system was used to measure the mean nuclear area, mean nuclear perimeter, nuclear circular rate, ratio of nuclear length to width (aspect ratio), and nuclear area coefficient of variation (NACV). Relationships between these parameters and nodal status were evaluated by t-test and logistic regression analysis. Results: Eighty-eight cases of squamous cell carcinoma (52 of the tongue, 25 of the gingiva, 4 of the buccal mucosa, and 7 of the mouth floor) were included: 46 with positive node classification and 42 with negative node classification. Nuclear area and perimeter were significantly larger in node-positive cases than in nodenegative cases; however, there were no significant differences in circular rate, aspect ratio, or NACV. We derived two risk models based on the results of multivariate analysis: Model 1, which identified age and mean nuclear area and Model 2, which identified age and mean nuclear perimeter. It should be noted that primary tumor site was not associated the pN-positive status. There were no significant differences in pathological nodal status by aspect ratio, NACV, or primary tumor site. Conclusion: Our method of preoperative nuclear morphometry may contribute valuable information to evaluations of the risk for lymph node metastasis in oral squamous cell carcinoma

Oligomerization-function relationship of EGFR on living cells detected by the coiled-coil labeling and FRET microscopy.

The epidermal growth factor receptor (EGFR) is a well-studied receptor tyrosine kinase and an important anticancer therapeutic target. The activity of EGFR autophosphorylation and transphosphorylation, which induces several cell signaling pathways, has been suggested to be related to its oligomeric state. However, the oligomeric states of EGFRs induced by EGF binding and the receptor-ligand stoichiometry required for its activation are still controversial. In the present study, we performed Förster resonance energy transfer (FRET) measurements by combining the coiled-coil tag-probe labeling method and spectral imaging to quantitatively analyze EGFR oligomerization on living CHO-K1 cell membranes at physiological expression levels. In the absence of its ligands, EGFRs mainly existed as monomers with a small fraction of predimers (~10%), whereas ~70% of the EGFRs formed dimers after being stimulated with the ligand EGF. Ligand-induced dimerization was not significantly affected by the perturbation of membrane components (cholesterol or monosialoganglioside GM3). We also investigated both dose and time dependences of EGF-dependent EGFR dimerization and autophosphorylation. The formation of dimers occurred within 20s of the ligand stimulation and preceded its autophosphorylation, which reached a plateau 90s after the stimulation. The EGF concentration needed to evoke half-maximum dimerization (~1nM) was lower than that for half-maximum autophosphorylation (~8nM), which suggested the presence of an inactive dimer binding a single EGF molecule

Purification Method of Extracellular Vesicles Derived from Human T-Cell Leukemia Virus Type 1-Infected Cells without Virions

To mediate intercellular communication, cells produce extracellular vesicles (EVs). These EVs transport many biomolecules such as proteins, nucleic acids, and lipids between cells and regulate pathophysiological actions in the recipient cell. However, EVs and virus particles produced from virus-infected cells are of similar size and specific gravity; therefore, the separation and purification of these two particles is often controversial. When analyzing the physiological functions of EVs from virus-infected cells, the presence or absence of virus particle contamination must always be verified. The human T-cell leukemia virus type 1 (HTLV-1)-infected cell line, MT-2, produces EVs and virus particles. Here, we validated a method for purifying EVs from MT-2 cell culture supernatants while avoiding HTLV-1 viral particle contamination. EV fractions were collected using a combination of immunoprecipitation with Tim-4, which binds to phosphatidylserine, and polymer precipitation. The HTLV-1 viral envelope protein, gp46, was not detected in the EV fraction. Proteomic analysis revealed that EV-constituted proteins were predominant in this EV fraction. Furthermore, the EVs were found to contain the HTLV-1 viral genome. The proposed method can purify EVs while avoiding virus particle contamination and is expected to contribute to future research on EVs derived from HTLV-1-infected cells