Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Ramucirumab plus docetaxel versus placebo plus docetaxel in patients with locally advanced or metastatic urothelial carcinoma after platinum-based therapy (RANGE): a randomised, double-blind, phase 3 trial

Few treatments with a distinct mechanism of action are available for patients with platinum-refractory advanced or metastatic urothelial carcinoma. We assessed the efficacy and safety of treatment with docetaxel plus either ramucirumab-a human IgG1 VEGFR-2 antagonist-or placebo in this patient population

Study on the Pathogenic and Molecular Evolution of Avian Influenza Viruses Subtype H6N1 in Experimental and Field chickens

在過去幾年中的病例我們經常可由野外雞群中分離到H6N1家禽流行性感冒病毒。由高病原性家禽流行性感冒爆發的歷史，幾乎所有事件皆源自於低病原性H5或H7病毒經由突變後產生。本實驗為瞭解台灣H6血清亞型病毒是否也有如H5、H7病毒因繼代而發生病原性改變，我們選擇三株H6N1病毒，兩株雞源一株鴨源經由極限稀釋（limiting dilution）及蔗糖梯度離心純化後將病毒個別接種在SPF雞及鴨，並在雞或鴨連續繼代十代。雖然繼代的雞及鴨並無顯現臨床症狀，但在以4-6週齡SPF雞隻做病原性試驗時部分雞隻因全身性感染剖檢下呈現肺炎、腎炎及胰臟炎病變，病毒接種在雞胚纖維芽母細胞（CEF）不加胰蛋白酶情況下會產生細胞病變效應（CPE）。在比較IVPI、ICPI、及HA切割位序列後發現繼代十代病毒致病力並無顯著增加，但病變發生比例隨著繼代而產生增加趨勢。由親緣關係分析，台灣H6N1病毒是屬於歐亞大陸病毒群和香港A/duck/HK/3461/99相似度最高。將原始病毒株及繼代後病毒株以點鼻方式接種在BALB/c小鼠作為哺乳動物模式，發現並不會引起全身性感染，白血球數並不受病毒接種影響而改變，體重也不受影響持續增加，可見台灣H6N1家禽流行性感冒病毒應不會直接感染哺乳動物。In last several years, the most frequent AIV isolated from chicken flocks was subtype H6N1. From the HPAI outbreak history, almost all the outbreaks were originated with the appearance of low virulent H5 or H7, then mutated to high pathogenic H5 or H7, respectively. In this study, we selected three isolates of H6N1, two chicken-origin and one duck-origin, after purify by means of limiting dilution and sucrose gradient ultracentrifugation, the viruses were inoculated into SPF chickens and ducks, respectively, and then were serially passed in SPF chickens/ducks for ten passages. Although the inoculated chickens/ducks showed no clinical signs, however, upon autopsy or necropsy pneumonia, nephosis and pancretitis of some inoculated chicken were noted, indicating possible systemic infection. The viruses could induce CPE in CEF cultures without adding trypsin. After ten passages, the virulence of the viruses did not changed significantly, ICPI, IVPI , rate of necropsy lesions, and the amino acid sequences at the HA cleavage site when were compared. Phylogenic analysis of the HA sequence revealed that these isolates belong to Eurasia lineages with high similarity to Hong Kong isolate A/duck/HK/3461/99. The AIV of different chicken-passage were intranasally inoculated into BALB/c mice. The AIV did not induce systemic infection in mice, and results of body weight gain and WBC counts did not show significant change among the mice inoculated with viruses of different passages.目錄 英文摘要.............................................. I 中文摘要.............................................. II 目錄.................................................. III 表次.................................................. V 圖次.................................................. VI 第一章 緒言........................................... 1 第二章 文獻探討....................................... 2 第一節 家禽流行性感冒病毒歷史背景................... 2 第二節 家禽流行性感冒病毒性狀....................... 5 一、病毒特性...................................... 5 二、病毒結構及基因體.............................. 5 三、病毒的複製.................................... 11 四、病毒抗原性改變特性...................... 11 第三節 宿主範圍與致病機制........................... 12 一、自然宿主範圍.................................. 12 二、家禽流行性感冒病毒致病機制.................... 14 第四節 家禽流行性感冒病毒感染人類................... 15 第五節 病毒實驗室診斷............................... 17 一、病原直接分離與同定............................ 17 二、 直接證實病毒之存在........................... 18 三、免疫螢光試驗病毒蛋白.......................... 18 四、證實病毒核酸之存在............................ 18 五、由家禽流行性感冒病毒血清抗體證實感染.......... 19 第六節 實驗室病原性評估............................. 20 一、病原性之評估.................................. 20 二、高病原性家禽流行性感冒之認定.................. 20 第三章 材料與方法.................................... 22 第一節 病毒來源..................................... 22 第二節 實驗動物.................................... 22 第三節 試驗方法.................................... 22 一、病毒增殖...................................... 22 二、病毒分離...................................... 23 三、病毒純化...................................... 23 四、病毒力價測定.................................. 24 五、病毒致病力試驗................................ 25 六、血清抗體檢測.................................. 25 七、病毒核酸的萃取................................ 26 八、聚合酶鏈反應人工引子與反應條件................ 26 九、反轉錄聚合酶鏈反應產物的確認.................. 28 十、反轉錄聚合酶鏈反應產物的純化.................. 29 十一、反轉錄聚合酶鏈反應產物選殖.................. 29 十二、反轉錄聚合酶鏈反應產物的定序反應............ 31 十三、定序結果分析................................ 31 第四節 動物試驗..................................... 31 一、病毒分佈試驗.................................. 31 二、家禽流行性感冒病毒繼代試驗.................... 33 三、家禽流行性感冒病毒哺乳動物模式試驗............ 34 第四章 結果........................................... 35 第一節 病毒增殖..................................... 35 第二節 病毒純化.................................... 35 第三節 病毒分佈與動物血清檢出試驗................... 35 第四節 台灣家禽流行性感冒病毒H6N1血清亞型致病力評估. 36 第五節 台灣家禽流行性感冒病毒H6N1血清亞型親緣分析... 37 第六節 台灣家禽流行性感冒病毒對哺乳動物致病力....... 40 第五章 討論......................................... 67 參 考 文 獻........................................... 7

Modulational instabilities and breaking strength for deep-water wave groups

Progression of nonlinear wave groups to breaking was studied numerically and experimentally. Evolution of such wave group parameters as distance to breaking and modulation depth was described. Numerical model demonstrated a good qualitative agreement with experimental results in describing the behaviour of distance to breaking and modulation depth as functions of initial wave steepnesses. It was shown that energy loss appears to be a function of modulation depth. Energy loss grows with modulation depth up to a certain threshold of the latter

Five-year outcome of conventional and drug-eluting transcatheter arterial chemoembolization in patients with hepatocellular carcinoma

Abstract Background Currently, no standard of care or therapies have been established for patients with advanced HCC. We evaluated the efficacy and safety of conventional transarterial chemoembolization using gelatin sponges or microspheres plus lipiodol-doxorubicin (cTACE) and TACE with doxorubicin-loaded drug eluting beads (DEB-TACE). Methods This retrospective study included 273 patients who received cTACE (n = 201) or DEB-TACE. Tumor response, survival, and adverse events were evaluated over a 5-year follow-up period. Results During 5-year follow-up, a greater percentage of patients treated with cTACE died than those treated with DEB-TACE (76.1% vs. 66.7%) (P = 0.045). At the last evaluation, all surviving patients had disease progression and no differences were seen between treatment groups. However, the time to disease progression differed between groups; median time to disease progression was 11.0 months for cTACE and 16.0 months for DEB-TACE (P = 0.019). The median survival time was 37 months in both treatment groups. No significant differences were observed between cTACE and DEB-TACE therapies in subgroups of patients with BCLC stage A or stage B + C either in survival time or time to disease progression (P values > 0.05). No significant differences were observed in survival status or disease progression between cTACE and DEB-TACE in patient subgroups with either tumor number > 5 or with the sum of the diameter of largest five HCC tumors being > 7 cm. Conclusions DEB-TACE demonstrates greater long-term benefits than cTACE in treating treatment-naïve patients with HCC. Results of this long-term study support the use of DEB-TACE in treating HCC

Effects of Coronavirus Persistence on the Genome Structure and Subsequent Gene Expression, Pathogenicity and Adaptation Capability

Coronaviruses are able to establish persistence. However, how coronaviruses react to persistence and whether the selected viruses have altered their characteristics remain unclear. In this study, we found that the persistent infection of bovine coronavirus (BCoV), which is in the same genus as SARS-COV-2, led to alterations of genome structure, attenuation of gene expression, and the synthesis of subgenomic mRNA (sgmRNA) with a previously unidentified pattern. Subsequent analyses revealed that the altered genome structures were associated with the attenuation of gene expression. In addition, the genome structure at the 5' terminus and the cellular environment during the persistence were responsible for the sgmRNA synthesis, solving the previously unanswered question regarding the selection of transcription regulatory sequence for synthesis of BCoV sgmRNA 12.7. Although the BCoV variants (BCoV-p95) selected under the persistence replicated efficiently in cells without persistent infection, its pathogenicity was still lower than that of wild-type (wt) BCoV. Furthermore, in comparison with wt BCoV, the variant BCoV-p95 was not able to efficiently adapt to the challenges of alternative environments, suggesting wt BCoV is genetically robust. We anticipate that the findings derived from this fundamental research can contribute to the disease control and treatments against coronavirus infection including SARS-CoV-2

The Impacts of Antivirals on the Coronavirus Genome Structure and Subsequent Pathogenicity, Virus Fitness and Antiviral Design

With the global threat of SARS-CoV-2, much effort has been focused on treatment and disease control. However, how coronaviruses react to the treatments and whether the surviving viruses have altered their characteristics are also unanswered questions with medical importance. To this end, bovine coronavirus (BCoV), which is in the same genus as SARS-CoV-2, was used as a test model and the findings were as follows. With the treatment of antiviral remdesivir, the selected BCoV variant with an altered genome structure developed resistance, but its pathogenicity was not increased in comparison to that of wild type (wt) BCoV. Under the selection pressure of innate immunity, the genome structure was also altered; however, neither resistance developed nor pathogenicity increased for the selected BCoV variant. Furthermore, both selected BCoV variants showed a better efficiency in adapting to alternative host cells than wt BCoV. In addition, the previously unidentified feature that the spike protein was a common target for mutations under different antiviral treatments might pose a problem for vaccine development because spike protein is a common target for antibody and vaccine designs. The findings derived from this fundamental research may contribute to the disease control and treatments against coronaviruses, including SARS-CoV-2

Preparation of Chicken Anemia Virus (CAV) Virus-Like Particles and Chicken Interleukin-12 for Vaccine Development Using a Baculovirus Expression System

Chicken infectious anemia (CIA) is a poultry disease that causes huge economic losses in the poultry industry worldwide. Commercially available CIA vaccines are derived from wild-type chicken anemia viruses (CAVs) by serial passage in cells or chicken embryos. However, these vaccinal viruses are not completely attenuated; therefore, they can be transmitted vertically and horizontally, and may induce clinical symptoms in young birds. In this study, we sought to eliminate these issues by developing a subunit vaccine exploiting the CAV structural proteins, engineering recombinant baculovirus-infected Spodoptera frugiperda (Sf9) cells that contained both the viral protein 1 (VP1) and VP2 of CAV. Moreover, we produced single-chain chicken interleukin-12 (chIL-12) in the same system, to serve as an adjuvant. The recombinant VP1 was recognized by chicken anti-CAV polyclonal antibodies in Western blotting and immunofluorescence assays, and the bioactivity of the recombinant chIL-12 was confirmed by stimulating interferon-γ (IFN-γ) secretion in chicken splenocytes. Furthermore, the ability of the recombinant VP1 to generate self-assembling virus-like particles (VLPs) was confirmed by transmission electron microscopy. Specific pathogen-free (SPF) chickens inoculated with VLPs and co-administered the recombinant chIL-12 induced high CAV-specific antibodies and cell-mediated immunity. Taken together, the VLPs produced by the baculovirus expression system have the potential to be a safe and effective CIA vaccine. Finally, we demonstrated the utility of recombinant chIL-12 as an adjuvant for poultry vaccine development