ARF1 and GBF1 Generate a PI4P-Enriched Environment Supportive of Hepatitis C Virus Replication

Cellular levels of phosphatidylinositol 4-phosphate (PI4P) have been shown to be upregulated during RNA replication of several viruses, including the HCV replicon model. However, whether PI4P is required in an infectious HCV model remains unknown. Moreover, it is not established whether the host transport machinery is sequestered by the generation of PI4P during HCV infection. Here we found that PI4P was enriched in HCV replication complexes when Huh7.5.1 cells were infected with JFH1. HCV replication was inhibited upon overexpression of the PI4P phosphatase Sac1. The PI4P kinase PI4KIII$\beta$ was also found to be required for HCV replication. Moreover, the vesicular transport proteins ARF1 and GBF1 colocalized with PI4KIIIβ and were both required for HCV replication. During authentic HCV infection, PI4P plays an integral role in virus replication

Management of granulomatous lobular mastitis: an international multidisciplinary consensus (2021 edition)

Granulomatous lobular mastitis (GLM) is a rare and chronic benign inflammatory disease of the breast. Difficulties exist in the management of GLM for many front-line surgeons and medical specialists who care for patients with inflammatory disorders of the breast. This consensus is summarized to establish evidence-based recommendations for the management of GLM. Literature was reviewed using PubMed from January 1, 1971 to July 31, 2020. Sixty-six international experienced multidisciplinary experts from 11 countries or regions were invited to review the evidence. Levels of evidence were determined using the American College of Physicians grading system, and recommendations were discussed until consensus. Experts discussed and concluded 30 recommendations on historical definitions, etiology and predisposing factors, diagnosis criteria, treatment, clinical stages, relapse and recurrence of GLM. GLM was recommended as a widely accepted definition. In addition, this consensus introduced a new clinical stages and management algorithm for GLM to provide individual treatment strategies. In conclusion, diagnosis of GLM depends on a combination of history, clinical manifestations, imaging examinations, laboratory examinations and pathology. The approach to treatment of GLM should be applied according to the different clinical stage of GLM. This evidence-based consensus would be valuable to assist front-line surgeons and medical specialists in the optimal management of GLM.Improving the Ability of Diagnosis and Treatment of Difficult Disease

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

Sac1 overexpression inhibits HCV replication.

<p>(A) Huh 7.5.1 cells transfected with pEGFP-Sac1 or mock and then cell lysates were analyzed by immunoblotting with the indicated antibodies. (B) Huh 7.5.1 cells transfected with pEGFP-Sac1 and infected with JFH1 were stained with antibodies against PI4P (red). (C) Huh 7.5.1 cells transfected with pEGFP-Sac1 and infected with JFH1 were stained with antibodies against HCV core (red). (D) Huh 7.5.1 cells transfected with pEGFP-Sac1 and infected with JFH1 were stained with antibodies against HCV NS5A (red). (E) Huh 7.5.1 cells were transfected with pEGFP-Sac1 and infected with JFH1. Total RNA was isolated and reversely transcribed and then quantitive PCR was performed. (F) Huh7.5.1 cells were transfected with pEGFP-Sac1 and infected with Jc1FLAG2(p7-nsGluc2A) for 3 days. Gaussia luciferase activity and cellular ATP levels were measured. The normalized luciferase activities were then divided by the normalized luciferase activity from mock treatment.</p

GBF1 is required for HCV replication.

<p>(A) Huh7.5.1 cells were treated with different dosages of BFA or GCA as indicated for 1 hour and then infected with JFH1 for 2 days. Total RNA was isolated and reverse transcribed and then quantitive PCR was performed. (B) Huh7.5.1 cells were treated with 20 µM GCA for 1 hour and then infected with JFH1 for 3 days. Cell lysates were analyzed by immunoblotting with the indicated antibodies. (C) Huh7.5.1 cells were treated with siRNA against GBF1 or control siRNA for 3 days and then infected with JFH1 for 3 days. Cell lysates were analyzed by immunoblotting with the indicated antibodies. (D) Huh7.5.1 cells were treated with siRNA against GBF1 or control siRNA for 3 days and then infected with JFH1 for 3 days. Total RNA was isolated and reverse transcribed and then quantitive PCR was performed.</p

ARF1 is required for HCV replication.

<p>(A) Huh7.5.1 cells were treated with siRNA against PI4KIIIβ or control siRNA for 3 days and then infected with JFH1 for 3 days. Cell lysates were analyzed by immunoblotting with the indicated antibodies. (B) Huh7.5.1 cells were treated with siRNA against ARF1 or control siRNA for 3 days and then infected with JFH1 for 3 days. Total RNA was isolated and reverse transcribed and then quantitive PCR was performed. (C) Huh7.5.1 cells were treated with siRNA against ARF1(#6 sequence or #8 sequence) or control siRNA and then infected with Jc1FLAG2(p7-nsGluc2A) for 3 days. Gaussia luciferase activity and cellular ATP levels were measured. The normalized luciferase activities were then divided by the normalized luciferase activity from mock treatment.</p

PI4KIIIβ colocalizes with ARF1 or GBF1.

<p>(A) Huh 7.5.1 cells transfected with a HA-tagged ARF1 construct and infected with JFH1 or mock were stained with antibodies against HA (red) or PI4KIIIβ (green). (B) Huh 7.5.1 cells infected with JFH1 or mock were stained with antibodies against PI4KIIIβ (red) or GBF1 (green).</p

Primers used for quantitative RT-PCR.

a<p>F, forward; R, reverse.</p

PI4P is rearranged during HCV infection.

<p>(A) Huh 7.5.1 cells infected or uninfected with JFH1 were stained with antibodies against PI4P (red) or β-COP (green). (B) Huh 7.5.1 cells infected with JFH1 were treated with 90 IU/ml IFN or mock infection and then stained with antibodies against PI4P (red) or β-COP (green). (C) Huh 7.5.1 cells infected or uninfected with JFH1 were stained with antibodies against PI4P (red) or NS-5A (green).</p