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Philadelphia virus is Stampado ransomware that encrypts the files on your computer, changes the name of the files, and adds the .locked extension to files it encrypts. Philadelphia ransomware will additionally leave various note files (.txt, .html) in every folder it encrypts files in and Windows Desktop. The note files contain a ransom note that explains what happened to the files and how to pay a ransom in order to acquire a key to decode the encrypted files.
Once a computer becomes infected with Philadelphia ransomware it will encrypt files, change the file names to something random, and replace Windows Desktop Wallpaper with an image of a ransom note. It will also dump files in every folder it encrypts files in that contain a ransom note. The ransomware is customizable depending on the distributor and is currently being sold for $400 USD by a malware developer named The Rainmaker. According to malware developer, Philadelphia ransomware is being sold as a low cost ransomware solution that allows any wannabe criminal to get an advanced ransomware campaign up and running with little expense or complexity.
Philadelphia ransomware is usually dispersed by malicious spam email attachments. The email content employs social engineering in order to trick unsuspecting victims into downloading a file under the guise that it is something it is not. Once the file is manually executed by the user ransomware will begin to advance on the computer system and carry through it's various functions.
It is not recommended to pay ransomware developers to decrypt your files. Instead you can use programs like Shadow Explorer, PhotoRec, or Recuva to restore corrupted files. Decryption software has also been created for this specific ransomware by Emsisoft and it is free to use.
Aliases: Philadelphia virus, Philadelphia ransomware
How to remove Philadelphia virus
1. Download and Install Recuva by Pirform or use the Emsisoft decryptor (and skip steps 2 – 6) to recover your files.
2. Run the program and start the Recuva Wizard.
3. Select All Files and click Next.
4. Select a file location. Click I'm not sure to search everywhere on your computer.
5. Click Start.
6. Select All Files with your mouse and click the Recover button. If you cannot restore your files with Recuva we recommend to try using Shadow Explorer to restore your files.
7. Download and Install Malwarebytes Anti-Malware software to detect and remove malicious files from your computer.
8. Open Malwarebytes and click the Scan Now button – or go to the Scan tab and click the Start Scan button.
9. Once the Malwarebytes scan is complete click the Remove Selected button.
10. To finish the Malwarebytes scan and remove detected threats click the Finish button and restart your computer if promoted to do so.
11. Download and Install HitmanPro by Surfright to perform a second-opinion scan.
12. Open HitmanPro and click Next to start scanning your computer. *If you are using the free version you may chose to create a copy or perform a one-time scan.
13. Once the HitmanPro scan is complete click the Next button.
14. To activate the free version of HitmanPro: enter your email address twice and click the Activate button.
15. Click the Reboot button.
16. Download and Install CCleaner by Piriform to cleanup junk files, repair your registry, and manage settings that may have been changed.
17. Open CCleaner and go to the main Cleaner screen. Click the Analyze button. When the process is complete, click the Run Cleaner button on the bottom right of the program interface.
18. Go to Tools > Startup and search for suspicious entries in each tab starting from Windows all the way to Content Menu. If you find anything suspicious click it and click the Delete button to remove it.
19. Go to the Registry window and click the Scan for Issues button. When the scan is complete click the Fix selected issues… button and click Fix All Selected Issues.
The key to staying protected against future infections is to follow common online guidelines and take advantage of reputable Antivirus and Anti-Malware security software with real-time protection.
Real-time security software
Security software like Malwarebytes and Norton Security have real-time features that can block malicious files before they spread across your computer. These programs bundled together can establish a wall between your computer and cyber criminals.
- Backup your computer and personal files to an external drive or online backup service
- Create a restore point on your computer in case you need to restore your computer to a date before infection
- Avoid downloading and installing apps, browser extensions, and programs you are not familiar with
- Avoid downloading and installing apps, browser extensions, and programs from websites you are not familiar with – some websites use their own download manager to bundle additional programs with the initial download
- If you plan to download and install freeware, open source software, or shareware make sure to be alert when you install the object and read all the instructions presented by the download manager
- Avoid torrents and P2P clients
- Do not open email messages from senders you do not know


text:
- Specification: Weight Capacity: 265 lbs. Weight: 8.8lbs. Dimension: 21.5''Wx24.8''Dx31.5''H . No need to install. Stackable and easy to store
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Product description
This is a garden chair, suitable for outdoor and indoor.
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subset: commoncrawl


text:
- Research article
- Open access
- Published:
Hepatocellular carcinoma-targeted drug discovery through image-based phenotypic screening in co-cultures of HCC cells with hepatocytes
BMC Cancer volume 16, Article number: 810 (2016)
Hepatocellular carcinoma (HCC) is one of the most common malignant cancers worldwide and is associated with substantial mortality. Because HCCs have strong resistance to conventional chemotherapeutic agents, novel therapeutic strategies are needed to improve survival in HCC patients.
Here, we developed a fluorescence image-based phenotypic screening system in vitro to identify HCC-specific drugs in co-cultures of HCC cells with hepatocytes. To this end, we identified two distinctive markers of HCC, CHALV1 and AFP, which are highly expressed in HCC cell lines and liver cancer patient-derived materials. We applied these markers to an HCC-specific drug screening system.
Through pilot screening, we identified three anti-folate compounds that had HCC-specific cytotoxicity. Among them, pyrimethamine exhibited the greatest HCC-specific cytotoxicity. Interestingly, pyrimethamine significantly increased the size and number of lysosomes and subsequently induced the release of cathepsin B from the lysosome to the cytosol, which triggered caspase-3-dependent apoptosis in Huh7 (HCC) but not Fa2N-4 cells (immortalized hepatocytes). Importantly, Fa2N-4 cells had strong resistance to pyrimethamine relative to Huh7 cells in 2D and 3D culture systems.
These results demonstrate that this in vitro image-based phenotypic screening platform has the potential to be widely adopted in drug discovery research, since we promptly estimated anticancer activity and hepatotoxicity and elucidated functional roles of pyrimethamine during the apoptosis process in HCC.
Hepatocellular carcinoma (HCC) is the seventh most common malignant cancer and the third leading cause of cancer-related deaths in the world [1–3]. Over the past decade, the advancements in medical device development, surgical techniques, radiology, liver transplantation, and other therapies have resulted in considerable improvements in HCC treatment [1, 4]. However, the numbers of incident cases and liver cancer deaths have still increased because most HCCs are detected at an advanced stage in patients with underlying liver dysfunction, making it a highly lethal cancer. Moreover, most HCCs are resistant to conventional chemotherapeutic agents, and patients with HCC usually have poor tolerance to systemic chemotherapy because of underlying liver dysfunction. In these situations, liver-targeted drugs with fewer side effects and high efficacy are a desired but unmet need for the treatment of liver cancer.
In this study, we used a fluorescence image-based phenotypic screening approach to specifically target HCC mixed with normal hepatocytes, because heterologous cell types within tumors can actively influence the therapeutic response and shape resistance. Specifically, we aimed to develop liver cancer-specific drugs that could induce cell death in HCC cells, while minimizing the damage to normal hepatocytes, in a mixed cell culture system containing hepatocytes and HCC cells. To distinguish between HCC cells and hepatocytes in this mixed cell culture system, we identified two well established markers of HCC, CHALV1 and AFP, which are highly expressed in HCC cell lines and liver patient samples [5, 6]. Given the heterogeneity of tumor tissues, our approach is expected to be positively utilized for drug discovery.
Anti-folate drugs are used in cancer chemotherapy and act through the inhibition of dihydrofolate reductase (DHFR). Since the inhibition of DHFR blocks nucleotide biosynthesis, anti-folate drugs reduce the proliferation of cancer cells [7]. In particular, pyrimethamine (2,4-diamino-5-p-chlorophenyl-6-ethyl-pyrimidine), a folic acid antagonist, is used to treat protozoal infections. It is also used as an antimalarial drug and a treatment for Toxoplasma gondii infections in immunocompromised patients [8–10]. Recent findings showed that pyrimethamine effectively induces apoptosis in pituitary adenoma cells, peripheral blood lymphocytes, and melanoma cells [11–13]. Although pyrimethamine has feasibility as an anticancer drug, its anticancer effects and functional roles have not been established in HCC. Here, we identified a hitherto unknown mechanism of pyrimethamine-induced apoptosis in HCC cells using fluorescence image-based phenotypic analysis. In order to assess pyrimethamine-induced phenotypic changes and cytotoxic effects in HCC, we applied various cell-based assay models in vitro to the High Content Screening system. We also applied a hepatocellular 3D culture method to this system, which is the appropriate culture model to maintain liver-specific functions and to validate drug efficiency.
Based on these applications, we established an image-based phenotypic screening platform for HCC-specific drug discovery and the functional study of interesting compounds. Additionally, we found that pyrimethamine induced HCC death via lysosome modification and activation of cathepsin B.
Cell culture and labeling
Fa2N-4 cells (an immortalized normal hepatocyte cell line) were purchased from Xenotech (Lenexa, KS, USA), and Huh7, Hep3B, PLC/PRF/5, SNU475 and SNU449 (human hepatocellular carcinoma cell line) were obtained from the Korean Cell Line Bank (KCLB). Huh7.5 [14] was kindly provided by Charles M. Rice (Rockefeller University, New York, USA), and Huh6 [15] was kindly provided by Dr. Ralf Bartenschlager (University of Heidelberg, Germany). Cells were maintained at 37 °C with 95 % humidity and 5 % CO2. After cell attachment (3–6 h), serum-containing plating medium (XenoTech, Lenexa, KS, USA) was replaced with MFE serum free supporting Fa2N-4 cells (SF) medium (XenoTech) which are nutrient rich medium for maintaining Fa2N-4 cells in culture. This is a serum free medium. Huh7 cells (a human HCC cell line) were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, Gaithersburg, MD, USA) supplemented with heat-inactivated 10 % fetal bovine serum (FBS; Gibco) and antibiotics (Gibco) at 37 °C in a humidified incubator under 5 % CO2. For the 3D culture, 8 μl of Matrigel (BD Biosciences, San Jose, CA, USA) was pipetted directly onto the surface and carefully spread to avoid bubbles in 384 well culture plates (Greiner Bio-One, Monroe, NC, USA), then incubated at 37 °C until the Matrigel solidified. Trypsinized single cells from a monolayer were centrifuged at 1,000 rpm, resuspended in 30 ml of supporting culture medium, and plated onto the Matrigel-coated plates at a density of 2 × 103 cells/well. Cells were incubated for 30 min at 37 °C to settle onto the Matrigel, then 10 % Matrigel-Medium was slowly added to each well. After maintaining for 5 days, the Matrigel-Medium was replaced every 2 days.
To distinguish between the Fa2N-4 and Huh7 cells in the mixed culture system, Fa2N-4 cells were labeled with CellLight® Nucleus-GFP (Thermo Fisher Scientific, Marietta, OH, USA). Fa2N-4 cells were infected with BacMam expression vectors encoding fusions of GFP with the SV40 nuclear localization sequence at 30 particles per cell, according to the manufacturer's instructions.
Primary cell culture
Isolated liver cancer tissues were cut into 3 mm3 pieces and washed with 4 °C Hank's balanced salt solution (Lonza, Walkersville, MD, USA) supplemented with 1× antibiotic antimycotic solution (Sigma, St Louis, MO, USA) and 1× penicillin streptomycin (Lonza) in a 100-mm petri dish, then moved to a 15-ml conical tube. Cells were washed three times with bovine serum solution (BS solution) consisting of Dulbecco's modified Eagle's medium: nutrient mixture F-12 (DMEM/F12; Gibco) supplemented with 1× antibiotic antimycotic solution (Sigma), 1× penicillin streptomycin (Lonza), and 10 % bovine serum (Gibco). Then, the cells were resuspended with 10 ml of BS solution and incubated at 4 °C for 16 h. After removing the BS solution and washing with fresh BS solution, tissues were incubated with 2 ml of 2× collagenase II (BD Biosciences) at 37 °C in a shaking chamber for 90 min. After incubation, 10 ml of BS solution was added and the sample was centrifuged at 600 rpm for 2 min. This washing step was performed several times until the supernatant became clear. The pellet was resuspended in hepatocyte basal medium (HBM; Lonza) containing 1× antibiotic antimycotic solution (Sigma), hepatocyte culture medium growth factor solution (Lonza), 10 % FBS (Gibco), and 5 μg/ml of hepatocyte growth factor (HGF; R&D Systems, Minneapolis, MN, USA), and the cells were plated in a collagen type I-coated T-25 flask (BD Biosciences). After incubation for 24 h at 37 °C in an incubator, the cells were washed with phosphate buffered saline (PBS; Lonza) containing 1× antibiotic antimycotic (Sigma) and were replaced with fresh HBM media containing supplements (Lonza).
Reagents and antibodies
Tetramethylrhodamine, methyl ester (TMRM), Hoechst 33342, LysoTracker® Red DND-99, LysoTracker® Green DND-26, 5-(and-6)-chloromethyl-2′,7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA), CellLight® Nucleus-GFP, Alexa Fluor® 633 phalloidin, goat anti-mouse Alexa Fluor® 633, goat anti-rabbit Alexa Fluor® 633, goat anti-mouse Alexa Fluor® 488, and goat anti-rabbit Alexa Fluor® 488 were purchased from Molecular Probes (Thermo Fisher Scientific). Resazurin, pyrimethamine, methotrexate, aminopterin, and most of the drugs were purchased from Sigma. The rabbit polyclonal anti-AFP (Dako, Denmark A/S, Denmark), mouse monoclonal anti-CHALV1 (Abcam, CSP, Cambridge, England), rabbit polyclonal anti-cleaved caspase-3 (Cell Signaling Technology, Danvers, MA, USA), mouse monoclonal anti-phospho-histone H2AX (γ-H2AX) (Millipore, Bedford, MA, USA), and rabbit polyclonal anti-cathepsin B (EMD/Calbiochem, San Diego, CA, USA) antibodies were purchased from each of the indicated companies.
Resazurin assay
Cells were seeded at 5 × 104 cells/well into 96 well microtiter plates (BD Biosciences) and cultured. On the next day, cells were treated with hepatotoxic or safety drugs. After 3 days, cells were supplemented with 44 μM resazurin in the medium. Eight hr later, resazurin reduction was measured colorimetrically (570/600 nm) using a Victor3 (Perkin Elmer, Waltham, MA, USA) plate reader.
High Content Screening (HCS) System
After being treated with the indicated concentrations of various drugs for 24 h, the cells were washed with PBS and stained by fluorescent probes, including TMRM, Hoechst 33342, and CM-H2DCFDA. The cells and probes were incubated together for the first 30 min. Automated live-cell multispectral image acquisition was performed on the Operetta® High Content Screening System using a 20× objective (Perkin Elmer). The fluorescence images were captured according to the following optimal excitation and emission wavelengths of each probe:
485 ± 20 and 515 ± 10 nm for CM-H2DCFDA (ROS)
532 ± 4 and 600 ± 12.5 nm for TMRM (MMP, mitochondrial membrane potential)
405 ± 25 and 455 ± 10 nm for Hoechst 33342 (nuclei)
To capture enough cells (> 100) for the analysis, four image fields were collected from each well, starting at the center. All of the image analysis was performed using Operetta and Harmony 3.5.1 software (Perkin Elmer). A series of measurements from the nuclei, lipids, ROS, and TMRM channel images were obtained for each drug.
Confocal immunofluorescence analysis
For immunofluorescence analysis, cells were fixed with 4 % paraformaldehyde (PFA, Sigma), permeabilized with 0.1 % Triton X-100 (Sigma) in PBS, and then washed three times with PBS. Cells were then incubated with anti-cleaved caspase-3 (Asp175) or anti-cathepsin B in PBS with 10 % normal goat serum (Vector Laboratories, Burlingame, CA, USA) for 12 h at 4 °C in a humidified chamber. Excess antibody was removed by washing three times with PBS. Cells were then incubated with fluorescein-conjugated secondary antibody (Molecular Probes) at a 1:200 dilution in PBS for 1 h at room temperature. Actin was visualized by Alexa Fluor® 633 phalloidin and nuclei were co-stained with Hoechst 33242 in 3D spheroids. After washing five times with PBS, automated cell multispectral image acquisition was performed on the Operetta® High Content Screening System at a 2 μm interval using a 20× objective (Perkin Elmer). The numbers of stacks varied according to the marker visualized, as follows:
405 ± 25 and 455 ± 10 nm for Hoechst 33342
488 ± 20 and 515 ± 10 nm for cleaved caspase-3 and cathepsin B (apoptosis markers)
532 ± 20 and 560 ± 10 nm for LysoTracker® Red DND-99 (lysosome)
655 ± 15 and 730 ± 25 nm for Alexa Fluor® 633 phalloidin (F-actin)
Various cancer tissues with surrounding normal tissues were arrayed from formal formalin-fixed and paraffin-embedded tissues on an AccuMax array (Petagen Inc., Seoul, Korea), and these arrayed slides were used for the immunostaining of CHALV1 and AFP. Deparaffinization and rehydration were performed using xylene and ethanol (Sigma), and pretreated slides were incubated in 3 % H2O2 (Sigma) for 13 min to remove endogenous peroxidase activity. The tissue was reacted with primary anti-CHALV1 and AFP for 16 h at 4 °C and washed with PBS for 10 min, and the sections were incubated for 2 h at room temperature with goat anti-rabbit Alexa Fluor® 633 and goat anti-mouse Alexa Fluor® 488 secondary antibodies. After washing three times with PBS, coverslips were mounted onto microscope slides using ProLong antifade mounting reagent (Molecular Probes). The slides were analyzed using the Operetta® High Content Screening System (Perkin Elmer).
Polyacrylamide gel electrophoresis (PAGE) and Western blot analysis
For PAGE and Western blot analysis, cells were solubilized with lysis buffer (Sigma), boiled for 5 min, and an equal amount of protein per well (50 μg) was analyzed by 10 % SDS-PAGE. After electrophoresis, proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (GE Healthcare Life Sciences, Piscataway, USA) and processed for immunoblotting. Blots were further incubated with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA) diluted at 1:5,000, and specific bands were visualized using a chemiluminescent substrate (ECL; Thermo Fisher Scientific). Autoradiographs were recorded onto X-Omat AR films (Eastman Kodak Co., Rochester, NY, USA).
Screening procedure
Step 1: Pilot screening
1.5×103 cells/well of Fa2N-4 and 0.8×103 cells/well of Huh7 were mixed and plated in 384 well plate. After 16 h, 10 μM of 43 compounds (see Additional file 1: Figure S5) were treated to each well in duplicate. After 3 days incubation, cells were fixed with 4 % PFA and permeabilized with 0.1 % Triton X-100. The cells were reacted with primary anti-CHALV1 and AFP for 16 h at 4 °C and washed with PBS for 10 min, and were incubated for 2 h at room temperature with goat anti-rabbit Alexa Fluor® 633 and goat anti-mouse Alexa Fluor® 488 secondary antibodies. After three times washing with PBS, cells were stained with Hoechst 33342 for nucleus. Cell images were obtained by Operetta® High Content Screening System and analyzed by Harmony 3.5.1® high content imaging and analysis software (Perkin Elmer).
Sorafenib was treated as positive control and Z' score was calculated by Harmony High-Content Imaging and Analysis Software using positive and negative (DMSO treatment) control. The compounds that > 50 % Huh7 and < 20 % Fa2N-4 inhibition were selected.
Step 2: Hit confirmation
The selected compounds were confirmed by resazurin assay after individual treatment to Huh7 and Fa2N-4 cells at 10 point concentrations (DMSO control, 1 pM to 100 μM). For statistical analysis of IC50 values, GraphPad Prism was used after resazurin assays. The values of IC50 were calculated by nonlinear regression model with a sigmoidal dose response. Experiments were performed in triplicate. Statistical analysis was performed by Student's t test (p < 0.05).
Step 3: Secondary assay
The selected compounds were re-confirmed by Step 1 screening system at 10 point concentrations.
Step 4: MoA (Mode of action) study
The mechanisms of anticancer effect on selected compounds were studied using HCS system and Western blot analysis.
Small interfering RNA (siRNA) transfection
Fa2N-4 and Huh7 cells were transfected with On-TARGET plus Human cathepsinB siRNA (siCTSB; Dharmacon, Lafayette, CO, USA). The sequences of siCTSB were as follows: siCTSB #1, 5'-GGAUCACUGUGGAAUCGAA-3', siCTSB #2, 5'-GCACAACUUCUACAACGUG-3', siCTSB #3, 5'-GAGGCUAUGUGGUACCUUC-3', siCTSB #4, 5'-GCACCGAUCAGUACUGGGA-3'. Cells were then transfected with these siRNAs for 48 h using Lipofectamine® RNAiMAX (Invitrogen).
Caspase 3/7 activity assay
Caspase-Glo® 3/7 Assay Systems (Promega, Madison, WI, USA) was utilized to detect DEVDase (caspase-3/7) activity. Briefly, cathepsin B knock-downed Huh7 and Fa2N-4 cells were treated with pyrimethamine, after which they were lysed in the lysis buffer provided in the kit. The protein content was then determined, after which the lysates were incubated with Ac-DEVD-pNA for 4 h at room temperature at 405 nm.
Statistical analysis
All experiments were performed at least three times. The results are expressed as the mean ± standard deviation (SD). Statistical analysis was performed using the Student's t-test.
CHALV1 and AFP are appropriate markers to distinguish between HCCs and hepatocytes
We aimed to develop liver cancer-specific compounds that induce cell death in HCC cells, while minimizing the damage to hepatocytes, by creating a mixed cell culture system. For HCC-specific drug screening, the co-culture system was composed of HCC cells and hepatocytes (Fig. 1). Because two different populations were present in the co-culture system, we first sought to find HCC-specific markers to distinguish between the HCC cells and hepatocytes.
In this study, we selected two distinctive markers of HCC, hepatocellular carcinoma monoclonal antibody (CHALV1) and α-fetoprotein (AFP). CHALV1 was raised against liver carcinoma cells of human origin and AFP is routinely used for liver cancer diagnosis. To confirm physiological relevancy, we detected CHALV1 and AFP in primary hepatocytes (83857) and primary hepatocellular carcinoma tumors (90768B, 34B, 647B, 108395B, and 110831B) which were isolated from liver resection specimens of liver cancer patients. Expression of CHALV1 and AFP was markedly higher in primary hepatocellular carcinomas than in primary hepatocytes (83857) (Fig. 2a). Immunohistochemical analysis revealed that HCC tissues had an abundance of CHALV1+/AFP+ cells relative to the surrounding normal tissues (Fig. 2b, Additional file 1: Figure S1). We observed that the expression of CHALV1 or AFP was also relatively higher in HCC lines than in Fa2N-4 cells, an immortalized human hepatocyte line (Fig. 2c, Additional file 1: Figure S2). Next, we sought to determine whether CHALV1 or AFP could distinguish between HCC cells and hepatocytes. To this end, we created a co-culture system using Huh7 HCC cells and Fa2N-4 cells that were labeled with CellLight® Nucleus-GFP (Additional file 1: Figure S3). Thus, the difference between HCC cells and hepatocytes was easily discernible in this co-culture culture system.
In Fig. 2d, the yellow arrows indicate cells with low expression of the HCC markers (CHALV1−/AFP−), which might be assumed to be the immortalized normal hepatocytes. Indeed, there was an overlap between the indicated cells and CellLight® Nucleus-GFP labeled-Fa2N-4 cells. Therefore, we concluded that CHALV1 and AFP are appropriate markers to distinguish between HCC cells and hepatocytes for the screening of HCC-specific compounds.
Identification of compounds targeting HCC cells versus normal hepatocytes in a mixed population
We designed a co-culture-based drug screening system, using Fa2N-4 (immortalized hepatocytes) and Huh7 cells (HCC cells) to identify compounds that selectively target HCC cells. Because this screening system was created by mixing of two kinds of cell lines which have different properties, we tried to find appropriate culture conditions that maintained the individuality of both cell types. Fa2N-4 cells are generally cultured in serum-free supporting culture medium (SF- medium). When Fa2N-4 cells were maintained in DMEM supplemented with heat-inactivated 10 % FBS, we observed a change in Fa2N-4 morphology. Despite the change in culture medium, Huh7 cells maintained their morphology (Additional file 1: Figure S4A). Expression of AFP in Huh7 cells was not changed when medium was changed from DMEM supplemented with heat-inactivated 10 % FBS to SF- medium (Additional file 1: Figure S4B).
Next, we estimated the doubling time of Huh7 cells and Fa2N-4 cells to decide the proper mixing ratio for the co-culture system. The doubling time of Huh7 cells was 23.8 h and the doubling time of Fa2N-4 cells as 43.5 h (Additional file 1: Figure S4C). Based on this, the cells were premixed at a ratio of Fa2N-4: Huh7 cells of 65:35 and plated in a randomly mixed state. Additionally, we confirmed that swapping of medium did not have an effect on the cell doubling time in both cell lines (data not shown). Therefore, we used SF- medium for the co-culture screening system.
Based on these results, we set up well-defined mixed HCC cell populations using Huh7 cells and Fa2N-4 cells in 384 well plates for image-based phenotypic screening. To confirm whether developed co-culture system detect selective effect between anticancer effect and drug induced hepatotoxicity, we configured the small screening library including largely two group. Class I includes approved anticancer drugs and cytotoxic compounds whereas Class II includes withdrawn or not marked due to hepatotoxicity and marketed with hepatotoxicity warnings in their labels. The mixed HCC cell populations were incubated for 3 days with 43 compounds (Fig. 3a, Additional file 1: Figure S5). After treatment, the cells were labeled with antibodies against CHALV1 and AFP and images were acquired. Small molecular weight compounds which only affected HCC cells were selected by the Harmony 3.5.1® high content imaging and analysis software. We determined the percentage of Huh7 cells to measure anticancer activity (as cancer selectivity) and the percentage of Fa2N-4 to detect hepatotoxicity based on the meaning Z' score = 0.73 (Fig. 3a). Through the pilot screening, we found HCC-specific drugs (methotrexate, pyrimethamine, and aminopterin, all anti-folate drugs) that significantly induced cell death in HCC (CHALV1+/AFP+) cells while minimizing the damage to hepatocytes (CHALV1−/AFP−) (Fig. 3b, Table 1). To confirm the HCC selectivity of anti-folate drugs, we estimated the growth inhibitory effects by a resazurin assay in an individual culture system. Among the drugs tested, pyrimethamine displayed the greatest cancer-specific cytotoxicity, with approximately 8-fold higher cytotoxicity in Huh7 cells than in Fa2N-4 cells (Table 1). When pyrimethamine was applied to co-cultures of HCC cells and hepatocytes, the reduction in CHALV1+/AFP + cells was significantly greater than the reduction in CHALV1−/AFP− cells (Fig. 3c). A dose response curve also showed that pyrimethamine increased cell death in HCC cells compared to normal hepatocytes. These results showed that pyrimethamine selectively suppressed the HCC population in a co-culture system of HCC cells with hepatocytes.
Pyrimethamine induces growth inhibition by DNA damage and S-phase cell cycle arrest without ROS accumulation and mitochondrial dysfunction
To determine the mechanism of pyrimethamine-induced tumor-specificity in HCC, we applied various cell-based assay models in vitro to the image-based phenotypic analysis system in order to visualize the pyrimethamine-induced phenotypic changes and cytotoxic effects. First, we compared the level of γ-H2AX in Huh7 and Fa2N-4 cells, because pyrimethamine can inhibit DNA synthesis by blocking the enzymatic activity of dihydrofolate reductase. Among the three anti-folate drugs, pyrimethamine displayed the highest intensity of γ-H2AX staining, because γ-H2AX did not appear as foci, but rather as a diffuse pattern (Fig. 4a). However, pyrimethamine induced similar amounts of H2AX phosphorylation in Huh7 and Fa2N-4 cells (Fig. 4b).
To investigate the mechanism of cell growth inhibition induced by pyrimethamine, pyrimethamine-treated Huh7 and Fa2N-4 cells were subjected to an EdU proliferation assay. The EdU proliferation assay revealed that pyrimethamine also similarly increased the number of EdU-positive cells in both cell lines (Fig. 4c). Thus, pyrimethamine did not have a differential effect on the level of DNA damage and S-phase cell cycle arrest between HCC cells and hepatocytes.
To further investigate the mechanism of HCC-specific cytotoxicity triggered by pyrimethamine, we analyzed phenotypic changes in cell organelles, because damaged organelles can trigger tumor cell death. Oxidative stress, mitochondrial damage, and lysosome distribution were measured using the image-based phenotypic analysis system in pyrimethamine-treated Huh7 and Fa2N-4 cells. Reactive oxygen species (ROS) are important signaling molecules in normal conditions, but their accumulation in pathological conditions leads to oxidative stress. Because ROS can provide important clues about the physiological status of the cell, its production was assessed. Compared with the cells treated with pyrimethamine, other anti-folate drugs, including methotrexate and aminopterin, induced a significantly high production of ROS in a dose-dependent manner, which was detected by the permeable and redox-sensitive dye CM-H2DCFDA after 24 h of anti-folate treatment. However, pyrimethamine did not enhance ROS accumulation at wide range of concentrations in both cell lines (Fig. 4d, Additional file 1: Figure S6A).
Next, we assessed mitochondrial function, which is a key factor in the regulation of apoptotic cell death. MMP (ΔΨm) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of ΔΨm renders cells depleted of energy, leading to subsequent death. We examined the effect of pyrimethamine on the ΔΨm by TMRM (tetramethylrhodamine, methyl ester) staining. However, as shown in Fig. 4d and Additional file 1: Figure S6B, the ΔΨm was not changed by pyrimethamine. These results showed that pyrimethamine-mediated HCC-specific cytotoxicity has no connection with growth inhibition by DNA damage, ROS accumulation, or mitochondrial dysfunction.
Pyrimethamine induces an increase in the size and number of lysosomes and subsequently induces strong activation of cathepsin B in Huh7 but not Fa2N-4 cells
Next, we detected morphological changes in lysosomes by LysoTracker, which labels acidic compartments in live cells. The untreated cells displayed numerous small LysoTracker-positive vesicles. Other anti-folate-treated cells showed few differences in the LysoTracker signal compared with the DMSO control. On the other hand, pyrimethamine treatment induced an enhancement in LysoTracker-labeled vesicle size, number, and LysoTracker intensity (Fig. 5a, b). Thus, we hypothesized that pyrimethamine induces apoptosis by causing lysosomal dysfunction in addition to the inhibition of DNA synthesis in HCC. Tumor cells have been reported to have larger lysosomes, which makes more susceptible to breakage. To determine whether the HCC-specific cytotoxicity of pyrimethamine was related to the number of lysosome vesicles, we compared the number of LysoTracker-positive vesicles in Huh7 and Fa2N-4 cells after pyrimethamine treatment. Interestingly, pyrimethamine more significantly increased the size and number of LysoTracker-positive vesicles in Huh7 cells than in Fa2N-4 cells (Fig. 5c). Recently, apoptotic stimuli have been shown to trigger lysosomal membrane permeability, leading to the release of cathepsins that can activate death signaling pathways in the cytosol. Specifically, the release of cathepsin B induces the activation of caspase-3 and −9, thereby leading to apoptosis. Based on this background, we analyzed cathepsin B localization in Huh7 cells, before and after the pyrimethamine treatment. The untreated cells displayed a yellow punctate pattern, because cathepsin B was localized to the lysosome vesicular compartment. However, pyrimethamine triggered the release of cathepsin B from the lysosome to cytosol (Fig. 5d). Next, we detected the active form of cathepsin B and the cleavage of caspase-3 in Fa2N-4 and Huh7 cells. Indeed, pyrimethamine treatment induced cathepsin B activation and caspase-3 cleavage in Huh7 cells to a greater extent than in Fa2N-4 cells (Fig. 5e).
To confirm whether pyrimethamine-mediated cathepsin B activation is responsible for the apoptosis in HCC, cathepsin B siRNA (siCTSB) was transfected to Fa2N-4 and Huh7 cells. Cathepsin B depletion significantly decreased the activity of caspase 3/7 by treatment of pyrimethamine in Huh7 cells (Fig. 5f). Additionally, the IC50 value of pyrimethamine was increased by cathepsin B depletion (from 3.4 to 8.8 μM) in Huh7, but not in Fa2N-4 (Fig. 5g). From these results, it appears that the tumor-specific cytotoxicity of pyrimethamine is mediated by the enhancement of cathepsin B activity in HCC cells.
Pyrimethamine exhibits an anti-tumor effect on Huh7 spheroids without toxicity to Fa2N-4 spheroids
In recent years, a paradigm shift from two-dimensional (2D) to 3D cell culture techniques has occurred, because 2D cell-based models fail to predict in vivo efficacy, contributing to the low success rate of translating an investigational new drug to clinical approval. In order to overcome the shortcomings of 2D culture during drug discovery, we used a 3D culture system, which is the appropriate culture model to sustain liver-specific functions that are more representative of in vivo models. Huh7 spheroids and Fa2N-4 spheroids were exposed to pyrimethamine for 7 days. For the analysis of cytotoxicity in both spheroids, we extracted the middle image from 50 of the 3D stack images. Typically, only a few cells became cleaved caspase-3-positive in untreated spheroids during the course of their maturation, whereas anti-folate drug-treated spheroids were composed mostly of cells with activated caspase-3 (Fig. 6a, Additional file 1: Figure S7). Of note, pyrimethamine induced a strong increase in caspase-3 in Huh7 spheroids compared with Fa2N-4 spheroids (Fig. 6a). Additionally, we investigated the effect of pyrimethamine on the F-actin pattern between Huh7 and Fa2N-4 spheroids, because bile canaliculi, which have an important role in the maintenance of liver function, contain many F-actin microfilaments. Pyrimethamine destroyed bile canaliculi-like architecture in Huh7 spheroids. However, Fa2N-4 spheroids maintained their pattern of F-actin staining after treatment with pyrimethamine (Fig. 6a). These results showed that pyrimethamine induced greater cytotoxicity on HCC cells than on hepatocytes in 3D culture.
HCC is the fourth most common form of cancer in the world, with an incidence of 2.1 in 100,000 in North America and 80 in 100,000 in China [16]. Because only 10–20 % of liver tumors can be removed surgically, the prognosis for this disease is very poor. The major causes of death are chronic infection by hepatitis B and C, and the metastases of tumors from elsewhere in the body [17]. For most liver cancer patients, treatment options are limited to palliative care, because liver cirrhosis causes the loss of main liver functions including detoxification, protein synthesis, and the production of biochemicals necessary for digestion.
Moreover, most HCCs are resistant to conventional chemotherapeutic agents. Cytotoxic drugs such as doxorubicin or cisplatin have no efficacy in the treatment of HCC. Recently, sorafenib, which targets the Raf kinase, VEGFR, and PDGFR signaling pathways, was approved for the treatment of patients with advanced HCC [2, 18]. Generally, sorafenib treatment costs US $5,400 per month, but it only extends lifespan by an average of 2.8 months with various side effects. Therefore, the search for new anticancer drugs for HCC is critical.
Previously, we developed target-based and phenotypic screening approaches for drug discovery and basic research into disease mechanisms [19, 20]. The phenotypic screening approach was the most successful approach for first-in-class drugs. Such screens can lead to the identification of novel targets, novel compound mechanisms of action, and new pathways of therapeutic value [21].
In present study, we found visual signatures of HCC cells and hepatocytes, and validated assays to screen for anti-HCC compounds. We obtained hepatocytes labeled with CellLight® Nucleus-GFP and mixed them with HCC cells. These mixtures were used as a training set for image-based supervised learning to develop the imaging analysis algorithms to detect HCC cells in a background of non-cancerous cells (Fig. 2d). Because our ultimate goal was to define visual parameters that could be used in the absence of a fluorescent marker, we subsequently identified two distinctive markers of HCC, CHALV1 and AFP, which were detected in HCC cell lines and primary HCC tumors (Fig. 2). CHALV1 and AFP had not yet been used for HCC drug screening, but we anticipate that they will become valuable biomarkers for predicting the risk of HCC in the foreseeable future. We developed heterotypic, mixed HCC cell populations using immortalized hepatocytes and HCC cells for HCC drug discovery (Fig. 3a). Cells were premixed at a Fa2N4:Huh7 cell ratio of 65:35, plated in a randomly mixed state, and incubated with cytotoxic compounds for 3 days.
Our HCC co-culture system represents a powerful method for detecting the selectivity index between anticancer activity and drug-induced liver toxicity. Moreover, this system can simply be used to elucidate heterotypic intercellular interactions during physiological and pathological processes. For the pilot screening, we treated with compounds from a small chemical library, which included class I compounds, consisting of clinically approved anticancer drugs and cytotoxic compounds, and class II compounds, consisting of hepatotoxic compounds (Additional file 1: Figure S5).
Through the pilot screening, we identified cancer-specific anti-folate drugs such as methotrexate, pyrimethamine, and aminopterin. Over the past few years, several studies have been done on the anticancer effects of pyrimethamine [11, 13, 22, 23]. However, the cancer-specific effects of pyrimethamine have never been studied in HCC. Here, pyrimethamine had the highest cancer selectivity among anti-folate drugs; however, it was only effective at high concentrations (Table 1).
We determined morphological changes of cell organelles, because organelle damage responses can be used to trigger tumor cell death. Oxidative stress, mitochondrial damage, lysosome distribution, and cell number were measured by the phenotypic analysis system in HCC.
Studies have shown that anti-folate drugs are involved in the de novo biosynthesis of purines and pyrimidines, thereby inducing an imbalance in the nucleotide pool, which leads to consequent DNA damage and S-phase cell cycle arrest. However, the level of DNA damage and S-phase cell cycle arrest was not significantly different between hepatocytes and HCC cells after pyrimethamine treatment (Fig. 4a, b, c). Moreover, because methotrexate induces ROS production [24, 25], we investigated the possibility that pyrimethamine was involved in ROS production. The accumulation of ROS was observed in cells treated with methotrexate and aminopterin. However, pyrimethamine treatment did not induce ROS production (Fig. 4d, Additional file 1: Figure S6A).
Lysosomes are promising potential drug targets and mediators of apoptosis signaling [26, 27]. Tumor cell lysosomes contain increased levels of cathepsins, and the release of these enzymes into the cytosol may result in apoptosis or necrosis [28, 29]. In Fig. 5a and c, we ascertained that pyrimethamine mediated lysosomal modification, which included alterations in the size and number of lysosomes. Of note, pyrimethamine mediated lysosomal modification, the release of cathepsin B from lysosomes to the cytoplasm, and the activation of caspase-3 to a greater extent in HCC cells than in hepatocytes (Fig. 5d).
To overcome the drawbacks of 2D culture, we built a cellular assay using 3D cell culture systems, which are more predictive of the in vivo and clinical responses. Liver cells in 3D culture, which remain viable and retain differentiated functions for a longer period of time, have a potential use for toxicity testing [30]. In the liver, hepatocytes polarize epithelial cells whose plasma membranes consist of two morphologically and functionally distinct domains: basolateral and bile canalicular membranes. Specifically, bile canaliculi (BC) play an important role in hepatic bile secretion. BC contraction is one of the functions of the liver at the tissue level, because it is needed to have communication and integration among neighboring hepatocytes [31, 32]. Therefore, we detected hepatic F-actin to estimate the effects of drugs on BC alteration and activated caspase-3, which is a hallmark of apoptosis in 3D spheroids. As shown in Fig. 6a, pyrimethamine also strongly induced cancer-specific cytotoxicity in 3D cell culture systems. A plausible mechanism for pyrimethamine-induced cancer selectivity in HCC is shown in Fig. 6b, which is mediated by lysosomal modification (increases in cathepsin B and cleaved caspase-3) and DNA damage. These results suggest that pyrimethamine may have a novel anticancer therapeutic use in liver cancer therapy.
In this paper, we have developed an in vitro co-culture system of HCC cells with hepatocytes to recapitulate tumor heterogeneity and have applied this co-culture system to an image-based phenotypic drug screening platform for the development of HCC therapeutics. We have also identified HCC-specific markers, CHALV and AFP, to distinguish between cell types in this platform. Based on the image-based phenotypic analysis, we promptly estimated anticancer activity and hepatotoxicity and elucidated functional roles of pyrimethamine during the apoptosis process in HCC. This in vitro screening platform has the potential to be widely adopted in drug discovery research due to its easy handling and efficiency.
Two dimensional
Three dimensional
bile canaliculi
- BS solution:
Bovine serum solution
5-(and-6)-chloromethyl-2′,7'-dichlorodihydrofluorescein diacetate, acetyl ester
Dihydrofolate reductase
Dose response curve
- EdU:
Fetal bovine serum
Green fluorescent protein
Hepatocellular carcinoma
High content screening
Lysosomal membrane permeability
Lysotracker red
- MMP (ΔΨm):
Mitochondria membrane potential
- MoA:
Mode of action
Phosphate buffered saline
Reactive oxygen species
Serum free supporting Fa2N-4 cells
Tetramethylrhodamine, methyl ester
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Not applicable.
This work was supported by National Research Foundation, Korea (NRF2014K1A4A7A0107 4647) and the Ministry of Health & Welfare, Korea (grant number: HI15C0972).
Availability of data and materials
The dataset supporting the conclusions of this article is included within the article and its additional files.
Authors' contributions
JJ and YS carried out the HCS screening to identify HCC-specific compounds and participated in the functional studies of pyrimethamine. JJ participated in HCC tissues immunohistochemistry and analysis. HS participated in design of the study as well as draft the manuscript, JK and KK coordinated and designed the study. JK and KK provided the HCC patient-derived primary cells and participated in HCC primary cells analysis and characterization. HS designed and was the overseer of the entire study. All authors have read and approved the final manuscript for publication.
Competing interests
The authors declare that they have no competing interests.
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Not applicable.
Ethics approval and Consent to participate
The study was conducted in accordance with the Declaration of Helsinki principles. It was approved by the Human Research Ethics Committee of ASAN medical center. The institute review board in ASAN medical center complies with the related laws such as ICH, KGCP or bioethics and safety act. Written informed consent for the use of tissues for research was taken from patients at the time of procurement of tumor specimens.
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Additional file
Additional file 1: Figure S1.
Immunocytochemistry images of CHALV1 and AFP in HCC cell lines. HCC cell lines were stained with CHALV1 (green) and AFP (red) antibody, and Fa2N-4 cells were used as negative control. Figure S2 Immunohistochemistry images of CHALV1 and AFP in various liver cancer tissues and their surrounding normal tissue. Slide was immunostained with CHALV1 and AFP in 32 cases Image acquisition was performed using high content screening system. Figure S3 Distinguishable staining of Fa2N-4 for more accurate analysis. Fluorescence images of Fa2N-4 labeled with CellLight® Nucleus-GFP (sky blue) and Hoechst33342 (deep blue). Figure S4 Examination of cell phenotype through swapping of medium. (A) Bright field images of Fa2N-4 and Huh7 cells were tested in serum free supporting Fa2N-4 cells (SF) culture medium and DMEM medium. (B) Western blot analysis of AFP expression of Fa2N-4 and Huh7 cell line, which were cultivated in SF and DMEM medium. (C) Doubling time of Fa2N-4 and Huh7 cell lines. Figure S5 List of 43 compounds used for image-based phenotypic screening. Class I are list of clinical approved anticancer drugs and cytotoxic compounds and Class II are list of hepatotoxic compounds. Figure S6 Level of ROS accumulation and mitochondrial membrane potential in Fa2N-4 and Huh7 cells with anti-folate drug. Examination of (A) ROS accumulation and (B) mitochondrial membrane potential were performed after 24 h.incubation with pyrimethamine. Based on the image, value was analyzed by Operetta. Experiments were performed in triplicate and error bars indicate standard deviation. (PPTX 16829 kb)
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Jang, JW., Song, Y., Kim, K.M. et al. Hepatocellular carcinoma-targeted drug discovery through image-based phenotypic screening in co-cultures of HCC cells with hepatocytes. BMC Cancer 16, 810 (2016). https://doi.org/10.1186/s12885-016-2816-x
DOI: https://doi.org/10.1186/s12885-016-2816-x


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Summer reading at Sno-Isle Libraries — an annual event for all children and teens — begins June 20 and continues through Sept. 11. After reading or being read to for 10 hours, each participant receives a free book provided through the generous support of the Sno-Isle Libraries Foundation and Friends of the Library groups.
The foundation has funded 1,500 books to be distributed to 14 Boys & Girls Clubs. This program directly supports the foundation's mission is to mobilize people and resources to expand the reach of library services, promote civic engagement, and foster a lifetime of learning.
With Sno-Isle Libraries expanding hours starting July 1, visiting the library has never been easier. Sno-Isle Libraries supports kids as they explore new places, meet new literary friends, and get swept up in stories which capture their imaginations.
"Reading or being read to during the summer months has so many positive benefits for kids and teens", said Jennifer Sullivan, Student Success Coordinator at Sno-Isle Libraries. "Our library staff are ready to help find print or digital books to engage every child or teen's interests and let them experience the joy of reading."
The program is designed to help children strengthen critical skills gained during the school year by engaging them in learning and growing opportunities during summer vacation. Parents and caregivers are invited to attend fun, interactive events to engage and entertain their children.
Young people are encouraged to visit sno-isle.org or their local library starting June 20 to begin logging hours for summer reading.


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IMOLA transports heavy and non-standard freight in Baltic States, Scandinavia and elsewhere in Europe.
We exam and plan safe, efficient and successful routes for your needs.
Our specialists will prepare the necessary documents and will coordinate the transportation with the respective authorities.
Oversized and non-standard cargo storage in a large, enclosed and guarded area of Riga.
We have specially equipped vehicles for oversized freight escort, which will ensure the safety of your freight and other road users.


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Canoodle quickly became a Square favorite with Chef Corbin Evans creations never failing to impress. Originally named Oxford Canteen, the side-alley restaurant rebranded to Canoodle. As the operation has grown, so will its location. In a social media post, Canoodle outlined what to expect in the coming weeks.
"We are super excited to announce the opening of a NEW Oxford Canteen in the near future. Located at 766 N. Lamar Blvd. at the intersection of Price Street. You may remember it as the Purple Garage Detailing Shop," stated Canoodle. "We will be serving food and beverage from both the original Oxford Canteen menu, like the Beef Brisket Grilled Cheese with Sriracha Mayo and Cheddar Cheese and the Breakfast Tacos, as well as some favorites from the Canoodle menu, like the Cheesy Chicken Ramen with Roasted Chicken, American Cheese and a Fried Egg, of course."
The new location does not have a definitive opening date, but will be posted to social media as soon as one is cemented. In the meantime, Canoodle plans to host, "a couple PopUps in the last 2 weeks of July to test out some new ideas, train some new staff and provide everyone one last chance to eat in the alley."
They are still looking to fill positions in front and back of house. Send resumes to email@example.com
Keep an eye out for PopUp dates and grand opening announcement. The full statement can be found embedded below.


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A common question asked for Office 365 users is "How do I configure my iPhone to access my Office 365 emails". The Office 365 help website has the answer here – http://help.outlook.com/en-au/140/dd125429.aspx
Basically, you will need to configure your iPhone with a Microsoft Exchange email account from the settings page. You will need the following information:
- Email – your Office 365 email address
- Username – this is the same as your email address above
- Password – your password
- Description – this is optional
- Server – this appears after you click next. See below for how to get the server name
To get the servername, you can follow the steps from my previous blog – http://blog.powerbiz.net.au/office/how-to-manually-configure-outlook-2010-for-office-365/ This involves point 1 and 2.
1. Log in to the Microsft Online Services portal – https://portal.microsoftonline.com and open up the Outlook Web App.
2. Note down the address of the server from the web browser address bar.
That should do the trick. You may have to restart the iPhone, as I have seen that issue before.
This solution should also work for connecting up your Android phones as well.


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If August in Melbourne arrives, lines of movie lovers aren't snaked around the Forum, rushing up and down Swanston Street isn't the city's favourite form of exercise, and "what did you just see?" isn't opening every conversation, is it really August in Melbourne? Technically, yes. Emotionally, for cinephiles who've flocked to the Melbourne International Film Festival for decades, no.
Thankfully, until Sunday, August 21 in 2022, it truly is August in Melbourne. MIFF is here. MIFF is underway. MIFF is happening in cinemas, projecting hundreds of films across the silver screen around town, right now in fact.
The highlights are many. The return of Melbourne's MIFF-going rituals, on pause for the past two years, ranks right up there — but every film festival is always about the movie marvels that grace its theatres. A selection will still stream online, too, in wonderful news for film fans who can't live and breathe the fest for its entire 18-day run, or reside outside of Victoria; however, MIFF's heart beats loudest inside a cinema.
If that's your happiest place as well, that's understandable. It is the most glorious spot for a movie buff to be. And, you have plenty to pack into MIFF's big in-person return, plus plenty of titles to pick from as well — including these ten must-sees that are only hitting the fest's big screens.
Flickering across a cinema screen, even the greatest of movies only engage two senses: sight and hearing. We can't touch, taste or smell films, even if adding scratch-and-sniff aromas to the experience has become a cult-favourite gimmick. British director Peter Strickland hasn't attempted that — but his features make you feel like you're running your fingers over an alluring dress (In Fabric), feeling the flutter of insect wings (The Duke of Burgundy) or, in his latest, enjoying the smells and tastes whipped up by a culinary collective that turns cooking and eating into performance art. Yes, if you've seen any of his movies before, Flux Gourmet instantly sounds like something only Strickland could make. While it's spinning that tale, it literally sounds like only something he could come up with as well, given that his audioscapes are always a thing of wonder (see also: the sound-focused Berberian Sound Studio). And, unsurprisingly due to his strong and distinctive sense of style and mood, everything about Flux Gourmet looks and feels like pure Strickland, too.
The setting: a culinary institute overseen by Jan Stevens (Gwendoline Christie, Game of Thrones), that regularly welcomes in different creative groups to undertake residencies. Her guests collaborate, percolate and come up with eye-catching blends of food, bodies and art — hosting OTT dinners, role-playing a trip to the supermarket, getting scatalogical and turning a live colonoscopy into a show, for instance. Watching and chronicling the latest stint by a 'sonic catering' troupe is journalist Stones (Makis Papadimitriou, Beckett), who also has gastrointestinal struggles, is constantly trying not to fart and somehow manages to keep a straight face as everything gets farcical around him. Asa Butterfield (Sex Education), Ariane Labed (The Souvenir: Part II) and Strickland regular Fatma Mohamed play the three bickering artists, and their time at the institute get messy and heated, fast — but this is a film that's as warm as it is wild, and stands out even among Strickland's inimitable work. Also crucial: riffing on This Is Spinal Tap.
What a delight it would be to trawl through Katia and Maurice Krafft's archives, sift through every video that features the French volcanologists and their work, and witness them doing their highly risky jobs against spectacular surroundings. That's the task that filmmaker Sara Dosa (The Seer and the Unseen) took up to make this superb documentary about the couple's lives — although, as magnificent as this incredibly thoughtful, informative and moving film is, it makes you wonder what a sci-fi flick made from the same footage would look like. There's a particular sequence that cements that idea, set to the also-otherworldly sounds of Air, and featuring the Kraffts walking around against red lava in their futuristic-looking protective silver suits. The entire enchanting score springs from Air's Nicolas Godin, and it couldn't better set the mood; that said, these visuals and this story would prove entrancing if nary a sound was heard, let alone a note or a word.
For newcomers to the Kraffts, their lives make quite the tale — one of two volcano-obsessed souls who instantly felt like they were destined to meet, then dedicated their days afterwards to understanding the natural geological formations. More than that, they were passionate about analysing what they dubbed 'grey volcanos', which produce masses of ash when they erupt, and often a body count. Attempting to educate towns and cities in the vicinity of volcanoes, so that they could react appropriately and in a timely way to avoid casualties, became a key part of their mission. This isn't the only doco about them — in fact, German director Werner Herzog is making his own, called The Fire Within: A Requiem for Katia and Maurice Krafft — but Fire of Love is a gorgeous, sensitive, fascinating and affecting ode to two remarkable people, their love, their passion and their impact. It's also benefits from pitch-perfect narration, too, courtesy of actor and Kajillionaire filmmaker Miranda July.
Thanks to everything from The Saddle Club and I Hate My Teenage Daughter to Sweet/Vicious and The Bold Type, Gold Coast-born Australian actor Aisha Dee knows what it's like to live life through screens. She's been acting since she was a teenager, and she's charted the highs of her chosen profession — all in front of a lens. In Sissy, she hops in front of a camera again, naturally, and not only once but twice. In this delightfully savvy and funny Aussie horror film, Dee turns in a wonderfully layered performance as the titular Instagram influencer, whose soaring follower count, non-stop flow of likes and adoring comments, and online fame all stems from her carefully poised and curated wellness videos. Also known as @SincerelyCecilia, the character's sense of self springs from that virtual attention too; however, when she reconnects with her childhood best friend Emma (co-director/co-writer Hannah Barlow), gets invited to her bachelorette weekend and finds old schoolyard dynamics bubbling up, that facade starts to shatter.
If Mean Girls was a slasher film set in a remote cabin in rural Australia, it might look something like Sissy — and that's a compliment multiple times over. Every horror movie wants to be smart and savage on multiple levels, but Barlow and fellow co-helmer/co-scribe Kane Senes (reteaming after 2017's For Now) weaponise everything from influencer culture and pastel, rainbow and glitter colour palettes to toxic friendships, all while spinning a clever, cutting and comedic take on the impact of bullying. They also fill their feature with as gloriously diverse a cast as Australian cinema has boasted, and with one helluva lead performance. If Carrie was set in today's always-online world, amid cancel culture and plentiful praise at the press of a button, it'd look like this, too, but this instant Aussie horror classic takes its own bold stab at plenty of genres.
When Normal People became the streaming sensation of the pandemic's early days, it made stars out of leads Paul Mescal and Daisy Edgar-Jones, and swiftly sparked another Sally Rooney adaptation from much of the same behind-the-scenes team. It wouldn't have been the hit it was if it hadn't proven an exercise in peering deeply, thoughtfully, lovingly and carefully, though, with that sensation stemming as much from its look as its emotion-swelling story. It should come as no surprise, then, that cinematographer Kate McCullough works the same magic on The Quiet Girl, a Gaelic-language coming-of-age film that sees the world as only a lonely, innocent, often-ignored child can. This devastatingly moving and beautiful movie also spies the pain and hardship that shapes its titular figure's world — and yes, it does so softly and with restraint, just like its titular figure, but that doesn't make the feelings it swirls up any less immense.
McCullough is just one of The Quiet Girl's key names; filmmaker Colm Bairéad, a feature first-timer who directs and adapts Claire Keegan's novella Foster, is another. His movie wouldn't be the deeply affecting affair it is without its vivid and painterly imagery — but it also wouldn't be the same without the helmer and scribe's delicate touch, which the 1981-set tale he's telling not only needs but demands. His focus: that soft-spoken nine-year-old, Cáit (newcomer Catherine Clinch), who has spent her life so far as no one's priority. With her mother (Kate Nic Chonaonaigh, Shadow Dancer) pregnant again, her father (Michael Patric, Smother) happiest drinking, gambling and womanising, and her siblings boisterously bouncing around their rural Irish home, she's accustomed to blending in and even hiding out. Then, for the summer, she's sent to her mum's older cousin Eibhlín (Carrie Crowley, Extra Ordinary) and her dairy farmer husband Seán (Andrew Bennett, Dating Amber). Now the only child among doting guardians, she's no less hushed, but she's also loved and cared for as she's never been before.
For fans of damn good coffee, and hot — lovers of black-and-white zigzag-patterned floors and fish in percolators, too — one type of documentary stands out above the rest. There aren't all that many of them, but they certainly do take viewers to a place both wonderful and strange. Those films? Docos about David Lynch, of course. Lynch/Oz is the latest, after Lynch (One), David Lynch: The Art Life and Blue Velvet Revisited, and it's essential viewing for devotees of the Twin Peaks, Eraserhead, Lost Highway and Mulholland Drive filmmaker. Director Alexandre O Philippe clearly adores Lynch, too, just as he adores making movies about movies (see also: Doc of the Dead, the Psycho-focused 78/52, Memory: The Origins of Alien and Leap of Faith: William Friedkin on The Exorcist. And, he's blatantly all in on the idea that the yellow brick road, and everything else in Victor Fleming's beloved big-screen favourite The Wizard of Oz, has been a big influence upon his latest subject.
"There is not a day that goes by when I don't think of The Wizard of Oz," Lynch has famously said — and Lynch/Oz explores the evident truth behind that comment. It traces Twin Peaks' red billowing curtains, and Blue Velvet's obviously blue version, back to The Wizard of Oz's green drapes. It spots Lost Highway's own version of a yellow brick road, Dune and Mulholland Drive's innocents whisked elsewhere, The Straight Story's focus on journeys and the wind rushing through Eraserhead. Philippe has the cannily edited filmic receipts to show all of the above, and to connect The Wizard of Oz to Lynch's constant interrogation of the gap between the American dream and the American myth, too. And, he has fellow filmmakers John Waters, Rodney Ascher (A Glitch in the Matrix), Karyn Kusama (Destroyer), and Justin Benson and Aaron Moorhead (Synchronic) among Lynch/Oz's cine-essay guides. We're definitely off to see the wizards here.
Sometimes, a comparison is so obvious that it simply has to be uttered and acknowledged. That's the case with You Won't Be Alone, the first feature from Macedonian Australian writer/director Goran Stolevski, who also helmed MIFF's 2022 opening-night pick Of an Age. His debut film's lyrical visuals, especially of nature, instantly bringing the famously poetic aesthetics favoured by Terrence Malick (The Tree of Life, A Hidden Life) to mind. Its musings on the nature of life, and human nature as well, easily do the same. Set centuries back, lingering in villages wracked by superstition and exploring a myth about a witch, You Won't Be Alone conjures up thoughts of Robert Eggers' The Witch as well. Indeed, if Malick had directed that recent favourite, the end product might've come close to this entrancing effort. Consider Stolevski's feature the result of dreams conjured up with those two touchstones in his head, though, rather than an imitator.
The place: Macedonia. The time: the 19th century. The focus: a baby chosen by the Wolf-Eateress (Anamaria Marinca, The Old Guard) to be her offsider. The feared figure has the ability to select and transform one protege, but she agrees to let her pick reach the age of 16 first. Nevena (Sara Klimoska, Black Sun) lives those formative years in a cave, in an attempt to stave off her fate. When the Wolf-Eateress comes calling, her initiation into the world — the world of humans, and of her physically and emotionally scarred mentor — is jarring. With Noomi Rapace (Lamb), Alice Englert (The Power of the Dog) and Carloto Cotta (The Tsugua Diaries) also among the cast, You Won't Be Alone turns Nevena's experiences of life, love, loss, desire, pain, envy and power into a haunting and thoughtful gothic horror fable. To say that it's bewitching is obvious, too, but also accurate.
Three years have passed since Nadav Lapid's Synonyms became the hit of the 2019 Berlinale — the prestigious festival's well-deserved Golden Bear-winner, in fact — and the filmmaker's eagerness to examine what it means to be Israeli hasn't faltered. Ahed's Knee, his latest feature, also wowed a big international fest, sharing the 2021 Cannes Film Festival's Jury Prize with Memoria. It's another moral drama as well, the director's preferred kind of narrative. It isn't afraid to be biting and blunt, either. Lapid's way with images, from probing closeups to pointed roving, proves powerful again. Ahed's Knee is also just as determined to see meaning in movement, thanks to arresting dance scenes that spring suddenly. They rush across the screen, and into the story, like no other alternative is possible for the movie's characters at those points to deal with everything that life has thrown and blown their way. Yes, Lapid is still blistering, too, and has plenty of ways to make his work resonate and sting.
The black-clad Y (Avshalom Pollak, Mountain), Ahed's Knee's protagonist, would clearly like the same kinds of descriptions thrown his way. When the movie opens, he's casting for a film about Palestinian activist Ahed Tamimi, who slapped a soldier and was arrested for it. That's all there in the title, as is the other reason that her story is known around the world: a member of the Israeli parliament said that she should've been shot in the knee as punishment. Lapid doesn't turn his feature into a making-a-movie movie, though, but follows Y to the dusty Arava Valley, where he's set to present a screening of a past film and answer questions afterwards. Ministry of Culture bureaucrat Yahalom (first-timer Nur Fibak) is eager to play host, but when she mentions that Y will need to sign a form agreeing only to speak on approved subjects, his sole day and night in town takes several turns.
Part of Manhattan since the 1880s, the Chelsea Hotel is as much a New York City icon as the Statute of Liberty or the Empire State Building, and as influential over the cultural landscape as well. It's where 2001: A Space Odyssey was written by Arthur C Clarke and Stanley Kubrick, where Janis Joplin and Allen Ginsburg have resided — Patti Smith, Madonna, Bob Dylan, Jimi Hendrix and Leonard Cohen as well — and a key factor in the Andy Warhol co-directed 1966 film Chelsea Girls. It's the last place that poet Dylan Thomas stayed, and where Nancy Spungen, girlfriend of the Sex Pistols' Sid Vicious, was found dead. All of these details could fuel a documentary, or several, but that's not the approach that the Martin Scorsese-produced Dreaming Walls: Inside the Chelsea Hotel takes. As the building undergoes extensive renovations that've been happening for years, upending long-term inhabitants and transforming historic apartments, filmmakers Amélie van Elmbt (The Elephant and the Butterfly) and Maya Duverdier spend time with the people determined not to leave.
Everyone who still calls the Chelsea home knows the ins and outs of its past; "the ghosts who haunt it," as one puts it. But Dreaming Walls considers those everyday dwellers — most linked to creative fields in one way or another, of course — the life and soul of the current joint. That might be easy when so much of the place, and its gorgeous gothic architecture, is a construction site in the documentary's frames. The contrast between stripped-bare walls and jam-packed apartments that've been occupied by the same people for decades is haunting as well. It's no wonder that this ethereal and evocative film is largely content to loiter, to listen and to bear witness to the folks who've been there, seen it all, heard what they didn't personally experience and aren't willing to simply move just because a boutique spot is poised to take over.
In the very first moments of her very first feature as a director — after working as an editor on films such as 2012's Post Tenebras Lux and 2014's Jauja — Natalia López demands her audience's attention. She earns it and ensures it as well, and looking away while Robe of Gems unfurls its story is impossible afterwards. To kick things off, a patient and painterly glimpse at the rural Mexican landscape comes into sight, fading up and bringing more and more dusty grey details with it with each second. Then, without the frame moving, a frenetic man is seen bashing and slashing through the plants. Next, it becomes apparent that there's a reflection as part of the image. And, it's also quickly evident that viewers are seeing someone else's vantage as they look on at the landscape. In fact, a couple peers out, in the middle of getting intimate (and immediately before flinging wooden furniture around, strewn pieces flying everywhere). With the 'start as you mean to go on' maxim in mind, it's a helluva opening.
López does indeed begin as she goes on, in a film that earned her this year's Berlinale's Silver Bear Jury Prize. The pivotal villa belongs to Isabel's (Nailea Norvind, Julia vs Julia) family, and offers somewhat of a respite from a marriage that's splintering like that thrown-about furniture, with the clearly well-to-do woman settling in with her children Benja (first-timer Balam Toledo) and Vale (fellow debutant Sherlyn Zavala Diaz). But tension inescapably lingers, given that the onsite caretaker María (newcomer Antonia Olivares) is unsettled by the disappearance of her sister, a plot point that makes a purposeful statement. The police are investigating, the cartel has a local presence, corruption is an ever-present force, and the gap between the wealthy and not-so is glaring. Progressing carefully from that powerhouse opening, Robe of Gems quickly seeps under your skin — and as its first visuals make abundantly clearly, every second is a marvel to look at.
If you somehow only knew two things about Japanese cinema — yes, just two — you'd likely be aware of its animation prowess thanks to the films of Studio Ghibli, and you'd probably also have crossed big-screen paths with a certain rampaging monster. Animated Godzilla movies exist, of course. They were always going to. That said, intriguing new feature Battlecry isn't one of them. Instead, it sees a different kind of creature, called shadow monsters, stalking an alternate 1980s vision of Tokyo that's clearly taken more than a few visual cues from Blade Runner. In this sci-fi vision, they're the result of a devastating drug called golden monkey, and they're starting to spread around the planet. That development sparks interest from two people: Soji, a soldier who can't escape the woes of his past, and Haya, a World Bank employee.
It should never cease to amaze anyone how diverse and inventive that Japanese animation constantly proves. Ghibli's output demonstrates that point again and again all by itself, but its adored films provide just one batch of examples. Everything from Akira, Ghost in the Shell and Neon Genesis Evangelion through to recent hits such as Your Name, Belle and Ride Your Wave makes the same point plain, too, and the list goes on. With Battlecry, first-time filmmaker Yanakaya crafted his gritty dystopia in a thoroughly DIY fashion solely on his computer. Although there's no literal fingerprints visible, obviously, there's still a handmade feel to the end result. That couldn't be more fitting for a film reimagines its setting with all the same lights, but with just as many shadows — and is determined to make its own imprint.


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Alan Bennett
His play: "Forty Years On" contains the line:
"And never more shall be so"
Which was used for the Girlfriend In A Coma vinyl matrix message.
Mentioned In
- Headful Of Heroes - NME (September 16, 1989)
- The Importance Of Being Morrissey - Channel 4 (June 8, 2003)
Discogs Information
Alan Bennett (born 9 May 1934, Armley, Leeds, Yorkshire, England) is an English playwright, screenwriter, author and actor.
External Links
Wikipedia Information
Alan Bennett (born 9 May 1934) is an English playwright, author, actor and screenwriter. Over his entertainment career he has received numerous awards and honours including two BAFTA Awards, four Laurence Olivier Awards, and two Tony Awards. He also earned an Academy Award nomination for his film The Madness of King George (1994). In 2005 he received the Society of London Theatre Special Award. Bennett was born in Leeds and attended Oxford University, where he studied history and performed with the Oxford Revue. He stayed to teach and research medieval history at the university for several years. His collaboration as writer and performer with Dudley Moore, Jonathan Miller and Peter Cook in the satirical revue Beyond the Fringe at the 1960 Edinburgh Festival brought him instant fame and later a Special Tony Award. He gave up academia, and turned to writing full time, his first stage play, Forty Years On, being produced in 1968. He also became known for writing dramatic monologues Talking Heads which ran in 1988, and 1999 on BBC1 earning a British Academy Television Award. Bennett gained acclaim with his various plays at the Royal National Theatre. He received the Laurence Olivier Award for Best Comedy Play for Single Spies in 1990. Next, he made his breakthrough with the play The Madness of George III in 1992. For this play, he received a nomination for the Laurence Olivier Award for Best New Play. The following year he staged a theatrical production of the BBC series Talking Heads in 1992. He continued receiving acclaim for his plays The Lady in the Van in 1999, The History Boys in 2004, and The Habit of Art in 2009. He won his second Tony Award for Best Play for The History Boys in 2005. The following plays were later adapted into films, The Madness of King George (1994), for which he received an Academy Award for Best Adapted Screenplay nomination, The History Boys (2005), and The Lady in the Van (2015). Bennett is also known for a wide variety of audio books, including his readings of Alice's Adventures in Wonderland and Winnie-the-Pooh.
Related Forum Threads
- [Morrissey Central] "SHAKE THE HAND THAT SHOOK THE HAND" (January 5, 2024) - Morrissey-solo (Jan 06, 2024)


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Careers Provision- Staff Information
Guidance to teaching and pastoral staff at Loreto Grammar School
This page is to help provide useful resources to help all teaching staff guide the students at Loreto Grammar in making informed choices about their future choices.
For up to date Labour Market Information (LMI) eClips is an excellent resource to use with your forms. Click here to access it. The password is the schools postcode, it is case sensitive and must have a space between the 4's. WA14 4AH
Further LMI for specific sectors:
Interactive research tool that students can utalise can be found here.
Students can also be made aware of the Careers page on the school website dedicated to various resources for;
Websites for Advice and Guidance
From September 2019 we as a school will be using Kloodle to capture the employability skills that students are developing within the curriculum and through enrichment activities. Registration can be completed via this link.


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Mobile health technology is proving to be a win-win solution for patient wellness and profitability for health care organizations. The development and adoption of mobile medical apps has helped to bridge the gap between patient and provider, and there are no signs of the market slowing down. In fact, it is estimated that by the end of 2017, demand for medical apps could outpace supply by as much as one-to-five.
Based on this demand, developers in the mobile health space and medical marketing services will have plenty of opportunity to provide tools for both consumers and medical companies that add value, improve patient engagement and deliver sustainable benefits. For health care organizations, the ability to truly capitalize on the health IT market starts with a comprehensive patient portal.
Online patient portals are an integral part of any online wellness infrastructure, and when integrated with electronic health records, can provide patients personalized access to their medical information 24/7, while also being a very efficient channel for communication. Access to view diagnostic results, schedule and make physician appointments, receive appointment reminders, request referrals and prescription refills and self monitor and communicate certain health aspects such as blood pressure encourages patients to take a more active role in their health. In short, portals engage patients in their own health condition and care.
Patients who are engaged and have ongoing access to their own personal health goals are also much more likely to be compliant. Patient compliance, especially around disease preventative screenings, is positively impacted with higher engagement levels and easy access to doctors and other health staff – key benefits of patient portals. Fast and easy access to providers also helps patients form better relationships, another key way to improve compliance.
One of the major benefits of online tools is the patient's ability to choose their method of communication – via email, text message or app notifications, while having up to the minute access to a range of information such as health recommendations and tips.
[related_content] This type of digital approach, personalized for users, is fast becoming the way of the future for new mobile apps too. Apple was recently reported to have eight new apps currently underway as part of a health care project to assist patients with specific conditions, disorders or other afflictions. Their development process includes thorough front-end research – finding out exactly what targeted users want in these apps – which leads to better patient engagement and support. Apple has also collaborated and worked with a range of medical researchers, universities and other resources to fully understand patient needs and requirements.
As more and more consumers become active in the medical app space and via patient portals, health care organizations can expect to see a boost to their bottom lines. Better operational efficiency, patient engagement and quality of care helps drive financial benefit. Many reliable studies point to significant financial gains from reduced no-shows, satisfied and happier patients and less paper and administration costs.
As health care IT continues to develop new technologies and improve on current ones, both patients and providers can look forward to reaping the benefits of improved wellness and better bottom lines.
Posted on July 06, 2020 by Jared Mauskopf
6 Questions To Ask When Partnering With A Medical App Development Company
Many healthcare organizations are seeking a means to develop their own healthcare or patient engagement solution, to positively impact patients' health, and streamline clinical processes. Patients perform many of their…Read more


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It's important to us that every dollar Bay Area Habitat for Humanity receives is spent wisely, and we know it's important to you too.
Please take a look at our latest financial reports and contact us if you have any questions about how we utilize the generous donations we receive to help families in need of decent, affordable homeownership.
Federal Tax ID Number: 76-0329145
View our 501c3 documentation here.


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A new agreement to develop 11 facilities that will use plastic waste for hydrogen production in the UK has been signed.
The £130 million deal between Peel Environmental, Waste2Tricity and PowerHouse Energy follows the announcement of the proposed "first of its kind" facility in the UK to be built in Cheshire.
The facilities will use an advanced thermal treatment technology to produce hydrogen from unrecyclable plastics – the clean source can then be used to power buses and heavy goods vehicles before being rolled out to hydrogen cars.
Almost 1.2 million tonnes of waste plastics are sent to landfill every year – the partnership, which is due to submit a planning application for the first site in the coming weeks, hopes to help support in diverting them.
Myles Kitcher from Peel Environmental, part of Peel L&P, said: "Hydrogen is increasingly being seen as a vital part of our journey to zero carbon. This deal could be transformational in delivering a UK first technology that can generate local sources of hydrogen but also provide a solution to plastic waste. As a business we're looking at solutions for all plastics with a vision for these facilities to sit alongside recycling and recovery.
"We're pioneering this solution in the North West but local authorities across the country could benefit from a more sustainable way to treat waste plastic, whilst also creating a local source of low carbon transport fuel which could help them meet their climate change targets."


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- Details
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Details - BOX BRAAI/BBQ GRILL - BY FRONT RUNNER
Camp cooking just got even more portable with this Box Braai / BBQ that keeps the flame off the ground for safe grilling anywhere. Packs flat to save precious cargo room and transport easily and packs neatly in Wolf Pack Pro or Wolf Pack*.
- A Box Braai/BBQ Grill that is designed to pack flat at 1.5 inches / 38mm thick.
- Features a large grill surface area.
- Sturdy base for safe grilling.
- Quick and easy 30-second setup.
- Sides of grill serve as a wind-break.
- Safely contains your fire and keeps the fire elevated off the ground.
- Constructed from durable 3CR12 stainless steel, providing high rust and corrosion resistance.
- Supplied with gird lifting tools.
- Stores neatly in Wolf Pack Pro or Wolf Pack*.
*Note: The Wolf Pack Pro or Wolf Pack are sold separately
Consists of:
1 x Braai/BBQ Assembly
1 x Ember Tray/Griddle
2 x Grid
2 x Grid Lifting Tools
Fitting & Assembly Instructions
Materials used:
3CR12 Steel
Product Dimensions:
Open Dimensions:
436mm (17.2") L x 436mm (17.2") W x 310mm (12.2") H
Close Dimensions:
415mm (16.3") L x 310mm (12.2") W x 50mm (1.9") H
7.9kg (17.4lbs)
Shipping & frieght
Whoa Free shipping!
We offer free shipping the contiguous US for order over $199! This obviously excludes super heavy and freight items. Alaska and Hawaii are sometimes difficult, so you most likely will receive a call from us, as we may incur extra shipping fees.
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All Items will be shipped as the customer chooses at checkout, and a tracking number will be provided. If there are special delivery instructions, please leave them in the notes.
All items will be properly padded and packed to minimize the risk of damage in transit. However, if a product does arrive damaged, it is up to the buyer to contact the shipper and file a claim, as all products are insured for the amount of purchase.
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We do our best to calculate Freight costs at checkout, but to get the most accurate shipping costs we suggest reaching out to us in advance. Over phone or email so we can give you the spot on costs, get more delivery details and make arrangements easier.
We want you to be satisfied! By now we all know we cannot control shipping carriers and delays, but we truly do our best to get your orders as soon as possible. If you have any questions or need assistance place contact us.
Return policy
GTFO is happy to offer a 30-day refund from the purchase date. Any returned product must be in new condition, in original packaging, for full refund. If a product is defective, we will defer to the manufacturer's warranty, and reach out to them on behalf of our customers. The warranty of any product purchased can be found in the product owner's manual or online.
- Buyer must supply the original sales receipt.
- All returned products must be in new condition, and in original packaging
- It is the buyer's responsibility to either bring or ship the product back to us at their cost before a refund is issued.
- We do not offer refunds on Gift cards.
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If the product purchased does not meet the needs of the customer, we will absolutely do our best to make it right for the customer. This may include an alternative product, or other means to create a positive experience for both the customer and ourselves.
OR just want a refund? No problem! As long as the above conditions are met, we will issue a refund. Please know that we have to charge a 3% fee on the transaction, to cover the charges that PayPal/Shopify/Mastercard charge us every time a transaction is made.
WARRANTY: For products that are being returned due to a warranty claim, GTFO will in some cases, replace the product out of dealer stock, and get credit from the manufacturer. However, in some cases, we will have to let the manufacturer of the product send a replacement.


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The NBA continues to take in on the chin after a social media post from Houston Rockets general manager Daryl Morey angered China.
Said post sided with pro-democracy protesters in Hong Kong, leading to the Far East's biggest nation threatening to end its relationship with the Association.
Fresh off the Lakers and Nets playing two preseason games in the communist nation, comes this report that several players met with NBA Commissioner Adam Silver.
According to ESPN's Rachel Nichols, said meeting was contentious. It led to some unnamed players questioning whether there's a double standard around the Association.
"During the meeting with the players, sources said, Silver was directly asked whether anything would happen to Morey, as several players said they believed that if a player had cost the NBA millions of dollars because of a tweet, there would be repercussions," the report read.
The NBA already announced that Morey will not face any discipline. Silver himself also went on record supporting free speech, a stance that has angered the oppressive regime in Beijing.
All the while, NBA players have been afraid to speak up about the hot-button topic. This comes at the same time that teams are preparing for a smaller salary cap moving forward due to the financial implications of the current spat with China.
Whether there is a double standard here is left up to interpretation. What we do know is that a usually boisterous NBA and its star players have been shockingly quiet on the topic.