Aster yomena. This study aimed to uncover the mechanisms underlying the potential therapeutic effects of Aster yomena callus pellet extract (AYC-P-E) on photoaging-induced skin pigmentation. Excessive melanogenesis was induced in B16F10 melanoma cells using α-melanocyte stimulating hormone (α-MSH). The effects of AYC-P-E treatment on melanin biosynthesis inducers and melanin synthesis inhibition were assessed. Based on the results, a clinical study was conducted in subjects with skin pigmentation. AYC-P-E inhibited melanogenesis in α-MSH-treated B16F10 cells, accompanied by decreased mRNA and protein expression of melanin biosynthesis inducers, including cyclic AMP response element-binding protein (CREB), tyrosinase, microphthalmia-associated transcription factor (MITF), tyrosinase related protein-1 (TRP-1), and TRP-2. This anti-melanogenic effect was mediated by mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) phosphorylation. Treatment of subjects with skin pigmentation with AYC-P-E-containing cream formulations resulted in 3.33%, 7.06%, and 8.68% improvement in the melanin levels at 2, 4, and 8 weeks, respectively. Our findings suggest that AYC-P-E inhibits excessive melanogenesis by activating MEK/ERK and AKT signaling, potentiating its cosmetic applications in hyperpigmentation treatment.Plant tissue culture holds immense potential for the production of secondary metabolites with various physiological functions. We recently established a plant tissue culture system capable of producing secondary metabolites from  Nature offers incredibly diverse natural substances that are capable of exerting various physiological effects In recent years, several studies have actively utilized natural materials possessing excellent physiological activity in functional cosmetics using plant tissue culture technology Aster yomena, a perennial herb found in the southern regions of South Korea, is widely used as a functional food ingredient in these regions. Recent studies have shown that extracts isolated from this plant using various methods possess various physiological properties, such as anti-obesity, anti-arthritic, and anti-inflammatory properties Aster yomena callus culture system and confirmed that metabolites with various functions can be produced using this system. Importantly, the metabolite composition of Aster yomena callus pellet extract (AYC-P-E) was found to alleviate skin photoaging in vitro findings, we conducted a clinical study of a cosmetic formulation that contains AYC-P-E in 22 healthy subjects with facial hyperpigmentation.Aster yomena roots were placed into Murashige and Skoog (MS) solid medium supplemented with 1 mg/l 2,4-dichlorophenoxyacetic acid , 3% (w/v) sucrose, and 0.4% (w/v) Gelrit for 2 weeks (at 25°C in the dark) to induce callus formation. The resultant cultured callus was inoculated into a 500 ml flask containing 100 ml of a liquid MS1D medium and cultured at 25°C in the dark on a rotary shaker at 80 rpm. Next, 100 ml cell suspensions for the exponential phase were transferred to a 2 l tank of a sterilized glass bioreactor, and the volume was adjusted to 1.5 l with the liquid MS1D medium. The bioreactor was maintained at 25°C in the dark, and the callus was incubated for 10 days. After 10 days, the pellets were collected following centrifugation at 12,000 rpm for 30 min at 4°C. The collected pellets (5 g) were suspended in 1 l sterile distilled water (DW) and incubated at 80°C for 2 h, and the supernatants were collected following centrifugation at 12,000 rpm for 30 min at 4°C. The collected solutions were dried in a vacuum freeze drier, and the freeze-dried powders were resuspended in PBS and filtered through a 0.22 μm filter. The final concentration of AYC-P-E was adjusted to 30 mg/ml.AYC-P-E was prepared according to previously described protocols 2 condition; Thermo Scientific, Waltham, MA, USA).A cryopreserved B16F10 murine melanoma cell line was obtained from Korea Cell Line Bank . B16F10 cells were grown in the presence of Dulbecco's Modified Eagle's Medium  supplemented with 10% fetal bovine serum (Gibco BRL) and 1% penicillin/streptomycin (Gibco BRL) at 37°C in a humidified incubator  was added. After 72 h, cells were harvested and washed twice with phosphate-buffered saline (PBS). The washed cells were stained with Annexin V  and propidium iodide  for 15 min at room temperature (20°C ± 5) in the dark and then washed with Annexin V binding buffer. Data were acquired using a Life Launch Attune Nxt Flow Cytometer (ThermoFisher Scientific) and analyzed using the FlowJo software . Next, to investigate the protective effect of AYC-P-E on melanoma cells against staurosporine (STS)-induced apoptotic damage. B16F10 cells (1×105 cells/well) were seeded in a 6-well plate for 12 h. Then, culture medium containing AYC-P-E (120 μl/ml) and STS  was added. After 72 h, cell viability and apoptotic cell death were analyzed using the EZ-Cytox kit  and annexin V/PI staining, respectively. Cell viability assay using EZ-Cytox kit was conducted according to the manufacturer's instructions.B16F10 cells at passage 5 were used for all the experiments. To investigate the cytotoxic effect of AYC-P-E in melanoma cells, B16F10 cells were seeded in a 6-well plate at a density of 1×104 cells/well for 12 h, and the media were removed. Then, culture medium (phenol red-free) containing α-MSH  was added for 1 h prior to treatment with AYC-P-E for an additional 72 h. To determine the extracellular melanin content, culture supernatants were analyzed at 405 nm using a microplate reader . To determine the intracellular melanin content, the cultured cells were lysed using RIPA buffer  and centrifuged at 12,000 × g for 1 h at 4°C. The collected pellets were then dissolved in 1 M NaOH containing 10% DMSO at 80°C for 1 h. The absorbance of the heated solution was analyzed at 405 nm using a microplate reader.B16F10 cells were seeded in a 6-well plate at a density of 5×105 cells/well in a 6-well plate) for 1 h prior to treatment with AYC-P-E. After 24 h of AYC-P-E treatment, the cells were harvested, washed twice with cold PBS, and total RNA isolation and qRT-PCR were performed as previously described Culture medium containing α-MSH (200 nM) was added to B16F10 cells (1×105 cells/well in a 6-well plate) for 1 h prior to treatment with AYC-P-E. After 24 h of AYC-P-E treatment, the cells were harvested, and washed twice with cold PBS. The washed cells were lysed using RIPA buffer, and proteins (20 µg) were subjected to 10% SDS-PAGE, and western blotting was performed as previously described 133), tyrosinase, microphthalamia-associated transcription factor (MITF), tyrosinase related protein-1 (TRP-1), TRP-2, mitogen-activated protein kinase 1/2 (MEK1/2), p-MEK1/2 (Ser218/Ser222), extracellular signal-regulated kinase 1/2 (ERK1/2), p-ERK1/2 (Thr202/Tyr204), protein kinase B , p-AKT (Ser473), and β-actin antibodies used were purchased from Santa Cruz Biotechnology .Culture medium containing α-MSH (200 nM) was added to B16F10 cells  for eight weeks. Patients with skin hyperpigmentation on the face  were included in the study. The exclusion criteria were as follows: pregnant or lactating women and women who may be pregnant, patients with a history of photoallergies or photosensitization, those who had been using steroid-containing skin external preparations for more than 1 month, those who had participated in a similar study within 6 months of the start of the current study, those with sensitive and irritable skin, those with skin abnormalities, such as spots, acne, erythema, and expansion of capillaries, those who used the same or similar cosmetics or drugs on the same skin area within three months of the beginning of the study, and those who consumed medicine or food claimed to have skin-whitening effects. The clinical research was approved (KDRI-IRB-20275) by the Institutional Review Board (IRB) of the Korea Dermatology Research Institute, and this study was conducted at Seongnam, South Korea, in July to September 2020 in accordance with the WHO guidelines for good clinical practice (GCP) for trials on pharmaceutical products. All protocols were initiated after receiving written consent in compliance with the principles of the Helsinki Declaration.The test samples were prepared in the form of a cream. In particular, the test sample contained 68.6% AYC-P-E, and the control group sample did not contain AYC-P-E. The samples were provided in the same container to ensure the double-blind design of the study. The test and control samples were randomly assigned to the left and right sides of the face of each subject, and the allocation was not disclosed to the subjects until the completion of the clinical research. The subjects applied each sample to the designated side of the face twice a day for 8 weeks. At each visit, all subjects washed the tested skin area and entered the constant temperature and humidity chamber  to rest for 30 min before participating in the test. The evaluation was conducted by measuring the melanin index (M-index) at the site of skin hyperpigmentation at each time point  before and after using the sample. The melanin index was defined as the average value obtained by measuring the skin hyperpigmentation five times using a Mexameter® MX 18 . in vitro data are expressed as the mean ± standard deviation (SD) of at least three independent experiments. The levels of significance for comparisons between samples were determined using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test using the GraphPad statistical software . The clinical study data were analyzed using the Statistical Package for the Social Sciences  program. All data are expressed as the mean ± standard error of measurement (SEM). Normality and prior homogeneity for data analysis were verified using paired t-tests. The interdependence of the same subject, comparison between groups, and confirmation of changes before and after the indicated time points were analyzed using repeated-measures analysis of variance (RM ANOVA). A P-value of <0.05 was considered statistically significant.The+/PI+; late apoptotic cells, Annexin V+/PI-; early apoptotic cells, or Annexin V-/PI+ cells; necrotic cells) in B16F10 cultures exposed to AYC-P-E  for prolonged periods of time (72 h). We also confirmed the cytoprotective effect of AYC-P-E on the apoptosis inducer STS-treated melanoma cells  for 1 h prior to AYC-P-E treatment . Subsequently, we measured the intracellular and extracellular melanin contents. As shown in Fig. Our group recently showed the effectiveness of AYC-P-E in improving various phenomena, such as skin damage, inflammatory reaction, decreased type I procollagen, and increased elastase, accompanied by photoaging in UVB-irradiated keratinocytes Melanogenesis is mediated by the activation of various signal transduction pathways, such as CREB, tyrosinase, MITF, TRP-1, and TRP-2 Activation of MEK/ERK and AKT signaling phosphorylates MITF, resulting in melanogenesis inhibition. For example, p-MEK-mediated ERK activation (an increase of p-ERK expression) phosphorylates MITF at serine 73, which is followed by MITF ubiquitination and proteasome-mediated degradation  in vitro results, the effectiveness of AYC-P-E in improving hyperpigmentation was examined in 22 subjects with hyperpigmentation in the facial area for 8 weeks after treatment with the test sample containing AYC-P-E and the control sample not containing AYC-P-E, and the change in the melanin index was analyzed. We found that the melanin index of the test group was significantly lower (P<0.05) than that of the control group 2 weeks after the initiation of AYC-P-E treatment and until the completion of the clinical study (8 weeks). The test group, treated with the test sample containing AYC-P-E, demonstrated 3.33, 7.06, and 8.68% hyperpigmentation improvement after 2, 4, and 8 weeks, respectively, whereas, the control group, treated with the control sample that did not contain AYCE, demonstrated 0.48, 1.41, and 2.5% hyperpigmentation improvement after 2, 4, and 8 weeks, respectively  in melanocytes In this study, inhibition of melanin synthesis by AYC-P-E was mediated by ERK and AKT activation, which resulted in improved melanin levels in patients with hyperpigmentation. This suggested the potential role of AYC-P-E as a functional cosmetic ingredient in skin whitening procedures or formulations. However, the whitening effects of each metabolite in AYC-P-E were not assessed. AYC-P-E contains a number of metabolites whose functionality has not been revealed. Therefore, further studies are required to identify the key metabolites of AYC-P-E possessing this whitening activity.In conclusion, AYC-P-E extracted using a plant tissue culture technique induced MITF degradation followed by downregulation of melanin-inducing factors  in melanocytes that promote melanogenesis. Importantly, AYC-P-E inhibited melanogenesis by activating the ERK and AKT signaling pathways. In conclusion, our previous findings as well as the results of the present study on the anti-photoaging effects of AYC-P-E indicate that AYC-P-E may be used as an ingredient in functional cosmetic products for the treatment of various symptoms  caused by photoaging.


Q-1 is a novel compound extracted from the Miao medicine Tiekuaizi. Although Q-1 is known to be a coumarin derivative, its structure has not been deposited in the ACX library. Our previous study showed that Q-1 inhibits the activity of inflammatory cells. This study explores the efficacy of Q-1 in regulating rheumatoid arthritis (RA). The findings show that Q-1 acts through the NF-−1) or low (15 mg·kg−1) doses of Q-1. The control group was administered with an equal volume of drinking water, while the positive control group was administered with Tripterygium glycoside (9.45 mg·kg−1) for 28 consecutive days. The arthritis indices and ankle joint swelling rates were determined. The levels of IL-1β, IL-6, monocyte chemoattractant protein-1 (MCP-1) in serum and sialic acid (SA) in liver homogenate were determined by enzyme-linked immunosorbent assay (ELISA). The pathological features of the ankle joint were analyzed by hematoxylin and eosin (HE) staining. The IκB, P-IκB, P65, and P-P65 protein levels in synovial tissue were assayed by western blotting.  The effects of Q-1 were explored using a bovine type II collagen-induced arthritis (CIA) rat model. The CIA rats were intragastrically administered with high  in the CIA model compared to the control group. RA was successfully replicated by the CIA model, as shown by the joint swelling results and histopathological sections of the ankle. Notably, all the above indicators decreased significantly (P < 0.01) after treatment with Q-1 compared to the model. In addition, animals treated with Q-1 showed lower inflammation in the ankle joints than the model rats.  The arthritis index, ankle joint swelling rate, IL-1κB, P-IκB, P65, and P-P65, inhibiting the excessive release of inflammatory factors, and inhibiting the overactivation of the NF-κB signaling pathway. The findings indicate that Q-1 effectively inhibited RA in rats by downregulating I Tripterygium glycosides have been shown to have significant immunosuppressive effects [Rheumatoid arthritis (RA) is a chronic inflammatory synovial disease that results in damage to the body's cartilage, eventually destroying bones and joints . RA is c effects .In China, the current incidence of RA is approximately 0.20%–0.37%. RA causes a high rate of disability and is extremely challenging to cure , 10. In Chimonanthus praecox (L.) Link and C. niters Oliv [TCM is based on herbs with a long history of use. Many herbs show promise for the treatment of RA , 14. Miaers Oliv , 16. Tieers Oliv  and can ers Oliv , 19. ComTripterygium glycosides [β, IL-6, and MCP-1  were purchased from Simbosheng Co., Ltd. (China). IκBα, phospho-IκBα (Ser32), NF-κB P65, and phospho-NF-κB (Ser536) were obtained from Cell Signaling Technology (USA). A hematoxylin and eosin staining kit (Lot 20180608), bicinchoninic acid (BCA) protein quantification kit (Lot 20190524), 5XTRIS glycine running buffer, bovine serum albumin, and Rainbow Broad Spectrum Protein MARKER (11-180KD) were obtained from Soleibao (China). Analytical reagents were purchased from China National Pharmaceutical Group Corporation.The novel compound Q-1 was isolated from Tiekuaizi in a previous study . Tripterycosides   and was performed in accordance to the animal use statement. Rats were housed under specific pathogen-free conditions with a 12 h/12 h dark/light cycle. All experiments were approved and performed in accordance with National Institutes of Health Guide for Care and Use of Laboratory Animals.Tripterygium glycosides (according to the ratio of human and rat dose of 6.13) [The CIA model was prepared following a previously described protocol , 23. In of 6.13) ; the Q-1of 6.13) . Continuβ, IL-6, and MCP-1. The ankle joints were also collected and fixed in 10% formalin followed by staining. The animals' liver and synovial tissues were collected for SA detection and western blot analysis, respectively.After the experiment was completed, the rats were anesthetized via the intraperitoneal injection of urethane. Blood was collected from the abdominal aorta and centrifuged to obtain serum for the detection of IL-1Wood's arthritis assessment criteria  were useThe volume under the body hair of the left ankle joint was calculated using the water dissolution replacement method based on the following formula: ankle joint swelling rate = volume measured at day 20/volume measured at day 48.β, IL-6, MCP-1, and SA following the manufacturer's instructions.Cells were lysed using RIPA lysis buffer following the manufacturer's protocol. Protein concentrations were determined using BCA protein assay. Commercial ELISA kits were used to determine the levels of IL-1μm sections, deparaffinized, and rehydrated for HE staining. Paraffin sections were stained with HE and analyzed.Histological examination was performed for human synovial tissues and rat knee joints. The tissues obtained from rat knee joints were fixed with 10% formalin. The tissues were then decalcified for 20 d. The samples were embedded in paraffin, cut into 4 β-actin as an internal control.Synovial tissues or cultured fibroblast-like synoviocytes (FLS) were treated with RIPA lysis buffer containing protease inhibitors. The supernatant was obtained, and the protein level in the lysate was determined by Bradford assay. Equal amounts of proteins (50 mg) were subjected to 10% sodium dodecyl-sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes and blocked with 5% skim milk in Tris-buffered saline. Membranes were probed with primary antibodies at 4°C overnight and incubated at 37°C with horseradish peroxidase-conjugated secondary antibodies for 2 h. Immunoreactive proteins were visualized using a Super Signal West Femto Trial Kit . Protein bands were scanned and quantified by densitometry using Image J software. The relative levels of target proteins were normalized using T-test or one-way analysis of variance followed by least significant difference post hoc test. Pearson's correlation test was used to explore the correlations between the expression levels of synovial IκB, P-IκB, P65, and P-P65 and the pathological parameters of rat CIA or synovial β-actin expression. Data are presented as mean ± standard error of the mean. P < 0.05 was considered to indicate statistical significance.Statistical analyses were performed using SPSS 22.0 software. Experimental data were analyzed using independent-sample P < 0.01). PCG, QHG, and QLG all exhibited significantly lower arthritis indices on day 41 compared to the model group (P < 0.05). These findings indicate that Q-1 can improve joint inflammation and reduce the arthritis index in CIA rats.No statistically significant differences in arthritis index were observed between the model group and each treatment group on day 1 . The modP < 0.01; P < 0.05 or P < 0.01). No significant differences in ankle joint swelling were observed between PCG, QHG, and QLG (P > 0.05).The model group exhibited significantly more ankle joint swelling compared to the control group . IκB, P-IκB, P65, and P-P65 protein expressions were downregulated in the synovial tissues of rats in the treatment groups compared to the model group (P < 0.05), indicating that Q-1 effectively inhibited the excessive activation of the NF-κB signaling pathway  and related genes. Monocytes invade synovial cells and persist for long time periods. This study established a CIA rat model of RA through the collagen-induced method.β, IL-6, MCP-1, and SA inflammation-related factors compared to the non-CIA rats. IκB, P-IκB, P65, and P-P65 were upregulated in the model group compared to the control group. HE staining showed that the rats' ankle joints had abundant inflammatory cells with extensive cell edema and degeneration in the model group, indicating a significant inflammatory response. Furthermore, analysis of the CIA model showed a significant activation of the NF-κB signaling pathway. Q-1 treatment significantly lowered the levels of IL-1β, IL-6, MCP-1, and SA inflammatory factors. Moreover, Q-1 led to the downregulation of IκB, P-IκB, P65, and P-P65 in the synovial tissues, and significantly lower levels of inflammatory cells were observed in the rat ankle joint after Q-1 treatment. These findings indicate that Q-1 has an anti-RA effect and can reduce the levels of inflammatory factors in CIA rats. The underlying mechanism by which Q-1 inhibits ankle joint inflammation involves the downregulation of IκB, P-IκB, P65, and P-P65 expression and the inhibition of the excessive activation of the NF-κB signaling pathway.The CIA model rats showed increased levels of IL-1κB, P-IκB, P65, and P-P65, in the synovial tissues, inhibiting the excessive release of inflammatory factors, and inhibiting the excessive activation of the NF-κB signaling pathway.In summary, Q-1 can significantly alleviate RA in rats by downregulating the expressions of I


PUF-A gene was knocked out by the Crispr/Cas9 method in human cervix epithelial HeLa cells. Loss of PUF-A in HeLa cells resulted in reduced clonogenic and lower transwell invasion capacity. Introduction of PUF-AY259F to PUF-A deficient HeLa cells was unable to restore colony formation. In addition, the unphosphorylated mutant of PUF-A, PUF-AY259F, attenuated PUF-A protein stability. Our results suggest the important role of Y259 phosphorylation of PUF-A in cell proliferation.Human PUF-A/PUM3 is a RNA and DNA binding protein participating in the nucleolar processing of 7S to 5.8S rRNA. The nucleolar localization of PUF-A redistributes to the nucleoplasm upon the exposure to genotoxic agents in cells. However, little is known regarding the roles of PUF-A in tumor progression. Phosphoprotein database analysis revealed that Y259 phosphorylation of PUF-A is the most prevalent residue modified. Here, we reported the importance of PUF-A’s phosphorylation on Y259 in tumorigenesis.  Drosophila melanogaster Pumilio and Caenorhabditis elegans fem-3 mRNA binding factors  and topoisomerase inhibitors [camptothecin (CPT) and etoposide]. PUF-A specifically interacts with the catalytic domain of PARP-1 and inhibits poly(ADP-ribosyl)ation of PARP-1 in vitro . We alsoand DDX3 . In thisHuman HeLa and HEK293T cells were obtained from the American Type Culture Collection  and cultured in DMEM medium  supplemented with 10% FBS. All cell lines were submitted to real time PCR for mycoplasma detection and short tandem repeats identification by capillary electrophoresis for cell line authentication.The mouse anti-PUF-A monoclonal antibody has been described . Mouse aAACTTCCATCGTAGCAACTC-3’ and sgRNA#2, 5’-TAACAGCCAAACACCCACAT-3’. Briefly, these two sgRNAs were cloned into an all-in-one sgRNA/Cas9 expression lentivector. HeLa cells were plated at 40–50% confluence and transfected with pU6-sgRNA/Cas9 plasmid using TrnasIT-LT1 reagent  for 96 h. A surrogate vector containing EGFP and mCherry was used to monitor the transfection efficiency. A serial dilution of HeLa cells was conducted to isolate a single cell colony. In total, fifty-five clones were grown and Immunofluorescent/immunoblotting analyses were performed to examine the knockout efficiency. Independent clones of PUF-A-ablated HeLa cells by CRISPR-Cas9 mediated gene deletion were isolated for the following experiments.Two sgRNA oligos were designed to delete exon 2 containing ATG translation start site using the following sequences, sgRNA#1, 5’-HeLa cells grown on coverslips were fixed in 4% paraformaldehyde in PBS for 20 min and permealized with 0.5% Triton for 5 min. Cells were incubated with anti-PUF-A or anti-HA primary antibodies and rhodamine-conjugated secondary antibodies. Immunostained images were recorded using a Leica upright microscope .Protein extracts were solubilized in RIPA buffer  on ice for 30 min and sonicated for 7 min using a Misonix S-4000 sonicator . The supernatants were collected by centrifugation at 13,500 rpm for 15 min at 4°C. Thirty micrograms of proteins were subjected to SDS-PAGE electrophoresis, transferred to nitrocellulose membranes, and immunoblotted with the indicated antibodies. The membranes were then incubated with enhanced chemiluminescent HRP substrate  for 3 min and followed by exposure to X-ray film.3 control and PUF-A knockout HeLa cells were seeded in DMEM complete growth media and allowed to grow for 12 days until visible colonies formed. Colonies were stained with 0.25% crystal violet in ddH2O, washed with PBS twice, and air dried. The stained colonies with diameter larger than 0.1 mm were counted.HeLa cells were trypsinized and number counted by a hemocytometer. Totally, 1x104 cells with 300 μl of serum-free medium containing 0.1% FBS were seeded into the upper chamber. 700 μl of medium containing 10% FBS was added into the lower wells. After 24 h for invasion assays, cells on the underside of the membrane of chambers were fixed in methanol and then stained with crystal violet. Invaded cells were recorded under a microscope and counted from three independent experiments.Forty-eight hours post transfection, matrigel invasion assays were conducted using 8 μm Transwell chambers. Matrigel was diluted in distilled water, added to the upper wells of Transwell chambers (2 mg/well), and dried in a sterile hood for 3 h at 37°C before the addition of cells. Cells at a concentration of 1x10Cells were seeded in a 24-well tissue culture plate and treated with CPT at the indicated concentrations. After 24 h, MTT (Sigma-Aldrich) solution (0.5 mg/mL) was added to each well. After incubation for 2 h at 37°C, formazan crystals in viable cells were solubilized in 200 mL of DMSO. The soluble formazan product was quantified using an ELISA reader at 570 nm.t-test (two-tailed) was used to calculate the statistical significance of differences between groups. The p < 0.05 was considered statistically significant.Analysis was carried out using GraphPad Prism 6 software. All values were expressed as mean ± SD. The paired Student’s PUF-A expression level with overall survival in cancer patients. Using the Kaplan Meier Plotter  [PUF-A expression is associated with poor survival in liver hepatocellular carcinoma using the cBioportal TCGA dataset analysis  , a publianalysis , supportTo illustrate whether PUF-A contributed to clonogenic formation ability, we knocked out PUF-A’s expression in HeLa cells by the Crispr-Cas9 gene editing method, which was conducted by the RNA Technology Platform and Gene Manipulation Core in Taiwan. Compared with control HeLa cells, the number of colonies was significantly reduced in PUF-A-ablated HeLa cells . HoweverTo examine whether PUF-A potentiates cell invasion, a transwell apparatus coating with Matrigel to mimic the microenvironment of invasion was conducted. The majority of control HeLa cells penetrated the Matrigel within 24 h; nonetheless, most of the PUF-A-depleted cells were unable to travel to the lower chamber . QuantitY257F and HA-PUF-AY259F were expressed in HeLa cells. Unlike HA-PUF-A and HA-PUF-AY257F, HA-PUF-AY259F remained in the nucleolus post CPT treatment  sites of PUF-A. Interestingly, the phosphorylation of PUF-A on Y259 displayed the highest score in many high-throughput references (Y259F). We also mutated tyrosine 257 with phenylalanine (PUF-AY257F) as a control and then expressed these mutants in HEK293T cells. Immunoprecipitation and immunoblotting analyses were carried out to determine the expression levels of PUF-AY259F and PUF-AY257F in response to CPT exposure. The expression levels of HA-PUF-A and HA-PUF-AY257F were similar in respective expressing cells. Intriguingly, the protein levels of HA-PUF-AY259F and HA-PUF-AY257F/Y259F were much lower than those of HA-PUF-A and HA-PUF-AY257F expressing cells in the presence of CPT  HeLa cells for immunofluorescence analysis. There was no nucleolar signal in PUF-A ablated cells. By contrast, HA-PUF-A transfection restored PUF-A’s localization in the nucleolus and relocated to the nucleoplasm post actinomycin D (ActD) treatment ation of PARP-1 after MNNG treatment as compared with HA-PUF-A ation activity of PARP1.In summary of our studies, PUF-A expression is associated with overall survival of cancer patients. PUF-A ablation reduces cell proliferation and transwell invasion in HeLa cells. The phosphorylation of PUF-A on Y259 residue is essential for PUF-A’s protein stability and clonogenic formation of HeLa cells. PUF-Ain vitro [Y259F mutant pulled down more PARP1 protein compared with wild-type PUF-A. However, this increased association did not further compromise PARP1’s PAR enzymatic activity, indicating that PUF-AY259F mutant may provide a physical interaction with PARP1 in the nucleolus instead of reducing PARP1 activity. Interestingly, the functions of PARP1 in the nucleolus not only modulate the rRNA transcription [Y259F protein remained in the nucleolus under genotoxic stress, more PARP1 could be entrapped in the nucleolus and this may result in the reduction of nucleolar DNA damage proteins redistributed to the nucleoplasm and subsequently impair downstream PARP1-mediated DNA damage repair, which results in an unfavorable condition for cells to survive under DNA genotoxic stress.Induction of DNA damage results in the rapid recruitment of PARP1 to DNA damage sites through its DNA-binding domain. This stimulates the catalytic activity of PARP1 to conduct the synthesis of polyADP-ribose (PAR) chains on itself, surrounding histones and non-histone proteins . DNA damin vitro . In S4 Fcription  but alsoThe eukaryotic ribosome consists of two subunits formed by the intricate association of ribosomal proteins with four distinct ribosomal RNAs (rRNAs). The small subunit (40S) comprises the 18S rRNA assembled to 33 ribosomal proteins, whereas the large subunit (60S) contains the 5S, 5.8S, and 28S rRNAs associated with 46 ribosomal proteins . PUF-A hY259F mutant was targeted to proteasomal degradation, it is most likely that dephosphorylated PUF-A mutant may alter its L-shaped structure and is destined to be polyubiquitinated for degradation.PUF-A contains eleven PUM repeats to form an L-shaped structure , which tY259F mutant was able to export from the nucleolus to the nucleoplasm. Since PUMY259F protein is unstable, it may be quickly degraded by the proteasomes in the nucleoplasm upon genotoxic exposure.Phosphorylation provides a dynamic mechanism to regulate protein activity, stability and subcellular localization, which is balanced through the reciprocal phosphorylation/dephosphorylation of kinases and phosphatases . For exaPUF proteins mainly participate in stem cell maintenance, organelle biogenesis, oogenesis, neuron function, and memory formation . We haveIn conclusion, our results imply that the tyrosine phosphorylation of PUF-A on Y259 residue is important for PUF-A stability and cell proliferation. Although we were unable to generate a specific antibody against phospho-Y259 of PUF-A, the phosphorylation of PUF-A on Y259 may become a useful marker in tumor progression.S1 FigY257F or HA-PUF-AY259F constructs and exposed to camptothecin for 1 h. Cells were immunostained with anti-HA antibody. Bar, 10 μm.(A) HeLa cells were exposed to camptothecin  for 1 h after the treatment of calycurin A (10 nM) or sodium orthovanadate (2 mM) for 1 h and then immunostained with anti-PUF-A mAb. Nucleoli were shown in dark spots by phase-contrast images. Scale bar, 10 μm. (B) HeLa cells were exposed to 5,6-dichlorobenzimidazole riboside  and CPT (5 μM) and whole-cell extracts were collected at the indicated times for Western blot analysis. Band intensity of phosphoproteins was measured by ImageJ software and normalized with internal PUF-A. All Western blots were processed in identical conditions and cropped from (DOCX)Click here for additional data file.S2 FigHA-Ub was transfected into HEK293T cells exposed to CPT (5 μM) for 18 h with or without the addition of MG132 (5 μM) for 6h. Total cell extracts were immunoprecipitated with anti-PUF-A monoclonal antibody and immunoblotted with anti-HA antibody. All Western blots were processed in identical conditions and cropped from (DOCX)Click here for additional data file.S3 FigY259F, and HA- PUF-AY257F/Y259F were transfected into HEK293T cells and exposed to CPT (5 μM) for 3 h. Cell extracts were immunoprecipitated by anti-HA beads and then immunoblotted by anti-HA and anti-PARP1 antibodies. All Western blots were processed in identical conditions and cropped from Y259F were transfected into PUF-A deficient HEK293T cells for 48 h and exposed to MNNG (5 μM) for indicated times. Cell extracts were immunoprecipitated by anti-PARP1 antibody and immunoblotted by anti-polyADP-ribose (PAR) antibody. All Western blots were processed in identical conditions and cropped from Y259F were transfected into PUF-A ablated HEK293T cells for 48 h and exposed to MNNG (2.5 μM) for 18 h and U2OS cells exposed to Etoposide (50 μM) for 18 h. Apoptotic cells were labeled with FITC-conjugated Annexin V for flow cytometry analysis. No significant difference in response to MNNG and etoposide was found.(A) HA-PUF-A, HA- PUF-A(DOCX)Click here for additional data file.S4 Fig(DOCX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.


The approval of combination therapies with direct-acting antiviral (DAA) regimens has led to significant progress in the field of hepatitis C virus (HCV) treatment. Although most patients treated with these agents achieve a virological cure, resistance to DAAs is a major issue. The rapid emergence of resistance-associated substitutions (RASs), in particular in the context of incomplete drug pressure, has an impact on sustained virological response (SVR) rates. Several RASs in NS3, NS5A and NS5B have been linked with reduced susceptibility to DAAs. RAS vary based on HCV characteristics and the different drug classes. DAA-resistant HCV variant haplotypes (RVs) are dominant in cases of virological failure. Viruses with resistance to NS3-4A protease inhibitors are only detected in the peripheral blood in a time frame ranging from weeks to months following completion of treatment, whereas NS5A inhibitor-resistant viruses may persist for years. Novel agents have been developed that demonstrate promising results in DAA-experienced patients. The recent approval of broad-spectrum drug combinations with a high genetic barrier to resistance and antiviral potency may overcome the problem of resistance. More specifically, 10The increased error rate of the HCV-RNA-dependent RNA polymerase combined with the increased virion production (100-fold higher than that of human immunodeficiency virus) .Viral quasispecies are presently defined as assortments of firmly related viral genomes exposed to a non-stop process of genetic variation, competition among the created variants and selection of the most suitable forms for a given environment . QuasispFitness is defined as the ability of a virus to produce infectious progeny. Subsequently, it reflects the intrinsic ability of a virus to adapt in a given environment. The continuous replication of large viral particles in a given environment leads to fitness acquisition and adaptation, whereas repeated bottlenecks that result in the accumulation of mutations lead to the opposite direction. The direct association between fitness and viral load has been linked to disease progression and responses to antiviral regimens .The existence of various diversity indices is of great importance in order to define viral quasispecies at a molecular level . A paramQuasispecies’ genetic diversity is estimated to be in the range of 1–3% and affects mainly structural rather than non-structural proteins . GenomicAll functional indices that are based on genetic distances assume that genomes that are far apart in sequence space are more likely to have functional differences compared to sequences that are close together. This assumption is not fully accepted for viral quasispecies since single mutations in genomes can influence pivotal functional aspects such as resistance against the immune system or antiviral agents . A majorWith the term classical indices, we refer to the indices used to describe viral quasispecies through the nucleotide sequencing process. Prior to the New Generation Sequencing, the description of the viral quasispecies was performed with the cloning and amplification of DNA or RNA, followed by Sanger sequencing. The information for deriving an estimate for the complexity of the viral quasispecies is located in the haplotypes that fully cover the amplicon and their observed frequencies A.In summary, indices from ecology can be used in order to characterize the complexity of viral quasispecies. However, the concept of complexity is multifaceted and subsequently no single index can describe in depth the actual meaning .The availability of a cell culture system for HCV has enabled the generation of HCV that exhibits different levels of fitness as a result of several passages in human hepatoma cells ,9. One oDAA therapy confers overall SVR rates above 90% . AlthougWild-type amino acids differ across the various HCV types (geno/subtype and geographic origin) . Geno2PhThe emergence and type of RAS depends upon the genotype/subtype and the drugs to which the virus has been exposed . RAS is The risk of RAS is higher in the context of virologic breakthrough during DAA therapy as opposed to relapse following DAA treatment . The durGenotypic analyses include next-generation sequencing (NGS) and population sequencing. Their main difference arises from their ability to detect single specific substitutions within the viral quasispecies of interest. Population sequencing is able to detect variants that represent over 15–20% of the quasispecies, whereas NGS can reach detection rates of 0.1–1%. Nevertheless, existing data suggest that only RASs present in >15% are clinically meaningful. Therefore, in clinical practice, HCV genotypic resistance testing can be carried out with population sequencing. If NGS is applied, a cut-off value of 15% for reporting RASs is recommended ,17.A recent systematic review of RAS sequencing protocols, alongside a public library of sequencing primers, provided guidance in the field of RASs screening and identification. Experts have identified several limitations of the existing methods and have highlighted areas requiring further development .NS3 protease inhibitors (PIs) are peptide-like inhibitors that compete with the intrinsic NS3 serine-protease substrates, thus preventing the viral polyprotein cleavage . With thFirst-generation PIs, such as vaniprevir, simeprevir, paritaprevir and asunaprevir, demonstrate increased antiviral potency. However, their genotypic coverage is suboptimal due to a limited genetic barrier to resistance and substantial cross-resistance at various amino acid locations . Second-In genotype 1 patients, simeprevir demonstrates increased cross-resistance with other first generation NS3 protease inhibitors . In patiMutations at position 168 (D168A/E/H/Q/T/V/Y) were observed in both GT1b replicons and in GT1b patients not responding to treatment with asunaprevir ,37. MutaThrough its different binding with the NS3 catalytic triad , grazoprRASs at various positions have been linked with paritaprevir failure. RAS at position 168 demonstrated an increased fold change for paritaprevir activity in vitro and is frequently detected in GT1a-1b and GT4 failing patients ,42,43,44Voxilaprevir is an EMA and FDA-approved second-generation PI with a broad-spectrum genotypic activity. A156T/V were the most frequently observed substitutions in GT1a and GT1b patients failing VOX treatment . Up untiGlecaprevir is another second-generation PI, approved by both the EMA and FDA. It remains efficacious in vitro against several RASs that are known to impair first generation PI efficacy, especially at positions 170, 155 and 54 in GT1 replicons . NeverthNS5A inhibitors bind to domain I of the NS5A protein, thus downregulating hyperphosphorylation and preventing its dimerization . By actiSeveral RASs have been linked to treatment failure in patients receiving daclatasvir. These include H58D, M28A/T, L31I/M/V, Q30E/H/K/R and Y93C/H/N in GT1a; Y93C/H/I/R and L31F/M/V in GT1b; L31I, Y93H and A30K in GT3; and L30H/R/S, L28M/V in GT4a and 4d ,52,53. TIn patients with elbasvir failure, the most commonly detected RASs were L31F/I/M/V, H58D, M28A/G/T/S, Y93C/H/N/S and Q30H/K/R/Y in GT1a; Y93H and L31F/M/V in GT1b; P58D, Y93C, L30H and L28M/S in GT4a ,54,55,56Y93H was the most frequently detected RAS in patients who failed ledipasvir-containing treatment. Several LDV RASs have been found in GT1a , GT1b  and GT4  ,57,58,59The RASs with the greatest impact in ombitasvir-treated patients were Q30E/H/K/L/R/Y, M28T/V, Y93C/F/H/L/N/S, L31V and H58D in GT1a; L31F/M/V, Y93H/S, L28M and R30Q in GT1b and L28S/V in GT4a or 4d ,60. In pPibrentasvir (PIB) is a second-generation NS5A inhibitor with a broad-spectrum genotypic activity, recently approved by the EMA and FDA. Data regarding pibrentasvir resistance are currently limited. RASs detected in GT3 patients failing pibrentasvir treatment included A30G/K, P58T, S24F, M28G/K, Y93H and L31F/I/M, while GT1a patients relapsed with one or more of these substitutions: M28A/G, K24R, L31M, Y93H, Q30K/R and H58D ,62,63. NVelpatasvir (VEL) is another second-generation NS5A inhibitor with enhanced genotypic activity, approved by both the FDA and EMA. NS5A RASs related to VEL-failure involve positions 24, 28, 30, 31, 32, 58, 92 and 93. In patients with velpatasvir failure, the most commonly detected RASs were Q30E/H/K/L/R, M28T/V, Y93H/N/R/S/W and L31I/M/V in GT1a patients; Y93C/H/N/S/T and L31M/V in GT-1b; L31I/M/V and Y93H in GT2; and A30K/V, E92K, Y93H/N/R and L31M/P/V in GT3 ,65,66. TThe RNA polymerase NS5B is structured in a “right-hand motif”, consisting of finger, palm and thumb domains. NS5B inhibitors include both nucleotide (NIs) and non-nucleotide inhibitors (NNIs). NIs act as NS5B substrates, occupying the NS5B active site, thus leading to direct chain termination. Due to the limited variability of the NS5B active site, NIs are efficacious against all HCV genotypes, while maintaining an increased tolerance to resistance . NNIs inSofosbuvir has been the backbone of HCV treatment due to its pangenotypic activity, as it targets the conserved active site of NS5B while demonstrating a high barrier to resistance. RAS S282T that is situated close to the enzyme’s active site has been shown in vitro to confer resistance to Sofosbuvir but is rarely found in patients who failed sofosbuvir-containing regimens (<4%) . The sigNS5B RASs related to dasabuvir treatment failure in GT1a infected patients were Y448C/H, G554S, A553T/V, G558R, C316Y, D559G, S556G/N and M414T/V. RASs in GT1b were M414I, S556G, D559Nand C316H//N/Y . OverallUntil recently, the appearance of RVs had an impact on the efficacy of second and third-line treatment options ,30,32,34Of note, 57% of GT1a, 93% of GT1b and 100% of GT4 patients who failed an NS5A inhibitor had no RAS in NS3 and were able to receive PI-based retreatment. Vice versa, 86% of GT1a and 83% of GT1b PI-experienced individuals had no NS5A RASs and, thus, were suitable to receive NS5A inhibitor-based treatment. The short half-life of NS3 RASs in patients with previous PI failure can explain the high SVR rates with retreatment regimens that included a PI plus sofosbuvir without an NS5A inhibitor. A study of 220 patients who failed DAA therapy from 39 hospitals in Spain showed that 88.6% had one or more RAS. The subtype-specific pattern of RAS emergence underscores the importance of accurate HCV subtyping . The Q80In everyday clinical practice, most patients that experienced DAA failure were treated with sofosbuvir plus a first-generation NS5A inhibitor. NS5A RASs patterns  were detected based on the NS5A inhibitor and the viral geno/subtype . In GT1aIn GT1b patients, similar NS5A RASs patterns were detected for daclatasvir and ledipasvir and, compared to GT1a patients, Y93H was highly prevalent. Since Y93H variants persist long-term and are associated with resistance to ombitasvir, for GT1b retreatment, the combination of a PI with a second-generation NS5A inhibitor is a good alternative. Y93H was the major RAS for ombitasvir in GT 1b and subsequently for all first-generation NS5A inhibitors. NS5B non-nucleoside RASs were rare in GT1a, but in GT1b, S556G was selected in combination with C316N.Treatment failure with daclatasvir/sofosbuvir in GT3 patients was linked to a strong increase in Y93H. Y93H on the other hand was not detected after ledipasvir/sofosbuvir treatment in the same genotype, possibly because of the limited antiviral efficacy of ledipasvir in GT3 . Second-In patients with GT4 infection, NS5A L28M followed by Y93C/H/S were the main RASs linked to daclatasvir/sofosbuvir and ledipasvir/sofosbuvir treatment failure, respectively. Notably, the moderate frequency of Y93 variants allows for retreatment with velpatasvir/sofosbuvir, as these variants produce low levels of velpatasvir resistance. Typical RASs detected after two-drug  failure in GT4 were L28V in NS5A and D168V in NS3, while Y93 variants were detected less frequently.Voxilaprevir/velpatasvir/sofosbuvir as multiple targeted rescue therapy is the first choice for patients who fail DAA therapy with rates of SVR above 90%, irrespective of the presence of RAS . DespiteA study of 177 GT1 patients who failed previous treatment with sofosbuvir and a NS5A inhibitor showed high SVR rates with retreatment with glecaprevir/pibrentasvir . NS5A RAThe European Society for the Study of the Liver (EASL) recommends that patients without cirrhosis or with compensated cirrhosis who failed after a DAA-containing regimen should be retreated with the fixed-dose combination of sofosbuvir/velpatasvir/voxilaprevir for 12 weeks. Patients who have predictors of lower response  can be retreated with the combination of sofosbuvir plus glecaprevir/pibrentasvir for 12 weeks. In patients with NS5A RASs who failed twice or more to achieve SVR, the triple combination of sofosbuvir/velpatasvir/voxilaprevir, or the triple combination of sofosbuvir with glecaprevir/pibrentasvir can be administered for 12 weeks with weight-based ribavirin and/or treatment duration can be prolonged to 16–24 weeks. Patients with decompensated (Child-Pugh B or C) cirrhosis have a contraindication for the use of protease inhibitors and should be retreated with sofosbuvir/velpatasvir with weight-based ribavirin for 24 weeks [Several DAA-based regimens are available for the treatment of chronic HCV infection. Despite their high efficacy, some patients demonstrate treatment failure which has been linked to the detection of RASs at baseline. In patients naïve to DAAs, RAS frequencies within the viral population either as transmitted or natural polymorphisms alongside several treatment options allow for a RASs-free treatment for all HCV genotypes. The presence of NS3 Q80K or NS5A RASs at baseline was linked to a reduced efficacy for several DAAs; however, this was overcome with intensified treatment schemes, such as the prolongation of treatment and/or the addition of ribavirin . For DAAIn summary, the identified resistance patterns for broadly used DAA regimens allow for the selection of specific retreatment options based on the result of resistance analysis. Resistance profiles for GT1b, more than the GT1a in Europe and 3a-infected patients largely overlap, thus limiting individualized RASs-free retreatment options considerably.


Transmembrane Protein 106A (TMEM106A), encoding a protein that blocks EV-A71 and CV-A16 infection. Combined approaches measuring viral infection, gene expression, and protein interactions uncovered that TMEM106A is required for optimal IFN-mediated viral inhibition and interferes with EV-A71 binding to host cells on the receptor scavenger receptor class B member 2 (SCARB2). Our findings reveal a new mechanism contributing to the IFN-mediated defense against EV-A71 and CV-A16 infection and provide a potential strategy for HFMD treatment by using the antiviral role of TMEM106A against enterovirus.Enterovirus A71 (EV-A71) and Coxsackievirus A16 (CV-A16) are the main causative agents of hand, foot and mouth disease (HFMD) worldwide. Studies showed that EV-A71 and CV-A16 antagonize the interferon (IFN) signaling pathway; however, how IFN controls this viral infection is largely unknown. Here, we identified an IFN-stimulated gene,  The symptoms include red rash on the hands and feet, painful red lesions on the inner cheeks and tongue, sore throat, headache, and fever . In rarereceptor , 11. Aftreceptor . Viral preceptor . Functioreceptor .Pattern recognition receptors (PRRs) are host cell-encoded proteins that sense viral infections through binding specifically to viral molecular patterns such as DNAs or RNAs and trigger downstream cascades through activating interferon (IFN) transcription. Secreted IFN proteins then serve as a signal to the host cells to launch antiviral responses, mostly through the activation of IFN-stimulated genes (ISGs) . StudiesTransmembrane Protein 106A (TMEM106A) is a type II transmembrane protein . It was Here, we present evidence showing that TMEM106A is an inhibitory factor against EV-A71 and CV-A16 infections. Expression of TMEM106A is stimulated upon type I IFN treatment. TMEM106A specifically blocks SCARB2-mediated viral infection. This mechanistic study suggests that TMEM106A associates with SCARB2, interfering with EV-A71 binding on the host cells. Thus, our data provide a new mechanism, triggered by the IFN signaling pathway, that inhibits SCARB2-mediated enterovirus infection.Vero cell, HEK293A cell (293A in short), 293A-SCARB2 cell (293A cell stably expressing SCARB2), rhabdomyosarcoma (RD) cell, JL-1 and JL-2 mAb (fluorescein isothiocyanate (FITC)-conjugated anti-SCARB2 mAb), pCAG-DsRed (a red fluorescent protein-expressing plasmid), and EV-A71-GFP viral packaging plasmids pWSK-T7-EV71-GFP and pCDNA3.1-T7RNAP (T7 RNA polymerase), were kindly provided by Dr. Liguo Zhang, Key Laboratory of Immunity and Infection, Institute of Biophysics, Chinese Academy of Sciences . All the cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)  supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco), 100 U/ml penicillin, and 100 μg/ml streptomycin, at 37°C in a 5% CO2 humidified atmosphere. To generate the cell line constitutively expressing tagged TMEM106A, 293A-SCARB2 cells were transfected with pcDNA4-TMEM106A as described below and selected with Zeocin (200 μg/ml). Resistant colonies were individually expanded and validated by western blotting. One positive clone was chosen and named 293A-SCARB2-TMEM106A. This process was applied to the empty vector and resulted in control cell 293A-SCARB2-Ctrl.BamHI and XbaI. To generate pcDNA3-mCherry, mCherry ORF was cloned into the pcDNA3.1 vector between XhoI and XbaI sites. pIRES2-mCherry was modified by replacing the enhanced green fluorescent protein (EGFP) coding sequence with mCherry coding sequence through cloning between BamHI and NotI sites introduced by fusion PCR. Human PSGL1 and SCARB2 cDNA were amplified from RD and 293A-SCARB2 cells, respectively, and subcloned to pIRES2-mCherry by inserting into EcoRI/SalI, BglII/EcoRI sites, resulting in pPSGL1-IRES-mCherry, pSCARB2-IRES-mCherry expression vectors. All primer sequences are listed in The plasmid pLPCX-TMEM106A is a lentiviral-based vector expressing TMEM106A . For the expression of myc-tagged TMEM106A full length and different truncated forms, DNAs were amplified from pLPCX-TMEM106A and cloned into pcDNA4/To/Myc-His B vector between TMEM106A transcript was designed according to the recommendation of Sigma-Aldrich  and named 106A-shRNA. To generate pSUPER- GFP-106A-shRNA, a pair of complementary oligonucleotides 5′-GATCCCCAAGTCAATCCTGTCCTCCATTCAAGAGATGGAGGACAGGATTGACTTTTTTA-3′ (sense) and 5′-AGCTTAAAAAAAGTCAATCCTGTCCTCCATCTCTTGAATGGAGGACAGGATTGACTTGGG-3′ (antisense) were synthesized with 5’ ends being BglII and HindIII restriction site overhangs, then annealed and then cloned into the BglII and HindIII sites of pSUPER.retro.neo+gfp . For each oligonucleotide, the target sequence was sense (underlined) followed by antisense orientation  separated by a nine-nucleotide spacer.The pSUPER RNAi System was used for knocking down the expression of TMEM106A. The sequence of the shRNA targeting the ® 633-conjugate donkey anti-Goat IgG (H+L) secondary antibodies were purchased from Life Technologies, and anti-SCARB2 aa27-432 , anti-SCARB2 aa339-437 , and anti-TMEM106A (kindly provided by Dr. Yingyu Chen) were used for flow cytometry and immunofluorescence assay.Rabbit anti-HA tag mAb , anti-6× His tag antibody , anti-LIMP II/SCARB2 antibody (D-3) , anti-VP2 mAb , rabbit anti-GAPDH polyclonal antibody , mouse anti-β-tubulin (MG7) monoclonal antibody , HRP-conjugated goat anti-mouse and anti-rabbit IgG antibodies (Sigma Aldrich) were used in western blotting. Alexa Fluor 555-conjugated donkey anti-Mouse IgG (H+L) and Alexa FluorEGFP reporter gene inserted between 5’-UTR and VP4 gene, was established by co-transfecting pcCNA3.1-T7-RNAP and pWSK-T7-EV71-GFP plasmids at a 10:1 ratio into 293T cells  in a humidified CO2 (5%) incubator at 37°C; one hour later, unbound viruses were washed away; cells were then cultured with fresh medium until tested. Viral titers were measured by plaque assay.EV-A71-MZ (GenBank accession no. KY582572), isolated from the throat swab of an ICU patient at Meizhou People’s Hospital , was pro3T cells . When GFDifferent kinds of cells were treated with 1000 IU/ml of recombinant human IFN-α2b (Prospec) for the indicated time, and then total RNAs were isolated and used to measure specific mRNA abundance by RT-qPCR. To examine the inhibitory effect of IFN on EV-A71, Vero, 293A-SCARB2 and RD cells were infected with EV-A71-GFP and treated with IFN-α2b at 0, 1000 and 10000 IU/ml for 8 h. The replication of the virus EV-A71-GFP was estimated by observing the GFP signal under a fluorescence microscope .5 cells/well. When reaching about 90% confluence, the cells were infected with culture supernatants containing viruses undiluted or diluted in 10-fold series for 1 h. Subsequently, the supernatants were aspirated, and cells were washed gently with PBS and then covered with DMEM containing 1% methylcellulose (Sigma-Aldrich) and 2% FBS. After incubation for 3 days, cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) and stained with 0.1% crystal violet. Plaques were then quantified by visual scoring.The viral plaque assay was performed as described previously . Briefly4 cells/well and incubated for 18~24 h, then co-transfected with either pPSGL1-IRES-mCherry or pSCARB2-IRES-mCherry along with pcDNA4-TMEM106A or pcDNA4 vectors at a mass ratio of 1:3 using Lipofectamine 2000 (Invitrogen). After incubation for 24 h, the cells were infected with EV-A71-GFP at a specific MOI of 0.1 for 1 h and incubated for 18 h. About 1×106 infected cells were collected and fixed in 4% paraformaldehyde for 15 min. After washing three times with PBS, cells were resuspended in 0.5 ml of PBS for flow cytometry  assay of GFP production from EV-A71-GFP.To assess the effect of the TMEM106A expression on the EV71-specific receptor function, 293A cells were seeded into a 24-well plate at a density of 1.5 × 10To estimate the effect of different TMEM106A truncated forms on EV-A71 replication, 293A-SCARB2 cells were seeded into a 24-well plate and incubated as described above, then co-transfected with the plasmids expressing full-length or truncated forms of TMEM106A together with a reporter plasmid pCAG-DsRed at a mass ratio of 3:1 and incubated for 24 h, followed by EV-A71-GFP infection at an MOI of 0.1. Twelve hours post-infection, the GFP production was measured by FCM as described above.TMEM106A mRNA level assay by RT-qPCR, the amount of protein was assessed by western blotting, or infected with EV-A71-MZ and incubated for another 12 h for EV-A71 2C mRNA level assay by RT-qPCR. The GAPDH mRNA level was used as an internal control. The relative level of target mRNA in the cells expressing 106A-shRNA was normalized to the cells transfected with the control vector.293A-SCARB2 and Vero cells were transfected individually with pSUPER-GFP-106A-shRNA or pSUPER-GFP. The latter one was used as a control. After incubation for 24 h, the cells expressing high levels of GFP were sorted by FACS (BD LSRFortessa). For testing the RNAi knockdown efficiency of TMEM106A, the sorted cells were treated with IFN-α2b (1000 IU/ml) for 12 h and used for GAPDH mRNA level. The primers used for qPCR analysis are listed in TRIzol reagent (Invitrogen) was used for cellular RNAs extraction, and TRIzol LS reagent (Invitrogen) was used for virus RNA extraction. Extracted RNAs were treated with DNase using the RQ1 Rnase-Free Dnase Kit . cDNA was synthesized with reverse transcriptase , then subjected to qPCR  using a LightCycler 480II (Roche). The target RNA level was calculated by the comparative cycle threshold (CT) method and normalized with 6 cells/well. 16-18 h later, when the cell monolayer was about 70% confluent, the cells were transfected with pcDNA4-TMEM106A-His using the Lipofectamine 2000. 48 h after transfection, the cells were transferred to a 10-cm dish and zeocin  was added to a final concentration of 200 μg/ml to select stably transfected cell lines. This selection took from one to two weeks. The medium was carefully changed every day. The resultant cells from this line were designated 293A-SCARB2-TMEM106A.293A-SCARB2 cells were seeded into a 6-cm dish at a density of 105 cells/well and cultured for 12 h. Then, cells were ice-chilled and incubated with EV-A71-MZ at an MOI of 100 for 1 h at 4°C, to allow viral attachment but impede viral entry. After three washes with ice-cold PBS, EV-A71 binding to the host cell surface was fixed in 250 ml ice-cold 4% paraformaldehyde for 20 min.293A-SCARB2-TMEM106A and 293A-SCARB2 cells were seeded in 24-well plates containing coverslips at a density of 10For entry assay, the cells were first incubated with EV-A71-MZ at 4°C for 1 h and then at 37°C for 30 min for viral entry. The cells were washed with PBS and then fixed. The fixed cells were permeabilized in PBS containing 0.2% TritonX-100 for 10 min, washed three times with PBS containing 0.1% TritonX-100 (PBST), and blocked in PBST containing 5.5% FBS for 30 min. Anti-VP2 antibody was used to detect the EV-A71 virions and then incubated with the secondary antibody Alexa Fluor 555-conjugated donkey anti-mouse IgG in the dark. Nuclei were stained with DAPI (Life Technologies). The results were analyzed by confocal microscopy .XhoI digestion and used as the DNA template. 293A-SCARB2-TMEM106A and 293A-SCARB2 cells were seeded in a 12-well plate at a density of 2.5 × 105 cells/well one day before transfection. For the transfection, 2 μg of EV-A71-GFP RNA mixed with 2.5 μl of Lipofectamine 2000 were added to each well. The expression of GFP was monitored at regular intervals under the microscope. After 18 h, the culture supernatant was collected to measure the infectious titer of extracellular viruses by plaque assay.EV-A71-GFP viral RNA was generated by using a T7 RiboMAX™ Large Scale RNA Production System-kit . The plasmid pWSK-T7-EV71-GFP was linearized by ®Transmembrane Protein Extraction Kit , according to the manufacturer’s instructions. The protein extracts were then analyzed by western blotting using anti-SCARB2 or anti-TMEM106A antibodies to show the expression of SCARB2 and TMEM106A proteins, with the expression of GAPDH protein as a control. After that, the PVDF membrane was stained to confirm the equal loading of two samples.Total, cytoplasmic, and membrane proteins of 293A-SCARB2 and 293A-SCARB2-TMEM106A cells were individually extracted by using the ProteoExtract6 cells were stained in 50 μl of FACS buffer (3% BSA in PBS) containing the FITC-conjugated JL-1 or JL-2 antibody  were co-incubated with anti-SCARB2  and anti-TMEM106A for 1 h at 4°C, followed by incubation with corresponding FITC-conjugated secondary antibodies. The cells were then resuspended in 50 μl of PBS and transferred to coverslips in a 24-well plate, fixed using ice-cold 4% paraformaldehyde in PBS for 30 min, and incubated at room temperature for 30 min. Until the cells were fixed, all steps were performed at 4°C. The cell membrane was permeabilized, and after blocking, nuclei staining was performed. Pictures were captured as described in the viral entry assay.293A-SCARB2-TMEM106A and 293A-SCARB2 cells were centrifuged, and the pellets (2 x 10t- test by performing SPSS software. Graphpad Prism (version 8.0) was used for two-way ANOVA with Bonferroni multiple comparisons statistical analysis. Before each statistical test, the normality of each data set was assessed.Each figure details the statistical tests used to analyze the data set presented. Comparison of the data was performed using Student’s TMEM106A mRNA was assessed in commonly used cell lines as well as peripheral blood mononuclear cells (PBMC) with or without IFN-α2b treatment. Among the tested cells, Vero cells produced the highest level of TMEM106A mRNA, whereas all other cells produced similar levels  were introduced into 293A-SCARB2 and Vero cells and collected by FACS sorting. All sorted cells were treated with IFN-α2b. About half of the TMEM106A RNAs were knocked down by shRNAs , a transmembrane region (a.a 96-115) and an extracellular region (amino acids 116-262) . We reasHere, we showed that TMEM106A is an ISG upregulated upon type I interferon treatment and is required for optimal IFN-mediated antiviral activity against EV-A71 infection Figure 1TMEM106A is not a robustly stimulated ISG compared to ISG54 but is more durable  cell lines but not in normal gastric tissues  can be found in the article/The Ethics Committee of Meizhou People’s Hospital waived the requirement for ethical approval and written informed consent for participants in this study because this application involves the use of de-identified swabs that could not be identified, in accordance with the national legislation and the institutional requirements.XG, XM, and SZ were major contributors in writing this manuscript. XG, SZ, and XM designed the experiments. XG, SZ, XBM, NL, XJ, and HY carried out the experiments and data collection. XG, SZ, and XBM performed the data analysis. All authors read and approved the final manuscript.This work was supported by the grants from the Natural Science Foundation of Guangdong Province (2019A1515012133), the Guangdong Provincial Key Laboratory of Precision Medicine and Clinical Translation Research of Hakka Population (2018B030322003), the Science and Technology Program of Meizhou , Key Scientific and Technological Project of Meizhou People’s Hospital (PY-A2019003).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


KRAS-activating mutations represent the most common oncogenic driver in non-small cell lung cancer (NSCLC), various attempts to inhibit KRAS failed in the past decade. KRAS mutations are associated with a poor prognosis and a poor response to standard therapeutic regimen. The recent development of new therapeutic agents  that target specifically KRAS G12C in its GDP-bound state has evidenced an unprecedented success in the treatment of this subgroup of patients. Despite providing pre-clinical and clinical efficacy, several mechanisms of acquired resistance to KRAS G12C inhibitors have been reported. In this setting, combined therapeutic strategies including inhibition of either SHP2, SOS1 or downstream effectors of KRAS G12C seem particularly interesting to overcome acquired resistance. In this review, we will discuss the novel therapeutic strategies targeting KRAS G12C and promising approaches of combined therapy to overcome acquired resistance to KRAS G12C inhibitors.Although  RAS  encodes a membrane-bound protein, initially described in 1960s by Harvey   isoform represents 75% of RAS mutant cancers (y Harvey  as a retll lines  and is cutation) . Notably cancers .KRAS alterations is identified in pancreatic adenocarcinoma (88%), colon and rectal adenocarcinoma (50%), and in lung adenocarcinoma (32%)  patients. However, in the latter, the location of mutations beyond the Q61 codon within the KRAS gene is more heterogeneous than in pancreatic adenocarcinoma cases  , 5. In pKRAS mutated oncogene was firstly demonstrated in 1984 , the clinical significance of  in 1984 . Since tKRAS mutations in NSCLC and discuss the pre-clinical and clinical development of sotorasib and adagrasib. Herein, we outline the main mechanisms of acquired resistance described to KRAS G12C inhibitors to present and summarize therapeutic strategies to overcome resistance.In this literature review, we focus on RAS encodes a membrane bound protein with a guanosine triphosphatase (GTPase), that is expressed in all mammalian cells  the G-domain – a highly conserved domain between RAS isoforms – which contains switch-I (aa 30-38) and switch-II (aa 59-76) loops is responsible for GTP-GDP exchange; (2) the C-terminal domain known as the hypervariable region and associated with significant variations among RAS isoforms and (3) the C-terminal CaaX box responsible for post translational modifications , 14–16. RAS mutant, RAS presents a reduced ability to hydrolyse GTP or to interact with GAPs, thereby leading to a permanent and constitutive activation of RAS and downstream effectors which promotes tumorigenesis   than squamous cell carcinoma (4%)  is the most frequent, as it constitutes 40% of all KRAS mutations in NSCLC  and younger patients  compared with other KRAS mutations (KRAS G12C mutations were more frequent in black and white patients than Asian patients (p<0.001)    . As wellarcinoma %  . Notably a NSCLC . The G12utations  and KRASp<0.001) , and arepatients , 34, KRAKRAS mutations (TP53 (about 40%), STK11  and KEAP1  are the most frequently reported  or ERBB4 mutations (p=0.025) compared with non- KRAS G12C patients  expression in NSCLC, thus contributing to exhausted T-cells  ≥50% was reported in a range of 34  (KRAS-mutant cells (KRAS mutant cells were found to markedly secrete granulocyte macrophage colony-stimulating factor (GM-CSF) which contributes to the recruitment and activation of myeloid-derived suppressor cells (MDSCs)  . Such re (Tregs) . Severalnt cells –55. Like (MDSCs) , 56. Theon MDSCs .KRAS mutations in NSCLC seem to be associated with worse prognosis  and progression-free survival    . The obj6-0.63]) . KRAS mu disease . On the patients .KRAS mutation sub-type as well as co-occurring mutations seem important to take into account as they might impact prognosis and response to treatment. Indeed, a better disease-free survival for patients with stage I lung adenocarcinoma harboring G12V/G12C mutations (p=0.0271) was reported, compared with other KRAS mutations  that express the A59G mutation – which abrogates RAS GTPase activity – ARS-853 failed to decrease KRAS GTP levels and ERK phosphorylation  reduced susceptibility to KRAS G12C inhibitors in vitro .More recently, AMG-510  and MRTXKRAS G12C cell lines   . Besidesografts) , 86 as e T-cells .KRAS G12C inhibitor to enter clinical development  whereas patients with alterations in effectors of cell cycle, WNT pathway and MAPK pathway are more prevalent in the late-progressor group   . Zhao etpatients . Based oKRAS G12C inhibitor to have entered clinical trials.Adagrasib, formerly MRTX849, is the second irreversible and selective KRAS G12C  and extensive tissue distribution , 92. SimRAS G12C Table 2 RAS G12C . ConsistRAS G12C . Pre-clit models . In line T-cells . Interes/nu mice .KRAS G12C mutation (NCT03785249) (STK11 (Adagrasib is currently being tested in the KRYSTAL-1 multi-cohort phase I/II study that enrolled patients with advanced or metastatic solid tumors harboring a 3785249) , 92. A r3785249) Table 2 3785249) . Similar3785249) . DespiteEGFR and ALK-driven NSCLC respectively   provokes a compensatory response involving the fibroblast growth factor receptor 1 (FGFR1), resulting in signaling rebound and adaptive drug resistance , 98. In  levels) . In this levels) .Interestingly, shortly after treatment, some cancer cells are sequestered in a quiescent state with low KRAS activity. The new KRAS G12C is maintained in its active, drug-insensitive state by epidermal growth factor receptor (EGFR) and aurora kinase (AURKA) signaling. In this setting, the synthesis of new KRAS G12C and its trading between the active or inactive states, modulates the divergent early response to KRAS G12C inhibition and allow cells to resume proliferation.KRAS G12C inhibition is also associated with induction of EGFR phosphorylation, following sotorasib treatment in NCI-H358 cells  mRNA levels were significantly higher in BEAS2BKRAS G12C cells treated with sotorasib, compared to control  on-target mechanisms; (2) by-pass alterations resulting in abnormal activation of downstream or connecting signaling pathways; and (3) phenotypic transformation   observed, followed by R68M and A59S . In adagrasib-resistant clones, KRAS Q99L was the most frequent secondary mutation , followed by Y96D and R68S   , KRAS G12C/Y96D NSCLC cells and the MGH1138-1 patient-derived model  . Secondactively) . Of notehibitors . Cross-rAS G12C) . Upon aded model . Moreovedagrasib . Secondasitivity . NotablyC allele . Consistin vitro .R68S, H95D/Q/R and Y96C mutant residues were found to disrupt non-covalent binding interactions between KRAS G12C and adagrasib  and sotorasib   , Tanaka et al. hypothesized that cis mutations resulting in G12C loss and conversion into another KRAS mutation might constitute an additional mechanism of acquired resistance  . This anfication . Consistpatients . Additiodagrasib , 105. Insistance . FinallyKRAS mutations were identified in 4 patients  while 3 patients presented KRAS amplification  were significantly upregulated in sotorasib-resistant cells , BRAF V600E and MAP2K1  , EGFR  and FGFR2  activating mutations  in plasma samples collected at disease progression on adagrasib revealed up to 10 distinct mutations affecting RAS-MAPK pathway effectors . In partI103del) . The impin vitro . Moreovedagrasib . Similardagrasib . In clinutations . MET and patient .KRAS G12C-targeted therapies, Awad et al. reported two cases of phenotypic changes to squamous-cell carcinoma at disease progression on adagrasib , compared to single monotherapy  was markedly inferior to those obtained with either sotorasib (IC50 = 69.33µM) or crizotinib (IC50 = 2.68µM) monotherapy, in MET-amplified, sotorasib-resistant cells (MET-amplified H23-sotorasib resistant xenograft models compared to monotherapy (50 = 0.07µM)   , encoded by d IRS1-4 –119. SHPd IRS1-4 , and intd IRS1-4 , therebyd IRS1-4 Figure 3in vivo studies highlight that KRAS-mutant NSCLC depends on SHP2 during carcinogenesis   and NSCLC (H358) , as a single-agent monotherapy (KRAS G12C patients with advanced solid tumors (NCT04699188).Pre-clinical ogenesis , 123. Baogenesis  or adagrogenesis  and RMC-ctively) . RMC-455C (H358) . Likewisotherapy . Notablyotherapy . Likewisotherapy  or in coKRAS G12C inhibitors, SHP099 , KRAS G12V (SW620 cells), KRAS G13D (LoVo cells) and KRAS G12S (A549 cells)  . Moreovein vitro .KRAS G12C inhibitors, when combined with treatments that target downstream KRAS G12C effectors. BI-3406 or TNO-155 monotherapy did not restore sensitivity to KRAS G12C inhibitors in Ba/F3 cells harboring KRAS G12C and Y96S or Y96D secondary mutations (KRAS G12C), modified to express KRAS Y96S or KRAS Y96D resistant mutations, were sensitive to the combination of BI-3406 and trametinib in both monolayer and 3D cell line models  and PI3K-AKT-mTOR pathway seem particularly relevant.Overall, these studies evidence promising pre-clinical rationales to revert or delay the emergence of acquired resistance to sotorasib and adagrasib Figure 3otherapy , 134 or otherapy , 136. Thhibitors  will be hibitors . As PI3KKRAS-mutant NSCLC patients, KRAS mutated NSCLC patients are expected to benefit from immune checkpoint inhibitors (ICIs) (KRAS G12C-mutant patients in particular (KRAS mutation subtypes in term of response or PFS (KRAS G12C patients tend to have higher response rate to ICIs (26.9%) and a longer mPFS (4.0 months) compared with non-KRAS G12C (response rate: 18.8% and mPFS 2.9 months) (KRAS mutations (versus KRAS wt) were associated with significant superior survival   , 138, anrticular –141. In rticular . No signe or PFS  although months) .TP53 co-mutation seems to be associated with a better response to ICIs, STK11 and KEAP1 co-mutations seem to impair response to ICIs. Indeed, TP53/KRAS co-mutated patients present higher PD-L1 expression, increased CD8+ TILs    . Indeed, pathway . It was p=0.039) . In partpression . Consistpression .KRAS G12C inhibitors, pre-clinical studies highlighted that either sotorasib or adagrasib have enhanced anti-tumor efficacy when combined with anti- PD-1/PD-L1 therapy. In contrast, although either AMG-510 or anti- PD-1 monotherapy caused tumor complete regression in only one out of ten mice, for each therapy; combined treatment achieved a complete and prolonged response in 9/10 CT-26 KRAS G12C mice . This work was supported by the Institut National du Cancer (INCa) (PRT-K program no. 2018-024 EMT-CoNCEPT).AS received honoraria from Amgen for participation in Board meetings.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


TGFBI, NID, LEPREL2, COL11A1, CYR61, PCOLCE, IGFBP7, COL4A2, CSPG2, and VTN) were identified using the CytoHubba plugin of Cytoscape. Seven of these hub genes showed significant differences in expression between chemotherapy-sensitive and chemotherapy-resistant samples. The prognostic value of these seven genes was evaluated using TCGA COAD  data. The results showed that TGFBI was highly expressed in chemotherapy-sensitive patients, and patients with high TGFBI expression have better survival.Colorectal cancer (CRC) is one of the most common malignant tumors. 5-fluorouracil (5-FU) has been used for the standard first-line treatment for CRC patients for several decades. Although 5-FU based chemotherapy has increased overall survival (OS) of CRC patients, the resistance of CRC to 5-FU based chemotherapy is the principal cause for treatment failure. Thus, identifying novel biomarkers to predict response to 5-FU based chemotherapy is urgently needed. In the present study, the gene expression profile of GSE3964 from the Gene Expression Omnibus database was used to explore the potential genes related to intrinsic resistance to 5-FU. A gene module containing 81 genes was found to have the highest correlation with chemotherapy response using Weighted Gene Co-expression Network Analysis (WGCNA). Then a protein-protein interaction (PPI) network was constructed and ten hub genes ( CRC is one of the most common malignant tumors and the second cause of tumor-related mortality worldwide , 2. By 2Over the last few decades, substantial progress has been made in the development of new treatment regimens that fundamentally increase the overall survival (OS) of CRC patients. Patients with stage III or high-risk stage II CRC benefit from the use of adjuvant chemotherapy with5-FU-based regimens , 10. Altin vivo studies , an enzyme encoded by  therapy , 15. Sevreatment . High DPreatment . In addireatment . It has  tissues . Thymidiotherapy . There iotherapy , 21. Howotherapy . Besidesotherapy , ABCC11 otherapy . Althoughttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE3964). This dataset contains expression profiling of clinical samples collected from CRC patients before the exposure to 5-FU based combined chemotherapy. Analysis of gene expression profiles between chemotherapy-sensitive patients and chemotherapy-resistant patients may identify biomarkers associated with innate tumor drug responses. Another gene expression profiles of CRC patients undergoing chemotherapy and corresponding clinical information were downloaded from TCGA COAD . Gene expression profiles of Dataset GSE19860 without z-score normalized (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE19860) was used to verify the expression patterns of the screened hub genes. This dataset contains responders and non-responders who received modified FOLFOX6 therapy.A flowchart of this study is presented in The gene expression profile of GSE3964 was constructed to gene co-expression networks using the WGCNA package in R to explore the modules of highly correlated genes among samples for relating modules to external sample traits . A weighGene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses for genes in functional modules were performed using an R package “clusterProfiler”. GO annotation is based on three categories, including biological processes (BP), cellular compartments (CC), and molecular functions (MF). Terms in GO and KEGG with a false discovery rate (FDR) < 0.05 were considered significantly enriched and were visualized by R package “ggplot2”.https://string-db.org), and an interaction with a combined score > 0.4 was considered as statistical significance. Cytoscape, an open-source bioinformatics software platform, was used to visualize molecular interaction networks. Hub genes were identified using the DMNC algorithm of cytoHubba plugin in Cytoscape.The protein-protein interaction (PPI) network of genes in functional modules was constructed with an online STRING database  according to the manufacturer’s instructions.HCT116 and DLD1 cells (purchased from ATCC) were cultured in DMEM medium (Cellmax) containing 10% fetal calf serum (BI), and 100 U/ml each of penicillin and strepcomycin (BI) at 37°C with 5% COTo establish 5-FU resistant cells, cells were treated with a high concentration of 5-FU for 24h, then the media was replaced with fresh media containing a low concentration of 5-FU. After 2 weeks of treatment at the low concentration, increase 1.5 times of the dose, and repeat the same. 5-FU resistant HCT116 cells were generated by treating its parental cells with 40µM 5-FU for 24h, then treating the cells with 0.3125 µM 5-FU and increasing 1.5 times of the dose. 5-FU resistant DLD1 cells were generated by treating its parental cells with 100µM 5-FU for 24h, then treating the cells with 1.25µM 5-FU and increasing 1.5 times of the dose. The 5-FU resistant HCT116 cells and 5-FU resistant DLD1 cells were obtained by continuous exposure to gradually increased concentrations of 5-FU for four months.HCT116 or DLD1 cells were washed with DMEM without phenol red and incubated with MTT -dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide) at the concentration of 0.5 mg/ml in DMEM without phenol red. Four hours after incubation, the media were dumped off and the formazan crystals were dissolved in dimethyl sulfoxide (DMSO). The optical density (OD) was measured by a photometer at 490 nm. The data were normalized to control and the ratios were presented as mean ± SE with three experiments.Proteins were separated by 9% SDS-PAGE and then transferred onto a nitrocellulose membrane . The following primary antibodies were used: anti-Beta Actin (Proteintech), anti-TGFBI (Proteintech). The following secondary antibody was used: Goat anti-mouse IgG-HRP antibody (Proteintech). The proteins were visualized using an ECL detection kit (GE).www.miaolingbio.com). pEGFP-C1 was linearized through EcoRI and BamHI. Then the TGFBI DNA was cloned into pEGFP-C1 through Gibson Assembly.Full-length TGFBI DNA was amplified by PCR with the primer 5’-GATCTCGAGCTCAAGCTTCGAATTCCATGGCGCTCTTCGTGCGG-3’ and 5’-GATCTCGAGCTCAAGCTTCGAATTCCATGGCGCTCTTCGTGCGGTCAGTTATCTAGATCCGGTGGATCCCTAATGCTTCATCCTCTCTAATAACTTTTGATAGACAG-3’ using pOTB7-TGFBI . The genes in the red module were highly correlated with the module (p = 6.7×10-10).To explore the functional clusters related to chemotherapy response, the weighted gene co-expression network was constructed from GSE3964 datasets which containing 10 chemotherapy-sensitive and 13 chemotherapy-resistant samples. The included samples were clustered with the average linkage hierarchical clustering method. The power of β = 4 was selected as the soft-thresholding parameter to conduct a scale-free network Figure 2To explore the potential function of the red module which had the highest correlation with chemotherapy response, GO and KEGG enrichment analysis was performed. For BP enrichment, the genes in the red module were mostly enriched in extracellular matrix organization and extracellular structure organization  were analyzed. In line with our expectations, among the CRC patients who received modified FOLFOX6 therapy, responders also showed a higher level of TGFBI than non-responders  showed significant differences between chemotherapy-sensitive and chemotherapy-resistant samples. To explore the prognostic value of these seven genes, Kaplan-Meier survival analysis was performed in CRC patients treated with 5-FU based chemotherapy from TCGA COAD. TGFBI was identified as a prognostic gene (p = 0.01), a high expression of which indicates a good prognosis , it contains an RGD (Arg-Gly-Asp) motif that can serve as a ligand recognition site for integrins (TGFBI (transforming growth factor β-induced protein), encoded by ntegrins . TGFBI mntegrins . Many rentegrins . TGFBI lntegrins . TGFBI pntegrins . Overexpntegrins . TGFBI entegrins . TGFBI cntegrins . TGFBI dntegrins . Besidesntegrins . These sntegrins . It has ntegrins . A studyntegrins .in vitro also support the conclusion. TGFBI levels were dramatically decreased in 5-FU resistant cell lines than that in the corresponding parental cells, both in HCT116 cells and DLD1 cells. Knocking down TGFBI in HCT116 cells led to increased resistance to 5-FU treatment compared with control cells. GFP-TGFBI overexpression dramatically restored the sensitivity of resistant HCT116 cells to 5-FU treatment compared with GFP overexpression.In addition to the dual role in tumor progression, the expression of TGFBI has also been associated with chemotherapeutic drug sensitivity. Loss of TGFBI induced specific resistance to paclitaxel in ovarian cancer cells, and paclitaxel-resistant cells treated with recombinant TGFBI protein show restoration of paclitaxel sensitivity . Immunohhttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE3964https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE19860.Publicly available datasets were analyzed in this study. This data can be found here: This study was approved by the Ethical Committee of Central South University (China).YW and KF conceived the concept, instructed data analysis, and revised manuscript. YW and QW conducted most data analysis and preparation of figures and table and wrote manuscript draft. YY, YC, and SL helped with some analysis and interpretation of data. KF reviewed the manuscript with input from all authors. All authors contributed to the article and approved the submitted version.This work was supported in part by grants 31900561 from National Natural Science Foundation of China, Innovation-driven Projects 2020CX016 from Central South University, Huxiang Talent Aggregation Project 2018RS3027 and 2019RS1010 from Hunan Provincial Science and Technology Department (China). YW was supported by Xiangya Hospital Central South University Postdoctoral Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Embryonated chicken eggs have been used since the mid-20th century to grow a wide range of animal viruses to high titers. However, eggs have found so far only limited use in the production of recombinant proteins. We now describe a system, based on a Sendai virus minigenome, to produce large amounts of heterologous viral glycoproteins in the allantoic cavity of embryonated eggs.Soluble forms of human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV) fusion (F) proteins, devoid of their transmembrane and cytoplasmic domains, were produced in allantoic fluids using the Sendai minigenome system. The first step was rescuing in cell cultures Sendai virus minigenomes encoding the proteins of interest, with the help of wild type Sendai virus. The second step was propagating such recombinant defective viruses, together with the helper virus, in the allantoic cavity of chicken embryonated eggs, and passage to optimize protein production. When compared with the production of the same proteins in the culture supernatant of cells infected with vaccinia recombinants, the yield in the allantoic fluid was 5–10 fold higher. Mutant forms of these soluble proteins were easily constructed by site-directed mutagenesis and expressed in eggs using the same approach.The simplicity and economy of the Sendai minigenome system, together with the high yield achieved in the allantoic fluid of eggs, makes it an attractive method to express soluble glycoproteins aimed for structural studies. E. coli is the simplest and most widely used organism for protein expression due to low cost and ease of use but it has serious limitations for expression of mammalian gene products, particularly glycoproteins [Over the past decades different expression systems have been developed for production of recombinant proteins. Each of these systems has strengths and weaknesses concerning yield, cost, speed, ease of manipulation and folding and post-translational modifications of the target proteins. proteins . Unmodifproteins . Howeverproteins .A repertoire of animal viruses has been grown in embryonated chicken eggs since the early 1930's . For exaParamyxoviridae family within the Mononegavirales order) replicates very efficiently in eggs, we contemplated the possibility of using this virus as a vector for large-scale production of heterologous glycoproteins in the allantoic fluid of embryonated eggs. Rescue of recombinant SeV [Since Sendai virus , including the transmembrane and cytoplasmic regions, has been expressed in HEp-2 cells infected with a recombinant vaccinia virus [TM- folds into trimers that are seen under the electron microscope as cone-shaped molecules [Two heterologous proteins were chosen for proof-of-principle experiments: i) a membrane-anchorless form of the human respiratory syncytial virus (HRSV) fusion (F) glycoprotein  and ii) ia virus  (rVV). Holecules . An analThe previously described HA-M-Svec-36-CB119 plasmid  here re. TranscrTM- forms of either HRSV F or HMPV F proteins  specific for the SeV F protein. The amount of allantoic fluid in the Western blots was standardized with an antibody specific for egg albumin Figure . In genea Figure . This isTM- was proteolitically processed to the F1 and F2 chains (only F1 was observed with the antibody used in the Western blot) about 50% of the HMPV/FTM- precursor remained unprocessed as F0 . This yield reduces the production cost of FTM- proteins about 10 times, considering only the cost of plastic and medium for growing animal cells in comparison with the cost of eggs. However, other significant advantages of embryonated eggs are: i) shorter incubation times, ii) simplicity of manipulations and iii) inexpensive equipment needed for production. These considerations are applicable at the laboratory scale, but other cost calculations may need to be done for industrial processes that require different manipulations and equipment.Figure TM- mutants could be easily obtained by site-directed mutagenesis of the minigenomes. As an example, Figure TM- mutant (lane 2), compared to FTM- wild type (lane 1), in which the basic amino acids of the two furin sites were replaced by Asn residues (NN mutant). Expression of this mutant in eggs generated an anchorless F protein in which all monomers remained uncleaved (F0), as shown previously for an equivalent mutant expressed from rVV [TM- cleavage may be important to maintain this soluble protein unaggregated since, as reported previously, extensive cleavage of its monomers leads to formation of aggregates [As mentioned previously, the small size of plasmids containing cDNA copies of SeV minigenomes facilitates genetic manipulation of the encoded foreign gene. Ffrom rVV . Inhibitgregates  that sedTM- protein produced in eggs has been found indistinguishable in a variety of tests (including reactivity with a panel of MAbs) from that produced in cells infected with rVV. As an example, the NN mutant has been purified to homogeneity  were grown in DMEM10, and G-418 (1 mg/ml) was added every third passage [Hep-2 and CV-1 cells were grown in DMEM medium containing Penicillin and Streptomycin  (Cambrex), supplemented with 10% fetal calf serum (DMEM10) (Linus) at 37°C in a 5% CO passage  to ensurTM-), lacking the transmembrane and cytoplasmic domains, has been described [TM- was obtained using the procedure of Blasco and Moss [TM- was engineered by substituting the amino acid G489 of the F protein by a stop codon. CV-1 monolayers were infected with the vaccinia virus vRB12 [TM-. vRB12 lacks most of the gene encoding vp37, the major protein component of the vaccinia virus envelope, and as a result is unable to form extracellular enveloped virions. Hence, vRB12 is defective in plaque formation under standard conditions. pRB21-derived plasmids carry a complete copy of vp37 and thus, via homologous recombination with vRB12, restore VP37 function. rVV-HMPV/FTM- was obtained from the supernatant of infected/transfected CV-1 cells and subjected to three consecutive rounds of plaque purification, and a virus stock was grown in this cell line.Wild type Sendai virus (SeV), strain Z, was propagated in embryonated 9-day-old chicken eggs, as described . Recombiand Moss . Brieflyand Moss . The plaus vRB12  and subsTM- and SVec/HMPV/FTM-, were obtained by replacing the HA-M gene in the vector SVec/HA-M by HRSV/FTM- and HMPV/FTM-. Briefly, HRSV/FTM- and HMPV/FTM- were PCR amplified with primers containing XmaI and EcoRI sites, and subcloned in XmaI-EcoRI digested SVec/HA-M. Sequencing of the plasmids confirmed the compliance of the "rule of six" [The plasmid HA-M-SVec-36-CB119 (here SVec/HA-M) contains a SeV-defective RNA genome cloned under the control of the T7 RNA polymerase promoter, and includes the SeV matrix protein gene tagged with the influenza virus haemagglutinin protein epitope  (HA-M).  of six" . Plasmid of six" ,29.TM- or SVec/HMPV/FTM- plasmids, along with pGEM-L (3.5 μg), pGEM-N (7 μg), and pGEM-P/Cstop (7 μg) using the MBS mammalian transfection kit (Stratagene), following the manufacturer's instructions. Twenty four hours later the cells were infected with SeV in DMEM supplemented with 2% FCS at a m.o.i. of 5, and after 24 h at 37°C the culture medium was harvested, clarified, and kept at -80°C.Eight hundred thousand BSR-T7/5 cells were seeded in a 35 mm Petri dish with DMEM10 medium. After 24 h, cells were co-transfected with 7 μg of SVec/HA-M, SVec/HRSV/FIn parallel, rescue of the defective minigenome was checked by immunofluorescence of BSR-T7/5 cells growing in microchamber slides. Transfection was done as mentioned in the previous paragraph. Forty eight hours after tranfection the cells were fixed and stained for immunofluorescence with the following primary antibodies: α-HA rabbit polyclonal antibody , α-HRSV F mouse monoclonal antibody 2F  or α-HMPRescue of the minigenomes in eggs was achieved by inoculating 200 μl of culture supernatant of transfected/infected BSR/T7-5 cells in the allantoic cavity of 9-day-old embryonated eggs. The embryos were incubated at 34°C for 48 h, and then transferred to 4°C. Allantoic fluid was harvested after 16 h in the cold and clarified by low-speed centrifugation. One hundred microliters of the allantoic fluids were used for serial passages in embryonated eggs. Allantoic fluids from the different passages were analyzed by Western blot.255–275 or HMPV/F468–484 (numbers refer to amino acids included in the peptides). After washing with 0,05%Tween-20 in PBS, the membranes were incubated with horseradish peroxidase-linked α-mouse, α-rat, or α-rabbit immunoglobulin secondary antibodies  and developed with ECL.Samples for protein analysis were subjected to 12% SDS-PAGE and electrotransferred to PVDF membranes . After blocking with I-Block agent (Tropix), membranes were incubated with one of the following primary antibodies: α-chicken egg albumin monoclonal antibody , α-HA high affinity rat monoclonal antibody , α-Sendai F monoclonal antibody (GB5) or rabbit polyclonal antibodies raised against synthetic peptides HRSV/FQuantification of band intensities in Western blots of figure Clarified allantoic fluids were centrifuged through a 25% glycerol cushion in PBS at 35,000 rpm for 3 h at 4°C in a SW40 rotor (Beckman). Pellets were then resuspended in PBS, and analyzed by Western blot.TM-NN was obtained by sequential mutagenesis of cleavage site II of HRSV/FTM- with the oligonucleotides F387-441+ (5'-CCAATGTAACATTAAGCAACAACAACAACAACAACTTTCTTGGTTTTTTGTTAGG-3') and F441-387- (5'-CCTAACAAAAAACCAAGAAAGTTGTTGTTGTTGTTGTTGCTTAATGTTACATTGG-3') and of cleavage site I with oligonucleotides F309-358+ (5'-GCACACCAGCAGCAAACAATAACGCCAACAACGAACTACCAAGG-3') and F358-309-(5'-CCTTGGTAGTTCGTTGTTGGCGTTATTGTTTGCTGCTGGTGTGC-3'), using the QuickChange Site-Directed Mutagenesis Kit (Stratagene) following the manufacturer's instructions. The plasmid obtained was checked by sequencing.SVec/HRSV/FTM-NN mutant was purified following procedures used previously for purification of analogous mutants from supernatants of Hep-2 cells infected with rVV [TM-NN protein was absorbed onto carbon films and stained with 1% sodium silicotungstate (pH 7.0). A Jeol 1200 electron microscope, operated at 100 kV, was used to view the samples. Micrographs were taken under minimum-dose, accurate defocus conditions to preserve details to ~1.5 nm[HRSV/Fwith rVV . Brieflywith rVV  covalento ~1.5 nm.TM- genes and established the expression system. LSV amplified and cloned the HMPV F protein gene. B G-B grew HMPV in tissue culture cells from the original stock and contributed to different cloning steps of HRSV/F and HMPV/F. GM and LR developed the original SeV supporting plasmids and help in setting up the rescue procedure. OC purified the recombinant proteins and LJC made the EM photographs. JJS and JAM were involved in concept development and experimental design.TC made the recombinant SVec plasmids carrying the FAll authors read and approved the final manuscript