Microstructure analysis, tribological correlation properties and strengthening mechanism of graphene reinforced aluminum matrix composites.

In this paper, graphene reinforced aluminum matrix composites are successfully prepared by high-energy ball milling. The results show that no graphene agglomeration is found in the mixed powder. The complex composites prepared by high energy ball milling and powder metallurgy have approximately 4-5 layers of graphene and the thickness of single-layer graphene is approximately 0.334 nm. The final experimental results confirm the formation of compound AlC3 in the microstructure, and its diffraction spot index is ([Formula: see text]00), ([Formula: see text]1[Formula: see text]) and (11[Formula: see text]). The maximum friction coefficient is 0.126, and the average friction coefficient is 0.027, suggesting good wear resistance and corrosion resistance. Additionally, the friction corrosion mechanism of the material is deeply analyzed. The results of strengthening mechanism analysis show that the main strengthening mechanism of the materials designed in this experiment is thermal mismatch strengthening. It can be concluded that the yield strength of the material calculated by the modified model is 227.75 MPa. This value is slightly lower than the calculated value of the general shear lag model (237.68 MPa). However, it is closer to the yield strength value of the actual material (211 MPa).

HBO1 overexpression is important for hepatocellular carcinoma cell growth.

Hepatocellular carcinoma (HCC) is a common primary liver malignancy lacking effective molecularly-targeted therapies. HBO1 (lysine acetyltransferase 7/KAT7) is a member of MYST histone acetyltransferase family. Its expression and potential function in HCC are studied. We show that HBO1 mRNA and protein expression is elevated in human HCC tissues and HCC cells. HBO1 expression is however low in cancer-surrounding normal liver tissues and hepatocytes. In HepG2 and primary human HCC cells, shRNA-induced HBO1 silencing or CRISPR/Cas9-induced HBO1 knockout potently inhibited cell viability, proliferation, migration, and invasion, while provoking mitochondrial depolarization and apoptosis induction. Conversely, ectopic overexpression of HBO1 by a lentiviral construct augmented HCC cell proliferation, migration and invasion. In vivo, xenografts-bearing HBO1-KO HCC cells grew significantly slower than xenografts with control HCC cells in severe combined immunodeficient mice. These results suggest HBO1 overexpression is important for HCC cell progression.

The efficacy and safety of the combination of axitinib and pembrolizumab-activated autologous DC-CIK cell immunotherapy for patients with advanced renal cell carcinoma: a phase 2 study.

OBJECTIVES: Although axitinib has achieved a preferable response rate for advanced renal cell carcinoma (RCC), patient survival remains unsatisfactory. In this study, we evaluated the efficacy and safety of a combination treatment of axitinib and a low dose of pembrolizumab-activated autologous dendritic cells-co-cultured cytokine-induced killer cells in patients with advanced RCC. METHODS: All adult patients, including treatment-naive or pretreated with VEGF-targeted agents, were enrolled from May 2016 to March 2019. Patients received axitinib 5 mg twice daily and pembrolizumab-activated dendritic cells-co-cultured cytokine-induced killer cells intravenously weekly for the first four cycles, every 2 weeks for the next four cycles, and every month thereafter. RESULTS: The 43 patients (22 untreated and 21 previously treated) showed a median progression-free survival (mPFS) of 14.7 months (95% CI, 11.16-18.30). mPFS in treatment-naive patients was 18.2 months, as compared with 14.4 months in pretreated patients (log-rank P-value = 0.07). Overall response rates were 25.6% (95% CI, 13.5-41.2%). Grade 3 or higher adverse events occurred in 5% of patients included hypertension (11.6%) and palmar-plantar erythrodysesthesia (7.0%). Peripheral blood lymphocyte immunophenotype and serum cytokine profile analyses demonstrated increased antitumor immunity after combination treatment particularly in patients with a long-term survival benefit, while those with a minimal survival benefit demonstrated an elevated proportion of peripheral CD8+TIM3+ T cells and lower serum-level immunostimulatory cytokine profile. CONCLUSIONS: The combination therapy was active and well tolerated for treatment of advanced RCC, either as first- or second-line treatment following other targeted agents. Changes in immunophenotype and serum cytokine profile may be used as prognostic biomarkers.

Functional Analysis of Sterol O-Acyltransferase Involved in the Biosynthetic Pathway of Pachymic Acid in Wolfiporia cocos.

Pachymic acid from Wolfiporia cocos possesses important medicinal values including anti-bacterial, anti-inflammatory, anti-viral, invigorating, anti-rejection, anti-tumor, and antioxidant activities. However, little is known about the biosynthetic pathway from lanostane to pachymic acid. In particular, the associated genes in the biosynthetic pathway have not been characterized, which limits the high-efficiency obtaining and application of pachymic acid. To characterize the synthetic pathway and genes involved in pachymic acid synthesis, in this study, we identified 11 triterpenoids in W. cocos using liquid chromatography tandem mass spectrometry (LC-MS/MS), and inferred the putative biosynthetic pathway from lanostane to pachymic acid based on analyzing the chemical structure of triterpenoids and the transcriptome data. In addition, we identified a key gene in the biosynthetic pathway encoding W. cocos sterol O-acyltransferase (WcSOAT), which catalyzes tumolusic acid to pachymic acid. The results show that silence of WcSOAT gene in W. cocos strain led to reduction of pachymic acid production, whereas overexpression of this gene increased pachymic acid production, indicating that WcSOAT is involved in pachymic acid synthesis in W. cocos and the biosynthesis of W. cocos pachymic acid is closely dependent on the expression of WcSOAT gene. In summary, the biosynthetic pathway of pachymic acid and the associated genes complement our knowledge on the biosynthesis of W. cocos pachymic acid and other triterpenoids, and also provides a reference for target genes modification for exploring high-efficiency obtaining of active components.

The incidence and prognostic effect of Fms-like tyrosine kinase 3 gene internal tandem and nucleolar phosphoprotein 1 genes in acute myeloid leukaemia: A PRISMA-compliant systematic review and meta-analysis.

BACKGROUND: Molecular genotyping is an important prognostic role in acute myeloid leukemia (AML) patients. We aimed to design this meta-analysis to discuss the incidence and prognostic effect of nucleolar phosphoprotein 1 (NPM1) and Fms-like tyrosine kinase 3 gene internal tandem (FLT3-ITD) gene in AML patients. METHODS: PubMed, Embase, Medline, and Cochrane library were systematically searched due to May 15, 2020. Four combinations of genotypes (FLT3-ITDneg/NPM1mut, FLT3-ITDpos/NPM1mut, FLT3-ITDneg/NPM1wt, FLT3-ITDpos/NPM1wt) were compared in association with the overall survival (OS) and leukemia-free survival (LFS) outcome, which expressed as pooled hazard ratio (HR) and 95% confidence intervals (CIs). RESULTS: Twenty-eight studies were included in our study. The incidence of FLT3-ITDneg/NPM1mut, FLT3-ITDpos/NPM1mut, FLT3-ITDneg/NPM1wt, and FLT3-ITDpos/NPM1wt was 16%, 13%, 50%, and 10%, respectively. The patients with FLT3-ITDneg/NPM1mut gene may have the best OS and LFS when comparing with FLT3-ITDpos/NPM1mut (HR = 1.94 and 1.70, P < .01), FLT3-ITDneg/NPM1wt (HR = 1.57 and 2.09, P < .01), and FLT3-ITDpos/NPM1wt (HR = 2.25 and 2.84, P < .001). CONCLUSION: AML patients with FLT3-ITDneg/NPM1mut gene type have the best survival outcome than the other 3 gene types, which should be an independent genotyping in AML classification.

Severe delayed pulmonary toxicity following PD-L1-specific CAR-T cell therapy for non-small cell lung cancer.

OBJECTIVES: This phase I study aimed to evaluate the antitumor effect and safety of programmed death-ligand-1 (PD-L1)-targeting autologous chimeric antigen receptor T (CAR-T) cells for patients with non-small cell lung cancer (NSCLC). METHODS: Programmed death-ligand-1-specific CAR-T cells were generated using lentiviral transduction. Four patients with NSCLC were recruited, but only one patient was finally involved. CAR-T cells were infused on three different days (total dose during therapy, 1 × 106 CAR-T cells kg-1 body weight). The date on which the patient received the first CAR-T cell infusion was designated as Day 0. RESULTS: Circulating CAR-T cells accounted for 3.30% of the patient's peripheral blood T cells detected by FACS analysis during the first follow-up (Day +29). The chest CT scan showed subtle tumor shrinkage (stable disease). On Day +43, the patient developed pyrexia without any known causes and dyspnoea that rapidly deteriorated to respiratory failure in 3 days. The chest X-ray and CT scan showed bilateral extensive pulmonary infiltration in addition to the tumor silhouette on the left upper lung. The interleukin (IL)-6 levels in serum dramatically increased (> 100-fold). The patient was immediately transferred to the ICU where he received oxygen and intravenous infusions of tocilizumab and methylprednisolone. His symptoms rapidly improved and the pulmonary inflammation gradually resolved. CONCLUSION: The clinical manifestations and test findings for this patient with NSCLC might represent unique clinical manifestations of solitary organ damage secondary to PD-L1-specific CAR-T cell therapy. The differential diagnosis, underlying mechanism and prevention and treatment strategies for such complications have also been discussed.

Influence of obesity on in-hospital and postoperative outcomes of hepatic resection for malignancy: a 10-year retrospective analysis from the US National Inpatient Sample.

OBJECTIVES: The influence of obesity on the outcomes of curative liver resection for malignancies remains controversial. We aimed to compare the in-hospital outcomes of liver resection for malignancy between obese and non-obese patients. DESIGN: This was a population-based, retrospective, observational study using data from the Nationwide Inpatient Sample (NIS), the largest all-payer US inpatient care database. SETTING: Hospitalisations of adults ≥18 years old with diagnoses of primary hepatobiliary malignancy or secondary malignant neoplasms of liver in the USA were identified from the NIS database between 2005 and 2014. PARTICIPANTS: Data of 18 398 patients ≥18 years old and underwent liver resection without pancreatic resection in the NIS were extracted. All included subjects had primary hepatobiliary malignancy or secondary malignant neoplasms of the liver. Patients were divided into obese and non-obese groups. These groups were compared with respect to postoperative complications, length of hospital stay and hospital cost according to surgical extent and approach. INTERVENTIONS: Patients were undergoing lobectomy of liver or partial hepatectomy. PRIMARY AND SECONDARY OUTCOME MEASURES: The primary endpoints of this study were postoperative complications, length of hospital stay and hospital cost. RESULTS: After adjustment, obese patients were significantly more likely to experience postoperative complications than were non-obese patients (adjusted OR 1.25, 95% CI 1.10 to 1.42), regardless of whether lobectomy or partial hepatectomy was performed. Furthermore, obesity was significantly associated with increased risk of postoperative complications in patients who underwent open liver resection, but not laparoscopic resection. No significant difference was observed in length of hospital stay or total hospital costs between obese and non-obese patients. CONCLUSIONS: After adjustment for preoperative comorbidities and other potential confounders, obesity is significantly associated with greater risk of complications in patients undergoing open liver resection for malignancy, but not laparoscopic resection.

BCL2L10/BECN1 modulates hepatoma cells autophagy by regulating PI3K/AKT signaling pathway.

The aim of this study was to investigate BCL2L10 and BECN1 expression and their effect on autophagy in hepatocellular carcinoma (HCC). We found that BCL2L10 expression was low in hepatoma tissues and cells. Overexpression of BCL2L10 decreased the activity of hepatoma cells. To analyze autophagic flux, we monitored the formation of autophagic vesicles by fluorescence protein method. Autophagy-related protein LC3B-II was accumulated and P62 was decreased, which indicated that autophagy was induced by BECN1, while BCL2L10 could suppress this trend. Immunofluorescence assay showed that BCL2L10 and Beclin 1 were co-located in hepatoma cells. Immunoprecipitation showed that BCL2L10 could inhibit the autophagy of hepatoma cells by combining with Beclin 1. ELISA and co-immunoprecipitation suggested that the combination between BCL2L10 and Beclin 1 reduced the bond between Beclin 1 and PI3KC3. Based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, the PI3K/AKT signaling pathway was significantly upregulated in HCC. In conclusions, BCL2L10 had a low expression in HCC tissues and cells, which could release BECN1 to induce autophagy of hepatoma cells by downregulating PI3K/AKT signaling pathway.

Identification of α-fetoprotein-specific T-cell receptors for hepatocellular carcinoma immunotherapy.

Hepatocellular carcinoma (HCC) is the major form of liver cancer for which there is no effective therapy. Genetic modification with T-cell receptors (TCRs) specific for HCC-associated antigens, such as α-fetoprotein (AFP), can potentially redirect human T cells to specifically recognize and kill HCC tumor cells to achieve antitumor effects. In this study, using lentivector and peptide immunization, we identified a population of cluster of differentiation 8 (CD8) T cells in human leukocyte antigen (HLA)-A2 transgenic AAD mice that recognized AFP158 epitope on human HCC cells. Adoptive transfer of the AFP158 -specific mouse CD8 T cells eradicated HepG2 tumor xenografts as large as 2 cm in diameter in immunocompromised nonobese diabetic severe combined immunodeficient gamma knockout (NSG) mice. We then established T-cell hybridoma clones from the AFP158 -specific mouse CD8 T cells and identified three sets of paired TCR genes out of five hybridomas. Expression of the murine TCR genes redirected primary human T cells to bind HLA-A2/AFP158 tetramer. TCR gene-engineered human T (TCR-T) cells also specifically recognized HLA-A2+ AFP+ HepG2 HCC tumor cells and produced effector cytokines. Importantly, the TCR-T cells could specifically kill HLA-A2+ AFP+ HepG2 tumor cells without significant toxicity to normal primary hepatocytes in vitro. Adoptive transfer of the AFP-specific TCR-T cells could eradicate HepG2 tumors in NSG mice. CONCLUSION: We have identified AFP-specific murine TCR genes that can redirect human T cells to specifically recognize and kill HCC tumor cells, and those AFP158 -specific TCRs have a great potential to engineer a patient's autologous T cells to treat HCC tumors. (Hepatology 2018).

SIRT1 in B[a]P-induced lung tumorigenesis.

Benzo[a]pyrene (B[a]P) is a carcinogen in cigarette smoke. We found that B[a]P induced SIRT1 in human bronchial epithelial BEAS-2B cell. SIRT1 was overexpressed in the lung of B[a]P-exposed mice and in human lung cancer biopsies. SIRT1 up-regulated TNF-α and ß-catenin and down-regulated the membrane fraction of E-cadherin. In addition, SIRT1 promoted invasion, migration and tumorigenesis of BEAS-2B cells in nude mice upon B[a]P exposure. Thus, SIRT1 is involved in B[a]P-induced transformation associated with activation of the TNF-α/ß-catenin axis and is as a potential therapeutic target for lung cancer.

Hyperthermia inhibits recombination repair of gemcitabine-stalled replication forks.

BACKGROUND: Gemcitabine is a potent nucleoside analogue against solid tumors, but development of drug resistance is a substantial problem. Removal of gemcitabine incorporated into DNA by repair mechanisms may contribute to resistance in chemo-refractory solid tumors. Human hepatocellular carcinoma (HCC) is usually very chemoresistant to gemcitabine. METHODS: We treated HCC in vitro and in vivo (orthotopic murine model with human Hep3B or HepG2 xenografts, 7-10 CB17SCID mice per group) with gemcitabine. The role of homologous recombination repair proteins in repairing stalled replication forks was evaluated with hyperthermia exposure and cell-cycle analysis. The Student t-test was used for two-sample comparisons. Multiple group data were analyzed using one-way analysis of variance. All statistical tests were two-sided. RESULTS: We demonstrated that Mre11-mediated homologous recombination repair of gemcitabine-stalled replication forks is crucial to survival of HCC cells. Furthermore, we demonstrated inhibition of Mre11 by an exonuclease inhibitor or concomitant hyperthermia. In orthotopic murine models of chemoresistant HCC, the Hep3B tumor mass with radiofrequency plus gemcitabine treatment (mean ± SD, 180±91mg) was statistically significantly smaller compared with gemcitabine alone (661±419mg, P = .0063). CONCLUSIONS: This study provides mechanistic understanding of homologous recombination inhibiting-strategies, such as noninvasive radiofrequency field-induced hyperthermia, to overcome resistance to gemcitabine in refractory human solid tumors.

Targeting SMARCAL1 as a novel strategy for cancer therapy.

SMARCAL1 is a SNF2 chromatin-remodeling protein with ATP-dependent annealing helicase activity. Recent studies have shown that SMARCAL1 is involved in DNA damage repair and cell cycle progression. Deficiency of SMARCAL1 enhances the anticancer activity of chemotherapy agents and reverses cancer cell resistance to these agents. Therefore, targeting SMARCAL1 is an attractive therapeutic approach for cancers with defects in DNA damage repair or cell cycle checkpoints. Here, we review advances in our understanding of the biochemical and cellular functions of SMARCAL1 made over the recent years and discuss the rationale for development of SMARCAL1 inhibitors as novel anticancer therapies.

Silencing thioredoxin induces liver cancer cell senescence under hypoxia.

AIM: Although thioredoxin 1 (TXN) has pleiotropic cellular functions as a redox-sensitive protein, very little is known about its role in tumor survival and growth under hypoxia. MHCC97H hepatocellular carcinoma cells have a high metastatic potential and high thioredoxin expression levels compared with their parent cell line, MHCC97. Thus, we used this cell line to explore the functional connections between TXN and hypoxia. METHODS: MHCC97H cells were cultured under normoxia and hypoxia for specific periods after nucleofection with TXN siRNA or control siRNA. We assessed the ß-phenylethyl isothiocyanate (PEITC) sensitivity of the cells, cell proliferation, cell cycle and senescence, and DNA damage response by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, colony formation assays, flow cytometry, ß-galactosidase staining, western blotting, and immunohistochemistry. RESULTS: ß-phenylethyl isothiocyanate treatment shifted reduced TXN to oxidized TXN in MHCC97H cells. Although silencing of TXN via siRNA had no effect on the PEITC sensitivity of the cells, it suppressed cell proliferation and colony formation under both normoxia and hypoxia. Under hypoxia, silencing TXN did not induce apoptosis but induced DNA damage response and cellular senescence. CONCLUSIONS: High TXN levels in MHCC97H cells protect them from DNA damage and cellular senescence under hypoxia. Targeting TXN might enhance the chemotherapeutic effects of some DNA-damaging agents against hepatocellular carcinoma.

Multiple kinase pathways involved in the different de novo sensitivity of pancreatic cancer cell lines to 17-AAG.

BACKGROUND: 17-Allylamino-17-demethoxygeldanamycin (17-AAG) specifically targets heat shock protein (HSP)90 and inhibits its chaperoning functions for multiple kinases involved in cancer cell growth and survival. To select responsive patients, the molecular mechanisms underlying the sensitivity of cancer cells to 17-AAG must be elucidated. MATERIALS AND METHODS: We used cytotoxicity assays and Western blotting to explore the effects of 17-AAG and sorafenib on cell survival and expression of multiple kinases in the pancreatic cancer cell lines AsPC-1 and Panc-1. Gene cloning and transfection, siRNA silencing, and immunohistochemistry were used to evaluate the effects of mutant p53 protein on 17-AAG sensitivity. RESULTS: AsPC-1 and Panc-1 responded differently to 17-AAG, with half maximal inhibitory concentration (IC(50)) values of 0.12 and 3.18 µM, respectively. Comparable expression of HSP90, HSP70, and HSP27 was induced by 17-AAG in AsPC-1 and Panc-1 cells. P-glycoprotein and mutant p53 did not affect 17-AAG sensitivity in these cell lines. Multiple kinases are more sensitive to HSP90 inhibition in AsPC-1 than in Panc-1 cells. After 17-AAG treatment, p-Bad (S112) decreased in AsPC-1 cells and increased in Panc-1 cells. Sorafenib markedly increased p-Akt, p-ERK1/2, p-GSK-3ß, and p-S6 in both cell lines. Accordingly, 17-AAG and sorafenib acted antagonistically in AsPC-1 and Panc-1 cells, except at high concentrations in AsPC-1 cells. CONCLUSIONS: Differential inhibition of multiple kinases is responsible for the different de novo sensitivity of AsPC-1 and Panc-1 cells to HSP90 inhibition. P-glycoprotein and mutant p53 protein did not play a role in the sensitivity of pancreatic cancer cells to 17-AAG.

Comparison of bolus remifentanil versus bolus fentanyl for blunting cardiovascular intubation responses in children: a randomized, double-blind study.

BACKGROUND: The authors found no study to compare the efficacy of bolus dose fentanyl and remifentanil blunting the cardiovascular intubation response in children, so they designed this randomized, double-blind clinical study to assess the effects of remifentanil 2 microg/kg and fentanyl 2 microg/kg by bolus injection on the cardiovascular intubation response in healthy children. METHODS: One hundred and two children, the American Society of Anesthesiologists (ASA) physical status 1-2 and scheduled for elective plastic surgery under general anesthesia, were randomly divided into one of two groups to receive the following treatments in a double blind manner: remifentanil 2 microg/kg (Group R) and fentanyl 2 microg/kg (Group F) when anesthesia was induced with propofol and vecuronium. The orotracheal intubation was performed using a direct laryngoscope. Blood pressure (BP) and heart rate (HR) were recorded before anesthesia induction (baseline values), immediately before intubation, at intubation and every minute for 5 minutes after intubation. The percent changes of systolic blood pressure (SBP) and HR relative to baseline values and the rate pressure product (RPP) at every observing point were calculated. The incidences of SBP and HR percent changes >30% of baseline values and RPP >22,000 during the observation were recorded. RESULTS: There were no significant differences between groups in the demographic data, baseline values of BP and HR and the intubation time. As compared to baseline values, BP, HR and RPP at intubation and their maximum values during observation increased significantly in Group F, but they all decreased significantly in Group R. BP, HR and RPP at all observed points, and their maximum values during the observation, were significantly different between groups. There were also significant differences between groups in the percent change of SBP and HR relative to baseline values at all observed points and their maximum percent changes during the observation. The incidences of SBP and HR percent increased >30% of the baseline values and RPP >22,000 during the observation, were significantly higher in Group F than in Group R, but the incidences of SBP and HR percent decreased >30% of baseline values were significantly lower in Group F compared with Group R. CONCLUSIONS: When used as part of routine anesthesia induction with propofol and vecuronium in children, fentanyl 2 microg/kg by bolus injection fails to effectively depress the cardiovascular intubation response. Remifentanil 2 microg/kg by bolus injection can completely abolish the cardiovascular intubation response, but also cause more adverse complications of temporary significant cardiovascular depression.

[Comparison of cardiovascular responses between orotracheal and nasotracheal intubation with the aid of GlideScope video laryngoscope].

OBJECTIVE: To compare the cardiovascular responses to orotracheal or nasotracheal intubation with the aid of GlideScope video laryngoscope (GSVL). METHODS: Sixty patients, American Society of Anesthesiologists (ASA) physical status I, aged 16-50 years, scheduled for elective plastic surgery under general anesthesia,were randomly allocated equally to the orotracheal intubation group (OTI group) and the nasotracheal intubation group (NTI group). After the routine anesthesia induction, orotracheal and nasotracheal intubation was respectively performed with the aid of GSVL. Non-invasive blood pressure and heart rate (HR) were recorded before (baseline values) and after anesthesia induction (postinduction values), during intubation and every minute for 5 minutes after intubation. Maximum values of blood pressure and HR during the observation periods were recorded. The product of HR and systolic blood pressure [rate pressure product (RPP)] at every time point was calculated. Duration for glottis exposure and duration for successful intubation were also noted. RESULTS: There were no significant differences between two groups in the demographic data (P>0.05). Glottis exposure time and intubation time in NTI group were significantly longer than those in OTI group [(52.2+/-13.5) seconds vs. (40.5+/-15.2) seconds, P<0.05]. After anesthesia induction, blood pressure and RPP in both groups decreased significantly compared with baseline values, but no significant change in HR was noted. Compared with their postinduction values, the blood pressure and RPP in both groups and HR in OTI group increased significantly at intubation. In OTI group,the maximum values of HR, diastolic blood pressure (DBP), mean arterial pressure (MAP), RPP exceeded their baseline values. But in NTI group,only maximal HR during the observation period was significantly higher than the baseline values. The blood pressure at every time point was not significantly different between two groups. But intubation in OTI group caused significant increases in HR and RPP compared with those in NTI group (both P<0.05). CONCLUSION: In anesthetized adult patients, orotracheal and nasotracheal intubations with the GSVL can result in a similar pressor response, however orotracheal intubation with GSVL causes more marked cardiovascular responses than nasotracheal intubation with the aid of GSVL.