Journal: Journal of Translational Medicine

Article Title: ALDH1L1 reverses CD8 + T cell exhaustion in the oral squamous cell carcinoma microenvironment by reprogramming L-glutamate metabolism

doi: 10.1186/s12967-026-07812-z

Figure Lengend Snippet: ALDH1L1 modulates CD8⁺ T-cell proliferation through the IL-15 signaling pathway. ( A ) Flow cytometric analysis of CD8⁺ T cell infiltration in clinical OSCC tissues ( n = 6 per group). ( B ) Expression distribution of ALDH1L1 across cell subtypes in OSCC from the TISCH2 database ( GSE172577 dataset). ( C ) Immunohistochemical staining of ALDH1L1 and CD8 in OSCC sections (ALDH1L1 low , n = 23; ALDH1L1 high , n = 29). ( D ) Immunohistochemical staining of ALDH1L1 and CD8 in xenograft tumor sections ( n = 6 per group). ( E ) Quantitative analysis of CD8⁺ T cell proportions in xenograft tissues by flow cytometry ( n = 6). ( F - G ) Correlation analysis between ALDH1L1 and chemokine mRNA expression levels in clinical OSCC samples (qRT-PCR, n = 12). H. qRT-PCR analysis of IL-15 mRNA expression in NC and ALDH1L1-KD groups of CAL 27 and SCC-25 cells ( n = 3). I. Extracellular IL-15 concentration in OSCC cell cultures with different ALDH1L1 expression levels ( n = 3). ( J ) Flow cytometric analysis of IL-15-mediated CD8⁺ T-cell proliferation in vitro, ( n = 3). (NC, negative control; OE, overexpression; KD, knockdown, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001)

Article Snippet: Recombinant Human ALDH1L1 Protein (Cat# bs-100230P, Biosschina) and Recombinant Human GLUL Protein (HY- P70294 , MedChemExpress) were obtained commercially.

Techniques: Expressing, Immunohistochemical staining, Staining, Flow Cytometry, Quantitative RT-PCR, Concentration Assay, In Vitro, Negative Control, Over Expression, Knockdown

Journal: Journal of Translational Medicine

Article Title: ALDH1L1 reverses CD8 + T cell exhaustion in the oral squamous cell carcinoma microenvironment by reprogramming L-glutamate metabolism

doi: 10.1186/s12967-026-07812-z

Figure Lengend Snippet: ALDH1L1 modulates CD8⁺ T-cell function through metabolic reprogramming. ( A ) Quantification of PD-1⁺ CD8⁺ T cells in clinical OSCC specimens by flow cytometry ( n = 6 per group). ( B ) Quantification of PD-1⁺ CD8⁺ T cells in mouse xenografts by flow cytometry ( n = 6 per group). ( C ) Top 20 enriched KEGG pathways identified from transcriptomic analysis. ( D ) Chord diagram illustrating 29 key metabolites identified by metabolomic profiling. ( E - H ) Flow cytometric evaluation of CD8⁺ T cell marker expression after stimulation with metabolites derived from ALDH1L1-KD or control CAL 27 cells and SCC-25 cells ( n = 3). (NC, negative control; OE, overexpression; KD, knockdown; ns, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001)

Article Snippet: Recombinant Human ALDH1L1 Protein (Cat# bs-100230P, Biosschina) and Recombinant Human GLUL Protein (HY- P70294 , MedChemExpress) were obtained commercially.

Techniques: Cell Function Assay, Flow Cytometry, Metabolomic, Marker, Expressing, Derivative Assay, Control, Negative Control, Over Expression, Knockdown

Journal: Journal of Translational Medicine

Article Title: ALDH1L1 reverses CD8 + T cell exhaustion in the oral squamous cell carcinoma microenvironment by reprogramming L-glutamate metabolism

doi: 10.1186/s12967-026-07812-z

Figure Lengend Snippet: L-glutamate accumulation mediates CD8⁺ T cell dysfunction in ALDH1L1-downregulated microenvironment. ( A - C ) Integrated multi-omics analysis reveals ALDH1L1-regulated metabolic signatures: Red boxes highlight co-enriched metabolic pathways from transcriptomic ( A ) and metabolomic ( B ) analysis, and key metabolites ( C ). ( D ) Extracellular L-glutamate concentration in conditioned media from CAL 27 and SCC-25 cell culture supernatants ( n = 3). ( E - F ) Flow cytometric analysis of L-glutamate effects on CD8⁺ T cell functional markers: ( E ) Proportion of IFN-γ⁺ CD8⁺ T cells; ( F ) Proportion of PD-1⁺ CD8⁺ T cells ( n = 3). ( G ) Cytotoxic activity of CD8⁺ T cells pretreated with conditioned medium from ALDH1L1-KD or NC OSCC cells medium with exogenous L-glutamate ( n = 3). (NC, negative control; KD, knockdown; Ctrl, control; Glu, L-glutamate; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001)

Article Snippet: Recombinant Human ALDH1L1 Protein (Cat# bs-100230P, Biosschina) and Recombinant Human GLUL Protein (HY- P70294 , MedChemExpress) were obtained commercially.

Techniques: Biomarker Discovery, Metabolomic, Concentration Assay, Cell Culture, Functional Assay, Activity Assay, Negative Control, Knockdown, Control

Journal: Journal of Translational Medicine

Article Title: ALDH1L1 reverses CD8 + T cell exhaustion in the oral squamous cell carcinoma microenvironment by reprogramming L-glutamate metabolism

doi: 10.1186/s12967-026-07812-z

Figure Lengend Snippet: ALDH1L1 transcriptionally regulates GLUL and interacts with GLUL to sustain L-glutamate metabolism. ( A ) Integrated multi-omics analysis of ALDH1L1-regulated metabolic network. Green circles: downregulated metabolites; Green squares: downregulated metabolic enzymes; Red squares: upregulated metabolic enzymes. ( B - C ) GLUL mRNA ( B ) and protein ( C ) expression in CAL 27 and SCC-25 cells assessed by qRT-PCR and western blot, respectively. ( D ) Protein-protein interaction (PPI) network between ALDH1L1 and glutamate-metabolizing enzymes predicted by the STRING database. ( E ) Three-dimensional structural model of ALDH1L1 (green)-GLUL (blue) interaction. Key residues are shown in stick representation; hydrogen bonds indicated by yellow dashed lines. ( F ) Co-immunoprecipitation analysis of endogenous interactions among ALDH1L1 and GLUL in CAL 27 cells. ( G ) ALDH1L1 enhances GLUL enzymatic activity in a dose-dependent manner in vitro ( n = 3). ( H ) Western blot validation of GLUL overexpression in plasmid-transfected CAL 27 cells. ( I ) Extracellular L-glutamate concentration following GLUL upregulation in ALDH1L1-KD CAL 27 cells ( n = 3). (NC: negative control; KD: ALDH1L1 knockdown; OE: GLUL overexpression; ns, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001)

Article Snippet: Recombinant Human ALDH1L1 Protein (Cat# bs-100230P, Biosschina) and Recombinant Human GLUL Protein (HY- P70294 , MedChemExpress) were obtained commercially.

Techniques: Biomarker Discovery, Expressing, Quantitative RT-PCR, Western Blot, Immunoprecipitation, Activity Assay, In Vitro, Over Expression, Plasmid Preparation, Transfection, Concentration Assay, Negative Control, Knockdown

Journal: Journal of Translational Medicine

Article Title: ALDH1L1 reverses CD8 + T cell exhaustion in the oral squamous cell carcinoma microenvironment by reprogramming L-glutamate metabolism

doi: 10.1186/s12967-026-07812-z

Figure Lengend Snippet: Stevioside enhances anti-PD-1 immunotherapy by targeting ALDH1L1. ( A ) Virtual screening workflow for identifying potential ALDH1L1-targeting compounds. ( B ) Two-dimensional chemical structures of the top 12 candidate compounds ranked by binding affinity to ALDH1L1 protein. ( C ) Western blot analysis demonstrating the effect of stevioside treatment on ALDH1L1 protein expression levels in CAL 27 cells (Ctrl, control). ( D ) Dose-response curve of stevioside (IC₅₀ = 195.3 µM). ( E ) Molecular docking analysis reveals the binding site of stevioside on ALDH1L1 protein. (Blue ribbon structure represents ALDH1L1 protein; green stick model represents stevioside molecule; stick structures indicate key interacting amino acid residues; yellow dashed lines denote hydrogen bond interactions.) ( F ) In vivo experiments confirm that stevioside combined with anti-PD-1 antibody treatment significantly enhances antitumor efficacy ( n = 5 per group). ( G - H ) Immunohistochemical staining of Ki-67 ( G ) and CD8 ( H ) in xenograft tumor sections ( n = 5 per group). (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001)

Article Snippet: Recombinant Human ALDH1L1 Protein (Cat# bs-100230P, Biosschina) and Recombinant Human GLUL Protein (HY- P70294 , MedChemExpress) were obtained commercially.

Techniques: Binding Assay, Western Blot, Expressing, Control, In Vivo, Immunohistochemical staining, Staining

Journal: Journal of Translational Medicine

Article Title: ALDH1L1 reverses CD8 + T cell exhaustion in the oral squamous cell carcinoma microenvironment by reprogramming L-glutamate metabolism

doi: 10.1186/s12967-026-07812-z

Figure Lengend Snippet: Schematic model illustrating the mechanism by which ALDH1L1 reverses CD8⁺ T cell exhaustion in OSCC

Article Snippet: Recombinant Human ALDH1L1 Protein (Cat# bs-100230P, Biosschina) and Recombinant Human GLUL Protein (HY- P70294 , MedChemExpress) were obtained commercially.

Techniques:

Journal: Experimental and Therapeutic Medicine

Article Title: Effect of Huaier on the proliferation and apoptosis of human gastric cancer cells through modulation of the PI3K/AKT signaling pathway

doi: 10.3892/etm.2015.2600

Figure Lengend Snippet: Effect of Huaier extract on the protein expression of AKT1, PI3K, PTEN, PDK1, caspase-9 and Bcl-2 in SGC7901 cells. The expression of GAPDH was used as an internal control. The SGC7901 cells were treated with 0, 2.5 and 5 mg/ml Huaier for 24 h, and (A) western blotting was used to analyze the protein expression. (B) Quantification of the data. The experiment was performed in triplicate, and the data are expressed as the mean ± standard deviation of the three separate experiments. *P<0.05 and **P<0.01, compared with the control. p-, phosphorylated-; PI3K, phosphatidylinositol 3-kinase; PTEN, phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase; PDK1, pyruvate dehydrogenase kinase isoform 1; Bcl-2, B-cell lymphoma 2; BAD, Bcl-2-associated death promoter.

Article Snippet: Primary monoclonal antibodies against AKT (1:1,000; 10176-2-AP), PDK1 (1:1,000; 10026-1-AP), Bcl-2-associated death promoter (BAD; 1:1,000; 10435-1-AP) and GAPDH (1:4,000; 60004-1-Ig) were purchased from Proteintech (Manchester, UK), while monoclonal antibodies against p-AKT (The-308; 1:800; 2118-1), p-AKT (Ser-473; 1:800; 2214-1), PI3K (1:1,000; 1593-S), PTEN (1:1,000; 5171-1), p-PTEN (1:1,000; 2134-1), Bcl-2 (1:1,000; 10026-1-AP), pro-caspase-9 (1:1,000; 1023-1) and cyclin B1 (1:2,000; 1495-1) were obtained from Abcam (Cambridge, UK), and monoclonal anti-cleaved-caspase-9 (1:1,000; sc-22182) was from Santa Cruz Biotechnology, Inc. (Shanghai, China).

Techniques: Expressing, Western Blot, Standard Deviation

Journal: Experimental and Therapeutic Medicine

Article Title: Effect of Huaier on the proliferation and apoptosis of human gastric cancer cells through modulation of the PI3K/AKT signaling pathway

doi: 10.3892/etm.2015.2600

Figure Lengend Snippet: Effect of Huaier extract on the protein expression of AKT1, PI3K, PTEN, PDK1, caspase-9 and Bcl-2. The expression of GAPDH was used as an internal control. MKN45 cells were treated with 0, 2.5 and 5 mg/ml for 24 h, and (A) western blotting was used to analyze the protein expression. (B) Quantification of the data. The experiment was performed in triplicate, and the data are expressed as the mean ± standard deviation of the three separate experiments. *P<0.05 and **P<0.01, compared with the control. p-, phosphorylated-; PI3K, phosphatidylinositol 3-kinase; PTEN, phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase; PDK1, pyruvate dehydrogenase kinase isoform 1; Bcl-2, B-cell lymphoma 2; BAD, Bcl-2-associated death promoter.

Article Snippet: Primary monoclonal antibodies against AKT (1:1,000; 10176-2-AP), PDK1 (1:1,000; 10026-1-AP), Bcl-2-associated death promoter (BAD; 1:1,000; 10435-1-AP) and GAPDH (1:4,000; 60004-1-Ig) were purchased from Proteintech (Manchester, UK), while monoclonal antibodies against p-AKT (The-308; 1:800; 2118-1), p-AKT (Ser-473; 1:800; 2214-1), PI3K (1:1,000; 1593-S), PTEN (1:1,000; 5171-1), p-PTEN (1:1,000; 2134-1), Bcl-2 (1:1,000; 10026-1-AP), pro-caspase-9 (1:1,000; 1023-1) and cyclin B1 (1:2,000; 1495-1) were obtained from Abcam (Cambridge, UK), and monoclonal anti-cleaved-caspase-9 (1:1,000; sc-22182) was from Santa Cruz Biotechnology, Inc. (Shanghai, China).

Techniques: Expressing, Western Blot, Standard Deviation

Journal: Experimental and therapeutic medicine

Article Title: PDK1 inhibition reduces autophagy and cell senescence through the PI3K/AKT signalling pathway in a cigarette smoke mouse emphysema model.

doi: 10.3892/etm.2023.11922

Figure Lengend Snippet: Figure 1. Evaluation of the mouse emphysema model. H&E staining of lung tissues from the (A) control, (B) emphysema, (C) PI3K inhibitor and (D) PDK1 inhibitor groups. Magnification, x400. (E) IL‑6 protein levels and (F) total cell count in BALF. Numbers of (G) neutrophils and (H) macrophages in BALF. (I) MLI and (J) DI were measured show the extent of airway remodelling in lung tissues. aP<0.05 vs. control. bP<0.05 vs. emphysema. BALF, bronchoalveolar lavage fluid; MLI, mean linear intercept; DI, destructive index; CS + CSE, emphysema group; CS3, PI3K inhibitor group; CS1, PDK1 inhibitor group; PI3K, phosphatidylinositol‑3‑kinase; PDK1, phosphoinositide dependent protein kinase 1.

Article Snippet: After the PVDF membrane was blocked with 5% BSA (cat. no. A8020; Beijing Solarbio Science & Technology Co., Ltd.) for 1 h at room temperature, it was incubated overnight at 4 ̊C with the following primary anti‐ bodies: PI3K (1:5,000; cat. no. 60225‐1‐1g); PDK1 (1:500; cat. no. 18262‐1‐AP); AKT (1:5,000; cat. no. 60203‐1‐1g); LC3B (1:500; cat. no. 18725‐1‐AP) (ProteinTech Group, Inc.) and p16 (1:2,000; cat. no. R22878; Zen BioScience Co., Ltd.).

Techniques: Staining, Control, Cell Counting

Journal: Experimental and therapeutic medicine

Article Title: PDK1 inhibition reduces autophagy and cell senescence through the PI3K/AKT signalling pathway in a cigarette smoke mouse emphysema model.

doi: 10.3892/etm.2023.11922

Figure Lengend Snippet: Figure 3. Expression of LC3B protein in airway epithelial tissues. (A) Immunofluorescence staining for LC3BII in airway epithelial tissue. Magnification, x400. (B) Comparison of LC3B protein expression among the experimental groups. aP<0.05 vs. control. bP<0.05 vs. emphysema. CS + CSE, emphysema group; CS3, PI3K inhibitor group; CS1, PDK1 inhibitor group; PDK1, phosphoinositide dependent protein kinase 1; MFI, mean fluorescence intensity.

Article Snippet: After the PVDF membrane was blocked with 5% BSA (cat. no. A8020; Beijing Solarbio Science & Technology Co., Ltd.) for 1 h at room temperature, it was incubated overnight at 4 ̊C with the following primary anti‐ bodies: PI3K (1:5,000; cat. no. 60225‐1‐1g); PDK1 (1:500; cat. no. 18262‐1‐AP); AKT (1:5,000; cat. no. 60203‐1‐1g); LC3B (1:500; cat. no. 18725‐1‐AP) (ProteinTech Group, Inc.) and p16 (1:2,000; cat. no. R22878; Zen BioScience Co., Ltd.).

Techniques: Expressing, Immunofluorescence, Staining, Comparison, Control, Fluorescence

Journal: Experimental and therapeutic medicine

Article Title: PDK1 inhibition reduces autophagy and cell senescence through the PI3K/AKT signalling pathway in a cigarette smoke mouse emphysema model.

doi: 10.3892/etm.2023.11922

Figure Lengend Snippet: Figure 2. Expression of PI3K, PDK1 and AKT proteins in the airway epithelial tissue. Immunohistochemical staining for (A) PI3K (B) PDK1 (C) AKT expression in the airway epithelial tissues. Magnification, x400. Quantification of (D) PI3K (E) PDK1 (F) AKT protein expression in the airway epithelial tissues in each group. aP<0.05 vs. control. bP<0.05 vs. emphysema. CS + CSE, emphysema group; CS3, PI3K inhibitor group; CS1, PDK1 inhibitor group; PDK1, phosphoinositide dependent protein kinase 1; AOD, average optical density.

Article Snippet: After the PVDF membrane was blocked with 5% BSA (cat. no. A8020; Beijing Solarbio Science & Technology Co., Ltd.) for 1 h at room temperature, it was incubated overnight at 4 ̊C with the following primary anti‐ bodies: PI3K (1:5,000; cat. no. 60225‐1‐1g); PDK1 (1:500; cat. no. 18262‐1‐AP); AKT (1:5,000; cat. no. 60203‐1‐1g); LC3B (1:500; cat. no. 18725‐1‐AP) (ProteinTech Group, Inc.) and p16 (1:2,000; cat. no. R22878; Zen BioScience Co., Ltd.).

Techniques: Expressing, Immunohistochemical staining, Staining, Control

Journal: Experimental and therapeutic medicine

Article Title: PDK1 inhibition reduces autophagy and cell senescence through the PI3K/AKT signalling pathway in a cigarette smoke mouse emphysema model.

doi: 10.3892/etm.2023.11922

Figure Lengend Snippet: Figure 4. Expression of p16 protein in the airway epithelial tissues. (A) Immunofluorescence staining for the analysis of p16 protein expression in airway epithelial tissues. Magnification, x400. (B) Comparison of p16 protein expression among the experimental groups. aP<0.05 vs. control. bP<0.05 vs. emphysema. CS + CSE, emphysema group; CS3, PI3K inhibitor group; CS1, PDK1 inhibitor group; PDK1, phosphoinositide dependent protein kinase 1; p16, cyclin‑dependent kinase inhibitor 2A.

Article Snippet: After the PVDF membrane was blocked with 5% BSA (cat. no. A8020; Beijing Solarbio Science & Technology Co., Ltd.) for 1 h at room temperature, it was incubated overnight at 4 ̊C with the following primary anti‐ bodies: PI3K (1:5,000; cat. no. 60225‐1‐1g); PDK1 (1:500; cat. no. 18262‐1‐AP); AKT (1:5,000; cat. no. 60203‐1‐1g); LC3B (1:500; cat. no. 18725‐1‐AP) (ProteinTech Group, Inc.) and p16 (1:2,000; cat. no. R22878; Zen BioScience Co., Ltd.).

Techniques: Expressing, Immunofluorescence, Staining, Comparison, Control

Journal: Experimental and therapeutic medicine

Article Title: PDK1 inhibition reduces autophagy and cell senescence through the PI3K/AKT signalling pathway in a cigarette smoke mouse emphysema model.

doi: 10.3892/etm.2023.11922

Figure Lengend Snippet: Figure 5. PI3K, PDK1, AKT, LC3B II and p16 protein expression levels. Western blotting for (A) PI3K, (B) PDK1, (C) AKT, (D) LC3B and (E) p16 expression in the airway epithelial tissues. GAPDH as an internal control. Semi‑quantification of PI3K, PDK1, AKT, LC3B and p16 protein expression in airway epithelial tissues in each group. aP<0.05 vs. control. bP<0.05 vs. emphysema. The protein expression levels were expressed as the ratio of band intensity for the target protein relative to that for the internal control GAPDH. Values are expressed as the mean ± standard deviation. CS + CSE, emphysema group; CS3, PI3K inhibitor group; CS1, PDK1 inhibitor group; PDK1, phosphoinositide dependent protein kinase 1; p16, cyclin‑dependent kinase inhibitor 2A.

Article Snippet: After the PVDF membrane was blocked with 5% BSA (cat. no. A8020; Beijing Solarbio Science & Technology Co., Ltd.) for 1 h at room temperature, it was incubated overnight at 4 ̊C with the following primary anti‐ bodies: PI3K (1:5,000; cat. no. 60225‐1‐1g); PDK1 (1:500; cat. no. 18262‐1‐AP); AKT (1:5,000; cat. no. 60203‐1‐1g); LC3B (1:500; cat. no. 18725‐1‐AP) (ProteinTech Group, Inc.) and p16 (1:2,000; cat. no. R22878; Zen BioScience Co., Ltd.).

Techniques: Expressing, Western Blot, Control, Standard Deviation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Glutamine deficiency promotes stemness and chemoresistance in tumor cells through DRP1-induced mitochondrial fragmentation

doi: 10.1007/s00018-021-03818-6

Figure Lengend Snippet: Cancer cells can survive and rearrange their metabolic phenotype in absence of glutamine. a Cell proliferation assay was done by counting cell number in 3 consecutive time point (24 h, 48 h, and 72 h) in glutamine-deprived condition (indicated as W/O GLN) in PA1 (n = 3) and HCT116 (n = 2) cells. b Histogram of cell cycle analysis and corresponding bar diagram of the histogram depicts that the cells were arrested in G0 phase upon glutamine deprivation in both PA1 and HCT116 (n = 3). c Apoptosis assay conducted in glutamine-deprived condition (24 h) and did not find any change in the live-cell population of PA1, and HCT116 (n = 3). d Red/green fluorescent population (%) was quantified for mitochondrial membrane potential using JC-1 dye in PA1, HCT116, and OAW42 upon 24 h of glutamine limiting condition, and presented in bar diagram (n = 3). e, f Extracellular flux analysis dictates that the cells became more glycolytic in nature while OXPHOS remained unchanged at 24 h time point in PA1 and OAW42 cells. g At 24 h of glutamine deprivation, glycolysis rate and glycolytic capacity increased in both PA1 and OAW42 cells. h Glucose uptake was increased in 24 h of glutamine deprivation in PA1 (n = 3). i Glucose oxidation through TCA cycle was increased in absence of glutamine at 24 h in PA1 (n = 3). j PDH phosphorylation increased in absence of glutamine at 24 h shown by Western blot in PA1 cells. k Glycostress assay kit was used to check the change in OCR after glucose injection in glutamine-deprived condition and silencing the PDHE1α in PA1 cells. l Heatmap of metabolites measured through NMR spectroscopy at 24 h of glutamine-starved condition in PA1. m Lactate level was increased in the medium when the cells are cultured in 24 h of glutamine limiting condition in PA1 (n = 3). Data are expressed in ± SEM, and statistical significance was calculated using two-tailed Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001). NS non-significant

Article Snippet: The siRNA against DRP1 (Cat#sc-43732), xCT (Cat#sc-76933) and PDHE1α (Cat#sc-91064) (Santa Cruz Biotechnology; Dallas, Texas,United State) were used at 20 nM concentration along with lipofectamine 2000 (Cat#11668019, Invitrogen) in the cell lines.

Techniques: Proliferation Assay, Cell Cycle Assay, Apoptosis Assay, Membrane, Phospho-proteomics, Western Blot, Injection, Structural Proteomics, Cell Culture, Two Tailed Test