BOP1 Silencing Suppresses Gastric Cancer Proliferation through p53 Modulation.

Block of proliferation 1 (BOP1) is a key protein involved in ribosome maturation and affects cancer progression. However, its role in gastric cancer (GC) remains unknown. This study aimed to explore the expression of BOP1 in GC and its potential mechanisms in regulating GC growth, and the relationship between BOP1 level in cancer tissues and survival was also analyzed. The expression of BOP1 was examined by immunohistochemistry (IHC) in a cohort containing 387 patients with primary GC. Cultured GC cells were treated by siRNA to knock down the BOP1 expression, and examined by CCK-8 assay and plate clone formation to assess cell proliferation in vitro. Apoptotic rate of cultured GC cells was detected by flow cytometry with double staining of AnnxinV/PI. The xenografted mouse model was used to assess GC cell proliferation in vivo. Western blot and IHC were also performed to detect the expression levels of BOP1, p53 and p21. Patients with higher level of BOP1 in cancer tissues had significantly poorer survival. BOP1 silencing significantly suppressed GC cell proliferation both in vitro and in vivo. It blocked cell cycle at G0/G1 phase and led to apoptosis of GC cells via upregulating p53 and p21. BOP1 silencing-induced suppression of cell proliferation was partly reversed by pifithrin-α (a p53 inhibitor). Our study demonstrated that BOP1 up-regulation may be a hallmark of GC and it may regulate proliferation of GC cells by activating p53. BOP1 might be considered a novel biomarker of GC proliferation, and could be a potential indicator of prognosis of GC patients. BOP1 might also be a potential target for the treatment of GC patients if further researched.

A nomogram based on glycomic biomarkers in serum and clinicopathological characteristics for evaluating the risk of peritoneal metastasis in gastric cancer.

BACKGROUND: Peritoneal metastasis (PM) in gastric cancer (GC) remains an untreatable disease, and is difficult to diagnose preoperatively. Here, we aim to establish a novel prediction model. METHODS: The clinicopathologic characteristics of a cohort that included 86 non-metastatic GC patients and 43 PMGC patients from Zhongshan Hospital were retrospectively analysed to identify PM associated variables. Additionally, mass spectrometry and glycomic analysis were applied in the same cohort to find glycomic biomarkers in serum for the diagnosis of PM. A nomogram was established based on the associations between potential risk variables and PM. RESULTS: Overexpression of 4 N-glycans (H6N5L1E1: m/z 2620.93; H5N5F1E2: m/z 2650.98; H6N5E2, m/z 2666.96; H6N5L1E2, m/z 2940.08); weight loss ≥ 5 kg; tumour size ≥ 3 cm; signet ring cell or mucinous adenocarcinoma histology type; poor differentiation; diffuse or mixed Lauren classification; increased CA19-9, CA125, and CA724 levels; decreased lymphocyte count, haemoglobin, albumin, and pre-albumin levels were identified to be associated with PM. A nomogram that integrated with five independent risk factors (weight loss ≥ 5 kg, CA19-9 ≥ 37 U/mL, CA125 ≥ 35 U/mL, lymphocyte count < 2.0 * 10 ~ 9/L, and H5N5F1E2 expression ≥ 0.0017) achieved a good performance for diagnosis (AUC: 0.892, 95% CI 0.829-0.954). When 160 was set as the cut-off threshold value, the proposed nomogram represented a perfectly discriminating power for both sensitivity (0.97) and specificity (0.88). CONCLUSIONS: The nomogram achieved an individualized assessment of the risk of PM in GC patients; thus, the nomogram could be used to assist clinical decision-making before surgery.

Relative Quantitation of Subclass-Specific Murine IgG Fc N-Glycoforms by Multiple Reaction Monitoring.

N-Linked glycosylation of the fragment crystallizable (Fc) domain of immunoglobulin G (IgG) is considered a significant modulator of antibody functions, which is known to be subclass-specific. As mice are the most widely used model organisms in immunological research, determining the variation in Fc glycosylation among each murine IgG subclass in different physiological or pathological statuses is beneficial for studying how the IgG subclass effector function is affected by Fc glycosylation. In this study, we established a method to quantify murine IgG Fc glycoforms normalized to the protein abundance at a subclass-specific level for various mouse strains using multiple reaction monitoring. The glycoform level was normalized to the subclass protein abundance (subclass-specific peptide intensity) in each IgG subclass to eliminate the contribution from the subclass protein abundance. Both good linearity and high repeatability of the method were validated by investigating a mixed mouse serum sample. The method was applied to quantify the differences in subclass-specific IgG Fc N-glycoforms between systemic sclerosis (SSc) mice and healthy control mice. The results demonstrated that each IgG subclass had its own characteristic-altered glycosylation, implying the close association of subclass-specific IgG Fc glycosylation with SSc in mice. This report demonstrates a method with great reliability and practicality that has promising potential for the relative quantitation of subclass-specific IgG Fc N-glycoforms in multiple mouse models.

Use of the serum glycan state to predict ovarian cancer patients' clinical response to chemotherapy treatment.

Ovarian cancer is the most lethal gynecologic carcinoma; because the tumor often relapses shortly after treatment. Glycosylation plays important roles in cancer drug resistance and could be used as biomarkers to predict the drug response of patients. We used MALDI-QIT-TOF MS to analyze the serum glycomic from patients with different drug responses. Samples were collected before treatment; follow-up visit were performed after 6 months. Forty-eight drug-sensitive patients and 16 drug-resistant patients were enrolled. Compared with drug-sensitive patients, 5 glyco-subclasses and 5 single glycans were significantly altered in drug-resistant patients. Lewis type, α2,3 sialic acid and multibranch glycans were increased, α2,6 sialic acid glycans were decreased. The peak at m/z 2986.44 showed stronger prediction abilities than other single glycans, with an AUC of 0.83. A panel of three increased glycans (m/z 2401.36, H5N4F1S2, a Lewis type biantennary glycan; m/z 2986.44, H6N5S3, a triantennary trisialylated glycan; m/z 3086.39, H6N5F1S3, a Lewis type triantennary glycan) combined with CA125 achieved an AUC value of 0.88, showing a strong discrimination performance. This study provides new insights into N-glycosylation patterns in ovarian cancer patients with different drug response. These altered glycans might serve as biomarkers to reflect patients' drug sensitivity and to guide clinical treatment. SIGNIFICANCE: A large number of ovarian cancer patients experience tumor relapse shortly after initial treatment. Glycosylation plays important roles in cancer drug resistance and could be used as a biomarker to predict the drug response of patients. However, the glycosylation expressed in patients with different drug response have not been elucidated. In the present study, we used MALDI-QIT-TOF MS to analyze the serum glycomic levels of patients with different drug responses. Several glycans were changed significantly between these two groups. A panel of three increased glycans (m/z 2401.36, a Lewis type biantennary glycan, 2986.44, a triantennary trisialylated glycan, and 3086.39, a Lewis type triantennary glycan) combined with CA125 performed better descrimination of these two groups with AUC of 0.88. These altered glycans might serve as biomarkers to reflect patients' drug sensitivity and to guide clinical treatment.

Prediction of neoadjuvant chemotherapeutic efficacy in patients with locally advanced gastric cancer by serum IgG glycomics profiling.

BACKGROUND: Neoadjuvant chemotherapy (NACT) could improve prognosis and survival quality of patients with local advanced gastric cancer (LAGC) by providing an opportunity of radical operation for them. However, no effective method could predict the efficacy of NACT before surgery to avoid the potential toxicity, time-consuming and economic burden of ineffective chemotherapy. Some research has been investigated about the correlation between serum IgG glycosylation and gastric cancer, but the question of whether IgG glycome can reflect the tumor response to NACT is still unanswered. METHOD: Serum IgG glycome profiles were analyzed by Ultra Performance Liquid Chromatography in a cohort comprised of 49 LAGC patients of which 25 were categorized as belonging to the NACT response group and 24 patients were assigned to the non-response group. A logistic regression model was constructed to predict the response rate incorporating clinical features and differential N-glycans, while the precision of model was assessed by receiver operating characteristic (ROC) analysis. RESULTS: IgG N-glycome analysis in pretreatment serum of LAGC patients comprises 24 directly detected glycans and 17 summarized traits. Compared with IgG glycans of non-response group, agalactosylated N-glycans increased while monosialylated N-glycans and digalactosylated N-glycans decreased in the response group. We constructed a model combining patients' age, histology, chemotherapy regimen, GP4(H3N4F1), GP6(H3N5F1), and GP18(H5N4F1S1), and ROC analysis showed this model has an accurate prediction of NACT response (AUC = 0.840) with the sensitivity of 64.00% and the specificity of 100%. CONCLUSION: We here firstly present the profiling of IgG N-glycans in pretreatment serum of LAGC. The alterations in IgG N-glycome may be personalized biomarkers to predict the response to NACT in LAGC and help to illustrate the relationship between immunity and effect of NACT.

Providing Bionic Glycome as internal standards by glycan reducing and isotope labeling for reliable and simple quantitation of N-glycome based on MALDI- MS.

Accurate, simple and economical methods for quantifying N-glycans are continuously required for discovering disease biomarkers and quality control of biopharmaceuticals. Quantitative N-glycomics based on MS using exogenous isotopic labeling internal standards is promising as it is simple and accurate. However, it is largely hampered by the lack of available glycan internal standard libraries with good coverage of the natural glycan structural heterogeneity as well as broad dynamic mass and ion abundance range. To overcome this limitation, we developed a novel method, providing 'Bionic Glycome' as internal standards for glycan quantitation by MALDI-MS. Bionic Glycome was produced using N-glycome from pooled samples to be analyzed as substrate by one step of glycan reducing and isotope labeling (Glycan-RAIL). Each bionic glycan has 3 Da mass increment over its corresponding glycan analyte based on hemiacetals/alditols and H/D mass difference. In addition, Bionic Glycome has the same glycome composition and similar glycome profile in abundance with N-glycome to be analyzed from biological sample. Through the investigation of single glycan standard and complex glycans released from model glycoprotein and serum, the results demonstrate that the method has good quantitative accuracy and high reproducibility. Lastly, this method was successfully used for discovery of lung cancer specific glycan markers by comparing the serum glycans from each sample in lung cancer group (n = 16) and healthy controls (n = 16), indicating its potential in clinical applications.

Response to immune complex vaccine in chronic hepatitis B patients is associated with lower baseline level of serum IgG galactosylation.

The composition of glycan in immunoglobulin G (IgG) has shown to affect various diseases and can be regulated by drugs and preventive vaccination. A hepatitis B surface antigen (HBsAg)-hepatitis B immunoglobulin (HBIG) immune complex (YIC) therapeutic vaccine for chronic hepatitis B (CHB) patients has undergone clinical trials. To explore for markers of CHB, which could be associated with responsiveness to YIC therapeutic vaccine, serum IgG glycosylation in CHB patients was analyzed.Kinetic changes of serum galactosylated IgG in 53 hepatitis Be antigen (HBeAg)-positive CHB patients treated with YIC were monitored by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) analysis. Whole blood cytokines were assayed by cytokine binding assay kits. All samples were back assayed before treatment, during therapy and follow-up for 6 months from a previous completed clinical trial.During YIC treatment, 26 patients with lower IgG galactosylation level at baseline [galactosylation level (Gal-ratio) = -0.29, 0.18 (mean, SD)] showed sustained increase of serum galactosylated IgG, and responded to YIC treatment by HBeAg seroconversion. While those who did not respond to YIC treatment [Gal-ratio = -0.40, 0.15 (mean, SD)] failed to show similar changes. Furthermore, this kinetic increase of galactosylated IgG correlated with marked up-regulated IL-2 level, confirming that effective cellular immune responses have participated in responsiveness.For HBeAg-positive CHB patients lower serum IgG galactosylation level may serve as an indicator for selecting a suitable subpopulation of candidates for YIC therapeutic vaccination.

The Value of Serum Immunoglobulin G Glycome in the Preoperative Discrimination of Peritoneal Metastasis from Advanced Gastric Cancer.

Background: Peritoneal metastasis, associated with poor prognosis in gastric cancer, is difficult to discriminate from advanced gastric cancer preoperatively. However, operative diagnosis could bring both mental and physical trauma and economic burden for patients. Consequently, a non-invasive biomarker is necessary to reduce the burden of operative diagnosis and improve survival quality of patients. This study aims to elucidate the correlation between Immunoglobulin G (IgG) N-glycome and peritoneal metastasis and find potential biomarkers in preoperative discrimination of peritoneal metastasis from advanced gastric cancer based on the comprehensive sample set. Methods: A total of 373 gastric cancer patients were enrolled and randomly sorted into training cohort (n=249) and validation cohort (n=124). The IgG N-glycome composition was analyzed by ultra-performance liquid chromatography. Results: Twenty-four glycan peaks were directly detected and 15 traits based on the same structures were evaluated between peritoneal metastasis group and advanced gastric cancer group. Several differences in IgG glycosylation were found: sialylation and fucosylation were increased in peritoneal metastasis, while neutral glycosylation, monogalacosylation and bisecting GlcNAc were decreased. Based on the significant glycomics profile, a glyco-model composed of five glycan peaks (GP6, GP9, GP11, GP21 and GP23) was established with area under the receiver operating characteristic curve (AUC) value of 0.80 (training cohort) and 0.77 (validation cohort), which showed good potential in discriminating peritoneal metastasis from advanced gastric cancer. The diagnostic performance of this model was further validated in a combined cohort (AUC=0.79). Two patients with gastric cancer were selected to perform and demonstrate the usage of the diagnostic workflow. Conclusions: Here we firstly present IgG glycome profiles in a large number of preoperative peritoneal metastasis serums. The IgG glycan was highly associated with peritoneal metastasis. These findings enhance the understanding of peritoneal metastasis. Besides, our results suggested that the newly established glyco-model could be a reliable predictor of the presence of peritoneal metastasis in patients with advanced gastric cancer.

Diagnostic Significance of Serum IgG Galactosylation in CA19-9-Negative Pancreatic Carcinoma Patients.

Background: Although Carbohydrate antigen 19-9 (CA19-9) is considered clinically useful and informative for pancreatic carcinoma (PC), false positive results, and false negative results have restricted its clinical use. Especially missed or delayed diagnosis of PC patients with negative CA19-9 value limited the utility. To improve prognosis of PC patients, the discovery of reliable biomarkers to assist CA19-9 is desired. Serum IgG galactosylation based on our previous report was altered in PC patients comparing to healthy controls. The objective of this study was to explore the diagnostic significance of IgG galactosylation in assisting CA19-9 for PC in a comprehensive way. Methods: Serum IgG galactosylation profiles were analyzed by MALDI-MS in cohort 1 (n = 252) and cohort 2 in which all CA19-9 levels were negative (n = 133). In each cohort, not only healthy controls and PC patients but also benign pancreatic disease (BPD) patients were enrolled. Peaks were acquired by the software of MALDI-MS sample acquisition, followed by being processed and analyzed by the software of Progenesis MALDI. IgG Gal-ratio, which was calculated from the relative intensity of peaks G0, G1, and G2 according to the formula (G0/(G1+G2×2)), was employed as an index for indicating the distribution of IgG galactosylation. Results: The Gal-ratio was elevated in PC comparing with that in non-cancer group (healthy controls and BPD). The area under the receiver operating characteristic curve (AUC) of IgG Gal-ratio was higher than that of CA19-9 (0.912 vs. 0.814). The performance was further improved when Gal-ratio and CA19-9 were combined (AUC: 0.928). Meanwhile, Gal-ratio also had great diagnostic value with a sensitivity of 92.31% (AUC: 0.883) in detection of PC at early stage. Notably, IgG Gal-ratio has great sensitivity (90.63%) and specificity (76.81%) in CA19-9-negative PC patients. Conclusions: IgG Gal-ratio had a great performance in detection of PC and could be used to assist CA19-9 in improving diagnosis performance through early stage detection, differentiation from BPD, and PC diagnosis with CA19-9-negative level.

Morphology Processing by Encapsulating GeP5 Nanoparticles into Nanofibers toward Enhanced Thermo/Electrochemical Stability.

Compared with elemental phosphorus, GeP5, with much better thermostability and super higher conductivity, can exhibit a comparable capacity (>2000 mA h g-1) with a much higher first Coulombic efficiency (95%) for lithium-ion batteries. However, such high capacity is accompanied by large volume expansions, leading to fast capacity fading. To improve the cycle stability, fabricating a special nanostructure to reduce the volume stress and compositing with a carbon matrix to buffer the volume change are highly required. However, nanostructured metal phosphides were rarely reported up to now because they are difficult to be synthesized via a normal wet chemistry method or gas phosphorization because of lack of proper reactants and poor thermostability of phosphides. Herein, we successfully achieve uniform carbon-encapsulated GeP5 nanofibers (GeP5@C-NF) by processing GeP5 nanoparticles into carbon nanofibers via electrospinning. After carbon encapsulation, the thermostability of GeP5 can be greatly improved to over 600 °C for higher battery safety. Such a nanofiber structure in which nanosized GeP5 is embedded in a carbon matrix can greatly accommodate the large volume changes during lithiation and provide fast electron transportation, thus contributing to a long cycle life (>1000 mA h g-1 after 200 cycles) and high rate performance (803 mA h g-1 at 2000 mA g-1). This morphology processing technique can be easily extended to other metal phosphide anodes which are limited by a lack of appropriate synthesis methods and poor thermostability.

Alteration of Serum IgG Galactosylation as a Potential Biomarker for Diagnosis of Neuroblastoma.

Background: Neuroblastoma (NB) is the most frequent pediatric malignant neoplasm that originates from embryonic neural crest cells. Urinary catecholamines in 24-h urine are most commonly analyzed for the diagnosis of neuroblastoma at good sensitivity; however, it is challenging to collect 24-h urine samples in a pediatric population. Therefore, development of more rapid, non-invasive and cost-effective tools for the diagnosis of NB remains needed. Serum immunoglobulin G (IgG) galactosylation have been found highly associated with adult cancers in our previous study. Methods: To explore the potential use of serum IgG galactosylation in aiding diagnosis of neuroblastoma, serum IgG galactosylation profiles of 26 neuroblastoma cases and 30 age-matched non-malignant controls were analyzed by MALDI MS. The alteration of IgG galactosylation in neuroblastoma patients was measured by a Gal-ratio formula: G0/(G1+G2×2), calculating the relative intensities of agalactosylated N-glycan (G0) vs mono-galactosyl N-glycan (G1) and digalactosyl N-glycan (G2). Results: The results showed that IgG Gal-ratios were significantly higher in neuroblastoma cases compared with non-malignant controls (p=5.0×10-4). And the Gal-ratio data generated sensitivity and specificity of 84.62% and 60.00%, combined with an AUC (area under the curve) of 0.80. Conclusions: The analysis of serum IgG galactosylation distribution may play a suggestive role for neuroblastoma diagnosis, or serve as a potential biomarker for NB diagnosis.

Discovery of Non-invasive Glycan Biomarkers for Detection and Surveillance of Gastric Cancer.

Purpose: Gastric cancer (GC), one of the world's top five most common cancers, is the third leading cause of cancer related death. It is urgent to identify non-invasive biomarkers for GC. The objective of our study was to find out non-invasive biomarkers for early detection and surveillance of GC based on glycomic analysis. Method: Ethyl esterification derivatization combined with MALDI-TOF MS analysis was employed for the comprehensive serum glycomic analysis in order to investigate glycan markers that would indicate the onset and progression of gastric cancer. Upon the discovery of the candidate biomarkers, those with great potential were further validated in an independent test set. Peaks were acquired by the software of MALDI-MS sample acquisition and processing and analyzed by the software of Progenesis MALDI. Results: The differences in glycosylation were found between non-cancer controls and gastric cancer samples: hybrid and multi-branched type (tri-, tetra-antennnary glycans) N-glycans were increased in GC, yet monoantennary, galactose, bisecting type and core fucose N-glycans were decreased. In training set, core fucose (AUC=0.923, 95%CI: 0.8485 to 0.9967) played an excellent diagnostic performance for the early detection of gastric cancer. The diagnostic potential of core fucose was further validated in an independent cohort (AUC=0.854, 95%CI: 0.7592 to 0.9483). Besides, several individual glycan structures reached both statistical criteria (p-values less than 0.05 and AUC scores that were at least moderately accurate) when comparing different stages of GC samples. Conclusion: We comprehensively evaluate the serum glycan changes in different stages of GC patients including peritoneal metastasis for the first time. We determined several N-glycan biomarkers, some of these have potential in distinguishing the early stage GC from healthy controls, and the others can help to monitor the progression of GC. The findings also enhance understanding of gastric cancer.

Lectin array and glycogene expression analyses of ovarian cancer cell line A2780 and its cisplatin-resistant derivate cell line A2780-cp.

BACKGROUND: Ovarian cancer is one of the most lethal gynecological malignancies, in which platinum resistance is a common cause of its relapse and death. Glycosylation has been reported to be involved in drug resistance, and glycomic analyses of ovarian cancer may improve our understanding of the mechanisms underlying cancer cell drug resistance and provide potential biomarkers and therapeutic targets. METHODS: The serous ovarian cancer cell line A2780 and its platinum-resistant counterpart A2780-cp were used in this study. We performed a lectin array analysis to compare the glycosylation patterns of the two cell lines, a gene expression array was employed to probe the differences in glycogenes. Furthermore, the results were verified by lectin blots. RESULTS: A2780-cp cell exhibited stronger intensities of Lens culinaris (LCA) Canavalia ensiformis (ConA), and Lycopersicon esculentum (LEL) and weaker intensities of Sambucus nigra (SNA) lectins. The gene expression array analysis revealed increased expression of Fut8, B3gnt4, B3gnt5, B4galt2 and decreased expression of Fut1 and ST6GalNAc 6 expression were evident in the A2780-cp cells. The lectin blot confirmed the differences in LCA, ConA, SNA and LEL between the A2780 and A2780-cp cells. CONCLUSIONS: The combination of the lectin and gene expression analyses showed that the levels of core fucosylation and poly-LacNAc were increased in the A2780-cp cells and the levels of Fuc α1-2(gal ß1-4) GlcNAc and α2-6-linked sialic structures were decreased in the A2780-cp cells. These glycans represent potential biomarkers and might be involved in the mechanism of drug resistance in ovarian cancer.

One-pot synthesis of Li3VO4@C nanofibers by electrospinning with enhanced electrochemical performance for lithium-ion batteries.

Electrospinning is firstly used to one-pot synthesis of Li3VO4@C nanofibers in a large scale. Although with the presence of organic sources in synthesis process, the pure phase Li3VO4 with superior nanofibrous morphology is still successfully obtained through adjusting different heat treatment processes and different vanadium sources. The prepared Li3VO4@C nanofibers exhibit a unique structure in which nanosized Li3VO4 particles are uniformly embedded in amorphous carbon matrix. Compared with Li3VO4/C powder, Li3VO4@C nanofibers display enhanced reversible capacity of 451mAhg-1 at 40mAg-1 with an increased initial coulombic efficiency of 82.3%, and the capacity can remain at 394mAhg-1 after 100 cycles. This superior electrochemical performance can be attributed to its unique structure which ensures a high reactivity by nanosized Li3VO4, more stable electrode/electrolyte interface by carbon encapsulation, improved electronic conductivity and buffered volume changes by flexible carbon matrix. The electrospinning technology provides an effective method to obtain high performance Li3VO4 as a promising anode material for lithium-ion batteries.

Integrated glycomic analysis of ovarian cancer side population cells.

BACKGROUND: Ovarian cancer is the most lethal gynecological malignancy due to its frequent recurrence and drug resistance even after successful initial treatment. Accumulating scientific evidence indicates that subpopulations of cancer cells with stem cell-like properties, such as so-called side population (SP) cells, are primarily responsible for these recurrences. A better understanding of SP cells may provide new clues for detecting and targeting these cancer-initiating cells and ultimately help to eradicate cancer. Changes in glycosylation patterns are remarkable features of SP cells. Here, we isolated SP cells from ovarian cancer cell lines and analyzed their glycosylation patterns using multiple glycomic strategies. METHODS: Six high-grade serous ovarian cancer cell lines were used for SP cell isolation. Among them, HO8910 pm, which contained the highest proportion of SP cells, was used for glycomic analysis of SP cells. Cell lysate of SP cells and main population cells was applied to lectin microarray and mass spectrometry for glycan profiling. Differently expressed glycan structures were further verified by lectin blot, flow cytometry, and real-time PCR analysis of their relevant enzymes. RESULTS: Expression of core fucosylated N-glycan and tumor-associated Tn, T and sT antigens were increased in SP cells. By contrast, SP cells exhibited decreased hybrid glycan, α2,3-linked sialic glycan and multivalent sialyl-glycan. CONCLUSIONS: Glycan structures, such as Tn, T, sT antigens, and core fucosylation may serve as biomarkers of ovarian cancer stem cells.