Erythroxylum cuneatum Prevented Cellular Adaptation in Morphineinduced Neuroblastoma Cells.

BACKGROUND: Chronic morphine stimulates prolonged stimulation of opioid receptors, especially µ-opioid subtype (MOR), which in turn signals cellular adaptation. However, the sudden termination of the use of morphine after chronic intake causes the withdrawal syndrome. OBJECTIVES: Hence, this study was designed to find an alternative treatment for morphine withdrawal using the alkaloid leaf extract of Erythroxylum cuneatum (E. cuneatum) for the treatment of morphine-exposed neuroblastoma cell lines. METHODS: SK-N-SH, a commercialised neuroblastoma cell line, was used in two separate study designs; the antagonistic and pre-treatment of morphine. The antagonistic treatment was conducted through concurrent exposure of the cells to morphine and E. cuneatum or morphine and methadone for 24 hrs. The pre-treatment design was carried out by exposing the cells to morphine for 24 hrs, followed by 24 hrs exposure to E. cuneatum or methadone. The cytosolic fraction was collected and assessed for proteins expression involved in cellular adaptation, including mitogen-activated protein (MAP)/extracellular signal-regulated (ERK) kinase 1/2 (MEK 1/2), extracellular signalregulated kinase 2 (ERK 2), cAMP-dependent protein kinase (PKA) and protein kinases C (PKC). RESULTS: The antagonistic treatment showed the normal level of MEK 1/2, ERK 2, PKA and PKC by the combination treatment of morphine and E. cuneatum, comparable to the combination of morphine and methadone. Neuroblastoma cells exposed to morphine pre-treatment expressed a high level of MEK 1/2, ERK 2, PKA and PKC, while the treatments with E. cuneatum and methadone normalised the expression of the cellular adaptation proteins. CONCLUSION: E. cuneatum exerted anti-addiction properties by lowering the levels of cellular adaptation proteins it's effects is comparable to that of methadone (an established anti-addiction drug).

Assessing the combined toxicity effects of three neonicotinoid pesticide mixtures on human neuroblastoma SK-N-SH and lepidopteran Sf-9 cells.

Neonicotinoid insecticides have been widely used in plant protection against pests worldwide. Generally, more than one neonicotinoids are detected in plants and foods, and such mixtures may show combined toxicity and increase the risk for both insects and higher organisms. In this study, the combined toxicity of imidacloprid (IM), acetamiprid (AC) and thiamethoxam (TH) was investigated using human neuroblastoma cell line (SK-N-SH) and lepidopteran cell line (Sf-9). Results showed that binary and ternary mixtures could enhance the inhibition of growth of both SK-N-SH and Sf-9 cells at low doses. In SK-N-SH cells, based on CompuSyn software analysis, all the mixtures of IM+AC, IM+TH, AC+TH and IM+AC+TH showed synergistic effects at concentrations < 50 mg/L, but IM+AC, IM+TH showed antagonistic effects at higher concentrations. For Sf-9 cells, all mixtures revealed synergistic effects at low concentrations (< 0.1 mg/L) except IM+AC, showing antagonism at higher concentrations (> 0.5 mg/L). The toxicity thresholds of mixtures denoted by BMDL10 values were all lower than those for single pesticides and the combined BMDL10 value of AC+TH was the lowest one. It is concluded that the co-occurrence of several neonicotinoid insecticides may enhance their toxicity and aggravate the health risk for both insects and human.

Differentially expressed long-chain noncoding RNAs in human neuroblastoma cell line (SH-SY5Y): Alzheimer's disease cell model.

A number of complex human diseases including neurological diseases is characterized by dysregulation of long-chain noncoding RNA (lncRNA). The pathogenesis of Alzheimer's disease (AD), a neurodegenerative disorder is believed to involve alterations in lncRNAs. However, the specific lncRNAs modified in AD remain to be determined. The aim of this study was to identify lncRNAs associated with AD using human neuroblastoma cell line (SH-SY5Y) treated with beta-amyloid (Aß) as a model of this disease. The differential expressions of lncRNA were compared between beta-amyloid (Aß) SH-SY5Y cells and normal SH-SY5Y cells utilizing Illumina X10 gene sequencing. The differential expression profiles of amyloid (Aß)-treated SH-SY5Y cells were determined and verified by qRT-PCR method. The expression levels of lncRNA were expressed by calculating the abundance of FPKM (measure gene expression). The differential expression of log2 (multiple change) >1 or log2 (multiple change) < -1 had statistical significance (P< .05). The differential expression profiles of amyloid (Aß)-treated SH-SY5Y cells showed 40 lncRNA were up-regulated, while 60 lncRNA were down-regulated. GO and KEGG analysis demonstrated that differentially expressed genes were predominantly involved in the mitogen-activated protein kinase (MAPK) signaling pathway, p53 signaling pathway, hepatitis B, cell cycle, post-translational protein modification, and regulation. In conclusion, approximately 100 dysregulated lncRNA transcripts were found in amyloid (Aß)-treated SH-SY5Y cells and these lncRNAs may play an important role in the occurrence and development of AD through altered signal pathways.

Treatment of human neuroblastoma cell line SH-SY5Y with HSP27 siRNA tagged-exosomes decreased differentiation rate into mature neurons.

Heat shock proteins (HSPs) participate in the regulation of different cell activities in response to stimuli. By applying different strategies, the modulation of heat shock proteins is at the center of attention. Conventional delivery approaches are not fully encouraged due to cytotoxicity and immunogenicity issues. Exosomes are touted as bio-shuttles for delivery of distinct biomolecules inside the cells. Here, we aimed to HSP27 small interfering RNA (siRNA)-tagged exosomes for the inhibition of Hsp27 in human neuroblastoma cell line SH-SY5Y and explored differentiation into neuron-like cells. Exosomes were isolated, characterized by scanning electron microscope (SEM) and CD63 then enriched with siRNA against Hsp27. Neuroblastoma cells were incubated with exosomes carrying siRNA for 48 hr. Exosome uptake was monitored by immunofluorescence assay. The cell viability and proliferation were analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and bromodeoxyuridine/5-bromo-2'-deoxyuridine incorporation assays. The ability of cells to form colonies was evaluated by clonogenic assay. The cell potential to express NeuN, a mature neuron factor, was studied by flow cytometry analysis. SEM showed the nano-sized particles and a high level of CD63 after enrichment. Immunofluorescence imaging revealed an appropriate transfection rate in cell exposed to Hsp27 siRNA tagged exosomes. The cell viability and proliferation were reduced compared to cells received nude exosomes ( p < 0.05). Clonogenic activity of cells was diminished by the inhibition of Hsp27. Flow cytometry analysis revealed that the inhibition of Hsp27 prohibited NeuN content, showing the maturation of SH-SY5Y cells to mature cells compared to control. These data confirmed that exosomes could be used as appropriate bio-shuttles for the inhibition of Hsp27-aborted cell differentiation toward mature neuron.

Heat shock protein 70 modulates neural progenitor cells dynamics in human neuroblastoma SH-SY5Y cells exposed to high glucose content.

In the current experiment, detrimental effects of high glucose condition were investigated on human neuroblastoma cells. Human neuroblastoma cell line SH-SY5Y were exposed to 5, 40, and 70 mM glucose over a period of 72 h. Survival rate and the proliferation of cells were analyzed by MTT and BrdU incorporation assays. Apoptosis was studied by the assays of flow cytometry and PCR array. In order to investigate the trans-differentiation capacity of the cell into mature neurons, we used immunofluorescence imaging to follow NeuN protein level. The transcription level of HSP70 was shown by real-time PCR analysis. MMP-2 and -9 activities were shown by gelatin Zymography. According to data from MTT and BrdU incorporation assay, 70 mM glucose reduced cell viability and proliferation rate as compared to control (5 mM glucose) and cells treated with 40 mM glucose (P < 0.05). Cell exposure to 70 mM glucose had potential to induced apoptosis after 72 h (P < 0.05). Our results also demonstrated the sensitivity of SH-SY5Y cells to detrimental effects of high glucose condition during trans-differentiation into mature neuron-like cells. Real-time PCR analysis confirmed the expression of HSP70 in cells under high content glucose levels, demonstrating the possible cell compensatory response to an insulting condition (pcontrol vs 70 mM group <0.05). Both MMP-2 and -9 activities were reduced in cells being exposed to 70 mM glucose. High glucose condition could abrogate the dynamics of neural progenitor cells. The intracellular level of HSP70 was proportional to cell damage in high glucose condition.

Effects of 1950 MHz radiofrequency electromagnetic fields on Aß processing in human neuroblastoma and mouse hippocampal neuronal cells.

Alzheimer's disease (AD) is a neurodegenerative disease leading to progressive loss of memory and other cognitive functions. One of the well-known pathological markers of AD is the accumulation of amyloid-beta protein (Aß), and its plaques, in the brain. Recent studies using Tg-5XFAD mice as a model of AD have reported that exposure to radiofrequency electromagnetic fields (RF-EMF) from cellular phones reduced Aß plaques in the brain and showed beneficial effects on AD. In this study, we examined whether exposure to 1950 MHz RF-EMF affects Aß processing in neural cells. We exposed HT22 mouse hippocampal neuronal cells and SH-SY5Y human neuroblastoma cells to RF-EMF (SAR 6 W/kg) for 2 h per day for 3 days, and analyzed the mRNA and protein expression of the key genes related to Aß processing. When exposed to RF-EMF, mRNA levels of APP, BACE1, ADAM10 and PSEN1 were decreased in HT22, but the mRNA level of APP was not changed in SH-SY5Y cells. The protein expression of APP and BACE1, as well as the secreted Aß peptide, was not significantly different between RF-EMF-exposed 7w-PSML, HT22 and SH-SY5Y cells and the unexposed controls. These observations suggest that RF-EMF exposure may not have a significant physiological effect on Aß processing of neural cells in the short term. However, considering that we only exposed HT22 and SH-SY5Y cells to RF-EMF for 2 h per day for 3 days, we cannot exclude the possibility that 1950 MHz RF-EMF induces physiological change in Aß processing with long-term and continuous exposure.

Lecithin-cholesterol acyltransferase in brain: Does oxidative stress influence the 24-hydroxycholesterol esterification?

24-Hydroxycholesterol (24OH-C) is esterified by the enzyme lecithin-cholesterol acyltransferase (LCAT) in the cerebrospinal fluid (CSF). We report here that the level of 24OH-C esters was lower in CSF of patients with amyotrophic lateral sclerosis than in healthy subjects (54% vs 68% of total 24OH-C, p=0.0005; n=8). Similarly, the level of 24OH-C esters in plasma was lower in patients than in controls (62% vs 77% of total 24OH-C; p=0.0076). The enzyme amount in CSF, as measured by densitometry of the protein band revealed by immunoblotting, was about 4-fold higher in patients than in controls (p=0.0085). As differences in the concentration of the LCAT stimulator Apolipoprotein E were not found, we hypothesized that the reduced 24OH-C esterification in CSF of patients might depend on oxidative stress. We actually found that oxidative stress reduced LCAT activity in vitro, and 24OH-C effectively stimulated the enzyme secretion from astrocytoma cells in culture. Enhanced LCAT secretion from astrocytes might represent an adaptive response to the increase of non-esterified 24OH-C percentage, aimed to avoid the accumulation of this neurotoxic compound. The low degree of 24OH-C esterification in CSF or plasma might reflect reduced activity of LCAT during neurodegeneration.

ACAT1 regulates the dynamics of free cholesterols in plasma membrane which leads to the APP-α-processing alteration.

Acyl-CoA:cholesterol acyltransferase 1 (ACAT1) is a key enzyme exclusively using free cholesterols as the substrates in cell and is involved in the cellular cholesterol homeostasis. In this study, we used human neuroblastoma cell line SK-N-SH as a model and first observed that inhibiting ACAT1 can decrease the amyloid precursor protein (APP)-α-processing. Meanwhile, the transfection experiments using the small interfering RNA and expression plasmid of ACAT1 indicated that ACAT1 can dependently affect the APP-α-processing. Furthermore, inhibiting ACAT1 was found to increase the free cholesterols in plasma membrane (PM-FC), and the increased PM-FC caused by inhibiting ACAT1 can lead to the decrease of the APP-α-processing, indicating that ACAT1 regulates the dynamics of PM-FC, which leads to the alteration of the APP-α-processing. More importantly, further results showed that under the ACAT1 inhibition, the alterations of the PM-FC and the subsequent APP-α-processing are not dependent on the cellular total cholesterol level, confirming that ACAT1 regulates the dynamics of PM-FC. Finally, we revealed that even when the Niemann-Pick-Type C-dependent pathway is blocked, the ACAT1 inhibition still obviously results in the PM-FC increase, suggesting that the ACAT1-dependent pathway is responsible for the shuttling of PM-FC to the intracellular pool. Our data provide a novel insight that ACAT1 which enzymatically regulates the dynamics of PM-FC may play important roles in the human neuronal cells.

Protective effect of the traditional Chinese medicine prescription, Jiu Nao Yi Zhi water extract, on human neuroblastoma SH-SY5Y cell line / 中国比较医学杂志

Objective To observe the protective effect of the traditional Chinese medicine prescription, Jiu Nao Yi Zhi water extract, on human neuroblastoma SH-SY5Y cell line, its effect on expression of insulin signal transduction pathway, and to explore the related mechanisms.Methods SH-SY5Y cells cultured in vitro, were divided into control group, Jiu Nao Yi Zhi No.1 prescription group and No.3 prescription group.The doses were 0.0625 mg/mL, 0.125 mg/mL, 0.25 mg/mL, 0.5 mg/mL and 1 mg/mL.The thiazolyl blue ( MTT) metabolic rate of each group was determined.The dose of 0.125 mg/mL was chosen for cell immunofluorescence analysis, and to observe the expression of insulin receptor substrates-1 ( IRS-1 ) , cAMP response element binding protein ( CREB ) , and the factors of insulin signal transduction pathway.Results Compared with the control group, MTT metabolic rates of the Jiu Nao Yi Zhi groups were significantly increased (P<0.05), and the cell morphology was much better in those groups, cell body more plump, well-adherent and neurite extensions were observed.The expressions of IRS-1 and CREB were higher than that in the control group.Conclusions The traditional Chinese medicine prescription Jiu Nao Yi Zhi water extract can protect neurons by promoting nerve cell growth, and improving the expression of IRS-1 and CREB, the factors of insulin signal transduction pathway.

The expression and correlation between neural nicotinic acetylcholine receptor subunit α3 and mitogen-activated protein kinase cell signaling transduction pathway in human neuroblastoma cell line SH-SY5Y overexposed to fluoride / 中华地方病学杂志

Objective To observe the expression of neural nicotinic acetylcholine receptor subunit α3 (α3nAChR) and extracellular regulated protein kinases (ERK1/2),c-Jun N-terminal kinase (JNK),p38 kinases of mitogen-activated protein kinase (MAPK) pathway in human neuroblastoma cell line SH-SY5Y overexposed to fluoride,and try to investigate the molecular mechanism of cell damage caused by overexposure of fluoride.Methods The SH-SY5Y cell with low expression of α3nAChR suppressed by silence interference RNA served as α3nAChR silence group;the normal SH-SY5Y cell served as control group,and the effect of silencing of αt3nAChR gene in SHSY5Y was detected by Western blotting and real-time PCR;SH-SY5Y cell was treated with different concentrations of fluoride (0.000,0.005,0.050,0.500,1.000,2.500,5.000 mmol/L),the safe concentration of fluoride in SHSY5Y cell was detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay;the SH-SY5Y cell of control group and α3nAChR silence group were treated with 4.000 mmol/L fluoride for 0,4,8,12,24,36,48 h according to the results of MTT assay;the expression of ERK1/2,JNK,p38 kinases of MAPK pathway in SH-SY5Y at protein levels was measured by Western blotting.Results The expression of α3nAChR mRNA (0.04 ± 0.03) and protein (12.0 ± 2.5) in α3nAChR silence group was decreased significantly compared with those of control group (1.00 ± 0.11,100.0 ± 11.3,t =24.58,28.80,all P ＜ 0.05).The viability of SH-SY5Y cell treated with 5.000 mmol/L fluoride (0.53 ± 0.15) was decreased significantly compared with that of SH-SY5Y cell treated with 0.000 mmol/L fluoride (1.05 ± 0.05,P ＜ 0.05).The increased expression of phospho-ERK1/2 was found in α3nAChR silence group and control group incubated with fluoride with time prolonged,and the expression of phospho-ERK1/2 increased significantly at time points 24,36 and 48 h (188.33 ± 7.33,200.00 ± 10.01,213.33 ± 11.55;125.33 ± 5.69,136.00 ± 4.52,155.33 ± 6.51) compared to 0 h in the same groups (100.00 ± 0.00,100.00 ± 0.00,all P ＜ 0.05),and the expression of phospho-ERK1/2 was higher significantly in α3nAChR silence group than those of control group (t =9.26,7.63,5.72,all P ＜ 0.05);no change of expression of total-ERK1/2 in the two groups was found with the passage of time.The gradually increased expression of phospho-JNK was found in α3nAChR silence group and control group,among which,the expression of phospho-JNK in o3nAChR silence group at time points 12,24,36 and 48 h (154.00 ± 6.25,149.00 ± 5.57,156.00 ± 6.08,141.67 ± 2.52) and in control group at 8,12,24,36,48 h (133.33 ± 10.69,173.00 ± 4.00,175.00 ± 11.79,200.67 ± 11.93,200.33 ± 18.58) was compared to those at 0 h in the same groups (100.00 ± 0.00,100.00 ± 0.00),and the difference was significant (all P ＜ 0.05);the higher expression of phospho-JNK was found in α3nAChR silence group other than control group at 8,12,24,36,48 h (t =-4.28,-5.02,-2.89,-8.33,-6.18,all P ＜ 0.05);no change of expression of total-JNK was found in the two groups (P ＞ 0.05).The increased expression of phospho-p38 was detected in control group at time points 24,36 and 48 h (120.33 ± 4.51,122.00 ± 7.55,119.67 ± 7.57) compared to 0 h in the same groups (100.00 ± 0.00,all P ＜ 0.05),and the expression of phospho-p38 was significantly higher than that in α3nAChR silence group at the same time points (93.33 ± 9.61,94.00 ± 5.01,98.33 ± 5.69,t =-4.01,-6.73,-5.59,all P ＜ 0.05);no change of expression of total-p38 was found in the two types of SH-SY5Y cells treated with fluoride (P ＞ 0.05).Conclusion When SH-SY5Y cells are exposed to fluoride;activation of ERK1/2 may be not depend on α3nAChR;α3nAChR may have protected the cell from apoptotic injury caused by activation of JNK pathway,and the activation of p38 may be depend on nAChRα3.

Haptoglobin increases with age in rat hippocampus and modulates Apolipoprotein E mediated cholesterol trafficking in neuroblastoma cell lines.

Alteration in cholesterol metabolism has been implicated in the pathogenesis of several neurodegenerative disorders. Apolipoprotein E (ApoE) is the major component of brain lipoproteins supporting cholesterol transport. We previously reported that the acute-phase protein Haptoglobin (Hpt) binds ApoE, and influences its function in blood cholesterol homeostasis. Major aim of this study was to investigate whether Hpt influences the mechanisms by which cholesterol is shuttled from astrocytes to neurons. In detail it was studied Hpt effect on ApoE-dependent cholesterol efflux from astrocytes and ApoE-mediated cholesterol incorporation in neurons. We report here that Hpt impairs ApoE-mediated cholesterol uptake in human neuroblastoma cell line SH-SY5Y, and limits the toxicity of a massive concentration of cholesterol for these cells, while it does not affect cholesterol efflux from the human glioblastoma-astrocytoma cell line U-87 MG. As aging is the most important non-genetic risk factor for various neurodegenerative disorders, and our results suggest that Hpt modulates ApoE functions, we evaluated the Hpt and ApoE expression profiles in cerebral cortex and hippocampus of adolescent (2 months), adult (5 and 8 months), and middle-aged (16 months) rats. Hpt mRNA level was higher in hippocampus of 8 and 16 month-old than in 2-month old rats (p < 0.05), and Hpt concentration increased with the age from adolescence to middle-age (p < 0.001). ApoE concentration, in hippocampus, was higher (p < 0.001) in 5 month-old rats compared to 2 month but did not further change with aging. No age-related changes of Hpt (protein and mRNA) were found in the cortex. Our results suggest that aging is associated with changes, particularly in the hippocampus, in the Hpt/ApoE ratio. Age-related changes in the concentration of Hpt were also found in human cerebrospinal fluids. The age-related changes might affect neuronal function and survival in brain, and have important implications in brain pathophysiology.

Cloning and characterization of the promoter regions from the parent and paralogous creatine transporter genes.

Interconversion between phosphocreatine and creatine, catalyzed by creatine kinase is crucial in the supply of ATP to tissues with high energy demand. Creatine's importance has been established by its use as an ergogenic aid in sport, as well as the development of intellectual disability in patients with congenital creatine deficiency. Creatine biosynthesis is complemented by dietary creatine uptake. Intracellular transport of creatine is carried out by a creatine transporter protein (CT1/CRT/CRTR) encoded by the SLC6A8 gene. Most tissues express this gene, with highest levels detected in skeletal muscle and kidney. There are lower levels of the gene detected in colon, brain, heart, testis and prostate. The mechanism(s) by which this regulation occurs is still poorly understood. A duplicated unprocessed pseudogene of SLC6A8-SLC6A10P has been mapped to chromosome 16p11.2 (contains the entire SLC6A8 gene, plus 2293 bp of 5'flanking sequence and its entire 3'UTR). Expression of SLC6A10P has so far only been shown in human testis and brain. It is still unclear as to what is the function of SLC6A10P. In a patient with autism, a chromosomal breakpoint that intersects the 5'flanking region of SLC6A10P was identified; suggesting that SLC6A10P is a non-coding RNA involved in autism. Our aim was to investigate the presence of cis-acting factor(s) that regulate expression of the creatine transporter, as well as to determine if these factors are functionally conserved upstream of the creatine transporter pseudogene. Via gene-specific PCR, cloning and functional luciferase assays we identified a 1104 bp sequence proximal to the mRNA start site of the SLC6A8 gene with promoter activity in five cell types. The corresponding 5'flanking sequence (1050 bp) on the pseudogene also had promoter activity in all 5 cell lines. Surprisingly the pseudogene promoter was stronger than that of its parent gene in 4 of the cell lines tested. To the best of our knowledge, this is the first experimental evidence of a pseudogene with stronger promoter activity than its parental gene.

Design and synthesis of novel quinazoline nitrogen mustard derivatives as potential therapeutic agents for cancer.

Thirteen novel quinazoline nitrogen mustard derivatives were designed, synthesized and evaluated for their anticancer activities in vitro and in vivo. Cytotoxicity assays were carried out in five cancer cell lines (HepG2, SH-SY5Y, DU145, MCF-7 and A549) and one normal human cell line (GES-1), in which compound 22b showed very low IC50 to HepG2 (the IC50 value is 3.06 µM), which was lower than Sorafenib. Compound 22b could inhibit cell cycle at S and G2/M phase and induce cell apoptosis. In the HepG2 xenograft model, 22b exhibited significant cancer growth inhibition with low host toxicity in vivo.

Fenretinide Induced Apoptosis in Human Neuroblastoma Cell Lines / 체질인류학회지

Retinoids play an important role in growth, reproduction and differentiation. Recently, retinoids have been used to both protect and treat from various animal models of carcinogenesis. In this study the effect of N-(4-hydroxyphenyl) retinamide (fenretinide) on viability of human neuroblastoma cell lines were evaluated. For the evaluation of apoptosis of human neuroblastoma cell lines by fenretinide. MTT assay, cytoplasmic DNA fragmentation, TUNEL stain, and Western blot analysis were performed. In MTT assay, fenretinide inhibited the proliferation of CHP134, IMR32 and SH-SY5Y but not in PC12 cells. Cytoplasmic DNA fragmentation was induced by treament of fenretinide (10 micrometer) for 48 h in IMR32 cells. PARP cleavage was detected by Western blot analysis after 16 h of treatment of fenretinide in CHP134, IMR32 and SH-SY5Y. These fenretinide effects on growth inhibition and increased apoptosis followed to the time dependent manner. The fenretinide treatment did not affect the phosphorylation of MAP kinases (ERK, JNK, p38). There was no change of Bcl-x and Bad expression after treatment of fenretinide (1 micrometer) in neroblastoma cell lines. Pretreatement of PD98059, SB203580, LY294002, or genistein also did not affect fenretinide-induced PARP cleavage in neuroblastoma cell lines. From these results, the fenretinide-induced apoptosis is due to the PARP cleavage which occured MAP kinase signal cascades independently.