Heavy metals in wild marine fish from South China Sea: levels, tissue- and species-specific accumulation and potential risk to humans.

Heavy metal pollution in marine fish has become an important worldwide concern, not only because of the threat to fish in general, but also due to human health risks associated with fish consumption. To investigate the occurrence of heavy metals in marine fish species from the South China Sea, 14 fish species were collected along the coastline of Hainan China during the spring of 2012 and examined for species- and tissue-specific accumulation. The median concentrations of Cd, Cr, Cu, Zn, Pb and As in muscle tissue of the examined fish species were not detectable (ND), 2.02, 0.24, 2.64, 0.025, and 1.13 mg kg(-1) wet weight, respectively. Levels of Cu, Zn, Cd and Cr were found to be higher in the liver and gills than in muscle, while Pb was preferentially accumulated in the gills. Differing from other heavy metals, As did not exhibit tissue-specific accumulation. Inter-species differences of heavy metal accumulation were attributed to the different habitat and diet characteristics of marine fish. Human dietary exposure assessment suggested that the amounts of both Cr and As in marine wild fish collected from the sites around Hainan, China were not compliant with the safety standard of less than 79.2 g d(-1) for wild marine fish set by the Joint FAO/WHO Expert Committee on Food Additives. Further research to identify the explicit sources of Cr and As in marine fish from South China Sea should be established.

Discovery of a Series of 4-Amide-thiophene-2-carboxyl Derivatives as Highly Potent P2Y14 Receptor Antagonists for Inflammatory Bowel Disease Treatment.

The P2Y14 receptor has been proven to be a potential target for IBD. Herein, we designed and synthesized a series of 4-amide-thiophene-2-carboxyl derivatives as novel potent P2Y14 receptor antagonists based on the scaffold hopping strategy. The optimized compound 39 (5-((5-fluoropyridin-2-yl)oxy)-4-(4-methylbenzamido)thiophene-2-carboxylic acid) exhibited subnanomolar antagonistic activity (IC50: 0.40 nM). Moreover, compound 39 demonstrated notably improved solubility, liver microsomal stability, and oral bioavailability. Fluorescent ligand binding assay confirmed that 39 has the binding ability to the P2Y14 receptor, and molecular dynamics (MD) simulations revealed the formation of a unique intramolecular hydrogen bond (IMHB) in the binding conformation. In the experimental colitis mouse model, compound 39 showed a remarkable anti-IBD effect even at low doses. Compound 39, with a potent anti-IBD effect and favorable druggability, can be a promising candidate for further research. In addition, this work lays a strong foundation for the development of P2Y14 receptor antagonists and the therapeutic strategy for IBD.

Differential responses to UVB irradiation in human keratinocytes and epidermoid carcinoma cells.

OBJECTIVE: To examine UVB-induced responses in normal human keratinocytes (HaCaT) and epidermoid carcinoma cells (A431) at the cellular and molecular level, and investigated the protective effect of salidroside. METHODS: Cells irradiated by UVB at various dosage and their viability was assessed by MTT assays, cell cycle was analysed by flow cytometry. The expression of NF-κB, BCL-2, and CDK6 after 50 J/m(2) UVB irradiation were detected by RT-PCR and western blotting. RESULTS: Our results confirmed greater tolerance of A341 cells to UVB-induced damage such as cell viability and cell cycle arrest, which was accompanied by differential expression changes in NF-κB, BCL-2, and CDK6. UVB exposure resulted in HaCaT cells undergoing G(1)-S phase arrest. When treated with salidroside, HaCaT survival was significantly enhanced following exposure to UVB, suggesting great therapeutic potential for this compound. CONCLUSION: Taken together, our study suggests that A431 respond differently to UVB than normal HaCaT cells, and supports a role for NF-κB, CDK6, and BCL-2 in UVB-induced cell G(1)-S phase arrest. Furthermore, salidroside can effectively protect HaCaT from UVB irradiation.

Differential expression profiles of microRNAs in NIH3T3 cells in response to UVB irradiation.

MicroRNAs (miRNAs) are known as a kind of small, noncoding RNA, which play an important role in mediating many biological processes such as development, cell proliferation and differentiation in plants and animals. Here we report the differential expression profiles of miRNAs and characterized putative target genes in NIH3T3 cells at a series of different time points after UVB irradiation (compared with no UVB irradiation). The relative expression of mature miRNA genes was determined by miRNA microarray technique and the results were confirmed by real-time reverse transcriptase polymerase chain reaction (qRT-PCR). Potential target genes of these miRNAs were classified into different function categories with the GOstat software (http://gostat.wehi.edu.au/cgi-bin/goStat.pl). Several miRNAs in this study expressed highly at different time points, especially mmu-miR-365 and mmu-miR-21. Three miRNAs were lowly expressed, of which mmu-miR-465 showed low levels of expression at all time points, whereas after 50 J m(-2) UVB irradiation mmu-miR-296 and mmu-miR-376c showed low levels of expression at 6 and 12 h, respectively. Our study provided a basis for the global characterization of UV-regulated miRNA expression.

[Role of long noncoding RNA MALAT1 promotes the occurrence and progression of cutaneous squamous cell carcinoma].

OBJECTIVE: To investigate the role of long noncoding RNA MALAT1 in the occurrence and progression of cutaneous squamous cell carcinoma (CSCC). METHODS: Fifty-five tissue samples of CSCC and 10 normal epidermal tissues were collected for examination of the expression of MALAT1 using q-PCR and in situ hybridization. Human CSCC A431 cells were transfected with small interfering RNAs (siNC, siMALAT1-1, and siMALAT1-2) using Lipofectamine2000 to knock down MALAT1 gene, and the changes in the cell migration, invasion, mobility and proliferation were analyzed using Transwell assay, wound healing assay, and CCK-8 assay; the changes in the expressions of the related factors of epithelial-mesenchymal transition (EMT), including E-cadherin, vimentin, and ß-catenin, were detected using qRT-PCR. RESULTS: Compared with normal tissues, CSCC tissues of different grades of differentiation all showed significantly increased expression of MALAT1 (P<0.001). In A431 cells, MALAT1 knockdown with siRNAs resulted in significantly lowered cell proliferation (P<0.001), migration (P<0.01), invasion (P<0.01), and mobility (P<0.01). Knocking down MALAT1 gene also caused significantly increased expressions of E-cadherin and ß-catenin (P<0.01) and lowered the expression of vimentin (P<0.01) in A431 cells. CONCLUSION: The long noncoding RNA MALAT1 promotes the occurrence and progression of CSCC and can potentially serve as a therapeutic target in treatment of CSCC.

Downregulation of HBx mRNA in HepG2.2.15 cells by small interfering RNA.

OBJECTIVE: Hepatitis B virus (HBV) infection is one of the most prevalent viral infectious diseases in humans. And it is still a challenge for the development of an effective therapy for HBV infection. Recently, the progress in RNA interference (RNAi) has shed some light on the inhibition of HBV expression and replication by RNAi specific for the various genes of the HBV genome. Some prior researches suggests that the HBV x protein (HBx) plays an important role in viral transcription, cell growth, and apoptotic cell death. METHODS: In the present study, we designed three siRNAs based on the X-protein of HBV sequences and tested their effects on the expression of HBx gene following sorting of siRNA-positive cells. The interference effect was tested in 24, 48, and 72 h. HBsAg in cultured media was assayed using western blot at various days post-transfection. The amount of HBx mRNA was quantitated by Real-time reverse-transcript PCR (RT-PCR). RESULTS: There was a decrease in the levels of HBV mRNA and HBsAg from the the transfected cells. Among these three siRNAs, siRNA-2 was found to be the most effective at suppressing HBV gene expression.

Distribution and ecological effect of mercury in Laogang landfill, Shanghai, China.

Laogang landfill near Shanghai is the largest landfill in China, and receives about 10000 t of daily garbage per day. Samples of topsoil and plants were analyzed to evaluate mercury pollution from the landfill. For topsoil samples, there were significant correlations among total mercury (HgT), combinative mercury (HgC) and gaseous mercury (HgG), and content of total organic carbon (TOC), but, no significantly relationship was found between Hg content and filling time. Hg content changes in vertical profiles with time showed that the average HgT of profiles 1992, 1996, and 2000 was similar, but their average HgG was quite different. HgT was significantly correlated with HgC in profile 1992 and 2000, and HgT was significantly correlated with HgG in profile 1996. HgG/HgT ratio in profile samples decreased in the order of (HgG/HgT)1992 > (HgG/Hg)1996 >> (Hg/HgT)2000. A simple outline of Hg release in landfill could be drawn: with increasing of filling time, degradation undergoes different biodegradation, accordingly, gaseous mercury goes through small, more, and small proportion to total mercury. Distribution of Hg in plants was inhomogeneous, following the order of leaf > root > stem. The highest value of leaf may be associated with higher atmospheric Hg from landfill. Ligneous plants (e.g. Phyllostachys glanca, Prunus salicina and Ligustrum lucidum) are capable of enriching more Hg than herbaceous plants.

[Advances in the studies of miRNAs and cell apoptosis].

MicroRNAs (miRNAs) are a recently discovered class of 18 - 24 nucleotide RNA molecules that negatively regulate target mRNAs. All studied multicellular eukaryotes utilise miRNAs to regulate basic cellular functions including proliferation, differentiation, and death. In this review, we will discuss the function of miRNAs that regulate cell proliferation and apoptosis. Antiapoptotic miRNAs include the miRNAs of the miR-17 cluster, miR-21, bantam and miR-14, Proapoptotic miRNAs include let-7, miR-15a and miR-16.

[Analgesic effect of huwentoxin-I, a new N-type voltage-sensitive calcium channel blocker, on acute visceral pain in rats].

OBJECTIVE: To study the antinociceptive effect of intrathecally administered huwentoxin-I (HWTX-I or HWAP-I) on visceral pain in rats with acute colon inflammation. METHODS: Nociceptive behaviors was induced by formalin injected into the submucosa of the sigmoid colon in rats and the typical behavioral patterns induced served as the indexes for visceral nociception scoring. HWAP-I (0.1-1.0 microg/kg x b x w.), SNX-111 (0.2, 0.5 and 0.75 microg/kg.b.w.) and morphine hydrochloride (0.05, 0.1 and 0.2 microg/kg x b x w.) were administered subarachnoidally 30 min before formalin injection. RESULTS: Similar to SNX-111 and hydrochloride morphine, HWAP-I administered subarachnoidally significantly reduced nociceptive responses in a dose-dependent manner in the rat model of visceral pain (P<0.05). Both HWAP-I and SNX-111 produced pain suppression effect at the dosage of 0.2 microg/kg x b x w., and in spite of the stronger antinociceptive effect of SNX-111 than HWAP-I at the same doses, SNX-111 caused obvious motor dysfunction in the rats at the doses higher than 0.5 microg/kg x b x w., which was not observed with HWAP-1 at such doses. The antinociceptive effect of morphine hydrochloride was initiated faster but lasted for a shorter period of time than the effects of HWAP-I and SNX-111. CONCLUSION: As a potent blocker of neuronal N-type voltage-sensitive calcium channel, HWAP-I induces remarkably dose-dependent inhibitory effect similar to SNX-111 and morphine on visceral pain induced by sigmoid colon submucosal injection of formalin in conscious rats.

Survival of mice with gamma-ray irradiation in hot and humid environments.

OBJECTIVE: To study the survival of the mice with gamma-ray irradiation in hot and humid environments (HHE) and determine the median lethal dose in 30 days (LD50/30). METHODS: One hundred mice were randomly divided into 2 groups (n=50 each) to receive gamma-ray irradiation at the dosage of either 7 or 9 Gy, and were then immediately transferred to the chambers simulating HHE for treatment for 30, 60, 90 or 120 min respectively, leaving one group at room temperature (n=10 in each group). The death rates of the mice within 30 days were observed. For LD50/30 determination, another 180 mice in 3 equal groups were subjected to irradiation at 5, 7 or 9 Gy, respectively and after further division of each group into 3 equal subgroups, the mice underwent treatment in HHE for 60 or 90 min respectively or at room temperature to observe their death rates within 30 days and calculate the LD50/30. RESULTS: Compared to the mice treated at room temperature, earlier onset and more cases of infections with much severity were observed in all the HHE groups, with reduced average survival time and obviously shortened median survival time (P<0.05), and LD50/30 tended to also decrease in the HHE groups. CONCLUSION: The survival indexes of irradiated mice are significantly decreased in hot and humid environments.

[Effect of hot and humid environment on bone marrow and spleen of mice exposed to gamma irradiation].

OBJECTIVE: To study the effect of hot and humid environment (HHE) on the bone marrow and spleen in mouse exposed to gamma irradiation. METHODS: After exposure to gamma irradiation at the dosage of 6.5 Gy, the mice were subjected to treatment in HHE for 1 h or at room temperature (RT) respectively. After exposure to HHE, the mice in HHE group were kept at RT. The bone marrow and spleen of the mice in both RT and HHE groups were examined at 1, 3, 5, 9 and 15 days after irradiation for bone marrow cellularity, DNA content in the bone marrow cells, and spleen/body weight ratio. RESULTS: Compared with the RT group, mice in the HEE group showed aggravation of decreases in bone marrow cells, DNA content and spleen/body weight ratio. CONCLUSIONS: HHE aggravates radiation injury of the bone marrow and spleen in mice.

[Effect of XK469 and adriamycin on the growth of H460 cells in vitro and its mechanism].

OBJECTIVE: To investigate the inhibitory effect of XK469 on the in vitro growth of H460 cells and its mechanism. METHODS: The survival curves of H460 cells treated with XK469, XN472 and adriamycin, respectively, were obtained by MTT analysis, and the effect of XK469 and adriamycin on the cell cycle of H460 cells examined by flow cytometry. Western blotting was adopted for detecting the expression of cdc2 and phos-cdc2 induced by XK469 and adriamycin. RESULTS: Different concentrations of XK469 and adriamycin could significantly inhibit the growth of H460 cells, induce their G2/M phase arrest, and increase phos-cdc2 expression; XN472 had a lesser effect on the growth of H460 cells. CONCLUSION: XK469 can increase phos-cdc2 expression and induce G2/M phase cell cycle arrest of H460 cells, resulting in inhibition of H460 cell growth. The inhibitory effect of XK469 on H460 cell growth is attributed to the chlorine in the 7-positon of its structure.

Irradiation-induced apoptosis of mouse bone marrow stromal cells.

OBJECTIVE: To study the apoptotic patterns of bone marrow stromal cells (BMSCs) after irradiation. METHODS: Apoptosis of BMSCs induced by (60)Co gamma irradiation was detected by both flow cytometry and electron microscopy. RESULTS: Compared with normal control group, no manifest changes in the apoptosis ratio occurred in BMSCs after 50 Gy irradiation (P>0.05) as demonstrated by flow cytometry. Four hours after 100 Gy irradiation, manifest changes in apoptosis ratio of BMSCs were observed (P<0.01), reaching the peak 20 h later. Similar apoptotic patterns occurred in BMSCs exposed to irradiation of 200 Gy (P>0.05), only the apoptosis ratio was slightly higher. CONCLUSION: Irradiation of proper dose may induce apoptosis in vitro in BMSCs that possess strong resistance to irradiation.

Relationships between degree of polymerization and antioxidant activities: a study on proanthocyanidins from the leaves of a medicinal mangrove plant Ceriops tagal.

Tannins from the leaves of a medicinal mangrove plant, Ceriops tagal, were purified and fractionated on Sephadex LH-20 columns. 13C nuclear magnetic resonance (13C-NMR), reversed/normal high performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDT-TOF MS) analysis showed that the tannins were predominantly B-type procyanidins with minor A-type linkages, galloyl and glucosyl substitutions, and a degree of polymerization (DP) up to 33. Thirteen subfractions of the procyanidins were successfully obtained by a modified fractionation method, and their antioxidant activities were investigated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging capacity and ferric reducing antioxidant power (FRAP) method. All these subfractions exhibited potent antioxidant activities, and eleven of them showed significantly different mean DP (mDP) ranging from 1.43±0.04 to 31.77±1.15. Regression analysis demonstrated that antioxidant activities were positively correlative with mDP when around mDP <10, while dropped and then remained at a level similar to mDP = 5 with around 95 µg ml(-1) for DPPH scavenging activity and 4 mmol AAE g(-1) for FRAP value.

[Mechanisms of ultraviolet B irradiation-induced injuries in 16HBE cells].

OBJECTIVE: To investigate the biological effects of ultraviolet B (UVB) irradiation on human bronchial epithelial cells (16HBE cells) and explore the possible mechanism. METHODS: The survival rates of 16HBE cells were detected by MTT assay at 12 h after UVB irradiation at different doses (0, 10, 30, 50, 70, and 100 J/m(2)) or at 50 J/m(2) for different durations (2, 4, 8, 12, and 24 h). The DNA ladder was detected by agarose gel electrophoresis, the cell cycle changes were analyzed by flow cytometry, and the expression of nuclear factor-κB (NF-κB)/p65 protein was assayed by Western blotting following the exposures. RESULTS: UVB irradiation of the cells resulted in lowered cell survival rates, DNA fragmentation, S phase arrest and up-regulation of NF-κB/p65 protein expression. CONCLUSIONS: UVB irradiation can induce growth inhibition and apoptosis of 16HBE cells, in which process NF-κB protein may play a key role.

[Preliminary study on the distribution and impact factors of methylmercury in surficial sediments from main mangrove wetlands of China].

Total mercury (THg), methylmercury (MeHg) and environmental factors were determined to study distributions of MeHg and Hg methylation in the sediments from 8 main mangrove areas of China. The results showed that it was not consistent for distributions of THg and MeHg in sediments. Concentrations of MeHg in sediments from Sanya, Dongzhaigang (Hainan Province), Techengdao, Leizhou, Gaoqiao, Futian (Guangdong Province), Daguansha (Guangxi Autonomous Region), Fugong (Fujian Province) were (0.24 +/- 0.04), (0.58 +/- 0.27), (0.52 +/- 0.23), (1.56 +/- 0.49), (0.50 +/- 0.25), (1.21 +/- 0.36), (1.86 +/- 1.04), (0.47 +/- 0.16) ng x g(-1) respectively. There were regional difference in MeHg contents which decreased in the order of Daguansha > Leizhou > Futian > Dongzhaigang > Techengdao > Gaoqiao > Fugong > Sanya. Input of Hg and organic matter from industry and aquiculture may lead to high level of MeHg. Compared with sediments from other estuaries of the world, serious pollution of MeHg was found in mangrove sediments of China. (2) % MeHg in mangrove sediments ranged from 0.11% to 7.13%, which decreased in the order of Daguansha > Techengdao > Leizhou > Futian > Gaoqiao > Dongzhaigang > Sanya > Fugong. There was significantly positive correlation between % MeHg and sandy fraction (p < 0.05), and significantly negative correlation between % MeHg and silt-clay fraction (p < 0.05). (3) Total bacteria (TB) in mangrove sediments ranged from 2.44 x 10(10) to 1.91 x 10(11) CFU/g (dry weight), TB decreased in the order of Fugong > Sanya > Gaoqiao > Dongzhaigang > Futian > Daguansha. Sulfate-reducing bacterium (SRB) ranged from 1.73 x 10(4) to 4.92 x 10(6) CFU/g, SRB decreased in the order of Futian > Fugong > Dongzhaigang > Sanya > Gaoqiao > Daguansha. Wastewater with high organic matters leads to high SRB. The types of surface sediments also had a great impact on the amount of SRB. There was no significant correlation among MeHg and environmental factors, which indicated that exogenous input is the main cause of MeHg pollution in mangrove sediments.

[Distribution of Hg in mangrove plants and correlation with Hg speciation in sediments].

Plant is an important role in biogeochemical cycle of Hg. The aim of this study is to ascertain Hg accumulation in several kinds of mangrove plants, and to discuss relationship among Hg concentrations in mangrove plants and different Hg speciation in sediments. Contents of total mercury (THg) in mangrove plants and sediments were determined. Hg speciation was determined with a modified Tessier's method. Contents of THg of the mangrove plants were in the range of 817.5-3 197.6 ng/g. In detail, Hg concentration was (1 579.4 +/- 1 326.8) ng/g in Kandelia candel, (2 115.1 +/- 1 892.3) ng/g in Aegiceras corniculatum, (2 159.3 +/- 1 678.7) ng/g in Avicennia marina, (2 566.5 +/- 821.6) ng/g in Bruguiera gymnorrhiza, (2 104.3 +/- 1 661.8) ng/g in Excoecaria agallocha, (3 197.6 +/- 2 782.8) ng/g in Sonneratia apetala, (817.5 +/- 632.3) ng/g in Acanthus ilicifolius, (1 801.8 +/- 1 255.4) ng/g in Rhizophora. stylosa, respectively. There are obvious interspecific variation, and organic variation in THg contents of mangrove plants, which is closely related to environment and physiological characteristics of mangrove plants. Enrichment of THg in mangrove plants was inhomogeneous, following the order of Sonneratia apetala > Bruguiera gymnorrhiza > Avicennia marina > Aegiceras corniculatum > Excoecaria agallocha > Rhizophora stylosa > Kandelia candel > Acanthus ilicifolius. Mercury exists mainly in volatile form in most mangrove wetlands, but mainly in the form of residue in sediments from Shenzhen mangrove wetlands. Significantly positive correlations were found among Hg concentrations in leaves and stems of Sonneratia apetala and volatile Hg, exchangeable Hg of sediments. Significantly positive correlations were also found among Hg concentrations in leaves of Excoecaria agallocha and volatile Hg, exchangeable Hg of sediments. But, there is no significant correlation between Hg concentrations of most mangrove plants and different Hg speciation in sediments. It showed that plants assimilate Hg from different sources, such as water, sediment and air, and that Hg assimilated by plants could transfer among different plant organics.

[Distribution of mercury in surficial sediments from main mangrove wetlands of China].

Surficial sediment samples were collected from main mangrove wetlands of China, mercury, pH, salinity, organic matters, grain size, Fe/Hg, Mn/Hg were analyzed. Mercury content ranges from 2.3 to 903.6 ng x g(-1), with a average of (197 +/- 137.6) ng x g(-1). Compared with local background level, serious Hg pollution with high Hg content was found in 7 areas, including Luoyangqiao [(467.5 +/- 68.8) ng x g(-1)], Fugong [(438.2 +/- 147.0) ng x g(-1)], Ewan [(264.3 +/- 89.2) ng x g(-1)], Yaojiayu [(125.4 +/- 27.1) ng x g(-1)], Fujian Province; Sanya [(164.8 +/- 143.9) ng x g(-1)], Dongzhaigang [(314.1 +/- 335.7) ng x g(-1)], Hainan Province, Shenzhen [(179.9 +/- 7.7) ng x g(-1)], Guangdong Province. Hg content was similar with background value in the other 6 areas, including Yunxiao [(63.3 +/- 43.9)ng x g(-1)], Fujian Province; Gaoqiao [(178.6 +/- 127.0) ng x g(-1)], Guangdong Province; Daguansha [(26.1 +/- 18.8) ng x g(-1)], Shankou [(73.8 +/- 21.1) ng x g(-1)], Beilun estuary [(117.8 +/- 51.4) ng x g(-1)], Qinzhou Bay [(147.5 +/- 107.6) ng x g(-1)], Guangxi Autonomous Region. Discrepancy of total Hg concentrations in mangrove wetlands is due to many environmental factors and human activities. Parameters such as grain size, pH, organic matter, Fe/Hg, Mn/Hg are significantly correlated with total Hg respectively. Clay and Fe/Hg are obvious factors influencing total Hg concentration. Impact of environmental factors on mercury can be showed by regression equation.

[Mechanisms of G2/M cycle arrest induced by topo IIalpha and II beta inhibitors in H460 cells].

OBJECTIVE: To compare the mechanisms of G(2)/M cycle arrest induced by topo IIalpha and IIbeta inhibitors in H460 cells. METHODS: The inhibitory effects of XK469, adriamycin and etoposide on H460 cell growth were analyzed by MTT assay. The changes in cell cycle and expressions of cdc2, phos-cdc2 and 14-3-3sigma proteins induced by these 3 topo II inhibitors were detected by flow cytometry and Western blotting, respectively. RESULTS: Both of the two types of topo II inhibitor resulted in dose-dependent G(2)/M phase arrest and growth inhibition of H460 cells, but XK469 failed to induce 14-3-3sigma protein expression as adriamycin and etoposide did. CONCLUSION: Topo IIalpha and topo IIbeta inhibitors induce growth inhibition of H460 cells possibly through two different mechanisms, namely the 14-3-3sigma-dependent pathway and the 14-3-3sigma-independent pathway, but further functional inhibition test of 14-3-3sigma is needed to confirm this hypothesis.