Increased emergency cases for out-of-hospital cardiac arrest due to cold spells in Shenzhen, China.

Cold spells have been associated with specific diseases. However, there is insufficient scientific evidence on the effects of cold spells on out-of-hospital cardiac arrest (OHCA). Data on OHCA cases and on meteorological factors and air pollutants were collected between 2013 and 2020. We adopted a quasi-Poisson generalized additive model with a distributed lag nonlinear model (DLNM) to estimate the effect of cold spells on daily OHCA incidence. Backward attributable risk within the DLNM framework was calculated to quantify the disease burden. We compared the effects and OHCA burden of cold spells using nine definitions. The risks of different cold spells on OHCA increased at higher intensities and longer durations. Based on Akaike's information criterion for the quasi-Poisson regression model and the attributable risk, the optimal cold spell was defined as a period in the cold month when the daily mean temperature was below the 10th percentile of the temperature distribution in the study period for at least 2 days. The single-day effect of the optimal cold spell on OHCA occurred immediately and lasted for approximately 1 week. The maximum single-day effect was 1.052 (95% CI: 1.018-1.087) at lag0, while the maximum cumulative effect was 1.433 (95% CI:1.148-1.788) after a 14-day lag. Men were more susceptible to cold spells. Young and middle-aged people were affected by cold spells similar to the elderly. Cold spells can increase the risk of OHCA with an approximately 1-week lag effect. Health regulators should take more targeted measures to protect susceptible populations during cold weather.

Associations between plasma metal mixture exposure and risk of hypertension: A cross-sectional study among adults in Shenzhen, China.

Background: Metal exposure affects human health. Current studies mainly focus on the individual health effect of metal exposure on hypertension (HTN), and the results remain controversial. Moreover, the studies assessing overall effect of metal mixtures on hypertension risk are limited. Methods: A cross-sectional study was conducted by recruiting 1,546 Chinese adults who attended routine medical check-ups at the Eighth Affiliated Hospital of Sun Yat-sen University, Shenzhen. The plasma levels of 13 metals were measured using inductively coupled plasma mass spectrometry. Multivariate logistic regression model, restricted cubic spline (RCS) model and the Bayesian Kernel Machine Regression (BKMR) model were applied to explore the single and combined effect of metals on the risk of HTN. Results: A total of 642 (41.5%) participants were diagnosed with HTN. In the logistic regression model, the adjusted odds ratios (ORs) were 0.71 (0.52, 0.97) for cobalt, 1.40 (1.04, 1.89) for calcium, 0.66 (0.48, 0.90), and 0.60 (0.43, 0.83) for aluminum in the second and third quartile, respectively. The RCS analysis showed a V-shaped or an inverse V-shaped dose-response relationship between metals (aluminum or calcium, respectively) and the risk of HTN (P for non-linearity was 0.017 or 0.009, respectively). However, no combined effect was found between metal mixture and the risk of hypertension. Conclusions: Plasma levels of cobalt, aluminum and calcium were found to be associated with the risk of HTN. Further studies are needed to confirm our findings and their potential mechanisms with prospective studies and experimental study designs.

Exosomal secretion may be a self-protective mechanism of its source cells under environmental stress: A study on human bronchial epithelial cells treated with hydroquinone.

Accumulating evidence reveals that exosome plays an important role in cell-to-cell communication in both physiological and pathological processes by transferring bioactive molecules. However, the role of exosomal secretion in the adaption of its source cells to the stimuli of environmental chemicals remains elusive. In this study, we revealed that the exposure of hydroquinone (HQ; the main bioactive metabolite of benzene) to human bronchial epithelial cells (16HBE) resulted in decreased ability of cell proliferation and migration, and simultaneously DNA damage and micronuclei formation. Interestingly, when exosomal secretion of HQ treated 16HBE cells was inhibited with the inhibitor GW4869, cellular proliferation and migration were further significantly reduced; concurrently, their DNA damage and micronuclei formation were both further significantly aggravated. Herein, we conclude that exosomal secretion of 16HBE cells may be an important self-protective function against the toxic effects induced by HQ.

Exosomal miR-221 derived from hydroquinone-transformed malignant human bronchial epithelial cells is involved in cell viability of recipient cells.

miR-221, an oncogenic microRNA, can promote cell proliferation and is highly expressed in various types of tumors. However, the role of exosomal miR-221 in benzene-caused carcinogenesis remains elusive. Our study was designed to investigate whether exosomes secreted by the hydroquinone (HQ; an active metabolite of benzene)-transformed malignant cells can transmit miR-221 to normal recipient cells and its possible effects on cell viability. Our investigation revealed that expression levels of miR-221 were significantly increased in HQ-transformed malignant cells relative to normal controls. Furthermore, exposure of control cells to exosomes that were derived from HQ-transformed malignant cells increased miR-221 levels and promoted their proliferation. Analyses of the biological potency of exosomes derived from HQ-transformed malignant cells in which miR-221 levels were decreased using an inhibitor, showed that both miR-221 levels and proliferation of recipient cells were decreased, but still were higher than those of normal 16HBE cells. Our study indicates that exosomal miR-221 derived from HQ-transformed malignant human bronchial epithelial cells is involved in the proliferation of recipient cells.

The relationship between extreme temperature and emergency incidences: a time series analysis in Shenzhen, China.

Extreme temperature has been reported to be associated with an increase in acute disease incidence in several cities. However, few similar studies were carried out in Shenzhen, which is a subtropical city located in the southern China. This study explored the relationship between the emergency incidences and extreme temperatures, and investigated the role of air pollutants played in the temperature-related effects on human health in Shenzhen. We conducted a distributed lag nonlinear model study on the effect of extreme temperatures on emergency incidences in Shenzhen city during 2013-2017. Here, only the total emergency incidences, emergency incidences for respiratory diseases, and cardiovascular diseases were taken into consideration. Air pollution, subgroups, and seasons were adjusted to investigate the impacts of extreme temperatures on emergency incidences. Relative risk (RR) and 95% confidence intervals were calculated with the R software. From lag 0 to 21 days, the RR of temperature-total emergency department visits, temperature-cardiovascular, and temperature-respiratory diseases was 1.09 (95% CI: 0.98-1.20), 1.22 (95% CI: 0.96-1.56), and 1.06 (95% CI: 0.70-1.60) at extremely low temperature (first percent of temperature, 10 °C), respectively. During the same lag days, the RR was 1.02 (95 % CI: 0.92-1.14), 0.64 (95% CI: 0.49-0.86), and 0.92 (95% CI: 0.56-1.53) between extremely high temperature and total emergency department visits, cardiovascular, and respiratory diseases, respectively. The cumulative effects gradually went up with time for all types of emergency incidences in warm seasons (5 days moving average of temperature < 22 °C). However, the cumulative effects of total emergency incidences and Cvd emergency incidences were increased within the first lag 5 days, and then decreased until lag 21 in hot seasons (5 days moving average of temperature ≥ 22 °C). The cumulative effects of Res emergency incidences showed a declined trend from lag 0 to lag 21. The elderly (≥ 65, P1: RR = 1.49, 95% CI (1.30, 1.71); P99: RR = 0.86, 95% CI (0.71, 1.04)) and men (P1: RR = 1.27, 95% CI (1.14, 1.42)) seemed to be more vulnerable to extreme temperature than the younger (≤ 64, P1: RR = 1.19, 95% CI (1.08, 1.32); P99: RR = 1.00, 95% CI (0.89, 1.12)) and women (P1: RR = 1.17, 95%CI (1.06, 1.30)). The effects of extremely low temperature on all types of emergency incidences were stronger than those of extremely high temperature in the whole year. In addition, impacts of cold weather lasted about several days while those of hot weather were acute and rapid. An increased frequency of emergency incidences is predicted by rising temperatures variations. These results have clinical and public health implications for the management of emergency incidences.

Effect on metabolic enzymes and thyroid receptors induced by BDE-47 by activation the pregnane X receptor in HepG2, a human hepatoma cell line.

2,2',4,4'-Tetra-bromodiphenyl ether (BDE-47), an important congener among polybrominated diphenyl ether (PBDE) compounds, has been predominantly in environmental samples and human tissue. Thyroid disruption is the most sensitive endpoint effect among a number of health effects of exposure to BDE-47 in animals and humans. However, the detailed underlying mechanisms in humans are not well understood. In the present study, human pregnane X receptor (hPXR)-overexpressing HepG2 cell model and a dual-luciferase reporter assay system were constructed to investigate the role of hPXR in BDE-47-induced alterations of expression of metabolic enzymes and TR in vitro. The results showed that hPXR was significantly activated by BDE-47, and expression levels of both mRNA and protein of the thyroid receptor (TR) isoforms TRα1 and TRß1 were decreased in hPXR-overexpressing HepG2 cells after BDE-47 treatment. However, the increased expression of hepatic microsomal phase I enzyme CYP3A4 and phase II enzymes, UGT1A3 and SULT2A1 were also found. Taken together, the results indicated that BDE-47 was a strong hPXR activator, activation of hPXR played an important role in BDE-47-induced down-regulation of TR, and up-regulations of CYP3A4, UGT1A3, and SULT2A1 participated in the process, which may provide more toxicological evidence on mechanisms of disruption of thyroid hormone induced by BDE-47.

Levels and profiles of PCDD/Fs, PCBs in mothers' milk in Shenzhen of China: estimation of breast-fed infants' intakes.

Sixty breast milk samples were collected in Shenzhen, China from July to November in 2007. The samples were analyzed of the concentrations of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and polychlorinated biphenyls (PCBs). The range of upper-bound for ∑TEQ-(PCDD/Fs+PCBs) in the samples was 4.10-35.3 pg TEQ g(-1) lipid (median: 10.6 pg TEQ g(-1) lipid; mean: 11.9 pg TEQ g(-1) lipid). The levels of the measured contaminants in the breast milk had significant correlations with the length of inhabitation period in Shenzhen (r=0.487, p<0.05 for PCDD/Fs, r=0.431, p<0.05 for PCBs and r=0.478, p<0.05 for ∑TEQ-(PCDD/Fs+PCBs)), and the consumption rate of fish (r=0.366, p<0.05 for PCDD/Fs, r=0.486, p<0.05 for PCBs and r=0.416, p<0.05 for ∑TEQ-(PCDD/Fs+PCBs)), respectively. Moreover, significant positive correlations were also detected between the participant's age (r=0.305, p<0.05 for ∑TEQ-PCBs and r=0.275, p<0.05 for ∑TEQ-(PCDD/Fs+PCBs)) and the body burdens of these contaminants respectively. It is estimated that the daily intake (EDI) of the sum of PCDD/Fs and DL-PCBs by the breast-fed infants was 5.60-161 pg TEQ kg(-1) bw per day (mean: 48.2 pg TEQ kg(-1) bw per day; median: 42.2 pg TEQ kg(-1) bw per day). The result showed that both the body burdens of PCDD/Fs and PCBs of the recruit population and the calculated EDI of the breast-fed infants were higher than those in the non-exposed areas in mainland China. This suggests that continuous surveillance on PCDD/Fs and PCBs levels in human milk is critical to more precisely evaluate the human health risk posed by the negative environmental impact in Shenzhen in the future.

[Screening and identification of differential serum proteins related to dermatitis medicamentosa-like of trichloroethylene].

OBJECTIVE: To screen and identify differential serum proteins which might be involved in dermatitis medicamentosa-like of trichloroethylene (DMLT). METHODS: Three groups of sera were collected from population exposed to trichloroethylene (TCE) (group I), patients suffering from DMLT (group II), and the healed cases (group III). After removing albumin and IgG in the three pools of sera, a comparative proteomic analysis was carried out. The images were analyzed using ImageMaster Platinum 2D 5.0 to screen the differentially expressed proteins. The protein spots were then subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and tandem mass spectrometry sequencing of tryptic peptides for further identification. RESULTS: The depletion of albumin and IgG greatly increased the number of protein spots to 300 ± 12.Five differential spots were identified, which were complement component C4b, apolipoprotein A-I, apolipoprotein C-III apolipoprotein C-II and transthyretin. Compared with group I, the expression levels of complement component C4b in group III and apolipoprotein C-II in group II were up-regulated (1.352 88-fold, 1.512 14-fold, respectively); compared with group I, the expression levels of apolipoprotein A-I, apolipoprotein C-III and transthyretin in group II were down-regulated (1.601 17-fold, 1.034 49-fold, 1.313 35-fold, respectively). CONCLUSION: The findings of this study show that most of the identified differential proteins are closely related to immunity and liver dysfunction, which provides some evidence on elucidating the mechanisms and screening of biomarkers of TCE intoxication.

[Effects of 2,2',4,4'-tetrabrominated diphenyl ethers on the TRalpha1 and TRbeta1 expression in liver in C57BL/6 mice].

OBJECTIVE: To study the effect of 2,2',4,4'-tetrabrominated diphenyl ethers (BDE-47) on the expression change of TRalpha1, TRbeta1 at both mRNA and protein levels. METHODS: C57BL/6 mice were divided randomly into 4 groups and administrated with corn oil, 0.5, 5 and 50 mg/kg/BW BDE-47 for 4 days through intraperitoneal injection respectively. GAPDH selected as internal standard, real time quantitative RT-PCR and Western blot were employed to detect the mRNA levels and protein of TRalpha1 and TRbeta1 in livers. RESULTS: Compared with the control group, the mRNA levels of TRalpha1 and TRbeta1 were up-regulated in the medium and high BDE-47 dose groups (P < 0.05). The protein level of TRalpha1 significantly up-regulated while TRbeta1 was significantly down-regulated. CONCLUSION: BDE-47 could change the expression of TRalpha1 and TRbeta1 in the mRNA and protein levels in liver.

[Study on skin sensitization as well as liver and kidney impairment in guinea pigs treated with trichloroethylene].

OBJECTIVE: To study skin sensitization as well as liver and kidney impairment in guinea pigs treated with trichloroethylene (TCE). METHODS: Guinea pig maximization test (GPMT) was applied in this study, guinea pigs were divided into 3 groups, namely negative control, positive control and TCE treatment. Animals of 3 groups were administrated with olive oil, 2, 4-dinitrochlorobenzene (DNCB), and TCE, respectively, by intradermal injection. The animal skin was observed and blood was collected after various treatment, the liver function tests were conducted, including detection of activities of ALT, AST, LDH and levels of creatinine, uric acid, and urea with automatic biochemical analyzer. RESULTS: Obvious skin impairment was observed in the groups of positive control and TCE treatment, the skin impairment included erythema and edema, the sensitization rate was 100% in positive control and 83.3% in TCE treatment group. Additionally, the activities of ALT, AST and LDH increased significantly in the groups of positive control and TCE treatment when compared with the negative control. CONCLUSIONS: Trichloroethylene is one of the strong hypersensitizing substances, it could induce skin allergic reaction and liver impairment in guinea pigs.

Elevated body burdens of PBDEs, dioxins, and PCBs on thyroid hormone homeostasis at an electronic waste recycling site in China.

A cross-sectional study of 25 sample sets (each set consisted of maternal serum and cord whole blood) from 50 pregnant women in zone A (n = 25 from exposed group) and zone B (n = 25 from reference group) was conducted to examine the association between thyroid hormone (TH) levels and PBDE, PCDD/F, and PCB exposures. Thyroid hormones TT3, TT4, and TSH levels were measured in maternal serum at 16 weeks of gestation. The concentrations of PBDEs, PCDD/Fs, and PCBs were determined by isotope dilution HRGC/HRMS in cord blood samples. Body burdens of the three contaminants in cord blood in zone A (median: summation sigma TEQ-PCDD/Fs 0.041, summation operator TEQ-PCBs 0.022 pg WHO-TEQ/g, summation operator PBDEs 23.4 pg/g whole weight, respectively) were significantly higher than those from the reference area (median: summation sigma TEQ-PCDD/Fs 0.014, summation sigma TEQ-PCBs 0.0041 pg WHO-TEQ/g, summation sigma PBDEs 16.15 pg/g, respectively) (p < 0.05). Levels of TT4 and TSH in serum in zone A were significantly lower than those in zone B (p < 0.05). A negative correlation was found between TT4 levels and body burdens of PCDD/Fs and PCBs. However, there was no significant association of concentration of PBDEs and levels of the three thyroid hormones. Our results suggest that electronic waste (e-waste) recycling contributes to high body burdens of PBDEs, PCDD/Fs, and PCBs and affects thyroid hormone homeostasis in humans. The potential health risk for neonates still needs further investigation.

[Effects and toxicity mechanism of 2,2',4,4'-tetrabrominated diphenyl ethers on the homeostasis of thyroid hormone system in C57BL/6 mice].

OBJECTIVE: To investigate the effects and of toxicity mechanism of 2,2',4,4'-TETRABDE (BDE-47) on TT3, TT4 and TSH in C57BL/6 mice. METHODS: C57BL/6 mice were divided randomly into 4 groups, and given with corn oil (control group) and 1 micromol/(kg x d), 10 micromol/(kg x day) and 100 micromol/(kg x day) BDE-47 for 4 days through intraperitoneal injection respectively. After collecting the bloods, livers and thyroids, the T3, T4 and TSH levels of serum were detected by electro chemiluminescenc (ECLI), thyroid peroxidase (TPO) activities in thyroid by guaiacol method and type-I deiodinase (DI) in liver by CHOPRA method. RESULTS: Compared with the control group, the level of TT4 decreased significantly in the three BDE-47 dose groups, TT3 in the medium and high dose groups decreased significantly, and the activities of DI increased in the medium and high dose groups significantly (P < 0.05), whereas TSH level in serum and activity of TPO in thyroid in the three dose groups weren't statistically significant comparing with control group. CONCLUSION: BDE-47 could affect the homeostasis of thyroid hormone system, and activity of type-I deiodinase (DI) in liver being changed might be the one of toxicity mechanisms.

Concentrations of PCDD/PCDFs and PCBs in retail foods and an assessment of dietary intake for local population of Shenzhen in China.

OBJECTIVES: Concentrations of 17 polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and 18 polychlorinated biphenyls (PCBs) were measured in 11 varieties of food groups collected from retail market of Shenzhen, Guangdong province, China from 2004 to 2006. PCDD/Fs and PCBs dietary intake from varies food for the local population was estimated in the study. METHODS: Concentrations of 17 polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and 12 dioxin-like polychlorinated biphenyls (PCBs) as well as 6 marker PCBs were measured in 11 varieties of food groups in total 110 food samples by isotope dilution HRGC/HRMS. PCDD/Fs and PCBs dietary intake for local population was estimated using total WHO-TEQ concentration and different food consumption amount of the local population. RESULTS: The median WHO-TEQ concentrations of sum of PCDD/Fs and DL-PCBs varied from 0.0093 pg/g (parts per trillion) in vegetable to 9.89 pg/g in fish. Fish was followed by egg 2.46, chicken 1.73, beef 0.94, mutton 0.82, duck 0.39, pork 0.37, milk powder 0.25, vegetable oil 0.076, cereals 0.022 pg/g and vegetable. The monthly intake of PCDD/Fs and PCBs were 40.9 pg WHO-TEQ/kg b.w. for local population. Sum of fish, livestock, and poultry contributed 77% to the Estimated Monthly Intake (EMI) in local population. CONCLUSION: Estimated dietary intake of PCDD/Fs and DL-PCBs for local population was below the provisional tolerable monthly intake(PTMI) set by Joint FAO/WHO Expert Committee on Food Additives (JECFA).

[Levels of PCDD/Fs, PCBs and PBDEs compounds in human placenta tissue].

OBJECTIVE: To establish the methods of Polychlorinated Dibenzo-p-Dioxins and Dibenzofurans (PCDD/Fs), polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) compounds determination by isotope dilution HRGC/HRMS simultaneously in human placenta tissue from mothers, and assess the human exposure risk to dioxins and PBDEs in study. METHODS: Concentrations of 17 PCDD/Fs and 12 dioxin-like PCBs as well as 7 PBDEs were measured in human placenta tissue samples by isotope dilution HRGC/HRMS. SigmaTEQ (PCDD + PCDFs + PCBs) concentration using WHO-TEF factor and PBDEs concentration was calculated respectively. Risk assessment of mother exposure to dioxins and PBDEs was evaluated. RESULTS: Median of SigmaTEQ (PCDD + PCDFs + PCBs) concentration for six samples was 18.15 WHO-TEQ pg/g lipid, ranging from 5.14 - 67.01 WHO-TEQ pg/g lipid. Although the median of SigmaTEQ (PCDD + PCDFs + PCBs) was lower than that of human blood of EU and Japan, and close to that of Korea and Taiwan non-exposure as reported in the literatures, the highest SigmaTEQ (PCDD + PCDFs + PCBs) concentration of placenta sample exceeded the value of high dioxins exposure area subjects in Taiwan. The dominant contributor congener for WHO-TEQ were 2, 3, 4, 7, 8-PeCDF, 1, 2, 3, 7, 8-PeCDD, PCB126, totally accounted for 65 percent of SigmaWHO-TEQ. Median and average of PBDE concentration for six samples were 2.73 ng/g lipid and 7.17 ng/g lipid, respectively, ranging from 0.95 - 25.99 ng/g lipid. BDE47 was the dominant contributor congener for the total concentration, accounted for 35 percent. CONCLUSION: The methods of PCDD/Fs, PCBs and PBDEs compounds determined by isotope dilution HRGC/HRMS simultaneously in human placenta tissue from mothers were established successfully, and the human exposure risk to PCDD/Fs, PCBs and PBDEs should be surveyed for the donor with the highest SigmaTEQ (PCDD + PCDFs + PCBs) and PBDEs concentration of placenta sample in the future.

Possible role of DNA polymerase beta in protecting human bronchial epithelial cells against cytotoxicity of hydroquinone.

OBJECTIVE: To explore the toxicological mechanism of hydroquinone in human bronchial epithelial cells and to investigate whether DNA polymerase beta is involved in protecting cells from damage caused by hydroquinone. METHODS: DNA polymerase beta knock-down cell line was established via RNA interference as an experimental group. Normal human bronchial epithelial cells and cells transfected with the empty vector of pEGFP-C1 were used as controls. Cells were treated with different concentrations of hydroquinone (ranged from 10 micromol/L to 120 micromol/L) for 4 hours. MTT assay and Comet assay [single-cell gel electrophoresis (SCGE)] were performed respectively to detect the toxicity of hydroquinone. RESULTS: MTT assay showed that DNA polymerase beta knock-down cells treated with different concentrations of hydroquinone had a lower absorbance value at 490 nm than the control cells in a dose-dependant manner. Comet assay revealed that different concentrations of hydroquinone caused more severe DNA damage in DNA polymerase beta knock-down cell line than in control cells and there was no significant difference in the two control groups. CONCLUSIONS: Hydroquinone has significant toxicity to human bronchial epithelial cells and causes DNA damage. DNA polymerase beta knock-down cell line appears more sensitive to hydroquinone than the control cells. The results suggest that DNA polymerase beta is involved in protecting cells from damage caused by hydroquinone.

[Construction of "pEGFP-C1-pU6-dsRNA" recombinant for human DNA polymerase beta RNA interference].

OBJECTIVE: To clone the "pEGFP-C1-pU6-dsRNA" recombinant for human DNA polymerase beta RNA interference, to provide research tool for the study on the function of DNA polymerase beta in repairing of human DNA damaged by environmental chemical pollutants (ECPs). METHODS: According to the gene sequence of polymerase beta cDNA published in Genbank, double strand RNA(dsRNA) sequence which was used in RNA interference was designed by dsRNA oligonucleotide designer and synthesized by chemical methods. DNA recombination technology was used to insert the up related dsRNA sequence into the vector of pSIREN-RetroQ, and then the "pSIREN-RetroQ-dsRNA" recombinant was obtained. After E. coli DH5alpha was transformed with the "pSIREN-RetroQ-dsRNA" recombinant and screened with ampicillin for positive clones, plasmid was extracted and digested by EcoR I and Bgl II , the fragment of"pU6-dsRNA"was purified. And then the "pU6-dsRNA"fragment was cloned into the vector of pEGFP-C1 by recombination technology, the recombinant of "pEGFP-C1-pU6-dsRNA" was obtained and identified by restriction endonuclease analysis and sequencing. RESULTS: The "pEGFP-C1-pU6-dsRNA" recombinant lied in the predicted band, and the sequence of insert was identical to the designed target fragment. CONCLUSION: The "pEGFP-C1-pU6-dsRNA" recombinant was successfully cloned for human DNA polymerase beta RNA interference, it was an important research tool for the further study.