ERK/PKM2 Is Mediated in the Warburg Effect and Cell Proliferation in Arsenic-Induced Human L-02 Hepatocytes.

This study aimed to investigate the potential role of pyruvate kinase M2 (PKM2) and extracellular regulated protein kinase (ERK) in arsenic-induced cell proliferation. L-02 cells were treated with 0.2 and 0.4 µmol/L As3+, glycolysis inhibitor (2-deoxy-D-glucose,2-DG), ERK inhibitor [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)-butadiene, U0126] or transfected with PKM2 plasmid. Cell viability, proliferation, lactate acid production, and glucose intake capacity were determined by CCK-8 assay, EdU assay, lactic acid kit and 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-D-glucose (2-NBDG) uptake kit, respectively. Also, levels of PKM2, phospho-PKM2S37, glucose transporter protein 1 (GLUT1), lactate dehydrogenase A (LDHA), ERK, and phospho-ERK were detected using Western blot and the subcellular localization of PKM2 in L-02 cells was detected by immunocytochemistry (ICC). Treatment with 0.2 and 0.4 µmol/L As3+ for 48 h increased the viability and proliferation of L-02 cells, the proportion of 2-NBDG+ cell and lactic acid in the culture medium, and GLUT1, LDHA, PKM2, phospho-PKM2S37, and phospho-ERK levels and PKM2 in nucleus. Compared with the 0.2 µmol/L As3+ treatment group, the lactic acid in the culture medium, cell proliferation and cell viability, and the expression of GLUT1 and LDHA were reduced in the group co-treated with siRNA-PKM2 and arsenic or in the group co-treated with U0126. Moreover, the arsenic-increased phospho-PKM2S37/PKM2 was decreased by U0126. Therefore, ERK/PKM2 plays a key role in the Warburg effect and proliferation of L-02 cells induced by arsenic, and also might be involved in arsenic-induced upregulation of GLUT1 and LDHA. This study provides a theoretical basis for further elucidating the carcinogenic mechanism of arsenic.

Association between arsenic (+3 oxidation state) methyltransferase gene polymorphisms and arsenic methylation capacity in rural residents of northern China: a cross-sectional study.

Arsenic is a toxic metal-like element. The toxic reaction of the body to arsenic is related to the ability of arsenic methylation metabolism. As the rate-limiting enzyme of arsenic methylation metabolism, the genetic single nucleotide polymorphisms (SNPs) of arsenic (+ 3 oxidation state) methyltransferase (AS3MT) gene are related to capacity of arsenic methylation. In this paper, we investigated the association of five SNPs (rs7085104, rs3740390, 3740393, rs10748835, and rs1046778) in AS3MT with arsenic methylation metabolizing using the data and samples from a cross-sectional case-control study of arsenic and Type 2 diabetes mellitus conducted in Shanxi, China. A total of 340 individuals were included in the study. Urinary total arsenic (tAs, µg/L) was detected by liquid chromatography-atomic fluorescence spectrometry (LC-AFS). According to "safety guidance value of urinary arsenic for population" as specified in WS/T665-2019 (China), participants were divided into the control group (tAs ≤ 32 µg/L, n = 172) and arsenic-exposed group (tAs > 32 µg/L, n = 168). iAs%, MMA%, and DMA% are as the indicator of arsenic methylation capacity. The genotypes of AS3MT SNPs were examined by Multiple PCR combined sequencing. Linear regression analysis showed that AG + GG genotype in rs7085104 was associated with decreased iAs% and increased DMA%. Moreover, AG + AA genotype in rs10748835 and TC + CC genotype in rs1046778 were associated with decreased iAs% and MMA% and increased DMA%. The interaction between rs7085104 and arsenic is associated with iAs% and DMA%. The interaction of rs3740390 and rs10748835 with arsenic is associated with iAs%. Haplotype CTAC (rs3740393-rs3740390-rs10748835-rs1046778) was associated with lower iAs% and higher DMA%, but this association disappeared after adjusting for age, gender, drink, smoking, BMI and tAs. Haplotype GCAC was associated with decreased MMA%. Our study provides additional support for revealing the factors influencing the metabolic capacity of arsenic methylation and might be helpful to identify the population susceptible to arsenic exposure through individualized screening in the future.

DELs enable the development of BRET probes for target engagement studies in cells.

DNA-encoded libraries (DELs) provide unmatched chemical diversity and starting points for novel drug modalities. Here, we describe a workflow that exploits the bifunctional attributes of DEL ligands as a platform to generate BRET probes for live cell target engagement studies. To establish proof of concept, we performed a DEL screen using aurora kinase A and successfully converted aurora DEL ligands as cell-active BRET probes. Aurora BRET probes enabled the validation and stratification of the chemical series identified from primary selection data. Furthermore, we have evaluated the effective repurposing of pre-existing DEL screen data to find suitable leads for BRET probe development. Our findings support the use of DEL workflows as an engine to create cell-active BRET probes independent of structure or compound SAR. The combination of DEL and BRET technology accelerates hit-to-lead studies in a live cell setting.

SIRT1/P53 pathway is involved in the Arsenic induced aerobic glycolysis in hepatocytes L-02 cells.

Arsenic is a known human carcinogen. Low doses of arsenic can induce cell proliferation, but the mechanism remains elusive. Aerobic glycolysis, also known as the Warburg effect, is one of the characteristics of tumour cells and rapidly proliferating cells. P53 is a tumour suppressor gene that has been shown to be a negative regulator of aerobic glycolysis. SIRT1 is a deacetylase that inhibits the function of P53. In this study, we found that P53 was involved in low dose of arsenic-induced aerobic glycolysis through regulating HK2 expression in L-02 cells. Moreover, SIRT1 not only inhibited P53 expression but also decreased the acetylation level of P53-K382 in arsenic-treated L-02 cells. Meanwhile, SIRT1 influenced the expression of HK2 and LDHA, which then promoted arsenic-induced glycolysis in L-02 cells. Therefore, our study demonstrated that the SIRT1/P53 pathway is involved in arsenic-induced glycolysis, thereby promoting cell proliferation, which provides theoretical basis for enriching the mechanism of arsenic carcinogenesis.

Malignant growth of arsenic-transformed cells depends on activated Akt induced by reactive oxygen species.

Arsenic is an identified carcinogen for humans.In this study, chronic exposure of human hepatocyte L-02 to low-doses of inorganic arsenic caused cell malignant proliferation. Meanwhile, compared with normal L-02 cells, arsenic-transformed malignant cells, L-02-As displayed more ROS and significantly higher Cyclin D1 expression as well as aerobic glycolysis. Moreover, Akt activation is followed by the upregulation of Cyclin D1 and HK2 expression in L-02-As cells, since inhibition of Akt activity by Ly294002 attenuated the colony formation in soft agar and decreased the levels of Cyclin D1 and HK2. In addition, scavenging of ROS by NAC resulted in a decreased expression of phospho-Akt, HK2 and Cyclin D1, and attenuates the ability of anchorage-independent growth ofL-02-As cells, suggested that ROS mediated the Akt activation in L-02-As cells. In summary, our results demonstrated that ROS contributes to the malignant phenotype of arsenic-transformed human hepatocyte L-02-As via the activation of Akt pathway.

The ROS/NF-κB/HK2 axis is involved in the arsenic-induced Warburg effect in human L-02 hepatocytes.

Arsenic has been identified as a carcinogen, although the molecular mechanism underlying itscarcinogenesis has not been fully elucidated. To date, only a few studies have attempted to confirm a direct link between oxidative stress and the Warburg effect . This study demonstrated that 0.2 µmol/L As3+ induced the Warburg effect to contribute to abnormal proliferation of L-02 cells, that was mediated by upregulation of hexokinase 2 (HK2), a key enzyme in glycolysis. Further study indicated that arsenic-induced accumulation of reactive oxygen species (ROS) activated the nuclear factor kappa B (NF-κB) signaling pathway by phosphorylation of p65 at the Ser536 and Ser276 sites, leading to upregulated expression of HK2. We therefore concluded that the ROS/NF-κB/HK2 axis contributes to the Warburg effect and cell proliferation induced by low doses of arsenic.AbbreviationsROS, Reactive oxygen species; NAC, N-acetyl-L-cysteine; 2-DG, 2-deoxy-D-glucose; 2-NBDG, 2-Deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose.

Exploring English for medical purposes (EMP) teacher cognition in the Chinese context.

It has been a growing trend in Chinese universities to shift from English for general purposes (EGP) to English for specific purposes (ESP) teaching. Against this background, large groups of teachers previously engaged in teaching EGP have become or are becoming ESP teachers, which means a complex process of learning for subject-specific information, transforming teaching practices and constructing new identities. Despite this, very little has been written about the ESP teacher cognition (TC) of language teaching or the factors influencing this shift in teaching. This study involved English for Medical Purposes (EMP) teachers in Chinese universities as participants, and a scale of EMP TC with 31 items was developed on the basis of questionnaire results. Data from exploratory factor analysis revealed six dimensions of the scale, namely, teacher attitude, teacher belief, teacher learning, teacher support, role identification, and teacher practice-that combine to constitute and influence EMP TC. While the identity factor has attracted wide attention in ESP teacher research, other factors have largely been neglected. Thus, this research highlights the importance of more factors in shaping and changing the language teaching cognition of EMP or ESP teachers in large, especially the teacher belief factor. In addition, results of independent samples t-tests indicated significant difference in EMP teacher learning in terms of gender, differences in EMP teacher attitude and teacher support in terms of EMP teaching experience. Suggestions for enhancing EMP TC are offered on the basis of the conclusions of this research.

Arsenic exposure elevated ROS promotes energy metabolic reprogramming with enhanced AKT-dependent HK2 expression.

Exposure to inorganic or organic arsenic compounds continues to pose substantial public health concerns for hundreds of millions of people around the globe. Highly exposed individuals are susceptible to various illnesses, including impairments and cancers of the lung, liver, skin and bladder. Long-term exposure to low-dose arsenic has been identified to induce aerobic glycolysis, which contributes to cells aberrant proliferation. However, the mechanism underlying arsenic-induced aerobic glycolysis is still unclear. Here, mtDNA copy number is enhanced in arsenic-exposed populations and a positive correlation between serum HK2 and urinary total arsenic was observed in the individuals with high urine arsenic (≥ 0.032 mg/L). In a rat model of trivalent arsenic (iAs3+) exposure, the levels of HK2, NDUFA9 and NDUFB8 were increased in the rats treated with iAs3+ daily by gavage for 12 weeks than those in the control rats. Subsequently, in a low-dose arsenic exposure cell model we found that 0.2 µmol/L iAs3+ induced aerobic glycolysis to promote L-02 cells proliferation and inhibit apoptosis, in which HK2 played an important role. Further studies showed accumulated ROS determined the metabolic reprogramming via activating AKT and then increasing HK2 expression. On the one hand, activated AKT induced aerobic glycolysis by increasing HK2 to promote L-02 cells viability and DNA synthesis; on the other hand, phosphorylated AKT induced HK2 mitochondrial outer-membrane location with VDAC1 to inhibit apoptosis. Taken together, our results indicated that ROS induced by low-dose arsenic exposure determined energy metabolic reprogramming and acted a critical regulator for AKT-dependent HK2 expression and aerobic glycolysis.

Association between fluoride exposure and kidney function in adults: A cross-sectional study based on endemic fluorosis area in China.

BACKGROUND: The kidney toxicity of fluoride exposure has been demonstrated in animal studies, and a few studies have reported kidney function injury in children with fluoride exposure. However, epidemiological information for the effects of long-term fluoride exposure on adult kidney function remains limited. METHODS: We conducted a cross-sectional investigation in Wenshui County, Shanxi Province to examine the association between fluoride exposure and kidney function in adults, and a total of 1070 adults were included in our study. Urinary fluoride concentrations were measured using the national standardized ion selective electrode method. And markers of kidney function injury (urinary NAG, serum RBP, serum Urea, serum C3, serum UA and serum αl-MG) were measured using automatic biochemical analyzer. Multivariate linear regression analysis and binary logistic regression model were used to assess the relationship between urinary fluoride and markers of kidney function injury. RESULTS: Urinary fluoride was positively correlated with urinary NAG and serum Urea, negatively correlated with serum C3. In multivariate linear regression models, every 1 mg/L increment of urinary fluoride was associated with 1.583 U/L increase in urinary NAG, 0.199 mmol/L increase in serum Urea, 0.037 g/L decrease in serum C3 after adjusting for potential confounding factors. In the binary logistic regression model, higher levels of urinary fluoride were associated with an increased risk of kidney function injury. Determination of kidney function based on urinary NAG, every 1 mg/L increment in the urinary fluoride concentrations was associated with significant increases of 22.8% in the risk of kidney function injury after adjusting for potential confounding factors. Sensitivity analysis for the association between urinary fluoride concentrations and markers of kidney function (urinary NAG, serum Urea, and serum C3) by adjusting for the covariates, it is consistent with the primary analysis. CONCLUSIONS: Our study suggests that long-term fluoride exposure is associated with kidney function in adults, and urinary NAG is a sensitive and robust marker of kidney dysfunction caused by fluoride exposure, which could be considered for the identification of early kidney injury in endemic fluorosis areas.

sKlotho is associated with the severity of brick tea-type skeletal fluorosis in China.

The change of serum soluble Klotho (sKlotho) content is related to a variety of osteoarthropathy. However, its association with the severity of skeletal fluorosis (SF) is not clear. Here, the association of tea fluoride exposure with serum sKlotho levels and the severity of SF were investigated and further verified in a rat model of fluorosis. A cross sectional case control study was conducted in residents over 50 years old from brick-tea drinking areas in Qinghai and Xinjiang Provinces, China. Concentrations of fluoride in brick tea water and urine were determined by ion selective electrode method, and the levels of serum sKlotho were determined by ELISA method. Linear regression and ordered logistic regression models were constructed to examine the relationship among fluoride exposure, serum sKlotho levels and the severity of SF. The kidney and small intestine of Wistar rats were isolated for detection of Klotho by immunohistochemistry (IHC), and femoral artery blood was sampled to measure the serum levels of sKlotho. An increase of 1 mg/day in tea fluoride intake (TFI) was associated with a 12.070 pg/mL (95% CI: 0.452-23.689) increase in serum sKlotho levels and a 1.163-fold (95% CI: 1.007-1.342) increase in the severity of SF after adjusting for age, gender, and ethnicity. Serum sKlotho levels were also positively associated with the severity of SF (P < 0.05). The mediation analysis showed that serum sKlotho levels mediated 17.76% of the increase in the severity of SF caused by an increase of 1 mg/day of TFI. Moreover, a significant increase of serum sKlotho levels in fluoride-exposed groups was also seen in the rat model. The present study suggests that serum sKlotho may be a potential mediator of SF in brick tea-type fluorosis endemic areas.

Modeling system for predicting enterococci levels at Holly Beach.

This paper presents a new modeling system for nowcasting and forecasting enterococci levels in coastal recreation waters at any time during the day. The modeling system consists of (1) an artificial neural network (ANN) model for predicting the enterococci level at sunrise time, (2) a clear-sky solar radiation and turbidity correction to the ANN model, (3) remote sensing algorithms for turbidity, and (4) nowcasting/forecasting data. The first three components are also unique features of the new modeling system. While the component (1) is useful to beach monitoring programs requiring enterococci levels in early morning, the component (2) in combination with the component (1) makes it possible to predict the bacterial level in beach waters at any time during the day if the data from the components (3) and (4) are available. Therefore, predictions from the component (2) are of primary interest to beachgoers. The modeling system was developed using three years of swimming season data and validated using additional four years of independent data. Testing results showed that (1) the sunrise-time model correctly reproduced 82.63% of the advisories issued in seven years with a false positive rate of 2.65% and a false negative rate of 14.72%, and (2) the new modeling system was capable of predicting the temporal variability in enterococci levels in beach waters, ranging from hourly changes to daily cycles. The results demonstrate the efficacy of the new modeling system in predicting enterococci levels in coastal beach waters. Applications of the modeling system will improve the management of recreational beaches and protection of public health.

Development of predictive models for determining enterococci levels at Gulf Coast beaches.

The US EPA BEACH Act requires beach managers to issue swimming advisories when water quality standards are exceeded. While a number of methods/models have been proposed to meet the BEACH Act requirement, no systematic comparisons of different methods against the same data series are available in terms of relative performance of existing methods. This study presents and compares three models for nowcasting and forecasting enterococci levels at Gulf Coast beaches in Louisiana, USA. One was developed using the artificial neural network (ANN) in MATLAB Toolbox and the other two were based on the US EPA Virtual Beach (VB) Program. A total of 944 sets of environmental and bacteriological data were utilized. The data were collected and analyzed weekly during the swimming season (May-October) at six sites of the Holly Beach by Louisiana Beach Monitoring Program in the six year period of May 2005-October 2010. The ANN model includes 15 readily available environmental variables such as salinity, water temperature, wind speed and direction, tide level and type, weather type, and various combinations of antecedent rainfalls. The ANN model was trained, validated, and tested using 308, 103, and 103 data sets (collected in 2007, 2008, and 2009) with an average linear correlation coefficient (LCC) of 0.857 and a Root Mean Square Error (RMSE) of 0.336. The two VB models, including a linear transformation-based model and a nonlinear transformation-based model, were constructed using the same data sets. The linear VB model with 6 input variables achieved an LCC of 0.230 and an RMSE of 1.302 while the nonlinear VB model with 5 input variables produced an LCC of 0.337 and an RMSE of 1.205. In order to assess the predictive performance of the ANN and VB models, hindcasting was conducted using a total of 430 sets of independent environmental and bacteriological data collected at six Holly Beach sites in 2005, 2006, and 2010. The hindcasting results show that the ANN model is capable of predicting enterococci levels at the Holly Beach sites with an adjusted RMSE of 0.803 and LCC of 0.320 while the adjusted RMSE and LCC values are 1.815 and 0.354 for the linear VB model and 1.961 and 0.521 for the nonlinear VB model. The results indicate that the ANN model with 15 parameters performs better than the VB models with 6 or 5 parameters in terms of RMSE while VB models perform better than the ANN model in terms of LCC. The predictive models (especially the ANN and the nonlinear VB models) developed in this study in combination with readily available real-time environmental and weather forecast data can be utilized to nowcast and forecast beach water quality, greatly reducing the potential risk of contaminated beach waters to human health and improving beach management. While the models were developed specifically for the Holly Beach, Louisiana, the methods used in this paper are generally applicable to other coastal beaches.

Synthesis and antibacterial evaluation of a series of oligorhamnoside derivatives.

A series of novel oligorhamnoside derivatives (1-10) and naturally occurring cleistrioside-5 were synthesized and evaluated for their in vitro antibacterial activities. Among them, dirhamnoside derivative 7 and cleistrioside-5 displayed similar antibacterial profiles and exhibited moderate to good inhibitory activities on bacterial growth against a panel of Gram-positive bacteria (MICs ≤ 4-32 µg/mL). The results revealed that these two compounds showed selectivity towards bacterial species strictly, without being affected by the antibiotic-resistant/susceptible properties of one species, which suggested that they might have the potential to avoid antibiotic cross-resistance. In addition, the preliminary SARs of this type of oligorhamnoside derivatives on the antibacterial activities were determined.

Total synthesis of caminoside B, a novel antimicrobial glycolipid isolated from the marine sponge Caminus sphaeroconia.

The first total synthesis of caminoside B, a novel marine antimicrobial glycolipid isolated from the marine sponge Caminus sphaeroconia, was developed. This marine small molecule inhibitor (IC(50)=20 microM) targeting type III secretory pathway of bacterial pathogenesis was assembled in good yield via a '2+2+1' strategy based on stereocontrolled construction of the four glycosidic linkages.

Discovery of the first series of small molecule H5N1 entry inhibitors.

The occurrence of highly pathogenic avian influenza virus H5N1 highlights the urgent need for new classes of antiviral drugs. Inhibition of H5N1 entry into cells may be an effective strategy. We report the first three small molecule inhibitors saponins with 3-O-beta-chacotriosyl residue, which showed potent inhibitory activity with IC(50) of 7.22-9.25 microM. The subsequent SAR studies showed the 3-O-beta-chacotriosyl residue was essential for the activity, and the aglycone structure also affected the activity.

Concise synthesis of clarhamnoside, a novel glycosphingolipid isolated from the marine sponge Agela clathrodes.

The first total synthesis of a novel alpha-galactoglycosphingolipid clarhamnoside has been achieved through a straightforward strategy. A thiogalactosyl donor with a benzylidene group at C-4 and C-6 and nonparticipating p-methoxybenzyl group at C-2 was successfully employed in the stereocontrolled syntheses of alpha-GalGSLs. The N-Phth-protected trifluoroacetimidate donor for terminal disaccharide was successfully applied in constructing the [GalNAc beta-(1-->6)-Gal] glycosidic linkage.

Total synthesis of cleistetroside-2, partially acetylated dodecanyl tetrarhamnoside derivative isolated from Cleistopholis patens and Cleistopholis glauca.

The total synthesis of a partially acetylated dodecanyl tetrarhamnoside derivative, cleistetroside-2, which was isolated from Cleistopholis patens and Cleistopholis glauca and showed significant in vitro antibacterial activity against the Gram-positive bacteria, was achieved for the first time.

Synthesis of the tetrasaccharide residue of clarhamnoside, a novel glycosphingolipid isolated from the marine sponge Agelas clathrodes.

A tetrasaccharide, alpha-l-Rhap-(1-->3)-beta-d-GalpNAc-(1-->6)-alpha-d-Galp-(1-->2)-alpha-d-Galp, the carbohydrate moiety of clarhamnoside isolated from the marine sponge Agelas clathrodes, was synthesized as its propyl glycoside via a convergent approach. The key steps to the synthetic strategy were the stereoselective construction of the reducing-end disaccharide alpha-d-Galp-(1-->2)-d-Galp (5) and efficient coupling with the terminal disaccharide alpha-l-Rhap-(1-->3)-d-GalpNAc building block, in which the N-phthalimido-protected trifluoroacetimidate 13 was proved to be an effective donor.