A practical study on the near-zero discharge of rainwater and the collaborative treatment and regeneration of rainwater and sewage.

Non-conventional water recovery, recycling, and reuse have been considered imperative approaches to addressing water scarcity in China. The objective of this study was to evaluate the technical and economic feasibility of Water Reclamation Plants (WRP) based on an anaerobic-anoxic-oxic membrane bioreactor (A2O-MBR) system for unconventional water resource treatment and reuse in towns (domestic sewage and rainwater). Rainwater is collected and stored in the rainwater reservoir through the rainwater pipe network, and then transported to the WRP for treatment and reuse through the rainwater reuse pumping station during the peak water demand period. During a year of operation and evaluation process, a total of 610,000 cubic meters of rainwater were reused, accounting for 10.4 % of the treated wastewater. In the A2O-MBR operation, the average effluent concentrations for COD (chemical oxygen demand), NH4+-N (ammonium), TN (total nitrogen), and TP (total phosphorus) were 14.23 ± 4.07 mg/L, 0.22 ± 0.26 mg/L, 11.97 ± 1.54 mg/L, and 0.13 ± 0.09 mg/L, respectively. The effluent quality met standards suitable for reuse in industrial cooling water or for direct discharge. The WRP demonstrates a positive financial outlook, with total capital and operating costs totaling 0.16 $/m3. A comprehensive cost-benefit analysis indicates a positive net present value for the WRP, and the estimated annualized net profit is 0.024 $/m3. This research has achieved near-zero discharge of wastewater and effective allocation of rainwater resources across time and space.

Acetylation of MLH1 by CBP increases cellular DNA mismatch repair activity.

The DNA mismatch repair (MMR) proteins recognize and repair DNA base pair mismatches and insertions/deletions of DNA that have occurred during DNA replication. Additionally, they are involved in regulation of the DNA damage response, including cell cycle checkpoints and apoptosis. Therefore, regulation of these proteins is essential for maintaining genomic integrity. It has been recognized that post-translational modifications, such as phosphorylation, ubiquitination, and acetylation, are being used as an important means to regulate the functions and stability of MMR proteins. Here, we report that a histone acetyltransferase CREB binding protein (CBP) interacts with and acetylates MLH1, a component of the MutLα complex (MLH1-PMS2). Moreover, CBP stabilizes MLH1 by preventing it from degradation via the ubiquitin-proteasome degradation pathway. Consistently, acetylation induced by a pan-histone deacetylase inhibitor, Trichostatin A, promotes the assembly between the MutSα (MSH2-MSH6) and MutLα complexes. Furthermore, overexpression of CBP enhances MMR activities in cells. Overall, our results suggest a novel role of CBP in prolonging MLH1 stability and enhancing MutSα-MutLα complex formation, leading to increased cellular MMR activity.

Enhanced Electrochemical Performance of Aprotic Li-CO2 Batteries with a Ruthenium-Complex-Based Mobile Catalyst.

Li-CO2 batteries are regarded as next-generation high-energy-density electrochemical devices. However, the greatest challenge arises from the formation of the discharge product, Li2 CO3 , which would accumulate and deactivate heterogenous catalysts to cause huge polarization. Herein, Ru(bpy)3 Cl2 was employed as a solution-phase catalyst for Li-CO2 batteries and proved to be the most effective one screened so far. Spectroscopy and electrochemical analyses elucidate that the RuII center could interact with both CO2 and amorphous Li2 C2 O4 intermediate, thus promoting electroreduction process and delaying carbonate transformation. As a result, the charge potential is reduced to 3.86 V and over 60 discharge/charge cycles are achieved with a fixed capacity of 1000 mAh g-1 at a current density of 300 mA g-1 . Our work provides a new avenue to improve the electrochemical performance of Li-CO2 batteries with efficient mobile catalysts.

ID1 inhibits foot-and-mouth disease virus replication via targeting of interferon pathways.

Inhibitor of DNA-binding 1 (ID1) protein has been studied intensively for its functions in tumorigenesis and maintenance of stem cell-like properties, but its roles in virus infection are less understood. In the present study, we have clearly shown that the foot-and-mouth disease virus (FMDV) promotes ID1 degradation via Cdh1-mediated ubiquitination to facilitate its replication. Mechanistic investigations reveal Forkhead Box O1 (FOXO1) as an ID1 partner, which suppresses interferon regulatory factors 3 expression and interferon (IFN) production. Further investigation identified that ID1 suppresses FOXO1 transcription activity through HDAC4-mediated deacetylation, promoting IFN production and antiviral immune response. These studies establish a prominent role for ID1 in suppressing FDMV replication, which may be extended to other viruses.

HDAC6 Regulates Radiosensitivity of Non-Small Cell Lung Cancer by Promoting Degradation of Chk1.

We have previously discovered that HDAC6 regulates the DNA damage response (DDR) via modulating the homeostasis of a DNA mismatch repair protein, MSH2, through HDAC6's ubiquitin E3 ligase activity. Here, we have reported HDAC6's second potential E3 ligase substrate, a critical cell cycle checkpoint protein, Chk1. We have found that HDAC6 and Chk1 directly interact, and that HDAC6 ubiquitinates Chk1 in vivo and in vitro. Specifically, HDAC6 interacts with Chk1 via the DAC1 domain, which contains its ubiquitin E3 ligase activity. During the cell cycle, Chk1 protein levels fluctuate, peaking at the G2 phase, subsequently resolving via the ubiquitin-proteasome pathway, and thereby allowing cells to progress to the M phase. However, in HDAC6 knockdown non-small cell lung cancer (NSCLC) cells, Chk1 is constitutively active and fails to resolve post-ionizing radiation (IR), and this enhanced Chk1 activity leads to preferential G2 arrest in HDAC6 knockdown cells accompanied by a reduction in colony formation capacity and viability. Depletion or pharmacological inhibition of Chk1 in HDAC6 knockdown cells reverses this radiosensitive phenotype, suggesting that the radiosensitivity of HDAC6 knockdown cells is dependent on increased Chk1 kinase activity. Overall, our results highlight a novel mechanism of Chk1 regulation at the post-translational level, and a possible strategy for sensitizing NSCLC to radiation via inhibiting HDAC6's E3 ligase activity.

Phosphazene-derived stable and robust artificial SEI for protecting lithium anodes of Li-O2 batteries.

A stable artificial solid electrolyte interphase (ASEI) containing phosphazene and perfluoroalkoxy groups was designed to protect Li anodes. The ASEI with high ionic conductivity and mechanical robustness successfully suppressed the growth of Li dendrites, significantly enhancing the electrochemical performance of the Li-O2 batteries.

Esophageal Histological Precursor Lesions and Subsequent 8.5-Year Cancer Risk in a Population-Based Prospective Study in China.

INTRODUCTION: Data on the associations between esophageal histological lesions and risk of esophageal squamous cell carcinoma (ESCC) in general populations are limited. We aimed to investigate these associations in a large Chinese general population to inform future Chinese ESCC screening guidelines. METHODS: We performed endoscopic screening of 21,111 participants aged 40-69 years from 3 high-risk areas of China in 2005-2009, and followed the cohort through 2016. Cumulative incidence and mortality rates of ESCC were calculated by baseline histological diagnosis, and hazard ratios of ESCC, overall and by age and sex, were assessed using the Cox proportional hazards models. RESULTS: We identified 143 new ESCC cases (0.68%) and 62 ESCC deaths (0.29%) during a median follow-up of 8.5 years. Increasing grades of squamous dysplasia were associated with the increasing risk of ESCC incidence and mortality. The cumulative ESCC incidence rates for severe dysplasia/carcinoma in situ, moderate dysplasia (MD), and mild dysplasia were 15.5%, 4.5%, and 1.4%, respectively. Older individuals (50-69 years) had 3.1 times higher ESCC incidence than younger individuals (40-49 years), and men had 2.4 times higher ESCC incidence than women. DISCUSSION: This study confirmed that increasing grades of squamous dysplasia are associated with increasing risk of ESCC and that severe dysplasia and carcinoma in situ require clinical treatment. This study suggests that in high-risk areas of China, patients with endoscopically worrisome MD should also receive therapy, the first screening can be postponed to 50 years, and endoscopic surveillance intervals for unremarkable MD and mild dysplasia can be lengthened to 3 and 5 years, respectively.

The USP10-HDAC6 axis confers cisplatin resistance in non-small cell lung cancer lacking wild-type p53.

Ubiquitin-specific peptidase 10 (USP10) stabilizes both tumor suppressors and oncogenes in a context-dependent manner. However, the nature of USP10's role in non-small cell lung cancer (NSCLC) remains unclear. By analyzing The Cancer Genome Atlas (TCGA) database, we have shown that high levels of USP10 are associated with poor overall survival in NSCLC with mutant p53, but not with wild-type p53. Consistently, genetic depletion or pharmacological inhibition of USP10 dramatically reduces the growth of lung cancer xenografts lacking wild-type p53 and sensitizes them to cisplatin. Mechanistically, USP10 interacts with, deubiquitinates, and stabilizes oncogenic protein histone deacetylase 6 (HDAC6). Furthermore, reintroducing either USP10 or HDAC6 into a USP10-knockdown NSCLC H1299 cell line with null-p53 renders cisplatin resistance. This result suggests the existence of a "USP10-HDAC6-cisplatin resistance" axis. Clinically, we have found a positive correlation between USP10 and HDAC6 expression in a cohort of NSCLC patient samples. Moreover, we have shown that high levels of USP10 mRNA correlate with poor overall survival in a cohort of advanced NSCLC patients who received platinum-based chemotherapy. Overall, our studies suggest that USP10 could be a potential biomarker for predicting patient response to platinum, and that targeting USP10 could sensitize lung cancer patients lacking wild-type p53 to platinum-based therapy.

HDAC6 regulates DNA damage response via deacetylating MLH1.

MutL homolog 1 (MLH1) is a key DNA mismatch repair protein, which plays an important role in maintenance of genomic stability and the DNA damage response. Here, we report that MLH1 is a novel substrate of histone deacetylase 6 (HDAC6). HDAC6 interacts with and deacetylates MLH1 both in vitro and in vivo Interestingly, deacetylation of MLH1 blocks the assembly of the MutSα-MutLα complex. Moreover, we have identified four novel acetylation sites in MLH1 by MS analysis. The deacetylation mimetic mutant, but not the WT and the acetylation mimetic mutant, of MLH1 confers resistance to 6-thioguanine. Overall, our findings suggest that the MutSα-MutLα complex serves as a sensor for DNA damage response and that HDAC6 disrupts the MutSα-MutLα complex by deacetylation of MLH1, leading to the tolerance of DNA damage.

Histone deacetylase 6 (HDAC6) deacetylates extracellular signal-regulated kinase 1 (ERK1) and thereby stimulates ERK1 activity.

Histone deacetylase 6 (HDAC6), a class IIb HDAC, plays an important role in many biological and pathological processes. Previously, we found that ERK1, a downstream kinase in the mitogen-activated protein kinase signaling pathway, phosphorylates HDAC6, thereby increasing HDAC6-mediated deacetylation of α-tubulin. However, whether HDAC6 reciprocally modulates ERK1 activity is unknown. Here, we report that both ERK1 and -2 are acetylated and that HDAC6 promotes ERK1 activity via deacetylation. Briefly, we found that both ERK1 and -2 physically interact with HDAC6. Endogenous ERK1/2 acetylation levels increased upon treatment with a pan-HDAC inhibitor, an HDAC6-specific inhibitor, or depletion of HDAC6, suggesting that HDAC6 deacetylates ERK1/2. We also noted that the acetyltransferases CREB-binding protein and p300 both can acetylate ERK1/2. Acetylated ERK1 exhibits reduced enzymatic activity toward the transcription factor ELK1, a well-known ERK1 substrate. Furthermore, mass spectrometry analysis indicated Lys-72 as an acetylation site in the ERK1 N terminus, adjacent to Lys-71, which binds to ATP, suggesting that acetylation status of Lys-72 may affect ERK1 ATP binding. Interestingly, an acetylation-mimicking ERK1 mutant (K72Q) exhibited less phosphorylation than the WT enzyme and a deacetylation-mimicking mutant (K72R). Of note, the K72Q mutant displayed decreased enzymatic activity in an in vitro kinase assay and in a cellular luciferase assay compared with the WT and K72R mutant. Taken together, our findings suggest that HDAC6 stimulates ERK1 activity. Along with our previous report that ERK1 promotes HDAC6 activity, we propose that HDAC6 and ERK1 may form a positive feed-forward loop, which might play a role in cancer.

Implications of the HDAC6-ERK1 feed-forward loop in immunotherapy.

The oncogene HDAC6 controls numerous cell processes that are related to tumorigenesis and metastasis, and has recently arisen as a target to treat malignancies. The ERK cascade is a classic pathway driving oncogenesis, and the components of this pathway are either highly mutated in cancers or are vital in cancer's pathological activity. The interactions between these important components of tumor proliferation have been examined, and our research has demonstrated that they regulate each other as evidenced by different posttranslational modifications. Preclinical evidence also supports clinical trials cotargeting these two pathways, which may provide better efficacy than single treatment. Furthermore, HDAC6 and ERK both participate in the regulation of T cell maturation and may have implications on the functions of immune cells. This leads to the possibility of connecting HDAC6 and ERK to immunotherapy. In this review, we summarize the published studies about the interaction of HDAC6 and ERK cascade and their relationship to cancers. We also include the association of HDAC6 and ERK to immune system and discuss the plausibility of linking these to immunotherapy.

RIPK1 binds to vitamin D receptor and decreases vitamin D-induced growth suppression.

Receptor interacting protein kinase 1 (RIPK1) is an enzyme acting downstream of tumor necrosis factor alpha to control cell survival and death. RIPK1 expression has been reported to cause drug resistance in cancer cells, but so far, no published studies have investigated the role of RIPK1 in vitamin D signaling. In the present study, we investigated whether RIPK1 plays any roles in 1,25-dihydroxyvitamin D3 (1,25D3)-induced growth suppression. In our studies, RIPK1 decreased the transcriptional activity of vitamin D receptor (VDR) in luciferase reporter assays independent of its kinase activity, suggesting a negative role of RIPK1 in 1,25D3 action. RIPK1 also formed a complex with VDR, and deletion analyses mapped the RIPK1 binding region to the C-terminal ligand-binding domain of the VDR. Subcellular fractionation analyses indicated that RIPK1 increased VDR retention in the cytoplasm, which may account for its inhibition of VDR transcriptional activity. Consistent with the reporter analyses, 1,25D3-induced growth suppression was more pronounced in RIPK1-null MEFs and RIPK1-knockdown ovarian cancer cells than in control cells. Our studies have defined RIPK1 as a VDR repressor, projecting RIPK1 depletion as a potential strategy to increase the potency of 1,25D3 and its analogs for cancer intervention.

Ubiquitin-specific Peptidase 10 (USP10) Deubiquitinates and Stabilizes MutS Homolog 2 (MSH2) to Regulate Cellular Sensitivity to DNA Damage.

MSH2 is a key DNA mismatch repair protein, which plays an important role in genomic stability. In addition to its DNA repair function, MSH2 serves as a sensor for DNA base analogs-provoked DNA replication errors and binds to various DNA damage-induced adducts to trigger cell cycle arrest or apoptosis. Loss or depletion of MSH2 from cells renders resistance to certain DNA-damaging agents. Therefore, the level of MSH2 determines DNA damage response. Previous studies showed that the level of MSH2 protein is modulated by the ubiquitin-proteasome pathway, and histone deacetylase 6 (HDAC6) serves as an ubiquitin E3 ligase. However, the deubiquitinating enzymes, which regulate MSH2 remain unknown. Here we report that ubiquitin-specific peptidase 10 (USP10) interacts with and stabilizes MSH2. USP10 deubiquitinates MSH2 in vitro and in vivo Moreover, the protein level of MSH2 is positively correlated with the USP10 protein level in a panel of lung cancer cell lines. Knockdown of USP10 in lung cancer cells exhibits increased cell survival and decreased apoptosis upon the treatment of DNA-methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and antimetabolite 6-thioguanine (6-TG). The above phenotypes can be rescued by ectopic expression of MSH2. In addition, knockdown of MSH2 decreases the cellular mismatch repair activity. Overall, our results suggest a novel USP10-MSH2 pathway regulating DNA damage response and DNA mismatch repair.

Fe65 Suppresses Breast Cancer Cell Migration and Invasion through Tip60 Mediated Cortactin Acetylation.

Fe65 is a brain-enriched adaptor protein known for its role in the action of the Aß amyloid precursor protein in neuronal cells and Alzheimer's disease, but little is known about its functions in cancer cells. The present study documents for the first time a role of Fe65 in suppressing breast cancer cell migration and invasion. Mechanistic studies suggest that the suppression is mediated through its phosphotyrosine binding domain 1 that mediates the recruitment of Tip60 to cortactin to stimulate its acetylation. The studies identify the Tip60 acetyltransferase as a cytoplasmic drug target for the therapeutic intervention of metastatic breast cancers.

HDAC6-Dependent Functions in Tumor Cells: Crossroad with the MAPK Pathways.

Histone deacetylase 6 (HDAC6) is emerging as a novel therapeutic target in cancer treatment. HDAC6 plays an important role in cell migration, cell transformation, and DNA damage response. Our and others' studies have linked HDAC6's functions and HDAC6's regulation to the mitogen-activated protein kinase (MAPK) pathways. In particular, HDAC6's activity has been found to be regulated by EGF-EGFR-Ras-Raf-MEK-ERK signaling. Inversely, HDAC6 has been reported to modulate the functions of EGFR and Ras. In this review, we summarize the literature on HDAC6 and MAPK pathways, and emphasize the interaction between HDAC6 and the ERK-MAPK signaling cascade.

Suppression of epithelial ovarian cancer invasion into the omentum by 1α,25-dihydroxyvitamin D3 and its receptor.

Epithelial ovarian cancer (EOC) is the leading cause of gynecological cancer death in women, mainly because it has spread to intraperitoneal tissues such as the omentum in the peritoneal cavity by the time of diagnosis. In the present study, we established in vitro assays, ex vivo omental organ culture system and syngeneic animal tumor models using wild type (WT) and vitamin D receptor (VDR) null mice to investigate the effects of 1α,25-dihydroxyvitamin D3 (1,25D3) and VDR on EOC invasion. Treatment of human EOC cells with 1,25D3 suppressed their migration and invasion in monolayer scratch and transwell assays and ability to colonize the omentum in the ex vivo system, supporting a role for epithelial VDR in interfering with EOC invasion. Furthermore, VDR knockdown in OVCAR3 cells increased their ability to colonize the omentum in the ex vivo system in the absence of 1,25D3, showing a potential ligand-independent suppression of EOC invasion by epithelial VDR. In syngeneic models, ID8 tumors exhibited an increased ability to colonize omenta of VDR null over that of WT mice; pre-treatment of WT, not VDR null, mice with EB1089 reduced ID8 colonization, revealing a role for stromal VDR in suppressing EOC invasion. These studies are the first to demonstrate a role for epithelial and stromal VDR in mediating the activity of 1,25D3 as well as a 1,25D3-independent action of the VDR in suppressing EOC invasion. The data suggest that VDR-based drug discovery may lead to the development of new intervention strategies to improve the survival of patients with EOC at advanced stages. This article is part of a Special Issue entitled "Vitamin D Workshop".

Potential role of the OPG/RANK/RANKL axis in prostate cancer invasion and bone metastasis.

Receptor activator of NF-κB (RANK), RANK ligand (RANKL) and osteoprotegerin (OPG) are key regulators of bone metabolism under both normal and pathological conditions, including prostate cancer (PCa) bone metastases. However, little is known concerning the expression and function of these regulators in prostate tumor samples and PCa cells and their correlation with invasion and bone metastasis. In the present study, we determined the expression of RANK, RANKL and OPG in 3 human PCa cell lines and 40 PCa patient samples by immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). As controls, samples from 20 patients with benign prostate hyperplasia (BPH) and normal prostate epithelial RWPE2 cells were also included in the analyses. The effects of soluble RANKL (sRANKL) and OPG as well as RANK knockdown on PCa invasion were examined in Transwell assays. Immunohistochemical staining detected little RANK, OPG and RANKL expression in hyperplasia prostate while the percentages of positivity were increased to 50, 45 and 52.5%, respectively, in prostate tumor tissues. OPG and sRANKL levels in the prostate tumor samples as measured by ELISA were ~10-fold that in the BPHs (P<0.01) and the levels were higher in aggressive tumors than non-aggressive ones (P<0.05). The sRANKL level in the serum of PCa patients was the same as that in the patients with BPH, yet the serum OPG levels correlated with the tissue levels (R2=0.620, P<0.01, which both showed a 10-fold increase in PCa over BPH (P<0.01) with higher levels in aggressive PCa than non-aggressive ones (P<0.05). Consistent with the tissue analyses, expression levels of RANK mRNA and protein were detected in multiple human PCa cell lines by RT-PCR and western blotting, respectively. The treatment of PCa cells with RANKL significantly increased the number of invaded cells (P<0.01), which was suppressed by the decoy receptor OPG. RANK siRNA transfection dramatically dampened the stimulatory effect of RANKL on PCa cell invasion. Our findings indicate that the expression of RANK, RANKL and OPG may be used as diagnostic markers to identify patients at high risk for aggressive PCa and that the effective suppression of PCa cell migration by OPG via the blockage of RANKL activity represents a potential therapeutic strategy for interfering with prostate tumor metastasis and progression to bone.

Vitamin D suppresses leptin stimulation of cancer growth through microRNA.

Obesity is a pandemic and major risk factor for cancers. The reduction of obesity would have been an effective strategy for cancer prevention, but the reality is that worldwide obesity has kept increasing for decades, remaining a major avoidable cancer risk secondary only to smoke. The present studies suggest that vitamin D may be an effective agent to reduce obesity-associated cancer risks in women. Molecular analyses showed that leptin increased human telomerase reverse transcriptase (hTERT) mRNA expression and cell growth through estrogen receptor-α (ERα) activation in ovarian cancer cells, which was suppressed by 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3]. The suppression was compromised when miR-498 induction by the hormone was depleted with microRNA (miRNA) sponges. In mice, high-fat diet (HFD) stimulation of ovarian tumor growth was remarkably suppressed by 1,25(OH)2D3 analogue EB1089, which was also compromised by miR-498 sponges. EB1089 did not alter HFD-induced increase in serum leptin levels but increased miR-498 and decreased the diet-induced hTERT expression in tumors. Quantitative RT-PCR analyses revealed an inverse correlation between hTERT mRNA and miR-498 in response to 1,25(OH)2D3 in estrogen-sensitive ovarian, endometrial, and breast cancers. The studies suggest that miR-498-mediated hTERT downregulation is a key event mediating the anti-leptin activity of 1,25(OH)2D3 in estrogen-sensitive tumors in women.

HDAC6 deacetylates and ubiquitinates MSH2 to maintain proper levels of MutSα.

MutS protein homolog 2 (MSH2) is a key DNA mismatch repair protein. It forms the MSH2-MSH6 (MutSα) and MSH2-MSH3 (MutSß) heterodimers, which help to ensure genomic integrity. MutSα not only recognizes and repairs mismatched nucleotides but also recognizes DNA adducts induced by DNA-damaging agents, and triggers cell-cycle arrest and apoptosis. Loss or depletion of MutSα from cells leads to microsatellite instability (MSI) and resistance to DNA damage. Although the level of MutSα can be reduced by the ubiquitin-proteasome pathway, the detailed mechanisms of this regulation remain elusive. Here we report that histone deacetylase 6 (HDAC6) sequentially deacetylates and ubiquitinates MSH2, leading to MSH2 degradation. In addition, HDAC6 significantly reduces cellular sensitivity to DNA-damaging agents and decreases cellular DNA mismatch repair activities by downregulation of MSH2. Overall, these findings reveal a mechanism by which proper levels of MutSα are maintained.