LAYN Serves as a Prognostic Biomarker and Downregulates Tumor-Infiltrating CD8+ T Cell Function in Hepatocellular Carcinoma.

Background: Layilin (LAYN) represents a valuable prognostic biomarker across various tumor types, while also serving as an innovative indicator of dysfunctional or exhausted CD8+ T cells and exhibiting correlation with immune context. However, the immune function and prognostic significance of LAYN in hepatocellular carcinoma (HCC) remain unexplored. Therefore, our objective is to investigate the role of LAYN in CD8+ T cell exhaustion, clinical prognosis, and the tumor microenvironment within HCC. Methods: TIMER or GEPIA databases were used to analyze LAYN expression level and its correlation with immune infiltration in HCC. Bioinformatics analysis was conducted on TCGA and scRNA-seq cohorts. The evaluation of LAYN expression level in fresh specimens was performed through IF, IHC, and ELISA assays. Flow cytometry and mRNA-seq were employed to investigate co-expressed genes of LAYN, the LAYN+CD8+ T cell exhaustion signature and immune function. Cell proliferation ability and killing activity were assessed using CCK8 and CFSE/PI. Results: The expression level of LAYN in HCC tumors was significantly higher compared to peri-tumors. Patients with high levels of LAYN exhibited poorer OS. GO or KEGG analysis confirmed that LAYN was involved in immune response and was positively associated with CD8+ T cell immune infiltration levels. Furthermore, LAYN negatively regulated the immune function of CD8+ T cells, leading to dysfunctional phenotypes characterized by elevated levels of CD39, TIM3 and reduced levels of perforin, TNF-α, Ki-67. CFSE/PI assays demonstrated that LAYN+CD8+ T cells displayed decreased cytotoxic activity. Additionally, there was a positive correlation between LAYN and CD146 levels, which are involved in adhesion and localization processes of CD8+ T cells. Interestingly, blocking LAYN partially restored the exhaustion properties of CD8+ T cells. Conclusion: LAYN exhibits a strong correlation with immune infiltration in the TME and represents a novel biomarker for predicting clinical prognosis in HCC. Moreover, targeting LAYN may hold promise as an effective strategy for HCC immunotherapy.

Significantly Enhanced Nitrate and Phosphorus Removal by Pyrite/Sawdust Composite-Driven Mixotrophic Denitrification with Boosted Electron Transfer: Comprehensive Evaluation of Water-Gas-Biofilm Phases during a Long-Term Study.

Further reducing total nitrogen (TN) and total phosphorus (TP) in the secondary effluent needs to be realized effectively and in an eco-friendly manner. Herein, four pyrite/sawdust composite-based biofilters were established to treat simulated secondary effluent for 304 days. The results demonstrated that effluent TN and TP concentrations from biofilters under the optimal hydraulic retention time (HRT) of 3.5 h were stable at <2.0 and 0.1 mg/L, respectively, and no significant differences were observed between inoculated sludge sources. The pyrite/sawdust composite-based biofilters had low N2O, CH4, and CO2 emissions, and the effluent's DOM was mainly composed of five fluorescence components. Moreover, mixotrophic denitrifiers (Thiothrix) and sulfate-reducing bacteria (Desulfosporosinus) contributing to microbial nitrogen and sulfur cycles were enriched in the biofilm. Co-occurrence network analysis deciphered that Chlorobaculum and Desulfobacterales were key genera, which formed an obvious sulfur cycle process that strengthened the denitrification capacity. The higher abundances of genes encoding extracellular electron transport (EET) chains/mediators revealed that pyrite not only functioned as an electron conduit to stimulate direct interspecies electron transfer by flagella but also facilitated EET-associated enzymes for denitrification. This study comprehensively evaluates the water-gas-biofilm phases of pyrite/sawdust composite-based biofilters during a long-term study, providing an in-depth understanding of boosted electron transfer in pyrite-based mixotrophic denitrification systems.

Epstein-Barr virus-driven metabolic alterations contribute to the viral lytic reactivation and tumor progression in nasopharyngeal carcinoma.

Metabolic reprogramming induced by Epstein-Barr virus (EBV) often mirrors metabolic changes observed in cancer cells. Accumulating evidence suggests that lytic reactivation is crucial in EBV-associated oncogenesis. The aim of this study was to explore the role of metabolite changes in EBV-associated malignancies and viral life cycle control. We first revealed that EBV (LMP1) accelerates the secretion of the oncometabolite D-2HG, and serum D-2HG level is a potential diagnostic biomarker for NPC. EBV (LMP1)-driven metabolite changes disrupts the homeostasis of global DNA methylation and demethylation, which have a significantly inhibitory effect on active DNA demethylation and 5hmC content. We found that loss of 5hmC indicates a poor prognosis for NPC patients, and that 5hmC modification is a restriction factor of EBV reactivation. We confirmed a novel EBV reactivation inhibitor, α-KG, which inhibits the expression of EBV lytic genes with CpG-containing ZREs and the latent-lytic switch by enhancing 5hmC modification. Our results demonstrate a novel mechanism of which metabolite abnormality driven by EBV controls the viral lytic reactivation through epigenetic modification. This study presents a potential strategy for blocking EBV reactivation, and provides potential targets for the diagnosis and therapy of NPC.

Aptamers: Promising Reagents in Biomedicine Application.

Nucleic acid aptamers, often termed "chemical antibodies," are short, single-stranded DNA or RNA molecules, which are selected by SELEX. In addition to their high specificity and affinity comparable to traditional antibodies, aptamers have numerous unique advantages such as wider identification of targets, none or low batch-to-batch variations, versatile chemical modifications, rapid mass production, and lack of immunogenicity. These characteristics make aptamers a promising recognition probe for scientific research or even clinical application. Aptamer-functionalized nanomaterials are now emerged as a promising drug delivery system for various diseases with decreased side-effects and improved efficacy. In this review, the technological strategies for generating high-affinity and biostable aptamers are introduced. Moreover, the development of aptamers for their application in biomedicine including aptamer-based biosensors, aptamer-drug conjugates and aptamer functionalized nanomaterials is comprehensively summarized.

miR-612 Enhances RSL3-Induced Ferroptosis of Hepatocellular Carcinoma Cells via Mevalonate Pathway.

Background: MicroRNA-612 (miR-612) has been proven to suppress the formation of invadopodia and inhibit hepatocellular carcinoma (HCC) metastasis by hydroxyacyl-CoA dehydrogenase alpha subunit (HADHA)-mediated lipid reprogramming. However, its biological roles in HCC cell ferroptosis remain unclear. Methods and Results: In this study, we found that HCC cells with high metastatic potential were more resistant to ferroptosis, indicating that ferroptosis is related to HCC metastasis. The levels of lipid reactive oxygen species (ROS) were found to be much lower in HCC cells with high metastatic potential by flow cytometry (FCM). We used HCC cells with miR-612 overexpression/knockout and HADHA overexpression/knockdown to test cell viability after stimulation with RSL3. HCC cells overexpressing miR-612 were more sensitive to ferroptosis, and miR-612 could increase lipid ROS levels. Furthermore, colony formation assays and Transwell assays showed that miR-612 could inhibit the proliferation and metastasis of HCC cells by promoting ferroptosis. We next confirmed that miR-612 influenced HCC cell ferroptosis by regulating HADHA. HADHA could upregulate the expression of key enzymes in the mevalonate (MVA) pathway. HADHA overexpression upregulated the expression of CoQ10 and decreased polyunsaturated fatty acid (PUFA) levels and lipid peroxide abundance. miR-612 also suppressed HCC cell proliferation and metastasis by enhancing RSL3- and lovastatin-induced ferroptosis in vivo. Conclusion: Overall, miR-612 promotes ferroptosis in HCC cells and affects HCC proliferation and metastasis by downregulating CoQ10 and increasing cellular PUFA levels and lipid peroxides via the HADHA-mediated MVA pathway.

Neoantigen-targeted TCR-engineered T cell immunotherapy: current advances and challenges.

Adoptive cell therapy using T cell receptor-engineered T cells (TCR-T) is a promising approach for cancer therapy with an expectation of no significant side effects. In the human body, mature T cells are armed with an incredible diversity of T cell receptors (TCRs) that theoretically react to the variety of random mutations generated by tumor cells. The outcomes, however, of current clinical trials using TCR-T cell therapies are not very successful especially involving solid tumors. The therapy still faces numerous challenges in the efficient screening of tumor-specific antigens and their cognate TCRs. In this review, we first introduce TCR structure-based antigen recognition and signaling, then describe recent advances in neoantigens and their specific TCR screening technologies, and finally summarize ongoing clinical trials of TCR-T therapies against neoantigens. More importantly, we also present the current challenges of TCR-T cell-based immunotherapies, e.g., the safety of viral vectors, the mismatch of T cell receptor, the impediment of suppressive tumor microenvironment. Finally, we highlight new insights and directions for personalized TCR-T therapy.

GPC3-IL7-CCL19-CAR-T primes immune microenvironment reconstitution for hepatocellular carcinoma therapy.

BACKGROUND: Chimeric antigen receptor (CAR)-T-cell therapy is a revolutionary treatment that has become a mainstay of advanced cancer treatment. Conventional glypican-3 (GPC3)-CAR-T cells have not produced ideal clinical outcomes in advanced hepatocellular carcinoma (HCC), and the mechanism is unclear. This study aims to investigate the clinical utility of novel GPC3-7-19-CAR-T cells constructed by our team and to explore the mechanisms underlying their antitumor effects. METHODS: We engineered a novel GPC3-targeting CAR including an anti-GPC3 scFv, CD3ζ, CD28 and 4-1BB that induces co-expression of IL-7 at a moderate level (500 pg/mL) and CCL19 at a high level (15000 pg /mL) and transduced it into human T cells. In vitro, cell killing efficacy was validated by the xCELLigence RTCA system, LDH nonradioactive cytotoxicity assay and was confirmed in primary HCC organoid models employing a 3D microfluid chip. In vivo, the antitumor capacity was assessed in a humanized NSG mouse xenograft model. Finally, we initiated a phase I clinical trial to evaluate the safety and effect of GPC3-7-19-CAR-T cells in the clinic. RESULTS: GPC3-7-19-CAR-T cells had 1.5-2 times higher killing efficiency than GPC3-CAR-T cells. The tumor formation rates in GPC3-7-19-CAR-T cells treated model were reduced (3/5vs.5/5), and the average tumor volumes were 0.74 cm3 ± 1.17 vs. 0.34 cm3 ± 0.25. Of note, increased proportion of CD4+ TEM and CD8+ TCM cells was infiltrated in GPC3-7-19-CAR-T cells group. GPC3-7-19-CAR-T cells obviously reversed the immunosuppressive tumor microenvironment (TME) by reducing polymorphonuclear (PMN)-myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells infiltration and recruiting more dendritic cells (DCs) to HCC xenograft tumor tissues. In one patient with advanced HCC, GPC3-7-19-CAR-T-cell treatment resulted in tumor reduction 56 days after intravenous infusion. CONCLUSIONS: In conclusion, GPC3-7-19-CAR-T cells achieved antitumor effects superior to those of conventional GPC3-CAR-T cells by reconstructing the TME induced by the dominant CD4+ TEM and CD8+ TCM cell subsets. Most importantly, GPC3-7-19-CAR-T cells exhibited good safety and antitumor efficacy in HCC patients in the clinic. ► Novel GPC3-7-19-CAR-T cells designed with mediate level of IL-7 secretion and high level of CCL19 secretion, which could recruit more mature DCs to assist killing on GPC3+HCCs. ►DC cells recruited by CCL19 could interact with CD4+ T cells and promote the differentiation of CD4+TEFF cells into CD4+TEM and CD8+TCM subsets, leading a better anti-tumor effect on GPC3+HCCs. ►Compared with conventional GPC3-CAR-T, GPC3-7-CCL19-CAR-T cells could reverse tumor immunosuppressive microenvironment by reducing PMN-MDSC and Treg cell infiltration.

Application of manganese oxides in wastewater treatment: Biogeochemical Mn cycling driven by bacteria.

Manganese oxides (MnOx) are recognized as a strongest oxidant and adsorbent, of which composites have been proved to be effective in the removal of contaminants from wastewater. This review provides a comprehensive analysis of Mn biochemistry in water environment including Mn oxidation and Mn reduction. The recent research on the application of MnOx in the wastewater treatment was summarized, including the involvement of organic micropollutant degradation, the transformation of nitrogen and phosphorus, the fate of sulfur and the methane mitigation. In addition to the adsorption capacity, the Mn cycling mediated by Mn(II) oxidizing bacteria and Mn(IV) reducing bacteria is the driving force for the MnOx utilization. The common category, characteristics and functions of Mn microorganisms in recent studies were also reviewed. Finally, the discussion on the influence factors, microbial response, reaction mechanism and potential risk of MnOx application in pollutants' transformation were proposed, which might be the promising opportunities for the future investigation of MnOx application in wastewater treatment.

Genomic and transcriptomic profiling reveals key molecules in metastatic potentials and organ-tropisms of hepatocellular carcinoma.

Metastasis is a landmark event for rapid postsurgical relapse and death of HCC patients. Although distinct genomic and transcriptomic profiling of HCC metastasis had been reported previously, the causal relationships of somatic mutants, mRNA levels and metastatic potentials were difficult to be established in clinic. Therefore, 11 human HCC cell lines and 7 monoclonal derivatives with definite metastatic potentials and tropisms were subjected to whole exome sequencing (WES) and whole transcriptome sequencing (WTS). TP53, MYO5A, ROS1 and ARID2 were the prominent mutants of metastatic drivers in HCC cells. During HCC clonal evaluation, TP53, MYO5A and ROS1 mutations occurred in the early stage, EXT2 and NIN in the late stage. NF1 mutant was unique in lung tropistic cell lines, RNF126 mutant in lymphatic tropistic ones. PER1, LMO2, GAS7, NR4A3 expression levels were positively associated with relapse-free survival (RFS) of HCC patients. The integrative analysis revealed 58 genes exhibited both somatic mutation and dysregulated mRNA levels in high metastatic cells. Altogether, metastatic drivers could accumulate gradually at different stages during HCC progression, some drivers might modulate HCC metastatic potentials and the others regulate metastatic tropisms.

Effect of Conventional and "Dental Truth or Dare" Board Game on Oral Hygiene Knowledge and Oral Hygiene Status of Preschool Children.

Aim: To compare the benefits of didactic versus board game-based oral health instruction on oral health knowledge (OHK) and oral hygiene of preschool students. Materials and Methods: Participants were selected through computer-assisted randomization. (Eighty students were selected in both the 3- to 4-year-old and 5- to 6-year-old age groups, respectively, for a total of 160 participants). Forty participants of each age group were assigned randomly to Group A (PowerPoint® presentation) and 40 to Group B ("Dental Truth or Dare" board game-based instruction). OHK and debris index-simplified (DI-S) were assessed at preintervention, and at 1-week, 1-month, and 3-month postintervention timepoints. Results: OHK scores increased significantly in the 3- to 4-year-old subset of Group A at the 1-week postintervention timepoint but declined and approximated the baseline value at the 3-month timepoint. In contrast, compared to baseline, significantly improved OHK scores were observed at all 3 timepoints in both age groups in Group B, and were especially pronounced in the 5- to 6-year-old subset. Although the 3-month scores were slightly lower than the 1-week scores, they were well above baseline values. Pre- and postintervention DI-S scores did not change significantly in the 3- to 4-year-old subset of Group A. However, significant increases in good DI-S scores and decreases in fair and poor scores were observed between baseline and 3-month timepoints in the 5- to 6-year-old subset of Group A and in both age subsets of Group B (P ≤ 0.05). OHK and DI-S scores were significantly higher among 5-6-year-olds than among the 3-4-year olds in both Groups A and B (P ≤ 0.05). Age and board game intervention were the main determinants of higher OHK and lower DI-S scores. The impact of intervention mode (board game) was greater than that of age. Conclusion: Board game-based oral hygiene education conferred significant short-term retention, enhanced OHK, and reduced DI-S. We conclude that gaming is an easily implemented and cost-effective educational tool for the improvement of oral hygiene in preschool children.

Integrated treatment of suburb diffuse pollution using large-scale multistage constructed wetlands based on novel solid carbon: Nutrients removal and microbial interactions.

In this study, an integrated treatment system was proposed and applied in situ, including detention tank, multistage constructed wetlands (CWs) and wastewater treatment plants (WWTPs), preventing nutrients flowing into Dianchi Lake, in which the treatment performance of multistage CWs were evaluated principally. Results skillfully realized the bypass purification of upstream river at dry reasons, as well as the effective management and treatment of the collected diffuse pollution at rainy reasons. The purified water flowing into water bodies could satisfy the Grade III of environmental quality standards for surface water in China with the average effluent concentrations of COD, NH4+-N, TN and TP decreased to 10 (51.2-72.7%), 0.5 (67.2-83.0%), 1.0 (71.2-79.6%) and 0.15 (72.3-89.4%) mg L-1, respectively. High-throughput sequencing results indicated that the application of poly-3-hydroxybutyrate-cohyroxyvelate-sawdust (PS) blends could enrich norank_f_Anaerolineaceae (7.95%) and Bradyrhizobium (10.2%), which were distinct from the dominant genera of Pleurocapsa (13.0%) in gravel-based CWs. Functional genes and metabolism analysis uncovered that the heterotrophic denitrification was the main pathway of nitrogen removal with the abundance of genes encoding TCA cycle, glycolysis and denitrification process up-regulated. In addition, molecular ecological network (MEN) analysis suggested the denitrification genes were positively correlated with the predominant microbes in PS-based CWs, favorable for denitrifiers to transfer and utilize electron donors during denitrification process. This study proved that the developed PS blends as carbon supplies in CWs and the proposed integrated treatment system are effective methods for watershed management, providing valuable reference to low-pollution wastewater treatment in practical engineering projects.

Erratum: Targeting Epstein-Barr virus oncoprotein LMP1-mediated high oxidative stress suppresses EBV lytic reactivation and sensitizes tumors to radiation therapy: Erratum.

[This corrects the article DOI: 10.7150/thno.46006.].

CPT1A-mediated fatty acid oxidation promotes cell proliferation via nucleoside metabolism in nasopharyngeal carcinoma.

As the first rate-limiting enzyme in fatty acid oxidation (FAO), CPT1 plays a significant role in metabolic adaptation in cancer pathogenesis. FAO provides an alternative energy supply for cancer cells and is required for cancer cell survival. Given the high proliferation rate of cancer cells, nucleotide synthesis gains prominence in rapidly proliferating cells. In the present study, we found that CPT1A is a determining factor for the abnormal activation of FAO in nasopharyngeal carcinoma (NPC) cells. CPT1A is highly expressed in NPC cells and biopsies. CPT1A dramatically affects the malignant phenotypes in NPC, including proliferation, anchorage-independent growth, and tumor formation ability in nude mice. Moreover, an increased level of CPT1A promotes core metabolic pathways to generate ATP, inducing equivalents and the main precursors for nucleotide biosynthesis. Knockdown of CPT1A markedly lowers the fraction of 13C-palmitate-derived carbons into pyrimidine. Periodic activation of CPT1A increases the content of nucleoside metabolic intermediates promoting cell cycle progression in NPC cells. Targeting CPT1A-mediated FAO hinders the cell cycle G1/S transition. Our work verified that CPT1A links FAO to cell cycle progression in NPC cellular proliferation, which supplements additional experimental evidence for developing a therapeutic mechanism based on manipulating lipid metabolism.

An Immune-Related Gene Signature Predicting Prognosis and Immunotherapy Response in Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is inflammation-associated cancer with high incidence and poor prognosis. In the last decade, immunotherapy has become an important strategy for managing HCC. OBJECTIVE: This study aimed to establish an immune-related gene signature for predicting prognosis and immunotherapy response in HCC. METHODS: We identified immune-related differentially expressed genes (IRDEGs) based on The Cancer Genome Atlas (TCGA) database and the Immunology Database and Analysis Portal (ImmPort) database. The weighted gene co-expression network analysis (WGCNA) and Cox proportional hazard model were utilized to determine hub immune-related genes (IRGs). The TIDE tool and R package pRRophetic were used to assess the correlation between the immune-related gene signature and the clinical responses to immunotherapy and chemotherapy. RESULTS: By using WGCNA combined with Cox proportional hazard model, PRC1, TOP2A, TPX2, and ANLN were identified as hub IRGs. The prognostic value of the newly developed gene signature (IRGPI) was demonstrated in both the TCGA database and the Gene Expression Omnibus (GEO) database. The TIDE tool showed that the high- and low-IRGPI groups presented significantly different tumor immune microenvironment and immunotherapy responses. Furthermore, the high-IRGPI group also had significantly lower chemoresistance to cisplatin than the low-IRGPI group. CONCLUSION: The IRGPI is a tool for predicting prognosis as well as responsiveness to immunotherapy and chemotherapy in HCC.

Simultaneous biological removal of nitrogen and phosphorus from secondary effluent of wastewater treatment plants by advanced treatment: A review.

With the advancement of water ecological protection and water control standard, it is the general trend to upgrade the wastewater treatment plants (WWTPs). The simultaneous removal of nitrogen and phosphorus is the key to improve the water quality of secondary effluent of WWTPs to prevent the eutrophication. Therefore, it is urgent to develop the applicable technologies for simultaneous biological removal of nitrogen and phosphorus from secondary effluent. In this review, the composition of secondary effluent from municipal WWTPs were briefly introduced firstly, then the three main treatment processes for simultaneous nitrogen and phosphorus removal, i.e., the enhanced denitrifying phosphorus removal filter, the pyrite-based autotrophic denitrification and the microalgae biological treatment system were summarized, their performances and mechanisms were analyzed. The influencing factors and microbial community structure were discussed. The advanced removal of nitrogen and phosphorus by different technologies were also compared and summarized in terms of performance, operational characteristics, disadvantage and cost. Finally, the challenges and future prospects of simultaneous removal of nitrogen and phosphorus technologies for secondary effluent were proposed. This review will deepen to understand the principles and applications of the advanced removal of nitrogen and phosphorus and provide some valuable information for upgrading the treatment process of WWTPs.

Simultaneously advanced removal of nitrogen and phosphorus in a biofilter packed with ZVI/PHBV/sawdust composite: Deciphering the succession of dominant bacteria and keystone species.

In this study, a biofilter was developed with a ZVI/PHBV/sawdust (ZPS) composite for treating simulative secondary effluent from wastewater treatment plants. Results showed that effluent concentrations of NO3--N and TP in the ZPS biofilter were stable below 2.0 mg/L and 0.1 mg/L, corresponding to 95% NO3--N removal and 99% TP removal, respectively. Microbial community analysis revealed that the transformation of dominant taxa from Dechloromonas to Clostridium sensu stricto_7 from 30 d to 120 d suggested that the ZVI-induced succession of dominant fermentation bacteria ensured the stable carbon supply for denitrification. Co-occurrence network analysis showed that the ZVI directly enhanced the interaction of microbial community. Fe-related bacteria occupied a key position in the rare species, which might maintain the function of iron-mediated organic matter decomposition and denitrification. These findings provide an alternative for advanced removal of nitrogen and phosphorus in biofilters packed with ZPS composites.

Insight into heavy metals (Cr and Pb) complexation by dissolved organic matters from biochar: Impact of zero-valent iron.

In this study, batch experiments were conducted to investigate the immobilization of HMs (Cr and Pb) by DOM derived from biochar in the presence and absence of zero-valent iron (Fe) in nitrate and HMs co-contaminated groundwater. Both Cr and Pb were removed effectively in biochar-Fe aqueous systems, while only Pb could be mitigated in biochar systems. Excitation-emission spectrophotometry combined with parallel factor analysis (EEM-PARAFAC) revealed that DOM released from biochar mainly contained human-like and tryptophan-like substances. Moreover, the fluorescence of hemic-like components could be quenched differently by the complexation of HMs, which proved the different removal efficiencies of Cr and Pb in biochar aqueous phase. In biochar-Fe aqueous systems, Fe-C micro-electrolysis was formed in prior to the complexation of DOM-Fe hydroxides. Thus, the chemical reduction was the primary way to removal HMs in batch-Fe systems, which was corresponding with the less variation of DOM components when adding Cr and Pb into aqueous systems. Besides, the observed DOM components with higher aromaticity and humification after adding Cr and Pb, further indicated the complexation of DOM-HMs through the analysis of adsorption and fluorescence indices. These results will provide new insights into the HMs retention on biochar, particularly for the role of Fe on the complexation process.

Enhanced pollutant removal from rural non-point source wastewater using a two-stage multi-soil-layering system with blended carbon sources: Insights into functional genes, microbial community structure and metabolic function.

A two-stage multi-soil-layering system with blended carbon sources (MSL-BCS) was constructed at pilot scale for treatment of rural non-point source wastewater. Results showed the MSL-BCS system had effective removal efficiencies with 64% of TN and 60% of TP, respectively. The addition of BCS could result in higher (1.6-3.1 fold) denitrification gene abundances (nirS and nosZ) for enhancing denitrification. High-throughput sequencing approach revealed that the higher abundance (>50%) of Epsilonbacteraeotra (Genus: Sulfuricurvum, Family: Thiovulaceae, Class: Campylobacteria, Phylum: Epsilonbacteraeota) enriched in the surface of BCS, which suggested that Epsilonbacteraeotra are the keystone species in achieving nitrogen removal through enhancing denitrification at oligotrophic level. KEGG analysis indicated that BCS might release some signaling molecules for enhancing the energy metabolism process, as well as stimulate the enzyme activities of histidine kinase, glycogen phosphorylase and ATPase, and thereby the denitrification processes were strengthened in MSL-BCS system. Consequently, this study could provide some valuable information on the removal performance and mechanism of engineering MSL systems packed with BCS to govern the rural wastewater treatment.

Pilot-scale two-stage constructed wetlands based on novel solid carbon for rural wastewater treatment in southern China: Enhanced nitrogen removal and mechanism.

Constructed wetlands (CWs) have been proved to be an alternative to the treatment of various wastewater. However, there are few studies focused on the removal performance and mechanisms of pollutants in pilot-scale CWs packed with novel solid carbon. In this study, we investigated the effect of poly-3-hydroxybutyrate-co-3-hydroxyvalerate/polyacetic acid (PHBV/PLA) blends as carbon source on pollutant's transformation, microbial communities and functional genes in pilot-scale aeration-anoxic two-stage CWs for polishing rural runoff in southern China. Results showed a striking improvement of TN removal in CWs with PHBV/PLA blends (64.5%) compared to that in CWs with ceramsite (52.9%). NH4+-N (61.3-64.6%), COD (40.4-53.8%) and TP (43.6-47.1%) were also removed effectively in both two CWs. In addition, the strains of Rhodocyclaceae and Bacteroidetes were the primary denitrifiers on the surface of PHBV/PLA blends. Further, the aerobic stage induced gathering of 16 S and amoA genes and the anoxic zone with PHBV/PLA blends increased the nirS genes, which fundamentally explained the better denitrification performance in CW based on PHBV/PLA blends. Consequently, this study will provide straightforward guidance for the operation of engineering CWs packed with polymers to govern the low-C/N rural wastewater.