Gas explosion characteristics and spray control mechanism in underground square.

The spray system mechanism during a gas explosion in an underground square pipeline is complex. In this paper, the underground square of Fuxin City is selected as the research object. FLACS numerical simulation software is used to analyze the spatial and temporal distribution characteristics of a gas explosion in an underground square pipeline with an unopened spray system using combustion and combustion rate models. Different spray pressures were compared and analyzed to determine the optimal spray control pressure, and the spray system mechanism was clarified. The results revealed that the gas explosion overpressure is divided into the overpressure gentle, overpressure rising, and overpressure decay stages, corresponding to a trend of rapid growth and slow decline. The influence of spray pressure on the gas explosion exhibits a promotion-inhibition-promotion trend, corresponding to 0-0.2 MPa, 0.2-0.6 MPa, and 0.6-1.6 Mpa, respectively. The peak overpressure and overpressure propagation rates are the lowest at 0.6 MPa, and the explosion suppression effect is the most pronounced. The spray system mechanism varies with the explosion overpressure stages. Generally, the time to peak value, that is, the peak time, the overall duration of the explosion, and the duration of the explosion stage decrease, whereas the peak explosion overpressure decreases.

A Carbon-Caged Rhodamine Generating Nitrosoperoxycarbonate for Photoimmunotherapy.

Photoimmunotherapy is a promising cancer treatment modality. While potent 1-e- oxidative species are known to induce immunogenic cell death (ICD), they are also associated with unspecific oxidation and collateral tissue damage. This difficulty may be addressed by post-generation radical reinforcement. Namely, non-oxidative radicals are first generated and subsequently activated into powerful oxidative radicals to induce ICD. Here, we developed a photo-triggered molecular donor (NPCD565) of nitrosoperoxycarbonate (ONOOCO2 -), the first of its class to our knowledge, and further evaluated its feasibility for immunotherapy. Upon irradiation of NPCD565 by light within a broad spectral region from ultraviolet to red, ONOOCO2 - is released along with a bright rhodamine dye (RD565), whose fluorescence is a reliable and convenient build-in reporter for the localization, kinetics, and dose of ONOOCO2 - generation. Upon photolysis of NPCD565 in 4T1 cells, damage-associated molecular patterns (DAMPs) indicative of ICD were observed and confirmed to exhibit immunogenicity by induced maturation of dendritic cells. In vivo studies with a bilateral tumor-bearing mouse model showcased the potent tumor-killing capability of NPCD565 of the primary tumors and growth suppression of the distant tumors. This work unveils the potent immunogenicity of ONOOCO2 -, and its donor (NPCD565) has broad potential for photo-immunotherapy of cancer.

Ultrafast and Multichannel Generation of the Triplet State in DNA-Intercalated Gilvocarcin V for an Enhanced Photoaddition Reaction.

Gilvocarcin V (GV) is a natural antibiotic exhibiting excellent antitumor activities and remarkably low toxicity in near-ultraviolet or visible light-dependent treatment. Notwithstanding, the [2 + 2] cycloaddition reaction between GV and thymine has been proven to be the key for its function in photodynamic therapy, and crucial mechanistic details about such a reaction are poorly understood. In this study, the electronic relaxation pathways and photoaddition reaction are characterized by femto- to nanosecond time-resolved spectroscopy combined with quantum chemical calculation. Our results reveal that ultrafast intersystem crossing (<3 ps) leads to the population of a local triplet excited state in DNA-intercalated GV. Such a state can further induce the formation of a biradical state, which is identified as the important reactive precursor for photoaddition between GV and thymine. The overall photoaddition quantum efficiency is determined to be 11.57 ± 1.0%. These results are essential to the elucidation of the DNA photoaddition mechanism of C-aryl glycoside-based artificial photocytotoxic agents and could help further development of those medicines.

Ultrafast spectroscopy study of DNA photophysics after proflavine intercalation.

Proflavine (PF), an acridine DNA intercalating agent, has been widespread applied as an anti-microbial and topical antiseptic agent due to its ability to suppress DNA replication. On the other hand, various studies show that PF intercalation to DNA can increase photogenotoxicity and has potential chances to induce carcinomas of skin appendages. However, the effects of PF intercalation on the photophysical and photochemical properties of DNA have not been sufficiently explored. In this study, the excited state dynamics of the PF intercalated d(GC)9 • d(GC)9 and d(AT)9 • d(AT)9 DNA duplex are investigated in an aqueous buffer solution. Under 267 nm excitation, we observed ultrafast charge transfer (CT) between PF and d(GC)9 • d(GC)9 duplex, generating a CT state with an order of magnitude longer lifetime compared to that of the intrinsic excited state reported for the d(GC)9 • d(GC)9 duplex. In contrast, no excited state interaction was detected between PF and d(AT)9 • d(AT)9. Nevertheless, a localized triplet state with a lifetime over 5 µs was identified in the PF-d(AT)9 • d(AT)9 duplex.

The identification of signature genes and their relationship with immune cell infiltration in age-related macular degeneration.

BACKGROUND: Age-related macular degeneration (AMD) is a prevalent source of visual impairment among the elderly population, and its incidence has risen in tandem with the increasing longevity of humans. Despite the progress made with anti-VEGF therapy, clinical outcomes have proven to be unsatisfactory. METHOD: We obtained differentially expressed genes (DEGs) of AMD patients and healthy controls from the GEO database. GO and KEGG analyses were used to enrich the DEGs. Weighted gene coexpression network analysis (WGCNA) was used to identify modules related to AMD. SVM, random forest, and least absolute shrinkage and selection operator (LASSO) were employed to screen hub genes. Gene set enrichment analysis (GSEA) was used to explore the pathways in which these hub genes were enriched. CIBERSORT was utilized to analyze the relationship between the hub genes and immune cell infiltration. Finally, Western blotting and RT‒PCR were used to explore the expression of hub genes in AMD mice. RESULTS: We screened 1084 DEGs in GSE29801, of which 496 genes were upregulated. These 1084 DEGs were introduced into the WGCNA, and 94 genes related to AMD were obtained. Seventy-nine overlapping genes were obtained by the Venn plot. These 79 genes were introduced into three machine-learning methods to screen the hub genes, and the genes identified by the three methods were TNC, FAP, SREBF1, and TGF-ß2. We verified their diagnostic function in the GSE29801 and GSE103060 datasets. Then, the hub gene co-enrichment pathways were obtained by GO and KEGG analyses. CIBERSORT analysis showed that these hub genes were associated with immune cell infiltration. Finally, we found increased expression of TNC, FAP, SREBF1, and TGF-ß2 mRNA and protein in the retinas of AMD mice. CONCLUSION: We found that four hub genes, namely, FAP, TGF-ß2, SREBF1, and TNC, have diagnostic significance in patients with AMD and are related to immune cell infiltration. Finally, we determined that the mRNA and protein expression of these hub genes was upregulated in the retinas of AMD mice.

Bacteria-loaded biochar for the immobilization of cadmium in an alkaline-polluted soil.

The combination of biochar and bacteria is a promising strategy for the remediation of Cd-polluted soils. However, the synergistic mechanisms of biochar and bacteria for Cd immobilization remain unclear. In this study, the experiments were conducted to evaluate the effects of the combination of biochar and Pseudomonas sp. AN-B15, on Cd immobilization, soil enzyme activity, and soil microbiome. The results showed that biochar could directly reduce the motility of Cd through adsorption and formation of CdCO3 precipitates, thereby protecting bacteria from Cd toxicity in the solution. In addition, bacterial growth further induces the formation of CdCO3 and CdS and enhances Cd adsorption by bacterial cells, resulting in a higher Cd removal rate. Thus, bacterial inoculation significantly enhances Cd removal in the presence of biochar in the solution. Moreover, soil incubation experiments showed that bacteria-loaded biochar significantly reduced soil exchangeable Cd in comparison with other treatments by impacting soil microbiome. In particular, bacteria-loaded biochar increased the relative abundance of Bacillus, Lysobacter, and Pontibacter, causing an increase in pH, urease, and arylsulfatase, thereby passivating soil exchangeable Cd and improving soil environmental quality in the natural alkaline Cd-contaminated soil. Overall, this study provides a systematic understanding of the synergistic mechanisms of biochar and bacteria for Cd immobilization in soil and new insights into the selection of functional strain for the efficient remediation of the contaminated environments by bacterial biochar composite.

Development of a ferroptosis-based molecular markers for predicting RFS in prostate cancer patients.

The goal of this study was to develop a ferroptosis-based molecular signature that can predict recurrence-free survival (RFS) in patients with prostate cancer (PCa). In this study, we obtained ferroptosis-related genes (FRGs) in FerrDb database and clinical transcriptome data in TCGA database and GEO database. Consensus cluster analysis was used to identify three molecular markers of ferroptosis in PCa with differential expression of 40 FRGs, including PD-L1 expression levels. We conducted a new ferroptosis-related signature for PCa RFS using four FRGs identified through univariate and multivariate Cox regression analyses. The signature was validated in the training, testing, and validation cohorts, and it demonstrated remarkable results in the area under the time-dependent receiver operating characteristic (ROC) curve of 0.757, 0.715, and 0.732, respectively. Additionally, we observed that younger patients, those with stage T III and stage T IV, stage N0, cluster 1, and cluster 2 PCa were more accurately predicted by the signature as independent predictors of RFS. DU-145 and RWPE-1 cells were successfully analyzed by qRT-PCR and Western blot for ASNS, GPT2, RRM2, and NFE2L2. In summary, we developed a novel ferroptosis-based signature for RFS in PC, utilizing four FRGs identified through univariate and multivariate Cox regression analyses. This signature was rigorously validated across training, testing, and validation cohorts, demonstrating exceptional performance as evidenced by its ROC curves. Notably, our findings indicate that this signature is particularly effective as an independent predictor of RFS in younger patients or those with stage T III and T IV, stage N0, and in clusters 1 and 2. Finally, we confirmed the expression of these four FRGs in DU-145 and RWPE-1 cell lines.

Mechanisms involved in the sequestration and resistance of cadmium for a plant-associated Pseudomonas strain.

Understanding Cd-resistant bacterial cadmium (Cd) resistance systems is crucial for improving microremediation in Cd-contaminated environments. However, these mechanisms are not fully understood in plant-associated bacteria. In the present study, we investigated the mechanisms underlying Cd sequestration and resistance in the strain AN-B15. These results showed that extracellular Cd sequestration by complexation in strain AN-B15 was primarily responsible for the removal of Cd from the solution. Transcriptome analyses have shown that the mechanisms of Cd resistance at the transcriptional level involve collaborative processes involving multiple metabolic pathways. The AN-B15 strain upregulated the expression of genes related to exopolymeric substance synthesis, metal transport, Fe-S cluster biogenesis, iron recruitment, reactive oxygen species oxidative stress defense, and DNA and protein repair to resist Cd-induced stress. Furthermore, inoculation with AN-B15 alleviated Cd-induced toxicity and reduced Cd uptake in the shoots of wheat seedlings, indicating its potential for remediation. Overall, the results improve our understanding of the mechanisms involved in Cd resistance in bacteria and thus have important implications for improving microremediation.

Catalytic enantioselective reductive alkynylation of amides enables one-pot syntheses of pyrrolidine, piperidine and indolizidine alkaloids.

The primary objective in synthetic organic chemistry is to develop highly efficient, selective, and versatile synthetic methodologies, which are essential for discovering new drug candidates and agrochemicals. In this study, we present a unified strategy for a one-pot, catalytic enantioselective synthesis of α-alkyl and α,α'-dialkyl pyrrolidine, piperidine, and indolizidine alkaloids using readily available amides and alkynes. This synthesis is enabled by the identification and development of an Ir/Cu/N-PINAP catalyzed highly enantioselective and chemoselective reductive alkynylation of α-unbranched aliphatic amides, which serves as the key reaction. This reaction is combined with Pd-catalyzed tandem reactions in a one-pot approach, enabling the collective, catalytic enantioselective total syntheses of eight alkaloids and an anticancer antipode with 90-98% ee. The methodology's enantio-divergence is exemplified by the one-step access to either enantiomer of alkaloid bgugaine.

Tracking the Early Stage of Triplet-Induced Photodamage in a DNA Dimer and Oligomer Containing 5-Methylcytosine.

Methylation at the C5 position of cytosine, a naturally occurring epigenetic modification on DNA, shows a high correlation with mutational hotspots in disease such as skin cancer. Due to its essential biological relevance, numerous studies were devoted to confirming that the methylated sites favor the formation of the cyclobutane pyrimidine dimer (CPD), a well-known UV-induced lesion. However, photophysical and photochemical properties of dinucleotides and polynucleotides containing 5-methylcytosine (5mC) remain elusive. Herein, a charge transfer (CT) triplet state, generated via intersystem crossing (ISC) from a CT singlet state that enhanced after methylation on cytosine, is directly observed by using femtosecond transient absorption (TA) and time-resolved mid-infrared (TRIR) spectroscopy together with quantum chemical calculations for the first time in the T5mC dimer. Such an ISC process is quenched due to limitations of the ground-state geometries in 5mC-containing single-strand oligomer d(T5mC)9. This mechanistic information is important for understanding the early stage of triplet state-induced CPD formation in 5mC containing DNA.

Excited State Kinetics of Benzo[a]pyrene Is Affected by Oxygen and DNA.

Benzo[a]pyrene is a widespread environmental pollutant and a strong carcinogen. It is important to understand its bio-toxicity and degradation mechanism. Herein, we studied the excited state dynamics of benzo[a]pyrene by using time-resolved fluorescence and transient absorption spectroscopic techniques. For the first time, it is identified that benzo[a]pyrene in its singlet excited state could react with oxygen, resulting in fluorescence quenching. Additionally, effective intersystem crossing can occur from its singlet state to the triplet state. Furthermore, the interaction between the excited benzo[a]pyrene and ct-DNA can be observed directly and charge transfer between benzo[a]pyrene and ct-DNA may be the reason. These results lay a foundation for further understanding of the carcinogenic mechanism of benzo[a]pyrene and provide insight into the photo-degradation mechanism of this molecule.

An ultrafast phototrigger of the Trp5CN-Trp motif in a ß-hairpin peptide.

Phototriggers are useful molecular tools to initiate reactions in enzymes by light for the purpose of photoenzymatic design and mechanistic investigations. Here, we incorporated the non-natural amino acid 5-cyanotryptophan (W5CN) in a polypeptide scaffold and resolved the photochemical reaction of the W5CN-W motif using femtosecond transient UV/Vis and mid-IR spectroscopy. We identified a marker band of ∼2037 cm-1 from the CN stretch of the electron transfer intermediate W5CN·- in the transient IR measurement and found UV/Vis spectroscopic evidence for the W·+ radical at 580 nm. Through kinetic analysis, we characterized that the charge separation between the excited W5CN and W occurs in 253 ps, with a charge-recombination lifetime of 862 ps. Our study highlights the potential use of the W5CN-W pair as an ultrafast phototrigger to initiate reactions in enzymes that are not light-sensitive, making downstream reactions accessible to femtosecond spectroscopic detection.

Distinct doorway states lead to triplet excited state generation in epigenetic RNA nucleosides.

N 6-Hydroxymethyladenosine (hm6A) and N6-formyladenosine (f6A) are two important intermediates during the demethylation process of N6-methyladenosine (m6A), which has been proven to show epigenetic function in mRNA. However, there is no knowledge about how the chemical integrity and stability could be altered when these two nucleosides are exposed to ultraviolet (UV) radiation. Herein, we report the first study on excited state dynamics of hm6A and f6A in solutions by using femtosecond time-resolved spectroscopy and quantum chemistry calculations. Surprisingly, triplet excited species are clearly identified in both hm6A and f6A after UV excitation, which is in sharp contrast to the 10-3 level triplet yield of adenosine scaffolds. Moreover, the doorway states leading to triplet states are found to be an intramolecular charge transfer state and a lower-lying dark nπ* state in hm6A and f6A, respectively. These discoveries pave the way to further study their effects on RNA strands and provide insight for understanding RNA photochemistry.

Rigid and Photostable Shortwave Infrared Dye Absorbing/Emitting beyond 1200 nm for High-Contrast Multiplexed Imaging.

The shortwave infrared (SWIR) spectral region beyond 1200 nm offers optimal tissue penetration depth and has broad potential in diagnosis, therapy, and surgery. Here, we devised a novel class of fluorochromic scaffold, i.e., a tetra-benzannulated xanthenoid (EC7). EC7 absorbs/emits maximally at 1204/1290 nm in CH2Cl2 and exhibits an unparalleled molar absorptivity of 3.91 × 105 cm-1 M-1 and high transparency to light at 400-900 nm. It also exhibited high resistance toward both photobleaching and symmetry breaking due to its unique structural rigidity. It is feasible for in vivo bioimaging and particularly suitable to couple with the shorter-wavelength analogues for high-contrast multiplexing. High-contrast dual-channel intraoperative imaging of the hepatobiliary system and three-channel in vivo imaging of the intestine, the stomach, and the vasculature were showcased. EC7 is a benchmark fluorochrome for facile biomedical exploitation of the SWIR region beyond 1200 nm.

Ultrafast Förster resonance energy transfer between tyrosine and tryptophan: potential contributions to protein-water dynamics measurements.

Ultrafast Förster Resonance Energy Transfer (FRET) between Tyrosine (Tyr, Y) and Tryptophan (Trp, W) in the model peptides Trp-(Pro)n-Tyr (WPnY) has been investigated using a femtosecond up-conversion spectrophotofluorometer. The ultrafast energy transfer process (<100 ps) in short peptides (WY, WPY and WP2Y) has been resolved. In fact, this FRET rate is found to be mixed with the rates of solvent relaxation (SR), ultrafast population decay (QSSQ) and other lifetime components. To further dissect and analyze the FRET, a spectral working model is constructed, and the contribution of a FRET lifetime is separated by reconciling the shapes of decay associated spectra (DAS). Surprisingly, FRET efficiency did not decrease monotonically with the growth of the peptide chain (as expected) but increased first and then decreased. The highest FRET efficiency occurred in peptide WPY. The kinetic results have been accompanied with molecular dynamics simulations that reconcile and explain this strange phenomenon: due to the strong interaction between amino acids, the distance between the donor and receptor in peptide WPY is actually closest, resulting in the fastest FRET. In addition, the FRET lifetimes (τcal) were estimated within the molecular dynamics simulations, and they were consistent with the lifetimes (τexp) separated out by the experimental measurements and the DAS working model. This benchmark study has implications for both previous and future studies of protein ultrafast dynamics. The approach taken can be generalized for the study of proximate tyrosine and tryptophan in proteins and it suggests spectral strategies for extracting mixed rates in other complex FRET problems.

Dexmedetomidine attenuates hippocampal neuroinflammation in postoperative neurocognitive disorders by inhibiting microRNA-329-3p and activating the CREB1/IL1RA axis.

RATIONALE: Due to its anti-inflammatory effect, dexmedetomidine (DEX) can confer neuroprotection in postoperative neurocognitive disorders (NCD). Here, the mechanism responsible for this effect of DEX is rarely ascertained. OBJECTIVES: Our research was implemented to figure out mechanism governing the protection of DEX against hippocampal neuroinflammation in postoperative NCD. METHODS: Exploratory laparotomy was applied for generating a postoperative NCD mouse model before bilateral hippocampal injection with microRNA (miR)-329-3p-agomir and intraperitoneal injection with DEX. Cognitive function of mice was evaluated by water maze test and fear conditioning test. Immunofluorescence was performed to assess microglial activation in hippocampus. After cell transfection and DEX treatment, mouse microglial cells (BV-2) were stimulated by lipopolysaccharide (LPS). IL-1ß, IL-6, and TNF-α levels and the number of phagocytes were assessed by ELISA and flow cytometry. Dual-luciferase reporter assay was adopted to assess the relationship between miR-329-3p and CREB1. RESULTS: miR-329-3p expression was reduced in the postoperative NCD mice after DEX treatment. DEX treatment or miR-329-3p downregulation caused attenuated cognitive dysfunction and microglia activation as well as reduced IL-1ß, IL-6, and TNF-α levels in the hippocampus of the postoperative NCD mice. Mechanistically, miR-329-3p inversely targeted CREB1 that activated IL1RA in LPS-induced BV-2 cells. DEX treatment, miR-329-3p inhibition, or CREB1 or IL1RA upregulation curtailed the release of proinflammatory proteins and the number of phagocytes in LPS-induced BV-2 cells. CONCLUSIONS: Collectively, our data provided the novel insight of the neuroprotective mechanism of DEX in postoperative NCD pertaining to the miR-329-3p/CREB1/IL1RA axis.

Excited State Dynamics of Methylated Guanosine Derivatives Revealed by Femtosecond Time-resolved Spectroscopy.

Methylated DNA/RNA nucleobases are important epigenetic marks in living species and play an important role for targeted therapies. Moreover, methylation could bring significant changes to the photo-stability of nucleic acid, leading these sites become mutational hotspots for disease such as skin cancer. While a number of studies have demonstrated the relationship between excited state dynamics and the biological function of methylated cytosine in DNA, investigations aimed at unraveling the excited state dynamics of methylated guanosine in RNA have been largely overlooked. In this work, influence of methylation on the excited state dynamics of guanosine is studied by using femtosecond time-resolved spectroscopy. Our results suggest that the effect of methyl substitution on the photophysical properties of guanosine is position sensitive. N1-methylguanosine shows very similar excited state dynamics as that in guanosine, while almost one order of magnitude longer lifetime of the La state is observed in N2, N2-dimethylguanosine. Notably, N7-methylation can lead to a new minimum on the La state and the excited state lifetime is two orders of magnitude longer than that of guanosine. These findings not only help understanding excited state dynamics of methylated guanosines, but also lay the foundation for further studying DNA/RNA strands incorporated with these bases.

Ultrasensitive Sensing of Volatile Organic Compounds Using a Cu-Doped SnO2 -NiO p-n Heterostructure That Shows Significant Raman Enhancement*.

Surface enhanced Raman scattering (SERS) based on chemical mechanism (CM) attracts tremendous attention for great selectivity and stability. However, low enhancement factor (EF) limits its practical applications for trace detection. Here, a novel sponge-like Cu-doping SnO2 -NiO p-n semiconductor heterostructure (SnO2 -NiOx /Cu), was first created as a CM-based SERS substrate with a significant EF of 1.46×1010 . This remarkable EF was mainly attributed to the enhanced charge-separation efficacy of p-n heterojunction and charge transfer resonance resulted from Cu doping. Moreover, the porous structure enriched the probe molecules, resulting in further SERS signals magnification. By immobilizing CuPc as an inner-reference element, SnO2 -NiOx /Cu was developed as a SERS nose for selective recognition of multiple lung cancer related VOCs down to ppb level. The information of VOCs was recorded in a barcode, demonstrating practical potential of a desktop SERS device for biomarker screening.

Ultrafast Fluorescence Spectroscopy via Upconversion and Its Applications in Biophysics.

In this review, the experimental set-up and functional characteristics of single-wavelength and broad-band femtosecond upconversion spectrophotofluorometers developed in our laboratory are described. We discuss applications of this technique to biophysical problems, such as ultrafast fluorescence quenching and solvation dynamics of tryptophan, peptides, proteins, reduced nicotinamide adenine dinucleotide (NADH), and nucleic acids. In the tryptophan dynamics field, especially for proteins, two types of solvation dynamics on different time scales have been well explored: ~1 ps for bulk water, and tens of picoseconds for "biological water", a term that combines effects of water and macromolecule dynamics. In addition, some proteins also show quasi-static self-quenching (QSSQ) phenomena. Interestingly, in our more recent work, we also find that similar mixtures of quenching and solvation dynamics occur for the metabolic cofactor NADH. In this review, we add a brief overview of the emerging development of fluorescent RNA aptamers and their potential application to live cell imaging, while noting how ultrafast measurement may speed their optimization.

Greenhouse gas emissions from constructed wetlands are mitigated by biochar substrates and distinctly affected by tidal flow and intermittent aeration modes.

Biochar substrates and tidal flow (TF) and intermittent aeration (IA) operation modes have recently been applied to improve the treatment performance of constructed wetlands (CWs), but their roles in regulating greenhouse gas (GHG) emissions from CWs are still unclear. In this preliminary study, CO2, CH4 and N2O fluxes and associated microbial characteristics in four groups of subsurface-flow CWs, i.e., ceramsite CWs (C-CWs), biochar-amended CWs (B-CWs), intermittently aerated B-CWs (AB-CWs) and tide-flow B-CWs (TB-CWs), were comparatively investigated. The results showed that biochar amendment significantly mitigated CH4 and N2O fluxes from the CWs by supporting higher abundances of mcrA and nosZ genes and higher ratios of pmoA/mcrA and nosZ/(nirK + nirS), thus reducing global warming potential (GWP, a decrease of 55.8%), in addition to promoting total nitrogen (TN) removal by 41.3%, mainly by increasing the abundances and activities of nitrifiers and denitrifiers. The TF mode efficiently improved nitrogen removal, but it greatly increased GHG fluxes since large amounts of GHGs escaped from the empty CW matrix after water draining. IA abated GHG emissions from the CWs, mainly after aeration. TF and IA decreased the abundances of functional bacteria and archaea related to C and N transformation, except nitrifiers, and shaped the microbial community structures. The application of a biochar substrate and IA mode can facilitate the design and operation of CWs in a more ecologically sustainable way.