A case report of the diagnosis and treatment of immune checkpoint inhibitor-related encephalitis induced by camrelizumab.

Background: Camrelizumab has been widely used in the treatment of various cancers, it is important to determine the side-effect of this drug and the corresponding treatment strategy. Case Description: The current case report describes the clinic, diagnosis, treatment and prognosis of camrelizumab-related encephalitis. Camrelizumab was administrated to a 67-year-old man with squamous cell carcinoma (SCC), a form of non-small cell lung cancer (NSCLC). One month after the treatment, the patient showed typical encephalitis symptoms including systemic fatigue, numbness of extremities and walking instability. Furthermore, the total protein in cerebrospinal fluid (CSF) was significantly elevated (1,399 vs. normal range 120-600 mg/L). Importantly, magnetic resonance imaging showed there was no brain metastasis. The patient did not get better after two days of intravenous injection of thioctic acid (1.2 g) and cobamamide (1.5 mg) once daily. Therefore, this patient was diagnosed as camrelizumab-related encephalitis. Then, we put him on one-month regimen: oral taper corticoids (methylprednisolone, MP) at 500 mg (days 1-4), 120 mg (days 5-10) and 60 mg (days 11-15); MP was replaced with oral prednisone acetate at 30 mg (days 16-30). After the treatment, the total protein in CSF was decreased to 873 mg/L, and all of encephalitis-related symptom was completely lost. About one year after the onset of encephalitis, the patient showed no recurrence of neurological symptoms. Conclusions: The present case proves the efficacy and safety of corticoids in the treatment of camrelizumab-related adverse effects.

Paenibacillus exopolysaccharide alleviates Malassezia-induced skin damage: Enhancing skin barrier function, regulating immune responses, and modulating microbiota.

Numerous studies have established a strong association between Malassezia and various skin disorders, including atopic dermatitis. Finding appropriate methods or medications to alleviate Malassezia-induced skin damage is of notable public interest. This study aimed to evaluate the therapeutic effect of the exopolysaccharide EPS1, produced by Paenibacillus polymyxa, on Malassezia restricta-induced skin damage. In vitro assays indicated that EPS1 reduced the expression of pro-inflammatory cytokine genes in TNF-α-induced HaCaT cells. In a murine model, EPS1 was found to mitigate clinical symptoms, reduce epidermal thickness and mast cell infiltration, improve skin barrier function, decrease pro-inflammatory cytokine levels associated with type 17 inflammation, enhance Tregs in the spleen, upregulate the transcription of Treg-related genes in skin lesions, and modulate the skin microbiota. This study is the first to report the alleviating effect of Paenibacillus exopolysaccharide on Malassezia-induced skin inflammation and its impact on the skin microbiota. These findings support the potential of Paenibacillus exopolysaccharides as consumer products and therapeutic agents for managing Malassezia-induced skin damage by improving skin barrier function, modulating immune responses, and influencing skin microbiota.

PET/CT Manifestations of a Case of Cavernous Hemangioma Misdiagnosed as Reproductive Tumor.

ABSTRACT: Cavernous hemangioma of mediastinum is a rare, benign tumor originating from vascular endothelial cells. It typically arises in the anterior mediastinum but has also been reported in the posterior mediastinum or the middle mediastinum, and occasionally involves both the anterior and middle compartments. We present the case of a 20-year-old man with a cavernous hemangioma of mediastinum initially misdiagnosed as a reproductive tumor by 18F-FDG PET/CT.

Podophyllotoxin derivatives-tubulin complex reveals a potential binding site of tubulin polymerization inhibitors in α-tubulin adjacent to colchicine site.

The colchicine site of ß-tubulin has been proven to be essential binding sites of microtubule polymerization inhibitors. Recent studies implied that GTP pocket of α-tubulin adjacent to colchicine sites is a potential binding site for developing tubulin polymerization inhibitors. However, the structural basis for which type of structural fragments was more beneficial for enhancing the affinity of α-tubulin is still unclear. Here, podophyllotoxin derivatives-tubulin complex crystals indicated that heterocyclic with the highly electronegative and small steric hindrance was conducive to change configuration and enhance the affinity of the residues in GTP pocket of α-tubulin. Triazole with lone-pairs electrons and small steric hindrance exhibited the strongest affinity for enhancing affinity of podophyllotoxin derivatives by forming two hydrogen bonds with αT5 Ser178. Pyrimidine with the secondary strong affinity could bind Asn101 to make the αH7 configuration deflection, which reduces the stability of tubulin result in its depolymerization. Conversely, 4ß-quinoline-podophyllotoxin with the weakest affinity did not interact with α-tubulin. The molecular dynamics simulation and protein thermal shift results showed that 4ß-triazole-podophyllotoxin-tubulin was the most stable mainly due to two hydrogen bonds and the higher van der Waals force. This work provided a structural basis of the potential binding sites for extending the α/ß-tubulin dual-binding sites inhibitors design strategy.

Characteristic cembrane-type diterpenoids with nitric oxide inhibitory activity from the gum resin of Boswellia carterii Birdw.

Five new characteristic cembrane-type diterpenoids (olibacartiols A-E, 1-5) were acquired from the gum resin of Boswellia carterii. The structures of these diterpenoids were characterized by detailed spectroscopic analysis, and compounds 1-3 were unambiguously confirmed by single-crystal X-ray diffraction experiments. The anti-inflammatory activities of the isolated compounds were evaluated using LPS-induced BV2 cell model and compounds 2-5 showed moderate NO inhibitory effects with IC50 values of 8.84 ± 1.02, 9.82 ± 1.95, 9.75 ± 2.24, and 7.39 ± 1.24 µM, respectively.

Management of biofilm by an innovative layer-structured membrane for membrane biofilm reactor (MBfR) to efficient methane oxidation coupled to denitrification (AME-D).

Aerobic methane oxidation coupled with denitrification (AME-D) has garnered significant attention as a promising technology for nitrogen removal from water. Effective biofilm management on the membrane surface is essential to enhance the efficiency of nitrate removal in AME-D systems. In this study, we introduce a novel and scalable layer-structured membrane (LSM) developed using a meticulously designed polyurethane sponge. The application of the LSM in advanced biofilm management for AME-D resulted in a substantial enhancement of denitrification performance. Our experimental results demonstrated remarkable improvements in nitrate-removal flux (92.8 mmol-N m-2 d-1) and methane-oxidation rate (325.6 mmol m-2 d-1) when using an LSM in a membrane biofilm reactor (L-MBfR) compared with a conventional membrane reactor (C-MBfR). The l-MBfR exhibited 12.4-, 6.8- and 3.4-fold increases in nitrate-removal rate, biomass-retention capacity, and methane-oxidation rate, respectively, relative to the control C-MBfR. Notably, the l-MBfR demonstrated a 3.5-fold higher abundance of denitrifying bacteria, including Xanthomonadaceae, Rhodocyclaceae, and Methylophilaceae. In addition, the denitrification-related enzyme activity was twice as high in the l-MBfR than in the C-MBfR. These findings underscore the LSM's ability to create anoxic/anaerobic microenvironments conducive to biofilm formation and denitrification. Furthermore, the LSM exhibited a unique advantage in shaping microbial community structures and facilitating cross-feeding interactions between denitrifying bacteria and aerobic methanotrophs. The results of this study hold great promise for advancing the application of MBfRs in achieving efficient and reliable nitrate removal through the AME-D pathway, facilitated by effective biofilm management.

Hyaluronan delays human amniotic epithelial stem cell senescence by regulating CD44 isoform switch to activate AKT/mTOR signals.

The replicative senescence of human amniotic epithelial stem cells (hAECs) is a major concern towards its clinical application. This study found that a 300-kDa hyaluronic acid (HA) could effectively delay the replicative senescence of hAECs, as indicated by the downregulation of cellular senescence markers and alteration of the cell cycle, and substantially improve the differentiation capacities of hAECs. HA was confirmed to regulate the CD44 isoform switch by upregulating the CD44s and downregulating the CD44v, thus exerting an anti-aging effect. We further found that HA induced the upregulation of hyaluronan synthase (HAS) 2, resulting in the activation of epithelial splicing regulatory protein 1 (ESRP1) and alternative splicing of CD44 mRNA, thereby promoting CD44s expression and inhibiting CD44v expression. Knockdown of HAS2 blocked ESRP1 expression and attenuated the anti-aging effects of HA. Hermes-1, a specific blocker of CD44, caused partial loss of the anti-aging effect of HA, upregulated senescence markers, and downregulated stemness markers. Furthermore, CD44s receptor activation was shown to initiate the AKT/mTOR downstream signaling. Conclusively, the study suggested that HA delayed hAEC senescence by regulating CD44 isoform switch to activate the AKT/mTOR signaling pathway, and there is potential for the clinical application of hAECs in combination with HA.

Integrated analysis of the potential roles of microRNA-messenger RNA networks in prostate adenocarcinoma.

Background: Prostate adenocarcinoma is a frequent cancer among men with high incidence and mortality rates. Biomarkers are useful for the treatment of cancers, so we need to explore the regulatory network of prostate adenocarcinoma. Method: The database from University of California Santa Cruz was used to determine expression of messenger RNAs and microRNAs. Weighted correlation network analysis was used for classifying genes. Search Tool for Recurring Instances of Neighboring Genes and Cytoscape were used for the construction of PPI network and selection of hub genes. The microRNAs were predicted in miRactDB. The relations between microRNAs and messenger RNAs were assessed by Statistical Product and Service Solutions. The prognostic value was evaluated through Kaplan-Meier method. Kyoto Encyclopedia of Genes and Genomes, Gene Ontology and Gene Set Enrichment Analysis were used for predicting potential function. Results: 10 hub genes were all overexpressed in tumor tissues compared to normal tissues, but only aurora kinase B and nucleolar and spindle associated protein 1 were both significantly related to disease-free interval and progression-free interval time.Aurora kinase B and nucleolar and spindle associated protein 1 were negatively related to hsa-miR-1-3p, hsa-miR-133a-3p, hsa-miR-133b and hsa-miR-221-3p but positively related to hsa-miR-15b-5p, hsa-miR-21-5p, hsa-miR-106b-5p, hsa-miR-183-5p, hsa-miR-191-5p, hsa-miR-210-3p, hsa-miR-425-5p and hsa-miR-653-5p. All microRNAs except has-miR-653-5p significantly were related to the disease-free interval and progression-free interval time. The functions of microRNAs were enriched in cell cycle. Conclusion: We identified hub messenger RNAs and core microRNAs and established a novel messenger RNA-microRNA network associated with the prognosis of prostate adenocarcinoma.

Risk factors for renal outcomes in children with antineutrophil cytoplasmic antibody-associated vasculitis: a nationwide retrospective study in China.

BACKGROUND: Pediatric antineutrophil cytoplasmic antibody-associated vasculitis (AAV) is a life-threatening systemic vasculitis featured by liability to renal involvement. However, there are few studies on the risk factors and predictive models for renal outcomes of AAV in children. METHODS: Data from 179 AAV children in multiple centers between January 2012 and March 2020 were collected retrospectively. The risk factors and predictive model of end-stage renal disease (ESRD) in AAV were explored. RESULTS: Renal involvement was the most typical manifestation (95.5%), and the crescent was the predominant pathological lesion (84.9%). The estimated glomerular filtration rate (eGFR) was evaluated in 114 patients, of whom 59.6% developed ESRD, and the median time to ESRD was 3.20 months. The eGFR [P = 0.006, odds ratio (OR) = 0.955, 95% confidence interval (CI) = 0.924-0.987] and the percentages of global glomerulosclerosis (pGGS; P = 0.018, OR = 1.060, 95% CI = 1.010-1.112) were independent risk factors for ESRD of renal biopsy. Based on the pGGS and eGFR at renal biopsy, we developed three risk grades of ESRD and one predictive model. The Kaplan‒Meier curve indicated that renal outcomes were significantly different in different risk grades (P < 0.001). Compared with serum creatinine at baseline, the predictive model had higher accuracy (0.86 versus 0.58, P < 0.001) and a lower coefficient of variation (0.07 versus 0.92) in external validation. CONCLUSIONS: Renal involvement is the most common manifestation of pediatric AAV in China, of which more than half deteriorates into ESRD. The predictive model based on eGFR at renal biopsy and the pGGS may be stable and accurate in speculating the risk of ESRD in AAV children. Supplementary file 2 (MP4 18937 KB).

Transplantation of human induced pluripotent stem cell derived keratinocytes accelerates deep second-degree burn wound healing.

BACKGROUND: Current evidence shows that human induced pluripotent stem cells (hiPSCs) can effectively differentiate into keratinocytes (KCs), but its effect on skin burn healing has not been reported. AIM: To observe the effects of hiPSCs-derived KCs transplantation on skin burn healing in mice and to preliminarily reveal the underlying mechanisms. METHODS: An analysis of differentially expressed genes in burn wounds based on GEO datasets GSE140926, and GSE27186 was established. A differentiation medium containing retinoic acid and bone morphogenetic protein 4 was applied to induce hiPSCs to differentiate into KCs. The expression of KCs marker proteins was detected using immunofluorescence staining. A model of a C57BL/6 mouse with deep cutaneous second-degree burn was created, and then phosphate buffered saline (PBS), hiPSCs-KCs, or hiPSCs-KCs with knockdown of COL7A1 were injected around the wound surface. The wound healing, re-epithelialization, engraftment of hiPSCs-KCs into wounds, proinflammatory factor level, and the NF-κB pathway proteins were assessed by hematoxylin-eosin staining, carboxifluorescein diacetate succinimidyl ester (CFSE) fluorescence staining, enzyme linked immunosorbent assay, and Western blotting on days 3, 7, and 14 after the injection, respectively. Moreover, the effects of COL7A1 knockdown on the proliferation and migration of hiPSCs-KCs were confirmed by immunohistochemistry, EdU, Transwell, and damage repair assays. RESULTS: HiPSCs-KCs could express the hallmark proteins of KCs. COL7A1 was down-regulated in burn wound tissues and highly expressed in hiPSCs-KCs. Transplantation of hiPSCs-KCs into mice with burn wounds resulted in a significant decrease in wound area, an increase in wound re-epithelialization, a decrease in proinflammatory factors content, and an inhibition of NF-κB pathway activation compared to the PBS group. The in vitro assay showed that COL7A1 knockdown could rescue the inhibition of hiPSCs-KCs proliferation and migration, providing further evidence that COL7A1 speeds up burn wound healing by limiting cell proliferation and migration. CONCLUSION: In deep, second-degree burn wounds, COL7A1 can promote KC proliferation and migration while also suppressing the inflammatory response.

A truncated DNA aptamer with high selectivity for estrogen receptor-positive breast cancer cells.

The estrogen receptor-positive (ER+) breast cancers constitute more than 50 % of breast cancers, seriously threatening the health of women. Unfortunately, the detection and targeted therapy of ER+ breast cancers remain a challenge. Here, a novel nucleic acid aptamer S1-4 was developed to specifically target ER+ breast cancer MCF-7 cells by using Cell-SELEX and nucleic acid truncation strategies. The affinity dissociation constant of the binding of aptamer S1-4 to MCF-7 cells was 97.6 ± 7.5 nM in vitro. Compared with HER2+ breast cells SK-BR-3 and triple-negative breast cancer cells MDA-MB-231, MCF-7 cells were selectively recognized and targeted by aptamer S1-4. Fluorescence tracing in vivo results also indicated that aptamer S1-4 selectively targeted the cell membrane of tumor tissues in MCF-7- but not in SK-BR3 or MDB-MA-231-bearing mice. This selectively developed novel aptamer probe S1-4 with high affinity could be used for the diagnosis and treatment of ER+ breast cancers.

Efficacy evaluation of neoadjuvant immunotherapy plus chemotherapy for non-small-cell lung cancer: comparison of PET/CT with postoperative pathology.

OBJECTIVES: To assess the value of positron emission tomography/computed tomography (PET/CT) in the efficacy evaluation of patients undergoing neoadjuvant immunotherapy plus chemotherapy, and to analyze its correlation with postoperative pathology. METHODS: The PET/CT metabolic parameters and CT size were retrospectively analyzed before and after neoadjuvant immunotherapy plus chemotherapy in 67 patients with resectable stage II/IIIA non-small-cell lung cancer (NSCLC). CT assessment based on immune response evaluation criteria in solid tumor criteria ((i)RECIST) was compared with PET/CT assessment based on the response criteria in solid tumors (PERCIST). The correlations between PET/CT metabolic parameters and postoperative pathology were analyzed. The value of PET/CT in the efficacy evaluation was assessed. RESULTS: The PET/CT assessment showed high consistency with postoperative pathological evaluation, yet the CT assessment showed low consistency with postoperative pathological evaluation. The (i)RECIST and PERCIST criteria showed statistically significant differences (p < 0.001). The postoperative pathological response was negatively associated with ΔSUVmax (%) (r = - 0.812, p < 0.001), ΔSUVmean (%) (r = - 0.805, p < 0.001), and ΔSUVpeak (%) (r = - 0.800, p < 0.001). The cut-off values of 75.8 for ΔSUVmax (%), 67.8 for ΔSUVmean (%), and 74.6 for ΔSUVpeak (%) had the highest sensitivity and specificity. CONCLUSION: The PERCIST criteria are more sensitive and accurate than (i)RECIST criteria to identify more responders when evaluating the response of neoadjuvant immunotherapy plus chemotherapy for NSCLC. PET/CT shows high accuracy in predicting postoperative pathological response. Our study shows the important role PET/CT plays in the efficacy evaluation of NSCLC patients undergoing neoadjuvant immunotherapy plus chemotherapy, as well as in predicting the prognosis and guiding postoperative treatment. CLINICAL RELEVANCE STATEMENT: Neoadjuvant immunotherapy plus chemotherapy is highly effective in the treatment of non-small-cell lung cancer. And PET/CT played an important role in the efficacy evaluation following neoadjuvant immunotherapy plus chemotherapy for non-small-cell lung cancer. KEY POINTS: • Neoadjuvant immunotherapy plus chemotherapy is highly effective in the treatment of NSCLC. • The PERCIST criteria are more sensitive and accurate than (i)RECIST criteria to identify more responders when evaluating the response of neoadjuvant immunotherapy plus chemotherapy for NSCLC. • PET/CT played an important role in the efficacy evaluation; ΔSUVmax (%), ΔSUVmean (%), and ΔSUVpeak (%) following neoadjuvant immunotherapy plus chemotherapy for NSCLC had high consistency and strong correlations with postoperative pathology.

Advances of antitumor drug discovery in traditional Chinese medicine and natural active products by using multi-active components combination.

The antitumor efficacy of Chinese herbal medicines has been widely recognized. Leading compounds such as sterols, glycosides, flavonoids, alkaloids, terpenoids, phenylpropanoids, and polyketides constitute their complex active components. The antitumor monomers derived from Chinese medicine possess an attractive anticancer activity. However, their use was limited by low bioavailability, significant toxicity, and side effects, hindering their clinical applications. Recently, new chemical entities have been designed and synthesized by combining natural drugs with other small drug molecules or active moieties to improve the antitumor activity and selectivity, and reduce side effects. Such a novel conjugated drug that can interact with several vital biological targets in cells may have a more significant or synergistic anticancer activity than a single-molecule drug. In addition, antitumor conjugates could be obtained by combining pharmacophores containing two or more known drugs or leading compounds. Based on these studies, the new drug research and development could be greatly shortened. This study reviews the research progress of conjugates with antitumor activity based on Chinese herbal medicine. It is expected to serve as a valuable reference to antitumor drug research and clinical application of traditional Chinese medicine.

Rational Design of Daunorubicin C-14 Hydroxylase Based on the Understanding of Its Substrate-Binding Mechanism.

Doxorubicin is one of the most widely used antitumor drugs and is currently produced via the chemical conversion method, which suffers from high production costs, complex product separation processes, and serious environmental pollution. Biocatalysis is considered a more efficient and environment-friendly method for drug production. The cytochrome daunorubicin C-14 hydroxylase (DoxA) is the essential enzyme catalyzing the conversion of daunorubicin to doxorubicin. Herein, the DoxA from Streptomyces peucetius subsp. caesius ATCC 27952 was expressed in Escherichia coli, and the rational design strategy was further applied to improve the enzyme activity. Eight amino acid residues were identified as the key sites via molecular docking. Using a constructed screening library, we obtained the mutant DoxA(P88Y) with a more rational protein conformation, and a 56% increase in bioconversion efficiency was achieved by the mutant compared to the wild-type DoxA. Molecular dynamics simulation was applied to understand the relationship between the enzyme's structural property and its substrate-binding efficiency. It was demonstrated that the mutant DoxA(P88Y) formed a new hydrophobic interaction with the substrate daunorubicin, which might have enhanced the binding stability and thus improved the catalytic activity. Our work lays a foundation for further exploration of DoxA and facilitates the industrial process of bio-production of doxorubicin.

Role of abnormal glycosylated IgA1 and interstitial transformation of glomerular endothelial cells in the development and progression of IgA nephropathy.

BACKGROUND: IgA nephropathy (IgAN) is a common primary renal disease in childhood. METHODS: Twenty blood samples and renal tissue from patients with IgAN, 20 blood samples from healthy children and 10 normal renal tissue were collected. Serum Gd-IgA1 and renal Gd-IgA1, CD31, α-SMA and vimentin were measured. RESULTS: The serum Gd-IgA1 concentration in the IgAN group was significantly higher. Gd-IgA1 was not expressed in normal kidneys, which was positive in the IgAN group. Gd-IgA1 levels in serum and renal tissue were not related. The expression of CD31 decreased significantly in IgAN group, while the expression of α-SMA and vimentin increased significantly. There was no significant correlation between the renal concentration of Gd-IgA1 and CD31, α-SMA and vimentin. CONCLUSION: The increased Gd-IgA1 in the serum and kidney may promote the pathogenesis of IgAN. The serum Gd-IgA1 cannot predict the extent of its deposition in the kidney. Endothelial mesenchymal transition (EndMT) may be involved in the pathogenesis of renal fibrosis in IgAN.

Steroid-resistant nephrotic syndrome caused by nuclear pore gene NUP133 variation.

Partially enlarged structure of NUP133: wild-type (upper) and mutant p.Lys966Asn (lower).

Advances in Pilot-Scale Stirred Bioreactors in Solid-State and Submerged Cultivations of Medicinal Mushrooms.

Great interest for large-scale production of medicinal mushroom biomass and various pharmaceutically active compounds production dictates the development of comprehensive technologies. Solid state and submerged cultivations in bioreactors represent the most promising technologies for fast and large amount production of medicinal fungi biomass and pharmaceutically active products for human and veterinary need. There are many stages from shaking culture studies to large-scale industrial production. Pilot-scale studies represent the bridge and the balance between the gap of laboratory and industrial scale. Therefore it is not a surprise that most of pilot-scale results and experiences remain uncovered industrial secrets. This chapter is an overview of available engineering achievements in submerged and solid-state cultivation experiences in pilot-scale bioreactors.

Advances in Production of Medicinal Mushrooms Biomass in Solid State and Submerged Bioreactors.

Production of mushroom fruit bodies using farming technology could hardly meet the increasing demand of the world market. During the last several decades, there have been various basic and applied studies on fungal physiology, metabolism, process engineering, and (pre)clinical studies. The fundamental aspects of solid-state cultivation of various kinds of medicinal mushroom mycelia in various types of bioreactors were established. Solid-state cultivation of medicinal mushrooms for their biomass and bioactive metabolites production appear very suitable for veterinary use. Development of comprehensive submerged technologies using stirred tank and airlift bioreactors is the most promising technology for fast and large-scale production of medicinal fungi biomass and their pharmaceutically active products for human need. The potentials initiate the development of new drugs and some of the most attractive over-the-counter human and veterinary remedies. This article is to overview the engineering achievements in solid state and submerged cultivations of medicinal mushrooms in bioreactors.

Sesquiterpenoids from the leaves of Chimonanthus nitens.

Two new sesquiterpenoids (1 and 3), one new natural product (2), and two known compounds (4 and 5) were isolated from the leaves of Chimonanthus nitens. Their structures were elucidated by spectroscopic analysis, and the absolute configuration of compound 3 was determined by the X-ray single-crystal diffraction analysis. The cytotoxicity of compounds 1-5 was evaluated at three concentrations on two human breast cancer cell lines (MDA-MB-468 and MDA-MB-231) by MTT assay. As a result, we found that the cytotoxicity was weak even with a concentration of these compounds up to 100 µM.

Biofilm stratification in counter-diffused membrane biofilm bioreactors (MBfRs) for aerobic methane oxidation coupled to aerobic/anoxic denitrification: Effect of oxygen pressure.

Aerobic methane oxidation coupled with denitrification (AME-D) executed in membrane biofilm bioreactors (MBfRs) provides a high promise for simultaneously mitigating methane (CH4) emissions and removing nitrate in wastewater. However, systematically experimental investigation on how oxygen partial pressure affects the development and characteristics of counter-diffusional biofilm, as well as its spatial stratification profiles, and the cooperative interaction of the biofilm microbes, is still absent. In this study, we combined Optical Coherence Tomography (OCT) with Confocal Laser Scanning Microscopy (CLSM) to in-situ characterize the development of counter-diffusion biofilm in the MBfR for the first time. It was revealed that oxygen partial pressure onto the MBfR was capable of manipulating biofilm thickness and spatial stratification, and then managing the distribution of functional microbes. With the optimized oxygen partial pressure of 5.5 psig (25% oxygen content), the manipulated counter-diffusional biofilm in the AME-D process obtained the highest denitrification efficiency, due mainly to that this biofilm had the proper dynamic balance between the aerobic-layer and anoxic-layer where suitable O2 gradient and sufficient aerobic methanotrophs were achieved in aerobic-layer to favor methane oxidation, and complete O2 depletion and accessible organic sources were kept to avoid constraining denitrification activity in anoxic-layer. By using metagenome analysis and Fluorescence in situ hybridization (FISH) staining, the spatial distribution of the functional microbes within counter-diffused biofilm was successfully evidenced, and Rhodocyclaceae, one typical aerobic denitrifier, was found to survive and gradually enriched in the aerobic layer and played a key role in denitrification aerobically. This in-situ biofilm visualization and characterization evidenced directly for the first time the cooperative path of denitrification for AME-D in the counter-diffused biofilm, which involved aerobic methanotrophs, heterotrophic aerobic denitrifiers, and heterotrophic anoxic denitrifiers.