mir-605-3p prevents liver premetastatic niche formation by inhibiting angiogenesis via decreasing exosomal nos3 release in gastric cancer.

BACKGROUND: Cancer-induced pre-metastatic niches (PMNs) play a decisive role in promoting metastasis by facilitating angiogenesis in distant sites. Evidence accumulates suggesting that microRNAs (miRNAs) exert significant influence on angiogenesis during PMN formation, yet their specific roles and regulatory mechanisms in gastric cancer (GC) remain underexplored. METHODS: miR-605-3p was identified through miRNA-seq and validated by qRT-PCR. Its correlation with the clinicopathological characteristics and prognosis was analyzed in GC. Functional assays were performed to examine angiogenesis both in vitro and in vivo. The related molecular mechanisms were elucidated using RNA-seq, immunofluorescence, transmission electron microscopy, nanoparticle tracking analysis, enzyme-linked immunosorbent assay, luciferase reporter assays and bioinformatics analysis. RESULTS: miR-605-3p was screened as a candidate miRNA that may regulate angiogenesis in GC. Low expression of miR-605-3p is associated with shorter overall survival and disease-free survival in GC. miR-605-3p-mediated GC-secreted exosomes regulate angiogenesis by regulating exosomal nitric oxide synthase 3 (NOS3) derived from GC cells. Mechanistically, miR-605-3p reduced the secretion of exosomes by inhibiting vesicle-associated membrane protein 3 (VAMP3) expression and affects the transport of multivesicular bodies to the GC cell membrane. At the same time, miR-605-3p reduces NOS3 levels in exosomes by inhibiting the expression of intracellular NOS3. Upon uptake of GC cell-derived exosomal NOS3, human umbilical vein endothelial cells exhibited increased nitric oxide levels, which induced angiogenesis, established liver PMN and ultimately promoted the occurrence of liver metastasis. Furthermore, a high level of plasma exosomal NOS3 was clinically associated with metastasis in GC patients. CONCLUSIONS: miR-605-3p may play a pivotal role in regulating VAMP3-mediated secretion of exosomal NOS3, thereby affecting the formation of GC PMN and thus inhibiting GC metastasis.

Identifying ADHD-Related Abnormal Functional Connectivity with a Graph Convolutional Neural Network.

Attention deficit hyperactivity disorder (ADHD) is a common neurodevelopmental disorder that is characterized by inattention, hyperactivity, and impulsivity. The neural mechanisms underlying ADHD remain inadequately understood, and current approaches do not well link neural networks and attention networks within brain networks. Our objective is to investigate the neural mechanisms related to attention and explore neuroimaging biological tags that can be generalized within the attention networks. In this paper, we utilized resting-state functional magnetic resonance imaging data to examine the differential functional connectivity network between ADHD and typically developing individuals. We employed a graph convolutional neural network model to identify individuals with ADHD. After classification, we visualized brain regions with significant contributions to the classification results. Our results suggest that the frontal, temporal, parietal, and cerebellar regions are likely the primary areas of dysfunction in individuals with ADHD. We also explored the relationship between regions of interest and attention networks, as well as the connection between crucial nodes and the distribution of positively and negatively correlated connections. This analysis allowed us to pinpoint the most discriminative brain regions, including the right orbitofrontal gyrus, the left rectus gyrus and bilateral insula, the right inferior temporal gyrus and bilateral transverse temporal gyrus in the temporal region, and the lingual gyrus of the occipital lobe, multiple regions of the basal ganglia and the upper cerebellum. These regions are primarily involved in the attention executive control network and the attention orientation network. Dysfunction in the functional connectivity of these regions may contribute to the underlying causes of ADHD.

Effect of stability of PTEN on hepatocellular carcinoma.

BACKGROUND: As an antioncogene gene, phosphataseandtensinhomolog (PTEN) is closely related to tumorigenesis. However, after mutation, PTEN will lose its function and no longer exert a tumor suppression effect. Through this research, we explored the impact of PTEN mutation on hepatic carcinoma (HCC) and the mechanism of PTEN for regulating HCC. METHODS: First, bioinformatics was used to analyze the prognosis of PTEN in HCC. PTEN-related genes were then further analyzed by the LinkedOmics database, and GO and KEGG functional enrichment analysis were performed. Next, databases were utilized to predict the mutation and mutation frequency of PTEN. Eventually, CRISPR-Cas12a was applied to detect the R130Q mutation on PTEN in clinical samples of HCC. Finally, the fact that miR-92a-3p targets PTEN was identified by dual luciferase reporter gene assays, RT-qPCR, western blot, and rescue experiments. RESULTS: Bioinformatics analysis indicated the high mutation frequency of R130Q/G/L* site on the PTEN gene. Through CRISPR-Cas12a, R130Q mutation was detected on PTEN in 26 out of 40 clinical samples of HCC. CONCLUSIONS: On the one hand, our study revealed that CRISPR-Cas12a might play an important role in the screening and prognosis of HCC as a new clinical method to detect PTEN mutation.

ATP-Independent Turnover of Dinitrogen Intermediates Captured on the Nitrogenase Cofactor.

Nitrogenase reduces N2 to NH3 at its active-site cofactor. Previous studies of an N2-bound Mo-nitrogenase from Azotobacter vinelandii suggest binding of three N2 species via asymmetric belt-sulfur displacements in the two cofactors of its catalytic component (designated Av1*), leading to the proposal of stepwise N2 reduction involving all cofactor belt-sulfur sites; yet, the evidence for the existence of multiple N2 species on Av1* remains elusive. Here we report a study of ATP-independent, EuII/SO3 2--driven turnover of Av1* using GC-MS and frequency-selective pulse NMR techniques. Our data demonstrate incorporation of D2-derived D by Av1* into the products of C2H2- and H+-reduction, and decreased formation of NH3 by Av1* concomitant with the release of N2 under H2; moreover, they reveal a strict dependence of these activities on SO3 2-. These observations point to the presence of distinct N2 species on Av1*, thereby providing strong support for our proposed mechanism of stepwise reduction of N2 via belt-sulfur mobilization.

Decoding emotion with phase-amplitude fusion features of EEG functional connectivity network.

Decoding emotional neural representations from the electroencephalographic (EEG)-based functional connectivity network (FCN) is of great scientific importance for uncovering emotional cognition mechanisms and developing harmonious human-computer interactions. However, existing methods mainly rely on phase-based FCN measures (e.g., phase locking value [PLV]) to capture dynamic interactions between brain oscillations in emotional states, which fail to reflect the energy fluctuation of cortical oscillations over time. In this study, we initially examined the efficacy of amplitude-based functional networks (e.g., amplitude envelope correlation [AEC]) in representing emotional states. Subsequently, we proposed an efficient phase-amplitude fusion framework (PAF) to fuse PLV and AEC and used common spatial pattern (CSP) to extract fused spatial topological features from PAF for multi-class emotion recognition. We conducted extensive experiments on the DEAP and MAHNOB-HCI datasets. The results showed that: (1) AEC-derived discriminative spatial network topological features possess the ability to characterize emotional states, and the differential network patterns of AEC reflect dynamic interactions in brain regions associated with emotional cognition. (2) The proposed fusion features outperformed other state-of-the-art methods in terms of classification accuracy for both datasets. Moreover, the spatial filter learned from PAF is separable and interpretable, enabling a description of affective activation patterns from both phase and amplitude perspectives.

Synthesis of a water-stable CsPbBr3 perovskite for selective detection of mercury ion in water.

By using the method of low-temperature crystallization, CsPbBr3 perovskite nanocrystals (PNCs) coated with trifluoroacetyl lysine (Tfa-Lys) and oleamine (Olam) were synthesized in aqueous solution. The structure of the CsPbBr3 PNCs was characterized by many methods, such as ultraviolet (UV)-visible absorption spectrophotometer, fluorescence spectrophotometer, transmission electron microscopy (TEM), and X-ray diffraction (XRD) pattern. The fluorescence emission of the CsPbBr3 PNCs is stable in water for about 1 day at room temperature. It was also found that the fluorescence of the PNCs could be obviously and selectively quenched after the addition of mercury ion (Hg2+ ), allowing a visual detection of Hg2+ by the naked eye under UV light illumination. The fluorescence quenching rate (I0 /I) has a good linear relationship with the addition of Hg2+ in the concentration range 0.075 to 1.5 mg/L, with a correlation coefficient (R2 ) of 0.997, and limit of detection of 0.046 mg/L. The fluorescence quenching mechanism of the PNCs was determined by the fluorescence lifetime and X-ray photoelectron spectroscopy (XPS) of the PNCs. Overall, the synthesis method for CsPbBr3 PNCs is simple and rapid, and the as-prepared PNCs are stable in water that could be conveniently used for selective detection of Hg2+ in the water environment.

Heterologous expression of a fully active Azotobacter vinelandii nitrogenase Fe protein in Escherichia coli.

The functional versatility of the Fe protein, the reductase component of nitrogenase, makes it an appealing target for heterologous expression, which could facilitate future biotechnological adaptations of nitrogenase-based production of valuable chemical commodities. Yet, the heterologous synthesis of a fully active Fe protein of Azotobacter vinelandii (AvNifH) in Escherichia coli has proven to be a challenging task. Here, we report the successful synthesis of a fully active AvNifH protein upon co-expression of this protein with AvIscS/U and AvNifM in E. coli. Our metal, activity, electron paramagnetic resonance, and X-ray absorption spectroscopy/extended X-ray absorption fine structure (EXAFS) data demonstrate that the heterologously expressed AvNifH protein has a high [Fe4S4] cluster content and is fully functional in nitrogenase catalysis and assembly. Moreover, our phylogenetic analyses and structural predictions suggest that AvNifM could serve as a chaperone and assist the maturation of a cluster-replete AvNifH protein. Given the crucial importance of the Fe protein for the functionality of nitrogenase, this work establishes an effective framework for developing a heterologous expression system of the complete, two-component nitrogenase system; additionally, it provides a useful tool for further exploring the intricate biosynthetic mechanism of this structurally unique and functionally important metalloenzyme. IMPORTANCE The heterologous expression of a fully active Azotobacter vinelandii Fe protein (AvNifH) has never been accomplished. Given the functional importance of this protein in nitrogenase catalysis and assembly, the successful expression of AvNifH in Escherichia coli as reported herein supplies a key element for the further development of heterologous expression systems that explore the catalytic versatility of the Fe protein, either on its own or as a key component of nitrogenase, for nitrogenase-based biotechnological applications in the future. Moreover, the "clean" genetic background of the heterologous expression host allows for an unambiguous assessment of the effect of certain nif-encoded protein factors, such as AvNifM described in this work, in the maturation of AvNifH, highlighting the utility of this heterologous expression system in further advancing our understanding of the complex biosynthetic mechanism of nitrogenase.

Rapid Genotyping of Campylobacter coli Strains from Poultry Meat by PFGE, Sau-PCR, and fla-DGGE.

The genotyping of Campylobacter coli was done using three methods, pulsed-field gel electrophoresis (PFGE), Sau-polymerase chain reaction (Sau-PCR), and denaturing gradient gel electrophoresis assay of flagellin gene (fla-DGGE) and the characteristics of these assays were compared. The results showed that a total of 53 strains of C. coli were isolated from chicken and duck samples in three markets. All isolates were clustered into 31, 33, and 15 different patterns with Simpson's index of diversity (SID) values of 0.972, 0.974, and 0.919, respectively. Sau-PCR assay was simpler, more rapid, and had higher discriminatory power than PFGE assay. Fla-DGGE assay could detect and illustrate the number of contamination types of C. jejuni and C. coli without cultivation, which saved more time and cost than Sau-PCR and PFGE assays. Therefore, Sau-PCR and fla-DGGE assays are both rapid, economical, and easy to perform, which have the potential to be promising and accessible for primary laboratories in genotyping C. coli strains.

Heterologous synthesis of the complex homometallic cores of nitrogenase P- and M-clusters in Escherichia coli.

Nitrogenase is an active target of heterologous expression because of its importance for areas related to agronomy, energy, and environment. One major hurdle for expressing an active Mo-nitrogenase in Escherichia coli is to generate the complex metalloclusters (P- and M-clusters) within this enzyme, which involves some highly unique bioinorganic chemistry/metalloenzyme biochemistry that is not generally dealt with in the heterologous expression of proteins via synthetic biology; in particular, the heterologous synthesis of the homometallic P-cluster ([Fe8S7]) and M-cluster core (or L-cluster; [Fe8S9C]) on their respective protein scaffolds, which represents two crucial checkpoints along the biosynthetic pathway of a complete nitrogenase, has yet to be demonstrated by biochemical and spectroscopic analyses of purified metalloproteins. Here, we report the heterologous formation of a P-cluster-containing NifDK protein upon coexpression of Azotobacter vinelandii nifD, nifK, nifH, nifM, and nifZ genes, and that of an L-cluster-containing NifB protein upon coexpression of Methanosarcina acetivorans nifB, nifS, and nifU genes alongside the A. vinelandii fdxN gene, in E. coli. Our metal content, activity, EPR, and XAS/EXAFS data provide conclusive evidence for the successful synthesis of P- and L-clusters in a nondiazotrophic host, thereby highlighting the effectiveness of our metallocentric, divide-and-conquer approach that individually tackles the key events of nitrogenase biosynthesis prior to piecing them together into a complete pathway for the heterologous expression of nitrogenase. As such, this work paves the way for the transgenic expression of an active nitrogenase while providing an effective tool for further tackling the biosynthetic mechanism of this important metalloenzyme.

Music-emotion EEG coupling effects based on representational similarity.

BACKGROUND: Music can evoke intense emotions and music emotion is a complex cognitive process. However, we know little about the cognitive mechanisms underlying these processes, and there are significant individual differences in the emotional responses to the same musical stimuli. NEW METHOD: We used the inter-subject representational similarity analysis (IS-RSA) method to investigate the shared music emotion responses across multiple participants. In addition, we extended IS-RSA to estimate the group cross-frequency coupling effects of music emotion. Based on the cross-frequency coupling IS-RSA, we analyzed the differences in cross-frequency coupling patterns under different music emotions using MI. Comparison of existing methods: most current IS-RSA analyses focus on within-frequency band analysis. However, the cognitive processing of music emotion involves not only activation and brain network connections differences within frequency bands but also information communication between frequency bands. RESULTS: The results of the within-frequency band IS-RSA analysis showed that the theta and gamma frequency bands play important roles in the inter-participant consistency of music emotion. The inter-frequency band IS-RSA analysis showed that the theta-beta coupling pattern exhibited stronger inter-participant consistency compared to the theta-gamma coupling pattern, and the theta-beta coupling had significant consistent representation across various music conditions. Through the significant regions of cross-frequency coupling representation similarity analysis, we performed phase-amplitude coupling analysis on FC4-C6 and FC4-Pz connections. For the theta-beta coupling pattern, we found that the MI of these two connections exhibited different coupling patterns under different music conditions, and they showed a significant decrease compared to the baseline period.

Light-driven Transformation of Carbon Monoxide into Hydrocarbons using CdS@ZnS : VFe Protein Biohybrids.

Enzymatic Fisher-Tropsch (FT) process catalyzed by vanadium (V)-nitrogenase can convert carbon monoxide (CO) to longer-chain hydrocarbons (>C2) under ambient conditions, although this process requires high-cost reducing agent(s) and/or the ATP-dependent reductase as electron and energy sources. Using visible light-activated CdS@ZnS (CZS) core-shell quantum dots (QDs) as alternative reducing equivalent for the catalytic component (VFe protein) of V-nitrogenase, we first report a CZS : VFe biohybrid system that enables effective photo-enzymatic C-C coupling reactions, hydrogenating CO into hydrocarbon fuels (up to C4) that can be hardly achieved with conventional inorganic photocatalysts. Surface ligand engineering optimizes molecular and opto-electronic coupling between QDs and the VFe protein, realizing high efficiency (internal quantum yield >56 %), ATP-independent, photon-to-fuel production, achieving an electron turnover number of >900, that is 72 % compared to the natural ATP-coupled transformation of CO into hydrocarbons by V-nitrogenase. The selectivity of products can be controlled by irradiation conditions, with higher photon flux favoring (longer-chain) hydrocarbon generation. The CZS : VFe biohybrids not only can find applications in industrial CO removal for high-value-added chemical production by using the cheap, renewable solar energy, but also will inspire related research interests in understanding the molecular and electronic processes in photo-biocatalytic systems.

Hierarchical Hybrid Networks for Automatic Pulmonary Blood Vessel Segmentation in Computed Tomography Images.

Pulmonary arterial hypertension (PAH) is considered the third most common cardiovascular disease after coronary heart disease and hypertension. The diagnosis of PAH is mainly based on the comprehensive judgment of computed tomography and other medical image examinations. Medical image processing based on deep learning has achieved significant success. However, the data belongs to the patient's privacy; therefore, the medical institutions as data custodians have the responsibility to protect the security of their data privacy. This situation makes medical institutions face a dilemma when building data-driven deep learning-assisted medical diagnosis methods. On the one hand, they need to pursue more high-quality data based on Big Data architecture for deep learning; on the other hand, they need to protect patient privacy to avoid data leakage. In response to the above challenges, we propose a hierarchical hybrid automatic segmentation model for pulmonary blood vessels based on local learning and federated learning approaches for segmenting the pulmonary blood vessels. The experiments prove the proposal could automatically segment the vessels from the original CT. It also indicates that the model based on a federated learning approach can achieve impressive performance under the premise of protecting data privacy for Big Data.

Interest-driven and legal supervision innovation of the capital pool model in Chinese banking financial products.

This paper studies the formation, interest-driven rationality and potential risks of the capital pool model in China's banking financial management business, as well as the correlation, coincidence and complexity of fund pool prohibition and the rigid payment strategy. Focusing on "The New Regulations on Asset Management" issued by the Chinese government in April 2018, this paper discusses the regulatory effects and existing problems of fund pool prohibition and rigid payment regulations. From the perspective of theoretical and empirical analysis, this paper studies the impact of the relationship between financial product yield and regulatory interest rate relationship on shadow banking. The paper also studies the capital pool model that is closely related to the shadow banking, rigid payment and unstandardized debts, so as to puts forward relevant policy suggestions on improving the external regulation and optimizing the internal control mechanism of shadow banking. This paper puts forward that pursuit of financial security value should not be independent from the development of the overall interests of asset management market. The reasonable and healthy development of the asset management industry should be guided by the principle of controlling the risks at an appropriate level. The regulations in relation to capital pool and rigid payment needs more flexibility and elasticity to help reduce or eliminate the negative impact on the efficiency of resource allocation in the asset management industry. Shadow banking plays an important role in the financing of small and medium-sized enterprises, which is the result of mutual competition and game between different banks in terms of yield rate. Moderate shadow banking scale plays a positive role in the macro economy. This argument has theoretical value and practical significance to ensure that the regulatory system is resilient to the financial system in the most effective way possible.

Dishevelled segment polarity protein 2 promotes gastric cancer progression through Wnt/ß-catenin pathway.

Dishevelled family proteins (DVL1-3), key scaffold proteins, act on canonical and non-canonical Wnt/ß-catenin signaling pathway. DVL has been implicated in various tumor progression. However, its role and underlying mechanisms in gastric cancer (GC) remain unclear. The aim of this study was to investigate the role of DVL in GC development using cell lines and 209 GC specimens. We analyzed three orthologs of DVL in GC tissues and paired adjacent non-tumor tissues, and only DVL2 is highly expressed in GC tissues. We also analyzed clinicopathological data on DVL2 expression in gastric cancer specimens. In immunohistochemistry, DVL2 expression was up-regulated in GC tissues compared with paired adjacent non-tumor tissues (153/209, 73.2%). DVL2 expression level was significantly correlated with many clinicopathological parameters such as T stage (P < 0.001) and N stage (P < 0.001). Survival analysis showed that the overall survival (OS) of patients with high expression of DVL2 was significantly shorter than those with low expression. Multivariate Cox regression analysis revealed that DVL2 expression was an important and independent prognostic factor for gastric cancer patients (P = 0.011, HR=1.78, 95%CI (1.14-2.79). Depletion of endogenous DVL2 using short hairpin RNA (shRNA) inhibited GC cell proliferation, migration, and invasion. The abnormal activation of Wnt/ß-catenin signaling pathway is mainly achieved through the abnormal expression of DVL2. DVL2 is highly expressed in gastric cancer tissues, which may be a new independent risk factor for the prognosis of gastric cancer patients. In gastric cancer, DVL2 overexpression plays a crucial role in the occurrence and development of gastric cancer, so it may become a new, effective and complementary therapeutic target for gastric cancer.

Enzymatic Fischer-Tropsch-Type Reactions.

The Fischer-Tropsch (FT) process converts a mixture of CO and H2 into liquid hydrocarbons as a major component of the gas-to-liquid technology for the production of synthetic fuels. Contrary to the energy-demanding chemical FT process, the enzymatic FT-type reactions catalyzed by nitrogenase enzymes, their metalloclusters, and synthetic mimics utilize H+ and e- as the reducing equivalents to reduce CO, CO2, and CN- into hydrocarbons under ambient conditions. The C1 chemistry exemplified by these FT-type reactions is underscored by the structural and electronic properties of the nitrogenase-associated metallocenters, and recent studies have pointed to the potential relevance of this reactivity to nitrogenase mechanism, prebiotic chemistry, and biotechnological applications. This review will provide an overview of the features of nitrogenase enzymes and associated metalloclusters, followed by a detailed discussion of the activities of various nitrogenase-derived FT systems and plausible mechanisms of the enzymatic FT reactions, highlighting the versatility of this unique reactivity while providing perspectives onto its mechanistic, evolutionary, and biotechnological implications.

Subtyping of Campylobacter coli isolated from raw poultry meat in retail markets using amplified intergenic locus polymorphism - A novel rapid subtyping method.

In order to provide more phylogenetic information of Campylobacter coli in large-scale epidemiological investigation, this work was undertaken to develop a novel genotyping method based on amplified intergenic locus polymorphism (AILP), by using pulsed-field gel electrophoresis (PFGE; using SmaI enzymes) as control. Eleven pairs of primers were selected to type C. coli strains for this purpose. A total of 68 C. coli isolates recovered from 51 retail raw chicken and 37 retail raw duck were subtyped. The Simpson's index of diversity (SID) of AILP and PFGE, as well as the adjusted Rand index (AR) and the adjusted Wallace coefficient (AW) between AILP and PFGE, were calculated. The new AILP method differentiated 68 C. coli isolates into 55 different subtypes (SID = 0.993), compared with 46 different profiles obtained from PFGE (SID = 0.980). The SID value of the AILP method was improved with the increasing number of primers, and a combination of 7 loci was selected as the optimal combination. The congruent analysis of the AILP method and PFGE showed moderate congruence between the two methods (AR = 0.462). The AW indicated that if AILP data is the available one can confidently predict the PFGE cluster. The results of this study showed that the AILP method had higher discrimination than PFGE and also allowed for significant reduction in time and cost.

Nitrogenase Fe Protein: A Multi-Tasking Player in Substrate Reduction and Metallocluster Assembly.

The Fe protein of nitrogenase plays multiple roles in substrate reduction and metallocluster assembly. Best known for its function to transfer electrons to its catalytic partner during nitrogenase catalysis, the Fe protein is also a key player in the biosynthesis of the complex metalloclusters of nitrogenase. In addition, it can function as a reductase on its own and affect the ambient reduction of CO2 or CO to hydrocarbons. This review will provide an overview of the properties and functions of the Fe protein, highlighting the relevance of this unique FeS enzyme to areas related to the catalysis, biosynthesis, and applications of the fascinating nitrogenase system.

Identification of Gene Coexpression Modules and Prognostic Genes Associated with Papillary Thyroid Cancer.

Thyroid cancer is a great part of the endocrine tumor with an increasing incidence. Papillary thyroid carcinoma (PTC) is the most common subtype. With the enormous pace taken in the microarray technology, bioinformatics is applied in data mining more frequently. Weighted gene coexpression network analysis (WGCNA) can perform analysis combining clinic information. We performed WGCNA for prognostic genes associated with PTC. From the GEO profile, we got ten modules. We identified a key module that was closest to patients' survival time. Then, we screened five hub genes (ATRX, BOD1L1, CEP290, DCAF16, and NEK1) from the key module based on the clinical information from TCGA. These five genes not only significantly differ between the normal and tumor groups but have prognostic value. The receiver operating characteristic (ROC) curve indicated that they had the potential to serve as prognostic genes. We performed next-generation sequencing using the PTC tissue to get more convincing evidence. Besides, we established a new signature and verified it through K-M plots and ROC. The signature could be an independent factor for the prognosis of PTC, and we built a nomogram to carry out a quantitative study. In a word, the hub genes we explored in the study deserved more basic and clinical research.

Evidence of substrate binding and product release via belt-sulfur mobilization of the nitrogenase cofactor.

The Mo-nitrogenase catalyses the ambient reduction of N2 to NH3 at the M-cluster, a complex cofactor that comprises two metal-sulphur partial cubanes ligated by an interstitial carbide and three belt-sulphurs. A recent crystallographic study suggests binding of N2 via displacement of the belt-sulphur(s) of the M-cluster upon turnover. However, the direct proof of N2 binding and belt-sulphur mobilization during catalysis remains elusive. Here we show that N2 is captured on the M-cluster via electron- and sulphur-depletion, and that the N2-captured state is catalytically competent in generating NH3. Moreover, we demonstrate that product release only occurs when sulphite is supplied along with a reductant, that sulphite is inserted as sulphide into the belt-sulphur displaced positions, and that there is a dynamic in-and-out of the belt-sulphurs during catalysis. Together, these results establish the mobilization of the cofactor belt-sulphurs as a crucial, yet overlooked, mechanistic element of the nitrogenase reaction.