Unconventional mechanism of action and resistance to rapalogs in renal cancer.

mTORC1 is aberrantly activated in renal cell carcinoma (RCC) and is targeted by rapalogs. As for other targeted therapies, rapalogs clinical utility is limited by the development of resistance. Resistance often results from target mutation, but mTOR mutations are rarely found in RCC. As in humans, prolonged rapalog treatment of RCC tumorgrafts (TGs) led to resistance. Unexpectedly, explants from resistant tumors became sensitive both in culture and in subsequent transplants in mice. Notably, resistance developed despite persistent mTORC1 inhibition in tumor cells. In contrast, mTORC1 became reactivated in the tumor microenvironment (TME). To test the role of the TME, we engineered immunocompromised recipient mice with a resistance mTOR mutation (S2035T). Interestingly, TGs became resistant to rapalogs in mTORS2035T mice. Resistance occurred despite mTORC1 inhibition in tumor cells and could be induced by coculturing tumor cells with mutant fibroblasts. Thus, enforced mTORC1 activation in the TME is sufficient to confer resistance to rapalogs. These studies highlight the importance of mTORC1 inhibition in nontumor cells for rapalog antitumor activity and provide an explanation for the lack of mTOR resistance mutations in RCC patients.

Impact of summer hypoxia on macrobenthic communities in a semi-enclosed bay: A long-term observation in the North Yellow sea of China.

The O2 content of the global ocean has been declining progressively over the past decades, mainly because of human activities and global warming. Despite this situation, the responses of macrobenthos under hypoxic conditions remain poorly understood. In this study, we conducted a long-term observation (2015-2022) to investigate the intricate impact of summer hypoxia on macrobenthic communities in a semi-enclosed bay of the North Yellow Sea. Comparative analyses revealed higher macrobenthos abundance (1956.8 ± 1507.5 ind./m2 vs. 871.8 ± 636.9 ind./m2) and biomass (8.2 ± 4.1 g/m2 vs. 5.6 ± 3.2 g/m2) at hypoxic sites compared to normoxic sites during hypoxic years. Notably, polychaete species demonstrated remarkable adaptability, dominating hypoxic sites, and shaping community structure. The decline in biodiversity underscored the vulnerability and diminished resilience of macrobenthic communities to hypoxic stressors. Stable isotope analysis provided valuable insights into food web structures. The average trophic level of macrobenthos measured 2.84 ± 0.70 at hypoxic sites, contrasting with the higher value of 3.14 ± 0.74 observed at normoxic sites, indicating the absence of predators at high trophic levels under hypoxic conditions. Moreover, trophic interactions were significantly altered, resulting in a simplified and more vulnerable macrobenthic trophic structure. The findings underscored the importance of comprehensive research to understand the complex responses of macrobenthic communities to hypoxia, thereby informing future conservation efforts in impacted ecosystems.

Targeting renal cell carcinoma with a HIF-2 antagonist.

Clear cell renal cell carcinoma (ccRCC) is characterized by inactivation of the von Hippel-Lindau tumour suppressor gene (VHL). Because no other gene is mutated as frequently in ccRCC and VHL mutations are truncal, VHL inactivation is regarded as the governing event. VHL loss activates the HIF-2 transcription factor, and constitutive HIF-2 activity restores tumorigenesis in VHL-reconstituted ccRCC cells. HIF-2 has been implicated in angiogenesis and multiple other processes, but angiogenesis is the main target of drugs such as the tyrosine kinase inhibitor sunitinib. HIF-2 has been regarded as undruggable. Here we use a tumourgraft/patient-derived xenograft platform to evaluate PT2399, a selective HIF-2 antagonist that was identified using a structure-based design approach. PT2399 dissociated HIF-2 (an obligatory heterodimer of HIF-2α-HIF-1ß) in human ccRCC cells and suppressed tumorigenesis in 56% (10 out of 18) of such lines. PT2399 had greater activity than sunitinib, was active in sunitinib-progressing tumours, and was better tolerated. Unexpectedly, some VHL-mutant ccRCCs were resistant to PT2399. Resistance occurred despite HIF-2 dissociation in tumours and evidence of Hif-2 inhibition in the mouse, as determined by suppression of circulating erythropoietin, a HIF-2 target and possible pharmacodynamic marker. We identified a HIF-2-dependent gene signature in sensitive tumours. Gene expression was largely unaffected by PT2399 in resistant tumours, illustrating the specificity of the drug. Sensitive tumours exhibited a distinguishing gene expression signature and generally higher levels of HIF-2α. Prolonged PT2399 treatment led to resistance. We identified binding site and second site suppressor mutations in HIF-2α and HIF-1ß, respectively. Both mutations preserved HIF-2 dimers despite treatment with PT2399. Finally, an extensively pretreated patient whose tumour had given rise to a sensitive tumourgraft showed disease control for more than 11 months when treated with a close analogue of PT2399, PT2385. We validate HIF-2 as a target in ccRCC, show that some ccRCCs are HIF-2 independent, and set the stage for biomarker-driven clinical trials.

An integrated approach using chemical ecological risk assessment and multi-integrated biomarker indexes approach to assess pollution: A case study of Ruditapes philippinarum in four bays on the Shandong Peninsula in China.

Considering the ecological risks of polycyclic aromatic hydrocarbons (PAHs) to the marine environment, it is urgent to find scientific and effective monitoring methods. In this study, an integrated approach combining chemical ecological risk assessment and multi-integrated biomarker indexes approach was used to assess the marine environment. Samples included seawater, sediments, and clam Ruditapes philippinarum were collected from four bays on the Shandong Peninsula, China in the four seasons of 2019. The concentrations, composition, potential sources, and ecological risk of PAHs were investigated in seawater and sediments. Risk quotient (RQ) and sediment quality guidelines (SQGs) were calculated to assess the ecological risks of PAHs in seawater and sediment, respectively. And then, clam Ruditapes philippinarum's multi-level biological response, including its ethoxyresorufin-O-deethylase (EROD), glutathione S-transferase (GST), superoxide dismutase (SOD), lipid peroxidation (LPO), and acetylcholinesterase (AChE) were investigated in-depth, by which multi-integrated biomarker indexes approach were calculated to evaluate marine environmental quality. Taken together, the results showed that the concentration of PAHs was in good agreement with the response of biomarkers, and the usefulness of the combined use of chemical ecological risk assessment and integrated biomarker indexes to assess PAHs pollution was verified.

Integration of chemical and biological methods: A case study of polycyclic aromatic hydrocarbons pollution monitoring in Shandong Peninsula, China.

Polycyclic aromatic hydrocarbons (PAHs), as persistent toxic substances (PTS), have been widely monitored in coastal environment, including seawater and sediment. However, scientific monitoring methods, like ecological risk assessment and integrated biomarker response, still need massive researches to verify their availabilities. This study was performed in March, May, August and October of 2018 at eight sites, Yellow River estuary (S1), Guangli Port (S2), Xiaying (S3), Laizhou (S4), Inner Bay (S5), Outer Bay (S6), Hongdao (S7) and Hongshiya (S8) of Shandong Peninsula, China. The contents of 16 priority PAHs in local seawater and sediment were determined, by which ecological risk assessment risk quotient (RQ) for seawater and sediment quality guidelines (SQGs) were calculated to characterize the PAHs pollution. Meanwhile, multiple biomarkers in the digestive gland of clam Ruditapes philippinarum were measured to represent different biological endpoints, including ethoxyresorufin-O-deethylase (EROD), glutathione S-transferase (GST), sulfotransferase (SULT), superoxide dismutase (SOD) and lipid peroxidation (LPO), by which integrated biomarker response (IBR) was calculated to provide a comprehensive assessment of environmental quality. Taken together, these results revealed the heaviest pollution at S2 as both PAHs concentrations and biomarkers responses reflected, and supported the integrated biomarker response as a useful tool for marine environmental monitoring, through its integration with SQGs.

How does T cell receptor clustering impact on signal transduction?

The essential function of the T cell receptor (TCR) is to translate the engagement of peptides on the major histocompatibility complex (pMHC) into appropriate intracellular signals through the associated cluster of differentiation 3 (CD3) complex. The spatial organization of the TCR-CD3 complex in the membrane is thought to be a key regulatory element of signal transduction, raising the question of how receptor clustering impacts on TCR triggering. How signal transduction at the TCR-CD3 complex encodes the quality and quantity of pMHC molecules is not fully understood. This question can be approached by reconstituting T cell signaling in model and cell membranes and addressed by single-molecule imaging of endogenous proteins in T cells. We highlight such methods and further discuss how TCR clustering could affect pMHC rebinding rates, the local balance between kinase and phosphatase activity and/or the lipid environment to regulate the signal efficiency of the TCR-CD3 complex. We also examine whether clustering could affect the conformation of cytoplasmic CD3 tails through a biophysical mechanism. Taken together, we highlight how the spatial organization of the TCR-CD3 complex - addressed by reconstitution approaches - has emerged as a key regulatory element in signal transduction of this archetypal immune receptor.

Investigating Spatial Heterogeneity of Nanoparticles Movement in Live Cells with Pair-Correlation Microscopy and Phasor Analysis.

How nanoparticles distribute in living cells and overcome cellular barriers are important criteria in the design of drug carriers. Pair-correlation microscopy is a correlation analysis of fluctuation in the fluorescence intensity obtained by a confocal line scan that can quantify the dynamic properties of nanoparticle diffusion including the number of mobile nanoparticles, diffusion coefficient, and transit time across a spatial distance. Due to the potential heterogeneities in nanoparticle properties and the complexity within the cellular environment, quantification of averaged auto- and pair-correlation profiles may obscure important insights into the ability of nanoparticles to deliver drugs. To overcome this issue, we used phasor analysis to develop a data standardizing method, which can segment the scanned line into several subregions according to diffusion and address the spatial heterogeneity of nanoparticles moving inside cells. The phasor analysis is a fit-free method that represents autocorrelation profiles for each pixel relative to free diffusion on the so-called phasor plots. Phasor plots can then be used to select subpopulations for which the auto- and pair-correlation analysis can be performed separately. We demonstrate the phasor analysis for pair-correlation microscopy for investigating 16 nm, Cy5-labeled silica nanoparticles diffusing across the plasma membrane and green fluorescent proteins (GFP) diffusing across nuclear envelope in MCF-7 cells.

Conformational States Control Lck Switching between Free and Confined Diffusion Modes in T Cells.

T cell receptor phosphorylation by Lck is an essential step in T cell activation. It is known that the conformational states of Lck control enzymatic activity; however, the underlying principles of how Lck finds its substrate over the plasma membrane remain elusive. Here, single-particle tracking is paired with photoactivatable localization microscopy to observe the diffusive modes of Lck in the plasma membrane. Individual Lck molecules switched between free and confined diffusion in both resting and stimulated T cells. Lck mutants locked in the open conformation were more confined than Lck mutants in the closed conformation. Further confinement of kinase-dead versions of Lck suggests that Lck confinement was not caused by phosphorylated substrates. Our data support a model in which confined diffusion of open Lck results in high local phosphorylation rates, and inactive, closed Lck diffuses freely to enable long-range distribution over the plasma membrane.

Clustering of the ζ-Chain Can Initiate T Cell Receptor Signaling.

T cell activation is initiated when ligand binding to the T cell receptor (TCR) triggers intracellular phosphorylation of the TCR-CD3 complex. However, it remains unknown how biophysical properties of TCR engagement result in biochemical phosphorylation events. Here, we constructed an optogenetic tool that induces spatial clustering of ζ-chain in a light controlled manner. We showed that spatial clustering of the ζ-chain intracellular tail alone was sufficient to initialize T cell triggering including phosphorylation of ζ-chain, Zap70, PLCγ, ERK and initiated Ca2+ flux. In reconstituted COS-7 cells, only Lck expression was required to initiate ζ-chain phosphorylation upon ζ-chain clustering, which leads to the recruitment of tandem SH2 domain of Zap70 from cell cytosol to the newly formed ζ-chain clusters at the plasma membrane. Taken together, our data demonstrated the biophysical relevance of receptor clustering in TCR signaling.

Human Indoleamine 2,3-Dioxygenase 1 Is an Efficient Mammalian Nitrite Reductase.

The heme enzyme indoleamine 2,3-dioxygenase-1 (IDO1) catalyzes the first reaction of l-tryptophan oxidation along the kynurenine pathway. IDO1 is a central immunoregulatory enzyme with important implications for inflammation, infectious disease, autoimmune disorders, and cancer. Here we demonstrate that IDO1 is a mammalian nitrite reductase capable of chemically reducing nitrite to nitric oxide (NO) under hypoxia. Ultraviolet-visible absorption and resonance Raman spectroscopy showed that incubation of dithionite-reduced, ferrous-IDO1 protein (FeII-IDO1) with nitrite under anaerobic conditions resulted in the time-dependent formation of an FeII-nitrosyl IDO1 species, which was inhibited by substrate l-tryptophan, dependent on the concentration of nitrite or IDO1, and independent of the concentration of the reductant, dithionite. The bimolecular rate constant for IDO1 nitrite reductase activity was determined as 5.4 M-1 s-1 (pH 7.4, 23 °C), which was comparable to that measured for myoglobin (3.6 M-1 s-1; pH 7.4, 23 °C), an efficient and biologically important mammalian heme-based nitrite reductase. IDO1 nitrite reductase activity was pH-dependent but differed with myoglobin in that it showed a reduced proton dependency at pH >7. Electron paramagnetic resonance studies measuring NO production showed that the conventional IDO1 dioxygenase reducing cofactors, ascorbate and methylene blue, enhanced IDO1's nitrite reductase activity and the time- and IDO1 concentration-dependent release of NO in a manner inhibited by l-tryptophan or the IDO inhibitor 1-methyl-l-tryptophan. These data identify IDO1 as an efficient mammalian nitrite reductase that is capable of generating NO under anaerobic conditions. IDO1's nitrite reductase activity may have important implications for the enzyme's biological actions when expressed within hypoxic tissues.

Functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination.

Antigen recognition by the T-cell receptor (TCR) is a hallmark of the adaptive immune system. When the TCR engages a peptide bound to the restricting major histocompatibility complex molecule (pMHC), it transmits a signal via the associated CD3 complex. How the extracellular antigen recognition event leads to intracellular phosphorylation remains unclear. Here, we used single-molecule localization microscopy to quantify the organization of TCR-CD3 complexes into nanoscale clusters and to distinguish between triggered and nontriggered TCR-CD3 complexes. We found that only TCR-CD3 complexes in dense clusters were phosphorylated and associated with downstream signaling proteins, demonstrating that the molecular density within clusters dictates signal initiation. Moreover, both pMHC dose and TCR-pMHC affinity determined the density of TCR-CD3 clusters, which scaled with overall phosphorylation levels. Thus, TCR-CD3 clustering translates antigen recognition by the TCR into signal initiation by the CD3 complex, and the formation of dense signaling-competent clusters is a process of antigen discrimination.

Time-Resolved Laurdan Fluorescence Reveals Insights into Membrane Viscosity and Hydration Levels.

Membrane viscosity and hydration levels characterize the biophysical properties of biological membranes and are reflected in the rate and extent of solvent relaxation, respectively, of environmentally sensitive fluorophores such as Laurdan. Here, we first developed a method for a time-resolved general polarization (GP) analysis with fluorescence-lifetime imaging microscopy that captures both the extent and rate of Laurdan solvent relaxation. We then conducted time-resolved GP measurements with Laurdan-stained model membranes and cell membranes. These measurements revealed that cholesterol levels in lipid vesicles altered membrane hydration and viscosity, whereas curvature had little effect on either parameter. We also applied the method to the plasma membrane of live cells using a supercritical angle fluorescence objective, to our knowledge the first time fluorescence-lifetime imaging microscopy images were generated with supercritical angle fluorescence. Here, we found that local variations in membrane cholesterol most likely account for the heterogeneity of Laurdan lifetime in plasma membrane. In conclusion, time-resolved GP measurements provide additional insights into the biophysical properties of membranes.

FSCS Reveals the Complexity of Lipid Domain Dynamics in the Plasma Membrane of Live Cells.

The coexistence of lipid domains with different degrees of lipid packing in the plasma membrane of mammalian cells has been postulated, but direct evidence has so far been challenging to obtain because of the small size and short lifetime of these domains in live cells. Here, we use fluorescence spectral correlation spectroscopy in conjunction with a probe sensitive to the membrane environment to quantify spectral fluctuations associated with dynamics of membrane domains in live cells. With this method, we show that membrane domains are present in live COS-7 cells and have a lifetime lower bound of 5.90 and 14.69 ms for the ordered and disordered phases, respectively. Comparisons to simulations indicate that the underlying mechanism of these fluctuations is complex but qualitatively described by a combination of dye diffusion between membrane domains as well as the motion of domains within the membrane.

Ultralow- and Low-Background Surfaces for Single-Molecule Localization Microscopy of Multistep Biointerfaces for Single-Molecule Sensing.

Single-molecule localization microscopy (SMLM) has created the opportunity of pushing fluorescence microscopy from being a biological imaging tool to a surface characterization and possibly even a quantitative analytical tool. The latter could be achieved by molecular counting using pointillist SMLM data sets. However, SMLM is especially sensitive to background fluorescent signals, which influences any subsequent analysis. Therefore, fabricating sensing surfaces that resist nonspecific adsorption of proteins, even after multiple modification steps, has become paramount. Herein is reported two different ways to modify surfaces: dichlorodimethylsilane-biotinylated bovine serum albumin-Tween-20 (DbT20) and poly-l-lysine grafted polyethylene glycol (PLL-PEG) mixed with biotinylated PLL-PEG (PLL-PEG/PEGbiotin). The results show that the ability to resist nonspecific adsorption of DbT20 surfaces deteriorates with an increase in the number of modification steps required after the addition of the DbT20, which limits the applicability of this surface for SMLM. As such, a new surface for SMLM that employs PLL-PEG/PEGbiotin was developed that exhibits ultralow amounts of nonspecific protein adsorption even after many modification steps. The utility of the surface was demonstrated for human influenza hemagglutinin-tagged mEos2, which was directly pulled down from cell lysates onto the PLL-PEG/PEGbiotin surface. The results strongly indicated that the PLL-PEG/PEGbiotin surface satisfies the criteria of SMLM imaging of a negligible background signal and negligible nonspecific adsorption.

The Chromatin Remodeling Protein Bptf Promotes Posterior Neuroectodermal Fate by Enhancing Smad2-Activated wnt8a Expression.

During vertebrate embryogenesis, the neuroectoderm is induced from dorsal ectoderm and then partitioned into anterior and posterior neuroectodermal domains by posteriorizing signals, such as Wnt and fibroblast growth factor. However, little is known about epigenetic regulation of posteriorizing gene expression. Here, we report a requirement of the chromatin remodeling protein Bptf for neuroectodermal posteriorization in zebrafish embryos. Knockdown of bptf leads to an expansion of the anterior neuroectoderm at the expense of the posterior ectoderm. Bptf functionally and physically interacts with p-Smad2, which is activated by non-Nodal TGF-ß signaling, to promote the expression of wnt8a, a critical gene for neural posteriorization. Bptf and Smad2 directly bind to and activate the wnt8a promoter through recruiting NURF remodeling complex. When bptf function or TGF-ß signal transduction is inhibited, the nucleosome density on the wnt8a promoter is increased. We propose that Bptf and TGF-ß/Smad2 mediate nucleosome remodeling to regulate wnt8a expression and hence neural posteriorization.

Self-calibrated line-scan STED-FCS to quantify lipid dynamics in model and cell membranes.

Only a limited number of noninvasive techniques are available to directly measure the dynamic behavior of lipids in model and cell membranes. Here, we explored whether a commercial instrument could be used for fluorescence correlation spectroscopy (FCS) under pulsed stimulated emission depletion (STED). To overcome issues with photobleaching and poor distinction between confocal and STED signals, we implemented resonant line-scan STED with filtered FCS, which has the additional benefit of autocalibrating the dimensions of the point-spread function and obtaining spatially resolved molecular mobility at subdiffraction resolution. With supported lipid bilayers, we achieved a detection spot radius of 40 nm, although at the expense of decreased molecular brightness. We also used this approach to map the dynamics of Atto646N-labeled sphingomyelin and phosphatidylethanolamine in the plasma membrane. Despite the reliability of the method and the demonstration that photobleaching and the photophysical properties of the dye did not influence diffusion measurements, we found great heterogeneities even within one cell. For both lipids, regions of high local density correlated with slow molecular diffusion, indicating trapping of Atto646N-labeled lipids. Future studies with new dyes are needed to reveal the origin of the trapping.

Typhoon-induced stormwater drives nutrient dynamics and triggers phytoplankton blooms in Laizhou Bay, China.

In this study, we investigated the hydrological and ecological impacts of heavy rainfall caused by the storm Rumbia and Typhoon Lekima on Laizhou Bay (LZB) through land‒sea synchronous field surveys, online remote sensors, and simulated enclosure experiments. Within two weeks of Rumbia, approximately 9% and 16% of the annual riverine total nitrogen (TN) and total phosphorus (TP) fluxes, respectively, were transported to the LZB and the proportions were 17% and 35%, respectively, for Lekima. The land use on the watersheds increased the rates of land-derived nutrient loading and altered their biogeochemical forms. Consequently, the average concentrations of dissolved inorganic nitrogen (DIN) and phosphorus (DIP) in the LZB increased by 2.6 and 1.0 times post-Rumbia and by 3.5 and 1.3 times post-Lekima, respectively. Relatively lower salinity and temperature, sudden increases in DIN, and strengthened coastal currents stimulated the growth of highly adaptable and small diatoms, resulting in the first diatom blooms. Subsequently, a bloom of Noctiluca scintillans formed.

Biodiversity and distribution patterns of blooming jellyfish in the Bohai Sea revealed by eDNA metabarcoding.

BACKGROUND: The mass occurrence of scyphozoan jellyfish severely affects marine ecosystems and coastal economies, and the study of blooming jellyfish population dynamics has emerged in response. However, traditional ecological survey methods required for such research have difficulties in detecting cryptic life stages and surveying population dynamics owing to high spatiotemporal variations in their occurrence. The environmental DNA (eDNA) technique is an effective tool for overcoming these limitations. RESULTS: In this study, we investigated the biodiversity and spatial distribution characteristics of blooming jellyfish in the Bohai Sea of China using an eDNA metabarcoding approach, which covered the surface, middle, and bottom seawater layers, and sediments. Six jellyfish taxa were identified, of which Aurelia coerulea, Nemopilema nomurai, and Cyanea nozakii were the most dominant. These three blooming jellyfish presented a marked vertical distribution pattern in the offshore regions. A. coerulea was mainly distributed in the surface layer, whereas C. nozakii and N. nomurai showed a upper-middle and middle-bottom aggregation, respectively. Horizontally, A. coerulea and C. nozakii were more abundant in the inshore regions, whereas N. nomurai was mainly distributed offshore. Spearman's correlation analysis revealed a strong correlation between the eDNA of the three dominant blooming jellyfish species and temperature, salinity, and nutrients. CONCLUSIONS: Our study confirms the applicability of the eDNA approach to both biodiverstiy evaluation of blooming jellyfish and investigating their spatial distribution, and it can be used as a supplementary tool to traditional survey methods.

Genomic and single-cell analyses reveal genetic signatures of swimming pattern and diapause strategy in jellyfish.

Jellyfish exhibit innovative swimming patterns that contribute to exploring the origins of animal locomotion. However, the genetic and cellular basis of these patterns remains unclear. Herein, we generated chromosome-level genome assemblies of two jellyfish species, Turritopsis rubra and Aurelia coerulea, which exhibit straight and free-swimming patterns, respectively. We observe positive selection of numerous genes involved in statolith formation, hair cell ciliogenesis, ciliary motility, and motor neuron function. The lineage-specific absence of otolith morphogenesis- and ciliary movement-related genes in T. rubra may be associated with homeostatic structural statocyst loss and straight swimming pattern. Notably, single-cell transcriptomic analyses covering key developmental stages reveal the enrichment of diapause-related genes in the cyst during reverse development, suggesting that the sustained diapause state favours the development of new polyps under favourable conditions. This study highlights the complex relationship between genetics, locomotion patterns and survival strategies in jellyfish, thereby providing valuable insights into the evolutionary lineages of movement and adaptation in the animal kingdom.

Carbon sequestration from refractory dissolved organic carbon produced by biodegradation of Saccharina japonica.

Using macroalgae cultures to sequester carbon has been proposed in recent years. Yet the key mechanism of carbon sequestration-how carbon in degrading biomass is converted into refractory dissolved organic carbon (RDOC) remains poorly understood. The process of producting RDOC via biomass degradation of Saccharina japonica, the most productive algae in China, was thus studied in the laboratory. Most of the carbon in the kelp biomass was converted to dissolved inorganic carbon (DIC) by bacterial respiration. Only 7.8% of the carbon in the kelp biomass was converted into labile DOC, semi-labile or semi-refractory DOC, and refractory DOC in turn. The enrichment of DIC resulted in hypoxic conditions in the water. For the hypoxia in the experiment, the sulfur-degrading bacteria Campylobacteria and Bacteroidia became the dominant bacterial classes, which were the key drivers for the transformation of labile DOC to semi-labile or semi-refractory DOC. Then, semi-labile or semi-refractory DOC was converted to RDOC, driven by the sulfite-reducing bacteria Clostridia and Kapabacteria. Finally, 0.3% of the carbon content in kelp was transformed into RDOC. The resulting RDOC, which was rich in sulfur and nitrogen elements, increased the molecular diversity and average molecular weight in the water. An important finding was that the production of RDOC may be accompanied by the environmental risk of hypoxia.