Significant accrual of soil organic carbon through long-term rice cultivation in paddy fields in China.

Paddy fields serve as significant reservoirs of soil organic carbon (SOC) and their potential for terrestrial carbon (C) sequestration is closely associated with changes in SOC pools. However, there has been a dearth of comprehensive studies quantifying changes in SOC pools following extended periods of rice cultivation across a broad geographical scale. Using 104 rice paddy sampling sites that have been in continuous cultivation since the 1980s across China, we studied the changes in topsoil (0-20 cm) labile organic C (LOC I), semi-labile organic C (LOC II), recalcitrant organic C (ROC), and total SOC. We found a substantial increase in both the content (48%) and density (39%) of total SOC within China's paddy fields between the 1980s to the 2010s. Intriguingly, the rate of increase in content and density of ROC exceeded that of LOC (I and II). Using a structural equation model, we revealed that changes in the content and density of total SOC were mainly driven by corresponding shifts in ROC, which are influenced both directly and indirectly by climatic and soil physicochemical factors; in particular temperature, precipitation, phosphorous (P) and clay content. We also showed that the δ13 CLOC were greater than δ13 CROC , independent of the rice cropping region, and that there was a significant positive correlation between δ13 CSOC and δ13 Cstraw . The δ13 CLOC and δ13 CSOC showed significantly negative correlation with soil total Si, suggesting that soil Si plays a part in the allocation of C into different SOC pools, and its turnover or stabilization. Our study underscores that the global C sequestration of the paddy fields mainly stems from the substantial increase in ROC pool.

Peatland Wildfires Enhance Nitrogen-Containing Organic Compounds in Marine Aerosols over the Western Pacific.

Peatland wildfires contribute significantly to the atmospheric release of light-absorbing organic carbon, often referred to as brown carbon. In this study, we examine the presence of nitrogen-containing organic compounds (NOCs) within marine aerosols across the Western Pacific Ocean, which are influenced by peatland fires from Southeast Asia. Employing ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) in electrospray ionization (ESI) positive mode, we discovered that NOCs are predominantly composed of reduced nitrogenous bases, including CHN+ and CHON+ groups. Notably, the count of NOC formulas experiences a marked increase within plumes from peatland wildfires compared to those found in typical marine air masses. These NOCs, often identified as N-heterocyclic alkaloids, serve as potential light-absorbing chromophores. Furthermore, many NOCs demonstrate pyrolytic stability, engage in a variety of substitution reactions, and display enhanced hydrophilic properties, attributed to chemical processes such as methoxylation, hydroxylation, methylation, and hydrogenation that occur during emission and subsequent atmospheric aging. During the daytime atmospheric transport, aging of aromatic N-heterocyclic compounds, particularly in aliphatic amines prone to oxidation and reactions with amine, was observed. The findings underscore the critical role of peatland wildfires in augmenting nitrogen-containing organics in marine aerosols, underscoring the need for in-depth research into their effects on marine ecosystems and regional climatic conditions.

Metagenomics and Stable Isotopes Uncover the Augmented Sulfide-Driven Autotrophic Denitrification in a Seasonally Hypoxic, Sulfate-Abundant Reservoir.

The mechanism governing sulfur cycling in nitrate reduction within sulfate-rich reservoirs during seasonal hypoxic conditions remains poorly understood. This study employs nitrogen and oxygen isotope fractionation in nitrate, along with metagenomic sequencing to elucidate the intricacies of the coupled sulfur oxidation and nitrate reduction process in the water column. In the Aha reservoir, a typical seasonally stratified water body, we observed the coexistence of denitrification, bacterial sulfide oxidation, and bacterial sulfate reduction in hypoxic conditions. This is substantiated by the presence of abundant N/S-related genes (nosZ and aprAB/dsrAB) and fluctuations in N/S species. The lower 15εNO3/18εNO3 ratio (0.60) observed in this study, compared to heterotrophic denitrification, strongly supports the occurrence of sulfur-driven denitrification. Furthermore, we found a robust positive correlation between the metabolic potential of bacterial sulfide oxidation and denitrification (p < 0.05), emphasizing the role of sulfide produced via sulfate reduction in enhancing denitrification. Sulfide-driven denitrification relied on ∑S2- as the primary electron donor preferentially oxidized by denitrification. The pivotal genus, Sulfuritalea, emerged as a central player in both denitrification and sulfide oxidation processes in hypoxic water bodies. Our study provides compelling evidence that sulfides assume a critical role in regulating denitrification in hypoxic water within an ecosystem where their contribution to the overall nitrogen cycle was previously underestimated.

Microbial Necromass, Lignin, and Glycoproteins for Determining and Optimizing Blue Carbon Formation.

Coastal wetlands contribute to the mitigation of climate change through the sequestration of "blue carbon". Microbial necromass, lignin, and glycoproteins (i.e., glomalin-related soil proteins (GRSP)), as important components of soil organic carbon (SOC), are sensitive to environmental change. However, their contributions to blue carbon formation and the underlying factors remain largely unresolved. To address this paucity of knowledge, we investigated their contributions to blue carbon formation along a salinity gradient in coastal marshes. Our results revealed decreasing contributions of microbial necromass and lignin to blue carbon as the salinity increased, while GRSP showed an opposite trend. Using random forest models, we showed that their contributions to SOC were dependent on microbial biomass and resource stoichiometry. In N-limited saline soils, contributions of microbial necromass to SOC decreased due to increased N-acquisition enzyme activity. Decreases in lignin contributions were linked to reduced mineral protection offered by short-range-ordered Fe (FeSRO). Partial least-squares path modeling (PLS-PM) further indicated that GRSP could increase microbial necromass and lignin formation by enhancing mineral protection. Our findings have implications for improving the accumulation of refractory and mineral-bound organic matter in coastal wetlands, considering the current scenario of heightened nutrient discharge and sea-level rise.

Dynamic changes, cycling and downward fate of dissolved carbon and nitrogen photosynthetically-derived from glaciers in upper Indus river basin.

Glaciers play key roles in capturing, storing, and transforming global carbon and nitrogen, thereby contributing markedly to their cycles. However, an integrated mechanistic approach is still lacking regarding glacier's primary producers (PP), in terms of stable dissolved inorganic carbon isotope (δ13C-DIC) and its relationship with dissolved carbon and nitrogen transformation d ynamic changes/cycling. Here, we sampled waters from glaciers, streams, tributaries, and the Indus River (IR) mainstream in the Upper IR Basin, Western Himalaya. Dissolved organic matter (DOM) appears to increase, on average, by ∼2.5-23.4% with fluctuations when passing from glaciers to streams-tributaries-IR mainstream (the upper and lower parts, respectively) continuum, implying that DOM originates from glaciers PP and is subsequently degraded. The corresponding fluctuations are observed for fluorescent DOM (FDOM), dissolved organic nitrogen (8.0-106.8%), NO3--N (-13.5/+16.6%), NH4+-N (-8.8/+13.0%), and NO2--N (70.7-217.5%). These variations are associated with overall DOM/FDOM transformations, with the production of ending byproducts (e.g. CO2/DIC). The δ13C-DIC values fluctuated from glaciers (-5.3 ± 2.5‰) to streams (-4.4 ± 2.1‰), tributaries (-4.3 ± 1.6‰), and IR mainstream (-4.2 ± 1.3‰). The δ13C-DIC data are consistent with C transformations that involve lighter CO2 emission into the atmosphere, whereas highly depleted DIC/CO2 is the signature of DOM degradation after its fresh production from glaciers PP which originated by photosynthetic activities (e.g. uptake/sink of atmospheric CO2: -8.4‰). Finally, glacier-fed meltwaters would simultaneously contribute to the biogeochemical characteristics of downward margins and specific ecosystems (lake/pond/groundwater/hot springs) via transformation dynamics/cycling of dissolved C and N with high photo/microbial lability. Our results highlight the substantial contribution of western Himalayan glaciers-derived DOM to the global C and N cycles.

Comprehensive source identification of heavy metals in atmospheric particulate matter in a megacity: A case study of Hangzhou.

Megacities face significant pollution challenges, particularly the elevated levels of heavy metals (HMs) in particulate matter (PM). Despite the advent of interdisciplinary and advanced methods for HM source analysis, integrating and applying these approaches to identify HM sources in PM remains a hurdle. This study employs a year-long daily sampling dataset for PM1 and PM1-10 to examine the patterns of HM concentrations under hazy, clean, and rainy conditions in Hangzhou City, aiming to pinpoint the primary sources of HMs in PM. Contrary to other HMs that remained within acceptable limits, the annual average concentrations of Cd and Ni were found to be 20.6 ± 13.6 and 46.9 ± 34.8 ng/m³, respectively, surpassing the World Health Organization's limits by 4.1 and 1.9 times. Remarkably, Cd levels decreased on hazy days, whereas Ni levels were observed to rise on rainy days. Using principal component analysis (PCA), enrichment factor (EF), and backward trajectory analysis, Fe, Mn, Cu, and Zn were determined to be primarily derived from traffic emissions, and there was an interaction between remote migration and local emissions in haze weather. Isotope analysis reveals that Pb concentrations in the Hangzhou region were primarily influenced by emissions from unleaded gasoline, coal combustion, and municipal solid waste incineration, with additional impact from long-range transport; it also highlights nuanced differences between PM1 and PM1-10. Pb isotope and PCA analyses indicate that Ni primarily stemmed from waste incineration emissions. This explanation accounts for the observed higher Ni concentrations on rainy days. Backward trajectory cluster analysis revealed that southern airflows were the primary source of high Cd concentrations on clean days in Hangzhou City. This study employs a multifaceted approach and cross-validation to successfully delineate the sources of HMs in Hangzhou's PM. It offers a methodology for the precise and reliable analysis of complex HM sources in megacity PM.

Machine Learning Predicts the Methane Clumped Isotopologue (12CH2D2) Distributions Constrain Biogeochemical Processes and Estimates the Potential Budget.

Methane (CH4) is a matter of environmental concern; however, global methane isotopologue data remain inadequate. This is due to the challenges posed by high-resolution testing technology and the need for larger sample volumes. Here, worldwide methane clumped isotope databases (n = 465) were compiled. We compared machine-learning (ML) models and used random forest (RF) to predict new Δ12CH2D2 distributions, which cover valuable and hard-to-replicate methane clumped isotope experimental data. Our RF model yields a reliable and continuous database including ruminants, acetoclastic methane, multiple pyrolysis, and controlled experiments. We showed the effectiveness of utilizing a new data set to quantify isotopologue fractionations in biogeochemical methane processes, as well as predicting the steady-state atmospheric methane clumped isotope composition (Δ13CH3D of +2.26 ± 0.71‰ and Δ12CH2D2 of +62.06 ± 4.42‰) with notable biological contributions. Our measured summer and winter water emitted gases (n = 6) demonstrated temperature-driven seasonal microbial community evolution determined by atmospheric clumped isotope temporal variations (Δ 13CH3D ∼ -0.91 ± 0.25 ‰ and Δ12CH2D2 ∼ +3.86 ± 0.84 ‰), which in turn is relevant for future models quantifying the contribution of methane sources and sinks. Predicting clumped isotopologues translates our methane geochemical understanding into quantifiable variables for modeling that can continue to improve predictions and potentially inform global greenhouse gas emissions and mitigation policy.

Multiple Sulfur Isotopic Evidence for Sulfate Formation in Haze Pollution.

The mechanism of sulfate formation during winter haze events in North China remains largely elusive. In this study, the multiple sulfur isotopic composition of sulfate in different grain-size aerosol fractions collected seasonally from sampling sites in rural, suburban, urban, industrial, and coastal areas of North China are used to constrain the mechanism of SO2 oxidation at different levels of air pollution. The Δ33S values of sulfate in aerosols show an obvious seasonal variation, except for those samples collected in the rural area. The positive Δ33S signatures (0‰ < Δ33S < 0.439‰) observed on clean days are mainly influenced by tropospheric SO2 oxidation and stratospheric SO2 photolysis. The negative Δ33S signatures (-0.236‰ < Δ33S < ∼0‰) observed during winter haze events (PM2.5 > 200 µg/m3) are mainly attributed to SO2 oxidation by H2O2 and transition metal ion catalysis (TMI) in the troposphere. These results reveal that both the H2O2 and TMI pathways play critical roles in sulfate formation during haze events in North China. Additionally, these new data provide evidence that the tropospheric oxidation of SO2 can produce significant negative Δ33S values in sulfate aerosols.

Chemodiversity of Dissolved Organic Matter Is Governed by Microbial Biogeography in Inland Waters.

Dissolved organic matter (DOM) is crucial for the carbon biogeochemical cycle and has a close link with microbiome in aquatic ecosystems; however, the causal relationship between DOM and microbial diversity in inland waters is not very clear so far. Therefore, a national survey of China's inland waters was conducted, and the DOM chemical composition and microbial community composition were determined by Fourier transform ion cyclotron resonance mass spectrometry and high-throughput sequencing to clarify the abovementioned question. Here, we found that DOM chemodiversity was governed by microbial community assembly in inland waters, not vice versa. Under the control of microbial biogeography, DOM chemodiversity showed a clear geographical distribution difference. Water DOM chemodiversity was mainly constrained by bacterial and archaeal community composition, whereas sediment DOM chemodiversity was mainly controlled by eukaryotic and fungal community composition. In addition, the sediment DOM chemical composition was also affected by the interaction of different microbial groups between waters and sediments. The study is the first to clarify the causal relationship and proposes a microbial regulatory mechanism on the geographical distribution pattern of DOM chemodiversity, thus further deepening the understanding of the DOM biogeochemical cycle.

Anthropogenic Disturbance Stimulates the Export of Dissolved Organic Carbon to Rivers on the Tibetan Plateau.

The impacts of human activities on the riverine carbon (C) cycle have only recently been recognized, and even fewer studies have been reported on anthropogenic impacts on C cycling in rivers draining the vulnerable alpine areas. Here, we examined carbon isotopes (δ13CDOC and Δ14CDOC), fluorescence, and molecular compositions of riverine dissolved organic matters (DOM) in the Bailong River catchment, the eastern edge of the Tibetan Plateau to identify anthropogenic impacts on the C cycle. Human activities show limited impact on dissolved organic carbon (DOC) concentration, but significantly increased the age of DOC (from modern to ∼1600 yr B.P.) and changed the molecular compositions through agriculture and urbanization despite in the catchment with low population density. Agricultural activities indirectly increased the leaching of N-containing aged organic matter from deep soil to rivers. Urbanization released S-containing aged C from fossil products into rivers directly through wastewater. The aged DOC from agricultural activity and wastewater discharge was partly biolabile and/or photolabile. This study highlights that riverine C is sensitive to anthropogenic disturbance. Additionally, the study also emphasizes that human activities reintroduce aged DOC into the modern C cycle, which would accelerate the geological C cycle.

Synthesis and anti-HIV activity of non-nucleoside reverse-transcriptase inhibitor DB02 phosphate derivatives based on water-soluble optimization.

To improve the water solubility of anti-human immunodeficiency virus (HIV) agent DB02, an excellent non-nucleoside reverse-transcriptase inhibitor (NNRTI) obtained in our previous efforts, we designed and synthesized four phosphate derivatives of DB02 based on the molecular model of DB02 with RT. Here, the antiviral activity of these four derivatives was detected, leading to the discovery of compound P-2, which possessed a superior potency to the lead compound DB02 against wild-type HIV-1 and a variety of HIV-resistant mutant viruses significantly. Furthermore, the water solubility of P-2 was nearly 17 times higher than that of DB02, and the pharmacokinetic test in rats showed that P-2 demonstrate significantly improved oral bioavailablity of 14.6%. Our study showed that the introduction of a phosphate ester group at the end of the C-2 side chain of DB02 was beneficial to the improvement of its antiviral activity and pharmacokinetic properties, which provided a promising lead for the further development of S-DACOs type of NNRTIs.

Identification of Trace Components in Sauce-Flavor Baijiu by High-Resolution Mass Spectrometry.

Sauce-flavor Baijiu is one of the most complex and typical types of traditional Chinese liquor, whose trace components have an important impact on its taste and quality. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) is one of the most favorable analytical tools to reveal trace molecular components in complex samples. This study analyzed the chemical diversity of several representative sauce-flavor Baijiu using the combination of electrospray ionization (ESI) and FT-ICR MS. The results showed that ESI+ and ESI- exhibited different chemical features characteristic of trace components. Overall, sauce-flavor Baijiu was dominated by CHO class compounds, and the main specific compound types were aliphatic, highly unsaturated with low oxygen, and peptide-like compounds. The mass spectral parameters resolved by FT-ICR MS of several well-known brands were relatively similar, whereas the greatest variability was observed from an internally supplied brand. This study provides a new perspective on the mass spectrometry characteristics of trace components of sauce-flavor Baijiu and offers a theoretical foundation for further optimization of the gradients in Baijiu.

Storage, patterns and influencing factors for soil organic carbon in coastal wetlands of China.

Soil organic carbon (SOC) in coastal wetlands, also known as "blue C," is an essential component of the global C cycles. To gain a detailed insight into blue C storage and controlling factors, we studied 142 sites across ca. 5000 km of coastal wetlands, covering temperate, subtropical, and tropical climates in China. The wetlands represented six vegetation types (Phragmites australis, mixed of P. australis and Suaeda, single Suaeda, Spartina alterniflora, mangrove [Kandelia obovata and Avicennia marina], tidal flat) and three vegetation types invaded by S. alterniflora (P. australis, K. obovata, A. marina). Our results revealed large spatial heterogeneity in SOC density of the top 1-m ranging 40-200 Mg C ha-1 , with higher values in mid-latitude regions (25-30° N) compared with those in both low- (20°N) and high-latitude (38-40°N) regions. Vegetation type influenced SOC density, with P. australis and S. alterniflora having the largest SOC density, followed by mangrove, mixed P. australis and Suaeda, single Suaeda and tidal flat. SOC density increased by 6.25 Mg ha-1 following S. alterniflora invasion into P. australis community but decreased by 28.56 and 8.17 Mg ha-1 following invasion into K. obovata and A. marina communities. Based on field measurements and published literature, we calculated a total inventory of 57 × 106 Mg C in the top 1-m soil across China's coastal wetlands. Edaphic variables controlled SOC content, with soil chemical properties explaining the largest variance in SOC content. Climate did not control SOC content but had a strong interactive effect with edaphic variables. Plant biomass and quality traits were a minor contributor in regulating SOC content, highlighting the importance of quantity and quality of OC inputs and the balance between production and degradation within the coastal wetlands. These findings provide new insights into blue C stabilization mechanisms and sequestration capacity in coastal wetlands.

Fungal-Mineral Interactions Modulating Intrinsic Peroxidase-like Activity of Iron Nanoparticles: Implications for the Biogeochemical Cycles of Nutrient Elements and Attenuation of Contaminants.

Fungal-mediated extracellular reactive oxygen species (ROS) are essential for biogeochemical cycles of carbon, nitrogen, and contaminants in terrestrial environments. These ROS levels may be modulated by iron nanoparticles that possess intrinsic peroxidase (POD)-like activity (nanozymes). However, it remains largely undescribed how fungi modulate the POD-like activity of the iron nanoparticles with various crystallinities and crystal facets. Using well-controlled fungal-mineral cultivation experiments, here, we showed that fungi possessed a robust defect engineering strategy to modulate the POD-like activity of the attached iron minerals by decreasing the catalytic activity of poorly ordered ferrihydrite but enhancing that of well-crystallized hematite. The dynamics of POD-like activity were found to reside in molecular trade-offs between lattice oxygen and oxygen vacancies in the iron nanoparticles, which may be located in a cytoprotective fungal exoskeleton. Together, our findings unveil coupled POD-like activity and oxygen redox dynamics during fungal-mineral interactions, which increase the understanding of the catalytic mechanisms of POD-like nanozymes and microbial-mediated biogeochemical cycles of nutrient elements as well as the attenuation of contaminants in terrestrial environments.

Molecular Trade-Offs between Lattice Oxygen and Oxygen Vacancy Drive Organic Pollutant Degradation in Fungal Biomineralized Exoskeletons.

Fungal-mineral interactions can effectively alleviate cellular stress from organic pollutants, the production of which are expected to rapidly increase owing to the Earth moving into an unprecedented geological epoch, the Anthropocene. The underlying mechanisms that may enable fungi to combat organic pollution during fungal-mineral interactions remain unclear. Inspired by the natural fungal sporulation process, we demonstrate for the first time that fungal biomineralization triggers the formation of an ultrathin (hundreds of nanometers thick) exoskeleton, enriched in nanosized iron (oxyhydr)oxides and biomolecules, on the hyphae. Mapped biochemical composition of this coating at a subcellular scale via high spatial resolution (down to 50 nm) synchrotron radiation-based techniques confirmed aromatic C, C-N bonds, amide carbonyl, and iron (oxyhydr)oxides as the major components of the coatings. This nanobiohybrid system appeared to impart a strong (×2) biofunctionality for fungal degradation of bisphenol A through altering molecular-level trade-offs between lattice oxygen and oxygen vacancy. Together, fungal coatings could act as "artificial spores", which enable fungi to combat physical and chemical stresses in natural environments, providing crucial insights into fungal biomineralization and coevolution of the Earth's lithosphere and biosphere.

High Molecular Diversity of Organic Nitrogen in Urban Snow in North China.

Snow serves as a vital scavenging mechanism to gas-phase and particle-phase organic nitrogen substances in the atmosphere, providing a significant link between land-atmosphere flux of nitrogen in the surface-earth system. Here, we used optical instruments (UV-vis and excitation-emission matrix fluorescence) and a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) to elucidate the molecular composition and potential precursors of snow samples collected simultaneously at four megacities in North China. The elemental O/N ratio (≥3), together with the preference in the negative ionization mode, indicates that the one and two nitrogen atom-containing organics (CHON1 and CHON2) in snow were largely in the oxidized form (as organic nitrates, -ONO2). This study assumed that scavenging of particle-phase and gas-phase organic nitrates might be significant sources of CHON in precipitation. A gas-phase oxidation process and a particle-phase hydrolysis process, at a molecular level, were used to trace the potential precursors of CHON. Results show that more than half of the snow CHON molecules may be related to the oxidized and hydrolyzed processes of atmospheric organics. Potential formation processes of atmospheric organics on a molecular level provide a new concept to better understand the sources and scavenging mechanisms of organic nitrogen species in the atmosphere.

Nitrate is an important nitrogen source for Arctic tundra plants.

Plant nitrogen (N) use is a key component of the N cycle in terrestrial ecosystems. The supply of N to plants affects community species composition and ecosystem processes such as photosynthesis and carbon (C) accumulation. However, the availabilities and relative importance of different N forms to plants are not well understood. While nitrate (NO3-) is a major N form used by plants worldwide, it is discounted as a N source for Arctic tundra plants because of extremely low NO3- concentrations in Arctic tundra soils, undetectable soil nitrification, and plant-tissue NO3- that is typically below detection limits. Here we reexamine NO3- use by tundra plants using a sensitive denitrifier method to analyze plant-tissue NO3- Soil-derived NO3- was detected in tundra plant tissues, and tundra plants took up soil NO3- at comparable rates to plants from relatively NO3--rich ecosystems in other biomes. Nitrate assimilation determined by 15N enrichments of leaf NO3- relative to soil NO3- accounted for 4 to 52% (as estimated by a Bayesian isotope-mixing model) of species-specific total leaf N of Alaskan tundra plants. Our finding that in situ soil NO3- availability for tundra plants is high has important implications for Arctic ecosystems, not only in determining species compositions, but also in determining the loss of N from soils via leaching and denitrification. Plant N uptake and soil N losses can strongly influence C uptake and accumulation in tundra soils. Accordingly, this evidence of NO3- availability in tundra soils is crucial for predicting C storage in tundra.

Universal Affinity Capture Liquid Chromatography-Mass Spectrometry Assay for Evaluation of Biotransformation of Site-Specific Antibody Drug Conjugates in Preclinical Studies.

Antibody drug conjugates (ADCs) can undergo in vivo biotransformation (e.g., payload metabolism, deconjugation) leading to reduced or complete loss of activity. The location/site of conjugation of payload-linker can have an effect on ADC stability and hence needs to be carefully optimized. Affinity capture LC-MS of intact ADCs or ADC subfragments has been extensively used to evaluate ADC biotransformation. However, the current methods have certain limitations such as the requirement of specific capture reagents, limited mass resolution of low mass change metabolites, low sensitivity, and use of capillary or nanoflow LC-MS. To address these challenges, we developed a generic affinity capture LC-MS assay that can be utilized to evaluate the biotransformation of any site-specific ADC independent of antibody type and site of conjugation (Fab and Fc) in preclinical studies. The method involves a combination of some or all of these steps: (1) "mono capture" or "dual capture" of ADCs from serum with streptavidin magnetic beads coated with a generic biotinylated antihuman capture reagent, (2) "on-bead" digestion with IdeS and/or PNGase F, and (3) reduction of interchain disulfide bonds to generate ∼25 kDa ADC subfragments, which are finally analyzed by LC-HRMS on a TOF mass spectrometer. The advantages of this method are that it can be performed using commercially available generic reagents and requires sample preparation time of less than 7 h. Furthermore, by reducing the size of intact ADC (∼150 kDa) to subfragments (∼25 kDa), the identification of conjugated payload and its metabolites can be achieved with excellent sensitivity and resolution (hydrolysis and other small mass change metabolites). This method was successfully applied to evaluate the in vitro and in vivo biotransformation of ADCs conjugated at different sites (LC, HC-Fab, and HC-Fc) with various classes of payload-linkers.

Synthesis and Biological Evaluation of a Carbamate-Containing Tubulysin Antibody-Drug Conjugate.

Antibody-drug conjugates (ADCs) use antibodies to deliver cytotoxic payloads directly into tumor cells via specifically binding to the target cell surface antigens. ADCs can enhance the anti-tumor effects of antibodies, and increase the delivery of cytotoxic payloads to cancer cells with a better therapeutic index. An ADC was prepared with a potent carbamate-containing tubulysin analogue attached to an anti-mesothelin antibody via a Cit-Val dipeptide linker. An aniline functionality in the tubulysin analogue was created to provide a site of linker attachment via an amide bond that would be stable in systemic circulation. Upon ADC internalization into antigen-positive cancer cells, the Cit-Val dipeptide linker was cleaved by lysosomal proteases, and the drug was released inside the tumor cells. The naturally occurring acetate of tubulysin was modified to a carbamate to reduce acetate hydrolysis of the ADC in circulation and to increase the hydrophilicity of the drug. The ADC bearing the monoclonal anti-mesothelin antibody and the carbamate-containing tubulysin was highly potent and immunologically specific to H226 human lung carcinoma cells in vitro, and efficacious at well-tolerated doses in a mesothelin-positive OVCAR3 ovarian cancer xenograft mouse model.

Control of Hydraulic Load on Bacterioplankton Diversity in Cascade Hydropower Reservoirs, Southwest China.

Hydroelectric reservoirs are highly regulated ecosystems, where the understanding on bacterioplankton has been very limited so far. In view of significant changes in river hydrological conditions by dam construction, hydraulic load (i.e., the ratio of mean water depth to water retention time) was assumed to control bacterioplankton diversity in cascading hydropower reservoirs. To evaluate this hypothesis, we investigated bacterioplankton composition and diversity using high-throughput sequencing and related environmental variables in eleven reservoirs on the Wujiang River, Southwest China. Our results showed a decrease of bacterioplankton diversity index with an increase of reservoir hydraulic load. This is because hydraulic load governs dissolved oxygen variation in the water column, which is a key factor shaping bacterioplankton composition in these hydroelectric reservoirs. In contrast, bacterioplankton abundance was mainly affected by nutrient-related environmental factors. Therefore, from a hydrological perspective, hydraulic load is a decisive factor for the bacterioplankton diversity in the hydroelectric reservoirs. This study can improve the understanding of reservoir bacterial ecology, and the empirical relationship between hydraulic load and bacterioplankton diversity index will help to quantitatively evaluate ecological effects of river damming.