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Abstract To unveil the nature of amorphous states, single‐element amorphous metals have been the perfect research subject due to the simplest composition. However, the extreme crystal nucleation and growth rate in single‐element metal make the synthesis of single‐element amorphous metals seemingly impossible in the past. Fortunately, benefited by several delicate synthetic strategies developed recently, the single‐element amorphous metals have been successfully demonstrated. This review aims to provide a systematic overview of the synthesis of single‐element amorphous metals covering the challenges in physics and recent achievements. In addition, current understanding of the atomic and electronic structures of single‐element amorphous metal has also been included. Finally, the challenges that worth further investigation are discussed. By identifying the potential avenues for further exploration, this review aims to contribute valuable insights that will propel the cognition of single‐element amorphous metals.

Fast and precise identification of microorganisms in the early diagnosis of sepsis is crucial for enhancing patient outcomes. Digital PCR (dPCR) is a highly sensitive approach for absolute quantification that can be utilized as a culture-independent molecular technique for diagnosing sepsis pathogens. We performed a retrospective investigation on 69 ICU patients suspected of sepsis. Our findings showed that a multiplex dPCR diagnostic kit outperformed blood culture in detecting the 15 most frequent bacteria that cause sepsis. Ninety-two bacterial strains were identified using dPCR at concentrations varying from 34 copies/mL to 105,800 copies/mL. The detection rate of dPCR was much greater than that of BC, with 27.53% (19/69) versus 73.91% (51/69). The sensitivity of dPCR was 63.2%. Our research indicated that dPCR outperforms blood culture in the early detection of sepsis-causing microorganisms. The diagnostic kit can detect a greater variety of pathogens with quantitative data, including polymicrobial infections, and has a quicker processing time. DPCR is a valuable technique that could aid in the proper management of sepsis.

International students play a crucial role in China’s talent development strategy. Thus, predicting overseas talent mobility is essential for formulating scientifically reasonable talent introduction policies, optimizing talent cultivation systems, and fostering international talent cooperation. In this study, we proposed a novel fractional-order grey model based on the Multi-Layer Perceptron (MLP) and Grey Wolf Optimizer (GWO) algorithm to forecast the movement of overseas talent, namely MGDFGM(1,1). Compared to the traditional grey model FGM(1,1), which utilizes the same fractional order at all time points, the proposed MGDFGM(1,1) model dynamically adjusts the fractional-order values based on the time point. This dynamic adjustment enables our model to better capture the changing trends in the data, thereby enhancing the model’s fitting capability. To validate the effectiveness of the MGDFGM(1,1) model, we primarily utilize Root Mean Square Error (RMSE) and Mean Absolute Percentage Error (MAPE) as the evaluation criteria for the prediction accuracy, as well as standard deviation (STD) as an indicator of the model stability. Furthermore, we perform experimental analysis to evaluate the predictive performance of the MGDFGM(1,1) model in comparison to NAÏVE, ARIMA, GM(1,1), FGM(1,1), LSSVR, MLP, and LSTM. The research findings demonstrate that the MGDFGM(1,1) model achieves a remarkably high level of prediction accuracy and stability for forecasting overseas talent mobility in China. The implications of this study offer valuable insights and assistance to government departments involved in overseas talent management.

Amorphous nanocatalysts have attracted significant attention due to their outstanding catalytic performance. Recent research has indicated that the performance of amorphous nanocatalysts is closely related to the short-to-medium-range order. This perspective aims to provide a comprehensive summary of the latest advances in understanding the significance of short-to-medium-range order in amorphous nanocatalysts and its correlation with catalytic performance. This perspective commences by presenting advanced methods employed for characterizing the short-to-medium-range order of amorphous nanocatalysts, including nanobeam electron diffraction, scanning transmission electron microscopy, atomic electron tomography, pair distribution function, and X-ray absorption fine structure spectroscopy. Next, the effect of short-to-medium-range order in determining the properties of amorphous nanocatalysts is discussed. Current challenges faced in amorphous nanocatalysts are eventually summarized with several prospective research directions. By identifying the obstacles and potential avenues for further exploration, this perspective aims to contribute valuable insights that will propel the development of high-efficient amorphous nanocatalysts.

SspABCD–SspE is a phosphorothioation-sensing bacterial defence system. Here, authors find that SspE exploits DNA phosphorothioation-binding preference to modulate its biochemical activities for the self/nonself discrimination and phage resistance.

While inducing strain to noble metal nanomaterials can modulate catalytic activities, the strain is often spatially dependent. Here, authors manipulate the planar strain in noble metal nanosheets for hydrogen evolution electrocatalysis by constructing amorphous–crystalline phase boundaries.

Abstract The conversion of CO2 into fuels and valuable chemicals is one of the central topics to combat climate change and meet the growing demand for renewable energy. Herein, we show that the formate dehydrogenase from Clostridium ljungdahlii (ClFDH) adsorbed on electrodes displays clear characteristic voltammetric signals that can be assigned to the reduction and oxidation potential of the [4Fe‐4S]2+/+ cluster under nonturnover conditions. Upon adding substrates, the signals transform into a specific redox center that engages in catalytic electron transport. ClFDH catalyzes rapid and efficient reversible interconversion between CO2 and formate in the presence of substrates. The turnover frequency of electrochemical CO2 reduction is determined as 1210 s−1 at 25 °C and pH 7.0, which can be further enhanced up to 1786 s−1 at 50°C. The Faradaic efficiency at −0.6 V (vs. standard hydrogen electrode) is recorded as 99.3% in a 2‐h reaction. Inhibition experiments and theoretical modeling disclose interesting pathways for CO2 entry, formate exit, and OCN− competition, suggesting an oxidation‐state‐dependent binding mechanism of catalysis. Our results provide a different perspective for understanding the catalytic mechanism of FDH and original insights into the design of synthetic catalysts.

Every generation of wireless technologies needs to bring a set of new system capabilities to enable future applications and services, the sixth generation mobile system (6G) is no exception. This paper provides an overview of the technology transformation from the communication-centric the forth generation mobile system (4G) and the fifth generation mobile system (5G) to the compute-centric 6G with the cloud-native system framework as the foundation of the next generation technologies. We explain what 6G plans to achieve, the fundamental reasons for this technology transformation, the architecture framework and enabling technologies to achieve 6G cloud-native technology objectives. This paper intends to provide a technical deep dive on the 6G cloud-native system to trigger more discussions, innovations and bring the technology transformation from concept to reality.

The SARS-CoV-2 Omicron outbreak is ongoing in Shanghai, home to 25 million population. Here, we presented a novel mathematical model to evaluate the Omicron spread and Zero-COVID strategy. Our model provided important parameters, the average quarantine ratio, the detection interval from being infected to being tested positive, and the spreading coefficient to understand the epidemic progression better. Moreover, we found that the key to a relatively accurate long-term forecast was to take the variation/relaxation of the parameters into consideration based on the flexible execution of the quarantine policy. This allowed us to propose the criteria for estimating the parameters and outcome for the ending stage that is likely to take place in late May. Altogether, this model helped to give a correct mathematical appraisal of the SARS-CoV-2 Omicron outbreak under the strict Zero-COVID policy in China.

ABSTRACT The compositional and physiological responses of autotrophic microbiotas to salinity in lakes remain unclear. In this study, the community composition and carbon fixation pathways of autotrophic microorganisms in lacustrine sediments with a salinity gradient (82.6 g/L to 0.54 g/L) were investigated by using metagenomic analysis. A total of 117 metagenome-assembled genomes (MAGs) with carbon fixation potentially belonging to 20 phyla were obtained. The abundance of these potential autotrophs increased significantly with decreasing salinity, and the variation of sediment autotrophic microbial communities was mainly affected by salinity, pH, and total organic carbon. Notably, along the decreasing salinity gradient, the dominant lineage shifted from Desulfobacterota to Proteobacteria. Meanwhile, the dominant carbon fixation pathway shifted from the Wood-Lungdahl pathway to the less-energy-efficient Calvin-Benson-Bassham cycle, with glycolysis shifting from the Embden-Meyerhof-Parnas pathway to the less-exergonic Entner-Doudoroff pathway. These results suggest that the physiological efficiency of autotrophic microorganisms decreased when the environmental salinity became lower. Metabolic inference of these MAGs revealed that carbon fixation may be coupled to the oxidation of reduced sulfur compounds and ferrous iron, dissimilatory nitrate reduction at low salinity, and dissimilatory sulfate reduction in hypersaline sediments. These results extend our understanding of metabolic versatility and niche diversity of autotrophic microorganisms in saline environments and shed light on the response of autotrophic microbiomes to salinity. These findings are of great significance for understanding the impact of desalination caused by climate warming on the carbon cycle of saline lake ecosystems. IMPORTANCE The Qinghai-Tibetan lakes are experiencing water increase and salinity decrease due to climate warming. However, little is known about how the salinity decrease will affect the composition of autotrophic microbial populations and their carbon fixation pathways. In this study, we used genome-resolved metagenomics to interpret the dynamic changes in the autotrophic microbial community and metabolic pathways along a salinity gradient. The results showed that desalination drove the shift of the dominant microbial lineage from Desulfobacterota to Proteobacteria, enriched autotrophs with lower physiological efficiency pathways, and enhanced coupling between the carbon cycle and other element cycles. These results can predict the future response of microbial communities to lake desalination and improve our understanding of the effect of climate warming on the carbon cycle in saline aquatic ecosystems.

ABSTRACT Phosphorothioate (PT) modification, a sequence-specific modification that replaces the nonbridging oxygen atom with sulfur in a DNA phosphodiester through the gene products of dndABCDE or sspABCD, is widely distributed in prokaryotes. DNA PT modification functions together with gene products encoded by dndFGH, pbeABCD, or sspE to form defense systems that can protect against invasion by exogenous DNA particles. While the functions of the multiple enzymes in the PT system have been elucidated, the exact role of DndE in the PT process is still obscure. Here, we solved the crystal structure of DndE from the haloalkaliphilic archaeal strain Natronorubrum bangense JCM10635 at a resolution of 2.31 Å. Unlike the tetrameric conformation of DndE in Escherichia coli B7A, DndE from N. bangense JCM10635 exists in a monomeric conformation and can catalyze the conversion of supercoiled DNA to nicked or linearized products. Moreover, DndE exhibits preferential binding affinity to nicked DNA by virtue of the R19- and K23-containing positively charged surface. This work provides insight into how DndE functions in PT modification and the potential sulfur incorporation mechanism of DNA PT modification. IMPORTANCE DndABCDE proteins have been demonstrated to catalyze DNA PT modification with the nonbridging oxygen in the DNA sugar-phosphate backbone replaced by sulfur. In the PT modification pathway, DndA exerts cysteine desulfurase activity capable of catalyzing the mobilization of sulfur from l-cysteine, which involves the ion-sulfur cluster assembly of DndC. This is regarded as the initial step of the DNA PT modification. Moreover, DndD has ATPase activity in vitro, which is believed to provide energy for the oxygen-sulfur swap, while the function of DndE is unknown. However, the exact function of the key enzyme DndE remains to be elucidated. By determining the structure of DndE from the haloalkaliphilic strain Natronorubrum bangense JCM10635, we showed that the archaeal DndE adopts a monomer conformation. Notably, DndE can introduce nicks to supercoiled DNA and exhibits a binding preference for nicked DNA; the nicking is believed to be the initial step for DNA to facilitate the sulfur incorporation.

The balance between anabolism and catabolism is disrupted with aging, with the rate of anabolism being faster than that of catabolism. Therefore, mTOR, whose major function is to enhance anabolism and inhibit catabolism, has become a potential target of inhibition for anti-aging therapy. Interestingly, it was found that the downregulation of the mTOR signaling pathway had a lifespan-extending effect resembling calorie restriction. In addition, the mTOR signaling pathway promotes cell proliferation and has been regarded as a potential anti-cancer target. Rapamycin and rapalogs, such as everolimus, have proven to be effective in preventing certain tumor growth. Here, we reviewed the basic knowledge of mTOR signaling, including both mTORC1 and mTORC2. Then, for anti-aging, we cited a lot of evidence to discuss the role of targeting mTOR and its anti-aging mechanism. For cancer therapy, we also discussed the role of mTOR signaling in different types of cancers, including idiopathic pulmonary fibrosis, tumor immunity, etc. In short, we discussed the research progress and both the advantages and disadvantages of targeting mTOR in anti-aging and anti-cancer therapy. Hopefully, this review may promote more ideas to be generated for developing inhibitors of mTOR signaling to fight cancer and extend lifespan.

Regulating the adsorption behaviour of the polysulfide species is the key to achieving highly stable Li-S batteries. Here, the authors show that amorphization-induced redistribution of d orbitals enable CoO to be a favourable candidate for polysulfide adsorption and conversion.

Summary: Achieving high selectivity and activity with the oxygen reduction reaction (ORR) is significant for developing efficient energy conversion techniques and chemical production. Here, we report that selective ORRs can be achieved by tuning short-range order in amorphous and crystalline NiO nanosheets (a-NiO NSs and c-NiO NSs, respectively). X-ray absorption spectroscopy analysis reveals that the short-range order of a-NiO NSs and c-NiO NSs mainly adopt the NiO5 pyramidal and NiO6 octahedral structures, respectively. The a-NiO NSs for electrochemical H2O2 production in 0.1 M KOH exhibits both high selectivity over 90% and high activity (1 mA cm−2 at 0.66 V versus RHE), while c-NiO NSs tends to catalyze ORRs through 4-electron pathways to generate H2O. Theoretical calculations indicate that the changed short-range order of a-NiO NSs leads to alteration of Ni d-orbital states, which can regulate the adsorption orientation and strength of ∗OOH intermediates to achieve high selectivity and activity of 2-electron ORRs.

While noble metal usage in catalysis is ubiquitous, the metals’ scarcity necessitates new materials designs for efficient utilization. Here, authors report a general strategy to prepare amorphous noble metal nanosheets and find the nanomaterials to act as efficient water-splitting electrocatalysts.

Investigating the distribution of hydrogen-producing bacteria (HPB) is of great significance to understanding the source of biological hydrogen production in geothermal environments. Here, we explored the compositions of HPB populations in the sediments of hot springs from the Daggyai, Quzhuomu, Quseyongba, and Moluojiang geothermal zones on the Tibetan Plateau, with the use of Illumina MiSeq high-throughput sequencing of 16S rRNA genes and hydA genes. In the present study, the hydA genes were successfully amplified from the hot springs with a temperature of 46–87°C. The hydA gene phylogenetic analysis showed that the top three phyla of the HPB populations were Bacteroidetes (14.48%), Spirochaetes (14.12%), and Thermotogae (10.45%), while Proteobacteria were absent in the top 10 of the HPB populations, although Proteobacteria were dominant in the 16S rRNA gene sequences. Canonical correspondence analysis results indicate that the HPB community structure in the studied Tibetan hot springs was correlated with various environmental factors, such as temperature, pH, and elevation. The HPB community structure also showed a spatial distribution pattern; samples from the same area showed similar community structures. Furthermore, one HPB isolate affiliated with Firmicutes was obtained and demonstrated the capacity of hydrogen production. These results are important for us to understand the distribution and function of HPB in hot springs.

Overseas students constitute the paramount talent resource for China, and, hence, overseas talent mobility prediction is crucial for the formulation of China’s talent strategy. This study proposes a new model for predicting the number of students studying abroad and returning students, based on the grey system theory, owing to the limited data and uncertainty of the influencing factors. The proposed model introduces change-point detection to determine the number of modeling time points, based on the fractional-order grey prediction model. We employed a change-point detection method to find the change points for determining the model length, based on the principle of new information priority, and used a fractional order accumulated generating operation to construct a grey prediction model. The two real data sets, the annual number of students studying abroad and returning students, were employed to verify the superiority of the proposed model. The results showed that the proposed model outperformed other benchmark models. Furthermore, the proposed model has been employed to predict the tendencies of overseas talent mobility in China by 2025. Further, certain policy recommendations for China’s talent strategy development have been proposed, based on the prediction results.

In recent years, the tree of life has expanded substantially. Despite this, many abundant yet uncultivated microbial groups remain to be explored.

DNA phosphorothioation (PT-DNA) is a DNA backbone sulfur modification that is recognized by the type-IV restriction endonuclease ScoMcrA. Here the authors provide insights into sulfur recognition by solving the crystal structure of the PT-DNA bound sulfur-binding domain (SBD) from ScoMcrA and they further show that SBD homologs are widely spread among prokaryotes.

Amino acid levels are known to regulate anabolic and catabolic pathways. Here, the authors report that amino acids also affect membrane trafficking by stimulating endosome-to-Golgi retrograde trafficking and regulating cell surface localization of certain Golgi proteins through Ragulator and Arl5.

ABSTRACT The underlying molecular mechanisms of flavin-dependent amine oxidases remain relatively poorly understood, even though many of these enzymes have been reported. The nicotine oxidoreductase NicA2 is a crucial enzyme for the first step of nicotine degradation in Pseudomonas putida S16 (DSM 28022). Here, we present the crystal structure of a ternary complex comprising NicA2 residues 21 to 482, flavin adenine dinucleotide (FAD), and nicotine at 2.25 Å resolution. Unlike other, related structures, NicA2 does not have an associated diacyl glycerophospholipid, wraps its substrate more tightly, and has an intriguing exit passage in which nine bulky amino acid residues occlude the release of its toxic product, pseudooxynicotine (PN). The replacement of these bulky residues by amino acids with small side chains effectively increases the catalytic turnover rate of NicA2. Our results indicate that the passage in wild-type NicA2 effectively controls the rate of PN release and thus prevents its rapid intracellular accumulation. It gives ample time for PN to be converted to less-harmful substances by downstream enzymes such as pseudooxynicotine amine oxidase (Pnao) before its accumulation causes cell damage or even death. The temporal metabolic regulation mode revealed in this study may shed light on the production of cytotoxic compounds. IMPORTANCE Flavin-dependent amine oxidases have received extensive attention because of their importance in drug metabolism, Parkinson’s disease, and neurotransmitter catabolism. However, the underlying molecular mechanisms remain relatively poorly understood. Here, combining the crystal structure of NicA2 (an enzyme in the first step of the bacterial nicotine degradation pathway in Pseudomonas putida S16 (DSM 28022)), biochemical analysis, and mutant construction, we found an intriguing exit passage in which bulky amino acid residues occlude the release of the toxic product of NicA2, in contrast to other, related structures. The selective product exportation register for NicA2 has proven to be beneficial to cell growth. Those seeking to produce cytotoxic compounds could greatly benefit from the use of such an export register mechanism.

The roles of long noncoding RNA FOXD2 adjacent opposite strand RNA 1 (FOXD2-AS1) in oral squamous cell carcinoma (OSCC) remain largely unknown. Here, the Atlas of Noncoding RNAs in Cancer online database was utilized to analyze the expression and clinical significance of FOXD2-AS1 in OSCC. Then, the cell proliferation of FOXD2-AS1-silenced OSCC cells (CAL-27) was assessed by MTT and clone formation experiments. FOXD2-AS1-coexpressed genes were enriched and analyzed via circlncRNAnet and Metascape tools. Finally, key molecules of the signal pathways of the aforementioned coexpressed genes were verified by western blotting. We found that FOXD2-AS1 was significantly highly expressed in OSCC tissues, and correlated with poor pathological grade and prognosis in patients with OSCC. Cell viability and clone formation ability were significantly inhibited after the knockdown of FOXD2-AS1. A total of 32 coexpressed genes of FOXD2-AS1 were identified, and those genes were enriched in the cell cycle. In conclusion, FOXD2-AS1 may be served as a potential prognostic indicator and therapeutic target for OSCC.

The lymphatic system is crucial for the regeneration of many tissues due to its fundamental role in immune cell trafficking, protein transport, and tissue homeostasis maintenance. Strategies stimulating lymphangiogenesis can provide new therapeutic approaches for tissue repair and regeneration (e.g., chronic wound healing). Here, we explored the effects of cerium-containing mesoporous bioactive glass nanoparticles (Ce-MBGNs) on lymphangiogenesis. The results showed that the extracts of Ce-MBGNs (1, 5, or 10 wt/v%) were non-cytotoxic toward lymphatic endothelial cells (LECs), while they enhanced the proliferation of LECs. Moreover, as evidenced by the scratch wound healing and Transwell migration assays, conditioned media containing the extract of Ce-MBGNs (1 wt/v%) could enhance the migration of LECs in comparison to the blank control and the media containing vascular endothelial growth factor-C (VEGF-C, 50 ng/mL). Additionally, a tube-formation assay using LECs showed that the extract of Ce-MBGNs (1 wt/v%) promoted lymphatic vascular network formation. Western blot results suggested that Ce-MBGNs could induce lymphangiogenesis probably through the HIF-1α/VEGFR-3 pathway. Our study for the first time showed the effects of Ce-MBGNs on stimulating lymphangiogenesis in vitro, highlighting the potential of Ce-MBGNs for wound healing.

Uncovering microbial response to salinization or desalinization is of great importance to understanding of the influence of global climate change on lacustrine microbial ecology. In this study, to simulate salinization and desalinization, sediments from Erhai Lake (salinity 0.3–0.8 g/L) and Chaka Lake (salinity 299.3–350.7 g/L) on the Qinghai–Tibetan Plateau were transplanted into different lakes with a range of salinity of 0.3–299.3 g/L, followed by in situ incubation for 50 days and subsequent geochemical and microbial analyses. Desalinization was faster than salinization in the transplanted sediments. The salinity of the transplanted sediment increased and decreased in the salinization and desalinization simulation experiments, respectively. The TOC contents of the transplanted sediments were lower than that of their undisturbed counterparts in the salinization experiments, whereas they had a strong negative linear relationship with salinity in the desalinization experiments. Microbial diversity decreased in response to salinization and desalinization, and microbial community dissimilarity significantly (P < 0.01) increased with salinity differences between the transplanted sediments and their undisturbed counterparts. Microbial groups belonging to Gammaproteobacteria and Actinobacteria became abundant in salinization whereas Bacteroidetes and Chloroflexi became dominant in desalinization. Among the predicted microbial functions, hydrogenotrophic methanogenesis, methanogenesis through CO2 reduction with H2, nitrate/nitrogen respiration, and nitrification increased in salinization; in desalinization, enhancement was observed for respiration of sulfur compounds, sulfate respiration, sulfur respiration, thiosulfate respiration, hydrocarbon degradation, chemoheterotrophy, and fermentation, whereas depressing was found for aerobic ammonia oxidation, nitrate/nitrogen respiration, nitrification, nitrite respiration, manganese oxidation, aerobic chemoheterotrophy, and phototrophy. Such microbial variations could be explained by changes of transplantation, salinity, and covarying variables. In summary, salinization and desalinization had profound influence on the geochemistry, microbial community, and function in lakes.

Summary: The size and shape control of amorphous nanomaterials has long been a bottleneck restricting their further development. Here, we present a general approach to synthesize more than twenty kinds of amorphous metal oxide nanosheets. The amorphous state of the nanosheets is determined using aberration-corrected high-resolution transmission electron microscopy and X-ray diffraction. By using extended X-ray absorption fine structure analysis, we demonstrate that amorphous nanosheets have a larger interatomic distance and a looser packing characteristic compared with crystalline counterparts. The as-prepared amorphous FeOx nanosheets are used as sodium-ion batteries anode materials, exhibiting notable performance with a specific capacity of 263.4 mAh g−1 at 100 mA g−1 and long-term cyclic stability. Significantly, a reversible amorphous-crystalline-amorphous structure transformation phenomenon during the cycling test is observed at the atomic scale.

ABSTRACT DNA phosphorothioate (PT) modification, in which the nonbridging oxygen in the sugar-phosphate backbone is substituted by sulfur, is catalyzed by DndABCDE or SspABCD in a double-stranded or single-stranded manner, respectively. In Dnd and Ssp systems, mobilization of sulfur in PT formation starts with the activation of the sulfur atom of cysteine catalyzed by the DndA and SspA cysteine desulfurases, respectively. Despite playing the same biochemical role, SspA cannot be functionally replaced by DndA, indicating its unique physiological properties. In this study, we solved the crystal structure of Vibrio cyclitrophicus SspA in complex with its natural substrate, cysteine, and cofactor, pyridoxal phosphate (PLP), at a resolution of 1.80 Å. Our solved structure revealed the molecular mechanism that SspA employs to recognize its cysteine substrate and PLP cofactor, suggesting a common binding mode shared by cysteine desulfurases. In addition, although the distance between the catalytic Cys314 and the substrate cysteine is 8.9 Å, which is too far for direct interaction, our structural modeling and biochemical analysis revealed a conformational change in the active site region toward the cysteine substrate to move them close to each other to facilitate the nucleophilic attack. Finally, the pulldown analysis showed that SspA could form a complex with SspD, an ATP pyrophosphatase, suggesting that SspD might potentially accept the activated sulfur atom directly from SspA, providing further insights into the biochemical pathway of Ssp-mediated PT modification. IMPORTANCE Apart from its roles in Fe-S cluster assembly, tRNA thiolation, and sulfur-containing cofactor biosynthesis, cysteine desulfurase serves as a sulfur donor in the DNA PT modification, in which a sulfur atom substitutes a nonbridging oxygen in the DNA phosphodiester backbone. The initial sulfur mobilization from l-cysteine is catalyzed by the SspA cysteine desulfurase in the SspABCD-mediated DNA PT modification system. By determining the crystal structure of SspA, the study presents the molecular mechanism that SspA employs to recognize its cysteine substrate and PLP cofactor. To overcome the long distance (8.9 Å) between the catalytic Cys314 and the cysteine substrate, a conformational change occurs to bring Cys314 to the vicinity of the substrate, allowing for nucleophilic attack.

Environmental concerns and depletion of traditional energy lead to the booming development of renewable distributed generation (RDG) in the past decade. However, due to intermittent nature of renewable energy sources, to what extent RDG could provide capacity to power systems becomes a critical issue to the utility company when implementing long-term system strategic (generation expansion) planning. On the other hand, in a smart-grid frame, the popularization of different varieties of demand-side resources enables the system to operate at more flexible modes ever before. The potential variability in load demand not only introduces additional dynamics and uncertainties to the system, but could also affect the reliability benefits of RDG. Therefore, in practice, to effectively estimate the reliability value of RDG in power systems, the potential interaction between generationand demand-side must be properly captured. In this paper, a study to assess the capacity credit (CC) of RDG in a context of distributed generation system is performed with consideration of the impacts of demand response (DR). A compound reliability model for DR is presented, which considers the uncertainties involved in both instant response and follow-on load recovery processes. On this basis, an assessment framework for the CC of RDG based on sequential Monte Carlo simulation is developed by which the inter-temporal characteristics of DR resources can be fully captured. The numerical study is implemented based on the IEEE-38 bus test case. The calculation results demonstrate that the CC of RDG would depend on a variety of factors, including penetration level, responsiveness of load demand and the correlations between RDG and DR availability. Also, it is shown that accounting for the underlying effect of DR is of absolute importance, otherwise the CC of RDG might be estimated erroneously in real practices.

The Hippo tumor suppressor pathway is essential for development and tissue growth control. Here the authors employ a multi-disciplinary approach to characterize the interactions of the three Hippo kinases with the signaling adaptor MOB1 and show how they differently affect development, tissue growth and tumor suppression.

Arthrobacter nicotinovorans decomposes nicotine through the pyridine pathway. 6-hydroxypseudooxynicotine 2-oxidoreductase (also named ketone dehydrogenase, Kdh) is an important enzyme in nicotine degradation pathway of A. nicotinovorans, and is responsible for the second hydroxylation of nicotine. Kdh belongs to the molybdenum hydroxylase family, and catalyzes the oxidation of 6-hydroxy-pseudooxynicotine (6-HPON) to 2,6-dihydroxy-pseudooxynicotine (2,6-DHPON). We determined the crystal structure of the Kdh holoenzyme from A. nicotinovorans, with its three subunits KdhL, KdhM, and KdhS, and their associated cofactors molybdopterin cytosine dinucleotide (MCD), two iron-sulfur clusters (Fe2S2), and flavin adenine dinucleotide (FAD), respectively. In addition, we obtained a structural model of the substrate 6-HPON-bound Kdh through molecular docking, and performed molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) calculations to unveil the catalytic mechanism of Kdh. The residues Glu345, Try551, and Glu748 of KdhL were found to participate in substrate binding, and Phe269 and Arg383 of KdhL were found to contribute to stabilize the MCD conformation. Furthermore, site-directed mutagenesis and enzymatic activity assays were performed to support our structural and computational results, which also revealed a trend of increasing catalytic efficiency with the increase in the buffer pH. Lastly, our electrochemical results demonstrated electron transfer among the various cofactors of Kdh. Therefore, our work provides a comprehensive structural, mechanistic, and functional study on the molybdenum hydroxylase Kdh in the nicotine degradation pathway of A. nicotinovorans.

The Drosophila ovary is recognized as a powerful model to study stem cell self-renewal and differentiation. Decapentaplegic (Dpp) is secreted from the germline stem cell (GSC) niche to activate Bone Morphogenic Protein (BMP) signaling in GSCs for their self-renewal and is restricted in the differentiation niche for daughter cell differentiation. Here, we report that Switch/sucrose non-fermentable (SWI/SNF) component Osa depletion in escort cells (ECs) results in a blockage of GSC progeny differentiation. Further molecular and genetic analyses suggest that the defective germline differentiation is partially attributed to the elevated dpp transcription in ECs. Moreover, ectopic Engrailed (En) expression in osa-depleted ECs partially contributes to upregulated dpp transcription. Furthermore, we show that Osa regulates germline differentiation in a Brahma (Brm)-associated protein (BAP)-complex-dependent manner. Additionally, the loss of EC long cellular processes upon osa depletion may also partly contribute to the germline differentiation defect. Taken together, these data suggest that the epigenetic factor Osa plays an important role in controlling EC characteristics and germline lineage differentiation.

The distribution and diversity of sulfate/sulfite reduction prokaryotic (SRP) communities in hot springs from the Quzhuomu and Daggyai Geothermal Zone of Tibetan, China, was reported for the first time. In hot springs that are naturally hyperthermal and anoxic, the sulfur cycle is one of the most active cycles of the elements. The distribution of SRP in response to temperature is of great importance to the understanding of biogeochemical cycling of sulfur in geothermal features. Little is known about the SRP in geothermal zone. In this study, the diversity of SRP was investigated in the sediments from the Daggyai and Quzhuomu geothermal zone using PCR amplification, cloning and sequencing of the dissimilatory sulfite reductase beta subunit gene (dsrB). The abundance of dsrB and 16S rRNA genes, were determined by quantitative polymerase chain reactions. In addition, correlations of the SRP assemblages with environmental factors were analyzed by the aggregated boosted tree (ABT) statistical analysis. The results showed that SRP populations were diverse, but were mainly composed of Desulfobacterales, Desulfovibrionales, Syntrophobacterales, Clostridia and Nitrospirales, and large fraction (25%) of novel sequences have branched groups in the dsrB phylogenetic tree. In Quzhuomu geothermal zone, sulfate-rich hot springs are characterized by thick bacterial mats that are green or red and the SRP populations mainly appear at mid-temperature (50 °C to 70 °C). In low-sulfate hot springs in the Daggyai geothermal zone, although gray or pink streamers are widely formed at 60 °C to 80 °C, they prefer to inhabit in green mat at lower temperature (30 °C to 50 °C). With increasing temperature, the diversity of the dsrB gene at the OTU level (cutoff 97%) decreased, while its relative abundance increased. This result suggests that temperature played an important role in affecting dsrB gene distribution.

ABSTRACT Nicotine, a toxic and addictive alkaloid from tobacco, is an environmental pollutant in areas near cigarette production facilities. Over the last decade, our group has studied, in depth, the pyrrolidine pathway of nicotine degradation in Pseudomonas putida S16. However, little is known regarding whole mechanism(s) regulating transcription of the nicotine degradation pathway gene cluster. In the present study, we comprehensively elucidate an overall view of the NicR2-mediated two-step mechanism regulating 3-succinoyl-pyridine (SP) biotransformation, which involves the association of free NicR2 with two promoters and the dissociation of NicR2 from the NicR2-promoter complex. NicR2 can bind to another promoter, Pspm, and regulate expression of the nicotine-degrading genes in the middle of nic2 gene cluster, which are not controlled by the previously reported Phsp promoter. We identified the function of the inverted repeat bases on the two promoters responsible for NicR2 binding and found out that the –35/–10 motif for RNA polymerase is overlapped by the NicR2 binding site. We clarify the exact role of 6-hydroxy-3-succinoyl-pyridine (HSP), which acts as an antagonist and may prevent binding of free NicR2 to the promoters but cannot release NicR2 from the promoters. Finally, a regulatory model is proposed, which consists of three parts: the interaction between NicR2 and two promoters (Pspm and Phsp), the interaction between NicR2 and two effectors (HSP and SP), and the interaction between NicR2 and RNA polymerase. IMPORTANCE We report the entire process underlying the NicR2 regulatory mechanism from association between free NicR2 and two promoters to dissociation of the NicR2-promoter complex. NicR2 can bind to another promoter, Pspm, which controls expression of nicotine-degrading genes that are not controlled by the Phsp promoter. We identified specific nucleotides of the Pspm promoter responsible for NicR2 binding. HSP was further demonstrated as an antagonist, which prevents the binding of NicR2 to the Pspm and Phsp promoters, by locking NicR2 in the derepression conformation. The competition between NicR2 and RNA polymerase is essential to initiate transcription of nicotine-degrading genes. This study extends our understanding of molecular mechanisms in biodegradation of environmental pollutants and toxicants.

Little is known about the distribution and ecological functions of abundant, intermediate, and rare biospheres and their correlations with environmental factors in hot springs. Here, we explored the microbial community composition of total, abundant, intermediate, and rare biospheres in 66 Tibetan hot springs (pairwise geographic distance 0–610 km, temperature 32–86°C, pH 3.0–9.5, and salinity 0.13–1.32 g/L) with the use of Illumina MiSeq high-throughput sequencing. The results showed that the abundant sub-communities were mainly composed of Chloroflexi, Proteobacteria, Deinococcus-Thermus, Aquificae, Bacteroidetes, and Firmicutes. In contrast, the rare sub-communities mainly consisted of most newly proposed or candidate phyla of Dictyoglomi, Hydrogenedentes, Atribacteria, Hadesarchaea, Aminicenantes, Microgenomates, Calescamantes, Omnitrophica, Altiarchaeales, and Chlamydiae. However, the abundant and rare sub-communities shared some common phyla (e.g., Crenarchaeota, Bathyarchaeota, and Chlorobi), which were composed of different OTUs. The abundant, intermediate, and rare sub-communities were mainly influenced by different environmental variables, which could be ascribed to the fact that they may have different growth and activity and thus respond differently to these variables. Spatial factors showed more contribution to shaping of the intermediate and rare communities than to abundant sub-community, suggesting that the abundant taxa were more easily dispersed than their rare counterparts among hot springs. Microbial ecological function prediction revealed that the abundant and rare sub-communities responded differently to the measured environmental factors, suggesting they may occupy different ecological niches in hot springs. The rare sub-communities may play more important roles in organic matter degradation than their abundant counterparts in hot springs. Collectively, this study provides a better understanding on the microbial community structure and potential ecological functions of the abundant and rare biospheres in hot spring ecosystems. The identified rare taxa provide new opportunities of ecological, taxonomic and genomic discoveries in Tibetan hot springs.

Time series data for decision problems such as energy demand forecasting are often derived from uncertain assessments, and do not meet any statistical assumptions. The interval grey number becomes an appropriate representation for an uncertain and imprecise observation. In order to obtain nonlinear interval grey numbers with better forecasting accuracy, this study proposes a combined model by fusing interval grey numbers estimated by neural networks (NNs) and the grey prediction models. The proposed model first uses interval regression analysis using NNs to estimate interval grey numbers for a real valued sequence; and then a grey residual modification model is constructed using the upper and lower wrapping sequences obtained by NNs. It turns out that two different kinds of interval grey numbers can be estimated by nonlinear interval regression analysis. Forecasting accuracy on real data sequences was then examined by the best non-fuzzy performance values of the combined model. The proposed combined model performed well compared with the other interval grey prediction models considered.

Thioarsenates are common arsenic species in sulfidic geothermal waters, yet little is known about their biogeochemical traits. In the present study, a novel sulfate-reducing bacterial strain Desulfotomaculum TC-1 was isolated from a sulfidic hot spring in Tengchong geothermal area, Yunnan Province, China. The arxA gene, encoding anaerobic arsenite oxidase, was successfully amplified from the genome of strain TC-1, indicating it has a potential ability to oxidize arsenite under anaerobic condition. In anaerobic arsenite oxidation experiments inoculated with strain TC-1, a small amount of arsenate was detected in the beginning but became undetectable over longer time. Thioarsenates (AsO4-xSx2- with x = 1–4) formed with mono-, di- and tri-thioarsenates being dominant forms. Tetrathioarsenate was only detectable at the end of the experiment. These results suggest that thermophilic microbes might be involved in the formation of thioarsenates and provide a possible explanation for the widespread distribution of thioarsenates in terrestrial geothermal environments.

Little is known about the relative importance of spatial and environmental factors to structuring aquatic and sedimentary microbial biogeography in lakes. Here, we investigated the microbial community composition (MCC) of the water (n = 35) and sediment (n = 35) samples from 16 lakes in western China (salinity: freshwater to salt saturation; pairwise geographical distance: 9 - 2027 km) using high-throughput sequencing and evaluated the relative importance of spatial and environmental factors to microbial (including total, abundant and rare) distributions. Our results showed that spatial factors were more important than environmental factors in shaping the biogeography of aquatic and sedimentary microbial communities in the studied lakes, and spatial factors on abundant microbial community was stronger than that on the total/rare microbial communities. Moreover, sedimentary rare MCC might be more sensitive to environmental factors than its aquatic counterpart. Such different biogeography responses of total, abundant and rare communities to environmental and spatial factors could be ascribed to different physiochemical properties between water and sediment. Collectively, this study expands our understanding of factors shaping microbial biogeography of total, abundant and rare communities between waters and sediments of lakes.

Enzyme promiscuity is a prerequisite for fast divergent evolution of biocatalysts. A phosphotriesterase-like lactonase (PLL) from Geobacillus kaustophilus HTA426 (GkaP) exhibits main lactonase and promiscuous phosphotriesterase activities. To understand its catalytic and evolutionary mechanisms, we investigated a "hot spot" in the active site by saturation mutagenesis as well as X-ray crystallographic analyses. We found that position 99 in the active site was involved in substrate discrimination. One mutant, Y99L, exhibited 11-fold improvement over wild-type in reactivity (kcat/Km) toward the phosphotriesterase substrate ethyl-paraoxon, but showed 15-fold decrease toward the lactonase substrate δ-decanolactone, resulting in a 157-fold inversion of the substrate specificity. Structural analysis of Y99L revealed that the mutation causes a ∼6.6 Å outward shift of adjacent loop 7, which may cause increased flexibility of the active site and facilitate accommodation and/or catalysis of organophosphate substrate. This study provides for the PLL family an example of how the evolutionary route from promiscuity to specificity can derive from very few mutations, which promotes alteration in the conformational adjustment of the active site loops, in turn draws the capacity of substrate binding and activity.

Microbe-mediated biogeochemical cycles contribute to the global climate system and have sensitive responses and feedbacks to environmental stress caused by climate change. Yet, little is known about the effects of microbial biodiversity (i.e., taxonmic and functional diversity) on biogeochemical cycles in ecosytems that are highly sensitive to climate change. One such sensitive ecosystem is Qinghai Lake, a high-elevation (3196 m) saline (1.4%) lake located on the Tibetan Plateau, China. This study provides baseline information on the microbial taxonomic and functional diversity as well as the associated stress response genes. Illumina metagenomic and metatranscriptomic datasets were generated from lake water samples collected at two sites (B and E). Autotrophic Cyanobacteria dominated the DNA samples, while heterotrophic Proteobacteria dominated the RNA samples at both sites. Photoheterotrophic Loktanella was also present at both sites. Photosystem II was the most active pathway at site B; while, oxidative phosphorylation was most active at site E. Organisms that expressed photosystem II or oxidative phosphorylation also expressed genes involved in photoprotection and oxidative stress, respectively. Assimilatory pathways associated with the nitrogen cycle were dominant at both sites. Results also indicate a positive relationship between functional diversity and the number of stress response genes. This study provides insight into the stress resilience of microbial metabolic pathways supported by greater taxonomic diversity, which may affect the microbial community response to climate change.

Lonicera confusa was a medical plant which could adapt to the Ca-rich environment in the karst area of China. The photosynthesis, relative chlorophyll content,differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) of L. confusa that cultivated in calcareous and sandstone soils were investigated. The results showed that the relative chlorophyll content and net photosynthesis rate of L. confusa in calcareous soil are much higher than that planted in sandstone soil, the higher content of calcium might play a role in keeping the chloroplast from harm and showed higher photosynthesis rate. The transpiration and stomata conductance were decreased in calcareous soil, which might result from the closure of stomata. The GeneFishing and proteomic results showed that the expression of DEGs and DEPs were critical for photosynthesis and stomata closure, such as RuBisCO, photosynthetic electron transfer c and malate dehydrogenase varied in the leaves of L. confusa that cultivated in different soils. These DEGs or DEPs were further found to be directly or indirectly regulated by calcium sensor proteins. This study enriched our knowledge of the molecular mechanism of high net photosynthesis rate and lower transpiration of L. confusa that cultivated in the calcareous soil in some degree.

The Tibetan Plateau in Northwest China hosts a number of hot springs that represent a biodiversity hotspot for thermophiles, yet their diversity and relationship to environmental conditions are poorly explored in these habitats. In this study we investigated microbial diversity and community composition in 13 Tibetan hot springs with a wide range of temperatures (22.1-75°C) and other geochemical conditions by using the 16S rRNA gene pyrosequencing approach. Bacteria (10(8)-10(11) copy/g; 42 bacterial phyla) in Tibetan hot springs were more abundant and far more diverse than Archaea (10(7)-10(10) copy/g; 5 archaeal phyla). The dominant bacterial phyla systematically varied with temperature. Moderate temperatures (75-66°C) favored Aquificae, GAL35, and novel Bacteria, whereas low temperatures (60-22.1°C) selected for Deinococcus-Thermus, Cyanobacteria, and Chloroflexi. The relative abundance of Aquificae was correlated positively with temperature, but the abundances of Deinococcus-Thermus, Cyanobacteria, and Chloroflexi were negatively correlated with temperature. Cyanobacteria and Chloroflexi were abundant in Tibetan hot springs and their abundances were positively correlated at low temperatures (55-43°C) but negatively correlated at moderate temperatures (75-55°C). These correlation patterns suggest a complex physiological relationship between these two phyla. Most archaeal sequences were related to Crenarchaeota with only a few related to Euryarchaeota and Thaumarchaeota. Despite the fact that microbial composition in Tibetan hot springs was strongly shaped by temperature, microbial diversity (richness, evenness and Shannon diversity) was not significantly correlated with temperature change. The results of this study expand our current understanding of microbial ecology in Tibetan hot springs and provide a basis for a global comparison.

The Rehai and Ruidian geothermal fields, located in Tengchong County, Yunnan Province, China, host a variety of geochemically distinct hot springs. In this study, we report a comprehensive, cultivation-independent census of microbial communities in 37 samples collected from these geothermal fields, encompassing sites ranging in temperature from 55.1 to 93.6°C, in pH from 2.5 to 9.4, and in mineralogy from silicates in Rehai to carbonates in Ruidian. Richness was low in all samples, with 21-123 species-level OTUs detected. The bacterial phylum Aquificae or archaeal phylum Crenarchaeota were dominant in Rehai samples, yet the dominant taxa within those phyla depended on temperature, pH, and geochemistry. Rehai springs with low pH (2.5-2.6), high temperature (85.1-89.1°C), and high sulfur contents favored the crenarchaeal order Sulfolobales, whereas those with low pH (2.6-4.8) and cooler temperature (55.1-64.5°C) favored the Aquificae genus Hydrogenobaculum. Rehai springs with neutral-alkaline pH (7.2-9.4) and high temperature (>80°C) with high concentrations of silica and salt ions (Na, K, and Cl) favored the Aquificae genus Hydrogenobacter and crenarchaeal orders Desulfurococcales and Thermoproteales. Desulfurococcales and Thermoproteales became predominant in springs with pH much higher than the optimum and even the maximum pH known for these orders. Ruidian water samples harbored a single Aquificae genus Hydrogenobacter, whereas microbial communities in Ruidian sediment samples were more diverse at the phylum level and distinctly different from those in Rehai and Ruidian water samples, with a higher abundance of uncultivated lineages, close relatives of the ammonia-oxidizing archaeon "Candidatus Nitrosocaldus yellowstonii", and candidate division O1aA90 and OP1. These differences between Ruidian sediments and Rehai samples were likely caused by temperature, pH, and sediment mineralogy. The results of this study significantly expand the current understanding of the microbiology in Tengchong hot springs and provide a basis for comparison with other geothermal systems around the world.

DNA phosphorothioation is widespread among prokaryotes, and might function to restrict gene transfer among different kinds of bacteria. There has been little investigation into the structural mechanism of the DNA phosphorothioation process. DndA is a cysteine desulfurase which is involved in the first step of DNA phosphorothioation. In this study, we determined the crystal structure of Streptomyces lividans DndA in complex with its covalently bound cofactor PLP, to a resolution of 2.4 Å. Our structure reveals the molecular mechanism that DndA employs to recognize its cofactor PLP, and suggests the potential binding site for the substrate L-cysteine on DndA. In contrast to previously determined structures of cysteine desulfurases, the catalytic cysteine of DndA was found to reside on a β strand. This catalytic cysteine is very far away from the presumable location of the substrate, suggesting that a conformational change of DndA is required during the catalysis process to bring the catalytic cysteine close to the substrate cysteine. Moreover, our in vitro enzymatic assay results suggested that this conformational change is unlikely to be a simple result of random thermal motion, since moving the catalytic cysteine two residues forward or backward in the primary sequence completely disabled the cysteine desulfurase activity of DndA.

The human Discs Large 1 (DLG1) protein uses two of its three PDZ domains to interact with the C-terminal peptide of the Adenomatous Polyposis Coli (APC) tumor suppressor protein. The DLG1/APC complex inhibits the cell cycle progression from the G0/G1 to the S phase, regulates epithelial cell migration and morphogenesis, and is required for polarization of the microtubule cytoskeleton. However, the molecular details of how DLG1 recognizes APC is not clear. In this study, we performed biochemical and biophysical assays to investigate the interactions between PDZ domains of DLG1 and the C-terminal peptide of APC. In addition, we determined the crystal structures of the PDZ1 and PDZ2 domains of DLG1 each in complex with the C-terminal 11-residue peptide of APC. Our biochemical, biophysical, and structural results revealed structural elements and residues on PDZ1 and PDZ2 domains of DLG1 and on APC crucial for their mutual interaction. In particular, our results show that the β2/β3 loops of PDZ1 and PDZ2 play important roles in contributing to the binding affinities between PDZ domains and APC, through interacting with the residues upstream of the canonical PDZ-binding S/T-X-V motif. The results provide new insights into the binding mode of a defined C-terminal segment of APC by the PDZ domains of DLG1.

Carbazole is a recalcitrant compound with a dioxin-like structure and possesses mutagenic and toxic activities. Bacteria respond to a xenobiotic by recruiting exogenous genes to establish a pathway to degrade the xenobiotic, which is necessary for their adaptation and survival. Usually, this process is mediated by mobile genetic elements such as plasmids, transposons, and insertion sequences.The genes encoding the enzymes responsible for the degradation of carbazole to catechol via anthranilate were cloned, sequenced, and characterized from a carbazole-degrading Sphingomonas sp. strain XLDN2-5. The car gene cluster (carRAaBaBbCAc) and fdr gene were accompanied on both sides by two copies of IS6100 elements, and organized as IS6100::ISSsp1-ORF1-carRAaBaBbCAc-ORF8-IS6100-fdr-IS6100. Carbazole was converted by carbazole 1,9a-dioxygenase (CARDO, CarAaAcFdr), meta-cleavage enzyme (CarBaBb), and hydrolase (CarC) to anthranilate and 2-hydroxypenta-2,4-dienoate. The fdr gene encoded a novel ferredoxin reductase whose absence resulted in lower transformation activity of carbazole by CarAa and CarAc. The ant gene cluster (antRAcAdAbAa) which was involved in the conversion of anthranilate to catechol was also sandwiched between two IS6100 elements as IS6100-antRAcAdAbAa-IS6100. Anthranilate 1,2-dioxygenase (ANTDO) was composed of a reductase (AntAa), a ferredoxin (AntAb), and a two-subunit terminal oxygenase (AntAcAd). Reverse transcription-PCR results suggested that carAaBaBbCAc gene cluster, fdr, and antRAcAdAbAa gene cluster were induced when strain XLDN2-5 was exposed to carbazole. Expression of both CARDO and ANTDO in Escherichia coli required the presence of the natural reductases for full enzymatic activity.We predict that IS6100 might play an important role in the establishment of carbazole-degrading pathway, which endows the host to adapt to novel compounds in the environment. The organization of the car and ant genes in strain XLDN2-5 was unique, which showed strong evolutionary trail of gene recruitment mediated by IS6100 and presented a remarkable example of rearrangements and pathway establishments.

The Wnt/beta-catenin signaling pathway plays essential roles in cell proliferation and differentiation, and deregulated beta-catenin protein levels lead to many types of human cancers. On activation by Wnt, the Wnt co-receptor LDL receptor related protein 6 (LRP6) is phosphorylated at multiple conserved intracellular PPPSPXS motifs by glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1), resulting in recruitment of the scaffolding protein Axin to LRP6. As a result, beta-catenin phosphorylation by GSK3 is inhibited and beta-catenin protein is stabilized. However, how LRP6 phosphorylation and the ensuing LRP6-Axin interaction lead to the inhibition of beta-catenin phosphorylation by GSK3 is not fully understood. In this study, we reconstituted Axin-dependent beta-catenin phosphorylation by GSK3 and CK1 in vitro using recombinant proteins, and found that the phosphorylated PPPSPXS peptides directly inhibit beta-catenin phosphorylation by GSK3 in a sequence and phosphorylation-dependent manner. This inhibitory effect of phosphorylated PPPSPXS motifs is direct and specific for GSK3 phosphorylation of beta-catenin at Ser33/Ser37/Thr41 but not for CK1 phosphorylation of beta-catenin at Ser45, and is independent of Axin function. We also show that a phosphorylated PPPSPXS peptide is able to activate Wnt/beta-catenin signaling and to induce axis duplication in Xenopus embryos, presumably by inhibition of GSK3 in vivo. Based on these observations, we propose a working model that Axin recruitment to the phosphorylated LRP6 places GSK3 in the vicinity of multiple phosphorylated PPPSPXS motifs, which directly inhibit GSK3 phosphorylation of beta-catenin. This model provides a possible mechanism to account, in part, for inhibition of beta-catenin phosphorylation by Wnt-activated LRP6.