Fertilized egg cells secrete endopeptidases to avoid polytubey.

Upon gamete fusion, animal egg cells secrete proteases from cortical granules to establish a fertilization envelope as a block to polyspermy1-4. Fertilization in flowering plants is more complex and involves the delivery of two non-motile sperm cells by pollen tubes5,6. Simultaneous penetration of ovules by multiple pollen tubes (polytubey) is usually avoided, thus indirectly preventing polyspermy7,8. How plant egg cells regulate the rejection of extra tubes after successful fertilization is not known. Here we report that the aspartic endopeptidases ECS1 and ECS2 are secreted to the extracellular space from a cortical network located at the apical domain of the Arabidopsis egg cell. This reaction is triggered only after successful fertilization. ECS1 and ECS2 are exclusively expressed in the egg cell and transcripts are degraded immediately after gamete fusion. ECS1 and ESC2 specifically cleave the pollen tube attractor LURE1. As a consequence, polytubey is frequent in ecs1 ecs2 double mutants. Ectopic secretion of these endopeptidases from synergid cells led to a decrease in the levels of LURE1 and reduced the rate of pollen tube attraction. Together, these findings demonstrate that plant egg cells sense successful fertilization and elucidate a mechanism as to how a relatively fast post-fertilization block to polytubey is established by fertilization-induced degradation of attraction factors.

Arsenic distribution and partitioning in multiple media in a typical catchment in the Qinghai-Tibetan plateau: A comparison between freshwater and saltwater lakes.

Arsenic (As) has been widely detected in surface media on the Qinghai-Tibetan Plateau (QTP); however, the differences in the As distribution and partitioning characteristics between freshwater and saltwater lakes remain poorly understood. To determine the distribution and partitioning characteristics of As, multimedia environmental samples were collected from a typical small watershed consisting of a river, wetland, and both freshwater and saltwater lakes on the QTP. Results showed that freshwater systems, represented by Hurleg Lake, were high in particulate arsenic (PAs) and low in dissolved arsenic (DAs), whereas the saltwater system represented by Tosen Lake, exhibited the reverse distribution. This discrepancy in As distribution was primarily attributed to evaporation enrichment, competitive adsorption of HCO3- and pH variations, as suggested by correlation analysis and stable isotopic composition of water. In the stratified Tosen Lake, an increasing trend of DAs in the water column was observed, potentially driven by the reductive dissolution of Fe (hydr)oxides and bacterial sulfate reduction in the anoxic bottom hypolimnion. Conversely, Hurleg Lake maintained oxic conditions with stable DAs concentrations. Notably, PAs was elevated in the bottom layer of both lakes, possibly due to uptake/adsorption by biogenic particles, as indicated by high levels of chl.α and suspended particulate matter. These findings offer insights into the potential future impact of climate change on As mobilization/redistribution in arid plateau lakes, with implications for management policies that regulate As pollution.

Enhanced extracellular production of laccase in Coprinopsis cinerea by silencing chitinase gene.

Laccase, a copper-containing polyphenol oxidase, is an important green biocatalyst. In this study, Laccase Lcc5 was homologous recombinantly expressed in Coprinopsis cinerea and a novel strategy of silencing chitinase gene expression was used to enhance recombinant Lcc5 extracellular yield. Two critical chitinase genes, ChiEn1 and ChiE2, were selected by analyzing the transcriptome data of C. cinerea FA2222, and their silent expression was performed by RNA interference (RNAi). It was found that silencing either ChiEn1 or ChiE2 reduced sporulation and growth rate, and increased cell wall sensitivity, but had no significant effect on mycelial branching. Among them, the extracellular laccase activity of the ChiE2-silenced engineered strain Cclcc5-antiChiE2-5 and the control Cclcc5-13 reached the highest values (38.2 and 25.5 U/mL, respectively) at 250 and 150 rpm agitation speeds, corresponding to productivity of 0.35 and 0.19 U/mL·h, respectively, in a 3-L fermenter culture. Moreover, since Cclcc5-antiChiE2-5 could withstand greater shear forces, its extracellular laccase activity was 2.6-fold higher than that of Cclcc5-13 when the agitation speed was all at 250 rpm. To our knowledge, this is the first report of enhanced recombinant laccase production in C. cinerea by silencing the chitinase gene. This study will pave the way for laccase industrial production and accelerate the development of a C. cinerea high-expression system. KEY POINTS: • ChiEn1 and ChiE2 are critical chitinase genes in C. cinerea FA2222 genome. • Chitinase gene silencing enhanced the tolerance of C. cinerea to shear forces. • High homologous production of Lcc5 is achieved by fermentation in a 3-L fermenter.

Polyoxometalates@Metal-Organic Frameworks Derived Bimetallic Co/Mo2 C Nanoparticles Embedded in Carbon Nanotube-Interwoven Hierarchically Porous Carbon Polyhedron Composite as a High-Efficiency Electrocatalyst for Al-S Batteries.

Al-S battery (ASB) is a promising energy storage device, notable for its safety, crustal abundance, and high theoretical energy density. However, its development faces challenges due to slow reaction kinetics and poor reversibility. The creation of a multifunctional cathode material that can both adsorb polysulfides and accelerate their conversion is key to advancing ASB. Herein, a composite composed of polyoxometalate nanohybridization-derived Mo2 C and N-doped carbon nanotube-interwoven polyhedrons (Co/Mo2 C@NCNHP) is proposed for the first time as an electrochemical catalyst in the sulfur cathode. This composite improves the utilization and conductivity of sulfur within the cathode. DFT calculations and experimental results indicate that Co enables the chemisorption of polysulfides while Mo2 C catalyzes the reduction reaction of long-chain polysulfides. X-ray photoelectron spectroscopy (XPS) and in situ UV analysis reveal the different intermediates of Al polysulfide species in Co/Mo2 C@NCNHP during discharging/charging. As a cathode material for ASB, Co/Mo2 C@NCNHP@S composite can deliver a discharge-charge voltage hysteresis of 0.75 V with a specific capacity of 370 mAh g-1 after 200 cycles at 1A g-1 .

The nuclear-localized RNA helicase 13 is essential for chloroplast development in Arabidopsis thaliana.

The chloroplast is a semi-autonomous organelle with a double membrane structure, and its structural stability is a prerequisite for its correct function. Chloroplast development is regulated by known nuclear-encoded chloroplast proteins or proteins encoded within the chloroplast itself. However, the mechanism of chloroplast development regulated by other organelles remains largely unknown. Here, we report that the nuclear-localized DEAD-box RNA helicase 13 (RH13) is essential for chloroplast development in Arabidopsis thaliana. RH13 is widely expressed in tissues and localized to the nucleolus. A homozygous rh13 mutant shows abnormal chloroplast structure and leaf morphogenesis. Proteomic analysis showed that the expression levels of photosynthesis-related proteins in chloroplasts were reduced due to loss of RH13. Furthermore, RNA-sequencing and proteomics data revealed decreases in the expression levels of these chloroplast-related genes, which undergo alternative splicing events in the rh13 mutant. Taken together, we propose that nucleolus-localized RH13 is critical for chloroplast development in Arabidopsis.

Adsorption Mechanism of Benzene Derivatives by Pagoda[n]arenes.

Despite the widespread use in industrial production, benzene derivatives are harmful to both human beings and the environment. The control of these substances has become an important subject of scientific research. This study introduces a new approach for adsorption and separation of benzene derivatives utilizing pagoda[n]arene based supramolecular materials. Density functional theory calculations were employed to investigate the molecular recognition mechanism of benzene derivatives by pagoda[4]arenes and pagoda[5]arenes (Pa[4]As and Pa[5]As). Results indicate that Pa[4]As and Pa[5]As can effectively accommodate benzene derivatives through non-covalent interactions, leading to the formation of stable host-guest complexes. Additionally, molecular dynamics simulations revealed that both crystalline and non-crystalline supramolecular aggregates of Pa[4]As and Pa[5]As possess the ability to adsorb benzene derivatives and maintain the stability of the adsorption. Moreover, increasing the temperature causes benzene derivatives to desorb from the adsorbing aggregates, and thus the material can be reutilized.

Inhibition of the hERG potassium ion channel by different non-nucleoside human cytomegalovirus polymerase antiviral inhibitor series and the exploration of variations on a pyrroloquinoline core to reduce cardiotoxicity potential.

Many non-nucleoside human cytomegalovirus (HCMV) inhibitors have been reported in patent and scientific literature, however, none have reached commercialization despite the urgent need for new HCMV treatments. Herein we report select compounds from different templates that all had low micromolar human ether-à-go-go (hERG) ion channel IC50 values. We also describe a series of pyrroloquinoline derivatives that were designed and synthesized to understand the effect of various substitution on human cytomegalovirus (HCMV) polymerase activity, antiviral activity, and hERG inhibition. These results demonstrated that hERG inhibition can be significantly altered based on the substitution on this template. An HCMV inhibitor with low hERG inhibition and reduced cytotoxicity is also described. The results suggest substitution can be fine tuned for the non-nucleoside polymerase inhibitors to reduce hERG inhibition and maintain HCMV antiviral potency.

The Role of TGF-ß Signaling in Saphenous Vein Graft Failure after Peripheral Arterial Disease Bypass Surgery.

Saphenous vein bypass grafting is an effective technique used to treat peripheral arterial disease (PAD). However, restenosis is the major clinical challenge for the graft vessel among people with PAD postoperation. We hypothesize that there is a common culprit behind arterial occlusion and graft restenosis. To investigate this hypothesis, we found TGF-ß, a gene specifically upregulated in PAD arteries, by bioinformatics analysis. TGF-ß has a wide range of biological activities and plays an important role in vascular remodeling. We discuss the molecular pathway of TGF-ß and elucidate its mechanism in vascular remodeling and intimal hyperplasia, including EMT, extracellular matrix deposition, and fibrosis, which are the important pathways contributing to stenosis. Additionally, we present a case report of a patient with graft restenosis linked to the TGF-ß pathway. Finally, we discuss the potential applications of targeting the TGF-ß pathway in the clinic to improve the long-term patency of vein grafts.

pH-Responsive Self-Assemblies from the Designed Folic Acid-Modified Peptide Drug for Dual-Targeting Delivery.

Targeting delivery is a promising technique for the therapy of cancers. A molecule FA-EEYSV-NH2, which consists of target recognition site folic acid (FA), dipeptide linker, and peptide drug, was designed as a novel anticancer prodrug. The molecules could self-assemble into nanoparticles at pH 7.0 and nanofibers at pH 5.0. By the aid of pH-responsiveness, the self-assemblies were used purposefully as targeted vehicles of self-delivery prodrugs. The results of cell toxicity and internalization assays have proved that the self-assemblies have good cancer cell selectivity. The selection was mainly attributed to the pH-responsive structure transition of self-assemblies and the FA active-targeting effect. We hope that our work could provide a useful strategy for finely tuning the properties and activities of peptide-based supramolecular nanomaterials, thus optimizing nanomedicines with enhanced performance.

Pathogen-secreted proteases activate a novel plant immune pathway.

Mitogen-activated protein kinase (MAPK) cascades play central roles in innate immune signalling networks in plants and animals. In plants, however, the molecular mechanisms of how signal perception is transduced to MAPK activation remain elusive. Here we report that pathogen-secreted proteases activate a previously unknown signalling pathway in Arabidopsis thaliana involving the Gα, Gß, and Gγ subunits of heterotrimeric G-protein complexes, which function upstream of an MAPK cascade. In this pathway, receptor for activated C kinase 1 (RACK1) functions as a novel scaffold that binds to the Gß subunit as well as to all three tiers of the MAPK cascade, thereby linking upstream G-protein signalling to downstream activation of an MAPK cascade. The protease-G-protein-RACK1-MAPK cascade modules identified in these studies are distinct from previously described plant immune signalling pathways such as that elicited by bacterial flagellin, in which G proteins function downstream of or in parallel to an MAPK cascade without the involvement of the RACK1 scaffolding protein. The discovery of the new protease-mediated immune signalling pathway described here was facilitated by the use of the broad host range, opportunistic bacterial pathogen Pseudomonas aeruginosa. The ability of P. aeruginosa to infect both plants and animals makes it an excellent model to identify novel immunoregulatory strategies that account for its niche adaptation to diverse host tissues and immune systems.

Crystal structure of TbEsa1 presumed Tudor domain from Trypanosoma brucei.

The essential SAS2-related acetyltransferase 1 (Esa1), as a acetyltransferase of MYST family, is indispensable for the cell cycle and transcriptional regulation. The Tudor domain consists of 60 amino acids and belongs to the Royal family, which serves as a module interacting with methylated histone and/or DNA. Although Tudor domain has been widely studied in higher eukaryotes, its structure and function remain unclear in Trypanosoma brucei (T. brucei), a protozoan unicellular parasite causing sleeping sickness in human and nagana in cattle in sub-Saharan Africa. Here, we determined a high-resolution structure of TbEsa1 presumed Tudor domain from T. brucei by X-ray crystallography. TbEsa1 Tudor domain adopts a conserved Tudor-like fold, which is comprised of a five-stranded ß-barrel surrounded by two short α-helices. Furthermore, we revealed a non-specific DNA binding pattern of TbEsa1 Tudor domain. However, TbEsa1 Tudor domain showed no methyl-histone binding ability, due to the absence of key aromatic residues forming a conserved aromatic cage.

Solution structure of TbUfm1 from Trypanosoma brucei and its binding to TbUba5.

Ubiquitin-like proteins are conserved in eukaryotes and involved in numerous cellular processes. Ufm1 is proved to play important roles in endoplasmic reticulum homeostasis, vesicle transportation and embryonic development. Enzyme cascade of Ufm1 is similar to that of ubiquitin. Mature Ufm1 is activated and conjugated to substrates by assistance of Ufm1 activating enzyme Uba5 (E1), Ufm1 conjugating enzyme Ufc1 (E2), and Ufm1 ligating enzyme Ufl1 (E3). Here, we determined the solution structure of TbUfm1 from Trypanosoma brucei (T. brucei) by NMR spectroscopy and explored the interactions between TbUfm1 and TbUba5/TbUfc1/TbUfl1. TbUfm1 adopts a typical ß-grasp fold, which partially wraps a central α-helix and the other two helixes. NMR chemical shift perturbation indicated that TbUfm1 interacts with TbUba5 via a hydrophobic pocket formed by α1α2ß1ß2. Although the structure and Uba5-interaction mode of TbUfm1 are conserved in Ufm1 proteins, there are also some differences, which might reflect the potential diversity of Ufm1 in evolution and biological functions.

Selective binding of mitophagy receptor protein Bcl-rambo to LC3/GABARAP family proteins.

Mitophagy regulates the metabolic level and cell fates by specifically degrading damaged or redundant mitochondria in these cells. During the formation of autophagosomes, autophagy receptors and adaptors, which usually contain a LC3-interacting region (LIR) domain, are recruited through their interactions with LC3/GABARAP family of proteins. Bcl-rambo is one of the mitophagy receptors that interact with LC3s/GABARAPs. In this study, we first measured the binding of Bcl-rambo to LC3s/GABARAPs in vitro and found Bcl-rambo has a selectivity to LC3C/GABARP/GABARAPL1. Further investigations with bioinformatics analyses and mutagenesis suggested that the interactions with the HP1 and HP2 sites of LC3s/GABARAPs and the residues at the X2 site of the LIR domain of Bcl-rambo are both critical for the selectivity. Moreover, assays in vivo showed that manipulating the selective binding of Bcl-rambo resulted in the changes of mitophagy inductions in the cells. Overall, our data revealed the selective binding between Bcl-rambo and LC3s/GABARAPs and its molecular mechanisms and biological significances, which will be helpful for future studies of mitophagy mediated by Bcl-rambo.

Mechanism of the salt activation of laccase Lac15.

Laccases (benzenediol: oxygen oxidoreductases, EC1.10.3.2) can oxidize various substrates, and those which are tolerant to and even activated by salts have attracted a lot of attention due to their application potential in certain industries. The mechanism of the salt activation of laccases is awaiting to be elucidated yet. Our previous study (Li, Xie et al. 2018) supposed that the salt activation of marine laccase Lac15 might be attributed to Cl- ion specifically binding to some local sites to interfere substrate binding and/or electron transfer. In this study, we found two sites whose mutations resulted in elimination of the salt activation of Lac15's activity towards catechol and dopamine respectively, and revealed that the mutations affected the activity by altering both Em and kcat, demonstrating the supposed mechanism. A model for the salt activation of laccases was accordingly proposed, albeit some details are to be elucidated.

Crystal structure of a hypothetical T2SS effector Lpg0189 from Legionella pneumophila reveals a novel protein fold.

Lpg0189 is a type II secretion system-dependent extracellular protein with unknown function from Legionella pneumophila. Herein, we determined the crystal structure of Lpg0189 at 1.98 Šresolution by using single-wavelength anomalous diffraction (SAD). Lpg0189 folds into a novel chair-shaped architecture, with two sheets roughly perpendicular to each other. Bioinformatics analysis suggests Lpg0189 and its homologues are unique to Legionellales and evolved divergently. The interlinking structural and bioinformatics studies provide a better understanding of this hypothetical protein.

An unusual GH1 ß-glucosidase from marine sediment with ß-galactosidase and transglycosidation activities for superior galacto-oligosaccharide synthesis.

A novel ß-glucosidase, BglD1 with high ß-galactosidase and transglycosidation activities, was screened and cloned from the deep-sea bacterium Bacillus sp. D1. BglD1 exhibited the maximal ß-glucosidase and ß-galactosidase activities at 55-60 °C and pH 5.5-6.0. The enzyme maintained approximately 50% of its original activity at 35 °C and pH 6.0 after 120-h incubation. When applied to synthesize galacto-oligosaccharides (GOS), BglD1 generated 118.3 g/L GOS (33.8% (w/w)) from 350 g/L lactose, with trisaccharide Gal-ß(1 → 3)-Lac and disaccharide Gal-ß(1 → 4)-Gal as the main components. Furthermore, BglD1 could hydrolyze lactose in milk and produce GOS simultaneously. Using milk as the substrate, BglD1 hydrolyzed 88.5% lactose and produced 3.3 g/L GOS after incubation at 30 °C for 1 h. To improve the transglycosidation activity, a mutant BglD1:E224T was generated based on the semi-rational design. The GOS yield of BglD1:E224T was 11.5% higher than that of BglD1 when using lactose solution as the substrate. Thus, BglD1 and the mutant could be used as beneficial alternatives of the existing ß-galactosidases for the production of GOS.

Solution structure of TbTFIIS2-2 PWWP domain from Trypanosoma brucei and its binding to H4K17me3 and H3K32me3.

Posttranslational modifications (PTMs) of core histones, such as histone methylation, play critical roles in a variety of biological processes including transcription regulation, chromatin condensation and DNA repair. In T. brucei, no domain recognizing methylated histone has been identified so far. TbTFIIS2-2, as a potential transcription elongation factors in T. brucei, contains a PWWP domain in the N-terminus which shares low sequence similarity compared with other PWWP domains and is absent from other TFIIS factors. In the present study, the solution structure of TbTFIIS2-2 PWWP domain was determined by NMR spectroscopy. TbTFIIS2-2 PWWP domain adopts a global fold containing a five-strand ß-barrel and two C-terminal α-helices similar to other PWWP domains. Moreover, through systematic screening, we revealed that TbTFIIS2-2 PWWP domain is able to bind H4K17me3 and H3K32me3. Meanwhile, we identified the critical residues responsible for the binding ability of TbTFIIS2-2 PWWP domain. The conserved cage formed by the aromatic amino acids in TbTFIIS2-2 PWWP domain is essential for its binding to methylated histones.

Improving the thermostability of a GH97 dextran glucosidase by rational design.

This study was aimed at improving the thermostability of dextran glucosidase PspAG97A, a member of the glycoside hydrolase family 97, from Pseudoalteromonas sp. K8. A total of 9 lysine residues were chosen using the TKSA-MC program based on the optimization of surface charge-charge interactions and were mutated to glutamate for shifting the enzyme's isoelectric point off its optimum pH value. Three mutants K75E, K363E and K420E showed enhanced thermostability. The triple mutant, K75E/K363E/K420E, was found to be the best with a 7.3-fold increase in half-life (t1/2) at 33 °C compared to that of the wild-type (WT). Most importantly, this mutant showed comparable enzymatic activity to that of the WT protein. Structural modelling demonstrated that increased surface charge-charge interactions and optimization of surface hydrophobic and electrostatic contacts contributed to the improved thermostability displayed by K75E/K363E/K420E.

Structure and allosteric coupling of type â ¡ antitoxin CopASO.

Toxin-antitoxin (TA) systems play critical roles in the environment adaptation of bacteria. Allosteric coupling between the N-terminal DNA-binding domain and the C-terminal toxin-binding domain of antitoxins contributes to conditional cooperativity in the functioning of type II TA. Herein, using circular dichroism (CD), nuclear magnetic resonance (NMR), X-ray crystallography, and size exclusion chromatography (SEC), the structure and DNA binding of CopASO, a newly identified type II antitoxin in Shewanella oneidensis, were investigated. Our data show that CopASO is a typical RHH antitoxin with an ordered N-terminal domain and a disordered C-terminal domain, and furthermore indicate that the C-terminal domain facilitates DNA binding of the N-terminal domain, which in turn induces the C-terminal domain to fold and associate.

Statistical coupling analysis uncovers sites crucial for the proton transfer in laccase Lac15.

Laccases (benzenediol: oxygen oxidoreductases, EC1.10.3.2) can oxidize wide range of compounds thus have great application potential in diverse industries. The catalytic mechanisms of laccases have been extensively studied, while the details of proton transfer remain to be fully elucidated. In this study, we tried to uncover the sites that are crucial for the proton transfer of microbial laccase Lac15. A residue near the trinuclear copper center, D396, was indicated by statistical coupling analysis (SCA) and structural alignment to be an important site like D93, which is conserved in laccases and believed crucial for the catalysis by facilitating proton transfer. A representative mutant at this site, D396A, similar to D93A, exhibited significantly impaired catalysis with the global structure and substrate binding slightly perturbed. The mutation resulted in stay of the intermediate I, which would accept a proton to proceed to next catalysis stage, suggesting D396 might play a critical role in the proton transfer. Our finding may help to completely elucidate the proton transfer mechanism in laccases.