Projecting the response of ecological risk to land use/land cover change in ecologically fragile regions.

Anthropogenic activities have dramatically altered land use/land cover (LULC), leading to ecosystem service (ES) degradation and further ecological risks. Ecological risks are particularly serious in ecologically fragile regions because trade-offs between economic development and ecological protection are prominent. Thus, ways in which to assess the response of ecological risks to LULC change under each development scenario in ecologically fragile regions remain challenging. In this study, future LUCC and its impact on ESs under four development scenarios in 2040 in western Jilin Province were predicted using a patch-generating land use simulation model and the Integrated Valuation of Ecosystem Services and Trade-offs (InVEST) model. Ecological risk was assessed based on future LUCC possibilities, and potential ES degradation and potential drivers of ecological risks were explored using a geographic detector. The results showed that the cropland development scenario (CDS) would experience large-scale urbanization and cropland expansion. Carbon storage (CS), habitat quality (HQ), and water purification (WP) degraded the most under the CDS, and grain yield (GY) and water yield (WY) degraded the most under the ecological protection scenario (EPS). The LUCC probability under the CDS (14.37 %) was the highest, while the LUCC probability under the comprehensive development scenario (CPDS) (8.68 %) was the lowest. The risk of WP degradation was greatest under the CDS, but the risk of soil retention (SR) degradation was greatest under the natural development scenario (NDS), EPS, and CPDS. Ecological risk coverage was the largest (98.04 %), and ecological risks were the highest (0.21) under the CDS, while those under the EPS were the opposite. Distance to roads and population density had a higher impact on ecological risks than other drivers. Further attention should be given to the ecological networks and pattern establishment in urbanized regions. This study will contribute to risk prevention and sustainable urban and agricultural development.

Efficient Biosynthesis of Acidic/Lactonic Sophorolipids and Their Application in the Remediation of Cyanobacterial Harmful Algal Blooms.

Cyanobacterial harmful algal blooms (CyanoHABs) pose significant threats to human health and natural ecosystems worldwide, primarily caused by water eutrophication, increased surface water temperature, and co-occurring microorganisms. Urgent action is needed to develop an eco-friendly solution to effectively curb the proliferation of CyanoHABs. Sophorolipids (SLs) are fully biodegradable biosurfactants synthesized by Starmerella bombicola. They can be classified into lactone and acid types. The lactone type displays strong antimicrobial activity, while the acid type exhibits good solubility, which make them ideal agents for mitigating CyanoHABs. Nevertheless, the broad utilization of SLs are hindered by their expensive production costs and the absence of effective genetic editing tools in the native host. In this study, we constructed recombinant strains capable of producing either acidic or lactonic SLs using the CRISPR-Cas9 gene editing system. The yields of acidic and lactonic SLs reached 53.64 g/L and 45.32 g/L in a shaking flask, respectively. In a 5 L fermenter, acidic SLs reached 129.7 g/L using low-cost glucose and rapeseed oil as substrates. The addition of 5 mg/L lactonic SLs effectively degraded cyanobacteria within 30 min, and a ratio of 8.25:1.75 of lactonic to acidic SLs showed the highest degradation efficiency. This study offers a safe and promising solution for CyanoHABs treatment.

Polymorphic KIR3DL3 expression modulates tissue-resident and innate-like T cells.

Most human killer cell immunoglobulin-like receptors (KIR) are expressed by natural killer (NK) cells and recognize HLA class I molecules as ligands. KIR3DL3 is a conserved but polymorphic inhibitory KIR recognizing a B7 family ligand, HHLA2, and is implicated for immune checkpoint targeting. The expression profile and biological function of KIR3DL3 have been somewhat elusive, so we searched extensively for KIR3DL3 transcripts, revealing highly enriched expression in γδ and CD8+ T cells rather than NK cells. These KIR3DL3-expressing cells are rare in the blood and thymus but more common in the lungs and digestive tract. High-resolution flow cytometry and single-cell transcriptomics showed that peripheral blood KIR3DL3+ T cells have an activated transitional memory phenotype and are hypofunctional. The T cell receptor (TCR) usage is biased toward genes from early rearranged TCR-α variable segments or Vδ1 chains. In addition, we show that TCR-mediated stimulation can be inhibited through KIR3DL3 ligation. Whereas we detected no impact of KIR3DL3 polymorphism on ligand binding, variants in the proximal promoter and at residue 86 can reduce expression. Together, we demonstrate that KIR3DL3 is up-regulated alongside unconventional T cell stimulation and that individuals may vary in their ability to express KIR3DL3. These results have implications for the personalized targeting of KIR3DL3/HHLA2 checkpoint inhibition.

Water quality pollution assessment and source apportionment of lake wetlands: A case study of Xianghai Lake in the Northeast China Plain.

Surface water pollution has always posed a serious challenge to water quality management. Improving water quality management requires figuring out how to comprehend water quality conditions scientifically and effectively as well as quantitatively identify regional pollution sources. In this study, Xianghai Lake, a typical lake-type wetland on the Northeast China Plain, was taken as the research area. Based on a geographic information system (GIS) method and 11 water quality parameters, the single-factor evaluation and comprehensive water quality index (WQI) methods were used to comprehensively evaluate the water quality of the lake-type wetland in the level period. Four key water quality parameters were determined by the principal component analysis (PCA) method, and more convenient comprehensive water quality evaluation models, the minimum WQI considering weights (WQImin-w) and the minimum WQI without considering weights (WQImin-nw) were established. The multiple statistical method and the absolute principal component score-multiple liner regression (APCS-MLR) model were combined to analyse the lake pollution sources based on the spatial changes in pollutants. The findings demonstrated that the WQImin-nw model's water quality evaluation outcome was more accurate when weights were not taken into account. The WQImin-nw model can be used as a simple and convenient way to comprehend the variations in water quality in wetlands of lakes and reservoirs. It was concluded that the comprehensive water quality in the study area was at a "medium" level, and CODMn was the main limiting factor. Nonpoint source pollution (such as agricultural planting and livestock breeding) was the most important factor affecting the water quality of Xianghai Lake (with a comprehensive contribution rate of 31.65%). The comprehensive contribution rates of sediment endogenous and geological sources, phytoplankton and other plants, and water diversion and other hydrodynamic impacts accounted for 25.12%, 19.65%, and 23.58% of the total impact, respectively. This study can provide a scientific method for water quality assessment and management of lake wetlands, and an effective support for migration of migratory birds, habitat protection and grain production security.

TOAC spin-labeled peptides tailored for DNP-NMR studies in lipid membrane environments.

The benefit of combining in-cell solid-state dynamic nuclear polarization (DNP) NMR and cryogenic temperatures is providing sufficient signal/noise and preservation of bacterial integrity via cryoprotection to enable in situ biophysical studies of antimicrobial peptides. The radical source required for DNP was delivered into cells by adding a nitroxide-tagged peptide based on the antimicrobial peptide maculatin 1.1 (Mac1). In this study, the structure, localization, and signal enhancement properties of a single (T-MacW) and double (T-T-MacW) TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid) spin-labeled Mac1 analogs were determined within micelles or lipid vesicles. The solution NMR and circular dichroism results showed that the spin-labeled peptides adopted helical structures in contact with micelles. The peptides behaved as an isolated radical source in the presence of multilamellar vesicles, and the electron paramagnetic resonance (EPR) electron-electron distance for the doubly spin-labeled peptide was ∼1 nm. The strongest paramagnetic relaxation enhancement (PRE) was observed for the lipid NMR signals near the glycerol-carbonyl backbone and was stronger for the doubly spin-labeled peptide. Molecular dynamics simulation of the T-T-MacW radical source in phospholipid bilayers supported the EPR and PRE observations while providing further structural insights. Overall, the T-T-MacW peptide achieved better 13C and 15N signal NMR enhancements and 1H spin-lattice T1 relaxation than T-MacW.

Expression and purification of the native C-amidated antimicrobial peptide maculatin 1.1.

Maculatin 1.1 (Mac1) is an antimicrobial peptide (AMP) from an Australian tree frog and exhibits low micromolar activity against Gram-positive bacteria. The antimicrobial properties of Mac1 are linked to its disruption of bacterial lipid membranes, which has been studied extensively by in vitro nuclear magnetic resonance (NMR) spectroscopy and biophysical approaches. Although in vivo NMR has recently proven effective in probing peptide-lipid interplay in live bacterial cells, direct structural characterisation of AMPs has been prohibited by low sensitivity and overwhelming background noise. To overcome this issue, we report a recombinant expression protocol to produce isotopically enriched Mac1. We utilized a double-fusion construct to alleviate toxicity against the Escherichia coli host and generate the native N-free and C-amidated termini Mac1 peptide. The SUMO and intein tags allowed native N-terminus and C-terminal amidation, respectively, to be achieved in a one-pot reaction. The protocol yielded 0.1 mg/L of native, uniformly 15 N-labelled, Mac1, which possessed identical structure and activity to peptide obtained by solid-phase peptide synthesis.

C-terminus amidation influences biological activity and membrane interaction of maculatin 1.1.

Cationic antimicrobial peptides have been investigated for their potential use in combating infections by targeting the cell membrane of microbes. Their unique chemical structure has been investigated to understand their mode of action and optimize their dose-response by rationale design. One common feature among cationic AMPs is an amidated C-terminus that provides greater stability against in vivo degradation. This chemical modification also likely modulates the interaction with the cell membrane of bacteria yet few studies have been performed comparing the effect of the capping groups. We used maculatin 1.1 (Mac1) to assess the role of the capping groups in modulating the peptide bacterial efficiency, stability and interactions with lipid membranes. Circular dichroism results showed that C-terminus amidation maintains the structural stability of the peptide (α-helix) in contact with micelles. Dye leakage experiments revealed that amidation of the C-terminus resulted in higher membrane disruptive ability while bacteria and cell viability assays revealed that the amidated form displayed higher antibacterial ability and cytotoxicity compared to the acidic form of Mac1. Furthermore, 31P and 2H solid-state NMR showed that C-terminus amidation played a greater role in disturbance of the phospholipid headgroup but had little effect on the lipid tails. This study paves the way to better understand how membrane-active AMPs act in live bacteria.

How do Self-Assembling Antimicrobial Lipopeptides Kill Bacteria?

Antimicrobial peptides are promising alternatives to traditional antibiotics. A group of self-assembling lipopeptides was formed by attaching an acyl chain to the N-terminus of α-helix-forming peptides with the sequence Cx-G(IIKK)yI-NH2 (CxGy, x = 4-12 and y = 2). CxGy self-assemble into nanofibers above their critical aggregation concentrations (CACs). With increasing x, the CACs decrease and the hydrophobic interactions increase, promoting secondary structure transitions within the nanofibers. Antimicrobial activity, determined by the minimum inhibition concentration (MIC), also decreases with increasing x, but the MICs are significantly smaller than the CACs, suggesting effective bacterial membrane-disrupting power. Unlike conventional antibiotics, both C8G2 and C12G2 can kill Staphylococcus aureus and Escherichia coli after only minutes of exposure under the concentrations studied. C12G2 nanofibers have considerably faster killing dynamics and lower cytotoxicity than their nonaggregated monomers. Antimicrobial activity of peptide aggregates has, to date, been underexploited, and it is found to be a very promising mechanism for peptide design. Detailed evidence for the molecular mechanisms involved is provided, based on superresolution fluorescence microscopy, solid-state nuclear magnetic resonance, atomic force microscopy, neutron scattering/reflectivity, circular dichroism, and Brewster angle microscopy.

The Location of the Antimicrobial Peptide Maculatin 1.1 in Model Bacterial Membranes.

Maculatin 1.1 (Mac1) is an antimicrobial peptide (AMP) from the skin secretions of Australian tree frogs. In this work, the interaction of Mac1 with anionic phospholipid bilayers was investigated by NMR, circular dichroism (CD) spectroscopy, neutron reflectometry (NR) and molecular dynamics (MD). In buffer, the peptide is unstructured but in the presence of anionic (DPC/LMPG) micelles or (DMPC/DMPG/DHPC) bicelles adopts a helical structure. Addition of the soluble paramagnetic agent gadolinium (Gd-DTPA) into the Mac1-DPC/LMPG micelle solution showed that the N-terminus is more exposed to the hydrophilic Gd-DTPA than the C-terminus in micelles. 2H and 31P solid-state NMR showed that Mac1 had a greater effect on the anionic lipid (DMPG). A deuterium labeled Mac1 used in NR experiments indicated that the AMP spanned across anionic (PC/PG) bilayers, which was compatible with MD simulations. Simulations also showed that Mac1 orientation remained transmembrane in bilayers and wrapped on the surface of the micelles regardless of the lipid or detergent charge. Thus, the peptide orientation appears to be more susceptible to curvature than charged surface. These results support the formation of transmembrane pores by Mac1 in model bacterial membranes.

Nitroxide spin-labeled peptides for DNP-NMR in-cell studies.

Antimicrobial peptides (AMPs) that target lipid membranes show promise as alternatives to conventional antibiotics. However, the molecular mechanisms of membrane perturbation, as most studies are performed in model systems and in-cell structural studies, have yet to be achieved. Solid-state NMR spectroscopy is a valuable technique to investigate peptide-membrane interactions and to determine the structure of peptides, but the short lifespan of bacteria, especially under magic angle spinning conditions, has not permitted in-cell structural studies. Here, we present the first dynamic nuclear polarization (DNP)-NMR in-cell studies of Escherichia coli bacteria incubated with the AMP maculatin 1.1 (Mac1) in combination with novel nitroxide spin-labeled peptides 2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid (TOAC)-[F3W]-Mac1 (MacW) and TOAC-TOAC-MacW. The in-cell 13C and 15N signal NMR enhancements, and 1H spin-lattice T1 relaxation times showed that TOAC-MacW and TOAC-TOAC-MacW performed better than the more hydrophilic biradical AMUPol used for DNP studies. Furthermore, the pores formed by the AMP increased the signal enhancements and decreased T1 values of specifically 13C- and 15N-labeled Mac1. This approach has a great potential for determining the first in situ structures of AMPs in bacteria.-Sani, M.-A., Zhu, S., Hofferek, V., Separovic, F. Nitroxide spin-labeled peptides for DNP-NMR in-cell studies.

In Situ Monitoring of Bacteria under Antimicrobial Stress Using 31P Solid-State NMR.

In-cell NMR offers great insight into the characterization of the effect of toxins and antimicrobial peptides on intact cells. However, the complexity of intact live cells remains a significant challenge for the analysis of the effect these agents have on different cellular components. Here we show that 31P solid-state NMR can be used to quantitatively characterize the dynamic behaviour of DNA within intact live bacteria. Lipids were also identified and monitored, although 31P dynamic filtering methods indicated a range of dynamic states for phospholipid headgroups. We demonstrate the usefulness of this methodology for monitoring the activity of the antibiotic ampicillin and the antimicrobial peptide (AMP) maculatin 1.1 (Mac1.1) against Gram-negative bacteria. Perturbations in the dynamic behaviour of DNA were observed in treated cells, which indicated additional mechanisms of action for the AMP Mac1.1 not previously reported. This work highlights the value of 31P in-cell solid-state NMR as a tool for assessing the antimicrobial activity of antibiotics and AMPs in bacterial cells.

Novel 2, 8-bit derivatives of quinolines attenuate Pseudomonas aeruginosa virulence and biofilm formation.

Signal molecules are stimulators of multiple quroum-sensing virulence and biofilm formation. Small molecule analogues have been suspected as a potent inhibitor in therapeutic strategy. Herein, we synthesized a series of small molecule compounds from the 2, 8-bit derivatives of quinoline by Suzuki coupling reaction. We found that these compounds have the biofilm inhibitory effect in normal condition instead of phosphate limitation state. Furthermore, lacZ reporter strain assay and rhamnolipids as well as pyocyanin experiments showed that these compounds did not affect las and pqs system but reduced the expression of rhl. All these results suggest that quinoline derivatives can be treated as potent inhibitors against biofilm and reduce virulence through the rhl system. This research will be useful in designing new quorum sensing inhibitors to attenuate the infection of bacteria.

Supramolecular arrays by the self-assembly of terpyridine-based monomers with transition metal ions.

Hierarchical construction of a highly ordered supramolecular array has been, in general, a challenge due to the complexation of building blocks and the hard-to-control weak interactions. Herein, we present a type of well-ordered nanoribbon, which was self-assembled via shape complimentary and hydrophobic effects from the bowl-shaped supramolecular components, which were synthesized by combining designer terpyridine-based monomers and two different metal ions (Ru2+, Zn2+). Interestingly, switching counter ions or changing monomer concentrations, a transformation between a uniform nanosphere and nanoribbon occurred. This opens a door to fabricate readily tailorable, large-scale, supramacromolecular materials.

Stepwise, multicomponent assembly of a molecular trapezoid possessing three different metals.

A novel terpyridine-based, trapezoidal architecture was synthesized by a coordination-driven multicomponent assembly and features three different tpy-M2+-tpy bonds (M2+ = Ru2+, Fe2+, and Zn2+) in the macrocyclic ring. This trimetallic macrocycle introduces the construction of polymetallosupramolecular assemblies possessing multiple, differing metal centers in an ordered, predetermined pattern. Characterization was accomplished by NMR spectroscopy, mass spectrometry, and UV-Vis spectroscopy.

RIP3 deficiency exacerbates inflammation in dextran sodium sulfate-induced ulcerative colitis mice model.

Ulcerative colitis (UC) is a chronic intestinal inflammatory disease. The receptor-interacting protein kinase 3 (RIP3) was reported to be involved in many inflammatory disease. However, the mechanism of RIP3 in the pathogenesis of UC is still unclear. To investigate the effects and possible mechanism of RIP3 in UC pathogenesis, RIP3-/- mice was used in dextran sulfate sodium (DSS)-induced colitis model. It was found that by DSS-induced colitis, RIP3-/- mice showed significantly enhanced colitis symptoms, including increased weight loss, colon shortening, and colonic mucosa damage and severity, but decreased production of interleukin 6 and interleukin 1ß. The results showed that RIP3 deficiency could not ameliorate but exacerbate the severity of colitis. On the mechanism, it was found that messenger RNA expressions of several repair-associated cytokines including interleukin 6, interleukin 22, cyclooxygenase 2, epithelial growth factor receptor ligand Epiregulin and matrix metalloproteinase 10 were siginificant decreased in RIP3-/- mice. Thus, RIP3-/- mice exhibited an impaired tissue repair in response to DSS. In a conclusion, RIP3 deficiency exerted detrimental effects in DSS induced colitis partially because of the impaired repair-associated cytokines expression.

E3 Ubiquitin Ligase Nedd4 Promotes Japanese Encephalitis Virus Replication by Suppressing Autophagy in Human Neuroblastoma Cells.

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes the most prevalent viral encephalitis in Asia. Since JEV is a neurotropic virus, it is important to identify key molecules that mediate JEV infection in neuronal cells and to investigate their underlying mechanisms. In this study, the critical role of Nedd4, an E3 ubiquitin ligase that is highly expressed in the central nervous system, was examined in JEV propagation. In SK-N-SH neuroblastoma cells, Nedd4 was up-regulated in response to JEV infection. Moreover, down-regulation of Nedd4 resulted in a significant decrease in JEV replication without alterations in virus attachment and internalization or in JEV pseudotyped virus infection, suggesting that Nedd4 participates in the replication but not in the entry stage of JEV infection. Further functional analysis showed that Nedd4 attenuated JEV-induced autophagy, which negatively regulates virus replication during infection. These results suggest that Nedd4 facilitates the replication of JEV by suppressing virus-induced autophagy. Taken together, our results indicate that Nedd4 plays a crucial role in JEV infection of neuronal cells, which provides a potential target for the development of novel treatment to combat JEV infection.

A Schisandra-Derived Compound Schizandronic Acid Inhibits Entry of Pan-HCV Genotypes into Human Hepatocytes.

Despite recent progress in the development of hepatitis C virus (HCV) inhibitors, cost-effective antiviral drugs, especially among the patients receiving liver transplantations, are still awaited. Schisandra is a traditional medicinal herb used to treat a range of liver disorders including hepatitis for thousands of years in China. To isolate the bioactive compounds of schisandra for the treatment of HCV infection, we screened a schisandra-extracts library and identified a tetracyclic triterpenoid, schizandronic acid (SZA), as a novel HCV entry inhibitor. Our findings suggested that SZA potently inhibited pan-HCV genotype entry into hepatoma cells and primary human hepatocytes without interfering virus binding on cell surface or internalization. However, virion-cell fusion process was impaired in the presence of SZA, along with the increased host membrane fluidity. We also found that SZA inhibited the spread of HCV to the neighboring cells, and combinations of SZA with interferon or telaprevir resulted in additive synergistic effect against HCV. Additionally, SZA diminished the establishment of HCV infection in vivo. The SZA target is different from conventional direct-acting antiviral agents, therefore, SZA is a potential therapeutic compound for the development of effective HCV entry inhibitors, especially for patients who need to prevent HCV reinfection during the course of liver transplantations.

Trachelogenin, a novel inhibitor of hepatitis C virus entry through CD81.

Although much progress has been made in antiviral agents against hepatitis C virus (HCV) in recent years, novel HCV inhibitors with improved efficacy, optimized treatment duration and more affordable prices are still urgently needed. Here, we report the identification of a natural plant-derived lignan, trachelogenin (TGN), as a potent entry inhibitor of HCV without genotype specificity, and with low cytotoxicity. TGN was extracted and purified from Caulis trachelospermi, a traditional Chinese herb with anti-inflammatory and analgesic effects. A crucial function of TGN was the inhibition of HCV entry during a post-binding step without affecting virus replication, translation, assembly and release. TGN blocked virus infection by interfering with the normal interactions between HCV glycoprotein E2 and the host entry factor CD81, which are key processes for valid virus entry. In addition, TGN diminished HCV cell-to-cell spread and exhibited additional synergistic effects when combined with IFN or telaprevir. In conclusion, this study highlights the effect of a novel HCV entry inhibitor, TGN, which has a target that differs from those of the current antiviral agents. Therefore, TGN is a potential candidate for future cocktail therapies to treat HCV-infected patients.

Serum adsorption, cellular internalization and consequent impact of cuprous oxide nanoparticles on uveal melanoma cells: implications for cancer therapy.

AIM: To investigate the biological fate of cuprous oxide nanoparticles (Cu2O-NPs) and to evaluate their potential in uveal melanoma therapy. MATERIALS & METHODS: The protein corona, cellular uptake mechanism and localization of Cu2O-NPs were investigated. Furthermore, the effect of Cu2O-NPs on uveal melanoma cell proliferation, migration and invasion, and possible mechanisms were studied in detail. RESULTS: Cu2O-NPs are able to adsorb serum proteins in cell culture medium, which are then internalized by uveal melanoma cells mainly through lipid raft-mediated endocytosis. Furthermore, Cu2O-NPs selectively inhibit cancer cell growth and impair the ability of uveal melanoma cell migration, invasion and the cytoskeleton assembly. The mechanism may be that Cu2O-NPs located in and damage mitochondria, autophagolysosomes and lysosomes, leading to elevated reactive oxygen species level and over-stimulated apoptosis and autophagy. CONCLUSION: The data provide detailed information of Cu2O-NPs for further application and indicate that Cu2O-NPs could be a potential agent for uveal melanoma therapy.

Anchoring of both PKA-RIIα and 14-3-3θ regulates retinoic acid induced 16 mediated phosphorylation of heat shock protein 70.

Our previous study reported that retinoic acid induced 16 (RAI16) could enhance tumorigenesis in hepatocellular carcinoma (HCC). However, the cellular functions of RAI16 are still unclear. In this study, by immunoprecipitation and tandem (MS/MS) mass spectrometry analysis, we identified that RAI16 interacted with the type II regulatory subunit of PKA (PKA-RIIα), acting as a novel protein kinase A anchoring protein (AKAP). In addition, RAI16 also interacted with heat shock protein 70 (HSP70) and 14-3-3θ. Further studies indicated that RAI16 mediated PKA phosphorylation of HSP70 at serine 486, resulting in anti-apoptosis events. RAI16 was also phosphorylated by the anchored PKA at serine 325, which promoted the recruitment of 14-3-3θ, which, in turn, inhibited RAI16 mediated PKA phosphorylation of HSP70. These findings offer mechanism insight into RAI16 mediated anti-apoptosis signaling in HCC.