Scale effects and spatial heterogeneity of driving factors in ecosystem services value interactions within the Tibet autonomous region.

Widespread land development, deforestation, and wetland degradation have disrupted the physical integrity and functional capacity of ecosystems, leading to a reduction in ecosystem service values (ESV). However, comprehensive research addressing ESV interactions that represent various ecosystem services from multifaceted angles is limited. Moreover, the relative significance and spatiotemporal diversity of natural and socio-economic variables influencing ESV demand further investigation. This study conducts both quantitative and qualitative assessments of the spatiotemporal dynamics and interrelationships of ESV in the Tibet autonomous region from 2000 to 2020. Geographical detector and geographically weighted regression models are applied to ascertain the relative importance and spatial heterogeneity of diverse ESV determinants. The findings reveal the following key insights: (1) Barren lands experienced the most substantial expansion from 2000 to 2020, indicating an exacerbation of desertification in the Tibet autonomous region. (2) Over the two decades, ESV exhibited an overall upward trajectory, with regulation of water flows, water bodies, and forests making the most significant contributions to ESV and its growth. (3) The quantitative and qualitative assessment of ESV interrelations has identified the number of trade-offs and synergies, along with spatial occurrences, offering a detailed foundation for the scientific management of ecosystems. Specifically, quantitative results portray ESV correlations as positive or negative, qualitative spatial mapping elucidates intricate local interactions among ESV. (4) The primary driver of ESV in the Tibet autonomous region is NDVI (0.072), with elevation following closely behind, underscoring the predominant influence of natural factors relative to socio-economic variables. This research serves as a scientific underpinning for the development of ecological conservation policies and the execution of ecological restoration initiatives.

The molecular basis for the pH-dependent calcium affinity of the pattern recognition receptor langerin.

The C-type lectin receptor langerin plays a vital role in the mammalian defense against invading pathogens. Langerin requires a Ca2+ cofactor, the binding affinity of which is regulated by pH. Thus, Ca2+ is bound when langerin is on the membrane but released when langerin and its pathogen substrate traffic to the acidic endosome, allowing the substrate to be degraded. The change in pH is sensed by protonation of the allosteric pH sensor histidine H294. However, the mechanism by which Ca2+ is released from the buried binding site is not clear. We studied the structural consequences of protonating H294 by molecular dynamics simulations (total simulation time: about 120 µs) and Markov models. We discovered a relay mechanism in which a proton is moved into the vicinity of the Ca2+-binding site without transferring the initial proton from H294. Protonation of H294 unlocks a conformation in which a protonated lysine side chain forms a hydrogen bond with a Ca2+-coordinating aspartic acid. This destabilizes Ca2+ in the binding pocket, which we probed by steered molecular dynamics. After Ca2+ release, the proton is likely transferred to the aspartic acid and stabilized by a dyad with a nearby glutamic acid, triggering a conformational transition and thus preventing Ca2+ rebinding. These results show how pH regulation of a buried orthosteric binding site from a solvent-exposed allosteric pH sensor can be realized by information transfer through a specific chain of conformational arrangements.

Protein-observed 19F NMR of LecA from Pseudomonas aeruginosa.

The carbohydrate-binding protein LecA (PA-IL) from Pseudomonas aeruginosa plays an important role in the formation of biofilms in chronic infections. Development of inhibitors to disrupt LecA-mediated biofilms is desired but it is limited to carbohydrate-based ligands. Moreover, discovery of drug-like ligands for LecA is challenging because of its weak affinities. Therefore, we established a protein-observed 19F (PrOF) nuclear magnetic resonance (NMR) to probe ligand binding to LecA. LecA was labeled with 5-fluoroindole to incorporate 5-fluorotryptophanes and the resonances were assigned by site-directed mutagenesis. This incorporation did not disrupt LecA preference for natural ligands, Ca2+ and d-galactose. Following NMR perturbation of W42, which is located in the carbohydrate-binding region of LecA, allowed to monitor binding of low-affinity ligands such as N-acetyl d-galactosamine (d-GalNAc, Kd = 780 ± 97 µM). Moreover, PrOF NMR titration with glycomimetic of LecA p-nitrophenyl ß-d-galactoside (pNPGal, Kd = 54 ± 6 µM) demonstrated a 6-fold improved binding of d-Gal proving this approach to be valuable for ligand design in future drug discovery campaigns that aim to generate inhibitors of LecA.

A Remote Secondary Binding Pocket Promotes Heteromultivalent Targeting of DC-SIGN.

Dendritic cells (DC) are antigen-presenting cells coordinating the interplay of the innate and the adaptive immune response. The endocytic C-type lectin receptors DC-SIGN and Langerin display expression profiles restricted to distinct DC subtypes and have emerged as prime targets for next-generation immunotherapies and anti-infectives. Using heteromultivalent liposomes copresenting mannosides bearing aromatic aglycones with natural glycan ligands, we serendipitously discovered striking cooperativity effects for DC-SIGN+ but not for Langerin+ cell lines. Mechanistic investigations combining NMR spectroscopy with molecular docking and molecular dynamics simulations led to the identification of a secondary binding pocket for the glycomimetics. This pocket, located remotely of DC-SIGN's carbohydrate bindings site, can be leveraged by heteromultivalent avidity enhancement. We further present preliminary evidence that the aglycone allosterically activates glycan recognition and thereby contributes to DC-SIGN-specific cell targeting. Our findings have important implications for both translational and basic glycoscience, showcasing heteromultivalent targeting of DCs to improve specificity and supporting potential allosteric regulation of DC-SIGN and CLRs in general.

The 3F Library: Fluorinated Fsp3 -Rich Fragments for Expeditious 19 F†NMR Based Screening.

Fragment-based drug discovery (FBDD) is a popular method in academia and the pharmaceutical industry for the discovery of early lead candidates. Despite its wide-spread use, the approach still suffers from laborious screening workflows and a limited diversity in the fragments applied. Presented here is the design, synthesis, and biological evaluation of the first fragment library specifically tailored to tackle both these challenges. The 3F library of 115 fluorinated, Fsp3 -rich fragments is shape diverse and natural-product-like with desirable physicochemical properties. The library is perfectly suited for rapid and efficient screening by NMR spectroscopy in a two-stage workflow of 19 F NMR and subsequent 1 H NMR methods. Hits against four diverse protein targets are widely distributed among the fragment scaffolds in the 3F library and a 67 % validation rate was achieved using secondary assays. This collection is the first synthetic fragment library tailor-made for 19 F NMR screening and the results demonstrate that the approach should find broad application in the FBDD community.

Allosteric Inhibition of a Mammalian Lectin.

Glycan-binding proteins are key components of central physiological and cellular processes such as self-/non-self-recognition, cellular tissue homing, and protein homeostasis. Herein, C-type lectins are a diverse protein family that play important roles in the immune system, rendering them attractive drug targets. To evaluate C-type lectin receptors as target proteins for small-molecule effectors, chemical probes are required, which are, however, still lacking. To overcome the supposedly poor druggability of C-type lectin receptors and to identify starting points for chemical probe development, we screened murine langerin using 1H and 19F NMR against a library of 871 drug-like fragments. Subsequently, hits were validated by surface plasmon resonance and enzyme-linked lectin assay. Using structure-activity relationship studies and chemical synthesis, we identified thiazolopyrimidine derivatives with double-digit micromolar activity that displayed langerin selectivity. Based on 1H-15N HSQC NMR and competitive binding and inhibition experiments, we demonstrate that thiazolopyrimidines allosterically inhibit langerin. To the best of our knowledge, this is the first report of drug-like allosteric inhibitors of a mammalian lectin.

Colwellia agarivorans sp. nov., an agar-digesting marine bacterium isolated from coastal seawater.

A novel Gram-stain-negative, facultatively anaerobic, yellowish and agar-digesting marine bacterium, designated strain QM50T, was isolated from coastal seawater in an aquaculture site near Qingdao, China. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the isolate represented a member of the genus Colwellia and exhibited the highest sequence similarity (97.4 %) to Colwellia aestuarii SMK-10T. Average nucleotide identity (ANI) values based on draft genome sequences between strain QM50T and C. aestuarii KCTC 12480T showed a relatedness of 72.0 % (ANIb) and 85.1 % (ANIm). Cells of strain QM50T were approximately 0.3-0.6×0.8-2.5 µm in size and motile by means of a polar flagellum. Growth occurred in the presence of 1.0-6.0 % (w/v) NaCl (optimum, 2.0-3.0 %), at pH 6.5-8.5 (optimum, pH 7.0) and at 4-37 °C (optimum, 28-30 °C). Strain QM50T was found to contain ubiquinone 8 (Q-8) as the predominant ubiquinone and summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH), C16 : 0 and C17 : 1ω8c as the main cellular fatty acids. Phosphatidylethanolamine and phosphatidylglycerol were found to be major polar lipids. The DNA G+C content of strain QM50T was determined to be 35.7 mol%. On the basis of phylogenetic and phenotypic data, strain QM50T represents a novel species of the genus Colwellia, for which the name Colwellia agarivorans sp. nov. is proposed. The type strain is QM50T (=KCTC 52273T=MCCC 1H00143T).

Marinifilum albidiflavum sp. nov., isolated from coastal sediment.

A novel Gram-stain-negative, facultatively anaerobic, filamentous, and yellowish-white-pigmented marine bacterium, designated strain FB208T, was isolated from marine sediment obtained off the coastal area of Weihai, China. Cells of strain FB208T were filamentous during exponential growth, fragmented to rods in the stationary growth phase and became spherical in aged cultures. It grew optimally at 33 °C, at pH 7.0-7.5 and in the presence of 2.0-3.0 % (w/v) NaCl. Based on the 16S rRNA gene sequence, strain FB208T was found to be closely related to Marinifilum flexuosum DSM 21950T (96.9 % similarity) and Marinifilum fragile JCM 15579T (96.4 %), with less than 90.0 % sequence similarity to other genera of the class Bacteroidia. Phylogenetic analysis, also based on 16S rRNA gene sequences, placed strain FB208T in the genus Marinifilum, family Marinifilaceae. The predominant isoprenoid quinone of strain FB208T was identified as menaquinone MK-7. The main cellular fatty acids were iso-C15 : 0, iso-C17 : 0 3-OH and iso-C17 : 1ω9c, and the major polar lipids were an unidentified lipid and aminophospholipid. The G+C content of the genomic DNA was 43.8 mol%. Based on these phylogenetic and phenotypic data, strain FB208T represents a novel species of the genus Marinifilum, for which the name Marinifilum albidiflavum sp. nov. is proposed. The type strain is FB208T (=KCTC 42591T=MCCC 1H00113T).

Kordiimonas sediminis sp. nov., isolated from a sea cucumber culture pond.

A marine bacterium, designated strain N39(T), was isolated from a sediment sample collected at a sea cucumber culture pond in Weihai, China. Cells of strain N39(T) were observed to be Gram-stain negative, facultatively anaerobic, motile rods showing catalase and oxidase negative reactions. Strain N39(T) was found to grow optimally at pH 8.0-8.5, 35-37 °C and in the presence of approximately 3.0 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain N39(T) belongs to the genus Kordiimonas in the family Kordiimonadaceae, appearing closely related to Kordiimonas lacus JCM 16261(T) (95.9 %), Kordiimonas aquimaris MEBiC06554(T) (95.1 %), Kordiimonas gwangyangensis JCM 12864(T) (94.2 %) and Kordiimonas aestuarii 101-1(T) (93.8 %). Ubiquinone 10 (Q-10) was found to be the major respiratory quinone. The dominant cellular fatty acids were identified as iso-C17:1 ω9c, iso-C17:0, iso-C15:0 and C17:1 ω6c. The predominant polar lipids were found to be phosphatidylglycerol, phosphatidylethanolamine and diphosphatidylglycerol. The DNA G + C content of strain N39(T) is 50.8 %. On the basis of genotypic, chemotaxonomic and phenotypic data, strain N39(T) is concluded to represent a novel species within the genus Kordiimonas, for which the name Kordiimonas sediminis sp. nov. is proposed. The type strain is N39(T) (=KCTC 42590(T) = MCCC 1H00112(T)).

Nocardioides gilvus sp. nov., isolated from Namtso Lake.

A Gram-stain positive, facultatively anaerobic, non-motile and rod to coccoid-shaped bacterium, designated XZ17(T), was isolated from Namtso Lake of Tibet, China. Strain XZ17(T) grew optimally at pH 8.0-9.0, at 30-33 °C and in the presence of 0-1.0 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences demonstrated that strain XZ17(T) belongs to the genus Nocardioides in the family Nocardioidaceae. Strain XZ17(T) shared pairwise 16S rRNA gene sequence similarities of 97.2, 96.8, 96.5, 96.4 and <96.0 % to Nocardioides solisilvae KCTC 39528(T), Nocardioides daejeonensis JCM 16922(T), Nocardioides jensenii NCIB 9770(T), Nocardioides dubius KCTC 9992(T) and other Nocardioides species, respectively. It contained MK-8 (H4) as the predominant menaquinone and C17:1 ω8c, C15:0, C17:0 and C18:1 ω9c as the major fatty acid. The strain had cell wall peptidoglycan based on LL-2,6-diaminopimelic acid. The polar lipids of strain XZ17(T) comprised diphosphatidylglycerol, phosphatidylglycerol, three unidentified phospholipids, three unidentified glycolipids and two unidentified lipids. The DNA G+C content of strain XZ17(T) was 68.9 mol%. Based on distinctive phenotypic characteristics, phylogenetic analysis and chemotaxonomic data, it can be concluded that strain XZ17(T) represents a novel species within the genus Nocardioides, for which the name Nocardioides gilvus sp. nov. is proposed. The type strain is strain XZ17(T) (=KCTC 39561(T) = MCCC 1H00114(T)).

The Effect of TWIK-1 Two Pore Potassium Channels on Cardiomyocytes in Low Extracellular Potassium Conditions.

BACKGROOUND: At low extracellular potassium ([K+]e) conditions, human cardiomyocytes can depolarize to -40 mV. This is closely related to hypokalemia-induced fatal cardiac arrhythmia. The underlying mechanism, however, is still not well understood. TWIK-1 channels are background K+ channels that are highly expressed in human cardiomyocytes. We previously reported that TWIK-1 channels changed ion selectivity and conducted leak Na+ currents at low [K+]e. Moreover, a specific threonine residue (Thr118) within the ion selectivity filter was responsible for this altered ion selectivity. METHODS: Patch clamp were used to investigate the effects of TWIK-1 channels on the membrane potentials of cardiomyocytes in response to low [K+]e. RESULTS: At 2.7 mM [K+]e and 1 mM [K+]e, both Chinese hamster ovary (CHO) cells and HL-1 cells ectopically expressed human TWIK-1 channels displayed inward leak Na+ currents and reconstitute depolarization of membrane potential. In contrast, cells ectopically expressed human TWIK-1-T118I mutant channels that remain high selectivity to K+ exhibited hyperpolarization of membrane potential. Furthermore, human iPSC-derived cardiomyocytes showed depolarization of membrane potential in response to 1 mM [K+]e, while the knockdown of TWIK-1 expression eliminated this phenomenon. CONCLUSIONS: These results demonstrate that leak Na+ currents conducted by TWIK-1 channels contribute to the depolarization of membrane potential induced by low [K+]e in human cardiomyocytes.

Drug-like Inhibitors of DC-SIGN Based on a Quinolone Scaffold.

DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin) is a pattern recognition receptor expressed on immune cells and involved in the recognition of carbohydrate signatures present on various pathogens, including HIV, Ebola, and SARS-CoV-2. Therefore, developing inhibitors blocking the carbohydrate-binding site of DC-SIGN could generate a valuable tool to investigate the role of this receptor in several infectious diseases. Herein, we performed a fragment-based ligand design using 4-quinolone as a scaffold. We synthesized a library of 61 compounds, performed a screening against DC-SIGN using an STD reporter assay, and validated these data using protein-based 1H-15N HSQC NMR. Based on the structure-activity relationship data, we demonstrate that ethoxycarbonyl or dimethylaminocarbonyl in position 2 or 3 is favorable for the DC-SIGN binding activity, especially in combination with fluorine, ethoxycarbonyl, or dimethylaminocarbonyl in position 7 or 8. Furthermore, we demonstrate that these quinolones can allosterically modulate the carbohydrate binding site, which offers an alternative approach toward this challenging protein target.

Identification of the Allosteric Binding Site for Thiazolopyrimidine on the C-Type Lectin Langerin.

Langerin is a mammalian C-type lectin expressed on Langerhans cells in the skin. As an innate immune cell receptor, Langerin is involved in coordinating innate and adaptive immune responses against various incoming threats. We have previously reported a series of thiazolopyrimidines as murine Langerin ligands. Prompted by the observation that its human homologue exhibits different binding specificities for these small molecules, we report here our investigations to define their exact binding site. By using structural comparison and molecular dynamics simulations, we showed that the nonconserved short loops have a high degree of conformational flexibility between the human and murine homologues. Sequence analysis and mutational studies indicated that a pair of residues are essential for the recognition of the thiazolopyrimidines. Taking solvent paramagnetic relaxation enhancement NMR studies together with a series of peptides occupying the same site, we could define the cleft between the short and long loops as the allosteric binding site for these aromatic heterocycles.

Synthesis and conformational analysis of linear homo- and heterooligomers from novel 2-C-branched sugar amino acids (SAAs).

Sugar amino acids (SAAs), as biologically interesting structures bearing both amino and carboxylic acid functional groups represent an important class of multifunctional building blocks. In this study, we develop an easy access to novel SAAs in only three steps starting from nitro compounds in high yields in analytically pure form, easily available by ceric (IV) mediated radical additions. Such novel SAAs have been applied in the assembly of total nine carbopeptoids with the form of linear homo- and heterooligomers for the structural investigations employing circular dichroism (CD) spectroscopy, which suggest that the carbopeptoids emerge a well-extended, left (or right)-handed conformation similar to polyproline II (PPII) helices. NMR studies also clearly demonstrated the presence of ordered secondary structural elements. 2D-ROESY spectra were acquired to identify i+1 NH ↔ i C 1 H, i C 2 H correlations which support the conformational analysis of tetramers by CD spectroscopy. These findings provide interesting information of SAAs and their oligomers as potential scaffolds for discovering new drugs and materials.