Linear resistivity at van Hove singularities in twisted bilayer WSe2.

Different mechanisms driving a linear temperature dependence of the resistivity ρ ∼ T at van Hove singularities (VHSs) or metal-insulator transitions when doping a Mott insulator are being debated intensively with competing theoretical proposals. We experimentally investigate this using the exceptional tunability of twisted bilayer (TB) WSe2 by tracking the parameter regions where linear-in-T resistivity is found in dependency of displacement fields, filling, and magnetic fields. We find that even when the VHSs are tuned rather far away from the half-filling point and the Mott insulating transition is absent, the T-linear resistivity persists at the VHSs. When doping away from the VHSs, the T-linear behavior quickly transitions into a Fermi liquid behavior with a T2 relation. No apparent dependency of the linear-in-T resistivity, besides a rather strong change of prefactor, is found when applying displacement fields as long as the filling is tuned to the VHSs, including D ∼ 0.28 V/nm where a high-order VHS is expected. Intriguingly, such non-Fermi liquid linear-in-T resistivity persists even when magnetic fields break the spin-degeneracy of the VHSs at which point two linear in T regions emerge, for each of the split VHSs separately. This points to a mechanism of enhanced scattering at generic VHSs rather than only at high-order VHSs or by a quantum critical point during a Mott transition. Our findings provide insights into the many-body consequences arising out of VHSs, especially the non-Fermi liquid behavior found in moiré materials.

Characterization of Bacillus amyloliquefaciens PM415 as a potential bio-preserving probiotic.

Animal feed is vulnerable to fungal infections, and the use of bio-preserving probiotics has received increasing attention. In contrast to Lactobacillus and Bifidobacteria spp., fewer Bacillus spp. have been recognized as antifungal probiotics. Therefore, our objective was to screen antifungal strains and provide more Bacillus candidates to bridge this gap. Here, we screened 56 bacterial strains for cyclic lipopeptide genes and conducted an antifungal assay with Aspergillus niger as a representative fungus. We found that a Bacillus strain Bacillus amyloliquefaciens PM415, isolated from pigeon manure, exhibited the highest fungal inhibition activity as demonstrated by the confrontation assay and morphological observation under scanning electron microscope (SEM). Preliminary safety assessment and probiotic characterization revealed its non-pathogenic feature and stress tolerance capability. Whole genome sequencing of Bacillus amyloliquefaciens PM415 revealed a genome size of 4.16 Mbp and 84 housekeeping genes thereof were used for phylogenetic analysis showing that it is most closely related to Bacillus amyloliquefaciens LFB112. The in silico analysis further supported its non-pathogenic feature at the genomic level and revealed potential biosynthetic gene clusters responsible for its antifungal property. RNA-seq analysis revealed genome-wide changes in transportation, amino acid metabolism, non-ribosomal peptides (NRPs) biosynthesis and glycan degradation during fungal antagonism. Our results suggest that Bacillus amyloliquefaciens PM415 is a safe and effective probiotic strain that can prevent fungal growth in animal feeds.

Evidence That DDR1 Promotes Oligodendrocyte Differentiation during Development and Myelin Repair after Injury.

Oligodendrocytes generate myelin sheaths vital for the formation, health, and function of the central nervous system. Mounting evidence suggests that receptor tyrosine kinases (RTKs) are crucial for oligodendrocyte differentiation and myelination in the CNS. It was recently reported that discoidin domain receptor 1 (Ddr1), a collagen-activated RTK, is expressed in oligodendrocyte lineage. However, its specific expression stage and functional role in oligodendrocyte development in the CNS remain to be determined. In this study, we report that Ddr1 is selectively upregulated in newly differentiated oligodendrocytes in the early postnatal CNS and regulates oligodendrocyte differentiation and myelination. Ddr1 knock-out mice of both sexes displayed compromised axonal myelination and apparent motor dysfunction. Ddr1 deficiency alerted the ERK pathway, but not the AKT pathway in the CNS. In addition, Ddr1 function is important for myelin repair after lysolecithin-induced demyelination. Taken together, the current study described, for the first time, the role of Ddr1 in myelin development and repair in the CNS, providing a novel molecule target for the treatment of demyelinating diseases.

Secondary structures, dynamics, and DNA binding of the homeodomain of human SIX1.

Human sine oculis homeobox homolog (SIX) 1 contains a homeodomain (HD), which is important for binding to DNA. In this study, we carried out structural studies on the HD of human SIX1 using nuclear magnetic resonance (NMR) spectroscopy. Its secondary structures and dynamics in solution were explored. HD is well-structured in solution, and our study shows that it contains three α-helices. Dynamics study indicates that the N- and C-terminal residues of HD are flexible in solution. HD of human SIX1 exhibits molecular interactions with a short double-strand DNA sequence evidenced by the 1 H-15 N-heteronuclear single quantum correlation (HSQC) and 19 F-NMR experiments. Our current study provides structural information for HD of human SIX1. Further studies indicate that this construct can be utilized to study SIX1 and DNA interactions.

Secondary Structures of the Transmembrane Domain of SARS-CoV-2 Spike Protein in Detergent Micelles.

Spike protein of SARS-CoV-2 contains a single-span transmembrane (TM) domain and plays roles in receptor binding, viral attachment and viral entry to the host cells. The TM domain of spike protein is critical for viral infectivity. Herein, the TM domain of spike protein of SARS-CoV-2 was reconstituted in detergent micelles and subjected to structural analysis using solution NMR spectroscopy. The results demonstrate that the TM domain of the protein forms a helical structure in detergent micelles. An unstructured linker is identified between the TM helix and heptapeptide repeat 2 region. The linker is due to the proline residue at position 1213. Side chains of the three tryptophan residues preceding to and within the TM helix important for the function of S-protein might adopt multiple conformations which may be critical for their function. The side chain of W1212 was shown to be exposed to solvent and the side chains of residues W1214 and W1217 are buried in micelles. Relaxation study shows that the TM helix is rigid in solution while several residues have exchanges. The secondary structure and dynamics of the TM domain in this study provide insights into the function of the TM domain of spike protein.

Mechanisms of Action for Small Molecules Revealed by Structural Biology in Drug Discovery.

Small-molecule drugs are organic compounds affecting molecular pathways by targeting important proteins. These compounds have a low molecular weight, making them penetrate cells easily. Small-molecule drugs can be developed from leads derived from rational drug design or isolated from natural resources. A target-based drug discovery project usually includes target identification, target validation, hit identification, hit to lead and lead optimization. Understanding molecular interactions between small molecules and their targets is critical in drug discovery. Although many biophysical and biochemical methods are able to elucidate molecular interactions of small molecules with their targets, structural biology is the most powerful tool to determine the mechanisms of action for both targets and the developed compounds. Herein, we reviewed the application of structural biology to investigate binding modes of orthosteric and allosteric inhibitors. It is exemplified that structural biology provides a clear view of the binding modes of protease inhibitors and phosphatase inhibitors. We also demonstrate that structural biology provides insights into the function of a target and identifies a druggable site for rational drug design.

Insights into Structures and Dynamics of Flavivirus Proteases from NMR Studies.

Nuclear magnetic resonance (NMR) spectroscopy plays important roles in structural biology and drug discovery, as it is a powerful tool to understand protein structures, dynamics, and ligand binding under physiological conditions. The protease of flaviviruses is an attractive target for developing antivirals because it is essential for the maturation of viral proteins. High-resolution structures of the proteases in the absence and presence of ligands/inhibitors were determined using X-ray crystallography, providing structural information for rational drug design. Structural studies suggest that proteases from Dengue virus (DENV), West Nile virus (WNV), and Zika virus (ZIKV) exist in open and closed conformations. Solution NMR studies showed that the closed conformation is predominant in solution and should be utilized in structure-based drug design. Here, we reviewed solution NMR studies of the proteases from these viruses. The accumulated studies demonstrated that NMR spectroscopy provides additional information to understand conformational changes of these proteases in the absence and presence of substrates/inhibitors. In addition, NMR spectroscopy can be used for identifying fragment hits that can be further developed into potent protease inhibitors.

A Practical Perspective on the Roles of Solution NMR Spectroscopy in Drug Discovery.

Solution nuclear magnetic resonance (NMR) spectroscopy is a powerful tool to study structures and dynamics of biomolecules under physiological conditions. As there are numerous NMR-derived methods applicable to probe protein-ligand interactions, NMR has been widely utilized in drug discovery, especially in such steps as hit identification and lead optimization. NMR is frequently used to locate ligand-binding sites on a target protein and to determine ligand binding modes. NMR spectroscopy is also a unique tool in fragment-based drug design (FBDD), as it is able to investigate target-ligand interactions with diverse binding affinities. NMR spectroscopy is able to identify fragments that bind weakly to a target, making it valuable for identifying hits targeting undruggable sites. In this review, we summarize the roles of solution NMR spectroscopy in drug discovery. We describe some methods that are used in identifying fragments, understanding the mechanism of action for a ligand, and monitoring the conformational changes of a target induced by ligand binding. A number of studies have proven that 19F-NMR is very powerful in screening fragments and detecting protein conformational changes. In-cell NMR will also play important roles in drug discovery by elucidating protein-ligand interactions in living cells.

Expression, purification of Zika virus membrane protein-NS2B in detergent micelles for NMR studies.

The Zika virus (ZIKV) genome encodes a polyprotein that can be post-translationally processed into functional viral proteins. The viral protease is indispensable in the maturation of viral proteins. The Zika protease comprises of two components crucial for catalysis. The N-terminal region of NS3 contains the catalytic triad and approximately 40 amino acids of NS2B are essential for folding and protease activity. NS2B is a membrane protein with transmembrane domains that are critical for the localization of NS3 to the membrane. In this study, we expressed and purified full-length NS2B from ZIKV in E. coli. Purified NS2B was then reconstituted into lyso-myristoyl phosphatidylglycerol (LMPG) micelles. It was found that compared to wild type NS2B, NS2B C11S mutation in LMPG exhibited dispersed cross peaks in the 1H15N-HSQC spectrum, thereby suggesting the feasibility for structural characterization using solution NMR spectroscopy.

Microbial production of rhamnolipids using sugars as carbon sources.

Rhamnolipids are a class of biosurfactants with effective surface-active properties. The high cost of microbial production of rhamnolipids largely affects their commercial applications. To reduce the production post, research has been carried out in screening more powerful strains, engineering microbes with higher biosurfactant yields and exploring cheaper substrates to reduce the production cost. Extensive refining is required for biosurfactant production using oils and oil-containing wastes, necessitating the use of complex and expensive biosurfactant recovery methods such as extraction with solvents or acid precipitation. As raw materials normally can account for 10-30% of the overall production cost, sugars have been proven to be an alternative carbon source for microbial production of rhamnolipids due to its lower costs and straightforward processing techniques. Studies have thus been focused on using tropical agroindustrial crop residues as renewable substrates. Herein, we reviewed studies that are using sugar-containing substrates as carbon sources for producing rhamnolipids. We speculate that sugars derived from agricultural wastes rich in cellulose and sugar-containing wastes are potential carbon sources in fermentation while challenges still remain in large scales.

Microbial production of rhamnolipids: opportunities, challenges and strategies.

Rhamnolipids are a class of biosurfactants which contain rhamnose as the sugar moiety linked to ß-hydroxylated fatty acid chains. Rhamnolipids can be widely applied in many industries including petroleum, food, agriculture and bioremediation etc. Pseudomonas aeruginosa is still the most competent producer of rhamnolipids, but its pathogenicity may cause safety and health concerns during large-scale production and applications. Therefore, extensive studies have been carried out to explore safe and economical methods to produce rhamnolipids. Various metabolic engineering efforts have also been applied to either P. aeruginosa for improving its rhamnolipid production and diminishing its pathogenicity, or to other non-pathogenic strains by introducing the key genes for safe production of rhamnolipids. The three key enzymes for rhamnolipid biosynthesis, RhlA, RhlB and RhlC, are found almost exclusively in Pseudomonas sp. and Burkholderia sp., but have been successfully expressed in several non-pathogenic host bacteria to produce rhamnolipids in large scales. The composition of mono- and di-rhamnolipids can also be modified through altering the expression levels of RhlB and RhlC. In addition, cell-free rhamnolipid synthesis by using the key enzymes and precursors from non-pathogenic sources is thought to not only eliminate pathogenic effects and simplify the downstream purification processes, but also to circumvent the complexity of quorum sensing system that regulates rhamnolipid biosynthesis. The pathogenicity of P. aeruginosa can also be reduced or eliminated through in vivo or in vitro enzymatic degradation of the toxins such as pyocyanin during rhamnolipid production. The rhamnolipid production cost can also be significantly reduced if rhamnolipid purification step can be bypassed, such as utilizing the fermentation broth or the rhamnolipid-producing strains directly in the industrial applications of rhamnolipids.

Membrane topology of NS2B of dengue virus revealed by NMR spectroscopy.

Non-structural (NS) proteins of dengue virus (DENV) are important for viral replication. There are four membrane proteins that are coded by viral genome. NS2B was shown to be one of the membrane proteins and its main function was confirmed to regulate viral protease activity. Its membrane topology is still not known because only few studies have been conducted to understand its structure. Here we report the determination of membrane topology of NS2B from DENV serotype 4 using NMR spectroscopy. NS2B of DENV4 was expressed and purified in detergent micelles. The secondary structure of NS2B was first defined based on backbone chemical resonance assignment. Four helices were identified in NS2B. The membrane topology of NS2B was defined based on relaxation analysis and paramagnetic relaxation enhancement experiments. The last three helices were shown to be more stable than the first helix. The NS3 protease cofactor region between α2 and α3 is highly dynamic. Our results will be useful for further structural and functional analysis of NS2B.

Selection of suitable detergents for obtaining an active dengue protease in its natural form from E. coli.

Dengue protease is a two-component enzyme and is an important drug target against dengue virus. The protease activity and protein stability of dengue nonstructural protein 3 (NS3) require a co-factor region from a four-span membrane protein NS2B. A natural form of dengue protease containing full-length NS2B and NS3 protease domain NS2BFL-NS3pro will be useful for dengue drug discovery. In current study, detergents that can be used for protease purification were tested. Using a water soluble protease construct, 39 detergents were selected for both NS2B and NS2BFL-NS3pro purification. The results showed that 18 detergents were able to sustain the activity of the natural dengue protease and 11 detergents could be used for NS2B purification. The results obtained in this study will be useful for biochemical and biophysical studies on dengue protease.

Secondary Structure and Membrane Topology of the Full-Length Dengue Virus NS4B in Micelles.

Dengue virus nonstructural protein 4B (NS4B) is a membrane protein consisting of 248 residues with a crucial role in virus replication and interference with the host innate immunity. The dengue virus serotype 3 NS4B was reconstituted into lyso-myristoyl phosphatidylglycerol (LMPG) micelles. Backbone resonance assignment of NS4B was obtained using conventional solution NMR experiments. Further studies suggested that NS4B contained eleven helices and six of them form five potential transmembrane regions. This study provides atomic level information for an important drug target to control flavivirus infections.

Solution structure of the transmembrane domain of the insulin receptor in detergent micelles.

The insulin receptor (IR) binds insulin and plays important roles in glucose homeostasis by regulating the tyrosine kinase activity at its C-terminus. Its transmembrane domain (TMD) is shown to be important for transferring conformational changes induced by insulin across the cell membrane to regulate kinase activity. In this study, a construct IR(940-988) containing the TMD was expressed and purified for structural studies. Its solution structure in dodecylphosphocholine (DPC) micelles was determined. The sequence containing residues L962 to Y976 of the TMD of the IR in micelles adopts a well-defined helical structure with a kink formed by glycine and proline residues present at its N-terminus, which might be important for its function. Paramagnetic relaxation enhancement (PRE) and relaxation experimental results suggest that residues following the TMD are flexible and expose to aqueous solution. Although purified IR(940-988) in micelles existed mainly as a monomeric form verified by gel filtration and relaxation analysis, cross-linking study suggests that it may form a dimer or oligomers under micelle conditions.

Structural analysis of the S4-S5 linker of the human KCNQ1 potassium channel.

KCNQ1 plays important roles in the cardiac action potential and consists of an N-terminal domain, a voltage-sensor domain, a pore domain and a C-terminal domain. KCNQ1 is a voltage-gated potassium channel and its channel activity is regulated by membrane potentials. The linker between transmembrane helices 4 and 5 (S4-S5 linker) is important for transferring the conformational changes from the voltage-sensor domain to the pore domain. In this study, the structure of the S4-S5 linker of KCNQ1 was investigated by solution NMR, circular dichroism and fluorescence spectroscopic studies. The S4-S5 linker adopted a helical structure in detergent micelles. The W248 may interact with the cell membrane.

Structural insight into the transmembrane domain and the juxtamembrane region of the erythropoietin receptor in micelles.

Erythropoietin receptor (EpoR) dimerization is an important step in erythrocyte formation. Its transmembrane domain (TMD) and juxtamembrane (JM) region are essential for signal transduction across the membrane. A construct compassing residues S212-P259 and containing the TMD and JM region of the human EpoR was purified and reconstituted in detergent micelles. The solution structure of the construct was determined in dodecylphosphocholine (DPC) micelles by solution NMR spectroscopy. Structural and dynamic studies demonstrated that the TMD and JM region are an ?-helix in DPC micelles, whereas residues S212-D224 at the N-terminus of the construct are not structured. The JM region is a helix that contains a hydrophobic patch formed by conserved hydrophobic residues (L253, I257, and W258). Nuclear Overhauser effect analysis, fluorescence spectroscopy, and paramagnetic relaxation enhancement experiments suggested that the JM region is exposed to the solvent. The structures of the TMD and JM region of the mouse EpoR were similar to those of the human EpoR.

Solution structure of the cyclic-nucleotide binding homology domain of a KCNH channel.

The carboxy-terminal region of the KCNH family of potassium channels contains a cyclic-nucleotide binding homology domain (CNBHD) that is important for channel gating and trafficking. The solution structure of the CNBHD of the KCNH potassium of zebrafish was determined using solution NMR spectroscopy. This domain exists as a monomer under solution conditions and adopts a similar fold to that determined by X-ray crystallography. The CNBHD does not bind cAMP because residue Y740 blocks the entry of cyclic-nucleotide to the binding pocket. Relaxation results show that the CNBHD is rigid except that some residues in the loop between ß6 and ß7 are flexible. Our results will be useful to understand the gating mechanism of KCNH family members through the CNBHD.

Isolation, characterization and application of a cellulose-degrading strain Neurospora crassa S1 from oil palm empty fruit bunch.

BACKGROUND: Oil palm empty fruit bunch (EFB) is a lignocellulosic waste produced in palm oil industry. EFB mainly consists of cellulose, hemicellulose (mainly xylan) and lignin and has a great potential to be reused. Converting EFB to fermentable sugars and value-added chemicals is a much better choice than treating EFB as waste. RESULTS: A cellulase-producing strain growing on oil palm empty fruit bunch (EFB) was isolated and identified as Neurospora crassa S1, which is able to produce cellulases using EFB as the sole carbon source. The strain started to secret cellulases into the medium after 24 h of cultivation at 30°C and reached its maximal cellulase activity at 240 h. Mass spectroscopy (MS) analysis showed that more than 50 proteins were secreted into the medium when EFB was used as the sole carbon source. Among them, 7 proteins were identified as putative enzymes associated with cellulose degradation. The whole cell culture of Neurospora crassa S1 was used to hydrolyze acid-treated EFB, giving a total sugar yield of 83.2%, which is comparable with that (82.0%) using a well-known cellulase producer Trichoderma reesei RUT-C30 (ATCC56765). CONCLUSION: Neurospora crassa S1 is a commercially promising native cellulase producer for EFB hydrolysis especially when the sugars obtained are to be fermented to products that require use of non-genetically engineered strains.

Structural insight into the transmembrane segments 3 and 4 of the hERG potassium channel.

The hERG (human ether-a-go-go related gene) potassium channel is a voltage-gated potassium channel containing an N-terminal domain, a voltage-sensor domain, a pore domain and a C-terminal domain. The transmembrane segment 4 (S4) is important for sensing changes of membrane potentials through positively charge residues. A construct containing partial S2-S3 linker, S3, S4 and the S4-S5 linker of the hERG channel was purified into detergent micelles. This construct exhibits good quality NMR spectrum when it was purified in lyso-myristoyl phosphatidylglycerol (LMPG) micelles. Structural study showed that S3 contains two short helices with a negatively charged surface. The S4 and S4-S5 linker adopt helical structures. The six positively charged residues in S4 localize at different sides, suggesting that they may have different functions in channel gating. Relaxation studies indicated that S3 is more flexible than S4. The boundaries of S3-S4 and S4-S4-S5 linker were identified. Our results provided structural information of the S3 and S4, which will be helpful to understand their roles in channel gating.