Stable peptide-assembled nanozyme mimicking dual antifungal actions.

Natural antimicrobial peptides (AMPs) and enzymes (AMEs) are promising non-antibiotic candidates against antimicrobial resistance but suffer from low efficiency and poor stability. Here, we develop peptide nanozymes which mimic the mode of action of AMPs and AMEs through de novo design and peptide assembly. Through modelling a minimal building block of IHIHICI is proposed by combining critical amino acids in AMPs and AMEs and hydrophobic isoleucine to conduct assembly. Experimental validations reveal that IHIHICI assemble into helical ß-sheet nanotubes with acetate modulation and perform phospholipase C-like and peroxidase-like activities with Ni coordination, demonstrating high thermostability and resistance to enzymatic degradation. The assembled nanotubes demonstrate cascade antifungal actions including outer mannan docking, wall disruption, lipid peroxidation and subsequent ferroptotic death, synergistically killing >90% Candida albicans within 10 min on disinfection pad. These findings demonstrate an effective de novo design strategy for developing materials with multi-antimicrobial mode of actions.

Clinical implications of CSF-ctDNA positivity in newly diagnosed diffuse large B cell lymphoma.

The clinical implications of CSF-ctDNA positivity in newly diagnosed diffuse large B cell lymphoma (ND-DLBCL) remains largely unexplored. One hundred ND-DLBCL patients were consecutively enrolled as training cohort and another 26 ND-DLBCL patients were prospectively enrolled in validation cohort. CSF-ctDNA positivity (CSF(+)) was identified in 25 patients (25.0%) in the training cohort and 7 patients (26.9%) in the validation cohort, extremely higher than CNS involvement rate detected by conventional methods. Patients with mutations of CARD11, JAK2, ID3, and PLCG2 were more predominant with CSF(+) while FAT4 mutations were negatively correlated with CSF(+). The downregulation of PI3K-AKT signaling, focal adhesion, actin cytoskeleton, and tight junction pathways were enriched in CSF(+) ND-DLBCL. Furthermore, pretreatment CSF(+) was significantly associated with poor outcomes. Three risk factors, including high CSF protein level, high plasma ctDNA burden, and involvement of high-risk sites were used to predict the risk of CSF(+) in ND-DLBCL. The sensitivity and specificity of pretreatment CSF-ctDNA to predict CNS relapse were 100% and 77.3%. Taken together, we firstly present the prevalence and the genomic and transcriptomic landscape for CSF-ctDNA(+) DLBCL and highlight the importance of CSF-ctDNA as a noninvasive biomarker in detecting and monitoring of CSF infiltration and predicting CNS relapse in DLBCL.

Genomic and immune microenvironment features influencing chemoimmunotherapy response in gastric cancer with peritoneal metastasis: a retrospective cohort study.

BACKGROUND: Patients with peritoneal metastasis (PM) from gastric cancer (GC) exhibit poor prognosis. Chemoimmunotherapy offers promising clinical benefits; however, its efficacy and predictive biomarkers in a conversion therapy setting remain unclear. The authors aimed to retrospectively evaluate chemoimmunotherapy efficacy in a conversion therapy setting for GC patients with PM and establish a prediction model for assessing clinical benefits. MATERIALS AND METHODS: A retrospective evaluation of clinical outcomes encompassed 55 GC patients with PM who underwent chemoimmunotherapy in a conversion therapy setting. Baseline PM specimens were collected for genomic and transcriptomic profiling. Clinicopathological factors, gene signatures, and tumor immune microenvironment were evaluated to identify predictive markers and develop a prediction model. RESULTS: Chemoimmunotherapy achieved a 41.8% objective response rate and 72.4% R0 resection rate in GC patients with PM. Patients with conversion surgery showed better overall survival (OS) than those without the surgery (median OS: not reached vs 7.82 m, P <0.0001). Responders to chemoimmunotherapy showed higher ERBB2 and ERBB3 mutation frequencies, CTLA4 and HLA-DQB1 expression, and CD8+ T cell infiltration, but lower CDH1 mutation and naïve CD4+ T cell infiltration, compared to nonresponders. A prediction model was established integrating CDH1 and ERBB3 mutations, HLA-DQB1 expression, and naïve CD4+ T cell infiltration (AUC=0.918), which were further tested using an independent external cohort (AUC=0.785). CONCLUSION: This exploratory study comprehensively evaluated clinicopathological, genomic, and immune features and developed a novel prediction model, providing a rational basis for the selection of GC patients with PM for chemoimmunotherapy-involved conversion therapy.

Single-molecule visualization determines conformational substate ensembles in ß-sheet-rich peptide fibrils.

An understanding of protein conformational ensembles is essential for revealing the underlying mechanisms of interpeptide recognition and association. However, experimentally resolving multiple simultaneously existing conformational substates remains challenging. Here, we report the use of scanning tunneling microscopy (STM) to analyze the conformational substate ensembles of ß sheet peptides with a submolecular resolution (in-plane <2.6 Å). We observed ensembles of more than 10 conformational substates (with free energy fluctuations between several kBTs) in peptide homoassemblies of keratin (KRT) and amyloidal peptides (-5Aß42 and TDP-43 341-357). Furthermore, STM reveals a change in the conformational ensemble of peptide mutants, which is correlated with the macroscopic properties of peptide assemblies. Our results demonstrate that the STM-based single-molecule imaging can capture a thorough picture of the conformational substates with which to build an energetic landscape of interconformational interactions and can rapidly screen conformational ensembles, which can complement conventional characterization techniques.

Experimental Insights into Conformational Ensembles of Assembled ß-Sheet Peptides.

Deciphering the conformations and interactions of peptides in their assemblies offers a basis for guiding the rational design of peptide-assembled materials. Here we report the use of scanning tunneling microscopy (STM), a single-molecule imaging method with a submolecular resolution, to distinguish 18 types of coexisting conformational substates of the ß-strand of the 8-37 segment of human islet amyloid polypeptide (hIAPP 8-37). We analyzed the pairwise peptide-peptide interactions in the hIAPP 8-37 assembly and found 82 interconformation interactions within a free energy difference of 3.40 kBT. Besides hIAPP 8-37, this STM method validates the existence of multiple conformations of other ß-sheet peptide assemblies, including mutated hIAPP 8-37 and amyloid-ß 42. Overall, the results reported in this work provide single-molecule experimental insights into the conformational ensemble and interpeptide interactions in the ß-sheet peptide assembly.

Delta-6 desaturase (Fads2) deficiency alters triacylglycerol/fatty acid cycling in murine white adipose tissue.

The Δ-6 desaturase (D6D) enzyme is not only critical for the synthesis of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from α-linolenic acid (ALA), but recent evidence suggests that it also plays a role in adipocyte lipid metabolism and body weight; however, the mechanisms remain largely unexplored. The goal of this study was to investigate if a D6D deficiency would inhibit triacylglycerol storage and alter lipolytic and lipogenic pathways in mouse white adipose tissue (WAT) depots due to a disruption in EPA and DHA production. Male C57BL/6J D6D knockout (KO) and wild-type (WT) mice were fed either a 7% w/w lard or flax (ALA rich) diet for 21 weeks. Energy expenditure, physical activity, and substrate utilization were measured with metabolic caging. Inguinal and epididymal WAT depots were analyzed for changes in tissue weight, fatty acid composition, adipocyte size, and markers of lipogenesis, lipolysis, and insulin signaling. KO mice had lower body weight, higher serum nonesterified fatty acids, smaller WAT depots, and reduced adipocyte size compared to WT mice without altered food intake, energy expenditure, or physical activity, regardless of the diet. Markers of lipogenesis and lipolysis were more highly expressed in KO mice compared to WT mice in both depots, regardless of the diet. These changes were concomitant with lower basal insulin signaling in WAT. Collectively, a D6D deficiency alters triacylglycerol/fatty acid cycling in WAT by promoting lipolysis and reducing fatty acid re-esterification, which may be partially attributed to a reduction in WAT insulin signaling.

ß-Sheet Assembly Translates Conservative Single-Site Mutation into a Perturbation in Macroscopic Structure.

Transferring structural information from amino acid sequence to macroscale assembly is a challenging approach for designing protein quaternary structure. However, the pathway by which the slight variations in sequence result in a global perturbation effect on the assembled structure is unknown. Herein, we design two synthetic peptides, QNL-His and QNL-Arg, with one amino acid substitution and use scanning tunneling microscopy (STM) to image individual peptides in the assembled state. The submolecular resolution of STM enables us to determine the folding structure and ß-sheet supramolecular organization of peptides. QNL-His and QNL-Arg differ in their ß-strand length distribution in pleated ß-sheet association. These structural variations lead to distinguishable outcomes in their ß-sheet assembled fibrils and phase transitions. The comparison of QNL-His versus QNL-Arg structures and macroscopic properties unveils the role of assembly to amplify the structural variations associated with a single-site mutation from a single-molecule scale to a macroscopic scale.

A reduction of skeletal muscle DHA content does not result in impaired whole body glucose tolerance or skeletal muscle basal insulin signaling in otherwise healthy mice.

Delta-6 desaturase (D6D), encoded by the Fads2 gene, catalyzes the first step in the conversion of α-linolenic acid to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The ablation of D6D in whole body Fads2-/- knockout (KO) mice results in an inability to endogenously produce EPA and DHA. Evidence supports a beneficial role for EPA and DHA on insulin-stimulated glucose disposal in skeletal muscle in the context of a metabolic challenge; however, it is unknown how low EPA and DHA levels impact skeletal muscle fatty acid composition and insulin signaling in a healthy context. The objective of this study was to examine the impact of ablating the endogenous production of EPA and DHA on skeletal muscle fatty acid composition, whole body glucose and insulin tolerance, and a key marker of skeletal muscle insulin signaling (pAkt). Male C57BL/6J wild-type (WT), Fads2+/- heterozygous, and Fads2-/- KO mice were fed a low-fat diet (16% kcal from fat) modified to contain either 7% w/w lard or 7% w/w flaxseed for 21 wk. No differences in total phospholipid (PL), triacylglycerol, or reactive lipid content were observed between genotypes. As expected, KO mice on both diets had significantly less DHA content in skeletal muscle PL. Despite this, KO mice did not have significantly different glucose or insulin tolerance compared with WT mice on either diet. Basal pAktSer473 was not significantly different between the genotypes within each diet. Ultimately, this study shows for the first time, to our knowledge, that the reduction of DHA in skeletal muscle is not necessarily detrimental to glucose homeostasis in otherwise healthy animals.NEW & NOTEWORTHY Skeletal muscle is the primary location of insulin-stimulated glucose uptake. EPA and DHA supplementation has been observed to improve skeletal muscle insulin-stimulated glucose uptake in models of metabolic dysfunction. Fads2-/- knockout mice cannot endogenously produce long-chain n-3 polyunsaturated fatty acids. Our results show that the absence of DHA in skeletal muscle is not detrimental to whole body glucose homeostasis in healthy mice.

Nanomicelles co-loading CXCR4 antagonist and doxorubicin combat the refractory acute myeloid leukemia.

Acute myeloid leukemia (AML) is featured with poor prognosis and high mortality, because chemo-resistance, nonspecific distribution and dose-limiting toxicity lead to a high rate of relapse and a very low 5-year survival percentage of less than 25%. CXCR4 is a highly expressed chemokine receptor in multiple types of AML cells and closely associated with the drug resistance and relapse. In this work, we integrate a chemically synthesized CXCR4 antagonistic peptide and doxorubicin using DSPE-mPEG2000 micelles (referred to as M-E5-Dox) that is applied to a very challenging refractory AML mouse model as well as human AML cell lines. Results showed that M-E5-Dox can effectively bind to the CXCR4-expressing AML cells, downregulating the signaling proteins mediated by CXCR4/CXCL12 axis and increasing the cellular uptake of Dox. Importantly, M-E5-Dox remarkably decreases the leukemic cells in the peripheral blood and bone marrow, as well as their infiltration in the spleen and liver of the AML mice, which in turn prolongs the survival significantly. Meanwhile, M-E5-Dox did not increase the cardiotoxicity of Dox. In conclusion, M-E5-Dox harnesses the functions of CXCR4 specific binding and CXCR4 antagonism of the peptide and the tumor cell killing capacity of Dox, which displays significant therapeutic effects and promising translational potentials for the treatment of refractory AML.

Peptide Binder with High-Affinity for the SARS-CoV-2 Spike Receptor-Binding Domain.

Rapid antigen detection tests are urgently needed for the early diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The discovery of a binder with high affinity and selectivity for the biomarkers presented by SARS-CoV-2 is crucial to the development of the rapid antigen detection method. We utilized the surface biopanning to identify a peptide binder R1 from a phage-displayed peptide library consisting of 109 independent phage recombinants. The R1 peptide exhibited high-affinity for specific binding with the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein with a dissociation constant KD of (7.5 ± 1.9) × 10-10 M, which maintained high binding affinity with the RBD derived from Gamma, Lambda, Delta, and Omicron variants. The composition and sequence dependence of binding characteristics in R1-RBD interactions was revealed by the binding affinity fluctuations between RBD and the scrambled sequences or single-site mutants of R1. The R1-functionalized gold nanoparticles possessed concentration-dependent response to RBD and selectivity over bovine serum albumin and human serum albumin. The peptide binder R1 shows the potential to be used for constructing a rapid detection method for the early-stage diagnostics for SARS-CoV-2.

Inhibition of Δ-6 desaturase reduces fatty acid re-esterification in 3T3-L1 adipocytes independent of changes in n3-PUFA cellular content.

Δ-6 desaturase (D6D) is a key enzyme in the synthesis of long-chain polyunsaturated fatty acids (LC-PUFA). Evidence suggests that reduced D6D activity not only disrupts LC-PUFA production, but also impacts whole body lipid handling and body weight; however, the mechanisms remain largely unexplored. Therefore, we investigated the effect of D6D inhibition on the regulation of lipid accumulation in 3T3-L1 adipocytes with and without changes in n-3 PUFA content. 3T3-L1 cells were treated with a D6D inhibitor (SC-26196) in the presence or absence of α-linolenic acid (ALA) throughout differentiation. We found that D6D inhibition blocked the conversion of ALA to eicosapentaenoic acid (EPA) and docosapentaenoic acid (DPAn-3) when ALA was supplemented, while no changes in n-3 PUFA content were observed in cells treated with the D6D inhibitor alone. D6D inhibited cells had reduced triacylglycerol (TAG) accumulation despite an EPA/DPA deficiency. In addition, analyses of cellular protein markers, as well as non-esterified fatty acids and glycerol release in medium, suggested an increase in lipolysis and a decrease in fatty acid re-esterification in D6D-inhibited cells, independent of n-3 PUFA changes. To provide further evidence, we treated cells with the D6D inhibitor in the presence or absence of EPA and compared them with ALA-treated cells. Although EPA further reduced TAG content, the reduced markers of fatty acid re-esterification were not affected by ALA or EPA. Collectively, this study provides new insight showing that D6D inhibition reduces TAG accumulation and fatty acid re-esterification in adipocytes independent of changes in n-3 PUFA cellular content.

Crystalline State Determines the Potency of Galectin-10 Protein Assembly to Induce Inflammation.

Protein crystallization is a prevalent phenomenon existing in the formation of intricate protein-assembled structures in living cells. Whether the crystallization of a protein would exert a specific biological function, however, remains poorly understood. Here, we reconstructed a recombinant galectin-10 (gal-10) protein and artificially engineered a gal-10 protein assembly in two distinguishable states: i.e., an insoluble crystalline state and a soluble state. The potency of the gal-10 protein in either the crystalline state or the soluble state to induce chemokine or cytokine release in the primary human nasal epithelial cells and nasal polyps derived from chronic rhinosinusitis patients with nasal polyps was investigated. The crystalline gal-10 upregulated the gene expression of chemokines or cytokines, including IL-1ß, IL-6, IL-8, TNF-α, and GM-CSF, in patient-derived primary cells and nasal polyps. In contrast, soluble gal-10 displayed a diminished potency to induce inflammation. Our results demonstrate that the gal-10 protein potency of activating inflammation is correlated with its crystalline state.

Plant and marine N3-PUFA regulation of fatty acid trafficking along the adipose tissue-liver axis varies according to nutritional state.

Marine sourced N3-PUFA regulate lipid metabolism in adipose tissue and liver; however, less is known about plant sourced N3-PUFA. The goal of this study was to investigate plant and marine N3-PUFA regulation of fatty acid trafficking along the adipose tissue-liver axis according to nutritional state. Mice were fed low-fat diets (7% w/w) containing either lard, flaxseed, or menhaden oils for 8 weeks, and were euthanized in either fed or fasted states. Substrate utilization and physical activity were assessed during the transition from a fed to fasted state. Plasma biomarkers (triacylglycerol [TAG], non-esterified fatty acids [NEFA]), as well as liver and epididymal adipose tissue (eWAT) lipogenic and lipolytic markers, were measured. Neither plant nor marine N3-PUFA influenced substrate utilization or activity during the transition from a fed to fasted state. In the fed state, marine N3-PUFA reduced plasma TAG levels compared to the other diets, with no further reduction seen in fasted mice. Hepatic lipogenic markers (Fasn, Acc, Scd1, and Elovl6) were reduced in the fed state with marine N3-PUFA, but not plant N3-PUFA. In the fasted state, mice fed either N3-PUFA accumulated less liver TAG, had lower plasma NEFA, and suppressed eWAT HSL activity compared to lard. Marine N3-PUFA are more potent regulators of lipogenesis than plant N3-PUFA in the fed state, whereas both N3-PUFA influence eWAT lipolysis and plasma NEFA in the fasted state. This work provides novel insights regarding N3-PUFA regulation of fatty acid trafficking along the adipose tissue-liver axis according to nutritional state.

Perturbation effect of single polar group substitution on the Self-Association of amphiphilic peptide helices.

As an important attempt towards creating hierarchical structures more like nature, the peptide is employed as a building block to build supramolecular architectures. An emerging question is whether the molecular mechanism of self-assembly obtained from the small molecule system, e.g., the driving forces of assembly are conventionally regarded as pairwise-additive, can be manifested in the self-association of biologically relevant amphiphilic peptides. A peptide, KRT-R, was derived from the 120-144 segment of keratin 14. The single cation-to-cation substitution with KRT-R at the site of 125 from arginine (R) to either lysine (K) or histidine (H) results in the peptide helices, KRT-K and KRT-H, sharing 96% sequence identity. These KRT-derived peptides possess similarities in the folding structures but exhibit divergent self-assembled structures. KRT-R and KRT-K self-assemble into sheets and fibrils, respectively. Whereas KRT-H associates into heterogeneous structures, including sheets, particles, and branched networks. The intrinsic tyrosine fluorescence spectroscopy measurements with the KRT-derived peptides within a temperature range of 25 °C to 95 °C reveal that the heating-triggered structural transitions of KRT-derived peptides are divergent. The alternation of single cationic residue changes the thermodynamic signature of peptide assemblies upon heating. A chemical denaturation experiment with KRT-derived peptides indicates that the intermolecular interactions that govern the supramolecular architectures formed by peptides are distinct. Overall, our work demonstrates the contribution of the interplay among various noncovalent interactions to supramolecular assembly.

Conservation and Identity Selection of Cationic Residues Flanking the Hydrophobic Regions in Intermediate Filament Superfamily.

The interplay between the hydrophobic interactions generated by the nonpolar region and the proximal functional groups within nanometers of the nonpolar region offers a promising strategy to manipulate the intermolecular hydrophobic attractions in an artificial molecule system, but the outcomes of such modulations in the building of a native protein architecture remain unclear. Here we focus on the intermediate filament (IF) coiled-coil superfamily to assess the conservation of positively charged residue identity via a biostatistical approach. By screening the disease-correlated mutations throughout the IF superfamily, 10 distinct hotspots where a cation-to-cation substitution is associated with a pathogenic syndrome have been identified. The analysis of the local chemical context surrounding the hotspots revealed that the cationic diversity depends on their separation distance to the hydrophobic domain. The nearby cationic residues flanking the hydrophobic domain of a helix (separation <1 nm) are relatively conserved in evolution. In contrast, the cationic residues that are not adjacent to the hydrophobic domain (separation >1 nm) tolerate higher levels of variation and replaceability. We attribute this bias in the conservation degree of the cationic residue identity to reflect the interplay between the proximal cations and the hydrophobic interactions.

Trends in the biological functions and medical applications of extracellular vesicles and analogues.

Natural extracellular vesicles (EVs) play important roles in many life processes such as in the intermolecular transfer of substances and genetic information exchanges. Investigating the origins and working mechanisms of natural EVs may provide an understanding of life activities, especially regarding the occurrence and development of diseases. Additionally, due to their vesicular structure, EVs (in small molecules, nucleic acids, proteins, etc.) could act as efficient drug-delivery carriers. Herein, we describe the sources and biological functions of various EVs, summarize the roles of EVs in disease diagnosis and treatment, and review the application of EVs as drug-delivery carriers. We also assess the challenges and perspectives of EVs in biomedical applications.

Opposite Regulatory Effects of Immobilized Cations on the Folding Vs. Assembly of Melittin.

Ions are crucial in modulating the protein structure. For the free ions in bulk solution, ammonium is kosmotropic (structure forming) and guanidinium is chaotropic (structure breaking) to the protein structure within the Hofmeister series. However, the effect of immobilized ions on a protein surface is less explored. Herein, we explored the influence of two immobilized cations (ammonium in the side chain of lysine and guanidinium in the side chain of arginine) on the folding and assembly of melittin. Melittin adopts an α-helix structure and is driven by hydrophobic interactions to associate into a helical bundle. To test the influence of immobilized cations on the peptide structure, we designed the homozygous mutants exclusively containing ammonium (melittin-K) or guanidinium (melittin-R) and compared the differences of melittin-K vs. melittin-R in their folding, assembly, and molecular functions. The side chains of lysine and arginine differ in their influences on the folding and assembly of melittin. Specifically, the side chain of R increases the α-helical propensity of melittin relative to that of K, following an inverse Hofmeister series. In contrast, the side chain of K favors the assembly of melittin relative to the side chain of R in line with a direct Hofmeister series. The opposite regulatory effects of immobilized cations on the folding and assembly of melittin highlight the complexity of the noncovalent interactions that govern protein intermolecular architecture.