Apatinib Plus Toripalimab (Anti-PD1 Therapy) as Second-Line Therapy in Patients With Advanced Gastric or Esophagogastric Junction Cancer: Results From a Randomized, Open-Label Phase II Study.

BACKGROUND: This study aimed to assess the activity of apatinib plus toripalimab in the second line for patients with advanced gastric or esophagogastric junction cancer (GC/EGJC). METHODS: In this open-label, phase II, randomized trial, patients with advanced GC/EGJC who progressed after first-line chemotherapy were enrolled and received 250 mg apatinib per day plus 240 mg toripalimab on day 1 per 3 weeks (arm A) or physician's choice of chemotherapy (PC, arm B). The primary endpoint of this study was the 1-year survival rate. Progression-free survival (PFS), overall survival (OS), overall response rate (ORR), and safety were assessed as secondary endpoints. RESULTS: Twenty-five patients received apatinib plus toripalimab while 26 were enrolled in arm B. The 1-year survival rates of the 2 groups were 43.3% and 42.3%, respectively (P = .903). The PFS was 2.77 versus 2.33 months (P = .660). The OS was 8.30 versus 9.88 months (P = .539). An objective response was reported in 20.0% of patients in arm A compared to 26.9% in arm B (P = .368), respectively. A total of 6 (24.0%) patients experienced adverse events of grade ≥ 3 in arm A, while 9 (34.6%) patients suffered from adverse events of grade ≥ 3 in arm B. No drug-related deaths occurred in either group. CONCLUSION: Toripalimab plus apatinib treatment in second-line therapy of advanced GC/EGJC showed manageable toxicity but did not improve clinical outcomes relative to PC treatment (ClinicalTrials.gov Identifier: NCT04190745).

Optical and Electrical Properties of AlxGa1-xN/GaN Epilayers Modulated by Aluminum Content.

AlGaN-based LEDs are promising for many applications in deep ultraviolet fields, especially for water-purification projects, air sterilization, fluorescence sensing, etc. However, in order to realize these potentials, it is critical to understand the factors that influence the optical and electrical properties of the device. In this work, AlxGa1-xN (x = 0.24, 0.34, 0.47) epilayers grown on c-plane patterned sapphire substrate with GaN template by the metal organic chemical vapor deposition (MOCVD). It is demonstrated that the increase of the aluminum content leads to the deterioration of the surface morphology and crystal quality of the AlGaN epitaxial layer. The dislocation densities of AlxGa1-xN epilayers were determined from symmetric and asymmetric planes of the ω-scan rocking curve and the minimum value is 1.01 × 109 cm-2. The (101¯5) plane reciprocal space mapping was employed to measure the in-plane strain of the AlxGa1-xN layers grown on GaN. The surface barrier heights of the AlxGa1-xN samples derived from XPS are 1.57, 1.65, and 1.75 eV, respectively. The results of the bandgap obtained by PL spectroscopy are in good accordance with those of XRD. The Hall mobility and sheet electron concentration of the samples are successfully determined by preparing simple indium sphere electrodes.

Efficacy relevance of PD-L1 expression on circulating tumor cells in metastatic breast cancer patients treated with anti-PD-1 immunotherapy.

PURPOSE: Breast cancer has become the leading cause of cancer mortality in women. Although immune checkpoint inhibitors targeting programmed death-1 (PD-1) are promising, it remains unclear whether PD-L1 expression on circulating tumor cells (CTCs) has predictive and prognostic values in predicting and stratifying metastatic breast cancer (MBC) patients who can benefit from anti-PD-1 immunotherapy. METHODS: Twenty six MBC patients that received anti-PD-1 immunotherapy were enrolled in this study. The peptide-based Pep@MNPs method was used to isolate and enumerate CTCs from 2.0 ml of peripheral venous blood. The expression of PD-L1 on CTCs was evaluated by an established immunoscoring system categorizing into four classes (negative, low, medium, and high). RESULTS: Our data showed that 92.3% (24/26) of patients had CTCs, 83.3% (20/26) of patients had PD-L1-positive CTCs, and 65.4% (17/26) of patients had PD-L1-high CTCs. We revealed that the clinical benefit rate (CBR) of patients with a cut-off value of ≥ 35% PD-L1-high CTCs (66.6%) was higher than the others (29.4%). We indicated that PD-L1 expression on CTCs from MBC patients treated with anti-PD-1 monotherapy was dynamic. We demonstrated that MBC patients with a cut-off value of ≥ 35% PD-L1-high CTCs had longer PFS (P = 0.033) and OS (P = 0.00058) compared with patients with a cut-off value of < 35% PD-L1-high CTCs. CONCLUSION: Our findings suggested that PD-L1 expression on CTCs could predict the therapeutic response and clinical outcomes, providing a valuable predictive and prognostic biomarker for patients treated with anti-PD-1 immunotherapy.

Single-Vesicle Infrared Nanoscopy for Noninvasive Tumor Malignancy Diagnosis.

Protein heterogeneity in molecular expression and structures determines tumorigenesis and is the diagnostic and therapeutic cancer biomarker. Small extracellular vesicles (sEVs) are cell-released nanoscaled membrane-bound vesicles transferring bioactive molecules for intercellular communication and playing essential roles in tumor progression and metastasis. Therefore, protein heterogeneity in tumor-derived sEVs indicates the degree of malignant transformation, providing a noninvasive biomarker for cancer diagnosis and malignancy evaluation. We employ near-field infrared (nano-FTIR) spectroscopy to investigate malignancy-related protein heterogeneity in a single sEV and demonstrate the discriminability of sEV protein heterogeneity to evaluate tumor malignancy and metastasis. We found that the amide I/II adsorption ratio of the sEVs increases with tumor malignancy, the proportion of α-helix + random coil (α-helix and random coil) in sEV proteins decreases with tumor malignancy, and the proportion of ß-sheet + ß-turn (ß-sheet and ß-turn) increases with tumor malignancy. These nano-FTIR spectral signatures of the sEVs from the primary tumor tissue of breast cancer patients show high sensitivity and specificity in evaluating tumor metastasis. This study shows the advantages of nano-FTIR in single sEV characterization and demonstrates the significance of sEV protein heterogeneity in cancer diagnosis. It provides a noninvasive solution to elucidate cancer development and facilitates the exploitation of potential cancer biomarkers.

Principles of Amino-Acid-Nucleotide Interactions Revealed by Binding Affinities between Homogeneous Oligopeptides and Single-Stranded DNA Molecules.

We have determined the binding strengths between nucleotides of adenine, thymine, guanine and cytosine in homogeneous single stranded DNAs and homo-octapeptides consisting of 20 common amino acids. We use a bead-based fluorescence assay for these measurements in which octapeptides are immobilized on the bead surface and ssDNAs are in solutions. Comparative analyses of the distribution of the binding energies reveal unique binding strength patterns assignable to each DNA nucleotide and amino acid originating from the chemical structures. Pronounced favorable (such as Arg-G, etc.) and unfavorable (such as Ile-T, etc.) binding interactions can be identified in selected groups of amino acid and nucleotide pairs that could provide basis to elucidate energetics of amino-acid-nucleotide interactions. Such interaction selectivity, specificity and polymorphism establish the contributions from DNA backbone, DNA bases, as well as main chain and side chain of the amino acids.

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.

Assessment of PD-L1 Expression on Circulating Tumor Cells for Predicting Clinical Outcomes in Patients with Cancer Receiving PD-1/PD-L1 Blockade Therapies.

BACKGROUND: Programmed death-1 (PD-1) and programmed death-ligand 1 (PD-L1) blockade immunotherapies have changed the landscape of cancer therapy. However, the main limitation of these therapies is the lack of definitively predictive biomarkers to predict treatment response. Whether PD-L1 expression on circulating tumor cells (CTCs) is associated with the clinical outcomes of immunotherapy remains to be extensively investigated. MATERIALS AND METHODS: One hundred fifty-five patients with different advanced cancers were enrolled in this study and treated with anti-PD-1/PD-L1 monoclonal antibodies. Using the Pep@MNPs method, CTCs were isolated and enumerated. The PD-L1 expression levels were analyzed by an immunofluorescence assay for semiquantitative assessment with four categories (negative, low, medium, and high). RESULTS: Prior to immunotherapy, 81.93% (127/155) of patients had PD-L1-positive CTCs, and 71.61% (111/155) had at least one PD-L1-high CTC. The group with PD-L1-positive CTCs had a higher disease control rate (DCR) (71.56%, 91/127), with a DCR of only 39.29% (11/28) for the remaining individuals (p = .001). The objective response rate and DCR in PD-L1-high patients were higher than those in the other patients (32.44% vs. 13.64%, p = .018 and 75.68% vs. 40.91%, p < .0001, respectively). The reduction in the counts and ratios of PD-L1-positive CTCs and PD-L1-high CTCs reflected a beneficial response to PD-1/PD-L1 inhibitors. Furthermore, patients with PD-L1-high CTCs had significantly longer progression-free survival (4.9 vs. 2.2 months, p < .0001) and overall survival (16.1 vs. 9.0 months, p = .0235) than those without PD-L1-high CTCs. CONCLUSION: The PD-L1 level on CTCs may serve as a clinically actionable biomarker for immunotherapy, and its dynamic changes could predict the therapeutic response. IMPLICATIONS FOR PRACTICE: This study was designed to investigate the role of programmed death-ligand 1 (PD-L1) expression on circulating tumor cells in predicting and monitoring response to programmed death-1 (PD-1)/PD-L1 blockade immunotherapies in patients with advanced cancer. The results of the study showed that PD-L1-high-expression circulating tumor cells (CTCs) were both a predictive biomarker and a prognostic factor in patients with advanced cancer treated with anti-PD-1/PD-L1 monoclonal antibodies. These observations suggest that PD-L1 level on CTCs is a potential clinical biomarker for immunotherapy.

Versatile Biosensing Toolkit Using an Electronic Particle Counter.

Development of a versatile biosensing toolkit is in urgent need for rapid and multiplexed detection applications. In this work, an electronic particle counter-implemented versatile biosensing toolkit has been developed for detecting a range of targets with high sensitivity, broad detection range, multiplexibility, simple operation, and low cost. The electrical resistance-based particle counter conventionally measuring the number of microspheres (1-100 µm) can quantify analytes. The versatility of this approach is verified by assaying small molecules, protein biomarkers, pathogen bacteria, and tumor cells using three strategies: (1) antigen-antibody interaction, (2) DNA hybridization, and (3) polypeptide recognition. More importantly, this biosensing toolkit allows the simultaneous detection of multiple targets with a broad detection range from pg mL-1 to µg mL-1, showing great potential as a powerful technique for food safety testing and biomedical diagnosis.

Ultrasensitive Gastric Cancer Circulating Tumor Cellular CLDN18.2 RNA Detection Based on a Molecular Beacon.

Gastric cancer (GC) is a major global cancer burden, and only HER2-targeted therapies have been approved in first line clinical therapy. CLDN18.2 has been regarded as a potential therapeutic target for gastrointestinal tumors, and global clinical trials have been in process. Hence, the precise, efficient, and noninvasive detection of CLDN18.2 expression is important for the effective application of this attractive target. A high similarity of protein sequence between CLDN18.1 and -18.2 made RNA become more suitable for the detection of CLDN18.2 expression. In this study, CLDN18.2 molecular beacon (MB) with a stem-loop hairpin structure was optimized by phosphorothioate and 2'-O-methyl for stability and efficiency. The MB could recognize CLDN18.2 RNA rapidly. Its resolution and selectivity has been verified in several model cells, demonstrating that MB can distinguish CLDN18.2 expression in several model cells. Furthermore, it was applied successfully to the circulating tumor cell (CTC) assay. The concordance in the expression of CLDN18.2 between CTCs and tissue biopsy is 100% (negative: 3 vs 3; positive: 7 vs 7), indicating that CLDN18.2 RNA detection in CTCs based on a MB will be a promising approach for searching potential patients to CLDN 18.2 targeted drug.

Composition-dependent multivalency of peptide-peptide interactions revealed by tryptophan-scanning mutagenesis.

We have examined in this contribution the composition dependence of binding characteristics in peptide-peptide interactions between an oligopeptide octa-glycine and a series of tryptophan-containing octapeptides. The binding energy associated with tryptophan-glycine interactions manifests pronounced stepwise binding characteristics as the number of tryptophan increases from 0 to 8 in the octapeptides consisting only of glycine and can be attributed to mono-, di-, and tri-valent peptide-peptide interactions. At the same time, only weak fluctuations in binding energy were observed as the number of tryptophan increases from 2 to 7. Such distinctive nonlinearity of composition-dependent tryptophan-glycine binding energy characteristics due to continuously varying tryptophan compositions in the octapeptides could be considered as a reflection of combinatorial contributions due to the hydrogen bonds originated from the indole moieties of tryptophan with the main chains of octapeptide of glycine containing N-H and C=O moieties and the van der Waals interactions (including π-π and π-CH interactions) between peptides.

Synthetic CXCR4 Antagonistic Peptide Assembling with Nanoscaled Micelles Combat Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is the most common adult acute leukemia with very low survival rate due to drug resistance and high relapse rate. The C-X-C chemokine receptor 4 (CXCR4) is highly expressed by AML cells, actively mediating chemoresistance and reoccurrence. Herein, a chemically synthesized CXCR4 antagonistic peptide E5 is fabricated to micelle formulation (M-E5) and applied to refractory AML mice, and its therapeutic effects and pharmacokinetics are investigated. Results show that M-E5 can effectively block the surface CXCR4 in leukemic cells separated from bone marrow (BM) and spleen, and inhibit the C-X-C chemokine ligand 12-mediated migration. Subcutaneous administration of M-E5 significantly inhibits the engraftment of leukemic cells in spleen and BM, and mobilizes residue leukemic cells into peripheral blood, reducing organs' burden and significantly prolonging the survival of AML mice. M-E5 can also increase the efficacy of combining regime of homoharringtonine and doxorubicin. Ribonucleic acid sequencing demonstrates that the therapeutic effect is contributed by inhibiting proliferation and enhancing apoptosis and differentiation, all related to the CXCR4 signaling blockade. M-E5 reaches the concentration peak at 2 h after administration with a half-life of 14.5 h in blood. In conclusion, M-E5 is a novel promising therapeutic candidate for refractory AML treatment.

Position-coded multivalent peptide-peptide interactions revealed by tryptophan-scanning mutagenesis.

We demonstrate in this contribution the evidence that significant cooperative binding effect can be identified for the amino acid sites that are determinant to the binding characteristics in peptide-peptide interactions. The analysis of tryptophan-scanning mutagenesis of the 14-mer peptide consisting only of glycine provides a mapping of position-dependent contributions to the binding energy. The pronounced tryptophan-associated peptide-peptide interactions are originated from the indole moieties with the main chains of 14-mer glycines containing N-H and CO moieties. Specifically, with the presence of two tryptophans as determinant amino acids, cooperative binding can be observed, which are dependent on relative positions of the two tryptophans with a "volcano"-like characteristics. An optimal separation of 6-10 amino acids between two adjacent binding sites can be identified to achieve maximal binding interactions.

Steric Dependence of Chirality Effect in Surface-Mediated Peptide Assemblies Identified with Scanning Tunneling Microscopy.

Amino acid chirality has been recognized as an important driving force in constructing peptide architectures, via interactions such as chirality-induced stereochemical effect. The introduction of site-specific chiral conversion of l- and d-amino acids in peptide sequences could enable the pursuit of the chirality effects in peptide assembly. In this work, we characterized the assemblies of heptapeptides with various side chain moieties and their chiral variants using STM. Specifically, two pairs of amino acids, Gln (Q) and Asn (N), Glu (E) and Asp (D), having one methylene difference in their side chains, are selected to elucidate the steric dependence of amino acid chiral effects on surface-bound peptide assemblies. The observed heptapeptide assembly structures reveal that chirality switching of a single amino acid is able to destabilize the surface-mediated peptide assemblies, and this disturbance effect can be positively correlated with the steric hindrance of amino acid side chains. Furthermore, the strength of the impact due to chiral conversion on heptapeptide assembly structure is noticeably dependent on the mutation sites, indicative of structural heterogeneity of chiral effects. These results could contribute to the molecular insights of chirality-induced stereochemical interactions in peptide assembly.

Diagnosis of Invasive Nonfunctional Pituitary Adenomas by Serum Extracellular Vesicles.

The invasiveness evaluation of nonfunctional pituitary adenoma (NFPAs) is crucial for the prediction of the malignant potential and for making surgical plans of NFPAs. Current invasiveness evaluation of NFPAs is based on neuroimaging, which can hardly predict the invasive potential and dynamically monitor disease progress. Here we used microbead-assisted flow cytometry to detect and analyze the serum extracellular vesicles (EVs) from 30 NFPAs patients (15 invasive and 15 noninvasive). Lower expressions of folate receptor 1 (FOLR1) and epithelial cell adhesion molecule (EpCAM) were found in serum EVs from the invasive NFPAs patients compared to the noninvasive ones [area under the curve (AUC) of 0.94 for FOLR1 and 0.88 for EpCAM]. Meanwhile, increased mRNA expression of vimentin and N-cadherin, two mesenchymal markers, was found in serum EVs from the invasive NFPAs patients compared to the noninvasive ones. Consistent results were observed in the tumor tissue that invasive NFPAs have lower expression of the epithelial markers while higher expression of the mesenchymal markers. These results suggested the possible role of epithelial-mesenchymal transition (EMT) in the invasiveness of NFPAs. Pituitary tumor transforming gene 1 (PTTG1) mRNA in serum EVs was also found to be an indicator for invasive NFPAs and is related with EMT. These results provide a method for the blood-based diagnosis and invasiveness evaluation of NFPAs and would be beneficial to the diagnosis, prognosis prediction, and surgical risk evaluation of NFPAs.

Identifying Terminal Assembly Propensity of Amyloidal Peptides by Scanning Tunneling Microscopy.

The abnormal accumulation of beta-amyloids (Aß) in brain is considered as a key initiating cause for Alzheimer's disease (AD) due to their richness in plaques and self-aggregate propensity. In recent studies, N-terminally extended Aß peptides (NTE-Aß) with the N-terminus originating prior to the canonical ß-secretase cleavage site were found in humans and suggested to have possible relevance to AD. However, the effects of the extended N-terminus on the amyloidegenic structure and aggregation propensity have not been fully elucidated. Herein, we characterized the assembly structures of Aß1-42, Aß(-5)-42, Aß(-10)-42 and Aß(-15)-42 with both normal and reversed sequences on highly oriented pyrolytic graphite (HOPG) surfaces with scanning tunneling microscopy (STM). The molecularly resolved surface-mediated peptide assemblies enable identification of amyloidegenic fragments. The observations reveal that the assembly propensity of the C-terminal strand of Aß1-42 is highly conserved and insensitive to N-terminal extensions. In contrast, different assembly structures of the N-terminal strand of Aß variants can be observed with possible assignment of varied amyloidegenic fragments in the extended N-termini, which may contribute to the varied aggregation propensities of Aß42 species.

Peptide Self-Assembly and Its Modulation: Imaging on the Nanoscale.

This chapter intends to review the progress in obtaining site-specific structural information for peptide assemblies using scanning tunneling microscopy. The effects on assembly propensity due to mutations and modifications in peptide sequences, small organic molecules and conformational transitions of peptides are identified. The obtained structural insights into the sequence-dependent assembly propensity could inspire rational design of peptide architectures at the molecular level.

Combined peripheral natural killer cell and circulating tumor cell enumeration enhance prognostic efficiency in patients with metastatic triple-negative breast cancer.

OBJECTIVE: Triple-negative breast cancer (TNBC) is a heterogeneous disease with poor prognosis. Circulating tumor cells (CTCs) are a promising predictor for breast cancer prognoses but their reliability regarding progression-free survival (PFS) is controversial. We aim to verify their predictive value in TNBC. METHODS: In present prospective cohort study, we used the Pep@MNPs method to enumerate CTCs in baseline blood samples from 75 patients with TNBC (taken at inclusion in this study) and analyzed correlations between CTC numbers and outcomes and other clinical parameters. RESULTS: Median PFS was 6.0 (range: 1.0-25.0) months for the entire cohort, in whom we found no correlations between baseline CTC status and initial tumor stage (P=0.167), tumor grade (P=0.783) or histological type (P=0.084). However, among those getting first-line treatment, baseline CTC status was positively correlated with ratio of peripheral natural killer (NK) cells (P=0.032), presence of lung metastasis (P=0.034) and number of visceral metastatic site (P=0.037). Baseline CTC status was predictive for PFS in first-line TNBC (P=0.033), but not for the cohort as a whole (P=0.118). This prognostic limitation of CTC could be ameliorated by combining CTC and NK cell enumeration (P=0.049). CONCLUSIONS: Baseline CTC status was predictive of lung metastasis, peripheral NK cell ratio and PFS in TNBC patients undergoing first-line treatment. We have developed a combined CTC-NK enumeration strategy that allows us to predict PFS in TNBC without any preconditions.

Antiamyloidogenic Activity of Aß42-Binding Peptoid in Modulating Amyloid Oligomerization.

The oligomerization and aggregation of amyloid ß (Aß) play central role in the pathogenesis of Alzheimer's disease (AD). Molecular binding agents for modulating the formation of Aß oligomers and fibrils have promising application potential in AD therapies. By screening a peptoid library using surface plasmon resonance imaging, amyloid inhibitory peptoid 1 (AIP1) that has high affinity to Aß42 is identified. AIP1 is demonstrated to inhibit Aß42 oligomerization and fibrillation and to rescue Aß42-induced cytotoxicity through decreasing the content of Aß42 oligomers that is related to cell membrane permeability. Molecular docking suggests that the binding sites of AIP1 may be at the N-terminus of Aß42. The blood-brain barrier (BBB) permeability of AIP1 using an in vitro BBB model is also revealed. This work provides a strategy for the design and development of peptoid-based antiamyloidogenic agents. The obtained amyloid inhibitory peptoid shows prospects in the therapeutic application in AD.

Stabilization Effect of Amino Acid Side Chains in Peptide Assemblies on Graphite Studied by Scanning Tunneling Microscopy.

An analysis is presented of the effects of amino acid side chains on peptide assemblies in ambient conditions on a graphite surface. The molecularly resolved assemblies of binary peptides are examined with scanning tunneling microscopy. A comparative analysis of the assembly structures reveals that the lamellae width has an appreciable dependence on the peptide sequence, which could be considered as a manifestation of a stabilizing effect of side-chain moieties of amino acids with high (phenylalanine) and low (alanine, asparagine, histidine and aspartic acid) propensities for aggregation. These amino acids are representative for the chemical structures involving the side chains of charged (histidine and aspartic acid), aromatic (phenylalanine), hydrophobic (alanine), and hydrophilic (asparagine) amino acids. These results might provide useful insight for understanding the effects of sequence on the assembly of surface-bound peptides.

Self-assembled chiral nanostructures of amphiphilic peptide: from single molecule to aggregate.

We report interesting hierarchical self-assembled architectures from a designed amphiphilic peptide. The bisignate cotton effects in circular dichroism spectra show typical peptide aggregation-induced. The observation of peptide assembly structures from initial particles and fibrils to ribbon structures is supported by microscopy (atomic force microscopy and transmission electron microscopy). The visualization of individual peptide at the single molecular level offered insights of the intermolecular interactions responsible for the formation of aggregates, which is investigated by scanning tunneling microscopy. The orientation of intermolecular bonds between carboxylic and amine group and the hydrophobic interactions between alanine residues could be the dominant driving force for the assembly chirality at near-neutral pH. The single molecular and aggregate level evidence in this manuscript will shed light on the understanding of hierarchical chiral self-assembly pathway and the underlying mechanism. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.