Layer-by-layer fabrication of alginate/polyethyleneimine multilayer on magnetic interface with enhanced efficiency in immuno-capturing circulating tumor cells.

BACKGROUND: The technology of capturing circulating tumor cells (CTCs) plays a crucial role in the diagnosis, evaluation of therapeutic efficacy, and prediction of prognosis in lung cancer. However, the presence of complex blood environment often results in severe nonspecific protein adsorption and interferences from blood cells, which negatively impacts the specificity of CTCs capture. There is a great need for development of novel nanomaterials for CTCs capture with prominent anti-nonspecific adsorptions from proteins or blood cells. RESULTS: We present a novel immune magnetic probe Fe3O4@(PEI/AA)4@Apt. The surface of Fe3O4 particles was modified with four layers of PEI/AA composite by layer-by-layer assembly. Furthermore, aptamers targeting epithelial marker EpCAM (SYL3C) and mesenchymal marker CSV (ZY5C) were simultaneously connected on Fe3O4@(PEI/AA)4 to improve the detection of different phenotypic CTCs and reduce false negatives. The results demonstrated that the (PEI/AA)4 coatings not only minimized non-specific protein adsorptions, but also significantly reduced the adsorption rate of red blood cells to a mere 1 %, as a result of which, the Fe3O4@(PEI/AA)4@Apt probe achieved a remarkably high capture efficiency toward CTCs (95.9 %). In the subsequent validation of clinical samples, the probe was also effective in capturing rare CTCs from lung cancer patients. SIGNIFICANCE AND NOVELTY: A (PEI/AA) polymerized composite with controllable layers was fabricated by layer-by-layer self-assembly technique, which displayed remarkable anti-nonspecific adsorption capabilities toward proteins and cells. Importantly, Fe3O4@(PEI/AA)4@Apt probe significantly improved CTCs capture purity in lung cancer patients to 89.36 %. For the first time, this study combined controllable (PEI/AA) layers with magnetic separation to innovatively build a resistant interface that significantly improves the specific capture performances of CTCs, broadening the application of this polymerized composite.

Metabolic classification suggests the GLUT1/ALDOB/G6PD axis as a therapeutic target in chemotherapy-resistant pancreatic cancer.

Metabolic reprogramming is known as an emerging mechanism of chemotherapy resistance, but the metabolic signatures of pancreatic ductal adenocarcinomas (PDACs) remain unclear. Here, we characterize the metabolomic profile of PDAC organoids and classify them into glucomet-PDAC (high glucose metabolism levels) and lipomet-PDAC (high lipid metabolism levels). Glucomet-PDACs are more resistant to chemotherapy than lipomet-PDACs, and patients with glucomet-PDAC have a worse prognosis. Integrated analyses reveal that the GLUT1/aldolase B (ALDOB)/glucose-6-phosphate dehydrogenase (G6PD) axis induces chemotherapy resistance by remodeling glucose metabolism in glucomet-PDAC. Increased glycolytic flux, G6PD activity, and pyrimidine biosynthesis are identified in glucomet-PDAC with high GLUT1 and low ALDOB expression, and these phenotypes could be reversed by inhibiting GLUT1 expression or by increasing ALDOB expression. Pharmacological inhibition of GLUT1 or G6PD enhances the chemotherapy response of glucomet-PDAC. Our findings uncover potential metabolic heterogeneity related to differences in chemotherapy sensitivity in PDAC and develop a promising pharmacological strategy for patients with chemotherapy-resistant glucomet-PDAC through the combination of chemotherapy and GLUT1/ALDOB/G6PD axis inhibitors.

Controlled Genetic Encoding of Unnatural Amino Acids in a Protein Nanopore.

Conventional protein engineering methods for modifying protein nanopores are typically limited to 20 natural amino acids, which restrict the diversity of the nanopores in structure and function. To enrich the chemical environment inside the nanopore, we employed the genetic code expansion (GCE) technique to site-specifically incorporate the unnatural amino acid (UAA) into the sensing region of aerolysin nanopores. This approach leveraged the efficient pyrrolysine-based aminoacyl-tRNA synthetase-tRNA pair for a high yield of pore-forming protein. Both molecular dynamics (MD) simulations and single-molecule sensing experiments demonstrated that the conformation of UAA residues provided a favorable geometric orientation for the interactions of target molecules and the pore. This rationally designed chemical environment enabled the direct discrimination of multiple peptides containing hydrophobic amino acids. Our work provides a new framework for endowing nanopores with unique sensing properties that are difficult to achieve using classical protein engineering approaches.

Health-related quality of life in patients with Kashin-Beck disease is lower than in those with osteoarthritis: a cross-sectional study.

BACKGROUND: Kashin-Beck disease (KBD) is an endemic deformable bone and joint disease, which affects the quality of life (QOL) of patients. We conducted a cross-sectional study of the QOL of KBD patients by a new KBD quality of life (KBDQOL) questionnaire. METHODS: A total of 252 KBD patients and 248 OA patients came from Northwest China, and 260 healthy people living in the same area as KBD and osteoarthritis (OA) patients served as the controls. KBDQOL questionnaire was used to evaluate the QOL of all objects. RESULTS: The average scores for physical functions, activity limitations, support of society, mental health and general health were significantly lower in KBD patients than that in OA patients and healthy people except for economics. Monofactor analysis showed that age, height, weight status, education level and grade of KBD had a significant effect on KBDQOL score. Multivariate analysis showed that grade of KBD was the influencing factor of physical function score; gender, age, height, grade of KBD and duration of symptoms were the influencing factors of activity restriction score; age and grade of KBD were factors affecting the general health score. CONCLUSION: The QOL of KBD patients was significantly lower than that of OA patients and healthy people. The KBDQOL questionnaire may be a promising tool for assessing the QOL of KBD patients.

Profiling the chemistry- and confinement-controlled sensing capability of an octameric aerolysin-like protein.

Octameric Aep1 was employed, for the first time to the best of our knowledge, as a nanopore to expand applications. After investigating the optimized conditions of Aep1 for single-channel recording, the sensing features were characterized. Cyclic and linear molecules of varying sizes and charges were employed to probe the radius and chemical environment of the pore, providing deep insights for expected future endeavors at predicting the structure of octameric Aep1. γ-CD showed unique suitability as an 8-subunit adapter in octameric Aep1, enabling the discrimination of ß-nicotinamide mononucleotide.

Protein nanopore reveals the renin-angiotensin system crosstalk with single-amino-acid resolution.

The discovery of crosstalk effects on the renin-angiotensin system (RAS) is limited by the lack of approaches to quantitatively monitor, in real time, multiple components with subtle differences and short half-lives. Here we report a nanopore framework to quantitatively determine the effect of the hidden crosstalk between angiotensin-converting enzyme (ACE) and angiotensin-converting enzyme 2 (ACE2) on RAS. By developing an engineered aerolysin nanopore capable of single-amino-acid resolution, we show that the ACE can be selectively inhibited by ACE2 to prevent cleavage of angiotensin I, even when the concentration of ACE is more than 30-fold higher than that of ACE2. We also show that the activity of ACE2 for cleaving angiotensin peptides is clearly suppressed by the spike protein of SARS-CoV-2. This leads to the relaxation of ACE and the increased probability of accumulation of the principal effector angiotensin II. The spike protein of the SARS-CoV-2 Delta variant is demonstrated to have a much greater impact on the crosstalk than the wild type.

Prevalence and radiographic features of atlantoaxial dislocation in adult patients with Kashin-Beck disease.

PURPOSE: Kashin-Beck disease (KBD) is an endemic osteoarthropathy affecting the epiphyseal growth plate of multiple joints in young and adolescent patients. Previous studies have focused on the visible deformed extremities instead of the spinal radiological features, especially the atlantoaxial joint. The aim of this study was to determine the prevalence and radiographic features of atlantoaxial dislocation (AAD) in adult patients with KBD. METHODS: This study was conducted on KBD patients in three typical endemic counties between October 2017 and November 2019. The patients were evaluated by collecting basic information, clinical signs and symptoms. They underwent dynamic cervical radiography, by which AAD was diagnosed. For those patients with confirmed or suspected AAD, computed tomography (CT) imaging was performed to observe the odontoid morphology and degenerative changes in the lateral atlantoaxial joints. Radiographic evaluations were reviewed to determine the prevalence and features of AAD. RESULTS: A total of 39 (14.6%) of 267 KBD patients were diagnosed with AAD. Compared with the non-AAD patients, the detection rate of AAD was associated with a longer disease duration and stage and was not associated with age, sex or BMI. Thirty-two patients had symptoms at the neck or neurological manifestations, while seven had no symptoms. There were three types of morphologies of the odontoid process in AAD patients: separating in 19 cases, hypoplastic in 15 cases and intact in five cases. Anterior dislocation was noted in 29 cases, and posterior dislocation was noted in ten cases. Thirty-four cases were reducible, and five were irreducible. The lateral atlantoaxial joints had different severities of degenerative changes in 17 cases. CONCLUSIONS: This study revealed that the prevalence of AAD was 14.6% in adult KBD patients. The radiographic features of AAD include manifestations of odontoid dysplasia and chronic degenerative changes in atlantoaxial joints. KBD patients with severe stages and longer disease duration were more vulnerable to the occurrence of AAD. We postulate that this atlantoaxial anomaly might originate from chondronecrosis of the epiphyseal growth plate of the odontoid process in young and adolescent individuals. This study may provide a clinical reference to help clinicians screen, prevent and treat AAD in adult patients with KBD.

Capture of Heterogeneous Circulating Tumor Cells in Colorectal Cancer Patients on an Immunomagnetic and Anti-Nonspecific Adsorption Platform.

Circulating tumor cells (CTC) have been represented by different phenotypes due to the epithelial-mesenchymal transition (EMT), which are epithelial CTC (E-CTC), mesenchymal CTC (M-CTC), and mixed (epithelial, mesenchymal) CTC (EM-CTC). Limited work has systematically discussed the associations of CTC number, especially proportions of E-CTC, M-CTC, and EM-CTC in total CTC, with colorectal cancer (CRC) progressions via a simple method with high performances. To achieve this goal, this paper presents the fabrication of a novel anti-nonspecific adsorption immunomagnetic platform called Fe3O4@SiO2@PTMAO@Aptamer, which was obtained by modifying polymeric trimethylamine N-oxide (PTMAO) on magnetic Fe3O4@SiO2, which was then linked with dual aptamers of an epithelial cellular adhesion molecule (EpCAM) and cell surface vimentin (CSV), targeting different CTC phenotypes. Results demonstrated that the abundant coating of PTMAO on Fe3O4@SiO2 improved the anti-nonspecific adsorption and noncell adhesion abilities of the immunomagnetic particles and could capture heterogeneous CTC with higher efficiency within 10 min. These excellent performances of Fe3O4@SiO2@PTMAO@Aptamer allowed us to inspect the correlations of numbers of E-CTC, M-CTC, EM-CTC, or proportions of them in total CTC with clinical information on CRC patients in detail. Our data innovatively and clearly revealed that the captured CTC, especially M-CTC proportion, displayed more close associations with progression, diagnosis, surgery, and chemotherapeutic effects for CRC patients. Overall, we believe that our approach will bring a new understanding of CTC-based liquid biopsy for cancer diagnosis and treatment.

Sanhuang xiexin decoction ameliorates secondary liver injury in DSS-induced colitis involve regulating inflammation and bile acid metabolism.

ETHNOPHARMACOLOGICAL RELEVANCE: SanHuang XieXin decoction (SXD) is a widely applicated traditional Chinese medicine (TCM) with a significant gut-liver axis regulation effect. AIM OF THE STUDY: To evaluate the therapeutic effect and elucidate the possible underlying molecular mechanisms of SXD on liver damage secondary to ulcerative colitis (UC) in mice. MATERIALS AND METHODS: A model of liver damage secondary to UC was induced by drinking 5% dextran sodium sulfate (DSS) in mice. These mice were treated with one of three doses of SXD or sulfasalazine (SASP), then liver samples were collected and tested. RESULTS: The results reveal that SXD treatment reduced liver cells swelling, and inhibited the accumulation of the hepatic-pro-inflammatory cytokines IL-1ß and tumor necrosis factor-α (TNF-α) in mice with colitis. In addition, SXD reduced the production of nitric oxide (NO) and malondialdehyde (MDA), and increased the activities of superoxide dismutase (SOD). In inflammation regulating, SXD significantly down regulated the protein expression of MyD88 and p-Iκα, but upregulated Iκα. In bile acid metabolism regulating, SXD significantly down regulated the protein expression of FXR, MRP2, BESP and SHP. Therefore, SXD treatment can regulate the TLR4-NF-κB and bile acid metabolism pathways to alleviate liver inflammation and cholestasis. CONCLUSIONS: These results demonstrate that SXD is a potential alternative therapeutic medicine for the treatment of liver damage secondary to colitis.

Sanhuang Xiexin decoction ameliorates DSS-induced colitis in mice by regulating intestinal inflammation, intestinal barrier, and intestinal flora.

ETHNOPHARMACOLOGICAL RELEVANCE: Sanhuang Xiexin decoction (SXD) is a widely applicated traditional Chinese medicine (TCM) with a significant intestinal anti-inflammatory effect. AIM OF THE STUDY: To evaluate the therapeutic effect and elucidate the possible underlying mechanisms of SXD on dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) in mice. METHODS: To model UC, 3% DSS was added to the drinking water for 7 days. The UC mice were grouped and treated with three doses of SXD (1.3, 2.6, and 6 g/kg) orally for 7 days. Mice body weight and disease activity index (DAI) scores were recorded daily. After treatment with SXD, the colon was removed, and the colon length and histopathological changes were recorded. Blood cells were counted and colonic inflammatory cytokines and oxidative stress indicators were examined. The key proteins in TLR4-MyD88-NF-κB signaling and the colonic barrier were determined by Western blot analysis. The restorative effect of SXD on intestinal flora was determined. RESULTS: Treatment with SXD reduced DAI scores, increased body weight, improved colon shortening, and decreased colonic damage. SXD decreased the numbers of white blood cells (WBCs), increased the numbers of red blood cells (RBCs), and inhibited the expression of inflammatory cytokines and oxidative stress indicators. In addition, SXD displayed an effective anti-inflammatory effect by inhibiting the expression levels of p-IκBα, TLR4, MyD88, and p65. Furthermore, SXD significantly restored the integrity of the colonic barrier and the abundance of beneficial flora. CONCLUSIONS: SXD significantly reduced DSS-induced colon damage when the dose was higher than 1.3 g/kg, and the middle dose group (2.6 g/kg) indicated the best effect. SXD effectively ameliorated DSS-induced UC in mice, possibly by inhibiting oxidative stress, protecting the mucosal barrier, inhibiting the TLR4-MyD88-NF-κB signaling pathway, and regulating the intestinal flora.

Full Width at Half Maximum of Nanopore Current Blockage Controlled by a Single-Biomolecule Interface.

A biological nanopore is one of the predominant single-molecule approaches as a result of its controllable single-biomolecule interface, which could reflect the "intrinsic" information on an individual molecule in a label-free way. Because the current blockage is normally treated as the most important parameter for nanopore identification of every single molecule, the fluctuation of current blockage for certain types of molecules, defined as full width at half maximum (fwhm) of current blockage, actually owns a dominant influence on nanopore resolution. Therefore, controlling the fwhm of current blockage of molecules is critical for the sensing capability of the nanopore. Here, taking an aerolysin nanopore as a model, by precisely controlling the functional group in this single-biomolecule interface, we could narrow the fwhm of nanopore current blockage for DNA identification and prolong the duration inside the nanopore. Moreover, a substantial correlation between fwhm of current blockage and duration is established, showing a non-monotonic variation. Besides, the mechanism is also clarified with studying the detailed current blockage events. This proposed correlation is further demonstrated to be applied uniformly across different mutant aerolysins for a certain DNA. This study proposes a new strategy for regulating molecular sensing from the duration of the analyte, which could guide the resolution of heterogeneity analysis using nanopores.

Long-term outcomes of arthroscopic debridement of the knee in adults with Kashin-Beck disease: an 18-year follow-up.

OBJECTIVE: Kashin-Beck disease (KBD) is an endemic degenerative joint disease with a high disability rate. We retrospectively evaluated the 18-year clinical follow-up outcomes of adult patients with KBD who underwent arthroscopic debridement for knee osteoarthritis. METHODS: Thirty-one patients with KBD (31 knees) underwent arthroscopy for knee osteoarthritis. The visual analog scale (VAS) score, walking distance, knee mobility, and patients' self-evaluated improvement in clinical symptoms were retrospectively evaluated before and 18 years after the operation. RESULTS: The patients' self-evaluated clinical symptoms showed considerable improvement at 2, 6, and 8 years after surgery but deteriorated at 10 and 18 years after surgery. Knee mobility was greater after than before arthroscopy but decreased from 6 to 18 years postoperatively. The VAS score for knee pain was high before the operation, decreased at 2 years postoperatively, increased at 6 years postoperatively, and was significantly lower at 18 years postoperatively than before surgery. The walking distance was significantly longer at 2, 6, and 8 years postoperatively than preoperatively. CONCLUSIONS: Arthroscopic treatment may be an effective therapy for adult patients with KBD who develop knee osteoarthritis. In this study, arthroscopy had a long-term effect on patients with KBD who had Kellgren-Lawrence grade

Proline-rich protein 11 overexpression is associated with a more aggressive phenotype and poor overall survival in ovarian cancer patients.

BACKGROUND: The proline-rich protein 11 (PRR11) is a newly identified oncogene associated with a poor prognosis in several human cancers. Nonetheless, research on its role in ovarian cancer (OC) remains largely understudied. Therefore, this study aims to evaluate the expression levels of PRR11 protein and its role in human ovarian cancer. METHODS: Immunohistochemistry analysis was used to evaluate the expression levels of PRR11 protein in human samples obtained from 49 patients diagnosed with OC and subjected to curative surgery in the First Affiliated Hospital of Wenzhou Medical University between 2007 and 2015. RESULTS: In total, 57.1% of the primary OC tumor tissue evaluated demonstrated overexpression of PRR11. Meanwhile, the survival analysis showed that the overall survival (OS) of patients presenting overexpression of PRR11 was significantly lower than the OS of the patients with negative PRR11. In subsequent experiments, it was found that silencing the expression of PRR11 expression inhibited the proliferation of tumor cells and the migration of cells in vitro. Further, cells subjected to PRR11 knockdown exhibited a decrease in tumor growth in vivo. The downregulation of PRR11 was coupled with a decrease in N-cadherin and downregulation in the expression of early growth response protein 1 (EGR1). CONCLUSIONS: The findings suggest that PRR11 might be considered as a potential target for prognostic assessment and gene therapy strategies for patients diagnosed with OC.

Unveiling the Heterogenous Dephosphorylation of DNA Using an Aerolysin Nanopore.

The simultaneous occurrence of multiple heterogeneous DNA phosphorylation statuses, which include 5' end phosphorylation, 5' end dephosphorylation, 3' end phosphorylation, and 3' end dephosphorylation, is crucial for regulating numerous cellular processes. Although there are many methods for detecting a single type of DNA phosphorylation, the direct and simultaneous identification of DNA phosphorylation/dephosphorylation on the 5' and/or 3' ends remains a challenge, let alone the unveiling of the heterogeneous catalysis processes of related phosphatases and kinases. Taking advantage of the charge-sensitive aerolysin nanopore interface, herein, an orientation-dependent sensing strategy is developed to enhance phosphorylation-site-dependent interaction with the nanopore sensing interface, enabling the direct and simultaneous electric identification of four heterogeneous phosphorylation statuses of a single DNA. By using this strategy, we can directly evaluate the heterogeneous dephosphorylation process of alkaline phosphatase (ALP) at the single-molecule level. Our results demonstrate that the ALP in fetal bovine serum preferentially catalyzes the 3' phosphate rather than both ends. The quantification of endogenous ALP activity in fetal bovine serum could reach the submilli-IU/L level. Our aerolysin measurements provide a direct look at the heterogeneous phosphorylation status of DNA, allowing the unveiling of the dynamic single-molecule functions of kinase and phosphatase.

Direct Quantification of Damaged Nucleotides in Oligonucleotides Using an Aerolysin Single Molecule Interface.

DNA lesions such as metholcytosine(mC), 8-OXO-guanine (OG), inosine (I), etc. could cause genetic diseases. Identification of the varieties of lesion bases are usually beyond the capability of conventional DNA sequencing which is mainly designed to discriminate four bases only. Therefore, lesion detection remains a challenge due to massive varieties and less distinguishable readouts for structural variations at the molecular level. Moreover, standard amplification and labeling hardly work in DNA lesion detection. Herein, we designed a single molecule interface from the mutant aerolysin (K238Q), whose sensing region shows high compatibility to capture and then directly convert a minor lesion into distinguishable electrochemical readouts. Compared with previous single molecule sensing interfaces, the temporal resolution of the K238Q aerolysin nanopore is enhanced by two orders, which has the best sensing performance in all reported aerolysin nanopores. In this work, the novel K238Q could discriminate directly at least three types of lesions (mC, OG, I) without labeling and quantify modification sites under the mixed heterocomposition conditions of the oligonucleotide. Such a nanopore electrochemistry approach could be further applied to diagnose genetic diseases at high sensitivity.

Durable Response After Combination Of Concurrent Chemoradiotherapy And Anti-PD-1 Therapy In HER2-Negative Advanced Gastric Adenocarcinoma: A Case Report.

Advanced gastric cancer (AGC) is difficult to treat with poor prognosis. Despite various possible treatments, the median survival time of such patients remains unsatisfactory. Therefore, new therapeutic approaches or combinations need to be further explored. We herein reported a case of a 54-year-old male patient who was initially diagnosed with HER2-negative advanced gastric cancer. Based on previous studies and patient's desire, we made a therapeutic plan: the combination of concurrent radiochemotherapy and immune checkpoint blockade therapy. After about 4 months of combined therapy, the patient showed satisfactory complete response to tumor lesions even metastatic lesions (CR, disappearance of all target lesions). In summary, the combination of concurrent SOX regimen chemotherapy, stomach radiotherapy and PD-1 antibody immunotherapy is effective in the treatment of advanced gastric cancer.

α-Ketoglutarate-Activated NF-κB Signaling Promotes Compensatory Glucose Uptake and Brain Tumor Development.

The rapid proliferation of cancer cells and dysregulated vasculature within the tumor leads to limited nutrient accessibility. Cancer cells often rewire their metabolic pathways for adaption to nutrient stress, and the underlying mechanism remains largely unknown. Glutamate dehydrogenase 1 (GDH1) is a key enzyme in glutaminolysis that converts glutamate to α-ketoglutarate (α-KG). Here, we show that, under low glucose, GDH1 is phosphorylated at serine (S) 384 and interacts with RelA and IKKß. GDH1-produced α-KG directly binds to and activates IKKß and nuclear factor κB (NF-κB) signaling, which promotes glucose uptake and tumor cell survival by upregulating GLUT1, thereby accelerating gliomagenesis. In addition, GDH1 S384 phosphorylation correlates with the malignancy and prognosis of human glioblastoma. Our finding reveals a unique role of α-KG to directly regulate signal pathway, uncovers a distinct mechanism of metabolite-mediated NF-κB activation, and also establishes the critical role of α-KG-activated NF-κB in brain tumor development.

Efficacy of apatinib on multiple advanced-stage nongastric cancers.

BACKGROUND: Apatinib has been approved for the treatment of advanced gastric adenocarcinoma and gastric-esophageal junctional adenocarcinoma, but its efficacy is unknown for other advanced solid tumors. AIMS AND OBJECTIVES: We retrospectively reviewed the use of apatinib for multiple advanced-stage non-gastric cancers. Ninety-two patients from 7 hospitals who received additional treatment except apatinib more than once were enrolled. MATERIALS AND METHODS: The primary end-point was the overall response rate (ORR), and the secondary end-points included progression-free survival (PFS), disease control rate (DCR), overall survival, and adverse reactions. We categorized all the patients into six groups according to their cancer type. RESULTS: In the lung cancer group, the ORR was 9% (95% confidence interval [CI], 3%-23%), DCR was 88% (95% CI, 74%-96%), and median PFS was 3 months (95% CI, 1.9-5.4 months). In the cervical cancer group, the ORR was 25% (95% CI, 3%-65%), DCR reached 100%, and median PFS was 3.5 months (95% CI, 0.6-9.0 months). There were different ORRs between the other cancer groups. In addition, the most common adverse effect of apatinib was palmar-plantar erythrodysesthesia syndrome (37%), followed by proteinuria (14%) and hypertension (13%). CONCLUSION: These results suggest that apatinib might be effective for not only gastric cancer but also other carcinomas including lung cancer, colorectal cancer, cervical cancer, liver cancer, breast cancer, and nasopharyngeal cancer. Thus, apatinib is a promising targeted drug for multiple types of cancer.

Simultaneous single-molecule discrimination of cysteine and homocysteine with a protein nanopore.

Discrimination between cysteine and homocysteine at the single-molecule level is achieved within a K238Q mutant aerolysin nanopore, which provides a confined space for high spatial resolution to identify the amino acid difference with a 5'-benzaldehyde poly(dA)4 probe. Our strategy allows potential detection and characterization of various amino acids and their modifications, and provides a crucial step towards developing nanopore protein sequencing devices.