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.

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.

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

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.

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.

Identification of Essential Sensitive Regions of the Aerolysin Nanopore for Single Oligonucleotide Analysis.

The aerolysin nanopore channel is one of the confined spaces for single molecule analysis which displays high spatial and temporal resolution for the discrimination of single nucleotides, identification of DNA base modification, and analyzing the structural transition of DNAs. However, to overcome the challenge of achieving the ultimate goal of the widespread real analytical application, it is urgent to probe the sensing regions of the aerolysin to further improve the sensitivity. In this paper, we explore the sensing regions of the aerolysin nanopore by a series of well-designed mutant nanopore experiments combined with molecular dynamics simulations-based electrostatic analysis. The positively charged lumen-exposed Lys-238, identified as one of the key sensing sites due to the presence of a deep valley in the electrostatic potentials, was replaced by different charged and sized amino acids. The results show that the translocation time of oligonucleotides through the nanopore can be readily modulated by the choice of the target amino acid at the 238 site. In particular, a 7-fold slower translocation at a voltage bias of +120 mV is observed with respect to the wild-type aerolysin, which provides a high resolution for methylated cytosine discrimination. We further determine that both the electrostatic properties and geometrical structure of the aerolysin nanopore are crucial to its sensing ability. These insights open ways for rationally designing the sensing mechanism of the aerolysin nanopore, thus providing a novel paradigm for nanopore sensing.

[Association of FasL-844T/C gene polymorphism with FasL expression in the nucleus pulposus of degenerative lumbar intervertebral discs].

OBJECTIVE: To investigate the association of FasL-844T/C gene polymorphism with the magnetic resonance imaging (MRI) findings and FasL expression in the nucleus pulposus of degenerative lumbar intervertebral discs. METHODS: Lumbar MRI data, venous blood and nucleus pulposus were collected from 105 patients with lumbar disc herniation. The genotypes of FasL-844T/C gene of the patients were determined using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Immunohistochemistry was used to detect the expression of FasL in the nucleus pulposus of the degenerative lumbar intervertebral discs. RESULTS: Compared with CC genotype, TT genotype of FasL-844T/C gene was associated with a significantly increased score of lumbar disc degeneration (P=0.003) as observed in MRI scan. FasL expression in the nucleus pulposus differed significantly between patients of FASL-844CC genotype and those of FASL-844TT genotype (P=0.048), but not between those of FASL-844CC and FASL-844CT genotypes (P=0.264). No significant association was found between MRI findings and FasL expression in the nucleus pulposus of the lumbar intervertebral discs. CONCLUSION: FasL-844T/C gene polymorphism is correlated with the expression of FasL in the nucleus pulposus of the intervertebral disc in patients with lumbar disc herniation. MRI findings of the lumbar intervertebral discs do not correlate with the expression of FasL in the nucleus pulposus of the intervertebral discs.

Hypoxia-induced apoptosis is blocked by adrenomedullin via upregulation of Bcl-2 in human osteosarcoma cells.

Adrenomedullin (ADM), a multifunctional regulatory peptide, is potentially induced by hypoxia in physiological and pathological tissues, including many types of malignant tumors. Recent research has demonstrated that ADM expression is highly associated with the prognosis and disease severity of human osteosarcoma. However, the effect of ADM on the apoptosis of osteosarcoma cells and its possible mechanism remain to be elucidated. In the present study, we observed that mRNA and protein levels of ADM were increased in human osteosarcoma SOSP-F5M2 cells under a hypoxic microenvironment induced by cobalt chloride (CoCl2) in a time-dependent manner. Treatment with ADM significantly blunted hypoxic-induced apoptosis, evaluated by Hoechst 33342 staining and Annexin V-FITC/PI labeling. The expression of B-cell lymphoma-2 (Bcl-2) was increased by administration of ADM; meanwhile, this effect was reversed by exogenously adding U0126, a selective inhibitor of MEK or ADM22-52 (ADM-specific receptor antagonist). These results demonstrated that ADM acted as a survival factor to inhibit hypoxic-induced apoptosis via interacting with its receptors CRLR-RAMP (2,3) in osteosarcoma cells. The anti-apoptotic function of ADM was found to be mediated by upregulation of the expression of Bcl-2 partially through activation of the MEK/ERK1/2 signaling pathway. Therefore, targeting of the ADM/ADM acceptors/ERK1/2/Bcl-2 pathway may provide a potential strategy through which to induce the apoptosis of osteosarcoma cells.

Mechanisms of tumor resistance to small-molecule vascular disrupting agents: treatment and rationale of combination therapy.

Small-molecule vascular disrupting agents (VDAs) target the established tumor blood vessels, resulting in rapidly and selectively widespread ischemia and necrosis of central tumor; meanwhile, blood flow in normal tissues is relatively unaffected. Although VDAs therapy is considered an important option for treatment, its use is still limited. The tumor cells at the periphery are less sensitive to vascular shutdown than those at the center, and subsequently avoid a nutrient-deprived environment. This phenomenon is referred to as tumor resistance to VDAs treatment. The viable periphery rim of tumor cells contributes to tumor regeneration, metastasis, and ongoing progression. However, there is no systematic review of the plausible mechanisms of repopulation of the viable tumor cells following VDAs therapy. The purpose of this review is to provide insights into mechanisms of tumor surviving small-molecule VDAs therapy, and the synergetic treatment to the remaining viable tumor cells at the periphery.

Posterior lumbar ring apophysis fracture.

Lumbar posterior ring apophysis fracture (PRAF) is an uncommon cause of low back pain in the pediatric age group, and a detailed understanding of this disease is important for the orthopaedic surgeon because it is easily misdiagnosed. However, to date no comprehensive review of PRAF has been published. The majority of published reports are in the form of cases report generally targeted at either diagnosis or therapy, or both. In this essay, we comprehensively review the pathogenesis, clinical presentation, diagnosis and treatment of PRAF.