Unwinding and spiral sliding of S4 and domain rotation of VSD during the electromechanical coupling in Nav1.7.

Voltage-gated sodium (Nav) channel Nav1.7 has been targeted for the development of nonaddictive pain killers. Structures of Nav1.7 in distinct functional states will offer an advanced mechanistic understanding and aid drug discovery. Here we report the cryoelectron microscopy analysis of a human Nav1.7 variant that, with 11 rationally introduced point mutations, has a markedly right-shifted activation voltage curve with V1/2 reaching 69 mV. The voltage-sensing domain in the first repeat (VSDI) in a 2.7-Å resolution structure displays a completely down (deactivated) conformation. Compared to the structure of WT Nav1.7, three gating charge (GC) residues in VSDI are transferred to the cytosolic side through a combination of helix unwinding and spiral sliding of S4I and ∼20° domain rotation. A conserved WNФФD motif on the cytoplasmic end of S3I stabilizes the down conformation of VSDI. One GC residue is transferred in VSDII mainly through helix sliding. Accompanying GC transfer in VSDI and VSDII, rearrangement and contraction of the intracellular gate is achieved through concerted movements of adjacent segments, including S4-5I, S4-5II, S5II, and all S6 segments. Our studies provide important insight into the electromechanical coupling mechanism of the single-chain voltage-gated ion channels and afford molecular interpretations for a number of pain-associated mutations whose pathogenic mechanism cannot be revealed from previously reported Nav structures.

Structural basis for high-voltage activation and subtype-specific inhibition of human Nav1.8.

The dorsal root ganglia-localized voltage-gated sodium (Nav) channel Nav1.8 represents a promising target for developing next-generation analgesics. A prominent characteristic of Nav1.8 is the requirement of more depolarized membrane potential for activation. Here we present the cryogenic electron microscopy structures of human Nav1.8 alone and bound to a selective pore blocker, A-803467, at overall resolutions of 2.7 to 3.2 Å. The first voltage-sensing domain (VSDI) displays three different conformations. Structure-guided mutagenesis identified the extracellular interface between VSDI and the pore domain (PD) to be a determinant for the high-voltage dependence of activation. A-803467 was clearly resolved in the central cavity of the PD, clenching S6IV. Our structure-guided functional characterizations show that two nonligand binding residues, Thr397 on S6I and Gly1406 on S6III, allosterically modulate the channel's sensitivity to A-803467. Comparison of available structures of human Nav channels suggests the extracellular loop region to be a potential site for developing subtype-specific pore-blocking biologics.

[Effect of needle knife on mTOR/Atg/ULK1/Beclin-1 axis and chondrocyte autophagy in rats with knee osteoarthritis].

OBJECTIVE: To observe the effect of needle knife on chondrocyte autophagy and expressions of autophagy-related protein and mammalian target of rapamycin (mTOR) in rats with knee osteoarthritis (KOA), and to explore the possible mechanism of needle knife for KOA. METHODS: A total of 42 SD rats were randomly divided into a normal group, a model group and a needle knife group, 14 rats in each group. Except for the normal group, the other two groups were injected with the mixture of papain and L-cysteine into the left hind knee joint to establish the KOA model. After modeling, the rats in the needle knife group were treated with needle knife at strip or nodule around the quadriceps femoris and medial and lateral collateral ligament on the affected side, once a week for 3 times (3 weeks). The changes of left knee circumference in each group were observed; the chondrocytes and ultrastructure of left knee joint were observed by HE staining and electron microscope; the mRNA and protein expressions of autophagy-related genes (Atg5, Atg12, Atg4a), Unc-51 like autophagy activated kinase 1 (ULK1), autophagy gene Beclin-1 and mTOR in left knee cartilage were detected by real-time fluorescence quantitative PCR and Western blot. RESULTS: After modeling, the left knee circumferences in the model group and the needle knife group were increased compared with those before modeling and in the normal group (P<0.05); after intervention, the left knee circumference in the needle knife group was smaller than that in the model group and after modeling (P<0.05). Compared with the normal group, the number of chondrocytes was decreased, and a few cells swelled, nuclei shrank, mitochondria swelled and autophagosomes decreased in the model group; compared with the model group, the number of chondrocytes was increased , and most cell structures returned to normal, and autophagosomes was increased. Compared with the normal group, the mRNA and protein expressions of Atg5, Atg12, Atg4a, Beclin-1 and ULK1 in the knee cartilage in the model group were decreased (P<0.05); compared with the model group, the expressions of the above indexes in the needle knife group were increased (P<0.05). Compared with the normal group, the mRNA and protein expressions of mTOR in the knee cartilage in the model group were increased (P<0.05); compared with the model group, the expressions of the above indexes in the needle knife group were decreased (P<0.05). CONCLUSION: The needle knife intervention could improve knee cartilage injury in rats with KOA, and its mechanism may be related to reducing the expression of mTOR and up-regulating the expressions of Atg5, Atg12, Atg4a, ULK1 and Beclin-1, so as to promote chondrocyte autophagy and delay the aging and degeneration of chondrocytes.

MBFFNet: Multi-Branch Feature Fusion Network for Colonoscopy.

Colonoscopy is currently one of the main methods for the detection of rectal polyps, rectal cancer, and other diseases. With the rapid development of computer vision, deep learning-based semantic segmentation methods can be applied to the detection of medical lesions. However, it is challenging for current methods to detect polyps with high accuracy and real-time performance. To solve this problem, we propose a multi-branch feature fusion network (MBFFNet), which is an accurate real-time segmentation method for detecting colonoscopy. First, we use UNet as the basis of our model architecture and adopt stepwise sampling with channel multiplication to integrate features, which decreases the number of flops caused by stacking channels in UNet. Second, to improve model accuracy, we extract features from multiple layers and resize feature maps to the same size in different ways, such as up-sampling and pooling, to supplement information lost in multiplication-based up-sampling. Based on mIOU and Dice loss with cross entropy (CE), we conduct experiments in both CPU and GPU environments to verify the effectiveness of our model. The experimental results show that our proposed MBFFNet is superior to the selected baselines in terms of accuracy, model size, and flops. mIOU, F score, and Dice loss with CE reached 0.8952, 0.9450, and 0.1602, respectively, which were better than those of UNet, UNet++, and other networks. Compared with UNet, the flop count decreased by 73.2%, and the number of participants also decreased. The actual segmentation effect of MBFFNet is only lower than that of PraNet, the number of parameters is 78.27% of that of PraNet, and the flop count is 0.23% that of PraNet. In addition, experiments on other types of medical tasks show that MBFFNet has good potential for general application in medical image segmentation.

Comparative structural analysis of human Nav1.1 and Nav1.5 reveals mutational hotspots for sodium channelopathies.

Among the nine subtypes of human voltage-gated sodium (Nav) channels, the brain and cardiac isoforms, Nav1.1 and Nav1.5, each carry more than 400 missense mutations respectively associated with epilepsy and cardiac disorders. High-resolution structures are required for structure-function relationship dissection of the disease variants. We report the cryo-EM structures of the full-length human Nav1.1-ß4 complex at 3.3 Å resolution here and the Nav1.5-E1784K variant in the accompanying paper. Up to 341 and 261 disease-related missense mutations in Nav1.1 and Nav1.5, respectively, are resolved. Comparative structural analysis reveals several clusters of disease mutations that are common to both Nav1.1 and Nav1.5. Among these, the majority of mutations on the extracellular loops above the pore domain and the supporting segments for the selectivity filter may impair structural integrity, while those on the pore domain and the voltage-sensing domains mostly interfere with electromechanical coupling and fast inactivation. Our systematic structural delineation of these mutations provides important insight into their pathogenic mechanism, which will facilitate the development of precise therapeutic interventions against various sodium channelopathies.

Mechanism of Action of Acupotomy in Inhibiting Chondrocyte Apoptosis in Rabbits with KOA through the PI3K/Akt Signaling Pathway.

OBJECTIVE: We examined the effects of acupotomy on the PI3K/Akt signaling pathway to elucidate the mechanism of action of acupotomy on articular chondrocyte apoptosis among rabbits with knee osteoarthritis (KOA). METHODS: New Zealand rabbits were randomly assigned to a healthy control group, placebo group, acupotomy group, and drug group, with 10 rabbits in each group. Changes in chondrocytes were examined by hematoxylin and eosin staining, and articular chondrocyte apoptosis was measured by electron microscopy and immunofluorescence. The mRNA and protein expression levels of PI3K and Akt were measured by real-time quantitative PCR and Western blot. RESULTS: In contrast, less chromatin margination and clear and smooth nuclear envelope boundary were visible in the acupotomy group and drug group. The number of apoptotic chondrocytes in the knee joint of rabbits was significantly higher in the placebo group than that in the acupotomy group and drug group (P < 0.05). The acupotomy group had a nonsignificantly lower number of apoptotic chondrocytes than the drug group (P > 0.05). Furthermore, the mRNA and protein expression levels of PI3K and Akt were significantly higher in the acupotomy group and drug group than those in the placebo group (P < 0.05) and were closer to normal levels in the acupotomy group than those in the drug group (P < 0.05). PI3K and Akt expression levels were negatively correlated with chondrocyte apoptosis in the knee joint of rabbits in all groups. CONCLUSION: Inhibiting chondrocyte apoptosis in the knee joint of KOA rabbits by upregulating the PI3K/Akt signaling pathway may be a possible mechanism of acupotomy in treating KOA.

Role of the PERK-eIF2α-CHOP Signaling Pathway in the Effect of Needle Knife Therapy on Knee Joint Chondrocyte Apoptosis.

Needle knife therapy, a form of acupuncture and moxibustion, has been widely used in the clinical treatment of knee osteoarthritis (KOA). However, the mechanism is not clear. Therefore, we studied the mechanisms of action of needle knife intervention on KOA in rabbits, with the PERK-eIF2α-CHOP pathway as a starting point, in order to determine the mechanism underlying knee joint chondrocyte apoptosis. Apoptosis and ultrastructural changes in the articular cartilage were examined by pathological study and transmission electron microscopy, and PERK, eIF2α, and CHOP mRNA and protein levels were detected by qRT-PCR and western blot, respectively. PERK, eIF2α, and CHOP protein levels were significantly higher in the model group than in the normal group (P < 0.01) and were considerably downregulated in the needle knife and the medicine groups compared to the model group (P < 0.01). The eIF2α, p-eIF2α, and CHOP protein levels were not significantly different between the needle knife and medicine groups. The PERK, eIF2α, and CHOP mRNA levels in the drug group were higher than those in the needle knife group (P < 0.01). Needle knife therapy can regulate PERK-eIF2α-CHOP signaling pathway, which could be one of the mechanisms by which it affects chondrocyte apoptosis in KOA rabbits.

Molecular basis for pore blockade of human Na+ channel Nav1.2 by the µ-conotoxin KIIIA.

The voltage-gated sodium channel Nav1.2 is responsible for the initiation and propagation of action potentials in the central nervous system. We report the cryo-electron microscopy structure of human Nav1.2 bound to a peptidic pore blocker, the µ-conotoxin KIIIA, in the presence of an auxiliary subunit, ß2, to an overall resolution of 3.0 angstroms. The immunoglobulin domain of ß2 interacts with the shoulder of the pore domain through a disulfide bond. The 16-residue KIIIA interacts with the extracellular segments in repeats I to III, placing Lys7 at the entrance to the selectivity filter. Many interacting residues are specific to Nav1.2, revealing a molecular basis for KIIIA specificity. The structure establishes a framework for the rational design of subtype-specific blockers for Nav channels.

Structure of the human voltage-gated sodium channel Nav1.4 in complex with ß1.

Voltage-gated sodium (Nav) channels, which are responsible for action potential generation, are implicated in many human diseases. Despite decades of rigorous characterization, the lack of a structure of any human Nav channel has hampered mechanistic understanding. Here, we report the cryo-electron microscopy structure of the human Nav1.4-ß1 complex at 3.2-Å resolution. Accurate model building was made for the pore domain, the voltage-sensing domains, and the ß1 subunit, providing insight into the molecular basis for Na+ permeation and kinetic asymmetry of the four repeats. Structural analysis of reported functional residues and disease mutations corroborates an allosteric blocking mechanism for fast inactivation of Nav channels. The structure provides a path toward mechanistic investigation of Nav channels and drug discovery for Nav channelopathies.