Ginsenoside Rg1 attenuates lipopolysaccharide-induced chronic liver damage by activating Nrf2 signaling and inhibiting inflammasomes in hepatic cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginseng (Panax ginseng C. A. Meyer) is a precious traditional Chinese medicine with multiple pharmacological effects. Ginsenoside Rg1 is a main active ingredient extracted from ginseng, which is known for its age-delaying and antioxidant effects. Increasing evidence indicates that Rg1 exhibits anti-inflammatory properties in numerous diseases and may ameliorate oxidative damage and inflammation in many chronic liver diseases. AIM OF THE STUDY: Chronic inflammatory injury in liver cells is an important pathological basis of many liver diseases. However, its mechanism remains unclear and therapeutic strategies to prevent its development need to be further explored. Thus, our study is to delve the protective effect and mechanism of Rg1 against chronic hepatic inflammatory injuries induced by lipopolysaccharide (LPS). MATERIALS AND METHODS: The chronic liver damage model in mice was build up by injecting intraperitoneally with LPS (200 µg/kg) for 21 days. Serum liver function indicators and levels of IL-1ß, IL-6 and TNF-α were examined by using corresponding Kits. Hematoxylin and Eosin (H&E), Periodic acid-Schiff (PAS), and Masson stains were utilized to visualize hepatic histopathological damage, glycogen deposition, and liver fibrosis. The nuclear import of p-Nrf2 and the generation of Col4 in the liver were detected by IF, while IHC was employed to detect the expressions of NLRP3 and AIM2 in the hepatic. The Western blot and q-PCR were used to survey the expressions of proteins and mRNAs of fibrosis and apoptosis, and the expressions of Keap1, p-Nrf2 and NLRP3, NLRP1, AIM2 inflammasome-related proteins in mouse liver. The cell viability of human hepatocellular carcinoma cells (HepG2) was detected by Cell Counting Kit-8 to select the action concentration of LPS, and intracellular ROS generation was detected using a kit. The expressions of Nuclear Nrf2, HO-1, NQO1 and NLRP3, NLRP1, and AIM2 inflammasome-related proteins in HepG2 cells were detected by Western blot. Finally, the feasibility of the molecular interlinking between Rg1 and Nrf2 was demonstrated by molecular docking. RESULTS: Rg1 treatment for 21 days decreased the levels of ALT, AST, and inflammatory factors of serum IL-1ß, IL-6 and TNF-α in mice induced by LPS. Pathological results indicated that Rg1 treatment obviously alleviated hepatocellular injury and apoptosis, inflammatory cell infiltration and liver fibrosis in LPS stimulated mice. Rg1 promoted Keap1 degradation and enhanced the expressions of p-Nrf2, HO-1 and decreased the levels of NLRP1, NLRP3, AIM2, cleaved caspase-1, IL-1ß and IL-6 in livers caused by LPS. Furthermore, Rg1 effectively suppressed the rise of ROS in HepG2 cells induced by LPS, whereas inhibition of Nrf2 reversed the role of Rg1 in reducing the production of ROS and NLRP3, NLRP1, and AIM2 expressions in LPS-stimulated HepG2 cells. Finally, the molecular docking illustrated that Rg1 exhibits a strong affinity towards Nrf2. CONCLUSION: The findings indicate that Rg1 significantly ameliorates chronic liver damage and fibrosis induced by LPS. The mechanism may be mediated through promoting the dissociation of Nrf2 from Keap1 and then activating Nrf2 signaling and further inhibiting NLRP3, NLRP1, and AIM2 inflammasomes in liver cells.

Repair of cervicothoracic skin defects with extra-long transverse cervical flaps by stepwise pressure packing in children: a technical innovation.

Objective: To investigate the clinical effect of prolonging predilated transverse cervical flap with stepwise pressure packing for neck and chest lesions in children. Methods: A retrospective review of children with large cervicothoracic lesions admitted to our department from January 2011 to June 2021 was conducted to compare stepwise pressure packing with normal dressing in the surgical method of transverse cervical pedicled flaps after expansion. Among 58 included children, 22 (14 males and 8 females) were allocated to the extended and expanded transverse cervical flap with stepwise compression dressing group, and 36 (19 males and 17 females) to the transverse cervical flap group. The causes of skin defects were: scars (37 cases) and giant nevus (21 cases). The course of the disease ranged from 0.5 to 8 years. The two groups were compared in terms of child satisfaction, the occurrence of infection, recurrence of the contracture, secondary operation, and repaired area. Results: In 22 cases of extended transverse cervical flaps, 8 cases were embedded with two expanders, resulting in a total of 30 expanded flaps, which were successfully transferred to the neck and chest without necrosis at the distal end of compression, with good effect. Comparison of pedicled transverse cervical flaps with stepwise pressure packing and pedicled transverse cervical flaps alone revealed no significant difference in child satisfaction, the occurrence of infection, recurrence of the contracture, and secondary surgery (all P > 0.05). Yet, there was a significant difference in the repair area between two groups (P < 0.05). Conclusion: Prolongation of pedicled cervical flaps after expansion with stepwise pressure packing resulted in an effective method for repairing the large skin defect of children's face and neck caused by various diseases. In terms of increasing neck repair area, the operation with stepwise pressure dressing was significantly superior to the simple packing.

Posterior Cranial Retraction Combined With Bilateral Parietal Distraction for Children With Nonsyndromic Craniosynostosis.

BACKGROUND: The nonsyndromic craniosynostosis is the most common of craniosynostoses in childhood. There are many treatments. We aim to treat 12 cases of nonsyndromic craniosynostosis via posterior cranial vault distraction osteogenesis combined with bilateral parietal distraction. METHODS: Data of a total of 12 patients (7 boys and 5 girls) with nonsyndromic sagittal synostosis who underwent distraction osteogenesis between January 2015 and August 2020 were retrospectively analyzed. Bilateral parietal bone flaps and posterior occipital flaps were designed and cut. Then, distraction device was placed, which was distracted at 5 days after surgery (twice per day, 0.4-0.6 mm/d, and lasting for 10-15 days). After 6 months of fixation, the secondary surgery was performed to remove the device. RESULTS: The scaphocephaly was corrected, and the appearance was satisfactory. Postoperative follow-up time was 6 to 14 months, with an average of 10 months, and the mean CI was 63.2 and 78.25 before and after surgery, respectively; the mean anterior-posterior skull diameter was shortened (12.63 ± 3.47) mm, the transverse diameter of both temporal regions was lengthened (15.4 ± 4.18) mm, and the scaphocephalic deformity was significantly improved. There was no detachment or rupture of the extender postoperatively. No severe complications, such as radiation necrosis or intracranial infection, were observed. CONCLUSION: Posterior cranial retraction combined with bilateral parietal distraction in children with nonsyndromic craniosynostosis, in which the proposed technique did not exhibit severe complications, and it is worthy of further promotion and application in clinical practice.

Treated Pierre Robin Sequence Using Placed Allogenic Acellular Bone Matrix and Mandibular Distraction Osteogenesis in the Neonate.

Objective: The aim of the study was to report our experience with placed allogenic acellular bone matrix and mandibular distraction osteogenesis in Pierre Robin sequence (PRS), and explore the role of distraction in the osteogenesis of acellular bone. Materials and Methods: A total of 428 neonates with severe PRS managed with placing allogenic acellular bone and bilateral mandibular distraction osteogenesis were included in the study. The procedure included using oblique-shaped osteotomy, fixing bilateral mandibular distractor, instantly extending a 4-6 mm gap, and placing allogenic acellular bone into the gap. The length of allogenic acellular bone was 4-5 mm. Although the surgical techniques, distraction, and consolidation periods were similar, the allogenic acellular bone matrix we placed was quite different from the traditional distraction. With the technology we used, tracheal intubation could be immediately removed, thus quickly improving breathing conditions compared to traditional methods after the surgery. The jaw extending and oral feeding could begin on the 5th day. The jaw was extended 0.6 mm twice a day until the mandible was overcorrected by 20%. Results: All 428 cases included in this study were successfully extubated after the operation, and the difficulty in breathing was instantly relieved. Total mandibular distraction was 15-20 mm. Oral feeding was started at 6 h to 6 days postoperatively, while hospital stay ranged from 18 to 20 days postoperatively. No major complications were reported. Medium to long-term results was good. Mandibular distractors were removed after 3 months. Conclusions: Bilateral mandibular distraction osteogenesis combined with placing allogenic acellular bone in the neonate are safe and accurate procedures, which are the primary treatment options for cases of severe PRS. It can be considered that the tension of distraction can promote osteogenesis in acellular bone and thus improve distractive effect of the mandible.

Celecoxib induces adipogenic differentiation of hemangioma-derived mesenchymal stem cells through the PPAR-γ pathway in vitro and in vivo.

Infantile hemangioma (IH) is a benign tumor that produces a permanent scar or a mass of fibro-fatty tissue after involution in 40-80% of cases. Celecoxib is an inhibitor of cyclooxygenase-2 (COX-2), and can inhibit angiogenesis and fibrosis. The present study aimed to clarify whether celecoxib is able to induce tumor regression with minimal side effects. For that purpose, the regulation of celecoxib in the involution of IH was investigated in an IH model. Hemangioma-derived mesenchymal stem cells (Hem-MSCs) were isolated from proliferating specimens, and an IH model was established by injecting these cells into nude mice. Celecoxib was administered in vitro and in vivo. Oil Red O staining and reverse transcription-quantitative-PCR were used to detect the adipogenic differentiation of Hem-MSCs. Histologic analysis and immunohistochemical staining of the tumor xenografts were performed to investigate the pathological evolution of the tumor. The results showed that celecoxib inhibited the proliferation and induced the adipogenic differentiation of Hem-MSCs in vitro. In vivo, adipocytes were only present in the celecoxib group at week 4, while a larger number of fibroblasts and collagenous fibers could be observed in the basic fibroblast growth factor group. Therefore, celecoxib may be a potential agent used for IH treatment by inducing adipogenesis and inhibiting fibroblast formation.

Tethered peptide activation mechanism of the adhesion GPCRs ADGRG2 and ADGRG4.

Adhesion G protein-coupled receptors (aGPCRs) constitute an evolutionarily ancient family of receptors that often undergo autoproteolysis to produce α and ß subunits1-3. A tethered agonism mediated by the 'Stachel sequence' of the ß subunit has been proposed to have central roles in aGPCR activation4-6. Here we present three cryo-electron microscopy structures of aGPCRs coupled to the Gs heterotrimer. Two of these aGPCRs are activated by tethered Stachel sequences-the ADGRG2-ß-Gs complex and the ADGRG4-ß-Gs complex (in which ß indicates the ß subunit of the aGPCR)-and the other is the full-length ADGRG2 in complex with the exogenous ADGRG2 Stachel-sequence-derived peptide agonist IP15 (ADGRG2(FL)-IP15-Gs). The Stachel sequences of both ADGRG2-ß and ADGRG4-ß assume a U shape and insert deeply into the seven-transmembrane bundles. Constituting the FXφφφXφ motif (in which φ represents a hydrophobic residue), five residues of ADGRG2-ß or ADGRG4-ß extend like fingers to mediate binding to the seven-transmembrane domain and activation of the receptor. The structure of the ADGRG2(FL)-IP15-Gs complex reveals the structural basis for the improved binding affinity of IP15 compared with VPM-p15 and indicates that rational design of peptidic agonists could be achieved by exploiting aGPCR-ß structures. By converting the 'finger residues' to acidic residues, we develop a method to generate peptidic antagonists towards several aGPCRs. Collectively, our study provides structural and biochemical insights into the tethered activation mechanism of aGPCRs.

Catalytic trajectory of a dimeric nonribosomal peptide synthetase subunit with an inserted epimerase domain.

Nonribosomal peptide synthetases (NRPSs) are modular assembly-line megaenzymes that synthesize diverse metabolites with wide-ranging biological activities. The structural dynamics of synthetic elongation has remained unclear. Here, we present cryo-EM structures of PchE, an NRPS elongation module, in distinct conformations. The domain organization reveals a unique "H"-shaped head-to-tail dimeric architecture. The capture of both aryl and peptidyl carrier protein-tethered substrates and intermediates inside the heterocyclization domain and L-cysteinyl adenylate in the adenylation domain illustrates the catalytic and recognition residues. The multilevel structural transitions guided by the adenylation C-terminal subdomain in combination with the inserted epimerase and the conformational changes of the heterocyclization tunnel are controlled by two residues. Moreover, we visualized the direct structural dynamics of the full catalytic cycle from thiolation to epimerization. This study establishes the catalytic trajectory of PchE and sheds light on the rational re-engineering of domain-inserted dimeric NRPSs for the production of novel pharmaceutical agents.

Treatment of Syndromic Craniosynostosis by Anterior and Posterior Vault Distraction Osteogenesis (A-PVDO).

OBJECTIVE: To explore the feasibility and therapeutic effect of anterior and posterior vault distraction osteogenesis (A-PVDO) in the treatment of infantile syndromic craniosynostosis. METHODS: Between January 2017 and December 2019, 7 children with syndromic craniosynostosis underwent osteotomy with Piezo-surgery at our hospital. The first step was to harvest the frontal bone flap and the orbitofrontal bone flap. The second step was to separate the scalp and expose the posterior occipital. Osteotomy was performed on the occipital tubercle. Thereafter, 2 distractors were horizontally installed on the upper edge of the anterior cranial orbit, 2 distractors of 3 cm were installed on the posterior cranial bone. Meanwhile, lambdoidal sutures were fixed by titanium plates. Bone distraction was initiated on postoperative day 5 at the rate of 0.4 to 0.6 mm/day, twice per-day, for a total of 10 to 15 days. After 6 months, the distractors and the titanium plates were removed by secondary surgery. RESULTS: The intracranial volume and posterior cranial morphology were recorded during the follow-up of 6 to 14 months (average = 12 months). The posterior craniums of 7 cases with lambdoidal sutures fixation were completely extended. The anterior cranial morphology was normal. All the cranial deformities were significantly improved. There were no severe complications, such as death, cranial necrosis, and intracranial infection. CONCLUSIONS: A-PVDO is an ideal method for the treatment of severe syndromic craniosynostosis, which can achieve more natural appearance than anterior vault distraction osteogenesis or posterior vault distraction osteogenesis. Moreover, A-PVDO causes no severe complications and is suitable for the treatment of severe syndromic craniosynostosis.

Mitochondrial Voltage-Dependent Anion Channel 1-Hexokinase-II Complex-Targeted Strategy for Melanoma Inhibition Using Designed Multiblock Peptide Amphiphiles.

Targeted therapies of melanoma are of urgent need considering the resistance of this aggressive type of cancer to chemotherapeutics. The voltage-dependent anion channel 1 (VDAC1)-hexokinase-II (HK-II) complex is an emerging target for novel anticancer therapies based on induced mitochondria-mediated apoptosis. The low cell membrane permeability of the anticancer 12-mer peptide N-Ter (RDVFTKGYGFGL) derived from the N-terminal fragment of the VDAC1 protein impedes the intracellular targeting. Here, novel multiblock VDAC1-derived cationic amphiphilic peptides (referred to as Pal-N-Ter-TAT, pFL-N-Ter-TAT, and Pal-pFL-N-Ter-TAT) are designed with a self-assembly propensity and cell-penetrating properties. The created multiblock amphiphilic peptides of partial α-helical conformations form nanoparticles of ellipsoid-like shapes and are characterized by enhanced cellular uptake. The amphiphilic peptides can target mitochondria and dissociate the VDAC1-HK-II complex at the outer mitochondrial membrane, which result in mitochondria-mediated apoptosis. The latter is associated with decrease of the mitochondrial membrane potential, cytochrome c release, and changes of the expression levels of the apoptotic proteins in A375 melanoma cells. Importantly, the mitochondrial VDAC1-derived amphiphilic peptides have a comparable IC50 value for melanoma cells to a small-molecule drug, sorafenib, which has been previously used in clinical trials for melanoma. These results demonstrate the potential of the designed peptide constructs for efficient melanoma inhibition.

Conformational dynamics of SARS-CoV-2 trimeric spike glycoprotein in complex with receptor ACE2 revealed by cryo-EM.

The recent outbreaks of SARS-CoV-2 pose a global health emergency. The SARS-CoV-2 trimeric spike (S) glycoprotein interacts with the human ACE2 receptor to mediate viral entry into host cells. We report the cryo-EM structures of a tightly closed SARS-CoV-2 S trimer with packed fusion peptide and an ACE2-bound S trimer at 2.7- and 3.8-Å resolution, respectively. Accompanying ACE2 binding to the up receptor-binding domain (RBD), the associated ACE2-RBD exhibits continuous swing motions. Notably, the SARS-CoV-2 S trimer appears much more sensitive to the ACE2 receptor than the SARS-CoV S trimer regarding receptor-triggered transformation from the closed prefusion state to the fusion-prone open state, potentially contributing to the superior infectivity of SARS-CoV-2. We defined the RBD T470-T478 loop and Y505 as viral determinants for specific recognition of SARS-CoV-2 RBD by ACE2. Our findings depict the mechanism of ACE2-induced S trimer conformational transitions from the ground prefusion state toward the postfusion state, facilitating development of anti-SARS-CoV-2 vaccines and therapeutics.

Sirolimus for Kaposiform Hemangioendothelioma With Kasabach-Merritt Phenomenon in Two Infants.

Kaposiform hemangioendothelioma is an aggressive vascular tumor with infiltrative growth that commonly occurs in infancy and is associated with a life-threatening consumptive coagulopathy, as well as Kasabach-Merritt phenomenon. Recently, promising results have shown that sirolimus had been successfully used to treat Kasabach-Merritt phenomenon without significant toxicity. However, the situation the authors encountered in treating infants was not so satisfactory. Here, the authors present 2 patients younger than 3 months with refractory Kaposiform hemangioendothelioma treated with sirolimus and experienced severe pneumonia. The outcomes suggest that it is necessary to keep an eye on any symptoms indicate the infection of respiratory tract and use the antibiotics in time. The 2 cases also remind us of the potential sign that indicate the recurrence of KMP, which refers to firmer lesion with deepen color, especially when it comes with complications.

The Postoperative Morphometrics of Orbital and Maxillary Area for Craniosynostosis.

PURPOSE: One of the most characteristic features in premature craniosynostosis is fronto-orbital retrusion. The standardized surgical technique of fronto-orbital advancement (FOA) can treat this (some) deformity, such as bilateral coronal synostosis. The purpose of the study is to investigate an available method to assess the postoperative outcome of the craniofacial surgery. METHODS: From 2010 to 2015, 6 pediatric patients were taken the FOA in the Department of Burn and Plastic Surgery in the Children's Hospital of Nanjing Medical University. All the patients were performed the computed tomography (CT) scan preoperatively and postoperatively. The CT databases were processed by DICOM files into MIMICS 16.0 software, which were automatically calculated into orbital volume and orbital roof and base surface area. T-test was used to compare measured values before and after surgery. P < 0.05 was considered statistically significant. RESULTS: The average preoperative orbital volume was 13930.70 mm, and the postoperative was 18578.67917 mm. After operation, the volume of orbital was significantly increased (P < 0.05). The mean area of the orbital roof surface was 753.989025 mm preoperatively, and the postoperative was 1122.074583 mm. The difference was statistically significant (P < 0.05). The average area of the orbital base (S2) was 334.94 ±â€Š91.76 mm. After the FOA, the orbital base was 356.99 ±â€Š114.21 mm. P(S2) = 0.6072 > 0.05, there was no significant statistical difference. CONCLUSIONS: Fronto-orbital advancement can successfully improve morphological orbital deformities in children with premature craniosynostosis, but much less for maxillary. The computer-assisted technique can present a measurement of FOA preoperatively and postoperatively, which make the evaluation intuitive.

High-resolution cryo-EM structure of the proteasome in complex with ADP-AlFx.

The 26S proteasome is an ATP-dependent dynamic 2.5 MDa protease that regulates numerous essential cellular functions through degradation of ubiquitinated substrates. Here we present a near-atomic-resolution cryo-EM map of the S. cerevisiae 26S proteasome in complex with ADP-AlFx. Our biochemical and structural data reveal that the proteasome-ADP-AlFx is in an activated state, displaying a distinct conformational configuration especially in the AAA-ATPase motor region. Noteworthy, this map demonstrates an asymmetric nucleotide binding pattern with four consecutive AAA-ATPase subunits bound with nucleotide. The remaining two subunits, Rpt2 and Rpt6, with empty or only partially occupied nucleotide pocket exhibit pronounced conformational changes in the AAA-ATPase ring, which may represent a collective result of allosteric cooperativity of all the AAA-ATPase subunits responding to ATP hydrolysis. This collective motion of Rpt2 and Rpt6 results in an elevation of their pore loops, which could play an important role in substrate processing of proteasome. Our data also imply that the nucleotide occupancy pattern could be related to the activation status of the complex. Moreover, the HbYX tail insertion may not be sufficient to maintain the gate opening of 20S core particle. Our results provide new insights into the mechanisms of nucleotide-driven allosteric cooperativity of the complex and of the substrate processing by the proteasome.

Internal Distraction Osteogenesis With Piezosurgery Oblique Osteotomy of Supraorbital Margin of Frontal Bone for the Treatment of Unilateral Coronal Synostosis.

PURPOSE: To assess the utility of internal distraction osteogenesis with Piezosurgery oblique osteotomy of supraorbital margin of frontal bone for the treatment of unilateral coronal synostosis and to study the outcome and complications of this procedure. Oblique osteotomy allows for entry into the cranial cavity, and along with parallel cut to the roof of the orbit, avoids the need to cut into the orbit which forms the frontal flap. METHODS: Oblique osteotomy was performed along the supraorbital rim to do a frontal suture of the glabella (ages of patients were less than 1 year) or on the opposite side of the supraorbital rim (ages of patients were older than 1 year) after performing a suturectomy of the effected coronal suture. Two internal distraction devices were subsequently placed across the osteotomized, fused coronal suture. Finally, the cranium pieces were divided in the middle and placed in the middle of the frontal bone using biological glue. Five days after the operation, a 0.6-mm distraction was done twice daily. The distraction was removed 6 months after reaching 2 to 3 cm. RESULTS: Internal distraction osteogenesis with supraorbital oblique osteotomy was performed in 9 patients suffering from unilateral coronal synostosis. Eight patients had no postoperative infections around the shaft puncture wounds. One patient had infection in the rods around the distraction during the period of fixed, but was cured with antibiotic treatment. During a mean follow-up period of 12 months (5-26 months), all patients were satisfied with the cosmetic and functional results. No complications, including fixed screw displacement, penetration of the cranium and dura mater or retraction of distraction devices, occurred. The devices were exposed in 1 patient, resulting in a postoperative scar. Despite these complications, the cranium was successfully expanded in all patients. CONCLUSIONS: Use of this procedure avoids the need for frontal osteotomy to move the orbit forward. Adding 2 cranium strips can be used to reconstruct the frontal nodule to make up for inadequacy of the frontal nodules highlighting to distract operation. Retracting a separated cranium is not easy after it has been fixed for 6 months. Thus, the management of unilateral coronal synostosis using internal distraction osteogenesis with supraorbital oblique osteotomy is safe and effective.

Evaluating Loading Deflection of Distraction Osteogenic Rib in a Rabbit Model.

BACKGROUND: The treatment of patients with partially atrophic rib and rib defects requires an ideal arc of the rib that has adequate bone length and width. To design and assemble a distraction device with a strain gauge, we need to establish an animal model for testing it during rib distraction osteogenesis. METHODS: Osteotomies were performed at the same position in the fifth rib in 8 rabbits. Customized distraction devices attached to strain gauges were used to distract the ribs. After a month of distraction and consolidation, loading deflection gauges were used, and specimens were examined histologically to record bone formation. RESULTS: Distraction osteogenesis was carried out successfully in all rabbits when the device used to distract the rib up to 4 cm. CONCLUSIONS: The device can be used for strain testing during rib distraction osteogenesis performed in a rabbit model. There was no significant difference in the loading deflection gauges of the bone between osteogenic and contralateral ribs. This animal model of costal distraction osteogenesis is successful.

Staggered ATP binding mechanism of eukaryotic chaperonin TRiC (CCT) revealed through high-resolution cryo-EM.

The eukaryotic chaperonin TRiC (or CCT) assists in the folding of 10% of cytosolic proteins. Here we present two cryo-EM structures of Saccharomyces cerevisiae TRiC in a newly identified nucleotide partially preloaded (NPP) state and in the ATP-bound state, at 4.7-Å and 4.6-Å resolution, respectively. Through inner-subunit eGFP tagging, we identified the subunit locations in open-state TRiC and found that the CCT2 subunit pair forms an unexpected Z shape. ATP binding induces a dramatic conformational change on the CCT2 side, thereby suggesting that CCT2 plays an essential role in TRiC allosteric cooperativity. Our structural and biochemical data reveal a staggered ATP binding mechanism of TRiC with preloaded nucleotide on the CCT6 side of NPP-TRiC and demonstrate that TRiC has evolved into a complex that is structurally divided into two sides. This work offers insight into how the TRiC nucleotide cycle coordinates with its mechanical cycle in preparing folding intermediates for further productive folding.

MicroRNA-497 regulates cell proliferation in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common primary cancer of the liver. MicroRNA-497 (miR-497) is known to be downregulated in several types of human cancer; however, the expression, function and underlying mechanisms of miR-497 in HCC remain unclear. Therefore, the present study investigated miR-497 expression in HCC samples and HCC-derived cell lines using reverse transcription-quantitative polymerase chain reaction. The protein expression of one of the predicted common targets of miR-497, insulin-like growth factor-1 receptor (IGF-1R), was assessed using western blot analyses and immunohistochemistry. The role of miR-497 in regulating the proliferation of HCC-derived cells was also investigated in vitro and in vivo. Of 60 paired specimens from HCC patients, miR-497 was downregulated in 42 cancer specimens compared with adjacent non-cancer tissues. Western blotting and immunohistochemical analyses revealed that IGF-1R expression was significantly increased in HCC compared to control tissues. In addition, overexpression of miR-497 was observed to inhibit colony formation and tumor growth in MHCC-97H human HCC cells. Conversely, SMMC-7721 human HCC cells transfected with a miR-497 inhibitor exhibited enhanced colony formation and tumor growth. Finally, IGF-1R protein, phosphoinositide 3-kinase/Akt signaling pathway-associated proteins and cyclin pathway-associated proteins were differentially expressed between miR-497-overexpressing cells and miR-497-silenced cells. These results indicate that miR-497 may be a potentially effective gene therapy target.

The microRNA-146a/b attenuates acute small-for-size liver graft injury in rats.

BACKGROUND & AIMS: A critical role of the Toll-like receptor (TLR)-4 and its downstream mediators in the pathogenesis of small-for-size liver graft injury has been documented. Recently, the microRNA-146 (miR-146) was identified as a potent negative regulator of the TLR4 signalling pathway. In this study, the role of miR-146a and miR-146b in the attenuation of TLR-4 signalling and small-for-size liver graft injury was investigated. METHODS: The expression levels of miR-146a and miR-146b during small-for-size liver graft injury were studied in vivo. In addition, the effects of miR-146a and miR-146b on the expression of IRAK1 and TRAF6 in the rat macrophage cell line NR8383 and rat liver kupffer cells were studied in vitro. The in vivo effect of miR-146a and miR-146b on small-for-size liver graft injury was studied by the tail vein injection of miR-146a mimics and miR-146b mimics. RESULTS: The levels of miR-146a and miR-146b decreased with a small-for-size liver graft. MiR-146a and miR-146b inhibited IRAK1 and TRAF6 expression by binding to the 3'UTR of IRAK1 or TRAF6, respectively, in the rat macrophage cell line NR8383. The administration of miR-146a mimics and miR-146b mimics prevented liver graft injury in small-for-size liver graft injury via the inactivation of IRAK1 and TRAF6 in vivo. CONCLUSIONS: miR-146a and miR-146b prevent liver injury in small-for-size liver graft injury via the inactivation of IRAK1 and TRAF6.

miR-146a ameliorates liver ischemia/reperfusion injury by suppressing IRAK1 and TRAF6.

A critical role of the Toll-like receptor(TLR) and its downstream molecules, including IL-1 receptor-associated kinase 1(IRAK1) and tumor necrosis factor receptor- associated factor 6(TRAF6), in the pathogenesis of liver ischemia/reperfusion (I/R) injury has been documented. Recently a microRNA, miR-146a, was identified as a potent negative regulator of the TLR signaling pathway. In this study, we investigated the role of miR-146a to attenuate TLR signaling and liver I/R injury in vivo and in vitro. miR-146a was decreased in mice Kupffer cells following hepatic I/R, whereas IRAK1 and TRAF6 increased. Overexpression of miR-146a directly decreased IRAK1 and TRAF6 expression and attenuated the release of proinflammatory cytokines through the inactivation of NF-κB P65 in hypoxia/reoxygenation (H/R)-induced macrophages, RAW264.7 cells. Knockdown experiments demonstrated that IRAK1 and TRAF6 are two potential targets for reducing the release of proinflammatory cytokines. Moreover, co-culture assays indicated that miR-146a decreases the apoptosis of hepatocytes after H/R. In vivo administration of Ago-miR-146a, a stable version of miR-146a in vivo, protected against liver injury in mice after I/R via inactivation of the TLR signaling pathway. We conclude that miR-146a ameliorates liver ischemia/reperfusion injury in vivo and hypoxia/reoxygenation injury in vitro by directly suppressing IRAK1 and TRAF6.