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1.
Nat Commun ; 12(1): 4880, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385444

RESUMO

Accurate and imperceptible monitoring of electrophysiological signals is of primary importance for wearable healthcare. Stiff and bulky pregelled electrodes are now commonly used in clinical diagnosis, causing severe discomfort to users for long-time using as well as artifact signals in motion. Here, we report a ~100 nm ultra-thin dry epidermal electrode that is able to conformably adhere to skin and accurately measure electrophysiological signals. It showed low sheet resistance (~24 Ω/sq, 4142 S/cm), high transparency, and mechano-electrical stability. The enhanced optoelectronic performance was due to the synergistic effect between graphene and poly (3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS), which induced a high degree of molecular ordering on PEDOT and charge transfer on graphene by strong π-π interaction. Together with ultra-thin nature, this dry epidermal electrode is able to accurately monitor electrophysiological signals such as facial skin and brain activity with low-motion artifact, enabling human-machine interfacing and long-time mental/physical health monitoring.


Assuntos
Eletrodos , Eletrofisiologia/métodos , Epiderme/fisiologia , Desenho de Equipamento/métodos , Monitorização Fisiológica/métodos , Dispositivos Eletrônicos Vestíveis , Artefatos , Compostos Bicíclicos Heterocíclicos com Pontes/química , Condutividade Elétrica , Eletrofisiologia/instrumentação , Eletrofisiologia/normas , Desenho de Equipamento/normas , Grafite/química , Humanos , Estrutura Molecular , Monitorização Fisiológica/instrumentação , Monitorização Fisiológica/normas , Movimento (Física) , Polímeros/química , Poliestirenos/química , Pele
3.
World J Surg Oncol ; 19(1): 226, 2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34330293

RESUMO

BACKGROUND: To investigate long-chain noncoding TM4SF1-AS1 in gastric cancer (GC) tissues and cells. METHODS: TM4SF1-AS1 in 40 GC tissues and adjacent tissues was detected and compared using real-time fluorescence quantitative PCR (qRT-PCR). TM4SF1-AS1 in MKN28 and SGC7901 GC cells was downregulated using small interfering RNA (shRNA). The cells were grouped into an interference group (shTM4SF1-AS1 group) and a control group (shControl group). MTT and Transwell tests were applied to determine the proliferation and invasion of the cells in both groups, and flow cytometry was performed to assess the apoptosis rate in the two groups. Western blotting was performed to determine changes in key proteins in cells during the epithelial-to-mesenchymal transition (EMT) and in the TM4SF1 and PI3K-AKT signalling pathways in response to the downregulation of TM4SF1-AS1. RESULTS: The proliferation of MKN28 and SGC7901 in the shTM4SF1-AS1 group was significantly inhibited at 48 h and 72 h compared to that in the shControl group (all P < 0.05). In the shTM4SF1-AS1 group, the number of invaded MKN28 and SGC7901 cells was significantly lower than that in the shControl group (all P < 0.05). Apoptosis in the MKN28 and SGC7901 shTM4SF1-AS1 groups was significantly higher than that in the shControl group (all P < 0.05). Compared to those in the shControl group, levels of E-cadherin in EMT-related proteins were significantly elevated (P < 0.01), while levels of N-cadherin, Snail and Twist1 were significantly decreased (all P < 0.01). After silencing the expression of LncTM4SF1-AS1, the expression levels of TM4SF1 in the shTM4SF1-AS1 group were downregulated compared to those in the shControl group, and the p-PI3K and p-AKT proteins in the PI3K-AKT signalling pathway in the shTM4SF1-AS1 group were downregulated compared to those of the shControl group. CONCLUSIONS: TM4SF1-AS1 is upregulated in gastric cancer tissues and cells. Interfering with and downregulating its expression inhibit cancer cell proliferation, invasion and the EMT and promote apoptosis. The underlying mechanism for these effects is related to silencing the TM4SF1 and PI3K-AKT signalling pathways. TM4SF1-AS1 may be a potential therapeutic target for gastric cancer.


Assuntos
RNA Longo não Codificante , Neoplasias Gástricas , Antígenos de Superfície , Apoptose , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Neoplasias/genética , Fosfatidilinositol 3-Quinases/genética , Prognóstico , RNA Longo não Codificante/genética , Neoplasias Gástricas/genética
4.
Brief Bioinform ; 2021 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-34180984

RESUMO

Targeting the interaction between severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2)-receptor-binding domain (RBD) and angiotensin-converting enzyme 2 (ACE2) is believed to be an effective strategy for drug design to inhibit the infection of SARS-CoV-2. Herein, several ultrashort peptidase inhibitors against the RBD-ACE2 interaction were obtained by a computer-aided approach based on the RBD-binding residues on the protease domain (PD) of ACE2. The designed peptides were tested on a model coronavirus GX_P2V, which has 92.2 and 86% amino acid identity to the SARS-CoV-2 spike protein and RBD, respectively. Molecular dynamics simulations and binding free energy analysis predicted a potential binding pocket on the RBD of the spike protein, and this was confirmed by the specifically designed peptides SI5α and SI5α-b. They have only seven residues, showing potent antiviral activity and low cytotoxicity. Enzyme-linked immunosorbent assay result also confirmed their inhibitory ability against the RBD-ACE2 interaction. The ultrashort peptides are promising precursor molecules for the drug development of Corona Virus Disease 2019, and the novel binding pocket on the RBD may be helpful for the design of RBD inhibitors or antibodies against SARS-CoV-2.

5.
J Mol Graph Model ; 106: 107938, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34020229

RESUMO

Syndecans (SDCs) are a family of four members of integral membrane proteins, which play important roles in cell-cell interactions. Dimerization/oligomerization generated by transmembrane domains (TMDs) appears to crucially regulate several functional behaviors of all syndecan members. The different levels of protein-protein interactions mediated by Syndecan TMDs may lead to a rather complicated function of Syndecans. The molecular mechanism of the different dimerization tendencies in each type of SDCs remains unclear. Here, the self-assembly process of syndecan TMD homodimers and heterodimers was studied in molecular details by molecular dynamics simulations. Our computational results showed that the SDC2 forms the most stable homodimer, which is consistent with previous experimental results. Detailed analysis suggests that instead of the conserved dimerizing motif G8XXXG12 in all four SDCs involved in homo- and hetero-dimerization of SDCs. The different locations of GXXXA motif affect the stability of SDC dimers. In addition, we found that A3XXXA7 can stabilize the dimerization, making the dimer of SDC2 the most stable among these SDC dimers. Our results shed light on the complex effect of multiple dimerizing motifs on the dimerization of transmembrane domains.


Assuntos
Sindecanas , Sequência de Aminoácidos , Dimerização , Domínios Proteicos , Estrutura Terciária de Proteína
6.
Hepatol Int ; 15(1): 155-165, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33385299

RESUMO

BACKGROUND AND AIMS: Rifaximin has been recommended as a prophylactic drug for hepatic encephalopathy (HE) and spontaneous bacterial peritonitis (SBP). This study aims to explore whether low-dose rifaximin can prevent overall complications and prolong survival in cirrhotic patients. METHODS: In this multi-centre randomized open-labelled prospective study, 200 patients with decompensated cirrhosis were randomly assigned at a ratio of 1:1. Patients in rifaximin group were administered 400 mg rifaximin twice daily for 6 months, and all other therapeutic strategies were kept unchanged in both groups as long as possible. The primary efficacy endpoints were the incidence of overall complications and liver transplantation-free survival. The secondary endspoints were the incidence of each major cirrhosis-related complication, as well as the Child-Pugh score and class. RESULTS: The major baseline characteristics were similar in the two groups except for HE. The cumulative incidence and frequency of overall complications were significantly lower in rifaximin group than in the control group (p < 0.001). Though liver transplantation-free survival was not significantly different between the two groups, subgroup analysis showed rifaximin markedly prolonged liver transplantation-free survival in patients with Child-Pugh score ≥ 9 (p = 0.007). Moreover, rifaximin markedly reduced the episodes of ascites exacerbation (p < 0.001), HE (p < 0.001) and gastric variceal bleeding (EGVB, p = 0.031). The incidence of adverse events was similar in the two groups. CONCLUSION: Low-dose rifaximin significantly decreases the occurrence of overall complications, leading to prolonged survival in patients with advanced stages of cirrhosis in this trail. Further study should be carried out to compare the effect of this low-dose rifaximin with normal dose (1200 mg/day) rifaximin in preventing cirrhosis-related complications. CLINICAL TRIAL NUMBER: NCT02190357.

7.
Infect Dis Ther ; 10(1): 241-252, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33111216

RESUMO

INTRODUCTION: This study aimed to analyze the diversity of intestinal flora in patients with chronic hepatitis B (CHB) and investigate the effect of entecavir on the intestinal flora in these patients. METHODS: Thirty patients with CHB and 30 healthy controls were recruited from the Department of Infectious Diseases and Department of Gastroenterology of Shanghai Tongji Hospital between January 2017 and December 2018. Stool samples were collected for the detection of intestinal flora by high-throughput sequencing. Patients with CHB received antivirus therapy with entecavir for 8 weeks. The biochemical and virological responses were assessed and the intestinal flora were compared. RESULTS: After entecavir treatment, the blood levels of alanine aminotransferase (ALT), interleukin-6 (IL-6), IL-8, tumor necrosis factor (TNF), and hepatitis B virus (HBV) DNA reduced significantly in patients with CHB and the species abundance of intestinal flora increased markedly. In patients with CHB, the unique genera included Butyrivibrio, Phaseolus acutifolius, and Prevotellaceae NK3B31 group before treatment and Howardella, Candidatus Stoquefichus, Citrobacter, Dysgonomonas, Faecalicoccus, Methanobrevibacter, Mitsuokella, Mobilitalea, Succinivibrio, Gluconobacter, and Plesiomonas after treatment. The abundance of the following genera increased significantly after entecavir treatment in patients with CHB: Clostridium sensu stricto 1, Erysipelotrichaceae UCG-007, and Intestinibacter. The abundance of Streptococcus, Atopobium, and Murdochiella reduced markedly after entecavir treatment in patients with CHB. CONCLUSION: After 8-week entecavir treatment, the blood biochemical, immunological, and virological responses improved significantly, the species abundance of intestinal flora increased markedly, and there were unique genera in patients with CHB before and after treatment.

8.
Adv Sci (Weinh) ; 7(23): 2002025, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33304756

RESUMO

Integration of diverse materials into 3D ordered structures is urgently required for advanced manufacture owing to increase in demand for high-performance products. Most additive manufacturing techniques mainly focus on simply combining different equipment, while interfacial binding of distinctive materials remains a fundamental problem. Increasing studies on macroscopic supramolecular assembly (MSA) have revealed efficient interfacial interactions based on multivalency of supramolecular interactions facilitated by a "flexible spacing coating." To demonstrate facile fabrication of 3D heterogeneous ordered structures, the combination of MSA and magnetic field-assisted alignment has been developed as a new methodology for in situ integration of a wide range of materials, including elastomer, resin, plastics, metal, and quartz glass, with modulus ranging from tens of MPa to over 70 GPa. Assembly of single material, coassembly of two to four distinctive materials, and 3D alignment of "bridge-like" and "cross-stacked" heterogeneous structures are demonstrated. This methodology has provided a new solution to mild and efficient assembly of multiple materials at the macroscopic scale, which holds promise for advanced fabrication in fields of tissue engineering, electronic devices, and actuators.

9.
Nanoscale ; 11(9): 3945-3951, 2019 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-30762052

RESUMO

Understanding the folding mechanism of knotted and slipknotted proteins has attracted considerable interest. Due to their topological complexity, knotted and slipknotted proteins are predicted to fold slowly and involve large topological barriers. Molecular dynamics simulation studies suggest that a slipknotted conformation can serve as an important intermediate to help greatly reduce the topological difficulty during the folding of some knotted proteins. Here we use a single molecule optical tweezers technique to directly probe the folding of a small slipknotted protein AFV3-109. We found that stretching AFV3-109 can lead to the untying of the slipknot and complete unfolding of AFV3-109. Upon relaxation, AFV3-109 can readily refold back to its native slipknot conformation with high fidelity when the stretching force is lower than 6 pN. The refolding of AFV3-109 occurs in a sharp two-state like transition. Our results indicate that, different from knotted proteins, the folding of a slipknotted protein like AFV3-109 can be fast, and may not necessarily involve a high topological barrier.


Assuntos
Pinças Ópticas , Proteínas/química , Cinética , Microscopia de Força Atômica , Simulação de Dinâmica Molecular , Método de Monte Carlo , Desnaturação Proteica , Dobramento de Proteína , Proteínas/metabolismo , Termodinâmica
10.
Int J Clin Exp Pathol ; 12(12): 4329-4337, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31933834

RESUMO

The purpose of the study was to analyze the prognostic value of metastatic lymph node ratio (LNR) in pancreatic cancer (PC) patients undergoing surgical operation. We retrospectively reviewed 615 patients with PC who underwent surgical operation. The clinic-pathologic factors related to lymph node metastasis (LNM) were analyzed. There are 251 patients with LNM (40.8%), 364 cases of patients without LNM (59.2%). We found that overall survival (OS) of PC was significantly correlated to histological type, degree of differentiation, clinical staging, LNM, and LNR (all P < 0.05). Multivariate Cox regression analysis demonstrated that LNR was an independent risk factor for postoperative survival rate of patients with PC (P=0.006), and the presence of LNM was not an independent factor for predicting poor prognosis. When age and gender were included in multivariate Cox proportional hazards, LNR was still the independent prognostic factor for the OS of patients with PC (P=0.013). The value of LNR in the prediction of the prognosis of PC was better than the number and presence of LNM. It provided some helpful advice for clinicians to formulating a reasonable treatment strategy.

11.
J Vis Exp ; (140)2018 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-30394393

RESUMO

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low cell retention and poor survival within the target zone. However, during the in vitro construction process, a solution for maintaining stem cell bioactivity and increasing the cell amount within the cell sheet is urgently needed. Here, this protocol presents a method for constructing a multilayered cell sheet with favorable stem cell bioactivity and optimal operability. Decellularized porcine pericardium (DPP) is prepared by phospholipase A2 (PLA2) decellularization method as the cell sheet scaffold, and rat bone marrow mesenchymal stem cells (BMSCs) are isolated and expanded as the seeded cells. The temporary multilayered cell sheet structure is constructed by using RAD16-I peptide hydrogel. Finally, the cell sheet is cultured with a dynamic perfusion system to stabilize the three-dimensional (3D) structure, and the cell sheet could be obtained following a 48-hour culture in vitro. This protocol provides an efficient and feasible method for constructing a multilayered stem cell sheet, and the cell sheet could be developed as a favorable stem cell therapy product in the future.


Assuntos
Regeneração Tecidual Guiada/métodos , Células-Tronco Mesenquimais/metabolismo , Engenharia Tecidual/métodos , Animais , Diferenciação Celular , Células Cultivadas , Masculino , Ratos , Ratos Sprague-Dawley
12.
Appl Opt ; 57(5): 1241-1246, 2018 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-29469870

RESUMO

Beads trapped in optical tweezers are aligned along the optical propagation direction, which makes it difficult to determine the number of beads with bright-field microscopy. This problem also dramatically influences the measurement of the optical trapping based single-molecule force spectroscopy. Here, we propose a video processing approach to count the number of trapped micro-objects in real time. The approach uses a normalized cross-correlation algorithm and image enhancement techniques to amplify a slight change of the image induced by the entry of an exotic object. As tested, this method introduces a ∼10% change per bead to the image similarity, and up to four beads, one-by-one falling into the trap, are identified. Moreover, the feasibility of the above analysis in a moving trap is investigated. A movement of the trap leads to a fluctuation of less than 2% for the similarity signal and can be ignored in most cases. The experimental results prove that image similarity measurement is a sensitive way to monitor the interruption, which is very useful, especially during experiments. In addition, the approach is easy to apply to an existing optical tweezers system.

13.
Langmuir ; 33(4): 1077-1083, 2017 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-28040904

RESUMO

Single-molecule force spectroscopy (SMFS) and molecular dynamics (MD) simulations have revealed that shear topology is an important structural feature for mechanically stable proteins. Proteins containing a ß-grasp fold display the typical shear topology and are generally of significant mechanical stability. In an effort to experimentally identify mechanically strong proteins using single-molecule atomic force microscopy, we found that staphylokinase (SAK), which has a typical ß-grasp fold and was predicted to be mechanically stable in coarse-grained MD simulations, displays surprisingly low mechanical stability. At a pulling speed of 400 nm/s, SAK unfolds at ∼60 pN, making it the mechanically weakest protein among the ß-grasp fold proteins that have been characterized experimentally. In contrast, its structural homologous protein streptokinase ß domain displays significant mechanical stability under the same experimental condition. Our results showed that the large malleability of native-state SAK is largely responsible for its low mechanical stability. The molecular origin of this large malleability of SAK remains unknown. Our results reveal a hidden complexity in protein mechanics and call for a detailed investigation into the molecular determinants of the protein mechanical malleability.


Assuntos
Fenômenos Mecânicos , Metaloendopeptidases/química , Fenômenos Biomecânicos , Estabilidade Enzimática , Metaloendopeptidases/metabolismo , Simulação de Dinâmica Molecular , Conformação Proteica em Folha beta , Domínios Proteicos , Desdobramento de Proteína , Solventes/química , Termodinâmica
14.
Angew Chem Int Ed Engl ; 56(22): 6117-6121, 2017 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-28026101

RESUMO

Single-molecule force spectroscopy (SMFS) has become a powerful tool in investigating the mechanical unfolding/folding of proteins at the single-molecule level. Polyproteins made of tandem identical repeats have been widely used in atomic force microscopy (AFM)-based SMFS studies, where polyproteins not only serve as fingerprints to identify single-molecule stretching events, but may also improve statistics of data collection. However, the inherent assumption of such experiments is that all the domains in the polyprotein are equivalent and one SMFS trajectory of stretching a polyprotein made of n domains is equivalent to n trajectories of stretching a single domain. Such an assumption has not been validated experimentally. Using a small protein NuG2 and its polyprotein (NuG2)4 as model systems, here we use optical trapping (OT) to directly validate this assumption. Our results show that OT experiments on NuG2 and (NuG2)4 lead to identical parameters describing the unfolding and folding kinetics of NuG2, demonstrating that indeed stretching a polyprotein of NuG2 is equivalent to stretching single NuG2 in force spectroscopy experiments and thus validating the use of polyproteins in SMFS experiments.


Assuntos
Proteínas/química , Imagem Individual de Molécula/métodos , Cinética , Dobramento de Proteína
15.
Int J Clin Exp Pathol ; 8(9): 9845-53, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26617694

RESUMO

OBJECTIVE: Aldosterone is related to the fibrosis of several organs, but the specific mechanism underlying the aldosterone induced hepatic fibrosis is still unclear. METHODS: Separation, culture and identification of primary hepatic stellate cells (HSCs): The fluids and digestives used in the present study were able to completely remove blood cells, digest hepatocytes and matrix, and effectively separate HSCs. The in situ perfusion was performed at 2 steps: in situ perfusion with pre-perfusion fluid and ex vivo perfusion with enzyme-containing perfusion fluid. Influence of Ald on PAI-1 and Smad expressions in HSCs: cells were divided into control group, Ald group (10(-6) M), spironolactone (SPI) group and Ald+SPI group, and the mRNA and protein expressions of PAI-1 and Smad were detected. Ald induced type I collagen expression in HSCs: Immunohistochemistry was performed to detect type I collagen expression in the supernatant of control group, Ald group (10(-6) M), TGF-ß1 group, and Ald+TGF-ß1 group. Influence of Ald and TGF-ß1 on PAI-1 expression in HSCs: cells were divided into control group, Ald group (10(-6) M), TGF-ß1 group, and Ald+TGF-ß1 group, and the mRNA and protein expressions of PAI-1 were determined by RT-PCR and Western blot assay, respectively. Synergistic effect of Ald and TGF-ß1 on PAI-1 expression in HSCs: cells were divided into control group, Ald group (10(-6)), TGF-ß1 group, Ald (10(-6) M)+TGF-ß1 group, Ald (10(-7) M)+TGF-ß1 group and Ald (10(-8) M)+TGF-ß1 group, and the mRNA and protein expressions of PAI-1 were detected by RT-PCR and Western blot assay, respectively. RESULTS: The survival rate, purity, markers and activation of HSCs were determined after separation. Influence of Ald on PAI-1 expression in HSCs: PAI-1 expression increased in HSCs of Ald group, SPI group and Ald+API group, and the PAI-1 expression in Ald group and Ald+SPI group was significantly higher than in control group (P<0.01). Influence of Ald on Smad expression in HSCs: Smad expression in Ald group, TGF-ß1 group and ALD+TGF-ß1 group was markedly higher than in control group (P<0.05). Smad expression in ALD+TGF-ß1 group increased significantly when compared with Ald group (P<0.01). Ald induced type I collagen expression in HSCs: type I collagen expression in Ald group, TGF-ß1 group and ALd+TGF-ß1 group was dramatically higher than in control group (P<0.05), and it in ALd+TGF-ß1 group was also significantly different from that in Ald group and TGF-ß1 group (P<0.01). Synergistic effects of Ald and TGF-ß1 on PAI expression in HSCs: PAI-1 expression in treated cells was markedly higher than in control group (P<0.01). PAI-1 expression in 10(-6) M Ald+5 ng/ml TGF-ß1 group increased dramatically as compared to Ald group and TGF-ß1 group (P<0.01), but the increased PAI-1 expression reduced after SPI treatment. Ald at different concentrations exerts synergistic effect with TGF-ß1 to increase PAI-1 expression in HSCs: PAI-1 expression in HSCs after different treatments increased markedly as compared to control group (P<0.01). Significant difference in PAI-1 expression was observed in 10(-6) M Ald+50 pg/ml TGF-ß1 group and 10(-6) M Ald group (P<0.01), PAI-1 expression in 10(-7) M Ald+50 pg/ml TGF-ß1 group was significantly higher than in 50 pg/ml TGF-ß1 group (P<0.01), but the PAI-1 expression in 10(-7) M Ald+50 pg/ml TGF-ß1 group was similar to that in 10(-6) M Ald group (P>0.05). CONCLUSION: Aldosterone is able to activate HSCs and increase PAI-1 expression during hepatic fibrosis, which may be inhibited by spironolactone. Aldosterone and TGF-ß1 may synergistically act on HSCs to increase PAI-1 expression as compared to treatment with aldosterone or TGF-ß1 alone. Aldosterone or TGF-ß1 alone may slightly increase PAI-1 expression in HSCs, which can be inhibited by spironolactone.


Assuntos
Aldosterona/metabolismo , Células Estreladas do Fígado/metabolismo , Cirrose Hepática/metabolismo , Inibidor 1 de Ativador de Plasminogênio/biossíntese , Fator de Crescimento Transformador beta1/metabolismo , Animais , Western Blotting , Células Cultivadas , Modelos Animais de Doenças , Imuno-Histoquímica , Masculino , Reação em Cadeia da Polimerase , Ratos , Ratos Sprague-Dawley
16.
Angew Chem Int Ed Engl ; 54(34): 9921-5, 2015 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-26136291

RESUMO

Directly observing protein folding in real time using atomic force microscopy (AFM) is challenging. Here the use of AFM to directly monitor the folding of an α/ß protein, NuG2, by using low-drift AFM cantilevers is demonstrated. At slow pulling speeds (<50 nm s(-1)), the refolding of NuG2 can be clearly observed. Lowering the pulling speed reduces the difference between the unfolding and refolding forces, bringing the non-equilibrium unfolding-refolding reactions towards equilibrium. At very low pulling speeds (ca. 2 nm s(-1)), unfolding and refolding were observed to occur in near equilibrium. Based on the Crooks fluctuation theorem, we then measured the equilibrium free energy change between folded and unfolded states of NuG2. The improved long-term stability of AFM achieved using gold-free cantilevers allows folding-unfolding reactions of α/ß proteins to be directly monitored near equilibrium, opening the avenue towards probing the folding reactions of other mechanically important α/ß and all-ß elastomeric proteins.


Assuntos
Redobramento de Proteína , Desdobramento de Proteína , Proteínas/química , Microscopia de Força Atômica , Estrutura Secundária de Proteína
17.
Am J Cancer Res ; 5(4): 1460-70, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26101710

RESUMO

Pancreatic cancer (PC) is one of the most malignant tumors. Rho GTPases can affect several types of human cancers, including PC. In this study, we investigated the role of Ras homolog family member T1 (RHOT1), a new member of Rho GTPases in PC. IHC results showed that RHOT1 was expressed significantly higher in PC tissues than paracancerous tissues (P<0.01) and SMAD family member 4 (SMAD4) was expressed lower in PC tissues (P<0.01). RHOT1 was widely expressed in PC cell lines analyzed by reverse transcription PCR (RT-PCR), real-time quantitative PCR (RT-qPCR) and western blotting (WB). SiRNA-RHOT1 significantly suppressed the proliferation and migration of SW1990 cells. Moreover, SMAD4 was identified as an effector of RHOT1. Our findings suggest that RHOT1 can regulate cell migration and proliferation by suppressing the expression of SMAD4 in PC, which may provide a novel sight to explore the mechanism and therapeutic strategy for PC.

18.
J Surg Oncol ; 111(7): 834-9, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25919911

RESUMO

BACKGROUND AND OBJECTIVES: CCAT2, a novel long non-coding RNAs (lncRNAs), is found to promote the metastasis and invasion of colon, lung, and breast cancers. This study aimed to investigate the level of CCAT2 in esophageal squamous cell carcinoma (ESCC) and to elucidate its clinical significance. METHODS: The expression level of CCAT2 and the status of MYC amplification were examined in 229 ESCC samples using quantitative real- time PCR. RESULTS: CCAT2 was upregulated in ESCC tissues, especially in cases with lymph node metastasis (LNM), advanced TNM stages, and MYC amplification. Furthermore, the level of CCAT2 was positively correlated with TNM stages, LNM, and the number of positive lymph nodes. High CCAT2 expression and MYC amplification were significantly associated with TNM stages and LNM. Survival analyses revealed that high CCAT2 expression and MYC amplification were significantly associated with poorer overall survival in ESCC patients. Furthermore, patients with high CCAT2 expression and MYC amplification had a 2.199-fold increased risk of death compared with those with low CCAT2 expression and MYC non-amplification. CONCLUSIONS: Our study provides the first evidence associating CCAT2 expression and poor survival in ESCC. CCAT2 may be a prognostic biomarker and therapeutic target for ESCC.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma Medular/genética , Carcinoma de Células Escamosas/genética , Neoplasias Esofágicas/genética , Proteínas Proto-Oncogênicas c-myc/genética , RNA Longo não Codificante/genética , Carcinoma Medular/mortalidade , Carcinoma Medular/secundário , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/secundário , Neoplasias Esofágicas/mortalidade , Neoplasias Esofágicas/patologia , Esôfago/metabolismo , Feminino , Seguimentos , Amplificação de Genes , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida
19.
Am J Cancer Res ; 4(5): 537-44, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25232495

RESUMO

Wnt signaling pathway plays an important role in physiological and pathological process, including in the occurrence and development of tumor. The purpose of this study is to determine whether Wnt2 and sFRP4, key molecules of signaling pathway, are of prognostic value for survival in patients with pancreatic cancer. We performed immunohistochemistry on tissue microarrays containing 90 pancreatic cancer specimens to evaluate the protein expression of Wnt2 and sFRP4. Our results showed that the cytoplasmic expression level of Wnt2 in pancreatic cancer tissues was significantly associated with LNM (P=0.029) and AJCC stage (P=0.008). Additionally, Kaplan-Meier analysis indicated that high Wnt2 expression was significantly correlated with poor clinical outcomes of patients with pancreatic cancer. In conclusion, Wnt2 may play an important role in the development of pancreatic cancer through activation of the Wnt pathways and serve as a potential candidate for treatment target of pancreatic cancer.

20.
J Am Chem Soc ; 136(34): 11946-55, 2014 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-25092607

RESUMO

The knotted/slipknotted polypeptide chain is one of the most surprising topological features found in certain proteins. Understanding how knotted/slipknotted proteins overcome the topological difficulty during the folding process has become a challenging problem. By stretching a knotted/slipknotted protein, it is possible to untie or tighten a knotted polypeptide and even convert a slipknot to a true knot. Here, we use single molecule force spectroscopy as well as steered molecular dynamics (SMD) simulations to investigate how the slipknotted protein AFV3-109 is transformed into a tightened trefoil knot by applied pulling force. Our results show that by pulling the N-terminus and the threaded loop of AFV3-109, the protein can be unfolded via multiple pathways and the slipknot can be transformed into a tightened trefoil knot involving ∼13 amino acid residues as the polypeptide chain is apparently shortened by ∼4.7 nm. The SMD simulation results are largely consistent with our experimental findings, providing a plausible and detailed molecular mechanism of mechanical unfolding and knot tightening of AFV3-109. These simulations reveal that interactions between shearing ß-strands on the threaded and knotting loops provide high mechanical resistance during mechanical unfolding.


Assuntos
Simulação de Dinâmica Molecular , Dobramento de Proteína , Proteínas/química , Sequência de Aminoácidos , Microscopia de Força Atômica , Dados de Sequência Molecular , Método de Monte Carlo , Conformação Proteica , Engenharia de Proteínas , Desdobramento de Proteína , Proteínas/genética , Termodinâmica
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