ABSTRACT
Native mass spectrometry (MS) is proving to be a disruptive technique for studying the interactions of proteins, necessary for understanding the functional roles of these biomolecules. Recent research is expanding the application of native MS towards membrane proteins directly from isolated membrane preparations or from purified detergent micelles. The former results in complex spectra comprising several heterogeneous protein complexes; the latter enables therapeutic protein targets to be screened against multiplexed preparations of compound libraries. In both cases, the resulting spectra are increasingly complex to assign/interpret, and the key to these new directions of native MS research is the ability to perform native top-down analysis, which allows unambiguous peak assignment. To achieve this, detergent removal is necessary prior to MS analyzers, which allow selection of specific m/z values, representing the parent ion for downstream activation. Here, we describe a novel, enhanced declustering (ED) device installed into the first pumping region of a cyclic IMS-enabled mass spectrometry platform. The device enables declustering of ions prior to the quadrupole by imparting collisional activation through an oscillating electric field applied between two parallel plates. The positioning of the device enables liberation of membrane protein ions from detergent micelles. Quadrupole selection can now be utilized to isolate protein-ligand complexes, and downstream collision cells enable the dissociation and identification of binding partners. We demonstrate that ion mobility (IM) significantly aids in the assignment of top-down spectra, aligning fragments to their corresponding parent ions by means of IM drift time. Using this approach, we were able to confidently assign and identify a novel hit compound against PfMATE, obtained from multiplexed ligand libraries.
Subject(s)
Ion Mobility Spectrometry , Membrane Proteins , Membrane Proteins/chemistry , Membrane Proteins/analysis , Ion Mobility Spectrometry/methods , Micelles , Mass Spectrometry/methods , Detergents/chemistry , Ions/chemistryABSTRACT
In this study, a self-responsive fluorescence aptasensor was established for the determination of lactoferrin (Lf) in dairy products. Herein, the aptamer itself functions as both a recognition element that specifically binds to Lf and a fluorescent signal reporter in conjunction with fluorescent moiety. In the presence of Lf, the aptamer preferentially binds to Lf due to its specific and high-affinity recognition by folding into a self-assembled and three-dimensional spatial structure. Meanwhile, its reduced spatial distance in the aptamer-Lf complex induces a FRET phenomenon based on the quenching of 6-FAM by amino acids in the Lf protein, resulting in a turn-off of the fluorescence of the system. As a result, the Lf concentration can be determined straightforwardly corresponding to the change in the self-responsive fluorescence signal. Under the optimized conditions, good linearities (R2 > 0.99) were achieved in an Lf concentration range of 2~10 µg/mL for both standard solutions and the spiked matrix, as well as with the desirable detection limits of 0.68 µg/mL and 0.46 µg/mL, respectively. Moreover, the fluorescence aptasensor exhibited reliable recoveries (89.5-104.3%) in terms of detecting Lf in three commercial samples, which is comparable to the accuracy of the HPCE method. The fluorescence aptasensor offers a user-friendly, cost-efficient, and promising sensor platform for point-of-need detection.
Subject(s)
Aptamers, Nucleotide , Biosensing Techniques , Dairy Products , Lactoferrin , Lactoferrin/analysis , Lactoferrin/chemistry , Aptamers, Nucleotide/chemistry , Biosensing Techniques/methods , Dairy Products/analysis , Fluorescence , Limit of Detection , Spectrometry, Fluorescence/methods , Food Analysis/methods , Fluorescence Resonance Energy Transfer/methodsABSTRACT
Benefitting from the rapid evolution of artificial intelligence and structural biology, an expanding collection of high-resolution protein structures has greatly improved our understanding of protein functions. Yet, proteins are inherently flexible, and these static structures can only offer limited snapshots of their true dynamic nature. The conformational and functional changes of calmodulin (CaM) induced by Ca2+ binding have always been a focus of research. In this study, the conformational dynamics of CaM and its complexes were investigated using a mobility capillary electrophoresis (MCE) and native mass spectrometry (native MS) based method. By analyzing the ellipsoidal geometries of CaM in the solution phase at different Ca2+ concentrations, it is interesting to discover that CaM molecules, whether bound to Ca2+ or not, possess both closed and open conformations. Moreover, each individual CaM molecule actively "jumps" (equilibrium exchange) between these two distinct conformations on a timescale ranging from milli- to micro-seconds. The binding of Ca2+ ions did not affect the structural dynamics of CaM, while the binding of a peptide ligand would stabilize CaM, leading to the observation of a single, compact conformation of the resulting protein complex. A target recognition mechanism was also proposed based on these new findings, suggesting that CaM's interaction with targets may favor a conformational selection model. This enriches our understanding of the binding principles between CaM and its numerous targets.
Subject(s)
Calcium , Calmodulin , Electrophoresis, Capillary , Mass Spectrometry , Calmodulin/chemistry , Calmodulin/metabolism , Electrophoresis, Capillary/methods , Calcium/chemistry , Calcium/metabolism , Mass Spectrometry/methods , Protein Conformation , Protein Binding , AnimalsABSTRACT
Fluid biopsy technology, characterized by its minimally invasive nature, speed, and continuity, has become a rapidly advancing and widely applied real-time diagnostic technique. Among various biomarkers, proteins represent the most abundant class of disease indicators. The sensitive and accurate detection of protein markers in bodily fluids is significantly influenced by the control exerted by recognition ligands. Aptamers, which are structurally dynamic functional oligonucleotides, exhibit high affinity, specific recognition of targets, and notable characteristics of high editability and modularity. These features make aptamer universal "recognition-capture" components, contribute to a significant leap in their applications within the biosensor domain. In this context, we provide a comprehensive review of the extensive application of aptamer-based biosensors in fluid biopsy. We systematically compile the characteristics and construction strategies of aptamer-based biosensors tailored for fluid biopsy, including aptamer sequences, affinity (KD), fluid background, sensing technologies, sensor construction strategies, incubation time, detection performance, and influencing factors. Furthermore, a comparative analysis of their advantages and disadvantages was conducted. In conclusion, we delineate and deliberate on prospective research trajectories and challenges that lie ahead in the realm of aptamer-based biosensors for fluid biopsy.
Subject(s)
Aptamers, Nucleotide , Biomarkers , Biosensing Techniques , Humans , Aptamers, Nucleotide/chemistry , Biosensing Techniques/methods , Biomarkers/analysis , Liquid Biopsy/methods , Proteins/analysis , Proteins/chemistry , Body Fluids/chemistryABSTRACT
Self-supervised pre-training and fully supervised fine-tuning paradigms have received much attention to solve the data annotation problem in deep learning fields. Compared with traditional pre-training on large natural image datasets, medical self-supervised learning methods learn rich representations derived from unlabeled data itself thus avoiding the distribution shift between different image domains. However, nowadays state-of-the-art medical pre-training methods were specifically designed for downstream tasks making them less flexible and difficult to apply to new tasks. In this paper, we propose grid mask image modeling, a flexible and general self-supervised method to pre-train medical vision transformers for 3D medical image segmentation. Our goal is to guide networks to learn the correlations between organs and tissues by reconstructing original images based on partial observations. The relationships are consistent within the human body and invariant to disease type or imaging modality. To achieve this, we design a Siamese framework consisting of an online branch and a target branch. An adaptive and hierarchical masking strategy is employed in the online branch to (1) learn the boundaries or small contextual mutation regions within images; (2) to learn high-level semantic representations from deeper layers of the multiscale encoder. In addition, the target branch provides representations for contrastive learning to further reduce representation redundancy. We evaluate our method through segmentation performance on two public datasets. The experimental results demonstrate our method outperforms other self-supervised methods. Codes are available at https://github.com/mobiletomb/Gmim.
Subject(s)
Imaging, Three-Dimensional , Humans , Imaging, Three-Dimensional/methods , Deep Learning , Algorithms , Supervised Machine LearningABSTRACT
Uphill walking is a common task encountered in daily life, with steeper inclines potentially imposing greater biomechanical and neuromuscular demands on the human body. The heel-to-toe drop (HTD) in footwear may influence the biomechanical and neuromuscular pattern of uphill walking; but the impact remains unclear. Adjustments in HTD can modulate biomechanical and neuromuscular patterns, mitigating the demands and optimizing the body's response to different inclinations. We hypothesize that adjustments in HTD can modulate biomechanical and neuromuscular patterns, mitigating the demands and optimizing the body's response to different inclinations. Nineteen healthy men walked on an adjustable slope walkway, with varied inclinations (6°, 12°, 20°) and HTD shoes (10mm, 25mm, 40 mm), while the marker positions, ground reaction forces and electromyography data were collected. Our study reveals that gait temporo-spatial parameters are predominantly affected by inclination over HTD. Inclination has a more pronounced effect on kinematic variables, while both inclination and HTD significantly modulate kinetic and muscle synergy parameters. This study demonstrates that an increase in the inclination leads to changes in biomechanical and neuromuscular responses during uphill walking and the adjustment of HTD can modulate these responses during uphill walking. However, the present study suggests that an increased HTD may lead to elevated loads on the knee joint and these adverse effects need more attention.
ABSTRACT
This paper serves as an annual review of capillary electrophoresis (CE) technology for 2023. The journals were selected based on their impact factor (IF), a universally recognized academic performance metric, combined with experimental work closely related to CE technology, to facilitate the rapid acquisition of significant research and application advancements in CE technology in 2023. A thematic search of the ISI Web of Science database yielded 669 research papers on CE technology published in 2023. This review highlights five experimental papers published in journals with IFs greater than 10.0, including Nature Communications, Nucleic Acids Research, Engineering, Journal of Medical Virology, and Carbohydrate Polymers, and 31 experimental papers from representative journals with IFs between 5.0 and 10.0, such as Analytical Chemistry, Analytica Chimica Acta, Talanta, and Food Chemistry. It also provides an overview of experimental research in journals with focused reporting on CE technology but with IFs less than 5.0, such as Journal of Chromatography A and Electrophoresis, as well as significant experimental research from key domestic Chinese core journals (Peking University). In 2023, all the latest scientific advancements reported in journals with an IF greater than 10.0 utilized previously reported CE methods, offering new breakthroughs for the promotion and application of CE technology. Additionally, new applications of CE in conjunction with mass spectrometry remained a hot topic. An increase in reports on the hardware aspects of CE, such as 3D printing and underwater systems, and significant breakthroughs in the analysis of non-solution samples, such as solid particles, cell vesicles, cells, viruses, and bacteria, was noted. CE is advantageous for the analysis of drugs and their components. In Chinese journals, the number of papers on CE applications exceeded that in previous years, with particular focus on the field of printing for new applications.
Subject(s)
Electrophoresis, Capillary , Electrophoresis, Capillary/methodsABSTRACT
Physiologic hand tremors are a critical factor affecting the aim of air pistol shooters. However, the extent of the effect of hand tremors on shooting performance is unclear. In this study, we aim to explore the relationship between hand tremors and shooting performance scores as well as investigate potential links between muscle activation and hand tremors. In this study, 17 male air pistol shooters from China's national team and the Air Pistol Sports Center were divided into two groups: the elite group and the sub-elite group. Each participant completed 40 shots during the experiment, with shooters' hand tremors recorded using three-axis digital accelerometers affixed to their right hands. Muscle activation was recorded using surface electromyography on the right anterior deltoid, posterior deltoid, biceps brachii (short head), triceps brachii (long head), flexor carpi radialis, and extensor carpi radialis. Our analysis revealed weak correlations between shooting scores and hand tremor amplitude in multiple directions (middle-lateral, ML: r2 = -0.22, p < 0.001; vertical, VT: r2 = -0.25, p < 0.001), as well as between shooting scores and hand tremor complexity (ML: r2 = -0.26, p < 0.001; VT: r2 = -0.28, p < 0.001), across all participants. Notably, weak correlations between shooting scores and hand tremor amplitude (ML: r2 = -0.27, p < 0.001; VT: r2 = -0.33, p < 0.001) and complexity (ML: r2 = -0.31, p < 0.001) were observed in the elite group but not in the sub-elite group. Moderate correlation were found between the biceps brachii (short head) RMS and hand tremor amplitude in the VT and ML directions (ML: r2 = 0.49, p = 0.010; VT: r2 = 0.44, p = 0.025) in all shooters, with a moderate correlation in the ML direction in elite shooters (ML: r2 = 0.49, p = 0.034). Our results suggest that hand tremors in air pistol shooters are associated with the skill of the shooters, and muscle activation of the biceps brachii (long head) might be a factor affecting hand tremors. By balancing the agonist and antagonist muscles of the shoulder joint, shooters might potentially reduce hand tremors and improve their shooting scores.
Subject(s)
Electromyography , Firearms , Hand , Tremor , Humans , Tremor/physiopathology , Male , Hand/physiology , Hand/physiopathology , Adult , Young Adult , Athletic Performance/physiology , Muscle, Skeletal/physiopathology , Muscle, Skeletal/physiologyABSTRACT
BACKGROUND: 2D/3D medical image registration is one of the key technologies for surgical navigation systems to perform pose estimation and achieve accurate positioning, which still remains challenging. The purpose of this study is to introduce a new method for X-ray to CT 2D/3D registration and conduct a feasibility study. METHODS: In this study, a 2D/3D affine registration method based on feature point detection is investigated. It combines the morphological and edge features of spinal images to accurately extract feature points from the images, and uses graph neural networks to aggregate anatomical features of different points to increase the local detail information. Meanwhile, global and positional information are extracted by the Swin Transformer. RESULTS: The results indicate that the proposed method has shown improvements in both accuracy and success ratio compared with other methods. The mean target registration error value reached up to 0.31 mm; meanwhile, the runtime overhead was much lower, achieving an average runtime of about 0.6 s. This ultimately improves the registration accuracy and efficiency, demonstrating the effectiveness of the proposed method. CONCLUSIONS: The proposed method can provide more comprehensive image information and shows good prospects for pose estimation and achieving accurate positioning in surgical navigation systems.
Subject(s)
Algorithms , Imaging, Three-Dimensional , Humans , X-Rays , Radiography , Imaging, Three-Dimensional/methods , Tomography, X-Ray Computed/methods , Image Processing, Computer-Assisted/methodsABSTRACT
CRISPR/Cas9 is a natural immune system of archaea and bacteria, which has been widely used in gene editing. In order to better control and improve the accuracy and safety of the system, inhibitors for SpyCas9 as "switches" have been selected for several years. The available inhibitors currently are all natural polypeptides inhibitors derived from phages, except one small molecule inhibitor. These natural inhibitors are challenging to obtain and are available in limited quantities, and the small molecule inhibitor is cytotoxic. Herein, we discover aptamers against the SpyCas9 protein, by coupling CE-SELEX within one-round pressure controllable selection strategy. One of the identified aptamers, Apt2, shows high affinity at the nanomolar level and leads for effective SpyCas9 enzymatic inhibition in vitro. It is predicted that Apt2 interacts with the HNH and RuvC domains of SpyCas9, competitively inhibiting the binding of substrate DNA to SpyCas9. The proposed aptamer inhibitor is the oligonucleotide inhibitor of SpyCas9, which has the potential in construction of the universal, simple and precise CRISPR-Cas9 system activity control strategy. Meanwhile, these aptamers could also be valuable tools for study of the functions of CRISPR/Cas9 and the related functional mechanisms.
Subject(s)
Aptamers, Nucleotide , Bacteriophages , Gene Editing , DNA/chemistry , Bacterial Proteins/metabolism , Aptamers, Nucleotide/metabolism , SELEX Aptamer TechniqueABSTRACT
High loading impact associated with heel strikes causes running injuries. This study aimed to investigate how loading impact is affected by midsole hardness and running surface type. Twelve young rear-foot runners ran at a fixed speed along an 18 m runway wearing shoes with different midsole hardness (Asker C-45, C-50, C-55, C-60, from soft to hard) and on two different surfaces (rubber and concrete). We quantified vertical average loading rate (VALR) and vertical impact peak force (VIPF). We conducted midsole × surface repeated-measures ANOVA on loading impact measures, and one-sample t-tests to compare VALR with a threshold value (80 BW·s-1). Midsole hardness and surface type mainly affected VALR. Although no significant effect of these variables was observed for VIPF magnitude, there were effects on time to VIPF and steps with VIPF. Several combinations of midsole and surface hardness reduced VALR below 80 BW·s-1: Asker C-45 with both surfaces, and Asker C-50 with a rubber surface. The combination of softer midsole and surface effectively reduced loading rates as shown by increased time to VIPF and reduced VALR. Combining softer midsole and surface results in the greatest cushioning, which demonstrates the benefit of considering both factors in reducing running injuries.
Subject(s)
Foot , Rubber , Hardness , Biomechanical Phenomena , ShoesABSTRACT
We aimed to examine the changes in balance performance, kinematic variables, and joint coordination of the lower extremities during the Y-balance learning task. Twenty female university students completed five consecutive blocks of Y-balance learning from days 3 to 7 (135 trials). Pre-tests and tests were performed on days 1 and 9. Maximum reach distance, peak joint angle, and joint coordination in the anterior (AL), posterolateral (PL), and posteromedial (PM) directions were measured to determine the efficacy of Y-balance performance. A repeated measures ANOVA was performed for the maximum reach distance across learning blocks to confirm whether learning had occurred. Our results indicated that the maximum reach distance on day 5 was longer than that on other learning days. The maximum reach distance significantly increased in the PL and PM directions after learning. The hip flexion (PL/PM), abduction (PM), internal rotation (PM), and external rotation (PL) angles increased after learning. The knee joint flexion angle increased in both AL and PL directions. Only the ankle dorsiflexion angle increased in the AL direction. Joint coordination indicated that the knee and hip joints performed simultaneously during internal rotation. Ankle-knee joint coordination was performed using dorsiflexion and flexion strategies. Statistical parametric mapping analysis indicated significant differences in the ankle sagittal plane in the AL direction, hip horizontal and hip/knee sagittal planes in the PL direction, and hip/knee sagittal and hip frontal/horizontal planes in the PM direction. These data suggest that the dynamic balance ability of the novice participants improved in relation to changes in coordination patterns after learning. The results of this study can be applied to other populations to improve their dynamic balance and prevent fall injuries.
Subject(s)
Knee Joint , Lower Extremity , Humans , Female , Movement , Ankle Joint , Range of Motion, Articular , Biomechanical Phenomena , Hip JointABSTRACT
This study investigated the effects of amygdalin (AMY, a cyanogenic glycoside widely distributed in the fruits and seeds of Rosaceae plants) on cardiac performance and ventricular remodeling in a rat model of myocardial infarction (MI). We also investigated whether the combination of AMY with exercise training (ExT) has a beneficial synergistic effect in treating MI rats. MI was induced by the ligation of the left anterior descending coronary artery in male SD rats. ExT or AMY treatment was started 1 week after MI and continued for 1 week (short-term) or 8 weeks (long-term). Cardiac function was evaluated by echocardiographic and hemodynamic parameters. Heart tissues were harvested and subjected to 2,3,5-triphenyl-tetrazolium chloride, Masson's trichrome, hematoxylin-eosin, and immunohistochemical staining. Gene expression was determined by quantitative polymerase chain reaction. Western blot gave a qualitative assessment of protein levels. AMY or ExT improved cardiac function and reduced infarct size in MI rats. AMY or ExT also suppressed myocardial fibrosis and attenuated inflammation in the infarct border zone of hearts from MI rats, as evidenced by inhibition of collagen deposition, inflammatory cell infiltration, and pro-inflammatory markers (interleukin 1ß, interleukin 6, tumor necrosis factor-α, and cyclooxygenase 2). Notably, the effects of AMY combined with ExT were superior to those of AMY alone or ExT alone. Mechanistically, these beneficial functions were correlated with the inhibition of MI-induced activation of the transforming growth factor-ß/Smad pathway. Collectively, AMY and ExT exert a synergistic effect on improving cardiac performance and ameliorating cardiac inflammation and fibrosis after MI, and the effects of long-term intervention were better than short-term intervention.
Subject(s)
Amygdalin , Myocardial Infarction , Animals , Rats , Rats, Sprague-Dawley , Amygdalin/pharmacology , Myocardial Infarction/therapy , Inflammation/therapy , FibrosisABSTRACT
Despite recent advances in cryo-electron microscopy and artificial intelligence-based model predictions, a significant fraction of structure determinations by macromolecular crystallography still requires experimental phasing, usually by means of single-wavelength anomalous diffraction (SAD) techniques. Most synchrotron beamlines provide highly brilliant beams of X-rays of between 0.7 and 2 Å wavelength. Use of longer wavelengths to access the absorption edges of biologically important lighter atoms such as calcium, potassium, chlorine, sulfur and phosphorus for native-SAD phasing is attractive but technically highly challenging. The long-wavelength beamline I23 at Diamond Light Source overcomes these limitations and extends the accessible wavelength range to λ = 5.9 Å. Here we report 22 macromolecular structures solved in this extended wavelength range, using anomalous scattering from a range of elements which demonstrate the routine feasibility of lighter atom phasing. We suggest that, in light of its advantages, long-wavelength crystallography is a compelling option for experimental phasing.
ABSTRACT
Introduction: Musculoskeletal simulation has been widely used to analyze athletes' movements in various competitive sports, but never in ski jumping. Aerodynamic forces during ski jumping take-off have been difficult to account for in dynamic simulation. The purpose of this study was to establish an efficient approach of musculoskeletal simulation of ski jumping take-off considering aerodynamic forces and to analyze the muscle function and activity. Methods: Camera-based marker-less motion capture was implemented to measure the take-off kinematics of eight professional jumpers. A suitable full-body musculoskeletal model was constructed for the simulation. A method based on inverse dynamics iteration was developed and validated to estimate the take-off ground reaction force. The aerodynamic forces, which were calculated based on body kinematics and computational fluid dynamics simulations, were exerted on the musculoskeletal model as external forces. The activation and joint torque contributions of lower extremity muscles were calculated through static optimization. Results: The estimated take-off ground reaction forces show similar trend with the results from past studies. Although overall inconsistencies between simulated muscle activation and EMG from previous studies were observed, it is worth noting that the activation of the tibialis anterior, gluteus maximus, and long head of the biceps femoris was similar to specific EMG results. Among lower extremity extensors, soleus, vastus lateralis, biceps femoris long head, gluteus maximus, and semimembranosus showed high levels of activation and joint extension torque contribution. Discussion: Results of this study advanced the understanding of muscle action during ski jumping take-off. The simulation approach we developed may help guide the physical training of jumpers for improved take-off performance and can also be extended to other phases of ski jumping.
ABSTRACT
Viruses with single-stranded, positive-sense (+) RNA genomes incur high numbers of errors during replication, thereby creating diversified genome populations from which new, better adapted viral variants can emerge. However, a definitive error rate is known for a relatively few (+) RNA plant viruses, due to challenges to account for perturbations caused by natural selection and/or experimental set-ups. To address these challenges, we developed a new approach that exclusively profiled errors in the (-)-strand replication intermediates of turnip crinkle virus (TCV), in singly infected cells. A series of controls and safeguards were devised to ensure errors inherent to the experimental process were accounted for. This approach permitted the estimation of a TCV error rate of 8.47 X 10-5 substitution per nucleotide site per cell infection. Importantly, the characteristic error distribution pattern among the 50 copies of 2,363-base-pair cDNA fragments predicted that nearly all TCV (-) strands were products of one replication cycle per cell. Furthermore, some of the errors probably elevated error frequencies by lowering the fidelity of TCV RNA-dependent RNA polymerase, and/or permitting occasional re-replication of progeny genomes. In summary, by profiling errors in TCV (-)-strand intermediates incurred during replication in single cells, this study provided strong support for a stamping machine mode of replication employed by a (+) RNA virus.
Subject(s)
Carmovirus , RNA Viruses , Carmovirus/genetics , RNA, Viral/genetics , RNA, Viral/metabolism , Mutation Rate , RNA Viruses/genetics , RNA-Dependent RNA Polymerase/metabolism , Virus Replication/geneticsABSTRACT
Superinfection exclusion (SIE) is an antagonistic interaction between identical or closely related viruses in host cells. Previous studies by us and others led to the hypothesis that SIE was elicited by one or more proteins encoded in the genomes of primary viruses. Here, we tested this hypothesis using Turnip mosaic virus (TuMV), a member of the genus Potyvirus of the family Potyviridae, with significant economic consequences. To this end, individual TuMV-encoded proteins were transiently expressed in the cells of Nicotiana benthamiana leaves, followed by challenging them with a modified TuMV expressing the green fluorescent protein (TuMV-GFP). Three days after TuMV-GFP delivery, these cells were examined for the replication-dependent expression of GFP. Cells expressing TuMV P1, HC-Pro, 6K1, CI, 6K2, NIa-VPg, NIb, or CP proteins permitted an efficient expression of GFP, suggesting that these proteins failed to block the replication of a superinfecting TuMV-GFP. By contrast, N. benthamiana cells expressing TuMV P3 or NIa-Pro did not express visible GFP fluorescence, suggesting that both of them could elicit potent SIE against TuMV-GFP. The SIE elicitor activity of P3 and NIa-Pro was further confirmed by their heterologous expression from a different potyvirus, potato virus A (PVA). Plants systemically infected with PVA variants expressing TuMV P3 or NIa-Pro blocked subsequent infection by TuMV-GFP. A +1-frameshift mutation in P3 and NIa-Pro cistrons facilitated superinfection by TuMV-GFP, suggesting that the P3 and NIa-Pro proteins, but not the RNA, are involved in SIE activity. Additionally, deletion mutagenesis identified P3 amino acids 3 to 200 of 352 and NIa-Pro amino acids 3 to 40 and 181 to 242 of 242 as essential for SIE elicitation. Collectively, our study demonstrates that TuMV encodes two spatially separated proteins that act independently to exert SIE on superinfecting TuMV. These results lay the foundation for further mechanistic interrogations of SIE in this virus.
Subject(s)
Potyviridae , Potyvirus , Superinfection , Potyvirus/genetics , Plant Diseases , NicotianaABSTRACT
BACKGROUND: Tendon-derived stem cells (TDSCs) are proposed as a potential cell-seed for the treatment of tendon injury due to their tenogenic differentiation potential. In this work, we defined the action of long non-coding RNA (lncRNA) muscle differentiation 1 (LINCMD1) in tenogenic differentiation of human TDSCs (hTDSCs). METHODS: Quantitative real-time PCR (qRT-PCR) was used to assess the levels of LINCMD1, microRNA (miR)-342-3p, and early growth response-1 (EGR1) mRNA. Cell proliferation was detected by the XTT colorimetric assay. Protein expression was quantified by western blot. hTDSCs were grown in an osteogenic medium to induce osteogenic differentiation, and the extent of osteogenic differentiation was assessed by Alizarin Red Staining (ARS). The activity of alkaline phosphatase (ALP) was measured by the ALP Activity Assay Kit. Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were used to evaluate the direct relationship between miR-342-3p and LINCMD1 or EGR1. RESULTS: Our results showed that enforced expression of LINCMD1 or suppression of miR-342-3p accelerated the proliferation and tenogenic differentiation and reduced osteogenic differentiation of hTDSCs. LINCMD1 regulated miR-342-3p expression by binding to miR-342-3p. EGR1 was identified as a direct and functional target of miR-342-3p, and knockdown of EGR1 reversed the effects of miR-342-3p suppression on cell proliferation and tenogenic and osteogenic differentiation. Furthermore, the miR-342-3p/EGR1 axis mediated the regulation of LINCMD1 on hTDSC proliferation and tenogenic and osteogenic differentiation. CONCLUSION: Our study suggests the induction of LINCMD1 in tenogenic differentiation of hTDSCs through miR-342-3p/EGR1 axis.