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Abstract The middle and lower reaches of the Yangtze River serve as principal rice production bases in China, yet the biodiversity and ecological processes of bacterial communities in paddy soils are not well understood. This study explores the diversity, composition, ecological function, and assembly processes of abundant and rare bacterial communities in paddy soils. A total of 129 paddy soil samples from 43 sites along the middle and lower reaches of the Yangtze River were collected and analyzed using NovaSeq sequencing. The results showed that the dominant phylum for both abundant and rare taxa was Proteobacteria, with a greater relative abundance of the abundant taxa. The diversity of the abundant community was lower than that of the rare community. Soil properties and geographic variables explained more of the variation in the abundant community than in the rare community. The rare community exhibited a significant distance‐decay relationship. The assembly of the abundant community was more influenced by stochastic processes, although both the abundant and rare communities were governed by stochastic processes. It is concluded that both abundant and rare bacterial communities exhibit differing biogeographic patterns, yet they undergo similar ecological processes in the paddy soils along the middle and lower reaches of the Yangtze River. These observations offer a theoretical framework for a deeper comprehension of the function of both abundant and rare bacteria, as well as the development and preservation of soil bacterial diversity within agricultural ecosystems.

Bare soil will cause soil erosion and contribute to air pollution through the generation of dust, making the timely and effective monitoring of bare soil an urgent requirement for environmental management. Although there have been some researches on bare soil extraction using high-resolution remote sensing images, great challenges still need to be solved, such as complex background interference and small-scale problems. In this regard, the Hybrid Attention Network (HA-Net) is proposed for automatic extraction of bare soil from high-resolution remote sensing images, which includes the encoder and the decoder. In the encoder, HA-Net initially utilizes BoTNet for primary feature extraction, producing four-level features. The extracted highest-level features are then input into the constructed Spatial Information Perception Module (SIPM) and the Channel Information Enhancement Module (CIEM) to emphasize the spatial and channel dimensions of bare soil information adequately. To improve the detection rate of small-scale bare soil areas, during the decoding stage, the Semantic Restructuring-based Upsampling Module (SRUM) is proposed, which utilizes the semantic information from input features and compensate for the loss of detailed information during downsampling in the encoder. An experiment is performed based on high-resolution remote sensing images from the China–Brazil Resources Satellite 04A. The results show that HA-Net obviously outperforms several excellent semantic segmentation networks in bare soil extraction. The average precision and IoU of HA-Net in two scenes can reach 90.9% and 80.9%, respectively, which demonstrates the excellent performance of HA-Net. It embodies the powerful ability of HA-Net for suppressing the interference from complex backgrounds and solving multiscale issues. Furthermore, it may also be used to perform excellent segmentation tasks for other targets from remote sensing images.

During long-duration dynamic loads, such as wind loads or seismic effects, the internal temperature and pressure of a damping cylinder escalate rapidly, which induce shifts in the mechanical attributes of viscous fluid dampers (VFDs). This study investigated the mechanical performance of VFD considering the coupling effects of temperature and pressure under long-duration loads. First, we analyzed the mechanical and energy-dissipation performances of the dampers based on the dynamic mechanical tests considering different loading frequencies, displacement amplitude, and loading cycles. The experimental results indicated that both temperature and pressure influenced the output of the dampers, and in the sealed environment of the damper pip, temperature and pressure exerted mutual influence. Furthermore, the relationship between the damping coefficient and temperature–pressure coupling effects was obtained. Subsequently, an improved mathematical model for the mechanical performance of a gap-type VFD was proposed by considering the macroscopic energy balance of the entire fluid within the damper. Finally, the accuracy of the mathematical model for VFD under long-duration dynamic loads was validated by comparing the computational results with the experimental data.

ABSTRACT Among various environmental stresses, the depletion of inorganic phosphate, which is an important signaling molecule that modulates virulence production, is notable. In our previous study, we demonstrated that lapA, encoding alkaline phosphatase, plays a key role in Pseudomonas aeruginosa biofilm formation in porcine skin explants, an ex vivo chronic wound model. However, the mechanism by which lapA participates in the virulence and biofilm formation of P. aeruginosa remains unclear. In the present study, the lapA deletion mutant was constructed in P. aeruginosa PAO1, and the phenotypic characterization was assessed. Our data demonstrated that phosphate depletion stress significantly enhanced elastase activity, hemolysis, rhamnolipid production, swarming and swimming motilities, and 3-oxo-C12-homoserine lactone (HSL) production in P. aeruginosa. Moreover, phosphate starvation evidently induced the virulence of P. aeruginosa in Caenorhabditis elegans (C. elegans) through fast-kill and slow-kill infections. Deletion of the lapA gene led to broad phenotypic changes, including reduced elastase activity, swimming motility, C4-HSL, and 3-oxo-C12-HSL production, as well as increased chitinase activity and rhamnolipid production under phosphate depletion stress, which was unrelated to C4-HSL. Deletion of the lapA gene also significantly inhibited PAO1 biofilm formation in porcine skin explants by reducing the expression levels of las and rhl quorum sensing systems and extracellular polymeric substance synthesis. Finally, lapA deletion also reduced the virulence of PAO1 in C. elegans in fast-kill and slow-kill infection assays. Thus, the findings suggest that targeting lapA can help control biofilm formation and reduce virulence in nonhealing-infected wounds, where P. aeruginosa is a persistent problem. IMPORTANCE Our previous study demonstrated that the expression of lapA was induced under phosphate depletion conditions, but its roles in virulence and biofilm formation by Pseudomonas aeruginosa remain largely unknown. This study presents a systematic investigation of the roles of lapA in virulence induction and biofilm formation by constructing a lapA-deficient strain with P. aeruginosa PAO1. The results showed that deletion of the lapA gene evidently reduced elastase activity, swimming motility, C4-HSL, and 3-oxo-C12-HSL production, and increased rhamnolipid production under phosphate depletion stress. Moreover, lapA gene deletion inhibited PAO1 biofilm formation in porcine skin explants by reducing the expression levels of las and rhl quorum sensing systems and extracellular polymeric substance synthesis. Finally, lapA gene deletion also reduced the virulence of PAO1 in Caenorhabditis elegans in fast-kill and slow-kill infection assays. This study provides insights into the roles of lapA in modulating P. aeruginosa virulence and biofilm formation under phosphate depletion stress.

This article presents a Lyapunov precise integral-based analysis method for seismic structures with added viscous fluid dampers. This study uses the full stress algorithm as the optimization method, considering the mean square of interstory drifts as the optimization objective, the position of the damper as the optimization object, and the random vibration analysis method as the calculation method to optimize seismic frame structures with viscous dampers. A precise integral solution is derived for the Lyapunov equation based on the general expression of the Lyapunov differential equation for the damping system under the excitation of a nonstationary stochastic process using two types of modulation functions: g(t)=1 and g(t)=t. Finally, the optimal damping arrangement is achieved using this method with a six-layer non-eccentric planar frame. In addition, the optimization results of this study are verified with those in the literature using time-history analysis, which verifies the feasibility and effectiveness of the proposed method. This study provides a method for the optimal configuration of dampers for seismic response of structures, which is beneficial for engineering applications and the protection of seismic structures.

The additional damping ratio (ADR) is an important indicator for evaluating the damping effect of structures with energy-dissipation devices. Most existing methods for determining the ADR require an analysis of the structural dynamic response and complex iterative calculations. An innovative simplified calculation method for determining the ADR of a structure supplemented by nonlinear viscous dampers is proposed. This method does not require the dynamic response of the structure to be calculated and only requires the structural characteristics, excitation frequency, and damper parameters. In this study, several typical calculation methods for the ADR were analysed. Then, a calculation formula for the ADR was derived with consideration of harmonic excitation under the condition where the excitation frequency is equal to the structural natural frequency, without calculation of the structural dynamic response or an iterative process. The effect of the excitation frequency on the calculated value of the ADR with different damping exponents was studied. Accordingly, the response spectrum average period (RSAP) was considered as the excitation period of ground motion to evaluate the excitation frequency, and a simplified calculation method for the ADR considering the effect of the excitation frequency characterised by the RSAP of the ground motion was established. Finally, the accuracy and effectiveness of the proposed method were verified by comparison with ADRs calculated using other methods.

In this paper, the finite-frequency fault detection filter design for discrete-time switched systems is investigated. The frequencies of the faults and the unknown disturbance input are assumed to be finite and in three known intervals, qualified as low-, middle-, and high-frequency intervals. Based on the switched Lyapunov function and the generalized Kalman-Yakubovic-Popov lemma, efficient conditions are obtained to guarantee the existence of a finite-frequency fault detection filter, such that the error system is asymptotically stable with an H∞/H- performance index. Finally, a chemical reactor control system is employed to illustrate the obtained techniques.

The accurate prediction of nonlinear structural behaviors under different seismic intensities is an important basis for seismic resilience assessments of building structures. The moment–rotation relationship is often used to characterize the seismic performance of connections, and is widely used in high-efficiency nonlinear structural analysis. In this paper, a method of calculating the curve using a four-linear equivalent model is presented, aiming to quantify the characteristic point parameters of the moment–rotation curves of monolithic precast beam–column (MPBC) connections for engineering design purposes. The method considered the contribution of the elastic flexure of beams and columns, the relative slip of beam longitudinal bars in the core zone, and the formation of plastic hinges at beam ends to the total deflection. Due to the presence of local complex configurations in MPBC connections, the fine fiber section method was used for moment–curvature analysis of critical beam sections. The determination of the sectional analysis processes was controlled by the strain of steel bars or concrete or their coupling effect. In addition, a two-step method was proposed to construct the moment–rotation relationship of cruciform beam–column connections for solving the deformation compatibility of beams on both sides of the column caused by asymmetric reinforcement and the strength difference between new and old concrete. To reflect the current manufacturing level of MPBC connections, 58 representative specimens reported in recent years were analyzed and classified as type 1–5. All types of MPBC connections and their 18 cast-in situ counterparts were calculated using the proposed method for both verification and quantification. The verification showed that the proposed method had good applicability to both cast-in situ and precast beam–column connections. The quantification showed that the characteristic point parameters were slightly different between these two connections. Accordingly, modification coefficients were suggested for MPBC connections to facilitate design.

The intensity non-stationarity is one of the basic characteristics of ground motions, the influences of which on the dynamic responses of structures is a pressing issue in the field of earthquake engineering. The BP neural network modified by the genetic algorithm was adopted in this research to investigate the influence of intensity nonstationary inputs on the structural dynamic responses from a new perspective. Firstly, many training data were generated from the prediction formula of dynamic response. The BP neural network was then pre-trained by sparsely selected data to optimize the initial weights and biases. Finally, the BP neural network was trained by all data, and the mean square error of predicted responses compared with the target response were less than 10−5. The calculation formula of sensitivity was also derived here to quantify the influence of the input change on the output. The presented method combines the advantages of neural networks in nonlinear multi-variable fitting and provides a new perspective for the study of earthquake nonstationary characteristics and their influence on the structural dynamic responses.

At present, the engineering designers generally design and analyze the precast structural models according to the equivalent cast-in-situ principle, and have a vague understanding of non-identical problems. However, these issues cannot be ignored, especially for high-intensity areas. This paper considers the differences of the hysteretic relationship between two typical precast joints and cast-in-situ (RC) joints, and researches the influence of these differences on the seismic response of frame structures. For the monolithic precast joint, the force mechanism was analyzed based on its assembly form, and the differences with the RC joint in the testing phenomena were explained accordingly. The dimensionless hysteresis models of two types of joints were proposed, and the rationality of the monolithic precast joint model was verified according to the existing experimental results. Different performances of joints were realized by assigning the constitutive models calculated from sectional reinforcement to the spring elements of analysis models. Considering two possible performance deficiencies of each type of precast joint separately, a total of seven structural analysis models were formed. Nonlinear static analysis and dynamic time-history analysis methods were adopted to reveal the differences between precast frames and the RC frame in terms of structural capacity curve, displacement response, ductility demands of components and structural residual deformation. The results showed that under strong seismic excitation, the response differences between precast frames and the RC frame were significant, so it is worthwhile to establish nonlinear models suitable for precast frames in seismic analysis. This study is valuable for understanding and distinguishing the nonlinear response of precast frames and traditional RC frames.

The improvement effect of a new strengthening strategy on dynamic action of masonry structure, by installing prefabricated concrete walls on the outer facades, is validated by shaking table test presented in this paper. We carried out dynamic tests of two geometrically identical five-story reduced scaled models, including an unstrengthened and a strengthened masonry model. The experimental analysis encompasses seismic performances such as cracking patterns, failure mechanisms, amplification factors of acceleration, and displacements. The results show that the strengthened masonry structure shows much more excellent seismic capacity when compared with the unstrengthened one.

The seismic response control of multistory frame structures using optimally placed viscoelastic dampers (VEDs) within consideration of soil-structure interaction (SSI) effect is investigated in this paper. The system is assumed to be elastic and responses of the system are obtained in frequency domain for stationary random seismic excitations. The optimal designs of VEDs in structures are achieved through genetic algorithm (GA) by minimizing the maximum response quantities of the system for a determined total amount of viscoelastic material. Two typical elastic multistory frame structures with different soil types and foundation embedment ratios are considered to demonstrate the optimization process. It is shown that the VEDs have the best control of the structural response for fixed base condition and the control efficiency decreases as the soil becomes softer. The optimal placement types of VEDs for different soil types differ from each other. With the decrease of soil stiffness, the optimal locations of VEDs have a tendency to shift to top floors.

Abstract Background Cytosolic calcium overload contributes to muscle degradation in Duchenne muscular dystrophy (DMD). The sarcoplasmic reticulum (SR) is the primary calcium storage organelle in muscle. The sarco-endoplasmic reticulum ATPase (SERCA) pumps cytosolic calcium to the SR during muscle relaxation. Calcium is kept in the SR by calcium-binding proteins. Methods Given the importance of the canine DMD model in translational studies, we examined transcriptional changes of SERCA (SERCA1 and SERCA2a), SERCA regulators (phospholamban, sarcolipin, myoregulin, and dwarf open reading frame), and SR calcium-binding proteins (calreticulin, calsequestrin 1, calsequestrin 2, and sarcalumenin) in skeletal muscle (diaphragm and extensor carpi ulnaris) and heart (left ventricle) in normal and affected male dogs by droplet digital PCR before the onset (≤ 2-m-old), at the active stage (8 to 16-m-old), and at the terminal stage (30 to 50-m-old) of the disease. Since many of these proteins are expressed in a fiber type-specific manner, we also evaluated fiber type composition in skeletal muscle. Results In affected dog skeletal muscle, SERCA and its regulators were down-regulated at the active stage, but calcium-binding proteins (except for calsequestrin 1) were upregulated at the terminal stage. Surprisingly, nominal differences were detected in the heart. We also examined whether there exists sex-biased expression in 8 to 16-m-old dogs. Multiple transcripts were significantly downregulated in the heart and extensor carpi ulnaris muscle of female dogs. In fiber type analysis, we found significantly more type I fiber in the diaphragm of 8 to 16-m-old affected dogs, and significantly more type II fibers in the extensor carpi ulnaris of 30 to 50-m-old affected dogs. However, no difference was detected between male and female dogs. Conclusions Our study adds new knowledge to the understanding of muscle calcium regulation in normal and dystrophic canines.

Excessive cytosolic calcium accumulation contributes to muscle degeneration in Duchenne muscular dystrophy (DMD). Sarco/endoplasmic reticulum calcium ATPase (SERCA) is a sarcoplasmic reticulum (SR) calcium pump that actively transports calcium from the cytosol into the SR. We previously showed that adeno-associated virus (AAV)-mediated SERCA2a therapy reduced cytosolic calcium overload and improved muscle and heart function in the murine DMD model. Here, we tested whether AAV SERCA2a therapy could ameliorate muscle disease in the canine DMD model. 7.83 × 1013 vector genome particles of the AAV vector were injected into the extensor carpi ulnaris (ECU) muscles of four juvenile affected dogs. Contralateral ECU muscles received excipient. Three months later, we observed widespread transgene expression and significantly increased SERCA2a levels in the AAV-injected muscles. Treatment improved SR calcium uptake, significantly reduced calpain activity, significantly improved contractile kinetics, and significantly enhanced resistance to eccentric contraction-induced force loss. Nonetheless, muscle histology was not improved. To evaluate the safety of AAV SERCA2a therapy, we delivered the vector to the ECU muscle of adult normal dogs. We achieved strong transgene expression without altering muscle histology and function. Our results suggest that AAV SERCA2a therapy has the potential to improve muscle performance in a dystrophic large mammal.

While adeno-associated viral (AAV) vectors are successfully used in a variety of in vivo gene therapy applications, they continue to be hampered by the immune system. Here, we sought to identify innate and cytokine signaling pathways that promote CD8+ T-cell responses against the transgene product upon AAV1 vector administration to murine skeletal muscle. Eliminating just one of several pathways (including DNA sensing via TLR9, IL-1 receptor signaling, and possibly endosomal sensing of double-stranded RNA) substantially reduced the CD8+ T-cell response at lower vector doses but was surprisingly ineffective at higher doses. Using genetic, antibody-mediated, and vector engineering approaches, we show that blockade of at least two innate pathways is required to achieve an effect at higher vector doses. Concurrent blockade of IL-1R1 > MyD88 and TLR9 > MyD88 > type I IFN > IFNaR pathways was often but not always synergistic and had limited utility in preventing antibody formation against the transgene product. Further, even low-frequency CD8+ T-cell responses could eliminate transgene expression, even in MyD88- or IL-1R1-deficient animals that received a low vector dose. However, we provide evidence that CpG depletion of vector genomes and including TLR9 inhibitory sequences can synergize. When this construct was combined with the use of a muscle-specific promoter, transgene expression in muscle was sustained with minimal local or systemic CD8+ T-cell response. Thus, innate immune avoidance/blockade strategies by themselves, albeit helpful, may not be sufficient to prevent destructive cellular responses in muscle gene transfer because of the redundancy of immune-activating pathways.

Adeno-associated virus (AAV) vectors are used for correcting multiple genetic disorders. Although the goal is to achieve lifelong correction with a single vector administration, the ability to redose would enable the extension of therapy in cases in which initial gene transfer is insufficient to achieve a lasting cure, episomal vector forms are lost in growing organs of pediatric patients, or transgene expression is diminished over time. However, AAV typically induces potent and long-lasting neutralizing antibodies (NAbs) against capsid that prevents re-administration. To prevent NAb formation in hepatic AAV8 gene transfer, we developed a transient B cell-targeting protocol using a combination of monoclonal Ab therapy against CD20 (for B cell depletion) and BAFF (to slow B cell repopulation). Initiation of immunosuppression before (rather than at the time of) vector administration and prolonged anti-BAFF treatment prevented immune responses against the transgene product and abrogated prolonged IgM formation. As a result, vector re-administration after immune reconstitution was highly effective. Interestingly, re-administration before the immune system had fully recovered achieved further elevated levels of transgene expression. Finally, this immunosuppression protocol reduced Ig-mediated AAV uptake by immune cell types with implications to reduce the risk of immunotoxicities in human gene therapy with AAV.

Some acute exercise effects are influenced by postexercise (PEX) diet, and these diet-effects are attributed to differential glycogen resynthesis. However, this idea is challenging to test rigorously. Therefore, we devised a novel genetic model to modify muscle glycogen synthase 1 (GS1) expression in rat skeletal muscle with an adeno-associated virus (AAV) short hairpin RNA knockdown vector targeting GS1 (shRNA-GS1). Contralateral muscles were injected with scrambled shRNA (shRNA-Scr). Muscles from exercised (2-hour-swim) and time-matched sedentary (Sed) rats were collected immediately postexercise (IPEX), 5-hours-PEX (5hPEX), or 9-hours-PEX (9hPEX). Rats in 5hPEX and 9hPEX experiments were refed (RF) or not-refed (NRF) chow. Muscles were analyzed for glycogen, abundance of metabolic proteins (pyruvate dehydrogenase kinase 4, PDK4; peroxisome proliferator-activated receptor γ coactivator-1α, PGC1α; hexokinase II, HKII; glucose transporter 4, GLUT4), AMP-activated protein kinase phosphorylation (pAMPK), and glycogen metabolism-related enzymes (glycogen phosphorylase, PYGM; glycogen debranching enzyme, AGL; glycogen branching enzyme, GBE1). shRNA-GS1 versus paired shRNA-Scr muscles had markedly lower GS1 abundance. IPEX versus Sed rats had lower glycogen and greater pAMPK, and neither of these IPEX-values differed for shRNA-GS1 versus paired shRNA-Scr muscles. IPEX versus Sed groups did not differ for abundance of metabolic proteins, regardless of GS1 knockdown. Glycogen in RF-rats was lower for shRNA-GS1 versus paired shRNA-Scr muscles at both 5hPEX and 9hPEX. HKII protein abundance was greater for 5hPEX versus Sed groups, regardless of GS1 knockdown or diet, and despite differing glycogen levels. At 9hPEX, shRNA-GS1 versus paired shRNA-Scr muscles had greater PDK4 and PGC1α abundance within each diet group. However, the magnitude of PDK4 or PGC1α changes was similar in each diet group regardless of GS1 knockdown although glycogen differed between paired muscles only in RF-rats. In summary, we established a novel genetic approach to investigate the relationship between muscle glycogen and other exercise effects. Our results suggest that exercise-effects on abundance of several metabolic proteins did not uniformly correspond to differences in postexercise glycogen.

The first generation of adeno-associated virus (AAV) vectors composed of the naturally occurring capsids and genomes, although effective in some instances, are unlikely to be optimal for gene therapy in humans. The use of the first generation of two different AAV serotype vectors (AAV9 and AAVrh74) in four separate clinical trials failed to be effective in patients with Duchenne muscular dystrophy, although some efficacy was observed in a subset of patients with AAVrh74 vectors leading to US Food and Drug Administration approval (Elevidys). In two trials with the first generation of AAV9 vectors, several serious adverse events were observed, including the death of a patient in one trial, and more recently, in the death of a second patient in an N-of-1 clinical trial. In a fourth trial with the first generation of AAVrh74 vectors, myositis and myocarditis were also observed. Here, we report that capsid- and genome-modified optimized AAVrh74 vectors are significantly more efficient in transducing primary human skeletal muscle cells in vitro and in all major muscle tissues in vivo following systemic administration in a murine model. The availability of optimized AAVrh74 vectors promises to be safe and effective in the potential gene therapy of muscle diseases in humans.

The intensity non-stationarity is one of the most important features of earthquake records. Modeling of this feature is significant to the generation of arti- ficial earthquake waves. Based on the theory of phase difference spectrum, an intensity non-stationary envelope function with log-normal form is proposed. Through a tremendous amount of earthquake records downloaded on Kik-net, a parameter fitting procedure using the genetic algorithm is conducted to obtain the value of model parameters under different magnitudes, epicenter distances and site conditions. A numerical example is presented to describe the procedure of generating fully non-stationary ground motions via spectral representation, and the mean EPSD (evolutionary power spectral density) of simulated waves is proved to agree well with the target EPSD. The results show that the proposed model is capable of describing the intensity non-stationary features of ground motions, and it can be used in structural anti-seismic analysis and ground motion simulation. [ABSTRACT FROM AUTHOR]

Abstract This study aims to develop a 4-limb canine gait analysis system using wireless inertial measurement units (IMUs). 3D printed sensor holders were designed to ensure quick and consistent sensor mounting. Signal analysis algorithms were developed to automatically determine the timing of swing start and end in a stride. To evaluate the accuracy of the new system, a synchronized study was conducted in which stride parameters in four dogs were measured simultaneously using the 4-limb IMU system and a pressure-sensor based walkway gait system. The results showed that stride parameters measured in both systems were highly correlated. Bland–Altman analyses revealed a nominal mean measurement bias between the two systems in both forelimbs and hindlimbs. Overall, the disagreement between the two systems was less than 10% of the mean value in over 92% of the data points acquired from forelimbs. The same performance was observed in hindlimbs except for one parameter due to small mean values. We demonstrated that this 4-limb system could successfully visualize the overall gait types and identify rapid gait changes in dogs. This method provides an effective, low-cost tool for gait studies in veterinary applications or in translational studies using dog models of neuromuscular diseases.

Background Cardiomyopathy is a leading health threat in Duchenne muscular dystrophy (DMD). Cytosolic calcium upregulation is implicated in DMD cardiomyopathy. Calcium is primarily removed from the cytosol by the sarcoendoplasmic reticulum calcium ATPase (SERCA). SERCA activity is reduced in DMD. Improving SERCA function may treat DMD cardiomyopathy. Dwarf open reading frame (DWORF) is a recently discovered positive regulator for SERCA, hence, a potential therapeutic target. Methods and Results To study DWORF's involvement in DMD cardiomyopathy, we quantified DWORF expression in the heart of wild‐type mice and the mdx model of DMD. To test DWORF gene therapy, we engineered and characterized an adeno‐associated virus serotype 9–DWORF vector. To determine if this vector can mitigate DMD cardiomyopathy, we delivered it to 6‐week‐old mdx mice (6×1012 vector genome particles/mouse) via the tail vein. Exercise capacity, heart histology, and cardiac function were examined at 18 months of age. We found DWORF expression was significantly reduced at the transcript and protein levels in mdx mice. Adeno‐associated virus serotype 9–DWORF vector significantly enhanced SERCA activity. Systemic adeno‐associated virus serotype 9‐DWORF therapy reduced myocardial fibrosis and improved treadmill running, electrocardiography, and heart hemodynamics. Conclusions Our data suggest that DWORF deficiency contributes to SERCA dysfunction in mdx mice and that DWORF gene therapy holds promise to treat DMD cardiomyopathy.

The Cas9-specific T cell response has been speculated to impair CRISPR therapy. Here, the authors show that local and systemic AAV CRISPR therapy induces cytotoxic killing and eliminates rescued dystrophin in canine models of Duchenne muscular dystrophy.

Abstract The University of Missouri (MU) has established a colony of dystrophin‐deficient dogs with a mixed breed background to mirror the variable pathologic effects of dystrophinopathies between persons of a given kindred to further the understanding of the genetic and molecular basis of the variable phenotype; thus to facilitate discovery of an effective therapeutic strategy. Herein we report the phenotype and genotype of a normal‐appearing 10‐month‐old colony female that died suddenly. At necropsy examination, there were reduced skeletal and laryngeal muscle volume and mild dilatation of the oesophagus. Microscopic findings consisted of extensive degeneration and regeneration of the axial skeletal, tongue, oesophageal, and laryngeal muscles that were characterized by considerable central nucleation, individual fibre mineralization and interstitial fibrosis. The myocardial findings were limited to infiltration of adipose cells in the interstitium. The female dog was a compound heterozygote with one X chromosome carrying a point mutation in intron 6 of the dystrophin gene and the other X chromosome carrying a repetitive element insertion in intron 13 of the dystrophin gene. Although the direct cause of death was uncertain, it might likely be due to sudden cardiac death as has been seen in Duchenne muscular dystrophy patients. This case demonstrated dystrophinopathy in female dogs that have no ameliorating normal X chromosome.

Vector production scale-up is a major barrier in systemic adeno-associated virus (AAV) gene therapy. Many scalable manufacturing methods have been developed. However, the potency of the vectors generated by these methods has rarely been compared with vectors made by transient transfection (TT), the most commonly used method in preclinical studies. In this study, we blindly compared therapeutic efficacy of an AAV9 micro-dystrophin vector generated by the TT method and scalable herpes simplex virus (HSV) system in a Duchenne muscular dystrophy mouse model. AAV was injected intravenously at 5 × 1014 (high), 5 × 1013 (medium), or 5 × 1012 (low) viral genomes (vg)/kg. Comparable levels of micro-dystrophin expression were observed at each dose in a dose-dependent manner irrespective of the manufacturing method. Vector biodistribution was similar in mice injected with either the TT or the HSV method AAV. Evaluation of muscle degeneration/regeneration showed equivalent protection by vectors made by either method in a dose-dependent manner. Muscle function was similarly improved in a dose-dependent manner irrespective of the vector production method. No apparent toxicity was observed in any mouse. Collectively, our results suggest that the biological potency of the AAV micro-dystrophin vector made by the scalable HSV method is comparable to that made by the TT method.

Adeno-associated virus (AAV) is one of the most important gene delivery vehicles for in vivo gene therapy. Intramuscular (i.m.) and intravascular (i.v.) injection are commonly used for AAV gene transfer. Unfortunately, the fate of AAV vectors following administration remains unclear at the histological level. Taking advantage of RNAscope, a recently developed in situ hybridization technique that can reveal high-resolution viral DNA localization information, in this study, we evaluated body-wide distribution of an AAV9 vector in the context of the cell and tissue microenvironments. We observed distinctive kinetics of cell and nuclear entry of the AAV DNA in striated muscle and liver following i.m. and i.v. injection. We also found characteristic distribution patterns of the AAV DNA in various histological structures in internal organs, including gonads and lymph nodes, following i.v. injection. Finally, we showed significantly body-wide spreading of the AAV DNA following i.m. injection. These results add a new dimension to our understanding of AAV transduction biology and provide a basis for assessing the full impact of AAV gene therapy.

Aged dystrophin-null canines are excellent models for studying experimental therapies for Duchenne muscular dystrophy, a lethal muscle disease caused by dystrophin deficiency. To establish the baseline, we studied the extensor carpi ulnaris (ECU) muscle in 15 terminal age (3-year-old) male affected dogs and 15 age/sex-matched normal dogs. Affected dogs showed histological and anatomical hallmarks of dystrophy, including muscle inflammation and fibrosis, myofiber size variation and centralized myonuclei, as well as a significant reduction of muscle weight, muscle-to-body weight ratio and muscle cross-sectional area. To rigorously characterize the contractile properties of the ECU muscle, we developed a novel in situ assay. Twitch and tetanic force, contraction and relaxation rate, and resistance to eccentric contraction-induced force loss were significantly decreased in affected dogs. Intriguingly, the time-to-peak tension and half-relaxation time were significantly shortened in affected dogs. Contractile kinetics predicted an unforeseen slow-to-fast myofiber-type switch, which we confirmed at the protein and transcript level. Our study establishes a foundation for studying long-term and late-stage therapeutic interventions in dystrophic canines. The unexpected myofiber-type switch highlights the complexity of muscle remodeling in dystrophic large mammals. This article has an associated First Person interview with the first author of the paper.

Abstract Background Delocalization of neuronal nitric oxide synthase (nNOS) from the sarcolemma leads to functional muscle ischemia. This contributes to the pathogenesis in cachexia, aging and muscular dystrophy. Mutations in the gene encoding dystrophin result in Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). In many BMD patients and DMD patients that have been converted to BMD by gene therapy, sarcolemmal nNOS is missing due to the lack of dystrophin nNOS-binding domain. Methods Dystrophin spectrin-like repeats 16 and 17 (R16/17) is the sarcolemmal nNOS localization domain. Here we explored whether R16/17 protein therapy can restore nNOS to the sarcolemma and prevent functional ischemia in transgenic mice which expressed an R16/17-deleted human micro-dystrophin gene in the dystrophic muscle. The palmitoylated R16/17.GFP fusion protein was conjugated to various cell-penetrating peptides and produced in the baculovirus-insect cell system. The best fusion protein was delivered to the transgenic mice and functional muscle ischemia was quantified. Results Among five candidate cell-penetrating peptides, the mutant HIV trans-acting activator of transcription (TAT) protein transduction domain (mTAT) was the best in transferring the R16/17.GFP protein to the muscle. Systemic delivery of the mTAT.R16/17.GFP protein to micro-dystrophin transgenic mice successfully restored sarcolemmal nNOS without inducing T cell infiltration. More importantly, R16/17 protein therapy effectively prevented treadmill challenge-induced force loss and improved muscle perfusion during contraction. Conclusions Our results suggest that R16/17 protein delivery is a highly promising therapy for muscle diseases involving sarcolemmal nNOS delocalizaton.

Duchenne muscular dystrophy (DMD) is an X-linked muscle-wasting disease caused by the loss of dystrophin. DMD is associated with muscle degeneration, necrosis, inflammation, fatty replacement, and fibrosis, resulting in muscle weakness, respiratory and cardiac failure, and premature death. There is no curative treatment. Investigations on disease-causing mechanisms offer an opportunity to identify new therapeutic targets to treat DMD. An abnormal elevation of the intracellular calcium (Cai2+) concentration in the dystrophin-deficient muscle is a major secondary event, which contributes to disease progression in DMD. Emerging studies have suggested that targeting Ca2+-handling proteins and/or mechanisms could be a promising therapeutic strategy for DMD. Here, we provide an updated overview of the mechanistic roles the sarcolemma, sarcoplasmic/endoplasmic reticulum, and mitochondria play in the abnormal and sustained elevation of Cai2+ levels and their involvement in DMD pathogenesis. We also discuss current approaches aimed at restoring Ca2+ homeostasis as potential therapies for DMD.