Unexpected extra exon skipping in the DYSF gene during restoring the reading frame by CRISPR/Cas9.

The DYSF gene encoding dysferlin protein is one of the largest and has many transcripts. Pathogenic variants in the gene can lead to various types of myopathies, which makes it a good object for studying the events occurring in it during genome editing by the CRISPR/Cas method. In this study, we evaluated the possibility of permanent skipping of exons 3-4, and 26-27 which deletion does not violate the reading frame and allows to eliminate truncated variants within exons. Editing was performed with simultaneous transfection of two sgRNA- and sa/spCas9-containing plasmids on HEK293T cell cultures and healthy donor myoblasts. Skipping of exons 3-4 was performed by destroying the splicing acceptor sites, and exons 26-27 by cuts in the flanking exons with the corresponding deletion in the DNA. Some unexpected results were obtained, when exons 26-27 were skipped, exon 30 was also absent in the transcript, although it is not alternatively spliced and is normally present in all transcripts. This event indicates that DNA changes near splicing sites can affect adjacent exons and the whole gene. However, this fact requires further study.

Dual Adeno-Associated Virus 9 with Codon-Optimized DYSF Gene Promotes In Vivo Muscle Regeneration and May Decrease Inflammatory Response in Limb Girdle Muscular Dystrophy Type R2.

Dysferlinopathy treatment is an active area of investigation. Gene therapy is one potential approach. We studied muscle regeneration and inflammatory response after injection of an AAV-9 with a codon-optimized DYSF gene. A dual-vector system AAV.DYSF.OVERLAP with overlapping DYSF cDNA sequences was generated. Two AAV vectors were separately assembled by a standard triple-transfection protocol from plasmids carrying parts of the DYSF gene. Artificial myoblasts from dysferlin-deficient fibroblasts were obtained by MyoD overexpression. RT-PCR and Western blot were used for RNA and protein detection in vitro. A dysferlinopathy murine model (Bla/J) was used for in vivo studies. Histological assay, morphometry, and IHC were used for the muscle tissue analysis. Dysferlin was detected in vitro and in vivo at subphysiological levels. RT-PCR and Western Blot detected dysferlin mRNA and protein in AAV.DYSF.OVERLAP-transduced cells, and mRNA reached a 7-fold elevated level compared to the reference gene (GAPDH). In vivo, the experimental group showed intermediate median values for the proportion of necrotic muscle fibers, muscle fibers with internalized nuclei, and cross-sectional area of muscle fibers compared to the same parameters in the control groups of WT and Bla/J mice, although the differences were not statistically significant. The inverse relationship between the dosage and the severity of inflammatory changes in the muscles may be attributed to the decrease in the number of necrotic fibers. The share of transduced myofibers reached almost 35% in the group with the highest dose. The use of two-vector systems based on AAV is justified in terms of therapeutic efficacy. The expression of dysferlin at a subphysiological level, within a short observation period, is capable of inducing the restoration of muscle tissue structure, reducing inflammatory activity, and mitigating necrotic processes. Further research is needed to provide a more detailed assessment of the impact of the transgene and viral vector on the inflammatory component, including longer observation periods.

Genetic screening of an endemic mutation in the DYSF gene in an isolated, mountainous population in the Republic of Dagestan.

BACKGROUND: Dysferlinopathy has a high prevalence in relatively isolated ethnic groups where consanguineous marriages are characteristic and/or the founder effect exists. However, the frequency of endemic mutations in most isolates has not been investigated. METHODS: The prevalence of the pathological DYSF gene variant (NM_003494.4); c.200_201delinsAT, p. Val67Asp (rs121908957) was investigated in an isolated Avar population in the Republic of Dagestan. Genetic screenings were conducted in a remote mountainous region characterized by a high level of consanguinity among its inhabitants. In total, 746 individuals were included in the screenings. RESULTS: This pathological DYSF gene variant causes two primary phenotypes of dysferlinopathy: limb-girdle muscular dystrophy (LGMD) type R2 and Miyoshi muscular dystrophy type 1. Results indicated a high prevalence of the allele at 14% (95% confidence interval [CI]: 12-17; 138 out of 1518 alleles), while the allele in the homozygous state was detected in 29 cases-3.8% (CI: 2.6-5.4). The population load for dysferlinopathy was 832.3 ± 153.9 per 100,000 with an average prevalence of limb-girdle muscular dystrophies ranging from 0.38 ± 0.38 to 5.93 ± 1.44 per 100,000. CONCLUSION: A significant burden of the allele was due to inbreeding, as evidenced by a deficiency of heterozygotes and the Wright fixation index equal to 0.14 (CI 0.06-0.23).

Small-scale fluctuations of laser beam fluence at the large B-integral in ultra-high intensity lasers.

Analytical expressions for the spatial spectrum of fluence fluctuations of a laser pulse propagating in a medium with Kerr nonlinearity have been obtained. It is shown that inhomogeneities with a spatial scale much larger than the critical scale of self-focusing grow insignificantly even at large values of the B-integral. Experiments using BK7 glass and a KDP crystal as a nonlinear medium confirm the obtained theoretical results. This may be interesting for pulse post-compression, frequency doubling, and other experiments using transmission optical elements in ultra-high intensity lasers.

Sublayer-Enhanced Growth of Highly Ordered Mn5Ge3 Thin Film on Si(111).

Mn5Ge3 epitaxial thin films previously grown mainly on Ge substrate have been synthesized on Si(111) using the co-deposition of Mn and Ge at a temperature of 390 °C. RMS roughness decreases by almost a factor of two in the transition from a completely polycrystalline to a highly ordered growth mode. This mode has been stabilized by changing the ratio of the Mn and Ge evaporation rate from the stoichiometric in the buffer layer. Highly ordered Mn5Ge3 film has two azimuthal crystallite orientations, namely Mn5Ge3 (001) [1-10] and Mn5Ge3 (001) [010] matching Si(111)[-110]. Lattice parameters derived a (7.112(1) Å) and c (5.027(1) Å) are close to the bulk values. Considering all structural data, we proposed a double buffer layer model suggesting that all layers have identical crystal structure with P63/mcm symmetry similar to Mn5Ge3, but orientation and level of Si concentration are different, which eliminates 8% lattice mismatch between Si and Mn5Ge3 film. Mn5Ge3 film on Si(111) demonstrates no difference in magnetic properties compared to other reported films. TC is about 300 K, which implies no significant excess of Mn or Si doping. It means that the buffer layer not only serves as a platform for the growth of the relaxed Mn5Ge3 film, but is also a good diffusion barrier.

Improving focusability of post-compressed PW laser pulses using a deformable mirror.

The use of the post-compression technique ensures gain in laser pulse peak power but at the same time degrades beam focusability due to the nonlinear wavefront distortions caused by a spatially nonuniform beam profile. In this paper a substantial focusability improvement of a post-compressed laser pulse by means of adaptive optics was demonstrated experimentally. The Strehl ratio increase from 0.16 to 0.43 was measured. Simulations showed that the peak intensity in this case reaches 0.52 of the theoretical limit.

11 fs, 1.5 PW laser with nonlinear pulse compression.

The PEARL laser output pulse with a duration of 60-70 fs was compressed to 11 fs after passing through a 5-mm thick silica plate and reflecting from two chirping mirrors with a total dispersion of -250 fs2. The experiments were carried out for the B-integral values up to 19 without damage of the optical elements, which indicates that small-scale self-focusing was suppressed. The results obtained show the possibility of further nonlinear compression scaling to multipetawatt power in pulses with duration commensurate with the field period.

Nitric oxide release and related light-induced cytotoxicity of ruthenium nitrosyls with coordinated nicotinate derivatives.

The synthetic approaches for the preparation of trans(NO,OH)-cis(NO2,NO2)-[RuNO(L)2(NO2)2OH], where L = ethyl nicotinate (I) and methyl nicotinate (II), are reported. The structures of the complexes are characterized by X-ray diffraction and analyzed by Hirshfeld surface analysis. Both compounds show a nitric oxide release reaction under 445 or 532 nm irradiation of dimethyl sulfoxide (DMSO) solutions, which is studied by combined ultraviolet-visible- (UV-vis), infrared- (IR), and electron paramagnetic resonance (EPR) spectroscopy and density functional theory (DFT) calculations. The charge transfer from the OH-Ru-NO chain and nitrite ligands to the antibonding orbitals of Ru-NO is responsible for the photo-cleavage of the ruthenium-nitrosyl bond. The elimination of NO leads to a side reaction, namely the protonation of the parent hydroxyl compound. The cytotoxicity and photo-induced cytotoxicity investigations of both compounds on the breast adenocarcinoma cell line MCF-7 reveal that (I) and (II) are cytotoxic with IC50 values of 27.5 ± 2.8 µM and 23.3 ± 0.3 µM, respectively. Moreover, (I) shows an increase of the toxicity after light irradiation by 7 times (IC50 = 4.1 ± 0.1), which makes it a prominent target for deeper biological investigations.

Magnetic resonance imaging pattern variability in dysferlinopathy.

The widespread use of magnetic resonance imaging (MRI) in the diagnosis of myopathies has made it possible to clarify the typical MRI pattern of dysferlinopathy. However, sufficient attention has not been given to the variability of MRI patterns in dysferlinopathy. MATERIALS AND METHODS: Twenty-five patients with the clinical manifestations of dysferlinopathy were examined. For all patients, creatine phosphokinase levels were measured and molecular genetics were examined. In two patients, immunohistochemical examinations of muscle biopsies were performed. MRI scanning was included T2 multi-slice multi-echo, T1 weighted, T2 weighted and Short Tau Inversion Recovery T2 weighted sequences. Quantitative and semi-quantitative evaluations of fatty replacement and swelling of the muscles were undertaken. RESULTS: Variability in the MRI patterns was lowest in the pelvis and leg muscles and highest in the thigh muscles. Three main types of MRI patterns were distinguished: posterior-dominant (80%), anterior-dominant (16%), and diffuse (4%). Among patients with the anterior-dominant pattern, the collagen-like variant (4%), proximal variant (4%) and pseudo-myositis (8%) were separately distinguished. CONCLUSIONS: Awareness of atypical MRI patterns in dysferlinopathy is important for increasing the efficiency of routine diagnostics and optimizing the search for causative gene mutations.

Asymmetric Interfaces in Epitaxial Off-Stoichiometric Fe3+xSi1-x/Ge/Fe3+xSi1-x Hybrid Structures: Effect on Magnetic and Electric Transport Properties.

Three-layer iron-rich Fe3+xSi1-x/Ge/Fe3+xSi1-x (0.2 < x < 0.64) heterostructures on a Si(111) surface with Ge thicknesses of 4 nm and 7 nm were grown by molecular beam epitaxy. Systematic studies of the structural and morphological properties of the synthesized samples have shown that an increase in the Ge thickness causes a prolonged atomic diffusion through the interfaces, which significantly increases the lattice misfits in the Ge/Fe3+xSi1-x heterosystem due to the incorporation of Ge atoms into the Fe3+xSi1-x bottom layer. The resultant lowering of the total free energy caused by the development of the surface roughness results in a transition from an epitaxial to a polycrystalline growth of the upper Fe3+xSi1-x. The average lattice distortion and residual stress of the upper Fe3+xSi1-x were determined by electron diffraction and theoretical calculations to be equivalent to 0.2 GPa for the upper epitaxial layer with a volume misfit of -0.63% compared with a undistorted counterpart. The volume misfit follows the resultant interatomic misfit of |0.42|% with the bottom Ge layer, independently determined by atomic force microscopy. The variation in structural order and morphology significantly changes the magnetic properties of the upper Fe3+xSi1-x layer and leads to a subtle effect on the transport properties of the Ge layer. Both hysteresis loops and FMR spectra differ for the structures with 4 nm and 7 nm Ge layers. The FMR spectra exhibit two distinct absorption lines corresponding to two layers of ferromagnetic Fe3+xSi1-x films. At the same time, a third FMR line appears in the sample with the thicker Ge. The angular dependences of the resonance field of the FMR spectra measured in the plane of the film have a pronounced easy-axis type anisotropy, as well as an anisotropy corresponding to the cubic crystal symmetry of Fe3+xSi1-x, which implies the epitaxial orientation relationship of Fe3+xSi1-x (111)[0-11] || Ge(111)[1-10] || Fe3+xSi1-x (111)[0-11] || Si(111)[1-10]. Calculated from ferromagnetic resonance (FMR) data saturation magnetization exceeds 1000 kA/m. The temperature dependence of the electrical resistivity of a Ge layer with thicknesses of 4 nm and 7 nm is of semiconducting type, which is, however, determined by different transport mechanisms.

Glu20Ter Variant in PLEC 1f Isoform Causes Limb-Girdle Muscle Dystrophy with Lung Injury.

Plectinopathies are orphan diseases caused by PLEC gene mutations. PLEC is encoding the protein plectin, playing a role in linking cytoskeleton components in various tissues. In this study, we describe the clinical case of a 26-year-old patient with an early onset plectinopathy variant "limb-girdle muscle dystrophy type 2Q," report histopathological and ultrastructural findings in m. vastus lateralis biopsy and a novel homozygous likely pathogenic variant (NM_201378.3:c.58G>T, NP_958780.1:p.Glu20Ter) in isoform 1f of the gene PLEC. The patient had an early childhood onset with retarded physical development, moderate weakness in pelvic girdle muscles, progressive weakening of limb-girdle muscles after the age of 21, pronounced atrophy of axial muscles, and hypertrophy of the gastrocnemius, deltoid, and triceps muscles, intermittent dyspnea, and no skin involvement. Findings included: non-infectious bronchiolitis and atelectasis signs, biopsy revealed myodystrophal pattern without macrophage infiltration, muscle fiber cytoskeleton disorganization resulted from the plectin loss, incomplete reparative rhabdomyogenesis, and moderate endomysial fibrosis. We have determined a novel likely pathogenic variant in PLEC 1f isoform that causes limb-girdle muscle dystrophy type 2Q and described the third case concerning an isolated myodystrophic phenotype of LGMD2Q with the likely pathogenic variant in PLEC 1f isoform. In addition, we have demonstrated the presence of severe lung injury in a patient and his siblings with the same myodystrophic phenotype and discussed the possible role of plectin deficiency in its pathogenesis.

Serum Cytokine Profile in a Patient Diagnosed with Dysferlinopathy.

Limb-girdle muscular dystrophy type 2 (LGMD2B) is a mild form of dysferlinopathy, characterized by limb weakness and wasting. It is an autosomal recessive disease, with currently 140 mutations in the LGMD2B gene identified. Lack of functional dysferlin inhibits muscle fiber regeneration in voluntary muscles, the main pathological finding in LGMD2B patients. However, the immune system has been suggested to contribute to muscle cell death and tissue regeneration. Serum levels of 27 cytokines were evaluated in a dysferlinopathy patient. Levels of 8 cytokines differed in patient serum compared to controls. Five cytokines (IL-10, IL-17, CCL2, CXCL10, and G-CSF) were higher while 3 were lower in the patient than in controls (IL-2, IL-8, and CCL11). Together, these data on serum cytokine profile of this dysferlinopathy patient suggest immune response activation, which could explain leukocyte infiltration in the muscle tissue.

Corrigendum: Twenty-Year Clinical Progression of Dysferlinopathy in Patients from Dagestan.

[This corrects the article on p. 77 in vol. 8, PMID: 28337173.].

Twenty-Year Clinical Progression of Dysferlinopathy in Patients from Dagestan.

To date, over 30 genes with mutations causing limb-girdle muscle dystrophy have been described. Dysferlinopathies are a form of limb-girdle muscle dystrophy type 2B with an incidence ranging from 1:1,300 to 1:200,000 in different populations. In 1996, Dr. S. N. Illarioshkin described a family from the Botlikhsky district of Dagestan, where limb-girdle muscle dystrophy type 2B and Miyoshi myopathy were diagnosed in 12 members from three generations of a large Avar family. In 2000, a previously undescribed mutation in the DYSF gene (c.TG573/574AT; p. Val67Asp) was detected in the affected members of this family. Twenty years later, in this work, we re-examine five known and seven newly affected family members previously diagnosed with dysferlinopathy. We observed disease progression in family members who were previously diagnosed and noted obvious clinical polymorphism of the disease. A typical clinical case is provided.

Single-shot laser pulse reconstruction based on self-phase modulated spectra measurements.

We report a method for ultrashort pulse reconstruction based only on the pulse spectrum and two self-phase modulated (SPM) spectra measured after pulse propagation through thin media with a Kerr nonlinearity. The advantage of this method is that it is a simple and very effective tool for characterization of complex signals. We have developed a new retrieval algorithm that was verified by reconstructing numerically generated fields, such as a complex electric field of double pulses and few-cycle pulses with noises, pedestals and dips down to zero spectral intensity, which is challenging for commonly used techniques. We have also demonstrated a single-shot implementation of the technique for the reconstruction of experimentally obtained pulses. This method can be used for high power laser systems operating in a single-shot mode in the optical, near- and mid-IR spectral ranges. The method is robust, low cost, stable to noise, does not require a priori information, and has no ambiguity related to time direction.

Adaptive acousto-optic technique for femtosecond laser pulse shaping.

We discuss the theoretical and experimental investigation of acousto-optic dispersive tunable filters, based on quasi-collinear geometry of light-sound interaction in a tellurium dioxide single crystal. The geometry uses the effect of strong acoustic anisotropy in the paratellurite as well as peculiarities of acoustic wave reflections at the free boundary of the crystal. A mathematical concept for determination of optical, electrical, and constructional parameters of the filters is developed. Different experimental acousto-optic filters intended for femtosecond pulse shaping are designed and tested. Preliminary experiments are performed in a subpetawatt optical parametric chirped pulse amplification laser system. The experimental data conform completely with the predicted data.