Myogenic Differentiation of Human Myoblasts and Mesenchymal Stromal Stem Cells under GDF11 on Poly-ɛ-Caprolactone-Collagen I-Polyethylene-Nanofibers

Primary myoblasts (Mb) and adipose derived mesenchymal stromal cells (ADSC) can be co-cultured and myogenically differentiated in the process of skeletal muscle tissue engineering. Electrospun composite nanofiber scaffolds represent suitable matrices for tissue engineering of skeletal muscle, combining biocompatibility and stability. Although growth differentiation factor 11 (GDF11) has been proposed as a rejuvenating circulating factor, restoring skeletal muscle function in aging mice, some studies have also described a harming effect of GDF11.Therefore the aim of the study was to analyze the effect of GDF11 on co-cultures of Mb and ADSC on poly-ε-caprolacton (PCL)-collagen I-polyethylene oxide (PEO)-nanofibers.Human Mb were co-cultured with ADSC two-dimensionally (2D) as monolayers or three-dimensionally (3D) on aligned PCL-collagen I-PEO-nanofibers. Differentiation media were either serum-free with or without GDF11, or serum containing as in a conventional differentiation medium. Cell viability was higher after conventional myogenic differentiation compared to serum-free and serum-free + GDF11 differentiation as was creatine kinase activity. Immunofluorescence staining showed myosin heavy chain expression in all groups after 28 days of differentiation. Gene expression of myosin heavy chain (MYH2) increased after serum-free + GDF11 stimulation compared to serum-free stimulation alone. The results of this study show that PCL-collagen I-PEO-nanofibers represent a suitable matrix for 3D myogenic differentiation of Mb and ADSC. In this context, GDF11 seems to promote myogenic differentiation of Mb and ADSC co-cultures compared to serum-free differentiation without any evidence of a harming effect.

An ultrapotent RBD-targeted biparatopic nanobody neutralizes broad SARS-CoV-2 variants

The wide transmission and host adaptation of SARS-CoV-2 have led to the rapid accumulation of mutations, posing significant challenges to the effectiveness of vaccines and therapeutic antibodies. Although several neutralizing antibodies were authorized for emergency clinical use, natural antibodies isolated from convalescent patients are vulnerable to SARS-CoV-2 Spike mutations. Here, we describe the screen of a panel of SARS-CoV-2 receptor-binding domain (RBD) targeted nanobodies (Nbs) from a synthetic library and the design of a biparatopic Nb dimer, named Nb1-Nb2, with tight affinity and super wide neutralization breadth against multiple SARS-CoV-2 variants of concern or interest. Deep-mutational scanning experiments identify the potential binding epitopes of the monomeric Nb1 and Nb2 on the RBD and demonstrate that bivalent Nb1-Nb2 has a strong escape resistant feature against more than 60 tested RBD amino acid substitutions. Using pseudovirion-based and trans-complementation SARS-CoV-2 tools, we determine that Nb1-Nb2 broadly neutralizes SARS-CoV-2, including variants Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Lambda (C.37), Kappa (B.1.617.1) and Mu (B.1.621). Furthermore, a heavy chain antibody is constructed by fusing the human IgG1 Fc to the biparatopic Nb (designated as Nb1-Nb2-Fc) to improve its neutralization potency, yield, stability and potential half-life extension. For the new Omicron variant (B.1.1.529) that harbors unprecedented multiple RBD mutations, Nb1-Nb2-Fc keeps a firm affinity (KD < 1.0*10E-12 M) and neutralizing activity (IC50 = 0.0017 nM). Together, we developed a biparatopic human heavy chain antibody with ultrapotent and broad-spectrum SARS-CoV-2 neutralization activity which highlights the potential clinical applications.

Let-7e-5p Regulates IGF2BP2, and Induces Muscle Atrophy

Background and AimsTo understand the role of microRNAs in muscle atrophy caused by androgen-depletion, we performed microarray analysis of microRNA expression in the skeletal muscles of Sham, orchiectomized (ORX), and androgen-treated ORX mice.MethodsTo clarify role and mechanisms of let-7e-5p in the muscle, the effect of let-7e-5p overexpression or knockdown on the expression of myosin heavy chain, glucose uptake, and mitochondrial function was investigated in C2C12 myotube cells. Moreover, we examined serum let-7e-5p levels among male subjects with type 2 diabetes.ResultsWe found that the expression of the miRNA, lethal (let)-7e-5p was significantly lower in ORX mice than that in Sham mice (p = 0.027); however, let-7e-5p expression in androgen-treated ORX mice was higher (p = 0.047). Suppression of let-7e-5p significantly upregulated the expression of myosin heavy chain, glucose uptake, and mitochondrial function. Real-time PCR revealed a possible regulation involving let-7e-5p and Igf2bp2 mRNA and protein in C2C12 cells. The serum let-7e-5p levels were significantly lower, which might be in compensation, in subjects with decreased muscle mass compared to subjects without decreased muscle mass. Let-7e-5p downregulates the expression of Igf2bp2 in myotube cells and inhibits the growth of the myosin heavy chain.ConclusionsBased on our study, serum level of let-7e-5p may be used as a potential diagnostic marker for muscle atrophy.

New cell biological explanations for kinesin-linked axon degeneration

Axons are the slender, up to meter-long projections of neurons that form the biological cables wiring our bodies. Most of these delicate structures must survive for an organism's lifetime, meaning up to a century in humans. Axon maintenance requires life-sustaining motor protein-driven transport distributing materials and organelles from the distant cell body. It seems logic that impairing this transport causes systemic deprivation linking to axon degeneration. But the key steps underlying these pathological processes are little understood. To investigate mechanisms triggered by motor protein aberrations, we studied more than 40 loss- and gain-of-function conditions of motor proteins, cargo linkers or further genes involved in related processes of cellular physiology. We used one standardised Drosophila primary neuron system and focussed on the organisation of axonal microtubule bundles as an easy to assess readout reflecting axon integrity. We found that bundle disintegration into curled microtubules is caused by the losses of Dynein heavy chain and the Kif1 and Kif5 homologues Unc-104 and Kinesin heavy chain (Khc). Using point mutations of Khc and functional loss of its linker proteins, we studied which of Khc's sub-functions might link to microtubule curling. One cause was emergence of harmful reactive oxygen species through loss of Milton/Miro-mediated mitochondrial transport. In contrast, loss of the Kinesin light chain linker caused microtubule curling through an entirely different mechanism appearing to involve increased mechanical challenge to microtubule bundles through de-inhibition of Khc. The wider implications of our findings for the understanding of axon maintenance and pathology are discussed.

Molecular Cloning of Dynein Heavy Chain and the Effect of Dynein Inhibition on the Testicular Function of Portunus trituberculatus

Dynein is a motor protein with multiple transport functions. However, dynein’s role in crustacean testis is still unknown. We cloned the full-length cDNA of cytoplasmic dynein heavy chain (Pt-dhc) gene and its structure was analyzed. Its expression level was highest in testis. We injected the dynein inhibitor sodium orthovanadate (SOV) into the crab. The distribution of Portunus trituberculatus dynein heavy chain (Pt-DHC) in mature sperm was detected by immunofluorescence. The apoptosis of spermatids was detected using a TUNEL kit; gene expression in testis was detected by fluorescence quantitative PCR (qPCR). The expression of immune-related factors in the testis were detected by an enzyme activity kit. The results showed that the distribution of Pt-DHC was abnormal after SOV injection, indicating that the function of dynein was successfully inhibited. Apoptosis-related genes p53 and caspase-3, and antioxidant stress genes HSP70 and NOS were significantly decreased, and anti-apoptosis gene bcl-2 was significantly increased. The activities of superoxide dismutase (SOD) and alkaline phosphatase (AKP) were significantly decreased. The results showed that there was no apoptosis in testicular cells after dynein function was inhibited, but the cell function was disordered. This study laid a theoretical foundation for the further study of apoptosis in testis and the function of dynein in testis and breeding of P. trituberculatus.

Disseminated Intravascular Coagulation Associated with Large Deletion of Immunoglobulin Heavy Chain

Although the majority of monogenic defects underlying primary immunodeficiency are microlesions, large lesions like large deletions are rare and constitute less than 10% of these patients. The immunoglobulin heavy chain (IGH) locus is one of the common regions for such genetic alterations. This study describes a rare case of autosomal recessive agammaglobulinemia with a homozygous large deletion in chromosome 14q32.33 (106067756-106237742) immunoglobulin heavy chain clusters with an unusual and severe skin infection and disseminated intravascular coagulopathy.

Mitochondrial mutational spectrum is associated with mammalian longevity: a novel signature of oxidative damage.

The mutational spectrum of the mitochondrial DNA (mtDNA) does not resemble any of the known mutational signatures of the nuclear genome and variation in mtDNA mutational spectra between different tissues and organisms is still incomprehensible. Since mitochondria is tightly involved in aerobic energy production, it is expected that mtDNA mutational spectra may be affected by the oxidative damage which is increasing with organismal aging. However, the well-documented mutational signature of the oxidative damage, G>T substitutions, is typical only for the nuclear genome while it is extremely rare in mtDNA. Thus it is still unclear if there is a mitochondria-specific mutational signature of the oxidative damage. Here, reconstructing mtDNA mutational spectra for 424 mammalian species with variable generation length which is a proxy for oocyte age, we observed that the frequency of AH>GH substitutions (H - heavy chain notation) is positively correlated with organismal longevity. This mutational bias from AH to GH significantly affected the nucleotide content of analyzed 650 complete mammalian mitochondrial genomes, where fourfold degenerative synonymous positions of long-lived species become more AH poor and GH rich. Because (i) A>G is a substitution, typical for mtDNA; (ii) it is characterized by very strong asymmetry: A>G is several-fold more frequent on a heavy chain as compared to the light one; (iii) it is sensitive to the time being single-stranded during mtDNA asynchronous replication; (iv) it is associated with oxidative damage of single-stranded DNA in recent experimental studies we propose that A>G is a novel mutational signature of age-associated oxidative damage of single-stranded mtDNA. The described association of the mtDNA mutational spectra with a species-specific life-history trait can significantly affect general patterns of molecular evolution of mtDNA.

Myofibre Hyper-Contractility in Horses Expressing the Myosin Heavy Chain Myopathy Mutation, MYH1E321G

Myosinopathies are defined as a group of muscle disorders characterized by mutations in genes encoding myosin heavy chains. Their exact molecular and cellular mechanisms remain unclear. In the present study, we have focused our attention on a MYH1-related E321G amino acid substitution within the head region of the type IIx skeletal myosin heavy chain, associated with clinical signs of atrophy, inflammation and/or profound rhabdomyolysis, known as equine myosin heavy chain myopathy. We performed Mant-ATP chase experiments together with force measurements on isolated IIx myofibres from control horses (MYH1E321G−/−) and Quarter Horses homozygous (MYH1E321G+/+) or heterozygous (MYH1E321G+/−) for the E321G mutation. The single residue replacement did not affect the relaxed conformations of myosin molecules. Nevertheless, it significantly increased its active behaviour as proven by the higher maximal force production and Ca2+ sensitivity for MYH1E321G+/+ in comparison with MYH1E321G+/− and MYH1E321G−/− horses. Altogether, these findings indicate that, in the presence of the E321G mutation, a molecular and cellular hyper-contractile phenotype occurs which could contribute to the development of the myosin heavy chain myopathy.

Diversity of Immunoglobulin Heavy Chain Repertoire in Patients With Rheumatoid Arthritis

Objectives Rheumatoid Arthritis (RA) is associated with polymorphism in major histocompatibility complex class II genes and dysregulations of CD4+ T cells which cause abnormalities in immune repertoire (iR) expression and intracellular signaling. We monitored nucleotide sequence changes in iR of immunoglobulin heavy chain (IGH), particularly complementarity determining region 3 (CDR3) during the course of treatments in RA patients using massively parallel sequencing technology.Methods CDR3 sequencing was carried out on clinical blood samples from RA patients for disease progress monitoring. The iR of each sample was measured using next generation sequencing (NGS) pipeline. Data analysis was done with a web-based iRweb server. Principal components analysis (PCA) was completed with commercial statistical pipeline. Results Datasets from 14 patients covered VDJ regions of IGH gene. D50 stayed low for all cases (mean D50 = 6.5). A pattern of shared CDR3 sequences was confirmed by a clustering pattern using PCA. Shared profile of 608 CDR3 sequences unique to the disease baseline was identified. D50 analyses revealed clonal diversity would remain low throughout the disease course even after treatment (mean D50 = 11.7 & 8.2 for csDMARD & bDMARD groups respectively) regardless of fluctuated disease activity. PCA has provided a correlation of change in immune diversity along the whole course of RA. Conclusion We have successfully constructed the experimental design, data acquisition, processing, and analysis pipeline of a high throughput massively parallel CDR3 sequences detection to be used to correlate RA disease activity and IGH CDR3 iR during disease progression with or without treatments.