MicroRNA-202 Induces Myoblast to Myocyte Differentiation through Targeting Rock-1

The expression patterns of microRNAs (small non-coding RNAs) are altered in many biological processes such as myogenesis. In this study, we aimed to investigate the impact of predicted miR-202, its target genes Akt2 and Rock-1 as a potential regulator of myoblast in the myocyte differentiation process using the C2C12 cell line. After confirmation of the differentiation process induced by 3% horse serum, the expression level of miRNA and its targets were evaluated. In the following, a luciferase assay was conducted to approve the effect of miRNA on its target. Our results indicated that miR-202 and Akt2 were significantly up-regulated during differentiation, while Rock-1 was downregulated. Co-transfection of miRNA with psiCHECK2-Rock-1 significantly presented that Rock-1 was directly targeted by miR-202. On the contrary, miR-202 has failed to enforce its inhibitory effect on Akt2 expression. In particular, miR-202 seems to be a regulator of muscle differentiation pathway thought targeting Rock-1.

THE EFFECT OF N-ACETYLCYSTEINE AND DEXAMETHASONE PARENTERAL ADMINISTRATION ON THE BIOCHEMICAL PROCESSES IN THE AQUEOUS HUMOR OF THE ANTERIOR CHAMBER OF THE EYES IN RABBITS WITH EXPERIMENTAL IMMUNOGENIC UVEITIS

Objective. To evaluate the impact and compare the efficiency of parenteral administration of N-acetylcysteine (NAC) and dexamethasone when used as monotherapy or combination therapy in the treatment of experimental immunogenic uveitis (EIU) in rabbits, as well as to explore the changes of biochemical parameters in the aqueous humor of the eyes in experimental animals. Material and Methods. An experimental study was performed on 45 rabbits (90 eyes). Of these 5 healthy intact rabbits (10 eyes) served as a control group. Acute immunogenic uveitis was caused in 40 rabbits by injecting normal horse serum subcutaneously (5 ml) and then intravitreally (0.07 ml). The animals with experimental uveitis were divided into 8 groups (5 animals each). The first 4 groups – control-1, experiment-1, control-3, experiment-3 – received daily intramuscular injections of placebo, NAC, dexamethasone or a combination of NAC and dexamethasone respectively for 3 days, and thereafter they were withdrawn from the experiment. The remaining 4 groups – control-2, experiment-2, control-4, experiment-4 received, respectively, daily intramuscular injections of placebo, NAC, dexamethasone, a combination of NAC and dexamethasone for 7 days, and after that they were also withdrawn from the experiment. The drugs in the aforementioned groups were used from the moment of the horse serum intravitreal injection. When withdrawing animals from the experiment, aqueous humor was taken from the anterior chamber of their eyes, followed by the evaluation of protein (albumin) concentration and the number of leukocytes. Results. A significant elevation of albumin and the number of leukocytes in the aqueous humor of the eyes in the rabbits with experimental immunogenic uveitis was noted. NAC effectively reduced the level of albumin and the number of leukocytes in the aqueous humor. Dexamethasone showed more efficacy in reducing the investigated aqueous humor biochemichal parameters than NAC. Nevertheless, a synergism of the pharmacological action of NAC and dexamethasone was detected, since their combination had the greatest potency in reduction of albumin level and the number of leukocytes in the aqueous humor of the eyes in the rabbits with experimental immunogenic uveitis, even though the dosage of dexamethasone in the groups with combined (NAC and dexamethasone) therapy was reduced by 50% (1 mg / kg body weight). Conclusion. Parenteral administration of NAC significantly reduces inflammation in EIU. Combination of NAC and dexamethasone showed synergy of action in reducing the intensity of inflammatory process in rabbits with EIU, which is an objective rationale for including NAC in the complex therapy of uveitis, which in turn will reduce a single or course dose of dexamethasone and lower the risks of side effects caused by glucocorticoids.

L-carnitine ameliorates the muscle wasting of cancer cachexia through the AKT/FOXO3a/MaFbx axis

Background Recent studies suggest potential benefits of applying L-carnitine in the treatment of cancer cachexia, but the precise mechanisms underlying these benefits remain unknown. This study was conducted to determine the mechanism by which L-carnitine reduces cancer cachexia. Methods C2C12 cells were differentiated into myotubes by growing them in DMEM for 24 h (hrs) and then changing the media to DMEM supplemented with 2% horse serum. Differentiated myotubes were treated for 2 h with TNF-α to establish a muscle atrophy cell model. After treated with L-carnitine, protein expression of MuRF1, MaFbx, FOXO3, p-FOXO3a, Akt, p-Akt, p70S6K and p-p70S6K was determined by Western blotting. Then siRNA-Akt was used to determine that L-carnitine ameliorated cancer cachexia via the Akt/FOXO3/MaFbx. In vivo, the cancer cachexia model was established by subcutaneously transplanting CT26 cells into the left flanks of the BALB/c nude mice. After treated with L-carnitine, serum levels of IL-1, IL-6 and TNF-α, and the skeletal muscle content of MuRF1, MaFbx, FOXO3, p-FOXO3a, Akt, p-Akt, p70S6K and p-p70S6K were measured. Results L-carnitine increased the gastrocnemius muscle (GM) weight in the CT26-bearing cachexia mouse model and the cross-sectional fiber area of the GM and myotube diameters of C2C12 cells treated with TNF-α. Additionally, L-carnitine reduced the protein expression of MuRF1, MaFbx and FOXO3a, and increased the p-FOXO3a level in vivo and in vitro. Inhibition of Akt, upstream of FOXO3a, reversed the effects of L-carnitine on the FOXO3a/MaFbx pathway and myotube diameters, without affecting FOXO3a/MuRF-1. In addition to regulating the ubiquitination of muscle proteins, L-carnitine also increased the levels of p-p70S6K and p70S6K, which are involved in protein synthesis. Akt inhibition did not reverse the effects of L-carnitine on p70S6K and p-p70S6K. Hence, L-carnitine ameliorated cancer cachexia via the Akt/FOXO3/MaFbx and p70S6K pathways. Moreover, L-carnitine reduced the serum levels of IL-1 and IL-6, factors known to induce cancer cachexia. However, there were minimal effects on TNF-α, another inducer of cachexia, in the in vivo model. Conclusion These results revealed a novel mechanism by which L-carnitine protects muscle cells and reduces inflammation related to cancer cachexia.

Impact of various preservation and storage methods on the viability of mycoplasma field strains isolated in Mali

The survival of five mycoplasma strains was studied in different storage media (Mycoplasma complet media without cryopreservative agent, Mycoplasma complete media with addition of horse serum, Mycoplasma complete media with addition of glycerol and lyophilized cultures without stabilizer) under different temperatures (+37°C, +4°C, -20°C, -85°C) during 24 months. Five Mycoplasma strains, Mycoplasma mycoides subsp mycoides (Mmm), Mycoplasma bovis (Mb), Mycoplasma agalactiae (Ma), Mycoplasma gallisepticum (Mg) and Mycoplasma synoviae (Ms) were isolated from various parts of the country. The initial titers of the strains determined by the agar plate count before storage were 42.4x10 7 UFC/ml (8.6 log UFC/ml) for Mmm strain; 32.4x10 8 UFC/ml (9.51 log UFC/ml) for M.bovis strain; 12.4x10 9 UFC/ml (10.09 log UFC/ml) for Ma strain; 2.4x10 9 UFC/ml (9.38 log UFC/ml) for Mg and 2.8x10 9 UFC/ml (9.45 log UFC/ml) for Ms strain. After 3 weeks of storage, no viable mycoplasmas were detected in all the conservation media at +37°C and after 3 months of storage at +4°C except for the lyophilized cultures in which an average viability rate of 17.81% was observed. Overall, the mycoplasma strains remained viable at freezing temperatures after 24 months regardless of the storage medium, but with decreasing titers, which was noticeable with mycoplasma complete media, and mycoplasma media with horse serum. Conversely, at -20°C the average viability rates after 24 months of storage were 84.36% (with glycerol) and 90.04% (lyophilized cultures). At -85°C after 24 months of storage, this was 87.98% (with glycerol) and 91.44% (lyophilized cultures). These findings suggest that, in the absence of the lysophylisation process, the addition of glycerol may be recommended for long-term storage of frozen mycoplasma isolates.

Myogenic differentiation potential of chicken mesenchymal stem cells from bone marrow

Background: Mesenchymal stem cells (MSCs) have the potential to multilineage differentiation, which can be used for a good model to provide critical insight of chicken muscle development. Differential adhesion method is one of the commonest methods to isolate MSCs based on the ability of plastic adhesion. 5-azacytidine (5-Aza), dexamethasone (DXMS), hydrocortisone (HC) and horse serum had been proved the potential to induce the myogenic differentiation of MSCs. However, the myogenic differentiation of MSCs is still poorly understood in chicken. In present study, we isolated chicken mesenchymal stem cells (cMSCs) from bone using 4-hour differential adhesion method and analyzed the myogenic effect of cMSCs treated with different method based on 5-Aza, DXMS, HC and horse serum.Results: cMSCs isolated by 4-hour differential adhesion method expressed MSCs special surface markers and presented normal growth characteristic. cMSCs showed great potential of myogenic differentiation by the treatment of 5-Aza and horse serum. RNA-sequence, GO and KGEE enrichment analysis revealed that this effect might be based on demethylation of 5-Aza and ECM-receptor interaction, focal adhesion, PI3K-Akt, p53, TGF-beta signaling pathways. Moreover, DXMS, HC and horse serum also presented potential of myogenic differentiation, but the effect was not as good as 5-Aza and horse serum method.Conclusions: cMSCs showed potential of myogenic differentiation by the treatment of 5-Aza and horse serum or DXMS, HC and horse serum.

Sero-Positivity of Japanese Encephalitis Virus in Equine Population of India Using IgG ELISA: Unraveling The Need for Vaccination

Japanese encephalitis (JE) is a mosquito borne flaviviral zoonoses, causing fatal disease in equines and humans. JE is endemic in most of the states of India with occurrence of human cases every year. The horses are not vaccinated against JE in India and thus they are at more risk of acquiring the disease. Due to non-availability of indigenously developed ELISA and high cost of imported kits, regular sero-surveillance is not being carried out to assess the true picture of JE virus in equine population of India. Therefore, a recombinant NS1 protein based indirect IgG ELISA was developed with the objective to assess the sero-positivity of JE virus in equine population of India. The diagnostic sensitivity and specificity of developed ELISA was 84.73 % and 86.70 %, respectively. The validation studies revealed good reproducibility of ELISA with kappa value ranging from 0.75 to 1 between the results of different laboratories. A total of 2069 horse serum samples were screened using the developed ELISA and 401 samples were positive for IgG against JEV with an overall sero-positivity of 19.38% in equine population of India. A sero-positivity of 25.90% and 12.22% was recorded in Himachal Pradesh and Jammu-Kashmir, both hill states of North zone of India for the first time, revealing the spread of virus to the non-endemic parts of the country. The high sero-positivity of JE virus recorded in equine population warrants the need for initiation of vaccination of horses in India to prevent the morbidity and mortality.

Normal equine serum for staphylomycosis

Studying the effect of normal horse serum in vivo and in vitro on staphylococci, Benedek (Zeit. F. 1mm. U. Exper. Ther., B. 47, H. 2) found that it has antitoxic, bactericidal and tissue immunizing effects.

Reversal of deficits in aged skeletal muscle during disuse and recovery in response to treatment with a secrotome product derived from partially differentiated human pluripotent stem cells

Aged individuals are at risk to experience slow and incomplete muscle recovery following periods of disuse atrophy. While several therapies have been employed to mitigate muscle mass loss during disuse and improve recovery, few have proven effective at both. Therefore, the purpose of this study was to examine the effectiveness of a uniquely developed secretome product (STEM) on aged skeletal muscle mass and function during disuse and recovery. Aged (22 months) male C57BL/6 were divided into PBS or STEM treatment (n = 30). Mice within each treatment were assigned to either ambulatory control (CON; 14 days of normal cage ambulation), 14 days of hindlimb unloading (HU), or 14 days of hindlimb unloading followed by 7 days of recovery (recovery). Mice were given an intramuscular delivery into the hindlimb muscle of either PBS or STEM every other day for the duration of their respective treatment group. We found that STEM-treated mice compared to PBS had greater soleus muscle mass, fiber cross-sectional area (CSA), and grip strength during CON and recovery experimental conditions and less muscle atrophy and weakness during HU. Muscle CD68 +, CD11b + and CD163 + macrophages were more abundant in STEM-treated CON mice compared to PBS, while only CD68 + and CD11b + macrophages were more abundant during HU and recovery conditions with STEM treatment. Moreover, STEM-treated mice had lower collagen IV and higher Pax7 + cell content compared to PBS across all experimental conditions. As a follow-up to examine the cell autonomous role of STEM on muscle, C2C12 myotubes were given STEM or horse serum media to examine myotube fusion/size and effects on muscle transcriptional networks. STEM-treated C2C12 myotubes were larger and had a higher fusion index and were related to elevated expression of transcripts associated with extracellular matrix remodeling. Our results demonstrate that STEM is a unique cocktail that possesses potent immunomodulatory and cytoskeletal remodeling properties that may have translational potential to improve skeletal muscle across a variety of conditions that adversely effect aging muscle.

On the use of normal horse serum for the treatment of diphtheria

The question of treating diphtheria with normal horse serum instead of antitoxic serum, which was much sensational in connection with the observations of Віlіng'a, has been repeatedly decided by both clinicians and bacteriologists in favor of antitoxic serum.