A modified animal-free serum technique for efficient isolation and proliferation of mesenchymal stem cells from Hoffa fat pad.

We focus on this study in designing an alternative technique for obtaining mesenchymal stem cells (MSCs) from residual tissue, Hoffa fat, in arthroscopic procedures. Two males and two females were included, and underwent knee arthroscopy; a sample of infrapatellar adipose tissue was obtained with basket forceps. The primary culture was made using the explant method and the culture media: DMEM-high glucose, supplemented with 10% of inactivated human allogeneic serum. All the cellular cultures remained under culture conditions for three weeks, after that by flow cytometry the cells were characterized by MSCs antibody panel: CD105, CD73 and CD90. Subsequently, in the first pass, the MSCs were cultured in commercial human chondrogenic, osteogenic and adipogenic mediums, respectively. After primary culture, we obtained on average 95,600.00 ± 7,233.26 cells/cm2, and the duplication time of MSCs isolate from Hoffa fat pad was established in 39 hours. By flow cytometry, we found that surface markers percentage for expanded MSCs (CD105, CD73, CD90) in primary culture significantly increased and its morphology was fibroblastic-like. After differentiation culture which was made in the first pass, by immunofluorescence, we obtained positive cell markers for three lineages of differentiation, adipocytes: LPL protein, osteocytes: RUNX2, Osteopontin, chondrocytes: SOX9, Aggrecan and COL2A1. We managed to isolate a significant number of MSCs from this source using an easy method to implement and minimal nutrient supplementation, with high potential for differentiation to mature mesenchymal tissues and potential use in basic experimental, preclinical and even clinical research.

Crosstalk between Exercise-Derived Endocannabinoidome and Kynurenines: Potential Target Therapies for Obesity and Depression Symptoms.

The kynurenine pathway (KP) and the endocannabinoid system (ECS) are known to be deregulated in depression and obesity; however, it has been recognized that acute physical exercise has an important modulating role inducing changes in the mobilization of their respective metabolites-endocannabinoids (eCBs) and kynurenines (KYNs)-which overlap at some points, acting as important antidepressant, anti-nociceptive, anti-inflammatory, and antioxidant biomarkers. Therefore, the aim of this review is to analyze and discuss some recently performed studies to investigate the potential interactions between both systems, particularly those related to exercise-derived endocannabinoidome and kynurenine mechanisms, and to elucidate how prescription of physical exercise could represent a new approach for the clinical management of these two conditions.

Characterization of Redox Environment and Tryptophan Catabolism through Kynurenine Pathway in Military Divers' and Swimmers' Serum Samples.

Endurance and resistance exercises, alone or in combination, induce metabolic changes that affect tryptophan (Trp) catabolism. The kynurenine pathway (KP) is the main route of Trp degradation, and it is modulated by the inflammatory and redox environments. Previous studies have shown that KP metabolites work as myokines that mediate the positive systemic effects related to exercise. However, it is poorly understood how different exercise modalities and intensities impact the KP. The aim of this study was to characterize the effect of two different exercise modalities, military diving and swimming, on the KP and the redox environment. A total of 34 healthy men from the Mexican Navy were included in the study, 20 divers and 14 swimmers, who started and stayed in military training consistently during the six months of the study; 12 Mexican men without fitness training were used as the control group. Physical fitness was determined at the beginning and after 6 months of training; criteria included body composition; serum levels of Trp, kynurenine (KYN), kynurenic acid (KYNA) and 3-hydroxykynurenine (3-HK); the glutathione ratio (GSH/GSSG); and malondialdehyde (MDA).. Results showed a significant loss of body fat in both the diver and swimmer groups. Compared with the control group, divers showed a decrease in Trp and 3-HK levels, but no changes were observed in the KYN/Trp, KYNA/Trp or 3-HK/Trp ratios, while swimmers showed a decrease in KYN levels and an increase in the KYNA and 3-HK levels. Additionally, divers showed a decrease in the GSH/GSSG ratio and an increase in MDA levels, in contrast to the swimmers, who showed a decrease in MDA levels and an increase in GSH/GSSG levels. Our findings suggest a differential shift in the KP and redox environment induced by diving and swimming. Swimming promotes an antioxidant environment and a peripheral overactivation of the KP.

Genetic analysis of muscular dystrophies: our experience in Mexico.

Muscular dystrophies are a group of well-defined genetic disorders characterized by the variable distribution of muscle wasting and progressive weakness. The diagnosis and treatment of these diseases remain challenging due to genetic heterogeneity and clinical overlapping. Herein, we describe our 10 years' experience with the diagnosis and management of muscular dystrophy patients. In total, 169 patients were screened for pathogenic variants in eleven genes linked to frequent muscular dystrophies using MLPA and NGS sequencing panels. Most frequent muscular dystrophies found in the Mexican population were dystrophinopathies, dysferlinopathies and calpainopathies. Novel variants were found in genes: DMD, CAPN3, DYSF, and FKRP. For Duchenne muscular dystrophy, improvements in early diagnosis and prolonged ambulation were achieved, on the contrary, for limb-girdle muscular dystrophies and congenital muscular dystrophies, uncomplimentary follow-up and lack of detection strategies were observed. For most common muscular dystrophies, improvements in diagnosis and management have been achieved in the last 10 years, due to a collaborative effort done nationwide.

Phenotypic Spectrum of Myopathies with Recessive Anoctamin-5 Mutations.

BACKGROUND: Biallelic variants in Anoctamin 5 (ANO5) gene are causative of limb-girdle muscular dystrophy (LGMD) R12 anoctamin5-related, non-dysferlin Miyoshi-like distal myopathy (MMD3), and asymptomatic hyperCKemia. OBJECTIVE: To describe clinic, histologic, genetic and imaging features, of ANO5 mutated patients. METHODS: Five patients, four from France (P1, P2, P3 and P4) and one from Mexico (P5), from four families were included. P1 and P2, belonging to group 1, had normal muscle strength; Group 2, P3, P4 and P5, presented with muscular weakness. Muscle strength was measured by manual muscle testing, Medical Research Council (MRC) grades 1/5 to 5/5. Laboratory exams included serum CK levels, nerve conduction studies (NCS)/needle electromyography (EMG), pulmonary function tests, EKG and cardiac ultrasound. ANO5 molecular screening was performed with different approaches. RESULTS: Group 1 patients showed myalgias with hyperCKemia or isolated hyperCKemia. Group 2 patients presented with limb-girdle or proximo-distal muscular weakness. Serum CK levels ranged from 897 to 5000 UI/L. Muscle biopsy analysis in P4 and P5 showed subsarcolemmal mitochondrial aggregates. Electron microscopy confirmed mitochondrial proliferation and revealed discontinuity of the sarcolemmal membrane. Muscle MRI showed asymmetrical fibro-fatty substitution predominant in the lower limbs.P1 and P2 were compound heterozygous for c.191dupA (p.Asn64Lysfs*15) and c.1898 + G>A; P3 was homozygous for the c.692G>T. (p.Gly231Val); P4 harbored a novel biallelic homozygous exons 1-7 ANO5 gene deletion, and P5 was homozygous for a c.172 C > T (p.(Arg 58 Trp)) ANO5 pathogenic variant. CONCLUSIONS: Our cohort confirms the wide clinical variability and enlarge the genetic spectrum of ANO5-related myopathies.

Lack of Delta-Sarcoglycan (Sgcd) Results in Retinal Degeneration.

Age-related macular degeneration (AMD) is the leading cause of central vision loss and severe blindness among the elderly population. Recently, we reported on the association of the SGCD gene (encoding for δ-sarcoglycan) polymorphisms with AMD. However, the functional consequence of Sgcd alterations in retinal degeneration is not known. Herein, we characterized changes in the retina of the Sgcd knocked-out mouse (KO, Sgcd-/-). At baseline, we analyzed the retina structure of three-month-old wild-type (WT, Sgcd+/+) and Sgcd-/- mice by hematoxylin and eosin (H&E) staining, assessed the Sgcd-protein complex (α-, ß-, γ-, and ε-sarcoglycan, and sarcospan) by immunofluorescence (IF) and Western blot (WB), and performed electroretinography. Compared to the WT, Sgcd-/- mice are five times more likely to have retinal ruptures. Additionally, all the retinal layers are significantly thinner, more so in the inner plexiform layer (IPL). In addition, the number of nuclei in the KO versus the WT is ever so slightly increased. WT mice express Sgcd-protein partners in specific retinal layers, and as expected, KO mice have decreased or no protein expression, with a significant increase in the α subunit. At three months of age, there were no significant differences in the scotopic electroretinographic responses, regarding both a- and b-waves. According to our data, Sgcd-/- has a phenotype that is compatible with retinal degeneration.

Ursolic and Oleanolic Acids Induce Mitophagy in A549 Human Lung Cancer Cells.

Ursolic and oleanolic acids are natural isomeric triterpenes known for their anticancer activity. Here, we investigated the effect of triterpenes on the viability of A549 human lung cancer cells and the role of autophagy in their activity. The induction of autophagy, the mitochondrial changes and signaling pathway stimulated by triterpenes were systematically explored by confocal microscopy and western blotting. Ursolic and oleanolic acids induce autophagy in A549 cells. Ursolic acid activates AKT/mTOR pathways and oleanolic acid triggers a pathway independent on AKT. Both acids promote many mitochondrial changes, suggesting that mitochondria are targets of autophagy in a process known as mitophagy. The PINK1/Parkin axis is a pathway usually associated with mitophagy, however, the mitophagy induced by ursolic or oleanolic acid is just dependent on PINK1. Moreover, both acids induce an ROS production. The blockage of autophagy with wortmannin is responsible for a decrease of mitochondrial membrane potential (Δψ) and cell death. The wortmannin treatment causes an over-increase of p62 and Nrf2 proteins promote a detoxifying effect to rescue cells from the death conducted by ROS. In conclusion, the mitophagy and p62 protein play an important function as a survival mechanism in A549 cells and could be target to therapeutic control.

Significant Association Between Variant in SGCD and Age-Related Macular Degeneration.

CFH and HTRA1 genes are traditional markers of increased risk of age-related macular degeneration (AMD) across populations. Recent findings suggest that additional genes-for instance, in the dystrophin-associated protein complex-might be promising markers for AMD. Here, we performed a case-control study to assess the effect of SGCD single nucleotide polymorphisms (SNPs), a member of this protein family, on AMD diagnosis and phenotype. We performed a case-control study of an under-studied population from Hispanics in Mexico City, with 134 cases with 134 unpaired controls. Cases were 60 years or older (Clinical Age-Related Maculopathy Staging (CARMS) grade 4⁻5, as assessed by experienced ophthalmologists following the American Association of Ophthalmology (AAO) guidelines), without other retinal disease or history of vitreous-retinal surgery. Controls were outpatients aged 60 years or older, with no drusen or retinal pigment epithelium (RPE) changes on a fundus exam and a negative family history of AMD. We examined SNPs in the SGCD gene (rs931798, rs140617, rs140616, and rs970476) by sequencing and real-time PCR. Genotyping quality checks and univariate analyses were performed with PLINK v1.90b3.42. Furthermore, logistic regression models were done in SAS v.9.4 and haplotype configurations in R v.3.3.1. After adjusting for clinical covariates, the G/A genotype of the SGCD gene (rs931798) significantly increases the odds of being diagnosed with AMD in 81% of cases (1.81; 95% CI 1.06⁻3.14; p = 0.031), especially the geographic atrophy phenotype (1.82; 95% CI 1.03⁻3.21; p = 0.038) compared to the G/G homozygous genotype. Moreover, the GATT haplotype in this gene (rs931798, rs140617, rs140616, and rs970476) is associated with lower odds of AMD (adjusted odds ratio (OR) 0.13; 95% CI 0.02⁻0.91; p = 0.041). SGCD is a promising gene for AMD research. Further corroboration in other populations is warranted, especially among other Hispanic ethnicities.

Pharmacogenetics of response to neoadjuvant paclitaxel treatment for locally advanced breast cancer.

Locally advanced breast cancer (LABC) cases have a varying five-year survival rate, mainly influenced by the tumor response to chemotherapy. Paclitaxel activity (response rate) varies across populations from 21.5% to 84%. There are some reports on genetic traits and paclitaxel; however, there is still considerable residual unexplained variability. In this study, we aimed to test the association between eleven novel markers and tumor response to paclitaxel and to explore if any of them influenced tumor protein expression. We studied a cohort of 140 women with LABC. At baseline, we collected a blood sample (for genotyping), fine needle aspirates (for Western blot), and tumor measurements by imaging. After follow-up, we ascertained the response to paclitaxel monotherapy by comparing the percent change in the pre-, post- tumor measurements after treatment. To allocate exposure, we genotyped eleven SNPs with TaqMan probes on RT-PCR and regressed them to tumor response using linear modeling. In addition, we compared protein expression, between breast tumors and healthy controls, of those genes whose genetic markers were significantly associated with tumor response. After adjusting for multiple clinical covariates, SNPs on the LPHN2, ROBO1, SNTG1, and GRIK1 genes were significant independent predictors of poor tumor response (tumor growth) despite paclitaxel treatment. Moreover, proteins encoded by those genes are significantly downregulated in breast tumor samples.

Dysferlin quantification in monocytes for rapid screening for dysferlinopathies.

INTRODUCTION: In this study, we determined normal levels of dysferlin expression in CD14+ monocytes by flow cytometry (FC) as a screening tool for dysferlinopathies. METHODS: Monocytes from 183 healthy individuals and 29 patients were immunolabeled, run on an FACScalibur flow cytometer, and analyzed by FlowJo software. RESULTS: The relative quantity of dysferlin was expressed as mean fluorescence intensity (MFI). Performance of this diagnostic test was assessed by calculating likelihood ratios at different MFI cut-off points, which allowed definition of 4 disease classification groups in a simplified algorithm. CONCLUSION: The MFI value may differentiate patients with dysferlinopathy from healthy individuals; it may be a useful marker for screening purposes. Muscle Nerve 54: 1064-1071, 2016.

Comparison of mutation profiles in the Duchenne muscular dystrophy gene among populations: implications for potential molecular therapies.

Novel therapeutic approaches are emerging to restore dystrophin function in Duchenne Muscular Dystrophy (DMD), a severe neuromuscular disease characterized by progressive muscle wasting and weakness. Some of the molecular therapies, such as exon skipping, stop codon read-through and internal ribosome entry site-mediated translation rely on the type and location of mutations. Hence, their potential applicability worldwide depends on mutation frequencies within populations. In view of this, we compared the mutation profiles of the populations represented in the DMD Leiden Open-source Variation Database with original data from Mexican patients (n = 162) with clinical diagnosis of the disease. Our data confirm that applicability of exon 51 is high in most populations, but also show that differences in theoretical applicability of exon skipping may exist among populations; Mexico has the highest frequency of potential candidates for the skipping of exons 44 and 46, which is different from other populations (p < 0.001). To our knowledge, this is the first comprehensive comparison of theoretical applicability of exon skipping targets among specific populations.

Immunodetection analysis of muscular dystrophies in Mexico.

INTRODUCTION: The muscular dystrophies (MDs) result from perturbations in the myofibers. These alterations are induced in part by mechanical stress due to membrane cell fragility, disturbances in mechanotransduction pathways, muscle cell physiology, and metabolism. METHODS: We analyzed 290 biopsies of patients with a clinical diagnosis of muscular dystrophy. Using immunofluorescence staining, we searched for primary and secondary deficiencies of 12 different proteins, including membrane, costamere, cytoskeletal, and nuclear proteins. In addition, we analyzed calpain-3 by immunoblot. RESULTS: We identified 212 patients with varying degrees of protein deficiencies, including dystrophin, sarcoglycans, dysferlin, caveolin-3, calpain-3, emerin, and merosin. Moreover, 78 biopsies showed normal expression of all investigated muscle proteins. The frequency rates of protein deficiencies were as follows: 52.36% dystrophinopathies; 18.40% dysferlinopathies; 14.15% sarcoglycanopathies; 11.32% calpainopathies; 1.89% merosinopathies; 1.42% caveolinopathies; and 0.47% emerinopathies. Deficiencies in lamin A/C and telethonin were not detected. CONCLUSION: We have described the frequency of common muscular dystrophies in Mexico.