Differentially expressed genes: OCT-4, SOX2, STAT3, CDH1 and CDH2, in cultured mesenchymal stem cells challenged with serum of women with endometriosis

Endometriosis is a common chronic gynecological disorder defined as the presence of ectopic functional endometrial tissues, outside uterine cavity, primarily on the pelvic peritoneum and the ovaries. Several studies revealed a correlation between aberrant stem-cell activity in the endometrium and endometriosis. Yet the molecular and cellular behaviors of mesnchymal stem cells in development of endometriosis are hampered by lack of invitro experiments. Our aim was to explore morphological and molecular changes associated with mesenchymal stem cells (MSCs) exposition to serum derived from women with severe endometriosis. Two cell cultures of MSCs isolated from endometrial tissues of two endometriosis-free women. Each cell culture was treated individually with the serum of women with endometriosis (experimental group/n =?7), and serum of women without endometriosis (control group/ n = 4) for 14?days. Quantitative Real-Time PCR was performed later to reveal expression of OCT-4, CDH1 and CDH2, STAT3 and SOX2 genes. Morphologically, cells showed no significant changes. However from molecular point of view, we found increased expression in OCT-4, CDH1 and CDH2. For STAT3 and SOX2 we did not find a significant difference. This study shows that endometriosis serum induced molecular changes in human endometrial MSCs (EnMSCs) that might be related to altered cell behavior which may be a step in differentiation that may be completed invivo by other factors to complete the process of transition. Further researches are needed for optimization to reach differentiation.     

Zinc finger protein binding to DNA: an energy perspective using molecular dynamics simulation and free energy calculations on mutants of both zinc finger domains and their specific DNA bases

Energy calculations based on MM-GBSA were employed to study various zinc finger protein (ZF) motifs binding to DNA. Mutants of both the DNA bound to their specific amino acids were studied. Calculated energies gave evidence for a relationship between binding energy and affinity of ZF motifs to their sites on DNA. ΔG values were ?15.82(12), ?3.66(12), and ?12.14(11.6) kcal/mol for finger one, finger two, and finger three, respectively. The mutations in the DNA bases reduced the value of the negative energies of binding (maximum value for ΔΔG = 42Kcal/mol for F1 when GCG mutated to GGG, and ΔΔG = 22 kcal/mol for F2, the loss in total energy of binding originated in the loss in electrostatic energies upon mutation (r = .98). The mutations in key amino acids in the ZF motif in positions-1, 2, 3, and 6 showed reduced binding energies to DNA with correlation coefficients between total free energy and electrostatic was .99 and with Van der Waal was .93. Results agree with experimentally found selectivity which showed that Arginine in position-1 is specific to G, while Aspartic acid (D) in position 2 plays a complicated role in binding. There is a correlation between the MD calculated free energies of binding and those obtained experimentally for prepared ZF motifs bound to triplet bases in other reports (), our results may help in the design of ZF motifs based on the established recognition codes based on energies and contributing energies to the total energy.     

Unbalancing the Phosphatidylinositol-4,5-bisphosphate–Cofilin Interaction Impairs Cell Steering

Cofilin is a key player in actin dynamics during cell migration. Its activity is regulated by (de)phosphorylation, pH, and binding to phosphatidylinositol-4,5-bisphosphate [PI(4,5)P₂]. Here, we here use a human cofilin-1 (D122K) mutant with increased binding affinity for PI(4,5)P₂ and slower release from the plasma membrane to study the role of the PI(4,5)P₂–cofilin interaction in migrating cells. In fibroblasts in a background of endogenous cofilin, D122K cofilin expression negatively affects cell turning frequency. In carcinoma cells with down-regulated endogenous cofilin, D122K cofilin neither rescues the drastic morphological defects nor restores the effects in cell turning capacity, unlike what has been reported for wild-type cofilin. In cofilin knockdown cells, D122K cofilin expression promotes outgrowth of an existing lamellipod in response to epidermal growth factor (EGF) but does not result in initiation of new lamellipodia. This indicates that, next to phospho- and pH regulation, the normal release kinetics of cofilin from PI(4,5)P₂ is crucial as a local activation switch for lamellipodia initiation and as a signal for migrating cells to change direction in response to external stimuli. Our results demonstrate that the PI(4,5)P₂ regulatory mechanism, that is governed by EGF-dependent phospholipase C activation, is a determinant for the spatial and temporal control of cofilin activation required for lamellipodia initiation.     

Caloric Restriction Shortens Lifespan through an Increase in Lipid Peroxidation,Inflammation and Apoptosis in the G93A Mouse,an Animal Model of ALS

Caloric restriction (CR) extends lifespan through a reduction in oxidative stress, delays the onset of morbidity and prolongs lifespan. We previously reported that long-term CR hastened clinical onset, disease progression and shortened lifespan, while transiently improving motor performance in G93A mice, a model of amyotrophic lateral sclerosis (ALS) that shows increased free radical production. To investigate the long-term CR-induced pathology in G93A mice, we assessed the mitochondrial bioenergetic efficiency and oxidative capacity (CS – citrate synthase content and activity, cytochrome c oxidase - COX activity and protein content of COX subunit- I and IV and UCP3- uncoupling protein 3), oxidative damage (MDA – malondialdehyde and PC – protein carbonyls), antioxidant enzyme capacity (Mn-SOD, Cu/Zn-SOD and catalase), inflammation (TNF-α), stress response (Hsp70) and markers of apoptosis (Bax, Bcl-2, caspase 9, cleaved caspase 9) in their skeletal muscle. At age 40 days, G93A mice were divided into two groups: Ad libitum (AL; n = 14; 7 females) or CR (n = 13; 6 females), with a diet equal to 60% of AL. COX/CS enzyme activity was lower in CR vs. AL male quadriceps (35%), despite a 2.3-fold higher COX-IV/CS protein content. UCP3 was higher in CR vs. AL females only. MnSOD and Cu/Zn-SOD were higher in CR vs. AL mice and CR vs. AL females. MDA was higher (83%) in CR vs. AL red gastrocnemius. Conversely, PC was lower in CR vs. AL red (62%) and white (30%) gastrocnemius. TNF-α was higher (52%) in CR vs. AL mice and Hsp70 was lower (62%) in CR vs. AL quadriceps. Bax was higher in CR vs. AL mice (41%) and CR vs. AL females (52%). Catalase, Bcl-2 and caspases did not differ. We conclude that CR increases lipid peroxidation, inflammation and apoptosis, while decreasing mitochondrial bioenergetic efficiency, protein oxidation and stress response in G93A mice.     

Markers of Skeletal Muscle Mitochondrial Function and Lipid Accumulation Are Moderately Associated with the Homeostasis Model Assessment Index of Insulin Resistance in Obese Men

Lower skeletal muscle mitochondrial oxidative phosphorylation capacity (OXPHOS) and intramyocellular lipid (IMCL) accumulation have been implicated in the etiology of insulin resistance (IR) in obesity. The purpose of this study was to examine the impact of endurance exercise on biochemical and morphological measures of IMCL and mitochondrial content, and their relationship to IR in obese individuals. We examined mitochondrial content (subunit protein abundance and maximal activity of electron transport chain enzymes), IMCL/mitochondrial morphology in both subsarcolemmal (SS) and intermyofibrillar (IMF) regions by transmission electron microscopy, and intracellular lipid metabolites (diacylglycerol and ceramide) in vastus lateralis biopsies, as well as, the homeostasis model assessment index of IR (HOMA-IR) prior to and following twelve weeks of an endurance exercise regimen in healthy age- and physical activity-matched lean and obese men. Obese men did not show evidence of mitochondrial OXPHOS dysfunction, disproportionate IMCL content in sub-cellular regions, or diacylglycerol/ceramide accretion despite marked IR vs. lean controls. Endurance exercise increased OXPHOS and mitochondrial size and density, but not number of individual mitochondrial fragments, with moderate improvements in HOMA-IR. Exercise reduced SS IMCL content (size, number and density), increased IMF IMCL content, while increasing IMCL/mitochondrial juxtaposition in both regions. HOMA-IR was inversely associated with SS (r = −0.34; P = 0.051) and IMF mitochondrial density (r = −0.29; P = 0.096), IMF IMCL/mitochondrial juxtaposition (r = −0.30; P = 0.086), and COXII (r = −0.32; P = 0.095) and COXIV protein abundance (r = −0.35; P = 0.052); while positively associated with SS IMCL size (r = 0.28; P = 0.119) and SS IMCL density (r = 0.25; P = 0.152). Our findings suggest that once physical activity and cardiorespiratory fitness have been controlled for, skeletal muscle mitochondrial and IMCL profile in obesity may only partially contribute to the development of IR.     

Vitamin D3 deficiency differentially affects functional and disease outcomes in the G93A mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neuromuscular disease characterized by motor neuron death in the central nervous system. Vitamin D supplementation increases antioxidant activity, reduces inflammation and improves motor neuron survival. We have previously demonstrated that vitamin D₃ supplementation at 10× the adequate intake improves functional outcomes in a mouse model of ALS.

Objective

To determine whether vitamin D deficiency influences functional and disease outcomes in a mouse model of ALS.

Methods

At age 25 d, 102 G93A mice (56 M, 46 F) were divided into two vitamin D₃ groups: 1) adequate (AI; 1 IU D₃/g feed) and 2) deficient (DEF; 0.025 IU D₃/g feed). At age 113 d, tibialis anterior (TA), quadriceps (quads) and brain were harvested from 42 mice (22 M and 20 F), whereas the remaining 60 mice (34 M and 26 F) were followed to endpoint.

Results

During disease progression, DEF mice had 25% (P = 0.022) lower paw grip endurance AUC and 19% (P = 0.017) lower motor performance AUC vs. AI mice. Prior to disease onset (CS 2), DEF mice had 36% (P = 0.016) lower clinical score (CS) vs. AI mice. DEF mice reached CS 2 six days later vs. AI mice (P = 0.004), confirmed by a logrank test which revealed that DEF mice reached CS 2 at a 43% slower rate vs. AI mice (HR = 0.57; 95% CI: 0.38, 1.74; P = 0.002). Body weight-adjusted TA (AI: r = 0.662, P = 0.001; DEF: r = 0.622, P = 0.006) and quads (AI: r = 0.661, P = 0.001; DEF: r = 0.768; P<0.001) weights were strongly correlated with age at CS 2.

Conclusion

Vitamin D₃ deficiency improves early disease severity and delays disease onset, but reduces performance in functional outcomes following disease onset, in the high-copy G93A mouse.