Investigation of oxidative stress parameters in different lifespan erythrocyte fractions in young untrained men after acute exercise.

NEW FINDINGS: What is the central question of this study? What is the influence of a single bout of exercise on the properties of erythrocyte fractions at different ages? What is the main finding and its importance? A single bout of exercise in untrained men induced oxidative stress in erythrocytes and had an influence on antioxidant defense in these cells. Old erythrocytes were more sensitive to oxidative damage than young and middle-aged cells. Higher levels of glutathione in old erythrocyte fractions did not protect them against oxidative stress. It seems that exercise may promote the removal of old erythrocytes from the circulation. The objective of this study was to establish the role of exercise-induced oxidative stress in the erythrocyte fractions [young (YF), middle-aged (MAF) and old (OF)] of young untrained men after acute exercise. Blood samples were collected before exercise, immediately after and 1 h after exercise. The maximal power generated was 292 ± 27 W, and exercise duration was 8.73 ± 0.9 min. Different optical properties and oxidative stress parameters were found in each erythrocyte fraction. Total thiols in YF and MAF after exercise and after 1 h rest were similar to values before exercise; however, in OF {32.7 ± 9.8 nmol [mg haemoglobin (Hb)]-1 } the concentration was lower in comparison to YF [55.5 ± 3.2 nmol (mg Hb)-1 ] and MAF [56.8 ± 7.7 nmol (mg Hb)-1 ] and increased 1 h later (P < 0.0002). The glutathione concentration was higher in OF [8.4 ± 0.4 nmol (mg Hb)-1 ] than in YF [4.5 ± 0.6 nmol (mg Hb)-1 ] and MAF [4.8 ± 0.5 nmol (mg Hb)-1 ; P < 0.0002] and did not change after exercise or 1 h later. In OF, the peroxide level was higher after exercise [1.2 ± 0.2 nmol (mg Hb)-1 ] and 1 h later [1.1 ± 0.2 nmol (mg Hb)-1 ], when compared with samples before exercise [0.9 ± 0.1 nmol (mg Hb)-1 ; P < 0.05]. Similar results were observed in YF and MAF. The level of thiobarbituric acid reactive substances (TBARS) was ∼2.5-fold higher in OF [0.19 ± 0.04 nmol (mg Hb)-1 ] when compared with YF [0.07 ± 0.01 nmol (mg Hb)-1 ] and MAF [0.08 ± 0.02 nmol (mg Hb)-1 ; P < 0.0002] and was increased after exercise, remaining unchanged 1 h later. In YF and MAF, no difference in the level of TBARS was detected after exercise or 1 h later. No difference in membrane fluidity was observed in all fractions. The erythrocyte OF appeared to be more sensitive to cellular oxidative damage.

Efficient episomal reprogramming of blood mononuclear cells and differentiation to hepatocytes with functional drug metabolism.

The possibility of converting cells from blood mononuclear cells (MNC) to liver cells provides promising opportunities for the study of diseases and the assessment of new drugs. However, clinical applications have to meet GMP requirements and the methods for generating induced pluripotent cells (iPCs) have to avoid insertional mutagenesis, a possibility when using viral vehicles for the delivery of reprogramming factors. We have developed an efficient non-integration method for reprogramming fresh or frozen blood MNC, maintained in an optimised cytokine cocktail, to generate induced pluripotent cells. Using electroporation for the effective delivery of episomal transcription factors (Oct4, Sox2, Klf4, L-Myc, and Lin28) in a feeder-free system, without any requirement for small molecules, we achieved a reprogramming efficiency of up to 0.033% (65 colonies from 2×10(5) seeded MNC). Applying the same cytokine cocktail and reprogramming methods to cord blood or fetal liver-derived CD34(+) cells, we obtained 148 iPS colonies from 10(5) seeding cells (0.148%). The iPS cell lines we generated maintained typical characteristics of pluripotent cells and could be successfully differentiated into hepatocytes with drug metabolic function.

Alterations in red blood cells and plasma properties after acute single bout of exercise.

The aim of this study was to investigate alterations in haemoglobin conformation and parameters related to oxidative stress in whole erythrocytes, membranes, and plasma after a single bout of exercise in a group of young untrained men. Venous blood samples from eleven healthy young untrained males (age = 22 ± 2 years, BMI = 23 ± 2.5 kg/m(2)) were taken from the antecubital vein before an incremental cycling exercise test, immediately after exercise, and 1 hour after exercise. Individual heart rate response to this exercise was 195 ± 12 beats/min and the maximum wattage was 292 ± 27 W. Immediately after exercise, significant increase in standard parameters (haemoglobin, haematocrit, lactate levels, and plasma volume) of blood was observed as well as plasma antioxidant capacity one hour after exercise. Reversible conformational changes in haemoglobin, measured using a maleimide spin label, were found immediately following exercise. The concentration of ascorbic acid inside erythrocytes significantly decreased after exercise. A significant decline in membrane thiols was observed one hour after exercise, but simultaneously an increase in plasma thiols immediately after and 1 h after exercise was also observed. This study shows that a single bout of exercise can lead to mobilization of defensive antioxidant systems in blood against oxidative stress in young untrained men.

Markers of sarcopenia increase 30-day mortality following emergency laparotomy: A systematic review.

BACKGROUND AND OBJECTIVE: Decreased skeletal muscle mass and quality are one of the several markers used for sarcopenia diagnosis and are generally associated with increased rates of post-operative infections, poorer recovery and increased mortality. The aim of this review was to evaluate methods applied to detect markers of sarcopenia and the associated outcomes for patients undergoing emergency laparotomy. METHODS: This review was conducted with reference to Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) guidelines. MEDLINE, Embase and Google Scholar databases were searched. Studies detecting patients with sarcopenia or skeletal muscle decline markers and the associated outcomes after emergency laparotomy surgery were considered. The Newcastle-Ottawa Scale was used to evaluate publication quality. RESULTS: Out of 103 studies, which were screened, 19 full-text records were reviewed and 7 studies were ultimately analyzed. The study cohort sizes ranged from n = 46 to n = 967. The age range was 36-95 years. There were 1107 females (53%) and 973 males (47%) across all 7 studies. All studies measured psoas muscle mass and three studies assessed psoas muscle quality using computerized tomography (CT) imaging. No study assessed muscle strength or function, while five studies showed an association between low muscle mass and increased mortality rates after emergency laparotomy. Among the three studies, which assessed muscle quality, two of three studies showed poorer 30-day survival rates. CONCLUSIONS: The existing literature is limited, however it indicates that low psoas muscle mass and quality markers are associated with increased 30-day mortality rates after emergency laparotomy. Therefore, muscle markers can be used as a new feasible tool to identify most at risk patients requiring further interventions.

The effect of sarcopenia on outcomes following orthopaedic surgery : a systematic review.

AIMS: Sarcopenia is characterized by a generalized progressive loss of skeletal muscle mass, strength, and physical performance. This systematic review primarily evaluated the effects of sarcopenia on postoperative functional recovery and mortality in patients undergoing orthopaedic surgery, and secondarily assessed the methods used to diagnose and define sarcopenia in the orthopaedic literature. METHODS: A systematic search was conducted in MEDLINE, EMBASE, and Google Scholar databases according to the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines. Studies involving sarcopenic patients who underwent defined orthopaedic surgery and recorded postoperative outcomes were included. The quality of the criteria by which a diagnosis of sarcopenia was made was evaluated. The quality of the publication was assessed using Newcastle-Ottawa Scale. RESULTS: A total of 365 studies were identified and screened, 26 full-texts were reviewed, and 19 studies were included in the review. A total of 3,009 patients were included, of whom 2,146 (71%) were female and 863 (29%) were male. The mean age of the patients was 75.1 years (SD 7.1). Five studies included patients who underwent spinal surgery, 13 included hip or knee surgery, and one involved patients who underwent fixation of a distal radial fixation. The mean follow-up was 1.9 years (SD 1.9; 5 days to 5.6 years). There was wide heterogeneity in the measurement tools which were used and the parameters for the diagnosis of sarcopenia in the studies. Sarcopenia was associated with at least one deleterious effect on surgical outcomes in all 19 studies. The postoperative rate of mortality was reported in 11 studies (57.9%) and sarcopenia was associated with poorer survival in 73% (8/11) of these. The outcome was most commonly assessed using the Barthel Index (4/19), and sarcopenic patients recorded lower scores in 75% (3/4) of these. Sarcopenia was defined using the gold-standard three parameters (muscle strength, muscle quantity or quality, and muscle function) in four studies (21%), using two parameters in another four (21%) and one in the remaining 11 (58%). The methodological quality of the studies was moderate to high. CONCLUSION: There is much heterogeneity in the reporting of the parameters which are used for the diagnosis of sarcopenia, and evaluating the outcome of orthopaedic surgery in sarcopenic patients. However, what data exist suggest that sarcopenia impairs recovery and increases postoperative mortality, especially in patients undergoing emergency surgery. Further research is required to develop processes that allow the accurate diagnosis of sarcopenia in orthopaedics, which may facilitate targeted pre- and postoperative interventions that would improve outcomes. Cite this article: Bone Joint J 2022;104-B(3):321-330.

Role of microRNA in muscle regeneration and diseases related to muscle dysfunction in atrophy, cachexia, osteoporosis, and osteoarthritis.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that have emerged as potential predictive, prognostic, and therapeutic biomarkers, relevant to many pathophysiological conditions including limb immobilization, osteoarthritis, sarcopenia, and cachexia. Impaired musculoskeletal homeostasis leads to distinct muscle atrophies. Understanding miRNA involvement in the molecular mechanisms underpinning conditions such as muscle wasting may be critical to developing new strategies to improve patient management. MicroRNAs are powerful post-transcriptional regulators of gene expression in muscle and, importantly, are also detectable in the circulation. MicroRNAs are established modulators of muscle satellite stem cell activation, proliferation, and differentiation, however, there have been limited human studies that investigate miRNAs in muscle wasting. This narrative review summarizes the current knowledge as to the role of miRNAs in the skeletal muscle differentiation and atrophy, synthesizing the findings of published data. Cite this article: Bone Joint Res 2020;9(11):798-807.

Alterations in the Properties of Red Blood Cells in Men with Coronary Artery Diseases after Comprehensive Cardiac Rehabilitation.

PURPOSE: Comprehensive cardiac rehabilitation (CCR) is a complex program aimed at improving the health status of patients with coronary artery disease (CAD), especially those who have been subjected to cardiac interventions (PCI and CABG).The aim of this study was to measure the changes in the properties of red blood cells (RBCs) in men with CAD after cardiac intervention and after participation in CCR program. METHODS: In this study, we have investigated the influence of the physical training-based CCR program in 12 men with CAD, after PCI or CABG. The characteristics of RBCs including the basic morphology of RBCs, the conformational state of RBC membrane protein and hemoglobin, acetylcholinesterase activity, membrane fluidity, the osmotic fragility, and thiol concentration in membrane and in hemolysate were measured. Ascorbate concentration and reduced glutathione were also determined. The analysis was performed in men, before and after participation in CCR. The properties of RBCs were observed in connection with the exercise test, and parameters were evaluated before, immediately after, and 1 hour after the exercise test. RESULTS: After CCR, a decrease in the mobility of erythrocyte membrane proteins was observed, which was accompanied by a decrease in lipid fluidity. In addition, immediately after the exercise test and 1 hour later, we measured a decrease in thiol level in hemolysate, but not in the plasma membrane. Unexpectedly, an increase in reduced glutathione concentration one hour after the exercise test after completing comprehensive cardiac rehabilitation was observed. CONCLUSION: CCR in men with CAD after cardiac intervention is connected with decreased membrane fluidity and decreased membrane protein mobility, which indicates that reduction of oxidative changes in these components occurs.

Validation of Reference Genes for Gene Expression Studies by RT-qPCR in HepaRG Cells during Toxicity Testing and Disease Modelling.

Gene expression analysis by quantitative real-time polymerase chain reaction (RT-qPCR) is routinely used in biomedical studies. The reproducibility and reliability of the data fundamentally depends on experimental design and data interpretation. Despite the wide application of this assay, there is significant variation in the validation process of gene expression data from research laboratories. Since the validity of results depends on appropriate normalisation, it is crucial to select appropriate reference gene(s), where transcription of the selected gene is unaffected by experimental setting. In this study we have applied geNorm technology to investigate the transcription of 12 'housekeeping' genes for use in the normalisation of RT-qPCR data acquired using a widely accepted HepaRG hepatic cell line in studies examining models of pre-clinical drug testing. geNorm data identified a number of genes unaffected by specific drug treatments and showed that different genes remained invariant in response to different drug treatments, whereas the transcription of 'classical' reference genes such as GAPDH (glyceralde- hyde-3-phosphate dehydrogenase) was altered by drug treatment. Comparing data normalised using the reference genes identified by geNorm with normalisation using classical housekeeping genes demonstrated substantial differences in the final results. In light of cell therapy application, RT-qPCR analyses has to be carefully evaluated to accurately interpret data obtained from dynamic cellular models undergoing sequential stages of phenotypic change.

Oxygen Plasma Substrate and Specific Nanopattern Promote Early Differentiation of HepaRG Progenitors.

Fully differentiated HepaRG™ cells are the hepatic cell line of choice for in vitro study in toxicology and drug trials. They are derived from a hepatoblast-like progenitor (HepaRG-P) that differentiates into a coculture of hepatocyte-like and cholangiocyte-like cells. This process that requires 2 weeks of proliferation followed by 2 weeks of differentiation using dimethyl sulfoxide (DMSO) can be time consuming and costly. Identifying a method to accelerate HepaRG-Ps toward a mature lineage would save both time and money. The ability to do this in the absence of DMSO would remove the possibility of confounding toxicology results caused by DMSO induction of CYP pathways. It has been shown that tissue culture substrates play an important role in the development and maturity of a cell line, and this is particularly important for progenitor cells, which retain some form of plasticity. Oxygen plasma treatment is used extensively to modify cell culture substrates. There is also evidence that patterned rather than planar surfaces have a positive effect on proliferation and differentiation. In this study, we compared the effect of standard tissue culture plastic (TCP), oxygen plasma coated (OPC), and nanopatterned substrates (NPS) on early differentiation and function of HepaRG-P cells. Since NPS were OPC we initially compared the effect of TCP and OPC to enable comparison between all three culture surfaces using OPC as control to asses if patterning further enhanced early differentiation and functionality. The results show that HepaRG-P's grown on OPC substrate exhibited earlier differentiation, proliferation, and function compared with TCP. Culturing HepaRG-P's on OPC with the addition of NPS did not confer any additional advantage. In conclusion, OPC surface appeared to enhance hepatic differentiation and functionality and could replace traditional methods of differentiating HepaRG-P cells into fully differentiated and functional HepaRGs earlier than standard methods. Impact statement We show significantly earlier differentiation and function of HepaRG progenitor cells when grown in dimethyl sulfoxide-free medium on oxygen plasma substrates versus standard tissue culture plastic. Further investigation showed that nanopatterning of oxygen plasma substrates did not confer any additional advantage over smooth oxygen plasma, although one pattern (DSQ120) showed comparable early differentiation and function.

Chlorpromazine toxicity is associated with disruption of cell membrane integrity and initiation of a pro-inflammatory response in the HepaRG hepatic cell line.

Chlorpromazine (CPZ) is a neuroleptic drug and prototype compound used to study intrahepatic cholestasis. The exact mechanisms of CPZ induced cholestasis remain unclear. Rat hepatocytes, or a sandwich culture of rat and human hepatocytes, have been the most commonly used models for studying CPZ toxicity in vitro. However, to better predict outcomes in pre-clinical trials where cholestasis may be an unwanted consequence, a human in vitro model, based on human HepaRG cells, capable of real-time, non-invasive and label free monitoring, alongside molecular investigations would be beneficial. To address this we used the human hepatic HepaRG cell line, and established concentrations of CPZ ranging from sub-toxic, 25 µM and 50 µM, to toxic 100 µM and compared them with untreated control. To assess the effect of this range of CPZ concentrations we employed electrical cell-substrate impedance sensing (ECIS) to measure viability and cell membrane interactions alongside traditional viability assays, immunocytostaining and qRT-PCR to assess genes of interest within adaptive and inflammatory pathways. Using these methods, we show a concentration dependant response to CPZ involving pro-inflammatory pathway, loss of tight junctions and membrane integrity, and an adaptive response mediated by Cytochrome P450 (CYP) enzyme activation and up-regulation of membrane phospholipid and xenobiotic transporters. In conclusion, structural changes within the membrane caused by sub-toxic and toxic concentrations of CPZ negatively impact the function of the cellular membrane. Damage to efflux transport proteins caused by CPZ induce cholestasis alongside downstream inflammation, which activates compensatory responses for cell survival. LAY SUMMARY: Chlorpromazine is a drug used to treat patients with schizophrenia, which has a known association with liver damage. Here we show that it causes inflammation and alters the cell membranes in liver and bile duct cells similar to what is seen within a human population. The initiation of the inflammatory response and changes to cellular structure may provide insight into the damage and disease process and inform medical treatment.

Alterations of erythrocyte structure and cellular susceptibility in patients with chronic renal failure: effect of haemodialysis and oxidative stress.

The aim of this study was to investigate erythrocytes rheological behaviour, membrane dynamics and erythrocytes susceptibility to disintegration upon strong oxidative stress induced by dialysis or by external H(2)O(2) among patients with CRF. EPR spectrometry was used to investigate alterations in physical state of cellular components. Generated ROS production induced: (1) significant increase of membrane fluidity in CRF erythrocytes treated with H(2)O(2) (p<0.005) and at 60 min of haemodialysis (p<0.05), (2) significant decrease of cytoskeletal protein-protein interactions (p<0.005) and (3) cellular osmotic fragility (p<0.0005). H(2)O(2) exacerbated these changes. Erythrocytes from CRF patients have changed rheological behaviour and present higher susceptibility to disintegration. Erythrocytes membrane characteristics indicate that CRF patients possess younger and more flexible cells, which are more susceptible to oxidative stress. This may contribute to the shortened survival of young erythrocytes in CRF patients.

Alterations in the in vitro and in vivo regulation of muscle regeneration in healthy ageing and the influence of sarcopenia.

BACKGROUND: Sarcopenia is defined as the age-related loss of skeletal muscle mass and function. While all humans lose muscle with age, 2-5% of elderly adults develop functional consequences (disabilities). The aim of this study was to investigate muscle myogenesis in healthy elderly adults, with or without sarcopenia, compared with middle-aged controls using both in vivo and in vitro approaches to explore potential biomarker or causative molecular pathways associated with sarcopenic versus non-sarcopenic skeletal muscle phenotypes during ageing. METHODS: Biomarkers of multiple molecular pathways associated with muscle regeneration were analysed using quantitative polymerase chain reaction in quadriceps muscle samples obtained from healthy elderly sarcopenic (HSE, n = 7) or non-sarcopenic (HENS, n = 21) and healthy middle-aged control (HMC, n = 22) groups. An in vitro system of myogenesis (using myoblasts from human donors aged 17-83 years) was used to mimic the environmental challenges of muscle regeneration over time. RESULTS: The muscle biopsies showed evidence of satellite cell activation in HENS (Pax3, P < 0.01, Pax7, P < 0.0001) compared with HMC. Early myogenesis markers Myogenic Differentiation 1 (MyoD1) and Myogenic factor 5 (Myf5) (P < 0.0001) and the late myogenesis marker myogenin (MyoG) (P < 0.01) were increased in HENS. In addition, there was a 30-fold upregulation of TNF-α in HENS compared with HMC (P < 0.0001). The in vitro system demonstrated age-related upregulation of pro-inflammatory cytokines (2-fold upregulation of interleukin (IL)-6, IL-8 mRNA, increased secretion of tumor necrosis factor-α (TNF-α) and IL-6, all P < 0.05) associated with impaired kinetics of myotube differentiation. The HSE biopsy samples showed satellite cell activation (Pax7, P < 0.05) compared with HMC. However, no significant upregulation of the early myogenesis (MyoD and Myf5) markers was evident; only the late myogenesis marker myogenin was upregulated (P < 0.05). Higher activation of the oxidative stress pathway was found in HENS compared with the HSE group. In contrast, there was 10-fold higher upregulation of HSPA1A a stress-induced chaperone acting upon misfolded proteins in HSE compared with the HENS group. CONCLUSIONS: Both pathological and adaptive processes are active in skeletal muscle during healthy ageing. Muscle regeneration pathways are activated during healthy ageing, but there is evidence of dysregulation in sarcopenia. In addition, increased cellular stress, with an impaired oxidative-stress and mis-folded protein response (HSPA1A), may be associated with the development of sarcopenia. The in vitro system of young and old myoblasts replicated some of the differences between young and old muscle.

Hepatic differentiation of human iPSCs in different 3D models: A comparative study.

Human induced pluripotent stem cells (hiPSCs) are a promising source from which to derive distinct somatic cell types for in vitro or clinical use. Existent protocols for hepatic differentiation of hiPSCs are primarily based on 2D cultivation of the cells. In the present study, the authors investigated the generation of hiPSC-derived hepatocyte-like cells using two different 3D culture systems: A 3D scaffold-free microspheroid culture system and a 3D hollow-fiber perfusion bioreactor. The differentiation outcome in these 3D systems was compared with that in conventional 2D cultures, using primary human hepatocytes as a control. The evaluation was made based on specific mRNA expression, protein secretion, antigen expression and metabolic activity. The expression of α-fetoprotein was lower, while cytochrome P450 1A2 or 3A4 activities were higher in the 3D culture systems as compared with the 2D differentiation system. Cells differentiated in the 3D bioreactor showed an increased expression of albumin and hepatocyte nuclear factor 4α, as well as secretion of α-1-antitrypsin as compared with the 2D differentiation system, suggesting a higher degree of maturation. In contrast, the 3D scaffold-free microspheroid culture provides an easy and robust method to generate spheroids of a defined size for screening applications, while the bioreactor culture model provides an instrument for complex investigations under physiological-like conditions. In conclusion, the present study introduces two 3D culture systems for stem cell derived hepatic differentiation each demonstrating advantages for individual applications as well as benefits in comparison with 2D cultures.

Cardiac rehabilitation improves the blood plasma properties of cardiac patients.

Cardiac rehabilitation (CR) improves exercise tolerance and general function. However, its effects on blood plasma in cardiac patients remain uncertain. Our aim was to examine the effect of comprehensive CR on the oxidative stress parameters and antioxidant plasma status in patients with coronary artery disease (CAD) after cardiac interventions. Exercise-based rehabilitation was established as ergometer training, adjusted for individual patients' physical efficiency. Training was repeated three times a week for two months. The standard biochemical (total cholesterol, HDL, LDL, triglycerides and erythrocyte sedimentation rate) and metabolic parameters (peak oxygen uptake [VO2] and peak workload) were determined. We assessed plasma viscosity, lipid peroxidation, carbonyl compounds levels, glutathione (GSH) and ascorbate (ASC) levels and the non-enzymatic antioxidant capacity of plasma in 12 patients with CAD before and after CR. Parameters were examined before exercise, immediately after exercise, and 1 h later. We also compared morphological and biochemical parameters of blood, as well as other parameters such as heart rate and blood pressure (resting and exercise), VO2max and peak workload (W) before and after CR. Before CR, a significant decrease in GSH concentration was observed 1 h after exercise. Conversely, after CR, GSH, and ASC levels remained unchanged immediately after exercise. However, ASC increased after CR after exercise and 1 h later in comparison to before CR. There was a significant increase in ferric reduction ability of plasma immediately after exercise after CR, when compared with before CR. CR improved several blood biochemical parameters, peak VO2, induced an increase in systolic blood pressure peak, and patients' peak workload. After CR, improvements were detected in oxidative stress parameters, except in the level of carbonyls. These changes may contribute to the increased functional heart capacity and better tolerance to exercise and functional capacity of the patients. These improvements could indicate better prognosis of future cardiac events and hospitalization and better quality of life.

Loss of oxidative defense and potential blockade of satellite cell maturation in the skeletal muscle of patients with cancer but not in the healthy elderly.

Muscle wasting in old age or cancer may result from failed myofiber regeneration and/or accelerated atrophy. This study aimed to determine from transcriptomic analysis of human muscle the integrity of the cellular stress response system in relation to satellite cell differentiation or apoptosis in patients with cancer (weight-stable (CWS) or weight-losing (CWL)) or healthy elderly (HE) when compared with healthy middle-aged controls (HMA). 28 patients with cancer (CWS: 18 and CWL: 10), HE: 21 and HMA: 20 underwent biopsy of quadriceps muscle. The expression of transcription factors for muscle regeneration (Pax3, Pax7 and MyoD) was increased in CWS and HE compared with HMA (p≤0.001). In contrast, the expression of the late myogenic differentiation marker MyoG was reduced in CWS and CWL but increased in HE (p≤0.0001). Bax was significantly increased in CWS, CWL and HE (p≤0.0001). Expression of the oxidative defense genes SOD2, GCLM, and Nrf2 was decreased in CWS and CWL but increased in HE (p≤0.0001). There is evidence for blockade of satellite cell maturation, upregulation of apoptosis and reduced oxidative defense in the muscle of cancer patients. In the healthy elderly the potential for differentiation and oxidative defense is maintained.

FGF, Insulin, and SMAD Signaling Cooperate for Avian Primordial Germ Cell Self-Renewal.

Precise self-renewal of the germ cell lineage is fundamental to fertility and reproductive success. The early precursors for the germ lineage, primordial germ cells (PGCs), survive and proliferate in several embryonic locations during their migration to the embryonic gonad. By elucidating the active signaling pathways in migratory PGCs in vivo, we were able to create culture conditions that recapitulate this embryonic germ cell environment. In defined medium conditions without feeder cells, the growth factors FGF2, insulin, and Activin A, signaling through their cognate-signaling pathways, were sufficient for self-renewal of germline-competent PGCs. Forced expression of constitutively active MEK1, AKT, and SMAD3 proteins could replace their respective upstream growth factors. Unexpectedly, we found that BMP4 could replace Activin A in non-clonal growth conditions. These defined medium conditions identify the key molecular pathways required for PGC self-renewal and will facilitate efforts in biobanking of chicken genetic resources and genome editing.

Molecular profile of organ culture-stored corneal epithelium: LGR5 is a potential new phenotypic marker of residual human corneal limbal epithelial stem cells.

Long-term preservation of corneal limbal epithelium may decrease its quality and change the molecular signature of the limbal epithelial stem cells. In this study we have investigated the molecular profile of isolated corneal epithelial cells that have been in storage for an extended time. Isolated cells were characterised by the expression profile of different cytokeratins and markers of squamous metaplasia (vimentin and α­actin). Furthermore, we examined global markers of adult stem cells including p63α and ABCG2 but also LGR5 as a novel stem cell marker. Immunocytochemical staining and PCR analysis of p63α, ABCG2 and LGR5 revealed the existence of side-population cells with a stem-cell phenotype and maintenance of corneal limbal stem cell properties. LGR5 expression can be related to cellular stemness and can be considered as a new phenotypic marker of residual human corneal limbal stem cells. However, the existence of CK10 together with co-expressed α-actin and vimentin suggests that the corneas investigated were under oxidative stress and showed evidence of squamous metaplasia.

Investigation of albumin properties in patients with chronic renal failure.

The aim of this study was the investigation of HSA properties and its structural changes after modification induced in vivo among patients with CRF who underwent haemodialysis. Application of different fluorescent dyes allowed the investigation of different regions of albumin molecule using ANS, bis-ANS, piren, piren maleimide and fluorescein isothiocyanate. As markers of oxidative modification, the total protein thiol, carbonyls, glycosylated plasma proteins and hydroperoxide were estimated in plasma. Additionally, this study investigated plasma viscosity and total antioxidant capacity (TAC) of the plasma. Results show that haemodialysis provoked significant changes in conformational properties of plasma albumin, which resulted in the loss of its biological functions. These findings suggest that oxidative stress and glycation of proteins in plasma are developed during haemodialysis. The results depict that one of the features of uraemia is the presence of signs of oxidative stress before haemodialysis. Nevertheless, oxidative stress and glycation of proteins in plasma are exacerbated during haemodialysis and are a complex process.

Nitric oxide induced oxidative changes in erythrocyte membrane components.

The effects of NO in its environment may vary considerably depending on various factors. This study shows oxidative mechanism of cellular membrane alterations, which is not associated with triggering of ONOOH generation but is induced by pure NO. Our investigation examined the influence of low concentration of NO (0.1; 0.2 mmol/l) on the qualitative changes of structure and dynamics of erythrocyte membrane. NO causes a statistically significant increase in membrane fluidity on different depths of lipid bilayer that is correlated with increase of lipids peroxidation. Statistically significant changes in the conformational state of cytoskeleton proteins were also detected. NO can be considered as a molecule responsible for determining rheological properties of erythrocytes membrane. Therefore, we propose that NO acts as pro-oxidant molecule at concentrations for which membrane appeared to be the first target before it entered the cytosol.