Oxidative stress and docosahexaenoic acid injury lead to increased necroptosis and ferroptosis in retinal pigment epithelium.

Age-related macular degeneration (AMD) is a complex disease caused by different genetic and environmental risk factors leading to loss of cells in the central part of the retina. Oxidative stress appears to be an important environmental risk factor that contributes to both the initiation and progression of AMD. Retinal pigment epithelium (RPE) plays an important role in regulating oxidative stress in the retina and is one of the main retinal cell types affected in AMD. A main function of RPE is to phagocytose photoreceptor outer segments (POS) which are rich in the polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA), making this cell type potentially more susceptible to oxidative stress-induced lipid peroxidation which can lead to cell death. RPE is known to undergo necrotic cell death in response to oxidative stress. The aim of this study was to determine if DHA in POS can increase oxidative damage to RPE. It was found that RPE undergo increased lipid peroxidation and decreased cell viability when stressed with hydrogen peroxide in combination with DHA or POS. H2O2-induced oxidative stress was found to cause both ferroptosis and necroptosis. However, the ferroptosis regulator acyl-CoA synthetase long-chain family member 4 (ACSL4) was found to be downregulated in RPE exposed to H2O2 and this effect was exacerbated when the RPE cells were simultaneously treated with DHA. Together, these results show a response of RPE when stressed which will likely be overwhelmed under disease conditions such as AMD resulting in cell death.

Effects of 56-Kilogram Kettlebell Swing Endpoint on Total Body Mechanics.

ABSTRACT: Ross, JA, Keogh, JWL, Lorenzen, C, and Lake, J. Effects of 56-kilogram kettlebell swing endpoint on total body mechanics. J Strength Cond Res 37(12): 2333-2338, 2023-In the past 2 decades, kettlebell training popularity has increased and the range of kettlebells has expanded to 2-92 kg. However, commercially available kettlebells above 56 kg have 12 kg increments, so alternatives to load are required to provide a suitable way of increasing training stimulus until the athlete is strong enough for a load increase. This study aimed to determine the differences in the force plate-derived biomechanical characteristics of heavy kettlebell swings to 3 different heights, as altering the height of the kettlebell swing may be one way to alter the mechanical demands with the same kettlebell mass. Fifteen resistance-trained men performed the kettlebell swing to acromion process height (AH), acromion process height + 20% (AH+20), and acromion process height-20% (AH-20). Swing height significantly affected vertical braking and propulsion phase net impulse and displacement, vertical braking velocity, and braking and propulsion work but not braking and propulsion duration. Altering kettlebell swing trajectory endpoint is a method to regress/progress the demands of kettlebell training. Coaches may be able to alter the acute demands and likely chronic adaptations of kettlebell training by prescribing different swing heights and kettlebell masses to their athletes.

Reliability of kettlebell swing one and five repetition maximum.

Background: Research into the kettlebell swing has increased in the last decade. There has been a paucity of literature assessing an individual's ability to perform the kettlebell swing exercise. The purpose of this study was to determine the test-retest reliability of the one and five repetition maximum (1RM and 5RM) kettlebell swing. Materials & Methods: Twenty four recreational resistance-trained participants performed an isometric mid-thigh pull (IMTP) and two familiarization sessions followed by three test sessions for each RM load approximately one week apart, using a custom-built plate-loaded kettlebell. On each test occasion, subjects completed a series of warm-up sets followed by 3-4 progressively heavier kettlebell swings to a standardized height until 1RM or 5RM was reached. Test-retest reliability was calculated using the intra-class correlation (ICC) and typical error was represented as the coefficient of variation (CV%) with 90% confidence limits (90% CL). The smallest worthwhile change (SWC%) representing the smallest change of practical importance, was calculated as 0.2 × between-subject standard deviation. The relationship of kettlebell swing performance and maximum strength was determined by Pearson correlation with ±90% CL between the absolute peak force recorded during IMTP and 1RM or 5RM. Results: Results demonstrated a high test-retest reliability for both the 1RM (ICC = 0.97, 90% CL [0.95-0.99]; CV = 2.7%, 90% CL [2.2-3.7%]) and 5RM (ICC = 0.98, 90% CL [0.96-0.99]; CV = 2.4%, 90% CL [1.9-3.3%]), respectively. The CV% was lower than the SWC for both the 1RM (SWC = 2.8%, 90% CL [1.9-3.5]) and 5RM (SWC = 2.9%, 90% CL [1.9-3.6]) kettlebell swing. The correlation between IMTP absolute peak force and the 1RM (r = 0.69, 90% CL 0.43-0.83) was large and very large for the 5RM (r = 0.75, 90% CL [0.55-0.87]). Conclusions: These results demonstrate the stability of 1RM and 5RM kettlebell swing performance after two familiarization sessions. Practitioners can be confident that changes in kettlebell swing 1RM and 5RM performance of >3.6 kg represent a practically important difference, which is the upper limit of the 90% CL.

The influence of hypoxia on the prostate cancer proteome.

Prostate cancer accounts for around 15% of male deaths in Western Europe and is the second leading cause of cancer death in men after lung cancer. Mounting evidence suggests that prostate cancer deposits exist within a hypoxic environment and this contributes to radio-resistance thus hampering one of the major therapies for this cancer. Recent reports have shown that nitric oxide (NO) donating non-steroidal anti-inflammatory drugs (NSAIDs) reduced tumour hypoxia as well as maintaining a radio-sensitising/therapeutic effect on prostate cancer cells. The aim of this study was to evaluate the impact of hypoxia on the proteome of the prostate and to establish whether NO-NSAID treatment reverted the protein profiles back to their normoxic status. To this end an established hormone insensitive prostate cancer cell line, PC-3, was cultured under hypoxic and normoxic conditions before and following exposure to NO-NSAID in combination with selected other common prostate cancer treatment types. The extracted proteins were analysed by ion mobility-assisted data independent acquisition mass spectrometry (MS), combined with multivariate statistical analyses, to measure hypoxia-induced alterations in the proteome of these cells. The analyses demonstrated that under hypoxic conditions there were well-defined, significantly regulated/differentially expressed proteins primarily involved with structural and binding processes including, for example, TUBB4A, CIRP and PLOD1. Additionally, the exposure of hypoxic cells to NSAID and NO-NSAID agents, resulted in some of these proteins being differentially expressed; for example, both PCNA and HNRNPA1L were down-regulated, corresponding with disruption in the nucleocytoplasmic shuttling process.

Hamstring Myoelectrical Activity During Three Different Kettlebell Swing Exercises.

Del Monte, MJ, Opar, DA, Timmins, RG, Ross, JA, Keogh, JWL, and Lorenzen, C. Hamstring myoelectrical activity during three different kettlebell swing exercises. J Strength Cond Res 34(7): 1953-1958, 2020-Kettlebell exercises have become an increasingly popular form of resistance training and component of lower-body rehabilitative training programs, despite a lack of scientific literature illustrating internal mechanisms and effectiveness of these approaches. Participants (n = 14) performed 3 different styles of kettlebell swings (hip hinge, squat, and double knee extension) and were assessed for medial hamstrings (MHs) and biceps femoris (BF) myoelectrical activity through surface electromyography (sEMG). Bipolar pregelled Ag/AgCl sEMG electrodes (10 mm diameter, 20 mm interelectrode distance) were placed on the participant's dominant limb after correct skin preparation. There was a main effect for swing type (p = 0.004), where the hip hinge swing elicited a greater overall MH and BF sEMG in comparison with the squat swing (mean difference = 3.92; 95% confidence interval [CI] = 1.53-6.32; p = 0.002) and the double knee extension swing (mean difference = 5.32; 95% CI = 0.80-9.83; p = 0.020). Across all swing types, normalized percentage of MH sEMG was significantly higher compared with the BF (mean difference = 9.93; 95% CI = 1.67-18.19; p = 0.022). The hip hinge kettlebell swing produced the greatest amount of hamstring sEMG for the 3 styles of kettlebell swings assessed. These findings have implications for the application of kettlebell swing exercises in strength and conditioning, injury prevention, and rehabilitation programs.

Plasma Metabolomics Identifies Lipid and Amino Acid Markers of Weight Loss in Patients with Upper Gastrointestinal Cancer.

Cachexia is a multifactorial wasting syndrome associated with high morbidity and mortality in patients with cancer. Diagnosis can be difficult and, in the clinical situation, usually relies upon reported weight loss. The 'omics' technologies allow us the opportunity to study the end points of many biological processes. Among these, blood-based metabolomics is a promising method to investigate the pathophysiology of human cancer cachexia and identify candidate biomarkers. In this study, we performed liquid chromatography mass spectrometry (LC/MS)-based metabolomics to investigate the metabolic profile of cancer-associated weight loss. Non-selected patients undergoing surgery with curative intent for upper gastrointestinal cancer were recruited. Fasting plasma samples were taken at induction of anaesthesia. LC/MS analysis showed that 6 metabolites were highly discriminative of weight loss. Specifically, a combination profile of LysoPC 18.2, L-Proline, Hexadecanoic acid, Octadecanoic acid, Phenylalanine and LysoPC 16:1 showed close correlation for eight weight-losing samples (≥5% weight loss) and nine weight-stable samples (<5%weight loss) between predicted and actual weight change (r = 0.976, p = 0.0014). Overall, 40 metabolites were associated with ≥5% weight loss. This study provides biological validation of the consensus definition of cancer cachexia (Fearon et al.) and provides feasible candidate markers for further investigation in early diagnosis and the assessment of therapeutic intervention.

Cell-Based Assays Using Differentiated Human Induced Pluripotent Cells.

This chapter describes the requirements and preconditions for using human induced pluripotent cell lines in assay development within the pharmaceutical industry. The joint collaborative effort between academic and pharma partners within the StemBANCC consortium which enabled the implementation of iPSC-derived cellular models for drug discovery is highlighted. This large collaborative scientific network has successfully derived a significant number of well-characterized patient-specific iPSC lines and established disease-relevant cellular assays, both of which are requirements for enabling pharmaceutical companies to develop more efficacious and safer medicines.

Human Induced Pluripotent Stem Cell-Derived Definitive Endoderm Bulk Culture and Hepatic Differentiation.

We have developed a method to bulk culture definitive endoderm cells generated from human iPSCs which can be stored and differentiated to hepatocytes. Human iPSC-derived definitive endoderm cells were sorted based on the expression of CXCR4. The sorted cells were able to proliferate for extended periods and can be cryopreserved. The definitive endoderm cells were subsequently utilized to generate functional hepatocytes expressing albumin and α-fetoprotein in different multiwell formats. This provides a method to reliably produce more consistent hepatocytes in greater quantities and has enabled the development of high-throughput screening strategies.

Identification of diagnostic upper gastrointestinal cancer tissue type-specific urinary biomarkers.

Several potential urinary biomarkers exhibiting an association with upper gastrointestinal tumour growth have been previously identified, of which S100A6, S100A9, rabenosyn-5 and programmed cell death 6-interacting protein (PDCD6IP) were further validated and found to be upregulated in malignant tumours. The cancer cohort from our previous study was subclassified to assess whether distinct molecular markers can be identified for each individual cancer type using a similar approach. Urine samples from patients with cancers of the stomach, oesophagus, oesophagogastric junction or pancreas were analysed by surface-enhanced laser desorption/ionization-time-of-flight mass spectrometry using both CM10 and IMAC30 (Cu2+-complexed) chip types and LC-MS/MS-based mass spectrometry after chromatographic enrichment. This was followed by protein identification, pattern matching and validation by western blotting. We found 8 m/z peaks with statistical significance for the four cancer types investigated, of which m/z 2447 and 2577 were identified by pattern matching as fragments of cathepsin-B (CTSB) and cystatin-B (CSTB); both molecules are indicative of pancreatic cancer. Additionally, we observed a potential association of upregulated α-1-antichymotrypsin with pancreatic and gastric cancers, of PDCD6IP, vitelline membrane outer layer protein 1 homolog (VMO1) and triosephosphate isomerase (TPI1) with oesophagogastric junctional cancers, and of complement C4-A, prostatic acid phosphatase, azurocidin and histone-H1 with oesophageal cancer. Furthermore, the potential pancreatic cancer biomarkers CSTB and CTSB were validated independently by western blotting. Therefore, the present study identified two new potential urinary biomarkers that appear to be associated with pancreatic cancer. This may provide a simple, non-invasive screening test for use in the clinical setting.

Urinary diagnostic proteomic markers for dynapenia in cancer patients.

Dynapenia is defined as the age-related loss of muscle strength, and plays a significant role in the loss of physical function and increased risk of disability among older individuals. The need for an early diagnosis supports the search for a biomarker that reflects muscle 'weakening'. This has previously proven difficult due to patient heterogeneity at presentation and lack of understanding of the underlying molecular mechanisms. The aim of the present study was to identify potential urinary biomarkers of dynapenia in patients undergoing potentially curative surgery for upper gastrointestinal cancer. Maximum isometric knee extensor strength (strain gauge) and maximum leg extensor power (Nottingham power rig) measurements were taken. Cut-off values for dynapenia were based on the Allied Dunbar national fitness survey. Values below the 5th percentile for the population matched for age and sex on the Allied Dunbar national fitness survey were used to stratify the cohort into dynapenic or normal. Urine samples taken at induction of anaesthesia were analysed by SELDI-TOF mass spectrometry using CM10 and IMAC30 chip-types to establish statistically significant m/z peak fingerprint patterns, followed by in-gel LC-MS/MS to identify molecular constituents. Statistical analysis of decision-tree calculations using Biomarker Pattern software resulted in models with sensitivities of 86 and 96%, specificities of 81 and 89%, and overall correctness of 84 and 93%, when applied to the entire cohort for power and strength measurement-based stratifications using the IMAC30 chip-type and the CM10 chip-type, respectively. The molecular identities of 10 peaks of interest were further investigated. After subtraction of potentially unrelated proteins, they were identified as fragments of Annexin A1, collagen α-1 (XV), perlecan and myotrophin. These results demonstrate that urinary screening can be used to define cancer-associated muscle weakness, and the identification of potential biomarkers could be invaluable in establishing a rapid test to measure and assess dynapenia in the clinical setting.

Proteomic identification of potential markers of myosteatosis in human urine.

Myosteatosis, the infiltration of fat in skeletal muscle, is associated with lower skeletal muscle density (SMD) as detected by computed tomography (CT). It increases with aging and obesity and is thought to play a role in the aetiology of insulin resistance and type II diabetes. The clinical significance of myosteatosis in cancer cachexia, however, remains to be determined. Along with demonstrable subcutaneous and visceral lipolysis, myosteatosis may also be a key component of the syndrome. We aimed to investigate the use of human urine as a non-invasive way to screen for molecular biomarkers of myosteatosis/reduced SMD using SELDI-TOF mass spectrometry. Pre-operative CT scans of patients undergoing surgery for upper gastrointestinal or hepatopancreaticobiliary cancer were analysed at the level of the third lumbar vertebrae. Myosteatosis was inferred as the presence of reduced SMD, which was defined as Hounsfield units for skeletal muscle <39.5 (two standard deviations below a normal healthy cohort). Urine was analysed by mass spectrometry using CM10 and IMAC30 SELDI-chips. Peaks observed in the CM10 and IMAC30 chip types, showed marked expressional differences between control and myosteatosis, were further investigated by mascot SELDI matrix matching. A total of 55 patients was recruited; 31 patients were found to be myosteatotic on CT scan. Application of the IMAC30-derived model to the entire cohort showed a sensitivity of 97%, specificity of 71% and an overall correctness of 85%. Application of the CM10 chipset-based model to the entire cohort, showed a 77% sensitivity, 67% specificity and 73% overall correctness. Analysis of the peaks of interest resulted in the identification of significant fragments of cathepsin C, argin, arylsulfatase A and glial fibrillary acidic protein. We identified several potential urinary molecular biomarkers associated with reduced SMD in cancer. Such markers are potentially useful in deriving a clinical screening test for myosteatosis.

The relationship between muscle protein content and CT-derived muscle radio-density in patients with upper GI cancer.

INTRODUCTION: Cancer cachexia is a multifactorial syndrome characterized by skeletal muscle loss. Cross-sectional analysis of CT scans is a recognized research method for assessing skeletal muscle volume. However, little is known about the relationship between CT-derived estimates of muscle radio-density (SMD) and muscle protein content. We assessed the relationship between CT-derived body composition variables and the protein content of muscle biopsies from cancer patients. METHODS: Rectus abdominis biopsies from cancer patients (n = 32) were analysed for protein content and correlated with phenotypic data gathered using CT body composition software. RESULTS: Skeletal muscle protein content varied widely between patients (median µg/mg wet weight = 89.3, range 70-141). There was a weak positive correlation between muscle protein content and SMD (r = 0.406, p = 0.021), and a weak positive correlation between protein content and percentage weight change (r = 0.416, p = 0.018). CONCLUSION: The protein content of skeletal muscle varies widely in cancer patients and cannot be accurately predicted by CT-derived muscle radio-density.

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.

Pluripotent stem cells to hepatocytes, the journey so far.

Over the past several years, there has been substantial progress in the field of regenerative medicine, which has enabled new possibilities for research and clinical application. For example, there are ongoing efforts directed at generating functional hepatocytes from adult-derived pluripotent cells for toxicity screening, generating disease models or, in the longer term, for the treatment of liver failure. In the present review, the authors summarise recent developments in regenerative medicine and pluripotent stem cells, the methods and tissues used for reprogramming and the differentiation of induced pluripotent stem cells (iPSCs) into hepatocyte-like cells. In addition, the hepatic disease models developed using iPSC technologies are discussed, as well as the potential for gene editing.

External kinetics of the kettlebell snatch in amateur lifters.

BACKGROUND: Kettlebell lifting has gained increased popularity as both a form of resistance training and as a sport, despite the paucity of literature validating its use as a training tool. Kettlebell sport requires participants to complete the kettlebell snatch continuously over prolonged periods of time. Kettlebell sport and weightlifting involve similar exercises, however, their traditional uses suggest they are better suited to training different fitness qualities. This study examined the three-dimensional ground reaction force (GRF) and force applied to the kettlebell over a 6 min kettlebell snatch set in 12 kettlebell-trained males. METHODS: During this set, VICON was used to record the kettlebell trajectory with nine infrared cameras while the GRF of each leg was recorded with a separate AMTI force plate. Over the course of the set, an average of 13.9 ± 3.3 repetitions per minute were performed with a 24 kg kettlebell. Significance was evaluated with a two-way ANOVA and paired t-tests, whilst Cohen's F (ESF) and Cohen's D (ESD) were used to determine the magnitude. RESULTS: The applied force at the point of maximum acceleration was 814 ± 75 N and 885 ± 86 N for the downwards and upwards phases, respectively. The absolute peak resultant bilateral GRF was 1,746 ± 217 N and 1,768 ± 242 N for the downwards and upwards phases, respectively. Bilateral GRF of the first and last 14 repetitions was found to be similar, however there was a significant difference in the peak applied force (F (1.11) = 7.42, p = 0.02, ESF = 0.45). Unilateral GRF was found have a significant difference for the absolute anterior-posterior (F (1.11) = 885.15, p < 0.0001, ESF = 7) and medio-lateral force vectors (F (1.11) = 5.31, p = 0.042, ESF = 0.67). DISCUSSION: Over the course of a single repetition there were significant differences in the GRF and applied force at multiple points of the kettlebells trajectory. The kettlebell snatch loads each leg differently throughout a repetition and performing the kettlebell snatch for 6 min will result in a reduction in peak applied force.

Recent early clinical drug development for acute kidney injury.

INTRODUCTION: Despite significant need and historical trials, there are no effective drugs in use for the prevention or treatment of acute kidney injury (AKI). There are several promising agents in early clinical development for AKI and two trials have recently been terminated. There are also exciting new findings in pre-clinical AKI research. There is a need to take stock of current progress in the field to guide future drug development for AKI. Areas covered: The main clinical trial registries, PubMed and pharmaceutical company website searches were used to extract the most recent clinical trials for sterile, transplant and sepsis-associated AKI. We summarise the development of the agents recently in clinical trial, update on their trial progress, consider reasons for failed efficacy of two agents, and discuss new paradigms in pre-clinical targets for AKI. Agents covered include- QPI-1002, THR-184, BB-3, heme arginate, human recombinant alkaline phosphatase (recAP), ciclosporin A, AB103, levosimendan, AC607 and ABT-719. Expert opinion: Due to the heterogenous nature of AKI, agents with the widest pleiotropic effects on multiple pathophysiological pathways are likely to be most effective. Linking preclinical models to clinical indication and improving AKI definition and diagnosis are key areas for improvement in future clinical trials.

Stem cell-derived models to improve mechanistic understanding and prediction of human drug-induced liver injury.

Current preclinical drug testing does not predict some forms of adverse drug reactions in humans. Efforts at improving predictability of drug-induced tissue injury in humans include using stem cell technology to generate human cells for screening for adverse effects of drugs in humans. The advent of induced pluripotent stem cells means that it may ultimately be possible to develop personalized toxicology to determine interindividual susceptibility to adverse drug reactions. However, the complexity of idiosyncratic drug-induced liver injury means that no current single-cell model, whether of primary liver tissue origin, from liver cell lines, or derived from stem cells, adequately emulates what is believed to occur during human drug-induced liver injury. Nevertheless, a single-cell model of a human hepatocyte which emulates key features of a hepatocyte is likely to be valuable in assessing potential chemical risk; furthermore, understanding how to generate a relevant hepatocyte will also be critical to efforts to build complex multicellular models of the liver. Currently, hepatocyte-like cells differentiated from stem cells still fall short of recapitulating the full mature hepatocellular phenotype. Therefore, we convened a number of experts from the areas of preclinical and clinical hepatotoxicity and safety assessment, from industry, academia, and regulatory bodies, to specifically explore the application of stem cells in hepatotoxicity safety assessment and to make recommendations for the way forward. In this short review, we particularly discuss the importance of benchmarking stem cell-derived hepatocyte-like cells to their terminally differentiated human counterparts using defined phenotyping, to make sure the cells are relevant and comparable between labs, and outline why this process is essential before the cells are introduced into chemical safety assessment. (Hepatology 2017;65:710-721).

New genetic signatures associated with cancer cachexia as defined by low skeletal muscle index and weight loss.

BACKGROUND: Cachexia affects the majority with advanced cancer. Based on current demographic and clinical factors, it is not possible to predict who will develop cachexia or not. Such variation may, in part, be due to genotype. It has recently been proposed to extend the diagnostic criteria for cachexia to include a direct measure of low skeletal muscle index (LSMI) in addition to weight loss (WL). We aimed to explore our panel of candidate single nucleotide polymorphism (SNPs) for association with WL +/- computerized tomography-defined LSMI. We also explored whether the transcription in muscle of identified genes was altered according to such cachexia phenotype METHODS: A retrospective cohort study design was used. Analysis explored associations of candidate SNPs with WL (n = 1276) and WL + LSMI (n = 943). Human muscle transcriptome (n = 134) was analysed using an Agilent platform. RESULTS: Single nucleotide polymorphisms in the following genes showed association with WL alone: GCKR, LEPR, SELP, ACVR2B, TLR4, FOXO3, IGF1, CPN1, APOE, FOXO1, and GHRL. SNPs in LEPR, ACVR2B, TNF, and ACE were associated with concurrent WL + LSMI. There was concordance between muscle-specific expression for ACVR2B, FOXO1 and 3, LEPR, GCKR, and TLR4 genes and LSMI and/or WL (P < 0.05). CONCLUSIONS: The rs1799964 in the TNF gene and rs4291 in the ACE gene are new associations when the definition of cachexia is based on a combination of WL and LSMI. These findings focus attention on pro-inflammatory cytokines and the renin-angiotensin system as biomarkers/mediators of muscle wasting in cachexia.

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.