Can period-related symptoms predict menstrual manipulation among Australian female cyclists?

This study explored the extent of menstrual manipulation and its associated impact on period-related symptoms and training disruptions in Australian Female Cyclists. 205 female cyclists, from recreational to elite level, participated in an online "Female Cyclist Questionnaire (FCQ)". The FCQ utilised a series of validated questionnaires to obtain demographic information and menstrual function of the respondents, and to investigate their menstrual manipulation habits and perceptions on how their period-related symptoms affected their well-being, mood, energy and training tolerance. More than 80% of the cyclists reported that their period-related symptoms impacted upon training and 41% made training adjustments based on these symptoms. Two-thirds of respondents thought their training should be phase-controlled yet only half discussed their hormonal cycles with their coaches. Menstrual manipulation was predicted by reduced "workout tolerance" in these cyclists (odds ratio = 0.632). Half of the respondents reported compromised ability to tolerate high-intensity interval training with period-related symptoms. Period pain, increased irritability, lower energy levels and more sugar cravings were commonly reported but did not predict menstrual manipulation. The data indicated that period-related symptoms are present in Australian female cyclists across all levels of participation. However, the perceived impact to training and subsequent behavioural changes varied among individuals.

Effects of In-Exercise Carbohydrate Supplementation on Prolonged High-Intensity Exercise Performance in Oral Contraceptive Users.

PURPOSE: To examine the impact of oral contraceptive (OC) phases on performance, physiological, and subjective responses to prolonged, intensive exercise when carbohydrate (CHO) stores are reduced. METHODS: Ten well-trained female cyclists using monophasic OC completed 4 identical trials (>150 min) under conditions of in-trial 60-g·h-1 CHO supplementation (CHO+) or placebo (CHO-) during the sugar- (SUG) and active-pill (ACT) phases of their OC cycle. Each trial comprised two 400-kcal time trials (TT) separated by 1 hour of submaximal cycling at first ventilatory threshold. RESULTS: Change in completion time from TT1 to TT2 was minimized in CHO+ compared with CHO- (4.06 [2.55] vs 6.08 [5.33] min; P = .019, effect size = -0.36). An interaction effect of OC and CHO was observed for time to complete TT (P = .006), mean TT power (P = .002), mean TT heart rate (P = .002), and posttrial emotional balance (P = .020) and negative emotional state (P = .033). In ACT, mean TT power and heart rate were higher in CHO+ when compared with CHO-, resulting in faster TTs in CHO+ and improved posttrial emotional well-being. When CHO was not supplemented, TT power and heart rate were higher in SUG when compared with ACT, resulting in faster TTs in SUG and improved posttrial emotional balance. CONCLUSION: CHO depletion during ACT negatively influenced TT performance and emotional well-being when compared with SUG. Irrespective of OC pill phase, CHO supplementation should be prioritized to sustain performance and improve postexercise recovery-stress balance.

Fatigue Resistance Is Altered during the High-Hormone Phase of Eumenorrheic Females but Not Oral Contraceptive Users.

PURPOSE: This study aimed to examine the effect of ovarian hormones and their synthetic equivalents on substrate utilization and fatigue resistance during a race-specific cycling protocol. METHODS: Seventeen well-trained female cyclists (nine eumenorrheic females, eight oral contraceptive users) completed two experimental trials, in a randomized order, in their low- (follicular/sugar pill) and high-hormone (luteal/active pill) phases. Each 91-min trial consisted of a 45-min moderate-intensity component (submaximal cycling, or SMC) followed by 6 min of high-intensity (HIT) and then a fatigue resistance test (FRT): 6 × 1-min all-out efforts with 1-min active recovery. Meals, comprising carbohydrate (CHO) intake of 8 g·kg -1 body mass, were standardized 24-h pretrial. An electrolyte-only solution was provided ad libitum during each trial. RESULTS: In eumenorrheic females, a large reduction in average power during FRT was observed in the luteal phase (277 ± 31 vs 287 ± 33 W; P = 0.032). Greater CHO ox (~ 4%, P = 0.020) during SMC and ventilatory inefficiencies during SMC and HIT (~7%, P < 0.001) were also observed in the luteal phase. In contraceptive users, despite some phasal changes in cardiorespiratory and metabolic data in SMC (~6% higher blood glucose and ~2% higher minute ventilation in active pill phase), none of the performance parameters in the FRT were different. CONCLUSIONS: Fatigue resistance was compromised only in high-hormone phase of the menstrual cycle, with eumenorrheic females likely susceptible because of increased CHO utilization during SMC. Hormone-induced ventilatory inefficiencies may also have increased metabolic demand. These findings emphasize the need to maintain CHO availability for power production, particularly in high-hormone phases.

Neutrophil extracellular traps facilitate cancer metastasis: cellular mechanisms and therapeutic strategies.

BACKGROUND: The formation of neutrophil extracellular traps (NETs) was initially discovered as a novel immune response against pathogens. Recent studies have also suggested that NETs play an important role in tumor progression. This review summarizes the cellular mechanisms by which NETs promote distant metastasis and discusses the possible clinical applications targeting NETs. METHOD: The relevant literature from PubMed and Google Scholar (2001-2021) have been reviewed for this article. RESULTS: The presence of NETs has been detected in various primary tumors and metastatic sites. NET-associated interactions have been observed throughout the different stages of metastasis, including initial tumor cell detachment, intravasation and extravasation, the survival of circulating tumor cells, the settlement and the growth of metastatic tumor cells. Several in vitro and in vivo studies proved that inhibiting NET formation resulted in anti-cancer effects. The biosafety and efficacy of some NET inhibitors have also been demonstrated in early phase clinical trials. CONCLUSIONS: Considering the role of NETs in tumor progression, NETs could be a promising diagnostic and therapeutic target for cancer management. However, current evidence is mostly derived from experimental models and as such more clinical studies are still needed to verify the clinical significance of NETs in oncological settings.

Cold Preservation of Human Hepatocytes with High Viability.

Freshly isolated human hepatocytes are an important model for translational research, validation of experiments done in animals, and preclinical studies. Human hepatocyte isolation often cannot be carried out easily on demand in common research laboratories, and researchers often collaborate to share hepatocytes or outsource hepatocyte isolations. As a prerequisite for such a strategy, hepatocytes have to maintain their phenotypes after transport. Therefore, this study aimed to determine if overnight storage or shipment of hepatocytes affects their quality when viability, adherence, and cytochrome P450 (CYP) activities are considered. Hepatocytes were stored overnight or shipped to a collaborator in a cold storage solution on wet ice. On the next day, viability of hepatocytes was assessed before plating the cells to determine adherence. Hepatocytes were also cultured in a sandwich culture to determine CYP activities and inducibility. The results showed that although viability (79% ± 0.7% on isolation) was significantly decreased by overnight storage or shipment by 11% (p < 0.001) or 15% (p < 0.001), respectively, the viability of hepatocytes the next day at above 64% ± 2.2% remained sufficiently high for further experiments. In addition, hepatocytes stored for 18 or 24 hours were adherent the next day, and a high confluence of 81% ± 10% to 91% ± 4% was achieved after 48 hours in culture when hepatocytes were adhered on collagen-coated plates. Furthermore, CYP enzyme activities were inducible and not affected by variables such as fibrosis, age, type of operation, steatosis, and body mass index. However, our data would suggest that the type of cancer (primary/secondary), sex (male/female), hypertension, glutamic oxaloacetic transaminase activity, partial thromboplastin time, and size of perfused liver had significant effects (p < 0.05) on induction of some CYP enzymes. In conclusion, human hepatocyte isolation can be carried out at a centralized site and shared between multiple researchers, increasing flexibility and access to a representative human liver in vitro model.

Perception of journal club seminars by medical doctoral students: results from five years of evaluation.

Objectives: A journal club is one of the well-established and popular methods of post-graduate education. In this work, we were interested to understand how the participants perceive journal club as a whole and how they evaluate their personal process of acquiring new scientific knowledge and development of soft-skills as an indispensable prerequisite of the lifelong learning. Project description: This study is a survey analysis examining perception of journal club sessions by post-graduate medical students. A checklist for journal club preparation as well as a questionnaire for evaluation of the journal club session by participants has been developed to determine if the journal club had met its aims. Data were collected by summing up all answers to each question of the questionnaire for each session. Qualitative data from a five-year evaluation period were compiled and analyzed. Results: The journal club checklist served as a guideline for the preparation of a journal club session as well as an evaluation questionnaire containing 24 items. Our work presents evidence that journal club seminars are well perceived by participants. Furthermore, a high percentage of participants deemed the working atmosphere to be constructive and found it worthwhile to participate in the sessions. The topics of the presentations have been positively evaluated, however only a minority of participants found that the topics of the journal club was related to their own specific research topic. Concerning the distribution of the journal article, we could show that distributing the paper one week before the journal club event provided sufficient time for preparation. Our evaluation revealed that two-thirds of the participants found discussions during journal club sessions rich and productive. The motivation to think more critically increased during journal club sessions. From our work, it is evident that the participants perceived the speakers´ soft-skills to have improved with the practice. Finally, we show a clear trend of improved perception of the value of journal club sessions from beginning to the end of the evaluation time. Conclusion: Based on the analyzed evaluations, we can conclude that journal club events are highly valued by participants and could be a good option for the development of certain soft-skills.

p70 Ribosomal Protein S6 Kinase Is a Checkpoint of Human Hepatic Stellate Cell Activation and Liver Fibrosis in Mice.

BACKGROUND & AIMS: Progression of chronic liver disease (CLD) to liver cirrhosis and liver cancer is a major global cause of morbidity and mortality. Treatment options capable of inhibiting progression of liver fibrosis when etiological treatment of CLD is not available or fails have yet to be established. We investigated the role of serine/threonine kinase p70 ribosomal protein S6 kinase (p70S6K) as checkpoint of fibrogenesis in hepatic stellate cells (HSCs) and as target for the treatment of liver fibrosis. APPROACH & RESULTS: Immunohistochemistry was used to assess p70S6K expression in liver resection specimen. Primary human or murine HSCs from wild-type or p70S6K-/- mice as well as LX-2 cells were used for in vitro experiments. Specific small interfering RNA or CEP-1347 were used to silence or inhibit p70S6K and assess its functional relevance in viability, contraction and migration assays, fluorescence-activated cell sorting, and Western blot. These results were validated in vivo by a chemical model of fibrogenesis using wild-type and p70S6K-/- mice. Expression of p70S6K was significantly increased in human cirrhotic vs noncirrhotic liver-tissue and progressively increased in vitro through activation of primary human HSCs. Conversely, p70S6K induced fibrogenic activation of HSCs in different models, including the small interfering RNA-based silencing of p70S6K in HSC lines, experiments with p70S6K-/- cells, and the pharmacological inhibition of p70S6K by CEP-1347. These findings were validated in vivo as p70S6K-/- mice developed significantly less fibrosis upon exposure to CCl4. CONCLUSIONS: We establish p70S6K as a checkpoint of fibrogenesis in vitro and in vivo and CEP-1347 as potential treatment option that can safely be used for long-term treatment.

Attribute Amnesia in the Auditory Domain.

Individuals are often unable to report an attribute of an object to which they recently attended, if they expected to report a different attribute, a phenomenon known as attribute amnesia (AA). To date, all AA studies have occurred in the visual domain. The purpose of this study was to explore the boundary conditions of AA by testing if AA also occurs in the auditory domain and, if so, for which attributes. It was found that AA was present when reporting the location (p = .003) and the number of tones (p < .001) of an auditory stimulus, but not when reporting its pitch (p = .383). These findings can be understood in terms of the organisation of the primary cortical areas and help explain the differences between visual working memory and auditory working memory.

Identification and characterization of novel splice variants of human farnesoid X receptor.

Farnesoid X receptor (FXR, NR1H4) is a ligand-activated nuclear receptor, which regulates bile acid, lipid and glucose metabolism. Due to these functions, FXR has been investigated as a potential drug target for the treatment of liver diseases, such as primary biliary cholangitis and non-alcoholic steatohepatitis. Based on the previously described four splice variants, it has been suggested that alternative promoter usage and splicing may have an impact on total FXR activity as a result of encoding functionally diverse variants. Here we aimed for a systematic analysis of human hepatic FXR splice variants. In addition to the previously described FXRα1-4, we identified four novel splice variants (FXRα5-8) in human hepatocytes, which resulted from previously undetected exon skipping events. These newly identified isoforms displayed diminished DNA binding and impaired transactivation activities. Isoform FXRα5, which suppressed the transactivation activity of the functional isoform FXRα2, was further characterized as deficient in heterodimerization, coactivator recruitment and ligand binding. These findings were further supported by molecular dynamics simulations, which offered an explanation for the behavior of this isoform on the molecular level. FXRα5 exhibited low uniform expression levels in nearly all human tissues. Our systematic analysis of FXR splice variants in human hepatocytes resulted in the identification of four novel FXR isoforms, which all proved to be functionally deficient, but one novel variant, FXRα5, also displayed dominant negative activity. The possible associations with and roles of these novel isoforms in human liver diseases require further investigation.

Nelfinavir and Its Active Metabolite M8 Are Partial Agonists and Competitive Antagonists of the Human Pregnane X Receptor.

The HIV protease inhibitor nelfinavir is currently being analyzed for repurposing as an anticancer drug for many different cancers because it exerts manifold off-target protein interactions, finally resulting in cancer cell death. Xenosensing pregnane X receptor (PXR), which also participates in the control of cancer cell proliferation and apoptosis, was previously shown to be activated by nelfinavir; however, the exact molecular mechanism is still unknown. The present study addresses the effects of nelfinavir and its major and pharmacologically active metabolite nelfinavir hydroxy-tert-butylamide (M8) on PXR to elucidate the underlying molecular mechanism. Molecular docking suggested direct binding to the PXR ligand-binding domain, which was confirmed experimentally by limited proteolytic digestion and competitive ligand-binding assays. Concentration-response analyses using cellular transactivation assays identified nelfinavir and M8 as partial agonists with EC50 values of 0.9 and 7.3 µM and competitive antagonists of rifampin-dependent induction with IC50 values of 7.5 and 25.3 µM, respectively. Antagonism exclusively resulted from binding into the PXR ligand-binding pocket. Impaired coactivator recruitment by nelfinavir as compared with the full agonist rifampin proved to be the underlying mechanism of both effects on PXR. Physiologic relevance of nelfinavir-dependent modulation of PXR activity was investigated in respectively treated primary human hepatocytes, which showed differential induction of PXR target genes and antagonism of rifampin-induced ABCB1 and CYP3A4 gene expression. In conclusion, we elucidate here the molecular mechanism of nelfinavir interaction with PXR. It is hypothesized that modulation of PXR activity may impact the anticancer effects of nelfinavir. SIGNIFICANCE STATEMENT: Nelfinavir, which is being investigated for repurposing as an anticancer medication, is shown here to directly bind to human pregnane X receptor (PXR) and thereby act as a partial agonist and competitive antagonist. Its major metabolite nelfinavir hydroxy-tert-butylamide exerts the same effects, which are based on impaired coactivator recruitment. Nelfinavir anticancer activity may involve modulation of PXR, which itself is discussed as a therapeutic target in cancer therapy and for the reversal of chemoresistance.

Microbiome Analysis from Paired Mucosal and Fecal Samples of a Colorectal Cancer Biobank.

The role of gut microbiota in colorectal cancer is subject to extensive research. Before usage of biorepositories for microbiome studies, it is crucial to evaluate technical feasibility of microbiome profiling from various biospecimens. The aim of this study was to assess the feasibility of DNA-extraction and microbiome profiling of samples from different sample sites, tissue sites and storage duration of a colorectal cancer biobank. Mucosa samples, mucosal scrapings and feces as well as different tissue sites (tumor, normal mucosa) were analyzed. 16S rRNA gene-based microbiome profiling with taxonomic assignment was performed on the Illumina MiSeq (Illumina, San Diego, USA) platform from stored snap frozen samples. For statistical analysis, α- and ß-diversity measures, PCoA, permutational multivariate analysis of variance and graphical representation were performed. Microbiome analysis could be successfully performed in most of the samples (overall 93.3%) with sufficient numbers of high-quality reads. There were no differences between sample sites, while in some measures significant differences were found between tumor and normal mucosa (α-diversity, Shannon/Simpson Indices p = 0.028/0.027, respectively). Samples stored for up to eight years were used and storage conditions had no significant influence on the results. Tumor and tissue samples of a biobank stored long term can be successfully used for microbiome analysis. As large sample sizes are needed for association studies to evaluate microbial impact on tumorigenesis or progression of colorectal cancer, an already established biorepository may be a useful alternative to prospective clinical studies.

Establishment of an Endoscopy-Guided Minimally Invasive Orthotopic Mouse Model of Colorectal Cancer.

Open orthotopic mouse models of colorectal cancer have disadvantages such as the requirement for advanced surgical skills or the trauma caused by laparotomy. To overcome these drawbacks, this study aimed to evaluate the establishment of a minimally invasive model using murine colonoscopy. CT26 and MC38 CRC cells of different concentrations were injected into BALB/C and C57BL/6J mice, respectively. Follow-up endoscopies were performed to assign an endoscopic score to tumor growth. Gross autopsy, histologic and immuno-histochemical evaluation, and immune scoring were performed. To describe the learning curve of the procedures, a performance score was given. Local tumor growth with colorectal wall infiltration, luminal ulceration, the presence of tumor-infiltrating lymphocytes, lympho-vascular invasion, and early spontaneous lymph node, peritoneal, and hepatic metastases were observed. The tumors showed cytoplasmic immuno-staining for CK20. Compared to the MC38/C57BL/6J model, tumorigenicity and immunogenicity of the CT26/BALB/C model were higher. Tumor volume correlated with the endoscopic score. This endoscopy-guided orthotopic mouse model is easy to learn and quick to establish. It features early metastasis and enables the study of interactions with the immune system. When specific cell concentrations and cell lines are applied, controlled local tumor growth and metastasis can be achieved within short observation periods.

Concurrent isolation of hepatic stem cells and hepatocytes from the human liver.

Hepatocytes differentiated from induced pluripotent stem cells or stem cells have the potential to be representative in vitro models of the human liver for research as well as early safety assessment programs. However, up until now, there has been no definitive proof that differentiated hepatocytes recapitulate the phenotype and functional characteristics of primary hepatocytes from the same individual. Thus, a method for the concurrent isolation of hepatocytes and hepatic stem cells is presented here to provide the cells necessary for the evaluation of the required benchmarking. The method presented here generated high-quality hepatocytes with a purity of 94 ± 1% and a high percentage viability of 79 ± 2%. Furthermore, the hepatic stem cells isolated were found to be actively proliferating and have a purity of 98 ± 1%. Thus, these isolated cells can be used as a powerful tool for the validation of differentiated hepatocyte in vitro models.

Susceptibility of Asialoglycoprotein Receptor-Deficient Mice to Lps/Galactosamine Liver Injury and Protection by Betaine Administration.

BACKGROUND: Work from our laboratory has shown that the ethanol-induced increase in apoptotic hepatocellular death is closely related to the impairment in the ability of the asialoglycoprotein receptor (ASGP-R) to remove neighboring apoptotic cells. In this study, we assessed the role of ASGP-R in fulminant liver failure and investigated whether prior treatment with betaine (a naturally occurring tertiary amine) is protective. METHODS: Lipopolysaccharide (LPS; 50 µg/kg BW) and galactosamine (GalN; 350 mg/kg BW) were injected together to wild-type and ASGP-R-deficient mice that were treated for two weeks prior with or without 2% betaine in drinking water. The mice were sacrificed 1.5, 3, or 4.5 h post-injection, and tissue samples were collected. RESULTS: LPS/GalN injection generate distinct molecular processes, which includes increased production of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), thus causing apoptosis as evident by increased caspase-3 activity. ASGP-R deficient animals showed increased liver caspase activities, serum TNF-α and IL-6 levels, as well as more pronounced liver damage compared with the wild-type control animals after intraperitoneal injection of LPS/GalN. In addition, prior administration of betaine was found to significantly attenuate the LPS/GalN-induced increases in liver injury parameters. CONCLUSION: Our work underscores the importance of normal functioning of ASGP-R in preventing severe liver damage and signifies a therapeutic role of betaine in prevention of liver injuries from toxin-induced fulminant liver failure.

Histone Deacetylase Expressions in Hepatocellular Carcinoma and Functional Effects of Histone Deacetylase Inhibitors on Liver Cancer Cells In Vitro.

Hepatocellular carcinoma (HCC) is a leading cause for deaths worldwide. Histone deacetylase (HDAC) inhibition (HDACi) is emerging as a promising therapeutic strategy. However, most pharmacological HDACi unselectively block different HDAC classes and their molecular mechanisms of action are only incompletely understood. The aim of this study was to systematically analyze expressions of different HDAC classes in HCC cells and tissues and to functionally analyze the effect of the HDACi suberanilohydroxamic acid (SAHA) and trichostatin A (TSA) on the tumorigenicity of HCC cells. The gene expression of all HDAC classes was significantly increased in human HCC cell lines (Hep3B, HepG2, PLC, HuH7) compared to primary human hepatocytes (PHH). The analysis of HCC patient data showed the increased expression of several HDACs in HCC tissues compared to non-tumorous liver. However, there was no unified picture of regulation in three different HCC patient datasets and we observed a strong variation in the gene expression of different HDACs in tumorous as well as non-tumorous liver. Still, there was a strong correlation in the expression of HDAC class IIa (HDAC4, 5, 7, 9) as well as HDAC2 and 8 (class I) and HDAC10 (class IIb) and HDAC11 (class IV) in HCC tissues of individual patients. This might indicate a common mechanism of the regulation of these HDACs in HCC. The Cancer Genome Atlas (TCGA) dataset analysis revealed that HDAC4, HDAC7 and HDAC9 as well as HDAC class I members HDAC1 and HDAC2 is significantly correlated with patient survival. Furthermore, we observed that SAHA and TSA reduced the proliferation, clonogenicity and migratory potential of HCC cells. SAHA but not TSA induced features of senescence in HCC cells. Additionally, HDACi enhanced the efficacy of sorafenib in killing sorafenib-susceptible cells. Moreover, HDACi reestablished sorafenib sensitivity in resistant HCC cells. In summary, HDACs are significantly but differently increased in HCC, which may be exploited to develop more targeted therapeutic approaches. HDACi affect different facets of the tumorigenicity of HCC cells and appears to be a promising therapeutic approach alone or in combination with sorafenib.

Identification of novel agonists by high-throughput screening and molecular modelling of human constitutive androstane receptor isoform 3.

Prediction of drug interactions, based on the induction of drug disposition, calls for the identification of chemicals, which activate xenosensing nuclear receptors. Constitutive androstane receptor (CAR) is one of the major human xenosensors; however, the constitutive activity of its reference variant CAR1 in immortalized cell lines complicates the identification of agonists. The exclusively ligand-dependent isoform CAR3 represents an obvious alternative for screening of CAR agonists. As CAR3 is even more abundant in human liver than CAR1, identification of its agonists is also of pharmacological value in its own right. We here established a cellular high-throughput screening assay for CAR3 to identify ligands of this isoform and to analyse its suitability for identifying CAR ligands in general. Proof-of-concept screening of 2054 drug-like compounds at 10 µM resulted in the identification of novel CAR3 agonists. The CAR3 assay proved to detect the previously described CAR1 ligands in the screened libraries. However, we failed to detect CAR3-selective compounds, as the four novel agonists, which were selected for further investigations, all proved to activate CAR1 in different cellular and in vitro assays. In primary human hepatocytes, the compounds preferentially induced the expression of the prototypical CAR target gene CYP2B6. Failure to identify CAR3-selective compounds was investigated by molecular modelling, which showed that the isoform-specific insertion of five amino acids did not impact on the ligand binding pocket but only on heterodimerization with retinoid X receptor. In conclusion, we demonstrate here the usability of CAR3 for screening compound libraries for the presence of CAR agonists.

Prediction of human drug-induced liver injury (DILI) in relation to oral doses and blood concentrations.

Drug-induced liver injury (DILI) cannot be accurately predicted by animal models. In addition, currently available in vitro methods do not allow for the estimation of hepatotoxic doses or the determination of an acceptable daily intake (ADI). To overcome this limitation, an in vitro/in silico method was established that predicts the risk of human DILI in relation to oral doses and blood concentrations. This method can be used to estimate DILI risk if the maximal blood concentration (Cmax) of the test compound is known. Moreover, an ADI can be estimated even for compounds without information on blood concentrations. To systematically optimize the in vitro system, two novel test performance metrics were introduced, the toxicity separation index (TSI) which quantifies how well a test differentiates between hepatotoxic and non-hepatotoxic compounds, and the toxicity estimation index (TEI) which measures how well hepatotoxic blood concentrations in vivo can be estimated. In vitro test performance was optimized for a training set of 28 compounds, based on TSI and TEI, demonstrating that (1) concentrations where cytotoxicity first becomes evident in vitro (EC10) yielded better metrics than higher toxicity thresholds (EC50); (2) compound incubation for 48 h was better than 24 h, with no further improvement of TSI after 7 days incubation; (3) metrics were moderately improved by adding gene expression to the test battery; (4) evaluation of pharmacokinetic parameters demonstrated that total blood compound concentrations and the 95%-population-based percentile of Cmax were best suited to estimate human toxicity. With a support vector machine-based classifier, using EC10 and Cmax as variables, the cross-validated sensitivity, specificity and accuracy for hepatotoxicity prediction were 100, 88 and 93%, respectively. Concentrations in the culture medium allowed extrapolation to blood concentrations in vivo that are associated with a specific probability of hepatotoxicity and the corresponding oral doses were obtained by reverse modeling. Application of this in vitro/in silico method to the rat hepatotoxicant pulegone resulted in an ADI that was similar to values previously established based on animal experiments. In conclusion, the proposed method links oral doses and blood concentrations of test compounds to the probability of hepatotoxicity.

A Standardized Collagen-Based Scaffold Improves Human Hepatocyte Shipment and Allows Metabolic Studies over 10 Days.

Due to pronounced species differences, hepatotoxicity of new drugs often cannot be detected in animal studies. Alternatively, human hepatocytes could be used, but there are some limitations. The cells are not always available on demand or in sufficient amounts, so far there has been only limited success to allow the transport of freshly isolated hepatocytes without massive loss of function or their cultivation for a long time. Since it is well accepted that the cultivation of hepatocytes in 3D is related to an improved function, we here tested the Optimaix-3D Scaffold from Matricel for the transport and cultivation of hepatocytes. After characterization of the scaffold, we shipped cells on the scaffold and/or cultivated them over 10 days. With the evaluation of hepatocyte functions such as urea production, albumin synthesis, and CYP activity, we showed that the metabolic activity of the cells on the scaffold remained nearly constant over the culture time whereas a significant decrease in metabolic activity occurred in 2D cultures. In addition, we demonstrated that significantly fewer cells were lost during transport. In summary, the collagen-based scaffold allows the transport and cultivation of hepatocytes without loss of function over 10 days.

Spontaneous Conventional Osteosarcoma Transformation of a Chondroblastoma: A Case Report and Literature Review.

Chondroblastomas are a primary benign cartilaginous tumor that accounts for approximately 1% of all benign bone tumors. Primarily they are treated by curettage. The patient presented 4 years after a successfully treated chondroblastoma (curettage and Bone cement). Wide resection of the proximal tibia with endoprosthesis replacement was done. Lung CT showed multiple lung metastasis and despite starting chemotherapy, he succumbed to the disease. We discuss regarding the possibilities of "aggressive" chondroblastoma and more recently termed chondroblastoma-like osteosarcoma which is a separate entity from chondroblastoma. Aggressiveness in chondroblastoma can be 1 of 3 types as follows: 1. benign chondroblastoma with lung metastasis. 2. malignant chondroblastoma. 3. subsequent malignant transformation of benign chondroblastoma. We have attempted to review the literature and describe the "aggressive" chondroblastoma and chondroblastoma-like osteosarcoma in this report.