A comparison of the nitrogen metabolic networks of Kluyveromyces marxianus and Saccharomyces cerevisiae

In grape must, nitrogen is available as a complex mixture of various compounds (ammonium and amino acids). Wine yeasts assimilate these multiple sources in order to suitably fulfil their anabolic requirements during alcoholic fermentation. Nevertheless, the order of uptake and the intracellular fate of these sources are likely to differ between strains and species. Using a two-pronged strategy of isotopic filiation and RNA sequencing, the metabolic network of nitrogen utilization and its regulation in Kluyveromyces marxianus were described, in comparison with those of Saccharomyces cerevisiae. The data highlighted differences in the assimilation of ammonium and arginine between the two species. The data also revealed that the metabolic fate of certain nitrogen sources differed, thereby resulting in the production of various amounts of key wine aroma compounds. These observations were corroborated by the gene expression analysis.     

Leukocyte-derived hepatic lipase increases HDL and decreases en face aortic atherosclerosis in LDLr-/- mice expressing CETP

In addition to hepatic expression, cholesteryl ester transfer protein (CETP) and hepatic lipase (HL) are expressed by human macrophages. The combined actions of these proteins have profound effects on HDL structure and function. It is not known how these HDL changes influence atherosclerosis. To elucidate the role of leukocyte-derived HL on atherosclerosis in a background of CETP expression, we studied low density lipoprotein receptor-deficient mice expressing human CETP (CETPtgLDLr(-/-)) with a leukocyte-derived HL deficiency (HL(-/-) BM). HL(-/-) bone marrow (BM), CETPtgLDLr(-/-) mice were generated via bone marrow transplantation. Wild-type bone marrow was transplanted into CETPtgLDLr(-/-) mice to generate HL(+/+) BM, CETPtgLDLr(-/-) controls. The chimeras were fed a high-fat, high-cholesterol diet for 14 weeks to promote atherosclerosis. In female HL(-/-) BM, CETPtgLDLr(-/-) mice plasma HDL-cholesterol concentration during high-fat feeding was decreased 27% when compared with HL(+/+) BM, CETPtgLDLr(-/-) mice (P < 0.05), and this was associated with a 96% increase in en face aortic atherosclerosis (P < 0.05). In male CETPtgLDLr(-/-) mice, leukocyte-derived HL deficiency was associated with a 16% decrease in plasma HDL-cholesterol concentration and a 25% increase in aortic atherosclerosis. Thus, leukocyte-derived HL in CETPtgLDLr(-/-) mice has an atheroprotective role that may involve increased HDL levels.     

Portrait of ependymoma recurrence in children: biomarkers of tumor progression identified by dual-color microarray-based gene expression analysis

Background

Children with ependymoma may experience a relapse in up to 50% of cases depending on the extent of resection. Key biological events associated with recurrence are unknown.

Methodology/Principal Findings

To discover the biology behind the recurrence of ependymomas, we performed CGHarray and a dual-color gene expression microarray analysis of 17 tumors at diagnosis co-hybridized with the corresponding 27 first or subsequent relapses from the same patient. As treatment and location had only limited influence on specific gene expression changes at relapse, we established a common signature for relapse. Eighty-seven genes showed an absolute fold change ≥2 in at least 50% of relapses and were defined as the gene expression signature of ependymoma recurrence. The most frequently upregulated genes are involved in the kinetochore (ASPM, KIF11) or in neural development (CD133, Wnt and Notch pathways). Metallothionein (MT) genes were downregulated in up to 80% of the recurrences. Quantitative PCR for ASPM, KIF11 and MT3 plus immunohistochemistry for ASPM and MT3 confirmed the microarray results. Immunohistochemistry on an independent series of 24 tumor pairs at diagnosis and at relapse confirmed the decrease of MT3 expression at recurrence in 17/24 tumor pairs (p = 0.002). Conversely, ASPM expression was more frequently positive at relapse (87.5% vs 37.5%, p = 0.03). Loss or deletion of the MT genes cluster was never observed at relapse. Promoter sequencing after bisulfite treatment of DNA from primary tumors and recurrences as well as treatment of short-term ependymoma cells cultures with a demethylating agent showed that methylation was not involved in MT3 downregulation. However, in vitro treatment with a histone deacetylase inhibitor or zinc restored MT3 expression.

Conclusions/Significance

The most frequent molecular events associated with ependymoma recurrence were over-expression of kinetochore proteins and down-regulation of metallothioneins. Metallothionein-3 expression is epigenetically controlled and can be restored in vitro by histone deacetylase inhibitors.     

Pak4 induces premature senescence via a pathway requiring p16INK4/p19ARF and mitogen-activated protein kinase signaling

Exposure of primary cells to mitogenic stimuli or oncogenes often causes them to undergo premature senescence. This is most likely a protective function that prevents uncontrolled proliferation. Pak4 is a target for the Rho GTPase Cdc42. Pak4 is overexpressed in human tumor cell lines, and it is the only member of the Pak family that is highly transforming in immortalized fibroblasts. Here we show that in primary fibroblasts, activated Pak4 inhibits cell proliferation and promotes premature senescence. Furthermore, Pak4 expression levels are upregulated in response to stimuli that promote senescence. Pak4-induced arrest appears to be mediated by a pathway that requires the ERK mitogen-activated protein kinase, as well as the cell cycle inhibitors p16^INK4 and p19^ARF. These new results describing a role for Pak4 in senescence are important for understanding why this protein is associated with cancer and how it promotes transformation in immortalized cells.     

Production of biologically active forms of recombinant hepcidin, the iron-regulatory hormone

Hepcidin is a liver produced cysteine-rich peptide hormone that acts as the central regulator of body iron metabolism. Hepcidin is synthesized under the form of a precursor, prohepcidin, which is processed to produce the biologically active mature 25 amino acid peptide. This peptide is secreted and acts by controlling the concentration of the membrane iron exporter ferroportin on intestinal enterocytes and macrophages. Hepcidin binds to ferroportin, inducing its internalization and degradation, thus regulating the export of iron from cells to plasma. The aim of the present study was to develop a novel method to produce human and mouse recombinant hepcidins, and to compare their biological activity towards their natural receptor ferroportin. Hepcidins were expressed in Escherichia coli as thioredoxin fusion proteins. The corresponding peptides, purified after cleavage from thioredoxin, were properly folded and contained the expected four-disulfide bridges without the need of any renaturation or oxidation steps. Human and mouse hepcidins were found to be biologically active, promoting ferroportin degradation in macrophages. Importantly, biologically inactive aggregated forms of hepcidin were observed depending on purification and storage conditions, but such forms were unrelated to disulfide bridge formation.     

The bounty of minor lakes: the role of small satellite water bodies in evolution and conservation of fishes in the Lake Victoria Region,East Africa

The Lake Victoria Region (LVR) encompasses the large lakes Victoria, Kyoga, Edward, George and Kivu, as well as scores of small satellite lakes within the parent catchments. Taken as a whole, the LVR originally harbored a unique fish fauna that included in excess of 600 endemic species of cichlid fishes. As a result of human influence, including a commercial fishery and the introduction of several exotic species, nearly 200 cichlid species and several endemic genera have become extinct from lakes Victoria and Kyoga. Recently, we have discovered that some of the apparently extinct taxa survive as extant representatives in the satellite lakes. Here, we summarize the findings of our ecological survey of the fish species of the satellite lakes. We also discuss the results of some preliminary genetic analyses, and highlight major genetic and ecological changes in the fish fauna that have taken place in the regional fishery. Minor lakes now play a crucial role in conserving the endangered species of the entire region, and also as living museums of East African ecological history. Our findings allude to the historical importance of minor satellite lakes as natural refugia for the fishes of the Lake Victoria Region, a region characterized by a history of geological and climatic instability.     

Contributions of the EMERALD project to assessing and improving microarray data quality

While minimum information about a microarray experiment (MIAME) standards have helped to increase the value of the microarray data deposited into public databases like ArrayExpress and Gene Expression Omnibus (GEO), limited means have been available to assess the quality of this data or to identify the procedures used to normalize and transform raw data. The EMERALD FP6 Coordination Action was designed to deliver approaches to assess and enhance the overall quality of microarray data and to disseminate these approaches to the microarray community through an extensive series of workshops, tutorials, and symposia. Tools were developed for assessing data quality and used to demonstrate how the removal of poor-quality data could improve the power of statistical analyses and facilitate analysis of multiple joint microarray data sets. These quality metrics tools have been disseminated through publications and through the software package arrayQualityMetrics. Within the framework provided by the Ontology of Biomedical Investigations, ontology was developed to describe data transformations, and software ontology was developed for gene expression analysis software. In addition, the consortium has advocated for the development and use of external reference standards in microarray hybridizations and created the Molecular Methods (MolMeth) database, which provides a central source for methods and protocols focusing on microarray-based technologies.     

Vegetarianism,female gender and increasing age,but not <Emphasis Type="Italic">CNDP1</Emphasis> genotype,are associated with reduced muscle carnosine levels in humans

Carnosine is found in high concentrations in skeletal muscles, where it is involved in several physiological functions. The muscle carnosine content measured within a population can vary by a factor 4. The aim of this study was to further characterize suggested determinants of the muscle carnosine content (diet, gender and age) and to identify new determinants (plasma carnosinase activity and testosterone). We investigated a group of 149 healthy subjects, which consisted of 94 men (12 vegetarians) and 55 women. Muscle carnosine was quantified in M. soleus, gastrocnemius and tibialis anterior using magnetic resonance proton spectroscopy and blood samples were collected to determine CNDP1 genotype, plasma carnosinase activity and testosterone concentrations. Compared to women, men have 36, 28 and 82% higher carnosine concentrations in M. soleus, gastrocnemius and tibialis anterior muscle, respectively, whereas circulating testosterone concentrations were unrelated to muscle carnosine levels in healthy men. The carnosine content of the M. soleus is negatively related to the subjects’ age. Vegetarians have a lower carnosine content of 26% in gastrocnemius compared to omnivores. In contrast, there is no difference in muscle carnosine content between omnivores with a high or low ingestion of β-alanine. Muscle carnosine levels are not related to the polymorphism of the CNDP1 gene or to the enzymatic activity of the plasma carnosinase. In conclusion, neither CNDP1 genotype nor the normal variation in circulating testosterone levels affects the muscular carnosine content, whereas vegetarianism, female gender and increasing age are the factors associated with reduced muscle carnosine stores.     

The guardians of inherited oncogenic vulnerabilities

Similar to seemingly maladaptive genes in general, the persistence of inherited cancer‐causing mutant alleles in populations remains a challenging question for evolutionary biologists. In addition to traditional explanations such as senescence or antagonistic pleiotropy, here we put forward a new hypothesis to explain the retention of oncogenic mutations. We propose that although natural defenses evolve to prevent neoplasm formation and progression thus increasing organismal fitness, they also conceal the effects of cancer‐causing mutant alleles on fitness and concomitantly protect inherited ones from purging by purifying selection. We also argue for the importance of the ecological contexts experienced by individuals and/or species. These contexts determine the locally predominant fitness‐reducing risks, and hence can aid the prediction of how natural selection will influence cancer outcomes.     

Medical Device Industry Approaches for Addressing Sources of Failing Cytotoxicity Scores

To ensure patient safety, medical device manufacturers are required by the Food and Drug Administration and other regulatory bodies to perform biocompatibility evaluations on their devices per standards, such as the AAMI-approved ISO 10993-1:2018 (ANSI/AAMI/ISO 10993-1:2018).However, some of these biological tests (e.g., systemic toxicity studies) have long lead times and are costly, which may hinder the release of new medical devices. In recent years, an alternative method using a risk-based approach for evaluating the toxicity (or biocompatibility) profile of chemicals and materials used in medical devices has become more mainstream. This approach is used as a complement to or substitute for traditional testing methods (e.g., systemic toxicity endpoints). Regardless of the approach, the one test still used routinely in initial screening is the cytotoxicity test, which is based on an in vitro cell culture system to evaluate potential biocompatibility effects of the final finished form of a medical device. However, it is known that this sensitive test is not always compatible with specific materials and can lead to failing cytotoxicity scores and an incorrect assumption of potential biological or toxicological adverse effects. This article discusses the common culprits of in vitro cytotoxicity failures, as well as describes the regulatory-approved methodology for cytotoxicity testing and the approach of using toxicological risk assessment to address clinical relevance of cytotoxicity failures for medical devices. Further, discrepancies among test results from in vitro tests, use of published half-maximal inhibitory concentration data, and the derivation of their relationship to tolerable exposure limits, reference doses, or no observed adverse effect levels are highlighted to demonstrate that although cytotoxicity tests in general are regarded as a useful sensitive screening assays, specific medical device materials are not compatible with these cellular/in vitro systems. For these cases, the results should be analyzed using more clinically relevant approaches (e.g., through chemical analysis or written risk assessment).

Medical devices are engineered to be of durable construction and to accommodate the functionality needed for proper device application. The biocompatibility of the materials, as well as their processing, is also important to ensure that the patients are not negatively affected by the devices when they enter the clinical setting. Certain materials of constructions used for medical devices (and manufacturing processes or processing aids) may contain chemicals that can lead to failing cytotoxicity scores using traditional, regulatory-mandated methodologies. Examples of common materials include plastics (e.g., polyethylene or polypropylene [co]polymers, polyvinyl chloride [PVC]) and metals (e.g., nitinol, copper [Cu]-containing alloys). Although providing stable and reliable materials for use in relation to performance parameters, various metals/alloys and plastics may evoke undesired cytotoxic effects. These effects might be observed as reduced cellular activity or decay in the in vitro assay, especially when standard methods and test parameters (e.g., extraction ratios) are used.1,2To prevent adverse effects (e.g., toxicity, or other types of biocompatibility-related issues) from occurring among patients and clinical end users, manufacturers are required to perform biocompatibility evaluations per guidance provided in e.g., ANSI/AAMI/ISO 10993-1:2018.3 This standard provides an overall framework for the biological evaluation, emphasizing a risk-based approach, as well as general guidance on relevant tests for specific types of contact to patients or users. Of note, traditional biocompatibility tests, within the battery of both in vivo and in vitro methods, could take up to 6 months (or take years, in the case of long-term systemic toxicity testing). Lengthy turnaround times stem from in vivo test methods, which are performed on animal models and include irritation, sensitization, systemic toxicity, genotoxicity, and carcinogenicity studies. Traditional in vitro tests involve exposure of cells or cellular material to device extracts in order to characterize toxicity in terms of cytotoxicity, genotoxicity, cellular metabolic activity, and aspects of hemocompatibility.3In recent years, as a complement to or a substitute for traditional testing methods, a risk-based approach using a chemical and materials characterization for evaluation of patient safety has become mainstream. The framework for this approach is provided in ISO 10993-18:2020.4 Moreover, the Association for the Advancement of Medical Instrumentation (AAMI) and, by extension, regulatory bodies (including the Food and Drug Administration [FDA] and International Organization for Standardization [ISO]) have driven the use of chemical and material characterization. Particularly for medical devices in long-term contact with patient (e.g., implantable devices), use of chemical and material characterization can reduce unnecessary animal testing and provide results that are scientifically sound and detailed, while being more cost and time efficient. For example, ISO 10993-13 highlights that a correctly conducted risk assessment can provide justification to exclude long-term biological testing, where the nature and extent of exposure confirms that the patient is being exposed to very low levels of chemicals that are below relevant toxicological thresholds.3Throughout the ISO 10993 series, it also is emphasized that conducting animal testing for biological risk evaluation should only be considered after all alternative courses of action (review of prior knowledge, chemical or physical characterization, in vitro evaluations, or alternative means of mitigation) have been exhausted. In addition, analytical chemistry used for chemical characterization can be used as a means for investigating possible culprits when traditional biocompatibility tests, such as cytotoxicity tests, fail, especially in cases where a known substance(s) in the material has cytotoxic potential (e.g., silver-infused wound dressing that provides antibacterial properties).However, it should be kept in mind that although chemistry can be a powerful tool in many cases, not all medical devices extracts are compatible with the analytical methods and instruments used, and these studies may not provide the full understanding of the toxicity profile of the device. In those cases, animal testing or further justification may still be needed to demonstrate a safe biocompatibility profile for the device.Cytotoxicity testing per AAMI/ISO 10993-5:2009/(R)20145 has historically been one of the most used (and is considered the most reactive) of the biocompatibility tests6,7 and can be efficiently used to detect abnormal effects to cells that may arise if harmful chemicals are present in device extracts. However, it also is recognized that cell-based test methods do not necessarily correlate to in vivo toxicological effects and actual clinical patient safety, often showing a reaction when no clinical adverse effects are known or expected to occur. For instance, some soluble metal ions (e.g., Cu, nickel [Ni]) are known to exert toxic effects on cells in an in vitro setting; however, their presence in surgical instruments and implants has demonstrated high patient tolerance and negligible effects upon clinical use.This article provides a brief evaluation of the clinical impact of metals and plasticizers commonly used in medical device materials that may lead to patient exposure during the use of devices, with emphasis given to those that may result in cytotoxicity failures in an in vitro setting. In addition, an approach to evaluating valid clinical risks using a toxicological risk assessment is discussed.