Hepatitis A Virus Infection in Cynomolgus Monkeys Confounds the Safety Evaluation of a Drug Candidate.

In a 3-month toxicity study in cynomolgus monkeys at a European contract laboratory, animals were infected with HAV, initially resulting in hepatic injury being incorrectly attributed to the test compound. Elevated serum ALT/AST/GLDH (5- to 10-fold) were noted in individual animals from all groups including controls, with no apparent dose, exposure, or time-related relationship. Liver histopathology revealed minimal to slight inflammatory cell accumulation in periportal zones of most animals, and minimal to slight hepatocyte degeneration/necrosis in 10/42 animals from all groups. As these findings were more pronounced in 6 drug-treated animals, including 2/6 in the low dose group, the draft report concluded: "treatment-related hepatotoxicity at all dose levels precluded determination of a NOAEL." However, the unusual pattern of hepatotoxicity suggested a factor other than drug exposure might have caused the hepatic effects. Therefore, snap-frozen liver samples were tested for hepatitis viruses using a PCR method. Tests for hepatitis B, C, and E virus were negative; however, 20/42 samples were positive for hepatitis A virus (HAV). Infection was strongly associated with increased serum ALT/GLDH, and/or hepatocyte degeneration/necrosis. Re-evaluation of the study in light of these data concluded that the hepatic injury was not drug-related. A subsequent 6-month toxicology study in HAV-vaccinated cynomolgus monkeys confirmed the absence of hepatotoxicity. Identification of HAV infection supported progression of the drug candidate into later clinical trials. Although rarely investigated, subclinical HAV infection has occasionally been reported in laboratory primates, including those used for toxicology studies and it may be more prevalent than the literature indicates.

The two faces of microorganisms in traditional brewing and the implications for no- and low-alcohol beers.

The production of alcoholic beverages is intrinsically linked to microbial activity. This is because microbes such as yeast are associated with the production of ethanol and key sensorial compounds that produce desirable qualities in fermented products. However, the brewing industry and other related sectors face a step-change in practice, primarily due to the growth in sales of no- and low-alcohol (NoLo) alternatives to traditional alcoholic products. Here we review the involvement of microbes across the brewing process, including both their positive contributions and their negative (spoilage) effects. We also discuss the opportunities for exploiting microbes for NoLo beer production, as well as the spoilage risks associated with these products. For the latter, we highlight differences in composition and process conditions between traditional and NoLo beers and discuss how these may impact the microbial ecosystem of each product stream in relation to microbiological stability and final beer quality.

The role of inorganic-phosphate, potassium and magnesium in yeast-flavour formation.

Inorganic-phosphate, potassium, and magnesium are key-minerals required for yeast growth, metabolism, and survival, the present work investigated its impact in yeast-flavour formation using a multi-factor experimental design, which was used to generate a range of phosphorous-potassium-magnesium resulting in a 28-point D-optimal design. Samples were evaluated using HPLC (ethanol), GC-MS (aroma), and CountStar (total yeast cell). Results revealed that yeast requires a minimal amount of inorganic-phosphate, potassium, and magnesium (250, 500, and 70 mg/L, respectively) to support yeast-growth and ethanol/flavour formation. Inorganic-phosphate was important for fatty acid esters formation/short chain fatty acid (SCFA) reduction. Potassium was important in the formation of acetate esters/higher alcohols. Magnesium was the most important inorganic element for ester formation/SCFA reduction; furthermore, ethanol production is magnesium-dependent. In conclusion, inorganic phosphate, potassium and magnesium play an important role in yeast-growth, esters and higher alcohols formation; and SCFA reduction. Ethanol formation is Mg-dependent.

Influence of essential inorganic elements on flavour formation during yeast fermentation.

The relative concentration of available inorganic elements is critical for yeast growth and metabolism and has potential to be a tool leading to directed yeast flavour formation during fermentation. This study investigates the influence of essential inorganic elements during alcoholic fermentation of brewers wort, fermented using three independent yeast strains, Saccharomyces pastorianus W34/70, and Saccharomyces cerevisiae strains M2 and NCYC2592 under a range of conditions replicated for each yeast strain. 10 treatments were applied: 1 control and 9 inorganic supplementations: standard brewers wort, ammonia-nitrogen, inorganic phosphate, potassium, magnesium, copper, zinc, iron, manganese and a composite mixture, Twenty-five chemical markers were evaluated by HPLC (ethanol, glycerol), and GC-MS (aroma). There was a significant change in volatile aroma compounds during fermentation, which was more prominent when supplemented with ammonia nitrogen, inorganic phosphate, potassium or magnesium (P < 0.05). Heavy metal ions mostly had a negative effect on the flavour formation.

Three-and-a-half to 18 year outcome data for costal osteochondral grafts for salvage of the proximal pole of the scaphoid.

Twelve patients who had undergone costal osteochondral graft reconstruction of the proximal pole of scaphoid were evaluated with clinical examination, patient-reported outcome scores and radiographs with an average follow-up of 10 years (range 3.5-18). The range of wrist motion was not significantly changed compared with the preoperative range of motion and functional outcomes scores were acceptable. The patients reported low pain scores despite the universal presence of radiographic changes of reduced carpal height and arthritis of the midcarpal and radiocarpal joints. Costal osteochondral graft reconstruction of the proximal pole of scaphoid offers good long-term pain relief and function.Level of evidence: IV.

Integrating Social Determinants of Health and Laboratory Data: A Pilot Study To Evaluate Co-Use of Opioids and Benzodiazepines.

As the opioid crisis continues to have devastating consequences for our communities, families, and patients, innovative approaches are necessary to augment clinical care and the management of patients with opioid use disorders. As stewards of health analytic data, laboratories are uniquely poised to approach the opioid crisis differently. With this pilot study, we aimed to bridge laboratory data with social determinants of health data, which are known to influence morbidity and mortality of patients with substance use disorders. For the purpose of this pilot study, we focused on the co-use of opioids and benzodiazepines, which can lead to an increased risk of fatal opioid-related overdoses and increased utilization of acute care. Using the laboratory finding of the copresence of benzodiazepines and opioids as the primary outcome measure, we examined social determinants of health attributes that predict co-use. We found that the provider practice that ordered the laboratory result is the primary predictor of co-use. Increasing age was also predictive of co-use. Further, co-use is highly prevalent in specific geographic areas or "hotspots." The prominent geographic distribution of co-use suggests that targeted educational initiatives may benefit the communities in which co-use is prevalent. This study exemplifies the Clinical Lab 2.0 approach by leveraging laboratory data to gain insights into the overall health of the patient.

Physiological and transcriptomic response of Saccharomyces pastorianus to cold storage.

Removal of yeast biomass at the end of fermentation, followed by a period of storage before re-inoculation into a subsequent fermentation, is common in the brewing industry. Storage is typically conducted at cold temperatures to preserve yeast quality, a practice which has unfavourable cost and environmental implications. To determine the potential for alleviating these effects, the transcriptomic and physiological response of Saccharomyces pastorianus strain W34/70 to standard (4°C) and elevated (10°C) storage temperatures was explored. Higher temperatures resulted in increased expression of genes associated with the production and mobilisation of intracellular glycogen, trehalose, glycerol and fatty acids, although these observations were limited to early stages of storage. Intracellular trehalose and glycerol concentrations were higher at 4°C than at 10°C, as a consequence of the cellular response to cold stress. However, significant changes in glycogen degradation or cellular fatty acid composition did not occur between the two sets of populations, ensuring that cell viability remained consistent. It is anticipated that this data may lead to changes in standard practice for handling yeast cultures, without compromising yeast quality. This work has significance not only for the brewing industry, but also for food and biofuel sectors requiring short-term storage of liquid yeast.

A Bayesian approach to analyzing phenotype microarray data enables estimation of microbial growth parameters.

Biolog phenotype microarrays (PMs) enable simultaneous, high throughput analysis of cell cultures in different environments. The output is high-density time-course data showing redox curves (approximating growth) for each experimental condition. The software provided with the Omnilog incubator/reader summarizes each time-course as a single datum, so most of the information is not used. However, the time courses can be extremely varied and often contain detailed qualitative (shape of curve) and quantitative (values of parameters) information. We present a novel, Bayesian approach to estimating parameters from Phenotype Microarray data, fitting growth models using Markov Chain Monte Carlo (MCMC) methods to enable high throughput estimation of important information, including length of lag phase, maximal "growth" rate and maximum output. We find that the Baranyi model for microbial growth is useful for fitting Biolog data. Moreover, we introduce a new growth model that allows for diauxic growth with a lag phase, which is particularly useful where Phenotype Microarrays have been applied to cells grown in complex mixtures of substrates, for example in industrial or biotechnological applications, such as worts in brewing. Our approach provides more useful information from Biolog data than existing, competing methods, and allows for valuable comparisons between data series and across different models.

Production of medium-chain volatile flavour esters in Pichia pastoris whole-cell biocatalysts with extracellular expression of Saccharomyces cerevisiae acyl-CoA:ethanol O-acyltransferase Eht1 or Eeb1.

Medium-chain volatile flavour esters are important molecules since they have extensive applications in food, fragrance, cosmetic, paint and coating industries, which determine different characteristics of aroma or taste in commercial products. Biosynthesis of these compounds by alcoholysis is catalyzed by acyl-CoA:ethanol O-acyltransferases Eht1 or Eeb1 in Saccharomyces cerevisiae. In this study, these two yeast enzymes were selected to explore their preparations as the form of whole cell biocatalysts for the production of volatile flavour esters. Here, the novel whole cell biocatalysts Pichia pastoris yeasts with functional extracellular expression of Eht1 or Eeb1 were constructed. Flavour production was established through an integrated process with coupled enzyme formation and ester biosynthesis in the recombinant yeasts in one pot, leading to the formation of volatile C6-C14 methyl and ethyl esters from wort medium. Interestingly, there is no significant difference between P. pastoris-EHT1 and P. pastoris-EEB1 in substrate preference during flavour biosynthesis, indicating a similar role of Eht1 and Eeb1 in P. pastoris cells, in contradiction with previous findings in S. cerevisiae to some extent. Consequently the study not only provides a greater understanding of these two enzymes in a heterogeneous host, but also demonstrated the positive effect of the recombinant Eht1 and Eeb1 in ester formation by P. pastoris live cells, potentially paving the way towards achieving efficient production of volatile flavour by an integrated biocatalytic system composed of recombinant enzyme production and flavour biosynthesis.

Screening of Non- Saccharomyces cerevisiae Strains for Tolerance to Formic Acid in Bioethanol Fermentation.

Formic acid is one of the major inhibitory compounds present in hydrolysates derived from lignocellulosic materials, the presence of which can significantly hamper the efficiency of converting available sugars into bioethanol. This study investigated the potential for screening formic acid tolerance in non-Saccharomyces cerevisiae yeast strains, which could be used for the development of advanced generation bioethanol processes. Spot plate and phenotypic microarray methods were used to screen the formic acid tolerance of 7 non-Saccharomyces cerevisiae yeasts. S. kudriavzeii IFO1802 and S. arboricolus 2.3319 displayed a higher formic acid tolerance when compared to other strains in the study. Strain S. arboricolus 2.3319 was selected for further investigation due to its genetic variability among the Saccharomyces species as related to Saccharomyces cerevisiae and availability of two sibling strains: S. arboricolus 2.3317 and 2.3318 in the lab. The tolerance of S. arboricolus strains (2.3317, 2.3318 and 2.3319) to formic acid was further investigated by lab-scale fermentation analysis, and compared with S. cerevisiae NCYC2592. S. arboricolus 2.3319 demonstrated improved formic acid tolerance and a similar bioethanol synthesis capacity to S. cerevisiae NCYC2592, while S. arboricolus 2.3317 and 2.3318 exhibited an overall inferior performance. Metabolite analysis indicated that S. arboricolus strain 2.3319 accumulated comparatively high concentrations of glycerol and glycogen, which may have contributed to its ability to tolerate high levels of formic acid.

Preparation of iron-enriched baker's yeast and its efficiency in recovery of rats from dietary iron deficiency.

OBJECTIVES: Iron is an important mineral, essential for the health and function of mammalian cells. Despite its key role, iron deficiency in humans is common worldwide, often leading to significant health issues within the population. The aim of this study was to evaluate the potential of using iron-enriched baker's yeast as a source of iron, especially for the protection and recovery from conditions related to anemia. METHODS: Iron-enriched yeast was prepared by cultivating cells on basal medium comprising different iron concentrations. The effects of iron supplementation on animal health were assessed by feeding anemic rats with a variety of diets containing either inorganic iron or iron-enriched yeast. Body weight, iron bioavailability, blood parameters, and the activity of iron-containing enzymes (catalase) were studied. RESULTS: Iron accumulation in yeast cells increased with iron concentration, reaching a maximum of 15 mg/g when 32 mM iron was applied. Rat groups fed iron-enriched yeast had the highest feed efficiency, iron bioavailability, and hemoglobin concentration. The source of iron supplementation influenced catalase activity in kidney tissues, increasing from 70 U/g tissue in anemic rats to 90 U/g tissue (inorganic iron salt), 110 U/g tissue (inorganic iron salt and non-enriched dry yeast), 145 U/g tissue (iron-enriched yeast 15 mg/g iron) and 115 U/g tissue (iron-enriched yeast 30 mg/g iron). The histologic study of tissues from liver, kidney, heart, and spleen of rats from different groups showed that the damage observed in tissues of anemic rats, was not observed after feeding with iron-enriched yeasts. CONCLUSION: The results demonstrated that ingestion of iron-enriched yeast is more efficient than inorganic treatment in recovery from iron deficiency, including tissue recovery in rats.

Effects of treatment with androgen receptor ligands on microRNA expression of prostate cancer cells.

Post-transcriptional regulation by microRNA (miRNA) is an important aspect of androgen receptor (AR) signalling in prostate cancer cells. However, the global profiling of miRNA expression in prostate cancer cells following treatment with AR ligands has not been reported so far. In this study we examined the effect of treatment with two AR agonists (mibolerone (MIB) and dihydrotestosterone (DHT)) and an AR antagonist (bicalutamide (BIC)) on miRNA expression in the human androgen-dependent LNCaP prostate cancer cell line using microarray technology and verification of selected miRNA using quantitative real-time PCR (qRT-PCR). No miRNA was identified as differentially expressed following treatment with the AR antagonist BIC. In contrast, a number of common and compound-specific alterations in miRNA expression were observed following treatment with AR agonists. Unexpectedly it was found that treatment with the AR agonists resulted in the repression of miR-221, a miRNA previously established to be involved with prostate cancer development. This observation indicates that this miRNA may have a more complex role in prostate cancer development than considered previously. Treatment with MIB led to an induction of miR-210 expression, a hypoxia-related miRNA. This miRNA is reported to be involved in cell adaptation to hypoxia and thus induction in conditions of normoxia may be important in driving metabolic changes observed in prostate cancer. Thus examining the effect of AR agonists and antagonists on miRNA expression can provide novel insights into the response of cells to AR ligands and subsequent downstream events.

EpiCollect+: linking smartphones to web applications for complex data collection projects.

Previously, we have described the development of the generic mobile phone data gathering tool, EpiCollect, and an associated web application, providing two-way communication between multiple data gatherers and a project database. This software only allows data collection on the phone using a single questionnaire form that is tailored to the needs of the user (including a single GPS point and photo per entry), whereas many applications require a more complex structure, allowing users to link a series of forms in a linear or branching hierarchy, along with the addition of any number of media types accessible from smartphones and/or tablet devices (e.g., GPS, photos, videos, sound clips and barcode scanning). A much enhanced version of EpiCollect has been developed (EpiCollect+). The individual data collection forms in EpiCollect+ provide more design complexity than the single form used in EpiCollect, and the software allows the generation of complex data collection projects through the ability to link many forms together in a linear (or branching) hierarchy. Furthermore, EpiCollect+ allows the collection of multiple media types as well as standard text fields, increased data validation and form logic. The entire process of setting up a complex mobile phone data collection project to the specification of a user (project and form definitions) can be undertaken at the EpiCollect+ website using a simple 'drag and drop' procedure, with visualisation of the data gathered using Google Maps and charts at the project website. EpiCollect+ is suitable for situations where multiple users transmit complex data by mobile phone (or other Android devices) to a single project web database and is already being used for a range of field projects, particularly public health projects in sub-Saharan Africa. However, many uses can be envisaged from education, ecology and epidemiology to citizen science.

Ubiquitin-conjugating enzyme E2C: a potential cancer biomarker.

The ubiquitin-conjugating enzymes 2C (UBE2C) is an integral component of the ubiquitin proteasome system. UBE2C consists of a conserved core domain containing the catalytic Cys residue and an N-terminal extension. The core domain is required for ubiquitin adduct formation by interacting with the ubiquitin-fold domain in the E1 enzyme, and contributes to the E3 enzyme binding. UBE2C N-terminal extension regulates E3 enzyme activity as a part of an intrinsic inhibitory mechanism. UBE2C is required for the destruction of mitotic cyclins and securin, which are essential for spindle assembly checkpoint and mitotic exit. The UBE2C mRNA and/or protein levels are aberrantly increased in many cancer types with poor clinical outcomes. Accumulation of UBE2C stimulates cell proliferation and anchorage-independent growth. UBE2C transgenic mice are prone to develop spontaneous tumors and carcinogen-induced tumor with evidence of chromosome aneuploidy.

Bringing the Field into Focus: User-centered Design of a Patient Expertise Locator.

Managing personal aspects of health is challenging for many patients, particularly those facing a serious condition such as cancer. Finding experienced patients, who can share their knowledge from managing a similar health situation, is of tremendous value. Users of health-related social software form a large base of such knowledge, yet these tools often lack features needed to locate peers with expertise. Informed directly by our field work with breast cancer patients, we designed a patient expertise locator for users of online health communities. Using feedback from two focus groups with breast cancer survivors, we took our design through two iterations. Focus groups concluded that expertise locating features proved useful for extending social software. They guided design enhancements by suggesting granular user control through (1) multiple mechanisms to identify expertise, (2) detailed user profiles to select expertise, and (3) varied collaboration levels. Our user-centered approach links field work to design through close collaboration with patients. By illustrating trade-offs made when sharing sensitive health information, our findings inform the incorporation of expertise locating features into social software for patients.

Yeast responses to stresses associated with industrial brewery handling.

During brewery handling, production strains of yeast must respond to fluctuations in dissolved oxygen concentration, pH, osmolarity, ethanol concentration, nutrient supply and temperature. Fermentation performance of brewing yeast strains is dependent on their ability to adapt to these changes, particularly during batch brewery fermentation which involves the recycling (repitching) of a single yeast culture (slurry) over a number of fermentations (generations). Modern practices, such as the use of high-gravity worts and preparation of dried yeast for use as an inoculum, have increased the magnitude of the stresses to which the cell is subjected. The ability of yeast to respond effectively to these conditions is essential not only for beer production but also for maintaining the fermentation fitness of yeast for use in subsequent fermentations. During brewery handling, cells inhabit a complex environment and our understanding of stress responses under such conditions is limited. The advent of techniques capable of determining genomic and proteomic changes within the cell is likely vastly to improve our knowledge of yeast stress responses during industrial brewery handling.

Peptide YY (PYY) is increased in elderly patients with femoral neck fractures: a prospective cohort study.

BACKGROUND: Peptide YY (PYY), a gut peptide, has recently been shown to inhibit appetite. The role of this peptide in elderly nutritionally-compromised patients with femoral neck fracture (FNF) has not been investigated. In this study, we investigated the longitudinal pattern of PYY levels during hospital stay and investigated the postprandial PYY response to a standard meal in patients with FNF and matched controls. METHODS: Fasting plasma concentrations of the PYY were measured on days 1, 4, 7, 14, 21 and 28 or on discharge from the hospital in 17 white patients with FNF. On the second week of stay, 13 patients with FNF consumed a standard breakfast following an overnight fasting. One fasting sample and one 45-minute postmeal sample were collected. A control group was made up of 17 matched healthy elderly patients. RESULTS: PYY concentrations were increased significantly over the length of hospital stay. Results of the test breakfast suggested a significant and exaggerated post-prandial PYY response, despite a smaller energy intake being consumed. CONCLUSIONS: This study shows PYY concentrations are increased during hospitalization and their post-prandial release exaggerated in this group of vulnerable patients, and suggests a role in the etiology of reduced appetite in this patient group.

Chitin scar breaks in aged Saccharomyces cerevisiae.

Ageing in budding yeast is not determined by chronological lifespan, but by the number of times an individual cell is capable of dividing, termed its replicative capacity. As cells age they are subject to characteristic cell surface changes. Saccharomyces cerevisiae reproduces asexually by budding and as a consequence of this process both mother and daughter cell retain chitinous scar tissue at the point of cytokinesis. Daughter cells exhibit a frail structure known as the birth scar, while mother cells display a more persistent bud scar. The number of bud scars present on the cell surface is directly related to the number of times a cell has divided and thus constitutes a biomarker for replicative cell age. It has been proposed that the birth scar may be subject to stretching caused by expansion of the daughter cell; however, no previous analysis of the effect of cell age on birth or bud scar size has been reported. This paper provides evidence that scar tissue expands with the cell during growth. It is postulated that symmetrically arranged breaks in the bud scar allow these rigid chitinous structures to expand without compromising cellular integrity.

The impact of brewing yeast cell age on fermentation performance, attenuation and flocculation.

Individual cells of the yeast Saccharomyces cerevisiae exhibit a finite replicative lifespan, which is widely believed to be a function of the number of divisions undertaken. As a consequence of ageing, yeast cells undergo constant modifications in terms of physiology, morphology and gene expression. Such characteristics play an important role in the performance of yeast during alcoholic beverage production, influencing sugar uptake, alcohol and flavour production and also the flocculation properties of the yeast strain. However, although yeast fermentation performance is strongly influenced by the condition of the yeast culture employed, until recently cell age has not been considered to be important to the process. In order to ascertain the effect of replicative cell age on fermentation performance, age synchronised populations of a lager strain were prepared using sedimentation through sucrose gradients. Each age fraction was analysed for the ability to utilise fermentable sugars and the capacity to flocculate. In addition cell wall properties associated with flocculation were determined for cells within each age fraction. Aged cells were observed to ferment more efficiently and at a higher rate than mixed aged or virgin cell cultures. Additionally, the flocculation potential and cell surface hydrophobicity of cells was observed to increase in conjunction with cell age. The mechanism of ageing and senescence in brewing yeast is a complex process, however here we demonstrate the impact of yeast cell ageing on fermentation performance.