Zebrafish Larvae as an Experimental Model of Cryptococcal Meningitis.

This chapter provides guidance for introducing Cryptococcus neoformans into the zebrafish larvae model system to establish a CNS infection phenotype that mimics cryptococcal meningitis as seen in humans. The method outlines techniques for visualizing different stages of pathology development, from initial to severe infection profiles. The chapter provides tips for real time visualization of the interactions between the pathogen and different aspects of the CNS anatomy and immune system.

Technology of Informative Feature Selection for Immunosignature Analysis.

The main difficulty in practical work with data obtained via immunosignature analysis is high dimensionality and the presence of a significant number of uninformative or false-informative features due to the specific character of the technology. To ensure practically relevant quality of data analysis and classification, it is necessary to take due account of this specific character. The aim of the study is to create and test the technology for effective reduction of immunosignature data dimensionality, which provides practically relevant and high quality of classification with due regard for the properties of the data obtained. MATERIALS AND METHODS: The study involved the use of two normalized data sets obtained from the public biomedical repository and containing the results of immunosignature analysis.The technology for selecting informative features was proposed within the framework of the study. It consisted of three successive steps: 1) breaking a multiclass task into a series of binary tasks using the "one vs all" strategy; 2) screening of false-informative features is performed for each binary comparison by comparing the values of the median of the sets "one" and "all"; 3) ranking of the remaining features according to their informative value and selection of the most informative ones for each binary comparison.To assess the quality of the proposed technology for informative feature selection, we used the results obtained after application of classification based on the filtered data. Support vector method that proved itself in the problems of high-dimensional data classification was used as a classification model. RESULTS: Effectiveness of the proposed technology for informative feature selection was determined. This technology allows us to provide high quality of classification while significantly reducing the feature space. The number of features eliminated in the second step is approximately 50% for each data set under consideration, which greatly simplifies subsequent data analysis. After the third step, when the feature space is reduced to 15 features, the quality of classification by the macro-average F1-score metric is assessed as 98.9% for the GSE52581 dataset. For the GSE52581 dataset, with the feature space reduced to 266 features, the quality of classification by the macro-average F1-score metric is 91.3%. CONCLUSION: The results of the work demonstrate the promising outlook of the proposed technology for informative feature selection as applied to the data of immunosignature analysis.

Experiences of rosacea and its treatment: an interpretative phenomenological analysis.

BACKGROUND: While rosacea is a chronic skin condition, it can often have a large psychosocial impact on the individual. There is therefore a need to understand the experience of living with rosacea from the patient perspective. OBJECTIVES: To examine the experience of living with rosacea and the experience of seeking and receiving treatment. METHODS: Nine participants took part in semistructured interviews, which were analysed using interpretative phenomenological analysis. RESULTS: Three superordinate themes were identified within the data: 'self-consciousness', which focused on the fear of others assigning blame to participants for having caused symptoms; 'avoidance, concealment and hiding emotions', referring to the coping strategies participants employed in response to rosacea; and 'inconsistencies in general practitioner treatment and guidance', which focused on the need for medical professionals to assess the psychosocial well-being of patients with rosacea. CONCLUSIONS: Rosacea can have a negative impact on the daily life of people with the condition, contributing to lowered self-esteem, embarrassment and feelings of shame. Engaging in emotion-focused and behavioural/avoidant-focused coping strategies increased participants' confidence and reduced their avoidance of social situations. However, such strategies might still serve to maintain underlying unhelpful cognitive processes. Consequently, it is important for medical professionals to assess for the presence of cognitive factors that might contribute to maintaining distress in patients with rosacea, and where unhelpful thoughts or beliefs are reported, patients may need to be referred for psychological support.

Interaction of Serum Antibodies from Breast Cancer Patients with Synthetic Peptides.

The blood serum of tumor patients contains antibodies recognizing tumor-associated antigens and other molecular products of tumor growth. We studied the interaction of blood antibodies from breast cancer patients with synthetic peptides that were applied on the microchip surface. The serum from healthy volunteers and breast cancer patients was shown to contain antibodies that interact with various peptides. Statistically significant between-group differences were observed in the level of binding with 122 informative peptides (0.01% of the total number of peptides on a microchip). Analysis of antibodies that interact with the peptide panel holds much promise for the diagnostics of breast cancer.

Minimally displaced tibial tuberosity avulsion fracture in nine skeletally immature large breed dogs.

OBJECTIVE: The objective of this study was to describe the clinical and radiographic features, as well as the treatment and outcome of minimally displaced tibial-tuberosity-avulsion-fractures (MDTTAF). MATERIALS AND METHODS: Signalment, history, diagnostics, therapy, and outcome were recorded. Follow-up was documented as re-examination, radiographic assessment or telephone conversation. RESULTS: Nine large breed dogs that were presented with lameness originating from the proximal tibia were included. All showed signs of pain when pressure was applied to the tibial tuberosity. There was no stifle instability or intra-articular disease. The main feature on mediolateral radiographs was a widened tibial-tuberosity-physis with reactive new bone and loss of edge definition of the epiphyseal and metaphyseal margins. Non-surgical treatment was chosen in eight dogs, and surgery in one dog. Radiographic follow-up showed progressive closure of the tibial-tuberosity-physis and healing. Clinical signs resolved at a median of 28 days (range: 14-120). DISCUSSION: Minimally displaced tibial-tuberosity-avulsion-fractures should be a differential diagnosis in skeletally immature large breed dogs older than nine months of age with signs of subtle pelvic-limb lameness, and signs of proximal tibial pain, but no evidence of stifle joint disease. Thorough clinical examination and critical review of bilateral radiographs are important to diagnose MDTTAF. The outcome in these cases suggests that the prognosis for MDTTAF is excellent. Age and size of the affected dogs in this study differ from an earlier publication that illustrated more severely displaced tibial tuberosity avulsion fractures, occurring mainly in terriers around five months of age.

Extent of variation within a laying flock: attainment of sexual maturity, double-yolked and soft-shelled eggs, sequence lengths and consistency of lay.

1. An experiment was conducted to determine the amount of variation that exists within a population of laying hens in age at first egg, the number of birds that did not come into lay, the prevalence of soft-shelled and double-yolked eggs, sequence characteristics and consistency of lay. 2. Oviducal problems accounted for most of the poor laying performance. About a third of the flock produced double-yolked or soft-shelled eggs at the onset of lay, the proportion being influenced by the age at photostimulation. In some instances these aberrations interrupted sequences. Differences in laying performance were particularly evident in mean sequence length, mean prime sequence length and mean pause length both within and between individuals. Furthermore, sequence length did not always decline in a regular fashion with advancing age. 3. The information gathered here has been used to develop a mechanistic, stochastic population model for laying hens.

Modelling the changes in the proportions of the egg components during a laying cycle.

1. As hens age, egg weight increases but the eggs contain proportionally more yolk and less albumen and shell. However, at a given age, larger eggs contain proportionally more albumen. When modelling the nutrient requirements of the hen over a production cycle, based on the daily outputs of each nutrient, egg weight needs to be predicted as the sum of the three components, since each has a unique chemical composition, and these proportional changes will therefore influence the nutrient requirements of the hen. 2. Yolk weight is related to hen age and may be calculated using a logistic or Gompertz function. Allometric functions are used to predict albumen weight from yolk weight and shell weight from the weight of the egg contents. 3. A mechanistic, stochastic population model for layers may be used to verify that these functions correctly reflect the proportional changes in the egg components with advancing hen age and at a given age, over a range of egg weights. 4. The various parameters used in the equations need to be defined for the available genotypes.

A mechanistic, stochastic, population model of egg production.

1. A mechanistic, stochastic egg production model is presented. Mean age at first egg may be predicted from the lighting programme applied during rearing, using the Bristol-Reading model (Lewis et al., 2002). 2. Rate of ovulation is determined by an amended version of the mathematical model of the ovulatory cycle, originally proposed by Etches and Schoch (1984). 3. Oviposition times are estimated from ovulation times. 4. Yolk, albumen and shell weights are calculated using allometric functions. 5. The model predicts egg production of a theoretical flock of laying hens for a full laying year, including random occurrences of double-yolked and soft-shelled eggs and internal ovulations.

An improved mathematical model of the ovulatory cycle of the laying hen.

1. The mathematical model of the hen's ovulatory cycle proposed by Etches and Schoch (British Poultry Science, 25: 65-76, 1984) predicts ovulation times for sequences of 2 to 9 ovulations only. 2. Continuous functions have been produced, representing the changes required to the parameters lambda1, lambda2, S1, S2, b1, b2 and b3, such that the prediction of any sequence length is now possible. 3. This improved ovulation model is capable of predicting ovulation times and intra-sequence ovulation intervals for any ovulation rate between 0.5 and 1.0. 4. The improved ovulatory model lends itself to stochasticity. The rate of lay of a population of hens at a time may be modelled with the use of means and standard errors for each of the parameters in the model. 5. Age-related changes in the ovulation rate of the population may be predicted using a combination of three methods, which are consistent with published theories that account for the decline in performance with time.

Immune responses in baboons vaccinated with HIV-2 genetic expression libraries.

Immunization using genetic expression libraries may be an improvement over conventional DNA immunization using a single gene because more epitopes are simultaneously presented to the immune system. In this study, we evaluated the effectiveness of an HIV-2 vaccine made from a genomic expression library in baboons. We found that HIV-2 expression library immunization induced HIV-2-specific memory responses but low levels of CD8+ cell anti-viral responses and neutralizing antibodies. After intravenous virus challenge using a homologous pathogenic variant, HIV-2UC2/9429, viral loads were similar in the HIV-2-immunized and control baboons. We conclude that although immunization using HIV-2 expression libraries induces immune responses, this approach does not provide protection in baboons against intravenous challenge with HIV-2.

Calvarial vault remodeling for sagittal synostosis.

Sagittal synostosis is the most common form of craniosynostosis (i.e., premature fusion of cranial sutures). Sagittal synostosis, which is the premature fusion of the sagittal suture, occurs in 56% to 58% of all reported cases of craniosynostosis. This article describes calvarial vault remodeling, the procedure of choice at Children's Hospital Los Angeles for surgical intervention to correct sagittal synostosis. Perioperative interventions to minimize patients' risks and maximize benefits are explained. A case study is presented to describe the experience of one patient and his family members.

Selective chemical inactivation of AAA proteins reveals distinct functions of proteasomal ATPases.

BACKGROUND: The 26S proteasome contains six highly related ATPases of the AAA family. We have developed a strategy that allows selective inhibition of individual proteasomal ATPases in the intact proteasome. Mutation of a threonine in the active site of Sug1/Rpt6 or Sug2/Rpt4 to a cysteine sensitizes these proteins to inactivation through alkylation by the sulfhydryl modifying agent NEM. Using this technique the individual contributions of Sug1 and Sug2 to proteasome function can be assessed. RESULTS: We show that both Sug1 and Sug2 can be selectively alkylated by NEM in the context of the intact 26S complex and as predicted by structural modeling, this inactivates the ATPase function. Using this technique we demonstrate that both Sug 1 and 2 are required for full peptidase activity of the proteasome and that their functions are not redundant. Kinetic analysis suggests that Sug2 may have an important role in maintaining the interaction between the 19S regulatory complex and the 20S proteasome. In contrast, inhibition of Sug1 apparently decreases peptidase activity of the 26S proteasome by another mechanism. CONCLUSIONS: These results describe a useful technique for the selective inactivation of AAA proteins. In addition, they also demonstrate that the functions of two related proteasomal AAA proteins are not redundant, suggesting differential roles of proteasomal AAA proteins in protein degradation.

A combination vaccine confers full protection against co-infections with influenza, herpes simplex and respiratory syncytial viruses.

Combined/composite vaccines should be useful in reducing the number of vaccinations and provide more flexibility in confronting biological warfare scenarios. We tested the effectiveness of a composite genetic vaccine designed from previously known protective antigens directed against influenza A virus (INF-A), herpes simplex virus type-1 (HSV-1) and respiratory syncytial virus (RSV) in a mouse-based challenge. Immunizing mice with a pool of four plasmids; INF-A haemagglutinin (HA), INF-A nucleoprotein (NP), HSV-1 glycoprotein D (gD) and RSV glycoprotein F, against the three pathogens provided full protection when mice were challenged with each pathogen. Remarkably, mice challenged with all three pathogens at once were also fully protected, even when a bacterial pathogen, Mycoplasma pulmonis, was included in the challenge. If these results are extendable to other combinations of vaccines in other hosts, it would support the development of gene vaccines as multi-component, combination vaccines.

Evidence that Gal11 protein is a target of the Gal4 activation domain in the mediator.

The mediator is an approximately 20 protein complex that is essential for the transcription of most genes in yeast. It is contacted by a number of gene-specific activators, but the details of these interactions are not well understood in most cases. Here, evidence is presented that the mediator component Gal11 represents at least one target of the Gal4 activation domain (AD). Deletion of Gal11 is shown to decrease the affinity of the Gal4 AD for the mediator, and direct binding of an N-terminal domain of Gal11 with the Gal4 AD is demonstrated. Quantitative studies, however, indicate that the K(D) of the 1:1 Gal4 AD--Gal11 complex is modest. Combined with in vivo data showing that Delta gal11 cells exhibit reduced, but still significant, Gal4-mediated gene expression, these results suggest that the dimeric activator might also contact another protein in the mediator in addition to Gal11.

The 19S regulatory particle of the proteasome is required for efficient transcription elongation by RNA polymerase II.

It is generally thought that the primary or even sole activity of the 19S regulatory particle of the 26S proteasome is to facilitate the degradation of polyubiquitinated proteins by the 20S-core subunit. However, we present evidence that the 19S complex is required for efficient elongation of RNA polymerase II (RNAP II) in vitro and in vivo. First, yeast strains carrying alleles of SUG1 and SUG2, encoding 19S components, exhibit phenotypes indicative of elongation defects. Second, in vitro transcription is inhibited by antibodies raised against Sug1, or by heat-inactivating temperature-sensitive Sug1 mutants with restoration of elongation by addition of immunopurified 19S complex. Finally, Cdc68, a known elongation factor, coimmunoprecipitates with the 19S complex, indicating a physical interaction. Inhibition of the 20S proteolytic core of the proteasome has no effect on elongation. This work defines a nonproteolytic role for the 19S complex in RNAP II transcription.

The 19S complex of the proteasome regulates nucleotide excision repair in yeast.

Previous studies suggest that the amino-terminal ubiquitin-like (ubl) domain of Rad23 protein can recruit the proteasome for a stimulatory role during nucleotide excision repair in the yeast Saccharomyces cerevisiae. In this report, we show that the 19S regulatory complex of the yeast proteasome can affect nucleotide excision repair independently of Rad23 protein. Strains with mutations in 19S regulatory subunits (but not 20S subunits) of the proteasome promote partial recovery of nucleotide excision repair in vivo in rad23 deletion mutants, but not in other nucleotide excision repair-defective strains tested. In addition, a strain that expresses a temperature-degradable ATPase subunit of the 19S regulatory complex manifests a dramatically increased rate of nucleotide excision repair in vivo. These data indicate that the 19S regulatory complex of the 26S proteasome can negatively regulate the rate of nucleotide excision repair in yeast and suggest that Rad23 protein not only recruits the 19S regulatory complex, but also can mediate functional interactions between the 19S regulatory complex and the nucleotide excision repair machinery. The 19S regulatory complex of the yeast proteasome functions in nucleotide excision repair independent of proteolysis.

The Gal4 activation domain binds Sug2 protein, a proteasome component, in vivo and in vitro.

An in vivo protein interaction assay was used to search a yeast cDNA library for proteins that bind to the acidic activation domain (AD) of the yeast Gal4 protein. Sug2 protein, a component of the 19 S regulatory particle of the 26 S proteasome, was one of seven proteins identified in this screen. In vitro binding assays confirm a direct interaction between these proteins. SUG2 and SUG1, another 19 S component, were originally discovered as a mutation able to suppress the phenotype of a Gal4 truncation mutant (Gal4(D)p) lacking much of its AD. Sug1p has previously been shown to bind the Gal4 AD in vitro. Taken together, these genetic and biochemical data suggest a biologically significant interaction between the Gal4 protein and the 19 S regulatory particle of the proteasome. Indeed, it is demonstrated here that the Gal4 AD interacts specifically with immunopurified 19 S complex. The proteasome regulatory particle has been shown recently to play a direct role in RNA polymerase II transcription and the activator-19 S interaction could be important in recruiting this large complex to transcriptionally active GAL genes.

Are all DNA binding and transcription regulation by an activator physiologically relevant?

Understanding how a regulatory protein occupies its sites in vivo is central to understanding gene regulation. Using the yeast Gal4 protein as a model for such studies, we have found 239 potential Gal4 binding sites in the yeast genome, 186 of which are in open reading frames (ORFs). This raises the questions of whether these sites are occupied by Gal4 and, if so, to what effect. We have analyzed the Saccharomyces cerevisiae ACC1 gene (encoding acetyl-coenzyme A carboxylase), which has three Gal4 binding sites in its ORF. The plasmid titration assay has demonstrated that Gal4 occupies these sites in the context of an active ACC1 gene. We also find that the expression of the ACC1 is reduced fourfold in galactose medium and that this reduction is dependent on the Gal4 binding sites, suggesting that Gal4 bound to the ORF sites affects transcription of ACC1. Interestingly, removal of the Gal4 binding sites has no obvious effect on the growth in galactose under laboratory conditions. In addition, though the sequence of the ACC1 gene is highly conserved among yeast species, these Gal4 binding sites are not present in the Kluyveromyces lactis ACC1 gene. We suggest that the occurrence of these sites may not be related to galactose regulation and a manifestation of the "noise" in the occurrence of Gal4 binding sites.

Evidence that proteolysis of Gal4 cannot explain the transcriptional effects of proteasome ATPase mutations.

The Gal system of Saccharomyces cerevisiae is a paradigm for eukaryotic gene regulation. Expression of genes required for growth on galactose is regulated by the transcriptional activator Gal4. The activation function of Gal4 has been localized to 34 amino acids near the C terminus of the protein. The gal4D allele of GAL4 encodes a truncated protein in which only 14 amino acids of the activation domain remain. Expression of GAL genes is dramatically reduced in gal4D strains and these strains are unable to grow on galactose as the sole carbon source. Overexpression of gal4D partially relieves the defect in GAL gene expression and allows growth on galactose. A search for extragenic suppressors of gal4D identified recessive mutations in the SUG1 and SUG2 genes, which encode ATPases of the 19S regulatory complex of the proteasome. The proteasome is responsible for the ATP-dependent degradation of proteins marked for destruction by the ubiquitin system. It has been commonly assumed that effects of SUG1 and SUG2 mutations on transcription are explained by alterations in the proteolysis of gal4D protein. We have investigated this assumption. Surprisingly, we find that SUG1 and SUG2 alleles that are unable to suppress gal4D cause a larger increase in gal4D protein levels than do suppressing alleles. In addition, mutations in genes encoding subunits of the proteolytic 20S sub-complex of the proteasome increase the levels of gal4D protein but do not rescue its transcriptional activity. Therefore, an alteration in the proteolysis of gal4D by the proteasome cannot explain the effects of mutations in SUG1 and SUG2 on expression of GAL genes. These findings suggest that the 19S regulatory complex may play a more direct role in transcription.