Comparative hybridization reveals extensive genome variation in the AIDS-associated pathogen Cryptococcus neoformans

Background

Genome variability can have a profound influence on the virulence of pathogenic microbes. The availability of genome sequences for two strains of the AIDS-associated fungal pathogen Cryptococcus neoformans presented an opportunity to use comparative genome hybridization (CGH) to examine genome variability between strains of different mating type, molecular subtype, and ploidy.

Results

Initially, CGH was used to compare the approximately 100 kilobase MAT a and MATα mating-type regions in serotype A and D strains to establish the relationship between the Log2 ratios of hybridization signals and sequence identity. Subsequently, we compared the genomes of the environmental isolate NIH433 (MAT a) and the clinical isolate NIH12 (MATα) with a tiling array of the genome of the laboratory strain JEC21 derived from these strains. In this case, CGH identified putative recombination sites and the origins of specific segments of the JEC21 genome. Similarly, CGH analysis revealed marked variability in the genomes of strains representing the VNI, VNII, and VNB molecular subtypes of the A serotype, including disomy for chromosome 13 in two strains. Additionally, CGH identified differences in chromosome content between three strains with the hybrid AD serotype and revealed that chromosome 1 from the serotype A genome is preferentially retained in all three strains.

Conclusion

The genomes of serotypes A, D, and AD strains exhibit extensive variation that spans the range from small differences (such as regions of divergence, deletion, or amplification) to the unexpected disomy for chromosome 13 in haploid strains and preferential retention of specific chromosomes in naturally occurring diploids.     

Identification of somatic chromosomal abnormalities in hypothalamic hamartoma tissue at the GLI3 locus

Hypothalamic hamartomas (HH) are rare, benign congenital tumors associated with intractable epilepsy. Most cases are sporadic and nonsyndromic. Approximately 5% of HH cases are associated with Pallister-Hall syndrome (PHS), which is caused by haploinsufficiency of GLI3. We have investigated the possibility that HH pathogenesis in sporadic cases is due to a somatic (tumor-only) mutation in GLI3. We isolated genomic DNA from peripheral blood and surgically resected HH tissue in 55 patients with sporadic HH and intractable epilepsy. A genome-wide screen for loss of heterozygosity (LOH) and chromosomal abnormalities was performed with parallel analysis of blood and HH tissue with Affymetrix 10K SNP microarrays. Additionally, resequencing and fine mapping with SNP genotyping were completed for the GLI3 gene with comparisons between peripheral blood and HH tissue pairs. By analyzing chromosomal copy-number data for paired samples on the Affymetrix 10K array, we identified a somatic chromosomal abnormality on chromosome 7p in one HH tissue sample. Resequencing of GLI3 did not identify causative germline mutations but did identify LOH within the GLI3 gene in the HH tissue samples of three patients. Further genotyping of 28 SNPs within and surrounding GLI3 identified five additional patients exhibiting LOH. Together, these data provide evidence that the development of chromosomal abnormalities within GLI3 is associated with the pathogenesis of HH lesions in sporadic, nonsyndromic patients with HH and intractable epilepsy. Chromosomal abnormalities including the GLI3 locus were seen in 8 of 55 (15%) of the resected HH tissue samples. These somatic mutations appear to be highly variable.     

Contribution of Copper Ion Resistance to Survival of Escherichia coli on Metallic Copper Surfaces

Bacterial contamination of touch surfaces poses a serious threat for public health. The use of bactericidal surface materials, such as copper and its alloys, might constitute a way to aid the use of antibiotics and disinfectants, thus minimizing the risk of emergence and spread of multiresistant germs. The survival of Escherichia coli on metallic copper surfaces has been studied previously; however, the mechanisms underlying bacterial inactivation on copper surfaces have not been elucidated. Data presented in this study suggest that bacteria are killed rapidly on dry copper surfaces. Several factors, such as copper ion toxicity, copper chelators, cold, osmotic stress, and reactive oxygen species, but not anaerobiosis, influenced killing rates. Strains deleted in copper detoxification systems were slightly more sensitive than was the wild type. Preadaptation to copper enhanced survival rates upon copper surface exposure. This study constitutes a first step toward understanding the reasons for metallic copper surface-mediated killing of bacteria.     

Rare Variants in PLXNA4 and Parkinson’s Disease

Approximately 20% of individuals with Parkinson’s disease (PD) report a positive family history. Yet, a large portion of causal and disease-modifying variants is still unknown. We used exome sequencing in two affected individuals from a family with late-onset familial PD followed by frequency assessment in 975 PD cases and 1014 ethnically-matched controls and linkage analysis to identify potentially causal variants. Based on the predicted penetrance and the frequencies, a variant in PLXNA4 proved to be the best candidate and PLXNA4 was screened for additional variants in 862 PD cases and 940 controls, revealing an excess of rare non-synonymous coding variants in PLXNA4 in individuals with PD. Although we cannot conclude that the variant in PLXNA4 is indeed the causative variant, these findings are interesting in the light of a surfacing role of axonal guidance mechanisms in neurodegenerative disorders but, at the same time, highlight the difficulties encountered in the study of rare variants identified by next-generation sequencing in diseases with autosomal dominant or complex patterns of inheritance.     

Stopped-flow dynamic light scattering as a method to monitor compaction during protein folding

Kinetic dynamic light scattering is a useful tool to follow compaction during protein folding. In contrast to measurements of the formation of secondary structure and side chain ordering, kinetic measurements of compactness are not well established up to now. This work describes the adaptation of a stopped-flow system (SFM-3) to a dynamic light scattering apparatus, which allows one to monitor the compaction of protein molecules by measuring the hydrodynamic Stokes radius R. The feasibility of such investigations was demonstrated by measuring R and the integrated scattered intensity I during refolding of ribonuclease A and phosphoglycerate kinase from yeast. Refolding was initiated by rapid mixing of protein solutions containing high concentrations of guanidine hydrochloride with buffer. Between 20 and 50 mixing events were performed in these experiments. Measuring both R and I in one and the same experiment is important to distinguish between true folding of individual molecules and cases where folding is accompanied by the appearance of transient oligomers or associated misfolded structures. On refolding of ribonuclease A (0.6 M GuHCl, 25 °C), after a fast phase the Stokes radius decreased from 2.26 nm to 1.95 nm with a time constant of 27 s without detectable aggregates. By contrast, transient and stable oligomers have been observed during the more complex folding of phosphoglycerate kinase. In general, the time-resolution of the method is of the order of 1 s. It can be extended to the subsecond time-range if the number of shots is not limited by the amount of protein available. Received: 8 August 1996 / Accepted: 18 October 1996     

IL-6 expression induced by adenosine A2b receptor stimulation in U373 MG cells depends on p38 mitogen activated kinase and protein kinase C

Adenosine binds to a class of G-protein coupled receptors, which are further distinguished as A(1), A(2a), A(2b) and A(3) adenosine receptors. As we have shown earlier, the stable adenosine analogue NECA (N6-(R)-phenylisopropyladenosine) stimulates IL-6 expression in the human astrocytoma cell line U373 MG via the A(2b) receptor. The mechanism by which NECA promotes astrocytic IL-6 expression has not been identified. By using various inhibitors of signal transduction, we found that p38 mitogen-activated protein kinases (MAPK) activation (inhibitor SB202190), but not extracellular signal-regulated kinase (ERK) (PD98059) and c-jun N-terminal kinase (JNK)(SP600125), is essential in the NECA-induced signalling cascade that leads to the increase in IL-6 synthesis in U373 MG cells. Results obtained with protein kinase C (PKC) inhibitors that have different substrate specificities, indicated that the PKC delta and epsilon isoforms are also involved in adenosine receptor A(2b) dependent upregulation of IL-6 expression. This is supported by the fact that NECA induced the activation of PKC delta and epsilon in U373 MG cells.     

Radial transport of water across cortical sleeves of excised roots ofZea mays L.

The stationary radial volume flows across maize (Zea mays L.) root segments without steles (sleeves) were measured under isobaric conditions. The driving force of the volume flow is an osmotic difference between the internal and external compartment of the root preparations. It is generated by differences in the concentrations of sucrose, raffinose or polyethylene glycol. The flows are linear functions of the corresponding osmotic differences ( ) up to osmotic values which cause plasmolysis. The straight lines obtained pass through the origin. No asymmetry of the osmotic barrier could be detected within the range of driving forces applied ( =±0.5 MPa), corresponding to volume-flow densities of j_{v, s}=±7·10^–8 m·s^–1. Using the literature values for the reflection coefficients of sucrose and polyethylene glycol in intact roots (E. Steudle et al. (1987) Plant Physiol.84, 1220–1234), values for the sleeve hydraulic conductivity of about 1·10^–7 m·s^–1 MPa^–1 were calculated. They are of the same order of magnitude as those reported in the literature for the hydraulic conductivity of intact root segments when hydrostatic pressure is applied.Abbreviations and symbols a _s outer surface of sleeve segment - c concentration of osmotically active solute - j _{v, s} radial volume flow density across sleeve segment - Lp_s hydraulic conductivity of sleeves - Lp_r hydraulic conductivity of intact roots - _N thickness of Nernst diffusion layer - reflection coefficient of root for solute - osmotic value of bulk phase - osmotic coefficient     

Neuromedin U-immunoreactivity in the nervous system of the small intestine of the pig and its coexistence with substance P and CGRP

Summary In the small intestine of the pig, neuromedin U (NMU)-immunoreactivity was mainly confined to the nerve plexus of the inner submucosal and mucosal regions. After colchicine treatment, a high number of immunoreactive nerve cell bodies was observed in the plexus submucosus internus (Meissner), whereas only a low number was found in the plexus submucosus externus (Schabadasch). The plexus myentericus as well as the aganglionic nerve meshworks in the circular and longitudinal smooth muscle layers almost completely lacked NMU-immunoreactivity. Double-labeling experiments demonstrated the occurrence of distinct NMU-containing neuron populations in the plexus submucosus internus: (1) relatively large type-II neurons revealing immunoreactivity for NMU and calcitonin gene-related peptide (CGRP) and/or substance P (SP); (2) a group of small NMU- and SP-immunoreactive neurons; (3) a relatively low number of small neurons displaying immunoreactivity for NMU but not for SP. Based on its distributional pattern, it is concluded that NMU plays an important role in the regulation and control of mucosal functions.