In vitro assessment of a collagen sponge for engineering urothelial grafts

OBJECTIVE: To investigate the deposition of urinary crystals and the growth characteristics of urothelial cells on a collagen sponge, as a preliminary step in engineering urothelial autologous grafts. MATERIALS AND METHODS: Collagen sponges were exposed to a continuous flow of urine at pH 5.3 and 6.3 for 1 week. The sponges were examined microscopically for crystal deposition and analysed for their calcium content. Two cell lines, RT112, derived from a well-differentiated transitional cell carcinoma, and UROtsa, an immortalized urothelial cell line, were seeded on the collagen sponges. Cells were cultured for 6, 12 and 21 days. The pattern of growth was analysed by histology and immunostaining with a pan-cytokeratin antibody. Growth was assayed to quantify cell proliferation on the sponges. RESULTS: No crystals were evident on any of the collagen sponges. Calcium deposition was negligible at pH 5.3. Although calcium levels were measurable at pH 6.3, the levels were very low. Both cell lines attached and grew in a stratified manner on the collagen sponge, RT112 forming a layer 6-8 cells thick, and UROtsa a layer 4-6 cells thick; cell proliferation was maximal at 5-10 days. The sponge remained easy to handle after 3 weeks in culture. CONCLUSION: These findings show that collagen sponges support the growth and stratification of urothelial cells, and indicate that the collagen sponge is a suitable substrate for developing urothelial autologous grafts.     

Compensatory neutral mutations and the evolution of RNA

There are many examples of RNA molecules in which the secondary structure has been strongly conserved during evolution, but the base sequence is much less conserved, e.g., transfer RNA, ribosomal RNA, and ribonuclease P. A model of compensatory neutral mutations is used here to describe the evolution of the base sequence in RNA helices. There are two loci (i.e., the two sides of the pair) with four alleles at each locus (corresponding to A, C, G, U). Watson-Crick base pairs (AU, CG, GC, and UA) are each assigned a fitness 1, whilst all other pairs are treated as mismatches and assigned fitness 1-s. A population of N diploid individuals is considered with a mutation rate of u per base. For biologically reasonable parameter values, the frequency of mismatches is always small but the frequency of the four matching pairs can vary over a wide range. Using a diffusion model, the stationary distribution for the frequency x of any of the four matching pairs is calculated. The shape depends on the combination of variables beta = 8Nu2/9s. For small beta, the distribution diverges at the two extremes, x = 0 and x = 1-z, where z is the mean frequency of mismatches. The population typically consists almost entirely of one of the four types of matching pairs, but occasionally makes shifts between the four possible states. The mean rate at which these shifts occur is calculated here. The effect of recombination between the two loci is to decrease the probability density at intermediate x, and to increase the weight at the extremes. The rate of transition between the four states is slowed by recombination (as originally shown by Kimura in a two-allele model with irreversible mutation). A very small recombination rate r approximately u2/s is sufficient to increase the mean time between transitions dramatically. In addition to its application to RNA, this model is also relevant to the 'shifting balance' theory describing the drift of populations between alternative equilibria separated by low fitness valleys. Equilibrium values for the frequencies of the different allele combinations in an infinite population are also calculated. It is shown that for low recombination rates the equilibrium is symmetric, but there is a critical recombination rate above which alternative asymmetric equilibria become stable.     

Experimental infection of pigs with the newly identified swine hepatitis E virus (swine HEV), but not with human strains of HEV

A novel virus of pigs, swine hepatitis E virus (swine HEV), was recently identified and shown to be antigenically and genetically related to human HEV. In the present study, we attempted to infect specific-pathogen-free (SPF) pigs experimentally with swine HEV or with human strains of HEV. Serum samples collected from naturally infected pigs were used as the source of swine HEV. Pigs inoculated intravenously with serum samples containing swine HEV seroconverted to anti-HEV 4 to 8 weeks postinoculation, and the virus spread to an uninoculated pig. Swine HEV was detected in nasal and rectal swab materials as early as 2 weeks postinoculation and for 4 to 8 weeks thereafter. Viremia appeared 4 to 6 weeks postinoculation and lasted 1 to 3 weeks. The inoculated pigs appeared clinically normal and serum liver enzymes were not significantly elevated. In contrast, pigs were not infected when inoculated intravenously with about 10(5) monkey infectious doses of one of two human strains of HEV (Sar-55 or Mex-14).     

Allelic loss at chromosomes 3p, 8p, 13q, and 17p associated with poor prognosis in head and neck cancer

BACKGROUND: Little is known about the molecular genetic events that contribute to the pathogenesis of squamous cell carcinoma of the upper aerodigestive tract. Previous molecular genetic studies have been limited to the identification of mutations of the p53 (also known as TP53) tumor suppressor gene, activation of a limited set of oncogenes, allelic loss at 3p and other locations, and occasional association with human papillomavirus infection. PURPOSE: Our purpose was to screen tumor tissue and blood from patients with squamous cell carcinoma of the upper aerodigestive tract for loss of heterozygosity at polymorphic loci corresponding to each of the autosomal chromosomes and to identify the locations of additional putative tumor suppressor genes, other than RB (also known as RB1) and p53, that may contribute to the pathogenesis of this disease. METHODS: Tumor tissue and blood were obtained from 68 consecutive patients with squamous cell carcinoma of the upper aerodigestive tract. In all cases, tumor tissue was obtained from the center of the surgical specimen. The relative absence of non-neoplastic tissue was confirmed by frozen-section histologic examination of immediately adjacent tissue. Initially, 30 paired tissue and blood samples were tested for loss of heterozygosity by polymerase chain reaction (PCR) to amplify 43 different highly polymorphic sequences containing small oligonucleotide repeats. After PCR amplification, with unique oligonucleotides flanking the repeat, visualization and sizing of the alleles on DNA sequencing gels were performed. Specific loss of heterozygosity was distinguished from random genetic loss due to generalized chromosomal instability if it occurred in more than 20% of specimens tested for a particular marker. RESULTS: Significant loss of heterozygosity (> 20%) occurred at alleles at chromosome bands 3p21 (32%), 3p25-26 (56%), 8pter-21.1 (31%), 13q14 (27%), and 17p12 (45%). Loss of heterozygosity at more than two loci was significant with a poor prognosis (P = .039). CONCLUSIONS: These findings demonstrate that squamous cell carcinoma of the upper aerodigestive tract exhibits genetic alterations at multiple loci and that allelic loss at more than two locations is indicative of a poor prognosis (the likelihood of the patient dying of disease). IMPLICATIONS: While tumor suppressor genes at 3p (VHL), 13q (RB), and 17p (p53) have been identified, altered genes at other loci on 3p and on 8p have not yet been characterized. Furthermore, the genotype at these loci for squamous cell carcinoma of the upper aerodigestive tract has prognostic importance and may identify the patients who should receive the most aggressive treatment.     

Industrial high‐rate (∼5 nm/s) deposited silicon nitride yielding high‐quality bulk and surface passivation under optimum anti‐reflection coating conditions

High‐quality surface and bulk passivation of crystalline silicon solar cells has been obtained under optimum anti‐reflection coating properties by silicon nitride (a‐SiN_x:H) deposited at very high deposition rates of ∼5 nm/s. These a‐SiN_x:H films were deposited using the expanding thermal plasma (ETP) technology under regular processing conditions in an inline industrial‐type reactor with a nominal throughput of 960 solar cells/hour. The low surface recombination velocities (50–70 cm/s) were obtained on p‐type silicon substrates (8·4 Ω cm resistivity) for as‐deposited and annealed films within the broad refractive index range of 1·9–2·4, which covers the optimum bulk passivation and anti‐reflection coating performance reached at a refractive index of ∼2·1. Copyright © 2005 John Wiley & Sons, Ltd.     

Foot dystonia and lumbar canal stenosis

We describe a patient who developed involuntary, painless, dystonic contraction of the toes of the right foot on standing or walking. The development of this abnormal movement had been preceded by sensory disturbance on the soles of both feet, triggered by dorsiflexion of the feet. Examination showed that weight bearing on the right foot and walking brought on clawing of the toes of the right foot, which was relieved within seconds of taking pressure off the right foot. There was sensory and reflex evidence of bilateral S1 root disturbance confirmed by electrophysiology. Magnetic resonance imaging of the lumbar spine showed marked stenosis of the lumbar canal with compression of the L5 and S1 nerve roots bilaterally. The patient underwent a lumbar laminectomy with nerve root exit foramina decompression, which abolished the foot dystonia and has considerably improved the sensory disturbance. This case demonstrates that lumbar canal stenosis and/or nerve root compression, may be responsible for foot dystonia. Amelioration of the abnormal movement by surgical decompression argues strongly in favour of this hypothesis.     

Physicochemical properties correlated with drug resistance and the reversal of drug resistance in Plasmodium falciparum

At high molar excess, verapamil can selectively increase the accumulation and cytotoxicity of structurally dissimilar natural product drugs in many multidrug-resistant tumor cell lines. Such concentrations of verapamil are also capable of increasing the accumulation and activity of chloroquine in chloroquine-resistant strains of the human malaria parasite Plasmodium falciparum. Despite such similarities, it is not clear why chloroquine-resistant P. falciparum is often susceptible to closely related compounds such as amodiaquine, whereas cancer cells are cross-resistant to many structurally unrelated drugs. For 13 aminoquinoline and aminoacridine compounds, relative drug resistance was negatively correlated with lipid solubility at physiological pH (r2 = 0.90, p < 0.0001). The ability of verapamil (5 microM) to reverse drug resistance was also negatively correlated with lipid solubility (r2 = 0.88, p < 0.0001). Furthermore, molar refractivity was weakly correlated with relative drug resistance (r2 = 0.46, p < 0.05) and reversal of drug resistance (r2 = 0.52, p < 0.005). Verapamil increases chloroquine accumulation by resistant parasites, a mechanism suggested to account for its selective chemosensitization effect. We show that the initial rate of chloroquine accumulation by resistant parasites is increased by verapamil. This effect of verapamil is abolished when deoxy-glucose is substituted for glucose. Therefore, verapamil produces an energy-dependent increase in the permeability of resistant parasites to chloroquine. For a panel of four chloroquine-resistant and two chloroquine-susceptible isolates, the effect of verapamil on the accumulation of chloroquine and monodesethyl amodiaquine was found to be correlated (r2 = 0.96, p < 0.001). Verapamil chemosensitization was also correlated for the two drugs (r2 = 0.92, p < 0.005), suggesting a common mechanism. In summary, the degree of drug resistance and the extent of verapamil chemosensitization for a particular drug seem to be dependent on general physical features such as lipid solubility and molar refractivity rather than on closely defined structural parameters. These studies provide insight into this important resistance mechanism of malaria parasites and may provide direction for the development of new drugs that are effective against resistant parasites.     

Application of HUMF13A01 (AAAG)n STR polymorphism to the genetic diagnosis of coagulation factor XIII deficiency

Deficiency of the A subunit of coagulation factor XIII causes a severe bleeding disorder requiring life long replacement therapy. The mutations causing A subunit deficiency appear to be very heterogeneous, and it is impractical to identify each mutation before genetic counselling or prenatal diagnosis can be attempted. In this study we have shown that a highly polymorphic short tandem repeat element, HUMF13A01 (AAAG)n that occurs in the 5' flanking sequence of the factor XIII A subunit gene, can be used to follow the segregation of deficiency causing mutations. We studied 6 families with factor XIII A subunit deficiency from 5 different ethnic groups. All parents were heterozygous for the repetitive element and therefore all the families were informative. The linked polymorphism was used to carry out the first prenatal diagnosis of factor XIII A subunit deficiency. The analysis of this polymorphism by the polymerase chain reaction is rapid, reliable, requires little DNA and is ideal for the genetic analysis of factor XIII A subunit deficiency.