Effect of nanoclay concentration level on the electrical properties of polypropylene under electron irradiation in a SEM.

For studying the electrical properties (charge trapping, transport and secondary electron emission) of the polypropylene-based nanocomposites with different contents of natural clay, the specimens were submitted to electron irradiation of a scanning electron microscope. A device, suitably mounted on the sample holder of the scanning electron microscope, was used to measure two currents (i.e. leakage and displacement currents) induced in the polypropylene-based nanocomposites (polymer nanocomposites) under electron irradiation. The evolution of trapped charge during irradiation for each type of studied polymer nanocomposites is deduced. The amount of trapped charge at the steady state is also determined by measuring the change of secondary electron image size associated to the electron trajectory simulation. It is found, surprisingly, that not only the leakage current increases as a function of clay loading level but also trapped charge. However, this could be related to the increase of conductivity in one hand and to proliferation of interfaces between nanoparticles and neighbouring materials on the other hand. These two processes play crucial role in controlling the carrier transport (through polymer nanocomposites or/and along its surface) closely related to the charge storage and leakage current. Additional experiment using dielectric spectroscopy were performed to show the effect of clay concentration in changing the dielectric relaxation behaviour and to evidence the existence of interfaces between nanoparticles and polymer. The secondary electron emission during electron irradiation is also studied through the total electron yield that is deduced by correlating the measured leakage and displacement currents.

[Hereditary variants of primary hyperparathyroidism--MEN1, MEN2, HPT-JT, FHH, FIHPT]. / Hereditäre Formen des primären Hyperparathyreoidismus. MEN1, MEN2, HPT-JT, FHH, FIHPT.

OBJECTIVE: The challenge in diagnosing primary hyperparathyroidism (HPT) is to detect hereditary cases before first surgery. About 5% of cases are hereditary and integral component of multiple endocrine neoplasia type 1 and 2 (MEN1/MEN2), hyperparathyroidism-jaw tumor syndrome (HPT-JT), familial hypocalciuric hypercalcemia (FHH), and familial isolated hyperparathyroidism (FIHPT). Aim of this study was to evaluate similarities and differences in hereditary varieties of HPT. PATIENTS: 80 patients with hereditary HPT were evaluated in a retrospective analysis between 1980 and 2010 concerning clinical findings, family history, therapy, biochemical and molecular-genetic findings and follow-up. RESULTS: 80 patients with hereditary HPT are described, 52 belonged to MEN1, 15 to MEN2, 7 to HPT-JT, 4 to FHH and 2 to FIHPT kindreds. Penetrance of HPT was highest in MEN1 (85%), followed by HPT-JT (64%), FHH (28.5%), and MEN2 (8%). Youngest age at diagnosis of HPT was 7 and 16 years in the MEN2/HPT-JT group. Serum Calcium was highest in the HPT-JT group (3.6 mM), recurrencies of HPT were highest in the MEN1 group (40.5%). Parathyroid cancer solely occurred in the HPT-JT group. In single cases HPT occurs in FHH. CONCLUSION: Among the different varieties of hereditary HPT MEN1-HPT is most frequent and carries the utmost recurrence rate. Early diagnosis of HPT-JT syndrome is important because of the occurrence of parathyroid cancer. Single cases of HPT in FHH are described. Preoperative diagnosis of hereditary HPT has therapeutic consequences concerning extent of surgery and implications concerning patient and family care.

New mutations in the RET protooncogene-L881V - associated with medullary thyroid carcinoma and -R770Q - in a patient with mixed medullar/follicular thyroid tumour.

UNLABELLED: Clinical studies are needed to classify rare and novel RET mutations associated with hereditary medullary thyroid carcinoma (MTC) into one of the clinical risk groups. Here we describe two new RET mutations/variants, R770Q and L881V, in patients with MTC and analyzed genotype-phenotype correlations associated with these RET mutations in the gene carriers. FAMILY 1: Calcitonin screening in a 42-year-old female patient with multinodular goiter showed elevated levels. RET mutation analysis revealed a new variant in exon 13 R770Q (CGA>CAA) in the patient. A thyroidectomy with central and lateral node dissection was done. Histology showed MTC in a mixed variance with follicular cancer of 2 cm diameter (T2N0M0). Postoperatively there was no increase of calcitonin after pentagastrin stimulation. The patient is biochemically cured concerning MTC and FTC after radioiodine therapy. In the sister of the index patient surprisingly another, previously not described amino-acid substitution Y791N (TAT><) in the RET protooncogene was found. In the parents the R770Q variant was detected in the mother, the Y791N mutation in the father. Another sister carries the R770Q variant. In all other gene carriers (aged 44-70 years), calcitonin levels were in the normal range, therefore, thyroidectomy had not yet been performed. FAMILY 2: In a 46-year-old female patient with nodular goiter thyroidectomy, central and left lateral lymph node dissection was done because of elevated calcitonin levels. Histology revealed a microcarcinoma with one lymph node metastasis (T1N1(1/8)Mx). RET analysis revealed a new mutation in exon 15 L881V (CTG>GTG). The L881V mutation was detected in five other family members. In the first generation stimulated calcitonin levels were in the normal range, therefore thyroidectomy had not yet been performed. In the sons of the index case thyroidectomy revealed CCH in the older one, no MTC in both. In a cousin thyroidectomy is intended because of elevated basal and stimulated calcitonin. CONCLUSION: Our clinical findings indicate that the L881V mutation may be associated with late-onset nonaggressive disease. If the germline RET R770Q variant has a causative role in the pathogenesis of the mixed medullar/follicular derived histology of the thyroid tumour in the index patient of family 1 has to be proven. The recommendations for prophylactic thyroidectomy in these mutations should be individualized depending on basal and stimulated calcitonin levels until more data are available.

Long-term outcome in 46 gene carriers of hereditary medullary thyroid carcinoma after prophylactic thyroidectomy: impact of individual RET genotype.

OBJECTIVE: In children with RET proto-oncogene mutation, curative treatment of medullary thyroid carcinoma (MTC) is possible by prophylactic thyroidectomy. Recommendations on the timing and extent of thyroidectomy are based upon a model that utilises genotype-phenotype correlations to stratify mutations into three risk groups. DESIGN: We evaluated the long-term outcome (mean follow-up 6.4 years, 15 patients more than 10 years, 26 patients more than 5 years) of operated gene carriers stratified into two risk groups (levels 1 and 2) based on the biological aggressiveness of MTC. RESULTS: In 46 RET gene carriers, prophylactic thyroidectomy was carried out between the ages of 4 and 21 years. Level 1 mutations were harboured by 11 patients (codons 790, 791, 804 and 891). Histology was completely normal in two patients; in seven patients C-cell hyperplasia (CCH) and in two patients T1 tumours were diagnosed. All patients with level 1 mutations were cured. Level 2 mutations were harboured by 35 patients (codons 618, 620, 630 and 634). Histology of these patients showed CCH in 11 patients, T1 tumours in 21, T2 tumour in 1, T3 tumour in 1 and Tx in 1 patient. Histology showed no lymph node involvement. Five patients with level 2 mutations failed to be cured; in two patients, persistence of MTC was diagnosed directly after thyroidectomy and in three during follow-up. In two patients carrying a 634 mutation, other endocrinopathies (hyperparathyroidism and bilateral pheochromocytoma) manifested during follow-up. CONCLUSIONS: If prophylactic thyroidectomy is done at early ages, cure rate is high. Timing and extent of prophylactic thyroidectomy can be modified by individual RET mutation.

Primary hyperparathyroidism as the leading symptom in a patient with a Y791F RET mutation.

Primary hyperparathyroidism (PHP; serum calcium 2.75 mmol/L, PTH 226 pg/ml) had been the first clinical manifestation of MEN-2A in a female patient (aged 55 years) with a mutation (Y791F, TAT-->TTT) in exon 13 of the RET proto-oncogene. The patient has a pentagastrin-induced rise in serum calcitonin (up to 57 pg/ml) considered normal for noncarriers but abnormal in family members of MEN-2 patients. This is the first case of MEN-2 due to this specific mutation with primary hyperparathyroidism as the first manifestation of the disease. In addition, the patient harbored, within the Menin gene, a polymorphism (D418D) reportedly associated with sporadic primary hyperparathyroidism. This case report indicates that molecular biological tests in MEN- 2 may only suggest a certain phenotype but cannot predict it with certainty. It may also suggest that genetic screening for MEN-2 may be advisable in patients with primary hyperparathyroidism and a borderline-high pentagastrin stimulation test, even in the absence of a positive family history.

[Hereditary medullary thyroid carcinoma--genotype-phenotype characterization]. / Hereditäres medulläres Schilddrüsenkarzinom--Genotyp-Phänotyp Charakterisierung.

BACKGROUND AND OBJECTIVE: Hereditary medullary thyroid carcinoma (MTC) is caused by germline mutations of the RET proto-oncogene. A genotype - phenotype correlation has been established, showing clustering of mutations in exons 10 and 11 in classical MEN 2 A syndrome, in exon 16 codon 918 in MEN 2 B syndrome and in exons 13-15 in familial MTC. A line of evidence suggested that the development and the aggressiveness of MTC in the different cancer syndromes is variable. Aim of this study was to compare the phenotype of exon 13-15 mutations with that of exon 11 mutation and possibly draw therapeutical consequences. PATIENTS AND METHODS: We compared the phenotype of 47 patients with mutations in exon 13-15 with 66 patients with exon 11, codon 634 mutation, the classical MEN2A. Patients were further subdivided as index and screening patients. RESULTS: Mean age of 19 index patients with codon 790, 791, 804 or 891 mutation was significant higher compared with 18 index patients with codon 634 mutation (mean age at diagnosis 50+/-12 years; range 30-69 y vs mean age 31+/-9 years; range 17-49 y), tumor stage at operation was favourable (C-cell hyperplasia n = 1; stage I n = 8; II n = 3; III n = 2; IV n = 2; no operation n = 1; no information n = 2 vs stage I n = 3; stage II n = 6; stage III n = 4, no information n =5), cure rate was better (56 % vs 38 %) and the death rate was lower (n = 2 vs n = 4). In screening patients no differences concerning the age, tumor stage, cure and death rate between patients with exons 13-15 and codon 634 mutations were seen. CONCLUSIONS: MTC in patients with exon 790, 791, 804, 891 mutations displayed a late onset and an indolent course compared to codon 634 mutation, this has to be taken into account when recommending timing and extent of prophylactic surgery.

Mutational spectrum of the steroid 21-hydroxylase gene in Austria: identification of a novel missense mutation.

This study attempted an analysis of the mutational spectrum of 21-hydroxylase deficiency in 79 unrelated Austrian patients with classical and nonclassical forms of congenital adrenal hyperplasia and their respective 112 family members. Apparent large gene deletions/conversions were present in 31% of the 158 unrelated congenital adrenal hyperplasia alleles, whereas the most frequent point mutations were intron 2 splice (22.8%), I172N (15.8%), V281L (12%), and P30L (7.6%), in line with the frequencies reported for other countries. In 5 of the 12 congenital adrenal hyperplasia alleles carrying a P30L mutation the aberration is based on a single base substitution, whereas the remaining 7 represent part of a CYP21B conversion (1 allele) or CYP21B/21A hybrid gene (6 alleles), the latter characterized by a junction site before intron 2 as indicated by Southern blot, PCR, and sequence analyses. Previously described mutations were not present in 1.2% of unrelated congenital adrenal hyperplasia alleles, including one female patient presenting with severe genital virilization. Sequence analysis of the complete functional 21-hydroxylase gene revealed an as yet undescribed mutation in exon 10-Arg(426)His, which has not yet been described to represent a common pseudogene sequence. In vitro expression experiments showed the Arg(426)His mutant to exhibit only low enzyme activity toward the natural substrate 17-hydroxyprogesterone corresponding to the degree of disease manifestation in the patient in whom it was found.

A helper phage to improve single-chain antibody presentation in phage display.

We show here that the number of single-chain antibody fragments (scFv) presented on filamentous phage particles generated with antibody display phagemids can be increased by more than two orders of magnitude by using a newly developed helper phage (hyperphage). Hyperphage have a wild-type pIII phenotype and are therefore able to infect F(+) Escherichia coli cells with high efficiency; however, their lack of a functional pIII gene means that the phagemid-encoded pIII-antibody fusion is the sole source of pIII in phage assembly. This results in an considerable increase in the fraction of phage particles carrying an antibody fragment on their surface. Antigen-binding activity was increased about 400-fold by enforced oligovalent antibody display on every phage particle. When used for packaging a universal human scFv library, hyperphage improved the specific enrichment factor obtained when panning on tetanus toxin. After two panning rounds, more than 50% of the phage were found to bind to the antigen, compared to 3% when conventional M13KO7 helper phage was used. Thus, hyperphage is particularly useful in stoichiometric situations, when there is little chance that a single phage will locate the desired antigen.

Epitopes fused to F-pilin are incorporated into functional recombinant pili.

In order to develop a system which allows infection by an epitope-specific phage-antibody via an F-pilus expressing that epitope, a study on the expression of foreign sequences on F-pilin was undertaken. Initially, a plasmid library was constructed with random sequences encoding one to five amino acid residues fused to the C terminus of F-pilin (traA) which was used to complement an F-plasmid with an amber mutation in traA. Functional F-pilin fusions were detected using the filamentous phage, fUSE2, which transduces tetracycline resistance, as well as immunoblots using a monoclonal antiserum specific for the acetylated N terminus of pilin. All the clones selected expressed the pilin-fusions and displayed full sensitivity towards fUSE2 infection, which was indistinguishable from the wild-type F-pilin. The sequences of fUSE2-sensitive clones when compared to randomly selected clones which were not fUSE2-sensitive, revealed no obvious pattern in the amino acid residues fused to the C terminus, except for a preference for a hydrophilic amino acid at position +1. Mutating the C-terminal Leu in wt (wild-type) pilin to Ser blocked pilus assembly and fUSE2 infection; the pilin was correctly processed but the level of acetylation at the N terminus appeared to decrease. Fusing a known epitope (myc) directly to the C terminus blocked processing of F-pilin leading to loss of F-pilus assembly and function. The introduction of random sequences between traA and this epitope yielded fully recombinant, functional F-pili but this appeared to be due to processing of the extension by an unidentified protease leading to loss of the epitope. Surface expression of another epitope (G2-10) was clearly demonstrated by immuno-electron microscopy of pili with a G2-10 monoclonal antibody. A different five amino acid residue spacer between the F-pilin C terminus and the G2-10 epitope produced a system that was transfer-proficient and fUSE2-sensitive, but the pili were barely detectable by immunoblots or by electron microscopy. While the underlying rules that govern successful epitope expression at the C terminus of F-pilin remain elusive, many types of foreign sequences can be displayed with varying degrees of success. Our results also suggest that pilin sequence determines a number of steps in the complex pathway for pilus assembly.

Following ion diffusion in solution.

An x-ray projection microscope equipped with a charge-coupled device camera allows direct observation of zinc (Zn(2+)) ions diffusing in aqueous hydrochloric acid solution during the corrosion of zinc foil and pellets. Time series of microradiographic images with a lateral resolution on the order of 10 micrometers allow observation of the time and spatial evolution of the colorless Zn(2+) ions in solution without any previous treatment. The concentration distribution of the ions can be quantified from these images. This technique should find applications in the biology, chemistry, and electrochemistry of aqueous solutions, allowing direct observation of the behavior and concentration fluctuations of medium or heavy ions moving in a weakly absorbing medium.