Quantitative modeling of near-field interactions incorporating polaritonic and electrostatic effects.

As scattering-scanning near-field optical microscopy (s-SNOM) continues to grow in prominence, there has been great interest in modeling the near-field light-matter interaction to better predict experimental results. Both analytical and numerical models have been developed to describe the near-field response, but thus far models have not incorporated the full range of phenomena accessible. Here, we present a finite element model (FEM), capable of incorporating the complex physical and spatial phenomena that s-SNOM has proved able to probe. First, we use electromagnetic FEM to simulate the multipolar response of the tip and illustrate the impact of strong coupling on signal demodulation. We then leverage the multiphysics advantage of FEM to study the electrostatic effect of metallic tips on semiconductors, finding that THz s-SNOM studies are most impacted by this tip-induced band-bending. Our model is computationally inexpensive and can be tailored to specific nanostructured systems and geometries of interest.

Protein disulphide isomerase (PDI) is protective against amyotrophic lateral sclerosis (ALS)-related mutant Fused in Sarcoma (FUS) in in vitro models.

Mutations in Fused in Sarcoma (FUS) are present in familial and sporadic cases of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). FUS is localised in the nucleus where it has important functions in DNA repair. However, in ALS/FTD, mutant FUS mislocalises from the nucleus to the cytoplasm where it forms inclusions, a key pathological hallmark of neurodegeneration. Mutant FUS also inhibits protein import into the nucleus, resulting in defects in nucleocytoplasmic transport. Fragmentation of the neuronal Golgi apparatus, induction of endoplasmic reticulum (ER) stress, and inhibition of ER-Golgi trafficking are also associated with mutant FUS misfolding in ALS. Protein disulphide isomerase (PDI) is an ER chaperone previously shown to be protective against misfolding associated with mutant superoxide dismutase 1 (SOD1) and TAR DNA-binding protein-43 (TDP-43) in cellular and zebrafish models. However, a protective role against mutant FUS in ALS has not been previously described. In this study, we demonstrate that PDI is protective against mutant FUS. In neuronal cell line and primary cultures, PDI restores defects in nuclear import, prevents the formation of mutant FUS inclusions, inhibits Golgi fragmentation, ER stress, ER-Golgi transport defects, and apoptosis. These findings imply that PDI is a new therapeutic target in FUS-associated ALS.

Rab-dependent cellular trafficking and amyotrophic lateral sclerosis.

Rab GTPases are becoming increasingly implicated in neurodegenerative disorders, although their role in amyotrophic lateral sclerosis (ALS) has been somewhat overlooked. However, dysfunction of intracellular transport is gaining increasing attention as a pathogenic mechanism in ALS. Many previous studies have focused axonal trafficking, and the extreme length of axons in motor neurons may contribute to their unique susceptibility in this disorder. In contrast, the role of transport defects within the cell body has been relatively neglected. Similarly, whilst Rab GTPases control all intracellular membrane trafficking events, their role in ALS is poorly understood. Emerging evidence now highlights this family of proteins in ALS, particularly the discovery that C9orf72 functions in intra transport in conjunction with several Rab GTPases. Here, we summarize recent updates on cellular transport defects in ALS, with a focus on Rab GTPases and how their dysfunction may specifically target neurons and contribute to pathophysiology. We discuss the molecular mechanisms associated with dysfunction of Rab proteins in ALS. Finally, we also discuss dysfunction in other modes of transport recently implicated in ALS, including nucleocytoplasmic transport and the ER-mitochondrial contact regions (MAM compartment), and speculate whether these may also involve Rab GTPases.

ALS-associated mutant FUS inhibits macroautophagy which is restored by overexpression of Rab1.

Amyotrophic lateral sclerosis (ALS) is characterised by the formation of intracellular misfolded protein inclusions that form in motor neurons. Autophagy is the major degradation pathway for aggregate-prone proteins within lysosomes. Autophagy begins by the production of the omegasome, forming the autophagosome membrane, which then fuses with the lysosome. Mutations in fused in sarcoma (FUS) cause 5% of familial ALS cases and FUS-positive inclusions are also formed in sporadic ALS tissues. In this study, we demonstrate that the expression of ALS-associated mutant FUS impairs autophagy in neuronal cells. In mutant FUS-expressing neuronal cells, accumulation of ubiquitinated proteins and autophagy substrates p62 and NBR1 was detected, and formation of both the omegasome and autophagosome was inhibited in these cells. However, overexpression of Rab1 rescued these defects, suggesting that Rab1 is protective in ALS. The number of LC3-positive vesicles was also increased in motor neurons from the spinal cord of an ALS patient carrying a FUS (R521C) mutation compared with a control patient, providing additional evidence that autophagy is dysregulated in mutant FUS-associated ALS. This study provides further understanding of the intricate autophagy system and neurodegeneration in ALS.

The role of s-nitrosylation and s-glutathionylation of protein disulphide isomerase in protein misfolding and neurodegeneration.

Neurodegenerative diseases involve the progressive loss of neurons, and a pathological hallmark is the presence of abnormal inclusions containing misfolded proteins. Although the precise molecular mechanisms triggering neurodegeneration remain unclear, endoplasmic reticulum (ER) stress, elevated oxidative and nitrosative stress, and protein misfolding are important features in pathogenesis. Protein disulphide isomerase (PDI) is the prototype of a family of molecular chaperones and foldases upregulated during ER stress that are increasingly implicated in neurodegenerative diseases. PDI catalyzes the rearrangement and formation of disulphide bonds, thus facilitating protein folding, and in neurodegeneration may act to ameliorate the burden of protein misfolding. However, an aberrant posttranslational modification of PDI, S-nitrosylation, inhibits its protective function in these conditions. S-nitrosylation is a redox-mediated modification that regulates protein function by covalent addition of nitric oxide- (NO-) containing groups to cysteine residues. Here, we discuss the evidence for abnormal S-nitrosylation of PDI (SNO-PDI) in neurodegeneration and how this may be linked to another aberrant modification of PDI, S-glutathionylation. Understanding the role of aberrant S-nitrosylation/S-glutathionylation of PDI in the pathogenesis of neurodegenerative diseases may provide insights into novel therapeutic interventions in the future.

Refinement of the Region for Split Hand/Foot Malformation 5 on 2q31.1.

Background: Deletions that encompass 2q31.1 have been proposed as a microdeletion syndrome with common clinical features, including intellectual disability/developmental delay, microcephaly, cleft palate, growth delay, and hand/foot anomalies. In addition, several genes within this region have been proposed as candidates for split hand-foot malformation 5 (SHFM5). Methods: To delineate the genotype-phenotype correlation between deletions of this region, we identified 14 individuals with deletions at 2q31.1 detected by microarray analysis for physical and developmental disabilities. Results: All subjects for whom detailed clinical records were available had neurological deficits of varying degree. Seven subjects with deletions encompassing the HOXD cluster had hand/foot anomalies of varying severity, including syndactyly, brachydactyly, and ectrodactyly. Of 7 subjects with deletions proximal to the HOXD cluster, 5 of which encompassed DLX1/DLX2, none had clinically significant hand/foot anomalies. In contrast to previous reports, the individuals in our study did not display a characteristic gestalt of dysmorphic facial features. Conclusion: The absence of hand/foot anomalies in any of the individuals with deletions of DLX1/DLX2 but not the HOXD cluster supports the hypothesis that haploinsufficiency of the HOXD cluster, rather than DLX1/DLX2, accounts for the skeletal abnormalities in subjects with 2q31.1 microdeletions.

Time-dependence of nuclear magnetic resonance quadrupole interactions for polymers under shear.

We consider the problem of performing NMR spectroscopy under conditions of flow, a central issue in Rheo-NMR. By way of example, the effects of rotational motion on the deuterium NMR spectrum are considered for Couette cell experiments involving deformation of polymers under shearing conditions. The polymer was modelled as a power law fluid and for each streamline, the spin Hamiltonian evolved to allow for flow reorientation. The gap-integral spectra are compared with the 'ideal' spectra for a polymer under shear, but without reorientation. It is found that flow does affect the shape of the deuterium spectra, as well as slightly perturbing the splittings.

Imprinting status of 11p15 genes in Beckwith-Wiedemann syndrome patients with CDKN1C mutations.

Beckwith-Wiedemann syndrome (BWS) is an imprinting disorder characterized by somatic overgrowth, congenital malformations, and predisposition to childhood tumors. Aberrant expression of multiple imprinted genes, including H19, IGF2, KCNQ1OT1, and CDKN1C, has been observed in BWS patients. It has been estimated that mutations in CDKN1C occur in 12-17% of BWS patients. We have screened 10 autosomal dominant pedigrees and 65 sporadic BWS cases by PCR/heteroduplex analysis and DNA sequencing and have identified four mutations, two of which were associated with biallelic IGF2 expression and normal H19 and KCNQ1OT1 imprinting. One patient demonstrated phenotypic expression of paternally transmitted mutation in this maternally expressed gene, a second proband is the child of one of a pair of monozygotic twin females who carry the mutation de novo, and a third patient exhibited unusual skeletal changes more commonly found in other overgrowth syndromes. When considered with other studies published to date, this work reveals the frequency of CDKN1C mutations in BWS to be only 4.9%. This is the first report of an analysis of the imprinting status of genes in the 11p15 region where CDKN1C mutations were associated with loss of IGF2 imprinting and maintenance of H19 and KCNQ1OT1 imprinting.

District nurses and home carers 3: project evaluation.

This article, the last in a series of three, discusses the evaluation of a project established to provide a programme of education for social services' home carers that focused on the development of skills relating to care work. This education was delivered by district nurses (DNs) to address a number of problems associated with the provision of personal care identified by the home carers and their DN colleagues. An action research framework was used to improve collaborative working between the staff of the two disciplines. The article outlines some of the key findings from the evaluation of the project in relation to the structure, the process and the outcome of the project. The project evaluation was mainly positive and this success can be partly attributed to the fact that an action research method was used to drive the project.

Denture satisfaction and clinical performance in a paediatric population.

OBJECTIVES: The objectives of this study were to investigate the clinical performance of partial upper dentures in children and to assess reported denture satisfaction in this young population. DESIGN: This was a retrospective study using patients' dental records in conjunction with a postal questionnaire. SETTING: The Paediatric Dentistry Clinic, School of Clinical Dentistry, Sheffield. SAMPLE AND METHODS: Fifty-eight children (aged 7-17 years) who had been provided with a partial upper denture to replace one or more missing permanent incisors were included in the study. The patients' dental records were examined for the following details: tooth type missing, presence of retained incisor roots, aetiology of tooth absence, age at which the denture was first provided, total time period dentures had been worn, and frequency and type of any denture repairs or replacements. Next a short questionnaire was sent to each child, which sought an evaluation of several parameters of denture satisfaction including: overall attitude towards wearing a denture, denture appearance, ease of eating, comfort and perceived degree of teasing from peers. Visual analogue scales (VAS) were employed to provide a graded response. RESULTS: 70.7% of subjects had a single missing incisor and trauma was the most common cause of tooth loss (77.3%). The mean age at which patients started wearing a denture was 11.6 years and the mean length of time dentures had been worn, at the time of the investigation, was 2.1 years. A 'T-shaped' denture was most commonly employed (77.5% of dentures). Overall, 36.2% of subjects had required at least one denture repair, 28.2% presenting within a year of denture provision. One or more denture replacements had been received by 60.3% of the patients, and 43.6% had required this within a year. Component failure was highest for the denture tooth (40% of repairs) and a change in the patient's dentition necessitated a denture remake in 66% of cases. VAS scores for parameters of denture satisfaction indicated an overall positive evaluation of the dentures, with the most positive response relating to ease of eating. However, the reported degree of teasing was high and increased with time. The only significant difference for denture satisfaction according to gender was for mastication: girls reportedly found eating easier than did boys. CONCLUSION: Children are generally accepting of upper partial dentures, but their poor clinical performance is of concern and there is need for improvement.

First-trimester prenatal sonographic findings associated with OEIS (omphalocele-exstrophy-imperforate anus-spinal defects) complex: a case and review of the literature.

First-trimester sonographic findings associated with omphalocele-exstrophy-imperforate anus-spinal defects (OEIS) complex and review of the literature regarding this rare congenital anomaly are presented.

Structural requirements for assembly of dimeric IgA probed by site-directed mutagenesis of J chain and a cysteine residue of the alpha-chain CH2 domain.

The structural features of J chain required for interaction with IgA in IgA dimer assembly were investigated by coexpression of wild-type and mutant forms of J chain with IgA1 in CHO cells. With wild-type J chain, a mixture of J chain-containing dimers and monomers was secreted. Substitution of Cys14 of J chain with Ser resulted in expression of only monomer IgA covalently associated with J chain. Similarly, mutation of Cys68 to Ser also resulted in expression predominantly of a monomer IgA-J chain species. These results suggest that Cys14 and Cys68 play critical roles in formation of J chain-containing IgA dimers, with each forming a disulfide bridge to an IgA monomer. Substitution of Asn48 with Ala, to prevent attachment of N-linked carbohydrate to J chain, also resulted in markedly reduced dimer assembly, suggesting a requirement for the sugar moiety in J chain function. We also mutated Cys311 on the C alpha2 domain of the IgA heavy chain to Ser. When coexpressed with wild-type J chain, this mutant was still capable of forming dimers, indicating that this residue was not involved in dimerization. Taken together, our results are consistent with an arrangement in which IgA monomers are linked end-to-end with J chain interposed.

Mutagenesis of the human IgA1 heavy chain tailpiece that prevents dimer assembly.

The structural features of the human IgA1 tailpiece required for interaction with J chain in IgA dimer assembly were investigated using a protein engineering approach. Wild-type and mutant forms of IgA1 were expressed in the mouse myeloma cell line, J558L, which endogenously expresses J chain. Wild-type IgA1 was secreted as a mixture of dimers and monomers. Deletion of the entire tailpiece by stop codon introduction completely prevented dimer formation. Similarly, substitution of the penultimate residue of the tailpiece, Cys471, with serine resulted in the secretion of IgA monomers alone. Substitution of Asn459 with alanine to prevent attachment of N-linked carbohydrate to the tailpiece also resulted in markedly reduced dimer assembly. These results indicate the critical role played by the tailpiece, and Cys471 in particular, in IgA dimerization. In addition, we found tailpiece-deleted IgA1 and the Cys to Ser471 mutant IgA1 were secreted as mixtures of covalently associated monomers (alpha 2L2) and alpha L half-molecules. The tailpiece may thus play some role in promoting the association of alpha-chains required for IgA monomer assembly.

Alterations in glomerular permeability in streptozotocin-induced diabetic rats.

The alteration in glomerular basement membrane permeability associated with microangiopathy in streptozotocin-induced diabetic rats was studied by determining the movement across the glomerular basement membrane of anionic ferritin probes injected into rats at different points in the development of the disease. Visualization of the concentration gradient of anionic ferritin and changes in ultrastructure was accomplished by electron microscopic examination of renal tissue prepared from both control and diabetic rats. In all control rats, the anionic ferritin did not leave the glomerular capillary lumen, nor were there any changes in the normal morphology of the glomerular capillary wall. In the diabetic animals, the concentration of anionic ferritin shifted from the capillary lumen in the abluminal direction. Distinct morphologic changes, such as widening of endothelial intercellular junctions, focal detachment of podocyte foot processes, and extensive thickening of the glomerular basement membrane, were noted in the diabetic rat, and these changes appear to correlate with the observed increase in permselectivity of anionic ferritin across the glomerular capillary wall.

Purification and characterization of chimeric human IgA1 and IgA2 expressed in COS and Chinese hamster ovary cells.

Ag-specific chimeric human IgA molecules, of the two human subclasses, IgA1 and IgA2, have been expressed in two mammalian cell systems. Analysis of the secreted IgA molecules, purified in milligram quantities from stable Chinese hamster ovary transfectants by Ag affinity chromatography, has allowed a direct comparison of the biologic properties of the two subclasses. HPLC gel filtration analysis revealed that in both subclasses, the IgA molecules associate predominantly into dimers. The monomer units are presumed to interact noncovalently, inasmuch as no dimers are evident when the antibodies are subjected to SDS-PAGE. The recombinant antibodies are glycosylated, inasmuch as a lectin blotting procedure revealed that the H chains of both subclasses are recognized by Con A. When subjected to digestion by preparations of IgA1-specific proteases secreted by two pathogenic streptococcal strains, Streptococcus sanguis and Streptococcus oralis, the recombinant IgA molecules behave just as their natural equivalents. Thus, only the chimeric IgA1 molecule is cleaved, with the IgA2 remaining intact. In terms of interaction with natural effector molecules, both recombinant IgA isotypes were shown to interact with Fc alpha receptors on calcitriol-stimulated HL-60 cells with similar affinity, but neither antibody was found to interact with human C1q. The expression system described readily permits manipulation of the human IgA genes, which should lead to a fuller molecular understanding of how this important antibody mediates its function.

Recombinant human antibodies: linkage of an Fab fragment from a combinatorial library to an Fc fragment for expression in mammalian cell culture.

The combinatorial phage library approach to immunoglobulin repertoire cloning recently made it possible to isolate gene fragments encoding human immunoglobulin G1 Fabs binding with high affinity to specific antigens. Here we describe the construction of genes encoding whole human anti-tetanus toxoid antibodies based on one of these gene fragments and the efficient expression of these constructs by co-transfection of separate heavy and light chain vectors into a Chinese hamster ovary cell line constitutively expressing a viral transactivator protein. This system will be generally useful for the rapid analysis of recombinant antibodies derived from combinatorial libraries.

Prenatally detected fragile X females: long-term follow-up studies show high risk of mental impairment.

The prenatal detection of a positive fragile X [fra(X)] female raises difficult counseling issues. In order to address questions regarding the long term outlook, we have conducted follow-up studies on 4 fra(X) positive females which were carried to term. Three were prenatally detected, and one was a false negative. The subjects were between 3 and 7 years old when follow-up investigation of mental status was conducted. The first case age, 6 and 9/12 years, had an IQ of 106. On measures of achievement she had some difficulty with arithmetic. The second and third cases were clearly affected. They were judged to be mildly to moderately mentally retarded. The fourth case was borderline normal. The prenatal amniocentesis cytogenetic frequencies had a mean of 3.74% (range 0-8.5%). On postnatal follow-up testing of blood, the mean cytogenetic frequency increased to 31.75% (range 24-47%), an 8.5 fold increase. Follow-up DNA samples from 3 of the 4 subjects were analyzed for underlying DNA mutations using probe StB12.3 which detects insertions and methylation status of the FMR-1 gene. All 3 showed an affected female genotype with a large insert (greater than 500bp) and complete CpG island methylation. We conclude: (1) prenatally detected cytogenetic frequencies of females increase by an average 8.5 fold on follow-up postnatal studies, (2) genetic counseling should indicate the risks to be affected are approximately 75% when a positive female is prenatally detected, (3) DNA testing can help determine carrier status but may not accurately predict whether a female will be mentally affected.