Long-term effects of the titanium butterfly implant on quality of life: a prospective cohort study.

PURPOSE: Nasal valve insufficiency is known to have a negative impact on both nasal patency and quality of life. The titanium butterfly implant is a surgical treatment proven to have a positive effect on these aspects up to 6 months postoperative. This study aimed to determine the long-term effects of the titanium butterfly implant on nasal obstruction symptoms and quality of life in adult patients with nasal valve insufficiency up to 5 years after procedure. METHODS: A prospective single cohort study was performed including 29 patients that underwent the titanium butterfly implant in one tertiary medical center. Data was obtained before and at least 5 years after surgery using three questionnaires: the Nasal Obstruction and Septoplasty Effectiveness questionnaire, the Sino-Nasal Outcome Test 22 and the Glasgow Benefit Inventory questionnaire. RESULTS: A significant decrease in total NOSE score was seen compared to baseline measurements. The SNOT-22 scores also showed a significant decrease, whereas the GBI scores showed no significant changes at the late follow-up. CONCLUSION: Seven years after placement the titanium butterfly implant still has a statistically significant improvement on otorhinologic-related quality of life compared to preoperative measurements.

Costs of complications after colorectal cancer surgery in the Netherlands: Building the business case for hospitals.

BACKGROUND: Healthcare providers worldwide are struggling with rising costs while hospitals budgets are under stress. Colorectal cancer surgery is commonly performed, however it is associated with a disproportionate share of adverse events in general surgery. Since adverse events are associated with extra hospital costs it seems important to explicitly discuss the costs of complications and the risk factors for high-costs after colorectal surgery. METHODS: Retrospective analysis of clinical and financial outcomes after colorectal cancer surgery in 29 Dutch hospitals (6768 patients). Detailed clinical data was derived from the 2011-2012 population-based Dutch Surgical Colorectal Audit database. Costs were measured uniform in all participating hospitals and based on Time-Driven Activity-Based Costing. FINDINGS: Of total hospital costs in this study, 31% was spent on complications and the top 5% most expensive patients were accountable for 23% of hospitals budgets. Minor and severe complications were respectively associated with a 26% and 196% increase in costs as compared to patients without complications. Independent from other risk factors, ASA IV, double tumor, ASA III, short course preoperative radiotherapy and TNM-4 stadium disease were the top-5 attributors to high costs. CONCLUSIONS: This article shows that complications after colorectal cancer surgery are associated with a substantial increase in costs. Although not all surgical complications can be prevented, reducing complications will result in considerable cost savings. By providing a business case we show that investments made to develop targeted quality improvement programs will pay off eventually. Results based on this study should encourage healthcare providers to endorse quality improvement efforts.

Comparison of different strategies of referral to a fall clinic: how to achieve an optimal casemix?

UNLABELLED: OBJECTIVE To study the potential differences in patient characteristics between two referral methods to a fall clinic, specifically: case-finding of patients admitted to an emergency department because of a fall, compared to direct referral to the fall clinic via the general practitioner. DESIGN: Cross-sectional study. SETTING: Fall clinics in two university teaching hospitals in the Netherlands. PARTICIPANTS: Three hundred community-dwelling older people aged 65 years or over currently attending the fall clinics in Nijmegen (Group 1, n=154) and in Amsterdam (Group 2, n=146). MEASUREMENTS: Patients were referred by a general practitioner (Group 1) or were selected using the Carefall Triage Instrument (CTI) after visiting the emergency department (Group 2). In all patients, modifiable risk factors for recurrent falls were assessed. RESULTS: Group 1 had less modifiable risk factors for falling (a mean of 4 (SD 1.6) vs. a mean of 5 (SD 1.5) in Group 2, p < 0.001). Compared to Group 2, Group 1 had more prevalent " recurrent falling (≥ 2 falls)" (p=0.001) and "assisted living in homes for the aged" (p=0.037). "Fear of falling", "mobility and balance problems", "home hazards" and "osteoporosis" were significantly less prevalent in Group 1. CONCLUSION: This study suggests that patients referred to a multidisciplinary fall prevention clinic by their general practitioner have a different risk profile than those selected by case finding using the CTI. These differences have consequences for the reach of secondary care for fall-preventive interventions and will probably influence the effectiveness and efficiency of a fall prevention program.

Visualization of fine-scale genomic structure by oligonucleotide-based high-resolution FISH.

The discovery of complex structural variations that exist within individual genomes has prompted a need to visualize chromosomes at a higher resolution than previously possible. To address this concern, we established a robust, high-resolution fluorescence in situ hybridization (FISH) method that utilizes probes derived from high complexity libraries of long oligonucleotides (>150 mers) synthesized in massively parallel reactions. In silico selected oligonucleotides, targeted to only the most informative elements in 18 genomic regions of interest, eliminated the need for suppressive hybridization reagents. Because of the inherent flexibility in our probe design methods, we readily visualized regions as small as 6.7 kb with high specificity on human metaphase chromosomes, resulting in an overall success rate of 94%. Two-color FISH over a 479-kb duplication, initially reported as being identical in 2 individuals, revealed distinct 2-color patterns representing direct and inverted duplicons, demonstrating that visualization by high-resolution FISH provides further insight in the fine-scale complexity of genomic structures. The ability to design FISH probes for any sequenced genome along with the ease, reproducibility, and high level of accuracy of this technique suggests that it will be powerful for routine analysis of previously difficult genomic regions and structures.

Identification of proximal 1p36 deletions using array-CGH: a possible new syndrome.

Monosomy 1p36 is the most common terminal deletion syndrome with an estimated occurrence of 1:5000 live births. Typically, the deletions span <10 Mb of 1pter-1p36.23 and result in mental retardation, developmental delay, sensorineural hearing loss, seizures, cardiomyopathy and cardiovascular malformations, and distinct facies including large anterior fontanel, deep-set eyes, straight eyebrows, flat nasal bridge, asymmetric ears, and pointed chin. We report five patients with 'atypical' proximal interstitial deletions from 1p36.23-1p36.11 using array-comparative genomic hybridization. Four patients carry large overlapping deletions of approximately 9.38-14.69 Mb in size, and one patient carries a small 2.97 Mb deletion. Interestingly, these patients manifest many clinical characteristics that are different from those seen in 'classical' monosomy 1p36 syndrome. The clinical presentation in our patients included: pre- and post-natal growth deficiency (mostly post-natal), feeding difficulties, seizures, developmental delay, cardiovascular malformations, microcephaly, limb anomalies, and dysmorphic features including frontal and parietal bossing, abnormally shaped and posteriorly rotated ears, hypertelorism, arched eyebrows, and prominent and broad nose. Most children also displayed hirsutism. Based on the analysis of the clinical and molecular data from our patients and those reported in the literature, we suggest that this chromosomal abnormality may constitute yet another deletion syndrome distinct from the classical distal 1p36 deletion syndrome.

DNA exhibits multi-stranded binding recognition on glass microarrays.

In the course of exploring the hybridization properties of glass DNA microarrays, multi-stranded DNA structures were observed that could not be accounted for by classical Watson-Crick base pairing. Non-denatured double-stranded DNA array elements were shown to hybridize to single-stranded (ss)DNA probes. Similarly, ssDNA array elements were shown to bind duplex DNA probes. This led to a series of experiments demonstrating the formation of multi-stranded DNA structures on the surface of microarrays. These structures were observed with a number of heterogeneous sequences, including both purine and pyrimidine bases, with shared sequence identity between the ssDNA and one of the duplex strands. Furthermore, we observed a strong binding preference near the ends of duplexes containing a 3'-homologous strand. We suggest that such binding interactions on cationic solid surfaces could serve as a model for a number of biological processes mediated through multi-stranded DNA.

Immobilised metal-ion affinity chromatography purification of histidine-tagged recombinant proteins: a wash step with a low concentration of EDTA.

Immobilised metal-ion affinity chromatography (IMAC) is widely used for the purification of recombinant proteins in which a poly-histidine tag is introduced. However, other proteins may also bind to IMAC columns. We describe the use of a washing buffer with a low concentration of EDTA (0.5 mM) for the removal of proteins without histidine tag from IMAC columns. Four histidine-tagged recombinant proteins/protein complexes were purified to homogeneity from cell culture medium of insect cells by using an EDTA washing buffer. The presence of a low concentration of EDTA in washing buffers during IMAC may have a general application in the purification of histidine-tagged proteins.

Domains of glycoprotein H of herpes simplex virus type 1 involved in complex formation with glycoprotein L.

The complex formation between glycoproteins H (gH) and L (gL) of herpes simplex virus type 1 (HSV-1) was studied by using five recombinant baculoviruses expressing open reading frames that contain deletions in the coding region of the extracellular domain of gH. In addition, the gH-deletion mutants contained a C-terminal tag. Complex formation of gL and the gH-deletion mutants was studied by immunoprecipitations with anti-tag monoclonal antibody (MAb) A16 and with the gH-specific MAbs 37S, 46S, and 52S. All gH-deletion mutants were complexed to gL when analyzed by MAb A16. MAb 37S precipitated complexes between gL and the two gH-deletion mutants that contain the epitope of this MAb. When the gH conformation-dependent MAbs 46S and 52S were used, gL was coprecipitated together with the gH-deletion mutant lacking amino acids 31-299, but gL was not coprecipitated with the gH-deletion mutant lacking amino acids 31-473. The data from the precipitation studies do allow at least two interpretations. There is either one site for gL binding on gH (residue 300-473) or gL contacts multiple regions of gH. We were unable to demonstrate gL-dependent cell surface expression of either of the gH-deletion mutants. This suggests that the coassociation of gH with gL is necessary but not sufficient for transport of gH to the cell surface.

Glycoprotein H of herpes simplex virus type 1 requires glycoprotein L for transport to the surfaces of insect cells.

In mammalian cells, formation of heterooligomers consisting of the glycoproteins H and L (gH and gL) of herpes simplex virus type 1 is essential for the cell-to-cell spread of virions and for the penetration of virions into cells. We examined whether formation of gH1/gL1 heterooligomers and cell surface expression of the complex occurs in insect cells. Three recombinant baculoviruses, expressing gL1, gH1, and truncated gH1 (gH1t), which lacks the transmembrane region, were constructed. It was shown that recombinant gH1/gL1 and gH1t/gL1 heterooligomers were produced in insect cells. As in mammalian cells, gH1 and gH1t were not detected on the surfaces of insect cells in the absence of gL1. When coexpressed with gL1, recombinant gH1 was displayed on the surfaces of insect cells. Coexpression of gH1t and gL1 resulted in secretion of the gH1t/gL1 complex into the cell culture medium, indicating that gH1t is also transported to the surfaces of insect cells. Our results indicate that the process of folding and intracellular transport of gH1 and gL1 is comparable in insect cells and mammalian cells and that the baculovirus expression system can be used to examine the complex formation and the intracellular transport of gH1 and gL1. The availability of secreted gH1t/gL1 complex offers the opportunity to further investigate the immunological properties of this complex.

Kinetic analysis of synthetic analogues of linear-epitope peptides of glycoprotein D of herpes simplex virus type 1 by surface plasmon resonance.

The interaction between mAb A16 and glycoprotein D (gD) of herpes simplex virus type 1 was analyzed by studying the kinetics of binding with a surface-plasmon-resonance biosensor. mAb A16 belongs to group VII antibodies, which recognize residues 11-19 of gD. In a previous study, three critical residues, Asp13, Arg16 and Phe17, of this epitope were identified by screening a phage display library that contained a random 15-amino-acid insert with the antibody. The contribution to binding of these residues in the motif DXXRF was further analyzed by an amino-acid-replacement study of the epitope gD-(9-19)-peptide and of a gD-(9-19)-peptide mimotope, previously obtained by screening the phage display library. Amino acid residues of the motif were replaced by a neutral amino acid residue, an amino acid residue with opposite charge and a corresponding D-amino acid residue. Kinetic parameters of peptide analogues were determined with a surface plasmon-resonance biosensor. The kinetic parameters of the peptide analogues were compared with the kinetic parameters of the interaction between mAb A16 and the epitope gD-(9-19)-peptide. The minimal size of the gD epitope for mAb A16 was also determined in this study. The kinetic constants of the resulting gD-(11-17)-peptide were found to be similar to those of entire gD. The kinetic analysis precisely defined the epitope on gD for mAb A16 to residues 11-17, identified Arg16 as an essential residue and suggested that Asp13 and Phe17 are mainly involved in stabilization of the secondary structure of the peptide.

Production and characterization of monoclonal antibodies against Panulirus interruptus hemocyanin.

Since the primary and higher-order structures of hemocyanin from the crustacean arthropod Panulirus interruptus have been elucidated completely, it should be possible to determine which regions of this immunogenic molecule are recognized most often by antibodies. Monoclonal antibodies were raised against subunits a and b of this hemocyanin, and fourteen of them were further characterized. The produced antibodies were of class IgG, subclasses 1 or 2a. Most of them had dissociation constants on the order of magnitude 10(-8)-10(-10), a few had lower affinities. Most clones showed no or negligible cross-reactivity with other crustacean hemocyanins. The reactivity of most other clones diminished with increasing sequence difference between the investigated hemocyanins. However, in a few instances a stronger reactivity with other hemocyanins was observed than with that from Panulirus interruptus. After complete denaturation of the hemocyanin there was no reaction with the monoclonal antibodies, indicating that the latter recognize conformational epitopes. Only one monoclonal antibody reacted with denatured hemocyanin. This antibody was also the only one which reacted with a CNBr digest, which means that it recognizes a sequential epitope. Several antibodies showed a faint reaction on Western blots, indicating the presence of some refolded native structure. Limited proteolysis of the hemocyanin molecule results in the formation of a 18 kDa fragment, representing domain 1, and a 55 kDa fragment representing domains 2 and 3. It was determined on Western blots of the digest on which fragment epitopes for eleven of the monoclonal antibodies were located.

Identification of the core residues of the epitope of a monoclonal antibody raised against glycoprotein D of herpes simplex virus type 1 by screening of a random peptide library.

Random peptide libraries (RPL) displayed on the surface of a filamentous bacteriophage can be used to identify peptide ligands that interact with target molecules. We have used a 15-amino acid residue RPL displayed on bacteriophage M13 to identify the core residues within the epitope of a monoclonal antibody (mAb) A16 which interacts with a continuous epitope restricted to amino acid residues 9 to 19 in the N-terminal region of glycoprotein D of herpes simplex virus type 1 (gD-1). The single peptide sequence obtained after three rounds of selection contained identical residues at three positions compared to the authentic gD-1 sequence. Synthetic peptides were prepared based on the sequence of the original epitope and the phage-derived epitope. The binding constants (Ka) with mAb A16 were determined using surface plasmon resonance (SPR) biosensor technology. The RPL-derived peptide and peptide 9-19 of gD-1 had approximately the same affinity for mAb A16. This suggests that those residues within the epitope that are essential for binding were identified. The synthesis of shorter versions of the RPL-derived peptide restricted the binding region to seven amino acid residues. These results show that minimal information retrieved from the screening of an RPL combined with peptide synthesis can characterize the epitope of an mAb with high resolution. Immunization of mice with the phage-derived peptide protected against a challenge with a lethal dose of herpes simplex virus type 1 equally well as the gD-1 derived peptide.

Determination of enzyme activity by high-performance liquid chromatography.

The application of high-performance liquid chromatography (HPLC) in the study of enzymatic reactions is reviewed. The rationale for using HPLC is given and whether the components of the reaction mixture should be derivatized prior to or after HPLC. An alphabetical list of enzymes assayed by HPLC is given. Substrate and product are included as well the derivatization reagent, detection method and biological matrix. Specific examples of these assays in a complex biological matrix viz. faeces are given. Future prospects are the detection of new enzymes using synthetic substrates and implementation of mass spectrometry to elucidate enzyme specificities.

Immunochemical detection of adducts of sulfur mustard to DNA of calf thymus and human white blood cells.

As part of a program to develop methods for dosimetry of exposure to sulfur mustard, we developed immunochemical methods for the detection of the major adduct, N7-[2-[(hydroxyethyl)thio]ethyl]guanine (N7-HETE-Gua), formed after alkylation of DNA with sulfur mustard. After immunization of rabbits with calf thymus DNA treated with sulfur mustard, we obtained the antiserum W7/10 with a high specificity for DNA adducts of sulfur mustard. With this serum, a competitive enzyme-linked immunosorbent assay was developed in which sulfur mustard adducts to DNA could be detected with a minimum detectable amount of 1-5 fmol per well and a selectivity that allows detection of one N7-HETE-Gua among 5 x 10(6) unmodified nucleotides in single-stranded DNA. The complications that arise to isolate double-stranded DNA from biological samples and to make the DNA single-stranded without destruction of the sulfur mustard adducts result in about a 20-fold higher limit for adduct detection in DNA from human blood than in single-stranded DNA. Presently, adducts in white blood cells can be detected after exposure of human blood to sulfur mustard concentrations > or = 2 microM. We synthesized N7-HETE-GMP for use as a hapten to generate monoclonal antibodies against this adduct. After immunization of mice with this adduct coupled to the carrier protein keyhole limpet hemocyanin we obtained several hybridomas producing monoclonal antibodies that recognize N7-HETE-Gua, containing an intact imidazolium ring. The sensitivity of the competitive ELISA with the monoclonal antibodies was comparable to that of the assays performed with the rabbit antiserum.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis, characterization, and quantitation of the major adducts formed between sulfur mustard and DNA of calf thymus and human blood.

As part of a program to develop methods for verification of alleged exposure to sulfur mustard, we synthesized and characterized the adducts most likely formed by alkylation of DNA with sulfur mustard: N7-[2-[(2-hydroxyethyl)thio]ethyl]guanine (1), bis[2-(guanin-7-yl)ethyl] sulfide (2), N3-[2-[(2-hydroxyethyl)thio]ethyl]adenine (3), and O6-[2-[(2-hydroxyethyl)thio]ethyl]-guanine and its 2'-deoxyguanosine derivative. Incubation of double-stranded calf thymus DNA and human blood with [35S]sulfur mustard in vitro followed by enzymatic degradation of the DNA and mild depurination afforded three major radioactive peaks upon HPLC analysis. These peaks were identified as 1-3 by coelution with the synthetic markers and mass spectrometric and electronic spectra. Compound 1 appeared to be the most abundant adduct, which is in agreement with previous investigations on DNA alkylation with sulfur mustard.

Analogues of peptide 9-21 of glycoprotein D of herpes simplex virus and their binding to group VII monoclonal antibodies.

Several analogues of the amino acid sequence of peptide 9-21 of glycoprotein D of herpes simplex virus type 1 (HSV-1) were synthesized and investigated for reactivity with different group VII monoclonal antibodies, Mabs LP14, ID3, 170, HD4, A16, EII-24 and Ev-10, in a competition enzyme-linked immunosorbent assay (ELISA). Replacement of Arg at position 16 by His resulted in a loss of binding with the group VII Mabs. Substitution of Pro at residue 14 by Leu gave a reduced binding for a number of Mabs and loss of binding for Mab A16. Substitution of Lys at position 10 by Glu gave reduced binding for three out of the seven Mabs. In addition substitutions of Met at position 11 by norleucine and oxidized Met were studied. The boundaries of the epitope cluster were mapped by studying synthetic variants of peptide 9-21 which were shorter either at the C-terminus or at the N-terminus, or both. Peptide 10-18 and peptide 9-17 were the shortest peptides, which were still reactive with the group VII Mabs. Mab HD4 requires the N-terminus of peptide 9-21 for effective binding, while for binding of other Mabs contribution of the residues in the C-terminal part of this peptide is more important.

T cell responses to synthetic peptides of herpes simplex virus type 1 glycoprotein D in naturally infected individuals.

To locate T cell determinants of glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1), proliferation assays of lymphocytes obtained from 10 healthy HSV-seropositive individuals were performed using 34 overlapping gD peptides as antigens. Despite large differences between individual responses to the peptides both in number of stimulating peptides and gD regions, three regions (1-54, 110-214, and 290-314) induced a response in 50% or more of the HSV-seropositives. T cells were less frequently stimulated by peptides of region 210-294. No correlation was found between serological data and proliferative responses to the peptides. The diversity in T cell response to the peptides suggests a lack of immunodominance, implying that a single peptide/region of gD, or a combination of peptides, will not be sufficient to serve as a basis for a future HSV-1 vaccine.

Thermal denaturation of plastocyanin: the effect of oxidation state, reductants, and anaerobicity.

The thermal stability of plastocyanin (PC) was determined as a function of oxidation state of the copper center and the presence of oxidants, reductants, oxygen, and EDTA. It was found that the copper center and its ligands play a crucial role in maintaining the stability of PC. Thermal denaturation was monitored by using far-uv circular dichroism (CD) spectra to monitor changes in secondary structure, the near-uv CD ellipticity at 280 nm to monitor changes in tertiary structure, and the absorbance at 597 nm and the 255-nm CD transition to monitor changes in the copper center. Reduced PC (Tm = 71 degrees C) was found to be more stable than the oxidized form (Tm = 61 degrees C). The Tm was increased by addition of reductants, removal of oxygen, or addition of EDTA. Two distinct denatured forms (designated D1 and D2) were separated by anion exchange fast protein liquid chromatography. Neither form contained a native copper center. Form D2 retained the characteristic 280-nm CD band but showed an altered far-uv CD spectrum. Its formation was inhibited by the addition of reductants or the removal of oxygen. It could be refolded to form native, Cu-PC upon incubation with copper plus a reductant such as dithionite. These results suggest that its formation involves the reversible oxidation of a group on the PC molecule, possibly a ligand to the copper such as Cys 84 or Met 92. Form D1 occurred in the presence of ferricyanide or at high temperatures in the presence of oxygen. EDTA inhibited its formation. Form D1 lost the 280-nm CD transition and its far-uv CD spectrum was altered. No renaturation was observed suggesting that Form D1 is the product of an irreversible oxidation step possibly involving a histidine ligand to the copper. Forms D1 and D2 are not interconvertible and represent the endpoints of two different denaturation pathways.