Data-driven directions for effective footwear provision for the high-risk diabetic foot.

AIMS: Custom-made footwear is used to offload the diabetic foot to prevent plantar foot ulcers. This prospective study evaluates the offloading effects of modifying custom-made footwear and aims to provide data-driven directions for the provision of effectively offloading footwear in clinical practice. METHODS: Eighty-five people with diabetic neuropathy and a recently healed plantar foot ulcer, who participated in a clinical trial on footwear effectiveness, had their custom-made footwear evaluated with in-shoe plantar pressure measurements at three-monthly intervals. Footwear was modified when peak pressure was ≥ 200 kPa. The effect of single and combined footwear modifications on in-shoe peak pressure at these high-pressure target locations was assessed. RESULTS: All footwear modifications significantly reduced peak pressure at the target locations compared with pre-modification levels (range -6.7% to -24.0%, P < 0.001). The metatarsal heads were most frequently targeted. Repositioning an existing (trans-)metatarsal pad in the shoe insole (-15.9% peak pressure relief), applying local cushioning to the insole (-15.0%) and replacing the insole top cover with Plastazote (-14.2%) were the most effective single modifications. Combining a new Plastazote top cover with a trans-metatarsal bar (-24.0% peak pressure relief) or with local cushioning (-22.0%) were the most effective combined modifications. CONCLUSIONS: In people with diabetic neuropathy and a recently healed plantar foot ulcer, significant offloading can be achieved at high-risk foot regions by modifying custom-made footwear. These results provide data-driven directions for the design and evaluation of custom-made footwear for high-risk people with diabetes, and essentially mean that each shoe prescribed should incorporate those design features that effectively offload the foot.

Collective protein dynamics in relation to function.

Several techniques for the analysis of the internal motions of proteins are available - separating large collective motions from small, presumably uninteresting motions. Such descriptions are helpful in the characterization of internal motions and provide insight into the energy landscape of proteins. The real challenge, however, is to relate large collective motions to functional properties, such as binding and regulation, or to folding. These issues have been recently addressed in several papers.

Resistance mechanisms in three human small cell lung cancer cell lines established from one patient during clinical follow-up.

Mechanisms for resistance were studied in three classic type, human small cell lung cancer cell lines, GLC14, GLC16, and GLC19, that were established from one patient during clinical follow-up. Clinically the tumor changed from sensitive (GLC14) to completely resistant to (chemo)therapy (GLC19) during this period. The stain with JSB-1 antibody, detecting the Mr 170,000 multidrug resistance associated glycoprotein, was most pronounced in GLC16 and absent in GLC19. Intracellular Adriamycin (Adr) concentrations were decreased in GLC16 and GLC19 versus GLC14. Glutathione levels were 12.9, 15.5, and 16.6 micrograms/mg protein; total sulfhydryl groups were 36.5, 45.7, and 48.8 micrograms/mg protein; and glutathione S-transferase activity was 13, 29, and 43 nmol I-chloro-2,4-dinitrobenzene/min/mg protein for GLC14, GLC16, and GLC19, respectively. Incubation with DL-buthionine-S,R-sulfoximine increased Adr and cisplatin induced cytotoxicity, whereas X-ray induced cytotoxicity remained the same. Catalase activity increased from 0.88 to 1.73 to 3.83 mumol H2O2/min/mg protein in, respectively, GLC14, GLC16, and GLC19. Compared to GLC14 and GLC16, Adr induced a higher amount of DNA strand breaks in GLC19. In none of the three cell lines could Adr induced DNA strand breaks be repaired. X-ray induced a comparable amount of DNA strand breaks in all three cell lines but all cell lines were capable of repairing the X-ray induced DNA strand breaks within 90 min. It is concluded that a number of different mechanisms are operative and that some but not all of the observed changes in mechanisms for drug resistance in these lines correlate with the clinical data.

Characterization of three small cell lung cancer cell lines established from one patient during longitudinal follow-up.

Three classic-type, small cell lung cancer cell lines (GLC-14, GLC-16, and GLC-19) have been established from one patient during longitudinal follow-up. During this period the tumor changed from sensitive to completely resistant to (chemo)therapy. A phenotypical and functional characterization of the different cell lines is given in combination with the matching clinical data. (a) The cell lines have been compared with the biopsies from which they were derived. There was a good match between the morphological, biochemical, and immunohistological findings in the cell lines as compared to those obtained in the biopsies. When the biopsy and cell line (GLC-14) obtained before the start of therapy were compared to the biopsies and cell lines (GLC-16 and GLC-19) acquired after the first and second reinduction therapy, respectively, no major changes could be observed. The only clear alteration was the loss of a neuroendocrine antigen (defined by monoclonal antibody MOC-51) in the posttherapy specimens. (b) The doxorubicin, melphalan, and etoposide sensitivity in vitro reflected the clinically observed development of resistance to treatment. The cell line (GLC-14) established before the start of therapy was more sensitive than the lines (GLC-16 and GLC-19) obtained after treatment. It is concluded that the cell lines described in this paper represent a well-characterized in vitro model in which the development of drug resistance in small cell lung cancer can be studied.

Molecular dynamics simulation of a phospholipase A2-substrate complex.

We have used knowledge of the three-dimensional structure of phospholipids and phospholipases A2 together with biochemical data, computer graphics modelling and a 48 ps molecular dynamics simulation to predict the structure of a phospholipase A2-substrate complex. There is remarkable similarity between this predicted structure of enzyme-substrate complex and the structure that can be deduced from the observed enzyme-inhibitor complex. Molecular dynamics simulation highlights the importance of the calcium-ion in substrate binding and the persistence of the His-48 to water-hydrogen bond is compatible with the proposed role of this water molecule as the nucleophile in catalysis.

Clinical characterization of non-small-cell lung cancer tumors showing neuroendocrine differentiation features.

In most cases of small-cell lung carcinomas (SCLC) phenotypic features compatible with a neuroendocrine differentiation status can be identified by monoclonal (MOC) antibody-based immunohistological procedures. Similar features can be recognized only in a minority of non-SCLC tumors. During a period of 30 months, all diagnostic non-SCLC biopsies (141 cases) were prospectively analysed for the presence of markers indicative for neuroendocrine differentiation. In 31% of all cases, such a presence could be noticed. Neuroendocrine differentiation (50% to 100% positive-staining tumor cells) was recognized more frequently in adenocarcinoma when compared to large-cell and squamous-cell carcinoma (chi 2 = 9.31, 2 degrees of freedom, P less than 0.01). To investigate whether the clinical behavior of these "neuroendocrine" non-SCLC cases mimics SCLC, a multivariate analysis for prognostic factors was performed. Among other prognostic factors, biopsies containing more than 50% positive-staining tumor cells with the MOC antibody-1 (MOC-1) were recognized as negative prognostic factors.

Interactions between 5-hydroxytryptamine receptor subtypes: is a disturbed receptor balance contributing to the symptomatology of depression in humans?

The purpose of this review is to describe the consequences of antidepressant treatment on the behaviour of rodents after activation of serotonin (5-hydroxytryptamine, 5-HT) receptor subtypes. In a summary table, the involvement of 5-HT receptors in inducing behavioural changes are described. It is emphasized that these effects are not always only exclusively linked to serotonergic functions nor that they are only initiated by central 5-HT receptors. Hereafter, the complex mutual inhibitory effects of 5-HT receptor subtype-mediated processes are discussed by interpreting effects of antagonists and describing the different effects of low and high doses of mixed 5-HT1C/5-HT2 receptor agonists. Mutual influences are seen particularly with 5-HT1A, 5-HT1C and 5-HT2, but not with 5-HT1B, 5-HT1D or 5-HT3 receptor-mediated effects. It is shown that the behavioural consequences of 5-HT1A, 5-HT1C and 5-HT2 receptor stimulation may be changed by brain lesions or chronic treatment with drugs. Among these drugs are the antidepressants. Finally, 5-HT receptor function in depressed patients is discussed, and the hypothesis is proposed that an important function of antidepressants is to restore a disturbed balance between 5-HT1A, 5-HT1C and 5-HT2 receptors in depressed patients.

Computer simulation as a tool for tracing the conformational differences between proteins in solution and in the crystalline state.

Knowledge about the architecture of macromolecules has been derived primarily from crystallography. Therefore, it has been a matter of concern whether the conformation of a macromolecule in solution, namely in vivo, might be different from that in the crystalline state. To determine the difference between the conformations, a protein (trypsin inhibitor) dissolved in water has been simulated using the method of molecular dynamics and the results are compared with those obtained from a simulation of the full crystalline unit cell. We report here that no significant difference was found for backbone atoms, except for two more or less flexible loops extending from the core of the protein and the very flexible carboxyterminal residues. The side-chains in which the conformation in solution differs considerably from that in the crystal all belong to polar residues.

Conformational changes in the chaperonin GroEL: new insights into the allosteric mechanism.

Conformational changes are known to play a crucial role in the function of the bacterial GroE chaperonin system. Here, results are presented from an essential dynamics analysis of known experimental structures and from computer simulations of GroEL using the CONCOORD method. The results indicate a possible direct form of inter-ring communication associated with internal fluctuations in the nucleotide-binding domains upon nucleotide and GroES binding that are involved in the allosteric mechanism of GroEL. At the level of conformational transitions in entire GroEL rings, nucleotide-induced structural changes were found to be distinct and in principle uncoupled from changes occurring upon GroES binding. However, a coupling is found between nucleotide-induced conformational changes and GroES-mediated transitions, but only in simulations of GroEL double rings, and not in simulations of single rings. This provides another explanation for the fact that GroEL functions a double ring system.

Dynamical structure of carboxypeptidase A.

Structural fluctuations of the apoenzyme form of carboxypeptidase A (EC 3.4.12.2) have been evaluated on the basis of molecular dynamics. The Konnert-Hendrickson refined coordinates of 2437 non-hydrogen atoms of the 307 amino acid residues derived from the X-ray structure of the holoenzyme served as the molecular model together with 548 calculated polar hydrogen atoms and 25 buried solvent molecules. Molecular dynamics simulations were carried out at 277 K, and the averaged structural properties of the protein were evaluated for the terminal 20 picosecond portion of a 48 picosecond trajectory. The average atomic displacement from the initial X-ray structure was 2.49 A for all atoms and 1.79 A for C alpha atoms. The average root-mean-square (r.m.s.) fluctuation of all atoms was 0.67 A as compared to 0.54 A evaluated from the X-ray-defined temperature factors. Corresponding r.m.s. fluctuations for backbone atoms were 0.56 A by molecular dynamics and 0.49 A by X-ray. On the basis of these molecular dynamics studies of the isolated molecule, it is shown that amino acid residues corresponding to intermolecular contact sites of the crystalline enzyme are associated with high amplitude motion. All eight segments of alpha-helix and eight regions of beta-strand were well preserved except for unwinding of the five C-terminal residues of the alpha-helix 112-122 that form part of an intermolecular contact in the crystal. Four regions of beta-strand and one alpha-helix with residues adjacent to or in the active site constitute a core of constant secondary structure and are shown not to change in relative orientation to each other during the course of the trajectory. The absence of the zinc ion does not markedly influence the stereochemical relationships of active site residues in the dynamically averaged protein. The extent of motional fluctuations of each of the subsites of substrate recognition in the active site has been evaluated. Active site residues responsible for specificity of substrate binding or splitting of the scissile bond exhibit low simulated motion. In contrast, residues in more distal sites of substrate recognition exhibit markedly greater motional fluctuations. This differential extent of dynamical motion is related to structural requirements of substrate hydrolysis.

A structure refinement method based on molecular dynamics in four spatial dimensions.

We have developed a method for structure refinement based on molecular dynamics in four spatial dimensions (4D-MD). The method was applied with success to the structure refinement of Cyclosporin A (CPA) and the lac-repressor headpiece (LAC) using atom-atom distance restraints derived from NMR data, two cases where conventional MD refinement methods failed. In the case of CPA we were able to refine seven out of nine substantially different structures, while conventional MD only refines three structures. Previously, it had appeared to be impossible to refine a given LAC structure with conventional MD methods, manual modifications were necessary in order to fulfil all the distance restraints. In this study we show that LAC can be refined without manual interference and using only 5000 steps (10 ps) of 4D-MD. The latter result is particularly interesting because it indicates that this method may be a very useful tool when modelling loops in proteins.

The high-resolution structure of the histidine-containing phosphocarrier protein HPr from Escherichia coli determined by restrained molecular dynamics from nuclear magnetic resonance nuclear Overhauser effect data.

The solution structure of the histidine-containing phosphocarrier protein HPr from Escherichia coli has been determined by NMR in combination with distance geometry and restrained molecular dynamics. The structure is based on 1520 experimental restraints identified from both three-dimensional 1H-1H-13C and 1H-1H-15N nuclear Overhauser effect multiple-quantum coherence spectroscopy and two-dimensional 1H-1H nuclear Overhauser effect spectra. Thirty-two four-dimensional coordinate frames were produced by metric matrix distance geometry, subjected to distance bounds driven dynamics, projected into three-dimensional space and again subjected to distance-bounds driven dynamics. These 32 distance geometry structures were refined further by restrained molecular dynamics (40 ps) in the GROMOS in vacuo force field. All 32 structures reached acceptable energy minima while satisfying the imposed restraints. Two of these structures were subjected to a further 200 ps of molecular dynamics simulation in water, using time-dependent distance restraining, followed by a 200 ps free simulation without any distance restraining. The resulting structure is very similar to the X-ray structure of Bacillus subtilis HPr, but differs mainly in the position of the two loops containing the active site histidine residue 15 and residues 53 to 57 relative to the rest of the structure. The unfavorable phi torsion angle that was found for residue 16 in the active center of unphosphorylated Streptococcus faecalis HPr was proposed to play a role in the activity of the protein. Although present at the early stages of the structure calculations, this torsion-angle strain disappeared in the final model obtained from molecular dynamics simulations in water using time-averaged distance restraining and upon releasing the distance restraints. This suggests that the strain may be an artifact of crystallization conditions instead of an essential element in the phosphorylation/dephosphorylation process.

Benchmark of Schemes for Multiscale Molecular Dynamics Simulations.

In multiscale molecular dynamics simulations the accuracy of detailed models is combined with the efficiency of a reduced representation. For several applications - namely those of sampling enhancement - it is desirable to combine fine-grained (FG) and coarse-grained (CG) approaches into a single hybrid approach with an adjustable mixing parameter. We present a benchmark of three algorithms that use a mixing of the two representation layers using a Lagrangian formalism. The three algorithms use three different approaches for keeping the particles at the FG level of representation together: 1) addition of forces, 2) mass scaling, and 3) temperature scaling. The benchmark is applied to liquid hexadecane and includes an evaluation of the average configurational entropy of the FG and CG subsystems. The temperature-scaling scheme achieved a 3-fold sampling speedup with little deviation of FG properties. The addition-of-forces scheme kept FG properties the best but provided little sampling speedup. The mass-scaling scheme yielded a 5-fold speedup but deviated the most from FG properties.

A molecular dynamics study of the 41-56 beta-hairpin from B1 domain of protein G.

The structural and dynamical behavior of the 41-56 beta-hairpin from the protein G B1 domain (GB1) has been studied at different temperatures using molecular dynamics (MD) simulations in an aqueous environment. The purpose of these simulations is to establish the stability of this hairpin in view of its possible role as a nucleation site for protein folding. The conformation of the peptide in the crystallographic structure of the protein GB1 (native conformation) was lost in all simulations. The new equilibrium conformations are stable for several nanoseconds at 300K (>10 ns), 350 K (>6.5 ns), and even at 450 K (up to 2.5 ns). The new structures have very similar hairpin-like conformations with properties in agreement with available experimental nuclear Overhauser effect (NOE) data. The stability of the structure in the hydrophobic core region during the simulations is consistent with the experimental data and provides further evidence for the role played by hydrophobic interactions in hairpin structures. Essential dynamics analysis shows that the dynamics of the peptide at different temperatures spans basically the same essential subspace. The main equilibrium motions in this subspace involve large fluctuations of the residues in the turn and ends regions. Of the six interchain hydrogen bonds, the inner four remain stable during the simulations. The space spanned by the first two eigenvectors, as sampled at 450 K, includes almost all of the 47 different hairpin structures found in the database. Finally, analysis of the hydration of the 300 K average conformations shows that the hydration sites observed in the native conformation are still well hydrated in the equilibrium MD ensemble.

Bending of the calmodulin central helix: a theoretical study.

The crystal structure of calcium-calmodulin (CaM) reveals a protein with a typical dumbbell structure. Various spectroscopic studies have suggested that the central linker region of CaM, which is alpha-helical in the crystal structure, is flexible in solution. In particular, NMR studies have indicated the presence of a flexible backbone between residues Lys 77 and Asp 80. This flexibility is related directly to the function of the protein because it enables the N- and C-terminal domains of the protein to move toward each other and bind to the CaM-binding domain of a target protein. We have investigated the flexibility of the CaM central helix by a variety of computational techniques: molecular dynamics (MD) simulations, normal mode analysis (NMA), and essential dynamics (ED) analysis. Our MD results reproduce the experimentally determined location of the bend in a simulation of only the CaM central helix, indicating that the bending point is an intrinsic property of the alpha-helix, for which the remainder of the protein is not important. Interestingly, the modes found by the ED analysis of the MD trajectory are very similar to the lowest frequency modes from the NM analysis and to modes found by an ED analysis of different structures in a set of NMR structures. Electrostatic interactions involving residues Arg 74 and Asp 80 seem to be important for these bending motions and unfolding, which is in line with pH-dependent NMR and CD studies.

Completion and refinement of 3-D homology models with restricted molecular dynamics: application to targets 47, 58, and 111 in the CASP modeling competition and posterior analysis.

A method is presented to refine models built by homology by the use of restricted molecular dynamics (MD) techniques. The basic idea behind this method is the use of structure validation software to determine for each residue the likelihood that it is modeled correctly. This information is used to determine constraints and restraints in an MD simulation including explicit solvent molecules, which is used for model refinement. The procedure is based on the idea that residues that the validation software identifies as correctly positioned should be strongly constrained or restrained in the MD simulations, whereas residues that are likely to be positioned wrongly should move freely. Two different protocols are compared: one (applied to CASP3 target T58) using full structural constraints with separate optimization of each short fragment and the other (applied to T47) allowing some freedom using harmonic restraining potentials, with automatic optimization of the whole molecule. Structures along the MD trajectory that scored best in structural checks were selected for the construction of models that appeared to be successful in the CASP3 competition. Model refinement with MD in general leads to a model that is less like the experimental structure (Levitt et al. Nature Struct Biol 1999;6:108-111). Actually, refined T47 was slightly improved compared to the starting model; changes in model T58 led not to further enhancement. After the X-ray structure of the modeled proteins became known, the procedure was evaluated for two targets (T47 and the CASP4 target T111) by comparing a long simulation in water with the experimental target structures. It was found that structural improvements could be obtained on a nanosecond time scale by allowing appropriate freedom in the simulation. Structural checks applied to fast fluctuations do not appear to be informative for the correctness of the structure. However, both a simple hydrogen bond count and a simple compactness measure, if averaged over times of typically 300 ps, correlate well with structural correctness and we suggest that criteria based on these properties may be used in computational folding strategies.

Single-agent oral etoposide for elderly small cell lung cancer patients.

Among new patients with small cell lung cancer (SCLC), 20% to 25% are over 70 years of age and account for 8,000 cases annually in the United States. For such patients, intensive, aggressive, cytotoxic, combination chemotherapy is not often used because of its association with unacceptable morbidity and mortality rates. However, treatment of these patients is often indicated, if not demanded. Etoposide is among the most active agents for the treatment of SCLC; both oral (PO) and intravenous preparations are available. The bioavailability of etoposide PO ranges from 15% to 75%. We carried out a phase II trial of etoposide PO in elderly patients (greater than or equal to 70 years of age) with a confirmed histologic diagnosis of SCLC. Patients were treated with etoposide capsules 800 mg/m2, with the total dose divided over 5 consecutive days. Cycles were repeated every 3 to 4 weeks for a total of six cycles. All treatment was administered on an outpatient basis. Staging procedures were confined to physical examination, chest x-ray, and laboratory investigations. Further staging procedures were carried out only if clinically indicated. In September 1985, 53 patients (35 male, 18 female) with a median age of 73 years (range, 70 to 95) were treated. After staging, 24 patients (45%) had limited disease (LD); the remaining 29 patients had extensive disease (ED). Overall response was 79% (complete response [CR], 17%; partial response, 62%). CRs were seen in 8 of 24 patients (33%) with LD and in 1 of 29 patients (3%) with ED. Median survival of all patients was 9.5 months (range, 1 to 22+) with 20 patients still alive; the toxicity rate was acceptable. Total alopecia developed in all patients, whereas significant neutropenia (8%) and thrombocytopenia (6%) were rare. There were no treatment-related deaths or hospital admissions. In this study, we showed activity of etoposide PO in patients with SCLC; these results were comparable with those reported for more intensive combination regimens. For elderly patients, etoposide PO provides excellent palliative treatment, with a high response rate and significant prolongation of survival. In addition, therapy is associated with minimal toxicity and can be administered entirely on an outpatient basis.(ABSTRACT TRUNCATED AT 400 WORDS)

Second-line carboplatin-based chemotherapy for small cell lung cancer: the Groningen experience.

In this report, the results of two phase II studies and one pilot study of second-line carboplatin-based chemotherapy for small cell lung cancer are described. Carboplatin plus vincristine given with or without ifosfamide resulted in response rates of 36% and 53%, respectively, in so-called chemotherapy-resistant patients. Toxicity of the carboplatin/vincristine regimen was mild (hematologic toxicity grade 4 was seen with 13% of the courses), whereas the combination including ifosfamide resulted in grade 4 thrombocytopenia in 57% of the courses and grade 4 leukocytopenia in 49%. A partial response was seen in one of nine patients with progression of brain metastases after chemotherapy, and in three patients the neurologic function score improved, with minor tumor reduction evident on computed tomography scan of the brain. We conclude that carboplatin is a useful agent for second-line chemotherapy in patients with an early relapse after induction chemotherapy.

Phase I study of oral teniposide (VM-26).

A phase I study of teniposide administered orally for 5 consecutive days was performed. The first dose was 60 mg/m2/d, with increments of 25 mg/m2/d. Nineteen patients entered the study and received a total of 77 courses with a median of two (range, 1 to 12). Dose-limiting toxicity occurred at 160 mg/m2/d and consisted of myelosuppression, mainly leukocytopenia, and gastrointestinal toxicity. Sufficient recovery of blood counts was seen by day 21 to allow for a repeat course. Two patients, treated with teniposide doses of 110 and 160 mg/m2/d, respectively, were considered toxic deaths. Partial alopecia was frequent at doses above 85 mg/m2/d. Retching and vomiting during administration of the drug were encountered in virtually all patients. Oral teniposide 135 mg/m2/d for 5 consecutive days on a 3-week schedule is recommended for evaluation of antitumor efficacy in phase II studies.

Treatment of brain metastases of small cell lung cancer with teniposide.

Over 50% of patients with small cell lung cancer (SCLC) will develop symptomatic brain metastases during the course of their disease. Results of whole brain radiotherapy, the standard treatment, are rather poor and relapses are frequent. Thus, new modes of therapy are urgently needed for these patients. In this study, the efficacy of teniposide was evaluated at a dose of 150 mg/m2 intravenously on days 1, 3, and 5 at 3-week intervals. In 11 of 26 evaluable patients an intracranial response was observed. Median response duration was 23 weeks (range, 9 to 50). Toxicity was acceptable, with grades 3/4 leukocytopenia and thrombocytopenia reported in 37% and 16%, respectively, of 123 courses. Therefore, teniposide is an effective agent against brain metastases of SCLC and is suitable for palliation of these patients.