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Whole brain quantitative CBF and CBV measurements using MRI bolus tracking: comparison of methodologies.

Three different deconvolution techniques for quantifying cerebral blood flow (CBF) from whole brain T*(2)-weighted bolus tracking images were implemented (parametric Fourier transform P-FT, parametric single value decomposition P-SVD and nonparametric single value decomposition NP-SVD). The techniques were tested on 206 regions from 38 hyperacute stroke patients. In the P-FT and P-SVD techniques, the tissue and arterial concentration time curves were fit to a gamma variate function and the resulting CBF values correlated very well (CBF(P-FT) = 1.02 x CBF(P-SVD), r(2) = 0.96). The NP-SVD CBF values (i.e., original unfitted curves were used) correlated well with the P-FT CBF values only when a sufficient number of time series volumes were acquired to minimize tracer time curve truncation (CBF(P-FT) x 0.92 x CBF(NP-SVD), r(2) = 0.88). The correlation between the fitted CBV and the unfitted CBV values was also maximized in regions with minimal tracer time curve truncation (CBV(fit) = 1.00 x CBV(unfit), r(2) = 0.89). When a sufficient number of time series volumes could not be acquired (due to scanner limitations) to avoid tracer time curve truncation, the P-FT and P-SVD techniques gave more reliable estimates of CBF than the NP-SVD technique.

Acute Disease↗

Combinatorial libraries: a tool to design antimicrobial and antifungal peptide analogues having lytic specificities for structure-activity relationship studies.

In the race for supremacy, microbes are sprinting ahead. This warning by the World Health Organization clearly demonstrates that the spread of antibiotic-resistant bacteria leads to a global health problem and that antibiotics never seen before by bacteria are urgently needed. Antimicrobial peptides represent such a source for novel antibiotics due to their rapid lytic activity (within minutes) through disruption of cell membranes. However, due to the similarities between bacterial, fungal, and mammalian plasma cell membranes, a large number of antimicrobial peptides have low lytic specificities and exhibit a broad activity spectrum and/or significant toxic effect toward mammalian cells. Mutation strategies have allowed the development of analogues of existing antimicrobial peptides having greater lytic specificities, although such methods are lengthy and would be more efficient if the molecular mechanisms of action of antimicrobial peptides were clearly elucidated. Synthetic combinatorial library approaches have brought a new dimension to the design of novel biologically active compounds. Thus, a set of peptide analogues were generated based on the screening of a library built around an existing lytic peptide, and on a deconvolution strategy directed toward activity specificity. These peptide analogues also served as model systems to further study the effect of biomembrane mimetic systems on the peptides structural behavior relevant to their biological activities.

Amino Acid Sequence↗

Differences in secondary structure of HAV-synthetic peptides induced by the sequential order of T- and B-cell epitopes.

The present study was undertaken to examine the structural features of two peptide constructs designed on the basis of linear combination of B and T-cell epitopes in different orientations (BT and TB) that may be important to explain the differences in the elicited antihepatitis A virus immune response and in the interaction with biological model membranes. A CD study was carried out and the corresponding quantitative analysis of the experimental data was done using deconvolution computer programs. Moreover, fluorescence experiments were performed to analyze differences in the fluorescence emission spectra of both molecules. The main conformational difference by CD studies was obtained working in aqueous medium. Although the TB sequence adopted a preferably random coil structure, the BT peptide was best fitted with beta-type structures. These results are further supported by fluorescence studies. These findings have relevance for the design of synthetic immunopeptides.

Amino Acid Sequence↗

Site-specific examination of secondary structure and orientation determination in membrane proteins: the peptidic (13)C=(18)O group as a novel infrared probe.

Detailed site-specific information can be exceptionally useful in structural studies of macromolecules in general and proteins in particular. Such information is usually obtained from spectroscopic studies using a label/probe that can reflect on particular properties of the protein. A suitable probe must not modify the native properties of the protein, and should yield interpretable structural information, as is the case with isotopic labels used by Fourier transform infrared (FTIR) spectroscopy. In particular, 1-(13)C=(18)O labels have been shown to relay site-specific secondary structure and orientational information, although limited to small peptides. The reason for this limitation is the high natural abundance of (13)C and the lack of baseline resolution between the main amide I band and the isotope-edited peak. Herein, we dramatically extend the utility of isotope edited FTIR spectroscopy to proteins of virtually any size through the use of a new 1-(13)C=(18)O label. The double-isotope label virtually eliminates any contribution from natural abundance (13)C. More importantly, the isotope-edited peak is further red-shifted (in accordance with ab initio Hartree-Fock calculations) and is now completely baseline resolved from the main amide I band. Taken together, this new label enables determination of site specific secondary structure and orientation in proteins of virtually any size. Even in small peptides 1-(13)C=(18)O is far preferable as a label in comparison to 1-(13)C=(18)O since it enables analysis without the need for any deconvolution or peak fitting procedures. Finally, the results obtained herein represent the first stage in the application of site-directed dichroism to the structural elucidation of polytopic membrane proteins.

Biopolymers↗

Carbon-detected NMR experiments to investigate structure and dynamics of biological macromolecules.

We have started to develop new NMR pulse sequences that detect carbon magnetization during the acquisition period. These experiments have become possible with the recent introduction of cryogenic probe heads. We show that a careful design of these carbon-detected experiments can at least partially compensate for the inherent lower sensitivity of carbon detection compared to proton detection. We discuss potential applications of carbon detection and demonstrate a deconvolution technique that removes the effects of carbon-carbon couplings from the spectra.

Biopolymers↗

Cross-talk filtering in four dye fluorescence-based DNA sequencing.

We have addressed two important issues of nonlinear cross-talk and baseline adjustment in DNA data processing. An important aspect in the processing of the four-dye fluorescence-based data is the cross-talk filtering. Typically, a matrix M, which is a function of the fluorophores and the fluorescence detection system, is used in the multicomponent analysis. In this deconvolution process the matrix is applied directly to the raw signal, on a linear cross-talk assumption. This necessitates the signal to be aligned to the baseline before the filter is applied. The various techniques used for aligning the raw data have the negative effect of adding distortion to the signal. An algorithm for cross-talk removal is presented in this paper. The algorithm uses the intensity difference of the signal rather than the actual value itself, thus making the cross-talk removal possible before the base line adjustment. In addition, a supplementary filtering step is proposed in order to account for the nonlinear nature of the cross-talk. This second step is based on a matrix T that accounts for the correlation of each of the signals with the other three. The overall result is a more precise presentation of the DNA data and less information loss through filtering.

Animals↗

Three-dimensional observation of electrophoretic migration of dsDNA in semidilute hydroxyethylcellulose solution.

Three-dimensional (3-D) video fluorescence microscopy is demonstrated for the investigation of biopolymer electrophoretic migration using double-stranded (ds)DNA in semidilute hydroxyethylcellulose (HEC) as a test system. It is shown that 3-D imaging enables visualization of segmental motion with greater detail than is available in conventional video microscopy. A high frame rate (50-110 frames per second (fps)) intensified progressive scan camera is used to acquire fifteen axial sections focused at different depths through the DNA molecule. A 3-D DNA image is generated from these sections using blind deconvolution image reconstruction and motion is represented as a succession of volume images. A 3-D extension of the Doi/Oana ellipsoidal model is used to fit the DNA envelope, allowing simple quantitative descriptions of the changing shape of the DNA as it interacts with the sieving polymer solution. With 3-D views of migrating DNA molecules we observe U-shaped conformations oriented at an angle to the microscope plane. We are also able to resolve ambiguities and artifacts resulting from loss of information from DNA segments that are not in focus.

Artifacts↗

Capillary electrophoresis investigation on the structure-enantioselectivity relationship in synthetic cyclopeptides as chiral selectors.

We recently reported the use of a deconvolution strategy to identify the best chiral selectors for Nalpha-2,4-dinitrophenyl (Dnp) amino acid racemates from a combinatorial library composed of thousands of homodetic cyclohexapeptides. Selection was based on the capillary electrophoresis (CE) enantioresolution for a set of Dnp-amino acids. The groups involved in the chiral discrimination were assessed by nuclear magnetic resonance (NMR) spectroscopy, which revealed a strong involvement of one of the aromatic rings of the cyclopeptide in the binding with the analyte. In order to better understand the recognition mechanism, and thus extend the applicability of the analytical system, modifications on both analyte and selector structure were introduced. The effects on separation were evaluated in terms of resolution values and mobility variation.

2,4-Dinitrophenol↗

Identification of structures of nitrogen-containing compounds in crude oils in conjunction with chemometric resolution.

A universal method was established for the systematically structural identification of nitrogen-containing compounds in crude oils. Pre-fractionation of the non-hydrocarbons in a crude oil sample into 7 fractions was performed by di-adsorption column chromatography using neutral aluminum oxide and silica gel; subsequent high-resolution separation of individual components was achieved by using capillary column gas chromatography, and compound types were detected by mass spectrometer. The two-dimensional data from the compounds in the fractions were further resolved by a chemometric method to obtained the deconvoluted chromatogram and mass spectrum of every compound, and then, the nitrogen-containing compounds were identified in combination with the retention indices. This method could relieve the difficulty of classical analysis in identifying those species with very low contents or incompletely separation, particularly in the cases where the authentic standards were not available for addition into the unknown samples in order to reveal what indeed existed in them. The structures of 168 nitrogen-containing compounds in a crude oil sample were determined by this method with satisfactory results.

Aluminum Oxide↗

Interictal and postictal alterations of pulsatile secretions of luteinizing hormone in temporal lobe epilepsy in men.

Mesial temporal lobe epilepsy has been associated with abnormalities of reproductive physiology, but the mechanisms of hormonal dysregulation are not clear. Chronic effects of the epileptic state and the acute impact of seizures could alter hypothalamic function, which is represented by the downstream pulsatile secretion of luteinizing hormone. This study evaluates the interictal and postictal secretion of luteinizing hormone in mesial temporal lobe epilepsy. We characterized luteinizing hormone secretion in patients with mesial temporal lobe epilepsy during two 24-hour epochs: an interictal baseline and a postictal interval initiated by an electrographically confirmed spontaneous seizure. Males, rather than females, were studied so that menstrual cycles could not account for differences between epochs. Blood luteinizing hormone and prolactin (as a positive control) were measured every 10 minutes. Deconvolution analysis defined luteinizing hormone secretion in terms of interpulse interval, amplitude, and mass. Approximate entropy quantitated relative degradation in the orderliness of serial luteinizing hormone release. Interictal baseline epochs were compared to those of healthy controls with unpaired Student's t tests and between interictal and postictal epochs within epileptic patients with paired t tests. Ten epileptic men completed both interictal and postictal epochs. Interictally, seizure patients had lower mean concentrations, slower pulse rates, and higher peak amplitudes than healthy male controls. Within epileptic patients, mean interpulse interval, pulse amplitude, and pulse mass were not affected by the occurrence of seizures, whereas the orderliness of pulse mass decreased postictally. Acute seizures induced timing irregularity in luteinizing hormone secretion, whereas chronic epilepsy was associated with changes in luteinizing hormone pulse frequency, amplitude, and mass. Altered timing and regularity of neuroendocrine pulse patterns may underlie other disorders of homeostasis in mesial temporal lobe epilepsy.

Adult↗

A fluorescent double-labeling method to detect and confirm apoptotic nuclei in Parkinson's disease.

In situ end-labeling (ISEL) has become a widely used method to determine whether cells die via apoptosis by detecting double-stranded DNA breaks that are the result of endonuclease digestion. The enzyme terminal deoxynucleotidyl transferase can be used to label the digested 3'-OH ends of DNA with biotin-, digoxigenin-, or fluorescent probe-conjugated nucleotides. However, both single-stranded and double-stranded DNA breaks can be labeled by this method and therefore ISEL cannot unequivocally demonstrate apoptosis when used alone. We have developed a fluorescent double-labeling method using ISEL combined with the cyanine dye YOYO-1 that binds to DNA. When combined with confocal laser microscopy and deconvolution analysis, YOYO-1 can demonstrate the presence or absence of nuclear chromatin condensation and thus confirm that ISEL-positive nuclei are indeed apoptotic. Preliminary findings indicate that dopaminergic neurons in the substantia nigra compacta die via apoptosis in Parkinson's disease.

Apoptosis↗

Cachexia in rheumatoid arthritis is not explained by decreased growth hormone secretion.

OBJECTIVE: Patients with rheumatoid arthritis (RA) lose body cell mass (BCM) by unknown mechanisms. Since the loss of BCM in normal aging individuals parallels the characteristic age-related decline in growth hormone (GH) secretion, this study was carried out to determine whether further decreased GH secretion plays a role in the pathogenesis of this loss of BCM in RA patients, termed "rheumatoid cachexia." METHODS: GH secretory kinetics were determined by deconvolution analysis in 16 patients with RA and 17 healthy controls matched for age (mean +/- SD 45.4 +/- 13.2 years and 47.1 +/- 14.6 years, respectively), sex, race, and body mass index. Blood samples were obtained every 20 minutes for 24 hours. Body composition was ascertained using total-body potassium (TBK) as a measure of BCM and dual x-ray absorptiometry to determine fat mass. RESULTS: BCM was reduced in patients with RA compared with healthy controls (mean +/- SD gm TBK 79.5 +/- 9.5 versus 94.9 +/- 11.9; P < 0.0005), but there was no difference in fat mass. GH kinetic parameters in patients with RA did not differ from those in controls. CONCLUSION: These findings suggest that GH kinetics are unaltered in RA patients compared with healthy subjects; thus, GH deficiency does not account for rheumatoid cachexia.

Adult↗

Muscle Tissue Transcriptome of Idiopathic Inflammatory Myopathy Reflects the Muscle Damage Process by Monocytes and Presence of Skin Lesions.

OBJECTIVE: We aim to investigate transcriptomic and immunophenotypic features of muscle specimens from patients with idiopathic inflammatory myopathy (IIM). METHODS: Bulk RNA-sequencing was performed on muscle biopsy samples from 16 patients with dermatomyositis (DM) and 9 patients with polymyositis (PM). Seven tested positive for anti-aminoacyl transfer RNA synthetase antibodies in the patients with DM (ARS-DM). We conducted weighted gene coexpression network analysis (WGCNA), differentially expressed gene (DEG) analysis, and gene set variation analysis to assess contributions of specific pathways. Cell proportions in muscle specimens were estimated using a deconvolution approach. RESULTS: WGCNA revealed significant positive correlations between serum creatine kinase (CK) levels and gene modules involved in cellular respiration, phagocytosis, and oxidative phosphorylation (OXPHOS). Significant positive correlations were also observed between CK levels and proportions of CD16-positive and negative monocytes and myeloid dendritic cells. Notably, patients with DM demonstrated enrichment of complement and interferon-&#x3b1; and &#x3b3; pathway genes compared with those with PM. Furthermore, ARS-DM demonstrated a higher proportion of Th1 cells and DEGs related to OXPHOS. Additionally, serum Krebs von den Lungen-6 levels correlated with gene modules associated with extracellular matrix and the transforming growth factor-&#x3b2; signaling pathway. CONCLUSION: Our study highlights a significant involvement of monocytes in muscle damage and delineates pathologic differences among IIM subtypes. DM was characterized by complement and interferon-&#x3b1; and &#x3b3; signaling, whereas ARS-DM was associated with OXPHOS. Distinctive gene expression variations in muscle specimens suggest that different pathologic mechanisms underlie muscle damage in each IIM phenotype.

Humans↗

Application of in vitro-in vivo correlations (IVIVC) in setting formulation release specifications.

An in vitro-in vivo correlation (IVIVC) was established for an osmotic controlled-release dosage form by deconvolution using data from an immediate-release treatment as the characteristic response. The established IVIVC was evaluated internally (predicting data used to develop the IVIVC) and externally (predicting data not originally included in developing the IVIVC), and the application of the IVIVC in product development was demonstrated. The estimated in vivo dissolution profile compared favorably with the in vitro drug release profile. Good agreement was demonstrated between the estimated and observed cumulative drug release across the entire time course (level A correlation) with low (<10%) predictive error for both C(max) and AUC(infinity). External validation using lots not initially included in the model development compared very well with the observed in vivo profile, with mean prediction errors less than 10% for both C(max) and AUC(infinity). The proposed method demonstrates a schema for developing IVIVCs using data from biostudies routinely conducted during formulation development. The method should facilitate product optimization and can support setting in vitro dissolution specification.

Algorithms↗

Absorption and disposition characteristics of nitrofurantoin in dogs.

This study reports the disposition kinetic properties of nitrofurantoin in dogs following single intravenous and oral administration of various formulations of nitrofurantoin. Also reported here is the effect of delaying gastric emptying by food and atropine on the absorption characteristics of nitrofurantoin. The drug absorption parameters calculated using a deconvolution computer program indicate that the rate and extent of enterohepatic recycling affects the elimination and absorption rate constants and thus confound the bioavailability calculations of nitrofurantoin, heretofore unrecognized in the literature. The plasma half-life following intravenous administration was 31 min (monoexponential equation) with little effect of enterohepatic recycling noted. Following oral administration, a biexponential equation with lag-time was used to fit the blood levels. The absorption half-lives were higher when nitrofurantoin was administered as a solid dosage form compared to a solution. The absorption half-lives following tablet administration ranged from 30 to 72 min and were not affected by food or atropine. The elimination half-lives following oral administration ranged from 19 to 87 min with significantly prolonged elimination when solid dosage forms were administered compared to solution. The extent of absorption ranged from 38 to 120 per cent. A direct correlation between the absorption and elimination half-life was established, indicating that increased biliary recycling direct affects the apparent disposition half-life. The three brands of nitrofurantoin tested for bioavailability showed that the use of blood levels without appropriate corrections for biliary recycling are not suitable for bioavailability testing of nitrofurantoin. The use of urinary excretion data in evaluating nitrofurantoin bioavailability is also questioned in the study.

Administration, Oral↗

Comparison of in vitro and in vivo release characteristics of acetaminophen from gradient matrix systems.

An effort was made to correlate the in vivo and in vitro release data of acetaminophen from two formulations of a recently developed controlled-release system, the Gradient Matrix System (GMS-1 and GMS-2). The in vivo release curves, obtained by deconvolution of the plasma concentration time plots, showed a small inter-subject variability. GMS-1 with fastest in vitro release also showed fastest in vivo release. A good relationship was only found after time-scaling of the release data.

Acetaminophen↗

In vitro/in vivo correlation of prolonged release dosage forms containing diltiazem HCI.

Six preparations were considered: three multiple unit dosage forms (micropellets in capsules) (D, E and G) and one matrix tablet (B) were experimental prolonged release formulations, two non-disintegrating tablets (A and C) were commercial products. The in vitro dissolution behaviour of the differing formulations was investigated using the USP XXII paddle apparatus. The in vivo study was effected on a panel of 12 healthy volunteers. The two commercial tablets (A and C) showed mean dissolution time (MDT) of 1.34 and 1.44 h and td of 91 and 92 min, respectively; for prolonged release formulations (B, E, D, and G) MDT ranged between 2.28 and 4.23 h and td between 149 and 291 min. The mean residence time (MRT) was 8.68 and 6.47 h for tablets A and C, respectively; it ranged between 9.62 and 10.24 h for the multiple unit formulations E, D, and G and was 11.27 h for matrix B. Formulation B also showed the higher apparent elimination half-life t1/2 (7.12 h), while apparent t1/2 for all the other formulations were very similar, ranging between 5.04 and 5.28 h. High variability between the various formulations was found for Cmax and AUC values, and no relationships could be established with the type of formulation. An in vitro/in vivo correlation was found for all the formulations examined on the basis of analogous parameters (MDT and MRT); (r = 0.83, p < 0.05). In a few cases the Wagner-Nelson deconvolution method was applied to individual plasma level versus time curves and the corresponding absorption curves were obtained. In these cases the in vitro/in vivo correlation was tested on the basis of the comparison of the in vivo absorption curves with the in vitro dissolution profiles. This was accomplished using the 'Levy's plot' (per cent released versus per cent absorbed) approach and provided further support for the correlation found.

Adult↗

Pharmacokinetics of intravenous and epidural ropivacaine in the rhesus monkey.

Ropivacaine is a new long-acting amide local anesthetic which is possibly less cardiotoxic than bupivacaine. The absorption and disposition of ropivacaine were characterized in six rhesus monkeys in an open two-way crossover study following intravenous and epidural administration. For these studies, animals were anesthetized for placement of intravenous and intraarterial catheters. For the epidural studies, a PE-10 catheter was also inserted 3 cm into the lumbar epidural space. After recovery from anesthesia, animals received ropivacaine 1 mg kg-1 intravenously over 1 min or 10 mg of ropivacaine epidurally (two 1 ml doses of 0.5%, 5 min apart), and arterial blood samples were obtained over 5 h. Serum ropivacaine concentrations were determined by gas chromatography with NP detection. Concentration-time data following i.v. and epidural administration were fitted simultaneously. Initial parameter estimates were obtained by analyzing each route separately. Input rates and their corresponding extent of absorption were estimated using deconvolution. Mean (+/- SD) disposition parameters included: Vss = 1.11 +/- 0.198 l kg-1; CL = 0.711 +/- 0.158 l h-1 kg-1; t1/2,z = 2.07 +/- 0.438 h. Mean (+/- SD) absorption parameters included: F1 = 0.506 +/- 0.221; t1/2,kal = 0.060 +/- 0.078 h; F2 = 0.444 +/- 0.182; t1/2,ka2 = 6.45 +/- 11.09 h. Ropivacaine's biphasic absorption and bioavailability are similar to those of other amide local anesthetics. The biphasic absorption may be related to partitioning into fat or regional changes in blood flow induced by the drug.

Amides↗