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Background: Iron overload is observed in neurodegenerative diseases, especially Alzheimer's disease (AD) and Parkinson's disease (PD). Homozygotes for the iron-overload (haemochromatosis) causing HFE p.C282Y variant have increased risk of dementia and PD. Whether brain iron deposition is causal or secondary to the neurodegenerative processes in the general population is unclear., Methods: We analysed 39,533 UK Biobank participants of European genetic ancestry with brain MRI data. We studied brain iron estimated by R2* and quantitative susceptibility mapping (QSM) in 8 subcortical regions: accumbens, amygdala, caudate, hippocampus, pallidum, putamen, substantia nigra, and thalamus. We performed genome-wide associations studies (GWAS) and used Mendelian Randomization (MR) methods to estimate the causal effect of brain iron on grey matter volume, and risk of AD, non-AD and PD. We also used MR to test whether genetic liability to AD or PD causally increased brain iron (R2* and QSM)., Findings: In GWAS of R2* and QSM we replicated 83% of previously reported genetic loci and identified 174 further loci across all eight brain regions. Higher genetically predicted brain iron, using both R2* and QSM, was associated with lower grey matter volumes in the caudate, putamen and thalamus (e.g., Beta-putamenQSM: -0.37, p = 2*10-46). Higher genetically predicted thalamus R2* was associated with increased risk of non-AD dementia (OR 1.36(1.16;1.60), p = 2*10-4) but not AD (p > 0.05). In males, genetically predicted putamen R2* increased non-AD dementia risk, but not in females. Higher genetically predicted iron in the caudate, putamen, and substantia nigra was associated with an increased risk of PD (Odds Ratio QSM ∼ substantia-nigra 1.21(1.07;1.37), p = 0.003). Genetic liability to AD or PD was not associated with R2* or QSM in the dementia or PD-associated regions., Interpretation: Our genetic analysis supports a causal effect of higher iron deposition in specific subcortical brain regions for Parkinson's disease, grey matter volume, and non-Alzheimer's dementia., Competing Interests: Declaration of competing interest All authors declare no conflicts of interest relevant to the manuscript., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Speech quality is one of the main foci of speech-related research, where it is frequently studied with speech intelligibility, another essential measurement. Band-level perceptual speech intelligibility, however, has been studied frequently, whereas speech quality has not been thoroughly analyzed. In this paper, a Multiple Stimuli With Hidden Reference and Anchor (MUSHRA) inspired approach was proposed to study the individual robustness of frequency bands to noise with perceptual speech quality as the measure. Speech signals were filtered into thirty-two frequency bands with compromising real-world noise employed at different signal-to-noise ratios. Robustness to noise indices of individual frequency bands was calculated based on the human-rated perceptual quality scores assigned to the reconstructed noisy speech signals. Trends in the results suggest the mid-frequency region appeared less robust to noise in terms of perceptual speech quality. These findings suggest future research aiming at improving speech quality should pay more attention to the mid-frequency region of the speech signals accordingly., (© 2024 Acoustical Society of America.)

Ganglion cysts are relatively common entities, but intraneural ganglia within peripheral nerves are rare and poorly understood. We present a case of a 51-year-old man who presented with acute left dropfoot. Initial magnetic resonance imaging (MRI) was misinterpreted as common peroneal neuritis consistent with a traction injury corroborated by the patient's history. However, after surgical decompression and external neurolysis were performed, the patient's symptoms worsened. Repeated MRI revealed an intraneural ganglion cyst of the common peroneal nerve with connection to the superior tibiofibular joint by means of its anterior recurrent branch that was evident retrospectively on preoperative MRI. It is crucial to carefully inspect atypical cases to further recognize and appreciate the dynamic aspect of this disease or "roller-coaster" phenomenon. Intraneural ganglion cysts rely heavily on intraneural and extraneural pressure gradients for propagation, which can be drawn from the expanded work of the unifying articular theory. This report emphasizes the importance of understanding the pathoanatomical and hydraulic factors to appropriately identify and treat intraneural ganglion cysts. Increased recognition of this pathologic entity as a differential diagnosis for acute onset dropfoot is also highlighted.

Inhibitors of leucine-rich repeat kinase 2 (LRRK2) and mutants, such as G2019S, have potential utility in Parkinson's disease treatment. Fragment hit-derived pyrrolo[2,3- d ]pyrimidines underwent optimization using X-ray structures of LRRK2 kinase domain surrogates, based on checkpoint kinase 1 (CHK1) and a CHK1 10-point mutant. (2 R )-2-Methylpyrrolidin-1-yl derivative 18 (LRRK2 G2019S c K i 0.7 nM, LE 0.66) was identified, with increased potency consistent with an X-ray structure of 18 /CHK1 10-pt. mutant showing the 2-methyl substituent proximal to Ala147 (Ala2016 in LRRK2). Further structure-guided elaboration of 18 gave the 2-[(1,3-dimethyl-1 H -pyrazol-4-yl)amino] derivative 32 . Optimization of 32 afforded diastereomeric oxolan-3-yl derivatives 44 and 45 , which demonstrated a favorable in vitro PK profile, although they displayed species disconnects in the in vivo PK profile, and a propensity for P-gp- and/or BCRP-mediated efflux in a mouse model. Compounds 44 and 45 demonstrated high potency and exquisite selectivity for LRRK2 and utility as chemical probes for the study of LRRK2 inhibition.

Objective metrics, such as the perceptual evaluation of speech quality (PESQ), short-time objective intelligibility (STOI), and signal-to-distortion ratio (SDR), are often used for evaluating speech. These metrics are intrusive since they require a reference (clean) speech signal to complete the evaluation. The need for a reference signal reduces the practicality of these metrics, since a clean reference signal is not typically available during real-world testing. In this paper, a two-stage approach is presented that estimates the objective score of these intrusive metrics in a non-intrusive manner, which enables testing in real-world environments. More specifically, objective score estimation is treated as a machine-learning problem, and the use of speech-enhancement residuals and convolutional long short-term memory (SER-CL) networks is proposed to blindly estimate the objective scores (i.e., PESQ, STOI, and SDR) of various speech signals. The approach is evaluated in simulated and real environments that contain different combinations of noise and reverberation. The results reveal that the proposed approach is a reasonable alternative for evaluating speech, where it performs well in terms of accuracy and correlation. The proposed approach also outperforms comparison approaches in several environments.

We describe our work to establish structure- and fragment-based drug discovery to identify small molecules that inhibit the anti-apoptotic activity of the proteins Mcl-1 and Bcl-2. This identified hit series of compounds, some of which were subsequently optimized to clinical candidates in trials for treating various cancers. Many protein constructs were designed to identify protein with suitable properties for different biophysical assays and structural methods. Fragment screening using ligand-observed NMR experiments identified several series of compounds for each protein. The series were assessed for their potential for subsequent optimization using 1 H and 15 N heteronuclear single-quantum correlation NMR, surface plasmon resonance, and isothermal titration calorimetry measurements to characterize and validate binding. Crystal structures could not be determined for the early hits, so NMR methods were developed to provide models of compound binding to guide compound optimization. For Mcl-1, a benzodioxane/benzoxazine series was optimized to a K d of 40 μM before a thienopyrimidine hit series was identified which subsequently led to the lead series from which the clinical candidate S 64315 (MIK 665) was identified. For Bcl-2, the fragment-derived series were difficult to progress, and a compound derived from a published tetrahydroquinone compound was taken forward as the hit from which the clinical candidate (S 55746) was obtained. For both the proteins, the work to establish a portfolio of assays gave confidence for identification of compounds suitable for optimization., Competing Interests: The authors declare no competing financial interest.

Mutations in leucine-rich repeat kinase 2 (LRRK2), such as G2019S, are associated with an increased risk of developing Parkinson's disease. Surrogates for the LRRK2 kinase domain based on checkpoint kinase 1 (CHK1) mutants were designed, expressed in insect cells infected with baculovirus, purified, and crystallized. X-ray structures of the surrogates complexed with known LRRK2 inhibitors rationalized compound potency and selectivity. The CHK1 10-point mutant was preferred, following assessment of surrogate binding affinity with LRRK2 inhibitors. Fragment hit-derived arylpyrrolo[2,3-b]pyridine LRRK2 inhibitors underwent structure-guided optimization using this crystallographic surrogate. LRRK2-pSer935 HEK293 IC 50 data for 22 were consistent with binding to Ala2016 in LRRK2 (equivalent to Ala147 in CHK1 10-point mutant structure). Compound 22 was shown to be potent, moderately selective, orally available, and brain-penetrant in wild-type mice, and confirmation of target engagement was demonstrated, with LRRK2-pSer935 IC 50 values for 22 in mouse brain and kidney being 1.3 and 5 nM, respectively.

Leucine-rich repeat kinase 2 (LRRK2) has attracted considerable interest as a therapeutic target for the treatment of Parkinson's disease. Compounds derived from a 2-aminopyridine screening hit were optimised using a LRRK2 homology model based on mixed lineage kinase 1 (MLK1), such that a 2-aminopyridine-based lead molecule 45, with in vivo activity, was identified., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Genetic variation in the leucine-rich repeat kinase 2 (LRRK2) gene is associated with risk of familial and sporadic Parkinson's disease (PD). To support clinical development of LRRK2 inhibitors as disease-modifying treatment in PD biomarkers for kinase activity, target engagement and kinase inhibition are prerequisite tools. In a combined proteomics and phosphoproteomics study on human peripheral mononuclear blood cells (PBMCs) treated with the LRRK2 inhibitor Lu AF58786 a number of putative biomarkers were identified. Among the phospho-site hits were known LRRK2 sites as well as two phospho-sites on human Rab10 and Rab12. LRRK2 dependent phosphorylation of human Rab10 and human Rab12 at positions Thr73 and Ser106, respectively, was confirmed in HEK293 and, more importantly, Rab10-pThr73 inhibition was validated in immune stimulated human PBMCs using two distinct LRRK2 inhibitors. In addition, in non-stimulated human PBMCs acute inhibition of LRRK2 with two distinct LRRK2 inhibitor compounds reduced Rab10-Thr73 phosphorylation in a concentration-dependent manner with apparent IC 50 's equivalent to IC 50 's on LRRK2-pSer935. The identification of Rab10 phosphorylated at Thr73 as a LRRK2 inhibition marker in human PBMCs strongly support inclusion of assays quantifying Rab10-pThr73 levels in upcoming clinical trials evaluating LRRK2 kinase inhibition as a disease-modifying treatment principle in PD.

In real-world situations, speech is masked by both background noise and reverberation, which negatively affect perceptual quality and intelligibility. In this paper, we address monaural speech separation in reverberant and noisy environments. We perform dereverberation and denoising using supervised learning with a deep neural network. Specifically, we enhance the magnitude and phase by performing separation with an estimate of the complex ideal ratio mask. We define the complex ideal ratio mask so that direct speech results after the mask is applied to reverberant and noisy speech. Our approach is evaluated using simulated and real room impulse responses, and with background noises. The proposed approach improves objective speech quality and intelligibility significantly. Evaluations and comparisons show that it outperforms related methods in many reverberant and noisy environments.

Time-frequency masking is a common solution for the single-channel source separation (SCSS) problem where the goal is to find a time-frequency mask that separates the underlying sources from an observed mixture. An estimated mask is then applied to the mixed signal to extract the desired signal. During signal reconstruction, the time-frequency-masked spectral amplitude is combined with the mixture phase. This article considers the impact of replacing the mixture spectral phase with an estimated clean spectral phase combined with the estimated magnitude spectrum using a conventional model-based approach. As the proposed phase estimator requires estimated fundamental frequency of the underlying signal from the mixture, a robust pitch estimator is proposed. The upper-bound clean phase results show the potential of phase-aware processing in single-channel source separation. Also, the experiments demonstrate that replacing the mixture phase with the estimated clean spectral phase consistently improves perceptual speech quality, predicted speech intelligibility, and source separation performance across all signal-to-noise ratio and noise scenarios.

Linkage and genome-wide association studies have identified a genetic risk locus for late-onset Parkinson's disease in chromosome 12, originally identified as PARK6. The causative gene was identified to code for a large multifunctional protein, LRRK2 (leucine-rich repeat kinase 2). The combined genetic and biochemical evidence supports a hypothesis in which the LRRK2 kinase function is causally involved in the pathogenesis of sporadic and familial forms of PD, and therefore that LRRK2 kinase inhibitors could be useful for treatment. Although LRRK2 has so far not been crystallised, the use of homology modelling and crystallographic surrogates has allowed the optimisation of chemical structures such that compounds of high selectivity with good brain penetration and appropriate pharmacokinetic properties are now available for understanding the biology of LRRK2 in vitro and in vivo. This chapter reviews LRRK2 biology, the structural biology of LRRK2 and gives an overview of inhibitors of LRRK2., (© 2017 Elsevier B.V. All rights reserved.)

Speech separation systems usually operate on the short-time Fourier transform (STFT) of noisy speech, and enhance only the magnitude spectrum while leaving the phase spectrum unchanged. This is done because there was a belief that the phase spectrum is unimportant for speech enhancement. Recent studies, however, suggest that phase is important for perceptual quality, leading some researchers to consider magnitude and phase spectrum enhancements. We present a supervised monaural speech separation approach that simultaneously enhances the magnitude and phase spectra by operating in the complex domain. Our approach uses a deep neural network to estimate the real and imaginary components of the ideal ratio mask defined in the complex domain. We report separation results for the proposed method and compare them to related systems. The proposed approach improves over other methods when evaluated with several objective metrics, including the perceptual evaluation of speech quality (PESQ), and a listening test where subjects prefer the proposed approach with at least a 69% rate.

As a means of speech separation, time-frequency masking applies a gain function to the time-frequency representation of noisy speech. On the other hand, nonnegative matrix factorization (NMF) addresses separation by linearly combining basis vectors from speech and noise models to approximate noisy speech. This paper presents an approach for improving the perceptual quality of speech separated from background noise at low signal-to-noise ratios. An ideal ratio mask is estimated, which separates speech from noise with reasonable sound quality. A deep neural network then approximates clean speech by estimating activation weights from the ratio-masked speech, where the weights linearly combine elements from a NMF speech model. Systematic comparisons using objective metrics, including the perceptual evaluation of speech quality, show that the proposed algorithm achieves higher speech quality than related masking and NMF methods. In addition, a listening test was performed and its results show that the output of the proposed algorithm is preferred over the comparison systems in terms of speech quality.

This study proposes an approach to improve the perceptual quality of speech separated by binary masking through the use of reconstruction in the time-frequency domain. Non-negative matrix factorization and sparse reconstruction approaches are investigated, both using a linear combination of basis vectors to represent a signal. In this approach, the short-time Fourier transform (STFT) of separated speech is represented as a linear combination of STFTs from a clean speech dictionary. Binary masking for separation is performed using deep neural networks or Bayesian classifiers. The perceptual evaluation of speech quality, which is a standard objective speech quality measure, is used to evaluate the performance of the proposed approach. The results show that the proposed techniques improve the perceptual quality of binary masked speech, and outperform traditional time-frequency reconstruction approaches.

The nitrilase from Pseudomonas fluorescens EBC191 converted 2-methyl-2-phenylpropionitrile, which contains a quaternary carbon atom in the α-position toward the nitrile group, and also similar sterically demanding substrates, such as 2-hydroxy-2-phenylpropionitrile (acetophenone cyanohydrin) or 2-acetyloxy-2-methylphenylacetonitrile. 2-Methyl-2-phenylpropionitrile was hydrolyzed to almost stoichiometric amounts of the corresponding acid. Acetophenone cyanohydrin was transformed to the corresponding acid (atrolactate) and amide (atrolactamide) at a ratio of about 3.4:1. The (R)-acid and the (S)-amide were formed preferentially from acetophenone cyanohydrin. A homology model of the nitrilase suggested that steric hindrance with amino acid residue Tyr54 could impair the binding or conversion of sterically demanding substrates. Therefore, several enzyme variants that carried mutations in the respective residues were generated and subsequently analyzed for the substrate specificity and enantioselectivity of the reactions. Enzyme variants that demonstrated increased relative activities for the conversion of acetophenone cyanohydrin were identified. The chiral analysis of these reactions demonstrated peculiar reaction kinetics, which suggested that the enzyme variants converted the nonpreferred (S)-enantiomer of acetophenone cyanohydrin with a higher reaction rate than that of the (preferred) (R)-enantiomer. Recombinant whole-cell catalysts that simultaneously produced the nitrilase from P. fluorescens EBC191 and a plant-derived (S)-oxynitrilase from cassava (Manihot esculenta) converted acetophenone plus cyanide at pH 4.5 to (S)-atrolactate and (S)-atrolactamide. These recombinant cells are promising catalysts for the synthesis of stable chiral quaternary carbon centers from ketones.

78 kDa glucose-regulated protein (Grp78) is a heat shock protein (HSP) involved in protein folding that plays a role in cancer cell proliferation. Binding of adenosine-derived inhibitors to Grp78 was characterized by surface plasmon resonance and isothermal titration calorimetry. The most potent compounds were 13 (VER-155008) with K(D) = 80 nM and 14 with K(D) = 60 nM. X-ray crystal structures of Grp78 bound to ATP, ADPnP, and adenosine derivative 10 revealed differences in the binding site between Grp78 and homologous proteins.

When clinicians are operating under a Life Participation Approach to Aphasia (LPAA) while treating persons with aphasia (PWAs), one measurement used to quantify outcomes is quality of life (QOL). Studies of QOL after stroke have identified multiple factors as cause agents. There is not an extensive body of research in the literature that compares the extent of aphasia and QOL and no literature as to how this applies in an LPAA. This article reports a comparison of aphasia quotients obtained from the Western Aphasia Battery-Revised with QOL scores obtained from the Stroke and Aphasia Quality of Life Scale-39 and discusses how the results are incorporated into long-term communication programs at a community-based center that employs an LPAA.

Geobacillus pallidus RAPc8 (NRRL: B-59396) is a moderately thermophilic gram-positive bacterium, originally isolated from Australian lake sediment. The G. pallidus RAPc8 gene encoding an inducible nitrilase was located and cloned using degenerate primers coding for well-conserved nitrilase sequences, coupled with inverse PCR. The nitrilase open reading frame was cloned into an expression plasmid and the expressed recombinant enzyme purified and characterized. The protein had a monomer molecular weight of 35,790 Da, and the purified functional enzyme had an apparent molecular weight of approximately 600 kDa by size exclusion chromatography. Similar to several plant nitrilases and some bacterial nitrilases, the recombinant G. pallidus RAPc8 enzyme produced both acid and amide products from nitrile substrates. The ratios of acid to amide produced from the substrates we tested are significantly different to those reported for other enzymes, and this has implications for our understanding of the mechanism of the nitrilases which may assist with rational design of these enzymes. Electron microscopy and image classification showed complexes having crescent-like, "c-shaped", circular and "figure-8" shapes. Protein models suggested that the various complexes were composed of 6, 8, 10 and 20 subunits, respectively.

Purpose: The anti-apoptotic function of the 70 kDa family of heat shock proteins and their role in cancer is well documented. Dual targeting of Hsc70 and Hsp70 with siRNA induces proteasome-dependent degradation of Hsp90 client proteins and extensive tumor specific apoptosis as well as the potentiation of tumor cell apoptosis following pharmacological Hsp90 inhibition., Methods: We have previously described the discovery and synthesis of novel adenosine-derived inhibitors of the 70 kDa family of heat shock proteins; the first inhibitors described to target the ATPase binding domain. The in vitro activity of VER-155008 was evaluated in HCT116, HT29, BT474 and MDA-MB-468 carcinoma cell lines. Cell proliferation, cell apoptosis and caspase 3/7 activity was determined for VER-155008 in the absence or presence of small molecule Hsp90 inhibitors., Results: VER-155008 inhibited the proliferation of human breast and colon cancer cell lines with GI(50)s in the range 5.3-14.4 microM, and induced Hsp90 client protein degradation in both HCT116 and BT474 cells. As a single agent, VER-155008 induced caspase-3/7 dependent apoptosis in BT474 cells and non-caspase dependent cell death in HCT116 cells. VER-155008 potentiated the apoptotic potential of a small molecule Hsp90 inhibitor in HCT116 but not HT29 or MDA-MB-468 cells. In vivo, VER-155008 demonstrated rapid metabolism and clearance, along with tumor levels below the predicted pharmacologically active level., Conclusion: These data suggest that small molecule inhibitors of Hsc70/Hsp70 phenotypically mimic the cellular mode of action of a small molecule Hsp90 inhibitor and can potentiate the apoptotic potential of a small molecule Hsp90 inhibitor in certain cell lines. The factors determining whether or not cells apoptose in response to Hsp90 inhibition or the combination of Hsp90 plus Hsc70/Hsp70 inhibition remain to be determined.

Antagonists of the human A(2A) receptor have been reported to have potential therapeutic benefit in the alleviation of the symptoms associated with neurodegenerative movement disorders such as Parkinson's disease. As part of our efforts to discover potent and selective antagonists of this receptor, we herein describe the detailed optimization and structure-activity relationships of a series of pyrimidine-4-carboxamides. These optimized derivatives display desirable physiochemical and pharmacokinetic profiles, which have led to promising oral activity in clinically relevant models of Parkinson's disease.

The design and synthesis of novel adenosine-derived inhibitors of HSP70, guided by modeling and X-ray crystallographic structures of these compounds in complex with HSC70/BAG-1, is described. Examples exhibited submicromolar affinity for HSP70, were highly selective over HSP90, and some displayed potency against HCT116 cells. Exposure of compound 12 to HCT116 cells caused significant reduction in cellular levels of Raf-1 and Her2 at concentrations similar to that which caused cell growth arrest.

Transient treatment with small molecule CDK inhibitors is toxic to cancer cells and leads to depletion of anti-apoptotic proteins and Chk1, coupled with DNA damage and induction of apoptosis. Here we have examined, which of these phenomena are necessary for CDK inhibitors to have an anti-proliferative effect. We find that 24 hours treatment with either a primarily CDK2-specific, or a primarily CDK7/9-specific, antagonist eliminates proliferative potential even if apoptosis is blocked and the tendency of CDK inhibition to result in DNA damage is overcome by expression of recombinant Chk1. Loss of proliferative potential is correlated with irreversible suppression of biomarkers of cell cycle progression. CDK inhibitors dramatically reduced levels of the anti-apoptotic proteins, Mcl-1 and XIAP, but siRNA-mediated suppression of Mcl-1 and XIAP did not induce cell death in the osteosarcoma cells used in this study. Finally, we found that many literature CDK inhibitors do not effectively suppress the CDK/cyclin complexes responsible for cell cycle progression at the minimum doses required to block proliferation: some are only effective after a substantial delay and may act via inhibition of CDK7.

The (-)-(11R,2'S)-enantiomer of the antimalarial drug mefloquine has been found to be a reasonably potent and moderately selective adenosine A(2A) receptor antagonist. Further investigation of this compound has led to the discovery of a series of keto-aryl thieno[3,2-d]pyrimidine derivatives, which are potent and selective antagonists of the adenosine A(2A) receptor. These derivatives show selectivity against the A(1) receptor. Furthermore, some of these compounds have been shown to have in vivo activity in a commonly used model, suggesting the potential for the treatment of Parkinson's disease.

We describe herein the discovery and development of a series of 4-arylthieno[3,2-d]pyrimidines which are potent adenosine A(2A) receptor antagonists. These novel compounds show high degrees of selectivity against the human A(1), A(2B) and A(3) receptor sub-types. Moreover, a number of these compounds show promising activity in vivo, suggesting potential utility in the treatment of Parkinson's disease.

A series of pyrazolo[3,4-d]pyrimidine, pyrrolo[2,3-d]pyrimidine and 6-arylpurine adenosine A(2A) antagonists is described. Many examples were highly selective against the human A(1) receptor sub-type and were active in an in vivo model of Parkinson's disease.

Virtual screening against a pCDK2/cyclin A crystal structure led to the identification of a potent and novel CDK2 inhibitor, which exhibited an unusual mode of interaction with the kinase binding motif. With the aid of X-ray crystallography and modelling, a medicinal chemistry strategy was implemented to probe the interactions seen in the crystal structure and to establish SAR. A fragment-based approach was also considered but a different, more conventional, binding mode was observed. Compound selectivity against GSK-3beta was improved using a rational design strategy, with crystallographic verification of the CDK2 binding mode.

Database searching led to the identification of potent A(2A) antagonists which do not contain the privileged furan moiety and which show selectivity over A(1) receptors. Simple substructure searching on a proprietary database identified compounds with activities in the low nM range. A targeted approach to the identification of non-furan containing compounds resulted in the identification of two novel series, with potency, selectivity and directional SAR from screening 113 compounds.

Crystallographic and modelling data, in conjunction with a medicinal chemistry template-hopping approach, led to the identification of a series of novel and potent inhibitors of human cyclin-dependent kinase 2 (CDK2), with selectivity over glycogen synthase kinase-3beta (GSK-3beta). One example had a CDK2 IC(50) of 120 nM and showed selectivity over GSK-3beta of 167-fold.

The protein structure guided design of a series of pyrazolo[1,5-a]pyrimidines with high potency for human cyclin-dependent kinase 2 (CDK2) is described. Some examples were shown to inhibit the growth of human colon tumour cells, were equipotent for CDK1 and were selective against GSK-3beta and other kinases.

The potent neuroexcitatory activity of kainoid amino acids in the mammalian CNS places new analogues in high demand as tools for neuropharmacological research. A range of 4-arylsulfanyl-substituted kainoids has been synthesised in a parallel fashion via mesylate displacement by a number of aromatic and heteroaromatic thiolates upon (2S,3S,4R)-1-benzoyl-3-tert-butoxycarbonylmethyl-4-methanesulfo nyloxy pyrrolidine-2-carboxylic acid methyl ester 8, which is obtainable in eight steps from trans-4-hydroxy-L-proline 5.

Lipid characterization of bone marrow in vivo with proton magnetic resonance spectroscopy was performed using Spin-Echo Planar Spectroscopic Imaging sequences. The methods are shown capable of rapidly generating two-dimensional chemical shift imaging data sets suitable for measuring lipid indices that reflect unsaturation levels among triglycerides, as demonstrated in oil phantoms and bone marrow from a healthy volunteer. The volume coverage, spatial resolution, acquisition speed, and spectral characteristics of Spin-Echo Planar Spectroscopic Imaging should make it attractive for clinical studies of diseases affecting normal lipid chemical composition.

Objective: To compare polytomography (PT) and computed tomography (CT) for visualizing fractures and arthrodeses, with and without metal hardware, to determine whether CT could adequately replace PT., Design and Patients: An ex vivo bovine model containing fractures in three planes, reduced with metal hardware, was created to compare fractures using PT and CT. The radiation dose at the skin surface was calculated for both examinations. For in vivo assessment, images of 14 patients who underwent both PT and CT (15 fractures, five arthrodeses) were coded, sorted, and independently read by four musculoskeletal radiologists. They rated the degree of certainty of their assessment. Time factors for patients and personnel and financial costs were also compared., Results: In the ex vivo model the fractures were well seen on both PT and CT. The radiation dose was higher for PT than for CT. In vivo, the degree of certainty in assessment of fractures and arthrodeses was higher for PT than CT in studies in which metal hardware was present, but there was no significant difference in studies without metal hardware or in the combined (with and without hardware) studies. The patient's and technologist's time required to perform a PT examination was greater than that for CT., Conclusion: In the assessment of fractures and arthrodeses containing metal hardware, PT is recommended. For studies without hardware, CT is equivalent and can replace PT.

Purpose: Numerous orthopedic disorders involve periaxial rotation of long bones. We have developed and evaluated a computer-assisted method that segments a bone from X-ray CT, graphically unwraps the bone around its long axis into a surface plot (signature landscape), and measures periaxial rotation as the translation shift of the landscape. Bones in known rotations and partially segmented surfaces were used, respectively, to test accuracy and problematic situations in bone segmentation., Method: CT images of three chicken femora at known rotations were analyzed to determine their relative periaxial rotations, which were compared with the known rotations., Results: The regression slope between measured and expected periaxial rotations was 1.005 +/- 0.003, with a maximum discrepancy of 2 degrees for same-bone and 3 degrees for interbone comparisons. Rasterization artifacts and the use of partial surfaces (with < 40% surface omission) resulted in a < 3 and < 1 degree error, respectively., Conclusion: The current method provides accurate and objective periaxial rotation measurements of a long bone.

Line scan Carr-Purcell-Meiboom-Gill spectroscopic imaging sequences have been used to extract lipid chemical composition indices in healthy adult bone marrow in the knee at 1.5 T. Since several spectroscopic echo readouts follow each excitation, the information acquired reflects a balance between spectral T2 decay processes and spectral resolution. To examine this balance in detail, data sets with two different echo spacings and spectral resolutions have been acquired to compare the information available from each in studies of bone marrow. Oils for which high field (7 T) proton spectra were recorded were used to evaluate the accuracy of lipid chemical composition indices extracted from the line scan Carr-Purcell-Meiboom-Gill spectroscopic imaging methods at 1.5 T. The extension of the method to fast spectroscopic imaging of bone marrow with multiple echoes is demonstrated.

The citrate signal at field strengths of whole body imagers arises from two sets of two strongly coupled methylene protons. This causes citrate spectra acquired with standard in vivo localization schemes like the double-echo point resolved echo spectroscopy (PRESS) sequence to have complicated dependencies on timing parameters. A homonuclear J-refocused version of the double-echo PRESS sequence that has previously been shown to completely remove J-modulations from weakly coupled AX systems is considered for its potential in acquiring signal from the strongly coupled AB system of citrate. An analytic solution to the problem is derived with the density matrix formalism and verified both numerically and experimentally for 7 T conditions. The general expression for the AB signal is applied to study the 1.5 T citrate signal where a substantial signal enhancement over conventional double-echo PRESS sequences is predicted and verified for echo times in the 150 to 300 ms range.

Repeated measures are reasonably common in injury research and thus tools are required for appropriate analysis in order to account for the correlated nature of this type of data. Three methods for analyzing repeated measures binary outcome data are presented and contrasted: generalized estimating equations (GEE), a survey sample methodology, and logistic regression. These methods are applied to data collected from a cohort study of rugby players, designed to examine the risk and protective factors for rugby injury. It is not, however, the purpose of this paper to present causal models of rugby injuries. The GEE approach is attractive because it is able to account for the correlation among a subject's outcomes and several covariates can be included in a model. The survey sample method approach, which also accounts for the correlation but is restrictive in terms of the number of covariates it can handle, is another approach which is described. These two methods are contrasted to logistic regression, which assumes independence among a subject's outcomes. Under certain circumstances, the three methods do not differ substantially from one another. Under other circumstances, since logistic regression ignores the correlated nature of the data, standard errors may be incorrectly estimated and thus certain covariates may be incorrectly identified as significant predictors in a model.

It is well known that when compared to conventional spin-echo (CSE) imaging for equivalent effective echo times, fast spin-echo (FSE) imaging experiments yield higher signal intensities for coupled spin systems, such as that for lipid. One hypothesis put forth for this phenomenon is the removal of scalar coupling-based echo amplitude modulation by the FSE pi pulse train. This would result in the maintenance of signal intensity in the late echoes, with an overall increase in image signal when the multiecho train data is combined to form the image data. It will be shown that in images and spectra obtained from the final echo of a Carr-Purcell-Meiboom-Gill (CPMG) pi pulse train, an increase in signal in coupled spin systems occurs, when compared to conventional single-echo images and spectra at identical echo times. One- and two-dimensional spectroscopy experiments confirm that it is the generation of an isotropic mixing Hamiltonian by the pi pulse train in FSE that is responsible for the increased signal in images of a simple AX system and of corn oil, a model for human fat. This relative increase in signal is due to the maintenance of in-phase magnetization in the coupled spin systems by this Hamiltonian. In CSE, the weak coupling Hamiltonian allows development of antiphase coherences which, in the presence of the line broadening due to the imaging gradients, result in signal loss.

Citrate detection and quantitation with proton spectroscopic methods are of current interest as potential tools in the diagnosis and staging of prostate cancer. The stimulated echo acquisition mode (STEAM) sequence is a commonly used volume-localization method for detecting citrate signal. Since the 1H citrate resonance at clinically available field strengths arises from a strongly coupled two-spin system, the 90 degrees RF pulses and localizing gradients used in STEAM sequences result in a complicated dependence of signal intensity on timing intervals and gradient amplitudes. The density-matrix formalism has been applied to arrive at a general solution to this problem. Citrate-signal properties at 1.5 T for different gradient localization schemes are examined with the solution. Optimal interpulse delays, deleterious gradient balances, zero-quantum oscillations with mixing time, and a low-frequency, large-amplitude oscillation with echo time are identified for signals acquired with the standard disposition of gradients in STEAM. The generality of the solution also allows for an examination of non-standard gradient disposition schemes for enhancing citrate signal and for quantifying the sensitivity of such approaches to both field inhomogeneities and off-resonance effects.

Objective: A line scan spectroscopic imaging method providing variable T2-weighted spectra from many small voxels along selected tissue columns was applied to study the chemical composition of hematopoietic and fatty marrow in the knees of adults and children., Materials and Methods: Line scan Carr-Purcell-Meiboom-Gill (CPMG) spectroscopic imaging sequences were implemented on a 1.5 T clinical scanner. Variable T2-weighted proton spectra from 128 locations along 20 cm long, 5 mm2 columns oriented superiorly to inferiorly through knees were collected from eight healthy adults and eight children., Results: In adult yellow marrow, olefinic protons, water, a composite lipid proton peak, and methyl/methylene protons contributed 6.4 +/- 0.4, 4.2 +/- 1.5, 7.2 +/- 0.5, and 82.2 +/- 1.9% (mean +/- SD) to the spectra, respectively. Marrow spectra were largely independent of position along the column. Marrow spectra of normal children showed distinct positional dependences. Epiphyseal marrow spectra of children (8-16 years old) resembled adult spectra but with more water (mean 15 vs. 4%). Metaphyseal marrow had higher, variable water content, reflecting the extent of marrow conversion and generally obscuring the olefinic proton peak., Conclusions: Spectroscopic imaging of columns is a time-efficient method for sampling extensive regions of bone marrow with high spatial resolution. It should prove useful for proton spectroscopic studies of hematologic pathologies and conditions requiring the monitoring of lipid composition.

Selective suppression of silicone or fat with chemical shift-selective (CHESS) pulses is difficult because of the small chemical shift difference between the primary lipid signal and the primary silicone signal at 1.5 T. Differentiation of these chemically distinct species is, however, an important clinical task in assessing implant rupture and silicone migration in breast tissue. A method uniquely suited for silicone-fat differentiation with fast spin-echo (FSE) sequences is reported. It is based on the dependence of fat signal on echo spacing in FSE imaging and results show that it may provide a clinically robust method for silicone-fat differentiation in magnetic resonance imaging of the breast.

The efficacy of using unenhanced head computed tomography (CT scans) as a routine screening procedure prior to lumbar puncture in the emergency department is studied retrospectively by comparing opening pressure during lumbar puncture to CT scan diagnosis in 42 patients. No correlation was found between CT scan findings and opening pressure.

The interaction of acrolein, an alpha,beta-unsaturated aldehyde, with polydeoxyadenylic acid and DNA has been investigated using 32P-postlabeling analysis. In preliminary experiments, polydeoxyadenylic acid was incubated with excess acrolein and then digested to 3' monophosphates prior to transfer of 32P from [gamma-32P]ATP with T4 polynucleotide kinase. The 3',5'-bisphosphates were 3'-dephosphorylated prior to two-dimensional thin layer chromatography on polyethyleneimine-cellulose layers. Autoradiography provided evidence for the formation of one extra spot of radioactivity, compared to the control. To determine the adduct structure, deoxyadenosine-5'-monophosphate was incubated with a 3-fold excess of acrolein. This material was mixed with a 32P-labeled digest of acrolein-polydeoxyadenylic acid, and the sample was analyzed by ion-pair high performance liquid chromatography. The spot of 32P observed by thin layer chromatography co-eluted with the major product of the acrolein nucleotide reaction mixture, which was purified by ion-pair high performance liquid chromatography. Two-dimensional nuclear magnetic resonance spectroscopy and mass spectrometry showed the adduct to be 3-(2'-deoxyribosyl-5'-monophosphatyl)-7,8,9-trihydro-9-hydro xy- pyrimido[2,3-i]purine(1,N6-propanodeoxyadenosine-5'-monophosphate) . High performance liquid chromatography was used to fractionate digests of acrolein-modified DNA prior to detection of this exocyclic adduct by 32P-postlabeling.

The in vitro effects of saccharin were evaluated in a transformed rat-bladder epithelial cell line. AY-27 cells were seeded in a 35-mm tissue culture well and incubated for 24 hr with saccharin or other test compounds dissolved in the media. The cytotoxic effects of saccharin were dependent on the salt form tested. Although dose-related decreases in cell viability and attachment were observed following exposure to sodium or potassium saccharin at concentrations of ≥50 mM, calcium saccharin decreased only cell viability. Sodium chloride, sodium ascorbate and sodium citrate decreased cell viability and attachment in a dose-related fashion, whereas neither potassium chloride nor calcium chloride had any effect. The finding that all of the tested sodium compounds were cytotoxic suggests that the sodium ion plays a role in the cytotoxicity of sodium saccharin. However, the maximum effect and potency of the sodium compounds differed, indicating that the anion moiety also contributes to the cytotoxic effect. Although effects were observed only at millimolar concentrations of the sodium salts, these concentrations corresponded to those in the urine that produced a urothelial proliferative response in rats fed these compounds in earlier studies.

Acrolein reacts with deoxycytidine 5'-monophosphate at physiological pH to form one major adduct. A second minor adduct can be detected when a 3-fold excess of acrolein is present in the reaction mixture. The products were separated by ion-pair HPLC on two reverse-phase columns connected in series using triethylammonium formate as ion-pair reagent. The major adduct was characterized as 3-(5'-monophospho-2'-deoxyribosyl)-7,8, 9-trihydro-7-hydroxy-pyrimido [3,4-c]pyrimidin-2-one (3,N4-propanodeoxcytidine 5'-monophosphate). This mixture of diastereomers was formed by addition of C1 of acrolein to the exocyclic amino group at the 4-position of cytidine, followed by ring closure between C3 of acrolein and N3 of the heterocyclic ring. In order to address the utility of 32P postlabeling for the detection of this exocyclic adduct in acrolein-modified nucleic acids, an acrolein-deoxycytidine 3'-monophosphate reaction mixture was subjected to 32P postlabeling. 3-Dephosphorylation with nuclease P1 and the 3'-phosphatase activity of T4 polynucleotide kinase yields a nucleotide 5'-[32P] monophosphate which cochromatographs with 3,N4-propanodeoxycytidine 5'-monophosphate. These data indicate that 32P postlabeling and 3'-dephosphorylation can be used in conjunction with ion-pair HPLC for the detection and quantitation of acrolein-modified nucleotides.

The sodium salt of saccharin is biologically more active as a urothelial cell mitogen in vivo, when fed to male rats, than are the potassium or calcium salts or the acid form, despite similar concentrations of saccharin excreted in the urine. The differences in bladder-mitogenic activity between sodium saccharin and the other salts of saccharin may be the result of known differences in the ionic composition of the urine of rats receiving these various forms of saccharin. These changes in the rat urine following administration of the different salts of saccharin could be responsible for the observed mitogenic responses to oral saccharin; alternatively the differences in the ionic composition of the urine could result in changes in the electronic structure of the saccharin molecule itself, allowing it to be more active in certain ionic environments. Since the pKa of saccharin is 1.8, essentially all of the saccharin in urine (pH greater than 5) will exist in the ionized form. We have used 17O, 15N, 13C and two-dimensional nuclear magnetic resonance (NMR) spectroscopy to explore the electronic structure of the saccharin molecule in aqueous solution. By observing the NMR spectra of the saccharinate ion in the presence of varying concentrations of hydrogen, potassium, sodium, calcium, magnesium, bicarbonate and urate, we have demonstrated that at physiological levels none of these ions significantly alters the electronic structure of the saccharin molecule. Hence the differences in the mitogenic response to the different saccharin salts cannot be explained by alterations in the structure of the saccharin molecule.

This paper reports an evaluation of an intergenerational consultation process designed to help people change significant relationships in the three-generational family system. The paper first describes the theory behind the consultation process that focuses on increasing personal authority in the family system; this is followed by a description of the consultation process. Clients who participated in the consultation process were compared to clients who participated in systems-oriented psychotherapy. The results indicate that clients who participated in the intergenerational consultation reported significantly more change in intergenerational triangulation and personal authority than did the control group. There were no differences between the groups in reported change in presenting problems, satisfaction with therapy, or helpfulness of therapy. Persons who had their parents in the office for a consultation also reported significantly more change in personal authority and less intergenerational intimidation than did clients who did not have their parents in the office for consultation. Implications of these findings for intergenerational theory and therapy as well as future research are discussed.