Findings from the Longitudinal CINRG Becker Natural History Study.

BACKGROUND: Becker muscular dystrophy is an X-linked, genetic disorder causing progressive degeneration of skeletal and cardiac muscle, with a widely variable phenotype. OBJECTIVE: A 3-year, longitudinal, prospective dataset contributed by patients with confirmed Becker muscular dystrophy was analyzed to characterize the natural history of this disorder. A better understanding of the natural history is crucial to rigorous therapeutic trials. METHODS: A cohort of 83 patients with Becker muscular dystrophy (5-75 years at baseline) were followed for up to 3 years with annual assessments. Muscle and pulmonary function outcomes were analyzed herein. Age-stratified statistical analysis and modeling were conducted to analyze cross-sectional data, time-to-event data, and longitudinal data to characterize these clinical outcomes. RESULTS: Deletion mutations of dystrophin exons 45-47 or 45-48 were most common. Subgroup analysis showed greater pairwise association between motor outcomes at baseline than association between these outcomes and age. Stronger correlations between outcomes for adults than for those under 18 years were also observed. Using cross-sectional binning analysis, a ceiling effect was seen for North Star Ambulatory Assessment but not for other functional outcomes. Longitudinal analysis showed a decline in percentage predicted forced vital capacity over the life span. There was relative stability or improved median function for motor functional outcomes through childhood and adolescence and decreasing function with age thereafter. CONCLUSIONS: There is variable progression of outcomes resulting in significant heterogeneity of the clinical phenotype of Becker muscular dystrophy. Disease progression is largely manifest in adulthood. There are implications for clinical trial design revealed by this longitudinal analysis of a Becker natural history dataset.

PLK1 inhibition selectively induces apoptosis in ARID1A deficient cells through uncoupling of oxygen consumption from ATP production.

Inhibitors of the mitotic kinase PLK1 yield objective responses in a subset of refractory cancers. However, PLK1 overexpression in cancer does not correlate with drug sensitivity, and the clinical development of PLK1 inhibitors has been hampered by the lack of patient selection marker. Using a high-throughput chemical screen, we discovered that cells deficient for the tumor suppressor ARID1A are highly sensitive to PLK1 inhibition. Interestingly this sensitivity was unrelated to canonical functions of PLK1 in mediating G2/M cell cycle transition. Instead, a whole-genome CRISPR screen revealed PLK1 inhibitor sensitivity in ARID1A deficient cells to be dependent on the mitochondrial translation machinery. We find that ARID1A knock-out (KO) cells have an unusual mitochondrial phenotype with aberrant biogenesis, increased oxygen consumption/expression of oxidative phosphorylation genes, but without increased ATP production. Using expansion microscopy and biochemical fractionation, we see that a subset of PLK1 localizes to the mitochondria in interphase cells. Inhibition of PLK1 in ARID1A KO cells further uncouples oxygen consumption from ATP production, with subsequent membrane depolarization and apoptosis. Knockdown of specific subunits of the mitochondrial ribosome reverses PLK1-inhibitor induced apoptosis in ARID1A deficient cells, confirming specificity of the phenotype. Together, these findings highlight a novel interphase role for PLK1 in maintaining mitochondrial fitness under metabolic stress, and a strategy for therapeutic use of PLK1 inhibitors. To translate these findings, we describe a quantitative microscopy assay for assessment of ARID1A protein loss, which could offer a novel patient selection strategy for the clinical development of PLK1 inhibitors in cancer.

Synthesis, characterization, method development, and validation of nor-ethylmorphine hydrochloride reference material using established analytical techniques for dope control analysis.

Ethylmorphine is permitted internationally for therapeutic purposes where morphine is not indicated across the globe. Nor-ethylmorphine a major metabolite of ethylmorphine. To differentiate the intake of morphine from ethylmorphine, nor-ethylmorphine stable reference material is desirable. There is no available commercial source and no data for reference material context for this substance. Therefore, nor-ethylmorphine HCl was synthesized and characterized, and purity and potency were assessed using nuclear magnetic resonance (NMR), high-resolution mass spectrometry (HRMS), Fourier transform infrared spectroscopy (FT-IR), thermogravimetry (TGA), and high-performance liquid chromatography (HPLC). Purity and potency were found to be 98.29% and 96.40%, respectively, providing a fit for purpose reference material for doping control analysis in sports.

Genome sequencing of herb Tulsi (Ocimum tenuiflorum) unravels key genes behind its strong medicinal properties.

BACKGROUND: Krishna Tulsi, a member of Lamiaceae family, is a herb well known for its spiritual, religious and medicinal importance in India. The common name of this plant is 'Tulsi' (or 'Tulasi' or 'Thulasi') and is considered sacred by Hindus. We present the draft genome of Ocimum tenuiflurum L (subtype Krishna Tulsi) in this report. The paired-end and mate-pair sequence libraries were generated for the whole genome sequenced with the Illumina Hiseq 1000, resulting in an assembled genome of 374 Mb, with a genome coverage of 61 % (612 Mb estimated genome size). We have also studied transcriptomes (RNA-Seq) of two subtypes of O. tenuiflorum, Krishna and Rama Tulsi and report the relative expression of genes in both the varieties. RESULTS: The pathways leading to the production of medicinally-important specialized metabolites have been studied in detail, in relation to similar pathways in Arabidopsis thaliana and other plants. Expression levels of anthocyanin biosynthesis-related genes in leaf samples of Krishna Tulsi were observed to be relatively high, explaining the purple colouration of Krishna Tulsi leaves. The expression of six important genes identified from genome data were validated by performing q-RT-PCR in different tissues of five different species, which shows the high extent of urosolic acid-producing genes in young leaves of the Rama subtype. In addition, the presence of eugenol and ursolic acid, implied as potential drugs in the cure of many diseases including cancer was confirmed using mass spectrometry. CONCLUSIONS: The availability of the whole genome of O.tenuiflorum and our sequence analysis suggests that small amino acid changes at the functional sites of genes involved in metabolite synthesis pathways confer special medicinal properties to this herb.

SInCRe-structural interactome computational resource for Mycobacterium tuberculosis.

We have developed an integrated database for Mycobacterium tuberculosis H37Rv (Mtb) that collates information on protein sequences, domain assignments, functional annotation and 3D structural information along with protein-protein and protein-small molecule interactions. SInCRe (Structural Interactome Computational Resource) is developed out of CamBan (Cambridge and Bangalore) collaboration. The motivation for development of this database is to provide an integrated platform to allow easily access and interpretation of data and results obtained by all the groups in CamBan in the field of Mtb informatics. In-house algorithms and databases developed independently by various academic groups in CamBan are used to generate Mtb-specific datasets and are integrated in this database to provide a structural dimension to studies on tuberculosis. The SInCRe database readily provides information on identification of functional domains, genome-scale modelling of structures of Mtb proteins and characterization of the small-molecule binding sites within Mtb. The resource also provides structure-based function annotation, information on small-molecule binders including FDA (Food and Drug Administration)-approved drugs, protein-protein interactions (PPIs) and natural compounds that bind to pathogen proteins potentially and result in weakening or elimination of host-pathogen protein-protein interactions. Together they provide prerequisites for identification of off-target binding.

Mechanistic Basis Of Peptide-Protein Interaction In AtPep1-PEPR1 Complex In Arabidopsis thaliana.

Peptide-mediated immunity against pathogens in plants can provide information on protein-peptide interactions and drug discovery in general. The molecular structure of AtPep1, a 23-amino acid signaling peptide isolated from Arabidopsis thaliana leaves and implicated in innate immunity, has evaded structural determination by biophysical methods. The details of molecular interaction of AtPep1 peptide with its receptor (PEPR1), a 170 kDa leucine-rich repeat (LRR) kinase is also unknown. We report a computational approach to the modeling AtPep1 by conformational sampling and its interaction with the receptor PEPR1. Molecular dynamics simulations were employed to sample and cluster energetically favorable conformations of AtPep1 and modeling of PEPR1 through homology. Docking of AtPep1 to PEPR1 and filtering of the biologically relevant poses were facilitated by the computational Ala-scanning mutations and binding energy analysis of the peptide-protein complex. This study provides the first independent in silico validation of the Structure-Activity- Relationship studies carried out on the AtPep1 and provides a molecular mechanism of the peptide-protein complex system.

Enriching the annotation of Mycobacterium tuberculosis H37Rv proteome using remote homology detection approaches: insights into structure and function.

The availability of the genome sequence of Mycobacterium tuberculosis H37Rv has encouraged determination of large numbers of protein structures and detailed definition of the biological information encoded therein; yet, the functions of many proteins in M. tuberculosis remain unknown. The emergence of multidrug resistant strains makes it a priority to exploit recent advances in homology recognition and structure prediction to re-analyse its gene products. Here we report the structural and functional characterization of gene products encoded in the M. tuberculosis genome, with the help of sensitive profile-based remote homology search and fold recognition algorithms resulting in an enhanced annotation of the proteome where 95% of the M. tuberculosis proteins were identified wholly or partly with information on structure or function. New information includes association of 244 proteins with 205 domain families and a separate set of new association of folds to 64 proteins. Extending structural information across uncharacterized protein families represented in the M. tuberculosis proteome, by determining superfamily relationships between families of known and unknown structures, has contributed to an enhancement in the knowledge of structural content. In retrospect, such superfamily relationships have facilitated recognition of probable structure and/or function for several uncharacterized protein families, eventually aiding recognition of probable functions for homologous proteins corresponding to such families. Gene products unique to mycobacteria for which no functions could be identified are 183. Of these 18 were determined to be M. tuberculosis specific. Such pathogen-specific proteins are speculated to harbour virulence factors required for pathogenesis. A re-annotated proteome of M. tuberculosis, with greater completeness of annotated proteins and domain assigned regions, provides a valuable basis for experimental endeavours designed to obtain a better understanding of pathogenesis and to accelerate the process of drug target discovery.

Analysis of designed ß-hairpin peptides: molecular conformation and packing in crystals.

The crystal structures of several designed peptide hairpins have been determined in order to establish features of molecular conformations and modes of aggregation in the crystals. Hairpin formation has been induced using a centrally positioned (D)Pro-Xxx segment (Xxx = (L)Pro, Aib, Ac6c, Ala; Aib = α-aminoisobutyric acid; Ac6c = 1-aminocyclohexane-1-carboxylic acid). Structures of the peptides Boc-Leu-Phe-Val-(D)Pro-(L)Pro-Leu-Phe-Val-OMe (1), Boc-Leu-Tyr-Val-(D)Pro-(L)Pro-Leu-Phe-Val-OMe (2, polymorphic forms labeled as 2a and 2b), Boc-Leu-Val-Val-(D)Pro-(L)Pro-Leu-Val-Val-OMe (3), Boc-Leu-Phe-Val-(D)Pro-Aib-Leu-Phe-Val-OMe (4, polymorphic forms labeled as 4a and 4b), Boc-Leu-Phe-Val-(D)Pro-Ac6c-Leu-Phe-Val-OMe (5) and Boc-Leu-Phe-Val-(D)Pro-Ala-Leu-Phe-Val-OMe (6) are described. All the octapeptides adopt type II' ß-turn nucleated hairpins, stabilized by three or four cross-strand intramolecular hydrogen bonds. The angle of twist between the two antiparallel strands lies in the range of -9.8° to -26.7°. A detailed analysis of packing motifs in peptide hairpin crystals is presented, revealing three broad modes of association: parallel packing, antiparallel packing and orthogonal packing. An attempt to correlate aggregation modes in solution with observed packing motifs in crystals has been made by indexing of crystal faces in the case of three of the peptide hairpins. The observed modes of hairpin aggregation may be of relevance in modeling multiple modes of association, which may provide insights into the structure of insoluble polypeptide aggregates.

Biochemical alterations induced by acute oral doses of iron oxide nanoparticles in Wistar rats.

Magnetic iron oxide nanoparticles with appropriate surface chemistry have been widely used with potential new applications in biomedical industry. Therefore, the aim of this study was to assess the size-, dose-, and time-dependent effects, after acute oral exposure to iron oxide-30 NP (Fe(2)O(3)-30), on various biochemical enzyme activities of clinical significances in a female Wistar rat model. Rats were exposed to three different doses (500, 1,000, and 2,000 mg/kg) of Fe(2)O(3)-30 and Fe(2)O(3)-Bulk along with control. Fe(2)O(3)-30 had no effect on growth, behavior, and nutritional performance of animals. Fe(2)O(3)-30 caused significant inhibition of acetylcholinestrase in red blood cells as well as in brains of treated rats. Further, more than 50% inhibition of total, Na(+)-K(+), Mg(2+), and Ca(2+)-ATPases activities, as observed in brains of exposed female rats, may be the result of disturbances in cellular physiology and the iono-regulatory process. Activation of the hepatotoxicity marker enzymes, aspartate aminotransferase and alanine aminotransferase, was recorded in serum and liver, whereas inhibition was observed in kidney. Similarly, enhancement of lactate dehydrogenase activity was observed in serum and liver; however, a decrease in enzyme levels was observed in kidneys of Fe(2)O(3)-30-treated rats. On the other hand, Fe(2)O(3)-Bulk did not depict any significant changes in these biochemical parameters, and alterations were near to control. Therefore, this study suggests that exposure to nanosize particles at acute doses may cause adverse changes in animal biochemical profiles. The use of the rat model signifies the correlation with the human system.

Repeated oral dose toxicity of iron oxide nanoparticles: biochemical and histopathological alterations in different tissues of rats.

Iron oxide (Fe2O3) nanoparticles are widely used in different fields of nanotechnology. However, studies on its toxicological effects in humans and the environment are scarce. Therefore in this investigation 28 days repeated dose oral toxicity studies were conducted on Fe2O3-30 nanoparticles and its counterpart Fe2O3-Bulk with special reference to target biochemical enzymes and histopathological changes in different tissues of female albino Wistar rats. The alterations observed after Fe2O3-30 treatment in various tissues of exposed rats were dose dependent. Low dose was less effective than medium and high doses with low dose demonstrating "no observed adverse effect" (NOAEL). Further, high dose treated rats showed toxic sign and symptoms but no mortality. Due to the repeated doses of Fe2O3-30 nanoparticles, significant inhibition was observed in total, Na(+)-K+, Mg2+ and Ca(2+)-ATPases in brain of exposed rats. Similarly, significant inhibition was recorded in RBC and brain acetylcholinesterase indicating that both synaptic transmission and nerve conduction were affected by this compound. Fe2O3-30 significantly increased aspartate amino transferase, alanine amino transferase and lactate dehydrogenase in serum and liver, whereas, these enzymes were significantly decreased in kidney indicating tissue necrosis and possible leakage of these enzymes into the blood stream. Increased levels of these enzymes in liver as well as in serum might be an adaptive mechanism due to the stress of iron nanoparticles. High dose treated rats of Fe2O3-30 showed dilated central vein, perivascular round cell collections in liver along with focal areas of necrosis, whereas kidney showed focal tubular damage and red pulp congestion, whereas prominent white pulp indices were observed in spleen. However, histopathological analysis of heart and brain tissues failed to show any adverse changes in their architecture exposed to repeated doses of Fe2O3-30 when compared with controls. Fe2O3-Bulk did not induce any adverse effects in either biochemical parameters or histopathology in the treated rats and the changes observed were near to controls and mostly insignificant, indicating that the counter part of nanoparticles i.e., bulk material is less potent than the nanoparticles in causing toxicity in the exposed animals. These results suggested that as particle size decreases, this iron nanoparticle showed increased toxicity, even though the same material is relatively inert in bulk form. The changes observed in these target enzyme activities could be useful as biomarkers of exposure to nanoparticles.

The ST Pinch: A side chain-to-side chain hydrogen-bonded motif.

The ST Pinch is a 12-membered hydrogen-bonded motif (Ser/Thr-Xaa-Ser/Thr) involving the side chain oxygen atoms of two Ser/Thr residues. We identified the ST Pinch in 104 proteins in a database containing high-resolution crystal structures. Conformational analysis of the ST Pinch in these proteins points to specific preferences for the Xaa residue and a high propensity of this residue to adopt positive φ angles. Our results suggest that this motif serves as a linker of secondary structural elements within proteins and is a new addition to the existing list of short hydrogen bond-stabilized motifs in proteins.

Hydrophobic peptide channels and encapsulated water wires.

Peptide nanotubes with filled and empty pores and close-packed structures are formed in closely related pentapeptides. Enantiomorphic sequences, Boc-(D)Pro-Aib-Xxx-Aib-Val-OMe (Xxx = Leu, 1; Val, 2; Ala, 3; Phe, 4) and Boc-Pro-Aib-(D)Xxx-Aib-(D)Val-OMe ((D)Xxx = (D)Leu, 5; (D)Val, 6; (D)Ala, 7; (D)Phe, 8), yield molecular structures with a very similar backbone conformation but varied packing patterns in crystals. Peptides 1, 2, 5, and 6 show tubular structures with the molecules self-assembling along the crystallographic six-fold axis (c-axis) and revealing a honeycomb arrangement laterally (ab plane). Two forms of entrapped water wires have been characterized in 2: 2a with d(O...O) = 2.6 A and 2b with d(O...O) = 3.5 A. The latter is observed in 6 (6a) also. A polymorphic form of 6 (6b), grown from a solution of methanol-water, was observed to crystallize in a monoclinic system as a close-packed structure. Single-file water wire arrangements encapsulated inside hydrophobic channels formed by peptide nanotubes could be established by modeling the published structures in the cases of a cyclic peptide and a dipeptide. In all the entrapped water wires, each water molecule is involved in a hydrogen bond with a previous and succeeding water molecule. The O-H group of the water not involved in any hydrogen bond does not seem to be involved in an energetically significant interaction with the nanotube interior, a general feature of the one-dimensional water wires encapsulated in hydrophobic environments. Water wires in hydrophobic channels are contrasted with the single-file arrangements in amphipathic channels formed by aquaporins.

Characterization of water wires inside hydrophobic tubular peptide structures.

The crystallographic observation of a hydrophobic, empty channel (diameter approximately 5.2 A) in the peptide Boc-(D)Pro-Aib-Leu-Aib-Val-OMe, prompted the investigation of the analog Boc-(D)Pro-Aib-Val-Aib-Val-OMe in which the side chain at position 3 was shortened, resulting in the structure of a channel (diameter approximately 7.5 A) containing a one-dimensional wire of water molecules. Crystallization in the space group P6(5) facilitates formation of a pore lined entirely by hydrocarbon side chains. Two forms of the entrapped water wires, with O...O separations of 3.5 and 2.6 A, are discussed. A lone hydrogen bond between the adjacent pairs of water molecules in the wire, with no strong interactions between the second water hydrogen and the hydrophobic walls of the channel, is a feature of the one-dimensional array. The structure provides the first crystallographic characterization of a water wire in a hydrophobic channel with implications in water and proton transport in membranes and carbon nanotubes.

Synthesis and conformational studies of peptides from new C-linked carbo-beta-amino acids (beta-Caas) with anomeric methylamino- and difluorophenyl moieties.

New C-linked carbo-beta-amino acids (beta-Caas), Cbz-(S)-beta-Caa-(NHBoc)-OMe (1) and Cbz-(R)-beta-Caa-(NHBoc)-OMe (2), with an additional amine group (methylamino group of NHBoc) at the C-1 position of the lyxofuranoside side chain and Boc-(S)-beta-Caa-(diFP)-OMe (3) and Boc-(R)-beta-Caa-(diFP)-OMe (4), with a C-difluorophenyl (diFP) moiety at the anomeric position of the lyxofuranoside side chain were prepared from D-mannose. Beta-peptides [tetra- and hexapeptides] were synthesized from these beta-Caas, 'epimeric' [at the amine stereocentre (C(beta))], using the concept of 'alternating chirality' to carry out their conformational studies [NMR (CDCl(3)), CD and MD]. In the monomer design, it was envisaged that the presence of an additional amine group in 1 or 2 would help in solubilizing the peptides in water, while, the C-difluorophenyl (diFP) moiety of 3 and 4 is expected to enhance the biological activity. The peptides having 1 and 2, though could not retain their 12-10-mixed helices in water, have shown moderate activity against gram positive and gram negative bacterial strains. The peptides prepared from 3 and 4, much against our expectations, did not display any biological activity.

Safety of intravenous terbutaline in acute severe asthma: a retrospective study.

OBJECTIVE: (1) To determine the effect of intravenous terbutaline in children with acute severe asthma on parameters like heart rate, blood pressure, electrocardiogram and serum electrolytes; (2) to assess the safety profile and to evaluate the outcome of children treated with intravenous terbutaline for acute severe asthma. DESIGN: Retrospective study of admission records of children admitted with acute severe asthma who needed intravenous terbutaline. SETTING: Children's Hospital at the Leicester Royal Infirmary, UK. PATIENTS: 77 children with acute severe asthma admitted between April 1999 and October 2002. RESULTS: There was a significant increase in heart rate and significant fall in diastolic blood pressure in this cohort. Four patients required inotropic support. None of the patients had cardiac arrhythmias. Potassium supplements were required in 10 patients due to hypokalaemia. All patients improved and none required initiation of ventilation after commencing terbutaline. There was no mortality in this cohort. CONCLUSIONS: Terbutaline was found to be safe for use in this patient group in doses ranging between 1 and 5 microg/kg/min. Intravenous terbutaline was found to be a useful adjunct in those who failed to respond to standard initial therapy.