Robust Devaney chaos in the two-dimensional border-collision normal form.

The collection of all non-degenerate, continuous, two-piece, piecewise-linear maps on R2 can be reduced to a four-parameter family known as the two-dimensional border-collision normal form. We prove that throughout an open region of parameter space, this family has an attractor satisfying Devaney's definition of chaos. This strengthens the existing results on the robustness of chaos in piecewise-linear maps. We further show that the stable manifold of a saddle fixed point, despite being a one-dimensional object, densely fills an open region containing the attractor. Finally, we identify a heteroclinic bifurcation, not described previously, at which the attractor undergoes a crisis and may be destroyed.

Multiple target-based pharmacophore design from active site structures.

Health care systems have benefitted from rational drug discovery processes like vHTS, virtual high throughput screening pharmacophores and quantitative structure-activity relationships, and many challenges have been explored using such techniques: decisions on specificity and selectivity are made after screening millions of molecules for multiple targets. Recent challenges in drug research emphasize the design of drugs that bind with more than one target of interest (multi-target) and do not bind with undesirable targets. This work attempts to use a three-dimensional interaction profile of the active site of a class of proteins, identify selective positions for the binding of functional groups, called features, and develop ensembles of multi-targeted pharmacophores that retain specificity and selectivity. The goal of this study is to develop multi-target pharmacophores by computational methods using protein structures alone to guide the discovery of novel inhibitors of plasmepsins, displaying selectivity over their human homologs, cathepsin D and pepsin. The development of such novel tools is attempted using a combination of different approaches such as the molecular interaction field, clique graph and inductive logic programming to identify and compare specific and selective complementary features. The identification of selective combinations of features has resulted in the design of multi-featured specific and selective pharmacophores that are validated using antimalarial compounds in ChEMBL that are known for their anti-plasmepsin II activity. This novel method is computationally less intensive and is applicable to any known class of target structures for finding specific and selective binders simultaneously.

Visible light C-H amidation of heteroarenes with benzoyl azides.

Benzoyl azides were used for the direct and atom economic C-H amidation of electron rich heteroarenes in the presence of phosphoric acid, a photocatalyst and visible light. Hetero-aromatic amides are obtained in good yields at very mild reaction conditions with dinitrogen as the only by-product. The reaction allows the use of aryl-, heteroaryl- or alkenyl acyl azides and has a wide scope for heteroarenes, including pyrroles, indole, furan, benzofuran and thiophene giving good regio-selectivities and yields.

A study of pulmonary tuberculosis in the elderly.

The incidence of tuberculosis (TB) is increasing globally. The diagnbsis of TB in elderly is often delayed due to the atypical presentation. There is also therapeutic problems because of presence of comorbidity, nutritional and socio-economic factors and increased incidence of adverse drug reactions and mortality. A prospective study was conducted among patients presenting with pulmonary tuberculosis at the department of TB and respiratory medicine in a tertiary care hospital between January, 2006 to July, 2006. Thirty patients aged more than 65 years were studied for their clinical characteristics, comorbid illnesses, sputum status, radiological features and adverse drug reactions. They were contrasted against the same parameters in 40 patients aged 65 years or less. The study showed that pulmonary TB in the elderly is characterised by (a) atypical symptoms, (b) more extensive radiological lesion with lower zone preponderance, (c) higher sputum positivity, (d) more frequent comorbidity, (e) more frequent side-effects and (f) higher mortality.

Extensive chronic graft-versus-host disease of skin successfully treated with thalidomide.

Thirty years female underwent allogenic peripheral blood stem cell transplantation for chronic myeloid leukaemia--chronic phase. She developed grade II acute skin graft-versus-host disease (GVHD) which was treated with cyclosporine and a short course of steroids. She developed extensive chronic GVHD of the skin and liver three hundred days post-transplant. She was managed with the standard immunosuppressants with partial response of liver dysfunction but no response of skin lesions. She showed a good response to therapy with resolution of skin lesions after treatment with thalidomide.

Quantitative target display: a method to screen yeast mutants conferring quantitative phenotypes by 'mutant DNA fingerprints'.

Whole genome sequencing of several microbes has revealed thousands of genes of unknown function. A large proportion of these genes seem to confer subtle quantitative phenotypes or phenotypes that do not have a plate screen. We report a novel method to monitor such phenotypes, where the fitness of mutants is assessed in mixed cultures under competitive growth conditions, and the abundance of any individual mutant in the pool is followed by means of its unique feature, namely the mutation itself. A mixed population of yeast mutants, obtained through transposon mutagenesis, was subjected to selection. The DNA regions (targets) flanking the transposon, until nearby restriction sites, are then quantitatively amplified by means of a ligation-mediated PCR method, using transposon-specific and adapter-specific primers. The amplified PCR products correspond to mutated regions of the genome and serve as 'mutant DNA fingerprints' that can be displayed on a sequencing gel. The relative intensity of the amplified DNA fragments before and after selection match with the relative abundance of corresponding mutants, thereby revealing the fate of the mutants during selection. Using this method we demonstrate that UBI4, YDJ1 and HSP26 are essential for stress tolerance of yeast during ethanol production. We anticipate that this method will be useful for functional analysis of genes of any microbe amenable to insertional mutagenesis.

DNA binding properties of basic helix-loop-helix fusion proteins of Tal and E47.

The basic helix-loop-helix (bHLH) transcription factor Tal has been shown to form heterodimers with the ubiquitously expressed bHLH transcription factor E47 and thereby modulate gene expression. The absence of homodimeric Tal-DNA complexes had been attributed to the inability of Tal to homodimerize, but subsequent studies have shown that the bHLH region of Tal does homodimerize. In order to correlate the contributions of both the basic region and the helix-loop-helix (HLH) domain to the lack of DNA binding by Tal homodimers, mutant and fusion proteins based on Tal and E47 were designed and synthesized. Size-exclusion chromatography established that all mutant and fusion proteins were dimeric. Point mutations were made within the basic region of Tal based on residues within E47 that are essential for DNA binding, but an affinity for DNA was not observed. Even complete replacement of the basic region in Tal with the basic region of E47, in an E47-Tal fusion protein, did not confer DNA binding upon the protein. However, when the dimerization domain in Tal was replaced with its E47 counterpart, in a Tal-E47 fusion protein, sequence specific DNA binding was observed with an apparent dissociation constant of 3.6 x 10(-9) M2. Furthermore, circular dichroism studies showed that the basic region of Tal in the Tal-E47 fusion protein underwent a random coil to helix transition in the presence of a specific DNA probe. These experimental observations indicate that the inability of Tal homodimers to recognize DNA stems from a misalignment of its basic region with respect to the HLH domain, rather than an intrinsic inability of the Tal basic region to bind DNA.

Zinc inhibition of protein trans-splicing and identification of regions essential for splicing and association of a split intein*.

Two important aspects of protein splicing were investigated by employing the trans-splicing intein from the dnaE gene of Synechocystis sp. PCC6803. First, we demonstrated that both protein splicing and cleavage at the N-terminal splice junction were inhibited in the presence of zinc ion. The trans-splicing reaction was partially blocked at a concentration of 1-10 microm Zn(2+) and completely inhibited at 100 microm Zn(2+); the inhibition by zinc was reversed in the presence of ethylenediaminetetraacetic acid. We propose that inactivation of Cys(160) at the C-terminal splice junction by the chelation of zinc affects both the N-S acyl rearrangement and the transesterification steps in the splicing pathway. Furthermore, in vivo and in vitro assays were established for the determination of intein residues and regions required for splicing or association between the N- and C-terminal intein halves. N-terminal truncation of the intein C-terminal segment inhibited both splicing and association activities, suggesting this region is crucial for the formation of an interface between the two intein halves. The replacement of conserved residues in blocks B and F with alanine abolished splicing but allowed for association. This is the first evidence showing that the conserved residues in block F are required for protein splicing.

A new synthesis of chlorins.

A new synthesis of chlorins has been developed, based upon the acid-catalyzed condensation of dialdehydes AB with dipyrromethanes CD.

Protein trans-splicing to produce herbicide-resistant acetolactate synthase.

Protein splicing in trans has been demonstrated both in vivo and in vitro by biochemical and immunological analyses, but in vivo production of a functional protein by trans-splicing has not been reported previously. In this study, we used the DnaE intein from Synechocystis sp. strain PCC6803, which presumably reconstitutes functional DnaE protein by trans-splicing in vivo, to produce functional herbicide-resistant acetolactate synthase II (ALSII) from two unlinked gene fragments in Escherichia coli. The gene for herbicide-resistant ALSII was fused in frame to DnaE intein segments capable of promoting protein splicing in trans and was expressed from two compatible plasmids as two unlinked fragments. Cotransformation of E. coli with the two plasmids led to production of a functional enzyme that conferred herbicide resistance to the host E. coli cells. These results demonstrate the feasibility of expressing functional genes from two unlinked DNA loci and provide a model for the design of nontransferable transgenes in plants.

Repetitive synchronized cyclical oscillations of multisystem parameters subsequent to high-dose thiopental therapy for status epilepticus secondary to herpes encephalitis.

We report a case of status epilepticus secondary to herpes encephalitis, treated with thiopental infusion and mechanical ventilation. The computerized storage and analysis of physiological data led to the detection of repetitive synchronized cyclical oscillations of arterial pressure, heart rate, EEG parameters, peripheral temperature and core temperature. Arterial pressure oscillations have been described in patients who are severely systemically unwell; cardiovascular and brain electrical activity may also oscillate in the presence of raised intracranial pressure. In contrast, this patient had no features of severe systemic illness or of raised intracranial pressure. Our hypothesis is that high-dose thiopental may have been a cause of our findings by producing autonomic dysfunction.

New syntheses of the C,D-ring pyrromethenones of phytochrome and phycocyanin.

Pyrromethenone 7, the C,D-ring segment of phytochrome (Pr, 4), has been prepared in an efficient fashion employing three new strategies. Each of these has potential advantages for the synthesis of labeled material. Our first approach is related to the Gossauer synthesis, with the difference that strong alkali is avoided in the condensation of the C- and D-ring components 8 and 17. The key silyloxypyrrole 17 was readily prepared on multigram scales beginning with inexpensive butyrolactone (10). A second synthesis began with 2-acetylbutyrolactone (41). The key steps involved conversion of 41 to the Z-enoltriflate 42, followed by Pd(0)-catalyzed coupling with trimethylsilylacetylene, p-chlorophenylselenide ring opening, and finally, amidation to afford the ring-D synthon 45 having the proper geometry and oxidation state for conversion to 7. Sonogashira coupling of 45 with the iodopyrrole 22, followed by oxidative elimination, and F(-)-induced 5-exo-dig cyclization of the resultant pyrroloalkyne 47, then completed the synthesis. In similar fashion, we have also prepared pyrromethenone 6, the C,D-ring segment of phycocyanin (2).

Statistical optimization of transdermal drug delivery system of terbutaline sulfate by factorial analysis.

A matrix type of transdermal drug delivery system of terbutaline sulfate was designed and developed by full 3(2) factorial method using polyvinyl alcohol (PVA) of medium molecular weight and polyvinyl pyrrolidone (PVP K-30) as matrix forming polymer and propylene glycol as plasticizer. Patches were designed to deliver 62 micrograms/cm2/hr of the drug into the systemic circulation. These were evaluated for in vitro skin permeation study through excised guinea pig skin. The permeation followed Higuchi kinetics, as its coefficients of correlation (r = 0.965-0.978) predominates over other permeation kinetics. Various physico-chemical parameters of the patches were investigated. Statistical optimization of in vitro permeation rate of the drug from the films were evaluated using two way analysis of variance (ANOVA), regression analysis.

Measuring community-oriented attitudes towards medical practice.

BACKGROUND: The measurement of attitude and attitudinal changes regarding community-oriented primary care (COPC) and the community-oriented principles of family medicine from the College of Family Physicians of Canada was a key component of this study involving family medicine residents. The Department of Family and Community Medicine at the Toronto Hospital initiated a new COPC curriculum in July 1997 for its first-year residents which was designed to teach the principles of family medicine which are community oriented. OBJECTIVE: This study was developed to provide an analysis and summary of the attitude and attitudinal changes of residents exposed to the programme and those of two cohort groups who were not exposed. METHODS: A quasi-experimental design was used. A 20-item questionnaire was administered pre- and post-intervention. Qualitative data were also collected from focus group sessions with the residents exposed to the programme. RESULTS: The questionnaire was found to have good reliability, with an alpha coefficient of 0.8. No significant differences were observed between the study and control groups pre- and post-intervention. Within the study group, two items from the questionnaire yielded significant differences (P < 0.05). These items dealt with lack of funding and impracticality issues of applying COPC in medical practice. They were also the prevalent themes generated from the focus group session analysis. CONCLUSIONS: The qualitative data corroborated the findings of the survey. These findings have helped in the evolution of the curriculum. Longitudinal studies to measure attitudes and the practice of COPC and community-oriented principles of family medicine after residency are recommended.

Protein trans-splicing and cyclization by a naturally split intein from the dnaE gene of Synechocystis species PCC6803.

A naturally occurring split intein from the dnaE gene of Synechocystis sp. PCC6803 (Ssp DnaE intein) has been shown to mediate efficient in vivo and in vitro trans-splicing in a foreign protein context. A cis-splicing Ssp DnaE intein construct displayed splicing activity similar to the trans-splicing form, which suggests that the N- and C-terminal intein fragments have a high affinity interaction. An in vitro trans-splicing system was developed that used a bacterially expressed N-terminal fragment of the Ssp DnaE intein and either a bacterially expressed or chemically synthesized intein C-terminal fragment. Unlike artificially split inteins, the Ssp DnaE intein fragments could be reconstituted in vitro under native conditions to mediate splicing as well as peptide bond cleavage. This property allowed the development of an on-column trans-splicing system that permitted the facile separation of reactants and products. Furthermore, the trans-splicing activity of the Ssp DnaE intein was successfully applied to the cyclization of proteins in vivo. Also, the isolation of the unspliced precursor on chitin resin allowed the cyclization reaction to proceed in vitro. The Ssp DnaE intein thus represents a potentially important protein for in vivo and in vitro protein manipulation.

Developmentally regulated expression of a unique small heat shock protein in Brugia malayi.

A screen of an expression library from the fourth larval stage (L4) of the parasitic nematode Brugia malayi resulted in the identification of a 727 bp full-length cDNA with 29-40% identity to members of the small heat shock family of proteins (Bm-hsp-s1). The open reading frame encoded a protein of approximately 18 kDA (Bm-HSP-s1). An alignment of the Bm-HSP-s1 sequence with the sequences of small HSPs from vertebrate and invertebrate species demonstrated that a majority of the identity was concentrated in the central alpha-crystallin domain. Bm-HSP-s1 was constitutively produced by L4 and adult parasites and at low levels by third-stage larvae (L3), but not by first-stage larvae (microfilariae). In adult parasites, Bm-HSP-s1 was localized to the body wall muscle cells and to the cells of the hypodermis/lateral cord. Bm-HSP-s1 production was induced in adult and L3 incubated at 42 degrees C and in L3s during the developmental transition from vector-stage to vertebrate-stage parasites at 37 degrees C. Neither increased nor decreased temperatures induced Bm-HSP-s1 production in microfilariae. Nitric oxide induced low-level, transient Bm-HSP-s1 synthesis in adults, but not in microfilariae. Bm-HSP-s1 did not function as a molecular chaperone to prevent heat-induced aggregation of a test substrate. The developmentally regulated expression and inducable nature of Bm-HSP-s1 suggests that it may have a stage-restricted role in maintaining parasite homeostasis.