Simulating the hydrodynamic conditions in the United States Pharmacopeia paddle dissolution apparatus

The objective of this work was to examine the feasibility of developing a high-performance computing software system to simulate the United States Pharmacopeia (USP) dissolution apparatus 2 (paddle apparatus) and thus aid in characterizing the fluid hydrodynamics in the method. The USP apparatus was modeled using the hydrodynamic package Fluent. The Gambit program was used to create a “wireframe” of the apparatus and generate the 3-dimensional grids for the computational fluid dynamics solver. The Fluent solver was run on an IBM RS/6000 SP distributed memory parallel processor system, using 8 processors. Configurations with and without a tablet present were developed and examined. Simulations for a liquid-filled vessel at a paddle speed of 50 rpm were generated. Large variations in fluid velocity magnitudes with position in the vessel were evident. Fluid velocity predictions were in good agreement with those previously published, using laser Doppler velocity measurements. A low-velocity domain was evident directly below the center of the rotating paddle. The model was extended to simulate the impact of the presence of a cylindrical tablet in the base of the dissolution vessel. The presence of the tablet complicated the local fluid flow, and large fluid shear rates were evident at the base of the compact. Fluid shear rates varied depending on the tablet surface and the location on the surface and were consistent with the reported asymmetrical dissolution of model tablets. The approach has the potential to explain the variable dissolution results reported and to aid in the design/prediction of optimal dissolution conditions for in vitro-in vivo correlations.     

The mechanism of dextransucrase action: Activation of dextransucrase from Streptococcus mutans OMZ 176 by dextran and modified dextran and the nonexistence of the primer requirement for the synthesis of dextran

Streptococcus mutans OMZ 176 was grown in a sucrose-free medium containing fructose as a carbohydrate source. Dextransucrase was precipitated from the culture supernatant with 40% saturated ammonium sulfate. The activity of dextransucrase was shown to be stimulated by exogenous dextran. Maximum activity was reached when the concentration of exogenous dextran was 2 mg/ml. Dextrans modified at the nonreducing ends by reaction with tripsyl chloride and/or by hydrolysis with an exodextranase also activated dextransucrase four to six times over that of a control. The exodextranasemodified dextrans have nonreducing chains that are very short in comparison with unmodified dextran and the tripsyl-modified dextrans have chains that are blocked at the nonreducing ends with a triisopropylbenzenesulfonyl group on the C₆ hydroxyl group. Because the nonreducing ends of the modified dextrans are not available for reaction, the activation of dextransucrase by these modified dextrans cannot be due to primer reactions with the nonreducing ends. The activation of dextransucrase, thus, must be by an alternate mechanism. Two alternative mechanisms discussed are an allosteric effect and nucleophilic displacement reactions by the added dextran. It was also found that the addition of increasing amounts of dextran shifted the synthesis from an insoluble dextran to a soluble dextran.     

HPRT Deficiency: Identification of Twenty-Four Novel Variants Including an Unusual Deep Intronic Mutation

Hypoxanthine phosphoribosyltranferase (HPRT) deficiency is an X-linked disorder of purine salvage that ranges phenotypically from hyperuricaemia to Lesch–Nyhan Syndrome. Molecular testing is necessary to identify female carriers within families as a prelude to prenatal diagnosis. During the period 1999–2010 the Purine Research Laboratory studied 106 patients from 68 different families. Genomic sequencing revealed mutations in 88% of these families, 24 of which were novel. In eight patients, exon sequencing was not informative. Copy-DNA analysis in one patient revealed an insertion derived from a deep intronic sequence with a genomic mutation flanking this region, resulting in the creation of a false exon. Carrier testing was performed in 21 mothers of affected patients, out of these, 81% (17) were found to be carriers of the disease-associated mutation. Our results confirm the extraordinary variety and complexity of mutations in HPRT deficiency. A combination of genomic and cDNA sequencing may be necessary to define mutations.     

A Point Mutation in the Gene for Asparagine-Linked Glycosylation 10B (Alg10b) Causes Nonsyndromic Hearing Impairment in Mice (Mus musculus)

The study of mouse hearing impairment mutants has led to the identification of a number of human hearing impairment genes and has greatly furthered our understanding of the physiology of hearing. The novel mouse mutant neurological/sensory 5 (nse5) demonstrates a significantly reduced or absent startle response to sound and is therefore a potential murine model of human hearing impairment. Genetic analysis of 500 intercross progeny localized the mutant locus to a 524 kilobase (kb) interval on mouse chromosome 15. A missense mutation in a highly-conserved amino acid was found in the asparagine-linked glycosylation 10B gene (Alg10b), which is within the critical interval for the nse5 mutation. A 20.4 kb transgene containing a wildtype copy of the Alg10b gene rescued the mutant phenotype in nse5/nse5 homozygous animals, confirming that the mutation in Alg10b is responsible for the nse5/nse5 mutant phenotype. Homozygous nse5/nse5 mutants had abnormal auditory brainstem responses (ABRs), distortion product otoacoustic emissions (DPOAEs), and cochlear microphonics (CMs). Endocochlear potentials (EPs), on the other hand, were normal. ABRs and DPOAEs also confirmed the rescue of the mutant nse5/nse5 phenotype by the wildtype Alg10b transgene. These results suggested a defect in the outer hair cells of mutant animals, which was confirmed by histologic analysis. This is the first report of mutation in a gene involved in the asparagine (N)-linked glycosylation pathway causing nonsyndromic hearing impairment, and it suggests that the hearing apparatus, and the outer hair cells in particular, are exquisitely sensitive to perturbations of the N-linked glycosylation pathway.     

Synthesis and in vitro toxicity of 4-MTA,its characteristic clandestine synthesis byproducts and related sulfur substituted α-alkylthioamphetamines

4-Methylthioamphetamine (4-MTA) is recognised as a 3,4-methylenedioxymethamphetamine (MDMA)-like drug of abuse. Such amphetamine-type drugs often contain byproducts of uncontrolled, illegal clandestine synthetic processes. We report the isolation and structural identification of a number of novel pyridines, dihydropyridone and N,N-di(1-aryl-2-propyl) amines as route-specific byproducts associated with clandestine synthesis of 4-MTA and related amphetamines. We report the in vitro cytotoxicity of 4-MTA, its synthesis byproducts together with some structurally related sulfur substituted α-alkyl phenethylamines in cell lines overexpressing human monoamine transporters as well as in a primary neuronal cell line model and a dopaminergic neuroblastoma cell line. 4-MTA along with a number of other structurally related amphetamine derivatives and synthetic impurities were found to be cytotoxic to these cells within pharmacologically defined concentrations implying that 4-MTA is a cytotoxic agent in vitro and therefore might have the potential to be a neurotoxic agent in vivo.     

Clinical Unity and Community Empowerment: The Use of Smartphone Technology to Empower Community Management of Chronic Venous Ulcers through the Support of a Tertiary Unit

Background

Chronic ulcers affect roughly 60,000 Irish people, at a total cost of €600,000,000, or €10,000 per patient annually. By virtue of their chronicity, these ulcers also contribute a significant burden to tertiary outpatient vascular clinics.

Objective

We propose utilizing mobile phone technology to decentralise care from tertiary centres to the community, improving efficiency and patient satisfaction, while maintaining patient safety.

Methods

Bespoke mobile software was developed for Apples iPhone 4 platform. This allowed for the remote collection of patient images prospectively and their transmission with clinical queries, from the primary healthcare team to the tertiary centre. Training and iPhones were provided to five public health nurses in geographically remote areas of the region. Data were uploaded securely and user end software was developed allowing the review and manipulation of images, along with two way communication between the teams. Establishing reliability, patients were reviewed clinically as well as remotely, and concordance analysed. Qualitative data were collected through focus group discussion.

Results

From October to December 2011 eight patients (61–83 yrs, mean 75.3 yrs) with chronic venous ulceration and their five public health nurses were recruited. Data were transmitted using 3 G, Edge, GPRS and WiFi, at a mean speed of 69.03 kps. Concordance was 100% for wound bed assessment, 80% for skin integrity/colour and 60% for exudate assessment. Focus group analysis explored the concept, practicalities and future applications of the system.

Conclusions

With an evolving national data network, the secure transmission of clinical images is a safe alternative to regular clinic appointments for patients with chronic venous ulceration. With further development, and packaged as a freely downloadable application, this has the potential to support the community care of chronic wounds.     

Automated tracking of mitotic spindle pole positions shows that LGN is required for spindle rotation but not orientation maintenance

Spindle orientation defines the plane of cell division and, thereby, the spatial position of all daughter cells. Here, we develop a live cell microscopy-based methodology to extract spindle movements in human epithelial cell lines and study how spindles are brought to a pre-defined orientation. We show that spindles undergo two distinct regimes of movements. Spindles are first actively rotated toward the cells’ long-axis and then maintained along this pre-defined axis. By quantifying spindle movements in cells depleted of LGN, we show that the first regime of rotational movements requires LGN that recruits cortical dynein. In contrast, the second regime of movements that maintains spindle orientation does not require LGN, but is sensitive to 2ME2 that suppresses microtubule dynamics. Our study sheds first insight into spatially defined spindle movement regimes in human cells, and supports the presence of LGN and dynein independent cortical anchors for astral microtubules.