Novel method for comparing coverage by future methods of ballistic facial protection.

The wearing of eye protection by United Kingdom soldiers in Afghanistan has reduced the morbidity caused by explosive fragments. However, the remaining face remains uncovered because there is a lack of evidence to substantiate the procurement of methods to protect it. Using a new computerised tool we entered details of the entry sites of surface wounds caused by explosive fragments in all UK soldiers who were injured in the face between 1 January 2010 and 31 December 2011. We compared clinical and predicted immediate and long term outcomes (as defined by the Abbreviated Injury Score (AIS) and the Functional Capacity Index (pFCI), respectively). We also used the tool to predict how additional protection in the form of a visor and mandible guard would affect outcomes. A soldier wearing eye protection was 9 times (1.03/0.12) less likely to sustain an eye injury than one without. However, 38% of soldiers in this series were not wearing eye protection at the time of injury. There was no significant difference between the AIS and pFCI scores predicted by the tool and those found clinically. There is limited evidence to support the use of a mandible guard; its greatest asset is better protection of the nose, but a visor would be expected to reduce long-term morbidity more than eye protection alone, and we recommend future trials to assess its acceptability to users. We think that use of this novel tool can help in the selection of future methods of ballistic facial protection.

Using computerised surface wound mapping to compare the potential medical effectiveness of Enhanced Protection Under Body Armour Combat Shirt collar designs.

INTRODUCTION: Protecting the neck from explosively propelled fragments has traditionally been achieved through a collar attached to the ballistic vest. An Enhanced Protection Under Body Armour Combat Shirt (EP-UBACS) collar has been identified as an additional method of providing neck protection but limited evidence as to its potential medical effectiveness exists to justify its procurement. METHOD: Entry wound locations and resultant medical outcomes were determined using Abbreviated Injury Scale (AIS) for all fragmentation neck wounds sustained by UK soldiers between 01 January 2010 and 31 December 2011. Data were prospectively entered into a novel computerised tool base and comparisons made between three EP-UBACS neck collar designs in terms of predicted reduction in AIS scores. RESULTS: All collars reduced AIS scores, with the greatest reduction provided by designs incorporating increased standoff from the neck and an additional semi-circle of ballistic material underneath the collar at the front and back. DISCUSSION: This technique confirms that reinforcing the neck collar of an EP-UBACS would be expected to reduce injury severity from neck wounds. However, without knowledge of entry wound locations for injuries to other body areas as well as the use of AIS scores without clinical or pathological verification its further use in the future may be limited. The ability to overlay any armour design onto a standardised human was potentially the most useful part of this tool and we would recommend developing this technique using underlying anatomical structures and not just the skin surface.

fdg-pet in two cases of neurofibromatosis type 1 and atypical malignancies.

Patients with neurofibromatosis type 1 (nf1) are at increased risk for both benign and malignant tumours, and distinguishing the malignant potential of an individual tumour is a common clinical problem in these patients. Here, we review two cases of uncommon malignancies (Hodgkin lymphoma and mediastinal germ-cell tumour) in patients with nf1. Although (18)F-fluorodeoxyglucose positron-emission tomography (fdg-pet) has been used to differentiate benign neurofibromas from malignant peripheral nerve sheath tumours, fdg-pet characteristics for more rare tumours have been poorly described in children with nf1. Here, we report the role of pet imaging in clinical decision-making in each case. In nf1, fdg-pet might be useful in the clinical management of unusual tumour presentations and might help to provide information about the malignant potential of uncommon tumours.

Porcine insulin biodegradable polyester microspheres: stability and in vitro release characteristics.

The stability of porcine insulin in biodegradable polymers, i.e., poly(DL-lactide-co-glycolide) 50:50 (50:50 DL-PLGA) and poly(L-lactide) (L-PLA) was investigated. Insulin encapsulated microspheres were fabricated from both polymers using double-emulsion-solvent evaporation and emulsion-solvent evaporation techniques and subjected to accelerated stability studies at 40 degrees C and 75% relative humidity. Porcine insulin was found to degrade in all microsphere formulations with an average of < 50% of the initial loading amount remaining intact at the end of 4 weeks. The two major degradation products observed in these formulations were determined to be A-21 desamido insulin and covalent insulin dimer with trace amounts of high molecular weight transformation products. In vitro release studies in phosphate buffered saline at 37 degrees C resulted in very slow and incomplete (< 30% in 30 days) release kinetics for all microsphere formulations. Extraction and analyses of the unreleased insulin within the microspheres revealed that an average of approximately 11% of the encapsulated insulin remained intact. The degradation products observed consisted of approximately 15% of three distinct deamidated hydrolysis products including A-21 desamido insulin, approximately 22% covalent insulin dimer, and trace amounts of high molecular weight transformation products. The degradation of porcine insulin within biodegradable polyester microspheres during stability and release studies can be attributed to the gradual decrease in the pH within the microspheres due to progressive polymer hydrolysis resulting in the production of DL-lactic and glycolic acids. The encapsulation of an acid-base indicator, bromophenol blue, in 50:50 PLGA microspheres (as a probe to estimate pH within the microspheres during accelerated stability studies) indicated that the pH decreased to approximately 3.8 after 3 weeks.

Stabilization of pH-induced degradation of porcine insulin in biodegradable polyester microspheres.

The purpose of this research project was to stabilize the pH-induced degradation of porcine insulin encapsulated within biodegradable polyester microspheres through the incorporation of a basic additive. Insulin microspheres fabricated using Poly(L-lactide) (L-PLA) and Poly(DL-lactide-co-glycolide) (50:50 DL-PLGA) were subjected to in vitro release studies and the stability of unreleased insulin encapsulated within microspheres was investigated. The intramicrosphere pH was estimated by encapsulating acid-base indicators covering a wide pH transition range within 50:50 DL-PLGA microspheres. Finally, a basic excipient sodium bicarbonate was incorporated in 50:50 DL-PLGA microspheres to minimize acid-induced insulin degradation. The in vitro release was slow and incomplete (< 30% in 30 days). Extraction and analyses of the unreleased insulin within the microspheres revealed that an average of approximately 11% remained intact. The degradation products observed consisted of approximately 15% of three distinct deamidated hydrolysis products including A-21 Desamido insulin, approximately 22% Covalent Insulin Dimer and trace amounts of High Molecular Weight Transformation Products. Comparison of the degradation profile of unreleased insulin contained in various microsphere formulations with the in vitro release kinetics indicated that an increase in covalent dimer formation within the microspheres prior to release is associated with a decrease in the cumulative percent insulin released during a 30-day incubation period. In an attempt to correlate insulin degradation with the drop in intra-microsphere pH due to polymer hydrolysis, it was determined that the pH within a degrading microsphere reaches a value of approximately 1.8 after 4 weeks. The incorporation of a basic excipient, sodium bicarbonate, in 50:50 DL-PLGA microspheres resulted in an improved in vitro release profile (cumulative release approximately 47.3% in 30 days) as well as a significant reduction in covalent dimerization of the unreleased insulin to barely detectable levels. The low pH microenvironment within a degrading microsphere is one of the major factors leading to protein instability, and the degradation of proteins encapsulated within polyester microspheres can be minimized by the incorporation of a basic excipient.

Evaluation of current compendial physiochemical test procedures for pharmaceutical elastomeric closures and development of an improved HPLC procedure.

The interaction between elastomeric container closures and the solutions they confine presents a potential hazard to the consumer due to extraction of closure ingredients into the dosage form. Each of the major Pharmacopeias, the United States, the European, and the Japanese, prescribe testing procedures for elastomeric closures. These consist of a series of non-specific wet chemical analyses performed on samples extracted into water or, in some cases, isopropanol (IPA) or the drug product vehicle. No consideration is given to the extracting potential of the drug product. Results from our testing on ten randomly selected closure samples indicated that these tests are not sensitive or specific enough to accurately measure the levels of extractables. Therefore, an HPLC gradient method was developed which had the required sensitivity and specificity. The prescribed compendial extractions, when performed on the various stopper types, proved inefficient and experiments were conducted in an attempt to improve them. These included increasing the time of the extractions, increasing the closure surface area, and increasing the strength of the extracting solvent (methylene chloride). The HPLC gradient method and the compendial wet chemical tests were then used to evaluate the stopper extractables. Results of the compendial analyses on the prescribed aqueous extractions were inconclusive as the number and relative amount of extractables in the closure could not be measured. The results of the compendial testing were only marginally improved using the stronger extraction conditions. Testing was dramatically improved, however, using the HPLC gradient method. As many as twenty extractables were detected in some of the samples and, unlike the compendial analysis, low level extractables were detected in the water samples. Identification of some of the extractables was accomplished via GC/MS.

Analysis of EST-driven gene annotation in human genomic sequence.

We have performed a systematic analysis of gene identification in genomic sequence by similarity search against expressed sequence tags (ESTs) to assess the suitability of this method for automated annotation of the human genome. A BLAST-based strategy was constructed to examine the potential of this approach, and was applied to test sets containing all human genomic sequences longer than 5 kb in public databases, plus 300 kb of exhaustively characterized benchmark sequence. At high stringency, 70%-90% of all annotated genes are detected by near-identity to EST sequence; >95% of ESTs aligning with well-annotated sequences overlap a gene. These ESTs provide immediate access to the corresponding cDNA clones for follow-up laboratory verification and subsequent biologic analysis. At lower stringency, up to 97% of annotated genes were identified by similarity to ESTs. The apparent false-positive rate rose to 55% of ESTs among all sequences and 20% among benchmark sequences at the lowest stringency, indicating that many genes in public database entries are unannotated. Approximately half of the alignments span multiple exons, and thus aid in the construction of gene predictions and elucidation of alternative splicing. In addition, ESTs from multiple cDNA libraries frequently cluster over genes, providing a starting point for crude expression profiles. Clone IDs may be used to form EST pairs, and particularly to extend models by associating alignments of lower stringency with high-quality alignments. These results demonstrate that EST similarity search is a practical general-purpose annotation technique that complements pattern recognition methods as a tool for gene characterization.

GAIA: framework annotation of genomic sequence.

As increasing amounts of genomic sequence from many organisms become available, and as DNA sequences become a primary reagent in biologic investigations, the role of annotation as a prospective guide for laboratory experiments will expand rapidly. Here we describe a process of high-throughput, reliable annotation, called framework annotation, which is designed to provide a foundation for initial biologic characterization of previously unexamined sequence. To examine this concept in practice, we have constructed Genome Annotation and Information Analysis (GAIA), a prototype software architecture that implements several elements important for framework annotation. The center of GAIA consists of an annotation database and the associated data management subsystem that forms the software bus along which other components communicate. The schema for this database defines three principal concepts: (1) Entries, consisting of sequence and associated historical data; (2) Features, comprising information of biologic interest; and (3) Experiments, describing the evidence that supports Features. The database permits tracking of annotation results over time, as well as assessment of the reliability of particular results. New framework annotation is produced by CARTA, a set of autonomous sensors that perform automatic analyses and assert results into the annotation database. These results are available via a Web-based query interface that uses graphical Java applets as well as text-based HTML pages to display data at different levels of resolution and permit interactive exploration of annotation. We present results for initial application of framework annotation to a set of test sequences, demonstrating its effectiveness in providing a starting point for biologic investigation, and discuss ways in which the current prototype can be improved. The prototype is available for public use and comment at http://www.cbil.upenn.edu/gaia.

Cloning a balanced translocation associated with DiGeorge syndrome and identification of a disrupted candidate gene.

DiGeorge syndrome (DGS), a developmental defect, is characterized by cardiac defects and aplasia or hypoplasia of the thymus and parathyroid glands. DGS has been associated with visible chromosomal abnormalities and microdeletions of 22q11, but only one balanced translocation--ADU/VDU t(2;22)(q14;q11.21). We now report the cloning of this translocation, the identification of a gene disrupted by the rearrangement and the analysis of other transcripts in its vicinity. Transcripts were identified by direct screening of cDNA libraries, exon amplification, cDNA selection and genomic sequence analysis using GRAIL. Disruption of a gene in 22q11.2 by the breakpoint and haploinsufficiency of this locus in deleted DGS patients make it a strong candidate for the major features associated with this disorder.

A gene for cleidocranial dysplasia maps to the short arm of chromosome 6.

Cleidocranial dysplasia (CCD) is an autosomal dominant generalized bone dysplasia characterized by mild-to-moderate short stature, clavicular aplasia or hypoplasia, supernumerary and ectopic teeth, delayed eruption of secondary teeth, a characteristic craniofacial appearance, and a variety of other skeletal anomalies. We have performed linkage studies in five families with CCD, with 24 affected and 20 unaffected individuals, using microsatellite markers spanning two candidate regions on chromosomes 8q and 6. The strongest support for linkage was with chromosome 6p microsatellite marker D6S282 with a two-point lod score of 4.84 (theta = .03). Furthermore, the multipoint lod score was 5.70 in the interval between D6S282 and D6S291. These data show that the gene for autosomal dominant CCD is located within a 19-cM interval on the short arm of chromosome 6, between D6S282 and D6S291.

Kinetic and mechanistic studies with bovine testicular hyaluronidase.

Bovine testicular hyaluronidase exhibits hydrolase and transglycosylase activity. To assess the magnitude of each type of reaction, the time-course of hyaluronidase catalysed hyaluronic acid degradation was followed using a sensitive and specific HPLC method. The kinetic parameters Km and Vmax were calculated for purified short chain hyaluronic acid oligomers and native hyaluronic acid based on the appearance of unreactive hyaluronic acid tetrasaccharide. For hyaluronic acid oligomers, as substrate size increased Km decreased from 2.06 to 1.09 mM while Vmax remained about the same, indicating a 5-fold increase in the enzyme-substrate association constant, k1 (kcat/Km). The values of k2 (kcat), the enzyme-substrate disassociation constant, for native hyaluronic acid and hyaluronic acid decasaccharide were similar. The value of k1 for native hyaluronic acid, however, was larger by 70-fold. Kinetic degradation mechanisms for each hyaluronic acid oligomer, using chemical-reaction kinetics, were proposed and evaluated by computer curve fitting analysis of the experimental time vs. concentration data. The derived rate constants, together with mass balance calculations, revealed that transglycosylation plays a significant role in the degradation of all hyaluronic acid oligomers studied.

Refinement of linkage of human severe combined immunodeficiency (SCIDX1) to polymorphic markers in Xq13.

The most common form of human severe combined immunodeficiency (SCID) is inherited as an X-linked recessive genetic defect, MIM 300400. The disease locus, SCIDX1, has previously been placed in Xq13.1-q21.1 by demonstration of linkage to polymorphic markers between DXS159 and DXS3 and by exclusion from interstitial deletions of Xq21.1-q21.3. We report an extension of previous linkage studies, with new markers and a total of 25 SCIDX1 families including female carriers identified by nonrandom X chromosome inactivation in their T lymphocytes. SCIDX1 was nonrecombinant with DXS441, with a lod score of 17.96. Linkage relationships of new markers in the SCIDX1 families were consistent with the linkage map generated in the families of the Centre d'Etude du Polymorphisme Humain (CEPH) and with available physical map data. The most likely locus order was DXS1-(DXS159,DXS153)-DXS106-DXS132-DXS4 53-(SCIDX1,PGK1, DXS325,DXS347,DXS441)-DXS447-DXS72-DXYS 1X-DXS3. The SCIDX1 region now spans approximately 10 Mb of DNA in Xq13; this narrowed genetic localization will assist efforts to identify gene candidates and will improve genetic management for families with SCID.

Optimization of the USP assay for hyaluronidase.

The current USP XXII assay for hyaluronidase (EC 3.2.1.35, HAse) determines activity indirectly by measuring the amount of undegraded hyaluronic acid (HA) substrate remaining after the enzyme is allowed to react with the HA for 30 min at 37 degrees C. To be acceptable as a substrate, the HA must pass a USP suitability test. In this study, seven HA samples, which differed in their anatomical origin, their commercial supplier, and their chondroitin sulphate content, were tested as substrates. One of these did not pass the USP suitability test and therefore would not be an officially acceptable substrate; however, it was carried through the investigation along with the others in order to demonstrate its effect on the analysis. All seven HAs were used as substrates to assay testicular hyaluronidases from three different suppliers. The standard by which the other hyaluronidase activities were measured was USP hyaluronidase reference standard. The activity values calculated for a particular hyaluronidase differed significantly depending on which HA was used as substrate in its assay. Optimal results, as judged on the bases of initial purity, suitability for the assay, linearity of the standard curve, and per cent relative standard deviation of the measured activity, were obtained with a HA substrate derived from vitreous humour.

Cloning of human and bovine homologs of SNF2/SWI2: a global activator of transcription in yeast S. cerevisiae.

We performed positional cloning of genes carried on yeast artificial chromosomes that span a human translocation breakpoint associated with a human disease and isolated by chance human and bovine genes with strong homology to the S. cerevisiae genes, SNF2/SWI2 and STH1, and the D. melanogaster gene brahma. We report here sequence analysis, expression data, and functional studies for this human SNF2-like gene (hSNF2L) and its bovine homolog (bovSNF2L). Despite strong homology at the amino acid level, hSNF2L is not capable of complementing the yeast mutations snf2 or sth1 in S. cerevisiae. Furthermore, in contrast to SNF2 itself, a fusion protein consisting of the DNA binding domain of LexA and hSNF2L did not transactivate a reporter gene downstream of LexA binding sites in a yeast expression system. The strong similarity between hSNF2L and these yeast and drosophila genes suggest that the mammalian genes are part of an evolutionarily conserved family that has been implicated as global activators of transcription in yeast and fruitflies but whose function in mammals remains unknown.

Simultaneous determination of phenolphthalein and phenolphthalein glucuronide from dog serum, urine and bile by high-performance liquid chromatography.

A procedure is described to simultaneously quantitate phenolphthalein and its glucuronide metabolite from dog serum, urine and bile using high-performance liquid chromatography. The major advantages of this over pre-existing methods include direct analysis of the parent compound and glucuronide metabolite without enzymatic hydrolysis, increased sensitivity and the potential for automation of a large number of samples. Analytes were extracted from serum and urine using a combination of liquid- and solid-phase extraction methodology. Bile samples were analyzed directly after a twenty-fold dilution with mobile phase. The components plus internal standard were separated by reversed-phase high-performance liquid chromatography using step gradient elution and quantitated by the absorbance of ultraviolet light at 230 nm. Limits of detection from 1 ml of serum, 0.1 ml of urine and 0.05 ml of bile were 0.1, 0.5 and 10 microgram/ml for phenolphthalein and 0.1, 10 and 50 microgram/ml for phenolphthalein glucuronide, respectively.