mTOR-driven glycolysis governs induction of innate immune responses by bronchial epithelial cells exposed to the bacterial component flagellin.

Human bronchial epithelial (HBE) cells play an essential role during bacterial infections of the airways by sensing pathogens and orchestrating protective immune responses. We here sought to determine which metabolic pathways are utilized by HBE cells to mount innate immune responses upon exposure to a relevant bacterial agonist. Stimulation of HBE cells by the bacterial component flagellin triggered activation of the mTOR pathway resulting in an increased glycolytic flux that sustained the secretory activity of immune mediators by HBE cells. The mTOR inhibitor rapamycin impeded glycolysis and limited flagellin-induced secretion of immune mediators. The role of the mTOR pathway was recapitulated in vivo in a mouse model of flagellin-triggered lung innate immune responses. These data demonstrate that metabolic reprogramming via the mTOR pathway modulates activation of the respiratory epithelium, identifying mTOR as a potential therapeutic target to modulate mucosal immunity in the context of bacterial infections.

DNA Commission of the International Society for Forensic Genetics: Recommendations on the validation of software programs performing biostatistical calculations for forensic genetics applications.

The use of biostatistical software programs to assist in data interpretation and calculate likelihood ratios is essential to forensic geneticists and part of the daily case work flow for both kinship and DNA identification laboratories. Previous recommendations issued by the DNA Commission of the International Society for Forensic Genetics (ISFG) covered the application of bio-statistical evaluations for STR typing results in identification and kinship cases, and this is now being expanded to provide best practices regarding validation and verification of the software required for these calculations. With larger multiplexes, more complex mixtures, and increasing requests for extended family testing, laboratories are relying more than ever on specific software solutions and sufficient validation, training and extensive documentation are of upmost importance. Here, we present recommendations for the minimum requirements to validate bio-statistical software to be used in forensic genetics. We distinguish between developmental validation and the responsibilities of the software developer or provider, and the internal validation studies to be performed by the end user. Recommendations for the software provider address, for example, the documentation of the underlying models used by the software, validation data expectations, version control, implementation and training support, as well as continuity and user notifications. For the internal validations the recommendations include: creating a validation plan, requirements for the range of samples to be tested, Standard Operating Procedure development, and internal laboratory training and education. To ensure that all laboratories have access to a wide range of samples for validation and training purposes the ISFG DNA commission encourages collaborative studies and public repositories of STR typing results.

D5S2500 is an ambiguously characterized STR: Identification and description of forensic microsatellites in the genomics age.

In the process of establishing short tandem repeat (STR) sequence variant nomenclature guidelines in anticipation of expanded forensic multiplexes for massively parallel sequencing (MPS), it was discovered that the STR D5S2500 has multiple positions and genomic characteristics reported. This ambiguity is because the marker named D5S2500 consists of two different microsatellites forming separate components in the capillary electrophoresis multiplexes of Qiagen's HDplex (Hilden, Germany) and AGCU ScienTech's non-CODIS STR 21plex (Wuxi, Jiangsu, China). This study outlines the genomic details used to identify each microsatellite and reveals the D5S2500 marker in HDplex has the correctly assigned STR name, while the D5S2500 marker in the AGCU 21plex, closely positioned a further 1643 nucleotides in the human reference sequence, is an unnamed microsatellite. The fact that the D5S2500 marker has existed as two distinct STR loci undetected for almost ten years, even with reported discordant genotypes for the standard control DNA, underlines the need for careful scrutiny of the genomic properties of forensic STRs, as they become adapted for sequence analysis with MPS systems. We make the recommendation that precise chromosome location data must be reported for any forensic marker under development but not in common use, so that the genomic characteristics of the locus are validated to the same level of accuracy as its allelic variation and forensic performance. To clearly differentiate each microsatellite, we propose the name D5S2800 be used to identify the Chromosome-5 STR in the AGCU 21plex.

Revision of the SNPforID 34-plex forensic ancestry test: Assay enhancements, standard reference sample genotypes and extended population studies.

A revision of an established 34 SNP forensic ancestry test has been made by swapping the under-performing rs727811 component SNP with the highly informative rs3827760 that shows a near-fixed East Asian specific allele. We collated SNP variability data for the revised SNP set in 66 reference populations from 1000 Genomes and HGDP-CEPH panels and used this as reference data to analyse four U.S. populations showing a range of admixture patterns. The U.S. Hispanics sample in particular displayed heterogeneous values of co-ancestry between European, Native American and African contributors, likely to reflect in part, the way this disparate group is defined using cultural as well as population genetic parameters. The genotyping of over 700 U.S. population samples also provided the opportunity to thoroughly gauge peak mobility variation and peak height ratios observed from routine use of the single base extension chemistry of the 34-plex test. Finally, the genotyping of the widely used DNA profiling Standard Reference Material samples plus other control DNAs completes the audit of the 34-plex assay to allow forensic practitioners to apply this test more readily in their own laboratories.

Forensic performance of two insertion-deletion marker assays.

Improving the amplification and analysis of highly degraded DNA extracts has been a longstanding area of research in forensic genetics. One of the most promising recent developments in analysis of degraded DNA is the availability of short, biallelic insertion-deletion length polymorphisms (InDels) in highly multiplexed assays. InDels share many of the favourable characteristics of single-nucleotide polymorphisms (SNPs) that make them ideal markers for analysis of degraded DNA, including: analysis in short amplicon size ranges, high multiplexing capability and low mutation rates. In addition, as length-based polymorphisms, InDels can be analysed with the same simple dye-labelled PCR primer methods as standard forensic short tandem repeats. Separation and detection of fluorescently dye-labelled PCR products by capillary electrophoresis eliminate the multiple step protocols required by SNP typing with single-base extension assays and provide a closer relationship between the input DNA and the profile peak height ratios. Therefore InDel genotyping represents an effective new approach for human identification that adds informative new loci to the existing battery of forensic markers. To assess the utility of InDels for forensic analysis, we characterised population variation with two InDel identification assays: the 30-plex Qiagen DIPplex panel and a 38-plex panel developed by Pereira et al. in 2009. Allele frequencies were generated for the 68 markers in US African American, Caucasian, East Asian and Hispanic samples. We made a thorough assessment of the individual and combined performance of the InDel sets, as well as characterising profile artifacts and other issues related to the routine use of these newly developed forensic assays based on artificially degraded DNA and mixed source samples.

Biology and Genetics of New Autosomal STR Loci Useful for Forensic DNA Analysis.

Short tandem repeats (STRs) are regions of tandemly repeated DNA segments found throughout the human genome that vary in length (through insertion, deletion, or mutation) with a core repeated DNA sequence. Forensic laboratories commonly use tetranucleotide repeats, containing a four base pair (4-bp) repeat structure such as GATA. In 1997, the Federal Bureau of Investigation (FBI) Laboratory selected 13 STR loci that form the backbone of the U.S. national DNA database. Building on the European expansion in 2009, the FBI announced plans in April 2011 to expand the U.S. core loci to as many as 20 STRs to enable more global DNA data sharing. Commercial STR kits enable consistency in marker use and allele nomenclature between laboratories and help improve quality control. The STRBase website, maintained by the U.S. National Institute of Standards and Technology (NIST), contains helpful information on STR markers used in human identity testing.

Predicting the human in vivo performance of different oral capsule shell types using a novel in vitro dynamic gastric model.

The disintegration of a capsule shell may determine the onset of drug dissolution from capsule formulations. In this study, the release of a rapidly dissolving model drug (paracetamol), from two hydroxypropyl methylcellulose capsules containing either carageenan (HPMC-C) or gellan gum (HPMC-G) and one hard gelatin (HG) capsule, were investigated using a conventional in vitro model, the USP dissolution apparatus I, and a novel in vitro model of the human gastric compartment, the dynamic gastric model (DGM). The results obtained in vitro were compared with in vivo gamma scintigraphy human data and in vivo gastric emptying profiles available in the literature. The drug release from HPMC-G capsules, observed with the USP dissolution apparatus I, was delayed with respect to the other two capsules, while the results obtained from the DGM in the fasted state were closer together, which was in agreement with data from the in vivo studies. In the fasted state, the capsule rupture times obtained from the DGM were similar to those observed by gamma scintigraphy in vivo studies. In the fed state, the 'apparent' rupture times observed with the DGM were delayed compared to fasted, and were even longer than those observed by scintigraphy in vivo for HPMC-G and HG capsules. However, these discrepancies can reasonably be explained by considering the impact of food upon dispersion of the capsule contents and the sampling from the DGM, when compared to the human scintigraphy experiments.

Comparative efficacy of four commercially available heartworm preventive products against the MP3 laboratory strain of Dirofilaria immitis.

A controlled laboratory study was conducted to evaluate the efficacy of four commercial products administered as a single treatment for the prevention of heartworm disease caused by Dirofilaria immitis in dogs. Forty-four commercially sourced Beagle dogs, 6-7 months of age, were received at the test site (Auburn University, Department of Pathobiology) on Study Day (SD) -72 to begin acclimation. On SD -30, each dog was inoculated subcutaneously with 100 infective, third-stage D. immitis larvae (MP3 strain, TRS Laboratories, Inc., Athens, GA). On SD -1, 40 dogs weighing 18.2-25.3 lbs were ranked by decreasing body weight and randomized to five groups of eight dogs each. On SD 0, the dogs assigned to Group 1 were treated orally with ivermectin/pyrantel pamoate chewable tablets, Group 2 dogs were treated orally with milbemycin oxime flavored tablets, Group 3 dogs were treated with selamectin topical solution, and Group 4 dogs were treated with imidacloprid/moxidectin topical solution. Group 5 dogs remained nontreated. Dosages for dogs in Groups 1-4 were based on the individual body weight of each dog and current labeled dose banding for each commercial product. All dogs were fasted overnight prior to treatment. Food was returned four hours after treatment. Animals were observed for abnormal clinical signs involving eyes, feces, respiration, behavioral attitude, locomotion/musculature, or skin conditions at prescribed intervals immediately after treatment and at twice daily intervals thereafter. On SD 90, whole blood was collected and tested for adult heartworm antigen. On SDs 119/120, the dogs were euthanized and subjected to necropsy examination for recovery of adult D. immitis and/or worm fragments. At necropsy, all 8 dogs in the nontreated group were infected with adult D. immitis (34-70 worms/dog, geometric mean (GM)=51.6 worms/dog). One or more adult D. immitis and/or worm fragments were recovered from 7 of 8 of the dogs each in Groups 1-3 (87.5% were heartworm positive). The respective GM worm burdens/dog for Groups 1-3 was 2.3, 2.4, and 2.3 which resulted in 95.6, 95.4 and 95.5% efficacy, respectively. No worms were recovered from any of the 8 dogs in Group 4 resulting in 100% efficacy.

MRI studies of the hydrodynamics in a USP 4 dissolution testing cell.

We present a detailed study of hydrodynamics inside the flow-through dissolution apparatus when operated according to USP recommendations. The pulsatile flow inside the flow-through cell was measured quantitatively using magnetic resonance imaging (MRI) at a spatial resolution of 234 × 234 µm(2) and slice thickness of 1 mm. We report the experimental protocols developed for in situ MRI studies and the effect that the operating conditions and tablet orientation have on the hydrodynamics inside commercial flow cells. It was found that the flow field inside the dissolution cells was, at most operating conditions, heterogeneous, rather than fully developed laminar flow, and characterised by re-circulation and backward flow. A model tablet was shown to be contacted by a wide distribution of local velocities as a function of position and orientation in the flow cell. The use of 1 mm beads acted as a distributor of the flow but did not suffice to ensure a fully developed laminar flow profile. These results emphasise the necessity to understand the influence of test conditions on dissolution behaviour in defining robust flow-through dissolution methods.

Forensic typing of autosomal SNPs with a 29 SNP-multiplex--results of a collaborative EDNAP exercise.

We report the results of an inter-laboratory exercise on typing of autosomal single nucleotide polymorphisms (SNP) for forensic genetic investigations in crime cases. The European DNA Profiling Group (EDNAP), a working group under the International Society for Forensic Genetics (ISFG), organised the exercise. A total of 11 European and one US forensic genetic laboratories tested a subset of a 52 SNP-multiplex PCR kit developed by the SNPforID consortium. The 52 SNP-multiplex kit amplifies 52 DNA fragments with 52 autosomal SNP loci in one multiplex PCR. The 52 SNPs are detected in two separate single base extension (SBE) multiplex reactions with 29 and 23 SNPs, respectively, using SNaPshot kit, capillary electrophoresis and multicolour fluorescence detection. For practical reasons, only the 29 SBE multiplex reaction was carried out by the participating laboratories. A total of 11 bloodstains on FTA cards including a sample of poor quality and a negative control were sent to the laboratories together with the essential reagents for the initial multiplex PCR and the multiplex SBE reaction. The total SNP locus dropout rate was 2.8% and more than 50% of the dropouts were observed with the poor quality sample. The overall rate of discrepant SNP allele assignments was 2.0%. Two laboratories reported 60% of all the discrepancies. Two laboratories reported all 29 SNP alleles in all 10 positive samples correctly. The results of the collaborative exercise were surprisingly good and demonstrate that SNP typing with SBE, capillary electrophoresis and multicolour detection methods can be developed for forensic genetics.

Effect of source and concentration of selenium on growth performance and selenium retention in broiler chickens.

A 6-wk experiment was conducted using 360 one-day-old Jumbo Cornish Cross broiler chicks to evaluate the effects of the source and concentration of Se on growth performance and Se retention. Broilers were fed corn-soy-based diets formulated to contain 0 (negative control), 0.1, 0.2, or 0.3 ppm of supplemental Se from SelenoSource AF (Se yeast A, Diamond V Mills, Cedar Rapids, IA), 0.3 ppm of Se from Sel-Plex (Se yeast B, Alltech, Nicholasville, KY), or 0.3 ppm of Se from sodium selenite. Starter diets were fed for the first 21 d (6 replicates of 10 broilers per treatment). On d 21, broilers were regrouped within dietary treatments to finisher cages (11 replicates of 5 broilers per treatment) and fed finisher diets for the following 21 d. Feed intake was measured daily. Body weights of the broilers were measured on d 1, 21, and 42. Excreta samples were collected for 4 consecutive days at the end of each 3-wk feeding period to estimate Se retention. Blood samples were collected from 11 broilers per treatment on d 42 to determine blood Se concentrations and glutathione peroxidase (GPX) activity. Selenium supplementation did not influence (P > 0.05) the growth performance of broilers at 42 d of age. Blood Se and GPX activities increased (P < 0.05) as the concentration of Se in diets increased. Selenium retention as a percentage of Se intake decreased linearly (P = 0.01) as the concentration of Se increased in the diets. At 0.3 ppm, blood Se and GPX activities were higher (P = 0.01) for broilers supplemented with Se yeast A compared with Se yeast B when expressed as relative to Se intake. Selenium retention was greater when organic Se was supplemented (P = 0.01), compared with inorganic Se. Results of the study suggest that Se retention had an inverse relationship with the concentration of Se supplemented and was influenced by the dietary source of Se. Whole blood Se relative to Se intake suggests a higher bioavailability of Se from the organic source compared with inorganic sodium selenite and that differences in bioavailability may exist between organic sources of Se.

The impact of additional Y-STR loci on resolving common haplotypes and closely related individuals.

Commercial Y-STR kits have permitted laboratories to go beyond the original nine minimal haplotype loci (MHL) and to discover the advantage of additional Y-STR loci in resolving common haplotypes. In an effort to examine the impact of Y-STR markers beyond the 17 loci now available in commercial kit form, new Y-STR loci are being investigated on a common set of samples representative of the major U.S. population groups. Additional Y-STRs can also increase the power of discrimination between closely related male individuals, which is important not only in forensics but also in the paternity and genetic genealogy communities.

DNA Commission of the International Society of Forensic Genetics (ISFG): an update of the recommendations on the use of Y-STRs in forensic analysis.

The DNA Commission of the International Society of Forensic Genetics (ISFG) regularly publishes guidelines and recommendations concerning the application of DNA polymorphisms to the problems of human identification. A previous recommendation published in 2001 has already addressed Y-chromosome polymorphisms, with particular emphasis on short tandem repeats (STRs). Since then, the use of Y-STRs has become very popular, and numerous new loci have been introduced. The current recommendations address important aspects to clarify problems regarding the nomenclature, the definition of loci and alleles, population genetics and reporting methods

DNA Commission of the International Society of Forensic Genetics (ISFG): an update of the recommendations on the use of Y-STRs in forensic analysis.

The DNA Commission of the International Society of Forensic Genetics (ISFG) regularly publishes guidelines and recommendations concerning the application of DNA polymorphisms to the problems of human identification. A previous recommendation published in 2001 has already addressed Y-chromosome polymorphisms, with particular emphasis on short tandem repeats (STRs). Since then, the use of Y-STRs has become very popular, and a numerous new loci have been introduced. The current recommendations address important aspects to clarify problems regarding the nomenclature, the definition of loci and alleles, population genetics and reporting methods.

Analysis of artificially degraded DNA using STRs and SNPs--results of a collaborative European (EDNAP) exercise.

Recently, there has been much debate about what kinds of genetic markers should be implemented as new core loci that constitute national DNA databases. The choices lie between conventional STRs, ranging in size from 100 to 450 bp; mini-STRs, with amplicon sizes less than 200 bp; and single nucleotide polymorphisms (SNPs). There is general agreement by the European DNA Profiling Group (EDNAP) and the European Network of Forensic Science Institutes (ENFSI) that the reason to implement new markers is to increase the chance of amplifying highly degraded DNA rather than to increase the discriminating power of the current techniques. A collaborative study between nine European and US laboratories was organised under the auspices of EDNAP. Each laboratory was supplied with a SNP multiplex kit (Foren-SNPs) provided by the Forensic Science Service, two mini-STR kits provided by the National Institute of Standards and Technology (NIST) and a set of degraded DNA stains (blood and saliva). Laboratories tested all three multiplex kits, along with their own existing DNA profiling technique, on the same sets of degraded samples. Results were collated and analysed and, in general, mini-STR systems were shown to be the most effective. Accordingly, the EDNAP and ENFSI working groups have recommended that existing STR loci are reengineered to provide smaller amplicons, and the adoption of three new European core loci has been agreed.