Demonstration of a mitochondrial DNA-compatible workflow for genetically variant peptide identification from human hair samples.

Hair is an evidentiary sample that typically does not provide sufficient nuclear DNA for forensic analysis. Therefore, state-of-the-art forensic examination for hair samples include subjective microscopic evaluation, mitochondrial DNA (mtDNA) analysis, and more recently, proteomic genotyping that uses protein variation in the form of genetically variant peptides (GVPs) to infer single nucleotide polymorphism (SNP) alleles. Since many cases involve limited sample amounts (approximately 2 cm or less), any additional destructive testing (besides mtDNA) would be excluded. If a mtDNA-compatible protein extraction workflow could be developed, GVPs would provide additional forensic value without sacrificing any portion of the original hair sample. Here, we demonstrate an optimized method that can be used to obtain both whole genome mtDNA and putative GVP profiles from a single limited hair sample. The method involves urea-based extraction of proteins from hair, followed by buffer exchange and protease digestion. Peptides are eluted through a 30 kDa membrane and analyzed using traditional proteomic techniques. DNA is subsequently extracted from the filter and analyzed using whole mt-genome analysis. The method was verified with a range of hair sample types (head, pubic, and arm hair) from a diverse cohort of 22 individuals. Specifically, putative GVP profiles and mtDNA haplotypes concordant with buccal swab samples from the same donor were obtained from 22 individuals. Further, the utility of the method was verified across two different laboratories. The method is applicable for proteomic-based GVP analysis and mt-genome analysis for forensic research applications.

Surface enhanced fluorescence by metallic nano-apertures associated with stair-gratings.

Metallic nano-apertures associated with stair-gratings are proposed for surface enhanced fluorescence with high excitation enhancement and narrow emission beaming effect. Fluorescence correlation spectroscopy method was utilized to analyze the fluorescence trace and fluorescence enhancement, and the angular patterns of fluorescent emission were measured with the back focal plane imaging method. The stair-grating presents a strong optical response which covering well both the excitation and the emission bands of the photoluminescence process. Such high enhancement and narrow directionality by the stair-gratings would enable the detection of single molecules with low numerical aperture objective effectively.

Post-mortem proteolysis and tenderisation are more rapid and extensive in female duck breast muscle.

The post-mortem proteolysis and tenderisation between male and female duck breast muscles were compared. The results showed that µ-calpain activity, desmin content and shear force decreased more quickly in female than in male samples stored at 5°C. It is suggested that the post-mortem proteolysis and tenderisation are more rapid and extensive in female duck breast muscle.

Targeting post-translational modifications of histones for cancer therapy.

Post-translational modifications (PTMs) on histones including acetylation, methylation, phosphorylation, citrullination, ubiquitination, ADP ribosylation, and sumoylation, play important roles in different biological events including chromatin dynamics, DNA replication, and transcriptional regulation. Aberrant histones PTMs leads to abnormal gene expression and uncontrolled cell proliferation, followed by development of cancers. Therefore, targeting the enzymes required for specific histone PTMs holds a lot of potential for cancer treatment. In this review article, we retrospect the latest studies in the regulations of acetylation, methylation, and phosphorylation of histones. We also summarize inhibitors/drugs that target these modifications for cancer treatment.

Saturated fatty acid induces cancer stem cell-like properties in human hepatoma cells.

Hepatic steatosis has been reported to be a risk factor for the development of liver cancer. The underlying mechanism for carcinogenesis remains to be elucidated. It has been postulated that cancer stem cells (CSCs) within tumor tissues are a subset of cells with stem cell properties of self-renewal and undifferentiation. The purpose of this study was to investigate the effects of a saturated fatty acid, palmitate (PA), on CSC-like properties of human hepatoma HepG2 cells. We investigated the effects of PA on HepG2 cells and primary rat hepatocytes (PRH) by exposing them to PA to induce lipid accumulation. Significant fat accumulation was observed by Oil Red O staining in cells exposed to PA, and it was accompanied by significant increase in NFκB (p65) nuclear translocation in HepG2 cells. Notably, PA significantly enhanced the sphere forming ability of HepG2 cells, but not PRH. Furthermore, PA significantly increased stemness gene expressions of Sox2 and Oct4, and sonic hedgehog (Shh) production. Notably, NFκB inhibitors, N-Acetyl-L-cysteine and pyrollidine dithiocarbamate, and a NOX inhibitor, diphenyleneiodonium, significantly attenuated PA-induced sphere forming ability of HepG2 cells. Our results suggest that lipid accumulation may not only induce pro-inflammatory responses in hepatocytes but may also activate CSC-like properties of hepatoma cells through NFκB activation.

Mapping QTL for an adaptive trait: the length of caudal fin in Lates calcarifer.

The caudal fin represents a fundamental design feature of fishes and plays an important role in locomotor dynamics in fishes. The shape of caudal is an important parameter in traditional systematics. However, little is known about genes involved in the development of different forms of caudal fins. This study was conducted to identify and map quantitative trait loci (QTL) affecting the length of caudal fin and the ratio between tail length and standard body length in Asian seabass (Lates calcarifer). One F1 family containing 380 offspring was generated by crossing two unrelated individuals. One hundred and seventeen microsatellites almost evenly distributed along the whole genome were genotyped. Length of caudal fin at 90 days post-hatch was measured. QTL analysis detected six significant (genome-wide significant) and two suggestive (linkage-group-wide significant) QTL on seven linkage groups. The six significant QTL explained 5.5-16.6% of the phenotypic variance, suggesting these traits were controlled by multiple genes. Comparative genomics analysis identified several potential candidate genes for the length of caudal fin. The QTL for the length of caudal fin detected for the first time in marine fish may provide a starting point for the future identification of genes involved in the development of different forms of caudal fins in fishes.

A standard panel of microsatellites for Asian seabass (Lates calcarifer).

Microsatellites are the most popular markers for parentage assignment and population genetic studies. To meet the demand for international comparability for genetic studies of Asian seabass, a standard panel of 28 microsatellites has been selected and characterized using the DNA of 24 individuals from Thailand, Malaysia, Indonesia and Australia. The average allele number of these markers was 10.82 +/- 0.71 (range: 6-19), and the expected heterozygosity averaged 0.76 +/- 0.02 (range: 0.63-1.00). All microsatellites showed Mendelian inheritance. In addition, eight standard size controls have been developed by cloning a set of microsatellite alleles into a pGEM-T vector to calibrate allele sizes determined by different laboratories, and are available upon request. Seven multiplex PCRs, each amplifying 3-5 markers, were optimized to accurately and rapidly genotype microsatellites. Parentage assignment using 10 microsatellites in two crosses (10 x 10 and 20 x 20) demonstrated a high power of these markers for revealing parent-sibling connections. This standard set of microsatellites will standardize genetic diversity studies of Asian seabass, and the multiplex PCR sets will facilitate parentage assignment.

Systematic reviews of low back pain prognosis had variable methods and results: guidance for future prognosis reviews.

OBJECTIVE: Systematic reviews of prognostic factors for low back pain vary substantially in design and conduct. The objective of this study was to identify, describe, and synthesize systematic reviews of low back pain prognosis, and explore the potential impact of review methods on the conclusions. STUDY DESIGN AND SETTING: We identified 17 low back pain prognosis reviews published between 2000 and 2006. One reviewer extracted and a second checked review characteristics and results. Two reviewers independently assessed review quality. RESULTS: Review questions and selection criteria varied; there were both focused and broad reviews of prognostic factors. A quarter of reviews did not clearly define search strategies. The number of potential citations identified ranged from 15 to 4,458 and the number of included prognosis studies ranged from 3 to 32 (of 162 distinct citations included across reviews). Seventy percent of reviews assessed quality of included studies, but assessed only a median of four of six potential biases. All reviews reported associations based on statistical significance; they used various strategies for syntheses. Only a small number of important prognostic factors were consistently reported: older age, poor general health, increased psychological or psychosocial stress, poor relations with colleagues, physically heavy work, worse baseline functional disability, sciatica, and the presence of compensation. We found discrepancies across reviews: differences in some selection criteria influenced studies included, and various approaches to data interpretation influenced review conclusions about evidence for specific prognostic factors. CONCLUSION: There is an immediate need for methodological work in the area of prognosis systematic reviews. Because of methodological shortcomings in the primary and review literature, there remains uncertainty about reliability of conclusions regarding prognostic factors for low back pain.

Pegylated interferons for chronic hepatitis C virus infection: an indirect analysis of randomized trials.

Dual therapy with pegylated interferon and ribavirin is recommended for patients with chronic hepatitis C virus infection who meet criteria for treatment, but it is unclear whether pegylated interferon alfa-2a or pegylated interferon alfa-2b is more effective or associated with fewer adverse events. Because data from head-to-head trials of pegylated interferon regimens are sparse, we performed adjusted indirect analysis using trials comparing dual therapy with pegylated interferon alfa-2a or pegylated interferon alfa-2b vs dual therapy with non-pegylated interferon. We searched for potentially relevant randomized controlled trials using electronic databases and reference lists. A total of 16 trials met inclusion criteria. Adjusted indirect comparisons found no statistically significant differences between dual therapy with pegylated interferon alfa-2a and dual therapy with pegylated interferon alfa-2b on the outcomes sustained virologic response [relative risk (RR) = 1.59, 95% CI: 0.56-4.46], withdrawal due to adverse events (RR = 0.86, 95% CI: 0.29-2.55), anaemia (RR = 1.67, 95% CI: 0.32-8.84), depression (RR = 1.09, 95% CI: 0.41-2.90) or flu-like symptoms (RR = 1.10, 95% CI: 0.53-2.29). Adjusting for potential publication bias and stratifying analyses by indicators of methodological quality, human immunodeficiency virus infection status, hepatitis C virus genotype, dose of ribavirin or dose of pegylated interferon did not change conclusions. There is insufficient evidence to support conclusions that dual therapy with one pegylated interferon is superior to the other. However, because estimates are imprecise, our results also do not rule out a clinically significant difference. Head-to-head trials are needed to verify the results of indirect analyses and provide additional guidance on optimal treatment choices.

The complete mitochondrial genome sequence and characterization of single-nucleotide polymorphisms in the control region of the Asian seabass (Lates calcarifer).

We determined the complete mtDNA nucleotide sequence of Lates calcarifer using the shotgun sequencing method. The mitochondrial DNA (mtDNA) was 16,535 base pairs (bp) in length, and contained 13 protein coding genes, 22 transfer RNAs, 2 ribosomal RNAs, and one major noncoding control region (CR). The CR was unusually short at only 768 bp. A striking feature of the mitochondrial genome was the high G+C content (46.1%), which is among the highest in fish. The gene order was identical to that of a typical vertebrate. Phylogenetic analyses using concatenated amino acid sequences of 12 protein-coding genes of 30 fish species representing 14 suborders clearly showed Lates calcarifer was located in the cluster of fish species from the order Perciformes, supporting the traditional systematic classification. We characterized single-nucleotide polymorphisms (SNPs) in the CR by sequencing the complete CR of 25 individuals obtained from Australia and Singapore. A total of 68 SNPs were detected. Eighteen SNPs were fixed with alternative nucleotides in Australian and Singapore seabass, and these SNPs could be used for differentiating fish from the two countries.

Changes in hospitality workers' exposure to secondhand smoke following the implementation of New York's smoke-free law.

OBJECTIVE: To assess the impact on hospitality workers' exposure to secondhand smoke of New York's smoke-free law that prohibits smoking in all places of employment, including restaurants, bars, and bowling facilities. DESIGN: Pre-post longitudinal follow up design. SETTINGS: Restaurants, bars, and bowling facilities in New York State. SUBJECTS: At baseline, 104 non-smoking workers in restaurants, bars, and bowling facilities were recruited with newspaper ads, flyers, and radio announcements. Of these, 68 completed a telephone survey and provided at least one saliva cotinine specimen at baseline. At three, six, and 12 month follow up studies, 47, 38, and 32 workers from the baseline sample of 68 completed a telephone survey and provided at least one saliva cotinine specimen. INTERVENTION: The smoke-free law went into effect 24 July 2003. MAIN OUTCOME MEASURES: Self reported sensory and respiratory symptoms and exposure to secondhand smoke; self administered saliva cotinine specimens. Analyses were limited to subjects in all four study periods who completed a telephone survey and provided at least one saliva cotinine specimen. RESULTS: All analyses were limited to participants who completed both an interview and a saliva specimen for all waves of data collection (n = 30) and who had cotinine concentrations < or = 15 ng/ml (n = 24). Hours of exposure to secondhand smoke in hospitality jobs decreased from 12.1 hours (95% confidence interval (CI) 8.0 to 16.3 hours) to 0.2 hours (95% CI -0.1 to 0.5 hours) (p < 0.01) and saliva cotinine concentration decreased from 3.6 ng/ml (95% CI 2.6 to 4.7 ng/ml) to 0.8 ng/ml (95% CI 0.4 to 1.2 ng/ml) (p < 0.01) from baseline to the 12 month follow up. The prevalence of workers reporting sensory symptoms declined from 88% (95% CI 66% to 96%) to 38% (95% CI 20% to 59%) (p < 0.01); there was no change in the overall prevalence of upper respiratory symptoms (p < 0.16). CONCLUSION: New York's smoke-free law had its intended effect of protecting hospitality workers from exposure to secondhand smoke within three months of implementation. One year after implementation, the results suggest continued compliance with the law.

Recent advances in radiotherapy for head and neck cancers.

Advancements in surgery have made it possible to resect cancers that had previously been regarded as incurable. Similarly, new developments in radiation oncology have helped improve the outlook for patients with locally advanced or recurrent head and neck cancers. Among these advancements are refinements in altered fractionation, three-dimensional conformal radiotherapy, intensity-modulated radiotherapy, stereotactic radiosurgery and fractionated stereotactic radiotherapy, neutron-beam radiotherapy, charged-particle radiotherapy, and intraoperative radiotherapy. These recent developments have allowed radiation oncologists to escalate the dose of radiation delivered to tumors while minimizing the dose delivered to surrounding normal tissue. Additionally, more continues to be learned about the optimum delivery of chemotherapy. This article provides an update on the status of these new developments in the treatment of head and neck cancers.

Acute urinary toxicity following transperineal prostate brachytherapy using a modified Quimby loading method.

PURPOSE: To examine the acute urinary toxicity following transperineal prostate implant using a modified Quimby loading method with regard to time course, severity, and factors that may be associated with a higher incidence of morbidity. METHODS AND MATERIALS: One hundred thirty-nine patients with prostate adenocarcinoma treated with brachytherapy from 1997 through 1999 had follow-up records available for review. Patients considered for definitive brachytherapy alone included those with prostate specific antigen (PSA) < or = 6, Gleason score (GS) < or = 6, clinical stage < T2b, and prostate volumes generally less than 40 cc. Patients with larger prostate volumes were given neoadjuvant antiandrogen therapy. Those with GS > 6, PSA > 6, or Stage > T2a were treated with external beam radiation therapy followed by brachytherapy boost. Sources were loaded according to a modified Quimby method. At each follow-up, toxicity was graded based on a modified RTOG urinary toxicity scale. RESULTS: Acute urinary toxicity occurred in 88%. Grade I toxicity was reported in 23%, grade II in 45%, and grade III in 20%, with 14% requiring prolonged (greater than 1 week) intermittent or indwelling catheterization. Overall median duration of symptoms was 12 months. There was no difference in duration of symptoms between patients treated with I-125 or Pd-103 sources (p = 0.71). After adjusting for GS and PSA, multivariate logistic regression analysis showed higher incidence of grade 3 toxicity in patients with larger prostate volumes (p = 0.002), and those with more seeds implanted (p < 0.001). Higher incidence of prolonged catheterization was found in patients receiving brachytherapy alone (p = 0.01), with larger prostate volumes (p = 0.01), and those with more seeds implanted (p < 0.001). CONCLUSION: Interstitial brachytherapy for prostate cancer leads to a high incidence of acute urinary toxicity, most of which is mild to moderate in severity. A prolonged need for catheterization can occur in some patients. Patients receiving brachytherapy alone, those with prostate volumes greater than 30 cc, and those implanted with a greater number of seeds have the highest incidence of significant toxicity.

Recurrent thromboembolic disease following splenectomy for pyruvate kinase deficiency.

We report a case of recurrent thromboembolic disease and chronic pulmonary hypertension in an adult patient with pyruvate kinase deficiency who underwent splenectomy as a child. Thromboembolism has been reported as a complication following splenectomy for various hereditary chronic hemolytic anemias. To our knowledge, this association has not been described in patients specifically with pyruvate kinase deficiency. Our patient presented at age 37 with recurrent pulmonary emboli, 36 years after splenectomy for severe hemolytic anemia. Work-up for other hypercoagulable states was negative. The mechanism for hypercoagulability in this condition is unclear but may involve a quantitative or qualitative change in disrupted thrombogenic red blood cell membranes that would normally be removed by the spleen. Clinicians should have a high index of suspicion for thrombotic events in these patients, as early diagnosis and treatment can reduce morbidity and mortality, and chronic anticoagulation may help prevent the sequelae of repeated thromboembolic events.

Piperidine renin inhibitors: from leads to drug candidates.

Non-peptidomimetic renin inhibitors of the piperidine type represent a novel structural class of compounds potentially free of the drawbacks seen with peptidomimetic compounds so far. Synthetic optimization in two structural series focusing on improvement of potency, as well as on physicochemical properties and metabolic stability, has led to the identification of two candidate compounds 14 and 23. Both display potent and long-lasting blood pressure lowering effects in conscious sodium-depleted marmoset monkeys and double transgenic rats harboring both the human angiotensinogen and the human renin genes. In addition, 14 normalizes albuminuria and kidney tissue damage in these rats when given over a period of 4 weeks. These data suggest that treatment of chronic renal failure patients with a renin inhibitor might result in a significant improvement of the disease status.

Measurement of myocardial blood flow with PET using 1-11C-acetate.

UNLABELLED: 11C-acetate has been used extensively for the noninvasive assessment of myocardial oxygen consumption and viability with PET. The use of early uptake of acetate by the heart to measure myocardial perfusion has been proposed. This study evaluated the application of 11C-acetate for absolute measurement of myocardial blood flow using a simple compartmental model that does not require blood sampling. METHODS: Eight healthy volunteers and 13 subjects with concentric left ventricular hypertrophy were studied under resting conditions with both 11Cacetate and 15O-water. Myocardial blood flow with 11C-acetate was obtained by fitting the first 3 min of the blood and tissue tracer activity curves to a two-compartment model. Flows obtained were compared with a validated approach using 15O-water. RESULTS: In healthy volunteers, regional myocardial perfusion at rest estimated with 11C-acetate was comparable with values obtained with 15O-water (1.06 +/- 0.25 and 0.96 +/- 0.12 mL/g/min, respectively). Perfusion in subjects with left ventricular hypertrophy was also comparable if the recovery coefficient (FMM) used was corrected for ventricular mass. If a fixed FMM was used, flow was greatly overestimated. FMM could be estimated from left ventricular mass (FMM = 0.46 + 0.002 x mass, r = 0.86, P < 0.0001). CONCLUSION: The results of this study suggest that 11C-acetate can be applied to quantitatively estimate myocardial perfusion under resting conditions using a two-compartment model without the need for blood sampling, provided that an appropriate FMM is chosen. This approach should increase the usefulness of this tracer and obviate administration of a separate tracer to independently measure perfusion.