Targeted next generation sequencing panel for HPV genotyping in cervical cancer.

Cervical cancer are generally caused by a persistent infection with the oncogenic virus, HPV. Patients with HPV integration are more prone to develop cervical cancer than patients without integration. In this proof-of-concept study, we aimed to develop a sensitive method based on targeted amplicon based NGS for early and precise detection of high-risk HPV-genotypes that are highly associated with the development of cervical cancer. Furthermore, we aimed to investigate if amplicon based NGS allowed for HPV genotyping in cervical lesions and whether it could detect HPV integration. The cohort included a group of CIN3+ biopsies (n = 64), CIN2 samples that progressed (n = 5), CIN2 samples that regressed (n = 3), healthy controls (n = 10), and plasma samples (n = 10) from cervical cancer patients. Sequencing was performed using a custom targeted NGS panel designed to detect all 25 high-risk and probably high-risk and two low-risk HPV genotypes. The method was validated by the SPF10 PCR-DEIA-LiPA25 assay. In the cohort, the following HPV genotypes were identified: HPV-16, 18, 31, 33, 35, 45, 51, 52, 56, 58, and 59. When comparing the results from the SPF10 PCR-DEIA-LiPA25 analyses with the NGS analyses, there was close to a perfect agreement (K = 0.92) among the genotyped HPV types, while in the two cases with complete disagreement, a third assay was applied, and here the results of the NGS analyses were confirmed. Whereas multiple HPV types were detected by the SPF10 PCR-DEIA-LiPA25 assay, the NGS analysis clearly suggest that there is one predomentant HPV type. The NGS assay was capable of detecting HPV-16 in a previous false-negative sample classified by the INNO-LiPA assay, emphasizing the importance of including multiple regions of the HPV genome when genotyping. For the 10 plasma samples, our NGS analyses showed full agreement with the digital droplet PCR (ddPCR) analyses of HPV positive as well as negative plasma samples. Lastly, the custom panel was capable of detecting the integration of HPV-16 in the SiHa cell line. The HPV panel provides a highly cost-effective method for HPV detection and genotyping, as exemplified by a list price of around 75 € per sample. In conclusion, the current study demonstrates that targeted NGS is capable of detecting and genotyping HPV in both FFPE biopsies and plasma samples. This method provides for early diagnosis and prognosis of cervical cancer disease progression, thereby optimizing the potential of recovery and survival for these patients.

Osteopathic Cranial Manipulative Medicine as a Proposed Addition to the Treatment Regimen for Idiopathic Sudden-Onset Unilateral Sensorineural Hearing Loss: A Case Report.

Adult sudden-onset unilateral sensorineural hearing loss is primarily idiopathic. There are few treatment options with high success rates, but the literature suggests there is equally moderate success with oral or intratympanic steroids, with some evidence to support the use of acupuncture as a salvage treatment. Here, we present a case of sudden unilateral sensorineural hearing loss with otolaryngology evaluation and audiograms before and after osteopathic manipulative treatment (OMT). Osteopathic cranial manipulative medicine (OCMM) directed at eustachian tube dysfunction elicited findings indicating resolution of symptoms was at least partly due to cranial treatment. This report explains a probable anatomic mechanism contributing to hearing loss and suggests treatment options that should be considered as part of the treatment of idiopathic unilateral hearing loss. Future research opportunities are also proposed.

Development of Kudzu (Pueraria Montana var. lobata) Reference Materials for the Determination of Isoflavones and Toxic Elements.

BACKGROUND: In collaboration with the Office of Dietary Supplements at the National Institutes of Health, the National Institute of Standards and Technology issued a suite of botanical matrix reference materials (RMs) and Standard Reference Material® (SRM) for determination of isoflavones and toxic elements in kudzu dietary supplement ingredients. OBJECTIVE: RM 8650 Pueraria montana var. lobata (Kudzu) Rhizome, SRM 3268 Pueraria montana var. lobata (Kudzu) Extract, and RM 8652 Kudzu-Containing Solid Oral Dosage Form were issued with values assigned for isoflavones (puerarin, daidzin, and daidzein), toxic elements (arsenic, cadmium, and lead), and selenium. METHODS: Isoflavone values were assigned using liquid chromatography with UV absorbance or mass spectrometry detection. Element values were assigned using inductively coupled plasma mass spectrometry and results from an interlaboratory comparison exercise. RESULTS: Mass fractions for puerarin were 32.2 ± 3.2 mg/g, 128 ± 13 mg/g, and 68.2 ± 6.9 mg/g in RM 8650, SRM 3268, and RM 8652, respectively. Arsenic increases from 156 ± 14 ng/g to 849 ± 83 ng/g and cadmium decreases from 348 ± 14 ng/g to 82.1 ± 4.9 ng/g from rhizome to extract. CONCLUSION: The kudzu RM/SRM suite complements previously issued soy-related SRMs with values assigned for isoflavones, which have been studied for their potential health benefits, and expands the analytical resource by providing values for puerarin, an isoflavone not found in soy. HIGHLIGHTS: The three new kudzurmaterials are for use in the determination of isoflavones, toxic elements, and selenium. For the isoflavones, these new kudzu materials provide higher levels of daidzin and daidzein than existing soy-related SRMs, and they provide a value for an isoflavone not in existing SRMs (puerarin). Toxic elements in RM 8650 and SRM 3268 provide new botanical matrixes for use by dietary supplement manufacturers for the verification of the safety of their raw materials.

[Brachycephaly in dog and cat: a "human induced" obstruction of the upper airways]. / Brachyzephalie bei Hund und Katze: eine "menschengemachte" Obstruktion der oberen Atemwege.

Selective breeding for exaggerated features caused in many brachycephalic dog and cat breeds virtually a loss of the nose, with serious anatomical and functional consequences. In addition to respiratory and olfactory tasks, in dogs the nose is of vital importance for thermoregulation. As obligatory nose breathers, dogs suffer far more than humans when their nasal ventilation is restricted. An open discussion in the broad public has to motivate authorities and kennel clubs to recognize extreme brachycephalic breeding as seriously affecting animal health and welfare.

[Geometry and function of the dog nose: how does function change when form of the nose is changed?]. / Geometrie und Funktion der Hundenase: Wie ändert sich die Funktion, wenn die Form verändert wird?

Nasal airflow resistance in brachycephalic dogs is significantly elevated compared to normal dogs. LaserAssisted TurbinEctomy (LATE)-surgery as well as xylometazolin were shown to reduce pathologically increased intranasal airway resistance in brachycephalic dogs by approximately 50 %. Impulse oscillometry provides a reliable and sensitive method to examine intranasal stenoses in the canine nose. Acoustic rhinometry allows assessment of changes in cross sectional area and volume of the canine nasal cavity.

Europium-155 in debris from nuclear weapons.

The lithium-drifted germanium detector enables determination of europium-155 on a routine basis in environmental samples contaminated with debris from nuclear weapons. From measurements of europium-155, cesium-144, and strontium-90 in air filters collected between 1961 and 1966, the yield of europium-155 from weapons was estimated at 1400 atoms per 10(6) fissions, which is close to the yield of europium-155 from fast fission of uranium-238.

Multimarker real-time reverse transcription-PCR for quantitative detection of melanoma-associated antigens: a novel possible staging method.

BACKGROUND: Detection of melanoma cells in peripheral blood is a promising method for monitoring haematogenous spread of melanoma cells. It enables us to detect early metastasis and to better stratify candidates for adjuvant immunotherapy. Inconsistent data on the sensitivity and clinical relevance of this method have been reported. STUDY DESIGN: We developed a multimarker real-time reverse transcription-PCR (RT-PCR) for quantification of five melanoma markers: Melan-A, gp100, MAGE-3, MIA and tyrosinase. In this prospective study, 65 patients with resected cutaneous melanoma stage IIB-III were screened. Peripheral blood samples were collected every 3 months for the following 18 months, and circulating melanoma cells were examined and compared with clinical staging results. RESULTS: Eighteen patients relapsed during the trial and showed different types of melanoma progression. All these patients experienced statistically significant tumour marker elevation in the period from 0 to 9 months before the disease progression. MAGE-3 was the most sensitive progression marker. In patients with progression, we observed three concordant positive markers in 39% of cases, two concordant positive markers in 28%, and finally one marker in 33%. CONCLUSIONS: This report describes a multiple-marker real-time RT-PCR, which is able to provide quantitative data on melanoma markers in the peripheral blood of melanoma patients. Measurement of the studied molecular markers in our hands represents a prognostic factor and a useful method for early detection of metastasis and treatment response of melanoma patients.

Applied Concepts in PBPK Modeling: How to Build a PBPK/PD Model.

The aim of this tutorial is to introduce the fundamental concepts of physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) modeling with a special focus on their practical implementation in a typical PBPK model building workflow. To illustrate basic steps in PBPK model building, a PBPK model for ciprofloxacin will be constructed and coupled to a pharmacodynamic model to simulate the antibacterial activity of ciprofloxacin treatment.

Good Practices in Model-Informed Drug Discovery and Development: Practice, Application, and Documentation.

This document was developed to enable greater consistency in the practice, application, and documentation of Model-Informed Drug Discovery and Development (MID3) across the pharmaceutical industry. A collection of "good practice" recommendations are assembled here in order to minimize the heterogeneity in both the quality and content of MID3 implementation and documentation. The three major objectives of this white paper are to: i) inform company decision makers how the strategic integration of MID3 can benefit R&D efficiency; ii) provide MID3 analysts with sufficient material to enhance the planning, rigor, and consistency of the application of MID3; and iii) provide regulatory authorities with substrate to develop MID3 related and/or MID3 enabled guidelines.

Laser-Raman investigation of lysozyme-phospholipid interactions.

The interaction of lysozyme with mixed 1,2-dipalmitoyl-L-phosphatidic acid/1,2-dimyristoyl-L-phosphatidylcholine liposomes was investigated by laser Raman spectroscopy. Substantial changes were observed i the spectra of both the lipid and protein in the mixed liposomes over the range 10-62 degrees C. At temperatures below 27 degrees C, interaction with lipid appears to slightly increase the amount of helical structure in lysozyme at the expense of random conformation. At temperatures above 30 degrees C, considerable beta-sheet is irreversibly formed. Onset of beta-formation appears to coincide with the formation of disordered lipid side-chains in the acidic component of the lipid. At all temperatures, the O-P-O diester stretching mode at 782 cm-1 is much more intense in the lipid/protein mixture than in lipid alone. It is observed that the dimyristoyl phosphatidylcholine chain-disorder transition is lowered by 3 degrees C, while that of the phosphatidic acid is lowered by 12 degrees C, yet the post-transition conformation contains a significantly higher proportion of trans-segments in the presence of lysozyme. These results are interpreted in terms of: (1) a polar interaction between acidic phospholipid and lysozyme at temperatures below either chain-disorder transition, in which lysozyme is essentially excluded from the hydrophobic portion of the lipid and (2) an interaction at higher temperatures which involves the lipid side-chains of dipalmitoyl phosphatidic acid in the disordered state and is manifested by a substantial conformational change.

A laser Raman spectroscopic investigation of phospholipid and protein configurations in hemoglobin-free erythrocyte ghosts.

Configurations of both the protein and lipid components of human red blood cell ghosts are examined by laser Raman spectroscopy. Protein configuration is estimated from bands observed in the Amide III region at 1240-1267 cm-1 in water and the Amide I' region at 1630-1670 cm-1 observed in 2H2O. The protein fraction appears to contain 40-55 percent alpha-helix with little beta-configuration. The hydrophobic side chains of the phospholipid component, as interpreted from the 1060-1130 cm-1 C-C stretching region, are estimated to contain 55-65 percent all-trans rigid configuration. These estimated are within the limits set by other physical techniques.

A threshold explains modulation of neural responses to opposite-contrast stereograms.

Disparity-sensitive neurons respond to contrast-inverted stereograms (aRDS) that do not evoke depth percepts. This is in conflict with the idea that such neurons are the direct correlate of depth perception. However, the output of neurons responding to aRDS may be further processed: neurons at later processing stages show weaker responses to aRDS than early stage neurons. Here, we show that such a response hierarchy emerges in a three-layered neural network. A numerical analysis demonstrates that threshold operations can largely explain the network's behavior as well as the electrophysiological data. An extension of the energy neuron model for disparity-sensitive neurons predicts increased responses to aRDS for an identifiable sub-class of cells and can thus be tested in electrophysiological experiments.

Luteinizing hormone-releasing hormone in the pigeon terminal nerve and olfactory bulb.

The presence of a terminal nerve in the avian brain has recently been reported. As the terminal nerve in other classes of vertebrates contains luteinizing hormone-releasing hormone (LHRH), we used immunocytochemistry to determine whether the pigeon terminal nerve also contained LHRH. We found LHRH-immunoreactivity in the olfactory nerve and in the olfactory bulb. The distribution of LHRH neurons was similar to the LHRH neuronal migration pathway during development.

Practical aspects of ultrahigh pressure capillary liquid chromatography.

A novel pressure-balanced injection valve was evaluated for use with ultrahigh pressure liquid chromatography (UHPLC) at pressures up to 120 MPa (1,200 bar). Fused-silica capillaries (30-33 cm x 100 microm I.D.) packed with nonporous 1.5 microm isohexylsilane-modified (C6) silica particles were employed to study maximum pressure, injection reproducibility, injection time, and sample amount consumed for an injection. The new valve was more reproducible, convenient, and required much less sample than previously used injection systems. The effect of column diameter on efficiency and sensitivity was studied. The 100 microm I.D. columns demonstrated approximately 40% lower efficiency but 10-fold higher sensitivity than the 29 microm I.D. columns. Columns packed with nonporous C6 particles produced higher efficiencies than columns packed with a 1.5 microm porous octadecylsilane-modified (C18) material.

Combretastatin A-1 phosphate potentiates the antitumour activity of cisplatin in a murine adenocarcinoma model.

BACKGROUND: The tubulin depolymerizing drug Combretastatin A-1 phosphate (CA1P), a water-soluble derivative of combretastatin A-1, has been recently shown to have a better efficacy in experimental models than the clinically active, close structural analogue, combretastatin A-4 phosphate (CA4P). Previous studies with CA4P in combination with standard anti-cancer agents have demonstrated improved efficacy relative to the standard agents. MATERIALS AND METHODS: In this study the synergistic effects of administering CA1P in combination with cisplatin (CPL) in a well-differentiated transplantable murine colon model (MAC 29) was evaluated. RESULTS: CA1P at 100 mgkg-1 significantly potentiated the anti-tumour effects of CPL. The effect with CPL was similar to that seen for CA1P at its maximum tolerated dose (MTD) alone. CONCLUSION: These data demonstrate that the combination of CA1P and CPL has significant preclinical antitumour activity against a transplantable murine adenocarcinoma model that is related to the antivascular effects of CA1P.

Combretastatin A-1 phosphate a novel tubulin-binding agent with in vivo anti vascular effects in experimental tumours.

In view of the clinical potential of a number of natural products, Combretastatin A-1 phosphate was developed as a water-soluble derivative of combretastatin A-1. This study examined the anti-tumour activity of this compound against an experimental colon tumour (MAC29) in mice. A comparison was made with the clinically active combretastatin A-4 phosphate. The new compound was well-tolerated up to a dose of 250 mg/kg and was more effective at producing tumour growth delays than the A-4 analogue. Significant growth delays were seen at a dose of 50 mg/kg whereas the A-4 phosphate produced no measurable growth delay until a dose of 150 mg/kg was administered. Histological examination of treated tumours indicated that combretastatin A-1 phosphate caused very severe haemorrhagic necrosis in the tumour tissue and analysis of the sections indicated that almost 94 percent of the tumour was dead within 24 hours of treatment. The mechanism of action of combretastatin A-1 phosphate appears to be similar to the A-4 phosphate in that tumour vascular shutdown occurs within 4 hours of treatment. In summary combretastatin A-1 phosphate, the water-soluble analogue of combretastatin A-1, is more potent against a well-vascularised murine colon tumour than its predecessor, combretastatin A-4 phosphate. These data suggest this compound may have potential for clinical development.

A multicenter, randomized, double-blind, placebo-controlled, two-year trial to study the effect of nitrendipine on chronic renal transplant function.

The ongoing multicenter, randomized, double-blind, placebo-controlled trial investigates the effect of nitrendipine on kidney function after renal transplantation. Renal transplant recipients (6th-12th postoperative week, serum creatinine < 3 mg/dl) were divided into a normotensive (diastolic blood pressure < 90 mmHg) and a hypertensive group (diastolic blood pressure > or = 90, < 115 mmHg). Normotensive patients are randomly treated for 104 weeks with nitrendipine 2 x 5 mg daily or placebo, hypertensive patients with 2 x 10 mg - 2 x 20 mg nitrendipine daily or placebo and in case of inefficacy with additional antihypertensive drugs. Primary end point of the study is the renal transplant function. The trial was started in June 1990. One hundred and eight patients were included into the normotensive and 138 patients into the hypertensive group. Renal allograft function, cyclosporine trough levels and the donor characteristics were not different between the normotensive and hypertensive groups at entry into the study. After 12 months there was no significant change of renal transplant function in both groups. Cyclosporine trough levels were also similar in the normotensive and hypertensive group after 12 months. As expected, blood pressure decreased significantly after 12 months from 150 +/- 17/95 +/- 11 mmHg to 141 +/- 16/90 +/- 9 mmHg in the hypertensive group (p < 0.01). In contrast, in the normotensive group blood pressure increased significantly from 128 +/- 12/80 +/- 6 mmHg to 135 +/- 15/86 +/- 8 mmHg (p < 0.001). No normotensive but 4 hypertensive patients developed graft failure during the first 12 months of the study.

Pulmonary capillary hemangiomatosis: radiographic appearance.

Pulmonary capillary hemangiomatosis is a rare disorder that is characterized pathologically by formation of capillaries along alveolar walls that may ultimately invade the pulmonary parenchyma and pulmonary arteries. The clinic presentation overlaps that of primary pulmonary hypertension, but the natural history of pulmonary capillary hemangiomatosis is one of rapid deterioration. The typical radiographic appearance is a diffuse bilateral reticulonodular pattern associated with enlarged central pulmonary arteries.

A Generic Integrated Physiologically based Whole-body Model of the Glucose-Insulin-Glucagon Regulatory System.

Models of glucose metabolism are a valuable tool for fundamental and applied medical research in diabetes. Use cases range from pharmaceutical target selection to automatic blood glucose control. Standard compartmental models represent little biological detail, which hampers the integration of multiscale data and confines predictive capabilities. We developed a detailed, generic physiologically based whole-body model of the glucose-insulin-glucagon regulatory system, reflecting detailed physiological properties of healthy populations and type 1 diabetes individuals expressed in the respective parameterizations. The model features a detailed representation of absorption models for oral glucose, subcutaneous insulin and glucagon, and an insulin receptor model relating pharmacokinetic properties to pharmacodynamic effects. Model development and validation is based on literature data. The quality of predictions is high and captures relevant observed inter- and intra-individual variability. In the generic form, the model can be applied to the development and validation of novel diabetes treatment strategies.CPT: Pharmacometrics & Systems Pharmacology (2013) 2, e65; doi:10.1038/psp.2013.40; published online 14 August 2013.