Development of an electrochemical immunosensor for the diagnostic testing of spotted fever using synthetic peptides

Publication date: 15 February 2018
Source:Biosensors and Bioelectronics, Volume 100
Author(s): Isis C. Prado, Mônica E.T.A. Chino, Antonia L. dos Santos, André L.A. Souza, Luciano G. Pinho, Elba R.S. Lemos, Salvatore G. De-Simone
Spotted fever is a rare acute and multisystemic febrile infectious disease with a mortality rate of ≥50% without adequate antibiotic treatment, and in diagnosed and treated cases, of approximately 2.5%. Currently, the applied test to diagnose this disease is the indirect immunofluorescence reaction, however two samples of paired sera are necessary to confirm the diagnosis, since using only one sample may allow for confusion with cross reactions. OmpA is an outer membrane protein present in the R. rickettsia, the etiological agent of spotted fever, able to activate dendritic and macrophage cells. It also presents immunogenicity properties, and is considered a target for the development of diagnostic tests for spotted fever. In this context, an amperometric immunosensor was developed for the identification of sera antibodies (human IgG) from patients with spotted fever aimed at improving sensitivity and minimize sample volume. The development of the immunosensor was conducted using a synthetic peptide, derivative from the H6PGA4 R. rickettsia protein, homologous to OmpA. Amperometric responses were generated at −0.6 to 0.6V, at a scan rate of 0.025Vs⁻¹ for 20 cycles, a limit of detection of approximately 10ngmL⁻¹ for the synthetic peptides and 0.01µgmL⁻¹ for the humam serum, a sensitivity of 2.59µA, adequate for the detection of spotted fever antibodies. The construction of this immunosensor, capable of identifying circulating antibodies in real time, can also be applied in the diagnosis of other infectious-parasitic diseases.



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Method optimization for heavy metal determination in milk powder: application to milk samples from Greece

Abstract

The scope of this study was the development, optimization and validation of an analytical method for the determination of selected heavy metals and trace elements (As, Hg, Se, Cd, Cu, Pb, Mn, Fe, Ni, Zn, Cr) in milk powder, using microwave-assisted digestion. A statistical experimental design approach using central composite design (CCD) was carried out, to investigate the effects of three independent pretreatment variables (final digestion temperature (°C), HNO₃ concentration (in % w/v), microwave hold time) on the heavy metal recovery of spiked undigested milk powder sample and to calculate the variable factor values which produce the optimum recovery. CCD results revealed that the optimum digestion conditions, with respect to maximum recovery were as follows: temperature 190 °C, HNO₃ 56.8% w/v, and digestion time of 8.47 min. The method was fully validated. Recoveries for all metals ranged between 92 and 108% while intra-day repeatability was below 6.59% (rsd). A certified reference material (ERM BD 150) that included 8 out of the total 11 heavy metals of the present study (Hg, Se, Cd, Cu, Pb, Mn, Ni, and Zn) was used to test the accuracy of the method where acceptable recovery values ranging between 96 and 107% were obtained. High heavy metal recoveries, short digestion time, and low acid consumption were the advantages of the pretreatment method. The analytical process was successfully applied for the determination of heavy metals in different milk samples from the Greek market. Heavy metal concentrations for Ni, Cr, Pb, Cd, Se, Mn, and Cu measured in this study reached 307, 102, 8.01, 5.96, 60.2, 519, and 438 μg/kg wet weight (ww), respectively. Zn and Fe were found at concentrations ranging 3.21–8.39 and 0.170–10.1 mg/kg ww, respectively. Risk assessment based on the WHO tolerable daily intake levels and the calculated target hazard quotients revealed that the consumption of the selected milk samples is considered safe.

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Evaluating the potential genotoxicity of phthalates esters (PAEs) in perfumes using in vitro assays

Abstract

We previously reported high levels of phthalate esters (PAEs) added as solvents or fixatives in 47 brands of perfumes. Diethyl phthalate was the most abundant compound (0.232–23,649 ppm), and 83.3% of the perfumes had levels >1 ppm, the threshold limit cited by a Greenpeace investigation. All samples had dimethyl phthalate levels higher than its threshold limit of 0.1 ppm, and 88, 38, and 7% of the perfumes had benzyl butyl phthalate, di(2-ethylhexyl) phthalate, and dibutyl phthalate levels, respectively, above their threshold limits. The role of PAEs as endocrine disruptors has been well documented, but their effect on genotoxic behavior has received little attention. We used in vitro single-cell gel electrophoresis (comet) and micronucleus (MN) assays with human lymphoblastoid TK6 cells to evaluate the genotoxic potency of 42 of the same perfumes and to determine its association with PAEs. All perfumes induced more DNA damage than a negative control (NEG), ≥ 90% of the samples caused more damage than cells treated with the vehicles possibly used in perfume's preparations such as methanol (ME) and ethanol (ET), and 11.6% of the perfumes caused more DNA damage than a positive control (hydrogen peroxide). Chromosome breakage expressed as MN frequency was higher in cells treated with 71.4, 64.3, 57.1, and 4.8% of the perfumes than in NEG, cells treated with ME or ET, and another positive control (x-rays), respectively. The genotoxic responses in the comet and MN assays were not correlated. The comet assay indicated that the damage in TK6 cells treated with five PAEs at concentrations of 0.05 and 0.2 ppm either individually or as a mixture did not differ significantly from the damage in cells treated with the perfumes. Unlike the comet assay, the sensitivity of the MN assay to PAEs was weak at both low and high concentrations, and MN frequencies were generally low. This study demonstrates for the first time the possible contribution of PAEs in perfumes to DNA damage and suggests that their use as solvents or fixatives should be regulated. Other ingredients with mutagenic/genotoxic properties, however, may also have contributed to the DNA damage. Future studies should focus on applying a series of assays that use different cellular models with various endpoints to identify the spectrum of genotoxic mechanisms involved.

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Relook on Mastoid Cavity Obliteration: A Prospective Study

Abstract

Canal wall down (CWD) mastoidectomy is the operation of choice for unsafe variety of chronic otitis media. But open mastoid cavity poses many problems. The solution of these problems is obliteration of mastoid cavity that is self-cleaning and easily maintained. In our study we aim to establish the effective technique for mastoid cavity obliteration in CWD mastoidectomy and review its efficacy in producing a dry, low maintenance cavity. This was a non-randomized longitudinal prospective study, performed over 2½ years in the department of ENT in a tertiary care hospital of Kolkata. Patients of chronic otitis media unsafe variety within the age group of 16–60 years were included in our study. Chronic otitis media unsafe variety with intratemporal or intracranial complications, and extensive cholesteatoma or granulation tissue that can't be cleared completely during operation were excluded. There was no statistical significance for hearing improvement between CWD mastoidectomy without obliteration and CWD mastoidectomy with obliteration. There was significant statistical significance for obliteration of cavity, epithelized cavity and dry cavity between CWD mastoidectomy without obliteration and CWD mastoidectomy with obliteration. Persistent discharge and granulation were significantly more in non-obliterated group. The time taken by the ear to become dry is much shorter after mastoid cavity obliteration. Moreover, lifelong aural toilet and dependence on an ENT surgeon is avoided. Inspite of all these, a few pre-conditions must be fulfilled before embarking on this type of surgery.

http://ift.tt/2eYoyrE

Relook on Mastoid Cavity Obliteration: A Prospective Study

Abstract

Canal wall down (CWD) mastoidectomy is the operation of choice for unsafe variety of chronic otitis media. But open mastoid cavity poses many problems. The solution of these problems is obliteration of mastoid cavity that is self-cleaning and easily maintained. In our study we aim to establish the effective technique for mastoid cavity obliteration in CWD mastoidectomy and review its efficacy in producing a dry, low maintenance cavity. This was a non-randomized longitudinal prospective study, performed over 2½ years in the department of ENT in a tertiary care hospital of Kolkata. Patients of chronic otitis media unsafe variety within the age group of 16–60 years were included in our study. Chronic otitis media unsafe variety with intratemporal or intracranial complications, and extensive cholesteatoma or granulation tissue that can't be cleared completely during operation were excluded. There was no statistical significance for hearing improvement between CWD mastoidectomy without obliteration and CWD mastoidectomy with obliteration. There was significant statistical significance for obliteration of cavity, epithelized cavity and dry cavity between CWD mastoidectomy without obliteration and CWD mastoidectomy with obliteration. Persistent discharge and granulation were significantly more in non-obliterated group. The time taken by the ear to become dry is much shorter after mastoid cavity obliteration. Moreover, lifelong aural toilet and dependence on an ENT surgeon is avoided. Inspite of all these, a few pre-conditions must be fulfilled before embarking on this type of surgery.

http://ift.tt/2eYoyrE

Long-term clinical safety of high-dose proton radiotherapy delivered with pencil beam scanning technique for extracranial chordomas and chondrosarcomas in adult patients: Clinical evidence of spinal cord tolerance

Publication date: Available online 4 September 2017
Source:International Journal of Radiation Oncology*Biology*Physics
Author(s): Sonja Stieb, James W. Snider, Lorenzo Placidi, Ulrike Kliebsch, Anthony J. Lomax, Ralf A. Schneider, Damien C. Weber
Purpose/ObjectivesTo assess the radiation dose tolerance of the spinal cord, we reviewed our institutional experience regarding the incidence of radiation-induced spinal cord toxicity following high dose, pencil beam scanning proton therapy (PBSPT).Materials/MethodsSeventy-six patients (median age, 53 years; range: 23–79), treated for spinal chordoma (n=55) or chondrosarcoma (n=21) met the following criteria and were retrospectively analyzed: PBSPT only, no re-irradiation or concomitant chemotherapy, maximum dose (Dmax) to the spinal cord of ≥45 Gy(RBE), ≥18 years of age, and follow-up of ≥12 months. The delivered dose was 59.4–75.2 Gy(RBE) (median, 73.9) delivered with conventional fractionation between 2000–2014. The Dmax, D2%, V40–V60 of the surface (sSC) and center (cSC) of the spinal cord were recorded. Toxicity was scored according to the Common Toxicity Criteria of Adverse Events (CTCAE v4.03).ResultsMedian follow-up was 65.5 months (range, 13–173). Patients received a mean Dmax and D2% to the sSC of 59.0 (median 58.7, range: 48.3 – 75.9), and 55.3 Gy(RBE) (median, 52.7, range: 43.1 – 73.8), respectively. The corresponding values for the cSC were 52.3 (median, 52.7, range: 32.3 – 73.3) and 51.1 Gy(RBE) (median, 52.0, range: 25.3 – 73.1), respectively. Four patients (5%) developed acute radiation-induced neurotoxicity (G1, n=1; G2, n=3). Twelve patients (16%) experienced late neurologic toxicities (G1, n=7; G2, n=4; G4, n=1). One patient with a history of pre-PBSPT symptomatic spinal cord compression redeveloped tetraplegia (G4) after receiving a Dmax of 57.8 Gy(RBE) to the sSC and 54.1 Gy(RBE) to the cSC. No significant correlation was found between sSC Dmax and D2%, cSC Dmax and D2%, or the length of CTV and toxicity.ConclusionsHigh-dose conformal PBSPT may be delivered safely in close proximity to the spinal cord with minimal neurotoxicity. Dose constraints of 64 Gy(RBE) as D2% for the sSC and 54 Gy(RBE) for the cSC appear appropriate for clinical use.

Teaser

Limited clinical data to define the radiotherapy tolerance of the spinal cord are available to clinicians. To add further clarity, we retrospectively evaluated our patients treated with proton therapy only for spinal chordomas and chondrosarcomas, who received a spinal cord dose in excess of 45 Gy(RBE) with special regard to neurotoxicity. Radiation doses reaching 64 Gy(RBE) to the surface of the spinal cord were associated with limited toxicity, defining this cut-off dose level as a safe threshold for appropriate clinical use.


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Serum microRNA Signature Predicts Response to High-dose Radiotherapy in Locally Advanced Non-Small-Cell Lung Cancer

Publication date: Available online 4 September 2017
Source:International Journal of Radiation Oncology*Biology*Physics
Author(s): Yilun Sun, Peter G. Hawkins, Nan Bi, Robert T. Dess, Muneesh Tewari, Jason W.D. Hearn, James A. Hayman, Gregory P. Kalemkerian, Theodore S. Lawrence, Randall K. Ten Haken, Martha M. Matuszak, Feng-Ming Kong, Shruti Jolly, Matthew J. Schipper
PurposeWhile efforts to improve control of locally-advanced non-small-cell lung cancer (NSCLC) by escalating radiation dose in unselected patients have been unsuccessful, we hypothesized that a subset of patients may derive benefit. Circulating serum microRNAs (c-miRNAs) have shown promise as prognostic and predictive biomarkers. We assessed the utility of c-miRNAs to predict response to high-dose radiotherapy.Methods and MaterialsData from 80 patients treated from 2004 to 2013 with definitive standard- or high-dose radiotherapy for stages II-III NSCLC as part of four prospective institutional clinical trials were evaluated. Pretreatment serum levels of 62 miRNAs were measured by quantitative reverse-transcription polymerase chain reaction array. We combined miRNA data and clinical factors to generate a Dose Response Score (DRS) for predicting overall survival (OS) following high-dose versus standard-dose radiotherapy. Elastic net Cox regression was used for variable selection and parameter estimation. Model assessment and tuning parameter selection were performed through full cross validation. DRS was also correlated with local progression, distant metastasis, and grade 3 or higher cardiac toxicity using Cox regression; and grade 2 or higher esophageal and pulmonary toxicity using logistic regression.ResultsEleven predictive miRNAs were combined with clinical factors to generate a DRS for each patient. In patients with low DRS, high-dose radiotherapy was associated with significantly improved OS compared to treatment with standard-dose radiotherapy (HR 0.22). In these patients, high-dose radiation also conferred lower risk of distant metastasis and local progression, although the latter association was not statistically significant. Patients with high DRS exhibited similar rates of OS regardless of dose (HR 0.78). DRS did not correlate with treatment-related toxicity.ConclusionsUsing c-miRNA signature and clinical factors, we developed a DRS that identified a subset of patients with locally-advanced NSCLC who derive an OS benefit from high-dose radiotherapy. This DRS may guide dose-escalation in a patient-specific manner.

Teaser

Escalation of radiation dose has not been beneficial in unselected patients with locally-advanced non-small-cell lung cancer. We hypothesized that a subset of patients may derive benefit and sought to identify a dose-response biomarker by analyzing patients treated with varying doses of radiotherapy on four institutional clinical trials. We identified a micro-RNA signature that, when combined with clinical factors, identified a subset of patients who exhibited an overall survival benefit following high-dose radiotherapy.


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