Detection of prolactin inducible protein mRNA, a biomarker for breast cancer metastasis, using a molecular beacon-based assay.

Mortality due to breast cancer is increasingly linked to early, undetected metastasis, making methods for earlier detection acutely necessary. We describe the development of an assay based on molecular beacon (MB) chemistry with fluorescence detection to monitor a breast cancer biomarker for the analysis of breast cancer metastasis. The MB assay is based on the complementary base-pairing interactions of the MB nucleic acid with mRNA indicative of breast cancer metastasis. The presence of mRNA is characterized by an increase in the fluorescence intensity of the molecular beacon. The assay gives a linear, reproducible response to prolactin inducible protein mRNA, with a limit of detection in the high picomolar range. This method sensitively and specifically identifies a biomarker directly in serum samples in minimal time and with a straightforward procedure, dramatically reducing the total time for sample analysis over current methods from days to hours. The potential impact of this work in detection and understanding of breast cancer metastasis lies in improvements in simplicity, accuracy, and speed over current methods, which could allow for improved patient treatment and prognoses. Ultimately, additional sample throughput will result in better understanding of disease progression.

Depigmentation therapy for vitiligo in patients with Fitzpatrick skin type VI.

Vitiligo is a depigmenting disorder characterized by the progressive loss of melanocytes. In cases of extensive vitiligo that is unresponsive to treatment and involves noticeable areas, such as the face and hands, total depigmentation is a clinical option. The choice to depigment is a difficult one for the patient given the irreversible nature of treatment and the psychosocial implications of skin color change. This issue can be particularly complex for black patients. Depigmentation has been practiced for decades and documented in the literature, but the practice in Fitzpatrick skin type VI is not well-documented. We present a case of depigmentation in a patient with Fitzpatrick skin type VI, as well as technical options for depigmentation, the clinical approach, patient preparation, and psychosocial issues involved with this treatment option.

Distinction of melanoma in situ from solar lentigo on sun-damaged skin using morphometrics and MITF immunohistochemistry.

Distinction between melanoma in situ (MIS) and solar lentigo (SL) on chronically sun-damaged skin (CSDS) by hematoxylin and eosin (H&E) criteria alone can be difficult and in frozen section (FS) material, may be virtually impossible without immunohistochemistry (IHC). In this study, we used microphthalmia-associated transcription factor (MITF) IHC-directed image analysis to compare melanocyte nuclear morphometrics of MIS, SL, and sections of sun-damaged skin from redundant tissue acquired during Mohs micrographic surgery. The mean nuclear diameter and melanocytic density figures for MIS were greater than those for SL and CSDS by both independent t-test and analysis of variance statistics. No significant differences in these parameters were found between SL and sun-damaged skin. Cutoff values that favored MIS over SL included melanocyte density ≥10 nuclei per 200 µm, nuclear diameter ≥9 µm, and a product of density and diameter of 80 or more, as each of these values was associated with 100% specificity of MIS diagnosis. Our results suggest that image analysis of melanocytes labeled with MITF IHC can be used to produce morphometric data that distinguish MIS from SL and CSDS. The study was conducted using permanent sections, but previous studies with FSs indicate that the findings would apply to FSs as well. Quantitative assessment of melanocytic parameters using image analysis will likely become increasingly important as an adjunct to conventional histopathology for the diagnosis and surgical management of MIS on sun-damaged skin.

Development and characterization of "push-pull" sampling device with fast reaction quenching coupled to high-performance liquid chromatography for pharmaceutical process analytical technologies.

A push-pull sampling system interfaced on-line to high-performance liquid chromatography (HPLC) was developed for micro-volume real-time monitoring of reaction mixtures. The device consists of concentric tubes wherein sample was continuously withdrawn through the outer tube and reaction quenchant continuously delivered through a recessed inner tube. The device allowed sampling rates of 0.1-6.0 µL/min from a reaction vessel and stopped the reaction by passive mixing with quenchant to preserve the conditions observed in the reaction vessel. A finite element model of the system showed that reaction mixtures could be completely mixed with quenchant within 4.3s at a flow rate of 1.0 µL/min. The model also showed that an offset distance of 1mm between the push capillary and sample capillary tips is sufficient to avoid leakage of quenchant/diluent into the bulk sample for push flow rates up to 95% of the pull flow rate. The maximum relative push flow rate was determined to be 90% of the pull flow rate experimentally. Delay between sampling and delivery to the HPLC was from 111±3s to 317±9s for pull flow rates from 1.0 to 3.0 µL/min in agreement with expected delays based on tubing volume. Response times were from 27±1s to 52±6s over the same flow rate range. The sampler was tested to determine the effects of sample viscosity. The sampler was also used to demonstrate periodic sampling capabilities. As a test of the system, it was used to monitor the base-catalyzed hydrolysis of aspirin for 1.5h, demonstrating its utility for monitoring an ongoing reaction.

Single-molecule FRET of protein-nucleic acid and protein-protein complexes: surface passivation and immobilization.

Single-molecule fluorescence spectroscopy reveals the real time dynamics that occur during biomolecular interactions that would otherwise be hidden by the ensemble average. It also removes the requirement to synchronize reactions, thus providing a very intuitive approach to study kinetics of biological systems. Surface immobilization is commonly used to increase observation times to the minute time scale, but it can be detrimental if the sample interacts non-specifically with the surface. Here, we review detailed protocols to prevent such interactions by passivating the surface or by trapping the molecules inside surface immobilized lipid vesicles. Finally, we discuss recent examples where these methods were applied to study the dynamics of important cellular processes at the single-molecule level.