A Practical Guide for the Quality Evaluation of Fluobodies/Chromobodies.

BACKGROUND: Fluorescent proteins (FPs) are pivotal reagents for flow cytometry analysis or fluorescent microscopy. A new generation of immunoreagents (fluobodies/chromobodies) has been developed by fusing recombinant nanobodies to FPs. METHODS: We analyzed the quality of such biomolecules by a combination of gel filtration and SDS-PAGE to identify artefacts due to aggregation or material degradation. RESULTS: In the SDS-PAGE run, unexpected bands corresponding to separate fluobodies were evidenced and characterized as either degradation products or artefacts that systematically resulted in the presence of specific FPs and some experimental conditions. The elimination of N-terminal methionine from FPs did not impair the appearance of FP fragments, whereas the stability and migration characteristics of some FP constructs were strongly affected by heating in loading buffer, which is a step samples undergo before electrophoretic separation. CONCLUSIONS: In this work, we provide explanations for some odd results observed during the quality control of fluobodies and summarize practical suggestions for the choice of the most convenient FPs to fuse to antibody fragments.

Biological Applications of Synthetic Binders Isolated from a Conceptually New Adhiron Library.

BACKGROUND: Adhirons are small (10 kDa) synthetic ligands that might represent an alternative to antibody fragments and to alternative scaffolds such as DARPins or affibodies. METHODS: We prepared a conceptionally new adhiron phage display library that allows the presence of cysteines in the hypervariable loops and successfully panned it against antigens possessing different characteristics. RESULTS: We recovered binders specific for membrane epitopes of plant cells by panning the library directly against pea protoplasts and against soluble C-Reactive Protein and SpyCatcher, a small protein domain for which we failed to isolate binders using pre-immune nanobody libraries. The best binders had a binding constant in the low nM range, were produced easily in bacteria (average yields of 15 mg/L of culture) in combination with different tags, were stable, and had minimal aggregation propensity, independent of the presence or absence of cysteine residues in their loops. DISCUSSION: The isolated adhirons were significantly stronger than those isolated previously from other libraries and as good as nanobodies recovered from a naïve library of comparable theoretical diversity. Moreover, they proved to be suitable reagents for ELISA, flow cytometry, the western blot, and also as capture elements in electrochemical biosensors.

Native Agarose Gels and Contact Blotting as Means to Optimize the Protocols for the Formation of Antigen-Ligand Complexes.

BACKGROUND: Protein complexes provide valuable biological information, but can be difficult to handle. Therefore, technical advancements designed to improve their manipulation are always useful. METHODS: We investigated the opportunity to exploit native agarose gels and the contact blot method for the transfer of native proteins to membranes as means for optimizing the conditions for obtaining stable complexes. As a simple model of protein-protein interactions, an antigen-ligand complex was used in which both proteins were fused to reporters. RESULTS: At each step, it was possible to visualize both the antigen, fused to a fluorescent protein, and the ligand, fused to a monomeric ascorbate peroxidase (APEX) and, as such, a way to tune the protocol. The conditions for the complex formation were adapted by modifying the buffer conditions, the concentration of the proteins and of the cross-linkers. CONCLUSIONS: The procedure is rapid, inexpensive, and the several detection opportunities allow for both the monitoring of complex stability and the preservation of the functionality of its components, which is critical for understanding their biomedical implications and supporting drug discovery. The overall protocol represents a handy alternative to gel filtration, uses very standard and ubiquitous equipment, and can be implemented rapidly and without specific training.

Isolation of Adhirons Specific for Plant Protoplast Membrane Biomarkers Is Simplified by Phagemid Design.

Phage display is an effective method to retrieve binders specific for a target epitope from a large clone library. Nevertheless, the panning process allows for the accumulation of some contaminant clones into the selected phage pool and, consequently, each clone requires individual screening to verify its actual specificity. This step is time-consuming, independently on the chosen method, and relies on the availability of reliable reagents. Since phages display a single binder responsible for the antigen recognition but their coat is formed by several repeats of the same proteins, the targeting of coat epitopes is often exploited to amplify the signal. Commercial anti-M13 antibodies are commonly labeled with peroxidase or FITC but customized antibodies might be necessary for specific applications. Here, we report a protocol describing the selection of anti-protoplast Adhirons that relies on the availability of nanobodies fused to a fluorescent protein to use during flow cytometry screening. Specifically, when preparing our Adhiron synthetic library, we designed a new phagemid that allows the expression of the clones fused to three tags. These can interact with a large variety of commercial and home-made reagents, selected according to the needs of the downstream characterization process. In the described case, we combined the ALFA-tagged Adhirons with an anti-ALFAtag nanobody fused with the fluorescent protein mRuby3.

Macroporous Epoxy-Based Monoliths Functionalized with Anti-CD63 Nanobodies for Effective Isolation of Extracellular Vesicles in Urine.

Extracellular vesicles (EVs) have enormous potential for the implementation of liquid biopsy and as effective drug delivery means, but the fulfilment of these expectations requires overcoming at least two bottlenecks relative to their purification, namely the finalization of reliable and affordable protocols for: (i) EV sub-population selective isolation and (ii) the scalability of their production/isolation from complex biological fluids. In this work, we demonstrated that these objectives can be achieved by a conceptually new affinity chromatography platform composed of a macroporous epoxy monolith matrix functionalized with anti-CD63 nanobodies with afflux of samples and buffers regulated through a pump. Such a system successfully captured and released integral EVs from urine samples and showed negligible unspecific binding for circulating proteins. Additionally, size discrimination of eluted EVs was achieved by different elution approaches (competitive versus pH-dependent). The physical characteristics of monolith material and the inexpensive production of recombinant nanobodies make scaling-up the capture unit feasible and affordable. Additionally, the availability of nanobodies for further specific EV biomarkers will allow for the preparation of monolithic affinity filters selective for different EV subclasses.

[Role of surgery in patients with metastases from melanoma. A case report]. / Ruolo della chirurgia in pazienti con metastasi da melanoma. Un caso clinico.

INTRODUCTION: The prognosis of patients with melanoma varies according to the staging of disease at the moment of diagnosis. Melanoma can metastasize to every organ or tissue, but the most common site involved is locoregional. In selected patients surgery plays a central role with the possibility of changing the prognosis at distance. CASE REPORT: A 65-year-old man with a diagnosis of neoplasm of the left thyroid lobe and metastatic melanoma with unknown primitive localization. Since 2003 the patient has undergone many operations to remove metastatic melanoma. Currently he is in good conditions and performs neoadjuvant palliative treatments. DISCUSSION: The indication for surgery in cases of patients with distant disease (IV stage) or metastases in transit (IIIc stage) is linked to the possibility of surgical removal of individual lesions. In particular are candidates for surgery patients who have a visceral localization less than or equal to 2 sites, a number less than or equal to 8 metastases, in good health and having a melanoma-specific survival estimated more than 3 months; surgical approach requires an accurate and early identification by imaging study. The case report shows that in selected cases, some patients may benefit from aggressive surgery, especially in terms of survival at distance.

Prognostic factors of insular versus papillary/follicular thyroid carcinoma.

The study aims were to characterize patients with insular thyroid cancer and to provide data on patient outcome after surgical therapy. We compared nine patients with insular thyroid cancer at the Department of Surgical Science of "La Sapienza" University of Rome with 27 patients of similar age and tumor size who had follicular and papillary cancer, for a minimum follow-up period of 24 months (range, 24-72 months). All of the patients examined underwent total thyroidectomy. Vascular invasion was observed in 44.4 per cent of insular carcinomas (P < 0.05 vs papillary carcinomas). No significant differences were observed regarding diagnostic method, multifocality, tumor nodes metastases (TNM), or stage. The death rate of patients with insular carcinoma (33.3%) was found to be higher than that of patients with follicular carcinoma (P < 0.05) and papillary carcinoma (P < 0.01). Relapsing lymph-node pathologies were observed in 4 patients (44.4%) with insular carcinoma (P < 0.05 vs those with follicular and papillary carcinomas). Distant metastases were observed in 66.6 per cent of insular carcinomas (P < 0.005 vs follicular carcinoma and P < 0.001 vs papillary carcinoma). At the end of follow-up, 2 patients (22.2%) with insular carcinoma were disease-free (P < 0.001 vs those with follicular and papillary carcinomas). Our study demonstrates an unfavorable prognostic role of the insular phenotype of thyroid cancer, such that this tumor can be classified as an autonomous clinical and pathological entity.

Control results on a sample of patients subjected to total thyroidectomy for papillary carcinoma: proposal for a personal diagnostic-therapeutic follow-up.

Well-differentiated thyroid neoplasms may be included among the most frequently occurring thyroid carcinomas. Papillary ca. is without doubt the best behaved type. The aim of the present work is to perform a retrospective case history study to assess patients with con papillary ca. who have been treated surgically over the last 17 years and have been subjected to periodic checks. A sample of patients was therefore extrapolated who had all undergone total thyroidectomy for papillary ca. of the thyroid. The incidence of local recurrence of the disease was verified, together with the results at distance. Furthermore, the assessments performed were evaluated and compared. From the sample of patients observed we inferred that papillary carcinoma of the thyroid can have a good prognosis over time provided periodic random checks are carried out.

Papillary microcarcinoma of the thyroid gland: analysis of prognostic factors including histological subtype.

OBJECTIVE: To evaluate the prognosis of papillary microcarcinoma (PMC) of the thyroid. DESIGN: Prospective study. SETTING: Teaching hospital, Italy. SUBJECTS: 89 patients treated for papillary micro carcinoma of thyroid and followed up for at least 6 years (range 6-12). INTERVENTION: Total thyroidectomy and, for lymph node metastases, laterocervical lymphadenectomy. MAIN OUTCOME MEASURES: Sex distribution, mode of onset of disease, diameter of tumour, histological type, histological stage, presence of lymph node metastases at the time of operation and type of operation. RESULTS: When the disease presented with laterocervical lymph nodes it was histologically more aggressive and included follicular (n = 3) and sclerosing (n = 4) types. CONCLUSION: Papillary microcarcinoma has an excellent prognosis if managed initially by total thyroidectomy.