Status quo of Extracellular Vesicle isolation and detection methods for clinical utility.

Extracellular vesicles (EVs) are nano-sized particles that hold tremendous potential in the clinical space, as their biomolecular profiles hold a key to non-invasive liquid biopsy for cancer diagnosis and prognosis. EVs are present in most bodily fluids, hence are easily obtainable from patients, advantageous to that of traditional, invasive tissue biopsies and imaging techniques. However, there are certain constraints that hinder clinical use of EVs. The translation of EV biomarkers from "bench-to-bedside" is encumbered by the methods of EV isolation and subsequent biomarker detection currently implemented in laboratories. Although current isolation and detection methods are effective, they lack practicality, with their requirement for high bodily fluid volumes, low equipment availability, slow turnaround times and high costs. The high demand for techniques that overcome these limitations has resulted in significant advancements in nanotechnological devices. These devices are designed to integrate EV isolation and biomarker detection into a one-step method of direct EV detection from bodily fluids. This provides promise for the acceleration of EVs into current clinical standards. This review highlights the importance of EVs as cancer biomarkers, the methodological obstacles currently faced in clinical studies and how novel nanodevices could advance clinical translation.

Oncogenic allelic interaction in Xiphophorus highlights hybrid incompatibility.

Mixing genomes of different species by hybridization can disrupt species-specific genetic interactions that were adapted and fixed within each species population. Such disruption can predispose the hybrids to abnormalities and disease that decrease the overall fitness of the hybrids and is therefore named as hybrid incompatibility. Interspecies hybridization between southern platyfish and green swordtails leads to lethal melanocyte tumorigenesis. This occurs in hybrids with tumor incidence following progeny ratio that is consistent with two-locus interaction, suggesting melanoma development is a result of negative epistasis. Such observations make Xiphophorus one of the only two vertebrate hybrid incompatibility examples in which interacting genes have been identified. One of the two interacting loci has been characterized as a mutant epidermal growth factor receptor. However, the other locus has not been identified despite over five decades of active research. Here we report the localization of the melanoma regulatory locus to a single gene, rab3d, which shows all expected features of the long-sought oncogene interacting locus. Our findings provide insights into the role of egfr regulation in regard to cancer etiology. Finally, they provide a molecular explainable example of hybrid incompatibility.

Impact of Heat on Coil Hydrodynamic Size Yields the Energetics of Denatured State Conformational Bias.

Conformational equilibria in the protein denatured state have key roles regulating folding, stability, and function. The extent of conformational bias in the protein denatured state under folding conditions, however, has thus far proven elusive to quantify, particularly with regard to its sequence dependence and energetic character. To better understand the structural preferences of the denatured state, we analyzed both the sequence dependence to the mean hydrodynamic size of disordered proteins in water and the impact of heat on the coil dimensions, showing that the sequence dependence and thermodynamic energies associated with intrinsic biases for the α and polyproline II (PPII) backbone conformations can be obtained. Experiments that evaluate how the hydrodynamic size changes with compositional changes in the protein reveal amino acid specific preferences for PPII that are in good quantitative agreement with calorimetry-measured values from unfolded peptides and those inferred by survey of the protein coil library. At temperatures above 25 °C, the denatured state follows the predictions of a PPII-dominant ensemble. Heat effects on coil hydrodynamic size indicate the α bias is comparable to the PPII bias at cold temperatures. Though historically thought to give poor resolution to structural details, the hydrodynamic size of the unfolded state is found to be an effective reporter on the extent of the biases for the α and PPII backbone conformations.

Standing thyroidectomy in 10 horses.

OBJECTIVE: To describe a surgical technique for thyroidectomy in horses with thyroid neoplasia under standing sedation and local anesthesia. STUDY DESIGN: Retrospective study. ANIMALS: Client-owned horses (n = 10). METHODS: Medical records of horses with a history of thyroid enlargement were included in the study if thyroid gland enlargement was treated surgically via hemi- or bilateral thyroidectomy, with the horse standing and sedated. Data derived from follow-up clinical examination, performance level, recurrence, and cosmetic outcome were evaluated. RESULTS: Thyroid enlargement was unilateral in 8 and bilateral in 2 horses. Histopathological findings included adenomas (5/10), adenocarcinomas (2/10), cystic hyperplasia (2/10), and C-cell adenoma (1/10). No major complications were encountered during or after surgery. All horses resumed their previous level of exercise within 6 weeks. Recurrence was diagnosed in 1 horse, 7 months after excision, and a second surgery was required. Recurrent laryngeal nerve neuropathy and seroma formation subsequent to surgery were not recorded in any of the cases. CONCLUSION: Thyroidectomy can safely be performed with the horse standing and sedated with local anesthesia. CLINICAL RELEVANCE: Performing standing thyroidectomy does not increase intraoperative or postoperative complications and could be considered for horses with thyroid enlargement amenable to surgery.

A national teleradiology programme in Mali: implementation and results.

We reviewed the national teleradiology programme in Mali to establish whether it improved diagnosis for patients and improved the referring doctor's ability to give an accurate diagnosis. The teleradiology programme connected the University Hospital in Bamako to all seven regional hospitals in Mali and one private health clinic. The pilot phase began in 2005 in three hospitals. Initially the implementation involved connections via broadband, but subsequently satellite antennae were provided at three remote hospitals in the north. Between 2005 and 2013, X-ray and mammogram images from 5628 patients were read by teleradiology. Radiologists provided the sole diagnosis for 29% of cases (i.e. the referrer did not make a diagnosis) and altered the regional doctor's diagnosis in 12% of cases. The proportion of cases for which the regional doctor gave no diagnosis decreased from 93% to 24% over the same period, indicating an increase in the doctors' confidence and incentive to test their own diagnosis. The percentage of cases for which regional doctors made an inaccurate diagnosis decreased to 3% in 2013. Use of the teleradiology service varied widely between hospitals. Successful implementation depended on local ownership of a network, which was developed in close collaboration with hospital leadership, national radiologists and other healthcare personnel.

Mitotic count by phosphohistone H3 immunohistochemical staining predicts survival and improves interobserver reproducibility in well-differentiated neuroendocrine tumors of the pancreas.

Well-differentiated neuroendocrine tumors (WDNETs) of the pancreas are graded on the basis of mitotic count or Ki67 index. Mitotic count has a narrow cutoff; its assessment is time consuming and carries poor interobserver reproducibility. Phosphohistone H3 (PHH3) is a mitosis-specific marker whose value has been validated in several tumor types. We sought to assess the utility of PHH3 in histologic grading of pancreatic WDNETs. Sixty-three cases of surgically resected primary pancreatic WDNETs were retrieved, and immunohistochemical analysis for PHH3 and Ki67 was performed. Mitotic rate was independently assessed by 4 pathologists on hematoxylin and eosin (HE; in 50 high-power fields [HPFs], expressed as mitoses/10 HPF) and PHH3 stains (in 50 HPFs, one 10×, and one 20× hotspot). PHH3 and Ki67 labeling indices were determined on a single 20× hotspot and expressed as the percentage of positive cells to total cells. We found that mitotic counts by various methods significantly correlated with each other and also with PHH3 and Ki67 indices, with the best correlation seen within the 3 different PHH3 counts (in 50 HPFs, one 10× and one 20× hotspot). Moreover, mitotic count on PHH3 was less time consuming than that on HE (1.68 vs. 3.67 min for 50 HPFs, P<0.0001). Histologic grade determined by PHH3 significantly correlated with disease-specific and disease-free survivals, with the best cutoffs of ≥4 mitoses/10 HPF (2 mm), ≥7 mitoses/10× hotspot, ≥5 mitoses/20× hotspot (log rank test, P<0.0001), and ≥0.16% for PHH3 labeling index (log rank test, P<0.0006). Tumor grades based on PHH3 stain also showed significant correlation with patient survivals in multivariate Cox proportional hazards models (P<0.05). Histologic grades by mitotic counts on PHH3 demonstrated high concordance and κ agreement with grades determined by mitotic count on HE. PHH3 stain also showed improved interobserver agreement in both original mitotic count (intraclass correlation 0.98 vs. 0.79) and final grade assignment (Fleiss κ 0.69 vs. 0.46) as compared with HE. Thus, our data confirmed that histologic grading by PHH3 stain has practical and prognostic values and offers reduced time and improved interobserver reproducibility in mitotic rate assessment and grade assignment. Although larger series are needed for validation, mitotic rate can potentially be determined by counting 1 hotspot, which will greatly facilitate the assessment of histologic grade in pancreatic WDNETs.