Potential of non-contrast spiral breast CT to exploit lesion density and favor breast cancer detection: A pilot study.

PURPOSE: To assess the density values of breast lesions and breast tissue using non-contrast spiral breast CT (nc-SBCT) imaging. METHOD: In this prospective study women undergoing nc-SBCT between April-October 2023 for any purpose were included in case of: histologically proven malignant lesion (ML); fibroadenoma (FA) with histologic confirmation or stability > 24 months (retrospectively); cysts with ultrasound correlation; and women with extremely dense breast (EDB) and no sonographic findings. Three regions of interest were placed on each lesion and 3 different area of EDB. The evaluation was performed by two readers (R1 and R2). Kruskal-Wallis test, intraclass correlation (ICC) and ROC analysis were used. RESULTS: 40 women with 12 ML, 10 FA, 15 cysts and 9 with EDB were included. Median density values and interquartile ranges for R1 and R2 were: 60.2 (53.3-67.3) and 62.5 (55.67-76.3) HU for ML; 46.3 (41.9-59.5) and 44.5 (40.5-59.8) HU for FA; 35.3 (24.3-46.0) and 39.7 (26.7-52.0) HU for cysts; and 28.7 (24.2-33.0) and 33.3 (31.7-36.8) HU for EDB. For both readers, densities were significantly different for ML versus EDB (p < 0.001) and cysts (p < 0.001) and for FA versus EDB (p=/<0.003). The AUC was 0.925 (95 %CI 0.858-0.993) for R1 and 0.942 (0.884-1.00) for R2 when comparing ML versus others and 0.792 (0.596-0.987) and 0.833 (0.659-1) when comparing ML versus FA. The ICC showed an almost perfect inter-reader (0.978) and intra-reader agreement (>0.879 for both readers). CONCLUSIONS: In nc-SBCT malignant lesions have higher density values compared to normal tissue and measurements of density values are reproducible between different readers.

Effects on Serum Inflammatory Cytokines of Cholecalciferol Supplementation in Healthy Subjects with Vitamin D Deficiency.

The effects of different cholecalciferol supplementation regimens on serum inflammatory cytokines in healthy subjects with vitamin D deficiency are still lacking. This is a single-center, open-label, randomized, parallel group study involving healthy subjects deficient in vitamin D (baseline 25OHD < 20 ng/mL) receiving oral cholecalciferol with three different dosing regimens: Group A: 10,000 IU/day for 8 weeks followed by 1000 IU/day for 4 weeks; Group B: 50,000 IU/week for 12 weeks and Group C: 100,000 IU every other week for 12 weeks. IL-17A, IL-6, IL-8, IL-10, IL-23 and TNFα were measured at baseline and at week 4, 8, 12, and 16. 75 healthy subjects were enrolled (58.7% female), with an average age of 34.1 ± 10.2 years. No statistical differences were observed among groups at baseline for either IL-6, IL-17A, IL-23, IL-8 or IL-10 at any time point; TNFα was indetectable. Concerning the whole sample, the time trend analysis showed a statistically significant linear trend for decreasing values over the treatment period for IL-6 (p = 0.016) and IL-17A (p = 0.006), while no significant time trends were observed for the other teste cytokines. No significant differences were found in the serum concentrations of the tested cytokines between week 12 and week 16. In young healthy individuals deficient in vitamin D, cholecalciferol administration showed a decrease in the serum IL-6 and IL-17A concentrations, without marked differences using the three regimens.

In silico reconstitution of DNA replication. Lessons from single-molecule imaging and cryo-tomography applied to single-particle cryo-EM.

DNA replication has been reconstituted in vitro with yeast proteins, and the minimal system requires the coordinated assembly of 16 distinct replication factors, consisting of 42 polypeptides. To understand the molecular interplay between these factors at the single residue level, new structural biology tools are being developed. Inspired by advances in single-molecule fluorescence imaging and cryo-tomography, novel single-particle cryo-EM experiments have been used to characterise the structural mechanism for the loading of the replicative helicase. Here, we discuss how in silico reconstitution of single-particle cryo-EM data can help describe dynamic systems that are difficult to approach with conventional three-dimensional classification tools.

A New Transgenic Mouse Line for Imaging Mitochondrial Calcium Signals.

Mitochondria play a key role in cellular calcium (Ca2+) homeostasis. Dysfunction in the organelle Ca2+ handling appears to be involved in several pathological conditions, ranging from neurodegenerative diseases, cardiac failure and malignant transformation. In the past years, several targeted green fluorescent protein (GFP)-based genetically encoded Ca2+ indicators (GECIs) have been developed to study Ca2+ dynamics inside mitochondria of living cells. Surprisingly, while there is a number of transgenic mice expressing different types of cytosolic GECIs, few examples are available expressing mitochondria-localized GECIs, and none of them exhibits adequate spatial resolution. Here we report the generation and characterization of a transgenic mouse line (hereafter called mt-Cam) for the controlled expression of a mitochondria-targeted, Förster resonance energy transfer (FRET)-based Cameleon, 4mtD3cpv. To achieve this goal, we engineered the mouse ROSA26 genomic locus by inserting the optimized sequence of 4mtD3cpv, preceded by a loxP-STOP-loxP sequence. The probe can be readily expressed in a tissue-specific manner upon Cre recombinase-mediated excision, obtainable with a single cross. Upon ubiquitous Cre expression, the Cameleon is specifically localized in the mitochondrial matrix of cells in all the organs and tissues analyzed, from embryos to aged animals. Ca2+ imaging experiments performed in vitro and ex vivo in brain slices confirmed the functionality of the probe in isolated cells and live tissues. This new transgenic mouse line allows the study of mitochondrial Ca2+ dynamics in different tissues with no invasive intervention (such as viral infection or electroporation), potentially allowing simple calibration of the fluorescent signals in terms of mitochondrial Ca2+ concentration ([Ca2+]).

A novel GTP-binding protein-adaptor protein complex responsible for export of Vangl2 from the trans Golgi network.

Planar cell polarity (PCP) requires the asymmetric sorting of distinct signaling receptors to distal and proximal surfaces of polarized epithelial cells. We have examined the transport of one PCP signaling protein, Vangl2, from the trans Golgi network (TGN) in mammalian cells. Using siRNA knockdown experiments, we find that the GTP-binding protein, Arfrp1, and the clathrin adaptor complex 1 (AP-1) are required for Vangl2 transport from the TGN. In contrast, TGN export of Frizzled 6, which localizes to the opposing epithelial surface from Vangl2, does not depend on Arfrp1 or AP-1. Mutagenesis studies identified a YYXXF sorting signal in the C-terminal cytosolic domain of Vangl2 that is required for Vangl2 traffic and interaction with the µ subunit of AP-1. We propose that Arfrp1 exposes a binding site on AP-1 that recognizes the Vangl2 sorting motif for capture into a transport vesicle destined for the proximal surface of a polarized epithelial cell.DOI:http://dx.doi.org/10.7554/eLife.00160.001.

Cryo-electron tomographic structure of an immunodeficiency virus envelope complex in situ.

The envelope glycoprotein (Env) complexes of the human and simian immunodeficiency viruses (HIV and SIV, respectively) mediate viral entry and are a target for neutralizing antibodies. The receptor binding surfaces of Env are in large part sterically occluded or conformationally masked prior to receptor binding. Knowledge of the unliganded, trimeric Env structure is key for an understanding of viral entry and immune escape, and for the design of vaccines to elicit neutralizing antibodies. We have used cryo-electron tomography and averaging to obtain the structure of the SIV Env complex prior to fusion. Our result reveals novel details of Env organisation, including tight interaction between monomers in the gp41 trimer, associated with a three-lobed, membrane-distal gp120 trimer. A cavity exists at the gp41-gp120 trimer interface. Our model for the spike structure agrees with previously predicted interactions between gp41 monomers, and furthers our understanding of gp120 interactions within an intact spike.