An MRI assessment of prostate cancer local recurrence using the PI-RR system: diagnostic accuracy, inter-observer reliability among readers with variable experience, and correlation with PSA values.

OBJECTIVES: The Prostate Imaging for Recurrence Reporting (PI-RR) system has been recently proposed to promote standardisation in the MR assessment of prostate cancer (PCa) local recurrence after radical prostatectomy (RP) and radiation therapy (RT). This study aims to evaluate PI-RR's diagnostic accuracy, assess the inter-observer reliability among readers with variable experience, and correlate imaging results with anatomopathological and laboratory parameters. METHODS: Patients who underwent a pelvic MRI for suspicion of PCa local recurrence after RP or RT were retrospectively enrolled (October 2017-February 2020). PI-RR scores were independently assessed for each patient by five readers with variable experience in prostate MRI (two senior and three junior radiologists). Biochemical data and histopathological features were collected. The reference standard was determined through biochemical, imaging, or histopathological follow-up data. Reader's diagnostic performance was assessed using contingency tables. Cohen's kappa coefficient (κ) and intraclass correlation coefficient (ICC) were calculated to measure inter-observer reliability. RESULTS: The final cohort included 120 patients (median age, 72 years [IQR, 62-82]). Recurrence was confirmed in 106 (88.3%) patients. Considering a PI-RR score ≥ 3 as positive for recurrence, minimum and maximum diagnostic values among the readers were as follows: sensitivity 79-86%; specificity 64-86%; positive predictive value 95-98%; negative predictive value 33-46%; accuracy 79-87%. Regardless of reader's level of experience, the inter-observer reliability resulted good or excellent (κ ranges across all readers: 0.52-0.77), and ICC was 0.8. Prostate-specific antigen (PSA) velocity, baseline-PSA, and trigger-PSA resulted predictive of local recurrence at imaging. CONCLUSIONS: The PI-RR system is an effective tool for MRI evaluation of PCa local recurrence and facilitates uniformity among radiologists. CLINICAL RELEVANCE STATEMENT: This study confirmed the PI-RR system's good diagnostic accuracy for the MRI evaluation of PCa local recurrences. It showed high reproducibility among readers with variable experience levels, validating it as a promising standardisation tool for assessing patients with biochemical recurrence. KEY POINTS: • In this retrospective study, the PI-RR system revealed promising diagnostic performances among five readers with different experience (sensitivity 79-86%; specificity 64-86%; accuracy 79-87%). • The inter-observer reliability among the five readers resulted good or excellent (κ ranges: 0.52-0.77) with an intraclass correlation coefficient of 0.8. • The PI-RR assessment score may facilitate standardisation and generalizability in the evaluation of prostate cancer local recurrence among radiologists.

Exploratory Analysis of the Role of Radiomic Features in the Differentiation of Oncocytoma and Chromophobe RCC in the Nephrographic CT Phase.

In diagnostic imaging, distinguishing chromophobe renal cell carcinomas (chRCCs) from renal oncocytomas (ROs) is challenging, since they both present similar radiological characteristics. Radiomics has the potential to help in the differentiation between chRCCs and ROs by extracting quantitative imaging. This is a preliminary study of the role of radiomic features in the differentiation of chRCCs and ROs using machine learning models. In this retrospective work, 38 subjects were involved: 19 diagnosed with chRCCs and 19 with ROs. The CT nephrographic contrast phase was selected in each case. Three-dimensional segmentations of the lesions were performed and the radiomic features were extracted. To assess the reliability of the features, the intraclass correlation coefficient was calculated from the segmentations performed by three radiologists with different degrees of expertise. The selection of features was based on the criteria of excellent intraclass correlation coefficient (ICC), high correlation, and statistical significance. Three machine learning models were elaborated: support vector machine (SVM), random forest (RF), and logistic regression (LR). From 105 extracted features, 41 presented an excellent ICC and 6 were not highly correlated with each other. Only two features showed significant differences according to histological type and machine learning models were developed with them. LR was the better model, in particular, with an 83% precision.

Gynaecological Causes of Acute Pelvic Pain: Common and Not-So-Common Imaging Findings.

In female patients, acute pelvic pain can be caused by gynaecological, gastrointestinal, and urinary tract pathologies. Due to the variety of diagnostic possibilities, the correct assessment of these patients may be challenging. The most frequent gynaecological causes of acute pelvic pain in non-pregnant women are pelvic inflammatory disease, ruptured ovarian cysts, ovarian torsion, and degeneration or torsion of uterine leiomyomas. On the other hand, spontaneous abortion, ectopic pregnancy, and placental disorders are the most frequent gynaecological entities to cause acute pelvic pain in pregnant patients. Ultrasound (US) is usually the first-line diagnostic technique because of its sensitivity across most common aetiologies and its lack of radiation exposure. Computed tomography (CT) may be performed if ultrasound findings are equivocal or if a gynaecologic disease is not initially suspected. Magnetic resonance imaging (MRI) is an extremely useful second-line technique for further characterisation after US or CT. This pictorial review aims to review the spectrum of gynaecological entities that may manifest as acute pelvic pain in the emergency department and to describe the imaging findings of these gynaecological conditions obtained with different imaging techniques.

Stabilization of 14-3-3 protein-protein interactions with Fusicoccin-A decreases alpha-synuclein dependent cell-autonomous death in neuronal and mouse models.

Synucleinopathies are a group of neurodegenerative diseases without effective treatment characterized by the abnormal aggregation of alpha-synuclein (aSyn) protein. Changes in levels or in the amino acid sequence of aSyn (by duplication/triplication of the aSyn gene or point mutations in the encoding region) cause familial cases of synucleinopathies. However, the specific molecular mechanisms of aSyn-dependent toxicity remain unclear. Increased aSyn protein levels or pathological mutations may favor abnormal protein-protein interactions (PPIs) that could either promote neuronal death or belong to a coping response program against neurotoxicity. Therefore, the identification and modulation of aSyn-dependent PPIs can provide new therapeutic targets for these diseases. To identify aSyn-dependent PPIs we performed a proximity biotinylation assay based on the promiscuous biotinylase BioID2. When expressed as a fusion protein, BioID2 biotinylates by proximity stable and transient interacting partners, allowing their identification by streptavidin affinity purification and mass spectrometry. The aSyn interactome was analyzed using BioID2-tagged wild-type (WT) and pathological mutant E46K aSyn versions in HEK293 cells. We found the 14-3-3 epsilon isoform as a common protein interactor for WT and E46K aSyn. 14-3-3 epsilon correlates with aSyn protein levels in brain regions of a transgenic mouse model overexpressing WT human aSyn. Using a neuronal model in which aSyn cell-autonomous toxicity is quantitatively scored by longitudinal survival analysis, we found that stabilization of 14-3-3 protein-proteins interactions with Fusicoccin-A (FC-A) decreases aSyn-dependent toxicity. Furthermore, FC-A treatment protects dopaminergic neuronal somas in the substantia nigra of a Parkinson's disease mouse model. Based on these results, we propose that the stabilization of 14-3-3 epsilon interaction with aSyn might reduce aSyn toxicity, and highlight FC-A as a potential therapeutic compound for synucleinopathies.

Pilot Study for the Assessment of the Best Radiomic Features for Bosniak Cyst Classification Using Phantom and Radiologist Inter-Observer Selection.

Since the Bosniak cysts classification is highly reader-dependent, automated tools based on radiomics could help in the diagnosis of the lesion. This study is an initial step in the search for radiomic features that may be good classifiers of benign-malignant Bosniak cysts in machine learning models. A CCR phantom was used through five CT scanners. Registration was performed with ARIA software, while Quibim Precision was used for feature extraction. R software was used for the statistical analysis. Robust radiomic features based on repeatability and reproducibility criteria were chosen. Excellent correlation criteria between different radiologists during lesion segmentation were imposed. With the selected features, their classification ability in benignity-malignity terms was assessed. From the phantom study, 25.3% of the features were robust. For the study of inter-observer correlation (ICC) in the segmentation of cystic masses, 82 subjects were prospectively selected, finding 48.4% of the features as excellent regarding concordance. Comparing both datasets, 12 features were established as repeatable, reproducible, and useful for the classification of Bosniak cysts and could serve as initial candidates for the elaboration of a classification model. With those features, the Linear Discriminant Analysis model classified the Bosniak cysts in terms of benignity or malignancy with 88.2% accuracy.

Microglia and astrocyte activation is region-dependent in the α-synuclein mouse model of Parkinson's disease.

Inflammation is a common feature in neurodegenerative diseases that contributes to neuronal loss. Previously, we demonstrated that the basal inflammatory tone differed between brain regions and, consequently, the reaction generated to a pro-inflammatory stimulus was different. In this study, we assessed the innate immune reaction in the midbrain and in the striatum using an experimental model of Parkinson's disease. An adeno-associated virus serotype 9 expressing the α-synuclein and mCherry genes or the mCherry gene was administered into the substantia nigra. Myeloid cells (CD11b+ ) and astrocytes (ACSA2+ ) were purified from the midbrain and striatum for bulk RNA sequencing. In the parkinsonian midbrain, CD11b+ cells presented a unique anti-inflammatory transcriptomic profile that differed from degenerative microglia signatures described in experimental models for other neurodegenerative conditions. By contrast, striatal CD11b+ cells showed a pro-inflammatory state and were similar to disease-associated microglia. In the midbrain, a prominent increase of infiltrated monocytes/macrophages was observed and, together with microglia, participated actively in the phagocytosis of dopaminergic neuronal bodies. Although striatal microglia presented a phagocytic transcriptomic profile, morphology and cell density was preserved and no active phagocytosis was detected. Interestingly, astrocytes presented a pro-inflammatory fingerprint in the midbrain and a low number of differentially displayed transcripts in the striatum. During α-synuclein-dependent degeneration, microglia and astrocytes experience context-dependent activation states with a different contribution to the inflammatory reaction. Our results point towards the relevance of selecting appropriate cell targets to design neuroprotective strategies aimed to modulate the innate immune system during the active phase of dopaminergic degeneration.

Haemodynamic-dependent arrest of circulating tumour cells at large blood vessel bifurcations as new model for metastasis.

Homing of circulating tumour cells (CTC) at distant sites represents a critical event in metastasis dissemination. In addition to physical entrapment, probably responsible of the majority of the homing events, the vascular system provides with geometrical factors that govern the flow biomechanics and impact on the fate of the CTC. Here we mathematically explored the distribution of velocities and the corresponding streamlines at the bifurcations of large blood vessel and characterized an area of low-velocity at the carina of bifurcation that favours the residence of CTC. In addition to this fluid physics effect, the adhesive capabilities of the CTC provide with a biological competitive advantage resulting in a marginal but systematic arrest as evidenced by dynamic in vitro recirculation in Y-microchannels and by perfusion in in vivo mice models. Our results also demonstrate that viscosity, as a main determinant of the Reynolds number that define flow biomechanics, may be modulated to limit or impair CTC accumulation at the bifurcation of blood vessels, in agreement with the apparent positive effect observed in the clinical setting by anticoagulants in advanced oncology disease.

Understanding the Potential Role of Sirtuin 2 on Aging: Consequences of SIRT2.3 Overexpression in Senescence.

Sirtuin 2 (SIRT2) has been associated to aging and age-related pathologies. Specifically, an age-dependent accumulation of isoform 3 of SIRT2 in the CNS has been demonstrated; however, no study has addressed the behavioral or molecular consequences that this could have on aging. In the present study, we have designed an adeno-associated virus vector (AAV-CAG-Sirt2.3-eGFP) for the overexpression of SIRT2.3 in the hippocampus of 2 month-old SAMR1 and SAMP8 mice. Our results show that the specific overexpression of this isoform does not induce significant behavioral or molecular effects at short or long term in the control strain. Only a tendency towards a worsening in the performance in acquisition phase of the Morris Water Maze was found in SAMP8 mice, together with a significant increase in the pro-inflammatory cytokine Il-1ß. These results suggest that the age-related increase of SIRT2.3 found in the brain is not responsible for induction or prevention of senescence. Nevertheless, in combination with other risk factors, it could contribute to the progression of age-related processes. Understanding the specific role of SIRT2 on aging and the underlying molecular mechanisms is essential to design new and more successful therapies for the treatment of age-related diseases.

Seeing through the smoke: Human and animal studies of cannabis use and endocannabinoid signalling in corticolimbic networks.

Public opinion surrounding the recreational use and therapeutic potential of cannabis is shifting. This review describes new work examining the behavioural and neural effects of cannabis and the endocannabinoid system, highlighting key regions within corticolimbic brain circuits. First, we consider the role of human genetic factors and cannabis strain chemotypic differences in contributing to interindividual variation in the response to cannabinoids, such as THC, and review studies demonstrating that THC-induced impairments in decision-making processes are mediated by actions at prefrontal CB1 receptors. We further describe evidence that signalling through prefrontal or ventral hippocampal CB1 receptors modulates mesolimbic dopamine activity, aberrations of which may contribute to emotional processing deficits in schizophrenia. Lastly, we review studies suggesting that endocannabinoid tone in the amygdala is a critical regulator of anxiety, and report new data showing that FAAH activity is integral to this response. Together, these findings underscore the importance of cannabinoid signalling in the regulation of cognitive and affective behaviours, and encourage further research given their social, political, and therapeutic implications.

Neuroprotective Effect of JZL184 in MPP(+)-Treated SH-SY5Y Cells Through CB2 Receptors.

Growing evidence suggests that the endocannabinoid system plays a role in neuroprotection in Parkinson's disease. Recently, we have shown the neuroprotective effect of monoacylglycerol lipase (MAGL) inhibition with JZL184 in the chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model. However, further investigation is needed to determine the neuroprotective mechanisms of the endocannabinoid system on the nigrostriatal pathway. The aim of this work was to investigate whether the neuroprotective effect of JZL184 in mice could be extended to an in vitro cellular model to further understand the mechanism of action of the drug. The SH-SY5Y cell line was selected based on its dopaminergic-like phenotype and its susceptibility to 1-methyl-4-phenylpyridinium iodide (MPP(+)) toxicity. Furthermore, SH-SY5Y cells express both cannabinoid receptors, CB1 and CB2. The present study describes the neuroprotective effect of MAGL inhibition with JZL184 in SH-SY5Y cells treated with MPP(+). The effect of JZL184 in cell survival was blocked by AM630, a CB2 receptor antagonist, and it was mimicked with JWH133, a CB2 receptor agonist. Rimonabant, a CB1 receptor antagonist, did not affect JZL184-induced cell survival. These results demonstrate that the neuroprotective effect of MAGL inhibition with JZL184 described in animal models of Parkinson's disease could be extended to in vitro models such as SH-SY5Y cells treated with MPP(+). This represents a useful tool to study mechanisms of neuroprotection mediated by MAGL inhibition, and we provide evidence for the possible involvement of CB2 receptors in the improvement of cell survival.

A laser-based technology for fabricating a soda-lime glass based microfluidic device for circulating tumour cell capture.

We developed a laser-based technique for fabricating microfluidic microchips on soda-lime glass substrates. The proposed methodology combines a laser direct writing, as a manufacturing tool for the fabrication of the microfluidics structures, followed by a post-thermal treatment with a CO2 laser. This treatment will allow reshaping and improving the morphological (roughness) and optical qualities (transparency) of the generated microfluidics structures. The use of lasers commonly implemented for material processing makes this technique highly competitive when compared with other glass microstructuring approaches. The manufactured chips were tested with tumour cells (Hec 1A) after being functionalized with an epithelial cell adhesion molecule (EpCAM) antibody coating. Cells were successfully arrested on the pillars after being flown through the device giving our technology a translational application in the field of cancer research.

GPR40 activation leads to CREB and ERK phosphorylation in primary cultures of neurons from the mouse CNS and in human neuroblastoma cells.

GPR40, the free fatty acid receptor 1, is expressed strongly in the primate pancreas and brain. While the role of pancreatic GPR40 in glucose homeostasis has been extensively studied, the absence of this G-protein-coupled receptor from the brain of rodents has hampered studies into its role in the central nervous system. However, we found intense GPR40 mRNA expression by in situ hybridization in mouse hippocampal and motor cortex neurons. Furthermore, in a neuroblastoma cell GPR40 was activated by docosahexaenoic acid and selective agonists, yet not by palmitic acid. Significantly, the activation of GPR40 provoked the phosphorylation of the cAMP response element-binding protein, CREB. The receptor was also functional in primary cultures of murine neurons, in which its activation by a selective agonist produced the phosphorylation of CREB and of extracellular signal-regulated kinases, ERK1/2. These results suggest that mice represent a suitable model for elucidating the role of GPR40 in brain function.

A simple and efficient method for the production of human glycosylated glial cell line-derived neurotrophic factor using a Semliki Forest virus expression system.

Human glial cell line-derived neurotrophic factor (hGDNF) is a very promising protein for the treatment of Parkinson's disease and other neurodegenerative disorders. The present work describes a quick and simple method to obtain a high amount of purified hGDNF using a mammalian cell-derived system. The method is based on the high expression level provided by a Semliki Forest virus vector and its ability to induce a strong shut-off of host-cell protein synthesis in mammalian cells. As a result, hGDNF is the only protein present in the supernatant and can be efficiently purified by a single chromatographic step. Using this system it was possible to eliminate other secreted proteins from the culture medium, like insulin-like growth factor-5, which are hard to remove using other hGDNF production methods. Purified hGDNF presents a complex glycosylation pattern typical of mammalian expression systems and is biologically active. This protocol could be extended to other secreted proteins and could be easily scaled up for industrial purposes.

Production of highly pure human glycosylated GDNF in a mammalian cell line.

The administration of glial cell line-derived neurotrophic factor (GDNF) has emerged as a promising strategy for the treatment of several diseases of the nervous system as Parkinson's disease, amyotrophic lateral sclerosis, spinal cord injury and nerve regeneration as well as ocular diseases and drug addictions. A procedure for the purification of human recombinant glycosylated GDNF using a mammalian expression system as the source of the protein is discussed in the present paper. The neurotrophic factor was purified using cation exchange chromatography and gel filtration. A human cell line was chosen as the source of therapeutic protein, since a recombinant protein with a structure and glycosylation pattern equivalent to the native form is desirable for its prospective therapeutic utilization. The activity of the highly pure protein obtained was confirmed with a cell-based bioassay. The purified protein is suitable for its in vivo evaluation in animals and for possible subsequent clinical application.

Two functional epitopes of pigment epithelial-derived factor block angiogenesis and induce differentiation in prostate cancer.

Pigment epithelial-derived factor (PEDF), an angiogenesis inhibitor with neurotrophic properties, balances angiogenesis in the eye and blocks tumor progression. Its neurotrophic function and the ability to block vascular leakage is replicated by the PEDF 44-mer peptide (residues 58-101). We analyzed PEDFs' three-dimensional structure and identified a potential receptor-binding surface. Seeking PEDF-based antiangiogenic agents we generated and tested peptides representing the middle and lower regions of this surface. We identified previously unknown antiangiogenic epitopes consisting of the 34-mer (residues 24-57) and a shorter proximal peptide (TGA, residues 16-26) with the critical stretch L19VEEED24 and a fragment within the 44-mer (ERT, residues 78-94), which retained neurotrophic activity. The 34-mer and TGA, but not the 44-mer reproduced PEDF angioinhibitory signals hinged on c-jun-NH2-kinase-dependent nuclear factor of activated T cell deactivation and caused apoptosis. Conversely, the ERT, but not the 34-mer/TGA induced neuronal differentiation. For the 44-mer/ERT, we showed a novel ability to cause neuroendocrine differentiation in prostate cancer cells. PEDF and the peptides bound endothelial and PC-3 prostate cancer cells. Bound peptides were displaced by PEDF, but not by each other, suggesting multiple receptors. PEDF and its active fragments blocked tumor formation when conditionally expressed by PC-3 cells. The 34- and 44-mer used distinct mechanisms: the 34-mer acted on endothelial cells, blocked angiogenesis, and induced apoptosis whereas 44-mer prompted neuroendocrine differentiation in cancer cells. Our results map active regions for the two PEDF functions, signaling via distinct receptors, identify candidate peptides, and provide their mechanism of action for future development of PEDF-based tumor therapies.

Striatal expression of GDNF and differential vulnerability of midbrain dopaminergic cells.

Glial cell line-derived neurotrophic factor (GDNF) is a member of the transforming growth factor-beta superfamily that when exogenously administrated exerts a potent trophic action on dopaminergic (DA) cells. Although we know a lot about its signalling mechanisms and pharmacological effects, physiological actions of GDNF on the adult brain remain unclear. Here, we have used morphological and molecular techniques, and an experimental model of Parkinson's disease in rats, to investigate whether GDNF constitutively expressed in the adult mesostriatal system plays a neuroprotective role on midbrain DA cells. We found that although all midbrain DA cells express both receptor components of GDNF (GFRalpha1 and Ret), those in the ventral tegmental area (VTA) and rostromedial substantia nigra (SNrm) also contain GDNF but not GDNFmRNA. The levels of GDNFmRNA are significantly higher in the ventral striatum (vSt), the target region of VTA and SNrm cells, than in the dorsal striatum (dSt), the target region of DA cells in the caudoventral substantia nigra (SNcv). After fluoro-gold injection in striatum, VTA and SNrm DA cells show triple labelling for tyrosine hydroxylase, GDNF and fluoro-gold, and after colchicine injection in the lateral ventricle, they become GDNF-immunonegative, suggesting that GDNF in DA somata comes from their striatal target. As DA cells in VTA and SNrm are more resistant than those in SNcv to intracerebroventricular injection of 6-OHDA, as occurs in Parkinson's disease, we can suggest that the fact that they project to vSt, where GDNF expression is significantly higher than in the dSt, is a neuroprotective factor involved in the differential vulnerability of midbrain DA neurons.