Diurnal variations of resting-state fMRI data : a graph-based analysis

Circadian rhythms (lasting approximately 24 h) control and entrain various physiological processes, ranging from neural activity and hormone secretion to sleep cycles and eating habits. Several studies have shown that time of day (TOD) is associated with human cognition and brain functions. In this study, utilizing a chronotype-based paradigm, we applied a graph theory approach on resting-state functional MRI (rs-fMRI) data to compare whole-brain functional network topology between morning and evening sessions and between morning-type (MT) and evening-type (ET) participants. Sixty-two individuals (31 MT and 31 ET) underwent two fMRI sessions, approximately 1 hour (morning) and 10 h (evening) after their wake-up time, according to their declared habitual sleep-wake pattern on a regular working day. In the global analysis, the findings revealed the effect of TOD on functional connectivity (FC) patterns, including increased small-worldness, assortativity, and synchronization across the day. However, we identified no significant differences based on chronotype categories. The study of the modular structure of the brain at mesoscale showed that functional networks tended to be more integrated with one another in the evening session than in the morning session. Local/regional changes were affected by both factors (i.e., TOD and chronotype), mostly in areas associated with somatomotor, attention, frontoparietal, and default networks. Furthermore, connectivity and hub analyses revealed that the somatomotor, ventral attention, and visual networks covered the most highly connected areas in the morning and evening sessions: the latter two were more active in the morning sessions, and the first was identified as being more active in the evening. Finally, we performed a correlation analysis to determine whether global and nodal measures were associated with subjective assessments across participants. Collectively, these findings contribute to an increased understanding of diurnal fluctuations in resting brain activity and highlight the role of TOD in future studies on brain function and the design of fMRI experiments

Serotoninergic receptor ligands improve Tamoxifen effectiveness on breast cancer cells

Background: Serotonin (or 5-Hydroxytryptamine, 5-HT) signals in mammary gland becomes dysregulated in cancer, also contributing to proliferation, metastasis, and angiogenesis. Thus, the discovery of novel compounds targeting serotonin signaling may contribute to tailor new therapeutic strategies usable in combination with endocrine therapies. We have previously synthesized serotoninergic receptor ligands (SER) with high affinity and selectivity towards 5-HT2A and 5-HT2C receptors, the main mediators of mitogenic effect of serotonin in breast cancer (BC). Here, we investigated the effect of 10 SER on viability of MCF7, SKBR3 and MDA-MB231 BC cells and focused on their potential ability to affect Tamoxifen responsiveness in ER+ cells. Methods: Cell viability has been assessed by sulforhodamine B assay. Cell cycle has been analyzed by flow cytometry. Gene expression of 5-HT receptors and Connective Tissue Growth Factor (CTGF) has been checked by RT-PCR; mRNA levels of CTGF and ABC transporters have been further measured by qPCR. Protein levels of 5-HT2C receptors have been analyzed by Western blot. All data were statistically analyzed using GraphPad Prism 7. Results: We found that treatment with SER for 72 h reduced viability of BC cells. SER were more effective on MCF7 ER+ cells (IC50 range 10.2 μM - 99.2 μM) compared to SKBR3 (IC50 range 43.3 μM - 260 μM) and MDA-MB231 BC cells (IC50 range 91.3 μM - 306 μM). This was paralleled by accumulation of cells in G0/G1 phase of cell cycle. Next, we provided evidence that two ligands, SER79 and SER68, improved the effectiveness of Tamoxifen treatment in MCF7 cells and modulated the expression of CTGF, without affecting viability of MCF10A non-cancer breast epithelial cells. In a cell model of Tamoxifen resistance, SER68 also restored drug effect independently of CTGF. Conclusions: These results identified serotoninergic receptor ligands potentially usable in combination with Tamoxifen to improve its effectiveness on ER+ BC patients

miR135a administration ameliorates brain ischemic damage by preventing TRPM7 activation during brain ischemia

Background: miRNA-based strategies have recently emerged as a promising therapeutic approach in several neurodegenerative diseases. Unregulated cation influx is implicated in several cellular mechanisms underlying neural cell death during ischemia. The brain constitutively active isoform of transient receptor potential melastatin 7 (TRPM7) represents a glutamate excitotoxicity-independent pathway that significantly contributes to the pathological Ca2+ overload during ischemia. Aims: In the light of these premises, inhibition of TRPM7 may be a reasonable strategy to reduce ischemic injury. Since TRPM7 is a putative target of miRNA135a, the aim of the present paper was to evaluate the role played by miRNA135a in cerebral ischemia. Therefore, the specific objectives of the present paper were: (1) to evaluate miR135a expression in temporoparietal cortex of ischemic rats; (2) to investigate the effect of the intracerebroventricular (icv) infusion of miR135a on ischemic damage and neurological functions; and (3) to verify whether miR135a effects may be mediated by an alteration of TRPM7 expression. Methods: miR135a expression was evaluated by RT- PCR and FISH assay in temporoparietal cortex of ischemic rats. Ischemic volume and neurological functions were determined in rats subjected to transient middle cerebral artery occlusion (tMCAo) after miR135a intracerebroventricular perfusion. Target analysis was performed by Western blot. Results: Our results demonstrated that, in brain cortex, 72 h after ischemia, miR135a expression increased, while TRPM7 expression was parallelly downregulated. Interestingly, miR135a icv perfusion strongly ameliorated the ischemic damage and improved neurological functions, and downregulated TRPM7 protein levels. Conclusions: The early prevention of TRPM7 activation is protective during brain ischemia

Prostate Health Index (Phi) and Prostate Cancer Antigen 3 (PCA3) Significantly Improve Prostate Cancer Detection at Initial Biopsy in a Total PSA Range of 2-10 ng/ml

Many efforts to reduce prostate specific antigen (PSA) overdiagnosis and overtreatment have been made. To this aim, Prostate Health Index (Phi) and Prostate Cancer Antigen 3 (PCA3) have been proposed as new more specific biomarkers. We evaluated the ability of phi and PCA3 to identify prostate cancer (PCa) at initial prostate biopsy in men with total PSA range of 2-10 ng/ml. The performance of phi and PCA3 were evaluated in 300 patients undergoing first prostate biopsy. ROC curve analyses tested the accuracy (AUC) of phi and PCA3 in predicting PCa. Decision curve analyses (DCA) were used to compare the clinical benefit of the two biomarkers. We found that the AUC value of phi (0.77) was comparable to those of %p2PSA (0.76) and PCA3 (0.73) with no significant differences in pairwise comparison (%p2PSA vs phi p = 0.673, %p2PSA vs. PCA3 p = 0.417 and phi vs. PCA3 p = 0.247). These three biomarkers significantly outperformed fPSA (AUC = 0.60), %fPSA (AUC = 0.62) and p2PSA (AUC = 0.63). At DCA, phi and PCA3 exhibited a very close net benefit profile until the threshold probability of 25%, then phi index showed higher net benefit than PCA3. Multivariable analysis showed that the addition of phi and PCA3 to the base multivariable model (age, PSA, %fPSA, DRE, prostate volume) increased predictive accuracy, whereas no model improved single biomarker performance. Finally we showed that subjects with active surveillance (AS) compatible cancer had significantly lower phi and PCA3 values (p < 0.001 and p = 0.01, respectively). In conclusion, both phi and PCA3 comparably increase the accuracy in predicting the presence of PCa in total PSA range 2-10 ng/ml at initial biopsy, outperforming currently used %fPSA

Novel USH1G homozygous variant underlying USH2-like phenotype of Usher syndrome

PURPOSE: Usher syndrome (USH) is an autosomal recessive disorder characterized by congenital sensorineural hearing impairment and retinitis pigmentosa. Classification distinguishes three clinical types of which type I (USH1) is the most severe, with vestibular dysfunction as an added feature. To date, 15 genes and 3 loci have been identified with the USH1G gene being an uncommon cause of USH. We describe an atypical USH1G-related phenotype caused by a novel homozygous missense variation in a patient with profound hearing impairment and relatively mild retinitis pigmentosa, but no vestibular dysfunction. METHODS: A 26-year-old female patient with profound congenital sensorineural hearing loss, nyctalopia and retinitis pigmentosa was studied. Audiometric, vestibular and ophthalmologic examination was performed. A panel of 13 genes was tested by next-generation sequencing (NGS). RESULTS: While the hearing loss was confirmed to be profound, the vestibular function resulted normal. Although typical retinitis pigmentosa was present, the age at onset was unusually late for USH1 syndrome. A novel homozygous missense variation (c.1187T>A, p.Leu396Gln) in the USH1G gene has been identified as causing the disease in our patient. CONCLUSIONS: Genetic and phenotypic heterogeneity are very common in both isolated and syndromic retinal dystrophies and sensorineural hearing loss. Our findings widen the spectrum of USH allelic disorders and strength the concept that variants in genes that are classically known as underlying one specific clinical USH subtype might result in unexpected phenotypes

Adenoviral gene transfer of PLD1-D4 enhances insulin sensitivity in mice by disrupting phospholipase D1 interaction with PED/PEA-15.

Over-expression of phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA-15) causes insulin resistance by interacting with the D4 domain of phospholipase D1 (PLD1). Indeed, the disruption of this association restores insulin sensitivity in cultured cells over-expressing PED/PEA-15. Whether the displacement of PLD1 from PED/PEA-15 improves insulin sensitivity in vivo has not been explored yet. In this work we show that treatment with a recombinant adenoviral vector containing the human D4 cDNA (Ad-D4) restores normal glucose homeostasis in transgenic mice overexpressing PED/PEA-15 (Tg ped/pea-15) by improving both insulin sensitivity and secretion. In skeletal muscle of these mice, D4 over-expression inhibited PED/PEA-15-PLD1 interaction, decreased Protein Kinase C alpha activation and restored insulin induced Protein Kinase C zeta activation, leading to amelioration of insulin-dependent glucose uptake. Interestingly, Ad-D4 administration improved insulin sensitivity also in high-fat diet treated obese C57Bl/6 mice. We conclude that PED/PEA-15-PLD1 interaction may represent a novel target for interventions aiming at improving glucose tolerance

Platelet-rich plasma counteracts detrimental effect of high-glucose concentrations on mesenchymal stem cells from Bichat fat pad

Diabetic patients display increased risk of periodontitis and failure in bone augmentation procedures. Mesenchymal stem cells (MSCs) and platelet-rich plasma (PRP) represent a relevant advantage in tissue repair process and regenerative medicine. We isolated MSCs from Bichat's buccal fat pad (BFP) and measured the effects of glucose and PRP on cell number and osteogenic differentiation potential. Cells were cultured in the presence of 5.5-mM glucose (low glucose [LG]) or 25-mM glucose (high glucose [HG]). BFP–MSC number was significantly lower when cells were cultured in HG compared with those in LG. Following osteogenic differentiation procedures, calcium accumulation, alkaline phosphatase activity, and expression of osteogenic markers were significantly lower in HG compared with LG. Exposure of BFP–MSC to PRP significantly increased cell number and osteogenic differentiation potential, reaching comparable levels in LG and in HG. Thus, high-glucose concentrations impair BFP–MSC growth and osteogenic differentiation. However, these detrimental effects are largely counteracted by PRP

Noninvasive diffusion magnetic resonance imaging of brain tumour cell size for the early detection of therapeutic response

Cancer cells differ in size from those of their host tissue and are known to change in size during the processes of cell death. A noninvasive method for monitoring cell size would be highly advantageous as a potential biomarker of malignancy and early therapeutic response. This need is particularly acute in brain tumours where biopsy is a highly invasive procedure. Here, diffusion MRI data were acquired in a GL261 glioma mouse model before and during treatment with Temozolomide. The biophysical model VERDICT (Vascular Extracellular and Restricted Diffusion for Cytometry in Tumours) was applied to the MRI data to quantify multi-compartmental parameters connected to the underlying tissue microstructure, which could potentially be useful clinical biomarkers. These parameters were compared to ADC and kurtosis diffusion models, and, measures from histology and optical projection tomography. MRI data was also acquired in patients to assess the feasibility of applying VERDICT in a range of different glioma subtypes. In the GL261 gliomas, cellular changes were detected according to the VERDICT model in advance of gross tumour volume changes as well as ADC and kurtosis models. VERDICT parameters in glioblastoma patients were most consistent with the GL261 mouse model, whilst displaying additional regions of localised tissue heterogeneity. The present VERDICT model was less appropriate for modelling more diffuse astrocytomas and oligodendrogliomas, but could be tuned to improve the representation of these tumour types. Biophysical modelling of the diffusion MRI signal permits monitoring of brain tumours without invasive intervention. VERDICT responds to microstructural changes induced by chemotherapy, is feasible within clinical scan times and could provide useful biomarkers of treatment response

po 292 breast cancer stem cell reprogramming deciphering the impact of glucose and the contribute of tumour microenvironment

Introduction Diabetes-associated hyperglycemia is linked to poorer prognosis and survival in breast cancer (BC). Indeed, glucose can affect both tumour and tumour-surrounding cells. BC cells are embedded in an adipocyte-rich microenvironment, which, beside adipocytes, contains Stromal-Vascular Fraction Cells (SVFCs). In this scenario, epithelial and stromal compartments communicate through the release of soluble factors and establish an intricate crosstalk. Here, we analysed whether glucose could directly affect the phenotype of ER+ MCF7 BC cells and interfere with their interaction with adipose-derived SVFCs, thereby promoting tumour progression. Material and methods MCF7 cell stemness markers were measured by qReal-Time PCR. Adipose-derived (Ad-)SVFCs were obtained by mammary adipose tissue specimens of women undergoing plastic surgery. The trascriptome of MCF7 exposed to either low (LG-5.5 mM) or high glucose concentration (HG-25 mM) was obtained by RNA-Sequencing (Illumina HiSeq3000). Results and discussions HG exposure of MCF7 determined a significant increase of SOX2 mRNA levels as compared to LG, suggesting the induction of stemness programming. Co-culture with Ad-SVFCs in HG increased SOX2, NANOG and OCT4 mRNA levels in MCF7, as compared to isolated culture, indicating the involvement of SVF-produced soluble factors in BC stem cell reprogramming. Moreover, in presence of Ad-SVFCs and HG, MCF7 produced a higher number of mammospheres, which also displayed larger size. However, both in LG and in HG, conditioned media (CM) obtained from Ad-SVFCs produced no relevant effect on MCF7 stemness. Nevertheless, when Ad-SVFCs were pre-incubated with CM obtained from HG-treated MCF7, their CM very effectively increased OCT4, NANOG and SOX2 mRNA levels in MCF7. Thus, HG likely perturbs MCF7, which produce soluble factors leading Ad-SVFCs to release, in turn, reprogramming factors for BC cell stemness. In this regard, we have observed that HG modification of MCF7 transcriptome includes deregulation of 17 genes (pval=0.05) encoding for secreted proteins involved in cancer progression-related pathways, which may potentially play a role in tumour-stroma interactions. Conclusion Glucose affects BC stem cell reprogramming both directly and through Ad-SVFCs. Deciphering the mechanisms that govern this intricate crosstalk will pave the way to new targeted strategies to improve BC control in conditions of metabolic derangement

Planar cell polarity genes Celsr1 and Vangl2 are necessary for kidney growth, differentiation, and rostrocaudal patterning

The mammalian kidney contains nephrons comprising glomeruli and tubules joined to ureteric bud–derived collecting ducts. It has a characteristic bean-like shape, with near-complete rostrocaudal symmetry around the hilum. Here we show that Celsr1, a planar cell polarity (PCP) gene implicated in neural tube morphogenesis, is required for ureteric tree growth in early development and later in gestation prevents tubule overgrowth. We also found an interaction between Celsr1 and Vangl2 (another PCP gene) in ureteric tree growth, most marked in the caudal compartment of the kidneys from compound heterozygous mutant mice with a stunted rump. Furthermore, these genes together are required for the maturation of glomeruli. Interestingly, we demonstrated patients with CELSR1 mutations and spina bifida can have significant renal malformations. Thus, PCP genes are important in mammalian kidney development and have an unexpected role in rostrocaudal patterning during organogenesis