Abdominal musculature segmentation and surface prediction from CT using deep learning for sarcopenia assessment.

PURPOSE: The purpose of this study was to build and train a deep convolutional neural networks (CNN) algorithm to segment muscular body mass (MBM) to predict muscular surface from a two-dimensional axial computed tomography (CT) slice through L3 vertebra. MATERIALS AND METHODS: An ensemble of 15 deep learning models with a two-dimensional U-net architecture with a 4-level depth and 18 initial filters were trained to segment MBM. The muscular surface values were computed from the predicted masks and corrected with the algorithm's estimated bias. Resulting mask prediction and surface prediction were assessed using Dice similarity coefficient (DSC) and root mean squared error (RMSE) scores respectively using ground truth masks as standards of reference. RESULTS: A total of 1025 individual CT slices were used for training and validation and 500 additional axial CT slices were used for testing. The obtained mean DSC and RMSE on the test set were 0.97 and 3.7 cm2 respectively. CONCLUSION: Deep learning methods using convolutional neural networks algorithm enable a robust and automated extraction of CT derived MBM for sarcopenia assessment, which could be implemented in a clinical workflow.

Diagnosis of focal liver lesions from ultrasound using deep learning.

PURPOSE: The purpose of this study was to create an algorithm that simultaneously detects and characterizes (benign vs. malignant) focal liver lesion (FLL) using deep learning. MATERIALS AND METHODS: We trained our algorithm on a dataset proposed during a data challenge organized at the 2018 Journées Francophones de Radiologie. The dataset was composed of 367 two-dimensional ultrasound images from 367 individual livers, captured at various institutions. The algorithm was guided using an attention mechanism with annotations made by a radiologist. The algorithm was then tested on a new data set from 177 patients. RESULTS: The models reached mean ROC-AUC scores of 0.935 for FLL detection and 0.916 for FLL characterization over three shuffled three-fold cross-validations performed with the training data. On the new dataset of 177 patients, our models reached a weighted mean ROC-AUC scores of 0.891 for seven different tasks. CONCLUSION: This study that uses a supervised-attention mechanism focused on FLL detection and characterization from liver ultrasound images. This method could prove to be highly relevant for medical imaging once validated on a larger independent cohort.

Detection and characterization of MRI breast lesions using deep learning.

PURPOSE: The purpose of this study was to assess the potential of a deep learning model to discriminate between benign and malignant breast lesions using magnetic resonance imaging (MRI) and characterize different histological subtypes of breast lesions. MATERIALS AND METHODS: We developed a deep learning model that simultaneously learns to detect lesions and characterize them. We created a lesion-characterization model based on a single two-dimensional T1-weighted fat suppressed MR image obtained after intravenous administration of a gadolinium chelate selected by radiologists. The data included 335 MR images from 335 patients, representing 17 different histological subtypes of breast lesions grouped into four categories (mammary gland, benign lesions, invasive ductal carcinoma and other malignant lesions). Algorithm performance was evaluated on an independent test set of 168 MR images using weighted sums of the area under the curve (AUC) scores. RESULTS: We obtained a cross-validation score of 0.817 weighted average receiver operating characteristic (ROC)-AUC on the training set computed as the mean of three-shuffle three-fold cross-validation. Our model reached a weighted mean AUC of 0.816 on the independent challenge test set. CONCLUSION: This study shows good performance of a supervised-attention model with deep learning for breast MRI. This method should be validated on a larger and independent cohort.

Predictors of responses to immune checkpoint blockade in advanced melanoma.

Immune checkpoint blockers (ICB) have become pivotal therapies in the clinical armamentarium against metastatic melanoma (MMel). Given the frequency of immune related adverse events and increasing use of ICB, predictors of response to CTLA-4 and/or PD-1 blockade represent unmet clinical needs. Using a systems biology-based approach to an assessment of 779 paired blood and tumor markers in 37 stage III MMel patients, we analyzed association between blood immune parameters and the functional immune reactivity of tumor-infiltrating cells after ex vivo exposure to ICB. Based on this assay, we retrospectively observed, in eight cohorts enrolling 190 MMel patients treated with ipilimumab, that PD-L1 expression on peripheral T cells was prognostic on overall and progression-free survival. Moreover, detectable CD137 on circulating CD8+ T cells was associated with the disease-free status of resected stage III MMel patients after adjuvant ipilimumab + nivolumab (but not nivolumab alone). These biomarkers should be validated in prospective trials in MMel.The clinical management of metastatic melanoma requires predictors of the response to checkpoint blockade. Here, the authors use immunological assays to identify potential prognostic/predictive biomarkers in circulating blood cells and in tumor-infiltrating lymphocytes from patients with resected stage III melanoma.

Ketamine alters cortical integration of GABAergic interneurons and induces long-term sex-dependent impairments in transgenic Gad67-GFP mice.

Ketamine, a non-competitive N-methyl-D-aspartate (NMDA) antagonist, widely used as an anesthetic in neonatal pediatrics, is also an illicit drug named Super K or KitKat consumed by teens and young adults. In the immature brain, despite several studies indicating that NMDA antagonists are neuroprotective against excitotoxic injuries, there is more and more evidence indicating that these molecules exert a deleterious effect by suppressing a trophic function of glutamate. In the present study, we show using Gad67-GFP mice that prenatal exposure to ketamine during a time-window in which GABAergic precursors are migrating results in (i) strong apoptotic death in the ganglionic eminences and along the migratory routes of GABAergic interneurons; (ii) long-term deficits in interneuron density, dendrite numbers and spine morphology; (iii) a sex-dependent deregulation of γ-aminobutyric acid (GABA) levels and GABA transporter expression; (iv) sex-dependent changes in the response to glutamate-induced calcium mobilization; and (v) the long-term sex-dependent behavioral impairment of locomotor activity. In conclusion, using a preclinical approach, the present study shows that ketamine exposure during cortical maturation durably affects the integration of GABAergic interneurons by reducing their survival and differentiation. The resulting molecular, morphological and functional modifications are associated with sex-specific behavioral deficits in adults. In light of the present data, it appears that in humans, ketamine could be deleterious for the development of the brain of preterm neonates and fetuses of addicted pregnant women.

Review of preparative and analytical procedures for the study of proteins in grape juice and wine.

Proteins have a great influence on wine quality as they exhibit a various range of properties. In fact, they are involved among others in white wine turbidity, organoleptic characteristics and foam formation in sparkling wines. These compounds could also be of major interest for varietal differentiation, regarding wine authentication and traceability issues. To provide a better understanding of the role played by these biomolecules in wine processing and explore their potential applications, there is a manifest need for the quantification and characterization of each individual one in terms of sequence, structure and intrinsic and functional properties. We thus present an overview of preparative and analytical methods for the study of proteins in grape juices and wines, from routine techniques to dedicated methodologies. They include sample preparation with chromatographic methods for the purification and identification of proteins, quantification protocols and characterization procedures such as electrophoretic techniques, immunological methods, sequencing, mass spectrometry, physico-chemical and structural analyses, and so on. We expose advantages and limits of each technique and focus on the different but complementary information they can provide. Despite the past years advances in the field proteins identification, the elucidation of the full protein profile for grape juices and wines remains strenuous. Their interactions with other wine compounds make the challenge even harder. We therefore emphasize the requirement of the techniques to be refined and suggest the developments to be expected.

Prolyl endopeptidase mRNA expression in the central nervous system during rat development.

Prolyl endopeptidase (PEP) is a serine protease that cleaves small peptides at the carboxyl side of L-proline. PEP has been reported to have important functions in the brain being implicated in learning and memory processes, psychological disorders and neurodegenerative diseases. Several PEP substrates have been shown to play a role during brain development and this observation led us to investigate the expression of PEP mRNA in the rat brain and spinal cord, from embryo to adult stages. In situ hybridization revealed that PEP mRNA is expressed early, from embryonic day 15, notably in germinative areas including the neocortical, hippocampal, pallidal, thalamic, anterior hypothalamic, tectal, cerebellar, pontine and medullary neuroepithelia. PEP mRNA was also found in the differentiating fields of the olfactory bulb, the orbital and cingulate cortex, the hippocampal formation, the cortical plate and the subventricular zone of the cortex. Quantitative RT-PCR analysis in various brain areas and the spinal cord showed that PEP mRNA levels are more abundant during the perinatal stages, coinciding with a period of neuronal migration and differentiation. From then on, PEP mRNA expression decreased, reaching its lowest levels at adulthood. Overall, the present data support the possibility that PEP exerts specific functions related to neurodevelopment besides those proposed to date.

Gastric electrical stimulation modulates hypothalamic corticotropin-releasing factor-producing neurons during post-operative ileus in rat.

High-frequency/low-energy gastric electrical stimulation (GES) is an efficient therapy to treat gastric emptying-related disorders but its mechanism of action remains poorly understood. We aimed to assess the effects of high-frequency/low-energy GES on corticotropin-releasing factor (CRF)-producing neurons in the paraventricular nucleus of the hypothalamus (PVN), which are involved in gastric ileus induced by laparotomy. Two electrodes were implanted in the rat gastric antrum during laparotomy, then stimulation (amplitude: 2 mA; pulse duration 330 micros; frequency: 2 Hz; 1 min ON/2 min OFF) or sham stimulation (control group) were applied. Using immunohistochemistry, the number of c-Fos protein-expressing neurons (c-Fos protein-immunoreactive cells, Fos-IR) was quantified in the PVN after 1 h of stimulation. The number of neurons expressing simultaneously c-Fos protein and CRF mRNA was measured by means of immunocytochemistry combined with in situ hybridization. Finally, c-Fos and CRF mRNA levels in the hypothalamus were determined by in situ hybridization or quantitative reverse transcriptase-polymerase chain reaction. Fos-IR in the PVN was significantly decreased 1 h after GES (P<0.05) but was not affected by sub-diaphragmatic vagotomy. The number of neurons containing c-Fos protein and CRF mRNA was lower in the GES group compared with the control group (P<0.05). In addition, c-Fos and CRF mRNA levels in the PVN were significantly decreased by GES (P

Pituitary adenylate cyclase-activating polypeptide directly modulates the activity of proopiomelanocortin neurons in the rat arcuate nucleus.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and the proopiomelanocortin (POMC)-derived peptide alpha-melanocyte-stimulating hormone (alpha-MSH) both regulate multiple neuroendocrine functions and feeding behavior. Two subtypes of PACAP receptor mRNAs, pituitary adenylate cyclase-activating polypeptide-specific receptor (PAC1-R) and pituitary adenylate cyclase-activating polypeptide/vasoactive intestinal polypeptide mutual receptor (VPAC2-R), are actively expressed in the arcuate nucleus of the hypothalamus, where POMC cell bodies are located. This observation led us to investigate the possible regulatory action of PACAP on rat POMC neurons. Double-labeling in situ hybridization histochemistry revealed that approximately 50% of POMC-producing neurons express PAC1-R and/or VPAC2-R mRNAs. The proportion of POMC neurons that also contain PAC1-R mRNA was homogeneous along the rostro-caudal axis of the arcuate nucleus while POMC-positive cell bodies expressing the VPAC2-R subtype were more abundant in the rostral region. Incubation of mediobasal hypothalamic explants with PACAP (10(-7) M; 30 min) increased POMC mRNA expression, and this effect was blocked by PACAP6-38 (10(-6) M). In contrast, incubation with vasoactive intestinal polypeptide (10(-7) M) did not affect POMC mRNA level. Incubation of hypothalamic fragments with PACAP (10(-7) M) caused a significant increase in alpha-MSH content in the tissue and in the incubation medium. Altogether, the present results reveal that exogenous PACAP, acting probably through PAC1-R, regulates the activity of POMC neurons in the rat hypothalamus. These data suggest that the effects of PACAP on the gonadotropin-releasing hormone neuroendocrine axis and the regulation of feeding behavior may be mediated, at least in part, through modulation of POMC neurons.

In situ hybridization localization of TRH precursor and TRH receptor mRNAs in the brain and pituitary of Xenopus laevis.

We examined the distribution of the mRNAs encoding proTRH and the three TRH receptor subtypes (xTRHR1, xTRHR2, and xTRHR3) in the Xenopus laevis CNS and pituitary. A positive correlation was generally observed between the expression patterns of proTRH and xTRHR mRNAs. xTRHRs were widely expressed in the telencephalon and diencephalon, where two or even three xTRHR mRNAs were often simultaneously observed within the same brain structures. In the pituitary, xTRHR2 was selectively expressed in the distal lobe, and xTRHR3 was found exclusively in the intermediate lobe of white background-adapted animals, indicating that, in amphibians, the effect of TRH on alpha-melanotropin (alpha-MSH) secretion from melanotrope cells is mediated through the novel receptor subtype xTRHR3.

Effect of prolyl endopeptidase inhibition on arginine-vasopressin and thyrotrophin-releasing hormone catabolism in the rat brain.

Compound S 17092 is a potent and selective inhibitor of prolyl endopeptidase (EC 3.4.21.26, PEP) that may be of therapeutic value for the treatment of memory impairment associated with neurodegenerative diseases. In the present study, we investigated the effects of S 17092 on the catabolism of the promnesic neuropeptides thyrotrophin-releasing hormone (TRH) and arginine-vasopressin (AVP) in the rat brain. In vitro, bacterial PEP hydrolysed both TRH and AVP, and the breakdown of the two peptides was almost completely prevented by 10(-5) M S 17092. In vivo, a single oral administration of S 17092 provoked a significant increase in TRH-like immunoreactivity (TRH-LI) in the cerebral cortex (+63% for a 10 mg/kg dose and +72% for a 30 mg/kg dose), as well as AVP-LI in the hippocampus (+54% for a 30 mg/kg dose), but did not affect TRH-LI in the amygdala nor AVP-LI in the cerebral cortex. Chronic administration of S 17092 (10 or 30 mg/kg daily) lead to a significant increase in THR-LI in the cerebral cortex (+55% and +56%, respectively), but did not modify AVP-LI in the hippocampus, nor in the cerebral cortex. These results show that the selective PEP inhibitor S 17092 increases TRH and AVP content in discrete regions of the rat brain. The present data suggest that the promnesic and antiamnesic effects of S 17092 can be accounted for, at least in part, by blockage of AVP and TRH degradation by PEP.

Adenosine A2A receptor gene disruption provokes marked changes in melanocortin content and pro-opiomelanocortin gene expression.

A2A receptor knockout (A2AR-/-) mice are more anxious and aggressive, and exhibit reduced exploratory activity than their wild-type littermates (A2AR+/+). Because alpha-melanocyte-stimulating hormone (alpha-MSH) influences anxiety, aggressiveness and motor activity, we investigated the effect of A2AR gene disruption on alpha-MSH content in discrete brain regions and pro-opiomelanocortin (POMC) expression in the hypothalamus and pituitary. No modification in alpha-MSH content was observed in the hypothalamus and medulla oblongata where POMC-expressing perikarya are located. In the arcuate nucleus of the hypothalamus, POMC mRNA levels were not affected by A2AR disruption. Conversely, in A2AR-/- mice, a significant increase in alpha-MSH content was observed in the amygdala and cerebral cortex, two regions that are innervated by POMC terminals. In the pars intermedia of the pituitary, A2AR disruption provoked a significant reduction of POMC mRNA expression associated with a decrease in alpha-MSH content. By contrast, in the anterior lobe of the pituitary, a substantial increase in POMC mRNA and adrenocorticotropin hormone concentrations was observed, and plasma corticosterone concentration was significantly higher in A2AR-/- mice, revealing hyperactivity of their pituitary-adrenocortical axis. Together, these results suggest that adenosine, acting through A2A receptors, may modulate the release of alpha-MSH in the cerebral cortex and amygdala. The data also indicate that A2A receptors are involved in the control of POMC gene expression and biosynthesis of POMC-derived peptides in pituitary melanotrophs and corticotrophs.

Galanin modulates the activity of proopiomelanocortin neurons in the isolated mediobasal hypothalamus of the male rat.

It has become apparent that galanin as well as proopiomelanocortin-derived peptides, such as beta-endorphin, play an important role in the hypothalamic circuitry that regulates neuroendocrine functions and appetite behavior. We have recently shown that GalR1 and GalR2 galanin receptor mRNAs are expressed in proopiomelanocortin neurons of the arcuate nucleus, suggesting a direct modulatory action of galanin on the proopiomelanocortin neuronal system. In the present study, we investigated the effect of galanin on beta-endorphin release and proopiomelanocortin mRNA expression from male rat mediobasal hypothalamic fragments incubated ex vivo. Galanin induced a decrease of spontaneous beta-endorphin release within the first 30-60 min of incubation and this effect was blocked by the galanin receptor antagonist galantide. Co-incubation of galanin with FK-506 (tacrolimus), a calcineurin inhibitor, suppressed the inhibitory effect of galanin on beta-endorphin release, suggesting that calcineurin is involved in the galanin-evoked decrease in beta-endorphin release. Measurement of beta-endorphin levels in the tissues at the end of the incubation period (120 min) revealed that galanin caused a two-fold increase of beta-endorphin peptide concentration in the mediobasal hypothalamic tissues. Concurrently, galanin induced an increase in the mean density of silver grains overlying proopiomelanocortin neurons after 60 min of incubation, an effect antagonized by galantide. Finally, reverse transcription-polymerase chain reaction analysis revealed that the mRNAs for the three galanin receptor subtypes (i.e. GalR1, GalR2, and GalR3) were expressed in the incubated mediobasal hypothalamic fragments. Taken as a whole, our results indicate that galanin plays a modulatory role on proopiomelanocortin neurons and this interrelation contributes to the elucidation of the neural circuitry that controls, among others, gonadotropin-releasing hormone function.

Evidence of the glycation and denaturation of LTP1 during the malting and brewing process.

The influence of malting and brewing processes on the chemical and structural modifications occurring on LTP1 was investigated by mass spectrometry and circular dichroism. Proteins were first purified from malt, and samples were collected at various steps of beer processing performed on two barley cultivars. The levels of LTP1 found in malt were not significantly different from the amounts in barley seed. However, in malt, both LTP1b, a post-translational form of LTP1, and a third isoform named LTP1c were isolated. Moreover, both of these proteins were found to be heterogeneously glycated but still exhibited an alpha-helix structure. Both glycated LTP1 and LTP1b were recovered during mashing. It was also shown that glycated LTP1 was unfolded during heat treatment of wort boiling, which is in agreement with the denatured form previously isolated from beer.

Evidence that TGF beta may directly modulate POMC mRNA expression in the female rat arcuate nucleus.

The purpose of the present study was to determine whether TGF beta, a cytokine secreted by hypothalamic astrocytes, was able to regulate POMC neurons in the arcuate nucleus. In a first set of experiments, mediobasal hypothalamic fragments were exposed to TGF beta(1), and the relative POMC mRNA expression was assessed by in situ hybridization using a radiolabeled POMC riboprobe. The results showed that 4 x 10(-10) M TGF beta(1) was efficient in decreasing significantly the amounts of POMC mRNA (P < 0.01). Interestingly, the decrease of relative POMC mRNA levels was higher in the rostral than in the caudal parts of the arcuate nucleus. In a second set of experiments, we examined the occurrence of TGF beta receptors expression in arcuate POMC neurons. Dual labeling in situ hybridization and in situ hybridization, coupled to immunohistochemical labeling, were performed to examine mRNA expression of the type I serine-threonine kinase receptor for TGF beta and the presence of type II receptor for TGF beta, respectively, in POMC neurons. The results indicated that TGF beta receptor I mRNA and TGF beta receptor II protein were expressed in numerous POMC neurons. Regional analysis revealed that the highest proportion of POMC neurons expressing TGF beta receptors was located in the rostral part of the arcuate nucleus. Using dual labeling immunohistochemistry, we also found that Smad2/3 immunoreactivity, a TGF beta(1) downstream signaling molecule, was present in the cytoplasm and nucleus of some POMC (beta-endorphin) neurons. We next examined whether the number of POMC neurons expressing TGF beta-RI mRNA was affected by sex steroids. Quantification of the number of POMC neurons expressing TGF beta receptor I mRNA in ovariectomized, ovariectomized E2-treated, and ovariectomized E2 plus progesterone-treated animals revealed that estrogen treatment decreased the expression of TGF beta receptor I mRNA in POMC neurons located in the rostral half of the arcuate nucleus, an effect reversed by progesterone in a subset of the most rostral cells. Taken together, these data reveal that TGF beta(1) may directly modulate the activity of POMC neurons through the activation of TGF beta receptors. Therefore, the present study provides additional evidence for the involvement of TGF beta(1) in the regulation of neuroendocrine functions and supports the existence of a glial-to-neurons communication within the arcuate nucleus.

Identification of a new form of lipid transfer protein (LTP1) in wheat seeds.

Recently, this laboratory has isolated from barley and beer extract an isoform of lipid transfer protein (LTP1), which was not fully sequenced (Jégou, S.; Douliez, J. P.; Mollé, D.; Boivin, P.; Marion, D. J. Agric. Food Chem. 2000, 48, 5023--5029). It was named LTP1b and exhibited a molecular weight 294 Da higher than that of the known LTP1. This paper reports the finding of an LTP1 isoform in wheat that also exhibits an excess of 294 Da compared to the native protein. Amino acid sequencing, reduction and alkylation, and mass spectrometry showed that this new LTP1b possesses the same N-terminal sequence as the native LTP1, suggesting that the difference resides in the binding of an adduct which has a molecular weight of 294 Da. The aim of the present paper is to highlight various biophysical techniques that afford the identification of such an isoform-like LTP1 and to correlate this finding with other isoforms of LTP1 that were isolated from other plants but not fully sequenced.

Specific expression of the urotensin II gene in sacral motoneurons of developing rat spinal cord.

The neuropeptide urotensin II (UII) is expressed in motoneurons of the brainstem and spinal cord in adults. Here, the expression pattern of the UII gene was studied in the developing rat spinal cord. UII mRNA was detected by reverse-transcription-polymerase chain reaction (RT-PCR) as early as E10. From E14 to E21, in situ hybridization revealed intense expression of the UII gene specifically in sacral motoneurons, while only faint expression was detected at cervical and thoracic levels. After birth (P0, P4), the expression of UII mRNA increased in motoneurons at all rostrocaudal levels. Thus, UII is the first gene reported to show expression limited to the sacral pool of motoneurons, which are known to have particular properties in terms of targets and programmed cell death.

Binding of two mono-acylated lipid monomers by the barley lipid transfer protein, LTP1, as viewed by fluorescence, isothermal titration calorimetry and molecular modelling.

The binding of two mono-acylated lipid monomers by plant lipid transfer proteins (LTP1s) presents an attractive field of research that could help our understanding of the functional role of this protein family. This task has been investigated in the case of barley LTP1 because it is known to exhibit a small cavity in its free state. The titration with lipids could not be followed by fluorescence with the native protein. Indeed, this LTP1 possesses a tyrosine residue on its C-terminus, Tyr91, which is not sensitive to lipid binding but mainly contributes to the fluorescence signal intensity. However, the binding of 1-myristoylglycerophosphatidylcholine (MyrGro-PCho) could be monitored by fluorescence after removal of Tyr91 by a carboxypeptidase. These experiments returned a dissociation constant of about 1 microM and showed that the protein can indeed bind two monomers. This result was corroborated by molecular modelling where the structure of the complex between barley LTP1 and MyrGro-PCho was derived from that determined in the case of wheat [Charvolin, D., Douliez, J.P., Marion, D., Cohen-addad, C. & Pebay-Peyroula, E. (1999) Eur. J. Biochem. 264, 562-568.]. Results from isothermal titration calorimetry experiments indicated non-classic titration behaviour but also suggested that two lipids could be bound by the protein. Finally, barley LTP1 binds two omega-hydroxypalmitic acid, a compound found in the family of cutin monomers. The fact that the binding of two lipids could be related to the physiological role of this protein family is discussed.

Purification and structural characterization of LTP1 polypeptides from beer.

We report on the purification of lipid transfer proteins (LTP) from barley seeds and beer with the aim of investigating the chemical modifications that occur during the brewing process. In seeds, the well-known LTP of 9 kDa (LTP1) has been found together with a second form named LTPb that displays comparable amino acid composition but was not fully sequenced. These two forms have been recovered in beer with marked chemical modifications including disulfide bond reduction and rearrangement and especially glycation by Maillard reaction. The glycation is heterogeneous with variable amounts of hexose units bound to LTPs. Circular dichroism shows that glycated LTP1 having all their disulfide bridges reduced are totally unfolded. These results provide a first basis for understanding how barley LTPs become foam-promoting agents during the malting and brewing process.