C. Mucignat

In the last couple of decades, the study of human living brain has benefitted of neuroimaging and non-invasive electrophysiological techniques, which are particularly valuable during development. A number of studies allowed to trace the usual stages leading from pregnancy to adult age, and relate them to functional and behavioral measurements. It was also possible to explore the effects of some interventions, behavioral or not, showing that the commonly followed pathway to adulthood may be steered by external interventions. These events may result in behavioral modifications but also in structural changes, in some cases limiting plasticity or extending/modifying critical periods. In this review, we outline the healthy human brain development in the absence of major issues or diseases. Then, the effects of negative (different stressors) and positive (music training) environmental stimuli on brain and behavioral development is depicted. Hence, it may be concluded that the typical deve...

Smell disorders are commonly reported with COVID-19 infection. Some patients show prolonged smell-related issues, even after the respiratory symptoms are resolved. To explore the concerns of patients, and to provide an overview for each specific smell disorder, we explored the longitudinal survey that was conducted by1, and contained self-reports on the changes of smell that participants experienced at two time points. People who still suffered from smell disorders at the second time point, hence named ‘longhaulers’, were compared to those who were not, hence named ‘non-longhaulers’. Specifically, three aims were pursued in this study. First, to classify smell disorders based on the participants’ self-reports. Second, to classify the sentiment of each self-report using a machine learning approach, and third, to find specific keywords that best describe the smell dysfunction in those self-reports. We found that the prevalence of parosmia and hyposmia was higher in longhaulers than in...

Introduction and aimDentistry is a highly demanding profession with a strong mental and physical involvement, possibly generating anxiety. Very few studies assessed psychophysiological activity in dentists, while none tried to relate it with gender during a routine working day. This study aims at evaluating correlations between gender, psychophysiological indexes, and psychological variables.Materials and methodsData were acquired at the Dental Clinic of the University of Padua on 20 healthy young dentists (10 M-10F) during a 24 h period of a working day. Physiological variables (measured with E4 Empatica) were electrodermal activity (EDA), heart rate variability (HRV) and heart rate (HR). Participants anxiety was measured through a self-reported scale on patient-relationship anxiety and through the Generalized Anxiety Disorder-7 Questionnaire (GAD-7).Results5 (3F, 2 M) participants over 20 had a GAD-7 score ≥ 10. Female gender, in comparison to Male, was associated with higher perc...

Chemosensory impairments have been established as a specific indicator of COVID-19. They affect most patients and may persist long past the resolution of respiratory symptoms, representing an unprecedented medical challenge. Since the SARS-CoV-2 pandemic started, we now know much more about smell, taste, and chemesthesis loss associated with COVID-19. However, the temporal dynamics and characteristics of recovery are still unknown. Here, capitalizing on data from the Global Consortium for Chemosensory Research (GCCR) crowdsourced survey, we assessed chemosensory abilities after the resolution of respiratory symptoms in participants diagnosed with COVID-19 during the first wave of the pandemic in Italy. This analysis led to the identification of two patterns of chemosensory recovery, limited (partial) and substantial, which were found to be associated with differential age, degrees of chemosensory loss, and regional patterns. Uncovering the self-reported phenomenology of recovery fro...

Background Olfaction is often affected in parkinsonian patients, but dopaminergic cells in the olfactory bulb are not affected by some Parkinson-inducing drugs. We investigated whether the drug MPTP produces the olfactory deficits typical of Parkinson and affects the olfactory bulb in mice. Findings Lesioned and control mice were tested for olfactory search, for motor and exploratory behavior. Brains and olfactory mucosa were investigated via immunohistochemistry for thyrosine hydroxylase, Olfactory Marker Protein and cyclic AMP-dependent protein kinase as an intracellular pathway involved in dopaminergic neurotransmission. MPTP induced motor impairment, but no deficit in olfactory search. Thyrosine hydroxylase did not differ in olfactory bulb, while a strong decrease was detected in substantia nigra and tegmentum of MPTP mice. Olfactory Marker Protein decreased in the olfactory bulb of MPTP mice, while a cyclic AMP-dependent protein kinase increased in the inner granular layer of M...

In the rodent brain, the central processing of ecologically relevant chemical stimuli involves many different areas located at various levels within the neuraxis: the main and accessory olfactory bulbs, some nuclei in the amygdala, the hypothalamus, and brainstem. These areas allow the integration of the chemosensory stimuli with other sensory information and the selection of the appropriate neurohormonal and behavioral response. This review is a brief introduction to the processing of intraspecific chemosensory stimuli beyond the secondary projection, focusing on the activity of the relevant amygdala and hypothalamic nuclei, namely the medial amygdala and ventromedial hypothalamus. These areas are involved in the appropriate interpretation of chemosensory information and drive the selection of the proper response, which may be behavioral or hormonal and may affect the neural activity of other areas in the telencephalon and brainstem. Recent data support the notion that the processing of intraspecific chemical signals is not unique to one chemosensory system and some molecules may activate both the main and the accessory olfactory system. Moreover, both these systems have mixed projections and cooperate for the correct identification of the stimuli and selection of relevant responses.

In eukaryotic cells, cAMP regulates many different cellular functions. Its effects are in most cases mediated by cAMP-dependent protein kinases. These consist of two regulatory and two catalytic subunits. In mammals, four different isoforms of cAMP-dependent protein kinases regulatory subunits have been characterized (RIa and ß, RIIa and ß). These four isoforms show a high level of homology and slightly

Papillary thyroid carcinoma (PTC) is the most common malignant tumor of the thyroid gland, accounting for 74-80% of all thyroid cancers. The 1799T>A transversion is an activating mutation of the BRAF oncogene that is common in and specific to conventional PTC. We studied the prevalence, tumorigenic role, and biochemical implications of rare BRAF variants in a large cohort of patients. A total of 2131 fine-needle aspiration biopsy samples were collected and subjected to BRAF mutation analysis. BRAF genetic variants were analyzed by Western blot, immunofluorescence, and in silico analysis. BRAF mutations were found in 50% (347/700) of thyroid cancers (644 PTCs, 22 anaplastic thyroid carcinomas, 34 follicular thyroid carcinomas). They were the classic (c.1799T>A, p.V600E) mutation in 96.8% (336/347) and rare genetic variants in 3.2% (11/347). In all, five infrequent BRAF alterations were detected: (i) c.1795_1797dupACA (p.T599dup); (ii) c.1801A>G (p.K601E); (iii) c.1799_1801delTGA (p.V600_K601>E); (iv) c.1799_1814>A (p.V600_S605>D); and (v) c.1798_1810delinsA (p.V600_W604>R). The last BRAF variant has never been described in the literature. Western blot analysis and immunofluorescence both revealed a variegated reactivity pattern, again emphasizing the peculiar role of every specific BRAF genetic alteration. In silico analysis of the samples studied revealed a stabilization of the…

A recombinant bovine herpesvirus 4 (BoHV-4EGFPDeltaTK), obtained by the insertion of an EGFP gene into the TK locus of DN 599 BoHV-4 strain, was injected into the lateral ventricle of the brain of mice and a clinical score was evaluated for 90 days. Although BoHV-4 was not neuro-pathogenic, BoHV-4EGFPDeltaTK transduction capability was analyzed. EGFP expression was localized in close proximity to the border of the ventricles and EGFP-positive cells were found to co-localize with ependymal cells. Although most of the cells had a polarized morphology, they were not neurons. EGFP-positive cells were seen to spread in tangentially oriented rows within the rostral migratory stream (RMS). Co-localization of EGFP signal with anti-GFAP antibody showed that they were glial cells. EGFP-positive cells were observed until 31 days post-injection and then disappeared completely. Virus isolation was possible at an early post-injection time (3 days), but then virus titer was below the detection limits at later times. Viral DNA, however, could be detected until 21 days post-injection. Thus, in this report we showed that (i) BoHV-4EGFPDeltaTK did not replicate in the mouse brain, (ii) is not pathogenic and (iii) gene transfer can be obtained in long-lived cells belonging to the RMS after BoHV-4EGFPDeltaTK injection within the lateral ventricle.

cAMP is a ubiquitous second messenger, which acts mainly through specific protein kinases that consist of two regulatory and two catalytic subunits. An unsolved problem in cAMP physiology is how it can regulate so many cellular functions through this simple enzymatic cascade. A tentative explanation is related to the different biochemical properties of the four regulatory subunit isoforms (RIα and RIβ, RIIα and RIIβ) and to their differential cell and tissue distribution. For example, detergent insoluble aggregates of RIα are present in some cholinergic neurons of the adult rat brain. Rat brains, from the embryonic stage to old age, were examined for the presence of highly concentrated clusters of RIα. They are present only in some neurons of restricted brain areas, for a limited time span. During development, labeled neurons appear in different brain areas after neuron migration, at a stage of advanced functional maturation. They have their greatest expression after birth but befor...

The intracellular second messenger adenosine 3′,5′-cyclic monophosphate (cAMP) acts mainly through cAMP-dependent protein kinases (PKA). In mammals and reptiles, the PKA regulatory isoforms (RI and RII) are differentially distributed among the various brain areas and cell types, according to the age of the animal. Since PKA distribution may be an additional marker for homologous areas, PKA regulatory subunit types RI and RII were examined in the chicken brain, a species not yet investigated. Chicken brains were examined from prehatching to adult age, by means of immunohistochemistry and biochemical characterization. Most PKA regulatory subunits were segregated in discrete non-soluble clusters that contained either RI or RII. While RII aggregates were present also in non-neuronal cells, RI aggregates were detected only in neurons of some brain areas that are mainly related to the telencephalon. They appeared later than RII aggregates; their presence and location varied during develop...

To contribute clarifying mechanisms operating in nose chemosensory epithelia and their developmental patterns, we analyzed the expression of different epithelial membrane transporters as well as the Clara cell secretory protein, CC26 in the olfactory, vomeronasal organ (VNO), and respiratory epithelia of embryonic (E13-E19) and postnatal (P1-P60) mice by means of immunohistochemistry and reverse transcriptase-polymerase chain reaction. Results showed that CC26, cAMP-activated chloride channel (CFTR), and the water channel protein aquaporin 2, 3, 4, and 5 (AQP2, AQP3, AQP4, and AQP5) are expressed in developing to adult chemosensory epithelia with differential timing; moreover, their pattern of expression is not identical in VNO and olfactory epithelia as well as the corresponding associated glands; co-localization experiments using olfactory marker protein showed that CFTR, CC26, and AQP4 are not expressed in olfactory neurones. CFTR is expressed in sustentacular cells of the VNO and olfactory epithelium as well as blood vessels of the underlying mucosa, and VNO (but not Bowman's) glands; a similar pattern (excluding blood vessels) is present for AQP2; AQP4 is found in the two chemosensory epithelia and in Bowman's glands. AQP3 is expressed in the olfactory epithelium and the associated Bowman's glands, but not in the VNO chemosensory epithelium and glands. AQP5 is expressed in the olfactory epithelium and both Bowman's and VNO glands. These results indicate that water/ions handling as well as antioxidant mechanisms operating at the surface and/or inside the nose chemosensory epithelia start developing in utero and are maintained up to sexual maturity.

Regulatory subunits of the cAMP dependent protein kinases are the most abundant receptor for cAMP in eukaryotic cells. Four isoforms of regulatory subunits (RIalpha and -beta, RIIalpha and -beta) have been distinguished. Distribution of the most abundant RII isoforms has been extensively studied in the brain, by immunohistochemistry and biochemical fractionation, while the least abundant RI isoforms have been neglected. In neurons most regulatory subunits are bound to the cytoskeleton. A protocol is presented that allows immunohistochemical and biochemical characterization of detergent-insoluble RI isoforms in the brain.

cAMP is a universal second messenger. In eucaryotes it acts mainly via protein kinases composed of regulatory (R) and catalytic subunits; their subcellular distribution may differ according to the cell type. In rodent brain, peculiar detergent-insoluble RIalpha aggregates were previously described in neurons of areas related to the limbic system, while RIIbeta is more evenly distributed also in non-nervous cells. It is unclear whether the regional distribution of regulatory subunits is typical of mammalian brain. Western blots and immunohistochemistry showed that in lizard brains a large fraction of the cAMP-dependent protein kinase regulatory isoforms is insoluble, as in mammals. Insoluble RIalpha and RII regulatory isoforms were not evenly distributed but organized in clearly separated aggregates. Numerous RII aggregates were present in almost all brain regions and were found also in non-nervous cells. As shown by immunohistochemistry and equilibrium binding of fluorescently tagged cAMP, RIalpha aggregates were restricted to neurons of some brain regions: telencephalon, particularly medial cortical areas, dorsal ventricular ridge, olfactory pathways, medial hypothalamus and cerebellar granular layer were intensely labelled. A very weak RIalpha labelling was detected in the brainstem reticular formation, in the periaqueductal gray and in the spinal cord dorsal horn. A similar distribution of RIalpha aggregates was also found in turtle brains. Their distribution is reminiscent of that observed in mammals, although with some differences in relative intensity and persistence. The supramolecular organization of the RIalpha isoform may help in establishing homologies and differences between brain areas involved in visceroemotional control.

An isocratic high-performance liquid chromatography (HPLC) method was developed and validated to determine Aloe Emodin (AE) in mouse plasma. The analysis required 0.3ml of plasma and involves extraction with dichloromethane. The HPLC separation was carried out on Symmetry Shield RP18, a mobile phase of methanol–water–acetic acid (65:35:0.2) and fluorescence detection at λex=410nm and λem=510nm. The retention time of AE was

During the first wave of infections, neurological symptoms in Coronavirus Disease 2019 (COVID-19) patients raised particular concern, suggesting that, in a subset of patients, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could invade and damage cells of the central nervous system (CNS). Indeed, up to date several in vitro and in vivo studies have shown the ability of SARS-CoV-2 to reach the CNS. Both viral and/or host related features could explain why this occurs only in certain individuals and not in all the infected population. The aim of the present study was to evaluate if onset of neurological manifestations in COVID-19 patients was related to specific viral genomic signatures. To this end, viral genome was extracted directly from nasopharyngeal swabs of selected SARS-CoV-2 positive patients presenting a spectrum of neurological symptoms related to COVID-19, ranging from anosmia/ageusia to more severe symptoms. By adopting a whole genome sequences approach,...

Chemosensory systems are deemed marginal in human pathology. In appraising their role, we aim at suggesting a paradigm shift based on the available clinical and experimental data that will be discussed. Taste and olfaction are polymodal sensory systems, providing inputs to many brain structures that regulate crucial visceral functions, including metabolism but also endocrine, cardiovascular, respiratory, and immune systems. Moreover, other visceral chemosensory systems monitor different essential chemical parameters of “milieu intérieur,” transmitting their data to the brain areas receiving taste and olfactory inputs; hence, they participate in regulating the same vital functions. These chemosensory cells share many molecular features with olfactory or taste receptor cells, thus they may be affected by the same pathological events. In most COVID-19 patients, taste and olfaction are disturbed. This may represent only a small portion of a broadly diffuse chemosensory incapacitation. I...

Chemosensory systems (olfaction, taste, trigeminus nerve, solitary chemoreceptor cells, neuroendocrine pulmonary cells, and carotid body, etc.) detect molecules outside or inside our body and may share common molecular markers. In addition to the impairment of taste and olfaction, the detection of the internal chemical environment may also be incapacitated by COVID-19. If this is the case, different consequences can be expected. (1) In some patients, hypoxia does not trigger distressing dyspnea (“silent” hypoxia): Long-term follow-up may determine whether silent hypoxia is related to malfunctioning of carotid body chemoreceptors. Moreover, taste/olfaction and oxygen chemoreceptors may be hit simultaneously: Testing olfaction, taste, and oxygen chemoreceptor functions in the early stages of COVID-19 allows one to unravel their connections and trace the recovery path. (2) Solitary chemosensory cells are also involved in the regulation of the innate mucosal immune response: If these ce...

ObjectiveOur aim was to estimate the prevalence of gingival overgrowth (hyperplasia) and to determine whether active molecules affect the severity of overgrowth in a group of epileptic patients.BackgroundThe effects of phenytoin on oral health have been explored in different studies, yet little information is available on other antiepileptic drugs.MethodsData were collected from 213 subjects of both sexes, from 5 to 80 years. Patients taking the same antiepileptic therapy for at least 1 year and meeting the inclusion criteria of the study (n = 162) were subjected to measurement of gingival overgrowth according to the modified Harris and Ewalt classification and O’Leary's plaque control record (OLR). Descriptive statistics were calculated. Data were analyzed using Pearson's r correlation coefficient and chi‐square test. Significance level was set at 5%.ResultsThe active drugs lamotrigine, oxcarbazepine, and phenobarbital were significantly associated with gingival overgrowth ...

Recent anecdotal and scientific reports have provided evidence of a link between COVID-19 and chemosensory impairments such as anosmia. However, these reports have downplayed or failed to distinguish potential effects on taste, ignored chemesthesis, generally lacked quantitative measurements, were mostly restricted to data from single countries. Here, we report the development, implementation and initial results of a multi-lingual, international questionnaire to assess self-reported quantity and quality of perception in three distinct chemosensory modalities (smell, taste, and chemesthesis) before and during COVID-19. In the first 11 days after questionnaire launch, 4039 participants (2913 women, 1118 men, 8 other, ages 19-79) reported a COVID-19 diagnosis either via laboratory tests or clinical assessment. Importantly, smell, taste and chemesthetic function were each significantly reduced compared to their status before the disease. Difference scores (maximum possible change ±100) ...

Despite cellular complexity, a limited number of small molecules act as intracellular second messengers. Protein kinase A (PKA) is the main transducer of the information carried by cyclic adenosine monophosphate (cAMP). Recently, cellular imaging has achieved major technical advancements, although the search for more specific and sensitive low‐molecular‐weight probes to explore subcellular events involving second messengers is still in progress. The convergent synthesis of a novel, fluorescent small molecule comprising the cAMP structure and a rhodamine‐based fluorescent residue, connected through a flexible linker, is described here. The interaction motif of this compound with PKA was investigated in silico using a blind docking approach, comparing its theoretical binding energy with the one calculated for cAMP. Moreover, the predicted pharmacokinetic properties were also computed and discussed. The new probe was tested on three areas of the mouse central nervous system (parietal cerebral cortex, hippocampus, and cerebellar cortex) with different fixation methods demonstrating remarkable selectivity towards the PKA RIα subunit. The probe showed overall better performances when compared to other commercially available fluorescent cAMP analogues, acting at lower concentrations, and providing stable labeling.

Male pheromones are believed to attract females and repel male mice in open field tests but, when tested in more complex environments, they can attract male mice in usually avoided areas. Females were tested in an apparatus with one dark and one light side, in the absence or presence of male urine or the major urinary proteins (MUPs) bearing the

Glioblastoma is the most lethal brain tumor. The poor prognosis results from lack of defined tumor margins, critical location of the tumor mass and presence of chemo- and radio-resistant tumor stem cells. The current treatment for glioblastoma consists of neurosurgery, followed by radiotherapy and temozolomide chemotherapy. A better understanding of the role of molecular and genetic heterogeneity in glioblastoma pathogenesis allowed the design of novel targeted therapies. New targets include different key-role signaling molecules and specifically altered pathways. The new approaches include interference through small molecules or monoclonal antibodies and RNA-based strategies mediated by siRNA, antisense oligonucleotides and ribozymes. Most of these treatments are still being tested yet they stay as solid promises for a clinically relevant success.

30 PROTEIN-BOUND ODORANTS AS FLAGS OF MALE MOUSE PRESENCE Carla Mucignat-Caretta1 and Antonio Caretta2 'istituto di Fisiologia Umana ... most abundant being 2-sec-butyl-4, 5-dihydrothiazole and 3, 4-dehydro-ero-brevicomin (Bacchini, Gaetani & Cavaggioni ...

Recently, two large multigene families of putative G-protein-linked receptors that are expressed in distinct subpopulations of neurones in the vomeronasal organ have been identified. These receptors probably mediate pheromone detection. The most surprising aspects of these findings are that there are so many receptors of two very different classes and that the receptors are unrelated to their counterparts in the main olfactory epithelium. This suggests that many active ligands are likely to exert effects through the vomeronasal organ. Parallel experiments addressing the nature of these ligands indicate a role for some proteins, as well as small molecules, as functional mammalian pheromones. In combination, these results begin to suggest a molecular basis for mammalian pheromone signalling.

Male rat chemosignals attract females and influence their reproductive status. Through the accessory olfactory bulb and its projection target, the posteromedial cortical nucleus of the amygdala (PMCo), species‐specific chemosignals detected by the vomeronasal organ (VNO) may reach the hypothalamus. To test this hypothesis in vivo, behavioural activation and neurotransmitter release in the PMCo were simultaneously monitored in freely moving female oestrus rats exposed to either rat or mouse urinary stimuli, or to odorants. Plasma levels of the luteinizing hormone were subsequently monitored. All stimuli induced an immediate behavioural activation, but only species‐specific chemosignals led to a delayed behavioural activation. This biphasic behavioural activation was accompanied by a VNO‐mediated release of the excitatory amino acids, aspartate and glutamate, in the PMCo. The late behavioural and neurochemical activation was followed by an increase in the levels of circulating luteini...