Processing of intraspecific chemical signals in the rodent brain.

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.

Gingival overgrowth induced by anticonvulsant drugs: A cross-sectional study on epileptic patients.

OBJECTIVE: Our 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. BACKGROUND: The effects of phenytoin on oral health have been explored in different studies, yet little information is available on other antiepileptic drugs. METHODS: Data 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%. RESULTS: The active drugs lamotrigine, oxcarbazepine, and phenobarbital were significantly associated with gingival overgrowth in 61%, 71%, and 53% of cases, respectively, and phenytoin, valproic acid, and carbamazepine in 50%, 44%, and 32% of cases, respectively. CONCLUSION: Different antiepileptic molecules may be related to gingival overgrowth. In addition to phenytoin, also lamotrigine, oxcarbazepine, and phenobarbital were associated with increased prevalence of gingival overgrowth. In the management of epileptic patients, dentists should take into account different drugs as possible causes for gingival overgrowth and warn for possible alternatives.

A new sensitive and subunit-selective molecular tool for investigating protein kinase A in the brain.

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.

Protein Kinase A Catalytic and Regulatory Subunits Interact Differently in Various Areas of Mouse Brain.

Protein kinase A (PKA) are tetramers of two catalytic and two regulatory subunits, docked at precise intracellular sites to provide localized phosphorylating activity, triggered by cAMP binding to regulatory subunits and subsequent dissociation of catalytic subunits. It is unclear whether in the brain PKA dissociated subunits may also be found. PKA catalytic subunit was examined in various mouse brain areas using immunofluorescence, equilibrium binding and western blot, to reveal its location in comparison to regulatory subunits type RI and RII. In the cerebral cortex, catalytic subunits colocalized with clusters of RI, yet not all RI clusters were bound to catalytic subunits. In stria terminalis, catalytic subunits were in proximity to RI but separated from them. Catalytic subunits clusters were also present in the corpus striatum, where RII clusters were detected, whereas RI clusters were absent. Upon cAMP addition, the distribution of regulatory subunits did not change, while catalytic subunits were completely released from regulatory subunits. Unpredictably, catalytic subunits were not solubilized; instead, they re-targeted to other binding sites within the tissue, suggesting local macromolecular reorganization. Hence, the interactions between catalytic and regulatory subunits of protein kinase A consistently vary in different brain areas, supporting the idea of multiple interaction patterns.

Molecular targets in glioblastoma.

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.

The Old Made New: Natural Compounds against Erectile Dysfunction.

The interest toward sex-related diseases keeps growing through the years. In this review, we focus our attention on erectile dysfunction (ED), a condition that caught much attention especially after the introduction on the market of phosphodiesterase 5 inhibitors such as the well-known sildenafil. Here, we briefly describe both the etiology of ED and the available treatments, examining then extensively some natural derivatives that, coming from traditional medicine, could represent promising starting points for the development of alternative remedies. In fact, herbal remedies from several parts of the world have been traditionally known for long, and were recently reconsidered and are now being studied to demonstrate their eventual potential in the treatment of ED. Among the various examples reported in the literature and reviewed here, plants and extracts containing polyphenols­especially a class of compounds called kraussianones­appear to be particularly effective and promising against ED.

The antiproliferative effects of ouabain and everolimus on adrenocortical tumor cells.

Ouabain is a cardiotonic steroid obtained from Strophanthus. Recently its role as antiproliferative agent has been investigated in tumor cells. Everolimus is a derivative of rapamycin and acts as a signal transduction inhibitor. Adrenocortical carcinoma is a rare cancer, with poor prognosis. This research focuses on antineoplastic properties of ouabain and its association with everolimus. We analyzed the effects of drugs on cells by MTT assay, by [(3)H] thymidine assay, by Wright's staining, by homogeneous caspases assay, by flow cytometry analysis and by Western blot analysis on H295R and SW13 cells and on primary adrenocortical tumor cells. Ouabain induced cell viability reduction in SW13, H295R and 5 primary adrenocortical tumor cells. Combination of ouabain with everolimus produced a stronger cytotoxic effect on cell proliferation and viability. Marked morphological changes were observed in both SW13 and H295R cell lines after ouabain treatment, with an increase in necrosis. Cell cycle distribution was altered by ouabain in SW13. Analysis of apoptosis demonstrated an increase in caspase activity, clearly evident for SW13 at 72h. FACS analysis by Annexin V-FITC kit and propidium iodide confirmed an increased level of necrosis at higher concentrations. Western blot analysis showed that PI3k/Akt signaling pathway was modified after ouabain treatments in SW13. Ouabain exerts antiproliferative effects on SW13 and H295R cell lines and on primary adrenocortical tumor cells. These data suggest that ouabain or ouabain derivatives may be potential anticancer agents.

Positive or negative environmental modulations on human brain development: the morpho-functional outcomes of music training or stress.

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 development follows a course strictly dependent from environmental inputs, and that external intervention can be designed to positively counteract negative influences, particularly at young ages. We also focus on the social aspect of development, which starts in utero and continues after birth by building social relationships. This poses a great responsibility in handling children education and healthcare politics, pointing to social accountability for the responsible development of each child.

Estimation of cAMP binding in hippocampus CA1 field by a fluorescent probe.

The hippocampus is an allocortex structure involved in many complex processes, from memory formation to spatial navigation. It starts developing during prenatal life but acquires its adult functional properties around the peripubertal age, in both humans and mice. Such prolonged maturation is accompanied by structural changes in microcircuitry and functional changes involving biochemical and electrophysiological events. Moreover, hippocampus undergoes plasticity phenomena throughout life. In murine rodents, the most relevant maturation steps in Cornu Ammonis 1 (CA1) hippocampal subfield occur during the third-fourth weeks of life. During this period, also the expression and localization of cAMP-dependent protein kinases (PKA) refines: many regulatory (R1A) PKA clusters appear, bound to the cytoskeleton. Here the binding characteristics of R1A are determined in CA1 by using confocal microscopy. Apparently, two binding sites are present with no evidence of cooperativity. Equilibrium dissociation constant is estimated around 22.9 nM. This value is lower from that estimated for R1A in soluble form, suggesting a different binding site conformation or accessibility in the tissue. The method described here may be useful to track the developmental changes in binding activity, which affects cAMP availability at selected intracellular microzones. Possible relations with functional consequences are discussed.

Age-Related In Vivo Structural Changes in the Male Mouse Olfactory Bulb and Their Correlation with Olfactory-Driven Behavior.

Olfactory areas in mammalian brains are linked to centers that modulate behavior. During aging, sensitivity to odors decreases and structural changes are described in olfactory areas. We explored, in two groups of male mice (young and elderly, 6 and 19 months old, respectively), the link between the changes in olfactory bulb structure, detected with magnetic resonance imaging, and behavioral changes in a battery of tests on motor, olfactory, cognitive performance, and emotional reactivity. The behavioral pattern of elderly mice appears less anxious, being less scared by new situations. Additionally, the olfactory bulb of young and elderly mice differed in two variables derived from magnetic resonance imaging (fractional anisotropy and T2 maps). A random forest approach allowed to select the variables most predictive of the differences between young and elderly mice, and correlations were found between three behavioral variables indicative of anxious behavior and the two magnetic resonance variables mentioned above. These data suggest that in the living mouse, it is possible to describe co-occurring age-related behavioral and structural changes in the olfactory bulb. These data serve as a basis for studies on normal and pathological aging in the mouse, but also open new opportunities for in vivo human aging studies.

Behavioral guidance for improving dental care in autistic spectrum disorders.

Introduction: Autism spectrum disorders (ASDs) impair many aspects of everyday life and may prevent access to dental care, often limiting it to emergencies. Impaired oral health has long-lasting negative consequences on health status and on the acquisition of oral habits (e.g., oral respiration and grinding) or competencies (e.g., proper speech production). Children with ASD may be scared in the dental setting, which is rich in sensory stimuli and requires physical contact. Due to their behavioral manifestations, they represent a challenge for dentists and hygienists. We created a dedicated pathway with behavioral support for children with ASD to allow dental care and possibly limit the use of general anesthesia. Methods: We evaluated the effects of behavioral support in a quasi-experimental design by comparing two groups of children with ASD. The first group (n = 84) was visited every 2 months for 3 years and received additional support (visual aids, caregiver training, and longer visit duration). A control group, matched for age and sex, was visited at least twice a year or more, if needed, according to standard healthcare guidelines. Results: Compliance with the schedule was high throughout the 3 years. The degree of collaboration significantly improved after 1 year in the supported group, while the control group did not change. At the end of the study, collaboration remained significantly higher than at the beginning in the supported group. Half of dental treatments were possible without general anesthesia in supported children. No adverse effect was apparent on collaboration due to COVID-19 restrictions. Discussion: Behavioral techniques improved the compliance of ASD children to regular dentistry visits and treatment. Furthermore, oral hygiene at home was similarly improved, addressing oral health from a lifelong perspective.

Effects of cysteinyl-leukotriene receptors' antagonism by montelukast on lung mechanics and olfactory system histology in healthy mice.

CONTEXT: At variance with steroid administration, the possible effects of leukotrienes inhibition on basal respiratory mechanics and olfactory system function are still unclear. OBJECTIVE: To investigate if interference with the leukotrienes activity may influence basal lung mechanics in healthy mammals, as well as the olfactory system. MATERIALS AND METHODS: We measured lung mechanics by the end-inflation occlusion method in control and in montelukast i.p. treated anaesthetised healthy mice (10 mg/kg/die for a week). A study of olfactory system histology was also conducted. RESULTS: Elastance and resistive properties of the lung were not affected by montelukast, while a significant increment of lung hysteresis was observed. The analysis of olfactory system histology revealed no significant effects of montelukast compared to controls. DISCUSSION AND CONCLUSIONS: Leukotrienes' antagonism does not affect respiratory mechanics in basal conditions, except for a hysteresis increment, which might counteract the increase in expiratory flow in asthmatic subjects assuming montelukast.

Not Only COVID-19: Involvement of Multiple Chemosensory Systems in Human Diseases.

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. Indeed, many COVID-19 peculiar symptoms may be explained by the impairment of visceral chemosensory systems, for example, silent hypoxia, diarrhea, and the "cytokine storm". Dysregulation of chemosensory systems may underlie the much higher mortality rate of COVID-19 Acute Respiratory Distress Syndrome (ARDS) compared to ARDSs of different origins. In chronic non-infectious diseases like hypertension, diabetes, or cancer, the impairment of taste and/or olfaction has been consistently reported. This may signal diffuse chemosensory failure, possibly worsening the prognosis of these patients. Incapacitation of one or few chemosensory systems has negligible effects on survival under ordinary life conditions but, under stress, like metabolic imbalance or COVID-19 pneumonia, the impairment of multiple chemosensory systems may lead to dire consequences during the course of the disease.

Traditional and Medical Applications of Fasting.

Fasting has been practiced for millennia, for religious, ethical, or health reasons. It is also commonplace among different species, from humans, to animals, to lower eukaryotes. Research on fasting is gaining traction based on recent studies that show its role in many adaptive cellular responses such as the reduction of oxidative damage and inflammation, increase of energy metabolism, and in boosting cellular protection. In this expert review, we recount the historical evolution of fasting and we critically analyze its current medical applications, including benefits and caveats. Based on the available data, we conclude that the manipulation of dietary intake, in the form of calorie restriction, intermittent fasting, dietary restriction with the exclusion of some nutrients, prolonged fasting, and so forth, is anthropologically engraved in human culture possibly because of its positive health effects. Indeed, many studies show that fasting ameliorates many biochemical parameters related to cardiovascular and cancer risk, and neurodegeneration. Mechanistic studies are plentiful, but largely limited to cell cultures or laboratory animals. Understandably, there are no controlled trials of any form of fasting that gauge the effects on [any cause] mortality. Physicians should be aware that misinformation is pervasive and that their patients often adopt dietary regimens that are far from being clinically validated. Moreover, doctors are often unaware of their patients' religious or traditional fasting and of its potential health effects. Based on current evidence, no long-term fasting should be undertaken without medical supervision until future research will hopefully help shed further light on fasting and its effects on human health.

Olfactory Dysfunction in COVID-19 Patients Who Do Not Report Olfactory Symptoms: A Pilot Study with Some Suggestions for Dentists.

BACKGROUND: Smell and taste dysfunction are frequently reported by SARS-CoV-2 positive patients. The degree of olfactory and gustatory dysfunction varies from a very mild reduction to their complete loss. Several studies have been performed to determine their prevalence in COVID-19 patients, mostly using subjective measurement methods. The literature lacks long-term studies regarding duration and recovery. METHODS: We assessed olfactory performance, using the Sniffin' Sticks olfactory test, in a group of patients who had not reported olfactory dysfunction, around 131 days after their COVID-19 diagnosis. RESULTS: 11 out of 20 subjects showed no olfactory reduction (65%), while 9 subjects showed reduced TDI score (45%). A total of 13 subjects (65%) scored above the cutoff point for Threshold, 16 subjects (80%) scored above the cutoff point for discrimination and 13 subjects (65%) scored above the cutoff point for identification. CONCLUSION: Objective measurement methods of olfactory performance show a higher prevalence of olfactory reduction compared to patients' self-reported questionnaires. Olfactory dysfunction can last even months after its onset and because of its high prevalence, it could be a screening symptom for suspect COVID-19 cases.

Aggregates of cAMP-dependent kinase isoforms characterize different areas in the developing central nervous system of the chicken, Gallus gallus.

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 development. RI aggregates were detected first in the olfactory bulb, around embryonic day 14; within 3 days they appeared in the hyperpallium and nidopallium, where the most intense labeling was observed in the perihatching period. Fainter RI aggregates persisted up to 3 years in the olfactory bulb and nidopallium caudale. Less intense RI aggregates were present for a shorter time, from 2 weeks to 3 months, in the septal nuclei, thalamic medial nuclei, periventricular hypothalamus, optic tectum periventricular area, brainstem reticular formation and spinal cord substantia gelatinosa. RI aggregates were not detected in many brain areas including the arcopallium, striatum and cranial nerve nuclei. RII distribution showed less variation during development. From embryonic day 12, some insoluble RII aggregates were detected in the brain; however, only minor modifications were observed in positive structures once they started to harbor insoluble RII aggregates. The present results suggest that the distribution of PKA aggregates may assist in characterizing phylogenetically homologous structures of the vertebrate central nervous system and may also unravel biochemical differences among areas considered homologous.

8-Hydroxynaphthalene-1,4-dione derivative as novel compound for glioma treatment.

Malignant gliomas continue to demand the search for improved chemotherapeutic solutions. In this work the results of a preliminary in vitro screening performed on a small library of compounds are disclosed. As a result 2-(2,4-dihydroxyphenyl)-8-hydroxy-1,4-naphthoquinone emerged as a promising therapeutic lead.

Are Multiple Chemosensory Systems Accountable for COVID-19 Outcome?

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 cells are affected in some COVID-19 patients, the mucosal innate immune response would be dysregulated, opening one up to massive infection, thus explaining why COVID-19 has lethal consequences in some patients. Similar to taste and olfaction, oxygen chemosensory function can be easily tested with a non-invasive procedure in humans, while functional tests for solitary chemosensory or pulmonary neuroendocrine cells are not available, and autoptic investigation is required to ascertain their involvement.

Cell Differentiation and Replication during Postnatal Development of the Murine First Molar.

Various signaling molecular pathways are involved in odontogenesis to promote cellular replication and differentiation. Tooth formation is controlled mainly by epithelial-mesenchymal interactions. The aim of this work was to investigate how cellular replication and differentiation ensue during the formation of the murine first molar in postnatal ages until eruption, focusing on morphogenesis, odontoblast differentiation and cellular replication. Wild-type CD1 mice were examined from birth to weaning. Morphogenesis and interaction between developing epithelial and mesenchymal tissues were evaluated in hematoxylin-eosin and Gomori trichome stained sections. Immunohistochemistry for nestin, which mediates the differentiation of odontoblasts, especially their polarization and elongation, showed that this intermediate filament was apparent already at postnatal day P1 in the apical region of odontoblasts and progressed apically from cusp tips, while it was not present in epithelial tissues. The expression of nuclear antigen Ki-67 highlighted dividing cells in both epithelial and mesenchymal tissues at P1, while one week later they were restricted to the cementoenamel junction, guiding root elongation. The link between odontoblast maturation and cellular replication in the different tooth tissues is essential to understand the development of tooth shape and dimension, to outline mechanisms of tooth morphogenesis and possibly eruption.

Differential effects of sodium chloride and monosodium glutamate on kidney of adult and aging mice.

Monosodium Glutamate (MSG) is used as flavour enhancer, with potential beneficial effects due to its nutritional value. Given the decline in kidney functions during aging, we investigated the impact of MSG voluntary intake on the kidney of male mice, aged 6 or 18 months. For 2 months, they freely consumed water (control group), sodium chloride (0.3% NaCl) or MSG (1% MSG) in addition to standard diet. Young animals consuming sodium chloride presented signs of proteinuria, hyperfiltration, enhanced expression and excretion of Aquaporin 2 and initial degenerative reactions suggestive of fibrosis, while MSG-consuming mice were similar to controls. In old mice, aging-related effects including proteinuria and increased renal corpuscle volume were observed in all groups. At an advanced age, MSG caused no adverse effects on the kidney compared to controls, despite the presence of a sodium moiety, similar to sodium chloride. These data show that prolonged MSG intake in mice has less impact on kidney compared to sodium chloride, that already in young animals induced some effects on kidney, possibly related to hypertension.