A follow-up on quantitative and qualitative olfactory dysfunction and other symptoms in patients recovering from COVID-19 smell loss.

BACKGROUND: Sudden smell loss is a specific early symptom of COVID-19, which, prior to the emergence of Omicron, had estimated prevalence of ~40% to 75%. Chemosensory impairments affect physical and mental health, and dietary behavior. Thus, it is critical to understand the rate and time course of smell recovery. The aim of this cohort study was to characterize smell function and recovery up to 11 months post COVID-19 infection. METHODS: This longitudinal survey of individuals suffering COVID-19-related smell loss assessed disease symptoms and gustatory and olfactory function. Participants (n=12,313) who completed an initial survey (S1) about respiratory symptoms, chemosensory function and COVID-19 diagnosis between April and September 2020, were invited to complete a follow-up survey (S2). Between September 2020 and February 2021, 27.5% participants responded (n=3,386), with 1,468 being diagnosed with COVID-19 and suffering co-occurring smell and taste loss at the beginning of their illness. RESULTS: At follow-up (median time since COVID-19 onset ~200 days), ~60% of women and ~48% of men reported less than 80% of their pre-illness smell ability. Taste typically recovered faster than smell, and taste loss rarely persisted if smell recovered. Prevalence of parosmia and phantosmia was ~10% of participants in S1 and increased substantially in S2: ~47% for parosmia and ~25% for phantosmia. Persistent smell impairment was associated with more symptoms overall, suggesting it may be a key marker of long-COVID illness. The ability to smell during COVID-19 was rated slightly lower by those who did not eventually recover their pre-illness ability to smell at S2. CONCLUSIONS: While smell ability improves for many individuals who lost it during acute COVID-19, the prevalence of parosmia and phantosmia increases substantially over time. Olfactory dysfunction is associated with broader persistent symptoms of COVID-19, and may last for many months following acute COVID-19. Taste loss in the absence of smell loss is rare. Persistent qualitative smell symptoms are emerging as common long-term sequelae; more research into treatment options is strongly warranted given that even conservative estimates suggest millions of individuals may experience parosmia following COVID-19. Healthcare providers worldwide need to be prepared to treat post COVID-19 secondary effects on physical and mental health.

Oral health status and management of autistic patients in the dental setting.

AIM: Dental treatment of patients with Autism Spectrum Disorders (ASD) is challenging due to difficulties in social interactions, hypersensitivity to sensory stimuli, various degrees of cognitive and developmental issues, poor collaboration, limited capacity to manage emotions, stereotyped movements, hyperactivity, limited adaptation to new situations and environments. Data on the type of treatment required are scarce. This study investigates the oral health status of ASD patients, the possibilities of chair treatment and the need for dental treatments under general anaesthesia (GA). METHODS: One hundred sixty-nine medical records, from 2005 to 2018, of patients with ASD were examined. Patients undergoing chair or general anaesthesia were assigned to two groups according age (younger or older than 14 years old). The level of cooperation was classified into four categories: none, poor, fair, good. CONCLUSION: Primary prevention, setting up a routine oral hygiene at home and regular dental examinations, internalised like a habit by patients, are the main instruments to maintain oral health in ASD patients. When non-cooperative patients need urgent treatment, safe and high-level dental therapies should be performed under GA.

Urinary volatile molecules vary in males of the 2 European subspecies of the house mouse and their hybrids.

Mice recognize other mice by identifying chemicals that confer a molecular signature to urinary marks. Such molecules may be involved in species recognition, and previous behavioral studies have related divergence of sexual preference between 2 subspecies of the house mouse (Mus musculus musculus and Mus musculus domesticus) to urinary odors. To characterize the differences between odors of males of the 2 subspecies and their first-generation offspring, the urinary volatile molecules were examined via gas chromatography coupled to mass spectrometry. Seven molecules were present in the samples from mice of at least one group. Their quantity varied among groups: M. m. domesticus showed a quantitatively richer panel of odorants in their urine when compared with M. m. musculus. The hybrids showed a more complex picture that was not directly related to one or the other parental subspecies. These quantitative differences may contribute to the specificity of the odorant bouquet of the 2 subspecies.

The olfactory system is affected by steroid aerosol treatment in mice.

Asthma needs continuous treatment often for years. In humans, some drugs are administered via aerosol, therefore they come in contact with both respiratory and olfactory mucosa. We explored the possibility that antiasthma corticosteroid treatment could influence the olfactory function by passage through the nose. A group of mice was exposed twice daily for 42 days to fluticasone propionate aerosol and was compared with a control group. Olfactory behavior, respiratory mechanics, histology, and immunoreactivity in the olfactory system were assessed. Fluticasone-treated mice were slower in retrieving a piece of hidden food, but both groups were similarly fast when the food was visible. When a clearly detectable odor was present in the environment, all mice behaved in a similar way. Respiratory mechanics indices were similar in all mice except for the viscose resistance, which was reduced in fluticasone-treated mice. Olfactory mucosa of fluticasone-treated mice was thicker than that of controls. Slight but consistent differences in staining were present for Olfactory Marker Protein but not for other proteins. A mild impairment of olfactory function is present in mice chronically treated with fluticasone aerosol, apparently accompanied by slight modifications of the olfactory receptor cells, and suggests monitoring of olfactory function modifications in long-term steroid users.

Mice recognize recent urine scent marks by the molecular composition.

Male mice mark the territory with urine scent marks that are frequently renewed to maintain the territory ownership. We measured the response of male mice to small spots of urine deposed either 0, 5, 11, 22, 45, 90 min, or 24 h before testing and show that mice loose interest in sniffing scent marks as they become older and older. We asked what scent features tell a mouse how recent a scent mark is, and therefore, we studied the molecule-to-behavior relationship by correlating 6 behavioral variables--the number of sniffing acts, the latency to the first sniff, the number of urine marks, the latency to the first mark, the area of the marks, and the number of fecal pellets-to 2,4-dehydro-exo-brevicomin, linalool, 2-sec-butyl-4,5-dihydrothiazole, 2,4-dimethylphenol, 4-ethylphenol, and 6,10-dimethyl-5,9-undecadien-2-one released from urine spots over the time, identified, and quantified by gas chromatography and mass spectrometry. Canonical correlation between the molecular and the behavioral principal components was strong (R(1) = 0.96, P = 0.026). The principal component based on 2,4-dehydro-exo-brevicomin, linalool, and 2-sec-butyl-4,5-dihydrothiazole correlated negatively with countermarking and positively with the sniffing behavior, suggesting a semantic feature of fresh male mouse urine.

Pheromone reception in mammals.

Pheromonal communication is the most convenient way to transfer information regarding gender and social status in animals of the same species with the holistic goal of sustaining reproduction. This type of information exchange is based on pheromones, molecules often chemically unrelated, that are contained in body fluids like urine, sweat, specialized exocrine glands, and mucous secretions of genitals. So profound is the relevance of pheromones over the evolutionary process that a specific peripheral organ devoted to their recognition, namely the vomeronasal organ of Jacobson, and a related central pathway arose in most vertebrate species. Although the vomeronasal system is well developed in reptiles and amphibians, most mammals strongly rely on pheromonal communication. Humans use pheromones too; evidence on the existence of a specialized organ for their detection, however, is very elusive indeed. In the present review, we will focus our attention on the behavioral, physiological, and molecular aspects of pheromone detection in mammals. We will discuss the responses to pheromonal stimulation in different animal species, emphasizing the complicacy of this type of communication. In the light of the most recent results, we will also discuss the complex organization of the transduction molecules that underlie pheromone detection and signal transmission from vomeronasal neurons to the higher centers of the brain. Communication is a primary feature of living organisms, allowing the coordination of different behavioral paradigms among individuals. Communication has evolved through a variety of different strategies, and each species refined its own preferred communication medium. From a phylogenetic point of view, the most widespread and ancient way of communication is through chemical signals named pheromones: it occurs in all taxa, from prokaryotes to eukaryotes. The release of specific pheromones into the environment is a sensitive and definite way to send messages to other members of the same species. Therefore, the action of an organism can alter the behavior of another organism, thereby increasing the fitness of either or both. Albeit slow in transmission and not easily modulated, pheromones can travel around objects in the dark and over long distances. In addition, they are emitted when necessary and their biosynthesis is usually economic. In essence, they represent the most efficient tool to refine the pattern of social behaviors and reproductive strategies.

Molecular aspects of pheromonal communication via the vomeronasal organ of mammals.

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.

Major urinary proteins, alpha(2U)-globulins and aphrodisin.

The major urinary proteins (MUPs) are proteins secreted by the liver and filtered by the kidneys into the urine of adult male mice and rats, the MUPs of rats being also referred to as alpha(2U)-globulins. The MUP family also comprises closely related proteins excreted by exocrine glands of rodents, independently of their sex. The MUP family is an expression of a multi-gene family. There is complex hormonal and tissue-specific regulation of MUP gene expression. The multi-gene family and its outflow are characterized by a polymorphism which extends over species, strains, sexes, and individuals. There is evidence of evolutionary conservation of the genes and their outflow within the species and evidence of change between species. MUPs share the eight-stranded beta-barrel structure lining a hydrophobic pocket, common to lipocalins. There is also a high degree of structural conservation between mouse and rat MUPs. MUPs bind small natural odorant molecules in the hydrophobic pocket with medium affinity in the 10(4)-10(5) M(-1) range, and are excreted in the field, with bound odorants. The odorants are then released slowly in air giving a long lasting olfactory trace to the spot. MUPs seem to play complex roles in chemosensory signalling among rodents, functioning as odorant carriers as well as proteins that prime endocrine reactions in female conspecifics. Aphrodisin is a lipocalin, found in hamster vaginal discharge, which stimulates male copulatory behaviour. Aphrodisin does not seem to bind odorants and no polymorphism has been shown. Both MUPs and aphrodisin stimulate the vomeronasal organ of conspecifics.

Binding of two fluorescent cAMP analogues to type I and II regulatory subunits of cAMP-dependent protein kinases.

Binding of two long wavelength fluorescent cAMP analogues, 8-thioacetamido-fluorescein-cAMP (SAF-cAMP) and 8-thioacetamido-rhodamine-cAMP (SAR-cAMP), to the RI (from bovine muscle) and RII (from bovine heart) regulatory subunits of cAMP dependent kinases has been studied. Displacement of [3H]cAMP from RI and RII and equilibrium dialysis measurements show that the fluorescent nucleotides are high affinity ligands for the cAMP binding sites. The binding is characterized by complex fluorescence spectral and fluorescence anisotropy changes, more evident for the fluorescein than for the rhodamine derivative. The fluorescence excitation spectrum of the bound SAF-cAMP is characterized by the appearance of a red shifted shoulder at 500-510 nm excitation wavelength region. Any change of the bound/free ratio in a solution equilibrium is accompanied by changes in fluorescence and anisotropy signals which are best detected at suitable wavelengths. It is proposed that fluorescence and anisotropy changes can distinguish between binding to type B (slow dissociating) and A (fast dissociating) cAMP binding sites of regulatory subunits. Applications of the fluorescent nucleotides to kinase localization and cAMP determination in living cells are discussed.

Aggregates of cAMP-dependent kinase RIalpha characterize a type of cholinergic neurons in the rat brain.

Acetylcholine is synthesized by different types of neurons, showing a distinct biochemical phenotype. Aggregates of RIalpha regulatory subunit of cAMP-dependent protein kinases are visualized by immunohistochemistry only in some cholinergic neurons, since they tightly colocalize with two different markers, choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT). These neurons are present mainly in brain areas related to the limbic system. None of the other regulatory subunits of cAMP dependent kinases colocalize with cholinergic markers.

Neuropeptide expression in the mouse vomeronasal organ during postnatal development.

The expression of selected regulatory neuropeptides was investigated by immunohistochemistry in nerves supplying the vomeronasal organ (VNO) of mice during postnatal development. Results show that neurons in the VNO are devoid of immunolabeling with any of the antibody used from 1 day to 2 months of age. In the non-receptor epithelium (NRE) and the vomeronasal vascular pump (VP) the timing of expression of regulatory neuropeptides differed among neuropeptides and the different VNO structures. Regulatory neuropeptides usually found in sensory nerves (substance P, calcitonin gene-related peptide) and efferent nerves (neuropeptide Y, atrial natriuretic peptide) are expressed in the NRE and the VP, respectively. These results support the view that the VNO is to some extent functional during postnatal development.

cAMP-dependent protein kinase type RI is found in clusters in the rat detergent-insoluble neuronal fraction.

Different types of cAMP dependent regulatory subunits have been characterized in the mammalian brain: RI alpha and beta, RII alpha and beta. The subcellular distribution of RI subunits has been examined in the rat brain. Partial amino acid sequencing of tryptic fragments from the Triton insoluble pellet of the rat brain shows that cAMP dependent regulatory subunits type RI alpha are found in this fraction. Immunohistochemistry shows that Triton-insoluble RI subunits are concentrated to form clusters and this distribution is distinct from RII subunits. Immunohistochemistry and fluorescent cAMP labeling show that the clusters bind fluorescent cAMP analogues. These results suggest that the high local concentration of RI subunits can modulate cAMP distribution among different cellular compartments.

Immunohistochemical investigation of the vomeronasal organ. Nitric oxide synthase expression in the mouse during postnatal development.

The expression of nitric oxide synthase type I (NOS-I), the key enzyme for the synthesis of the gaseous neurotransmitter nitric oxide, was investigated by means of immunohistochemistry in the vomeronasal organ (VNO) of mice from postnatal day 1 for 2 months. The results show that NOS is expressed in extrinsic nerve supplying the developing erectile tissue of VNO (the so-called VNO pump) as well as blood vessels in the connective tissue laying under the receptor epithelium at postnatal day 1. At 8, 15 and 21 postnatal days, and at 2 months the density of NOS-1-immunoreactive nerves goes along with the development of the erectile tissue. From postnatal day 8 onwards, NOS-1-immunoreactive fibers are found also in the vicinity of the VNO glands. These data suggest that nitric oxide (NO) modulates VNO activity early after birth in the mouse.

Changes in female cognitive performance after energetic drink consumption: a preliminary study.

1. A group of males and a group of females consumed either no drink, a placebo or a caffeine-containing energy drink. Subjects were requested to perform a simple reaction times task and a go-no-go reaction times task. 2. Males responded significantly faster than females. The effect of drink was apparent only for females performing the go-no-go task, while it was undetectable in female simple reaction times and in males. 3. A mild effect due to the drink, but not to the placebo, appears only when the task requires a certain degree of cognitive processing and is evident only in subjects possibly adopting a cognitive strategy that allows an improvement of performance.

Visualization of detergent insoluble cyclic AMP-dependent protein kinase RIalpha aggregates in the rat brain.

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.

Pheromonally accelerated puberty is enhanced by previous experience of the same stimulus.

The influence of rearing conditions on pheromone-induced puberty acceleration was tested on Swiss mice. Litters were reared in one of three conditions: with either both parents, or with two females, or finally with two females in the presence of urinary pheromonal cues from adult males, which are known to induce puberty acceleration. Nine days after weaning the females were exposed to either prepubertal or adult male urine. In the groups reared with either both parents or with two females and the pheromonal cues from stranger males, females treated with adult male urine presented heavier uteri and more cornified vaginal smears than females reared in the same conditions but subsequently treated with prepubertal males urine. In the group reared simply with two females, the differences in both uterus weight and vaginal smears did not reach statistical significance. The data support the notion that early experience of pheromonal cues may influence the response to pheromones in a later period, even if the preweaning exposure to males had no direct influence on early signs of puberty onset.

N-butanol olfactory threshold and nasal patency before and after palatal expansion in children. A preliminary study.

OBJECTIVES: Olfaction is based on the function of the nasal olfactory receptors. Children can well detect and respond to odors in order to have information about food and environment. Rapid maxillary expansion seems to improve dental class and increase nasal patency correcting oral respiration in children. Nevertheless, there are no studies demonstrating that expansion in pediatric patients could influence olfactory sensitivity. The aim of this study was to evaluate olfactory threshold and nasal patency in children aged from 6 to 12 years before and after rapid maxillary expansion. METHOD: N-butanol olfactory thresholds, anterior active rhinomanometry, and peak nasal inspiratory flow were measured in 12 children (6-12 years) before (T0), 20 days (T1), and 6 months after rapid maxillary expansion application (T2). RESULTS: A significant lower olfactory threshold was found comparing T2 and T0 N-butanol olfactory threshold values (p=0.038). Peak nasal inspiratory flow showed a significant improvement both at T1 and T2, with respect to T0 values (p=0.043 and p=0.0001, respectively). T2 nasal resistances showed a trend towards a significant reduction when compared with T1 values (p=0.15). CONCLUSION: This pilot study suggested that rapid maxillary expansion may lead to improved N-butanol olfactory thresholds, at least 6 months after palatal expansion. Furthermore, rapid maxillary expansion seems to improve peak nasal inspiratory flow values, and finally although with lower sensitivity, reduce nasal resistances as measured by rhinomanometry.

Localization of Triton-insoluble cAMP-dependent kinase type RIbeta in rat and mouse brain.

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 (RIalpha and beta, RIIalpha and beta). These four isoforms show a high level of homology and slightly different biochemical properties. In addition to biochemical properties, a different anatomical distribution of the regulatory isoforms may contribute to determine the specificity of diverse cAMP effects. By immunohistochemistry, the distribution of the detergent-insoluble fraction of RIbeta isoform has been examined in rat and mouse brain. Biochemical fractionation shows that a large fraction of both RIalpha and RIbeta isoforms is bound to the cytoskeleton. RIbeta labelling can be observed only in few locations: Purkinje cells, olfactory mitral cells, lateral thalamic neurons, superior olivary complex neurons. These cell populations are involved in the so called Purkinje cell degeneration. On the other hand, RIalpha aggregates have a more widespread distribution, in brain areas involved in visceroemotional control. At the subcellular level, these two subunits show a different pattern of labelling: in most cells a sharply defined clustered labelling is observed for RIalpha isoforms, while the RIbeta isoform presents a weaker, diffuse intracytoplasmic distribution. Competition experiments point to the presence of, as yet unidentified, different and selective anchoring proteins for the two similar RIalpha and beta isoforms. It is suggested that, as is the case for structural proteins, a different supramolecular organization of similar regulatory proteins may be crucial in order to fulfill different functions.

Protein-bound male urinary pheromones: differential responses according to age and gender.

The attractive properties of male urinary pheromones were tested on adult or prepubertal male and female mice. An androgen-dependent protein is present in adult male urine (major urinary protein, MUP) which has been suggested to be a pheromone-binding protein. We tested the pheromonal properties of the protein-bound volatiles in a test of attractiveness. These molecules, that co-purify with MUP, attract females and repel adult males. In prepubertal animals, females are repelled and males are attracted by the same stimuli. These results are similar to those obtained by others with adult male whole urine. Therefore MUP binds molecules with a pheromonal activity, and these molecules are sufficient to act as male signals.

Acceleration of puberty onset in female mice by male urinary proteins.

1. Puberty onset in female mice is accelerated by exposure to conspecific adult male urine, which acts through the vomeronasal organ and the accessory olfactory system. A distinctive component of adult male mouse urine is the major urinary protein complex (MUP), which is a lipocalin; it has a hydrophobic pocket that binds small endogenous volatile molecules. The MUP gene family also codes for a hexapeptide, which has four residues in common with the N-terminal region of MUP. 2. MUP, the volatiles bound to MUP and the MUP-related hexapeptide have been tested for the induction of puberty acceleration by measuring the increase in uterus weight related to the first pro-oestrus phase. MUP, together with its bound volatiles, induces puberty acceleration. Its activity is retained even when the volatiles have been removed either by organic extraction or competition displacement with a high-affinity ligand. 3. MUP-related hexapeptide also induces puberty acceleration in female mice. In contrast, the odorants bound to MUP do not exert this effect. 4. It is proposed that the vomeronasal organ contains receptors that recognize the short N-terminal consensus sequence, N-Glu-Glu-Ala-X-Ser (where X is a polar residue), common to both MUP and the hexapeptide.