Impact of Rehydration Following Systemic Dehydration on Vocal Fold Gene Expression.

OBJECTIVE: Biological data on the beneficial effects of vocal fold rehydration are lacking. This study aimed to examine the effects of acute systemic dehydration on vocal fold gene expression and determine whether rehydration would reverse these changes. METHODS: Male New Zealand White rabbits (N = 24, n = 8/group) provided the animal model. Systemic dehydration was induced by 5 days of water volume restriction. Rehydration was provided by ad-lib water for 3 days following dehydration. Euhydrated rabbits were used as the control group. Vocal fold tissue was dissected. Seventeen genes were selected based on physiological function and role in supporting vocal fold structure, oxidative stress, hemodynamics, and extracellular matrix turnover. Relative gene expression was assessed by RT-qPCR. RESULTS: Rehydration following systemic dehydration can modulate gene expression, with expression patterns suggesting that rehydration reverses dehydration-induced changes in over half of the tested genes. CLIC5 (chloride intracellular channel 5) and EFEMP1 (EGF containing fibulin extracellular matrix protein 1) genes were significantly upregulated in the dehydration group compared with the euhydrated control. A1BG (alpha-1B-glycoprotein) and IL1RAP (interleukin 1 receptor accessory protein) were downregulated by rehydration compared with the dehydration group. CONCLUSION: This study provides molecular evidence for a transcriptional response to rehydration following acute systemic dehydration in the vocal folds. These data are the first to study gene expression following realistic dehydration and rehydration paradigms and provide biological data to support clinical recommendations to increase water intake after acute dehydration. LEVEL OF EVIDENCE: NA Laryngoscope, 133:3499-3505, 2023.

Comparative proteomic changes in rabbit vocal folds undergoing systemic dehydration and systemic rehydration.

BACKGROUND: A considerable body of clinical evidence suggests that systemic dehydration can negatively affect voice production, leading to the common recommendation to rehydrate. Evidence for the corrective benefits of rehydration, however, is limited with mixed conclusions, and biological data on the underlying tissue changes with rehydration is lacking. In this study, we used a rabbit model (n = 24) of acute (5 days) water restriction-induced systemic dehydration with subsequent rehydration (3 days) to explore the protein-level changes underlying the molecular transition from euhydration to dehydration and following rehydration using LC-MS/MS protein quantification in the vocal folds. We show that 5-day water restriction led to an average 4.3% decrease in body weight with relative increases in anion gap, Cl-, creatinine, Na+, and relative decreases in BUN, iCa2+, K+, and tCO2 compared to control (euhydrated) animals. A total of 309 differentially regulated (p < 0.05) proteins were identified between the Control and Dehydration groups. We observed a noteworthy similarity between the Dehydration and Rehydration groups, both well differentiated from the Control group, highlighting the distinct timelines of resolution of the clinical symptoms of systemic dehydration and the underlying molecular changes. SIGNIFICANCE: Voice disorders are a ubiquitous problem with considerable economic and psychological impact. Maintenance of proper hydration is commonly prescribed as a general vocal hygiene practice. There is evidence that dehydration negatively impacts phonation, but our understanding of the state of vocal folds in the context of systemic dehydration are limited, particular from a molecular perspective. Further, ours is a novel molecular study of the short-term impact of rehydration on the tissue. Given the relatively minimal difference in vocal fold proteomic profiles between the Dehydration and Rehydration groups, our data demonstrate a complex physiological response to acute systemic dehydration, and highlight the importance of considering persistent underlying molecular pathology despite the rapid resolution of clinical measures. This study sets a foundation for future research to confirm the nature of potential beneficial outcomes of clinical recommendations related to hydration.

Furosemide-induced systemic dehydration alters the proteome of rabbit vocal folds.

Whole-body dehydration (i.e., systemic dehydration) leads to vocal fold tissue dehydration. Furosemide, a common diuretic prescribed to treat hypertension and edema-associated conditions, induces systemic dehydration. Furosemide also causes voice changes in human speakers, making this method of systemic dehydration particularly interesting for vocal fold dehydration studies. Our objective was to obtain a comprehensive proteome of vocal folds following furosemide-induced systemic dehydration. New Zealand White rabbits were used as the animal model and randomly assigned to euhydrated (control) or furosemide-dehydrated groups. Systemic dehydration, induced by injectable furosemide, was verified by an average body weight loss of -5.5% and significant percentage changes in blood analytes in the dehydrated rabbits compared to controls. Vocal fold specimens, including mucosa and muscle, were processed for proteomic analysis using label-free quantitation LC-MS/MS. Over 1600 proteins were successfully identified across all vocal fold samples; and associated with a variety of cellular components and ubiquitous cell functions. Protein levels were compared between groups showing 32 proteins differentially regulated (p ≤ 0.05) in the dehydrated vocal folds. These are mainly involved with mitochondrial translation and metabolism. The downregulation of proteins involved in mitochondrial metabolism in the vocal folds suggests a mechanism to prevent oxidative stress associated with systemic dehydration. SIGNIFICANCE: Voice disorders affect different population demographics worldwide with one in 13 adults in the United States reporting voice problems annually. Vocal fold systemic hydration is clinically recognized for preventing and treating voice problems and depends on optimal body hydration primarily achieved by water intake. Herein, we use the rabbit as a translatable animal model, and furosemide as a translatable method of systemic dehydration, to reveal a comprehensive proteomic profile of vocal fold mucosa and muscle in response to systemic dehydration. The significant subset of proteins differentially regulated due to furosemide-induced dehydration offer novel insights into the molecular mechanisms of systemic dehydration in the vocal folds. These findings also deepen our understanding of changes to tissue biology after diuretic administration.

Recurring exposure to low humidity induces transcriptional and protein level changes in the vocal folds of rabbits.

Voice disorders are an important human health condition. Hydration is a commonly recommended preventive measure for voice disorders though it is unclear how vocal fold dehydration is harmful at the cellular level. Airway surface dehydration can result from exposure to low humidity air. Here we have induced airway surface dehydration in New Zealand White rabbits exposed to a recurring 8-h low humidity environment over 15 days. This model mimics an occupational exposure to a low humidity environment. Exposure to moderate humidity was the control condition. Full thickness soft-tissue samples, including the vocal folds and surrounding laryngeal tissue, were collected for molecular analysis. RT-qPCR demonstrated a significant upregulation of MUC4 (mucin 4) and SCL26A9 (chloride channel) and a large fold-change though statistically non-significant upregulation of SCNNA1 (epithelial sodium channel). Proteomic analysis demonstrated differential regulation of proteins clustering into prospective functional groups of muscle structure and function, oxidative stress response, and protein chaperonin stress response. Together, the data demonstrate that recurring exposure to low humidity is sufficient to induce both transcriptional and translational level changes in laryngeal tissue and suggest that low humidity exposure induces cellular stress at the level of the vocal folds.

Restricted Water Intake Adversely Affects Rat Vocal Fold Biology.

OBJECTIVES: A holistic understanding of the many ways that systemic dehydration affects vocal fold biology is still evolving. There are also myriad physiologically relevant methodologies to induce systemic dehydration. To untangle the effects of systemic dehydration on vocal fold biology, we need to utilize realistic, clinically translatable paradigms of systemic dehydration in lab animals. Restricted access to water accommodates clinical translation. We investigated whether systemic dehydration via reduced water intake would negatively affect vocal fold biology. STUDY DESIGN: Prospective, in vivo study design. METHODS: Male Sprague Dawley rats (N = 13) were provided 4 mL/100 g of water/day for 5 days, whereas male control rats (N = 8) were given ad lib access to water. Following euthanasia, tissues were processed for histological staining, gene expression, and protein assays. RESULTS: Renin gene expression level in kidneys increased significantly (P ≤ .05), validating dehydration. Dehydration induced by restricted water access downregulated the gene expression of interleukin-1α and desmoglein-1 (P ≤ .05). Hyaluronidase-2 gene expression increased after dehydration (P ≤ .05). The protein level of desmoglein-1 decreased after dehydration (P ≤ .05). Histological analyses suggested decreased hyaluronan (P ≤ .05) in the water-restricted rat vocal fold. CONCLUSION: Reduced daily water intake for just 5 days impairs vocal fold biology by disrupting inflammatory cytokine release, reducing plasma membrane integrity, and disrupting the hyaluronan network. This is the first study investigating the dehydrating effects of restricted water intake on vocal fold tissue in an in vivo model. LEVEL OF EVIDENCE: NA (prospective animal study). Laryngoscope, 131:839-845, 2021.

RNA sequencing identifies transcriptional changes in the rabbit larynx in response to low humidity challenge.

BACKGROUND: Voice disorders are a worldwide problem impacting human health, particularly for occupational voice users. Avoidance of surface dehydration is commonly prescribed as a protective factor against the development of dysphonia. The available literature inconclusively supports this practice and a biological mechanism for how surface dehydration of the laryngeal tissue affects voice has not been described. In this study, we used an in vivo male New Zealand white rabbit model to elucidate biological changes based on gene expression within the vocal folds from surface dehydration. Surface dehydration was induced by exposure to low humidity air (18.6% + 4.3%) for 8 h. Exposure to moderate humidity (43.0% + 4.3%) served as the control condition. Ilumina-based RNA sequencing was performed and used for transcriptome analysis with validation by RT-qPCR. RESULTS: There were 103 statistically significant differentially expressed genes identified through Cuffdiff with 61 genes meeting significance by both false discovery rate and fold change. Functional annotation enrichment and predicted protein interaction mapping showed enrichment of various loci, including cellular stress and inflammatory response, ciliary function, and keratinocyte development. Eight genes were selected for RT-qPCR validation. Matrix metalloproteinase 12 (MMP12) and macrophage cationic peptide 1 (MCP1) were significantly upregulated and an epithelial chloride channel protein (ECCP) was significantly downregulated after surface dehydration by RNA-Seq and RT-qPCR. Suprabasin (SPBN) and zinc activated cationic channel (ZACN) were marginally, but non-significantly down- and upregulated as evidenced by RT-qPCR, respectively. CONCLUSIONS: The data together support the notion that surface dehydration induces physiological changes in the vocal folds and justifies targeted analysis to further explore the underlying biology of compensatory fluid/ion flux and inflammatory mediators in response to airway surface dehydration.

Natural Infection by SARS-CoV-2 in Companion Animals: A Review of Case Reports and Current Evidence of Their Role in the Epidemiology of COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the coronavirus disease 2019 (COVID-19), is the causative infectious agent of the current pandemic. As researchers and health professionals are still learning the capabilities of this virus, public health concerns arise regarding the zoonotic potential of SARS-CoV-2. With millions of people detected with SARS-CoV-2 worldwide, reports of companion animals possibly infected with the virus started to emerge. Therefore, our aim is to review reported cases of animals naturally infected with SARS-CoV-2, particularly companion pets, shedding light on the role of these animals in the epidemiology of COVID-19.

Development of a quantitative PCR for the detection of Rangelia vitalii.

The aim of this study was to develop and validate a SYBR Green qPCR assay to detect and quantify a fragment of the 18S rRNA gene of Rangelia vitalii in canine blood. Repeatability of the qPCR was determined by the intra- and inter-assay variations. The qPCR showed efficiency of E=101.30 (r(2)=0.996), detecting as few as one copy of plasmid containing the target DNA. Specificity of the assay was performed using DNA samples of Babesia canis, B. gibsoni, Ehrlichia canis, E. ewingii and Leishmania sp. No cross-reactivity was observed. Field samples consisting of blood from 265 dogs from Porto Alegre, Brazil were also tested. A total of 24 (9.05%) samples were positive for R. vitalii. Amplicons of 50% of positive samples were confirmed to be R. vitalii by Sanger sequencing. The positive samples had an average of 3.5×10(5) organisms/mL of blood (range: 1.27×10(3)-1.88×10(6)) based on the plasmid-generated standard curve. In conclusion, the SYBR Green qPCR assay developed herein is sensitive and specific and can be used as a diagnostic tool for detection and quantification of R. vitalii in canine blood samples.

Occurrence and identification of hemotropic mycoplasmas (Hemoplasmas) in free ranging and laboratory rats (Rattus norvegicus) from two Brazilian zoos.

BACKGROUND: Hemotropic mycoplasmas (hemoplasmas), bacteria belonging to the class Mollicutes, are obligatory red blood cell pathogens of a variety of animal species. They may cause acute anemia that is life-threatening or chronic disease that is clinically silent, but may interfere with results of experimental studies when using infected animals. Since these bacteria cannot be cultivated, molecular techniques are the gold standard for diagnosing an infection, investigating its prevalence, and describing new species. Mycoplasma coccoides and M. haemomuris are the most commonly recognized hemoplasmas in the blood of wild and laboratory rodents. Neither the epidemiology nor clinical and molecular characterization of hemoplasma infection in free-ranging rodents in Brazil has been previously reported. The aims of this study were to investigate the occurrence of hemoplasmas in free-ranging rats (Rattus norvegicus) captured in the Passeio Público and Curitiba Zoo and compare hematologic parameters of infected and non-infected animals. RESULTS: Anti-coagulated blood samples collected from 43 free-ranging and 20 nursery rats were included in the study. Overall 63.5% were positive using SYBR® Green quantitative PCR (qPCR) of the 16S rRNA gene to screen for hemoplasma infection (72% among free-ranging rats; 45% among laboratory-raised rats). Sequencing of the qPCR products showed that all but one sample had >98% identity to M. haemomuris. Phylogenetic analysis based on a fragment of approximately 1300 bp of the 16S rRNA gene showed 99% identity to a new hemoplasma from European rats and 98% identity to a hemotropic mycoplasma described infecting a European harvest mouse (Micromys minutus). No statistically significant changes in hematologic parameters between infected and non-infected rats were found, confirming the low pathogenicity and/or silent characteristics of the infection. CONCLUSIONS: Our findings suggest that hemoplasmas are likely endemic in rodent species in this region. The epidemiology, especially as it relates to the mode of transmission, needs to be further investigated as well as the possibility that other animal species, including humans, might become infected.

Draft Genome Sequence of Mycobacterium bovis Strain SP38, a Pathogenic Bacterium Isolated from a Bovine in Brazil.

We report a draft genome sequence of Mycobacterium bovis strain SP38, isolated from the lungs of a cow in Brazil. The assembly of reads resulted in 36 contigs in a total of approximately 4.37 Mb. Comparison of M. bovis strains sequenced to date will aid in understanding bovine tuberculosis in Brazil.

Accumulation of brachycerine, an antioxidant glucosidic indole alkaloid, is induced by abscisic acid, heavy metal, and osmotic stress in leaves of Psychotria brachyceras.

Psychotria brachyceras Muell. Arg. produces the antioxidant monoterpene indole alkaloid (MIA) brachycerine, which, besides retaining a glucose residue, has its terpenoid moiety derived not from secologanin, but probably from epiloganin, representing a new subclass of MIAs. In this work we showed that osmotic stress agents, such as sodium chloride, sorbitol and polyethylene glycol (PEG), induced brachycerine accumulation in leaf disks of P. brachyceras. Other oxidative stress inducers, such as exposure to aluminum and silver, also increased brachycerine content. Abscisic acid (ABA) treatment was shown to increase brachycerine yield, suggesting its involvement in brachycerine induction during osmotic stress. Ascorbate peroxidase activity was induced in PEG-treated leaf disks, whereas superoxide dismutase (SOD) activity remained unaltered. Assays with specific inhibitors of the cytosolic mevalonate (MVA) and plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways showed that the terpenoid moiety of brachycerine derived predominantly from the MEP pathway. These results suggest a potential involvement of brachycerine in plant defense against osmotic/oxidative stress damage, possibly contributing to detoxification of hydroxyl radical and superoxide anion as a SOD-like molecule.

Early changes in gene expression induced by acute UV exposure in leaves of Psychotria brachyceras, a bioactive alkaloid accumulating plant.

UV-B radiation can damage biomolecules, such as DNA, RNA, and proteins, halting essential cellular processes; this damage is partly due to ROS generation. Plant secondary metabolites may protect against UV-B. Psychotria brachyceras Müll. Arg. (Rubiaceae), a subtropical shrub, produces brachycerine, a monoterpene indole alkaloid mainly accumulated in leaf tissues, which displays antioxidant and antimutagenic activities. Exposure of P. brachyceras cuttings to UV-B radiation significantly increases leaf brachycerine concentration. It has been suggested that this alkaloid might contribute to protection against UV-B damage both through its quenching activity on ROS and as UV shield. To identify differentially expressed genes of P. brachyceras in response to UV-B and investigate a possible influence of this stimulus on putative brachycerine-related genes, suppressive subtractive hybridization was applied. Complementary DNA from UV-B-treated leaves for 24 h was used as tester, and cDNA from untreated leaves, as driver. After BLASTX alignments, 134 sequences matched plant genes. Using quantitative RT-PCR, selected genes potentially related to brachycerine showed significant increases in transcription after UV-B exposure: tryptophan decarboxylase, ACC oxidase, UDP-glucose glucosyltransferase, lipase, and serine/threonine kinase. Results suggest a possible involvement of brachycerine in acute UV-B responses and show that alkaloid accumulation seems at least partly regulated at transcriptional level.

Antioxidant and antimutagenic properties of the monoterpene indole alkaloid psychollatine and the crude foliar extract of Psychotria umbellata Vell.

Psychollatine is a monoterpene indole alkaloid produced and accumulated by Psychotria umbellata Vell. (Rubiaceae) leaves in relatively high amounts (approximately 3% of the dry weight). The alkaloid has been shown to display opioid-like analgesic, anxiolytic, antidepressive and antipsychotic activities in rodents. In vitro assays suggested a protective role for this molecule in plant oxidative stress responses. This work reports antioxidant properties of psychollatine and the crude foliar extract from P. umbellata in strains of Saccharomyces cerevisiae proficient and deficient in antioxidant defenses exposed to H2O2 and paraquat. The antimutagenic activity of P. umbellata and its main alkaloid were assayed in S. cerevisiae N123 strain in presence of H2O2. Moreover, the antioxidant capacity of these substances on the hydroxyl radical (OH.) was investigated, using the hypoxanthine/xanthine oxidase assay. Psychollatine and the crude foliar extract of P. umbellata showed protective effect against oxidative stress in yeast, acting both as antioxidant and antimutagenic agents.

Antioxidant and antimutagenic effects of the crude foliar extract and the alkaloid brachycerine of Psychotria brachyceras.

The monoterpene indole alkaloid brachycerine from Psychotria brachyceras has been shown to be induced by UV and to have in vitro antioxidant activity, indicating a possible protective role against the secondary effects of this radiation. In this work, we have studied the antioxidant properties of brachycerine and a crude foliar extract from P. brachyceras by using Saccharomyces cerevisiae strains proficient and deficient in antioxidant defenses. The mutagenic and antimutagenic potential of these substances were assayed in S.cerevisiae N123 strain in the presence and absence of H2O2. In addition, we tested the antioxidant capacity of brachycerine and a crude foliar extract from P. brachyceras on hydroxyl radicals (OH) using the hypoxanthine/xanthine oxidase assay. The results show that brachycerine and the crude foliar extract of P. brachyceras have antioxidant and antimutagenic effects in yeast and probably this action is mainly due to the scavenging of OH radicals.

Environmental and ontogenetic control of accumulation of brachycerine, a bioactive indole alkaloid from Psychotria brachyceras.

Brachycerine is a monoterpenoid indole alkaloid accumulated in Psychotria brachyceras plants (Rubiaceae). To better understand the accumulation patterns of this alkaloid, we investigated its content in different plant organs from field-grown trees, throughout the seasons, during seedling development, and in response to potential biotic factors regulating its biosynthesis. Quantification by RP-HPLC showed that aerial vegetative organs (green stems, young and old leaves) yielded similar amounts of brachycerine [0.1-0.2% dry weight (DW)]. Brachycerine was not detected in roots. In reproductive structures, the highest brachycerine amounts (0.3% DW) were found in inflorescences. Alkaloid concentration decreased in mature fruits (0.045% DW). The lowest concentration in reproductive organs was observed in quiescent seeds (0.004% DW). Apparently, brachycerine content dropped during radicle emission in germinating seeds. During seedling development, an increase in leaf content from 0.02 to 0.1% DW was observed between the stages of 2 and 14 leaves, respectively. Salicylic acid did not affect brachycerine content. A doubling of alkaloid content was observed in wounded plants, and a threefold induction occurred with jasmonic acid treatment, suggesting that brachycerine biosynthesis is regulated by jasmonate production.