In this study, the GTP cyclohydrolase I (GTPCH) gene was overexpressed to investigate the role of BH4 in lipogenesis in oleaginous fungus Mortierella alpina. Transcriptome data analysis reveal that GTPCH expression was upregulated when nitrogen was exhausted, resulting in lipid accumulation. Significant changes were also found in the fatty acid profile of M. alpina grown on medium that contained a GTPCH inhibitor relative to that of M. alpina grown on medium that lacked the inhibitor. GTPCH overexpression in M. alpina (the MA-GTPCH strain) led to a sevenfold increase in BH4 levels and enhanced cell fatty acid synthesis and poly-unsaturation. Increased levels of nicotinamide adenine dinucleotide phosphate (NADPH) and upregulated expression of NADPH-producing genes in response to enhanced BH4 levels were also observed, which indicate a novel aspect of the NADPH regulatory mechanism. Increased BH4 levels also enhanced phenylalanine hydroxylation and nitric oxide synthesis, and the addition of an NOS or a PAH inhibitor in the MA-GTPCH and control strain cultures decreased fatty acid accumulation, NADPH production, and the transcript levels of NADPH-producing genes. Our research suggests an important role of BH4 in lipogenesis and that the phenylalanine catabolism and arginine–nitric oxide pathways play an integrating role in translating the effects of BH4 on lipogenesis by regulating the cellular NADPH pool. Thus, our findings provide novel insights into the mechanisms of efficient lipid biosynthesis regulation in oleaginous microorganisms and lay a foundation for the genetic engineering of these organisms to optimize their dietary fat yield.Tetrahydrobiopterin (BH PAH is ide (NO) . BH4 is e stress . Studiesholipids . The rolMortierella alpina is a well-known lipid-producing fungus that produces a high level of PUFAs  is responsible for the conversion of GTP to dihydroneopterin triphosphate, which is the rate-determining step for BHynthesis . GTPCH oclerosis , accelerclerosis , repair clerosis , restoreclerosis , and attclerosis . HoweverM. alpina to enhance the availability of BH4 and enable the assessment of the roles of BH4 in lipogenesis. NADPH generation, phenylalanine hydroxylation, and NO synthesis were also investigated to explore the regulatory mechanism of intracellular BH4 biosynthesis on lipogenesis. This study provides novel insights into the mechanisms of efficient lipid biosynthesis in oleaginous microorganisms and lays a foundation for the genetic engineering of these organisms to optimize the production of dietary fat.In this study, GTPCH overexpression was induced in Mortierella alpina (ATCC 32222) was cultured in Kendrick medium for 8 days as described earlier   . The reaescribed . Transcrescribed .4  inhibits GTPCH by direct competition with substrate GTP, which has been extensively used to investigate the function of BH4 . In this4 . Fatty aAgrobacterium tumefaciens C58C1, which was grown in MM medium containing 100 μg/mL kanamycin and rifampicin  was used to extract total RNA. RNA was reverse transcribed to cDNA using the iScript Select cDNA synthesis kit (Bio-Rad) according to the manufacturer’s instructions. The GTPCH gene (JF746874) was amplified using the primers (GTPCH-F/R) presented in fampicin . Cell pe00 = 0.3 . The celembranes . After iembranes , M. alpieviously , and theeviously . The PCRqPCR was performed on a CFX96 qPCR System (Bio-Rad) with SYBR Green Supermix (Bio-Rad) as described previously . The amp4 and dihydrobiopterin (BH2) levels were measured by liquid chromatography and mass spectrometry (LC–MS) after iodine oxidation in acidic or alkaline conditions as described previously  by acid oxidation, 10 μL of 1% iodine in 2% KI solution was added to 90-μL supernatant. To determine BH2 and biopterin levels by alkali oxidation, 10 μL of 1M NaOH was added to 80 μL supernatant, followed by the addition of 10 μL iodine/KI solution. For folate extraction, the mycelia were immediately collected and frozen in liquid nitrogen and homogenized with a pestle in liquid nitrogen to extract folate. The homogenate was resuspended and mixed in 1 mL of extraction buffer , transferred to a 1.5 mL tube, and kept at −20°C for 15 min. The sample was then spun in a microcentrifuge at 12,000 g for 5 min at 4°C, and the supernatant was taken as the first extract. The pellet was then resuspended in the extraction buffer and stored at −20°C for 15 min. This sample was spun at 12,000 g for 15 min at 4°C, and the supernatant was taken as the second extract. The combined extract was subjected to LC–MS analysis to determine the folate level  (Tyrosine levels in eviously . BrieflyoVision) . NADPH lLiquid chromatography and mass spectrometry was performed using a Thermo Scientific Dionex UltiMate 3000 RSLC system coupled online with the Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer . Chromatographic separation was performed on a TSK-Gel Amide-80 column  maintained at 35°C. Aliquots of reaction mixtures were then injected into the column in 5 mM ammonium acetate and acetonitrile following the gradient elution program shown in Previous transcriptome data were analyzed to determine the changes in the expression levels of GTPCH during the course of lipogenesis, which was induced by nitrogen exhaustion . TranscrM. alpina  accumulation levels than those grown in a medium without DAHP by approximately 36%  relative to that in M. alpina cultures grown in a medium without DAHP   were increased significantly to approximately 276% compared with those in the M. alpina CCFM 501 strain that contained pBIG2-ura5s-IT1 (the control strain) (The binary vector pBIG2-ura5s-GTPCH was constructed to transfer the GTPCH gene into the trophic) . This vetrophic) . The T-D1/TrpCR1 . The pre1/TrpCR1 . qPCR al strain) .4 levels in M. alpina  and 6-phosphogluconate dehydrogenase (PGD) in the PPP is one of the major determinants of efficient lipid biosynthesis in oleaginous microorganisms . In our . alpina , whereas+-dependent IDH (IDH1 and IDH2) was upregulated (285 and 111%) upon GTPCH overexpression  cycle . In our pression .MA-GTPCH strain in our study, the expression of NADPH-producing genes in folate metabolism, including methylenetetrahydrofolate dehydrogenase (MTHFD) 1, MTHFD2, and MTHFDL, was upregulated   .M. alpina , a PAH inhibitor in higher organisms , an NOS inhibitor in higher organisms   . DHFR isnditions . Thus, t4 plays an important role in lipogenesis in higher organisms. Mutations in the BH4-dependent PAH result in the metabolic disease called phenylketonuria, the patients of which present lower concentrations of AA and eicosapentaenoic acid (M. alpina also showed reduced TFA and AA levels when grown on a medium with PAH inhibitor (4 in M. alpina was suggested to play a role in contributing NADPH for lipogenesis via phenylalanine metabolism by PAH (4 in lipogenesis. In our study, obvious changes were observed in the fatty acid content and status in M. alpina grown on a medium with GTPCH inhibitor (4 can control lipogenesis either on its own or via interactions with other as yet unrecognized regulatory factors. Collectively, our results suggest an important role of BH4 in lipogenesis in M. alpina. Because the effect of BH4 is probably extensive in lipogenesis, the role of BH4 should be deeply investigated and explored in other oleaginous microorganisms.It has been suggested that BHoic acid . In our nhibitor . BH4 in m by PAH . Howevernhibitor . In addinhibitor . These rM. alpina, the NADPH-generating pathway was constructed to map the regulatory pathway of NADPH (4 levels in the MA-GTPCH strain suggests a novel aspect of intracellular NADPH regulation by BH4. Compared with the samples from the control strain, those from the MA-GTPCH strain showed upregulated expression of all NADPH-producing genes when the lipid had accumulated for a long period of time. Thus, BH4 likely promotes an elegant coordination of glycolysis, PPP, TCA cycle, and one carbon pool by folate to enhance lipid synthesis and poly-unsaturation via NADPH regulation in M. alpina (NADPH is the limiting factor and a critical reducing agent in lipid biosynthesis . Unsaturof NADPH . Our obs. alpina .4 level was significantly increased upon GTPCH overexpression, which enhanced the function of phenylalanine catabolism and the arginine–NO pathway. The addition of an NOS or a PAH inhibitor in the MA-GTPCH and control strain cultures decreased fatty acid accumulation, NADPH production, and the transcript levels of NADPH-producing genes, which confirmed the transduction effects of phenylalanine catabolism and arginine–NO pathway (The intracellular BH pathway . The met pathway . Fumarat pathway , which c pathway . This in pathway .4 is also a major determinant of whether NOS produces NO or superoxide and thus plays a key role in regulating cellular oxidative stress (In vivo studies have demonstrated that increased BH4 levels could enhance the NO content and inhibit vascular oxidative stress in transgenic mouse models of GTPCH overexpression (4 level enhanced the function of NOS in the MA-GTPCH strain, resulting in the generation of NO rather than superoxide, thus suppressing oxidative stress (+-reducing enzymes to the NADPH pool has been shown to be altered by oxidative stress (M. alpina may also increase the contributions of NADP+-reducing enzymes to the NADPH pool by suppressing oxidative stress (BHe stress . In vivopression . In our e stress  (Schulz e stress . Oxidatie stress . The cone stress . Enhancee stress . In highe stress . NO can e stress .4 levels and revealed that the availability of BH4 is critical for lipogenesis in M. alpina. Phenylalanine catabolism and NO synthesis may play an integrating role in translating the effects of BH4 on lipogenesis by regulating the intracellular NAPDH pool. RNAi of the PAH or NOS gene should be adopted in future studies to explore the mechanisms of BH4-induced lipogenesis. Our findings provide novel insights into the mechanisms of efficient lipid biosynthesis regulation in oleaginous microorganisms and lay a foundation for the genetic engineering of these organisms to optimize the production of dietary fat.Our study established the utility of GTPCH overexpression as a specific and novel approach to enhance intracellular BHThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.All authors contributed to the conception and planning of the study. HW and CZ performed the experiments and drafted the manuscript. HC, JZ, WC, and ZG supervised the experimental work. HZ, YC, and WC reviewed the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Most sudden-onset hearing loss is due to otolaryngologic- and very rarely to cerebrovascular disease. We report a woman with sudden bilateral sensorineural hearing loss. This case suggests that even in the absence of brainstem or cerebellar signs, magnetic resonance imaging (MRI) and MR angiography (MRA) should be performed since such studies may reveal signs of life-threatening vertebrobasilar artery occlusion.A 73-year-old Japanese woman with a history of hypertension, hyperlipidemia, and atrial fibrillation who suffered bilateral deafness with vertigo and vomiting was transferred from a local hospital to our department. On admission her consciousness was clear and vertigo was absent. Neurological examination revealed only bilateral sensorineural hearing loss. Head computed tomography (CT) returned no significant findings. The next morning she gradually developed severe drowsiness. Diffusion-weighted MRI demonstrated acute cerebral infarction in the brainstem and bilateral cerebellum; MRA showed basilar artery occlusion due to a cardioembolic thrombus. Revascularization was obtained by endovascular treatment. However, her condition worsened progressively during the following hours. CT revealed new brainstem lesions, massive cerebellar swelling, and obstructive hydrocephalus. She died on the second day after her admission.When hearing loss is due to vertebrobasilar occlusive disease, the prognosis is very poor. We suggest that vertebrobasilar stroke be suspected in patients with bilateral sensorineural hearing loss who present with risk factors for stroke such as atrial fibrillation and other neurologic signs. The prognosis of patients with sudden-onset sensorineural hearing loss, an inner ear disorder, is relatively good. It tends to be due to idiopathic sudden deafness, Meniere's disease, or a perilymphatic fistula. More rarely it is attributable to vertebrobasilar ischemia. The incidence of vertebrobasilar ischemia in sudden sensorineural hearing loss is approximately 1.2% [When hearing loss is due to vertebrobasilar occlusion, the prognosis is very poor. We report a woman who presented with only bilateral hearing loss without vertigo and vomiting. Consequently, initially we did not suspect specific anomalies including vertebrobasilar impairment. However, angiography revealed occlusion of lower third of the BA. Based on the experience reported here, we suggest that patients with sudden bilateral hearing loss be subjected to MRI and MRA studies because they may reveal signs of life-threatening vertebrobasilar artery occlusive disease.A 73-year-old Japanese woman with a history of hypertension, hyperlipidemia, and atrial fibrillation (AF) with a CHA2DS2-VASC score of 3 developed bilateral hearing loss with vertigo and vomiting and was brought to a local hospital. Her home medications included 5 mg daily of atorvastatin. The diagnosis was sudden deafness and she was transferred to our emergency department. At that time her consciousness was clear. She had no significant family, social, environmental, or employment history. She did not smoke or take alcohol. Her blood pressure was 147/72 mmHg, her pulse rate was 78 beats/minute with AF, and her temperature 36.8 °C. One year earlier she interrupted anticoagulant therapy because she experienced recurrent epistaxis.She reported only bilateral hearing loss without vertigo and vomiting. Dysarthria, weakness, ataxia, diplopia, dysphagia, and Horner syndrome were absent. As computed tomography (CT) returned no specific findings Fig.  she retu9/l, hemoglobin of 14.7 g/dl, platelet count of 268 × 109/l, blood urea nitrogen 11.3 mg/dl, and creatinine of 0.58 mg/dl, as well as a normal liver function test result. Diffusion-weighted imaging (DWI) revealed acute multifocal lesions involving the bilateral cerebellar hemispheres and pons and the posterior circulation. The DWI posterior circulation Acute Stroke Prognosis Early CT Score (DWI-pc-ASPECTS) was 7  score was 7 E2V1M4) and the National Institute of Health Stroke Scale (NIHSS) score was 33. We found the normal pupil reflexes, symmetrical facial responses and withdrawal of both arms and legs to painful stimuli. Her general physical examination was unremarkable. Her blood pressure was 170/90 mmHg, her pulse rate was 80/minute with AF, and her temperature 36.4 °C. Her laboratory test results showed a white blood cell count of 8.8 × 10V1M4 and s 7 Fig. d, e. As Transfemoral cerebral angiography showed occlusion of the V4 segment of the left VA just proximal to its union with the BA Fig. a, b. LefHer past history of untreated AF and current angiographic findings strongly suggested a cardioembolic thrombus-induced basilar artery occlusion; we did not perform echocardiography. Postoperatively she was comatose; brain CT showed a large brainstem infarct and upward herniation Fig.  and she We showed a woman who presented with only bilateral hearing loss without vertigo and vomiting caused by embolic occlusion of lower third of the BA. When hearing loss is due to vertebrobasilar occlusion, the prognosis is very poor. We suggest that patients with sudden bilateral hearing loss be subjected to MRI and MRA studies because they may reveal signs of life-threatening vertebrobasilar artery occlusive disease.et al. [Sudden-onset sensorineural hearing loss usually suggests an inner ear disorder, e.g. Meniere disease, acute labyrinthitis, autoimmune inner ear disease, or a perilymphatic fistula. Most sudden-onset sensorineural hearing loss is unilateral. The reported incidence of vertebrobasilar ischemia in patients with sudden sensorineural hearing loss is approximately 1.2–8.0% ; 1.4% ofOur patient had no symptoms of brainstem and cerebellar involvement. Among vascular causes, ischemic stroke in the territory of the AICA, a branch of the BA, is the leading cause of sensorineural hearing loss , 6. UsuaMisery perfusion in the bilateral AICA territories was due to insufficient supply from the PcomA-mediated retrograde collateral pathway. Because the inner ear requires a high-energy metabolism and the IAA is an end artery with little collateral circulation from the otic capsule, the inner ear is particularly vulnerable to ischemia –14. The et al. [et al. [Many patients with vertebrobasilar occlusive disease have a poor outcome and experience truncal ataxia, quadriplegia, locked-in syndrome, coma, and death , 18, 19.et al.  and Kim  [et al.  reportedOurs is a rare case of sudden bilateral sensorineural hearing loss due to embolic occlusion of the vertebrobasilar artery, the AICA, and the PICA. The delayed diagnosis of ischemic stroke in the posterior circulation can be life-threatening. Therefore, an early diagnosis and the proper management of hearing impairment may provide a window to prevent the progression of infarction to larger areas of the brainstem and cerebellum. Clinicians must consider the possibility of vertebrobasilar occlusive disorder especially in patients with sudden bilateral hearing impairment, with risk factors for stroke and the manifestation of other neurologic signs.


Ixodes) transmitted spirochete, Borreliella burgdorferi. Here we report on the immunogenicity and efficacy of VANGUARD®crLyme (Zoetis), the most recent canine Lyme disease vaccine to be approved by the United States Department of Agriculture. VANGUARD®crLyme is a subunit vaccine consisting of outer surface protein A (OspA) and a recombinant outer surface protein C (OspC) based-chimeric epitope protein (chimeritope) that consists of at least 14 different linear epitopes derived from diverse OspC proteins. The combination of OspA and the OspC chimeritope (Ch14) in the vaccine formulation allows for the development of humoral immune responses that work synergistically to target spirochetes in both ticks and in mammals. Immunogenicity was assessed in purpose-bred dogs. A two-dose vaccination protocol resulted in high antibody titers to OspA and Ch14 and vaccinal antibody reacted with 25 different recombinant OspC variants. Efficacy was demonstrated using an Ixodes scapularis -purpose bred dog challenge model. Vaccination with VANGUARD®crLyme provided protection against infection and prevented the development of clinical manifestations and histopathological changes associated with Lyme disease.Lyme disease, a public health threat of significance to both veterinary and human medicine, is caused by the tick ( B. burgdorferi, B. afzelli, B. garinii, and B. bavariensis are causative agents in Europe and Asia. Additional species have been identified-but have not been clearly associated with overt clinical disease. In 2014, the LD spirochetes were assigned separate genus status and classified as BorreliellaLyme disease (LD) is the most common vector-borne disease in the northern hemisphere Ixodes tick populations Ixodes ticks and a diverse array of mammalian reservoirs Ixodes scapularis and I. pacificus are the dominant tick vectors while I. ricinus and I. persulcatus dominate in EuropeThe incidence of LD in humans and companion animals in N. America B. burgdorferiClinical manifestations of canine LD typically develop slowly and are initially non-descript B. burgdorferi proteins in I. scapularis nymphs (8th) and mammals (1st) Subunit and bacterin vaccines are available for the prevention of LD in dogs B. burgdorferi infection and prevented the development of clinical manifestations associated with LD.Distinct variants of OspC, referred to as OspC types (differentiated by letter or other designations), have been identified 22.1Bordetella bronchiseptica were administered to all study dogs. Four dogs were randomly selected to be sentinels , 16 dogs were assigned to the T01 placebo group  and 16 were assigned to the T02 vaccine group (henceforth referred to as ‘vaccinate dogs’). VANGUARD®crLyme (Zoetis), henceforth referred to as ‘vaccine’, was manufactured using practices in accordance with USDA-approved Outline of Production  at the minimum immunizing dose. Placebo and vaccine were administered on Day 0  and Day 21 . On Day 0, dogs were tested for Ab to the B. burgdorferi C6 peptide using the SNAP® 4Dx Test (IDEXX). Note that subsequent C6 Ab analyses conducted in this study employed the SNAP4Dx® Plus Test (IDEXX). After each treatment, dogs were closely monitored for adverse events as detailed below. A study timeline is presented in Antibody profile defined laboratory-purposed beagles  were obtained and randomly allocated to treatment groups, rooms, and pens at the study site using a statistical software suite (SAS Institute). Dogs were maintained at Zoetis research sites in accordance with USDA Animal Welfare Regulations  and approved Institutional Animal Care and Use Committee (IACUC) protocols (Zoetis). Prophylactic vaccines for parvovirus and 2.2I. scapularis ticks collected from a collection site in Wakefield, Rhode Island (latitude and longitude: 41.438171 and −71.501556) . Prior to infestation, ticks were stored in vented vials  and the percentage of B. burgdorferi infected ticks (56.7%) determined by PCR using B. burgdorferi flaB specific primers. On Day 42, 30 ticks per dog  were placed in infestation chambers, placed on placebo and vaccinate dogs, and the ticks were fed to repletion. The ticks were collected on study Day 51. For the remaining course of the study, the dogs were monitored for clinical signs of LD including ataxia, lameness, depression, lethargy, head pressing, myalgia, and other manifestations.Tick challenge was performed using adult 2.3405-490. ELISAs were run in duplicate and antigen specific titers calculated from the average plus three standard deviations of the negative control A405-490 value. The reciprocal test sample dilution above the average negative control OD represented the endpoint titer. Vaccination induced antibody responses to diverse OspC type proteins were assessed by immunoblot using recombinant OspC proteins exactly as decribed in an earlier study Blood was collected from each dog on Days 0, 21, 35, 77, 117, and 153, and sera harvested using standard methods. The SNAP® 4Dx and SNAP4®Dx Plus tests were performed as instructed by the manufacturer (IDEXX). ELISA assays for OspA and Ch14 were performed using standard methods. In brief, detection antigens  were immobilized onto 96-well microtiter plates , the plates contents were removed and the wells were washed with blocking buffer  followed by a final wash with PBST . Serially diluted sera were added and the plates were incubated at 37 °C for 1 hr. After washing, horseradish peroxidase conjugated goat anti-dog IgG(H + L) was added , the plates were washed, ABTS substrate was added  and absorbance read at A2.4Tissue from the area of the tick feeding site, left and right (L/R) shoulder synovial joint tissue, L/R elbow synovial joint tissue, L/R carpus synovial joint tissue, L/R stifle synovial joint tissue, L/R tarsus synovial joint tissue, kidneys, femur, and humerus were collected. Samples were blinded and then formalin fixed, processed, embedded, sectioned, mounted, and stained with hematoxylin and eosin on a fee for service basis by an independent party (Charles River). Sections were examined by a blinded board-certified Veterinary Pathologist (Zoetis Inc.) using light microscopy. A severity grade assessing the type, distribution, and severity of inflammation was assigned based on a scoring system where 0 = absent, 1 = minimal, 2 = mild, 3 = moderate, 4 = marked, and 5 = severe; scores >= 2 were considered abnormal.2.5P ≤ 0.05). Data were analyzed using SAS® software . A Cochran-Armitage test adjusting for post-infestation room was used to analyze C6 Ab ever being positive post-tick challenge, having at least one joint graded 2 or higher, and the presentation of disease.All hypothesis tests were carried out at the 0.05 level of significance  placebo dogs at one or more timepoints post-tick feeding . No clin3.2B. burgdorferi and that vaccination with VANGUARD®crLyme resulted in a significant difference in the prevention of B. burgdorferi infection compared to placebo (P < 0.0001).All sentinel dogs were C6 Ab (-) at all timepoints. All placebo dogs (16/16) were C6 Ab (+) on two or more occasions post-tick feeding. Seven of sixteen placebo dogs were C6 Ab (+) on Day 77 and all were (+) on Days 117 and 153. Fifteen of sixteen vaccinates were C6 Ab (-) at all timepoints. A single vaccinate (dog WAR2) was C6 Ab (+) on Day 77, but (-) on Days 117 and 153. In total, of the 48 C6 Ab tests conducted per study group after tick infestation, 97.9% and 18.8% of the tests done on vaccinate and placebo dogs were (-), respectively. Results are summarized in Ab titers to the OspA and Ch14 were determined for each individual dog . All vacTo determine if vaccination with VANGUARD®crLyme induces broadly cross-reactive antibodies to diverse OspC proteins, immunoblots consisting of twenty-five recombinant OspC variants were screened with sera collected on Day 0 and Day 35. All OspC variants tested were immunoreactive with sera from vaccinate dogs B.3.3B. burgdorferi was present in the hypodermis/subcutis of 14/16 placebo dogs , but was absent from the hypodermis/subcutis of vaccinates and sentinels. Mononuclear inflammation with decreased numbers of hair follicles  and subsequent dermal collagen condensation was identified in 10/16 placebos, 6/16 vaccinates, and 0/4 sentinels. Results are summarized in Histopathology changes were noted at the tick bite sites and within and surrounding joints among study groups , Fig. 4.B. burgdorferi infection (p < 0.0001).Within and around joints, histopathological changes included synovial hypertrophy and hyperplasia (including papillary projections), mononuclear and neutrophilic inflammation within the joint and local subcutis, and fibrin deposition . Synovia3.4B. burgdorferi infection; 3) abnormal histopathology. Based on these criteria, 16/16 placebo dogs were LD (+) while all sentinel dogs and vaccinates were LD (-). In summary, vaccination with VANGUARD®crLyme resulted in a significant difference (P < 0.0001) in the prevention of disease caused by B. burgdorferi (Dogs were scored as (+) for LD if they seroconverted to C6 Ab and met one of the following clinical criteria: 1) stiffness, limping, or lameness on three or more consecutive days or three or more events of intermittent lameness; 2) multiple episodes of clinical signs suggestive of gdorferi .3.5The temperature of each dog was monitored over time. One sentinel (not vaccinated) dog (XXR2) presented with a temperature of 39.9 °C on day 23. Two placebo dogs (SER2 and XKR2) had temperatures of 39.3 °C on three occasions. One vaccinate (SJQ2) had a temperature of 39.4 °C on Day 23. Elevated temperatures were not associated with the administration of placebo or vaccine. In summary, VANGUARD®crLyme is safe when administered subcutaneously as a two-dose series (three weeks apart) starting at approximately 8 weeks of age.4B. burgdorferi strains in both ticks and mammals. In this study, we demonstrated the immunogenicity and broad protective efficacy of VANGUARD®crLyme in a purpose bred dog-Ixodes scapularis challenge model.VANGUARD®crLyme is a USDA approved canine LD vaccine. It is a subunit vaccine consisting of non-lipidated OspA and an OspC chimeritope (referred to Ch14). As detailed above, Ch14 is a recombinant protein consisting of seven L5 and seven H5 OspC epitopes from diverse OspC type proteins. This design strategy allows for the removal of conserved segments of OspC that do not elicit protective Ab responses and the generation of a protein consisting primarily of well-characterized linear epitopes from diverse OspC types In a recent study, Grosenbaugh et al B. burgdorferi strains that produce different OspC types ospC types from tissue and tissue derived cultures from dogs infected using field collected ticks. One of the most frequently amplified OspC types in that study was OspC type F. OspC type F specific antibody responses have been detected in horses and wild canids (eastern coyotes) To assess protective efficacy, placebo and vaccinates were challenged using field collected ticks. The use of field collected ticks, as opposed to laboratory infected ticks, is important for several reasons. First, the use of naturally infected ticks circumvents the inherent limitations associated with the use of ticks infected with laboratory adapted strains. Second, field collected ticks typically carry a heterogenous population of P < 0.0001) against B. burgdorferi infection. Importantly, vaccination did not result in adverse events demonstrating that VANGUARD®crLyme is safe for use in dogs 8 wks of age or older.All dogs that received the placebo seroconverted to the C6 peptide after tick challenge while fifteen of sixteen VANGUARD®crLyme vaccinated dogs were C6 Ab (-) at all time points. One vaccinate was C6 Ab (+) at a single timepoint. However, that dog was C6 Ab (-) at all other timepoints and did not develop clinical or histopathological changes suggestive of LD. Vaccination with VANGUARD®crLyme also prevented the development of clinical manifestations commonly associated with LD. Consistent with earlier studies, some of the placebo dogs developed clinically apparent disease with single or multiple episodes of lameness post-tick feeding B. burgdorferi in ticks and inhibit transmission to mammals. Consistent with this, Ab titers to OspA and OspC did not increase in the VANGUARD®crLyme vaccinated dogs after tick feeding. If transmission had occurred, an increase in Ab titers to OspC (as measured using Ch14) would have been expected due to the expression of OspC by B. burgdorferi in mammals As discussed above, Ab to both OspA and OspC can target B. mayoniiB. mayonii raises the possibility that the vaccine could potentially protect against newly emerging species in North America and LD species and strains present in Eurasia. This remains to be demonstrated.Previous studies revealed that immunization with prototype OspC chimeritopes triggers antibody responses to all OspC types represented in the vaccine construct B. burgdorferi infection and sub-clinical arthritis. As described in the 2018 American College of Veterinary Internal Medicine Lyme Borreliosis consensus statement, VANGUARD®crLyme is the only canine LD vaccine on the market with a USDA-approved 15-month duration of immunity label VANGUARD®crLyme was specifically designed to provide protection through two synergistic mechanisms of action. Vaccination induced Ab to OspA can target spirochetes in the tick Richard T Marconi is an inventor of VANGUARDcrLyme and a consultant for Zoetis. RTM receives royalties and speaking fees from Zoetis. All other authors were employees of Zoetis at the time the studies were conducted.


Oxidative stress is an important mechanism underlying cellular damage of the inner ear, resulting in hearing loss. In order to prevent hearing loss, several types of antioxidants have been investigated; several experiments have shown their ability to effectively prevent noise-induced hearing loss, age-related hearing loss, and ototoxicity in animal models. Exogenous antioxidants has been used as single therapeutic agents or in combination. Antioxidant therapy is generally administered before the production of reactive oxygen species. However, post-exposure treatment could also be effective. Preconditioning refers to the phenomenon of pre-inducing a preventative pathway by subtle stimuli that do not cause permanent damage in the inner ear. This renders the inner ear more resistant to actual stimuli that cause permanent hearing damage. The preconditioning mechanism is also related to the induction of antioxidant enzymes. In this review, we discuss the mechanisms underlying antioxidant-associated therapeutic effects and preconditioning in the inner ear. In addition to hearing, distinguishing sounds is also a key function of the inner ear. However, the inner ear may sometimes be slowly damaged from birth. Typical damage of the normal function of the inner ear is caused by noise exposure, ototoxic drugs, aging, and autoimmune damage; sometimes, sudden loss of inner ear function may also be caused by unknown reasons. The incidence of impaired hearing is currently increasing . HearingThe major mechanisms of inner ear damage induced by noise (noise-induced hearing loss) are physical damage , decreasOne of the factors that intensifies noise-induced hearing loss is smoking. Smoking directly supplies exogenous ROS and also promotes the generation of endogenous ROS, thereby increasing ROS concentration in tissues. In an animal model, smoking before noise exposure aggravated noise-induced hearing loss and induced a permanent threshold shift, even at the noise level that normally resulted in a temporary threshold shift . Age-related hearing loss or presbycusis is a phenomenon caused by the loss of hair cells and spiral ganglion cells in the cochlea. Studies have shown that environmental and genetic factors were involved in the degeneration of cochlear hair cells . MoreoveNAT) gene, which is involved in the metabolism and detoxification of free radical species, age-related hearing loss occurs more frequently than in healthy subjects [ROS play an essential role in such age-related hearing loss. Indeed, ROS cause genetic and cell mutations inhibiting cellular functioning, and consequently play an important role in senescence or age-related deafness . With agsubjects .Previous studies have also reported the association between metabolic syndrome and ROS production. It is generally acknowledged that ROS affect tissue integrity, and thus the progression of metabolic syndrome. In particular, excessive salt consumption has been reported to be a relevant factor in this process . MetabolIn addition, in a longitudinal study, which recruited 1381 people, hearing loss was found to be more prominent in women over 50 years of age affected by metabolic syndrome . ElevateAminoglycoside antibiotics and cisplatin are known to cause ototoxic hearing loss. In particular, cisplatin causes excessive free radical production in cells , resultiSudden hearing loss is a sensorineural process that occurs suddenly, within hours or from two to three days, without a well-known cause. As the treatment prognosis varies, the causes are thought to be multivariate, including viral infections, vascular disorders, and autoimmune diseases ,41,42,43Immune-mediated hearing loss is a rare condition associated with autoimmune problems, characterized by frequent, recurrent, and chronic progress, which is known to account for approximately 1% of all hearing loss cases or hearing loss in approximately five of every 100,000 people per year . HoweverSudden hearing loss and autoimmune hearing loss have a common factor, i.e., they respond to steroid treatment when associated with rapid hearing loss. The use of steroids or immunosuppressants has been attempted to control the process of immune-related hearing loss. TNF-α is a pro-inflammatory cytokine that contributes to the maintenance of cellular homeostasis. TNF-α is rarely observed in normal internal organs; however, its levels increase with age , inflammFor vestibular schwannoma-related sensorineural hearing loss, in particular, the TNF-α level within vestibular schwannoma secretions have been reported to be associated with a degree of hearing loss . In factAnti-TNF-α therapy was studied to prevent such damage. For example, recent studies have reported improved hearing upon intratympanic injection of infliximab, a TNF-α inhibitor, in refractory and recurrent patients with hearing loss . In an aStudies focusing on antioxidant therapies in ROS-related hearing loss have been reported ,54. ConvROS-related damage in the inner ear can be prevented by reducing ROS generation or enhancing the antioxidant system. Specifically, the administration of exogenous antioxidants, the upregulation of endogenous antioxidant production, and the promotion of the ROS scavenger system can prevent inner ear damage .N-acetylcysteine (NAC), sodium thiosulfate (STS), amifostine, d-methionine, vitamin E, flunarizine, lipoic acid, and ebselen [Previous studies have identified a variety of agents that could protect the inner ear from oxidative stress, including  ebselen ,58. Antil-cysteine that acts as an antioxidant in two ways. As a thiol, NAC can act directly as an antioxidant. Moreover, as an l-cysteine precursor, NAC stimulates the endogenous antioxidant system [eq)), impulse (75 pairs of 155 dB peak sound pressure level (pSPL) impulses), or continuous  for 6 h). The protective effects of NAC against high-kurtosis noise were evident even at low doses. In another study by Lorito et al. [NAC is an acetylated t system . Previout system ,63,64,65t system . Moreovet system , chinchio et al. , rats wed-methionine and N-acetyl-l-cysteine (12.5 mg/kg each), (2) a group receiving 12.5 mg/kg of d-methionine, (3) a group receiving 12.5 mg/kg of N-acetyl-l-cysteine, and (4) saline-treated controls. The chinchillas were exposed to 6 h of continuous 105 ± 0.5 dB SPL octave-band noise centered at 4 kHz. Notably, the combination of d-methionine and NAC led to a significant reduction of inner ear damage at most frequencies. Conversely, the d-methionine-only treated group showed significant recovery of hearing only in the middle frequencies. Another study investigated the effect of intraperitoneal injection of 325 mg/kg NAC combined with 25, 50, or 75 mg/kg sodium salicylate or saline as the control in the recovery from a 6 h exposure to a 105 dB SPL octave-band noise centered at 4 kHz [Some investigators have examined the effects of concomitant administration of other antioxidants together with NAC. Indeed, concomitant administration of antioxidants can produce higher antioxidative effects at lower dosages. For instance, Clifford et al.  designatat 4 kHz . Intrigun = 566) were administered NAC or a placebo during weapon training [Two randomized clinical trials have assessed the effect of NAC in textile workers  and the training ; althougA recent meta-analysis also reported the otoprotective effects of NAC (when administered for more than six weeks) in patients receiving aminoglycoside antibiotics . However2. Because a significantly lower incidence of hearing loss was observed in the cisplatin plus STS-treated group, the authors indicated STS as a potential otoprotective agent.STS quenches ROS and preserves the activity of antioxidant enzymes . The otod-methionine, the enantiomer of the amino acid l-methionine, is another potent antioxidant that has been considered in preclinical studies [d-methionine can directly function as an antioxidant and as an adjuvant of the endogenous antioxidant system [d-methionine increases intracellular GSH levels [d-methionine, administered before or after noise exposure, could rescue noise-induced hearing loss in mice by protecting the cochlear morphology, inhibiting apoptosis, and maintaining the expression levels of connexin 26 and 30 [d-methionine could be observed in the rescue of noise-induced permanent threshold shift [d-methionine or sterile 0.9% saline, the rescue of noise-induced permanent threshold shift depended on the dose of administered d-methionine, with 200 mg/kg not significantly reducing the mean permanent threshold shift but 600 mg/kg mediating efficient rescue. Similarly, noise-induced decreases in the activities of the Na+, K+-ATPase, and Ca2+-ATPase, as well as in mean lipid peroxidation and nitric oxide levels, were also recovered in a d-methionine dose-dependent manner. studies ,91. d-met system . In factH levels , specifiH levels , therebyH levels . Moreove6 and 30 . Moreoveld shift . In factd-methionine, combined with radiation and chemotherapy, protected the hearing threshold at frequencies of 10 kHz and higher [d-methionine for 30 min before cisplatin infusion, or orally treated with 1000 mg/kg (200 mg/mL) d-methionine delivered by gavage 2 h before infusion of cisplatin, were found to be protected from cisplatin-induced auditory brainstem response threshold shift at all tested frequencies .The use of d higher . In factAlpha-lipoic acid also contains thiol groups and has been shown to protect the cochlea from cisplatin-induced ototoxicity and noise in animal models ,76. NeveIn another animal study of kanamycin-induced ototoxicity, kanamycin-triggered increased auditory brainstem response threshold shifts in BALB/c mice were significantly inhibited by subcutaneous injection of α-lipoic acid. Immunohistochemical staining and Western blot analysis of mouse cochleae showed decreased expression of phosphorylated p38 mitogen-activated protein kinase (MAPK) and phosphorylated c-Jun N-terminal kinase (JNK) .Furthermore, in an age-related hearing loss A/J mouse model, measurement of the auditory brainstem response thresholds revealed greater hearing loss attenuation in the group treated with 50 μg/g α-lipoic acid than in the controls . AdditioQuaranta et al. conducted a preliminary clinical trial on 30 healthy subjects and measured a temporary threshold shift of hearing of 6 kHz 2 min after the end of noise exposure (90 dB hearing loss (HL) 3 kHz pure tone for 10 min) . Interes2 amifostine immediately before or 3 h after each cisplatin infusion could alleviate hearing loss [Amifostine is a prodrug that provides selective protection for healthy tissues. Its active metabolite, WR-1065, protects the cochlea by scavenging ROS. In animal models, systemic administration of amifostine protected outer hair cells of the cochlea ,95 and ting loss . Althouging loss .Ebselen is a selenium-containing compound. It serves as an antioxidant, scavenges ROS by mimicking the GSH peroxidase, is an anti-inflammatory agent, and an activator of nuclear factor, erythroid 2-like 2 (NRF2) ,97. In aGuinea pigs receiving an oral dose of 10 mg/kg ebselen, 1 h before exposure to 115 dB SPL 4 kHz octave-band noise for 3 h, were protected from temporary threshold shift . In the Flunarizine decreases proinflammatory cytokine secretion by downregulating the expression of nuclear factor-kappa B (NF-κB) and MAPK through the activation of NRF2 . HoweverOther antioxidants that may potentially play a protective role against cochlear oxidative stress include ginseng , coenzymKorean red ginseng has anti-ROS and anti-apoptotic properties, and therefore may play a role in the prevention of noise-induced hearing loss. For example, Kang et al. fed Korean red ginseng (200 mg/kg) to BALB/c mice for three days after exposure to 110 dB noise for 3 h to induce temporary threshold shift . These aCoQ10 is a component of the mitochondrial respiratory chain. It inhibits mitochondrial lipid peroxidation, induces ATP production, and is involved in ROS scavenging and prevention of oxidative stress-induced apoptosis. Fetoni et al. injected guinea pigs intraperitoneally with water-soluble CoQ10 at a dose of 100 mg/kg and subjected them to 120 dB SPL pure tone sound at a frequency of 6 kHz for 60 min . CoQ10 sMitoquinone, a mitochondria-targeted derivative of the antioxidant ubiquinone, was also suggested as an otoprotective agent . NeverthAspirin reportedly exhibits a preventive effect on cisplatin-induced ototoxicity . Similard-methionine [2O2 and HO− scavenger and is a major contributor to cellular GSH synthesis. ACAR improves the maintenance of mitochondrial membrane energetics and integrity. The endogenous antioxidant enzymatic system has evolved to control the detrimental effects of free radicals in the ear, including increased levels of antioxidant enzymes, such as copper/zinc and manganese superoxide dismutases , catalase, GSH-related enzymes (glutathione peroxidases (GPxs), glutathione transferases (GSTs), and glutathione reductase), and peroxiredoxins (PRDXs). SODs can dismutate two O2− anions into H2O2 and molecular oxygen. In addition to differential requirements for metal ions as cofactors, SODs have distinct subcellular localizations; SOD1 is localized in the cytoplasm and extracellularly, while SOD2 is localized in the mitochondria. Spontaneous and enzymatically catalyzed reactions, including plasma membrane-associated NADPH oxidases, the mitochondrial electron transport chain, the cytosolic xanthine oxidase, and cytochrome p450 monooxygenases mainly acting in the endoplasmic reticulum, can give rise to O2.−. Therefore, SODs are regarded as the first line of endogenous antioxidant enzymes to convert toxic ROS to less harmful H2O2 [Since excessive free radical buildup in the inner ear is clearly a key factor in the pathogenesis of various types of hearing loss, increasing cochlear antioxidant supplies could substantially prevent hair cell damage and hearing loss. Exogenous antioxidant agents, such as various free radical scavengers including GSH , ebselenthionine  have shothionine . Furtherthionine  and the thionine , respectthionine . Moreovethionine . NAC funful H2O2 .2O2 or organic hydroperoxides to water or corresponding alcohols using reduced GSH as an electron donor (H2O2 + 2GSH = GS-SG → 2H2O). GPxs display a selenium dependent GSH peroxidase activity, relying on a selenocysteine residue encoded by an opal TGA codon. In mammalian tissues, at least four types of selenium dependent GPx isozymes have been identified; a classical GPx (GPx1), a gastrointestinal GPx (GPx2), a plasma GPx (GPx3), and a phospholipid GPx (GPx4). GPx1–3 act as homotetramers, while GPx4 functions as a monomer. These isozymes have distinct subcellular locations, such as cytosol, nucleus, and mitochondria for GPx1; cytosol and nucleus for GPx2; cytosol and extracellular space for GPx3; and nucleus, cytosol, mitochondria, and membranes for GPx4 [The tetrameric enzyme catalase is, then, responsible for detoxification through the breakdown of two hydrogen peroxide molecules into one molecule of oxygen and two molecules of water. According to the differences in their sequences and structures, catalases are divided in three types. Monofunctional heme-containing catalases are the most widespread type, present in all aerobic organisms. Bifunctional catalases-peroxidases carrying a heme group belong to the second type, which is relatively less abundant in nature. The third type consists of Mn-containing catalases that lack the heme group . GPxs arfor GPx4 . PRDXs bfor GPx4 . These afor GPx4 ,118,119.The preconditioning effect is an interesting phenomenon observed during hair loss-prevention studies. Preconditioning refers to the process of pre-inducing a preventative pathway by subtle stimuli that do not cause permanent damage in the inner ear. After preconditioning, the inner ear is more resistant to the actual major stimuli that are supposed to cause permanent damage in the inner ear. In other words, preconditioning refers to providing the cells with continuous weak stress, and thereby allowing them to develop the ability to protect themselves against significant stress stimuli. In the inner ear, several types of preconditioning have been studied, i.e., hyperthermia , restraiThe effector mechanisms of such preconditioning stimuli have been identified as increased levels of heat shock proteins , glucocoSound conditioning is a strategy to protect hearing damage by creating resistance to harmful intense noise stimuli through pretreatment with low-level noise stimuli . The mecHSP70 mRNA in cochlea, suggesting that the corresponding protein is involved in the protective mechanism of heat stress.The preconditioning effect is not only caused by sound (sound conditioning). It has also been reported that the incidence of noise-induced hearing loss was reduced when systemic stress was increased . Both hyAfter restraining mice in plastic tubes for 12 h, Wang et al. exposed them to a noise level that caused hearing loss . The res2). In CoCl2-treated mice, noise exposure, which was supposed to induce a permanent threshold shift of hearing, could not induce permanent hearing loss [2. A subsequent study revealed that CoCl2 pretreatment protected auditory hair cells (HEI-OC1) from H2O2-mediated cytotoxicity via the activation of the redox-sensitive transcription factors HIF-1α and NRF2, as well as their target gene PRDX6, indicating the protective roles of antioxidant enzymes against noise-triggered oxidative damage [In addition, hypoxic preconditioning conferred significant protection against broadband noise exposure via the activation of hypoxia-inducible factor 1 subunit alpha (HIF-1α) within the organ of Corti . Under ning loss . In conte damage . The inve damage , as showThe unfolded protein response (UPR) is responsible for protein quality control through the upregulated expression of ER chaperones, the inhibition of de novo protein synthesis, and the removal of misfolded proteins when overaccumulated in the ER ,139,140.XBP1 mRNA, leading to the production of an active XBP1 transcriptional factor [In mammalian cells, the UPR is induced by IRE1 , PERK 1, and ATFl factor . XBP1 enl factor .CHOP and genes coding for b-ZIP transcription factors, PERK triggers apoptosis in excessive ER-stress environments. Moreover, ATF6, as an active transcriptional factor, induces the expression of genes involved in UPR by releasing its cytoplasmic domain via proteolytic cleavage. A study of the association between hearing loss and ER stress showed that, in cells carrying mutated GJB2, the corresponding mutant protein accumulated in the ER and failed to move to the plasma membrane, causing hearing loss [GJB2 mutations are found in patients with nonsyndromic hearing loss. Additionally, in mice that suffered from hearing loss by intracochlear injection of chemicals inducing mitochondrial dysfunction, CHOP, one of the ER stress sensors, was expressed in the lateral wall of the cochlea before the occurrence of apoptosis [Because PERK can also induce genes that cause apoptosis, such as ing loss . Notablypoptosis . These f2O2 decreased [When weak ER stress was preconditioned using ER stress inducers in a renal epithelial cell line, cellular injury induced by Hecreased . Similarecreased . Furtherecreased . SimilarFinally, when ischemic preconditioning was induced, AKT phosphorylation and the levels of HIF-1α and GRP78 were increased, while the levels of PERK and ATF4 were reduced, resulting in protection from renal ischemia-reperfusion injury . MoreoveAs ROS-related tissue damage is the major pathway underlying hearing loss; several pieces of evidence could potentially prove the efficiency of antioxidant therapies against noise-, aging-, and ototoxicity-induced hearing loss. Several compounds have shown a protective or rescuing effect against hearing loss in clinical studies, namely NAC , alpha-lipoic acid, amifostine, ebselen, and coenzyme Q10. However, further well-designed clinical studies should be conducted to confirm the protective effects of these compounds.


Transient evoked otoacoustic emissions (TEOAEs) are a validated technique in newborn hearing screening that is regularly used in many countries. It reflects normal hearing or at least no more than 30 dB HL hearing loss. Breastfeeding has many advantages and some studies have demonstrated that it prevents otitis media by means of opening the Eustachian tube and clearing mucus in the middle ear which is perhaps also combined with immunological effects. A few studies have related how newborn feeding can vary the pass rate to TEOAE. The goal of this study was to investigate the relationship between newborn feeding and TEOAE newborn hearing screening results.st 48 h of life.Data were retrospectively collected from healthy vaginally delivered newborns of gestational age >37 weeks and body weight > 2.5 kg at the maternity ward. Newborn feeding history was compared with the pass rate to TEOAE performed within the 1P<0.0001).The study group included 12,866 newborns. In this group, significant differences were found based on the feeding method (breastfeeding was found to be better than formula, Breastfeeding improves newborn hearing screening results with TEOAE.Lies in the fact that breastfed children respond better to the test and need to repeat it fewer times, avoiding problems such as loss to follow-up and additional work. Universal newborn hearing screening is routinely performed because only 50% of babies born with hearing loss carry a hearing loss risk factor. Early detection leads to an efficient treatment of the affected neonates, resulting in a better final prognosis -3.There are several techniques used in newborn hearing screening. Otoacoustic emissions (OAEs) are low-level acoustic signals generated by the cochlea and passed through the middle ear into the external ear canal. OAEs are an objective indication of normal cochlear function, unlike pure-tone audiometry; OAE-based screening does not require any behavioral cooperation from the testee which makes it a very good screening method for infants. OAEs occur in nearly all ears with normal hearing and middle ear function. Transient evoked otoacoustic emission (TEOAE) testing is one of the most frequently used techniques due to its accuracy, simplicity, speed, and low cost, as described in diverse studies ,4-6.Researchers have compared the sensitivity of evoked OAE testing with pure-tone audiometry and concluded that OAE testing is more sensitive in detecting the early onset of cochlear pathologies before a change in hearing thresholds occurs .A major drawback of TEOAE testing as a screening technique for newborns relates to the middle ear status, which can severely affect its pass rate. In addition, the presence of debris and vermix in the external ear meatus of the newborn can result in false-positive screenings. This factor can lead to an overestimation of the actual hearing loss failure rate.nd day of life. For this reason, the TEOAE test is done as near as possible to discharge. The average stay in our hospital for mothers following a vaginal delivery is 48 h, and for cesarean section, it is more than 72 h, this allowed for a successful hearing screening implementation program [Another crucial factor is the newborn’s age at the moment of testing. Data strongly suggest that the prime testing window is beyond 24-48 h of life, as fluid in the middle ear and in the external meatus is normally significantly reduced on the 2Many sources, including the Joint Committee on Infant Hearing and the Commission for the Early Detection of Hearing Loss (CODEPEH) based in Spain, define well-known hearing loss risk factors -10. HoweThe objective of this study is to answer the question of whether newborn feeding can truly influence the newborn TEOAE screening results and modify the TEOAE pass rate.Significant differences in TEOAE amplitudes between groups can alter the pass rate of screening tests; therefore, the goal of this study was to compare newborn feeding history with influences on the pass rate to the TEOAE test as a method for newborn hearing screening during the first 48 h of life with the hypothesis that breastfeeding might help us obtain PASS results on the first couple of days since the Eustachian tube might take longer to open and provide middle ear aeration in bottle-fed newborns.Data were collected between 2000 and 2019 from all healthy newborns without any known hearing loss risk factor in the maternity ward. This retrospective study was approved by the ethical committee of this hospital on July 15, 2019 with code 12/2019.The focus of the study was limited to healthy newborns without any syndrome or known disease. In addition, newborns with Apgar lower than 7 at 5 min were excluded from the study. Only vaginally delivered newborns were included because timing is a crucial factor in response, and neonates delivered by cesarean section stay in the hospital 72 h, so the TEOAE test is done around this age.To eliminate other possible confounding factors , only neThe bilateral TEOAE screening was performed as close as possible to 48 h of life. Sometimes an initial TEOAE test near discharge at 48 h was done, even though the baby was a little fussy because the baby and the screener were both available at that moment. However, if the baby “failed,” it is assumed that it was because the baby was fussy and another test was done a few hours later, immediately before discharge; most of these babies passed the test. Thus, in this case, the second test was considered a more valid result and was the one included in this study. If the baby passed, no more testing was done.All nurses performed the screening, on every shift, every day of the week, based on availability. The screening was performed in the newborn room with as little background noise as possible after parental verbal consent was obtained. Testing took place after feeding time to ensure the newborn was calm and also to avoid inherent noises related to feeding. No sedation was administered.The TEOAEs were recorded with an ECHOCHECK OAE Screener based on the ILO88  system and connected to the ILO ECP neonatal probe. This emits a standard click-type non-linear stimulus of 1 ms duration. The intensity of which is 84 ± 3 dB SPL (sound pressure level) 80 times/s receives and averages the responses produced by the cochlea to OAEs from 1-4 kHz with a primary response band of 1.6-3.2 kHz., the 1.6 kHz frequency is filtered to avoid noise contamination.The device is small and portable. Its settings automatically adapt to the size of the external auditory canal. It has luminous signals that confirm that the stimulus is reaching the ear correctly and that the noise level is admissible for the test . “Pass” results indicate that there are TEOAEs. A normal result (pass) requires a signal/noise level response above 6 dB with a minimum of 512 valid responses for at least 5 s. The duration of the test usually oscillates between 45 s and a maximum of 5 min.A newborn with normal bilateral response was accepted as a pass; otherwise, it was deemed a fail .The dependent variable is the TEOAE result before discharge at 48 h of life (pass/refer).The independent variable is the newborn’s feeding type registered in maternal history (breast/formula).Following frequency analysis of the variables, a univariate analysis was completed between the TEOAE results and the study variables with the Chi-squared test and risk estimate with odds ratio.Statistical analyses were only conducted on patients that had data available for either of the study variables (feeding vs. TEOAE results).The significance level was established at p < 0.05. The data were analyzed using Excel 2016 and SPSS version 20.The percentage of breastfed babies in our study (which does not include 3% mixed feeding) was 73.16%. Although this appears not to be very high, the large study period (19 years) must be considered. Over the last years, education on breastfeeding has raised figures to around 75%-80%, which is more in line with current trends. The advantage being that the comparison group of formula-fed babies was therefore sufficiently large.Testing took place following feeding time, because adequate TEOAE response needs the baby to be calm to avoid as much noise as possible and also because it seems that babies that are bottle-fed make more noises (gulping and gasping), and therefore, results would be more difficult to obtain compared to breastfeeding babies.There are some studies that show a better pass rate to TEOAE screening in newborns fed with breast milk. In a former study, in a different group of newborns, about diverse perinatal factors influencing TEOAE results, we have informed in a preliminary way about a significant difference in response between breast and formula-fed newborns .The objective of this study is to analyze pass rate to TEOAE newborn hearing screening depending on feeding type in a selected group of healthy newborns, which were vaginally delivered, to term and at a normal weight, without hearing risk factors, and as close as possible to 48 h of life before discharge from the maternity ward to avoid some confounding factors cited in other studies -16.rd day, and it is known that TEOAE’s results are more accurate beyond the 3rd day.An age approaching 48 h was selected because it is clear that before 24 h of life, the fail rate is much higher than later . That isst OAE (up to 42 h of life) and needed repeated hearing tests [Healthy term babies in the maternity ward were selected because some studies show that immaturity, low birthweight, and therapy in the intensive care ward are important factors that disturb the OAE  and compng tests .Our results show that there are very significant differences in TEOAE results depending on the type of feeding. There was a significantly lower proportion of failing TEOAE results in the breastfed group compared with the formula-fed one  and an odds ratio (OR) of 1.43 (CI 1.25-1.65) for failing the test was calculated in the formula group.If we look at all the babies that have TEOAE done, you realize that formula-fed represents 26.8% of all TEOAE, and in this special group, one can see that there are more fails (33.8%) than pass (26.3%). The contrary occurs with breastfed babies, they represent 73.2% of all TEOAE done and the percentage of fails (66.2%) is lower than the pass (73.7%).These results agree with a higher rate of hearing loss in formula-fed infants, as shown recently by Van Kerschaver  in a stuThe rationale for this difference is probably better explained based on middle ear status since diverse studies have demonstrated that breastfeeding alone can be considered a protection factor against middle ear changes. For example, Garcia  publisheThe mechanism for these differences lies in the theory that an earlier opening of the Eustachian tube and/or a better middle ear clearance due to the position of the baby or the suction movements for breastfeeding can explain this.There has also been the suggestion that it is the method of feeding (bottle vs. breast) that creates an increased risk of otitis media (OM), regardless of whether the bottled milk is formula or expressed breastmilk. The position of the infant during feeding (supine or semi-upright) has been proposed as an explanation.Boone  showed tet al. [In addition, Tully et al.  reportedet al.  has arguIt has also been established that the mechanics of infant sucking for bottle-fed or mixed-fed babies are different from that of breast-fed babies, with fewer sucks and longer pauses observed for bottle-fed babies . Infant There are also physiological mechanisms that could explain the association between breastfeeding and reduced risk of OM. Strong negative pressure is generated by breastfeeding, in contrast to bottle-feeding. Suck, swallow, and breathing patterns are also different from bottle-fed infants ,28.All this can explain why breastfed newborns show a better response to TEOAE newborn hearing screening since they will have a better middle ear status from the beginning and, as discussed above, this is correlated with the good evidence from systematic reviews and meta-analysis showing a protective effect of breastfeeding on the risk of OM in the first 2 years of life . In addiThe Echocheck screener results do not provide actual TEOAE response amplitude values. The TEOAE test without normal results indicates a hearing loss greater than 30 dB HL. Additional studies using actual response amplitude data are needed to consider the amount of difference in response.The non-linear protocol used in the current study is the most common method used to record TEOAEs . This meThe Echocheck screener explores a frequency range from 0 to 4 kHz. Further studies are required to determine if there is any effect in some of the frequencies outside of this range, such as differences in higher frequencies that cannot be detected with this device.Given that healthy newborns were examined for this study, it remains unknown if formula feeding increases the susceptibility to other neonatal hearing loss factors. In addition, perhaps there are other unknown perinatal factors that can vary response in formula-fed newborns. More studies in this area are needed.Breastfeeding is an important factor related to a normal response in OAEs test. It may improve the final results of newborn hearing screening reducing the number of neonates who need to be rescheduled for a repeat test, as well as the associated anxiety and the possibility of losing patients during follow-up. These are major problems in neonatal hearing screening. This is another good reason to encourage newborn breastfeeding.


Pithecia, on the presence of modified nails on the second toe. These observationssuggest that a grooming claw or a grooming claw-like nail occurs indifferent Pithecia species, but that it does not consistently occur in allindividuals.The presence of a grooming claw on the second toe is acharacteristic of Strepsirrhini and tarsiers. There is also some evidencefor the presence of a grooming claw in Platyrrhini. Here we reportqualitative findings from different species of saki monkeys, genus Strepsirrhini possess grooming claws on thesecond toe, tarsiers also on the third toe. Grooming claws differ in shapefrom other nails and are specifically used for scratching and grooming thefur of the head and neck region . Theabsence of a grooming claw is regarded as a synapomorphy of Simiae(Anthropoidea), delimiting them from Strepsirrhini and tarsiers.Aotus, Pithecia and Saimiri and described thenails on their second pedal digits as wider and projecting steeper andfurther above the digital pad. Maiolino et al. (2011) also interpreted thesecond nail from individuals of the genus Aotus as grooming claws. Furthermore, theydescribed tegulae with a grooming-claw-like morphology in Callicebus but did not findevidence for a grooming claw in Pithecia. They noted that the third phalanges of the second pedal digit of several platyrrhine species weredorsally more canted; i.e., they project dorsally and at a steep anglecompared to the third phalanges of the third pedal digit.Grooming claws are a form of unguis, the keratinized structure found on thedistal phalanges of the digits in most mammals . Inprimates, nails (ungulae), claw-like nails (tegulae) and grooming claws canbe distinguished. Strepsirrhini possess ungulae, tegulae and a grooming clawon the second pedal digit .Within Haplorrhini, tarsiers possess ungulae and a grooming claw on thesecond and third pedal digit; Catarrhini only possess ungulae, whereaswithin Platyrrhini Callitrichidae also possess tegulae . The latter are oftenregarded as claws but differ from the claws of non-primate mammals by thepresence of an ungual or apical tuft . These expansions are present on ungular,tegular as well as grooming phalanges . Platyrrhiniretain several plesiomorphic primate traits, e.g., the presence of threepremolars and the fusion of the ectotympanic ring with the side of theauditory bulla . Bluntschli (1929) was the first to suggestthat Platyrrhini may also retain a grooming claw. He reported modified nailson the second toe of wild individuals of the genera Pithecia monachus , and by the contrasting conclusions ofBluntschli (1929) and Maiolino et al. (2011). We observed livingindividuals, revised museum specimens and reviewed photographic material toexamine whether – as suggested by Bluntschli (1929) – a grooming clawmight be present in the genus Pithecia, the smallest genus of Pitheciinae.Our study aims to contribute to the discussion on the presence of agrooming claw in Platyrrhini. It was stimulated by the depiction of whatlooks like a grooming claw in a naturalistic engraving ,observations on a living 22.1Pithecia by matching the collection number or the geographicorigin where available.The first author examined 26 specimens in the collections of the Museumfür Naturkunde Berlin (ZMB) and the Zoologisches ForschungsmuseumAlexander Koenig in Bonn (ZFMK) (Table S1 in the Supplement). For taxonomic identification, wecompared the name given on the label with names given by Marsh (2014) in herrevision of the genus 2.2We browsed scientific and popular books and articles that include photos ofprimates in the library of the Deutsches Primatenzentrum – Leibniz-Institutfür Primatenforschung (DPZ).2.3\opttitle{Observations on living animals }Pithecia monachus was temporarily housed at the Estación BiológicaQuebrada Blanco (EBQB), a field research site in the north-eastern PeruvianAmazonia. The animal was very tame and could be handled and observed atclose range. A juvenile female P. monachus, confiscated by the forest police and housedat the Centro de Reproducción y Conservación de Primates no Humanos(CRCP) in Iquitos, could also be examined at close range. For details seeBartecki and Heymann (1987).A juvenile male Pithecia pithecia  was observed and photographs taken on 27 January 2014 at the Cologne Zoo.A family group of three 2.4Our principal criterion was whether the nail was dorsally canted, i.e.,whether the nail formed a larger ankle with the apical pad, since a steep anklewas considered the most noticeable characteristic of grooming claws . The ankle can be estimatedboth in museum specimens and in living animals. We considered longitudinalcurvature, bilateral compression, pointedness, and projection beyond theapical pad, always in comparison to the nails on the other digits, asadditional but weaker criteria . We measured thelength and width of nails in museum specimens with a digital caliper fromthe tip of the nail to the edge of the skin covering the proximal part ofthe nail. The width-to-length ratio was calculated and used as a proxy tobilateral compression of the nail on the second digit in comparison to thenails on the third and fourth digit. All measurements are available in theSupplement (Table S2).3Pithecia showsvariation in shape and size .The nail on the second digit of the foot of various species of 3.1P. monachus the nail on the second digit was strongly dorsallycanted, protruded beyond the apical pad and was slightly narrower and morelongitudinally curved than the other nails . In the juvenilefemale P. monachus, the nail on the second digit was only slightly dorsally canted, didnot protrude and was not notably narrower or longitudinally curved.In the juvenile male P. pithecia observed at the Cologne Zoo, the nail on the second digit wasslightly dorsally canted, protruded strongly beyond the apical pad and wasslightly narrower than the other nails, but it was not notably longitudinallycurved .In all three 3.2In all museum specimens, the nail on the second digit was canted dorsally,but to a varying degree, and in several cases it was also more longitudinallycurved than the other nails. The most notable cases are depicted in Fig. 1e–g. While the nail of the second digit seemed to protrude beyond the apicalpad in many specimens, this cannot be reliably evaluated, as the soft tissueof the fingertips may have receded in the dried skin, thus giving a falseimpression.Measurements of nail length and width and the width-to-length ratio revealconsiderable variation . A visual inspection of thespecimens' individual profiles for length, width and the width-to-length ratioacross digits 2–5 reveals no pattern .3.3Pithecia inusta, the nail on the second digitprotrudes more strongly beyond the apical pad than the other nails; it isneither dorsally canted nor notably longitudinally curved . In anadult male P. pithecia, the nail on the second digit is dorsally canted and alsoprotrudes beyond the apical pad, different from the nails on the otherdigits .We found two photographs in the literature that were detailed enough toallow for an evaluation. In a juvenile female 4Pithecia. In several museum specimens andliving individuals the nail on the second pedal digit does not conform tothe description of a typical ungula, which lies flat on the apical pad andis barely bilaterally compressed . Rather, compared to a typical ungula, it is generally canted dorsally and narrower and occasionally pointed, eventhough less longitudinally curved and pointed than a typical falcula andbilaterally less compressed than a tegula. According to Soligo andMüller (1999) and Maiolino et al. (2011) the most noticeablecharacteristic of a grooming claw is the steep angle which it forms dorsallywith the apical pad. This is also the most obvious characteristic of theseveral supposed grooming claws shown in Fig. 1. Furthermore, the apical padis positioned more proximally, which is typical for a grooming claw phalanx so that the nail is protruding beyond the apicalpad. This protrusion could be the result of less abrasion compared to thenails on the other digits, particularly since the functional axis of thefoot goes through the fourth digit in platyrrhines .Our qualitative study provides support for the notion by Bluntschli (1929)of the presence of a grooming claw in Pithecia show strong similarities to the grooming claw of strepsirrhines andtarsiers. Nevertheless not all individuals exhibit the same nailmorphologies leading to the assumption that a grooming claw or a groomingclaw-like nail on the second pedal digit is not a consistent trait withinthe genus Pithecia. If more detailed and quantitative studies reveal that it wereindeed a grooming claw, the question then emerges of whether it represents anoccasional atavism or an unstable plesiomorphy of the Platyrrhini that hasalready disappeared not only from several lineages  but also from a fraction of thoselineages where it still occurs. Mapping the presence/supposed presence of agrooming claw on a primate phylogenetic tree suggests that it mightrepresent a plesiomorphy of Platyrrhini that was lost independently inseveral lineages . The assumption of a plesiomorphy predicts that agrooming claw should have been present in the last common ancestor ofPlatyrrhini and Catarrhini.In summary, the nails on the second pedal digits of individuals from thegenus Indri indri, possesses agrooming claw although it is much larger than all other primates withgrooming claws .It is noticeable that there is only evidence for grooming claws within thesmaller genera of Platyrrhini . Butbody size alone cannot be a limiting factor; the indri, In summary, the answer to the question “Do saki monkeys possess a groomingclaw?” is some perhaps do; others do not. We hope that this paperstimulates further research into this question. A more conclusive answerwould contribute to the understanding of the evolution and the loss oftraits in primate phylogeny.10.5194/pb-7-19-2020-supplementThe supplement related to this article is available online at: https://doi.org/10.5194/pb-7-19-2020-supplement.


Ear (tympanostomy) tube (TT) placement is a common ambulatory surgery in children. Despite the commonality of this treatment, the long-term effects are unknown. The objective of this study was to determine the rate of permanent hearing loss, as measured by use of a rehabilitative hearing device.n = 193,880), No-TT –recurrent visits to a physician for middle ear disease, did not undergo ear tubes , and Control – an age/sex matched group who had not undergone ear tubes and who didn’t have repeat physician visits for middle ear disease . The main outcome measures were risk and odds ratio (OR) of rehabilitative hearing devices.A retrospective comprehensive population-based cohort study was performed, evaluating all hospitals in the Canadian province of Ontario. Three cohorts of children were constructed: TT – at least one ear tube procedure , an FM system , and an implanted bone conduction device .The TT cohort had a higher risk of obtaining a hearing aid (OR 4.53 vs. No-TT, An association between ear tube placement and long-term need for a rehabilitative hearing device was found. This association warrants future prospective research in this area. Ear (tympanostomy) tube (TT) placement is a common ambulatory surgery that children undergo . PersistInsertion of TT has been shown to be associated with development of eardrum pathologic abnormalities, including segmental atrophy, perforation, and cholesteatoma . FurtherThe consistent feature of these studies is the small sample size, and as such, the risk of type-2 error is present. The current study was designed to evaluate in a large comprehensive population long-term hearing loss in TT patients of a severity that merited a rehabilitative hearing device, and to compare this population to patients with recurrent middle ear disease managed without TT, and to healthy age/sex-matched controls.Queen’s University Health Sciences & Affiliated Teaching Hospitals Research Ethics Board approved this study, project #6017300.Ontario has a population of over 14 million residents, the largest Canadian province. Canadian hospitals must report all same-day and inpatient operative procedures. The Canada Health Act governs the Canadian healthcare system, which is a publically funded and administered. This Act mandates comprehensive universal coverage for all medically necessary services, including TT and bone-conducting hearing aids. Private health insurance for these procedures is prohibited. Furthermore, all residents are eligible  for partial financial coverage of assistive hearing devices (such as hearing aids and FM systems) through the Assistive Devices Program (ADP). These surgical procedures and assistive devices are available to all persons equally. The present study defined children as persons aged 0 to 18 years. Exclusion criteria were: a valid patient identifier was not available in the dataset, if age/sex information was not available, or if they had undergone a TT procedure  in the 2 years prior to January 1, 1994. To further characterize the cohorts, the diagnosis of cleft palate/lip was sought  contains demographic information for all Ontario residents who are eligible for the Ontario Health Insurance Plan (OHIP). RPDB data is maintained by the Ontario Ministry of Health and Long Term Care. RPDB data includes: health card number, date of birth, gender, address, and deceased date (if applicable). At ICES, all personal identifying information in RPDB is removed. An anonymous unique identifier, the ICES Key Number (IKN) is generated from each health number. The IKN is used to link data sources within ICES. Diagnosis and fee-for-service claims submitted by physicians, and paid by the universal health care system, are contained within the OHIP database. The Assistive Devices Program (ADP) database contains (among many other variables) information regarding attainment of an assistive hearing device, including a hearing aid and/or FM system.TT cohort was constructed of all children who had received at least one TT surgery during the study period . The No-TT cohort had more than five visits to a physician with an Eustachian tube dysfunction diagnosis (Serous Otitis Media or Suppurative Otitis Media) within 1 year  and obtainment of an assistive hearing device  claimed in patients between the TT cohort and the No-TT cohort, and between the TT cohort and the Control cohort.p-value< 0.05.Statistical analyses were performed at ICES using SAS Enterprise Guide software, version 7.1 . Two-sided statistical significance was a Three cohorts of children were studied: TT cohort , No-TT cohort , and Control cohort  , 0.221% (No-TT cohort), and 0.072% (Control cohort). Evaluating the risks of cleft palate/lip in the subgroup of each cohort who obtained “Any Hearing Rehabilitative Device” revealed that only a minority of patients in each cohort who had a hearing device were patients with cleft palate/lip: 6.780% (TT cohort), 2.088% (No-TT cohort), and 1.292% (Control cohort).In a comprehensive dataset, this study reports the need for assistive hearing devices in ear tube patients, recurrent ear disease patients managed non-operatively, and healthy control patients. Furthermore, there is an association between TT and needing an assistive hearing device. This study is not able to comment on causation, and the authors believe causation should not be inferred.Controversy exists regarding the role of TT in pediatric patients with recurrent/persistent middle ear disease. While studies support short term resolution of fluid and improvement of hearing in children with otitis media with effusion –11, and This study demonstrated higher odds ratios of rehabilitative hearing devices in the TT cohort, compared both to the No-TT and Control cohorts. The TT comparison to the Control provides information as to the risk on hearing of the combined effect of the underlying middle ear disease and a possible role of the tubes themselves. The results obtained in this respect are expected, as recurrent/persistent middle ear disease itself is known to be a risk factor for hearing loss . InteresThis study used an operational definition of disease severity. This was the number of presentations to a physician in one year in which the child received a diagnosis of middle ear disease. Both the TT and No-TT cohorts had greater than 5 visits in the year prior to the index date. There is no agreed upon measure of severity of Eustachian tube dysfunction or middle ear disease. The authors believe that within the limitations of administrative data, this measure is the most appropriate and objective measure to match the groups.A possible explanation for the average 6 year delay (after the index date) in obtaining amplification is that children with mild/moderate hearing loss may not be identified until significant speech and language delays are recognized in school. In Ontario, it is common for children with permanent hearing impairment to be identified to their school boards upon entry to school (age 4–5). Permanent hearing loss may not be identified until academic performance issues arise or behavioral issues are noted. More thorough audiological testing is requested when these issues are identified, and it is at this time that the diagnosis of permanent hearing loss is made, and personal devices (hearing aids and FM systems) are obtained. This delayed identification of permanent hearing loss could account for the 6 year gap in the studied groups. It is furthermore important to note that the differences between the groups in time to first hearing aid/FM system or time to first implanted hearing device are not statistically significant. This prevents further conclusions from being drawn regarding this data.The strengths of this study include a large comprehensive population-based design, which includes all patients and all surgeons that perform TT in Ontario. Additionally, the ability to use two control groups and have long-term follow-up greatly enhanced the design. The size of the odds ratios between the groups are notable.Administrative data has limitations. This study design does not permit an assessment of causation, just an association. Factors that could influence the outcomes such as the indications for surgery, indications for choosing conservative management with serial examinations, secondary smoke exposure, daycare attendance, use of pacifiers, developmental delay, family history of otitis media, audiometric testing results, OHIP diagnostic code data quality, and findings at surgery are not available. Data quality is dependent on accurate coding by the physician and hospital coders. A comparison of administrative data with hospital chart data concluded that major events  are accurately coded . The diaThis study revealed the actual prevalence of hearing assistive device usage in children in Ontario . While the overall risks are low, recurrent/persistent ear disease and ear tube placement are associated with higher risks. This association requires further evaluation in prospectively-designed studies.


Hearing loss among older adults is prevalent and associated with dementia and health care utilization. However, cross-sectional data suggest less than 20% of adults with hearing loss use hearing aids. There is a paucity of studies examining trends in hearing aid ownership over time. This study analyzed data from the 2011, 2015, and 2018 cycles of the National Health Aging and Trends Study (NHATS), a nationally-representative longitudinal study of Medicare Beneficiaries. Participants were asked “in the last month, [have you/has [he/she]] use a hearing aid or other hearing device?” (“yes” or “no”). Among a weighted sample of Medicare Beneficiaries 70 years and older , the overall proportion who own and use hearing aids rose from 14.96% in 2011 to 16.90% in 2015 to 18.45% in 2018. As age increased so did the proportion of older adults who used hearing aids. A smaller proportion of Black Americans used hearing aids across time and experienced a smaller overall increase in the proportion in hearing aid ownership over the 8-year period compared to White Americans (+0.78% vs. +4.30%). Black women had the lowest rates of hearing aids use across the 8-year period. Notably, older adults at less than 100% of the federal poverty level experienced an overall decrease in proportion of hearing aid ownership and use. This study lays the groundwork to examine the impact of the Over-the-Counter Hearing Aid Act of 2017 across subpopulations when it takes effect in 2021.