Objective: The aim of this study was to investigate the relation between visual and vestibular hypersensitivity, and Depersonalization/Derealization symptoms in patients with chronic dizziness.Materials and Methods: 319 adult patients with chronic dizziness for more than 3 months  were included in this prospective cross-sectional study. Patients underwent a complete audio-vestibular workup and 3 auto questionnaires: Hospital Anxiety and Depression (HAD), Depersonalization/Derealization Inventory (DDI), and an in-house questionnaire  assessing 9 activities with a score ranging from 0 (no difficulty) to 10  and 11 (avoidance) to detect patients with visual and vestibular hypersensitivity .Results: DDI scores were higher in case of VVH , migraine , and motion sickness . Women scored DDI higher than men . DDI scores were also related to depression and anxiety. DDI score was also higher during spells than during the basal state.Conclusion: During chronic dizziness, Depersonalization/Derealization symptoms seem to be related to anxiety and depression. Moreover, they were prominent in women, in those with visual and vestibular hypersensitivity, migraine, and motion sickness. Chronic dizziness in patients with a unilateral stable vestibular weakness or even normal inner ear function and no neurological abnormality is a diagnostic and therapeutic challenge. In an attempt to define at least a part of this population, a syndrome designated as phobic postural vertigo was proposed to define those with a hypersensitivity to visual stimuli and movements . Other mChronic dizziness is typically associated with anxiety and depression, but whether balance disorders are a consequence, or a contributing factor to these disorders remains unclear , 6. PsycDepersonalization/Derealization (DD) symptoms as evaluated by DRD inventory (DDI) , are preIdentifying subgroups of patients with chronic vertigo who are present DD symptoms will potentially lead to a better understanding of the phenomenon and targeted therapeutic actions. To our knowledge, the relation between visual and vestibular hypersensitivity (VVH), and DD symptoms has not been studied. We hypothesized that patients with chronic dizziness and VVH had higher anxiety, depression, and DDI scores.The principal objective of this study was to investigate the relation between VVH and DD symptoms. In addition, we also analyzed the relation between several clinical parameters  and the extent of VVH and DD symptoms.This cross-sectional study included 319 consecutive patients with a spontaneous dizziness. The group was selected from a population of 500 consecutive patients examined for balance disorders in one tertiary referral center during 5 months. The study was conducted during a routine follow-up and data acquisition and analyses were not blinded. The inclusion criteria were: patients complaining of spontaneous dizziness according to International Classification of Vestibular Disorders I (ICVD-I V 1.0)  lasting The possible triggering disease was classified into the following categories:n = 58 (18%)Recent benign paroxysmal positional vertigo Cured BPPV (>12 months before inclusion): n = 36 (11%)Stress defined by anxiety or traumatic stress associated to spontaneous dizziness without abnormality of clinical and instrumental vestibular examination : n = 36 n = 33 (10%)Probable Ménière's disease according to the Ménière's disease diagnostic criteria : n = 33 n = 30 (9%)Vestibular migraine according to Barany Society criteria : n = 30 n = 11 (3%)Otolithic dysfunction defined as vertigo or postural unsteadiness, normal canal function, and abnormality of sacculocolic or utriculoocular myogenic evoked potentials : n = 11 n = 8 (3%)Unilateral vestibular loss defined by a canal paresis on bicaloric test (>30% asymmetry of the sum of 2 the stimulations measured by the slow-phase velocity of the nystagmus on videonystagmography) and video Head Impulse test : n = 11 (3%)Central disorders were defined as vertigo, dizziness, or unsteadiness associated to abnormal ocular pursuit control and/or gaze nystagmus and/or dysmetric saccades, and/or absent ocular fixation, and/or abnormalities of central vestibular pathways on MRI : n = 11 n = 8 (2%)Age-related dizziness defined by age >75 years-old and spontaneous dizziness, and no evident deficit of canal or otolith function, and no identifiable neurologic abnormality: n = 3 (1%)Vestibular paroxysmia was diagnosed according to Strupp et al. . In thisn = 4 (1%)Perilymphatic fistula diagnosed according to Portmann et al. . In thisn = 2 (1%).Drug-related group defined by patients with orthostatic dizziness, no clinical or instrumental signs of vestibular deficit and anti-hypertensive medication : n = 2 (n = 30 (10%).Undetermined triggering event: In addition to this routine workup, patients responded to 3 self-assessment questionnaires: Dizziness in Daily Activity (DDA), Hospital Anxiety Depression Scale (HAD) , and DDIIn the DDA questionnaire, the patients were asked if they were dizzy in the 9 following situations: (1) Rapid head movements when dish-washing; (2) Sport and house-keeping; (3) Looking both ways before crossing the street; (4) Moving visual scene ; (5) Climbing or coming down ; (6) Moving images on screens; (7) Undergoing acceleration and break ; (8) Bending forward ; (9) Open spaces . For each item, the patient indicated whether he/she was concerned by the activity and scored the dizziness from 0 (none) to 10 . A score of 11 was assigned to the activities which were avoided due to unbearable discomfort. A global score was calculated as the sum of the scores ranging from 0 to 99.n = 7) and depression (n = 7). Each item was scored from 0 to 3. A score was calculated for anxiety (aHAD) and depression (dHAD) separately ranging from 0 to 21. Characterized anxiety and depression were defined as a score > 8/21 for each subgroup of questions . Values were expressed as mean ± SD. Continuous variables were analyzed by a paired or an unpaired n = 210 vs. 15.2± 14.84, n = 102, p < 0.0001, unpaired t-test). The rate of avoidance was also higher in women . Age and etiology categories were similar in these subgroups and did not appear as confounding factors (data not shown).In total, 312 patients responded to the DDA questionnaire . All then = 111 vs. 19.1 ± 17.23, n = 201 patients without migraine, p < 0.001, unpaired t-test). The effect of migraine on the score appeared to be separate from the effect of sex .DDA score was also higher in migraineurs . This effect appeared to be independent from the effect of sex , and there was no difference of DDA scores between younger (<60 years) and senior patients . Similarly, etiology categories did not seem to influence DDA assessment (data not shown).Age did not seem to influence the DDA score. Indeed, there was no correlation between age and DDA score . The group scoring 0 on DDA did not include any case of motion sickness while this symptom was noted in a significantly higher proportion of individuals among those with a DDA score above 0 .Forty-one patients (13%) declared no discomfort for all proposed activities . This group was composed of 20 males and 21 females with a mean age of 61 ± 16.1 years. This group was more masculine in comparison to the group scoring > 0 on DDA .This score was higher in women  and in young patients . Etiology also seemed to influence the anxiety scores. Higher scores were recorded in stress-related dizziness and otolithic syndrome (Anxiety (aHAD) score in our population with chronic dizziness was 8.6 ± 4.54 . Similarly, those with acquired motion sickness had higher aHAD ratings .Patients with a history of migraine reported higher anxiety levels than others . There was no difference between men and women . In the same manner as aHAD, scores in young patients tended to be higher than those in senior patients . dHAD did not seem to be influenced by a history of migraine or acquired motion sickness (data not shown).Depression (dHAD) score in the population was 5.0 ± 3.94 (Patients with characterized anxiety or depression (scores > 8) had higher DDA scores than those with scores below 8 , suggestn = 318). Women scored higher than men . Also, patients below 60 years had higher scores than seniors . As for the HAD questionnaire, etiology also affected DDI score independently from the sex with higher levels in central disorders and in women . Patients with acquired motion sickness had also higher DDI scores than those who did not suffer from it .Patients with a personal history of migraine also provided higher DDI ratings in comparison to those without a migraine history .Patients with characterized anxiety and depression (aHAD and Dhad > 8) had higher DDI scores .The analysis of the parameters which influenced DDA score in our population by a multiple regression model showed that the combination of dHAD, aHAD, DDI, and age as independent factors were correlated to DDA . This significant increase concerned 25 items out of 28 . Younger patients (<60 years) had higher DDI score shifts during the spells than the seniors . But the sex did not seem to influence the amplitude of the shift . History of migraine and acquired motion sickness did not influence the amplitude of the shift . Etiology did not seem to alter the DDI score shift during spells (When asked to score their perception during past vertigo spells by the DDI, patients estimated their DD symptoms higher than the one at the basal state (9.6 ± 6.67, ut of 28 . The meag spells .In this study, we showed that patients with chronic balance disorder complaining from vestibular and visual discomfort in daily life activities were frequently young women with a history of migraine or acquired motion sickness. The extent of the discomfort was related not only to depression and anxiety levels, but also to the depersonalization/derealization sensations. The intensity of these latter increased during vertigo spells suggesting the impact of vertigo on the perception of self, environment, and time.The relation between anxiety, depression, and vertigo has been already reported in several studies . This asAn extensive literature has suggested the sex difference in the integration of visual inputs into the balance function and motion sickness , 29. WomThe same female preponderance has been observed for the incidence of migraine . In thisCurrent questionnaires evaluating vertigo such as Dizziness Handicap Inventory do not assess VVH . SituatiConsequently, to the aim of investigating the relation between VVH and DD symptoms, we designed an in-house questionnaire especially targeting situations in which VVH is incapacitating and assessed the handicap by a Likert scale. The validity of the Likert method as a psychometric tool has been demonstrated in many domains specially in chronic vertigo  The addiThe relation between self-awareness and vestibular function was investigated as early as the beginning of twentieth century . Since iThe relation between DD symptoms and vestibular disorders has been previously reported by Sang et al. . These aIn conclusion, chronic dizziness can entail not only anxiety and depression but also sensations of depersonalization, and derealization independently from the etiology. The observation that DDI scores increase during vertigo spells suggests that balance disorders enhance depersonalization and derealization. This possible causality can be explained by the disturbances of our internal body scheme and the environment representation during vestibular disorders, and the uncertainty on the validity of sensory inputs that they generate. DD complaints were more frequent and intense in young female patients and in those suffering from migraine and motion sickness. These patients also reported incapacitating symptoms related to visual and vestibular hypersensitivity.The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.MT and AB analyzed data and prepared the manuscript. CV, CH, and UD participated in the study design. MT, AC, and SH included and examined the patients.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.


The development of biocompatible composite materials is in high demand in many fields such as biomedicine, bioengineering, and biotechnology. In this study, two series of poly  and poly (ε-caprolactone)-based films filled with neat and modified with poly (glutamic acid) (PGlu) nanocrystalline cellulose (NCC) were prepared. An analysis of scanning electron and atomic force microscopies’ results shows that the modification of NCC with poly (glutamic acid) favored the better distribution of the nanofiller in the polymer matrix. Investigating the ability of the developed materials to attract and retain calcium ions led to the conclusion that composites containing NCC modified with PGlu induced better mineralization from model solutions than composites containing neat NCC. Moreover, compared to unmodified NCC, functionalization with PGlu improved the mechanical properties of composite films. The subcutaneous implantation of these composite materials into the backs of rats and the further histological investigation of neighboring tissues revealed the better biocompatibility of polyester materials filled with NCC–PGlu. Currently, aliphatic polyesters are one of the most widely used biodegradable polymers. They have found a wide range of applications in fields such as food industry, surgery, drug delivery, and regenerative medicine . One of Biological inertness and a lack of reactive functionality are among the poor properties of aliphatic polyesters. As a result, more and more research is devoted to their preparation for advanced materials for medical and regenerative applications to overcome the drawbacks arising from potential inflammatory side effects, including their high hydrophobicity, and low cell adhesion ,10,11. TTo reinforce the mechanical properties of polyester materials, different kinds of mineral nanoparticles are typically applied as fillers ,13,16,17It has been demonstrated recently that the modification of PLA/poly(lactic acid-co-glycolic acid) (PLGA) nanofibers with glutamic acid peptide induces effective calcium phosphate nucleation and the osteogenic differentiation of marrow stromal cells . The abiwM = 2100 (weight average molecular weight), nM = 2000 (number average molecular weight), Ɖ = 1.05 (dispersity), and the amount of residual benzyl groups was 22% (according to NMR) /[Sn(Oct)2] = 2). Synthesis was conducted in vacuum-processed Schlenk tubes over 7 h for PDLLA and 20 h for PCL. After synthesis, polymers were dissolved in chloroform, precipitated in cold methanol, filtered and washed with a fresh portion of methanol, and dried in vacuum. The molecular weights (wM) and dispersity (Ɖ) were determined by size exclusion chromatography (SEC) with the use of a Shimadzu HPLC system  consisting of a pump LC-10AD VP, system controller SCL-10A VP, and refractometric detector RID-10A  supplied with a Rheodyne 725i injection valve  and two columns of Agilent PLgel MIXED-D  . Analyses were performed at 40 °C and at a flow rate of 1.0 mL/min with the use of THF as an eluent and PS standards for calibration. Data acquisition and processing was performed using LC solution software . Viscosities of sample solutions (in CHCl3) with different concentrations were measured using Ostwald’s capillary viscosimeter. The synthesis of PDLLA and PCL was carried out in bulk at 130 °C via the ring-opening polymerization of D,L-lactide and ε-caprolactone, respectively. The composition of polymerization mixture and polymerization time was optimized to obtain polymer samples with close intrinsic viscosity. The monomer-to-Sn (Oct)4 was added to the suspension containing 1 g of NCC. The reaction was carried out at 4 °С for 24 h. The oxidized NCC was purified by dialysis against water with the use of the membrane bag with MWCO (molecular weight cut off.) 1000. The product was stored in suspension at 4 °C. Prior to use, the product was concentrated on a rotary evaporator. The concentrated suspension was added to an excessive amount of buffer solution of choice, so that the pH was not changed in the tenths place value. Before modification, the NCC suspension (8 mass%) was diluted 10 times with water and cooled to 4 °C. Then, the aqueous solution containing 300 mg of NaIOThen, 250 mg of PGlu in 10 mL of water was added to 100 mL of suspension containing 1.0 g of oxidized NCC in 0.01 M sodium borate buffer (pH 8.5). The reaction was carried out for 3 h at 22 °C under stirring. To reduce imine bonds and free aldehyde groups, 200 mg of sodium borohydride was added to reaction medium that was left for 1 h. Cellophane, moistened with water, was fixed on the glass cylinder (i.d. = 75 mm) and then dried for 24 h. A 5% polymer solution in chloroform (6.5 mL) was poured onto cellophane inside the glass cylinder and left for 12 h in air for chloroform evaporation. After that, the cellophane was removed, and the obtained polymer films were dried in air thermostat at 50 °C until constant weight was reached . To prepare composite films, original or modified NCC was dispersed in polymer solution in chloroform under ultrasonication. The amount of NCC was equal to 5%, 10%, and 15% regarding the weight of polymer in solution. Other manipulations were done as described above for preparations of pure polymer films.Mechanical characteristics of the films were determined under uniaxial extension using band-like specimens of 2 mm × 20 mm using the AG-100kNX Plus Shimadzu universal mechanical system . The extension speed was 10 mm/min. Surface morphologies and topographies were analyzed by scanning electron (SEM) and atomic force microscopy (AFM) using Zeiss AURIGA Laser  and INTEGRA-Aura Spectrum Instruments , respectively. Before the registration of SEM images, the surfaces of film specimens were additionally deposited with conductive carbon layers. The AFM images were analyzed with the WSxM software  . Polariz2, 37 °C). The round-shaped films with a diameter of 6 mm were stuck into 96-well plates and MSC (5 × 103 cells per well) were seeded. After 24 hours of incubation, the non-attached dead cells were washed out, and the amount of attached cells was evaluated using an MTT test. The data from cell culture experiments are presented as average ± SD (n = 4). Cell adhesion on the surfaces of the obtained films was evaluated using bone marrow mesenchymal stem cells (MSC). Mesenchymal bone marrow stromal cells were isolated from newborn rabbit bones. The rabbits were purchased from the Federal State Unitary Company “Nursery Rappolovo”. The procedure of obtaining MSC from rabbits was approved in an Institute of Phthysiopulmonology Ethical Committee session and protocoled (Statement #46 from 18.04.2018). MSCs were cultivated in αMEM medium  supplemented with 10% fetal bovine serum (FBS) , 100 U/mL penicillin , and 100 μg/mL streptomycin . In experiments, the cells of 2–6 passages were used. Cell cultivation was performed in a thermostatic incubator (5% CO2+ and PO43− ions at concentrations known for body fluids . . 34]. The optimal modification route includes three steps. First, NCC oxidation is carried out to generate highly reactive aldehydes. Second, the reaction between aldehydes and the terminal amino group of PGlu with the formation of imine (Schiff’s base) bonds is performed. Finally, the imine bonds and the unreacted aldehyde groups are reduced with sodium borohydride. The modification efficiency was evaluated  to be 22As expected, the dispersion of the neat NCC in the polymer solution prepared in chloroform was followed by an aggregation of hydrophilic filler. Extensive suspension ultrasonication before film casting was required in order to obtain a more or less homogeneous dispersion of crystals in the polyester matrix. In spite of this, aggregates were still present in the final films due to the poor compatibility of the hydrophobic matrix polymer and hydrophilic NCC. In the case of NCC modified with PGlu, aggregation was much less pronounced, and NCC distribution in the polymer matrix was much more even. The formation of pores is provoked by crystallization of the polymer during the volatile organic solvent evaporation. The comparison of images registered for films manufactured from pure polymer and polymer containing 15 mass% of non-modified NCC  shows coThe casting of composite films from polymer solution containing the same amount of modified NCC provided much better surface uniformity comparable with polymer films without filler. Additionally, the obtained pure polyester and composite materials were investigated by optical transmitted light and reflected microscopy . For botSurface topography of the prepared materials was evaluated by AFM . For the(E), tensile strengths (bσ), and elongations at break (bε) were determined for pure PDLLA and PCL films as well as their composites with NCC and NCC–PGlu . All PCL-based implants demonstrated low values very close to the average number of lymphocytes. The same was observed for PDLLA films filled with NCC–PGlu. In turn, pure PDLLA and PDLLA filled with unmodified NCC demonstrated the increased number of lymphocytes (p < 0.01). As to neutrophils, the lowest average numbers were detected for PCL and PDLLA composites filled with modified NCC (p < 0.05). Some amounts of giant multinucleated cells were also detected in all specimens. However, their level in pure PDLLA and all PCL-based materials was much lower than in PDLLA-based composites. The highest number of giant multinucleated cells was detected in PDLLA filled with 10% and 15% NCC (p < 0.01). However, there were no statistical differences between the PDLLA and PDLLA–NCC–PGlu (p = 0.2).In general, it can be concluded that pure PCL as well as its composites demonstrated lower inflammatory response than PDLLA-based materials. The higher inflammation effect detected for PDDLA-based materials can be a result of their faster degradation and stronger acidification of the environment in comparison with PCL matrices. A comparison of the average numbers of lymphocytes, neutrophils, and giant multinucleated cells for PDLLA composites allows the conclusion that films containing neat NCC induced a higher inflammation response regarding PDLLA filled with NCC–PGlu. 2+ ions. The in vitro and in vivo studies indicate the high biocompatibility and suitability of the composites for biomedical applications. It is important to note that none of the composite materials caused any necrotic processes in the surrounding tissues, and did not demonstrate an elevated expression of inflammatory cells in comparison with pure aliphatic polyester. Moreover, the composites based on aliphatic polyester and NCC–PGlu showed lower inflammatory response than pure aliphatic polyester or composites filled with neat NCC. We have developed polyester-based materials filled with NCC–PGlu, and demonstrated their improved mechanical characteristics compared to neat NCC as a filler. These data are explained by the better distribution of modified NCC in the hydrophobic aliphatic polyester matrix and interactions between modified crystals and the polymer matrix. This explanation is also supported by the surface morphology and topography results. Moreover, the manufactured composites of PE and NCC–PGlu demonstrated better uptake and retention of Ca


Qinj) capacity and long-term stability. Boron-doped diamond (BDD) electrodes have shown promise for neural stimulation applications, but their practical applications remain limited due to the poor charge transfer capability of diamond. In this work, we present an attractive approach to produce BDD electrodes with exceptionally high surface area using porous titanium nitride (TiN) as interlayer template. The TiN deposition parameters were systematically varied to fabricate a range of porous electrodes, which were subsequently coated by a BDD thin-film. The electrodes were investigated by surface analysis methods and electrochemical techniques before and after BDD deposition. Cyclic voltammetry (CV) measurements showed a wide potential window in saline solution (between −1.3 and 1.2 V vs. Ag/AgCl). Electrodes with the highest thickness and porosity exhibited the lowest impedance magnitude and a charge storage capacity (CSC) of 253 mC/cm2, which largely exceeds the values previously reported for porous BDD electrodes. Electrodes with relatively thinner and less porous coatings displayed the highest pulsing capacitances (Cpulse), which would be more favorable for stimulation applications. Although BDD/TiN electrodes displayed a higher impedance magnitude and a lower Cpulse as compared to the bare TiN electrodes, the wider potential window likely allows for higher Qinj without reaching unsafe potentials. The remarkable reduction in the impedance and improvement in the charge transfer capacity, together with the known properties of BDD films, makes this type of coating as an ideal candidate for development of reliable devices for chronic neural interfacing.Robust devices for chronic neural stimulation demand electrode materials which exhibit high charge injection ( Porous TiN coatings have long been employed for pacemaker electrodes and have also been used for fabrication of neural stimulation and recording electrodes , voltage transient (VT) measurements, and electrochemical impedance spectroscopy (EIS).6Al4V electrode pin, which belongs to a system intended for genital nerve stimulation , the N2 flow was varied from 30 to 300 standard cubic centimeters per min (sccm), while the deposition time was kept constant at 27.5 × 103 s. In another set of samples , the flow rate of N2 was kept at 180 sccm while the deposition time was modified. In both cases, the Ar flow was kept constant at 180 sccm.The test samples were fabricated using a monopolar Ti4 was added to the chamber at a total flow rate of 500 sccm. Tri-methyl boron was added to the gas as the dopant source, at boron to carbon concentrations of 10,000 ppm. The substrate temperature was maintained at ~750°C by using a pressure of 25 Torr (3.33 kPa) and a microwave power of 2,500 W.BDD thin films were synthesized on the TiN layers using an Astex AX6500 microwave plasma enhanced CVD system. The TiN-coated electrodes were first immersed in a 0.33 g/L solution of diamond nanoparticles (3.8 ± 0.7 nm) from Shinshu University to seed the surface for diamond growth. Hydrogen gas with an addition of 1% CHThe TiN thin-films were investigated using scanning electron microscopy (SEM) . Detailed images of all electrodes were recorded at 80,000× magnification. For the assessment of film thickness, flat substrates (10 × 10 mm) obtained from silicon wafers were coated during deposition of each batch of electrodes. The silicon substrates were placed in a manner that ensured an even coating thickness. The coated substrates were subsequently broken and analyzed by cross sectional SEM. The thickness was measured via analysis of the SEM micrographs using Image J . Each sample was measured at several locations along the cleavage to assess for thickness variations. Only small variations were observed and a unique thickness could unambiguously be assigned to each sample. The surface morphology of the BDD/TiN films was analyzed by a FERA3 GM SEM  with Schottky field emission cathode (FEG-SEM). Images were taken in the high-resolution mode at the accelerating voltage of 5 kV to minimize the interaction volume.3 Raman peak position.Raman spectroscopy of BDD/TiN films was carried out at room temperature using an InVia Raman Microscope  with the following conditions: wavelength = 325 nm, ×40 Olympus objective, 65 μm slits, spot focus, grating = 2,400 L/mm. A high pressure, high temperature Ib single crystal diamond was used as a reference for the sp2) was investigated by an optical profilometer . In addition, surface roughness and topography over a small area (5 × 5 μm2) were investigated by atomic force microscopy (AFM) using a Dimension Icon ambient AFM  in peak force tapping mode using Tap150AL-g tips .Topography and surface roughness over a large area (220 × 280 μm2), a platinum foil counter electrode (50 cm2), and a Ag|AgCl reference electrode (1.6 cm2). Measurements were performed in Ringer's solution at room temperature.All electrochemical measurements were carried out in a three-electrode set-up, using the either the TiN or the BDD/TiN electrodes as working electrodes  ratio was calculated by dividing the CSC of the porous coatings by the CSC of the corresponding smooth coating at a sweep rate of 0.05 V/s.Cpulse) was calculated for each pulse using the following equation:Voltage transient measurements were made using a cathodic-first bipolar symmetric current pulse with an interphase, during which no current was applied. Each phase had a phase width of 200 μs and the duration of the inter-phase was 40 μs. For analysis of the VTs, the OCP was set to 0 V and the IR drop was subtracted. The IR-drop was calculated for each phase by subtracting the potential at 20 μs after pulse cessation from the last data point of the respective phase. The pulsing capacitance .Cyclic voltammetry and VT measurements were performed with VersaSTAT 3 potentio-galvanostat . The impedance spectrum was measured from 0.1 Hz to 100 kHz, five points/decade using a sinusoidal measurement current of 5.0 μA. Impedance spectroscopy was performed using Solartron, Model 1294 in conjunction with 1260 Impedance/gain-phase Analyzer . Linear regression analyses of the CSC and 2 while keeping the deposition time at 27.5 × 103 s. The partial pressure of N2 in the deposition chamber was modified by varying the N2 flow rate. At the lowest flow rate, the TiN films displayed a relatively smooth surface . The column size .Diamond thin-films were synthesized on all types of TiN coating (I–VII). SEM images Figure  along wi2, was governed by grooves on the underlying TiAlV substrate, which are ~20 μm wide and up to 400 nm high. The roughness on this scale was around 200–350 nm and it was not significantly influenced by the TiN or the BDD coating. However, the small-scale topography measured by AFM over an area of 5 × 5 μm2 was mostly governed by the topography of the TiN pyramidal structure. The roughness of the diamond layer, which measured on a flat surface was around 30 nm, had only a minimal influence on the topography of the BDD/TiN films. Figure The large-scale topography of the diamond films, as measured using an optical profilometer over an area of 220 × 280 μm−1 as well as broad features related to sp2 at 1,360 and 1,585 cm−1, i.e., the D and G bands.Raman spectroscopy confirmed the synthesis of diamond films in all TiN substrates. Figure 2, respectively. Table 2.μm for the bare TiN films (r2 = 0.98) and 14.2 mC/cm2.μm for the BDD/TiN films (r2 = 0.99), but the slopes are not significantly different (P > 0.05).The water window potentials were obtained by CV and their values were typically −0.6 to 0.9 V for TiN and −1.3 to 1.2 V for BDD/TiN electrodes (vs. Ag|AgCl). Table Cpulse for all electrode types, which was derived from the VT measurements. Except for electrode type I, the Cpulse of the BDD/TiN electrodes was consistently lower than the Cpulse of the corresponding TiN electrodes. Figure Cpulse of BDD/TiN electrodes leads to larger electrode potentials. However, as the safe potential window for BDD is larger than for TiN, the amount of charge that can safely be injected will be higher for BDD/TiN than for TiN. Cpulse displays a negative trend for both TiN and BDD/TiN electrodes as a function of TiN coating thickness  and −0.052 mF/cm2.μm for the BDD/TiN films (r2 = 0.77).Table s Figure . The CpuAs anticipated, increased film porosity significantly reduced the impedance of the electrodes Figure . The imp2 to ensure a stoichiometric Ti/N ratio below 0.6, which has been shown to be unfavorable for columnar growth  led to higher thickness and higher porosity, which was reflected as an increased ESA/GSA ratio. Further increase in N2 (sample IV) still gave an increase in area due to increased porosity, although the thickness was smaller. Going to higher N2 the growth rate was slower, so that the lower thickness dominated over the increased porosity and an overall decrease in the ESA/GSA was obtained. For the samples grown at constant gas flow rate , film thickness, and ESA/GSA ratio were directly correlated.A range of porous TiN films, to be used as templates, was deposited onto test electrodes by means of a physical vapor deposition. The smooth TiN reference films were fabricated using a low partial pressure of N Ohring, . For the3/sp2 changes with measurement position. This apparent variation in sp3/sp2 is related to the fact that the BDD coating is very thin and therefore the grain boundary content is high.Surface analysis of the BDD/TiN samples by SEM and AFM revealed homogeneous and high quality BDD films. The overall structure and topography of the films appeared similar to the bare TiN samples, suggesting that CVD deposition did not significantly affect the morphology of the TiN template. The uniform coverage of diamond crystallites indicates a highly cohesive diamond film. This is in agreement with previous studies, which have shown that TiN possesses several favorable properties for nucleation and growth of good quality CVD diamond films, including low diffusivity of carbon, compatible interatomic potential, and small lattice mismatch . This means that the amount of charge that can be injected without reaching unsafe potentials is doubled by applying a BDD thin-film onto a porous TiN coating. It is important to view these results in the light of the intended application of the BDD/TiN electrodes, which is in vivo chronic neurostimulation. It has been shown that the stimulation performance of TiN electrodes deteriorates after implantation , the production parameters are of critical importance, as the extra coating increases the pore resistance, which may deteriorate Qinj. This study suggests that specific deposition parameters are optimal for stimulation electrodes, as increased thickness and N2 flow only result to a certain extent in larger Cpulse and Qinj. The data suggests that BDD deposited onto thinner coatings with smaller columnar size results in better stimulation performance. A thicker coating, however, results in a high CSC and low impedance, which could be exploited for other purposes, such as electrical and electrochemical sensing. This highlights the versatility of the novel coating combination presented in this work.To achieve an increased charge injection (Cpulse would make them more suited for sensing applications. On the other hand, relatively higher Cpulse were obtained with thinner films with small column size, which would result more favorable for stimulation applications. Although BDD/TiN electrodes displayed a higher impedance magnitude and a lower Cpulse as compared to the bare TiN electrodes, the wider potential window likely allows for higher Qinj without reaching unsafe potentials. These remarkable improvements, together with the known mechanical stability, resistance to biofouling and long-term in vivo stability of BDD films, makes this coating combination an ideal candidate for development of reliable devices for chronic neural interfacing. This novel type of coating is particularly attractive for the development of commercially viable electrodes due to the simplicity and the scalability of the approach.In this work, we have fabricated a range of BDD/porous TiN electrodes with very high surface area, which exhibit a broad safe potential window and CSC-values which are superior to those reported in the literature for porous BDD electrodes. Electrodes with more porous and thick coatings were associated with higher CSC and lower impedance magnitudes, but the relatively limited AT, MN, and CP conceived and designed the study. SM, MM, SS, and KR prepared the samples. MM, SS, KR, LF, LK, and AT obtained the data and performed the surface analysis of the films. SM and VP acquired and analyzed the electrochemistry data under supervision of NR. SM and CP drafted the manuscript and designed the figures. All authors contributed to the critical revision of the draft and approved the submitted version.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.


An unperturbed Baseline phase was followed by a gradual Ramp to a 30% upward F1 shift stimulating a compensatory response, a Hold phase where the perturbation was repeatedly presented with alternating blocks of masking trials to probe adaptation, and an End phase with masking trials only to measure persistence of any adaptation. AOS participants showed normal compensation to unexpected F0 perturbations, indicating that auditory feedback control of low-level, non-segmental parameters is intact. Furthermore, individuals with AOS displayed an adaptive response to sustained F1 perturbations, but age-matched controls and APH participants did not. These findings suggest that older healthy adults may have less plastic motor programs that resist modification based on sensory feedback, whereas individuals with AOS have less well-established and more malleable motor programs due to damage from stroke.Two auditory perturbation experiments were used to investigate the integrity of neural circuits responsible for speech sensorimotor adaptation in acquired apraxia of speech (AOS). This has implications for understanding the nature of AOS as well as normal speech motor control. Two experiments were conducted. In Experiment 1, compensatory responses to unpredictable fundamental frequency (F0) perturbations during vocalization were investigated in healthy older adults and adults with acquired AOS plus aphasia. F0 perturbation involved upward and downward 100-cent shifts versus no shift, in equal proportion, during 2 s vocalizations of the vowel /a/. In Experiment 2, adaptive responses to sustained first formant (F1) perturbations during speech were investigated in healthy older adults, adults with AOS and adults with aphasia only (APH). The F1 protocol involved production of the vowel /ε/ in four consonant-vowel words of Australian English ( The model proposes that left vPMC is crucial for the readout of finely tuned speech motor programs for frequently produced speech sequences such as syllables from the native language; these motor programs constitute the feedforward component of speech motor commands. Damage to left vPMC impairs the readout of these motor programs, resulting in the primary symptoms that characterize AOS  is a disorder of speech motor control subsequent to damage in the left inferior frontal cortex, particularly the ventral premotor cortex (vPMC), in adults who previously had normal speech production . Accordisee also .Although some past studies suggest that auditory feedback control mechanisms may be intact in AOS, the situation is less clear than for feedforward control mechanisms that are widely believed to be impaired in AOS. Past studies have relied primarily on masking noise to investigate auditory feedback control mechanisms in AOS, and these studies have produced mixed results. compensations. Similarly, perturbing auditory parameters results in compensatory movements generated by the auditory feedback control system  responses are seen  of specific vowels in the acoustic signal. F1 maps tongue height in the oral cavity such that an upward perturbation  during production of the vowel /ε/, for example, is interpreted as a drop in tongue height . This leads the speaker to oppose the perturbation by elevating the tongue and shifting production toward /i/, with the response typically being 10–50% of the magnitude of the original perturbation e.g., . PresentWhile studies of compensation and adaptation to perturbations are well established in healthy young speakers, the impact of neurological damage to the cortical speech network on feedback and feedforward control processes is not well understood. Furthermore, very few auditory perturbation studies have been performed on healthy adults over 50 years of age  and an F1 sustained perturbation study (Experiment 2) aimed at testing the DIVA model hypotheses regarding feedforward and feedback control mechanisms in AOS laid out above. Healthy older adults and adults with acquired AOS participated in a single testing session that included diagnostic testing followed by Experiments 1 and 2. The order of the two experiments was randomized across participants. Given that adults with AOS typically have some degree of co-existing aphasia, we also recruited a group of patients with aphasia only  to the F1 perturbation study (Experiment 2) to test whether any differences might be due to a general effect of neurological damage.M = 63.3 years, SD = 9.1 years, range 50–80 years) secondary to single left hemisphere middle cerebral artery stroke and 10 age-matched healthy older control adults . Diagnosis of AOS was based on consensus between two expert judges, using diagnostic criteria of Participants were 12 individuals diagnosed with AOS plus aphasia . All participants provided written informed consent.Participants attended a single testing session that included diagnostic testing followed by Experiments 1 and 2, in random order. Participants were seated in a sound-attenuated booth in front of a computer monitor. They were told that “ah” would appear on the screen and they were to produce that vowel sound until the word disappeared (2 s), take a breath and be ready for the next stimulus . They were asked to maintain an even habitual pitch, clear vocal quality and a comfortable loudness level. If vocal intensity was outside 70–75 dB, an error signal was displayed after the trial . Participants were fitted with circum-aural headphones (AKG HSC171) with integrated condenser microphone positioned at 10 cm from the mouth. They were informed that they would hear their voice through the headphones and that sometimes it might sound odd but they were to continue vocalizing regardless. A total of 108 trials were presented under three conditions of equal frequency : normal auditory feedback , 100 cent upward pitch-shift of 400 ms duration, and 100 cent downward shift of 400 ms. After the initial five non-perturbed trials, the order of the conditions was randomized. Latency of the perturbation was 12–15 ms and onset of F0 shift randomly varied between 200 and 400 ms, in 50 ms steps, from onset of vocalization.The apparatus included a Motu Microbook II USB Audio Interface and Behringer Xenyx 502 mixer connected to a Lenovo laptop running PitchPresent software  to control timing, direction and magnitude of F0 shifts and recording of vocal responses. Vocalization was recorded at 48 kHz. Auditory feedback was delivered through the headphones at 80–85 dB. The 10 dB gain between voice and feedback channels was used to mask air-born and bone-conducted voice feedback.Due to a tendency to falling F0 in the pre-perturbation time window of vocalizations for many participants, a difference method was used to assess F0 trajectory in each trial /mean pitch frequency of pre-perturbation baseline (Hz))].100 × . For the AOS group, response latency and area under the curve were not significantly correlated with AOS severity  or with aphasia severity .Two participants were excluded from analysis due to poor quality audio files. There were no missing trials for remaining participants. There was no significant effect of group using independent samples pars orbicularis. This latter region is more anterior to area 44 (pars opercularis) and vPMC traditionally associated with concomitant Broca’s aphasia and AOS.Findings for response latency of older healthy adults to the F0 perturbation were similar to those reported by M = 62.88 years, SD = 7.77, range: 54–76; see Table M = 61.5 years, SD = 8.5, range: 52–79) were recruited. An additional three older healthy adults  were excluded for failing the audiology screening. Inclusionary criteria are described in Experiment 1. Also, a group of eight adults diagnosed with aphasia only  were recruited as a brain-damaged control .Eight of the 12 participants with AOS plus aphasia from Experiment 1 were able to participate in Experiment 2 , /bε/ (bear), /kε/ (care), /dε/ (dare), and /pƆ/ (paw). The words pear, bear, and care were used for training, being presented in baseline and in F1 perturbed conditions. The words pear, dare, and paw were presented under masking noise to test for adaptation , transfer of the adaptive response to the trained vowel in an untrained phonetic context (dare), and for vowel-specificity of any adaptive response (paw).Speech stimuli were five monosyllabic consonant-vowel (CV) words of Australian English: /pε/ (pear) is associated with lowering of tongue height and shifting of the vowel toward /a/ . The vowel /e/ has been used in previous studies, . An upward perturbation of F1 for /ε/  or noise masking feedback . Five blocks of each were provided, for a total of 150 trials. Comparison between the participant’s productions of training words during Baseline and each block of perturbed trials in the Hold phase assessed their sensorimotor compensation, while comparison of the masked productions from Baseline and Hold phases tested for sensorimotor adaptation. Finally, the End phase presented 60 trials under noise masking  to test for persistence of any adaptation effect. Note that, with a repeated perturbation over consecutive trials, performance in the perturbed trials of the Hold phase actually reflects a combination of compensation to the immediate perturbation and some short-term adaptation to the perturbation from preceding trial blocks. For convenience, we refer to this here as compensation to differentiate it from the longer-term adaptation seen in the absence of perturbation.The adaptation protocol comprised four phases with a total of 320 trials involving the participant reading aloud each stimulus word as it appeared on a computer screen, at a rate of 1 stimulus per 5 s. The Baseline phase comprised an initial 50 trials, presenting the five stimulus words 10 times each with normal auditory feedback and noise masking randomized, followed by an additional 40 trials with normal auditory feedback. The Baseline established the participant’s habitual F1 in the stimulus words and accounted for any potential variations due to increased vocal intensity with masking . During Verbal feedback regarding vocal loudness and quality was provided during the four experimental phases only if vocal intensity or quality were notably out of range . Participant responses were digitally recorded at 48,000 samples/sec for later analysis.pear substituted for paw), large formant-tracking errors , poor vocal quality or non-speech noises  were discarded from further analysis. For each recorded stimulus, the most stable artifact-free region of the vowel was manually selected based on visual inspection of the spectrogram. Mean F1 frequency for each extracted vowel segment was then estimated using LPC analysis in MATLAB . Data for individual participants are presented in Supplementary Table pear for paw), or paraphasia .A graphical user interface (GUI) was developed in MATLAB to process each participant’s recorded speech productions. Trials that contained off-target responses ] for three participants each. The intra-rater reliability was ICC = 0.83 to 0.97 and the inter-rater reliability was ICC = 0.81 to 0.96.The produced trajectories of F1 versus time  were averaged across the vowel selection. To allow comparisons among participants with differing F1 trajectories, especially in relation to group and sex, each participant’s F1 values were normalized to their mean baseline values as shown in Eq. (1) below, with the dependent variable expressed as a participant’s average response to the perturbation (% F1 change from the Baseline reference of 100%) for each phase or block of trials.To account for potential vocal intensity differences between masked and unmasked trials, which could influence F1, the Mean F1 obtained from the masked Baseline trials was used to normalize the masked trials in the Hold and End phases while the Mean F1 obtained from the normal feedback Baseline trials was used to normalize the perturbed feedback trials in the Hold phase for each training word. Normality of data was checked and confirmed using probability plots.t-tests using the 100% baseline reference value were performed. This analysis established, for each participant group, which blocks within each phase differed significantly from the baseline. To evaluate the differences between groups within each phase, a series of linear mixed model analyses was undertaken as this approach is robust to missing data points in the patient datasets. Factors included Group (three levels), Block . Participant was entered as a repeated factor. LSD post hoc testing was undertaken to explore significant effects. Given the exploratory nature of this study, α level was set at 0.05.To determine the significance of compensatory responses for each group and stimulus type at each block within Hold and End phases, one-sample Performance of the groups is shown in Figures t-tests considering the perturbed trial blocks of the Hold phase, showed that older controls clearly compensated to the shift in all five blocks relative to baseline , with up to a 7% average drop in F1 , with up to a 10% average drop in F1  and 3 (p = 0.034) with up to 9% average drop in F1  = 9.694, p = 0.004], but did not alter the outcome of the models.Comparing across groups, an Unstructured linear mixed model including the fixed effect of Group (three levels), the repeated effect of Block , and the Group by Block interaction, covarying for average F1 on unperturbed trials during the baseline period, was the model with best fit, compared against a first-order regressive covariance structure with or without the covariate , with average percent shift ranging between 100.31% (SD = 5.63) and 101.06% (SD = 5.21)1. Consistently, there was no evidence of F1 change for the transfer word  in the first masked trial block . The AOS group showed significant adaptation on pear for masked trial blocks 2 , 3 , 4 , and 5  but not for block 1 . There was also a significant change in F1 for the transfer word  in the first masked trial block . Similar to controls, the APH group showed no significant adaptation on pear , with average percent shift ranging from 93.52 (SD = 8.25) to 100.50% (SD = 6.83). Consistently, there was no evidence of F1 change for the transfer word  in the first masked trial block . No group showed a change in F1 for the control word paw in the masked trials of the Hold phase.One sample pear, an Unstructured model including the fixed effect of Group (three levels), the repeated effect of Block (five levels), and the Group by Block interaction was the model with best fit, compared against a first-order regressive covariance structure with or without the covariate of average baseline F1  = 0.000, p = 0.983] and inclusion in the Unstructured model did not alter the outcome. Residuals were normally distributed. The main effect of group was significant (p = 0.032), as well as the Group by Block interaction (p = 0.023), but Block was not significant (p = 0.223). LSD pairwise comparisons for group showed that the AOS group differed from controls (p = 0.010), with the AOS group tending to have percent shift F1 values below 100% and the controls close to 100%. Compared to the Control group, the AOS group showed significantly lower F1 shift in blocks 2, 3, and 5 . Compared to the APH group, the AOS group showed significantly lower F1 shift in block 2 only (p = 0.001). No other comparisons were significant. Average vocal intensity  in each block was also considered as a covariate but was not significant  and inclusion in the model did not alter the outcome.Comparing across groups for the perturbed word t-tests considering the perturbed word pear in the masked trial blocks of the End phase, showed that older controls showed no evidence of adaptation to the shift relative to baseline with percent shift values at or above 100%  and 106.25 , respectively. For the first masked trial block for transfer word dare, there was a similar trend of increasing F1 shift relative to baseline but this was not significant . The AOS group showed retention of the Hold phase adaptation effect for pear through End blocks 1  and 2 . While mean percent shift values did not change across the remaining blocks, ranging from 92.55 to 93.93, some participants did not produce sufficient on-target productions for analysis and there was likely insufficient power to reach significance. For the first block for the transfer word dare, the F1 shift approached significance . Similar to controls, the APH group showed no significant adaptation for pear in the End phase , with average percent shift ranging from 102.91 to 106.30. Also there was no change on the transfer word dare . No group showed a change in F1 for the control word paw in the masked trials of the End phase.One sample r = 0.963, p = 0.002). This was not the case for the AOS and APH groups . We also explored whether baseline instability (SD of F1) might explain the degree of adaptation (percent change in F1) in the End phase across groups. AOS participants showed significantly more individual variability than controls, with the APH participants being intermediate between these two groups . Considering the whole participant sample, irrespective of group, there was no significant correlation between individual variability in baseline and degree of adaptation in the End phase ; this also was not significant when considering the AOS group alone , though this analysis was under-powered.To explore whether the rising trend in percent change of F1 for older controls may be related to vocal intensity with the extended delivery of masking, we performed Pearson’s correlations. For older controls, vocal intensity  was highly correlated with percent shift across blocks (p = 0.010), block (p = 0.021), and the group by block interaction (p = 0.041).Comparing across groups, an Unstructured model including the fixed effect of Group (three levels), the repeated effect of Block , and the Group by Block interaction, covarying for average F1 during the baseline period, was the model with best fit, compared against a first-order regressive covariance structure with or without the covariate  to test whether any differences in speech motor control might be due to a general effect of neurological damage. It was hypothesized that (a) compensatory feedback responses to unexpected perturbation of a low-level non-segmental auditory parameter, F0 during steady state vowel production, would be similar between healthy older adults and adults with AOS, and (b) with sustained F1 perturbation, older controls and the APH group would demonstrate adaptation that persisted after the perturbation signal was replaced with masking noise, but AOS participants would demonstrate no adaptation. The first hypothesis regarding feedback control was supported. However, the second hypothesis regarding adaptation of feedforward motor programs was not supported: healthy older adults and those with APH showed no clear adaptation within the short time frame tested, while performance of those with AOS suggested adaptation.The data from Experiment 1 indicate that individuals with AOS do show immediate compensation to an auditory perturbation of F0, similar to older healthy adults. The magnitude of the F0 compensatory response in both older healthy and AOS participants was similar to that reported by Recall that performance in the F1 perturbed trials of the Hold phase in Experiment 2 reflects a combination of compensation to the immediate perturbation and some short-term adaptation to the perturbation from preceding trial blocks. We have referred to this as compensation to differentiate it from the longer-term adaptation seen in the absence of perturbation. This experiment suggests that all groups showed some compensation to the F1 perturbation; although, findings for the patient groups should be interpreted with caution due to their baseline variability.dare that was only presented under the masking condition. In contrast to healthy older adults and those with APH, the AOS group showed significant adaptation during masked trials in the Hold phase and this persisted into the first blocks of the End phase. The effect was also observed for the transfer word dare that was only presented under the masking condition.Previous work with healthy younger adults e.g.,  has showThe unexpected finding of adaptation to a sustained F1 perturbation in AOS patients but not age-matched healthy controls could be interpreted as follows. In light of this negative finding, it is noteworthy that we found significant adaptation using the exact protocol with healthy young speakers  motor programs due to partial damage to left vPMC ; and thaWe suggest a note of caution in the interpretation of results of Experiment 2, until replication. Visual inspection of the normal-feedback trials in Figure Considering the absence of adaptive response in the APH group, one might have expected abnormal F1 compensation if the sample had been weighted toward patients with posterior lesions and impaired speech perception  and displayed motor adaptation to a sustained perturbation of a segmental parameter (F1). The latter finding contrasted with age-matched controls and individuals with aphasia without AOS, who showed no adaptation to the F1 perturbation. These findings suggest that older healthy adults may have less plastic motor programs that resist modification based on sensory feedback, whereas individuals with AOS have less well-developed motor programs due to damage from stroke. Furthermore, they indicate that individuals with AOS can improve their speech motor programs with practice, a capability that is crucial to the success of speech therapies aimed at improving speaking skills in AOS.The finding of adaptation in the AOS group contrasts with the DIVA model prediction that left vPMC damage resulting in AOS should also impair the readout of auditory targets for ongoing speech sequences, which in turn should impair motor adaptation to a sustained auditory perturbation. Computational modeling, systematically manipulating extent of damage to left hemisphere vPMC/speech sound maps may shed light e.g., . MethodoKB, MH, PS, AK, DR, JT, and FG contributed to hypotheses and experimental designs. KB, MH, PS, AK, and AD analyzed the data and setup. KB and AK conducted the experiments and wrote the manuscript. All authors edited the 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.


In conclusion, this study shows that MGP gene rs1800802 polymorphism is associated with increased risk for knee OA in Chinese Han population and the rs1800802 polymorphism may be a diagnostic marker of radiographic knee OA.Several studies have explored the association between matrix Gla protein (MGP) gene polymorphism and knee osteoarthritis (OA) risk; however, they obtained conflicting findings. The present study aims to explore the association of MGP gene polymorphism and OA risk in a Chinese Han population. A total of 256 patients with radiographic knee OA and 327 control subjects were recruited in this case–control study. The genotypes of MGP gene rs1800802 polymorphism was determined by standard PCR and restriction fragment length polymorphism (PCR-RLFP). In this case–control study, we observed that MGP gene rs1800802 polymorphism increased the risk of knee OA. Subgroup analyses also found that rs1800802 polymorphism was related to the elevated risk for knee OA among the female, smoker, drinker, and body mass index (BMI) ≥25 kg/m Osteoarthritis (OA), is the most prevalent type of arthritis and main reason for the chronic disability of the elderly . AccordiMatrix Gla protein (MGP) gene is located at 12p12.3, consisting of four exons . MGP is Evidence suggests that a nonsense mutation of MGP polymorphic sites could produce a truncated protein, which could not bind calcium, resulting in cartilage calcification abnormity and extensive vascular calcification . The effFrom June 2014 to April 2018, 256 knee OA patients and 327 healthy controls were selected from the Shandong Provincial Hospital affiliated to Shandong University And Suichang People’s Hospital. The study was approved by the Ethics Committee of the above two hospitals. We obtained written informed consent from all included patients and controls. Knee OA diagnosis was based on the the American College of Rheumatology Clinical Criteria [The demographic data and information on all the established risk factors including sex, age, smoking, drinking, body mass index (BMI) and K–L grading were collected from medical records. Alcoholics were defined as someone who consumed alcohol at least once per week for more than 6 months. Individuals who smoked up to 1 year were considered as smokers.2, 2.5 U of Taq-DNA-polymerase , 0.2 mM dNTPs , and 0.2 mM primers (Biosynthesis). The PCR cycling conditions: an initial denaturation period of 5 min at 95°C, followed by 35 cycles of 30 s at 94°C, 30 s at 60°C, and 1 min at 72°C, and with a final extension at 72°C for 10 min. The digested PCR products were subjected to gel electrophoresis and visualized by ethidium bromide staining. To control the quality of genotyping, the PCR-RFLP method was performed without knowing the status of the cases or controls.Blood samples were collected using vacutainer tubes containg EDTA and stored at −20°C until use. Genomic DNA was isolated from whole blood using the QIAamp DNA Blood Mini Kit . The concentration and quality of extracted DNA were measured using Nano Drop 2000. Genotyping was carried out by standard PCR and restriction fragment length polymorphism (PCR-RLFP). PCRs were performed in a 50 μl volume containing 50 ng of DNA template, 5 μl of 10× PCR buffer, 2.0 mM MgClt test (for continuous variables) or Chi-squared (χ2) test . The associations between genotype of rs1800802 polymorphism and the risk of knee OA were examined by computing the odds ratios (ORs) and 95% confidence intervals (CIs) using logistic regression analysis. The Hardy–Weinberg equilibrium (HWE) was tested to evaluate whether the control group could represent the whole population by a goodness-of-fit χ2 test. Statistical significance was assumed at P-values at <0.05 level. Above statistical analyses were performed using the SAS software package .We evaluated the differences in demographics variables and genotype frequencies of rs1800802 polymorphism using Student’s P>0.05). According to the radiographic K–L grading system, approximately 57.4% of OA patients had a KL grading of 3 or 4.P=0.035). Similarly, GG genotype was associated with a statistically significant increased risk for knee OA in the recessive model . Taking the A allele as reference, a significant association was observed between the presence of G allele and a higher risk of developing OA .The genotype distribution and allele frequencies of rs1800802 polymorphism were provided in P=0.044) rather than in males. The significant differences were also found to be existed in the comparison of GG genotype versus combined genotype (AA + AG). This significant association also held true for smokers, alcoholics, and subjects with ≥25 kg/m2 groups.Subgroup analyses were conducted according to sex, age, smoking, alcoholism, and BMI . Stratif2 groups.In the present study, we evaluated the association between MGP gene rs1800802 polymorphism and the risk of knee OA in a Chinese population and found that rs1800802 polymorphism increased the risk of knee OA. Subgroup analyses obtained similar findings among the females, smokers, drinkers, and BMI ≥25 kg/mMGP, a kind of secreted protein, acts as an inhibitor of bone morphogenetic protein signaling and has high affinity to calcium ions . Luo et 2 groups, suggesting that OA patients were more likely to expose to these factors.Misra et al.  first foSeveral potential limitations of this study should be considered. First, the relationship between the MGP gene polymorphism and knee OA susceptibility could not be fully revealed by a single case–control study because of the relatively small sample and it might underpower the facticity. Second, the cases and volunteers were selected from hospitals, which may not exactly represent the general population. Third, we did not obtain detailed information about severity of OA and its response to treatment respectively, which restricted our further analyses of the role MGP gene polymorphism played in OA. Fourth, we only investigated one SNP of MGP gene. Fifth, we did not know how the gene variant of MGP affects the development of knee OA. Sixth, the interacting between MGP gene polymorphisms and other environmental factors was not explored. Last but not the least, like other studies, we only evaluated the effects of MGP gene polymorphism among Chinese population, which may not generalize to other ethnic groups; so further studies with larger sample sizes in other races are warranted to confirm our findings.To sum up, this study shows that MGP gene rs1800802 polymorphism may contribute to the increased risk for knee OA. However, this is only a preliminary conclusion as our results were obtained in Chinese Han population. Better designed and more multicenter case–control studies from diverse ethnic populations are needed to validate our findings.


The successful usage of 10 w/v % mc as support ink was proven by multichannel plotting of the support together with a plottable calcium phosphate cement (CPC) acting as main structure. CPC scaffolds displaying critical overhangs or a large central cavity could be plotted accurately with the newly developed mc support ink. The dissolution properties of mc allowed complete removal of the gel without residuals, once CPC setting was finished. Finally, we fabricated a scaphoid bone model by computed tomography data acquisition and co-extrusion of CPC and the mc support hydrogel.3D plotting is an additive manufacturing technology enabling biofabrication, thus the integration of cells or biologically sensitive proteins or growth factors into the manufacturing process. However, most (bio-)inks developed for 3D plotting were not shown to be processed into clinically relevant geometries comprising critical overhangs and cavities, which would collapse without a sufficient support material. Herein, we have developed a support hydrogel ink based on methylcellulose (mc), which is able to act as support as long as the co-plotted main structure is not stable. Therefore, 6  Low-t3) . These sFirst attempts to overcome shape limitations of 3D plotting were developed by usage of a support bath ,7,8. HowMultichannel plotting of a temporary supporting material together with the actual main ink could extend both, material variety and shape diversity of 3D plotted constructs. An ideal ink should comprise two functionalities: (i) the material ink acts as support for the main ink, preventing plotted strands from collapsing and (ii) the material ink acts as sacrificial material, which means that it is vanishing after post-processing and stabilization of the main scaffold. Additionally, material inks applicable for 3D plotting of support structures must not only meet the requirements mentioned, but also those of viscosity, plottability, and post-plotting stability. Within this manuscript, the term support ink is used, however the developed biomaterial ink also meets the requirement acting as sacrificial material.® F127 . Previous studies investigated its use to create channel-like structures or sacrificial molds [® F127 is also used to create bioinks acting as a main structure and is needed in high concentrations to show enhanced plotting properties [Hydrogels have been developed for several applications of 3D plotting and their viscoelastic properties make them ideal for usage not just as bioink but also as cell-free support ink . The inkal molds ,11,12. Hoperties ,14. ThusInactive Ingredients Search for Approved Drug Products by the FDA [Water-soluble methylcellulose (mc) could be a promising candidate. mc is a synthetic cellulose ether with methyl groups substituting hydrogen atoms of the cellulose molecules. It showed unique viscosity enhancement of aqueous solutions, as well as increased dissolution properties at decreasing temperatures  ,16,17. A the FDA , minimizFor our study, we have chosen to use a ceramic calcium phosphate cement (CPC) as the main ink. CPCs are composites of inorganic precursors and an aqueous solution. In contact with water molecules, the setting process of CPC is initiated and, for example just like within this work, α-tricalcium phosphate is precipitating to nanocrystalline hydroxyapatite (HAp). Nonetheless, the usage of the classical cements does not allow fabrication by 3D plotting , as the fast setting process leads to needle clogging. Therefore, organic carrier liquids were developed substituting the aqueous phase. They were shown to retard the hydroxyapatite precipitation reaction ,22,23 anWithin this paper, we investigated the suitability of mc as support ink for CPC constructs, carried out by multichannel plotting of mc and CPC. We aimed to: (i) develop a mc paste fitting the support ink requirements; (ii) which was characterized for its extrusion and plotting properties. Based on these results, CPC constructs were fabricated with (iii) critical overhangs and (iv) cavities in order to enable fabrication of complex shaped CPC constructs, suitable as scaffolds for bone reconstruction or tissue engineering purposes.w/v %, 8 w/v % and 10 w/v %; therefore, the resulting support inks within this manuscript are referred to as mc6, mc8, and mc10, respectively. Higher concentrations of mc were excluded from this study, as it was not possible to achieve homogeneous gels with uniform viscosity. Furthermore, a low concentration was intended to be advantageous in terms of the sacrificial behaviour of mc inks. It was decided to dissolute mc at 4 °C to ensure full hydration of the mc chains. After complete dissolution, all three combinations, mc6, mc8, and mc10, were almost transparent, homogeneous and viscous pastes, which were used for further characterization.Methylcellulose was dissolved in water in three different concentrations: 6 −1, which is in our experience applicable to assess the plotting behavior of a material  higher mass flow of the mc6 paste was observed in comparison to mc8 and mc10. For example, at an air pressure of 200 kPa the mass flow of the mc6 paste was 12.72 ± 1.0 mg·s−1, whereas the mass flows of mc8 and mc10 were calculated as 0.75 ± 0.12 and 0.19 ± 0.02 mg·s−1. Based on these results, the following air pressure values were set for good extrusion with a plotting speed of 10 mm·s−1: mc6—100 kPa, mc8—200 kPa, and mc10—250 kPa.The extrusion properties were tested using a cylindrical needle with an inner diameter of 250 μm. Firstly, mass flow through the needle was evaluated as function of the applied air pressure . All thrz-direction with smallest collapse of the mc10 paste.Shape fidelity of the mc inks was evaluated by a filament fusion test, as proposed by Ribeiro and co-workers . ShortlyBased on the results of rheology and extrusion, mc10 was chosen to be the favorite support ink for 3D plotting of complex shaped CPC structures.Firstly, the shape fidelity of CPC constructs with overhangs was compared to structures of the same geometry, which were plotted without supporting material. The setup of the plotting process is shown in Then, the pyramid was plotted in an inverted orientation displaying critical overhangs which would collapse without supporting material. The principle of plotting is shown in 3. The inner cube plotted with mc10 (plotted without strand distance/macropores) had a volume of 15 × 15 × 15 mm3. After plotting, stereomicroscopical images showed clearly the presence of the non-porous mc10 cube  and cannot be applied to more complex geometries like cavities. Moreover, residues of mc are likely to remain. Therefore, the hydration process of mc should be the favorable method to remove mc after the main structure is post-processed and stable.® F127 (so it can be easily dissolved at 4 °C within a few hours), but additionally can dissolve at 37 °C, as it is still a non-crosslinked sol. For example, mc was shown to be released out of an alginate matrix at 37 °C, without harming cell viability of embedded cells [® F127 [® F127 is stable.Herein, we successfully have developed a support ink based on methylcellulose, specifically applicable for a self-setting CPC. mc10 has comparable thermoresponsive properties like the commonly used Pluroniced cells . In a reed cells  or, as sed cells . This is [® F127 , and migFor the first time, CPC was plotted with a support ink to obtain complex shaped constructs of clinical relevance. In the past, complex shaped calcium phosphate scaffolds were manufactured by powder printing ,46,47, oFirstly, the influence of plotting a support structure together with a CPC main structure was evaluated. The inner structure of the tested pyramids was not affected, whereas the outer borders were changed distinctly. The pyramid plotted without support comprised more roundish strands, as it is expected for strands at a surface of every plotted scaffold. In contrast, the pyramid plotted with mc10 as support ink in reversed orientation revealed flattened strands at the interface to mc. Both, roundish and flattened strands are approximations of the real geometry of the pyramid. In the case of a possible implant, there are neither right angles nor complete convex surfaces. Therefore, the geometry of a single strand might never mimic the real geometry of a human bone specimen. As the resorption of the nanocrystalline HAp by osteoclasts is part of the natural bone remodeling process ,55, the However, the rough geometry of anatomical structures can be plotted: the example of the human scaphoid bone showed sufficient shape reproduction in comparison with the three-dimensional image file. Parameters influencing the plotted shape were on the one hand the resolution of the computed tomography data set and the precision of the generation of the stl-file. On the other hand, the strand distance and orientation, the used needle diameters for extrusion of CPC and mc and plotting properties like the layer thickness, applied air pressure and plotting velocity influence the outcome significantly. For CPC, these parameters were optimized in our group, achieving filigree strands and scaffolds with high inner complexity .Crucially, CPC scaffolds could be fabricated containing large inner cavities. Such closed inner pores can only by generated by additive manufacturing methods. The dissolution properties of mc allowed fast vanishing of the internal mc cube after CPC was set (3 days), leaving behind a cavity of the desired cubic shape. This might enable the fabrication of scaffolds with highly controlled (macro)porosity to improve the ingrowth of cells and vascular structures, both accepted as main challenges in bone tissue engineering.The usage of a newly developed methylcellulose-based support ink was sufficient to enable fabrication of structures with critical overhangs and to produce controlled cavities inside a CPC construct. For the first time, it was shown that CPC can be used to fabricate constructs of any clinical and anatomical relevant shape by 3D plotting. Furthermore, a human scaphoid bone model of high shape-fidelity could be produced. Further work should concentrate on more control of the dissolution properties of mc, which basically could be tailored for any application by addition of gel-strengthening and gel-weakening substances. The principle of biphasic plotting of the support ink and CPC will facilitate the production of patient-individual HAp implants, fabricated by 3D plotting.w/v%, 8 w/v% and 10 w/v%, respectively). To ensure full binding of water to mc, solutions were stirred every 2 h. The resulting support inks are referred to as mc6, mc8, and mc10.CPC was manufactured by InnoTERE GmbH . For the support material, methylcellulose powder  was dissolved for 12 h in deionized water at 4 °C in different concentrations  at 25 °C. Shear thinning was characterized by increasing the shear rate from 0–100 s−1 over 1200 s (increment 0.08 s−1) and viscosity was measured (n = 3). Shear recovery of the mc pastes (n = 3) was characterized orientating at the protocols proposed by Paxton and co-workers and Kesti and co-workers [−1 was applied for 200 s, followed by a hundred-fold higher shear rate of 500 s−1 for 100 s. This procedure was repeated two times. Then, again a shear rate of 5 s−1 was applied for 200 s. For the whole time of observation, viscosity was measured and shear recovery was obtained. Stress ramps of mc6, mc8, and mc10 were performed to evidence flowing of the inks at high shear rates (data not shown).Rheological examinations of the mc pastes were performed with a plate-plate rheometer  were allowed to flow through the needle for 50 s at different air pressures , then the mass of the extruded volume was measured. Resulting mass flow The mass flow of mc6, mc8, and mc10 was conducted through a conical needle with an inner diameter of 250 μm ; needle and plate were at room temperature. The inks (n = 3) were performed following the protocol of Ribeiro and co-workers [Filament fusion tests of the mc inks  to scaffolds  utilizing a three-axis multichannel plotter . The needle (Globaco) dependent plotting parameters are summarized in Micro X-ray computed tomography was conducted by a custom-built setup called TomoTU of TU Dresden, Institute of Fluid Mechanics, Chair of Magnetofluiddynamics, Measurement and Automation Technology. The grey value images of the sample were generated with 0.5° angular increment  for a tube current of 170 μA and an acceleration voltage of 90 kV. The size of the photodiode array and therefore of the images was 2304 × 2940  pixel. A magnification of 3.55 was chosen, which resulted in a resolution of 1 pixel = 13.95 μm. Reconstructions of the 3D-images was created by grey value analysis (threshold = 120) with Paraview 5.5 software . Macropo


Cats tend to retain food preferences across their lifetime, but intrinsic variables could affect their diet choices so as to preserve internal homoeostasis. This work intended to study the feeding behavior of cats in relation to diet composition and some intrinsic variables  by analyzing data from a 10-year database of two-feeder food preference tests (2007–2017). Diet mineral components  affected food preferences negatively. In addition, the influence of body weight and sex manifested as lower food intake in both females and the heaviest cats (relative to bodyweight). However, only body weight affected food preferences, where the heaviest animals presented the higher preferences overall. During the cold season, animals  displayed higher food intake, whereas hot seasons increased male cat preferences for palatable diets, hence we observed an interaction between sex and climate season. In conclusion, understanding the relationship that both food composition and some intrinsic variables cats have with their diet preference could help in improving the formulation of specific pet food diets, so that these adequately satisfy the physiological and hedonic needs of domestic cats.n = 1021; period 2007−2017) was used to explore the feeding behavior of domestic cats. Principal component (PC) analysis and linear regression between food nutrients and preferences  were performed. Intake and preference for Diet A were analyzed by intrinsic cats’ variables and climate season. The PC1 , phosphorus (P), and ash), PC2 (lipids and ether extract) and PC4  had borderline significance . Ash and CF contents presented negative correlations with food preference , and Ca had borderline significance and negative correlation with food preference . Body weight and sex influenced the intake of Diet A, being lower for females  and heaviest cats . However, only body weight affected food preferences, where the heaviest cats had greater preferences for Diet A. Hot season decreased food intake , mostly in females . Males had greater preferences for Diet A during hot seasons  and females presented similar preferences throughout the year (p = 0.950). Mineral contents, body weight and sex affected food intake and preferences of cats under the influence of climate season, probably explained by adaptive changes in food detection.A ten-year database of food preference tests ( Felis silvestris catus) display feeding behavior that stems from specific nutritional requirements [Felis silvestris lybica), who hunted small-sized prey [Domestic cats  was provided by the Research Centre for Pet Feeding Behaviour , located in the metropolitan region of Chile , and then used to analyze the effect of diet composition and intrinsic variables of cats on their food intake and preference for commercial diets. Experimental procedures were approved by the bioethical committee of “Facultad de Ciencias Veterinarias y Pecuarias”, Universidad de Chile (N°042013).A total of 1021 preference tests using 24 different mongrel domestic cats (Felis silvestris catus)  were used in this study over the complete period (2007–2017). An average of 15 ± 2 animals was maintained per year at the Center. These animals were sourced from local shelters at an average age of 6 months and then submitted for clinical examination by veterinarians of the Clinical Sciences Department of FAVET before being registered as experimental animals. Once housed, they were trained for 3 to 6 months to ease their adaptation to the experimental procedures. During this period, they were adapted to 2-feeder preference trials with highly palatable foods in both feeders to stimulate the intake of the two options. Health of cats was assessed prior to the start of the experiments through clinical, hematologic and biochemical serum profiles, and all cats that participated in preference trials were considered healthy. The age of the cats ranged from 1 to 13 years old and females usually represented a greater percentage of the total animals. Cats were housed individually in single kennels that were split into inner and outer areas of 94 × 90 × 105 cm and 94 × 90 × 90 cm, respectively.Cats had access to a sandbox and fresh water on the outer area of each kennel, while on the inner area animals were fed a single ration (3% of their body weight) of a commercial diet (daily at 09:30 a.m.), as well as ad libitum access to water and a comfortable bed. This ration was adjusted monthly accordingly to body weight records of each cat to ensure their body condition remained stable. Environmental conditions were partially controlled by the Centre by means of door and window management. However, average temperature varied across different seasons during the year, ranging between a maximum of 22 °C in summer and a minimum of 7.7 °C in winter . Animals0.75). As for the nutritional composition of the experimental diets, these were subjected to a proximate chemical analysis, acid hydrolysis, and a calorimetric pump . Diets were analyzed following standard Association of Official Analytical Chemist (AOAC) methods [Food trials comprised a series of two-bowl food preference tests that were performed within each kennel to assess the variability of their nutritional, physical, and/or organoleptic properties. Each trial lasted 4 days (1 test/day) in which diets were counterbalanced in position (left or right) to diminish the effect of possible side bias over preferences. Every test began on the afternoon (02:00 pm) and ended on the next morning (10:00 am). Each cat was individually tested over this 20-hour period by offering two different diets, each on one bowl of the same color, shape, and size. Rations offered were adjusted according to their body weight (3%) at all times, whether animals were participating in a trial or not. On test days, cats were fed only the experimental diets and water was freely available for them. Feeders were weighed at the beginning and the end of each test, to estimate the amount of food that the animals consumed from each diet. In order to better equate intake across animals and diminish size effects, consumption was subsequently corrected by their metabolic weight  without having the same standard diet across tests, the most preferred diet in each test was labeled as Diet A, whereas the less preferred one was labeled as Diet B, and the degree of preference was expressed as the relative consumption of Diet A over Diet B + Diet A allowing evaluation of the effect of variables on the selection of the most preferred diet (see Equation (1)). To this end, our data for consumption and preference of Diet A were sorted by sex, age, and body weight of each cat, as well as the current climate season at the time of performing each test.The evaluation of how nutritional components of diets  may explain food preferences and if the components were grouped, was carried out using a principal component analysis (PCA) and ‘devtools’ and ‘ggbiplot’ packages of the statistical software R . All nutkmY corresponds to the intake (corrected for metabolic weight) or to Diet A preference, β0 is the intercept, βn is the vector of fixed effects, bk corresponds to the random effect of the kth cat and εm(k) is the error term or the elements that were not included in the analysis. This model assumes that random effects and errors are independent at the nesting level and follow an approximately normal distribution, with means equal to zero and variance equal to σ2. The Likelihood Ratio Test (LRT) was used to select fixed effects from the initial model (full model), removing variables whose regression coefficients were not significant (p > 0.05) using a stepwise backward elimination procedure [p < 0.05 [p-values were between 0.1 and 0.05 [Intrinsic variables effects on intake and preference of Diet A was examined using linear mixed models, also known as variance component models, which are characterized by the consideration of the nested structure of the data, providing a chance to decompose variance into a number of different components which can then be given a useful interpretation ,27,28. Trocedure . An adjurocedure . Dummy vp < 0.05 . A bordeand 0.05 . All theand 0.05 , plus ‘lp = 0.059, p = 0.055 and p = 0.053, respectively). Variances  for the first four principal components (PC1−PC4) were greater than one (>1) and, altogether, they explained more than 85% of the variability found in the original variable . These components allowed us to summarize our data into multivariate linear regression analyses, without losing information or minimizing such loss. In particular, the values for these components (expressed as percentages) were 34.98%, 29.70%, 13.87%, and 10.37%, respectively. Eigenvectors  from therho = −0.269, p = 0.031; rho = −0.338, p = 0.006, respectively). In the case of Ca, its contents in the diet had a negative correlation with food preference (rho = −0.241) and a borderline significance (p = 0.054).In p < 0.001). Specifically, we observed that food intake (corrected by metabolic weight) decreased by 5.490 units for every increase of one unit of body weight. Sex also had a significant effect as males increased their food intake by 11.758 units . As for the season, dummy variables signaled winter as the reference category, where autumn was significantly different than winter . Thus, food intake (corrected by metabolic weight) increased by 7.864 units in autumn vs. winter. Also, we observed a seasonal effect on food intake when reclassifying seasons simply as either hot (summer and spring) or cold (winter and autumn), as cats ate less from Diet A during the hot season . Meanwhile, males tended to decrease food intake as their body weight increased , whereas the interaction between sex and season did not significantly affect the intake of Diet A (p = 0.149). In particular, females were the only sex that significantly increased their intake under cold temperatures  and third slopes (β = 1.165 and p = 0.001) were observed. As for the sex variable, our analysis revealed (p = 0.023).The mixed model analysis revealed that food preferences data was not normally distributed, hence data were transformed using the decimal logarithmic function to ensure its normality for the residual analysis. This analysis revealed that the preference for Diet A was affected by only two intrinsic variables: body weight and sex. The first one, body weight, had a complex behavior that evidenced a non-linear relation with food preference. In particular, at least two slope changes were detected, hence three relations could be observed between food preference and body weight, and clearly indicated that heaviest cats did present a greater preference for Diet A. Specifically, significant results for the first  when it was compared against data for winter (via dummy variables). Also, the sex*season interaction did have an effect over food preference for Diet A, as males preferred Diet A more often during summer (β = 10.216 and p = 0.023), whereas females presented similar degree of preference for Diet A throughout the year (p = 0.950). The interaction between sex and season when food preferences were reclassified as either hot (summer and spring) or cold (winter and autumn) is expressed in Meanwhile, the season of the year affected food preference for Diet A, as it decreased in summer . In this regard, it is worth noting that the content of Ca and P in most diets used for the preference tests were higher than the percentages recommended by The Association of American Feed Control Officials , hence i2 g−1h−1), reflecting a lower requirement of calories per unit of weight to maintain their daily energy requirements. Another interesting feature of the data is that heavier cats could prefer Diet A more due to some perception changes for certain macronutrients, which may arise due to failures in the physiological regulation of food intake , as it has been reported for obese or overweight humans and rats [Interestingly, the consumption of preferred diets was affected by body weight and sex in cats, but not by their age. In the case of body weight, the lower consumption of Diet A (preferred diet) that was observed in heavier animals could stem from the fact that a smaller body surface area would generate a lower specific metabolic rate , as well as the contents of crude fiber have a negative effect on food preferences. On the contrary, body weight decreased food consumption but increased the preference of our cats for palatable diets (Diet A). Additionally, while males usually ate more than females, sex did not affect food preferences. As for the effect of seasons, the fact that Diet A was preferred in the cold season was due to a higher consumption by females, which could relate to their reproductive cycle. Yet males tended to prefer this diet more during the hot season, probably because higher temperatures may aid them to better discriminate palatable foods by their volatile cues. Consequently, accounting for these factors when performing food preference tests—to a homogeneous feline population and under controlled environmental conditions—would help to eliminate possible biases and to reduce data variability. Such results would more reliably reveal the food preferences and feeding behavior of domestic cats when offered commercial diets. In turn, this may improve formulation and development of cat foods adjusting to fit animal’s specific nutrient requirements as well as to an adequate palatability level increasing both, cats’ health and wellbeing.