Accurate information about type, degree, and configuration of hearing loss are necessary for successful audiological early interventions. Auditory brainstem response with tone burst stimuli (TB ABR) and auditory steady-state response (ASSR) exams provide this information.To analyze the clinical applicability of TB ABR and ASSR at 2 kHz in infants, comparing responses in full-term and premature neonates.The study was cross-sectional, clinical and experimental. Subjects consisted of 17 premature infants and 19 full-term infants. TB ABR and ASSR exams at 2000 Hz were done during natural sleep.The electrophysiological minimum response obtained with TB ABR was 32.4 dBnHL (52.4 dBSPL); the ASSR minimum was 13.8 dBHL (26.4 dBSPL). The exams required 21.1 min and 22 min, respectively. Premature and full-term infant responses showed no statistically significant differences, except for auditory steady-state response duration.Both exams have clinical applicability at 2 kHz in infants, with 20 min of duration, on average. In general, there are no differences between premature and full-term individuals. Audiologic diagnosis within the first few weeks or months of life has become more frequent with neonatal screening programsElectroacoustic and electrophysiologic tests, in particular otoacoustic emissions (OAE) and the auditory brainstem response (ABR), are recommended for defining the behavioral audiogram in children aged below 6 monthsThe ABR with clicks is commonly used in children. Although less used in clinical settings due to increased testing time, tone burst stimuli are very useful because it is frequency specific, and therefore can be used to assess low, middle, and high frequenciesA similar advantage may be attained with the auditory steady-state response (ASSR), in which continuous, amplitude and/or frequency modulated tones evoke electrophysiologic responses that make possible a detailed and objective evaluation of hearingMany researchers in this area have become interested in ASSR and tone burst ABR testing; these are promising tests, about which there is no consensus as to which is the best and fastest for clinical use. Existing studies have been made in adults; the Brazilian literature is particularly poor in describing these tests in term and preterm infants.Thus, the purpose of this study was to investigate the clinical applicability of tone burst ABR and 2 kHz ASSR in infants by evaluating the time required for each test and comparing the responses of term and preterm infants.A cross-sectional, clinical, and experimental study was carried out at the clinic of the Speech Therapy School. The institutional review board of the university approved this study (process no. 0713/07). Parents and caretakers authorized the children to participate and the results to be divulged by signing a free informed consent form.The inclusion criteria were: preterm or term infants, with normal meatoscopy in both ears, bilaterally present transient evoked otoacoustic emissions, presence of the cochlear-palpebral reflex at 100 dBSPL, and click stimulus BAPE within normal limits for each child's age. Absence of one or more of these criteria excluded of the subject from this study. A Welch Allyn Pocket Junior otoscope was used for performing meatoscopy, and a Smart EP (Intelligent Hearing Systems) device was used for OAE and click stimulus BAPE. The cochlear-palpebral reflex was investigated using an agogo. Thus, middle ear conditions, poor outer ear cell function, and neural/central hearing loss were excluded.A statistics professional defined the minimum sample number at 30 subjects. Initially, 68 infants were selected for the study from October 2007 to July 2008. Twenty-three subjects were excluded by not meeting one or more of the inclusion criteria; testing was not concluded in a single session in nine subjects, who did not return to complete the tests.Thus, the sample comprised 36 infants that were allocated into two groups according to the gestational age. The preterm group  comprised 17 infants . The term group  comprised 19 infants .The Smart EP device was also used in tone burst ABR and ASSR testing. Before testing the skin was prepared with Nuprep (an abrasive paste). ECG conductive adhesive Medi-Trace electrodes  were placed so that recording was ipsilateral to the stimulated ear; the impedance was < 5 kΩ. The electrodes were placed as follows: active electrode on Fpz, reference electrode on M1 or M2, ground electrode on the contralateral mastoid to the stimulated ear. Subjects remained sleeping naturally on their caretaker's lap during testing.The order and ear for starting the test were random; tone burst ABR was done first on one infant, and ASSR was done first on the next infant, on the free ear depending on how each child was positioned on the lap of the caretaker. The test parameters were those of the manual and the pertinent literature , Frame 2This choice was also based on a pilot project with 10 infants that checked test feasibility relative to the number of frequencies and intensities that were studied.In this pilot project, tone burst ABR and ASSR were recorded at 500, 1000, 2000, and 4000 Hz, and difficulties resulting from test duration (about 3 hours) were checked. This difficulty was still found when reducing the test to the frequencies 500 and 4000 Hz; we then decided for 2000 Hz, which was considered feasible to apply in a single session in the pilot project.The chosen intensity was 20 dBSPL in the pilot projects, as most infants presented ASSR at this intensity; furthermore, the V wave (tone burst ABR) could not be identified accurately at 20 dBHL in any of the infants. This experiment helped define our test protocol.The test duration was measured by two methods - a microcomputer clock and a KD-1069® chronometer . The former was used to measure absolute time (Δt abs) - the test duration from the beginning (after placing the electrodes) to the end (before removing the electrodes). Eventual pauses to control test conditions and/or the infant were also computed. The chronometer was used to measure relative time (Δt rel) - it was paused each time the test was stopped.Rarefied stimuli were presented with ER 3A in-ear phones decreasingly at 80 dBnHL, 60 dBnHL, 40 dBnHL, and 30 dBnHL. The minimum electrophysiologic response at 2 kHz was measured by visual analysis of the V wave and reproducibility in two tracings.ASSR testing was started at 20 dBSPL with 10 dBSPL increments until obtaining the minimum response, since this was a hearing sample. ASSR testing was done with a maximum presentation number of 400 sweeps. If a response was attained with a smaller number of sweeps, the test was continued for two extra 40 sweep updates to check whether the responses persisted; this procedure avoided false positive results.Fast Fourier transformation was applied to analyze frequency domain responses. The equipment detected the responses automatically based on F statistics, which assesses the response amplitude at the modulation frequency in relation to noise amplitude at adjacent frequencies. A response was considered positive if the signal-to-noise ratio was higher than 6.13 at a p-value below 0.05.Descriptive statistics and non-parametric tests (Wilcoxon's test and Mann-Whitney's test) were applied in the analysis. Wilcoxon's test was applied to verify differences or similarities between left and right ears; the Mann-Whitney test was used for comparing the term and preterm groups. The significance level was 0.05 (5%); values without statistical significance were marked with an asterisk (*).44.4% males. The mean gestational age in GT subjects was 39 1/7 weeks; at the evaluation, the mean chronological age of infants was 8 2/7 weeks. The mean gestational age in the GPT was 32 6/7 weeks; the post-conception mean age was 47 3/7 weeks. The analysis of both groups showed that there were no significant differences between right and left ears; thus, both ears were considered in the subsequentThe study sample consisted of 55.6% females and In 72 ears, the mean tone burst ABR minimum responses in nHL and SPL were 32.4 and 52.4 dB. The ASSR means in HL and SPL were 13.8 and 26.4 dB .Table 1DComparing GT and GPT revealed no statistically significant differences except for the absolute and relative ASSR times; in this case, the GT had lower times for carrying out the procedures .Table 2CThe absolute time was significantly higher than the relative time in test duration , showingBefore starting the discussion, it is necessary to point out that comparisons between the tone burst ABR and ASSR is difficult to perform accurately, as there are differences in stimulus calibration  and the response detection method in these proceduresSimilarities in the side or ears - as noted in the present study - have also been reported in the literatureIn the complete series n = 72 ears) the mean minimum response was 32.4 dBnHL (52.4 dBSPL) at 2 kHz for tone burst ABR in the term and preterm groups. Studies with similar groups at the same frequency have reported lower results - in other words, better threholds2 ears thVery few studies have compared thresholds measured with tone burst ABR and ASSR in the same populationThe choice of two measurements of test duration was made to observe the true clinical application of these tools. Sedation for tone burst ABR and ASSR testing in infants is not a common practice in Brazil, as opposed to what occurs in other countiresth to the 47th weeks was responsible for similar findings in preterm and term infants in the present study, which differs from previous workExcept for the duration of the ASSR, there were no statistically significant differences between the preterm and term groups in the comparative analysis. The literature contains some differences in comparisons of similar infants, in which preterm infants have worse thresholds, slower responses, and longer latencies compared to term infantsFurther studies of different population groups with other methods to study ASSR and tone burst ABR will help understand these techniques in greater detail to expand their clinical applicability.The results of this study led to the conclusion that the two methods - tone burst ABR and ASSR - may be applied clinically at 2 kHz in infants; the mean test duration was 20 minutes. Preterm infants require more time for ASSR, but in general showed no differences compared to term infants.


SGCA, SGCB, SGCG and SGCD genes. Delta-sarcoglycanopathy (LGMDR6) is the least frequent and is considered an ultra-rare disease. Our aim was to characterize the clinical and genetic spectrum of a large international cohort of LGMDR6 patients and to investigate whether or not genetic or protein expression data could predict a disease’s severity.Sarcoglycanopathies include four subtypes of autosomal recessive limb-girdle muscular dystrophies  that are caused, respectively, by mutations in the This is a retrospective study collecting demographic, genetic, clinical and histological data of patients with genetically confirmed LGMDR6 including protein expression data from muscle biopsies.We contacted 128 paediatric and adult neuromuscular units around the world that reviewed genetic data of patients with a clinical diagnosis of a neuromuscular disorder. We identified 30 patients with a confirmed diagnosis of LGMDR6 of which 23 patients were included in this study. Eighty-seven per cent of the patients had consanguineous parents. Ninety-one per cent of the patients were symptomatic at the time of the analysis. Proximal muscle weakness of the upper and lower limbs was the most common presenting symptom. Distal muscle weakness was observed early over the course of the disease in 56.5% of the patients. Cardiac involvement was reported in five patients (21.7%) and four patients (17.4%) required non-invasive ventilation. Sixty per cent of patients were wheelchair-bound since early teens (median age of 12.0 years). Patients with absent expression of the sarcoglycan complex on muscle biopsy had a significant earlier onset of symptoms and an earlier age of loss of ambulation compared to patients with residual protein expression.This study confirmed that delta-sarcoglycanopathy is an ultra-rare neuromuscular condition and described the clinical and molecular characteristics of the largest yet-reported collected cohort of patients. Our results showed that this is a very severe and quickly progressive disease characterized by generalized muscle weakness affecting predominantly proximal and distal muscles of the limbs. Similar to other forms of sarcoglycanopathies, the severity and rate of progressive weakness correlates inversely with the abundance of protein on muscle biopsy. et al. describe the largest cohort to date of patients with the ultra-rare neuromuscular disorder delta-sarcoglycanopathy, and show that the severity and rate of progressive weakness correlates inversely with the abundance of protein expression on muscle biopsy.Alonso-Pérez  Among them, sarcoglycanopathies are one of the most frequent forms especially when symptoms onset during childhood.2-7 There are four sarcoglycan genes  causing autosomal recessive LGMD . The frequency of each type of sarcoglycanopathy varies depending on the studied population, although LGMDR3/LGMD2D and LGMDR5/LGMD2C are the two most frequent forms.8Limb-girdle muscular dystrophies (LGMD) are a heterogeneous group of genetic diseases that affect skeletal muscle causing progressive loss of muscle fibres leading to muscle weakness predominantly affecting the pelvic and shoulder girdle.SGCD gene and was originally described in 1996,9–11 and is thought to be the least common type of sarcoglycanopathy although the number of existing cases is not known. There are only a few reports describing the clinical features of single cases or short cohorts and therefore neither the clinical features nor the disease progression over time is well known.12–14Delta-sarcoglycanopathy (LGMDR6/LGMD2F) is caused by recessive mutations in the 15–18 We have recently observed that symptoms’ onset before the age of 10 years, protein expression in the muscle biopsy of <30% or mutations leading to absence of protein expression were independent risk factors associated with a more severe phenotype characterized by early loss of ambulation.8 However, it is not known whether these are also risk factors of quick progression in patients with LGMD-R6.Sarcoglycanopathies (LGMDR3–6) are in general severe disorders characterized by weakness onset at first decade of life leading to loss of ambulation during adolescence or early adulthood. However, patients with a milder phenotype with onset of symptoms after the second decade of life, a slowly progressive course and still ambulant after the age of 50 or 60 years have also been described.SGCD. Our aims were to describe the main clinical and genetic features, investigate potential genotype–phenotype correlations and identify factors influencing the progression of the disease.To answer these questions, we collected demographic, genetic, clinical and muscle protein data of a large international cohort of patients with mutations in SGCD gene. Anonymized data from clinical reports were collected in a survey and stored in a secure server in the Hospital de la Santa Creu i San Pau (HSCSP). The study was approved by the Ethics Committee of the HSCSP. To identify patients for the study, we used two different strategies. On the one hand we contacted 128 paediatric and/or adult hospitals from 38 countries. Only 11 neuromuscular centres from nine different countries had patients with delta-sarcoglycanopathy and agreed to participate in the study. On the other hand, we also contacted colleagues that lead next-generation sequencing (NGS) studies in large cohorts of neuromuscular disease patients without diagnosis. These studies actually included 9610 patients. None of these patients had mutations in the SGCD gene.This is an observational retrospective cross-sectional study reviewing clinical and genetic data of patients with confirmed pathogenic mutations in the SGCD gene; and (ii) enough data available in the clinical records to answer ∼70% of the variables of the survey about disease onset and progression, presence or absence of cardiac and respiratory involvement and muscle function status at last clinical assessment.The inclusion criteria for the study were: (i) genetically confirmed diagnosis of delta-sarcoglycanopathy by identification of two heterozygous or one homozygous pathogenic mutation in the 20 The need for ventilatory support was also collected as well as the age at which it was started. A copy of the survey is provided in the Participating centres completed a survey for each of the patients included in the study. The survey collected demographic, clinical and genetic data of all patients. We also collected information about the muscle biopsy if performed, including morphological features and levels of protein expression measured by immunohistochemistry or immunofluorescence. Cardiac involvement was defined according to international guidelines based on the presence of left ventricular ejection fraction lower than 50% and/or fractional shortening lower than 25% and/or the existence of morphological abnormalities in the ventricular walls evaluated by echocardiography or the existence of cardiac conduction defects identified using ECG or Holter monitoring.SGCD gene were centrally reviewed by experienced geneticists from HSCSP (L.G.-Q. and P.G.) to confirm pathogenicity and predict protein expression. We considered nonsense, frameshift and canonical splice site mutations as non-protein producing since these mutations will cause a disruption of the reading frame or a considerable shortening of the transcript resulting in mRNA elimination by nonsense-mediated decay or aberrant protein degradation. A sequence-based prediction of impact the of the missense mutations on protein function and/or expression was performed using different computational approaches assessing sequence conservation; for example, SIFT21 and PANTHER-PSEP,22 as well as sequence and structural features such as PolyPhen-223 and MutationTaster.25 Deep intronic variants were considered compatible with protein production since these variants even when resulting in abnormal splicing they usually produce a residual amount of wild-type transcript.27Mutations in the t-test or Mann–Whitney test for quantitative variables. We used a Cox proportional hazard regression model for the analyses of time to wheelchair. P was considered significant if lower than 0.05. Hierarchical analysis and graphical representation as a heatmap of muscle strength measured using the MRC scale was performed using R software v.3.1.3 as previously described.28 Statistical analysis was performed by J.D.-M. and J.A.-P. using SPSS® Statistics software v.21 from IBM®.Quantitative variables were analysed using the Shapiro–Wilk test to verify the normal distribution. Comparison between the different subgroups of patients was performed using the Chi-squared test for categorical variables and the Student's The data that support the findings of this study are available from the corresponding author on reasonable request.29–34 After reviewing the results of 9610 patients, no patients with delta-sarcoglicanopathy were found.We contacted 128 paediatric and adult neuromuscular units and/or neurology departments around the world and identified a total of 30 patients with a confirmed genetic diagnosis of LGMDR6. We excluded seven patients because clinical data were not available or updated. The 23 patients included were from nine different countries: Spain, UK, Italy, Hungary, Turkey, Iran, Pakistan, Brazil and Canada. Additionally, we also contacted international centres that have lead research projects using NGS to sequence large cohorts of patients with neuromuscular diseases.Among the 23 identified patients with confirmed diagnosis of LGMDR6, 10 were males and 13 were females from 18 different families. Consanguinity was present in 20 patients (87%) and 12 patients (52.2%) had another relative affected by the disease.n = 21, 91.3%) were symptomatic at the time of data collection. However, two patients were considered presymptomatic as they had neither symptoms nor signs of neuromuscular involvement on clinical examination. These two patients were 4 and 5 years old, respectively, and relatives of other affected patients. Among the 21 symptomatic patients, median age at onset of symptoms was 5.0 years (range: 2–24 years). Diagnostic delay, defined as the time from onset of symptoms to the confirmed genetic diagnosis of the disease range from 1 to 37 years (median: 6.5 years). Median diagnostic delay in patients with onset of symptoms before 2010, when genetic testing was done using Sanger sequencing was of 10.0 years , while diagnostic delay in patients with onset of symptoms after 2010, when NGS studies started to be used in the diagnosis process of LGMD was of 2.0 years . These differences were statistically significant .Most of the patients  and distal  weakness was also present from early stages of the disease, especially in patients with no remaining protein expression , calf hypertrophy  and generalized muscle atrophy  were also described. Tiptoe walking due to Achilles tendon contractures was reported in 15 patients (71.4%) at a median age of 9.5 years (range 3–16). Foot deformities, mainly pes cavus due to early lower limb distal muscle weakness was also observed  with a median age of diagnosis of 13.0 years (range 11–17). Dilated cardiomyopathies were present in three patients (60%) and heart rhythm abnormalities were present in two patients. The last cardiac assessment showed median left ventricle ejection fraction of 40% (range 30–58). Four out of these five patients were under treatment because of the cardiac involvement. Median forced vital capacity at last assessment was of 62.0% (range 44–84). Four patients (19%) required ventilatory support from a median age of 20.5 years (range 13–24) and were using it a median of 9 h per night (range 6–15). None of the patients had a tracheostomy.P = 0.295, Mann–Whitney test) or in the time of progression of the disease  between patients with residual protein expression and those with no protein expression.A muscle biopsy was performed in 14 patients (60.9%) at a median age of 10.0 years (range 2–34) and a median time of disease duration of 4.5 years (range 1–30). Increase in the amount of fibrotic tissue and presence of necrotic muscle fibres were the most frequent features . Inflammatory infiltrates were observed in 28.6% of biopsies . ResiduaSGCD gene  no protein expression if patients had an undetectable expression of the sarcoglycan complex measured by muscle immunohistochemistry or immunofluorescence or carried two frameshift or nonsense mutations; and (ii) residual protein expression detected by immunohistochemistry or immunofluorescence.To study the potential genotype–phenotype correlation in delta-sarcoglycanopathies, we studied the impact of the mutations on the residual protein expression in 14 muscle biopsies. Based on these data we estimated the residual protein expression in the nine remaining patients without muscle biopsy but who shared the same variants in the P = 0.49, Student’s t-test).We did not observe differences in the duration of the disease, defined as the time from onset of symptoms to last clinical evaluation, between both groups of patients   than patients with residual protein expression    and lostpression . Consequox test) . The corP = 0.09) or the needed for respiratory support  between both groups.There were not significant differences in the prevalence of cardiac involvement  was supposed to be a very rare disease. In recent years, a series of studies have analysed large cohorts of patients with undiagnosed genetic muscle diseases using genes panels and/or whole exome sequencing. The frequency of LGMDR6 in these studies is very low or absent.40 Furthermore, published LGMDR6 reports so far have just included a few cases or isolated patients.41 A recent publication analysing a large cohort of European sarcoglycanopathies showed that only 1.5% of the cases were delta-sarcoglycanopathy.8 This previous evidence confirms that delta-sarcoglycanopathy can be considered an ultra-rare disease that it is defined as having a prevalence lower than 20 patients per million of population or one case per 50 000 habitants. In the case of delta-sarcoglycanopathy, the prevalence could be much lower probably in the order of one case per million of habitants. We do not have a clear explanation why patients with delta-sarcoglycanopathy are so infrequent compared to patients with other forms of sarcoglycanopathy. One potential explanation could be that mutations in this gene may be lethal during foetal development. If that were the case, this could be more probably related to a cardiac problem than to a skeletal muscle problem as there are no patients with congenital myopathy described so far. On the contrary, it is well known that mutations in the SGCD gene can be associated to developmental problems of the heart in preclinical animal models, such as zebrafish, and also to progressive cardiomyopathy both in humans and animal models.42–45 From a functional point of view, both the beta and the delta subunits are the core of the sarcoglycan complex suggesting than lack of beta-sarcoglycan should be as deleterious as the delta subunit for development, but patients with mutations in the SGCB are much more frequent than patients with mutations in the SGCD gene.46–48 Another reason could be that SCGD is devoid of sequence more prone to mutate compared to other sarcoglycan genes, but we observed that mutations in the SGCD were located in all the exons, same as the other sarcoglycan genes, suggesting that no particular exons are lethal if mutated.8Sarcoglycanopathies, as a group, are one of the most common recessive LGMDSGCA, SGCB and SGCG genes have been classically associated with a rapid progression of muscle weakness leading to severe disability, loss of ambulation in the second decade of life and frequently presenting cardiac and respiratory involvement.49 However, disease progression can be more heterogeneous as several patients, especially those with mutations in the SGCA gene, have a later onset of the disease and can loss ambulation later in life or even remain ambulant.18 To broaden the clinical presentation, patients with alpha-sarcoglycanopathy and isolated hyperCKaemia have been described.50 In contrast, patients with beta and gamma sarcoglycanopathies have in general a more homogeneous clinical picture with an early loss of ambulation.51 Our data shows that delta-sarcoglycanopathy is similar to other sarcoglycanopathies, with early onset in the first decade of life, rapid progression and loss of ambulation in the second decade of life in a high percentage of cases. However, we have observed that residual protein expression could influence clinical progression and severity of the disease. Indeed, 77.8% of patients with no remaining protein expression and 87.5% of patients with symptoms onset before the age of 5 years were wheelchair-bound at the age of 15 years. On the other hand, 21.4% of patients with residual protein expression and 25% of patients with symptom onset after the age of 5 years lose ambulation before adolescence. Moreover, only 42.9% of patients with residual protein expression ended up in a wheelchair and in all cases after the age of 10 years. These data support that residual protein expression is associated with a later onset of the disease and better prognosis as is shown in LGMDs produced by mutations in 47 Mutations in the SGCD gene should therefore translate into a complete disruption of the complex leading to absence of expression of all sarcoglycan subunits in the muscle membrane and, consequently, to a more fragile membrane suffering more damage after every contraction, as has been described in the murine model of the disease.52 In fact, the delta-sarcoglycan murine model is characterized by severe muscle weakness and cardiac involvement.54 However, in this cohort some of the patients for whom a muscle biopsy was available (8 of 14) did have residual protein expression of the sarcoglycan subunits identified by immunohistochemistry. These patients presented with a later onset and less severe disease, suggesting that residual expression of the different components of the complex was enough to produce a better prognosis.Beta and delta-sarcoglycan subunits form the core of the sarcoglycan complex and therefore are essential for its assembly.56All patients were homozygous for one pathogenic variant and 87% had a history of consanguinity in the family. The most frequent types of mutation were frameshift mutations (up to 39.1% of the patients); however, 30.4% of patients carried a large deletion affecting one or more exons, which might be missed by Sanger sequencing and could only be confirmed by a quantitative technique such as multiplex ligation-dependent probe amplification (MLPA). It is therefore important to use MLPA in those cases with a high suspicion of the disease based on clinical and/or muscle biopsy data if Sanger sequencing does not identify any pathogenic mutation.58 However, there are differences in the frequency and the type of cardiac or respiratory involvement depending on the mutated gene. For example, it is known that cardiac involvement in the form of a cardiomyopathy is more frequent in patients with beta-sarcoglycanopathy, than in patients with alpha or gamma-sarcoglycanopathy.59 Loss of delta-sarcoglycan is associated with cardiomyopathy is several preclinical animal models. For example, the different BIO14.6 hamster strains that harbour a deletion in the SCGD gene can develop either dilated or hypertrophic cardiomyopathy.60 In mice, delta-sarcoglycan deficiency leads to cardiomyopathy that is aggravated as a result of coronary artery vascular irregularities probably as a consequence of the absence of delta-sarcoglycan in the smooth muscle.61 Delta-sarcoglycan is key for normal cardiac development in zebrafish, leading to left–right asymmetry of the heart and disorganization of the intracellular myofibrils.62 There has been a long discussion related with the existence of isolated dominant cardiomyopathy produced by heterozygous mutations in the SGCD gene. Tsubata et al.63 found a single missense mutation  associated with severe dominant cardiomyopathy in a family with three patients carrying this mutation. However, the pathogenic relevance of the p.Ser151Ala variant was challenged when this mutation was discovered in a large consanguineous family homozygous for p.Ala131Pro suffering from LGMDR6/2F but lacking any signs of cardiac disease in family members carrying the p.Ser151Ala mutation.64 Additionally, the knock-in of p.Ser151Ala caused a rather mild phenotype of cardiomyopathy in mice.45 Based on these data, the pathogenic potential of the p.Ser151Ala missense mutation to cause familial cardiomyopathy is unlikely. Surprisingly, we have only observed cardiac involvement in five patients in our cohort (23.8%), which was not related to a longer disease duration or with the level of residual protein expression. None of the patients had previous history of coronary heart disease. These data indicate that, in humans, delta-sarcoglycan defiency is not always associated to cardiac problems, although a longer follow-up of the patients included here is needed to confirm that they do not develop cardiac problems with age. Respiratory insufficiency requiring ventilatory support was only observed in four patients (19.0%) and was also not related with a longer disease duration or remaining protein expression. These data confirm that periodic assessment of cardiac and respiratory involvement is needed in all cases regardless of a patient’s age and the clinical status.Cardiac and respiratory involvement requiring ventilatory support is frequent in patients with sarcoglycanopathy.8 Moreover, for some patients, protein expression was estimated based on the results observed in patients sharing the same mutations in whom protein expression was studied in muscle biopsy. Although it is probable that protein expression is similar between patients with the same mutations, this is speculative and therefore these results should be interpreted cautiously. Despite our effort in contacting as many neuromuscular units as possible, we could have missed some delta-sarcoglycanopathy patients for inclusion into our study. An international registry for patients with mutations in the sarcoglycan genes, specifically in this case for patients with mutations in the SGCD gene could be useful to identify more patients. However, our series is the largest described so far and includes patients from different countries and ethnicities although there were no patients from Africa or South-East Asia.Our study has some limitations. First, data were collected retrospectively and there were some missing data in all cases. Second, quantification of protein expression was carried out through immunofluorescence performed at each centre with different antibodies and techniques including immunohistochemistry or immunofluorescence. Quantification of protein expression using western blot would have been better and could have helped us to identify a cut-off point predicting a more severe progression, as has been described in other types of sarcoglycanopathy.SGCD gene. We have identified that residual protein expression is associated with a later onset of disease and milder phenotype with later loss of ambulation. These data should be useful for the design of natural history studies and for clinical trial design, including gene replacement therapies, that are currently under development for other types of sarcoglycanopathy.In conclusion, our study provides new and relevant information that widens the knowledge on the clinical and genetic features of patients with mutations in the This investigation was sponsored by a grant from the Spanish Ministry of Health, Fondos FEDER-ISCIII PI18/01525 to J.D.-M. J.A.P. was supported by the ‘Rio Hortega’ grant (CM19/00178), Acción Estratégica de Salud (EAS), Instituto de Salud Carlos III (Spain). B.M. and K.H. were supported by grants NKFIH 119540 and EFOP-3.6.1–16-2016–00004. L.G.-Q. and P.G. received funding from FIS PI8/01585, funded by ISCIII and FEDER, ‘Una manera de hacer Europa’.All authors report no competing interests.Brain online.awab301_Supplementary_DataClick here for additional data file.


Advanced Parkinson’s disease (APD) cannot be treated efficiently using the classical medications however, in recent decades invasive therapeutical methods were implemented and confirmed as effective. One of these methods makes it possible to continue the levodopa (LD) supplementation as a gel administered directly into the upper intestine. However, there are a number of unanswered questions regarding this method. Therefore, we retrospectively analyzed a 10-year period of selected patients that were treated with levodopa/carbidopa intestinal gel (LCIG). We included all APD patients with motor fluctuations and dyskinesia at presentation. LCIG treatment was started in 150 patients: on average these patients received LD for 10.6 ± 4.4 years with a frequency of 5.2 ± 1.0/day until the introduction of LCIG. The estimated and the real LCIG dose differed significantly (mean: 1309 ± 321 mg vs. 1877 ± 769 mg). The mean duration of LCIG administration was 19.8 ± 3.6 h, but in a number of 62 patients we had to administer it for 24 h, to maximize the therapeutic benefit. A carefully and individually adjusted LCIG treatment improves the quality of life of APD patients, but questions remain unresolved even after treating a large number of patients. It is important to share the ideas and observations based on the real-life experience related to the optimal timing, the appropriate dose and duration of administration of the LCIG. Parkinson’s disease (PD) is a neurodegenerative disease with significantly improved therapy in recent decades; therefore even patients with advanced PD (APD) now have access to effective treatment options, even though these represent invasive methods ,2. In thThe levodopa/carbidopa intestinal gel (LCIG), containing levodopa (20 mg/mL) and carbidopa monohydrate 5 mg/mL) is a treatment developed for patients with advanced PD (APD). The LCIG delivered continuously into the upper intestine via percutaneous endoscopic gastro-jejunostomy (PEG-J) provides more stable plasma levels compared to oral LD  mg/mL is,7. Timelon and off phase) as measured on the Hoehn and Yahr scale, and on the MMSE  score. The exact doses of LD, dopamine agonists, monoamine oxidase B inhibitor (MAO-Bi), catechol-O-methyl transferase inhibitor (COMTi) and/or amantadine and any kind of motor fluctuations and dyskinesias were recorded as well. For testing the efficiency of LCIG therapy and also for the adjustment of titration of doses patients were hospitalized. Calculation of estimated LCIG doses  was performed according to literature recommendations [We retrospectively evaluated all APD patients with motor fluctuations and dyskinesia  at presentation, that were treated between June 2011 and June 2021 with LCIG by the multidisciplinary team of the 2nd Neurology Clinic in Târgu Mureș. In this tertiary center the only available device-aided therapy during the evaluated period was LCIG. We recorded the demographic and clinical aspects, disease and treatment duration, severity .Statistical analysis was performed using the Prism 8.0 software package . Depending on the type of data, descriptive statistics, Fisher’s exact and Mann—Whitney tests were used, the level of statistical significance was During the aforementioned 10 years period we started LCIG treatment in a total of 150 patients out of which 81 (54%) were male. The mean age of the patients was 63.97 ± 8.16 years, and the MMSE score was 26.1 ± 2.39 points. Between the onset of the disease and the introduction of LCIG 10.95 ± 4.44 years elapsed, with half of the patients deciding to accept therapy in less than one month. The spectrum of motor complications (fluctuations and different subtypes of dyskinesia) is presented in The last utilized add-on therapy is detailed in on and off states, according to the Hoehn and Yahr scale (p < 0.001) are all presented in Before the introduction of LCIG patients received LD treatment for a period of 10.6 ± 4.4 years until; in the same period levodopa was divided in 5.2 ± 1.0 doses per day. The last LD dose before PEG-J, the calculated and final LCIG dose which offer a significant improvement of clinical status, both during the The initial parameters and dosing of LCIG therapy  are shown in The difference between the theoretically calculated LCIG and the actual dose used  is presented in Parkinson’s disease is the most common movement disorder, besides mitochondrial dysfunction and age-related synuclein aggregation the role of neuroinflammation and increased oxidative stress can also be suspected ,13. In PImproving the adverse pharmacokinetics of levodopa is a constant challenge ,16,17, wAs is often observed in a hospital setting, different add-on agents have different degrees and qualities of beneficial  effects on each patient. The extent to which the newer preparations improve the clinical picture in the late stages  and the extent to which they are suitable for delaying the introduction of various device aided therapies is not yet clear .There are few data in the literature on the incidence and severity of various motor and non-motor complications in patients with APD and the proportion of patients considered to be eligible for device-aided therapies in movement disorders centers ,27. In tWhat are the upper limits of conventional dopaminergic medication, especially LD doses , without compromising patient/relative compliance, or how are these influenced by access to various add-on therapies or device-aided therapies?How long is it advisable to “insist” on available combination therapy options and what doses should be used in case of these treatments (the concept of “optimized medication”); also, in what way are these treatment options influenced by access (or lack of) to different device-aided therapy options?How can the severity and profile of various motor and non-motor complications be accurately assessed and what is their real impact on quality of life?How can one recognize the time when motor complications can no longer be controlled or ameliorated with standard oral/transdermal therapy ?What is the best time to start LCIG so that the benefits are maximized ?Similarly, only a small number of publications aim to evaluate the real-life data of patients with APD (the proportion of patients considered eligible for LCIG in tertiary centers serving a large number of patients). Under these conditions, the clinician who provides long-term monitoring of patients with advanced disease and is forced to constantly adjust the therapeutic strategy may face the several clinical practice challenges:off and/or 1 h of severe dyskinesia despite 5 times daily combination levodopa therapy, one of the available device-aided therapies should be considered [off period in 150 patients and 2.96 ± 0.84 h of dyskinesia in 95 cases (out of which 44 patients presented an average of 3 ± 0.82 h of diphasic dyskinesia) can be found. This system of criteria, which became well-known as the “5-2-1 rule”, provided a good basis for general neurologists working in non-specific centers. However, due to the lack of clear-cut protocols, the “5-2-1 rule” was supplemented by later resolutions [Although LCIG therapy was registered in Romania in the fall of 2009 , we usednsidered . In the olutions ,34. Our olutions ) shows tIn our previous publications we also showed that available add-on therapies were used significantly more frequently in our patients immediately prior to the introduction of LCIG . This trPrevious studies have shown that nearly two-thirds of patients with PD have taken less than 80% of the medications prescribed by their physician . FurtherWhen discussing the optimal use of LCIG, everybody should be aware that there is a lack of therapeutic recommendations to select the appropriate device-aided therapy. Analyzing the data in the literature, we can see that even under the conditions of highly developed healthcare (ex. in Norway), where all three device-aided therapies are available, there are significant differences between regions regarding the use of these therapies . SimilarIn light of all this, it is conceivable that in the selection of each device-aided therapies , more emphasis may be placed on the alternative that allows monotherapy (in appropriately selected patients) .At the start of clinical use of LCIG, the recommended treatment duration was a minimum of 16 h . Over thRegarding the time of initiation, the titration of LCIG  and the management of previous dopaminergic medication is largely based on the expertise of the treating physician. The introduction of LCIG and trial dosing should be timed in a way that the patient does not feel rushed, but should not be delayed so that the expected efficacy is appropriate. In this way the number of subsequent interruptions can be reduced. One goal would be for the patient to receive the correct dose of LCIG monotherapy because, as COSMOS has demonstrated, this treatment alone can have a sufficiently effective effect and improve compliance. For our patients, the titration time was approximately one week in hospital settings. The COSMOS study  provides evidence that LCIG monotherapy is a feasible and effective long-term option for the treatment of patients with APD ,44.A retrospective analysis of patients enrolled in the GLORIA study showed that the adverse event rate was higher in patients requiring doses greater than 2000 mg/day of LCIG, compared with those on doses below 2000 mg/day, although the safety profile and efficacy were acceptable . RegardiIn our previous article, we followed for 18 months the effectiveness of LCIG in 40 patients with severe disabling dyskinesia and we showed that 4 of the 6 patients in whom treatment was discontinued (including a patient with repeated psychotic episodes) received ≥2000 mg/day LCIG . The tolIn a recent analysis of the reasons for discontinuation of treatment, we pointed out that when initiating patients on LCIG, it would be useful to identify a “risk profile” for fallout (which is supposed to increase over time). Thus, in patients with multiple comorbidities, in those with cognitive impairment, in those who require significantly higher doses compared to the calculated doses and/or continuous administration for 24 h, in those with dopaminergic side-effects, etc., additional measures such as more frequent evaluations, greater involvement of the aftercare surveillance could extend the long-term therapeutic benefit of LCIG. These observations are consistent with the idea previously held by other experts that the benefits of LCIG treatment might be greater in patients with less advanced disease (in terms of disease duration and severity of motor complications). .In the present survey, we present the experience of a University Teaching Hospital where the only available device-aided therapy is LCIG. Our previous analyses have demonstrated that Parkinson’s patients in our center are treated according to international guidelines and with a strategy similar to that followed by professionals in other countries ,49,50. DA carefully and individually adjusted LCIG treatment significantly improves the quality of life of APD patients, but questions remain unresolved even after treating a large number of patients for 10 years. We believe that sharing the ideas and observations based on the real-life experience of high-traffic centers can further refine and improve patient selection criteria as well as the practical considerations of the implementation of LCIG treatment .


Coronavirus (SARS-CoV-2) is a respiratory infection virus that was first detected in Wuhan, China. The virus causes COVID-19 disease and the outbreak was recognised as a pandemic by the World Health Organization (WHO) in March 2020. SARS-CoV-2 virion was first imaged using cryo-electron microscopy by the Chinese Center for Disease Control and Prevention (CDC). Atomic Force Microscopy is a unique technique that can allow imaging of biomolecules under different conditions. In this work, we used Atomic Force Microscopy to characterize SARS-CoV-2 on tissue culture polystyrene (TCPS) and glass coverslip surfaces. We isolated SARS-CoV-2 and drop casted it on coverslip glass and tissue culture polystyrene surfaces. We analyzed height profiles, density, and aggregation behavior of the virion on glass and polystyrene surfaces. We observed the coffee ring effect on the drop casted samples and close packing of virions near the coffee rings on both surfaces with relatively higher virion distribution on the tissue culture polystyrene (TCPS) substrates. We compare virion agglomeration on the two types of surfaces. Finally, we applied ethanol disinfectant to virions on the surface to visualize the effect of ethanol and image the ultrastructure of SARS-CoV-2. We studied the density and aggregation behavior of virions on TCPS and glass substrates, surface functionality, coffee ring effect on both surfaces and the effect of ethanol-based disinfectant on the virion structure using atomic force microscopy. While virologists and other health professional desperately seek to develop a vaccine for the virus, understanding the three dimensional structure of the virus has become a crucial factor in understanding how the different components of the virus interact with cells and other materials and environmental conditions. The ability to control and manipulate interactions with the virus can greatly assist us in designing procedures and products to improve public health by limiting and halting the spread of the disease.COVID-19 is an infectious disease caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2).20 The gray surface is a spherical envelope that surrounds the nucleus of the virus, containing genetic material. The grey envelope includes structural proteins called envelope (E), membrane (M) and spike (S) glycoproteins.21 Spike proteins (S) which are ∼20 nm in length have been shown to be extremely important for entrance of the virus into the cells.22–24 The diameter of the virion varies between 70 to 140 nm and average diameter is 100 nm.25–28 There is a review on surface behavior of corona virus family29 but the surface behavior of SARS-CoV-2 is different and is still under investigation. SARS-CoV-2 has been found to be more stable on plastic and stainless steel surfaces.30 SARS-CoV-2 has been found to be more stable on smoother surfaces,31 but the surface functionality perspective was missing. In another study, the stability of SARS-CoV-2 at different temperatures, surfaces and their inactivation using ethanol based and other disinfectants were investigated.32 The lack of any suitable molecular visualization techniques have restricted investigations at the virion/molecular level. To date, due to its miniscule size range, SARS-CoV-2 virus has been mostly observed with cryo-electron microscopy.1,21,33,34 However, this technique has several disadvantages viz, (a) the samples are usually in the form of thin slices of infected tissue and require a conductive coating (b) the imaging is performed at very low cryogenic temperatures and (c) erroneous identification of coronavirus directly in tissues have been reported35 and (d) the images are 2 dimensional.The SARS-CoV-2 virus is the newest member belonging to corona virus family. We can utilize the popular colour-coded, illustrated visualization model of the SARS-CoV-2 virus provided by the U.S. Centers for Disease Control and Prevention (CDC) as shown in in situ experiments to observe dynamic biological activity at high resolution.36 In this technique, the sample do not require to be coated and the rendered images have the added advantage of being 3 dimensional. Previously, atomic force microscopy was used to characterize the ultra-structure of SARS coronavirus,37 and in a parallel study by Kiss et al.,38 still to be published, the authors claim to have characterized SARS-CoV-2 virus in different environments using Atomic Force Microscopy. They claim to have obtained high resolution topographical structural information and data on the thermal sensitivity and nanomechanical dynamics of SARS-CoV-2.Atomic Force Microscope (AFM) is a unique technique that enables imaging of biological molecules in their native environment at the molecular scale, and in some cases, has even allowed researchers to perform 39 on both surfaces and effect of ethanol-based disinfectant on virion structure.In this work, we isolated SARS-CoV-2 virions to investigate the topographical and phase images of the virion on tissue culture polystyrene (TCPS) and glass cover slip substrates using atomic force microscopy. We studied the density and aggregation behavior of virions on both substrates, surface functionality and coffee ring effect2.2.1−1 penicillin, 50 μg mL−1 streptomycin added  Dulbecco's modified Eagle's medium . Since the clinical samples were obtained via RT-PCR, only the samples showing the smallest Ct (cycle threshold) were selected for virus isolation stage. The virus isolation step was performed in a class 3 biosafety cabinet  in a biosafety level 3 (BSL-3) laboratory in the Virology Department, Faculty of Veterinary Medicine at Fırat University, Turkey. Following filtration through a 0.22 μm sterile membrane filter , nasopharyngeal swab specimens were inoculated into African green monkey kidney  cells. Cells were maintained at 37 °C in 5% CO2 in the presence of Dulbecco's Modified Eagle's medium containing 2% fetal bovine serum, l-glutamine, 100 U mL−1 penicillin and 100 μg mL−1 streptomycin. The infected cells were monitored daily for cytopathic effect. Cytopathic effects were observed in Vero E6 cells 36 hours after inoculation and 80% of the cells were lysed at 72 hours. Before performing the centrifugation step, the cell suspension was frozen at −80 °C and thawed several times. The nuclear fraction was then separated from the lysed by centrifugation  at 4500 rpm for 20 min at 4 °C. The virus containing supernatant was collected and stored at −80 °C as SARS-CoV-2 virus stock solution.Informed consents were obtained from human participants of this study. Nasopharyngeal swabs taken at an authorized diagnostic laboratory for COVID-19 from individual patients in Turkey, who were confirmed COVID-19 positive, by real-time reverse transcription polymerase chain reaction (RT-PCR) and transferred with a mixture of 10% heat-inactivated fetal bovine serum , 50 U mL2.2All sample preparations and imaging studies were carried out in a BSL3 Class Laboratory.2.2.140 Both types of substrates were rinsed with a copious amounts of Milli-Q water .For AFM sample preparation we used cover glasses from Thermo Scientific and CELL Star tissue culture polystyrene cell culture dishes as substrates. For the polystyrene (PS) surface substrates, oxygen containing plasma treatment was performed to efficiently oxidized the surface. This treatment has been shown to enable better cell adhesion by introducing biologically relevant chemistry.2.2.2A 4 μL drop of Milli-Q water was placed on surfaces to measure the water contact angle. Three different positions on the sample were tested to assure the accuracy. TCPS and glass substrate water contact angle was measured to be 49.8° and 31.9°, respectively. We measured the water contact angle of polystyrene surface without any treatment as 74.6°, confirming that the TCPS surfaces have active functional groups on their surfaces.2.2.320 μL of purified SARS-CoV-2 solution was drop casted on to coverslip and TCPS dishes. Each sample was incubated for 15 minutes and then rinsed three times with a copious amount Milli-Q water while holding each sample about 30° angle to allow the excess water to flow evenly over the surface.The samples were dried overnight in Petri dishes and were then inactivated by exposing the samples to UV light for 30 minutes. The samples were loaded to AFM after drying overnight.2.2.4−1 force constant. We used 70% amplitude setpoint and 0.5 Hz scanning rate for imaging. After completion of our experiments, the samples were bleached with 5% NaOCl solution for chemical destruction of the virus and safe disposal of samples.Atomic force microscope imaging was carried out using a portable ezAFM  also located in a BSL3 Class laboratory. For general imaging, dynamic mode scanning was performed using Nanosensors PPP-XYNCSTR cantilevers with 7.4 N m2.2.5Data was analyzed using Image Analyzer Software . To avoid any loss of any of finer details in the images, no filters or smoothing operations were applied to the original data. Heights of virions were measured at the center of the particles relative to the substrates.3.3.1We imaged the dried samples on TCPS and glass substrate with the AFM in dynamic mode which provided the added advantage of phase imaging. Phase images in AFM refers to the phase lag between cantilever driving signal (at the back of cantilever where the drive piezo is located) and the signal from cantilever oscillation at the tip (where the cantilever tip interacts with the surface). Phase lag reflects the changes in mechanical properties of the surface as qualitative data.We obtained high resolution SARS-CoV-2 phase images that could be linked to qualitative local mechanics of virions and corresponding topography image on TCPS substrates as in 3.2We had to adopt different approach for height measurements of the virions. One, which took into account (a) the possibility of compression of the low modulus of the virions by the AFM tip as it scanned across the surface and (b) the regions surrounding the virions may have a layer of residual products from sample preparation process.et al.41 Although this is considerably smaller than the force applied in contact mode AFM there is still a possibility of some compression of the low-modulus virions by the AFM tip as it scanned across if we consider force–distances curves captured by Kiss et al.38 The height differences from middle top of the virion to the base were reported to range between 9 nm to 15 nm for glass substrate and 14 nm to 38 nm for TCPS substrate. These values are much smaller than diameter of SARS-CoV-2 virus, which were to vary from 80 to 120 nm.25 It therefore possible that (a) the drying of samples, causes the virions to flop flatter on to the surface and (b) the surface in the areas surrounding the virions could be coated with a by-product layer of intracellular content like proteins that are smaller than the virions. In order confirm the existence of such a by-product layer and obtain a more accurate measurement of the height of the virions, we employed a technique called nanoshaving, a method that was introduced by Liu et al.42The force applied by the AFM tip on the sample in dynamic mode scanning was calculated to be 0.2 nN by using a method proposed by Zhong Using the same AFM tip, we nanoshaved the sample by operating the AFM in contact mode and applying ∼250 nN force to the surface. This ensured any residues covered the substrate surface during incubation process was removed from the substrate surface as the tip scanned across the selected area in the image as illustrated in 2 area varied from 39 nm to 50 nm and 45 nm to 71 nm on the glass and TCPS surface, respectively and the height distribution for glass and TCPS surface are given in Representative AFM topography images of SARS-CoV-2 on glass substrate and TCPS substrate shown given in 3.32 area was counted as 153 ± 30 and 39 ± 30, respectively. The density of virions adsorbed onto TCPS surfaces was found to be ∼3.84 times higher than glass surfaces. SARS-COV-2 is an enveloped virus in which the surrounding envelope consists of a lipid bilayer that includes membrane proteins that are envelope (E), membrane (M) and the spike (S).21 We think active groups on the TCPS surface interacts with SARS-CoV-2 membrane proteins to give better adhesion compared to the glass substrates.We scanned at least 10 images on the samples to obtain a measure of the virion density on glass and TCPS surfaces from their topography images. The number of virions on TCPS and glass surface in 5 μm3.443 Virus aggregation-disaggregation is a complicated system and some studies have shown that virions tend to aggregate in order to increase their effective life under different environmental conditions.44 It is quite possible that the formation of aggregate structures of virions could lead to significant increases in the virulence transmission, as well as, prolong their effective life by acting as a protective barrier from environmental antiviral effects. It is also possible that greater aggregation of virions could be related to infecting a host cell with a higher virus load.Virions are known to agglomerate due to changes in environmental conditions such as virus particles which are released from cells.In this study, we tried to investigate SARS-CoV-2 aggregation behavior by analyzing the AFM topography images of uncoated substrate surface and virion coated surfaces in greater detail. SARS-CoV-2 exhibited dense aggregation behavior on TCPS substrate showing the presence of dimers, trimers, and larger grain multimers as marked in We were able to confirm the formation of aggregated virions and eliminate surface effects by comparing the surface morphologies before and after deposition of virion solution as shown in Agglomerated virions have larger mass compared to individual virions and, in the absence of any chemical interaction between surface and virions, as would be the case of the glass surface, the larger aggregates are more likely to be washed away surface during washing process. TCPS surfaces, on the other hand, have active groups on the surfaces that can interact and bind with virus membrane proteins. Consequently, the washing process is less likely to flush away the agglomerated virions from the surface. Another factor, TCPS substrate has a much rougher surface it could promote greater aggregation/cluster sites where virions get more surface area to interact/bind with the surface leading to stronger binding. This would explain why larger virion particles on TCPS surface would resist being flushed away in the washing process.3.5When it comes to deposition by drop casting, we can consider two different scenarios for the evaporation process (a) as constant contact area evaporation on hydrophilic surface and (b) as constant contact angle evaporation on hydrophobic surface. From our contact angle measurements (Section 2.2.2), both glass and TCPS surfaces, were shown to have water contact angles of less than 90°, from which we can consider both hydrophilic surfaces. Droplet evaporation mechanism on TCPS and glass substrate is based on constant contact area evaporation where evaporated on the solution-philic surface that causes coffee ring effect at the edge boundaries of droplet. An illustrative image that shows distribution of virions is given in 45 This causes more material to accumulate at the droplet edges as illustrated in 39Zone I represents large agglomeration region of virions due to coffee ring effect, Zone II represents close packing of virions along the surface and Zone III represents individual and agglomerated groups of virions bound to on surface during incubation period. We observed that the evaporation of droplets shows coffee ring effect on both surfaces. TCPS substrate exhibited less material accumulated to edge of the droplets visually. However, in the Zone III region, the virion density is higher on TCPS surface than glass surface, this as we mentioned earlier in Section 3.2, 3.4 was probably caused by the presence of active chemical groups on the TCSP surface that interact with virus membrane proteins resulting in a more evenly distribution across the 3 zones. A stronger coffee ring effect at the droplet boundaries with closely packed SARS-CoV-2 virions near the edge boundaries (Zone I) on glass substrates as shown as in The coffee ring model is based on the fact that the evaporation rate in a droplet is higher along the edges of the droplet and the liquid from the interior is replaced by the evaporating liquid.3.646 In order to eliminate probability of contamination, we used ultra-pure ethanol and Milli-Q water for preparation of 80% ethyl alcohol.Finally, we tried to observe the inactivation of SARS-CoV-2 virions by an ethanol-based disinfectant. Ethanol at 80% has been reported to be highly effective against SARS-CoV-2 by dissolving lipid bilayer membrane which holds the virus structure and exposing the intracellular content.We used the same dried samples used in early experiments to drop cast a 5 μL of 80% ethyl alcohol solution onto virion surface. The sample was left to dry overnight before imaging. We observed that all globular virion structures had ruptured and flattened, leaving and virion substructures exposed on surface. SARS-CoV-2 AFM topography images on TCPS surface before and after exposing to 80% ethyl alcohol is given in AFM topography image in 4.In this study, we have demonstrated it is possible to effectively use atomic force microscopy to visually investigate the effects of TCPS and glass substrates on the deposition and distribution on SARS-CoV-2. We drop casted solution that included SARS-CoV-2 onto both glass and TCPS substrates without any modification. We observed virion distribution on the surface to be denser and more uniform on TCPS substrate compared to glass and recorded density and height differences on the two surfaces. We found that, due to their biofriendly surface functionality, TCPS surfaces attracted around 4 times higher virion density on the surface compared to the glass surface. Virions deposition on both surfaces showed coffee ring effect and we observed increasing virion distribution gradient from centre of the droplet to boundary edges and closely packed virion distribution near to coffee ring. We also observed that SARS-CoV-2 on TCPS substrate showed denser multimer aggregation on the surface, but we were unable to observe clear aggregation behaviour in samples cast on glass substrates. The heights of SARS-CoV-2 virions on glass and TCPS substrates were measured in range of 42 to 47 nm and 62 to 67 nm respectively. Finally, we have obtained visual confirmation that exposing the virions to 80% ethyl alcohol inactivates viral particles by rupturing their protective spherical lipid bilayer envelope.There are no conflicts to declare.


Grm5) on an ASD-like phenotype using a rat model to better understand the role of mGluR5 signaling in behavior patterns and clinical manifestations of ASD. We observed that mGluR5 Ko rats exhibited exaggerated self-grooming and increased marble burying, as well as deficits in social novelty. Our results suggest that mGluR5 Ko rats demonstrate an ASD-like phenotype, specifically impaired social interaction as well as repetitive and anxiety-like behavior, which are correlates of behavior symptoms observed in individuals with ASD. The mGluR5 Ko rat model characterized in this study may be explored to understand the molecular mechanisms underlying ASD and for developing effective therapeutic modalities.Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by deficits in communication, and social skills, as well as repetitive and/or restrictive interests and behaviors. The severity of ASD varies from mild to severe, drastically interfering with the quality of life of affected individuals. The current occurrence of ASD in the United States is about 1 in 44 children. The precise pathophysiology of ASD is still unknown, but it is believed that ASD is heterogeneous and can arise due to genetic etiology. Although various genes have been implicated in predisposition to ASD, metabotropic glutamate receptor 5 (mGluR5) is one of the most common downstream targets, which may be involved in autism. mGluR5 signaling has been shown to play a crucial role in neurodevelopment and neural transmission making it a very attractive target for understanding the pathogenesis of ASD. In the present study, we determined the effect of genetic ablation of mGluR5 ( Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by deficits in behavior and communication –4. The cASD is a complex disorder, and a wide arsenal of factors have been involved in the pathophysiology of this neurodevelopmental disorder. Therefore, a multidisciplinary approach is the key to understand its etiology and for the design of rational interventions. Studies in animal models are aimed at simulating the core phenotypes associated with ASD to identify the mechanisms that underscore the entire spectrum of the disorder –15. Due SHANK3, MECP2, NLGN3, NRXN1 and FMR1  were subjected to a comprehensive battery of ASD-associated behavior tests at 12 weeks of age. Animals were group housed in a room with 12-hr light /12-hr dark Light/Dark cycle. Food and water were provided ad libitum. To control potential litter effect, one animal each of WT and Ko per litter was randomly selected for behavior phenotyping. This experimental design allows the use of standard statistical methods, such as t tests for analysis [Ko rats. Genotyping was performed by Transnetyx company  using custom designed probes having primer sequence, mGluR5 F CTTCATGAGGGTTGTACCTTCC; mGluR5 R GTGTGCACAGCTGAGACATAAG. Behavioral analyses were conducted by the trained observers that were blinded to the rat genotype. The study protocol was approved by the Animal Care and Use Committee of the University of Miami and was in full compliance with the NIH guidelines for the care and use of laboratory animals.Heterozygous breeders of mGluR5 analysis , 67. TheRats were brought to the testing room at least 30 minutes before the test. Each animal was videotaped for 10 minutes undisturbed after a 20-minute habituation time in their own cage (46 cm length × 23.5 cm wide × 20 cm high). The dimension of the open field (OF) chamber was 85 x 50 x 50 cm. Animals were placed in the middle of the arena at the start of the test. They remained in the arena for 10 minutes and their positions were tracked using Ethovision Version 11.5 . The center region was defined as an area that covered 42.35% of the total arena . Time spent in the center  and in the periphery of the arena  as well as the proportion of time spent in the perimeter of the box was used as an index of anxiety –70. We aAfter a 20-minute habituation period, animals were videotaped undisturbed for 10 minutes. The number of bouts of grooming sessions and the time spent in grooming was determined by two trained observers who were blinded to the experimental conditions. Self-grooming behaviors included wiping the nose, face, head, and ears with forepaws, as well as licking the body, anogenital area and tail , 73, 74.The social interaction test was performed using a three-chamber compartment comprising of three phases. In the first phase, the test animal was introduced to the middle compartment of the three- chambered apparatus and was allowed 5 minutes to freely explore left and right chambers each containing an empty Plexiglas cage. In the second phase, a non-familiar wild-type rat matched for sex and age (“stranger rat”) was placed in the cage in the right compartment. The test rat was placed in the middle chamber with closed connecting doors. The experiment started when the operator opened the doors, and the rat behavior was recorded over 10 minutes. Sociability was assessed by recording the time spent in the “stranger rat” chamber (in seconds), and in the empty cage chamber (in seconds) , 75, 76.Immediately following the second phase of the test, a novel wild-type rat matched for sex and age (“novel rat”) was placed in the cage in the left chamber while the “stranger rat” (from the second phase of the test) became familiar (“familiar rat”) to the test animal in this third phase. The search for social novelty was assessed by recording the time spent in the “familiar rat” chamber (in seconds) and in the “novel rat” chamber (in seconds) , 75, 76.MBT was used to measure repetitive and anxiety-related behaviors , 78. Thet-test. A Mann-Whitney’s test was performed for nonparametric samples. The threshold of statistical significance was set with a p < 0.05.Statistical analyses were conducted with XLSTAT and GraphPad Prism version 28. Quantitative variables were compared using a Student’s Ko rats self-groomed significantly more frequently and for longer durations compared to the WT animals (p < 0.01) . The mean grooming frequency was 3.31 ± 1.35 for the Ko rats compared to 1.16 ± 0.89 for the WT rats. Average grooming time was 15.25 ± 8.37 seconds and 7.42 ± 2.25 seconds in the Ko and WT rats, respectively. However, there was no statistically significant difference in grooming frequency and grooming time between Ko males and Ko females (p > 0.05).Excessive self-grooming has been observed in preclinical animal models of ASD representing repetitive behaviors and as expression of anxiety , 80. We Ko group than in the WT rats  . We did not find any statistically significant difference in rearing frequency between Ko males and Ko females (p > 0.05).Rearing frequency is the frequency with which the rodent stands on its hind legs in the open field, which might be a direct measure of anxiety , 81, 82.Grm5) on MBT in a rat model is still not known. Therefore, we subjected WT and Ko rats to MBT. We determined the number of marbles buried as well as the total digging time in Ko and WT rats. We observed that the Ko rats significantly buried more marbles compared to the WT animals   .The MBT allows in assessing anxiety-like, and repetitive behaviors. The effect of genetic ablation of mGluR5  . These results suggest that were no significant differences in the locomotor activity of Ko and WT rats. We then determined time spent in the center and periphery by the Ko and WT rats . There was no statistical difference between the Ko and WT rats in time spent in periphery and center (p > 0.05).The open-field test is used to measure locomotor activity and anxious behavior of animal models. We observed that there was no significant difference in the total distance travelled between the Figs 6A and 7A). We observed that there was a statistically significant difference in sociability between Ko and WT rats. The WT rats spent significantly more time in the stranger rat chamber compared to the empty chamber  (p < 0.001). On the contrary, the Ko rats showed no preference for stranger rat and empty (p > 0.05). These findings indicate that Ko rats show deficits in sociability. However, there was no statistically significant difference in sociability between Ko males and Ko females (p > 0.05).Using the three-chamber test, we assessed the sociability and social novelty behaviors . The WT rats spent more time in the novel rat chamber compared to time spent in the familiar rat chamber (p < 0.001). On the other hand, Ko rats showed no preference and there was no statistically significant difference in time spent in familiar and novel rat chambers (p > 0.05) . These results suggest the reluctance to social novelty of Ko rats as compared to WT animals. However, there was no statistically significant difference in social reluctance between Ko males and Ko females (p > 0.05).During the third phase of the three-chambers test assessing the search for social novelty, a novel rat age and sex-matched (“novel rat”) was inserted in the cage of the left chamber while the “familiar rat” remained in the right chamber (Grm5) on ASD-associated behavioral manifestations. In the present study, we subjected mGluR5 Ko rats to a battery of ASD-associated behavior phenotypes such as repetitive behavior, anxiety-like phenotype, social preference, locomotor activity and digging behavior. We observed that Ko rats several behavior deficits congruent with an ASD-like phenotype, suggesting the critical role of mGluR5 signaling in determining increased susceptibility to autism.Metabotropic glutamate receptor mediated signaling, especially through mGluR5, has been hypothesized to play a crucial role in the pathophysiology of ASD. In support of this concept, the pharmacological enhancement of mGluR5 ameliorates behavioral deficits in preclinical animal models of ASD . As mGluKo rats self-groomed significantly more frequently and for longer durations than the WT animals, thus exhibiting ASD-like phenotype.Aberrant self-grooming has been associated with ASD-like phenotype in animal models representing stereotyped repetitive behavior , 78, 83.Ko rats buried more marbles and spent more time in digging the marbles compared to WT animals showing repetitive and anxiety-like behavior. On par with these findings, increased marble burying has been observed in various ASD animal models. The loss of Tsc2 in Purkinje cells and deficiency of cyclooxygenase-2 (COX2) have been associated with ASD-like phenotype in rodent models [Marble burying test is commonly used to determine the repetitive and anxiety-like phenotype, which is one of the most important hallmarks of ASD. Both the number of the marbles buried, as well as the amount of time the animal spends exploring the marbles or digging are often studied, similar to the type of analyses performed in our study. We observed that t models , 87, 88.t models .Ko and WT rats. Therefore, we determined whether there are differences in the time spent in the center and periphery zones between the Ko and WT rats in the open-field test. However, we observed no significant differences in the time spent in the different zones. These results are in agreement with previous findings where no perimeter preference was observed in other animal models of ASD [Grm5), which needs to be explored in future studies.An important parameter in the open-field test is the total distance travelled. If there are no differences in total distance traversed between different strains, it facilitates making valid comparisons for various behavior tests, as locomotor activity is no longer a confounding variable in the data analysis. The significant differences in locomotor activity may skew the data by preventing analyses for time spent in certain designated zones of the maze as differences may be due to inactivity instead of due to the genotype effect. In the present study, we observed no statistically significant difference in the locomotor activity of the s of ASD . It is pShank2, Shank3, and Sarm1 Ko mouse models of ASD [Sarm1 knock-out mouse model of ASD and prevented ASD-like alterations in rats exposed to cannabinoid during the prenatal period [Ko rats exhibited sociability deficits as well as impaired social behavior thus emphasizing the importance of mGluR5signaling in normal brain functioning.Impairments in social behavior is one of the characteristic hallmarks that is observed in individuals with ASD. mGluR5 signaling has been shown to play an important role in social interaction. As a proof of this concept, the mGluR5 positive allosteric modulator (PAM) CDPPB has been demonstrated to ameliorate social interaction deficits in the s of ASD , 90, 91.l period . These iKo mice. It has been shown that mGluR5 ko mice show impairments in social interaction and altered marble burying activity compared to WT controls [Ko mice. Interestingly, mGluR5 Ko mice showed altered locomotor activity which may have confounded behavioral measurements. We do not observe such alteration in locomotor activity of Ko rats compared to WT animals thus suggesting this rat model can be used to understand the molecular underpinnings of ASD as well as testing the efficacy of future novel therapeutics for autism.In addition, our findings are in agreement with the data obtained from mGluR5 controls . HoweverKo rats display ASD-like phenotype. Our results highlight the crucial role of mGluR5 signaling in ASD-associated behavior deficits. One of the limitations of our study is the constitutive deletion of mGluR5 expression. Future studies using conditional Ko rats with deletion of mGluR5 only in the brain or specific areas of the brain can shed further light on the role of mGluR5 signaling in the pathophysiology of ASD.In summary, our findings suggest that mGluR5


Infections caused by multidrug-resistant microorganisms have become increasingly common in hospital environments around the world. Gram-negative bacilli stands out among multidrug-resistant bacteria mostly due to the production of carbapenemase enzymes which lead to resistance to most β-lactam antibiotics including the carbapenems. As a consequence, polymyxins have been reintroduced in the clinic as a last resort to treat infections caused by Gram-negative bacilli resistant to carbapenems. However, the only reliable method to evaluate the susceptibility to polymyxins is the broth microdilution, a laborious and time-consuming technique. Among infections caused by multidrug-resistant bacteria, bloodstream infections are the most worrisome as they can lead to sepsis and septic shock with high mortality rates.in vitro, this work aimed to evaluate a rapid method of polymyxins susceptibility either from colonies grown on agar or directly from positive blood culture bottles using the technology of MALDI-TOF.Considering the severity of sepsis and the need for a treatment guided for the susceptibility test Enterobacterales and non-fermenting Gram-negative bacilli resistant to carbapenems were obtained from patients attending a tertiary care hospital in southern Brazil and tested as follows: 122 isolates from colonies grown on agar plates and 117 isolates directly from spiked positive blood cultures.The method was based on the “direct on target microdroplets growth assay” (DOT-MGA) originally developed by Idelevich and collaborators with some modifications (Adapted DOT-MGA). Isolates of The adapted DOT-MGA presented 95 and 100% of categorical agreement considering the colonies grown on agar plates and directly from positive blood cultures, respectively.The adapted DOT-MGA test proved to be a reliable technique to evaluate the susceptibility to polymyxins to be used in microbiology laboratories with the MALDI-TOF equipment. Currently, antibiotic resistance is considered one of the main global threats, which directly impacts the public health and economy of nations. In the last decades, infections caused by multidrug-resistant (MDR) microorganisms have caused a significant increase in morbidity and mortality rates in patients, as well as an important impact on hospitalization costs . The extGram-negative bacilli stands out among the MDR bacteria mostly due to the production of carbapenemase enzymes which lead to resistance to most β-lactam antibiotics including the carbapenems. As a consequence, polymyxins have been reintroduced in the clinic as a last resort to treat infections caused by Gram-negative bacilli resistant to carbapenems. However, the increased use of polymyxins has led to the emergence of Gram-negative bacilli resistant to this group of antibiotics .Due to factors inherent to the polymyxins, such as an altered diffusion in agar, the methods used for the evaluation of the susceptibility to these antibiotics are limited. The disk diffusion and the gradient strips tests, widely used to determine susceptibility to antimicrobials in routine laboratories of microbiology, are not reproducible for polymyxins. In fact, the latter are not recommended by the main guidelines of antimicrobial susceptibility tests, the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing . The broThe MALDI-TOF technology was incorporated in the recent past years in the microbiology laboratories for the rapid and reliable identification of bacteria. More recently, the MALDI-TOF technology has been evaluated as a tool to perform the antimicrobial susceptibility test . In thisEnterobacterales and non-fermenters) resistant to carbapenems. We have evaluated the adapted DOT-MGA from colonies grown on agar plates and directly from spiked positive blood cultures.We aimed to evaluate an adapted version of the DOT-MGA using the conventional steel MALDI-TOF target (reusable) with other modifications to determine the susceptibility of Polymyxin B for Gram-negative bacilli  were obtained from patients (only one isolate per patient) attending a tertiary care hospital in southern Brazil. For the evaluation of the colonies grown on agar plates, 122 isolates were selected: 93 Enterobacterales resistant to carbapenems  and 29 non-fermenters resistant to carbapenems: 18 Pseudomonas aeruginosa, 9 Acinetobacter calcoaceticus-baumannii complex e 2 Pseudomonas fluorescens.All isolates  and 30 non-fermenters resistant to carbapenems: 15 Pseudomonas aeruginosa and 15 Acinetobacter calcoaceticus-baumannii complex.For evaluation of isolates directly from blood cultures bottles, 117 isolates were selected: 88 Enterobacterales) or Blood Agar plates (non-fermenters). Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as quality control.The reference method of BMD, according to the 4 UFC/mL. One milliliter of this suspension was inoculated with 4 ml of sterile human blood  into blood cultures vials and incubated in the automated BactAlert® system until positivity.Bacterial suspensions were prepared at 0.5 McFarland standard turbidity and diluted 1:1 with saline to obtain a final inoculum of around 10A volume of 3 ml obtained from the positive blood cultures was centrifuged at 3,000 rpm for 5 min, the supernatant was discarded and the pellet was mixed with 3 ml of saline. The procedure was repeated and the final bacterial pellet with saline was used to prepare the bacterial suspension for the Adapted DOT-MGA.Enterobacterales and to 1.5 MacFarland for non-fermenters. Subsequently, a dilution  was made with CA-MHB. A volume of 50 μl of the bacterial suspension was pipetted in a 96 wells microtiter plate and added of the same volume of a polymyxin B solution to obtain a final concentration of 2 μl/ml, which is the breakpoint for susceptibility according to EUCAST. Afterwards, 6 μl of the bacterial suspension with antibiotic, as well as the growth control without antibiotic, were spotted onto a reusable steel MALDI-TOF target plates , in triplicate.Adapted DOT-MGA was performed as previously described  with modTargets plates were incubated for 4 h at 35 ± 1°C in a plastic transport box  with approximately 3 ml of water in the bottom to allow a humidity chamber in order to prevent the microdroplets from evaporating. After incubation, the remaining liquid of the target plates was carefully removed from the spots using a tissue (Softy’s Elite®) by touching the top of the microdroplets. After a drying period (5–10 min), 0.5 μl of formic acid 70% and 1 μl of HCCA  were added onto the spots. MALDI-TOF MS analysis was performed in a Bruker MicroFlex LT mass spectrometer  using the Bruker MALDI BioTyper System . To interpret the results, the software MBT Compass – (Version 4.1.80) was used. The interpretation was performed as follows: when MALDI-TOF identified the bacteria in the spot containing 2 μg/ml of Polymyxin B (score ≥ 1.7), the isolate was considered resistant to the antibiotic, and when the identification was not achieved the isolate was considered susceptible. The test was considered invalid when the Adapted DOT-MGA did not identify the positive control in at least two repeated experiments.Results of Adapted DOT-MGA were interpreted as resistant or susceptible to Polymyxin B when at least two of the triplicates presented the same result. Discrepant results of Adapted DOT-MGA in comparison to BMD were repeated once and whether the result was the same, the result was considered final. Whether the repetition presented a result different from the first test, a third repetition was performed and the final result was considered for two equal repetitions.The results were classified in Categorical Agreement (CA) which indicates the number of isolates grouped in the same susceptibility category (susceptible or resistant) in Adapted DOTA-MGA and the standard BMD. Results of nonagreement were classified as Very Major Errors (VMEs) when isolates were susceptible to Polymyxin B in Adapted DOT-MGA and resistant in BMD, and Major Errors (MEs) when isolates were categorized as resistant in Adapted DOT-MGA and susceptible in BMD.Enterobacterales, 100% for Pseudomonas sp., and 88.1% for Acinetobacter calcoaceticus-baumannii complex) as compared with the standard BMD method  and susceptible by the Adapted DOT-MGA. The Adapted protocol also presented 4 ME (5.5%): 3 Escherichia coli (MIC = 2 μg/ml by BMD) and 1 Acinetobacter calcoaceticus-baumannii complex (MIC = 1 μg/ml by BMD) presented resistance to Polymyxin B by the Adapted DOT-MGA  in comparison to BMD, considering the valid tests (Pseudomonas aeruginosa and 1 Enterobacter sp) presented invalid results in the Adapted DOT-MGA.The Adapted DOT-MGA directly from positive blood cultures presented 100% of CA  to obtain the best results considering the maximum of 4 h of incubation. It is important to highlight that the Adapted DOT-MGA protocol uses the steel reusable MALDI-TOF target plate for the microdroplets incubation and the MBT Compass software for the interpretation of results, the same software used for bacterial identification, in contrast to the disposable MALDI-TOF targets and the MBT FAST prototype software used in the original DOT-MGA  in order to obtain the fastest protocol and we found that non-fermenters needed an increased bacterial concentration (1.5 McFarland and 1:1 dilution) in comparison to  DOT-MGA , 2021. TEscherichia coli which had MIC = 2 μg/ml by BMD, which is the breakpoint to distinguish between susceptibility and resistance to Polymyxin B, which could explain the false resistance results by DOT-MGA.The results of this study indicated a very satisfactory concordance between the Adapted DOT-MGA and the BMD, with more than 97% of general categorical agreement. Low rates (2.3%) of VME were found (only two isolates considering colonies from agar plates and directly from blood cultures) which indicates that the Adapted DOT-MGA presented a considerable improvement in comparison to the original protocol proposed by Idelivich for colistin. It has to be considered that these two isolates presented growth in one of the triplicates which may indicates that they may present subpopulations which lead to inconsistent results in the Adapted DOT-MGA method. We think that the result of the Adapted DOT-MGA should not be reported before the confirmation of the MIC by BMD when one of the triplicate indicate resistance. The ME found in the DOT-MGA method was mostly due to three Enterobacterales resistant to carbapenems but also non-fermenters resistant to carbapenems, which encompass all pathogens of Priority 1 for Research and Development of new antibiotics according to the World Health Organization  make difficult the protein extraction for the MALDI-TOF analysis.The Adapted DOT-MGA demonstrated to be an excellent and feasible alternative to rapidly evaluate the susceptibility to Polymyxin B either from colonies grown on agar plates as well as directly from positive blood cultures, with no additional costs or extra expertise for the laboratories that are equipped with the MALDI-TOF, reducing the turnaround time of the exam for up to 1 day and potentially contributing to the correct treatment of the patients with infections caused by MDR bacteria.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The study was approved at the ethical committee of Clinical Hospital of Porto Alegre (CAE) under the number: 49112821100005327 in August 25, 2021.PB conceptualized the study, designed the methodology, conducted the tests, made project administration, performed the investigation, the data analysis, and wrote the original paper. AB provided resources, made the review, as well as were the leadership and responsible for funding acquisition. FV, PW, NM, and AS helped to conduct the experimental tests. All authors contributed to the article and approved the submitted version.This study received a grant of “Fundo de Incentivo à Pesquisa e Eventos do Hospital de Clínicas de Porto Alegre (FIPE/HCPA) as well as of “INPRA—Instituto Nacional de Pesquisa em Resistência Antimicrobiana—Brazil (INCT/CNPq).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.